Durham E-Theses
Heavy metals and aquatic bryophytes: accumulation and
their use as monitors
Kelly, Martyn G.
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Kelly, Martyn G. (1986)
Heavy metals and aquatic bryophytes: accumulation and their use as monitors,
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HEAVY METALS AND AQUATIC BRYOPHYTES:
ACCUMULATION AND THEIR USE AS MONITORS.
by
Martyn G. K e l l y
B.Sc.
(London)
A t h e s i s s u b m i t t e d f o r t h e degree o f Doctor o f Philosophy I n t h e
U n i v e r s i t y o f Durham, England.
Department o f Botany, October 1986
The copyright of this thesis rests with the author.
No quotation from it should be published without
his prior written consent and information derived
from it should be acknowledged.
^5. FEB. 1987
This t h e s i s r e s u l t s e n t i r e l y from niy own work and has not p r e v i o u s l y
o f f e r e d i n c a n d i d a t u r e f o r any o t h e r degree or diploma
M.G.
been
Kelly
1986
Water i s the essence of e a r t h
P l a n t s are the essence of water
Chandagya Upanishad
c i r c a 600 - 400 B.C.
ABSTRACT
An e x p e r i m e n t a l study was made o f accumulation and l o s s of heavy metals
by t h e a q u a t i c moss Rhynchostegium r l p a r l o i d e s and o f t h e processes
The
involved.
i n f o r m a t i o n gained were used t o assess t h e e f f e c t i v e n e s s of t h i s species
as a m o n i t o r .
Growth o f Rhynchostegium c o n t i n u e d t h r o u g h o u t t h e year w i t h peaks i n
s p r i n g and autumn.
There were p o s i t i v e c o r r e l a t i o n s between growth and water
and a i r t e m p e r a t u r e s .
Strong d i f f e r e n c e s i n growth r a t e s i n t h e f o u r
streams
were n o t r e l a t e d t o n u t r i e n t or heavy metal c o n c e n t r a t i o n s .
V a r i o u s p a t t e r n s o f mesh bag were t e s t e d as c o n t a i n e r s f o r t r a n s p l a n t e d
Rhynchostegium t o be used as a m o n i t o r .
No s i g n i f i c a n t d i f f e r e n c e s i n
a c c u m u l a t i o n by moss were found between boulders or bags, or i n accumulation
w i t h d i f f e r e n t p a t t e r n s o f bags.
Accumulation was reduced s l i g h t l y a t t h e
c e n t r e o f bags packed w i t h l a r g e q u a n t i t i e s o f moss.
The p h y s i o l o g y o f Zn accumulation was a l s o s t u d i e d .
A large part of
a c c u m u l a t i o n (> 70%) i n t h e e a r l y stages (< 12 h) was i n a form r e a d i l y
exchanged f o r competing c a t i o n s such as Ca and N i ; over longer time p e r i o d s
t h e r e was s i g n i f i c a n t accumulation i n t o a more t i g h t l y bound compartment.
There was no evidence t h a t uptake i n t o t h i s l a t t e r compartment was under t h e
d i r e c t control of the plant's
metabolism.
There was d i f f e r e n t i a l accumulation o f C r ( I I I ) and C r ( V I )
in
l a b o r a t o r y experiments; d u r i n g a case study i t was p o s s i b l e t o " p r e d i c t " t h e
s p e c i a t i o n o f Cr i n t h e water by t h e c o n c e n t r a t i o n s accumulated
by t h e moss.
These r e s u l t s c o n f i r m t h a t bryophytes a r e u s e f u l as monitors of heavy
metal p o l l u t i o n i n a wide range o f circumstances.
A range of such
a p p l i c a t i o n s a r e o u t l i n e d , a l o n g w i t h recommendations f o r standard methods
f o r u s i n g moss bags.
Abbreviations
t
s
min
h
d
wk
time
second
minute
hour
day
week
year
pm
mm
cm
dm
m
km
mi crometre
millimetre
centimetre
decimetre
metre
kilometre
ug
mg
kg
t
microgram
milligram
gram
kilogram
tonne
ml
1
millilitre
litre
meq
umol
mmol
uM
M
N
mi 1 1 i e q u i v a l e n t
micromole
millimole
micromoJar
molar
normal
°C
kPa
uS
degrees C e l s i u s
kiloPascal
microSiemen
CCCP
DCMU
DNP
EDTA
FRP
HA
HEPES
c a r b o n y l cyanide-m-chlorophenylhydrazone
3-(3.4-dichlorophenyl)-l,1-dimethylurea
2 , 4-dinjtrophenol
e t h y l e n e d i a m i n e t e t r a - a c e t i c a c i d (disodium s a l t )
f i l t r a b l e r e a c t i v e phosphate
humic acids
.\^-2-hydroxypiperazine-N'-2-ethanesulphonic a c i d
n
df
X
SD
p
r
F
.\'.S.
number o f measurements
degrees o f freedom
mean
standard d e v i a t i o n
probability
correlation coefficient
variance r a t i o
not s i g n i f i c a n t
AAS
AFS
ANOVA
atomic a b s o r p t i o n s p e c t r o p h o t o m e t r y
apparant f r e e space
analysis of variance
&
DPS
PAR
Q
STw
cond.
curr.
temp.
Donnan f r e e space
photosynthetically active radiation
temperature q u o t i e n t
sewage t r e a t m e n t works
conductivity
c u r r e n t speed
temperature
Conventions
N.S.
*
**
***
p < 0.05
p > 0.05 < 0.01
p > 0.01 < 0.001
p < 0.001
Amblysteg
bbrevialed
na.ne f o r Amblystegium r i p a r i u m .
Fontinalis
a b b r e v i a t e d name f o r F o n t i n a l i s a n t i p y r e t i c a .
Rhynchostegium
a b b r e v i a t e d name f o r Rhynchostegium r i p a r i o i d e s .
Acknowledgements
I am v e r y g r a t e f u l t o the Resources and Ecology D i v i s i o n ,
BP
I n t e r n a t i o n a l L i m i t e d f o r t h e i r generous f u n d i n g of t h i s p r o j e c t and i n
p a r t i c u l a r t o Drs C. G i r t o n and W.J.
S y r a t t f o r t h e i r continued i n t e r e s t .
Durham, P r o f . D. B o u l t e r p r o v i d e d research f a c i l i t i e s and Dr. B.A.
In
IJhitton
gave much encouragement and c r i t i c i s m a t a l l stages i n h i s s u p e r v i s i o n of the
project.
Collegues i n t h e A l g a l Research Group helped i n many ways but e s p e c i a l l y
I wish t o thank C.R.
Maine and P. Masters f o r many hours of a s s i s t a n c e i n
f i e l d and l a b o r a t o r y .
L.ll.
Lauchlan c h e e r f u l l y performed many of the atomic
a b s o r p t i o n analyses and Dr M.T.
microcomputer.
Gibson gave advice and a s s i s t a n c e w i t h the
Other members of t h e Department have been w i l l i n g t o share
t h e i r e x p e r t i s e , i n p a r t i c u l a r Dr C.E.
mention f o r her h e l p .
Deane-Drummond deserves s p e c i a l
Drs N. Chaffey and N. H a r r i s helped w i t h e l e c t r o n
microscopy.
I t was
t h r o u g h BP t h a t I met Dr R.P.
Gemmell who
i n t r o d u c e d me t o the
case study s i t e and I am g r a t e f u l both t o him and t o Dr D.G.
West Water A u t h o r i t y ) f o r d i s c u s s i o n s about the s i t e .
Holland (North
M e t e o r o l o g i c a l and
d i s c h a r g e data f o r t h e N o r t h e r n Pennines was made a v a i l a b l e by I . Hudson and
Dr M. Storey (Northumbrian Water A u t h o r i t y ) and I am g r a t e f u l t o both water
a u t h o r i t i e s f o r a l l o w i n g me t o quote d a t a .
Several ideas developed
i n t h i s t h e s i s are the r e s u l t of d i s c u s s i o n s w i t h
o t h e r b i o l o g i s t s , i n p a r t i c u l a r Mr E. Mycock ( N o r t h West Water A u t h o r i t y ) and
Dr P.J.
Say and I.G. Burrows ( N o r t h e r n Environmental Consultants L i m i t e d ) .
N o r t h e r n Environmental
C o n s u l t a n t s a l s o k i n d l y allowed use of t h e i r i o n
chromatograph.
F i n a l l y I wish t o thank my p a r e n t s f o r t h e i r encouragement over the t h r e e
years and my f i a n c e e . Heather Ohnstad, n o t o n l y f o r able a s s i s t a n c e i n f i e l d
and l a b o r a t o r y and f o r p r o o f - r e a d i n g t h i s t h e s i s but a l s o f o r constant
s u p p o r t and encouragement.
CONTENTS
TITLE
1
ABSTRACT
4
ABBREVIATIONS
CONVENTIONS
5
ACKNOWLEDGEMENTS
6
CONTENTS
8
LIST OF TABLES
13
LIST OF FIGURES
17
LIST OF APPENDICES
21
1. INTRODUCTION
22
1.1 GENERAL INTRODUCTION
22
1.2 HEAVY METALS IN FRESHWATERS
1.21
Sources o f heavy metals i n f r e s h w a t e r s
1.22
Environmental c h e m i s t r y o f heavy metals i n f r e s h w a t e r s
1.221 C o n c e n t r a t i o n s i n f r e s h w a t e r s
1.222 Chemical behaviour
1.223 A d s o r p t i o n and t r a n s p o r t o f heavy metals
23
23
24
1.3 HEAVY METALS AND THE AQUATIC BIOTA
1.31
Introduction
1.32
E f f e c t o f heavy metals on a q u a t i c b i o t a
1.321 D i f f e r e n t i a l t o l e r a n c e and t o x i c i t y
1.322 Movement o f heavy metals through f r e s h w a t e r ecosystems
1.33
P h y s i o l o g y o f heavy metal uptake i n p l a n t s
1.331 Heavy metals as " e s s e n t i a l " n u t r i e n t s
1.332 Mechanism o f c a t i b n uptake
27
27
28
28
29
30
30
30
1.4 HEAVY METALS AND BRYOPHYTES
1.41
Introduction
1.42
Heavy metals and t e r r e s t r i a l bryophytes
1.43
A q u a t i c bryophytes and heavy metals
1
431 Accumulation s t u d i e s
1.432 T o x i c i t y o f heavy metais t o a q u a t i c bryophytes
1.433 Mechanism o f uptake o f heavy metals by bryophytes
1.5 BIOLOGICAL MONITORS OF HEAVY METAL POLLUTION
1.51
D e f i n i t i o n s and scope o f b i o l o g i c a l m o n i t o r i n g
1.52
B i o l o g i c a l m o n i t o r s o f heavy metals
1 .521
Introduction
1.522
Invertebrates
1.523 P l a n t s
1.524 Use o f moss-bags
1.6 GROWTH OF AQUATIC BRYOPHYTES
24
25
26
53
33
34
34
35
36
37
37
39
39
39
40
42
43
1.7 AIM
45
2. METHODS
47
2.1 ANALYTICAL METHODS
2.11
Routine a n a l y t i c a l procedures
2.12
Routine atomic a b s o r p t i o n spectophotometry procedure
2.13
C o l l e c t i o n and a n a l y s i s o f water
2.14
A n a l y s i s o f anion samples
2.15
C o l l e c t i o n and a n a l y s i s o f moss samples
47
47
48
51
51
53
2.2 GROWTH MEASUREMENTS
54
2.3 FIELD EXPERIMENTS
2.31
D e s i g n a t i o n o f sampling s i t e s
2.32
Experimental regime
57
57
58
2.4 LABORATORY EXPERIMENTS
2.41
Experimental regime and media
2.42
E l u t i o n methods
2.421
Background
2.422 .Meas-.ircnient o f membrane damage
2.423 Routine procedure f o r s e p a r a t i n g exchangeable f r a c t i o n
2.43
Preparation of c e l l walls
59
59
60
60
61
62
62
2.5 ELECTRON MICROSCOPY METHODS
63
2.6 STATISTICS AND COMPUTING
2.61
Hardware and s o f t w a r e
2.62
P r e l i m i n a r y a n a l y s i s of data
2.63
S t a t i s t i c a l a n a l y s i s o f data
66
66
67
67
3. STUDY SITES
69
3.1 INTRODUCTION
69
3.2 GEOGRAPHICAL LOCATION AND PHYSICAL CHARACTERISTICS
3.21
The Northern Pennine O r e f i e l d
3.211
I n t r o d u c t i o n and g e o l o g i c a l background
3.2:2 A l s t o n Moor
3.213 Weardale
3.22
Durham C o a l f i e l d
3.23
Croal V a l l e y
72
72
72
73
74
75
79
3.3 ENVIRONMENTAL BACKGROUND TO SITES IN N-E. ENGLAND
3.31
Introduction
3.32
Water c h e m i s t r y
3.33
Macrophytes
80
80
80
85
3.4 DETAILS OF SITES USED FOR GROWTH STUDY
3.41
Introduction
3.42
Environment o f streams
3.421
Introduction
3.422 A i r temperature
3.423 P r e c i p i t a t i o n
3.424 Flow
3.42
D e t a i l s of study s i t e s
3.421
F o r e s h i e l d Burn, reach 0091-05
87
37
37
87
87
90
90
90
90
10
3.422
3.423
3.424
B l a g i l l Burn, reach 0288-70
D a d d r y s h i e l d Burn, reach 0309-80
"Race F e l l Burn", reach 0310-90
93
93
93
3.5 DETAILS OF SITES USED FOR CASE STUDY
3.51
Introduction
3.52
Environmental background
3.521 R e l a t i o n s h i p between f l o w and chromium c o n c e n t r a t i o n s
3.522 D e s c r i p t i o n o f study s i t e s
98
98
98
98
103
4. GROWTH OF RHYNCHOSTEGIUM
107
4.1 INTRODUCTION
107
4.2 PRELIMINARY STUDIES
4.21
Introduction
4.22
Shoot e x t e n s i o n a t apex
4.23
R e l a t i o n s h i p between mass and shoot l e n g t h
107
107
108
108
4.3 ENVIRONMENT OF STREAMS
4.31
Water temperature
4.32
Light
4.33
Water c h e m i s t r y
108
108
113
117
4.4 GROWTH OF MOSSES IN STREAMS
Introduction
4.41
F o r e s h i e l d Burn, reach 0091-05
4.42
B l a g i l l Burn, reach 0288-70
4.43
D a d d r y s h i e l d Burn, reach 0309-80
4 .44
"Race F e l l Burn", reach 0310-90
4.45
4 .46 Synthesis o f data
118
118
120
120
120
125
125
4.5 ANNUAL GROWTH RATES
133
4.6 SPATIAL DENSITY AND ANNUAL PRODUCTION OF DRY MATTER
139
5. DEVELOPMENT OF METHOD FOR USE OF MOSS-BAGS
141
5.1 INTRODUCTION
141
5.2 TYPES OF BAGS
5.21 E f f e c t upon p h y s i c a l environment w i t h i n bag
141
142
5.3 EXPERIMENTS ON METAL ACCUMULATION WITHIN MESH BAGS
5.31
Comparison o f a c c u m u l a t i o n by Rhynchostegium on
boulders and i n bags
5.32
Comparison o f d i f f e r e n t p a t t e r n s o f bags
5.33
V a r i a t i o n i n Zn a c c u m u l a t i o n between r e p l i c a t e bags
5.34
Homogeneity o f metal a c c u m u l a t i o n w i t h i n l a r g e
packed bags
5.35
Accumulation o f Zn by F o n t i n a l i s on boulders and i n bags
143
156
159
6. EXPERIMENTAL STUDIES ON METAL ACCUMULATION AND LOSS
163
6.1 INTRODUCTION
163
6.2 ACCUMULATION OF HEAVY METALS OVER THREE WEEKS
163
143
149
154
11
6.3 ROLE OF CELL WALL EXCHANGE IN ZINC ACCUMULATION
6.31
Introduction
6.32
Methodology
6.321
Comparison o f e l u e n t s
6.322 E f f e c t o f i n c r e a s i n g exposure times
6.323 Removal o f z i n c from i s o l a t e d c e l l w a l l s
6.33
Accumulation o f Zn over 24 h
6.34
Accumulation o f Zn over 14 d
165
165
168
168
168
172
172
175
6.4 ROLE OF METABOLISM IN Zn ACCUMULATION
6.41
Introduction
6.42
Effect of l i g h t
6.421
In laboratory
6.422 I n f i e l d
6.423 E f f e c t o f dark p r e i n c u b a t i o n on Zn uptake
6.43
E f f e c t o f temperature
6.44
Effect of metabolic i n h i b i t o r s
6.441
Methodoiogy
6.442 L a b o r a t o r y experiment
176
176
181
181
182
187
189
189
189
190
6.5 LOSS OF HEAVY METALS
6.51
Introduction
6.52
Rate o f l o s s o f Zn
6.53
Loss o f Zn under f i e l d c o n d i t i o n s
6.54
E f f e c t o f l e n g t h o f exposure t o Zn
195
195
196
196
199
6.55
201
E f f e c t o f e x t e r n a l medium
7. ZINC ACCUMULATION BY ISOLATED CELL WALLS
204
7.1 INTRODUCTION
7.2 PRELIMINARY STUDIES
7.21
Ultrastructure of c e l l walls
7.22
R e l a t i v e mass o f c e l l w a l l s
7.23
I o n i c c o m p o s i t i o n o f c e l l w a l l s : l a b o r a t o r y experiment
7.24
Nature o f Mn and Fe i n c e l l w a l l s
7.25
I o n i c c o m p o s i t i o n o f c e l l ^ a l l s : f i e l d experiment
7.26
K i n e t i c s o f exchange o f Zn
for H
204
2
204
205
207
210
214
215
7.3 STOICHIOMETRY OF EXCHANGE OF ZN FOR DIFFERENT CATIONS
7.31
Exchange o f Zn " f o r H \
7.32
Exchange o f Zn
f o r K and Ca
222
222
222
7.4 ACCUMULATION OF ZN BY CELL WALLS
7.41
Accumulation over 6 h
7.42
Accumulation by whole t i p s and by c e l l w a l l s
225
225
230
7.5 ROLE OF CA IN CONTROLLING ZN ADSORPTION
7.51 Accumulation o f Zn by c e l l w a l l s e q u i l i b r a t e d i n Ca
solutions
7.52
C o m p e t i t i o n f o r exchange s i t e s between Zn and Ca
233
233
233
8. CASE STUDY
236
8.1 INTRODUCTION
236
8.2 OUTLINE OF STUDY
236
12
8.3 FIELD STUDIES
8.31
Uptake o f Cr by Rhynchostegium i n 0267-90
8.32 Cr i n moss over six-week p e r i o d
8.321
Indigenous moss
8.322 T r a n s p l a n t e d moss
8.323 Comparison w i t h N.W.W.A. data
236
236
240
240
240
241
8.4 LABORATORY EXPERIMENTS
8.41
Introduction
8.42 I n i t i a l uptake r a t e s i n l a b o r a t o r y
8.43
Uptake o f Cr a t d i f f e r e n t aqueous Cr c o n c e n t r a t i o n s
246
246
246
246
8.5 DISCUSSION
8.51
Introduction
8.52 Comparison o f l a b o r a t o r y and f i e l d r e s u l t s
8.53
Comparison o f a q u a t i c and accumulated c o n c e n t r a t i o n s
247
247
247
252
9. DISCUSSION
255
9.1 INTRODUCTION
255
9.2 GROWTH EXPERIMENTS
9.21
Introduction
9.22 S e a s o n a l i t y i n growth o f Rhynchostegium
9.23 D i f f e r e n c e s between streams
9.24 Comparison w i t h o t h e r species
9.25 E f f e c t o f heavy metals on growth o f Rhynchostegiun
255
255
255
257
257
259
9.3 MECHANISM OF ACCUr-IULATION 0? ZINC
9.31
A c c u m u l a t i o n and l o s s by whole t i p s
9.32 Role o f t h e c e l l w a l l
9.33 Growth and heavy m e t a l uptake and l o s s
260
260
266
269
9.4 USE OF AQUATIC BRYOPHYTES AS MONITORS
9.41
Moss-bag methodology
9.42 Experience o f case study
9.43 A q u a t i c b r y o p h y t e s as m o n i t o r s
270
270
273
274
9.5 CONCLUDING REMARKS
277
SUMMARY
279
REFERENCES
282
APPENDIX 1
APPENDIX 2
302
305
13
LIST OF TABLES
table
2.1
2.2
2.3
page
D e t e c t i o n l i m i t s , or lowest measured
c o n c e n t r a t i o n s f o r AAS.
49
Random e r r o r s a s s o c i a t e d t-jith d e t e r m i n a t i o n
of metals i n vjater samples by flame AAS.
50
C o n c e n t r a t i o n o f elements and o t h e r
parameters i n Chu lOE.
60
physico-chemical
3.1
Sampling s i t e s i n A l s t o n Moor.
74
3.2
Sampling s i t e s i n Weardale.
75
3.3
Sampling s i t e s i n Durham C o a l f i e l d .
76
3.4
Zn c o n c e n t r a t i o n s i n 0024-20 and 0024-22 between
1980 and 1986.
79
3.5
Sampling s i t e s i n Croal V a l l e y .
80
3.6
Physico-chemical v a r i a b l e s i n study reaches
i n N-E. England.
Anion c h e m i s t r y i n study reaches i n N-E. England
on 24-07-86.
3.7
3.8
82
83
Metal c h e m i s t r y i n study reaches i n N-E. England
on 24-07-86.
84
3.9
Macrophytes i n study reaches i n N-E. England.
86
3.10
P h y s i c a l and chemical v a r i a b l e s a t case study s i t e s
on 01-07-86.
101
3.11
Water c h e m i s t r y i n R. C r o a l , 13-06-86 t o 20-07-86.
106
4.1
C o r r e l a t i o n s and r e g r e s s i o n s between shoot mass and
l e n g t h f o r f o u r p o p u l a t i o n s o f Rhynchostegiurr..
C o r r e l a t i o n s between spot water temperature
r e a d i n g s i n study reaches and mean monthly maximu.'n
and minimum temperatures a t Burnhope r e s e r v o i r .
T y p i c a l values f o r photon f l u x d e n s i t y a t each
s i t e used i n growth study.
4.2
4.3
4.4
4.5
113
116
117
Ranking o f environmental v a r i a b l e s d u r i n g growth
study.
119
C o r r e l a t i o n c o e f f i c i e n t s between growth r a t e o f
Rhynchostegium i n f o u r streams.
130
14
table
4.6
4.7
4.8
4.9
4.10
4.11
4.12
page
Comparisons of growth r a t e of Rhynchostegium i n
f o u r streams u s i n g p a i r w i s e t - t e s t s .
131
Spearman's Rank C o r r e l a t i o n (Rho) c a l c u l a t e d between
growth r a t e of Rhynchostegium i n f o u r streams.
131
K e n d a l l ' s Tau-B c o e f f i c i e n t c a l c u l a t e d between growth
r a t e o f Rhynchostegium i n f o u r streams.
132
C o r r e l a t i o n s between environmental v a r i a b l e s and
growth r a t e of Rhynchostegiuni i n f o u r streams.
132
Time-series a n a l y s i s of r e l a t i o n s h i p s between growth
r a t e o f Rhynchostegium and environmental v a r i a b l e s .
134
Increases i n l e n g t h and mass of Rhynchostegium over
12 months, c a l c u l a t e d from mean monthly growth r a t e
w i t h and w i t h o u t adjustment f o r m i s s i n g data.
139
S p a t i a l d e n s i t y and annual r a t e of d r y matter
p r o d u c t i o n of Rhynchostegium shoots on b o u l d e r s .
140
5.1
Mesh bags used i n t r a n s p l a n t experiments.
142
5.2
5.3
E f f e c t o f mesh bags on p h y s i c a l environment of moss.
Selected environmental v a r i a b l e s during transplant
experiment i n 0012-90.
143
5.4
5.5
5.6
5.7
5.8
5.9
5.10
144
Comparison o f c o n c e n t r a t i o n s of metals i n in s i t u moss
and i n moss t r a n s p l a n t e d i n t o 0012-45 on boulders and i n
bags.
147
Regression e q u a t i o n s and s i g n i f i c a n c e l e v e l s f o r Zn,
Cd and Pb accumulation d u r i n g t r a n s p l a n t experiment i n
0012-45.
148
A n a l y s i s o f v a r i a n c e between t r e a t m e n t s (boulders v
bags) d u r i n g t r a n s p l a n t experiment.
149
S e l e c t e d e n v i r o n m e n t a l v a r i a b l e s d u r i n g moss-bag
t r a n s p l a n t i n 0024-22.
150
A n a l y s i s o f v a r i a n c e between accumulation of Zn i n
d i f f e r e n t p a t t e r n s of mesh bag d u r i n g moss-bag
t r a n s p l a n t experiment i n 0024-22.
153
C o n c e n t r a t i o n s o f z i n c i n water and Rhynchostegium
d u r i n g t r a n s p l a n t experiment i n 0024-22.
154
A n a l y s i s o f v a r i a n c e w i t h i n and between r e p l i c a t e bags
of Rhynchostegium t r a n s p l a n t e d from 0005-44 t o 0024-22.
155
15
table
5.11
5.12
5.13
5.14
6.1
6.2
6.3
6.4
6.5
6.6
6.7
6.8
6.9
6.10
6.11
6.12
page
S e l e c t e d e n v i r o n m e n t a l v a r i a b l e s i n 0055-30 and
0048-80 d u r i n g t r a n s p l a n t experiment.
156
Comparison o f metal accumulation a t c e n t r e and edge
of moss-bags u s i n g "Student" t - t e s t s .
159
Regressions and c o r r e l a t i o n s between Zn c o n c e n t r a t i o n s
i n F o n t i n a l i s and time d u r i n g t r a n s p l a n t experiment
between 0014-40 and 0024-22.
162
A n a l y s i s o f v a r i a n c e between s i g n i f i c a n t r e g r e s s i o n s
f o r data c o l l e c t e d d u r i n g F o n t i n a l i s t r a n s p l a n t experiment.
162
S e l e c t e d e n v i r o n m e n t a l v a r i a b l e s d u r i n g long-term
(= 23 d) t r a n s p l a n t experiment.
164
L i n e a r c o r r e l a t i o n s between c o n c e n t r a t i o n s of Zn,
Cd and Pb i n water samples and i n s i t u moss over 23 d.
165
C o n c e n t r a t i o n s o f K and Zn (as percent o f t o t a l ) i n
v a r i o u s f r a c t i o n s of Rhynchostegium a f t e r t r e a t m e n t
with three eluents.
169
Removal o f Zn (as percent of t o t a l ) from i s o l a t e d
c e l l w a l l s by N i C l g and EDTA.
172
C o n c e n t r a t i o n o f Zn (+ SD) r e m a i n i n g i n s o l u t i o n d u r i n g 14-d
b a t c h c u l t u r e experiment.
176
Comparison, u s i n g "Student" t - t e s t s , of Zn accumulation
by Rhynchostegium i n c u b a t e d i n l a b o r a t o r y a t 15 °C i n
l i g h t and dark.
182
S e l e c t e d e n v i r o n m e n t a l v a r i a b l e s i n 0288-90
d u r i n g f i e l d comparison of Zn uptake i n l i g h t
and dark.
187
E f f e c t o f l i g h t on Zn accumulation by Rhynchostegius
t r a n s p l a n t e d i n t o 0288-90 ( a c c u m u l a t i o n i n dark i s
g r e a t e r than i n l i g h t a t a l l sampling occasions)
188
E f f e c t o f dark p r e t r e a t m e n t on subsequent Zn accumulation
by Rhynchostegium t r a n s p l a n t e d i n t o 0288-90 i n l i g h t
and dark.
188
"Student" t - t e s t s between t r e a t m e n t s i n l i g h t / d a r k
t r a n s p l a n t experiment i n 0288-90. See Table 6.9.
189
E f f e c t of temperature on Zn accumulation by
Rhynchostegium i n l a b o r a t o r y batch c u l t u r e ;
comparison of sample means.
190
Metabolic i n h i b i t o r s tested.
191
16
table
6.13
6.14
6.15
6.16
7.1
7.2
7.3
7.4
7.5
7.6
8.1
8.2
page
C o n c e n t r a t i o n s o f K r e l e a s e d by t r e a t m e n t o f
R h y n c h o s t e g i u m f o r 1 h a t 15 °C w i t h d i f f e r e n t
metabolic i n h i b i t o r s .
192
Effect of 1 h pretreatment with metabolic i n h i b i t o r s
on Zn a c c u m u l a t i o n by R h y n c h o s t e g i u m a t 15 °C i n
l a b o r a t o r y b a t c h c u l t u r e ; c o m p a r i s o n o f sample means.
195
C r o s s - c o r r e l a t i o n s computed between Zn a c c u m u l a t i o n
by R h y n c h o s t e g i u m and aqueous Zn i n 0024-20 o v e r
11 d f o r t h r e e i n c r e a s i n g d a i l y l a g s .
199
S i g n i f i c a n c e o f d i f f e r e n c e s ( c a l c u l a t e d by " S t u d e n t "
t - t e s t s ) b e t w e e n c o n c e n t r a t i o n s o f Zn i n R h y n c h o s t e g i u m
a t t = 48 h and a t t i m e s p r e v i o u s t o t h i s .
200
Concentrations of metals i n e e l ] w a j l s of
R h y n c h o s t e g i u m b e f o r e and a f t e r e q u i l i b r a t i o n
media.
209
in various
Comparison o f metal c o n c e n t r a t i o n s , u s i n g "Student"
t - t e s t s i n c e l l w a l l s o f R h y n c h o s t e g i u m f r o m 0310-90
and 0288-90 b e f o r e and a f t e r e q u i l i b r a t i o n i n v a r i o u s
media.
209
E f f e c t o f EDTA and humic a c i d s on a d s o r p t i o n o f m e t a l s
by R h y n c h o s t e g i u m : c o m p a r i s o n u s i n g ANOVA and " S t u d e n t "
t-tests.
211
Selected environmental data f o r study s i t e s
s e c t i o n 7.24.
214
used i n
M e t a l c o m p o s i t i o n o f c e l l w a J l s o f Rhynchostegiu.m
e q u i l i b r a t e d i n d i f f e r e n t water samples.
216
C o r r e l a t i o n s between w a t e r c h e m i s t r y v a r i a b l e s and
c o n c e n t r a t i o n s of metals adsorbed onto c e l l w a l l s ;
combined d a t a f o r c e l l w a l l s f r o m p o p u l a t i o n s f r o m
0288-90 and 0310-90.
217
Environmental
i n 02S7-90.
237
variables during transplant
C o n c e n t r a t i o n o f Cr i n w a t e r and
experiment
i n mosses a t 0267-80.
241
17
L I S T OF FIGURES
figure
2.1
2.2
3.1
3.2
3.3
3.4
page
The e f f e c t o f s a m p l e s i z e on mean a n d 95% c o n f i d e n c e
i n t e r v a l s i n groiirth measurements.
56
Concentration of c h l o r o p h y l l i n successive e x t r a c t i o n s of
Rhynchostegium d u r i n g c e l l - w a l l e x t r a c t i o n s .
65
Map o f N-E. E n g l a n d s h o w i n g l o c a t i o n s o f s a m p l i n g
regions.
71
Diurnal fluctuations
on 25-08-83.
78
i n Zn c o n c e n t r a t i o n s i n 0024-22
W e e k l y mean maximum a n d minimum t e m p e r a t u r e s a t Burnhope
Reservoir.
M o n t h l y mean p r e c i p i t a t i o n
89
a t Burnhope R e s e r v o i r i n
1984-85 a n d 1985-86.
92
3.5
M o n t h l y mean d i s c h a r g e a t Harwood Beck f o r 1985-86.
92
3.6
F o r e s h i e l d B u r n , r e a c h 0091-05.
95
3.7
Blagill
95
3.8
D a d d r y s h i e l d B u r n , 0309-80.
97
3.9
"Race F e l l B u r n " , r e a c h 0310-90.
97
3.10
R e l a t i o n s h i p b e t w e e n mean d a i l y f l o w a n d t o t a l
B u r n , r e a c h 0288-70.
chromium
i n R. C r o a l b e t w e e n J a n u a r y 1982 a n d December 1984.
100
3.11
R. C r o a l a t D a r c y L e v e r , r e a c h 0267-80.
103
3.12
R. C r o a l a t Nob End, r e a c h 0267-90.
103
3.13
B l a c k s h a w B r o o k , r e a c h 0378-90.
105
3.14
B l a c k s h a w B r o o k , r e a c h 0378-90, s h o w i n g N-W.
edge o f s p o i l heap.
S h o o t o f R h y n c h o s t e g i u m s h o w i n g zone o f a p i c a l
elongation.
105
4.1
4.2
110
R e l a t i o n s h i p b e t w e e n s h o o t mass a n d l e n g t h o f
Rhynchostegium i n f o u r reaches.
112
4.3
Water t e m p e r a t u r e i n f o u r reaches d u r i n g g r o w t h s t u d y .
115
4.4
Seasonal changes i n g r o w t h r a t e o f Rhynchostegium
i n 0091-05.
121
18
f igure
4.5
4.6
4.7
4.8
4.9
4.10
4.11
4.12
4.13
4.14
5.1
5.2
5.3
5.4
6.1
6.2
6.3
page
R e l a t i o n s h i p between g r o w t h r a t e o f Rhynchostegium
and w a t e r t e m p e r a t u r e i n 0091-05.
121
S e a s o n a l changes
i n 0288-70.
124
i n growth r a t e o f Rhynchostegium
R e l a t i o n s h i p between g r o w t h r a t e o f Rhynchostegium
and w a t e r t e m p e r a t u r e i n 0288-70.
124
Seasonal changes
i n 0309-80.
127
i n growth r a t e o f Rhynchostegium
R e l a t i o n s h i p between g r o w t h r a t e o f Rhynchostegium
and w a t e r t e m p e r a t u r e i n 0309-80.
127
Seasonal changes
i n 0310-90.
129
i n g r o w t h r a t e o f Rhynchostegium
R e l a t i o n s h i p between g r o w t h r a t e o f Rhynchostegium
and w a t e r t e m p e r a t u r e i n 0310-90.
129
R e l a t i o n s h i p between g r o w t h r a t e o f Rhynchostegiun
and w a t e r t e m p e r a t u r e : summed d a t a .
136
R e l a t i o n s h i p between g r o w t h r a t e o f Rhynchostegium
and mean maximum a i r t e m p e r a t u r e : summed d a t a .
138
R e l a t i o n s h i p between g r o w t h r a t e o f Rhynchostegium
and mean minimum a i r t e m p e r a t u r e : summed d a t a .
138
A c c u m u l a t i o n o f Zn, Cd a n d Pb b y R h y n c h o s t e g i u m
t r a n s p l a n t e d i n t o 0012-45 on b o u l d e r s and i n bags
( p a t t e r n I I , 5.2).
146
A c c u m u l a t i o n o f Zn b y R h y n c h o s t e g i u m t r a n s p l a n t e d
i n t o 0024-22 i n t h r e e p a t t e r n s o f mesh b a g .
152
A c c u m u l a t i o n o f Zn, Cd a n d Pb b y R h y n c h o s t e g i u m a t
t h e c e n t r e a n d edge o f moss-bags t r a n s p l a n t e d i n t o
0048-80.
158
A c c u m u l a t i o n o f Zn b y F o n t i n a l i s t r a n s p l a n t e d
0024-22 on b o u l d e r s a n d i n bags.
161
into
A c c u m u l a t i o n o f Zn, Cd a n d Pb b y R h y n c h o s t e g i u m
t r a n s p l a n t e d f r o m 0091-05 i n t o 0288-90.
167
E f f e c t o f i n c r e a s i n g l e n g t h s o f e x p o s u r e on e l u t i o n o f
K a n d Zn f r o m R h y n c h o s t e g i u m b y N i C l ^ a n d EDTA.
171
A c c u m u l a t i o n o f Zn i n e x c h a n g e a b l e and r e s i d u a l
f r a c t i o n s o f R h y n c h o s t e g i u m o v e r 12 h a t 15 °C i n
laboratory batch c u l t u r e .
178
19
figure
6.4
6.5
6.6
6.7
6.8
6.9
6.10
6.11
6.12
6.13
6.14
7.1
7.2
7.3
7.4
7.5
7.6
D o u b l e - r e c i p r o c a l p l o t o f d a t a p r e s e n t e d i n F i g . 6.3.
page
178
A c c u m u l a t i o n o f Zn i n e x c h a n g e a b l e and r e s i d u a l
f r a c t i o n s o f R h y n c h o s t e g i u m o v e r 14 d a t 15 °C I n
laboratory batch c u l t u r e .
180
D o u b l e - r e c i p r o c a l p l o t o f Zn a c c u m u l a t e d i n r e s i d u a l
f r a c t i o n o f R h y n c h o s t e g i u m o v e r 14 d.
180
A c c u m u l a t i o n o f Zn by R h y n c h o s t e g i u m i n c u b a t e d
i n l a b o r a t o r y a t 15 °C in l i g h t and d a r k .
184
A c c u m u l a t i o n o f Zn b y R h y n c h o s t e g i u m
i n t o 0288-90 i n l i g h t and d a r k .
184
transplanted
A p p a r a t u s a s s e m b l e d i n 0288-90 f o r f i e l d c o m p a r i s o n
o f Zn a c c u m u l a t i o n i n l i g h t and d a r k ( 6 . 4 2 2 and 6 . 4 2 3 ) .
186
A c c u m u l a t i o n o f Zn by R h y n c h o s t e g i u m a t 15 "C i n
l a b o r a t o r y batch c u l t u r e a f t e r pretreatment with
metabolic i n h i b i t o r s .
194
L o s s o f Zn f r o m R h y n c h o s t e g i u m t r a n s p l a n t e d
0288-90 t o 0091-05.
198
C o n c e n t r a t i o n s o f Zn i n w a t e r and
i n 0 0 2 4 - 2 0 ; 07-08-84 t o 16-08-84.
from
Rhynchostegium
198
L o s s o f Zn f r o m R h y n c h o s t e g i u m i n 0310-90 a f t e r
d i f f e r e n t l e n g t h s o f e x p o s u r e t o Zn i n 0012-28
and 0 3 0 9 - 8 0 .
203
L o s s o f Zn f r o m two p o p u l a t i o n s o f R h y n c h o s t e g i u m
i n c u b a t e d a t 15 °C i n a r a n g e o f m e d i a .
203
Transverse section of a leaf c e l l
xlOOOO.
207
o f Rhynchostegium
Removal o f Mn, Fe and Zn f r o m c e l l w a l l s o f
R h y n c h o s t e g i u m b y sM09ssiv& e x t r a c t i o n s .
213
R e l a t i o n s h i p b e t w e e n Zn i n s o l u t i o n and Zn i n c e l l
w a l l o f Rhynchostegium.
219
R e l a t i o n s h i p b e t w e e n Ca i n s o l u t i o n and Ca i n c e l l
w a l l o f Rhynchostegium.
219
R e l a t i o n s h i p b e t w e e n Ca i n s o l u t i o n and Zn i n c e l l
w a l l o f Rhynchostegium.
219
E f f l u x of
from c e l l w a l l s o f Rhynchostegium
i n c u b a t e d i n 50 mg l " Zn.
221
20
figure
7.7
7.8
7.9
7.10
page
Efflux of H
from c e l l w a l l s o f Rhynchostegium
i n c u b a t e d i n 50 mg 1
Zn + 0.1 M NaKO .
221
R e l a t i o n s h i p b e t w e e n Zn i n media a n d Zn a d s o r b e d
o n t o p r o t o n a t e d c e l l w a l l s o f R h y n c h o s t e g i u m a t 15 °C
i n l a b o r a t o r y batch c u l t u r e .
223
E f f e c t o f i n c r e a s i n g c o n c e n t r a t i o n s o f Zn on
by p r o t o n a t e d c e l l w a l l s o f Rhynchostegium.
223
release
R e l a t i o n s h i p b e t w e e n Zn i n medium and Zn a d s o r b e d
o n t o c e l l w a l l s o f R h y n c h o s t e g i u m i n K- and Ca-forms
a t 15 °C i n l a b o r a t o r y b a t c h c u l t u r e .
227
7.11
Double-reciprocal p l o t of data presented
227
7.12
R e l e a s e o f K a n d Ca f o l l o w i n g i n c u b a t i o n i n d i f f e r e n t
c o n c e n t r a t i o n s o f Zn. S o l i d l i n e s i n d i c a t e d i f f e r e n t
r a t i o s o f e x c h a n g e o f Ca o r K f o r Zn.
7.13
7.14
7.14
7.15
8.1
8.2
8.3
8.4
8.5
i n F i g . 7.10.
229
Zn a c c u m u l a t i o n b y c e ^ l w a l l s o f R h y n c h o s t e g i u m
i n c u b a t e d i n 50 mg 1
Zn a t 15 °C i n l a b o r a t o r y
batch c u l t u r e .
232
Zn a c c u m u l a t i o n b y w h o l e t i p s a n d c e l ^ i v a l l s o f
R h y n c h o s t e g i u m i n c u b a t e d i n 50 mg 1
Zn a t 15 °C
i n l a b o r a t o r y batch c u l t u r e ,
i = uncorrected data,
i i = c o r r e c t e d d a t a ; see 7.42 f o r f u r t h e r d e t a i l s .
232
I n t e r a c t i o n s b e t w e e n a d s o r b e d Ca a n d Zn: e f f e c t
o f i n c r e a s i n g c o n c e n t r a t i o n s o f Ca i n c e l l w a l l
on s u b s e q u e n t Zn a d s o r p t i o n .
Samples i n c u b a t e d
a t 15 °C u n d e r s t a n d a r d b a t c h c u l t u r e c o n d i t i o n s .
235
I n t e r a c t i o n s b e t w e e n a d s o r b e d Ca a n d Zn: e f f e c t
o f i n c r e a s i n g c o n c e n t r a t i o n s o f Ca i n medium on
s u b s e q u e n t Zn a d s o r p t i o n .
Samples i n c u b a t e d
a t 15 °C u n d e r s t a n d a r d b a t c h c u l t u r e c o n d i t i o n s .
235
A c c u m u l a t i o n o f Cr b y R h y n c h o s t e g i u m t r a n s p l a n t e d
i n t o 0267-90.
239
C o n c e n t r a t i o n s o f Cr i n w a t e r a n d moss i n 0267-80
and 0267-90 d u r i n g s i x - w e e k s t u d y p e r i o d .
243
C o n c e n t r a t i o n s o f Cr i n w a t e r a n d moss i n 0378-90
d u r i n g six-week study p e r i o d .
245
A c c u m u l a t i o n o f C r ( I I I ) and C r ( V I ) by Rhynchostegium
o v e r 24 h a t 15 °C i n l a b o r a t o r y b a t c h c u l t u r e .
249
A c c u m u l a t i o n o f C r ( I I I ) and C r ( V I ) by Rhynchostegium
a t d i f f e r e n t aqueous c o n c e n t r a t i o n s o f Cr a t 15 "C and
i n l a b o r a t o r y batch c u l t u r e .
251
21
L I S T OF APPENDICES
Al.
A2.
Method f o r u s i n g moss-bags t o m o n i t o r
pollution.
heavy
R e l a t i o n s h i p between Langmuir i s o t h e r m
Michael is-Menton equation.
and
metal
302
305
22
1.
1.1
INTRODUCTION
GENERAL INTRODUCTION
The
t o x i c e f f e c t s o f h e a v y m e t a l s have been known f o r a l o n g t i m e .
d e l e t e r i o u s e f f e c t s o f l e a d on h e r b a g e a r o u n d
was
The
s m e l t m i l l s o f C o u n t y Durham
d e s c r i b e d by C a l v e r t ( 1 8 8 4 ) who went on t o s a y : " I t
i s a source o f
r e g r e t t h a t w h e r e m a n u f a c t u r i n g i n d u s t r i e s a r e c a r r i e d on t h e r e a r e u s u a l l y
emanations
The
o f smoke a n d g a s " .
e x t e n t o f t h e problems
c a u s e d by heavy m e t a l s has s i n c e s p r e a d
l o c a l i z e d a r e a s where o r e s w e r e m i n e d and s m e l t e d
most d e v e l o p e d
parts of the world.
p o l l u t i o n as a s o u r c e o f p o l l u t i o n
their
t o include regions of
They a r e now second o n l y t o o r g a n i c
i n British
rivers
(Hargreaves,
i n c r e a s e i n i m p o r t a n c e h a s been m i r r o r e d by g r o w i n g p u b l i c
Techniques
f o r measuring
1981) and
concern.
c o n c e n t r a t i o n s o f h e a v y m e t a l s a r e becoming
i n c r e a s i n g l y r e f i n e d and i n c l u d e s o p h i s t i c a t e d
a b s o r p t i o n s p e c t r o p h o t o m e t r y (AAS).
spectrophotometry, d i r e c t
from
t e c h n i q u e s such as a t o m i c
i n d u c t i v l y c o u p l e d plasma e m i s s i o n
p o t e n t i o m e t r y and a n o d i c
There i s a l s o c o n s i d e r a b l e i n t e r e s t
i n t h e d e v e l o p m e n t o f methods t o a s s e s s
t h e i m p a c t o f h e a v y m e t a l s on t h e b i o t a ,
e c o l o g i s t s a n d as an a d d i t i o n a l
stripping voltametry.
both f o r i t s inherent
m.onitoring technique.
interest to
A t one l e v e l
this
may be an " i n t e r m e d i a t e t e c h n o l o g y " ( s o n s u Schumacher. 1973) a p p r o a c h t o
monitoring c o n c e n t r a t i o n s , w h i l s t a t another
additional
to straightforward
This thesis
i s concerned
a q u a t i c mosses and w i t h
chemicai
with
analyses.
s t u d i e s on t h e u p t a k e o f h e a v y m e t a l s by
using this
information
t h e s e as m o n i t o r s o f h e a v y m e t a l p o l l u t i o n
or e p i s o d i c heavy m e t a l
i t may p r o v i d e i n f o r m a t i o n
pollution.
t o d e v e l o p methods f o r u s i n g
and. i n p a r t i c u l a r ,
intermittent
23
The
term
"heavy m e t a l " h a s been r e t a i n e d t h r o u g h o u t d e s p i t e c r i t i c i s m s
(Nieboer & Richardson,
1980),
which
d e f i n i t i o n and i s o f t e n extended
and
l i g h t e r metals
p o i n t o u t t h a t t h e t e r m h a s no r i g i d
t o i n c l u d e m e t a l l o i d s ( s u c h as s e l e n i u m )
( s u c h as a l u m i n i u m
of t o x i c i t y .
Say & W h i t t o n
arouse p u b l i c
i n t e r e s t and s c i e n t i f i c
albeit not i n a s t r i c t l y
considered
i n this
(1981)
biological
and barium)
with similar
connotations
argue t h a t w h i l s t t h e term continues t o
d i s c u s s i o n t h e n i t has a p l a c e ,
o r chemical
context.
The e l e m e n t s
s t u d y ; c h r o m i u m , z i n c , cadmium a n d l e a d , a l l f i t t h e
"classical" definition
o f a heavy m e t a l by h a v i n g a d e n s i t y g r e a t e r t h a n
five.
The
remainder
sources,
of this
environmental
systems.
i n t r o d u c t i o n g i v e s a b r i e f overviei-j o f t h e
c h e m i s t r y and b i o l o g y o f heavy metals
I t deals mainly w i t h zinc, the p r i n c i p a l
b u t c o n t a i n s some r e f e r e n c e s t o o t h e r m e t a l s .
detailed
i n freshwater
element i n t h i s
study,
T h i s i s f o l l o w e d by a more
c o n s i d e r a t i o n o f t h e aims and scopes o f b i o l o g i c a l m o n i t o r i n g and
the c o r r e c t choice o f organisms f o r p a r t i c u l a r
circumstances.
1.2 HEAVY METALS I N FRESHWATERS
1.21
Sources o f heavy metals
The
i n freshwaters
p r o d u c t i o n a n d c o n s u m p t i o n o f h e a v y m e t a l s have shown a r a p i d
i n c r e a s e i n t h i s c e n t u r y ( N r i a g u , 1979),
total
1979).
emissions
t o t h e environment
The p r o b l e m
accompanied by i n c r e a s e s i n t h e
( R i i h l i n g & T y l e r , 1968; L i v e t t e t a l . ,
may be c o n s i d e r e d
i n two p a r t s :
problems a s s o c i a t e d
w i t h t h e " p r o d u c t i o n " o f h e a v y m e t a l s , e.g. m i n i n g a n d s m e l t i n g and
problems a s s o c i a t e d w i t h t h e i r use.
The
principal
o r e - p r o d u c i n g c o u n t r i e s o f t h e w o r l d a r e U.S.A.,
Canada, U.S.S.R., A u s t r a l i a a n d S o u t h A f r i c a
remainder
o f t h e resources
( G o v e t t , 1976).
Much o f t h e
a r e c o n c e n t r a t e d i n a s m a l l number o f A s i a n ,
24
A f r i c a n a n d L a t i n A m e r i c a n c o u n t r i e s ; f o r e x a m p l e 70% o f t h e w o r l d ' s
copper reserves are concentrated
and
Zambia a n d t h e p r i n c i p a l
Bolivia
( G o v e t t , 1976).
i n C h i l e , P e r u , U.S.A., U.S.S.R., Z a i r e
t i n deposits are
l o c a t e d i n S-E. A s i a a n d
U.K. d e p o s i t s o f h e a v y m e t a l s
copper, t i n , a r s e n i c and a s s o c i a t e d
upland
(principally
"gangue" m i n e r a l s ) a r e c o n f i n e d t o
s i n c e Roman t i m e s
(Raistrick
seams h a v e been e x h a u s t e d o r r e n d e r e d
abroad.
Streams and r i v e r s
i nthese
& Jennings,
The
present
o f uses.
for galvanizing steel;
(Webb,
Zinc,
are coupled
heavy metals
of
l e a d have been r e l e a s e d
the environment.
The p r o d u c t i o n , u s e
are a l l r e s p o n s i b l e f o r the
I t i s estimated
t o t h e e n v i r o n m e n t by a n t h r o p o g e n i c
50% o f a l l anthropogenic
emissions
although
i n petroleum
recent
has caused a s i g n i f i c a n t
the M i s s i s s i p p i River which,
(Trefry
1.22
The
causes
o f lead a t the
present
(Moore &
i n U.S.A. J i m i t i n g
lead
d e c l i n e i n lead transported
w i t h t r i b u t a r i e s , d r a i n s 4 0 % o f t h e U.S.A.
e t a l . , 1985).
Environmental
1.221
legislation
release
t h a t 19.6 x 10*^ t
time are d e r i v e d from combustion o f petroleum-based f u e l s
additives
w i t h an
more r e c e n t u s e s i n c l u d e m a n u f a c t u r e o f such
of
Ramamoorthy, 1 9 8 4 ) ,
contain
f o r e x a m p l e , was t r a d i t i o n a l l y used
eventual d i s p o s a l o f these products
1979).
from
1978).
widespread commodities as b a t t e r i e s and d e t e r g e n t s .
into
Many o f t h e s e
r e g i o n s , however, o f t e n s t i l l
problems a s s o c i a t e d w i t h heavy metals
increasing diversity
(Nriagu,
1965).
u n e c o n o m i c by cheap i m p o r t s
e l e v a t e d c o n c e n t r a t i o n s o f heavy m e t a l s
by
lead,
r e g i o n s o f N. E n g l a n d , N. Wales a n d S-W. E n g l a n d and have been
exploited
and
known
c h e m i s t r y o f heavy metals
Concentrations
i n freshwaters
i n freshwaters
u b i q u i t y o f many h e a v y m e t a l s
background c o n c e n t r a t i o n s
( 1 . 2 1 ) makes r e l i a b l e
estimates o f
( i . e . u n i n f l u e n c e d by a n t h r o p o g e n i c
input) very
25
difficult
t o measure, due
r e g i o n s and
analysis
both
to the problems o f c o n t a m i n a t i o n
( M a r t i n e t al^. , 1980).
contained
t o the problems of d e f i n i n g
0.011
mg
Zn,
( A b d u l l a h & Royle.
1972).
d u r i n g sample c o l l e c t i o n
"Clean" stream
0.00041 mg
1~^
S h i l l e r and
uncontaminated
Cd
waters
and
i n N.
0.0007 mg
and
Wales
1~^
Pb
B o y l e ( 1 9 8 5 ) measured d i s s o l v e d ( =
of a'/xc
0.4
pm
f i l t r a b l c ) c o n c e n t r a t i o n s ^ o f b e t w e e n 0.00002 and
rivers draining r e l a t i v e l y undisturbed
regions of the world.
m e a s u r e m e n t s w i t h i n t h e U.S.A. r a n g e d f r o m 0.000071 mg
^;
however h e a v i l y p o l l u t e d
(Whitton,
1980).
r e a c h e s may
Chemical
behaviour
is
(e.g. Laxen,
very d i l u t e .
T h i s has
in determining
two
i n n a t u r a l waters
Silver,
1972).
The
makes c h l o r i d e
the s p e c i a t i o n of d i s s o l v e d s i l v e r
(Whitlow
1985).
solubility
o f t h e common m e t a l
s a l t s w i t h p h o s p h a t e s , s i l i c a t e s and
c o l d water,
complexes i n n a t u r a l waters
w h i l s t n i t r a t e s and
h o w e v e r , i s pH.
o t h e r two
Solubility
common s p e c i e s
Of
the major anions,
carbonates
sulphates
s i n g l e most i m p o r t a n t f a c t o r c o n t r o l l i n g
are
t e n d t o be
metal
insoluble in
the s p e c i a t i o n of metal
o f z i n c d e c r e a s e s as
pH
i n n a t u r a l waters,
ZnCO
zinc s o l u b i l i t y unless
heavy
is well
r a t h e r more s o l u b l e .
r i s e s , up
and
O
control
are,
f o r example,
(Cotton & Wilkinson.
ions i n n a t u r a l waters
known ( C h e m i c a l Rubber Company, 1 9 8 5 ) .
The
concentrations
important implications for their
by c h l o r i d e i o n s
r e l a t i v e c o n c e n t r a t i o n s of these
The
1
1984b).
w i t h respect to other ions, ligands etc..
readily precipitated
Kicc.
Their
behaviour
terms,
a major f a c t o r
in
^ t o 0.0156 mg
c o n t a i n much h i g h e r
Even t h e h i g h e s t c o n c e n t r a t i o n s o f h e a v y m e t a l s
i n chemical
1
1
Ranges o f s e v e r a l o r d e r s o f m a g n i t u d e have a l s o been
shown f o r o t h e r h e a v y m e t a l s
1.222
0.0018 mg
The
ions,
t o pH
Zn(OH) ,
c.
t h e c o n c e n t r a t i o n o f d i s s o l v e d carbon
9.5.
cannot
&
26
d i o x i d e i s v e r y h i g h (Hem, 1 9 7 2 ) .
Williemite
s o l u b l e than these b u t t h e g e n e r a l l y
preclude t h i s
1972).
onto p a r t i c u l a t e s .
(Shiller
& Boyle.
c o n t r o l on s o l u b i l i t y
forms
An i n c r e a s e i n pH i n c r e a s e s t h e amount o f z i n c
p r e c i p i t a t e s w i t h oxides, carbonates
an a n i o n .
behaviour
1.223
The
The
These t w o f o r m s
involved
(CrO^ ) and behaves as
1984).
metals
i s f u r t h e r a d j u s t e d by t h e t y p e
i n c l u d e exchange a d s o r p t i o n o n t o charged
sites
c o m p l e x a t i o n b y o r g a n i c compounds such as humic
a c i d s ( T i p p i n g e t a_l. , 1 9 8 3 ) a n d a d s o r p t i o n and c o - p r e c i p i t a t i o n
p r o p o r t i o n s adsorbed
oxides
vary with
( L i o n e t a j . - , 1982; L a x e n , 1 9 8 4 a ) .
The
t h e m e t a l a n d t h e c o n c e n t r a t i o n ; t h e mean
o f 0.22 pm f i l t r a b l e m e t a l
i n N. E n g l a n d
metals
(Cr(III)
o f l i g a n d s a n d s u r f a c e s on w h i c h a d s o r p t i o n can o c c u r .
o n t o manganese a n d i r o n
(calculated
chromium i s a l s o
1980; Moore & Ramamoorthy,
a q u a t i c c h e m i s t r y o f heavy m e t a l s
fulvic
ratio
f o u n d as c h r o m a t e
A d s o r p t i o n and t r a n s p o r t o f heavy
(O'Connor & Renn, 1 9 6 4 ) ,
and
upon
have v e r y d i f f e r e n t c h e m i c a l and b i o l o g i c a l
(Cranston & Murray,
processes
(Pb(II));
two o x i d a t i o n s t a t e s f r e q u e n t i n n a t u r a l w a t e r s
Cr(VI) i s usually
and a v a i l a b i l i t y
and p h o s p h a t e s d e p e n d i n g
Lead i s a p o l y v a l e n t c a t i o n w i t h one
o x i d a t i o n s t a t e s t a b l e i n n a t u r a l waters
Cr(VI)).
similar aquatic
however l e a d (Group I V b ) i s l e s s s o l u b l e and r e a d i l y
redox c o n d i t i o n s and a l k a l i n i t y .
and
adsorbed
1985) .
(Hem, 1 9 7 2 ) ;
polyvalent, with
(Hem,
e f f e c t o f pH i s t o a f f e c t a d s o r p t i o n o f m e t a l s
Cadmium, v e r y c l o s e t o z i n c i n Group l i b has a f a i r l y
chemistry
i s less
low c o n c e n t r a t i o n s o f d i s s o l v e d s i l i c a
f r o m a c t i n g as a s i g n i f i c a n t
The s e c o n d m a j o r
(zinc s i l i c a t e )
t o " t o t a l " metal
i n a s u r v e y o f 105 s i t e s
was 0.88 f o r z i n c , 0.81 f o r cadmium and 0.61 f o r l e a d
from data
i n Wehr & W h i t t o n , 1 9 8 3 a ) .
t h a t may be t r a n s p o r t e d i n t h i s
The t o t a l
amount o f
way i s c o n s i d e r a b l e ; t h e Susquehanna
27
River,
S-E. U.S.A. t r a n s p o r t s
3048.2 t y r ~ ^
nickel
d i s c h a r g e s 84 t
(Turekian
y r ^ zinc,
Michigan
(Romano, 1 9 7 6 ) .
greatest
a t high
1.3
flows
88.4 t y r ~ ^ c h r o m i u m , 45.7 t y r ~ s i l v e r a n d
& Scott,
1967)
a n d t h e Grand C a l m u t R i v e r
1.2 t y r ^ cadmium a n d 24 t y r ^ l e a d
The t o t a l
(Bradley
i n t o L.
amounts o f m e t a l s t r a n s p o r t e d a r e
& Lewin,
1982).
HEAVY METALS AND THE AQUATIC BIOTA
1.31
Introduction
This section
i s a brief
freshwater biota.
review o f the biology
The f i r s t
part
o f heavy m e t a l s i n
( 1 . 3 2 ) r e v i e w s t h e e f f e c t s o f heavy
m e t a l s a t t h e o r g a n i s m i c a n d c o m m u n i t y l e v e l s , t h e i r movement t h r o u g h
c h a i n s and the h e a l t h
followed
i m p l i c a t i o n s where man i s t h e t o p consumer.
by a r e v i e w o f t h e c u r r e n t
accumulation by a q u a t i c
marine systems w i l l
It
i suseful
be i n c l u d e d
a t this
throughout t h e thesis:
defines
plants
generally
where
relevant.
to define
t w o terms t h a t w i l l
" u p t a k e " and " a c c u m u l a t i o n " .
refers
physiologist
t a k e s up s o l u t e s
prior to
t o t h e w h o l e s u i t e o f p r o c e s s e s by w h i c h an o r g a n i s m
and o t h e r
"bioaccumulation"
i s o f t e n used
compounds f r o m
instead
" b i o " may l a y t o o much s t r e s s
i t s environment.
The t e r m
o f " a c c u m u l a t i o n " ; however t h e
on a c t i v e
b i o l o g i c a l processes,
whereas p a s s i v e p r o c e s s e s a r e o f t e n more i m p o r t a n t
"e.xchange" r e f e r s t o r e v e r s i b l e a d s o r p l i v e
rather
The p l a n t
recur
" A c c u m u l a t i o n " has a Jess c l e a r d e f i n i t i o n b u t
concentrates ions
prefi.x
R e f e r e n c e s t o t e r r e s t r i a l and
u p t a k e as t h e p r o c e s s e s by w h i c h a p l a n t
their assimilation.
This i s
knowledge o f the p h y s i o l o g y o f metal
(1.33).
point
food
(1.33).
The t e r m
p r o c e s s e s a t t h e c o l l wa3]
t h a n t o s p e c i f i c e x c h a n g e by i o n - c a r r i e r mechanisms.
28
1.32
E f f e c t o f h e a v y m e t a l s on a q u a t i c
1.321
Differential
t o l e r a n c e and
E x a m p l e s o f b e t w e e n - and
biota
toxicity
within-species variation
i n tolerance of
o r g a n i s m s t o heavy m e t a l s a r e w e l l documented ( A n t o n o v i c s
As
the t o t a l
total
c o n c e n t r a t i o n of zinc i n the environment increases
number o f s p e c i e s
decreases (Whitton & Diaz,
however, i s not c o n f i n e d
species
to i n d i v i d u a l
selection
adaptations
(Bradshaw & M c N e i l l y ,
Whitton,
1976;
Stigeoclonium
zinc concentrations
to high
Say
( > 0.2
e t a^.,
t o l e r a n c e i n the blue-green
culturing
metal
i n t o higher
aquatic
mg
1
decrease,
o f z i n c ( W h i t t o n , 1980).
1981)
and
t e n u e and
indeed
few
I t is
acquired
by
populations of
H o r m i d i u m spp.
^ ) concentrations
1977).
of zinc
Shehata & W h i t t o n
alga Anacystis
from
the
streams
of z i n c .
1979;
(Harding
&
(1982) induced
n i d u l a n s by
have a l s o been o b s e r v e d
i n v e r t e b r a t e s (Armitage,
the
a s s a y e d f o r t o l e r a n c e showed g e n e t i c
concentrations
concentrations
This
t o l e r a n c e t o heavy m e t a l s i s a t r a i t
f i l a m e n t o u s green algae
with elevated
1980).
so
taxa; a l l l a r g e r taxa contain a
tolerant to elevated concentrations
g e n e r a l l y assumed t h a t
natural
e t a l . , 1971).
repeatedly
sub-
S i m i l a r e f f e c t s of
in field
populations
Armitage & Blackburn,
zinc
increased
of
1985;
LaPoint
e t a l . , 1984).
T h e r e i s a l s o good d o c u m e n t a t i o n f o r d i f f e r e n t i a l
s t u d i e s on
depending
upon w a t e r c h e m i s t r y
at particular
sites.
described
e t al_. , 1977)
were e x t e n d e d t o i n c l u d e t h e e f f e c t s
above (Say
The
toxicity
various other chemical
factors
Magnesium, c a l c i u m
phosphate a i l reduced s i g n i f i c a n t l y
and
z i n c , w h i l s t y j p H had
the reverse
i n t h e media (Say
Hormidium
effect.
The
c o m p l e x i n g a g e n t s s u c h as h u m i c , f u . l v i c and
the t o x i c i t y
increased
of zinc
& Whitton,
concentrations
o f humic a c i d s
1977).
the t o x i c i t y
effect
of various
phenolic
a c i d s was
to Elodea n u t t a l ] i i (van der W e r f f ,
reduced the
of
1984)
toxicity
of
natural
t o reduce
and
o f cadmium
and
29
c o p p e r t o D a p h n i a spp.
(Winner,
1984).
apparent amelioration of t o x i c i t y
the organism
and
this
The
t o speciation of ions outside
1984;
Laegreid et a l . ,
1984).
Movement o f h e a v y m e t a l s t h r o u g h f r e s h w a t e r e c o s y s t e m s
relative
o f t e n an
may
t o be due
two s t u d i e s show t h e
i s an o p i n i o n o f a l a r g e number o f w o r k e r s a t t h e
present time (Langston & Bryan,
1.322
These l a t t e r
insolubility
o f heavy m e t a l s
i m p o r t a n t s i n k f o r heavy metals
( 1 . 2 2 2 ) means t h a t s e d i m e n t s
( H a r d i n g & W h i t t o n , 1978).
be s u b s e q u e n t l y r e l e a s e d f r o m t h e s e d i m e n t s
(Hamilton-Taylor et a l . ,
by b i o g e o c h e m i c a l
are
They
cycling
1 9 8 4 ) , by t u r b u l e n c e ( E v e r a r d & Denny, 1985a) o r
by u p t a k e by r o o t e d m a c r o p h y t e s
( W e l s h & Denny, 1976;
p r o d u c e r s h a v e been shown t o be
important f a c t o r s i n the r e c y c l i n g of
metals
1984;
(Hamilton-Taylor et a l . ,
B a c c i n i , 1985).
1980).
Primary
High
concentrations
o f h e a v y m e t a l s h a v e been m e a s u r e d i n a w i d e r a n g e o f p r i m a r y p r o d u c e r s :
planktonic algae
Whitton,
which
and,
1981)
(Lund, 1957), f i l a m e n t o u s r i v e r i n e algae
and
aquatic bryophytes
form i m p o r t a n t food sources
i n turn,
for fish.
(Harding &
(Wehr & W h i t t o n , 1 9 8 3 a ) ,
f o r aquatic invertebrates
some o f
(Cummins,
1975)
Some " b i o c o n c e n t r a t i o n " o f m e t a l s o c c u r s a l o n g t h e
f o o d c h a i n ( E v e r a r d & Denny, 1 9 8 4 ) ; h o w e v e r t h e r e i s a l s o e v i d e n c e f o r
d i r e c t a b s o r p t i o n of metals across g i l l s
( D a v i e s e t al^. , 1976).
contains r e l a t i v e l y
Muscle t i s s u e ,
( V o y e r e t a l . , 1975)
t h e m a i n human f o o d f r o m
low c o n c e n t r a t i o n s o f heavy metals
t h e r i s k s t o human h e a l t h a r e l o w e x c e p t w h e r e f i s h
the
diet.
or i n t o
eggs
fish,
(Phillips,
1980)
form a large p a r t of
and
30
1.33 P h y s i o l o g y
o f heavy metal uptake i n p l a n t s
1.331 Heavy m e t a l s as " e s s e n t i a l " n u t r i e n t s
T h e r e i s good e v i d e n c e t h a t z i n c , c o p p e r and p o s s i b l y c o b a l t a r e
of
essential
Kirkby,
i n small
q u a n t i t i e s f o r the growth^all higher
p l a n t s (Mengel &
1982; S a l i s b u r y & Ross, 1 9 7 8 ) .
1.332 Mechanism o f c a t i o n u p t a k e
In c o n t r a s t t o major n u t r i e n t s ,
d e t a i l e d s t u d i e s on t h e p h y s i o l o g y
u p t a k e o f m i n o r n u t r i e n t s and h e a v y m e t a l s a r e r e l a t i v e l y
worth
considering
briefly
a c c u m u l a t e d and f r o m
h a v e been
onto c e l l
A number o f mechanisms f o r c a t i o n a c c u m u l a t i o n
walls.
t h e i o n s do n o t c r o s s
potential
gradient
concentration
(ii
This
t h e plasmalemma; a d s o r p t i o n
and i i i
below) i n t o t h e c e l l
(Liittge & Higinbotham,
i i . d i f f u s i o n down c o n c e n t r a t i o n
diffusion
i s not s t r i c t l y
down e l e c t r o c h e m i c a l
by a d e n o s i n e t r i p h o s p h a t e
gradients.
(Liittge & Higinbotham,
The n a t u r e
The i n t e r i o r
of c e l l s i s
p o t e n t i a l may be m a i n t a i n e d
This
by an
i s fuelled
1978);
transported
t o the i o n , d i f f u s e through
side
i n turn, create a
by i n c r e a s i n g t h e
( e s p e c i a l l y hydrogen).
(ATP) ( P o o l e ,
c a t i o n s may be " a c t i v e l y "
the other
1.31)
gradients;
i o n pump a c t i v e l y e x t r u d i n g c a t i o n s
which bind
may,
(sensu
1979);
u s u a l l y s t r o n g l y e l e c t r o n e g a t i v e and t h i s
iv.
"uptake"
o f c a t i o n s and t h e o s m o t i c p r e s s u r e i n t h e r e g i o n a d j a c e n t t o
t h e plasmalemma
iii.
on mechanisms o f u p t a k e t h a t may be
identified:
i. adsorption
as
I t is
t h e p r o c e s s e s by w h i c h m a j o r n u t r i e n t s a r e
these t o speculate
r e l e v a n t t o heavy m e t a l s .
scarce.
of
i n t o c e l l s by " c a r r i e r "
t h e membrane and r e l e a s e
molecules
t h e i o n on
1979).
o f t r a n s p o r t o f any p a r t i c u l a r
c i r c u m s t a n c e i s d e d u c e d by a p p l y i n g e q u a t i o n s
ion in a
given
s u c h as t h e N e r n s t
equation
31
to
t h e measured c o n c e n t r a t i o n s i n s i d e and o u t s i d e t h e c e l l and t o t h e
measured p o t e n t i a l
(Liittge & Hlglnbotham,
1 9 7 9 ) ; however t h e r e a r e
few
e s t i m a t e s o f t h e c o n c e n t r a t i o n s o f heavy m e t a l s i n t h e c y t o p l a s m w i t h
which
t h e s e methods may
cells
for
What i s c l e a r ,
i s t h a t the requirement of
t h e " e s s e n t i a l " h e a v y m e t a l s i s l o w ( S a l i s b u r y & Ross, 1978)
that
required
in particular,
1982) and
for
be u s e d .
i s s u b s e q u e n t l y bound i n t o enzymes.
carbonic anhydrase,
superoxide dismutase
a p p r o x i m a t e l y 17%
of c e l l u l a r
Zinc i s e s s e n t i a l f o r ,
found i n c h l o r o p l a s t s
(Hewitt,
zinc
1983).
(Mengel
and
cytochromes
I and
a and a^.
nearly a l l c e l l u l a r
therefore,
I I (Mengel
i n s p i n a c h and p a r s l e y .
& Kirkby,
& Kirkby,
These two enzymes a c c o u n t
c o n s t i t u e n t of plastocyanin, a c o n s t i t u e n t of the electron
between photosystems
and
Copper i s a
transport chain
1982), superoxide
dismutase
These t h r e e enzymes g e n e r a l l y a c c o u n t f o r
copper
(Hewitt,
1983).
the c o n c e n t r a t i o n s of " f r e e "
Under n o r m a l
z i n c and
copper
conditions,
will
be v e r y low.
Under m o t a l - G t r o s s o d c o n d i t i o n s ,
p r o d u c t i o n o f low m o l e c u l a r w e i g h t
p r o t e i n s such as m e t a l l o t h i o n i n s
( R a u s e r & C u r v e t t o , 1980)
complexing agents
Grill
(Mathys,
again a c t t o decrease
Low
of
1977;
the c o n c e n t r a t i o n
et a l . , 1985)
a c t t o make c o n d i t i o n s g e n e r a l l y
i n the maintenance
f a v o u r a b l e f o r the "passive"
of these c o n d i t i o n s ,
r e l i a n c e o f u p t a k e on m e t a b o l i s m .
a s t u d y o f z i n c u p t a k e by
Similarly,
a r t e f a c t s o f pK c h a n g e s i n t h e media due
the
wall
t h e r e i s an e x p e n d i t u r e
t h e r e may
be an
apparent
P a r r y and Hayward ( 1 9 7 3 ) , i n
"active"
t r a n s p o r t may
concluded
in fact
be
t o metabolism.
s t u d i e s d e a l i n g w i t h h e a v y m e t a l s t h e r e has
r o l e of the c e l l
As
the v o l v o c a l e D u n a l i e l l a t e r t i o l e c t a
t h a t some e x p e r i m e n t s t h a t d e m o n s t r a t e d
In
induced: these
cytoplasmic c o n c e n t r a t i o n s , coupled w i t h the e l e c t r o n e g a t i v i t y
c e l l s may
energy
be
i n the cytoplasm.
a c c u m u l a t i o n o f h e a v y m e t a l s by a q u a t i c p l a n t s .
of
may
and o t h e r
i n the preliminary
been much emphasis upon
stages of uptake.
This i s not
32
always
a v i e w h e l d b y c o n v e n t i o n a l p l a n t p h y s i o l o g i s t s , however Mengel a n d
Kirkby
(1982) noted t h a t t h e c a l c i u m c o n t e n t o f p l a n t s I s c a u s a l l y
connected
analogue
with
their
c a t i o n exchange c a p a c i t y .
o f heavy m e t a l s
more i n t e r e s t
cell
i s a useful
i n many ways; i t i s a m i n o r n u t r i e n t w h i c h i s
g e n e r a l l y excluded from t h e cytoplasm
w i t h a s t r o n g tendency
Calcium
t o adsorb.
(Marme, 1 9 8 3 ) and i t i s a l s o d i v a l e n t
C a t i o n s s u c h as p o t a s s i u m , w h i l s t o f
t o plant physiologists, are less l i k e l y
t o adsorb onto t h e
wall.
An
e x t e n s i o n t o t h i s h y p o t h e s i s i s p r o v i d e d by P e t e r s o n
showed t h a t t h e c o n c e n t r a t i o n o f z i n c
( 1 9 6 9 ) who
i n the c e l l walls of Agrostis
i n c r e a s e d as t h e p l a n t s t o l e r a n c e t o z i n c
increased.
S i m i l a r r e s u l t s were
o b t a i n e d by T u r n e r and M a r s h a l l ( 1 9 7 2 ) , i n d i c a t i n g t h a t t h e c e l l
t h e s e p l a n t s may be p h y s i o l o g i c a l l y a d a p t e d
these t h e o r i e s r e g a r d i n g t h e r o l e of t h e c e l l
t h e y may, i n t u r n ,
accumulation.
walls of
as a t o l e r a n c e mechanism.
E x c l u s i o n mechanisms h a v e a l s o been f o u n d i n some a l g a e
If
(Foster,
1977).
wall are correct
Wehr a n d W h i t t o n ( 1 9 8 3 a )
found calcium c o n c e n t r a t i o n s i n
c o n t r o l l i n g m e t a l a c c u m u l a t i o n by
R h y n c h o s t e g i u m ; s u c h a f i n d i n g may r e l a t e t o c o m p e t i t i o n a t c e l l
wall
sites.
R i t c h i e a n d Larkum
tracers
then
h e l p t o e x p l a i n t h e r o l e o f calcium i n m e d i a t i n g metal
p l a n t s a n d w a t e r t o be a m a j o r f a c t o r
exchange
tenuis
(1984) used e f f l u x - t y p e e x p e r i m e n t s w i t h
t o study the size of the c e l l
intestinalis.
These i n d i c a t e d t h a t
radioactive
w a l l compartment i n Enteromorpha
i n this
species the c e l l
wall
c o m p a r t m e n t was l a r g e a n d e x c h a n g e o c c u r r e d o v e r a l o n g e r t i m e p e r i o d
m i g h t h a v e been p r e d i c t e d .
T h i s was an i n t e r e s t i n g r e s u l t ; many p r e v i o u s
w o r k e r s h a d assumed t h a t c e l l
m a t t e r o f a few minutes
than
wall
exchange was a r a p i d p r o c e s s , l a s t i n g a
and i n some s p e c i e s such as Sphagnum (Clymo, 1963)
t h i s was shown t o be s o .
The r e l e v a n c e o f R i t c h i e a n d Larkum's r e s u l t i s
33
that
i t enables
interpreted
processes
e x t e n d i n g o v e r much l o n g e r p e r i o d s t o be
i n terms o f c e l l - w a l l
cation-exchange
p e r se.
There i s good agreement t h a t
due
exchange, a l t h o u g h n o t a l l o f t h i s i s
the p r e l i m i n a r y stages o f metal uptake a r e
t o e x c h a n g e a d s o r p t i o n ( P i c k e r i n g & P u i a , 1969;
J a s t r o w & Koeppe, 1980;
Mengel & K i r k b y , 1982); however t h e r e i s d i s a g r e e m e n t
subsequent
interpret
accumulation i s "active" or "passive".
the e f f e c t s
as t o w h e t h e r
C u t l e r and R a i n s
or not
(1974)
o f t e m p e r a t u r e , a n a e r o b i c c o n d i t i o n s and m e t a b o l i c
i n h i b i t o r s as a f f e c t i n g
t h e s e q u e s t r a t i o n o f cadmium s u b s e q u e n t t o
d i f f u s i o n a c r o s s t h e plasmalemma.
The t e r m " m e t a b o l i c a l l y c o n t r o l l e d " may
be a s u i t a b l e c o m p r o m i s e b e t w e e n t h e a p p a r e n t l y f a v o u r a b l e c o n d i t i o n s f o r
passive uptake
with
1.4
(see above) and t h e m e t a b o l i c processes
which are a s s o c i a t e d
this.
HEAVY METALS AND BRYOPHYTES
1.41
Introduction
A l a r g e p r o p o r t i o n o f s t u d i e s on h e a v y m e t a l s and b r y o p h y t e s a r e
concerned
with
terrestrial
consider b r i e f l y
existing
1.42
species; i t i s worthwhile, therefore, t o
t h e s e s t u d i e s b e f o r e m a k i n g a more d e t a i l e d a n a l y s i s o f
literature
on h e a v y m e t a l s a n d a q u a t i c b r y o p h t y e s .
Heavy m e t a l s a n d t e r r e s t r i a l
bryophytes
T h e r e h a s been c o n s i d e r a b l e i n t e r e s t
by b r y o p h y t e s a n d t h e i r
conspicuous
elements
on m i n o r a ] v e i n s ( S h a c k l e t t e , 1965;
One genus o f moss, M i e l i c h h o f e r l a .
the so-called
moss" was c o n f i n e d t o c o p p e r - r i c h s u b s t r a t e s ( H a r t m a n .
a l t h o u g h subsequent
metals
C r y p t o g a m s were f r e q u e n t l y f o u n d t o be
of the flora
P u r v i s & James, 1 9 8 5 ) .
"copper
effect.
i n t h e a c c u m u l a t i o n o f heavy
r e s u l t s have i n d i c a t e d
that
1969),
i t i sprobably high
34
c o n c e n t r a t i o n s o f s u l p h u r compounds r a t h e r t h a n c o p p e r
determines the d i s t r i b u t i o n
interest
i n the responses
(Hawksworth
pollution
1977).
of cryptogams
& Rose, 1976)
(Gilbert,
(Wilkins,
and
1968;
The
h a v e been u n i f i e d
pollution
Again,
b r y o p h y t e and
(Goodman e t a l . ,
1977;
Rao
pollution
t h e s e encompass b o t h b i o g e o c h e m i c a l p r o s p e c t i n g and
C o m p o s i t e samples o f up t o f i v e
p e a t samples suspended i n t h e r i v e r
Streams d r a i n i n g
MacLean and
mineralization
l e a d - z i n c m i n e s i n W.
J o n e s ( 1 9 7 5 ) , B u r t o n and
C o n c e n t r a t i o n s o f z i n c and
z i n c and
1.7
mg
ng g
1 ''^ J e a d ) .
i n Scapania
" in Philonotis
14825 pg g
and
metals
samples o f s t r e a m w a t e r were u n r e l i a b l e
g
1974)
(3.433).
i n d i c a t o r s of uranium
up t o 1950
& Rao,
studies
pollution monitoring.
(1985).
LeBlanc
s t u d i e s h a v e d o c u m e n t e d heavy m e t a l a c c u m u l a t i o n by a q u a t i c
bryophytes.
reliable
lichens
by r e c e n t t h e o r i e s c o n c e r n i n g t h e mechanism o f
Accumulation
Severa]
Both
s t u d i e s o f t h e s e two c o n t r a s t i n g s o u r c e s o f
A q u a t i c b r y o p h y t e s and h e a v y
1.431
a l s o been much
to a i r pollution.
S y r a t t & W a n s t a l l , 1969;
a c c u m u l a t i o n o f heavy m e t a l s
1.43
T h e r e has
which
b r y o p h y t e s w e r e u s e d t o m o n i t o r gaseous
s u b s e q u e n t l y a l s o a i r b o r n e h e a v y metaJ
e t al.. , 1977).
itself
Higher
t h e R.
Etherow,
i n t h e catchment,
(Whitehead
& Brooks,
whilst
1969).
Wales w e r e t h e s u b j e c t o f s t u d i e s
Peterson
( 1 9 7 9 ) and Jones e t
by
al.
l e a d i n w a t e r were h i g h ( u p t o 27.5
mg
C o n c e n t r a t i o n s o f z i n c i n b r y o p h y t e s were
u n d u l a t a (MacLean & J o n e s , 1975)
unduJata
i n Jungermannia g r a c i l l i m a
1979).
i n mesh bags were b o t h
f o n t a n a ( B u r t o n & P e t e r s o n , 1979)
i n Scapania
species of aquatic
and
o f l e a d up t o
(MacLean & J o n e s , 1975)
(as Solenostoma g r a c i l l i m a ;
and 7023 ug
and
15940 pg g
Burton & Peterson,
c o n c e n t r a t i o n s were f o u n d i n R h y n c h o s t e g i u m r i p a r i o i d e s i n
N.
England,
(Say e t a l . ,
1981)
and
by Empain ( 1 9 7 6 )
and
^
35
M o u v e t ( 1 9 8 5 ) w o r k i n g on i n d u s t r i a l l y p o l l u t e d
al.
(1981) a t t e m p t e d t o c o r r e l a t e
c o n c e n t r a t i o n s i n w a t e r and
s e d i m e n t were e i t h e r
Subsequent
Say e t
c o n c e n t r a t i o n s f o u n d i n mosses w i t h
i n s e d i m e n t s and w h i l s t c o r r e l a t i o n s
mosses and w a t e r w e r e g e n e r a l l y
and
r i v e r s i n France.
significant, correlations
between
between
mosses
i n s i g n i f i c a n t or only s l i g h t l y s i g n i f i c a n t .
r e s e a r c h has e x t e n d e d t h e above w o r k t o d e v e l o p d a t a b a s e s on t h e
a c c u m u l a t i o n o f a r a n g e o f h e a v y m e t a l s by S c a p a n i a u n d u l a t a ( W h i t t o n e t
al.,
1982), F o n t i n a l i s
Rhynchostegium
at
l o w pH's
antipyretica
riparioides
(Say & W h i t t o n , 1983)
(Wehr & W h i t t o n , 1 9 8 3 a ) .
and
Decreased a c c u m u l a t i o n
has been d e m o n s t r a t e d i n a c i d - ^ e t r e s s e d s t r e a m s i n S c o t l a n d
(Caines e t a l . , 1985).
1.432
T o x i c i t y o f heavy m e t a l s t o a q u a t i c b r y o p h y t e s
T h e r e a r e r e l a t i v e l y f e w s t u d i e s on m e t a l t o x i c i t y
Rhynchostegium
Fontinalis
r i p a r i o i d e s , S c a p a n i a u n d u l a t a and, t o a l e s s e r
antipyretica
metal concentrations
(1.431), although populations i n heavily
be g e n e t i c a l l y
transplants
of Fontinalis
polluted
r e a c h e s o f t h e R.
pigments
(measured
adapted t o these environments.
as c h l o r o p h y l l
r i p a r i o i d e s and A m h l y s t e g i u m
was
Field
Y s t w y t h caused d e g r a d a t i o n o f t h e
1975).
chlorophyll
: p h a e o p h y t i n r a t i o ) a f t e r exposure f o r
F a s t e r c h a n g e s were o b s e r v e d i n
experiments using three species of F o n t i n a l i s .
Rhynchostegium
polluted
squamosa and S c a p a n i a u n d u l a t a f r o m u n p o l l u t e d t o
weeks (MacLean & J o n e s ,
laboratory
extent,
h a v e been r e c o r d e d o v e r a w i d e r a n g e o f a q u a t i c
r e a c h e s may
ten
t o aquatic bryophtes.
fluviatile
Rhynchostegium
( G l i m e & Keen, 1 9 8 4 ) .
t h e most t o l e r a n t , f o l l o w e d
by A m b l y s t e g i u m and t h e
Fontinalis species.
The
critical
compared w i t h
c o n c e n t r a t i o n s a r e r e l a t i v e l y h i g h when b r y o p h y t e s a r e
o t h e r groups of p l a n t s ;
Weise e t a l . ( 1 9 8 5 ) measured
36
reductions i n the natural
rates of photosynthesis i n Fontinalis
a n t i p y r e t i c a o n l y a b o v e 1.0 mg 1 ^ z i n c a n d Sommer and W i n k l e r
(1982)
n o t e d r e d u c t i o n s i n p h o t o s y n t h e s i s i n F. a n t i p y r e t i c a a t s i m i l a r
c o n c e n t r a t i o n s o f cadmium a n d c o p p e r .
on t o x i c i t y
(Sommer & W i n k l e r ,
1.433 Mechanism
of uptake
Calcium
had an a m e l i o r a t i n g
1982).
o f h e a v y m e t a l s by b r y o p h y t e s
A l a r g e c a t i o n e x c h a n g e c a p a c i t y has been w i d e l y q u o t e d as a
factor
c o n t r o l l i n g heavy m e t a l a c c u m u l a t i o n by b r y o p h y t e s
1970; R i c h a r d s o n ,
significant
1981).
The p r o b l e m
the cell
of particulate
metal uptake.
t r a p p i n g i s fundamental
t o s t u d i e s on heavy
b r y o p h y t e s have
f o rparticulate trapping"
: volume r a t i o s
discrete
a r e merely a r t e f a c t s o f inadequate
Mats o f t e r r e s t r i a l
e f f e c t i v e morphologies
(Brown,
1984).
with
particularly
large
Methods f o r " d e t e c t i n g "
c o n t a m i n a t i o n i n c l u d e measurement
o f a s h w e i g h t as a p r o p o r t i o n
t h e d r y w e i g h t ; S h a c k l e t t e ( 1 9 6 5 ) u s e d a s h w e i g h t s o f g r e a t e r t h a n 10%
as i n d i c a t i v e o f c o n t a m i n a t i o n .
of
i n and between l e a v e s ;
w a l l and i n s i d e t h e c e l l .
How many " r e s u l t s "
washing o f samples?
of
(Riihling & Tyler,
p a r t o f t h e h e a v y m e t a l measured i n p l a n t s may be i n o t h e r
precipitates within
particulate
major
T h e r e i s i n c r e a s i n g e v i d e n c e , however, t h a t a
c o m p a r t m e n t s : t r a p p e d as p a r t i c u l a t e s
surface area
effect
i r o n and t i t a n i u m ,
particulate
An a l t e r n a t i v e
high concentrations of titanium
contamination (Mitchell,
1960; R i c h a r d s o n ,
" c o n t a m i n a t i o n " may, h o w e v e r , be t h e f i r s t
Observations
of bryophyte c e l l
indicating
1981).
stage o f true
e l e c t r o n dense d e p o s i t s i n t h e c e l l
Such
uptake.
w a l l s from metal-enriched
u s i n g t r a n s m i s s i o n e l e c t r o n microscopy
confirmed
Js measurement o f t h e r a t i o
environments
has r e v e a l e d t h e p r e s e n c e o f
wall.
These have been
subsequently
t o be m e t a l - r i c h d e p o s i t s u s i n g e l e c t r o n m i c r o p r o b e
techniques
37
(Mouvet,
1984; S a t a k e & M i y a s a k a , 1 9 8 4 ) .
particles
Satake e t a l . (1983)
i n Jungermannia v u l c a n l c o l a from mercury-enriched
s u l p h i d e s o f mercury,
confirmed
streams
a l t h o u g h t h e r e a s o n why t h e s e a r e d e p o s i t e d
t o be
remains
unclear.
The
importance
(1.332).
o f cation-exchange
Clymo's ( 1 9 6 3 )
above
i n v e s t i g a t i o n a l s o showed b r y o p h y t e s t o have h i g h
concentrations of unesterifled
ion-exchange
i n b r y o p h y t e s has been m e n t i o n e d
i nthe cell
p o l y u r o n l c a c i d s , t h e most l i k e l y
walls,
compared w i t h h i g h e r p l a n t s .
sites of
T h i s emphasis
upon e x t r a c e l l u l a r a c c u m u l a t i o n by subsequent i w r k e r s has tended t o p l a y
down t h e r o l e o f i n t r a c e l l u l a r u p t a k e
e f f e c t s h a v e been i n v e s t i g a t e d ;
a c c u m u l a t i o n by b r y o p h y t e s .
antipyretica
squarrosus
metabolic
l i g h t h a d no i n f l u e n c e on m e t a l
N e i t h e r z i n c a c c u m u l a t i o n by F o n t i n a l i s
( B r o w n & B e c k e t t , 1 9 8 5 ) w e r e a f f e c t e d , a l t h o u g h cadmium
( B e c k e t t & Brown, 1 9 8 4 ) was r e d u c e d
by dark p r e t r e a t m e n t .
uptake
Different
( P i c k e r i n g & P u i a , 1 9 6 9 ) o r cadmium u p t a k e b y R h y t i d i a d e l p h u s
by t h e l i c h e n P e l t i g e r a
and
o f heavy m e t a l s .
by F o n t i n a l i s
uptake
i n the dark
I n c r e a s e d t e m p e r a t u r e caused i n c r e a s e d z i n c
antipyretica
( P i c k e r i n g & Puia,
1969).
1.5 BIOLOGICAL MONITORS OF HEAVY EIETAL POLLUTION
1.51
D e f i n i t i o n s and scope o f b i o l o g i c a l m o n i t o r i n g
The
t e r m " b i o l o g i c a l m o n i t o r i n g " h a s been a p p l i e d t o a l a r g e number o f
s i t u a t i o n s where a b i o l o g i c a l
changing
workers
response
i s used t o d e t e r m i n e
the effect of
e n v i r o n m e n t a l c o n d i t i o n s ( H e r r i c k s & S c h a e f f e r , 1985).
have a t t e m p t e d t o f o r m a l i z e t e r m i n o l o g y .
A fevj
There i s , f o r example, a
d i s t i n c t i o n between " i n d i c a t o r s " which
a r e e s s e n t i a l l y q u a l i t a t i v e and
"monitors" which
(Bonde, 1977; W h i t t o n , 1 9 8 4 ) .
third
major
group
a r e more q u a n t i t a t i v e
a r e t e s t organisms
which a r e used p r i m a r i l y i n t h e
l a b o r a t o r y t o e v a l u a t e and p r e d i c t p o s s i b l e e n v i r o n m e n t a l
effects.
A
38
Herricks
and
Schaeffer
( 1 9 8 5 ) d e f i n e t h e f i e l d - o r i e n t a t e d and
orientated
techniques
as
Biological
a s s e s s m e n t may
b i o a s s e s s e m e n t and
be
further
(to define patterns of v a r i a t i o n
bioassays r e s p e c t i v e l y .
subdivided
(Mason, 1981)
t o d e t e c t t r e n d s ) and
i n t o m e t h o d s w h i c h s u p p l e m e n t c h e m i c a l and
and
conventional
d e p e n d s i n p a r t upon t h e
analyses.
(1984) l i s t s h i s t o r i c a l
i n c l u d e the king's
respectively.
"biological"
techniques
use
canaries
t o x i n s and
b a s e d on
last century
s y s t e m o f K o l k w i t z and
associations
r i v e r s are
Hargreaves et aj^.,
1979)
d i f f e r e n t categories
distorted
new.
monitoring
i n c o a l mines, to
is biological
approach.
of p a r t i c u l a r
which
detect
in origin
from
biological
The
and
t h e two
Plectoptera
Other forms of p o l l u t i o n ,
r e s u l t s ( H e l l a w e l l , 1978).
s u c h as
The
organic
Jarvae
may
Biotic
1970;
tolerance to
heavy m e t a l s ,
in
pollution
(Chandler,
( l e a s t t o l e r a n t ) to chironomid
1975).
techniques
most common (Mason, 1 9 8 1 ) .
of organisms r e f l e c t d i f f e r i n g
the
biotic
i n d i c e s , the Trent
Index
such
o x y g e n demand
e f f e c t s of organic
the Chandler B i o t i c
being
monitoring
and
Examples o f
taxonomic groups (Wilhm,
assessed u s i n g a v a r i e t y of
( W o o d i w i s s , 1964)
cut
available.
Marsson (1908, 1909), a
based upon c o m m u n i t y s t r u c t u r e .
tolerant).
i s less clear
(Bonde, 1 9 7 7 ) , b i o c h e m i c a l
are
pollution,
methods
i n c l u d e the a n a l y s i s of water f o r pathogens, d a t i n g from
m a j o r i t y of f i e l d - o r i e n t a t e d
Index
is
n a t u r a l l y o c c u r r i n g c a r b o n monoxide
The
i n U.K.
techniques
the environment are not
m e t h o d s h a v e been t h e t r a d i t i o n a l
the saprobic
index
division
A l a r g e p r o p o r t i o n of p o l l u t i o n
w o r k o f Koch i n t h e
and
repeated
a n a l y s e s and
examples o f b i o l o g i c a l
w i n e t a s t e r and
anthropogenically-introduced
This
l e v e l s of technology
B i o l o g i c a l approaches t o m o n i t o r i n g
Cairns
physical
surveys
monitoring.
Another fundamental d i v i s i o n of b i o l o g i c a l monitoring
replace
into
i n space); s u r v e i l l a n c e (the
measurement o f v a r i a b l e s i n o r d e r
w h i c h may
laboratory-
(most
give
D i v e r s i t y i n d i c e s , r e f l e c t i n g a more
39
general
"level"
situations
o f e n v i r o n m e n t a l s t r e s s , a r e b e t t e r s u i t e d t o these
(Hellawell,
been d e v e l o p e d
1978; Mason, 1981) and a number o f t h e s e have a l s o
(Armitage e t al..,
o t h e r forms o f p o l l u t i o n ,
1983).
B i o l o g i c a l methods f o r d e t e c t i n g
a l t h o u g h common i n t h e s c i e n t i f i c
h a v e h a d l e s s i m p a c t on W a t e r A u t h o r i t y
1.52
literature,
biologists.
B i o l o g i c a l m o n i t o r s o f heavy metals
1.521
Introduction
The r e m a i n d e r
of t h i s
section w i l l
consider biological monitoring of
heavy m e t a l s w i t h r e f e r e n c e t o f r e s h w a t e r s b u t , a g a i n , i n c l u d i n g o t h e r
r e f e r e n c e s where r e l e v a n t .
As commented a b o v e ( 1 . 5 1 ) t h e s e methods have
n o t been so r e a d i l y a d o p t e d
as m e t h o d s d e a l i n g w i t h o r g a n i c p o l l u t i o n .
order t o adopt
these b i o l o g i c a l
In
t e c h n i q u e s as " s t a n d a r d " methods a l o n g s i d e
c o n v e n t i o n a l c h e m i c a l a n a l y s e s and t h e f e w b i o l o g i c a l methods t h a t have
been w i d e l y a c c e p t e d , g e n u i n e
advantages
t h e c o s t and t h e l e v e l o f t e c h n i c a l
must be d e m o n s t r a t e d .
The t i m e ,
( i n c l u d i n g taxonomic) a b i l i t y
required
are a l l important considerations.
1.522
Invertebrates
D e s p i t e r e s e r v a t i o n s as t o t h e a p p l i c a b i l i t y
m o n i t o r i n g heavy metal p o l l u t i o n
(Hellawell,
( 1 9 7 7 ) f o u n d b o t h t h e T r e n t and C h a n d l e r
monitoring zinc p o l l u t i o n
have used d i v e r s i t y
indices
i n d i c e s t o be s u i t a b l e f o r
Northamptonshire.
Other
tolerances of p a r t i c u l a r
familiar with
workers
taxonomic
( A r m i t a g e , 1980; A r m i t a g e & B l a c k b u r n , 1985; N o r r i s e t a j . . ,
The n a t u r e o f t h i s
Solbe
( C a i r n s , 1 9 7 9 ) o r h a v e p e r f o r m e d more d e t a i l e d
s t u d i e s c o n c e n t r a t i n g on t h e r e l a t i v e
groups
indices i n
1977; Mason, 1 9 8 1 ) ,
biotic
i n W i l l o w Brook,
of b i o t i c
type of study l i m i t s
1982).
i t s p r a c t i c a l use t o i n d i v i d u a l s
i n v e r t e b r a t e taxonomy a t t h e f a m i l y
l e v e l or
below.
40
Several
(Burrows
s t u d i e s , both marine
( B r o o k s & Rumsby, 1 9 6 5 ) a n d f r e s h w a t e r
& W h i t t o n , 1984; E v e r a r d & Denny, 1 9 8 4 ) have r e p o r t e d t h e a b i l i t y
of i n v e r t e b r a t e s t o accumulate heavy m e t a l s .
in
R o u t i n e use o f i n v e r t e b r a t e s
t h i s way, h o w e v e r , i s l i m i t e d b y t h e n e e d t o a l l o w them a p e r i o d t o v e n t
t h e i r g u t contents
(Say e t a l . ,
1986).
W h i l s t t h i s may be a c o n s t r a i n t ,
t h e r e a r e some s i g n i f i c a n t a d v a n t a g e s o f c e r t a i n t y p e s o f i n v e r t e b r a t e based a c c u m u l a t i o n
s t u d i e s e x e m p l i f i e d b y B r y a n a n d Hummerstone's
s t u d y i n t h e Looe e s t u a r y , C o r n w a l l
different
chemical
1.523
" n i c h e s " t o i n d i c a t e s i l v e r and l e a d p o l l u t i o n
from
i n different
f r a c t i o n s o f t h e environment.
Plants
Increasing environmental
species
stress i s often associated with
r i c h n e s s ( W h i t t a k e r , 1975);
c o n c e n t r a t i o n s o f heavy metals
Say
T h i s made u s e o f o r g a n i s m s
(1977)
& W h i t t o n , 1980).
t h i s h a s been o b s e r v e d
i n t h e environment
Such a t e c h n i q u e
m o n i t o r f o r heavy metals
( W h i t t o n & D i a z , 1980;
approach i s t o observe
Whitton
( 1 9 8 4 ) , f o r example,
t h a t b o t h S t i g e o c l o n i u m tenue and Cladophora g l o m e r a t a
eutrophic rivers
i n temperate
i n t o l e r a n t t o heavy m e t a l s
increased
i s n o t , h o w e v e r , an e x c l u s i v e
a n d a more p r a c t i c a l
changes i n s e l e c t e d items o f t h e b i o t a .
with
decreased
regions; the l a t t e r ,
notes
a r e common i n
however, i s v e r y
and t h e presence o f S t i g e o c l o n i u m alone i s
f r e q u e n t l y a s s o c i a t e d w i t h e l e v a t e d c o n c e n t r a t i o n s o f heavy m e t a l s .
W h i l s t t h e r e a r e few s t u d i e s l i k e t h a t o f Foster
a n a l y s i s o f p l a n t community s t r u c t u r e ,
quantify colonization
(1986)
of a r t i f i c i a l
(1982) w i t h
detailed
t h e r e have been s e v e r a l a t t e m p t s t o
s u b s t r a t e s by a l g a e .
Deniseger e t al_.
r e p o r t a r e c e n t e x a m p l e o f t h i s u s i n g g l a s s m i c r o s c o p e s l i d e s as t h e
substratum.
B a c i l l a r i o p h y t a were d o m i n a n t above and b e l o w a l e a d , z i n c and
copper mine b u t C h l o r o p h y t a
(Mougeotia
a n d U l o t h r i x ) were v i r t u a l l y
absent
41
a t t h e downstream s i t e ;
an
i n t e r e s t i n g observation I n the l i g h t
P a t t e r s o n and W h i t t o n ( 1 9 8 1 ) .
dissimilarity
b e e n r e p o r t e d t o be
literature
i s t o use
on t h i s .
sediments
p l a n t s as a c c u m u l a t o r s and
There are s e v e r a l advantages
and
i n some i n s t a n c e s t h e y may
to affect
biota.
not require a d d i t i o n a l
m e t a l s by v i r t u a l l y
include algae
Say
pollution
there i s a very
of plants
over
be more p r a c t i c a b l e
1981).
Amongst t h e s e
collection,
P l a n t s a r e l e s s m o b i l e t h a n i n v e r t e b r a t e s and
time t o vent gut contents.
every group
(Keeney e t a l . ,
1981), bryophytes
( H e i s e y and
of
i n d i c a t i o n of the f r a c t i o n of a metal
o f a q u a t i c p l a n t has been r e p o r t e d ;
1976;
Johnson e t a l • ,
(MacLean & J o n e s , 1975;
Damman, 1982;
Fayed & A b d - E l - S h a f y ,
Lower p l a n t s h a v e an o b v i o u s a d v a n t a g e
do
A c c u m u l a t i o n o f heavy
1978;
these
Harding &
Burton & Peterson,
e t a l . , 1 9 8 1 ) , p t e r i d o p h y t e s ( P a s c h a l & McNamara, 1975)
1985).
algal
t r a n s p o r t compared w i t h w a t e r s a m p l e s , p o s s i b l e i n d i c a t i o n
i n t e r m i t t e n t p o l l u t i o n e v e n t s and
Whitton,
D i v e r s i t y of
a r e t h e i r h i g h l e v e l s o f a c c u m u l a t i o n , t h e i r ease o f
s t o r a g e and
likely
t h e s e showed
lowest c l o s e s t t o the source of
than s t r a i g h t f o r w a r d w a t e r samples ( W h i t t o n e t a l . ,
advantages
a
1985).
A second approach
a n i m a l s and
s p e c i e s e v e n n e s s and
b e t w e e n s i t e s d u r i n g t h e summer.
(Austin & Deniseger,
large
diversity,
i n d e x w e r e a l s o c a l c u l a t e d t o compare t h e s i t e s ;
strong differences
s p e c i e s has
Species
of
1985;
and
1979;
angiosperms
S t a v e s & Knaus,
i n t h a t a l l m i n e r a l s may
be
assumed t o d e r i v e f r o m t h e w a t e r , w h i l s t
i n rooted higher plants there i s
evidence
d e r i v e f r o m b o t h t h e s u b s t r a t e and
(Denny, 1972)
t h e w a t e r and
themselves
may
be
t h a t m i n e r a l s may
translocated within
the plant.
These f e a t u r e s
be u s e d i n a more s o p h i s t i c a t e d m o n i t o r i n g s y s t e m ;
(1980) n o t e d
that whilst
s h o o t , l e a d was
not.
copper
was
Entry of lead
translocated
may
Welsh & Denny
from the r o o t t o the
i n t o t h e s h o o t was
shown t o be
the
42
result of s t i r r i n g
Denny, 1985a;
It
o f t h e sediments under t u r b u l e n t c o n d i t i o n s (Everard
1985b).
i s p e r t i n e n t t o end t h i s
where p l a n t s have p l a y e d
these
s e c t i o n w i t h some examples o f s i t u a t i o n s
a positive role
i n aquatic monitoring.
s t u d i e s c o n c e r n a q u a t i c mosses a n d t h e t h i r d
Enteromorpha.
Say a n d W h i t t o n
Two o f
deals w i t h estuarine
( 1 9 8 3 ) c o l l e c t e d s a m p l e s o f w a t e r and
F o n t i n a l i s a n t i p y r e t i c a f r o m b e l o w an i n t e r m i t t e n t z i n c d i s c h a r g e
Wear, n e a r F r o s t e r l e y .
showed l i t t l e
&
on t h e R.
The c o n c e n t r a t i o n o f a q u a t i c z i n c was l o w and
v a r i a t i o n f o r some 50 km b e l o w t h e e f f l u e n t .
The
c o n c e n t r a t i o n o f z i n c i n 2-cm t i p s o f F o n t i n a l i s was much h i g h e r n e a r t h e
s o u r c e o f t h e p o l l u t i o n and d e c r e a s e d g r a d u a l l y d o w n s t r e a m .
concentrations
i n t h e moss p r e s u m a b l y r e f l e c t
c o n c e n t r a t i o n i n t h e w a t e r was much h i g h e r .
The h i g h
p e r i o d s when t h e z i n c
A similar
type of study
c o n d u c t e d by Mouvet (1985) u s i n g C i n c l i d o t u s d a n u b i c u s t o d e t e c t
of copper a t f o u r s i t e s along
t h e R. B i e n n e , F r a n c e .
monitors
i n estuaries i s equally effective.
chemical
composition
interpret,
makes c o n v e n t i o n a l
however H a r d i n g
was
discharges
The use o f b i o l o g i c a l
F l u c t u a t i n g w a t e r l e v e l s and
w a t e r samples d i f f i c u l t t o
( 1 9 8 0 ) u s e d a n a l y s e s o f E n t e r o m o r p h a t o g i v e an
integrated record of p o l l u t i o n
i n t h e Mersey
estuary.
1.524 Use o f moss-bags
The
u s e o f mesh bags f o r e x p e r i m e n t a l
h a v e been u s e d w i d e l y
studies of organic
i n decomposition
pollution
(Standing
t r a n s p l a n t s i s n o t new.
studies
(Sharma & G o e l , 1986) and i n
Committee o f A n a l y s t s ,
s t u d i e s o f a i r p o l l u t i o n t h e y w e r e p a c k e d w i t h Sphagnum
out
t o increase
They
1983).
w h i c h was
In
teased
t h e s u r f a c e a r e a a n d s u s p e n d e d a t known h e i g h t s above t h e
ground f o r long periods
constitutes a truly
of time
(Goodman e t a l . , 1 9 7 7 ) .
b i o l o g i c a l method i s d e b a t a b l e
Whether
this
as t h e Sphagnum was
43
oven-dried
and
washed p r i o r t o u s e .
I t was,
however, a n o v e l t e c h n i q u e
and
q u i t e w i d e l y used.
Similarly,
t h e i r use
i n a q u a t i c m o n i t o r i n g i s n o t new.
a l r e a d y been made o f W h i t e h e a d and
t h a t several water
Brooks'
management b o d i e s
developed
bryophyte
The
monitoring techniques
( e . g . Wehr & W h i t t o n ,
w h e r e t h e r e a r e no
( 1 9 6 9 ) s t u d y and
indigenous
populations of bryophytes.
in
the r i v e r ,
1.6
GROWTH OF
The
few
growth
the c o n d i t i o n s of exposure
t o d i g e s t i o n and a n a l y s i s .
AQUATIC BRYOPHYTES
r a t e of aquatic bryophytes
1981).
(Wehr & W h i t t o n ,
importance
has
been t h e s u b j e c t o f
in nutrient cycling
Of more p a r t i c u l a r
1 9 8 3 b ) and
necessary t o consider
and
in tufa-formation
s m o o t h mat
with
little
i t s shoot
the
year
a v a i l a b l e f o r m o n i t o r i n g purposes.
the e c o l o g i c a l forms o f t e r r e s t r i a l
Rhynchostegium
as
i n t e r e s t to t h i s study i s the
Gimmingham & B i r s e ( 1 9 5 7 ) d i s c u s s
bryophytes.
offering
t o remain
relatively
(Meyer, 1 9 7 9 ) ,
d i s c u s s i n g t h e d i f f e r e n t methods o f m e a s u r i n g g r o w t h
species.
using
collection
of populationj/is of Rhynchostegium t o p e r s i s t throughout
Before
sites,
be b e t t e r c o l l e c t e d
s u b s t r a t a f o r i n v e r t e b r a t e s ( G l l m e & Clemens, 1972)
ability
At other
of technique, from
t h e l e n g t h o f e x p o s u r e and
studies given t h e i r
(Pentecost,
in situations
Another major advantage i s i n
standardize a l l aspects
o f t h e moss, t h r o u g h
are
t h a t have a l r e a d y been
1983a; Wehr e t a l . , 1983)
r a t h e r t h a n i n s i t u mosses.
being able to f u l l y
i t i s probable
p r i n c i p l e advantage i s i n being
i n f o r m a t i o n r e g a r d i n g t h e n a t u r e o f p o l l u t i o n may
transplanted
has
i n d i f f e r e n t p a r t s of the world
u s i n g o r have used s i m i l a r t e c h n i q u e s .
a b l e t o use
Mention
aquatic
the d i f f e r e n t growth
(as Eurhynchium r i p a r i o i d e s )
s y s t e m p r o s t r a t e and
r e s i s t a n c e t o the water.
and
i t is first
is classified
adpressed t o the
This species
forms of
as
rock,
along with
others
a
44
s u c h as F o n t i n a l i s a n t i p y r e t i c a and
f o u n d as
flora
Amblystegium
"wefts" attached to the " t r a i l i n g
i s more l i k e l y t o d e v e l o p
h i g h f l o w and
which
riparlum
i s also often
edge" o f b o u l d e r s .
A lush bryo-
on b o u l d e r s l e a s t s u b j e c t t o movement u n d e r
a r e c o n s e q u e n t l y more d i f f i c u l t
t o remove f o r
measurements.
A number o f t e c h n i q u e s h a v e been u s e d i n s t u d i e s o f g r o w t h r a t e s o f
terrestrial
bryophytes.
two o r more d i f f e r e n t
Clymo ( 1 9 7 0 )
suggests
t h a t c l o s e agreement between
methods i s t h e b e s t e v i d e n c e
d i v e r s e t h e methods t h e b e t t e r t h e i r m u t u a l
o f a c c u r a c y and t h e more
support.
Few
s u b s e q u e n t t o Clymo ( 1 9 7 0 ) do t h i s , a l t h o u g h t h e r e a s o n
s a m p l i n g d i f f i c u l t i e s as n e g l i g e n c e .
t h a t had
been u s e d s u c c e s s f u l l y and
in the sub-Antarctic.
satisfactory
of the s t u d i e s
i s as
l i k e l y t o be
R u s s e l l ( 1 9 8 4 ) r e v i e w e d t h e methods
a p p l i e d these t o a v a r i e t y of
Of e i g h t t e c h n i q u e s t h a t were t r i e d , none were
f o r a l l o f t h e 16 s p e c i e s u n d e r c o n s i d e r a t i o n .
Four o f t h e
t e c h n i q u e s were s u i t e d o n l y t o b r y o p h t y e s w i t h v e r t i c a l l y g r o w i n g
forms
and
one,
photography,
forming r a d i a l l y expanding
consistently
(1970),
was
c o l o n i e s on r o c k s u r f a c e s .
s u c e s s f u l - method was
a cushion p a r a l l e l
amount o f w i r e s t i l l
technique suited
s e g m e n t s may
t o t h e stems.
most
(Longton,
situations
The
1970;
handle i s
G r o w t h i s measured f r o m t h e
only to v e r t i c a l l y growing species.
f e w s p e c i e s show s u c h v a r i a t i o n .
water
The
t h e " c r a n k e d - w i r e " t e c h n i q u e o f Clymo
p r o j e c t i n g above t h e s u r f a c e .
be u s e d d i r e c t l y
growth
s u i t a b l e o n l y f o r saxicolous species
i n w h i c h a p i e c e o f w i r e shaped l i k e a c a r s t a r t i n g
pushed i n t o
species
Again,
is a
W e l l - d e f i n e d annual
B a k e r , 1972)
o n l y method r e a l l y
i s " t a g s " of t h r e a d t i e d around
this
but
relatively
suited to flowing-
t h e t i p a known d i s t a n c e
from the t i p .
"Tags" h a v e been u s e d i n a number o f s t u d i e s ( O v e r b a c h
Tallis,
1959;
Benson-Evens & B r o u g h , 1966)
& Happach, ] 9 5 6 ;
b u t t h e method does have some
45
s i g n i f i c a n t drawbacks.
Tags may i n t e r f e r e w i t h e x t e r n a l c a p i l l a r y
i n ectohydric species
( R u s s e l l , 1984) b u t t h i s
in aquatic habitats.
R i e l e y e t a l . (1979) noted
liable
t o d i s t u r b t h e normal growth
relatively
action
i s u n l i k e l y t o be a p r o b l e m .
t h a t a p p l y i n g t h e t a g s was
o f t h e moss, b u t t h e t a g s a r e
e a s y t o a t t a c h i n t h e open m a t s a n d w e f t s t y p i c a l o f
Rhynchostegium and t h e water
previous positions.
consuming ( T a l l i s ,
c u r r e n t v e r y q u i c k l y s e t t l e s t h e moss i n t o i t s
The c h a r g e t h a t t h e m e t h o d i s t e d i o u s and t i m e 1 9 5 9 ; R u s s e l l , 1 9 8 4 ) h a s no answer.
1.7 A I M
This study f o l l o w s e a r l i e r p r o j e c t s funded
b y t h e European Economic
Community ( c o n t r a c t 0 7 4 - 7 4 - 1 ENV.UK) a n d b y t h e D e p a r t m e n t o f E n v i r o n m e n t
( c o n t r a c t DGR/480/571 " B r y o p h y t e s
f o r M o n i t o r i n g R i v e r Water Q u a l i t y " ) .
These s t u d i e s c o n c e n t r a t e d upon d e v e l o p i n g methods f o r p r o c e s s i n g and
analyzing aquatic bryophytes
metal
and e s t a b l i s h i n g broad
c o n c e n t r a t i o n s i n t h e water
relationships
a n d i n a number o f s p e c i e s .
between
The p r e s e n t
p r o j e c t c o n t i n u e d t h i s w o r k a n d c o n c e n t r a t e d on d e v e l o p i n g methods f o r
u s i n g moss-bags t o m o n i t o x
The
species
i n t e r m i t t e n t heavy metal
c h o s e n f o r t h e s t u d y was R h y n c h o s t e g i u m
(synonyms Eurhynchium r i p a r i o i d e s ,
s e v e r a l advantages over
in particular
the country
pollution.
riparioides
Platyhipnidium riparioides).
o t h e r common a q u a t i c p l a n t s ( W h i t t o n e t a l . ,
i t i s probably
t h e m o s t common a q u a t i c b r y o p h y t e
(N.T.H. H o l m e s ^ u n p u b l i s h e d ) .
metals
of aquatic
( 1 . 4 3 1 , Say e t a l . , 1 9 8 1 ; Wehr & W h i t t o n , 1 9 8 3 b ) , i s r o b u s t ,
p e r e n n i a l and r e l a t i v e l y
was
1981),
species i n
I t a l s o a c c u m u l a t e s heavy
t o h i g h e r c o n c e n t r a t i o n s t h a n s e v e r a l o t h e r common s p e c i e s
bryophytes
T h i s has
made w i t h a n o t h e r
easy t o i d e n t i f y .
common s p e c i e s ,
In addition, a brief
Fontinalis
antipyretica.
comparison
46
The
review of the l i t e r a t u r e
fundamental
and
r e v e a l e d a number o f a r e a s ,
both
a p p l i e d , w h e r e f u r t h e r s t u d y w o u l d be v a l u a b l e .
t h e s p e c i f i c alms o f t h e t h i s
s t u d y w e r e as
follows:
i . t o s t u d y t h e e f f e c t s o f mesh-bags on m e t a l a c c u m u l a t i o n
R h y n c h o s t e g i u m and
Therefore
by
t o compare t h i s w i t h s i m u l t a n e o u s u p t a k e by p o p u l a t i o n s
on b o u l d e r s ;
i i . t o i n v e s t i g a t e the p h y s i o l o g i c a l processes i n v o l v e d
a c c u m u l a t i o n and
the c e l l u l a r
on t h e r o l e o f t h e c e l l - w a l l
iii.
t o make an
p o l l u t i o n and
practical
l o c a t i o n of metals.
i n metal
i n metal
T h i s was
to concentrate
accumulation;
i n - d e p t h s t u d y o f a s i t e o f i n t e r m i t t e n t heavy m e t a l
t o use
aquatic bryophytes
to monitor t h i s .
Particular
p r o b l e m s a s s o c i a t e d w i t h d e p l o y i n g moss-bags w e r e t o be
investigated;
iv.
t o study the growth
r a t e o f R h y n c h o s t e g i u m i n a number o f s t r e a m s
a
12-month p e r i o d and
t o compare t h e e f f e c t s o f d i f f e r e n t c o n c e n t r a t i o n s
of heavy m e t a l s .
over
47
2. METHODS
2.1
2.11
ANALYTICAL METHODS
Routine
All
laboratory
procedures
l a b o r a t o r y glassware,
made o f b o r o s i l i c a t e
w i t h t h e e x c e p t i o n o f snap-cap v i a l s , was
("Pyrex") g l a s s .
absorption spectrophotometry
(AAS)
Volumetric glassware
was o f "A" g r a d e .
used f o r a t o m i c
Snap-cap v i a l s , made
f r o m soda g l a s s , w e r e u s e d f o r a l l d i g e s t s a m p l e s and f o r some w a t e r
samples.
Previous
workers
i n Durham ( e . g . Wehr, 1983)
contamination a r i s i n g from these
tubes.
f o u n d no p r o b l e m s o f
P l a s t i c apparatus
was c h o s e n
f o l l o w i n g r e c o m m e n d a t i o n s o f B a t l e y a n d G a r d n e r ( 1 9 7 7 ) a n d was made o f
either polypropylene
or polyethylene.
e i t h e r 30 ml s n a p - c a p v i a l s
("Azlon").
Water s a m p l e s w e r e c o l l e c t e d
(see above),
o r 60 m l p o l y p r o p y l e n e
into
bottles
Moss s a m p l e s w e r e s t o r e d i n 250 m l b o t t l e s made f r o m h i g h -
density polyethylene.
Two
grades o f water
most g e n e r a l
water
were used: s i n g l e - d i s t i l l e d
water
was used f o r
l a b o r a t o r y purposes and f o r r i n s i n g o f glassware
was u s e d f o r a n a l y t i c a l
a n a l y t i c a l glassware
To December 1984;
and d e i o n i z e d
p u r p o s e s , f o r media and f o r f i n a l
(see b e l o w ) .
rinsing of
Two s o r t s o f d e i o n i z e d w a t e r were u s e d :
d o u b l e - d i s t i l l e d water
p a s s e d t h r o u g h a Houseman model C
ion°exchange c o l u m n .
From J a n u a r y 1985;
"Milli-Q"
reagent
Both are termed " d e i o n i z e d " water
Inorganic metal
"Analar"
Poole,
grade.
Dorset,
salts
(with
grade water
(Millipore
Corp.).
throughout.
t h e e x c e p t i o n o f Na^SiO^. 5H2O) were o f
A l l w e r e o b t a i n e d t h r o u g h B r i t i s h D r u g Houses (BDH),
a s w e r e most o t h e r r e a g e n t s
hydroxyethylpiperazine-N'-2-ethanesulphonic
hydrochloride
system
w i t h t h e e x c e p t i o n o f N-2a c i d (HEPES),
hydroxylamine
( b o t h f r o m Sigma) and humic a c i d ( f r o m A l d r i c h ) .
Standards
48
f o r AAS were BDH " S p e c t r o s o l " 1000 mg 1 ^ s t a n d a r d s o l u t i o n s prepared I n
1 N acid.
Working standards were prepared from these f o r each batch o f
analyses.
HNO
f o r a n a l y t i c a l purposes was o f atomic a b s o r p t i o n grade
( F i s o n s ) , o t h e r a c i d s were "Analar" grade.
The f o l l o w i n g procedure was adopted f o r washing apparatus; very d i r t y
v e s s e l s were g i v e n a p r e l i m i n a r y scrub i n t a p water p l u s d e t e r g e n t and
r i n s e d i n t a p water.
distilled
A l l apparatus was g i v e n a p r e l i m i n a r y r i n s e i n
water, immersed i n 4% HNO
f o r a minimum o f 30 minutes (Laxen &
H a r r i s o n , 1981), r i n s e d s i x times i n d i s t i l l e d
water and s e t t o d r y .
Apparatus used i n t h e c o l l e c t i o n and a n a l y s i s o f anion samples was r i n s e d
u s i n g t h e above p r o t o c o l except t h a t 10% H^SO^
was used i n place of HNO^.
A n a l y t i c a l glassware was g i v e n an a d d i t i o n a l two r i n s e s i n d e i o n i z e d water
prior to drying.
2.12
Routine
atomic
absorption spectrophotometry
procedure
A l l analyses o f metals were performed on a Perkin-Elmer (PE) model
5000 Atomic A b s o r p t i o n Spectrophotometer w i t h a PE model 5000 Automatic
Burner C o n t r o l U n i t .
Low l e v e l d e t e r m i n a t i o n s were performed on t h e above
w i t h a g r a p h i t e f u r n a c e (PE model HGA 500) and autosampler
(PE model AS 40)
r e p l a c i n g t h e burner u n i t .
Most d e t e r m i n a t i o n s were performed u s i n g an a i r - C ^ H ^
flame. For
analyses o f Mg, Ca, Fe, Cd and Pb, s t a n d a r d s o l u t i o n s contained the same
m o l a r i t y o f a c i d as t h e sample s o l u t i o n s .
r e q u i r e d , except f o r K, Mg and Ca.
No p r e t r e a t m e n t of samples was
For K, an a d d i t i o n of 15000 mg 1 "
NaCl t o a 4 ml a l i q u o t o f sample ( f i n a l c o n c e n t r a t i o n = 10% v/v) was made,
t o suppress i o n i z a t i o n i n t e r f e r e n c e s (Perkin-Elmer C o r p o r a t i o n , 1982) and
f o r Mg and Ca an a d d i t i o n o f 7% L a C l ^ was made ( f i n a l c o n c e n t r a t i o n =
0.51% v/v) t o suppress chemical i n t e r f e r e n c e s from A l , Be, P, S i , T i , Va
49
and Z i (Perkin-Elmer C o r p o r a t i o n , 1982).
A deuterium-arc
lamp was used t o
c o r r e c t f o r background a b s o r p t i o n ( l i g h t s c a t t e r i n g by p a r t i c l e s and
m o l e c u l a r a b s o r p t i o n o f l i g h t by molecules
i n t h e flame) f o r a l l
d e t e r m i n a t i o n s on t h e f u r n a c e and f o r a n a l y s i s o f Cd and Pb u s i n g t h e
flame.
The d e t e c t i o n l i m i t f l u c t u a t e d s l i g h t l y between batches, depending upon
the
machine set-up b u t t y p i c a l d e t e c t i o n l i m i t s a r e g i v e n i n Table 2.1.
A n a l y t i c a l q u a l i t y c o n t r o l f o l l o w e d g u i d e l i n e s g i v e n by Cheeseman and
Wilson
(1978); p r e l i m i n a r y s t u d i e s p r o v i d e d e s t i m a t e s o f random e r r o r s and
these d a t a were used t o c o n s t r u c t t a b l e s g i v i n g values o f each v a r i a b l e
+ 1 S.D.
( 6 7 % c o n f i d e n c e i n t e r v a l ) and + 1.98 S.D.
(98% confidence
i n t e r v a l ) (Table 2 . 2 ) .
Table 2.1
for
D e t e c t i o n l i m i t s , or lowest measured c o n c e n t r a t i o n s ( i n mg 1 ^ )
AAS.
Graphite
furnace
flame
Na
-
0.005
K
-
0.05
.Mg
-
< 0.20
Ca
-
< 0.20
Cr
< 0.002
0.010
Mn
< 0.001
0.005
Fe
0.005
Zn
< 0.001
0.003
Cd
< 0.0005
0.003
Pb
< 0.0010
0.02
0.03
50
Table 2.2
Random e r r o r s a s s o c i a t e d w i t h d e t e r m i n a t i o n o f metals i n water
samples (mg 1
metal
Na
K
) by flame A.A.S..
concentration
+ 1 SD
+ 1.96 SD
0.200
0.007
0.014
2.000
0.015
0.029
2.000
0.072
0.141
0.108
0.212
0.200
0.005
0.010
2.00
0.02
0.04
0.08
0.16
0.200
0.013
0.026
2.00
0.02
0.04
0.08
0.16
0.20
0.06
0.12
1.00
0.09
0.18
0.050
0.002
0.004
0.200
0.006
0.012
1.00
0.025
0.049
0.0500
0.0016
0.0032
0.200
0.002
0.004
1.00
0.01
0.02
0.050
0.001
0.002
0.200
0.002
0.004
1.00
0.01
0.02
0.200
0.005
0.010
1.000
0.007
0.014
4.000
0.067
0.013
10.00
Mg
10.00
Ca
10.00
Mn
Fe
Zn
Cd
Pb
51
2.13 C o l l e c t i o n and a n a l y s i s o f water
Water was c o l l e c t e d from r i v e r s and streams i n a 2 - l i t r e
polypropylene
beaker, which had been r i n s e d s e v e r a l times i n the water before t h e sample
was t a k e n .
Two f r a c t i o n s o f water were c o l l e c t e d .
" T o t a l " water i s water
decanted from t h e beaker a f t e r being a l l o w e d t o stand f o r approximately
f i v e mins t o a l l o w l a r g e r p a r t i c l e s (> 20 pm diameter; Black, 1965) t o
s e t t l e out.
" F i l t r a b l e " water i s water t h a t was passed f i r s t through a
0.22 pm Nuclepore f i l t e r
(Sterilin).
The water was c o l l e c t e d from t h e
beaker u s i n g a 20 ml p o l y p r o p y l e n e s y r i n g e (Faber S a n i t a s ) and f o r c e d
through a Swinnex f i l t e r h o l d e r ( M i l l i p o r e Corp.) c o n t a i n i n g t h e f i l t e r ,
a c i d washed as d e s c r i b e d i n 2.11.
The f i r s t few drops through t h e f i l t e r
were shaken i n t h e sample b o t t l e and d i s c a r d e d and approximately 25 ml of
sample c o l l e c t e d .
replaced.
For s e v e r a l s i t e s t h e f i l t e r became clogged and was
Again, t h e f i r s t few drops through were discarded.
Nuclepore f i l t e r s were used f o r one experiment
0.4 pm
(7.25) t o f a c i l i t a t e
c o l l e c t i o n o f a l a r g e volume (250 ml) o f stream water.
Water samples were
s t o r e d i n an i c e box u n t i l r e t u r n t o t h e l a b o r a t o r y , where 2 drops of HN0_,
were added t o each b o t t l e .
Samples were s t o r e d a t 4 °C u n t i l a n a l y s i s .
Samples t h a t were t o be used f o r anion a n a l y s i s were f i l t e r e d through a
GF/C f i l t e r
i n t o an H^SO^-washed, i o d i z e d p o l y e t h y l e n e b o t t l e and s t o r e d
at 4 °C u n t i l a n a l y s i s .
The methods used t o measure physico-chemical
v a r i a b l e s are summarized
below:
L i g h t was measured u s i n g e i t h e r a Skye I n s t r u m e n t s sensor and meter or a
Macam QlOl sensor and meter.
designed
Both had "cosine c o r r e c t e d " r a d i a t i o n
t o measure p h o t o s y n t h e t i c a l l y a c t i v e r a d i a t i o n (PAR).
sensors
The Macam
52
sensor was
not w a t e r p r o o f so readings were taken a t water l e v e l but not
below.
Temperature was
measured i n s i t u u s i n g a mercury thermometer or a
t h e r m i s t o r a t t a c h e d t o a WTW
Current speed was
experiments
model LF 91 meter.
measured on a c a l i b r a t e d O t t c u r r e n t meter.
t h e i m p e l l e r was
WTW
For most
placed i n the f a s t e s t c u r r e n t i n the reach,
about 10 cm below t h e s u r f a c e and p e r p e n d i c u l a r t o the d i r e c t i o n of f l o w
( P a t t e r s o n , 1983).
D u r i n g the growth experiments
b e i n g used, t h e i m p e l l e r was
C o n d u c t i v i t y was
and where moss-bags were
placed about 50 cm downstream of the sample.
recorded e i t h e r u s i n g an E l e c t r o n i c Switchgear
mark V c o n d u c t i v i t y meter o r , i n s i t u , u s i n g a WTW
model MC
model LF 91 c o n d u c t i v i t y
meter.
pH was
recorded u s i n g an Orion model 407A meter w i t h an Orion model
combination
e l e c t r o d e , or u s i n g a WTW
91-05
model PH 91 meter and I n g o l d
combination e l e c t r o d e .
A l k a l i n i t y was
measured by a p o t e n t i o m e t r i c t i t r a t i o n a g a i n s t 0.02
(American P u b l i c H e a l t h A s s o c i a t i o n ,
D i s s o l v e d oxygen was
WTW
model OXI 91 meter and
The e l e c t r o d e r e q u i r e d a steady c u r r e n t of
15 cm s •'" past t h e membrane and,
the e l e c t r o d e was
H_SO,
Z
4
1981).
measured i n s i t u u s i n g a WTW
e l e c t r o d e model EG 90.
M
i f t h e stream d i d not provide t h i s ,
s t i r r e d u n t i l i t gave a steady
reading.
then
53
2.14 A n a l y s i s o f a n i o n samples
PO^-P
(as F i l t r a b l e Reactive Phosphate (FRP)) was determined
c o l o r i m e t r i c a l l y a c c o r d i n g t o t h e method o f Murphy and R i l e y (1952),
m o d i f i e d by E i s e n r e i c h e t a l . (1975).
CI , NOg-N and SO^-S were analyzed
u s i n g a L a b o r a t o r y Data C o n t r o l (LDC) i o n chromatograph w i t h a Chrompack
SPH 125 autosampler
and an LDC ConductoMonitor
I I I c o n d u c t i v i t y meter
a t t a c h e d t o a Kipp and Zonen BD40 c h a r t r e c o r d e r .
This i s a high-pressure
l i q u i d chromatograph w i t h a 25 cm Hamilton X-100 ion-exchange column packed
w i t h PRP X-100 r e s i n ( H a m i l t o n ) .
A 2-cm guard column c o n t a i n i n g t h e same
r e s i n luas used t o p r o t e c t t h e s e p a r a t i o n column by removing
poisonous
substances.
The e l u e n t was 4 mM P-hydroxybenzoic a c i d a t pH 8.5
-1
w i t h a f l o w r a t e o f 2 mm min
kPa.
4
and a pump pressure o f about 1.59 x 10
Peak h e i g h t s were recorded a t f u l l - s c a l e d e f l e c t i o n s of between 1 and
10 uS cm ^.
2.15
potentially
C o n c e n t r a t i o n s were c a l c u l a t e d from peak h e i g h t s .
C o l l e c t i o n and a n a l y s i s o f moss samples
The procedure
f o r c o l l e c t i n g moss was adapted t o s u i t t h e f i n a l use:
t o p r o v i d e samples d i r e c t l y f o r a n a l y s i s , o r t o p r o v i d e m a t e r i a l f o r
t r a n s p l a n t experiments
or l a b o r a t o r y use.
For samples t h a t were t o be
analyzed d i r e c t l y ( i . e . w i t h no f u r t h e r e x p e r i m e n t a t i o n ) , moss was
c o l l e c t e d from a t l e a s t f i v e separate l o c a t i o n s w i t h i n a reach (Nehr &
W h i t t o n , 1983a).
For m a t e r i a l t h a t was t o be experimented
upon f u r t h e r ,
l a r g e r c o l l e c t i o n s were u s u a l l y r e q u i r e d and, i n order t o preserve e x i s t i n g
p o p u l a t i o n s , moss was c o l l e c t e d from j u s t above and below, as v j e l l as
w i t h i n t h e d e s i g n a t e d reach (Wehr, 1983).
I t was picked o f f b o u l d e r s ,
r i n s e d s e v e r a l times i n t h e stream water t o remove l o o s e l y a t t a c h e d
sediment,
i n v e r t e b r a t e s and algae, and placed i n sample b o t t l e s .
Moss was
s t o r e d i n an i c e box u n t i l r e t u r n t o t h e l a b o r a t o r y and t h e r e a f t e r i n a
54
r e f r i g e r a t o r u n t i l required.
A l l samples f o r a n a l y s i s were processed
w i t h i n 48 h by r i n s i n g i n d i s t i l l e d water and then t r a n s f e r r i n g shoots t o a
p e t r i d i s h where 2-cm t i p s were removed and r i n s e d s e v e r a l times i n
d e i o n i z e d water.
cap v i a l s .
These were then b l o t t e d and p u t i n t o HNO^-washed snap-
F i v e r e p l i c a t e sub-samples o f each sample were taken, each
c o n t a i n i n g 25-30 t i p s (Wehr & W h i t t o n , 1983a).
These were d r i e d a t 105 °C
b e f o r e b e i n g weighed and t r a n s f e r r e d t o b o i l i n g tubes w i t h 5 ml o f 2 M
HNO , placed on a Tecam " d r i - b l o c k " a t 120 °C f o r 45 mins and allowed t o
cool.
The samples were c e n t r i f u g e d t o separate the c l e a r s o l u t i o n from the
slurry.
the
The c l e a r s o l u t i o n was poured i n t o a 25 ml v o l u m e t r i c f l a s k and
s l u r r y was r e c e n t r i f u g e d w i t h d e i o n i z e d water.
The c l e a r s o l u t i o n from
t h i s was added t o t h e v o l u m e t r i c f l a s k and made up t o volume.
were s t o r e d i n t h e i r o r i g i n a l v i a l s u n t i l
2.2
The samples
analysis.
GROWTH MEASUREMENTS
Measurements o f growth r a t e were performed on p o p u l a t i o n s o f
Rhynchostegium a t t a c h e d t o l a r g e , b u t n o t immovable, bouJders t o reduce the
p r o b a b i l i t y o f t h e boulder b e i n g washed away d u r i n g p e r i o d s o f high f l o w ,
yet
still
October
enable t h e boulders t o be removed f o r measurements.
After
1985 two b o u l d e r s were marked a t each s i t e t o f u r t h e r reduce the
p r o b a b i l i t y o f a l l data f o r one month b e i n g l o s t .
Growth r a t e was measured by the attachment o f t h r e a d ("tags") t o
i n d i v i d u a l shoots a t a known d i s t a n c e from t h e t i p and measuring the shoot
e x t e n s i o n a f t e r one month.
Gf the e i g h t techniques reviewed by R u s s e l l
(1984), o n l y the attachment o f l a g s was r e a l l y s u i t e d t o the growth form o f
Rhynchostegium ( 1 . 6 ) .
A p r e l i m i n a r y experiment i n 0310-90 t e s t e d the
e f f e c t o f i n c r e a s i n g sample s i z e on the sample mean ( F i g . 2.1).
data a sample s i z e o f 35 shoots was chosen.
From these
55
Fig.
2.1
The e f f e c t o f sample s i z e on mean and 95% c o n f i d e n c e i n t e r v a l s i n
measurements of g r o w t h r a t e of Rhynchostegium i n 0310-90.
56
20
15
E
E
c
o
10
h
5
h
0)
E
CL
E
o
w
10
20
30
sample
40
size
50
57
Boulders were removed from t h e water, placed i n an enamel t r a y and tags
of t h r e a d c u t and looped over shoots, 10 mm from t h e t i p .
Black and w h i t e
t h r e a d was used i n a l t e r n a t e months t o a v o i d c o n f u s i o n w i t h the p r e v i o u s
month's growth.
35 shoots were l a b e l l e d per s i t e and the boulder r e t u r n e d
to t h e water i n t h e same p o s i t i o n as b e f o r e .
a p p r o x i m a t e l y one month i n t e r v a l s .
The s i t e s were v i s i t e d a t
At each v i s i t boulders were removed as
b e f o r e , t h e l e n g t h from t h e t h r e a d t o the t i p was measured f o r each shoot
and a f r e s h p i e c e o f t h r e a d looped on 10 mm from t h e t i p .
of
The t o t a l number
l a b e l l e d shoots was counted and f r e s h shoots were l a b e l l e d t o b r i n g t h e
t o t a l up t o 35 a g a i n .
The boulder was r e p l a c e d as b e f o r e , water samples
c o l l e c t e d and p h y s i c o - c h e m i c a l v a r i a b l e s measured.
The r a t e o f growth was c a l c u l a t e d as:
- 1
growth r a t e (mm wk " ) = (L
n
where;
- L ) x (7/n)
•
n = number o f days s i n c e p r e v i o u s sample
L
= l e n g t h o f shoot a t s t a r t (= 10 mm)
o
L
= l e n g t h o f shoot a t time = n
n
Occasions where the l e n g t h o f the shoot was l e s s than 10 mm were a l s o
recorded, hence t h e f i n a l f i g u r e r e f e r s t o " n e t " , n o t " t o t a l " growth.
2.3 F I E L D
EXPERIMENTS
2.31 D e s i g n a t i o n o f s a m p l i n g
sites
A l l sample s i t e s were a l l o c a t e d a unique s i x f i g u r e code i n
accordance w i t h s t a n d a r d p r a c t i c e i n Durham ( W h i t t o n e t aj.., 1976).
The
f i r s t f o u r f i g u r e s r e f e r t o t h e stream and t h e l a s t two t o a p a r t i c u l a r
10 m s t r e t c h , termed a "reach".
No s i g n i f i c a n c e i s a t t a c h e d t o stream
58
numbers, they serve s o l e l y t o enable records t o be s t o r e d on a computer.
The
reach number a l l o w s any 10 m reach t o be r e l o c a t e d a t f u t u r e dates
i s k e p t , a l o n g w i t h d e t a i l s of landmarks,
g r i d references e t c . .
and
Reach 01
i s r e s e r v e d f o r the source and 99 f o r the reach immediately above a
confluence.
A f u l l e r d e s c r i p t i o n of the system may
be found i n Holmes and
W h i t t o n (1981).
2.32
E x p e r i m e n t a l regime
Moss f o r f i e l d experiments was
c o l l e c t e d (2.15) and
t o the e x p e r i m e n t a l s i t e i n beakers enclosed i n an i c e box.
a t t a c h e d t o b o u l d e r s was
transferred
Where moss
r e q u i r e d then medium-sized boulders w i t h a good
cover of moss were chosen.
I n both i n s t a n c e s moss was kept moist but not
immersed i n stream water d u r i n g t r a n s p o r t .
Stakes t o s u p p o r t moss-bags were placed a p p r o x i m a t e l y mid-stream
hammered secureJy
i n t o the stream bed.
alongside the stakes.
Moss was
Transplanted boulders were placed
placed i n moss-bags ( f o r q u a n t i t i e s ,
Chapter 5 ) , which were looped over stakes u s i n g s t o u t s t r i n g .
the
see
The necks of
bags were k e p t loose t o f a c i l i t a t e the removal of samples.
of t h r e e bags was
and
A maximum
a t t a c h e d t o each s t a k e .
Sample i n t e r v a l s were spaced a c c o r d i n g t o experience.
An
initial
phase of r a p i d uptake, f o l l o w e d by a longer p e r i o d of slow accumu]ation
expected, so the sample i n t e r v a l s were c l o s e l y spaced (30-min
for
the f i r s t f o u r hours and subsequently
was
intervals)
longer time i n t e r v a l s were used.
At each sampling o c c a s i o n , s m a l l amounts of moss were withdrawn from the
bags and pooled t o g i v e a sample which was
for
stored i n a polyethylene b o t t l e
t r a n s p o r t to the l a b o r a t o r y and t r e a t m e n t as d e s c r i b e d i n 2.15.
Physico-chemical
measurements and water samples were c o l l e c t e d a t h o u r l y
59
i n t e r v a l s f o r t h e f i r s t f o u r hours and subsequently each time a moss sample
was
taken.
2.4
LABORATORY ESPERIMEHTS
2.41
Escperisaental regime and media
As f a r as p o s s i b l e , a l l experiments were performed on a s i n g l e
p o p u l a t i o n o f Rhynchostegium (from 0310-90).
This i s a low-zinc s i t e and
f o r experiments where moss from a z i n c - e n r i c h e d s i t e was r e q u i r e d t h i s was
c o l l e c t e d from 0288-70.
The t i p s were prepared as d e s c r i b e d i n 2.15 and
s t o r e d m o i s t , i n t h e dark a t 4 °C u n t i l used.
The n u t r i e n t medium f o r l a b o r a t o r y s t u d i e s was a m o d i f i c a t i o n o f t h a t
d e s c r i b e d by Chu (1942), known i n Durham as Chu lOE.
The medium was
b u f f e r e d w i t h 2.5 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulphonic
(HEPES) and a d j u s t e d t o pH 7 u s i n g 2 M NaOH.
acid
I r o n was c h e l a t e d w i t h 3.15
mM e t h y l e n e d i a m i n e t e t r a - a c e t i c a c i d (EDTA). Physico-chemical parameters and
t h e c o n c e n t r a t i o n s o f elements a r e l i s t e d i n Table 2.3.
The medium was prepared a t l e a s t 12 h i n advance o f each experiment
and l e f t t o e q u i l i b r a t e t o the c o r r e c t temperature.
added i m m e d i a t e l y b e f o r e t h e moss was i n t r o d u c e d .
Metal stocks were
Ten t i p s ( d r y weight =
+ 10 mg) were added t o 200 ml o f medium i n a 250 ml Ehlenmeyer f l a s k and
p l a c e d i n a shaker t a n k (Gallenkamp)
-2
photon m
s e t u s u a l l y a t 15 °C and w i t h 100 praol
-1
s
c o n t i n u o u s subsurface i l l u m i n a t i o n .
Preliminary
experiments had shown t h a t p r o v i d e d the f l a s k s were shaken, the medium
remained
s a t u r a t e d w i t h r e s p e c t t o d i s s o l v e d gases under these c o n d i t i o n s .
60
Table 2.3
Concentrations
o t h e r physico-chemical
o f elements (mg 1 ^, b e f o r e pH adjust.Tient) and
parameters i n Chu lOE.
N
6..84
Na
4.,53
Mg
2,.47
Si
1,,44
P
0..89
S
3.,27
K
1,.12
CI
1..13
Ca
9,.78
Jin
0.,012
Fe
0 .50
Co
0..002
Cu
0,.005
Zn
0..013
Mo
0 .003
B
0,.125
EDTA 3..15 1
HEPES 2 .5 mM
pH
7.0
conductivity
144 pS cm ^
total alkalinity
2.5 meq 1 ^
At the end o f the i n c u b a t i o n p e r i o d t i p s were washed i n d e i o n i z e d
water b e f o r e b e i n g e i t h e r d r i e d and d i g e s t e d (2.15) o r f u r t h e r t r e a t e d .
2.42
Elution
2.421
Background
methods
A method which a l l o w s r a p i d c o m p a r t m e n t a l i z a t i o n o f ions w i t h i n a
plant i s , obviously, highly desirable.
Several methods e x i s t f o r t h e
p r e c i s e c o m p a r t m e n t a l i z a t i o n o f i o n s , y e t these are g e n e r a l l y too time
consuming (e.g. a u t o r a d i o g r a p h y )
o r i n v o l v e the use o f techniques t h a t i t
i s n o t p o s s i b l e t o adapt t o l a r g e numbers o f f i e l d p o p u l a t i o n s (e.g. t r a c e r
-flux analysis).
interest
(1.332):
For t h e purposes o f t h i s study two compartments were o f
61
a. t h e " c e l l w a l l " , "exchangeable", o r "Donnan Free Space"
(DFS)
f r a c t i o n and
b. t h e " i n t r a c e l l u l a r "
fraction.
The p r i n c i p l e requirement
o f a method, t h e r e f o r e , was t h a t i t allowed
r a p i d d i f f e r e n t i a t i o n between these two f r a c t i o n s .
Several workers have
used e i t h e r competing ions ( R i i h l i n g & T y l e r , 1970;
Brown & Buck, 1979) o r
c h e l a t i n g agents such as EDTA ( E r n s t & van der Werff, 1978;
W h i t t o n , 1982)
t o remove the exchangeable component.
Shehata &
The p r i n c i p l e o f both
i s t o e x t r a c t the exchangeable component w i t h o u t damaging the plasmalemma
i n t h e process.
T h i s p r e c l u d e s the use o f t r a d i t i o n a l s o i l e l u e n t s such as
a c e t i c a c i d ( A g r i c u l t u r a l Development and A d v i s o r y S e r v i c e ,
1981).
The s e q u e n t i a l e l u t i o n method o f Brown and Buck (1979) was used as a
framework around which o t h e r methods were t e s t e d .
The f u l l
treatment
e x t r a c t s f o u r separate components, o f which o n l y the exchangeable and t o t a l
i n t r a c e l l u l a r were o f i n t e r e s t .
made:
The f o l l o w i n g m o d i f i c a t i o n s were t h e r e f o r e
o n l y a s i n g l e r i n s e i n d e i o n i z e d water was performed
a t the s t a r t
and step 3 ( s t e e p i n g i n b o i l i n g , d e i o n i z e d water) was o m i t t e d .
The e l u e n t s
were: 20 mM (= 1000 mg l""'') N i C l ^ (Brown & Buck, 1979), 0.138 mM (= 40 mg
1~")
EDTA (Shehata, 1982;
S l i n g s b y , 1972).
Shehata & W h i t t o n , 1982)
P r e l i m i n a r y experiments
and 0.75 M HCl (Brown &
t o check f o r membrane damage, t o
c o n s i d e r t h e e f f e c t o f i n c r e a s e d exposure times and t o examine t h e i r
on Zn removal from i s o l a t e d c e l l w a l l s were performed,
effect
p r i o r t o the
s e l e c t i o n o f a method.
2.422 Measurement o f membrane damage
In order t o assess the methods o u t l i n e d above a simple "probe" f o r
checking menbrane i n t e g r i t y was used.
This was based upon the a n a l y s i s o f
the c o n c e n t r a t i o n o f K r e l e a s e d by the t r e a t m e n t s .
K i s known t o be
62
s e l e c t i v e l y accumulated by p l a n t c e l l s and t o be l o c a t e d
i n s i d e the c e l l
(Flowers & L a u c h l i , 1983).
Release of K, t h e r e f o r e ,
i m p l i e s damage t o the membrane (Brown & S l i n g s b y , 1972;
1977)
predominately
Puckett e t a l . ,
and a breakdown i n the m e t a b o l i c processes which m a i n t a i n the Na^/K^
pump s i n the membrane.
P u c k e t t e t a l . (1977), f o r example, noted a good
c o r r e l a t i o n between the t o t a l K l o s s from U m b i l i c a r i a muhlenbergii
Cladonia
The
r a n g i f e r i n a and r e d u c t i o n i n ''"^C
e x t e n t o f K leakage was
fixation.
assessed q u a l i t a t i v e l y by comparing the K
c o n c e n t r a t i o n washed out of the moss by the v a r i o u s t r e a t m e n t s .
An
wash i n d e i o n i z e d water d i s p l a c e d K i n the "Apparent Free Space"
served as a c o n t r o l .
s o l u t i o n s and
and
The moss was
then washed i n the
i n d e i o n i z e d water again.
initial
(AFS)
and
experimental
I f t h e r e was no damage t o the
membrane, then t h e subsequent washes would be expected t o c o n t a i n low
c o n c e n t r a t i o n s of K, whereas i f the membrane was
continue to leak out.
damaged, then K would
A K c o n c e n t r a t i o n s i g n i f i c a n t l y h i g h e r than i n the
i n i t i a l s o l u t i o n i n d i c a t e s some damage t o the membrane.
2.423 Routine procedure f o r s e p a r a t i n g exchangeable f r a c t i o n
N i C l ^ was
(see 6.32).
s e l e c t e d as the most a p p r o p r i a t e e l u t i n g agent f o r z i n c
A r o u t i n e procedure was
d e r i v e d , as f o l l o w s ;
t i p s were g i v e n a p r e l i m i n a r y r i n s e i n d e i o n i z e d water (1 h ) , f o l l o w e d by
two r i n s e s i n 20 mM
NiCl^
t o volume and analyzed;
( 1 h each); the s o l u t i o n s were pooled, made up
f i n a l l y the t i p s were r i n s e d i n d e i o n i z e d water ( 1
h),
d r i e d , weighed and d i g e s t e d
2.43
Preparation of c e l l walls
(2.15).
The method used t o prepare c e l l w a l l s was
Larkum (1982).
adapted from R i t c h i e and
C e l l c o n t e n t s were e x t r a c t e d u s i n g T r i t o n X-100
and
63
methanol t o y i e l d a t i p which r e t a i n e d i t s gross morphology, y e t was
l a r g e l y f r e e from c e l l o r g a n e l l e s .
This they d e s c r i b e d as a "crude
cell
w a l l p r e p a r a t i o n " , t o d i s t i n g u i s h i t from t h e purer p r e p a r a t i o n s based upon
homogenized t i s s u e which have been devised (e.g. Demarty e t a l . , 1978).
For the purposes o f t h i s study t h e b e n e f i t s t h a t would have been gained
from t h e l a t t e r t e c h n i q u e were outweighed
and
form o f t h e l i v i n g
by t h a t o f r e t e n t i o n o f t h e shape
tissue.
T i p s t h a t were used i n t h e p r e p a r a t i o n o f c e l l w a l l s were g i v e n a
p r e l i m i n a r y wash i n 0.1 M hydroxylamine
i n 0.01 M HNO^ , t o remove
e n c r u s t i n g manganese and i r o n oxides (Gupta & Chen, 1975) and were then
shaken a l t e r n a t e l y i n 0.5% T r i t o n X-100 and 90% methanol ( 1 h each) u n t i l
all
p i g m e n t a t i o n was removed.
as c h l o r o p h y l l
The c o n c e n t r a t i o n o f c h l o r o p h y l l (measured
by t h e method o f Marker e t al,. , 1980) i n successive
Si
methanol e x t r a c t i o n s i s g i v e n i n F i g . 2.2.
I n one experiment an a d d i t i o n a l
e l u e n t , 0.2 M ammonium o x a l a t e i n 0.2 M o x a l i c a c i d ( T i p p i n g e t al., 1985),
was i n c l u d e d t o s e l e c t i v e l y s o l u b i l i z e Fe o x i d e s .
I n o r d e r t o demonstrate
t h e c a t i o n exchange behaviour of t h e c e l l
w a l l s , t h e crude c e l l w a l l p r e p a r a t i o n s were converted t o t h e H^ form by
f o u r washes, each o f f i v e minutes d u r a t i o n f o l l o w e d by one wash o v e r n i g h t ,
i n 0.1 M HNO^
(Wainwright & B e c k e t t , 1975).
successive washes i n d e i o n i z e d water.
T h i s was f o l l o w e d by
A p r e l i m i n a r y experiment showed a t
l e a s t e i g h t washes t o be r e q u i r e d u n t i l no f u r t h e r drop i n pH, caused by
H^ i o n s b e i n g washed o u t o f t h e AFS, was observed.
2.5
ELSCTROM MICROSCOPY METHODS
I n d i v i d u a l leaves o f Rhynchostegium were f i x e d i n a s o l u t i o n of 2.5%
g l u t a r a l d e h y d e , 1.5% formaldehyde
and 0.05 M c a c o d y l a t e b u f f e r a t pH 7.0
f o r 2.5 h, p o s t f i x e d i n 1 % osmium t e t r o x i d e and r i n s e d i n two 10 minute
64
Figure
2.2 C o n c e n t r a t i o n of c h l o r o p h y l l i n s u c c e s s i v e e x t r a c t i o n s o f
Rhynchostegium d u r i n g c e l l - w a l l
preparation
65
0.3
if)
'c
D
QJ
O
C
0.2
a
o
o
0.1
J
1
1
2
n u m b e r of
3
^
5
extractions
66
changes o f d e i o n l z e d water.
They were then passed through a d e h y d r a t i o n
s e r i e s c o n s i s t i n g o f two 10 min changes i n 10%, 20%. 25%, 30%, 50% and 75*
e t h a n o l , t h r e e 10 min changes i n 100% e t h a n o l and 24 h i n a 50:50 mix o f
100% ethano] and Spurr's
low v i s c o s i t y r e s i n (Spurr, 1969).
A f t e r two 24 h
changes i n r e s i n the sample was polymerized a t 80% °C o v e r n i g h t .
80 - 250 nm i n t h i c k n e s s were c u t u s i n g a diamond k n i f e .
Sections
The s e c t i o n s were
s t a i n e d w i t h u r a n y l a c e t a t e and lead c i t r a t e , mounted on copper g r i d s and
viewed u s i n g a P h i l i p s EM 400 t r a n s m i s s i o n e l e c t r o n microscope.
2.6
STATISTICS AND COMPUTING
2.61 Hardware and s o f t w a r e
Computing f a c i l i t i e s c o n s i s t e d o f t h e Northumbrian U n i v e r s i t i e s
M u l t i p l e Access Computer (NUMAC) and a microcomputer.
The NUMAC machine
c o n s i s t G d o f an IBM 360/370 m a i n f r a m e ( r e p l a c e d by an Ahmdahl 470/V8 i n
September 1985) r u n n i n g under the Michigan
Terminal System (MTS). The
microcomputer was a Research Machines (RM) Nimbus, capable o f connecting t o
NUMAC, b u t more f r e q u e n t l y operated
independently.
This used the MS-DOS
o p e r a t i n g system.
Most data were analyzed
on t h e mainframe, u s i n g the Michigan
I n t e r a c t i v e Data A n a l y s i s Software
(MIDAS) package (Fox & Quire, 1976) o r
the MIiMITAB package (Ryan e t aj.. , 1982).
i n these
C e r t a i n procedures n o t a v a i l a b l e
(e.g. some forms o f a n a l y s i s o f v a r i a n c e (ANOVA)) were
performed
on the RM Nimbus u s i n g t h e " M u l t i p l a n " spreadsheet package ( M i c r o s o f t ,
1982) .
This t h e s i s was prepared
u s i n g the "Word" word processing package
( M i c r o s o f t , 1983) on the RM Nimbus w i t h a Toshiba P1351 p r i n t e r .
67
.2.62 P r e l i m i n a r y a n a l y s i s o f data
Raw data was e n t e r e d
i n t o d a t a f l l e s and transformed
w i t h i n KIDAS o r
M u l t i p l a n t o g i v e metal c o n c e n t r a t i o n s as pg g ^ d r y weight.
Descriptive
measures were o b t a i n e d f o r each sample (= f i v e sub-samples i n f i e l d
experiments and f o u r i n l a b o r a t o r y experiments) and sample means were
a b s t r a c t e d and used t o c r e a t e new d a t a f i l e s arranged by t r e a t m e n t .
C o e f f i c i e n t s o f v a r i a t i o n w i t h i n samples were g e n e r a l l y < 15%. I n
soire i n s t a n c e s , however, they rose t o 50% and even 100%.
I n such
i n s t a n c e s , t h e i n d i v i d u a l sub-samples were examined and very o f t e n one subsample was o b v i o u s l y r e s p o n s i b l e f o r t h e b u l k o f t h e v a r i a t i o n .
There i s
no u n i v e r s a l l y accepted procedure f o r d e a l i n g w i t h maverick values b u t i n
t h i s work a s i n g l e value was removed from a sample mean o n l y i f by doing so
t h e c o e f f i c i e n t o f v a r i a t i o n was reduced by 50% o r more.
The p r e l i m i n a r y a n a l y s i s o f data i n v o l v e d simple t e s t s t o s a t i s f y
t h a t t h e data f i t t e d a normal d i s t r i b u t i o n and, where a p p r o p r i a t e ,
t r a n s f o r m a t i o n s were a p p l i e d .
square-root
MIDAS was used t o compute l o g ^ , l o g ^ ^ and
values f o r each case and t o produce c o e f f i c i e n t s o f skewness
and kuir-tosis, h i s t o g r a m s and c o r r e l a t i o n m a t r i c e s f o r each v a r i a b l e .
These
were compared and t h e most a p p r o p r i a t e t r a n s f o r m a t i o n chosen.
2.63 S t a t i s t i c a l a n a l y s i s o f data
The m a j o r i t y o f t h e data c o u l d be analyzed by r e l a t i v e l y
s t r a i g h t f o r w a r d b i v a r i a t e s t a t i s t i c s , e.g. product-moment c o r r e l a t i o n
c o e f f i c i e n t s , ANOVA and "Student" t - t e s t s , a l l a v a i l a b l e w i t h i n t h e MIDAS
package.
Regressions were compared u s i n g a form o f ANOVA (Parker, 1979;
Mead & Curnow, 1983) c a l c u l a t e d w i t h i n t h e M u l t i p l a n spreadsheet
( M i c r o s o f t , 1982).
The r e s i d u a l mean squares a r i s i n g from each i n d i v i d u a l
r e g r e s s i o n were compared u s i n g a " 2 - t a i l e d " F - t e s t and, i f t h e d i f f e r e n c e
68
was
n o t s i g n i f i c a n t , then an ANOVA, comparing the sum
of the two i n d i v i d u a l
r e g r e s s i o n s w i t h a s i n g l e r e g r e s s i o n composed of the two sets of data
combined, was
performed.
Non-parametric t e s t s were c a l c u l a t e d w i t h i n MIDAS; the t e s t s used were
Spearman's Rank C o r r e l a t i o n , Kendall's Tau c o e f f i c i e n t and
Kendall's
c o e f f i c i e n t of concordance ( S t a t i s t i c a l Research Laboratory, 1976;
1977 ) .
Elliott,
69
3. STUDY SITES
3.1
INTRODUCTION
T h i s c h a p t e r i n t r o d u c e s t h e areas and streams where f i e l d
experiments
were performed and m a t e r i a l f o r l a b o r a t o r y experiments was c o l l e c t e d .
S i t e s were chosen f o r a number o f reasons; nearness t o Durham,
a v a i l a b i l i t y o f h e a l t h y Rhynchostegium and presence or absence o f heavy
metals and o t h e r e n v i r o n m e n t a l f e a t u r e s were t h e main ones.
The study
was w e l l l o c a t e d i n t h a t t h e N o r t h e r n Pennine O r e f i e l d , t h e l a r g e s t
o r e f i e l d i n B r i t a i n and once t h e most i m p o r t a n t lead mining r e g i o n i n t h e
w o r l d , i s w i t h i n 30 km o f Durham ( F i g . 3.1), p r o v i d i n g a wide range o f
sites.
Some s t u d i e s , however, were performed on lowland s i t e s c l o s e r t o
Durham and f o r t h e case study (Chapter 8 ) , a s i t e a t B o l t o n , N-W. England
provided a s t i m u l a t i n g
problem.
The g e n e r a l geography and geology c h a r a c t e r i s t i c o f t h e r e g i o n i s
o u t l i n e d f i r s t , under f o u r headings; A l s t o n Moor and Weardale r e p r e s e n t
t h e N o r t h e r n Pennine O r e f i e l d , s i t e s c l o s e r t o Durham a r e l o c a t e d on t h e
Durham C o a l f i e l d and t h e case study s i t e i s t h e Croal V a l l e y .
account
This
i s based upon G e o l o g i c a l Survey Memoirs (Dunham, 1949; Smith &
F r a n c i s , 1967; Tonks e t al,. , 1931) a l o n g w i t h o t h e r m a t e r i a l .
The
q u a l i t y o f t h e water i s recorded as R i v e r Chemical Class ( N a t i o n a l Water
C o u n c i l , 1981; see Toms, 1975, f o r d e s c r i p t i o n s o f t h e f o u r c l a s s e s ) . A
summary o f e n v i r o n m e n t a l data a t s i t e s i n N-E. England i s presented n e x t ,
f o l l o w e d by a more d e t a i l e d c o n s i d e r a t i o n o f t h e s i t e s used f o r t h e
growth s t u d i e s (Chapter 4) and t h e case study (Chapter 8 ) .
70
F i g . 3.1
Map o f N-E.
England showing l o c a t i o n o f sampling r e g i o n s ,
1 = 0005, R. Deerness;
2 = 0012, Rookhope Burn;
3 = 0014, R.
Browney;
4 = 0024, R. Team;
5 = 0048, R. Nent;
6 = 0055, R. South Tyne.
71
72
3.2
GEOGRAPHICAL LOCATION AND
PHYSICAL CHARACTERISTICS
3.21 The N o r t h e r n Pennine O r e f i e l d
3.211
I n t r o d u c t i o n and g e o l o g i c a l background
The N o r t h e r n Pennine O r e f i e l d extends over a l a r g e area of Northern
England, from the Tyne V a l l e y south t o the Craven d i s t r i c t of Y o r k s h i r e
and i s d i v i d e d i n t o two p a r t s by the Stainmore
and Brough.
The p r e s e n t study i s concerned
of t h e o r e f i e l d .
T h i s may
Gap
between Barnard
Castle
o n l y w i t h the n o r t h e r n p a r t
be v i s u a l i z e d as a p l a t e a u u p l i f t e d along the
Pennine f a u l t a t i t s western margins, t i l t e d t o the east ( T a y l o r e t a l . ,
1971) and d i s s e c t e d by t h r e e main r i v e r s ; the Tyne, Wear and Tees and
their tributaries.
I t r e p r e s e n t s the h i g h e s t l a n d i n England o u t s i d e of
t h e La'.<e D i s t r i c t , e x t e n d i n g up t o 893 m a t the summit of Cross F e l l .
As
much of t h e geology of t h e r e g i o n i s common t o both A l s t o n Moor and
Weardale i t seems p e r t i n e n t t o i n c l u d e a few g e n e r a l comments here.
The g r e a t e r p a r t of t h e upland r e g i o n i s u n d e r l a i n by the Lower
C a r b o n i f e r o u s S e r i e s (Johnson,
1981).
The Carboniferous rocks i n t u r n
r e s t upon a basement of Lower Palazeoic rocks i n t o which t h e Weardale
G r a n i t e was
i n t r u d e d d u r i n g t h e Devonian P e r i o d (Dunham, 1981).
At the
end of t h e C a r b o n i f e r o u s P e r i o d these were u p l i f t e d t o form the A l s t o n
Block and a t t h e same time v o l c a n i c a c t i v i t y i n the r e g i o n caused the
Great Whin S i l l of q u a r t z d o l e r i t e t o be i n t r u d e d i n t o t h e Carboniferous
s t r a t a and, subsequently, f o r m i n e r a l i z a t i o n t o occur.
was
g i v e n i t s eastwards
tilt
The whole area
i n t h e T e r t i a r y P e r i o d (Johnson,
1981).
The Lower C a r b o n i f e r o u s Limestone S e r i e s are composed of a l t e r n a t i n g
l i m e s t o n e s , s h a l e s , sandstones and t h i n c o a l seams l a i d doi-m i n a wide
d e l t a , t h e r e l a t i v e p r o p o r t i o n s of each changing i n a N-S
direction.
Upper C a r b o n i f e r o u s Great Limestone was d e p o s i t e d on t o p of t h i s ,
f o l l o w e d by an a l t e r n a t i n g s e r i e s of limestones and o t h e r s t r a t a .
The
The
73
l i u e s t o n e i s g r a d u a l l y d e p l e t e d and by the t o p of the sequence a
sandstone known as M i l l s t o n e G r i t ( r e l a t e d , but not i d e n t i c a l t o , t h e
M i l l s t o n e G r i t of Y o r k s h i r e and Lancashire) dominates (Johnson,
3.212
1981).
A l s t o n Moor
The A l s t o n Moor area covers t h e R. South Tyne v a l l e y south from
A l s t o n a l o n g w i t h i t s t r i b u t a r y v a l l e y s and i s n e a r l y a l l of an a l t i t u d e
above
300 m (Dunham, 1949).
The western edges are d r a i n e d by the R.
Eden but t h e m a j o r i t y of the r e g i o n i s d r a i n e d by the R. South Tyne
(0055) which r i s e s a t Tynehead a t t h e f o o t of Cross F e l l and f l o w s f i r s t
west and then n o r t h t o Hexham and the confluence w i t h the R. North Tyne.
The
p r i n c i p a l s e t t l e m e n t s are a t A l s t o n , Nenthead and
The
area was
f o r m e r l y a v e r y p r o d u c t i v e lead mining area, e s p e c i a l l y
i n t h e Nenthead r e g i o n and was
Company.
Garrigill.
e x t e n s i v e l y worked by the London Lead
There are abundant m i n e r a l v e i n s and a r t e f a c t s of t h i s
mining
i n t h e r e g i o n i n c l u d i n g a l a r g e number of s p o i l heaps and many a d i t s
shafts.
Several streams i n the area d r a i n through these and
consequently
e n r i c h e d i n heavy metals
(Say, 1977).
and
are
The R. Nent (0048),
which c o l l e c t s these a l o n g w i t h t h e drainage waters from s e v e r a l l e v e l s ,
a l s o c o n t a i n s h i g h c o n c e n t r a t i o n s (Armitage,
1979).
0091 and 0288 a r e both s m a l l r i g h t - b a n k t r i b u t a r i e s of 0048,
approximately
and severed
1 km a p a r t ; 0288 f l o w s past t h e entrance t o drainage
s p o i l heaps and s u r f a c e workings and i s consequently
e n r i c h e d ; 0091 has r e l a t i v e l y low c o n c e n t r a t i o n s of heavy metals.
levels
metal0102
i s a r i g h t - b a n k t r i b u t a r y of 0055 c o n t a i n i n g e l e v a t e d c o n c e n t r a t i o n s of
heavy m e t a l s .
0055, d e s p i t e numerous t r i b u t a r i e s such as 0102 which are
m e t a l - e n r i c h e d , c o n t a i n s o n l y s l i g h t l y e l e v a t e d c o n c e n t r a t i o n s of heavy
metals u n t i l
i t s confluence w i t h 0048 a t A l s t o n (Wehr, 1983).
74
Table 3.1 Sampling s i t e s i n A l s t o n Moor ( b l a n k = n o t sampled)
stream-reach
name
grid ref.
altitude
river
(m)
class
0048-80
R. Nent
NY 739469
320
2
0055-30
R. South Tyne
NY 716468
280
lA
0091-05
F o r e s h i e l d Burn
NY 751469
330
0102-85
G a r r i g i l l Burn
NY 743419
350
0288-70
B l a g i l l Burn
NY 739471
320
0288-90
B l a g i l l Burn
NY 739473
330
3.213 i'Jeardale
The R. Wear s t a r t s a t Wearhead by t h e confluence
o f K i l l h o p e Burn and
Burnhope Burn and f l o w s f o r 107 km t o j o i n t h e North Sea a t Wearmouth.
Weardale, t h e upland p a r t o f t h e catchment, extends down as f a r as
Wolsingham, 20 km from Wearhead.
A l a r g e area o f Weardale i s m i n e r a l i z e d
and t h e r e were many l a r g e mines i n t h e p a s t , both i n t h e main v a l l e y and
in i t s tributaries.
0012 i s an i m p o r t a n t l e f t bank t r i b u t a r y o f t h e Wear w i t h a catchment
which i n c l u d e s Rookhope, p r o b a b l y
t h e most i m p o r t a n t
lead-mining
s e t t l e m e n t i n Weardale ( R a i s t r i c k & Jennings, 1965); consequently i t
contains
r e l a t i v e l y h i g h c o n c e n t r a t i o n s o f heavy metals.
0012-28 i s i n
the upper p a r t o f t h e v a l l e y and 0012-45 i s close t o i t s confluence
the R. Wear.
with
0310 i s a r i g h t - b a n k t r i b u t a r y o f 0012, about 2 km long.
0309 i s a r i g h t bank t r i b u t a r y o f t h e Wear which c o l l e c t s water from
Chapel F e l l and from Greenhaws Hush (a "hush" i s a type o f surface
75
w o r k i n g ) and f l o w s t h r o u g h t h e hamlet o f D a d d r y s h i e l d b e f o r e i t s
confluence.
The reach used i n t h e study (0309-80) i s 100 m below t h e
hamlet o f D a d d r y s h i e l d .
Table 3.2 Sampling s i t e s i n Weardale ( b l a n k = n o t sampled).
stream-
name
grid ref.
altitude
river
(m)
class
0012-28
Rookhope Burn
NY 919428
340
0012-45
Rookhope Burn
NY 953387
230
0309- 30
D a d d r y s h i e l d Burn
NY 895380
290
0310- 90
"Race F e l l Burn"
NY 918427
350
3.22 Durham
Coalfield
The f i n a l
Measures".
chemical
d i v i s i o n o f t h e Upper C a r b o n i f e r o u s i s known as t h e "Coal
A c y c l e o f s h a l e , sandstone and c o a l seams was repeated i n a
broad c o a s t a l d e l t a covered by a l u x u r i a n t swamp v e g e t a t i o n (Johnson,
1981).
period
T h i s was u p l i f t e d and t i l t e d a t t h e end o f t h e Carboniferous
(3.211) such t h a t t h e p r e s e n t c o a l seams extend eastwards
N o r t h Sea.
into the
The o n l y o t h e r major d e p o s i t i n t h e r e g i o n i s t h e Permian
Reef o f Magnesium Limestone ( d o l o m i t e ) (Smith & F r a n c i s , 1967).
The
physiography o f t h e r e g i o n i s v e r y d i f f e r e n t t o t h e North Pennine
O r e f i e l d ; lower, w i t h a g e n t l e r r e l i e f and a f e r t i l e
c l a y l e f t by t h e l a s t g l a c i a t i o n .
farmed and e x p l o i t e d f o r i n d u s t r y .
for
t h e ecology o f s u r f a c e waters.
Consequently
cover of boulder
i t i s densely populated,
These a l l have i m p o r t a n t i m p l i c a t i o n s
76
The main r i v e r s i n t h e r e g i o n are the Wear and the Tyne.
le;H-bank t r i b u t a r y of the Wear r i s i n g near Consett and
0014
is a
flowing
a p p r o x i m a t e l y 30 km t o a confluence 7.5 km above Durham C i t y .
0014-40 i s
a p p r o x i m a t e l y 10 km above t h i s confluence and 1 km below a Sewage
Treatment
0014
Works (STW)
a t Langley Park.
0005 i s a r i g h t - b a n k t r i b u t a r y of
which f l o w s t h r o u g h a v a l l e y once i n t e n s i v e l y e x p l o i t e d f o r c o a l .
I n t h e p a s t i t c o n t a i n e d h i g h c o n c e n t r a t i o n s of suspended s o l i d s as a
r e s u l t of m i n i n g a c t i v i t i e s which have now ceased.
0014
The confluence w i t h
i s 2 km above t h e confluence between 0014 and the R. Wear.
The o t h e r study s i t e on the Durham C o a l f i e l d was a r i g h t - b a n k
t r i b u t a r y of t h e R. Tyne.
t h e "yne.
0024 r i s e s near S t a n l e y and f l o w s f o r 27 kz to
There are a l a r g e number of e f f l u e n t s e n t e r i n g along i t s
l e n g t h (Wehr e t a l . , 1981)
i n c l u d i n g some heavy metal e f f l u e n t s .
The
reaches used (0024-20 and 0024-22) a r e both l o c a t e d c l o s e t o Causey Arch,
2 km downstream of a heavy metal i n p u t from a b a t t e r y f a c t o r y as w e l l as
e f f l u e n t s from a STW
colliery.
and, i n t h e p a s t , pumped groundwater
from a
There are b o t h d i u r n a l ( F i g . 3.2) and longer term (Table
f l u c t u a t i o n s i n t h e c o n c e n t r a t i o n of z i n c i n t h e water.
Table 3.3 Sampling s i t e s i n t h e Durham C o a l f i e l d .
stream-
name
grid ref.
altitude
(m)
reach
river
class
0005-44
R. Deerness
NZ 226421
80
IB
0014-40
R. Browney
NZ 222455
90
IB
0024-20
R. Team
NZ 202560
130
3
0024-22
R. Team
NZ 203560
130
3
chemical
3.4)
77
F i g . 3.2
id 2200
Changes i n z i n c c o n c e n t r a t i o n i n 0024-22 on 25-08-83 between 0900
78
10.0
r
8.0
h
-6.0
• =
total
o =
filtrable
2100
2i;00
-
E
Q)
g
^.0
C
2.0
h
0900
1200
1500
1800
t i m e (h)
79
Table 3.4
Zinc c o n c e n t r a t i o n s (mg 1-1) measured i n R. Team between 1980
and 1986.
reach
Zn
5.80
20
0 28
Wehr S t a l . (1981)
21.5.81
20
0 30
Wehr a Whitton (1983a)
5.81
20
0 19 - 6 90
Wehr & W h i t t o n (1983b)
25.8.83
22
3 40 - 7 07
t h i s t h e s i s ( F i g . 3.2)
1.3.84
22
1 36 - 1 88
t h i s t h e s i s (Table 5.7)
6.8.84
20
4 34
t h i s t h e s i s ( F i g . 6.13)
8.8.84
20
3 09
t h i s t h e s i s ( F i g . 6.13)
10.8.84
20
2 75
t h i s t h e s i s ( F i g . 6.13)
12.8.84
20
1 65
t h i s t h e s i s ( F i g . 6.13)
14.8.84
20
1 22
t h i s t h e s i s ( F i g . 6.13)
16.8
84
20
1 30
t h i s t h e s i s ( F i g . 6.13)
11.6.86
22
0 87
t h i s t h e s i s (Table 7.4)
24.7.86
20
0 34
t h i s t h e s i s (Table 3.8)
date
- 6.82
reference
filtrable
3.23 Croal V a l l e y
The C r o a l V a l l e y l i e s i n an area o f N-W.
focus o f i n t e n s e i n d u s t r i a l
activity.
England t h a t has been t h e
The r i v e r c o l l e c t s water from a
number o f t r i b u t a r i e s which r i s e on t h e W. Pennine moors and f l o w s
t h r o u g h B o l t o n t o a c o n f l u e n c e w i t h t h e R. I r w e l l , i t s e l f a t r i b u t a r y of
t h e Mersey.
The area i s s i t u a t e d on t h e Upper Carboniferous Coal
Measures, o v e r l a i n by a deep d e p o s i t o f boulder c l a y .
i t s e l f has d e p o s i t s o f d e l t a g r a v e l s , sands and s i l t s
The Croal V a l l e y
(Tonks e t a j . . ,
80
1931).
General f e a t u r e s o f t h e Mersey catchment are g i v e n by Holland and
Harding
(1984).
Three s i t e s were used; two on the R. Croal (0267) and one (0378-90)
on ii s m a l l r i g h t - b a n k t r i b u t a r y , Blackshaw Brook (Table 3.5).
Table 3.5 Sampling s i t e s i n Croal V a l l e y ( b l a n k = not
stream-reach
name
grid ref.
sampled).
altitude
river
(m)
class
0267-80
R. Croal
SD 736075
60
2
0267-90
R. C r o a l
SD 749066
50
2
0378-90
Blackshaw Brook
SD 744075
50
3.3
chemical
ENVIRONMENTAL BACKGROUND TO SITES IN N-E. ENGLAND
3.31
Introduction
A wide range o f water c h e m i s t r i e s were encountered
d u r i n g t h e study;
these a r e summarized i n t h i s s e c t i o n , along w i t h d e t a i l s o f macrophytes
found i n t h e study
3.32
Water
reaches.
Chemistry
The a v a i l a b i l i t y o f s u i t a b l e s u b s t r a t e s f o r c o l o n i z a t i o n i n r i f f l e
s e c t i o n s meant t h a t most o f the reaches s t u d i e d had moderately
fast
c u r r e n t speeds (Table 3.6); t h e t u r b u l e n c e i n these areas acted t o keep
them c l o s e t o s a t u r a t i o n w i t h r e s p e c t t o d i s s o l v e d oxygen, even i n
reaches w i t h c o n s i d e r a b l e i n p u t s o f o r g a n i c p o l l u t i o n (e.g. 0014-40,
0024-20; 0024-22; see Table 3.3). T y p i c a l l y pH values were > 7.0
( e x c e p t i o n =^ 0012-28), i n d i c a t i n g t h e pH p r e f e r e n c e o f the species
81
recorded i n p r e v i o u s s t u d i e s (Merry e t a l . , 1981; Wehr, 1983; Wehr &
W h i t t o n , 1983a).
D u r i n g a more i n t e n s i v e study of f o u r of these reaches
(0091-05; 0288-70; 0309-80; 0310-90; see Chapter 4) these values
f l u c t u a t e d by up t o + 0 . 3
pH
units.
On t h e b a s i s of anion and c a t i o n analyses, the study s i t e s i n
England r e s o l v e i n t o two d i s t i n c t groups (Table 3.7;
C o a l f i e l d (FRP
> 100 yg g~^;
M-E.
Table 3.8): Durham
NO„-N > 10 mg l""^; Na > 40 mg 1~-^; X > 10
mg l " " ^ ) and t h e N o r t h e r n Pennines (FRP
Na < 10 mg l " " ^ ; K < 10 mg l " ' ^ ) .
< 30 pg
; NO
-N < 2 mg l"'^;
These r e f l e c t both d i f f e r e n c e s i n
bedrock geology and the d i f f e r e n t i n t e n s i t i e s of l a n d use and p o p u l a t i o n
etc.
(3.2).
Those streams i n t h e N o r t h e r n Pennines w i t h r e l a t i v e l y h i g h
c o n c s n t r a t i o n s of n u t r i e n t s (0012-45; 0288-70; 0288-90) are a s s o c i a t e d
w i t h l o c a l c o n c e n t r a t i o n s o f p o p u l a t i o n o r more i n t e n s i v e a g r i c u l t u r a l
activity.
There i s a wide range o f c o n c e n t r a t i o n s o f hardness components;
c o n c e n t r a t i o n s of Ca range from 17.6 mg 1 ^ (0310-90) t o 85 mg 1 "
(0005-44).
Most a r e , however, over 40 mg 1 ^ and t h e r e i s no c l e a r
d i s t i n c t i o n between upland and lowland
streams.
The h i g h e s t c o n c e n t r a t i o n of z i n c measured d u r i n g t h e survey was
rag I
(0012-28) a l t h o u g h c o n c e n t r a t i o n s of up t o 7.07
measured i n 0024-22 (Table 3.4).
2.05
mg l"""^ have been
Concentrations i n the metal-enriched
streams of t h e N o r t h e r n Pennines were g e n e r a l l y l e s s than 0.5 mg 1 ^
w i t h two e x c e p t i o n s : 0012-28 (Zn.
. . (Zn. ) = 2.06
X O L Q. 1
Zn
(Zn^) = 2.05
mg 1
mg l"'^;
L
) and 0048-80 (Zn^ = 1.39
mg 1
; Zn^
f iltrab
0.475 mg 1 ""^) . There i s a l a r g e d i f f e r e n c e i n t h e r a t i o s of Zn
Zn
between these streams; t h e mean r a t i o was
f
0.82
(SD =
0.25)
to
82
Table 3.6
Physico-chemical v a r i a b l e s i n study reaches i n K-E. England.
stream
date
-reach
total
dissolved
speed
alkalinity
oxygen
(m s ^ ) ( p S cm ^ )
(neq 1 ^ )
current
cond.
pH
{% s a t ) (mg 1 ^ )
0005-44
24.7.86
0.502
1397
7.7
10.0
108
11.5
0012-28
26.5.86
0.316
159
6.5
1.2
97
10.3
0012-45
21.6.84
0.280
285
7.8
4.7
121
13.1
0014-40
24.7.86
0.609
674
7.8
4.5
86
9.1
0024-20
9.8.84
0.719
740
7.1
2.0
98
10.3
3024-22
11.6.86
0.435
718
7.7
3.3
95
9.9
0048-80
15.8.84
0.083
491
8.0
5.9
106
10.3
0055-30
24.7.86
0.433
288
8.1
3.7
107
10.5
0091-05
13.6.86
0.483
234
7.7
4.3
110
11.7
0102-85
11.6.86
0.345
124
7.5
1.8
103
11.3
0288-70
13.6.86
0.381
280
8.1
4.5
101
11.3
0288-90
7.8.86
0.789
211
7.6
2.7
104
10.9
0309- 80
13.6.86
0.635
360
7.8
5.5
104
11.0
0310- 90
13.6.86
0.479
136
7.4
2.2
102
10.4
83
Table 3.7 Anion c h e m i s t r y i n study reaches i n N-E. England on 24-7-1986
(AM = A l s t o n Moor; DC = Durham C o a l f i e l d ; WD = Weardale).
stream-reach
area
FRP
NOg-N
,-1
(yg 1 ^ )
0005-44
DC
0012-28
WD
3.9
0012-45
WD
23.7
0014-40
DC
0024-20
101
-1
(mg 1 ^ )
148
SO^-S
(Bg 1 ^ )
CI
(ng 1 ^ )
190
66.7
0.5
173
9.4
0.6
76
8.8
1215
10.7
124
62.2
DC
3654
55.3
86
71.1
0024-25
DC
3683
44.8
67
62.2
0048 -80
AM
3.6
0.8
113
5.7
0055-30
AM
6.1
0.4
19
6.2
0091-05
AM
9.3
0.1
2
0.6
0102-85
AM
8.7
0.1
9
0.9
0288-70
12.6
1.1
68
6.8
0288-90
10.6
0.1
55
7.2
7.6
0.3
10
6.5
0309-80
WD
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3.33
Macrophytes
S i x t e e n macrophytes (sensu Holmes & W h i t t o n , 1977) were encountered
d u r i n g t h i s s t u d y (Table 3.9).
The s e l e c t i o n of reaches was
weighted
towards those w i t h p o p u l a t i o n s o f Rhynchostegium; t w e l v e out of f o u r t e e n
reaches c o n t a i n e d t h i s s p e c i e s .
The n e x t most abundant species were
F o n t i n a l i s a n t i p y r e t i c a ( t h r e e r e a c h e s ) , Lemanea f l u v i a t i l i s
r e a c h e s ) , Cladophora
g l o m e r a t a , S t i g e o c l o n i u m tenue and
r i p a r i u m ( t h r e e reaches each).
(four
Amblystegium
The r i c h e s t a q u a t i c bryophyte f l o r a s were
found i n upland streams such as 0055-30, 0091-05 and 0310-90; some
species (e.g. Hygrohypnum spp., C i n c l i d o t u s f o n t i n a l o i d e s and
a c i c u l a r e ) were found o n l y here.
Racomitrium
The l a t t e r two species were
c h a r a c t e r i s t i c o f t h e upper p a r t s o f b o u l d e r s s u b j e c t t o o n l y p e r i o d i c
immersion.
Amblystegium
r i p a r i u m was c o n f i n e d t o lowland s i t e s ,
r e f l e c t i n g i t s observed p r e d i l e c t i o n f o r r e l a t i v e l y n u t r i e n t r i c h waters
( W h i t t o n e t al,., 1981; H o l l a n d & Harding, 1984).
Cladophora
glomerata i s
a l s o c h a r a c t e r i s t i c o f e u t r o p h i c waters but i s i n t o l e r a n t of heavy metals
( W h i t t o n , 1970); i t i s found a t t h e two s i t e s on Durham C o a l f i e l d w i t h
low c o n c e n t r a t i o n s o f heavy metals (0005-44 and 0012-40) but a l s o a t
0091-05 i n A l s t o n Moor which i s r e l a t i v e l y n u t r i e n t poor.
v a s c u l a r p l a n t s were observed; Equisetum
arvense a t two s i t e s i n the
N o r t h e r n Pennines and Ranunculus p e n i c i l l a t u s a t 0014-40.
p e c t i n a t u s was
observed
Two a q u a t i c
i n 0024 a p p r o x i m a t e l y 1 km upstream
Potamogeton
of 0024-20.
86
Table 3.9
Macrophytes p r e s e n t i n study reaches i n N-E. England ( L f =
Lemanea f l u v i a t i l i s ;
Ba = Batrachospermum sp.; Cg = Cladophora glomerata;
Mo = Mougeotia sp.; Bp = Bryum p s e u d o t r i q u e t r u m ; Cf = C i n c l i d o t u s
f o n t i n a l o i d e s ; Ra = Racomitrium a c i c u l a r e ; Rr = Rhynchostegium
r i p a r i o i d e s ; Ar = Amblystegium r i p a r i u m ; Af = A. f l u v l a t i l e ;
Fontinalis antipyretica;
Fa =
Ho = Hygrohypnum ochraceum; HI = H. l u r i d u m ;
Ea = Equisetum arvensc; Rp = Ranunculus p e n i c i l l a t u s var p e n i c i l l a t u s ) .
stream-reach
L f Ba Cg St Mo Bp Cf Ra Rr Ar Af Fa Ho HI Ea Rp
0005-44
1
1
1
0012-28
0012-45
1
1
1
1
0024-20
1
0024-22
1
0048-80
1
1
1
1
1
1
1
1
1 1 1
1
1
1
1
0288-70
1
0288-90
:
1
J
1
0102-85
0310- 90
1
1
0091-05
0309- 80
1
1
0014-40
0055-30
1
1
1
1
1
1
]
1
1
1
87
3.4 DETAILS OF SITES USED FOR GROWTH STUDY
3.41
Introduction
Four s i t e s i n t h e N o r t h e r n Pennines were chosen f o r the growth study
such t h a t a l l c o u l d be sampled i n one day.
C r i t e r i a used t o s e l e c t s i t e s
i n c l u d e d a good growth o f Rhynchostegium a t t a c h e d t o movable boulders ( i n
practice
the s i z e o f b o u l d e r s was determined u l t i m a t e l y by the f l o w
regime w i t h i n the s t r e a m ) .
s i t e s i n the immediate
A l l s i t e s were i n upland areas; no lowland
v i c i n i t y were a b l e t o f u l f i l
the above c r i t e r i a ;
and they i n c l u d e d s i t e s w i t h and w i t h o u t metal c o n t a m i n a t i o n and w i t h
different nutrient
3.42
3.421
Environment
regimes.
o f streams
Introductioi!
I t was not p o s s i b l e t o c o l l e c t a l l o f the environmental data from the
immediate
l o c a l i t y o f the study s i t e s .
M e t e o r o l o g i c a l and discharge data
were s u p p l i e d by t h e Northumbrian Water A u t h o r i t y
(N.W.A.) from a
m e t e o r o l o g i c a l s t a t i o n a t Burnhope R e s e r v o i r i n Weardale (N'Y 846393, 357
m) ar.d a f l o w - g a u g i n g s t a t i o n a t Harwood Beck i n Upper Teesdale (NY
849309, 390 m).
These s i t e s are both w i t h i n 20 km o f the f i e l d s i t e s and
are o f a s i m i l a r a l t i t u d e (Table 3.1; Table 3.2).
These data are
presented t o g i v e a broad i n d i c a t i o n o f seasonal changes i n the
environment d u r i n g t h e study p e r i o d .
3.422 A i r temperature
Maximum and minimum a i r temperatures c o l l e c t e d
are p l o t t e d as weekly means ( F i g . 3.3).
from Rurnhope Reservoir
These i n d i c a t e
the r e l a t i v e l y
cool c]i."nale experienced a t these a l t i t u d e s ; o n l y t h r e e weeks had a
weekly mean maximum temperature o f 20 °C o r more, w h i l s t the minimum
88
F i g . 3.3 Weekly mean maximum and minimum temperatures a t Burnhope
R e s e r v o i r i n 1985-86.
89
25
20
o
o
15
q;
D
"o
i_
a;
Q.
10
O
o
o o o
o
%
o
e
CD
O
Q
^ o
' < 9
O
o
0
^
•
m
O (
pcD
o
Q
o o
O
o
O
O
0%
O
o
-5
J
A
1985
S
O
N
D
J
F
M
A
M
J
1986
•
=
maximunn
o = minimum
J
90
temperature was a t or below 0 °C f o r f o u r t e e n weeks.
The c o l d e s t month
was February and t h e warmest was J u l y .
3.423 P r e c i p i t a t i o n
P r e c i p i t a t i o n was c o l l e c t e d i n a r a i n gauge, 12.5 cm i n diameter and
.30 cm above t h e ground a t Burnhope R e s e r v o i r , a t 0900 each morning.
.Monthly means f o r 1984-85 and 1985-86 are presented as a histogram ( F i g .
3.4).
The maximum p r e c i p i t a t i o n i n 1985-86 f e l l d u r i n g t h e w i n t e r
months; t h e w e t t e s t month was January 1986 and t h e d r i e s t was October
1985.
Note a l s o t h e p a r t i c u l a r l y wet August i n 1985 compared w i t h data
f o r 1984.
3.424 Flow
Flow data from Harwood Beck a r e presented as a histogram ( F i g . 3.5) t o
g i v e an i n d i c a t i o n o f l i k e l y v a r i a t i o n s i n t h e reaches under
consideration.
There i s a s i g n i f i c a n t c o r r e l a t i o n ( r = 0.905 ***)
between mean f l o w and mean r a i n f a l l ; maximum f l o w s were recorded d u r i n g
w i n t e r b u t , as i n 3.423, t h e v a l u e recorded f o r August 1985 was
p a r t i c u l a r l y high.
3.43 D e t a i l s o f s t u d y s i t e s
3.43]
F o r e s h i e l d Burn, reach 0091-05
T h i s s i t e d r a i n s open moorland
and some improved pasture and c o n t a i n s
r e l a t i v e l y low c o n c e n t r a t i o n s of n u t r i e n t s (Table 3.7) and heavy metals.
The reach was h e a v i l y shaded by Acer pseudoplatanus
( F i g . 3.6).
and Sorbus aucuparia
Rhynchostegium was f a i r l y abundant, p r o v i d i n g about 30%
cover, and was t h e dominant macrophyte a l t h o u g h s e v e r a l others were
observed
(Table 3.9).
and 400 cm i n diameter.
I t was found on cobbles and boulders between 200
91
Fig.
3.4
1985-86.
Fig.
3.5
Monthly mean p r e c i p i t a t i o n a t Burnhope R e s e r v o i r i n 1984-85 and
^VA
- m 5 - s . ^ ^ cKgoW Vvm, ' v'^m-'^s.
Monthly mean d i s c h a r g e a t Harwood Beck i n 1985-86.
92
60
o
X)
50
E
E
40
c
o
30
20
c
o
10
E
J
J
1985
N
D
J
F
1986
M
M
2.0 h
0)
oi
i_
o
o
(/)
1.0
c
o
01
£
J
J
1985
0
D
J
F
1986
M
M
93
3.432 B l a g i l l Burn, reach 0288-70
0288-70 i s s i t u a t e d a t the f o o t of a steep wooded bank which caused
heavy shading (see Table 4.3), again by Acer and Sorbus ( F i g . 3.7).
was
immediately upstream of a disused a d i t .
RhynchosteHium was
It
D i s t r i b u t i o n of
patchy; on a s m a l l (about 2 m h i g h ) w a t e r f a l l and
on
an a r t i f i c i a l w e i r , both below the study reach, cover was between 80%
90%; however i n o t h e r p a r t s i t was v i r t u a l l y n o n - e x i s t e n t .
and
I n 0288-70 i t
had a cover of between 10% and 20% on cobbles and boulders between 150
and 350
i n dia.meter.
I t was
the o n l y macrophyte i n the reach (Table
3.9) .
3.433 D a d d r y s h i e l d Burn, reach 0309-80
T h i s reach was
p a r t i a l l y shaded by Acer, Sorbus and Betula pubescens;
however the p a r t where the b o u l d e r s were s i t u a t e d was shaded o n l y by
herbaceous v e g e t a t i o n and by t h e bank ( F i g . 3.8).
The reach c o n s i s t e d of
a s e r i e s of s m a l l (about 20 cm h i g h ) w a t e r f a l l s composed of l a r g e
b o u l d e r s ( s i z e = 250 t o 600 cm i n d i a m e t e r ) and bedrock blocks w i t h
cobbles and pebbles between.
The w a t e r f a l l s were covered
Rhynchoslegjum and Lemanea f1uv1 a t i I i s
3 . 4 3 4 "Race F e l l Burn",
with
i n approximately equal abundance.
reach 0310-90
0330-90 d r a i n s a Pinus p l a n t a t i o n and open moorland before j o i n i n g
0012.
Although t h e r e are s e v e r a l mines i n the area, the stream
does not have e l e v a t e d c o n c e n t r a t i o n s of heavy metals.
itself
The reach i s 2 m
above and 8 m below a s m a l l (about 1 m h i g h ) w a t e r f a l l and i s unshaded
except by herbaceous v e g e t a t i o n ( F i g . 3.9).
I t has a very dense cover of
Rhynchostegium a l l year round
(Wehr & W h i t t o n , 1983b), p a r t i c u l a r l y on
the
cobbles and boulders between 150 and 500
w a t e r f a l l and on bedrock,
in diameter.
The p l a n t s are p a r t i c u l a r l y r o b u s t , w i t h l a r g e
cm
94
F i g . 3.6
F o r e s h i e l d Burn, reach 0091-05.
F i g . 3.7
B l a g i l l Burn, reach 0288-70.
95
96
F i g . 3.8
D a d d r y s h i e l d Burn, reach 0309-80.
F i g . 3.9
"Race F e l l Burn", reach 0310-90.
97
98
leaves and shoots o f above average l e n g t h and weight (Wehr, 1983). Other
macrophytes found I n t h e reach i n c l u d e F o n t l n a l i s a n t i p y r e t i c a , Bryum
pseudotriquetrum
and Hygrohypnum l u r i d u m (Table 3.9).
3.5 DETAILS OF SITES USED FOR CASE STUDY
3.51
Introduction
The H a l l Chemical Works smelted
1968.
chromate i n B o l t o n between 1880 and
The s p o i l heap r e s u l t i n g from these a c t i v i t i e s was subsequently
landscaped and r e v e g e t a t e d
(Gemmell, 1972; 1973: 1974).
Before
r e v e g e t a t i o n t h e r e was c o n s i d e r a b l e l e a c h i n g o f chromium i n t o the R.
Croal (3 - 5 t y r ^ ) ,
1973).
w i t h pronounced e f f e c t s on t h e b i o t a
A f t e r w a r d s , a p p r o x i m a t e l y h a l f o f t h e r a i n f a l l on t h e s i t e was
r e t u r n e d t o t h e atmosphere as
e v a p o t r a n s p i r a t i o n (Gemmell, 1977) and two
s e t t l i n g tanks c o l l e c t e d much o f the remaining drainage.
has
(Breeze,
improved as a r e s u l t o f t h i s b u t the R. Croal s t i l l
The s i t u a t i o n
receives
s i g n i f i c a n t i n p u t s o f chromium (unpublished computer records o f North
West Water A u t h o r i t y (N.W.W.A.)) and these a r e i n v e s t i g a t e d i n t h i s
study.
3.52
Environmental
3.521
Background
R e l a t i o n s h i p betweer; f l o w and chromium c o n c e n t r a t i o n s
The
v a r i a t i o n i n chromium c o n c e n t r a t i o n s i n the Croai below the H a l l
Chemical Works was s t u d i e d u s i n g data s u p p l i e d by the N.W.W.A.. The
maximum c o n c e n t r a t i o n "recorded between 1982 and 1984 was 0.215 mg 1
56%
o f records f o r t h i s p e r i o d were below 0.053 mg I - l
of range) and o n l y ~% were above C.]C8 mg ]
range).
.
(lowest q u a r t i l e
(upper two q u a r t i i e s o f
The g r e a t e s t v a r i a t i o n i n the chromium c o n c e n t r a t i o n occurred a t
p e r i o d s o f low f l o w ( F i g . 3.10) which tended
August; t h i s p e r i o d was chosen f o r the study.
t o occur d u r i n g J u l y and
99
F i g . 3.10
R e l a t i o n s h i p between mean d a i l y f l o w and t o t a l chromium i n R.
Croal between January 1982 and December 1984.
100
200
R. CROAL.
NOB
END
150
O
O
O
0)
o 100
O
O
O
o
<o o
O
O
(DO^
°o op
50
o
o
o
oo o
- o — -e
O
OOO-
O
O
-O-O
O-
5
flow
Im^s"''
D.L.
101
3.522 D e s c r i p t i o n o f study s i t e s
Three s i t e s were chosen f o r study (Table 3.10).
Two were on the
C r o a l , above (0267-80) and below (0267-85) the H a l l Chemical Works.
0267-80 was a f a s t - f l o w i n g c a n a l i z e d s t r e t c h of the r i v e r ( F i g . 3.11) and
0267-85 was a r i f f l e a p p r o x i m a t e l y 2 km downstream
( F i g . 3.12).
Both'
s i t e s had abundant growths of Cladophora glomerata; other macrophytes a t
the two s i t e s were F o n t i n a l i s a n t i p y r e t i c a and Ranunculus f l u i t a n s a t
0267-80 and Amblystegium r i p a r i u m , t r a c e s of F o n t i n a l i s a n t i p y r e t i c a ,
Ranunculus f l u i t a n s and Rorippa n a s t u r t i u m - a q u a t i c u m a t 0267-85.
t h i r d s i t e (0378-90) was on a small t r i b u t a r y along the N-W.
The
edge of the
s p o i l heap ( F i g . 3.13), i m m e d i a t e l y before t h e stream entered a c u l v e r t
( F i g . 3.14).
The water a t t h i s s i t e was s i g n i f i c a n t l y harder than a t the
o t h e r s i t e s (Table 3.10) r e f l e c t i n g the l o c a l e f f e c t s of t h e s p o i l
(Gemmell, 1977).
and s i l t ,
In addition,
i t had an u n s t a b l e s u b s t r a t e of f i n e sand
which supported v i s u a l l y - o b v i o u s growths of the blue-green alga
Flectonema b u t no macrophytes.
Table 3.10 P h y s i c a l and chemical v a r i a b l e s a t case study s i t e s on 1-7-85
site
current
cond.
pH
total
Cr
alkalinity
speed
(m s"-^ )
Ca
(meq l""^ ) (mg 1
(pS cm"-^ )
) (mg 1
3.8
35.5
0.043
o
4.1
$6.3
<0.017
8.6
7.4
75.3
0.504
0267-80
0.84
368
7.6
0257-85
0.38
593
I .
0378-90
0.36
652
102
F i g . 3.11
R. Croal a t Darcy Lever, reach 0267-80,
F i g . 3.12
R. Croa] a t Nob End, reach 0267-90.
103
'
^ ^ ^ ^
104
F i g . 3.13
Blackshaw Brook, reach 0378-90.
F i g . 3.14
Blackshaw Brook, reach 0378-90, showing
edge of s p o i l heap.
N-W.
105
106
Some a d d i t i o n a l water c h e m i s t r y data f o r t h e p e r i o d o f t h e study was
o b t a i n e d from t h e N.W.W.A., from a sampling p o i n t about 1 km downstream o f
0267-85.
T h i s i n c l u d e d b i o c h e m i c a l oxygen demand (B.O.D.), chemical oxygen
demand ( C C D . ) and anion analyses n o t made d u r i n g the study (Table 3.11).
Values f o r B.O.D. and C C D . r e f l e c t t h e o r g a n i c a l l y - p o l l u t e d s t a t e o f the
r i v e r ; values f o r t h e n u t r i e n t analyses are t y p i c a l
eutrophic
o f a moderately
river.
Table 3.11 Water c h e m i s t r y i n R. C r o a l , 13-6-85 t o 20-7-85 ( a l l v a l u e s ,
except pH, as mg 1 ^ ) .
n
min
max
mean
pH
13
7.3
7.5
B.O.D.
13
2.2
24.0
10.5
6.8
CCD.
13
21.0
141.0
65.4
38.0
NH -N
4
13
0.20
2.85
1.65
0.77
NO -N
13
0.40
1.75
0.82
0.36
NO -N
2
13
0.08
0.24
0.14
0.05
Cl"
13
17.0
44.0
FRP
13
<0.05
0.30
26.8
SD
8.1
107
4. GROWTH OF RHYNCHOSTEGIUM
4.1
INTRODUCTION
T h i s chapter p r e s e n t s r e s u l t s o f a seasonal survey of t h e r a t e of
growth o f Rhynchostegium a t f o u r s i t e s i n t h e Northern Pennines.
p r o v i d e s a fundamental background
a g a i n s t which seasonal v a r i a t i o n s i n t h e
a v a i l a b i l i t y o f moss f o r m o n i t o r i n g purposes and i t s subsequent
of metals may be i n t e r p r e t e d .
meteorological
This
accumulation
D e t a i l s o f these s i t e s , along w i t h some
and e n v i r o n m e n t a l data a r e g i v e n i n Chapter 3.
S i t e s were
sampled a t monthly i n t e r v a l s between J u l y 1985 and June 1986. Due t o a
reduced growth r a t e i n w i n t e r months, temperature was measured but no growth
measurements were made i n February 1986.
Two p r e l i m i n a r y l a b o r a t o r y experiments are described f i r s t
T h i s i s f o l l o w e d by a d e s c r i p t i o n of t h e environmental background
(4.2).
t o the four
streams i n s e c t i o n 4.3 and growth r a t e s a t t h e f o u r s i t e s i n s e c t i o n 4.4. I n
s e c t i o n 4.46, r e s u l t s from t h e f o u r s i t e s are com.pared and c o r r e l a t i o n s w i t h
e n v i r o n m e n t a l v a r i a b l e s made.
production
F i n a l i y , e s t i m a t e s of annual d r y matter
a t t h e f o u r s i t e s a r e made (4.5, 4.6).
4.2 PRELIMINARY EXPERIMENTS
4.21
Introduction
Two b r i e f l a b o r a t o r y experiments are d e s c r i b e d t o t e s t an i n i t i a l
assumption
r e g a r d i n g growth of shoots - t h a t t h e r e i s a s h o r t region of
a p i c a l e l o n g a t i o n beJow which t h e r e i s a l i n e a r r e l a t i o n s h i p between l e n g t h
and d r y w e i g h t .
108
4.22
Shoot e x t e n s i o n a t apex
The zone o f e l o n g a t i o n o f shoot t i p s o f Rhynchosteglum from 0310-90 was
determined u s i n g a low-power b i n o c u l a r microscope.
Leaves were c u t o f f from t h e apex i n t h r e e planes ( F i g . 4.1); these a r e
c l o s e l y bunched t o form a c l u s t e r a t the t i p and i n a zone o f e l o n g a t i o n
e x t e n d i n g 3 mm back from t h e t i p .
Below th.^s leaves are spaced
equidistantly.
4.23
R e l a t i o n s h i p between mass and shoot l e n g t h
A second p r e l i m i n a r y experiment was performed t o r e l a t e increases i n
l e n g t h t o c o r r e s p o n d i n g i n c r e a s e s i n shoot mass.
Shoots c o l l e c t e d from each o f the f o u r reaches were c u t i n t o a v a r i e t y
of l e n g t h s , from 0.5 cm t o 3.0 cm and samples c o n s i s t i n g o f known numbers
of each l e n g t h d r i e d and weighed.
There were s i g n i f i c a n t c o r r e l a t i o n s between mass and l e n g t h f o r a l l
f o u r s i t e s ( F i g . 4.2, Table 4.1), w i t h r e g r e s s i o n c o e f f i c i e n t s of between
0.87
and 1.65.
Three o f the p o p u l a t i o n s had slopes o f > 1.0 and p o s i t i v e
i n t e r c e p t s ; t h i s i n d i c a t e s t h a t mass i n c r e a s e s f a s t e r than l e n g t h along the
stem and t h a t the a p i c a l 0.5 cm i n these p o p u l a t i o n s has a s l i g h t l y h i g h e r
d r y weight than the r e g i o n below.
4.3
ENVIRONMENT OF STREAMS
4.31 Water Temperature
V a r i a t i o n s i n monthly spot temperature readings are c l o s e l y c o r r e l a t e d
(Table 4.2), w i t h an average range between the f o u r s i t e s o f 1.9 °C ( F i g .
4.3).
These d i f f e r e n c e s are c o r r e l a t e d w i t h the a l t i t u d e o f the streams:
0309- 80 i s the lowest and on 10 out o f 12 occasions was the warmest w h i l s t
0310- 90 i s the h i g h e s t and g e n e r a l l y the c o o l e s t .
109
F i g . 4.1
Shoot of Rhynchostegium from 0310-90, showing zone of a p i c a l
elongation.
Scale bar = 1 mm.
110
Ill
F i g . 4.2
R e l a t i o n s h i p between shoot mass and l e n g t h of Rhynchostegium i n
f o u r reaches i n N-E.
England used i n growth study.
112
5
0091-05
o
o
U
0288-70
U
o
o
3
3
o
o
2
8
2
8
8
1
CO
O
E
5
r 0309-80
-
8
0310-90
U
3
8
o
o
2
1
o
8 o
1
l e n g t h (cm)
2
3
113
Spot temperature r e a d i n g s a l s o c o r r e l a t e d c l o s e l y t o mean monthly
maximum
and minimum, temperatures c o l l e c t e d a t Burnhope R e s e r v o i r (Table
4.2).
Table 4.1 C o r r e l a t i o n s and r e g r e s s i o n s between shoot mass and l e n g t h f o r
f o u r p o p u l a t i o n s o f Rhynchostegium.
stream-reach
regression equation
correlation
significance
coefficient
0091-05
y = 1 .45 X - 0,.43
0,.993
***
0288-70
y = 1 . 14X - 0.. 21
0..991
***
0309-80
y = 1 .65 X - 0.. 14
0,.988
***
0310-90
y = 0,.87 X - 0..20
0..979
***
4.32 L i g h t
I t was n o t p o s s i b l e t o c o l l e c t l i g h t f l u x data throughout t h e 12 month
p e r i o d ; however a s e t o f f i g u r e s f o r comparative purposes was c o l l e c t e d on
13.6.86.
These g i v e an i n d i c a t i o n of t h e degree of shading experienced a t
each o f t h e f o u r s i t e s by comparing photon f l u x a t water l e v e l w i t h photon
f l u x i n a nearby unshaded area.
Readings were c o l l e c t e d a t t h e two p o s i t i o n s a t each s i t e over t h e
course o f a p p r o x i m a t e l y 1 h.
These were very v a r i a b l e as t h e weather
a l t e r n a t e d between b r i g h t and o v e r c a s t .
T y p i c a l values (Table 4.3) show
a t t e n u a t i o n o f l i g h t t o be g r e a t e s t a t 0091-05
( 7 0 % ) , 0309-80 (26%) and 0310-90 ( 2 1 % ) .
, f o l l o w e d by 0288-70
There were few t r e e s or shrubs a t
0309-80 and none a t 0310-90 and t h e shading a t these s i t e s probably
r e p r e s e n t s shading by t h e stream banks.
114
F i g . 4.3
reaches
Seasonal v a r i a t i o n s i n spot t e m p e r a t u r e measurements i n f o u r
i n N-E.
England.
115
0091-05
10
0288-70
r
5
o
o
a;
0309-80
D
a
10
tern
-4—'
5
i_
CD
Q.
0310-90
J J A S O N D J F M A M J
1985
1986
116
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117
Table 4.3 T y p i c a l v a l u e s f o r photon f l u x d e n s i t y a t each s i t e (pmol
-2
photon
-1
m
s
) used i n growth
site
study.
photon f l u x
time
water l e v e l
0091-05
1100 - 1200
0288-70
1000 - 1100
0309- 80
0310- 90
4.33 Water
240 + 118
85 +
% attenuation
control
1300 + 680
81
23
284 +
73
70
1400 - 1500
776 + 400
1054 +
32
26
1500 - 1600
724 + 145
918 + 121
21
Chemistry
S e v e r a l aspects o f water c h e m i s t r y measured over t h e 12 month p e r i o d
r e f l e c t t h e c o n t r i b u t i o n s o f d i f f e r e n t catchment geologies t o t h e streams
and,
i n p a r t i c u l a r , t h e i n f l u e n c e o f t h e Carboniferous Limestone.
Water
c h e m i s t r y i s summarized i n S e c t i o n 3.32 and i n Table 4.4.
A l l o f t h e streams showed l a r g e ranges of pH measurements.
A pH o f 6.9
was measured i n 0288-70 i n August 1985 and one of 8.4 i n 0309-80 i n June
1985.
These were t h e extremes recorded; a l l of t h e means were i n t h e range
7.6 t o 7.8.
The pH i n 0091-05 tended t o be the h i g h e s t , f o l l o w e d by 0309-
80, 0288-70 and 0310-90.
i n 0310-90.
T o t a l a l k a l i n i t y was h i g h e s t i n 0309-80 and l e a s t
T o t a l a l k a l i n i t i e s a t 0091-05 and 0288-70 were very s i m i l a r .
C o n d u c t i v i t y and t h e c o n c e n t r a t i o n of hardness components (Mg and Ca)
a l l r e f l e c t t h e i n f l u e n c e of background geology;
0309-80 and t h e " s o f t e s t " a t 0310-90.
the "hardest" water was a t
0091-05 and 0288-70 were, again,
v e r y s i m i l a r (Table 3.6; Table 3.8).
0288-70 g e n e r a l l y c o n t a i n e d h i g h e r c o n c e n t r a t i o n s of n u t r i e n t elements
than o t h e r streams and 0091-05 c o n t a i n e d lower c o n c e n t r a t i o n s (Table 4.4).
118
0310-90 had a h i g h c o n c e n t r a t i o n o f FRP (30.7 mg 1 ''^) compared w i t h o t h e r
streams (Table 3.7).
The
c o n c e n t r a t i o n s o f both Mn and Fe showed considerable
variability,
however c o n c e n t r a t i o n s o f both were h i g h e r i n 0091-05 and 0288-70 than i n
0309-80 and 0310-90 (Table 4.4).
0288-70 and 0309-80 were chosen t o r e p r e s e n t Zn-enriched
0091-05 and 0310-90 t o r e p r e s e n t low-Zn streams.
of Zn i n the Zn-enriched
mg
1 •
'
^ (0309-80).
magnitude lower.
streams and
The mean c o n c e n t r a t i o n s
steams were 0.230 mg l"'^ (0288-70) and 0.216
Concentrations
The demarcation
i n t h e low-Zn streams were an order o f
between e n r i c h e d and unenriched
sites for
Cd and Pb was l e s s c l e a r (Table 3.8); the range o f mean Cd c o n c e n t r a t i o n s
was
from 0.0010 mg 1 " t o 0.0015 mg 1
and the range of mean Pb
c o n c e n t r a t i o n s was between 0.0028 mg 1 "'^ and 0.0139 mg 1
4.4
4.41
.
GROWTH OF MOSSES IN STREAMS
Introduction
Although
the p r o p o r t i o n o f streams and r i v e r s i n which Rhynchostegium i s
found i s h i g h , t h e m i c r o h a b i t a t a t any s i t e which i t may occupy i s q u i t e
s p e c i f i c ; a l l p o p u l a t i o n s were a t t a c h e d t o a s t a b l e s u b s t r a t a , bedrock i n
0310-90, w a t e r f a l l s a t 0310-90 and 0288-70 and boulders a t 0091-05 and
0309-80.
D u r i n g the course o f the study s e v e r a l boulders were l o s t , presumably
due
t o h i g h f l o w s and these were r e p l a c e d by l a r g e r boulders.
On b o u l d e r s ,
Rhynchostegium was found m a i n l y on t h e " t r a i l i n g edge" and on the t o p o f
the boulder.
That on the t r a i l i n g edge was o f t e n i n a l a r g e weft w h i l s t
moss on the t o p grew i n an i n t r i c a t e mat p a t t e r n which appeared t o be an
e f f e c t i v e sediment t r a p .
A t 0310-90 t h i s process l e d , d u r i n g the course o f
119
Table 4.4
Ranking o f e n v i r o n m e n t a l v a r i a b l e s measured i n f o u r reaches i n
N-E. England d u r i n g growth study.
variable
rank
physico-chemical
variables
c u r r e n t speed
0310-90 > 0309-80 = 0288-70 > 0091-05
conductivity
0309-80 > 0288-70 > 0091-05 > 0310-90
pH
0091-05 > 0309-80 > 0288-70 > 0310-90
total
alkalinity
oxygen s a t u r a t i o n
0309-80 > 0091-05 > 0288-70 > 0310-90
0309-80 > 0091-05 > 0288-70 > 0310-90
anions
FRP
0310-90 > 0288-70 > 0091-05 > 0309-80
NO„-N
o
0288-70 > 0310-90 = 0309-80 > 0091-05
S04-S
0288-70 > 0309-80 > 0310-90 > 0091-05
CI
0288-70 > 0310-90 > 0309-80 > 0091-05
cations
Na
K
filtrable
filtrable
"^^f i l t r a b l e
Ca
fiItrable
"^"f i l t r a b l e
0309-80 > 0310- 90 > 0091-05 > 0288-70
0309-80 > 0288- 70 > 0091-05 > 0310-90
0309-80 > 0288- 70 > 0091-05 > 0310-90
0309-80 > 0288- 70 > 0091-05 > 0310-90
0288-70 > 0091- 05 > 0309- 80 > 0310-90
0091-05 > 0288- 70 > 0310- 90 > 0309-80
Fp
filtrable
Zn
Cd
Pb
filtrable
f
iItrable
fIltrable
0288-70 > 0309- 80 > 0091-05 > 0310-90
0288-70 > 0309- 80 > 0091-05 > 0310-90
0288-70 > 0309-80 > 0310- 90 > 0091-05
120
t h e s t u d y , t o t h e e s t a b l i s h m e n t of a sedge and of t h e acrocarpous moss Bryu-Ti
p s e u d o t r i q u e t r u m on t h e b o u l d e r .
4.42
F o r e s h i e l d Burn, reach 0091-05
Boulders i n t h i s reach proved r e l a t i v e l y u n s t a b l e ; they were recovered
from t h e streams on j u s t n i n e out of t w e l v e occasions.
The r a t e of growth of
moss was q u i t e v a r i a b l e ; the r a t e i n J u l y and August being of the same order
as r a t e s f o r November t o March.
was
observed
i n June 1986
September and December.
( F i g . 4.4); s m a l l e r peaks were ireasured i n
The low r a t e of growth was not s i g n i f i c a n t l y
c o r r e l a t e d w i t h temperature
4.43
The maximum r a t e of growth of Rhynchostegium
( F i g . 4.5;
r = 0.652).
B l a g i l l Burn, reach 0288-70
The b o u l d e r s were recovered from t h i s reach on a l l but one
(January 1986).
occasion
The growth r a t e i n J u l y 1985 was v e r y r a p i d (1.40 mm wk
^ );
however t h i s r a t e subsequently d e c l i n e d and remained a t a p p r o x i m a t e l y
1.0 mm wk
^ u n t i l November 1985
( F i g . 4.6).
There was
still
some growth
d u r i n g t h e w i n t e r months (0.31 mm week " i n December 1985 and 0.38
i n February/March 1986).
By s p r i n g the growth r a t e was
t h e maximum growth r a t e d u r i n g t h i s study was
(1.94 mm wk
i n c r e a s i n g again and
i n June
1986
^) .
There was
no s i g n i f i c a n t r e l a t i o n s h i p ( r = 0.609) when growth r a t e
c o n s i d e r e d as a f u n c t i o n of temperature
4.44
observed
mm week
was
( F i g . 4.7).
D a d d r y s h i e l d Burn, reach 0309-80
T h i s was
t h e lowest (Table 3.2) and t h e warmest ( F i g . 4.3) of the f o u r
streams and the growth r a t e of Rhynchostegium was c o r r e s p o n d i n g l y f a s t e r than
i n t h e o t h e r streams ( F i g . 4.8).
Boulders were recovered on a l l but one
121
F i g . 4.4
Seasonal changes i n growth r a t e of Rhynchostegium i n 0091-05.
F i g . 4.5
R e l a t i o n s h i p between growth r a t e of Rhynchostegium and water
temperature i n 0091-05.
122
3.0
CD
0)
S 2.0
E
E
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1.0
J
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1985
M
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1986
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• 7/85
•
8/85
•3/86
•
1/86
Z.
6
water
8
10
12
temperature
(°C)
123
F i g . 4.6
Seasonal changes i n growth r a t e of Rhynchostegium i n 0288-70,
F i g . 4.7
R e l a t i o n s h i p between growth r a t e o f Rhynchostegium and water
temperature i n 0288-70.
124
3.0
% 2.0
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1986
1986
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water temperature
I
12
(°C)
M
125
one occasion; however i n March 1986 o n l y one l a b e l l e d t i p was found.
maximum r a t e of growth was observed
i n J u l y 1985
(2.31 mm wk
^) and there
was a g r a d u a l decrease i n the r a t e u n t i l December (0.40 mm wk
r a t e p i c k e d up a g a i n i n the S p r i n g and by June 1986 was 2.15
r a t e of growth was
The
The
mm wk
^.
The
s i g n i f i c a n t l y c o r r e l a t e d w i t h water temperature ( r =
0.842 **; F i g . 4.9) and i n d i c a t e s r e l a t i v e l y r a p i d growth a t warm water
temperatures i n the Summer and slower growth i n the Winter.
4.45
"Race F e l l Burn", reach 0310-90
0310-90 i s both the h i g h e s t and the most exposed of the f o u r
streams.
T h i s i s r e f l e c t e d both i n the lower temperatures (4.31) and i n
t h e r e l a t i v e l y slow r a t e s of growth ( F i g . 4.10).
h i g h e s t i n June 1986
The r a t e of growth
was
(1.44 mm week ^) and the growth r a t e i n the w i n t e r
months was v e r y slow (0.12 mm wk
i n February/March).
The stream does,
however, appear t o have a s m a l l e r range of discharges; Rhynchostegium i s
found a t t a c h e d t o r e l a t i v e l y s m a l l boulders and on no occasion was
the
b o u l d e r l o s t due t o h i g h f l o w s .
This stream d i f f e r e d from the o t h e r s i n t h a t t h e r e was a w e l l
developed peak of growth i n Autumn, t o a maximum of 1.2 mm wk
^ : otherwise
t h e r e was no c l e a r r e l a t i o n s h i p ( r = 0.521) between water temperature and
growth r a t e ( F i g . 4.11).
4.46
S y n t h e s i s of data
A n a l y s i s o f data was
performed
i n two p a r t s ; a n a l y s i s of the
r e l a t i o n s h i p between growth r a t e and environmental data f o r each stream,
f o l l o w e d by a n a l y s i s of the combined data from the four
reaches.
Data c o l l e c t e d from d i f f e r e n t reaches were compared by both parametric and
non-parametric t e s t s .
Two
s o r t s of p a r a m e t r i c t e s t s were performed;
126
F i g . 4.8
Seasonal changes i n growth r a t e of Rhynchostegium i n 0309-80.
F i g . 4.9
R e l a t i o n s h i p between growth r a t e of Rhynchostegjum and water
temperature i n 0309-80.
127
3.0 h
I
2.0
0)
0)
£
E
|,.o
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05
J
0
A
D
J
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M
F
1986
1985
3.0 r
y - 0.264X - 0 . 9 4 4
• 7/85
9/85 2
2.0
•6/86
5/86^
0)
•10/85
E
E
• 11/85
1.0
o
3/86
•,4/86
cn
•12/85
_L
2
4
water
6
8
10
temperature
12
(°C)
128
Fig.
4.10
Seasonal changes
Fig.
4.11
R e l a t i o n s h i p between
temperature
i n 0310-90.
i n growth
growth
r a t e of Rhynchostegium
r a t e of Rhynchostegium
i n 0310-90.
and
water
129
I
0)
CP
3.0
r
2.0
h
E
e
£
1.0
o
cn
J
N
0
A
J
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F
1986
1985
2.0
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0)
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10/85.
E
E
liO h
11/85
»9/85
••5/86
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•
5/85
•12/85
4/86 •
•8/85
• 1/86
• 3/86
2
I
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\
\
I
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6
8
10
12
water
temperature
(°C)
M
130
p r o d u c t - m o m e n t c o r r e l a t i o n c o e f f i c i e n t s and p a i r w i s e
pair of variables.
three
The r a t e o f g r o w t h was c o r r e l a t e d
t - t e s t s between each
s i g n i f i c a n t l y between
p a i r s o f s t r e a m s ; 0091-05 and 0310-90, 0288-70 and 0309-80 and
70 and 0310-90
(Table 4.5); these i n d i c a t e s t r o n g
0288-
linear relationships
b e t w e e n t h e r a t e o f g r o w t h i n t h e s e p a i r s o f s t r e a m s and no such r e l a t i o n s h i p
between o t h e r p a i r s o f streams.
monthly rates
by
o f g r o w t h between s e v e r a l
the pairwise
> 0.05
80.
T h e r e were s t r o n g
t-tests.
differences
p a i r s of streams.
These were shown
T h r e e o u t o f s i x c o m p a r i s o n s were s i g n i f i c a n t a t p
( T a b l e 4 . 6 ) ; t h e s e were b e t w e e n 0091-05 and 0310-90, 0288-70 and 0309-
and 0288-70 and 0310-90.
Differences
i n the monthly rates of growth
b e t w e e n 0091-05 and 0288-70. b e t w e e n 0091-05 and 0309-80
and
0310-90 w e r e n o t s i g n i f i c a n t .
significant
and between 0309-80
The same p a i r s o f s t r e a m s showed
r e l a t i o n s h i p s i n each c a s e : however \^ i s u n l i k e l y t h a t p a i r s o f
s t r e a m s w h i c h do n o t have s i g n i f i c a n t l i n e a r c o r r e l a t i o n s w i l l
differences
T a b l e 4.5
four
between
i n monthly rates
Correlation
have r e s o v a b l e
of growth.
c o e f f i c i e n t s between g r o w t h r a t e o f Rhynchostegium i n
streams.
0091-05
1 .000
0288-70
0.736
1 .000
0 3 0 9 - 80
0.426
0.853"
0310- 90
0.366^
0.772"
0091-05
0288-70
.000
1 .000
0309-80
0310-90
131
Table 4.6
Comparisons o f growth r a t e o f Rhynchosteglum I n f o u r
streams
using pairwise t-tests.
0091-05
1.000
0288-70
-0.889
1.000
0 3 0 9 - 80
-2.318
-3.237^
1.000
0.964^
3.012^
3.921
1.000
0091-05
0288-70
0309-80
0310-90
0 3 1 0 - 90
A v a r i e t y of non-parametric
t e s t s were p e r f o r m e d ; Spearman's
rank
correlation
(Rho) and K e n d a l l ' s Tau-B b o t h gave v e r y s i m i l a r r e s u l t s w i t h
significant
differences
and
0288-70 and 0310-90 ( T a b l e 4.7; T a b l e 4 . 8 ) , as f o r t h e p a r a m e t r i c
tests.
K e n d a l l ' s c o e f f i c i e n t o f c o n c o r d a n c e , a measure o f a g r e e m e n t
calculated
b e t w e e n a l l s a m p l i n g s i t e s gave a v a l u e o f 0.754; a s i g n i f i c a n t
value which
four
b e t w e e n 0091-05 and 0310-90, 0288-70 and 0309-80
indicated
t h a t t h e r e was some d e g r e e o f s i m i l a r i t y
between t h e
sites.
T a b l e 4.7
Spearman's r a n k c o r r e l a t i o n (Rho) c a l c u l a t e d
of Rhynchostegium a t f o u r
s i t e s i n growth
between g r o w t h
study.
0091-05
1.000
0288-70
0.536
1.000
0 3 0 9 - 80
0.429
0.842^
1.000
0310- 90
0.881^
0.754^
0.503
1.000
0091-05
0288-70
0309-80
0310-90
rate
132
T a b l e 4.B
K e n d a l l ' s Tau-B c o e f f i c i e n t c a l c u l a t e d
Rhynchostegium a t f o u r
s i t e s i n growth
between g r o w t h r a t e o f
study.
0091-05
1.000
0288-70
0.536
1.000
0 3 0 9 - 80
0.429
0.842^
1.000
0310- 90
0.714^
0.600^
0.503
1.000
0091-05
0288-70
0309-80
0310-90
The p o s s i b l e
growth
e f f e c t o f seasonal
r a t e a t one s i t e ,
(negative),
Table
4.7
0310-90 was c o r r e l a t e d
a t t w o s i t e s w i t h mean d i s c h a r g e
maximum a i r t e m p e r a t u r e
temperature
changes was a l s o
(negative)
Correlations
Rhynchostegium i n f o u r
investigated.
w i t h mean
(negative),
rainfall
a t two w i t h
( p o s i t i v e ) and a t one, 0309-80, w i t h minimum a i r
(Table 4.9).
between e n v i r o n m e n t a l v a r i a b l e s
and g r o w t h r a t e o f
streams.
0309-80
0288-70
0310-90
stream-reach
0091-05
mean
•0.741
-0.734
mean d i s c h a r g e
0.671
-0.910
air
0.572
0.864^
0.929^^
0.452
0.383
0.725
0.922' "
0.288
rainfall
¥:¥r
temp.
The
-0.366
-0.817
-0.641
-0.850^^
max
air
temp•^ . ^
Time-series
a n a l y s i s u s i n g a c r o s s c o r r e l a t i o n was a p p l i e d
t e s t was w h e t h e r i n any
population
d e l a y between changes i n t h e environment
each, c r o s s - c o r r e l a t i o n s
t h e r e was
t o the data
evidence of a c o n s t a n t
and t h e g r o w t h r a t e o f moss.
between v a r i a b l e s
were c a l c u l a t e d
using
In
increasing
133
monthly
tended
l a g s f r o m z e r o t o t h r e e months ( T a b l e 4 . 1 0 ) .
t o decrease
response
with
increasing lags.
t o a i r and w a t e r
Overall,
correlations
0310-90 showed a one month l a g i n
t e m p e r a t u r e s ; however o n l y one o f t h e s e
t e m p e r a t u r e ) was s i g n i f i c a n t a n d none o f t h e o t h e r s t r e a m s
(water
showed a s i m i l a r
lag.
Only
one s i t e
h a d a s i g n i f i c a n t r e l a t i o n s h i p b e t w e e n t h e r a t e o f moss
g r o w t h and w a t e r
all
temperature
s i t e s were p o o l e d
(0309-80,
see 4 . 4 4 ) ;
r a t e and w a t e r
growth
r a t e a n d mean maximum a i r t e m p e r a t u r e
4.14).
Using
temperature
( r = 0.627 ***, F i g . 4 . 1 2 ) , between
( r = 0.507 ***, F i g .
t h e s i g n i f i c a n t r e g r e s s i o n s t o e x t r a p o l a t e back, then i t i s
.Tioss does n o t g r o w ;
This corresponds
minimum t e m p e r a t u r e b e l o w w h i c h t h e
t o a w a t e r t e m p e r a t u r e o f 1.7 °C, a
mean maximum a i r t e m p e r a t u r e o f -1.6 °C and a w e e k l y
temperature
light
( r = 0.513 **, F i g . 4.13) and
r a t e a n d mean minimum a i r t e m p e r a t u r e
p o s s i b l e t o c a l c u l a t e an a p p r o x i m a t e
weekly
o f -5.6 °C; however no a l l o w a n c e
on g r o w t h
rate
4.5 A n n u a l g r o w t h
estimated
minimum a i r
i s made f o r t h e e f f e c t o f
i n this.
rates
E s t i m a t e s w e r e a l s o made o f t o t a l
Approximate
g r o w t h o v e r t h e 12 month p e r i o d .
r a t e s f o r t h e months where no d a t a were c o l l e c t e d were
from water
t e m p e r a t u r e a n d F i g . 4.12.
T h r e e such
replacements
were n e c e s s a r y
f o r 0091-05 and one each f o r 0288-70 and 0309-80.
in
t h e year ranged
length
77.6 mm
total
from
t h e n t h e r e were s i g n i f i c a n t r e l a t i o n s h i p s b e t w e e n
growth
between g r o w t h
however when d a t a
over
i n 0309-80
f r o m 33.4 mm
i n 0310-90 ( u n c o r r e c t e d ) t o
( u n c o r r e c t e d ) o r 73.3 ( c o r r e c t e d ) ( T a b l e 4 . 1 1 ) .
i n c r e a s e i n 0309-80 was more t h a n d o u b l e
The r e g r e s s i o n e q u a t i o n s c a l c u l a t e d
Increases
that
The
i n 0310^90,
i n 4.23 ( T a b l e 4.1) were used t o
c o n v e r t these values from increases i n l e n g t h per u n i t time t o increases i n
134
T a b l e 4.10
T i m e - s e r i e s a n a l y s i s o f r e l a t i o n s h i p s between g r o w t h r a t e o f
R h y n c h o s t e g i u m and e n v i r o n m e n t a l v a r i a b l e s .
variable
l a g (months)
0
1
2
3
0. 741
0. 300
0. 120
-0. 030
-0. 671
0. 218
0. 051
0. 183
0091-05
rainfall
flow
max.
a i r temp.
-0. 572
0. 311
0. 006
-0. 318
min.
a i r temp.
0. 383
0. 348
0. 104
-0. 542
0. 664
0. 269
0. 090
-0. 637
-0. 734
0. 242
0. 174
-0. 282
-0 .101
0. 022
-0. 420
0. 383
0. 009
-0. 169
water
temp.
0288-70
rainfall
max.
a i r temp.
**
910
0. 864*
min.
a i r temp.
0..725
0. 289
-0. 026
-0. 301
0..548
0. 273
-0..022
-0. 350
-0,.366
0,.226
0,.065
-0,.693
0,.184
0 .543
0 .468
-0 .030
0.
f low
water
temp.
0309-80
rainfall
0,.022
0 .641
flow
*
**
max.
min.
water
a i r temp.
a i r temp.
temp.
0 .736
0 .929
0
0
*
*
0 .660
0 .212
-0 .205
*
0 .700
0 .248
-0 . 187
0 .033
0 .276
-0 .080
-0 . 126
0 . 166
0 .070
0 . 553
0 .434
0 .303
.922
.859
0310-90
*
rainfall
-0 .817
f low
-0
*
max.
a i r temp.
.850
0 .452
min.
a i r temp.
0 .288
0 .536
0 .528
0 .222
0 .482
0 .683
0 .560
0 . 176
water
temp.
135
Fig.
in
4.12
R e l a t i o n s h i p between g r o w t h r a t e o f Rhynchostegium, measured
f o u r r e a c h e s i n N-E.
E n g l a n d o v e r 12 m o n t h s , and s p o t
t e m p e r a t u r e measurements:
summed d a t a .
water
136
•=
0091-05
o = 0288-70
• = 0309-80
• = 0310-90
y=0.170x-0.318
3.0
<D
O)
E
E
I
2.0
o
<_
cn
8
water
10
temperature
12
(°C]
137
Fig.
in
4.13
four
Relationship
reaches
temperature:
Fig.
in
4.14
four
i n N-E.
sum.Tied
te^'.perature.
growth r a t e o f Rhynchostegium,
England
o v e r 12 m o n t h s ,
measured
and mean maximum a i r
data.
Relationship
reaches
between
i n N-E.
between
growth r a t e o f Rhynchostegium,
England
o v e r 12 m o n t h s ,
measured
and mean maximum a i r
138
• = 0091-05
o = 0288-70
• = 0 3 0 9 - 80
• = 0310-90
y = 0.093x - 0.060
5
10
15
maximum air t e m p e r a t u r e
-
20
(°C)
3.0
y = 0.087x + 0.639
w
?
E
E
2.0
o
1.0
10
15
minimum air t e m p e r a t u r e
20
(°C)
139
mass ( T a b l e 4 . 1 1 ) .
120.8
I n c r e a s e s i n mass o f b e t w e e n 29.2 mg ( 0 3 1 0 - 9 0 ) and
mg ( c o r r e c t e d v a l u e , 0 3 0 9 - 8 0 ) p e r s h o o t p e r y e a r were r e c o r d e d .
T a b l e 4 . 1 1 I n c r e a s e i n l e n g t h a n d mass o f R h y n c h o s t e g i u m o v e r 12 m o n t h s ,
calculated
f r o m mean m o n t h l y g r o w t h r a t e w i t h and w i t h o u t c o r r e c t i o n f o r
missing data.
0091-05
0288-70
0309-80
0310-90
uncorrected
39.5
54.3
77.6
33.4
corrected
43.3
51.3
73.3
uncorrected
56.9
61.7
127.9
corrected
62.4
58.2
120.8
length
(mm y r ^ )
mass (mg y r
)
29.2
4.6 S h o o t d e n s i t y a n d a n n u a l p r o d u c t i o n o f d r y m a t t e r
M e a s u r e m e n t s o f s h o o t d e n s i t y w e r e made u s i n g a q u a d r a t o f a r e a
cm
.
T h i s was p l a c e d on p a t c h e s
number o f t i p s was c o u n t e d .
6.7
o f R h y n c h o s t e g i u m on b o u l d e r s and t h e
From t h e s e i t was p o s s i b l e t o e s t i m a t e a n n u a l
d r y m a t t e r p r o d u c t i o n b y m u l t i p l y i n g by t h e i n c r e a s e i n mass p e r s h o o t p e r
year
( c o r r e c t e d v a l u e s ; Table 4.11).
s h o o t s cm"^
(0288-70)
t o 6.4 s h o o t s cm~^
g r e a t e s t d e n s i t y was a s s o c i a t e d w i t h
comparison
with
Spatial
other sites
d e n s i t y ranged
(0310-90)
f r o m 4.4
( T a b l e 4.12); t h e
the slowest growth rate
(0310-90) b u t
showed no c l e a r r e l a t i o n s h i p between
growth
-2
r a t e and s h o o t d e n s i t y .
Dry m a t t e r p r o d u c t i o n i s expressed
as g dm
-1
yr
( T a b l e 4.12) and showed t h e s e r i e s 0310-90 < 0288-70 < 0091-05 < 0309-80.
The
rate
i n 0309-80 was t h r e e t i m e s f a s t e r
t h a n ^ r a t e i n 0310-90.
140
T a b l e 4.12
S p a t i a l d e n s i t i e s and a n n u a l r a t e s o f d r y m a t t e r p r o d u c t i o n o f
R h y n c h o s t e g i u m s h o o t s on
stream-reach
boulders..
spatial
density
( s h o o t s cm
mean
)
dry-matter
( g dm
SD
0091-05
5.8
0.94
36.2
0288-70
4.4
0.26
25.6
0 3 0 9 - 80
4.8
0.29
58.0
0310- 90
6.4
0.31
18.7
production
-2
yr
-1
)
141
5. DEVELOPMENT OF METHOD FOR USE OF MOSS-BAGS
5.1
INTRODUCTION
A l t h o u g h moss-bags h a v e been used i n t h e p a s t f o r m o n i t o r i n g heavy m e t a l
pollution
reached
i n f r e s h w a t e r s ( 1 . 5 2 4 ) , r e s u l t s f r o m t h e s e s t u d i e s have
the literature.
Individual
workers
have d e v e l o p e d
a c c o r d i n g t o s p e c i f i c needs a n d c i r c u m s t a n c e s
t o p r o d u c e a s t a n d a r d method.
This chapter
rarely
methods
and t h e r e has been no a t t e m p t
i s concerned
with the
development o f such a method.
The
experiments
known w a t e r
experiments;
a l l i n v o l v e d m a n i p u l a t i o n s o f moss between s t r e a m s o f
chemistries.
i n t h e one e x c e p t i o n F o n t i n a l i s a n t i p y r e t i c a was u s e d .
I n t e r e s t was f o c u s s e d
on t h e m e t a l Zn, a l t h o u g h a n a l y s e s o f Cd and Pb were
a l s o made, w h e r e r e l e v a n t .
effects,
i n terms
The p r i n c i p l e q u e s t i o n a s k e d was what a r e t h e
o f r a t e s o f a c c u m u l a t i o n e t c . , o f t r a n s f e r r i n g moss f r o m
b o u l d e r s t o mesh b a g s ?
environment
R h y n c h o s t e g i u m was used f o r a l l b u t one o f t h e
Experiments
a r e concerned
w i t h i n bags, t h e r a t e o f uptake
t h a t on b o u l d e r s a n d t h e e f f e c t s
c o n c e n t r a t i o n s o f heavy
with
the physical
by moss i n bags compared
with
o f b a g s t y l e and p a c k i n g on f i n a l
metals.
5.2 TYPES OF BAGS
Three t y p e s o f bag were t e s t e d .
readily available
shops.
Features
t o f u t u r e workers
of their
Types I and I I I
fishing
line
The m a t e r i a l s were chosen so as t o be
and were a l l o b t a i n e d f r o m
design are l i s t e d
i n Table 5.1.
were assembled from t h e f a b r i c ;
(breaking strain
high-street
nylon
monofilament
= 5.4 k g ) was used t o sew p a t t e r n
I and s t o u t
142
sewing thread
of the
often
type
for pattern I I I .
i n which bulbs,
Type I I was
fruit
and
obtained
v e g e t a b l e s and
Mesh bags u s e d i n t r a n s p l a n t
pattern
fabric
with nylon
mesh
I IT
are
size
(cm)
colour
15 x
15
green
30
x
30
orange
15 x
15
white
bag
muslin,
stout
sewn up
moss-bags a r e
examine t h e
moss.
t o be
e f f e c t s of
environment w i t h i n
effect
stream tank
tank
and
m e a s u r e d 50
light
of the
and
then
t h e bag
environment around
upon t h e p h y s i c a l
cm
e f f e c t upon t h e
the e f f e c t
c u r r e n t meter
current
speed was
(2.13).
speed t h r o u g h
d o w n s t r e a m o f t h e bag
o f each t y p e
The
bag
tested using
was
interest
o f bag
the
on
obstacle.
Pattern
an
artificial
held across the w i d t h
a double l a y e r of f a b r i c
(Table
5.2).
Pattern
s t r e a m c u r r e n t w h i l s t p a t t e r n I I I , due
a co.-;siderable
i t i s of
penetration.
bags on
the c u r r e n t
bag
used f o r extended p e r i o d s
E x p e r i m e n t s w e r e c o n d u c t e d on
w a t e r c u r r e n t and
The
with
thread
E f f e c t upon p h y s i c a l
If
was
tennis b a l l s etc.
monofilament
manufactured nylon
II
the
bag
experiments.
g a r d e n n e t t i n g , sewn t o g e t h e r
to
is a
sold.
T a b l e 5.1
5.21
made-up and
I I fell
was
I had
little
to i t s close
between p a t t e r n s
of
weave,
I and I I I .
143
Light penetration through
t h e bags was a l s o m e a s u r e d .
l e a s t a t t e n u a t i o n and p a t t e r n s
I I and I I I b o t h
Pattern
removed a t h i r d
I caused t h e
of incident
light.
T a b l e 5.2
E f f e c t o f mesh-bags on p h y s i c a l e n v i r o n m e n t o f moss.
Pattern
mesh s i z e
c u r r e n t speed
(meshes cm ^ )
(cm s ^)
before
after
(S a t t e n u a t i o n )
% diff.
0. .9
0. ,246
0. .209
15
13.3
II
0, .6
0, .213
0 ,086
49
33.3
0, .07
0. .225
0 .001
99.5
33.3
EXPERIMENTS ON METAL ACCUMULATION WITHIN mSE
5.31
flux
I
III
5.3
light
BAGS
C o m p a r i s o n o f a c c u m u l a t i o n b y R h y n c h o s t e g i u n on b o u l d e r s a n d i n bags.
U p t a k e o f Zn, Cd a n d Pb b y R h y n c h o s t e g i u m on b o u l d e r s and i n mesh
bags ( p a t t e r n I , 5.2) was f o l l o w e d , u s i n g
transplanted
given
i n t o 0012-45.
i n T a b l e 5.3.
throughout
t h e experiment ( ? i g .
Two p h a s e s o f u p t a k e o f Zn a n d Pb w e r e a p p a r e n t ; an i n i t i a l ,
phase l a s t i n g a p p r o x i m a t e l y
accumulation.
rapid
6 h, f o l l o w e d by a p e r i o d o f slower
The d e m a r c a t i o n i n t o t w o p h a s e s was n o t a p p a r e n t f o r Cd,
however t h e c o n c e n t r a t i o n s ,
orders
f r o m 0310-90
Environmental v a r i a b l e s i n t h e two streams a r e
Uptake o f a l l t h r e e metals continued
5.1).
a population
a n d t h e r e f o r e t h e f l u x e s , a r e t w o and t h r e e
o f magnitude r e s p e c t i v e l y lower than
f o r Zn a n d Pb.
144
T a b l e 5.3
Selected
experiment
(metals
environmental
as mg 1
-1
v a r i a b l e s i n 0012-45 d u r i n g t r a n s p l a n t
forfiltrable
fraction;
blanks
missing
values).
date
time
curr.
temp.
pH
Ca
Zn
Pb
(m s " l ) (°C)
19.6.84
0950
0.294
15.5
7.6
43.0
0.149
0.0181
1050
0.372
16.0
7.7
42.7
0.139
0.0135
1150
0.350
17.0
7.8
43.6
0.116
0.0183
1250
0.280
17.5
7.8
43.6
0.100
0.0171
1350
0.315
18.0
7.8
43.3
0.101
0.0142
1450
0.339
18.5
7.8
43.1
0.110
0.0107
1550
0.375
18.5
7.8
42.4
0.112
0.0097
1750
0.366
19.0
7.8
49.3
0.095
0.0167
2150
0.282
18.0
7.9
42.9
0.088
0.0087
20.6.84
1010
0.326
15.0
7.8
43.9
0.143
0.0207
21.6.84
0950
0.319
13.0
7.8
23.6.84
1200
0.219
11.0
7.7
56.4
0.148
0.0084
Samples o f j^n s i t u
for
R h y n c h o s t e ^ i u m were a l s o c o l l e c t e d
c o m p a r i s o n w i t h t r a n s p l a n t e d moss ( T a b l e
experiment
( t = 101 h ) , t h e c o n c e n t r a t i o n
was a p p r o x i m a t e l y
o f Cd was s l i g h t l y
significantly
below t h a t
A t t h e end o f t h e
o f Zn i n t h e t r a n s p l a n t e d
equal t o the concentration
concentration
5.4).
d u r i n g the study
i n t h e in s i t u
moss, t h e
b e l o w , and t h e c o n c e n t r a t i o n
i n t h e i n s i t u .moss.
moss
o f Pb was
145
Fig.
5.1
Accumulation
bags ( p a t t e r n
I , 5.2)
o f Zn, Cd and Pb by R h y n c h o s t e g i u m
transplanted
i n t o 0012-45 on ^ ^ 1
on b o u l d e r s and
146
AOOO r
3000
-2- 2000
(/>
o
E
1000
c
N
20
80
60
iO
100
t i m e (h)
U
12
10
8
VI
o
E
TP
O
6
i,
2
20
40
80
60
100
time
300
o> 200
3.
(h)
h
o
h
0O,
6>'
o
E
100
Q.
20
/iO
80
60
100
t i m e (h)
•
:
m o s s on
boulders
o
-
moss
bags
in
147
Table
5.4
Comparison o f c o n c e n t r a t i o n s o f metals
moss t r a n s p l a n t e d i n t o
I n Pg g ^. f i g u r e s
i n i n s i t u moss a n d i n
0012-45 on b o u l d e r s and i n bags ( m e t a l
i n brackets = 95^ confidence
limits).
t r a n s p l a n t e d moss
Time
(h)
metal
4.0
Zn
Cd
Pb
101 .0
Zn
Cd
Pb
for
each t r e a t m e n t , b o t h w i t h
t;ransf o r m a t i o n .
("slow p h a s e " ) .
without
The r a p i d
whilst
that
for
the slow
situ
boulder
875 .8
939 .7
2851 .4
( 3 1 .0)
(49, .8)
(879,.0)
3 .80
2,.34
10,.38
( 0 ,.53)
( 0 ,.31)
moss
( 2 .,09)
94 .0
108. .0
426, .0
(9..8)
( 1 1 . .4)
(255..0)
3775 .3
3385,.9
3537 .2
,
(234..4)
(587. V
(556. 6)
7 .49
,
9..97
( 0 ..67)
( 1 . ,38)
11, .40
( 2 .,06)
333, .0
235. ,0
462 ,
.0
(35. ,5)
(101 ..3)
(113..0)
were c a l c u l a t e d ; on a l l sample means
and w i t h o u t a n a t u r a l l o g a r i t h m
a n d on t h p s a m p l e s < 6.0 h ( " r a p i d p h a s e " ) and > 6.0 h
R e g r e s s i o n s c a l c u l a t e d f o r a l l s a m p l e s , b o t h w i t h and
l o g ^ t r a n s f o r m a t i o n , were s i g n i f i c a n t
5.5).
in
bag
Four s e t s o f r e g r e s s i o n e q u a t i o n s
accumulation
phase o f Zn a c c u m u l a t i o n
on b o u l d e r s
phase.
was n o t .
F o r Cd u p t a k e
f o r Zn, Cd and Pb
(Table
by moss i n bags was
significant
B o t h t r e a t m e n t s were h i g h l y
significant
the i n i t i a l
6 h i s significant f o r
148
t a b l e 5.5
Regression equations
accumulation
and,
during transplant
therefore,
and s i g n i f i c a n c e
e x p e r i m e n t i n 0012-45 ( d e g r e e s o f f r e e d o m
levels of significance
v a r y between t r e a t m e n t s ) ; see 5 . 3 1 .
bags v t i m e
ZINC
a l l data, normal
l e v e l s f o r Zn, Cd and Pb
boulders v time
y
r
31.03 x 4 835.88 **
0.983 **
y = 25.32 x + 712.36 **
r = 0.974 **
y
r
522 X + 4 3 6 . 3 1 **
0.881 **
y = 402 X + 426.25 **
r = 0.823 **
< 6.0 h
y
r
78.05 X + 668.40 **
0.816 **
y = 47.92 X -i 641.15
r = 0.577 N.S.
> 6.0 h
y
r
28.94 X + 977.74 **
0.988 **
y = 2 4 . 9 1 X + 737.97 **
r = 0.976 **
y
r
0.057 X + 3.028 **
0.722 **
y = 0.074 X + 2.868 **
r = 0.879 **
y
r
1.237 X + 1.852 **
0.815 **
y = 1.352 X + 1.763 **
r = 0.844 **
< 6.0 h
y
r
0.65 X + 1.026 *
0.697 *
y = 0.41 X + 1.787
r = 0.505 N.S.
> 6.0 h
y
r
0.027 X + 5.206 N.S,
0.558 N.S.
y = 0.068 X + 3.234 **
r = 0.998 **
y
r
2.017 X + 154.41 **
0.828 **
y = 1.424 X + 9 9 . 8 1 **
r = 0.832 **
y
r
36.25 X + 125.47 **
778 **
y = 26.78 X - 77.33 **
r = 0.818 **
< 6.0 h , n o r m a l
y
r
2.27 X -f 146.29
0.097 N.S.
y = J .32 X + 93.08
r = 0.092 N.S.
> 6.0 h , n o r m a l
y
r
1.59 X + 185.23 *
0.948 **
all
data,
log
CADMIUM
a l l data, normal
all
data,
log
LEAD
a l l data, normal
all
data,
log
N.S
y = 1.19 X - 116.04
r = 0.849 *
N.S
N.S.
N.S
N.S.
149
"bags" y e t n o t s i g n i f i c a n t
not s i g n i f i c a n t
uptake,
f o r "boulders".
f o r "bags" and h i g h l y s i g n i f i c a n t
The
i nthe i n i t i a l
significant
(Table 5.6).
significantly
f o r t h e s l o w phase.
phases o f uptake
phase
There i s c o n s i d e r a b l e
of a l l three
r e g r e s s i o n s were compared
f o rthe rapid
F o r Pb
metals.
( b a g v b o u l d e r ) u s i n g ANOVA
Of t h e s i x c o m p a r i s o n s o n l y one, Zn w i t h no t r a n s f o r m a t i o n ,
was s i g n i f i c a n t
w h e r e moss i n bags a p p e a r e d t o a c c u m u l a t e Zn a t a
higher rate
t h a n moss on b o u l d e r s .
h o w e v e r , on t h e s a m p l e means o n l y .
point
f o r "boulders".
n e i t h e r "bags" o r " b o u l d e r s " a r e s i g n i f i c a n t
and o n l y "bags" a r e s i g n i f i c a n t
"noise"
The s l o w phase i s t h e o p p o s i t e ,
( F i g . 5.1) a r e a l s o t a k e n
T h i s ANOVA was p e r f o r m e d ,
I f t h e 95% c o n f i d e n c e
i n t o account,
l i m i t s f o r each
then the v a r i a t i o n w i t h i n t h e
s a m p l e s a p p e a r s t o be g r e a t e r t h a n t h e v a r i a t i o n between t h e t w o
treatments.
Table
5.6
transplant
A n a l y s i s o f v a r i a n c e between t r e a t m e n t s
( b o u l d e r s v bags) d u r i n g
experiment
Zn
Cd
Pb
no t r a n s f o r m a t i o n
6.23*
0.631
1.402
log^
1.397
0.088
transformation
5.32 C o m p a r i s o n o f d i f f e r e n t
The e f f e c t
of d i f f e r e n t
0.822
p a t t e r n s o f bags
meshes on a c c u m u l a t i o n
o f Zn by R h y n c h o s t e g i u m
i n moss-bags was t e s t e d b y t r a n s p l a n t i n g a p o p u l a t i o n f r o m 0310-90 t o
0024-22.
T h r e e p a t t e r n s o f b a g were u s e d ( 5 . 2 ) , each c o n t a i n i n g s u f f i c i e n t
moss f o r one s a m p l e t o be t a k e n
were a t t a c h e d t o t h e s t a k e s
controls.
the
Environmental
experiment
with
f o ranalysis.
Additional
lengths of s t r i n g .
v a r i a b l e s were measured
(Table 5.7).
samples o f moss
These a c t e d as
throughout
t h e course o f
150
T a b l e 5.7
Selected environmental
experiment
i n 0024-22
missing
v a r i a b l e s d u r i n g moss-bag t r a n s p l a n t
( m e t a l s i n mg 1 ^ f o r f i l t r a b l e
date
time
curr.
temp.
(m s- 1)
Ca
Zn
CC)
1.6.84
0900
10 .0
5 .9
20 .1
0.03
0024-22
1.6.84
1120
12 .0
7 .0
56 . 1
1.56
„
It
t l
1220
0 .291
12 .3
7 .0
60 .1
1.86
1320
0 .232
13. 0
7 0
58 6
1.81
1420
0 267
13 .5
7 1
58 6
1.50
1520
0 .261
13. 8
7 0
67 2
1.47
1620
0 268
14. 0
7 0
74 0
1.61
1720
0 238
13. 8
7 0
73 9
1.71
1930
0 215
13. 0
7 0
72 3
1.81
11. 0
6 9
67 8
1.65
13. 5
6 9
60 1
1.36
13. 0
6 8
65 0
1.67
13. 0
6 6
49 0
1.80
2320
Unlike
5 h.
2.6.84
1120
3.6.84
1120
4.6.84
1950
0 274
0 245
the previous experiment,
saturation
all
pH
0310-90
n
is
after
4 - 6 h(Fig
the
uptake curves
5.2) , and t h e
rapid"
T h i s was b a s e d upon a v i s u a l e x a m i n a t i o n
slightly
different
t o t h a t used i n 5.31.
o f t h e samples were a l l s i g n i f i c a n t ;
higher
blanks
values).
site
for
fraction;
levels of significance
coefficients.
appeared
phase o f
o f t h e u p t a k e c u r v e s and
The r e g r e s s i o n s based upon
the l o g ^ transformed
i n t h e r e g r e s s i o n and h i g h e r
A l l of the regressions calculated
d a t a had b o t h
correlation
f o r t h e r a p i d phase were
151
Fig.
5.2
Accumulation
three patterns
o f mesh bag.
i = control
ii
iii
iv
= pattern I
= pattern I I
= pattern
o f Zv. b y R h y n c h o s t e g i u m
III.
\
.
transplanted
i n t o 0024-22 i n
152
CN
00
ID
0)
E
O
o
o
O
O
O
CN
(j^_6 5n)
ssoiii ui uz
153
also
highly significant
a n d h i g h l y c o r r e l a t e d ; however two o f t h e
r e g r e s s i o n s f o r t h e s l o w phase w e r e n o t s i g n i f i c a n t .
t i m e and p a t t e r n
I I bags a n d t i m e a n d p a t t e r n
These were between
I I I bags.
ANOVA was p e r f o r m e d b e t w e e n a l l p a i r s o f s i g n i f i c a n t
5.8);
h o w e v e r none o f t h e c o m p a r i s o n s were s i g n i f i c a n t .
instance
t h e v a l u e o f F was l e s s
patterns
Analysis
o f mesh b a g d u r i n g
1 . a l l d a t a , no
transplant
regressions.
e x p e r i m e n t i n 0024-22.
pattern
I
pattern I I
transformation
pattern
I
0.378
pattern
I I
0.171
0.003
pattern
I I I
0.652
0.077
a l l data,
loge
0.075
transformation
pattern
I
0.544
pattern
I I
0.002
0.360
pattern
I I I
0.381
0.00004
0.289
0.557
0.028
3. < 5.0 h , no
transformation
pattern
I
0.021
pattern
I I
0.463
pattern
I I I
0.372
4. > 5.0 h , no
residual
o f v a r i a n c e b e t w e e n a c c u m u l a t i o n o f Zn i n d i f f e r e n t
control
2.
(Table
I n a l l b u t one
t h a n 1.0, i n d i c a t i n g t h a t
v a r i a t i o n exceeded t h e v a r i a t i o n o f t h e combined
T a b l e 5.8
regressions
transformation
pattern
I
0.512
pattern
I I
0.968
pattern
I I I
1.846
0.319
154
5.33 V a r i a t i o n
i n Zn a c c u m u l a t i o n b e t w e e n r e p l i c a t e
bags.
The w i t h i n - a n d b e t w e e n - b a g v a r i a t i o n was t e s t e d on d a t a
d u r i n g a t r a n s p l a n t experiment
bags
(pattern
each s a m p l i n g
II,
Table
c o n c e n t r a t i o n s measured
Table
5.9
t h e c o n c e n t r a t i o n o f Zn i n 0024-22 was v e r y h i g h
i s reflected
i n the high
i n b o t h t r a n s p l a n t e d ( T a b l e 5.9) and i n s i t u
(29764 pg g
^).
C o n c e n t r a t i o n s o f Zn i n w a t e r and i n R h y n c h o s t e g i u m d u r i n g
t r a n s p l a n t experiment
Time
i n 0024-22.
from
Zn i n w a t e r
start (h)
(mg 1-1)
Zn i n moss
(ug g-1)
2
4.04
5274
4
5.65
7815
6
7.07
9874
8
4.75
10796
12
3.40
10328
ANOVA perfor.med
on t h e d a t a
between samples o f t h e i n s i t u
= 6 h.
replicate
5.1) o f moss were suspended i n t h e r i v e r and a t
( + SD) = 6.34 ( + 1.20) mg 1 ~ \ n = 6 ) and t h i s
Rhynchostegium
Five
o c c a s i o n a sample was c o l l e c t e d f r o m each.
During t h e experiment
(x
b e t w e e n 0005-44 a n d 0024-22.
collected
(TabJe 5.10) showed
variation
p o p u l a t i o n and b e t w e e n bags a t t = 2 h and t
T h e r e were n o t s i g n i f i c a n t
bags a t o t h e r t i m e s .
significant
amounts o f v a r i a t i o n
between
replicate
155
T a b l e 5.10
transplanted
source
Analysis of variance within
into
and between r e p l i c a t e
bags o f moss
0024-22.
degrees o f
freedom
sum o f
squares
mean
square
t = 0 h
between
within
4
18
1.24 X lO"^
2.27 X lO"^
3.09 X lo;:
1.26 X lO'
2.448
N.S.
t = 2 h
between
within
4
18
3.07 X lO:
8
9.97 X 10
8
7.67 X 10.
5.54 X lO'
13.84
t = 4 h
between
within
4
19
8
3.87 X 10^
1.42 X 10^
9.67 X lO:
8
7.47 X 10
1.30
N.S.
t = 6 h
between
within
4
18
3.21 X 10^
1.20 X 10"
8.03 X l o ;
6.65 X 10'
12.07
**
t = 8 h
between
within
4
20
3.16 X 10^
10
3.34 X 10
8
7.89 X 10^
1.51 X l o '
0.52
N.S.
t = 12 h
between
within
3
15
2.46 X l o :
10
1.71 X 10
8.19 X 10^
1.14 X 10^
0.72
N.S.
in situ
between
within
3
14
11
3.07 X 10
11
2.85 X 10
11
1.02 X 10
10
2.04 X 10
5.03
*
156
5.34 H o m o g e n e i t y o f m e t a l a c c u m u l a t i o n w i t h i n
The e f f e c t
l a r g e p a c k e d bags.
o f bag p a c k i n g on t h e r a t e o f m e t a l a c c u m u l a t i o n was
on a p o p u l a t i o n f r o m 0055-30 t r a n s p l a n t e d t o 0048-80 i n l a r g e
(pattern
II,
carefully
moss-bags
f o r f o u r d a y s . A f t e r f o u r days t h e bags were
removed f r o m t h e r i v e r and s a m p l e s were t a k e n f r o m t h e c e n t r e and
t h e edge.
Physico-chemical
collected
v a r i a b l e s and c a t i o n s a m p l e s w e r e a l s o
(Table 5.11).
T a b l e 5.11
moss-bag
5.2) and l e f t
tested
Selected environmental variables
t r a n s p l a n t experiment
filtrable
i n 0055-30 and 0048-80 d u r i n g
( m e t a l c o n c e n t r a t i o n s i n mg
1 ''^ f o r
fraction).
reach
date
time
c u r r e n t speed
(m
pH
Ca
Zn
s"^
0055-30
10.8.84
1340
0.679
7.6
25.8
0.075
0048-80
15.8.84
1345
0.166
8.0
65.4
1.62
The t w o t r e a t m e n t s w e r e n o t s i g n i f i c a n t l y d i f f e r e n t f o r any o f t h e
three metals
contained
(Table 5.12).
h i g h e r c o n c e n t r a t i o n s t h a n s a m p l e s f r o m t h e c e n t r e , w h i l s t f o r Cd
t h e o p p o s i t e was
noticed,
F o r Zn and Pb t h e s a m p l e s f r o m t h e edge
true.
are very large
The 9 5 % c o n f i d e n c e l i m i t s
( F i g . 5.3).
f o r Cd, i t s h o u l d be
157
Fig.
5.3
Accumulation
edge o f moss-bags
confidence l i m i t s .
o f Zn, Cd and Pb by R h y n c h o s t e g i u m a t t h e c e n t r e
transplanted
(\(>
'W)
into
0048-44.
Vertical
b a r s = 9596
and
158
Zn
T
12,000
cn
cn
10,000
8000
o
e
6000
o
c
o
o
4000
2000
c
centre
E
edge
Cd
159
Table
5.12
C o m p a r i s o n o f m e t a l a c c u m u l a t i o n a t " c e n t r e " and "edge" o f
moss-bags u s i n g " S t u d e n t "
20032
Cd
1.16
Pb
2182
An e x p e r i m e n t
demonstrate
0.23
N.S.
0.84
1.10
N.S.
2347
0.33
N.S.
21318
5.35 A c c u m u l a t i o n
significance
"Student" t
edge
centre
Zn
t-test.
o f Zn b y F o n t i n a l i s on b o u l d e r s and i n bags
similar
t o 5.31 was p e r f o r m e d
using Fontinalis
common f e a t u r e s o f u p t a k e b y t h e two s p e c i e s .
i n order t o
This involved a
t r a n s p l a n t b e t w e e n 0014-40 a n d 0024-22 u s i n g b o u l d e r s a n d mesh bags
(pattern
I I , 5.2).
U p t a k e b o t h by moss on b o u l d e r s and i n bags a p p e a r e d t o r e a c h a p l a t e a u
a f t e r about
well
24 h ( F i g . 5 . 4 ) . Samples c o l l e c t e d
correlated with
demarcation
l o g ^ time
(Table 5.13).
i n t o a " r a p i d " and "slow"
chosen f o r f u r t h e r
analysis;
phase.
over
t h e e n t i r e p e r i o d were
T h e r e was no c l e a r
Two l i n e a r s e c t i o n s were
< 4.0 h and > 12.0 h and c o m p a r i s o n s were made
b e t w e e n b o u l d e r s and bags f o r each o f t h e s e and t h e w h o l e d a t a .
comparison
was s i g n i f i c a n t
(Table
5.14).
No
160
Fig.
5.4
Accumulation
b o u l d e r s and
bags.
o f Zn by F o n t i n a l i s
Vertical
transplanted
b a r s = 95% c o n f i d e n c e
into
intervals.
0024-22 i n
161
•
= m o s s on
boulders
o
= moss
bags
in
3000
cn
cn
2000
?
i
(/)
o
£
9
c
1000
o
f
?
10
20
30
^0
time
50
(h)
162
T a b l e 5.13
R e g r e s s i o n s and c o r r e l a t i o n s b e t w e e n Zn c o n c e n t r a t i o n s i n
F o n t i n a l i s and t i m e d u r i n g t r a n s p l a n t e x p e r i m e n t between 0014-40 and 002422.
treatment
regression
equation
correlation
coefficient
all
data,
no t r a n s f o r m a t i o n
boulder
y = 26.7 x + 9 6 1
10.55*
bag
y = 23.0 x + 979
5.47*
all
data,
0.754**
0.637*
I n transformation
boulder
y = 365 x -i- 679
73.89**
0.950***
bag
y = 333 x + 687
25.14**
0.871***
< 4.0 h , no t r a n s f o r m a t i o n
boulder
y = 312 x + 212
109.13**
0.986**
bag
y = 189 x + 338
18.80**
0.928**
> 12.0 h , no t r a n s f o r m a t i o n
boulder
bag
T a b l e 5.14
collected
Analysis
y = 2.25 x + 1796
0.10
N.S.
0.214
y = 14.0 x + 2261
2.33 N.S.
-0.734
of variance
during Fontinalis
between s i g n i f i c a n t
t r a n s p l a n t experiment
treatment
regressions
N.S.
N.S.
f o r data
(5.35).
significance
all
data,
no t r a n s f o r m a t i o n
0.082
N.S.
all
data,
log transformation
e
0.083
N.S.
5.45
X.S.
< 4.0 h , no t r a n s f o r m a t i o n
> 12.0 h , no t r a n s f o r m a t i o n
163
6.
EXPERIMENTAL STUDIES ON METAL ACCUMULATION AND LOSS
6.1 INTRODUCTION
E x p e r i m e n t a l s t u d i e s on m e t a l a c c u m u l a t i o n a n d l o s s were
both
i n t h e l a b o r a t o r y and t h e f i e l d .
s t u d i e s on p r a c t i c a l
Field
experiments
These p r o v i d e a b a c k g r o u n d
involved
experiments
accumulation.
extended
cell
t r a n s p l a n t s between streams
r e p o r t e d here concern
o f known w a t e r
They d e a l f i r s t
and o f m e t a b o l i s m
w i t h a c c u m u l a t i o n o f h e a v y m e t a l s o v e r an
i n t h i s process
t o consider therole of the
(6.3, 6,4).
F i n a l l y the rates
a t which heavy metals a r e l o s t and the f a c t o r s which a f f e c t
considered
for
(6.5).
Experiments
conditions.
t h e mechanism o f m e t a l
p e r i o d o f t i m e (6.2) and then proceed
wall
for
a s p e c t s o f m o n i t o r i n g t e c h n i q u e s (Chapters 5 and 8 ) .
c h e m i s t r y t o examine r a t e s o f u p t a k e and l o s s under n a t u r a l
The
performed
this are
w e r e f o c u s s e d on Zn, a l t h o u g h a f e w r e s u l t s
Cd a n d Pb a r e i n c l u d e d .
6.2 ACCUMULATION OF HEAVY METALS OVER THREE WEEKS
The
t i m e c o u r s e o f h e a v y m e t a l a c c u m u l a t i o n by R h y n c h o s t e g i u m was
investigated
by t r a n s p l a n t i n g moss t o a m e t a l - e n r i c h e d s t r e a m a n d s a m p l i n g
over a 23-d p e r i o d .
Moss was c o J l e c t e d f r o m 0091-05 and t r a n s p l a n t e d t o 0288-90 i n mesh
bags ( 5 . 2 , p a t t e r n
II).
Samples o f moss were c o l l e c t e d f r o m b o t h
t r a n s p l a n t e d a n d i n d i g e n o u s p o p u l a t i o n s a t 0. 1 , 3, 7, 1 1 , 17 a n d 23 d f r o m
the s t a r t
o f t h eexperiment.
W a t e r samples were c o l l e c t e d and p h y s i c o -
c h e m i c a l v a r i a b l e s m e a s u r e d on e a c h s a m p l i n g o c c a s i o n ( T a b l e 6 . 1 ) .
164
Table
6.1
Selected environmental v a r i a b l e s during long-term
t r a n s p l a n t experiment
fraction;
(= 23 d )
( m e t a l c o n c e n t r a t i o n s i n mg 1 ''^ f o r t h e f i l t r a b l e
blanks = n o t measured).
stream
date
-reach
Zn
Ca
t e m p . pH
Cd
Pb
(°C)
0091 -05
23. .7
12
7. 6
50. 8
0. 036
0. 0002
0. 0011
0288 -90
23,,7
12
8,,2
6 1 , .6
0, 273
0..0008
0.,0022
0288 -90
24. .7
12
8.,2
50,.4
0. 268
0. 0013
0.,0070
0288 -90
26. .7
12
8,.2
6 1 , ,4
0,.250
0..0018
0 .0040
0288 -90
30,,7
13
8,.2
60..6
0,,276
0..0064
0..0027
0288 -90
3 ,8
13
7,.6
26 .4
0 .155
0 .0014
0 .0054
0288 -90
9,.8
11
7,.7
43,.6
0,.337
0 .0017
0 .0057
0288 -90
15 .8
12
7 .7
63 .8
0 .296
The mean c o n c e n t r a t i o n o f Zn i n 0288-90 was a l m o s t
an o r d e r o f
m a g n i t u d e g r e a t e r t h a n i n 0091-05 ( T a b l e 6 . 1 ) ; t h e minimum Zn c o n c e n t r a t i o n
i n 0288-90 (3.8.84)
coincided w i t h a spate.
C o n c e n t r a t i o n s o f Cd a n d Pfa i n
0288-90 w e r e n o t so h i g h c o m p a r e d w i t h 0091-05; Cd i n 0288-90 r a n g e d
from
0.0008 mg l " " ^ t o 0.0064 mg 1 ~ ^ compared w i t h 0.0002 i n 0091-05 and Pb
c o n c e n t r a t i o n s i n 0288-90 r a n g e d
f r o m 0.0022 mg l " " ^ t o 0.0070 mg l " " ^
c o m p a r e d w i t h 0.0011 mg 1 ^ i n 0091-05.
Cd a n d Pb a l l o c c u r r e d on d i f f e r e n t
Zn a c c u m u l a t e d
The maximum c o n c e n t r a t i o n s o f Zn,
days ( T a b l e 6 . 1 ) .
throughout t h e experiment
( F i g . 6.1).
The
final
c o n c e n t r a t i o n was s i g n i f i c a n t l y g r e a t e r t h a n t h e c o n c e n t r a t i o n measured i n
in
s i t u moss.
The a c c u m u l a t i o n
o f Cd a p p e a r s t o be c o m p l e t e
after
d a y s ( F i g . 6.1) when t h e c o n c e n t r a t i o n i n t h e t r a n s p l a n t e d moss was
seven
still
165
significantly
of
lower
t h a n t h e c o n c e n t r a t i o n i n i n s i t u moss.
Pb was a l s o c o m p l e t e b y t h e e n d o f t h e e x p e r i m e n t
c o n c e n t r a t i o n was a p p r o x i m a t e l y
i n t h e c o n c e n t r a t i o n o f Zn, Cd a n d Pb i n
i n s i t u moss; none o f t h e s e , h o w e v e r , c o r r e l a t e d w i t h t h e c o r r e s p o n d i n g
concentrations
and
( F i g . 6.1) and t h e
t h e same as t h a t w i t h i n t h e i n s i t u moss.
There were c o n s i d e r a b l e v a r i a t i o n s
the
Accumulation
i n water
samples ( T a b l e 6.2).
A f t e r 4-d t h e s e changes i n Zn
Pb c o n c e n t r a t i o n s i n i n s i t u moss w e r e m i r r o r e d b y changes i n t h e
c o n c e n t r a t i o n s i n t r a n s p l a n t e d moss; t h e r e were n o t enough d a t a ,
for
a valid
however,
s t a t i s t i c a l comparison o f these.
Table
6.2
L i n e a r c o r r e l a t i o n s b e t w e e n c o n c e n t r a t i o n s o f Zn, Cd a n d Pb i n
water
samples and i n s i t u
moss o v e r
metal
23 d.
correlation
coefficient
Zn
0.055
N.S.
Cd
0.500
N.S.
Pb
0.067
N.S.
6.3 ROLE OF CELL WALL EXCHANGE I N ZINC ACCUMULATION
6.31
Introduction
The
time course
s h o r t term
of accumulation
( > 48 h , c h a p t e r
i n d i c a t e s that accumulation
ion-exchange processes alone
processes r e l a t i v e
o f metals
5) and l o n g e r term
i n t o moss s a m p l e s b o t h
over
( > 23 d, 6.2) e x p e r i m e n t s ,
c o n t i n u e s f o r l o n g e r t h a n w o u l d be e x p e c t e d i f
w e r e i n v o l v e d . The r o l e o f i o n - e x c h a n g e
t o o t h e r p r o c e s s e s was i n v e s t i g a t e d by r e m o v i n g
166
Fig.
6.1
Accumulation
o f Zn, Cd and Pb by R h y n c h o s t e g i u m
f r o m 0091-05 t o 0288-90 i n p a t t e r n
Vertical
b a r s = 95?^ c o n f i d e n c e
transplanted
I I moss-bags d u r i n g A u g u s t
intervals.
1984.
167
•o,
6000
r
5000
h
O
- transplanted
O
= in
situ
moss
moss
in
o
e
c
15
20
time
10
12
16
U
(days)
18
time
-
V)
600
J-
400
h
20
(days)
o
E
CL
200
15
time
20
(days)
22
168
ions adsorbed onto t h ec e l l
the p l a n t
6.32
w a l l and a n a l y z i n g
separately.
Methodology
6.321
Comparison
The
ability
of eluents
of three eluents
(2.421,
2.422) t o remove K a n d Zn f r o m
R h y n c h o s t e g i u m was t e s t e d on t h r e e p o p u l a t i o n s :
f r a c t i o n a t e d w i t h no f u r t h e r
mg 1 ^ Zn f o r
treatment
treatment,
\ h a n d 0288-90,
(Table
o n l y 9% - 25%
agreement
although
c o l l e c t e d and
in 1
c o l l e c t e d a n d f r a c t i o n a t e d w i t h no f u r t h e r
6.3).
b y e l u t i n g w i t h HCl w h i l s t
was e l u t e d b y N i C l ^ a n d EDTA ( T a b l e
between N i C l
6.3).
a n d EDTA f o r t h e p e r c e n t a g e o f Zn
theproportions
recovered
There i s l e s s
recovered,
by H C l , w h i c h damages t h e membranes, a r e
excess o f each.
6.322 E f f e c t o f i n c r e a s i n g e x p o s u r e
The
( F i g . 6.2).
o f Zn removed
first
times
e f f e c t o f i n c r e a s i n g exposure times
Rhynchostegium
Zn
0310-90,
0310-90 t h a t h a d been shaken
8 2 % - 9 0 % o f K i n t h e p l a n t was r e c o v e r e d
in
t h e s e and t h o s e r e m a i n i n g i n
from
0310-90 t h a t h a d p r e v i o u s l y been exposed t o 1
E l u t i o n was i n i t i a l l y
r a p i d ; 67% o f t h e f i n a l
by N i C l ^ a n d 6 5 % o f t h a t removed
t e n minutes.
intervals of thefinal
value.
o f K removed
both w i t h i n
T h e r e was l i t t l e
throughout
t h e experiment.
i n the
2 h, 1 0 8 % and
the 95% confidence
variation
mg 1 ^
concentration
by EDTA was removed
The r a t e s u b s e q u e n t l y d e c r e a s e d and a f t e r
8 5 % r e s p e c t i v e l y , h a d been removed,
concentration
t o N i C l ^ a n d EDTA was t e s t e d on
i n the
169
T a b l e 6.3
Concentrations
fractions after
treatment
o f K and Zn ( a s p e r c e n t
w i t h three eluents
of t o t a l )
I n various
(A & D = d e i o n i z e d
water
w a s h e s ; B & C = washes i n e l u e n t ; E = r e s i d u a l d i g e s t ; see 6.321 f o r
further
details).
eluent
metal
A
B + C
D
E
0310-90
K
Zn
1.43
1.78
11.00
45.11
5.74
2.44
81.82
50.67
0310-90 + Zn
K
Zn
8.06
0.80
24.95
84.32
5.99
0.90
61.00
13.98
0288-90
K
Zn
3.40
1.56
9.23
53.97
6.22
0.55
81.15
43.92
0310-90
K
Zn
1.86
2.60
9.25
75.14
1.46
3.47
87.43
18.79
0310-90 + Zn
K
Zn
7.50
0.55
23.24
65.17
2.36
0.90
66.90
33.38
0288-90
K
Zn
6.27
1.06
10.78
82.66
1.54
0.58
81.41
15.69
0310-90
K
Zn
1.81
2.09
92.10
82.66
0.77
1.12
5.31
14.12
0310-90 + Zn
K
Zn
6.85
0.56
82.51
96.23
1.70
0.31
8.93
2.90
0288-90
K
Zn
2.40
0.72
93.50
94.54
0.70
0.31
3.40
4.43
N1C1„
EDTA
HCl
170
Fig.
6.2
Effect
o f i n c r e a s i n g e x p o s u r e t i m e on e l u t i o n
R h y n c h o s t e g i u m by N i C l ^ ( t o p ) and EDTA ( b o t t o m ) .
confidence
limits.
o f K and Zn
Vertical
bars
from
= 95?i
171
6000
5000
^^000
3000
2000
time (h)
6000
5000
o. -iOOO
>
o
E
a>
3000
c
o
2000 Hc
<u
u
c
o
o
1000
h
time
(h)
172
6.323 Removal o f Zn f r o m
In
order
isolated
t o remove a m b i g u i t y
whole p l a n t s , t h e i r a b i l i t y
was a l s o t e s t e d .
rinsed
several
The c e l l
times
cell
walls
a b o u t t h e e f f e c t o f N i C l ^ a n d EDTA on
t o remove Zn f r o m
w a l l s were shaken i n 10 mg 1
a n d washed i n e l u e n t f o r t h r e e
r e m o v e d 9 5 . 7 % o f Zn a f t e r one wash ( T a b l e
washes.
EDTA was l e s s s u c c e s s f u l ,
washes.
Following
N i C l ^ was s e l e c t e d
isolated cell
walls
Zn f o r 1 h ,
1 h periods.
6.4) a n d 9 9 . 4 % a f t e r
as t h e most a p p r o p r i a t e
NiCl^
three
o n l y 8 8 . 4 % was removed a f t e r
t h i s e x p e r i m e n t and t h o s e r e p o r t e d
(2.43)
three
i n 6.321 a n d 6.322,
e l u e n t f o r subsequent
experiments.
T a b l e 6.4
by N i C l g
Removal o f Zn ( a s p e r c e n t a g e o f t o t a l ) f r o m
a n d EDTA ( s e e 6.323 f o r f u r t h e r
isolated cell
walls
details).
eluent
elution
EDTA
NiCl,
1
75.7
95.7
2
8.4
3.2
3
4.3
0.6
residual
0.6
11.6
6.33 A c c u m u l a t i o n o f Zn o v e r 12 h
The
cellular
l o c a t i o n o f a c c u m u l a t e d Zn was i n v e s t i g a t e d i n an
e x p e r i m e n t p e r f o r m e d i n t h e l a b o r a t o r y u s i n g N i C I ^ as an e l u e n t
Rhynchostegium from
(6.323).
0310-90 was exposed t o 1.0 mg 1 ^ Zn and samples
w e r e c o l l e c t e d o v e r a 12-h p e r i o d .
shaken i n N i C l ^ f o r two p e r i o d s
Tips
were r i n s e d i n d e i o n i z e d
of 1 h before
being
w a t e r and
d r i e d , w e i g h e d and
173
digested.
for
the s t a r t
was
o f t h e e x p e r i m e n t , 29%
i n the r e s i d u a l d i g e s t
t h e moss s a m p l e s was
a c c o u n t e d f o r 77.5%
was
t h e r e s i d u a l d i g e s t were a n a l y z e d
Zn.
At
1\%
B o t h t h e N i C l ^ f r a c t i o n and
r e m o v e d by
o f Zn
( F i g . 6.3).
(SD
= 2.6%)
of the t o t a l
The
total
i n the r e s i d u a l
equation
This
also a small
t h e s e d a t a was
max
" ^"
[Zn
] = maximum p o s s i b l e
t
=
max
in
fraction
study.
2%
remained i n
increased
i n t h e amount
a
double-reciprocal
The
be:
] t
accumulated at time
K = time
converts
23%
t h e L i n e w e a v e r - B u r k e p l o t o f enzyme k i n e t i c s .
f^^t"'
[Zn
t h e 12-h
increase
t o use
d e s c r i b i n g u p t a k e i s t h e r e f o r e assumed t o
where:
accumulated
exchangeable
w a t e r and
and
fraction.
[Zn^]
t
This
t h a t had
amount o f e x c h a n g e a b l e Zn
t h e r e was
A second method o f p r e s e n t i n g
from
Zn
removed by NIC]
throughout
t h e p r e l i m i n a r y wash i n d e i o n i z e d
t h e e x p e r i m e n t and
p l o t adapted
The
l a r g e l y removed by N i C l ^ .
the r e s i d u a l f r a c t i o n .
throughout
o f t h e Zn was
t
concentration
of
Zn
r e q u i r e d f o r h a l f maximum a c c u m u l a t i o n
time.
to a s t r a i g h t
[Zn^]
t'
line
form:
[Zn
I t
max'
[Zn
I t
max-
2
175
[Zn^]
t
Conversion o f t h e data
time
[Zn
I
max'
(Zn
[Zn
]
max
i n t h i s way makes i t p o s s i b l e t o e s t i m a t e t h e
f o r h a l f maximum a c c u m u l a t i o n t o o c c u r
accumulation
t
( K ) a n d t h e maximum
possible
) as t h e i n t e r c e p t on t h e y a x i s and t h e s l o p e
IIIQX
multiplied
by t h e i n t e r c e p t , r e s p e c t i v e l y .
A c c u m u l a t i o n by b o t h
exchangeable and r e s i d u a l f r a c t i o n s f i t t h e s e equations
time
of t h e experiment
well within the
( F i g . 6.4) and t h e f o l l o w i n g c o n s t a n t s
were
calculated:
i . exchangeable
[Zn^^^]
fraction
= 4348 y g g " ^
K = 1.0 h
Hid A
i i . residual
[Zn
max
fraction
1 - 1052 p g g""^
K = 1.6 h
6.34 A c c u m u l a t i o n o f Zn o v e r 14 d
The c e l l u l a r
longer
time
l o c a t i o n o f a c c u m u l a t e d Zn was a l s o s t u d i e d o v e r a
period using
t h e same a p p r o a c h as 6.33.
Samples were c o l l e c t e d
o v e r a p e r i o d o f 14 d.
T h e r e was l i t t l e
and
change i n t h e c o n c e n t r a t i o n o f Zn o v e r b e t w e e n 24-h
14-d ( F i g . 6 . 5 ) ; h o w e v e r t h e p r o p o r t i o n t h a t was removed b y N i C l g
d e c r e a s e d and t h e r e s i d u a l p r o p o r t i o n
T h e r e was a l s o an e x p o n e n t i a l
solution
the t o t a l
(Table
increased.
decrease i n t h e c o n c e n t r a t i o n
6 . 5 ) , w h i c h was i n v e r s e l y c o r r e l a t e d
Zn a c c u m u l a t e d by t h e t i p s .
( r = -0.973 * * * ) w i t h
The c o n c e n t r a t i o n o f Zn i n c o n t r o l
f l a s k s w i t h no t i p s added was n o t s i g n i f i c a n t l y d i f f e r e n t
the concentration
a t the start
(Table
o f Zn i n
6.5).
a t t = l - i - d from
D a t a were a g a i n
tested
against
176
tho c o n c o n t r a t i o n a t t h e s t a r t (Table 6 . 5 ) . — B a t a
again t e s t e d
agaiits4
a d o u b l e - r e c i p r o c a l p l o t ; however o n l y t h e r e s i d u a l f r a c t i o n was
s i g n i f i c a n t l y correlated
( F i g . 6.6) and t h e f o l l o w i n g constants were
calculated:
Zn
max
Table 6.5
=- 2778 ug g""^ K = 0.81 d - 19 h
C o n c e n t r a t i o n o f Zn (+ SD) r e m a i n i n g i n s o l u t i o n dui^ing 14 d
batch c u l t u r e experiment ( 1 4 * = c o n t r o l s o l u t i o n a t 1^^-d).
time
(d)
Zn i n media
(mg
0
1.086 (0.077)
1
0.306 (0.043)
2
0.230 (0.022)
3
0.198 (0.074)
4
0.182 (0.015)
6
0.148 (0.033)
8
0.129 (0.019)
14
0.074 (0.021)
14*
1.024 (0.023)
6.4 THE ROLE OF METABOLISM IN ZN ACCUMULATION
6.41
Introduction
The p r e v i o u s s e c t i o n
(6.3) demonstrated t h a t t h e r e was s i g n i f i c a n t
a c c u m u l a t i o n over p e r i o d s o f s e v e r a l hours i n t o a compartment
e l u t a b l e u s i n g a competing c a t i o n .
t h a t was n o t
The q u e s t i o n posed by these r e s u l t s i s
by what mechanism i s t h i s f r a c t i o n accumulated?
I s i t a p u r e l y passive
177
Fig.
6.3
AccumuDation o f Zn i n exchangeable and r e s i d u a l
f r a c t i o n s of
Rhynchostegium over 12 h a t 15 °C i n l a b o r a t o r y b a t c h c u l t u r e .
bars = 95% c o n f i d e n c e
Fig.
6.4
intervals.
D o u b l e - r e c i p r o c a l p l o t of data presented i n F i g . 6.3
Vertical
178
5000 rtotal
1,000 h
3000 h
exchangeable
cn
c
o
o
o
2000 h
res idual
N
1000
time
0.0020
0.0015 h
cn
?
0.0010
c
0.0005 h
1
time
(h)
h)
179
F i g . 6.5
Accumulation of Zn i n exchangeable
and r e s i d u a l f r a c t i o n s of
Rhynchostegium over 14 d a t 15 °C i n l a b o r a t o r y
batch c u l t u r e .
Vertical
bars = 95% c o n f i d e n c e l i m i t s .
F i g . 6.6
over 14 d.
Double r e c i p r o c a l p l o t of Zn accumu]atjon i n r e s i d u a l f r a c t i o n
180
5000
total
^000
residual
3000
u
2000
exchangeable
1000
2
i,
6
8
time
10
12
(days)
0.0000
0.0006
^
h
0.0005
c
0.0004 h
0.0003
t i m e (days)
U
181
are c o n s i d e r e d i n t h i s s e c t i o n by comparing metal accumulation by t i p s i n
d i f f e r e n t m e t a b o l i c s t a t e s ; under d i f f e r e n t l i g h t regimes, a t d i f f e r e n t
temperatures
and i n the presence and absence of v a r i o u s metabolic
inhibitors.
6.42
E f f e c t of l i g h t
6.421
In laboratory
The
i n f l u e n c e of l i g h t was
t e s t e d by comparing Zn accumulation
by
-2
exposed t o f u l l
l i g h t i n i n c u b a t i o n tanks (= 100 pmol photon m
a c c u m u l a t i o n by t i p s i n f l a s k s darkened w i t h aluminium
of b l a c k p o l y e t h y l e n e .
The
experiment
-1
s
lOE a t pH 7.0
) with
f o i l and two l a y e r s
l i g h t f l u x i n the l a t t e r treatment was
T i p s were p r e i n c u b a t e d i n Chu
tips
zero.
(2.41) f o r 7 d p r i o r t o the
t o p r e c o n d i t i o n them t o the l a b o r a t o r y environment.
They were
then placed i n f l a s k s as d e s c r i b e d above and harvested ( f o u r f l a s k s per
t r e a t m e n t ) a f t e r 1 h, 24 h and 168 h (= 7 d ) .
Accumulation
(Fig.
6.7,
of Zn f o l l o w e d s i m i l a r k i n e t i c s to previous
see a l s o 5.3,
experiments
6.2), w i t h a c o n c e n t r a t i o n of approximately 4000
pg g""^ a f t e r 1 h, 10000 ug g"^^ a f t e r 24 h and 12000 ug g"'^ a f t e r 168 h.
There was
no s i g n i f i c a n t d i f f e r e n c e between the l i g h t and dark t r e a t m e n t s
a t any of t h e t h r e e sample times (Table 6.6).
182
Table 6.6
Comparison, u s i n g "Student" t - t e s t , o f Zn accumulation by
Rhynchostegium i n c u b a t e d i n l a b o r a t o r y batch c u l t u r e at 15 "C i n l i g h t
dark.
Zn i n moss (yg g
time (h)
light
1
)
"Student" t
dark
4305
4208
0.445
N.S.
24
10561
10568
0.019
^\S.
168
11599
13026
1.390
y.s.
6.422 I n f i e l d
A second experiment t o i n v e s t i g a t e t h e e f f e c t o f l i g h t was conducted i n
the
field.
Moss was t r a n s p l a n t e d between 0310-90 and 0288-90.
A t 0288-90
an area o f t h e stream o f a p p r o x i m a t e l y 1.5 m x 1.5 m was covered w i t h
" f e r t i l i z e r " - t y p e bags ( b l a c k or w h i t e w i t h b l a c k i n t e r i o r s ) c o n s t r u c t e d
from heavy d u t y p o l y e t h y l e n e .
These were supported on a framework o f t e n t
poles ( F i g . 6.9).
Moss was p l a c e d i n 0288-90 i n moss-bags (5.2, p a t t e r n I I ) , e i t h e r
i n s i d e t h e darkened area o r o u t s i d e .
an 8-h p e r i o d .
Samples o f each were c o l l e c t e d
over
Water samples were a l s o c o l l e c t e d and physico-chemical
v a r i a b l e s measured.
Values o f some physico-chemical v a r i a b l e s and o f
l i g h t - f l u x a r e g i v e n i n Table 6.7.
L i g h t f l u x w i t h i n t h e darkened area was
-2 - 1
-2 -1
c o n s i s t e n t l y 0 pmol photon m
s
(one r e a d i n g o f 1 ymol photon m
s ).
-2 - 1
O u t s i d e , t h e f l u x ranged from 120 ymol photon m
-2
pmol photon
m
s
( o v e r c a s t ) t o 1200
-1
s
( s u n n y ) , t h e maximum b e i n g recorded a t 1240.
D u r i n g t h e course o f t h e experiment, moss i n t h e darkened area
c o n s i s t e n t l y accumulated
more Zn than moss i n t h e l i g h t ( F i g . 6.8). The
d i f f e r e n c e s were s i g n i f i c a n t a t a l l times except t = 1 and t = 4 (Table
6.8) .
183
F i g . 6.7
A c c u m u l a t i o n o f Zn by Rhynchostegium i n c u b a t e d i n l a b o r a t o r y a t
15 °C i n l i g h t and d a r k .
F i g . 6.8
V e r t i c a l bars = 95% c o n f i d e n c e l i m i t s .
A c c u m u l a t i o n of Zn by Rhynchostegium t r a n s p l a n t e d i n t o 0288-90 i n
l i g h t and dark.
V e r t i c a l bars = 95% c o n f i d e n c e l i m i t s .
(^ ^
184
L = light
D = dark
15000
-
10000
-
L D
L D
L D
1
24
168
cn
cn
if)
I/)
o
E
5000
N
time
1000
{h)
-
cn
3
U)
o
= dark
E
o
= light
c
N
time
(h)
185
F i g . 6.9
Apparatus assembled i n 0288-90 f o r f i e l d comparison of Zn
a c c u m u l a t i o n i n l i g h t and dark.
186
187
Table 6.7 S e l e c t e d e n v i r o n m e n t a l v a r i a b l e s i n 0288-90 d u r i n g f i e l d
comparison o f Zn uptake
time
light
i n l i g h t and dark.
flux
temp
(pmol photon m
PH
Ca
(°C)
Zn
(mg 1
' )
1040
125
5. 1
7 .3
30 .7
0 . 192
1140
166
5 3
7 .3
27 .5
0 .187
1240
1200
6 0
7 4
28 .9
0 . 179
1440
157
6 5
7 4
27 .7
0 .176
1640
170
6 3
7 4
27 .3
0 . 182
1840
120
6. 3
7 4
27 2
0 181
6.423 E f f e c t o f dark p r e i n c u b a t i o n on Zn uptake
A second t r a n s p l a n t experiment
the
was performed
i n t h e f i e l d t o compare
e f f e c t s o f dark and l i g h t p r e t r e a t m e n t s on subsequent r a t e s o f Zn
accumulation.
Rhynchostegium from 0310-90 was p r e i n c u b a t e d i n t h e dark and
l i g h t a t 0310-90 f o r 48 h ( F i g . 6.9) and then t r a n s f e r r e d t o 0288-90 f o r a
further
24 h.
There was no s i g n i f i c a n t d i f f e r e n c e between any o f t h e t r e a t m e n t s a f t e r
the
p r e i n c u b a t i o n p e r i o d i n 0310-90 (F = 1.68, N.S.).
T r a n s f e r t o 0288-90
r e s u l t e d i n Zn accumulation by t h e moss i n a l l f o u r t r e a t m e n t s (Table 6.9)
a l t h o u g h t h e r e were s e v e r a l s i g n i f i c a n t d i f f e r e n c e s between treatments (F =
44.21
***).
The sample p r e i n c u b a t e d i n t h e dark accumulated s i g n i f i c a n t l y
more when t r a n s p l a n t e d i n t o t h e l i g h t b u t t h e r e was no s i g n i f i c a n t
d i f f e r e n c e i n f i n a l Zn c o n c e n t r a t i o n s i n moss preincubated i n t h e l i g h t
(Table 6.10) .
188
Table 6.8
E f f e c t o f l i g h t on Zn accumulation by Rhynchostegium
t r a n s p l a n t e d i n t o 0288-90 ( a c c u m u l a t i o n was g r e a t e r i n t h e dark on a l l
sampling o c c a s i o n s ) .
time
"Student" t
(h)
Table 6.9
0.5
4.786
**
1
1.377
N.S.
2
3.400
**
4
2.265
N.S.
6
3.497
**
8
4.436
**
Zn a c c u m u l a t i o n by Rhynchostegium t r a n s p l a n t e d i n t o 0288-90 i n
l i g h t and d a r k .
pre-treatment
post-treatment
mean
95% confidence
dark
dark
1123
1154 - 1092
light
1367
1420 - 1314
dark
1417
1500 - 1334
light
1486
1568 - 1404
light
limits
189
Table 6.10
"Student" t - t e s t s between t r e a t m e n t s i n l i g h t / d a r k
experiment i n 0288-90; See t a b l e
dark - dark
6.9.
1.00
dark - l i g h t
11.05***
l i g h t - dark
1.00
9.16***
light - light
1.38
11.54***
dark - dark
6.43
transplant
1.00
3.37**
dark - l i g h t
1.64
l i g h t - dark
1.00
light - ]ight
E f f e c t o f temperature
The e f f e c t o f temperature was
i n v e s t i g a t e d by comparing accumulation o f
Zn i n t h e l a b o r a t o r y a t two temperatures; 10 °C and 25 °C.
Otherwise
parameters were as b e f o r e (2.41) and t i p s were again e q u i l i b r a t e d a t 15 °C
f o r seven days p r i o r t o the experiment.
There was
s i g n i f i c a n t l y g r e a t e r accumulation a t 25 "C a f t e r 1 h, 24 h
and 168 h (Table 6.11).
and 25% a f t e r 168 h.
The d i f f e r e n c e was 35% a f t e r 1 h, 24% a f t e r 24 h
These were a d j u s t e d t o g i v e the p r e d i c t e d Q^^
values
( t h e e f f e c t of a 10 °C r i s e i n temperature, assuming the e f f e c t of
t e m p e r a t u r e t o be l i n e a r ) .
These a d j u s t e d values were 23% a f t e r 1 h,
a f t e r 24 h and 16.5% a f t e r 168 h. g i v i n g
^ values of 1.23,
1.16
and
16%
1.16
respectively.
6.44
E f f e c t of metabolic i n h i b i t o r s
6.441
Methodology
A p r e l i m i n a r y l a b o r a t o r y experiment compared the e f f e c t of a range of
m e t a b o l i c i n h i b i t o r s on K e f f l u x from t i p s of Rhynchostegium (2.422).
D e t a i l s o f the s i x i n h i b i t o r s are g i v e n i n Table
6.12.
190
Table 6.11
E f f e c t o f temperature on Zn accumulation by Rhynchostegium i n
l a b o r a t o r y batch c u l t u r e ;
comparison
o f sample means.
Zn i n moss ( y g g ^)
time
10 °C
25 °C
"Student" t
1 h
3578
4838
7.056
24 h
7553
9404
7.970
8915
11119
7.740
168 h
Three f r a c t i o n s
***
were c o l l e c t e d : a f t e r 1 h r i n s e s i n each o f d e i o n i z e d
w a t e r , t h e m e t a b o l i c i n h i b i t o r and d e i o n i z e d water again.
was
Each f r a c t i o n
subsequently analyzed f o r K and the t i p s were d r i e d and weighed.
The c o n c e n t r a t i o n s o f K r e l e a s e d by the p r e l i m i n a r y wash i n d e i o n i z e d
water ranged from 43.4 yg g"'^ t o 100.4
y g g""^ (Table 6.13).
i n h i b i t o r s r e l e a s e d some K, r a n g i n g from 159.1 yg g
A l l o f the
(HK) t o 8696 yg g
(DCIVIU) and s i g n i f i c a n t K was a l s o d e t e c t e d i n the second r i n s e i n d e i o n i z e d
water.
The r a n k i n g o f t h e i n h i b i t o r s was:
control
< KH < KaM
< CCCP = CH
O
< DNP < DCIVIU.
6.442 L a b o r a t o r y experiment
Two m e t a b o l i c i n h i b i t o r s were used i n t h i s experiment: 2,4-DNP and
DCMU.
Both o f these caused s i g n i f i c a n t K leakage i n the p r e v i o u s
experiment
(6.441) which suggested t h a t membrane t r a n s p o r t systems were
s t r o n g l y a f f e c t e d by t h e i n h i b i t o r (2.422).
The i n h i b i t o r s were chosen f o r
t h i s and because t h e r e i s a r e l a t i v e l y l a r g e l i t e r a t u r e on the biochemical
effects
o f each.
Samples were p r e i n c u b a t e d i n t h e i n h i b i t o r a t the
c o n c e n t r a t i o n s used i n 6.441
transferred straight
f o r 90 min p r i o r t o the experiment, r i n s e d and
t o the e x p e r i m e n t a l media.
191
-—
cn c-
00
00
0 5
0 5
0 )
'-•1
col
""I
K J I
a> CO
T-l
«i
c05
iH
CSl
G
0)
u
(31
c-
^
03
03
U
C 03
«l
(ul
u
u
03
XI
03
0)
»
U
00
0 5
r-(
^—•
c
o o
to .Q
u
<D
0 5
*
+-"1
•'-'1 (Ul
<u!
O
0}
•H
O
3
O
CO
CM
in
g
s
O
03 .Q
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T3
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Q
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T-l
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c-
CO
m
lO
1
o
o
CO
03
o
I
3
o
o
in
CO
o>
bA
•H
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CO
00
rH
1
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CO
>-l +j
cu
to
o
1
03
00
<M
1
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rH
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SH
03
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T3
03 a;
c
e
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CO
r-H
>>
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rH
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O
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u
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«
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c
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03
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1 to
£
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•rH >>
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>> ?>>
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&}
1—1
XI
a
c o
o
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i-H
CO X I
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•^•^
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o
s
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'—^
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•H
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x:
o
rH
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>>
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N
CO
G
3
3
>> u
c n
d)
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—
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O
0
u
u
O
3
rH r-H
X 3
?>>
o x:
•rH ^
•a (u
1
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•rH
CO
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•—• ri
CO
,
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192
Table 6.13 C o n c e n t r a t i o n s o f K r e l e a s e d by t r e a t m e n t o f Rhynchostegiua f o r
1 h a t 15 °C with d i f f e r e n t m e t a b o l i c i n h i b i t o r s .
K i n fraction
treatment
d e i o n i z e d water
(yg g
metabolic i n h i b i t o r
45.8
)
•ionized water
57.6
control
54.1
DNP
96.9
2148
HH
69.2
159.1
82.8
CCCP
58.6
680.8
514.0
CH
43.4
743.0
389.0
NaN„
63.1
369.3
131.2
DCMU
100.4
8696
829.5
1784.3
The e f f e c t o f t h e i n h i b i t o r s on t h e growth o f t h e moss was apparent
a f t e r 168 h. A t t h i s time " c o n t r o l " t i p s showed s i g n s o f f r e s h growth
( b r i g h t green shoots a p p e a r i n g from t h e o r i g i n a l t i p s ) ; DNP-treated moss
looked l i t t l e d i f f e r e n t t o t h e s t a r t and t h e DCMU-treated moss was a brown
colour.
There were s e v e r a l s i g n i f i c a n t d i f f e r e n c e s i n Zn accumulation between
t r e a t m e n t s ( F i g . 6.10).
These were a t t = 1 h between c o n t r o l and DMP and
between c o n t r o l and DCMU and a t t = 24 h between c o n t r o l and DCr^U and
between DNP and DCMU.
(Table 6.14).
There were no s i g n i f i c a n t d i f f e r e n c e s a t t = 168 h
193
F i g . 6.10
Accumulation
of Zn by Rhynchostegiuin a t 15 °C i n l a b o r a t o r y
batch c u l t u r e a f t e r p r e t r e a t m e n t w i t h m e t a b o l i c i n h i b i t o r s .
= 95% c o n f i d e n c e
limits.
Vertical
bars
194
1 =
2
=
control
2.U
3 =
1
2
-DNP
DCMU
3
1
2
3
1
2
15000
CD
10000
o
E
N
5000
24
time
168
(h)
3
195
6.5 LOSS OF HEAVY METALS
6.51
Introduction
The s u c c e s s f u l m o n i t o r i n g o f i n t e r m i t t e n t heavy metal p o J l u t i o n r e l i e s upon
t h e a b i l i t y o f Rhynchostegium t o both accumulate heavy metals and t o r e t a i n
e l e v a t e d c o n c e n t r a t i o n s o f these a f t e r t h e " p u l s e " o f p o l l u t i o n has passed
downstream.
Experiments were c a r r i e d o u t t o i n v e s t i g a t e t h e f a c t o r s which
a f f e c t t h e r a t e a t which heavy metals a r e l o s t from Rhynchostegium.
P r e l i m i n a r y experiments a r e d e s c r i b e d which c h a r a c t e r i z e these
Table 6.14 E f f e c t o f 1 h p r e t r e a t m e n t w i t h m e t a b o l i c i n h i b i t o r s on Zn
a c c u m u l a t i o n by Rhynchostegium a t 15 °C i n l a b o r a t o r y batch c u l t u r e ;
comparison
o f sample means (C = c o n t r o l ) .
time (h)
1 h
24 h
168 h
treatments
"Student" t
C V DNP
4.732
**
C V DCMU
3.770
**
DNP V DCMU
0.000
N.S.
C V DNP
0.218
N.S.
C V DCMU
8.688
***
DNP V DCMU
8.455
***
C V DNP
2.354
N.S.
C V DCMU
1.129
N.S.
DNP V DCMU
0.307
N.S.
processes: an e x p e r i m e n t a l t r a n s p l a n t from a Zn-enriched stream i n t o a lowZn stream ( 6 . 5 2 ) , a s e r i e s o f samples o f Rhynchostegium c o l l e c t e d from a
196
stream i n which t h e c o n c e n t r a t i o n of Zn i n the water was
(6.53) and
s l o w l y decreasing
the e f f e c t of i n c r e a s i n g l e n g t h s of exposure t o Zn on
subsequent r a t e s of l o s s ( 6 . 5 4 ) .
the
I n a d d i t i o n a l a b o r a t o r y experiment
performed t o i n v e s t i g a t e the e f f e c t of v a r i o u s washing treatments
loss
6.52
was
on metal
(6.55).
Rate of l o s s of
An experiment was
Zn
designed i n order t o i n v e s t i g a t e the l o s s of Zn from
moss f o l l o w i n g a t r a n s p l a n t between 0288-90, a stream w i t h e l e v a t e d
concentrations
of Zn, and
Rhynchostegium was
p a t t e r n I I ) and
0091-05.
t r a n s p l a n t e d both on boulders and
samples were c o l l e c t e d from these and
Loss of Zn from moss was
i n moss-bags (5.2,
from i n s i t u moss.
e v i d e n t over a longer p e r i o d than uptake ( F i g .
6.11), the m a j o r i t y o c c u r r i n g w i t h i n the f i r s t f o u r days of the experiment.
The
r a t e of l o s s subsequently decreased and a f t e r 23 d the c o n c e n t r a t i o n i n
t h e t r a n s p l a n t e d moss remained above t h a t i n the i n s i t u Rhynchostegium.
6.53
Loss of Zn under f i e l d
An experiment was
designed i n order t o i n v e s t i g a t e the e f f e c t of a
changing Zn c o n c e n t r a t i o n
Rhynchostegium.
conditions
i n water on the c o n c e n t r a t i o n of Zn i n i n s i t u
Samples of water and of Rhynchostegium were c o l l e c t e d over
a p e r i o d of 10 d from 0024-20.
The
mg
c o n c e n t r a t i o n of Zn i n the water d e c l i n e d from 3.70
1 "'^ d u r i n g the course of the experiment.
of Zn from Rhynchostegium ( F i g . 6.12).
( r = 0.89
**) when the c o n c e n t r a t i o n s
This was
There was
mg
to
accompanied by
a significant
i n the moss and
1
1.30
loss
correlation
i n the water were
compared; however a t i m e - s e r i e s a n a l y s i s , u s i n g c r o s s - c o r r e l a t i o n s showed
197
F i g . 6.11
0091-05.
F i g . 6.12
Loss of Zn from Rhynchostegium t r a n s p l a n t e d from 0288-90 t o
V e r t i c a l bars = 95% c o n f i d e n c e l i m i t s .
^ ScjptenvW
C o n c e n t r a t i o n s of Zn i n water and Rhynchostegium i n 0024-20,
07-08-84 to 16-08-84.
V e r t i c a l bars = 95% c o n f i d e n c e
limits.
198
® = moss on b o u l d e r
O = m o s s in bag
6000
= in s i t u
r
moss
4000
2000
8
10
time
(days)
n 7000
6000
N
-\ 5000
3
o
2
4.00
c
3.00
A
4000
3000
01
^
U3
M
2.00
8
time
9
10
(days)
A 2000
-
4 1000
O
11
199
the s t r o n g e s t c o r r e l a t i o n t o be a s s o c i a t e d
(Table
w i t h a l a g p e r i o d of one
day
6.15).
Table 6.15
Cross-correlations
computed between Zn accumulation by
Rhynchostegium and aqueous Zn i n 0024-20 over 11 d f o r t h r e e
increasing
d a i l y lags (maximum p o s i t i v e c o r r e l a t i o n i s u n d e r l i n e d ) .
6.54
l a g (d)
0
1
2
3
n
10
10
9
8
r
0.890
0.976
0.879
0.754
E f f e c t of l e n g t h of exposure t o Zn
The
e f f e c t s of i n c r e a s i n g l e n g t h of exposure t o Zn was
f i e l d t r a n s p l a n t i n which Rhynchostegium from 0310-90 was
0012-28, approxim.ately
t e s t e d by a
transplanted
30 m away, f o r d i f f e r e n t l e n g t h s of time
into
before
being r e t u r n e d t o 0310-90 along w i t h Rhynchostegium from a nearby metalenriched
stream, 0309-80.
p e r i o d of 48
The
Sa.mples of moss and water were c o l l e c t e d over a
h.
t o t a l Zn accumulated was
as expected from p r e v i o u s
dependent upon the l e n g t h of i t s exposure,
experiments; hence Rhynchostegium w i t h a s h o r t
p e r i o d o f exposure ( 1 h) accumulated 1130
1660
pg g
The
and a f t e r 24 h i t c o n t a i n e d
r a t e of l o s s was
exposure, Zn was
pg g ^ Zn, a f t e r 4 h i t contained
2373 pg g ^ Zn.
dependent upon the l e n g t h of the o r i g i n a l
l o s t v e r y r a p i d l y ( i . e . w i t h i n 2 h) from moss which
been exposed f o r s h o r t p e r i o d s
( F i g . 6.13).
That which had had
exposure r e t a i n e d Zn f o r longer p e r i o d s ; the c o n c e n t r a t i o n was
24 h
still
had
200
s i g n i f i c a n t l y above background a f t e r 12 h i n 0330-90.
0309-80 l o s t Zn over the e n t i r e experimental
The p o p u l a t i o n
from
period.
The e f f e c t i v e n e s s o f Rhynchostcgium as a monitor depends upon being
a b l e t o determine e n r i c h e d c o n c e n t r a t i o n s a number of hours a f t e r exposure.
The l e n g t h o f t h i s p e r i o d was examined by performing
"Student"
t-tests
between the c o n c e n t r a t i o n a t t = 48 h. assumed t o be the background, and
the c o n c e n t r a t i o n a t times p r e v i o u s t o t h i s .
I f the c r i t e r i o n of a
s i g n i f i c a n t d i f f e r e n c e represents a q u a n t i f i a b l e
e f f e c t then a 1 h exposure
t o Zn i s d e t e c t a b l e o n l y f o r about 1 h a f t e r being r e t u r n e d t o a low-Zn
environment (Table 6.16).
Exposures of 4 h and 24 h were d e t e c t a b l e f o r
longer p e r i o d s o f t i m e , between 12 h and 24 h.
Table 6.16
S i g n i f i c a n c e o f d i f f e r e n c e s ( c a l c u l a t e d by "Student"
t-test)
between c o n c e n t r a t i o n o f Zn i n .Rhynchostegium a t t = 48 h and a t times
previous
to t h i s .
time
1
-4
-24
0309
0.0
0 . 0000
0.0000
0.0000
0 .0000
0.5
0 .0027
0,0000
0.0000
0 .0000
] .0
0 .0129
0.0000
o.ooco
0 ,0000
2.0
0 , 64 54
0,0000
0,0000
0 .0000
4 .0
0 . 0044
0.0001
0,0000
0,.0000
6 .0
0 .8191
0.0001
0.0000
0,,0000
8 .0
r\
yj
, 3211
0.0000
0.0112
0. 0000
0 .9024
0.0000
0.0085
0. 0003
24 .0
r\
. 6360
0.ncoo
0.2779
0 . 0005
48 . 0
1 .0000
1.0000
1.0000
1 0000
12,0
201
6.55 E f f e c t o f e x t e r n a l medium
The e f f e c t o f t h e e x t e r n a ] medium on t h e l o s s of Zn from RhYnchost^gi^um
was t e s t e d i n a l a b o r a t o r y batch c u l t u r e experiment.
were used: Chu lOF, + 3.15 mg
mg 1 ^ Ca; 5 mg 1
u s i n g HEPES.
The f o l l o w i n g media
EDTA; Chu lOE - EDTA; 3.15 mg 1~^ EDTA; 20
PO^-P and d e i o n i z e d water.
A i i were b u f f e r e d a t pH 7.0
Two p o p u l a t i o n s o f Rhynchostegium were used; a p o p u l a t i o n
from 0288-90, a metal e n r i c h e d stream and a p o p u l a t i o n from 0310-90. an
uncontaminated stream, which had been exposed t o 1 mg 1 ^ Zn f o r 1 h p r i o r
to the experiment.
Shoots were incubated i n the experimental media f o r
24 h.
Zn was more e a s i l y removed from the p o p u l a t i o n from 0310-90 t h a t had
been exposed b r i e f l y
6.14).
t o Zn. than from the p o p u l a t i o n from 0288-90 ( F i g .
The general order o f l o s s from the two p o p u l a t i o n s was s i m i l a r ; f o r
t h e p o p u l a t i o n from 0310-90. t h e treatment w i t h PO^-P released the l e a s t
Zn, f o l l o w e d by d e i o n i z e d water, Chu lOE -EDTA. Ca. Chu lOE ^ EDTA and
EDTA.
The order o f l o s s f o r Rhynchostegiurn
from 0288-90 was the same,
except
t h a t d e i o n i z e d water removed s l i g h t l y l e s s Zn than PO -P.
4
The g r e a t e s t d i f f e r e n c e between the two p o p u l a t i o n s was observed i n t h e
Chu lOE + EDTA ( d i f f e r e n c e = 0.40), f o l l o w e d by Chu lOE - EDTA ( 0 . 3 9 ) , Ca
( 0 . 3 5 ) . EDTA ( 0 . 3 1 ) , d e i o n i z e d water
(0.11) and PO^-P (-0.05).
202
F i g . 6.13
Loss o f Zn from Rhynchostegium i n 0310-90 a f t e r
l e n g t h s of exposure t o Zn i n 0012-28 and 0309-80.
F i g . 6.14
different
<.:i5)s|"?t - X(. s\%C)
Loss o f Zn from two p o p u l a t i o n s of Rhynchostegium i n c u b a t e d a t
15 °C i n a range o f media.
See, bt^Oc 9-of WtV(lr
dsto^.
203
3000
ro
cn
=
1h
pretreatment
=
4h
pretreatment
= 24h
pretreatment
C71
=1.
= moss
from
0 3 0 9 - 80
2000
I/)
o
e
N
1000
t i m e (h)
1 o
1.2
m c
o
—1
> o
I
D
•D
01
1.0
iOB
0)
(_
c 0.6
N
'^0.4
o
0.2
i
•
o
m c
o
-1
> o
m
o
>
o
Q
•D
I
0288
I
Tl
0310
+ Zn
204
7. ZINC ACCUMULATION BY ISOLATED CELL WALLS
7.1
INTRODUCTION
T h i s chapter r e p o r t s a s e r i e s of l a b o r a t o r y
experiments c a r r i e d o u t
i n an a t t e m p t t o examine the p o s s i b l e c o n t r i b u t i o n of t h e c e l l w a l l i n Zn
a c c u m u l a t i o n by Rhynchostegium.
Previous chapters (5 and 6 ) , noted r a p i d
uptake i n t o a phase which was r e a d i l y exchangeable;
r e p r e s e n t i n g ion-exchange
onto t h e c e l l w a l l ( 6 . 3 ) .
t h i s was i n t e r p r e t e d as
As t h i s phase was
c l e a r l y l a r g e and played an i m p o r t a n t r o l e i n t h e e a r l y stages of uptake, a
more d e t a i l e d i n v e s t i g a t i o n i n t o i t s p r o p e r t i e s
Preliminary
was c a r r i e d out.
experiments a r e concerned w i t h t h e s t r u c t u r e and gross
morphology of t h e c e l l w a l l
(7.2).
These a r e f o l l o w e d
by a d e s c r i p t i o n of
the i o n i c c o m p o s i t i o n o f t h e c e l l w a l l i n d i f f e r e n t media and n a t u r a l
waters and t h e k i n e t i c s of exchange o f Zn i n t h e presence
(7.3,
7.4).
investigated
of o t h e r ions
F i n a l l y , t h e r o l e o f Ca i n m e d i a t i n g Zn accumulation i s
(7.5).
7.2 PRELIMINARY STUDIES
7.21
U l t r a s t r u c t u r e of c e l l
Material
walls
from 0310-90 was prepared f o r e l e c t r o n microscopy t o
examine t h e f i n e s t r u c t u r e o f t h e c e l l w a l l of Rhynchostegium.
The c e l l has a very t h i c k c e l l w a l l ( F i g . 7.1), a c c o u n t i n g f o r one t h i r d
of i t s t o t a l diameter.
Two d i s t i n c t areas of t h e c e l l w a l l are v i s i b l e ; a
l i g h t e r c o l o u r e d r e g i o n c o m p r i s i n g t h e primary and secondary
cell
walls,
w h i l s t d a r k e r , wedge-shaped areas ( l a b e l l e d "U" on F i g . 7.1) appear t o be
e x t e n s i o n s of the middle l a m e l l a .
These areas may be composed o f p e c t i c
205
materials.
No p a t t e r n t h a t corresponded
t o these areas was v i s i b l e i n
s u r f a c e views o f t h e c e l l under a l i g h t microscope when a s t a i n s p e c i f i c
f o r p e c t i n s , r u t h e n i u m r e d . was used t o s t a i n u n f i x e d
7.22 R e l a t i v e
mass o f c e l l
cells.
walls
In o r d e r t o complement t h e above v i s u a l examination, t h e mass o f t h e
c e l l w a l l r e l a t i v e t o t h e mass o f t h e whole t i p was estimated a t t h e s t a r t
of t h e s t u d y , u s i n g m a t e r i a l from 0310-90.
D u p l i c a t e samples of f r e s h t i p s (from 5 t o 50 t i p s per sample) were
b l o t t e d and d r i e d a t 50 °C f o r 1 h and weighed.
One s e t of t i p s was d r i e d
a t 105 °C f o r 24 h and reweighed and t h e second s e t e x t r a c t e d
(2.43),
a t 50 °C f o r 1 h and weighed, then d r i e d a t 105 °C and weighed.
dried
The f i r s t
1 h d r y i n g p e r i o d removed water from s u r f a c e s and t h e subsequent weight i s
r e f e r r e d t o as " f r e s h w e i g h t " .
There were s i g n i f i c a n t l i n e a r c o r r e l a t i o n s between f r e s h weights and
d r y w e i g h t s f o r a l l combinations o f whole t i p s and c e l l w a l l s .
The d r y
w e i g h t s o f both whole t i p s and c e l l w a l l s were 0.93 of t h e i r f r e s h weight
(SD = 0.04 and 0.03 r e s p e c t i v e l y ) .
The f r e s h weight of t h e c e l l w a l l s was
0.77 (SD = 0.12) o f t h e f r e s h weight of t i p s and t h e i r d r y weight was 0.72
(SD = 0.12) o f t h e f r e s h weight o f t i p s .
From these data i t was p o s s i b l e t o e s t i m a t e t h e r e l a t i v e mass o f t h e
cell wall:
(f.wt
= f r e s h weight and d.wt = d r y w e i g h t ; s u b s c r i p t s
t and w r e f e r t o
whole 2-cm t i p s and c e l l w a l l p r e p a r a t i o n s r e s p e c t i v e l y . )
if
d.wt
Ul
and
then
= 0.93 f . w t
Ui
f . w t = 0.77 f . w t
hi
t
d.wt = 0.93 x 0.77 f . w t ^
w
t
d.wt
= 0.72 f . w t
w
t
206
F i g . 7.1
Transverse s e c t i o n of a l e a f c e l l o f Rhynchostegium,
C = c e l l w a l l ; M = m i d d l e l a m e l l a ; U = unknown (see 7.21).
xlOOOO.
Scale bar =
1pm
207
208
if
d . w t ^ = 0.93 f . w t ^
then
f.wt
= (d.wt
L
•
d.wt
/ 0.72) = ( d . w t .
W
= ( 0 . 7 2 / 0.93)
t
d.wt^
7.23
The n e x t
f r o m 0310-90
the c e l l
of c e l l
w a l l s : l a b o r a t o r y experiment
In order
the protonated
to obtain c e l l
state with walls at equilibrium with
w a l l s a t e q u i l i b r i u m w i t h media,
25 t i p s w e r e s h a k e n i n 50 ml o f medium f o r 1 h.
A Chu
and w i t h and w i t h o u t 20 mg
T h e r e were i n c r e a s e s
( T a b l e 7 . 1 ) ; Ca was
i n the concentrations of several of the ions
O t h e r m a j o r c o n t r i b u t o r s were Na
0.01)
SD = 0 . 0 0 5 ) .
samples
( x = 0.12,
Fe a c c o u n t e d f o r 0.21
however t h e r e were h i g h e r c o n c e n t r a t i o n s o f t h i s
( 0 . 8 2 i n 0288 and 0.81
i n 0310) i n d i c a t i n g
f o r 0.55
ions i n the w a l l a t the
end o f t h e e x p e r i m e n t .
the t o t a l ;
mg 1 " EDTA
c o n s i s t e n t l y t h e most a b u n d a n t i o n , a c c o u n t i n g
( x = 0.10,
was
1 ^ humic a c i d (HA).
f o r a l l t r e a t m e n t s ; SD = 0.02) o f t h e t o t a l
and Mg
samples
lOE medium
u s e d w i t h t h e f o l l o w i n g m o d i f i c a t i o n s ; w i t h and w i t h o u t 3.15
(mean
wall
e x p e r i m e n t examined t h e c o n c e n t r a t i o n s o f c a t i o n s i n c e l l
w a l l s and compared
of
d.wt^
77% o f t h e d r y w e i g h t .
Ionic composition
medium.
= 0.77
t h e sample o f moss c o l l e c t e d
constituted
0.93)
d.wt^
W
In
/
L
(SD
SD =
= 0.01) o f
i n control
t h a t Fe was
actually
l o s t during the incubation.
T h e r e were s i g n i f i c a n t d i f f e r e n c e s b e t w e e n t h e c o n c e n t r a t i o n s
w a l l s o f t h e two p o p u l a t i o n s f o r s e v e r a l i o n s
c o n c e n t r a t i o n o f Zn i n t h e two p o p u l a t i o n s was
all
four treatments
and K and Mn
populations f o r three of the f o u r
showed
i n the
(Table 7.2); the
significantly different for
s i g n i f i c a n t d i f f e r e n c e s between
treatments.
209
T a b l e 7.1 C o n c e n t r a t i o n s o f m e t a l s ( I n p g g
Rhynchostegium b e f o r e and a f t e r e q u i l i b r a t i o n
) i nc e l l walls of
i n v a r i o u s m e d i a , see 7 . 2 3 .
sample
treatment
Na
K
Mg
Ca
Mn
Fe
Zn
total
0288 -90
control
368
192
113
155
26
4138
38
5030
(144)
(21)
(6)
(56)
(4)
(132)
(1)
424
166
90
139
12
3857
98
(148)
(17)
(2)
(27)
(12)
(155)
(12)
2016
241
1511
8886
15
3446
104
(251)
(14)
(84)
(616)
(2)
(473)
(20)
1978
398
1604
9201
32
3409
46
(643)
(112)
(123)
(662)
(5)
(401 ) (4)
1886
256
1515
9052
16
3160
84
(423)
(51)
(64)
(506)
(2)
(667)
(22)
2271
299
1531
8607
33
3324
47
(463)
(31)
(87)
(487)
(10)
(397)
(6)
2318
260
1447
8302
15
3665
450
(334)
(31)
(27)
(199)
(2)
(450)
(11)
1830
348
1548
9060
29
3404
57
(247)
(55)
(63)
(1034)(12)
(557)
(8)
1827
172
1424
8223
13
2973
102
(136)
(24)
(41)
(261)
(2)
(737)
(17)
1847
290
1611
9059
26
3524
46
(201)
(32)
(64)
(336)
(17)
(295)
(6)
0 3 1 0 -90
0 2 8 8 -90
0 3 1 0 -90
0 2 8 8 -90
0 3 1 0 - -90
0 2 8 8 --90
0 3 1 0 - -90
0288- 90
0310- 90
control
-t-EDTA-HA
+EDTA-HA
-EDTA-HA
-EDTA-HA
-^EDTA+HA
-HEDTA+HA
-EDTA-KHA
-EDTA+HA
4786
16115
16668
15969
16112
16457
16276
14734
16403
Table 7 . 2 Comparison, u s i n g "Student" t - t e s t s , o f metal c o n c e n t r a t i o n s i n
c e l l w a l l s o f Rhynchostegium from 0 2 8 8 - 9 0 and 0 3 1 0 - 9 0 b e f o r e and a f t e r
e q u i l i b r a t i o n i n v a r i o u s media.
treatment
Na
K
conrrois
0.537
1.879
-EDTA-HA
0.110
2.781
*
EDTA- HA
1.228
1.433
4-EDTA+HA
2.349
+EDTA-HA
0.152
Mg
Ca
Mn
Fe
Zn
0.502
2. 1 7 0
2.769
10.364
] .239
0.698
5.947
***
0. 1 2 0
5.560
***
0.303
1.270
3.250
*
0.421
3.237
2.781
2.939
*
1.439
2.490
*
0.729
5.318
**
5.965
***
4.932
*«
3.926
**
1.578
1.387
6.233
**«
210
ANOVA was u s e d t o compare t r e a t m e n t s ( T a b l e 7 . 3 ) .
significant
There were
d i f f e r e n c e s w i t h i n t h e p o p u l a t i o n f r o m 0288-90 f o r K and Ca b u t
no s i g n i f i c a n t
d i f f e r e n c e s between t r e a t m e n t s f o r p o p u l a t i o n s from
The v a r i o u s t r e a t m e n t s were a l s o compared u s i n g " S t u d e n t " t - t e s t s
7.3);
s i x o u t o f 84 t r e a t m e n t s w e r e s i g n i f i c a n t ;
within
had
t h e p o p u l a t i o n f r o m 0288-90.
significant
7.24
Nature
A brief
Fe i n c e l l
the
initial
1).
and
(Table
occurred
C o n t r o l samples f r o m t h e t w o r e a c h e s
d i f f e r e n c e s b e t w e e n Mg, Fe and Zn ( T a b l e 7 . 2 ) .
o f Mn a n d Fe i n c e l l
and
a l l o f these
0310-90.
experiment
walls
rinse
wall
was c o n d u c t e d
i n 7.23.
T i p s were p r e p a r e d as u s u a l
i n hydroxylamine
They w e r e t h e n t r e a t e d
t o i n v e s t i g a t e t h e n a t u r e o f Mn
h y d r o c h l o r i d e and were p r o t o n a t e d ( s t a g e
i n hydroxylamine
h y d r o c h l o r i d e ( 1 h; stage 2)
i n 0.2 M ammonium o x a l a t e / 0.2 M o x a l i c a c i d
d a r k a t room t e m p e r a t u r e .
(2.43), omitting
( 4 h; s t a g e 3 ) , i n t h e
Samples w e r e t a k e n a f t e r
each s t a g e and a n a l y z e d
f o r Mn, Fe a n d Zn ( F i g . 7 . 2 ) .
A t t h e s t a r t t h e r e was s i g n i f i c a n t l y more Mn i n t h e c e l l
0288-90 c o m p a r e d w i t h 0310-90
removed a f t e r
contained
t h e wash
significantly
("Student"
i n hydroxylamine.
and
0310-90 a f t e r
The p o p u l a t i o n f r o m
more Fe a t t h e s t a r t
treatment with
o x a l a t e ("Student"
significantly different
treatment
("Student"
from
t = 7.15 * * * ) . N e a r l y a l l was
("Student"
Fe was removed a t each s t a g e ; however t h e r e was s t i l l
in
walls
both hydroxylamine
0310-90
t = 14.44 * * * ) .
s i g n i f i c a n t l y more Fe
("Student"
t = 6.32 * * )
t = 11.54 * * * ) . The c o n c e n t r a t i o n o f Zn was
between p o p u l a t i o n s o n l y a f t e r
the oxalate
t = 3.57 * ) ; however some Zn was removed by e a c h .
211
Table
7.3
Effect
Rhynchostegium
o f EDTA a n d h u m i c a c i d s on a d s o r p t i o n o f m e t a l s by
cell
walls;
c o m p a r i s o n s u s i n g ANOVA a n d " S t u d e n t "
t-tests.
site
comparison
Na
K
0288-90
ANOVA
2.051
6.223 2.483 3.705 1.832 1.056 1.063
**
*
"Student"
0310-90
Mg
Ca
Mn
Fe
Zn
t-tests
4EDTA-HA V
-EDTA-HA
0.527 0.563 0.059 0.418 0.579 0.699 1 383
4EDTA-HA V
+EDTAI-HA
1.445 1.125 1.469 1.803 0.151 0.668 0.887
+EDTA-HA V
-EDTA+HA
1.312 5.038 1.877 1.982 1.501 1.080 0.192
-EDTA--HA V
+EDTA-fHA
1.602 0.142 1.953 2.760 0.876 1.253 0.845
-EDTA-HA V
-EDTA+HA
0.260 2.982 2.390 2.915 2.409 0.377 1.316
*
*
+EDTA+HA V
-EDTA+HA
2.713
*
ANOVA
0.914 2.266 0.833 0.578 0.274 0.151 2.576
"Student"
*
4.515 0.931 0.484 1.644 1.602 0.771
**
t-tests
+EDTA-HA V
-EDTA-HA
0.740 1.704 0.967 1.448 0.175 0.301 0 ^44
+EDTA-HA V
+EDTA+HA
0.429 0.794 0 . 8 1 1 0.230 0.385 0.016 2.168
EDTA-HA V
-EDTAf.HA
0.388 J . 8 5 6 0.107 0.383 0.672 0.460 0.047
-EDTA-HA V
i-EDTA+HA
1.683 1.558 0.311 0.794 0.445 0.232 1.845
-EDTA-HA V
-EDTA+HA
1.681
+EDTA+HA V
-EDTAf-HA
0.109 1.833 1.408 0.016 0.335 0.381 2.015
0.398 1.485 1.530 0.701 0.806 0.252
212
Fig.
7.2
Removal o f Mn,
Fe and Zn f r o m c e l l
walls
o f Rhynchostegium.
1 = control;
2 = a f t e r treatment with
hydroxylamine
hydrochloride;
3 = a f t e r treatment with
ammonium o x a l a t e
/ oxalic
acid.
213
o
CO
600
O
o
ro
00
CD
o
o
CO K)
O
CP
o
CO
o
o
ro
00
00
I
tD CD
O
O
Mn
^00
200
7
4000
Fe
2000
c
o
c
80
0)
Zn
o
c
o
o
40
214
7.25
I o n i c c o m p o s i t i o n of c e l l w a l l s : f i e l d
The
stream
experiment
c o n c e n t r a t i o n s of c a t i o n s adsorbed from a v a r i e t y of r i v e r
waters
and
were compared u s i n g s i m i l a r m e t h o d o l o g y t o t h a t a b o v e .
Water
samples c o l l e c t e d from streams w i t h a v a r i e t y of background c h e m i s t r i e s
(Table 7.4),
filtered
ice-box u n t i l
15
°C.
Table
approximately
Protonated
7.4
t h r o u g h 0.45
cell
site
w a l l s were e q u i l i b r a t e d
cm
pH
-1 ,
i n these waters
Mg
Ca
Zn
7 .7
3 .3
13 .70
56 .9
0 .870
0091-05
159
7 .4
3 .0
2 .75
21 .7
0,.017
0102-85
124
7,.5
3,.2
2,. 14
14 .8
0..393
0288-80
214
7. 5
3..2
3..88
27 ,.3
0..191
0309-80
284
7.
,7
5.,4
6.,06
38. .5
0. 153
0310-90
122
7 .2
1. 8
1. 89
14 .,4
0. 019
f o l l o w e d a s i m i l a r p a t t e r n t o 7.23
t h e l a r g e s t p r o p o r t i o n ( x = 0.61;
= 0 . 0 0 5 ) , Na
( x = 0.04;
p r o p o r t i o n s o f Mg
and
SD
= 0.01)
SD
controls
( x = 0.25;
(Table 7.1).
(Table 7.5);
Ca
= 0 . 0 3 ) , f o l l o w e d by Mg
and
(metal
K ( x = 0.02;
SD
accounted
(x =
= 0.005).
0.06;
The
K i n w a l l s are t h e r e f o r e e l e v a t e d w i t h respect t o t h e
c o n c e n t r a t i o n s a d s o r b e d f r o m media ( 7 . 2 3 ) .
proportion
f o r 1 h.
1 ^)
718
SD
to
alkalinity
0024-20
for
up
fraction).
total
(meq
Adsorption
s t o r e d i n an
d a t a f o r s t u d y s i t e s used i n 7.24
1 ^ for filtrable
conductivity
(pS
N u c l e p o r e f i l t e r s and
2 h p r i o r t o use when t h e y were b r o u g h t
Selected environmental
c o n c e n t r a t i o n s as mg
pm
SD
= 0.03)
The
Fe a g a i n a c c o u n t e d
although t h i s
is little
for a large
different
c o n c e n t r a t i o n o f Zn a d s o r b e d was
quite
to
variable.
215
Correlations
between c o n c e n t r a t i o n s i n c e l l
walls
were computed f o r a l l o f t h e metals (Table 7 . 6 ) .
b e t w e e n most i o n s i n t h e c e l l
solution.
variables
T h e r e were
correlations
and t h e i r c o r r e s p o n d i n g i o n i n
The e x c e p t i o n s were K and Fe; no v a r i a b l e s
either.
any
walls
and a q u a t i c
Fe i n t h e c e l l
w a l l was n o t c o r r e l a t e d
other metals i n t h ec e l l
in the cell
walls
walls;
correlated
with
with the concentrations o f
o t h e r w i s e c o n c e n t r a t i o n s o f most m e t a l s
were c o r r e l a t e d w i t h s e v e r a l
others.
t h e s e c o r r e l a t i o n s a r e p r e s e n t e d as g r a p h s ; Zn i n c e l l
Some examples o f
walls
plotted
a g a i n s t Zn i n w a t e r shows a v e r y c l o s e r e l a t i o n s h i p a t a l l v a l u e s ( F i g .
7.3);
Ca i n t h e c e l l
cell
walls
were a l s o
7.26
Kinetics
w a l l and Ca i n t h e w a t e r a n d Ca i n w a t e r and Zn i n
significantly correlated
( F i g . 7.4; F i g . 7 . 5 ) .
o f exchange o f Z n ^ * f o r
T h i s was measured i n two ways: w i t h a n d w i t h o u t
strength
of
adjuster.
Samples o f 25 p r o t o n a t e d
50 mg 1 ^ Zn i n a b e a k e r .
no f u r t h e r c h a n g e .
Exchange was r a p i d
t h e u s e o f an i o n i c
t i p s were d r o p p e d i n t o 50 ml
pH was measured a t i n t e r v a l s u n t i l
Samples were s t i r r e d
continuously
i t showed
b e t w e e n measurements.
( F i g . 7.6), 95% t a k i n g place w i t h i n f o u r minutes.
A
s i m i l a r c u r v e was o b t a i n e d when 0.1 M NaNO_, was added t o t h e Zn s o l u t i o n
as a n i o n i c s t r e n g t h
adjuster
(Fig. 7.7).
T i p s from t h e f i r s t
w e r e a n a l y z e d f o r t h e i r Zn c o n t e n t ; t h e mean o f f o u r
(SD = 6 0 1 ) \ig g ^ •
pH a t s t a r t
The e x p e c t e d c o n c e n t r a t i o n
r e p l i c a t e s was 12839
was c a l c u l a t e d
= 4.89
[ H ' ] = 1.29 x l O " ^ M
pH a f t e r 15 m i n u t e s = 3.77
[ H " ] = 1.70 x 1 0 ~ ^ M
change
[ H + ] = 1.57 x lo"'^ M
i n pH
=1.12
experiment
as f o l l o w s :
216
T a b l e 7.5
Metal
equilibrated
section
composition
o f c e l l w a l l s o f Rhynchostegium
i n d i f f e r e n t n a t u r a l water
( i n pg g
samples f o r 1 h a t 15 °C.
).
See
7.25.
Ca
Mn
384
1350
(34) (55)
10242
(434)
98 4130 480 17648
(5) (300) (48)
272
1347
(63) (31)
9960
(226)
80 3300
(1) (92)
Mg
Fe
Zn
total
population
site
Na
K
0288-90
0024-20
964
(282)
0310-90
0024-20
729
(130)
0288-90
0091-05
746
(122)
400
(169)
983
10007
(41) (247)
0310-90
0091-05
711
(99)
164
(44)
934
(41)
9674 12 3759
(300) (1) (418)
0288-90
0102-85
584
(168)
368
(65)
922
(70)
8075 45 4214
302
14510
(882) (11)(377) (23)
0310-90
0102-85
587
(296)
216
(32)
882
(43)
7876
(602)
0288-90
0288-90
579
(182)
337
1014
(48) ( 7 )
0310-90
0283-90
606
(169)
268
(73)
0288-90
0309-80
530
(48)
346
1170
(43) (19)
11031
(87)
0310-90
0309-80
595
(152)
182
(9)
10771 12 3495
180
16359
(382) (0.5)(279) (11)
0288-90
0310-90
561
(47)
437
(98)
923
(30)
8740
(533)
0310-90
0310-90
579
(92)
226
(23!
832
(24)
7920
10 3 5 2 j
71 13159
(230)(0.5)(387) (14)
10055
(269)
997 10041
(25) (351)
1124
(35)
18 4176
(5) (424)
460 16148
(9)
58 16388
(6)
84
15338
(12)
13 3743 310
13627
(4) (241) (20)
31 4298
175
(14)(222) (4)
16489
12 3662
194
15780
(2) (216) (37)
26 4210
156
(8) (279) (8)
126^
33
(11)(239)
17469
52
15009
(5)
217
T a b l e 7.6
C o r r e l a t i o n s b e t w e e n w a t e r c h e m i s t r y v a r i a b l e s and
c o n c e n t r a t i o n s o f metals adsorbed
f r o m 0288-90 and 0310-90 c o m b i n e d .
cell
onto c e l l
w a l l s ; data f o r populations
Aq = aqueous m e t a l s ; cw = m e t a l s i n
wall.
variable
Na
K
cw
cw
Mg
^cw
Ca
Mn
cw
Fe
cw
Zn
cw
cw
conductivity
0.704 0.096 0.946 0.468 0.868 0.793 0.793
*
**
**
**
pH
0.313 0.102 0.816 0.655 0.477 0.144 0.691
**
total
Na
alkalinity
*
*
0.021 0.168 0.592 0.904 0.002 0.096 0.036
*
**
0.753 0.145 0.842 0.250 0.917 0.243 0.816
aq
0.699 0.106 0.933 0.424 0.882 0.251 0.810
^
*
0.0-
Mg,
'"J
0.675 0.078 0.964 0.513 0.845 0.251 0.785
*
**
**
**
Ca
0.598 0.028 0.987 0.693 0.730 0.238 0.683
*
* * * * *
*
^•3
Mn
Fe
0.754 0.148 0.830 0.229 0.920 0.238 0.829
**
**
**
**
0.428 0.030 0.151 0.083 0.007 0.114 0.060
aq
I
Zn
0.605 0.107 0.752 0.092 0.884 0.195 0.983
*
**
**
**
-4
1.57 X 10
therefore
m o l s r e l e a s e d i n t o 50 ml s o l u t i o n
1.57 x 1 0 ~ ^ / 1000 = y / 50
y = 7.85 X lO'^ m o l s
-3
+
= 7.85 X 10
mmol H
r e l e a s e d by 25.27 mg c e l l
_3
therefore
7.85 x 10
/ 25.27 = z / 1000
wall
218
Fig.
7.3
R e l a t i o n s h i p b e t w e e n Zn i n s o l u t i o n and Zn i n c e l l
Rhynchostegium.
15
7.4
R e l a t i o n s h i p b e t w e e n Ca
Rhynchostegium.
i n natural
w a t e r samples f o r 1 h a t
Cell walls
i n s o l u t i o n and Ca
suspended i n n a t u r a l
in cell
wall
of
w a t e r samples f o r 1 h a t
°C.
Fig.
7.5
R e l a t i o n s h i p b e t w e e n Ca
Rhynchostegium.
15
suspended
of
"C.
Fig.
15
Cell walls
wall
°C.
Cell wails
i n s o l u t i o n a n d Zn i n c e l l
suspended i n n a t u r a l
wall
of
w a t e r samples f o r 1 h a t
219
600
y = 4 7 i x » 80.5
=
NJ
300
200
0.2
0.4
•
=0288-90
O
=0310-90
0.5
Zn in w a t e r
0.8
(mg
11000
10000
9000
8000
7000
h
_L
10
_L
20
30
iO
50
60
Ca in water (mg 1"^)
500
O
-
400
I
o
300
S
c
N
200
100
10
20
_L
30
J
40
50
60
Co in water
(mg
("1)
I'M
220
Fig.
50 mg
Fig.
50 mg
7.6
Efflux
l"""^
7.7
of
cell
w a l l s o f Rhynchostegium
incubated i n
from
cell
w a l l s of Rhynchostegium
incubated i n
Zn.
E f f l u x o f H"^
1"-^ Zn
from
0.1 M NaNO
221
•
= + eel I w a l Is
o = - cell
walls
pH
5
10
time
(minutes)
5.0
pH
4.0
3.0
5
10
time
(minutes)
15
222
solve
f o r z t o g i v e mmols
released
p e r gram c e l l
wall
z = 0.310 mmols g"^'^
assume 2 H
if
+
are replaced
0.310 mmols g
then
H'
by 1 Zn
2+
released
0.310 / 2 = 0.155 mmols g " " Zn a r e a d s o r b e d
1 mmol Zn = 0.0654 g; t h e r e f o r e 0.155 .T.mol = 0.01014 g = 10.14 mg g'"^.
The mean e x p e c t e d c o n c e n t r a t i o n
1700) y g g
i n t h e f o u r r e p l i c a t e s was 13000 (SD =
; t h i s was n o t s i g n i f i c a n t l y d i f f e r e n t f r o m
that actually
measured i n t h e t i p s .
7.3 STOICHIQMETRY OF EXCHANGE OF ZN FOR DIFFERENT CATIONS
7.31 Exchange o f Z n ^ ' f o r
2+
Exchange o f Zn
-I
f o rH
0 t o 100 mg 1 " , u s i n g
was t e s t e d o v e r a r a n g e o f Zn c o n c e n t r a t i o n s ,
t h e same e x p e r i m e n t a l
c o n d i t i o n s as above
w i t h no i o n i c s t r e n g t h a d j u s t o r wi*fe a 1 h i n c u b a t i o n
Adsorption
(Fig.
followed saturation kinetics,
7 . 8 ) . The r a t e o f a d s o r p t i o n
5 mg 1 " Zn i n s o l u t i o n .
adsorption
typical
from
(7.23)
time.
of a Langauir
isotherm
s t a r t e d t o d e c l i n e above a b o u t
T h e r e was a l i n e a r r e l a t i o n s h i p b e t w e e n Zn
and t h e c h a n g e i n pH o b s e r v e d i n t h e s o l u t i o n o v e r t h e r a n g e 0
t o 25 mg l "'^ Zn ( F i g . 7.9; r = 0.979 * * * ) .
7.32 Exchange o f Zn
2+
+
for K
2+
and Ca
The a b o v e e x p e r i m e n t was r e p e a t e d
using K
+
and Ca
2+
+
instead of K .
C e l l w a l l s w e r e e q u i l i b r a t e d i n 0.1 M s o l u t i o n s o f KCl o r Ca(WO )
c> C:
Zn
223
Fig.
7.8
Relationship
between
cell
w a l l s of Rhynchostegium
Zn i n medium and Zn a d s o r b e d o n t o
a t 15 °C i n l a b o r a t o r y b a t c h
protonated
culture.
2Fig.
7.9
E f f e c t of increasing concentrations
cell
w a l l s o f R h y n c h o s t e g 1 uni.
o f Zn
on H
release
by
224
12000
a.
8000
o
5
N
^000
-J
20
40
60
Zn
1.6
\
I
80
100
supplied
(mg
1"^)
r
1.2
ApH
0.8
0.4
-
5000
Zn
adsorbed
10000
(yg
g"M
225
was
s u p p l i e d i n 2.5 mM HEPES a t pH 7.0.
and
Ca-form c e l l
A d s o r p t i o n o f Zn o n t o b o t h K - f o r m
w a l l s showed t h e same k i n e t i c s as 7.31 ( F i g . 7 . 1 0 ) ;
h o w e v e r t h e c o n c e n t r a t i o n s a d s o r b e d were a p p r o x i m a t e l y d o u b l e .
w e r e t e s t e d a g a i n s t b o t h L a n g m u i r and F r e u n d l i c h i s o t h e r m s ;
a g a i n s t both forms d e v i a t e d from
isotherm but f i t t e d
the remainder
( F i g . 7.11;
n o t e t h a t t h i s and
s e c t i o n u s e s pmol g ^ r a t h e r t h a n p g g
mode o f r e p r e s e n t a t i o n i s f o r m a l l y
e q u a t i o n o f enzyme k i n e t i c s
of the Michaelis-Menton
Zn a d s o r p t i o n
l i n e a r i t y when p l o t t e d on a F r e u n d l i c h
a Langmuir i s o t h e r m w e l l
of this
The d a t a
identical
t o the Lineweaver-Burke
(see A p p e n d i x 2 ) .
equation t o t h i s
This
Applying the
interpretation
i t becomes p o s s i b l e t o e s t i m a t e
t h e e q u i l i b r i u m c o n c e n t r a t i o n o f Zn a n d t h e d i s s o c i a t i o n c o n s t a n t as t h e
i n t e r c e p t and s l o p e
respectively;
K-f orm:
s l o p e = -126.9; i n t e r c e p t = 413.7 pmol g ^
Ca-form:
s l o p e = -225.7; i n t e r c e p t = 457.2 umol g
Exchange o f Zn f o r b o t h c a t i o n s had s l o p e s o f < 1.0 a n d n e g a t i v e
intercepts
( F i g . 7 . 1 2 ) , i n d i c a t i n g some r e l e a s e o f each as soon as t h e t i p s
were suspended
ina dilute
solution.
L i n e s drawn on F i g . 7.12 w i t h
o f 2.0, 1.0 and 0.5 i n d i c a t e t h e e x p e c t e d
are
released f o r every
curves
if,
respectively,
slopes
two ions
Zn i o n a d s o r b e d ; one i s r e l e a s e d f o r e v e r y one i o n
a d s o r b e d ; o r one i s r e l e a s e d f o r e v e r y t w o i o n s a d s o r b e d .
7.4
7.41
ACCUMULATION OF Zn BY CELL WALLS
Accumulation
This
experiment
over
6 h
tests
t h e a s s u m p t i o n made i n p r e v i o u s e x p e r i m e n t s
e x c h a n g e p r o c e s s e s were c o m p l e t e d
within
1 h.
Accumulation
o f Zn by
that
226
Fig.
7.10
R e l a t i o n s h i p b e t w e e n Zn i n medium and Zn a d s o r b e d o n t o
walls of RMnchosteglum
i n K- and C a - f o r m s a t 15 °C i n l a b o r a t o r y
culture.
Fig.
7.11
Double-reciprocal plot
of data presented
i n F i g . 7.10.
cell
batch
227
30000
.-
o
cn
o
• o o
• o
20000
<u
t_
o
in
-a
a
c
M
o
•
o
=
O
10000
20
K*-fornn
++
form
= Ca
60
40
Zn
80
supplied
100
(mg
1"^ )
0.008
o
cn
0.006
o
E
o
o
X)
<D
X3
t_
O
0.004
in
•D
8
a
S8
o
c
0.002
-L
0.002
0.004
0.006
1
Z n s u p p i ied (jjM)
J
0.008
0.010
228
Fig.
7.12
R e l e a s e o f K and Ca f o l l o w i n g
incubation
c o n c e n t r a t i o n s o f Zn a t 15 °C i n l a b o r a t o r y
indicate
in different
batch c u l t u r e .
d i f f e r e n t r a t i o s o f exchange o f K o r Ca f o r Zn.
Solid
lines
229
= K*
O = Co
200
h
150
U
100
h
0
h
-form
-form
cn
o
E
3-
w
o
0)
1/1
c
o
•50
100
Zn
adsorbed
200
(jjmol
300
g"^ )
230
protonated
c e l l w a l l s was
f o l l o w e d o v e r 6 h.
A l l other
c o n d i t i o n s were
as
before.
85%
of uptake a f t e r
7 . 1 3 ) , 96%
7.42
after
6 h was
1 h and
the
completed w i t h i n
remainder w i t h i n
A c c u m u l a t i o n by w h o l e t i p s and
by
cell
2
protonated
cell
a t a more r e a l i s t i c
w a l l s and
and
t i p s per
7.14
treatment
samples o f c e l l
168
h
(= 7 d ) .
1) and
after
concentration
tip.
This
weight
w a l l s and
( F i g . 7.14
at
1 h
no
A c c u m u l a t i o n by
different
but
to relate
i n 200
ml medium p l u s
c e l l w a l l s was
24 h and
after
been c o r r e c t e d
w a l l s a c c o u n t e d f o r 71%
significant
cell
cell
1 h and
more Zn
w a l l s a t 24
accumulation at
than
h was
168
168
h was
significantly
to
mg
1
^
1 h,
24
h
(Fig.
the
i n the
whole
of the t o t a l
d i f f e r e n c e between
walls after
1.0
calculated before
dry
corrected
24 h b u t a t 168
the c e l l w a l l s
h
h was
higher
not
than
significantly
A c c u m u l a t i o n by
than
the
(Fig.
s i g n i f i c a n t l y higher
f r o m a c c u m u l a t i o n a t e i t h e r 1 h o r 24 h.
t i p s at both
observations
a p r o p o r t i o n of the c o n c e n t r a t i o n
T h e r e was
( p = > 0.05)
compared i n
whole t i p s were h a r v e s t e d
whole p l a n t accumulated s i g n i f i c a n t l y
ii).
was
shoots.
i i ) r e s u l t s had
assumed t h a t c e l l
(7.22).
t o whole
A c c u m u l a t i o n by
o f z i n c as
i n order
were i n c u b a t e d
v a l u e s f o r w h o l e t i p s and
7.14
have u s e d c e l l w a l l s i n a
concentration
i n whole t i p s
made u n d e r l a b o r a t o r y c o n d i t i o n s
and
i n excess of
s t a t e t h a t w o u l d be u n u s u a l u n d e r n a t u r a l c o n d i t i o n s .
protonated
Zn
h.
o f Zn
f o u n d i n n a t u r a l c o n d i t i o n s and
A c c u m u l a t i o n o f Zn
Ten
15 m i n u t e s ( F i g .
walls
P r e v i o u s e x p e r i m e n t s a l l used c o n c e n t r a t i o n s
concentrations
the f i r s t
a t 1 h.
whole
231
Fig.
mg
1
7.13
at
by c e l l
w a l l s of Rhynchostegium
Zn a t 15 °C i n l a b o r a t o r y b a t c h c u l t u r e .
confidence
Fig.
Zn a c c u m u l a t i o n
7.14
confidence
i n 50
bars = 95%
limits.
Zn a c c u m u l a t i o n
by w h o l e
t i p s and c e l l
15 °C i n l a b o r a t o r y b a t c h c u l t u r e .
corrected
Vertical
incubated
d a t a , see 7.42
limits.
for further
w a l l s o f Rhyp.chostegium
i .= uncorrected data:
details.
Vertical
bars
ii. =
=
95%
20000
232
r
cn
in
in
o
E
10000
c
N
^
J
L
1
2
3
4
time
C =
cell
w c
5
6
(h)
wall
W = whole
w c
L
tip
wc
W C
4000
4000
3000
3000
2000
2000
W C
W C
24
168
r
cn
in
in
o
E
c
N
1000
H
1000
24
168
h
time
(h)
233
7.5
ROLE OF
7.51
CALCIUM I N CONTROLLING ZINC ADSORPTION
A c c u m u l a t i o n o f Zn
The
previous
protonated.
by c e l l w a l l s e q u i l i b r a t e d i n Ca
e x p e r i m e n t s used c e l l
In n a t u r a l waters,
w a l l s t h a t had
w h e r e Ca
and
Mg
are
solutions
been f u l l y
the dominant
t h e s e w i l l o c c u p y a l a r g e p r o p o r t i o n o f e x c h a n g e s i t e s and
in determining
the concentration
C e l l w a l l s were f i r s t
o f Zn
protonated
)
a t a range of c o n c e n t r a t i o n s .
50
of
10 mg
ml
for
both
The
limits
Ca
1
and
Zn
Zn
b u f f e r e d a t pH
mean c o n c e n t r a t i o n
w e r e 15046 pg g
a b o u t 15 mg
"'^ and
o f Zn
7.52
trend.
1
^ Ca
Competition
then
f o r 1 h.
14198
14622 pg
pg g
a d s o r b e d a t any
The
and
concentration
.
as
adsorption
solution
Ca
before
and
t h e 95%
T h e r e was
little
confidence
variation
of
in
Ca.
f o u r w e r e b e l o w ; however t h e r e
o f Ca
adsorbed increased
and
ions.
then
from
In t h i s
up
was
to
a r a n g e o f Ca
Zn
t h e r e was
Zn
s o l u t i o n s onto
experiment the c e l l
( r = 0.995 * * * ) .
a significant
adsorption, w i t h a slope
cell
walls
ions: a
concentrations.
o f Zn w e r e a d s o r b e d a t l o w
and
Ca
s u p p l i e d w i t h s o l u t i o n s o f two
f i t t e d the Langmuir i s o t h e r m
( F i g . 7.16)
and
and
o f Zn
concentrations
adsorption
g
f o r e x c h a n g e s i t e s b e t w e e n Zn and
single concentration
highest
Samples were a n a l y z e d
supplied.
walls equilibrated to different
Ca
suspended i n
equilibrium concentration
P r e v i o u s e x p e r i m e n t s examined a d s o r p t i o n
were p r o t o n a t e d
e q u i l i b r a t e d i n s o l u t i o n s of
These w e r e r i n s e d and
7.0
o f Zn was
Three samples exceeded t h e s e l i m i t s
systematic
role
( F i g . 7.15).
the c o n c e n t r a t i o n
no
play a
t h a t i s adsorbed.
and
Ca(NO
may
cations
concentrations
The
o f Ca
linear correlation
in
between
o f -0.155 ( r = 0.920 * * * ) .
234
Fig.
7.15
Interactions
between
c o n c e n t r a t i o n s o f Ca i n c e l l
a d s o r b e d Ca a n d Zn: e f f e c t o f
wall
on s u b s e q u e n t Zn a d s o r p t i o n .
i n c u b a t e d a t 15 °C u n d e r s t a n d a r d b a t c h c u l t u r e
Fig.
7.16
Interactions
between
a d s o r b e d Ca and Zn: e f f e c t o f
a t 15 °C u n d e r s t a n d a r d
batch c u l t u r e
Samples
conditions.
c o n c e n t r a t i o n s o f Ca i n -nedium on s u b s e q u e n t Zn a d s o r p t i o n .
incubated
increasing
conditions.
increasing
Samples
235
500
Ca + Z n
400
"7
300
20
40
60
80
e q u i l i b r i u m Co c o n c e n t r a t i o n
a t s t a r t ( m g 1"'')
2000
r
y = - 0 . 1 5 5 X +167/.
1500
cn
X3
0)
1000
XI
t_
o
in
o
c
N
500
2000
J
L
4000
6000
Ca a d s o r b e d
(yg g'')
100
236
8. CASE STUDY
8.1
INTRODUCTION
P r e v i o u s c h a p t e r s have d e m o n s t r a t e d
those f e a t u r e s which a f f e c t t h e
a c c u m u l a t i o n o f m e t a l s by a q u a t i c b r y o p h y t e s , p a r t i c u l a r l y w i t h r e f e r e n c e
to
t h e u s e o f moss-bags ( C h a p t e r 5 ) .
will
be used i n a s t u d y o f a r e a l
pollution.
Both
be
situation
with
intermittent
i n d i g e n o u s a n d t r a n s p l a n t e d mosses w i l l
m o n i t o r s o f Cr p o l l u t i o n
effectiveness,
I n t h i s chapter aquatic bryophytes
i n t h e R. C r o a l
heavy
be s t u d i e d as
( 0 2 6 7 ) . N-W. E n g l a n d .
compared w i t h c o n v e n t i o n a l w a t e r
metal
Their
sampling techniques,
will
considered.
8.2 OUTLINE OF STUDY
The
s t u d y was c o n d u c t e d
i nthree parts:
i. preliminary
t r a n s p l a n t o f R h y n c h o s t e g i u m b e l o w t h e H a l l C h e m i c a l Works
to
i t s p r o p e r t i e s o f metal accumulation
investigate
i i . weekly
visits,
o v e r a s i x week p e r i o d ,
t r a n s p l a n t e d moss a n d w a t e r
iii.
experiments
i n which
samples were c o l l e c t e d
i n 0267-90,
samples o f i_n s i t u a n d
from t h r e e
sites,
i n t h e l a b o r a t o r y t o c o n f i r m t h e s p e c i a t i o n o f Cr i n 0267,
8.3 FIELD STUDIES
8.31
U p t a k e o f Cr by R h y n c h o s t e g i u m i n 0267-90
U p t a k e o f Cr by R h y n c h o s t e g i u m i n t h e C r o a l was i n v e s t i g a t e d by
t r a n s p l a n t i n g a p o p u l a t i o n f r o m 0310-90 i n t o
Environmental
(Table 8.1).
0267-90 u s i n g moss-bags.
v a r i a b l e s were measured d u r i n g t h e c o u r s e o f t h e e x p e r i m e n t
237
The
concentration
3.5 pg g ^.
o f Cr i n t h e moss a t t h e s t a r t
Accumulation
continued throughout t h e experiment
w i t h no e v i d e n c e o f an a s y m p t o t e
for
the f i r s t
(yg
g • " ) / concentration
this,
of t h e experiment
b e i n g approached.
12 h ; t h e c o n c e n t r a t i o n
i n water
ratio
(mg 1
The c o n c e n t r a t i o n
T a b l e 8.1
oxygen
)) at this
p o i n t was 286.
o f Cr i n R h y n c h o s t e g i u m showed
temp.
significant
total alkalinity
(0.963
(-0.962 * * ) .
pH
cond.
Ca
i n 0267-90.
Cr
start
(h)
After
r a t i o a t 24-h was
Environmental v a r i a b l e s d u r i n g t r a n s p l a n t experiment
time from
slow
( c o n c e n t r a t i o n i n moss
c o r r e l a t i o n s w i t h c o n d u c t i v i t y ( r = 0.934 * * )
* * ) and d i s s o l v e d
( F i g . 8.1),
A c c u m u l a t i o n was
a c c u m u l a t i o n was more r a p i d and t h e c o n c e n t r a t i o n
1260.
was
(°C)
(pS cm'^)
(mg
T^)
(mg
T^)
0
14.0
7.6
405
33.9
0.037
1
M.3
7.3
397
25.9
0.033
2
14.5
7.4
388
30.3
0.028
3
14.9
7.5
381
36.3
0.035
4
15.3
7.6
378
31.1
0.041
5
15.6
7.5
381
38.4
0.046
6
16.0
7.6
379
38.7
0.049
8
16.3
7.5
382
37.2
0.051
10
16.3
7.5
388
37.3
0.047
12
16.4
7.5
393
38.1
0.056
18
15.6
7.3
423
39.5
0.040
24
15.6
7.5
421
39.6
0.049
238
Fig.
8.1
June
19S5.
.Accumulation
Vertical
o f Cr by R h y n c h o s t e g i u m
b a r s = 95"?!; c o n f i d e n c e
transplanted
limits.
into
0267-90 i n
239
70
r
10
12
U
16
18
20
time (h)
22
2U
240
8.32
Cr I n moss o v e r s i x - w e e k
8.321
Indigenous
period
moss
Samples o f t h e most a b u n d a n t I n d i g e n o u s s p e c i e s a t 0267-80 and
were c o l l e c t e d o v e r a six-week
period.
These s p e c i e s were
a n t i p y r e t l c a a t 0267-80 and A m b l y s t e g i u m
Fontlnalls
r i p a r i u m a t 0267-90.
Aqueous Cr was d e t e c t a b l e a t 0267-90 u s i n g f l a m e AAS,
six
on f o u r o f t h e
o c c a s i o n s . The maximum c o n c e n t r a t i o n r e c o r d e d was 0.047 mg
Cr was d e t e c t e d a t 0267-80
0267-90
l '^.
No
( F i g . 8.2).
D e s p i t e l o w Cr c o n c e n t r a t i o n s i n t h e w a t e r , Cr was d e t e c t e d i n
F o n t i n a l i s on t h r e e o f t h e s i x s a m p l i n g o c c a s i o n s .
c o n c e n t r a t i o n r e c o r d e d was 50 pg g
Amblystegium
on a ] ] o c c a s i o n s
.
The m a x i mum
Cr was d e t e c t e d i n i n s i t u
a t 0267-90.
T h i s i n c l u d e d one o c c a s i o n when
no Cr was d e t e c t a b l e i n w a t e r samples and once when i t was a t d e t e c t i on
limit
pg g
( 0 . 0 2 0 mg
^.
1
).
The c o n c e n t r a t i o n s r a n g e d
f r o m 130 p g g
t o 298
T h e r e was no d i r e c t c o r r e l a t i o n w i t h t h e c o n c e n t r a t i o n s measured
in
the water.
The c o n c e n t r a t i o n s measured on t h e o c c a s i o n s
or
below d e t e c t i o n
limit
when Cr was a t
i n t h e w a t e r were b o t h h i g h e r t h a n t h e h i g h e s t
c o n c e n t r a t i o n m e a s u r e d a t 0267-80.
On 2 5 t h J u l y , one o f t h e two d a t e s
no Cr was measured i n t h e w a t e r , t h e c o n c e n t r a t i o n i n A m b l y s t e g i u m
when
was t h e
h i g h e s t r e c o r d e d a t any p o i n t d u r i n g t h e s t u d y .
8.322 T r a n s p l a n t e d moss
.Moss-bags were p u t o u t on a l l o c c a s i o n s b u t were o n l y r e c o v e r e d
three occasions
detected
8.2).
a t 0267-80, and n o t a t a l l a t 0267-90.
A t 0267-80, Cr
i n t r a n s p l a n t e d R h y n c h o s t e g i u m on each o f t h e s e o c c a s i o n s
C o n c e n t r a t i o n s ranged
c o n c e n t r a t i o n s were s l i g h t l y
on
f r o m 43.3 pg g ^ t o 35.8 pg g
was
(Table
; these
lower than c o r r e s p o n d i n g c o n c e n t r a t i o n s i n
241
indigenous
with
F o n t i n a l l s b u t do show s i g n i f i c a n t
the concentration
i n 0310-90 ( 1 . 9 p g g ^ ; SD = 0 . 2 ) .
T a b l e 8.2 C o n c e n t r a t i o n
date
o f Cr i n w a t e r and i n mosses a t 0267-80.
concentration
concentration
in
Fontinalis
Rhynchostegium
(mg 1 ^ )
(in situ)
(transplanted)
25.7.85
< 0.010
50.0
(SD = 4.7)
43.3 (SD = 2.8)
1.8.85
< 0.010
44.0
(SD = 5.4)
35.8 (SD = 2.9)
< 25.0 (SD = 1.2)
37.0 (SD = 6.4)
8.8.85
water
0.021
T h e r e was no i n d i g e n o u s
were r e c o v e r e d
although
on a l l o c c a s i o n s .
the concentrations
concentrations
8.323
On each o c c a s i o n ,
however, t h e s e bags had
substratum.
Consequently,
o f Cr i n t h e w a t e r were v e r y h i g h , t h e
i n t h e t r a n s p l a n t e d moss were l o w ( F i g . 8 . 3 ) .
C o m p a r i s o n w i t h N.W.W.A. d a t a
The N.W.W.A. c o J l e c t e d
was a p p r o x i m a t e l y
highest
Cr c o n t e n t
1 h prior
a r o u t i n e w a t e r sample on 25.7.85 a t 1115; t h i s
to the collection
found d u r i n g t h e study.
t h e moss sample had a l o w c o n c e n t r a t i o n
1 h earlier
between
state
i n moss ( p g g~ )
moss i n B l a c k s h a w B r o o k ; however t h e moss-bags
been c o v e r e d b y t h e s h i f t i n g sand and s i l t
for
a c c u m u l a t i o n o f Cr compared
concentration
0.030 mg 1 ''^ Cr.
( 0 . 0 1 0 mg 1
c o n c l u s i v e l y whether t h i s elevated
the high
Water samples c o l l e c t e d w i t h
o f Cr ( < 0.020 mg 1 ^ ) ; however
t h e N.W.W.A. sample c o n t a i n e d
t h e two samples i s s m a l l
o f t h e moss sample w i t h t h e
The d i f f e r e n c e
) and i t i s n o t p o s s i b l e t o
Cr c o n c e n t r a t i o n was
i n Amblystegium r i p a r i u m .
responsible
242
Fig.
8.2
during
Concentrations
six-week
confidence
o f Cr i n w a t e r
study period
limits.
a n d mosses
i n 0267-80 a n d 0267-90
i n J u l y and A u g u s t 1985.
Vertical
bars
= 95%
243
cn
Fontinalis.
200
0 2 6 7 - 80
=1.
100
O
0
^
0.10
^
0.05
-
water,
0~
o
O
^00
U
300
3
200
O
o
0 2 6 7 - 80
cr D.L.
Amblystegium.
0267-90
100
D.L.
T
0.10
water.
02 6 7 - 9 0
cn
^
0.05
D.L.
i_
o
CD
00
ID
CD
244
Fig.
8.3
during
Concentrations
six-week
o f Cr
study period
i n water
i n J u l y and
and
in transplanted
August
1985.
moss i n 0378-90
245
cn
3
Rhynchostegm m
transplant
300
to
o
^
200
c
100
1.0 ,-
water
1
CD
E
CD
a
0.5
c
t_
o
LO
00
in
CO
CN
in
LO
00
5
i n
CO
CN
LO
00
CD
in
246
8.4 LABORATORY EXPERIMENTS
8.41
Introduction
The
v a r i o u s o x i d a t i o n s t a t e s o f Cr a r e known t o behave b i o l o g i c a l l y i n
v e r y d i f f e r e n t ways.
p r e l i m i n a r y experiments
accumulation
in
Initial
Cr(VI)
were c o n d u c t e d i n t h e l a b o r a t o r y t o compare
found
and C r ( V I ) .
o f Cr f r o m
10 mg 1
solutions of C r ( I I I )
(as CrK(S0^)2)
( a s KCrO^) was f o l l o w e d o v e r a 24-h p e r i o d .
Accumulation
o f b o t h f o r m s o f Cr c o n t i n u e d
T h e r e was a d i f f e r e n c e o f a p p r o x i m a t e l y
throughout
t h e experiment.
two o r d e r s o f magnitude between t h e
c o n c e n t r a t i o n s o f t h e two forms ( F i g . 8.4).
had
t h e r e f o r e , some
uptake r a t e s i n t h el a b o r a t o r y
Accumulation
and
experiments,
by R h y n c h o s t e g i u m o f t h e t w o f o r m s o f Cr m o s t commonly
nature, C r ( I I I )
8.42
In addition to thef i e l d
a c c u m u l a t e d 2600 p g g ''^ o f Cr, w h i l s t
A f t e r 24 h t h e moss i n C r ( I I I )
that
i n C r ( V I ) had accumulated
-1
a b o u t 20 p g g " .
3.8 f o r C r ( I I I )
8.43
( r = 0.930 * * * )
6 h t h e slopes
results
and 0.20 f o r C r ( V I )
( r = 0.810 * * ) .
i n 8.42 show a l a r g e d i f f e r e n c e i n t h e c o n c e n t r a t i o n s o f
two f o r m s o f Cr a c c u m u l a t e d by R h y n c h o s t e g i u m o v e r 24 h.
unknown how t h i s
r e l a t i o n s h i p continued,
c o n c e n t r a t i o n s expected
i n the f i e l d ) ,
0.01
As i t was
e i t h e r b e l o w 1 mg 1 ( i . e .
o r a b o v e , an
conducted using concentrations of C r ( I I I )
and C r ( V I )
experiment
was
from
- 10.0 mg T"^ .
R h y n c h o s t e g i u m was i n c u b a t e d
of
( = Cr i n moss / t i m e ) were
U p t a k e o f Cr a t d i f f e r e n t aqueous Cr c o n c e n t r a t i o n s
The
the
Over t h e f i r s t
Cr(III)
treatment.
o r C r ( V I ) f o r 24 h.
i n media c o n t a i n i n g a known c o n c e n t r a t i o n
T h e r e were f o u r r e p l i c a t e s o f each
247
As i n 8.42, h i g h e r c o n c e n t r a t i o n s o f Cr w e r e f o u n d
incubated
i n C r ( I I I ) than
i n that
c o n c e n t r a t i o n range s t u d i e d .
8.5)
incubated
i n t h e moss
I n Cr(VI),
When c o n s i d e r e d
throughout t h e
on a l o g ^ ^ / l o g ^ Q
t h e r e l a t i o n s h i p between t h e c o n c e n t r a t i o n s o f metal
moss was l i n e a r
f o r both chemical
basis (Fig.
i n medium a n d i n
forms.
8.5 DISCUSSION
8.51
Introduction
The
and
case s t u d y r e p r e s e n t s a r e l a t i v e l y
self-contained unit of results
i t i s t h e r e f o r e r e l e v a n t t o i n c l u d e some d i s c u s s i o n o f t h e s e
F u r t h e r comments o f a more g e n e r a l n a t u r e a r e p r e s e n t e d
discussion
8.52
results
P a r t o f t h e o b j e c t o f t h e s t u d y was t o i n v e s t i g a t e
predict
i n t h e main
(9.4).
Comparison o f l a b o r a t o r y and f i e l d
accumulation
here.
differential
o f C r ( I I I ) and C r ( V I ) i n t h e l a b o r a t o r y a n d , f r o m
t h e chemical
s p e c i a t i o n o f Cr i n t h e C r o a l .
this,
In thelaboratory
t h e r e was a marked d i f f e r e n c e
i n t h e c o n c e n t r a t i o n s o f t h e two f o r m s o f Cr
a c c u m u l a t e d by R h y n c h o s t e g i u m
(8.4).
concentrations approximately
(Fig.
8.5);this
C r ( I I I ) was a c c u m u l a t e d t o
one o r d e r o f m a g n i t u d e g r e a t e r t h a n
greater accumulation
Cr(VI)
o f C r ( I I I ) meant t h a t r e l a t i v e l y l o w
a q u e o u s c o n c e n t r a t i o n s ( < 0.01 mg 1 " ) w e r e r e a d i l y d e t e c t e d w i t h o u t use
of
t h e graphite furnace or preconcentration
The
techniques.
c o n c e n t r a t i o n s o f aqueous Cr d u r i n g t h e f i e l d
s t u d y were
l o w b u t i t was r e a d i l y a c c u m u l a t e d by R h y n c h o s t e g i u m t o a
c o n c e n t r a t i o n o f 63 p g g
magnitude, b u t s t i l l
( F i g . 8.1).
significantly
lower
This
relatively
final
i s i n t h e same o r d e r o f
t h a n t h e c o n c e n t r a t i o n s measured
248
Fig.
8.4
Accumulation of C r ( I I I )
15 °C i n l a b o r a t o r y b a t c h
culture.
and C r ( V I ) b y R h y n c h o s t e g i u m
o v e r 24 h a t
249
20
10
CD
Cr
VI
Cr
i n
12
time
24
(h)
!2 3000
o
E
o
2000
1000
-
10
250
Fig.
8.5
Accumulation
of C r ( I I I )
and C r ( V I ) by R h y n c h o s t e g i u m
at different
a q u e o u s c o n c e n t r a t i o n s o f Cr a t 15 "C i n l a b o r a t o r y b a t c h c u l t u r e .
b a r s = 95%
confidence
limits.
Vertical
251
10.000
1000
r = 0.79
Cr VI
-
r = 0.88
cn
(/)
o
E
100
o
_L
o
o
O
o
Cr in m e d i u m
(mg I
)
«
252
In
t h e l a b o r a t o r y a t s i m i l a r aqueous c o n c e n t r a t i o n s o f C r ( I I I )
( F i g . 8.5).
There were, however, c o n s i d e r a b l e d i f f e r e n c e s between c o n d i t i o n s i n t h e
l a b o r a t o r y and i n t h e f i e l d .
calcium
The
and n u t r i e n t c o n c e n t r a t i o n s i n t h e f i e l d
field
r e s u l t s support
the Croal
Cr(VI)
These i n c l u d e l o w e r
is Cr(III),
a hypothesis
although
(Gemmell, 1973).
This
temperatures
and h i g h e r
( T a b l e 3.10; T a b l e 3 . 1 1 ) .
t h a t t h e p r e d o m i n a n t f o r m o f Cr i n
t h e n a t u r e o f Cr i n t h e o r i g i n a l w a s t e was
i s p a r t l y e x p l a i n e d b y t h e mechanism o f
r e c l a m a t i o n u s e d , w h i c h i n c l u d e d a d d i t i o n o f FeSO^ t o r e d u c e C r ( V I ) t o t h e
less toxic
Cr(III)
encountered
Cr(III)
form
(Gemmell, 1974; 1 9 7 7 ) .
i n n a t u r a l waters
(Cranston
Both forms a r e f r e q u e n t l y
& M u r r a y , 1980) and o x i d a t i o n from
back t o C r ( V I ) i s slow ( C o t t o n & W i l k i n s o n ,
1972; E a r l e y & Cannon,
1965) .
8.53
Comparison o f a q u a t i c and accumulated
The
r e l a t i o n s h i p b e t w e e n aqueous c o n c e n t r a t i o n s a n d c o n c e n t r a t i o n s i n
aquatic bryophytes
other metals
1983a).
h a s n o t been e s t a b l i s h e d f o r Cr i n t h e same way as f o r
( W h i t t o n e t a l . , 1982; Say & W h i t t o n ,
intermittent pollution
scatter plot
then
This
concentrations
i n the river,
Cr a n d o t h e r
expected
ina
When p l a n t s a r e u s e d as m o n i t o r s o f
s u c h r e l a t i o n s h i p s do n o t h o l d and a b i v a r i a t e
o f aqueous Cr v Cr i n moss d u r i n g t h i s
relationship.
i s more l i k e l y
study
t o be a r e f l e c t i o n
shows no
on t h e f l u c t u a t i n g
r a t h e r t h a n a f u n d a m e n t a l d i f f e r e n c e between
metals.
There i s evidence o f s l i g h t
occasions,
1983; Wehr & W h i t t o n ,
Such w o r k g i v e s an i n d i c a t i o n o f t h e a c c u m u l a t i o n
g i v e n aqueous c o n c e n t r a t i o n o f m e t a l .
Cr c o n t a m i n a t i o n
b o t h m i s s e d b y f l a m e AAS
have o r i g i n a t e d
site
concentrations
( F i g . 8 . 2 ) . Such p u l s e s
f r o m one o f s e v e r a l s t o r m
(D.J. Holland,
pers.
a t 0267-80 on t w o
as t h e s e
may
sewer o v e r f l o w s u p s t r e a m o f t h e
comm.) and i t w o u l d h a v e been p o s s i b l e a t t h e
253
t i m e t o use
pollution,
in situ a q u a t i c b r y o p h y t e s
as has
t o t r a c e the source
been shown p r e v i o u s l y (Say & W h i t t o n ,
of
the
1983;
Mouvet,
1984) .
0 2 6 7 - 9 0 i s d o w n s t r e a m o f t h e H a l l C h e m i c a l Works; h o w e v e r i t was
a l w a y s p o s s i b l e t o d e t e c t Cr
i n the water.
On
two o c c a s i o n s
c o n c e n t r a t i o n o f Cr was
below t h e d e t e c t i o n l i m i t
concentrations recorded
i n t h e moss on b o t h o c c a s i o n s
higher than
explained
i n moss f r o m 0267-80 ( F i g . 8 . 2 ) .
i n terms of d i f f e r e n t accumulation
F o n t i n a l i s a c c u m u l a t e d more Zn
Wehr & W h i t t o n ,
then
1 9 8 3 b ) and
i t i s reasonable
i f t h e Cr
occasion
and
Cr
prior
T h i s d i f f e r e n c e cannot
r a t e s by
(Wehr,
this
i s evidence
t h e N.W.W.A. one
This i s the o n l y reason
( 0 . 0 3 0 mg
i n t h e moss.
other
t o these
to
The
water
samples d i d
1 "^ ) b u t t h i s
was
p r o b a b l y n o t h i g h enough t o a c c o u n t f o r a l l o f t h e a c c c u m u l a t i o n
i n Amblystegium
The
of a p e r i o d of h i g h c o n c e n t r a t i o n s
hour p r e v i o u s
e l e v a t e d c o n c e n t r a t i o n o f Cr
1983;
Cr(III)
s i g n i f i c a n t l y h i g h e r t h a n on any
a c c o u n t f o r t h e h i g h e r Cr c o n c e n t r a t i o n s r e c o r d e d
c o n t a i n an
species.
i s i n the form of c a t i o n i c
t o t h e sample b e i n g c o l l e c t e d .
s a m p l e c o l l e c t e d by
t h e two
be
t o presume t h a t t h i s h o l d s t r u e a l s o f o r Cr.
on 2 5 t h J u l y was
of
the
significantly
p e r u n i t mass t h a n A m b l y s t e g i u m
c o n c e n t r a t i o n o f Cr
( F i g . 8.2)
the
f o r f l a m e AAS;
were
not
observed
(8.323).
More q u a n t i t a t i v e c o n c l u s i o n s c o u l d have been d r a w n , h o w e v e r , i f t h e r e
w e r e more d a t a on
Such d a t a
Cr
i s more d i f f i c u l t t o c o l l e c t due
encountered
Whitton
water
t h e r e l a t i o n s h i p b e t w e e n aqueous and
and
(1983a),
s a m p l e s and
d a t a were a n a l y z e d
i t was
excluded
from
d u r i n g the present
Cr.
t o the lower c o n c e n t r a t i o n s of
t o t h e s c a r c i t y o f s i t e s o f Cr
f o r e x a m p l e , d e t e c t e d Cr
accumulated
pollution.
Wehr &
i n o n l y 27 o u t o f 105
their
"total"
subsequent a n a l y s i s .
s t u d y and
t h e r e was
a
These
significant
254
p o s i t i v e r e l a t i o n s h i p between l o g
10
Cr i n w a t e r
( C r ) and l o g , . Cr i n
aq
10
2-cm t i p s o f R h y n c h o s t e g i u m ( C r ^ ^ ^ ) w i t h t h e f o l l o w i n g
^°SlO ^ ^ i p =
(I^^IO
F = 25.20 ***
parameters:
^^aq) * 3
r = 0.782 ***
C a l c u l a t i o n o f p r e d i c t e d values
i n Wehr and W h i t t o n ' s
Cr a q = 0.020 mg 1
i s n e c e s s a r i l y crude because t h e d a t a
(1983a) study concerns Rhynchostegium o n l y .
t h e expected c o n c e n t r a t i o n
At t h e
i n Rhynchostegium i s
13 pg g ^ w h i c h i s an o r d e r o f m a g n i t u d e l o w e r
than
concentrations
measured i n A m b l y s t e g i u m i n 0267-90 b u t c o r r e s p o n d s t o t h e l o w e s t
concentrations
measured i n F o n t i n a l i s a t 0267-80.
An i n c r e a s e
i n Cr
of
ac
0.010 mg 1
tips
t o 0.030 mg 1 ' causes an i n c r e a s e
t o 20.5 pg g ' . s t i l l
i n Amblystegium.
an o r d e r
I ti s unlikely
i n the concentration i n
of magnitude lower
than
concentrations
t h a t t h e aqueous c o n c e n t r a t i o n s
.measured
by N.W.W.A. ( 8 . 3 2 3 ) w o u l d have been r e s p o n s i b l e
f o rthe high
in
been a p e r i o d o f h i g h e r
t h e moss ( 2 9 8 p g g~^)
and t h e r e had p r o b a b l y
concentrations
prior
to c o l l e c t i o n
concentrations
p r e d i c t e d by t h e above e q u a t i o n
i n Rhynchostegium i s a p p r o x i m a t e l y
o f t h e samples.
concentrations
measured
0267-80 i n d i c a t e p e r i o d s
0.070 mg 1 ~ ' .
The aqueous
t o g i v e such a c o n c e n t r a t i o n
0.300 mg 1 ', d o u b l e t h e h i g h e s t
c o n c e n t r a t i o n measured by Wehr and W h i t t o n
may be e x p e c t e d a t p e r i o d s
concentrations
(1983a) b u t i n t h e range
o f l o w f l o w i n 0267 ( F i g . 3 . 1 0 ) .
that
The
i n F o n t i n a l i s and t r a n s p l a n t e d R h y n c h o s t e g i u m a t
w i t h aqueous c o n c e n t r a t i o n s
o f 0.030 nig 1
to
255
9.
9.1
DISCUSSION
INTRODUCTION
This chapter discusses fundamental
and
will
metal
9.2
relate
t h e s e t o t h e use
and a p p l i e d a s p e c t s o f t h e
results
o f a q u a t i c mosses as m o n i t o r s o f h e a v y
pollution.
GROlfTH EXPERIMENTS
9.21
Introduction
The
s u c c e s s f u l use
o f a moss f o r m o n i t o r i n g p u r p o s e s r e q u i r e s
c o n s i d e r a b l e i n f o r m a t i o n on
has
gone some way
i t s growth b i o l o g y (1.6).
to provide this
will
be d i s c u s s e d
9.22
Seasonality i n growth r a t e of
f o r Rhynchostegium.
The
present study
Some a s p e c t s o f
here.
Rhynchostegiuo
A l t h o u g h t h e r e were d i f f e r e n c e s between t h e g r o w t h r a t e i n t h e
streams
growth
studied
occurred i n late
t h r e e of t h e f o u r streams
t h e r e was
s p r i n g and
but
The
temperatures
temperature
e f f e c t and
( S m i t h , 1978;
i s t h e r e f o r e more t h a n j u s t
Wehr &
a
this
i n d i c a t e t h a t t h e r e i s some p h e n o l o g i c a l c o n t r o l
w-.thin t h e p l a n t s i m i l a r t o t h a t i n F o n t i n a l i s d a l e c a r l i c a
Time-series analysis, using cross-correlations
evidence
which
r e l a t i v e l y warm
t h e c o i n c i d e n c e o f g a m e t o p h o r e f o r m a t i o n and
i n c r e a s e d g r o w t h r a t e may
little
(4.4)
Watson, 1982;
a t t h i s t i m e were s t i l l
becoming c o o l e r ( F i g . 4.2); t h i s
Peak
e a r l y summer, a l t h o u g h i n
a s e c o n d peak i n O c t o b e r
c o i n c i d e d w i t h the f o r m a t i o n of capsules
1983b).
four
( c h a p t e r 4 ) i t i s p o s s i b l e t o make a f e w comments.
i n a l l streams
Whitton,
this
o f a l a g b e t w e e n a v a r i a b l e and
(Glime,
(Table 4.10),
1984).
showed
i t s e f f e c t on t h e r a t e o f
256
g r o w t h e x c e p t i n 0310-90 w h e r e t h e t h r e e t e m p e r a t u r e v a r i a b l e s a l l had
l a g o f one m o n t h i n t h e i r e f f e c t .
The
at
e f f e c t of temperature i t s e l f
a range
is interesting.
o f t e m p e r a t u r e s , f r o m 3.1
"C
made i n F e b r u a r y when t e m p e r a t u r e s had
some g r o w t h was
observed
t o 12.3
dropped
a t each o c c a s i o n .
"C
O b s e r v a t i o n s w e r e made
(no measurements w e r e
t o 0.6
T h e r e was
°C i n 0091-05) and
a
significant
r e l a t i o n s h i p b e t w e e n g r o w t h r a t e and w a t e r t e m p e r a t u r e w i t h i n t h i s
(Fig.
4.12)
and
air
t e m p e r a t u r e o f -1.7
variations
in light
w i n t e r months.
compared w i t h
1967;
t h a t Drepanocladus
1 °C
this
still
had
( F o r n w a l l & Glime,
but i s not unusual
glomerata
(Whitton,
f o r an a q u a t i c b r y o p h y t e ;
i n b o t h C a l l i e r g o n sarmentosum
and
indicates
the t h e o r e t i c a ] capacity t o r e s p i r e at 0
1982).
low
°C.
c o l d - a d a p t e d t o t e m p e r a t u r e s of between 0
T h e r e was
no e v i d e n c e o f a
temperature
s t u d y ( F i g . 4 . 1 3 ) ; maximum g r o w t h r a t e s h a v e been
f o r a q u a t i c b r y o p h y t e s a t t e m p e r a t u r e s w e l l above t h o s e f o u n d i n
ability
explain
(Wehr, 1983;
Glime,
1982;
Maberley,
i n N-E.
1985a;
1985b).
o f R h y n c h o s t e g i u m t o s u r v i v e and grow a t l o w
i t s p e r s i s t e n c e t h r o u g h w i n t e r months.
England.
1981)
temperatures
In a previous study
Wehr & W h i t t o n , 1983b) e s t i m a t e s w e r e made o f
a b u n d a n c e ( s e n s u Holmes & W h i t t o n , 1977;
sites
the
°C and b a c k - e x t r a p o l a t i o n o f h i s r e s u l t s
s t u d y ( S a n f o r d , 1979:
The
may
1984)
F o n t i n a l i s d u r i a e i was
ceiling during this
observed
a l s o be a t a minimum d u r i n g
f o r growth of Cladophora
(1980. ) n o t e d r e s p i r a t i o n
Similarly,
"C and a mean w e e k l y maximum
Minimum t e m p e r a t u r e s f o r g r o w t h o f R h y n c h o s t e g i u m a r e
sp. a t 1.2
would
Such a h y p o t h e s i s p a y s no r e g a r d t o s e a s o n a l
those r e q u i r e d
Drepanocladus
and
°C.
q u a l i t y which would
Cambridge e t a l . ,
Priddle
range
e x t r a p o l a t i o n of the r e g r e s s i o n i n d i c a t e s t h a t growth
c e a s e a l t o g e t h e r a t a w a t e r t e m p e r a t u r e o f 1.7
a
relative
o f a q u a t i c mosses a t s e v e n
A t a l l s i t e s R h y n c h o s t e g i u m showed l e s s
t h a n a number o f o t h e r a q u a t i c b r y o p h y t e s
( i n c l u d i n g Amblystegium
seasonality
riparium
257
and F o n t i n a l l s a n t i p y r e t i c a ) a n d m a c r o p h y t i c a l g a e
S t i g e o c l o n i u m t e n u e and Lemanea f l u v i a t i l i s ) .
9.23 D i f f e r e n c e s b e t w e e n
(4.46).
t h i s was b o t h t h e l o w e s t
The g r o w t h
gron'th r a t e s o f R h y n c h o s t e g i u m
r a t e was f a s t e s t
i n 0309-80 ( 4 . 4 4 ) ;
( T a b l e 3.2) and t h e warir.est ( F i g . 4.3) o f t h e f o u r
s t r e a m s a n d , w i t h 0 3 1 0 - 9 0 , t h e l e a s t shaded ( T a b l e 4 . 3 ) .
have c o n t r i b u t e d t o t h e r e l a t i v e l y
concentrations of nitrate
o t h e r streams
(4.45)
which
These f a c t o r s
l o w compared w i t h
The s l o w e s t r a t e o f g r o w t h was f o u n d
was t h e h i g h e s t
i n 0310-90
( T a b l e 3 . 2 ) , c o o l e s t ( F i g . 4.3) and most
a l t h o u g h i t had h i g h e r c o n c e n t r a t i o n s o f n u t r i e n t
t h a n 0309-80 ( T a b l e 4 . 4 ) .
may
r a p i d r a t e o f g r o v j t h ; hovjever t h e
and p h o s p h a t e were r e l a t i v e l y
(Table 4.4).
exposed stream,
glomerata,
streams
There were s e v e r a l d i f f e r e n c e s i n monthly
between streams
(Cladophora
I n 0091-05 a l o w g r o w t h
low c o n c e n t r a t i o n s o f n u t r i e n t s
(4.42).
anions
r a t e was a s s o c i a t e d
with
The l o w e s t c o n c e n t r a t i o n s o f t h e s e
were measured i n 0091-05, a s s o c i a t e d w i t h a f a i r l y
low r a t e o f growth
(4.42) .
A f u r t h e r f a c t o r which
streams i s d e s i c c a t i o n .
may c a u s e d i f f e r e n t i a l g r o w t h
The d e p t h
o f water
r a t e s between
at the sites varied
c o n s i d e r a b l y a n d some p o p u l a t i o n s s p e n t more t i m e o u t o f t h e w a t e r
others.
R h y n c h o s t e g i u m h a s been shown t o be r e l a t i v e l y
emersion
(Penuelas,
growth
rates with
1984) and t h i s
r a t e o f growth
study
may e x p l a i n t h e c o - i n c i d e n c e
o f low
species
o f R h y n c h o s t e g i u m i s much s l o w e r
number o f o t h e r a q u a t i c p l a n t s .
during this
tolerant to
l o w f l o w s i n t h e summer.
9.24 C o m p a r i s o n w i t h o t h e r
The
than
(4.44)
than recorded
The maximum r a t e o f 2.31 mm wk ~
i s v e r y l o w compared w i t h g r o w t h
a n g i o s p e r m s s u c h as H y d r l l l a v e r t i c i l l a t a
(Barko,
1982),
ina
recorded
rates of aquatic
Elodea
nuttallii
258
( K u n i i , 1 9 8 4 ) , R a n u n c u l u s f l u i t a n s ( E i c h e n b e r g e r & W e i l e n m a n n , 1982) and
P o t a m o g e t o n p e c t l n a l u s ( H o w a r d - W i l l i a m s , 1978) and a l g a e such as C l a d o p h o r a
( W h i t t o n , 1967; C a m b r i d g e e t a l , . , 1984) and E n t e r o m o r p h a f l e x u o s a ( A l l e n ,
1970).
The r a t e i s , h o w e v e r , c o m p a r a b l e w i t h t h o s e f o r a v a r i e t y o f o t h e r
bryophytes.
T h e r e a r e p r o b l e m s in ."naking d i r e c t c o m p a r i s o n s w i t h t h e s e
r e c o r d s p a r t l y b e c a u s e a l a r g e number o f s u c h r e c o r d s a r e based on s i n g l e
a n n u a l nieans r a t h e r t h a n t h e more d e t a i l e d s t u d y c o n d u c t e d h e r e .
Such
f i g u r e s h a v e t o be a d j u s t e d b e f o r e a c o m p a r i s o n may be made. Clymo ( 1 9 7 0 )
n o t e s l a r g e d i f f e r e n c e s i n t h e r a t e o f g r o w t h o f Sphagnum b e t w e e n l o w l a n d
and u p l a n d s i t e s i n G r e a t B r i t a i n and s l o w r a t e s have been r e c o r d e d f o r
Racomitrium lanuginosum a t upland s i t e s ( T a l l i s , 1959), P o l y t r i c h u m
( L o n g t o n , 1970) and D i c r a n u m ( a s C h o r i s o d o n t i u a ; B a k e r , 1972) i n A n t a r c t i c a
and Sphagnum i n A l a s k a ( L u k e n , 1 9 8 5 ) ; t h e s e r a t e s w e r e a l l b e l o w
0.5 mm wk ^ ( c o r r e c t e d f o r l e n g t h o f g r o w i n g s e a s o n ) .
Rates were h i g h e r
( b u t s t i l l a v e r a g e d b e l o w 1 mm wk ^ o v e r t h e w h o l e y e a r ) f o r t h r e e s p e c i e s
a t l o w l a n d s i t e s i n Wales ( B e n s o n - E v a n s & B r o u g h , 1 9 6 5 ) .
The r a t e o f
g r o w t h o f R h y n c h o s t e g i u m may be sloi-u compared w i t h o t h e r a q u a t i c p l a n t s b u t
i t i s o f t h e same o r d e r as f o r a r a n g e o f o t h e r b r y o p h y t e s .
O t h e r measurements o f g r o w t h r a t e s o f a q u a t i c b r y o p h y t e s i n c l u d e t h a t of
M a r s d e n ( 1 9 7 9 ) who
0.35
mm
wk
measured t h e g r o w t h r a t e o f Scapania
^ ; a l o w f i g u r e compared w i t h t h e s e r e s u l t s ,
surprisingly
so as M a r s d e n ' s s t u d y was
e x p l a n a t i o n may
in
and
w h i c h may
this
hypnoides
observed
i n t h e summer.
S.
An
undulata
o r " c u s h i o n s " ( s e n s u Gimmingham &
cause c o n s i d e r a b l e s e l f - s h a d i n g
t y p e o f g r o w t h f o r m may
scour.
perhaps
l i e i n t h e d i f f e r e n c e s between growth forms.
grows i n c l o s e l y packed " t u r f s "
1957)
conducted
u n d u l a t a t o be
(Harper, 1977);
relate to i t s ability
Growth r a t e s o f F o n t i n a l i s d a l e c a r l i c a
Birse,
advantages
to r e s i s t abrasion
(Johnson,
1978)
and
F.
( G l i m e , 198?,) i n t h e f i e l d w e r e two t o t h r e e t i m e s t h e maximum
h e r e and
t h e growth r a t e of Amblystegium
riparium
i n the
259
l a b o r a t o r y was a l s o c o n s i d e r a b l y f a s t e r ( S a n f o r d , 1 9 7 9 ) .
Semi-aouatic
p o p u l a t i o n s o f Sphagnum c u s p i d a t u m
showed g r o w t h r a t e s o f up t o
3.5 mm wk ~ b o t h i n t h e l a b o r a t o r y ( a t 15 °C) and i n t h e f i e l d ( P r e s s e t
a l . , 1986).
A t e n t a t i v e hypothesis concerning the differences l i e s i n the
"niches" of these d i f f e r e n t species i n streams.
Several aquatic bryophytes
h a v e w e l l d e f i n e d " n i c h e s " i n s t r e a m s ( T u t i n , 1949; G l i m e , 1970; S l a c k &
G l i m e , 1985) w h i c h may r e l a t e t o t h e p h y s i o l o g i c a l l i m i t s o f each
(Penuelas, 1984).
One s u c h may be b o u n d a r y l a y e r r e s i s t a n c e s t o a q u a t i c
COg d i f f u s i o n ( J e n k i n s & P r o c t o r , 1 9 8 5 ) .
S t r e a m l i n e d morphology
enables
F o n t i n a l i s t o m a x i m i s e t h i s , w h i l s t m a t - f o r m i n g s p e c i e s such as
R h y n c h o s t e g i u m a r e , p e r h a p s , r e s t r i c t e d t o more t u r b u l e n t a r e a s .
This i n
t u r n may i n c r e a s e t h e l o s s e s due t o a b r a s i o n and s c o u r (Conboy & G l i m e ,
1971) and t h e l e n g t h o f t i m e t h a t t h e y a r e n o t immersed ( 9 . 2 2 ) .
9.25
E f f e c t o f h e a v y m e t a l s on r a t e o f g r o w t h o f R h y n c h o s t e g i u m
D u r i n g t h e s t u d y t h e r e were no s i g n s o f a d v e r s e
i n e i t h e r o f t h e s t r e a m s w h i c h had
0 3 0 9 - 8 0 , a c t u a l l y had
e l e v a t e d c o n c e n t r a t i o n s . One
by I ' J h i t t o n and D i a z
c o n c e n t r a t i o n s has
d e s c r i b e d as a s p e c i e s o f i n t e r m e d i a t e t o l e r a n c e t o z i n c
( 1 9 8 0 ) and
been n o t e d .
i t s o c c u r r e n c e over a wide range of z i n c
Work by N.G.W. F e n t o n
n a t u r a l l y t o l e r a n t of c o n c e n t r a t i o n s of zinc w e l l
copper
(4.44).
t o l e r a n c e s t o heavy m e t a l s .
d a t a ) showed R h y n c h o s t e g i u m f r o m a l o w - z i n c s t r e a m
experienced here.
of these,
t h e f a s t e s t growth r a t e of t h e f o u r streams
T h i s r a i s e s q u e s t i o n s r e g a r d i n g a d a p t a t i o n s and
R h y n c h o s t e g i u m was
e f f e c t s o f heavy m e t a l s
(Durham, u n p u b l i s h e d
(0310-90)
t o be
i n excess of those
G l i m e & Keen ( 1 9 8 4 ) showed i t t o be more t o l e r a n t t o
t h a n t h r e e s p e c i e s o f F o n t i n a l i s , a s p e c i e s on w h i c h t h e r e has
some w o r k on t o x i c i t y
These w o r k e r s
relatively
(Sommler and W i n k l e r , 1982;
Weise e t a^.,
been
1985).
showed F o n t i n a l i s t o c o n t i n u e t o p h o t o s y n t h e s i z e a t
h i g h c o n c e n t r a t i o n s o f h e a v y m e t a l s compared w i t h f i l a m e n t o u s
260
a l g a e s u c h as C l a d o p h o r a ( W h i t t o n . 1 9 7 0 ) , S t i g e o c l o n l u m ( H a r d i n g & W h i t t o n ,
1976) a n d H o r m l d l u m (Say e t a l . , 1 9 7 7 ) ; however t h e l a t t e r t w o s p e c i e s
were c a p a b l e o f d e v e l o p i n g t o l e r a n c e s r e l a t i v e l y r a p i d l y .
I t i s unlikely
t h a t Rhynchostegium r e q u i r e s a g e n e t i c a d a p t a t i o n t o s u r v i v e a t these
concentrations.
9.3 MECHANISM OF ACCUMULATION OF ZINC
9.31
Accumulation
and l o s s by whole
tips
S e v e r a l a q u a t i c p l a n t s have been shown t o a c c u m u l a t e
heavy m e t a l s
l o n g p e r i o d s o f t i m e ( M a r s d e n , 1979; H a r d i n g & W h i t t o n , 1 9 8 1 ;
1985;
over
Bond e t e d . ,
E v e r a r d & Denny, 1985b) a n d e x i s t i n g d a t a f o r z i n c a c c u m u l a t i o n by
R h y n c h o s t e g i u m i m p l i e s t h a t a p l a t e a u was r e a c h e d
within
12 h (Wehr,
1983).
However t h i s s t u d y showed t h a t a c c u m u l a t i o n o f z i n c , cadmium a n d l e a d
c o n t i n u e d over a p e r i o d o f s e v e r a l days ( 6 . 2 ) .
These c o n f l i c t i n g
results
r e q u i r e some e x p l a n a t i o n : s a m p l e s o f i n s i t u moss were c o l l e c t e d a n d
analyzed
f o r t h e i r metal c o n t e n t over
theexperimental period.
Changes i n
c o n c e n t r a t i o n s o f z i n c and cadmium i n t h e in s i t u moss a r e m i r r o r e d by
fluctuations
i n t h e c o n c e n t r a t i o n s i n t r a n s p l a n t e d moss f r o m a b o u t
onwards ( 6 . 2 ) .
T h e i r a c c u m u l a t i o n p e r i o d l a s t s f o r about
s u b s e q u e n t changes r e f l e c t
6.1).
accumulation
s i x days a n d
changes i n d i s s o l v e d z i n c a n d cadmium
The d a t a f o r l e a d does n o t f o l l o w s u c h a p a t t e r n :
day s i x
(Table
therate of
i s s l o w e r a n d c o n t i n u e s o v e r a p e r i o d o f 16 d a y s ( F i g . 6 . 1 ) .
Z i n c and l e a d a c c u m u l a t i o n by t r a n s p l a n t e d S c a p a n i a u n d u l a t a showed an
initial
phase o f a c c u m u l a t i o n o f a s i m i l a r d u r a t i o n
Rhynchostegium
(6.2) (Marsden, 1979);
t h e s e were f o l l o w e d b y changes i n
c o n c e n t r a t i o n s i n t r a n s p l a n t e d moss w h i c h
concentrations
i n i^n s i t u m a t e r i a ] .
to that of
r e f l e c t e d changes i n
261
A second p o i n t o f i n t e r e s t i n t h e r e s u l t s from t h e l o n g - t e r m t r a n s p l a n t
experiments i s t h a t t h e f i n a l c o n c e n t r a t i o n f a c t o r o f zinc i n transplanted
moss i s s i g n i f i c a n t l y h i g h e r t h a n t h e c o n c e n t r a t i o n i n i n s i t u moss ( F i g
6.1) a n d v i c e v e r s a f o r cadmium and l e a d . T h i s may r e f l e c t v a r i e t a l
d i f f e r e n c e s b e t w e e n p o p u l a t i o n s ; Wehr ( 1 9 8 3 ) f o u n d s i m i l a r marked
d i f f e r e n c e s w i t h r e s p e c t t o z i n c a c c u m u l a t i o n . Wehr ( 1 9 8 3 ) f o u n d v a r i e t a l
d i f f e r e n c e s between p o p u l a t i o n s i n a t r a n s p l a n t i n v o l v i n g f i v e p o p u l a t i o n s
b e t w e e n l o w - z i n c s t r e a m s a n d 0048-90.
The d i f f e r e n c e s i n 6.2 a r e more
l i k e l y t o r e f l e c t these i n t e r - p o p u l a t i o n d i f f e r e n c e s than t h e development
o f an t o l e r a n c e mechanism a n d t h i s c o n c l u s i o n i s s u b s t a n t i a t e d by t h e s t u d y
on z i n c t o l e r a n c e i n R h y n c h o s t e g i u m r e f e r r e d t o above ( 9 . 2 4 )
The
extended
processes
involved
exchange s i t e s .
adsorption
interpreted
i n accumulation than simply a d s o r p t i o n onto c e l l
these r e s u l t s
The a p p a r e n t
i n terms
location
o f o t h e r processes
( F i g . 6.3) w h i c h
f o r by t h e r e s i d u a l
fraction.
accounted
indicated the
Throughout t h e
f o r 77.5% o f
centrifugation
copper e x t r a c t e d
(Brown & House, 1 9 7 8 ) .
roots of higher plants are s i m i l a r
accumulated
Brown ( 1 9 8 2 ) a n d c o - w o r k e r s
wall of
(Burton & Peterson,
from t h e c e l l
wall of
Results from analyses of
( T u r n e r , 1970).
The r e s i d u a l
z i n c t h a t was n o t removed by N i C l ^ i s assumed t o r e p r e s e n t
uptake.
(Brown,
compares w i t h 8 0 % o f z i n c f o u n d i n t h e c e l l
and 93% o f a c c u m u l a t e d
have
( F i g 6.5) b u t a s i g n i f i c a n t
F o n t i n a l i s a n t i p y r e t i c a by d i f f e r e n t i a l
Solenostoma crenulatum
as w e l l
of zinc during accumulation
12 h , t h e e x c h a n g e a b l e f r a c t i o n
1979),
( R u h l i n g & T y l e r , 1970;
1972; Brown & House, 1 9 7 8 ) , a l t h o u g h r e c e n t l y w o r k e r s
p r o p o r t i o n was a c c o u n t e d
zinc
wall
Much e m p h a s i s has been p l a c e d on t h e r o l e o f exchange
important r o l e of exchange-adsorption
first
i m p l i e s t h a t t h e r e a r e more
i n m e t a l a c c u m u l a t i o n by b r y o p h y t e s
Brown & B a t e s .
1984).
period of accumulation
24.5% o f
intracellular
w e r e a b l e t o r e l e a s e much o f t h i s by
r u p t u r i n g t h e plasmalemma u s i n g d i l u t e a c i d s .
The p o t a s s i u m
released a t
262
this
stage
(2.422)
i n d i c a t e d t h a t t h e metals o r i g i n a t e d from i n s i d e t h e c e l l
rather
been o b s e r v e d
than from d i s c r e t e p r e c i p i t a t e s outside t h e c e l l
f o r some s p e c i e s
(Silverberg,
which
have
1975; Sharpe & Denny, 1976;
S a t a k e & M i y a s a k a , 1984; M o u v e t , 1984) a l t h o u g h t h e s e have been o b s e r v e d t o
form w i t h i n a minute
concentrations
o f contact w i t h metal s a l t s ,
(Sharpe
& Denny,
Over a 14-d e x p o s u r e
decreased
artefact
which
proportion
t h e exchange s i t e s .
Less t h a n 5 0 % o f z i n c
i n the l a t t e r
over extended
intracellular
uptake.
Moreover, a comparison
period
i s located
constituted
here
a significantly
Accumulation
The
larger fraction
and c o n s e q u e n t l y w i l l
this
i n v e s t i g a t e d d u r i n g the study.
zinc accumulation
more t y p i c a l
walls
b y b o t h was v e r y
( F i g . 7.14).
tips
The d i f f e r e n c e
wall
represents a
have more exchange s i t e s
intracellular
p e r u n i t mass
The r e s u l t s
f r a c t i o n was a c c u m u l a t e d
show ( 6 . 4 2 1 )
experiment
i n the light
(6.422)
was
no e f f e c t o f l i g h t
i n the laboratory but the results of t h i s
were i n c o n t r a s t w i t h a f i e l d
decreased
f o r by
plants.
mechanism by w h i c h
zinc accumulation
accumulation
24 h a n d 168 h w h o l e
i s n o t so p r o n o u n c e d as i n F i g . 6.5 b u t t h e c e l l
compared w i t h whole
on
t h a t metal
o f a c c u m u l a t i o n by w h o l e t i p s and by c e l l
1 h (as i n Fig. 6.5), w h i l s t a f t e r
"model" system
i n t h e exchangeable
p e r i o d s o f t i m e may be a c c o u n t e d
(7.42) provides support f o r t h i s hypothesis.
similar after
( T a b l e 6.5)
( F i g . 6 . 5 ) . Such a h i g h
provides s t r o n g evidence
by b r y o p h y t e s
i n flasks
b e i n g e s t a b l i s h e d b e t w e e n t h e media and
by t h e end o f t h e e x p o s u r e
proportion
( s e e 7:25) s i n c e t h e r e was an
i n the concentration of zinc
l e a d t o new e q u i l i b r i a
zinc
i n c r e a s e d ( F i g . 6 . 5 ) . T h i s may be an
of the batch c u l t u r e conditions
would
fraction
1976).
period the proportion of extracellular
and t h e r e s i d u a l
e x p o n e n t i a l decrease
albeit at high
experiment
i n wb«4ie t h e r e was
( F i g . 6 . 8 ) . The f o r m e r g i v e s t h e
r e s u l t s compared w i t h d a t a f o r a c c u m u l a t i o n o f h e a v y
metals
263
i n t o a l g a e ( G u t n e c h t , 1961; S k o w r o n s k i , 1 9 8 4 b ) , b r y o p h y t e s ( P i c k e r i n g *
P u i a , 1969; Brown & B e c k e t t , 1985)
and l i c h e n s ( B e c k e t t & Brown, 1984) and
f o r t h e i n f l u x o f m a j o r i o n s i n t o mosses ( S i n c l a i r , 1 9 6 8 ) .
Dark
p r e i n c u b a t i o n l e d t o decreased accumulation of z i n c (Table 6.9); t h i s
e f f e c t was g r e a t e s t when t h e moss was t r a n s p l a n t e d i n t o t h e z i n c - e n r i c h e d
s t r e a m i n t h e d a r k ( 6 . 4 2 3 ) . These t h r e e e x p e r i m e n t s g i v e a p p a r e n t l y
c o n f l i c t i n g r e s u l t s w h i c h s u g g e s t t h a t t h e s t a t e o f t h e moss p r i o r t o t h e
e x p e r i m e n t s may have been d i f f e r e n t i n each c a s e .
T h e r e w e r e s i g n i f i c a n t e f f e c t s o f t e m p e r a t u r e a t a l l t h r e e sample t i m e s
(Table 6 . 1 1 ) ; corresponding temperature q u o t i e n t
after
I h and
1.16 after
( 1 9 7 9 ) q u o t e Q^^
24 h and
168
h (6.43).
v a l u e s o f b e t w e e n 1.2 and
t h e r m a l a g i t a t i o n and
1.6
2 t o 3 as b e i n g t y p i c a l
(Q.,Q) v a l u e s w e r e
L i i t t g e and
1.23
Higinbotham
as b e i n g c h a r a c t e r i s t i c
o f membrane d i f f u s i o n .
v a l u e s f o u n d h e r e a r e t h e r e f o r e l o w even i f t h e p r o c e s s
of
The
i s assumed t o be
p a s s i v e ; h o w e v e r t h e y compare f a v o u r a b l y w i t h v a l u e s f o r z i n c a c c u m u l a t i o n
by F o n t i n a l i s a n t i p y r e t i c a
(Q^Q
= 1 - 2 ; P i c k e r i n g & P u i a , 1 9 6 9 ) and f o r
n i c k e l a c c u m u l a t i o n by t h e l i c h e n U m b i l i c a r i a m u h l e n b e r g i i (Q. . = 1 . 1 :
J.
Nieboer
0
et^ a J . , 1 9 7 6 ; b o t h v a l u e s c a l c u i a t e d f r o m d a t a i n t h e s e p a p e r s )
f o r a c c u m u l a t i o n of major
(Clarkson,
1974).
m e t a b o l i c energy
H i g h Q^^
root
tissues
v a l u e s a l o n e do n o t n e c e s s a r i l y i m p l y t h a t
i s i n v o l v e d s i n c e any p r o c e s s w i t h an a p p r e c i a b l e e n e r g y
b a r r i e r t o s u r m o u n t may
t h e s e may
i o n s s u c h as p h o s p h a t e i n t o
give similar
results
(Nobel, 1 9 7 4 ; Bowling,
i n c l u d e p a s s i v e d i f f u s i o n a c r o s s a membrane.
The
results
1976);
shown
i n 6 . 4 3 d i d n o t s e p a r a t e t h e e f f e c t s o f exchange a d s o r p t i o n and
the
intracellular
reduce
o v e r a l l Q^^
still
a b s o r p t i o n and
value.
"active"
a l a r g e e x c h a n g e a b l e componant may
Even i f t h e p r e s e n t Q^^
below v a l u e s quoted
of t r u l y
and
uptake.
by L i i t t g e and
values are doubled
Higinbotham
the
they are
( 1 9 7 9 ) as b e i n g
typical
264
The r e s u l t s f r o m o t h e r s t u d i e s u s i n g m e t a b o l i c i n h i b i t o r s a r e f a r f r o m
c o n s i s t e n t and t e n d t o r e i n f o r c e t h e d i f f i c u l t i e s i n i n t e r p r e t a t i o n which
m u s t be o v e r c o m e i f i n h i b i t o r s a r e t o be u s e d e f f e c t i v e l y (Webb, 1 9 6 3 ;
Bowling, 1976).
I n t h i s s t u d y i n h i b i t o r s were used t o s e p a r a t e t h e e f f e c t s
of o x i d a t i v e - and photo- p h o s p h o r y l a t i o n (6.442).
T h e r e was no c o n s i s t e n t
d i f f e r e n c e b e t w e e n t r e a t m e n t s ( F i g . 6 . 1 0 ) ; s i g n i f i c a n t d i f f e r e n c e s were
o b s e r v e d o n l y w h e r e s a m p l e s t r e a t e d w i t h t h e i n h i b i t o r h a d a c c u m u l a t e d more
z i n c t h a n t h e c o n t r o l s a n d t h i s may i n d i c a t e " l e a k y " membranes.
Neither
i n h i b i t o r caused reduced a c c u m u l a t i o n .
O t h e r w o r k e r s have r e p o r t e d a
d e c r e a s e i n o v e r a l l membrane p o t e n t i a l i n b r y o p h y t e s u s i n g a z i d e , 2,4-DNP
and ammonium c h l o r i d e .
These a l l a f f e c t o x i d a t i v e p h o s p h o r y l a t i o n w h i c h
may i m p l y t h a t r e s p i r a t i o n r a t h e r t h a n p h o t o s y n t h e s i s , was t h e s o u r c e o f
e n e r g y ( C o n t a r d i & D a v i e s , 1978); however o t h e r e q u a l l y v a l i d
i n t e r p r e t a t i o n s o f t h e s e r e s u l t s a r e p o s s i b l e because o f t h e q u e s t i o n a b l e
s p e c i f i c i t y of the i n h i b i t o r s .
DNP h a s been o b s e r v e d t o cause a r e d u c t i o n
i n a c c u m u l a t i o n o f z i n c by F o n t i n a l i s a n t i p y r e t i c a o v e r l o n g p e r i o d s o f
t i m e ( P i c k e r i n g & P u i a , 1969) a n d t o r e d u c e cadmium a c c u m u l a t i o n by t h e
green a l g a Stichococcus b a c i l l a r i s (Skowronski, 1984a).
Skowronski (1984a)
a l s o r e p o r t e d r e d u c e d a c c u m u l a t i o n i n t h e p r e s e n c e o f CCCP. an u n c o u p l e r o f
p h o t o p h o s p h o r y l a t i o n and F e l l e a n d B c n t r u p ( 1 9 7 7 ) n o t e d a r e d u c e d p o t e n t i a l
d i f f e r e n c e i n t h e l i v e r w o r t R i c c i a f l u i t a n s i n t h e p r e s e n c e o f CCCP.
It
i sonly possible t o speculate
intracellular
mechanism o f
z i n c u p t a k e by R h y n c h o s t e g i u m ; t h e r e a r e s t i l l
fundamental questions
(1979) discuss
on t h e p r o b a b l e
t h a t need t o be r e s o l v e d .
some
L u t t g e and H i g i n b o t h a m
e x a m p l e s o f p r o c e s s e s s u c h as e n z y m i c r e a c t i o n s w h i c h may
r e d u c e i n t r a c e l l u l a r c o n c e n t r a t i o n s o f i o n s a n d t h u s enhance d i f f u s i o n .
Such p r o c e s s e s may t h e r e f o r e a p p e a r t o be " a c t i v e " as changes i n m e t a b o l i s m
affect
rates o f passive
diffusion.
r e q u i r e s a c a l c u l a t i o n o f the Nernst
The f o r m a l
potential
proof t h a t uptake i s a c t i v e
f o r zinc which,
i n turn.
265
r e q u i r e s measurement o f t h e
membrane p o t e n t i a l .
The
components i n s e v e r a l
normal c o n d i t i o n s
i n t r a c e l l u l a r zinc concentration
intracellular
r o l e o f z i n c i s as
enzyme s y s t e m s , n o t a b l y
z i n c w h i c h does e n t e r
t h e s e p r o t e i n s and
carbonic
anhydrase.
c e l l s w o u l d be
l a r g e l y bound
concentration
gradient
(Nobel,
1974;
L i i t t g e & H i g i n b o t h a m , 1979)
usually
s t r o n g l y e l e c t r o n e g a t i v e , w h i c h w o u l d a l s o cause d i f f u s i o n
down an
electrochemical
Consideration
provide
gradient
of the r a t e of
f u r t h e r i n f o r m a t i o n on
streams l o s t
Moss f r o m
mosses t h a t had
a l s o Wehr, 1 9 8 3 ) .
the assumption t h a t a l a r g e p r o p o r t i o n of zinc
compartment of
the c e l l
w h i c h enhanced z i n c
cations
f r o m where i t i s n o t
loss
(e.g.
EDTA and
f r o m e x c h a n g e s i t e s and
P opulation with only a b r i e f
had
exposure
f r o m i^n s i t u R h y n c h o s t e g i u m
into
o n l y had
This
a
brief
tends t o
confirm
is localized in a
easily lost.
Those f a c t o r s
c a l c i u m ) were e x p e c t e d t o remove
(= 1 h )
to zinc
(6.54).
A
the
gradual
resulted i n a closely-matched
observed d u r i n g
and
t r a n s p l a n t (6.2)
b r o a d c o r r e l a t i o n s between m e t a l c o n c e n t r a t i o n s
(Whitton
overall
e t a l . , 1982;
Impression,
Say
& Whitton,
1983;
t h e r e f o r e , i s o f an
e x t r a - and
intracellular
short-term
f l u c t u a t i o n s represented
loss
(6.53) i n d i c a t i n g slow e q u i l i b r a t i o n
S i m i l a r changes were
long term
compartments.
the
may
R h y n c h o s t e g i u m t o changes i n i t s e n v i r o n m e n t .
the
are
metal-enriched
t h e most p r o n o u n c e d e f f e c t on
decrease i n ambient zinc c o n c e n t r a t i o n s
of zinc
established
loss of metals from Rhynchostegium
see
a
t h a t the i n t e r i o r s of c e l l s
localization.
( 6 . 5 4 ; 6.55;
be
( S h a r p e & Denny, 1 9 7 6 ) .
zinc a t a slower r a t e than
exposure t o zinc
t h a t t h e r e may
In addition i t i s well
into
the
concentration
i n t o the c e l l .
low and
Under
intracellular
cell
i s very
the
structural
t h e r e f o r e i t i s r e a s o n a b l e t o assume t h a t
of zinc
and
are
reflected in
i n b r y o p h y t e s and
Wehr & W h i t t o n ,
of
the
i n water
1983a).
The
e q u i l i b r i u m between w a t e r and
both
This
for
hypothesis
by changes i n t h e
accounts both
exchangeable
266
c o m p a r t m e n t and
compartment.
9.32
longer-term
Role of the c e l l
The
cell
some two
gains
and
t h i r d s of the t o t a l
dry weight
intracellular
of the c e l l .
f o r exchange a d s o r p t i o n o n t o
(7.26,
the
i s s u b s t a n t i a l (7.21), accounting
the absence o f competing ions i n excess o f
adsorbed
from
wall
w a l l of Rhynchostegium
great potential
losses
7.31).
Zinc
i s normally
a cell
11000
There i s o b v i o u s l y
w a l l of t h i s
pg g-1
size; in
o f z i n c may
a m i n o r component o f t h e
the dominant c a t i o n s are
be
ionic
balance of freshwaters
( 1 . 2 2 1 ) and
magnesium and
which account f o r the g r e a t e s t p r o p o r t i o n of
calcium,
a d s o r b e d m e t a l s i n t h e r a n g e o f w a t e r s t e s t e d h e r e (7.23
w e r e s i g n i f i c a n t c o r r e l a t i o n s between c o n c e n t r a t i o n s
w a l l s f o r a number o f i o n s and
finding;
calcium
Whitton,
o t h e r w o r k e r s have f o u n d
1983a).
The
number o f samples and
visited:
t h e r e was
concentrations
Carboniferous
The
calcium,
(3.211).
relationship
e f f e c t of calcium
on
e x p e r i m e n t was
cell
This
c o r r e l a t e d w i t h the
is a surprising
h o w e v e r , based on a r e l a t i v e l y
may
be due
The
on
to the type of
small
stream
between d i s s o l v e d
the o r e - v e i n s
being
intruded into
the
l a b o r a t o r y experiment using a s i n g l e
concentrations
zinc adsorption
showed t h e more
is that
there i s a
( F i g . 7.15).
zinc accumulation
i n v e s t i g a t e d by Wehr ( 1 9 8 3 ) and
in
There
t i p s o f R h y n c h o s t e g i u m (Wehr &
a range of c a l c i u m
e f f e c t o f aqueous c a l c i u m
strongly negative
7.6).
by whole 2-cm
t h i s discrepancy
limestone
7.25).
a s t r o n g n e j j a t i v e i n f l u e n c e o f aqueous
e x p e r i m e n t was.
o f z i n c and
and
i n w a t e r and
positively
also a positive c o r r e l a t i o n
z i n c c o n c e n t r a t i o n and
likely
i n water (Table
on z i n c a c c u m u l a t i o n
usually
t h e r e were a l s o s e v e r a l c r o s s - c o r r e l a t i o n s ;
t h e c o n c e n t r a t i o n o f z i n c , f o r e x a m p l e , was
c o n c e n t r a t i o n of calcium
for
Wehr and
of a s i m i l a r design
by R h y n c h o s t e g i u m
Whitton
t o 7.52;
(1983a).
accumulation
was
also
Wehr's
of a s i n g l e zinc
267
c o n c e n t r a t i o n was t e s t e d o v e r a r a n g e o f c a l c i u m
concentrations.
e f f e c t was r a p i d a n d a l m o s t l i n e a r o v e r t h e r a n g e 0 - 20 mg 1 ^
at
higher concentrations
in
this
study
t h e e f f e c t decreased.
o b s e r v e d by Wehr
wall;
calcium
this
being
( F i g . 7.16) I n d i c a t i n g t h a t t h e " e f f e c t "
i s i n keeping with the generally apoplastic location of
w a l l s were e q u i l i b r a t e d
s u p p l i e d w i t h z i n c ; indeed
concentrations
7.15).
of e i t h e r calcium
The most p r o b a b l e
t h e r e was l i t t l e
effects
concentrations
variation
i n the
o r z i n c over t h e e n t i r e range t e s t e d ( F i g .
explanation
introduced t o the hypotonic
No s i m i l a r
t o various
i s t h a t new e q u i l i b r i a
w a l l and t h e m e d i a w e r e s e t up when e q u i l i b r a t e d
(1983a),
found
( 1 9 8 3 ) may h a v e been l a r g e l y due t o c o m p e t i t i o n a t t h e
w e r e o b s e r v e d when c e l l
cell
A s i m i l a r r e s u l t was
i n p l a n t s (Marme, 1983; D e m a r t y e t a j . . , 1 9 8 4 ) .
before
calcium;
( 7 . 5 2 ) when z i n c a d s o r p t i o n was p l o t t e d a g a i n s t t h e c a l c i u m
c o n c e n t r a t i o n i n t h e media
cell
The
media.
cell
between t h e .
w a l l s were
I n t h e s t u d y by Wehr and W h i t t o n
m u l t i v a r i a t e s t a t i s t i c s were u s e d t o i n v e s t i g a t e t h e f a c t o r s
a f f e c t i n g metal
accumulation
by R h y n c h o s t e g i u m and d i s s o l v e d c a l c i u m
strong negative
i n f l u e n c e on z i n c a c c u m u l a t i o n .
had a
I t was n o t p o s s i b l e t o
make a d i r e c t c o m p a r i s o n o f t h e r e g r e s s i o n c o e f f i c i e n t s as Wehr and
Whitton's
likely
wall.
s u c h as t h e c h l o r o c o c c a l e
Competitive
however i t i s
e f f e c t s may r e l a t e
Golenkinia
(Bachmann,
1981) and H y l o c o m i u m s p l e n d e n s
but n o t f o r Scapania u n d u l a t a
problems i n t h i s
data;
to competition a t
S i m i l a r e f f e c t s have been o b s e r v e d f o r a number o f o t h e r
(Harding & Whitton,
calcium
log^^-transformed
t h a t a l a r g e component o f t h e s e
the c e l l
species
was c a l c u l a t e d u s i n g
study
inhibition
1 9 6 3 ) , Lemanea
( R u h l i n g & T y l e r , 1970)
( W h i t t o n e t al.. , 1982) because t h e r e were
i n s e p a r a t i n g t h e e f f e c t s o f pH and aqueous
of intracellular
was a l s o o b s e r v e d
cadmium u p t a k e by magnesium and
i n Rhytidiadelphyus
1 9 8 5 ) ; t h i s was i n t e r p r e t e d as i n d i c a t i n g
u p t a k e s y s t e m was i n v o l v e d f o r t h i s
calcium.
metal.
squarrosus
(Brown &
that a relatively
Beckett,
non-specific
268
O t h e r e n v i r o n m e n t a l f a c t o r s t e s t e d d u r i n g t h e p r e s e n t s t u d y were
p h o s p h a t e , EDTA a n d h u m i c a c i d .
P h o s p h a t e was t e s t e d o n l y i n one
e x p e r i m e n t ( 6 . 5 5 ) a n d h a d l i t t l e e f f e c t on m e t a l l o s s a f t e r e i t h e r a l o n g er a s h o r t - t e r m exposure t o z i n c .
Wehr ( 1 9 8 3 ) a l s o f o u n d p h o s p h a t e t o have
l i t t l e e f f e c t on z i n c a c c u m u l a t i o n a l t h o u g h i t h a s been n o t e d t o h a v e an
a m e l i o r a t i n g e f f e c t on z i n c t o x i c i t y t o H o r m i d i u m r i v u l a r e (Say & W h i t t o n ,
1 9 7 7 ) , A n a c y s t i s n i d u l a n s ( S h e h a t a & W h i t t o n , 1982) a n d z i n c - t o l e r a n t
s t r a i n s o f S t i g e o c l o n i u m tenue (Harding & W h i t t o n , 1977).
I t h a s been
s u g g e s t e d t h a t f a c t o r s i n t h e e n v i r o n m e n t w h i c h a f f e c t a c c u m u l a t i o n may
a l s o a f f e c t t o x i c i t y ( W h i t t o n & Say, 1 9 7 5 ) ; t h i s does n o t a p p e a r t o be t h e
c a s e f o r p h o s p h a t e on z i n c a c c u m u l a t i o n o v e r t h e r a n g e s e x p e r i e n c e d i n
n a t u r e (Wehr, 1 9 8 3 ) .
EDTA d i d h a v e a s i g n i f i c a n t
e f f e c t on l o s s o f z i n c
study i n v o l v i n g a d s o r p t i o n onto c e l l
c o n c e n t r a t i o n s used were s i m i l a r
d i f f e r e n c e s were s t a t i s t i c a l l y
walls
concentrations of iron
wall
significant
i n thec e l l
I n 7.23 o n l y s i x o f t h e
( T a b l e 7 . 3 ) ; f o u r o f t h e s e were
I t i spossible that the high
w a l l masked t h e e f f e c t s o f EDTA.
Cell
p r e p a r a t i o n s w e r e g i v e n a p r e l i m i n a r y wash i n h y d r o x y l a m i n e
hydrochloride
1975)
may
(7.23) a l t h o u g h t h e
i n each c a s e .
b e t w e e n s o l u t i o n s w i t h a n d w i t h o u t F.DTA.
(6.55) b u t n o t i n t h e
( 2 . 4 3 ) t o remove i r o n a n d manganese o x i d e s ( G u p t a & Chen,
but a recent publication
n o t have s o l u b i l i z e d
additional
right
( T i p p i n g e t a j . . , 1985)
a l l the iron deposits;
a d s o r p t i o n and c o - p r e c i p i t a t i o n
1981a; 1 9 8 1 b ) w h i c h w o u l d f u r t h e r
potential
binding sites
( T i p p i n g e t al^. , 1985)
o f h e a v y m e t a l s i n t h e i r own
remained
after
Treatment
with
approximately half of the iron
treatment w i t h hydroxylamine
substances
i n c r e a s e t h e number o f
( T i p p i n g e t al,., 1983).
removed
this
t h e s e i n t u r n may cause
( L a x e n , 1984a; 1 9 8 4 c ) a n d i n a d d i t i o n may a d s o r b humic
(Tipping,
effect
has shown t h a t
(7.24).
This,
on t h e c o n c e n t r a t i o n o f z i n c r e m a i n i n g i n t h e p l a n t
oxalate
which
i n t u r n , had an
( F i g . 7.2).
269
R e s u l t s i n 6.55 a n d Wehr ( 1 9 8 3 ) i n d i c a t e t h a t t h e p r e s e n c e o r absence o f
l o w c o n c e n t r a t i o n s o f EDTA may have a s i g n i f i c a n t e f f e c t upon z i n c
accumulation.
S e v e r a l o f t h e a b o v e comments c o n c e r n i n g EDTA a p p l y a l s o t o h u m i c a c i d s .
A g a i n , no c o n c l u s i v e s t a t e m e n t may be made f r o m t h e r e s u l t s p r e s e n t e d
(7.23);
s e v e r a l other workers
heavy m e t a l s
have n o t e d a d e c l i n e i n t h e accui^.ulation o f
( v a n d e r W e r f f , 1984)
(Laegreid e t aj,.,
1984)
o r i n t h e i r t o x i c i t y t o both
and a n i m a l s
(Winner,
1984)
these a r e t h e r e s u l t o f r e a c t i o n s o u t s i d e c e l l s
The
high a f f i n i t y
m.ay
h a v e masked t h e e x p e c t e d
9.33
here
plants
and i t i s l i k e l y
(Langston & Bryan,
that
1984).
o f i r o n o x i d e s f o r humic m a t e r i a l s ( T i p p i n g i 9 8 1 a ; 1982b)
effect
(7.23,
7.24).
Growth and heavy m e t a l a c c u m u l a t i o n and l o s s
I n a d d i t i o n t o t h e p h y s i o l o g i c a l processes
a c c u m u l a t i o n and l o s s , each p r o c e s s
i n v o l v e d i n heavy m e t a l
may a l s o be i n f l u e n c e d by g r o w t h ,
r e s p e c t i v e l y e x p o s i n g new s i t e s f o r m e t a l a d s o r p t i o n o r a b s o r p t i o n o r
"diluting"
e x i s t i n g metal concentrations.
these views
f r o m d a t a i n c h a p t e r s 4 a n d 6.
Rhynchostegium i s r e l a t i v e l y
a l g a e and macrophytes;
slow-growing
The c o n c l u s i o n i n 9.2 was t h a t
compared w i t h o t h e r f r e s h w a t e r
a v e r a g e r a t e s were a b o u t
week ^ a t peak p e r i o d s ( 9 . 2 3 ) .
the process
1 mm week ^ r i s i n g t o 2 ram
The r a t e s o f l o s s o f h e a v y m e t a l s were
dependent upon t h e l e n g t h o f i n i t i a l
e n r i c h e d streams
I t i s p o s s i b l e t o t e s t both of
exposure;
i nmaterial
fro.T. m e t a l -
c o u l d t a k e s e v e r a l d a y s ( S . 5 1 ; r i a r s d e n , 1979;
Wehr, 1 9 8 3 ) .
Growth o f Rhynchostegium i n such a s h o r t l e n g t h o f t i m e
be v e r y l o w .
1 mm o f s h o o t w o u l d
r e p r e s e n t 5% o f t o t a l
would
mass ( 4 . 2 3 ) ; i f 4 0 %
o f a c c u m u l a t i o n a f t e r 23 d o c c u r s w i t h i n 24 h t h e n t h e r e m a i n i n g 6 0 % w o u l d
be a c c o u n t e d
f o r b y 2 - 3 mm o f g r o w t h o r 1 0 % - 1 5 % o f t h e f i n a l
mass.
270
9.4 USE OF AQUATIC BRYOPHYTES AS MONITORS
9.41 Moss-bag m e t h o d o l o g y
The
results
i n c h a p t e r 5 showed t h e a c c u m u l a t i o n o f heavy m e t a l s
R h y n c h o s t e g i u m i n moss-bags t o p r o c e e d
boulders
( 5 . 3 1 ) and
(5.32).
These a r e e n c o u r a g i n g
I t was
f e a t u r e s i f a s t a n d a r d method f o r u s i n g
Some f e a t u r e s o f t h e r e s u l t s ,
"noisy".
that the i n i t i a l
b o t h b o u l d e r s and
(y
bags ( l e a d ) .
seen i n bags ( z i n c and
I n 5.32
t h e r e was
phase
c a d n i u n ) and f o r
no s u c h i n i t i a l
"noise"
5.2).
The
the
stages of accumulation of a l l
F o u r o f t h e s i x r e g r e s s i o n s based on t h e r a p i d
w e r e n o t s i g n i f i c a n t and t h i s was
(Fig.
however,
comment.
n o t e d i n 5.31
m e t a l s was
on
t o be r e l a t i v e l y u n a f f e c t e d by t h e p a t t e r n o f t h e bags
moss-bags i s t o be p r o p o s e d .
require further
a t t h e sa.'ne r a t e as by r.oss
by
kinetics
o f a c c u m u l a t i o n o f z i n c were a l s o v e r y d i f f e r e n t
two e x p e r i m e n t s .
5.31
fitted
the s t r a i g h t f o r w a r d
= m X H c) c l o s e r t h a n t h e l o g -normal
5.6)), whilst
function
t h e a c c u m u l a t i o n c u r v e i n 5.32
linear
between
function
(y = l o g
x + c;
the log
-normal
fitted
Table
e
f u n c t i o n b e t t e r than the "normal".
arbitrarily
split
for
5.31
and
not
i n 5.31.
into
" r a p i d " and
5 h f o r 5.32.
S i m i l a r l y , when t h e c u r v e s
"slow"
An a p p a r e n t
sections the d i v i s i o n f e l l
s a t u r a t i o n was
When t h e r e g r e s s i o n e q u a t i o n s f o r t h i s
compared t h e n t h e i n i t i a l
s l o p e s i n 5.32
were
a r e 2.5
observed
initial
at 6 h
i n 5.32
but
phase a r e
t i m e s more r a p i d t h a n i n
5.31.
The
streams
different:
i n which
0024-22 ( 5 . 3 2 )
water, w h i l s t water
( T a b l e 3.7;
and
t h e s e e x p e r i m e n t s were conducted
Table
i n t y p e s and
i s a l o w l a n d s t r e a m , v e r y e u t r o p h i c and w i t h
i n 0012-45 ( 5 . 3 1 ) i s l e s s h a r d and
3.8).
ijere very
less eutronhic
There are c o n s i d e r a b l e d i f f e r e n c e s i n
c o n c e n t r a t i o n s of metal inputs.
f u n d a m e n t a l l y s i m i l a r d e s i g n s and
hard
catchments
Both experiments
had
s i m i l a r q u a n t i t i e s o f moss were used i n
271
e a c h bag.
T h a t t h e d i f f e r e n c e s f r o m 5.32 were o b s e r v e d i n b o t h t r e a t : r . e n t s
i n 5.31 i m p l i e s t h a t i t i s a f e a t u r e o f t h e t r a n s p l a n t and n o t o f t h e mossbag and may be r e l a t e d t o d i f f e r e n c e s i n w a t e r c h e m i s t r y .
Although
t r e n d and
the r e s u l t s
c o n t e n t s o f t h e bag
bags ( 5 . 2 2 )
and
i s due
Why
p e r c o l a t i o n t h r o u g h moss and
( + 100
g wet
weight).
enough o n l y f o r one
e t al.,
with
and
the
an e f f e c t when t y p e
III
between
I t is
s h o u l d be r e s t r i c t e d t o 4 - 5 samples
Fewer bags a r e r e q u i r e d t h a n i f each bag
contained
s a m p l e y e t t h e q u a n t i t y o f moss i n each bag
teased
does n o t
5.34.
(1983) adopted t h e p r a c t i c e of workers
1977)
on a i r p o l l u t i o n
(Goodman
o u t t h e moss i n t h e bags t o f o r m c i r c u l a r
increased s u r f a c e area
the
through
not through muslin i s not clear.
a p p r o a c h t h e v o l u m e o f moss u s e d i n
Everard
a t the c e n t r e of
t h e r e s h o u l d be a d i f f e r e n c e
t h a t t h e c o n t e n t s o f a bag
a
much moss
t o reduced p e r c o l a t i o n of water
i t i s s u r p r i s i n g n o t t o see
are considered.
illustrate
c a u t i o n on t h e s u b j e c t o f how
I f t h e r e i s reduced accumulation
then presumably t h i s
suggested
a r e n o t c o n c l u s i v e t h e y do
i t i s w o r t h w h i l e t o urge
t o p a c k i n t o a bag.
bag
i n 5.34
: volume r a t i o s .
T h i s may
p r e c a u t i o n i n l a k e s o r i n s l o w - f l o w i n g r i v e r s and
discs
be a s e n s i b l e
streams;
however i n
r i v e r s or streams t h e e f f e c t of the c u r r e n t i s both t o p e r c o l a t e water
t h r o u g h t h e bag
The
stakes
both
and
technique
(2.32).
i n lakes
t o aggregate
used here
t h e moss i n t o a s t r e a m l i n e d f o r m .
f o r f i x i n g bags i n r i v e r s was
t o a t t a c h them t o
Suspended bags h a v e been u s e d s u c c e s s f u l l y by o t h e r
(Denny, 1 9 8 1 ;
Everard
& Denny, 1985b) and
in rivers
B u r r o w s , u n p u b l i s h e d ) ; however i n r i v e r s t h e y a r e s u s c e p t i b l e t o
and
to high flows.
The
securely attached well
technique
i s most s u c c e s s f u l when t h e bag
out of reach
o f t h e bank and
use
o t h e r s u p p o r t i n g s t r u c t u r e s s u c h as f e n c e s ,
The
techniques
i t may
comm.) i n v o l v e a n c h o r i n g
(I.G.
vandalism
can
be
be p o s s i b l e t o
i f these cross
u s e d by Mouvet ( 1 9 8 4 ) , P r i g g ( 1 9 7 7 ) and
workers
streams.
E. Tvlycock ( p e r s .
t h e moss-bags s e c u r e l y t o t h e s t r e a m
bed
272
u s i n g stones or c o n c r e t e b l o c k s . I t i s t h e a u t h o r ' s experience t h a t such
b l o c k s have t o be q u i t e s u b s t a n t i a l t o p r e v e n t t h e i r removal d u r i n g h i g h
flows.
I t i s p o s s i b l e t h a t the s i z e o f t h e b l o c k r e q u i r e d c o u l d be
p r e d i c t e d from the e m p e r i c a l r e l a t i o n s h i p g o v e r n i n g the s i z e of the boulder
moved by a p a r t i c u l a r f l o w r a t e . A s t a k e which i s s e c u r e l y hammered i n t o
the stream bed can r e s i s t v e r y h i g h f l o w s a l t h o u g h i n the process i t may
t r a p suspended m a t e r i a l ; i t i s p r o b a b l y more secure under such c o n d i t i o n s
than a boulder but i t i s f a r more conspicuous t o passers-by.
The most
widespread problem a s s o c i a t e d w i t h t r a n s p l a n t s i s vandalism. This appears
t o be a f a i r l y u n i v e r s a l problem a s s o c i a t e d w i t h the use of unattended
f i e l d equipment and i s exacerpated by use of m a t e r i a l s which a t t r a c t
a t t e n t i o n , reaches which are c l o s e t o h a b i t a t i o n and by experiments d u r i n g
school h o l i d a y s . E f f o r t s may be made t o minimise these problems; p a t t e r n
I I bags were made from a b r i g h t orange m a t e r i a l which made them conspicuous
w h i l s t p a t t e r n I moss-bags were c o l o u r e d green (Table 5.1). No data were
c o l l e c t e d on the r e l a t i v e q u a l i t i e s of each w i t h r e s p e c t t o vandalism but
the d a r k e r c o l o u r i s l i k e l y t o a t t r a c t fewer casual passers-by.
Anglers
w i l l t a k e a g r e a t e r i n t e r e s t i n the stream and are l i k e l y t o spot any
o b j e c t , p a r t i c u l a r l y one which i s l i a b l e t o snag a l i n e ; they w i l l a l s o be
equipped w i t h waders e t c . t o remove them. Anglers are a l s o l i k e l y t o have
an above-average i n t e r e s t i n water q u a l i t y and may be e n t h u s i a s t i c i f they
know why the bags have been placed t h e r e .
The main d i f f e r e n c e i n the way
i n which the moss i s exposed t o water
between boulders and bags i s t h a t i n the l a t t e r method the moss l i e s
t o the bed of the stream and i s t h e r e f o r e c l o s e r t o i t s n a t u r a l
closer
position.
R e s u l t s i n 5.31 show t h a t t h e r e i s no s i g n i f i c a n t d i f f e r e n c e i n
a c c u m u l a t i o n r a t e s between moss i n these two p o s i t i o n s .
A f i n a ] c o n s i d e r a t i o n i f t h e method i s t o be proposed f o r r o u t i n e
m o n i t o r i n g purposes, i s t o determine the p o i n t a t which the t r a n s p l a n t e d
273
moss i s s u i t a b l e f o r use as a m o n i t o r . This would be a t t h e p o i n t when
f l u c t u a t i o n s i n t h e c o n c e n t r a t i o n i n t h e water w i l l be r e f l e c t e d by changes
i n t h e c o n c e n t r a t i o n i n t h e moss. This i s o b v i o u s l y n o t d u r i n g t h e r a p i d
phase; however once t h e r a t e o f accumulation has slowed down then i t i s
p o s s i b l e t h a t a change i n t h e c o n c e n t r a t i o n o f metal i n t h e water may be
d e t e c t e d by a change i n the c o n c e n t r a t i o n i n t h e moss. ^ The r a p i d phase
l a s t e d f o r a p p r o x i m a t e l y 6 h ( c h a p t e r 5) and accumulation o f z i n c and
cadmium c o n t i n u e d a t a slow r a t e over f i v e or s i x days and accumulation o f
l e a d c o n t i n u e d over a longer p e r i o d o f time ( 6 . 2 ) . At t h e end of t h i s
p e r i o d moss responded t o changes i n aqueous metal c o n c e n t r a t i o n s i n the
same way as indigenous p o p u l a t i o n s ( 9 . 3 1 ) . This may be a long time i f a
r o u t i n e survey u s i n g moss-bags i s envisaged; however i n s i t u a t i o n s where
two s i t e s (e.g. above and below a d i s c h a r g e ) are being compared then
accumulation over a s h o r t e r p e r i o d o f time (e.g. 24 h) may be s u f f i c i e n t .
In 6.2 t h e d i f f e r e n c e s i n f i n a l c o n c e n t r a t i o n s o f z i n c accumulated were
a s c r i b e d t o v a r i e t a l d i f f e r e n c e s ( 9 . 3 1 ) . This i s i t s e l f a p o t e n t i a l
of
source
c o n f u s i o n ; i n 6.2 t h e d i f f e r e n c e a t t = 23 d was s i g n i f i c a n t a t
p > 0.05 and i f s i g n i f i c a n t d i f f e r e n c e s a r e t o be taken as evidence of
metal c o n t a m i n a t i o n , then e f f o r t s must be made t o overcome such v a r i e t a l
differences.
S i g n i f i c a n t d i f f e r e n c e s between p o p u l a t i o n s were a l s o found
by Wehr (1983).
The s i m p l e s t method i s t o c o l l e c t a l l Rhynchostegium from
a s i n g l e source and t o use t h i s i n a l l t r a n s p l a n t experiments.
9.42 Experience
o f case study
P a r t of t h e preceding d i s c u s s i o n i s i l l u s t r a t e d by t h e experience of t h e
case study ( c h a p t e r 8 ) . On s e v e r a l occasions
stakes and bags were l o s t ,
p r o b a b l y due t o i n t e r f e r e n c e by passers-by (8.322); however t h i s study
demonstrated
the f e a s a b i l i t y o f u s i n g a q u a t i c bryophytes
i n conjunction
w i t h s t r a i g h t f o r w a r d water samples f o r m o n i t o r i n g i n t e r m i t t e n t p o l l u t i o n .
274
e s p e c i a l l y when t h e c o n c e n t r a t i o n s i n t h e water approach the d e t e c t i o n
l i m i t f o r a p a r t i c u l a r method (8.321). I n t e r p r e t a t i o n a t t h r e e l e v e l s was
p o s s i b l e . C o n c e n t r a t i o n s a t 0267-80 i n d i c a t e d the presence of an i n p u t
between the two reaches ( F i g . 8.2); an e x t e n s i o n o f t h i s t o i n c l u d e s i t e s
upstream o f 0267-80 would have been u s e f u l i n d e t e c t i n g sources of the low
chromium c o n c e n t r a t i o n s measured i n p l a n t s i n these reaches. The second
approach used t h e computer database t o p r e d i c t a q u a t i c c o n c e n t r a t i o n s
( 8 . 5 3 ) . The l a t t e r approach was hampered by lack of a s u i t a b l y s i z e d
database f o r each o f the t h r e e species which might have a l l o w e d
m u l t i v a r i a t e s t a t i s t i c s t o be used and a more a c c u r a t e p r e d i c t i o n t o be
made. F i n a l l y , by i n c l u d i n g l a b o r a t o r y experiments i t was p o s s i b l e t o
p r e d i c t the chemical species of chromium i n the r i v e r (8.4. 8.52).
9.43
A q u a t i c bryophytes as m o n i t o r s
Both indigenous and t r a n s p l a n t e d p o p u l a t i o n s of Rhynchostegium are
s u i t a b l e f o r use as m o n i t o r s .
With indigenous p o p u l a t i o n s t h e r e may
be
problems a s s o c i a t e d w i t h v a r i e t a l d i f f e r e n c e s (9.31); Rhynchostcgium
is
f r e q u e n t l y observed w i t h capsules (Smith, 1978) and i t has been suggested
t h a t these p l a y a s i g n i f i c a n t r o l e i n spore d i s p e r s a l (Wehr, 1983).
There
i s a p o s s i b i l i t y of v a r i e t a l d i f f e r e n c e s i n p o p u l a t i o n s along a r i v e r or
stream compared w i t h p o p u l a t i o n s of F o n t i n a l i s a n t i p y r e t i c a , f o r example,
which o n l y r a r e l y produces spores (Smith, 1978) and whose d i s p e r s a l i s
p r o b a b l y l a r g e l y e f f e c t e d by v e g e t a t i v e fragments c a r r i e d downstream (Glime
e t al.. , 1979).
There are no isoenzyme or s i m i l a r s t u d i e s on a q u a t i c
bryophytes t o c o n f i r m t h i s .
Any such d i f f e r e n c e s may be a f u n c t i o n of the
d i s t a n c e downstream between p o p u l a t i o n s which, i n the case o f many
m o n i t o r i n g s t u d i e s i s l i k e l y t o be q u i t e s m a l l .
There are a number of ways i n which these bryophytes may be used; i n the
t e r m i n o l o g y o f Goltermann
e t a l . (1978) these may
divided into d i f f e r e n t
275
" l e v e l s " depending upon t h e needs and t h e f a c i l i t i e s of d i f f e r e n t water
m o n i t o r i n g bodies. The f i r s t " l e v e l " i s as a r e c o r d o f a suspected
p o l l u t i o n source by a concerned member o f t h e p u b l i c . A s c e n a r i o i s
envisaged where a member o f an a n g l i n g c l u b observes a f i s h k i l l and
c o n t a c t s t h e water a u t h o r i t y . A sample o f moss c o l l e c t e d a t t h e time may
s u b s e q u e n t l y be analyzed i f heavy metals a r e suspected. A second " l e v e l "
would a p p l y t o a suspected p o l l u t i o n source known t o a Water A u t h o r i t y ; a
p r e l i m i n a r y f i e l d assessment o f t h e s i t e would be f o l l o w e d by a planned
s e r i e s o f t r a n s p l a n t s o r samples and t h e i r subsequent a n a l y s i s . Such
s t u d i e s were c a r r i e d o u t by Harding (1979; 1980) i n Wincham Brook,
Cheshire. I t may be necessary a t t h i s stage t o take d u p l i c a t e o r
t r i p l i c a t e samples i n t h e manner o f "Formal Samples" (Toms, 1975), which
are a v a i l a b l e f o r independent a n a l y s i s . The f i n a l " l e v e l " i s an e x t e n s i o n
of t h e second l e v e l , feedback from which may lead t o a d d i t i o n a l samples or
a f i e l d o r l a b o r a t o r y experiment t o c o n f i r m t h e chemical species (e.g. 8.4)
or t h e accumulation o r l o s s p r o p e r t i e s o f a p a r t i c u l a r p o p u l a t i o n .
I t is
a t t h i s stage t h a t a knowledge of t h e l o c a l i z a t i o n of t h e metal ( c h a p t e r s 6
& 7) may p l a y a s i g n i f i c a n t r o l e .
The p r e v i o u s paragraph presupposes
t h a t t h e study i s t o be c a r r i e d out
i n an area where f a c i l i t i e s e t c . a r e a v a i l a b l e ; t h i s may n o t be t h e case i n
remote o r t h i r d w o r l d r e g i o n s .
W h i t t o n e t al. ( 1 9 8 J ) l i s t t e n advantages
of p l a n t samples over water samples; these i n c l u d e t h e i r ease o f
collection,
storage and t r a n s p o r t which may make them p o t e n t i a l l y
useful
f o r Environmental Impact Assessments and p i l o t s t u d i e s i n these r e g i o n s .
E f f e c t i v e use of such techniques would r e q u i r e an understanding of t h e
r e l a t i o n s h i p between d i s s o l v e d and accumulated
c o n c e n t r a t i o n s o f heavy
metals f o r t h e species i n q u e s t i o n ; t h i s i s p r e s e n t l y known f o r o n l y a few
species ( W h i t t o n e t a l . , 1982; Say & W h i t t o n , 1983; Wehr & W h i t t o n , 1983a)
and t h e r e may be enough data i n t h e l i t e r a t u r e t o e s t a b l i s h
relationships
276
f o r a few more.
The v a l u e o f t h e data may be enhanced by a knowledge o f pH
and a q u a t i c c a l c i u m c o n c e n t r a t i o n a l t h o u g h i t may be p o s s i b l e t o s u b s t i t u t e
c o n d u c t i v i t y f o r the l a t t e r .
I n such cases t h e mosses are being used
s i m p l y as " c o n c e n t r a t o r s " o f a q u a t i c metals and i t may be p o s s i b l e t o
d e v i s e s i m p l e methods f o r t h e e x t r a c t i o n and subsequent c o l o r i m e t r i c
a n a l y s i s which would r e p r e s e n t a genuine
" I n t e r m e d i a t e Technology"
approach
t o water q u a l i t y m o n i t o r i n g .
Some s t u d i e s have i g n o r e d t h e taxonomy o f bryophyte samples and i n s t e a d
analyzed aggregates c o m p r i s i n g s e v e r a l species (Whitehead & Brooks, 1969;
P r i g g , 1977).
This i s a f u r t h e r p o t e n t i a l s i m p l i f i c a t i o n of t h e method
which may have some v a l u e i n p a r t i c u l a r circumstances; d i f f e r e n t i a l
a c c u m u l a t i o n by d i f f e r e n t species ( B u r t o n & Peterson, 1979; Say e t a l . ,
1981; Wehr & W h i t t o n , 1983b) means t h a t non-homogeneous samples may g i v e
variable
results.
An example o f t h e use of bryophytes t o supplement a n a l y s i s of water
samples i s g i v e n i n chapter 8; data on chromium accumulation by
Rhynchostegium t r a n s p l a n t e d i n t o 0267-90 (8.31) were compared w i t h s i m i l a r
data c o l l e c t e d i n t h e l a b o r a t o r y (8.42) t o g i v e some i n d i c a t i o n o f t h e
s p e c i a t i o n o f t h e chromium; i n t h i s study i t was i n t h e form of
C r ( I I I ) ( 8 . 5 2 , 9.42).
A second use i s t o i n v e s t i g a t e r a t e s o f accumulation
and l o s s by a p a r t i c u l a r p o p u l a t i o n i n a p a r t i c u l a r reach.
This may f o l l o w
a sample o f indigenous moss which c o n t a i n e d e l e v a t e d c o n c e n t r a t i o n s o f
heavy m e t a l s , p o s i n g t h e q u e s t i o n s how l o n g ago d i d t h e episode occur and
what was i t s c o n c e n t r a t i o n ?
I t may n o t be p o s s i b l e t o answer these
a c c u r a t e l y ; an e s t i m a t e o f t h e d i s s o l v e d metal c o n c e n t r a t i o n corresponding
to the c o n c e n t r a t i o n i n the plant i s possible.
Results i n 6.54 I n d i c a t e
t h a t t h e r a t e o f l o s s was dependent upon t h e l e n g t h of time of exposure t o
the metal which may n o t be known; however t h e approximate time o f t h e event
may be and c o n c e n t r a t i o n s e s t i m a t e d by b a c k - e x t r a p o l a t i o n from F i g . 6.13
277
f o r d i f f e r e n t l e n g t h s o f exposure g i v e a range o f values i n d i c a t i n g e i t h e r
r a p i d l o s s from a b r i e f exposure t o a h i g h c o n c e n t r a t i o n or slower l o s s
from l o n g e r exposure t o lower c o n c e n t r a t i o n s .
Everard and Denny (1985b) used a " l e v e l I I I " approach
f l u x e s of lead i n Ullswater.
approach
This fundamental
to investigate
study r e q u i r e d a d i f f e r e n t
to straightforward monitoring studies.
I t was not p o s s i b l e , f o r
example, s i m p l y t o r e l a t e metal c o n c e n t r a t i o n s i n moss t o metal
c o n c e n t r a t i o n s i n water; however they were a b l e t o show the r o l e o f
t u r b u l e n c e i n i n f l u e n c i n g metal accumulation.
9.5
CONCLUDING REMARKS
The range o f a p p l i c a t i o n s of a q u a t i c bryophytes f o r m o n i t o r i n g heavy
metal p o l l u t i o n i s p o t e n t i a l l y wide (9.43) and may
applied i n nature.
be both fundamental
and
The accumulation of heavy metals by Rhynchostegium i s
r a p i d ( c h a p t e r s 5 & 6) and i s uncomplicated by i n t e r a c t i o n s w i t h o t h e r
components of the food chain and movement (compared w i t h accumulation by
a n i m a l s ) or w i t h accumulation through r o o t s (compared w i t h h i g h e r p l a n t s ) .
Rhynchostegium i s a l s o r e l a t i v e l y p e r e n n i a l ( c h a p t e r 4 ) .
W h i l s t the l a c k
o f a s u i t a b l e substratum may be a l i m i t a t i o n t o c o l o n i z a t i o n by
Rhynchostegium under n a t u r a l c o n d i t i o n s , the use of moss-bags extends the
p o s s i b l e range of s i t u a t i o n s where i t may
be used.
deploy and the basic technique ( c h a p t e r 5) may
range of l o c a l circumstances
a c c u m u l a t i o n , comparing
( 9 . 4 1 , 9.43).
Moss-bags are cheap t o
be adapted t o s u i t a wide
The b i v a r i a t e models of metal
metaJs i n streamwater w i t h metals i n mosses i s a
u s e f u l s t a r t i n g p o i n t f o r case s t u d i e s o f i n t e r m i t t e n t heavy metal
pollution.
These r e l a t i o n s h i p s are maintained by a range of p h y s i o l o g i c a l phenomena
( 9 . 3 1 ; 9.32).
I n t r a c e l l u l a r accumulation i s as i m p o r t a n t as exchange-
a d s o r p t i o n (6.3) and i s fundamental
t o the r e t e n t i o n o f heavy metals a f t e r
278
i n t e r m i t t e n t p o l l u t i o n events (6.55). Knowledge of these processes i s
s t i l l l i m i t e d by the lack of a fundamental p h y s i o l o g i c a l examination of
heavy metal uptake i n these species ( 9 . 3 1 ) ; however these w i l l c o n t r i b u t e
more t o fundamental botany than t o a p p l i e d m o n i t o r i n g where knowledge of
the heavy metai accumulation p r o p e r t i e s of v a r i o u s species w i l l p r o v i d e a
s u i t a b l e framework f o r a wide range of a p p l i c a t i o n s .
279
SUTvIMARY
1.
A study,
combining both f i e l d
and l a b o r a t o r y experiments, was made o f
heavy metal a c c u m u l a t i o n and l o s s by a q u a t i c .9.osses w i t h p a r t i c u l a r
reference
t o t h e i r s u i t a b l i t y f o r use as i n s i t u or t r a n s p l a n t e d
monitors.
2.
The growth r a t e o f Rhynchostegium r i p a r i o i d e s was s t u d i e d a t f o u r s i i c s
i n t h e N o r t h e r n Pennines u s i n g c o t t o n tags t o measure shoot
Growth c o n t i n u e d
extension.
t h r o u g h o u t t h e 12-oonth p e r i o d v j i t h maxisua growth
r a t e i n t h e s p r i n g and a s m a l l e r peak i n t h e autumn.
Growth r a t e was
r e l a t e d s t r o n g l y t o water temperature and maxi-ium and nini.T!UE a i r
t e m p e r a t u r e s , o f a number o f e n v i r o n m e n t a l v a r i a b l e s measured.
There
were a l s o d i f f e r e n c e s between growth r a t e s i n d i f f e r e n t streams,
apparently
it
o f n u t r i e n t s o r heavy metals;
i s suggested t h a t these may r e l a t e t o d i f f e r e n c e s i n t h e l e n g t h o f
time
3.
not r e l a t e d t o concentrations
emersed.
F i e l d experiments were used t o e v a l u a t e
t r a n s p l a n t e d a q u a t i c mosses.
mesh-bags as c o n t a i n e r s f o r
There were no s i g n i f i c a n t d i f f e r e n c e s
between a c c u m u l a t i o n o f heavy metals by moss on boulders o r i n bags or
between a c c u m u l a t i o n o f z i n c by moss i n t h r e e d i f f e r e n t p a t t e r n s o f
bag.
There were some s i g n i f i c a n t d i f f e r e n c e s between r e p l i c a t e bags o f
Rhynchostegium over a 12-h p e r i o d and t h e r e was a l s o s l i g h t l y reduced
a c c u m u l a t i o n a t t h e c e n t r e o f bags packed w i t h l a r g e q u a n t i t i e s of
moss.
The t e c h n i q u e was, however, g e n e r a l l y r o b u s t and a p p l i c a b l e t o a
range o f s i t u a t i o n s .
280
4.
An experiment u s i n g F o n t i n a l i s a n t i p y r e t i c s i n place of Rhynchostegium
was
a l s o performed.
There was
no s i g n i f i c a n t d i f f e r e n c e i n
a c c u m u l a t i o n of z i n c between ? o n t i n a l i s on boulders or i n bags.
t h i s species may
5.
Use
of
extend the range of a p p l i c a t i o n of moss-bags.
An e l u t i n g agent, t o remove z i n c from exchange s i t e s , K E S used t o study
i n t r a c e l l u l a r l o c a t i o n s of z i n c .
was
chosen i n p r e f e r e n c e t o EDTA.
accumulated d u r i n g the f i r s t
and
A f t e r p r e l i m i n a r y expsrir.3nts
T h i s removed about 70Ss of z i n c
12-h.
T h i s p r o p o r t i o n decreased over t i s e
a f t e r 14 d l e s s than 50% of z i n c was
f r a c t i o n ; however t h i s may,
conditions.
^^Cl^
l o c a t e d i n the exchangeable
i n p a r t , be an a r t e f a c t of batch c u l t u r e
Accumulation by both exchangeable and
f i t t e d s a t u r a t i o n k i n e t i c s over the f i r s t
residual fractions
12 h; t h e r e a f t e r o n l y
a c c u m u l a t i o n by the r e s i d u a l f r a c t i o n d i d .
6.
The
p o s s i b l e r o l e of m e t a b o l i c a c t i v i t y was
manipulating
the p l a n t ' s environment.
tested i n d i r e c t l y
by
R e s u l t s of s t u d i e s on the e f f e c t
o f t e m p e r a t u r e and m e t a b o l i c i n h i b i t o r s i n d i c a t e d no d i r e c t c o n t r o l by
metabolism on z i n c a c c u m u l a t i o n by Rhynchostegium.
Studies on
the
e f f e c t of l i g h t gave ambiguous r e s u l t s .
7.
The
s u b s t a n t i a l c e l l w a l l of Rhynchostegium was
capable of adsorbing
l a r g e q u a n t i t i e s of i o n s ( > 12000 yg g " z i n c from a s i n g l e - s a l t
s o l u t i o n ) ; calcium
8.
was
the dominant i o n adsorbed from n a t u r a l waters
and
t h i s had
a s t r o n g i n f l u e n c e on z i n c a d s o r p t i o n .
was
bound more t i g h t l y by the oxide d e p o s i t on the surface of the
Exchange of z i n c f o r d i f f e r e n t ions was
c o m p l e t i o n w i t h i n t h r e e minutes.
+
s i n g l e i o n s o l u t i o n s of
1:1.
for
H
cell.
r a p i d and reached 90?o
On a molar b a s i s exchange of z i n c f o r
+
, K
A further fraction
2+
and
Ce
was
i n the r a t i o of less than
There were no s i g n i f i c a n t d i f f e r e n c e s between exchange of zinc
monovalent or d i v a l e n t c a t i o n s .
281
9.
Three species o f a q u a t i c moss were used as monitors of i n t e r m i t t e n t
chromium p o l l u t i o n i n t h e R. C r o a l , N-W.
s i t e o f a disused s m e l t e r .
England, above and below t h e
There was evidence
of s l i g h t c o n t a m i n a t i o n
i n t h e r i v e r above t h e s m e l t e r ; however c o n c e n t r a t i o n s of chromium i n
moss below t h e s m e l t e r were c o n s i d e r a b l y h i g h e r .
There was no
r e l a t i o n s h i p between chromium c o n c e n t r a t i o n s i n water and i n moss which
supports o t h e r evidence o f p e r i o d i c f l u c t u a t i o n s i n t h e c o n c e n t r a t i o n
of chromium i n t h e r i v e r .
10. L a b o r a t o r y s t u d i e s showed d i f f e r e n t i a l accumulation of C r ( I I I ) and
O r ( V I ) by Rhynchostegium i n l a b o r a t o r y batch c u l t u r e .
These r e s u l t s
were a p p l i e d t o t h e case study o f chromium p o l l u t i o n and confirmed t h e
presence o f C r ( I I I ) i n water.
11. A s t a n d a r d method f o r u s i n g a q u a t i c bryophytes
p o l l u t i o n i s suggested.
This involves t r a n s p l a n t i n g
above and below a suspected
predetermined
t o monitor heavy metal
discharge.
moss i n moss-bags
These are l e f t f o r a
l e n g t h of time b e f o r e b e i n g c o l l e c t e d and analyzed
a c c o r d i n g t o standard methods.
An e l e v a t e d c o n c e n t r a t i o n o f metal i n
the moss below t h e discharge i s an i n d i c a t i o n t h a t t h e r e has been a
r e l e a s e o f heavy metals d u r i n g t h e p e r i o d o f exposure.
12. I t i s argued s t r o n g l y
t h a t a q u a t i c bryophytes are s u i t a b l e as monitors
f o r a wide range o f heavy metal p o l l u t i o n problems.
From t h e e x i s t i n g
framework o f knowledge, m o n i t o r i n g techniques may be adapted f o r many
s i t u a t i o n s and a t a v a r i e t y o f " l e v e l s " o f s o p h i s t i c a t i o n .
These
i n c l u d e s i t u a t i o n s o f i n t e r m i t t e n t heavy metal p o l l u t i o n where
accumulation o f heavy metals i n t o a compartment which i s n o t r a p i d l y
exchangeable may p r o v i d e a r e c o r d of t h e event f o r some time a f t e r i t
has
occurred.
282
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r i v e r s w i t h e l e v a t e d z i n c . pp. 457-463. I n : Hemphill D.D. (Ed.) Trace
Substances i n Environmental Health-XIV.
U n i v e r s i t y of M i s s o u r i ,
Columbia.
M.T.H.
W h i t t o n B.A., Diaz B.M. & Holmesi(1976).
A computer o r i e n t a t e d r e c o r d i n g
system f o r p l a n t s i n f l o w i n g waters. 52pp. D u p l i c a t e d r e p o r t .
Department of Botany, U n i v e r s i t y of Durham, England.
W h i t t o n B.A. & Say P.J. (1975). Heavy Metals. pp. 286-311.
B.A. (Ed.) R i v e r Ecology. B l a c k w e l l , Oxfo'^d.
I n : Whitton
W h i t t o n B.A., Say P.J. & Jupp B.P. (1982). Accumulation of z i n c , cadmium
and lead by t h e a q u a t i c l i v e r w o r t Scapania. Environmental P o l l u t i o n
S e r i e s B 3, 299-316.
W h i t t o n B.A., Say P.J. & Wehr J.D. (1981). Use of p l a n t s t o monitor heavy
metals i n r i v e r s , pp.135-146. I n : Say P.J. & W h i t t o n B.A. (Eds) Heavy
Metals i n N o r t h e r n England: Environmental And B i o l o g i c a l Aspects.
Department of Botany, U n i v e r s i y of Durham, England.
Wilhm J,F. (1975). B i o l o g i c a l i n d i c a t o r s of p o l l u t i o n .
pp. 375-402.
W h i t t o n B.A. (Ed.) R i v e r Ecology. B l a c k w e l l , Oxford.
In:
W i l k i n s P. (1977). Observations on ecology of M i e l i c h h o f e r i a elongata and
o t h e r "copper mosses" i n the B r i t i s h I s l e s . B r y o l o g i s t 80, 175-181.
Winner R.W.
(1984). The t o x i c i t y and b i o a c c u m u l a t i o n of cadmium and copper
as a f f e c t e d by humic a c i d . Aquatic T o x i c o l o g y 5, 267-274.
Woodiwiss F.S. (1964), The b i o l o g i c a l system of stream c l a s s i f i c a t i o n used
by the T r e n t R i v e r Board, Chemistry and I n d u s t r y
443-447,
302
APPENDIX 1
METHOD FOR USING MOSS-BAGS TO MONITOR HEAVY METAL POLLUTION
INTRODUCTION
T h i s method i s f o r comparison o f metal c o n c e n t r a t i o n s i n samples o f
Rhynchostegium r i p a r i o i d e s o r F o n t i n a l i s a n t i p y r e t i c a from above and below a
discharge.
I t i s p a r t i c u l a r l y u s e f u l i f t h e discharge i s suspected t o be
i n t e r m i t t e n t and thus may be missed i f water analyses alone a r e performed.
The method i n v o l v e s t r a n s p l a n t i n g moss i n t o t h e reach.
I f there i s a large
p o p u l a t i o n o f moss i n t h e reach then a t r a n s p l a n t may be unnecessary and i n
s i t u moss may be used i n s t e a d . I t i s based upon work performed i n Durham,
from 1979 t o 1986. F u r t h e r d e t a i l s o f metal accumulation by Rhynchostegium
T.ay be found i n t h i s t h e s i s , Wehr (1983) and Wehr & Whitton (1983a; :983b)
and by F o n t i n a l i s a n t i p y r e t i c a i n Say & W h i t t o n (1983). A comparison o f
methods f o r p r o c e s s i n g mosses i s r e p o r t e d i n Wehr e t a l . (1983) and a study
on methods f o r u s i n g moss-bags i n t h i s t h e s i s .
PRINCIPLE OF METHOD
A q u a t i c mosses a r e t r a n s p l a n t e d i n t o reaches above and below a suspected
d i s c h a r g e . A f t e r a s t a n d a r d p e r i o d o f immersion they a r e removed, d i g e s t e d
i n n i t r i c a c i d and t h e i r metal c o n c e n t r a t i o n deter.-nined.
APPARATUS
field
acid-washed c o n t a i n e r f o r c o l l e c t i o n o f moss
ice-box
stakes
moss-bags
twine
c l u b hammer
p o l y e t h y l e n e b o t t l e t o t r a n s f e r moss t o l a b o r a t o r y
laboratory
p e t r i dishes
c r y s t a l l i s i n g dishes
forceps
scalpel
d r y i n g oven, s e t a t 105 "C
heating block
b o i l i n g tubes
bench-top c e n t r i f u g e
25 ml v o l u m e t r i c f l a s k s
atomic a b s o r p t i o n spectrophotometer
reagents
d e i o n i z e d water
2 M KNO (atomic a b s o r p t i o n grade)
s t a n d a r d s o l u t i o n s f o r atomic a b s o r p t i o n
spectrophotometry
METHOD
1 C o l l e c t i o n o f moss
1.1 The reach from which moss i s c o l l e c t e d should be f r e e fron; any
p o s s i b i l i t y o f c o n t a m i n a t i o n by heavy metals.
303
1.2 The reach i s d e f i n e d w i t h r e f e r e n c e t o one or more e a s i l y r e l o c a t a b l e landmarks
1.3 The p o p u l a t i o n o f Rhynchostegium a t t h e reach should n o t be
s i g n i f i c a n t l y reduced by removing moss f o r t r a n s p l a n t i n g . The moss
should be a v a i l a b l e t h r o u g h o u t t h e year.
1.4 C o l l e c t i o n o f moss i s r e s t r i c t e d t o p l a n t s which a r e f u l l y submerged
and, as much as p o s s i b l e , l o c a t e d w i t h i n t h e area o f maximum c u r r e n t
velocity.
P l a n t s a r e c o l l e c t e d from a t l e a s t f i v e separate l o c a t i o n s
w i t h i n t h e reach.
1.5 The moss i s r i n s e d and shaken s e v e r a l times i n stream vjatsr t o remove
a s s o c i a t e d sediments, i n v e r t e b r a t e s and e n t a n g l e d f i l a m e n t o u s algae.
Excess water i s squeezed from t h e p l a n t s , vjhich a r e s t o r e d i n a
p o l y e t h y l e n e beaker r i n s e d s e v e r a l times i n stream water.
1.6 Mosses a r e s t o r e d i n an ice-box and a r e t r a n s f e r r e d immediately t o
the m o n i t o r i n g s i t e .
2 Use o f moss-bags
2.1 A p p r o x i m a t e l y 15 - 20 g (wet w e i g h t ) o f moss i s p u t i n t o each bag.
T h i s p r o v i d e s about t w i c e as .many 2-cm t i p s as i s r e q u i r e d f o r one
sample f o r a n a l y s i s . One o r two samples may be r e t a i n e d f o r
independent a n a l y s i s a t t h i s stage.
2.2 S t e e l stakes a r e p o s i t i o n e d above and below t h e d i s c h a r g e , away from
t h e bank, i n an area w i t h a f a s t c u r r e n t and a r e d r i v e n i n t o t h e
stream bed. The bags a r e a t t a c h e d t o these by t w i n e so t h a t they
remain submerged.
2.3 A suggested sampling program i s t o analyze samples from each reach
every f o r t n i g h t , unless t h e r e i s a suspected p o l l u t i o n i n c i d e n t , when
a sample s h o u l d be taken as soon as p o s s i b l e and a t i n t e r v a l s o f
a p p r o x i m a t e l y 12 h a f t e r t h i s .
2.4 The sample i s removed from, t h e bag, washed s e v e r a l times i n stream
water, squeezed f r e e o f excess m o i s t u r e and placed i n a c o n t a i n e r .
I t i s t r a n s f e r r e d t o t h e l a b o r a t o r y i n an ice-box.
3 Laboratory treatment
3.1 I f samples cannot be t r e a t e d immediately then they a r e s t o r e d i n a
r e f r i g e r a t o r o v e r n i g h t and t r e a t e d t h e f o l l o w i n g morning.
3.2 The moss i s r i n s e d t h o r o u g h l y i n a stream o f d i s t i l l e d water t o
re.move obvious a t t a c h e d sediment and i s t r a n s f e r r e d t o d e i o n i z e d
water where 2-cm t i p s a r e removed. T h i s u s u a l l y i n c l u d e s t h e m.ain
a x i s and s e v e r a l branches.
3.3 A p p r o x i m a t e l y 125 t i p s a r e taken t o g i v e a f i n a l d r y weight o f about
200 mg. These a r e washed two t o t h r e e times i n d e i o n i z e d water and
d i v i d e d i n t o f i v e sub-samples which a r e placed i n acid-washed v i a l s
and d r i e d t o a c o n s t a n t w e i g h t . I t i s suggested t h a t a d d i t i o n a l
samples f o r independent a n a l y s i s a r e s t o r e d a t t h i s stage.
3.4 D r i e d sub-sa.mples a r e d i g e s t e d i n 5 ml o f 2 M riNO^ a t about 120 °C f o r
45 minutes and a l l o w e d t o c o o l . Sub-samples a r e c e n t r i f u g e d t o
s e p a r a t e t h e c l e a r s o l u t i o n from t h e s l u r r y and t h e c l e a r s o l u t i o n i s
poured i n t o a 25 ml v o l u m e t r i c f l a s k . The s l u r r y i s r e - c e n t r i f u g e d
w i t h d e i o n i z e d water and t h e s u p e r n a t a n t i s added t o t h e v o l u m e t r i c
f l a s k and made up t o volume.
3.5 Samples a r e analyzed by atomic a b s o r p t i o n spectrophotometry a g a i n s t
acid-matched b l a n k s and standards. The r e s u l t s a r e converted from .T.ass
of metal per u n i t volume t o mass o f metal per u n i t mass as f o l l o w s :
304
cone.
i n sample
(yg g~ )
cone, i n d i g e s t
(mg 1 M
X
25 m]
X
1000
mass o f sample (mg)
3.6 The mean, s t a n d a r d d e v i a t i o n a n d 9 5 % c o n f i d e n c e l i m i t s a r e c a l c u l a t e d
f o r e a c h s a m p l e a n d t h e s a m p l e s f r o m above a n d b e l o w t h e d i s c h a r g e
compared.
COST PER SAMPLE
These c o s t s a r e c a l c u l a t e d assumming o n l y v e r y b a s i c l a b o r a t o r y
f a c i l i t i e s are available.
C o s t s a r e based upon p r i c e s i n O c t o b e r 1986,
e x c l u s i v e o f V.A.T. a t 15%.
1. C a p i t a l
items t o equip f o r process
cost
centrifuge
£300
heating block
£290
automatic p i p e t t e
£ 70
moss- bags ( 1 )
05 p
stakes ( 2 )
.50 D
£660.00
Recurrent items
a c i d (AAS g r a d e )
staff (3)
replacement o f c a p i t a l items
overheads ( e l e c t r i c i t y , gas)
3. A n a l y s i s
AAS ( a t c o m m e r c i a l
rates)
.02 p
£ 12.00
.10 p
.30p
£ 12.42
£ 20.00
NOTES
Mesh b a g s ^ w e r e made f r o m a g a r d e n mesh f r o m a l o c a l n u r s a r y a t a c o s t
o f 40p m
f o r a 2 m wide r o l l .
N y l o n m o n o f i l a m e n t f i s h i n g l i n e was
used t o sew t h i s i n t o bags o f a p p r o x i m a t e l y 15 x 20 cm ( 5 . 2 ) .
I fi t is
assumed t h a t 20 bags may be made f r o m 1 m o f mesh t h e n t h e c o s t p e r bag
i s n e g l i g a b l e . Bags may be r e - u s e d .
S t a k e s w e r e made f r o m m i l d s t e e l ar^d c o s t 50 p e a c h .
L a b o u r i s assumed t o c o s t £3.85 h
305
APPENDIX 2
RELATIONSHIP BETWEEN LANGMUIR ISOTHERM AND
MICHAELIS-MENTON EQUATION
The M i c h a e l i s - M e n t o n e q u a t i o n o f enzyme k i n e t i c s and t h e L a n m u i r i s o t h e r m
o f p h y s i c a l c h e m i s t r y b o t h r e l a t e r e a c t i o n s t o t h e number o f a v a i l a b l e
r e a c t i o n s i t e s and h a v e t h e same b a s i c f o r m .
The M i c h a e l i s - M e n t o n e q u a t i o n i s u s u a l l y w r i t t e n as:
where:
Xni
^
S
=
V
^
V
=
max
and
1
Km
1
V
V
S
V
max
(1)
M i c h a e l i s Menton c o n s t a n t
substrate concentration
velocity
maximum v e l o c i t y
the Langmuir I s o t h e r m
1
x/m
where:
x/m
^
C
=
b
=
k
=
max
1
as:
1 1
1
C
kb
b
(2)
amount a d s o r b e d p e r u n i t mass
e q u i l i b r i u m c o n c e n t r a t i o n of adsorbate
a d s o r p t i o n maximum
term r e l a t i n g t o bonding energy.
Khen I h e y a r e u s e d t o e x a m i n e Zn u p t a k e t h e n 1/Zn may r e p l a c e 1/V o r
i / ( x / m ) and Zn
i s u s e d t o d e s c r i b e t h e maximum v a l u e o f Zn (= Vmax o r b ) .
max
Tnen:
1
1
1
1
1
kbC
Zn
Zn
max
Let b =
C
max
1
Km
kb
Zn max
1
Km
Zn
max
kZn
max
Zn
1
Km
max
Zn
max