Phosphatase activities of mosses,
algae and cyanobacteria as
biomonitoring tools for upland water
nutrient status
Water Framework Directive and uplands
The WFD derived from gaps in previous legislation to protect Europe’s water.
Two key requirements:
• To classify ecological status and, where necessary, restore the structure and
function of aquatic ecosystems.
• Introduction of a river basin management planning system.
• Biological classification systems have been intercalibrated at EU level for comparability
between member states.
• Present biomonitoring methods can contribute to ecological status classification and
the restoration but mainly for lowland rivers with high inorganic nutrient loads.
• Planned management measures (basic measures) have been set out to tackle pollution
problems
• In upland areas however, ‘basic measures’ are mostly not applicable and so
‘supplementary measures’ need developing.
• Classification using standard monitoring methods of water chemistry can be misleading.
• The implementation of the diatom methodology for the WFD is suited for more
downstream sites (useful for inorganic nutrients, less environment variation).
• Need to develop a new biomonitoring system for uplands
Biological monitoring and upland streams
• The large surface area of upland catchments and the associated meteorological
conditions and exposure to climate warming and atmospheric N deposition all result in
a highly vairable and shifting stream water quality.
• Another confounding feature of upland areas is that much of the filtrable nutrient
fractions/species in flowing waters can be organic, deriving from organic rich soils (e.g.
peat, forested soils).
• At present the ‘basic measures’ of biological classification under the WFD does not
take organic P into consideration.
• Ideal organisms for this purpose should be widespread and tolerant of a range of
nutrient levels, as opposed to species with narrow ecological ranges.
• Organisms should "integrate" the environment over much longer period, such as longlived algal colonies and, ideally, also aquatic bryophytes.
Bioavailability of organic phosphorus
•
Most, if not all, phototrophs can utilize inorganic phosphate in their environment,
and it is commonly assumed that the ability to utilize organic phosphate is more
restricted.
•
Yet there is a very large and increasing body of evidence proving that many
organisms can use organic compounds as a P source.
•
And it has also been shown that organisms can utilize organic phosphates as
their sole phosphorus source, much of the evidence has come indirectly from
studies on phosphatases and phosphatase activity….
•
….and research has shown that many organisms from upland regions have high
surface phosphatase activity and, where tested, can grow on organic P sources,
so organic P is clearly important.
•
Surveys of species distributions and water chemistry have shown that some
organisms are actually favoured by organic P.
S h oot L en gth
(m m )
Growth of organisms using organic phosphorus
0.25 mg L-1 P
90
0.5 mg L-1 P
1 mg L-1 P
60
30
-2
B iofilm D W (m g cm )
Growth of the aquatic moss Warnstorfia fluitans (Hedw.) Loeske from an acidic stream in North-East
England in media containing inorganic P (), PME (G-1-P;  ) and PDE (DNA; ) over 31 days.
1 .2
Organic P type
1 .0
0 .8
Nos of strains
growing
para-nitrophenyl phosphate
50
0 .6
β-glycerophosphate
50
0 .4
bis-para-nitrophenyl phosphate
47
DNA
49
ATP
40
phytic acid
35
0 .2
0 .0
01
05
09
13
17
Biofilms consisting of Chlorococcum sp., Phormidium
sp. and Synechocystis sp. medium containing KH2PO4
at 3.68 mg L-1 P () and 0.368 mg L-1 P and organic P
only (glucose-6-phosphate) at 0.368 mg L-1 P (□).
A survey of 50 cyanobacterial strains cultured in
media containing organic P (Whitton et al. 1991).
Bryophytes and nutrient status of upland areas
• mosses are non-motile, evergreen, relatively tolerant to various pollutants and easy to
find and identify.
• Assessment of atmospheric N deposition studied using the internal N content and
phosphatase activities of terrestrial mosses .
• Mosses growing at terrestrial and aquatic sites display ‘surface’ phosphatase activity,
which varies in response to the concentrations of N and P in the moss tissue. This in
turn reflects the relative availability of these nutrients in the ambient environment.
• Mosses possess the ability to use a wide range of organic phosphate esters present in
the ambient environment.
• Whole catchment monitoring – as broadly the same methods can be used for
terrestrial and aquatic mosses, and similar sets of samples can be used for monitoring
phosphorus status and heavy metal contamination.
Phosphatases: background information
• Phosphatases are enzymes that hydrolyse organic P
compounds:
– Phosphomonoesterases (PMEase) hydrolyse phosphomonoesters
• e.g. sugar phosphates, inositol hexaphosphates
– Phosphodiesterases (PDEase) hydrolyse phosphodiesters
• e.g. DNA, RNA, phospholipids
• Activity can often be demonstrated simply by staining (BCIPNBT, ELF®97).
Phosphatases: background information
Substrate
Product
Enzyme
para-nitrophenyl phosphate (pNPP)
para-nitrophenol
PMEase
4-methyumbelliferyl phosphate (MUP)
methylumbelliferone
PMEase
bis-para-nitrophenyl phosphate (bis-pNPP)
para-nitrophenol
PDEase
bis-4-methylumbelliferyl phosphate (bis-MUP)
methylumbelliferone
PDEase
ELF®97 phosphate
ELF®97 alcohol
PMEase
• Phosphatases are represented by a "whole bunch of enzymes" (Boavida, 1990),
characterized by different half-saturation constants, temperature and pH optima
and substrate specificity.
Phosphatase activity: characterisation
14
C ladoph ora sp .
12
10
8
(mmol pNP mg-1d.wt h-1)
6
4
0.6
A ctivity v T im e
2
0.5
0
3
0.4
4
5
6
40
8
9
10
11
pH
0.3
0.2
7
30
0.1
20
R hynchostegium riparioides
0.0
0
2
4
6
8
10
T im e (h )
12
14
16
10
0
3
4
5
6
7
8
9
10
11
PMEase
PDEase
H an es-W oolf
lin ear tran sform ation
2400
0.5
2000
0.4
[S /V ]
-1
P h osp h atase activity
-1
(m m ol p N P m g d .w t h )
Phosphatase activity: characterisation
0.3
0.2
1600
1200
800
0.1
400
0.0
0
0
100
200
300
400
0
500
100
200
300
400
S u b stra te C o n cen tra tio n (m M )
PMEase and PDEase ctivities of
phytobenthos dominated by Cladophora
glomerata in Lake Albano, Rome, Italy
Vmax
Km
R2
PMEase
0.46
100
0.98
 PDEase
0.33
139
0.93

500
Cranecleugh Burn (CB)
N. T. W. Ellwood, S. M. Haile, B. A.Whitton 2008. Aquatic plant nutrients,
moss phosphatase activities and tissue composition in four upland
streams in northern England. Journal of Hydrology. 350, 246– 260
Cranecleugh Burn (CB)
Yet Burn (YB)
Stonesdale Beck (SD)
Stonesdale Beck
Stony Gill (SG)
Moss species
• Fontinalis antipyretica
(CB, YB, SG)
• Rhynchostegium riparioides
(SG)
• Fontinalis squamosa
(SD)
Moss species
• Fontinalis antipyretica
(CB, YB, SG)
• Rhynchostegium riparioides (SG)
• Fontinalis squamosa
(SD)
Moss species
• Fontinalis antipyretica
(CB, YB, SG)
• Rhynchostegium riparioides
(SG)
• Fontinalis squamosa
(SD)
300
C ra n ecleu g h B u rn
Seasonal streamwater
phosphorus
concentrations
200
100
FRP
FOP
0
300
Y et B u rn
-1
P hosphorus (mg L )
200
100
0
300
S to n esd a le B eck
200
100
0
300
Filtrable Reactive Phosphorus
and
Filtrable Organic Phosphorus
concentrations of the four
sample streams (n = 3)
S to n y G ill
200
100
0
M
M
1999
J
S
N
J
M
2000
M
J
S
N
J
2001
Water quality variability of two upland streams
Stream
Stonesdale
Beck
Stony Gill
Year 1
min
mean
-1
Current (m s )
0.2
0.7
Temp (°C)
0.5
7.7
Cond (µS cm-1) 53.0 115.0
pH
5.9
7.2
NO3-N
18.1 250.5
NH4-N
12.1
35.1
FON
TN
FRP
4.8
55.8
FOP
4.4
88.3
TP
58.4 256.7
Flow (m s-1)
0.09
0.42
Temp (°C)
1.0
8.0
Cond (µS cm-1) 90.0 159.0
pH
7.5
8.1
NO3-N
369.0 802.3
NH4-N
9.1
39.0
FON
TN
FRP
< 2.0
14.7
FOP
6.8
88.8
TP
19.6 167.2
181.8
188.9
539.6
Year 2
min
0.2
1.0
94.0
6.4
13.0
< 2.0
197.0
383.0
2.7
90.8
2.6
72.2
63.9 22.9
(12 samples)
mean max
CV
0.7
1.5 63.9
7.0
13.8 58.1
152.0
266.0 33.0
7.1
7.8
6.0
127.0
371.0 79.4
15.0
60.0 111.2
473.5
929.0 55.5
781.3 1140.0 34.8
7.7
31.1 100.9
39.1
82.0 78.5
88.4
174.2 54.5
0.75
16.4
293.0
8.5
1643.0
86.0
46.6
58.2
38.0
3.7
48.1
51.7
v
0.05
1.4
73.0
7.2
< 2.0
< 2.0
< 2.0
310.0
2.2
99.4
62.3 < 2.0
61.8 10.0
0.34
1.9 147.7
8.0
13.6 50.5
180.0
267.0 35.0
8.0
8.4
4.0
201.7
500.0 69.2
33.3
240.0 202.8
v
293.3
870.0 100.1
738.3 1120.0 36.5
6.3
18.3 72.0
47.4
272.8 160.3
66.8
316.2 127.1
max
1.2
15.3
306.0
8.0
748.1
74.8
47.3
171.8
309.6
CV
44.2
66.2
64.0
7.6
89.9
47.2
Seasonal variation of environmental
and biological variables
160
C o e ffic ie n c y o f va ria tio n (% )
140
120
100
80
60
40
20
e
e
as
as
P
D
E
E
M
P
is
T
is
T
su
su
e
e
P
N
-N
4
H
N
O
N
3
-N
P
O
F
F
R
P
0
Seasonal coefficients of variation of streamwater P status, and moss tissue nutrient
status and surface PMEase and PDEase activities (%)
Relationships between cellular and
ambient water nitrogen and phosphorus
Population
N
P
N:P
F. antipyretica SG
N
P
N:P
F. antipyretica YB N
P
N:P
F. squamosa SD
N
P
N:P
Rhynchostegium
N
P
N:P
Water P fractions
FRP
FOP TP
F. antipyretica CB
TOP
FTP
-0.60 -0.72** -0.80** -0.66*
-0.47**
Water N fractions
NO3
NH3
TIN
0.62*
0.60*
0.60*
-0.45*
-0.41*
0.44*
-0.41*
-0.61*
-0.56**
FON
TFN
TON
TN
-0.56**
-0.47*
-0.68**
-0.69**
0.64* -0.72** 0.61*
Water N : P ratio
TIN:FRP FON:FOP
N:P
0.49*
-0.45*
-0.38*
-0.45*
-0.61** -0.54** -0.45* -0.52**
0.60** 0.57** 0.46* 0.52**
-0.38
-0.36 -0.43* -0.41* -0.39* -0.52**
0.39* 0.47*
-0.79**
-0.48*
0.44*
-0.79**
-0.39*
0.48*
-0.53**
0.39*
-0.40*
180
160
-1
-1
P h osp h atase activity (m m ol p rod u ct g d .w t h )
140
120
MUP
()
80
pNPP
()
60
bis-pNPP ()
100
40
20
0
8
10
12
14
16
18
20
22
24
26
28
T issu e N : P
180
160
MUP
()
pNPP
()
bis-pNPP ()
140
120
100
80
60
40
20
0
1 .0
1 .5
2 .0
2 .5
3 .0
-1
T issu e P (m g g d .w t)
3 .5
4 .0
Relationship
between
phosphatase
activity and tissue
nutrient status
River transect study
Christmas, M & Whitton, B.A. (1998b) Phosphorus and
aquatic bryophytes in the Swale - Ouse river system, northeast England. 2. Phosphomonoesterase and
phosphodiesterase activities of Fontinalis antipyretica.
Science of the Total Environment 210/211: 401-409.
Didymosphenia geminata an
invasive species or and
bioindicator of global warming or
catchment disturbance?
River Coquet
Northumberland
FO P as % FTP
100
90
80
70
60
50
Changes in FOP as % FTP from Jan to Aug
2000.
Stony Gill
North Yorkshire
Global distribution of Didymosphenia geminata
(m m ol p N P m g C h l a -1 h -1 )
P h osp h atase activity
40
P M E ase
40
30
30
20
20
10
10
5
P D E ase
P D E ase : P M E ase
4
3
2
1
0
0
M
A
M
J
J
0
M
A
M
J
J
M
A
M
J
J
Conclusions
• Nutrients fractions in upland environments can be dominated by organic forms
• Increasing evidence indicates that organisms can obtain nutrition from organic
compounds
• Basic biological measures do not take this into consideration
• Phosphatases are ubiquitously produced and simple assays give much information of
nutrient status of environment (PMEase and PDEase)
• Relationships between activity and tissue and environment nutrient status
• Indication of organic nutrient dynamics
• Surveys using longer living organisms that tolerate a wide range of nutrient
conditions, i.e. with a wide distribution, are particularly useful for both spatial and
temporal monitoring
• Changes in activities integrate large ambient variation and better indicate trends.
• Relative concentrations of organic P compared to inorganic P offers competative
advantages to certain species.
• A simple measure, avoiding lengthy taxonomic identification
• Similar methodology for aquatic and terrestrial mosses for whole catchment surveys
Thank you !
Surface phosphatase activity, cellular and ambient
nitrogen and phosphorus
F. antipyretica CB
PMEase PDEase
FRP
FOP
TP
TOP
PP
NO3
NH4
TIN
FON
TON
TN
-0.655*
TIN: FRP
FON:FOP
TN:TP
0.608*
F. antipyretica YB
PMEase PDEase
F. squamosa SD
PMEase PDEase
F. antipyretica SG
PMEase PDEase
R. riparioides SG
PMEase PDEase
0.531** 0.737**
0.427* 0.729**
0.390* 0.560**
0.677** 0.703**
0.666** 0.655**
0.608** 0.632**
0.505**
0.537*
0.676*
0.671*
0.736**
0.695** 0.664**
0.529* 0.488*
0.640** 0.551**
Tissue N
Tissue P
Tissue N:P
* P < 0.05 and ** P < 0.01
0.594*
0.393*
-0.711*
-0.379**
-0.500** -0.495**
0.562** 0.365*
0.523** 0.399*
-0.338*
-0.344*
-0.536**
-0.566**
-0.352*
-0.392*
Terrestrial
and semi-aquatic
Relationships between PMEase
activity and tissue N, tissue P and
tissue N: P ratio of four species
sampled from contrasting
environments (terrestrial and
semi-aquatic) from Nov 1999 to
Oct 2000 from Widdybank Fell.
B.L. TURNER, R. BAXTER, N.T.W. ELLWOOD and B. A.
WHITTON (2003) Seasonal phosphatase activities of
mosses from Upper Teesdale, northern England.
Journal of Bryology 25: 189–200
Phosphatase, tissue N and P and environment
realtionships
The interrelations between tissue N and P concentrations, tissue N : P
ratio, phosphatase activities and aqueous variables showed :
• 1) Significant +ve relationship between tissue N and aqueous NO3-N in
some populations, but not between tissue P and aqueous P
concentration;
• 2) Significant +ve relationships between phosphatase activities and
aqueous organic N, but none with aqueous organic P;
• 3) Significant +ve relationships between phosphodiesterase :
phosphomonoesterase activities and aqueous organic N;
• 4) Significant -ve relationships between phosphatase activities and
tissue P concentration;
• 5) Significant +ve relationships between phosphatase activities and
tissue N : P.
Both types of biological measurement are valuable for monitoring ambient
nutrients in upland streams. Neither method is clearly better than the
other, so both should be included in surveys.
Phosphorus in freshwater
• Natural sources of phosphorus that supply freshwaters derive from the weathering of
the catchment parent rock and soil, and from atmospheric input.
• Particulate P can be composed of many mineral, amorphous precipitates and sorbed
reaction products.
• Dissolved P is comprised of orthophosphate, inorganic polyphosphates and organic P.
• Dissolved organic P generally occurs as phosphomonoesters (PME) and
phosphodiesters (PDE)......
• .......and can be the dominant P form in Upland water courses (> 90%).
FO P as % FTP
100
90
80
70
60
50
Changes in FOP as %
FTP from Jan to Aug
2000. Stony Gill,
North Yorkshire, UK
S u b s tra te s a tu ra tio n a c tivity
-1
-1
(m m o l p ro d u c t g d ry w t h )
4
0 .6
0 .5
3
0 .4
2
0 .3
1
0 .2
0
0 .1
4
6
8
10
Assay pH
S u b s tra te s a tu ra tio n (1 0 0 m M )
E n viro n m e n ta l s u b s tra te (1 m M )
S u b stra te sa tu ra tio n a ctivity
-1
-1
(mm o l p ro d u ct g d ry w t h )
R h y n c h o s te g iu m rip a rio id e s (a x e n ic )
0 .7
25
20
0 .5
15
0 .4
0 .3
10
0 .2
5
0 .1
0
0 .0
4
6
8
A ssa y p H
10
E n viro n m e n ta l su b stra te a ctivity
-1
-1
(mm o l p ro d u ct g d ry w t h )
5
E n viro n m e n ta l s u b s tra te a c tivity
-1
-1
(m m o l p ro d u c t g d ry w t h )
Phosphatase activity and pH
F o n tin a lis a n tip y re tic a
0 .6
River transect study
Christmas, M & Whitton, B.A. (1998a) Phosphorus and aquatic
bryophytes in the Swale - Ouse river system, north-east
England. 1. Relationship between ambient phosphorus, internal
N:P ratio and surface phosphatase activity. Science of the Total
Environment 210/211: 389-399.