;
;.
^
MUSSEL STRESS EXPERIMENT - FINAL REPORT
I
^
by
P.M.J. Herman, M. Habets & C_._Heip
Rijksuniversiteit Gent
histochemical analyses
.
.
and
J.M. Bouquegneau & C.Dopagne
<^
Universite de Liege--: physiological measurements, heavy metal
analysis
»
This^study was sponsored through the BMM-UGMM research contract
nr.
85/17.
/
{,'.'
;
-.1 .'.
./
//^
Both^laboratories retain the responsability for the respective
parts _ of the work they car-ried out. The general interpretation
and discussion of the results was a cooperative effort.
Gent / Liege, 31-12-85
.-
»
Summary.
A
of
an
feasability
study
integrated
pollution
monitoring
strategy
using different tests of
stress
with
the
blue
mussel,
Hyt11 us edulis, was carried out
in
the
zone.
Belgian coastal
The tests
Included
physiological
in the
measurements, integrated
scope for
and
growth,
histological
and chemical analyses of the digest!ve glands
0-f
the
musselsg
of
analysis
metal
heavy
content,
\
biochemical characterization of metallothioneins, Shikata ^
test
for
histochemical demonstration
of
metalloproteins,
test
and
stability
demonstrat i on
of
NADPH -
lysosomal
neotetrazolium reductase activity
Native
intertidal
mussel populations were sampled
in
four
stations along the Belgian coast.
In addition mussels
were transplanted to two experimental stations at sea
(one
.
at _the west coast, one off the harbour of Zeebrugge)
-^
.
station
had an upper cage (depth -3 <n) and a lower cage (3<n
above the sediment)
In
none of the stations we could show pollution stress
Each
.
due to heavy metals.
Metallothioneins were not formed above
the background_level. In contrast, we found a varying degree
of pollution due to organic compounds, as shown by the
NTR
measurement
5
t ^
the
In
two
apparent
stability.
the coastal stations pollution was most severe
harbours
Zeebrugge and Oostende This effect
.
.
in
#,
15
in scope for growth, NTR - reaction, and lysosomal
Relatively
good
conditions
-found
were
»
in
Nieuwpoort. In Breskens scope for growth is relatively high,
but the lysosomal stability is low, probably pointing to
a
combination of goad food conditions and the presence of some
stress factor.
In
the
in
NTR
experimental stations a clear pattern is
activity (which indicates organic pollution).
.found
The
stress is higher in the upper cages than in the lower ones,
implying
a
vertical gradient even in these shallow
(10
m)
stations. It is higher off Zeebrugge than off Nieuwpoort.
The
station
off
Ni euwpoort has a
higher
scope
and a higher realized shell growth than the station
Zeebrugge. Lysosomal stability is much influenced by a
.for
growth,
off
sometimes extensive
t i ssue
We think
degenerati on
this
has developed in
reaction
degeneration
to
the
heavy
infestations
with
a
FIytilicola intestinalis,
parasitic commensal copepod
this
factor
Apparently
does not
influence
the
as
scope -for growth to the same extent
the
.
.
1 ysosomal stability
a
<f
Introduction
1.
the
with
of
deals
an
feasability
report
monitoring
strategy
using
different
pollution
integrated
The
tests
of stress with the blue mussel, Hytilus edulis
Marine
was
mainly
developed
at
the
Institute
for
strategy
Envi ronmental
Research (Plymouth, UK), and proposed by the
This
>
for
tool
the
continuous
as
a
Research Centre"
its
o-F
the
Southern
Bight
o-f
the
North
Sea
and
monitoring
its
studied
we
as
a
estuaries.
utility
Specifically,
"Water
pollution indicator in the Belgian coastal zone
a
both
method
The
physi ologi
incorporates
at the whole organism level (integrated in the
measur emen t s
histochemical
and
analyses of
Scope For Growth)
cal
the
t
digestive gland.
1.1.
Scope -for growth
.
variables
envi ronmental
Many
physiological
temperature
anthropogenic
However,
heavy metals, pesticides,
(hydrocarbons,
variables
natural
1971).
a
important
such
as
(Widdows
the
.
are
&
.
They may
factor (Akberalli & Trueman, 1985),
stress
Bayne,
stress
pollution
. ) may also exert
on metabolism.
influence
considerable
can
influence
may
condition o-f marine animals, among which
be
an
that
be measured by several physiological stress indicators:
Cr i
the
Scope -for Growth (Warren & Davi 5 » 1967;
Widdows & Bayne, 1971; Thompson & Bayne, 1974),
sp,
1<?71;
the
growth
1971; Thompson & Bayne, 1974), the ratio
(Crisp,
oxygen to excreted nitrogen <OsN ratio) (Comer
respired
rate
0+
s<
Cowey, 1968; Bayne, 1973; Widdows, 1978)
The
Scope -for Gr-ow-th is defined as the energy that can
.
and
reproductive
devoted .to the production o-f somatic
the
as
It
is
determined
1978).
al
et
tissues
(Bayne
>
that
15
the
effectively
between
difference
energy
and
respiration
and the
energy lost through
assimilated
these
energetic
The
rationale
of
integrating
excretion
measurements in the Scope for Growth is that it provides an
be
that
can
metabolic
the
of
activity,
index
overal 1
envi
ronmental
including
conditions,
the
with
correlated
lives
in a
when an animal
In
stress
fact,
pollution
for
it
will
have
to
use
some
energy
environment
polluted
will
Consequently, this energy
detoxi-fication
processes
be
»
.
.
»
N
.
no longer be available -for growth.
of
authors
Several
have studied the Scope for Growth
multitude
of
edulis in relation to
a
the
mussel Hytilus
&
(Widdows
environmental
Bayne,
parameters: t emp er at ur e
nutrition
8(
1973),
Bayne,
1973; Gabbott
1971; Bayne,
Gabbott 8e Bayne, 1973;
Thompson &c Bayne,
1973;
(Bayne,
1974;
Hawk ins
et
1981), etc..
al
1982;
8; Randlov, 1981; Navarro and Winter,
Ri isgard
.
r
1985), suspended matter (Kiorboe
et
al. ,
(l<?78a,b) has constructed a mathematical model
to
for Growth and its constituting -factors
Widdows
Scope
relating
toad quantity, mussel weight, and season
1.2
.
Histochemical methods.
N
-from
the scope for growth measurements we used
the
method
+ or
demonstration
of
s
histochemical
.
Shikata-test
for
the
stress
metalloproteins,
pollution
of
the
of
determi nati on
stability
lysosomes, and
reductase)
(NADPH-neotetrazoli um
NTR
of
demonstrati on
Apart
three
.
These methods were mainly developed by Moore and
UK) and are
IMER (Plymouth,
extensively
(1985).
described in Bayne et al
activity.
at
coworkers
.
The
rationale behind the combination o-f these
methods
measurements is double.
growth
scope
at
measur
ements
reveal
integrated responses
Physi ologi cal
that
level
It is hypothesized
the
of an entire organism.
.far
the
with
alterations at the cellular level
structural-functi onal
themselves
manifest
earlier
at
or
may
levels.
of
stress
lower
Secondly, the different tests probe for different -forms
e g
stress
test,
heavy metals in the Shikata
pol I ut ion
in
the
and
probably
other
organic
compounds
hydrocarbons
NTR test)
It should therefore be possible to interpret an
specific
overall
o-f scope -far growth in terms o-f
lowering
pollution factors.
(1985) thoroughly review the principles,
Bayne et al
results
of the
and
lysosomal destabi1isat ion
methodology
.
.
.
.
5
NTR method
We will only
Extensive
reference
here
aspects
and
.
.
.
in
Bayne et al
The
(1985)
.
summarize these
briefly
-found
lists may also be
.
.
1 ysosomal-phagosomal complex
system consisting
conditions)
mostly
membranes
of
stress
membranes, thus
intracellular
is an
which
normal
bound.
(in
inactive hydrolytic enzymes are
-factors
Several
to
may
destabi1ize
lysosomal
the
enzymes which
the hydralytic
certain
cellular
both
components and
catabolize
sequestrati on
Destabilizatian is preceded by
xenobiotics.
This
accumulation o-F the xenobiotics in the lysosomes
and
of
been
shown
to
operate
with
a
large
variety
has
process
viruses,
aromatic
hydrocarbons,
metal
ions
compounds, e-g
activating
.
.
etc.
a
Overloading
of
the
capacity
storage
activation of the degradative enzymes
for
test
The
1ysosomal stability
membranesin
the
1ysosomal
destabilising
results
.
in
.
solution
5,
10,
an
perforfned
acid
by
bu-f-fer
(pH = 4.5) during succesively longer intervals (2,
15,
histochemical
lysosomal
20,
25
staining
enzymes.
min. ) .
15
Colour
As
the
the
performed
destabi1izati on
of
for
one
intensity is
proportional
After
enzymes themselves
activity
acid, a peak col or ati on is
by
that time when the membranes have just been,stabilized.
fenzyme
destabilized
As,
.
IS
.
t
are
found
a
the
to
also
at
the lysosomal destabi1ization time
has
result o-f
a wide
of
stress
variety
conditions:
hyperthermia,
hypoxia,
starvation,
spawning,
injection
of aromatic hydrocarbons and water-solubIe
crude
oil
fractions.
The level of the response correlates
wel 1
with
the level o-f the stress stimulus.
On the other
hand,
it
correlates
also
with
the
physiological
measurements
(e. g.
scope -for growth) .
been
A
decrease
of
observed
as
a
.
NADPH-neotetrazoliurn
reductase
(NTR) 15
linked
with
microsomal
(smooth
reticulum)
endop1asmatic
.
the
mixed-function
important
s
compound
oxidases.
These
enzyme
systems
play
an
role in oxidizing (and detoxi-fying) toxic organic
in
the
NTR
cytoplasm
th e
represents
0-f
the
activity
micrasomal respiratory
chains
terminating
in cytochrome P-450.
Experiments have
shown that
its
IS
stimulated
activity
by aromatic
.
NADPH-ox idi z i ng
.
^
hydrocarbons,
/
of
crude
ail.
phenqrbita 1 ,
and the water-soluble
is believed that
a stimulation
It
fraction
of
its
activity results only -from this group o-f stressors.
The Shikata test -for metal loproteins
(Shikata
et
1974)
is somewhat less validated than the
other
two
methods.
An
fixed
acid permanganate solution is used (on
sections)
to
axidise
in
sulphydryl groups
metal-binding
The
ions
proteins,
thereby
removing the metal
sulphonic
acid
residues remaining are stained by orcein, -forming blue
(Jain
et
al.
1978).
- purple
granules
IS
Staining
al.,
.
.
.
recorded
It
on presence - absence basis of these granules
should
be noted
that
in contrast to the
claims
in
some
Johnson et al. ,
1984) this
method is not
(e.g.
papers
metal 1othi oneins.
-For
In
specific
all
principle
(and in addition
other
metal-binding
proteins
compounds,
such as elastic fibres) are stained.
One can only expect a
dif-ference between metal lothioneins and
other
quantitative
.
.
proteins.
Relatively
high
recorded
above the
coastal
are
zone,
well
concentr-ations o-f heavy metals,
in the Belgian
total concentrations
needed to activate
the
formation
in n.
edul is.
metallothioneins above the background level
1.3.
Sampling and the experimental setup
The
of
.
observations and experiments described here are o-f
four
native
First, we sampled simultaneously
of
M.
edul
is
the
populations
along
Belgian
coast.
Secondly, we set out three o-f-f shore buoys
in the
coastal
zone
Two cages (at different depths)
containing
50 mussel s
the
from
Eastern
Scheldt
(S.W
originating
Holland)
commercial
cultures were attached to
each buoy
After
52 days the mussels were recovered at
two
stations
the
third
one
was
lost).
For
a report
(un-fortunatel y,
of
the
The condition of these
cruises, see annex 1.
mussel s
two
typ
es.
intertidal
.
.
.
compared to the
experiment
(except
was
condition o-f the mussels prior to
for scope for growth measurements,
below) and internally between the two recovered stations.
-it-
the
see
.
2'30
t
.
3'00
I
.
3'30
0
5f30'
51'30
A
Fig. 1: location of coastal stations (^- )
Bresken
t
^
and experimental stations at sea (A)
*
^r
+
+.
»
<
a
»
+'
x
<
I*
+
r
t<
Zeebrugge
>.
1»
+
^
0
0
51'15
51'15'J
^
.
A
Oostende
10km
Nieuwpoort
^
t
f
BELGIE
De Panne
>
*.
*
+
f
»
+
^
<s>
51'00
T
I
*
51*0'
t»
£
2*30'
3°00'
3'30'
1
»
f
--.-^
MUSSEL STRESS EXPERIMENT ANCHORING SYSTEM
UG"JED_SfAnEWL5£LH)7.
.
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MLLWS
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MUSSEL CAGE
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2.
MATERIAL AND METHODS.
2 1.
.
Sampling sites and experimental setup.
We sampled
September 17,
native
in-tertidal mussel
populations
1985 at four stations (Breskens,
Oostende and
Nieuwpoort
.
on
Zeebrugge,
D within
map in Fig.
hal-f
hour
be-fore
an
or after low tide
-The largest
possible
animals
were
collected
in
the deepest accessible
places
see
.
.
The precise locations are;
- Breskens:
clumps of mussels were collected -fr
om
a
very
m -from the sand, just
protruding about 0.5
east
o-f the yacht harbour
- Zeebrugge
mussels were collected from crevices
between
the
rocks
serving as surf-breakers at the
west
side
(=
sea-ward side) o-f the harbour pier.
old
wreck
.
.
- Oostende:
mussel s
to the harbour.
nearest
were collected
on
the
beach, known to be heavily polluted.
- Nieuwpoort:
mussels were collected from
a
located approximately 800 m west o-F the harbour.
At
for
the
for
surf-breaker
This sur-f-breaker protects a small
surf-breaker
each
site 10 mussels were collected
and prepared
analysis; 10 mussels were also used
for
scope -for growth measurements.
The remainder were used
histochemical
the
determination
content
heavy metal
and
th e
biochemical analysis o-f metalloproteins.
The
1 also shows the
Fig.
map in
location
of
the
three
stations
experimental
due
to
Station 2 was lost,
the
cable
a
breakage of
upon
2
recovery.
Fig.
shows the
schematically
mouring system. Two cages were
crf
.
.
attached
to the cable connecting the subsurface buoy to the
concrete
bottom.
block:
one
m»
one at 3 m above the
at depth -3
As the
water
(Station
depth was limited
off
Zeebrugge: 9 3 m» Station off Nieuwpoort: 10.4
these
m) ,
were about 3-4
m apart
The 4 cages will be
cages
denoted
.
.
9
by the following abbreviations:
N up
upper cage, station off Nieuwpoort
N lo ; lower cage, station o-f-f Nieuwpoort
z up
upper cage, station off Zeebrugge
Z lo : lower cage, station off Zeebrugge
The cages were constructed from plastic-coated
(1
w1 re
cm2 maze width)
They were subdivided
.
.
.
.
»
compartments, each containing 5 mussels.
metal
in 10
mussels used for this experiment
were
purchased
a
commercial
mussel
culture
at
Yerseke (The
Netherlands)
and
They were freshly fished, not watered,
not
treated
in
the machines that clear
fouling
organlms
from
the
shells.
Due to a defect in the
sea
circulating
water
system of the research vessel, the mussels were
The
-from
.
.
.
»
in
moist
(Station
air and at +- 10 "C durin
3) .
This
kept
h
g 15 (Station 1) to 20
treatment
have
caused
may
bad
considerable mortality, and should be avoided in the future.
c.
/
Ten
mussel s
were
-f i;-; ed
h after purchase.
w i th i rs 2
the
for
histochemical
analysi s
However, for practical
reasons,
scope -for- growth measurements could only start the next
day,
i mplyi ng
the same
harsh
treatment
for
as
the
experimental mussels.
'->
-»
.*
-it-^
Scope For Growth fneasurements.
It
~~7~
he
experimental
protocol
measurements has
been
?y.
(Ui ddows
Bayne, 1971;
-For the scope
described
by
.for
growth
authors
numerous
Thompson 8< Bayne, 1974;
al . ,
1978; Widdows, 1978b; Widdows et al . ,
Hawk i ns
et
al .
Winter,
1985).
1982;
Bay ne et
1981; Navarro s<
We
have
cl osel y
nFollowed the method described by URC Environment (1985).
rate
15 determined on
Respiration
individual
in closed containers
placed
-filled
with
500
ml
?
sea
water.
the
ex peri ment.
<=
mu
sel
s
fiItered
The sea water is saturated with oxygen prior to
electrode
oxygen
8012-100)
duri ng
Oxygen consumption is measured wi th
an
(Di ssolved
Probe
Model no.
0>; y gen
It is
approximately one hour.
expressed
in J/h by the conversion equation;
R = 0^ consumed (mg/h) x 14.23 J/mg
(Crisp, 1971).
Ammani a e;-;creti on
i ndi vi dual
animals in
directly
15
measured
by
placing
250 ml flasks containing filtered sea
water. A control flask with sea water, but without an animal
1 S
run along
After approximately 3 h ammonia concentration
1 S
determi ned
with
th e
method of
phenol-hypochlorite
1969).
Solarzano,
The energetic equivalent of the
ammoni
.
a
e;-;cretion is calculated as
(Elliot
?<
Davison, 1975)
a
.
.
E = (NH^-N) excreted (mg/h) x 24.8 (J/mg)
Assi mi I at i on
rate
is measured -form
the
"clearance
of
suspension of planktonic algae
Phaeodacty1 urn
tricornutum
Bohlin
A
(Baci11ariophyceae).
monaspeci + i c
culture
was established in the laboratory, based on a spike
{obtained
the
Universite
from
de
Louvai n
la
Catholique
Neuve.
The culture medium was that described by Gui Hard &
rate"
.
.
Ryther (1982).
For
placed
water
the measurement o-f clearance rate the animals
in a -flow-through system through which filtered
(with
rate
0-Jmeasured
are
sea
the algal suspension added),
ml/min).
ij 0
before
and
The density of
-flows at a known
cells
the algal
1 5
the
passage through
their
chamber with a Turner Fluorometer Model
111.
fluori metric
measurements were previously
calibrated
after
e>;peri mental
The?
with
of
di rect
the
LJLg)
.
counts of the algal cells (see interim
The
formul a
then
-fol1owi ng
clearance rate;
report
yields
*
-. <B
the
CR =
CI -
Co
Fl
x
(1/h)
CI
where CI = algal concentration before passage through the
experimental chamber (no.
o+ cells/ml)
.
Co = algal concentration after passage through the
experimental chamber (no
Fl = flux
.
of cells/ml).
(1/h).
In energetic units we have for the energy consumption rate:
c =
CR x
(N) x (16.3)
(J/h)
where <N) = quantity of organic matter ingested <mg)
16.3 =
content
energetic
determined by
Bayne et
(1978b)
.
o-f the
as
algae (J/mg)
al.
(1978)
and
Widdows
.
assimilation
The
product
the
efficiency
ratio
of
The
.
as
rate is subsequently calculated
and
the
absorption
Conover (1966)
latter is defined by the
the consumption rate
.
.
F -
Absorption efficiency 7 e 7
E
F (1 - E)
where F = percentage o-f organic matter in the food
E = percentage o-f organic matter in the -faeces
The assimilation is thus calculated as
A
c
e.
x
The scope for growth is then defined ass
p = A - (R
A
+
E)
minimum o-f
(J/h)
10 mussels was used for the
scope
for
growth measurements in each station.
refer
details o-f the experimental methods we
the interim report "Mussel stress experiment - Scope for
Growth", Contrat UGMM-ULg Ref 85/16
For
the
to
N
2 3
.
N
Histochemical methods.
thick
sections
of
the
transverse (cryostat)
digest!ve
gland were cut with a cryostat American
Opticals
The tissues
(hand-operated5
cabinet temperature < - 25 C)
were
fixed in aromate-free frozen hexane, cooled in
liquid
N2.
Size
limitations
of
the
tissue
holders
cryostat
far
al lowed
and
maximum three mussels to be cut together,
placed on the same object glass
10
urn
.
.
CT
The
dissected
mussels
-from
coastal
the
populations
were
air at
transfer to the laboratory in damp
ambient
h between collection and
temperature (duration; 1-3
fixation).
were
The mussels -from the o-f-f shore experiments
fixed
aboard
of
the
the ship, immediately a-fter recovery
upon
cages.
For
closel y
are
the
the
histochemical
we
followed
stainings
very
which
protocols, established
by IMER and WRC,
f
in
given
App.
relevant details.
+
0-f
Fixation
We will mention here
.
only
the sections -for the Shikata-test
.few
a
not
was
were
Instead the sections
by submersion in Baker's formal
placed in a closed container at 55 C for 2 h, together
with
a
done
.
sufficient amount o-f formaldehyde in
powder -form.
comm
and
This method gives better -Fixation (Moore, pers.
prevents loosening o-f the sections -from the object glasses.
+
from
Orcein (o-f which several -forms exist) was purchased
.
UCB.
+
The destabi1isation
detected
by staining
lysosomal
ronidase
should
o-f the lysosomal membranes
for
either
of
be
can
different
three
N-acetyl-/3 -hexosaminidase, /?-glucu-
enzymes:
In principle the three methods
are
yield the same results, since all three enzymes
and arylsulfatase
.
destabi1ization o-f the membranes
We have
activated
upon
methods -for two series
of
tried
the
three
mussels, and
con-firm that the results are identical.
coul d
The staining
intense
of N-acetyl-/^-heK05aminida5e gives by -Far the mos
This
three
enzyme is the most prevalent o-f -the
staining.
comm. ) .
There-fore this method
H.
in
edulis (Moore, pers
.
.
N
was adopted -for the rest o-f the samples.
+
Shikata-test
The stability o-f the colour is good for the
and the lysosomal stability test
However, the purple-blue
colour
o-f the NTR - test is only stable when the section is
.
mounted
(DIFCO).
mounting
*
water-soluble
"UV-free
a
mounting medium"
all
other
The colour reacted very quickly with
3
media we tried.
Un-fortuna-bely, we had to wait
in
months between ordering and delivery a-f the good
mounting
medium.
compan sons
For that reason we have only relative
(from
the
of
NTR-reaction between the coastal populations
them
immediate inspections) but we are not able to compare
fr
with the experimental stations.
^
+
be
~L
to
the
The scaling of the NTR-reaction on a 0-5 scale proved
We have been able to
difficult.
stations
in
relatively
di-f-ferent
staining
ordering)
order
of
intensity
decreasing order
(two observers arriving independently at the
of
p
but
are
not able to define the
scale
same
that
so
possible
scaling of the individual sections is
this
automatic
in
A test has been performed with an
way
Aerts
and
w
p.
Decoster
by
i mage-analyser
with
RUG) .
this
It is possible
centre,
(image-analysis
blue from
the
method to
separate the
purple colour
and
extent
of
measure the intensity
background, and to
A
on
measurement
will
be
per-f
armed
objectively
staining
unequi vocal
»
.
.
.
all
sections
analysis
as
soon
as
the time schedule
centre will permit it.
the results will be communicated
~ 1 n-
.
of
the
»
image
(planned early February a separate short note) .
in
with the
A general staining o-f the sections
Papanicaloau
(see
1985
183 for
Bayne et al.,
a
pprotocol)
was
These sections were used to
performed.
measure the
proliferation
which
of an aberrant connective-type tissue,
we
reaction
in
to
suppose developed
infestation
by
Hytilicola
a parasitic - commensal
intestinalis,
copepod.
The prol i-feration o-f this tissue was
of
expressed as the 7.
+
method
.
.
.
the
surface
dsgenerative
o-f normal tissue (with digestive
tissue
that
Surfaces
were meass^ured
with a camera lucida.
was
occupied
by
tubuli)
and
the
latter.
with a pl ani meter an drawings
t-3-.
~*
j». -^ -
made
**
3.
RESULTS.
../,
3.1. Measurements on'the whole organism,level
.
3.1.1. Mortality of mussels exposed in experimental stations
at
sea
^^
A-fter the 52 days o-f eKposure at sea, the [mor-t.ali'ty J3-F
The results
has been recorded in each cage.
3.
observed
Fig.
The highest mortality was
in the cage Z lo, the lowest in the N up cage
the
mussel s
shown in
.
are
.
.
Zlo > Nlo
>
Z up
>
Nup
Mortality (%)
100
50 J
04
lo
Fig
*
lo
up
Zeebrugge
up
Nieuwpoort
3: percentage mortality of the mussels in the cages.
3.1.2.
3 1.2. 1.
.
Scope for growth and biometric measurements.
Coastal stations,
2
Table
summarizes the physiological measurements on
mussel s collected
in the four coastal
stations.
We
used animals o-f about the same shell
have
size in all
-Four
(the
mean size o-f the mussels was 48.7 +3.2
populations
The results
in Table 2 are
the
mm)
real
experimental
uncorrected
of
the
values,
.for differences in dry weight
mussels.
We assumed that the mussels of the
same
shell
the
.
length
were of about equal age
In doing sa, the scope -for
and
reflect
the
other physiological measures also
dif-ferences
in body condition (the degree o-f filling of the
.
growth
12-
shells by the so-ft body parts).
Oxygen consumption is significantly lower in Zeebr
than
in
the other stations (t-test, P < 0.01).
three
stations
do
not
differsignificantly
another.
the
lower
Presumably,
respiration
ugge
9
Zeebrugge
The other
from
one
rate
in
is a direct reflection of the lower dry weight of
the soft body parts.
.'^
;..-^-
Table 2.
f
Breskens
R
(2.82)
0.19 (0.12)
13. 15
E
CR
c
/
r
i
3.23 (0.57)
23.21 (4.04)
Zeebrugge
Oostende
Nieuwpoort
8.49
0. 19
14.71
0.66
14.85 (4.93)
0.17 (0.09)
3.44 (0.76)
22.79 (5.01)
91
e
21 14 (3.68)
p
7 79 (3.74)
.
12.99 (2.85)
4.31
.
(12.0)
(13.2)
17. 1
30. 1
(4.13)
(0.07)
2.55 (0.48)
16.86 (3.19)
70
11.82 (3.24)
57
A
GG 32.7
NG 35.9
(1.47)
(0.06)
-3.95
(3.51)
(11. D
»
(4.51)
-21 2
-30.3
7 26 (8.14)
.
(30.8)
(43.9)
.
(19.5)
3 30 (0.70)
23.86 (5.04)
93
22.29 (4.70)
26.0
27.8
(28.5)
(30.5)
1.454
(0.152)
(3.28)
=
DM
1
.
221
(0.368)
S/D 44.62 (14.18)
Table
1.125 (0.265)
44.54 (10.31)
0 657 (0.101)
71.72 (10.72)
.
35.14
2s Physiological measurements on the mussels -from the
coastal
animals.
stati ons.
Mean values (Standard Deviations) o-f
n
R:
E:
excretion
rate
respiration
rate (J/h);
CR:
c
e
(J/h);
clearance rate (1/h);
consumption (J/h);
A
Assimi1 ati an
absorption
efficiency
(dimensi onless, 7.) ;
Pl
rate
(J/h);
Scope for growth <J/h); GG:
gross
growth
NG: net
efficiency
(dimensionless,
./.);
growth
efficiency
.
.
II
.
.
.
.
(dimension- less, 7.) ;
DW
Dry weight o-f the soft body parts
divided
by dry weight
(mm/g)
providing an index o-f shell -filling
(g);
S/D
.
.
shell
.
»
length
.
Ammonia excretion
in
the
is higher in Oostende than
other
stations
p
<
0.05) .
No significant
(t-test,
di-f-ferences exist between the other three.
Clearance
rate
and
are
also
energy
consumption
p < 0.05), and
do
significantly
lower in Oostende <t-test,
not differ between the other stations
is more than 90 7.
in
Absorption
Breskens
efficiency
and Nieuwpoort,
whereas in Oostende (70 7.) and
especially
in
For
this
Zeebrugge (57 7.) it is considerably lower.
measurement all
animals
are pooled in one
so
container,
that no standard deviaton
can be given.
.
.
»
»
-1^-
*
Assimilation
rates
similar
are
Nieuwpoort;
they are significantly
than in the other two.stations.
rf/
^? ^-
»
Breskens
in
and
higher (t-test, P< 0.01)
rf-
The \ scope .for^<3r',OWJ!:]2/Jl..s above 7 J/h in
Breskens
and
It
15
lower
Nieuwpoort.
significantly
in
Zeebrugge
(t-test, P < 0.05), and has a negative value in Oostende.
.
Thus
f
we arrive at the following sequence in scope
for
growth values:
0 «
z « N < B
Gross
and net growth efficiency were calculated
from
the
physiological measurements on the individual mussels at
each station.
They are given by the equations:
gross growth efficiency =
K
1
K
2
net growth efficiency
Th&
stations
»
differences
_ A - (R+E)
c
_ A - (R+E)
A
growth
in
efficiency
between the
very similar to the di-f-ferences in scope
Only the Zeebrugge values are aberrant,
are
growth.
they approach the
high
values
-found
in
Nieuwpoort.
The
of
dry
weight
the soft body is
Zeebrugge than in the three other stations.
for
in that
Breskens and
much
lower
in
a
The ratio between the length of the shells and the dry
of
of
the soft body reflects the degree of -filling
the
shells
th e
by the soft body.
The higher this
ratio,
In
lower
the
the
mussels
-fi11 ing.
Nieuwpoort
are
better filled
p < 0.01) than
in the
significantly
(t-test,
other
stations.
similar
IS
in Breskens
and
Filling
weight
^
Oostende,
where,
turn, the mussels
in
better filled than in Zeebrugge (t-test,
significantly
are
p < 0 01)
.
.
^
3 I
.
.
2.2.
Experimental stations at sea
Table 3
summarizes the physiological measurements on
to
that
the
mussels
were transplanted
experimental
As these mussels come -from the same population,
stations.
the
the measurements are readily comparable between one another
The oxygen consumption
is
different
significantly
p
z
(t-test,
< 0.05) between i) Z up and Z low and ii)
up
and
N up.
No signi-ficant difference is -found between N
up
.
and N lo
1
Ammoni a excretion
1 ower
cages in both
tr-
*
similar between the
upper and
it
IS
stations, but
significantly
IS
»
higher at Zeebrugge than at Nieuwpoort (t-test, P < 0.01).
Clearance
all
rate
and energy consumption are similar
stations, encept at N up, where they are lower (t-test,
p < 0.05) .
14-
at
Table 3.
Zeebrugge
z
z
up
(2.72)
R
E
11.87
0.50
CR
c
3. 13
(1 13)
23. 16
(9.69)
57 7.
78 7.
e
A
p
3. 13
.
(19.2)
(33.8)
19.94
2.34
(1.15)
16.28 (8.01)
93 7.
78 7.
15.14 (7.45)
12.70 (6.25)
(7.46)
4.90 (5.21)
2.42 (4.37)
.
10
n
(1.20)
-3.68 (8.59)
1 62 (11.75)
-25.4
-44.6
N la
9.90 (4.85)
0.34 (0.12)
25.57 <9.56)
57 ./.
78 7.
14.57 (5.45)
13.46 (5.52)
18.42 (7.56)
1. OB (6.20)
5 64 (8.48)
N up
lo
17.84 (7.41)
0.42 (0.19)
(0.15)
.
GG -4.8
8.4
NG
Nieuwpoort
(51.7)
25.3
27 2
(90.6)
(37.4)
(40.2)
.
9
9 27
(4.96)
0.33 (0.11)
.
3.24
24.85
93
78
23. 11
19.38
(1.12)
(8.44)
7.
7.
(7.85)
(6.58)
13.52 (9.59)
9 66 (8.04)
»
48.8
32.5
10
(30.4)
(32.7)
12
3: Physiological measurements on the mussels from the
stations
at
Mean values
sea.
(Standard
experimental
of
n animals
R: respiration rate
deviations)
E
(J/h);
CR
rate
clearance
rate
excretion
Cs
(J/h);
(1/h);
consumption <J/h); e: absorption efficiency (dimensionless,
7.)
A:
to two different assumptions (see
text) ;
according
rate
GG
Assi mi 1 ati on
(J/h);
P: Scope for growth
(J/h);
NG: net growth
rate
growth rate (dimension less, 7.) ;
gross
Table
9
.
.
.
.
.
.
(dimension- less,
values
7.)
The first row for A
.
and
p
<
gives
the
calcu- lated with absorption efficiencies from
the second row with a constant absorption
coastal
stations;
GG and
rate.
efficiencies.
NG
for
are
Due to
estimate
the
coastal
absorption
able
technical difficulties, we have not been
the
-four
the absorption efficiency in any of
In
Table 3 we have given two
alternative
populations.
have
taken
we
the
val Lies
of
approximations
First,
in
the
efficiency
absorption
corresponding coastal
have
stations.
we
taken
the
mean absorption
Second,
the
of
four
coastal
stations
for
the
four
efficiency
experimental cages.
to
.
»
"a";-
Consumption rates
are similar in the -four cages.
is lower in N up, but not significantly.
Assimilation
depends, of course, on the assumption
It
W9~
^<
on
the
With
absorption
the
efficiency
absorption
efficiencies
lo
o-f the coastal stations, it is higher in N
than
in the other stations
(p < 0.01), which do not
differ
from one another
significantly
With a constant absorption
the
assimilation
values.
do
efficiency,
not
differ
.
.
significantly between stations
the
Taking
.
absorjE»t_^on _ _ef f i ci ency
the
of
stations, thefscoEe^for^gTowthjLs significantly higher at N
lo
than
at
N up (p < 0.05) .
be'tween z up and z lo.
different
It
is not
significantly
The sequence o-f scope -for
growth is then given by;
.~'\
~^.
z lo
coastal
< z
up< Nup« N lo
^
.
IS
With
a
somewhat
di-Fference
have:
constant absorption e-fficiency, this
the
changed
However,
only
sequence
significant
.
still is between N lo and the other stations.
^r
^
z
Table
mussels.
lo
4
^
N up
<
Z up « N lo
<
shows the
in the
mortality
also
includes
the
biometric
It
at
We
experimental
data
of
the
and after
the onset of the experiment (to)
the recovery o-f the cages
Mortality is lower in the upper cages than in the lower
population
.
ones. The dif-ference is more pronounced in Zeebrugge than in
Nieuwpoort.
rt^
The length Jgrowth of
than
Nieuwpoort
of
the
mm)
»
.
Zeebrugge.
observed on the empty shells
f
at
shells were individually measured
.
erosion
in
the
The five
shells
in
a
cage
either of the same
were
size (to
the
nearest
differed so much in size that
could
be
they
compartment
0. 1
shell 5
15
in
higher
-^- _/
The growth
values
were
-n,
in
corrected
-for the
the
All
cages.
onset
the
or
experiment.
»
recognized after the experiment.
Upon recovery we observed
that
all the empty shells had decreased in length
(average
decreases
were
N
-0.294
mm >
.
z
up:
la .
in the lower
.
.
-0.305
lo
The
.
z
mm
»
-0.361 mm) .
.
.
-0.427
decrease
N
.
»
up:
is mar
e
and was interpreted as due to
erosion
corrected
of the fragile shell fringes. Growth was
.for erosion by assuming that growing shells had suffered the
important
cages,
^
same
erosion as the empty ones, and that most mortality had
occurred
at
the
onset
of
the
experiment
.
The
latter
assumption is reasonable in view of the bad treatment of the
animals just prior to the experiment. The growth values thus
give the -following sequence
.
.
z up < z la
<
N lo
<
Ih-
N up
Ye rs eke
Z up
Zlo
N up
N lo
M
38 7,
60 7,
34 7,
46 7,
G
0.26 (0.48)
0.34 (0.40)
0.46 ( 0.74)
0.45 (0.51)
DW
2.032 (0.415)
1.994 (0.614)
2.034 (0.613)
2.417 (0.486)
2.061 (0.839)
S/D
31.1
33.51 (10.17)
31.91 (8.84)
26.16 (9.01)
32.78 (9.28)
n
9
9
10
12
0
Table 4: Mytilus edulis experiment at sea.
M-. Mortality (% o-f the 50 e:<posed animals that died during
the 5 '-> day experiment); G: growth (mm/52 days) of the surDW dry weight o-f the so-Ft body parts (g) ;
viving animals;
S/D: shell length divided by dry weight o-f the soft body
(mm/g) providing an index o-f shell -filling. The latter two
measurements are based onn animals. All values, except
mortality, are means (standard deviation).
^_
.
.
*
3.2. Measurements on the digest!ve gl and
3.2. 1.
N
Analysis of the heavy metals content
.
Table 5 shows the cadmium, copper and zinc content
of
the
mussels collected at the coastal stations, purchased at
Yerseke
and recovered from the experimental stations
a^ter
52
The
days.
heavy metal contents
were
determined
by
atomic
absorption
a
spectrophotometry
model
»
using
370A
Perkin-Elmer
-fl ame
spectrophotometer after
mineralization
for
B h in HN03 65 7. and dilution.
They are relatively low
and no
correlation could be detected between these
values
and
the
scope
for
growth
measurements
.
During
the
experiment, a slight contamination by cadmium has occurred,
whereas the
decreased
9
copper
and
zinc concentrations
s omewhat
could not be
performed
on
Significance
test
these data, since all tissues were pooled for the analysis
.
Table 5.
Intertidal populations
Nieuwpoort
Cd
Cu
Zn
0 3
4.1
4 0
3 0
5 0
20.9
21.7
23.2
31 2
.
Oostende
Zeebrugge
0.4
0 2
Breskens
0.7
.
.
.
.
=ssss
Yerseke
as
=
0.0
4.0
sc
3.2
22 0
23.1
20.8
.
3 0
3 3
.
0.4
Table
s=
»
0.6
0.3
N lo
z up
30.0
ssas
w*
Experimental stations
N up
.
ssa:
.
Cadmium, copper and zinc contents
wet
(ug/g
weight)
4
the digestive glands o-f mussels -from
native
the
and
populations
of
along
Belgian
the
coast,
5:
of
experimental
mussels before exposure ("Yerseke"), and a-fter
exposur-e in three o-f the four cages
3.2.2.
will
Histological analysis.
The results of the Shikata - test for
not
be discussed
in
detail
.
metal 1oprotei ns
for
the
different
In
populations
all cases the results were very
similar
coloured
Faintly
granules are seen at high
magnification.
However, these granules are not comparable to the intensil
a
tf *0
17
.
y
stained
granules
from
received
.
present
IMER.
in
the
reference
slides
we
All the sect ijoos should therefore
be
-r^
c1assi -f i ed as_ negat i y.e_T'oi- this test
The problem posed by
.
these
faint granules resides in the characteristics at both
the
test
n.
edulis
principle and the test animal.
only
starts
metallothioneins at
producing
elevated
relatively
metal
which are well
above envi ronmental
concentrations,
concentrations
in
On the other
zone.
the Belgian coastal
hand, the test is not specific for metallothioneins.
The
coloured
faintly
granules may therefore be interpreted
as
different -from metallothioneins.
metalloproteins
Moreover,
biochemical
characterization
has
been
at
th e
performed
of
University
described
Liege, according to a
previously
method
1983)
No metallothionein
(Bouquegneau et al.
was
.
»
.
^
-A
»*'
^-h
^
<» UL^A
detected (above the background level)~by that method either.
^It
<rf
^,
3.2 2 1.
.
Coastal stations
.
Table
6
.
shows the
mean
labilization
time
of
the
lysosomal
-from
membranes in the -four populations, arranged
to
West.
East
a
Friedman
Non - parametric comparison in
XXXX) showeda
multiple
comparison test (Sokal & Rohl-f,
p
difference between the
Nieuwpoort_ population
significant
.
-^
th
^
.^
*Jf
< 0.05) .
.three (P
The difference
between
and
Oostende
Breskens is almost
Zeebrugge
significant
(0.05 < p < 0.10)
and
^
at h
e
er
»^>*,
Table 6
.
Population
mean (min.)
S.E
7 5
6 I
Breskens
1 1
0 7
1.5
.
Zeebrugge
.
Ni euwpoort
.
.
It
9.7
18 0
Oostend
e
n
.
1.3
.
10
9
10
10
Table 6s Labilisation time (min.) of the lysosomal membranes in
Mean and Stanfour coastal populations o-f Mytilus edulis
.
dard Error o-f n observations.
'T
almost
- test
yielded
and a faint reaction in Breskens.
The I NTR
Ni euwpoort
^
no
reaction
in
*
The
reaction
in
strongly positive in Zeebrugge, but most pronounced
This
the
population.
gives us
-following
ordering:
was
the
Oostende
0 > z
» B > N
Due to the stability problems discussed earlier,
ordering can, unfortunately, not be refined
any
-1R
this
more.
^
//
3 2.2.2
.
f-
..;.'-. ff
- ^
7
^
Experimental stations at sea.
.
v^
^»
-^
<9W<
^
v *^
Table 7
shows thejlysosome labilisation time,
in
the
Yerseke population
(i -e.
t tie" musse 1's" at' C h'e ' st ar t of
the
and
the
mussels from the
experiment)
-four
experimental
.»
The labilisation time is generally depressed,
cages.
N
*
but
up and in Z lo the values are not much lower than
the Yerseke population.
in
.
in
A Friedman multiple comparison test showed that the
Yerseke population is significantly different fr
am the
others. N up and Z lo are not significantly different -Fr
one another, but are significantly higher than N lo and Z
up, which in turn are not different. This yields the
following sequence:
u
om
^
r
v
» N up
Y
> z
la »
z
up >
lo
Table 7.
Cage
z
mean (min.)
lo
S.E.
z up
N lo
15.0
5.2
4. 1
2 6
0 6
0.5
N up
18.1
2.6
7. degeneration
n
9
.
22
47
10
10
9
.
73
39
Table 7: Labilisation time (min.) of the lysosomal membranes in
four experimental stations at sea. Mean and Standard
Error of
observat i ons.
n
Also indicated
is the
ratio of tissue degeneration (see teKt)
mean
a
f
f
r\
^
^
\
f1
( The
NTR -
test
shows
a
^""
distinct
pattern:
animals -from the upper cages show a much
.than those o-f the lower cages, while the
in the
generally
stronger o-f-f Zeebrugge, than
IS
of-f Nieuwpoort
>
Zup
The
.
stronger
reaction
station
.
.
N up
> z
lo
> N lo
by
infestation
n.
in-fluence on our results.
serious
..'
both
at
stations
reaction
/.-
<
/','
u
intestinalis
had
IS
Campbel1,
1970):
the
a
In the literature on
n.
a particular reaction of the mussels to
sometimes described (e.g.
Korringa,
in+estation
»
intestinalis may have
intestinal gland changes
heavy
1952,
in colour
typical
We .found
green to very pale, whitish
this
change back in many (heavily invested)
mussel5
.from
our
experimental cages.
Microscopically, the white
tissue
kind
a
o-f connect!ve
corresponds to
where the
tissue,
digestive
The extent
tubuli have completely regressed
to
.from
the
.
.
which
greatly
table
this
apparent degeneration had taken
between
individuals, but also between
7
this is expressed as the ratio of the
_. 1 0_
place
differed
In
cages
surface
xn
.
»
.f
the
section
occupied
by the degenerated tissue
surface
occupied
by normal tissue, containing tubuli,
th
to
e
and
degenerated tissue.
4
.
DISCUSSION.
Table
8
summarizes the
results
the
of
different
measurements, in the form of an ordering of the stations.
Table 8
.
Coastal Stations
Stations at sea
Zlo>Nla>Zup >Nup1^
Mortality
^*»
^"<
^_
Scope for growth
Shell growth
0 «
metal Iothi onei ns
NTR
not detected
'y
z « N< B
z lo < z up < N up « N 1 o -^
z up < z lo < N la < N up «not detected
z up > N up > z lo > N la (/.
N lo > z up » z lo > N up
/
0 > z » B > N
Z <= 0= B < N
Lysosomal stability
.V'
Table 8: Summary of the result of the monitoring of 4 coastal stations
and 4 experimental cagesat sea. In each class the stations
are arranged in increasing or decreasing order of the
relevant parameters.
/..
-"
_/
.yw
^
ati
/ ...
.(f
,- S..f...h I
f
CL
\
//'
.-1-
./.
4,
^
c-'"'
>.'
w
^ .
^
/
ir
/
f
.<.
Jt
t
The ordering of the stations is more meaningful than a
absolute
for
the
the
of
discussion
values,
especially
measurements
Many natural parameters may
physiological
these
influence
and we do not dispose of
values,
enough
to
the
our
in
in-formation
baseline
classify
region
"low"
As
for
the
either
or
as
measur emen t s
"high"
histochemical
measurements, some form of stress is
present
.
.
.
.
NTR is higher, and lysosomal stability
all populations
the
Yerseke cbhtr-61
lower
in
all populations than in
metal
contents
can
In
the heavy
contrast,
population
nowhere be described as abnormally high.
»
in
.
*
IS
-.h,
.
.i
It
is clear -from this table that the stations Oostende
the
mussels than
Zeebrugge are less favourable for
Breskens
and Nieuwpoort. Pollution stress in Zeebrugge and
.found back in scope for growth an in NTR
It
Oostende IS
metallothionein
contents.
in
not
IS
seen
Presumably
from organic
stress
along the coast is mostly
pollution
and
»
.
.
#
and
compounds,
highest
1ysosomal stability
near
does
or
in
indicate
a
the
harbours.
form
of
stress
in
Breskens which is not reflected
the scope for
Due to stress conditions which act on both
crf ten
well
SF6 and lysosomal
stability are
.
)
1
However,
different
provoke
v
as
1 5
things.
different
th
.
e
case
in
Breskens,
The
»
in
growth.
variables,
they
correlated.
do measure
In particular the feeding conditions may
reactions.
-20
The
Breskens
station
.
IS
.favourable
to
The
mussels in several respects.
structure
which
far
the animals were collected protrudes
enough
from
the
sediment
to eliminate the erodi
influence
of
ng
sand.
Moreover, as the nutrient load of the Scheldt
water
food
15
high,
-Favourable
conditions may be very
in
the
mouth.
We interpret
our
estuary
measurements as
an
indication
of
the presence of (pollution) stress
in
what
on
.
would otherwise be very good growing conditions.
/
A
/
priori, we had expected a pllution gradient fr
the
(low
am
/
Scheldt
river (high pollution) towards the west coast
pallution).
The
harbours
influence
as
Such a gradient is not apparent from the data.
of Oostende and Zeebrugge clearly had too much
(point
?> pollution sources
It
.
would
tie
y
interesting to study coastal populations in between the
harb^urs^
^i^n^rder~t'o'7aet^ " i')""^fr the^ ^
can-Ge
shown,...and ii). how far the
polluting influence in the coastal waters.
As indicated
infestation
with
too
^^-
exert
on
the results o-F our
sea»
at least on the lysosomal stability measurements.
on
clearly
point
the
a
experiment
data
shell
their
f*^
in the results section, we +eel that
Hytilicola intestinalls might have had
influence
1 arge
harbours
r*
at
The
growth, SFG measurements and NTR activity
to
a
influence
higher
pollution
in th
e
This
station
is near
Zeebrugge station.
the
Zeebrugge
and
harbour,
Scheldt
in the e-f fluent plume o-f the
nver.
However, the pattern in lysosomal stability might have been
without
clearer
the
factor
of
the
interfering
n.
.
intestinal is in-fectian.
A
is that
interpretation
the
described are
in accordance
with
older
but
contrast
with
observations,
some recent
assessments of
the e-f-fects o-f
n.
i ntest i na1i s on
mussels
Moore et al
(1977) did not find
(e. g.
Dare, 1985)
any
pathological
with
thi
conditions
histological
changes
problem
5
.
.
.
in
Mytilus edulis as a
<
.
IS
»
direct
absent).
to
response
Mytilicola
intestinalis, except for a limited damage to the
intestinalis
epitheliurn
at the places where M
gut
in
it
(here the cilia
are
reduced or
additional
studies have shown that
n.
does
nat
have serious deleterious
usually
with
contact
Several
intestinalis
e-f-feet
on
FIytilus edulis (Davey et al
1978; Gee et al.,
»
1977).
are
when
Bayne et al . ,
The only exceptions
high i.nfestation levels coincide with other stress factors.
(1985) to the
This
led
Dare
conclusion
that Mytilicola
intestinalis
harmless
should be considered as a relatively
than a parasite of Hytilus
edul is.
In
commensal, rather
these
of
vi ew
our
conclusion
that
studies,
Hytilicola
intestinalis
might be responsible for the apparent
tissue
.
1977;
*.
<
^
.
degeneration
based
must
be
viewed with some
caution
absence
.
-further (experimental) research.
to perform similar experiments in the
with Mytilicola-free mussels.
needs
surely
better
seems
A
qual ity
This
-factor
»
It
was
the
subject
Anyway, it
-future
of unknown importance in this study
an
is the
and quantitiy o-f the food available to the mussels
factor
IS
for
the
not only important.
measurements
.
.
performed
in
.
the reports of similar observations, and
af
This
any other apparent causal agent
on
vi ew
here, but has considerable importance in its own,
of
the
eutrophication problems
21.-
in the
Belgian
In our experiment the different reactions o-f
-for growth on the one hand, and NTR on the other hand
due
to this factor
be
Eutrophication may have a
effect
on
positive
scope -for growth, whereas it
may not
induce
It
I S
a particular reaction in the NTR experiments.
an
advantage o-f the integrated monitoring strategy that the
different
measurements allow one at least to hypothesize on
coastal
zone.
scope
could
m
this kind of mechanisms
differenc.e
The
in
t
1 ower
all,
.
NTR - reaction between the upper and
in these shallow stations.
cages is remarkable
the
The
apart
cages were only a few meter
.
After
shell
fr
and
data also indicate di-f-ferences
erosion
in the
conditions
between
In
the upper and lower cages.
deeper
- 50
m) Johnson et al.
(1984)
water
(40
also
found
a
marked difference
NTR between upper and lower cages in
growth
activity.
g
one
provoke the NTR - reaction have a low speci-fic
that
e.
this difference was at least as
large
« 10 m>
The
shallow station
substances
However,
their
.
in
aromatic
.
more
influence
The -fact that
hydrocarbons).
at lower depths would indicate
weight
they
exert
their
could
be
that
This
higher near the surface.
the
case
or
if they are not completely in dissolved form,
if
and
by some mechanism they are skimmed out of the water
surface (e g
th e
taken
to
by adsorption to
rising air
concentration
<
IS
.
.
bubbles)
then
m
even
explicitly
-. ^^f
.
I-f this tentative conclusion could be
confirmed
coastal
waters
should
three-dimensional
for
any
concentration.
It
would be
shallow, well-mixed
be-~ consi d ered
as
^1
'-.^
pollytipn
concentration
to
study i-F vertical
interesting
could directly be found for organic pollutants
measurement of
^
t
.
.
f
22~
gradients
\
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-2A-