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The effects of acidic seawater on three species of lamellibranch mollusc
J. Exp. Mar. Biol. Ecol., 1990, Vol. 143, pp. 181-191 Elsevier
JEMBE
01501
The effects of acidic seawater on three species of lamellibranch mollusc R.N.
Bamber
NPTEC Marine Biology Unit, Fawley Power Station, Fawley. Southampton. UK (Received
13 June 1990; accepted
4 August
1990)
Three species of commercial bivalve mollusc, Ostrea edulis, Crassostreu gigas and Mytilus edulis, were maintained in seawater over a pH range of 5.4-8.2 (control), normally for 30 days. Significant mortalities occurred in C. gigas at pH < 6, in M. edulis at pH < 6.6, in 0. edulir young at pH < 6.9 (after 60 days) and spat at pH < 7 after 18 days. Survival at a given pH level reduced with time of exposure and with increased temperature, and increased with size of animal. Growth suppression, tissue weight loss, reduced shell size, shell dissolution and suppressed feeding activity occurred at pH < 7. Abnormal behaviour analogous to narcosis (excessive shell gaping, torpor) occurred at pH ~6.5, possibly attributable to CO, excess. These and other collated results confirm that seawater at pH ,<7 is intolerable to bivalve molluscs. Abstract:
Key words: Acidic
seawater;
Bivalve mollusc;
Crassostrea; Flue-gas
pH; Mytilus; Ostrea
desulphurization;
INTRODUCTION
There are a number of methods under consideration for the reduction of power station flue-gas emissions and their contribution to acidic precipitation. The seawater-washing process for flue-gas desulphurization (FGD) has, to date, only been applied in the world to smaller generating plants (up to 500 MW). The technology (of pumping seawater) is well known to power industries, and the process is potentially the most cost-effective of those currently under consideration for coastal power stations. The feasibility of seawater-washing
FGD,
particularly
balance between environmental of achieving that acceptability.
for larger power stations,
acceptability
of the discharge
will depend
upon the
and the potential
costs
The most important characteristic of the effluent seawater from such an FGD plant is its reduced pH. While natural pH levels, to which marine and estuarine organisms have evolved, range between 7.6 and 8.4 for fully marine seawater (occasionally dropping towards 7.0 in certain estuaries), an FGD effluent may leave the process plant at a pH ~6.0, There are two obvious ways to bring the pH back towards normal seawater levels, viz., to lime the effluent or to increase the quantity of seawater pumped
Correspondence address: R.N. Bamber, Southampton SO4 ITW, UK. 0022-0981/90/$03.50
0
1990 Elsevier
NPTEC
Science
Marine
Publishers
Biology Unit, Fawley
B.V. (Biomedical
Power Station,
Division)
Fawiey,
182
through
R.N. BAMBER the system
environmental limestone
cost:
(by up to four-fold). liming
to the eflluent,
owing to transport
would
Both methods
introduce
as well as increasing
requirements;
increased
heavy
have an economic
metal
contaminants
the environmental seawater
impact
flow will increase
and an from
the
of the plant the rate of
abstraction and impingement of marine organisms at the intake. Reduced pH has been so infrequent an occurrence naturally in the sea that there has been little research on its potential environmental effects (Knutzen, 198 1). In a previous paper (Bamber, 1987) an experimental protocol was described which was designed to investigate the tolerance of marine organisms to acidic seawater. Results were given from an initial series of experiments on the carpet-shell clam Ve/enerupisdecussata (L.); significant deleterious effects were found on this species at pH ~7. The present paper describes further experiments on three more species of lamellibranch mollusc, the native oyster Ostrea edulis L., the Pacific oyster Crassostrea gigas (Thunberg) and the native mussel Myths edulis L.; all these species are of commercial importance as human food. The relevance of such sessile benthic species in the context of an acidic effluent is that, unlike pelagic or errant forms, they would be unable to move away from the discharge water if they were to find it intolerable.
METHODS The experimental apparatus is described and figured by Bamber (1987). Essentially, test animals are maintained in 2-l aquaria in a continuous through-flow of fully aerated, sand-filtered seawater, acidified by the addition of Analar sulphuric acid to give a range of (normally) eight pH levels including the control, with up to four replicate test aquaria per pH level. From the experience of the Venerupis experiments, the tested pH range was from 5.5 up to seawater normal (usually z 8. l), with emphasis on pH 6.0-7.5. pH levels were stable within + 0.1 U over the duration of the experiments. Salinity ( z 34x,) and temperature were the ambient levels for Southampton water. Experiments undertaken at different times of the year allowed the testing of animals at different (seasonal) temperatures. lo-20 animals (depending on size) were used in each test aquarium. Experiments normally ran for 30 days, and were repeated for each species tested. 0. edulis was tested at three different ages: newly settled spat were supplied by Southern Sea Fisheries District; small (Z 1 cm across) and larger (4 cm) young native oysters were supplied by the Ministry of Agriculture, Fisheries and Food Fisheries Laboratory, Conwy. Young (Z 1 cm) C. gigas were obtained from hatchery-reared stock at Seasalter Shellfish, Whitstable. M. edulis was collected from the lower R. Adur Estuary, Sussex, and the material segregated into large (Z 5 cm, probably 5-6 yr old) and small (up to 2.5 cm, l-2 yr old) individuals. All animals were acclimated to Fawley seawater for a minimum of 1 wk prior to experimental exposure. The test animals were observed for activity (feeding, movement, etc.) throughout the experiment; measurements of valve gape during feeding were made directly with a ruler
BIVALVES
to the nearest
IN ACIDIC
1 mm. At the end of an experiment
24 h, when the test animals were fixed and preserved analysis.
Survival was determined
183
SEAWATER
the acid dosing was turned in absolute
alcohol prior to further
as the number of animals showing movement
on stimulus) within 24 h return to normal (non-acidified) seawater survivors were showing evident activity at the end of an experiment). weights were determined smaller individuals hydrochloric acid.
as dry weights:
larger molluscs
off for (closing
(in practice all Flesh and shell
were dissected,
while with
the shells were removed (after weighing) by dissolution in 6”/, Size was determined as shell length in M. edulis and 0. edulis,
measured with vernier calipers to the nearest 0.1 mm; owing to its asymmetry and irregular shape, size of C. gigas was measured as the area of the shell, determined by comparing the weight of a photocopy of the shell to that of a known-area-standard photocopy. Growth (in shell) was measured in 0. edulis as the width of new shell outside the pallial line (“postpallial shell edge”) expressed as a proportion of the remaining length of the shell on the same axis (through the umbo); growth in C. gigus was determined simply as the presence or absence of finger-like extensions of the shell edge at the end of the experiment (see Fig. 5; no individuals began the experiments with such extensions). Where individual molluscs were measured and weighed, log-log correlations were determined between size and shell and flesh weights, giving equations of the form: log W = A log size + B where Wis shell or flesh weight. Where the weight approaches a cubic function of length, the constant A approaches 3; in practice, A is usually < 3, especially for flesh weight (e.g., Nielsen, 1985) and can be (and is here) used as a direct index of flesh condition (condition factor). Samples of the relevant test species were fixed and retained at the beginning of each experiment, and analysed as above to act as a comparative “start control”. Feeding rate was measured as in Bamber (1987) by quantifying (by weight or by volume) the settled sedimentary material in each test aquarium at the end of each experiment,
on the basis that the majority
of such material
resulted
from faecal and
pseudofaecal production. Other convenient behavioural aspects, such as byssal attachment, were also quantified at the end of the experiment. Significant departures from “normal” condition for the various measurements were based on ANOVAR and t tests, in comparison to both nonacidified controls and “start controls”.
RESULTS
With the exception of 0. edulis spat, no mortalities occurred during the experiments at pH > 7.5 (Fig. 1); significant mortalities occurred at pH < 6.6 in small 0. edulis and small and large M. edulis, and at pH < 6.0 in C. gigas. Larger 0. edulis showed no
184
mortality
R.N. BAMBER
at any pH level within 30 days, and single replicates
at this time, mortalities mortality
had occurred
at all levels of pH ~7.0
(Fig. lA-C)
at pH 66.9.
were run on for 60 days;
The 0. edulis spat showed
after 18 days. It is evident
that survival improves
heavy
in the case of 0. edulis
with size. Equally, increasing
mortality
with time is
shown by the larger 0. edulis (Fig. lC), by C. gigus (Fig. 1D) and in more detail by large M. edulis (Fig. 2). An effect of temperature on survival was only shown by the large M. edulis (Fig. lE), where mortalities for all pH levels ~6.5.
at 14 “C were significantly
100
0
D
A
% 20
.. . . . .
LALt0
60
greater than at 9.2 “C
0
t
+
I
60 % 20
loo-
0000
0
C
.
.
.
. . .
60 .
%’ 20..
PH
5
6
7
0
5
6
7
6
Fig. 1. Mean percentage survival against pH of (A-C) 0. edulis (A) spat after 18 days, (B) small oysters after 30 days, (C)large oysters after 30 days (open circles) and 60 days (filled circles), (D) C. gisas after 14 days (open circles) and 30 days (tilled circles), (E) large M. edulis after 30 days at 9.2 “C (filled circles) and 14 “C (open circles), (F) small M. edulis after 30 days. Vertical bars are ranges where present from multiple replicates; 100% survival points are coincident for open and filled circles on C-E.
Shell length of M. edulis showed a significant positive correlation with pH, notably pH ~7.5, but, with much intrasample variation in size, there was no significant distinction found between individual pH levels based on ANOVAR (Fig. 3A). Shell sizes of large (Fig. 3B) and small 0. edulis and C. gigas (Fig. 3C) also showed a positive
BIVALVES
IN ACIDIC
I
185
20
15
IO
5
SEAWATER
25
-jr,
daya Fig. 2. Percentage
survival
over time for large M. edulis at four levels of pH (means
L
for four replicates).
6
2
L_
5
.
P
z3.5
__
______e__
____
+ #
_
______
d’ *
5
.
5
3
D
F-20, E
___
_-mm_
__
_______
_-em
.;15. = 2 10’ *
p,,5
6
7
8
p,,5
+
t
t
li
t
+a
6
7
8
Fig. 3. Condition (means with ranges) of bivalve molluscs after 30 days at a range of pH: shell length (mm) of(A) M. edulis and (B) 0. edulis, (C) shell area (mm*) of C. gigas, (D) shell weight (mg) of”small” 0. edulis, (E) flesh weight (mg) of C. gigas, (F) condition factor (power relationship between flesh weight and shell length) of large M. edulis. Horizontal dashed lines on B, D and E represent levels for control animals at beginning of experiment.
186
R.N. BAMBER
trend with pH, and after 30 days the animals were significantly smaller than nonacidified controls at pH < 7.0; 0. eliulis were smaller than start controls at pH < 6.0. Shell weights of M. edulis showed a trend of weight loss but no significant relationship with pH. Both oyster species showed a positive correlation to pH; being significantly lighter than controls at pH < 7, and 0. edzdis specimens again being hghter than start controls at pH < 6 (e.g., Fig. 3D). When related to shell size, the 0. edzdis relationship was not significant, but C. gigus “shell density” (weight per cube of the length) was significantly suppressed at pH t7. These significantly less dense C. gigus shells, and the 0. edulis specimens which were smaller than start controls after 30 days, must be a result of shell dissolution at these pH levels. The mussel and C. gigas shells appeared to be dissolving over their whole surface, leading to reduced weight for dimension of shell; the 0. eduiti on the other hand lost shell material most noticeably from the more fragile shell edge, resulting in “smaller” shells. Flesh weights showed a significant positive trend with pH in C. gigas (Fig. 3E), being lower than those of start controls at pH ~6; there was a less clear but significant positive correlation for 0. edulis, large animals having a lower flesh weight after 30 days at pH 6.5 than start controls; the flesh weights for M. edulis, expressed as condition factor (Fig. 3F), again showed a significant positive correlation but with much intrasample variance. .
.
Fig. 4. Growth (means with ranges) over a range of pH of (A) large 0. e&&s (postpallial shell edge as a proportion of prepallial shell length) and (B) C. gigus (T/, showing extension growth after 14 days).
Growth was not measured for M. edulis. The proportions of post-pallial shell edge of 0. edulis showed a significant suppression of growth at pH < 7 (Fig. 4A), while extension growth of C. gigus was significantly suppressed at pH < 7.1 and absent at pH < 6.5 (Figs. 4B, 5). Feeding rate, expressed as faecal and pseudofaecal production, showed a consistent positive correlation with pH in all the species tested, with significant reduction in feeding at pH < 7.2 (Fig. 6), irrespective of the size of the animal, water temperature or duration of the experiment. All three species exhibited behavioural inhibition analogous to narcosis at pH d 6.6; they showed conspicuously increased gaping of the shells at these levels (Fig. 7) and would close only slowly in response to repeated stimuli (tapping the a~u~ia~, while
BIVALVES
IN ACIDIC
I cm
,
Fig. 5. C. gigus, diagrammatic,
30-
SEAWATER
187
4
showing degree of development of “fingers” of extension at pH 6, 7 and 8 (control).
growth after 30 days
D
A t
20
10.
65
60 I
201” PH
5
6
7
8
Fig. 6. Faecal plus pseudofaecal production (as weight or volume of residual sediment in test aquaria, means with ranges in arbitrary units) over a range of pH after 30 days for (A) small 0. edulis, (B) large 0. edtdis, (C and D) C. g&s. (E) large (open circles) and small (filled circles) M. edulis at 9.2 “C, (F) large M. edulis at 14 “C.
R.N.BAMBER
18X
individuals at higher levels showed a normal response of rapid closure after a single stimulus. Byssal attachment is the normal behaviour for both M, edulis and young C. gigas: such behaviour was absent in mussels at pH < 6.6 and oysters at pH < 6.9. These behavioural responses showed absolute changes at the critical pH levels indicated rather than trends with pH.
* I
10 t
A
PH
B
+
E5 0
20
5
6
15.
I. ***+ v 7
IO.
8
Ii it
it+
t
PH
Fig. 7.Shell gape (means with ranges, mm) after 11 days at a range of pH for (A) 0. edulis and (B) M. edulis.
DISCUSSION
The responses of the tested molluscs to reduced pH in seawater are summarized in Fig. 8. Critical pH levels for significant mortality ranged from 6.9 for 0. edzdis (after 60 days), 6.6 for M. edulis (30 days) and 6.0 for C. gigas (30 days), levels increasing with time and temperature and decreasing with increasing size. All species showed similar responses to acidity of growth suppression, shell dissolution, tissue weight loss and feeding activity suppression at pH G7.0, and abnormal (“narcotic”) behaviour at pH 66.5. All responses other than the last showed a trend (negative correlation for mortality, positive correlation for the others), with pH, at least over the range of response significantly deviating from that of the controls. These responses are attributed to a direct effect of acidity, and indicate that pH levels below those occurring naturally in the sea are deleterious to these species at least. The critical pH level of 6.5 causing abrupt changes in irritability and behaviour was not part of a pH-related trend, and may be attributable to disruption of the CO,carbonate buffe~ng system in the seawater (see ~it~eld & Turner, 1986). It is significant that the pH threshold for significant avoidance of acidic seawater by fish is also at this pH level (Davies, 1989) again attributed to change in the CO, equilibrium. Though Carter’s (1964) work on fish showed an initial narcotic effect of increased CO, followed by recovery, it is not clear whether this reflected a readjustment of the carbonate equilibrium or some adaptation by the fish. If either of these were to apply to the tested invertebrates, then increased CO, is probably not the cause of the behavioural responses. Dissolved CO, was measured during experiments, and found
BIVALVES IN ACIDIC SEAWATER
189
to be at full saturation in all aquaria at all pH levels; unfortunately, the methods of measuring CO, levels in seawater involve acidification, and are thus inapplicable to
these experiments. Alternative techniques are being explored.
Increasing
Feeding
mortality
suppressed
Reduced growth, size, flesh and shell weight
Behovioural
PH
I 5
Range of ‘normal
Inhibition
I
6
I 7
sea-water
I 8
Fig. 8. Summary of effects of acidic seawater on 0. edulis (open bars), C. gigas (particoloured bars) and M. edulis (black bars) after 30 days (large 0. edulis after 60 days).
Estuarine animals were initially expected to be more tolerant to reduced pH than stenohaline marine species. It was thus somewhat surprising that, for the only response which showed some marked interspecific variation, viz., mortality, 0. e&&s survived much longer than estuarine M. e&&s; research into stress assessment of 0. edulis (e.g., Hutchinson & Hawkins, 1989) has generally shown that this species is more sensitive to such stresses as tidal exposure and salinity change (L. E. Hawkins, pers. comm.). Couion et al. (1987) suggest that the survival of M. edulis -c 50 m from a TiO, industry effluent, of intermittent low pH, demonstrates its ability to adapt to a low pH environment. They quote 24-h TL,, levels, from other studies, of pH 1.8 for M. edulis and 4.5 for the oyster. There will inevitably be variability in tolerance to environmental characters between ~~~~~~~populations (e.g., Mallet et al., 1987); nonetheless, these data say more about the inadequacy of 24-h tolerance tests than about the differential sensitivity of mussels and oysters. The time scale of the experiments was determined by the maximum time before such factors as siltation in and algai growth on the apparatus might be compromising the integrity of the experimental design, assessed to be z 30 days (the 60-day 0. edulis experiment involved an entire cleaning of the apparatus after the first 30 days). From the evidence both of increasing mortality with time at all levels of pH < 7.0 and of significant sublethal effects at higher subnormal levels, it must be assumed that reduced pH will lead to significant mortalities at these higher subnormal levels in the longer term. The tested species take 1 yr to reach maturity. While larger animals have been shown
R.N. BAMBER
190
to be more tolerant would
survive
unfortunately
to acidity, the ultimate
to produce impractical
viable
offspring
test of significant (i.e., a time
effect is whether
animals
scale of two generations,
for these experiments).
TABLE I
Other responses of bivalves to low pH reported in literature Species
Effect
M. edulis Pinrada fucata
Reduced gamete respiration Increased adult mortality Increased weight loss attributed to shell dissolution Adult reduced pumping rate Abnormal adult shell movement Increased larval mortality Inhibited larval development Reduced larval growth Increased larval mortality Inhibited larval development Reduced larval growth Inhibition of feeding Inhibition of shell growth Flesh weight reduction Increased shell dissolution Behavioural inhibition Increased mortality
C. virghica
M. mercenaria
V. decussata
Critical pH 1.6 1.48 7.66 7.0 7.0 7.0 6.15 6.15 6.5 7.0 6.15 7.0 7.0 7.0 1.5 6.0 6.1-6.4
Authority Akberali et al. (1985) Kawatani & Nishii (1969) Kawatani & Nishii (1969) Loosanoff & Tommers (1947) Loosanoff & Tommers (1947) Calabrese & Davis (1966) Calabrese & Davis (1966) Calabrese & Davis (1966) Calabrese & Davis (1966) Calabrese & Davis (1966) Calabrese & Davis (1966) Bamber (1987) Bamber (1987) Bamber (1987) Bamber (1987) Bamber (1987) Bamber (1987)
The results of other studies on low pH effects on bivalve molluscs are summarized in Table I; the experiments of Loosanoff & Tommers (1947) and of Calabrese & Davis (1966) were conducted in static seawater, of drifting pH. While some significant deleterious effects are found at pH z 7.5 (surprisingly close to the lower limit of natural seawater), it is clear from these data and the present results that seawater at pH d 7.0 is intolerable to bivalve molluscs. This is above the levels currently recommended as “Environmental
Quality
Standards”
(e.g., EPA, 1976; EEC, 1979).
ACKNOWLEDGEMENTS I am grateful to Southern Sea Fisheries District and Peter Millican of the MAFF Conwy Laboratory for the 0. edulis material and to Nigel Bridgwater for assistance in the collection of the M. edulis; also to various members of staff at the Fawley Marine Biology Unit for helping to maintain the continuity of these longer term experiments.
BIVALVES
IN ACIDIC
SEAWATER
191
REFERENCES Akberali, H. B., M. J. Earnshaw & K. R. M. Marriott, 1985. The action of heavy metals on the gametes of the marine mussel, Myrilus edulis (L.) - II. Uptake of copper and zinc and their effect on respiration in the sperm and unfertilized egg. Mar. Environ. Res., Vol. 16, pp. 37-59. Bamber, R.N., 1987. The effects of acidic sea water on young carpet-shell clams Venerupis decussata (L.) (Mollusca: Veneracea). /. Exp. Mar. Biol. Ecol., Vol. 108, pp. 241-260. Calabrese, A., & H. C. Davis, 1966. The pH tolerance of embryos and larvae of Mercenaria mercenaria and Crassostrea virginica. Biol. Bull. (Woods Hole, Mass.), Vol. 13 1, pp. 427-436. Carter, L., 1964. Effects of acidic and alkaline efluents on fish in sea-water. Effluent Water Treat. J., No. October 1964, pp. 484-486. Coulon, J., M. Truchet & R. Martoja, 1987. Chemical features of mussels (Mytilus edulis) in situ exposed to an eflluent of the titanium dioxide industry. Ann. Insf. Oceanogr. Paris, Vol. 63, pp. 89-100. Davies, J. K., 1989. Reactions of marine fish to low pH sea water I. sand smelt (Atherina boyeri Risso). Cent. Electr. Gener. Board Intern. Rep., No. ESTD/L/0046/R89, 16 pp. EEC, 1979. Council directive on the quality required of shell fish waters. Off: J. Eur. Commun., No. 79/923/EEC. EPA, 1976. Quality criteria for water. U.S. Environ. Prof. Agency Rep., No. EPA-44019-76-023, 501 pp. Hutchinson, S. & L.E. Hawkins, 1989. Stress assessment of Ostrea edulis. Porcupine Newsl., Vol. 4, pp. 106-l 12. Knutzen, J., 1981. Effects of decreased pH on marine organisms. Mar. Pollut. Bull., Vol. 12, pp. 25-29. Kuwatani, Y. & T. Nishii, 1969. Effects of pH of culture water on the growth of the Japanese pearl oyster. Bull. Jpn Sot. Sci. Fish., Vol. 35, pp. 342-350. Loosanoff, V. L. & F. D. Tommers, 1947. Effect of low pH upon rate of water pumping of oysters, Ostrea virginica. Anat. Rec., Vol. 99, pp. 668-669. Mallett, A. L., C. E. A. Carver, S. S. Coffen & K. R. Freeman, 1987. Mortality variations in natural populations of the blue mussel, Mytilus edulis. Can J. Fish. Aquaf. Sci., Vol. 44, pp. 1589-1594. Nielsen, M.V., 1985. Increase in shell length as a measure of production and ingestion ofMytilus edulis (L.). J. Exp. Mar. Biol. Ecol., Vol. 88, pp. 101-108. Whitfield, M. & D. R. Turner, 1986. The carbon-dioxide system in estuaries - an inorganic perspective. Sci. Total Environ., Vol. 49, pp. 235-255.
JEMBE
01501
The effects of acidic seawater on three species of lamellibranch mollusc R.N.
Bamber
NPTEC Marine Biology Unit, Fawley Power Station, Fawley. Southampton. UK (Received
13 June 1990; accepted
4 August
1990)
Three species of commercial bivalve mollusc, Ostrea edulis, Crassostreu gigas and Mytilus edulis, were maintained in seawater over a pH range of 5.4-8.2 (control), normally for 30 days. Significant mortalities occurred in C. gigas at pH < 6, in M. edulis at pH < 6.6, in 0. edulir young at pH < 6.9 (after 60 days) and spat at pH < 7 after 18 days. Survival at a given pH level reduced with time of exposure and with increased temperature, and increased with size of animal. Growth suppression, tissue weight loss, reduced shell size, shell dissolution and suppressed feeding activity occurred at pH < 7. Abnormal behaviour analogous to narcosis (excessive shell gaping, torpor) occurred at pH ~6.5, possibly attributable to CO, excess. These and other collated results confirm that seawater at pH ,<7 is intolerable to bivalve molluscs. Abstract:
Key words: Acidic
seawater;
Bivalve mollusc;
Crassostrea; Flue-gas
pH; Mytilus; Ostrea
desulphurization;
INTRODUCTION
There are a number of methods under consideration for the reduction of power station flue-gas emissions and their contribution to acidic precipitation. The seawater-washing process for flue-gas desulphurization (FGD) has, to date, only been applied in the world to smaller generating plants (up to 500 MW). The technology (of pumping seawater) is well known to power industries, and the process is potentially the most cost-effective of those currently under consideration for coastal power stations. The feasibility of seawater-washing
FGD,
particularly
balance between environmental of achieving that acceptability.
for larger power stations,
acceptability
of the discharge
will depend
upon the
and the potential
costs
The most important characteristic of the effluent seawater from such an FGD plant is its reduced pH. While natural pH levels, to which marine and estuarine organisms have evolved, range between 7.6 and 8.4 for fully marine seawater (occasionally dropping towards 7.0 in certain estuaries), an FGD effluent may leave the process plant at a pH ~6.0, There are two obvious ways to bring the pH back towards normal seawater levels, viz., to lime the effluent or to increase the quantity of seawater pumped
Correspondence address: R.N. Bamber, Southampton SO4 ITW, UK. 0022-0981/90/$03.50
0
1990 Elsevier
NPTEC
Science
Marine
Publishers
Biology Unit, Fawley
B.V. (Biomedical
Power Station,
Division)
Fawiey,
182
through
R.N. BAMBER the system
environmental limestone
cost:
(by up to four-fold). liming
to the eflluent,
owing to transport
would
Both methods
introduce
as well as increasing
requirements;
increased
heavy
have an economic
metal
contaminants
the environmental seawater
impact
flow will increase
and an from
the
of the plant the rate of
abstraction and impingement of marine organisms at the intake. Reduced pH has been so infrequent an occurrence naturally in the sea that there has been little research on its potential environmental effects (Knutzen, 198 1). In a previous paper (Bamber, 1987) an experimental protocol was described which was designed to investigate the tolerance of marine organisms to acidic seawater. Results were given from an initial series of experiments on the carpet-shell clam Ve/enerupisdecussata (L.); significant deleterious effects were found on this species at pH ~7. The present paper describes further experiments on three more species of lamellibranch mollusc, the native oyster Ostrea edulis L., the Pacific oyster Crassostrea gigas (Thunberg) and the native mussel Myths edulis L.; all these species are of commercial importance as human food. The relevance of such sessile benthic species in the context of an acidic effluent is that, unlike pelagic or errant forms, they would be unable to move away from the discharge water if they were to find it intolerable.
METHODS The experimental apparatus is described and figured by Bamber (1987). Essentially, test animals are maintained in 2-l aquaria in a continuous through-flow of fully aerated, sand-filtered seawater, acidified by the addition of Analar sulphuric acid to give a range of (normally) eight pH levels including the control, with up to four replicate test aquaria per pH level. From the experience of the Venerupis experiments, the tested pH range was from 5.5 up to seawater normal (usually z 8. l), with emphasis on pH 6.0-7.5. pH levels were stable within + 0.1 U over the duration of the experiments. Salinity ( z 34x,) and temperature were the ambient levels for Southampton water. Experiments undertaken at different times of the year allowed the testing of animals at different (seasonal) temperatures. lo-20 animals (depending on size) were used in each test aquarium. Experiments normally ran for 30 days, and were repeated for each species tested. 0. edulis was tested at three different ages: newly settled spat were supplied by Southern Sea Fisheries District; small (Z 1 cm across) and larger (4 cm) young native oysters were supplied by the Ministry of Agriculture, Fisheries and Food Fisheries Laboratory, Conwy. Young (Z 1 cm) C. gigas were obtained from hatchery-reared stock at Seasalter Shellfish, Whitstable. M. edulis was collected from the lower R. Adur Estuary, Sussex, and the material segregated into large (Z 5 cm, probably 5-6 yr old) and small (up to 2.5 cm, l-2 yr old) individuals. All animals were acclimated to Fawley seawater for a minimum of 1 wk prior to experimental exposure. The test animals were observed for activity (feeding, movement, etc.) throughout the experiment; measurements of valve gape during feeding were made directly with a ruler
BIVALVES
to the nearest
IN ACIDIC
1 mm. At the end of an experiment
24 h, when the test animals were fixed and preserved analysis.
Survival was determined
183
SEAWATER
the acid dosing was turned in absolute
alcohol prior to further
as the number of animals showing movement
on stimulus) within 24 h return to normal (non-acidified) seawater survivors were showing evident activity at the end of an experiment). weights were determined smaller individuals hydrochloric acid.
as dry weights:
larger molluscs
off for (closing
(in practice all Flesh and shell
were dissected,
while with
the shells were removed (after weighing) by dissolution in 6”/, Size was determined as shell length in M. edulis and 0. edulis,
measured with vernier calipers to the nearest 0.1 mm; owing to its asymmetry and irregular shape, size of C. gigas was measured as the area of the shell, determined by comparing the weight of a photocopy of the shell to that of a known-area-standard photocopy. Growth (in shell) was measured in 0. edulis as the width of new shell outside the pallial line (“postpallial shell edge”) expressed as a proportion of the remaining length of the shell on the same axis (through the umbo); growth in C. gigus was determined simply as the presence or absence of finger-like extensions of the shell edge at the end of the experiment (see Fig. 5; no individuals began the experiments with such extensions). Where individual molluscs were measured and weighed, log-log correlations were determined between size and shell and flesh weights, giving equations of the form: log W = A log size + B where Wis shell or flesh weight. Where the weight approaches a cubic function of length, the constant A approaches 3; in practice, A is usually < 3, especially for flesh weight (e.g., Nielsen, 1985) and can be (and is here) used as a direct index of flesh condition (condition factor). Samples of the relevant test species were fixed and retained at the beginning of each experiment, and analysed as above to act as a comparative “start control”. Feeding rate was measured as in Bamber (1987) by quantifying (by weight or by volume) the settled sedimentary material in each test aquarium at the end of each experiment,
on the basis that the majority
of such material
resulted
from faecal and
pseudofaecal production. Other convenient behavioural aspects, such as byssal attachment, were also quantified at the end of the experiment. Significant departures from “normal” condition for the various measurements were based on ANOVAR and t tests, in comparison to both nonacidified controls and “start controls”.
RESULTS
With the exception of 0. edulis spat, no mortalities occurred during the experiments at pH > 7.5 (Fig. 1); significant mortalities occurred at pH < 6.6 in small 0. edulis and small and large M. edulis, and at pH < 6.0 in C. gigas. Larger 0. edulis showed no
184
mortality
R.N. BAMBER
at any pH level within 30 days, and single replicates
at this time, mortalities mortality
had occurred
at all levels of pH ~7.0
(Fig. lA-C)
at pH 66.9.
were run on for 60 days;
The 0. edulis spat showed
after 18 days. It is evident
that survival improves
heavy
in the case of 0. edulis
with size. Equally, increasing
mortality
with time is
shown by the larger 0. edulis (Fig. lC), by C. gigus (Fig. 1D) and in more detail by large M. edulis (Fig. 2). An effect of temperature on survival was only shown by the large M. edulis (Fig. lE), where mortalities for all pH levels ~6.5.
at 14 “C were significantly
100
0
D
A
% 20
.. . . . .
LALt0
60
greater than at 9.2 “C
0
t
+
I
60 % 20
loo-
0000
0
C
.
.
.
. . .
60 .
%’ 20..
PH
5
6
7
0
5
6
7
6
Fig. 1. Mean percentage survival against pH of (A-C) 0. edulis (A) spat after 18 days, (B) small oysters after 30 days, (C)large oysters after 30 days (open circles) and 60 days (filled circles), (D) C. gisas after 14 days (open circles) and 30 days (tilled circles), (E) large M. edulis after 30 days at 9.2 “C (filled circles) and 14 “C (open circles), (F) small M. edulis after 30 days. Vertical bars are ranges where present from multiple replicates; 100% survival points are coincident for open and filled circles on C-E.
Shell length of M. edulis showed a significant positive correlation with pH, notably pH ~7.5, but, with much intrasample variation in size, there was no significant distinction found between individual pH levels based on ANOVAR (Fig. 3A). Shell sizes of large (Fig. 3B) and small 0. edulis and C. gigas (Fig. 3C) also showed a positive
BIVALVES
IN ACIDIC
I
185
20
15
IO
5
SEAWATER
25
-jr,
daya Fig. 2. Percentage
survival
over time for large M. edulis at four levels of pH (means
L
for four replicates).
6
2
L_
5
.
P
z3.5
__
______e__
____
+ #
_
______
d’ *
5
.
5
3
D
F-20, E
___
_-mm_
__
_______
_-em
.;15. = 2 10’ *
p,,5
6
7
8
p,,5
+
t
t
li
t
+a
6
7
8
Fig. 3. Condition (means with ranges) of bivalve molluscs after 30 days at a range of pH: shell length (mm) of(A) M. edulis and (B) 0. edulis, (C) shell area (mm*) of C. gigas, (D) shell weight (mg) of”small” 0. edulis, (E) flesh weight (mg) of C. gigas, (F) condition factor (power relationship between flesh weight and shell length) of large M. edulis. Horizontal dashed lines on B, D and E represent levels for control animals at beginning of experiment.
186
R.N. BAMBER
trend with pH, and after 30 days the animals were significantly smaller than nonacidified controls at pH < 7.0; 0. eliulis were smaller than start controls at pH < 6.0. Shell weights of M. edulis showed a trend of weight loss but no significant relationship with pH. Both oyster species showed a positive correlation to pH; being significantly lighter than controls at pH < 7, and 0. edzdis specimens again being hghter than start controls at pH < 6 (e.g., Fig. 3D). When related to shell size, the 0. edzdis relationship was not significant, but C. gigus “shell density” (weight per cube of the length) was significantly suppressed at pH t7. These significantly less dense C. gigus shells, and the 0. edulis specimens which were smaller than start controls after 30 days, must be a result of shell dissolution at these pH levels. The mussel and C. gigas shells appeared to be dissolving over their whole surface, leading to reduced weight for dimension of shell; the 0. eduiti on the other hand lost shell material most noticeably from the more fragile shell edge, resulting in “smaller” shells. Flesh weights showed a significant positive trend with pH in C. gigas (Fig. 3E), being lower than those of start controls at pH ~6; there was a less clear but significant positive correlation for 0. edulis, large animals having a lower flesh weight after 30 days at pH 6.5 than start controls; the flesh weights for M. edulis, expressed as condition factor (Fig. 3F), again showed a significant positive correlation but with much intrasample variance. .
.
Fig. 4. Growth (means with ranges) over a range of pH of (A) large 0. e&&s (postpallial shell edge as a proportion of prepallial shell length) and (B) C. gigus (T/, showing extension growth after 14 days).
Growth was not measured for M. edulis. The proportions of post-pallial shell edge of 0. edulis showed a significant suppression of growth at pH < 7 (Fig. 4A), while extension growth of C. gigus was significantly suppressed at pH < 7.1 and absent at pH < 6.5 (Figs. 4B, 5). Feeding rate, expressed as faecal and pseudofaecal production, showed a consistent positive correlation with pH in all the species tested, with significant reduction in feeding at pH < 7.2 (Fig. 6), irrespective of the size of the animal, water temperature or duration of the experiment. All three species exhibited behavioural inhibition analogous to narcosis at pH d 6.6; they showed conspicuously increased gaping of the shells at these levels (Fig. 7) and would close only slowly in response to repeated stimuli (tapping the a~u~ia~, while
BIVALVES
IN ACIDIC
I cm
,
Fig. 5. C. gigus, diagrammatic,
30-
SEAWATER
187
4
showing degree of development of “fingers” of extension at pH 6, 7 and 8 (control).
growth after 30 days
D
A t
20
10.
65
60 I
201” PH
5
6
7
8
Fig. 6. Faecal plus pseudofaecal production (as weight or volume of residual sediment in test aquaria, means with ranges in arbitrary units) over a range of pH after 30 days for (A) small 0. edulis, (B) large 0. edtdis, (C and D) C. g&s. (E) large (open circles) and small (filled circles) M. edulis at 9.2 “C, (F) large M. edulis at 14 “C.
R.N.BAMBER
18X
individuals at higher levels showed a normal response of rapid closure after a single stimulus. Byssal attachment is the normal behaviour for both M, edulis and young C. gigas: such behaviour was absent in mussels at pH < 6.6 and oysters at pH < 6.9. These behavioural responses showed absolute changes at the critical pH levels indicated rather than trends with pH.
* I
10 t
A
PH
B
+
E5 0
20
5
6
15.
I. ***+ v 7
IO.
8
Ii it
it+
t
PH
Fig. 7.Shell gape (means with ranges, mm) after 11 days at a range of pH for (A) 0. edulis and (B) M. edulis.
DISCUSSION
The responses of the tested molluscs to reduced pH in seawater are summarized in Fig. 8. Critical pH levels for significant mortality ranged from 6.9 for 0. edzdis (after 60 days), 6.6 for M. edulis (30 days) and 6.0 for C. gigas (30 days), levels increasing with time and temperature and decreasing with increasing size. All species showed similar responses to acidity of growth suppression, shell dissolution, tissue weight loss and feeding activity suppression at pH G7.0, and abnormal (“narcotic”) behaviour at pH 66.5. All responses other than the last showed a trend (negative correlation for mortality, positive correlation for the others), with pH, at least over the range of response significantly deviating from that of the controls. These responses are attributed to a direct effect of acidity, and indicate that pH levels below those occurring naturally in the sea are deleterious to these species at least. The critical pH level of 6.5 causing abrupt changes in irritability and behaviour was not part of a pH-related trend, and may be attributable to disruption of the CO,carbonate buffe~ng system in the seawater (see ~it~eld & Turner, 1986). It is significant that the pH threshold for significant avoidance of acidic seawater by fish is also at this pH level (Davies, 1989) again attributed to change in the CO, equilibrium. Though Carter’s (1964) work on fish showed an initial narcotic effect of increased CO, followed by recovery, it is not clear whether this reflected a readjustment of the carbonate equilibrium or some adaptation by the fish. If either of these were to apply to the tested invertebrates, then increased CO, is probably not the cause of the behavioural responses. Dissolved CO, was measured during experiments, and found
BIVALVES IN ACIDIC SEAWATER
189
to be at full saturation in all aquaria at all pH levels; unfortunately, the methods of measuring CO, levels in seawater involve acidification, and are thus inapplicable to
these experiments. Alternative techniques are being explored.
Increasing
Feeding
mortality
suppressed
Reduced growth, size, flesh and shell weight
Behovioural
PH
I 5
Range of ‘normal
Inhibition
I
6
I 7
sea-water
I 8
Fig. 8. Summary of effects of acidic seawater on 0. edulis (open bars), C. gigas (particoloured bars) and M. edulis (black bars) after 30 days (large 0. edulis after 60 days).
Estuarine animals were initially expected to be more tolerant to reduced pH than stenohaline marine species. It was thus somewhat surprising that, for the only response which showed some marked interspecific variation, viz., mortality, 0. e&&s survived much longer than estuarine M. e&&s; research into stress assessment of 0. edulis (e.g., Hutchinson & Hawkins, 1989) has generally shown that this species is more sensitive to such stresses as tidal exposure and salinity change (L. E. Hawkins, pers. comm.). Couion et al. (1987) suggest that the survival of M. edulis -c 50 m from a TiO, industry effluent, of intermittent low pH, demonstrates its ability to adapt to a low pH environment. They quote 24-h TL,, levels, from other studies, of pH 1.8 for M. edulis and 4.5 for the oyster. There will inevitably be variability in tolerance to environmental characters between ~~~~~~~populations (e.g., Mallet et al., 1987); nonetheless, these data say more about the inadequacy of 24-h tolerance tests than about the differential sensitivity of mussels and oysters. The time scale of the experiments was determined by the maximum time before such factors as siltation in and algai growth on the apparatus might be compromising the integrity of the experimental design, assessed to be z 30 days (the 60-day 0. edulis experiment involved an entire cleaning of the apparatus after the first 30 days). From the evidence both of increasing mortality with time at all levels of pH < 7.0 and of significant sublethal effects at higher subnormal levels, it must be assumed that reduced pH will lead to significant mortalities at these higher subnormal levels in the longer term. The tested species take 1 yr to reach maturity. While larger animals have been shown
R.N. BAMBER
190
to be more tolerant would
survive
unfortunately
to acidity, the ultimate
to produce impractical
viable
offspring
test of significant (i.e., a time
effect is whether
animals
scale of two generations,
for these experiments).
TABLE I
Other responses of bivalves to low pH reported in literature Species
Effect
M. edulis Pinrada fucata
Reduced gamete respiration Increased adult mortality Increased weight loss attributed to shell dissolution Adult reduced pumping rate Abnormal adult shell movement Increased larval mortality Inhibited larval development Reduced larval growth Increased larval mortality Inhibited larval development Reduced larval growth Inhibition of feeding Inhibition of shell growth Flesh weight reduction Increased shell dissolution Behavioural inhibition Increased mortality
C. virghica
M. mercenaria
V. decussata
Critical pH 1.6 1.48 7.66 7.0 7.0 7.0 6.15 6.15 6.5 7.0 6.15 7.0 7.0 7.0 1.5 6.0 6.1-6.4
Authority Akberali et al. (1985) Kawatani & Nishii (1969) Kawatani & Nishii (1969) Loosanoff & Tommers (1947) Loosanoff & Tommers (1947) Calabrese & Davis (1966) Calabrese & Davis (1966) Calabrese & Davis (1966) Calabrese & Davis (1966) Calabrese & Davis (1966) Calabrese & Davis (1966) Bamber (1987) Bamber (1987) Bamber (1987) Bamber (1987) Bamber (1987) Bamber (1987)
The results of other studies on low pH effects on bivalve molluscs are summarized in Table I; the experiments of Loosanoff & Tommers (1947) and of Calabrese & Davis (1966) were conducted in static seawater, of drifting pH. While some significant deleterious effects are found at pH z 7.5 (surprisingly close to the lower limit of natural seawater), it is clear from these data and the present results that seawater at pH d 7.0 is intolerable to bivalve molluscs. This is above the levels currently recommended as “Environmental
Quality
Standards”
(e.g., EPA, 1976; EEC, 1979).
ACKNOWLEDGEMENTS I am grateful to Southern Sea Fisheries District and Peter Millican of the MAFF Conwy Laboratory for the 0. edulis material and to Nigel Bridgwater for assistance in the collection of the M. edulis; also to various members of staff at the Fawley Marine Biology Unit for helping to maintain the continuity of these longer term experiments.
BIVALVES
IN ACIDIC
SEAWATER
191
REFERENCES Akberali, H. B., M. J. Earnshaw & K. R. M. Marriott, 1985. The action of heavy metals on the gametes of the marine mussel, Myrilus edulis (L.) - II. Uptake of copper and zinc and their effect on respiration in the sperm and unfertilized egg. Mar. Environ. Res., Vol. 16, pp. 37-59. Bamber, R.N., 1987. The effects of acidic sea water on young carpet-shell clams Venerupis decussata (L.) (Mollusca: Veneracea). /. Exp. Mar. Biol. Ecol., Vol. 108, pp. 241-260. Calabrese, A., & H. C. Davis, 1966. The pH tolerance of embryos and larvae of Mercenaria mercenaria and Crassostrea virginica. Biol. Bull. (Woods Hole, Mass.), Vol. 13 1, pp. 427-436. Carter, L., 1964. Effects of acidic and alkaline efluents on fish in sea-water. Effluent Water Treat. J., No. October 1964, pp. 484-486. Coulon, J., M. Truchet & R. Martoja, 1987. Chemical features of mussels (Mytilus edulis) in situ exposed to an eflluent of the titanium dioxide industry. Ann. Insf. Oceanogr. Paris, Vol. 63, pp. 89-100. Davies, J. K., 1989. Reactions of marine fish to low pH sea water I. sand smelt (Atherina boyeri Risso). Cent. Electr. Gener. Board Intern. Rep., No. ESTD/L/0046/R89, 16 pp. EEC, 1979. Council directive on the quality required of shell fish waters. Off: J. Eur. Commun., No. 79/923/EEC. EPA, 1976. Quality criteria for water. U.S. Environ. Prof. Agency Rep., No. EPA-44019-76-023, 501 pp. Hutchinson, S. & L.E. Hawkins, 1989. Stress assessment of Ostrea edulis. Porcupine Newsl., Vol. 4, pp. 106-l 12. Knutzen, J., 1981. Effects of decreased pH on marine organisms. Mar. Pollut. Bull., Vol. 12, pp. 25-29. Kuwatani, Y. & T. Nishii, 1969. Effects of pH of culture water on the growth of the Japanese pearl oyster. Bull. Jpn Sot. Sci. Fish., Vol. 35, pp. 342-350. Loosanoff, V. L. & F. D. Tommers, 1947. Effect of low pH upon rate of water pumping of oysters, Ostrea virginica. Anat. Rec., Vol. 99, pp. 668-669. Mallett, A. L., C. E. A. Carver, S. S. Coffen & K. R. Freeman, 1987. Mortality variations in natural populations of the blue mussel, Mytilus edulis. Can J. Fish. Aquaf. Sci., Vol. 44, pp. 1589-1594. Nielsen, M.V., 1985. Increase in shell length as a measure of production and ingestion ofMytilus edulis (L.). J. Exp. Mar. Biol. Ecol., Vol. 88, pp. 101-108. Whitfield, M. & D. R. Turner, 1986. The carbon-dioxide system in estuaries - an inorganic perspective. Sci. Total Environ., Vol. 49, pp. 235-255.