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Combined effects of salinity and cadmium chloride upon embryos and larvae of the Japanese oyster, Crassostrea gigas
Marine Environmental Research 15 (1985) 303-312
Combined Effects of Salinity and Cadmium Chloride upon Embryos and Larvae of the Japanese Oyster,
Crassostrea gigas R. R o b e r t & E. His Institut Franqais de Recherche pour rExploration de la Mer, 63 boulevard Deganne 33120 Arcachon, France (Received: 21 January, 1985)
ABSTRACT Following the observation of high concentrations of cadmium in Gironde estuarine waters, the response of embryos and larvae of the Japanese oyster, Crassostrea gigas to this heavy metal at various salinity regimes was studied for 10 days in the laboratory. The experimental design was a 4 x 4factorial experiment using concentrations of cadmium of 0, 10, 20 and 50 pg litre -1 and salinities of 20, 25, 30 and 35%0. Statistical analysis indicated that the salinity changes have important effects on the embryonic development, survival and growth of C. gigas larvae. No deleterious effect of the toxin at all cadmium concentrations and no interaction between cadmium and salinity were found.
INTRODUCTION Situated 15 km above Le Verdon, the left part of the Gironde estuary, in south-west France (see Fig. 1), was, until recently, the largest oyster reef on the Atlantic French coast. Formerly, Crassostrea angulata spat accounted for 3 0 ~ of French oyster production. In 1970, disease destroyed these beds, which have, since 1973, been replaced by the Japanese oyster, Crassostrea gigas. In spite of the destruction of the most productive zone, due to the recent extension of Le Verdon Harbour, the spawning stock was large enough 303 Marine Environ. Res. 0141-1136/85/$03"30 © ElsevierApplied Science Publishers Ltd, England, 1985. Printed in Great Britain
304
R. Robert, E. His
The
~ A
ROYAN
NCE GIRONDE
z
Le
RCACHOI
Verdon
,,¢ uJ
o 0
I-z
0
I0
I
i
20kin
I
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Ambes s p i t
Fig. 1.
Map of the Gironde estuary showing the position of the Crassostrea gigas oyster reef (hatched area).
to mitigate the lack of spat from the Marennes-Oleron and Arcachon centers. Since 1979, high concentrations of cadmium have been found in oysters from the mouth of the Gironde (Boutier, 1981). The toxicity of this heavy metal and its concentration along trophic chains are known (Flatau & Aubert, 1979). The results of Calabrese et al. (1973) and Martin et al. (1981) suggest that cadmium is not very toxic to Crassostrea larvae. On the other hand, Watling (1978) stated that concentrations of cadmium as low as 20 #g litre-1 affect the growth of Crassostrea gigas larvae and cause mortality. In addition, the influence of salinity on the larval development of bivalves has been recorded (Hrs-Brenko et al.,
Effects of salinity and cadmium chloride upon Crassostrea gigas larvae
305
1977) and it is known that heavy metal toxicity could increase with salinity changes (Maclnnes & Calabrese, 1979). Important salinity fluctuations occur in the Gironde estuary. Outside Le Verdon, in its oceanic part, the following salinity variations were recorded during the summer of 1979:17"6%0 (June) and 32"5%0 (August); 17%o was the lowest salinity found in June, 1980, with an increase to 32"6%0 in August. These observations led us to investigate the combined effects of cadmium and salinity on the embryonic and larval development of the Japanese oyster, Crassostrea gigas.
METHODS Adult oysters were thermally conditioned in the laboratory for 3 weeks at 20 + 1 °C in a recirculating water system (Robert et al., 1982). They were induced to spawn by thermal stimulation and the addition of sperm from one sacrificed male. The eggs were fertilized in treated seawater. After incubation, 16 000 larvae were put into each of a series of 2-1itre hard glass beakers which contained filtered (0.2 #m) natural seawater prepared at the desired test conditions. The larvae were fed on a mixed algal diet of Isochrysis aft. galbana, Chaetoceros calcitrans and Platymonas suecica to give a final concentration of 100 cells per microlitre as previously described by Helm & Millican (1977). All experimental solutions were changed daily for the first 2 days after fertilization and then renewed at 48-h intervals, at which time samples of the larvae were taken over a 10-day period. Behaviour and structure were studied microscopically. Mortalities and abnormalities were assessed by counting the number of dead larvae in a sample of a hundred individuals from each beaker. The size was determined by measuring the width across the valve from photographs. Fifty individuals were measured from each replicate, giving a total of a hundred for each treatment. The measurement of growth rate ceased when 50 ~o mortality was reached. Natural seawater formed the basis for the various treatment solutions and was obtained in bulk 5 miles offshore from the Cap-Ferret peninsula (Arcachon Bay, see Fig. 1). Four salinities, ranging from 20 to 35%0 in 5%0 steps, were made up by the addition of NaC1 or distilled water. The cadmium concentrations of 0, 10, 20 and 50 #g litre- ~were prepared by adding aliquots of cadmium chloride solution (100mglitre -1) to
306
R. Robert, E. His
seawater for each salinity treatment. These concentrations were chosen because of the results of Watling (1978) who found a 40 ~ reduction in the size of spat at 20/~g litre- 1. Duplicate cultures were established for each of the experimental treatments (thirty-two beakers for the trial).
RESULTS Effect of cadmium chloride on the embryonic development and formation of D larvae
In all beakers the D larval stage was reached within 24 h of fertilization. Less than 10 ~o of abnormal larvae were found in the control and treated cultures, except those exposed to 35%0 salinity. In that case, 36 ~o of the larvae were abnormal in the control beaker. The most easily detected and frequently seen abnormalities were an enlarged velum, or irregular shell shapes, such as a concave hinge or inducted shell margins, as previously described by Le Pennec & Leroux (1979). Abnormal development of embryos decreased as cadmium concentrations increased--26 ~ at 10 pg litre - 1, 22 ~o at 20/lg litre- 1 and 10 ~o at 50 #g litre-1. This relationship was confirmed on the second day. Effect of cadmium chloride on larval mortality
High mortalities occurred in all beakers exposed to 35%o salinity. After 2 days, 14 ~o mortality was found in the control culture, reaching 40 ~ and 70 ~ on days 6 and 8, respectively. In the treated cultures half the larval population died after a week's exposure and mortalities were more than 80 ~ at the end of the experiment. The other treatments showed the percentage mortalities of larvae to be less than 6 ~ , even at the highest cadmium concentration tested. Effect of cadmium chloride on larval growth
The effect of cadmium on the mean size at the 95 ~ confidence interval, and on the growth of living C. gigas larvae, is illustrated in Table 1. The dynamics of the phenomenon were studied by means of a three-way analysis of variance (Table 2). As previously stated, because of the mortality at 35%0 salinity from the eighth day, this ANOVA was
TABLE 1
81-11 +_2.55 105-70 -+5-86
113.35 -+2.21
74.71 77"19 _+0.90 -+0-97
88-75 -+2'66
99"75 +_3.38
75-44 -+ 1"35
76-40 +2.17
97.66 110-76 127.84 +_5.38 +_6-26 -+3"48
67.11 _+1.12
67'08 -+0'86
6
8
10
96'96 -+ 1"96
81.66 _+0.94
67"58 _+0.57
71-80 -+ 1.09
72.16 -+0.91
66"60 +_0'50
76"22 -+ 1.67
73.94 _+0.93
68"69 -+0"45
60.89 _+0'65
4
68.97 -+0-52
60.79 +0-61
70-06 -+0"55
60.02 +_0-55
63.06 -+0"62
61.07 _+0'58
2
30%°
61.59 +0.51
3570o
57.15 +0'85
20'f~
1
25%0
30%0
10
35%0
0
(days)
Age oJ the larvae Jrom ]ertilization
60.82 -+0.70
20%0
30'~oo
91"10 -+ 1.90
77.88 _+1-01
122.57 -+4.90
I 13-84 -+3-08
99.61 102.36 +_3-75 _+2.51
88"72 -+2"37
79-53 -+0-91
20
60.77 61.23 +_0"70 - + 0 - 6 9
35%o~
72.13 -+0"88
72.31 -+0.74
92.02 -+6.47
89.42 _+3.19
77"35 -+ 1'29
75.59 _+0.97
69'34 68"56 67.40 69-44 +_0-57 _+0"66 - + 0 " 7 4 -+0'60
60-69 -+0"58
25'~
Cadmium concentration (lag litre- l )
20700
35%°
92"18 -+2"23
I 13.27 -+4.84
114.64 -+3.50
73"20 _+1"35
78"21 73.66 _ + 0 . 9 2 +_0.70
102.25 103.03 +_4.09 +_2.77
85'84 _+2'38
81.17 _+0.91
69'11 68'22 68'21 +_0"55 _+0-48 -+0"55
61-30 61-02 60.33 _+0.60 +_0-64 -+0"67
25~
25%0
20%o
114.47 -+5.05
94.25 +_3.67
82"06 -+ 1"79
76.20 -+0.83
80"46 _+1.01
123.06 -+4.19
116.87 -+3.64
97.25 106-12 _+3.93 -+3.29
93"82 94"05 +_2"11 _+2"12
79.40 + 1.01
68-51 69"53 67"45 +_0"58 +_0"67 -+0"59
62.02 62.74 60.89 _+0-58 _+0"56 _+0"55
30700
50
Mean Shell Width (/~m + 95 % Confidence Limit) of Crassostrea gigas Larvae Grown in Various Concentrations of Cadmium Chloride and Various Rearing Salinities in a 10-Day Period
,-o
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"
e~ ~-, .q
~--~" ga ~.
R. Robert, E. His
308
TABLE 2 The Effect of Salinity and Cadmium Chloride on the Growth of Crassostrea gigas Larvae in a 6-Day Period. Three-way Analysis of Variance with Transformed Data (log x). The Residual Includes Time x Salinity and Time × Cadmium Concentration Interactions which are Near Zero
Source of variation
Time Salinity Cadmium concentration Salinity x cadmium concentration Residual (error) Total
Degrees of freedom
Mean square
F ratio
p
3 3 3 9
0.159 734 325 9 0.022 242 698 3 0.001 484 445 3 0.002 385 600 7
21.48 2.99 0-20 0-11
0"001 0'041 NS NS
45 63
0.023 256 425 0 0.209 103 490 8
NS = Not significant.
conducted over a 6-day period. It confirmed that time was highly significant (P < 0.001) (which is obvious in a growth study) and showed that the effect of different salinity regimes on the subsequent growth of larvae during the 6-day period was significant (P < 0.05). On the other hand, cadmium chloride, at the different concentrations tested, had no effect and showed no interaction whatsoever between toxicity and salinity. The effects of salinity and cadmium toxicity on growth rate were also tested by a two-way analysis of variance every second day (Table 3). After the eighth day, these ANOVA did not include the 35700osalinity data because of mortalities. They confirmed the significant effect of salinity from the second day and the harmlessness of cadmium chloride.
DISCUSSI ON The various stages in the life history of marine species have different tolerances to pollutants; thus, the development stages are often the most sensitive (Brown & Ahsanullah, 1971; Bryan, 1971; Granmo, 1972). Crassostrea gigas embryos proved to be less tolerant to toxic materials than older larvae, especially when fertilization occurred in treated waters (Brereton et al., 1973; His & Robert, 1981), Cadmium chloride up to 50 #g litre-1 has no effect on the embryonic development of C. gigas. On the other hand, 63 ~o of abnormal larvae were found (His & Robert,
Effects of salinity and cadmium chloride upon Crassostrea gigas larvae
309
TABLE 3 The Effect of Salinity and Cadmium Chloride on the Mean Size of Crassostrea gigas Larvae in a 10-Day Period. A Two-way Analysis of Variance was Made Every 2 Days
Age oJ the larvae jJ'om Jertilization (days)
10
Source oj variation
Degrees of jt'eedom
Mean square
F ratio
p
Salinity Cadmium concentration Residual Total
3 3 9
7-65 5.07 7.88 20-60
2.91 1.93
NS NS
Salinity Cadmium concentration Residual Total
3 3 9
22.51 3.15 14.85 40.51
4.55 0-64
0.05 NS
Salinity Cadmium concentration Residual Total
3 3 9
184.11 12.02 33-60 229.82
16.40 1.07
<0.001 NS
Salinity Cadmium concentration Residual Total
3 3 9
1 277-58 47.12 55.68 1 380.37
68.84 2-54
<0,001 NS
Salinity Cadmium concentratton Residual Total
2 3 6
975.68 44-78 286.15 1 306.61
10.23 0.31
0,01
Salinity Cadmium concentration Residual Total
2 3 6
633.47 205.09 327.93 1 166.50
5-80 1.25
0.04 NS
NS = Not significant.
310
R. Robert, E. His
1982) when embryos were exposed at a concentration of 50 ~g litre- 1 of copper sulphate under similar experimental conditions. Calabrese et al. (1973) showed that some toxicants could retard growth drastically at lower concentrations than are required to prevent normal embryonic development. Exposure of C. gigas larvae to 20/~g litre-1 of copper chloride for a period of 7 days resulted in a 20 ~ reduction in size (His & Robert, 1981). Even at 50/~g litre-1, cadmium chloride does not affect larval growth. Embryos and larvae of C. gigas are more tolerant to cadmium than to copper, which confirms the results obtained with other marine species (Martin et al., 1981 ; Sullivan et al., 1983). Cadmium seems to be of intermediate toxicity to Crassostrea gigas larvae, as stated by Martin et al. (1981). Our data contrast with those reported by Watling (1978, 1982)who observed a growth reduction at 20~uglitre -~ and considered that cadmium is fairly toxic to Crassostrea gigas. We think that her results could be explained by the poor quality of her brood larvae or the experimental conditions in respect of salinity (S%o = 34"7%0). Indeed, 30 ~ of mortality affected her control culture after 7 days. The experiments described in this paper prove that cadmium chloride affects neither embryonic development nor larval growth for concentrations up to 50/~g litre-1. On the other hand, the effect of salinity on maximum growth and survival were obtained at 20%° and 25%0 (temperature = 24 + I°C) as has been established by Walne & Helm (1974), Helm & Millican (1977) and Wilson (1981). Lastly, there is no significant interaction between salinity and cadmium. In other words, there are no synergistic or antagonist effects of salinity and cadmium at the tested concentrations. By extrapolating our results to field conditions we may assume that the repercussions of a moderate cadmium pollution (up to 50 pg litre-1) on the life cycle of C. gigas in a zone where the salinity of the water ranges from 20%0 to 30%0 are very small, as stated by Zaroogian & Morrisson (1981). Therefore, the cadmium content of the water of the Gironde estuary (1 pg litre-1) cannot affect the reproductive potential of the oyster reef.
ACKN OWLEDGEMENTS The authors are grateful to Dr A. Dinet for his kind help in the statistical analysis and to Professor J. M. P~r6s for reading the manuscript and suggesting significant improvements.
Effects of salinity and cadmium chloride upon Crassostrea gigas larvae
311
REFERENCES Boutier, B. (1981). Synth6se des r6sultats de la surveillance des micropolluants dans la mati6re vivante. Bull. R.N.O., 15, 115-74. Brereton, A., Lord, H., Thornton, I. & Webb, J. S. (1973). Effect of zinc on growth and development of larvae of the Pacific oysters, Crassostrea gigas. Marine Biology, 19, 96-101. Brown, B. E. & Ahsanullah, M. (1971). Effect of heavy metals on mortality and growth. Mar. Poll. Bull., 2, 182-7. Bryan, G. W. (1971). The effects of heavy metals (other than mercury) on marine and estuarine organisms. Proc. Roy. Soc. (ser. B), 117, 389-410. Calabrese, A., Collier, R. S., Nelson, D. A. & Maclnnes, J. R. (1973). The toxicity of heavy metals to embryos of the American oyster, Crassostrea virginica. Mar. Biol., 18, 162-6. Flatau, G. & Aubert, M. (1979). Etude de la toxicit6 directe et indirecte du cadmium en milieu marin. Rev. Int. Ocean. M~dic., 53, 51-9. Granmo, A. (1972). Development and growth of eggs and larvae of Mytilus edulis exposed to a linear dodecylbenzenesulphon LAS. Mar. Biol., 15, 356-8. Helm, M. M. & Millican, F. P. (1977). Experiments in the hatchery rearing of the Pacific oyster larvae (Crassostrea gigas Thunberg). Aquaculture, 11, 1-12. His, E. & Robert, R. (1981). Effects of copper chloride on the eggs and D larvae of Crassostrea gigas (Thunberg). Preliminary results. Cons. inter. Explor. Mer, comm. F/43, 13 pp. His, E. & Robert, R. (1982). Le danger des traitements par le sulfate de cuivre en zone conchylicole: Toxicit6 vis ~ vis des oeufs et des jeunes larves de Crassostrea gigas. Rev. Tray. Ir~t. P~ches marit., 45(2), 117-25. Hrs-Brenko, M., Claus, C. & Bubic, S. (1977). Synergistic effects of lead, salinity and temperature on embryonic development of the mussel, Mytilus galloprovincialis. Mar. Biol., 44, 109-15. Le Pennec, M. & Leroux, S. (1979). Effets d'un p6trole brut sur la formation de la coquille de Mytilus edulis (L.) (Mytilidae, Bivalvia). Rev. Int. Ocean. M~dic., 55, 49-55. Maclnnes, J. R. & Calabrese, A. (1979). Combined effect of salinity, temperature and copper on embryos and early larvae of the American oyster, Crassostrea virginica. Arch. Environm. Contam. Toxicol., 8, 553-62. Martin, M., Osborn, K., Billig, P. & Glickstein, N. (1981). Toxicities of ten metals to Crassostrea gigas and Mytilus edulis embryos and Cancer magister larvae. Mar. Poll. Bull., 12(9), 305-8. Robert, R., His, E. & Maurer, D. (1982). L'unit~ d'~cophysiologie et de molysmologie larvaire des bivalves d'int~r~t commercial du laboratoire de rlnstitut Scientifique et Technique des P~ches Maritimes. Rev. Tray. Inst. P~ches marit., 45(3), 197-209. Sullivan, B. K., Buskey, E., Miller, D. C. & Ritacco, P. J. (1983). Effects of copper and cadmium on growth, swimming and predator avoidance in Eurytemora aJfinis (Copepoda). Mar. Biol., 77, 299-306.
312
R. Robert, E. His
Walne, P. R. & Helm, M. M. (1974). The routine culture of the Pacific oyster (Crasssostrea gigas) at Conway during 1973. Shellfish information leaflet, 32, 1-10. Watling, H. R. (1978). Effect of cadmium on larvae and spat of the oyster, Crassostrea gigas (Thunberg). Trans. roy. soc. South Africa, 43(2), 125-34. Watling, H. R. (1982). Comparative study of the effect of zinc, cadmium and copper on the larval growth of three oyster species. Bull.Environm. Contam. Toxicol., 28, 195-201. Wilson, J. (1981). Hatchery rearing of Ostrea edulis and Crassostrea gigas. Aquaculture Technical Bulletin, 4, 1-34. Zaroogian, C. E.-& Morrisson, G. (1981). Effect of cadmium body burdens in adult Crassostrea virginica on fecundity and viability of larvae. Bull. Environm. Contam. Toxicol., 334-48.
Combined Effects of Salinity and Cadmium Chloride upon Embryos and Larvae of the Japanese Oyster,
Crassostrea gigas R. R o b e r t & E. His Institut Franqais de Recherche pour rExploration de la Mer, 63 boulevard Deganne 33120 Arcachon, France (Received: 21 January, 1985)
ABSTRACT Following the observation of high concentrations of cadmium in Gironde estuarine waters, the response of embryos and larvae of the Japanese oyster, Crassostrea gigas to this heavy metal at various salinity regimes was studied for 10 days in the laboratory. The experimental design was a 4 x 4factorial experiment using concentrations of cadmium of 0, 10, 20 and 50 pg litre -1 and salinities of 20, 25, 30 and 35%0. Statistical analysis indicated that the salinity changes have important effects on the embryonic development, survival and growth of C. gigas larvae. No deleterious effect of the toxin at all cadmium concentrations and no interaction between cadmium and salinity were found.
INTRODUCTION Situated 15 km above Le Verdon, the left part of the Gironde estuary, in south-west France (see Fig. 1), was, until recently, the largest oyster reef on the Atlantic French coast. Formerly, Crassostrea angulata spat accounted for 3 0 ~ of French oyster production. In 1970, disease destroyed these beds, which have, since 1973, been replaced by the Japanese oyster, Crassostrea gigas. In spite of the destruction of the most productive zone, due to the recent extension of Le Verdon Harbour, the spawning stock was large enough 303 Marine Environ. Res. 0141-1136/85/$03"30 © ElsevierApplied Science Publishers Ltd, England, 1985. Printed in Great Britain
304
R. Robert, E. His
The
~ A
ROYAN
NCE GIRONDE
z
Le
RCACHOI
Verdon
,,¢ uJ
o 0
I-z
0
I0
I
i
20kin
I
,,.,J t-
<
Ambes s p i t
Fig. 1.
Map of the Gironde estuary showing the position of the Crassostrea gigas oyster reef (hatched area).
to mitigate the lack of spat from the Marennes-Oleron and Arcachon centers. Since 1979, high concentrations of cadmium have been found in oysters from the mouth of the Gironde (Boutier, 1981). The toxicity of this heavy metal and its concentration along trophic chains are known (Flatau & Aubert, 1979). The results of Calabrese et al. (1973) and Martin et al. (1981) suggest that cadmium is not very toxic to Crassostrea larvae. On the other hand, Watling (1978) stated that concentrations of cadmium as low as 20 #g litre-1 affect the growth of Crassostrea gigas larvae and cause mortality. In addition, the influence of salinity on the larval development of bivalves has been recorded (Hrs-Brenko et al.,
Effects of salinity and cadmium chloride upon Crassostrea gigas larvae
305
1977) and it is known that heavy metal toxicity could increase with salinity changes (Maclnnes & Calabrese, 1979). Important salinity fluctuations occur in the Gironde estuary. Outside Le Verdon, in its oceanic part, the following salinity variations were recorded during the summer of 1979:17"6%0 (June) and 32"5%0 (August); 17%o was the lowest salinity found in June, 1980, with an increase to 32"6%0 in August. These observations led us to investigate the combined effects of cadmium and salinity on the embryonic and larval development of the Japanese oyster, Crassostrea gigas.
METHODS Adult oysters were thermally conditioned in the laboratory for 3 weeks at 20 + 1 °C in a recirculating water system (Robert et al., 1982). They were induced to spawn by thermal stimulation and the addition of sperm from one sacrificed male. The eggs were fertilized in treated seawater. After incubation, 16 000 larvae were put into each of a series of 2-1itre hard glass beakers which contained filtered (0.2 #m) natural seawater prepared at the desired test conditions. The larvae were fed on a mixed algal diet of Isochrysis aft. galbana, Chaetoceros calcitrans and Platymonas suecica to give a final concentration of 100 cells per microlitre as previously described by Helm & Millican (1977). All experimental solutions were changed daily for the first 2 days after fertilization and then renewed at 48-h intervals, at which time samples of the larvae were taken over a 10-day period. Behaviour and structure were studied microscopically. Mortalities and abnormalities were assessed by counting the number of dead larvae in a sample of a hundred individuals from each beaker. The size was determined by measuring the width across the valve from photographs. Fifty individuals were measured from each replicate, giving a total of a hundred for each treatment. The measurement of growth rate ceased when 50 ~o mortality was reached. Natural seawater formed the basis for the various treatment solutions and was obtained in bulk 5 miles offshore from the Cap-Ferret peninsula (Arcachon Bay, see Fig. 1). Four salinities, ranging from 20 to 35%0 in 5%0 steps, were made up by the addition of NaC1 or distilled water. The cadmium concentrations of 0, 10, 20 and 50 #g litre- ~were prepared by adding aliquots of cadmium chloride solution (100mglitre -1) to
306
R. Robert, E. His
seawater for each salinity treatment. These concentrations were chosen because of the results of Watling (1978) who found a 40 ~ reduction in the size of spat at 20/~g litre- 1. Duplicate cultures were established for each of the experimental treatments (thirty-two beakers for the trial).
RESULTS Effect of cadmium chloride on the embryonic development and formation of D larvae
In all beakers the D larval stage was reached within 24 h of fertilization. Less than 10 ~o of abnormal larvae were found in the control and treated cultures, except those exposed to 35%0 salinity. In that case, 36 ~o of the larvae were abnormal in the control beaker. The most easily detected and frequently seen abnormalities were an enlarged velum, or irregular shell shapes, such as a concave hinge or inducted shell margins, as previously described by Le Pennec & Leroux (1979). Abnormal development of embryos decreased as cadmium concentrations increased--26 ~ at 10 pg litre - 1, 22 ~o at 20/lg litre- 1 and 10 ~o at 50 #g litre-1. This relationship was confirmed on the second day. Effect of cadmium chloride on larval mortality
High mortalities occurred in all beakers exposed to 35%o salinity. After 2 days, 14 ~o mortality was found in the control culture, reaching 40 ~ and 70 ~ on days 6 and 8, respectively. In the treated cultures half the larval population died after a week's exposure and mortalities were more than 80 ~ at the end of the experiment. The other treatments showed the percentage mortalities of larvae to be less than 6 ~ , even at the highest cadmium concentration tested. Effect of cadmium chloride on larval growth
The effect of cadmium on the mean size at the 95 ~ confidence interval, and on the growth of living C. gigas larvae, is illustrated in Table 1. The dynamics of the phenomenon were studied by means of a three-way analysis of variance (Table 2). As previously stated, because of the mortality at 35%0 salinity from the eighth day, this ANOVA was
TABLE 1
81-11 +_2.55 105-70 -+5-86
113.35 -+2.21
74.71 77"19 _+0.90 -+0-97
88-75 -+2'66
99"75 +_3.38
75-44 -+ 1"35
76-40 +2.17
97.66 110-76 127.84 +_5.38 +_6-26 -+3"48
67.11 _+1.12
67'08 -+0'86
6
8
10
96'96 -+ 1"96
81.66 _+0.94
67"58 _+0.57
71-80 -+ 1.09
72.16 -+0.91
66"60 +_0'50
76"22 -+ 1.67
73.94 _+0.93
68"69 -+0"45
60.89 _+0'65
4
68.97 -+0-52
60.79 +0-61
70-06 -+0"55
60.02 +_0-55
63.06 -+0"62
61.07 _+0'58
2
30%°
61.59 +0.51
3570o
57.15 +0'85
20'f~
1
25%0
30%0
10
35%0
0
(days)
Age oJ the larvae Jrom ]ertilization
60.82 -+0.70
20%0
30'~oo
91"10 -+ 1.90
77.88 _+1-01
122.57 -+4.90
I 13-84 -+3-08
99.61 102.36 +_3-75 _+2.51
88"72 -+2"37
79-53 -+0-91
20
60.77 61.23 +_0"70 - + 0 - 6 9
35%o~
72.13 -+0"88
72.31 -+0.74
92.02 -+6.47
89.42 _+3.19
77"35 -+ 1'29
75.59 _+0.97
69'34 68"56 67.40 69-44 +_0-57 _+0"66 - + 0 " 7 4 -+0'60
60-69 -+0"58
25'~
Cadmium concentration (lag litre- l )
20700
35%°
92"18 -+2"23
I 13.27 -+4.84
114.64 -+3.50
73"20 _+1"35
78"21 73.66 _ + 0 . 9 2 +_0.70
102.25 103.03 +_4.09 +_2.77
85'84 _+2'38
81.17 _+0.91
69'11 68'22 68'21 +_0"55 _+0-48 -+0"55
61-30 61-02 60.33 _+0.60 +_0-64 -+0"67
25~
25%0
20%o
114.47 -+5.05
94.25 +_3.67
82"06 -+ 1"79
76.20 -+0.83
80"46 _+1.01
123.06 -+4.19
116.87 -+3.64
97.25 106-12 _+3.93 -+3.29
93"82 94"05 +_2"11 _+2"12
79.40 + 1.01
68-51 69"53 67"45 +_0"58 +_0"67 -+0"59
62.02 62.74 60.89 _+0-58 _+0"56 _+0"55
30700
50
Mean Shell Width (/~m + 95 % Confidence Limit) of Crassostrea gigas Larvae Grown in Various Concentrations of Cadmium Chloride and Various Rearing Salinities in a 10-Day Period
,-o
o~ 0~"
?,
e..
"
e~ ~-, .q
~--~" ga ~.
R. Robert, E. His
308
TABLE 2 The Effect of Salinity and Cadmium Chloride on the Growth of Crassostrea gigas Larvae in a 6-Day Period. Three-way Analysis of Variance with Transformed Data (log x). The Residual Includes Time x Salinity and Time × Cadmium Concentration Interactions which are Near Zero
Source of variation
Time Salinity Cadmium concentration Salinity x cadmium concentration Residual (error) Total
Degrees of freedom
Mean square
F ratio
p
3 3 3 9
0.159 734 325 9 0.022 242 698 3 0.001 484 445 3 0.002 385 600 7
21.48 2.99 0-20 0-11
0"001 0'041 NS NS
45 63
0.023 256 425 0 0.209 103 490 8
NS = Not significant.
conducted over a 6-day period. It confirmed that time was highly significant (P < 0.001) (which is obvious in a growth study) and showed that the effect of different salinity regimes on the subsequent growth of larvae during the 6-day period was significant (P < 0.05). On the other hand, cadmium chloride, at the different concentrations tested, had no effect and showed no interaction whatsoever between toxicity and salinity. The effects of salinity and cadmium toxicity on growth rate were also tested by a two-way analysis of variance every second day (Table 3). After the eighth day, these ANOVA did not include the 35700osalinity data because of mortalities. They confirmed the significant effect of salinity from the second day and the harmlessness of cadmium chloride.
DISCUSSI ON The various stages in the life history of marine species have different tolerances to pollutants; thus, the development stages are often the most sensitive (Brown & Ahsanullah, 1971; Bryan, 1971; Granmo, 1972). Crassostrea gigas embryos proved to be less tolerant to toxic materials than older larvae, especially when fertilization occurred in treated waters (Brereton et al., 1973; His & Robert, 1981), Cadmium chloride up to 50 #g litre-1 has no effect on the embryonic development of C. gigas. On the other hand, 63 ~o of abnormal larvae were found (His & Robert,
Effects of salinity and cadmium chloride upon Crassostrea gigas larvae
309
TABLE 3 The Effect of Salinity and Cadmium Chloride on the Mean Size of Crassostrea gigas Larvae in a 10-Day Period. A Two-way Analysis of Variance was Made Every 2 Days
Age oJ the larvae jJ'om Jertilization (days)
10
Source oj variation
Degrees of jt'eedom
Mean square
F ratio
p
Salinity Cadmium concentration Residual Total
3 3 9
7-65 5.07 7.88 20-60
2.91 1.93
NS NS
Salinity Cadmium concentration Residual Total
3 3 9
22.51 3.15 14.85 40.51
4.55 0-64
0.05 NS
Salinity Cadmium concentration Residual Total
3 3 9
184.11 12.02 33-60 229.82
16.40 1.07
<0.001 NS
Salinity Cadmium concentration Residual Total
3 3 9
1 277-58 47.12 55.68 1 380.37
68.84 2-54
<0,001 NS
Salinity Cadmium concentratton Residual Total
2 3 6
975.68 44-78 286.15 1 306.61
10.23 0.31
0,01
Salinity Cadmium concentration Residual Total
2 3 6
633.47 205.09 327.93 1 166.50
5-80 1.25
0.04 NS
NS = Not significant.
310
R. Robert, E. His
1982) when embryos were exposed at a concentration of 50 ~g litre- 1 of copper sulphate under similar experimental conditions. Calabrese et al. (1973) showed that some toxicants could retard growth drastically at lower concentrations than are required to prevent normal embryonic development. Exposure of C. gigas larvae to 20/~g litre-1 of copper chloride for a period of 7 days resulted in a 20 ~ reduction in size (His & Robert, 1981). Even at 50/~g litre-1, cadmium chloride does not affect larval growth. Embryos and larvae of C. gigas are more tolerant to cadmium than to copper, which confirms the results obtained with other marine species (Martin et al., 1981 ; Sullivan et al., 1983). Cadmium seems to be of intermediate toxicity to Crassostrea gigas larvae, as stated by Martin et al. (1981). Our data contrast with those reported by Watling (1978, 1982)who observed a growth reduction at 20~uglitre -~ and considered that cadmium is fairly toxic to Crassostrea gigas. We think that her results could be explained by the poor quality of her brood larvae or the experimental conditions in respect of salinity (S%o = 34"7%0). Indeed, 30 ~ of mortality affected her control culture after 7 days. The experiments described in this paper prove that cadmium chloride affects neither embryonic development nor larval growth for concentrations up to 50/~g litre-1. On the other hand, the effect of salinity on maximum growth and survival were obtained at 20%° and 25%0 (temperature = 24 + I°C) as has been established by Walne & Helm (1974), Helm & Millican (1977) and Wilson (1981). Lastly, there is no significant interaction between salinity and cadmium. In other words, there are no synergistic or antagonist effects of salinity and cadmium at the tested concentrations. By extrapolating our results to field conditions we may assume that the repercussions of a moderate cadmium pollution (up to 50 pg litre-1) on the life cycle of C. gigas in a zone where the salinity of the water ranges from 20%0 to 30%0 are very small, as stated by Zaroogian & Morrisson (1981). Therefore, the cadmium content of the water of the Gironde estuary (1 pg litre-1) cannot affect the reproductive potential of the oyster reef.
ACKN OWLEDGEMENTS The authors are grateful to Dr A. Dinet for his kind help in the statistical analysis and to Professor J. M. P~r6s for reading the manuscript and suggesting significant improvements.
Effects of salinity and cadmium chloride upon Crassostrea gigas larvae
311
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312
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Walne, P. R. & Helm, M. M. (1974). The routine culture of the Pacific oyster (Crasssostrea gigas) at Conway during 1973. Shellfish information leaflet, 32, 1-10. Watling, H. R. (1978). Effect of cadmium on larvae and spat of the oyster, Crassostrea gigas (Thunberg). Trans. roy. soc. South Africa, 43(2), 125-34. Watling, H. R. (1982). Comparative study of the effect of zinc, cadmium and copper on the larval growth of three oyster species. Bull.Environm. Contam. Toxicol., 28, 195-201. Wilson, J. (1981). Hatchery rearing of Ostrea edulis and Crassostrea gigas. Aquaculture Technical Bulletin, 4, 1-34. Zaroogian, C. E.-& Morrisson, G. (1981). Effect of cadmium body burdens in adult Crassostrea virginica on fecundity and viability of larvae. Bull. Environm. Contam. Toxicol., 334-48.