- Email: [email protected]
PCB accumulation and alterations of lipids in two length classes of the oyster Crassostrea angulata and of the clam Ruditapes decussatus
PII:
Marine Environmental Research, Vol. 45, No. 3, pp. 259-268. 1998 % 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0141-ll36/98 $19.00+0.00 SOl41-1136(97)00130-X
ELSEVIER
PCB Accumulation and Alterations of Lipids in Two Length Classes of the Oyster Crassostrea angulata and of the Clam Ruditapes decussatus A. M. Ferreira* and C. Vale Instituto (Received
de Investigaqgo
30 October
das Pescas e do Mar, Av. Brasilia,
1996; revised version received 10 October published June 1998)
1400 Lisboa,
1997; accepted
Portugal
1 November
1997;
ABSTRACT Polychlorinated biphenyls (PCB) were analysed in two length classes of the oyster Crassostrea angulata and the clam Ruditapes decussatus collected in Sado estuary and Ria Formosa, respectively. Oysters showed no signtjicant differences ( p < 0.05) in accumulation with size, while smaller clams presented higher PCB levels than the larger ones. The PCB accumulation in same size classes of the two species, and the effect of PCB-exposure on their lipids were investigated during 60d under laboratory controlled conditions. Again, no signtficant diflerences ( p
biphenyls (PCB), C. angulata, R. decussatus, accu-
INTRODUCTION Polychlorinated biphenyls (PCB) are ubiquitous pollutants, and have been widely used by a large variety of industries over the past 50 years (McManus et al., 1983). These *To whom correspondence
should be addressed. 259
260
A. M. Ferreira. C. Vale
compounds are efficiently sequestered in lipid-rich tissues of aquatic organisms, the lipids, thus, playing an important role in PCB bioaccumulation (Addison, 1982; Kawai et al., 1988). In bivalves, beyond environmental conditions and seasonal fluctuations of lipids, size is also an important factor influencing PCB accumulation (Phillips, 1980). The mechanism influencing age-dependent accumulation of organochlorines in biota corresponds, in general, to the change in lipid content (Phillips, 1986). In spite of the independence of lipid content with age in bivalves (Galtsoff, 1964; Bayne, 1976) there are examples of varying PCB accumulation with age/size: organochlorine accumulation in the oyster Crassostrea virginica was not dependent upon size (Vreeland, 1974); the blue mussel, Mytilus edulis, showed a positive correlation between PCB levels and body length (Kuwabara et al., 1986); and the freshwater mussel Lampsilis radiata showed a negative relationship between the two variables (Muncaster et al., 1990). The obtained relationships were explained by changes in metabolic processes related to size differences in assimilation rates (Winter, 1978). PCB exposure has been associated with biochemical, physiological and pathological changes in organisms (Phillips, 1986; Cosper et al., 1987; Walker and Johnston, 1989). The kinetics of PCB accumulation in bivalves is rapid (Pruell et al., 1986), and when a certain level is reached, consumption of reserve lipids or alteration of lipid metabolism may occur (Addison, 1982; Reddy and Rao, 1989). The objectives of this study were to compare the PCB accumulation of two length classes of the oyster Crassostrea angulata and the clam Ruditapes decussatus, and to evaluate the effects on lipids under drastic contamination conditions.
MATERIALS
AND METHODS
Collection of bivalves
In order to determine the PCB concentrations in the clam Ruditapes decussatus and in the oyster Crassostrea angulata of two length classes, a sampling programme was carried out: in 1987/88 clams of two size classes, ~25 and > 35mm, were collected monthly from intertidal growth banks of Ria Formosa; in 1990 oysters (< 50 and > 60 mm) were collected biweekly from natural banks of the upper Sado estuary. In each sampling, a composite sample of 25 individuals was done for each length class. In addition, a larger number of clams and oysters of the same size classes were collected from the same banks in May 1990 in order to perform a laboratory experiment of PCB contamination. Preparation of algae cocktail
A short-term experiment was performed to determine the time for PCB contamination of the algae cocktail (Isochrisis a# galbana and Tetraselmis suecica) used as bivalve diet. Each species came from an algae laboratory culture. The algae were mixed in a glass beaker and kept aerated for 72 h. Aroclor 1254 in methanol (0.1 ~g+t-l), was then added. During the first six hours, samples were taken hourly and then a sample after 72 h. The samples were filtered and the algae analysed for PCB. The concentration of PCB in the cocktail reached the maximum value (1000 ng g-‘) 1 h after exposure, which indicates a
PCB in Crassostrea 1600
T ---__
---___
// 0
Fig. 1.
261
and Ruditapes
2
4
'Ike(h)*
'/
--__
-e
I 70
72
PCB concentrations in algae (Isochrisis aff. galbana and Tetraselmis suecica) during 72 h exposure.
fast incorporation into the algae (Fig. 1). Based on these results, a daily contamination protocol was defined: the algae mixture was treated with PCB by adding to a lo-litre phytoplankton culture (concentration approximately lo6 cells litre-‘) of 2 ml solution of 100 pgml-i PCB (Aroclor 1254, Alltech Associates); the exposed cocktail was allowed to settle for 1 h before adding as bivalve diet. Bivalve PCBexposure
Two independent 60-d experiments were performed with clams and oysters. Each bivalve species was placed in two independent closed water systems. Each system consists of a fiberglass tank (5Ox5Ox3Ocm) containing 50 litre of natural seawater, a water circulating system in PVC, and a pump with a porcelain chamber. During the experiments 500ml min-i of seawater was pumped into the tanks, and the water was maintained at 20°C at salinity 35% and pH 8.2. PCB level in natural seawater of the circulating system was approximately 6nglitre-i. The seawater in the tanks was continuously aerated at a rate of 1000 ml min-‘, and the accumulated biodeposits were removed daily. The clams and oysters were placed in two separated tanks, containing small and large individuals, and acclimated to the system for 1 week. During this period, they were fed daily with a lo6 cellslitre-’ of the algae cocktail. These bivalves from each tank were divided in two tanks, keeping together the two size classes. In the contaminated tanks, oysters and clams were fed daily with the algae mixture previously contaminated with PCB, while in the control tanks they continued to be fed with the non-contaminated algae cocktail. The maximum amount of methanol added to the contaminated tank per day was 30 ,ul litre-‘. Clams were collected from the two tanks at the start (day 0) and after 5, 15, 30, 47, 50 and 60d, and the oysters from the other two at days 0, 3, 5,9, 15, 20, 30, 40, 50 and 60. Each sample consisted of a pool of 10 individuals of similar size. The samples of the two length classes were frozen and subdivided into two aliquots for PCB and lipid analyses.
262
A. M. Ferreira. C. Vale
PCB and lipid analysis For PCB determinations the composite samples of soft tissues were extracted with n-hexane, cleaned up with Florisil and analysed by electron capture gas chromatography. Details of the methodology have been described elsewhere (Ferreira and Vale, 1995). Lipids were extracted with chloroform:methanol (2:l) as described by Folch et al. (1957). The lipid classes (polar lipids, triglycerides, sterols and free fatty acids) were separated by thin layer chromatography on silica-gel 60 plates and quantified by scanning densitometry (Madureira et al., 1993). The precision of lipid determinations was 0.5%. PCB concentrations were the sum of 17 PCB congeners 18, 26, 44, 49, 52, 101, 118, 128, 138, 149, 151, 153, 170, 180, 183, 187 and 194 (IUPAC numbers; Ballschmiter and Zell, 1980). All results are expressed on a dry weight basis. Statistical analysis of the data was performed using the two-way analysis of variance (ANOVA).
RESULTS Field results The concentrations of PCB in two length classes of oysters ( < 50 and > 60 mm) and of clams ( < 25 and > 35 mm) collected from Sado estuary and Ria Formosa, respectively, are presented in Fig. 2. In both plots, levels in smaller individuals are represented in the y-axis and concentrations in larger ones in the x-axis. The points corresponding to oysters are plotted close to the bisectrix, meaning that individuals of the two size classes showed approximately the same PCB levels; e.g. (3; 3) and (33; 41) according to the sampling period. Lipids of oysters from the two length classes were also not statistically different (p < O-05) and, thus, the representation on a lipid weight basis showed a similar lack of size variation. A different situation was recorded for clams, most points being plotted along the y-axis; i.e. smaller clams contained more PCB than larger clams (e.g. 52; 4).
l
Cl
10
20
30
40
[PCBIlarger ind.(ng.g-I)
50
R. decussstus l
0
20
40
60
[PCB]larger ind.(ng.g-1)
Fig. 2. Concentrations of PCB (ngg-’ dry wt) in oysters (Crassostrea angulata) of ~50 and of > 60 mm length and clams (Ruditapes decussatus) of < 25 and > 35 mm length collected in Sado estuary and Ria Formosa, respectively.
263
PCB in Crassostrea and Ruditapes
0
10
20
30
40
50
60
Days Fig. 3. PCB concentrations (ngg-’ dry wt) in exposed (m,A) and control oysters (lJ,A) length classes ( < 50 and > 60 mm), during the 60-d experiment.
Since smaller clams contained lower lipids than larger clams, these differences accentuated when concentrations are expressed on a lipid weight basis.
of two
are more
Laboratory experiment+ysters Concentrations of PCB in contaminated and control oysters of the same two length classes ( < 50 and > 60 mm) are presented in Fig. 3. In the control oysters of both classes, residues remained relatively constant (50 ngg-‘) during the 60d. In the PCB-exposed oysters, levels increased with time: after 3 d concentrations increased until 174ngg-’ ( < 50 mm) and 115 ng g-’ ( > 60 mm); then it remained at relatively constant values until day 30; and, thereafter, residues increased again displaying a broad maximum (> 1500 and > 2500 ng g-’ for smaller and larger oysters, respectively). Levels in the two length classes were not statistically different (p < 0.05); however, PCB residues were, in general, slightly higher in the smaller than in the larger ones. When levels are expressed on a lipid weight basis, similar accumulation curves were observed. The time-course of polar lipid, triglyceride, sterol and sterol ester concentrations in PCB-exposed and in control oysters of the two sizes was compared (Figs 4, 5). In larger oysters ( > 60 mm) exposed to PCB, the levels of polar lipids, triglycerides and sterol esters, remained within narrow intervals, and no significant differences (p < 0.05) were found between control groups. In contrast, the concentrations of triglycerides in exposed smaller oysters ( < 50 mm) were significantly different (p < 0.04) from those found in control. In the contaminated oysters, triglyceride concentrations decreased considerably during 30 d and remained at low values until the end of the experiment. In control oysters, the levels were relatively high. Concentrations of the other lipid classes displayed little decrease with time. Composition and lipid content of the two size classes were not statistically different (p < 0.05).
A. M. Ferreira. C. Vale
0
10
20
30
40
50
60
0
10
20
10
20
30
40
50
60
40
50
60
Days
Days
0
30
40
50
60
0
10
20
30
Days
Days
Fig. 4. Percentage composition of polar lipids, triglycerides, sterols and sterol esters in PCB-exposed (+) and control (0) oysters of < 50 mm length.
0
10
20
30
40
50
60
0
10
20
10
20
30
Days Fig. 5. Percentage
40
50
60
40
50
60
Days
Days
0
30
40
50
60
0
10
20
30
Days
composition of polar lipids, triglycerides, sterols and sterol esters in contaminated (+) and control (0) oysters of > 60mm length.
PCB in Crassostrea and Ruditapes
265
2500
2000 5 z? -O 1500 F‘;p E 1000 H
clams
PC&exposed
>35 mm
500
Control clams 0 0
10
20
40
30
50
60
Days Fig. 6. Concentrations
in exposed (A,+)
and in control (&m) length.
clams of < 25 mm and of > 35 mm
i/ijip.J+$ ~~~_~~ 1
0
IO
20
30
Days
Days Fig. 7. Percentage
40
50
60
1
0
10
20
30
40
50
60
Days
Days
composition of lipid classes (polar lipids, triglycerides, sterols and free fatty acids) in PCB-exposed (+) and control (0) clams of < 25 mm length.
A. M. Ferreira, C. Vale
266
Laboratory experiment--clams Concentrations of PCB in contaminated and control clams ( < 25 and > 35 mm), along a 60-d experiment, are presented in Fig. 6. While the residues in control clams remained low (15 ng g-l), levels in exposed clams were substantially higher. In larger individuals, values increased gradually with the time and reached 360ngg-’ after 50d of the experiment. In smaller individuals, the levels were much higher than those of larger ones and displayed a maximum of 2400ngg-’ at day 30. The differences between the two length classes indicates that accumulation of PCB in R. decussatus is size dependent. This dependence was also recorded when levels were normalized to lipids. Concentrations of lipid classes in PCB-exposed and control clams are presented in Fig. 7 (< 25 mm) and Fig. 8 (> 35mm). In general, for the two length classes, differences between contaminated and control organisms were small and non-significant (p < 0.05). However, concentrations of triglycerides in contaminated smaller clams were significantly different (p ~0.02) from those found in control. Triglyceride residues in these clams showed a reduction by the end of the experiment, which was accompanied by an increase of sterol and free fatty acid levels.
DISCUSSION The results obtained, both in the field and in PCB-exposure laboratory experiments, illustrate two contrasting situations: PCB accumulation was similar in small and large
Days
Days
3T
8
31
yj?ygtg
!
Days Fig. 8. Percentage
O ’
0
I;
i.
~
30
I
40
50
60
Days
composition of lipid classes (polar lipids, triglycerides, sterols and free fatty acids) in PCB-exposed (+) and control (0) clams of > 35 mm length.
PC3 in Crassostrea and Ruditapes C. anguluta, whereas smaller R. decussatus accumulated
267
more PCB than the larger ones. In the laboratory, under high PCB contamination, reserve lipids of C. ungulutu from two length classes were progressively consumed over time, while only smaller R. decussutus displayed a pronounced alteration in lipid composition. Variations in PCB accumulation among organisms and within the same species are often explained by the differences of lipid contents (Phillips, 1986). However, the different PCB accumulation of the two length classes of R. decussatus can not be attributed to lipids, since PCB and lipids in clams from the field varied inversely, and the two clam classes in the experiment displayed similar lipid content and composition. Muncaster et al. (1990) found that PCB accumulation in the freshwater mussel Lumpsifis rudiuta varied inversely with the body size, which was attributed to alterations in assimilation rates. In fact, Winter (1978) showed that, for many bivalve species, the percentage of food ingested, with respect to tissue weight, increases very rapidly with a decrease in body size. Since the food ingestion is much higher in smaller R. decussutus than in larger ones (Massapina, personal communication) the allometric relationship referred to by Winter (1978) is presumably valid in this clam species. Changes in PCB accumulation between the two size classes of R. decussutus appear, thus, to be related to alterations in assimilation rate. However, not all bivalve species follow such a size dependence assimilation rate. For example, the assimilation efficiency in Crassostreu gigus was not significantly dependent on body size (Gerdes, 1983). This may be also valid for Crussostreu ungulutu. Uniformity of lipid content and size independence with respect to assimilation rate, may both explain the comparable PCB levels recorded in the two body size classes of C. ungulata. A gradual consumption of reserve lipids in oysters, and an alteration of lipid metabolism in smaller clams that accumulated higher residues of PCB, was registered. These modifications can not be attributed to spawning, since sharp decreases in lipids were not recorded, which would be observed if they occur during the experimental period (Galtsoff, 1964; Ferreira, 1993). In studies with other marine organisms, it was found that lipids change as a consequence of organochlorine exposure (Addison, 1982; Reddy and Rao, 1989). In an experiment with C. angulutu, we observed a pronounced consumption of triglycerides in PCB-exposed oysters, as well as a decrease of energetic adenylic charge (Madureira et al., 1993). This confirms the degradation of physiological state of the C. angulutu when exposed to high PCB levels. Only in smaller clams, which accumulated high quantities of PCB in the experiment, effects on lipids were detected. The response of clams to high residues of PCB was a reduction in triglycerides and an increase in sterol and free fatty acids. Clearly, these changes reflected an alteration on lipid metabolism of the clams. The shift of lipids observed in the two studied bivalve species suggests the mobilisation of energy-rich lipids for production of energy during toxic stress caused by PCB. Such a response to organochlorine contamination was also observed in the shrimp Metapenueus monoceros (Reddy and Rao, 1989). Peterson et al. (1994) studied the behaviour of the clam Corbiculufluminea in two sites with different PCB contamination, and concluded that a pronounced reduction of valve movement was related to the contamination. In our experimental study, oysters and small clams showed a substantial decrease of PCB residues, after a prolonged period of PCB accumulation and when triglycerides were at low levels. These reductions are probably related to modifications of food ingestion rate. It appears, thus, that PCB causes physiological or behavioural stress in C. ungulata of both sizes and in smaller R. decussutus, resulting in a decrease of filtering/feeding activity.
A. M. Ferreira. C. Vale
268
REFERENCES Addison, R. F. (1982) Organochlorine 47-71.
compounds and marine lipids. Progress in Lipid Research 21,
Ballschmiter, K. and Zell, M. (1980) Analysis of polychlorinated biphenyls (PCB) by glass capillary gas chromatography. Zeitschrift fur Analytische Chemie 302, 2&31. Bayne, B. L. (1976) Marine Mussels: Their Ecology and Physiology. Cambridge University Press, Cambridge. Cosper, E. M., Snyder, B. J., Arnold, L. M., Zaikowski, L. A. and Wurster, C. F. (1987) Induced resistance to polychlorinated biphenyls confers cross-resistance and altered environmental fitness in a marine diatom. Marine Environmental Research 23, 207-222. Ferreira, A. M. (1993) Bioacumulacao de organoclorados na ostra Crassostrea angulata e na am&ijoa Ruditapes decussatus. Thesis, Instituto National de Investigacgo das Pescas, Portugal. Ferreira, A. M. and Vale, C. (1995) The importance of runoff to DDT and PCB inputs to the Sado estuary and Ria Formosa. Netherlands Journal of Aquatic Ecology 29, 211-216. Folch, J., Lees, M. and Stanley, G. H. (1957) A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226, 497-509. Galtsoff, P. S. (1964) The American oyster Crassostrea virginica Gmelin. US Fish Wildlife Service, Fish Bull. 64. Gerdes, D. (1983) The pacific oyster Crassostrea gigas. Part I. Feeding behaviour of larvae and adults. Aquaculture 31, 195-219. Kawai, S., Fukushima, M., Miyazaki, N. and Tatsukawa, R. (1988) Relationship between lipid composition and organochlorine levels in the tissues of striped dolphin. Marine Pollution Bulletin 19, 129133.
Kuwabara, K., Fukuhima, S., Tanaka, H. M., Miyata, H. and Kashimoto, T. (1986) Relationship between shell length of blue mussels and levels of residual PCBs within the body. Journal of the Food Society of Japan 27, 565568.
Madureira, M. J., Picado, A. M., Ferreira, A. M., Mendonca, E., Le Gal, Y. and Vale C. (1993). PCB contamination in the oyster Crassostrea angulata: effects on lipids and adenylic energetic charge. Science of the Total Environment (Suppl.), 599605. McManus, G. B., Wyman, K. D., Peterson, W. T. and Wurster, C. F. (1983) Factors affecting the elimination of PCB in the marine copepod Acartia tonsa. Estuarine Coastal and Shelf Science 17,421430.
Muncaster, B. W., Hebert, P. D. N. and Lazar, R. (1990) Biological and physical factors affecting the body burden of organic contaminants in freshwater mussels. Archives of Environmental Toxicology 19, 25-34.
Peterson, M. J., Southworth, G. R. and Ham, K. D. (1994) Effect of sublethal chlorinated discharges on PCB accumulation in transplanted asiatic clams (Corbiculafluminea). Water Air and Soil Pollution 73, 169-178. Phillips, D. J. H. (1980) Quantitative Aquatic Biological Indicators. Applied Science Publishers, London. Phillips, D. J. H. (1986) Use of organisms to quantify PCBs in marine and estuarine environments. In PCBs and the Environment, ed. J. S. Waid, Vol. 2, pp. 127-181. CRC Press, Boca Raton, Florida. Pruell, R. J., Lake, J. L., Davis, W. R. and Quinn, J. G. (1986) Uptake and depuration of organic contaminants by blue mussels (Mytilus edulis) exposed to environmentally contaminated sediments. Marine Biology 91, 497-507. Reddy, M. S. and Rao, K. V. (1989) Zn vivo modification of lipid metabolism in response to phosphamidon, methylparathion and lindane exposure in the penaeid prawn, Metapenaeus monoceros. Bulletin of Environmental Contamination and Toxicology 43, 603-610.
Vreeland, V. (1974) Uptake of chlorobiphenyls by oysters. Environmental Pollutution 6, 135-140. Walker, C. H. and Johnston, G. 0. (1989) Interactive effects of pollutants at the toxicokinetics level-implications for the marine environment. Marine Environmental Research 28, 521-525. Winter, J. E. (1978) A review on the knowledge of suspension-feeding in lamellibranchiate bivalves, with special reference to artificial aquaculture systems. Aquaculture 13, l-33.
Marine Environmental Research, Vol. 45, No. 3, pp. 259-268. 1998 % 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0141-ll36/98 $19.00+0.00 SOl41-1136(97)00130-X
ELSEVIER
PCB Accumulation and Alterations of Lipids in Two Length Classes of the Oyster Crassostrea angulata and of the Clam Ruditapes decussatus A. M. Ferreira* and C. Vale Instituto (Received
de Investigaqgo
30 October
das Pescas e do Mar, Av. Brasilia,
1996; revised version received 10 October published June 1998)
1400 Lisboa,
1997; accepted
Portugal
1 November
1997;
ABSTRACT Polychlorinated biphenyls (PCB) were analysed in two length classes of the oyster Crassostrea angulata and the clam Ruditapes decussatus collected in Sado estuary and Ria Formosa, respectively. Oysters showed no signtjicant differences ( p < 0.05) in accumulation with size, while smaller clams presented higher PCB levels than the larger ones. The PCB accumulation in same size classes of the two species, and the effect of PCB-exposure on their lipids were investigated during 60d under laboratory controlled conditions. Again, no signtficant diflerences ( p
biphenyls (PCB), C. angulata, R. decussatus, accu-
INTRODUCTION Polychlorinated biphenyls (PCB) are ubiquitous pollutants, and have been widely used by a large variety of industries over the past 50 years (McManus et al., 1983). These *To whom correspondence
should be addressed. 259
260
A. M. Ferreira. C. Vale
compounds are efficiently sequestered in lipid-rich tissues of aquatic organisms, the lipids, thus, playing an important role in PCB bioaccumulation (Addison, 1982; Kawai et al., 1988). In bivalves, beyond environmental conditions and seasonal fluctuations of lipids, size is also an important factor influencing PCB accumulation (Phillips, 1980). The mechanism influencing age-dependent accumulation of organochlorines in biota corresponds, in general, to the change in lipid content (Phillips, 1986). In spite of the independence of lipid content with age in bivalves (Galtsoff, 1964; Bayne, 1976) there are examples of varying PCB accumulation with age/size: organochlorine accumulation in the oyster Crassostrea virginica was not dependent upon size (Vreeland, 1974); the blue mussel, Mytilus edulis, showed a positive correlation between PCB levels and body length (Kuwabara et al., 1986); and the freshwater mussel Lampsilis radiata showed a negative relationship between the two variables (Muncaster et al., 1990). The obtained relationships were explained by changes in metabolic processes related to size differences in assimilation rates (Winter, 1978). PCB exposure has been associated with biochemical, physiological and pathological changes in organisms (Phillips, 1986; Cosper et al., 1987; Walker and Johnston, 1989). The kinetics of PCB accumulation in bivalves is rapid (Pruell et al., 1986), and when a certain level is reached, consumption of reserve lipids or alteration of lipid metabolism may occur (Addison, 1982; Reddy and Rao, 1989). The objectives of this study were to compare the PCB accumulation of two length classes of the oyster Crassostrea angulata and the clam Ruditapes decussatus, and to evaluate the effects on lipids under drastic contamination conditions.
MATERIALS
AND METHODS
Collection of bivalves
In order to determine the PCB concentrations in the clam Ruditapes decussatus and in the oyster Crassostrea angulata of two length classes, a sampling programme was carried out: in 1987/88 clams of two size classes, ~25 and > 35mm, were collected monthly from intertidal growth banks of Ria Formosa; in 1990 oysters (< 50 and > 60 mm) were collected biweekly from natural banks of the upper Sado estuary. In each sampling, a composite sample of 25 individuals was done for each length class. In addition, a larger number of clams and oysters of the same size classes were collected from the same banks in May 1990 in order to perform a laboratory experiment of PCB contamination. Preparation of algae cocktail
A short-term experiment was performed to determine the time for PCB contamination of the algae cocktail (Isochrisis a# galbana and Tetraselmis suecica) used as bivalve diet. Each species came from an algae laboratory culture. The algae were mixed in a glass beaker and kept aerated for 72 h. Aroclor 1254 in methanol (0.1 ~g+t-l), was then added. During the first six hours, samples were taken hourly and then a sample after 72 h. The samples were filtered and the algae analysed for PCB. The concentration of PCB in the cocktail reached the maximum value (1000 ng g-‘) 1 h after exposure, which indicates a
PCB in Crassostrea 1600
T ---__
---___
// 0
Fig. 1.
261
and Ruditapes
2
4
'Ike(h)*
'/
--__
-e
I 70
72
PCB concentrations in algae (Isochrisis aff. galbana and Tetraselmis suecica) during 72 h exposure.
fast incorporation into the algae (Fig. 1). Based on these results, a daily contamination protocol was defined: the algae mixture was treated with PCB by adding to a lo-litre phytoplankton culture (concentration approximately lo6 cells litre-‘) of 2 ml solution of 100 pgml-i PCB (Aroclor 1254, Alltech Associates); the exposed cocktail was allowed to settle for 1 h before adding as bivalve diet. Bivalve PCBexposure
Two independent 60-d experiments were performed with clams and oysters. Each bivalve species was placed in two independent closed water systems. Each system consists of a fiberglass tank (5Ox5Ox3Ocm) containing 50 litre of natural seawater, a water circulating system in PVC, and a pump with a porcelain chamber. During the experiments 500ml min-i of seawater was pumped into the tanks, and the water was maintained at 20°C at salinity 35% and pH 8.2. PCB level in natural seawater of the circulating system was approximately 6nglitre-i. The seawater in the tanks was continuously aerated at a rate of 1000 ml min-‘, and the accumulated biodeposits were removed daily. The clams and oysters were placed in two separated tanks, containing small and large individuals, and acclimated to the system for 1 week. During this period, they were fed daily with a lo6 cellslitre-’ of the algae cocktail. These bivalves from each tank were divided in two tanks, keeping together the two size classes. In the contaminated tanks, oysters and clams were fed daily with the algae mixture previously contaminated with PCB, while in the control tanks they continued to be fed with the non-contaminated algae cocktail. The maximum amount of methanol added to the contaminated tank per day was 30 ,ul litre-‘. Clams were collected from the two tanks at the start (day 0) and after 5, 15, 30, 47, 50 and 60d, and the oysters from the other two at days 0, 3, 5,9, 15, 20, 30, 40, 50 and 60. Each sample consisted of a pool of 10 individuals of similar size. The samples of the two length classes were frozen and subdivided into two aliquots for PCB and lipid analyses.
262
A. M. Ferreira. C. Vale
PCB and lipid analysis For PCB determinations the composite samples of soft tissues were extracted with n-hexane, cleaned up with Florisil and analysed by electron capture gas chromatography. Details of the methodology have been described elsewhere (Ferreira and Vale, 1995). Lipids were extracted with chloroform:methanol (2:l) as described by Folch et al. (1957). The lipid classes (polar lipids, triglycerides, sterols and free fatty acids) were separated by thin layer chromatography on silica-gel 60 plates and quantified by scanning densitometry (Madureira et al., 1993). The precision of lipid determinations was 0.5%. PCB concentrations were the sum of 17 PCB congeners 18, 26, 44, 49, 52, 101, 118, 128, 138, 149, 151, 153, 170, 180, 183, 187 and 194 (IUPAC numbers; Ballschmiter and Zell, 1980). All results are expressed on a dry weight basis. Statistical analysis of the data was performed using the two-way analysis of variance (ANOVA).
RESULTS Field results The concentrations of PCB in two length classes of oysters ( < 50 and > 60 mm) and of clams ( < 25 and > 35 mm) collected from Sado estuary and Ria Formosa, respectively, are presented in Fig. 2. In both plots, levels in smaller individuals are represented in the y-axis and concentrations in larger ones in the x-axis. The points corresponding to oysters are plotted close to the bisectrix, meaning that individuals of the two size classes showed approximately the same PCB levels; e.g. (3; 3) and (33; 41) according to the sampling period. Lipids of oysters from the two length classes were also not statistically different (p < O-05) and, thus, the representation on a lipid weight basis showed a similar lack of size variation. A different situation was recorded for clams, most points being plotted along the y-axis; i.e. smaller clams contained more PCB than larger clams (e.g. 52; 4).
l
Cl
10
20
30
40
[PCBIlarger ind.(ng.g-I)
50
R. decussstus l
0
20
40
60
[PCB]larger ind.(ng.g-1)
Fig. 2. Concentrations of PCB (ngg-’ dry wt) in oysters (Crassostrea angulata) of ~50 and of > 60 mm length and clams (Ruditapes decussatus) of < 25 and > 35 mm length collected in Sado estuary and Ria Formosa, respectively.
263
PCB in Crassostrea and Ruditapes
0
10
20
30
40
50
60
Days Fig. 3. PCB concentrations (ngg-’ dry wt) in exposed (m,A) and control oysters (lJ,A) length classes ( < 50 and > 60 mm), during the 60-d experiment.
Since smaller clams contained lower lipids than larger clams, these differences accentuated when concentrations are expressed on a lipid weight basis.
of two
are more
Laboratory experiment+ysters Concentrations of PCB in contaminated and control oysters of the same two length classes ( < 50 and > 60 mm) are presented in Fig. 3. In the control oysters of both classes, residues remained relatively constant (50 ngg-‘) during the 60d. In the PCB-exposed oysters, levels increased with time: after 3 d concentrations increased until 174ngg-’ ( < 50 mm) and 115 ng g-’ ( > 60 mm); then it remained at relatively constant values until day 30; and, thereafter, residues increased again displaying a broad maximum (> 1500 and > 2500 ng g-’ for smaller and larger oysters, respectively). Levels in the two length classes were not statistically different (p < 0.05); however, PCB residues were, in general, slightly higher in the smaller than in the larger ones. When levels are expressed on a lipid weight basis, similar accumulation curves were observed. The time-course of polar lipid, triglyceride, sterol and sterol ester concentrations in PCB-exposed and in control oysters of the two sizes was compared (Figs 4, 5). In larger oysters ( > 60 mm) exposed to PCB, the levels of polar lipids, triglycerides and sterol esters, remained within narrow intervals, and no significant differences (p < 0.05) were found between control groups. In contrast, the concentrations of triglycerides in exposed smaller oysters ( < 50 mm) were significantly different (p < 0.04) from those found in control. In the contaminated oysters, triglyceride concentrations decreased considerably during 30 d and remained at low values until the end of the experiment. In control oysters, the levels were relatively high. Concentrations of the other lipid classes displayed little decrease with time. Composition and lipid content of the two size classes were not statistically different (p < 0.05).
A. M. Ferreira. C. Vale
0
10
20
30
40
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60
0
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20
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40
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60
40
50
60
Days
Days
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60
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10
20
30
Days
Days
Fig. 4. Percentage composition of polar lipids, triglycerides, sterols and sterol esters in PCB-exposed (+) and control (0) oysters of < 50 mm length.
0
10
20
30
40
50
60
0
10
20
10
20
30
Days Fig. 5. Percentage
40
50
60
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60
Days
Days
0
30
40
50
60
0
10
20
30
Days
composition of polar lipids, triglycerides, sterols and sterol esters in contaminated (+) and control (0) oysters of > 60mm length.
PCB in Crassostrea and Ruditapes
265
2500
2000 5 z? -O 1500 F‘;p E 1000 H
clams
PC&exposed
>35 mm
500
Control clams 0 0
10
20
40
30
50
60
Days Fig. 6. Concentrations
in exposed (A,+)
and in control (&m) length.
clams of < 25 mm and of > 35 mm
i/ijip.J+$ ~~~_~~ 1
0
IO
20
30
Days
Days Fig. 7. Percentage
40
50
60
1
0
10
20
30
40
50
60
Days
Days
composition of lipid classes (polar lipids, triglycerides, sterols and free fatty acids) in PCB-exposed (+) and control (0) clams of < 25 mm length.
A. M. Ferreira, C. Vale
266
Laboratory experiment--clams Concentrations of PCB in contaminated and control clams ( < 25 and > 35 mm), along a 60-d experiment, are presented in Fig. 6. While the residues in control clams remained low (15 ng g-l), levels in exposed clams were substantially higher. In larger individuals, values increased gradually with the time and reached 360ngg-’ after 50d of the experiment. In smaller individuals, the levels were much higher than those of larger ones and displayed a maximum of 2400ngg-’ at day 30. The differences between the two length classes indicates that accumulation of PCB in R. decussatus is size dependent. This dependence was also recorded when levels were normalized to lipids. Concentrations of lipid classes in PCB-exposed and control clams are presented in Fig. 7 (< 25 mm) and Fig. 8 (> 35mm). In general, for the two length classes, differences between contaminated and control organisms were small and non-significant (p < 0.05). However, concentrations of triglycerides in contaminated smaller clams were significantly different (p ~0.02) from those found in control. Triglyceride residues in these clams showed a reduction by the end of the experiment, which was accompanied by an increase of sterol and free fatty acid levels.
DISCUSSION The results obtained, both in the field and in PCB-exposure laboratory experiments, illustrate two contrasting situations: PCB accumulation was similar in small and large
Days
Days
3T
8
31
yj?ygtg
!
Days Fig. 8. Percentage
O ’
0
I;
i.
~
30
I
40
50
60
Days
composition of lipid classes (polar lipids, triglycerides, sterols and free fatty acids) in PCB-exposed (+) and control (0) clams of > 35 mm length.
PC3 in Crassostrea and Ruditapes C. anguluta, whereas smaller R. decussatus accumulated
267
more PCB than the larger ones. In the laboratory, under high PCB contamination, reserve lipids of C. ungulutu from two length classes were progressively consumed over time, while only smaller R. decussutus displayed a pronounced alteration in lipid composition. Variations in PCB accumulation among organisms and within the same species are often explained by the differences of lipid contents (Phillips, 1986). However, the different PCB accumulation of the two length classes of R. decussatus can not be attributed to lipids, since PCB and lipids in clams from the field varied inversely, and the two clam classes in the experiment displayed similar lipid content and composition. Muncaster et al. (1990) found that PCB accumulation in the freshwater mussel Lumpsifis rudiuta varied inversely with the body size, which was attributed to alterations in assimilation rates. In fact, Winter (1978) showed that, for many bivalve species, the percentage of food ingested, with respect to tissue weight, increases very rapidly with a decrease in body size. Since the food ingestion is much higher in smaller R. decussutus than in larger ones (Massapina, personal communication) the allometric relationship referred to by Winter (1978) is presumably valid in this clam species. Changes in PCB accumulation between the two size classes of R. decussutus appear, thus, to be related to alterations in assimilation rate. However, not all bivalve species follow such a size dependence assimilation rate. For example, the assimilation efficiency in Crassostreu gigus was not significantly dependent on body size (Gerdes, 1983). This may be also valid for Crussostreu ungulutu. Uniformity of lipid content and size independence with respect to assimilation rate, may both explain the comparable PCB levels recorded in the two body size classes of C. ungulata. A gradual consumption of reserve lipids in oysters, and an alteration of lipid metabolism in smaller clams that accumulated higher residues of PCB, was registered. These modifications can not be attributed to spawning, since sharp decreases in lipids were not recorded, which would be observed if they occur during the experimental period (Galtsoff, 1964; Ferreira, 1993). In studies with other marine organisms, it was found that lipids change as a consequence of organochlorine exposure (Addison, 1982; Reddy and Rao, 1989). In an experiment with C. angulutu, we observed a pronounced consumption of triglycerides in PCB-exposed oysters, as well as a decrease of energetic adenylic charge (Madureira et al., 1993). This confirms the degradation of physiological state of the C. angulutu when exposed to high PCB levels. Only in smaller clams, which accumulated high quantities of PCB in the experiment, effects on lipids were detected. The response of clams to high residues of PCB was a reduction in triglycerides and an increase in sterol and free fatty acids. Clearly, these changes reflected an alteration on lipid metabolism of the clams. The shift of lipids observed in the two studied bivalve species suggests the mobilisation of energy-rich lipids for production of energy during toxic stress caused by PCB. Such a response to organochlorine contamination was also observed in the shrimp Metapenueus monoceros (Reddy and Rao, 1989). Peterson et al. (1994) studied the behaviour of the clam Corbiculufluminea in two sites with different PCB contamination, and concluded that a pronounced reduction of valve movement was related to the contamination. In our experimental study, oysters and small clams showed a substantial decrease of PCB residues, after a prolonged period of PCB accumulation and when triglycerides were at low levels. These reductions are probably related to modifications of food ingestion rate. It appears, thus, that PCB causes physiological or behavioural stress in C. ungulata of both sizes and in smaller R. decussutus, resulting in a decrease of filtering/feeding activity.
A. M. Ferreira. C. Vale
268
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Vreeland, V. (1974) Uptake of chlorobiphenyls by oysters. Environmental Pollutution 6, 135-140. Walker, C. H. and Johnston, G. 0. (1989) Interactive effects of pollutants at the toxicokinetics level-implications for the marine environment. Marine Environmental Research 28, 521-525. Winter, J. E. (1978) A review on the knowledge of suspension-feeding in lamellibranchiate bivalves, with special reference to artificial aquaculture systems. Aquaculture 13, l-33.