Distribution of Polychlorinated biphenyls in dab (Limanda limanda) from the baie de seine (Eastern Channel)

Marine Environmental Research 38 (1994) 77-91

ELSEVIER

Distribution of Polychlorinated Biphenyls in Dab (Limanda limanda) from the Baie de Seine (Eastern Channel) V6ronique Loizeau & Alain Abarnou IFREMER, Centre de Brest, Direction de l'Environnement Littoral, B.P. 70, 29280 Plouzane, France (Received 17 November 1992; revised version received 9 July 1993; accepted 13 July 1993)

A BSTRA CT Polychlorinated biphenyls (PCBs) were determined in liver, muscle and gonad tissues of dab from the Baie de Seine. Very similar PCB patterns were observed in all samples; the main components possess the -245, -234, -2345 chlorine substitution. Contamination levels varied with the lipid content of the tissues. The highest concentrations were found in liver in the range 100-700 ng g~ dry weight jor the PCB congener No. 153 (22'44'55' hexachlorobiphenyl). The concentrations in liver and muscle increased with age for both male and female fish. Gonads from male fish showed continued low contamination. However, the contamination in gonads from mature female fish (> 2 years) varied during the year: the highest concentrations were observed in early spring, just before the spawning which is an important pathway for the elimination of these contaminants.

INTRODUCTION The bioaccumulation of persistent and toxic compounds represents a major hazard to marine life and hence to humans through seafood and fish consumption. Many authors have investigated the processes leading to the enrichment of xenobiotic compounds within foodwebs, particularly in order to build predictive models of bioaccumulation (Thomann and Connolly, 1984; de Vries and Goossens, 1987; O ' C o n n o r and Pizza, 1987; Connolly, 1991). 77

78

Vkronique Loizeau, Alain Abarnou

Fish take up contaminants both from the water and from their food. In coastal and estuarine areas, the superficial sediment is continuously enriched by hydrophobic contaminants of terrestrial origin. Consequently, benthic species are heavily exposed to these potentially toxic compounds (Schafer et al., 1982; Cossa et al., 1992). In our study, dab (Limanda limanda) was chosen as the top predator of a very simple benthic foodweb. This species, commonly found in European coastal waters, has already been studied for PCBs (Knickmeyer and Steinhart, 1989) and its biology is well documented (Deniel, 1981; Quiniou, 1986; Tassel, 1988). Monitoring programmes have stressed the high levels of PCB contamination in mussels from the Seine estuary and its surrounding area (Abarnou and Simon, 1986; Claisse, 1989). Obviously, these man-made chemicals come from the river Seine and are transported into the marine environment mainly with suspended particulate material (Abarnou et aL, 1987). The authors have been conducting ongoing research into this problem of PCB contamination, in the Seine estuary and in the Baie de Seine. This work on the distribution of PCBs in dab contributes to our assessment of the fate of these persistent chemicals in biota from a polluted ecosystem. The objectives of the study were to obtain qualitative and quantitative information on the distribution of PCBs in various organs of the dab and to assess the variation of the contamination in relation to biological and physiological processes.

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Sampling sites of the dabs.

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Distribution of PCBs in dab

79

MATERIALS A N D METHODS

Sampling Fish were caught using a beam trawl in the eastern part of the Baie de Seine (Fig. 1). Strictly speaking, dab cannot be considered as a sedentary fish since they reach deeper waters as they get older. Therefore, the study has been limited to specimens from the four-year-old classes, with the assumption that they all belong to the same population from the Baie de Seine, living in coastal waters down to 40 m (Tassel, 1988). The biological characteristics of the dab, such as length and weight were measured, as well as the wet weights of their liver and gonads. Otoliths were removed for age determination. The stomach contents were also observed for confirmation of the diet of the dab. All samples of liver, gonad and muscle tissues were kept frozen in pre-cleaned glass jars until analysis.

Chemical analyses The material to be analysed was freeze-dried and the water content determined. Total lipids were measured according the protocol of Bligh and Dyer (1959). PCBs were determined on the saponified extract following the method described by de Boer (1988). Approximately I-2 g of dry material, carefully weighed, was treated with a solution of potassium hydroxide in ethanol for 4 h at 90°C. After cooling, the remaining solution was extracted by pentane. This extract was then concentrated for the clean-up on a florisil microcolumn. The eluate was evaporated under a nitrogen stream and re-dissolved in an exactly known volume of iso-octane for gas chromatography. GC-ECD analyses were carried out on a Hewlett Packard 5880 chromatograph equipped with a 50 m CP Sil8 CB capillary column (0.25 mm internal diameter and 0.20/xm film thickness) and with a CP Sill9 CB of the same dimensions. Two separate analyses on columns of different polarity increase the accuracy of the data because several congeners cannot be precisely determined on a single apolar column (Duinker et al., 1988; Bowadt and Larsen, 1992). Improvement in the results was obtained by a second analysis of the extracts on a slighty more polar column, such as CP Sil 19, which appears a good second choice for the analysis of the congeners CB 149/CB 118 and CB 153/CB 105 (de Boer et al., 1992). The instrumental conditions were set as shown in Table 1. The PCBs were quantified by individual components on the basis of a

VOronique Loizeau, Alain Abarnou

80

TABLE 1

Instrumental Conditions Parameter

Injection Initial oven temperature Temperature program Detector Make up gas Carrier gas

Conditions On column 1 /zl-iso-octane 120°C; 4 min 30°C/min--170°C 3°C/min--280°C 63 N i - - E C D at 300°C Argon methane Hydrogen at a linear velocity of 0.5 m/s

set of eight congeners from tri- to heptachlorobiphenyls, namely CB 28, 52, 101, 105, 118, 138, 153 and 180 according to the usual nomenclature (Ballschmiter and Zell, 1980). These representative PCB congeners are dominant in environmental samples and have been recommended for pollution monitoring studies. From this list, only the 'mono-ortho' congeners CB 105 and 118 present toxic properties, but to a lesser extent than the 'non-ortho' compounds. All the steps in the preparation of the extracts and the instrumental conditions were optimized. Under these conditions, congeners were quantified at concentrations higher than 0.5 ng g l dry weight with a precision (repeatability) of 10%. During the period of this study, our laboratory participated in the ICES/IOC/OSPARCOM intercomparison exercise on the analysis of chlorobiphenyls congeners in marine media (lst and 2nd step). Statistical treatments

Multivariate analyses were carried out to facilitate the interpretation of the large set of data obtained throughout this study. Principal component analysis is a data transformation technique that facilitates the search for patterns within a set of variables. The statistical background and techniques are described in many texts and references (Benzecri, 1980). Principal component analyses (PCA) were performed using the PC software package A D D A D . RESULTS A N D DISCUSSION Nature of the contaminants

The chromatogram (Fig. 2) obtained from an extract of muscle tissues of dab gives an example of the usual observations made on all the samples.

81

Distribution of PCBs in dab TCN

153 138

187 149 101

52 .

28 .l L _ L

180

i

128

I

170

Fig. 2. Chromatogramof an extractof dab muscleon a CB Sil8 CB column(numberingof peaks accordingto Ballschmiterand Zell, 1980). Injectioncorresponds to an approximate amount of 5 mg, i.e. 1 mg on a lipid basis. From a qualitative point of view, very similar chromatograms were obtained independently of either the analysed organ or the age of the fish. The PCB fingerprints were characterized by the importance of the hexachlorobiphenyls, notably CB 138 and CB 153 which represent by far the major compounds of this extract. These two chlorobiphenyls were also found at high concentrations in water samples from the Baie de Seine (Abarnou et al., 1987; Abarnou, 1988). In all samples from dabs, the main chlorobiphenyls possess four or more chlorine atoms per molecule. Moreover, most of them possess the same chlorine substitution with at least one of the following half structure, either - 245 (CBs Nos 101, 118, 138, 149, 153, 180, 183, 187) or - 2345 (CBs Nos 170, 180, 194). Similar findings were observed in flatfishes from the North Sea (Boon et al., 1984; Knickmeyer and Steinhart, 1989).

Principal component analysis of the PCB concentrations A spreadsheet of the full data set was built using the determination of eight PCB congeners in tissues of three organs from 113 dabs of various age and sex collected all through the year. In this table, the PCB concentrations in one sample constitute a row whereas each column is made up of all the results for the same congener. The statistical treatment is made on this matrix.

82

VOronique Loizeau, Alain Abarnou 4 years 3 years

[ ~ - ] - ] 2 years 1 year ~Male

gonads

I Axis 2

(a)

i-1 (b) Fig. 3. Principal component analysis of PCB concentrations in liver, muscle and gonad of 4 year-classes of dabs. (a) Plot of observations in the principal plan of the first two components; L, liver; M, muscle, G, gonad. (b) Projection of the standardised variables (PCB congeners) in the dual rulspace. Numbers associated with the arrows refer to the IUPAC nomenclature of PCB congeners.

The data are plotted in the first principal plane (Fig. 3(a)) defined by the first two principal components which together represent 91.5% o f the total variance. In this display, several clusters appear depending on the biological and physiological characteristics: observations on livers are all distributed above the first axis, while observations on muscle fall below. F o r both organs, we can observe clusters o f specimens o f the same age group.

Distribution of PCBs in dab

83

The distribution of observations in gonads appears differently; indeed we observe on the left a large grouping of observations on all male gonads of any age group, whereas, data from female gonads are scattered in the plot. Figure 3(b) illustrates the projection of vectors which represent the initial variables, the eight congeners, in the first principal plane. The more these vectors are close together, the more they are correlated. Thus, all variables are hightly correlated, and pointing in the same direction. Since the first axis, which represents 81.6% of the total variance, and all the variables are closely related, one can say that this axis is a size factor. In other words, the PCB concentrations increase along this axis from left to right. The significance of the second axis, which retains only 9.9% of the total variance, remains unclear. The hydrophobic character of the various congeners or their metabolization rate may be possible explanations. The relative distribution of the variou.s congeners on each side of the first axis gives an indication in favour of the first hypothesis because the highly chlorinated compounds (CB 153 and CB 138) follow the distribution of the representative points of the liver samples, a lipid rich organ. On the other hand, the less chlorinated and slighty more polar congeners, below two pentachlorobiphenyls (CB 101 and CB 118) having similar hydrophobic properties as indicated by comparable octanol-water partition coefficient (log Ko,v = 7.07-7.12, Rapaport and Eisenreich, 1984), are far from each other. Thus the ordering of the CBs along the second axis does not fully endorse only an effect of the bioconcentration potential. Metabolization may also affect the relative distribution of CBs between the organs of dab. The CBs 52 and 28, each with three of four chlorine atoms, are more easily excreted than the other compounds. The other congeners possess one ring with a similar 245 substitution. According to its structure, the CB 101 (22'455') would be partially metabolized, and probably to a greater extent than CB 118 (23'44'5). Similar observations have already been made on the relative persistance of CB 118 in crustaceans compared with molluscs (Boon et al., 1992). Hence, both these initial assumptions on a possible effect of hydrophobicity and metabolism may partially explain the second axis which represents only a minor part of the total variance. Referring back to the PC plot (Fig. 3(a)), the relationships between the samples may be further discussed. Thus, for the liver and muscle tissues, the concentration of the major hydrophobic congeners increases with the age of the fish. For the gonads, the distribution of the observations remains unclear.

84

V~ronique Loizeau, Alain Abarnou

Variation of PCB concentration in gonads As observed previously, the contamination in dab gonad tissues is rather low, particularly in male fish. The first PC analysis failed to reveal any clear groupings for these tissues from female fish. Figure 4 represents the results of a second PC analysis carried out using only the data from the gonads. The distribution of the eight variables differs from that obtained with all the observations. However, it is again the first axis, a size factor, which by far retains the major part of the total variance (88.2%). The second axis is probably explained by the relative contribution of each of the eight congeners. However this observation will not be discussed further as the contribution of this axis (3.7%) remains of minor importance. In the PC plot, all observations made on gonads from males fall within the same group which includes the samples with the lowest contamination. For females, the distribution of the observations is more complicated. Several groupings appear to be related to the age of the dab and the period of sampling. Considering the example of three year old fish and following the first axis, we find the lowest concentrations in June (CB 153:35-50 ng g 1 D W ) . Then the concentrations in the ovaries increase in specimens caught in October (CB 153:50-80 ng g l DW) reaching higher concentrations in March (CB 153:90-100 ng g-~ DW) and April (CB 153:90-120 ng g i DW). The same seasonal variations in concentrations are observed for older fishes. However, this type of cyclic variation has not been found for dab at younger stages of development. Clearly, it is the reproductive cycle of fish which explains the low level of contaminants observed in spring. According to Tassel (1988), for the dabs from the eastern Channel, the spawning period is from March to April. This author has summarized the development of the ovaries during the year: the spawning period is from March to April at the end of which the dabs are very much thinner due to loss of lipid. This is followed by a resting period from May to September when lipid reserves are built up again. From October to February there is a period of growth of ovaries which utilizes lipids from reserves stored in the liver. Tassel (1988) has also determined that for female dabs the first spawning occurs in the third year. The data reported in this paper for dabs sampled in October, March, April and June follow this cycle. In June the spawning is achieved for all fish and gonads reach their lowest lipid content; consequently the contamination level is at a minimum. After this period, the increase in lipids during summer and particularly just before the spawning period is accompanied by a similar variation of PCB concentration in the gonads.

Distribution of PCBs in dab

85

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118

(b) Fig. 4. Principal component analysis of PCB concentrations in gonad of four yearclasses of dabs. (a) Plot of observations in the rulspace of the first two components; IIl, March; IV, April; VI, June; X, October. (b) Projection of the standardised variables (PCB congeners) in the dual rulspace. Numbers associated with the arrows refer to the IUPAC nomenclature of PCB congeners.

Kamman et aL (1990) has already mentioned the effect of reproduction on the distribution of PCBs in the ovaries of dab from the North Sea. The transfer of lipids from livers to ovaries is also accompanied by an increase in polar lipids compared to neutral lipids. A change in the composition of the lipids would modify the relative distribution of the CBs in the various tissues according to the solubilities of these contaminants in lipid rich tissues. Indeed Kamman et al. (1990) have measured higher CB

86

V~ronique Loizeau, Alain Abarnou

138/CB 153 ratios in ovaries than in livers and also an increase in the slighty more polar compounds like CB 128 (22'33'44'). Unfortunately the lipid composition of the three organs we have analyzed for PCB has not been determined. However, our results for a limited number of samples, approximately 3-6 for each group of the same year and the same sampling period, suggest a more efficient transfer of CB 138 from livers to ovaries. The transfer of hydrophobic contaminants from the lipid reserves to the gonads and ultimately to the eggs has been discussed previously (Solbakken et al., 1984; Giesy et al., 1986; Spies and Rice, 1988; Ankley et al., 1989). The potential toxic effect of this process on the hatching success and consequently on the recruitment of the species needs further investigation. A very approximate calculation of the amount of contaminants which are excreted during the spawning period, may be done on the basis of the loss of weight of gonads and the decrease of PCB concentrations of the contaminants. According to Tassel (1989), for female dabs between 3-5 years, the loss of the weight of the ovaries is around 10-100 g (fresh weight), the larger loss being observed for older fish. We have determined concentrations of PCBs in the gonads before and after spawning. For the CB 153, concentration decreases from 100 ng g-t to 50 ng g-~ (dry weight). With these assumptions the amount of CB 153 eliminated from gonads during spawning would represent approximately 0.5 /zg for the youngest fishes and 15 /zg for older fishes (some 8-12% of the whole body contamination). However, depending upon the hatching success of contaminated eggs, these contaminants would normally be reintroduced into the water column. The male sexual products eliminated in this way represent a minor factor, since gonads represent only 1-2% of the whole body weight and do not contain much contaminant. PCB concentrations in dab

The above PC analyses have clearly demonstrated the effects of biological parameters on the PCB contamination in dab. The most convincing results are the groupings of observations according to organs and age, the increase of PCB concentration with age and the influence of reproduction cycle on the contamination levels particularly in female dabs. Consequently, the outcome of this statistical interpretation allows us to present a summary of PCB measurements in dab (Table 2). Mean concentrations have been calculated for each of the groupings observed in the PC plots. The results are given for two congeners, CB 138 and CB 153, because they are the predominant components in all the samples and because the PCB patterns in the various tissues are very similar.

TABLE 2

Muscle Liver Gonad"

Muscle Liver Gonad a

Muscle Liver Gonad a

Muscle Liver Gonad"

1 year

2 years

3 years

4 years

8 8 2

13 13 5

21 21 12

11 11 7

N samples

244 692 45

233 457 9

181 408 15

24 188 11

(40) (7) (5)

(36) (40) (1)

(27) (36) (4)

(6) (9) (2)

CB 138 mean (SD)

236 668 38

225 432 14

163 408 18

22 141 7

(26) (14) (5)

(41) (58) (2)

(21) (23) (4)

(6) (11) (1)

CB 153 mean (SD)

Males

13-6 39.0 1.9

14-5 45.4 4

9.4 37.9 1.3

13.8 51.2 1.2

(1.1) (1.9) (0.4)

(1-2) (5.7) (0.7)

(2.6) (8) (0.5)

(1.1) (1.2) (0.5)

Lipids mean (SD)

a For gonads, only data for fish collected during the early spring are given.

Organ

Age

14 14 4

26 26 3

12 12 12

7 7 7

N samples

260 641 155

225 404 132

185 371 109

21 184 57

(39) (97) (13)

(56) (92) (3)

(30) (91) (13)

(1) (14) (5)

CB 138 mean (SD)

265 622 137

213 394 97

187 368 101

19 170 59

(26) (117) (14)

(56) (84) (5)

(22) (83) (14)

(3) (20) (6)

CB 153 mean (SD)

Females

15.0 42.4 15.1

13-5 41.4 14.4

10.1 31.6 9.9

8.7 49.2 4.8

(1.4) (5.9) (0.6)

(0-7) (8) (1.2)

(1.4) (2.4) (0.7)

(1.4) (1.7) (0.5)

Lipids mean (SD)

Concentrations of the Two Main CBs (ng n ~ dry weight) and Lipid Contents in Three Organs of Dab from the Baie de Seine

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VOronique Loizeau, Alain Abarnou

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Distribution of PCBs in dab

89

The similarity of the PCB patterns is presented (Fig. 5) using mean ratios of congeners concentrations to that of CB 153, a method previously adopted by several authors (Duinker et al., 1983; Boon et al., 1992). Examination of the PCB patterns in dabs indicate a slightly higher contribution of the highly chlorinated components in the youngest fishes. As these specimens live in coastal waters, this probably reflects the change in PCB composition and the relative enhancement of more chlorinated compounds as PCBs are transported from the freshwater to the marine environment. The data shown in Table 2 are very similiar to the results of Knickmeyer and Steinhardt (1989) for dab from the North Sea, where concentrations of CB 153 in liver vary in the range 120-600 ng g i dry weight. Cossa et al. (1992) have published data for flounder (Platichtysflesus) from the Bale de Seine. They measured muscle concentrations of CB 138 in the range 70-900 ng g l dry weight which are comparable to our results (20-250 ng g-l) observed for dabs. In a macrotidal estuary, like the Seine estuary, high concentrations of suspended particular material and associated contaminants are present in the mixing zone (Allen et al., 1980; Salomon and Allen, 1983; Abarnou et al., 1987). Higher PCB concentrations observed in flounders may reflect their euryhaline nature and the potentially long-term exposure to hydrophobic contaminants carried by suspended particles. This work has shown that the contamination level in the organs of dab increases with age. It also highlights the importance of lipid content on the level of PCB contamination in the various tissues and its subsequent variation during the reproductive cycle. In this respect, the utilization of lipid reserves for the maturation of gonads and their eventual loss with eggs during the spawning represents an important mechanism for the elimination of contaminants, This process should be taken into account when modelling the fate of persistent hydrophobic contaminants in the foodweb. The transfer of PCBs from adult to eggs should also be considered when sampling dab or similar flat fishes for monitoring purposes, or when conducting an ecotoxicological assessment of these toxic compounds.

ACKNOWLEDGEMENTS The authors wish to thank Louis Quiniou from UBO Brest who helped them during the sampling cruises and gave precious advice on the biology of fish, and their colleague from IFREMER, Philippe Gros, who instructed them in the use of multivariate analyses. Alistair Lane from France Aquaculture is gratefully acknowledged for improving the English text.

90

V6ronique Loizeau, Alain Abarnou REFERENCES

Abarnou, A. (1988). Les polychlorobiph6nyles en baie de Seine. Rapport IFREMER DERO-88.06-EL, 119 pp. Abarnou, A. & Simon, S. (1986). Contamination de restuaire et de la baie de Seine par les PCB. Bilan des 6tudes. In: La Baie de Seine. Actes de Colloques No 4. IFREMER, 530 pp. Abarnou, A., Avoine, J., Dupont, J. P., Lafite, R. & Simon, S. (1987). Role of suspended sediments on the distribution of PCB in the Seine estuary (France). Continent. ShelfRes., 7, 1345-50. Allen, G. P., Salomon, J. C., Bassoullet, P., du Penhoat, Y. & de Granpre (1980). Effects of tides on mixing and suspended sediment transport in macrotidal estuaries. Sediment. Geol., 26, 69-90. Ankley, G. T., Tillitt, D. E. & Giesy, J. P. (1989). Maternal transfer of bioactive polychlorinated aromatic hydrocarbons in spawning chinook salmon (Oncorhynchus tschawytscha). Mar. Environ. Res., 28, 231-4. Ballschmiter, K. H. & Zell, M. (1980). Analysis of PCB by glass capillary gas chromatography. Fresenius Z. Anal, Chem., 302, 20-31. Benzecri, F. (1980). Introduction ~ l'analyse des correspondances d'apr~s un exemple de donnbes m6dicales. Cahier de l'analyse des donn6es, 3, 283-310. Bligh, E. G. & Dyer, W. (1959). A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol., 37, 911-17. Boon, J. P., Oudejans, R. C. H. M. & Duinker, J. C. (1984). Kinetics of individual polychlorinated biphenyl (PCB) components in juvenile sole (Solea solea) in relation to their concentrations in food and to lipid metabolism. Comp. Biochem. Physiol., 79 C, 131-42. Boon, J. P., Van Arnhem, E., Jansen, S., Kannan, N., Petrick, G., Goksoyr, A., Schulz, D., Duinker, J. C. & Reijnders, P. J. H. (1992). The toxicokinetics of PCBs in marine mammals with special reference to possible interactions of individual congeners with the cytockrome P450 - dependent monoxygenase system - - an overview. In persistent polluants in marine ecosystems. A SETAC Special publication. Ed. C.H. Walker & D.R. Livingston. Pergamon Press, Oxford, 272 pp. Bowadt, S. & Larsen, B. (1992). Improved congener-specific GC analysis of chlorobiphenyls on coupled CP Sil 8 and HT 5 columns. J. High Res. chrom., 15, 377-80. Claisse, D. (1989). Chemical contamination of french coasts: The results of ten years mussel watch. Mar. Pollut. Bull., 20, 523-8. Connolly, J. P. (1991). Application of a food chain model to polychlorinated biphenyl contamination of the lobster and winter flounder food chains in New Bedford harbor. Environ. Sci. Technol., 25, 760-70. Cossa, D., Auger, D., Averty, B., Lu~on, M., Masselin, P. & Noel, J. (1992). Flounder (Platichthys flesus) muscle as an indicator of metal and organochlorine contamination of French Atlantic coastal waters. Ambio, 21, 176-82. de Boer, J. (1988). Chlorobiphenyls in bound and non-bound lipids of fishes; comparison of different extraction methods. Chemosphere, 17, 1803-10. de Boer, J., Dao, Q. T. & van Dortmond, R. (1992). Retention times of fifty one chlorobiphenyl congeners on seven narrow bore capillary columns coated with different stationary phase. J. High Res. Chrom., 15, 249-55.

Distribution of PCBs in dab

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