Biomagnification Study on Organochlorine Compounds in Marine

Marine fish culture is an increasing activity in the western Mediterranean. ...... Lake Kerkini, a North-Eastern Mediterranean Wetland of Internationa...
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Environ. Sci. Technol. 2003, 37, 3375-3381

Biomagnification Study on Organochlorine Compounds in Marine Aquaculture: The Sea Bass (Dicentrarchus labrax) as a Model ROQUE SERRANO,* AÄ N G E L A S I M A L - J U L I AÄ N , ELENA PITARCH, AND F EÄ L I X H E R N AÄ N D E Z Analytical Chemistry, Department Experimental Sciences, University Jaume I, P.O. Box 8029 AP 12080, Castello´n, Spain INMACULADA VARO Ä AND JUAN C. NAVARRO Institute of Aquaculture of Torre la Sal, C.S.I.C., 12595 Ribera de Cabanes, Castello´n, Spain

Biomagnification of organochlorine compounds (pesticides and polychlorinated biphenyls) through the marine aquaculture food chain is investigated. From first-feeding, specimens of sea bass were exposed to commercial fish feed (that contained DDTs and PCBs residues) for ca. 24 months, and selected tissues (white and red muscle, liver, and visceral fat) were analyzed after 6 and 24 months of diet exposure. Data obtained showed that experimental fish tissues presented a similar contamination pattern to that of fish feed, and biomagnification processes of these compounds were proved. Additionally, commercial sea bass cultured in farms from the western Mediterranean were analyzed, their organochlorine concentrations being significantly lower than those of the 24 month old experimental fish. Thus, the exposition of human population to OCs through consumption of cultured fish would be lower than expected from experimental biomagnification studies, although red muscle presented similar OC levels in both cases, which were much higher than those of white muscle. Although levels of organochlorine compounds were found to be low, the persistence, ubiquity, and toxicity of these compounds, together with their presence in fish feed, make it necessary to monitor OC residues in the routine quality assurance programs of aquaculture activities, as this food chain is a source of these toxic compounds for human consumers. The development of sensitive analytical methodology based on GC-MS/MS has allowed for the reaching of low detection limits required to carry out the present study.

Introduction Currently, the contamination pattern in the marine environment includes, together with other pollutants, a variety of organochlorine compounds (OCs), which are worldwide dispersed as demonstrated by their detection in tropical and * Corresponding author phone: +34-964-728094; fax: +34-964728066; e-mail: [email protected]. 10.1021/es020229+ CCC: $25.00 Published on Web 07/03/2003

 2003 American Chemical Society

subtropical species (1) and polar marine organisms (2, 3). This group of pollutants includes industrial products such as polychlorinated biphenyls (PCBs) and insecticides such as diphenyldichlorotrichloroetane (pp′-DDT), hexachlorobenzene, and lindane (γ-HCH), which are still a matter of major concern, despite the restrictions in their use. These compounds are considered among the most dangerous pollutants because of their toxicity and stability, long biological half-life, and high liposolubility, which results in its high bioaccumulation and biomagnification along the food chain involving a wide range of trophic levels, as has been demonstrated by several authors (4-6). As a consequence, they are often detected in fish (7-9), birds (10), and marine mammals (11, 12). Because of the bioaccumulation ability of these compounds, they have reached the top level of the ecosystem and have been detected in human adipose tissues and fluids (13-18). Dietary intake, and specially marine organisms, is considered as one of the most important sources of OCs for the human population (13, 19). Therefore, it is essential to know the levels of these contaminants in foods processed from marine raw materials. Organochlorine compounds have been detected in salt boiled and smoked fish (20), fish oils (21), and fish feed and cultured fish (22-24). The bioaccumulation of these compounds through a diet composed of a high percentage of marine products can result in an important contribution to the human body burden of organochlorines, as has been observed in populations with predominantly marine products in the diet (13, 19). Aquaculture has been developed in past decades as a consequence of the increase of consumption by the world population, since fisheries have possibly reached their maximum level of production, due to overexploitation. Human population is at the top of an artificial food chain composed of fish feed and fish specimens, that can result in a possible new way for the accumulation of OCs in human beings. Marine fish culture is an increasing activity in the western Mediterranean. It is mainly focused on two species: gilthead sea bream (Sparus aurata) and sea bass (Dicentrarchus labrax), working in parallel with traditional fisheries. Both cultured and wild fish are important components of the Mediterranean diet. Wild fish tend to be leaner than farmed fish (25, 26). Factors such as lipid level in the diet (27), ration level (28, 29), and the weight of the fish (30, 31) increase the body lipid composition and, consequently, can account for an increase in the accumulation of lipophilic contaminants. The aim of this paper is to contribute to a better understanding of bioaccumulation and biomagnification of OCs through the artificial food chain from aquaculture activities composed of fish foods, cultured fish, and human consumers as the top trophic level. Thus, concentration levels of OCs in fish feed used in aquaculture activities have been determined, and biomagnification of these pollutants from fish feed to cultured fish is investigated in pilot plant conditions. Moreover, the levels of organochlorine compounds in commercial culture sea bass have been determined and compared to those of samples from this experimental study.

Materials and Methods Samples. Fish feed used in the biomagnification experiment was obtained from a commercial supplier. As usual in fish culture, the nominal compositions of fish feed used during the first and second years of the experiment were in 50/20 VOL. 37, NO. 15, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Program Used for MS/MS Detection of OCs and Extracted Ions Selected for Quantitative Determinatione compound

parent ion

time (min)

window

mass range

voltage (V)

Rta (min)

HCB HCB-13C6d lindane endosulfan ether heptachlor aldrin heptachlor epoxide R-endosulfan p,p′-DDE p,p′-DDE-D8d β-endosulfan p,p′-DDD endosulfan sulfate p,p′-DDT metoxychlor mirex PCB 28 PCB 52 PCB 101 PCB 118 PCB 138 PCB 138-13C12d PCB 153 PCB 180

288b 288b 219 241 337 293 355 241 250c 250c 241 235 289 235 227 272 258 292 326 326 360 372 360 396

3.50 3.50 7.50 8.40 9.25 10.30 11.30 12.65 13.60 13.60 14.60 15.20 16.47 16.75 18.00 20.00 3.50 9.80 11.50 11.50 15.60 15.60 15.60 18.50

10 10 5 8 8 10 8 8 8 8 8 8 8 8 4 8 4 4 4 4 8 8 8 8

170-295 170-295 140-225 165-245 200-345 145-300 175-360 165-245 145-260 145-260 165-245 160-240 175-295 160-240 110-230 200-280 140-262 180-295 170-330 170-330 200-380 200-280 200-380 220-402

3.50 3.50 1.05 1.56 1.50 1.75 1.60 2.50 3.00 3.00 2.50 1.30 1.60 1.70 1.50 1.50 2.00 1.50 2.00 2.00 1.75 1.75 1.75 2.25

7.31 7.31 7.85 8.84 9.56 10.57 11.80 12.99 13.82 13.82 15.02 15.44 16.63 16.83 19.21 20.5 9.16 10.09 12.85 15.09 16.99 16.99 15.93 19.66

extracted ions selected 214, 249 220, 225 181, 183 204-208, 237-241 265 220 265 170, 172, 204-208 176 183, 184 170, 172, 204-208 165, 199 217, 219, 251-255 165, 199 152, 169 237 186 220, 222 254, 256 254, 256 288, 290 302, 337, 372, 374 288, 290 322,324,326

a Rt: retention time. b The total m/z range selected (283-293) covers the individual parent ions of HCB (284) and HCB-13C (290). c The total m/z 6 range selected (245-255) covers the individual parent ions of pp′-DDE (246) and pp′-DDE-D8 (254). d The entries in italic letters represent the isotopically labeled compounds used as surrogate internal standards. e OC pesticides and PCBs were analyzed with different MS/MS programs.

and 45/12 proteins/lipids percentages, respectively. Feed was manufactured from triturated fish obtained from fisheries in the Peru-Chile coast area and blended with fish oils. Samples were collected whenever a new batch was used for fish feeding and stored at -20 °C until analysis. Analysis of each batch sample was made in sixtuplicate. Sea bass (Dicentrarchus labrax) specimens for biomagnification experiments were obtained from spawnings of a captive breeding group in the “Institute of Aquaculture of Torre la Sal, CSIC, Castello´n, Spain (IATS)” (hatching on March 1999). IATS belongs to the Spanish Council for Scientific Research, and it can be considered as a pilot plant representative of marine fish inland aquaculture in Spain. During the biomagnification experiment, 10 fish were sacrificed at two sampling times, 6th month (weight: 26 ( 4 g, length: 13 ( 1 cm) and 24th month (weight: 533 ( 40 g, length: 33 ( 10 cm), after anesthesia by immersion in ice. The tissues selected for analysis were liver, white muscle (the bulk of a fish body), red muscle (small areas at the root of the fins and in the strip along the center of each flank, more irrigated and with higher fat content than white muscle), and visceral fat (accumulation of fat surrounding the viscera). Fish tissues were pooled after dissection and stored at -20 °C until analysis (n ) 5). Each replicate used for analysis was composed of two specimens. Cultured sea bass specimens from western Mediterranean farms were obtained from the market in Summer 2001 (weight range 250-500 g), dissected, and stored at -20 °C until analysis (n ) 5). Each replicate was composed of two specimens. Bioaccumulation Experiment. One hundred sea bass (Dicentrarchus labrax), cultured at the IATS, were randomly distributed into two 90 L circular aquaria. After 6 months of diet exposure they were transferred to 2500 L circular aquaria. Densities were similar to the usual ones used in aquaculture in inland farming. All aquaria were supplied with running seawater (salinity 37‰, pH ) 8.3) under continuous aeration, and fish were maintained at a natural temperature and photoperiod (40 °N and 0 °E). Seawater was previously analyzed and OCs were not detected (limit of detection between 0.01 and 0.1 µg/L). 3376

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Fish were hand fed daily “ad libitum” with the fish feed described above. Fish feed was stored at 4 °C until use. To monitor biomagnification of organochlorine compounds present in fish feed, each new batch was analyzed. Ten fish specimens were sampled randomly after 6 and 24 months of feeding. Biomagnification factors (BMF) were calculated as the ratio between lipid-based concentrations of organochlorine compounds in the fish tissues and in the fish feed after 6 or 24 months of diet exposure (31). As three different fish feed batches were used with the experiment, calculations of BMF at 24 months were made taking into account the time of exposure to each batch. Thus, the pondered arithmetic mean of organochlorine concentrations in the diet was calculated on the basis of months of exposure to each batch and on their concentration in each fish feed batch. Analytical Methodology. Organochlorine compounds, including pesticides and derivatives (DDTs -p,p′-DDT, p,p′DDE, p,p′-DDD-, HCB, aldrin, heptachlor, heptachlor epoxide, lindane, mirex, metoxychlor, endosulfan (R and β isomers), ensodulfan sulfate, and endosulfan ether) and polychlorinated biphenyls (IUPAC nos. 28, 52, 101, 118, 138, 153, 180), were analyzed in fish feed and fish tissues (white and red muscle and liver) following the method described by Serrano et al. (24). Visceral fat was analyzed based on the method reported by Herna´ndez et al. (17) for human adipose tissue. Briefly, extraction in fish feed, muscle, and liver was carried out by refluxing ca. 8 g of homogenized fresh sample in n-hexane during 4 h. In visceral fat samples, the extraction was performed by dissolving directly the sample in n-hexane. Cleanup of fish tissues was performed by means of normal phase liquid chromatography (NPLC) injecting 1 mL of hexanic extract (4 g sample per mL) into a silica gel HPLC column. Cleanup of fish feed was made in two steps. First, acid digestion (sulfuric acid) was applied to 1 mL of hexanic extract (4 g sample per mL) in order to remove most of the fats, and then a NPLC cleanup was applied to the acid-treated extracts. Analysis of the fatfree LC fractions was performed by gas chromatography with tandem mass spectrometry (GCMS/MS) using an ion trap mass spectrometer, under conditions shown in Table 1 (details reported in ref 24).

TABLE 2. Concentration of Organochlorine Compounds (Mean and Relative Standard Deviation in Brackets, n ) 6) Detected in Three Batches of Fish Feed Used along the Biomagnification Study batch of fish feed and period of use concn (ng‚g-1 fresh wt) 1a

concn (ng‚g-1 lipid wt)

batch 1-6/ compound 2000

2a

3b

batch batch 7/2000- 2-12/ 1/2001 2001

HCB pp′-DDT pp′-DDE pp′-DDD lindane aldrin PCB 28 PCB 52 PCB 101 PCB 118 PCB 138 PCB 153 PCB 180

1.1 (8) 3.9 (12) 8.3 (4) 4.8 (11) c c d 0.9 (14) 3.1 (3) 1.7 (18) 3.4 (8) 3.2 (10) 0.6 (38)

0.4 (12) 1.7 (10) 6.6 (7) 4.5 (10) d d 0.3 (14) 0.3 (15) 1.0 (7) 0.8 (5) 1.8 (12) 2.7 (5) 0.7 (9)

3.6 (16) 2.8 (17) 9.8 (13) 4.1 (9) c c d 1.4 (17) 3.8 (9) 1.7 (20) 5.1 (4) 5.7 (8) 0.4 (14)

batch 1 batch 2 batch 3 1-6/ 7/2000- 2-12/ 2000 1/2001 2001 22.8 17.8 61.4 25.6 c c d 9.1 23.7 10.7 32.1 35.8 2.8

5.7 20.8 43.7 22.5 c c d 4.7 16.1 9.0 18.2 17.0 2.9

4.3 18.6 73.5 49.6 d d 3.1 3.4 11.2 9.4 20.1 30.2 8.3

a Nominal composition 50/20 (proteins/lipids, percentage). b Nominal composition 45/12 (proteins/lipids, percentage). c Not detected (below 0.1 ng.g-1). d Detected (estimated concentration between 0.1 and 0.3 ng.g-1).

The analytical method applied showed excellent sensitivity and selectivity, as a consequence of the use of tandem mass spectrometry. It was validated by recovery experiments down to 5 ng‚g-1, as lower spiked levels were not checked due to the presence of DDTs and PCBs in the blank samples (24). However, the powerful analytical characteristics of GC-MS/ MS together with the efficiency of the HPLC cleanup has allowed us in the present paper to detect the analytes at concentrations as low as 0.1 ng‚g-1 (estimated as limit of detection, except for endosulfan ether with 0.2 ng‚g-1). The limit of quantification has been calculated as three times the detection limit (i.e. nine times the background noise in the chromatograms), resulting in values of 0.3 ng‚g-1, except for endosulfan ether (0.6 ng‚g-1). All analytes detected in the samples analyzed were confirmed by tandem mass spectrometry. For the quality control in analyses of real samples, 50 ng of isotopically labeled standards was added before extraction as surrogates. This allowed us to correct the possible analytical errors in every sample analysis. Labeled standards used were HCB-13C6 (for the determination of HCB, lindane, endosulfan ether, heptachlor, aldrin, heptachlor epoxide, and R-endosulfan); pp′-DDE-D8 (for DDTs, β-endosulfan, endosulfan sulfate, metoxychlor, and mirex); and PCB138-13C12 (for PCBs). Determination of Fat. The total fat content in the sample extracts was determined by gravimetry, evaporating at 95 °C until constant weight. Data Analysis. Tissue OCs concentrations are expressed on a lipid weight basis (ng‚g-1 lipid weight). For Principal Components Analysis data were transformed as Log (ng‚g-1 lipid weight +1) in order to correct dependence between arithmetic means and standard deviations. Nondetected records were considered as 0, and detected as 0.1 (detection limit). Homoscedasticity of variances was tested by means of Barlett’s test (P < 0.05). Statistical tests were conducted using STATGRAPHICS version 7.0 (Statistical Graphics Corporation).

Results and Discussion Fish Feed Contamination Pattern. Table 2 shows the concentrations of organochlorine compounds (OCs) detected in the three fish feed batches used during the biomagnifi-

cation experiment. The predominant pesticide was pp′-DDT and its derivatives, with the highest concentrations corresponding to the metabolite pp′-DDE, which reached 10 ng‚g-1 fresh weight. This pattern is in agreement with the pollution pattern of different marine products (20, 22). HCB was also quantified in the three fish feed samples analyzed. Lindane and aldrin were detected in only one batch, while heptachlor, heptachlor epoxide, metoxychlor, mirex, and endosulfan and derivatives were not detected in any sample. With regards to the seven polychlorinated biphenyls (PCBs) investigated, all of them were detected in the three samples analyzed, with the higher concentrations corresponding to the 6-chlorinated congeners (up to 5.7 ng‚g-1 fresh weight). Fish feed used in this study was prepared from trash fish fishmeal blended with fish oils which are added for polyunsaturated fatty acids (ω3) enrichment, using fishery harvest from the Peru-Chile coast. The contamination pattern observed in the fish feed analyzed in a certain degree reveals the pollution present in that area. The relatively low DDE/ DDT ratios found, between 2 and 4, in comparison with values around 10 in other areas (11), might suggest the recent use of DDT in the area. Moreover, the low values of the ratio sum of PCBs:total DDTs (between 1.1 and 1.6) also suggest the relevance of DDT in the ecosystem and the low level of industrialization in that area (see ref 11). Tissue Contamination Patterns. Table 3 shows the concentration levels of OCs determined in different tissues of experimental fish after 6 and 24 months of dietary exposure. As expected, all the OCs detected in fish feed were also found in the tissues of cultured sea bass. It must be taken into account that experimental fish were cultured in clean seawater free of pesticides and raised from spawnings in the IATS, where the experiment was carried out, without any other known exposure source of organochlorines except diet. Therefore, the contamination pattern detected in experimental fish might be taken as “imported” from the Peru-Chile coast to the western Mediterranean area. The presence of aldrin in some samples (specially the concentration levels in cultured sea bass red muscle) is unusual, as aldrin is efficiently metabolized by fish to dieldrin, which was not analyzed in this work. The reliability of GC/ MS-MS for peak identification suggests that this finding was not a false positive. More research should be required to confirm this finding. The long-term distribution (24 months) of the OCs in the tissues were visceral fat > liver > red muscle > white muscle as could be expected attending to their lipid content and similar to the distribution found by other authors in different organisms (see ref 33). The higher lipid-based concentrations in tissues corresponded to DDT derivatives and 6-chlorinated PCB congeners, reaching concentrations in most cases higher than 50 ng‚g-1 lipid weight. It is remarkable that levels found in visceral fat in the 6th month are higher than in the 24th month. This fact is in agreement with observations made by Smith (33) in rat adipose tissue, who noticed a peak of pp′-DDT after 6 months of dietary exposure, followed by a plateau. In our study, concentrations of organochlorine compounds found in fish tissues in the 24th month of the experiment could be considered as representative of long-term dietary exposure. The highest PCB concentrations correspond to PCB 138 and 153 which agree with most of the authors’ findings in marine organisms. Additionally, we found relative high concentrations of congeners 118 (5-chlorinated coplanar mono-ortho-substituted) and 180 (7-chlorinated coplanar di-ortho-substituted) that have been identified as dioxinlike PCBs as competitively bound aryl hydrocarbon receptors, the cause of dioxin toxicity (34). VOL. 37, NO. 15, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 3. Average Concentrations of Organochlorine Compounds (Minimum and Maximum Values in Brackets, n ) 5)a Found in Experimental and Cultured Commercial Sea Bass white muscle compound

6th month

24th months

red muscle cultured sea bassb

6th month

24th months

liver cultured sea bass (ng‚g-1

HCB DDT DDE DDD lindane aldrin PCB 28 PCB 52 PCB 101 PCB 118 PCB 138 PCB 153 PCB 180

d 0.8 (0.6-1.0) 2.6 (2.4-2.8) 1.9 (1.5-2.3) c d d d 0.8 (0.7-0.9) 0.7 (0.5-0.9) 1.5 (1.2-1.8) 1.9 (1.6-2.2) 0.5 (0.3-0.7)

c d 1.0 (0.8-1.2) 0.8 (0.7-0.9) c c d d 0.3(d-0.4) 0.4(d-0.6) 0.7 (0.5-0.9) 0.9 (0.6-2.2) 0.3 (d-2.5)

d c d d c d d d d d d d d

0.6 (0.5-0.7) 1.9 (1.5-2.3) 6.7 (4.6-8.8) 4.3 (3.1-4.5) d d d 0.7 (0.5-0.9) 2.1 (1.3-2.9) 1.9 (1.2-2.6) 4.6 (3.2-6.0) 5.7 (3.7-7.7) 1.5 (1.2-1.8)

Concentration 1.1 (0.7-1.5) 9.2 (8.1-10.3) 3.4 (2.3-4.5) 5.4 (4.2-6.6) 11.2 (9.2-13.2) 11.9 (9.9-13.9) 8.0 (7.3-8.7) 7.6 (6.9-8.5) c d c 9.1 (7.5-10.7) 0.8 (0.6-1.0) 1.0 (0.8-1.2) 1.0 (0.7-1.3) 0.9 (0.7-1.1) 3.3 (2.7-3.9) 2.6(2.2-3.0) 3.2 (2.8-3.6) 2.4 (2.0-2.8) 6.7 (5.7-7.7) 5.0 (4.5-5.5) 8.7 (7.8-9.8) 8.8 (7.5-10.1) 2.0 (1.7-2.3) 2.0 (1.5-2.5)

HCB DDT DDE DDD lindane aldrin PCB 28 PCB 52 PCB 101 PCB 118 PCB 138 PCB 153 PCB 180

d 51.3 174.0 124.7 c d d d 52.7 48.0 100.7 128.7 36.0

c d 84.6 64.1 c c d d 27.4 35.9 58.1 74.4 25.6

d c d d c d d d d d d d d

15.8 47.8 168.3 107.5 d d d 17.8 52.0 46.3 114.8 143.5 36.3

10.5 33.7 112.1 79.8 c c 8.4 9.9 32.6 31.8 66.7 86.5 20.3

visceral fat

6th month

24th months

cultured sea bass

6th month

24th months

fresh weight) 1.1 (0.8-1.4) 2.8 (2.2-3.4) 9.9 (7.9-11.9) 6.0 (2.8-9.2) d 1.9 (1.5-2.3) 1.6 (1.1-2.1) 2.3 (1.7-2.4) 6.5 (5.2-7.8) 5.7 (4.8-6.6) 12.5 (10.0-15.0) 16.1 (14.8-17.4) 4.2 (3.2-5.2)

2.5 (1.8-3.2) 2.9 (2.2-3.6) 16.9 (16.6-17.2) 12.0 (9.7-14.3) c c 1.9 (1.4-2.4) 2.1 (1.6-2.6) 5.2 (4.2-6.2) 4.8 (3.6-6.0) 9.1 (8.1-10.1) 12.4 (11.2-13.6) 3.1 (2.2-4.0)

d 1.7 (1.3-2.1) 5.1 (4.2-6.0) 3.1 (2.7-3.5) c 1.3 (1.0-1.6) d d 0.9 (0.5-1.3) 0.7 (0.5-0.9) 2.4 (2.0-2.8) 2.9 (2.1-3.7) 0.6 (0.5-0.7)

49.0 (38.7-59.3) 35.8 (28.8-42.8) 373.6 (235.6-510.6) 343.2 (309.2-356.2) 6.4 (4.1-8.7) c 8.4 (7.6-9.2) 16.0 (9.2-22.8) 52.9 (48.5-57.3) 31.2 30.6-31.8) 96.9 (66.9-1266.9) 139.0 (92.0-186.0) 36.9 (26.5-48.3)

7.0 (4.6-9.4) 22.0 (17.6-28.4) 78.3 (49.3-97.3) 53.4 (49.7-57.1) 1.0 (0.8-1.2) c 4.3 (3.9-4.7) 5.8 (5.1-6.5) 19.0 (14.4-23.8) 19.6 (14.5-24.7) 42.8 (28.7-56.9) 66.4 (43.8-82.0) 15.7 (9.7-21.7)

14.6 16.8 99.4 70.6 c c 11.2 12.1 30.5 28.5 53.7 72.9 18.1

d 34.2 101.0 61.0 c 26.4 d d 17.2 14.8 48.4 57.8 11.2

70.1 51.1 533.7 490.3 9.2 c 12.0 22.8 75.6 44.6 138.4 198.6 52.7

Concentration (ng‚g-1 lipid weight) 102.7 5.7 60.0 14.2 131.7 49.6 83.9 30.0 d d 100.9 9.4 10.7 8.1 10.2 11.5 29.3 32.6 27.0 28.7 55.6 62.7 97.8 80.6 21.9 21.1

a Each replicate was composed of two specimens. b Commercial cultured sea bass from aquaculture factories sited in the western Mediterranean area. c Not detected (below 0.1 ng.g-1). concentration between 0.1 and 0.3 ng.g-1).

17.5 55.1 195.8 133.5 2.5 c 10.7 14.4 47.6 49.0 107.0 166.1 39.2 d

Detected (estimated

TABLE 4. Physical and Ecotoxicological Parameters for OCs and PCBs Detected in Samples compound aldrin HCB lindane pp′-DDT pp′-DDE pp′-DDD PCB no. 28 PCB no. 52 PCB no. 101 PCB no. 118 PCB no. 138 PCB no. 153 PCB no. 180

Log water solubilitya Log (25 °C/mg/L) Kowb BCF fishc 0.027 f 7.3 not soluble f f 0.65 0.26 0.099 0.099 0.038 0.038 0.014

6.5 5.6 3.7 6.4 6.2 f 5.5 5.9 6.3 6.3 6.7 6.7 7.1

f 4.0 2.5 5.1 4.9 f 4.2 4.6 5.0 5.0 5.4 5.4 5.8

BMFd

TDIe (mg/Kg)

1.6 (1.9) f 0.9 (2.9) 1.6 (3.2) 2.0 (3.7) f 2.3 (2.8) 1.9 (3.0) 3.0 (5.1) 2.4 (4.7) 2.6 (6.0) 3.4 (7.3)

0.0001 f 0.008 0.02 f f 0.001 0.001 0.001 0.001 0.001 0.001 0.001

a From ref 35. b Mean values from data in ref 36. c Data for PCBs from ref 32, and data for OCs and derivatives from Kow according to ref 33. d BMF calculated in this paper for liver after 24 months of diet exposure (BMF values for visceral fat in brackets). e Tolerable diary intake from refs 35 and 22. f Not calculated or data not available.

Biomagnification. Table 4 shows the physicochemical characteristics and the ecotoxicological parameters for all the organochlorine compounds detected in the samples. The biomagnification factor (BMF) for liver should be representative as it is the main lipid storage organ in teleostei. However, the presence of visceral fat in experimental animals must be taken into account in this study. The fact that experimental fish were fed until satiation together with the sedentary conditions in experimental aquaria could have produced an excessive intake of food and the storage of lipids in visceral fat. This hypothesis is supported by the high lipid content found in liver of experimental fish (15-20%). Therefore, BMF calculated in fat are also shown in Table 4 (in brackets), as representative of lipid storage tissue. Figures 1 and 2 show the BMFs calculated for each compound in the different tissues investigated after 6 and 24 months of dietary exposure. In general, BMF values were higher than 1, indicating biomagnification of the organochlorines present in the diet, as it could be expected attending to their Kow values and the low biotransformation rate of OCs in living organisms. It can be seen that BMF values in the 6th month were higher than in the 24th month in white and red muscle as well as in visceral fat for most of the compounds studied, which could be associated with the period of time necessary to reach the steady state in each tissue. However, in liver this trend was inverted, probably as a consequence of the metabolic activity of this organ. The highest BMF corresponded to the PCB 180 congener, especially after the 6th month of exposure, with values between 7 and 20, which could be due to the high lipophilic character of the 7-chlorinated PCB congeners (Figure 2). Nevertheless, BMF of PCB-180 at the 24th month significantly decrease. This fact is possibly caused by the metabolic adaptation of organisms, leading to higher biotransformation rates. Moreover, BMFs in visceral fat were higher than in liver for all the compounds studied, suggesting a direct storage of nonpolar contaminants in visceral fat with low biotransformation rate. This is supported by the difference in BMF values for DDT in visceral fat (2.9) and in liver (0.9) (Table 4). Commercial Sea Bass Analysis. The analysis of commercial sea bass samples cultured in farms from the western Mediterranean area showed a similar contamination pattern than fish feed analyzed in this paper (Table 3). In general, organochlorine concentrations were lower than in specimens cultured in aquaria with similar weights (300-500 g). Moreover, experimental sea bass had visceral fat, while

FIGURE 1. Biomagnification factors for organochlorine pesticides and metabolites at the 6th and the 24th month of the experiment. commercial sea bass did not present appreciable amounts of fat in viscera. The absence of visceral fat in commercial samples could be interpreted in terms of lower food intake, in comparison with experimental fish. Additionally this lower fat presence in viscera would lead to a decrease in OCs concentrations. Thus, Table 3 shows, in general, lower OC levels in commercial fish than in experimental ones, except in red muscle where similar levels were found in both types of samples. This fact could be explained by a higher lipid content of this tissue. Commercial fish are cultured offshore in large cages and fed with measured amounts of food, while experimental samples were fed until satiation in small aquaria. This could be an explanation for the different OCs concentration levels found in commercial and experimental fish, supported by the higher lipid content found in experimental fish livers as compared to those of commercial sea bass livers (5% and up to 20%, respectively). Data Analysis. The results of the Principal Components Analysis (PCA) applied to the lipid based OCs concentrations in the tissues studied showed that the first and second components explained 75.3 and 11.1% of the variance contained in the data, respectively. Figure 3 shows the position of the different variables studied with respect to the two first Principal Components. As can be observed, fish feed contamination patterns calculated at 6 and 24 months of exposure are located close together with respect to Principal Component 1 (PC1), like the patterns of the tissues analyzed from experimental fish. These variables are separated along the PC1 from the position of culture sea bass tissues patterns. This is in agreement with the fact indicated above about the differences in culture conditions between commercial and experimental fish, inducing the presence of visceral fat in experimental fish. VOL. 37, NO. 15, 2003 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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hand, the proximity between visceral fat patterns suggests the direct storage of OCs in visceral fat with low biotransformation rates, as stated above.

Acknowledgments We thank Generalitat Valenciana (Conselleria de Cultura y Educacio´n, Project GV 00-019-2) for financial support of this study.

Literature Cited

FIGURE 2. Biomagnification factors for PCBs congeners at 6 and 24 months of the experiment.

FIGURE 3. Principal components plot. Variable codes: wm6wm24: white muscle contamination pattern at 6 or 24 months of diet exposure, respectively. rm6-rm24: id red muscle. liver6liver24: id liver. fat6-fat24: id visceral fat. feed 6-feed24. id fish feed (at 24 months it was calculated as pondered arithmetic mean from the three batch used). cult- from cultured fish. In relation to the Principal Component 2 (PC2), Figure 3 shows the separation of patterns determined at 6 and 24 months of dietary exposure. The highest difference is obtained for liver patterns, visceral fat patterns being located, however, close together. The distances between obtained for liver patterns at 6 and 24 months are perhaps the consequence of the high metabolic activity of this organ. On the other 3380

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Received for review November 15, 2002. Revised manuscript received May 16, 2003. Accepted May 20, 2003. ES020229+

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