ment 377 (2007) 36–44www.elsevier.com/locate/scitotenv

Science of the Total Environ

Evidence for higher biomagnification factors of lower chlorinatedPCBs in cultivated seabass

Paulo Antunes a,b,⁎, Odete Gil a, Maria Armanda Reis-Henriques b

a INIAP/IPIMAR – Instituto Nacional de Investigação Agrária e das Pescas, Av. Brasília, 1449-006 Lisboa, Portugalb ICBAS, Instituto de Ciências Biomédicas de Abel Salazar, Largo Professor Abel Salazar, 2, 4099-003 Porto, Portugal

Received 6 June 2006; received in revised form 25 January 2007; accepted 26 January 2007Available online 2 March 2007

Abstract

Thirty three PCB congeners were analyzed in three size classes of seabass (Dicentrarchus labrax), from a semi-intensive fishfarm, in food and in abiotic compartment. Water and suspended particulated matter (SPM) showed higher concentrations of lowerchlorinated PCBs, whereas fish tissues and food showed higher concentrations of CB153 and CB138. In order to examine theinfluence of different inputs to seabass contamination, bioaccumulation factors (BAF), biota-suspended particulate matterbioaccumulation factors (BSMAF) and biomagnification factors (BMF) were calculated. In general, the log BAF and log BSMAFshowed good correlations with log Kow, for congeners with log KowN6.1. In three fish size classes, PCB congeners with logKowb6.1 were found at concentrations higher than what was expected based on the BAF calculated for the more hydrophobiccongeners, suggesting a different contribution of sources. The application of a mass balance model for accumulation of somecongeners in seabass estimated concentrations in fish 3- to 38-fold higher than the measured values, probably by overestimatinggill absorption. The model was applied with other scenarios in order to maximize uptake from food, which may have a significantinfluence in accumulation, and not only from diet pellets. Its contribution to fish contamination for highly chlorinated PCBs mayreach a maximum of 54–64%, and for lower chlorinated PCBs its contribution is lower than 20%. In this work it was proved that toreduce fish contamination the quality of the water should be controlled.© 2007 Elsevier B.V. All rights reserved.

Keywords: PCB; Dicentrarchus labrax; Farmed fish; Bioaccumulation

1. Introduction

The presence of polychlorinated biphenyls (PCBs) inthe global ecosystem has been recognized since Riseb-rough et al. (1968), in particular in marine organisms(Jensen et al., 1969). PCB usage in Portugal was restricted

⁎ Corresponding author. INIAP/IPIMAR – Instituto Nacional deInvestigação Agrária e das Pescas, Departamento de ambiente aquático,Av.Brasília, 1449-006Lisboa,Portugal.Tel.:+351213027170; fax:+351213015948.

E-mail address: pantunes@ipimar.pt (P. Antunes).

0048-9697/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.scitotenv.2007.01.094

to close systems in 1976 (Decreto-Lei no. 378/76, 20May),and banned in all new industrial equipment in 1988(Decreto-Lei no. 221/88, 28 June).However, their chemicalstability, that made them so interesting for industry, alsoleads to the concept of global pollutant: “compounds thatpossess toxic properties, resist degradation, bioaccumulateand are transported through air, water and migratoryspecies, across international boundaries and deposited farfrom their place of release, where they accumulate interrestrial and aquatic ecosystems” (UNEP, 2001).

In recent years there has been a great concern aboutthe fish consumption risk for human health, in particularwith farmed fish (Hites et al., 2004). The levels of PCBs

37P. Antunes et al. / Science of the Total Environment 377 (2007) 36–44

in fish are reported in innumerable studies, includingwild and farmed seabass in Portugal (Antunes et al.,2001; Antunes and Gil, 2004). The most commonapproach to evaluate bioaccumulation is to compare thelevels in the organism with those in contaminationsources (Kucklick et al., 1996). The possible sources ofPCBs to fish are food, water and sediment (Moermondet al., 2004). The diet is recognized to be the majorsource of PCBs in farmed fish (Hites et al., 2004), theusual strategy used to reduce fish contamination is tocontrol the diet's quality. But if the diet has low levels ofcontamination, the influence of water contaminationmay overcome the influence of food. Seabass do notfeed on sediment, but the water usually has a highcontent of suspended particulated matter (SPM). SPM ina fish farm may be constituted by a considerableproportion of food and fish excretion; however, it couldbe useful to consider its influence as an independentsource of contaminants. For comparison of levels in theorganism and in the sources, three processes can bestudied: bioaccumulation — the process which causesan increased chemical concentration in an aquaticorganism compared to that in water, due to uptake byall exposure routes including dietary absorption,transport across respiratory surfaces and dermal absorp-tion (Mackay and Fraser, 2000); biomagnification —can be regarded as a special case of bioaccumulation inwhich the chemical concentration in the organismexceeds that in the organism's diet due to dietaryabsorption (Mackay and Fraser, 2000); and biota-SPMaccumulation — process of transference of contami-nants from SPM to the organism, either from gill passivediffusion or gut adsorption. The significance of thisprocess will be evaluated in this paper.

Octanol is the most commonly used lipid surrogate,and most bioconcentration measurements have beenshown to correlate well with octanol/water partitioncoefficients (Kow). Empirical models can be deducedcorrelating the bioaccumulation factors with log Kow.Despite that some studies produce similar correlations(Oliver and Niimi, 1985; Gil, 1997), other studies haveled to very diverse conclusions according to theenvironment and species behaviour. Studies of wildspecies showed a negative correlation of bioaccumula-tion factors to compounds with log Kowb6, that changedwith fish size, and a positive correlation when log Kow

was higher than 6 (Antunes et al., 2001).Exposure of organochlorines solely from one source

only occurs in laboratorial experiments. In nature, fishare always exposed to different sources of contaminants,and therefore the field results must be carefullydiscussed. Simple tools that allow us to evaluate the

various uptake and loss processes are the mass balancemodels (Mackay and Fraser, 2000). In general, thesemodels consider the organism as a single “box”, andquantify uptake from food, respiration and dermaldiffusion, and elimination through respiration, dermaldiffusion, egestion of feces and urine, metabolicconversion, reproductive losses, and growth dilution.These models require information about the chemicalsand the organism, in particular: respiration, feeding andgrowth rates, kinetics information on metabolisation anddiffusion, and environmental parameter (Mackay, 2001;Arnot and Gobas, 2004).

In the present study PCB concentrations in three sizeclasses of seabass and in the semi-intensive fish farmenvironment were analyzed. Accumulation factors werecalculated to compare behaviour of different congeners. Asimple model was used to evaluate the transference ofPCBs from food, water and SPM to cultivated seabass andto allow the aquaculture industry to manage contamina-tion risk. Selecting a controlled ecosystem also allows toapply a more complete model (mass balance model) andto calculate the relative importance of each source.

2. Materials and methods

2.1. Sampling

Sea bass (Dicentrarchus labrax) of three differentlength classes were collected in a semi-intensive fishfarm located in the south of Portugal, in 2003 (Table 1).Fish of each length class were collected in independenttanks, the two smaller classes from glass fibber tanks,with constant water circulation, and larger specimens in3-ha land tanks with water circulation controlled by thetides. Seabass were dissected and the muscle and liverfrozen for chemical analysis. Samples were analyzedindividually, or in pools of two individuals for thesmaller size class. All tanks had the same water supplyand fish fed with similar commercial formulation of dietpellets only distinct in the size. In each tank water wassampled. Samples were filtered through pre-washed(hexane) and pre-combusted (350 °C, 10 h) Gelman A/Efilters to separate dissolved and particulate fractions.The dissolved fraction was collected in glass flasks andanalyzed within 2 days. Filters were stored frozen andthen dried at 40 °C for analysis.

2.2. Materials

The following materials were used for sampleextraction and aliquot purification: n-hexane distilledin the laboratory, dichloromethane p.a. (Merck), sodium

Table 1Range of length (cm), and weight (g), and mean±standard deviation oflipid content (%) and total PCB (ng g−1, dry weight) concentration ofmuscle and liver of seabass and supplied diet pellets of the three lengthclasses

Size class I II III

n of samples 7 a 12 6Fish length (cm) 10.1–11.5 13.1–15.4 26.2–29.3Fish fresh weight (g) 11.9–18.2 27.3–43.5 219.2–308.0Liver Lipid content

(%)59.3±7.7 60.4±9.2 60.4±7.0

tPCB (ng g−1) 151.2±35.0 156.9±31.3 117.5±19.9Muscle Lipid content

(%)14.2±2.5 9.3±2.7 10.3±1.4

tPCB (ng g−1) 31.0±5.2 31.3±10.1 24.8±7.8Diet pellets Lipid content

(%)17.4±4.1 17.4±4.1 17.4±4.1

tPCB (ng g−1) 22.0±4.7 22.0±4.7 22.0±4.7a Composed samples with 2 individuals.

38 P. Antunes et al. / Science of the Total Environment 377 (2007) 36–44

sulphate – anhydrous (Merck), heated at 440 °Covernight, Florisil – 60–100 mesh (Merck), activatedat 440 °C overnight, and partially deactivated with 1%distilled water, and sulphuric acid p.a. (Merck).

PCBs standard was obtained as a certified solutionfrom AccuStandard Inc. (Q-CME-01) containing 40individual congeners.

2.3. Analysis

Analyses were performed according to Antunes andGil (2004). Diet pellets and fish tissues were extractedwith n-hexane for 6 h. SPM samples were Soxhletextracted for 16 h, and 2 L of water was liquid–liquidextracted with n-hexane also. Fat content was deter-mined gravimetrically from aliquots of tissue extracts.The remaining portion of the extracts was purified in aFlorisil column and further with sulphuric acid. After

Table 2Quantified congeners (n) and average concentrations in water (ng L−1) and SP

No. Cl Dissolved

I II III

n Conc. (ng L−1) n Conc. (ng L−1) n Conc. (ng L−

Tri 5 1.7 5 1.5 5 1.3Tetra 4 1.4 4 0.89 4 1.0Penta 6 2.0 6 1.1 6 0.81Hexa 5 0.54 4 0.46 5 0.51Hepta 2 0.12 1 0.07 2 0.11Octa 0 nd 0 nd 0 ndNona 0 nd 0 nd 0 ndDeca 0 nd 0 nd 0 ndTotal PCB 5.8 4.0 3.8

nd — Not detected.

concentration each sample was injected into a HewlettPackard 5890 series II, equipped with a DB5 (J&WScientific) capillary Column (60 m, 0.25 mm I.D.,0.25 μm film thickness) and an electron capturedetector (ECD). A mixture of individual CBs was usedas external standard for quantification. CBs 30, 65 and204 were used as recovery standards. Detection limit,calculated as three times the standard deviation ofblanks, ranged from 0.01 to 0.04 ng g−1. Generallycompounds presented clear peaks. Seven of the PCBcongeners presented some analytical interferences(abnormally high or unresolved peaks) in a largenumber of samples, so they were not considered in thiswork.

2.4. Statistical analysis

One-way analysis of variance or Student's t-testwas used to compare concentrations (Zar, 1996). Anoverall statistical test was performed to test whetherregressions were identical and pairwise slope compar-isons were performed using the Student's t-test (Zar,1996). A 5% significance level was used for allstatistical tests.

3. Results and discussion

3.1. PCBs in water and SPM

Levels of PCB in water and SPM of the tanks arepresented in Table 2. The most abundant congeners inwater were CBs 33 (trichloro-), 52 and 49 (tetrachlor-obiphenyls), and congeners with more than 6 chlorinesare almost all below detection limit. SPM contained ahigher number of quantified congeners, with a highrelative abundance of the lower chlorinated CBs. The

M (ng g−1) grouped by chlorination degree, and total PCB, in each tank

SPM

I II III1) n Conc. (ng g−1) n Conc. (ng g−1) n Conc. (ng g−1)

5 4.3 5 2.4 5 2.43 2.0 3 1.4 3 1.57 3.7 5 1.5 5 1.26 4.6 5 2.2 4 1.45 1.8 2 0.63 2 0.351 0.13 0 nd 0 nd0 nd 0 nd 0 nd0 nd 0 nd 0 nd

16.5 8.0 6.9

39P. Antunes et al. / Science of the Total Environment 377 (2007) 36–44

most abundant were CBs 18 (trichloro-), 118 (penta-chloro-) and 149 (hexachlorobiphenyl), and the highestchlorinated CBs are still below detection limit in almostall samples.

In this semi-intensive fish farm all tanks have thesame water supply, so water and SPM did not showsignificant differences of concentrations between thetanks.

3.2. PCBs in sea bass

Lipid content and total PCB levels detected in muscleand liver of sea bass of the three length classes arepresented in Table 1. The total PCB concentrationsshowed higher levels in liver than in muscle (Table 1).Good correlations between lipid content and the PCBconcentrations were obtained for the three size classesand two tissues all together (r2 =0.850), this suggeststhat differences among tissues may be explained bylipids, which was also found in other marine organisms

Fig. 1. Concentrations of individual PCB congeners normalized for CB153, iAsterisks represent significant differences between the three size classes (AN

(Hebert and Keenleyside, 1995; Hop et al., 2002).However, total PCB in liver of Class III is significantlylower than the level in Class II, and this cannot beattributed to lipids, since the lipid content did not changein these samples. This lower level may be due todifferences in metabolic capacity of adult seabass or bythe presence of a large amount of visceral fat in adultswhich may produce a different distribution of PCBswithin the organism. No other significant differences inlipid content and total PCB levels were found amonglength classes (Table 1).

When examining individual congeners some differ-ences were observed. For the evaluation of thesedifferences, the concentrations were normalized forCB153. This congener was chosen for normalizationbecause it was the most abundant, with few variationsbetween samples and most likely had the smallermetabolic rate (Goerke and Weber, 1990). Fig. 1presents the normalized concentrations in muscle andliver. The highest congener contributions correspond to

n muscle of seabass of the three length classes (mean±standard error).OVA test, 95% confidence).

40 P. Antunes et al. / Science of the Total Environment 377 (2007) 36–44

CB153 followed by CB138, which agrees with thefindings of other studies in marine organisms (Loizeauet al., 2001). These two congeners are predominant alsoin diet pellets.

Despite the similarity in total PCB concentrations, inthe muscle the congener differences among the sizeclasses were recorded. The smaller individuals showedhigher concentrations of CBs with log Kowb6.1 (Fig. 1),which have higher contribution than in food. A prefer-ential accumulation of tri- and tetrachlorobiphenyls by thesmaller individuals was also observed in wild seabassfrom Ria de Aveiro (Antunes et al., 2001). Smaller fishhave higher contact with water so it is probable that theyuptake a higher amount of these compounds from waterthan larger fish. Some differences were also observed, thepenta- to heptachlorobiphenyls presented higher contri-butions in individuals of Class II. Liver presentedirregular differences in composition pattern. Short-termvariations of CB content, due to smaller mass of liver in

Fig. 2. Bioaccumulation factors (BAF), biota-suspended particulate matterversus log Kow for seabass of the three length classes. Kow from Hawker an

relation to muscle (3–8%) and the higher metabolism ofthis organ (Sarasquete et al., 2001), cause elevatedchanges in CB concentrations in the liver.

3.3. Bioaccumulation factors

An easy way to evaluate the influence of thedifferent sources of contaminants is by comparing thelevels in the organism with those in the sources. Forthat we calculated several accumulation factors:bioaccumulation factor (BAF), defined as the ratiobetween the compound concentration in fish's tissues(lipid basis) and in water; and the biota-suspendedparticulate matter accumulation factor (BSMAF), asthe ratio between concentration in fish's tissues (lipidbasis) and in SPM; and biomagnification factor (BMF),as the ratio between the concentration in fish's tissues(lipid basis) and in diet (lipid basis). The logarithms ofthese factors were plotted against log Kow (Fig. 2). For

accumulation factors (BSMAF) and biomagnification factors (BMF)d Connell (1988).

Table 3Parameters used in the FISH model simulation

Chemical properties

Compound CB18 CB52 CB101 CB138 CB153

Molecular mass (g/mol) a 257.5 292.0 326.3 360.9 360.9Log Kow

b 5.24 6.1 6.38 6.83 6.92Henry's law constant

(Pa m3/mol) a58.1 47.6 32.7 48.6 42.9

Metabolism half-life (h) c 10000 10000 10000 10000 10000

Environmental and fish properties

Seabass size class I II III

Fish volume (cm3) d 15 33 260Fish lipids (fraction) d 0.142 0.093 0.103Food lipids (fraction) d 0.15 0.15 0.15Feeding rate (%body volume) d 2 2 2Growth rate (fraction body volume/day) d 0.023 0.023 0.023Concentration of SPM (g/m3) d 33 33 33Organic carbon content (%) d 0.015 0.015 0.015a Campfens and Mackay (1997).b Hawker and Connell (1988).c Defined higher than 600 days, for negligible influence (Gobas,

1992).d Field measure.

41P. Antunes et al. / Science of the Total Environment 377 (2007) 36–44

both BAF and BSMAF similar values for compoundswith log Kowb6.1 were observed. The values increasewhen log Kow were greater than 6.1 which could be dueto the higher hydrophobic character of these congeners.In general, the BMF were higher than 1 and the values

Fig. 3. Estimated and measured data of log BAF (A), and estimated uptakeseabass of the three length classes. Assuming gut absorption only from diet

were not significantly different, except for compoundswith log Kowb6.1, which presented higher BMF.

The PCBs' accumulation pathway from SPM toorganism may be through their gills or gut. In order toasses which of these pathways have more influence therelationships between BAF and BSMAF and log Kow

were compared. Similar tendency was observed: constantvalues until log Kow=6.1, and linear growth afterward.The calculation of the fugacity (Campfens and Mackay,1997) confirmed that CBs in SPM, despite beingpresumably constituted by a considerable proportion offood, were equilibrated with CBs in water. Therefore, theaccumulation pathway from both SPM and water must beidentical. The higher biomagnification factors for lowerchlorinated PCBs and the differences between accumu-lation of congeners with log Kow lower than 6.1 andhigher than 6.1, suggest different contribution of sourcesof PCBs. Based on these results we may formulate twohypothesis: (a) uptake from water column, trough gills,has high influence in the total accumulation of PCBs withlog Kowb6.1; (b) uptake from SPM follows the samepathway as dissolved fraction.

3.4. Mass balance model

To test these hypotheses we applied a mass balancemodel to the system, assuming that each fish class is in asteady state. Our assumptions are the same as the modelFISH presented byClark et al. (1990) andMackay (2001),

through gills and gut (B) for congeners 18, 52, 101, 153 and 138, inpellets.

Fig. 4. Estimated and measured data of log BAF (A), and uptake through gills and gut (B) for congeners 18, 52, 101, 153 and 138, in seabass of thethree length classes. Assuming gut absorption only from diet pellets, and adjusting gill uptake resistance to fit measured values.

42 P. Antunes et al. / Science of the Total Environment 377 (2007) 36–44

and can be downloaded from http://www.trentu.ca/cemc.We evaluated, as example, five PCB congeners withdifferent Kow. In Table 3 are presented the compound andfish properties used to perform the simulation. Chemicalproperties were obtained from the literature (Table 3), andfish and environmental concentrations were measured inthis work. The metabolism half-lives were set as beinghigher than 600 days for all congeners such thatmetabolism is negligible (Gobas, 1992). The kineticfactors are unknown for the studied environment,therefore the parameters established by the authors of

Fig. 5. Estimated and measured data of log BAF (A), and uptake through gillthree length classes. Assuming gut absorption from diet pellets, and conside

the model, in their study area, were used. Results wereanalyzed assuming that the overall fish concentration issimilar to muscle concentration.

First approach was made assuming that fish do notingest a significant amount of SPM. We estimatedconcentrations, considering the gut uptake only from dietpellets. With these results we calculated the BAF, whichwere compared with measured data for seabass of thethree size classes (Fig. 3A). The estimated values are 3-to 38-fold higher than measured values, and seabass ofClass III presented higher deviation from the estimated

s and gut (B) for congeners 18, 52, 101, 153 and 138, in seabass of thering ingestion of SPM in equal amount as diet pellets.

43P. Antunes et al. / Science of the Total Environment 377 (2007) 36–44

results. This is probably due to the presence of higheramount of visceral fat that may affect gut absorption. Theassumption that fish concentration is similar to muscleconcentration may be not quite right in this weight classseabass. The mass balance model estimated also theuptake and elimination from the different pathwaysindependently (Fig. 3B) and verified that water is themajor source for PCBs uptake. Using these kineticparameters, water contribution is higher than 97% forCBs 18, 52 and 101 and 84–89% for CBs 153 and 138.These values are probably overestimated. Performing asensitivity analysis to evaluate the effect of the kineticparameter error in fish's final concentration, we verifiedthat, in this steady-state model, the only kineticparameter with significant influence in fish's concentra-tion is gill uptake resistance. Borga and Di Guardo(2005) had applied the same model in an arctic marineecosystem and obtained PCB concentrations in zoo-plankton and fish two orders of magnitude lower thanthose of the measured values. The water temperature inPortugal is generally higher than in the Great Lakes, andmuch higher than in the arctic. Increase of temperaturemust produce an increase of gill uptake resistance.

By adjusting gill uptake resistance in order to fit themeasured PCB concentrations, we obtained the value0.01 h (one order of magnitude higher than establishedby the model authors), the uptake from water will be82–94% for CBs 18, 52 and 101, and 36–46% for CBs153 and 138 (Fig. 4B). This approach minimises the gillabsorption but still indicates that water contaminationhad a higher contribution to PCBs accumulation than thefish's diet.

To verify if uptake from SPM follows the samepathway as dissolved fraction we tested another sce-nario, assuming that fish ingested equal amounts ofSPM and diet pellets. Estimated concentrations are 4- to84-fold higher than measured values (Fig. 5A). Theingestion of SPM does not change the gill uptake, butgut uptake increases and values will be very differentaccording to Kow. Uptake from water will vary from66% (CB18) to 8% (CB153) of total uptake (Fig. 5B).The hypothesis that the accumulation pathway of SPMcontaminants is similar to the dissolved fraction was notconfirmed. However, this increase in gut absorption isfrom SPM, fish food had a small contribution to PCBaccumulation.

4. Conclusions

Accumulation of PCBs in this farmed species dependsnot only on the different sources of contamination but alsoon interaction between added food and abiotic compart-

ments. SPM, including left over food, had equilibratedPCB concentrations with water. Considering ingestion ofSPM, food uptake will have a significant influence inaccumulation, as usually recognized (Moermond et al.,2004). But this accumulation is not only from diet pellets,they had the lowest relative contamination and itscontribution to fish contamination is low. Only for highlychlorinated PCBs it may contribute with a maximum of54–64%, for lower chlorinated PCBs its contribution islower than 20%. Congener's properties affect substan-tially the accumulation pathways. As the relativeimportance of the pathways changeswith chlorine contentof PCBs and the empirical models do not explain thedifferences, once they can reflect only one of theaccumulation mechanisms.

In this work it was proved that to reduce fish con-tamination the quality of the water should be controlledand if necessary improved.

Acknowledgements

The manuscript has benefited greatly from the reviewsof the anonymous referees. Paulo Antunes acknowledgesthe PhD fellowship from the Portuguese Foundation forScience and Technology.

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