Persistent organic pollutants in edible marine species from the Gulf of Naples, Southern Italy

Science of the Total Environment 343 (2005) 83 – 95 www.elsevier.com/locate/scitotenv

Persistent organic pollutants in edible marine species from the Gulf of Naples, Southern Italy Barbara Naso*, Daniele Perrone, Maria Carmela Ferrante, Marcella Bilancione, Antonia Lucisano Dipartimento di Patologia e Sanita` Animale, Universita` degli Studi di Napoli Federico II, Via Veterinaria 1, 80137 Napoli, Italy Received 22 May 2004; accepted 7 October 2004

Abstract Edible tissues from 10 marine species, collected from the Gulf of Naples in the Southern Tyrrhenian Sea (Italy) between February and July 2003, were analysed for the presence of organochlorine pesticides hexachlorobenzene (HCB) and DDTs ( p,pV-DDT, p,pV-DDE, and p,pV-DDD), and 20 polychlorinated biphenyls (PCBs). The PCB levels (calculated as the sum of all the determined congeners) were found to be the highest (from 56.8 to 47909.5 ng/g on lipid basis), followed by the DDTs (sum of p,pV-DDT and its metabolites; bdl–2095.5 ng/g) and HCB (bdl–165.4 ng/g). There were marked differences in residue levels of DDTs and PCBs among the various species under investigation (from Pb0.05 to Pb0.001). Since the presence of organochlorine pollutants was most evident in the strictly resident species which inhabit shallow coastal waters, contamination of the Gulf of Naples by these compounds probably derives from local agricultural, industrial, and municipal sources. Concentrations of DDTs and PCBs detected in this study were generally comparable or higher than those found in studies of similar species from other Mediterranean and non-Mediterranean regions subject to a high anthropogenic impact. From the human health point of view, the residue levels of HCB and DDTs detected in this study are well below the Maximum Residue Limits for some foods of animal origin (0.2 and 1 mg/kg fat weight for HCB and DDTs calculated as the sum of p,pV-DDT, p,pV-DDE, p,pV-DDD, and o,pV-DDT, respectively). However, the concentrations of PCBs (calculated as the sum of the seven btargetQ congeners IUPAC nos. 28, 52, 101, 118, 138, 153, and 180) detected in all the analysed samples far exceed the action limit of 200 ng/g fat weight recommended by the European Union for eggs, fresh pig meat, fresh poultry meat, and derived products. D 2004 Elsevier B.V. All rights reserved. Keywords: Organochlorine pesticides; Polychlorinated biphenyls; Edible marine species; Gulf of Naples; Campania region; Bioaccumulation

1. Introduction * Corresponding author. Dipartimento di Patologia e Sanita` Animale-Sezione di Tossicologia, Facolta` di Medicina Veterinaria, Via Veterinaria 1, 80137 Napoli, Italy. Tel.: +39 81 297820; fax: +39 81 297122. E-mail address: [email protected] (B. Naso). 0048-9697/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.scitotenv.2004.10.007

Organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) have been produced and used for agricultural and industrial purposes for a long time

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B. Naso et al. / Science of the Total Environment 343 (2005) 83–95

and on a large scale. Because of their high chemical stability, lipophilicity, and persistence, these chemicals tend to bioconcentrate and biomagnify in the food chains and persist in the environment for many years, representing a definite health hazard for both wildlife and humans. Long-term chronic exposure to OCPs and PCBs has been correlated with severe injury to the nervous, endocrine, reproductive, and immune systems in birds, fish, and mammals (Ratcliffe, 1967; Kelce et al., 1995; Skaare et al., 2000; Toft et al., 2003). Ingestion is the main source of human exposure to organochlorine pollutants, and, in particular, the consumption of seafood from contaminated areas (Schlummer et al., 1998; Alcock et al., 1998). Moreover, several studies have recently demonstrated a clear correlation between the frequency of fish consumption and the levels of organochlorines in human tissues, serum, and milk (Kiviranta et al., 2002; Fitzgerald et al., 2004). In Italy, the production and use of most of the OCPs was banned in the 1970s, whereas the use of PCBs has been strongly restricted since 1985. However, considerable quantities of both groups of chemicals have recently been detected in the Italian marine environment (Bayarri et al., 2001; Storelli and Marcotrigiano, 2001; Di Muccio et al., 2002; Stefanelli et al., 2002; Binelli and Provini, 2003; Stefanelli et al., 2004). The Gulf of Naples, off the coast of the Campania region (Southern Italy), is an area of vital environmental importance; many resident marine species live, feed, and die in this area, and many of the pelagic species reproduce there. Moreover, in 1997, the bPunta CampanellaQ Marine Reserve (1128 ha) was established within the study area to protect the wild fauna. However, the Gulf of Naples is subject to OCP and PCB pollution from the intensively cultivated areas, the highly populated urban centres, and the large industrial complexes clustered along the coast. The Gulf of Naples is also the outlet of one of the most, if not the most, contaminated rivers in Italy, the Sarno, which receives untreated effluent from a rich in agriculture, densely populated, and heavily industrialised inland area (1300–2000 inhabitants/km 2 ) (Ministero dell’Ambiente e della Tutela del Territorio, 2004). Despite the presence of all these possible contaminants, little is known about the accumulation of persistent organochlorine pollutants in marine species

from the Gulf of Naples apart from a recent study on birds from coastal areas of the Campania region which demonstrated undesiderably high mean levels of PCBs in the liver of two fish-eating bird species at the top of marine food chain, the yellow-legged herring gull (Larus cachinnans) and the blackheaded gull (Larus ridibundus) (about 72 and 99 Ag/g lipid weight, respectively) (Naso et al., 2003). To acquire further data on the state of contamination of the Gulf of Naples and to assess potential risks for fish consumers, this study investigated the residue levels of persistent organochlorine pollutants in some of its edible marine species. The marine species were selected on the following basis: previous use as bioindicators for chemical monitoring, wide distribution in the monitored area, easy sampling, wide commercial diffusion, and, hence, a reliable indication of human exposure to the investigated pollutants. The influence of biological factors, such as habitat, feeding habits, migration, and capacity to metabolise organochlorine pollutants, on the levels of contamination was also examined.

2. Materials and methods 2.1. Sampling During the period February–July 2003, 10 different edible marine species were caught by local professional fishermen from the Gulf of Naples off the coast of the Campania region (40852VN, 14816VE), Southern Italy (Fig. 1). After capture, care was taken to avoid any contamination of the specimens that were immediately packed in ice and sent to our laboratory, where their weight and length were recorded. Table 1 shows the biometric data and the main information about the habitat, the feeding, and the migratory habits of the species under investigation. The fish and cephalopods were dissected, and the edible parts were homogenized. For anchovy and red mullet, edible parts from 20 specimens were pooled together and considered as a single sample; the mussels were opened with stainless steel knives, and the soft tissues were removed, pooled together (60 specimens per pool), then homogenized. All the homogenized samples were stored at 20 8C until chemical analysis was performed.

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Fig. 1. Map of the Campania region.

2.2. Chemical analysis The concentrations of hexachlorobenzene (HCB), p,pV-DDT, p,pV-DDE, p,pV-DDD, and the 20 polychorinated biphenyls, IUPAC nos. 28, 52, 66, 74, 99, 101, 105, 118, 128, 138, 146, 153, 170, 177, 180, 183, 187, 196, 194, and 201 were determined for each sample. The abovementioned PCBs included the seven btargetQ congeners (IUPAC nos. 28, 52, 101, 118, 138, 153, and 180) recommended by the European Union as indicators of PCB contamination. The 13 remaining congeners were chosen because they are known to be markedly bioaccumulated in fish-eating birds from the Mediterranean Sea and the Atlantic Ocean (Renzoni et al., 1986; Walker, 2001). Moreover, these refractory congeners were the most frequently detected PCBs in the yellow-legged herring gulls and black-headed gulls from the Gulf of Naples (unpublished data from the Authors). The extraction of the samples, the separation of the analytes from the lipids, and the fractionation of the purified extract were carried out following the method described by

Di Muccio et al. (2002), with some modifications. Homogenized edible parts of marine species (approximately 3 g) were cold-extracted with petroleum ether/ acetone (1:1, v/v). The extract was passed through a glass tube packed with anhydrous sodium sulphate and then evaporated to dryness by rotavapor. The extracted lipid content was determined gravimetrically. Extrelut glass columns prepacked with widepore kieselguhr (Merck, Darmstadt, Germany) were used as a solid support to carry out the liquid–liquid partition of OC residues from the lipidic material. In particular, the dried extract was first resuspended in nhexane and then it was transferred into an ExtrelutNT3 column, and the solvent was removed by a nitrogen stream. An Extrelut-NT1 column was emptied until 1 cm of the Extrelut material remained; then, C18 material (0.36 g) was added, followed by 1.5 cm of Extrelut material. This column was positioned under the Extrelut-NT3, and the combined column system was eluted with acetonitrile. The C18 material was used since it is able to substantially reduce the carry over of lipid material into the acetonitrile eluate.

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Table 1 Biometric data (mean and, in parenthesis, range) of the analyzed specimens and few ecological notes about them Species

Number of samples

Weight (g)

Length (cm)

Habitat

Feeding habits

Migratory habits

Blue mussel (Mytilus galloprovincialis) Musky octopus (Eledone moschata) Common octopus (Octopus vulgaris)

8 (60)a

17b (13–24) 140 (120–150) 212 (160–270)

5c (4–6)

Benthic species: coastal rocks in surface waters (0–2 m depth) Benthic species: muddy and slimy bottoms (15–90 m depth) Benthic, neritic species: from the coastline to outer edge of the continental shelf (few to 200 m depth) Demersal, neritic species: sandy or muddy bottoms of the continental shelf (few to 200 m depth) Pelagic and euryhaline species (few-150 m depth)

Water filtering organisms: plankton and suspended organic matter Bivalve molluscs and crustaceans

Sessile species

Mainly meso- and macrozooplankton

6 7

31d (29–33) 38d (33–42)

Bivalves, crustaceans, and organic remains

12

164 (110–225)

21d (16–25)

Anchovy (Engraulis encrasicholus)

6 (20)a

17 (6–20)

10 (9–11)

Mackerel (Scomber scombrus)

10

99 (56–150)

22 (18–28)

Pelagic species (few to 200 m depth)

Zooplankton, zoobenthos and nekton

Red mullet (Mullus barbatus)

11 (20)a

35 (21–40)

13 (11–14)

Benthic invertebrates (crustaceans, worms, and molluscs)

European hake (Merluccius merluccius)

13

162 (110–280)

28 (24–37)

Grey mullet (Mugil cephalus)

6

327 (110–600)

30 (22–38)

Sea bass (Dicentrarchus labrax)

7

212 (180–270)

21 (13–25)

Benthic species: gravely and muddy bottoms of the continental shelf (5–300 m depth) Demersal, neritic species: sandy and muddy bottoms of the continental shelf (usually 70–370 m depth) Benthic and euryhaline species: sandy and muddy bottoms close to shore (few to 120 m depth), harbors Benthic and euryhaline species: muddy and slimy bottoms close to shore (few to 100 m depth), harbors

a b c d

Number of specimens which constituted the pooled samples. Shell and soft tissue weight. Shell length. Total length (mantle and tentacles).

Small molluscs, crustaceans, other cuttlefishes and juvenile demersal fishes

Small fish (small hakes, anchovies, sardines, and gadoid species) and crustaceans Zooplankton, benthic organisms, detritus, diatom algae, and invertebrates Top predator fish: suprabenthic species (fishes, crustaceans and molluscs)

Limited seasonal vertical migrations: deeper waters in winter and shallower waters in spring and summer Limited seasonal vertical migrations: deeper waters in winter and shallower waters in spring Long-range horizontal and vertical migrations: deep waters in winter, and coastal and surface waters in summer Long-range horizontal and vertical migrations: deep waters in winter, and coastal and surface waters in March–April Strictly resident species

Limited seasonal vertical migrations: deep water in winter and more inshore in summer Resident species: often enters estuaries and rivers

Resident species: often enters estuaries

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Cuttlefish (Sepia officinalis)

Strictly resident species

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The eluate was evaporated, and then the dry residue was dissolved in n-hexane and transferred to a glass column packed with activated Florisil. The column was eluted with n-hexane and n-hexane/toluene (80:20, v/v). The first fraction contained PCBs, HCB, and p,pV-DDE; the second fraction contained p,pV-DDD and p,pV-DDT. Each fraction was concentrated to a small volume, and PCB 209 was added as an internal standard. Gas chromatographic analysis of the OCPs and PCBs was carried out by a Carlo Erba HRGC 5160 Mega Series equipped with a 63Ni electron capture detector (ECD). The cold on-column mode was used for the injection. A 25 m0.32 mm id, 0.25-Am film thickness CP-SIL 5 CB fused silica capillary column coated with a 100% dimethyl polysiloxane stationary phase (Chrompack, Middelburg, The Netherlands) was used to separate and quantify the residues. All the samples were also analysed on a confirmation column of different polarity from the quantitation column, a 30 m0.32 mm id, 0.25-Am film thickness Rtx-1701 coated with a 14% cyanopropylphenyl/86% dimethyl polysiloxane stationary phase (Restek, Bellefonte, PA), to verify chemical identity. The ECD was kept at 310 8C. Hydrogen and nitrogen were used as carrier gas and make-up gas, respectively. Organochlorines were identified by comparing the retention times on the two columns with those of the standards and then quantified by comparing the individually resolved peak areas with those of the corresponding standards. The concentrations of resolved peak of each of the 20 individual PCB congeners were summed to obtain the congener sum PCB levels. DDTs were calculated as the sum of p,pV-DDT, p,pV-DDE, and p,pV-DDD. The method detection limits for OCPs and PCBs ranged from 0.1 to 0.5 ng/g. Pure reference standard solutions were used for instrument calibration, recovery determination, and quantification (Dr. Ehrenstorfer laboratory). All the solvents were pesticide residue analysis grade, purchased from Pestiscan (Labscan, Dublin, Ireland). Fish tissue blanks were extracted and analysed to check for cross-contamination. In addition, aliquots of the fish tissue blanks, spiked with standard mixtures at three concentration levels, were extracted and analysed in triplicate to evaluate the recovery. The recovery rate of the organochlorines was between 80% and 110%. Quality assurance for the measure-

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ment of OCPs and PCBs was confirmed by analyzing standard reference materials (SRM). Organochlorine concentrations, not corrected for recovery, are expressed as nanogram per gram on a lipid weight basis to avoid intraspecies and interspecies variability due to differences in lipid content and to allow a more accurate comparison of bioaccumulation in the different marine species. 2.3. Statistical analysis Statistical analysis was carried out on the lipidnormalized organochlorine concentrations. OCP and PCB concentrations were log 10 -transformed to approximate a normal distribution of the data. Organochlorine levels below the detection limit were replaced with half the value of the respective detection limit. Differences in concentrations of organochlorines among the individual marine species were evaluated by one-way analysis of variance (ANOVA). When significant differences were observed among the species, the Tukey–Kramer Multiple Comparisons Test was applied to the ANOVA to determine which means were significantly different. All analyses and calculations were performed by GraphPad Software (GraphPad InStat).

3. Results and discussion 3.1. Contamination levels and sources The concentrations of OCPs and PCBs in the edible parts of marine species from the Gulf of Naples are shown in Table 2. With the sole exception of the musky octopus, in which the mean concentration of HCB was higher than that of the DDTs, the levels of organochlorines were as follows: PCBsNDDTsNHCB. Despite the considerable length of time that has passed since 1978 when legal restrictions were introduced for the use of DDT in Italy, its main metabolite p,pV- DDE was the most frequently detected OCP in the marine species investigated (81% of the samples) and was present in much higher concentrations than the other OCPs (ranging from bdl to 11426.3 ng/g). p,pV-DDD was found in 25% of the samples from blue mussel, red mullet, European hake, grey mullet, and sea bass at levels ranging from bdl to 669.2 ng/g; p,pV-DDT was

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Fat (%) HCB p,pT-DDT p,pT-DDE p,pT-DDD DDTs

Blue mussel (n=8)a

Musky octopus (n=6)

Common octopus (n=7)

Cuttlefish (n=12)

Anchovy (n=6)a

Mackerel (n=10)

Red mullet (n=11)b

European hake (n=13)

Grey mullet (n=6)

Sea bass (n=7)

1.00F0.48 12.2 (bdl–25.7) 76.1 (bdl–204.4) 91.2 (32.1–125.6) 9.9 (bdl–43.4) 177.2 (32.1–308.8)

1.29F0.23 70.0 (bdl–165.4) ndd

0.38F0.22 27.2 (bdl–119.5) nd

1.09F0.19 10.4c (bdl–34.9) nd

1.25F0.41 13.7 (bdl–49.8) nd

4.47F5.79 15.6 (bdl–63.4) nd

39.8 (bdl–152.5) nd

9.1 (bdl–50.8) nd

223.8 (135.3–311.1) nd

180.9 (bdl–532.2) nd

43.8e (bdl–80.7)

39.8f (bdl–152.5)

9.1g (bdl–50.8)

223.8 (135.3–311.1)

180.9h (bdl–532.2)

0.52F0.24 29.7 (bdl–93.1) 124.1 (bdl–406.8) 487.3 (188.5–1163.6) 33.7 (bdl–198.6) 645.1 (188.5–1769.0)

1.30F0.90 11.0 (bdl–32.6) nd

43.8 (bdl–80.7) nd

1.96F0.80 28.0 (bdl–65.8) 33.6 (bdl–125.6) 165.9 (145.4–217.8) 21.0 (bdl–54.6) 220.6 (153.0–318.0)

1.42F0.89 46.7 (16.8–72.1) 36.6 (bdl–156.9) 733.6 (274.4–1257.5) 201.2 (bdl–334.2) 971.3 (469.3–1257.5)

10.8 (bdl–32.4) 5.0 (bdl–15.2) nd

nd

nd

nd

nd

nd

nd

nd

nd

14.7 (bdl–26.8) nd

nd

nd

nd

nd

nd

5.9 (bdl–36.8) 26.8 (bdl–123.6) nd

nd

nd

nd

nd

nd

6.7 (bdl–20.1) 4.2 (bdl–12.5) 28.6 (bdl–52.7) 4.5 (bdl–13.3) 115.9 (bdl–217.0) 75.4 (bdl–133.2) 15.4 (bdl–26.4)

356.7 (bdl–900.0) nd

134.2 (bdl–347.1) nd

238.8 (bdl–456.2) nd

15.0 (bdl–133.3) nd

813.1 (230.6–1769.6) 51.2 (bdl–213.0) 2196.3 (659.7–4797.8) 1524.0 (486.0–3543.5) 297.2 (90.1–591.3)

305.4 (31.2–640.0) 95.9 (bdl–229.6) 2334.8 (147.4–4351.4) 395.1 (27.9–1004.2) 212.3 (17.8–478.7)

573.2 (bdl–1142.7) 59.4 (bdl–321.8) 1216.0 (329.3–2442.7) 1124.0 (312.0–2250.4) 170.4 (86.7–294.4)

114.5 (bdl–593.1) nd

8.4 (bdl–47.8) 13.8 (bdl–42.6) 36.2 (bdl–110.8) 165.3 (bdl–212.8) 18.1 (bdl–66.0) 400.8 (311.8–509.1) 299.1 (210.4–503.7) 71.2 (bdl–141.4)

78.1 (bdl–473.6) 281.4 (bdl–1629.4) 86.9 (bdl–306.8) 762.6 (bdl–1923.4) 73.4 (bdl–347.8) 1527.3 (391.1–4780.4) 1304.3 (281.9–4397.2) 251.2 (bdl–918.6)

23.0 (bdl–84.0) 159.9 (bdl–495.5) 22.3 (bdl–120.5) nd

PCB congeners 28 9.4 (bdl–44.8) 52 24.7 (bdl–42.2) 74 4.2 (bdl–12.6) 66 91,7 (bdl–139.2) 101 139.2 (bdl–233.0) 99 86.5 (bdl–123.2) 118 293.1 (135.8–403.6) 105/146 24.0 (bdl–45.4) 153 556.2 (244.1–818.1) 138 430.4 (192.1–655.8) 128/187 94.6 (48.7–121.2)

396.5 (27.5–1445.6) 273.2 (bdl–1178.2) 62.6 (bdl–305.8)

438.9 (bdl–1426.3) 264.5 (bdl–669.2) 703.3 (bdl–2095.5)

1248.8 (205.5–3909.0) 257.8 (bdl–896.9) 2087.9 (265.0–6448.1) 229.1 (bdl–830.1) 4067.6 (568.5–11705.3) 3729.1 (511.9–11899.3) 589.2 (bdl–1634.6)

97.7 (bdl–166.6) 126.0 (bdl–319.8) 50.3 (bdl–209.6) 508.0 (bdl–1245.0) 1398.7 (207.3–2177.3) 243.2 (bdl–599.2) 2783.8 (493.7–5200.8) 494.9 (69.3–1371.0) 5504.2 (799.3–8880.7) 5028.6 (561.5–8596.9) 784.7 (bdl–1852.8)

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Table 2 Organochlorine concentrations (mean and range, nanogram per gram lipid weight) in edible parts of marine species from the Gulf of Naples, Southern Italy

183

196

nd

194

nd

10.5 (bdl–21.1) 3.3 (bdl–9.8) 76.4 (63.1–102.5) 24.1 (bdl–43.6) 11.7 (bdl–35.2) 14.5 (bdl–32.1) nd

1519.0 (694.3–2186.2) 1928.2j (787.5–2679.0)

318.9 (63.1–572.9) 407.0k (130.4–726.2)

177 180 170

Target PCBs i

PCBs a

81.1 (bdl–221.2) 47.3 (bdl–178.5) 791.3 (238.1–1372.7) 232.6 (bdl–513.0) 53.4 (bdl–130.3) 55.6 (bdl–142.4) 72.5 (bdl–239.4) 5681.5 (1139.2–11445.6) 6572.6 (1541.7–12550.0)

194.5 (12.6–435.2) nd 881.6 (63.7–1536.3) 30.9 (bdl–90.9) 52.9 (bdl–98.5) 55.8 (bdl–110.9) 57.4 (bdl–113.4) 4051.1 (295.6–7707.1) 4750.9 (326.0–8804.3)

93.1 (bdl–162.0) 107.1 (33.0–719.9) 445.3 (253.6–749.4) 159.0 (72.5–273.0) 46.2 (24.3–51.1) 55.7 (29.0–68.1) 49.0 (4.2–54.5) 2834.4 (1141.6–6827.7) 3666.7 (1584.7–7907.4)

Pool of 60 specimens.

b

Pool of 20 specimens.

c

Pb0.05 vs. sea bass.

d

Not detected.

e

Pb0.05 vs. European hake, Pb0.01 vs. sea bass.

f

Pb0.05 vs. blue mussel, Pb0.01 vs. anchovy and grey mullet, Pb0.001 vs. red mullet, European hake and sea bass.

g

Pb0.01 vs. mackerel, Pb0.001 vs. blue mussel, anchovy, red mullet, European hake, grey mullet and sea bass.

h

Pb0.05 vs. European hake, Pb0.01 vs. sea bass.

i

Sum of all the determined congeners.

j

Pb0.01 vs. sea bass.

k

l m n

Pb0.01 vs. common octopus, cuttlefish and European hake, Pb0.001 vs. grey mullet and sea bass. Pb0.05 vs. anchovy, Pb0.01 vs. common octopus, Pb0.001 vs. cuttlefish, European hake, grey mullet and sea bass. Pb0.01 vs. red mullet. Pb0.05 vs. anchovy, Pb0.001 red mullet.

22.5 (bdl–185.1) 24.0 (bdl–148.3) 206.1 (791.9) 87.2 (bdl–326.4) 18.7 (bdl–113.8) 19.8 (bdl–120.7) 14.1 (bdl–110.3) 1005.3 (56.8–4009.2) 1254.1l (56.8–5319.5)

26.4 (bdl–63.7) 32.9 (bdl–51.9) 226.1 (132.3–339.4) 53.3 (bdl–92.2) 19.8 (bdl–57.1) 19.2 (bdl–65.5) 15.6 (bdl–57.1) 1119.8 (865.6–1565.0) 1421.0 (1024.2–2270.7)

139.4 (bdl–591.2) 94.7 (bdl–452.4) 569.7 (253.8–1155.2) 238.4 (90.1–780.4) 35.8 (bdl–101.6) 56.2 (bdl–161.8) 40.3 (bdl–100.7) 4478.0 (1109.3–15387.0) 5572.4 (1293.4–19489.8)

451.0 (bdl–1272.2) 428.8 (51.2–1310.5) 1922.8 (269.7–3991.9) 648.4 (26.7–1993.2) 246.3 (33.7–603.2) 290.5 (bdl–730.9) 112.9 (bdl–276.4) 13239.1 (1921.9–39406.5) 16515.2m (3134.6–47909.5)

525.2 (bdl–1496.9) 523.5 (bdl–1327.3) 2319.9 (325.3–5954.3) 1014.5 (105.0–3120.4) 300.6 (bdl–1040.9) 286.7 (bdl–1124.1) 296.2 (38.4–1006.8) 17259.0 (2387.1–31273.8) 22286.6n (3022.6–45735.6)

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201

41.9 (bdl–65.9) 50.2 (bdl–84.8) 65.9 (35.2–122.3) 16.4 (8.4–44.5) nd

89

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only found in blue mussel, red mullet, European hake, and sea bass (22% of the samples) at concentrations between bdl and 406.8 ng/g. Thus, p,pV-DDE was the principal form of DDT in all the species studied, constituting from 51.5% to 100% of the DDTs (Table 2). These findings are not surprising considering the high chemical stability and hydrophobicity of p,pVDDE (log K ow value of 6.36) and its long half-life and, hence, persistence in both abiotic and biotic components of the aquatic ecosystems (Walker, 2001). The DDE/DDT ratio is commonly used to assess the chronology of DDT input into the ecosystems (Bordajandi et al., 2003). The absence of DDT in cephalopods, anchovy, mackerel, and grey mullet and the DDE/DDT ratio greater than 1 in the other species would suggest that there has been no recent input of technical DDT from the Campania region into the Gulf of Naples. The exposure of the marine species to DDTs is likely due to the large amount of DDT that was used in the coastal areas of the Campania region previous to the ban, as well as to atmospheric input from other contaminated areas. HCB was found in 69% of the samples at relatively low concentrations ranging between bdl and 165.4 ng/g. The present finding of HCB in marine organisms from the Gulf of Naples may be ascribed not only to its previous use as a fungicide treatment for seeds but also to the fact that it is a by-product in the manufacturing processes of various chlorine-containing chemicals and an impurity in several pesticides (Bailey, 2001). PCBs were found in all the marine species (100% of the samples) at concentrations ranging between 56.8 and 47909.5 ng/g (calculated as the sum of all the investigated congeners). The sum of the seven btargetQ congeners constituted from 77.3% to 86.4% of the PCBs (Table 2). Although there were differences among the marine species, PCB patterns were always dominated by a large contribution from the hepta-, hexa-, and pentachlorinated PCBs 180, 153, 138, and 118, which collectively accounted for from 69.8% to 91.6% of the PCBs. PCB 153 was the dominant congener in all the species (Table 2). The aforementioned congeners are the most abundant in commercial PCB mixtures, such as Aroclor 1254 and 1260, which are the most common used in European countries. The 180, 153, 138, and 118 congeners turned out to be the most abundant also due both to their high lipophilicity, stability, and persistence that

facilitate the adsorption to sediments and the accumulation in the aquatic ecosystem, and to their molecular structure. PCBs 180, 153, 138, and 118 tend to be refractory to metabolic attack by monooxygenases and, consequently, to be more slowly eliminated because of their high degree of chlorination and the lack of adjacent unsubstituted H-atoms in ortho–meta and/or meta–para positions on the aromatic ring (Walker, 2001). The tri- and tetrachlorobiphenyls (PCB 28, 52, 74, and 66) and octachlorobiphenyls (PCB 201, 196, and 194), when present, were detected at noticeably lower concentrations than the hepta-, hexa-, and pentachlorinated congeners. The less chlorinated homologues have a lower logK ow and, as a consequence, they have a lower propensity to leave the aqueous environment for organic compartments. Moreover, once inside the organisms, they are usually more rapidly metabolised than the higher chlorinated congeners because of the presence of more unsubstituted ring positions on their biphenyl rings available for the metabolic attack. As far as the octachlorobiphenyls are concerned, they have a high logK ow (from 7.62 to 7.80), and, therefore, they are readily adsorbed to sediments, and only a small fraction remains in the dissolved form. These homologues are therefore less available for passive bioconcentration process (Gray, 2002). The organochlorine pollution of the Gulf of Naples ecosystem is attributable to the many sources of agricultural, municipal, and industrial contamination in the Campania region. In particular, these chemicals mainly arrive in the Gulf of Naples as a consequence of evaporation, atmospheric fallout, surface run-off, and wastewater discharges from the intensively cultivated areas, the densely populated urban centres, the large industrial complexes, and the many waste dumps clustered along the coast. The totally untreated agrochemical, industrial, and domestic effluents drained by the river Sarno into the sea (Fig. 1) also play an important role in the organochlorine contamination of the aquatic environment. This hypothesis is confirmed by the presence of the highest concentrations of organochlorine pollutants in the sea bass and the grey mullet, two strictly resident and benthic species, which inhabit nearshore marine areas (Tables 1 and 2). In Table 3, the concentrations of DDTs and PCBs detected in the edible parts of marine organisms from

Table 3 Mean concentrations of DDTs and PCBs (nanogram per gram lipid weight) from this study and in edible parts of similar species from other Mediterranean and non-Mediterranean regions Species

Fat %

DDTs

PCBs

References

Gulf of Naples, Italy Adriatic Sea, Italy Adriatic Sea, Italy Ariake Sea, Japan Philippines Pearl River Estuary, China Norvegian coasts Gulf of Naples, Italy Embro Delta, Spain Adriatic Sea, Italy Adriatic Sea, Italy Gulf of Naples, Italy Adriatic Sea, Italy Adriatic Sea, Italy Gulf of Naples, Italy Adriatic Sea, Italy Gulf of Naples, Italy Embro Delta, Spain Gulf of Naples, Italy Embro Delta, Spain Ria de Aveiro, Portugal

Blue mussel (Mytilus galloprovincialis) Blue mussel (Mytilus galloprovincialis) Blue mussel (Mytilus galloprovincialis) Mussel Green mussel (Perna viridis) Green mussel (Perna viridis) Blue mussel (Mytilus edulis) Red mullet (Mullus barbatus) Red mullet (Mullus barbatus) Red mullet (Mullus barbatus) Red mullet (Mullus barbatus) Anchovy (Engraulis encrasicholus) Anchovy (Engraulis encrasicholus) Anchovy (Engraulis encrasicholus) European hake (Merluccius merluccius) European hake (Merluccius merluccius) Grey mullet (Mugil cephalus) Grey mullet (Mugil cephalus) Sea bass (Dicentrarchus labrax) Sea bass (Dicentrarchus labrax) Sea bass (Dicentrarchus labrax)

1.00F0.23 1.4; 1.4; 1.6a 1.16; 0.90; 1.51a 0.6 na na na 1.96F0.80 3.3F1.2 4.3; 4.4; 4.9a 3.36; 3.52; 3.53a 1.25F0.41 1.8; 3.2; 3.3a 1.27; 2.32; 3.53a 0.52F0.24 na; 1.02; 1.07a 1.30F0.90 1.9F0.5 1.42F0.89 0.8F0.2 5.0

177.2 (32.1–308.8) 1.6; 1.9; 3.0b,c,d 37.8; 46.5; 141.7c nae 69 (4–200) 51.4 20.9F10.1 220.6 (153.0–318.0) 542F337 9.8; 8.1; 8.5b, c, d 228.0; 95.5; 157.1c 223.8 (135.3–311.1) 6.4; 8.6; 11.9b, c, d 967.7; 417.2; 317.6c 645.1 (188.5–1769.0) na; 281.0; 399.1c 703.3 (bdl–2095.5) 388F107 971.3 (469.3–1257.5) 513F97 108F43

1928.2 (787.5–2679.0) 5.9; 1.3; 18.5c,d 245.6; 465.4; 955.6c 590.0 660 (22–2100) 615.1 79.5F59.3f 1421.0 (1024.2–2270.7) 20.3F9.0f 19.8; 22.3; 43.4c, d 369.2; 414.3; 863.4c 3666.7 (1584.7–7907.4) 16.4; 19.8; 62.7c, d 1045.2; 620.6; 1105.0c 5572.4 (1293.4–19489.8) na; 2091.2; 2047.9c 13239.1 (1921.9–39406.5)f 109F43f 17259.0 (2387.1–31273.8)f 800F50f 275F84

Present study Bayarri et al., 2001 Stefanelli et al., 2004 Nakata et al., 2002 Monirith et al., 2003 Fu et al., 2003 Green and Knutzen, 2003 Present study Pastor et al., 1996 Bayarri et al., 2001 Stefanelli et al., 2004 Present study Bayarri et al., 2001 Stefanelli et al., 2004 Present study Stefanelli et al., 2004 Present study Pastor et al., 1996 Present study Pastor et al., 1996 Antunes and Gil, 2004

a b c d e f

Lipid content (%) in samples from Southern, Central and Northern Adriatic Sea, respectively. p,pV-DDE. Mean concentrations in samples from Southern, Central, and Northern Adriatic Sea, respectively. Nanogram per gram wet weight. No data available. Sum of the seven target congeners IUPAC nos. 28, 52, 101, 118, 138, 153, and 180.

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Location

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the Gulf of Naples are reviewed and compared with those found in similar species from other Mediterranean and non-Mediterranean regions. It can be seen that the mean levels of DDTs detected in specimens from the Gulf of Naples are generally comparable to those reported for specimens from other Mediterranean areas (Pastor et al., 1996; Bayarri et al., 2001; Stefanelli et al., 2004). The only exception is sea bass from Ria de Aveiro, Portugal (Antunes and Gil, 2004), in which concentrations of DDTs were substantially lower than those detected in sea bass from the Gulf of Naples. Otherwise, the PCB levels found in this study are generally higher than those reported from the other Mediterranean regions. Lastly, levels of DDTs and PCBs found in mussels from the Gulf of Naples are both considerably higher than those recently reported for similar species from the coastal areas of Norway, Japan, China and the Philippines, subject to high anthropogenic impact (Nakata et al., 2002; Green and Knutzen, 2003; Fu et al., 2003; Monirith et al., 2003). 3.2. Interspecies differences in organochlorine accumulation The concentrations of organochlorines in gillbreathing aquatic organisms are governed by both bioconcentration and biomagnification (Hamelink, 1977; Randall et al., 1998; Gray, 2002). Direct uptake from water or sediments by passive diffusion through body surfaces is the major route of organochlorine intake for small organisms at lower trophic levels (e.g., plankton, bivalves and crustaceans) and is positively correlated with the logK ow of the chemicals and the amounts of lipids within the organisms (McKay and Fraser, 2000; Gray, 2002). As the body mass and the trophic level in the food chain of marine organisms increase, the biomagnification process plays an increasing role in the accumulation of organic pollutants, especially of the more hydrophobic ones (Loizeau et al., 2001; Binelli and Provini, 2003). In this study, there were significant differences in the residue levels of organochlorine pollutants among the investigated species (from Pb0.05 to Pb0.001), and the PCB congener profiles varied in the different species (Table 2). These differences may be attributable to various abiotic and biotic factors, such as the degree of chlorination and logK ow of the chemicals, as

well as recent habitat and feeding habits of the specimens. Variations in migratory patterns and in the metabolic capacity to detoxify may also have an effect on the magnitude and the pattern of accumulated organochlorines in the investigated species (Ashley et al., 2003). The highest levels of both DDTs and PCBs were found in sea bass and grey mullet (Table 2). This probably reflects the nature of the habitat of these benthic and euryhaline species which usually inhabit shallow waters with sandy or muddy bottoms along the coast, and ports and estuaries, which are generally considered to be more heavily polluted than open waters (Lewis et al., 2002). Therefore, sea bass and grey mullet probably receive large quantities of organochlorine pollutants present in the water and in the sediments through a process of bioconcentration. However, the high contamination levels detected in these species may also be ascribed to their feeding habits; sea bass is a top predator, and hence the dietary uptake is an important route of entry for organochlorines in this species, especially the higher chlorinated PCB homologues (Loizeau et al., 2001). Grey mullet is an omnivorous and detritivorous scavenger feeding mainly on organic matter by means of the ingestion of sediments, as well as plankton, benthic organisms, and diatom algae (Moreira et al., 1992). As previously mentioned, organochlorines tend to be adsorbed to the organic fraction of the sediments, which consequently act as a final reservoir and source of these pollutants in the aquatic systems (Nakata et al., 2002). Therefore, in spite of its low trophic position in the food chain, grey mullet acquires high concentrations of organochlorines, especially compounds with high logK ow values, by ingesting sediments. The strong interaction with sea bed and its sediments may also have an important influence on the degree of contamination detected in the other benthic and neritic fish and cephalopods studied (Tables 1 and 2). Anchovy and mackerel generally had lower organochlorine levels compared with the neritic fish species (Table 2). In particular, the levels of DDTs found in mackerel were significantly lower than those detected in European hake and sea bass ( Pb0.05), whilst concentrations of PCBs in mackerel were lower than those in European hake, grey mullet, and sea bass ( Pb0.001; Table 2). In addition, both anchovy and mackerel showed a PCB pattern in which the levels of

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the lighter PCB congeners 28, 52, 74, 66, and 99 were lower than the detection limit in all the samples (Table 2). These results may be ascribed to the pelagic and long-range horizontal and vertical migrating nature of mackerel and anchovy, which overwinter in open sea and only move into surface waters closer to shore in spring and summer, respectively (Collette and Nauen, 1983; Whitehead et al., 1988; Table 1). Elimination rates for lipophilic contaminants generally decrease with increasing logK ow (Gray, 2002); hence, during the period spent in less contaminated open sea water, these species probably undergo a selective loss of lighter congeners by passive diffusion across the gills, whilst the more lipophilic and heavier congeners are retained. This hypothesis is supported by the fact that the mackerels and anchovies analyzed in this study were caught in early March and July, respectively, when they had only just ventured close to shore. Consequently, these two species cannot be expected to offer a reliable picture of the level of pollution at the site where they are caught. Surprisingly, musky octopus, common octopus, and cuttlefish were much less contaminated by DDTs than the other marine organisms investigated. The concentrations of DDTs detected in these cephalopods were often significantly lower than those found in the other species (Table 2). However, cuttlefish and common octopus had relatively high PCB levels. Neither the nature of the habitat nor feeding habits offer a satisfactory explanation for these results (Table 1). The substantial differences in levels of DDTs and PCBs detected in octopus and cuttlefish could be ascribed to differences in their ability to metabolize these xenobiotics. In aquatic invertebrates, especially molluscs, the weak development of the different forms of cytochrome P450, belonging to gene family 2, which are the main responsible for the oxidative attack of PCBs, may explain the high concentration of these chemicals in octopus and cuttlefish (Walker, 2001). Limited data are available on the biotransformation and detoxification of OCPs in cephalopods. However, it has been demonstrated that glutathione-Stransferase from the digestive gland of the cephalopods has one of the highest enzymatic activities ever recorded for glutathione-S-transferases (Tomarev et al., 1993; Ji et al., 1995). Cephalopods should therefore be able to rapidly convert both p,pV-DDT and p,pV-DDE into water-soluble metabolites and

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conjugates and, consequently, promote their rapid excretion. Although blue mussels occupy the lowest position in the food chain among the marine organisms investigated, these bivalves often had higher levels of organochlorines than those found in the other species (Table 2). The congeners 153, 138, and 118 were also predominant in the mussels and contributed to more than 65% of the PCBs. However, the PCB accumulation profile differed from the other species in containing a relatively higher contribution of tri-, tetra-, and pentachlorinated biphenyls 28, 52, 66, 74, 99, and 101 (approximately 15% of the PCBs), a smaller concentration of PCB 180, and the absence of the octachlorobiphenyls 201, 196, and 194 (Table 2). Since mussels are sessile filter feeders, they accumulate organic pollutants almost exclusively by passive diffusion through the gills (Binelli and Provini, 2003). Passive diffusion also represents the main mechanism for the elimination of these chemicals. In fact, the bivalves show a very limited capacity to metabolise PCBs through the cytochrome P450 system (Cajaraville et al., 2000), and, hence, the concentrations of each of the PCB congeners, as well as the total congener pattern, are scarcely modified by biotrasformation. For these reasons, the levels of organochlorines and the accumulation profile of PCBs detected in mussels likely reflect the state of pollution of the coastal waters of the Gulf of Naples and, in particular, of the surface waters that they filter. 3.3. Risk assessment The concentrations of HCB and DDTs found in all the analysed samples are well below the Maximum Residue Limits (MRLs) for organochlorine pesticides in some food products of animal origin (0.2 and 1 mg/ kg fat weight for HCB and DDTs calculated as the sum of p,pV-DDT, p,pV-DDE, p,pV-DDD, and o,pV-DDT, respectively; Decreto Ministeriale 19 maggio, 2000; Table 2). With regard to PCBs, the European Commission set an action limit of 200 ng/g fat weight for the sum of the concentrations of the seven btargetQ congeners in eggs, fresh pig meat, fresh poultry meat, and derived products (Commission of the European Communities, 1999), but no similar limits are available for seafood. As shown in Table 2, in all the marine species from the Gulf of Naples the mean

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PCB concentrations, calculated as the sum of the seven btargetQ congeners indicated by the EU, far exceeded the abovementioned limit.

4. Conclusions The results from this study confirm that the high levels of DDE and PCBs previously detected in fisheating birds from the Campania region are mainly due to heavy pollution of the Gulf of Naples aquatic ecosystem. The presence of the highest levels of OCPs and PCBs in the littoral (benthic and neritic) marine species clearly points to local sources of contamination mainly located along the coast of the Campania region. Since PCB concentrations in the marine species collected from the Gulf of Naples always exceeded the limits set for human consumption, we suggest that seafood consumption patterns for the study area and vicinity should be determined to allow a more accurate assessment of the risk for consumer health. Moreover, as soon as possible, the European Commission should also set limits for PCBs in fish, crustaceans, and molluscs, which are the main source of human exposure to these pollutants within the European Union (Commission of the European Communities, 2004). It is also necessary to implement measures to control industrial waste discharge and to avoid dispersal of these persistent toxic contaminants into the environment. Finally, additional studies should be carried out to monitor the levels of organochlorine pollutants in edible fish from the Gulf of Naples, especially in the marine species which were found to be more highly contaminated in this study.

Acknowledgments The authors are grateful to Dr Ciro Sbarra for his important help in collecting specimens.

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