Environ. Sci. Technol. 2005, 39, 4421-4430
Bioaccumulation of Organochlorines in Crows from an Indian Open Waste Dumping Site: Evidence for Direct Transfer of Dioxin-Like Congeners from the Contaminated Soil MICHIO X. WATANABE, HISATO IWATA,* MAFUMI WATANABE, SHINSUKE TANABE, ANNAMALAI SUBRAMANIAN, KUMIKO YONEDA, AND TAKUMA HASHIMOTO Center for Marine Environmental Studies (CMES), Ehime University, Bunkyo-cho 2-5, Matsuyama 790-8577, Japan, and Japan Wildlife Research Center, Shitaya 3-10-10, Taito-ku, Tokyo 110-8676, Japan
To assess the significance of waste dumping sites as a source of chemical contamination to ecosystems, we analyzed the residue levels of polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs), and other organochlorines in the breast muscle of crows from a dumping site in the south of Chennai city, South India. Crows from the dumping site contained significantly higher total TEQs (60 ( 27 pg/g lipid wt) than those from the reference sites (26 ( 18 pg/g lipid wt). Especially, certain dioxin-like coplanar PCB congeners (Co-PCBs), such as CB-77 and CB-105, whose source is commercial PCBs, were significantly higher in crows from the dumping site than those from the reference sites. Profiles of PCDDs/DFs and Co-PCBs in crows from the dumping site were similar to those of soil at the same site, which was confirmed by principal component analysis. Furthermore, significant positive correlations were obtained between the congener-specific bioconcentration factors (BCFs) of PCDDs/DFs estimated from concentrations in crows and soil from the dumping site and the theoretical BCFs calculated from water-particle and lipid-water partitioning coefficients. On the other hand, the estimated BCFs had significant negative correlations with the molecular weight of PCDDs/DFs, indicating that molecular size limits their bioaccumulation. These results suggest that dioxin-like congeners in the soil of the dumping site were transferred directly to the crows through the ingestion of on-site garbage contaminated with soil, rather than through trophic transfer in the ecosystem. The present study provides insight into the ecological impacts of dumping sites.
Introduction Contamination by organochlorines (OCs), such as polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs), * Corresponding author phone/fax:+81-89-927-8172; e-mail:
[email protected]. 10.1021/es050057r CCC: $30.25 Published on Web 05/18/2005
2005 American Chemical Society
polychlorinated biphenyls (PCBs), and DDTs, is of growing concern due to their greater persistency, potential for magnification in the food chain, and production of toxic effects on human health and wildlife. Open landfill dumping areas of municipal wastes in Asian developing countries are ubiquitous and have received particular attention recently with regard to chemical accumulation. In the dumping sites, a variety of municipal wastes are dumped and burned under low temperature by spontaneous combustion or intentional incineration. It can be anticipated that dioxins and related compounds are formed by such low-temperature conditions and the ambient environment may be polluted by these contaminants. In addition, dioxin-like coplanar PCBs (Co-PCBs) may leak out from electrical appliances containing technical PCBs that are disposed in these dumping sites. Our group conducted a survey of PCDDs/DFs and Co-PCBs in soils collected from the Philippines, Cambodia, Vietnam, and India and found that the residue levels of dioxin-like congeners in soils from the dumping sites were apparently higher than those from agricultural and urban soils (1). Furthermore, concentrations of these contaminants in human breast milk collected from mothers living near a dumping site in India were higher than those in reference sites in India, the Philippines, Vietnam, and Cambodia (2). These results indicate that the dumping sites are potential sources of PCDDs/DFs and Co-PCBs and these congeners released from the dumping site may enter the ambient environment. The use of OC pesticides has been restricted already in most developed countries since the 1970s due to their great persistency and high toxicity. However, they are still being used for public health purposes and agriculture in some developing countries. During the past decade, we have conducted comprehensive investigations on OC pollution in developing Asian countries and found relatively high residue levels of HCHs and DDTs in air, water, sediment, and biological samples from some developing countries (38). Especially, India is one of the greatest consumers of HCHs and the most contaminated nation in the world (9). The estimated cumulative consumptions of HCHs and DDTs in India until 1995 were 1 000 000 and 500 000 t, respectively (9, 10). Our previous study showed the accumulation of OCs in birds collected from India (5-8). To our knowledge, however, no study has reported the residue levels of these contaminants in wildlife around open dumping sites. Crows can be used as a bioindicator species for the monitoring of environmental contaminants in local areas because of their omnivorous and residential nature. Jungle crows in Tokyo are known to move within 1 and 4 km from their roosts per day in the breeding and nonbreeding season, respectively (11). In Indian waste dumping sites, house crows and jungle crows feed on the raw garbage. Therefore, biomagnification of chemical compounds through the contaminated garbage and further biochemical and adverse effects on crows are speculated. This study clarifies the contamination levels of PCDDs/ DFs, Co-PCBs, and other OCs in the breast muscle of crows collected from a dumping site in South India that were compared with those of crows from the reference sites (South India and Tokyo, Japan). The origin of dioxin-like compounds was inferred from the results of PCA analysis as well as based on the profiles of PCDDs/DFs and Co-PCBs. Furthermore, the bioconcentration factors (BCFs) of individual congeners were estimated from the concentrations in crows and soils from the dumping site and compared to the theoretical BCF values, which were calculated from water-particle and lipidVOL. 39, NO. 12, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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water partitioning coefficients. In addition, the molecular weight of each congener as a factor that affects the efficiency of gastrointestinal absorption was discussed in association with BCFs.
Materials and Methods Sample Collection. The information on the location where samples were collected is given in the Supporting Information. For analysis of dioxin-like compounds, five house crows (HCs, Corvus splendens) were collected from a dumping site in Perungudi and a reference site (Muttukadu) near Chennai City, South India. As for jungle crows (JCs, Corvus macrorhynchos), 5 from the dumping site and 13 from Tokyo, Japan as a reference site were collected. For the analysis of other OCs, 10 and 12 JCs from the dumping site and the reference site were collected, respectively. In India, crows are not endangered species. Even some nomadic tribes catch them regularly for their food. So, we preferred to use crows for our work. Japanese crows were captured with permission from the Tokyo Metropolitan Government. Composition of the waste was as follows: organic compounds such as raw garbage, green waste, timber, paper, textiles, and rags (over 50%) > inert (about 30%) > others including plastic, metal, rubber, and leather (12). The dumping site is about 1 400 000 m2 (1). Specimens from India and Japan were collected in August-September 2000, and December 2002, respectively. After collection, crows from India were immediately iced and transported to a laboratory in Japan with cold insulators. Crows from Japan were immediately dissected and iced in the sampling site. Breast muscle samples were removed in the laboratory of Ehime University and stored in a freezer at -20 °C until chemical analysis was performed. Chemical Analysis. Chemical analysis of PCDDs/DFs and Co-PCBs was carried out following the method described previously (13) and summarized in the Supporting Information. Briefly, the muscle (about 20 g) was grounded with anhydrous sodium sulfate and extracted in a Soxhlet apparatus for 9 h with dichloromethane (DCM). The extract was concentrated to 20 mL, and a 2-ml aliquot of the extract was used for the determination of lipid content by the gravimetric method. 13C12-labeled PCDDs/DFs and 13C12labeled Co-PCBs were spiked to the extract as internal standards. The extract, after adding internal standards, was then applied to a gel permeation chromatography column (GPC) for lipid removal. The fraction containing the PCDDs/ DFs and Co-PCBs was collected, concentrated, and passed through a 3-g-activated silica gel packed glass column. PCDDs/DFs and Co-PCBs were eluted with 220 mL of hexane. After concentration, the cleaned-up extract was spiked into 10-g-activated basic alumina packed in a glass column. The first fraction was eluted with hexane, which contained PCB isomers including most of the mono-ortho congeners, and the second fraction was eluted with 50% of DCM in hexane, which contained the remaining mono-ortho Co-PCBs, nonortho Co-PCBs, and PCDDs/DFs. The second fraction eluted from the activated alumina column was then passed through activated carbon-dispersed silica gel (Kanto Chemical, Japan) packed in a glass column. The first fraction from the carbondispersed silica gel column was collected with 25% of DCM in hexane for mono-ortho Co-PCBs and was combined with the first fraction from the alumina column. The second fraction from the carbon-dispersed silica gel column eluted with 220 mL of toluene contained non-ortho Co-PCBs and PCDDs/DFs. Both fractions were concentrated to near dryness, and a 13C12-labeled standard was added to each fraction as a syringe spike. Analytical conditions of highresolution gas chromatography (HRGC) and high-resolution mass spectroscopy (HRMS) are shown in the Supporting Information. Recoveries for the 13C12-labeled PCDDs/DFs and Co-PCBs, which were added prior to GPC column, were within 4422
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60-120%. The 2378-tetrachlorodibenzo-p-dioxin toxic equivalencies (TEQs) were calculated using toxic equivalency factors determined for avian species (14). The total PCBs, DDTs, HCHs, CHLs, HCB, HP epoxide, tris(4-chlorophenyl)methane (TCPMe), and tris(4-chlorophenyl)methanol (TCPMOH) were analyzed following the method reported previously (15). Briefly, the muscle (about 10 g) was ground with anhydrous sodium sulfate and extracted in a Soxhlet apparatus for 7-8 h with a mixture of diethyl ether and hexane. After concentration, the lipid content was determined from the 2-ml aliquot of the 10-ml extract. Another 5-ml aliquot of the extract was added to GPC. The fraction containing OCs was concentrated and passed through an activated Florisil column for fractionation. The identification and quantification of PCBs and most of the OC pesticides were performed using a GC (Agilent 6980N) equipped with an electron capture detector and an autoinjection system (Agilent 7683 Series Injector). The GC column used was a fused silica capillary DB-1 (J&W Scientific, 0.25-µm film thickness, 0.25-mm i.d., 30-m length). Identification and quantification of HP epoxide, TCPMe, and TCPMOH were performed using a GC-MSD (Agilent 5973N) in selective ion monitoring mode (SIM) equipped with an autoinjection system (Agilent7683 Series Injector) and a fused silica capillary DB-1. The OC concentrations were calculated by comparing the peak area of the sample to the corresponding external standard. The PCB standard used for quantification was an equivalent mixture of 62 PCB isomers and congeners (BP-MS) obtained from Wellington Laboratories Inc., Ontario, Canada. The concentrations of individually resolved peaks of PCB isomers and congeners were summed to obtain the total PCB concentrations. Procedural blanks were analyzed simultaneously with the samples to check for interferences of contamination from solvents and glassware. Statistical Analysis. Correlation analyses were performed by Spearman’s rank correlation. The Mann-Whitney U test was used for the detection of statistical differences between groups. These statistical analyses were performed using StatView version 5.0 (SAS Institute Inc., NC). To compare the congener profiles, we performed a principal component analysis (PCA) with Excel Toukei 2000 for Windows (Social Survey Research Information Co., Ltd., Tokyo, Japan). For PCA, each congener concentration in the individual animals was normalized as a contribution ratio to the total concentrations of PCDDs/DFs, non-ortho Co-PCBs, or mono-ortho Co-PCBs. For samples with values below the detection limit, half of the respective limit of quantification was substituted to perform statistical analysis. As for congeners for which concentrations were less than the detection limit in more than half of the specimens, the bioconcentration factors (BCF) were not calculated. The value of p < 0.05 was regarded as statistically significant.
Results and Discussion Concentrations and TEQs of PCDDs/DFs and Coplanar PCBs. Because the residue levels of PCDDs/DFs, Co-PCBs, and other OCs in JCs and HCs showed no significant difference between males and females, the data from specimens of both sexes were therefore pooled for the discussion below. Concentrations of PCDDs/DFs in the crows from the dumping site ranged from 130 to 320 in JCs and 120 to 500 pg/g lipid wt in HCs, whereas concentrations of PCDDs/DFs in HCs from the Indian reference site were 37110 pg/g lipid wt (Table 1). The JCs from the Japanese reference site (Tokyo) contained a range of concentrations (30-190 pg/g lipid wt) similar to the HCs from the Indian reference site. The contamination levels of PCDDs/DFs in JCs and HCs from the dumping site were significantly higher than those from the reference sites (p < 0.01). A previous
TABLE 1. Concentrations of PCDDs/DFs and Coplanar PCBs (pg/g Lipid wt) and other Organochlorines (ng/g Lipid wt) in the Breast Muscle of House Crows and Jungle Crows from the Indian Dumping and Reference Sitesa species
house crow
sampling site
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collected year n lipid (%) PCDDs 2378-T4CDD 12378-P5CDD 123478-H6CDD 123678-H6CDD 123789-H6CDD 1234678-H7CDD O8CDD PCDFs 2378-T4CDF 12378-P5CDF 23478-P5CDF 123478-H6CDF 123678-H6CDF 123789-H6CDF 234678-H6CDF 1234678-H7CDF 1234789-H7CDF O8CDF non-ortho Co-PCBs CB-77 CB-81 CB-126 CB-169 mono-ortho Co-PCBs CB-105 CB-114 CB-118 CB-123 CB-156 CB-157 CB-167 CB-189
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total PCDDs total PCDFs total PCDDs/DFs total non-ortho Co-PCBs total mono-ortho Co-PCBs total Co-PCBs
dumping site (India) 2000 5 3.0 ( 0.47 6.1 ( 4.2 18 ( 10* 13 ( 8.1* 36 ( 22* 6.4 ( 5.2c 66 ( 37** 81 ( 39**
