Analysis of Polycyclic Aromatic Hydrocarbons in Tursiops truncatus. Center for Coastal Environmental Health Final Report P.O. Number EU133C06SE3543
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1 Analysis of Polycyclic Aromatic Hydrocarbons in Tursiops truncatus Center for Coastal Environmental Health Final Report P.O. Number EU133C06SE3543 M. YE MARINE LABORATORY (JAR Submitted to: Patricia Fair, Ph.D. 219 Ft. Johnson Road Charleston, South Carolina Dana L. Wetzel, Ph. D. Mote Marine Laboratory, 1600 Ken Thompson Pkwy, Sarasota, FL January 2007 Mote Marine Laboratory Technical Report Number 1153
2 Objective: The objective of this contract was to assess levels of polycyclic aromatic hydrocarbons in blubber collected from bottlenose dolphins (Tursiops truncatus) from the Indian River Lagoon, Florida, and inshore waters off Charleston, South Carolina. Methods: Sample collection: Twenty-two blubber samples were collected via biopsy from bottlenose dolphins, Tursiops truncatus, near Charleston, S.C. and in the Indian River Lagoon, Florida, in 2004 and The location on the body from which samples were taken was not provided. The samples were wrapped in aluminum foil and frozen in a standard freezer at the Charleston Laboratory (NOAA). Sub-samples were sent, frozen, to Mote Marine Laboratory, where they were archived in a freezer for analyses of polycyclic aromatic hydrocarbons (PAHs). P AH analyses: An aliquot from each sample was removed for moisture content analysis. Blubber was weighed out to the nearest 0.1 g (sample sizes ranged from approximately 0.5 g to 1.5 g) and appropriate polycyclic aromatic hydrocarbon (P AH) internal standards were then added. All samples were extracted using a Dionex ASE 300, extraction system following modified EPA methods (EPA 1998). The extracts were evaporated to near dryness and re-dissolv~d in 1 rnl hexane. Total lipid content was determined gravimetrically for each sample. The extracts were further purified by silica gel-alumina column chromatography. Field blanks, laboratory blanks, matrix spikes and duplicates were included. All extracts were analyzed by a Thermo Finnigan DSQ quadrapole gas chromatograph-mass spectrometer (GCMS) equipped with a 30 m DB-5 fused silica column for qualitative and quantitative identification of 37 individual P AH' s, including parent compounds and homologs (Table 1). Oven temperature program was held at 50 C for two minutes and then programmed from 50 C to 280 C at 6 C min-i and held at 280 C for 15 minutes with helium as the carrier gas. The mass spectrometer scanned from mass in 0.5 sec at an ionization potential of70 e V. All mass spectral data were compared to spectra produced by authentic standards and to previously published library spectra and by quantifying the base peak ion of each P AH against the base peak of the internal standard. The laboratory minimum detectable amounts have been calculated at long/g. Laboratory analytical precision has been previously determined in our laboratory by making replicate injections ofpah standards to ascertain reproducibility. Standard deviations for the standards used indicated a maximum laboratory error of ± 11 %, and an average standard deviation of ± 3%. 2
3 TABLE 1. SELECTED P AHS MONITORED ~ naphthalene (C O -C4) ~ benzo( a )anthracene* ~ fluorene (C O -C4) ~ chrysene (Co-C4) ~ dibenzothiophene (C O -C4) ~ benzo(a)pyrene (BAP) ~ anthracene * ~ benzo( e )pyrene ~ phenanthrene (Co-C4) ~ benzo(k)fluoranthene ~ fluoranthene* ~ benzo(b )fluoranthene ~ pyrene (C O -C4) The C,-C 4 homologs of these PAHs are reported as combined C,-C 4 homologs with the succeeding PAHs (e.g., C,-C 4 homologs of phenanthrene reported as combined C,-C 4 homologs for phenanthrene + anthracene). The unsubstituted parent homolog is Co, and C,-C 4 are the substituents containing from one to four carbon atoms in the attached side chains. Results Blubber samples from 22 dolphins (Table 2) were analyzed for P AHs. These data are reported in wet weight, dry weight, and per gram lipid in order to facilitate comparisons with other studies, which may have only reported concentrations in one of these ways. The major P AH constituent found in all the blubber samples was the naphthalene parent compound. In some of the samples, there were detectable levels of the C 1 and C 2 substituted homo logs of naphthalene in addition to the parent compound. Using the wet weight values, the concentration of total P AHs (only naphthalenes were found in any of the samples) ranged from below detection limits (BDL) to 0.34 ug/g and the dry weight values ranged from BDL to a high of 0.92 ug/g. The values in ug/g oflipid ranged from BDL to Aguilar (1985) has noted that organic pollutants are distributed among the different tissues and organs of cetaceans in proportion to their lipid content. As a result, residue levels of pollutants must be expressed in relation to the lipid content of the tissue rather than fresh weight in order to establish comparisons between different organs in the same individual, different individuals in a population or different species. Calculating the amount of contaminant per gram of lipid, therefore helps to normalize sample matrices. 3
4 Table 2: Results ofpah analyses are reported in ug/g total PAHs, wet weight (w/w), dry weight (d/w) and per g lipid. BDL= below detection limits. Sample # Ug/g w/w ug/g d/w ug/g lipid % lipid BDL BOL BOL BDL BDL BDL BDL BOL BDL BDL BDL BDL BDL BDL BDL BOL BDL BOL BDL BDL BDL BDL BDL BDL BDL BDL BDL BDL BOL BDL BDL BDL BDL The percent lipid in the dolphin blubber samples ranged from 5.50% to 40.70% (see Table 2). The observed differences in percent lipid can help to explain why a sample such as FB# , with a percent lipid of only 11.50%, can have low wet weight and dry weight values for P AHs, but can have much higher concentrations when reported as contaminant per gram of lipid than other samples with higher values reported in wet and dry weights. Discussion Polycyclic aromatic hydrocarbons are the most toxic components of petroleum and are considered genotoxic. Evidence suggests that here is a correlation between high levels of certain P AHs in the environment and an increase of carcinogenesis and mutagenesis in exposed organisms (NRCC 1983). Low molecular weight PAHs such as naphthalenes, tend to remain in solution where they are bioavailable to organisms through ingestion or respiration. Such P AHs are readily taken up by marine organisms and are more toxic than other P AHs for biota (Marsili et al. 2001). 4
5 There are few studies on P AH accumulation in marine mammals. However, marine mammals such as cetaceans are unusual in that they have limited options for flushing toxins from their systems. Fat-soluble contaminants such as P AHs may accumulate in the lipid-rich blubber in the body, then metabolize during illness, pregnancy, lactation or starvation, thereby releasing the lipid-bound organic contaminants (Marsili et al2001). Recent studies on the metabolism of cetaceans indicate that their detoxifying capacity is limited (Watanabe et al. 1989; Tanabe and Tatsukawa 1992; Fossi et al. 1992, 1997a, 1997b; Fossi and Marsili 1997). Comparisons of naphthalene concentrations found in the cetaceans of this study can be made with a few studies on cetaceans from impacted regions. Studies of P AH concentrations in the striped dolphin (Stenella coeruleoalba) from the petroleum impacted Mediterranean Sea suggest that naphthalene concentrations alone ranged from to ug/g wet weight (Marsili et al 2001). Law and Whinnett (1992) found levels ofnaphathlene averaging ug/g wet weight in muscle tissue from the harbor porpoise (Phocoena phocoena) from the coastal areas of the United Kingdom. In the waters off highly industrialized areas of Hong Kong, Leung et al. (2005) found naphthalene concentrations of to ug/g wet weight in the Indo-Pacific humpback dolphin (Sousa chinensis). None of the reported values in these studies were nonnalized to lipid content. The values for naphthalene (wet weight) in the bottlenose dolphins examined in this study are very low in comparison to results of the studies noted above suggesting minimal exposure to and accumulation ofpahs. However, measurements of body burdens alone may not be sufficient to indicate the likelihood of effects from exposure. Using biomarkers of exposure such as mixed function oxidase (MFO) activity, cytochrome P450 induction, and DNA adducts to further evaluate the health of these cetaceans could be a important next step for evaluating and conserving a population of protected marine mammals. References Aguilar, A Compartrnentation and reliability of sampling procedures in organochlorine pollution surveys of cetaceans. Residue Reviews. 95 : EPA Method 3545A, Pressurized Fluid Extraction, Test Methods for Evaluating Solid Waste, EPA, Washington DC, January Fossi, M.C., L. Marsili, C. Leonzio, G.N. Disciara, M. Zanardelli, and S. Focardi The use of non-destructive biomarker in Mediterranean cetaceans: preliminary data on MFO activity in skin biopsy. Marine Pollution Bulletin 24: Fossi, M.C. and L. Marsili The use of non-destructive biomarkers in the study of marine mammals. Biomarkers 2:
6 Fossi, M.C., e. Savelli, L. Marsili, S. Casini, B. Jimenez, M. Junin, H. Castello, J.A. Lorenzani. 1997a. The use of non-destructive biomarkers and residue analysis to assess the health status of endangered species of pinnipeds in the southwest Atlantic. Marine Pollution Bulletin. 34: Fossi, M.C., L. Marsili, M. Junin, H. Castello, J.A. Lorenzani, S. Casini, C. Savelli, and C. Leonzio. 1997b. Skin biopsy as a non-destructive tool for the toxicological assessment of endangered populations of pinnipeds: preliminary results on mixed function oxidase in Otariaflavescens. Chemosphere.35: Law, R.J. and J.A. Wbinnett Polycyclic aromatic hydrocarbons in muscle tissue of harbour porpoises (Phocoena phocoena) from UK waters. Marine Pollution Bulletin 24: Leung, C.e.M, T.A. Jefferson, S.K. Hung, G.J. Zheng, L.W.Y. Yeung, B.J. Richardson and P.K.S. Lam Petroleum hydrocarbons, polycyclic aromatic hydrocarbons, organochlorine pesticides and polychlorinated biphenyls in tissues of Indo-Pacific humback dolphins from south China waters. Marine Pollution Bulletin. 42: Marsili, L., A. Caruso, M.e. Fossi, M. Zanardelli, E. Politi, and S. Focardi Polycyclic aromatic hydrocarbons (P AHs) in subcutaneous biopsies of Mediterranean cetaceans. Chemosphere. 44: NRCC (National Research Council of Canada) Polycyclic Aromatic Hydrocarbons in the Aquatic Environment: Formation, Sources, Fate and Effects on Aquatic Biota, NRC Associate Committee on Scientific Criteria for Environmental Quality, Publication No. NRCC 18981, Ottawa, Ont., 209 p. (1983). Tanabe, S., and R. Tatsukawa Chemical modernization and vulnerability of cetaceans: increasing toxic threat of organochlorine contaminants. In: Walker, e.h. Livingstone, D.R. (Eds.), Persistent Pollutants in Marine Ecosystems. Pergamon Press, Oxford, pp Watanabe, S., T. Shimada, S. Nakamuru, N. Nishiyama, N. Yamashita, S. Tanabe, and R. Tatsukawa Specific profile of liver microsomal cytochrome P-450 in dolphin and whales. Marine Environmental Research. 27 :
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