Search

Organochlorine (OC) contaminant concentrations in tissues and lipid profiles in blubber are summarised for 101 gray whales (Eschrichtius robustus) from the eastern North Pacific stock. Samples were obtained from presumably healthy gray whales during a 1994 subsistence hunt in the Russian Arctic (n = 17) and also from biopsy sampling of live animals from the Washington coast (n = 38). In addition, tissues were collected from two groups of animals (1988-1991, n = 22; and 1999, n = 24) that stranded along the west coast of the USA. These whales represent a diverse group of animals with respect to lipid stores, age, gender, health and reproductive status. Information about these biological factors is necessary before contaminant concentration data can be properly interpreted. Differences in blubber lipid levels and profiles were examined among these groups of whales. Significantly higher lipid levels were found in the blubber of subsistence animals that were sampled following summer feeding in the Bering and Chukchi Seas, compared to lipid levels in the biopsied and stranded animals. Lipid class profiles from blubber of presumably healthy gray whales (i.e. from subsistence and biopsy sampling) contained primarily triglycerides and were very different from those of stranded animals that showed lipid decomposition (increased proportions of free fatty acids, cholesterol and phospholipids). Furthermore, lipid class profiles were found to be a means of estimating the quality of a blubber sample from stranded cetaceans. An examination of how biological factors (e.g. gender, reproductive status, age) contribute to interpreting the differences found in contaminant concentrations among the gray whales was also undertaken. Although not statistically significant, higher (OC) concentrations were found in males compared to females, thus suggesting the tendency of the mother to shift her contaminant burden to her calf during gestation and lactation. Results also indicated that there was no significant increase in concentrations of contaminants in the blubber with increase in length (surrogate for age). Higher concentrations of OC contaminants were found in stranded juvenile gray whales, compared to juvenile subsistence whales, and were thought to result from retention of OCs in blubber of the stranded animals as lipid stores are mobilised for energy and total lipid levels decrease, rather than from a difference in diet or feeding areas. OC concentrations in various tissues (blubber, liver, kidney, muscle, brain) were similar on a lipid weight basis, except for brain, which had lower lipid-adjusted OCs because the blood-brain barrier limits contaminant transfer.

This paper reviews current scientific literature to provide information for avoiding errors commonly made in comparing and interpreting datasets from laboratories measuring organochlorine contaminants in cetaceans. Before making comparisons and interpretations using heterogeneous datasets (e.g. those from different laboratories or those from different methods in the same laboratory), it is essential to consider specific information about the animals sampled (e.g. age, sex, reproductive status, body condition and health status), sampling procedures (e.g. necropsy of subsistence, stranded or bycaught individuals; remote or surgical biopsy), methods for measuring and conventions for expressing analytical results for lipids and contaminants (e.g. percent lipid, percent dry weight, contaminant concentration units, totals of contaminant groups such as PCB congeners) and quality assurance performance. Reformatting should be carried out, as necessary, to unify the datasets (e.g. into like units and weight basis) and allow a critical evaluation of the data to be made. As part of the data interpretation, caveats or limits in the comparability of the datasets (based on quality assurance results) should be provided. In addition, the biological relevance of the data must be considered in interpreting the datasets.

The biopsy 2 via dart, trocar or surgery 2 is becoming the preferred protocol for sampling skin and blubber of many cetacean species, because a small sample from a healthy animal may provide better information than a larger sample collected via necropsy from an ill or emaciated animal. Furthermore, the biopsy is often the only means of obtaining samples (e.g. for threatened or endangered species). Because biopsy darts collect only a small sample of tissue 2 and blubber can be heterogeneous in structure and composition 2 it is essential to compare the results obtained from biopsies to those found by analysing full-thickness blubber samples obtained via necropsy. This manuscript compares blubber stratification in two odontocete species, white whales (Delphinapterus leucas) and killer whales (Orcinus orca). Five parameters (i.e. lipid percent and classes, contaminant concentrations and profiles, fatty acid profiles) were measured by blubber depth. Results of these comparisons strongly suggest that biopsy results must be interpreted with caution and in conjunction with results from species-specific blubber depth profiling. For example, lipid classes measured in biopsy samples of white whales and killer whales were similar to those for equivalent-depth samples obtained by necropsy. In addition, lipid-adjusted contaminant concentrations measured in dart or trocar samples adequately represented those obtained by necropsy of both species. Conversely, the lipid content in biopsy samples was lower than that found in same-depth necropsied samples due to loss of lipid during sampling. Also, because of the high level of fatty acid stratification observed, fatty acid profiles from the outer blubber layer collected via biopsy from both species are less likely than the metabolically active inner layer to be useful in determining the prey species consumed by these odontocetes. This study demonstrates, for white and killer whales, that properly interpreted results from blubber biopsies can provide valuable information about the body condition, health and life history of individual animals.

Following the 2010 Deepwater Horizon oil spill, petroleum-related compounds and chemical dispersants were detected in the waters of the Gulf of Mexico. As a result, there was concern about the risk to human health through consumption of contaminated seafood in the region. Federal and Gulf Coast State agencies worked together on a sampling plan and analytical protocols to determine whether seafood was safe to eat and acceptable for sale in the marketplace. Sensory and chemical methods were used to measure polycyclic aromatic hydrocarbons (PAHs) and dispersant in >8,000 seafood specimens collected in federal waters of the Gulf. Overall, individual PAHs and the dispersant component dioctyl sodium sulfosuccinate were found in low concentrations or below the limits of quantitation. When detected, the concentrations were at least two orders of magnitude lower than the level of concern for human health risk. Once an area closed to fishing was free of visibly floating oil and all sensory and chemical results for the seafood species within an area met the criteria for reopening, that area was eligible to be reopened. On April 19, 2011 the area around the wellhead was the last area in federal waters to be reopened nearly 1 y after the spill began. However, as of November 9, 2011, some state waters off the Louisiana coast (Barataria Bay and the Delta region) remain closed to fishing.

The ability to determine the age of individual humpback whales Megaptera novaeangliae and estimate population age distributions is fundamental to assessments of status and long-term viability. Existing methods of ageing large whales rely either on limited longitudinal sighting studies of individual whales from their year of birth, or on post-mortem procedures to extract tissues suitable for determining age. Here we describe a potential method for ageing live free-ranging humpback whales using low-impact biopsy sampling techniques. Shallow outer-blubber samples were obtained from known-age whales from 2 distinct populations (North Atlantic, Gulf of Maine, n = 39; North Pacific, Southeast Alaska, n = 31), and analyzed for their fatty acid (FA) compositions. Multilinear FAage models were derived for these known-age whales, and serve as the basis from which the age of unknown-age whales can be estimated. Four FA-age models were developed; one for each humpback population analyzed separately, and an additional 2 by combining both populations into a single dataset and deriving models based on ‘exact’ and ‘exact’ plus ‘minimum’ known-age whales independently. Each of these empirical models was based on a linear combination of 2 FA ratios rather than individual FA compositions, and shown to be largely independent of sex, diet and nutritional status. Although the precision (σ) of these models was somewhat variable (ranging between 3.1 and 5.3 yr for the specific populations modeled), the results suggest that it may be possible to estimate the age of individual humpback whales from any population with better than decadal resolution using this approach.

Measuring chemical tracers in tissues of marine predators provides insight into the prey consumed and the predator's contaminant exposure. In this study, samples from Type C killer whales {Orcinus area) biopsied in Antarctica were analyzed for chemical tracers (i.e., stable isotopes of carbon and nitrogen, fatty acids, and persistent organic pollutants [POPs]). Profiles of these individual tracers were very different from those of killer whale populations that have been studied in the eastern North and eastern Tropical Pacific. For example, δ 13 C and δ 15 N stable isotope values and most POP concentrations were significantly lower in the Antarctic population. In addition, multivariate statistical analyses of both fatty acid and POP profiles found distinctly different patterns for Antarctic Type C whales compared to those from whales in the other populations. Similar assays were conducted on four species of Antarctic marine fish considered potential prey for Type C killer whales. Results were consistent with a diet of fish for Type C whales, but other species (e.g., low trophic-level marine mammals or penguins) could not be eliminated as supplemental prey.

Investigations into the cause of the Steller sea lion population decline have focused on numerous factors, including exposure to toxic contaminants such as organochlorines (OCs). OCs, such as polychlorinated biphenyls (PCBs), 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane, or dichlorodiphenyltrichloroethane (DDT), have been associated with various biological effects in marine mammals. We measured these compounds in whole blood of free-ranging Steller sea lion pups in order to determine the extent and magnitude of contamination across their geographical range. Of 212 pups analyzed for OCs, 76 pups (36 females and 40 males) were from western Alaska and the other 136 (63 females and 73 males) were from the Russian Far East. Concentrations of summation SigmaPCBs in the whole blood of pups from western Alaska ranged from 0.21 to 13 ng/g wet weight with a mean of 2.1+/-0.27 ng/g wet weight. In the Russian animals, summation SigmaPCB concentrations in the whole blood of pups ranged from 0.33 to 36 ng/g wet weight with a mean of 4.3+/-0.44 ng/g wet weight. summation SigmaDDT concentrations in the whole blood of pups from western Alaska ranged from 0.18 to 11 ng/g wet weight with a mean of 1.6+/-0.23 ng/g wet weight. In Russia, summation SigmaDDT in the whole blood of pups ranged from undetectable to 26 ng/g wet weight with a mean of 3.3+/-0.36 ng/g wet weight. Average OC concentrations were significantly higher in the blood of Russian animals compared to western Alaska (for PCBs and DDTs, p0.001) and in both areas females had higher concentrations than males. Male pups from western Alaska had significantly lower levels of summation SigmaPCBs and summation SigmaDDT when compared to male pups from Russia (for PCBs and DDTs p0.001). Female pups from western Alaska were significantly lower in summation SigmaPCBs than Russian female pups (for PCBs p=0.009) as were female pups for summation SigmaDDT levels between areas (for DDTs p=0.026). OC contaminants data indicate that Steller sea lion pups have measurable concentrations of these synthetic chemicals. While any physiological effect and the specific role these chemicals may have in either the decline or the failure of the endangered Steller sea lion population to recover needs to be further investigated, this study indicates specific areas and animals that may be most at risk.

We analyzed blubber and blood samples for organochlorines (OCs) from 158 Hawaiian monk seals ( Monachus schauinslandi ) at four of their six primary breeding colonies in the Northwestern Hawaiian Islands. Levels of OCs in blubber were lower in adult females compared to juveniles or adult males, evidently owing to the transfer of these lipophilic compounds to pups by pregnant and lactating females. Concentrations of ∑PCBs and p,p′ -DDE in blubber generally increased with age until seals were sexually mature and then continued to increase with age in males after puberty. Average levels of PCBs and PCB toxic equivalents (TEQs) in blubber were significantly higher in adult male and juvenile seals at Midway Atoll than the same age class of seals at the other colonies. Unlike concentrations of OCs in blubber, circulating levels of OCs in blood did not vary consistently among age classes of seals or among breeding colonies. Though the concentrations of OCs measured in Hawaiian monk seals were generally equal to or lower than those reported for other pinniped species in the North Pacific Ocean, they were high enough in a few seals to potentially affect their health.

"Southern Resident" killer whales include three "pods" (J, K and L) that reside primarily in Puget Sound/Georgia Basin during the spring, summer and fall. This population was listed as "endangered" in the US and Canada following a 20% decline between 1996 and 2001. The current study, using blubber/epidermis biopsy samples, contributes contemporary information about potential factors (i.e., levels of pollutants or changes in diet) that could adversely affect Southern Residents. Carbon and nitrogen stable isotopes indicated J- and L-pod consumed prey from similar trophic levels in 2004/2006 and also showed no evidence for a large shift in the trophic level of prey consumed by L-pod between 1996 and 2004/2006. Sigma PCBs decreased for Southern Residents biopsied in 2004/2006 compared to 1993-1995. Surprisingly, however, a three-year-old male whale (J39) had the highest concentrations of Sigma PBDEs, Sigma HCHs and HCB. POP ratio differences between J- and L-pod suggested that they occupy different ranges in winter.

Since 1987, the Alaska Marine Mammal Tissue Archival Project (AMMTAP) has collected tissues from 18 marine mammal species. Specimens are archived in the National Institute of Standards and Technology's National Biomonitoring Specimen Bank (NIST-NBSB). AMMTAP has collected blubber, liver and/or kidney specimens from a number of ringed seals (Phoca hispida) from the areas near Nome and Barrow, Alaska and walruses (Odobenus rosmarus) from several locations in the Bering Sea. Thirty-three ringed seal and 15 walrus blubber samples from the NIST-NBSB were analyzed for persistent organic pollutants (POPs). The compounds determined included PCBs (28 congeners or congener groups), DDT and related compounds, hexachlorobenzene (HCB), hexachlorocyclohexane isomers (HCHs), chlordanes, dieldrin, and mirex. POP concentrations in ringed seal blubber were significantly higher in Barrow than in Nome when statistically accounting for the interaction of age and gender; HCB, however, was not statistically different between the two locations. Unlike males, POP concentrations and age were not significantly correlated in females probably as a result of lactational loss. POP concentrations in walrus blubber were lower than in ringed seal blubber for SigmaPCBs, chlordanes, and HCHs, but higher for dieldrin and mirex. POP concentrations in ringed seals and walrus from Alaska provide further evidence that the western Arctic tends to have lower or similar POP concentrations compared to the eastern Canadian Arctic.

Blubber biopsy samples from eastern North Pacific killer whales Orcinus orca were ana- lyzed for fatty acids, carbon and nitrogen stable isotopes and organochlorine contaminants. Fatty acid profiles were sufficiently distinct among the 3 reported ecotypes ('resident,' 'transient' or 'offshore') to enable individual animals to be correctly classified by ecotype and also by mitochondrial DNA (mtDNA) haplotype. Profiles of PCBs also enabled unambiguous classification of all 3 killer whale ecotypes, but stable isotope values lacked sufficient resolution. Fatty acid, stable isotope and PCB profiles of the resident and transient ecotypes were consistent with those expected for these whales based on their reported dietary preferences (fish for resident whales, marine mammals for transients). In addition, these ecotype profiles exhibited broad similarity across geographical regions, suggesting that the dietary specialization reported for resident and transient whales in the well-studied eastern North Pacific populations also extends to the less-studied killer whales in the western Gulf of Alaska and Aleutian Islands. Killer whales of the same ecotype were also grouped by region of sample col- lection. The mean stable isotope ratios of various regional groups differed considerably, suggesting that the prey preferences of these North Pacific killer whales may be both region and ecotype spe- cific. Furthermore, 3 specific ecotypes of killer whales were found to have measured stable isotope values that were consistent with dietary preferences reported in the literature. Finally, although the offshore population had blubber fatty acid profiles implicating fish as its primary prey, contaminant and stable isotope results were equally congruent with predation on marine mammals.

After nearly 3 decades of decline, the western stock of Steller sea lions (SSL; Eumetopias jubatus) was listed as an endangered species in 1997. While the cause of the decline in the 1970s and 1980s has been attributed to nutritional stress, recent declines are unexplained and may result from other factors including the presence of environmental contaminants. SSL tissues show accumulation of butyltins, mercury, PCBs, DDTs, chlordanes and hexachlorobenzene. SSL habitats and prey are contaminated with additional chemicals including mirex, endrin, dieldrin, hexachlorocyclohexanes, tetrachlorodibenzo-p-dioxin (TCDD) and related compounds, cadmium and lead. In addition, many SSL haulouts and rookeries are located near other hazards including radioactivity, solvents, ordnance and chemical weapon dumps. PCB and DDT concentrations measured in a few SSL during the 1980s were the highest recorded for any Alaskan pinniped. Some contaminant exposures in SSL appear to be elevated in the Gulf of Alaska and Bering Sea compared to southeast Alaska, but there are insufficient data to evaluate geospatial relationships with any certainty. Based on very limited blubber data, current levels of PCBs may not pose a risk to SSL based on comparison to immunotoxicity tissue benchmarks, but SSL may have been at risk from pre-1990 PCB exposures. While exposure to PCBs and DDTs may be declining, SSL are likely exposed to a multitude of other contaminants that have not been monitored. The impacts of these exposures on SSL remain unknown because causal effects have not been established. Field studies with SSL have been limited in scope and have not yet linked contaminant exposures to adverse animal health or population effects. Several biomarkers may prove useful for monitoring exposure and additional research is needed to evaluate their utility in SSL. We conclude that there are insufficient data to reject the hypothesis that contaminants play a role in the continued decline of SSL, and suggest that a coordinated monitoring program be developed which can be related to key biological, ecological and laboratory toxicity data.

Developing methods that accurately and rapidly measure aromatic components of oil spills in marine biota and habitats is a challenge for analytical chemists.

The National Benthic Surveillance Project (NBSP), a component of NOAA's National Status and Trends Program, monitored sediment and bottomfish for chlorinated hydrocarbon pesticides (CHs), PCBs and polycyclic aromatic hydrocarbons (PAHs) at selected urban and nonurban sites along the west coast of the USA from 1984 to 1993. This project successfully generated an extensive data set to evaluate the recent status and trends of environmental quality in coastal waters. This article summarizes and interprets the status of selected PAHs, PCBs and CHs in surficial sediments and selected species of bottom-feeding fish for 50 sites in Alaska, Washington, Oregon and California for the first 7 years (1984–1990) of the NBSE. The highest concentrations of most sediment-associated organic contaminants were present in the most highly urbanized areas, and many of the organic contaminants were bioaccumulated by indigenous marine fish species. The concentrations of PAHs and CHs in sediments generally correlated with levels of these compounds or their derivatives in bottom-dwelling fish. Assessment of trends in the concentrations of chlordanes, dieldrin, DDTs, PAHs and PCBs for the 7 year period from 1984–1990 suggest that, since the mid-1980s, concentrations of the persistent CHs, such as PCBs and DDTs in sediment and fish, show no consistent temporal trends, whereas levels of PAHs, which are nonpoint source contaminants, showed consistent increases at both nonurban and urban near-coastal sites.

Starry flounder (Platichthys stellatus), white croaker (Genyonemus lineatus) and sediments were collected annually from selected sites within San Francisco Bay, and a reference site in Bodega Bay between 1984--1991. Fish livers were examined for toxicopathic lesions and analysed for selected chlorinated hydrocarbons (CHs) such as PCBs, DDTs, chlordanes and dieldrin; sediment and fish stomach contents were analysed for polycyclic aromatic hydrocarbons (PAHs) and CHs; and bile was analysed for PAH metabolites. Sediment concentrations of PAHs, PCBs and DDTs; bile concentrations of PAH metabolites; and liver concentrations of PCBs, dieldrin and chlordanes were generally significantly higher at all San Francisco Bay sites compared to the Bodega Bay reference site. For both species, hydropic vacuolation of biliary epithelial cells was the most prevalent liver lesion detected and was statistically associated with sediment and tissue concentrations of PAHs or their metabolites, PCBs, DDTs, chlordanes and dieldrin. Temporal trends analyses showed that at Hunters Point, sediment PAHs and CHs increased between 1984--1991, while liver concentrations of CHs decreased. Liver concentrations of dieldrin in starry flounder decreased at all three San Francisco Bay sites

A rapid method has been developed to screen for planar chlorobiphenyl (CB) congeners, as well as certain other CBs and DDTs, in tissue samples from marine biota. The analytes were extracted from tissue matrices with 1:1 hexane/pentane (v/v) and interfering compounds were separated from the CBs on a gravity-flow column packed with acidic, basic and neutral silica gel eluted with 1:1 hexane/methylene chloride (v/v). Subsequently, the planar CB congeners were resolved from the DDTs and other CBs by HPLC on Cosmosil PYE analytical columns cooled to 9 degrees C and were measured by an ultraviolet (UV) photodiode array (PDA) detector. Two important advantages of PDA over conventional UV detection were the ability to identify individual analytes by comparing their UV spectra to those of reference standards and the ability to establish the spectral homogeneity (purity) of the analytes by comparing spectra within a peak to the apex spectrum. The HPLC/PDA method was tested with tissue samples from fish, shellfish and marine mammals; concentrations of certain CBs and DDTs in samples determined by screening compared favorably with those in the same samples analyzed by a comprehensive method (e.g., gas chromatography/high resolution mass spectrometry). However, the HPLC/PDA method was about an order of magnitude less sensitive than determinations by comprehensive methods.

In order to monitor the quality of coastal waters that provide habitats for living marine resources, samples of sediment and biota must be analyzed to assess the degree and distribution of anthropogenic contamination. Analytical time and costs can be greatly reduced by first employing methods that screen for contaminants before selecting samples for rigorous analyses. In this paper, we review the applications of rapid high-performance liquid chromatographic (HPLC) methods to screen for aromatic compounds in sediment, bile and tissue samples. These methods have been used to assess damage to natural resources after the Exxon Valdez oil spill. In addition, the bile screening method has also been used to evaluate contaminants in fish sampled for a national monitoring program. The rapid screening of sediment or bile provides an estimate of contaminant concentrations that can then be confirmed in selected samples by more complicated and expensive analyses by gas chromatography/3-mass spectrometry (GC-MS). Furthermore, HPLC and GC-MS chromatographic patterns from sediment and bile can provide information about the source of contamination, e.g., crude oil, diesel fuel or pyrogenic contaminants. We also discuss the important role screening methods will play in the future in assessing the quality of aquatic habitats, the safety of seafood, and other important issues related to anthropogenic contamination.

After the grounding of the Exxon Valdez, sediment samples were collected to determine the degree and distribution of the oiling. Sixty sediments from 10 sites in Prince William Sound, AK, were analyzed for Prudhoe Bay crude oil (PBCO) using a rapid HPLC screening method that measured fluorescence at wavelength pairs specific for two- and three-ring petroleum-related aromatic compounds (ACs). Concentrations of individual ACs in the sediments were also determined by GC/MS to compare the results of the two methods. Concentrations of ACs measured by HPLC screening were highly correlated with the sums of ACs determined by GC/MS, thus validating the screening method as an effective tool for estimating concentrations of petroleum-related ACs in sediments. Moreover, differences in HPLC chromatographic patterns among sediments suggested different sources of contamination, e.g., crude oil or diesel fuel. Finally, GC/MS analyses confirmed that PBCO was a primary source of contamination in many sediments. 21 refs., 5 figs., 2 tabs.

The assessment of oil exposure and resulting biological effects in subtidal fish following marine oil spills is a rather challenging task, due to difficulty in sampling and a lack of methodologies that allow direct determination of petroleum exposure. Because fish can rapidly metabolize petroleum hydrocarbons, standard chemical analyses are of little use for assessing exposure of fish to oil. However, methods are now available for measuring the metabolites of petroleum hydrocarbons in fish, as described elsewhere by Krahn and her colleagues. There have also been advances in determining early biochemical effects—such as induction of the cytochrome P450 system—and more severe biological effects—such as histopathological alterations and reproductive dysfunction—in fish species following exposure to petroleum hydrocarbons and related compounds. Accordingly, following the Exxon Valdez oil spill, we have been using these approaches and methodologies to measure oil exposure in subtidal fish species, together with associated biological effects. These studies have been conducted at sites inside Prince William Sound and along the Kenai and Alaska Peninsulas. Our results show a continuing exposure of several fish species, including benthic species, which indicates petroleum contamination of subtidal areas. We have also assessed reproductive function and histopathological alterations in several of these species; and while major effects have not been documented, some suggestion of histopathological alterations of gill exists in one species of benthic fish. Still to be determined are the potential impacts on fishery resources of long-term exposure to petroleum, albeit at moderate to low levels.

Approximately 1 year after Kuwaiti crude oil was spilled and burned during the Gulf War, scientists on board the NOAA R/V Mt Mitchell sampled sediment and biota in the Gulf to look for evidence of petroleum contamination. Subtidal sediments and fish bile were analysed for petroleum-related aromatic compounds (ACs) on board the Mt Mitchell using cost-effective and rapid high-performance liquid chromatographic (HPLC) screening methods. Parent ACs and their alkyl homologs were measured in sediments. However, because fish extensively metabolize ACs in their livers to form polar metabolites that are concentrated in bile for excretion, metabolites of ACs were measured in bile. HPLC screening analyses found ACs in many of the sediment and fish bile samples collected. The results of HPLC screening were confirmed in selected samples by gas chromatography/mass spectrometry (GC/MS) analyses that identified petroleum-related ACs (e.g., alkyl phenanthrenes or dibenzothiophenes) in sediments and their metabolites in bile.

The determination of polycyclic aromatic hydrocarbon (PAH) metabolites in bile can serve as a tool for assessing environmental PAH exposure in fish. Biliary PAH metabolite levels can be measured using several analytical methods, including simple fluorescence assays (fixed fluorescence detection or synchronous fluorescence spectrometry); high-performance liquid chromatography with fluorescence detection (HPLC-F); gas chromatography–mass spectrometry (GC–MS) after deconjugation, extraction and derivatization of the bile sample, and finally by advanced liquid chromatography-tandem mass spectrometry (LC–MS/MS) and gas chromatography-tandem mass spectrometry (GC–MS/MS) methods. The method alternatives are highly different both with regard to their analytical performance towards different PAH metabolite structures as well as in general technical demands and their suitability for different monitoring strategies. In the present review, the state-of-the-art for these different analytical methods is presented and the advantages and limitations of each approach as well as aspects related to analytical quality control and inter-laboratory comparability of data and availability of certified reference materials are discussed., Financial support for different parts of the current work was provided by the EU – Standards, Measurements and Testing program (contract SMT4-CT98-2250), the Norwegian Research Council (projects 653879, 152449/720 and 153898/S40) and Total E&P Norge AS (TCP project). M.O. Sydnes (IRIS), G. Ylitalo and J. Stein (NOAA) are acknowledged for their valuable critical comments on the manuscript.

Chemical pollution at sites in or near San Diego Bay was investigated between 1984 and 1988. The mean concentrations of selected polychlorinated biphenyls (PCBs), metals (e.g., copper and lead), and aromatic hydrocarbons in sediments from sites in the southern and central portions of the bay were significantly higher (p≤0.05) than those in sediment samples from nearby nonurban sites

High concentrations of toxic chemicals in sediment and white croaker (Genyonemus lineatus), as well as liver diseases (e.g., carcinomas) in this species, were found in the Los Angeles area. The highest concentrations of aromatic hydrocarbons (AHs) in the sediment were in San Pedro Bay, and the highest concentrations of 1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) derivatives were in sediment from near the White Point sewer outfall. Concentrations of AHs, polychlorobiphenyls (PCBs), and DDT derivatives were generally higher in food organisms from the croaker's stomach than in sediment. Moreover, croaker from San Pedro Bay and White Point were substantially contaminated with DDT derivatives and metabolites of aromatic compounds, compared to croaker from the Hyperion outfall and Dana Point. The evidence suggest that the observed pathological conditions of the liver were associated with exposure of the croaker to toxic chemicals, which occurred, at least in part, through the ingestion of contaminated food organisms.

Awareness of contamination from anthropogenic sources in the environment has led to a demand for increasing numbers of analyses to evaluate the extent of the problem. As a result, there is a need for rapid, low cost methods to screen for contaminants in environmental samples to allow priorities to be set for the more expensive, detailed analyses. In this paper, we report a method to screen sediments for aromatic hydrocarbons. The method uses sonic extraction followed by size-exclusion HPLC with fluorescence detection to estimate the concentrations of fluorescent aromatic compounds in sediment samples. The concentrations of aromatic compounds determined by the screening method are highly correlated with the sum of the aromatic hydrocarbons determined by a standard analytical method which uses HPLC cleanup and GC/MS quantitation.

‘‘Southern Resident” killer whales (Orcinus orca) that comprise three fish-eating ‘‘pods” (J, K and L) were listed as ‘‘endangered” in the US and Canada following a 20% population decline between 1996 and 2001. Blubber biopsy samples from Southern Resident juveniles had statistically higher concentrations of certain persistent organic pollutants than were found for adults. Most Southern Resident killer whales, including the four juveniles, exceeded the health-effects threshold for total PCBs in marine mammal blubber. Maternal transfer of contaminants to the juveniles during rapid development of their biological systems may put these young whales at greater risk than adults for adverse health effects (e.g., immune and endocrine system dysfunction). Pollutant ratios and field observations established that two of the pods (K- and L-pod) travel to California to forage. Nitrogen stable isotope values, supported by field observations, indicated possible changes in the diet of L-pod over the last decade. Published by Elsevier Ltd.

Livers of wild English sole (Parophrys vetulus) from polluted waterways and embayments of Puget Sound, Washington, are affected by a spectrum of multiple, co-occurring idiopathic hepatic lesions, including neoplasms, putative preneoplastic foci of cellular alteration, and unique degeneration conditions. Results from a statistical analysis of the patterns of co-occurrence of these lesions in wild English sole indicate that these lesions represent morphologically identifiable steps leading to the development of hepatic neoplasms. This sequence parallels the lesion progression in experimental models of chemically induced liver carcinogenesis in rodents. The hypothesis that these lesions in wild English sole can be caused by exposure to certain xenobiotic hepatotoxic and hepatocarcinogenic compounds in Puget Sound is based on: a) statistical associations between levels of aromatic hydrocarbons (Σ AHs) an sediment and prevalences of these idiopathic liver lesions, b) the contribution of Σ AHs in accounting for the variability in hepatic neoplasm prevalence in a logistic regression model, c) elevated odds ratios for several idiopathic hepatic lesion types in sole from polluted sites in Puget Sound, d) significant correlations between prevalences of idiopathic hepatic lesions and levels of fluorescent metabolites of aromatic compounds (FACs) in bile of English sole, and e) experimental induction of putatively preneoplastic focal lesions in English sole injected with a PAH-enriched fraction of an extract from a contaminated urban sediment from Puget Sound, that were morphologically identical to lesions found in wild English sole from the same site.

This study reports the analysis of polycyclic aromatic compound (PAC) metabolites, as biomarkers of exposure to PACs in marine environment. PAC metabolites were measured in bile samples from 14 species of demersal fish caught in the Sao Sebastiao Channel (SSC), SE Brazilian coastline. Naphthalene (NPH) equivalents, phenanthrene (PHN) equivalents, and benzo[a]pyrene (BaP) equivalents were quantified using a reverse-phase high-performance liquid chromatography coupled with fluorescence detection method. For all samples, the means (±standard deviation, n = 37) of concentrations obtained for NPH, PHN, and BaP equivalents were, respectively, 290,000 ± 200,000 ng/g, 18,000 ± 14,000 ng/g, and 970 ± 1900 ng/g. These results indicate recent exposure of these fish to PACs in their environment. In addition, two species (Cyclichthys spinosus and Prionotus nudigula) of fish were analyzed in order to investigate local sources of PAC contamination in the SSC and the influence of the petroleum terminal in fish caught in remote areas. The results showed that these fish species potentially migrate along the channel, especially P. nudigula. Correlations among groups of PAC metabolites indicate the same petrogenic source for NPH and PHN equivalents and a combustion source (e.g., automobile, ships) for BaP equivalents. The ratio BaP/PHN equivalents (0.05 ± 0.07, n = 37) confirms the predominance of petrogenic PACs for contamination by these chemicals in this region.

Top predators in the marine environment integrate chemical signals acquired from their prey that reflect both the species consumed and the regions from which the prey were taken. These chemical tracers—stable isotope ratios of carbon and nitrogen; persistent organic pollutant (POP) concentrations, patterns and ratios; and fatty acid profiles—were measured in blubber biopsy samples from North Pacific killer whales (Orcinus orca) (n = 84) and were used to provide further insight into their diet, particularly for the offshore group, about which little dietary information is available. The offshore killer whales were shown to consume prey species that were distinctly different from those of sympatric resident and transient killer whales. In addition, it was confirmed that the offshores forage as far south as California. Thus, these results provide evidence that the offshores belong to a third killer whale ecotype. Resident killer whale populations showed a gradient in stable isotope profiles from west (central Aleutians) to east (Gulf of Alaska) that, in part, can be attributed to a shift from off-shelf to continental shelf-based prey. Finally, stable isotope ratio results, supported by field observations, showed that the diet in spring and summer of eastern Aleutian Island transient killer whales is apparently not composed exclusively of Steller sea lions.

Existing methods for analyzing methylsulfonyl-PCBs in animal tissues require multiple laborious clean-up steps prior to quantitation. In this paper, we report a new, efficient method for the isolation and determination of methylsulfonyl-PCB metabolites in the blubber, liver, kidney, lung, and milk from mammals of marine origin using: solvent extraction, GPC fractionation, and clean-up on an adsorption chromatography column prior to analysis by gas chromatography–mass spectrometry. The method is rapid and can be completed in about half the analysis time required by most other reported methods. The method exhibits excellent analyte recoveries (89–95%), and good reproducibilities with CV's ranging from 3–12% depending on analyte concentration. Detection limits are approximately 1 ng/g lipid. The method is further validated by comparing its quantitative results to that of an independent reference analytical method for several different marine mammal blubber samples. Finally, the method is applied to profile the distribution of methylsulfones among the various tissue types studied for a single bottle-nose dolphin (Tursiops truncatus) sample. In addition, the extent of their transplacental transfer from mother to unborn fetus for several pilot whale (Globicephala melaena) mother-fetus pairs is examined.

Organochlorine (OC) levels in liver and blubber of 20 bowhead whales (Balaena mysticetus) collected during the Eskimo subsistence harvest at Barrow (Alaska, USA) in 1992 and 1993 are presented. Liver sum DDT (lipid weight) was significantly greater in male whales than in females. Most of the organochlorines measured were at higher levels in longer (older) than in shorter (younger) males. For female bowhead whales, hexachlorobenzene and lipid levels decreased and other OC levels did not change significantly with increasing length. Most organochlorine contaminants have low concentrations in tissues of the bowhead whale compared to concentrations in tissues of other cetaceans, especially Odontocetes. Based on allowable daily intakes (ADI) levels established by the Canadian Northern Contaminants Program (Ottawa, Ontario, Canada) "safe" levels of blubber to consume were calculated. Chlordane levels in bowhead whale blubber results in the most restrictive consumption amount (50 g blubber/day). We expect no adverse effects related to these organochlorine contaminants to occur in bowhead whales or in consumers of their tissues. However, investigation of low level chronic exposure effects and a more rigorous assessment of histopathology, biomarkers, and immune status in the bowhead whale would be required to conclude "no effect" with more certainty.

Levels of organochlorine contaminants in blood of northern fur seal (Callorhinus ursinus) pups and the blood and milk of their dams early in the lactation period are reported here. The contaminants included 15 selected individual polychlorinated biphenyl (PCB) congeners and DDT metabolites identified through high-performance liquid chromatography. Congeners CB-77, -81, -126, -169 and -189 were below the limits of detection in milk and blood samples analyzed. Congener-specific concentrations of PCBs in the blood of pups were compared based on the age of their dam (or = 5 years or7 years). Pups of young (presumably primiparous) dams had significantly elevated levels of CBs-101, -118, -128, -138, -153/87, -170/194, and -180 than pups of older (multiparous) dams. Congeners CB-128 and -170/194 were detected in the blood of pups of young dams but not in the blood of pups of older dams nor in any of the dams blood. Additionally, pups had higher blood levels in seven of 10 detected PCB congeners as compared to the levels measured in milk when adjusted for lipid content. Levels of DDT metabolites and toxic equivalency quotients of dioxin-like congeners followed similar trends. Lipid-normalized concentrations of CB-101 and total PCBs were significantly higher in the blood of dams than in their milk. CB-128, -156, -157, -170/194, p,p'-DDT, o,p'-DDT, p,p'-DDD and o,p'-DDD were not detected in dam blood samples, but were detected in milk samples. Calculation of 'biomagnification factors' from milk to pup blood indicated a biomagnification of CB-101, -105, -118, -138, -153/87, and -180. Significant mean accumulation factors ranged from 1.5 to 7.5. Inter-annual differences in exposure levels and specific congener concentrations in both milk and blood were apparent. Northern fur seal pups, especially first-born, have a substantial exposure to organochlorine contaminants at a critical developmental stage.

Blubber samples from four Alaska seal species (bearded seal, Erignathus barbatus; harbor seal, Phoca vitulina; northern fur seal, Callorhinus ursinus; ringed seal, P. hispida) were collected for inclusion in the US National Biomonitoring Specimen Bank, as well as for immediate analysis as part of the contaminant monitoring component of the US National Marine Fisheries Service's Marine Mammal Health and Stranding Response Program. The blubber samples were analyzed for organochlorine (OC) contaminants (e.g., PCB congeners, pesticides, DDTs). Results for bearded and ringed seals from the Alaska Arctic revealed low blubber concentrations of OC contaminants. Harbor seals from Prince William Sound, Gulf of Alaska, had somewhat higher blubber concentrations of OC contaminants. In contrast, northern fur seals sampled from the Pribilof Islands had blubber concentrations of certain OC contaminants that were about an order of magnitude higher than those found in the other seal species. Differences in contaminant concentrations among the Alaska seals may be explained by differences in feeding habits and migratory patterns; age or gender did not appear to account for the differences observed. The highest concentrations of OCs were found in harbor seals stranded along the northwestern US mainland, which is consistent with higher concentrations of anthropogenic contaminants being found in urban coastal areas than in more remote Arctic environments. The integration of real-time contaminant monitoring with specimen banking provides important baseline data that can be used to plan and manage banking activities. This includes identifying appropriate specimens that are useful in assessing temporal trends and increasing the utility of the banked samples in assessing chemical contaminant accumulation and relationships to biological effects.

English sole (Parophrys vetulus) inhabiting polluted waterways and embayments of Puget Sound, Washington, are affected with a variety of multiple, co-occurring idiopathic hepatic lesions, including unique degenerative conditions, putatively preneoplastic foci of cellular alteration, and neoplasms. Results of a statistical analysis of the patterns of co-occurrence of these lesions in wild English sole are consistent with the concept that these lesions represent morphologically identifiable steps forming a sequence of progression ultimately leading to the development of hepatic neoplasms. This progressive sequence parallels the pattern identified in experimental models of chemically induced hepatocarcinogenesis in rodents. The rationale for the hypothesis that these lesions in wild English sole can be caused by exposure to certain hepatoxic and hepatocarcinogenic xenobiotic compounds in the marine environment is based on the demonstration of significant and consistent statistical associations between levels of aromatic hydrocarbons (AHs) in sediment and prevalences of these idiopathic liver lesions; a significant contribution by sediment AHs to the variability in hepatic neoplasm prevalence in a logistic regression model; significantly increased probabilities for several idiopathic lesions in sole from chemically contaminated sites in Puget Sound; significant correlations between uptake of polycyclic aromatic hydrocarbons, as measured by levels of fluorescent metabolites of aromatic compounds in bile of sole, and prevalences of several hepatic lesion types; and experimental induction of unique degenerative, proliferative, and putatively preneoplastic focal lesions in English sole injected with either benzo(a)pyrene or a polycyclic aromatic hydrocarbons (PAH) enriched fraction of an extract from a contaminated urban sediment from Puget Sound.(ABSTRACT TRUNCATED AT 250 WORDS)

A study was conducted to assess the potential for uptake of toxic chemicals by down-stream migrant salmon in an urban estuary. Juvenile chinook salmon (Oncorhynchus tshawytscha) were collected from the Duwamish Waterway (located in Seattle, Washington) and from the Nisqually River (a reference site). The mean concentrations of aromatic hydrocarbons and polychlorinated biphenyls (PCBs) in the stomach contents (food organisms) of salmon from the Duwamish Waterway were approximately 650 times and 4 times, respectively, higher than those in salmon from the Nisqually River. Similarly, the mean concentration of bile metabolites of aromatic compounds which fluoresce at benzo[a]pyrene wavelengths was 24 times higher in the urban salmon compared to the reference salmon, whereas the mean concentration of PCBs in liver of urban salmon was 3 times higher than that in reference salmon. The study clearly demonstrated that, during their residency in this urban estuary, juvenile chinook salmon bioaccumulate substantial levels of toxic chemicals. The possible effects of these chemical exposures on the health and survival of this species are not presently known.

Winter flounder (Pseudopleuronectes americanus) were sampled from three sites located near Norwalk, New Haven, and Niantic, Connecticut, in Long Island Sound during February 1987, to evaluate the degree of chemical contamination and to determine possible effects of contaminant exposure. At each site, sediment and infaunal invertebrates were also collected and analyzed for trace metals and organic chemicals. Specimens of liver and kidney from winter flounder were examined for histopathological conditions, including the presence of macrophage aggregates in liver tissue. Liver samples were also analyzed for DNA damage (i.e., the formation of adducts between DNA and chemical contaminants). Blood samples were collected and analyzed for erythrocyte micronuclei. The sampling site near New Haven was determined to be the most affected site, from the standpoints of greater chemical contamination and possible effects on winter flounder. Concentrations of aromatic hydrocarbons (AHs) and polychlorinated biphenyls (PCBs) were highest in sediment from this site, and the highest prevalences of the histopathological changes and DNA alterations were also found in the livers of winter flounder from this site. No differences in the concentrations of contaminants in fish or in frequencies of erythrocyte micronuclei in fish blood were found between sites. None of the sites sampled had contaminant levels or prevalences of lesions as high as previously found at other East Coast locations (e.g., Boston Harbor, Massachusetts, Raritan Bay, New York). Overall, our results indicate moderate levels of pollution at two of the urban sites in Long Island Sound and provide a framework for expanded studies to better define the extent and impact of chemical pollution in Long Island Sound.

A series of field studies was conducted between 1979 and 1985 in Puget Sound, Washington State, to investigate etiological relationships between prevalences of hepatic neoplasms in bottom-dwelling marine fish species, with emphasis on English sole (Parophrys vetulus), and concentrations of toxic chemicals in sediments and affected fish. Statistically significant (p less than or equal to 0.05) correlations have been found between the prevalences of hepatic neoplasms in English sole and the following parameters: sediment concentrations of aromatic hydrocarbons, and concentrations of the metabolites of aromatic compounds in the bile of affected sole. A significant difference (p less than 0.001) was also found between the relative concentrations of aromatic free radicals in the liver microsomes of English sole with liver lesions compared to sole without liver lesions. Laboratory studies designed to evaluate the etiology of the liver neoplasms in English sole have also yielded evidence that is consistent with the view that high molecular weight aromatic hydrocarbons, e.g., benzo[a]pyrene (BaP), are hepatocarcinogens in English sole. The current status of a series of long-term (up to 18 months) exposures of English sole and rainbow trout (Salmo gairdneri) to selected fractions of Puget Sound sediment extracts, enriched with aromatic hydrocarbons and nitrogen-containing aromatic compounds, and to individual carcinogens (e.g., BaP) is discussed. Images FIGURE 6. FIGURE 7. FIGURE 8. FIGURE 9. FIGURE 10. FIGURE 11. FIGURE 12.

A rapid procedure for the determination of naphthalene and its metabolites in bile of rainbow trout and mice is described. The integrated analytical techniques combine high-performance liquid chromatography/ultraviolet fluorescence detection and plasma desotption/chemical ionization mass spectrometry for identification and quantitation. After separation by reverse-phase liquid chromatography, naphthalene and its metablolites are detected and quantitated by ultraviolet fluoresence spectometry. Identification of two metabolites is confirmed by mass spectometry. A direct insertion probe tip for a conventional chemical ionization mass spectometer was modified to obtain spectra of thermally labile compounds. A spectrum of less than 100 ng of naphthyl glucuronide, a labile glucuronic acid conjugate of 1-naphthol, was obtained with this system.

A bacterial bioluminescence assay was evaluated for assessing the toxicity of contaminated marine sediments. Preliminary assays established the feasibility of testing methanol–dichloromethane sediment extracts and demonstrated the advantages of solvent-exchanging the extracts into ethanol before testing. Bioluminescence assays were then conducted on extracts of 18 marine sediments varying in nature and degree of chemical contamination. Statistical analyses revealed significant associations between acute toxicity expressed as a 15-min EC50 (the concentration of extract causing a 50% reduction in bioluminescence after a 15-min exposure) and concentrations of sums of measured aromatic hydrocarbons, naphthalenes, and chlorinated hydrocarbons. We conclude that the bioluminescence assay is useful as a rapid method of comparing and ranking the toxicity of organic extracts of contaminated sediments.

Petroleum is composed of a complex mixture of hydrocarbons that readily undergo chemical and biological conversions on entering aquatic environments. These conversions lead to the formation of a host of oxygenated products, some of which are potentially toxic to marine life and to the consumer of fishery products. State-of-the-art analytical methods, as employed in our laboratories, utilize glass-capillary gas chromatography in conjunction with mass spectrometry to analyze environmental samples containing trace amounts of aliphatic and aromatic petroleum hydrocarbons. These procedures are applied on a routine basis to the analysis of seawater, sediments and tissues of marine organisms. Despite this analytical proficiency, a need exists for analyzing oxygenated and other polar petroleum products in environmental samples. For example, techniques such as high-performance liquid chromatography (HPLC), in conjunction with on-line fluorometric assay techniques and mass spectrometry, make possible the analysis of polar oxygenated compounds resulting from both chemical and biological conversions. These methodologies are first steps toward the development of routine assay procedures for environmental samples. Current techniques for hydrocarbon analyses and new methods for analyzing polar aromatic compounds are discussed.

On March 19, 1984, more than 170000 gallons of oil were spilled into the Columbia River. We had recently developed analytical methods for estimating the exposure of fish to aromatic compounds by measuring the concentrations of metabolites of these contaminants in fish bile. The oil spill provided an opportunity to field test our methods in assessing the exposure of fish to petroleum aromatic compounds from the spilled oil. Our findings indicated that, within 5 days after the spill, mean concentrations of metabolites of aromatic compounds in the bile of white sturgeon (Acipenser transmontanus) captured 57 miles downstream from the spill were significantly higher than those of sturgeon caught upriver.

Statistical comparisons were made between the relative mean concentrations of metabolites of aromatic compounds in bile (as estimated by a high-performance liquid chromatography/fluorescence detection method) and idiopathic liver lesions in English sole (Parophrys vetulus) from eleven Puget Sound sites. A significant positive correlation was found between prevalences of neoplasms, foci of cellular alteration, megalocytic hepatosis and total hepatic lesions and the concentrations of metabolites of aromatic compounds. These findings provide further evidence of the putative relationship between aromatic compounds and serious idiopathic liver diseases in bottom-dwelling fish.

Interest in the assessment of the anthropogenic contamination of the marine environment has accelerated in recent years. Existing methods to analyze environmental samples (e.g., sediments or tissues) for trace amounts of organic contaminants such as aromatic hydrocarbons and chlorinated compounds are tedious and costly. We report a rapid, efficient high-performance liquid chromatography (HPLC) procedure which uses a size-exclusion column to separate the analytes of interest from interfering compounds in the sample matrix. Analytical results from the HPLC method were, in general, comparable to a gravity-column method which had been used for several years. The HPLC method had several other advantages: improved precision; the ability to monitor chromatographic conditions; the potential for automating analyses; and reduced consumption of solvents and other materials.

Field studies were conducted over a 5-year period in Puget Sound to investigate etiological relationships between prevalences of hepatic neoplasms in bottom-dwelling marine fish, with emphasis on English sole (Parophrys vetulus), and concentrations of toxic chemicals in sediments and bottom fish. Statistically significant correlations were found between prevalences of hepatic neoplasms and (1) sediment concentrations of aromatic hydrocarbons, and (2) concentrations of metabolites of aromatic compounds in the bile. A significant difference was also found between the concentrations of N-oxyl derivatives of nitrogen heterocycles (free radicals) in liver microsomes of English sole with liver lesions compared to sole without liver lesions. These N-oxyl free radicals were apparently metabolically derived from complex suites of nitrogen heterocycles present in Puget Sound environments. Recent evidence suggests that aromatic free radicals (possibly derivatives of aromatic hydrocarbons) may also be bound to hepatic DNA isolated from English sole with liver neoplasms collected in several polluted areas of Puget Sound. Such evidence for xenobiotic-DNA interactions has thus far not been obtained for fish from reference areas and for lesion-free fish from polluted areas. Various laboratory studies designed to evaluate the etiology of the liver neoplasms have also yielded evidence that is consistent with the view that high molecular weight aromatic hydrocarbons play a significant role in the etiology of hepatic tumors in the bottom-dwelling fish. Associations between chemical exposures and diseases in other marine species from southern California and Boston Harbor are also discussed. Similar relationships between liver and skin neoplasms in selected bottom feeding fresh water species and sediment-associated chemicals are also presented.