Cyst-based toxicity tests. XVIII. Application of ostracodtoxkit microbiotest in a bioremediation project of oil-contaminated sediments: sensitivity comparison with Hyalella azteca solid-phase assay

To further validate the scope of use of the 6-day sediment contact microbiotest conducted with the ostracod Heterocypris incongruens, we compared the sensitivity of this small-scale culture/maintenance-free assay with the 14-day solid-phase Hyalella azteca test. The present study was undertaken within the framework of a Canadian bioremediation project on oil-contaminated freshwater sediments along an intertidal shoreline of the Saint-Lawrence River near the town of Sainte-Croix, Quebec, Canada. Sediment subsamples, collected during three sampling periods over 21 weeks from five plots (each with four replicates) contaminated with different treatments, were analyzed for their toxic effects on the two test species. Sediment samples taken immediately after the plots were spread with oil were very toxic to both crustaceans (mortality between 80% and 100%), but the mortality of the amphipods was substantially lower than that of the ostracods for samples collected after 6 weeks. Fifteen weeks after the onset of the controlled oil spill experiment, the sediments of all plots were still quite toxic to Heterocypris but not to Hyalella. Statistical analysis of the mortality figures was performed by Blaise et at. (2003) and revealed a statistically significant correlation (R = 0.584 at the P = 0.001 level) between data pairs. Analysis of the precision of the two assays showed a substantially higher uniformity (lower variation coefficients between the four replicas) of the ostracod results over that of the amphipod assay. This study corroborated the findings of two previous investigations conducted in Canada and in Belgium with the same test species. All three investigations concur in pointing out the potential of the new ostracod microbiotest as a reliable and sensitive ecotoxicological test for routine and low-cost monitoring of contaminated sediments.     

Toxicity assessment of oil-contaminated freshwater sediments

The performance of four microscale toxicity bioassays conducted on whole sediments was evaluated during a bioremediation project undertaken in 1999-2000 on a crude oil-contaminated freshwater shoreline of the St. Lawrence River, Quebec, Canada. The toxicity tests assessed included: (1) the Microtox solid-phase assay (MSPT), (2) the Biotox Flash solid-phase test (Flash), (3) the algal solid-phase assay (ASPA), and 4) the Ostracodtoxkit solid-phase assay. Data generated with these assays were compared with those obtained using the standard endobenthic amphipod (Hyalella azteca) bioassay. Bioanalytical comparisons indicated that all five solid-phase tests were useful in detecting the toxicity of oiled sediments; however, statistical analyses distinguished a difference in response between the invertebrate (amphipod and Ostracodtoxkit) and bacterial luminescence tests (MSPT and Flash). Based on these results, it is recommended that careful selection of biotests be made in the design of the test battery for assessment of residual oil sediment toxicity. Time-series toxicity data generated with ASPA indicated that oiled sediments in the freshwater wetlands of the St. Lawrence River remained toxic to phytoplankton for at least 65 weeks and that remediation treatment was able to accelerate detoxification by 16 weeks.     

Regulatory influence of light and temperature on petroleum toxicity to Anabaena doliolum

Effects of light and temperature, the most important variables in natural environments, on the toxicity of Assam crude oil extract were examined. Illumination of oil extract increased the degree of inhibition of growth, photosynthesis, and respiration of Anabaena doliolum. Higher light intensities were more effective in inhibiting the test organism. This may result from the formation of highly toxic photooxidation products. The toxicity of oil extract also showed an increase with increase in temperature, even though it might be envisioned that elevated temperature would facilitate the loss of highly toxic volatile fractions. It is possible, therefore, that the toxic effects of petroleum might be more severe in tropical than in temperate climates.     

Adaptation of microalgae to a gradient of continuous petroleum contamination

In order to study adaptation of microalgae to petroleum contamination, we have examined an environmental stress gradient by crude oil contamination in the Arroyo Minero River (AMR), Argentina. Underground crude oil has constantly leaked out since 1915 as a consequence of test drilling for possible petroleum exploitation. Numerous microalgae species proliferated in AMR upstream of the crude oil spill. In contrast, only four microalgal species were detected in the crude oil spill area. Species richness increases again downstream. Microalgae biomass in the crude oil spill area is dominated by a mesophile species, Scenedesmus sp. Effects of oil samples from AMR spill on photosynthetic performance and growth were studied using laboratory cultures of two Scenedesmus sp. strains. One strain (Se-co) was isolated from the crude oil spill area. The other strain (Se-pr) was isolated from a pristine area without petroleum contamination. Crude oil has undetectable effects on Se-co strain. In contrast crude oil rapidly destroys Se-pr strain. However, Se-pr strain can adapt to low doses of petroleum (≤3% v/v total hydrocarbons/water) by means of physiological acclimatization. In contrast, only rare crude oil-resistant mutants are able to grow under high levels of crude oil (≥10% v/v total hydrocarbons/water). These crude oil-resistant mutants have arisen through rare spontaneous mutations that occur prior to crude oil exposure. Species richness in different areas of AMR is closely connected to the kind of mechanism (genetic adaptation vs. physiological acclimatization) that allows adaptation. Resistant-mutants are enough to assure the survival of microalgal species under catastrophic crude oil spill.     

A Population Genetic Analysis of the Potential for a Crude Oil Spill to Induce Heritable Mutations and Impact Natural Populations

The primary environmental impact following an oil spill typically is acute toxicity to fish and wildlife. However, multigenerational effects through toxicant-induced heritable mutations might also occur. Some polycyclic aromatic hydrocarbon (PAH) components of crude oil are potentially mutagenic, although specific components and doses that induce mutations are poorly known. We applied population genetics concepts to assess the extent of mortality and the persistence of deleterious heritable mutations resulting from exposure to potential mutagens, such as crude oil. If lethal mutations are induced, the population will experience some mortality, but the mutations are quickly removed or reduced to low frequency by natural selection. This occurs within one or a few generations when mutations are dominant or partially recessive. Totally recessive alleles persist in low frequency for many generations, but result in relatively little impact on the population, depending on the number of mutated loci. We also applied population genetics concepts to assess the potential for heritable mutations induced by the Exxon Valdez oil spill in Prince William Sound, Alaska, to affect pink salmon populations. We stress that breeding units (e.g., streams with distinct spawning populations of salmon) must be considered individually to assess heritable genetic effects. For several streams impacted by the oil spill, there is inconsistency between observed egg mortality and that expected if lethal heritable mutations had been induced by exposure to crude oil. Observed mortality was either higher or lower than expected depending on the spawning population, year, and cohort considered. Any potential subtle effect of lethal mutations induced by the Exxon Valdez oil spill is overridden by natural environmental variation among spawning areas. We discuss the need to focus on population-level effects in toxicological assessments because fish and wildlife management focuses on populations, not individuals.     

A combined subchronic (90-day) toxicity and neurotoxicity study of a single-cell source of docosahexaenoic acid triglyceride (DHASCO oil).

Docosahexaenoic acid (DHA), a 22-carbon long-chain polyunsaturated fatty acid of the omega-3 family, is a major structural component of neural membranes and is a particularly important nutrient during infant development. New safe and well-defined sources of DHA are required for infant formula fortification and dietary supplementation. DHASCO oil is an algal-derived triglyceride containing 40-50% DHA. Previous studies have shown that DHASCO oil is neither mutagenic nor toxic in acute or 28-day subchronic tests. To further establish the safety of this oil, a 90-day subchronic toxicity study in rats which included haematology, clinical chemistry, pathology and ophthalmologic, neurobehavioural and neuropathological assessments, using doses of 0.5 and 1.25g/kg body weight/day was performed. There were no treatment-related adverse effects in any of the parameters measured at either dose. Based on these results, the no-adverse-effect level (NOAEL) for DHASCO oil under the conditions of this study corresponds to the highest dose level. The DHA in the DHASCO oil was bioavailable, resulting in significant elevations in the levels of this fatty acid in liver, heart and brain after 90 days of administration. In conclusion, this 90-day subchronic toxicity study provides additional evidence that DHASCO oil is a safe and bioavailable source of dietary DHA.     

Ingestion and sublethal effects of physically and chemically dispersed crude oil on marine planktonic copepods

Planktonic copepods play a key function in marine ecosystems, however, little is known about the effects of dispersants and chemically dispersed crude oil on these important planktonic organisms. We examined the potential for the copepods Acartia tonsa, Temora turbinata and Parvocalanus crassirostris to ingest crude oil droplets and determined the acute toxicity of the dispersant Corexit^® 9500A, and physically and chemically dispersed crude oil to these copepods. We detected ingestion of crude oil droplets by adults and nauplii of the three copepod species. Exposure to crude oil alone (1 µL L^?1, 48 h) caused a reduction of egg production rates (EPRs) by 26–39 %, fecal pellet production rates (PPRs) by 11–27 %, and egg hatching (EH) by 1–38 % compared to the controls, depending on the species. Dispersant alone (0.05 µL L^?1, 48 h) produced a reduction in EPR, PPR and EH by 20–35, 12–23 and 2–11 %, respectively. Dispersant-treated crude oil was the most toxic treatment, ~1.6 times more toxic than crude oil alone, causing a reduction in EPR, PPR and EH by 45–54, 28–41 and 11–31 %, respectively. Our results indicate that low concentrations of dispersant Corexit 9500A and chemically dispersed crude oil are toxic to marine zooplankton, and that the ingestion of crude oil droplets by copepods may be an important route by which crude oil pollution can enter marine food webs.     

Chemical dispersant potentiates crude oil impacts on growth, reproduction, and gene expression in Caenorhabditis elegans

The economic, environmental, and human health impacts of the deepwater horizon (DWH) oil spill have been of significant concern in the general public and among scientists. This study employs parallel experiments to test the effects of crude oil from the DWH oil well, chemical dispersant Corexit 9500A, and dispersant-oil mixture on growth and reproduction in the model organism Caenorhabditis elegans. Both the crude oil and the dispersant significantly inhibited the reproduction of C. elegans. Dose-dependent inhibitions of hatched larvae production were observed in worms exposed to both crude oil and dispersant. Importantly, the chemical dispersant Corexit 9500A potentiated crude oil effects; dispersant-oil mixture induced more significant effects than oil or dispersant-alone exposures. While oil-alone exposure and dispersant-alone exposure have none to moderate inhibitory effects on hatched larvae production, respectively, the mixture of dispersant and oil induced much more significant inhibition of offspring production. The production of hatched larvae was almost completely inhibited by several high concentrations of the dispersant-oil mixture. This suggests a sensitive bioassay for future investigation of oil/dispersant impacts on organisms. We also investigated the effects of crude oil/dispersant exposure at the molecular level by measuring the expressions of 31 functional genes. Results showed that the dispersant and the dispersant-oil mixture induced aberrant expressions of 12 protein-coding genes (cat-4, trxr-2, sdhb-1, lev-8, lin-39, unc-115, prdx-3, sod-1, acr-16, ric-3, unc-68, and acr-8). These 12 genes are associated with a variety of biological processes, including egg-laying, oxidative stress, muscle contraction, and neurological functions. In summary, the toxicity potentiating effect of chemical dispersant must be taken into consideration in future crude oil cleanup applications.     

The safety of synthetic zeolites used in detergents

Synthetic zeolites are replacing phosphates as builders in laundry detergents; workers and consumers may, therefore, increasingly be exposed to these materials and it is important to assess their safety. This article puts mechanistic, toxicological and exposure data into context for a safety assessment. Zeolites are hygroscopic compounds with ion-exchanging properties. They may partially decompose under acidic conditions such as in the stomach releasing sodium ions, silicic acid and aluminum salts. The intact molecule is not bioavailable after oral intake or exposure through the dermal and inhalational routes. Under current conditions of manufacture and use, no systemic toxicity is to be expected from neither the intact molecule nor the degradation products; a significant effect on the bioavailability of other compounds is not likely. Zeolites may cause local irritation. It is, therefore, important to minimise occupational exposure. The co-operation of detergent manufacturers with the manufacturers of washing machines is necessary to find the right balance between environmental aspects such as energy and water savings and the occurrence of detergent residues on textiles due to insufficient rinsing.     

Effects of purification on the bioavailability of botulinum neurotoxin type A

Botulinum neurotoxins (BoNTs) are among the most potent biological toxins for humans. They are primarily produced by the gram-positive, anaerobic spore-forming bacterium, Clostridium botulinum. In bacterial cultures, secreted BoNTs are associated with non-toxic accessory proteins forming large complexes. Neurotoxin-associated proteins have been shown to play an important role in the oral toxicity of BoNTs by protecting them from degradation and digestion by gastric acid and enzymes. Most toxicity studies using BoNTs have been performed using highly purified toxin. In this study, the toxicities of purified and crude BoNT/A toxin preparations were compared. Protein components secreted into culture supernatants along with BoNT/A were identified by mass spectrometry and the contribution of extra proteins found in the soluble crude toxin extracts to the toxicity of BoNTs was determined in mouse models of oral and parenteral botulinum intoxication. Analysis of crude toxin composition permitted assessment of the impact of accessory proteins on the oral bioavailability of BoNT/A toxin in food matrices.     

A contribution to safety assessment of veterinary drug residues: in vitro/ex vivo studies on the intestinal toxicity and transport of covalently bound residues.

1. The gastrointestinal fate of protein-bound residues of the model compound furazolidone (FZD) was investigated in vitro and ex vivo. Protein-bound residues were generated in rat liver microsomes, isolated by solvent extraction and digested with 0.5% hydrochloric acid and Pronase E. 2. During digestion, 3-amino-2-oxazolidinone (AOZ), the side chain of furazolidone, was partly released from bound residues. 3. The absorption of free AOZ and digested protein-bound residues was tested in isolated perfused rat gut segments (IPGS) and in the intestinal cell line Caco-2. Free AOZ was transfered both in the IPGS model and in Caco-2 monolayer cultures, while no indications for passage of bound residues were obtained. 4. No acute toxicity of AOZ or digested food residues respectively was observed in gut segments and Caco-2 cells at concentrations that were substantially above maximum residue levels to be expected in food of animal origin after administration of therapeutic doses. 5. The results demonstrate that digestive processes can alter the chemical nature of drug residues and yield degradation products that may be bioavailable for the consumer. Thus, the covalent binding of xenobiotics to macromolecular tissue constituents cannot necessarily be regarded as an irreversible endpoint of residue bioavailability and toxicity.     

Essentiality and toxicity of selenium and its status in Australia: a review

Selenium (Se) is an essential trace element for animals and humans because of its role in an antioxidant enzyme glutathione peroxidase. This enzyme protects cell membranes from damage caused by the peroxidation of lipids. The paper provides an overview of the effects of Se toxicity and deficiency in humans and animals. It is well established that Se deficiency causes health implications in humans and animals. Se is also very toxic and can cause Se poisoning (selenosis) in humans and animals. In Australia, Se deficiency has caused health problem to livestock; however, the problems were eliminated after the introduction of Se supplementation. Se toxicity has also been reported in some regions of Australia as a result of livestock feeding on Se accumulative plant species. The major source of Se is diet, and in many regions of the world the levels of Se in the soils generally reflect the Se status in human populations. In foods, the bioavailability and toxicity of Se depend on its chemical forms. Generally, organic forms of Se are more bioavailable and less toxic than the inorganic forms (selenites, selenates). The Se status in the Australian population and how this is compared with the rest of the world is also discussed.     

Responses of sympatric Karenia brevis,Prorocentrum minimum,and Heterosigma akashiwo to the exposure of crude oil

Impacts of the Deepwater Horizon oil spill on phytoplankton, particularly, the tolerability and changes to the toxin profiles of harmful toxic algal species remain unknown. The degree to which oil-affected sympatric Karenia brevis, Prorocentrum minimum, and Heterosigma akashiwo, all of which are ecologically important species in the Gulf of Mexico, was investigated. Comparison of their tolerability to that of non-toxic species showed that the toxin-production potential of harmful species does not provide a selective advantage. Investigated toxin profiles for K. brevis and P. minimum demonstrated an increase in toxin productivity at the lowest crude oil concentration (0.66 mg L^?1) tested in this study. Higher crude oil concentrations led to significant growth inhibition and a decrease in toxin production. Findings from this study could assist in the assessment of shellfish bed closures due to high risk of increased toxin potential of these phytoplankton species, especially during times of stressed conditions.     

Biological effects and toxicity of diluted bitumen and its constituents in freshwater systems

Approximately 50 billion cubic meters of bitumen resides within the oil sands region of Alberta, Canada. To facilitate the transport of bitumen from where it is extracted to where it is processed, the bitumen is diluted with natural gas condensate (‘dilbit’), synthetic crude from hydrocracking bitumen (‘synbit’), or a mixture of both (‘dilsynbit’). A primary consideration for the effects of diluted bitumen products on freshwater organisms and ecosystems is whether it will float on the water surface or sink and interact with the stream or lake sediments. Evidence from a spill near Kalamazoo, MI, in 2010 and laboratory testing demonstrate that the nature of the spill and weathering of the dilbit, synbit or dilsynbit prior to and during contact with water will dictate whether the product floats or sinks. Subsequent toxicological data on the effects of dilbit and other diluted bitumen products on freshwater organisms and ecosystems are scarce. However, the current literature indicates that dilbit or bitumen can have significant effects on a wide variety of toxicological endpoints. This review synthesizes the currently available literature concerning the fate and effects of dilbit and synbit spilled into freshwater, and the effects of bitumen and bitumen products on aquatic organisms and ecosystems. Dilbit is likely to provide ecological impacts that are similar to and extend from those that follow from exposure to lighter crude oil, but the prospect of bitumen settling after binding to suspended sediments elevates the risk for benthic impacts in streams and lakes. Copyright © 2015 John Wiley & Sons, Ltd.     

Toxic effects of chemical dispersant Corexit 9500 on water flea Daphnia magna

In 2010, approximately 2.1 million gallons of chemical dispersants, mainly Corexit 9500, were applied in the Gulf of Mexico to prevent the oil slick from reaching shorelines and to accelerate biodegradation of oil during the Deepwater Horizon oil spill. Recent studies have revealed toxic effects of Corexit 9500 on marine microzooplankton that play important roles in food chains in marine ecosystems. However, there is still little known about the toxic effects of Corexit 9500 on freshwater zooplankton, even though oil spills do occur in freshwater and chemical dispersants may be used in response to these spills. The cladoceran crustacean, water flea Daphnia magna , is a well‐established model species for various toxicological tests, including detection of juvenile hormone‐like activity in test compounds. In this study, we conducted laboratory experiments to investigate the acute and chronic toxicity of Corexit 9500 using D. magna . The acute toxicity test was conducted according to OECD TG202 and the 48 h EC₅₀ was 1.31 ppm (CIs 0.99–1.64 ppm). The reproductive chronic toxicity test was performed following OECD TG211 ANNEX 7 and 21 days LOEC and NOEC values were 4.0 and 2.0 ppm, respectively. These results indicate that Corexit 9500 has toxic effects on daphnids, particularly during the neonatal developmental stage, which is consistent with marine zooplankton results, whereas juvenile hormone‐like activity was not identified. Therefore, our findings of the adverse effects of Corexit 9500 on daphnids suggest that application of this type of chemical dispersant may have catastrophic impacts on freshwater ecosystems by disrupting the key food chain network. Copyright © 2016 John Wiley & Sons, Ltd.     

Toxicity assessment and bioaccumulation in zebrafish embryos exposed to carbon nanotubes suspended in Pluronic® F-108

Carbon nanotubes (CNTs) are often suspended in Pluronic® surfactants by sonication, which may confound toxicity studies because sonication of surfactants can create degradation products that are toxic to mammalian cells. Here, we present a toxicity assessment of Pluronic® F-108 with and without suspended CNTs using embryonic zebrafish as an in vivo model. Pluronic® sonolytic degradation products were toxic to zebrafish embryos just as they were to mammalian cells. When the toxic Pluronic® fragments were removed, there was little effect of pristine multi-walled CNTs (pMWNTs), carboxylated MWNTs (cMWNTs) or pristine single-walled carbon nanotubes (pSWNTs) on embryo viability and development, even at high concentrations. A gel electrophoretic method coupled with Raman imaging was developed to measure the bioaccumulation of CNTs by zebrafish embryos, and dose-dependent uptake of CNTs was observed. These data indicate that embryos accumulate pMWNTs, cMWNTs and pSWNTs yet there is very little embryo toxicity.     

Consumer product in vitro digestion model: Bioaccessibility of contaminants and its application in risk assessment

This paper describes the applicability of in vitro digestion models as a tool for consumer products in (ad hoc) risk assessment. In current risk assessment, oral bioavailability from a specific product is considered to be equal to bioavailability found in toxicity studies in which contaminants are usually ingested via liquids or food matrices. To become bioavailable, contaminants must first be released from the product during the digestion process (i.e. become bioaccessible). Contaminants in consumer products may be less bioaccessible than contaminants in liquid or food. Therefore, the actual risk after oral exposure could be overestimated. This paper describes the applicability of a simple, reliable, fast and relatively inexpensive in vitro method for determining the bioaccessibility of a contaminant from a consumer product. Different models, representing sucking and/or swallowing were developed. The experimental design of each model can be adjusted to the appropriate exposure scenarios as determined by the risk assessor. Several contaminated consumer products were tested in the various models. Although relevant in vivo data are scare, we succeeded to preliminary validate the model for one case. This case showed good correlation and never underestimated the bioavailability. However, validation check needs to be continued.     

Flow Cytometric Determination of Genotoxic Effects of Exposure to Petroleum in Mink and Sea Otters

Three experiments were conducted to investigate the genotoxic effects of crude oil on mink and sea otters. In the first experiment, the effects on mink of chronic exposure to weathered Prudhoe Bay crude oil were studied. Female mink were fed a diet that included weathered crude oil for a period of 3 weeks prior to mating, during pregnancy and until weaning. Kits were exposed through lactation and by diet after weaning until 4 months of age. Kidney and liver tissues of the kits were examined using flow cytometry (FCM) and it was found that the genome size was increased in kidney samples from the experimental group compared to the control group. This effect was probably due to some type of DNA amplification and it could have been inherited from the exposed mothers or have been a somatic response to oil exposure in the pups. No evidence of clastogenic effects, as measured by the coefficient of variation (CV) of the G1 peak, was found in kidney or liver tissue. In the second experiment, yearling female mink were exposed either by diet or externally to crude oil or bunker C fuel oil. Evidence for clastogenic damage was found in spleen tissue for the exposure groups, but not in kidney tissue. No evidence of increased genome size was observed. In the third experiment, blood was obtained from wild-caught sea otters in Prince William Sound. The sea otters represented two populations: one from western Prince William Sound that was potentially exposed to oil from the Exxon Valdez oil spill and a reference population from eastern Prince William Sound that did not receive oil from the spill. The spill had occurred 1.5 years prior to obtaining the blood samples. Although the mean CVs did not differ between the populations, the exposed population had a significantly higher variance of CV measurements and five out of 15 animals from the exposed population had CVs higher than the 95% confidence limits of the reference population. It is concluded that FCM is a sensitive indicator of the clastogenic effects of oil exposure and that haematopoietic tissues and blood are best for detecting clastogenic damage. Moreover, the observed differences in the genome size of the kidney cells were possibly heritable effects, but this needs further investigation. Lastly, sea otters exposed to spilled oil 1.5 years earlier showed evidence of clastogenic damage in one-third of the individuals sampled.     

巴豆发酵品与生巴豆、巴豆霜中毒性成分的含量比较

目的:解决炮制对巴豆减毒存在的缺陷问题。方法:采用重量分析法和考马斯亮蓝G-250染色法分别测定2种真菌(灵芝菌和白僵菌)的巴豆发酵品(分别被称为"灵巴菌质"和"白巴菌质")、生巴豆和巴豆霜中毒性成分脂肪油和总蛋白的含量;并利用气相色谱-质谱(GC-MS)联用法对所得4种样品的脂肪油进行化学成分分析。结果:巴豆经灵芝菌和白僵菌发酵后,其毒性成分脂肪油和总蛋白的含量明显降低,且均低于巴豆霜;生巴豆、巴豆霜、灵巴菌质和白巴菌质的脂肪油中分别鉴定出39、24、36和42种成分。与生巴豆相比,2种发酵品脂肪油成分的组成和相对含量产生了明显变化;且灵巴菌质和白巴菌质中各出现了5种新成分。结论:巴豆经发酵后毒性成分含量明显降低,其组成成分也发生较大变化。本试验可为发酵对巴豆的"去毒存效"奠定物质基础。     

K-ras oncogene DNA sequences in pink salmon in streams impacted by the Exxon Valdez oil spill: no evidence of oil-induced heritable mutations

It was hypothesized in previous studies that the Exxon Valdez oil spill in Prince William Sound, Alaska, induced heritable mutations and resulted in mortality of pink salmon (Oncorhynchus gorbuscha) embryos. In one of these studies, laboratory exposure of pink salmon embryos to crude oil resulted in apparent mutation-induction in exon 1 and exon 2 of the K-ras oncogene, but no fish from the area impacted by the oil spill were analyzed. We assessed K-ras exon 1 and exon 2 DNA sequences in pink salmon from five streams that were oiled and five streams that were not oiled by the Exxon Valdez oil spill in Prince William Sound, and two streams with natural oil seeps and one stream without seeps on the Alaska Peninsula. Of the 79 fish analyzed for exon 1 and the 89 fish analyzed for exon 2, none had the nucleotide substitutions representing the mutations induced in the laboratory study. Other variable nucleotides occurred in similar proportions in oiled and non-oiled streams and probably represent natural allelic variation. These data do not support the hypothesis that heritable mutations in the K-ras gene were induced by the Exxon Valdez oil spill or oil seeps.