Characterization of basic immune function parameters in the fathead minnow (Pimephales promelas), a common model in environmental toxicity testing.

The fathead minnow (Pimephales promelas) is an environmental sentinel species, commonly used in toxicity testing. However, there is a lack of data regarding basic immune function in this species. To improve the usefulness of the fathead minnow as a model for basic immune function and immunotoxicity, this study sought to 1) compare the differential expression of immune function genes in naïve fathead minnows and 2) determine the effects of pathogen exposure on immune gene expression and spleen index. To accomplish this, kidney, spleen and liver tissue were collected three days post injection (dpi) from adult male fathead minnows from each of the following groups: 1) uninjected control 2) sham-injected (Hank's balanced salt solution) and 3) pathogen-injected (Yersinia ruckeri). Spleen tissue was also collected at seven and 14 dpi. Differential tissue expression of immune function genes was evaluated in naïve minnows and expression patterns were similar to those found in other fish species, with liver tissue generally having the highest amount of expression. Following pathogen injection, the expression of complement component 3 (c3) (4.4-fold, kidney; 2.5-fold, liver), interleukin 11 (il11) (4.8-fold, kidney; 15.2-fold, liver) and interleukin 1ß (il1ß) (8.2-fold, kidney; 17.2-fold, spleen; 2.6-fold, liver) were significantly upregulated. Elastase 2 (elas2) was significantly downregulated (5.8-fold) in liver tissue. A significant increase in spleen index at seven dpi was also observed in pathogen-injected minnows. This study has identified endpoints that are part of the normal response to pathogen in fathead minnows, an essential step toward the development of the fathead minnow as a model for immunotoxicity evaluations.

Methylmercury Bioaccumulation in Stream Food Webs Declines with Increasing Primary Production.

Opposing hypotheses posit that increasing primary productivity should result in either greater or lesser contaminant accumulation in stream food webs. We conducted an experiment to evaluate primary productivity effects on MeHg accumulation in stream consumers. We varied light for 16 artificial streams creating a productivity gradient (oxygen production =0.048-0.71 mg O2 L(-1) d(-1)) among streams. Two-level food webs were established consisting of phytoplankton/filter feeding clam, periphyton/grazing snail, and leaves/shredding amphipod (Hyalella azteca). Phytoplankton and periphyton biomass, along with MeHg removal from the water column, increased significantly with productivity, but MeHg concentrations in these primary producers declined. Methylmercury concentrations in clams and snails also declined with productivity, and consumer concentrations were strongly correlated with MeHg concentrations in primary producers. Heterotroph biomass on leaves, MeHg in leaves, and MeHg in Hyalella were unrelated to stream productivity. Our results support the hypothesis that contaminant bioaccumulation declines with stream primary production via the mechanism of bloom dilution (MeHg burden per cell decreases in algal blooms), extending patterns of contaminant accumulation documented in lakes to lotic systems.

A comparison of commercially-available automated and manual extraction kits for the isolation of total RNA from small tissue samples.

BACKGROUND: This study compared the performance of five commercially available kits in extracting total RNA from small eukaryotic tissue samples (<15 mg). Total RNA was isolated from fathead minnow (Pimephales promelas) tissues (spleen, blood, kidney, embryo, and larvae) using the Qiagen RNeasy® Plus Mini, Qiagen RNeasy® Plus Universal, Promega Maxwell® 16 LEV simplyRNA, Ambion MagMAX™-96 and Promega SimplyRNA HT kits. Kit performance was evaluated via measures of RNA quantity (e.g., total RNA amount) and quality (e.g., ratio of absorbance at 260 and 280 nm, RNA integrity number (RIN), presence of gDNA). RESULTS: With the exception of embryos, each kit generally extracted ≥5 µg of total RNA from each sample. With regard to RNA quality, the RINs of RNA samples isolated via the Plus Mini and Maxwell® 16 kits were consistently higher than those of samples extracted via the remaining three kits and for all tissues, these kits produced intact RNA with average RIN values ≥7. The Plus Universal and SimplyRNA HT kits produced moderately degraded (RIN values <7, but ≥5), while the RNA recovered via the MagMAX™ kit tended to exhibit a high degree of degradation (RIN values <5). CONCLUSIONS: Each kit was generally capable of extracting the amount of RNA required for most downstream gene expression applications suggesting that RNA yield is unlikely to be a limiting factor for any of the kits evaluated. However, differences in the quality of RNA extracted via each of the kits indicate that these kits may differ in their ability to yield RNA acceptable for some applications. Overall, the findings of this study demonstrate that there are practical differences between commercially available RNA extraction kits that should be taken into account when selecting extraction methods to be used for isolating RNA designated for gene expression analysis.

Quantitative risk model for polycyclic aromatic hydrocarbon photoinduced toxicity in Pacific herring following the Exxon Valdez oil spill.

Phototoxicity occurs when exposure to ultraviolet radiation increases the toxicity of certain contaminants, including polycyclic aromatic hydrocarbons (PAHs). This study aimed to (1) develop a quantitative model to predict the risk of PAH phototoxicity to fish, (2) assess the predictive value of the model, and (3) estimate the risk of PAH phototoxicity to larval and young of year Pacific herring (Clupea pallasi) following the Exxon Valdez oil spill (EVOS) in Prince William Sound, Alaska. The model, in which median lethal times (LT50 values) are estimated from whole-body phototoxic PAH concentrations and ultraviolet A (UVA) exposure, was constructed from previously reported PAH phototoxicity data. The predictive value of this model was confirmed by the overlap of model-predicted and experimentally derived LT50 values. The model, along with UVA characterization data, was used to generate estimates for depths of de minimiz risk for PAH phototoxicity in young herring in 2003/2004 and immediately following the 1989 EVOS, assuming average and worst case conditions. Depths of de minimiz risk were estimated to be between 0 and 2 m deep when worst case UVA and PAH conditions were considered. A post hoc assessment determined that <1% of the young herring population would have been present at depths associated with significant risk of PAH phototoxicity in 2003/2004 and 1989.

Ultraviolet radiation affects invasibility of lake ecosystems by warm-water fish.

Predicting where species invasions will occur remains a substantial challenge in ecology, but identifying factors that ultimately constrain the distribution of potential invaders could facilitate successful prediction. Whereas ultraviolet radiation (UVR) is recognized as an important factor controlling species distribution and community composition, the role of UVR in a habitat invasibility context has not been explored. Here we examine how underwater UVR can regulate warm-water fish invasion. In Lake Tahoe, California and Nevada, USA, established populations of exotic bluegill sunfish (Lepomis macrochirus) are currently limited to turbid, low-UVR embayments. An in situ incubation experiment that manipulated incident UVR exposure of larval bluegill, combined with an assessment of UVR exposure levels in nearshore habitats around Lake Tahoe, demonstrates that UVR can mediate habitat invasibility. Our findings suggest that the susceptibility to invasion by UVR sensitive species may increase in transparent aquatic systems threatened by declining water quality, and they highlight the importance of abiotic factors as regulators of invasion risk in ecosystems.

CYP1A expression in caged rainbow trout discriminates among sites with various degrees of polychlorinated biphenyl contamination.

It has become increasingly apparent that resident fish can develop resistance to chemicals in their environment, thus compromising their usefulness as sentinels of site-specific pollution. By using a stream system whose resident fish appear to have developed pollutant resistance (Brammell et al., Mar Environ Res 58:251-255, 2005), we tested the hypothesis that the pollutant-inducible biomarker, cytochrome P4501A (CYP1A), as measured in field-caged juvenile rainbow trout (Oncorhynchus mykiss), would reflect relative pollution differences between reference and polychlorinated biphenyl (PCB)-contaminated sites. Trout were caged in the Town Branch/Mud River system (Logan County, KY), a stream system undergoing remediation for PCBs. Fish were held in remediated (Town Branch), unremeditated (Mud River), and reference sites for 2 weeks during spring 2002. At the end of this period, gill and hepatic CYP1A expression were measured. To evaluate the relative PCB exposure of caged trout and provide a reference point against which to calibrate CYP1A response, PCB levels were quantified in sediments from each site. Hepatic CYP1A expression in caged trout clearly detected the presence of PCBs in the Town Branch/Mud River stream system. Sediment PCB levels and hepatic CYP1A expression in caged trout produced identical pollution rankings for the study sites. Gill CYP1A expression, although suggestive of site differences, was not statistically different among sites. Unlike resident fish, which failed to show site differences in hepatic CYP1A expression in this waterway (Brammell et al. 2005), caged fish proved to be a sensitive discriminator of relative PCB contamination in this system. In summary, we determined that CYP1A expression in caged fish reflected relative in situ pollutant exposure. The exposure paradigm confirmed that 2 weeks was a sufficient caging period for evaluating CYP1A response in this species at these temperatures (13-19 degrees C). In addition, these studies demonstrate that tissue-specific CYP1A expression can provide insights into likely routes of exposure. We conclude that CYP1A expression in caged trout is a reliable and inexpensive first-pass determination of relative environmental pollutant exposure and bioavailability in aqueous systems.

Gene sequences for cytochromes p450 1A1 and 1A2: the need for biomarker development in sea otters (Enhydra lutris).

There has been recent public concern regarding the impacts of environmental pollution on populations of otters. Population level impacts have been seen with otter (Lutra lutra) populations in Europe due to polychlorinated biphenyls, and with some segments of the Prince William Sound, AK, sea otter (Enhydra lutris) population following the Exxon Valdez oil spill. Despite public interest in these animals and their ecological significance, there are few tools that allow for the study of otter's response to contaminant exposure. Cytochrome p450 1A (CYP1A) performs the first step in metabolizing many xenobiotics, including many polychlorinated biphenyls and polycyclic aromatic hydrocarbons. CYP1A induction is a frequently used biomarker of exposure to these compounds. Despite the potential importance of this gene in ecological risk assessment, the complete coding sequence has not been published for any otter species. This study's objective was to isolate the gene for CYP1A1 and CYP1A2 in sea otters using a series of PCR-based approaches. The coding sequences from CYP1A1 and CYP1A2 from sea otters were identified and published in GenBank. Both CYP1A sequences are homologous to those obtained from marine mammals and other carnivores. These sequences will be useful as tools for researchers assessing contaminant exposure in mustelid populations.

Atrazine and increased male production by Daphnia: the importance of combining field and laboratory approaches.

Atrazine is one of the most commonly applied herbicides in North America and annually pulses through many midwestern stream and reservoir systems. Previous studies have yielded conflicting results regarding the ability of atrazine to stimulate male production by Daphnia, an effect hypothesized to lower population growth rates during a period of intense larval fish predation. In the present study, populations of Daphnia parvula and Daphnia ambigua exhibited high proportions of males but no ephippial females when atrazine pulsed into Acton Lake, a small midwestern reservoir. Field results thus supported the hypothesis of excess male production by Daphnia during the spring herbicide pulse. In laboratory studies, dose-response studies, and population-level assays revealed no effect of atrazine on male production or population growth rate of multiple clones differing in reproductive strategy and exposure history. However, D. parvula increased male production in response to an endogenous crustacean hormone (methyl farnesoate). Excess male production observed in the field population was therefore not likely caused by atrazine, although we cannot rule out the possibility of interactive effects of atrazine and some other stressor. Apparent signs of endocrine disruption in the presence of high concentrations of a suspected agent should be viewed with caution in the absence of parallel laboratory studies involving individuals from the populations of interest.

Estimating incident ultraviolet radiation exposure in the northern Gulf of Mexico during the Deepwater Horizon oil spill.

Millions of barrels of oil were released into the Gulf of Mexico following the 2010 explosion of the Deepwater Horizon oil rig. Polycyclic aromatic hydrocarbons (PAHs) are toxic components of crude oil, which may become more toxic in the presence of ultraviolet (UV) radiation, a phenomenon known as photo-induced toxicity. The Deepwater Horizon spill impacted offshore and estuarine sites, where biota may be co-exposed to UV and PAHs. Penetration of UV into the water column is affected by site-specific factors. Therefore, measurements and/or estimations of UV are necessary when one is assessing the risk to biota posed by photo-induced toxicity. We describe how estimates of incident UV were determined for the area impacted by the Deepwater Horizon oil spill, using monitoring data from radiometers near the spill, in conjunction with reference spectra characterizing the composition of solar radiation. Furthermore, we provide UV attenuation coefficients for both near- and offshore sites in the Gulf of Mexico. These estimates are specific to the time and location of the spill, and fall within the range of intensities utilized during photo-induced toxicity tests performed in support of the Deepwater Horizon Natural Resource Damage Assessment (NRDA). These data further validate the methodologies and findings of phototoxicity tests included in the Deepwater Horizon NRDA, while underscoring the importance of considering UV exposure when assessing possible risks following oil spills. Environ Toxicol Chem 2018;37:1679-1687. © 2018 SETAC.

An International Perspective on the Tools and Concepts for Effluent Toxicity Assessments in the Context of Animal Alternatives: Reduction in Vertebrate Use.

Since the 1940s, effluent toxicity testing has been used to assess potential ecological impacts of effluents and help determine necessary treatment options for environmental protection prior to release. Strategic combinations of toxicity tests, analytical tools, and biological monitoring have been developed. Because the number of vertebrates utilized in effluent testing is thought to be much greater than that used for individual chemical testing, there is a new need to develop strategies to reduce the numbers of vertebrates (i.e., fish) used. This need will become more critical as developing nations begin to use vertebrates in toxicity tests to assess effluent quality. A workshop was held to 1) assess the state of science in effluent toxicity testing globally; 2) determine current practices of regulators, industry, private laboratories, and academia; and 3) explore alternatives to vertebrate (fish) testing options and the inclusion of modified/new methods and approaches in the regulatory environment. No single approach was identified, because of a range of factors including regulatory concerns, validity criteria, and wider acceptability of alternatives. However, a suite of strategies in a weight-of-evidence approach would provide the flexibility to meet the needs of the environment, regulators, and the regulated community; and this "toolbox" approach would also support reduced reliance on in vivo fish tests. The present Focus article provides a brief overview of wastewater regulation and effluent testing approaches. Alternative methodologies under development and some of the limitations and barriers to regulatory approaches that can be selected to suit individual country and regional requirements are described and discussed. Environ Toxicol Chem 2018;37:2745-2757. © 2018 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Statistical analysis of cytochrome P4501A biomarker measurements in fish.

Induction of the cytochrome P4501A (CYP1A) enzyme system in fish is a common biomarker of exposure to aromatic hydrocarbons. Induction of CYP1A can be measured at a number of steps in the transcription-translation-functional protein pathway using a variety of techniques. The present study examined the range of these measurements from 94 published papers in an attempt to examine the statistical characteristics of each method. Cytochrome P4501A induction, as measured by catalytic ethoxyresorufin-O-deethylase (EROD) activity, protein levels (enzyme-linked immunosorbent assay, Western blot analysis, and immunohistochemistry), and mRNA levels (Northern blot analysis and reverse transcription-polymerase chain reaction), was analyzed. When possible, the variance structure, effect size determination, and dose-response modeling of each method of measurement in the laboratory and field were examined. Conclusions from this analysis include: 1) Because of interlaboratory and interspecies variability, general end-point determinations will need to be defined in terms of the statistically detectable fold-change of measurements relative to control or reference values, and 2) fold-change in EROD activity provides the most robust measure of the dose responsiveness of aromatic hydrocarbons within specific chemical classes (e.g., polycyclic aromatic hydrocarbons). The relationship between the ability to measure statistical differences in induction level and the biological significance of those measurements has yet to be defined. To utilize these biomarkers in a risk assessment context, this relationship must be addressed at the scientific and management levels.

Review of the photo-induced toxicity of environmental contaminants.

Solar radiation is a vital component of ecosystem function. However, sunlight can also interact with certain xenobiotic compounds in a phenomenon known as photo-induced, photo-enhanced, photo-activated, or photo-toxicity. This phenomenon broadly refers to an interaction between a chemical and sunlight resulting in increased toxicity. Because most aquatic ecosystems receive some amount of sunlight, co-exposure to xenobiotic chemicals and solar radiation is likely to occur in the environment, and photo-induced toxicity may be an important factor impacting aquatic ecosystems. However, photo-induced toxicity is not likely to be relevant in all aquatic systems or exposure scenarios due to variation in important ecological factors as well as physiological adaptations of the species that reside there. Here, we provide an updated review of the state of the science of photo-induced toxicity in aquatic ecosystems.

Altered gene expression: a mechanism for reproductive toxicity in zebrafish exposed to benzo[a]pyrene.

Exposure of fish to polycyclic aromatic hydrocarbons (PAHs) has consistently been shown to have a negative impact on reproduction (e.g. decreased spawning success, decreased ovarian somatic index (OSI) and lower circulating levels of 17beta-estradiol and vitellogenin). While an understanding of the mechanism behind these changes has yet to be fully elucidated, it has been proposed that PAHs can alter the expression of genes important in regulating reproduction. The objectives of this study were to: (1) determine the effect of exposure to waterborne benzo[a]pyrene (B[a]P) (0, 1.5 and 3.0 microg/L) for 56 days on egg production and OSI in female zebrafish (Danio rerio) and (2) determine the effect of B[a]P on transcription of genes involved in reproduction including gonadotropins (follicle stimulating hormone (FSH) and luteinizing hormone (LH)), steroidogenic enzymes (CYP11A1, CYP17, CYP19A1, CYP19A2 and 20beta-HSD), estrogen receptor beta (ERbeta) and vitellogenin. Cytochrome P4501A1 (CYP1A1) was also measured in the liver and heads as an indicator of exposure to B[a]P. A reduction in total egg output was observed in B[a]P exposed fish as well as a decrease in OSI in fish exposed to 3.0 microg/L B[a]P. A significant increase in CYP1A1 expression in the heads as compared to the control was observed whereas no significant difference in CYP1A1 was detected in livers. A significant increase in 20beta-HSD mRNA occurred in heads and pre-vitellogenic oocytes from fish exposed to 1.5 and 3.0 microg/L as compared to the controls. CYP19A2 and vitellogenin were significantly increased following exposure to 3.0 microg/L in the heads and liver, respectively. No effects on the expression of FSH, LH, CYP19A1 or ERbeta were observed. Results from this study demonstrate that reproduction in zebrafish is affected by waterborne exposure to B[a]P and that altered transcription of genes important in regulating reproduction (20beta-HSD, CYP19A2 and vitellogenin) may be one of the underlying mechanisms of B[a]P-induced reproductive toxicity.

Increased ovarian follicular apoptosis in fathead minnows (Pimephales promelas) exposed to dietary methylmercury.

Exposure to environmentally relevant concentrations of dietary methylmercury impairs the reproduction of fish. Although specific mechanisms are unknown, recent research has linked altered reproduction in fish to the suppression of circulating levels of sex steroid hormones by methylmercury. We hypothesize that methylmercury induces apoptosis in steroidogenic gonadal cells in fish, thereby interfering with the synthesis of sex steroid hormones critical for the regulation of reproduction. To test this hypothesis, we chronically exposed fathead minnows (Pimephales promelas) to one of three diets contaminated with methylmercury: 0.06 microg Hg g(-1) (control), 0.87 microg Hg g(-1), and 3.93 microg Hg g(-1) dry weight. Apoptosis was evaluated histologically in ovaries of female fathead minnows by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL). Methylmercury significantly increased the number of apoptotic follicular cells in primary growth and cortical alveolus stage ovarian follicles. Ovarian follicular cells (i.e., granulosa, theca) are responsible for the production of 17beta-estradiol and other sex steroid hormones. Increased ovarian follicular apoptosis was related to suppressed 17beta-estradiol concentrations and smaller ovary size of female fathead minnows. Our results suggest increased apoptosis of steroidogenic gonadal cells as a possible mechanism for the suppression of sex steroid hormones and ultimately the impairment of reproduction in fish exposed to methylmercury.

Gene expression in caged fish as a first-tier indicator of contaminant exposure in streams.

The development of sensitive, biologically based indicators of contaminant exposure (i.e., biomarkers) is an ongoing topic of research. These indicators have been proposed as a first-tier method of identifying contaminant exposure. The primary objective of this research was to implement a biomarker-based method of exposure assessment using caged fish and real-time reverse-transcriptase polymerase chain reaction (rtRT-PCR) measurements of gene expression. Primers were developed for the CYPIA, metallothionein, and vitellogenin genes in rainbow trout (Oncorhynchyus mykiss), cutbow trout (Oncorhynchyus clarkii x mykiss), and Atlantic salmon (Salmo salar). Each of these genes has been shown to respond specifically to planar aromatic compounds, heavy metals, and environmental estrogens, respectively. Juvenile fish were placed in cages and exposed in situ at reference and contaminated sites on the Cache la Poudre River (CO, USA), the Arkansas River (CO, USA), the St. John River (NB, Canada), and two urban creeks near Dayton (OH, USA). Quantitative gene expression was determined using rtRT-PCR. Biomarker expression profiles were obtained that demonstrated differences in CYPIA, metallothionein, and vitellogenin mRNA production unique to each site, indicating that specific types of compounds were bioavailable and present in sufficient concentrations to elicit transcriptional responses in the organism. These findings support the use of a biomarker-based approach to exposure identification and assessment.

Photo-induced toxicity of Deepwater Horizon slick oil to blue crab (Callinectes sapidus) larvae.

The 2010 Deepwater Horizon oil spill resulted in the accidental release of approximately 700 million L of crude oil into the Gulf of Mexico. Photo-induced toxicity after co-exposure to ultraviolet (UV) radiation is 1 mechanism by which polycyclic aromatic hydrocarbons (PAHs) from oil spills may exert toxicity. Blue crab are an important commercial and ecological resource in the Gulf of Mexico, and their largely transparent larvae may make them sensitive to PAH photo-induced toxicity. The goal of the present study was to examine the sensitivity of early lifestage blue crab (Callinectes sapidus) zoea to slick oil collected during the Deepwater Horizon spill. Blue crab zoea were exposed to 1 of several dilutions of water accommodated fractions from 1 of 2 sources of oil and gradations of natural sunlight in a factorial design. Two 7-h solar exposures were carried out with a recovery period (dark) in between. Survival was found to be UV- and PAH-dependent. Toxicity was observed within the range of surface PAH concentrations reported in the Gulf of Mexico during the Deepwater Horizon spill. These findings indicate that early lifestage blue crab are sensitive to photo-induced toxicity from Deepwater Horizon slick oil.

The fish embryo toxicity test as a replacement for the larval growth and survival test: A comparison of test sensitivity and identification of alternative endpoints in zebrafish and fathead minnows.

The fish embryo toxicity (FET) test has been proposed as an alternative to the larval growth and survival (LGS) test. The objectives of the present study were to evaluate the sensitivity of the FET and LGS tests in fathead minnows (Pimephales promelas) and zebrafish (Danio rerio) and to determine if the inclusion of sublethal metrics as test endpoints could enhance test utility. In both species, LGS and FET tests were conducted using 2 simulated effluents. A comparison of median lethal concentrations determined via each test revealed significant differences between test types; however, it could not be determined which test was the least and/or most sensitive. At the conclusion of each test, developmental abnormalities and the expression of genes related to growth and toxicity were evaluated. Fathead minnows and zebrafish exposed to mock municipal wastewater-treatment plant effluent in a FET test experienced an increased incidence of pericardial edema and significant alterations in the expression of genes including insulin-like growth factors 1 and 2, heat shock protein 70, and cytochrome P4501A, suggesting that the inclusion of these endpoints could enhance test utility. The results not only show the utility of the fathead minnow FET test as a replacement for the LGS test but also provide evidence that inclusion of additional endpoints could improve the predictive power of the FET test.

Assessment of the toxicity of anthracene photo-modification products using the topminnow (Poeciliopsis lucida) hepatoma cell line (PLHC-1).

Many polycyclic aromatic hydrocarbons (PAHs) are acutely toxic to fish and other aquatic organisms in the presence of environmentally realistic intensities of solar ultraviolet radiation (SUVR). In this study, the cytoxicity/phototoxicity of anthracene photo-modified products to aquatic animals was assessed based on in vitro toxicity assays using a fish hepatoma cell line (PLHC-1 cell line). The results from this study showed that pre-exposure of anthracene/cell culture media to SUVR caused a significant amount of photo-modification and reduced the phototoxicity of parent anthracene compound. SUVR pre-exposed anthracene did not induce cytotoxicity in the absence of SUVR. It was shown that in comparison with anthracene, two major anthracene photo-modified products (anthraquinone, 1,2-dihydroxyanthraquinone) were significantly less phototoxic to PLHC-1 cells. Also in the absence of SUVR, these chemicals did not induce any detectable cytotoxicity to PLHC-1 cells. In conclusion, this study demonstrated that photo-modification reduced the phototoxicity of anthracene to PLHC-1 cells because major anthracene photo-modified products are not phototoxic. Also it was found that these photo-modified anthracene products were not cytotoxic in the absence of SUVR. This study indicated the need for separate toxicity assessments of individual photo-modified PAH products in both animal and plant models.

Laboratory and field validation of multiple molecular biomarkers of contaminant exposure in rainbow trout (Oncorhynchus mykiss).

Novel primers for three different genes (CYP1A1, vitellogenin, and metallothionein) for use in reverse transcription-polymerase chain reaction (RT-PCR) were developed and tested in laboratory flow-through experiments using model compounds benzo[a]pyrene, estradiol, and cadmium. Field validations of the molecular biomarkers were performed using 8-d or 48-h in situ, cagedexposure of juvenile rainbow trout (Oncorhynchus mykiss). Expression levels of genetranscription(messengerRNA [mRNA]) were monitored in gill and liver tissue after exposure at sites downstream of a known source of creosote contamination in the Little Scioto River (Marion, OH, USA) and metals and hydrocarbon contamination in Dick's Creek (Middletown, OH, USA). Significant increases of liver cytochrome P4501A1 gene (CYP1A1) and vitellogenin were measured in both streams at sites downstream of contaminant sources. Significant increases of gill CYP1A1 were also observed. Significant increases of gill and livermetallothionein occurred in Dick's Creek. A sample of gill tissue taken from each fish prior to placement in the Little Scioto River provided a pre-exposure control measurement for each individual. Expression of CYP1A1 and vitellogenin in trout at both streams indicated that exposure to bioavailable hydrocarbons and endocrine-disrupting compounds had taken place. Previous to this study, estrogenic compounds had not been detected in the Little Scioto River. Metallothionein expression confirmed the presence of metals in Dick's Creek. These results suggest a mixed contaminant environment that poses physiological hazard to existing teleost populations. Multiple molecular biomarkers provided a sensitive, physiologically based method of determining contaminant presence and bioavailability to aquatic organisms.

Acute photo-induced toxicity and toxicokinetics of single compounds and mixtures of polycyclic aromatic hydrocarbons in zebrafish.

The present study examined photo-induced toxicity and toxicokinetics for acute exposure to selected polycyclic aromatic hydrocarbons (PAHs) in zebrafish. Photo-enhanced toxicity from co-exposure to ultraviolet (UV) radiation and PAHs enhanced the toxicity and exhibited toxic effects at PAH concentrations orders of magnitude below effects observed in the absence of UV. Because environmental exposure to PAHs is usually in the form of complex mixtures, the present study examined the photo-induced toxicity of both single compounds and mixtures of PAHs. In a sensitive larval life stage of zebrafish, acute photo-induced median lethal concentrations (LC50s) were derived for 4 PAHs (anthracene, pyrene, carbazole, and phenanthrene) to examine the hypothesis that phototoxic (anthracene and pyrene) and nonphototoxic (carbazole and phenanthrene) pathways of mixtures could be predicted from single exposures. Anthracene and pyrene were phototoxic as predicted; however, carbazole exhibited moderate photo-induced toxicity and phenanthrene exhibited weak photo-induced toxicity. The toxicity of each chemical alone was used to compare the toxicity of mixtures in binary, tertiary, and quaternary combinations of these PAHs, and a predictive model for environmental mixtures was generated. The results indicated that the acute toxicity of PAH mixtures was additive in phototoxic scenarios, regardless of the magnitude of photo-enhancement. Based on PAH concentrations found in water and circumstances of high UV dose to aquatic systems, there exists potential risk of photo-induced toxicity to aquatic organisms.