Anne E. McElroy

A review of the tissue residue approach for organic and organometallic compounds in aquatic organisms

This paper reviews the tissue residue approach (TRA) for toxicity assessment as it applies to organic chemicals and some organometallic compounds (Sn, Hg, and Pb) in aquatic organisms. Specific emphasis was placed on evaluating key factors that influence interpretation of critical body residue (CBR) toxicity metrics including data quality issues, lipid dynamics, choice of endpoints, processes that alter toxicokinetics and toxicodynamics, phototoxicity, species- and life stage-specific sensitivities, and biotransformation. The vast majority of data available on TRA is derived from laboratory studies of acute lethal responses to organic toxicants exhibiting baseline toxicity. Application of the TRA to various baseline toxicants as well as substances with specific modes of action via receptor-mediated processes, such as chlorinated aromatic hydrocarbons, pesticides, and organometallics is discussed, as is application of TRA concepts in field assessments of tissue residues. In contrast to media-based toxicity relationships, CBR values tend to be less variable and less influenced by factors that control bioavailability and bioaccumulation, and TRA can be used to infer mechanisms of toxic action, evaluate the toxicity of mixtures, and interpret field data on bioaccumulated toxicants. If residue-effects data are not available, body residues can be estimated, as has been done using the target lipid model for baseline toxicants, to derive critical values for risk assessment. One of the primary unresolved issues complicating TRA for organic chemicals is biotransformation. Further work on the influence of biotransformation, a better understanding of contaminant lipid interactions, and an explicit understanding of the time dependency of CBRs and receptor-mediated toxicity are all required to advance this field. Additional residue-effects data on sublethal endpoints, early life stages, and a wider range of legacy and emergent contaminants will be needed to improve the ability to use TRA for organic and organometallic compounds.

ACUTE TOXICITY OF RESMETHRIN, MALATHION AND METHOPRENE TO LARVAL AND JUVENILE AMERICAN LOBSTERS (HOMARUS AMERICANUS) AND ANALYSIS OF PESTICIDE LEVELS IN SURFACE WATERS AFTER SCOURGE™, ANVIL™AND ALTOSID™APPLICATION

Abstract Acute toxicity and immune response, combined with temperature stress effects, were evaluated in larval and juvenile American lobsters (Homarus americanus) exposed to malathion, resmethrin and methoprene. These pesticides were used to control West Nile virus in New York in 1999, the same year the American lobster population collapsed in western Long Island Sound (LIS). Whereas the suite of pesticides used for mosquito control changed in subsequent years, a field study was also conducted to determine pesticide concentrations in surface waters on Long Island and in LIS after operational applications. The commercial formulations used in 2002 and 2003—Scourge, Anvil and Altosid—contain the active ingredients resmethrin, sumithrin and methoprene, respectively. Concentrations of the synergist piperonyl butoxide (PBO) were also measured as a proxy for pesticide exposure. Acute mortality in Stage I-II larval lobsters demonstrated that they are extremely sensitive to continuous resmethrin exposure. Resmethrin LC50s for larval lobsters determined under flow-through conditions varied from 0.26–0.95 μg L−1 in 48- and 96-h experiments at 16°C, respectively. Increased temperature (24°C) did not significantly alter resmethrin toxicity. Malathion and methoprene were less toxic than resmethrin. The 48-h LC50 for malathion was 3.7 μg L−1 and methoprene showed no toxicity at the highest (10 μg L−1) concentration tested. Phenoloxidase activity was used as a measure of immune response for juvenile lobsters exposed to sublethal pesticide concentrations. In continuous exposures to sublethal doses of resmethrin (0.03 μg L−1) or malathion (1 μg L−1) for 7 d at 16 or 22°C, temperature had a significant effect on phenoloxidase activity (P ≤ 0.006) whereas pesticide exposure did not (P = 0.880). The analytical methods developed using high performance liquid chromatography coupled to time-of-flight mass spectroscopy (LC-TOF-MS) provided high sensitivity with mass detection limits of 0.1–0.3 ng L−1. Pesticide levels were often detected in the ng L−1 range in Long Island surface waters and western LIS (except in open waters), but rarely at concentrations found to be toxic in flow-through laboratory exposures, even immediately after spray events.

Severe Inbreeding and Small Effective Number of Breeders in a Formerly Abundant Marine Fish

In contrast to freshwater fish it is presumed that marine fish are unlikely to spawn with close relatives due to the dilution effect of large breeding populations and their propensity for movement and reproductive mixing. Inbreeding is therefore not typically a focal concern of marine fish management. We measured the effective number of breeders in 6 New York estuaries for winter flounder (Pseudopleuronectes americanus), a formerly abundant fish, using 11 microsatellite markers (6–56 alleles per locus). The effective number of breeders for 1–2 years was remarkably small, with point estimates ranging from 65–289 individuals. Excess homozygosity was detected at 10 loci in all bays (FIS = 0.169–0.283) and individuals exhibited high average internal relatedness (IR; mean = 0.226). These both indicate that inbreeding is very common in all bays, after testing for and ruling out alternative explanations such as technical and sampling artifacts. This study demonstrates that even historically common marine fish can be prone to inbreeding, a factor that should be considered in fisheries management and conservation plans.

Investigation of Epizootic Shell Disease in American Lobsters (Homarus americanus) from Long Island Sound: I. Characterization of Associated Microbial Communities

ABSTRACT Epizootic shell disease (ESD) is a degradative process of the carapace in the American lobster, (Homarus americanus), putatively caused by bacterial infection, and potentially responsible for serious economic losses to the lobster fishery. In Long Island Sound (LIS), ESD is prevalent in lobsters from eastern LIS (ELIS), but almost absent in western LIS (WLIS), presenting a unique opportunity to examine the influence of microbial communities on the disease process among these subpopulations. Bacterial community compositions in diseased shell, healthy shell subsamples from lobsters exhibiting signs of disease, and carapace subsamples of healthy lobsters from ELIS, WLIS, and a coastal Maine reference site were profiled using terminal restriction fragment length polymorphism (TRFLP). Although overall bacterial community membership in diseased shell was not significantly different from healthy shell and healthy lobsters, prevalence of some individual terminal restriction fragments (TRFs) varied among disease state. Several TRFs were more abundant within lesions, whereas representation of other members appeared to be diminished, particularly members of the &bgr;- and &ggr;-proteobacteria. One TRF linked to anaerobic bacteria was enriched in lesions, suggesting anoxic microenvironments within diseased tissues. Activities of 4 ectohydrolases among communities were also measured in replicate excised shell samples. Chitinase potentials were high in all samples, and were indistinguishable among sample types. In contrast, proteinase and cellulase potentials were significantly higher in diseased shell than healthy shell and healthy lobster. Lipase potentials in LIS samples were significantly higher than those from Maine, but similar among disease states. The absence of site-specific differences in microbial communities suggests that biogeographic variation in colonizing microbes is not a factor in disease susceptibility. Lesion development appears to induce compositional shifts in normal carapace microflora, with displacement of some community members as others become more prevalent. Protein and cellulose appear to be more important targets than chitin for bacterial degradation within lesions. Furthermore, lipase activity, degrading the epicuticle lipid layer, may play a key role in regions with high disease prevalence.

Ontogenesis of gonadal aromatase gene expression in atlantic silverside (Menidia menidia) Populations with genetic and temperature‐dependent sex determination

Cytochrome P450 aromatase (P450arom), an enzyme that converts testosterone to 17beta-estradiol, is an important mediator of sex determination in teleosts with genetic sex determination (GSD) and temperature-dependent sex determination (TSD). We compared the ontogenetic expression of P450arom in two populations of Atlantic silversides, Menidia menidia, which exhibit TSD (South Carolina) or GSD (Nova Scotia, Canada) using quantitative, real-time polymerase chain reaction (qRT-PCR). Embryos and newly hatched larvae were reared at an intermediate sex ratio-producing temperature (21 degrees C), and older larvae and juveniles were reared at temperatures that feminize (15 degrees C) and masculinize (28 degrees C) to assess the temperature response of P450arom during development. Before sex determination, embryos and newly-hatched larvae displayed negligible P450arom expression, indicating minimal upregulation of this gene before sex determination. Gene expression increased in both populations during sex differentiation. Nova Scotia fish with GSD exhibited presumptive male- and female-like expression levels during early sex differentiation that were not influenced by temperature. South Carolina fish displayed low levels of expression at 28 degrees C with significantly heightened expression in some individuals at 15 degrees C, indicating that P450arom is temperature sensitive in the population with TSD. Populations also differed in the timing and maximal levels of P450arom expression, with fish from Nova Scotia exhibiting both the highest and earliest increase in expression in presumptive females. Our results support the hypothesis that P450arom is involved in female sex differentiation in this species, but is only responsive to temperature in M. menidia populations that exhibit TSD.

Investigation of Epizootic Shell Disease in American Lobsters (Homarus americanus) from Long Island Sound: II. Immune Parameters in Lobsters and Relationships to the Disease

ABSTRACT Epizootic shell disease (ESD) affects lobsters (Homarus americanus) in eastern Long Island Sound (ELIS) and the near-shore waters of southern New England. Marring the shell of individuals, ESD decreases the economic value of infected lobsters and can lead to mortality in severely affected individuals. ESD tends to be most common in areas around Buzzards Bay, RI, and ELIS, and least prevalent in offshore canyons, off Maines coast, and in western Long Island Sound (WLIS). To investigate the potential role of the immune system in determining an individuals or populations susceptibility to ESD, the immunocompetence and disease status of lobsters with and without signs of ESD from ELIS, WLIS, and Boothbay Harbor, ME, were assessed during late spring (June) 2007. We also measured internal defense parameters of ELIS lobsters during midsummer (August 2007), early fall (October 2007), and the following spring (June 2008) to assess how lobster immune systems respond temporarily. Despite high interindividual variability in defense-related factors, multivariate analyses showed that lobsters from ELIS presented significantly reduced immune responses relative to lobsters from either WLIS or Maine. Disease severity correlated negatively with several immune parameters, suggesting that the higher prevalence of ESD in ELIS may be related, at least in part, to reduced immunocompetency of ELIS lobsters.

Interactions between hypoxia and sewage-derived contaminants on gene expression in fish embryos

Fish embryos were used to evaluate the interaction among common environmental and chemical stressors found in urban coastal environments, namely hypoxia, aryl hydrocarbon receptor (AhR) agonists, and estrogenic compounds. At the molecular level, the systems responding to these stressors share common response factors, and evidence exists for cross-talk between them. Biomarkers of exposure to these stressors, cytochrome P4501a (Cyp1a), estrogen receptor alpha (ERα), brain cytochrome P450 aromatase (Cyp19a2 or AromB), and hypoxia inducible factor 1 alpha (Hif-1α) mRNA expression were examined using qRT-PCR simultaneously in embryos of two well studied species, the Atlantic killifish, Fundulus heteroclitus, and the zebrafish Danio rerio. Embryos of both species were exposed to the model Cyp1a inducer β-naphthoflavone (BNF) or 17-β estradiol (E2) under either normoxic or hypoxic (5% oxygen atmosphere) conditions and harvested prior to hatch at 9 days post fertilization (dpf) for the killifish, and 48h post fertilization (hpf) for the zebrafish. BNF significantly induced Cyp1a expression in embryos of both species with killifish embryos being more responsive (700-fold>control) than zebrafish embryos (7-100-fold>control). AromB was also significantly influenced by treatment, but to a lesser extent, with mean expression levels increased by less than two-fold over control values in response to E2, and in one case upregulated by BNF. ERα and Hif-1α were constitutively expressed in embryos of both species, but expression was unaffected by exposure to either BNF or E2. Hypoxic conditions downregulated AromB expression strongly in killifish but not in zebrafish embryos. The impact of hypoxia on expression of other genes in either species was inconsistent, although an interactive effect between hypoxia and BNF on several of the genes evaluated was observed. These data are the first to examine expression patterns of these important environmental response genes together in embryos of two important model fish species. The results support the use of Cyp1a expression as a biomarker of AhR agonists in fish embryos, and indicate that AromB may be more responsive than ERα to estrogenic chemicals at this stage in development. Hif-1α expression was not found to be a good biomarker of hypoxic exposure in either killifish or zebrafish embryos. The interaction observed between BNF and co-exposure to hypoxia warrants further investigation. Finally killifish embryos are generally more sensitive than zebrafish embryos at this stage of development supporting their use in environmental assessments.

Hypoxia depresses CYP1A induction and enhances DNA damage, but has minimal effects on antioxidant responses in sheepshead minnow (Cyprinodon variegatus) larvae exposed to dispersed crude oil

The growing incidence of hypoxic regions in coastal areas receiving high volumes of anthropogenic discharges requires more focused risk assessment of multiple stressors. One area needing further study is the combined effect of hypoxia and oil exposure. This study examined the short-term sublethal effects of co-exposure to hypoxia and water accommodated fractions (WAF) and chemically enhanced WAFs (CEWAFs) of Southern Louisiana Crude oil on detoxification, antioxidant defenses and genotoxicity in early life stage sheepshead minnow (Cyprinodon variegatus). CYP1A induction (evaluated by measuring EROD activity), activity of a number of key antioxidant enzymes (GST, GR, GPx, SOD, CAT, and GCL), levels of antioxidants (tGSH, GSH, and GSSG), evidence of lipid peroxidation (evaluated using the TBARS assay), and DNA damage (evaluated using the comet assay) provided a broad assessment of responses. Contaminant detoxification pathways induced by oil exposure were inhibited by co-exposure to hypoxia, indicating a maladaptive response. The interactive effects of oil and hypoxia on antioxidant defenses were mixed, but generally indicated less pronounced alterations, with significant increases in lipid peroxidation not observed. Hypoxia significantly enhanced DNA damage induced by oil exposure indicating the potential for significant deleterious effects post exposure. This study demonstrates the importance of considering hypoxia as an enhanced risk factor in assessing the effects of contaminants in areas where seasonal hypoxia may be prevalent.

Hypoxia Enhances the Toxicity of Corexit EC9500A and Chemically Dispersed Southern Louisiana Sweet Crude Oil (MC-242) to Sheepshead Minnow (Cyprinodon variegatus) Larvae

Oil exploration and production activities are common in the northern Gulf of Mexico as well as many other coastal and near coastal areas worldwide. Seasonal hypoxia is also a common feature in the Northern Gulf, and many other coastal areas, which is likely to increase in severity and extent with continuing anthropogenic nutrient inputs. Hypoxia has well established physiological effects on many organisms, and it has been shown to enhance the toxicity of polycyclic aromatic hydrocarbons (persistent components of petroleum) in fish. The goal of this study was to examine the combined effects of hypoxia and exposure to contaminants associated with oil spills. We evaluated the effects of short term (48 hr) exposures to Corexit EC9500A, water accommodated fractions (WAF), and chemically enhanced water accommodated fractions (CEWAF) prepared from Southern Louisiana Sweet Crude Oil (MC 242) on survival of sheepshead minnow (Cyprinodon variegatus) larvae held under normoxic (ambient air) or hypoxic (2 mg/L O2) conditions. Results demonstrated that hypoxia significantly enhances mortality observed in response to Corexit or CEWAF solutions. In the latter case, significant interactions between the two stressors were also observed. Our data supports the need to further evaluate the combined stresses imparted by hypoxia and exposure to petroleum hydrocarbons and dispersants.

Metals, Organic Compounds, and Nutrients in Long Island Sound: Sources, Magnitudes, Trends, and Impacts

Long Island Sound (LIS) is a relatively shallow estuary with a mean depth of 20 m (maximum depth 49 m) and a unique hydrology and history of pollutant loading. These factors have contributed to a wide variety of contamination problems in its muddy sediments, aquatic life, and water column. The LIS sediments are contaminated with toxic compounds and elements related to past and present wastewater discharges and runoff. These include nonpoint and stormwater runoff and groundwater discharges, whose character has changed over the years along with the evolution of its watershed and industrial history. Major impacts have resulted from the copious amounts of nutrients discharged into LIS through atmospheric deposition, domestic and industrial waste water flows, fertilizer releases, and urban runoff. All these sources and their effects are in essence the result of human presence and activities in the watershed, and the severity of pollutant loading and their impacts generally scales with total population in the watersheds surrounding LIS. Environmental legislation passed since the mid-to-late 1900s (e.g., Clean Air Act, Clean Water Act) has had a beneficial effect, however, and contaminant loadings for many toxic organic and inorganic chemicals and nutrients have diminished over the last few decades (O’Shea and Brosnan 2000; Trench et al. 2012; O’Connor and Lauenstein 2006; USEPA 2007). Major strides have been made in reducing the inflow of nutrients into LIS, but cultural eutrophication is still an ongoing problem and nutrient control efforts will need to continue. Nonetheless, LIS is still a heavily human impacted estuary (an “Urban Estuary,” as described for San Francisco Bay by Conomos 1979), and severe changes in water quality and sediment toxicity as well as ecosystem shifts have occurred since the European colonization in the early 1600s (Koppelman et al., 1976). The Sound has seen the most severe environmental changes over the last 400 years during its 10,000 year history (Lewis, this volume), suggesting that human impacts have overwhelmed the natural forces at play.