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Ecotoxicity testing refers both to the assessment of chemical effects on fish, birds, or other wildlife species and the testing of soil, sediment, or effluents for the presence of toxic compounds. To fulfill their mandates to protect the environment, several ICCVAM member agencies, including DOI and EPA, require those that manufacture, handle, and discharge pesticides and other chemical products to conduct ecotoxicity testing.
Ecotoxicity testing can require animal testing using either the species of interest or animal models of the species of interest. ICCVAM member agencies are exploring ways to reduce or replace animal use for ecotoxicity studies and expect strong progress in the area through 2018 and 2019.
Anticoagulant rodenticides are used in conservation biology management to control invasive rodents, which can devastate colonies of ground-nesting birds on island refuges. Fish in coral reefs surrounding island refuges may be exposed to anticoagulant rodenticides during the treatment of the islands. However, there has been virtually no investigation into the sensitivity of fish toward anticoagulant rodenticides. DOI scientists use the up-and-down procedure (OECD TG 425; EPA OPPTS 870.1100) to minimize animal use when determining the sensitivity of fish species to anticoagulant rodenticide exposure. A modified version of the up-and-down procedure extends the observation period and adds clinical pathology endpoints (histopathology and blood-clotting time).
In 2016 and 2017, these studies focused on the effect of anticoagulant rodenticides on Hawaiian triggerfish and surrogate species. Data from these studies will be used to associate lethal and sublethal effects with anticoagulant rodenticide exposure and to determine half-lives of these substances in fish tissues. These data will also be used to understand the potential for human exposure from consumption of fish.
Fisheries managers use toxicants to control invasive and undesirable fish species. These substances need to be screened for effects on nontarget species.
USGS developed a screening process to minimize and replace animal testing while developing new chemical control agents for invasive species. The goal is to identify compounds that are potentially toxic to target species while posing minimal risk to native species. The three integrated phases of the screening process include identifying physical and chemical properties of compounds that affect bioavailability in fish; prescreening of a publicly available chemical databank to prioritize candidate compounds; and screening of selected compounds for cytotoxicity using in vitro biological assays, ecotoxicity modeling, and fish cell lines. Although in vivo testing continues to be utilized in the development of new fish toxicants, the screening process enables minimization of animal testing while developing a new chemical control agent for invasive species.
Ecotoxicity modeling uses a quantitative structure-activity relationship modeling system that determines species-specific responses to chemical exposures using existing toxicity data and chemical properties. These in silico assessment methods can be used to prioritize candidate compounds and estimate cytotoxicity, and can predict more than 2,000 endpoints for nearly 200 species. The models will be available in 2018.
Promising novel toxicants identified in the ecotoxicity modeling step are tested using cellular assays to identify which toxicants have the biological activity required. USGS has developed multiple endpoint assays that measure cell viability based on quantitation of adenosine triphosphate. These assays use cell lines from native species including fathead minnow, bluegill sunfish, rainbow trout, lake sturgeon, and paddlefish, as well as invasive species, including silver carp and bighead carp. Compounds that demonstrate potent cytotoxic effects are then selected for in vivo assays for toxicity screening.
As part of ongoing assessments of wildlife health, DOI is investigating potential cardiovascular effects on fish from pesticides and pharmaceuticals frequently detected in surface waters and fish tissues. USGS conducts high-content screening of compounds to formulate hypotheses and prioritize compounds for further toxicity testing. This approach reduces animal use, test compound needed, and waste by utilizing pre-feeding fish embryos in a microtiter plate format. These assays can provide evidence to justify larger-scale studies to determine actual risk versus perceived risk of contaminants.
The current high-content screening assay is a developmental cardiotoxicity assay that assesses total body length, pericardial area, intersegmental vessel area, circulation, and heart rate after a 72-hour exposure. This array of endpoints allows for a targeted assessment of toxicity. In addition to an LC50 estimate, the assay provides a relatively rapid mode of action information, allowing formation of hypotheses on sublethal impacts of contaminants. Data derived from these studies on acute toxicity and mode of action for pesticides and pharmaceuticals will support a better understanding of potential effects on wildlife species.