This dissertation explores subcellular, organismal and community level effects induced by pesticides of concern in agricultural surface water, and evaluates the use of molecular methods; i.e., environmental DNA (eDNA) metabarcoding, in watershed-wide, multitrophic assessments of freshwater biodiversity. I sought to characterize the impacts of chemical disturbance events occurring at multiple levels of biological organization in anthropogenically impacted freshwater ecosystems. I first evaluated the lethality and sublethal effects of two pesticides: imidacloprid (IMI) and chlorantraniliprole (CHL), as single compounds and binary mixtures, on invertebrates (Daphnia magna) and fish (Pimephales promelas). To explore the effects of complex mixtures, I also conducted exposures as described above on contaminated surface water samples collected near agricultural fields associated with the Salinas River Watershed CA (USA). Analytical chemistry data from surface water samples showed chemicals of emerging concern as common analytes at levels expected to cause detrimental effects on aquatic life. I measured acute toxicity in invertebrates exposed to field-collected surface water, and fish exposed to these water samples had significant changes in expression of genes (RT-qPCR) involved with detoxification and neuromuscular function. Exposure of fish to single compounds or binary mixtures of IMI and CHL led to increased relative gene expression of ryanodine receptors (RyR) in fish. Furthermore, IMI targeted the postsynaptic nicotinic acetylcholine receptor (nAChR) in aquatic invertebrates and CHL caused overactivation of RyR in invertebrates and fish. Overall, high levels of invertebrate toxicity and impacts to neuromuscular health in fish are occurring, and pesticides of emerging concern result in detrimental effects in both invertebrates and fish.
Based on these findings, I examined behavioral endpoints in the fathead minnow (Pimephales promelas) after the surface water exposures outlined above. I detected differences in both light-induced startle responses and average total distance moved (mm/s), as well as the duration and/or frequency of cruising, bursting and freezing endpoints. These behaviors directly relate to factors influencing survival, feeding and growth, as well as potential for predator avoidance, and thus changes induced by chemical exposure contribute to ecological risk. I detected sublethal and environmentally relevant effects from exposure to contaminated surface waters, which would likely be missed in standard toxicology assessments based on mortality, illustrating the importance of incorporating sublethal endpoints in risk assessments.
I then examined behavioral effects of exposure to contaminated surface water before and after a disturbance event (a “first flush” rainstorm at the end of a dry period). I postulated that the swimming behavior of D. magna would be a sensitive bioindicator of exposure to environmentally relevant concentrations of pesticides of concern (IMI and CHL) under laboratory conditions as well as within complex mixtures in contaminated surface waters. I determined that average total distance moved is a sensitive endpoint for pesticide exposure. Daphnia magna response to light stimulus was the most sensitive endpoint measured. In exposures conducted before the first flush event, I detected strong dose-response patterns, with exposed organisms showing a significantly reduced response compared to controls. After first flush, I measured hypoactivity for all sites. I detected different response patterns to light stimulus for each site tested: negative dose-response, non-monotonic, and positive dose-response patterns, with significantly different responses from controls at all concentrations tested.
Having determined sublethal and toxic effects from organismal exposure to water samples collected from the Salinas River Watershed, I sought to evaluate how aquatic biodiversity may be distributed across a chemically impacted watershed, and how diversity estimates obtained from both eDNA metabarcoding and morphological identification would correlate with biotic index scores. I collected eDNA from sediment at sites throughout the Salinas River Watershed, across a range of habitat qualities, and compared the resulting taxonomy with morphological data from a subset of high-diversity sites. I detected sensitive invertebrate taxa (Ephemeroptera, Plecoptera, Trichoptera; EPT) from morphology and eDNA, with significant overlap (> 76.67%) between methods, but some taxa were missing from the sequence database, highlighting the importance of taxonomic database development. Sequencing detected more benthic macroinvertebrate taxa than morphology when compared at the genus and species levels of taxonomic resolution. Metabarcoding of sampled eDNA detected rare species of concern and invasive species. Impacted sites contained greater numbers of species known to be tolerant to poor water quality, whereas I only detected several sensitive EPT taxa from least impacted reference sites. Hydrologic distance (waterbody) and biotic index score both accounted for > 27% of the dissimilarity in taxa measured between sites. These findings suggest that biotic indices obtained from eDNA metabarcoding data can be effectively incorporated into watershed-wide, multitrophic assessments of freshwater biodiversity.
Freshwater ecosystems in urban and agriculturally developed watersheds are simultaneously exposed to chemical mixtures often include new and emerging contaminants of concern, for which toxicological data may be limited. As the complexity of mixtures increases, non-targeted, effect-based evaluations become necessary for determining potential detrimental outcomes. Through my dissertation work I demonstrate that subcellular, organismal and community level effects are induced by pesticides of concern present in surface water.
