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In the context of the European Union (EU) Drinking Water Directive, freshwater mussels (Order Unionoida: Bivalvia) can help us face the challenges of safe drinking water provisions for all citizens in the EU. Specifically, the implementation of high frequency noninvasive (HFNI) valvometers allows the early detection of eventual pollution events in drinking water treatment plants. Currently real-time behavioral analysis is deployed in a number of EU countries, and we foresee a bright future as new technological advances are developed concerning HFNI valvometers.

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The utilization of molecular endpoints in ecotoxicology can provide rapid and valuable information on immediate organismal responses to chemical stressors and is increasingly used for mechanistic interpretation of effects at higher levels of biological organization. This study contributes knowledge on the sublethal effects of a commonly used insecticide, the phenylpyrazole fipronil, on larval fathead minnow (Pimephales promelas), utilizing a quantitative transcriptomic approach. Immediately after 24h of exposure to fipronil concentrations of ≥31 µg.L(-1), highly significant changes in gene transcription were observed for aspartoacylase, metallothionein, glucocorticoid receptor, cytochrome P450 3A126 and vitellogenin. Different mechanisms of toxicity were apparent over the course of the experiment, with short-term responses indicating neurotoxic effects. After 6 days of recovery, endocrine effects were observed with vitellogenin being up-regulated 90-fold at 61 µg.L(-1) fipronil. Principal component analysis demonstrated a significant increase in gene transcription changes over time and during the recovery period. In conclusion, multiple mechanisms of action were observed in response to fipronil exposure, and unknown delayed effects would have been missed if transcriptomic responses had only been measured at a single time-point. These challenges can be overcome by the inclusion of multiple endpoints and delayed effects in experimental designs.

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The delta smelt (Hypomesus transpacificus) is an endangered pelagic fish species endemic to the Sacramento-San Joaquin estuary (CA, USA), and considered an indicator of ecosystem health. Copper is a contaminant of concern in Californian waterways that may affect the development and survival of this endangered species. The experimental combination of molecular biomarkers with higher level effects may allow for interpretation of responses in a functional context that can be used to predict detrimental outcomes caused by exposure. A delta smelt microarray was developed and applied to screen for candidate molecular biomarkers that may be used in monitoring programs. Functional classifications of microarray responses were used along with quantitative polymerase chain reaction determining effects upon neuromuscular, digestive, and immune responses in Cu-exposed delta smelt. Differences in sensitivity were measured between juveniles and larvae (median lethal concentration = 25.2 and 80.4 µg/L Cu(2+), respectively). Swimming velocity declined with higher exposure concentrations in a dose-dependent manner (r = -0.911, p < 0.05), though was not statistically significant to controls. Genes encoding for aspartoacylase, hemopexin, α-actin, and calcium regulation proteins were significantly affected by exposure and were functionally interpreted with measured swimming responses. Effects on digestion were measured by upregulation of chitinase and downregulation of amylase, whereas downregulation of tumor necrosis factor indicated a probable compromised immune system. Results from this study, and many others, support the use of functionally characterized molecular biomarkers to assess effects of contaminants in field scenarios. We thus propose that to attribute environmental relevance to molecular biomarkers, research should concentrate on their application in field studies with the aim of assisting monitoring programs.

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Toxic effect concentrations of insecticides are generally determined using the technical grade or pure active ingredient. Commercial insecticide formulations, however, contain a significant proportion (>90%) of so-called inert ingredients, which may alter the toxicity of the active ingredient(s). This study compares the sublethal toxicity of two insecticides, the pyrethroid bifenthrin, and the phenylpyrazole fipronil, to their commercial formulations, Talstar and Termidor. Both insecticides are used for landscape treatment and structural pest control, and can be transported into surface water bodies via stormwater and irrigation runoff. We used larval fathead minnow (Pimephales promelas), to determine effects on growth and swimming performance after short-term (24h) exposure to sublethal concentrations of pure insecticides and the respective formulations. Significantly enhanced 7d growth was observed at 10% of the 24h LC(10) (53 microg L(-)(1)) fipronil. Swimming performance was significantly impaired at 20% of the 24h LC(10) (0.14 microg L(-)(1)) of bifenthrin and 10% of the 24h LC(10) of Talstar (0.03 microgL(-)(1)). Fipronil and Termidor led to a significant impairment of swimming performance at 142 microgL(-)(1) and 148 microgL(-)(1) respectively, with more pronounced effects for the formulation. Our data shows that based on dissolved concentrations both formulations were more toxic than the pure active ingredients, suggesting that increased toxicity due to inert ingredients should be considered in risk assessments and regulation of insecticides.

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The present study determined the effects of environmentally relevant, short-term (4-h) exposure to the pyrethroid insecticide esfenvalerate on mortality, food consumption, growth, swimming ability, and predation risk in larvae of the fathead minnow (Pimephales promelas). Acute effect concentrations were determined, and in subsequent experiments, fish were exposed to the following measured sublethal concentrations: 0.072, 0.455, and 1.142 microg/L of esfenvalerate. To measure growth rates (% dry wt/d), 8-d-old fathead minnows were exposed to esfenvalerate for 4 h, then transferred to control water and held for 7 d. Food consumption and abnormal swimming behavior were recorded daily. Additional behavioral experiments were conducted to evaluate how esfenvalerate affects the optomotor response of the fish. To quantify predation risk, esfenvalerate-exposed fathead minnow larvae were transferred to 9.5-L aquaria, each containing one juvenile threespine stickleback (Gasterosteus aculeatus). Sticklebacks were allowed to feed for 45 min, after which the number of surviving minnows was recorded. No mortality occurred during 4-h exposures to esfenvalerate, even at nominal concentrations of greater than 20 microg/L. Delayed mortality (50%) was observed at 2 microg/L after an additional 20 h in clean water. Fish exposed to 0.455 and 1.142 microg/L of esfenvalerate exhibited impaired swimming and feeding ability as well as reduced growth compared to fish exposed to 0.072 microg/L and controls. Predation risk also was significantly increased for larvae exposed to 0.455 and 1.142 microg/L of esfenvalerate. These results demonstrate that larval fish experiencing acute exposures to sublethal concentrations of this insecticide exhibit significant behavioral impairment, leading to reduced growth and increased susceptibility to predation, with potentially severe consequences for their ecological fitness.

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