Multi- and Trans-Generational Effects on Daphnia Magna of Chlorpyrifos Exposures.

Chlorpyrifos, a broad-spectrum neurotoxic organophosphate insecticide, is subject to atmospheric and hydrolytic transport from application sites to aquatic ecosystems. Across the landscape, concentrations in surface water can vary spatially and temporally according to seasonal use practices. Standardized bioassays can provide a screening-level understanding of aquatic receptor acute and chronic toxicity. However, these bioassays do not address ecologically relevant exposure patterns that may impact fitness and survival within and across generations. The aim of the present study was to evaluate the utility of a second-tier, screening-level methodology employing Daphnia magna multi- and transgenerational bioassays spanning four generations to investigate the effect of variable chronic chlorpyrifos exposure. The multigenerational assay consisted of continuous chlorpyrifos exposure across four consecutive 21-day bioassays using progeny from the previous assay for each successive generation. In the transgenerational assay, only the parent (F0) generation was exposed. For both assays, survival and reproduction were assessed across treatments and generations. Results indicated that (1) following continuous chlorpyrifos exposure at ecologically relevant concentrations to four generations of D. magna, the highest treatment showed an apparent tolerance response for both survival and reproductive success in the F3 generation, and (2) chlorpyrifos exposure to the F0 generation did not result in treatment effects in the unexposed F1, F2, and F3 generations in the apical endpoints of survival and reproduction. Employing a suite of acute and chronic bioassays, including chronic exposures spanning multiple generations, allows for a more robust screening-level evaluation of the potential impact of chlorpyrifos on aquatic receptors for variable periods of exposure. Environ Toxicol Chem 2022;41:1054-1065. © 2021 SETAC.

Neurotoxicity of chlorpyrifos and chlorpyrifos-oxon to Daphnia magna.

Chlorpyrifos (CPF) is a widely used broad-spectrum organophosphate insecticide. CPF elicits neurotoxic effects in exposed organisms by inhibiting the activity of acetylcholinesterase enzymes (AChE), which prolongs nerve transmission and results in neurotoxic symptoms and death at high doses. While CPF is capable of eliciting neurotoxic effects, chlorpyrifos-oxon (CPFO) is the primary neurotoxicant agent. Aquatic organisms bioactivate CPF to CPFO through the Cytochrome P450 phase I metabolic pathway following exposure to CPF. Additionally, in the environment, CPF transforms to CPFO, primarily through photo-oxidation. As both compounds can be transported in air and water to aquatic ecosystems, there is the potential for exposure to non-target organisms. The potential for adverse impacts on aquatic receptors depends on patterns of exposure and toxicity of individual compounds and the mixture. To study the neurotoxicity of these compounds, a 48 h acute and 21 d chronic Daphnia magna bioassay was conducted independently with CPF and CPFO. Acute bioassay results show a median lethal concentration (LC50) of 0.76 µg L-1 for CPF and 0.32 µg L-1 for CPFO, suggesting that CPFO is 2.4 times more acutely toxic to D. magna. Acute assay results were also used to derive Benchmark Dose Levels of 0.58 µg L-1 for CPF and 0.25 µg L-1 for CPFO. However, neither compound elicited an effect on reproduction or growth at relevant chronic exposures. As D. magna are a small and relatively sensitive species, and the AChE inhibition adverse outcome pathway is highly conserved, these results may be cautiously extrapolated in assessing adverse impacts on aquatic receptors.1.

Developing Global Leaders for Research, Regulation, and Stewardship of Crop Protection Chemistry in the 21st Century.

To provide sufficient food and fiber to the increasing global population, the technologies associated with crop protection are growing ever more sophisticated but, at the same time, societal expectations for the safe use of crop protection chemistry tools are also increasing. The goal of this perspective is to highlight the key issues that face future leaders in crop protection, based on presentations made during a symposium titled "Developing Global Leaders for Research, Regulation and Stewardship of Crop Protection Chemistry in the 21st Century", held in conjunction with the IUPAC 13th International Congress of Pesticide Chemistry in San Francisco, CA, USA, during August 2014. The presentations highlighted the fact that leaders in crop protection must have a good basic scientific training and understand new and evolving technologies, are aware of the needs of both developed and developing countries, and have good communication skills. Concern is expressed over the apparent lack of resources to meet these needs, and ideas are put forward to remedy these deficiencies.

Longitudinal trends in organophosphate incidents reported to the National Pesticide Information Center, 1995-2007.

BACKGROUND: Regulatory decisions to phase-out the availability and use of common organophosphate pesticides among the general public were announced in 2000 and continued through 2004. Based on revised risk assessments, chlorpyrifos and diazinon were determined to pose unacceptable risks. To determine the impact of these decisions, organophosphate (OP) exposure incidents reported to the National Pesticide Information Center (NPIC) were analyzed for longitudinal trends. METHODS: Non-occupational human exposure incidents reported to NPIC were grouped into pre- (1995-2000) and post-announcement periods (2001-2007). The number of total OP exposure incidents, as well as reports for chlorpyrifos, diazinon and malathion, were analyzed for significant differences between these two periods. The number of informational inquiries from the general public was analyzed over time as well. RESULTS: The number of average annual OP-related exposure incidents reported to NPIC decreased significantly between the pre- and post-announcement periods (p < 0.001). A significant decrease in the number of chlorpyrifos and diazinon reports was observed over time (p < 0.001). No significant difference in the number of incident reports for malathion was observed (p = 0.4), which was not phased-out of residential use. Similar to exposure incidents, the number of informational inquiries received by NPIC declined over time following the phase-out announcement. CONCLUSION: Consistent with other findings, the number of chlorpyrifos and diazinon exposure incidents reported to NPIC significantly decreased following public announcement and targeted regulatory action.

Clutch morphology and the timing of exposure impact the susceptibility of aquatic insect eggs to esfenvalerate.

We investigated Baetis spp. (mayfly), Hesperoperla pacifica (stonefly), and Brachycentrus americanus (caddisfly) susceptibility at the egg stage to esfenvalerate, a synthetic pyrethroid insecticide. Eggs were obtained from the field or from field-collected gravid females at sites near Corvallis (OR, USA) and the Metolius River at Camp Sherman (OR, USA) for static exposures under controlled conditions for temperature and light. Eggs were exposed to esfenvalerate for 48 h at concentrations ranging from 0.025 to 4.0 microg/L. No effect on mortality or posthatch growth was detected in H. pacifica eggs exposed to esfenvalerate concentrations up to 1.0 microg/L. Exposure to 0.07 microg/L of esfenvalerate, however, caused a significant increase in Baetis spp. egg mortality, and exposure of near-eclosion eggs to lower concentrations (0.025 and 0.05 microg/L) resulted in behavioral effects and reduced survivorship in newly hatched Baetis nymphs. Early stage B. americanus eggs were 10-fold more sensitive to esfenvalerate when removed from the gelatinous clutch before exposure, an indication that the gelatin affords protection from toxicant exposure. Exposures of near-hatch B. americanus clutches to esfenvalerate concentrations ranging between 0.035 and 0.2 microg/L, however, resulted in significant clutch death within clutches resulting from behavioral aberrations of first-instar larvae. The results of the present study suggest that aquatic insect egg clutch morphology can be a strong influence on susceptibility of embryos to esfenvalerate exposure.

Effects of dietary esfenvalerate exposures on three aquatic insect species representing different functional feeding groups.

Given the chemical properties of synthetic pyrethroids, it is probable that compounds, including esfenvalerate, that enter surface waters may become incorporated into aquatic insect food sources. We examined the effect of dietary esfenvalerate uptake in aquatic insects representing different functional feeding groups. We used three field-collected aquatic insect species: A grazing scraper, Cinygmula reticulata McDunnough (Ephemeroptera: Heptageniidae); an omnivorous filter feeder, Brachycentrus americanus Banks (Trichoptera: Brachycentridae); and a predator, Hesperoperla pacifica Banks (Plecoptera: Perlidae). Laboratory-cultured algae were preexposed for 24 h to esfenvalerate concentrations of 0, 0.025, 0.05, and 0.1 microg/L and provided to two C. reticulata age classes (small and final-instar nymphs). Reduction in small nymph growth was observed following three weeks of feeding on algae exposed to 0.05 and 0.1 microg/L of esfenvalerate, and the highest dietary exposure reduced egg production in final-instar nymphs. The diet for B. americanus and H. pacifica consisted of dead third-instar Chironomus tentans larvae preexposed for 24 h to esfenvalerate concentrations ranging between 0.1 and 1.0 microg/L. Consumption of larvae exposed to 0.5 to 1.0 microg/L of esfenvalerate caused case abandonment and mortality in B. americanus caddisfly larvae. Although H. pacifica nymphs readily consumed esfenvalerate-exposed larvae, no adverse effects were observed during the present study. Furthermore, no evidence of esfenvalerate-induced feeding deterrence was found in any of the species tested, suggesting that aquatic insects may not be able to distinguish between pyrethroid-contaminated and uncontaminated food sources. These findings indicate that feeding deterrence is not a factor in regulating aquatic insect dietary exposures to synthetic pyrethroids.

Impact of aquatic insect life stage and emergence strategy on sensitivity to esfenvalerate exposure.

We investigated the impact of aquatic insect life stage and emergence strategy on sensitivity to esfenvalerate, a synthetic pyrethroid insecticide, using field-collected Brachycentrus americanus Banks (Trichoptera: Brachycentridae) and Cinygmula reticulata McDunnough (Ephemeroptera: Heptageniidae) insects. Final-instar C. reticulata emergence was observed for one week following three environmentally relevant, 48-h esfenvalerate exposures (0.005, 0.01, and 0.015 microg/L). Emergence was significantly depressed following exposure to esfenvalerate and resulted from an increase in nymph mortality during the emergence process. This experiment was duplicated for late-instar C. reticulata nymphs, which were similar in size to the final-instar nymphs but were not near emergence. Late-instar C. reticulata mayflies were approximately fivefold less sensitive to esfenvalerate exposures as gauged by one-week mortality rates. Brachycentrus americanus pupal mortality was significantly increased over that in controls following 48-h esfenvalerate exposures of 0.1 and 0.2 microg/L. These response concentrations correlated closely with those for case-abandonment rates of fourth-instar B. americanus larvae (a sublethal effect of esfenvalerate exposure). Pupal mortality rates were approximately 16-fold higher than those observed in larvae. Adult female egg weight as a percentage of total body weight was significantly decreased following pupal esfenvalerate exposures of 0.05, 0.1, and 0.2 microg/L. These findings suggest that exposure to esfenvalerate may impair hemimetabolous insect emergence behaviors and may decrease fecundity in holometabolous aquatic insects.

Esfenvalerate-induced case-abandonment in the larvae of the caddisfly (Brachycentrus americanus).

Field-collected Brachycentrus americanus Banks (Trichoptera: Brachycentridae) larvae were used to investigate the relationship between esfenvalerate exposure and case-abandonment response, determine larval ability to construct a new case, and measure the change in predation risk to insects in rebuilt cases. We evaluated case-abandonment following four environmentally relevant esfenvalerate exposures, 0.05, 0.1, 0.2, and 0.4 microg/L; 48-h exposures to 0.2 and 0.4 microg/L (nominal) esfenvalerate both resulted in over 60% of larvae abandoning cases and were statistically indistinguishable. Propensity to engage in building behaviors was significantly diminished in 0.2 and 0.4 microg/L esfenvalerate-exposed insects that had abandoned cases, with less than 20% of exposed insects producing cases. Cases built by intoxicated larvae were characterized by a disorganized composition, and required half the pressure to crush versus cases built by nonexposed larvae. Pre-exposing case-building material to 1 microg/L esfenvalerate also reduced the physical strength of rebuilt cases. Larvae inhabiting weaker rebuilt cases and larvae without cases were significantly more susceptible to predation by second year Hesperoperla pacifica Banks (Plecoptera: Perlidae) stonefly nymphs than those in original cases. Overall, we concluded that small behavioral responses can have profound consequences for survival of species and reveal susceptible stages in life-cycles that can be overlooked by conventional approaches to ecological risk assessment.

A sensory system at the interface between urban stormwater runoff and salmon survival.

Motor vehicles are a major source of toxic contaminants such as copper, a metal that originates from vehicle exhaust and brake pad wear. Copper and other pollutants are deposited on roads and other impervious surfaces and then transported to aquatic habitats via stormwater runoff. In the western United States, exposure to non-point source pollutants such as copper is an emerging concern for many populations of threatened and endangered Pacific salmon (Oncorhynchus spp.) that spawn and rear in coastal watersheds and estuaries. To address this concern, we used conventional neurophysiological recordings to investigate the impact of ecologically relevant copper exposures (0-20 microg/L for 3 h) on the olfactory system of juvenile coho salmon (O. kisutch). These recordings were combined with computer-assisted video analyses of behavior to evaluate the sensitivity and responsiveness of copper-exposed coho to a chemical predation cue (conspecific alarm pheromone). The sensory physiology and predator avoidance behaviors of juvenile coho were both significantly impaired by copper at concentrations as low as 2 microg/L. Therefore, copper-containing stormwater runoff from urban landscapes has the potential to cause chemosensory deprivation and increased predation mortality in exposed salmon.

Effects of 17alpha-ethinylestradiol and bisphenol A on vertebral development in the fathead minnow (Pimephales promelas).

Growth, reproductive ability, and metabolic functions may be impaired by disruption of early endocrine patterning. Natural and synthetic estrogens detected in surface waters have been linked to reproductive endocrine signaling disruption in several species. The present study characterizes the nonreproductive morphological endpoint of vertebral anomalies in fish exposed to environmental estrogens. Estrogen is a proliferation-inducing compound in osteoblasts, regulating cartilage and bone deposition during development in vertebrates. The hypothesis for the present work is that xenobiotics with estrogenic activity adversely impact vertebral bone formation. Fathead minnows (Pimephales promelas) were exposed to 0.1 to 100 microg/L 17alpha-ethinylestradiol (EE2) and 0.1 to 1,000 microg/L bisphenol A (BPA) from egg stage (24 h postfertilization) to 25 to 26 d posthatch. Fish were measured for length and analyzed microscopically to determine degree of skeletal development (developmental score) and the occurrence of spinal abnormalities, including vertebral compression, bone fusion, and spinal curvatures. Fish length and developmental score were inversely related to vertebral malformations in exposed fish. Skeletal developmental was affected significantly in EE2-exposed fish: Vertebral malformations were observed in up to 62% of fish in a nonmonotonic dose-response. However, BPA did not significantly impair skeletal development or induce vertebral malformations. The bioassay results suggest vertebral bone development is a potential endpoint of endocrine disruption from potent estrogenic compounds in surface waters.

Environmental stresses and skeletal deformities in fish from the Willamette River, Oregon.

The Willamette River, one of 14 American Heritage Rivers, flows through the most densely populated and agriculturally productive region of Oregon. Previous biological monitoring of the Willamette River detected elevated frequencies of skeletal deformities in fish from certain areas of the lower (Newberg pool [NP], rivermile [RM] 26 - 55) and middle (Wheatland Ferry [WF], RM 72 - 74) river, relative to those in the upper river (Corvallis [CV], RM 125-138). The objective of this study was to determine the likely cause of these skeletal deformities. In 2002 and 2003, deformity loads in Willamette River fishes were 2-3 times greater at the NP and WF locations than at the CV location. There were some differences in water quality parameters between the NP and CV sites, but they did not readily explain the difference in deformity loads. Concentrations of bioavailable metals were below detection limits (0.6 - 1 microg/ L). Concentrations of bioavailable polychlorinated biphenyls (PCBs) and chlorinated pesticides were generally below 0.25 ng/L. Concentrations of bioavailable polycyclic aromatic hydrocarbons were generally less than 5 ng/L. Concentrations of most persistent organic pollutants were below detection limits in ovary/oocyte tissue samples and sediments, and those that were detected were not significantly different among sites. Bioassay of Willamette River water extracts provided no evidence that unidentified compounds or the complex mixture of compounds present in the extracts could induce skeletal deformities in cyprinid fish. However, metacercariae of a digenean trematode were directly associated with a large percentage of deformities detected in two Willamette River fishes, and similar deformities were reproduced in laboratoryfathead minnows exposed to cercariae extracted from Willamette River snails. Thus, the weight of evidence suggests that parasitic infection, not chemical contaminants, was the primary cause of skeletal deformities observed in Willamette River fish.

Comparative thresholds for acetylcholinesterase inhibition and behavioral impairment in coho salmon exposed to chlorpyrifos.

Chlorpyrifos is a common organophosphate insecticide that has been widely detected in surface waters that provide habitat for Pacific salmon in the western United States. Although chlorpyrifos is known to inhibit acetylcholinesterase (AChE) in the brain and muscle of salmonids, the relationship between sublethal AChE inhibition and more integrative indicators of neuro-behavioral impairment are poorly understood. This is particularly true for exposures that reflect the typical range of pesticide concentrations in the aquatic environment. To directly compare the effects of chlorpyrifos on AChE activity and salmon behavior, we exposed juvenile coho salmon (Oncorhynchus kisutch) to chlorpyrifos (0-2.5 microg/L) for 96 h. A computer-assisted, three-dimensional video imaging system was used to measure spontaneous swimming and feeding behaviors in control and chlorpyrifos-exposed fish. After the behavioral trials, brain and muscle tissues were collected and analyzed for AChE activity. Chlorpyrifos inhibited tissue AChE activity and all behaviors in a dose-dependent manner. Moreover, brain AChE inhibition and reductions in spontaneous swimming and feeding activity were significantly correlated. Benchmark concentrations for sublethal neurotoxicity (statistical departure values) were <0.5 microg/L and were similar for both neurochemical and behavioral endpoints. Collectively, these results indicate a close relationship between brain AChE inhibition and behavioral impairment in juvenile coho exposed to chlorpyrifos at environmentally realistic concentrations.

Temperature influences on water permeability and chlorpyrifos uptake in aquatic insects with differing respiratory strategies.

Aquatic insects have evolved diverse respiratory strategies that range from breathing atmospheric air to breathing dissolved oxygen. These strategies result in vast morphological differences among taxa in terms of exchange epithelial surface areas that are in direct contact with the surrounding water that, in turn, affect physiological processes. This paper examines the effects of acute temperature shifts on water permeability and chlorpyrifos uptake in aquatic insects with different respiratory strategies. While considerable differences existed in water permeability among the species tested, acute temperature shifts raised water influx rates similarly in air-breathing and gill-bearing taxa. This contrasts significantly with temperature-shift effects on chlorpyrifos uptake. Temperature shifts of 4.5 degrees C increased 14C-chlorpyrifos accumulation rates in the gill-bearing mayfly Cinygma sp. and in the air-breathing hemipteran Sigara washingtonensis. However, the temperature-induced increase in 14C-chlorpyrifos uptake after 8 h of exposure was 2.75-fold higher in Cinygma than in Sigara. Uptake of 14C-chlorpyrifos was uniformly higher in Cinygma than in Sigara in all experiments. These findings suggest that organisms with relatively large exchange epithelial surface areas are potentially more vulnerable to both osmoregulatory distress as well as contaminant accumulation. Temperature increases appear more likely to impact organisms that have relatively large exchange epithelial surface areas, both as an individual stressor and in combination with additional stressors such as contaminants.

Treatment of atrazine in nursery irrigation runoff by a constructed wetland.

To investigate the treatment capability of a surface flow wetland at a container nursery near Portland, Oregon, atrazine was introduced during simulated runoff events. Treatment efficiency was evaluated as the percent atrazine recovered (as percent of applied) in the water column at the wetland's outlet. Atrazine treatment efficiency at the outlet of the constructed wetland during a 7-d period ranged from 18-24% in 1998 (experiments 1-3) and 16-17% in 1999 (experiments 4 and 5). Changes in total flow, or frequency and intensity of runoff events did not affect treatment. For experiment 6 in 1999, where the amount, frequency, and duration of runoff events exceeded all other experiments, treatment was compromised. For all experiments, deethylatrazine (DEA) and deisopropylatrazine (DIA) accounted for 13-21% of the initial application. Hydroxyatrazine (HA) was rarely detected in the water. Organic carbon adsorption coefficients (Koc) were determined from batch equilibrium sorption isotherms with wetland sediment, and they decreased in the order of HA > DIA > atrazine > DEA. Static water-sediment column experiments indicated that sorption is an important mechanism for atrazine loss from water passing through the constructed wetland. The results of the MPN assay indicated the existence in the wetland of a low-density population of microorganisms with the potential to mineralize atrazine's ethyl side chain.

Pacific steelhead (Oncorhynchus mykiss) exposed to chlorpyrifos: benchmark concentration estimates for acetylcholinesterase inhibition.

Steelhead trout (Oncorhynchus mykiss) were exposed for 96 h to the organophosphate chlorpyrifos to establish benchmark concentration (BMC) values in the low-effect range of brain acetylcholinesterase (AChE) inhibition. The U.S. Environmental Protection Agency (U.S. EPA) benchmark dose software was used to model the data. Benchmark concentrations were determined for a range of inhibition levels at 5, 10, and 20%, at 1 and 2 control standard deviations (SD), and at an experimental limit-of-detection level of 2.5%. One contributing difficulty in establishing precise inhibition BMCs in the lower effect region is the variability associated with the AChE analytical method. To minimize this variability, the Ellman method was modified specifically for analysis of O. mykiss brain tissue. Laboratory-established BMCs for chlorpyrifos were then compared with the U.S. EPA 96-h water quality criteria and with the concentration levels detected in Northwest surface waters that are home to threatened steelhead trout. The U.S. EPA 96-h water quality criteria of 0.083 microg/L is below the BMC(02.5), the limit-of-detection value for this study. The average chlorpyrifos concentration detected during a two-week period in one monitored stream was 0.127 microg/L, which approaches the BMC(02.5). The peak chlorpyrifos concentration detected at 0.482 microg/L is near the BMC(1SD) estimate.