Challenges with the current methodology for conducting Endangered Species Act risk assessments for pesticides in the United States.

The US Environmental Protection Agency (USEPA or the Agency) is responsible for administering the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). The Agency is also required to assess the potential risks of pesticides undergoing registration or re-registration to threatened and endangered (i.e., listed) species to ensure compliance with the Endangered Species Act. To assess potential risks to listed species, a screening-level risk assessment in the form of a biological evaluation (BE) is undertaken by the Agency for each pesticide. Given the large number of registration actions handled by the USEPA annually, efficient tools for conducting BEs are desirable. However, the "Revised Method" that is the basis for the USEPA's BE process has been ineffective at filtering out listed species and critical habitats that are at de minimis risk to pesticides. In the USEPA's BEs, the Magnitude of Effect Tool (MAGtool) has been used to determine potential risks to listed species that potentially co-occur with pesticide footprints. The MAGtool is a highly prescriptive, high-throughput compilation of existing FIFRA screening-level models with a geospatial interface. The tool has been a significant contributor to risk inflation and ultimately process inefficiency. The ineffectiveness of the tool stems from compounding conservatism, unrealistic and unreasonable assumptions regarding usage, limited application of species-specific data, lack of consideration of multiple lines of evidence, and inability to integrate higher-tier data. Here, we briefly describe the MAGtool and the critical deficiencies that impair its effectiveness, thus undermining its intention. Case studies are presented to highlight the deficiencies and solutions are recommended for improving listed species assessments in the future. Integr Environ Assess Manag 2023;19:817-829. © 2022 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Natural and anthropogenic factors and their interactions drive stream community integrity in a North American river basin at a large spatial scale.

Urbanization, agriculture, and other human activities can exert considerable influence on the health and integrity of stream ecosystems. These influences vary greatly over space, time, and scale. We investigated trends in stream biotic integrity over 19 years (1997-2016) in relation to natural and anthropogenic factors in their spatial context using data from a stream biomonitoring program in a region dominated by agricultural land use. Macroinvertebrate and fish diversity and abundance data were used to calculate four multimetric indices (MMIs) that described biotic integrity of streams from 1997 to 2016. Boosted regression trees (BRT), a machine learning technique, were used to model how stream integrity responded to catchment-level natural and anthropogenic drivers including land use, human population density, road density, runoff potential, and natural factors such as latitude and elevation. Neither natural nor anthropogenic factors were consistently more influential on the MMIs. Macroinvertebrate indices were most responsive to time, latitude, elevation, and road density. Fish indices were driven mostly by latitude and longitude, with agricultural land cover among the most influential anthropogenic factors. We concluded that 1) stream biotic integrity was mostly stable in the study region from 1997 to 2016, although macroinvertebrate MMIs had decreased approximately 10% since 2010; 2) stream biotic integrity was driven by a mix of factors including geography, human activity, and variability over yearly time intervals; 3) MMI responses to environmental drivers were nonlinear and often nonmonotonic; 4) MMI composition could influence causal inferences; and 5) although our findings were mostly consistent with the literature on drivers of stream integrity, some commonly seen patterns were not evident. Our findings highlight the utility of large-scale, publicly available spatial data for understanding drivers of stream biodiversity and illustrate some potential pitfalls of large scale, integrative analyses.

Thiamethoxam: Long-term effects following honey bee colony-level exposure and implications for risk assessment.

Neonicotinoid insecticides have been used in a wide range of crops through seed treatment, soil and foliar applications and a large database exists on both their lethal and sub-lethal effects on honey bees under controlled laboratory conditions. However, colony-level studies on the effects of neonicotinoids in field studies are limited, primarily due to their complexity and the resources required. This paper reports the combined results of two large-scale colony-feeding studies, each with 6 weeks of continuous dosing of 12 colonies per treatment (24 control) to 12.5, 25, 37.5, 50 or 100 ng thiamethoxam/g sucrose solution. Exposure continued beyond dosing with residues present in stored nectar and bee-bread. The studies were conducted in an area with limited alternative forage and colonies were required to forage for pollen and additional nectar The studies provide colony-level endpoints: significant effects (reductions in bees, brood) were observed after exposure to the two highest dose rates, colony loss occurred at the highest dose rate, but colonies were able to recover (2-3 brood cycles after the end of dosing) after dosing with 50 ng thiamethoxam/g sucrose. No significant colony-level effects were observed at lower dose rates. The data reported here support the conclusions of previous colony-level crop-based field studies with thiamethoxam, in which residues in pollen and nectar were an order of magnitude below the colony-level NOEC of 37.5 ng thiamethoxam/g sucrose. The feeding study data are also compared to the outcomes of regulatory Tier 1 risk assessments conducted using guidance provided by the USA, Canada, Brazil and the EU regulatory authorities. We propose an adaptation of the European chronic adult bee risk assessment that takes into account the full dataset generated in laboratory studies while still providing an order of magnitude of safety compared with the colony feeding study NOEC.

Thiamethoxam honey bee colony feeding study: Linking effects at the level of the individual to those at the colony level.

Neonicotinoid insecticides have been used globally on a wide range of crops through seed treatment as well as soil and foliar applications and have been increasingly studied in relation to the potential risk to bees because of their detection in pollen and nectar of bee-attractive crops. The present article reports the results of laboratory studies (10-d adult and 22-d larval toxicity studies assessing the chronic toxicity of thiamethoxam to adult honey bees and larvae, respectively) and a colony feeding study, with 6 wk of exposure in an area with limited alternative forage, to provide a prewintering colony-level endpoint. The endpoints following exposure of individuals in the laboratory (10-d adult chronic no-observed-effect concentration [NOEC] for mortality 117 µg thiamethoxam/kg sucrose solution, 141 µg thiamethoxam/L sucrose solution; 22-d larval chronic NOEC 102 µg thiamethoxam/kg diet) are compared with those generated at the colony level, which incorporates sublethal effects (no-observed-adverse-effect concentration [NOAEC] 50 µg thiamethoxam/L sucrose solution, 43 µg thiamethoxam/kg sucrose solution). The data for sucrose-fed honey bee colonies support the lack of effects identified in previous colony-level field studies with thiamethoxam. However, unlike these field studies demonstrating no effects, colony feeding study data also provide a threshold level of exposure likely to result in adverse effects on the colony in the absence of alternative forage, and a basis by which to evaluate the potential risk of thiamethoxam residues detected in pollen, nectar, or water following treatment of bee-attractive crops. Environ Toxicol Chem 2018;37:816-828. © 2017 SETAC.

Initial recommendations for higher-tier risk assessment protocols for bumble bees, Bombus spp. (Hymenoptera: Apidae).

Global declines of bumble bees and other pollinator populations are of concern because of their critical role for crop production and maintenance of wild plant biodiversity. Although the consensus among scientists is that the interaction of many factors, including habitat loss, forage scarcity, diseases, parasites, and pesticides, potentially plays a role in causing these declines, pesticides have received considerable attention and scrutiny. In response, regulatory agencies have introduced more stringent pollinator testing requirements for registration and reregistration of pesticides, to ensure that the risks to pollinators are minimized. In this context, guidelines for testing bumble bees (Bombus spp.) in regulatory studies are not yet available, and a pressing need exists to develop suitable protocols for routine higher-tier studies with these non-Apis sp., social bees. To meet this need, Bayer CropScience LP, Syngenta Crop Protection LLC US, and Valent USA. Corporation organized a workshop bringing together a group of global experts on bumble bee behavior, ecology, and ecotoxicology to discuss and develop draft protocols for both semi-field (Tier II) and field (Tier III) studies. The workshop was held May 8-9, 2014, at the Bayer Bee Care Center, North Carolina, USA. The participants represented academic, consulting, and industry scientists from Europe, Canada, the United States, and Brazil. The workshop identified a clear protection goal and generated proposals for basic experimental designs, relevant measurements, and endpoints for both semifield (tunnel) and field tests. These initial recommendations are intended to form the basis of discussions to help advance the development of appropriate protocol guidelines.

Assessment of the toxicological interaction of sertraline with cholinesterase inhibiting insecticides in aquatic insects using the black fly, Simulium vittatum IS-7.

Sertraline is a selective serotonin reuptake inhibitor (SSRI) prescribed as an antidepressant. Although SSRIs are known to block serotonin reuptake sites on cell membranes, they also have been shown to inhibit acetylcholinesterase (AChE) activity. Thus, the interaction of these chemicals with other AChE inhibitors, namely, organophosphate and carbamate insecticides, is of interest. In addition, these insecticides have been shown to interact with serotonergic neuronal pathways creating questions as to how these chemicals might interact. In this study, the interactive effect of sertraline (SSRI) in binary combinations with carbaryl (carbamate insecticide) and diazinon (organophosphate insecticide) was assessed using a 48-h acute toxicity test with black fly larvae, Simulium vittatum IS-7. Results showed that observed mortality was bracketed by the independent action model and concentration addition model with the independent action model slightly underestimating mortality and the concentration addition model slightly overestimating mortality. Varying the concentration of the chemicals in the mixture did not indicate that sertraline was interacting with the insecticides to make them more toxic or vice versa. These results indicate that sertraline and the insecticides are likely eliciting toxicity at separate neuronal pathways since no interaction was observed.

Mechanistic approach to understanding the toxicity of the azole fungicide triadimefon to a nontarget aquatic insect and implications for exposure assessment.

Mechanistic and stereoselective based in vitro metabolism assays were utlilized to gain insight into the toxic mode of action of the 1,2,4-triazole fungicide, triadimefon, with black fly (Diptera: Simuliidae) larvae. Based on results from enzyme inhibitor studies, the metabolism of triadimefon in black fly larvae microsomes was found to occur predominantly via an oxidative P450-mediated pathway; triadimenol was formed via the stereoselective reduction of the prochiral carbonyl group of triadimefon. The relatively minor contribution of carbonyl reduction suggests that triadimefon may inhibit ecdysone 20-monooxygenase and disrupt insect molting hormone biosynthesis. 48-h LC50 tests for triadimefon and triadimenol with black fly larvae yielded median values (with 95% confidence intervals) of 6.1 (5.8-6.4) and 22.3 (20.3-24.1) mg/L respectively. The exposure of black fly larvae to sublethal concentrations of triadimefon resulted in increased microsomal P450 activity and affected the microsomal rates of both triadimefon depletion and triadimenol formation. In contrast to trout, black fly larvae produced a higher fraction of the more toxic triadimenol stereoisomers, which may explain in part why triadimefon exhibited a significantly greater toxicity with black fly larvae than trout. These results illustrate that while LC50 tests conducted with commercial triadimenol would presumably expose each organism to the same relative abundance of the four triadimenol stereoisomers, LC50 tests with triadimefon ultimately expose each organism to a unique set of triadimenol stereoisomers depending upon the organism's stereoselective metabolism.

Fractionation and metabolic turnover of carbon and nitrogen stable isotopes in black fly larvae.

Diet-tissue fractionation factors and metabolic turnover rates of delta15N and delta13C were assessed in laboratory-reared black fly (Simulium vittatum IS-7) larvae fed isotopically distinct diets. Five treatments consisted of using food with different delta15N signatures throughout the experiments (19-26 days), a sixth shifted from a low to high delta15N signature diet (uptake) on day 14, and the last shifted from a high to low delta15N signature diet (elimination) on day 14. In the larvae, diet-tissue fractionation factors for delta13C, which were in steady state with food, ranged from -0.61 to 2.0, with a median of 1.87. The delta15N diet-tissue fractionation factors were mostly negative, ranging from +2.85 to -24.96 per thousand, with a single positive value from the elimination treatment in which larval delta15N did not achieve steady state with the food. Diet-tissue fractionation factors also had a significant negative relationship (r2 = 0.98) with delta15N values in the food suggesting that nitrogen diet-tissue fractionation factors are 15N concentration-dependent. The delta15N of shed head capsules and feces were enriched in 15N and could be mechanisms for elimination of 15N by the larvae. For delta15N, metabolic turnover values based on the Hesslein model were highly consistent (0.40 to 0.43 delta15N*day(-1)) between uptake and elimination phases and across experiments and were an order of magnitude greater than growth rates. The rapid turnover of nitrogen in black fly larvae, which was orders of magnitude greater than measured in vertebrates, makes them an excellent indicator of short-term changes in nitrogen inputs to aquatic systems.

Toxicity of fipronil and its enantiomers to marine and freshwater non-targets.

Fipronil is a phenylpyrazole insecticide used in agricultural and domestic settings for controlling various insect pests in crops, lawns, and residential structures. Fipronil is chiral; however, it is released into the environment as a racemic mixture of two enantiomers. In this study, the acute toxicity of the (S,+) and (R,-) enantiomers and the racemic mixture of fipronil were assessed using Simulium vittatum IS-7 (black fly), Xenopus laevis (African clawed frog), Procambarus clarkii (crayfish), Palaemonetes pugio (grass shrimp), Mercenaria mercenaria (hardshell clam), and Dunaliella tertiolecta (phytoplankton). Results showed that S. vittatum IS-7 was the most sensitive freshwater species to the racemic mixture of fipronil (LC50 = 0.65 microg/L) while P. pugio was the most sensitive marine species (LC50 = 0.32 microg/L). Procambarus clarkii were significantly more sensitive to the (S,+) enantiomer while larval P. pugio were significantly more sensitive to the (R,-) enantiomer. Enantioselective toxicity was not observed in the other organisms tested. Increased mortality and minimal recovery was observed in all species tested for recovery from fipronil exposure. These results indicate that the most toxic isomer of fipronil is organism-specific and that enantioselective toxicity may be more common in crustaceans than in other aquatic organisms.

Mitigating the effects of the green alga Scenedesmus quadricauda on the efficacy of Bacillus thuringiensis var. israelensis against larval black flies.

The green alga Scenedesmus quadricauda has been shown to decrease the efficacy of Bacillus thuringiensis var. israelensis (Bti) to black fly larvae in laboratory experiments. In this study, 2 approaches to mitigating the effects of S. quadricauda were tested, increasing the dose of Bti exposed to the larval black flies and increasing the length of the exposure period. Results showed that increasing the dose as well as increasing the exposure time were effective methods for mitigating the effects of S. quadricauda on Bti efficacy. Assessment of larval mortality at different exposure periods to Bti indicate that the manifestation of toxicity or acquisition of toxic doses of the Bti formulation takes place approximately 2-3 times slower when S. quadricauda is present. These results provide information that might be useful in developing a plan of action to combat this complex situation in field operations.

Impacts of lawn-care pesticides on aquatic ecosystems in relation to property value.

To determine the potential impacts of lawn-care pesticides on aquatic ecosystems, the macroinvertebrate communities of six streams were assessed using a multimetric approach. Four streams flowed through residential neighborhoods of Peachtree City, GA, USA, with differing mean property values and two reference streams were outside the city limits. A series of correlation analyses were conducted comparing stream rank from water quality and physical stream parameters, habitat assessments, benthic macroinvertebrate metric, pesticide toxicity and metal toxicity data to determine relationships among these parameters. Significant correlations were detected between individual analyses of stream rank for pesticide toxicity, specific conductance, turbidity, temperature and dissolved oxygen with benthic macroinvertebrate metrics.

Effects of algae on the efficacy of Bacillus thuringiensis var. israelensis against larval black flies.

Personnel from several black fly control programs have reported that the efficacy of Bacillus thuringiensis var. israelesis (Bti) is reduced during periods when algal concentrations are high in the waterways. Although the reduction in Bti-induced mortality in black fly larvae is presumed to be related to the presence of algae, no scientific data support this theory. In this study, 4 genera of algae (Microcytis, Scenedesmus, Dictrosphaerium, and Chlorella) commonly detected in Pennsylvania rivers where Bti-induced mortality in black fly larvae has been reduced were assessed to determine their respective effects on Bti-induced mortality by using an orbital shaker bioassay with laboratory-reared black fly larvae (Simulium vittatum cytospecies IS-7). A significant reduction in Bti-induced mortality was observed when Scenedesmus was present in the flasks at concentrations > or = 16,000 cells/ml. The Bti-induced mortality of larvae was not significantly reduced when Chlorella, Dictyosphaerium, or Microcytis was present in the flasks, even at concentrations > or = 250,000 cells/ml. These results indicate that the presence of certain types of algae can reduce the mortality of black flies exposed to Bti. Although not clearly defined, the mechanisms involved may be related to algal morphology due to overall size and structures associated with certain types of algae, and possible interference with feeding.

Influence of developmental stage on susceptibilities and sensitivities of Simulium vittatum IS-7 and Simulium vittatum IIIL-1 (Diptera: Simuliidae) to chlorpyrifos.

Determination of the most sensitive and susceptible organismal life stage is important for use of the organism in toxicity assessments of environmental pollutants. In the present study, the sensitivities and susceptibilities of larval developmental stages of two black fly sibling species, Simulium vittatum Zetterstedt cytospecies IS-7 and S. vittatum Zetterstedt cytospecies IIIL-1, were determined using the organophosphate-insecticide chlorpyrifos. Differences in sensitivity and susceptibility were determined through analysis of slopes and median lethal concentration values produced from 24-h orbital shaker toxicity tests, respectively. The results showed no difference in sensitivity or susceptibility between sibling species. However, early instar (second and third) as well as mid-instar (fourth and fifth) groupings of S. vittatum IS-7 were significantly more susceptible than late-instar (sixth and seventh) larval groupings. Likewise, mid-instar groupings of S. vittatum IIIL-1 were more susceptible than late-instar larval groupings. However, neither species showed significant differences in sensitivity among instar groupings. The results of the present study indicate that the mid-instar groupings of black fly larvae are the best choice for use in toxicity tests, both because of their increased susceptibility compared to late-instar groupings and because of their ease in handling and manipulating in the laboratory compared to early instar groupings.

Susceptibility of black fly larvae (Diptera: Simuliidae) to lawn-care insecticides individually and as mixtures.

Urban and suburban watersheds have the potential to be highly impacted by chemicals, especially insecticides to control insect pests on lawns, ornamental plants, and home gardens. Three of the most common lawn-care insecticides detected in urban watersheds, carbaryl, chlorpyrifos, and malathion, have been evaluated using an acute orbital shaker toxicity test to determine their respective concentrations that produce 50% mortality (LC50) in Simulium vittatum Zetterstedt cytospecies IS-7 larvae. Results of the 48-h LC50 tests show chlorpyrifos to be the most toxic to black fly larvae (LC50 = 0.28 microg/L) followed by carbaryl (LC50 = 23.72 microg/L) and malathion (LC50 = 54.20 microg/L). These insecticides were also tested as binary and ternary mixtures using the toxic unit (TU) approach. Toxicity was shown to be greater than additive for the ternary mixture of chlorpyrifos-carbaryl-malathion (LC50 = 0.56 TU) and the binary mixtures of chlorpyrifos-malathion (LC50 = 0.72 TU) and carbaryl-malathion (LC50 = 0.78 TU). The binary combination of chlorpyrifos and carbaryl was shown to be additive (LC50 = 0.98 TU). These results indicate that aquatic invertebrate populations in urban and suburban streams may experience a higher-than-expected increase in toxicity-related effects when all three chemicals are present in the waterway.