The Roles of Diet and Habitat Use in Pesticide Bioaccumulation by Juvenile Chinook Salmon: Insights from Stable Isotopes and Fatty Acid Biomarkers.

Stable isotopes (SI) and fatty acid (FA) biomarkers can provide insights regarding trophic pathways and habitats associated with contaminant bioaccumulation. We assessed relationships between SI and FA biomarkers and published data on concentrations of two pesticides [dichlorodiphenyltrichloroethane and degradation products (DDX) and bifenthrin] in juvenile Chinook Salmon (Oncorhynchus tshawytscha) from the Sacramento River and Yolo Bypass floodplain in Northern California near Sacramento. We also conducted SI and FA analyses of zooplankton and macroinvertebrates to determine whether particular trophic pathways and habitats were associated with elevated pesticide concentrations in fish. Relationships between DDX and both sulfur (δ34S) and carbon (δ13C) SI ratios in salmon indicated that diet is a major exposure route for DDX, particularly for individuals with a benthic detrital energy base. Greater use of a benthic detrital energy base likely accounted for the higher frequency of salmon with DDX concentrations > 60 ng/g dw in the Yolo Bypass compared to the Sacramento River. Chironomid larvae and zooplankton were implicated as prey items likely responsible for trophic transfer of DDX to salmon. Sulfur SI ratios enabled identification of hatchery-origin fish that had likely spent insufficient time in the wild to substantially bioaccumulate DDX. Bifenthrin concentration was unrelated to SI or FA biomarkers in salmon, potentially due to aqueous uptake, biotransformation and elimination of the pesticide, or indistinct biomarker compositions among invertebrates with low and high bifenthrin concentrations. One FA [docosahexaenoic acid (DHA)] and DDX were negatively correlated in salmon, potentially due to a greater uptake of DDX from invertebrates with low DHA or effects of DDX on FA metabolism. Trophic biomarkers may be useful indicators of DDX accumulation and effects in juvenile Chinook Salmon in the Sacramento River Delta.

Assessing the Influence of Organic Carbon, Aging Time and Temperature on Bioaccessibility of Bifenthrin.

Tenax extraction, a measure of chemical desorption rates from sediments, was used to evaluate the bioaccessibility of bifenthrin in two different sediments exposed to three temperatures aged over a 56-d holding period. A 24-h single-point Tenax extraction was used and parent 14C-bifenthrin and polar metabolites were quantified in the sediment and Tenax. Bioaccessibility of bifenthrin was inversely related to the organic carbon (OC) content in the sediment, holding time, and temperature. Sequestration of the bifenthrin into slowly desorbing fractions within the sediment appears to have decreased degradation of the parent compound into metabolites and decreased the amount of parent compound bioaccessible for uptake by the Tenax. These results suggest that the environmental risk of bifenthrin to aquatic species is greatest immediately after the pesticide enters a waterbody after runoff, for low-OC content sediments, and in areas or seasons where water temperatures are colder.

Predicting Resistance: Quantifying the Relationship between Urban Development, Agricultural Pesticide Use, and Pesticide Resistance in a Nontarget Amphipod.

Resistance alleles within the voltage-gated sodium channel (vgsc) have been correlated with pyrethroid resistance in wild populations of the nontarget amphipod, Hyalella azteca from California (CA), U.S.A. In the present study, we expand upon the relationship between land use and the evolution of pesticide resistance in H. azteca to develop a quantitative methodology to target and screen novel populations for resistance allele genotypes in a previously uninvestigated region of the U.S. (New England: NE). By incorporating urban land development and toxicity-normalized agricultural pesticide use indices into our site selection, we successfully identified three amino acid substitutions associated with pyrethroid resistance. One of the resistance mutations has been described in H. azteca from CA (L925I). We present the remaining two (vgsc I936F and I936V) as novel pyrethroid-resistance alleles in H. azteca based on previous work in insects and elevated cyfluthrin resistance in one NE population. Our results suggest that urban pesticide use is a strong driver in the evolution of resistance alleles in H. azteca. Furthermore, our method for resistance allele screening provides an applied framework for detecting ecosystem impairment on a nationwide scale that can be incorporated into ecological risk assessment decisions.

Dietary Exposure to Bifenthrin and Fipronil Impacts Swimming Performance in Juvenile Chinook Salmon (Oncorhynchus tshawytscha).

Two commonly used insecticides, bifenthrin and fipronil, can accumulate in the prey of juvenile Chinook salmon, yet the effects of dietary exposure are not understood. Therefore, to better characterize the effect of a dietary exposure route, juvenile Chinook salmon were fed chironomids dosed with a concentration of 9 or 900 ng/g of bifenthrin, fipronil, or their mixture for 25 days at concentrations previously measured in field-collected samples. Chinook were assessed for maximum swimming performance (Umax) using a short-duration constant acceleration test and biochemical responses related to energetic processes (glucose levels) and liver health (aspartate aminotransferase (AST) activity). Chinook exposed to bifenthrin and bifenthrin and fipronil mixtures had a significantly reduced swimming performance, although not when exposed to fipronil alone. The AST activity was significantly increased in bifenthrin and mixture treatments and glucose levels were increased in Chinook following a mixture treatment, although not when exposed to fipronil alone. These findings suggest that there are different metabolic processes between bifenthrin and fipronil following dietary uptake that may influence toxicity. The significant reductions in swimming performance and increased levels of biochemical processes involved in energetics and fish heath could have implications for foraging activity and predator avoidance in wild fish at sensitive life stages.

The Use of Non-targeted Lipidomics and Histopathology to Characterize the Neurotoxicity of Bifenthrin to Juvenile Rainbow Trout (Oncorhynchus mykiss).

Due to the detection frequencies and measured concentrations in surface water, the type I pyrethroid insecticide, bifenthrin, has been of particular concern within the Sacramento-San Joaquin Delta in California. Concentrations have been detected above levels previously reported to impair neuroendocrine function and induce neurotoxicity to several species of salmonids. Metabolomic and transcriptomic studies indicated impairment of cellular signaling within the brain of exposed animals and potential alteration of lipid metabolism. To better understand the potential impacts of bifenthrin on brain lipids, juvenile rainbow trout (Oncorhynchus mykiss) were exposed to mean bifenthrin concentrations of 28 or 48 ng/L for 14 days, and non-targeted lipidomic profiling in the brain was conducted. Brain tissue sections were also assessed for histopathological insult following bifenthrin treatment. Bifenthrin-exposed trout had a concentration-dependent decrease in the relative abundance of triglycerides (TGs) with levels of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) significantly altered following 48 ng/L bifenthrin exposure. An increased incidence of histopathological lesions, such as focal hemorrhages and congestion of blood vessels, was noted in the brains of bifenthrin-treated animals, suggesting an association between altered lipid metabolism and neuronal cell structure and integrity.

Taking the Bite out of Mosquito Bites: The Role of Perceived Risk.

The present study examined how dimensions of perceived risk contributed to mosquito avoidance behaviors in a flood-prone area. Mosquito avoidance behaviors were classified into proactive (i.e. used repellant sources) and withdrawal/reactive (i.e. reduced outside activities) behaviors. After controlling for level of mosquito bites, increased scores for above normal perceived risk were associated with withdrawal/reactive avoidance behaviors; whereas, increased scores for normal perceived risk were associated with proactive avoidance behaviors. Efforts to improve mosquito avoidance behaviors should distinguish the type of perceived risk and the type of avoidance behavior. Greater congruence between perceived risk (i.e. normal risk of mosquito-borne illnesses) and avoidance behaviors (i.e. planning avoidance behaviors) will increase the effectiveness of education programs for disease prevention.

Effects of dietary cypermethrin exposure on swimming performance and expression of lipid homeostatic genes in livers of juvenile Chinook salmon, Oncorhynchus tshawytscha.

The increased use of pyrethroid insecticides raises concern for exposure to non-target aquatic species, such as Chinook salmon (Oncorhynchus tshawytscha). Cypermethrin, a type II pyrethroid, is frequently detected in surface waters and sediments at concentrations that exceed levels that induce toxicity to several invertebrate and salmonid species. To better understand the effects of cypermethrin to salmonids following dietary exposure, juvenile Chinook salmon were dietarily exposed to a 0, 200, or 2000 ng/g cypermethrin diet for a duration of 7, 14, or 21 days and assessed for body burden residues, swimming performance, lipid content, and lipid homeostatic gene expression. The average cypermethrin concentrations in fish dietarily exposed to cypermethrin for 21 days were 155.4 and 952.1 ng cypermethrin/g lipid for the 200 and 2000 ng/g pellet treatments, respectively. Increased trends of fatty acid synthase (fasn, r2 = 0.10, p < 0.05) and ATP citrate lyase (acly, r2 = 0.21, p < 0.001) mRNA expression were found in the fish livers relative to increasing cypermethrin body burden residues, though no significant changes in the mRNA expression of farnesoid X receptor or liver X receptor were observed. Furthermore, Chinook salmon dietarily exposed to cypermethrin did not have a significantly altered burst swimming performance (Umax). These results support studies that have suggested Umax may not be a sensitive endpoint when assessing the effects of certain pesticide classes, such as pyrethroids, but that dysregulation of fasn and acly expression may alter lipid homeostasis and energy metabolism in the liver of fish dietarily exposed to cypermethrin.

Pyrethroid bioaccumulation in field-collected insecticide-resistant Hyalella azteca.

Wild-type Hyalella azteca are highly sensitive to pyrethroid insecticides and typically do not survive exposure; however, pyrethroid bioaccumulation by insecticide-resistant H. azteca is an important potential risk factor for the transfer of pyrethroids to higher trophic species in aquatic systems. In the current study, four populations of pyrethroid-resistant H. azteca with corresponding sediment samples were sampled throughout the year, and nine-current use pyrethroids (tefluthrin, fenpropathrin, bifenthrin, cyhalothrin, permethrin, cyfluthrin, cypermethrin, esfenvalerate and deltamethrin) were measured. Bifenthrin was detected in every pyrethroid-resistant H. azteca tissue sample, up to 813 ng/g lipid, while cyhalothrin and permethrin were detected in fewer (18 and 28%, respectively) samples. Concurrent sampling of the sediment showed total pyrethroid concentrations exceeding toxic unit thresholds for non-resistant H. azteca survival, and confirmed the ubiquitous presence of bifenthrin at each site and sampling event. Bifenthrin concentrations in H. azteca tended to be higher in samples collected in winter months, and seasonal factors, such as temperature and rainfall, may have contributed to the noted differences in bioaccumulation. Finally, the bifenthrin and permethrin biota-sediment accumulation factors (BSAF) for pyrethroid-resistant H. azteca were similar to the BSAF values for less sensitive invertebrates, and therefore the development of resistance may enable an additional pathway for trophic transfer of pyrethroids in species that would otherwise be too sensitive to survive the exposure.

Analysis of RNA Interference (RNAi) Biopesticides: Double-Stranded RNA (dsRNA) Extraction from Agricultural Soils and Quantification by RT-qPCR.

Double-stranded RNA (dsRNA) molecules are used as a novel class of biopesticides. To enable assessments of the ecological risk associated with their release to receiving environments, we developed an approach to quantify dsRNA in agricultural soils using quantitative reverse transcription-polymerase chain reaction (RT-qPCR). To allow quantification of dsRNA adsorbed to particles, we also developed a protocol to transfer dsRNA from particles to the extraction buffer by changing particle surface charge and adding constituents to compete with dsRNA for adsorption sites. Our approach could quantify dsRNA amounts as low as 0.003 ngdsRNA/gsoil. This approach is the first available field-applicable approach able to quantify dsRNA biopesticides down to environmentally relevant concentrations. We applied this approach to investigate dsRNA dissipation (including dilution, degradation, and adsorption) in two agricultural soils. When we applied a low amount of dsRNA (1 ngdsRNA/gsoil) to the soils, we observed that a greater fraction of dsRNA was adsorbed to and extractable from soil particles in a silty clay loam soil than in a fine sandy loam soil. In both soils, dsRNA dissipated on the timescale of hours. Overall, these results demonstrate that our approach can be applied to assess the environmental fate of dsRNA biopesticides at concentrations relevant to their release to soils.

Lifelong Exposure to Dioxin-Like PCBs Alters Paternal Offspring Care Behavior and Reduces Male Fish Reproductive Success.

Offspring survival, cohort performance, and ultimately population dynamics are strongly influenced by maternal characteristics (e.g., fecundity), whereas paternal contribution is often considered limited to genetic-driven fitness of males through sexual selection. However, male contribution to reproductive success can be particularly influential in species exhibiting paternal offspring care. Polychlorinated biphenyls (PCBs) are widespread, persistent contaminants that can disrupt maternal reproductive processes and negatively affect offspring. In contrast, how PCBs affect paternal reproductive success is largely unknown, but could ultimately affect population dynamics. We examined the effects of lifelong PCB exposure on the reproductive processes of male fathead minnows (Pimephales promelas), a species exhibiting sole paternal offspring care, by examining endocrine-associated gene expression, testes histology, secondary sexual characteristics, courtship ability, offspring care, and offspring survival. PCBs minimized male secondary sexual characteristics, but did not affect gonadal end points or inhibit ability to court females. Fathers exposed to high concentrations of dioxin-like PCBs had changes in gene expression, reduced offspring care behavior, and higher embryo mortality, possibly due to fathers spending less time within nests and less frequently tending to embryos. Through complex interactions among gene expression, physical characteristics, and behavior, PCBs inhibit paternal reproductive success and have the potential to suppress population size.

Evaluating toxicity risk in sediments after remediation at a Superfund megasite using a Triad approach.

The Pine River downstream of the Velsicol Superfund site has been contaminated with various hydrophobic organic pollutants for more than 50 years. Remediation and sediment dredging near the site began in spring of 1999, and was completed in 2006. In 2011, the Michigan Department of Environmental Quality completed a baseline assessment report long-term monitoring plan for the Pine River. However, there has been limited assessment of the benthic community since this evaluation. The objective of this research was to evaluate the risk of Pine River sediment to aquatic macroinvertebrates downstream from the Superfund site after decades of degradation and dredging using the Triad approach. Three sites were selected downstream from the Superfund site, and an upstream reference site was used. At each site, macroinvertebrates surveys were conducted and sediments were collected for chemical analysis of DDT (1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane) and its degradation products and for laboratory toxicity testing for mortality and sublethal effects using Hyalella azteca and Chironomus dilutus. Sediment concentrations of DDT, DDD, and DDE were below levels expected to cause toxicity, and there was no observed toxicity in laboratory tests. Additionally, there were no statistically significant differences in richness, richness of Ephemeroptera, Plecoptera, and Trichoptera (EPT) species, total EPT abundance, percent EPT, or percent dominant taxa between the reference site and the downstream sites. There was an observed decrease in abundance of macroinvertebrate taxa at all downstream sites and a shift in macroinvertebrate structure when comparing the reference with most impaired sites. Although the sites downstream of the Superfund site remain different than the upstream control, there are improvements in species composition and abundance. However, more research is needed to evaluate the potential effects on ecosystem function.

Using Mutations for Pesticide Resistance to Identify the Cause of Toxicity in Environmental Samples.

Traditional Toxicity Identification Evaluations (TIE) are applied to identify causal agents in complex environmental samples showing toxicity and rely upon physical or chemical manipulation of samples. However, mutations conferring toxicant resistance provide the opportunity for a novel biologically based TIE. Populations within the Hyalella azteca complex from pesticide-affected waterways were 2 and 3 orders of magnitude more resistant to the pyrethroid cyfluthrin and the organophosphate chlorpyrifos, respectively, than laboratory-cultured H. azteca widely used for toxicity testing. Three resistant populations, as well as laboratory-cultured, nonresistant H. azteca, were exposed to urban and agricultural runoff. Every sample causing death or paralysis in the nonresistant individuals had no effect on pyrethroid-resistant individuals, providing strong evidence that a pyrethroid was the responsible toxicant. The lack of toxicity to chlorpyrifos-sensitive, but pyrethroid-resistant, individuals suggested chlorpyrifos was not a likely toxicant, a hypothesis supported by chemical analysis. Since these mutations that confer resistance to pesticides are highly specific, toxicity to wild-type, but not resistant animals, provides powerful evidence of causality. It may be possible to identify strains resistant to even a wider variety of toxicants, further extending the potential use of this biologically based TIE technique beyond the pyrethroid and organophosphate-resistant strains currently available.

Advancing the Use of Passive Sampling in Risk Assessment and Management of Sediments Contaminated with Hydrophobic Organic Chemicals: Results of an International Ex Situ Passive Sampling Interlaboratory Comparison.

This work presents the results of an international interlaboratory comparison on ex situ passive sampling in sediments. The main objectives were to map the state of the science in passively sampling sediments, identify sources of variability, provide recommendations and practical guidance for standardized passive sampling, and advance the use of passive sampling in regulatory decision making by increasing confidence in the use of the technique. The study was performed by a consortium of 11 laboratories and included experiments with 14 passive sampling formats on 3 sediments for 25 target chemicals (PAHs and PCBs). The resulting overall interlaboratory variability was large (a factor of ∼10), but standardization of methods halved this variability. The remaining variability was primarily due to factors not related to passive sampling itself, i.e., sediment heterogeneity and analytical chemistry. Excluding the latter source of variability, by performing all analyses in one laboratory, showed that passive sampling results can have a high precision and a very low intermethod variability (

Improvements and cost-effective measures to the automated intermittent water renewal system for toxicity testing with sediments.

The push to make bioassays more sensitive has meant an increased duration of testing to look at more chronic endpoints. To conduct these longer bioassays through the use of traditional bioassay methods can be difficult, as many traditional bioassays have employed manual water changes, which take considerable time and effort. To that end, static-renewal systems were designed to provide researchers a technique to ease the manual water change burden. One of the most well-known static-renewal designs, the static intermittent renewal system (STIR) was produced by the United States Environmental Protection Agency in 1993. This system is still being used in laboratories across the globe today. However, these initial designs have become rather dated as new technologies and methods have been developed that make these systems easier to build and operate. The following information details changes to the initial design and a proof of concept experiment with the benthic invertebrate, Chironomus tepperi, to validate the modifications to the original system.

The Value of Using Multiple Metrics to Evaluate PCB Exposure.

Current methods for evaluating exposure in ecosystems contaminated with hydrophobic organic contaminants typically focus on sediment exposure. However, a comprehensive environmental assessment requires a more holistic approach that not only estimates sediment concentrations, but also accounts for exposure by quantifying other pathways, such as bioavailability, bioaccumulation, trophic transfer potential, and transport of hydrophobic organic contaminants within and outside of the aquatic system. The current study evaluated the ability of multiple metrics to estimate exposure in an aquatic ecosystem. This study utilized a small lake contaminated with polychlorinated biphenyls (PCBs) to evaluate exposure to multiple trophic levels as well as the transport of these contaminants within and outside of the lake. The PCBs were localized to sediments in one area of the lake, yet this area served as the source of PCBs to aquatic invertebrates, emerging insects, and fish and terrestrial spiders in the riparian ecosystem. The Tenax extractable and biota PCB concentrations indicated tissue concentrations were localized to benthic invertebrates and riparian spiders in a specific cove. Fish data, however, demonstrated that fish throughout the lake had PCB tissue concentrations, leading to wider exposure risk. The inclusion of PCB exposure measures at several trophic levels provided multiple lines of evidence to the scope of exposure through the aquatic and riparian food web, which aids in assessing risk and developing potential future remediation strategies.

Fate and transport of furrow-applied granular tefluthrin and seed-coated clothianidin insecticides: Comparison of field-scale observations and model estimates.

The transport of agricultural insecticides to water bodies may create risk of exposure to non-target organisms. Similarly, widespread use of furrow-applied and seed-coated insecticides may increase risk of exposure, yet accessible exposure models are not easily adapted for furrow application, and only a few examples of model validation of furrow-applied insecticides exist using actual field data. The goal of the current project was to apply an exposure model, the Pesticide in Water Calculator (PWC), to estimate the concentrations of two in-furrow insecticides applied to maize: the granular pyrethroid, tefluthrin, and the seed-coated neonicotinoid, clothianidin. The concentrations of tefluthrin and clothianidin in surface runoff water, sampled from a field in central Illinois (USA), were compared to the PWC modeled pesticide concentrations in surface runoff. The tefluthrin concentrations were used to optimize the application method in the PWC, and the addition of particulate matter and guttation droplets improved the models prediction of clothianidin concentrations. Next, the tefluthrin and clothianidin concentrations were calculated for a standard farm pond using both the optimized application method and the application methods provided in PWC. Estimated concentrations in a standard farm pond varied by a factor of 100 for tefluthrin and 50 for clothianidin depending on the application method used. The addition of guttation droplets and particulate matter to the model increased the annual clothianidin concentration in a standard farm pond by a factor of 1.5, which suggested that these transport routes should also be considered when assessing neonicotinoid exposure.

Methodological and Environmental Impacts on Bioaccessibility Estimates Provided by Single-Point Tenax Extractions.

Single-point Tenax extractions (SPTEs) of hydrophobic organic contaminants provide estimates of bioaccessibility through consistent measures of the chemical concentration initially in the rapidly desorbing fraction in sediment (C rapT0), such that a constant ratio is expected between SPTE and C rapT0 (C T /C rapT0, where T is the duration of the SPTE). As environmental factors (i.e., aging time and organic carbon content) and contaminant hydrophobicity can affect the C rapT0, the utility of the SPTEs as exposure estimates hinges on the consistency of the C T /C rapT0 ratio. Individually these factors have little impact on the ability of SPTEs to represent bioaccumulation, but the effect of these factors in combination, as well as SPTE methodological variation on the C T /C rapT0 ratio is poorly understood. The current study evaluated how environmental and methodological variation-expressed as varying Tenax to organic carbon mass (Tenax:OC) ratios-impacts the C 24h/C rapT0 ratio of pyrethroids in laboratory-spiked sediments. A multiple regression analysis was used to examine the impact of organic carbon, pyrethroid hydrophobicity, Tenax mass, and aging time on the C 24h/C rapT0 ratio. Only aging time of the pyrethroids in sediment significantly affected the C 24h/C rapT0 ratio with a slight decline of -0.0027/d in the C 24h/C rapT0 ratio, and this decline was considered negligible as a consistent C 24h/C rapT0 ratio of 1.46 ± 0.03 was observed across all experimental treatments. This result further demonstrates the consistency of SPTEs to estimate bioaccessibility of hydrophobic contaminants in sediment and subsequent exposure.

Multiple origins of pyrethroid insecticide resistance across the species complex of a nontarget aquatic crustacean, Hyalella azteca.

Use of pesticides can have substantial nonlethal impacts on nontarget species, including driving evolutionary change, often with unknown consequences for species, ecosystems, and society. Hyalella azteca, a species complex of North American freshwater amphipods, is widely used for toxicity testing of water and sediment and has frequently shown toxicity due to pyrethroid pesticides. We demonstrate that 10 populations, 3 from laboratory cultures and 7 from California water bodies, differed by at least 550-fold in sensitivity to pyrethroids. The populations sorted into four phylogenetic groups consistent with species-level divergence. By sequencing the primary pyrethroid target site, the voltage-gated sodium channel, we show that point mutations and their spread in natural populations were responsible for differences in pyrethroid sensitivity. At least one population had both mutant and WT alleles, suggesting ongoing evolution of resistance. Although nonresistant H. azteca were susceptible to the typical neurotoxic effects of pyrethroids, gene expression analysis suggests the mode of action in resistant H. azteca was not neurotoxicity but was oxidative stress sustained only at considerably higher pyrethroid concentrations. The finding that a nontarget aquatic species has acquired resistance to pesticides used only on terrestrial pests is troubling evidence of the impact of chronic pesticide transport from land-based applications into aquatic systems. Our findings have far-reaching implications for continued uncritical use of H. azteca as a principal species for monitoring and environmental policy decisions.

A simultaneous extraction method for organophosphate, pyrethroid, and neonicotinoid insecticides in aqueous samples.

A method was developed for the extraction and analysis of 2 organophosphate, 8 pyrethroid, and 5 neonicotinoid insecticides from the same water sample. A salted liquid-liquid extraction (LLE) was optimized with a solid-phase extraction (SPE) step that separated the organophosphates (OPs) and pyrethroids from the neonicotinoids. Factors that were optimized included volume of solvent and amount of salt used in the LLE, homogenization time for the LLE, and type and volume of eluting solvent used for the SPE. The OPs and pyrethroids were quantified using gas chromatography-mass spectrometry, and the neonicotinoids were quantified using liquid chromatography-diode array detector. Results showed that the optimized method was accurate, precise, reproducible, and robust; recoveries in river water spiked with 100 ng L(-1) of each of the insecticides were all between 86 and 114 % with RSDs between 2 and 8 %. The method was also sensitive with method detection limits ranging from 0.1 to 27.2 ng L(-1) depending on compounds and matrices. The optimized method was thus appropriate for the simultaneous extraction of 15 widely applied insecticides from three different classes and was shown to provide valuable information on their environmental fate from field-collected aqueous samples.

Mixture toxicity of imidacloprid and cyfluthrin to two non-target species, the fathead minnow Pimephales promelas and the amphipod Hyalella azteca.

Two species, the fathead minnow Pimephales promelas and the amphipod Hyalella azteca, were tested to examine acute toxicity to two insecticides, cyfluthrin and imidacloprid individually and as a mixture. Cyfluthrin was acutely toxic to P. promelas and H. azteca with EC50 values and 95 % confidence intervals of 0.31 µg L(-1) (0.26-0.35 µg L(-1)) and 0.0015 µg L(-1) (0.0011-0.0018 µg L(-1)), respectively. Imidacloprid was not acutely toxic to P. promelas at water concentrations ranging from 1 to 5000 µg L(-1), whereas it was toxic to H. azteca with a EC50 value of 33.5 µg L(-1) (23.3-47.4 µg L(-1)). For the P. promelas mixture test, imidacloprid was added at a single concentration to a geometric series of cyfluthrin concentrations bracketing the EC50 value. A synergistic ratio (SR) of 1.9 was found for P. promelas, which was calculated using the cyfluthrin-only exposure and mixture-exposure data. Because cyfluthrin and imidacloprid were toxic to H. azteca, the mixture test was designed based on an equipotent toxic unit method. Results from the mixture test indicated a model deviation ratio (MDR) of 1.7 or 2.7 depending on the model. Mixture test results from the simultaneous exposure to cyfluthrin and imidacloprid with both species indicated a greater than expected toxic response because the SR or MDR values were >1. Because these two insecticides are commonly used together in the same product formulations, nontarget species could be more affected due to their greater-than-additive toxicity observed in the current study.