Neotropical stingless bees have frequently been reported to possess high biodiversity, ecological significance, and sensitivity to insecticides. Surprisingly, few studies have been conducted so far to assess their sensitivity to neonicotinoid insecticides, although there are indications that this insecticide class is especially toxic to stingless bees. The aim of this study was therefore to evaluate the acute oral and topical toxicity of two neonicotinoids, imidacloprid and thiamethoxam, to the neotropical stingless bee Melipona scutellaris. Besides these active ingredients, commercial products containing them were also evaluated. The commercial products were more toxic to the bees than the active ingredients, which may be due to direct toxicity of co-formulants and, indirectly, through their higher biological activity and facilitation of uptake by organisms. The neonicotinoids were more toxic through topical contact than oral exposure. This is the opposite trend as previously reported for honeybees, which is explained through differences in life-history traits with stingless bees. M. scutellaris was more sensitive to the test substances than standard bee test species commonly used in (temperate) toxicity assessments. This thus stresses the need to include stingless bees in neotropical risk assessments. Nevertheless, the relatively high mortality occasionally observed in control groups highlights the biological sensitivity of stingless bees to laboratory conditions rather than a methodological flaw. This finding reinforces the importance of refining experimental setups by minimizing handling stress and improving cage microclimate to enhance control survival and ensure even greater robustness in future toxicity assessments involving native species.

In acute fish toxicity tests, mortality has traditionally served as the primary endpoint. However, in accordance with the "3Rs" principle-replacement, reduction, and refinement-there is a growing need to minimize the suffering and pain experienced by test fish. In this study we aimed to establish a behavioral framework for identifying the moribund state in zebrafish (Danio rerio), providing a humane and ethically refined alternative endpoint. Continual observation of zebrafish exposed to ten representative chemicals allowed the documentation of twelve clinical signs, with severities of the signs evaluated using the death/clinical-sign ratio, which represents the proportion of fish exhibiting a given sign that subsequently died. The signs "immobility," "immobility at surface," and "lethargy" emerged as strong predictors of imminent death, each exhibiting a death/clinical-sign ratio of 1.0 across all tested chemicals and concentrations, indicating that all fish exhibiting these signs died within the 96-hr test period. Furthermore, the survival times from the onset of these signs to death were sufficiently short to justify their definition as moribund states. Accordingly, we defined these signs as moribund endpoints and propose that fish exhibiting any of them should be euthanized during the test period. Notably, these findings align with previously reported results in Japanese medaka, indicating the potential cross-species applicability of these moribund endpoints.

The per and polyfluoroalkyl substance (PFAS) precursor alternative, 6:2 fluorotelomer sulfonate (6:2 FTS), has been detected globally. The central aim of this work was to evaluate the chronic toxicity of 6:2 FTS for this understudied PFAS. Using the amphibian African clawed-frog Xenopus laevis, we tested the main hypothesis that, regardless of sex, the sensitivity of this model to 6:2 FTS would be comparable to that of North American amphibians. Larvae were exposed to 6:2 FTS (1.2 to 1200 ppb) from Niewkoop and Faber (NF) stage 51 to 65 which took a range of 24 to 42 days. We found significant growth stimulation at 120 ppb (26% mass increase, 6% snout vent length increase) without traditional dose-dependency. This growth stimulation coincided with a non-significant developmental delay at 120 ppb (38.0 ± 2.9 vs 35.5 ± 1.8 days to NF 65). The non-monotonic response yielded dual NOEC/LOEC interpretations: 1) Growth stimulation with a NOEC = 12 ppb and a LOEC = 120 ppb; and 2) Adverse effects with a NOEC = 1200 ppb). X. laevis sensitivity to 6:2 FTS appears comparable to North American native amphibians (reported NOECs: 800-1,800 ppb), though the stimulation response and lack of a dose-response complicates their application for assessing ecological risks. While genetic sexing enabled sex-specific analysis in this species, no differences in sensitivity or accumulation rates were detected. These findings highlight the critical importance of endpoint selection in PFAS risk evaluation and supports previous findings with other amphibians showing that exposure to environmentally-relevant 6:2 FTS concentrations should not adversely impact growth and development.

Aqueous film-forming foams (AFFFs) are widely used fire suppression products that have been identified as a direct source of environmental per- and polyfluoroalkyl substances (PFAS). Per- and polyfluoroalkyl substance exposure has demonstrated chronic and sub-lethal effects on biota. Ongoing efforts aim to reduce and, ideally, eliminate PFAS use in AFFF products. However, there is little known about the potential toxic effects of the new PFAS-free AFFFs, specifically on benthic organisms. The objective of this study is to quantify the effects of seven AFFFs on growth in the hard clam, Mercenaria mercenaria, over a 21-day exposure period with juvenile animals. Additionally, AFFF effects are reported from algal toxicity assays and a feeding study. Five of the PFAS-free AFFFs negatively impacted growth over the exposure period while one PFAS-free AFFF and the reference PFAS-containing AFFF had no observable effect. Median effect concentrations (EC50) for shell growth ranged from 5.81 mg/L to >100 mg/L. Clam dry and wet weights also decreased with increasing exposure concentration (p <0.05). Algal growth was impacted over a 96-hr exposure. Impacts were observed to final standing biomass and overall growth rates at the highest exposure concentrations. However, complete lethality was only observed for one PFAS-free product, suggesting lack of food availability was likely not the primary driver of growth inhibition for all products. Net particle clearance rates in AFFF exposed clams were not found to be impacted, suggesting there was no obvious AFFF influence on organismal feeding ability. The presented results identify chronic effects of exposure to these AFFFs in this economically and ecologically important bivalve species and is expected to inform decisions regarding PFAS replacement AFFF products.

The increasing anthropogenic impact on ecosystems highlights the growing importance of studying vegetation responses to inorganic and organic pollutants in deltaic and coastal bay environments. This study evaluates morphometric indicators of ecological stress in vegetation from different ecological groups, specifically helophytes and mesophytes, within the super- and subaqueous landscapes of the lower Don River in Russia, including its delta and the coasts of the Taganrog Bay. Analysis of plant species revealed a high level of metals and polycyclic aromatic hydrocarbons (PAHs) accumulation in the following cosmopolitan plants: common reed (Phragmites australis (Cav.) Trin. ex Steud), couch grass (Elytrigia repens (L.) Nevski), and eastern cocklebur (Xanthium orientale L.). Using analysis of variance, factor analysis, and correlation analysis, it was shown that the accumulation of metals and PAHs in plant organs depends on the sampling area, the content of pollutants in soils, their properties, as well as the species-specific characteristics of the plants. The accumulation of pollutants in roots is higher than in stems. The concentration of metals in plants followed the order: Mn > Cr > Ni > Zn > Pb > Cu > Cd. The degree of pollutant accumulation in the roots and stems of cosmopolitan plants decreased in the sequence: X. orientale > E. repens > P. australis. Plants growing in the northern part of the Don River delta and along the northern shore of the Taganrog Bay were exposed to the highest total toxic load. Metal accumulation in plants depends mainly on anthropogenic load and species traits, while PAH accumulation is driven by their physicochemical properties, especially water solubility. Using dispersion analysis, it was shown that in habitats with increased anthropogenic pressure, the studied plant species exhibited changes in growth characteristics. Significant reductions in stem length were observed in X. orientale, while stem length increased in E. repens, indicating an active response triggered by changes in environmental conditions along a gradient of rising metals and PAHs levels in the soil.

Microplastic-derived dissolved organic matter (MP-DOM) has attracted widespread attention due to their adverse effects on ecological health. However, the dynamic formation of MP-DOM at molecular level is not yet fully understood. Herein, the molecular level formation characteristics and mechanism of polyamide-microplastic-derived dissolved organic matter (MPPA-DOM) during irradiation were explored using fluorescence spectroscopy. Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS), and parallel factor analysis (PARAFAC). The results showed that the time-dependent fluorescence signatures revealed a dominant tyrosine-like component whose relative abundance increased from 49.63% to 89.62% during irradiation, suggesting a gradual accumulation of protein-related substances. Molecular element analyses of MPPA-DOM revealed the predominance of CHON molecules (78.82%-89.30%), which was attributed to the degradation of the C-N backbone structure. In contrast, CHO molecules exhibited a lower proportion (9.45%) under prolonged irradiation. Aliphatic/peptide-like compounds in MPPA-DOM remained the dominant component with percentage range of 66.4%-68.7%, while lignin-like compounds slightly increased with the increase of irradiation time. The reduced molecules were dominated in MPPA-DOM with percentage range of 96.4%-99.1%. As irradiation increased, the saturated compounds decreased from 91.53% to 82.45% and the unsaturated compounds increased from 7.6% to 14.1%. This study proposed a molecular-level formation mechanism of MPPA-DOM under irradiation. Nitrogen-rich molecules were persistent and highly stable during irradiation, indicating that they could play a more important role in the migration and transformation of MPPA-DOM. The findings in this study will provide support for assessing the potential ecological risks of MP-DOM in water systems.

Octamethylcyclotetrasiloxane (D4) is a cyclic volatile methylsiloxane compound associated with the production of polydimethylsiloxanes (PDMS). Depending on processing conditions, silicone fluids made by equilibration polymerization can contain residual D4 at parts-per-million to parts-per-hundred levels. When silicone fluids enter the environment through use or disposal, aquatic organisms may be exposed to residual D4. To accurately assess the contribution of D4 to the aquatic hazard of silicone fluids, knowledge of the partitioning behavior of D4 is needed. In this study, PDMS-to-air partition coefficients (K  PDMS-air) for D4 were directly measured at 21 °C using a static equilibration method. The influence of various factors on K  PDMS-air was explored, including the PDMS fluid viscosity (molecular weight), the D4 concentration in the polymer, addition of hydrophobized fumed silica to PDMS, and the presence of amine functional groups within the PDMS structure. For permethylated PDMS, log K  PDMS-air values varied between 4.39 and 4.53. Incorporation of treated silica filler at up to 25%w/w had no impact on K  PDMS-air, while introduction of (aminopropyl)methyl-dimethylsiloxy units to the polymer (3.7 wt% nitrogen) produced modestly lower log K  PDMS-air, 4.19. The K  PDMS-air values were combined with reported air-water partition coefficients (K  air-water) for D4 to calculate values of K  PDMS-water based on the thermodynamic cycle. The resulting log K  PDMS-water values ranged from 6.88 to 7.22, which were used to estimate the maximum attainable aqueous D4 concentrations for different polymer/water phase ratios. For a D4 content of 0.025% w/w in the polymer, the current threshold for classification of the polymer as hazardous to the aquatic environment, the maximum aqueous D4 concentration was ≤ 0.032 μg/L. These concentrations were at least 100-fold less than chronic aquatic toxicity thresholds derived from studies with D4, suggesting that the 0.025% w/w threshold is overly stringent in assigning aquatic hazard classifications to PDMS materials like those tested here.

Environmental chemicals impact health through diverse pathways, underscoring the need for approaches beyond traditional animal-based testing to capture their transport, transformation, and mechanisms for risk assessment. Network toxicology offers an in silico framework to elucidate such exposure pathways and toxicity mechanisms, thereby supporting New Approach Methodologies for toxicity assessment. This study utilized network-based approaches to comprehensively characterize the impacts of inorganic arsenic and cadmium contamination in India from a One Health perspective. First, Aggregate Exposure Pathways were constructed by systematically curating India-specific data on the presence of inorganic arsenic and cadmium across environmental and biological states to understand their transport and transformation. Next, inorganic arsenic- and cadmium-induced toxicities were explored by mapping their toxicity endpoints from six exposome-relevant databases to key events across Adverse Outcome Pathways (AOPs) cataloged in AOP-Wiki, revealing 51 and 78 associated AOPs, respectively. Construction of AOP networks further aided in inferring novel taxonomic applicability information. Moreover, the combined AEP-AOP cosntructs facilitated mechanistic case studies of human and ecological health-relevant pathways, and highlighted critical knowledge gaps in terms of human exposures and bioaccumulation within tissues. Further, stressor-species networks constructed using ECOTOX data identified vulnerable species and those with bioaccumulative potential. Additionally, species sensitivity distributions and toxicity-normalized SSDs provided a comparative framework for prioritizing these compounds, and their integration into stressor-species networks highlighted sensitive species, enhancing their relevance for ecological risk assessment. Finally, the risk quotient analysis for Indian rivers revealed that many regions exhibited elevated ecological risks. Overall, this study underscores the value of network toxicology in supporting a One Health-based framework to inform regulatory decision-making and enhance mitigation strategies for inorganic arsenic and cadmium in India.

Saponins are natural plant metabolites with surface-active and bioactive properties against plant pests, making them promising biopesticides. However, their environmental fate in soil remains unclear. This study investigated the sorption properties of three triterpenoid saponins: two monodesmosidic α-hederin and hederacolchiside A1 saponins, and the bidesmosidic hederacoside C saponin, on common soil constituents including clay minerals (kaolinite, montmorillonite), metal oxides (gibbsite, goethite), black carbon, and topsoil. Batch sorption experiments assessed influences of structures, sorbent properties and environmental factors. All saponins exhibited unexpectedly strong sorption (distribution coefficient (Kd)> 10³ L/kg on topsoil), with α-hederin showing the highest affinity (Kd = 229 × 10³ L/kg on goethite), attributed to its moderate hydrophobicity (octanol-water partition coefficient, log Kow ∼ 4.4), short sugar chain, and interactions involving carboxyl (-COOH) and hydroxyl (-OH) functional groups. In contrast, more polar hederacoside C (log Kow ∼ -1.2) showed weaker sorption with Kd of 1.56 × 10³ to 22.7 × 10³ L/kg. Sorption isotherms followed Freundlich behavior and increased by ∼50% at acidic pH for α-hederin and hederacolchiside A1 due to protonation of carboxylic acid groups (acid dissociation constant, pKa ≈ 4.7-4.9), whereas hederacoside C lacking carboxylic acid groups remained unaffected. Salts and fulvic acid reduced α-hederin sorption (up to 80%), likely due to ion exchange and competitive complexation. Desorption studies showed α-hederin was strongly retained (<20% desorption), particularly on metal oxides. Scenario-based modeling indicates that at realistic saponin biopesticide doses (50 micromolar (µM)), α-hederin and hederacolchiside A1 remain largely immobile, whereas hederacoside C may slightly leach in low-sorption soils. These findings highlight the combined role of saponin structure and soil mineralogy in regulating environmental mobility, with implications for biopesticide design and risk assessment.

The presence of emerging contaminants (ECs), associated with the widespread use of pharmaceuticals, personal care products, illicit drugs, microplastics, and other compounds derived from modern consumption, constitutes a growing environmental threat. Their continuous entry through wastewater and inefficient treatment systems generates endocrine disruptions, reproductive effects on wildlife, and potential risks to human health. In contrast, traditional contaminants (TPs), such as heavy metals, have been more studied and partially regulated, although their toxicity persists as a challenge for water quality and public health. Therefore, systematic monitoring of ECs and TPs is essential to identify sources of contamination, assess impacts, and guide remediation and environmental management strategies. The objective of this systematic review was to compile and analyze scientific literature on the incidence and effects of ECs and TPs in water resources, focusing on their most common types, environmental pathways, and biological models used for toxicity testing. The review was based on 200 research articles from the Americas. The search was conducted in ScienceDirect, Web of Science, PubMed, and Scopus®. databases, using combinations of keywords related to etiologies, TPs, and their presence in water resources. The inclusion criteria considered only publications in English, published between 2005 and 2024. Studies from other regions, articles outside the time range, and duplicates were excluded, and only one version of each work was retained. The final selection was made by reviewing titles, abstracts, and references, allowing for independent extraction of relevant information. The findings provide a fundamental framework for improving water resource management and underscore the urgent need to integrate etiologies into regulation and monitoring programs to ensure the sustainability of aquatic ecosystems.

During crop growth cycle, several different plant protection products (PPPs) are often applied in combination or sequentially. Such sequential applications result in unintentional mixtures of residues that may affect ecosystem services supported by non-target organisms such as soil microbes and nematodes. This scenario of sequential PPP application is frequent in agricultural practice but rarely addressed experimentally at field scale with regard to environmental impacts. The objective of this study was to evaluate the effect of individual and sequential application of three PPPs (the herbicide clopyralid, the insecticide zeta-cypermethrin, and the fungicide pyraclostrobin) on soil microbial communities, and on the abundance of free-living nematode. Single applications (at 1× or 10× the agronomical dose) were made to triplicated field plots with each one of the PPPs or all three PPPs in sequence, with untreated plots serving as controls. Plots were sampled prior to each application, and 7 and 28 days thereafter. The composition and abundance of the fungal community were found to be more affected compared to the bacterial community by PPP applications, while the bacterial community structure was mainly influenced by soil properties. Only transient effects of PPP applications were detected on nematode abundance. Higher-tier ecotoxicological tests such as the present field study offer greater ecological relevance compared to laboratory tests but are challenged by environmental variations that should be accounted for when evaluating the ecotoxicity of pesticides on soil micro-organisms.

Applying euthanasia before death in acute fish toxicity (AFT) tests contributes significantly to animal welfare by serving as a "refinement" of the test method. A previous study defined three severe signs as moribund states in Japanese medaka (Oryzias latipes), based on the severity classification of clinical signs, for the purpose of euthanizing fish during the AFT testing before death. However, this approach limits the range of sign that can be used to justify euthanasia. It has also been suggested that predicting the time of death can help determine the appropriate timing for euthanasia in such tests, if survival time (time from the onset of a clinical sign to death) for each clinical sign is known. In this study, we used video recording equipment to assess the survival times for 12 clinical signs observed in response to eight chemical substances, with the aim of ensuring timely euthanasia for fish during acute fish toxicity tests using Japanese medaka. We found that euthanasia could be applied to a broader range of signs and was appropriate for many fish exhibiting clinical signs, by predicting the latest time of death from the maximum survival time for each clinical sign. Case studies with five chemicals showed that this method allowed euthanasia to be applied to many individuals exhibiting clinical signs in a timely manner, without any deviation from the LC50 values obtained using the original test method.

In the European framework for assessing the ecological effects of plant protection products on aquatic organisms, standard tests usually rely on constant laboratory exposure. This Tier 1 approach may lead to overly conservative assessments for short term exposures lasting only a few hours under realistic environmental conditions. To address this, the European Food Safety Authority (EFSA) proposed a Tier 2C assessment to incorporate more realistic dynamic exposure profiles through refined tests and toxicokinetic/toxicodynamic (TKTD) modelling. Currently, the only accepted TKTD model for macrophyte growth inhibition relates to the duckweed Lemna sp. We hypothesise that this model can be adapted for other macrophytes including sediment rooted macrophytes. We propose a Modelling Approach for Growth inhibition of MAcrophytes, MAGMA, to simulate both, constant and dynamic exposure tests under laboratory conditions. The model was calibrated using experimental data from Myriophyllum spicatum exposed to two herbicides with different modes of action. Validation against single and two-pulse exposure experiments showed good agreement between model predictions and observed data. MAGMA can therefore serve as a valuable Tier 2C tool for predicting macrophyte growth rates under dynamic exposure conditions.

Commercial beekeepers transport colonies across the United States to provide pollination services for over 100 different crops, especially for almond production in California. In these agricultural settings, honey bees are exposed to adjuvant compounds that are included as "inert ingredients" in pesticide formulations or are added as a separate adjuvant product to pesticides to improve application characteristics. However, evidence suggests that some of these inert ingredients pose risks to bees. This study used a Potter Spray Tower to determine the 48-hr acute toxicities (Lethal Concentration 50 [LC50]) of 37 different pesticide inert ingredients or adjuvant principal functioning agents. Additionally, toxicity trends of 15 alcohol ethoxylates were determined for structural and physical metrics, including moles of ethoxylation, number of carbons in the alcohol group, and Hydrophilic-Lipophilic Balance (HLB). Lastly, Lethal Dose 50 (LD50) values for an organo-silicone (Silwet Eco®) and a non-ionic surfactant (Makon 10®) were determined when applied to different parts of the adult honey bee. The results show that 25 of the 37 tested inert ingredients demonstrated a significant 48-hr dose-response, and 15 of these 25 inert ingredients with a significant dose-response demonstrated an LC50 below the maximum concentration tested. For alcohol ethoxylates, moles of ethoxylation and number of carbons in the alcohol group were not significant predictors for toxicity, but HLB did show a significant trend with toxicity. The body placement assay indicated that application to the dorsal part of the head and the ventral thorax were generally the most toxic, but that the two adjuvants produced differing results. The findings from this study can guide the selections of adjuvants and pesticide inerts to reduce risks to honey bees.

Aquatic and terrestrial food webs are linked by movements of adult aquatic insects from aquatic to terrestrial ecosystems. Contaminants can affect these linkages by reducing insect survival and increasing tissue concentrations through adult metamorphosis, thus changing contaminant flux from water to land. Most anthropogenically influenced freshwater ecosystems are exposed to multiple contaminants. To better understand the combined effects of contaminants on aquatic-terrestrial linkages, we investigated how contaminants affect these linkages, focusing on the combined effects of mercury and pesticides on insect-mediated contaminant flux from wetlands. In a field survey of 15 wetlands in the Prairie Pothole Region, an important agricultural region of North America, we found that insect-mediated mercury flux was 3.5 times more strongly predicted by emergence biomass than tissue mercury. Thus, factors that were previously found to influence aquatic insect emergence biomass in this system, including insecticide tissue concentrations in adult insects, open water surface area, and agricultural land use, were the most likely to drive insect-mediated mercury flux. Pesticide flux, on the other hand, was 3.3 times more strongly predicted by tissue concentration of pesticides than by emergence. Thus, factors that influence pesticide exposure, and to a smaller extent emergence biomass, were the most likely to drive pesticide flux. Our results show how factors driving contaminant fluxes by adult aquatic insects differ by contaminant class and suggest one mechanism by which toxic effects of one contaminant (pesticides) could influence the flux of another (mercury). Predicting contaminant fluxes in areas where different mixtures of contaminants are present can aid in identifying risk to insectivores.

Per- and polyfluoroalkyl substances (PFAS) are thousands of toxic synthetic chemicals that bioaccumulate and persist in the environment. They are known to cause immunotoxicity, organ damage, endocrine disruption, and reproductive impairments in wildlife such as sea otters (Enhydra lutris). However, there is limited information on the distribution of these chemicals across the northeastern Pacific, and baseline data are missing to assess their potential impacts on sea otters in regions such as British Columbia (BC), Canada. We analyzed liver (n = 11) and skeletal muscle samples (n = 5) from 11 deceased sea otters from coastal BC using the U.S. Environmental Protection Agency method 1633 with ultrahigh performance liquid chromatography coupled to a triple quadrupole mass spectrometer. We found 8 of the 40 tested PFAS were present in all sampled sea otters, although concentrations of each PFAS varied between individuals. Sea otter livers contained more PFAS compounds at higher total average concentrations than skeletal muscle (i.e., 8 PFAS totaling 10.38 ng/g wet wt vs. 1 PFAS totaling 0.38 ng/g wet wt). Only perfluorooctanesulfonamide (PFOSA) was identified in both liver and muscle tissues, whereas the remaining 7 PFAS were unique to the liver. The three PFAS that dominated the liver PFAS composition (perfluorononanoic acid, PFOSA, and perfluorooctanesulfonic acid) accounted for 84% of the contaminant load in the livers. Geographically, PFAS concentrations were more than three times higher on average in sea otters recovered near major cities and shipping routes. Identifying the contaminants accumulating in sea otters provides insights into the health threats confronted by recovering sea otter populations. Our study also establishes baseline PFAS contamination levels in BC sea otters, which can be used to monitor and regulate the presence of PFAS on marine environments in western Canada.

Florpyrauxifen-benzyl (ProcellaCOR®) is an aquatic herbicide commonly used to control Eurasian watermilfoil (Myriophyllum spicatum) and other invasive aquatic plants. Previous studies have demonstrated effective Eurasian watermilfoil control under low aqueous concentrations (<10 µg L-1) and short exposure times (<24 hr). While florpyrauxifen-benzyl possesses an excellent environmental profile and its acute toxicity has been assessed in freshwater model organisms, there has been no work to examine toxicity of this herbicide in salmonids. Therefore, the objective of this study was to evaluate the acute toxicity to Endangered Species Act (ESA)-listed Chinook salmon (Oncorhynchis tshawytscha). Chinook fry and smolts were exposed to florpyrauxifen-benzyl at 50 and 100 µg L-1 for 96 hr under a 24-hr static renewal protocol at 12 °C. Daily observations included fish startle response, position in the water column, and signs of overt toxicity. No adverse effects of the herbicide were observed at either concentration in both Chinook fry and smolts. Our results indicate that the maximum US-labeled application rate of florpyrauxifen-benzyl (48 µg L-1 ai) did not result in overt toxicity to juvenile salmonids under the exposure scenarios used in this study.

The buildup of pollutants on impervious surfaces, and their subsequent flush into the environment within stormwater, could worsen with expected increases in prolonged dry periods and extreme rain events due to climate change. As such, the monitoring and treatment of urban stormwaters is becoming a high priority. Of particular interest is road runoff in urban areas, which has been found to be acutely lethal to salmonids and frequently contains elevated concentrations of metals and organic contaminants. In this study, samples of road runoff were collected in the Metro Vancouver area of British Columbia, Canada, and assessed for acute lethality to rainbow trout (Oncorhynchus mykiss). Three of the four stormwaters tested exhibited 100% mortality in the 96-hr test. Stormwater toxicity was demonstrated to be reduced by treatment in a rain garden. Phase I Toxicity Identification Evaluation (TIE) techniques initially identified a metal as the cause of toxicity in one stormwater, which was determined to be zinc after Phase II/III TIE testing. The second stormwater sample revealed an organic constituent to be responsible for toxicity, and subsequent TIE testing implicated N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine-quinone (6PPD-Q). The potential contribution of 6PPD-Q to toxicity was assessed by performing TIE techniques on a standard solution of 6PPD-Q in parallel with the stormwater. Chemical analysis of 6PPD-Q using Condensed-Phase Membrane Introduction Mass Spectrometry (CP-MIMS) was used to support toxicity assessments. This is the first study to use the TIE approach to provide a toxicity profile for 6PPD-Q.

To protect marine environments, standardized whole effluent toxicity (WET) testing methods using a diverse range of aquatic organisms have been developed. A predominant method of WET testing is the fish larval growth and survival (LGS) test, which employs sheepshead minnow (Cyprinodon variegatus) or inland silverside (Menidia beryllina) larvae. Though fish LGS tests have been effective in the identification of potentially toxic effluents, animal welfare concerns have spurred efforts to develop and implement new approach methods (NAMs), such as the fish embryo toxicity (FET) and the mysid (Americamysis bahia) survival and growth tests, which feature fish embryos and invertebrates, respectively. The goal of this study was to advance the development of marine testing alternatives by assessing the sensitivity of marine FET and mysid tests relative to that of standardized fish tests (ie, fish LGS tests) for two environmentally relevant chemicals (ie, nickel [Ni] and phenanthrene [Phe]). In addition, the utility of sublethal endpoints as additional FET test metrics was explored. The findings of this study indicate that the mysid test is more sensitive than the other test methods evaluated for the assessment of Ni and Phe acute toxicity. Further, the mysid test showed comparable, or greater, sensitivity compared to the standardized fish LGS tests for the evaluation of chronic toxicity. Though the marine FET tests were less sensitive than the most sensitive fish LGS test for Ni and Phe, the inclusion of sublethal endpoints (ie, pericardial edema and hatchability) increased FET test sensitivity. These findings indicate the predictive power of marine FET and mysid tests relative to fish LGS tests for the chemicals tested and suggest that FET test performance can be improved through the inclusion of additional endpoints. The results of this study lay the groundwork for future studies that aim to compare the performance of these test types using complex whole effluent mixtures.

Fish are exposed to countless chemicals over their lifetime, with the totality of internal exposure termed the eco-exposome. In vitro bioassays can be used to complement targeted chemical analysis to quantify the mixture effects of chemicals in fish and relate them to the effects of extracts water and sediments. Fathead minnows (Pimephales promelas) were exposed in cages for 48 hr and 21 days at four field sites with diverse chemical profiles. Fish, water and sediments were collected, extracted and analyzed with four cell-based reporter gene assays. Water from all sites activated xenobiotic metabolism in vitro, whereas only water from a site near a wastewater treatment plant activated the estrogen receptor. Only 5% of water samples were above their effect-based trigger values (EBTs) for surface water, suggesting a low overall chemical burden. In contrast, 77% of bioactive sediment samples exceeded tentative sediment EBTs, suggesting the mixture effects of chemicals in the sediment are likely to be more problematic. Whole fish extracts activated the arylhydrocarbon receptor and oxidative stress response, with the greatest effect observed at a site impacted by both legacy and more recent contamination. Interpretation of mixture effects in extracts from caged fish versus laboratory controls was confounded by background contamination of fish food, as well as endogenous chemicals. Comparison of measured mixture effects with mixture effects predicted from detected chemical concentrations and their relative effect potencies indicated that mixture effects in fish extracts were mainly dominated by chemicals detected in sediment. Sediment and water did not reliably serve as a proxy for the eco-exposome. Bioanalytical investigation of whole fish extracts provides a novel approach to comprehensively characterize the fish exposome.