Molecular and biochemical evaluation of effects of malathion, phenanthrene and cadmium on Chironomus sancticaroli (Diptera: Chironomidae) larvae.

In-vitro effects of sub-lethal concentrations of malathion, phenanthrene (Phe) and cadmium (Cd) were tested on Chironomus sancticaroli larvae in acute bioassays by measuring biochemical and molecular parameters. Malathion was evaluated at 0.001, 0.0564 and 0.1006 mg L-1; Phe at 0.0025, 1.25 and 2.44 mg L-1; and Cd at 0.001, 3.2 and 7.4 mg L-1. The recovery test carried out at the highest concentration of each compound showed that survival of larvae exposed to Phe ranged from 4% to 5%, while the effects of malathion and Cd were irreversible, not allowing the emergence of adults. Results showed that malathion and Cd inhibited AChE, EST-α and ES-ß activities at the two highest concentrations. Phe at 0.0025, 1.25 and 2.44 mg L-1; and Cd at 3.2 and 7.4 mg L-1 inhibited glutathione S-transferase activity. Oxidative stress was exclusively induced by the lowest concentration of malathion considering SOD activity once CAT was unaffected by the stressors. Lipid peroxidation was registered exclusively by malathion at the two highest concentrations, and total hemoglobin content was only reduced by Cd at the two highest concentrations. The relationship among biochemical results, examined using the PCA, evidenced that malathion and Cd concentrations were clustered into two groups, while Phe only formed one group. Four hemoglobin genes of C. sancticaroli were tested for the first time in this species, with Hemoglobin-C being upregulated by malathion. The toxicity ranking was malathion > Phe > Cd, while biochemical and molecular results showed the order malathion > Cd > Phe. Our results highlight the importance of combining different markers to understand the effects of the diverse compounds in aquatic organisms.

The Toxicogenome of Hyalella azteca: A Model for Sediment Ecotoxicology and Evolutionary Toxicology.

Hyalella azteca is a cryptic species complex of epibenthic amphipods of interest to ecotoxicology and evolutionary biology. It is the primary crustacean used in North America for sediment toxicity testing and an emerging model for molecular ecotoxicology. To provide molecular resources for sediment quality assessments and evolutionary studies, we sequenced, assembled, and annotated the genome of the H. azteca U.S. Lab Strain. The genome quality and completeness is comparable with other ecotoxicological model species. Through targeted investigation and use of gene expression data sets of H. azteca exposed to pesticides, metals, and other emerging contaminants, we annotated and characterized the major gene families involved in sequestration, detoxification, oxidative stress, and toxicant response. Our results revealed gene loss related to light sensing, but a large expansion in chemoreceptors, likely underlying sensory shifts necessary in their low light habitats. Gene family expansions were also noted for cytochrome P450 genes, cuticle proteins, ion transporters, and include recent gene duplications in the metal sequestration protein, metallothionein. Mapping of differentially expressed transcripts to the genome significantly increased the ability to functionally annotate toxicant responsive genes. The H. azteca genome will greatly facilitate development of genomic tools for environmental assessments and promote an understanding of how evolution shapes toxicological pathways with implications for environmental and human health.

Contaminant exposure effects in a changing climate: how multiple stressors can multiply exposure effects in the amphipod Hyalella azteca.

Global climate change (GCC) is likely to intensify the synergistic effects between altered physicochemical parameters [of changing habitats] and other anthropogenic threats, such as water pollution, posing increased risks to aquatic biodiversity. As such, it is critical to understand how organisms will respond to changes in water temperature and salinity in the presence of contaminants. We exposed the epibenthic amphipod Hyalella azteca to a 3 × 3 factorial treatment design of three temperatures and three salinities ranging from 12 to 18 °C and 0 to 8 parts per thousand (ppt), respectively, in combination with a low-level environmentally relevant concentration of the pyrethroid insecticide bifenthrin (1 ng/L). Effects on survival and swimming behavior were evaluated after 96 h exposure. Transcription of a select suite of genes was monitored at 24, 48, and 96 h using quantitative polymerase chain reaction (qPCR). Our results not only demonstrate that the changes in salinity and temperature result in negative effects to invertebrate survival, behavior, and gene response, but that the effects were significantly more pronounced in the presence of bifenthrin. This is particularly important since greater thermal fluctuations, changes in timing and extent of glacial melt, and changes in precipitation, could result in H. azteca experiencing lower temperatures at times that coincide with increased spraying of pyrethroids. These environmentally relevant exposures using the standard test species H. azteca provide essential information for understanding effects caused by GCC in conjunction with increasing pesticide use, further highlighting the need to incorporate GCC impacts into risk assessments of contaminants of concern.

LC- and GC-QTOF-MS as Complementary Tools for a Comprehensive Micropollutant Analysis in Aquatic Systems.

Efficient strategies are required to implement comprehensive suspect screening methods using high-resolution mass spectrometry within environmental monitoring campaigns. In this study, both liquid and gas chromatography time-of-flight mass spectrometry (LC-QTOF-MS and GC-QTOF-MS) were used to screen for >5000 target and suspect compounds in the Sacramento-San Joaquin River Delta in Northern California. LC-QTOF-MS data were acquired in All-Ions fragmentation mode in both positive and negative electrospray ionization (ESI). LC suspects were identified using two accurate mass LC-QTOF-MS/MS libraries containing pesticides, pharmaceuticals, and other environmental contaminants and a custom exact mass database with predicted transformation products (TPs). The additional fragment information from the All-Ions acquisition improved the confirmation of the compound identity, with a low false positive rate (9%). Overall, 25 targets, 73 suspects, and 5 TPs were detected. GC-QTOF-MS extracts were run in negative chemical ionization (NCI) for 21 targets (mainly pyrethroids) at sub-ng/L levels. For suspect screening, extracts were rerun in electron ionization (EI) mode with a retention time locked method using a GC-QTOF-MS pesticide library (containing exact mass fragments and retention times). Sixteen targets and 42 suspects were detected, of which 12 and 17, respectively, were not identified by LC-ESI-QTOF-MS. The results highlight the importance of analyzing water samples using multiple separation techniques and in multiple ionization modes to obtain a comprehensive chemical contaminant profile. The investigated river delta experiences significant pesticide inputs, leading to environmentally critical concentrations during rain events.

Changes in Menidia beryllina Gene Expression and In Vitro Hormone-Receptor Activation After Exposure to Estuarine Waters Near Treated Wastewater Outfalls.

Fishes in estuarine waters are frequently exposed to treated wastewater effluent, among numerous other sources of contaminants, yet the impacts of these anthropogenic chemicals are not well understood in these dynamic and important waterways. Inland silversides (Menidia beryllina) at an early stage of development [12 days posthatch (dph)] were exposed to waters from two estuarine wastewater-treatment outfall locations in a tidal estuary, the Sacramento/San Joaquin Delta (California, USA) that had varied hydrology and input volumes. The genomic response caused by endocrine-disrupting compounds (EDCs) in these waters was determined using quantitative polymerase chain reaction on a suite of hormonally regulated genes. Relative androgenic and estrogenic activities of the waters were measured using CALUX reporter bioassays. The presence of bifenthrin, a pyrethroid pesticide and known EDC, as well as caffeine and the anti-inflammatory pharmaceutical ibuprofen, which were used as markers of wastewater effluent input, were determined using instrumental analysis. Detectable levels of bifenthrin (2.89 ng L(-1)) were found on one of the sampling dates, and caffeine was found on all sampling dates, in water from the Boynton Slough. Neither compound was detected at the Carquinez Strait site, which has a much smaller effluent discharge input volume relative to the receiving water body size compared with Boynton Slough. Water samples from both sites incubated in the CALUX cell line induced estrogenic and androgenic activity in almost all instances, though the estrogenicity was relatively higher than the androgenicity. Changes in the abundance of mRNA transcripts of endocrine-responsive genes and indicators of general chemical stress were observed after a 96-h exposure to waters from both locations. The relative levels of endocrine response, changes in gene transcript abundance, and contaminant concentrations were greater in water from the Boynton Slough site despite those effluents undergoing a more advanced treatment process. The availability of a widely geographically distributed estuarine model species (M. beryllina) now allows for improved assessment of treated effluent impacts across brackish, estuarine, and marine environments.

The use of growth and behavioral endpoints to assess the effects of pesticide mixtures upon aquatic organisms.

Aquatic communities are often subject to complex contaminant mixtures, usually at sublethal concentrations, that can cause long-term detrimental effects. Chemicals within mixtures can effectively interact, resulting in synergism, antagonism or additivity. We investigated the tertiary mixture effects of two pyrethroids, lambda-cyhalothrin and permethrin, and the organophosphate chlorpyrifos, evaluating sublethal endpoints; immobility and growth, on Chironomus dilutus in 10-day exposures. We utilized a toxic units (TU) approach, based on median lethal concentrations (LC50) for each compound. The concepts of independent action and concentration addition were used to compare predicted mixture toxicity to observed mixture toxicity. Increased immobility resulted from mixture concentrations ≥1 TU (7.45 ng/L lambda-cyhalothrin × 24.90 ng/L permethrin × 129.70 ng/L chlorpyrifos), and single pesticides concentrations ≥0.25 TU (5.50 ng/L lambda-cyhalothrin, 24.23 ng/L permethrin, 90.92 ng/L chlorpyrifos, respectively). Growth was inhibited by pesticide mixtures ≥0.125 TU (1.04 ng/L lambda-cyhalothrin × 3.15 ng/L permethrin × 15.47 ng/L chlorpyrifos), and singly by lambda-cyhalothrin ≥0.25 TU (5.50 ng/L), and permethrin ≥0.167 TU (18.21 ng/L). The no observed effect concentrations (NOEC) for immobility and growth, for both mixture and single-pyrethroid exposure, were up to 8.0 and 12.0 times respectively lower than the corresponding NOEC for survival. The median effective concentrations (EC50) for growth (mixture and single-pyrethroid exposure) were up to 7.0 times lower than the respective LC50. This study reinforces that the integration of sublethal endpoints in monitoring efforts is powerful in discerning toxic effects that would otherwise be missed by solely utilizing traditional toxicity assessments.

Occurrence and distribution of emerging micropollutants in the central part of the German Bight.

Micropollutants (MPs) are transported via rivers from industrial and urban areas to the German Bight (G.B.). In contrast to the mounting rivers less information is available on the occurrence of MPs and their transformation products (TPs) in the marine environment of the G.B. In this study 83 compounds, including 26 metabolites of pharmaceuticals and environmental TPs were measured in water at 46 sampling sites in estuaries of Ems, Weser, Elbe, and the G.B. 36 MPs were even detected in the open sea areas (salinity > 34 psu) at 0.07-5.1 ng/L and to the best of our knowledge 10 MPs were detected in the marine environment for the first time. Concentrations of 8 MPs exceeded PNEC values suggesting a potential risk for sea life. Spatial distribution and relation of MPs with salinity allowed identifying emission paths for certain compounds and revealed the emissions from the River Elbe and Rhine.

Investigation of potential metal emissions from galvanic anodes in offshore wind farms into North Sea sediments.

To evaluate potential metal emissions from offshore wind farms (OWFs), 215 surface sediment samples from different German North Sea OWFs taken between 2016 and 2022 were analyzed for their mass fractions of metals and their isotopic composition of Sr. For the first time, this study provides large-scale elemental data from OWFs of the previously proposed galvanic anode tracers Cd, Pb, Zn, Ga and In. Results show that mass fractions of the legacy pollutants Cd, Pb and Zn were mostly within the known variability of North Sea sediments. At the current stage the analyzed Ga and In mass fractions as well as Ga/In ratios do not point towards an accumulation in sediments caused by galvanic anodes used in OWFs. However, further investigations are advisable to evaluate long-term effects over the expected lifetime of OWFs, especially with regard to the current intensification of offshore wind energy development.

Agricultural surface water, imidacloprid, and chlorantraniliprole result in altered gene expression and receptor activation in Pimephales promelas.

The toxicity of single pesticides is likely underestimated when considering complex pesticide mixtures found in agricultural runoff and this is especially true for newer pesticides with little toxicity data on non-target species. The goal of our study was to compare the toxicity of two newer pesticides, imidacloprid (IMI) and chlorantraniliprole (CHL), when an invertebrate and fish were exposed to single compounds, binary mixtures or surface water collected near agricultural fields. A secondary goal was to determine whether changes in select subcellular molecular pathways correspond to the insecticides' mechanisms of activity in aquatic organisms. We conducted acute (96 h) exposures using a dilution series of field water and environmentally relevant concentrations of single and binary mixtures of IMI and CHL. We then evaluated survival, gene expression and the activity of IMI toward the n-acetylcholine receptor (nAChR) and CHL activity toward the ryanodine receptor (RyR). Both IMI and CHL were detected at all sampling locations for May 2019 and September 2019 sampling dates and exposure to field water led to high invertebrate but not fish mortality. Fish exposed to field collected water had significant changes in the relative expression of genes involved with detoxification and neuromuscular function. Exposure of fish to single compounds or binary mixtures of IMI and CHL led to increased relative gene expression of RyR in fish. Furthermore, we found that IMI targets the nAChR in aquatic invertebrates and that CHL can cause overactivation of the RyR in invertebrates and fish. Overall, our finding suggests that IMI and CHL may impact neuromuscular health in fish. Expanding monitoring efforts to include sublethal and molecular assays would allow the detection of subcellular level effects due to complex mixtures present in surface water near agricultural areas.

Developmental exposure to environmentally relevant concentrations of bifenthrin alters transcription of mTOR and ryanodine receptor-dependent signaling molecules and impairs predator avoidance behavior across early life stages in inland silversides (Menidia beryllina).

Altered transcription of calcium-dependent signaling cascades involving the ryanodine receptor (RyR) and mechanistic target of rapamycin (mTOR) in response to environmental exposures have been described in model vertebrates, including zebrafish, while the relevance for wild fishes remains unknown. To address this knowledge gap, we exposed the euryhaline model species Menidia beryllina (inland silversides) to the insecticide bifenthrin, a known modulator of calcium signaling. The main objectives of this study were to determine: (1) whether exposure of developing silversides to environmentally relevant concentrations of bifenthrin alters their behavior; and (2) whether behavioral changes correlate with altered expression of genes involved in RyR and mTOR-dependent signaling pathways. At six hours post fertilization (hpf), inland silversides were exposed to bifenthrin at 3, 27 and 122 ng/L until 7 days post fertilization (dpf, larvae hatched at 6dpf), followed by a 14-day recovery period in uncontaminated water. Transcriptional responses were measured at 5, 7 and 21 dpf; locomotor behavior following external stimuli and response to an olfactory predator cue were assessed at 7 and 21 dpf. Bifenthrin elicited significant non-monotonic transcriptional responses in the majority of genes examined at 5 dpf and at 21 dpf. Bifenthrin also significantly altered predator avoidance behavior via olfactory mechanisms with main effects identified for animals exposed to 3 and 27 ng/L. Behavioral effects were not detected in response to visual stimuli during acute exposure, but were significant in the predator-cue assessment following the recovery period, suggesting delayed and long-term effects of early developmental exposures to bifenthrin. Our findings demonstrate that at picomolar (pM) concentrations, which are often not represented in ecotoxicological studies, bifenthrin perturbs early development of inland silversides. These developmental impacts are manifested behaviorally at later life stages, specifically as altered patterns of predator avoidance behavior, which have been correlated with population decline. Collectively, these data suggest that bifenthrin may be negatively impacting wild fish populations.

An assessment of direct and indirect effects of two herbicides on aquatic communities.

Herbicides are often detected in watersheds at concentrations that are toxic to phytoplankton, potentially causing indirect effects on higher trophic organisms. The long-term effects of 5 applications over 30 d of binary mixtures of the herbicides diuron and hexazinone were assessed at "low" and "high" concentrations typically found in the environment, using mesocosms. Sixteen of 95 phytoplankton taxa, 3 of 18 zooplankton taxa, and 6 of 14 macroinvertebrate taxa responded negatively to contaminant exposures. Herbicide applications altered the phytoplankton community structure. Relative abundance of Cyanophyceae decreased following 5 applications from 52.1% in the control to 37.3% in the low treatment and to 25.9% in the high treatment, while Chlorophyceae increased to 50.6% in the low treatment and to 61.7% in the high treatment compared with the control (39.7%). Chlorophyceae had the greatest number of affected species (8), whereas 1 species within the Cyanophyceae was negatively affected on more than 1 sampling day. Further, chlorophyll a was reduced on 4 and 5 d out of the 8 total sampling days in the low and high treatments, respectively, compared with the control. These results highlight that integrating multiple taxa and contaminants with long-term exposures in ecological risk assessments of herbicides can facilitate the ability to make predictive and mechanistic generalizations about the role of herbicides in shaping patterns of species abundance in natural systems. Environ Toxicol Chem 2017;36:2234-2244. © 2017 SETAC.

Environmentally relevant concentrations of herbicides impact non-target species at multiple sublethal endpoints.

There is concern over herbicide exposure effects on aquatic primary production and zooplankton as herbicides are found in aquatic ecosystems at concentrations potentially toxic to phytoplankton. We first aimed to determine the effect concentrations (growth inhibition) and mixture interactions of the herbicides diuron (0.5 to 50µg/L) and hexazinone (0.5 to 40µg/L) on the green algae Pseudokirchneriella subcapitata. Secondly, we evaluated chronic effects on Daphnia magna that were periodically fed on P. subcapitata that had been exposed to low, medium, and high concentrations. We hypothesized that based on the mode of action of the herbicides we would observe additive growth inhibition in algae, and sublethal effects on D. magna. Growth inhibition in P. subcapitata following mixture exposure was most consistent with the concentration addition (CA) concept; while the independent action (IA) model underestimated the combined effect. The lowest observed effect concentrations (LOEC) were 1.50µg/L hexazinone, 1.18µg/L diuron, and 0.125 TU (0.30µg/L diuron×0.12µg/L hexazinone) in the single and binary mixture exposures, respectively. High hexazinone exposure decreased D. magna survival (80% vs. 55.6%). Neonate number was reduced by 13.9% in high mixture and 23.5% in high hexazinone treatments. Gravid body length was reduced by 9.5% following exposure to the high mixture. Herbicide exposure decreased neonate size, especially in later broods. Herbicides decreased the phototaxic responses of neonates in most treatments. Herbicide exposure effects were detected at environmentally relevant concentrations, levels considered to be safe according to current USEPA aquatic life benchmarks, suggesting that these benchmarks need to be updated to improve ecological risk assessment. As herbicides are some of the most applied pesticides worldwide, sublethal endpoints can serve as sensitive early warning tools to indicate their presence and can support regulatory assessments and monitoring to protect aquatic life.

A long-term assessment of pesticide mixture effects on aquatic invertebrate communities.

To understand the potential effects of pesticide mixtures on aquatic ecosystems, studies that incorporate increased ecological relevance are crucial. Using outdoor mesocosms, the authors examined long-term effects on aquatic invertebrate communities of tertiary mixtures of commonly used pesticides: 2 pyrethroids (permethrin, λ-cyhalothrin) and an organophosphate (chlorpyrifos). Application scenarios were based on environmentally relevant concentrations and stepwise increases of lethal concentrations from 10% (LC10) to 50% (LC50) based on laboratory tests on Hyalella azteca and Chironomus dilutus; repeated applications were meant to generally reflect runoff events in a multiple-grower or homeowner watershed. Pyrethroids rapidly dissipated from the water column, whereas chlorpyrifos was detectable even 6 wk after application. Twelve of 15 macroinvertebrate and 10 of 16 zooplankton taxa responded to contaminant exposures. The most sensitive taxa were the snail Radix sp., the amphipod H. azteca, the water flea Daphnia magna, and copepods. Environmentally relevant concentrations had acute effects on D. magna and H. azteca (occurring 24 h after application), whereas lag times were more pronounced in Radix sp. snails and copepods, indicating chronic sublethal responses. Greatest effects on zooplankton communities were observed in environmentally relevant concentration treatments. The results indicate that insecticide mixtures continue to impact natural systems over multiple weeks, even when no longer detectable in water and bound to particles. Combinations of indirect and direct effects caused consequences across multiple trophic levels.

Transcriptomic changes underlie altered egg protein production and reduced fecundity in an estuarine model fish exposed to bifenthrin.

Pyrethroid pesticides are a class of insecticides found to have endocrine disrupting properties in vertebrates such as fishes and in human cell lines. Endocrine disrupting chemicals (EDCs) are environmental contaminants that mimic or alter the process of hormone signaling. In particular, EDCs that alter estrogen and androgen signaling pathways are of major concern for fishes because these EDCs may alter reproductive physiology, behavior, and ultimately sex ratio. Bifenthrin, a pyrethroid with escalating usage, is confirmed to disrupt estrogen signaling in several species of fish, including Menidia beryllina (inland silverside), an Atherinid recently established as a euryhaline model. Our main objective was to broadly assess the molecular and physiological responses of M. beryllina to the ng/L concentrations of bifenthrin typically found in the environment, with a focus on endocrine-related effects, and to discern links between different tiers of the biological hierarchy. As such, we evaluated the response of juvenile Menidia to bifenthrin using a Menidia-specific microarray, quantitative real-time polymerase chain reaction (qPCR) on specific endocrine-related genes of interest, and a Menidia-specific ELISA to the egg-coat protein choriogenin, to evaluate a multitude of molecular-level responses that would inform mechanisms of toxicity and any underlying causes of change at higher biological levels of organization. The sublethal nominal concentrations tested (0.5, 5 and 50ng/L) were chosen to represent the range of concentrations observed in the environment and to provide coverage of a variety of potential responses. We then employed a 21-day reproductive assay to evaluate reproductive responses to bifenthrin (at 0.5ng/L) in a separate group of adult M. beryllina. The microarray analysis indicated that bifenthrin influences a diverse suite of molecular pathways, from baseline metabolic processes to carcinogenesis. A more targeted examination of gene expression via qPCR demonstrated that bifenthrin downregulates a number of estrogen-related transcripts, particularly at the lowest exposure level. Choriogenin protein also decreased with exposure to increasing concentrations of bifenthrin, and adult M. beryllina exposed to 0.5ng/L had significantly reduced reproductive output (fertilized eggs per female). This reduction in fecundity is consistent with observed changes in endocrine-related gene expression and choriogenin production. Taken together, our results demonstrate that environmental concentrations of bifenthrin have potential to interfere with metabolic processes, endocrine signaling, and to decrease reproductive output.

A comparison of the sublethal and lethal toxicity of four pesticides in Hyalella azteca and Chironomus dilutus.

Laboratory toxicity testing is the primary tool used for surface water environmental risk assessment; however, there are critical information gaps regarding the sublethal effects of pesticides. In 10-day exposures, we assessed the lethal and sublethal (motility and growth) toxicities of four commonly used pesticides, bifenthrin, permethrin, cyfluthrin, and chlorpyrifos, on two freshwater invertebrates, Chironomus dilutus and Hyalella azteca. Pyrethroids were more toxic than the organophosphate chlorpyrifos in both species. Bifenthrin was most toxic to H. azteca survival and growth. Cyfluthrin was most toxic to C. dilutus. However, cyfluthrin had the greatest effect on motility on both H. azteca and C. dilutus. The evaluated concentrations of chlorpyrifos did not affect C. dilutus motility or growth, but significantly impacted H. azteca growth. Motility served as the most sensitive endpoint in assessing sublethal effects at low concentrations for both species, while growth was a good indicator of toxicity for all four pesticides for H. azteca. The integration of sublethal endpoints in ambient water monitoring and pesticide regulation efforts could improve identification of low-level pesticide concentrations that may eventually cause negative effects on food webs and community structure in aquatic environments.