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Assessing the harm caused by pollutants in urban ecosystems remains a significant challenge. Traditional ecotoxicological endpoints are often not sensitive enough to detect the effects of toxicants at environmentally relevant concentrations (≤ng/L). A potential solution is using molecular biology methods to look at small biochemical changes caused by exposure to ng/L concentrations of contaminants. This has been tested in the lab but not conclusively demonstrated in the field. We exposed the freshwater amphipod (Austrochiltonia subtenuis) to water from an urban wetland containing known concentrations of per-and polyfluoroalkyl substances (as well as very low concentrations of pesticides) for 14 days and analyzed their metabolite profiles. Mannose, Myo-inositol, and Isopropyl propionate were found to change in PFAS exposed amphipods, a similar response to that previously observed in laboratory exposures to the same PFAS, but not pesticides. The results give a better understanding of PFAS toxicity at environmentally relevant concentrations and conditions., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Oliver A.H. Jones has received research funding from the Environment Protection Authority Victoria and the Defense Science Institute for general research into PFAS., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Temperature and mineralization are among the most important environmental factors affecting all processes of aquatic ecosystems, including geographical distribution of water animals. Previously we showed that a brackish water population of Gammarus lacustris, a widespread amphipod, demonstrates substantially higher thermotolerance than a freshwater population. A possible reason for this difference is the fact that brackish water conditions are closer to internal media mineralization. Here we aimed to test this hypothesis and relate the observed effects in animal survival under the heat shock to the status of cellular defence systems. We acclimated four groups of amphipods from the same freshwater population to 0.5 ‰ and 15 ‰ at the temperatures of 6°С or 15°С. Acclimation at 6°С, but not at 15°С, to 15 ‰ significantly increased resistance of the amphipods to heat shock at 30°C. At 6°С activities of antioxidant enzymes and levels of the lipid peroxidation products in G. lacustris did not react to the increase in mineralization and the heat shock, while the level of HSP70 elevated two-fold in amphipods acclimated to mineralization of 15 ‰ compared to animals acclimated to 0.5 ‰. Thus, the observed increase in thermotolerance could be explained by the higher initial level of HSP70 and potentially other heat shock proteins caused by a less energy-demanding, more isotonic level., Competing Interests: Declaration of competing interest The authors declare no conflict of interest exists., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The past decades have marked the rise of metabolomics and lipidomics as the -omics sciences which reflect the most phenotypes in living systems. Mass spectrometry-based approaches are acknowledged for both quantification and identification of molecular signatures, the latter relying primarily on fragmentation spectra interpretation. However, the high structural diversity of biological small molecules poses a considerable challenge in compound annotation. Feature-based molecular networking (FBMN) combined with database searches currently sets the gold standard for annotation of large datasets. Nevertheless, FBMN is usually based on collision-induced dissociation (CID) data, which may lead to unsatisfying information. The use of alternative fragmentation methods, such as electron-activated dissociation (EAD), is undergoing a re-evaluation for the annotation of small molecules, as it gives access to additional fragmentation routes. In this study, we apply the performances of data-dependent acquisition mass spectrometry (DDA-MS) under CID and EAD fragmentation along with FBMN construction, to perform extensive compound annotation in the crude extracts of the freshwater sentinel organism Gammarus fossarum. We discuss the analytical aspects of the use of the two fragmentation modes, perform a general comparison of the information delivered, and compare the CID and EAD fragmentation pathways for specific classes of compounds, including previously unstudied species. In addition, we discuss the potential use of FBMN constructed with EAD fragmentation spectra to improve lipid annotation, compared to the classic CID-based networks. Our approach has enabled higher confidence annotations and finer structure characterization of 823 features, including both metabolites and lipids detected in G. fossarum extracts., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Metal pollution is a major concern for aquatic environments. Widespread contamination by various trace metal ions has been described in freshwater streams as well as their subsequent bioaccumulation, potentially leading to toxicity and trophic transfer. Metallothioneins constitute an evolutionary conserved family of low molecular weight, cysteine-rich, metal-chelating proteins, whose known physiological functions are the maintenance of the homeostasis of essential metals, the detoxification of non-essential metals, and the protection against oxidative stress and free radicals. In this study, we identified two metallothionein-coding transcripts, mt1 and mt2, in the transcriptome of the amphipod Gammarus fossarum, a sentinel species widely used to assess the quality of watersheds. For the first time, we investigated the organ-specific patterns of expression of these two mt transcripts at the individual level in the gills and the caeca of this small crustacean. In silico analysis and experimental exposures to environmentally relevant concentrations of cadmium, zinc and silver showed that G. fossarum mt1 induction is stronger after Cd exposure compared to the other tested metals. G. fossarum mt1 was more significantly induced in the caeca than in the gills of exposed organisms for any metal exposure, while G. fossarum mt2 was, at least at the individual level, more inducible in the gills than in the caeca of G. fossarum exposed to Cd and Zn. Our results provide new genetic resources that will help to improve the understanding of metal homeostasis in this sentinel species., Competing Interests: Declaration of Competing Interest The authors declare that they have no known conflict of interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Temperature is a crucial environmental factor affecting the distribution and performance of ectothermic organisms. This study introduces a new temperature damage model to interpret their thermal stress. Inspired by the ecotoxicological damage model in the General Unified Threshold model for Survival (GUTS) framework, the temperature damage model assumes that damage depends on the balance between temperature-dependent accumulation and constant repair. Mortality due to temperature stress is driven by the damage level exceeding a threshold. Model calibration showed a good agreement with the measured survival of Gammarus pulex exposed to different constant temperatures. Further, model simulations, including constant temperatures, daily temperature fluctuations, and heatwaves, demonstrated the model's ability to predict temperature effects for various environmental scenarios. With this, the present study contributes to the mechanistic understanding of temperature as a single stressor while facilitating the incorporation of temperature as an additional stressor alongside chemicals in mechanistic multistressor effect models.

The multixenobiotic resistance (MXR) mechanism has been demonstrated to be present in a wide range of species, including aquatic organisms. However, amphipods (Crustacea: Malacostraca: Amphipoda), which constitute a large order of arthropods, are extremely poorly studied in this regard. Information on MXR proteins in these animals would be highly relevant, as some amphipods are important models in ecotoxicology due to their roles in many freshwater environments, including the ancient Lake Baikal. In this work, we studied the diversity of ABC transporters in the available transcriptomes of over 60 endemic Baikal amphipods in comparison to other related species. This showed that most classes of ABC transporters are present in all analyzed species and that most Baikal amphipods detectably express no more than one complete ABCB full transporter. We also showed that these sequences were conservative across different species, and their phylogeny was congruent with the species phylogeny. Thus, we chose the abcb1 coding sequence from Eulimnogammarus verrucosus, a widespread species playing an important role in the lake ecosystem, to establish the first heterologous expression system for an amphipod Abcb1/P-glycoprotein based on the Drosophila melanogaster S2 cell line. The resulting stably transfected S2 cell line was expressing the abcb1 of E. verrucosus about 1000 times higher than the homologous fly genes, and the target protein, Abcb1, showed to confer a high MXR-related efflux activity. Our results indicate the suitability of the S2-based expression systems for the study of arthropod ABCB1 homologs., Competing Interests: Declaration of competing interest The authors declare no conflict of interest exists., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Bioaccumulation of organic contaminants from contaminated food sources might pose an underestimated risk toward shredding invertebrates. This assumption is substantiated by monitoring studies observing discrepancies of predicted tissue concentrations determined from laboratory-based experiments compared with measured concentrations of systemic pesticides in gammarids. To elucidate the role of dietary uptake in bioaccumulation, gammarids were exposed to leaf material from trees treated with a systemic fungicide mixture (azoxystrobin, cyprodinil, fluopyram, and tebuconazole), simulating leaves entering surface waters in autumn. Leaf concentrations, spatial distribution, and leaching behavior of fungicides were characterized using liquid chromatography coupled with high-resolution tandem mass spectrometry (LC-HRMS/MS) and matrix-assisted laser desorption ionization-mass spectrometric imaging. The contribution of leached fungicides and fungicides taken up from feeding was assessed by assembling caged (no access) and uncaged (access to leaves) gammarids. The fungicide dynamics in the test system were analyzed using LC-HRMS/MS and toxicokinetic modeling. In addition, a summer scenario was simulated where water was the initial source of contamination and leaves contaminated by sorption. The uptake, translocation, and biotransformation of systemic fungicides by trees were compound-dependent. Internal fungicide concentrations of gammarids with access to leaves were much higher than in caged gammarids of the autumn scenario, but the difference was minimal in the summer scenario. In food choice and dissectioning experiments gammarids did not avoid contaminated leaves and efficiently assimilated contaminants from leaves, indicating the relevance of this exposure pathway in the field. The present study demonstrates the potential impact of dietary uptake on in situ bioaccumulation for shredders in autumn, outside the main application period. The toxicokinetic parameters obtained facilitate modeling of environmental exposure scenarios. The uncovered significance of dietary uptake for detritivores warrants further consideration from scientific as well as regulatory perspectives. Environ Toxicol Chem 2023;42:1993-2006. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC., (© 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.)

Delayed toxicity is a phenomenon observed for aquatic invertebrates exposed to nicotinic acetylcholine receptor (nAChR) agonists, such as neonicotinoids. Furthermore, recent studies have described an incomplete elimination of neonicotinoids by exposed amphipods. However, a mechanistic link between receptor binding and toxicokinetic modeling has not been demonstrated yet. The elimination of the neonicotinoid thiacloprid in the freshwater amphipod Gammarus pulex was studied in several toxicokinetic exposure experiments, complemented with in vitro and in vivo receptor-binding assays. Based on the results, a two-compartment model was developed to predict the uptake and elimination kinetics of thiacloprid in G. pulex . An incomplete elimination of thiacloprid, independent of elimination phase duration, exposure concentrations, and pulses, was observed. Additionally, the receptor-binding assays indicated irreversible binding of thiacloprid to the nAChRs. Accordingly, a toxicokinetic-receptor model consisting of a structural and a membrane protein (including nAChRs) compartment was developed. The model successfully predicted internal thiacloprid concentrations across various experiments. Our results help in understanding the delayed toxic and receptor-mediated effects toward arthropods caused by neonicotinoids. Furthermore, the results suggest that more awareness toward long-term toxic effects of irreversible receptor binding is needed in a regulatory context. The developed model supports the future toxicokinetic assessment of receptor-binding contaminants.

Talitrid amphipods are an important component of detritus web, playing a key role in the fragmentation of organic matters of marine and terrestrial origin, and it is well known that sandhoppers ingest microplastics. To assess the effective consumption of bioplastics and their effects on survival rate and on pollutants transfer (i.e. phthalates) on supralittoral arthropods, laboratory experiments were conducted by feeding adult T. saltator with two different types of bioplastic commonly used in the production of shopping bags. Groups of about 20 individuals were fed with 10 × 10 cm sample sheets of the two types of bioplastic for four weeks. The results show that amphipods ingest bioplastics even in the absence of microbial film and that ingestion of bioplastic can have effects on talitrid amphipods. Microtomographic analyses of faecal pellets seem consistent with this finding. The high phthalate concentrations in freshly collected individuals suggest the presence in the environment of these compounds, and the ability of amphipods to assimilate them, while the decrease in phthalate concentrations in bioplastic-fed individuals could be attributed to the scavenging effect of virgin plastic, as already observed in a previous study. In summary, the results indicate that different bioplastics may have effects on T. saltator (i.e. survival rate and faecal pellets structure) and confirm a potential role of amphipods in the degradation of bioplastics in supralittoral zone of marine sandy beaches, even when bioplastics are not colonized by bacterial biofilm that seems to improve palatability., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Microplastics are widespread pollutants in the environment and are considered a global pollution problem. Microplastics mostly originate from larger plastics and due to environmental conditions are undergoing constant fragmentation processes. It is important to understand the fragmentation pathways, since they play a key role in the fate of the particles, and also directly influence toxicity. Amphipods are potential inducers of plastic debris fragmentation. Here, Hyalella azteca was exposed to different concentrations (540, 2700, 5400 items/L) of 24.5 µm polystyrene microplastics (PS-MP) for 7 days. After exposure, oxidative stress, particle size reduction, and mortality were checked. No significant mortality was seen in any of the treatments, although changes were recorded in all enzymatic biomarkers analyzed. It was observed that throughout the ingestion and egestion of PS-MP by H. azteca, particles underwent intense fragmentation, presenting a final size up to 25.3% smaller than the initial size. The fragmentation over time (24, 72, 120, 168 h) was verified and the results showed a constant reduction in average particle size indicating that H. azteca are able to induce PS-MP fragmentation. This process may facilitate bioaccumulation and trophic particle transfer., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Bioconcentration tests using the freshwater amphipod Hyalella azteca as an alternative to conventional fish tests have recently received much attention. An appropriate computational model of H. azteca could help in understanding the mechanisms behind bioconcentration, in comparison to the fish as test organism. We here present the first mechanistic model for H. azteca that considers the single diffusive processes in the gills and gut. The model matches with the experimental data from the literature quite well when appropriate physiological information is used. The implementation of facilitated transport was essential for modeling. Application of the model for superhydrophobic compounds revealed binding to organic matter and the resulting decrease in bioavailable fraction as the main reason for the observed counterintuitive decrease in uptake rate constants with increasing octanol/water partition coefficient. Furthermore, estimations of the time needed to reach steady state indicated that durations of more than a month could be needed for compounds with a log K ow  > 8, limiting the experimental applicability of the test. In those cases, model-based bioconcentration predictions could be a preferable approach, which could be combined with in vitro biotransformation measurements. However, our sensitivity analysis showed that the uncertainty in determining the octanol/water partition coefficients is a strong source of error for superhydrophobic compounds., (© 2023. The Author(s).)

Amphipods are ideal indicators for biomonitoring and ecotoxicological studies of environmental contaminants because they are extensively distributed in aquatic environments, are easy to collect and are important in nutrient cycling. Marine amphipods (Allorchestes compressa) were exposed to two concentrations of copper and pyrene, and their mixtures, for 24 and 48 h. Changes in polar metabolites were assessed using Gas Chromatography Mass Spectrometry (GC-MS)-based untargeted metabolomics. Generally, limited metabolite changes were observed for copper and pyrene single exposures (eight and two significant metabolites, respectively), while 28 metabolites had changed following exposures to mixtures. Furthermore, changes were mainly observed after 24 h but had seemingly returned to control levels after 48 h. Multiple types of metabolites were affected including amino acids, Tricarboxylic acid (TCA) cycle intermediates, sugars, fatty acids, and hormones. This study highlights the sensitivity of metabolomics in assessing the impacts of low concentrations of chemicals compared to traditional ecotoxicological endpoints., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Information on the relationship between the exposure concentrations of metals and their biodistribution among organs remained scarce in invertebrates. The objective of this study was to investigate the effects of Cd concentration on the organotropism, toxico-kinetic and fate of this metal in different organs of gammarids exposed to dissolved radioisotope 109 Cd. Gammarids male were exposed for 7 days to three environmental Cd concentrations (i.e. 4, 52 and 350 ng.L -1 ) before being placed in depuration conditions (i.e. uncontaminated water). At several sampling times, Cd concentrations were determined by 109 Cd γ-counting in water, caeca, cephalon, gills, intestine and remaining tissues. Bioconcentration Factors (BCF) and Cd relative proportions in organs were calculated to assess the exposure concentration effect on the bioaccumulation capacities. The dependance of the organ-specific kinetic parameters to Cd water concentrations were estimated by fitting nested one-compartment toxico-kinetic (TK) models to both the accumulation and depuration data, by Bayesian inference. Then, for each Cd concentrations, the metal exchanges among organs over time were formalized by a multi-compartments TK model fitted to all organ data simultaneously. Our results highlighted that, at the end of the exposure phase, BCF and Cd relative proportions, in each organ, were not significantly modulated by water concentrations. Kinetically, Cd accumulation rates in all organs (except intestines) were depended on the exposure concentration, but not the elimination rates. The in vivo management of Cd (i.e. metal exchanges among organs) remained qualitatively unchanged according to exposure concentration. All these results also highlighted key role of that organs in the management of Cd: bioconcentration by caeca, storage by gills and main entry pathway by intestine. This study shows the interest of implementing TK approaches to test the effect of environmental factors on bioaccumulation, inter-organ exchanges and fate of contaminants in invertebrate body to enhance the understanding of the toxicity risk., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)

Some macroalgae drift on the ocean and are stranded on coasts, and these stranded brown macroalgae are regarded to be degraded by organisms. Alginate is a major component of brown macroalgae. An uncovering of how carbon is cycled through brown macroalgae is needed to deeply understand coastal ecosystems. In this study, to gain insights into metabolism of brown macroalgae and alginate in the organisms, we initially confirmed that supralittoral talitrid amphipods (beach fleas or sandhoppers collected on the Shibagaki coast in Ishikawa Prefecture, Japan) fed on the brown macroalgae. We then isolated bacteria such as Vibrio sp. with alginate-assimilating capability from the gut of the amphipods. Metagenomic analysis of the gut of amphipods housed in several conditions (e.g. macroalgae or paper as feed, non-sterilized or sterilized environment) showed no condition-dependent compositions of bacteria and fungi, but Vibrio sp. were detected at high frequency, in good agreement with the isolation of Vibrio sp. An intervention study using antibiotics showed that amphipods fed on algae or paper at about the same rate in the presence or absence of antibiotics, and that the antibiotics had no effects on the life span. Moreover, intervention with antibiotics completely killed Vibrio sp. and some other bacteria, and had significant effects on the composition of the flora in the gut, with elimination of the variations observed in the guts of amphipods housed without antibiotics. These data suggest that microbes that were killed by antibiotics, including Vibrio sp., in the gut of talitrid amphipods are not essential for assimilation of brown macroalgae., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2022 Nakamura et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Multiple reaction monitoring (MRM) mass spectrometry is emerging as a relevant tool for measuring customized molecular markers in freshwater sentinel species. While this technique is typically used for the validation of protein molecular markers preselected from shotgun experiments, recent gains of MRM multiplexing capacity offer new possibilities to conduct large-scale screening of animal proteomes. By combining the strength of active biomonitoring strategies and MRM technologies, this study aims to propose a new strategy for the discovery of candidate proteins that respond to environmental variability. For this purpose, 249 peptides derived from 147 proteins were monitored by MRM in 273 male gammarids caged in 56 environmental sites, representative of the diversity of French water bodies. A methodology is here proposed to identify a set of customized housekeeping peptides (HKPs) used to correct analytical batch effects and allow proper comparison of peptide levels in gammarids. A comparative analysis performed on HKPs-normalized data resulted in the identification of peptides highly modulated in the environment and derived from proteins likely involved in the environmental stress response. Overall, this study proposes a breakthrough approach to screen and identify potential proteins responding to relevant environmental conditions in sentinel species., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Understanding the changes in the temporal and spatial concentrations of chemical substances in sediment toxicity tests facilitates interpretation of their toxicity and accumulation in benthic organisms because benthic organisms are affected by chemicals via multiple exposure pathways. However, such investigations using chronic sediment toxicity tests have rarely been performed. To examine the concentration profiles of a hydrophobic organic chemical using chronic spiked-sediment toxicity tests, we performed 28-day sediment toxicity tests of fluoranthene with a freshwater amphipod, Hyalella azteca, using a semi-flow-through system and compared the results with those of 10-day tests. In these experiments, we measured various types of fluoranthene concentrations over the test periods: total dissolved (C diss ) and freely dissolved (C free ) concentrations in overlying water and porewater as well as sediment concentrations. We also examined which concentration correlated with the amphipod bioconcentration factor (BCF). We found that both overlying water and porewater C free did not differ significantly on days 10 and 28. Sediment concentrations remained almost stable for 28 days, whereas C diss in overlying water varied temporally. These results suggest that the 28-day test provides almost constant concentrations of fluoranthene, particularly in porewater, even in a semi-flow-through system. In addition, the comparison of BCF of fluoranthene on day 10 in the present study with that obtained from water-only tests reported in the literature suggested that C free in pore water was the most representative indicator of bioaccumulation in H. azteca. Our findings support the possible use of a water-exchange system in chronic spiked-sediment toxicity tests of hydrophobic organic chemicals. However, further studies using sediments and chemicals with different properties are warranted to generalize the findings of the present study. Environ Toxicol Chem 2022;41:2679-2687. © 2022 SETAC., (© 2022 SETAC.)

Internal phosphorus (P) loading can increase the P level in the water column and further sustains cyanobacterial blooms. This study focused on the role of benthic fauna bioturbation in affecting the sediment P release and the P level of water column in a eutrophic lake, Lake Taihu. The macrofauna density decreased from 4766.56 ± 10541.80 ind/m 2 in 2007 to 345 ± 447.63 ind/m 2 in 2020 due to the frequent bottom-water hypoxia in Lake Taihu. The reduced macrofauna density majorly resulted from Grandidierella taihuensis, Limnodrilus hoffmeisteri, and Tanypus chinensis larvae, and their total density decreased by approximately 97% in 2020 compared to 2007. G. taihuensis, one of the major benthic faunas, was further used as a representative to investigate the effects of bioturbation on sediment P release using high-resolution sampling and imaging techniques. The results show that G. taihuensis can increase the O 2 penetration depth by more than 20 mm through bio-irrigation, and causes the redox conditions in burrows and surrounding sediments to change dramatically within a few minutes due to the intermittent ventilation. Subsequent oxidation of the soluble Fe(II) led to the formation of Fe-oxide bound P in the surface sediments, thereby increasing the P retention in the sediments. When the G. taihuensis density was 1563 ind/m 2 at the sampling site, approximately 0.12 g m -2 yr -1 P can be retained in sediments. As previous studies have shown that L. hoffmeisteri and T. chinensis played a similar role in increasing the P retention in sediments through their bioturbation activities, the sharp decline in benthic fauna density and burrowing activities in Lake Taihu should be an important reason for maintaining the high P level in the water column by decreasing the P retention in sediments., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:, (Copyright © 2022. Published by Elsevier Ltd.)

Arsenic (As) is a toxic metalloid present in high levels in diverse regions of Argentina. The aim of this study was to determine acute As-mediated toxicity in two different populations of autochthonous Hyalella curvispina amphipods from a reference site (LB) and an agricultural one (FO) within North Patagonia Argentina. Previously, both populations exhibited significant differences in pesticide susceptibility. Lab assays were performed to determine acute lethal concentrations, as well as some biochemical parameters. Lethal concentration (LC 50 ) values obtained after 48 and 96 hr As exposure were not significantly different between these populations, although FO amphipods appeared slightly less susceptible. LC 50 -48 hr values were 3.33 and 3.92 mg/L As, while LC 50 -96 hr values were 1.76 and 2.14 mg/L As for LB and FO amphipods. The no observed effect concentration (NOEC) values were 0.5 mg/L As. Cholinesterase (ChE) activity was significantly diminished by As acute exposure (0.5-1.5 mg/L As), indicative of a significant neurotoxic action for this metalloid in both amphipod populations. Activities of catalase (CAT) and glutathione S-transferase (GST) and levels of reduced glutathione (GSH) were differentially altered following As exposure. CAT activity was increased after 96 hr As exposure. GST activity and GSH levels were significantly elevated followed by either a decrease or a return to control values after 96 hr treatment. However, additional studies are necessary to understand the mechanisms underlying the As-mediated oxidative effects in H. curvispina . Our findings suggest that measurement of ChE activity in H. curvispina amphipods might serve as a useful biomarker of As exposure and effect.

Parhyale hawaiensis has emerged as the crustacean model of choice due to its tractability, ease of imaging, sequenced genome, and development of CRISPR/Cas9 genome editing tools. However, transcriptomic datasets spanning embryonic development are lacking, and there is almost no annotation of non-protein-coding RNAs, including microRNAs. We have sequenced microRNAs, together with mRNAs and long non-coding RNAs, in Parhyale using paired size-selected RNA-seq libraries at seven time-points covering important transitions in embryonic development. Focussing on microRNAs, we annotate 175 loci in Parhyale, 88 of which have no known homologs. We use these data to annotate the microRNAome of 37 crustacean genomes, and suggest a core crustacean microRNA set of around 61 sequence families. We examine the dynamic expression of microRNAs and mRNAs during the maternal-zygotic transition. Our data suggest that zygotic genome activation occurs in two waves in Parhyale with microRNAs transcribed almost exclusively in the second wave. Contrary to findings in other arthropods, we do not predict a general role for microRNAs in clearing maternal transcripts. These data significantly expand the available transcriptomics resources for Parhyale, and facilitate its use as a model organism for the study of small RNAs in processes ranging from embryonic development to regeneration., (© 2022. The Author(s).)

The importance of aquaporins (AQPs) in the transport of water and solutes through cell membranes is well recognized despite being relatively new. To date, despite their abundance, diversity, and presence in disparate environments, amphipods have only been mentioned in studies about the AQPs of other animals and have never been further investigated. In this work, we aimed to recover from public data available AQPs of these crustaceans and reconstruct phylogenetic affinities. We first performed BLAST searches with several queries of diverse taxa against different NCBI databases. Then, we selected the clades of AQPs retrieving the amphipod superfamily Gammaroidea as monophyletic and ran phylogenetic analyses to assess their performances. Our results show how most of the AQPs of amphipods are similar to those of other crustaceans, despite the Prip-like displayed different paralogs, and report for the first time a putative Aqp8-like for arthropods. We also found that the candidate genes of Prip-like, Bib-like, Aqp12-like, and Glp-like help solve deeper relationships in phylogenies of amphipods while leaving uncertainties in shallower parts. With our findings, we hope to increase attention to the study of amphipods as models for AQP functioning and evolution.

Given extensive use of pesticides in agriculture, there is concern for unintended consequences to non-target species. The non-target freshwater amphipod, Hyalella azteca has been found to show resistance to the organophosphate (OP) pesticide, chlorpyrifos, resulting from an amino acid substitution in acetylcholinesterase (AChE), suggesting a selective pressure of unintended pesticide exposure. Since resistant organisms can survive in contaminated habitats, there is potential for them to accumulate higher concentrations of insecticides, increasing the risk for trophic transfer. In the present study, we estimated the uptake and elimination of chlorpyrifos in non-resistant US Lab, and resistant Ulatis Creek (ULC Resistant), H. azteca populations by conducting 24-h uptake and 48-h elimination toxicokinetic experiments with 14 C-chlorpyrifos. Our results indicated that non-resistant H. azteca had a larger uptake clearance coefficient (1467 mL g -1 h -1 ) than resistant animals (557 mL g -1 h -1 ). The half-life derived from the toxicokinetic models also estimated that steady state conditions were reached at 13.5 and 32.5 h for US Lab and ULC, respectively. Bioaccumulation was compared between non-resistant and resistant H. azteca by exposing animals to six different environmentally relevant concentrations for 28 h. Detection of chlorpyrifos in animal tissues indicated that resistant animals exposed to high concentrations of chlorpyrifos were capable of accumulating the insecticide up to 10-fold higher compared to non-resistant animals. Metabolite analysis from the 28-h concentration experiments showed that between 20 and 50 % parent compound was detected in H. azteca. These results imply that bioaccumulation potential can be more significant in chlorpyrifos resistant H. azteca and may be an essential factor in assessing the full impacts of toxicants on critical food webs, especially in the face of increasing pesticide and chemical runoff., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

This study aimed to reveal the toxic characteristics of di-(2-ethylhexyl) phthalate (DEHP) by examining the biochemical and histopathological changes in Gammarus pulex, exposed to different doses of DEHP. For this purpose, the lethal concentration 50 (LC 50 ) value of the DEHP was determined by using a static test and found to be 0.079 ± 0.01 ppm. Three subletal doses of DEHP were applied to the G. pulex for 24 and 96 h. Superoxide dismutase (SOD), catalase (CAT), cytochrome P450 1A1 (CYP1A1), and glutathione S-transferase (GST) activities were measured using commercial ELISA kits. The caspase method, which is an immunohistochemical analysis method, was used to determine the apoptosis that occurred in the G. pulex. The results showed that the CYP1A1 activities decreased in the groups exposed to different doses of DEHP compared to the control group (p > 0.05). CAT activity was found to increase in the application groups at the 24 and 96 h compared to the control group. In addition, it was found that SOD and GST activities increased at the 96 h compared to the control group. In light of the microscope examination of the model organism, hemolymphatic lacunae filled with hemolymph and reduction or absence of hemolymphatic ducts were observed especially in the G. pulex gills. Collapse of the gills and hyperplasia were observed after 96 h. As a result, it is suggested that changes in SOD, CAT, and GST activities can potentially be used as sensitive biomarkers for risk assessment in the environment and increased immunoreactivity in G. pulex caused by DEHP depending on increased application doses and application times., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Omics approaches are continuously providing new clues on the mechanisms of action of contaminants in species of environmental relevance, contributing to the emergence of molecular ecotoxicology. Co-expression network approaches represent a suitable methodological framework for studying the rich content of omics datasets. This study aimed to find evidence of key pathways and proteins related to the testicular toxicity in the sentinel crustacean species Gammarus fossarum exposed to endocrine disruptors using a weighted protein co-expression network analysis. From a shotgun proteomics dataset of male gonads of G. fossarum organisms exposed to cadmium (Cd), pyriproxyfen (Pyr) and methoxyfenozide (Met) in laboratory conditions, four distinct modules were identified as significantly correlated to contaminants' exposure. Protein set enrichment analysis identified modules involved in cytoskeleton organization and oxidative stress response associated with the Cd exposure. The module associated with Pyr exposure was associated with endoplasmic reticulum stress (ER) response, and the module correlated with Met exposure was characterized by a significant proportion of amphipod-restricted proteins whose functions are still not characterized. Our results show that co-expression networks are efficient and adapted tools to identify new potential mode of actions from environmental sentinel species, such as G. fossarum, using a proteogenomic approach, even without an annotated genome., (Copyright © 2021. Published by Elsevier B.V.)

Species with effective thermal adaptation mechanisms allowing them to thrive within a wide temperature range can benefit from climatic changes as they can displace highly specialized species. Here, we studied the adaptive capabilities of the Baikal endemic amphipods Eulimnogammarus verrucosus (Gerstfeld, 1858) and Eulimnogammarus cyaneus (Dybowsky, 1874) compared to the potential Holarctic Baikal invader Gammarus lacustris Sars, 1863 at the cellular level including the energy metabolism and the antioxidant system. All species were long-term exposed to a range of temperatures (1.5 °C to mimic winter conditions and the three species-specific preferred temperatures (i.e., 6 °C for E. verrucosus, 12 °C for E. cyaneus and 15 °C for G. lacustris). At 1.5 °C, we found species-specific metabolic alterations (i.e., significantly reduced ATP content and lactate dehydrogenase activity) indicating limitations on the activity level in the Holarctic G. lacustris. Although the two Baikal endemic amphipod species largely differ in thermal tolerance, no such limitations were found at 1.5 °C. However, the cold-stenothermal Baikal endemic E. verrucosus showed changes indicating a higher involvement of anaerobic metabolism at 12 °C and 15 °C, while the metabolic responses of the more eurythermal Baikal endemic E. cyaneus may support aerobic metabolism and an active lifestyle at all exposure temperatures. Rising temperatures in summer may provide a competitive advantage for G. lacustris compared to the Baikal species but the inactive lifestyle in the cold is likely preventing G. lacustris from establishing a stable population in Lake Baikal.

Toxicokinetic (TK) models refer to the process of contaminant bioaccumulation as a balance between rate of uptake from different sources (e.g., water or food), and rate of elimination via different processes such as excretion, growth and/or biotransformation. Biotransformation can considerably modify the fate of chemicals in an organism, especially their bioavailability, residence time, and toxicity. Invertebrate models generally neglect this process as they assume a low metabolic activity. However, some species such as Gammarus sp. amphipods are able to metabolize a vast range of organic compounds. Some recent TK models include biotransformation, but they prove limited for estimating related parameters by giving negative values and/or large uncertainties for biotransformation rate(s). Here we propose a generic TK model accounting for biotransformation using a Bayesian framework for simultaneously estimating the parameters. We illustrated the added value of our method by fitting this generic TK model to 22 published datasets of several benthic invertebrate species exposed to different chemicals. All parameters are estimated simultaneously for all datasets and showed narrow estimates. Furthermore, the median model predictions and their 95% credibility intervals showed that the model confidently fitted the data. In most cases the uncertainties around biotransformation rate(s) were reduced in comparison to the original studies. From a methodology standpoint, this paper reflects that Bayesian inference has real added value for simultaneously estimating all TK parameters for parent chemicals and their metabolite(s) based on all available data, while accounting for different types of data and the correlation between parameters. Bayesian inference was able to overcome the limits of previous methods, since no parameters were fixed and no irrelevant negative values were obtained. Moreover, the 95% credibility intervals around model predictions, which are core uncertainties for Environmental Risk Assessment, were easily acquired., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Gammarid amphipods are a crucial link connecting primary producers with secondary consumers, but little is known about their nutritional ecology. Here we asked how starvation and subsequent feeding on different nutritional quality algae influences fatty acid retention, compound-specific isotopic carbon fractionation, and biosynthesis of ω-3 and ω-6 polyunsaturated fatty acids (PUFA) in the relict gammarid amphipod Pallaseopsis quadrispinosa . The fatty acid profiles of P. quadrispinosa closely matched with those of the dietary green algae after only seven days of refeeding, whereas fatty acid patterns of P. quadrispinosa were less consistent with those of the diatom diet. This was mainly due to P. quadrispinosa suffering energy limitation in the diatom treatment which initiated the metabolization of 16:1ω7 and partly 18:1ω9 for energy, but retained high levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) similar to those found in wild-caught organisms. Moreover, α-linolenic acid (ALA) from green algae was mainly stored and not allocated to membranes at high levels nor biosynthesized to EPA. The arachidonic acid (ARA) content in membrane was much lower than EPA and P. quadrispinosa was able to biosynthesize long-chain ω-6 PUFA from linoleic acid (LA). Our experiment revealed that diet quality has a great impact on fatty acid biosynthesis, retention and turnover in this consumer.

Mercury (Hg) is a global contaminant resulting of both natural processes and human activities. In aquatic environments, studies conducted on vertebrates highlighted changes of gene expression or activity of antitoxic and oxidative enzymes. However, although Hg is a highly toxic compound in aquatic environments, only a few studies have evaluated the lethal and sublethal effects of inorganic Hg on Gammarus sp. Therefore, this study aimed at evaluating the effects of inorganic Hg (HgCl 2 ) on the expression of 17 genes involved in crucial biological functions or mechanisms for organisms, namely respiration, osmoregulation, apoptosis, immune and endocrine system, and antioxidative and antitoxic defence systems. The study was performed in males of the freshwater amphipod Gammarus pulex exposed to two environmentally relevant concentrations (50 and 500 ng/L) at two temperature regime fluctuations (16 °C and 20 °C +/-2 °C) for 7 and 21 days. Results showed that G. pulex mortality was dependent on Hg concentration and temperature; the higher the concentration and temperature, the higher the mortality rate. In addition, the Integrated Biomarker Response emphasized that HgCl 2 toxicity was dependent on the concentration, time and temperature of exposure. Overall, antioxidant and antitoxic defences, as well as the endocrine and immune systems, were the biological functions most impacted by Hg exposure (based on the concentration, duration, and temperature tested). Conversely, osmoregulation was the least affected biological function. The results also demonstrated a possible adaptation of G. pulex after 21 days at 500 ng/L, regardless of the exposure temperature. This study allowed us to show that Hg deregulates many crucial biological functions after a short exposure, but that during a long exposure, an adaptation phenomenon could occur, regardless of temperature., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Rationale: (Eco-)toxicological effects are mostly derived empirically and are not correlated with metal uptake. Furthermore, if the metal content is determined, mostly bulk analysis of the whole organism population is conducted; thus, biological variability is completely disregarded, and this may lead to misleading results. To overcome this issue, we compared two different solid sampling techniques for the analysis of single organisms., Methods: In this study, complementary electrothermal vaporization/inductively coupled plasma mass spectrometry (ETV/ICP-MS) ⇔ laser ablation/inductively coupled plasma mass spectrometry (LA/ICP-MS)-based methods for the analysis of individual organisms were developed and the results obtained were compared with the concentrations obtained after digestion and measured using ICP-MS. For this purpose, a common (eco-)toxicological test organism, the mud shrimp Corophium volutator, was selected. As proof-of-concept application, these organisms were incubated with environmentally relevant metals from galvanic anodes, which are often used for protection against metal corrosion in, for example, offshore wind farms., Results: The bulk analysis revealed that large quantities of the incubated elements were detectable. Using the ETV/ICP-MS method, we could identify a high biovariability within the population of organisms tested. Using the LA/ICP-MS method, it could be determined that the large quantities of the elements detected were due to adsorption of the metals and not due to uptake, which correlates well with the absence of (eco-)toxicological effects., Conclusions: The results obtained imply the efficiency of complementary methods to explain the absence or presence of (eco-)toxicological effects. In particular, methods that allow for single-organism analysis or provide even a spatial resolution support the interpretation of ecotoxicological findings., (© 2020 John Wiley & Sons, Ltd.)

Standardized experimental approaches for the quantification of the bioaccumulation potential of nanomaterials in general and in (benthic) invertebrates in particular are currently lacking. We examined the suitability of the benthic freshwater amphipod Hyalella azteca for the examination of the bioaccumulation potential of nanomaterials. A flow-through test system that allows the generation of bioconcentration and biomagnification factors was applied. The feasibility of the system was confirmed in a 2-lab comparison study. By carrying out bioconcentration and biomagnification studies with gold, titanium dioxide and silver nanoparticles as well as dissolved silver (AgNO 3 ) we were able to assess the bioaccumulation potential of different types of nanomaterials and their exposure pathways. For this, the animals were examined for their total metal body burden using inductively coupled mass spectroscopy (ICP-MS) and for the presence of nanoparticulate burdens using single-particle ICP-MS. The role of released ions was highlighted as being very important for the bioavailability and bioaccumulation of metals from nanoparticles for both examined uptake paths examined (bioconcentration and biomagnification). In 2018 a tiered testing strategy for engineered nanomaterials was proposed by Handy et al. that may allow a waiver of bioaccumulation fish studies using inter alia invertebrates. Data gained in studies carried out with invertebrates like the developed Hyalella azteca test may be included in this proposed tiered testing strategy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The effects of cyanobacteria (Aphanizomenon flos-aquae (90%), Microcystis aeruginosa) and dense Elodea canadensis beds on the health endpoints of the amphipod Gmelinoides fasciatus and bivalve mollusc Unio pictorum were examined in mesocosms with simulated summer conditions (July-August 2018) in the environment of the Rybinsk Reservoir (Volga River Basin, Russia). Four treatments were conducted, including one control and three treatments with influencing factors, cyanobacteria and dense elodea beds (separately and combined). After 20 days of exposure, we evaluated the frequency of malformed and dead embryos in amphipods, heart rate (HR) and its recovery (HRR) after stress tests in molluscs as well as heat tolerance (critical thermal maximum or CTMax) in both amphipods and molluscs. The significant effect, such as elevated number of malformed embryos, was recorded after exposure with cyanobacteria (separately and combined with elodea) and presence of microcystins (MC) in water (0.17 μg/l, 40% of the most toxic MC-LR contribution). This study provided evidence that an elevated number (>5% of the total number per female) of malformed embryos in amphipods showed noticeable toxicity effects in the presence of cyanobacteria. The decreased oxygen under the influence of dense elodea beds led to a decrease in HR (and an increase in HRR) in molluscs. The notable effects on all studied biomarkers, embryo malformation frequency and heat tolerance in the amphipod G. fasciatus, as well as the heat tolerance and heart rate in the mollusc U. pictorum, were found when both factors (elodea and cyanobacteria) were combined. The applied endpoints could be further developed for environmental monitoring, but the obtained results support the importance of the combined use of several biomarkers and species, especially in the case of multi-factor environmental stress., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Bioaccumulation assessment predominantly relies on the bioconcentration factor (BCF) as the sole decisive metric. The test guideline 305 by the Organisation for Economic Co-operation and Development (OECD) provides the standard procedure for deriving this in vivo fish BCF, which is not only expensive and labor-intensive, but also requires many animals. Accordingly, there is a great need for and interest in alternative methods that can help to reduce, replace, and refine vertebrate tests, as described in the 3R principles. Two alternative approaches have been developed: the bioconcentration test with the freshwater amphipod Hyalella azteca and the OECD test guideline 319 which provides a method to determine experimentally derived in vitro metabolism rates that can then be incorporated into in silico prediction models for rainbow trout BCF calculation. In the present study both alternative methods were applied to 5 substances of different physicochemical characteristics. The results were compared with literature values of fish in vivo BCFs and additional BCFs obtained with the alternative methods, if available. Potential differences between the results of the test methods are discussed utilizing information such as in vivo metabolism rates. The currently available data set suggests that these 2 alternative methods pose promising alternatives to predict bioaccumulation in fish, although defined applicability domains have yet to be determined. Environ Toxicol Chem 2020;39:1813-1825. © 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC., (© 2020 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.)

The research study was about revealing the biochemical response of Gammarus pulex related to insecticide methomyl before and after bioremediation by two soil bacteria species, Ochrobactrum thiophenivorans and Sphingomonas melonis. Catalase (CAT), glutathione S-transferase.(GST), cytochrome. P4501A1 (CYP1A1) activities in G. Pulex related to methomyl solution were investigated in 24 h and 96 h. ELISA method was used for test studies. CAT enzyme was decreased in Gammarus pulex that was exposed to methomyl after all exposure period (P < 0.05). CAT activities were returned to control results after bioremediation assays. GST enzyme activity was decreased depending on methomyl exposure during 24 h but increased during 4 days (P < 0.05). After 8 days of bioremediation period, GST activity increased again during 24 h while decreased during 4 days (P < 0.05). CYP1A1 activity increased in Gammarus pulex that was exposed to methomyl after all exposure period (P > 0.05). After bioremediation, statistically significant changes were not revealed in CYP1A1 activities (P > 0.05). According to the results of our study, CYP1A1, CAT, and GST activities in G. pulex sanctioned the capability of Ochrobactrum thiophenivorans and Sphingomonas melonis in methomyl bioremediation. Isolated and enriched Ochrobactrum thiophenivorans and Sphingomonas melonis that were added to 2.5 ppb concentrations of methomyl for 8 days. Each day, chemical oxygen demand (COD) and biochemical oxygen demand (BOD 5 ), pH and dissolved oxygen parameters were monitored. At the final phase of the bioremediation step, it was determined that these bacteria have efficient methomyl bioremediation properties in a mixed corsortia at a rate of 86%. These results show that these bacteria can be used for bioremediate the receiving environments that are polluted by these kinds of insecticides.

The ability of organisms to cope with environmental stressors depends on the duration and intensity of the stressor, as well as the type of stress. For aquatic organisms, oxygen limitation has been implicated in limiting heat tolerance. Here we examine how starvation affects heat tolerance in the amphipod Gammarus fossarum (Koch, 1836) and whether observed changes can be explained from alterations in oxidative metabolism, depletion of energy reserves, upregulation of heat shock proteins or susceptibility to oxygen limitation. Starved amphipods showed impaired survival compared to fed amphipods during prolonged exposure to mild heat. In contrast, under acute, high-intensity heat exposure they actually showed improved survival. We observed a lower demand for oxygen in starved amphipods which could make them less susceptible to oxygen limitation. Such a role for oxygen in limiting heat tolerance was verified as hypoxia impaired the heat tolerance of amphipods, especially starved ones. Fed amphipods likely rely more on anaerobic metabolism to maintain energy status during heat stress, whereas for starved amphipods aerobic metabolism appears to be more important. The depletion of their energy reserves constrains their ability to maintain energy status via anaerobic metabolism. We did not find evidence that alterations in heat tolerance following starvation were related to the upregulation of heat shock proteins. In conclusion, starvation can have opposite effects on heat tolerance, acting via pathways that are operating on different time scales., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Antibiotics may constitute a risk for aquatic detritivorous macroinvertebrates (i.e., shredders) via waterborne and dietary antibiotic exposure. In addition, antibiotics can alter the food quality for shredders mediated by shifts in leaf-associated decomposer (i.e., aquatic fungi and bacteria) communities. However, little is known about the relative importance of the waterborne and dietary effect pathway. Therefore, we followed a tiered testing approach aimed at assessing the relative importance of these effect pathways. We employed the antibiotic ciprofloxacin (CIP) and the shredder Gammarus fossarum as model stressor and test species, respectively. In a first step, we assessed the short-term waterborne toxicity of CIP using survival and leaf consumption of G. fossarum as response variables. Alterations in the leaf-associated decomposer community, which may be reflected by their palatability, were assessed using food choice assays. Finally, we conducted a 2 × 2-factorial experiment over 24 days assessing the pathways individually and combined using energy processing (i.e., leaf consumption and feces production), growth and energy storage (i.e., neutral lipid fatty acids) as variables. Short term waterborne exposure indicated low toxicity with LC 50 and EC 50 values of 13.6 and 6.4 mg CIP/L, respectively. At the same time, shredders did not prefer any leaf material during the food choice assay. However, the fungal community was significantly affected in the highest CIP-treatments (0.5 and 2.5 mg/L) suggesting an altered food quality for shredders. This assumption is supported by the results of the long-term assay. At 0.5 mg CIP/L, gammarids' leaf consumption, growth and energy storage were increased when subjected via the dietary pathway, which was linked to changes in the leaf-associated microbial community. Our data highlight the importance of dietary effect pathways for effects on shredders, potentially impacting energy dynamics in detritus-based stream ecosystems., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Prey reconfigure their physiology to avoid costs of prolonged predator pressure. However, these changes might not occur under periodic predation risk, with repeating acute phases. To test the effect of predation risk continuity on changes in prey physiology, we exposed amphipods: Dikerogammarus villosus and Gammarus jazdzewskii to periodic and constant predation cue. After one week, we measured: cellular defence systems: total antioxidant status (TAS), heat shock proteins (Hsp70); intracellular damage marker: lipid peroxidation (TBARS); condition index: glycogen concentration. Predator presence reduced TAS level in G. jazdzewskii independent of its continuity and in D. villosus after periodic exposure. Amphipods showed downregulation of Hsp70 when exposed to periodic (D. villosus) or constant (G. jazdzewskii) predation risk. Exposure to predators reduced TBARS level in D. villosus (irrespective of the continuity) and G. jazdzewskii (periodic exposure). Glycogen concentration in both species was not affected by predator presence. Thus, the continuity of the predator cue shaped prey physiology reconfiguration, optimizing costs of physiological adjustments under challenging conditions. Nevertheless, the lack of negative consequences of the prolonged exposure to the predator cue, whether constant or periodic, shows that amphipods can thrive under chronic predation risk, which is a constant part of the wild environment.

Carotenoids are diverse lipophilic natural pigments which are stored in variable amounts by animals. Given the multiple biological functions of carotenoids, such variation may have strong implications in evolutionary biology. Crustaceans such as Gammarus amphipods store large amounts of these pigments and inter-population variation occurs. While differences in parasite selective pressure have been proposed to explain this variation, the contribution of other factors such as genetic differences in the gammarid ability to assimilate and/or store pigments, and the environmental availability of carotenoids cannot be dismissed. This study investigates the relative contributions of the gammarid genotype and of the environmental availability of carotenoids in the natural variability in carotenoid storage. It further explores the link of this natural variability in carotenoid storage with major crustacean immune parameters. We addressed these aspects using the cryptic diversity in the amphipod crustacean Gammarus fossarum and a diet supplementation protocol in the laboratory. Our results suggest that natural variation in G. fossarum storage of dietary carotenoids results from both the availability of the pigments in the environment and the genetically-based ability of the gammarids to assimilate and/or store them, which is associated to levels of stimulation of cellular immune defences. While our results may support the hypothesis that carotenoids storage in this crustacean may evolve in response to parasitic pressure, a better understanding of the specific roles of this large pigment storage in the crustacean physiology is needed., Competing Interests: The authors have declared that no competing interests exist.

This study aimed to assess how bioturbation by freshwater benthic macroinvertebrates with different biological traits alone or in combination could modify trace elements (TE) fate between sediment and water, and if water TE concentration and animal TE content impair their body stores. Three macroinvertebrate species were exposed to TE contaminated sediment for 7 days: the omnivorous Echinogammarus berilloni (Amphipoda), the sediment feeding Tubifex tubifex (Oligochaeta) and the filter feeding Pisidium sp. (Bivalvia). Treatments were one without invertebrates (control), two with amphipods or mussels alone, and the combinations amphipod-mussel, and amphipod-mussel-worms. Water TE concentration increased significantly in 2 or 3 species mesocosms, concerning mainly Rare Earth Elements, Cr, U and Pb, known to be associated to the colloidal phase. By contrast, water soluble TE were not affected by animals. For both, amphipods and mussels, TE body content increased with the number of coexisting species. For amphipods, this increase concerned both, soluble and colloid-associated TE, possibly due to intense contact and feeding from sediment and predation on tubificids. TE bioaccumulation in mussel was less important and characterized by soluble TE, with water filtration as most plausible uptake route. Protein, triglyceride and Whole Body Energy Budget increased in amphipods with the number of coexisting species (probably by feeding on mussels' feces and tubificids) whereas triglycerides declined in mussels (presumably filtration was disturbed by amphipods). This study highlights interspecific interactions as key drivers explaining both: TE bioturbation, depending on their water solubility or colloidal association, and the exposure/contamination of species through another species activity., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Regulatory assessment of the bioaccumulation from water is commonly based on bioconcentration factors (BCFs) derived from fish flow-through tests. Such experiments require many laboratory animals and are time-consuming and costly. An alternative test setup for organic, neutral compounds using the amphipod Hyalella azteca was recently suggested, resulting in BCF values which show a strong correlation with fish BCF data. In the present study, the bioconcentration potential of the ionic compound laurate was elucidated in H. azteca. The sodium salt of 1- 14 C laurate was applied to H. azteca in a flow-through and a semistatic approach. Because of rapid biodegradation, a semistatic approach with frequent medium replacements was required to ensure a stable medium concentration. Laurate was also rapidly metabolized by H. azteca. A large proportion of the total radioactivity measured in the amphipod tissue was not extractable, suggesting that mineralized laurate was accumulated in the calcified exoskeleton of H. azteca. This was confirmed in a further study using carbonate [ 14 C]. A lipid-normalized (5.0%) Hyalella BCF of 8.9 was calculated for laurate, measured as free fatty acids. The results of the bioconcentration studies with H. azteca confirm the low bioaccumulation potential of the test item previously observed in fish. However, more organic ionic compounds with various properties need to be tested to assess whether a general correlation between fish and Hyalella BCF data exists. Environ Toxicol Chem 2020;39:310-322. © 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC., (© 2019 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.)

Aquatic media are ultimate recipients of various contaminants including pesticides pervasively applied in agrosystems. Characterizing the ecotoxicity of pesticides and their mixtures to aquatic wildlife at field-realistic levels is thus crucial for environmental risk assessment. This study aims at assessing the effects of two current-use insecticides, imidacloprid and chlorpyrifos, on Gammarus fossarum using multi-level biomarkers. In microcosms, gammarids were exposed for 72 h to insecticides tested individually or in mixture at 0.01, 0.1 and 1 μg/L of each chemical. Multi-metric responses were assessed at the individual level (behavioural traits: locomotion, respiration and amplexus formation) and the cellular level (enzymes involved in growth, moulting, digestion and cell stress). The results showed insecticide-elicited behavioural and biochemical responses from the lowest concentration of 0.01 μg/L. Overall, single exposures stimulated behavioural traits and inhibited enzymatic activities, highlighting subtle impacts at different organizational levels but these were not dose related. For binary mixtures, antagonistic effects (i.e. less-than-additive) on biomarkers were mainly observed when compared with single exposures. Multi-variable analyses indicated the complementarity of behavioural and biochemical biomarkers in identifying sublethal biological alterations and dose-dependent multiple action sites of insecticides. Besides, the mortality observed only for the mixture at 1 μg/L demonstrated a high lethal potential of insecticides in a simple binary combination. To conclude, this study demonstrates disturbances in individual performances and cellular impairments occurring at environmentally realistic exposure levels in a non-target wild species. Since the sublethal effects, such as those identified with this multi-biomarker approach, could lead to long-term alterations in population dynamics of agricultural areas, they constitute promising early endpoints for risk assessment of insecticides., (Copyright © 2019 Elsevier B.V. All rights reserved.)

A former mining site has been the subject of extensive remediation and restoration, with a significant focus on disconnecting mine spoils from groundwater and managing the quantity and quality of runoff. A remaining task is ensuring concentrations of zinc (Zn) in the stream outflow of a pit lake are reduced below water quality standards. The efficacy of multiple capping materials for decreasing Zn dissolution from sediments was conducted under natural and reasonable worst-case conditions (pH = 5.5). Capping materials included AquaBlok™, limestone, and limestone-bone char. Field exposures were conducted in limnocorrals that isolated overlying water columns above the sediment and capping treatments. Simultaneous in situ and ex situ toxicity tests were conducted using Daphnia magna, Hyalella azteca, and Chironomus dilutus. In situ caged organisms were protected from temperature shock (warm epilimnetic waters) by deploying within a Toxicity Assessment Container System (TACS). Organisms were exposed to surficial sediments, caps, and hypolimnetic overlying waters for 4 d. Ex situ testing was conducted in core tube mesocosms containing sediments and caps at similar temperatures (15-19 °C). Results demonstrated the usefulness of TACS deployment in stratified lake systems. There were no differences in responses between treatments involving sediment capping materials in both in situ and ex situ tests. The lack of differences was likely due to dissolved Zn in surface water being below the hardness-adjusted threshold effects levels (164 μg L -1 ). This field- and laboratory-based weight-of-evidence study provided site-specific data to support the selection of an effective remedy, with reduced uncertainty compared to laboratory and chemistry-only approaches. Environ Toxicol Chem 2019;39:240-249. © 2019 SETAC., (© 2019 SETAC.)

Mercury (Hg) is a global contaminant that poses a human health risk in its organic form, methylmercury (MeHg), through consumption of fish and fishery products. Bioaccumulation of Hg in the aquatic environment is controlled by a number of factors expected to be altered by climate change. We examined the individual and combined effects of temperature, sediment organic carbon, and salinity on the bioaccumulation of MeHg in an estuarine amphipod, Leptocheirus plumulosus, when exposed to sediment from two locations in the Gulf of Maine (Kittery and Bass Harbor) that contained different levels of MeHg and organic carbon. Higher temperatures and lower organic carbon levels individually increased uptake of MeHg by L. plumulosus as measured by the biota-sediment accumulation factor (BSAF), while the effect of salinity on BSAF differed by sediment source. Multi-factor statistical modeling using all data revealed a significant interaction between temperature and organic carbon for both sediments, in which increased temperature had a negative effect on BSAF at the lowest carbon levels and a positive effect at higher levels. Our results suggest that increased temperature and carbon loading, of a magnitude expected as a result from climate change, could be associated with a net decrease in amphipod BSAF of 50 to 71%, depending on sediment characteristics. While these are only first-order projections, our results indicate that the future fate of MeHg in marine food webs is likely to depend on a number of factors beyond Hg loading., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Toxicokinetic (TK) models are relevant and widely used to predict chemical concentrations in biological organisms. The importance of dietary uptake for aquatic invertebrates has been increasingly assessed in recent years. However, the model parameters are estimated on limited specific laboratory data sets that are bounded by several uncertainties. The aim of this study was to implement a Bayesian framework for simultaneously estimating the parameters of a generic TK model for benthic invertebrate species from all data collected. We illustrate our approach on the bioaccumulation of PCB153 by two species with different life traits and therefore exposure routes: Chironomus riparius larvae exposed to spiked sediment for 7 days and Gammarus fossarum exposed to spiked sediment and/or leaves for 7 days and then transferred to a clean media for 7 more days. The TK models assuming first-order kinetics were fitted to the data using Bayesian inference. The median model predictions and their 95% credibility intervals showed that the model fit the data well. From a methodological point of view, this paper illustrates that simultaneously estimating all model parameters from all available data by Bayesian inference, while considering the correlation between parameters and different types of data, is a real added value for TK modeling. Moreover, we demonstrated the ability of a generic TK model considering uptake and elimination routes as modules to add according to the availability of the data measured. From an ecotoxicological point of view, we show differences in PCB153 bioaccumulation between chironomids and gammarids, explained by the different life traits of these two organisms., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In Western Europe, river water quality can be assessed using sentinel species such as the amphipod Gammarus fossarum. In this work of environmental metabolomics, the objective was to develop suitable chemometrics methods, using a limited number of individuals, to assess the modification of the metabolism of G. fossarum exposed to two human pharmaceuticals. Males and females gammarids were exposed to a mixture of the anxiolytic oxazepam and the antiepileptic carbamazepine (1000 ng L -1 ) for 14 days under laboratory conditions according to a full factorial design 2² (repeated 5 times). They were analyzed at the single individual scale using a method including a μQuEChERS type extraction followed by a nanoliquid chromatography analysis coupled to high-resolution mass spectrometry. The molecular fingerprints obtained were investigated using XCMS. Several corrections of experimental drifts (by using lock mass and Quality Control samples) were tested prior to using APCA + method for the exploitation of the unbalanced designed data. Signal reproducibility was greatly improved by the lock mass normalisation. From the experimental design, a significant effect of both experimental factors "exposure to the mixture" and "gammarid gender" on the signals measured were highlighted by APCA+. Finally, the results obtained made it possible to identify variables responsible for each of the factor effects., (Copyright © 2019 Elsevier B.V. All rights reserved.)

It was assessed the efficiency of the electrocoagulation (EC) in slaughterhouse wastewater (SW) treatment by using antioxidant parameters of Gammarus pulex. The SW was treated by EC. Superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) activities and malondialdehyde (MDA) levels in G. pulex exposed to pre- and post-treated of the SW during 24 h and 96 h were analysed. Standard methods were applied during the analysing process of the physicochemical quality parameters for both untreated and treated SW. All measured physicochemical parameters were decreased following the treatment process via EC. After the treatment process, it was observed that while SOD activities and MDA levels were decreased, CAT activities were increased and GPx activities did not exhibit any change. In conclusion, the present study demonstrated the abilities of untreated SW to promote oxidative stress in model organism. The SOD, CAT activities and MDA levels in G. pulex revealed that EC process were efficient in the SW treatment.

High-throughput proteomics can be performed on animal sentinels for discovering key molecular biomarkers signing the physiological response and adaptation of organisms. Ecotoxicoproteomics is today amenable by means of proteogenomics to small arthropods such as Gammarids which are well known sentinels of aquatic environments. Here, we analysed two regional Gammarus pulex populations to characterize the potential proteome divergence induced in one site by natural bioavailable mono-metallic contamination (cadmium) compared to a non-contaminated site. Two RNAseq-derived protein sequence databases were established previously on male and female individuals sampled from the reference site. Here, individual proteomes were acquired on 10 male and 10 female paired organisms sampled from each site. Proteins involved in protein lipidation, carbohydrate metabolism, proteolysis, innate immunity, oxidative stress response and lipid transport were found more abundant in animals exposed to cadmium, while hemocyanins were found in lower abundance. The intrapopulation proteome variability of long-term exposed G. pulex was inflated relatively to the non-contaminated population. These results show that, while remaining a challenge for such organisms with not yet sequenced genomes, taking into account intrapopulation variability is important to better define the molecular players induced by toxic stress in a comparative field proteomics approach., (Copyright © 2019 Elsevier B.V. All rights reserved.)

It has been suggested that giant Antarctic marine invertebrates will be particularly vulnerable to declining O 2 levels as our ocean warms in line with current climate change predictions. Our study provides some support for this oxygen limitation hypothesis, with larger body sizes being generally more sensitive to O 2 reductions than smaller body sizes. However, it also suggests that the overall picture is a little more complex. We tested predictions from three different, but overlapping, O 2 -related hypotheses accounting for gigantism, using four Antarctic amphipod species encompassing a wide range of body sizes. We found a significant effect of body size, but also of species, in their respiratory responses to acutely declining O 2 tensions. The more active lifestyle of intermediate-sized Prostebbingia brevicornis was supported by a better respiratory performance than predicted by the oxygen limitation hypothesis alone, but consistent with the symmorphosis hypothesis. We suggest that giant polar amphipods are likely to be some of the first to fare badly in an O 2 -poor ocean. However, the products of past evolutionary innovation, such as respiratory pigments that enhance O 2 -transport and novel gas exchange structures, may in some species offset any respiratory disadvantages of either large or small body size. This article is part of the theme issue 'Physiological diversity, biodiversity patterns and global climate change: testing key hypotheses involving temperature and oxygen'.

Sublethal effects of water-accommodated fraction (WAF) from crude oil of Neuquén basin, Northern Patagonia-Argentina, were examined on both antioxidant and detoxification system of Hyalella curvispina adults collected in Los Barreales (LB) lake and in an oil-polluted stream (DS). The effects of WAF exposure during 6, 24 and 48 h were evaluated in the glutathione content (GSH) and glutathione S-transferase (GST), catalase (CAT) and cytochrome P450 (CYP450) activities. Populations from DS and LB showed not only different basal GSH content and enzyme activities but also different behavior to WAF exposure. LB population exposed to WAF showed a significant increase in GSH content, CAT and CYP450 activities, compared to control group. DS population presented high basal levels in CAT and CYP activity compared with LB population, but their response to WAF exposure was minor. Amphipods from DS, chronically exposed to hydrocarbons, were adapted to their environment., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The Tenax technique was used as an alternative exposure metric to assess the bioavailability of polychlorinated biphenyls (PCBs) from contaminated marine sediments. The sediments used were collected from 2 Superfund sites, New Bedford Harbor (MA, USA) and Gould Island (RI, USA). No sieving was conducted for either sediment after arrival, and sediments were stored in stainless steel drums at 2.8 to 4.0 °C in the dark until use. Exhaustive chemical extractions, single-point 24-h Tenax extractions, and 14-d bioaccumulation tests using the amphipod Leptocheirus plumulosus were conducted for both sediments. The sum of 119 PCB congeners from total exhaustive chemical extraction in the New Bedford Harbor and Gould Island sediments were 1084 and 188.2 µg/g organic carbon, respectively. The PCB concentrations from the bioaccumulation tests and Tenax extractions showed that both exposure metrics followed a similar trend in amount and distribution of PCB congeners. The results from both exposure metrics were fit into a log-log linear regression, and then compared with a previously developed log-log linear model for freshwater organisms. The results showed that although the marine data fell within the prediction intervals of the freshwater linear model, the marine regression followed a lower trajectory due to the differences in both the slopes and intercepts between the marine and freshwater regressions. The present study showed a strong relationship between Tenax and marine invertebrate PCB concentrations. Environ Toxicol Chem 2019;38:1188-1197. © 2019 SETAC., (© 2019 SETAC.)

Anthropogenic pressures, such as contaminant exposure, may affect stable isotope ratios in biota. These changes are driven by alterations in the nutrient allocation and metabolic pathways induced by specific stressors. In a controlled microcosm study with the amphipod Gammarus spp., we studied effects of the β-blocker propranolol on stable isotope signatures (δ15N and δ13C), elemental composition (%C and %N), and growth (protein content and body size) as well as biomarkers of oxidative status (antioxidant capacity, ORAC; lipid peroxidation, TBARS) and neurological activity (acetylcholinesterase, AChE). Based on the known effects of propranolol exposure on cellular functions, i.e., its mode of action (MOA), we expected to observe a lower scope for growth, accompanied by a decrease in protein deposition, oxidative processes and AChE inhibition, with a resulting increase in the isotopic signatures. The observed responses in growth, biochemical and elemental variables supported most of these predictions. In particular, an increase in %N was observed in the propranolol exposures, whereas both protein allocation and body size declined. Moreover, both ORAC and TBARS levels decreased with increasing propranolol concentration, with the decrease being more pronounced for TBARS, which indicates the prevalence of the antioxidative processes. These changes resulted in a significant increase of the δ15N and δ13C values in the propranolol-exposed animals compared to the control. These findings suggest that MOA of β-blockers may be used to predict sublethal effects in non-target species, including inhibited AChE activity, improved oxidative balance, and elevated stable isotope ratios. The latter also indicates that metabolism-driven responses to environmental contaminants can alter stable isotope signatures, which should be taken into account when interpreting trophic interactions in the food webs., Competing Interests: The authors have declared that no competing interests exist.

We aimed to show how the predicted pH decrease in the ocean would alter the toxicity, bioconcentration and dietary transfer of trace metal copper on seagrass ecosystems, on a short-term basis. Seagrass Zostera noltei was exposed to two pH levels (8.36 and 8.03) and three copper levels (nominal concentrations, <3, 30 and 300 μg Cu L -1 ) in a factorial design during 21 days, while Gammarus locusta amphipods were continuously fed with the treated seagrass leaves. We found that the toxicity and bioconcentration of copper in seagrasses were not affected by pH, yet complex copper-pH interactions were observed in the seagrass photosynthesis. We demostrated that seagrasses can act as a copper source in the food web via direct consumption by herbivores. Future research need to investigate the interactive effects on a long-term basis, and to include biochemical and molecular endpoints to provide additional insights to the complex phisiological interactions observed., (Copyright © 2019. Published by Elsevier Ltd.)