Toxicological effects of di-(2-ethylhexyl) phthalate in Gammarus pulex: a biochemical and histopathological assessment.

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 (LC50) 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.

Biomarkers in bivalve mollusks and amphipods for assessment of effects linked to cyanobacteria and elodea: Mesocosm study.

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.

Comparison of Alternative Methods for Bioaccumulation Assessment: Scope and Limitations of In Vitro Depletion Assays with Rainbow Trout and Bioconcentration Tests in the Freshwater Amphipod Hyalella azteca.

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.

Laboratory and Field-Based Assessment of the Effects of Sediment Capping Materials on Zinc Flux, Bioavailability, and Toxicity.

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.

Ecotoxicoproteomic assessment of the functional alterations caused by chronic metallic exposures in gammarids.

Very few ecotoxicological studies have been performed on long-term exposure under controlled conditions, hence limiting the assessment of the impact of chronic and diffuse chemical pressures on the health of aquatic organisms. In this study, an ecotoxicoproteomic approach was used to assess the integrated response and possible acclimation mechanisms in Gammarus fossarum following chronic exposures to Cd, Cu or Pb, at environmentally realistic concentrations (i.e. 0.25, 1.5 and 5 µg/L respectively). After 10-week exposure, changes in protein expression were investigated in caeca of control and exposed males. Gel-free proteomic analyses allowed for the identification of 35 proteins involved in various biological functions, for which 23 were significantly deregulated by metal exposures. The protein deregulation profiles were specific to each metal, providing evidence for metal-specific action sites and responses of gammarids. Among the tested metals, Cu was the most toxic in terms of mortality, probably linked with persistent oxidative stress. Moulting and osmoregulation were the major biological functions affected by Cu in the long-term. In Pb-exposed gammarids, significant deregulations of proteins involved in immune response and cytoskeleton were observed. Reproduction appears to be strongly affected in gammarids chronically exposed to Cd or Pb. Besides, modified expressions of several proteins involved in energy transfer and metabolism highlighted important energetic reshuffling to cope with chronic metal exposures. These results support the fact that metallic pressures induce a functional and energetic cost for individuals of G. fossarum with potential repercussions on population dynamics. Furthermore, this ecotoxicoproteomic study offers promising lines of enquiry in the development of new biomarkers that could make evidence of long-term impacts of metals on the health of organisms.

Comparison of whole animal costs of protein synthesis among polar and temperate populations of the same species of gammarid amphipod.

Protein synthesis can account for a substantial proportion of metabolic rate. Energetic costs of protein synthesis, should in theory, be the same in marine invertebrates from a range of thermal habitats, and yet direct measurements using inhibitors produce widely differing values, especially in the cold. The present study aimed to remove any potential confounding interspecific effects by determining costs of protein synthesis in two latitudinally separated populations of the same species (amphipod, Gammarus oceanicus) living in two different thermal regimes; polar vs cold-temperate. Costs of protein synthesis were determined in summer acclimatised G. oceanicus from Svalbard (79°N) at 5°C and from Scotland (58°N) at 13°C. Amphipods were injected with the protein synthesis inhibitor, cycloheximide (CHX), at 9mmoll-1 in crab saline to give a tissue concentration of 0.05mgCHXg-1FW and left for 60min before the injection of [3H] phenylalanine. After incubation for 120min (180min in total from initial injection), both whole-animal rates of oxygen uptake and absolute rates of protein synthesis were significantly reduced in CHX-treated amphipods vs controls injected with saline. Both populations exhibited similar costs of protein synthesis of ~7µmolO2mg-1protein which is close to the estimated theoretical minimum for peptide bond formation, and similar to the values obtained in cell-free systems. The study demonstrates that in G. oceanicus, costs of protein synthesis rates were not elevated in the cold but were fixed among polar and cold-temperate populations.

NMR-based metabolomics for the environmental assessment of Kaohsiung Harbor sediments exemplified by a marine amphipod (Hyalella azteca).

Inflow of wastewater from upstream causes a large flux of pollutants to enter Kaohsiung Harbor in Taiwan daily. To reveal the ecological risk posed by Kaohsiung Harbor sediments, an ecological metabolomic approach was employed to investigate environmental factors pertinent to the physiological regulation of the marine amphipod Hyalella azteca. The amphipods were exposed to sediments collected from different stream inlets of the Love River (LR), Canon River (CR), Jen-Gen River (JR), and Salt River (SR). Harbor entrance 1 (E1) was selected as a reference site. After 10-day exposure, metabolomic analysis of the Hyalella azteca revealed differences between two groups: {E1, LR, CR} and {JR, SR}. The metabolic pathways identified in the two groups of amphipods were significantly different. The results demonstrated that NMR-based metabolomics can be effectively used to characterize metabolic response related to sediment from polluted areas.

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.

Functional characterization of the oxidative capacity of mitochondria and glycolytic assessment in benthic aquatic organisms.

The metabolism of benthic aquatic invertebrates, populating transitional water ecosystems, is influenced by both physiological and environmental factors, thus involving an adjustment of physiological processes which has a metabolic cost. In order to discover changes in metabolic pathways in response to specific factors, it's firstly necessary characterizing the principal cellular metabolic activities of the small benthic aquatic organisms. We approach here the bioenergetic state issue of two benthic organisms, i.e. Lekanesphaera monodi and Gammarus insensibilis, evidencing that no apparent and statistically significative differences between them in aerobic as well in glycolytic capacities are detected, except for COX activity.

Multibiomarker assessment of cerium dioxide nanoparticle (nCeO2) sublethal effects on two freshwater invertebrates, Dreissena polymorpha and Gammarus roeseli.

Cerium nanoparticles (nCeO2) are widely used in everyday products, as fuel and paint additives. Meanwhile, very few studies on nCeO2 sublethal effects on aquatic organisms are available. We tried to fill this knowledge gap by investigating short-term effects of nCeO2 at environmentally realistic concentrations on two freshwater invertebrates; the amphipod Gammarus roeseli and the bivalve Dreissena polymorpha, using an integrated multibiomarker approach to detect early adverse effects of nCeO2 on organism biology. Differences in the behaviour of the organisms and of nanoparticles in the water column led to differential nCeO2 bioaccumulations, G. roeseli accumulating more cerium than D. polymorpha. Exposure to nCeO2 led to decreases in the size of the lysosomal system, catalase activity and lipoperoxidation in mussel digestive glands that could result from nCeO2 antioxidant properties, but also negatively impacted haemolymph ion concentrations. At the same time, no strong adverse effects of nCeO2 could be observed on G. roeseli. Further experiments will be necessary to confirm the absence of severe nCeO2 adverse effects in long-term environmentally realistic conditions.

Comparative analysis of two weight-of-evidence methodologies for integrated sediment quality assessment.

The results of sediment quality assessment by two different weight-of-evidence methodologies were compared. Both methodologies used the same dataset but as criteria and procedures were different, the results emphasized different aspects of sediment contamination. One of the methodologies integrated the data by means of a multivariate analysis and suggested bioavailability of contaminants and their spatial distribution. The other methodology, used in the dredged material management framework recently proposed in Spain, evaluated sediment toxicity in general by assigning categories. Despite the differences in the interpretation and presentation of results, the methodologies evaluated sediment risk similarly, taking into account chemical concentrations and toxicological effects. Comparison of the results of different approaches is important to define their limitations and thereby avoid implications of potential environmental impacts from different management options, as in the case of dredged material risk assessment. Consistent results of these two methodologies emphasized validity and robustness of the integrated, weight-of-evidence, approach to sediment quality assessment. Limitations of the methodologies were discussed.

Cost of reproduction. Changes in metabolism and endosulfan lethality caused by reproductive behavior in Hyalella curvispina (Crustacea: Amphipoda).

Biocides are periodically applied in agricultural activities, reaching aquatic systems and acting upon the biota. Amphipods are widely used in toxicity tests because of their sensitivity to a wide range of pollutants. In this work, we report the differential lethality of a widely used pesticide, endosulfan, on the amphipod Hyalella curvispina at two life stages and in three different adult groups, males and females separated by sex and both sexes grouped together. In addition, oxygen consumption of adult groups was determined as a way to estimate the role of behavioral activities and exposure to endosulfan in metabolism shifts. There were no differences between the LC(50) of juveniles and the adults when they were separated by sex (p>0.05). Nevertheless, the LC(50) of adults without sexual differentiation was significantly lower than the LC(50) of juveniles and adults separated by sex (p<0.05). The oxygen consumption rate was higher when adults were grouped without sexual differentiation in the control group. The exposure to low concentrations of endosulfan causes an increase in oxygen consumption in all the treatments. The sexual behavior increased the metabolism and the sensitivity to endosulfan. In future evaluations, adults grouped without sexual differentiation, which were the most sensitive group, should be included in order to mimic the environmental conditions. Using only juveniles or adults separated by sex in toxicity tests may inaccurately estimate the lethality of biocides, especially in species with constant reproductive activities.

DNA damage in caged Gammarus fossarum amphipods: a tool for freshwater genotoxicity assessment.

The aim of this study was to propose a tool for freshwater environmental genotoxicity assessment using Gammarus fossarum, a high ecologically relevant species. In a first part, gammarids were caged upstream and downstream wastewater treatment plant effluent output. The sensitivity of genotoxic responses of haemocytes, oocytes and spermatozoa was compared using the Comet assay. Spermatozoa appeared to be the most sensitive, suitable and relevant cell type for genotoxicity risk assessment. In a second part, a watershed-scale study was conducted over 2 years to evaluate the applicability of our caging procedure. The genotoxic impact of a contamination was followed, taking into account seasonal variability. DNA damage in spermatozoa exhibited low basal level and low variability in control upstream sites, providing a reliable discrimination of polluted sites. Finally, DNA damage in caged G. fossarum has been proved to be a sensitive and reproducible tool for freshwater genotoxicity assessment.

Bioavailability assessment of a contaminated field sediment from Patrick Bayou, Texas, USA: toxicity identification evaluation and equilibrium partitioning.

Contaminated sediments are commonly found in urbanized harbors. At sufficiently high contaminant levels, sediments can cause toxicity to aquatic organisms and impair benthic communities. As a result, remediation is necessary and diagnosing the cause of sediment toxicity becomes imperative. In the present study, six sediments from a highly industrialized area in Patrick Bayou (TX, USA) were subjected to initial toxicity testing with the mysid, Americamysis bahia, and the amphipod, Ampelisca abdita. All sediments were toxic to the amphipods, while sites PB4A, PB6A, and PB9 were the only sites toxic to mysids. Due to its toxicity to both test organisms, site PB6A was chosen for a marine whole sediment phase I toxicity identification evaluation (TIE). Results of the TIE found toxicity to amphipods was primarily due to nonionic organic contaminants (NOCs), rather than cationic metals or ammonia. Causes of mysid toxicity in the TIE were less clear. An assessment of metal bioavailability using equilibrium partitioning (EqP) approaches supported the results of the TIE that cationic metals were not responsible for observed toxicity in PB6A for either organism. Toxic units (TU) calculated on measured concentrations of NOCs in the sediment yielded a total TU of 1.25, indicating these contaminants are contributing to the observed sediment toxicity. Using a combination of these TIE and EqP assessment tools, this investigation was capable of identifying NOCs as the likely class of contaminants causing acute toxicity to amphipods exposed to Patrick Bayou sediment. The cause of mysid toxicity was not definitively determined, but unmeasured NOCs are suspected.

Mixture toxicity assessment of wood preservative pesticides in the freshwater amphipod Gammarus pulex (L.).

All over the world, insecticides and fungicides are used to protect wood against pathogens. To document the environmental toxicity of wood preservative mixtures, freshwater amphipods Gammarus pulex (L.) were submitted to organic pesticides given independently or in mixtures. When given independently at environmentally realistic concentrations, propiconazole and tebuconazole (triazoles fungicides) were not toxic for G. pulex, 3-iodo-2-propinyl butyl carbamate (IPBC, fungicide) was moderately toxic, and cypermethrin (pyrethroid insecticide) was extremely toxic. 96-h LC50 were, respectively, 4703, 1643, 604, and 0.09 microg L(-1). When amphipods were submitted to a mixture mimicking the composition of a commercial solution (18.2% of cypermethrin, 45.8% propiconazole, 17.2% tebuconazole, 18.8% IPBC), the overall toxicity was equal to that of the most toxic component, namely cypermethrin. But, when organisms were submitted to the real commercial mixture containing pesticides, solvents and additives, the toxic effects were markedly higher. Moreover, a third mixture with only 0.002% cypermethrin showed lethality 2.5-18-fold higher than those predicted by the commonly used models. The present results show that toxicity of wood preservative mixtures cannot be assessed starting only from the toxicities of each single component. Furthermore, the present data strongly suggest that the environmental impacts of wood preservative mixtures might be frequently underestimated.

Alterations in the energy budget of Arctic benthic species exposed to oil-related compounds.

We studied cellular energy allocation (CEA) in three Arctic benthic species (Gammarus setosus (Amphipoda), Onisimus litoralis (Amphipoda), and Liocyma fluctuosa (Bivalvia)) exposed to oil-related compounds. The CEA biomarker measures the energy budget of organisms by biochemically assessing changes in energy available (carbohydrates, protein and lipid content) and the integrated energy consumption (electron transport system activity (ETS) as the cellular aspect of respiration). Energy budget was measured in organisms subjected to water-accommodated fraction (WAF) of crude oil and drill cuttings (DC) to evaluate whether these compounds affect the energy metabolism of the test species. We observed significantly lower CEA values and higher ETS activity in G. setosus subjected to WAF treatment compared to controls (por=0.19). Different responses to oil-related compounds between the three test species are likely the result of differences in feeding and burrowing behavior and species-specific sensitivity to petroleum-related compounds.

Assessment of sediment toxicity during anaerobic biodegradation of vegetable oil using Microtox and Hyalella azteca bioassays.

The potential ecological impacts of anaerobic degradation of vegetable oil on freshwater sediments were investigated. Sediment toxicity was evaluated using two regulatory biotests: the Microtox Solid Phase Test and an amphipod (Hyalella azteca) bioassay. The results of the Microtox test showed that the toxicity of the vegetable-oil-contaminated sediments (about 17-33 g oil/kg dry sediments) increased after 2 weeks incubation and then decreased to near background levels after incubation for 8 weeks under anaerobic conditions. The amphipod toxicity bioassay showed that the toxicity of fresh contaminated sediments decreased over time and returned to background levels within 8 weeks. These results suggest that the impact of vegetable oils on organisms within sediments may be limited. To account for the significance of environmental conditions, additional studies over a wide range of incubation conditions (e.g., temperature, nutrient concentration) and other test organisms at various trophic levels are recommended for both acute and chronic toxicity assessment.

Multi-level assessment of chronic toxicity of estuarine sediments with the amphipod Gammarus locusta: I. Biochemical endpoints.

We report on biomarker responses conducted as part of a multi-level assessment of the chronic toxicity of estuarine sediments to the amphipod Gammarus locusta. A companion article accounts for organism and population-level effects. Five moderately contaminated sediments from two Portuguese estuaries, Sado and Tagus, were assessed. Three of them were muddy and two were sandy sediments. The objective was to assess sediments that were not acutely toxic. Three of the sediments met this criterion, the other two were diluted (50% and 75%) with clean sediment until acute toxicity was absent. Following 28-d exposures, the amphipods were analysed for whole-body metal bioaccumulation, metallothionein induction (MT), DNA strand breakage (SB) and lipid peroxidation (LP). Two of the muddy sediments did not cause chronic toxicity. These findings were consistent with responses at organism and population levels that showed higher growth rates and improvement of reproductive traits for amphipods exposed to these two sediments. Two other sediments, one muddy and one sandy, exhibited pronounced chronic toxicity, affecting SB, MT induction (in muddy sediment), survival and reproduction. Potential toxicants involved in these effects were identified. The last sandy sediment exhibited some loss of DNA integrity, however growth was also enhanced. Present results, together with the organism/population-level data, and also benthic communities information, were analysed under a weight-of-evidence approach. By providing evidence of exposure (or lack of it) to contaminants in sediments, the biomarkers here applied assisted in distinguishing toxicants' impacts in test organisms from the confounding influence of other geochemical features of the sediments.