Freshwater Mussels as Sentinels for Safe Drinking Water Supply in Europe.

In the context of the European Union (EU) Drinking Water Directive, freshwater mussels (Order Unionoida: Bivalvia) can help us face the challenges of safe drinking water provisions for all citizens in the EU. Specifically, the implementation of high frequency noninvasive (HFNI) valvometers allows the early detection of eventual pollution events in drinking water treatment plants. Currently real-time behavioral analysis is deployed in a number of EU countries, and we foresee a bright future as new technological advances are developed concerning HFNI valvometers.

Pollution of the Niger Delta with total petroleum hydrocarbons, heavy metals and nutrients in relation to seasonal dynamics.

The African Niger Delta is among the world's most important wetlands in which the ecological effects of intensive oil exploitation and global change are not well documented. We characterized the seasonal dynamics and pollution with total-petroleum-hydrocarbons (TPHs), heavy-metals (HMs) and nutrient-loads in relation to climate-driven variables. High TPH concentrations up to 889 mg/L and HMs up to 13.119 mg/L were found in water samples, with pronounced spatio-temporal variation throughout the year. HM pollution index and contamination factor indicate serious ecological and human health hazards, especially for Cd, Cu, Hg, and Ni. Significant differences in TPHs/HMs were observed between sites and seasons, with correlations between TPHs-HMs, and climate-variables and TPHs-HMs. Nutrient levels, turbidity, salinity, temperature, and SO42- were high and interlinked with the variability of TPHs/HMs being greatest during wet season. These findings suggest an urgent need for improved pollution control in the Niger Delta taking into account the observed spatio-temporal variation and the exacerbation of effects in light of climate change. Given the high levels of contamination, further assessments of exposure effects and bioaccumulation in biota should include future climate change scenarios and effects on humans who intensively depend on the system for drinking water, food supply and livelihood.

Moving Toward Standardized Toxicity Testing Procedures with Particulates by Dietary Exposure of Gammarids.

Ecotoxicological effect assessment of particulate materials and sparingly soluble substances is an emerging field. Current standard toxicity tests of aquatic organisms are based on soluble substances which are added to the aqueous phase. Although soluble substances distribute homogeneously, particles can form aggregates, resulting in inhomogeneous distribution and unpredictable exposure. Therefore, test scenarios need to be adapted to overcome these uncertainties. We present a dietary particle exposure tool for the toxicity testing of sparingly soluble substances or particles in combination with a standardizable food source for gammarids based on decomposition and consumption tablets (DECOTABs). Four food supplements in the DEOCOTAB formulation were compared to test their influence on the energy reserves of gammarids. Although feeding rate was constant for most supplements, mortality and energy reserves revealed clear differences. Tabs supplemented with algae-based phyll or animal protein-based trout food best met all of the requirements. Fluorescent plastic microparticles (10-65 µm) were homogenously distributed and stable in the DECOTABs. Constant feeding was observed, and the number of ingested microparticles by Gammarus roeseli was quantified in relation to the consumed food. The developed method provides a realistic and methodologically reliable uptake from the oral pathway and allows the quantification of inner exposition via feeding rate, providing a promising tool for standardized dietary exposure scenarios with particles. Environ Toxicol Chem 2021;40:1463-1476. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Modulation of PAH toxicity on the freshwater organism G. roeseli by microparticles.

Polycyclic aromatic hydrocarbons are widespread and environmentally persistent chemicals that readily bind to particles in air, soil and sediment. Plastic particles, which are also an ubiquitous global contamination problem, may thus modulate their environmental fate and ecotoxicity. First, the acute aqueous toxicity of phenanthrene in adult Gammarus roeseli was determined with a LC50 of 471 µg/L after 24 h and 441 µg/L after 48 h. Second, considering lethal and sublethal endpoints, effects of phenanthrene concentration on G. roeseli were assessed in relation to the presence of anthropogenic and natural particles. The exposure of gammarids in presence of either particle type with phenanthrene resulted after 24 and 48 h in reduced effect size. Particle exposure alone did not result in any effects. The observed reduction of phenanthrene toxicity by polyamide contradicts the discussion of microplastics acting as a vector or synergistically. Especially, no difference in modulation by plastic particles and naturally occurring sediment particles was measured. These findings can most likely be explained by the similar adsorption of phenanthrene to both particle types resulting in reduced bioavailability.

Does environmental stress affect cortisol biodistribution in freshwater mussels?

As of today, regulation and physiological purpose of steroid hormones in invertebrates such as mussels are not completely understood. Many studies were able to show their presence, but their origin and genesis are not clear. Nevertheless, knowledge about changes in steroid hormone biodistribution in reaction to treatments could improve our understanding of their physiological functions in these species. Cortisol is a corticosteroid, which is frequently used as a stress biomarker in vertebrates, like fish or higher organisms. The aim of the study was to optimize cortisol extraction from various tissues of mussels, to develop a quantitative ELISA test system, and to study changes in biodistribution of cortisol in reaction to negative and positive stimulation treatments. As model organism, we used Anodonta anatina, a widespread freshwater mussel species native to Europe. We quantified cortisol concentrations in hepatopancreas, mantle, gills, gonads and the foot muscle. Tissue-specific reactions to environmental influences, simulated with the chemical stressors copper (II) chloride and sodium chloride, were assessed. During the 24-hours treatment, we additionally observed changes in cortisol regulation in response to feeding activity of the mussels. Besides, we found highly significant variations in the biodistribution of cortisol in different tissues, with a peak in the hepatopancreas. Whole body cortisol did not increase in the treated groups. However, balancing of all measured tissues showed redistribution of more than 10% of total body cortisol from the hepatopancreas to all other tissues during copper (II) chloride stressor treatment, but also when mussels ingested feed, compared to the non-fed control group. No redistribution was observed during sodium chloride treatment. We conclude that there can be a redistribution of cortisol in mussels, depending on external influences.

Exposure of zebra mussels to extracorporeal shock waves demonstrates formation of new mineralized tissue inside and outside the focus zone.

The success rate of extracorporeal shock wave therapy (ESWT) for fracture nonunions in human medicine (i.e. radiographic union at 6 months after ESWT) is only approximately 75%. Detailed knowledge regarding the underlying mechanisms that induce bio-calcification after ESWT is limited. We analyzed the biological response within mineralized tissue of a new invertebrate model organism, the zebra mussel Dreissena polymorpha, after exposure with extracorporeal shock waves (ESWs). Mussels were exposed to ESWs with positive energy density of 0.4 mJ/mm2 (A) or were sham exposed (B). Detection of newly calcified tissue was performed by exposing the mussels to fluorescent markers. Two weeks later, the A-mussels showed a higher mean fluorescence signal intensity within the shell zone than the B-mussels (P<0.05). Acoustic measurements revealed that the increased mean fluorescence signal intensity within the shell of the A-mussels was independent of the size and position of the focal point of the ESWs. These data demonstrate that induction of bio-calcification after ESWT may not be restricted to the region of direct energy transfer of ESWs into calcified tissue. The results of the present study are of relevance for better understanding of the molecular and cellular mechanisms that induce formation of new mineralized tissue after ESWT.

A systematic approach to evaluate the influence of environmental conditions on eDNA detection success in aquatic ecosystems.

The use of environmental DNA (eDNA) to determine the presence and distribution of aquatic organisms has become an important tool to monitor and investigate freshwater communities. The successful application of this method in the field, however, is dependent on the effectiveness of positive DNA verification, which is influenced by site-specific environmental parameters. Factors affecting eDNA concentrations in aquatic ecosystems include flow conditions, and the presence of substances that possess DNA-binding properties or inhibitory effects. In this study we investigated the influence of different environmental parameters on the detection success of eDNA using the invasive goby Neogobius melanostomus. In a standardized laboratory setup, different conditions of flow, sediment-properties, and fish density were compared, as well as different potential natural inhibitors such as algae, humic substances, and suspended sediment particles. The presence of sediment was mainly responsible for lower eDNA detection in the water samples, regardless of flow-through or standing water conditions and a delayed release of eDNA was detected in the presence of sediment. Humic substances had the highest inhibitory effect on eDNA detection followed by algae and siliceous sediment particles. The results of our study highlight that a successful application of eDNA methods in field surveys strongly depends on site-specific conditions, such as water flow conditions, sediment composition, and suspended particles. All these factors should be carefully considered when sampling, analyzing, and interpreting eDNA detection results.

Miniature circulatory systems: A new exposure system for ecotoxicological effect assessments in riverine organisms.

Long-term effect assessments in ecotoxicological investigations are important, yet there is a lack of suitable exposure systems for these experiments that can be used for riverine species. A cost-efficient miniature circulatory system was developed that was evaluated for its applicability in long-term exposures in 2 stream-dwelling species: brown trout (Salmo trutta) and an amphipod (Gammarus roeseli). In an egg-to-fry exposure of S. trutta, the toxicity of 2 reverse osmosis concentrates was investigated as examples. Control hatching rate of yolk sac fry was 75 ± 7% and thus complies with the Organisation for Economic Co-operation and Development validity criterion (≥66%). The reverse osmosis concentrates did not impair the hatching rate in any tested concentration. In G. roeseli, mortality rates remained below 20% during a 21-d cultivation, fulfilling the common validity criterion in ecotoxicological testing. Mortality was significantly lower when the species was fed with conditioned alder leaves instead of an artificial shrimp food. Finally, a toxicity test on G. roeseli using copper as the test substance revealed median lethal concentration (LC50) values of 156 µg/L after 96 h and 99 µg/L after 264 h, which is in line with literature findings using other accepted exposure units. In conclusion, the miniature circulatory system provides a novel and cost-efficient exposure system for long-term investigations on riverine species that may also be applicable for other species of fishes and macroinvertebrates. Environ Toxicol Chem 2016;35:2827-2833. © 2016 SETAC.

Establishing mussel behavior as a biomarker in ecotoxicology.

Most freshwater mussel species of the Unionoida are endangered, presenting a conservation issue as they are keystone species providing essential services for aquatic ecosystems. As filter feeders with limited mobility, mussels are highly susceptible to water pollution. Despite their exposure risk, mussels are underrepresented in standard ecotoxicological methods. This study aimed to demonstrate that mussel behavioral response to a chemical stressor is a suitable biomarker for the advancement of ecotoxicology methods that aids mussel conservation. Modern software and Hall sensor technology enabled mussel filtration behavior to be monitored real-time at very high resolution. With this technology, we present our method using Anodonta anatina and record their response to de-icing salt pollution. The experiment involved an environmentally relevant 'pulse-exposure' design simulating three subsequent inflow events. Three sublethal endpoints were investigated, Filtration Activity, Transition Frequency (number of changes from opened to closed, or vice versa) and Avoidance Behavior. The mussels presented a high variation in filtration behavior, behaving asynchronously. At environmentally relevant de-icing salt exposure scenarios, A. anatina behavior patterns were significantly affected. Treated mussels' Filtration Activity decreased during periods of very high and long de-icing salt exposure (p<0.001), however, increased during short de-icing salt exposure. Treated mussels' Transition Frequency increased during periods of very high and long de-icing salt exposure (p<0.001), which mirrored the Avoidance Behavior endpoint observed only by mussels under chemical stress. Characteristics of Avoidance Behavior were tighter shell closures with repeated and irregular shell movements which was significantly different to their undisturbed resting behavior (p<0.001). Additionally, we found that mussels were sensitive to a chemical stressor even when the mussel's valves were closed. Due to the effects of de-icing salt pollution on freshwater mussel behavior, we suggest better management practices for de-icing salt use be implemented. Our experimental method demonstrated that, with the application of current technologies, mussel behavioral response to a chemical stressor can be measured. The tested sublethal endpoints are suitable for mussel ecotoxicology studies. Avoidance Behavior proved to be a potentially suitable endpoint for calculating mussel behavior effect concentration. Therefore we recommend adult mussel behavior as a suitable biomarker for future ecotoxicological research. This method could be applied to other bivalve species and for physical and environmental stressors, such as particulate matter and temperature.

Acute effects of salinity exposure on glochidia viability and host infection of the freshwater mussel Anodonta anatina (Linnaeus, 1758).

Freshwater mussels of the Unionida are globally in decline and knowledge of the sensitivity of their vulnerable life stages to stressors is crucial for their conservation. Increasing salinity, e.g., due to road deicing, has been proposed as an important stressor, but its impact on the complex interaction between mussel larvae and their obligate attachment to fish hosts remains largely unknown. This study tested the acute toxicity effects of environmentally relevant chloride concentrations on larvae of European Anodonta anatina mussel as well as the impacts on attachment rates of the larvae to their fish host. Chloride concentrations above 727 mg⋅L(-1) significantly affected glochidia viability and the 24h EC50 value was determined at 2,505 mg⋅L(-1). Successful attachment of glochidia to the host fish Phoxinus phoxinus was negatively correlated with increasing chloride concentration and became significant at concentrations > 2,909 mg⋅L(-1). Comparable responses could be observed by separately counting fin and gill attached glochidia, while gill attachment showed the highest correlation with overall attachment rates. These results indicate a potential threat from short-term elevated chloride concentrations during runoff events on sensitive life stages of freshwater mussels. Consequently, we propose additional chloride sensitivity tests on other mussel species as well as the reduction of salt peak input loading into freshwater bodies through a 3R-principle (restriction of use, retention of runoff for peak concentration avoidance and replacement by alternatives) in areas where endangered mussels occur. CAPSULE: Freshwater mussels of the Unionoida are globally in decline and knowledge on the sensitivity of the most vulnerable larval stages to salinity is crucial for their conservation.

Increased RO concentrate toxicity following application of antiscalants - acute toxicity tests with the amphipods Gammarus pulex and Gammarus roeseli.

In reverse osmosis, a frequently used technology in water desalination processes, wastewater (RO concentrate) is generated containing the retained solutes as well as so-called antiscalants (AS), i.e. chemical substances that are commonly applied to prevent membrane-blocking. In this study, a risk assessment of a possible discharge of concentrate into a small stream was conducted. The acute toxicity of two concentrates containing two different ASs and of concentrate without AS to the amphipods Gammarus pulex and Gammarus roeseli was studied. Mortality of gammarids exposed to the concentrate without AS was not different to the control, whereas concentrates including ASs caused mortality rates up to 100% at the highest test concentrations after 168 h. Resulting EC50-values were 36.2-39.4% (v/v) after 96 h and 26.6-58.0% (v/v) after 168 h. These results suggest that the ecotoxicological relevance of antiscalants is greater than currently assumed.

Triclosan impairs swimming behavior and alters expression of excitation-contraction coupling proteins in fathead minnow (Pimephales promelas).

Triclosan (TCS), a high volume chemical widely used in consumer products, is a known aquatic contaminant found in fish inhabiting polluted watersheds. Mammalian studies have recently demonstrated that TCS disrupts signaling between the ryanodine receptor (RyR) and the dihydropyridine receptor (DHPR), two proteins essential for excitation-contraction (EC) coupling in striated muscle. We investigated the swimming behavior and expression of EC coupling proteins in larval fathead minnows (Pimephales promelas) exposed to TCS for up to 7 days. Concentrations as low as 75 µg L(-1) significantly altered fish swimming activity after 1 day; which was consistent after 7 days of exposure. The mRNA transcription and protein levels of RyR and DHPR (subunit CaV1.1) isoforms changed in a dose and time dependent manner. Crude muscle homogenates from exposed larvae did not display any apparent changes in receptor affinity toward known radioligands. In nonexposed crude muscle homogenates, TCS decreased the binding of [(3)H]PN20-110 to the DHPR and decreased the binding of [(3)H]-ryanodine to the RyR, demonstrating a direct impact at the receptor level. These results support TCS's impact on muscle function in vertebrates further exemplifying the need to re-evaluate the risks this pollutant poses to aquatic environments.

Impacts of the phenylpyrazole insecticide fipronil on larval fish: time-series gene transcription responses in fathead minnow (Pimephales promelas) following short-term exposure.

The utilization of molecular endpoints in ecotoxicology can provide rapid and valuable information on immediate organismal responses to chemical stressors and is increasingly used for mechanistic interpretation of effects at higher levels of biological organization. This study contributes knowledge on the sublethal effects of a commonly used insecticide, the phenylpyrazole fipronil, on larval fathead minnow (Pimephales promelas), utilizing a quantitative transcriptomic approach. Immediately after 24h of exposure to fipronil concentrations of ≥31 µg.L(-1), highly significant changes in gene transcription were observed for aspartoacylase, metallothionein, glucocorticoid receptor, cytochrome P450 3A126 and vitellogenin. Different mechanisms of toxicity were apparent over the course of the experiment, with short-term responses indicating neurotoxic effects. After 6 days of recovery, endocrine effects were observed with vitellogenin being up-regulated 90-fold at 61 µg.L(-1) fipronil. Principal component analysis demonstrated a significant increase in gene transcription changes over time and during the recovery period. In conclusion, multiple mechanisms of action were observed in response to fipronil exposure, and unknown delayed effects would have been missed if transcriptomic responses had only been measured at a single time-point. These challenges can be overcome by the inclusion of multiple endpoints and delayed effects in experimental designs.

Changes in gene transcription and whole organism responses in larval fathead minnow (Pimephales promelas) following short-term exposure to the synthetic pyrethroid bifenthrin.

The combination of molecular and whole-organism endpoints in ecotoxicology provides valuable information about the ecological relevance of sublethal stressor effects in aquatic ecosystems such as those caused by the use of insecticides and translocation of their residues into surface waters. This study contributes knowledge about the sublethal effects of a common use insecticide, the synthetic pyrethroid bifenthrin, on larval fathead minnow (Pimephales promelas). Transcriptomic responses, assessed by quantitative real-time PCR, combined with individual effects on swimming performance were used to estimate the ecological relevance of insecticide impacts. Significant transcriptomic responses were observed at 0.07 µg L(-1) bifenthrin (lowest observed effect concentration, LOEC) but mostly followed a biphasic rather than a linear dose-response with increasing concentration. Transcript patterns for genes involved in detoxification, neuromuscular function and energy metabolism were linked to an impairment of swimming performance at ≥0.14 µg L(-1) bifenthrin. With increasing treatment concentration, a significant down-regulation was observed for genes coding for cyp3a, aspartoacylase, and creatine kinase, whereas metallothionein was up-regulated. Additionally, bifenthrin induced endocrine responses as evident from a significant up-regulation of vitellogenin and down-regulation of insuline-like growth factor transcripts. Recovery occurred after 6 days and was dependent on the magnitude of the initial stress. During the recovery period, down-regulation of vitellogenin was observed at lowest exposure concentrations. The data presented here emphasize that links can be made between gene transcription changes and behavioral responses which is of great value for the evaluation and interpretation of biomarker responses.

Linking molecular biomarkers with higher level condition indicators to identify effects of copper exposures on the endangered delta smelt (Hypomesus transpacificus).

The delta smelt (Hypomesus transpacificus) is an endangered pelagic fish species endemic to the Sacramento-San Joaquin estuary (CA, USA), and considered an indicator of ecosystem health. Copper is a contaminant of concern in Californian waterways that may affect the development and survival of this endangered species. The experimental combination of molecular biomarkers with higher level effects may allow for interpretation of responses in a functional context that can be used to predict detrimental outcomes caused by exposure. A delta smelt microarray was developed and applied to screen for candidate molecular biomarkers that may be used in monitoring programs. Functional classifications of microarray responses were used along with quantitative polymerase chain reaction determining effects upon neuromuscular, digestive, and immune responses in Cu-exposed delta smelt. Differences in sensitivity were measured between juveniles and larvae (median lethal concentration = 25.2 and 80.4 µg/L Cu(2+), respectively). Swimming velocity declined with higher exposure concentrations in a dose-dependent manner (r = -0.911, p < 0.05), though was not statistically significant to controls. Genes encoding for aspartoacylase, hemopexin, α-actin, and calcium regulation proteins were significantly affected by exposure and were functionally interpreted with measured swimming responses. Effects on digestion were measured by upregulation of chitinase and downregulation of amylase, whereas downregulation of tumor necrosis factor indicated a probable compromised immune system. Results from this study, and many others, support the use of functionally characterized molecular biomarkers to assess effects of contaminants in field scenarios. We thus propose that to attribute environmental relevance to molecular biomarkers, research should concentrate on their application in field studies with the aim of assisting monitoring programs.

Sublethal toxicity of commercial insecticide formulations and their active ingredients to larval fathead minnow (Pimephales promelas).

Toxic effect concentrations of insecticides are generally determined using the technical grade or pure active ingredient. Commercial insecticide formulations, however, contain a significant proportion (>90%) of so-called inert ingredients, which may alter the toxicity of the active ingredient(s). This study compares the sublethal toxicity of two insecticides, the pyrethroid bifenthrin, and the phenylpyrazole fipronil, to their commercial formulations, Talstar and Termidor. Both insecticides are used for landscape treatment and structural pest control, and can be transported into surface water bodies via stormwater and irrigation runoff. We used larval fathead minnow (Pimephales promelas), to determine effects on growth and swimming performance after short-term (24h) exposure to sublethal concentrations of pure insecticides and the respective formulations. Significantly enhanced 7d growth was observed at 10% of the 24h LC(10) (53 microg L(-)(1)) fipronil. Swimming performance was significantly impaired at 20% of the 24h LC(10) (0.14 microg L(-)(1)) of bifenthrin and 10% of the 24h LC(10) of Talstar (0.03 microgL(-)(1)). Fipronil and Termidor led to a significant impairment of swimming performance at 142 microgL(-)(1) and 148 microgL(-)(1) respectively, with more pronounced effects for the formulation. Our data shows that based on dissolved concentrations both formulations were more toxic than the pure active ingredients, suggesting that increased toxicity due to inert ingredients should be considered in risk assessments and regulation of insecticides.