Nature-based solutions (NbS): A management paradigm shift in practitioners' perspectives on riverbank soil bioengineering.

Nature-based Solutions (NbS) are promoted as practical and theoretical solutions that simultaneously provide human well-being and biodiversity benefits. One example is soil bioengineering using construction techniques based on living vegetation, and is frequently used for riverbank stabilization, flood protection, and erosion control. Compared with civil engineering, NbS offer many advantages such as cost reduction, limited impact on the environment, and production of ecosystem services. However, their use is still marginal for riverbank control, especially in urban areas. In this paper, we focus on soil bioengineering techniques for riverbank protection in an urban context from the practitioners' perspective. We question to what extent NbS require a shift in management paradigm. We used qualitative methods to interview 17 practitioners working in the Rhone Alps basin (France). Our results reveal that switching from civil engineering to soil bioengineering is not only a technical change, but also requires a shift from a "predict and control" paradigm to an "adaptive management" paradigm because of three major reasons. First, soil bioengineering techniques require redefinition of the performance of engineering structures with the inclusion of ecological and social dimensions. Second, the adoption of soil bioengineering techniques requires that practitioners, elected people and inhabitants reconsider risk sharing and acceptance. Third, the techniques require practitioners to adopt a new posture, with new soft skills (humility and daring) and a new collective organization (collective feedback). Finally, we identify three levers for a broader use of such techniques: (i) systematic assessment of the ecological, economical, and social benefits of such techniques; (ii) improving risk acceptance and sharing; (iii) fostering of social learning among practitioners through collective or technical feedback.

Multisubstance Indicators Based on Caged Gammarus Bioaccumulation Reveal the Influence of Chemical Contamination on Stream Macroinvertebrate Abundances across France.

Most anthropogenic stressors affecting freshwater systems are qualitatively known. However, the quantitative assessment of contaminant exposure and effects to aquatic communities is still difficult, limiting the understanding of consequences on aquatic ecosystem functioning and the implementation of effective management plans. Here, multisubstance indicators based on caged gammarid bioaccumulated contamination data are proposed (for metals and persistent organic pollutants, POPs) to map the bioavailable contamination level of freshwater ecosystems at a large spatial scale. We assessed the ability of these indicators to highlight the relationships between chemical exposure gradients and alteration in the abundance of macroinvertebrate populations on a data set of 218 watercourses distributed throughout France. We identified spatial regional heterogeneities in the levels of bioavailable contamination of metals (18 compounds) and POPs (43 compounds). Besides this, a degradation of Gammaridae, Ephemeridae, and Hydrobiidae densities with increasing levels of metal contamination are identified relative to Baetidae, Chironomidae, and Hydropsychidae. We show here that active biomonitoring allows the establishment of multisubstance indicators of bioavailable contamination, which reliably quantify chemical exposure gradients in freshwater ecosystems. Our ability to identify species-specific responses to chemical exposure gradients demonstrates the promising possibility to further decipher the effects of chemical contamination on macroinvertebrate assemblages through this type of indicator.

Ecotoxico-Proteomics for Aquatic Environmental Monitoring: First in Situ Application of a New Proteomics-Based Multibiomarker Assay Using Caged Amphipods.

As a proof of principle, a selected reaction monitoring (SRM) mass spectrometry-based methodology was applied to the simultaneous quantification of dozens of protein biomarkers in caged amphipods (Gammarus fossarum). We evaluated the suitability of the methodology to assess complex field contaminations through its application in the framework of a regional river monitoring network. Thanks to the high throughput acquisition of biomarker levels in G. fossarum exposed in four reference and 13 contaminated sites, we analyzed the individual responses of 38 peptides reporting for 25 proteins of interest in 170 organisms. Responses obtained in contaminated sites included inductions of vitellogenin-like proteins in male organisms, inductions of Na+K+/ATPases, and strong inhibitions of molt-related proteins such as chitinase and JHE-carboxylesterase. Proteins from detoxification and immunity processes were also found modulated in abundance. Summarizing, the results presented here show that the SRM strategy developed for multibiomarker measurement paves a very promising way to define multiple indicators of the health status of sentinel organisms for environmental hazard assessment.

Bioaccumulation of Toxoplasma and Cryptosporidium by the freshwater crustacean Gammarus fossarum: Involvement in biomonitoring surveys and trophic transfer.

The protozoa Toxoplasma gondii and Cryptosporidium parvum are public health priorities because their oocysts can persist in recreational, surface, drinking, river, and sea water sources for a long time. To evaluate the capacity of the freshwater crustacean Gammarus fossarum to accumulate T. gondii and C. parvum oocysts, gammarids were exposed to 200, 2000 or 20,000 oocysts per gammarid and per day for 21 days followed by 5 days of depuration. C. parvum DNA was detected by qPCR in G. fossarum in only one out of four pools for the highest concentration and after 14 days of exposure, and T. gondii DNA was detected after 7 days of exposure to the two highest concentrations. Our results document the capacity of G. fossarum to accumulate T. gondii in its tissues proportionally to the ambient concentration; the maximum number of oocysts was detected in gammarid tissues after exposure to 20,000 oocysts per day. Mean values of 3.26 (±3), 21.71 (±15.18), and 17.41 (±10.89) oocysts were detected in gammarids after 7, 14, and 21 days, respectively, and after 5 days of depuration, T. gondii oocysts were still present in gammarid tissues. These results show for the first time that a freshwater crustacean can bioaccumulate T. gondii oocysts, suggesting that G. fossarum is a potential effective bioindicator of protozoan contamination in biomonitoring studies. Moreover, due to its key position in freshwater food webs, G. fossarum could also play a role in the trophic transfer of protozoa.

Identifying the impact of toxicity on stream macroinvertebrate communities in a multi-stressor context based on national ecological and ecotoxicological monitoring databases.

In situ bioassays are used to measure the harmful effects induced by mixtures of toxic chemicals in watercourses. In France, national-scale biomonitoring data are available including invertebrate surveys and in-field chemical toxicity measures with caged gammarids to assess environmental toxicity of mixtures of chemicals. The main objective of our study is to present a proof-of-concept approach identifying possible links between in-field chemical toxicity, stressors and the ecological status. We used two active biomonitoring databases comprising lethal toxicity (222 in situ measures of gammarid mortality) and sublethal toxicity (101 in situ measures of feeding inhibition). We measured the ecological status of each active biomonitoring site using the I2M2 metric (macroinvertebrate-based multimetric index), accounted for known stressors of nutrients and organic matter, hydromorphology and chemical toxicity. We observed a negative relationship between stressors (hydromorphology, nutrients and organic matter, and chemical toxicity) and the good ecological status. This relationship was aggravated in watercourses where toxicity indicators were degraded. We validated this hypothesis for instance with nutrients and organic matter like nitrates or hydromorphological conditions like percentage of vegetation on banks. Future international assesments concerning the role of in-field toxic pollution on the ecological status in a multi-stressor context are now possible via the current methodology.

Interest of a multispecies approach in active biomonitoring: Application in the Meuse watershed.

A biomonitoring approach based on a single model species cannot be representative of the contaminations impacts on the ecosystem overall. As part of the Interreg DIADeM program ("Development of an integrated approach for the diagnosis of the water quality of the River Meuse"), a study was conducted to establish the proof of concept that the use of a multispecies active biomonitoring approach improves diagnostic of aquatic systems. The complementarity of the biomarker responses was tested in four model species belonging to various ecological compartments: the bryophyte Fontinalis antipyretica, the bivalve Dreissena polymorpha, the amphipod Gammarus fossarum and the fish Gasterosteus aculeatus. The species have been caged upstream and downstream from five wastewater treatment plants (WWTPs) in the Meuse watershed. After the exposure, a battery of biomarkers was measured and results were compiled in an Integrated Biomarker Response (IBR) for each species. A multispecies IBR value was then proposed to assess the quality of the receiving environment upstream the WWTPs. The effluent toxicity was variable according to the caged species and the WWTP. However, the calculated IBR were high for all species and upstream sites, suggesting that the water quality was already downgraded upstream the WWTP. This contamination of the receiving environment was confirmed by the multispecies IBR which has allowed to rank the rivers from the less to the most contaminated. This study has demonstrated the interest of the IBR in the assessment of biological impacts of a point-source contamination (WWTP effluent) but also of the receiving environment, thanks to the use of independent references. Moreover, this study has highlighted the complementarity between the different species and has emphasized the interest of this multispecies approach to consider the variability of the species exposition pathway and sensibility as well as the mechanism of contaminants toxicity in the final diagnosis.

In Situ Reproductive Bioassay with Caged Gammarus fossarum (Crustacea): Part 2-Evaluating the Relevance of Using a Molt Cycle Temperature-Dependent Model as a Reference to Assess Toxicity in Freshwater Monitoring.

Active biomonitoring approaches are now recognized as relevant for monitoring water contamination and toxicity. Nevertheless, due to the confounding influence of variable and uncontrolled environmental conditions such as temperature, biological markers measured on transplanted individuals to assess water quality are difficult to interpret. The purpose of the present study is to propose a methodology for adapting a laboratory test of chronic sublethal toxicity based on the molting cycle of Gammarus fossarum to in situ assays. To this end, we 1) adapted the molt cycle temperature-dependent model developed in Part 1 (Chaumot et al. 2020, this issue) to the fluctuating temperatures measured in the field; 2) assessed the predictive power of our approach as a "reference value" from gammarids caged in 9 nonimpacted sites at different seasons; and 3) tested the relevance of our tool to interpret in situ reproductive bioassays from 5 upstream/downstream studies and a large-scale deployment in 12 sites. Our approach based on modeling the progress of gammarid molting cycle as a function of temperature appeared to be a relevant and robust tool for interpreting in situ observations in different environmental contexts in time and space. By avoiding using a "reference" or upstream situation as a baseline from which water quality could be assessed, this approach provides a real added value to water quality diagnosis in biomonitoring programs. Environ Toxicol Chem 2020;39:678-691. © 2019 SETAC.

How to quantify the links between bioavailable contamination in watercourses and pressures of anthropogenic land cover, contamination sources and hydromorphology at multiple scales?

Active biomonitoring permits the quantification of biological exposure to chemicals through measurements of bioavailable concentrations in biota and biological markers of toxicity in organisms. It enables respective comparison of the levels of contamination between sites and sampling campaigns. Caged gammarids are recently proposed as relevant probes for measuring bioavailable contamination in freshwater systems. The purpose of the present study was to develop a multi-pressure and multiscale approach, considering metallic contamination levels (from data based on active biomonitoring) as a response to pressures (combination of individual stressors). These pressures were anthropogenic land cover, industry density, wastewater treatment plant density, pressures on stream hydromorphological functioning, riverside vegetation and bioavailability factors. A dataset combining active biomonitoring and potentially related pressures was established at the French national scale, with 196 samplings from 2009 to 2016. The links between pressures and metallic contamination were defined and modelled via structural equation modeling (more specifically partial least squares - path modeling). The model enabled the understanding of the respective influences of pressures on metallic bioconcentration in caged sentinel organisms. Beyond validating the local influence of industries and wastewater treatment plants on metallic contamination, this model showed a complementary effect of driving forces of anthropogenic land cover (leading to human activities). It also quantified a significant influence of pressures on stream hydromorphological functioning, presence of vegetation and physico-chemical parameters on metal bioconcentration. This hierarchical multi-pressure approach could serve as a concept on how pressures and contamination (assessed by active biomonitoring) can be connected. Its future application will enable better understanding of environmental pressures leading to contamination in freshwater ecosystems.

High-multiplexed monitoring of protein biomarkers in the sentinel Gammarus fossarum by targeted scout-MRM assay, a new vision for ecotoxicoproteomics.

Ecotoxicoproteomics employs mass spectrometry-based approaches centered on proteins of sentinel organisms to assess for instance, chemical toxicity in fresh water. In this study, we combined proteogenomics experiments and a novel targeted proteomics approach free from retention time scheduling called Scout-MRM. This methodology will enable the measurement of simultaneously changes in the relative abundance of multiple proteins involved in key physiological processes and potentially impacted by contaminants in the freshwater sentinel Gammarus fossarum. The development and validation of the assay were performed to target 157 protein biomarkers of this non-model organism. We carefully chose and validated the transitions to monitor using conventional parameters (linearity, repeatability, LOD, LOQ). Finally, the potential of the methodology is illustrated by measuring 277-peptide-plex assay (831 transitions) in sentinel animals exposed in natura to different agricultural sites potentially exposed to pesticide contamination. Multivariate data analyses highlighted the modulation of several key proteins involved in feeding and molting. This multiplex-targeted proteomics assay paves the way for the discovery and the use of a large panel of novel protein biomarkers in emergent ecotoxicological models for environmental monitoring in the future. BIOLOGICAL SIGNIFICANCE: The study contributed to the development of Scout-MRM for the high-throughput quantitation of a large panel of proteins in the Gammarus fossarum freshwater sentinel. Increasing the number of markers in ecotoxicoproteomics is of most interest to assess the impact of pollutants in freshwater organisms. The development and validation of the assay enabled the monitoring of a large panel of reporter peptides of exposed gammarids. To illustrate the applicability of the methodology, animals from different agricultural sites were analysed. The application of the assay highlighted the modulation of some biomarker proteins involved in key physiological pathways, such as molting, feeding and general stress response. Increasing multiplexing capabilities and field test will provide the development of diagnostic protein biomarkers for emergent ecotoxicological models in future environmental biomonitoring programs.

Comparative proteomics in the wild: Accounting for intrapopulation variability improves describing proteome response in a Gammarus pulex field population exposed to cadmium.

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.

Shotgun proteomics datasets acquired on Gammarus pulex animals sampled from the wild.

This data article associated with the manuscript "Comparative proteomics in the wild: accounting for intrapopulation variability improves describing proteome response in a Gammarus pulex field population exposed to cadmium" refers to the shotgun proteomics analysis performed on 40 Gammarus pulex animals sampled from the wild. Proteins were extracted, digested with trypsin, and the resulting peptides were identified by tandem mass spectrometry. Here, we present the list of proteins from males and the list of proteins from females that are differentially detected between the Brameloup and the Pollon populations. Data are available via ProteomeXchange with identifiers PXD013656 and PXD013712, respectively.

De novo transcriptomes of 14 gammarid individuals for proteogenomic analysis of seven taxonomic groups.

Gammarids are amphipods found worldwide distributed in fresh and marine waters. They play an important role in aquatic ecosystems and are well established sentinel species in ecotoxicology. In this study, we sequenced the transcriptomes of a male individual and a female individual for seven different taxonomic groups belonging to the two genera Gammarus and Echinogammarus: Gammarus fossarum A, G. fossarum B, G. fossarum C, Gammarus wautieri, Gammarus pulex, Echinogammarus berilloni, and Echinogammarus marinus. These taxa were chosen to explore the molecular diversity of transcribed genes of genotyped individuals from these groups. Transcriptomes were de novo assembled and annotated. High-quality assembly was confirmed by BUSCO comparison against the Arthropod dataset. The 14 RNA-Seq-derived protein sequence databases proposed here will be a significant resource for proteogenomics studies of these ecotoxicologically relevant non-model organisms. These transcriptomes represent reliable reference sequences for whole-transcriptome and proteome studies on other gammarids, for primer design to clone specific genes or monitor their specific expression, and for analyses of molecular differences between gammarid species.

Evaluation of psychiatric hospital wastewater toxicity: what is its impact on aquatic organisms?

The primary source of pharmaceuticals to the aquatic environment is the discharge of wastewater effluents. Pharmaceuticals are a large and diverse group of compounds. Among them, psychotropic substances are particularly interesting to study due to their specific known mode of action. The present study was performed to investigate the effects of wastewater effluents from a psychiatric hospital wastewater treatment plant (WWTP) on several aquatic organisms. All the analyzed pharmaceuticals (10 compounds) were detected in WWTP effluents as well as in the receiving river. Although the environmental concentrations were generally at trace levels (ng L-1 to µg L-1), induce toxic effects were observed. This study showed the effects of the WWTP effluents on the oogenesis and/or embryogenesis of amphipod crustacean Gammarus fossarum, Japanese fish medaka Oryzias latipes, mollusk Radix peregra, and planarian Schmidtea polychroa. A decrease of the number of oocytes and produced embryos was observed for G. fossarum and S. polychroa. Similarly, the hatching rate of R. peregra was affected by effluents. In the receiving river, the macroinvertebrate community was affected by the wastewater effluents discharge.

Application of a multidisciplinary and integrative weight-of-evidence approach to a 1-year monitoring survey of the Seine River.

Quality assessment of environments under high anthropogenic pressures such as the Seine Basin, subjected to complex and chronic inputs, can only be based on combined chemical and biological analyses. The present study integrates and summarizes a multidisciplinary dataset acquired throughout a 1-year monitoring survey conducted at three workshop sites along the Seine River (PIREN-Seine program), upstream and downstream of the Paris conurbation, during four seasonal campaigns using a weight-of-evidence approach. Sediment and water column chemical analyses, bioaccumulation levels and biomarker responses in caged gammarids, and laboratory (eco)toxicity bioassays were integrated into four lines of evidence (LOEs). Results from each LOE clearly reflected an anthropogenic gradient, with contamination levels and biological effects increasing from upstream to downstream of Paris, in good agreement with the variations in the structure and composition of bacterial communities from the water column. Based on annual average data, the global hazard was summarized as "moderate" at the upstream station and as "major" at the two downstream ones. Seasonal variability was also highlighted; the winter campaign was least impacted. The model was notably improved using previously established reference and threshold values from national-scale studies. It undoubtedly represents a powerful practical tool to facilitate the decision-making processes of environment managers within the framework of an environmental risk assessment strategy.

Caged Gammarus as biomonitors identifying thresholds of toxic metal bioavailability that affect gammarid densities at the French national scale.

Bioaccumulated concentrations of toxic elements in biomonitor invertebrate species have already been used to successfully link metal bioavailability and impairments of stream macroinvertebrate communities at the scale of the watershed. However, implementing this empirical comparative approach at a greater spatial scale remains a challenge due to the diversity of biogeographical contexts encompassed by regional and national scales. We showed in previous studies that the use of standard organisms caged permits the use of a common biomonitor over a far greater geographical range, while limiting the influence of confounding factors on levels of bioavailable contamination. In this study, levels of Cd, Hg, Ni and Pb contamination assessed by active biomonitoring with caged Gammarus fossarum were compared to abundances of on-site gammarids on 94 sites in France. Based on this national dataset of in situ bioassays, we first re-determined bioavailable background assessment concentrations (BBACs), i.e. concentrations measured in caged G. fossarum indicating a significant bioavailable contamination, which we had previously defined at a regional scale for these four metallic elements. On-site gammarid abundances were retrieved from monitoring programs implemented by French water agencies for the evaluation of ecological status for the European Water Framework Directive. These abundances were corrected for the influence of stream physico-chemical typology in order to permit a reliable comparison of gammarid densities between sites at the national scale. Clear trends of degradation of gammarid densities with increasing levels of bioaccumulated concentrations were identified for three of the four elements (Cd, Ni and Pb). Threshold concentrations in caged organisms above which the numbers of free-ranging gammarids were abnormally low - namely bioavailable ecological assessment concentrations (BEACs) - were determined. The reliability and validity of the BEACs, their comparison with BBACs and their usefulness in terms of prioritisation of contaminants, sites in freshwater management, are discussed.

Gammarus fossarum as a sensitive tool to reveal residual toxicity of treated wastewater effluents.

Wastewater treatment plants (WWTPs) are one of the main sources of freshwater pollution eventually resulting in adverse effects in aquatic organisms. Treated effluents can contain many micropollutants at concentrations often below the limit of chemical quantification. On a regulatory basis, WWTP effluents have to be non-toxic to the aquatic environment, wherefore not only chemical abatement but also ecotoxicological evaluation through relevant bioassays is required. Standardized bioassays currently used are often not sensitive enough to reveal a residual toxicity in treated effluents. Therefore, attention must be paid to the development of better-adapted approaches implementing more sensitive organisms and relevant endpoints. In this study, the toxicity of two differently treated effluents (activated sludge treated effluents with and without ozonation) towards the ecologically relevant species Gammarus fossarum was evaluated. Organism fitness traits such as reproduction and sperm DNA integrity were followed in exposed organisms. In complement, enzymatic biomarkers were measured indicating the presence of neurotoxic compounds (acetylcholinesterase activity), the presence of pathogens likely to increase the toxic effects of chemical compounds (phenol-oxidase activity), and the presence of toxic compounds inducing detoxification mechanisms (glutathione-S-transferase activity). Enzymatic activities were not modified, but significant sub-lethal effects were observed in exposed organisms. In both effluents, females showed a retarded molt cycle, a reduced fecundity and fertility, and >90% of developed embryos exhibited developmental malformations. In addition, a slight but significant genotoxic effect was measured in gammarid sperm. In a whole, no difference in toxicity was found between both effluents. Coupling reproduction impairment and genotoxicity assessment in Gammarus fossarum seems to be a valuable and sensitive tool to reveal residual toxicity in effluents containing a mixture of micropollutants at very low concentrations. Finally, a direct relationship between the observed toxic responses and the quantified micropollutant concentrations could not be evidenced.

Seasonal variability and inter-species comparison of metal bioaccumulation in caged gammarids under urban diffuse contamination gradient: implications for biomonitoring investigations.

Although caging of Gammarus species offers promising lines of inquiry to monitor metal bioavailability in freshwaters, the interspecies responsiveness to metal exposures is still unclear. In addition, abiotic factors inherent to transplantation can hamper the interpretation of field bioaccumulation data. To assess the relevance of using gammarids as biomonitors, we investigated the seasonal influence on metal bioaccumulation in two common species, Gammarus pulex and Gammarus fossarum. During four seasons, caged gammarids were deployed on three sites along the Seine River exhibiting a diffuse gradient of multi-metal contamination: a site upstream and two sites downstream from the Paris megacity. For each seasonal deployment, metal concentrations in animals were determined after 7d-exposure in situ (Ag, Cd, Co, Cu, Mn, Ni, Pb and Zn). Results show that the seasonal patterns of metal contaminations are similar between both Gammarus species, and closely related to the river axis' contamination gradient. Statistical analyses indicate that bioaccumulation of essential metals in both species is influenced by season, especially by water temperature. This highlights the necessity to consider this climatic factor inherent to the deployment period for a reliable interpretation of bioaccumulation data in the field. The comparison of accumulation factors suggests that these two species coming from different geochemical origins display similar abilities to internalize metals. This generic responsiveness of caged gammarids supports their use as sentinel organisms to quantify low spatiotemporal variations in metal bioavailabilities.

Linking feeding inhibition with reproductive impairment in Gammarus confirms the ecological relevance of feeding assays in environmental monitoring.

The in situ feeding bioassay in Gammarus fossarum is recognized as a reliable tool for monitoring the toxicity of freshwater contamination. However, whether recorded feeding inhibitions can potentially provoke population-level adverse outcomes remains an open question. In the present study, the authors present an experimental study in G. fossarum, which contributes to the quantitative description of the links between feeding inhibitions and impacts on female reproductive performance. The authors studied the impacts of food deprivation on reproductive endpoints (i.e., fecundity, fertility, molt cycle) during 2 successive molting cycles. Among the main results, the authors found that food deprivation triggered a slowdown of the molting process and a reduction in fertility but no alteration to embryonic development. These reproductive impairments appeared for feeding inhibition values usually recorded in monitoring programs of environmental pollution. Using a population model translating Gammarus life-history, the authors predicted that the observed reproductive alterations predict a strong degradation of population dynamics. The present study underlines the importance of feeding inhibition in population-level risk assessment and discusses how establishing upscaling schemes based on quantitative mechanistic links between impacts at different levels of biological organization can be applied in environmental monitoring to propose an ecotoxicological assessment of water quality, which would be sensitive, specific, and ecologically relevant.