The use of copper as plant protection product contributes to environmental contamination and resulting impacts on terrestrial and aquatic biodiversity and ecosystem functions.

Copper-based plant protection products (PPPs) are widely used in both conventional and organic farming, and to a lesser extent for non-agricultural maintenance of gardens, greenspaces, and infrastructures. The use of copper PPPs adds to environmental contamination by this trace element. This paper aims to review the contribution of these PPPs to the contamination of soils and waters by copper in the context of France (which can be extrapolated to most of the European countries), and the resulting impacts on terrestrial and aquatic biodiversity, as well as on ecosystem functions. It was produced in the framework of a collective scientific assessment on the impacts of PPPs on biodiversity and ecosystem services in France. Current science shows that copper, which persists in soils, can partially transfer to adjacent aquatic environments (surface water and sediment) and ultimately to the marine environment. This widespread contamination impacts biodiversity and ecosystem functions, chiefly through its effects on phototrophic and heterotrophic microbial communities, and terrestrial and aquatic invertebrates. Its effects on other biological groups and biotic interactions remain relatively under-documented.

Recommendations to reduce the streetlight effect and gray areas limiting the knowledge of the effects of plant protection products on biodiversity.

Preserving biodiversity against the adverse effects of plant protection products (PPPs) is a major environmental and societal issue. However, despite intensive investigation into the ecotoxicological effects of PPPs, the knowledge produced remains fragmented given the sheer diversity of PPPs. This is due, at least in part, to a strong streetlight effect in the field of ecotoxicology. Indeed, while some PPPs have been investigated in numerous ecotoxicological studies, there are many for which the scientific literature still has little or no information on their ecotoxicological risks and effects. The PPPs under the streetlight include a large variety of legacy substances and a more limited number of more recent or currently-in-use substances, such as the herbicide glyphosate and the neonicotinoid insecticides. Furthermore, many of the most recent PPPs (including those used in biocontrol) and PPP transformation products (TPs) resulting from abiotic and/or biotic degradation are rarely addressed in the international literature in the field of ecotoxicology. Here, based on a recent collective scientific assessment of the effects of PPPs on biodiversity and ecosystem services in the French and European contexts, this article sets out to illustrate the limitations and biases caused by the streetlight effect and numbers of gray areas, and issue recommendations on how to overcome them.

Impacts of neonicotinoids on biodiversity: a critical review.

Neonicotinoids are the most widely used class of insecticides in the world, but they have raised numerous concerns regarding their effects on biodiversity. Thus, the objective of this work was to do a critical review of the contamination of the environment (soil, water, air, biota) by neonicotinoids (acetamiprid, clothianidin, imidacloprid, thiacloprid, thiamethoxam) and of their impacts on terrestrial and aquatic biodiversity. Neonicotinoids are very frequently detected in soils and in freshwater, and they are also found in the air. They have only been recently monitored in coastal and marine environments, but some studies already reported the presence of imidacloprid and thiamethoxam in transitional or semi-enclosed ecosystems (lagoons, bays, and estuaries). The contamination of the environment leads to the exposure and to the contamination of non-target organisms and to negative effects on biodiversity. Direct impacts of neonicotinoids are mainly reported on terrestrial invertebrates (e.g., pollinators, natural enemies, earthworms) and vertebrates (e.g., birds) and on aquatic invertebrates (e.g., arthropods). Impacts on aquatic vertebrate populations and communities, as well as on microorganisms, are less documented. In addition to their toxicity to directly exposed organisms, neonicotinoid induce indirect effects via trophic cascades as demonstrated in several species (terrestrial and aquatic invertebrates). However, more data are needed to reach firmer conclusions and to get a clearer picture of such indirect effects. Finally, we identified specific knowledge gaps that need to be filled to better understand the effects of neonicotinoids on terrestrial, freshwater, and marine organisms, as well as on ecosystem services associated with these biotas.

Main conclusions and perspectives from the collective scientific assessment of the effects of plant protection products on biodiversity and ecosystem services along the land-sea continuum in France and French overseas territories.

Preservation of biodiversity and ecosystem services is critical for sustainable development and human well-being. However, an unprecedented erosion of biodiversity is observed and the use of plant protection products (PPP) has been identified as one of its main causes. In this context, at the request of the French Ministries responsible for the Environment, for Agriculture and for Research, a panel of 46 scientific experts ran a nearly 2-year-long (2020-2022) collective scientific assessment (CSA) of international scientific knowledge relating to the impacts of PPP on biodiversity and ecosystem services. The scope of this CSA covered the terrestrial, atmospheric, freshwater, and marine environments (with the exception of groundwater) in their continuity from the site of PPP application to the ocean, in France and French overseas territories, based on international knowledge produced on or transposable to this type of context (climate, PPP used, biodiversity present, etc.). Here, we provide a brief summary of the CSA's main conclusions, which were drawn from about 4500 international publications. Our analysis finds that PPP contaminate all environmental matrices, including biota, and cause direct and indirect ecotoxicological effects that unequivocally contribute to the decline of certain biological groups and alter certain ecosystem functions and services. Levers for action to limit PPP-driven pollution and effects on environmental compartments include local measures from plot to landscape scales and regulatory improvements. However, there are still significant gaps in knowledge regarding environmental contamination by PPPs and its effect on biodiversity and ecosystem functions and services. Perspectives and research needs are proposed to address these gaps.

Collective scientific assessment as a relevant tool to inform public debate and policymaking: an illustration about the effects of plant protection products on biodiversity and ecosystem services.

Several sustainable development goals cannot be achieved without implementing a new generation of environmental measures to better preserve or restore biodiversity and ecosystem services. However, understanding and addressing biodiversity loss and ecosystem degradation is a challenging problem that is not solvable without integrating the best and latest science. It is crucial to enhance the legibility of this knowledge for decision-makers and policymakers following good-practice standards of scientific assessment. This is the main objective of collective scientific assessments (CSAs), as carried out by the French National Research Institute for Agriculture, Food and the Environment (INRAE) since the early 2000s following a documented procedure to inform public policy and foster public debate on complex interdisciplinary issues. This article describes the main steps of the CSA procedure designed by INRAE's Directorate for Collective Scientific Assessment, Foresight and Advanced Studies, from formulation of the initial question asked by public or para-public bodies (typically ministry divisions or environmental agencies) to wider dissemination of the results and conclusions. This process description is then illustrated through the example of a CSA recently commissioned by three French Ministries (for Ecology, for Research, and for Agriculture) regarding (i) contamination of terrestrial, freshwater, and marine ecosystems by plant protection products (PPPs); (ii) the resulting effects on biodiversity and ecosystem services; and (iii) possible prevention and mitigation strategies. The capacity of this kind of CSA to inform public debate and policymaking is then exemplified through a description of the main outcomes generated by the latest CSA dealing with the adverse effects of PPPs. We also provide a short overview of some key expectations from the current CSA, with a focus on the recent development of the ecosystem service approach in ecological risk assessments of PPPs in the European Union. This illustration demonstrates that CSAs, which are applicable to a wide variety of complex interdisciplinary questions that are not limited to environmental issues, are a relevant tool to inform public debate and policymaking.

Biomarkers as tools for monitoring within the Water Framework Directive context: concept, opinions and advancement of expertise.

The Water Framework Directive (WFD) currently supports chemical and ecological monitoring programmes in order to achieve the good water surface status. Although chemical and ecological assessments are necessary, they have some limitations. Chemical approaches focus on certain substances identified as priorities, but they do not take into account other potentially harmful substances and also ignore the hazards related to contaminant cocktails. On the other hand, while ecological approaches provide holistic information on the impairment of biological communities in ecosystems, they do not distinguish the role of contaminants in these alterations, and consequently do not allow the establishment of contaminant impact reduction plans. Consequently, ecotoxicologists suggest the use of effect-based tools such as biomarkers. Biomarkers highlight the effect of potentially harmful substances (or a cocktail), and their specificity towards the chemicals makes it possible to properly discriminate the role of toxicants within biological community impairments. Thus, the integration of such tools (besides existing chemical and ecological tools) in the WFD could considerably improve its biomonitoring strategy. The B n' B project (Biomarkers and Biodiversity) exposes key objectives that will allow to (i) establish an inventory of the biomarkers developed by French laboratories; (ii) determine their methodological advancement and limits and, on this basis, formulate recommendations for biomonitoring use and future research needs; (iii) discuss the biomarkers' ecological significance, specificity to contaminants and interpretation capacity; (iv) establish, in fine, a selection of valuable biomarkers to enter the WFD; and (iv) propose integrative tools to facilitate the decision-taking by stakeholders.

An integrative approach to assess ecological risks of surface water contamination for fish populations.

Contamination of aquatic ecosystems is considered as one of the main threats to global freshwater biodiversity. Within the European Water Framework Directive (EU-WFD) a particular attention is dedicated to assess ecological risks of surface water contamination and mitigation of chemical pressures on aquatic ecosystems. In this work, we evaluated ecological risks of surface water contamination for fish populations in four EU-WFD rivers through an integrative approach investigating three Lines of Evidence (chemical contamination, biomarker responses as early warning signals of contamination impacting individuals and ecological analyses as an indicator of fish community disturbances). This work illustrates through 4 case studies the complementary role of biomarkers, chemical and ecological analyses which, used in combination, provide fundamental information to understand impacts of chemical pressures that can affect fish population dynamics. We discuss the limitations of this approach and future improvements needed within the EU-WFD to assess ecological risk of river contamination for fish populations.

Acclimation capacity of the three-spined stickleback (Gasterosteus aculeatus, L.) to a sudden biological stress following a polymetallic exposure.

To get closer to the environmental reality, ecotoxicological studies should no longer consider the evaluation of a single pollutant, but rather combination of stress and their interaction. The aim of this study was to determine if responses of a fish to a sudden biological stress could be modified by a prior exposure to a chemical stress (a polymetallic contamination). For this purpose, in situ experiment was conducted in three ponds in the Haute-Vienne department (France). One pond was chosen for its high uranium concentration due to uranium mine tailings, and the two other ponds, which were not submitted to these tailings. Three-spined sticklebacks (Gasterosteus aculeatus) were caged in these ponds for 14 days. After this period, fish were submitted to a biological stress, exerted by lipopolysaccharides injection after anesthesia, and were sacrificed 4 days after these injections for multi-biomarkers analyses (leucocyte viability, phagocytic capacity and reactive oxygen species production, antioxidant peptide and enzymes, lipid peroxidation and DNA damage). The pond which received uranium mine tailings had higher metallic concentrations. Without biological stress, sticklebacks caged in this pond presented an oxidative stress, with increasing of reactive oxygen species levels, modification of some parts of the antioxidant system, and lipid peroxidation. Caging in the two most metal-contaminated ponds resulted in an increase of susceptibility of sticklebacks to the biological stress, preventing their phagocytic responses to lipopolysaccharides and modifying their glutathione contents and glutathione-S-transferase activity.

In situ effects of metal contamination from former uranium mining sites on the health of the three-spined stickleback (Gasterosteus aculeatus, L.).

Human activities have led to increased levels of various pollutants including metals in aquatic ecosystems. Increase of metallic concentrations in aquatic environments represents a potential risk to exposed organisms, including fish. The aim of this study was to characterize the environmental risk to fish health linked to a polymetallic contamination from former uranium mines in France. This contamination is characterized by metals naturally present in the areas (manganese and iron), uranium, and metals (aluminum and barium) added to precipitate uranium and its decay products. Effects from mine releases in two contaminated ponds (Pontabrier for Haute-Vienne Department and Saint-Pierre for Cantal Department) were compared to those assessed at four other ponds outside the influence of mine tailings (two reference ponds/department). In this way, 360 adult three-spined sticklebacks (Gasterosteus aculeatus) were caged for 28 days in these six ponds before biomarker analyses (immune system, antioxidant system, biometry, histology, DNA integrity, etc.). Ponds receiving uranium mine tailings presented higher concentrations of uranium, manganese and aluminum, especially for the Haute-Vienne Department. This uranium contamination could explain the higher bioaccumulation of this metal in fish caged in Pontabrier and Saint-Pierre Ponds. In the same way, many fish biomarkers (antioxidant and immune systems, acetylcholinesterase activity and biometric parameters) were impacted by this environmental exposure to mine tailings. This study shows the interest of caging and the use of a multi-biomarker approach in the study of a complex metallic contamination.

Juvenile roach (Rutilus rutilus) increase their anaerobic metabolism in response to copper exposure in laboratory conditions.

This study aims to determine the potential impairment of cell energy synthesis processes (glycolysis and respiratory chain pathways) by copper in juvenile roach at different regulation levels by using a multi-marker approach. Juvenile roach were exposed to 0, 10, 50, and 100 µg/L of copper for 7 days in laboratory conditions. The glycolysis pathway was assessed by measuring the relative expression levels of 4 genes encoding glycolysis enzymes. The respiratory chain was studied by assessing the electron transport system and cytochrome c oxidase gene expression. Muscle mitochondria ultrastructure was studied, and antioxidant responses were measured. Furthermore, the main energy reserves-carbohydrates, lipids, and proteins-were measured, and cellular energy was evaluated by measuring ATP, ADP, AMP and IMP concentrations. This study revealed a disturbance of the cell energy metabolism due to copper exposure, with a significant decrease in adenylate energy charge in roach exposed to 10 µg/L of copper after 1 day. Moreover, ATP concentrations significantly decreased in roach exposed to 10 µg/L of copper after 1 day. This significant decrease persisted in roach exposed to 50 µg/L of copper after 7 days. AMP concentrations increased in all contaminated fish after 1 day of exposure. In parallel, the relative expression of 3 genes encoding for glycolysis enzymes increased in all contaminated fish after 1 day of copper exposure. Focusing on the respiratory chain, cytochrome c oxidase gene expression also increased in all contaminated fish at the two time-points. The activity of the electron transport system was not disturbed by copper, except in roach exposed to 100 µg/L of copper after 1 day. Copper induced a metabolic stress. Juvenile roach seemed to respond to the ensuing high energy demand by increasing their anaerobic metabolism, but the energy produced by the anaerobic metabolism is unable to compensate for the stress induced by copper after 7 days. This multi-marker approach allows us to reach a greater understanding of the effects of copper on the physiological responses of juvenile roach.

In situ experiments to assess effects of constraints linked to caging on ecotoxicity biomarkers of the three-spined stickleback (Gasterosteus aculeatus L.).

The aim of this study was to evaluate the effects of caging constraints on multiple fish biomarkers used during ecotoxicological studies (biometric data, immune and antioxidant systems, and energetic status). Two of these constraints were linked to caging: starvation and fish density in cages, and one in relation to the post-caging handling: a short transport. Three in situ experiments were conducted with three-spined sticklebacks (Gasterosteus aculeatus). The first experiment compared the effects of three densities (low, medium, and high). The second experiment compared effects of starvation in fish fed every two days with fish that were not fed. Finally comparisons between sticklebacks which have suffered a short car transport after caging and sticklebacks killed without preliminary transport were made. The lack of food had no effect on fish energetic reserves but negatively affected their condition index and their immune system. Transport and high density induced oxidative stress, defined as an overproduction of reactive oxygen species and a stimulation of the antioxidant system. These two constraints also harmed the leucocyte viability. In order not to have any impact on ecotoxicity biomarkers during in situ experiments, it is preferable to decrease fish density in cages, prevent transport before dissections, and feed fish when the caging lasts more than two weeks.

Characterization of a genotoxicity biomarker in three-spined stickleback (Gasterosteus aculeatus L.): Biotic variability and integration in a battery of biomarkers for environmental monitoring.

As a large array of hazardous substances exhibiting genotoxicity are discharged into surface water, this work aimed at assessing the relevance of adding a genotoxicity biomarker in a battery of biomarkers recently developed in the model fish three-spined stickleback (Gasterosteus aculeatus). First the confounding influence of gender, body length, and season (used as a proxy of age and of the fish reproductive status, respectively) on the level of primary DNA damage in erythrocytes was investigated in wild sticklebacks. Then, the genotoxity biomarker was included in a large battery of biomarkers assessing xenobiotic biotransformation, oxidative stress and neurotoxicity, and implemented in five sites. Gender, age and reproductive status did not influence DNA damage level in fish from the reference site. A significant relationship between the level of primary DNA damage and fish length (as a proxy of age also correlated to the season) was highlighted in the contaminated site. Among all biomarkers investigated in the field, the level of DNA damage was one of the four most discriminating biomarkers with EROD, catalase activity and the level of lipid peroxidation representing together 75.40% of the discriminating power in sampled fish. The level of DNA damage was correlated to the EROD activity and to the level of peroxidation, which mainly discriminated fish from sites under urban pressure. Finally, Integrated Biomarker Response indexes (IBRv2), which were calculated with the whole biomarker response dataset exhibited higher values in the Reveillon (9.62), the Scarpe and Rhonelle contaminated sites (5.11 and 4.90) compared with the two reference sites (2.38 and 2.55). The present work highlights that integration of a genotoxicity biomarker in a multiparametric approach is relevant to assess ecotoxicological risk in freshwater aquatic organisms.

Involvement of fish immunomarkers in environmental biomonitoring approach: Urban and agri-viticultural context.

The Champagne region (France) is characterized by various chemical environmental pressures which could interfere with the immune status of natural populations of European bullhead, Cottus sp. Some adult fish were caught by electrofishing in spring, summer and autumn to determined immune effect of urban (Muizon), intensive agricultural (Bouy; Prunay) or viticultural (Serzy; Prunay) influences. The major results demonstrated an increase of cellular mortality and a decrease of phagocytosis activity in the stations impacted by agri-viticultural chemicals. These immunomodulations followed the temporal variability due to different treatments (agricultural impacts on spring; viticultural effects on autumn). At the present time, not enough data was provided to confirm the impact of agri-viticultural chemicals on fish immune system without interaction with other environmental factors. For example, in summer, the immunomarkers seems to be not only correlated with water contamination but also with other environmental factors (pathogens, physical field degradation, nutrients, temperature …). Nevertheless, immune parameters give a global view of organism and ecosystem health explaining growing interest for these biomarkers in environmental risk assessment.

Recommendations to design environmental monitoring in the European bullhead, Cottus sp., based on reproductive cycle and immunomarker measurement.

European bullhead is a relevant fish species to assess adverse effects of environmental stress on wild fish. Nevertheless, their complex reproductive cycle is very different between sites and could interfere with many physiological processes. Thus, prior to use biomarker to statute on environmental quality of rivers, we wanted to characterize reproductive profile (spawn number, GSI, gonad development). The major results demonstrated that the two types of reproductive cycle shown were strongly correlated to water temperature variation. In a second time, even if innate immunomarkers are highly relevant on biomonitoring program, hormonal variation seems to impact severely their responses. Thus, the link between reproductive status and immune activity (leucocyte distribution, cellular mortality, respiratory burst, phagocytosis activity) must also be study. Nonetheless, in the present work, immune capacities seems to be more correlated with season and environmental factors than reproduction.

Coupling of OECD standardized test and immunomarkers to select the most environmentally benign ionic liquids option--towards an innovative "safety by design" approach.

This paper proposed a potential industrial accompaniment to reduce ionic liquid harmfulness by a novel combination of OECD Daphnia magna standardized test and fish immunomarkers. The combination of these two tests allowed multicriteria examination of ILs impacts in different organisms and trophic levels. The work provided new data for legislation and opened a door towards an integrative environmental evaluation due to direct implications of immune system in fish and ecosystem health. Whatever the species, each IL tested induced deleterious effects suggesting that toxic impact was especially due to IL lipophilicity properties. Nevertheless, cation moieties of ILs seemed to draw overall toxicity of ILs to significant extent as supported by lower cell mortality shown with imidazolium-based ILs compared to phosphonium-based ILs. However, the anions moieties have some additional effect, as revealed by quite dissimilar toxicity within same IL family. Concerning the more integrative biomarkers, the cationic-based ILs tested possessed also dissimilar effect on immune system of fish, especially on leucocyte distribution, lysosomal membrane integrity and phagocytosis activity. These results confirm that ILs toxicity could be influenced by design and that chemical engineering processes can integrate ecological footprint reduction strategies for successful IL utilization in the future.

Former uranium mine-induced effects in caged roach: a multiparametric approach for the evaluation of in situ metal toxicity.

To characterize environmental risks linked to former uranium mines in the Limousin region of France, a study was conducted on fish health effects from uranium releases. Two private ponds were compared in this study, one with uranium contamination and one background site, upstream of the mining zone. Roach, Rutilus rutilus, were caged for 28 days in both ponds. Physico-chemical parameters of water and sediments and bioaccumulation of metals in several organs were determined. After 14 and 28 days of caging, immune, oxidative stress, biotransformation, neurotoxicity and physiological parameters were measured. Iron and aluminium were quantified in the water of both sites; however, barium and manganese were only present in the water of the uranium contaminated site. Uranium was present in both sites but at very different concentrations. The sediments from the uranium contaminated site contained high levels of radioactive elements coming from the disintegration chain of uranium. Results of biological parameters indicated stimulation of immune parameters and of oxidative stress and a decrease of AChE in fish caged in the uranium contaminated pond compared to the uranium-free pond. Overall, the results determined roach health status in the context of pollution from poly-metallic mining. The data strengthen our knowledge of the environmental risk assessment associated with radioactive substances in the environment.

Assessment of the European flounder responses to chemical stress in the English Channel, considering biomarkers and life history traits.

A multi-biomarker approach was developed to evaluate responses of European flounder (Platichthys flesus) in three contrasted estuaries over the English Channel: the Canche (pristine site), Tamar (heavy metals and PAHs contamination) and Seine (heavily pollution with a complex cocktail of contaminants). The condition factor and several biomarkers of the immune system, antioxidant enzymes, energetic metabolism and detoxification processes were investigated in young-of-the-year (0+) and one-year-old (1+) flounder. Results underlined the difference between the pristine site and the Seine estuary which showed a lower condition factor, a modulation of the immune system, a higher Cytochrome C oxidase activity, and an up-regulation of BHMT expression. The moderate biomarker responses in the Tamar fish could be linked to the specific contamination context of this estuary. Flounder life history traits were analyzed by otolith microchemistry, in order to depict how the fish use their habitat and thus respond to chemical stress in estuaries.