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Biosorption is a sustainable biological treatment for remediating environmental hazardous matrices. The shells of the invasive bivalve Corbicula fluminea were tested for their potential as a natural, abundant sorption material to cope with extremely toxic olive oil mill wastes (OOMW). Shells of C. fluminea were exposed for 14 days to OOMW and variations in the chemical composition of the waste and the shells were monitored by FTIR analysis. In general, the results showed the inefficacy of the shells in adsorbing chemicals from OOMW, namely regarding predominant aromatic structures. A significant slight increase in the 1750–1180 cm−1 region was found, relating to a mild reduction of the peak areas in the same FTIR region noted in shells, which suggests partial shell dissolution; this trend was further supported by the parallel increase of the levels of both pH and Chemical Oxygen Demand. Although the shells of C. fluminea were found inadequate as a sink for the organic components present in OOMW, the abundance of the resource and previous evidence of its absorption capacity for different contaminants support the need for extended research on the use of this biomaterial as a sorption agent in bioremediation.

Wildfires are an environmental concern due to the loss of forest area and biodiversity, but also because their role as drivers of freshwater systems contamination by metals. In this context, the fish Gambusia holbrooki was used as a model, deployed for in situ exposure in watercourses standing within a recently burnt area and further assessment of toxic effects. The fish were exposed during 4 days at four different sites: one upstream and another downstream the burnt area and two within the burnt area. Biochemical biomarkers for oxidative stress and damage were assessed. The extent of lipoperoxidative damage was monitored by quantifying malondialdehyde and DNA damage evaluated through erythrocyte nuclear abnormalities observation. Chemical analysis revealed higher metal levels within the burnt area, and exposed fish consistently showed pro-oxidative responses therein, particularly an increase of gill glutathione peroxidase and glutathione reductase activity, the records doubling compared to samples from sites in the unburnt area; also the activity of glutathione-S-transferases comparatively increased (by 2-fold in the liver) in samples from the burnt area, and malondialdehyde was produced twice as much therein and in samples downstream the burnt area reflecting oxidative damage. Consistently, the frequency of erythrocyte nuclear abnormalities was higher at sites within and downstream the burnt area. This study supports the use of sensitive oxidative stress and genotoxicity biomarkers for an early detection of potentially noxious ecological effects of wildfires runoff.

Soil provides innumerable valuable ecosystem services, such as the production of food and the direct support of wildlife, by ensuring the availability of adequate habitat. However, unsustainable human activities are resulting in degradation of soils worldwide. Hence, it is of utmost importance to raise awareness about this often-overlooked environmental issue. This article presents an inquiry-based activity that challenges students to assess the ecological quality of soil in the surroundings of their classroom. Plus, students and teachers are invited to become citizen scientists by sharing their data with researchers, thus contributing to a future endeavor to map soil quality through broad geographic ranges.

The classification of wastes regarding hazardous property HP 14 (ecotoxicity) is essential for proper waste management. In the EU, HP 14 has been estimated based on waste chemical composition rather than using biotests, and guidelines for experimental assessment are still lacking. This study aims at evaluating the potential ecotoxicological impacts of weathered coal fly ash (CFA) from a landfill, as a case study to assess the current EU methodology used to classify wastes regarding HP 14. A large amount of CFA is still landfilled, but its valorisation would be of interest. The analysis was based on the chemical composition of CFA (in ClassifyMyWaste software), and on a battery of five biotests applied to eluates, with Lepidium sativum, Aliivibrio fischeri, Raphidocelis subcapitata, Lemna minor and Daphnia magna. Through chemical analysis, most of the simulations with data from the literature indicated "Possible Hazard", including the sample of this work. Biotests revealed low impairment for most endpoints. D. magna was the most sensitive organism, but the inhibitory effect was significantly reduced after pH adjustment of the eluate. The test with A. fischeri does not seem to be adequate to assess CFA due to the high variability observed in results. The methodology involving a simple battery of bioassays was proven to be enlightening, providing relevant results for HP 14 assessment. The chosen battery of biotests (excluding the A. fischeri test) may be a good starting point to represent the aquatic environment in this context. In short, it seems that weathered CFA can be considered non-hazardous, and therefore the material under analysis could be valorised in practical applications without significant ecotoxic effect on the environment.

Groundwater is an indispensable resource for humankind and sustainable biomes functioning. Anthropogenic disturbance threatens groundwater ecosystems globally, but to which extent groundwater organisms respond to stressors remains poorly understood. Groundwater animals are rare, with small populations, difficult to find and to breed in the lab, which poses a main challenge to the assessment of their responses to pollutants. Despite the difficulties, assessing the toxicity of a large spectrum of stressors to groundwater organisms is a priority to inform towards appropriate environmental protection of these ecosystems. We tested the sensitivity to CuSO4, diclofenac, and NaCl of a groundwater population of the copepod Diacyclops crassicaudis crassicaudis and compared its sensitivity with the model organism Daphnia magna. We ranked its sensitivity using a species sensitivity distribution (SSD) approach using the feasible data available for groundwater and surface crustaceans. Our results show that the most toxic compound was CuSO4 for which higher amount of data was recorded and wider variability in response was observed. It was followed by diclofenac, largely lacking data for groundwater-adapted organisms, and the least toxic compound was NaCl. The differential sensitivity between D. crassicaudis and D. magna was contaminant-dependent. As a general trend D. crassicaudis was always distributed in the upper part of the SSD curves together with other groundwater-adapted organisms. Our results highlight that the widespread groundwater populations of the D. crassicaudis species complex, which can be successfully breed in the lab, may provide a reasonable approach to assess the ecological effects of anthropogenic stressors in groundwater ecosystems.

Anthropogenic activities such as agriculture, industry and climate change have been creating an enormous pressure on freshwater ecosystems leading to its degradation. Because the Water Framework Directive (WFD; main EU instrument for ecological water quality assessment) is costly and time-consuming, the main goal of this study was to use the scoring by flow cytometry (FCM) of bacterial communities with high DNA content (HNA) and low DNA content (LNA), a fast and easy methodology, as bioindicators. To portray this study, 3 sampling sites of Caima river with different levels of environmental impact (site 1 – no anthropogenic impacted site; site 2 – downstream wastewater treatment plant and site 3 – downstream a deactivated mine) were analysed along the 4 seasons of the year 2017 (winter, spring, summer and autumn). The ecological status of the sampling sites was accessed following the methodology described by WFD and biotic index results obtained for macroinvertebrate and periphyton communities, as well as the BOD5, were compared with the bacterial community scored as HNA and LNA. Bacteria community analysis showed high bacteria density at site 2 corresponding to high amounts of organic input by the wastewater treatment plant. Also, HNA bacteria were found to be in higher quantities at site 2, related to an increase of nutrients, while LNA bacteria were more prominent in river headwater, corresponding to an oligotrophic environment. Correlations between biological indices and bacteria community composition were very strong, showing that bacteria communities may serve as indicators of water quality assessment. Although this FCM technique provide good responses, further investigations are needed to confirm the feasibility of this method.

Mixtures containing ether sulfate based surfactants (SLEnS) and linear alkylbenzene sulfonic acid (LAS) are relatively common in personal care and household products. When in mixture, they form mixed micelles, which act as reservoirs for the cleaning process. The increase of ethylene oxide (EO) units in the head of SLEnS lowers the critical micelle concentration, meaning that less quantity of each surfactant is needed to form the micelles. Within an eco-friendly perspective this is advantageous since less chemicals are expected to be released into the environment. But, this advantage will only be effective if variations with the higher number of EO units exhibit a lower toxicity as well. Despite its wide use in commercial products, the ecotoxicity of these micelles and the influence of the EO units on their toxicity still did not receive the necessary attention. In this context, the present study aimed at assessing the influence of the number of ethylene oxide (EO) units in the head groups of SLEnS on the toxicity of the SLEnS-LAS mixed micelles to the bacterium Vibrio fischeri (Microtox® assay, here used as a fast and preliminary ecotoxicological indicator). The SLEnS variants with fewer EO units showed higher toxicity relatively to those with more EO units - EC50 range (0–50 EO units): 0.56–8.59 mg L−1, thus they can be suggested as environmentally safer variants to be used in personal care and household products. Provided the consistency of Microtox® results as obtained here, this quick and cost-effective procedure can be an important tool towards the development of eco-friendlier surfactant-based formulations.

The interest on deep eutectic solvents (DES) has been increasing. However, the ecotoxicological profile of DES is scarcely known. Also, despite previous studies showed that DES components dissociate in water, none assessed DES toxicity using the classical and adequate models for mixture toxicity prediction - concentration addition (CA) and independent action (IA). This study evaluates the ecotoxicological profile of DES based on [N1111]Cl, [N2222]Cl and [N3333]Cl as hydrogen bond acceptors (HBA) combined with hydrogen-bond donors (HBD) vis. ethylene glycol and 1-propanol, through the Microtox® Acute Toxicity Test. CA and IA with deviations describing synergism/antagonism, dose-ratio and dose-level effects were fitted to the toxicity data. Neither the starting materials nor DES were found hazardous to Aliivibrio fischeri, in this specific case agreeing with the claimed "green character" of DES. Among the starting materials, ethylene glycol was the least toxic, whereas [N3333]Cl was the most toxic (30 min-EC50 = 96.49 g L-1 and 0.5456 g L-1, respectively). DES toxicity followed the same trend as observed for the salts: [N1111]Cl-based DES

Wildfires are a complex environmental problem worldwide. The ashes produced during the fire bear metals and PAHs with high toxicity and environmental persistence. These are mobilized into downhill waterbodies, where they can impair water quality and human health. In this context, the present study aimed at assessing the toxicity of mimicked wildfire runoff to human skin cells, providing a first view on the human health hazardous potential of such matrices. Human keratinocytes (HaCaT) were exposed to aqueous extracts of ashes (AEA) prepared from ash deposited in the soil after wildfires burned a pine or a eucalypt forest stand. Cytotoxicity (MTT assay) and changes in cell cycle dynamics (flow cytometry) were assessed. Cell viability decreased with increasing concentrations of AEA, regardless of the ash source, the extracts preparation method (filtered or unfiltered to address the dissolved or the total fractions of contaminants, respectively) or the exposure period (24 and 48 h). The cells growth was also negatively affected by the tested AEA matrices, as evidenced by a deceleration of the progress through the cell cycle, namely from phase G0/G1 to G2. The cytotoxicity of AEA could be related to particulate and dissolved metal content, but the particles themselves may directly affect the cell membrane. Eucalypt ash was apparently more cytotoxic than pine ash due to differential ash metal burden and mobility to the water phase. The deceleration of the cell cycle can be explained by the attempt of cells to repair metal-induced DNA damage, while if this checkpoint and repair pathways are not well coordinated by metal interference, genomic instability may occur. Globally, our results trigger public health concerns since the burnt areas frequently stand in slopes of watershed that serve as recreation sites and sources of drinking water, thus promoting human exposure to wildfire-driven contamination.

The EU Water Framework Directive (WFD) assessment scheme has been putting in force the evaluation of freshwater ecosystems in Europe, including a new paradigm of ecological status. After almost 20 years since the WFD implementation, it is imperative to evaluate the efficiency of its standard assessment scheme and to explore the possibility of learning how to improve its effectiveness. That is the spirit of this review, aiming (i) to explore the existing literature on the WFD bioassessment scheme for assessing freshwater ecosystem health, particularly in lotic ecosystems (where the WFD scheme is most consolidated); (ii) to document which paths are suggested by the scientific community to improve the efficiency of the bioassessment in tackling current challenges. In the specific arena of bioassessment, we first identify the major constraints to the WFD full implementation in rivers. Second, we analyse retrospective Ecological Risk Assessment (ERA) as an evaluation approach supporting management actions that could inspire improvements in the WFD bioassessment scheme. Third, we review the advances and debate on complementary metrics to improve WFD evaluation protocols and/or the feasibility of the evaluation outcome. Fourth, a conceptual scheme for an improved evaluation strategy is presented. Our proposal essentially merges the WFD bioassessment scheme with the ERA philosophy, proposing a tiered approach of increasing complexity and spatial resolution, where expert judgement is included surgically at all decision stages. This scheme requires true integration of chemical, ecological and ecotoxicological LoE for a quantitative estimation of risks, and provides a comprehensive framework that accommodates tools and perspectives already suggested by other authors. Besides providing a literature review on the strengths and weaknesses of the current WFD bioassessment scheme, we wish to open way for the scientific discussion towards an improved conceptual scheme for the evaluation of ecosystem health., CESAM - Centro de Estudos Ambientais e Marinhos, Universidade de Aveiro(UIDP/50017/2020 + UIDB/50017/2020)

Current farm systems rely on the use of Plant Protection Products (PPP) to secure high productivity and control threats to the quality of the crops. However, PPP use may have considerable impacts on human health and the environment. A study protocol is presented aiming to determine the occurrence and levels of PPP residues in plants (crops), animals (livestock), humans and other non-target species (ecosystem representatives) for exposure modelling and impact assessment. To achieve this, we designed a cross-sectional study to compare conventional and organic farm systems across Europe. Environmental and biological samples were/are being/will be collected during the 2021 growing season, at 10 case study sites in Europe covering a range of climate zones and crops. An additional study site in Argentina will inform the impact of PPP use on growing soybean which is an important European protein-source in animal feed. We will study the impact of PPP mixtures using an integrated risk assessment methodology. The fate of PPP in environmental media (soil, water and air) and in the homes of farmers will be monitored. This will be complemented by biomonitoring to estimate PPP uptake by humans and farm animals (cow, goat, sheep and chicken), and by collection of samples from non-target species (earthworms, fish, aquatic and terrestrial macroinvertebrates, bats, and farm cats). We will use data on PPP residues in environmental and biological matrices to estimate exposures by modelling. These exposure estimates together with health and toxicity data will be used to predict the impact of PPP use on environment, plant, animal and human health. The outcome of this study will then be integrated with socio-economic information leading to an overall assessment used to identify transition pathways towards more sustainable plant protection and inform decision makers, practitioners and other stakeholders regarding farming practices and land use policy. EEA Balcarce Fil: Silva, Vera. Wageningen University and Research. Soil Physics and Land Management Group; Países Bajos. Fil: Alaoui, Abdallah. University of Bern. Institute of Geography. Centre for Development and Environment; Suiza. Fil: Schlünssen, Vivi. Aarhus University. Department of Public Health; Dinamarca. National Research Centre for the Working Environment; Dinamarca. Fil: Vested, Anne. Aarhus University. Department of Public Health; Dinamarca. Fil: Graumans, Martien. Radboud Institute for Health Sciences; Países Bajos. Fil: Van Dael, Maurice. Radboud Institute for Health Sciences; Países Bajos. Fil: Trevisan, Marco. Università Cattolica del Sacro Cuore. Department for Sustainable Food Process; Italia. Fil: Suciu, Nicoleta. Università Cattolica del Sacro Cuore. Department for Sustainable Food Process; Italia. Fil: Mol, Hans. Wageningen University and Research. Wageningen Food Safety Research; Países Bajos. Fil: Beekmann, Karsten. Wageningen University and Research. Wageningen Food Safety Research; Países Bajos. Fil: Figueiredo, Daniel. Utrecht University. Institute for Risk Assessment Sciences; Países Bajos. Fil: Harkes, Paula. Wageningen University and Research. Soil Physics and Land Management Group; Países Bajos. Fil: Hofman, Jakub. Masaryk University. Faculty of Science. Research Centre for Toxic Compounds in the Environment; Republica Checa. Fil: Kandeler, Ellen. University of Hohenheim. Institute of Soil Science and Land Evaluation; Alemania. Fil: Abrantes, Nelson. University of Aveiro. Centre for Environmental and Marine Studies and Department of Environment and Planning; Portugal. Fil: Campos, Isabel. University of Aveiro. Centre for Environmental and Marine Studies and Department of Environment and Planning; Portugal. Fil: Martínez, María Ángeles. Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas; España. Fil: Pereira, Joana Luísa. Centre for Environmental and Marine Studies and Department of Environment and Planning; Portugal. Fil: Goossens, Dirk. Wageningen University and Research. Soil Physics and Land Management Group. Países Bajos. KU Leuven Department of Earth and Environmental Sciences; Belgica. Fil: Gandrass, Juergen. Institute of Coastal Environmental Chemistry; Alemania. Fil: Debler, Freya. Institute of Coastal Environmental Chemistry; Alemania. Fil: Huerta Lwanga, Esperanza. Wageningen University and Research. Soil Physics and Land Management Group; Países Bajos. Fil: Jonker, Marlot. Dutch Mammal Society; Países Bajos. Fil: Van Langevelde, Frank. Wageningen University and Research. Wildlife Ecology and Conservation Group; Países Bajos. Fil: Sorensen, Martin. Aarhus University. Department of Animal Science; Dinamarca. Fil: Wells, Jerry. Wageningen University and Research. Host-Microbe Interactomics, Animal Sciences Group; Países Bajos. Fil: Boekhorst, Jos. Wageningen University and Research. Host-Microbe Interactomics, Animal Sciences Group; Países Bajos. Fil: Huss, Anke. Utrecht University. Institute for Risk Assessment Sciences; Países Bajos. Fil: Mandrioli, Daniele. Ramazzini Institute. Cesare Maltoni Cancer Research Center; Italia. Fil: Sgargi, Daria. Ramazzini Institute. Cesare Maltoni Cancer Research Center; Italia. Fil: Nathanail, Paul. Land Quality Management; Reino Unido. Fil: Nathanail, Judith. Land Quality Management; Reino Unido. Fil: Tamm, Lucius. Research Institute of Organic Agriculture; Suiza. Fil: Fantke, Peter. Technical University of Denmark. Department of Technology, Management and Economics; Dinamarca. Fil: Mark, Jennifer. Research Institute of Organic Agriculture; Suiza. Fil: Grovermann, Christian. Research Institute of Organic Agriculture; Suiza. Fil: Larsen, Ana Frelih. Ecologic Institute; Alemania. Fil: Herb, Irina. Ecologic Institute; Alemania. Fil: Chivers, Charlotte Anne. University of Gloucestershire. Countryside and Community Research Institute; Reino Unido. Fil: Mills, Jane. University of Gloucestershire. Countryside and Community Research Institute; Reino Unido. Fil: Alcon, Francisco. Universidad Politécnica de Cartagena; España. Fil: Contreras, Josefina. Universidad Politécnica de Cartagena; España. Fil: Baldi, Isabelle. Bordeaux University. INSERM U1219; Francia. Fil: Pasković, Igor. Institute of Agriculture and Tourism, Department of Agriculture and Nutrition; Croacia. Fil: Matjaz, Glavan. University of Ljubljana. Biotechnical Faculty; Eslovenia. Fil: Norgaard, Trine. Aarhus University. Department of Agroecology; Dinamarca. Fil: Aparicio, Virginia Carolina. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Fil: Ritsema, Coen. Wageningen University and Research. Soil Physics and Land Management Group; Países Bajos. Fil: Geissen, Violette. University and Research. Soil Physics and Land Management Group; Países Bajos. Fil: Scheepers, Paul. Radboud Institute for Health Sciences; Países Bajos.

Knowledge on the molecular basis of ionic liquids' (ILs) ecotoxicity is critical for the development of these designer solvents as their structure can be engineered to simultaneously meet functionality performance and environmental safety. The molecular effects of ILs were investigated by using RNA-sequencing following Daphnia magna exposure to imidazolium- and cholinium-based ILs: 1-ethyl-3-methylimidazolium chloride ([C2mim]Cl), 1-dodecyl-3-methylimidazolium chloride ([C12mim]Cl) and cholinium chloride ([Chol]Cl)-; the selection allowing to compare different families and cation alkyl chains. ILs shared mechanisms of toxicity focusing e.g. cellular membrane and cytoskeleton, oxidative stress, energy production, protein biosynthesis, DNA damage, disease initiation. [C2mim]Cl and [C12mim]Cl were the least and the most toxic ILs at the transcriptional level, denoting the role of the alkyl chain as a driver of ILs toxicity. Also, it was reinforced that [Chol]Cl is not devoid of environmental hazardous potential regardless of its argued biological compatibility. Unique gene expression signatures could also be identified for each IL, enlightening specific mechanisms of toxicity. published

The toxicity of Ionic Liquids (ILs) to aquatic organisms has been a matter of substantial interest, involving the toxicity assessment for a small number of species, which is limitative given the variation in species sensitivity to different classes of ILs. Therefore, the main objective of this study was to validate, using an integrative approach (i.e., integrating the responses of several species), the heuristic rules that have been assumed for the ecotoxicity of ILs, namely the effects of the cation, elongation of the cation alkyl chain and anion moiety. For this purpose, four ILs were selected as models and their toxicity was determined for a wide variety of species, which allowed the development of species sensitivity distribution curves. The analysis of the distribution curves enables the determination of hazard concentrations affecting 5% of the represented communities (HC5) and also the establishment of structure-ecotoxicity relationships for ILs. The median effect concentration (EC50) values varied widely and the species most sensitive to each IL was variable. The integrative HC5 values varied by five orders of magnitude, between 3.020 × 10-3 mg L-1 representing the 1-dodecyl-3-metilimidazolium chloride, which raises concerns on the environmental hazardous potential of this IL, and 106.9 mg L-1 for cholinium chloride, which confirms its low environmental toxicity. The SSD approach showed cholinium chloride as the least toxic IL, followed by cholinium dihydrogenocitrate, 1-ethyl-3-methylimidazolium and 1-dodecyl-3-methylimidazolium chloride, as the most toxic. This illustrates the minor effect of the anion on toxicity for this set of ILs, whereas the cation and the cation alkyl chain length had pronounced effects, validating the heuristic rules defining structure-ecotoxicity relationships of ILs. A very strong linear correlation between hydrophobicity and HC5 was found (ρ =-0.9991). This approach allows a more efficient prediction of the potential environmental effects of ILs, thus preventing the need to comprehensively assess to the ecotoxicity of all ILs, which can be many within each family and each cation/anion possible combination. Ultimately, this will sustain the development of ILs posing less environmental hazards nonetheless retaining the desired performance. published

Cyanobacteria can cause ecological, economic and human health problems, climate change trends giving them advantage over other phytoplanktonic groups. Management strategies exist to deal with the nuisance, but many are not effective due to the likelihood of cell lysis and toxin release. This study explores the suitability of the use of the invasive and widespread bivalve Corbicula fluminea for cyanobacterial control. The capacity of clams to filter and ingest cyanobacteria was evaluated using a set of bloom-forming strains comprising different morphological features potentially affecting edibility and palatability. Results generally showed limited filtration of the majority of cyanobacteria, compared with green microalgae used as the reference for optimal filtration (ca. 60 μg Chl a removed in 120 min), except for Pseudanabaena (ca. 90 μg Chl a removed) and Anabaena (ca. 120 μg Chl a removed). The specific attention given to the binomial filtration-ingestion exposed that filtration often directly relates to the deposit of cyanobacteria as pseudofaeces rather than reflects assimilation through effective ingestion. Allocation to pseudofaeces accounted for more than 50% of the Chl a removed from water for all cyanobacteria tested. Adding to effective filtration by the clam, the related accumulation of cyanobacterial biomass in pseudofaeces has the potential for further exploitation since it can be functionally paralleled with the use of synthetic flocculants for cyanobacterial (villain) removal in settings where a natural treatment alternative using an (bad) invasive bivalve could be suitable yet controlled to avoid side ecosystem effects.

The development of environmentally friendly plant protection products (PPPs), including pesticides, is a challenge nowadays. A commercial herbicide combining terbuthylazine and nicosulfuron as active substances (a.s.) was selected as a model PPP. The suitability of manipulating the ratio between a.s. towards alternative formulations with reduced impacts in a non-target indicator (Lemna minor) was assessed. The efficacy of such eco-friendlier a.s. ratios was then assessed using a target weed, Portulaca oleracea. Single and mixture toxicity testing with L. minor revealed an antagonistic joint action of the a.s., suggesting an environmentally protective effect of the combination compared to single dosing of a.s. The efficacy testing against the target weed of single and combined treatments of the a.s. showed that (i) the a.s. behave antagonistically throughout the whole P. oleracea response surface; (ii) there were no environmentally safe a.s. combinations ensuring target-efficacy; (iii) terbuthylazine alone was effective in controlling P. oleracea with no environmental hazardous potential, dosed at concentrations 10-fold lower than those involved in commercially recommended application doses. Overall, this case-study suggests that modelling tools widely used in the field of environmental risk assessment of PPPs may also have application in PPP design stages for a more efficient meeting of efficacy and environmental friendliness requirements.

Wildfires are an important environmental problem in forested watersheds and can significantly alter water quality. Besides the reported ecotoxicological effects on pelagic species, the accumulation of post-fire contaminants in river sediments can also impair the benthic species. In this study, three sediment-dwelling species, Chironomus riparius, Atyaephyra desmarestii and Echinogammarus meridionalis, with different sensitivities, habitats, behaviours and/or feeding strategies, were exposed to water and sediments, in in-situ and in laboratory. Four sites were selected in a partially burnt basin (Alfusqueiro river basin), within and upstream the burnt area. The sites within the burnt area showed higher metal burden in both water and sediment, as well as changes in water physico-chemistry, consistently with the typical effects of incoming post-fire runoff. Both in-situ and laboratory exposures to water and sediments affected by the wildfire induced post-exposure feeding inhibition in the three tested macroinvertebrates. In fact, laboratory and field bioassays have produced generally consistent post-exposure feeding inhibition responses, but the most impactful response could be recognised after in-situ bioassays at the river site within the burnt area, where the species respond to the physico-chemical fluctuations during the exposure period. This comparative perspective supports the importance of using in-situ bioassays as a more realistic approach when dealing with complex and intermittent natural samples such as those affected by post-fire runoff. Overall, our results reinforce the awareness about the negative effects of wildfires on benthic biota, with significant feeding depression and consequent reduction in the available energy budget to ensure successful detoxification, growth and reproduction signalling potential trophic and functional disruption at the ecosystem level. In addition, the duality conditions of sediments as a sink and source of contaminants reinforce concerns, as the exposure of benthic organisms may persist in the long term, even after runoff income ceases due to the resuspension of contaminated sediments.

The number of wildfires has markedly increased in Mediterranean Europe, including in Portugal. Wildfires are environmentally concerning, not only due to the loss of biodiversity and forest area, but also as a consequence of environmental contamination by specific compounds including metals and polycyclic aromatic compounds (PAHs). These contaminants, mostly bound to ashes, can reach downstream water bodies, namely through surface runoff, being ultimately dispersed by vast areas and contacting with aquatic biota. Being toxicologically noteworthy, the potential toxic outcomes of the input of such chemicals across the aquatic compartment must be characterized. In this context, the present study used a biomarker-based approach to find early-warning signals of toxicity triggered by the exposure of the mosquitofish, Gambusia holbrooki, to affected aqueous runoff and stream water samples collected from a forest burnt area. The chemical analysis revealed concerning levels of metals and polycyclic aromatic hydrocarbons in both runoff and stream water samples. Biological responses elicited by the collected samples showed the occurrence of pro-oxidative modifications, specifically driven by enzymatic forms involved in the metabolism of glutathione. Despite these effects, no further signs of involvement of metals and PAHs were elicited in terms of neurotoxicity. The overall set of data implicates chemicals resulting from wildfires in clear deleterious effects in exposed fish. published

Generally, Antarctica is considered to be an untouched area of the planet; however, the region's ecosystems have been subject to increased human pressure for at least the past half-century. This study assessed soils of Fildes Peninsula, where trace element pollution is thought to prevail. Four soil samples were collected from different locations and assessed following tier 1 methodologies for chemical and ecotoxicological lines of evidence (LoE) used in typical soil Environmental Risk Assessment (ERA). Trace element quantification was run on soil samples and sequential extracts, and elutriates were used to address their ecotoxicity using a standard ecotoxicological battery. The highest levels of trace elements were found for Cr, Cu, Ni and Zn, which were well above baseline levels in two sites located near previously identified contamination sources. Trace element concentrations in soils were compared with soil quality guidelines to estimate the contribution of the chemical LoE for integrated risk calculations; risk was found high, above 0.5 for all samples. Total concentrations in soil were consistent with corresponding sequentially extracted percentages, with Cu and Zn being the most bioavailable elements. Bacteria did not respond consistently to the elutriate samples and cladocerans did not respond at all. In contrast, the growth of microalgae and macrophytes was significantly impaired by elutriates of all soil samples, consistently to estimated trace element concentrations in the elutriate matrix. These results translated into lower risk values for the ecotoxicological compared to the chemical LoE. Nevertheless, integrated risk calculations generated either an immediate recommendation for further analysis to better understand the hazardous potential of the tested soils or showed that the soils could not adequately sustain natural ecosystem functions. This study suggests that the soil ecosystem in Fildes has been inadequately protected and supports previous claims on the need to reinforce protection measures and remediation activities.

The aim of this special issue was to bring together papers dealing with current challenges and trends in pesticide ecotoxicology. The special issue is composed of 25 papers into various topics, ranging from the development of more ecological ecotoxicological test approaches to an evaluation of citation bias in pesticide ecotoxicology literature. This paper provides a general introduction and an index of the various papers in the special issue. This special issue is trusted to provide a valuable contribution in addressing current challenges in pesticide ecotoxicology.

Mediterranean forests are highly susceptible to wildfires, which can cause several impacts not only within burnt areas but also on downstream aquatic ecosystems. The ashes’ washout from burnt areas by surface runoff can be a diffuse source of toxic substances, such as metals, when reaching the nearby aquatic systems, and can be noxious to aquatic organisms. The present work aimed at assessing the ecotoxicological effects of post-fire contamination on two aquatic producers (the microalgae Raphidocelis subcapitata and the macrophyte Lemna minor) through in-situ bioassays, validating the obtained results with the outcomes of laboratory bioassays with surface water collected simultaneously. Four distinct sites were selected in a basin partially burnt (Ceira river basin; Coimbra district, Portugal) for bioassay deployment: one site upstream the burnt area in the Ceira river (RUS); three sites located under the influence of the burnt area, one immediately downstream of the burnt area in the Ceira river (RDS) and the other two in tributary streams within the burnt area (BS1 and BS2). The in-situ bioassays lasted for 13 days and began following the first post-fire major rain events. Results showed that the microalgae growth rate was able to distinguish the three sites within and downstream of the burnt area (BS1, BS2, RDS) from the site upstream (RUS). By contrast, the macrophytes growth rate only allowed to differentiate between the sites within the burnt area (BS1 and BS2) and those up- and downstream of the burnt area (RUS and RDS). The in-situ results for both species were corroborated with the results of the laboratory experiments, supporting the use of laboratory surrogates for a screening assessment of wildfire impacts in aquatic ecosystems. Direct causal relationships between the observed ecotoxicological effects on R. subcapitata and L. minor and the physical-chemical parameters of the water samples were difficult to establish, although the results suggest (i) a role of differential major and trace metal load in explaining species growth variation; (ii) interaction between metals and/or between metals and other field parameters are likely to modulate the biological responses to the challenges deriving from wildfire runoff.

Freshwater ecosystems face widespread diffuse and point-source contamination. Species Sensitivity Distributions (SSDs) have been used as a tool to determine chemical concentration benchmarks that represent protective levels for most species in the environment. Here we used a SSD approach to assess on the adequacy of standard planktonic organisms to reflect the response of benthic communities, critically supporting the structure and function of lotic ecosystems. For the purpose, SSDs reflecting non-lethal responses of standard planktonic and selected benthic organisms were built based on EC50 values (collected in the literature or estimated following testing herein) regarding three model contaminants: potassium dichromate (PD), 3,5-dichlorophenol (DCP) and lead chloride (LC). The derived HC5 estimates were discriminatory between chemicals and the uncertainty associated with the estimate was remarkably low. The HC5 estimates with corresponding uncertainty were generally within the same order of magnitude for the three chemicals tested, with better discrimination between chemicals regarding their hazardous potential being achieved for benthic organisms: DCP was clearly less hazardous than PD, but LC tends to be as hazardous as PD and DCP (assuming the confidence interval ranges). Moreover, benthic communities were more sensitive to both DCP and PD, in this later case the HC5 being lower by more than one order of magnitude than that found for planktonic communities; for LC, confidence intervals overlapped, preventing a feasible assumption regarding differential sensitivity of the compared communities. Microphytobenthos was highlighted as the most sensitive group to the three tested chemicals in SSDs covering the benthic compartment, while SSDs with planktonic organisms did not consistently show trends in sensitivity ordering. Overall, our results suggest that protective benchmarks retrieved from SSDs built with the responses of standard planktonic organisms (which are the most commonly used for regulation purposes) do not adequately protect benthic communities.

Metal contamination, as well as pesticides, organic matter and nutrient input are main factors leading to freshwater ecosystems degradation. The Water Framework Directive (WFD) was implemented within the European Union with the ultimate goal of promoting a good ecological status in all European waterbodies. However, the broad implementation of the bioassessment behind WFD is costly and time-consuming and the search for complementary methodologies has been given significant attention. In this context, the main goal of this study was to evaluate whether flow cytometry (FCM) and denaturing gradient gel electrophoresis (DGGE) can be used as cellular/molecular tools to efficiently assess riverine bacterioplankton communities and relevantly inform on the ecological quality of these ecosystems. Caima river was chosen as case study using three sampling sites reflecting different levels and types of contamination (point-source organic and metal input). Both bacterioplankton community assessment approaches (DGGE and FCM), as well as macroinvertebrate and periphyton communities were consistent in signaling organic contamination. The putatively metal-loaded site bears some contradictory results depending on the community focused, possibly due to the overall low levels of metals actually found and seasonality. When comparing the two bacterioplankton community analysis tools, DGGE and FCM, the results obtained were essentially coherent, with FCM being simpler, faster and still accurate for screening bacteria communities via quantification of bacteria of high and low DNA content. This highlights the suitability of the FCM approach for prioritization of contaminated sampling sites and reinforces the suitability of using bacterioplankton communities as the focus of rapid tools to complement bioassessment sensu the WFD methodology, e.g. assisting the prioritization of potentially impacted areas.

Caenorhabditis elegans has been an invaluable model organism in research fields such as developmental biology and neurobiology. Neurotoxicity is one of the subfields greatly profiting from the C. elegans model within biomedical context, while the corresponding potential of the organism applied to environmental studies is relevant but has been largely underexplored. Within the biomedical scope, the implication of metals and organic chemicals with pesticide activity (hereinafter designated as pesticides) in the etiology of several neurodegenerative diseases has been extensively investigated using this nematode as a primary model organism. Additionally, as a well-known experimental model bearing high sensitivity to different contaminants and representing important functional levels in soil and aquatic ecosystems, C. elegans has high potential to be extensively integrated within Environmental Risk Assessment (ERA) routines. In spite of the recognition of some regulatory agencies, this actual step has yet to be made. The purpose of this review is to discuss the major advantages supporting the inclusion of C. elegans in lower tiers of ERA. Special emphasis was given to its sensitivity to metals and pesticides, which is similar to that of other model organisms commonly used in ERA (e.g. Daphnia magna and Eisenia sp.), and to the large array of endpoints that can be tested with the species, both concerning the aquatic and the soil compartments. The inclusion of C. elegans testing may hence represent a relevant advance in ERA of chemicals, providing ecologically relevant insights towards improvement of the regulatory capacity for establishing appropriate environmental protection benchmarks.

International audience; Ionic Liquids (ILs) are generically regarded as environmentally “harmless” and thus, assumed as “non-toxic”. However, due to the endless design possibilities, their ecotoxicological profile is still poorly known. An accurate knowledge on the toxicity of a substance is required, under the scope of environmental regulation worldwide, before their application and commercialization. Knowledge on the relationship between the chemical structure and toxic effects is essential for the future design of more biocompatible solvents. Focusing on the use of ILs as base lubricants, lubricant additives, or even as potential working fluids for absorption heat pumps, the knowledge on its environmental impact is of great importance, due to the possibility of spills. In this specific context, four analogues of glycine-betaine-based ILs (AGB-ILs) and four glycine-betaine based ILs (GB-ILs) were synthesized and characterized. Their ecotoxicity was assessed using representatives of two trophic levels in aquatic ecosystems, the bacteria Allivibrio fischeri (commonly used as a screening test organism) and the microalgae Raphidocelis subcapitata (as an alternative test organism that has been proven very sensitive to several IL families). The microalgae were more sensitive than the bacteria, hence, following a precautionary principle, we recommend considering the toxicity towards microalgae as an indicator in future studies regarding the focused ILs. Although four of the studied ILs were derived from a natural amino acid, all were considered hazardous for the aquatic environment, disproving the primary theory that all ILs derived from natural compounds are benign. Furthermore, the modification in the structure of anion and the cation can lead to the increase of toxicity.

As a consequence of the growing global dependence on groundwater resources, environmental risk assessments (ERA) for groundwater are increasingly required and, with that, ecotoxicological studies with groundwater fauna (stygofauna). However, the literature on the ecotoxicological studies with stygobiotic species (i.e. species that complete their life cycle exclusively in groundwater) has not expanded significantly since the first paper published in this field. The limitations regarding the use of stygobiotic species for ecotoxicological testing are clear and consistent across the globe; stygobiotic species are often 1) naturally present in low numbers, 2) difficult to collect, and 3) difficult to culture under laboratory conditions. This paper reviews the methods used in ecotoxicological studies performed with stygobiotic species, and provides ten recommendations for Good Laboratory Practice (GLP) for such tests. The recommendations focused on the following 10 points: 1) the taxonomic identification, the life stage/size and gender of the test organisms; 2) collection methodology of the organisms, including collection location, conditions and methods; 3) holding and acclimation conditions in the laboratory; 4) exposure conditions such as test set up and exposure time, number of replicates and densities of organisms in tests and in test vessels; 5) range-finding test set up and schedule; 6) final test design, including details of controls and treatments, and replication options; 7) incubation conditions, specifying temperature, pH and oxygenation levels throughout the test; 8) test duration; 9) observations and endpoints; 10) test validity criteria and compliance. The recommendations were developed for the purpose of supporting future short-term ecotoxicological testing with stygofauna through providing consistency in the protocols. A discussion of the potential implications for groundwater managers and decision-makers committed to ERA for groundwater is included.

Benthic diatoms constitute keystone assemblages in riverine ecosystems, and their structure is used to support regulatory water quality assessment. However, no standard ecotoxicological tests exist using integrated responses of the benthic diatom assemblages. This work aimed to assess whether benthic diatom assemblages are responsive to different riverine contaminants through a previously developed rapid toxicity test, supporting future attempts towards its standardization and integration in both prospective and retrospective Environmental Risk Assessment (ERA) schemes. We selected two benthic diatoms assemblages likely responding similarly to pollution (similar IPS diatom index score), collected from two rivers in Northern-Central Portugal (sites: Palhal and Cabreia). Fresh whole diatom assemblages were exposed for 48 h to five model contaminants (glyphosate, imidacloprid, SDS, CuSO4, and Pb). At the end of the test, changes induced by the exposures in overall yield and in the yield of each diatom genus were assessed. The assemblage collected at Palhal was invariably more responsive and sensitive than that collected at Cabreia, both considering overall and genus-specific yields, regardless of the tested contaminant. Achnanthes, Fragilaria and Navicula were the most responsive genus, regardless of the tested contaminant or assemblage. The distinct response profiles observed for the two assemblages to the same contaminants at the same concentration ranges suggest that using this test method to support prospective ERA is inadequate. However, the method can be an asset supporting retrospective ERA, as the responses seem to be shaped by the interplay of resilience drivers promoted by the local conditions, e.g. adaptive changes in assemblage structure.

Wildfires, increasingly frequent and severe in the Mediterranean countries, can cause serious impacts on ecosystems. As a diffuse source of contaminants – namely inorganic elements (metals, metalloids and non-metallic elements) and polycyclic aromatic hydrocarbons (PAHs) – post-fire runoff may cause toxic effects on the aquatic biota. Despite the recognition of these likely impacts, this issue has been still largely neglected. This study explores the impacts of ash from wildfires on the freshwater clam Corbicula fluminea and the ability of this filter feeder to concentrate the contaminants associated with ash in post-fire runoff. The bio-concentration potential of the clam was assessed in a 7-day filtration experiment, using aqueous extract of ashes (AEA) to represent post-fire runoff. Despite the noticeable mortality of the clams exposed to the AEA (∼30% higher in AEA than in control after 7 days of exposure), there was a clear reduction on the concentration of almost all pyrolytic substances in the water linked to bio-filtration and bio-concentration activity. Along 7 days the clams operated decreases of more than 70% for some chemicals (Cu, Mn and Zn) while others were no longer detected in the water at the end of the experiment (Cr, NAP and PHE). The removal from water was accompanied by contaminants’ accumulation in clams’ soft tissues and, less extensively, in shells. Lower pollutant loads of filtered AEA translated into decreased toxicity of the matrix towards standard aquatic species (microalgae, macrophytes and cladocerans) confirming an improvement as well in the biological quality of the water through bio-filtration. Although post-fire loads can compromise C. fluminea survival, this study evidenced the potential of this invasive species to contribute to water quality improvement.

Among the various environmental disturbances caused by wildfires, their impacts within burnt areas and on the downhill aquatic ecosystems has been receiving increased attention. Post-fire rainfalls and subsequent runoffs play an important role in transporting ash and soil to aquatic systems within the burnt areas. These runoffs can be a diffuse source of toxic substances such as metals. The present work aims at assessing the effects of ash-loaded runoff on feeding rates of three representative aquatic invertebrates (Daphnia magna, Corbicula fluminea and Atyaephyra desmarestii) and the mosquitofish, Gambusia holbrooki, through post-exposure feeding inhibition bioassays carried out in-situ and in the laboratory using water collected from the experimental field sites. Four sites were selected in a partially burnt basin for bioassay deployment and sample collection: one site upstream of the burnt area (RUS); three sites receiving runoff directly from the burnt area, one immediately downstream of the burnt area (RDS) and two in permanent tributary streams within the burnt area (BS1 and BS2). The in-situ exposure lasted four days and began following the first post-fire major rain events. At sites affected by the wildfire, post-exposure feeding rates for D. magna, A. desmarestii and G. holbrooki were lower, which is consistent with the highest levels of metals found at these sites compared to the unaffected site, although the individual concentrations of each metal were generally below corresponding ecological safety benchmarks. Thus, interactions between metals and/or between metals and other environmental parameters certainly played a role in modulating the ecotoxic effects of the runoffs; this was further supported by a Toxic Units Summation exercise. Even if direct causal links between the ecotoxicological effects observed in D. magna, A. desmarestii and G. holbrooki and the physicochemical parameters of the water samples could not be established, the results suggest an important role of major and trace elements in explaining post-exposure feeding rate variation.

The responses of non-target organisms to pesticide exposure are still poorly explored in what concerns the development of adjustments favouring population success. Owing to the vital role of DNA integrity, it is important to identify genome-maintenance skills and their determinant factors. Thus, the major aims of the present study were: (i) to assess the genotoxicity of the penoxsulam-based herbicide (Viper®) to the crayfish Procambarus clarkii; (ii) to understand the influence of gender and contamination history in the genotoxic responses following exposure to this herbicide; (iii) to investigate the damage mechanisms involved in putative adjustments shown by P. clarkii. Two populations were tested, one from a reference site and the other from a historically contaminated site. Specimens from both populations were exposed to Viper®, considering environmentally relevant penoxsulam concentrations (20 and 40 µg L−1) and to a model genotoxicant (EMS). Comet assay was adopted to assess the genetic damage in gills. The results disclosed the genotoxicity of the herbicide to crayfish (a non-target organism). Additionally, organisms exposed to the highest concentration of penoxsulam signalized the influence of factor “population” towards the genotoxic pressure (measured as effective DNA breaks): P2 males from the historically impacted population displayed a significantly higher susceptibly (by up to 53.98%) when compared to control, while the homologous group from the reference population presented levels similar to its respective control. When DNA lesion-repair enzymes were considered, DNA oxidation patterns suggested an increased ability of this gender (39.75% lower than negative control) to deal with this particular type of damage, namely considering pyrimidines oxidation. It is worth remarking that the influence of the exposure history on the protection/vulnerability to the penoxsulam-based herbicide was only evident in males, despite depending on the type of DNA damage: when the non-specific damage was considered, organisms from the impacted population seemed to be more vulnerable while regarding to the oxidative damage, males from the impacted population appeared to be more protected than organisms that have never been exposed to penoxsulam. Overall, the influence of factors “gender” and “contamination history” was demonstrated as well as its dependence on DNA damage type was evident. EMS groups did not present the differences between populations, reinforcing the agent-specific adjustment hypothesis. These findings highlighted the importance of considering differential physiological backgrounds in ecogenotoxicological analysis, hence favouring the elaboration of more plausible and holistic approaches integrating the environmental risk assessment of pesticides.

Formulants used in Plant Protection Products (PPPs) to promote their efficiency are normally undisclosed in the PPP documentation, unless they bear a human health or environmental hazardous potential per se. PPP regulation also demands the assessment of putative interactions among formulants within each product recipe and consequent effects, but these results are often unavailable. Such a case is that of the herbicide Winner Top (Selectis®, Portugal), which we selected as a model commercial formulation in the present study specifically aiming at (i) characterising its aquatic toxicity towards sensitive eco-receptors (Raphidocelis subcapitata, Chlorella vulgaris, Lemna minor and Lemna gibba), as well as that of its active substances (a.s.) nicosulfuron and terbuthylazine; (ii) comparing the ecotoxicity among the commercial formulation, the corresponding mixture of its a.s. and this a.s.'s mixture increasingly enriched with the formulants. Single chemical testing revealed that terbuthylazine was the strongest microalgae growth inhibitor and nicosulfuron was the strongest macrophyte growth inhibitor. On the other hand, the commercial formulation was consistently less toxic than the corresponding mixture of the a.s., suggesting that Winner Top formulants (72.9% of the commercial formulation) interact with the a.s., promoting less than additive effects in the selected non-target species. Importantly, this environmentally protective effect of the formulation can be apparent. Because macrophytes share most physiological features with the weeds targeted by the studied herbicide, it is likely that increased application doses are required to reach desired efficacy levels with the consequent detrimental increase of PPP residues load in edge-of-field freshwater ecosystems.

Mercury naturally contaminated environments, like Deception Island (Antarctica), are field labs to study the physiological consequences of chronic Hg-exposure at the community level. Deception Island volcanic vents lead to a continuous chronic exposure of the phytoplanktonic communities to potentially toxic Hg concentrations. Comparing Hg-contaminated areas (Fumarolas Bay - FB, Gabriel de Castilla station - GdC station), no significant differences in chlorophyll a concentrations were detected, indicating that biomass production was not impaired by Hg-exposure despite the high Hg levels found in the cells. Moreover, the electron transport energy, responsible for energy production, also presented rather similar values in phytoplankton from both locations. Regarding FB communities, although the cells absorbed and trapped lower amounts of energy, the effect of Hg was not relevant in the photochemical work produced by the electronic transport chain. This might be due to the activation of alternative internal electron donors, as counteractive measure to the energy accumulated inside the cells. In fact, this alternative electron pathway, may have allowed FB communities to have similar electron transport energy fluxes without using respiration as photoprotective measure towards excessive energy. Hg-exposed cells also showed a shift from the energy flux towards the PS I (photosystem I), alleviating the excessive energy accumulation at the PS II (photosystem II) and preventing an oxidative burst. Our findings suggest a higher energy use efficiency in the communities exposed to volcanic Hg, which is not observable in cultured phytoplankton species grown under Hg exposure. This may constitute a metabolic adaptation, driven from chronic exposure allowing the maintenance of high levels of primary productivity under the assumingly unfavourable conditions of Deception Island.

Bivalves are commonly used in biomonitoring programs to track pollutants. Several features, including its filter feeding abilities, cumulatively argue in favour of the use of the Asian clam (Corbicula fluminea) as a biosentinel and an ecotoxicological model. Filtration in bivalves is very sensitive to external stimuli and its control is dictated by regulation of the opening/closure of the valves, which may be used as an avoidance defence against contaminants. Here, we investigate the filter-feeding behaviour of the Asian clam as an endpoint for assessing exposure to pollutants, driven by two complementary goals: (i) to generate relevant and sensitive toxicological information based on the ability of C. fluminea to clear an algal suspension, using the invasive species as a surrogate for native bivalves; (ii) to gain insight on the potential of exploring this integrative response in the refinement of chemical control methods for this pest. Clearance rates and proportion of algae removed were measured using a simple and reproducible protocol. Despite some variation across individuals and size classes, 50-90% of food particles were generally removed within 60-120 min by clams larger than 20 mm. Removal of algae was sensitive to an array of model contaminants with biocide potential, including fertilizers, pesticides, metals and salts: eight out of nine tested substances were detected at the mu g l(-1) or mg l(-1) range and triggered valve closure, decreasing filter-feeding in a concentration-dependent manner. For most toxicants, a good agreement between mortality (96 h - LC50 within the range 0.4-5500 mg l(-1)) and feeding (2 h - 1050 within the range 0.005-2317 mg l(-1)) was observed, demonstrating that a 120-min assay can be used as a protective surrogate of acute toxicity. However, copper sulphate was very strongly avoided by the clams (IC50 = 5.3 mu g l(-1)); on the contrary, dichlorvos (an organophosphate insecticide) did not cause feeding depression, either by being undetected by the clams' chemosensors and/or b, This work was supported by the strategic programs UID/AMB/50017/2013 (POCI-01-0145-FEDER-007638) and UID/BIA/04050/2013 (POCI-01-0145-FEDER-007569), funded by the European Regional Development Fund through COMPETE2020 and PT2020, and by National Funds through the Portuguese Science Foundation (FCT). Funding entities had no role in the study design, in the collection, analysis and interpretation of data, in the writing of the paper, or in the decision to submit the article for publication. JL Pereira is a recipient of an individual research grant by FCT (SFRH/BPD/101971/2014)., info:eu-repo/semantics/publishedVersion

Cationic acrylamide-based polyelectrolytes (cPAM) are widely used in industry. They can be designed for optimal performance in a specific application, but this opportunity means the environmental safety of all different alternatives needs to be addressed. Both the inclusion of environmental toxicity as a design variable and the establishment of relationships between structure and ecotoxicity are thus current challenges. The aim of this study was to assess whether structural variables such as molecular weight, charge density and the integrative intrinsic viscosity parameter can be used to predict the environmental safety of cPAMs, as well as if these relationships are stable when the biological models change. Five cPAMs comprising molecular weight and charge density gradients were tested against bacteria, microalgae, macrophytes and daphnids. While correlations were found between physical properties of cPAMs as expected, no clear ecotoxicity patterns could be identified. All cPAMs can be classified as harmful to aquatic life on the basis of the responses elicited in the most sensitive organisms, microalgae and daphnids. Unicellular bacteria were the least sensitive eco-receptors possibly due to cell wall structure or the protective effect of the ionic strength of the test medium. The macrophytes were also tolerant to cPAMs exposure, which may be related to exposure avoidance mechanisms. The order of toxicity of cPAMs depended on the test organism, preventing the establishment of stable structure-ecotoxicity relationships. Therefore, the study leads to the overall generalist recommendation of relying on the most sensitively responding test organisms when developing new (eco)safe-by-design cPAMs.

Corbicula fluminea, is one of the most successful invasive species in fresh and brackish waters. Dispersal is one of the most determinant steps in the invasive process, and the full understanding of the mechanisms involved in this step is critical for adequate pest management both in the wild and in industries affected by this species' biofouling activity. A mucous drogue line produced by mucocytes packed along the inner demibranchs of the clams' gills seem to play an important role in assisting drifting and hence dispersal. Two Asian clam populations geographically separated (one in the USA and the other in Portugal), in- vestigated at different times of the year, were reported to differ in terms of mucous drogue line production and floating. In this study, genetics and seasonality effects were hypothesized to explain the difference between the populations. To test these hypotheses, the two populations were genetically compared, and the Portuguese one was followed for 14 months to record the animals' mucous drogue line production and flotation capabilities and locate the population reproductive periods. Our results signal a possible scenario of micro- evolution with consequences on the production of the clams' mucilaginous drogue line. Although some authors advocate a link between mucous threads formation and reproduction events, such a relationship was not observed in this study. By contributing to the understanding of a physiological trait of the Asian clam that is important for dispersal, this study may be of practical relevance for pest monitoring and control.

While the biofouler Corbicula fluminea (Muller, 1774) is known to cause great economic losses in North America, studies reporting the problem in Europe are much scarcer. This paper explores the industrial effects of the species in Portugal, the gateway by which the bivalve entered Europe around 30 years ago. National waterworks, major power stations, cement plants, pulp and paper mills and irrigation systems were surveyed. The industrial impacts of the pest were shown to remain relatively mild; irrigation systems are those that seem to be facing more significant economic losses due to infestation. Possible reasons for the apparent discrepancy between this result and the species dispersal in natural ecosystems are discussed, and recommendations on adequate responses to the latent threat are provided. This study may assist the implementation of integrated pest management policies in countries at risk of invasion or recently invaded, and contribute to an understanding of the species’ progression in industrial environments.

The commercial herbicide formulation Betanal® Expert and its active ingredients (a.i.s) ethofumesate, phenmedipham and desmedipham were focused in this study. Following questions yielding from a previous study, an in-depth analysis of the reproductive toxicity of the pesticide was made. Long-term exposures of Daphnia magna and Daphnia longispina to Betanal® Expert, to each a.i. and to a customised mixture matching the a.i.s ratio within the commercial formulation were carried out, and deleterious effects in the offspring were recorded. This intended to clarify whether (1) the tested compounds induce reproductive injury; (2) there is interspecific variation in daphnids tolerance to the compounds; (3) there is an interaction between chemicals in combined treatments; and (4) the so-called inert ingredients added to the commercial formulation contribute to the toxicity of the herbicide. Generally, developmental impair was observed in both species (egg abortion and release of undeveloped embryos or dead offspring) at concentrations of any of the a.i.s below 1 mg L(-1). Ethofumesate was invariably the least toxic pesticide, and D. magna tended to be of slightly higher sensitivity to the exposures compared to D. longispina. Joint exposures indicated that the a.i.s can interact, inducing more than and less than additive effects for Betanal® Expert and the customised a.i. mixture, respectively. This indicates that inert ingredients co-formulating the commercial pesticide (which are absent from the customised a.i. mixture) actually contribute to its overall toxicity. This study constitutes an add-on to the discussion on the ecotoxicological framework required for authorisation of pesticide trade and usage. The results support the need to consider test species, long-term hazardous potential and toxicity of commercial formulations rather than solely that of active ingredients, as relevant variables in pesticide regulation.

The availability of rapid and effective methodologies for assessing lotic systems with microphytobenthos is still quite scarce. Hence, the primary goal of this study was to optimize the growth conditions of the sensitive and ubiquous benthic diatom Navicula libonensis for laboratorial and field assessments. The effect of different conditions of temperature, photoperiod, initial cell density, test duration and cell encapsulation into calcium alginate beads was evaluated in a first set of experiments. There was a slight increase in the growth of free and immobilized cells at 23 °C, at lower initial cell densities and at the shortest experimental period (6 days). Through all the conditions, the growth profiles of free versus immobilized were fairly variable. A second experimental trial involved the validation of selected conditions, applied to the ecotoxicological testing of N. libonensis to two reference chemicals—3,5-dichlorophenol and potassium dichromate. A similar response of free and immobilized cells was observed between exposures to spiked stream water and synthetic medium, and through the conditions tested. This outcome suggests that N. libonensis may potentially provide reliable responses under direct in situ exposures.

The biofouling Asian clam Corbicula fluminea greatly affects freshwater-dependent facilities in Europe and North America, including in the Great Lakes region. As chlorination has become increasingly restricted, finding alternative control agents is a priority. Due to the species' epifaunal nature, the body of knowledge on Dreissena polymorpha is larger than that on the Asian clam, and there is a tendency to assume that mitigation methods should work similarly for both species. However, this generalisation is inaccurate, and the optimisation of Asian clam control relies on species-specific toxicological data. This paper reports information on the potential of three candidate biocides for C. fluminea control: (i) the cationic polydiallyldimethylammonium chloride (polyDADMAC), (ii) potassium chloride and (iii) aluminum sulphate. While these chemicals may be employed in a range of contexts, they are particularly suitable for the highly regulated drinking water industry. LT 50 values ranging from 284.3 h, for polyDADMAC applied at 10 mg/L, to 855.1 h, for an aluminum sulphate concentration of 11 mg/L, were obtained. Ecotoxicological standard tests with Pseudokirchneriella subcapitata and Daphnia magna suggested that, amongst the three biocides, potassium chloride is the one representing lower potential environmental hazard (with, for example, 48 h-EC 50 higher than 1 g/L for Daphnia immobilisation, which compares to values of 20.2 and 112.5 mg/L for polyDADMAC and aluminum sulphate, respectively). The three chemicals are promising control agents at dosages compatible with waterworks' operational requirements with polyDADMAC having the highest biocidal activity, but also posing more environmental risks.

Soil avoidance by earthworms has been generally considered a relevant and sensitive endpoint for assessing soil contamination by xenobiotics. However, when pesticide ecotoxicological assessment is concerned, the sensitivity of the recently standardized avoidance assay has been questioned. We hypothesized that this controversy may be due to the specific pesticide mode of action of the chemicals used rather than reveal inconsistencies in the test feasibility, i.e. provided that no pesticides interfering with neuronal pathways are tested, this bioassay should keep expected high levels of sensitivity. In this study, the avoidance behaviour of the earthworm Eisenia andrei under exposure to the carbamate insecticide methomyl [S-methyl N-(methylcarbamoyloxy)thioacetimidate] was linked to the corresponding acetylcholinesterase (AChE) inhibition. Significant AChE inhibition occurred at lower concentrations (from 0.86 mg Kg(-1) onwards) than significant avoidance of spiked soil (from 5.62 mg Kg(-1) onwards). This indicates that assessments regarding pesticides that have neurotoxic activity may be biased if behavioral endpoints are selected. Despite theoretical hypothesis that have been raised, this should be the first study providing preliminary experimental evidence on such a link between avoidance behavior and neuronal impairment levels in earthworms. Further studies are ongoing that should refine conclusions of this study. published

Zinc pyrithione (ZnPT) is an organometallic biocide with bactericide, algaecide, and fungicide activity. Considering this biological activity, ZnPT has been used in anti-fouling paints, and also in human therapeutics and cosmetics, in shampoos to treat dandruff and seborrhoea. Despite its potential uses and consequent presence in the aquatic environment, the ecotoxicological effects of ZnPT are poorly understood. This work aims to characterise the effects of ZnPT in biochemical parameters of the Asian clam, one of the most invasive bivalves known for its biofouling action in hydro-dependent industries, using a classical (LC50 determination) and a biomarker-based approach (quantification of the activities of catalase, GSTs, and acetylcholinesterase, and also the muscle glycogen content). The here determined LC50-96 h for zinc pyrithione was 2.17 mg/L. ZnPT caused significant increases in the activity of catalase and of cholinesterases. These findings evidence the pro-oxidative effects caused by the metabolism of ZnPT. Despite the absence of clear effects, it is important to stress that the presence of ZnPT in the wild is usually accompanied by other pyrithiones, whose co-existence can contribute to the exertion of considerable toxic effects. © 2018 Elsevier Bruno Nunes was hired through the Investigator FCT program (IF/01744/2013). Joana L. Pereira and Sara Antunes are recipients of individual post-doctoral grant ( SFRH/BPD/101971/2014 and SFRH/BPD/109951/2015 , respectively) by the Portuguese Foundation for Science and Technology (FCT) . Thanks also due to CESAM (UID/AMB/50017) for financial support and FCT through national funds and co-funding FEDER , within the PT2020 Partnership Agreement.