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Sustainable development is a key challenge for contemporary human societies; failure to achieve sustainability could threaten human survival. In this review article, we illustrate how Machine Learning (ML) could support more sustainable development, covering the basics of data gathering through each step of the Environmental Risk Assessment (ERA). The literature provides several examples showing how ML can be employed in most steps of a typical ERA.A key observation is that there are currently no clear guidance for using such autonomous technologies in ERAs or which standards/checks are required. Steering thus seems to be the most important task for supporting the use of ML in the ERA of nano- and smart-materials. Resources should be devoted to developing a strategy for implementing ML in ERA with a strong emphasis on data foundations, methodologies, and the related sensitivities/uncertainties. We should recognise historical errors and biases (e.g., in data) to avoid embedding them during ML programming.

Silver (Ag) nanomaterials (NMs) are used in many products, eventually reaching the environment at some life stage and as they can be harmful their impact should be assessed. Although research has focused on Ag NM toxicity, less focus has been on toxicokinetics. The aim of this study was to assess the kinetics of Ag nanomaterial (Ag NM300K) and AgNO3in the soil invertebrateEnchytraeus crypticus. Tests followed OECD guideline 317, with 14 days uptake followed by 14 days elimination in LUFA 2.2 soil. Two sub-lethal concentrations were selected based on enchytraeid sensitivity in a reproduction test (6 and 60 mg Ag per kg for Ag NM300K, and 5 and 45 mg Ag per kg for AgNO3), and spiked soil aged for 3 and 14 days after spiking. Total and 0.01 M CaCl2extractable soil concentrations were evaluated at day 0, 1 and 14 for all the exposures. Overall, enchytraeids showed increasing Ag uptake with time, followed by a decrease when transferred to clean soil. For the lowest exposure concentrations, the difference in Ag uptake rate constants between 3 and 14 days aging was larger (10-fold) for AgNO3than for NM300K (uptake rates being highest for soil aged for 3 days), which was in line with the higher CaCl2-extractable Ag concentrations in AgNO3spiked soil. At the higher exposure concentrations, for AgNO3the difference in Ag uptake rate constants between 3 and 14 days aged soils was 2-fold, with the bioaccumulation factor (BAF) being highest at 3 days aging. For Ag NM300K, the uptake rate constant was low with virtually no elimination, suggesting that body Ag concentrations may keep on increasing with time leading to a higher risk of longer-term exposure compared to the Ag ions. These findings show the importance of understanding the toxicokinetics of ionic and nano forms of silver and other elements, and the key role of aging in determining NM bioavailability.

Simple Summary Earthworms have been used for centuries in traditional medicine, and more than a century ago were praised by Charles Darwin as one of the most important organisms in the history of the world. These worms are well-studied with a wealth of information available, for example on the genome, the gene expression, the immune system, the general biology, and ecology. These worms live in many habitats, and they had to find solutions for severe environmental challenges. The common compost worm, Eisenia fetida, has developed a unique mechanism to deal with intruding (nano)materials, bacteria, and viruses. It deals with the intruders by covering these with a defence toxin (lysenin) targeted to kill the intruder. We outline how this mechanism probably can be used as a therapeutic model for human COVID-19 (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2) and other corona viruses. Abstract Earthworms have been used for centuries in traditional medicine and are used globally as an ecotoxicological standard test species. Studies of the earthworm Eisenia fetida have shown that exposure to nanomaterials activates a primary corona-response, which is covering the nanomaterial with native proteins, the same response as to biological invaders such as a virus. We outline that the earthworm Eisenia fetida is possibly immune to COVID-19 (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2), and we describe the likely mechanisms of highly receptor-specific pore-forming proteins (PFPs). A non-toxic version of this protein is available, and we hypothesize that it is possible to use the earthworm’s PFPs based anti-viral mechanism as a therapeutic model for human SARS-CoV-2 and other corona viruses. The proteins can be used as a drug, for example, delivered with a nanoparticle in a similar way to the current COVID-19 vaccines. Obviously, careful consideration should be given to the potential risk of toxicity elicited by lysenin for in vivo usage. We aim to share this view to activate its exploration by the wider scientific community while promoting a potential therapeutic development.

Safer by design (SBD)modifications of nanomaterials (NMs)have been pursued, aiming to maintain functionality and yet reduce hazard and support sustainable nanotechnology. The present case study involves copper oxide nanomaterials (CuO NMs)used in paint that have been surface modified by a SBD approach to particles coated with citrate (CIT − ), ascorbate (ASC − ), polyethylenimine (PEI + ), and polyvinylpyrrolidone (PVP). We assessed the effect of the 4 different surface modified (CIT, ASC, PVP and PEI)NMs plus the pristine non-coated (PRI NM)and a Cu salt (CuCl 2 ), using the soil multispecies test system (samples at 28–56–84 days). Further, the species were tested individually, and Cu was measured in the test media (soil and soil solution)and organisms. There was a potential relationship with zeta potential, and toxicity of CuO NMs was as follows: -PEI (+28 mV)caused the least impact, -ASC and -CIT (-17 mV, -18 mV)the most, while PVP and PRI (-8 mV, -9 mV)caused an intermediate response. Differences were not explained by the contribution of soluble Cu. Coating interfered with the release of Cu 2+ and/or the activation of copper regulators and detoxification mechanisms in the organisms, i.e. time to reach some kind of stability in organisms' uptake was shorter for -ASC and longer for -PVP during prolonged time. Thus, one of the main findings is that NMs hazard assessment requires long term testing to understand predicted effects across materials. Further, the coverage using a multispecies approach offers increased relevance and a more ecosystem qualified response.

In standard toxicity tests one generation of test organisms is used, and they are usually exposed only during a fraction of their life-cycle. This approach is very important but does not cover the potential effects of multigenerational (MG) exposure and may underestimate risks. Hence, the main aim of this study was to assess the MG impact of the veterinary pharmaceutical ivermectin (IVM) on Folsomia candida during three generations (F1–F3). Ivermectin is a veterinary medicine, persistent in the environment and toxic to non-target soil invertebrates. A suite of different endpoints was used including avoidance, survival, reproduction, size and cellular biomarkers (catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione S-transferase (GST), acetylcholinesterase (AChE) and lipid peroxidation (LPO)). Survival and reproduction were affected (LC50: 40 mg/kg; EC50: 5 mg/kg), but no avoidance occurred, which poses additional ecological concern. Exposure throughout the generations showed similar toxicity in terms of survival and reproduction. Regarding size there was an impact, e.g., organisms were smaller and more abundant in F2 and larger and less abundant in F3. This can have implications in terms of risk as e.g. smaller organisms can respond differently to stress compared to larger organisms in future generations. The antioxidant mechanisms were dynamically activated along the generations, e.g. in F1 CAT was increased whereas in F3 there was increased GST activity, which resulted in damage (LPO) for F1 and F2 organisms but not for the F3 generation. The multi-endpoint approach proved to be beneficial for the interpretation of results and we recommend it, especially for persistent and/or highly adsorptive chemicals, but also endocrine disruptors. Moreover, the evaluation of size as an additional sub-lethal endpoint has significantly added to the relevance of this test. The relative proportion of small, medium and large animals may be an even more relevant aspect of this endpoint. This does not require guideline modifications and is hence easily implementable.

The potential implications of long-term exposure to contaminants are not covered by current standard toxicity guidelines, usually referring to one generation and a fraction of the life cycle of the test species. Hence, in the present study, we aimed to assess the effects of the multigenerational exposure (generation 1–3: F1-F3) of Folsomia candida to an insect growth regulator (IGR) compound: teflubenzuron (TFB). The selected endpoints included both the standard ones as in the OECD and ISO guidelines (survival, reproduction, and avoidance) as well as additional ones (organisms size and cellular oxidative stress markers: acetylcholinesterase, glutathione S-transferase, catalase, glutathione peroxidase, lipid peroxidation). Although no avoidance behaviour was recorded at field-relevant concentrations (PEC (Predicted Environmental Concentration) = 0.06 mg/kg soil dry weigh (dwt)), survival and reproduction were impacted (LC50 = 0.1 mg/kg soil dwt; EC50 = 0.05 mg/kg soil dwt). Multigenerational exposure to TFB caused increased toxicity in F. candida in F3 in terms of survival and reproduction. This could be related to the mode of action of TFB which does not seem to activate some of the general stress mechanisms of response like oxidative stress. In addition, TFB causes a reduction of the organisms’ size, with a reduction of the number of large-sized juveniles, which has potential adverse consequences in terms of organisms’ performance, e.g. change in age structure and hence population dynamics. Hence, both observations may increase the environmental concern and associated risk of this insecticide.

Cobalt and cobalt nanoparticles have many applications, for example, in the hard metal industry and in tires. The assessment of long term effects is crucial, as these materials are persistent. For many organism groups, multigenerational (MG) exposure is a highly relevant scenario for persistent materials. In this study, the biological effect of CoCl2 (salt) and Tungsten Carbide Cobalt nanoparticles (WCCo NPs) exposure was assessed in an MG test (4 generations in spiked + 2 generations in clean soil) using the OECD/ISO standard soil test species Enchytraeus crypticus. To ensure transgenerational survival, sublethal concentrations were used to assess the MG impact. MG exposure did not increase toxicity (survival, reproduction). There was an increase in reproduction at low concentrations of Co. Materials were characterized in the exposure media and the organisms in terms of Co content. Uptake of Co-occurred from exposure to both CoCl2 and WCCo, although without toxicity for WCCo. Cobalt from CoCl2 exposure seemed to be stored, whereas for WCCo it was eliminated.

Submitted by Vivian Rosa Storti (vivian.storti@unesp.br) on 2019-08-01T19:31:06Z No. of bitstreams: 1 Environmental science nano.pdf: 1327166 bytes, checksum: f97ab255a890aff30553739e39d30b76 (MD5) Approved for entry into archive by Luana Priscila Costa (luana@reitoria.unesp.br) on 2019-08-05T14:57:38Z (GMT) No. of bitstreams: 1 Environmental science nano.pdf: 1327166 bytes, checksum: f97ab255a890aff30553739e39d30b76 (MD5) Made available in DSpace on 2019-08-05T14:57:38Z (GMT). No. of bitstreams: 1 Environmental science nano.pdf: 1327166 bytes, checksum: f97ab255a890aff30553739e39d30b76 (MD5) Previous issue date: 2019-05-23 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The use of nanotechnology in the agrochemical sector aims to increase pesticide efficiency, and at the same time provide more targeted delivery, reducing the application volume and thus its environmental footprint. However, the possible risks of these new nanopesticides to non-target organisms are still sparsely investigated. The aim of the present study was to investigate the effects of a nanoformulation of atrazine (nano_ATZ) to non-target soil invertebrates. The effect was compared with the commercial formulation (Gesaprim®) and atrazine (the pure active ingredient, a.i.), using the a.i. in a field concentration range using the soil invertebrate, Enchytraeus crypticus (Oligochaeta) as the non-target organisms. The endpoints evaluated included avoidance behaviour (2d), hatching success (11d), survival and reproduction (based on both the standard enchytraeid reproduction test (28d) and on the full life cycle test (46d)). Results showed that enchytraeids avoided soil spiked with Gesaprim and atrazine (a.i.), but not nano_ATZ. While all tested atrazine forms affected the hatching success (11d, early development stage), the toxicity in later stages, as measured in terms of survival and reproduction (46d) showed that Gesaprim was the least toxic (EC10 ca. 200 mg kg−1), followed by nano_ATZ (EC10 ca. 180 mg kg−1) and atrazine (a.i.) (EC10 ca. 100 mg kg−1). These findings are important to nanopesticide regulatory purposes, showing the potential effects of nanoformulation compared to the current commercial non-nano ATZ in a.i. field concentrations, and that information on additional test species and exposure routes are missing, as well as the longer term consequences. Preprint Department of Biology & CESAM, University of Aveiro Department of Bioscience, Aarhus University, Vejlsovej São Paulo State University (UNESP), Institute of Science and Technology, Sorocaba, Brazil Human and Natural Sciences Center, Federal University of ABC, Santo André, Brazil São Paulo State University (UNESP), Department of Physics and Chemistry, School of Engineering, Ilha Solteira, Brazil FAPESP: 2017/21004-5

The main aim of the study is to evaluate the effects of the pharmaceutical diphenhydramine (DPH) on embryo-larvae Danio rerio across distinct levels of organization - individual and subcellular - and correlate those effects with the DPH mode of action (MoA) assessed by in silico analysis. An embryos heartbeat rate reduction was observed at 10 mg/L DPH, but 0.001 to 10 mg/L did not significantly affect the zebrafish survival, hatching and morphology. Larvae swimming distance decreased (hypoactivity) at 1 and 10 mg/L DPH. Moreover, the straightforward movements decrease and the increase in the zigzag movements or movements with direction changes, shown an erratic swimming behavior. Energy budgets decreased for lipid (0.01 mg/L DPH) and carbohydrate (10 mg/L DPH) contents. Cholinesterase (neural function) and glutathione S-transferase (Phase II biotransformation/antioxidant processes) increased their activities at 10 mg/L DPH, where a decrease in the total glutathione content (antioxidant system) was observed. DNA damage was found at 0.01 and 10 mg/L DPH. However, a DNA repair occurred after subsequent 72 h in clean media. The in silico study revealed a relevant conservation between human and zebrafish DPH target molecules. These data provide a valuable ecotoxicological information about the DPH effects and MoA to non-target organisms.

Once in the aquatic ecosystems, nanoplastics (NPls) can interact with other contaminants acting as vectors of transport and altering their toxicological effects towards organisms. Thus, the present study aims to investigate how polystyrene NPls (44 nm) interact with the herbicide phenmedipham (PHE) and affect its toxicity to zebrafish embryos. Single exposures to 0, 0.015, 0.15, 1.5, 15 and 150 mg/L NPls and 0.02, 0.2, 2 and 20 mg/L PHE were performed. Embryos were also exposed to the binominal combinations: 0.015 mg/L NPls + 2 mg/L PHE, 0.015 mg/L NPls + 20 mg/L PHE, 1.5 mg/L NPls + 2 mg/L PHE and 1.5 mg/L NPls + 20 mg/L PHE. Due to the low solubility of PHE in water, a solvent control was performed (0.01% acetone). PHE was quantified. Mortality, heartbeat and hatching rate, malformations appearance, locomotor behavior and biomarkers related to oxidative stress, neurotransmission and energy budgets were analyzed. During 96 h, NPls and PHE single and combined exposures did not affect embryos development. After 120 h, NPls induced hyperactivity and PHE induced hypoactivity. After 96 h, NPls increased catalase activity and PHE increased glutathione S-transferases activity. On the combination 0.015 mg/L NPls + 20 mg/L PHE, hyperactivity behavior was found, similar to 0.015 mg/L NPls, and cholinesterase activity was inhibited. Additionally, the combination 1.5 mg/L NPls + 20 mg/L PHE increased both catalase and glutathione S-transferases activities. The combination NPls with PHE affected more biochemical endpoints than the single exposures, showing the higher effect of the binominal combinations. Dissimilar interactions effects - no interaction, synergism and antagonism - between NPls and PHE were found. The current study shows that the effects of NPls on bioavailability and toxicity of other contaminants (e.g. PHE) cannot be ignored during the assessment of NPls environmental behavior and risks.

Gene expression can vary with the organisms' life stage. It is known that embryos can be more sensitive to toxicant exposure, as previously demonstrated for Enchytraeus crypticus (Oligochaeta) exposed to cadmium (Cd), known to cause embryotoxicity and hatching delay. It was shown that Ca enters embryos via the L-type Ca channels in the cocoon membrane, this being affected in Cd exposed embryos (Cd-Ca competition is well-known). In the present study, the embryotoxic mechanisms of Cd were studied via high-throughput gene expression for E. crypticus. Cocoons (1-2 days old), instead of the adult organism, were exposed in Cd spiked LUFA 2.2 soil during 1 day. Results showed that Cd affected Ca homeostasis which is implicated in several other molecular processes. Several of the major modulators of Cd toxicity (e.g., impaired gene expression, cell cycle arrest, DNA and mitochondrial damage) were identified in the embryos showing its relevancy as a model in ecotoxicogenomics. The draft Adverse Outcome Pathway was improved. Previously was hypothesized that gene regulation mechanisms were activated to synthesize more Ca channel proteins - this was confirmed here. Further, novel evidences were that, besides the extracellular competition, Cd competes intracellularly which causes a reduction in Ca efflux, and potentiates Cd embryotoxicity. (C) 2018 Elsevier Ltd. All rights reserved.

Biocidal products represent mixtures that might be released into the environment at application and continuously during service life. Concentration addition (CA) has been proposed as default model to calculate theoretical mixture toxicity. However, the suitability of CA for chronic toxicity towards soil organisms has so far rarely been evaluated and therefore needs further experimental evidence. The present study investigated the toxicity of a wood preservative product and the individual active substances (tebuconazole and IPBC) therein with the aim to evaluate the compliance with the CA prediction for the product. Folsomia candida was selected as test organism for this purpose using the endpoints reproduction and avoidance behaviour. Both endpoints were increasingly impacted by increasing concentrations of the wood preservative product as well as its active substances tested individually. The chronic effects of the product could be predicted by CA with less than 4-fold deviation, while the assessment for avoidance behaviour indicated a strong underestimation. This underestimation could not be attributed to the one known formulation additive, an organic solvent. Overall, the present study provides some more evidence that CA could be applied as default model for standard endpoints of soil organisms, but warns against using CA for behavioural responses.

Despite that silver (Ag) is among the most studied nanomaterials (NM) in environmental species and Ag's embryotoxicity is well known, there are no studies on Ag NMs embryotoxicity in soil invertebrates. Previous Full Life Cycle (FLC) studies in Enchytraeus crypticus, a standard soil invertebrate, showed that Ag materials decreased hatching success, which was confirmed to be a hatching delay effect for silver nitrate (AgNO3) and mortality for Ag NM300K. In the present study, we aimed to investigate if the impact of Ag takes place during the embryonic development, using histology and immunohistochemistry. E. crypticus cocoons were exposed to a range of concentrations of Ag NM300K (0–10–20–60–115 mg Ag/kg) and AgNO3 (0–20–45–60–96 mg Ag/kg) in LUFA 2.2 soil, in an embryotoxicity test, being sampled at days 1, 2, 3 and 6 (3, 4, 5 and 7 days after cocoon laying). Measured endpoints included the number of embryonic structures, expression of transferrin receptor (TfR) and L type calcium channels (LTCC) through histological and immunohistochemistry analysis, respectively. Results confirmed that Ag materials affected the embryonic development, specifically at the blastula stage (day 3). The expression and localization of TfR in E. crypticus was shown in the teloblasts cells, although this transcytosis mechanism was not activated. Ag affected calcium (Ca) metabolism during embryonic development: for AgNO3, LTCC was initially activated, compensating the impact, for Ag NM300K, LTCC was not activated, hence no Ca balance, with irreversible consequences, i.e. terminated embryonic development. An Adverse Outcome Pathway was drafted, integrating the mechanisms here discovered with previous knowledge.

Recent years have seen the development of various colloidal formulations of pesticides and other agrochemicals aimed at use in sustainable agriculture. These formulations include inorganic, organic or hybrid particulates, or nanocarriers composed of biodegradable polymers, that can provide a better control of the release of active ingredients. The very small particle sizes and high surface areas of nanopesticides may however also lead to some unintended (eco)toxicological effects due to the way in which they interact with the target and non-target species and the environment. The current level of knowledge on ecotoxicological effects of nanopesticides is scarce, especially in regard to the fate and behaviour of such formulations in the environment. Nanopesticides will however have to cross a stringent regulatory scrutiny before marketing in most countries for health and environmental risks under a range of regulatory frameworks that require pre-market notification, risk assessment and approval, followed by labelling, post-market monitoring and surveillance. This review provides an overview of the key regulatory and ecotoxicological aspects relating to nanopesticides that will need to be considered for environmentally-sustainable use in agriculture.

DNA methylation is an epigenetic mechanism of particular importance in developmental biology, but methylation also varies along organisms' life cycle. Recent studies have deliberated copper (Cu) exposure induced epigenetic changes in Enchytraeus crypticus, a standard species belonging to one of the most common and important genera of soil invertebrates in many ecosystems. There is however no information on how DNA methylation levels change within the life cycle of this species. We here investigate the global DNA methylation profile along the life cycle of E. crypticus and compare this to the expression of target genes involved in methylation. Results showed that after the lowest DNA methylation level at day 3 (early embryonic stage, blastula) there was an increase by day 7 (organogenesis) after which levels were maintained at days 11, 18 and 25. DNA methyltransferase associated protein 1 (DMPA1) and Methyl Binding Domain 2 (MBD2) gene expression was highest during embryo stages (3 to 7 days), then decreasing (11, 18 days) and finally unregulated in adults (25 days). Hence, we here show that DNA methylation in E. crypticus changes among the different life stages, from cocoons to adults. Such information is a key knowledge to use this endpoint and tool in an ecotoxicology context. This means that it is almost implicit that gene expression levels are age specific for a given stressor. It seems logic to recommend to always compare individuals with the same age between treatments, and to be careful when extrapolating results among life stages. Once, we understand more of these effects we may even be able to predict which life stage is more sensitive to specific stressors. An experimental design that aims to cover epigenetics of stressors in a multigenerational exposure, including transgenerational effects, should ensure the synchronous age of organisms for sampling analysis purposes.

The European Medicines Agency (EMA) regards the potential risks of human medicinal products to the environmentand their impacts are assessed, as well as management to limit this impact. Hazard assessment of novel materials,which differ from conventional chemicals, e.g. nanobiomaterials, poses testing challenges and represents a work in-progress with much focus on the optimization of required methodologies. For this work-in-progress, we herehighlight where changes/updates are required in relation to the main elements for international testing based onOECD guidelines, supported by knowledge from the nanotoxicity area. The outline describes two major sections,nanobiomaterials and environmental hazards, including its challenges and learned lessons, with recommendationsfor implementation in OECD guidelines. Finally, the way forward via a testing strategy is described.

Veterinary pharmaceuticals are widely used as food additives in the poultry industry, and the unknown consequences of releasing these compounds into the environment are of concern. The purpose of the present study was to determine the direct impact of 2 veterinary pharmaceuticals (nicarbazin and monensin), commonly used in the poultry industry, on nontarget invertebrates and plant species. Ecotoxicological tests were used to evaluate the acute and chronic toxicity in earthworms (Eisenia andrei), collembolans (Folsomia candida), and 2 plant species (Brassica rapa and Triticum aestivum). Chemical analytical measurements were in good agreement with the nominal concentrations used, although some variability was seen. The results obtained showed no effects of nicarbazin at the highest nominal tested concentration of 1000 mg a.i./kg soil dry weight on any of the organisms, whereas exposure to monensin caused a concentration-specific response pattern. Species sensitivity to monensin decreased in the following rank order: B. rapa > T. aestivum > E. andrei > F. candida, with measured median effect concentrations (based on soil exposure) ranging between approximately 10 and 120 mg/kg. Our results emphasize the importance of using a test battery when assessing ecotoxicological effects by using different ecophysiological endpoints and species from different trophic levels. Environ Toxicol Chem 2018;37:3145-3153. © 2018 SETAC.

Silver (Ag) is amongst the most well studied nanomaterials (NMs), although most studies have only dealt with a single AgNM at a time and one biological endpoint. We here integrate the results of various testing-tools (endpoints) using a terrestrial worm, the standard ecotoxicological model organism Enchytraeus crypticus. Exposure spanned both water and soil exposure, it covered all life stages (cocoons, juveniles and adults), varying exposure durations (1-2-3-4-5-21 days), and covered 5 biological endpoints: hatching success, survival, reproduction, avoidance and gene expression (qPCR target genes GABA and Acetyl cholinesterase). We tested 4 Ag materials: PVP coated (PVP-AgNM), non-coated (NC-AgNM), the JRC reference Ag NM300K and AgNO3. Results showed that short-term exposure via water to assess impact on cocoons’ hatching predicted longer term effects such as survival and reproduction. Moreover, if we extended the exposure from 11 to 17 day this allowed discrimination between hatch delay and impairment. Exposure of juveniles and adults via water showed that juveniles were most sensitive with survival affected. Across materials the following toxic ranking was observed: AgNO3 ≥ Ag NM300K ≫ NC-AgNM ≥ PVP-AgNM. E. crypticus avoided AgNO3 in a dose-response manner, avoiding most during the first 24 h. Avoidance of Ag NM300K and NC-AgNM only occurred during the first 24 h and the PVP coated AgNM were not avoided at all. The up-regulation of the GABA triggering anesthetic effects, indicated the high ecological impact of Ag materials in soil: Ag affects the GABAergic system hence organisms were not able to efficiently avoid and became intoxicated, this caused impacts in terms of survival and reproduction. Multi-endpoint approach advances the understanding of effects of (nano)materials: towards a mechanistic based risk assessment strategy.

Effects of pollutants are mostly assessed using standard testing procedures, which cover a fraction of the animals' life cycle. Although, in nature species are exposed during multiple generations of sub-lethal doses of persistent chemicals. In the present study, we focused on the multigenerational (MG) effects of silver in Folsomia candida during 6 generations using the EC50 for reproduction as exposure concentration. We tested 9 different exposure scenarios, going from continuous 6 generations Ag exposure over pulse exposure ( i.e. one generation clean, next contaminated, next clean etc.) to gradually increasing the number of exposure generations, with a final transfer to clean media. The biological endpoints assessed included survival, reproduction and size, with reproduction being the most sensitive. The biological response depended on the specific MG scenario. e.g. the 6 Ag MG caused a decreased number of juveniles from F4, whereas the pulse exposure experienced an increase in reproductive output when in dean soil. It is uncertain whether Ag causes transgenerational effects, but the reproduction levels in both pulse exposures are lower than in continuous control over the 6 generations which could be due to transference of Ag by the maternal generation. Overall, population size distribution seemed to indicate a delay in time for egg laying, with close relationship between adult survival, organisms size and reproduction output. Size monitoring allowed significant added interpretation possibilities and we strongly recommend the addition of this endpoint to the standard guideline. The smaller observed size range can have implications in terms of adaptation potential, carrying associated increased risk. (C) 2018 Elsevier B.V. All rights reserved.

The current testing of nanomaterials (NMs) via standard toxicity tests does not cover many of the NMs specificities. One of the recommendations lays on understanding the mechanisms of action, as these can help predicting long-term effects and safe-by-design production. In the present study, we used the high-throughput gene expression tool, developed for Enchytraeus crypticus (4 × 44k Agilent microarray), to study the effects of exposure to several copper (Cu) forms. The Cu treatments included two NMs (spherical and wires) and two copper-salt treatments (CuNO3 spiked and Cu salt field historical contamination). To relate gene expression with higher effect level, testing was done with reproduction effect concentrations (EC20, EC50), using 3 and 7 days as exposure periods. Results showed that time plays a major role in the transcriptomic response, most of it occurring after 3 days. Analysis of gene expression profiles showed that Cu-salt-aged and Cu-nanowires (Nwires) differed from CuNO3 and Cu-nanoparticles (NPs). Functional analysis revealed specific mechanisms: Cu-NPs uniquely affected senescence and cuticle pattern formation, which can result from the contact of the NPs with the worms' tegument. Cu-Nwires affected reproduction via male gamete generation and hermaphrodite genitalia development. CuNO3 affected neurotransmission and locomotory behavior, both of which can be related with avoidance response. Cu salt-aged uniquely affected phagocytosis and reproductive system development (via different mechanisms than Cu-Nwires). For the first time for Cu (nano)materials, the adverse outcome pathways (AOPs) drafted here provide an overview for common and unique effects per material and linkage with apical effects.

There is a growing need to recover degraded soils to restore their essential ecosystem services and limit damages of anthropic activities onto these systems. Safe and sustainable solutions for long-term recovery must be designed, ideally by recycling existing resources. Using ash from combustion of residual forest biomass at the pulp and paper industry is an interesting and sustainable strategy to recover mining soils. However, formulations must be found to limit the potential toxicity associated with soluble salts and chloride that ash contains. Here, we assessed the effectiveness of three field ash-based amendments for the recovery of three highly acidic soils from Portuguese abandoned mines. Three amendments were tested: an un-stabilized mixture of ash and biological sludge, granulated ash, and granulated ash mixed with composted sludge. One year after application in open field plots (in the scope of LIFE No_Waste project), soil health restoration was evaluated through (i) soil physico-chemical characterization and (ii) soil habitat functions though standardized ecotoxicological tests. This study highlights that stabilized materials provided nutrients, organic matter and alkalinity that corrected soil pH and decreased metal bioavailability, while controlling the release of soluble salts and chloride from ash. This soil improvement correlated with improved soil model organisms’ reproduction and survival. For similar amendment, the native soil properties studied (as soil native electrical conductivity) affected the level of organism response. This work provides evidence that ash stabilization, formulation and supplementation with organic matter could be sustainable strategies to restore highly degraded mining soils and to recover their ecological functions. It further highlights the importance of analyzing combined effects on soil physico-chemical properties and ecological function recovery to assess restoration strategy efficiencies in complex multi-stressor environments.

Silver nanomaterials (AgNMs) are broadly used and among the most studied nanomaterials. The underlying molecular mechanisms (e.g. protein and metabolite response) that precede phenotypical effects have been assessed to a much lesser extent. In this paper, we assess differentially expressed proteins (DEPs) and metabolites (DEMs) by high-throughput (HTP) techniques (HPLC-MS/MS with tandem mass tags, reversed-phase (RP) and hydrophilic interaction liquid chromatography (HILIC) with mass spectrometric detection). In a time series (0, 7, 14 days), the standard soil model Enchytraeus crypticus was exposed to AgNM300K and AgNO3 at the reproduction EC20 and EC50. The impact on proteins/metabolites was clearly larger after 14 days. NM300K caused more upregulated DEPs/DEMs, more so at the EC20, whereas AgNO3 caused a dose response increase of DEPs/DEMs. Similar pathways were activated, although often via opposite regulation (up vs down) of DEPs, hence, dissimilar mechanisms underlie the apical observed impact. Affected pathways included e.g. energy and lipid metabolism and oxidative stress. Uniquely affected by AgNO3 was catalase, malate dehydrogenase and ATP-citrate synthase, and heat shock proteins (HSP70) and ferritin were affected by AgNM300K. The gene expression-based data in Adverse Outcome Pathway was confirmed and additional key events added, e.g. regulation of catalase and heat shock proteins were confirmed to be included. Finally, we observed (as we have seen before) that lower concentration of the NM caused higher biological impact. Data was deposited to ProteomeXchange, identifier PXD024444.

An imbalance between reactive oxygen species (ROS) and antioxidants in a living organism results in oxidative stress. Measures of such imbalance can be used as a biomarker of stress in ecotoxicology. In this study, we implemented the ROS detection method based on the oxidant-sensing probe dichloro-dihydro-fluorescein diacetate (DCFH-DA), detected by fluorescence microscopy, in Enchytraeus crypticus adults and cocoons, i.e., also covering the embryo stage. Hydrogen peroxide (H2O2), a well-known ROS inducer, was used both to optimize the method and as positive control. Implementation was successful, and the method was used to assess ROS formation in E. crypticus cocoons and adults when exposed to the reference silver nanomaterial Ag NM300K, at two effect concentrations (EC20 and EC50) for both hatching and reproduction over 3 and 7 days. The measured ROS levels varied with time, concentration, and developmental stage, with higher levels detected in adults compared with cocoons. In cocoons, ROS levels were higher at the EC20 than the EC50, which could be explained by non-monotonic concentration-response curve for hatching and reproduction, as previously observed. The increase in ROS levels at day 3 preceded the oxidative damage, as reported to occur later (day 7) in adults. The DCFH-DA method was successfully implemented here and can be further used as a new tool to detect ROS formation in E. crypticus, especially after short-term exposure to chemicals, including nanomaterials. We recommend the use of 3 and 7 days in the exposure design for this assessment.

Nanoparticles (NPs) such as nickel (Ni) are widely used in several applications. Nevertheless, the environmental effects of Ni NPs are still poorly understood. In the present study, the toxicity of Ni NPs and nickel nitrate (NiNO3 ) was assessed using the standard test species in soil ecotoxicology, Enchytraeus crypticus (Oligochaeta), in a full life cycle test, adding the endpoints hatching, growth, and time to reach maturity, besides survival and reproduction as in the standard Organisation for Economic Co-operation and Development Guideline 220 and/or International Organization for Standardization 16387. For Ni NPs, the Ni in soil and in soil solution was concentration- and time-dependent, with a relatively higher soil solution content in the lower and shorter exposure concentrations and times. Overall, NiNO3 was more toxic than Ni NPs, and toxicity seemed to occur via different mechanisms. The former caused reduced hatching (50% effect concentration [EC50] = 39 mg Ni/kg soil), and the negative effects remained throughout the life cycle, in all measured endpoints (growth, maturation, survival, and reproduction). For Ni NPs, hatching was the most sensitive endpoint (EC50 = 870 mg Ni/kg soil), although the organisms recovered; that is, additional endpoints across the life cycle showed that this effect corresponded to a delay in hatching because organisms survived and reproduced at concentrations up to 1800 mg Ni/kg soil. On the other hand, the lowest tested concentration of Ni NPs (100 mg Ni/kg soil) caused reproduction effects similar to those at higher concentrations (1000 and 1800 mg Ni/kg soil). The present results show that the potential implications of a nonmonotonic dose response should be considered when assessing the risks of Ni NP exposure in soil. Environ Toxicol Chem 2017;36:2934-2941. © 2017 SETAC.

Energy budget studies offer important information in terms of organisms' fitness and this has become an increasingly common stress marker. The method is well developed but the issue of food supply (addition or not) during exposure is not consensual. Moreover, chemicals such as cadmium (Cd) are known to affect food uptake, e.g. via feeding inhibition, hence energy budget results could be because of decreased uptake and not direct toxicity. On the other hand, food deprivation can be a stressor itself and bias results. In the present study we compared exposure with (F) and without food (NF) along time (2, 4 and 8 days). The Cellular Energy Allocation (CEA) method was used to study the effects of Cd in the soil standard species Enchytraeus albidus (Oligochaeta). In control conditions (un-spiked soil), carbohydrate and lipid budgets were mobilized in NF. When testing Cd, variations in carbohydrates and lipid budgets depend on the presence of food and time of exposure. The main hypothesized mechanisms triggered by Cd exposure were similar (i.e. induction of protein synthesis and increase in energy consumption). Differences between F and NF over time indicate that the process of mobilization/preservation of energy reserves depends on the food/energy intake (e.g. in Cd exposed organisms, mobilization of proteins occurred within 2 days when food was present, while in the absence of food carbohydrate and protein budgets were mobilized from 2 to 4 days). Comparisons between F-NF studies should not be done directly. Moreover, we recommend exposure without food because it allows a better discrimination of effects (in particular within periods of exposure).

Toxicokinetics information is key to understanding the underlying intoxication processes, although this is often lacking. Hence, in the present study the toxicokinetics of copper (Cu) and cadmium (Cd) was assessed in the soil invertebrate Enchytraeus crypticus. The animals were exposed in LUFA 2.2 natural soil spiked to the estimated EC20 for reproduction effects in the Enchytraeid Reproduction Test (ERT), i.e. 80 mg Cu/kg soil Dry Weight (DW) and 20 mg Cd/kg soil DW. Tests followed the OECD guideline 317, including a 14-day uptake phase in spiked soil followed by 14 days elimination in clean soil, with samplings at days 0, 1, 2, 4, 7, 10, and 14. Exposure to Cu showed fast uptake, reaching a steady state after approx. 7 days, whereas for Cd, internal concentration increased and did not reach a clear steady state even after 14 days. When transferred to clean soil, Cu was rapidly eliminated returning to initial levels, while Cd-exposed animals still contained increased residue levels after 14 days. These differences in toxicokinetics have consequences for the toxicity and toxicodynamics and are indicative of the way essential and non-essential elements are handled by enchytraeids, likely also other soil invertebrates. This argues for the relevancy of longer exposure testing for elements like Cd compared to Cu, where phenotypical effects can well occur later at non-tested periods, e.g. after the 21 days’ duration of the standard ERT using E. crypticus.

Once in the environment, nanoplastics (NPls) may interact with other contaminants, such as pharmaceuticals, potentially acting as carriers and modulating their toxicity. Thus, the main aim of the current study is to investigate how polystyrene (PS) NPls (mean diameter: 60 nm) interact with simvastatin (SIM), an anticholesterolemic drug, and modulate its toxicity to zebrafish (Danio rerio) embryos. PS NPls were carboxyl group functionalized, to promote the interaction/binding of NPls with SIM (worst-case scenarios) and it was fluorescently dyed, allowing to detect the intake. Exposure was 96 h to 0–150 mg/L NPls or 0–150 µg/L SIM, as well as to dual combinations (NPls 0.015 or 1.5 mg/L and SIM 12.5 or 15 µg/L). PS NPls alone did not exert effects whereas SIM (≥ 12.5 µg/L) significantly delayed the hatching, decreased the heartbeat, induced edemas and mortality. The combination of NPls (1.5 mg/L) and SIM (12.5 or 15 µg/L) had significant effects on the survival of the organisms while the correspondent NPls and SIM single exposures did not have significant effects on this endpoint. Concerning the malformations appearance, SIM alone had similar effects than when in co-exposures (0.015 mg/L NPls plus 12.5 or 15 µg/L SIM). Hatching and heartbeat increased after the co-exposures SIM and NPls comparing with SIM single exposures, showing that 0.015 mg/L NPls plus 12.5 or 15 µg/L SIM did not cause significant effects on these endpoints. This study shows that NPls effects on bioavailability and toxicity of other contaminants cannot be ignored when assessing the environmental behavior and risks of NPls.

Environmental risk assessment (ERA) based on effects caused by chronic and longer term exposure is highly relevant. Further, if mechanistic based approaches (e.g. omics) can be included, beyond apical endpoints (e.g. reproduction), the prediction of effects increases. For Cu NMs (and CuCl2) this has been studied in detail, covering multi-omics and apical effects using the soil standard species Enchytraeus crypticus. The intermediate level effects like cell/tissue and organ alterations represent a missing link. In the present study we aimed to: 1) perform long term exposure to Cu materials (full life cycle and multigeneration, 46 and 224 days) to collect samples; 2) perform histology and immunohistochemistry on collected samples at 12 time points and 17 treatments; 3) integrate all levels of biological organization onto an adverse outcome pathway (AOP) framework. CuO NMs and CuCl2 caused both similar and different stress response, either at molecular initiating events (MIE) or key events (KEs) of higher level of biological organization. Cell/Tissue and organ level, post-transcriptional and transcriptional mechanisms, through histone modifications and microRNA related protein, were similarly affected. While both Cu forms affected the Notch signalling pathway, CuCl2 also caused oxidative stress. Different mechanisms of DNA methylation (epigenetics) were activated by CuO NMs and CuCl2 at the MIE. published

Certain invertebrates (and vertebrates) are able to regenerate lost body parts. In the present study we investigated if Enchytraeus crypticus (Oligocheata), a species used for standard ecotoxicological testing, shows regeneration ability. Artificial amputation was induced. The results showed that regeneration occurs for this species, this being promoted by the presence of a mass of undifferentiated cells (blastema) in the wound site. This only occurred in the anterior fragment resulting in the posterior regeneration (formation of the tail), i.e. the posterior fragment does not regenerate the head and degenerates. The regenerated organisms were tested for fertility, which was confirmed. This is an important knowledge and ecological feature, E. crypticus has an additional survival strategy. Further, given this is an ecotoxicological model species and the possible implications thereof – E. crypticus can fragment and regenerate hence survival results may include this occurrence.

Effects of nickel and copper on Enchytraeus albidus (Oligochaeta) were investigated using the cellular energy allocation approach. This methodology is used to evaluate the energetic status of an organism and is indicative of its overall condition. Enchytraeids were exposed to the reproduction Effect Concentrations (EC50 and EC90), and the parameters measured were the total energy reserves available (protein, carbohydrate and lipid budgets) and the energy consumption [based on electron transport system activity] which were further integrated to obtain the cellular energy allocation over different periods of exposure (0–2, 2–4 and 4–8 days). Carbohydrates (in comparison to lipids and proteins) were the only energy source mobilized in the case of nickel within 8 days of exposure. For copper exposure, protein budgets were also strongly reduced. Energy consumption increased in a time and dose-dependent way for copper and in the longer exposure period (4–8 days) at the EC90 for Ni exposure, indicating that this is a good biomarker for effects of short-time metal exposure, while cellular energy allocation was only significantly reduced for the EC90 of copper (4–8 days) and EC50 of nickel (2–4 days). The effects of nickel at concentrations causing 50 and 90 % decrease in reproduction were likely not due to the changes in cellular energy allocation within 8 days of exposure.

Climate changes are expected to be greatest in the polar and temperate areas, where predictions point to an increase in freeze–thaw events and changes in precipitation, evaporation, and salinity patterns. These events will therefore affect biological activity of the soil compartment that may result in large impacts in ecosystem functioning and dynamics therein. This concern becomes even more important when considering the presence of contaminants due to intense anthropogenic activity, which may lead to synergistic or antagonistic effects and increase or decrease the impact on natural ecosystems. This paper reviews the effect of physical and chemical stressors on enchytraeids, with special emphasis on Enchytraeus albidus because most relevant studies have involved this species. E. albidus is a freeze-tolerant and euryhaline organism and several studies suggest that the absence of salinity may have important (negative) consequences not only for their freeze tolerance ability but also for their reproduction and capacity to deal with the presence of contaminants, such as metals and fungicides. Single and joint effects of constant freezing or freeze–thaw cycles and surfactants, such as 4-nonylphenol, affected negatively E. albidus freeze tolerance by decreasing the levels of cryoprotectants, membrane fluidity and interfering with cellular energy allocation. Because enchytraeids are of ecological significance in many important habitats along the Artic and cold-temperate environments, a reduction in abundance may result in disturbances of the decomposition processes in soils. The knowledge of the biological, physiological, and biochemical limits of enchytraeids to combined effect of physical and chemical stressors are crucial to provide a scientific basis for improving the setting of safety factors when extrapolating from controlled (and optimal) laboratory conditions to natural soil ecosystems. Therefore, there is a need to expand and evolve experiments that more realistically mimic the situation in the field, where interactions between factors are highly relevant. The synergistic or antagonistic interactions identified in the present review may also represent a stepping stone in the evaluation and possible inclusion of natural factors, like cold and salinity, in standardized enchytraeid test guidelines and consequently in risk assessment of chemicals.

Ivermectin (IVM) is a broad acting antihelmintic used in various veterinary pharmaceuticals. It has been shown that IVM enters the aquatic compartment and adversely affects organisms including fish. This study is based on the hypothesis that long term exposure to IVM affects fish and thus, the main objective was to assess the chronic effects of 0.25 and 25 µg IVM/L to zebrafish using multiple endpoints representative of several levels of biological organization: weight, behaviour (swimming and feeding) and subcellular markers including biomarkers for oestrogenicity (vitellogenin-VTG), oxidative stress (catalase-CAT and glutathione-S-transferase-GST) and neurotransmission (cholinesterase-ChE). Concentrations as low as 0.25 µg IVM/L disrupted the swimming behaviour, causing fish to spend more time at the bottom of aquaria. Such reduction of the swimming performance affected the feeding ability which is likely responsible for the weight loss. The effects on weight were gender differentiated, being more pronounced in males (0.25 µg IVM/L) than in females (25 µg IVM/L). Fish exposed to 25 µg/L exhibited darker coloration and mild curvature of the spine. No effects on VTG and AChE were observed, but a reduction on CAT and GST levels was observed in fish exposed to 25 µg IVM/L, although these alterations probably only reflect the general condition of the fish which was significantly compromised at this concentration. Despite that predicted environmental concentrations of IVM are below 0.25 µg/L, the behavioural effects may be translated into important ecological impacts, e.g. at predator-prey interactions where fish competitive advantage can be decreased. Future work should address the link between behaviour disruption and population fitness. The current study was based on a one experiment and multiple endpoint (anchored) approach, allowing the results to be integrated and linked towards a mechanistic understanding.

Tungsten carbide cobalt (WCCo) nanoparticles (NPs) are widely used in hard metal industries. Pulmonary diseases and risk of cancer are associated with occupational exposure, but knowledge about the environmental fate and effects is virtually absent. In this study, the fate and effects of crystalline WCCo NPs, WC, and Co2+ were assessed in the soil model Enchytraeus crypticus, following the standard Enchytraeid Reproduction Test (ERT). An additional 28 day exposure period compared to the ERT (i.e., a total of 56 days) was performed to assess longer-term effects. WCCo NPs affected reproduction at a concentration higher than the corresponding Co based (EC50 = 1500 mg WCCo/kg, equivalent to 128 mg Co/kg). WC showed no negative effect up to 1000 mg W/kg. Maximum uptake of Co was 10-fold higher for CoCl2 compared to WCCo exposed organisms. Overall toxicity seems to be due to a combined effect between WC and Co. This is supported by the soil bioavailable fraction and biological tissue measurements. Last, results highlight the need to consider longer exposure period of NPs for comparable methods standardized for conventional chemicals.

Toxicity of pollutants is known to have a different impact depending on the organisms' life stage. Standard tests are often based on one life stage, i.e. effects could be underestimated. We aimed here to develop and optimize a test system using eggs of Folsomia candida (4–5 days) instead of the juveniles (10–12 days old) required by the OECD standard test guideline No. 232 (2009). Accordingly, the exposure time and thus the test duration was extended. Tests with “standard” juveniles (10–12 days old) and, adults (21 and 28 days old) were also performed. Cadmium (Cd) was used as test substance. The extension to the test guideline starts as follows: 1) synchronization of eggs in a thin soil layer on plaster of Paris, 2) selection of viable eggs, 3) burying these eggs in groups of 5 in soil. Afterwards, the test procedure will follow the standard procedure as described in the OECD standard test. Cadmium caused ca. 50% effects on reproduction at 60 mg Cd/kg soil dry weight (DW) when exposing juveniles or adults. There was no significant impact of Cd on the eggs, the hatching process or the latter life stages until ca. 250 mg Cd/kg DW (Cd is stable during this exposure period). Hence, Cd seems to affect reproduction before egg laying, i.e., during egg formation or during juvenile-adult stages. In order to clarify whether other chemicals do act in a similar way testing with different chemicals is highly recommended. Testing of different life stages does provide insight on the mechanisms and effects of contaminants and offers important insight.

Although environmental effects are mostly assessed via standard individual species the ecological relevance of multispecies testing is well-recognized and highly recommended. Hence, the effect of copper oxide nanomaterials (CuONM) and CuCl2 were assessed using the validated soil multispecies system (SMS). Besides the individual species (IS) "standard" tests for all, a predation study was done. Toxicity was higher in the SMS than in the IS, and longer exposure showed increased toxicity. The predator ( Hypoaspis aculeifer) preyed most on smaller species, but the net biomass consumed was similar across species. Internal Cu in Folsomia candida reached ca. 140 μg Cu/g dry weight, fluctuating over time, especialy for CuCl2. Copper was mostly bound to soil components for both Cu forms (

Avoidance behaviour offers a highly relevant information as it reveals the ability to avoid (or not) possible toxic compounds in the field, hence it provides information on reasons for the presence/absence in the field. The earthworm Eisenia fetida was used to study avoidance behaviour to four silver forms (three nanomaterials (NMs) and one salt) over four time points (24, 48, 72 and 96 h), using OECD standard soil. Avoidance behaviour depended on both exposure material and concentration, but in general changed little with exposure duration. Avoidance was highest for the salt (AgNO3) for all exposure durations and showed a continuous higher avoidance with time (based on EC50 values). The AgNMs avoidance was in the order NM300K

Scientific Reports 7: Article number: 42460; published online: 09 March 2017; updated: 12 February 2018. The Acknowledgements section in this Article is incomplete: “This work was supported by the European Union FP7 projects MODERN (Ref. 309314-2, to C.P.R., J.J.S.-F.), MARINA (Ref. 263215, to J.J.S.-F.

Nanoenabled products (NEPs) have numerous outdoor uses in construction, transportation or consumer scenarios, and there is evidence that their fragments are released in the environment at low rates. We hypothesized that the lower surface availability of NEPs fragment reduced their environmental effects with respect to pristine nanomaterials. This hypothesis was explored by testing fragments generated by intentional micronisation ("the SUN approach"; Nowack et al. Meeting the Needs for Released Nanomaterials Required for Further Testing: The SUN Approach. Environmental Science & Technology, 2016 (50), 2747). The NEPs were composed of four matrices (epoxy, polyolefin, polyoxymethylene, and cement) with up to 5% content of three nanomaterials (carbon nanotubes, iron oxide, and organic pigment). Regardless of the type of nanomaterial or matrix used, it was observed that nanomaterials were only partially exposed at the NEP fragment surface, indicating that mostly the intrinsic and extrinsic properties of the matrix drove the NEP fragment toxicity. Ecotoxicity in multiple assays was done covering relevant media from terrestrial to aquatic, including sewage treatment plant (biological activity), soil worms (Enchytraeus crypticus), and fish (zebrafish embryo and larvae and trout cell lines). We designed the studies to explore the possible modulation of ecotoxicity by nanomaterial additives in plastics/polymer/cement, finding none. The results support NEPs grouping by the matrix material regarding ecotoxicological effect during the use phase. Furthermore, control results on nanomaterial-free polymer fragments representing microplastic had no significant adverse effects up to the highest concentration tested.

Due to climate change and intense anthropogenic activity, organisms from cold regions are often exposed to combined effects of temperature fluctuations and contaminants. In this investigation, we assessed the lipid, protein, and carbohydrate energy budgets; the energy available (Ea); consumed (Ec); and cellular energy allocation (CEA) of the freeze-tolerant Enchytraeus albidus, when exposed to sublethal concentrations of 4-nonylphenol (a lipophilic contaminant) for 7 days, followed by exposure to different temperature regimes (continuous 2 °C, continuous -4 °C, and daily freeze-thaw cycles (FTC) (2 to -4 °C) for additional 10 days. Results showed that a pre-exposure to 4-nonylphenol (4-NP) induced important changes in the worms' energy budgets and CEA and increased mortality with most severe effects observed for the FTC events. For FTC, lipids were the most accumulated energy source, whereas during freezing (-4 °C), proteins were the most used. FTC caused the highest Ec, indicating the higher energy requirements for organisms when shifting between freezing and thawing events. This is also in line with the higher mortality observed in FTC compared to continuous -4 °C or 2 °C. Worms exposed to continuous freezing presented relatively stable and positive levels of Ea and low levels of Ec, possibly related with the decrease in metabolism.

Nearly 80% of all the nano-powders produced worldwide are metal oxides, and among these materials titanium dioxide (TiO2) is one of the most produced. Titanium dioxide's toxicity is estimated as low to soil organisms, but some studies have shown that TiO2 nanoparticles can cause oxidative stress. Additionally, it is known that TiO2 is activated by ultraviolet (UV) radiation, which can promote photocatalytic generation of reactive oxygen species, which is seldom taken into account in toxicity testing. In the present study, the authors investigated the effects of different TiO2 and zirconium materials on the soil oligochaete Enchytraeus crypticus, using exposure via soil, water, and soil:water extracts, and studied the effects combined with UV radiation. The results showed that zirconium dioxide (bulk and nano) was not toxic, whereas zirconium tetrachloride reduced enchytraeid reproduction in soil (50% effect concentration = 502 mg/kg). The TiO2 materials were also not toxic via soil exposure or under UV radiation. However, pre-exposure to TiO2 and UV radiation via aqueous media caused a lower reproductive output post-exposure in clean soil (20–50% less but only observed at the lowest concentration tested, 1 mg/L); that is, the effect of TiO2 in water was potentiated by the UV radiation and measurable as a decrease in reproduction in soil media. Environ Toxicol Chem 2015;34:2409–2416. © 2015 SETAC

Supralittoral ecosystems are among the most challenging environments for soil organisms, particularly when salinity fluctuations are involved, frequently combined with the presence of contaminants as a result of intense anthropogenic activities. Knowledge of how salinity influences the effect of contaminants in supralittoral species is crucial for determining the safety factors required when extrapolating results from optimal laboratory conditions to these natural ecosystems. The present study therefore evaluated the effects of 2 metals (copper and cadmium) and 2 organic compounds (carbendazim and 4-nonylphenol) in the absence or presence of 15‰ NaCl in the potworm Enchytraeus albidus, a model organism for ecotoxicology studies commonly found in supralittoral ecosystems, The potworms had a higher reproduction in saline soil than in control soil. Moreover, the effects of copper and carbendazim on reproduction were smaller than when they were tested in nonsaline soil. Potworms exposed to nonsaline soils also had significantly higher tissue concentrations of metals, which partly explains the effects on reproduction. The influence of salinity on effects of 4-nonylphenol was, however, less clear; effects on survival decreased in saline soil, but effects on reproduction were highest in saline soil. The latter slightly correlated with tissue concentrations of the chemical. The present study provides the first evidence that soil salinity has a significant influence on the impact of contaminants evaluated with the enchytraeid reproduction test.

Gene expression studies provided a major step forward in ecotoxicology and ecological functional genomics, among other areas. How to classify changes in gene expression to be significant compared to its normal operating range (NOR) requires a number of biological and statistical criteria. Doubts have been casted about when a change in gene expression has ecologically relevant meaning and what is the normal biological variation or NOR. Because genes are highly sensitive and self-regulating pieces, how can we design our experiments to control normal variation? The main aim of the present paper is to discuss such questions, while providing suggestions for improvement and interpretation. We have analysed several gene control data sets of Enchytraeus albidus comparing standard control test conditions, i.e. (1) from experiments performed at different occasions in time, (2) to culture media, (3) to acetone control, (4) from exposure time series (2, 4 and 8 days) and (5) between replicates. We discriminated between some normal variation sources. For instance, the transference of organisms from cultures to standard testing condition suggests a lowered general metabolism. Chemical carriers like acetone seem to cause changes related with soil structure and hypoxia conditions. Organisms have a “time-like” transcription signature when sampled along a time series. The coefficient of variance (CV) between biological replicates was 0.1 E. albidus gene expression was made which represents a naturally varying ecological window – this contributes for the discrimination between stress responses and natural ecological variations in soil studies.

It is here shown how partial order can be used to provide a robust and consistent implementation of the DF4 approach which provides unbiased information enabling comparison and open up the possibility for grouping. The approach is based on few assumptions, works well with the data, can include different types of input parameters, and can provide fundamental information about the ranks of tested materials. It is shown that the materials in many cases are below one threshold within a tier, but above another threshold within the same tier. It is also observed that the ranks of the materials can differ between tiers, although this is less relevant for DF4 since parameters evaluation may be hierarchical.

Enchytraeids have been used in standard ecotoxicity testing for about 20 years now. Since adopting the standard test for survival and reproduction, a number of additional tools have been developed, including transcriptomics and enzymatic biomarkers. So far, a genotoxicity tool and endpoint have not been used; hence, the goals of the current study included the optimization of the in vivo alkaline comet assay in Enchytraeus crypticus. Further, the effect of silver nanomaterial (Ag NM300K, dispersed, 15 nm) was tested and compared with AgNO3 . Hydrogen peroxide (H2 O2 ) was used as a positive control. The various steps were optimized. The fully detailed standard operating procedure (SOP) is presented here. Ag materials caused genotoxicity, this being differentiated for the nano and non-nano forms. AgNO3 caused genotoxicity after 3 days (d) of exposure in a dose related manner, although after 7d the effects were either reduced or repaired, whereas Ag NM300K caused higher genotoxicity after 7d for the lowest concentration (EC20), highlighting a potential non-monotone dose-response effect. Overall, the comet assay showed the power to discriminate effects between materials and also toxicity at low relevant doses. This article is protected by copyright. All rights reserved.

Climate change has serious impacts on ecosystems, e.g. species diversity and abundance. It is well known that changes in temperature may have a pronounced influence on the reproductive output, growth and survival of various terrestrial species. However, much less is known on to how changes in temperature combined with exposure to pollution will influence biodiversity, the interaction between species, and the resulting change in species composition. In order to understand the effects of changes in temperature and copper pollution (individually and in combination) on soil communities and processes, a factorial multispecies experiment was performed. Six animal species (representing different functional groups) were exposed in control (30 mg Cu/kg) and copper-contaminated soil (1000 mg Cu/kg) to four temperatures (10, 14, 19, and 23 °C) representing the “summer” range (low to high) for Denmark, and three exposure periods (28, 61, and 84 days). The species composition, feeding activity and OM turnover were assessed throughout. Multivariate analysis displayed significant changes in the food-web both with different copper levels and temperatures, resulting in different species composition for each exposure scenario. The most important species were Enchytraeus crypticus (most sensitive to copper and temperature) and Folsomia candida (most abundant). Major changes in abundance due to temperature occurred in the first 28 days of exposure, where population growth was higher. A temperature dependent population growth rate could be modeled for an exposure period of 28 days, whereas after 61 and 84 days of exposure the data did not fit the model. Especially for treatments that also included Cu, modeling of the population growth was no longer possible. The results of our study indicate that when climate change occurs in polluted areas, the consequences on populations cannot be predicted based on data from non-polluted areas. The risk may be synergistic for certain species, as indicated in the present study, and the final balance may depend on the particular species composition of that ecosystem.

Polyoxometalates (POMs) are metal oxo clusters that have been investigated for several applications in material sciences, catalysis, and biomedicine; these gained increasing interest in the field of nanotechnology as nanocarriers for drug delivery. Associated to the increasing applications, there is the need for information regarding the effects on the environment of these compounds, which is completely absent in the literature. In the present study, the effects of europium polyoxometalates encapsulated into silica nanoparticles (Eu-POM/SiO2 NPs) were assessed on the soil representative Enchytraeus crypticus. The individual materials were also assessed (Eu-POMs and SiO2 NPs). Toxicity was evaluated in various test media with increasing complexity: water, soil/water extracts, and soil. Toxicity was only observed for Eu-POM/SiO2 NPs and in the presence of soil components. Despite the fact that effects were observed for concentrations higher than current predicted environmental concentration (PEC), attention should be given to the growing use of these compounds. The present study shows the importance of assessing the effects in soil media, also compared to water. Moreover, results of \"no effect\" are critically needed and often unpublished. The present study can contribute to the improvement of the OECD guidelines for safety of manufactured nanomaterials on environmental toxicity in the soil compartment providing an improved test alternative.

Enchytraeus albidus is a freeze-tolerant enchytraeid found in diverse habitats, from supra-littoral to terrestrial, and spanning temperate to arctic regions. Thus, this worm is often exposed to sub-zero temperatures and fluctuating salinity regimes that can lead to physiological stress. We therefore studied the oxidative stress by measuring lipid peroxidation, anti-oxidant defenses and neurotransmission activity in E. albidus from arctic (Greenland) and temperate (Germany) regions during a short-term exposure to saline conditions (0, 15, 35 and 50‰ NaCl) and low temperatures (+ 2, − 2 and − 5 °C). Various enzymatic and non-enzymatic oxidative stress markers were analyzed. Results have shown that both salt and freezing caused oxidative stress in E. albidus, particularly from Germany, as confirmed by catalase, glutathione-S-transferase and superoxide dismutase activities and lipid peroxidation levels. Neurotransmission (as judged from acetylcholinesterase activity) was reduced by saline conditions at + 2 °C, but stimulated at − 2 and − 5 °C. Worms from Greenland had relatively higher and more stable levels of antioxidants than worms from Germany, reflecting their higher tolerance of freezing and saline conditions.

Cellular energy allocation (CEA) is a methodology developed to evaluate the effects of toxic stress on the metabolic balance of organisms. It consists of the integration of the energy reserves available (Ea; total carbohydrate, protein and lipid content) and energy consumption (Ec) estimated by measuring electron transport system (ETS) activity. The main goal of the present study was to evaluate the effects on the energy budget of the soil invertebrate Enchytraeus albidus (Oligochaeta) after exposure to dimethoate, atrazine, and carbendazim (by testing the reproduction 10% effective concentration, 20% effective concentration, 50% effective concentration and 90% effective concentration) over periods of time from 0 d to 2 d, 4 d, and 8 d. Significant changes in energy reserves were observed with all pesticides, together with effects on energy consumption. Carbohydrates were the first energy source to be used, and clear depletions occurred with all pesticides. Energy consumption increased generally over longer exposures and with higher concentrations of the pesticides. Although clear changes were seen in the individual energy reserve budgets and on Ec, CEA was only significantly reduced with atrazine exposures longer than 4 d. The nearly absent effects on CEA at concentrations known to affect reproduction indicate that the reduction in reproduction is not likely to be caused by a reduction in the total energy budget during the first 8 d of exposure. The present study showed the importance of complementing CEA interpretation with the individual Ea and Ec parameters, in particular if these show opposite balances. The Ea and Ec results were in good agreement with gene transcription results from a parallel study, hence suggesting translation and showing the advantage of combining various effect levels to advance the understanding of mechanisms. Environ Toxicol Chem 2013;32:2800–2807. © 2013 SETAC

The transcriptomic effects of Ibuprofen (IBU) in the digestive gland tissue of Mytilus galloprovincialis Lam. specimens exposed at low environmental concentrations (250 ng L-1) are presented. Using a 1.7 K feature cDNA microarray along with linear models and empirical Bayes statistical methods 225 differentially expressed genes were identified in mussels treated with IBU across a 15-day period. Transcriptional dynamics were typical of an adaptive response with a peak of gene expression change at day 7 (177 features, representing about 11% of sequences available for analysis) and an almost full recovery at the end of the exposure period. Functional genomics by means of Gene Ontology term analysis unraveled typical mussel stress responses i.e. aminoglycan (chitin) metabolic processes but also more specific effects such as the regulation of NF-kappa B transcription factor activity. (C) 2016 Elsevier Ltd. All rights reserved.