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Abstract Nickel nanoparticles (NiNPs) have an estimated production of ca. 20 tons per year in the US. Nickel has been risk-assessed for long in Europe, but not NiNPs, hence the concern for the environment. In the present study, we focused on investigating the mechanisms of toxicity of NiNPs and the comparison to NiNO 3. The high-throughput microarray for the soil ecotox model Enchytraeus crypticus (Oligochaeta) was used. To anchor gene to phenotype effect level, organisms were exposed to reproduction effect concentrations EC20 and EC50, for 3 and 7 days. Results showed commonly affected pathways between NiNPs and NiNO 3 , including increase in proteolysis, apoptosis and inflammatory response, and interference with the nervous system. Mechanisms unique to NiNO 3 were also observed (e.g. glutathione synthesis). No specific mechanisms for NiNPs were found, which could indicate that longer exposure period (>7 days) is required to capture the peak response to NiNPs. A mechanisms scheme is assembled, showing both common and unique mechanisms to NiNO 3 and NiNPs, providing an important framework for further, more targeted, studies. Graphical abstract Image 1 Highlights • High-throughput gene expression results for NiNPs and NiNO 3 were obtained. • Gene expression was anchored to higher effect level (reproduction EC20/50). • Common mechanisms were observed between the two Ni forms. • Mechanisms unique to NiNO 3 were observed, but not for NiNPs. • Mechanistic scheme (Adverse Outcome Pathway like) was drafted for Ni materials. An Adverse Outcome Pathway for nickel based NM was built, providing an important framework for further, more targeted, studies. [ABSTRACT FROM AUTHOR]

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 (AgNO 3 ) for all exposure durations and showed a continuous higher avoidance with time (based on EC 50 values). The AgNMs avoidance was in the order NM300K

Nanoagrochemicals have the potential to revolutionize agriculture towards a precision farming system, able to reduce application rates and consequently their environmental footprint, while keeping efficacy. Several nanoagrochemicals (including nanopesticides (Npes) and nanofertilizers (Nfer)) are already commercialized but the environmental risk assessment of these advanced materials is often lacking. In the present study, we studied the commercial fertilizer WELGRO® Cu + Zn and assessed its ecotoxicity to the soil invertebrate species Enchytraeus crypticus (Oligochaeta), further comparing it to its individual active substances CuO and ZnO. To get a comprehensive picture of possible effects, we used four types of highly relevant tests in LUFA 2.2 soil: 1) avoidance behaviour (2 days), 2) reproduction (OECD standard, 28 d), 3) its extension (56 d), and 4) the full life cycle (FLC) (46 d) – this high level of hazard screening allows for increased interpretation. The results confirmed the nano-features of WELGRO® and a higher toxicity than the mixture of the individual components CuO + ZnO. E. crypticus avoided the soil spiked with WELGRO® and CuO + ZnO, this being the most sensitive endpoint - avoidance behaviour. Both WELGRO® and the active substances were little to non-toxic based on the OECD standard test. However, the toxicity dramatically increased in the tests focussing on longer-term sustainability measures, i.e., 56 days, ca. 170 for WELGRO®. This seems related to the nano-features of WELGRO®, e.g., slow release of ions from the nanoparticles throughout time. The FLCt results showed WELGRO® affected hatching and juveniles' survival, being these the most sensitive life stages. Hence, under actual real world field usage scenarios, i.e., based on the recommended application rates, nanoenabled WELGRO® can affect oligochaete populations like enchytraeids, both via the immediate avoidance behaviour and also via prolonged exposure periods. [Display omitted] • WELGRO®Cu + Zn (commercial) represents a nanoenabled fertilizer (150 nm NPs). • Effects of WELGRO® and its active substances, a mixture of CuO and ZnO were assessed. • WELGRO® was more toxic than the mixture of its active substances. • Avoidance behaviour was the most sensitive endpoint. • The toxicity increased with prolonged exposure (from 28 to 56 d), ca. 170x for WELGRO®. [ABSTRACT FROM AUTHOR]

Nanoemulsions (NEs) have been extensively studied as carriers for drug delivery, since these provide a good alternative to the existing non-nano systems, while promoting their target delivery and controlled release. NEs are considered safe drug carriers from a pre-clinical perspective, but there is currently no information on their ecotoxicological effects. In the present study we investigated the toxicity of a NE material (lecithin, sunflower oil, borate buffer) designed to be used as a liposomal excipient for eye drops, further referred to as (Lipid Particle:LP) LP_Eye and its dispersant (borate buffer) (LP_Eye disp.). Effects were assessed using two model species in soil ecotoxicology in LUFA 2.2 soil: Enchytraeus crypticus (Oligochaeta) and Folsomia candida (Collembola), based on the OECD standard guideline (28 days) and its extension, a longer-term exposure (56 days). The endpoints evaluated included survival, reproduction, and size. LP_Eye and LP_Eye disp. were toxic to E. crypticus and F. candida , affecting all measured endpoints. The toxicity of LP_Eye in E. crypticus seemed to be induced by the dispersant, whereas for F. candida , more sensitive, this was less explanatory. There were no indications that toxicity increased with longer exposure. Current results provide ecotoxicological data for a group of NMs that was absent, revealing toxicity to relevant environmental species. Indications were that the dispersant contributed to most of the observed effects, thus there is room to improve the formulation and achieve lower environmental impact. Graphical abstract. [Display omitted] • Nanoemulsions (NEs) ecotoxicity is virtually unknown. • Hazardss of NE with medical application were assessed in E. crypticus and F. candida. • NE was toxic to these soil invertebrates (survival, reproduction, size). • The NE dispersant seems to explain most of the toxicity. • The NE formulation could be improved to reduce environmental impacts. [ABSTRACT FROM AUTHOR]

Engineered Nanoparticles (ENPs) present novel/added challenges to the established effect assessment modus operandi , requiring an update of used methods. ENPs are dimensionally and physically different from conventional chemicals, which imply that the metrics with which we relate effect and the type of effect responses are different from that of the conventional approach. Effects on organisms are often preceded by changes on the sub-organismal level (cell, genes), dedicated tools have vast potential to detect earlier (and link to) effects on higher levels of organization. High-throughput screening (HTS) is rapid, cost-effective and specific. One way forward is to link HTS to population outcomes, targeting a systems toxicology approach. Although the benefits of integrating various levels of information may seem obvious, this is an even more decisive aspect when rapid answers are needed for ENPs. Here we rank the available tools/methods, highlight main study gaps and list priority needs and the way forward. [ABSTRACT FROM AUTHOR]

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-κB transcription factor activity. [ABSTRACT FROM AUTHOR]

Metals are among the most common contaminants in soils in Europe. Although their effects are relatively well known regarding survival and reproduction to soil invertebrates, their mode of action is poorly understood. Enchytraeus albidus is a model organism in ecotoxicology and with the development of a gene library for this species, transcriptomic studies are now possible. The main aim of this study is to understand the Cu and Ni mechanisms of response in E. albidus, in comparison with Cd and Zn (already studied). E. albidus were exposed to Cu and Ni for 4 days to the reproduction effect concentrations EC50 and EC90. Results indicate that Cu and Ni have similar mechanisms of toxicity. When comparing four elements (hierarchical clustering) it was possible to observe a clear separation of Cd from all other metals. This separation correlates with the available information from other species regarding the toxicokinetics of the tested elements. [Copyright &y& Elsevier]

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. [Display omitted] • Nanoplastics (NPls) alone were not severely harmful to zebrafish embryos. • NPls presence modified the toxicity of phenmedipham (PHE). • NPls presence appears to increase the uptake of PHE towards zebrafish embryos. • NPls + PHE exposure disturbed more biochemical endpoints than the single exposures. • Diverse interaction effects (none/synergism/antagonism) found at NPls + PHE exposure. [ABSTRACT FROM AUTHOR]

To better understand chemical modes of action, emphasis has been given to stress responses at lower levels of biological organization. Cholinesterases and antioxidant defenses are among the most used biomarkers due to their crucial role in the neurocholinergic transmission and in cell homeostasis preventing DNA damage, enzymatic inactivation and lipid peroxidation. The main goal of this study was to investigate the effects of zinc and cadmium on survival and reproduction of E. albidus and to assess metals oxidative stress potential and neurotoxic effects at concentrations that affected reproduction. Both metals affected the enchytraeids’ survival and reproduction and induced significant changes in the antioxidant defenses as well as increased lipid peroxidation, indicating oxidative damage. This study demonstrates that determining effects at different levels of biological organization can give better information on the physiological responses of enchytraeids in metal contamination events and further unravel the mechanistic processes dealing with metal stress. [Copyright &y& Elsevier]

Abstract: Triclosan (TCS) is an antimicrobial agent which is used as a broad-spectrum bacteriostatic and found in personal care products, and due to this it is spread in the environment. Aquatic toxicity studies have been reported, but little data is available for its toxicity to terrestrial organisms. The main purpose of this study was to assess the toxicity of TCS in the terrestrial environment, using a battery of soil species, belonging to different taxonomic levels. The species included the invertebrates Eisenia andrei, Enchytraeus albidus and Folsomia candida and the plants Triticum aestivum (monocotyledonous) and Brassica rapa (dicotyledonous). For the invertebrate species the reproduction EC10s were 0.6 (E. andrei), 2 (E. albidus) and 7(F. candida) mg TCS/kg soil dry weight (DW). For plants'' emergence EC10s are 0.1 (B. rapa) and 142 (T. aestivum) TCS/kg soil DW. The calculated PNEC (Predicted No Effect Concentration) ranged between 0.0008 and 0.004mg TCS/kg (95% certainty) and between 0.04 and 0.2mg TCS/kg soil DW (50% certainty) when using the SSD (Species Sensitivity Distribution) approach; applying a safety factor to the lowest EC10 resulted in a PNEC of 0.06mg TCS/kg soil DW. [Copyright &y& Elsevier]

Abstract: Contamination problems are often characterized by complex mixtures of chemicals. There are two conceptual models usually used to evaluate patterns of mixture toxicity: Concentration Addition (CA) and Independent Action (IA). Deviations from these models as synergism, antagonism and dose dependency also occur. In the present study, single and mixture toxicity of atrazine, dimethoate, lindane, zinc and cadmium were tested in Porcellionides pruinosus and Enchytraeus albidus, using avoidance as test parameter. For both species patterns of antagonism were found when exposed to dimethoate and atrazine, synergism for lindane and dimethoate exposures (with the exception of lower doses in the isopod case study) and concentration addition for cadmium and zinc occurred, while the exposure to cadmium and dimethoate showed dissimilar patterns. This study highlights the importance of dose dependencies when testing chemical mixtures and that avoidance tests can also be used to asses the effects of mixture toxicity. [Copyright &y& Elsevier]

Abstract: Enchytraeids are ecologically relevant soil species and are commonly used in standardized toxicity tests. Their rapid reaction to a chemical exposure can be used as a toxicological measurement endpoint that assesses the avoidance behavior. The objectives of this investigation were to determine the effects of soil properties on the avoidance behavior of Enchytraeus albidus and to optimize the duration of avoidance test. The avoidance tests included (1) exposures in OECD artificial soil with standard or modified properties (pH, clay or peat content), and (2) exposures to copper chloride, cadmium chloride, and to the organic pesticides dimethoate and phenmedipham for different time periods. Results showed that alteration of OECD soil constituents significantly affected the avoidance behavior of enchytraeids, and that the 48-h exposure was the optimal duration of the test. Consideration of soil properties is important for selecting appropriate experimental design and interpreting the results of the enchytraeid avoidance test. [Copyright &y& Elsevier]

Abstract: Enchytraeids (Enchytraeus albidus) directly improve the pore structure of the soil and are indirectly involved in regulating the degradation of organic matter. Due to their behavior they are able to avoid unfavorable environmental conditions. Avoidance tests allow a first assessment of toxicity of a contaminated or spiked soil within 48 h, by using the reaction of the enchytraeids as measurement endpoint. In this period, the organisms can choose between the control soil and the test soil. In the tests reported here, enchytraeids were exposed to LUFA 2.2 soil spiked with the following set of toxic substances: copper chloride, zinc chloride, cadmium chloride, phenmedipham, benomyl, carbendazim, dimethoate, atrazine, pentachlorophenol, chlorpyriphos, lindane, TBTO, Linear Alkylbenzene Sulfonates (LAS) and boric acid. Different chemical concentrations were tested. EC50s ranged from 8 mg/kg (Carbendazim) to >1000 mg/kg (e.g. LAS). While the tested heavy metals showed clear dose-response relationships, the effect pattern differed considerably in the tests with organic chemicals, e.g. no avoidance behaviour was observed in LAS, even at very high doses. Here we proposed to standardize the Enchytraeid avoidance test in a way similar to what is currently done for the earthworm and collembolan avoidance tests by the International Standardisation Organisation (ISO). [Copyright &y& Elsevier]

Abstract: Soil ecotoxicology studies are usually performed in standard soils, such as the OECD (Organization for Economic Cooperation and Development) artificial soil or the LUFA St. 2.2, a natural soil. When assessing the toxic effects in the environment, soil properties are often different from those in standard soils, which might lead to a different exposure situation for the test species and, therefore, to a different evaluation of the risk of the test substance. Selected to cover a broad range of properties and based on the Euro-Soils concept, 18 different soils were studied regarding their suitability to two test species: Enchytraeus albidus and Enchytraeus luxuriosus (Enchytraeidae). In reproduction tests, the test species reacted differently to the tested soils, but both enchytraeids did not survive in acid soils (i.e. pH⩽5). Results indicate that although a combined effect of soil properties seems to occur, certain soil properties are causing specific toxic effects. In the present study, EC50s in enchytraeids changed by a factor of 9 for juveniles and nearly 30 for the adults of E. luxuriosus (maximum values; slightly lower values were found for E. albidus), which shows how important the test soil can become for the environmental risk assessment of chemicals. More data using more soils and species are required to understand the effect of soil properties in soil toxicology. Nevertheless, it was clear that certain soil properties such as OM (Organic Matter) and WHC (Water Holding Capacity) or pH, CEC (Cation Exchange Capacity), C/N (Carbon/Nitrogen ratio) and clay content did interact with the chemical and the organisms. [Copyright &y& Elsevier]

Abstract: Enchytraeids are typical inhabitants of many soils, contributing to vital processes of this environmental compartment. Indirectly they are involved in regulating the degradation of organic matter, as well as improving the pore structure of the soil. Due to their behaviour, they are able to avoid unfavourable environmental conditions. Avoidance tests with enchytraeids, initially developed with earthworms by several authors, are quick and easy to perform. With these tests a first assessment of the toxicity of a (contaminated or spiked) soil is possible in just 48h by using the reaction of the enchytraeids as measurement endpoint. In this period of time the organisms can choose between the control soil and the other soil (a contaminated or spiked or another soil with different physico-chemical properties). In the tests reported here, the enchytraeids were exposed to control soils spiked with the fungicides Benomyl and Carbendazim and the herbicide Phenmedipham. Several chemical concentrations were tested in order to evaluate the avoidance behaviour to toxic substances. In fact, often these short-term screening tests gave results showing avoidance at concentrations in a range similar to the acute test results but, higher than in chronic tests. Further tests are needed to decide whether the results gained in this study can be extrapolated to other chemicals. It is proposed to standardize the Enchytraeid Avoidance Test as it is currently done for the Earthworm Avoidance Test by the International Standard Organization (ISO). [Copyright &y& Elsevier]

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. • Ash-based formulations improved physico-chemical conditions in highly degraded mining soils. • Invertebrate reproduction benefits from stabilized ash granules and composted sludge application. • Organic carbon addition with composted sludge promotes E. crypticus reproduction. • Ash granulation reduced EC and soluble chloride-related hazard for model invertebrates. Ash-based amendments are a promising strategy to contribute to the recovery of mining soils' capacity to support ecosystems function and to recycling nutrients in relevant waste streams. [ABSTRACT FROM AUTHOR]

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 AgNO 3 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 AgNO 3 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 AgNO 3 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. [Display omitted] • One of the most complete high-throughput data set was obtained for the environment. • Good agreement between the 3 omics layers: proteomics, metabolomics, transcriptomics. • Activated pathways were oppositely (up vs down) regulated for AgNM300K and AgNO 3. • Integration of high quality "omics" in Risk Assessment will be beneficial. [ABSTRACT FROM AUTHOR]

The concern about microplastic (a group of polymers) in the environment may cause us to overlook a more substantial problem: microplastics will fragment into nanoplastics. This fragmentation will lead to a high number of nanoplastics particles. Such nanoplastic can be taken up by cells, as opposed to microscale particles that are either not or to much less extend taken up. Fragmentation into nano will also release materials previously safely embedded in the polymer. We here present results from 25 OECD/ISO in vivo hazard tests, and beyond, e.g. extended exposure duration, with Enchytraeus crypticus , using pristine nano-scale materials (NMs) [CuO, Fe 2 O 3 , Organic Pigment, MWCNT], fragmented products (polymers) with these NMs embedded in the matrices (FP_NM), and fragmented polymers without NMs (FP) [covering the 4 major plastic types: Acrylic, Polyethylene, Polypropylene and Epoxy]. For example, MWCNTs induced a highly significant population decrease after extended period of 60 days, despite having no impact after 28 days' exposure, the standard OECD duration. We conclude, that the standard tests were not suitable to evaluate hazards of these plastic fragments, weathering/ageing of materials is recommended, and extension of test duration can add value to the testing of NMs. We must refocus the concern to testing with polymers (not only "plastics"), from micro-to nano-polymers, and from aquatic to terrestrial environments. Image 1 • Because microplastics fragment, nanoplastics (NPl) are estimated to greatly increase. • Results of 25 in vivo OECD tests showed guidelines need update for NPl testing. • Update recommendation include relevant NPls weathering/aging. • Update recommendation include extended test duration for testing of vPvB like NPls. Thorough evaluation of the current terrestrial guidelines shows they are not adequate to identify nanoplastic related hazard, weathering of materials and extension of test duration are recommended. [ABSTRACT FROM AUTHOR]

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 AgNO 3. 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: AgNO 3 ≥ Ag NM300K ≫ NC-AgNM ≥ PVP-AgNM. E. crypticus avoided AgNO 3 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. Image 1 • Novel multi-endpoint approach for mechanistic based hazard assessment in E. crypticus. • Ag exposed organisms cannot avoid Ag after 24 h. • Ag affects the GABBAergic correlating to avoidance. • Ag affects primarily embryo development and juvenile stages. • High ecological impact of Ag long term exposure expected. Multi-endpoint approach advances the understanding of effects of (nano)materials: towards a mechanistic based risk assessment strategy. [ABSTRACT FROM AUTHOR]

Nanopesticides (Npes) offer improved efficacy compared to their conventional forms while reducing the usage/application rates, hence being more sustainable options. However, there is still a knowledge gap on the Npes environmental impacts. To support the safety of nano-enabled pesticides, the present study aimed at assessing the toxicity of the commercial Npe NUCOP-M and the active substance copper oxychloride, using the ecotoxicological soil model Enchytraeus crypticus and LUFA 2.2 soil. Bioassays were performed to assess various endpoints within short-to longer-term exposures: avoidance behaviour (2 d), hatching (13 d), survival, reproduction and organisms' size (based on the standard OECD test (28 d), the OECD extension (56 d), and the Full Life Cycle test – FLCt (46 d)). Based on the standard OECD test and its extension, NUCOP-M had a similar level of toxicity as copper oxychloride without indications of increase in toxicity over time (28 versus 56 d). The shorter-term exposures (2 and 13 d) showed higher toxicity for copper oxychloride. The exposure from cocoon stage (FLCt) seemed to provide an adaptative advantage (reduced toxicity) to NUCOP-M. The differences might be related to a slower release of Cu2+ ions from NUCOP-M, which seems to account for the toxicity at longer-term. Based on the recommended application doses (ca. 1.72 mg NUCOP-M kg−1, i.e. 0.62 mg Cu kg−1 in the topsoil) there is no unacceptable risk of NUCOP-M on the enchytraeid population. [Display omitted] • Environmental impacts of nanopesticides (Npes) should be studied. • Ecotoxicity of commercial Npe NUCOP-M was assessed in E. crypticus. • High screening level (endpoints and exposure time) are relevant. • NUCOP-M presented lower risks to enchytraeids than Cu-oxychloride. • Differences might be related to a slower release of Cu2+ ions from NUCOP-M. [ABSTRACT FROM AUTHOR]

The widespread production and use of silver nanomaterials (AgNMs) in consumer and medical products have been raising environmental concerns. Once in the environment, the soil is one of the major sinks of AgNMs due to e.g. sewage sludge applications, and invertebrates are directly exposed. In this study, we investigate the potential of N-acetylcysteine (NAC) to reduce the toxic effects of Ag NM300 K (and AgNO 3) on the soil invertebrate Enchytraeus crypticus. Ag NM300 K induces mortality, reproduction impairment, and avoidance. The addition of NAC to the soil showed a remarkable reduction in the toxicity of Ag, indicating that NAC can act as a detoxifying agent for terrestrial organisms exposed to Ag materials. That the reduction in toxicity likely is caused by thiol groups, was confirmed by GSH and GSSH studies. Identifying the mechanisms and hence alternatives that allow the recovery of contaminated soils is an important mitigation measure to promote environmental safety and reduce the associated risks to human health. Further, it may inform on strategies to implement in safe-by-design industry development. Image 1 • Thiol antioxidants (N-acetylcysteine (NAC), glutathione (GSH)) reduce Ag toxicity. • E. crypticus was exposed to Ag materials after spiking soil with NAC (and GSH). • Added NAC remarkably reversed the toxic effects caused by Ag materials. • Added GSH corroborates that thiol groups must play a key role in reducing Ag toxicity. N-acetylcysteine (NAC) and reduced glutathione (GSH), thiol-containing compounds, remarkably reversed the toxic effects caused by Ag materials. [ABSTRACT FROM AUTHOR]

[Display omitted] • Aging and its interaction with toxicity is seldom assessed in standards. • Toxicity of Cd was assessed in aged E. crypticus : young (19d), old (92d) and oldest (128d). • Age influenced Cd toxicity: oldest > young > old. • Oldest and youngest were most sensitive to Cd. Aging is a natural process for all living beings and there is often a decline of overall performance after a certain age tipping point. The response to stressors, e.g. exposure to chemicals can differ depending on the individuals' age. Standard guidelines do not always include synchronized age animals and are often optimized based on healthy animals of homogeneous populations, not aiming to cover age aspects. Hence, in the present study we aimed to assess the effects of aging on Enchytraeus crypticus (Oligochaeta) response to cadmium (Cd) exposure. For that, cultures with 3 synchronized ages [young: 18–20 days old, old: 91–93 days old and oldest: 127–129 days old] plus the standard (non-synchronized adults), were exposed to CdCl 2 (0, 1, 3.2, 10, 32, 100, 320 mg Cd/kg) in LUFA 2.2 soil. Results showed that Cd toxicity varied depending on the animal's age, with the young and oldest being most sensitive to Cd (reproduction EC 50 _old = 138 > EC 50 _young = 69 > EC 50 _oldest = 46 mg Cd/kg soil). This highlights the importance of age when assessing toxicity. Testing animals with synchronized age should improve results comparability but will not necessarily capture the most sensitive age. Toxicity to Cd has been shown to be higher in younger animals compared to standard cultures, although this may not be a general pattern with other chemicals or stressors. When using standardized guidelines, if cultures are not synchronized results will suffer from variability due to various ages of the animals. For a standard context we recommend the start with young (18–20 days) animals instead of non-synchronized adults. On the other hand, the study of populations on the edge windo w is highly relevant and can be much more explored in an ecotoxicology frame, as this can also enlighten impacts of aging in a wider species context. [ABSTRACT FROM AUTHOR]

Amongst climate change's impacts a major concern is salinization of soils, for example due to saltwater intrusion. The aim of the present study was to investigate in a standard field soil the impacts of soil salinity increase. The purpose was to study this in two soil invertebrates that are key model ecotoxicology test-species, Enchytraeus crypticus and Folsomia candida as surrogate representatives of the soil ecosystem. The exposure followed the standard ecotoxicity OECD (Organization for Economic Cooperation and Development) test guidelines, and the assessed endpoints were survival, reproduction and size. Exposure done in LUFA 2.2 soil, spiked with 0,0.6,1,2,3,4,5,6,8 g NaCl/kg soil dry weight (DW) for E. crypticus (21 days) and 0,0.06,0.6,1,2,3,4,5,6 g NaCl/kg soil DW for F. candida (28 days). There was a similar impact for both species in terms of survival (LC 50 =4.2 g NaCl/kg soil DW), whereas at the reproductive output level of F. candida (EC 50 =2.1 g NaCl/kg soil DW) was more sensitive than E. crypticus (EC 50 =2.4 g NaCl/kg soil DW). Further, size was impacted for F. candida in a monotonic dose-response curve for both adults (EC 50 =3.5 g NaCl/kg soil DW) and juveniles (EC 50 =2 g NaCl/kg soil DW), whereas for E. crypticus there was an increase in reproductive output at lower concentrations (0.6–1 g NaCl/kg soil DW). This increased reproduction was associated with a larger size of adults within the same concentration range. Considering the prediction from the climate models, the soil invertebrate community will be affected. As upper soils are likely to have the highest salinity increase due to evaporation, soil surface species, such as the collembolan tested here, are at higher risk. Negative population effects were occurring within salinity levels predicted by climate change models. [Display omitted] • The risk of soil salinity increase was assessed in invertebrate model fauna. • Impact on survival was similar between F. candida and E. crypticus. • Higher impact on F. candida reproduction, a soil surface species. • Soil surface is predicted to be more affected by salinity than deeper soil layers. • Predictions (2060) are much higher than the tolerance for these species. [ABSTRACT FROM AUTHOR]

There is an urgent need for sufficient knowledge to allow reliable assessment of the risks associated with engineered nanomaterials (ENPs). Significant advances in basic understanding of nano safety have been made, but there is still no clear systematic basis for risk-related research, and major uncertainties remain in the absence of uniform procedures. The following papers provide the guidance on how to proceed within the area of fate and hazard assessment, and how this links into grouping, testing and risk assessment of nanomaterials. This guidance is coupled with an industrial view on the most important research areas for nanomaterials. [ABSTRACT FROM AUTHOR]

Abstract: Enchytraeids are important inhabitants of a wide variety of soil types and suitable test organisms for environmental risk assessment through the determination of effects on survival and reproduction. In this study, the effect of pesticides with different modes of action is assessed in the standard test species Enchytraeus albidus. Main goals were: (a) to test the toxicity of a range of pesticides to E. albidus in a natural soil (LUFA 2.2), (b) to assess whether avoidance gave a prediction of effects on the survival and reproduction, and (c) to investigate if the organisms’ response to different toxics can be grouped into the respective chemical classes. Pesticides selected were the: (1) herbicides: phenmedipham and atrazine; (2) fungicides: carbendazim and pentachlorophenol; and (3) insecticides: dimethoate and lindane. All tested pesticides caused effects in the survival and reproduction of E. albidus and the compounds that showed a higher toxicity were carbendazim, dimethoate and atrazine. The effect concentrations were not chemical class dependent. In general, survival and reproduction showed similar response patterns. Avoidance behaviour showed trends of response similar to these other measured endpoints, but was less sensitive and more variable (data from a previous study). It was not possible to establish a clear correlation between survival, reproduction and avoidance endpoints. From an ecological point of view, avoidance tests are relevant but due to lower sensitivity and higher variability it is recommended that the enchytraeid avoidance test should not be used for risk assessment purposes. [Copyright &y& Elsevier]

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 EC 20 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. [ABSTRACT FROM AUTHOR]

Abstract 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. Graphical abstract Image 1 Highlights • E. crypticus embryos are more sensitive to Cd than adults. • Embryos' mechanisms were studied using high-throughput gene expression. • Major modulators of Cd toxicity were identified in the embryos. • Cd competes with Ca inside the cells (novel), besides extracellular (known). • Adverse Outcome Pathway (AOP) was improved. [ABSTRACT FROM AUTHOR]

Accumulating evidence shows that epigenetics-mediated phenotypic plasticity plays a role in an organism’s ability to deal with environmental stress. However, to date, the role of epigenetic modifications in response to stress is hardly investigated in soil invertebrates. The main objective of this proof of principle study was to explore whether total cytosine and locus-specific CpG methylation are present in two important ecotoxicological model organisms, the springtail Folsomia candida and the potworm Enchytraeus crypticus , and if so, whether methylation patterns might change with increased toxicant exposure. LC–MS/MS analyses and bisulfite sequencing were performed to identify the CpG methylation state of the organisms. We show here, for the first time, a total level of 1.4% 5-methyl cytosine methylation in the genome of E. crypticus , and an absence of both total cytosine and locus-specific CpG methylation in F. candida . In E. crypticus , methylation of CpG sites was observed in the coding sequence (CDS) of the housekeeping gene Elongation Factor 1α, while the CDS of the stress inducible Heat Shock Protein 70 gene almost lacked methylation. This confirms previous observations that DNA methylation differs between housekeeping and stress-inducible genes in invertebrates. DNA methylation patterns in E. crypticus were not affected by exposure to copper (II) sulfate pentahydrate (CuSO 4 ·5H 2 O) mixed in with LUFA 2.2 soil at sublethal effect concentrations that decreased reproduction by 10%, 20% and 50%. Although, differences in CpG methylation patterns between specific loci suggest a functional role for DNA methylation in E. crypticus , genome-wide bisulfite sequencing is needed to verify whether environmental stress affects this epigenetic hallmark. [ABSTRACT FROM AUTHOR]