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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.

The hazards of nanomaterials/nanoparticles (NMs/NPs) are mostly assessed using individual NMs, and a more systematic approach, using many NMs, is needed to evaluate its risks in the environment. Libraries of NMs, with a range of identified different but related characters/descriptors allow the comparison of effects across many NMs. The effects of a custom designed Fe-doped TiO2 NMs library containing 11 NMs was assessed on the soil model Enchytraeus crypticus (Oligochaeta), both with and without UV (standard fluorescent) radiation. Effects were analyzed at organism (phenotypic, survival and reproduction) and gene expression level (transcriptomics, high-throughput 4x44K microarray) to understand the underlying mechanisms. A total of 48 microarrays (20 test conditions) were done plus controls (UV and non-UV). Unique mechanisms induced by TiO2 NPs exposure included the impairment in RNA processing for TiO2_10nm, or deregulated apoptosis for 2%FeTiO2_10nm. Strikingly apparent was the size dependent effects such as induction of reproductive effects via smaller TiO2 NPs (≤12 nm) - embryo interaction, while larger particles (27 nm) caused reproductive effects through different mechanisms. Also, phagocytosis was affected by 12 and 27 nm NPs, but not by ≤11 nm. The organism level study shows the integrated response, i.e. the result after a cascade of events. While uni-cell models offer key mechanistic information, we here deliver a combined biological system level (phenotype and genotype), seldom available, especially for environmental models.

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.

Dpto. Medio Ambiente y Agronomía (INIA), The current environmental hazard assessment is based on the testing of the pristine substance. However, it cannot be excluded that (nano)pharmaceuticals are excreted into sewage during the use phase followed by entry into wastewater treatment plants (WWTPs). Sorption to sewage sludge or release via effluent can result in modified ecotoxicological effects which possibly can only be detected with a modified test approach. The objective of our study was to investigate a realistic exposure scenario for metallic nanoparticles (NPs) in pharmaceutical products, excreted into effluent, and released into the environment after treatment in WWTPs. The test approach was illustrated by using gold (Au) NPs. Effluent from model WWTPs were investigated in aquatic tests (Daphnia magna, fish cell lines). Sewage sludge was used as a sole food source (Eisenia fetida) or mixed with soil and used as test medium (soil microorganisms, Folsomia candida, Enchytraeus crypticus). To cover the aspect of regulation, the test systems described in OECD-test guidelines (OECD TG 201, 211, 220, 232, 249, 317) were applied. Modifications and additional test approaches were included to meet the needs arising out of the testing of nanomaterials and of the exposure scenarios. The results were assessed regarding the suitability of the test design and the toxicity of Au-NPs. Except for activated sludge as a sole food source for E.fetida, the selected test approach is suitable for the testing of nanomaterials. Additional information can be gained when compared to the common testing of the pristine nanomaterials in the standardized test systems. Effects of Au-NPs were observed in concentrations exceeding the modeled environmental., This project has received funding from the European Union’s Horizon 2020 research and innovation programme under BIORIMA grant agreement No 760928 for the testing and under NANORIGO grant agreement No 814530 regarding risk governance. Further, to FCT (Fundação para a Ciência e Tecnologia) / MCTES (Ministério da Ciência e Tecnologia do Ensino Superior) for the financial support to CESAM (UIDP/50017/2020+UIDB/50017/2020). S.I.L. Gomes is funded by National Funds (OE), through FCT, I.P., in the scope of the framework contract foreseen in the numbers 4, 5 and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law 57/2017, of July 19.

Enchytraeids (Annelida) are soil invertebrates with worldwide distribution that have served as ecotoxicology models for over 20 years. We present the first high-quality reference genome of Enchytraeus crypticus, assembled from a combination of Pacific Bioscience single-molecule real-time and Illumina sequencing platforms as a 525.2 Mbp genome (910 gapless scaffolds and 18,452 genes). We highlight isopenicillin, acquired by horizontal gene transfer and conferring antibiotic function. Significant gene family expansions associated with regeneration (long interspersed nuclear elements), the innate immune system (tripartite motif-containing protein) and response to stress (cytochrome P450) were identified. The ACE (Angiotensin-converting enzyme) — a homolog of ACE2, which is involved in the coronavirus SARS-CoV-2 cell entry — is also present in E. crypticus. There is an obvious potential of using E. crypticus as a model to study interactions between regeneration, the innate immune system and aging-dependent decline., Enchytraeus crypticus is a soil-dwelling annelid worm that has been used over the past two decades as an ecotoxicology model. Here, Mónica Amorim and colleagues present the first genome for E. crypticus. The authors identify a number of expanded gene families, including several involved with innate immunity.

The outstanding work performed by standardization organizations for guidelines to assess hazards, e.g., OECD (Organisation for Economic Co-operation and Development), is clearly visible by the currently available number and broad coverage, from aquatic to terrestrial organisms dealing with environmental relevant issues. Nevertheless, novel materials challenge the adequateness and fit-for-purpose of such standards, as the standards were developed to assess hazards of “conventional” chemical substances and not advanced materials (e.g. materials that may deliberately change behaviour). While standardization is a well-known process that requires extended time before reaching implementation stage, there is strong support from regulatory bodies for the development of New Approach Methodologies (NAMs) (e.g., updating of current guidelines, development of novel omics-, in vitro-, and in silico- tests including modelling and read-across) that meet regulatory preparedness (i.e. have considered issues important for regulatory testing). There are currently several NAMs available, complying with high quality standards and relevancy, which should be adopted. In the current review, we collected the available literature on NAMs to assess hazards of Nanomaterials (NMs), focusing on the terrestrial environment, and critically discuss the advantages, challenges and gaps. Tests were grouped into 1) Standard tests (OECD/ISO), 2) Standard tests (OECD/ISO) extensions: time course or prolonged exposures and/or multigenerational, and 3) Alternative tests, beyond current OECD/ISO: omics, biomarkers, in vitro, in silico and modelling. The goal is to provide guidance on the best practices and test designs focusing on the specificities of testing NMs, outlining recommendations and way forward.

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.

Assessing the risks of nanomaterials/nanoparticles (NMs/NPs) under various environmental conditions requires a more systematic approach, including the comparison of effects across many NMs with identified different but related characters/descriptors. Hence, there is an urgent need to provide coherent (eco)toxicological datasets containing comprehensive toxicity information relating to a diverse spectra of NPs characters. These datasets are test benches for developing holistic methodologies with broader applicability. In the present study we assessed the effects of a custom design Fe-doped TiO2 NPs library, using the soil invertebrate Enchytraeus crypticus (Oligochaeta), via a 5-day pulse via aqueous exposure followed by a 21-days recovery period in soil (survival, reproduction assessment). Obviously, when testing TiO2, realistic conditions should include UV exposure. The 11 Fe-TiO2 library contains NPs of size range between 5-27 nm with varying %Fe (enabling the photoactivation of TiO2 at energy wavelengths in the visible-light range). The NPs were each described by 122 descriptors, being a mixture of measured and atomistic model descriptors. The data were explored using single and univariate statistical methods, combined with machine learning and multiscale modelling techniques. An iterative pruning process was adopted for identifying automatically the most significant descriptors. TiO2 NPs toxicity decreased when combined with UV. Notably, the short-term water exposure induced lasting biological responses even after longer-term recovery in clean exposure. The correspondence with Fe-content correlated with the band-gap hence the reduction of UV oxidative stress. The inclusion of both measured and modelled materials data benefitted the explanation of the results, when combined with machine learning. This journal is

Engineered nanoparticles (NPs) are emerging contaminants of concern and it is important to understand their environmental behavior and ecological risks to exposed organisms. Despite their ubiquitous presence in the environment, there is little information about the hazards of certain NPs, such as boron (BNPs) and vanadium (VNPs). The aim of the present research was to investigate the effects of commercial BNPs and VNPs (80 to 100 nm) to zebrafish embryos, at different levels of biological organization. A range of nominal concentrations for both NPs (0, 0.01, 0.1, 1, and 10 mg/L) was tested. Due to the presence of triton X-100 in the NPs’ stock dispersions, an additional control group was included (0.001% triton X-100). Survival, hatching, and malformations of embryos were assessed for 96 hours (h) exposure. Locomotor behavior was evaluated at 120 h. Furthermore, embryos were exposed to 0, 1, and 10 mg/L of NPs to evaluate a set of biomarker responses after 96 h: cholinesterase (ChE) and glutathione S-transferase (GST) activities, total glutathione (TG) and energy budgets levels. VNPs induced malformations (10 mg/L), hyperactivity (10 mg/L), erratic swimming (0.01 mg/L), altered swimming pattern (>0.01 mg/L), delayed hatching (10 mg/L) and altered biochemical responses involved in antioxidant defense (GST and TG at >1 mg/L), neurotransmission (ChE at 10 mg/L) and energy metabolism (lipids at >1 mg/L and carbohydrates at 10 mg/L). BNPs caused malformations (10 mg/L), affected swimming pattern (>0.01 mg/L), induced erratic swimming (10 mg/L) and decreased TG content and GST activity (>1 mg/L). At the same concentrations, VNPs affected a greater number of endpoints than BNPs, demonstrating a greater toxicity to zebrafish embryos. The present study shows that BNPs and VNPs may affect aquatic organisms, albeit at relatively great non-environmentally relevant concentrations, reinforcing the importance of the risk assessment of different NPs.

From the start of the 21st century, nanoecotoxicological research has been growing in fast steps due to the need to evaluate the safety of the increasing use of engineered nanomaterials. Boron (B) and vanadium (V) nanoparticles (NPs) generated by anthropogenic activities are subsequently released in the environment, therefore, organisms can be continuously exposed to these NPs for short or long periods. However, the short and long-term effects of BNPs and VNPs on soil organisms are unknown. This work aimed to recognize and describe their potential toxicological effects on the model species Enchytraeus crypticus, assessing survival and reproduction, through a longer-term exposure (56 days (d)—OECD test extension of 28 d), and avoidance behavior, through a short-term exposure (48 hours (h)). After 28 d, BNPs did not induce a significant effect on E. crypticus survival, whereas they decreased the organisms’ reproduction at 500 mg/kg. From 10 to 500 mg/kg, VNPs decreased the E. crypticus survival and/or reproduction. After 56 d, 100 to 500 mg/kg BNPs and 50 to 500 mg/kg VNPs, decreased the reproduction output of E. crypticus. The estimated Effect Concentrations (ECx) based on reproduction, for BNPs, were lower at 56 d compared with 28 d, for VNPs, an opposite pattern was found: ECx 28 d &lt, ECx 56 d. BNPs did not induce an avoidance behavior, but organisms avoided the soil contaminated with 10 mg VNPs/kg. The tested NPs showed different E. crypticus apical effects at 28 d from the ones detected at 56 d, dependent on the type of NPs (B vs. V). In general, VNPs showed to be more toxic than BNPs. However, the effects of VNPs were alleviated during the time of exposure, contrarily to BNPs (which became more toxic with extended duration). The present study adds important information about NPs toxicity with ecological significance (at the population level). Including long-term effects, the obtained results contributes to the improvement of NPs risk assessment.

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.

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.

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

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 the high usage of pesticides in current agricultural practices, its effects to humans and to the environment (non-target species) are a continuous concern. Soil dwelling organisms are among the first in line of exposure to pesticides, however their risks are often based on the pure active ingredient (a.i.) and not on the commercial formulated products (FPs) actually applied in the fields. In the present study, we investigated the effects of two fungicide FPs versus its a.i. (s): Amistar® XTRA and the respective a.i. (s) azoxystrobin and cyproconazole, and Prosaro® 250 EC and the respective a.i. (s) prothioconazole and tebuconazole, to the non-target soil oligochaete Enchytraeus crypticus. The standard Enchytraeid Reproduction Test was used to assess effects on survival and reproduction. Results showed that Amistar was more toxic than Prosaro, particularly for reproduction (EC50 = 161 mg Amistar/kg soil, EC50 = 350 mg Prosaro/kg soil). For both FPs, reproductive effects were mainly related to one of its a.i. (s) (azoxystrobin [EC50 = 37 mg azosxystrobin/kg soil] for Amistar, and tebuconazole [EC50 = 41 mg tebuconazole/kg soil] for Prosaro), while lethal effects were not predicted by the toxicity of its a.i. (s) (particularly in the case of Prosaro, which was more toxic than its a.i. (s)). These findings highlight the need to further explore the toxicity data of the FPs compared to the a.i. (s), aiming to predict a more realistic environmental hazard of pesticides.

International audience; The convergence of nanotechnology and biotechnology has led to substantial advancements in nano-biomaterials (NBMs) used in medical devices (MD) and advanced therapy medicinal products (ATMP). However, there are concerns that applications of NBMs for medical diagnostics, therapeutics and regenerative medicine could also pose health and/or environmental risks since the current understanding of their safety is incomplete. A scientific strategy is therefore needed to assess all risks emerging along the life cycles of these products. To address this need, an overarching risk management framework (RMF) for NBMs used in MD and ATMP is presented in this paper, as a result of a collaborative effort of a team of experts within the EU Project BIORIMA and with relevant inputs from external stakeholders. The framework, in line with current regulatory requirements, is designed according to state-of-the-art approaches to risk assessment and management of both nanomaterials and biomaterials. The collection/generation of data for NBMs safety assessment is based on innovative integrated approaches to testing and assessment (IATA). The framework can support stakeholders (e.g., manufacturers, regulators, consultants) in systematically assessing not only patient safety but also occupational (including healthcare workers) and environmental risks along the life cycle of MD and ATMP. The outputs of the framework enable the user to identify suitable safe(r)-by-design alternatives and/or risk management measures and to compare the risks of NBMs to their (clinical) benefits, based on efficacy, quality and cost criteria, in order to inform robust risk management decision-making.

Nanoplastics (NPls) are ubiquitous in terrestrial environments, with numerous consequences for biodiversity and ecosystems. Research is urgently required to clarify the NPls environmental behaviour, fate and ecotoxicological effects to soil ecosystems. The aim of this research was to assess and comprehend the effects of polystyrene NPls to the terrestrial species Enchytraeus crypticus using survival, reproduction and avoidance behaviour as endpoints. A range of concentrations, 0.015 to 1500 mg NPls/kg LUFA 2.2 (Landwirtschaftliche Untersuchungs- und Forschungsanstalt Speyer, Germany) soil, was tested. Due to the effect of tween 20 and sodium azide (NaN3) on the NPls dispersion, the effects of these compounds were also assessed separately. After 21 d, 1200 and 1500 mg/kg NPls dispersion had significant effects on the organism survival and/or reproduction. However, these effects may be mainly associated with tween 20 and NaN3 present in the NPls dispersion and not with NPls themselves. After 48 h, there was a tendency of the organisms to avoid the NPls spiked soils, being this response significant at 0.015 mg/kg although a reduced avoidance behaviour was observed as NPls concentration increased. The present study provides screening data on the effects of NPls, alone and considering the presence of other compounds like the solvents, which is essential for regulators and strategic management of plastic pollution.

It has become clear how important it is to assess longer term effects of (nano) materials in the environment given the current evidence showing how epigenetics drives response mechanisms. Here we studied global DNA methylation in standard soil invertebrate Enchytraeus crypticus over 224 days when exposed to nanostructured tungsten carbide cobalt (WCCo nanomaterials (NMs)) and to cobalt (CoCl2) in a multigenerational experiment. In order to assess the transgenerational effect, we used a multigenerational (MG) test design consisting of four generations in spiked soil followed by two generations in clean soil. Results showed that MG exposure to WCCo NMs caused global DNA methylation to increase, which continued in unexposed generations and was associated with an increase in reproduction (phenotypic effect). In general, WCCo NMs caused more (and more consistent) methylation than CoCl2.

Chemical stressors can induce epigenomic changes, i.e., changes that are transferred to the next generation, even when the stressor is removed. Literature on chemical induced epigenetic effects in environmental species is scarce. We here provide the first results on epigenetic effects caused by nanomaterials with an environmental OECD standard soil model species Enchytraeus crypticus species. We assessed the epigenetic potential in terms of global DNA methylation, gene-specific methylation via bisulfite sequencing and MS-HRM (Methylation Sensitive - High Resolution Melting), and gene expression qPCR for genes involved in DNA methylation, histone modifications, non-coding RNA and stress response mechanisms).We have exposed E. crypticus in a multigenerational (MG) test design to Cu (copper oxide nanomaterials (CuO NMs) and copper salt (CuCl2)). To link possible epigenetic effects to population changes, we used exposure concentrations (ECx) that caused a 10% and 50% reduction in the reproductive output (10% and 50% are the standards for regulatory Risk Assessment), the organisms were exposed for five consecutive generations (F1–F5) plus two generations after transferring to clean media (F5–F7), 7 generations in a total of 224 days. Results showed that MG exposure to Cu increased global DNA methylation and corresponded with phenotypic effects (reproduction). Gene expression analyses showed changes in the epigenetic, stress and detoxification gene targets, depending on the generation and Cu form, also occurring in post-exposure generations, hence indicative of transgenerational effects. There were in general clear differences between organisms exposed to different Cu-forms, hence indicate nanoparticulate-specific effects. Keywords: Methylation, Transgenerational, Methylation sensitive high resolution melting

Silica nanoparticles (SiO2NPs) are one of the most used in commercial products and biomedical tools, however, their environmental effects have not been fully described. Although negative effects of SiO2NPs on the behavior of freshwater invertebrates have been reported, the knowledge is limited, especially the effect of nanopowders in terrestrial organisms. Accordingly, the aim of the present study is to understand the effects of SiO2NPs on the avoidance behavior of five soil species, whose niche may differ thus contributing to differential harmful SiO2NPs effects. Hence, avoidance assays testing SiO2NPs concentrations of 0, 10, 100, 250, 500 and 1000 mg/kg were performed with Enchytraeus crypticus, Folsomia candida, Tenebrio molitor, Porcellionides pruinosus and Eisenia fetida. SiO2NPs induced different behavioral effects, depending on the invertebrate ecology/habitat, exposure route and physiology. T. molitor, P. pruinosus and F. candida did not avoid contaminated soil, however, E. crypticus and E. fetida significantly avoided SiO2NPs spiked soil. Since these terrestrial worms (oligochaetes) live mostly burrowed in the soil, this can provide greater opportunity for SiO2NPs&rsquo, uptake. On the other hand, the other tested organisms mainly living on the upper part of the soil did not avoid the SiO2NPs spiked soil. The avoidance data obtained here also highlight the need for further studies to understand whether (or not) the detected behavioral responses are linked to either neurotransmission processes or sensorial aspects of the biological models.

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.

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.

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.

Soil invertebrates have been widely used in ecotoxicology studies for decades, although their use as in vitro models, albeit promising, has not been pursued as much. The immune cells of earthworms (coelomocytes) and the coelomic fluid can be used, and are a highly relevant in vitro system. Although it has been tested before, to cover the testing of nanomaterials (NMs), several challenges should be considered. NMs characteristics (dispersibility, agglomeration, etc.) can interfere with the common in vitro methodologies, not only during exposure, but also during the measurements. Here, we have assessed the effect of a CuO NMs case study using surface-modified particles, functionalized for safe-by-design strategies with ascorbate, citrate, polyethylenimine, and polyvinylpyrrolidinone, plus the pristine CuO NMs and copper chloride (CuCl2) for comparison. Eisenia fetida&rsquo, s coelomocytes were exposed for 24 h via the coelomic fluid. Changes in cell viability were evaluated using flow cytometry. All materials affected the cells in a dose-related manner, where CuCl2 was the most toxic followed by the citrate-coated CuO NM. There was a strong correlation between NM characteristics, e.g., the hydrodynamic size, and the EC50 (50% Effect Concentrations) values. This screening further confirms the potential for the usage of the standard earthworm model as an in vitro standard. Further detailed in vitro studies are needed using other NMs aiming toward their implementation and standardization. Additional cell endpoints can also be assessed, making it a high content tool for mechanistic understanding.

Graphene-based nanomaterials (GBNs) possess unique physicochemical properties, allowing a wide range of applications in physical, chemical, and biomedical fields. Although GBNs are broadly used, information about their adverse effects on ecosystem health, especially in the terrestrial environment, is limited. Therefore, this study aims to assess the toxicity of two commonly used derivatives of GBNs, graphene oxide (GO) and reduced graphene oxide (rGO), in the soil invertebrate Enchytraeus crypticus using a reduced full life cycle test. At higher exposure concentrations, GO induced high mortality and severe impairment in the reproduction rate, while rGO showed little adverse effect up to 1000 mg/kg. Collectively, our body of results suggests that the degree of oxidation of GO correlates with their toxic effects on E. crypticus, which argues against generalization on GBNs ecotoxicity. Identifying the key factors affecting the toxicity of GBNs, including ecotoxicity, is urgent for the design of safe GBNs for commercial purposes.

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.

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.

Studies have been showing how changes in ultraviolet (UV) affect the terrestrial system, mostly focusing on higher plants and indirect effects, e.g. UV changed food quality/decomposition. Much less attention has been given to direct effect on terrestrial species, although the negative effects have been recognized for some earthworms. Further, the actual mechanisms of UV toxicity to soil invertebrates are even less understood. We here studied the effect of UV on the soil oligochaete Enchytraeus crypticus, and attempted to identify the possible mechanisms of toxicity using high-throughput gene expression. Applying a UV dose equivalent to UV during the winter months in northern Europe we observed an 80% decrease in reproduction. For these organisms, approximately 5% of the genes were differentially expressed. Among the observations was an activation of the DNA repair mechanisms, nucleotide excision repair, which correlated with survival of the organisms. An observed repressing of apoptosis seems to have deleterious effects (e.g. because it may lead to the accumulation of aberrant cells) leading to a decline in reproduction. The mechanisms activated by UV were similar to those mechanisms activated in humans, showing conservation across species.

Poly-ε-caprolactone (PCL) based medical devices are increasingly produced and thus, their presence in the environment is likely to increase. The present study analysed the biodegradation of PCL electro-spun scaffolds (alone) and PCL electro-spun scaffolds coated with human recombinant (hR) collagen and Bovine Achilles tendon (BAT) collagen in sewage sludge and in soil. Additionally, an eco-toxicological test with the model organism Enchytraeus crypticus was performed to assess environmental hazard of the produced materials in soils. The electro-spun scaffolds were exposed to activated sludge and three different soils for various time periods (0-7-14-21-28-56-180 days); subsequently the degradation was determined by weight loss and microscopical analysis. Although no toxicity occurred in terms of Enchytraeus crypticus reproduction, our data indicate that biodegradation was dependent on the coating of the material and exposure condition. Further, only partial PCL decomposition was possible in sewage treatment plants. Collectively, these data indicate that electro-spun PCL scaffolds are transferred to amended soils.

Enchytraeus albidus is a terrestrial earthworm widespread along the coasts of northern Europe and the Arctic. This species tolerates freezing of body fluids and survives winters in a frozen state. Their acclimatory physiological mechanisms behind freeze tolerance involve increased fluidity of membrane lipids during cold exposure and accumulation of cryoprotectants (glucose) during the freezing process. Gene regulatory processes of these physiological responses have not been studied, partly because no gene expression tools were developed. The main aim of this study was to understand whether the freeze tolerance mechanisms have a transcriptomic basis in E. albidus. For that purpose, first the transcriptome of E. albidus was assembled with RNAseq data. Second, two strains from contrasting thermal environments (Germany and Greenland) were compared by mapping barcoded RNAseq data onto the assembled transcriptome. Both of these strains are freeze tolerant, but Greenland is extremely freeze tolerant. Results showed more plastic responses in the Greenland strain as well as higher constitutive expression of particular stress response genes. These altered transcriptional networks are associated with an adapted homeostasis coping with prolonged freezing conditions in Greenland animals. Previously identified physiological alterations in freeze‐tolerant strains of E. albidus are underpinned at the transcriptome level. These processes involve anion transport in the hemolymph, fatty acid metabolism, metabolism, and transport of cryoprotective sugars as well as protection against oxidative stress. Pathway analysis supported most of these processes, and identified additional differentially expressed pathways such as peroxisome and Toll‐like receptor signaling. We propose that the freeze‐tolerant phenotype is the consequence of genetic adaptation to cold stress and may have driven evolutionary divergence of the two strains.

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.