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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]

Polycyclic aromatic hydrocarbons (PAHs) continue to cause environmental challenges as a result of their release into the environment by a great variety of anthropogenic activities and their accumulation in soils. Studies were conducted on the toxicological effect of the model PAH phenanthrene using the soil invertebrate model Enchytraeus crypticus at the individual, tissue, and molecular levels. Animals were exposed for 2 d and 21 d to phenanthrene concentrations corresponding to the (previously estimated) 3-wk effective concentration, 10% (EC10) and EC50 for effects on reproduction. Gene expression profiling did not reveal a typical phenanthrene-induced biotransformation signature, as it usually does in arthropods and vertebrates. Instead, after 2 d of exposure, only general metabolic processes were affected, such as translation and adenosine triphosphate synthesis-coupled electron transport. Histological sections of tissues of 2-d exposed animals showed no deviations from control. In contrast, after prolonged exposure of up to 21 d, histopathological effects were found: chloragogenous cells were highly vacuolated and hypertrophic. This was corroborated by differential expression of genes related to immune response and oxidative stress at the transcriptomic level. The data exemplify the complexity and species-specific features of PAH toxicity among soil invertebrate communities, which restricts read-across and extrapolation in the context of soil ecological risk assessment. Environ Toxicol Chem 2016;35:2713-2720. © 2016 SETAC [ABSTRACT FROM AUTHOR]

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

The aim of this study was to evaluate the toxicokinetics-toxicodynamics (TKTD) of Cu and Cd in the soil model organism Enchytraeus crypticus , and assess the development of internal effect concentrations over time. Animals were exposed in LUFA 2.2 soil spiked with increasing concentrations of Cu and Cd. Survival, reproduction and internal metal concentrations in the animals were evaluated at different points in time over a period of 21 days. Internal concentrations increased with time, for Cu reaching a steady state after c. 10 days, except for the highest test concentration, and for Cd continuing to increase after 21 days. Applying a one-compartment model to all data together, estimated uptake and elimination rate constants for Cu and Cd were 0.08 and 0.45 kg soil/kg organism/day and 0.4 and 0.04 per day, respectively. Median lethal concentrations, based on total soil concentrations, decreased with time for Cu and did not reach a steady state level, but they did not change with time for Cd. The LC50 inter (based on internal concentrations) was 75 mg Cu/kg body DW and > 800 mg Cd/kg body weight. Animals were able to regulate Cu internal concentrations, keeping them low, while for Cd internal concentrations continued to increase showing lack of regulation and also the importance of exposure time. This study highlights the advantages of using a TKTD approach to understand the relation between organism survival and internal Cu or Cd concentrations over time. [Display omitted] • Cu and Cd present distinct toxicokinetic-toxicodynamic patterns in E. crypticus. • For the essential element Cu, internal concentration was kept low and regulated. • High concentrations of Cd were internalized before phenotypical effects occurred. • Internal concentration is highly relevant for predicting environmental hazards. [ABSTRACT FROM AUTHOR]

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. [Display omitted] • Proposal of a higher test design for nanopharmaceuticals exemplified for nano gold. • Simulation of wastewater treatment plants followed by soil and aquatic tests is feasible. • Low risk of medical applied Au expected for soil invertebrates, daphnids. • Effects on microbial community structure unlike microbial function and fish cells are not excluded. • Aging of Au-NPs during the process in the WWTP induces toxicity in the fish cells. [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]

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'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. [ABSTRACT FROM AUTHOR]

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]

Information on effects of silver nanoparticles on soil invertebrates, especially using long-term exposures, is scarce. In this study we investigated the effects of the reference Ag (NM300K) (compared to AgNO 3 ) using the full life cycle test (FLCt) of the soil invertebrate Enchytraeus crypticus . Results showed that effects were higher compared to the standard reproduction test, which is shorter and does not cover the FLC. Both Ag forms caused a reduction on hatching success, juvenile and adult survival and reproduction with similar ECx. Differences between AgNO 3 and Ag NM300K could be discriminated using the FLCt: AgNO 3 decreased hatching success was shown to be a delay in the process, whereas Ag NM300K caused irreversible effects during the same time frame. These effects may have occurred during the embryo development, hatching (inhibition) or survival of hatched juveniles. Ag NM300K caused non-monotonic concentration-response effect as observed by the high effect of the lowest concentration (20 mg kg-1). It is known that dispersion is higher at lower concentrations – this could explain the increased effect at low concentration. Non monotonic responses are well described in the literature, where effects of high cannot predict for low concentrations, hence special attention should be given for NMs low concentration effects. [ABSTRACT FROM AUTHOR]

• Organisms' tissues sampled along full life cycle and multigenerational exposure to Cu. • Immunohistochemistry confirmed genetics and epigenetics effects. • Differentiated molecular initiating events between Cu NM and CuCl 2. • Time series exposure allowed to collect time of events for building AOPs. 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 CuCl 2) 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 CuCl 2 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, CuCl 2 also caused oxidative stress. Different mechanisms of DNA methylation (epigenetics) were activated by CuO NMs and CuCl 2 at the MIE. [ABSTRACT FROM AUTHOR]