Toxicokinetics and toxicodynamics of Ag nanomaterials (NM300K) in the soil environment-impact on Enchytraeus crypticus (Oligochaeta).

Silver (Ag) is one of the most used elements in the nanomaterials (NMs) form, which upon release to the environment can be harmful to organisms. We compared the toxicokinetics (TK) and toxicodynamics (TD) of Ag from AgNO3 (0, 15, 45, 135, 405 mg Ag/kg soil) and AgNM300K (0, 75, 150, 300, 600, 1200 mg Ag/kg soil) in the model organism Enchytraeus crypticus. Organisms were exposed in LUFA 2.2 soil, and besides body Ag concentrations, survival and reproduction were determined, in a time series (for 21 days). In the soil, the available (CaCl2 extractable) Ag fraction from Ag NM300K increased from 0 to 21 days but did not consistently change for AgNO3. Internal concentrations reached equilibrium in most exposures to both Ag forms. The organisms were able to internalize and eliminate Ag, but less when exposed to Ag NM300K. The overall uptake rate constants for Ag from AgNO3 and Ag NM300K exposures were 0.05 and 0.06 kg soil/kg organism/day, respectively, the elimination rate constants 0.2 and 0.1 day-1, respectively. For AgNO3 the median lethal concentrations decreased steadily with time, while for Ag NM300K they remained constant during the first 10 days of exposure followed by a 2-fold decline in the last 7 days. The 21-d LC50s for both Ag forms were similar but the LC50inter (based on internal concentrations) were 63 and 121 mg Ag/kg body DW (Dry Weight) for AgNO3 and Ag NM300K, respectively, showing higher toxicity of AgNO3. These results show the importance of assessing time to toxicity, a relevant factor in toxicity assessment, especially for NMs.

Toxicokinetics and toxicodynamics of copper and cadmium in the soil invertebrate Enchytraeus crypticus (Oligochaeta).

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 LC50inter (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.

Nanopharmaceuticals (Au-NPs) after use: Experiences with a complex higher tier test design simulating environmental fate and effect.

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.

The Proteome of Enchytraeus crypticus-Exposure to CuO Nanomaterial and CuCl2 -in Pursue of a Mechanistic Interpretation.

The transcriptome of the ecotoxicological model Enchytraeus crypticus (known as potworm) is well studied, but the downstream changes at the protein level remained a gap. Changes in the protein regulation following exposure to CuO nanomaterial (NM) and Cu salt (CuCl2 ) are investigated. HPLC-MS/MS using tandem mass tags is used. CuO NM elicits higher number of differentially expressed proteins (DEPs) compared to CuCl2 with little to no overlap of proteins. CuO NM causes more stress response mechanisms, with good agreement between DEPs, genes, and metabolites. CuCl2 causes higher impact in shorter time periods, but organisms have conserved mechanisms (constitutive genes) that allow Cu handling and detoxification. CuO NM causes higher impact after a longer exposure period, inducing regulation of facultative genes with a whole differentiated paradigm and cascade. This could be due to different issues: 1) the cell uptake route is different for Cu NM and Cu ions, 2) internalized Cu NM can result in a "Trojan-horse" effect, 3) the cascade of events occurs in a different time order, and 4) the organism uptake is different between life stages, i.e., cocoons thickened surface protects the entry of NM and juveniles have facilitated entry via tegument. The data have been deposited to the ProteomeXchange with identifier PXD010647.

Interactions of Soil Species Exposed to CuO NMs are Different From Cu Salt: A Multispecies Test.

Although environmental effects are mostly assessed via standard individual species the ecological relevance of multispecies testing is well-recognized and highly recommended. Hence, the effect of copper oxide nanomaterials (CuONM) and CuCl2 were assessed using the validated soil multispecies system (SMS). Besides the individual species (IS) "standard" tests for all, a predation study was done. Toxicity was higher in the SMS than in the IS, and longer exposure showed increased toxicity. The predator ( Hypoaspis aculeifer) preyed most on smaller species, but the net biomass consumed was similar across species. Internal Cu in Folsomia candida reached ca. 140 µg Cu/g dry weight, fluctuating over time, especialy for CuCl2. Copper was mostly bound to soil components for both Cu forms (<0.2% of the total Cu in soil solution, < 0.007% on the ionic form, but the soil solution content increased with the total added concentration). Hazard Concentrations (HC5) showed higher toxicity and more similarity between Cu forms at longer-term exposure. Risk of NMs is relatively limited at present (IS based instead of SMS) with short exposure times (i.e., longer is required) and too few IS tested. The impact of species interactions is highlighted and is of key importance to include in ecosystem hazard prediction.

Fate and Effect of Nano Tungsten Carbide Cobalt (WCCo) in the Soil Environment: Observing a Nanoparticle Specific Toxicity in Enchytraeus crypticus.

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

Environmental Impacts by Fragments Released from Nanoenabled Products: A Multiassay, Multimaterial Exploration by the SUN Approach.

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

Implementing the DF4 in a robust model, allowing for enhanced comparison, prioritisation and grouping of Nanomaterials.

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

Enchytraeus crypticus fitness: effect of density on a two-generation study.

Organisms' density can influence physiological processes related with fitness. In the present study we assessed the influence of organisms' density on the life-history parameters in two consecutive generations in Enchytraeus crypticus (Oligochaeta), a standard model in soil ecotoxicology. The densities tested were 1 (N1) and 20 (N20) organisms per replicate and 10 vs. 20 g of soil (for the 2nd generation test only). Results showed that reproductive output was affected by density, with organisms in N1 producing three times more juveniles per adult than when at N20. Organisms' length was affected by both density and space, i.e., organisms were smaller when less space available. Further, the density of parental generation (F0) had no influence on the endpoints reproduction and length assessed in F1, hence there was no transference of effects. These findings have potential implications in the standard Enchytraeid Reproduction Test, i.e. early mortality of the adults during toxicant exposure can affect the number and size of the offspring and the final results will also reflect the density related changes in reproduction.

Effect of Cu and Ni on cellular energy allocation in Enchytraeus albidus.

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

Combined effect of temperature and copper pollution on soil bacterial community: climate change and regional variation aspects.

The aim of this study was to assess the combined effects of temperature and copper (Cu) contamination in the structure of soil bacterial community. For this, contaminated or spiked and control soils from two different geographic origins (PT-Portugal and DK-Denmark) were used. The DK soil was from a historically contaminated study field, representing a long-term exposure to Cu while the PT soil was from a clean site and freshly spiked with Cu. Soil bacterial communities were exposed in mesocosms during 84 days to 3 different temperatures based on values typically found in each geographic region and temperature conditions that simulated a warming scenario. Obtained results indicate that Cu stress alters the structure of bacterial community and that this effect is, to some extent, temperature-dependent. Effects on bacterial diversity for both soils were also observed. Differences in the DK and PT communities' response were apparent, with the community from the historically contaminated soil being more resilient to temperature fluctuations. This study presents evidence to support the hypothesis that temperature alters the effect of metals on soils. Further, our results suggest that the definition of soils quality criteria must be based on studies performed under temperatures selected for the specific geographic region. Studies taking into account temperature changes are needed to model and predict risks, this is important to e.g. future adjustments of the maximum permissible levels for soil metal contamination.

Enchytraeid Reproduction Test(PLUS): hatching, growth and full life cycle test--an optional multi-endpoint test with Enchytraeus crypticus.

Soil ecotoxicity standard tests for invertebrates are usually limited to the assessment of endpoints like survival and reproduction. Adverse effects may occur at other developmental stages, e.g. embryo development, hatching or growth. The species Enchytraeus crypticus is a model organism in the standard soil ecotoxicology test, where survival and reproduction are assessed. In the present study we optimized the test method to include additional endpoints. The proposed test started with synchronized age organisms', and included additionally hatching success, growth, maturation status and full life cycle. This allowed for the calculation of cocoon production and population growth rate. Results indicated that Cd is embryotoxic, main effect occurs on the embryo developmental stage and maturity. Further, the full life cycle test can discriminate between pre- and post-embryo formation. The increased sensitivity and full life cycle detail level makes it potentially useful for novel materials e.g. nanomaterials where the mode of action and hence effect target is unknown.

Transcriptome assembly and microarray construction for Enchytraeus crypticus, a model oligochaete to assess stress response mechanisms derived from soil conditions.

BACKGROUND: The soil worm Enchytraeus crypticus (Oligochaeta) is an ecotoxicology model species that, until now, was without genome or transcriptome sequence information. The present research aims at studying the transcriptome of Enchytraeus crypticus, sampled from multiple test conditions, and the construction of a high-density microarray for functional genomic studies. RESULTS: Over 1.5 million cDNA sequence reads were obtained representing 645 million nucleotides. After assembly, 27,296 contigs and 87,686 singletons were obtained, from which 44% and 25% are annotated as protein-coding genes, respectively, sharing homology with other animal proteomes. Concerning assembly quality, 84% of the contig sequences contain an open reading frame with a start codon while E. crypticus homologs were identified for 92% of the core eukaryotic genes. Moreover, 65% and 77% of the singletons and contigs without known homologs, respectively, were shown to be transcribed in an independent microarray experiment. An Agilent 180 K microarray platform was designed and validated by hybridizing cDNA from 4 day zinc- exposed E. crypticus to the concentration corresponding to 50% reduction in reproduction after three weeks (EC50). Overall, 70% of all probes signaled expression above background levels (mean signal + 1x standard deviation). More specifically, the probes derived from contigs showed a wider range of average intensities when compared to probes derived from singletons. In total, 522 significantly differentially regulated transcripts were identified upon zinc exposure. Several significantly regulated genes exerted predicted functions (e.g. zinc efflux, zinc transport) associated with zinc stress. Unexpectedly, the microarray data suggest that zinc exposure alters retro transposon activity in the E. crypticus genome. CONCLUSION: An initial investigation of the E. crypticus transcriptome including an associated microarray platform for future studies proves to be a valuable resource to investigate functional genomics mechanisms of toxicity in soil environments and to annotate a potentially large number of lineage specific genes that are responsive to environmental stress conditions.

Importance of freeze-thaw events in low temperature ecotoxicology of cold tolerant enchytraeids.

Due to global warming it is predicted that freeze-thaw cycles will increase in Arctic and cold temperate regions. The effects of this variation becomes of particular ecological importance to freeze-tolerant species when it is combined with chemical pollutants. We compared the effect of control temperature (2 °C), daily freeze-thaw cycles (2 to -4 °C) and constant freezing (-2 °C) temperatures on the cold-tolerance of oligochaete worms (Enchytraeus albidus) and tested how survival was influenced by pre-exposure to 4-nonylphenol (4-NP), a common nonionic detergent found in sewage sludge amended soils. Results showed that combined effect of 4-NP and daily freeze-thaw cycles can cause higher mortality to worms as compared with sustained freezing or control temperature. Exposure to 4-NP caused a substantial depletion of glycogen reserves which is catabolized during freezing to produce cryoprotective concentrations of free glucose. Further, exposure to freeze-thaw cycles resulted in higher concentrations of 4-NP in worm tissues as compared to constant freezing or control temperature (2 °C). Thus, worms exposed to combined effect of freeze-thaw cycles and 4-NP suffer higher consequences, with the toxic effect of the chemical potentiating the deleterious effects of freezing and thawing.

Oxidative stress biomarkers and metallothionein in Folsomia candida--responses to Cu and Cd.

Folsomia candida (Collembola) is a standard soil ecotoxicological species; effect assessment includes survival and reproduction as endpoints. In the present study, and for the first time, a range of oxidative stress biomarkers measurement was optimized and validated. The antioxidant capacity was measured by the activities of catalase (CAT), glutathione reductase (GR), glutathione-s-transferase (GST) and content of total glutathione (TG). The oxidative damage in the lipid membranes was estimated by lipid peroxidation (LPO) and metallothionein (MT) levels. The exposure included the essential and non-essential metals Cu and Cd, in LUFA 2.2 natural standard soil, using a series of sampling times along a 10 days period (0, 2, 4, 6 and 10 days). Exposure concentrations were selected based on their reproduction EC50 values, 60 and 1000 mg/kg soil DW, for Cd and Cu respectively. The protocols were optimized and results show that oxidative stress biomarkers can be successfully used in F. candida, this being highly relevant as complementary information to the mechanistic level. The selected sampling times gave a good indication of the markers dynamic and can be reduced/adapted in future testing. Results showed that both metals caused an increase in the MT levels after 6 days but Cd acted as a stronger oxidant agent compared to Cu, i.e. causing higher damage. In sum, Cd mobilized/activated more antioxidant enzymes, but the increased activities were not enough to prevent LPO. This study confirms that the oxidative stress caused by Cd is higher despite the use of same reproduction EC50 indicating that toxicity seems more reversible for Cu than for Cd. Among others, GST and MT would be a good selection of biomarkers for Cd effect.

Antioxidant and neurotoxicity markers in the model organism Enchytraeus albidus (Oligochaeta): mechanisms of response to atrazine, dimethoate and carbendazim.

The present study aimed to investigate the effects of dimethoate, atrazine and carbendazim on the antioxidant defences and neuronal function of the soil organism Enchytraeus albidus. Effects were studied at concentrations known to affect their reproduction (EC20, EC50 and EC90) and along time (2, 4, 8, 14 and 21 days). In general, responses were more pronounced at periods of exposure longer than 8 days and at the highest concentrations. Multivariate statistics (RDA-PRC) clearly displayed that exposure duration had an effect itself, biomarkers' responses showed interaction for all pesticides and catalase scored consistently high, indicating its relevancy in the group of measured markers. Univariate analysis indicated oxidative stress for all pesticides and atrazine induced oxidative damage in lipids. Atrazine seems to be effectively metabolized by GST of the biotransformation system, as its activity significantly increased after exposure to this pesticide. Dimethoate caused ChE inhibition, indicating an impairment of the neuronal function. Carbendazim impaired the antioxidant system, but no oxidative damage was observed, along with any effects on the ChE activity. The integrated biomarker response analysis was performed but we suggest modifications due to limiting artefacts.

Iron Oxide (Magnetite)-Based Nanobiomaterial with Medical Applications-Environmental Hazard Assessment Using Terrestrial Model Species.

Nanobiomaterials (NBMs) have tremendous potential applications including in cancer diagnosis and treatment. However, the health and environmental effects of NBMs must be thoroughly assessed to ensure safety. Fe3O4 (magnetite) nanoparticles coated with polyethylene glycol (PEG) and poly (lactic-co-glycolic acid) (PLGA) were one of the focus NBMs within the EU project BIORIMA. Fe3O4 PEG-PLGA has been proposed to be used as a contrast agent in magnetic resonance imaging for the identification of solid tumors and has revealed low cytotoxicity in several cell lines. However, the effects of Fe3O4 PEG-PLGA have not been assessed in terrestrial environments, the eventual final sink of most materials. In the present study, the effects of Fe3O4 PEG-PLGA and its precursor, (un-coated) Fe3O4 NMs, were assessed in soil model invertebrates Enchytraeus crypticus (Oligochaeta) and Folsomia candida (Collembola). The endpoints were survival, reproduction, and size, based on the standard OECD test (28 days) and its extension (56 days). The results showed no toxicity for any of the endpoints evaluated, indicating that the NBM Fe3O4 PEG-PLGA poses no unacceptable risk to the terrestrial environment.

Multigenerational exposure of Ag materials (nano and salt) in soil - environmental hazards in Enchytraeus crypticus (Oligochaeta).

Because of its properties, silver is among the most used metals both as salt and as nanomaterials (NMs), hence reaching the environment. Multigenerational (MG) exposure testing is scarce, and especially so for NMs and soil invertebrates. In this study the MG effects of Ag NMs (Ag NM300K) and Ag salt (AgNO3) were assessed, using Enchytraeus crypticus in LUFA 2.2 soil. Survival, reproduction and internal Ag concentration in the animals were measured throughout 7 generations (5 generations (F0-F4) in spiked soil plus 2 (F5-F6) in clean soil) exposed to sublethal concentrations corresponding to the reproduction EC10 and EC50 obtained in standard toxicity tests (45 and 60 mg Ag per kg soil DW for AgNO3; 20 and 60 mg Ag per kg soil DW for Ag NM300K). MG exposure caused a dose-related decrease in reproduction for both Ag forms. Ag uptake peaked in the F1 (64 days) for AgNO3 and F2 (96 days) for Ag NM300K, after which it decreased. In agreement with toxicokinetic studies, a maximum body Ag concentration was reached (20 mg Ag per kg body DW (AgNO3) and 70 mg Ag per kg body DW (Ag NM300K)) and after which detoxification mechanisms seem to be activated with elimination of Ag accompanied by a decrease in reproduction. Transfer to clean soil allowed Ag to be (fully) eliminated from the animals. This MG study confirmed the effects determined in standard reproduction toxicity tests but further allowed to monitor the dynamics between exposure and effects of the Ag materials, and how the animals seem to cope with Ag for 7 generations by compensating between detoxification and reproductive output.

Safe and sustainable by design Ag nanomaterials: A case study to evaluate the bio-reactivity in the environment using a soil model invertebrate.

Safe-and-sustainable-by-design (SSbD) nanomaterials (NMs) or NM-containing products are a priority. Silver (Ag) NMs have a vast array of applications, including biomedical and other products, even as nanopesticides. Thus, their release to the environment is expected to increase. The aim of the present study was to assess the ecotoxicity of the SSbD Ag NM to the soil model species Enchytraeus crypticus (Oligochaeta). The Ag NM tested consists in a SSbD Ag with biomedical applications, a hydroxyethyl cellulose (HEC) coated Ag NMs (AgHEC) and its toxicity was compared to the naked Ag NMs (Ag-Sigma), an Ag-based biomedical product (PLLA-Ag: Poly l-Lactide microfibers doped with Ag), and AgNO3. Effects were assessed both in soil and aqueous media, following the standard OECD guideline in soil (28 days) and the OECD extension (56 days), and short-term pulse (5 days) in aqueous media: reconstituted water (ISO water) and soil:water (S:W) extracts, followed by a 21-days recovery period in soil. Ag materials were thoroughly characterized as synthesized and during the test in media and animals. Results in S:W showed AgHEC was more toxic than Ag-Sigma (ca. 150 times) and PLLA-Ag (ca. 2.5 times), associated with a higher Ag uptake. Higher toxicity was related to a smaller hydrodynamic size and higher suspension stability, which in turn resulted in a higher bioavailability of Ag NMs and released ions, particularly in S:W. Toxicity was correlated with the main physicochemical features, providing useful prediction of AgNMs bioactivity. The ability to test E. crypticus in a range of media with different and/or increasing complexity (water, S:W extracts, soil) provided an excellent source to interpret results and is here recommended.

Toxicity mechanisms of plastic nanoparticles in three terrestrial species: antioxidant system imbalance and neurotoxicity.

The detrimental impacts of plastic nanoparticles (PNPs) are a worldwide concern, although knowledge is still limited, in particular for soil mesofauna. This study investigates the biochemical impact of 44 nm polystyrene PNPs on three soil models-Enchytraeus crypticus (Oligochaeta), Folsomia candida (Collembola) and Porcellionides pruinosus (Isopoda). Exposure durations of 3, 7 and 14 days (d) were implemented at two concentrations (1.5 and 300 mg kg-1 PNPs). Results revealed PNPs impact on the activities of the glutathione-dependent antioxidative enzyme, glutathione S-transferase (GST) and on the neurotransmitter acetylcholinesterase (AChE) for all three species. Catalase (CAT) played a minor role, primarily evident in F. candida at 300 mg kg-1 PNPs (CAT and GST response after 14 d), with no lipid peroxidation (LPO) increase. Even with the antioxidant defence, P. pruinosus was the most sensitive species for lipid oxidative damage (LPO levels increased after 7 d exposure to 300 mg kg-1 PNPs). Significant AChE inhibitions were measured already after 3 d to both PNP concentrations in F. candida and E. crypticus, respectively. Significant AChE inhibitions were also found in P. pruinosus but later (7 d). Overall, the toxicity mechanisms of PNPs involved antioxidant imbalance, being (mostly) the glutathione-associated metabolism part of that defence system. Neurotoxicity, linked to AChE activities, was evident across all species. Sensitivity to PNPs varied: P. pruinosus > F. candida ≅ E. crypticus. This pioneering study on PNPs toxicity in soil invertebrates underscores its environmental relevance, shedding light on altered biochemical responses, that may compromise ecological roles and soil ecosystem fitness.