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.

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.

Advanced materials - Food grade melatonin-loaded Lipid Surfactant Submicron Particles (LSSP)-environmental impacts.

Lipid-based nanoparticles (LNPs) are advanced materials (AdMa), particularly relevant for drug delivery of poorly water-soluble compounds, while also providing protection, stabilization, and controlled release of the drugs/active substances. The toxicological data available often focus on the specific applications of the LNPs-drug tested, with indication of low toxicity. However, the ecotoxicological effects of LNPs are currently unknown. In the present study, we investigated the ecotoxicity of a formulation of Lipid Surfactant Submicron Particles (LSSPs) loaded with melatonin at 1 mg/mL. The LSSPs formulation has been developed to be fully compliant with regulatory for its potential use in the market and all components are food additives. The same formulation without the thickening agent xanthan gum (stabilizer in water phase) designated as LSSP-xg, was also tested. Two soil model invertebrate species were tested in LUFA 2.2 soil: Enchytraeus crypticus (Oligochaeta) and Folsomia candida (Collembola). Effects were assessed based on the OECD standard guideline (28 days) and its extension, the longer-term exposure (56 days). Assessed endpoints were survival, reproduction, and size. LSSPs and LSSP-xg were toxic to E. crypticus and F. candida reducing their survival and reproduction in a dose-dependent way: e.g., 28-day exposure: E. crypticus: LC/EC50 = 30/15 mg LSSPs/kg soil and F. candida LC/EC50 = 55/44 mg LSSPs/kg soil, with similar values for LSSP-xg. Size was also reduced for F. candida but was the least sensitive endpoint. There were no indications that toxicity increased with longer term exposure. The results provide relevant information on ecotoxicity of a AdMa and highlights the need for awareness of the potential risks, even on products and additives usually used in food or cosmetic industry. Further information on single components and on their specific assembly is necessary for the interpretation of results, as it is not fully clear what causes the toxicity in this specific AdMa. This represents a typical challenge for AdMa hazard assessment scenario.

Environmental hazards of WELGRO® Cu+Zn: A nano-enabled fertilizer.

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.

Environmental hazards of nanopesticides to non-target soil species - commercial nanoformulation versus its active substance (Karate Zeon® and lambda-cyhalothrin).

Nanopesticides (Npes) carry the potential of increased efficacy while reducing application rates, hence increasing agricultural productivity in a more sustainable way. However, given its novelty, the environmental risk assessment of these advanced materials is mostly absent. In the present study we investigated the ecotoxicity of a commercial insecticide, with reported nanofeatures, Karate Zeon®, and compared it to its active substance lambda-cyhalothrin. It is hypothesised that the use of the nanopesticide Karate Zeon® poses lower risk to enchytraeids than its active substance. The standard non-target soil invertebrate Enchytraeus crypticus was used, and exposure was done in LUFA 2.2 soil in 4 tests (endpoints: days): avoidance test [avoidance behaviour: 2 days], OECD standard reproduction test [survival, reproduction plus adults' size: 28 days] and its extension [total number organisms: 56 days], and Full Life Cycle (FLC) test [hatching and juveniles' size: 13 days; survival, reproduction and adults' size: 46 days]. Results showed that enchytraeids did not avoid Karate Zeon® nor its active substance lambda-cyhalothrin, which could be due to neurotoxicity. There was no indication of increased toxicity with prolonged exposure (46, 56d) compared to the standard (28d) for neither of the materials, being overall equally toxic in terms of hatching, survival, and reproduction. The FLCt results indicated that the juvenile stage was the most sensitive, resulting in higher toxicity for the adult animals when exposed from the cocoon stage. Although toxicity was similar between Karate Zeon and lambda-cyhalothrin, different patterns of uptake and elimination cannot be excluded. The benefits of using Karate Zeon will rely on reduced application rates.

Nanoemulsion carriers for drug delivery: Assessment of environmental hazards.

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.

TiO2 nanoparticles' library toxicity (UV and non-UV exposure) - High-throughput in vivo transcriptomics reveals mechanisms.

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.

Using Machine Learning to make nanomaterials sustainable.

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.

Environmental Hazards of Nanobiomaterials (Hydroxyapatite-Based NMs)-A Case Study with Folsomia candida-Effects from Long Term Exposure.

Hydroxyapatite (HA) is a calcium phosphate used in many fields, including biomedical applications. In particular, ion-doped HA nanomaterials (nHA) are developed for their increased bioactivity, particularly in the fields of regenerative medicine and nanomedicine. In this study, we assessed the ecotoxicological impact of five nHA materials: a synthesized calcium hydroxyapatite (CaP-HA), superparamagnetic iron-doped hydroxyapatite (Fe-HA), titanium-doped hydroxyapatite (Ti-HA), alginate/titanium-doped hydroxyapatite hybrid composite (Ti-HA-Alg), and a commercial HA. The soil ecotoxicology model species Folsomia candida (Collembola) was used, and besides the standard reproduction test (28 days), an extension to the standard for one more generation was performed (56 days). Assessed endpoints included the standard survival and reproduction, and additionally, growth. Exposure via the standard (28 days) did not cause toxicity, but reproduction increased in commercial HA (significantly at 320 mg HA/kg) whereas via the extension (56 days) it decreased in all tested concentrations. Juveniles' size (56 days) was reduced in all tested nHA materials, except commercial HA. nHA materials seem to trigger a compromise between reproduction and growth. Long-term effects could not be predicted based on the standard shorter exposure; hence, the testing of at least two generations (56 days) is recommended to assess the toxicity of nanomaterials, particularly in F. candida. Further, we found that the inclusion of size as additional endpoint is highly relevant.

Safer and Sustainable-by-Design Hydroxyapatite Nanobiomaterials for Biomedical Applications: Assessment of Environmental Hazards.

Developments in the nanotechnology area occur ensuring compliance with regulatory requirements, not only in terms of safety requirements, but also to meet sustainability goals. Hence, safer and sustainable-by-design (SSbD) materials are also aimed for during developmental process. Similar to with any new materials their safety must be assessed. Nanobiomaterials can offer large advantages in the biomedical field, in areas such as tissue repair and regeneration, cancer therapy, etc. For example, although hydroxyapatite-based nanomaterials (nHA) are among the most studied biomaterials, its ecotoxicological effects are mostly unknown. In the present study we investigated the toxicity of seven nHA-based materials, covering both different biomedical applications, e.g., iron-doped hydroxyapatite designed for theragnostic applications), hybrid collagen/hydroxyapatite composites, designed for bone tissue regeneration, and SSbD alternative materials such as titanium-doped hydroxyapatite/alginate composite, designed as sunscreen. The effects were assessed using the soil model Enchytraeus crypticus (Oligochaeta) in the natural standard LUFA 2.2 soil. The assessed endpoints included the 2, 3 and 4 days avoidance behavior (short-term), 28 days survival, size and reproduction (long term based on the OECD standard reproduction test), and 56 days survival and reproduction (longer-term OECD extension). Although overall results showed little to no toxicity among the tested nHA, there was a significant decrease in animals' size for Ti-containing nHA. Moreover, there was a tendency for higher toxicity at the lowest concentrations (i.e., 100 mg/kg). This requires further investigation to ensure safety.

Avoidance Behaviour of Six Collembolan Species Shows Species-Specific Sensitivity-Impact of Ag NM300K.

Although standard testing guidelines use a species as a representative surrogate, species-specific sensitivity is well-known. The aim of this study was to investigate the species-specific difference in avoidance behaviour among Collembola species exposed to silver (Ag) nanomaterials (NM) (Ag NM300K). The avoidance test was performed with Folsomia candida, an international standard species in laboratory tests, and five widely distributed species with different life history traits, commonly used in small multispecies systems (Folsomia fimetaria, Proisotoma minuta, Mesaphorura macrochaeta, Protaphorura fimata and Ceratophysella denticulata). There was higher avoidance in euedaphic species, such as F. candida and F. fimetaria, compared to the epiedaphic species C. denticulata, which showed the least avoidance behaviour. An explanation may be that euedaphic species (living in deeper soil layers) are more directly exposed within the soil pores and have developed a pronounced avoidance behaviour. In contrast, species living on the surface are likely less directly exposed and hence only avoid at higher total concentrations. Additionally, difference in cuticula between the groups, providing different degrees of protection against exposure, can explain the different behaviours. The present results highlight the importance of biodiversity for the ecosystem and raise awareness on species sensitivity.

Impacts of Longer-Term Exposure to AuNPs on Two Soil Ecotoxicological Model Species.

The production, use and disposal of nanoparticles (NPs) has been increasing continuously. Due to its unique properties, such as a high resistance to oxidation, gold NPs (AuNPs) are persistent in the environment, including the terrestrial, one of the major sinks of NPs. The present study aimed to assess the effects of AuNPs (from 10 to 1000 mg/kg) on two OECD standard ecotoxicological soil model species, Enchytraeus crypticus and Folsomia candida, based on the reproduction test (28 days) and on a longer-term exposure (56 days), and survival, reproduction, and size were assessed. AuNPs caused no significant hazard to F. candida, but for E. crypticus the lowest tested concentrations (10 and 100 mg AuNPs/kg) reduced reproduction. Further, AuNPs' toxicity increased from the 28th to the 56th day mainly to F. candida, as observed in animals' size reduction. Therefore, longer-term exposure tests are recommended as these often reveal increased hazards, not predicted when based on shorter exposures. Additionally, special attention should be given to the higher hazard of low concentrations of NPs, compared to higher concentrations.

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.

Reactive Oxygen Species Detection Using Fluorescence in Enchytraeus crypticus-Method Implementation through Ag NM300K Case Study.

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.

The Curious Case of Earthworms and COVID-19.

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.

Annelid genomes: Enchytraeus crypticus, a soil model for the innate (and primed) immune system.

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.

Machine learning and materials modelling interpretation of in vivo toxicological response to TiO2 nanoparticles library (UV and non-UV exposure).

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.

Multiomics assessment in Enchytraeus crypticus exposed to Ag nanomaterials (Ag NM300K) and ions (AgNO3) - Metabolomics, proteomics (& transcriptomics).

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.