Cationic surfactants are used in many industrial processes and in consumer products with concurrent release into the aquatic environment, where they may accumulate in aquatic organisms to regulatoryly relevant thresholds. Here, we aimed to better understand the bioconcentration behavior of three selected cationic surfactants, namely N,N-dimethyldecylamine (T10), N-methyldodecylamine (S12), and N,N,N-trimethyltetradecylammonium cation (Q14), in the cells of fish liver (RTL-W1) and gill (RTgill-W1) cell lines. We conducted full mass balances for bioconcentration tests with the cell cultures, in which the medium, the cell surface, the cells themselves, and the plastic compartment were sampled and quantified for each surfactant by HPLC MS/MS. Accumulation in/to cells correlated with the surfactants' alkyl chain lengths and their membrane lipid–water partitioning coefficient, D_MLW. Cell-derived bioconcentration factors (BCF) of T10 and S12 were within a factor of 3.5 to in vivo BCF obtained from the literature, while the cell-derived BCF values for Q14 were >100 times higher than the in vivo BCF. From our experiments, rainbow trout cell lines appear as a suitable conservative in vitro screening method for bioconcentration assessment of cationic surfactants and are promising for further testing.

Tire and road wear particles (TRWP) contain complex mixtures of chemicals and release them to the environment, and potential toxic effects of these chemicals still need to be characterized. We used a standardized surrogate for TRWP, cryogenically milled tire tread (CMTT), to isolate and evaluate effects of tire-associated chemicals. We examined organic chemical mixtures extracted and leached from CMTT for the toxicity endpoints genotoxicity, estrogenicity, and inhibition of bacterial luminescence. The bioassays were performed after chromatographic separation on high-performance thin-layer chromatography (HPTLC) plates. Extracts of CMTT were active in all three HPTLC bioassays with two estrogenic zones, two genotoxic zones, and two zones inhibiting bacterial luminescence. Extracts of CMTT artificially aged with thermooxidation were equally bioactive in each HPTLC bioassay. Two types of aqueous leachates of unaged CMTT, simulating either digestion by fish or contact with sediment and water, contained estrogenic chemicals and inhibitors of bacterial luminescence with similar profiles to those of CMTT extracts. Of 11 tested tire-associated chemicals, two were estrogenic, three were genotoxic, and several inhibited bacterial luminescence. 1,3-Diphenylguanidine, transformation products of N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine, and benzothiazoles were especially implicated through comparison to HPTLC retention factors in the CMTT samples. Other bioactive bands in CMTT samples did not correspond to any target chemicals. Tire particles clearly contain and can leach complex mixtures of toxic chemicals to the environment. Although some known chemicals contribute to estrogenic, genotoxic, and antibacterial hazards, unidentified toxic chemicals are still present and deserve further investigation. Overall, our study expands the understanding of potential adverse effects from tire particles and helps improve the link between those effects and the responsible chemicals.

Tire and road wear particles (TRWP) resulting from tire abrasion while driving raise concerns due to their potential contribution to aquatic toxicity. Our study aimed to assess cryogenically milled tire tread (CMTT) particle toxicity, used as a proxy for TRWP, and associated chemicals to fish using two Rainbow Trout (Oncorhynchus mykiss) cell lines representing the gill (RTgill-W1) and the intestinal (RTgutGC) epithelium. CMTT toxicity was evaluated through several exposure pathways, including direct contact, leaching, and digestion, while also assessing the impact of particle aging. Following OECD TG249, cell viability was assessed after 24 h acute exposure using a multiple-endpoint assay indicative of cell metabolic activity, membrane integrity and lysosome integrity. In vitro EC50 values for the fish cell lines exceeded river TRWP concentrations (2.02 g/L and 4.65 g/L for RTgill-W1 and RTgutGC cell lines, respectively), and were similar to in vivo LC50 values estimated at 6 g/L. Although toxicity was mainly driven by the leaching of tire-associated chemicals, the presence of the particles contributed to the overall toxicity by inducing a continuous leaching, highlighting the importance of considering combined exposure scenarios. Aging and digestion conditions were also found to mediate CMTT toxicity. Thermooxidation resulted in a decreased chemical leaching and toxicity, while in vitro digestion under mimicked gastrointestinal conditions increased leaching and toxicity. Specific chemicals, especially Zn, 2-mercaptobenzothiazole, 1,3-diphenylguanidine, and N-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD) were identified as contributors to the overall toxicity. Although 6PPD-quinone was detected in CMTT digestate, cytotoxicity assays with RTgill-W1 and RTgutGC cell lines showed no toxicity up to 6 mg/L, supporting the notion of a specific mode of action of this chemical. This study provides insights into the toxicological mechanisms induced by tire particles and their associated chemicals and can help in the evaluation of potential risks to aquatic life associated with TRWP.

Aquatic ecosystems continue to be threatened by chemical pollution. To what extent organisms are able to cope with chemical exposure depends on their ability to display mechanisms of defense across different organs. Among these mechanisms, biotransformation processes represent key physiological responses that facilitate detoxification and reduce the bioaccumulation potential of chemicals. Biotransformation does not only depend on the ability of different organs to display biotransformation enzymes but also on the affinity of chemicals towards these enzymes. In the present study, we explored the ability of different organs and of two freshwater fish to support biotransformation processes through the determination of in vitro phase I and II biotransformation enzyme activity, and their role in supporting intrinsic clearance and the formation of biotransformation products. Three environmentally relevant pollutants were evaluated: the polycyclic aromatic hydrocarbon (PAH) pyrene (as recommended by the OECD 319b test guideline), the fungicide azoxystrobin, and the pharmaceutical propranolol. Comparative studies using S9 sub-cellular fractions derived from the liver, intestine, gills, and brain of brown trout (Salmo trutta) and rainbow trout (Oncorhynchus mykiss) revealed significant phase I and II enzyme activity in all organs. However, organ- and species-specific differences were found. In brown trout, significant extrahepatic biotransformation was observed for pyrene but not for azoxystrobin and propranolol. In rainbow trout, the brain appeared to biotransform azoxystrobin. In this same species, propranolol appeared to be biotransformed by the intestine and gills. Biotransformation products could be detected only from hepatic biotransformation, and their profiles and formation rates displayed species-specific patterns and occurred at different magnitudes. Altogether, our findings further contribute to the current understanding of organ-specific biotransformation capacity, beyond the expression and activity of enzymes, and its dependence on specific enzyme-chemical interactions to support mechanisms of defense against exposure.

Regulation of chemicals requires knowledge of their toxicological effects on a large number of species, which has traditionally been acquired through in vivo testing. The recent effort to find alternatives based on machine learning, however, has not focused on guaranteeing transparency, comparability and reproducibility, which makes it difficult to assess advantages and disadvantages of these methods. Also, comparable baseline performances are needed. In this study, we trained regression models on the ADORE "t-F2F" challenge proposed in [Schür et al., Nature Scientific data, 2023] to predict acute mortality, measured as LC50 (lethal concentration 50), of organic compounds on fishes. We trained LASSO, random forest (RF), XGBoost, Gaussian process (GP) regression models, and found a series of aspects that are stable across models: (i) using mass or molar concentrations does not affect performances; (ii) the performances are only weakly dependent on the molecular representations of the chemicals, but (iii) strongly on how the data is split. Overall, the tree-based models RF and XGBoost performed best and we were able to predict the log10-transformed LC50 with a root mean square error of 0.90, which corresponds to an order of magnitude on the original LC50 scale. On a local level, on the other hand, the models are not able to consistently predict the toxicity of individual chemicals accurately enough. Predictions for single chemicals are mostly influenced by a few chemical properties while taxonomic traits are not captured sufficiently by the models. We discuss technical and conceptual improvements for these challenges to enhance the suitability of in silico methods to environmental hazard assessment. Accordingly, this work showcases state-of-the-art models and contributes to the ongoing discussion on regulatory integration.

In the aquatic environment, microplastic particles (MP) can accumulate in microbial communities that cover submerged substrata, i.e. in periphyton. Despite periphyton being the essential food source for grazers in the benthic zones, MP transfer from periphyton to benthic biota and its ecotoxicological consequences are unknown. Therefore, in this study, we investigated the effects of 1) MP on embryonal development of freshwater gastropod Physa acuta embryos, 2) MP on adult Physa acuta individuals through dietary exposure and 3) on the MP surface properties. Embryonal development tests were carried out with spherical polyethylene MP in the size of 1–4 μm (MP). Over a period of 28 days, embryonal development and hatching rate were calculated. In the feeding experiments, periphyton was grown in the presence and absence of MP and was then offered to the adult Physa acuta for 42–152 h. The snails readily ingested and subsequently egested MP, together with the periphyton as shown by MP quantification in periphyton, snail soft body tissue and feces. No selective feeding behavior upon MP exposure was detected. The ingestion of MP had no effect on mortality, feeding and defecation rate. Yet, the reproductive output of snails, measured as the number of egg clutches and numbers of eggs per clutch, decreased after the ingestion of MPs, while the hatching success of snail embryos those parents were exposed remained unaffected. In contrast, hatching rate of snail embryos was significantly reduced upon direct MP exposure. MP optical properties were changed upon the incorporation into the periphyton and the passage through the digestive tract. Our results indicate that MP incorporated in periphyton are bioavailable to aquatic grazers, facilitating the introduction of MP into the food chain and having direct adverse effects on the grazers' reproductive fitness.

Permanent rainbow trout (Oncorhynchus mykiss) cell lines represent potential in vitro alternatives to experiments with fish. We here developed a method to assess the bioaccumulation potential of anionic organic compounds in fish, using the rainbow trout liver-derived RTL-W1 cell line. Based on the availability of high quality in vivo bioconcentration (BCF) and biomagnification (BMF) data and the substances’ charge state at physiological pH, four anionic compounds were selected: pentachlorophenol (PCP), diclofenac (DCF), tecloftalam (TT) and benzotriazol-tert-butyl-hydroxyl-phenyl propanoic acid (BHPP). The fish cell line acute toxicity assay (OECD TG249) was used to derive effective concentrations 50 % and non-toxic exposure concentrations to determine exposure concentrations for bioaccumulation experiments. Bioaccumulation experiments were performed over 48 h with a total of six time points, at which cell, medium and plastic fractions were sampled and measured using high resolution tandem mass spectrometry after online solid phase extraction. Observed cell internal concentrations were over-predicted by K_OW-derived predictions while pH-dependent octanol–water partitioning (D_OW) and membrane lipid-water partitioning (D_MLW) gave better predictions of cell internal concentrations. Measured medium and cell internal concentrations at steady state were used to calculate RTL-W1-based BCF, which were compared to D_OW- or D_MLW-based model approaches and in vivo data. With the exception of PCP, the cell-derived BCF best compared to D_OW-based model predictions, which were higher than predictions based on D_MLW. All methods predicted the in vivo BCF for diclofenac well. For PCP, the cell-derived BCF was lowest although all BCF predictions underestimated the in vivo BCF by ≥ 1 order of magnitude. The RTL-W1 cells, and all other prediction methods, largely overestimated in vivo BMF, which were available for PCP, TT and BHPP. We conclude that the RTL-W1 cell line can supplement BCF predictions for anionic compounds. For BMF estimations, however, in vitro-in vivo extrapolations need adaptation or a multiple cell line approach.

The use of machine learning for predicting ecotoxicological outcomes is promising, but underutilized. The curation of data with informative features requires both expertise in machine learning as well as a strong biological and ecotoxicological background, which we consider a barrier of entry for this kind of research. Additionally, model performances can only be compared across studies when the same dataset, cleaning, and splittings were used. Therefore, we provide ADORE, an extensive and well-described dataset on acute aquatic toxicity in three relevant taxonomic groups (fish, crustaceans, and algae). The core dataset describes ecotoxicological experiments and is expanded with phylogenetic and species-specific data on the species as well as chemical properties and molecular representations. Apart from challenging other researchers to try and achieve the best model performances across the whole dataset, we propose specific relevant challenges on subsets of the data and include datasets and splittings corresponding to each of these challenge as well as in-depth characterization and discussion of train-test splitting approaches.

Climate change, biodiversity loss, and chemical pollution are planetary-scale emergencies requiring urgent mitigation actions. As these "triple crises" are deeply interlinked, they need to be tackled in an integrative manner. However, while climate change and biodiversity are often studied together, chemical pollution as a global change factor contributing to worldwide biodiversity loss has received much less attention in biodiversity research so far. Here, we review evidence showing that the multifaceted effects of anthropogenic chemicals in the environment are posing a growing threat to biodiversity and ecosystems. Therefore, failure to account for pollution effects may significantly undermine the success of biodiversity protection efforts. We argue that progress in understanding and counteracting the negative impact of chemical pollution on biodiversity requires collective efforts of scientists from different disciplines, including but not limited to ecology, ecotoxicology, and environmental chemistry. Importantly, recent developments in these fields have now enabled comprehensive studies that could efficiently address the manifold interactions between chemicals and ecosystems. Based on their experience with intricate studies of biodiversity, ecologists are well equipped to embrace the additional challenge of chemical complexity through interdisciplinary collaborations. This offers a unique opportunity to jointly advance a seminal frontier in pollution ecology and facilitate the development of innovative solutions for environmental protection.

Effluents of wastewater treatment plants can impact microbial communities in the receiving streams. However, little is known about the role of microorganisms in wastewater as opposed to other wastewater constituents, such as nutrients and micropollutants. We aimed therefore at determining the impact of wastewater microorganisms on the microbial diversity and function of periphyton, key microbial communities in streams. We used a flow-through channel system to grow periphyton upon exposure to a mixture of stream water and unfiltered or ultra-filtered wastewater. Impacts were assessed on periphyton biomass, activities and tolerance to micropollutants, as well as on microbial diversity. Our results showed that wastewater microorganisms colonized periphyton and modified its community composition, resulting for instance in an increased abundance of Chloroflexi and a decreased abundance of diatoms and green algae. This led to shifts towards heterotrophy, as suggested by the changes in nutrient stoichiometry and the increased mineralization potential of carbon substrates. An increased tolerance towards micropollutants was only found for periphyton exposed to unfiltered wastewater but not to ultra-filtered wastewater, suggesting that wastewater microorganisms were responsible for this increased tolerance. Overall, our results highlight the need to consider the role of wastewater microorganisms when studying potential impacts of wastewater on the receiving water body.

We present a decision support system using a Bayesian network to predict acute fish toxicity from multiple lines of evidence. Fish embryo toxicity testing has been proposed as an alternative to using juvenile or adult fish in acute toxicity testing for hazard assessments of chemicals. The European Chemicals Agency has recommended the development of a so-called weight-of-evidence approach for strengthening the evidence from fish embryo toxicity testing. While weight-of-evidence approaches in the ecotoxicology and ecological risk assessment community in the past have been largely qualitative, we have developed a Bayesian network for using fish embryo toxicity data in a quantitative approach. The system enables users to efficiently predict the potential toxicity of a chemical substance based on multiple types of evidence including physical and chemical properties, quantitative structure-activity relationships, toxicity to algae and daphnids, and fish gill cytotoxicity. The system is demonstrated on three chemical substances of different levels of toxicity. It is considered as a promising step towards a probabilistic weight-of-evidence approach to predict acute fish toxicity from fish embryo toxicity.

Tire and road wear particles (TRWP) account for an important part of the polymer particles released into the environment. There are scientific knowledge gaps as to the potential bioaccessibility of chemicals associated with TRWP to aquatic organisms. This study investigated the solubilization and bioaccessibility of seven of the most widely used tire-associated organic chemicals and four of their degradation products from cryogenically milled tire tread (CMTT) into fish digestive fluids using an in vitro digestion model based on Oncorhynchus mykiss. Our results showed that 0.06-44.1% of the selected compounds were rapidly solubilized into simulated gastric and intestinal fluids within a typical gut transit time for fish (3 h in gastric and 24 h in intestinal fluids). The environmentally realistic scenario of coingestion of CMTT and fish prey was explored using ground Gammarus pulex. Coingestion caused compound-specific changes in solubilization, either increasing or decreasing the compounds’ bioaccessibility in simulated gut fluids compared to CMTT alone. Our results emphasize that tire-associated compounds become accessible in a digestive milieu and should be studied further with respect to their bioaccumulation and toxicological effects upon passage of intestinal epithelial cells.

Periphyton is a freshwater biofilm composed of prokaryotic and eukaryotic communities that occupy rocks and sediments, forming the base of the food web and playing a key role in nutrient cycling. Given the large surface that periphyton comprises, it may also act as a sink for a diverse range of man-made pollutants, including microplastics (MP). Here we investigated the effect of 1–4 μm and 63–75 µm sized, spherical polyethylene MP with native and ultraviolet (UV)-weathered surface on developing natural stream periphyton communities over 28 days. In order to ensure proper particle exposure, we first tested MP suspension in water or in water containing either Tween 80, extracellular polymeric substances – EPS, fulvic acids, or protein. We found the extract of EPS from natural periphyton to be most suitable to create MP suspensions in preparation of exposure. Upon exposure, all tested types of MP were found to be associated with the periphyton, independent of their size and other properties. While biomass accrual and phenotypic community structure of the photoautotrophs remained unchanged, the prokaryotic and eukaryotic communities experienced a significant change in composition and relative abundances. Moreover, alpha diversity was affected in eukaryotes, but not in prokaryotes. The observed changes were more prominent in periphyton exposed to UV-treated as compared with native surface MP. Mechanical properties, as assessed by compression rheology, showed that MP-exposed periphyton had longer filamentous streamers, higher stiffness, lower force recovery and a higher viscoelasticity than control periphyton. Despite the observed structural and mechanical changes of periphyton, functional parameters (i.e., photosynthetic yield, respiration and nutrient uptake efficiencies) were not altered by MP, indicating the absence of MP toxicity, and suggesting functional redundancy in the communities. Together, our results provide further proof that periphyton is a sink for MP and demonstrate that MP can impact local microbial community composition and mechanical properties of the biofilms. Consequences of these findings might be a change in dislodgement behavior of periphyton, a propagation through the food chains and impacts on nutrient cycling and energy transfer. Hence, taking the omnipresence, high persistence and material and size diversity of MP in the aquatic environment into account, their ecological consequences need further investigation.

Out of the >107 million chemicals already registered with the Chemical Abstracts Services, less than 0.5% are being regulated, and even fewer are evaluated for their safety. Consequently, a new paradigm in risk assessment is urgently needed. It should encompass faster and less costly methods and reduce the number of animals needed for testing. One proposal is to combine computational modeling with small-scale bioassay methods. This chapter describes the methods that link in vitro bioassays using fish cells with physiologically based toxicokinetic (PBTK) modeling in order to predict the acute toxicity, bioaccumulation, and impact of chemicals on fish growth. The main focus is on PBTK modeling; thus all the model equations and parameters available for eight fish species as well as suggestions for possible software implementation will be provided. The PBTK model described here can account for respiratory and dietary uptake routes and for chemical biotransformation processes.

Metal-based nanoparticles (NPs) are considered detrimental to aquatic organisms due to their potential accumulation. However, little is known about the mechanisms underlying these effects and their species-specificity. Here we used stable silver (Ag) NPs (20 nm, from 10 to 500 μg/L) with a low dissolution rate (≤2.4%) to study the bioaccumulation and biological impacts in two freshwater gastropods: Lymnaea stagnalis and Planorbarius corneus. No mortality was detected during the experiments. Ag bioaccumulation showed a dose-related increase with an enhanced concentration in both species after 7d exposure. L. stagnalis displayed a higher accumulation for AgNPs than P. corneus (e.g., up to 18- and 15-fold in hepatopancreas and hemolymph, respectively) which could be due to the more active L. stagnalis having greater contact with suspended AgNPs. Furthermore, the hepatopancreas and stomach were preferred organs for bioaccumulation compared to the kidney, mantle and foot. Regarding biological responses, the hemolymph rather than hepatopancreas appeared more susceptible to oxidative stress elicited by AgNPs, as shown by significantly increasing lipid peroxidation (i.e., formation of malondialdehyde). Neurotoxicity was detected in L. stagnalis when exposed to high concentrations (500 μg/L). Comparison with impacts elicited by dissolved Ag revealed that the effects observed on AgNPs exposure were mainly attributable to NPs. These results highlighted the relationship between the physiological traits, bioaccumulation, and toxicity responses of these two species to AgNPs and demonstrated the necessity of species-specificity considerations when assessing the toxicity of NPs.

We applied machine learning methods to predict chemical hazards focusing on fish acute toxicity across taxa. We analyzed the relevance of taxonomy and experimental setup, showing that taking them into account can lead to considerable improvements in the classification performance. We quantified the gain obtained throught the introduction of taxonomic and experimental information, compared to classification based on chemical information alone. We used our approach with standard machine learning models (K-nearest neighbors, random forests and deep neural networks), as well as the recently proposed Read-Across Structure Activity Relationship (RASAR) models, which were very successful in predicting chemical hazards to mammals based on chemical similarity. We were able to obtain accuracies of over 93% on datasets where, due to noise in the data, the maximum achievable accuracy was expected to be below 96%. The best performances were obtained by random forests and RASAR models. We analyzed metrics to compare our results with animal test reproducibility, and despite most of our models "outperform animal test reproducibility" as measured through recently proposed metrics, we showed that the comparison between machine learning performance and animal test reproducibility should be addressed with particular care. While we focused on fish mortality, our approach, provided that the right data is available, is valid for any combination of chemicals, effects and taxa.

Wastewater treatment plants (WWTPs) play an important role in retaining organic matter and nutrients but to a lesser extent micropollutants. Therefore, treated wastewater is recognized as a major source of multiple stressors, including complex mixtures of micropollutants. These can potentially affect microbial communities in the receiving water bodies and the ecological functions they provide. In this study, we evaluated in flow-through channels the consequences of an exposure to a mixture of stream water and different percentages of urban WWTP effluent, ranging from 0% to 80%, on the microbial diversity and function of periphyton communities. Assuming that micropollutants exert a selective pressure for tolerant microorganisms within communities, we further examined the periphyton sensitivity to a micropollutant mixture extracted from passive samplers that were immersed in the wastewater effluent. As well, micropollutants in water and in periphyton were comprehensively quantified. Our results show that micropollutants detected in periphyton differed from those found in water, both in term of concentration and composition. Especially photosystem II inhibitors accumulated in periphyton more than other pesticides. Although effects of other substances cannot be excluded, this accumulation may have contributed to the observed higher tolerance of phototrophic communities to micropollutants upon exposure to 30% and 80% of wastewater. On the contrary, no difference in tolerance was observed for heterotrophic communities. Exposure to the gradient of wastewater led to structural differences in both prokaryotic and eukaryotic communities. For instance, the relative abundance of cyanobacteria was higher with increasing percentage of wastewater effluent, whereas the opposite was observed for diatoms. Such results could indicate that differences in community structure do not necessarily lead to higher tolerance. This highlights the need to consider other wastewater constituents such as nutrients and wastewater-derived microorganisms that can modulate community structure and tolerance. By using engineered flow-through channels that mimic to some extent the required field conditions for the development of tolerance in periphyton, our study constitutes a base to investigate the mechanisms underlying the increased tolerance, such as the potential role of microorganisms originating from wastewater effluents, and different treatment options to reduce the micropollutant load in effluents.

Understanding the ability of fish intestinal cells to act as a barrier for nanoparticle (NP) uptake and their effects is of significance from an environmental perspective as well as for human health, for which fish serves as an important nutrient source. We used an in vitro intestinal barrier model, based on rainbow trout intestinal (RTgutGC) cells, to elaborate the toxicity and translocation of five types of metal-based NPs. The NPs were polyvinylpyrrolidone (PVP)-coated Ag NPs, uncoated Ag NPs, CuO NPs, ZnO NPs and TiO₂ NPs. In conventional monolayers on impermeable supports, cell viability declined according to classical sigmoidal dose-response curves with EC50 values between 0.28 mg L^-1 and >100 mg L^-1 in the following rank order, from the most toxic (lowest EC50) to the least toxic (highest EC50): PVP–Ag NPs < uncoated Ag NPs < CuO NPs < ZnO NPs < TiO₂ NPs. When cells were cultured on permeable membranes to mimic an intestinal lumen and a blood-facing side, however, a much higher resistance of the cells towards NP-induced stress was noted with little to no impact on cell viability or barrier integrity. Yet, increased levels of Ag, Cu and Zn but not Ti were measured in the blood-side mimicking (basolateral) compartment, indicating translocation of Ag, Cu, and Zn-based NPs or ions liberated from them through the epithelial cell layer. Since especially CuO NPs appeared to be translocated as intact particles, they were investigated in more detail. A time- and temperature-dependent analysis, involving different endocytosis inhibitors, suggested that CuO NPs were translocated through the epithelium by apical caveolae-mediated endocytosis followed by delayed export onto the basolateral side. These data give valuable insights into NP uptake by, and translocation through, the fish intestinal epithelium and will be of value for future research on the molecular mechanisms of NPs that enter the fish via this critical uptake route.

The number of new psychoactive substances (NPS) on the illicit drug market increases fast, posing a need to urgently understand their toxicity and behavioural effects. However, with currently available rodent models, NPS assessment is limited to a few substances per year. Therefore, zebrafish (Danio rerio) embryos and larvae have been suggested as an alternative model that would require less time and resources to perform an initial assessment and could help to prioritize substances for subsequent evaluation in rodents. To validate this model, more information on the concordance of zebrafish larvae and mammalian responses to specific classes of NPS is needed. Here, we studied toxicity and behavioural effects of opioids in zebrafish early life stages. Synthetic opioids are a class of NPS that are often used in pain medication but also frequently abused, having caused multiple intoxications and fatalities recently. Our data shows that fentanyl derivatives were the most toxic among the tested opioids, with toxicity in the zebrafish embryo toxicity test decreasing in the following order: butyrfentanyl>3-methylfentanyl>fentanyl>tramadol> O-desmethyltramadol>morphine. Similar to rodents, tramadol as well as fentanyl and its derivatives led to hypoactive behaviour in zebrafish larvae, with 3-methylfentanyl being the most potent. Physico-chemical properties-based predictions of chemicals' uptake into zebrafish embryos and larvae correlated well with the effects observed. Further, the biotransformation pattern of butyrfentanyl in zebrafish larvae was reminiscent of that in humans. Comparison of toxicity and behavioural responses to opioids in zebrafish and rodents supports zebrafish as a suitable alternative model for rapidly testing synthetic opioids.

Tire and road wear particles (TRWP) have been shown to represent a large part of anthropogenic particles released into the environment. Nevertheless, the potential ecological risk of TRWP in the different environmental compartments and their potential toxic impacts on terrestrial and aquatic organisms remain largely underinvestigated. Several heavy metals compose TRWP, including Zn, which is used as a catalyst during the vulcanization process of rubber. This study investigated the solubilization potential of metals from cryogenically milled tire tread (CMTT) and TRWP in simulated gastric fluids (SF_GASTRIC) and simulated intestinal fluids (SF_INTESTINAL) designed to mimic rainbow trout (Oncorhynchus mykiss) gastrointestinal conditions. Our results indicate that the solubilization of heavy metals was greatly enhanced by gastrointestinal fluids compared to that by mineral water. After a 26 h in vitro digestion, 9.6 and 23.0% of total Zn content of CMTT and TRWP, respectively, were solubilized into the simulated gastrointestinal fluids. Coingestion of tire particles (performed with CMTT only) and surrogate prey items (Gammarus pulex) demonstrated that the animal organic matter reduced the amount of bioavailable Zn solubilized from CMTT. Contrastingly, in the coingestion scenario with vegetal organic matter (Lemna minor), high quantities of Zn were solubilized from L. minor and cumulated with Zn solubilized from CMTT.

Silver nanoparticles (Ag NPs) are widely used in consumer products especially because of their antimicrobial properties. However, this wide usage of Ag NPs is accompanied by their release into the environment where they will be rapidly transformed to other silver species - especially silver sulfide (Ag₂S). In the present study, we synthesized Ag NPs and sulfidized them to obtain a core-shell system Ag@Ag₂S NPs. Both types of particles form stable dispersions with hydrodynamic diameters of less than 100 nm when diluted in water, but tend to form micrometer-sized agglomerates in biological exposure media. Application of Ag and Ag@Ag₂S NPs to rainbow trout intestinal cells (RTgutGC) resulted in a concentration-dependent cytotoxicity for both types of particles, as assessed by a three-endpoint assay for metabolic activity, membrane integrity and lysosomal integrity. The Ag NPs were shown to be slightly more toxic than the Ag@Ag₂S NPs. Adding Ag or Ag@Ag₂S NPs to RTgutGC cells, grown on a permeable membrane to mimic the intestinal barrier, revealed considerable accumulation of silver for both types of particles. Indeed, the cells significantly attenuated the NP translocation, allowing only a fraction of the metal to translocate across the intestinal epithelium. These findings support the notion that the intestine constitutes an important sink for Ag NPs and that, despite the reduced cytotoxicity of a sulfidized NP form, the particles can enter fish where they may constitute a long-term source for silver ion release and cytotoxicity.

Information about acute fish toxicity is routinely required in many jurisdictions for environmental risk assessment of chemicals. This information is typically obtained using a 96-hour juvenile fish test for lethality according to OECD test guideline (TG) 203 or equivalent regional guidelines. However, TG 203 has never been validated using criteria currently required for new test methods including alternative methods. Characterization of the practicality and validity of TG 203 is important to provide a benchmark for alternative methods. This contribution systematically summarizes the available knowledge about limitations and uncertainties of TG 203, based on methodological, statistical, and biological considerations. Uncertainties stem from the historic flexibility (e.g. use of a broad range of species) and constraints of the basic test design (e.g., no replication). Other sources of uncertainty arise from environmental safety extrapolation based on TG 203 data. Environmental extrapolation models, combined with data from alternative methods, including mechanistic indicators of toxicity, may provide at least the same level of environmental protection. Yet, most importantly the 3R advantages of alternative methods allow a better standardization, characterization and an improved basic study design. This can enhance data reliability and thus facilitate the comparison of chemical toxicity, as well as the environmental classifications and prediction of no-effect concentrations of chemicals. Combined with the 3R gains and the potential for higher throughput, a reliable assessment of more chemicals can be achieved, leading to improved environmental protection.

We derived two novel cell lines from rainbow trout (RT) proximal (RTpi-MI) and distal intestine (RTdi-MI) and compared them with the previously established continuous cell line RTgutGC. Intestinal stem cells, differentiating and differentiated epithelial cells, and connective cells were found in all cell lines. The cell lines formed a polarized barrier, which was not permeable to large molecules and absorbed proline and glucose. High seeding density induced their differentiation into more mature phenotypes, as indicated by the downregulation of intestinal stem cell-related genes (i.e., sox9, hopx and lgr5), whereas alkaline phosphatase activity was upregulated. Other enterocyte markers (i.e., sglt1 and pept1), however, were not regulated as expected. In all cell lines, the presence of a mixed population of epithelial and stromal cells was characterized for the first time. The expression by the stromal component of lgr5, a stem cell niche regulatory molecule, may explain why these lines proliferate stably in vitro. Although most parameters were conserved among the three cell lines, some significant differences were observed, suggesting that characteristics typical of each tract are partly conserved in vitro as well.

1.1 The assay presented in this report
We here present the development and validation of the RTgill-W1 cell line assay to predict fish acute toxicity of chemicals in vitro. In this test, confluent monolayers of the gill cells are exposed in a completely defined, protein-free exposure medium, L-15/ex, for 24 h in 24-well tissue culture plates. Chemical concentrations in the exposure medium are quantified at the onset and upon termination of exposure. Cell viability after exposure is analysed by three fluorescent indicator dyes, measured on the same set of cells, and compared to unexposed cells, which serve as control. Combining the cell viability measurements and the measured chemical exposure concentrations, effective concentrations impacting 50 % of the gill cells (EC50 value) are calculated based on concentration-response modelling. [...]

Quantification of chemical toxicity in small-scale bioassays is challenging owing to small volumes used and extensive analytical resource needs. Yet, relying on nominal concentrations for effect determination maybe erroneous because loss processes can significantly reduce the actual exposure. Mechanistic models for predicting exposure concentrations based on distribution coefficients exist but require further validation with experimental data. Here we developed a complementary empirical model framework to predict chemical medium concentrations using different well-plate formats (24/48-well), plate covers (plastic lid, or additionally aluminum foil or adhesive foil), exposure volumes, and biological entities (fish, algal cells), focusing on the chemicals' volatility and hydrophobicity as determinants. The type of plate cover and medium volume were identified as important drivers of volatile chemical loss, which could accurately be predicted by the framework. The model focusing on adhesive foil as cover was exemplary cross-validated and extrapolated to other set-ups, specifically 6-well plates with fish cells and 24-well plates with zebrafish embryos. Two case study model applications further demonstrated the utility of the empirical model framework for toxicity predictions. Thus, our approach can significantly improve the applicability of small-scale systems by providing accurate chemical concentrations in exposure media without resource- and time-intensive analytical measurements.

We developed phospho-ERK1/2 ELISA for human and rainbow trout liver cells, employing HepG2 and RTL-W1 cell lines as models. The assay was applied to detect changes in ERK1/2 activity for nine chemicals, added over a wide concentration range and time points. Cell viability was measured to separate ERK1/2 regulation from cytotoxicity. Perfluorooctane sulfonate and carbendazim did not change ERK1/2 activity; influence on ERK1/2 due to cytotoxicity was indicated for tributyltin and cypermethrin. Mancozeb, benzo[a]pyrene, and bisphenol A stimulated ERK1/2 up to ∼2- (HepG2) and 1.5 (RTL-W1)-fold, though the kinetics differed between chemicals and cell lines. Bisphenol A and benzo[a]pyrene were the most potent concentration-wise, altering ERK1/2 activity in pM (HepG2) to nM (RTL-W1) range. While atrazine and ibuprofen increased ERK1/2 activity by ∼2-fold in HepG2, they did not initiate an appreciable response in RTL-W1. This assay proved to be a sensitive, medium- to high-throughput tool for detecting unrecognized ERK1/2-disrupting chemicals.

Substituted para-benzoquinones and hydroquinones are ubiquitous transformation products that arise during oxidative water treatment of phenolic precursors, for example through ozonation or chlorination. The benzoquinone structural motive is associated with mutagenicity and carcinogenicity, and also with induction of the oxidative stress response through the Nrf2 pathway. For either endpoint, toxicological data for differently substituted compounds are scarce. In this study, oxidative stress response, as indicated by the AREc32 in vitro bioassay, was induced by differently substituted para-benzoquinones, but also by the corresponding hydroquinones. Bioassays that indicate defense against genotoxicity (p53RE-bla) and DNA repair activity (UmuC) were not activated by these compounds. Stability tests conducted under incubation conditions, but in the absence of cell lines, showed that tested para-benzoquinones reacted rapidly with constituents of the incubation medium. Compounds were abated already in phosphate buffer, but even faster in biological media, with reactions attributed to amino- and thiol-groups of peptides, proteins, and free amino acids. The products of these reactions were often the corresponding substituted hydroquinones. Conversely, differently substituted hydroquinones were quantitatively oxidized to p-benzoquinones over the course of the incubation. The observed induction of the oxidative stress response was attributed to hydroquinones that are presumably oxidized to benzoquinones inside the cells. Despite the instability of the tested compounds in the incubation medium, the AREc32 in vitro bioassay could be used as an unspecific sum parameter to detect para-benzoquinones and hydroquinones in oxidatively treated waters.

Background: The advent of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology marked the beginning of a new era in the field of molecular biology, allowing the efficient and precise creation of targeted mutations in the genome of every living cell. Since its discovery, different gene editing approaches based on the CRISPR/Cas9 technology have been widely established in mammalian cell lines, while limited knowledge is available on genetic manipulation in fish cell lines. In this work, we developed a strategy to CRISPR/Cas9 gene edit rainbow trout (Oncorhynchus mykiss) cell lines and to generate single cell clone-derived knock-out cell lines, focusing on the phase I biotransformation enzyme encoding gene, cyp1a1, and on the intestinal cell line, RTgutGC, as example.
Results: Ribonucleoprotein (RNP) complexes, consisting of the Cas9 protein and a fluorescently labeled crRNA/tracrRNA duplex targeting the cyp1a1 gene, were delivered via electroporation. A T7 endonuclease I (T7EI) assay was performed on flow cytometry enriched transfected cells in order to detect CRISPR-mediated targeted mutations in the cyp1a1 locus, revealing an overall gene editing efficiency of 39%. Sanger sequencing coupled with bioinformatic analysis led to the detection of multiple insertions and deletions of variable lengths in the cyp1a1 region directed by CRISPR/Cas9 machinery. Clonal isolation based on the use of cloning cylinders was applied, allowing to overcome the genetic heterogeneity created by the CRISPR/Cas9 gene editing. Using this method, two monoclonal CRISPR edited rainbow trout cell lines were established for the first time. Sequencing analysis of the mutant clones confirmed the disruption of the cyp1a1 gene open reading frame through the insertion of 101 or 1 base pair, respectively.
Conclusions: The designed RNP-based CRISPR/Cas9 approach, starting from overcoming limitations of transfection to achieving a clonal cell line, sets the stage for exploiting permanent gene editing in rainbow trout, and potentially other fish cells, for unprecedented exploration of gene function.

The use of omics is gaining importance in the field of nanoecotoxicology; an increasing number of studies are aiming to investigate the effects and modes of action of engineered nanomaterials (ENMs) in this way. However, a systematic synthesis of the outcome of such studies regarding common responses and toxicity pathways is currently lacking. We developed an R-scripted computational pipeline to perform reanalysis and functional analysis of relevant transcriptomic data sets using a common approach, independent from the ENM type, and across different organisms, including Arabidopsis thaliana, Caenorhabditis elegans, and Danio rerio. Using the pipeline that can semiautomatically process data from different microarray technologies, we were able to determine the most common molecular mechanisms of nanotoxicity across extremely variable data sets. As expected, we found known mechanisms, such as interference with energy generation, oxidative stress, disruption of DNA synthesis, and activation of DNA-repair but also discovered that some less-described molecular responses to ENMs, such as DNA/RNA methylation, protein folding, and interference with neurological functions, are present across the different studies. Results were visualized in radar charts to assess toxicological response patterns allowing the comparison of different organisms and ENM types. This can be helpful to retrieve ENM-related hazard information and thus fill knowledge gaps in a comprehensive way in regard to the molecular underpinnings and mechanistic understanding of nanotoxicity.

Testing chemicals for fish acute toxicity is a legal requirement in many countries as part of environmental risk assessment. To reduce the number of fish used, substantial efforts have been focused on alternative approaches. Prominently, the cell viability assay with the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, has proven to be highly predictive and robust. Like the gills, the intestine is considered a major site of chemical uptake and biotransformation, but, in contrast to the gills, it is expected to be exposed to more hydrophobic chemicals, which enter the fish via food. In the present study, we therefore aimed to extend the cell bioassay to the rainbow trout epithelial cell line from intestine, RTgutGC. Using 16 hydrophobic and volatile chemicals from the fragrance palette, we show that also the RTgutGC cell line can be used to predict fish acute toxicity of chemicals and yields intra-laboratory variability in line with other bioassays. By comparing the RTgutGC toxicity to a study employing the RTgill-W1 assay on the same group of chemicals, a fragrance-specific rela­tionship was established that reflects an almost perfect 1:1 relationship between in vitro and in vivo toxicity results. Thus, both cell lines can be used to predict fish acute toxicity, either by extrapolating based on the in vivo-in vitro relationship or by taking the in vitro results at face value. We moreover demonstrate the derivation of non-toxic concentrations for down­stream applications that rely on a healthy cell state, such as the assessment of biotransformation or chemical transfer.

Zebrafish (Danio rerio) early life stages offer a versatile model system to study the efficacy and safety of drugs or other chemicals with regard to human and environmental health. This is because, aside from the well-characterized genome of zebrafish and the availability of a broad range of experimental and computational research tools, they are exceptionally well suited for high-throughput approaches. Yet, one important pharmacokinetic aspect is thus far only poorly understood in zebrafish embryo and early larvae: their biotransformation capacity. Especially biotransformation of electrophilic compounds is a critical pathway because they easily react with nucleophile molecules, such as DNA or proteins, potentially inducing adverse health effects. To combat such adverse effects, conjugation reactions with glutathione and further processing within the mercapturic acid pathway have evolved. We here explore the functionality of this pathway in zebrafish early life stages using a reference substrate (1-chloro-2,4-dinitrobenzene, CDNB).

With this work we show that zebrafish embryos can biotransform CDNB to the respective glutathione conjugate as early as 4 hours post fertilization. At all examined life stages, the glutathione conjugate is further biotransformed to the last metabolite of the mercapturic acid pathway, the mercapturate, which is slowly excreted.

Being able to biotransform electrophiles within the mercapturic acid pathway shows that zebrafish early life stages possess the potential to process xenobiotic compounds through glutathione conjugation and the formation of mercapturates. The presence of this chemical biotransformation and clearance route in zebrafish early life stages supports the application of this model in toxicology and chemical hazard assessment.

 

In view of the steadily increasing number of chemical compounds used in various products and applications, high-throughput toxicity screening techniques can help meeting the needs of 21st century risk assessment. Zebrafish (Danio rerio), especially its early life stages, are increasingly used in such screening efforts. In contrast, cell lines derived from this model organism have received less attention so far. A conceivable reason is the limited knowledge about their overall capacity to biotransform chemicals and the spectrum of expressed biotransformation pathways. One important biotransformation route is the mercapturic acid pathway, which protects organisms from harmful electrophilic compounds. The fully functional pathway involves a succession of several enzymatic reactions. To investigate the mercapturic acid pathway performance in the zebrafish embryonic cell line, PAC2, we analyzed the biotransformation products of the reactions comprising this pathway in the cells exposed to a nontoxic concentration of the reference substrate, 1-chloro-2,4-dinitrobenzene (CDNB). Additionally, we used targeted proteomics to measure the expression of cytosolic glutathione S-transferases (GSTs), the enzyme family catalyzing the first reaction in this pathway. Our results reveal that the PAC2 cell line expresses a fully functional mercapturic acid pathway. All but one of the intermediate CDNB biotransformation products were identified. The presence of the active mercapturic acid pathway in this cell line was further supported by the expression of a large palette of GST enzyme classes. Although the enzymes of the class alpha, one of the dominant GST classes in the zebrafish embryo, were not detected, this did not seem to affect the capacity of the PAC2 cells to biotransform CDNB. Our data provide an important contribution toward using zebrafish cell lines, specifically PAC2, for animal-free high-throughput screening in toxicology and chemical hazard assessment.

1-Chloro-2,4-dinitrobenzene (CDNB) is widely used as a model substrate for measuring enzyme activity of glutathione S-transferases in toxicity studies and in studies focusing on the metabol-ic capacity of different test systems. To allow the quantification of CDNB at low, non-toxic concentra-tions, we developed a sensitive liquid chromatography-mass spectrometry (LC-MS) technique, which is based on electron capture ionization using atmospheric pressure chemical ionization (APCI) in negative ion mode. Gas phase reactions occurring under atmospheric pressure produce specific ions that allow direct CDNB quantification down to 17 ng/ml in water. Using the new technique, we were able to verify CDNB exposure concentrations applied in two typical toxicity studies with early life stages of the com-mon model organisms, zebrafish (Danio rerio) and a zebrafish embryonic cell line (Pac2).

Marine mammals, such as whales, have a high proportion of body fat and so are susceptible to the accumulation, and associated detrimental health effects, of lipophilic environmental contaminants. Recently, we developed a wild-type cell line from humpback whale fibroblasts (HuWa). Extensive molecular assessments with mammalian wild-type cells are typically constrained by a finite life span, with cells eventually becoming senescent. Thus, the present work explored the possibility of preventing senescence in the HuWa cell line by transfection with plasmids encoding the simian virus large T antigen (SV40T) or telomerase reverse transcriptase (TERT). No stable expression was achieved upon SV40 transfection. Transfection with TERT, on the other hand, activated the expression of telomerase in HuWa cells. At the time of manuscript preparation, the transfected HuWa cells (HuWa_TERT) have been stable for at least 59 passages post-transfection. HuWa_TERT proliferate rapidly and maintain initial cell characteristics, such as morphology and chromosomal stability. The response of HuWa_TERT cells to an immune stimulant (lipopolysaccharide (LPS)) and an immunotoxicant (Aroclor1254) was assessed by measurement of intracellular levels of the pro-inflammatory cytokines interleukin (IL)-6, IL-1β and tumour necrosis factor (TNF)-α. HuWa_TERT cells constitutively express IL-6, IL-1β and TNFα. Exposure to neither LPS nor Aroclor1254 had an effect on the levels of these cytokines. Overall, this work supports the diverse applicability of HuWa cell lines in that they display reliable long-term preservation, susceptibility to exogenous gene transfer and enable the study of humpback whale-specific cellular response mechanisms.

Microscopic plastic (MP) particles are a ubiquitous contaminant in aquatic environments, which may bind hydrophobic chemicals, such as polycyclic aromatic hydrocarbons (PAHs), altering their environmental fate and interactions with biota. Using rainbow trout gill (RTgill-W1) and intestinal (RTgutGC) epithelial cells we investigated the effects of polystyrene microbeads (PS-MBs; 220 nm) on the cyto- and genotoxicity of the environmental pollutants benzo[a]pyrene (BaP) and 3-nitrobenzanthrone (3-NBA) over 48 h (0, 0.1, 1 and 10 μM). The Alamar Blue bioassay, used to assess cytotoxicity, showed that both pollutants significantly decreased cell viability by 10–20% at 10 μM in both cell lines after 48 h whereas PS-MBs (5 or 50 μg mL⁻¹) were non-toxic. Cytotoxicity in cells treated with PS-MBs together with BaP or 3-NBA were similar to those observed after exposure to BaP or 3-NBA alone. Using the formamidopyrimidine-DNA glycosylase (FPG)-modified comet assay 3-NBA, but not BaP, induced DNA damage in RTgutGC cells at 10 μM (∼10% tail DNA in the absence and ∼15% tail DNA in the presence of FPG versus ∼1% in controls), whereas PS-MBs alone showed no detrimental effects. Interestingly, comet formation was substantially increased (∼4-fold) when RTgutGC cells were exposed to PS-MBs (50 μg mL⁻¹) and 10 μM 3-NBA compared to cells treated with 3-NBA alone. Further, using ³²P-postlabelling we observed strong DNA adduct formation in 3-NBA-exposed RTgutGC cells (∼900 adducts/10⁸ nucleotides). 3-NBA-derived DNA adduct formation was significantly decreased (∼20%) when RTgutGC cells were exposed to MB and 3-NBA compared to cells treated with 3-NBA alone. Our results show that PS-MBs impact on the genotoxicity of 3-NBA, causing a significant increase in DNA damage as measured by the comet assay in the intestinal cell line, providing proof of principle that MPs may alter the genotoxic potential of PAHs in fish cells.

In this study we present the first fish-gut-on-chip model. This model is based on the reconstruction of the intestinal barrier by culturing two intestinal cell lines from rainbow trout, namely epithelial RTgutGC and fibroblastic RTgutF, in an artificial microenvironment. For a realistic mimicry of the interface between the intestinal lumen and the interior of the organism we i) developed ultrathin and highly porous silicon nitride membranes that serve as basement membrane analogues and provide a culture interface for the fish cells; ii) constructed a unique micro-well plate-based microfluidic bioreactor that enables parallelization of experiments and creates realistic fluid flow exposure scenarios for the cells; iii) integrated electrodes in the reactor for non-invasive impedance sensing of cellular well-being. In a first approach, we used this reactor to investigate the response of epithelial fish cells to in vivo-like shear stress rates of 0.002–0.06 dyne per cm², resulting from fluid flow within the intestinal lumen. Moreover, we investigated the interplay of epithelial and fibroblast cells under optimal flow conditions to carefully evaluate the benefits and drawbacks of the more complex reconstruction of the intestinal architecture. With our fish-gut-on-chip model we open up new strategies for a better understanding of basic fish physiology, for the refinement of fish feed in aquaculture and for predicting chemical uptake and bioaccumulation in fish for environmental risk assessment. The basic principles of our reactor prototype, including the use of ultrathin membranes, an open microfluidic circuit for perfusion and the micro-well plate-based format for simplified handling and avoidance of air-bubbles, will as well be of great value for other barrier-on-chip models.

An in vitro model of the fish intestine is of interest for research and application in diverse fields such as fish physiology, aquaculture and chemical risk assessment. The recently developed epithelial barrier model of the fish intestine relies on the RTgutGC cell line from rainbow trout (Oncorhynchus mykiss), cultured in inserts on permeable membranes. Our aim was to extend the current system by introducing intestinal fibroblasts as supportive layer in order to reconstruct the epithelial–mesenchymal interface as found in vivo. We therefore initiated and characterized the first fibroblast cell line from the intestine of rainbow trout, which has been termed RTgutF. Co-culture studies of RTgutGC and RTgutF were performed on commercially available electric cell substrate for impedance sensing (ECIS) and on newly developed ultrathin, highly porous alumina membranes to imitate the cellular interaction with the basement membrane. Cellular events were examined with non-invasive impedance spectroscopy to distinguish between barrier tightness and cell density in the ECIS system and to determine transepithelial electrical resistance for cells cultured on the alumina membranes. We highlight the relevance of the piscine intestinal fibroblasts for an advanced intestinal barrier model, particularly on ultrathin alumina membranes. These membranes enable rapid crosstalk of cells cultured on opposite sides, which led to increased barrier tightening in the fish cell line-based epithelial–mesenchymal model.
 

Predicting fish acute toxicity of chemicals in vitro is an attractive alternative method to the conventional approach using juvenile and adult fish. The rainbow trout (Oncorhynchus mykiss) cell line assay with RTgill-W1 cells has been designed for this purpose. It quantifies cell viability using fluorescent measurements for metabolic activity, cell- and lysosomal-membrane integrity on the same set of cells. Results from over 70 organic chemicals attest to the high predictive capacity of this test. We here report on the repeatability (intralaboratory variability) and reproducibility (interlaboratory variability) of the RTgill-W1 cell line assay in a round-robin study focusing on 6 test chemicals involving 6 laboratories from the industrial and academic sector. All participating laboratories were able to establish the assay according to preset quality criteria even though, apart from the lead laboratory, none had previously worked with the RTgill-W1 cell line. Concentration-response modeling, based on either nominal or geometric mean-derived measured concentrations, yielded effect concentrations (EC50) that spanned approximately 4 orders of magnitude over the chemical range, covering all fish acute toxicity categories. Coefficients of variation for intralaboratory and interlaboratory variability for the average of the 3 fluorescent cell viability measurements were 15.5% and 30.8%, respectively, which is comparable to other fish-derived, small-scale bioassays. This study therefore underlines the robustness of the RTgill-W1 cell line assay and its accurate performance when carried out by operators in different laboratory settings.

Humpback whales, like other polar wildlife, accumulate persistent organic pollutants. In Southern hemisphere populations, hexachlorobenzene (HCB) dominates the contaminant profiles. HCB is linked to a variety of health effects and is classified as a group 2B carcinogen, but the mechanism of action is a matter of contention. Potential toxicological effects to humpback whales remain entirely unknown. The recently established humpback whale fibroblast cell line (HuWa) offers an in vitro model for toxicological investigations. We here combine this novel cell line with a passive dosing strategy to investigate whale-specific toxicity of HCB. The relevant partitioning coefficients were determined to produce stable and predictable exposure concentrations in small-scale bioassays. The system was used to assess acute toxicity as well as genotoxicity of HCB to the HuWa cell line. While we found some transient reductions in metabolic activity, measured with the indicator dye alamarBlue, no clear acute toxic effects were discernible. Yet, a significant increase in DNA damage, detected in the alkaline comet assay, was found in HuWa cells exposed to 10 μg L^-1 HCB during the sensitive phase of cell attachment. Collectively, this work provides a ready-to-use passive dosing system and delivers evidence that HCB elicits genotoxicity in humpback whale cells.

Silver nanoparticle toxicity has been extensively studied in several vertebrate cells. Its interaction with cellular essential metals homeostasis, however, has largely been overlooked. In this study, we used a novel in vitro model of the fish enterohepatic system to investigate the effect of citrate-coated AgNP (cit-AgNP) and AgNO₃ on the homeostasis of copper, iron and zinc. The intestine and the liver are key tissues for whole body absorption and processing of metals. The enterohepatic system is based on a co-culture of intestinal cells (rainbow trout gut, RTgutGC) grown on permeable supports and hepatic cells (rainbow trout liver, RTL-W1) grown in the sub-located well. We have investigated early responses to sub-toxic and toxic doses of cit-AgNP and AgNO₃. Viability assays indicated that lysosomes were a target of cit-AgNP. Moreover, in comparison to AgNO₃, cit-AgNPs elicit a similar but attenuated metal stress response (induction of MT mRNA and ATP7A protein trafficking). Metal quantification revealed that, while intestinal cells accumulated similar amounts of silver following non-toxic exposure to equivalent amounts of either AgNO₃ or cit-AgNP, cells exposed to AgNO₃ excreted significantly more Ag to the basolateral chamber resulting in higher Ag accumulation in RTL-W1 cells. In addition, the application of toxic doses of AgNO₃ resulted in a reduction of intracellular zinc and iron. Silver nanoparticles were detected by STEM/EDX in RTgutGC after 3 hours of exposure but not after 24 hours suggesting rapid intracellular dissolution. Thus, Ag is a potent disruptor of essential metals homeostasis and cit-AgNP, which tend to be more difficult to excrete by the cell, can prolong this effect.

We studied the role of the fish intestine as a barrier for organic chemicals using the epithelial barrier model built on the rainbow trout (Oncorhynchus mykiss) intestinal cell line, RTgutGC and the newly developed exposure chamber, TransFEr, specifically designed to work with hydrophobic and volatile chemicals. Testing 11 chemicals with a range of physicochemical properties (logK_OW: 2.2 to 6.3, logHLC: 6.1 to 2.3) and combining the data with a mechanistic kinetic model enabled the determination of dominant processes underlying the transfer experiments and the derivation of robust transfer rates. Against the current assumption in chemical uptake modeling, chemical transfer did not strictly depend on the logK_OW but resulted from chemical-specific intracellular accumulation and biotransformation combined with paracellular and active transport. Modeling also identified that conducting elaborate measurements of the plastic parts, including the polystyrene insert and the PET filter, is unnecessary and that stirring in the TransFEr chamber reduced the stagnant water layers compared to theoretical predictions. Aside from providing insights into chemical uptake via the intestinal epithelium, this system can easily be transferred to other cell-based barrier systems, such as the fish gill or mammalian intestinal models and may improve in vitro–in vivo extrapolation and prediction of chemical bioaccumulation into organisms.

The fish intestine comprises an important environment-organism interface that is vital to fish growth, health and pathogen defense. Yet, knowledge about the physiology and defense mechanisms toward environmental stressors, such as bacterial or viral cues, is limited and depends largely on in vivo experiments with fish. On this background, we here explore the immune competence of a recently established in vitro intestinal barrier model based on the rainbow trout (Oncorhynchus mykiss) intestinal epithelial cell line, RTgutGC. We demonstrate that the RTgutGC cell barrier reacts to two immune stimuli, the bacterial lipopolysaccharide (LPS) from Escherichia coli and the viral Poly(I:C), by regulating the mRNA abundance of selected genes in a partly time- and concentration dependent manner. The immune stimuli activated the Myd88-and Ticam-dependent signalling cascades, which resulted in downstream activation of pro-inflammatory cytokines and interferon, comparable to the regulatory patterns known from in vivo. Stimuli exposure furthermore influenced the regulation of epithelial barrier markers and resulted in slightly impaired barrier functionality after long-term exposure to LPS. Collectively, we provide proof of the usefulness of this unique cell culture model to further gain basic understanding of the fish innate immune system and to apply it in various fields, such as fish feed development and fish health in aquaculture or the evaluation of immuno-toxicity of chemical contaminants.

Nanomaterial risk governance requires models to estimate the material flow, fate and transport as well as uptake/bioavailability, hazard and risk in the environment. This study assesses the fit of such available models to different stages during the innovation of nano-enabled products. Through stakeholder consultations, criteria were identified for each innovation stage from idea conception to market launch and monitoring. In total, 38 models were scored against 41 criteria concerning model features, applicability, resource demands and outcome parameters. A scoring scheme was developed to determine how the models fit the criteria of each innovation stage. For each model, the individual criteria scores were added, yielding an overall fit score to each innovation stage. Three criteria were critical to stakeholders and incorporated as multipliers in the scoring scheme; the required time/costs and level of expertise needed to use the model, and for risk assessment models only, the option to compare PEC and PNEC. Regulatory compliance was also identified as critical, but could not be incorporated, as a nanomaterial risk assessment framework has yet to be developed and adopted by legislators. In conclusion, the scoring approach underlined similar scoring profiles across stages within model categories. As most models are research tools designed for use by experts, their score generally increased for later stages where most resources and expertise are committed. In contrast, stakeholders need relatively simple models to identify potential hazards and risk management measures at early product development stages to ensure safe use of nanomaterials without costs and resource needs hindering innovation.

Neonicotinoids are widely used insecticides that have frequently been found in freshwater with concentrations ranging from ng to μg/L. It is known that these compounds impact non-target invertebrates, such as bees and gammaridae, in terms of toxicity and behavior, but impacts and species differences on vertebrates such as fish are little explored. The aim of this study was to investigate and compare the effects of one widely used neonicotinoid, imidacloprid, on development and behavior of two fish model species: Zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes). Fish were exposed for 5 (zebrafish) and 14 (medaka) days from 0.2 to 2000 μg/L imidacloprid by aqueous exposure. Survival, development, behavior and histological features were monitored and organism-internal concentrations and biotransformation products measured. Imidacloprid caused sublethal effects in both species but the effects were much stronger in medaka with deformities, lesions and reduced growth being the most prominent impacts. Due to the overall longer time of development, time-integrated exposure of medaka was about 2-fold higher compared to zebrafish, potentially accounting for parts of the sensitivity differences. Our results underline the importance of taking species sensitivity differences into account especially when considering that medaka responded at imidacloprid concentrations that have been measured in the environment.

The objective of this study was to evaluate the suitability of the rainbow trout intestinal epithelial cell line (RTgutGC) as an in vitro model for studies of gut immune function and effects of functional feed ingredients. Effects of lipopolysaccharide (LPS) and three functional feed ingredients [nucleotides, mannanoligosaccharides (MOS), and beta-glucans] were evaluated in RTgutGC cells grown on conventional culture plates and transwell membranes. Permeation of fluorescently-labeled albumin, transepithelial electrical resistance (TEER), and tight junction protein expression confirmed the barrier function of the cells. Brush border membrane enzyme activities [leucine aminopeptidase (LAP) and maltase] were detected in the RTgutGC cells but activity levels were not modulated by any of the exposures. Immune related genes were expressed at comparable relative basal levels as these in rainbow trout distal intestine. LPS produced markedly elevated gene expression levels of the proinflammatory cytokines il1b, il6, il8, and tnfa but had no effect on ROS production. Immunostaining demonstrated increased F-actin contents after LPS exposure. Among the functional feed ingredients, MOS seemed to be the most potent modulator of RTgutGC immune and barrier function. MOS significantly increased albumin permeation and il1b, il6, il8, tnfa, and tgfb expression, but suppressed ROS production, cell proliferation and myd88 expression. Induced levels of il1b and il8 were also observed after treatment with nucleotides and beta-glucans. For barrier function related genes, all treatments up-regulated the expression of cldn3 and suppressed cdh1 levels. Beta-glucans increased TEER levels and F-actin content. Collectively, the present study has provided new information on how functional ingredients commonly applied in aquafeeds can affect intestinal epithelial function in fish. Our findings suggest that RTgutGC cells possess characteristic features of functional intestinal epithelial cells indicating a potential for use as an efficient in vitro model to evaluate effects of bioactive feed ingredients on gut immune and barrier functions and their underlying cellular mechanisms.

The gut of fish belongs to the most essential barriers that mark the border between the organism and its surrounding environment. The pivotal barrier function, allowing absorption of nutrients from the diet while simultaneously protecting the organism from pathogens and contaminants, is accomplished by a single layer of epithelial cells lining the intestinal lumen. In vitro, this barrier has been mimicked by culturing fish epithelial intestinal cells on conventional permeable membranes within a two-chamber system, which creates an upper and a lower compartment representing the intestinal lumen and blood circulation, respectively. This simplified approach, however, has at least three important limitations: The first being restricted diffusion through the several micrometer thick commercial membranes, which moreover have quite limited porosity; the second being a lack of interaction with other intestinal cell types; and the third being absence of mechanical stimulation, such as shear forces from fluid flow to better simulate the physiology of the intestine. To overcome these limitations, this thesis focuses on the recreation of the piscine intestinal microenvironment by combining cells derived from the intestine of fish, precisely epithelial and fibroblast cell lines from rainbow trout (Oncorhynchus mykiss), with engineered microsystems. The applied stepwise approach encompasses (a) the development of an ultrathin permeable membrane as novel support for barrier forming cells, (b) followed by combining epithelial cells and fibroblasts for intestinal architecture reconstruction, and (c) exposure to fluid flow to mimic the mechanical forces occurring on the epithelial-lumen interface.

Artificial ultrathin membranes for intestinal cell culture were found to better mimic features of the delicate, highly permeable basement membrane that underlines the epithelial cells in vivo compared to conventional porous supports. Two types of membranes were fabricated in this study. The first was an anodized aluminum oxide membrane that features densely packed pores in the nanometer range and allows for fast diffusion of small molecules. This type of membrane is ideal for high quality microscopy and supports epithelial polarization. However, membranes release, albeit non-cytotoxic, concentrations of aluminum ions, which might be critical for toxicological investigations. Therefore, a second type of an ultrathin membrane, namely a silicon nitride membrane, was fabricated. It has pores in the micrometer range, is optically transparent and has been applied beyond static exposures for microfluidic studies.

To initiate co-culture of epithelial cells with fibroblasts, an intestinal fibroblast cell line from rainbow trout needed to be characterized first. The cells feature typical fibroblast morphology and behavior and appear to be infinite. The combination of epithelial and fibroblast cells, when in direct contact, had no beneficial effect on barrier tightness. Cell culture on opposite sides of ultrathin alumina membranes, however, resulted in increased trans-epithelial electrical resistance, suggesting enhanced barrier tightness from cellular cross-talk.

A uniquely designed microfluidic bioreactor with integrated ultrathin silicon nitride membranes as substrate for cell growth was finally developed to allow for realistic flow conditions on epithelial cells. The arising fluid shear stress on epithelial monolayers and epithelial-fibroblast co-cultures positively affected barrier resistance when applied at a moderate rate. This physiological adaptation allows for better comparability to the fish intestine in vivo compared to cells cultured under static conditions. This finding highlights the importance of mechanical stimulation for realistic organ mimicry within in vitro systems.

To conclude, this thesis demonstrates the benefits of recreating a more physiologically realistic microenvironment for epithelial cell cultures from fish intestine. By introducing ultrathin permeable membranes as novel culture substrate, adding a fibroblast cell line and shear stress, the novel in vitro intestinal barrier model now better reflects intestinal properties of the in vivo counterpart and allows for improved exposure and transport phenomena during experimental uptake studies, e.g. for chemical pollutants. This thesis therefore paves the way for improved understanding of normal and impaired physiology of the fish intestine and better in vitro to in vivo extrapolation while contributing to a reduced need of animal experiments.

Silver nanoparticles (AgNPs) are widely used as antimicrobial agents. During their life cycle, some of the AgNPs are released into natural environments, where chronic exposure to them can continue to cause harmful effects on microorganisms. However, very little is known about long-term impacts on important ecosystem compartments such as periphyton, a microbial community of algae and bacteria that covers submerged surfaces and contributes importantly to primary production and other ecosystem processes. Thus, the present study focused on assessing the accumulation of citrate-coated AgNPs and dissolved Ag⁺ in periphyton and on testing chronic effects on periphyton community structure and a range of functional endpoints. Stream periphyton grown in microcosms was exposed to 0.1, 1 and 10 μM AgNPs and 0.1 μM AgNO₃ (a source of Ag⁺ ions) for up to 21 days. By that time, 84 to 98% of the total available silver in the microcosms was strongly associated with the periphyton. The strongest and broadest impacts on functional endpoints were observed at the highest AgNP concentration (10 μM), which caused a decline in algal primary production and microbial respiration but a simultaneous increase in bacterial secondary production and total biomass accrual. The community structure of both phototrophs and heterotrophs was also changed. Overall, our results reveal that periphyton strongly accumulates AgNPs, leading to a shift of community metabolism towards heterotrophy, with possible consequences for trophic transfer in aquatic food webs exposed to Ag contamination.

The number of new psychoactive substances (NPS) increases rapidly, harming society and fuelling the need for alternative testing strategies. These should allow the everincreasing number of drugs to be tested more effectively for their toxicity and psychoactive effects. One proposed strategy is to complement rodent models with zebrafish (Danio rerio) larvae. Yet, our understanding of the toxicokinetics in this model, owing to the waterborne drug exposure and the distinct physiology of the fish, is incomplete. We here explore the toxicokinetics and behavioral effects of an NPS, meta-chlorophenylpiperazine (mCPP), in zebrafish larvae. Uptake kinetics of mCPP, supported by toxicokinetic modeling, strongly suggested the existence of active transport processes. Internal distribution showed a dominant accumulation in the eye, implying that in zebrafish, like in mammals, melanin could serve as a binding site for basic drugs. We confirmed this by demonstrating significantly lower drug accumulation in two types of hypo-pigmented fish. Comparison of the elimination kinetics between mCPP and previously characterized cocaine demonstrated that drug affinities to melanin in zebrafish vary depending on the structure of the test compound. As expected from mCPP-elicited responses in rodents and humans, zebrafish larvae displayed hypoactive behavior. However, significant differences were seen between zebrafish and rodents with regard to the concentration-dependency of the behavioral response and the comparability of tissue levels, corroborating the need to consider the organism-internal distribution of the chemical to allow appropriate dose modeling while evaluating effects and concordance between zebrafish and mammals. Our results highlight commonalities and differences of mammalian versus the fish model in need of further exploration.

The present review covers developments in studies of nanomaterials (NMs) in the environment since our much cited review in 2008. We discuss novel insights into fate and behavior, metrology, transformations, bioavailability, toxicity mechanisms, and environmental impacts, with a focus on terrestrial and aquatic systems. Overall, the findings were that: 1) despite substantial developments, critical gaps remain, in large part due to the lack of analytical, modeling, and field capabilities, and also due to the breadth and complexity of the area; 2) a key knowledge gap is the lack of data on environmental concentrations and dosimetry generally; 3) substantial evidence shows that there are nanospecific effects (different from the effects of both ions and larger particles) on the environment in terms of fate, bioavailability, and toxicity, but this is not consistent for all NMs, species, and relevant processes; 4) a paradigm is emerging that NMs are less toxic than equivalent dissolved materials but more toxic than the corresponding bulk materials; and 5) translation of incompletely understood science into regulation and policy continues to be challenging. There is a developing consensus that NMs may pose a relatively low environmental risk, but because of uncertainty and lack of data in many areas, definitive conclusions cannot be drawn. In addition, this emerging consensus will likely change rapidly with qualitative changes in the technology and increased future discharges.

The intestinal epithelium acts as vital gate keeper between the fish and its surrounding environment. A single layer of connected epithelial cells forms a selective barrier, which, among other essential functions, is important for nutrient uptake and defence against pathogens and putatively toxic chemicals. Despite its recognised significance, large knowledge gaps exist regarding the functionality of the fish intestine in general and the epithelium in specific, in large part due to the unavailability of suitable model systems. This thesis exploits a recently established in vitro fish intestinal cell barrier model from rainbow trout (Oncorhynchus mykiss), which is based on the so far only available fish intestinal epithelial cell line, RTgutGC. Motivated by the fact that the RTgutGC-based barrier model allows unprecedented opportunities for experimentation, this thesis engaged in embedding this cell line model into an in vitro testing strategy for assessing chemical transfer into fish. The focus was set on generally difficult-to-test chemicals, i.e. chemicals that are hydrophobic and/or volatile. For this purpose fragrances were selected, which combine chemicals in regular use with the described properties. Moreover, the response of the model epithelium to pathogenic stimulation was explored, in order to lay the groundwork for extending the in vitro testing strategy to different states of immune challenge in the future. [...]

Das Darmepithel fungiert als selektive Barriere zwischen dem Fisch und seiner Umgebung. Es besteht aus einer einfachen Schicht miteinander verbundener Epithelzellen und ist besonders für die Nährstoffaufnahme und die Abwehr von Krankheitserregern und potentiell toxischen Chemikalien von großer Bedeutung. Trotz dieser Relevanz bestehen noch immer große Wissenslücken in Bezug auf die Funktionalität des Fischdarms und insbesondere des Epithels. Der Grund dafür ist hauptsächlich das Fehlen von Modellsystemen. In dieser Arbeit wird ein neu etabliertes in vitro Modell verwendet, das auf der bislang einzigen verfügbaren Darmepithelzelllinie der Regenbogenforelle (Oncorhynchus mykiss), RTgutGC, basiert. Da dieses Barrieremodell beispiellose Experimentiermöglichkeiten bietet, wurde es zur Entwicklung einer in vitro Teststrategie zur Bewertung der Chemikalienaufnahme in den Fisch herangezogen. Der Fokus lag hierbei auf eher schwer zu testenden, d.h. hydrophoben und flüchtigen Chemikalien. Zu diesem Zweck wurden Duftstoffe ausgewählt, da diese regelmäßig verwendet werden und die oben genannten Eigenschaften besitzen. Darüber hinaus wurde die Reaktion des Modells auf Immunstimulation untersucht, um die Grundlage für eine Ausweitung der Teststrategie auf verschiedene Zustände der Immunsystemsbeeinträchtigung zu schaffen. [...]

Transfer of compounds across cellular barriers is a critical step of compound uptake into organisms. Using in vitro barrier systems to evaluate such transfer is attractive because of the higher throughput and reduced resource needs compared to animal studies. Thus far, however, studying the transfer of hydrophobic and volatile compounds was hampered by the unavailability of in vitro exposure systems that allow for stable and predictable chemical exposure concentrations. To overcome this limitation, we constructed a novel exposure chamber, TransFEr, and tested it with an in vitro epithelial barrier model using the rainbow trout (Oncorhynchus mykiss) intestinal cell line, RTgutGC. Key features of the chamber are its closed design and rotatable silicon segments, which can serve for chemical dosing and sampling. Using the fragrance damascone beta (log K_OW: 3.7, log HLC: −3.9) as a pilot chemical, we were able to demonstrate that our exposure chamber provides for stable chemical exposure concentrations and full mass balance. The RTgutGC epithelium served as barrier for damascone beta transfer, which we attribute to chemical retention and biotransformation in the intestinal cells. Nevertheless, substantial transfer of the chemical across the epithelium occurred. When a chemical sink, i.e. a silicon segment, was included in the basolateral chamber to mimic blood constituents binding in vitro, transfer was about three-fold enhanced. We suggest that the presented methodology can help to obtain insights into chemical uptake mechanisms via the intestinal or other epithelia of fish and other animals for hydrophobic and volatile chemicals.

The maximal chemical concentration that causes an acceptably small or no effect in an organism or isolated cells is an often - sought - after value in toxicology. Existing approaches to derive this value have raised several concerns; thus, it is often chosen case - by - case based on personal experience. To overcome this ambiguity, we propose an approach for choosing the non - toxic concentration (NtC) of a chemical in a rational, tractable way. We developed an algorithm that identifies the highest chemical concentration which causes no more than 10% effect (≤ EC10) including the modeled 95% confidence intervals and considering each of the measured biological replicates; and whose toxicity is not significantly different from no effect.The developed algorithm was validated in two steps: by comparing its results with measured and modeled data for 91 dose - response experiments with fish cell lines and/or zebrafish embryos; and by measuring actual effects caused by NtCs in a separate set of experiments using a fish cell line and zebrafish emb ryos. The algorithm provided an NtC that is more protective than NOEC (No - Observed - Effect - Concentration), NEC (modeled No - Effect Concentration), EC10 and Benchmark Dose (BMD). Despite focusing on small scale bioassays here, this study indicates that the NtC algorithm could be used in various systems. Its application on the survival of zebrafish embryos and on metabolic activity in cell lines showed that NtCs can be applied to different effect measurements, time points and levels of biological organization. The algorithm is available as Matlab and R code, and as a free, user friendly online application.

Permanent fish cell lines constitute a promising complement or substitute for fish in the environmental risk assessment of chemicals. We demonstrate the potential of a set of cell lines originating from rainbow trout (Oncorhynchus mykiss) to aid in the prediction of chemical bioaccumulation in fish, using benzo[a]pyrene (BaP) as a model chemical. We selected three cell lines from different tissues to more fully account for whole-body biotransformation in vivo: the RTL-W1 cell line, representing the liver as major site of biotransformation, and the RTgill-W1 (gill) and RTgutGC (intestine) cell lines, as important environment-organism interfaces, which likely influence chemical uptake. All three cell lines were found to effectively biotransform BaP. However, rates of in vitro clearance differed, with the RTL-W1 cell line being most efficient, followed by RTgutGC. Co-exposures with α-naphthoflavone as potent inhibitor of biotransformation, assessment of CYP1A catalytic activity, and the progression of cellular toxicity upon prolonged BaP exposure revealed that BaP is handled differently in the RTgill-W1 compared to the other two cell lines. Application of the cell-line-derived in vitro clearance rates into a physiology-based toxicokinetic model predicted a BaP bioconcentration factor (BCF) of 909-1057 compared to 920 reported for rainbow trout in vivo.

Zebrafish is a widely used animal model in biomedical sciences and toxicology. Although evidence for the presence of phases I and II xenobiotic defense mechanisms in zebrafish exists on the transcriptional and enzyme activity level, little is known about the protein expression of xenobiotic metabolizing enzymes. Given the important role of glutathione S-transferases (GSTs) in phase II biotransformation, we analyzed cytosolic GST proteins in zebrafish early life stages and different organs of adult male and female fish, using a targeted proteomics approach. The established multiple reaction monitoring-based assays enable the measurement of the relative abundance of specific GST isoenzymes and GST classes in zebrafish through a combination of proteotypic peptides and peptides shared within the same class. GSTs of the classes alpha, mu, pi and rho are expressed in zebrafish embryo as early as 4 h postfertilization (hpf). The majority of GST enzymes are present at 72 hpf followed by a continuous increase in expression thereafter. In adult zebrafish, GST expression is organ dependent, with most of the GST classes showing the highest expression in the liver. The expression of a wide range of cytosolic GST isoenzymes and classes in zebrafish early life stages and adulthood supports the use of zebrafish as a model organism in chemical-related investigations.

If adverse outcome pathways (AOPs) are to become the new standard predictive tool for chemical risk assessment in ecotoxicology, substantial effort will be required to construct AOPs for exposures to different chemical groups making sure that we have enough representation of different test species to adequately cover the tree of life. This should include plants, which have not yet received sufficient attention from the AOP community. In this chapter, we present Chlamydomonas reinhardtii, a unicellular green microalga that serves as a model organism for, among others, photosynthesis and the circadian rhythm. We review C. reinhardtii as a model organism for ecotoxicology and summarize different publicly available genomic and OMICS resources for the species. We also present a new putative AOP for C. reinhardtii exposed to silver, constructed based on integration of transcriptomic and proteomic datasets. Finally, we present the current state-of-the-art bioinformatics procedures that can be used for constructing AOPs from OMICS type of datasets and evaluate whether the approaches are suitable for C. reinhardtii.

The objective of the present study was to review the current knowledge regarding the bioaccumulation potential of ionizable organic compounds (IOCs), with a focus on the availability of empirical data for fish. Aspects of the bioaccumulation potential of IOCs in fish that can be characterized relatively well include the pH dependence of gill uptake and elimination, uptake in the gut, and sorption to phospholipids (membrane–water partitioning). Key challenges include the lack of empirical data for biotransformation and binding in plasma. Fish possess a diverse array of proteins that may transport IOCs across cell membranes. Except in a few cases, however, the significance of this transport for uptake and accumulation of environmental contaminants is unknown. Two case studies are presented. The first describes modeled effects of pH and biotransformation on the bioconcentration of organic acids and bases, while the second employs an updated model to investigate factors responsible for accumulation of perfluorinated alkyl acids. The perfluorinated alkyl acid case study is notable insofar as it illustrates the likely importance of membrane transporters in the kidney and highlights the potential value of read-across approaches. Recognizing the current need to perform bioaccumulation hazard assessments and ecological and exposure risk assessment for IOCs, the authors provide a tiered strategy that progresses (as needed) from conservative assumptions (models and associated data) to more sophisticated models requiring chemical-specific information

Der vorliegende Bericht fasst die Ergebnisse des Forschungsprojektes "Biologische Nachbehandlung von kommunalem Abwasser nach Ozonung", kurz "ReTREAT", zusammen. Das Projekt wurde durch das Bundesamtes für Umwelt (BAFU) im Rahmen der Umwelttechnologieförderung (UTF) finanziert. Zudem wurde das Projekt durch hohe Eigen- und Cash-Leistungen der Industriepartner finanziell und technisch unterstützt.
Ziel des Projektes war es Untersuchungen zur Leistungsfähigkeit und Effizienz verschiedener Verfahren der biologischen Nachbehandlung zur Reduktion der ökotoxikologischen Wirkung und zur Reduktion von Transformationsprodukten von ozonten, kommunalem Abwassers durchzuführen.

Permeable membranes are indispensable for in vitro epithelial barrier models. However, currently available polymer-based membranes are low in porosity and relatively thick, resulting in a limited permeability and unrealistic culture conditions. In this study, we developed an ultrathin, nanoporous alumina membrane as novel cell culture interface for vertebrate cells, with focus on the rainbow trout (Onchorynchus mykiss) intestinal cell line RTgutGC. The new type of membrane is framed in a silicon chip for physical support and has a thickness of only 1 µm, with a porosity of 15% and homogeneous nanopores (Ø = 73 ± 21 nm). Permeability rates for small molecules, namely lucifer yellow, dextran 40 and bovine serum albumin, exceeded those of standard polyethylene terephthalate (PET) membranes by up to 27 fold. With the final goal to establish a representative model of the fish intestine for environmental toxicology, we engineered a simple culture set-up, capable to test the cellular response towards chemical exposure. Herein, cells were cultured in a monolayer on the alumina membranes and formed a polarized epithelium with apical expression of the tight junction protein ZO-1 within 14 days. Impedance spectroscopy, a non-invasive and real time electrical measurement, was used to determine cellular resistance during epithelial layer formation and chemical exposure to evaluate barrier functionality. Resistance values during epithelial development revealed different stages of epithelial maturity and were comparable with the in vivo situation. During chemical exposure, cellular resistance changed immediately, when barrier tightness or cell viability was affected. Thus, our study demonstrates nanoporous alumina membranes as promising novel interface for alterative in vitro approaches, capable to allow cell culture in a physiologically realistic manner and to enable high quality microscopy and sensitive measurement of cellular resistance.

Die Identifizierung und Überwachung von Chemikalieneffekten auf Organismen in der Umwelt beruht auf einem breiten Spektrum von Herangehensweisen und Messpunkten. Allerdings wurden bisher viele der existierenden Untersuchungsmethoden lediglich für wenige Modellorganismen entwickelt. Die Berücksichtigung der Artenvielfalt und der Vielfalt an Reaktionen auf Umweltstressoren ist daher nach wie vor eine grosse Herausforderung. Die Analyse von messengerRNA (mRNA), welche für ausgewählte Biomarkermoleküle kodieren, ist dagegen ein vielversprechender Ansatz, da er auf ein weites Spektrum an Organismen und biologische Antworten übertragbar ist.
In diesem Projekt sollten die Einflüsse von Mikroschadstoffeintrag von Abwässern auf Flussökosysteme untersucht werden. Es wurde ein Biomarker-Genset für die Bachforelle (Salmo trutta) und die Regenbogenforelle (Oncorhynchus mykiss) etabliert, um die Wirkung von Mikroschadstoffen, wie Pharmaka und Pestizide, im Fisch nachweisen zu können. Ein typisches Biomarker-Set besteht aus 20-25 spezifischen Genen, welche verschiedene zelluläre Stressantworten, wie allgemeinen Stress, Metall- und oxidativen Stress, Biotransformation, Immunregulation usw. reflektieren. Die Regulation dieser Gene wurde in Leber und Nierengewebe in Bachforellen analysiert, welche ober- und unterströmig von vier verschieden Abwasserreinigungsanlagen (ARA) gefangen wurden. Zwei Standorte wurden doppelt untersucht: Herisau (Appenzell) vor und nach der Aufschaltung der Pulveraktivkohle (PAK)- Abwasserreinigungs-einheit und Steinach (St. Gallen) vor und nach dem Stopp der Abwassereinleitung. An diesen Standorten sollte überprüft werden ob mit dieser Methode Veränderungen der Wasserqualität und der Effekte auf die Fische erfasst werden können. Die Daten zeigen, dass die mRNA Regulation standortspezifisch ist. Fische, welche Unterhalb der Kläranalagen gefangen wurden, zeigen eine erhöhte Stressantwort an. Zudem konnten wir aufgrund der Genexpression das Einwirken von spezifischen Gruppen von Chemikalien identifizieren, wie Schwermetalle, endokrine Disruptoren, Pharmaka oder Pestizide. Tatsächlich bestätigte die chemische Analytik das Vorhandensein solcher Chemikalien im Wasser an den Probenahmestandorten. Ebenfalls konnten positive Effekte auf die Wasserqualität durch die Umbauten der ARAs in Herisau und Steinach mittels Genexpressionsanalyse in den untersuchten Fischen bestätigt werden.
Im Projekt ReTREAT auf der ARA Neugut wurden verschiedene Nachbehandlungen, im Anschluss an die Ozonung von Abwasser, auf deren Leistungsfähigkeit untersucht, toxische Transformations- oder Oxidationsnebenprodukte zu reduzieren. Zur Erfassung der ökotoxikologischen Wirkung wurden zahlreiche Biotests durchgeführt, darunter auch ein Fish Early Life Stage-Test (FELST) mit Regenbogenforellen (Oncorhynchus mykiss). Als Ergänzung zu den Standardendpunkten dieses Tests wurden normalerweise nicht erfassbare subletale Effekte mittels einem spezifischen Biomarker-Genset untersucht. Die Ergebnisse bestätigen die Effizienz der Ozonung, organische Mikroverunreinigungen aus dem Abwasser zu eliminieren und hervorgerufene subletale Effekte auf die Fische zu verringern. In Übereinstimmung mit den meisten anderen Tests konnte die gute Eignung der Nachbehandlung des ozonierten Abwassers durch den frischen Aktivkohlefilter (GAK2) bestätigt werden.
Im Projekt ReTREAT auf der ARA Neugut wurden verschiedene Nachbehandlungen, im Anschluss an die Ozonung von Abwasser, auf deren Leistungsfähigkeit untersucht, toxische Transformations- oder Oxidationsnebenprodukte zu reduzieren. Zur Erfassung der ökotoxikologischen Wirkung wurden zahlreiche Biotests durchgeführt, darunter auch ein Fish Early Life Stage-Test (FELST) mit Regenbogenforellen (Oncorhynchus mykiss). Als Ergänzung zu den Standardendpunkten dieses Tests wurden normalerweise nicht erfassbare subletale Effekte mittels einem spezifischen Biomarker-Genset untersucht. Die Ergebnisse bestätigen die Effizi-enz der Ozonung, organische Mikroverunreinigungen aus dem Abwasser zu eliminieren und hervorgerufene subletale Effekte auf die Fische zu verringern. In Übereinstimmung mit den meisten anderen Tests konnte die gute Eignung der Nachbehandlung des ozonierten Abwassers durch den frischen Aktivkohlefilter (GAK2) bestätigt werden.
Damit ist dies eine vielversprechende Methode für die Überwachung der Wasserqualität und der Fischgesundheit. Die Biomarker-Gensets sind sehr flexibel; weitere Anpassung für spezifische Effektmessungen, Übertragung auf andere Fischarten sowie weitere Anwendungen sind möglich.
Zudem wird versucht das Umweltmonitoring auf etablierte Zelllinien vom Fisch zu erweitern, welche eine tierfreie Alternative für die Umweltüberwachung darstellen könnten. Dabei werden permanente Zelllinien der Regenbogenforelle eingesetzt. Diese werden direkt im Labor mit Wasserproben, die an den Feldstandorten gewonnen wurden, exponiert. Die danach in diesen Zellen gemessene Expression wurde mit der Genexpression von im Fluss gefangenen Fischen und der chemischen Analytik verglichen. Stimmen die biologischen Antworten überein, könnten die Fischzellen zukünftig die Umweltüberwachung "übernehmen". Bisher konnte eine gute Übereinstimmung mit einigen Markergenen gefunden werden.

Synthetic glucocorticoids (GCs) are potential endocrine disrupting compounds that have been detected in the aquatic environment around the world in the low ng/L (nanomolar) range. GCs are used as immunosuppressants in medicine. It is of high interest whether clobetasol propionate (CP), a highly potent GC, suppresses the inflammatory response in fish after exposure to environmentally relevant concentrations. Bacterial lipopolysaccharide (LPS) challenge was used to induce inflammation and thus mimic pathogen infection. Zebrafish embryos were exposed to ≤1000 nM CP from ~1 h post fertilization (hpf) to 96 hpf, and CP uptake, survival after LPS challenge, and expression of inflammation-related genes were examined. Our initial experiments were carried out using 0.001% DMSO as a solvent vehicle, but we observed that DMSO interfered with the LPS challenge assay, and thus masked the effects of CP. Therefore, DMSO was not used in the subsequent experiments. The internal CP concentration was quantifiable after exposure to ≥10 nM CP for 96 h. The bioconcentration factor (BCF) of CP was determined to be between 16 and 33 in zebrafish embryos. CP-exposed embryos showed a significantly higher survival rate in the LPS challenge assay after exposure to ≥0.1 nM in a dose dependent manner. This effect is an indication of immunosuppression. Furthermore, the regulation pattern of several genes related to LPS challenge in mammals supported our results, providing evidence that LPS-mediated inflammatory pathways are conserved from mammals to teleost fish. Anxa1b, a GC-action related anti-inflammatory gene, was significantly down-regulated after exposure to ≥0.05 nM CP. Our results show for the first time that synthetic GCs can suppress the innate immune system of fish at environmentally relevant concentrations. This may reduce the chances of fish to survive in the environment, as their defense against pathogens is weakened.

Principles for determining uptake kinetics and bioconcentration factors for nanoparticles and test organisms have only been cursorily explored. Here we report the derivation of bioconcentration factors (BCFs) and the role of surface functionalization in the interactions between the nematode Plectus aquatilis and titanium dioxide nanoparticles (TiO₂ NPs) dispersed in freshwater. Because of the high background concentration of titanium in natural waters, TiO₂ NPs irradiated to produce ⁴⁸V labeled TiO₂ NPs, or doped with 1% niobium, were used to determine BCFs taking either an equilibrium partitioning or a kinetic modelling approach. The BCFs based on equilibrium partitioning increased from 71 (±17) L kg⁻¹ at the highest exposure concentrations to 5.1 (±3.2) × 103 L kg⁻¹ at the lowest exposure concentration, indicating that this approach is not valid. A kinetic modeling approach, based on the uptake rate and elimination rates for a two-phase elimination, best reflected the experimental data and was then used to determine BCFs. To further rationalize the kinetic interactions between the TiO₂ NPs and the nematode, organisms were exposed to surface-functionalized TiO₂ NPs with positively charged, negatively charged, steric stabilizing and environmentally relevant coatings. Correlations between the extent of TiO₂ NP association (i.e. attached and internalized) with the nematode, and TiO₂ NP properties, were examined. For all parameters considered, association of the surface functionalized TiO₂ NPs with the nematode best correlated with the TiO₂ NP sedimentation rate. These results indicate that concepts developed for hydrophobic contaminants are not applicable to nanoparticles and concepts specific to nanoparticles will be of greater utility.

The intestine of fish is a multifunctional organ: lined by only a single layer of specialized epithelial cells, it has various physiological roles including nutrient absorption and ion regulation. It moreover comprises an important barrier for environmental toxicants, including metals. Thus far, knowledge of the fish intestine is limited largely to in vivo or ex vivo investigations. Recently, however, the first fish intestinal cell line, RTgutGC, was established, originating from a rainbow trout (Oncorhynchus mykiss). In order to exploit the opportunities arising from RTgutGC cells for exploring fish intestinal physiology and toxicology, we present here the establishment of cells on commercially available permeable membrane supports and evaluate its suitability as a model of polarized intestinal epithelia. Within 3 weeks of culture, RTgutGC cells show epithelial features by forming tight junctions and desmosomes between adjacent cells. Cells develop a transepithelial electrical resistance comparable to in vivo measured values, reflecting the leaky nature of the fish intestine. Immunocytochemistry reveals evidence of polarization, such as basolateral localization of Na⁺/K⁺-ATPase (NKA) and apical localization of the tight junction protein ZO-1. NKA mRNA abundance was induced as physiological response toward a saltwater buffer, mimicking the migration of rainbow trout from fresh to seawater. Permeation of fluorescent molecules proved the barrier function of the cells, with permeation coefficients being comparable to those reported in fish. Finally, we demonstrate that cells on permeable supports are more resistant to the toxicity elicited by silver ions than cells grown the conventional way, likely due to improved cellular silver excretion.

Opioid drugs, such as morphine (MO), detected in aquatic environments worldwide, may harm fish due to their semi-persistence and ability to potently interact with molecular targets conserved across vertebrates. Here, we established a waterborne bacterial lipopolysaccharide (LPS) challenge assay with zebrafish embryos as a model to investigate chemically-induced disruption of the innate immune system, and used it to study the effects of MO exposure. Exposure to 1 mg/L MO resulted in pronounced immunosuppression, reflected in downregulation of several inflammation-related genes, including myd88, trif, traf6, p38, nfκb2, il-1β, il-8 and ccl34a. Fish exposed to 1 mg/L MO accumulated 11.7 ng/g (wet weight) of MO, a concentration comparable to that reported in blood of chronic drug abusers subject to higher infection rates. Surprisingly, exposure to lower MO concentrations (100 ng/L–100 μg/L) led to exacerbation of LPS-induced inflammation. Two ATP-binding cassette (ABC) transporters known to be involved in the xenobiotic efflux - abcb4 and abcc2, also known as multixenobiotic resistance (MXR) transporters - were downregulated at 100 ng/L MO. We hypothesized that ABC/MXR transporters could modulate the severity of inflammation by being involved in efflux of LPS, thus regulating its accumulation in the organism. Indeed, we could demonstrate that blocking of ABC/MXR transporters by an inhibitor, cyclosporine A, results in stronger inflammation, coinciding with higher LPS accumulation, as visualized with fluorescently labeled LPS. Our work demonstrates that MO can disrupt fish innate immune responses at environmentally relevant concentrations. We also provide evidence for a role of ABC/MXR transporters in LPS efflux in fish. These finding may be applicable across other taxa, as ABC transporters are evolutionary conserved. Since diverse environmentally present chemicals are known to interfere with ABC/MXR transporters' expression or activity, our discovery raises concerns about potential adverse effects of such compounds on the immune system responses in aquatic organisms.

Zebrafish larvae exposed to cocaine accumulate cocaine in the eye. Here we used electroretinography (ERG) to assess the effect of accumulated cocaine on the outer retina function of zebrafish larval eyes. We found a statistically significant increase of the ERG responses at moderate to bright light levels, showing that the presence of cocaine increased retinal responses to light, especially in the bright light range. This increase may be linked to dopamine in the retina, since cocaine is known to increase effective dopamine concentrations in the nervous system.

The significant increase of contaminants entering fresh water bodies calls for the development of rapid and reliable methods to monitor the aquatic environment and to detect water toxicity. Cell culture-based biosensing techniques utilise the overall cytotoxic response to external stimuli, mediated by a transduced signal, to specify the toxicity of aqueous samples. These biosensing techniques can effectively indicate water toxicity for human safety and aquatic organism health. In this review we account for the recent developments of the mainstream cell culture-based biosensing techniques for water quality evaluation, discuss their key features, potentials and limitations, and outline the future prospects of their development.

Growing concern about the adverse environmental and human health effects of a wide range of micropollutants requires the development of novel tools and approaches to enable holistic monitoring of their occurrence, fate and effects in the aquatic environment. A European-wide demonstration program (EDP) for effect-based monitoring of micropollutants in surface waters was carried out within the Marie Curie Initial Training Network EDA-EMERGE. The main objectives of the EDP were to apply a simplified protocol for effect-directed analysis, to link biological effects to target compounds and to estimate their risk to aquatic biota. Onsite large volume solid phase extraction of 50 L of surface water was performed at 18 sampling sites in four European river basins. Extracts were subjected to effect-based analysis (toxicity to algae, fish embryo toxicity, neurotoxicity, (anti-)estrogenicity, (anti-)androgenicity, glucocorticoid activity and thyroid activity), to target analysis (151 organic micropollutants) and to nontarget screening. The most pronounced effects were estrogenicity, toxicity to algae and fish embryo toxicity. In most bioassays, major portions of the observed effects could not be explained by target compounds, especially in case of androgenicity, glucocorticoid activity and fish embryo toxicity. Estrone and nonylphenoxyacetic acid were identified as the strongest contributors to estrogenicity, while herbicides, with a minor contribution from other micropollutants, were linked to the observed toxicity to algae. Fipronil and nonylphenol were partially responsible for the fish embryo toxicity. Within the EDP, 21 target compounds were prioritized on the basis of their frequency and extent of exceedance of predicted no effect concentrations. The EDP priority list included 6 compounds, which are already addressed by European legislation, and 15 micropollutants that may be important for future monitoring of surface waters. The study presents a novel simplified protocol for effect-based monitoring and draws a comprehensive picture of the surface water status across Europe.

Background: Silver nanoparticles (AgNP) are widely applied and can, upon use, be released into the aquatic environment. This raises concerns about potential impacts of AgNP on aquatic organisms. We here present a side by side comparison of the interaction of AgNP with two contrasting cell types: algal cells, using the algae Euglena gracilis as model, and fish cells, a cell line originating from rainbow trout (Oncorhynchus mykiss) gill (RTgill-W1). The comparison is based on the AgNP behavior in exposure media, toxicity, uptake and interaction with proteins.
Results: (1) The composition of exposure media affected AgNP behavior and toxicity to algae and fish cells. (2) The toxicity of AgNP to algae was mediated by dissolved silver while nanoparticle specific effects in addition to dissolved silver contributed to the toxicity of AgNP to fish cells. (3) AgNP did not enter into algal cells; they only adsorbed onto the cell surface. In contrast, AgNP were taken up by fish cells via endocytic pathways. (4) AgNP can bind to both extracellular and intracellular proteins and inhibit enzyme activity.
Conclusion: Our results showed that fish cells take up AgNP in contrast to algal cells, where AgNP sorbed onto the cell surface, which indicates that the cell wall of algae is a barrier to particle uptake. This particle behaviour results in different responses to AgNP exposure in algae and fish cells. Yet, proteins from both cell types can be affected by AgNP exposure: for algae, extracellular proteins secreted from cells for, e.g., nutrient acquisition. For fish cells, intracellular and/or membrane-bound proteins, such as the Na⁺/K⁺-ATPase, are susceptible to AgNP binding and functional impairment.

We introduce a novel in vitro rainbow trout intestinal barrier model and demonstrate its suitability for investigating nanoparticle transport across the intestinal epithelium. Rainbow trout (Oncorhynchus mykiss) intestinal cells (RTgutGC) were grown as monolayers on permeable supports leading to a two-compartment intestinal barrier model consisting of a polarized epithelium, dividing the system into an upper (apical) and a lower (basolateral) compartment, and thereby mimicking the intestinal lumen and the portal blood, respectively. The cells express the tight junction protein ZO-1 and build up a transepithelial electrical resistance comparable to the in vivo situation. Fluorescent polystyrene nanoparticles (PS-NPs; average hydrodynamic diameter: 73 ± 18 nm) were accumulated by RTgutGC cells in a time-, temperature- and concentration-dependent manner. Uptake of PS-NPs was confirmed using fluorescence microscopy. Cells formed an efficient barrier largely preventing the translocation of PS-NPs to the basolateral compartment. Taken together, these data demonstrate the suitability of the in vitro barrier model to study the effects of nanoparticles in fish intestinal epithelial cells.

This work focuses on Tonalide, a small fragrance molecule with an octanol-water partitioning coefficient of 5.7. Fragrances are an environmentally relevant substance class because they are produced in high amounts and strongly utilized for a multitude of products. The input into the aquatic environment is significant due to continuous use and low removal rates in waste water treatment plants. Their partly high hydrophobicity makes dietary uptake and bioaccumulation very likely. In this work, a cell line-based model for the intestinal barrier in fish and its application for compound permeability testing is presented and tested with Tonalide in a recently developed setup.
The model is based on intestinal epithelial cells from rainbow trout that were seeded into permeable supports. This creates a separation into an apical, representing the luminal side, and a basolateral, representing the blood-facing side, compartment, which generates a system in which permeation can be precisely studied. Complementary, an airtight stainless steel chamber was used for the compound exposure to reduce loss through evaporation and material binding. Furthermore, the system allowed the use of a passive dosing system for the exposure of hydrophobic substances as well as apical and basolateral stirring. For the basolateral compartment two setups were used: Medium without sink which leads to compound concentration gradient decrease over time and medium with sink used to maintain the concentration gradient. This setup is well suited for the assessment of fragrance permeation because it reduces compound loss, a common problem caused by their relative hydrophobicity and volatility.
It was found that experimental system and methodology are suitable to test the Tonalide permeation across the RTgut-GC cell layer. The accumulation in the cells and the permeation from the apical into the basolateral compartment was clearly detected through measurement of Tonalide mass in the different compartments. The permeation through permeable supports with and without cells was not significantly different. The same is true for the permeation with an without passive sink. The Tonalide permeability for experiments without sink is 4.95*10^-6 ± 8.3*10^-7 cm/s for cell-free and 6.595*10^-6 ± 7.2*10^-7 cm/s for cell-containing permeable supports, for experiments with sink the corresponding values are 6.667*10^-6 ± 3.3*10^-7 cm/s and 6.537*10^-6 ± 1.6*10^-6 cm/s. The accumulation in the cells increased twofold through the use of the passive sink. The measured permeation was compared to and evaluated with the help of permeabilities of other compounds. This revealed a dependency of substance lipophilicity and permeability across cell layers in permeable supports.

Within the FP7 EU project NanoValid a consortium of six partners jointly investigated the hazard of silver nanoparticles (AgNPs) paying special attention to methodical aspects that are important for providing high-quality ecotoxicity data. Laboratories were supplied with the same original stock dispersion of AgNPs. All partners applied a harmonised procedure for storage and preparation of toxicity test suspensions. Altogether ten different toxicity assays with a range of environmentally relevant test species from different trophic levels were conducted in parallel to AgNP characterisation in the respective test media. The paper presents a comprehensive dataset of toxicity values and AgNP characteristics like hydrodynamic sizes of AgNP agglomerates and the share (%) of Ag⁺-species (the concentration of Ag⁺-species in relation to the total measured concentration of Ag). The studied AgNP preparation (20.4 ± 6.8 nm primary size, mean total Ag concentration 41.14 mg/L, 46–68% of soluble Ag⁺-species in stock, 123.8 ± 12.2 nm mean z-average value in dH₂O) showed extreme toxicity to crustaceans Daphnia magna, algae Pseudokirchneriella subcapitata and zebrafish Danio rerio embryos (EC50 < 0.01 mg total Ag/L), was very toxic in the in vitro assay with rainbow trout Oncorhynchus mykiss gut cells (EC50: 0.01–1 mg total Ag/L); toxic to bacteria Vibrio fischeri, protozoa Tetrahymena thermophila (EC50: 1–10 mg total Ag/L) and harmful to marine crustaceans Artemia franciscana (EC50: 10–100 mg total Ag/L). Along with AgNPs, also the toxicity of AgNO₃ was analyzed. The toxicity data revealed the same hazard ranking for AgNPs and AgNO₃ (i.e. the EC50 values were in the same order of magnitude) proving the importance of soluble Ag⁺-species analysis for predicting the hazard of AgNPs. The study clearly points to the need for harmonised procedures for the characterisation of NMs. Harmonised procedures should consider: (i) measuring the AgNP properties like hydrodynamic size and metal ions species in each toxicity test medium at a range of concentrations, and (ii) including soluble metal salt control both in toxicity testing as well as in Ag⁺-species measurements. The present study is among the first nanomaterial interlaboratory comparison studies with the aim to improve the hazard identification testing protocols.

Zebrafish (Danio rerio) larvae have been suggested as vertebrate model to complement or even replace mammals for rapidly assessing behavioral effects of psychoactive drugs. Yet, divergent responses have been reported in mammals and fish despite the conservation of many drug targets. Cocaine, eg, acts as stimulant in mammals but no such response has been documented for zebrafish larvae. We hypothesized that differences in exposure routes (inhalation or injection in mammals vs waterborne in fish) may be a reason for differences in behavioral responses. We characterized cocaine toxicokinetics by liquid chromatography-mass spectrometry and found its rapid uptake into larvae. We used Matrix-assisted laser desorption ionization-mass spectrometry imaging for the first time to characterize internal distribution of cocaine in zebrafish larvae. Surprisingly, eyes accumulated the highest amount of cocaine and retained most of it even after 48 h depuration. We attribute this to trapping by pigment melanin, a thus far little explored mechanism that may also be relevant for other basic drugs. Cocaine also reached the brain but with levels similar to those in trunk indicating simple passive diffusion as means of distribution which was supported by toxicokinetic models. Although brain levels covered those known to cause hyperactivity in mammals, only hypoactivity (decreased locomotion) was recorded in zebrafish larvae. Our results therefore point to cocaine’s anesthetic properties as the dominant mechanism of interaction in the fish: upon entry through the fish skin and gills, it first acts on peripheral nerves rapidly overriding any potential stimulatory response in the brain.

The aim of the research was to explore the contribution of hepatic cytochrome CYP1A and metallothionein (MT) mRNA expression to biological effect monitoring. The study was conducted in the European flounder (Platichthys flesus) from the Gulf of Gdańsk. mRNA abundance was measured using reverse transcriptase polymerase chain reaction (RT-PCR) in liver RNA of fish sampled from three coastal stations and from one offshore station in the inner Gulf. The contribution of the mRNA-based biomarkers to the assessment of the environment was determined in conjunction with a selection of commonly applied biochemical markers: 7-ethoxyresorufin-O-deethylase (EROD), glutathione-S-transferase (GST), catalase (CAT), metallothioneins (MT), fluorescent aromatic compounds (FACs), all measured in the same individual fish. The mRNA biomarkers contributed to the separation between the sampling sites, but no correlations between CYP1A mRNA and EROD nor between MT mRNA and MT proteins were found, which should be attributed to the different levels these biomarkers correspond to and to the differences in factors that may affect them. One case of strong correlation between CYP1A mRNA and FACs was encountered. The overall results of this study suggest that biomarkers measured at the mRNA abundance level constitute a valuable addition to biomonitoring studies by providing additional information and contributing to the differentiation of results.

The need for alternative approaches to the use of vertebrate animals for hazard assessment of chemicals and pollutants has become of increasing importance. It is now the first consideration when initiating a vertebrate ecotoxicity test, to ensure that unnecessary use of vertebrate organisms is minimized wherever possible. For some regulatory purposes, the use of vertebrate organisms for environmental risk assessments has been banned; in other situations, the number of organisms tested has been dramatically reduced or the severity of the procedure refined. However, there is still a long way to go to achieve a complete replacement of vertebrate organisms to generate environmental hazard data. The development of animal alternatives is based not just on ethical considerations but also on reducing the cost of performing vertebrate ecotoxicity tests and in some cases on providing better information aimed at improving environmental risk assessments. The present Focus article provides an overview of the considerable advances that have been made toward alternative approaches for ecotoxicity assessments over the last few decades.

The removal of emerging contaminants during water treatment is a current issue and various technologies are being explored. These include UV- and ozone-based advanced oxidation processes (AOPs). In this study, AOPs were explored for their degradation capabilities of 25 chemical contaminants on the US Environmental Protection Agency's Contaminant Candidate List 3 (CCL3) in drinking water. Twenty-three of these were found to be amenable to hydroxyl radical-based treatment, with second-order rate constants for their reactions with hydroxyl radicals (·OH) in the range of 3–8 × 10⁹ M⁻¹ s⁻¹. The development of biological activity of the contaminants, focusing on mutagenicity and estrogenicity, was followed in parallel with their degradation using the Ames and YES bioassays to detect potential changes in biological effects during oxidative treatment. The majority of treatment cases resulted in a loss of biological activity upon oxidation of the parent compounds without generation of any form of estrogenicity or mutagenicity. However, an increase in mutagenic activity was detected by oxidative transformation of the following CCL3 parent compounds: nitrobenzene (·OH, UV photolysis), quinoline (·OH, ozone), methamidophos (·OH), N-nitrosopyrolidine (·OH), N-nitrosodi-n-propylamine (·OH), aniline (UV photolysis), and N-nitrosodiphenylamine (UV photolysis). Only one case of formation of estrogenic activity was observed, namely, for the oxidation of quinoline by ·OH. Overall, this study provides fundamental and practical information on AOP-based treatment of specific compounds of concern and represents a framework for evaluating the performance of transformation-based treatment processes.

To understand conditions affecting bioavailability and toxicity of citrate-coated silver nanoparticles (cit-AgNP) and dissolved silver at the luminal enterocyte interface, we exposed rainbow trout (Oncorhynchus mykiss) gut cells (RTgutGC) in media of contrasting composition: two amino acid-containing media, one of which was supplemented with proteins, as can be expected during digestion; and two protein and amino acid-free media contrasting low and high chloride content, as can be expected in the lumen of fish adapting to freshwater or seawater, respectively. Dose–response curves were generated measuring cell metabolic activity, membrane and lysosome integrity over a period of 72 hours. Then, nontoxic doses were applied and total silver accumulation, metallothionein and glutathione reductase mRNA levels were determined. The presence of proteins stabilized cit-AgNP keeping them in suspension. Conversely, in protein-free media, cit-AgNP agglomerated and settled, resulting in higher cellular accumulation of silver and toxicity. Chloride concentrations in exposure media modulated the toxicity of AgNO₃ but not of cit-AgNP. Moreover, while amino acid-containing media are protective against AgNO₃, likely due to the formation of thiolate complexes, they are only partially protective against cit-AgNP. Viability assays indicated that lysosomes are targets of cit-AgNP, supporting the hypothesis that cit-AgNP exert toxicity intracellularly. Metallothionein, a sensor of metal bioavailability, was induced by cit-AgNP in high chloride medium but not in low chloride medium, indicating that chloride might have a role in mobilizing silver from intercellular vesicles. Overall, this study shows that AgNP bioavailability and toxicity in the intestine is linked to its luminal content.

While short-term exposures of vertebrate cells, such as from fish, can be performed in defined, serum-free media, long-term cultures generally require addition of growth factors and proteins, normally supplied with a serum supplement. However, proteins are known to alter nanoparticle properties by binding to nanoparticles. Therefore, in order to be able to study nanoparticle–cell interactions for extended periods, the rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, was adapted to proliferate in a commercial, serum-free medium, InVitrus VP-6. The newly adapted cell strain was named RTgill-W1-pf (protein free). These cells proliferate at a speed similar to the RTgill-W1 cells cultured in a fully supplemented medium containing 5% fetal bovine serum. As well, they were successfully cryopreserved in liquid nitrogen and fully recovered after thawing. Yet, senescence set in after about 10 passages in InVitrus VP-6 medium, revealing that this medium cannot fully support long-term culture of the RTgill-W1 strain. The RTgill-W1-pf cell line was subsequently applied to investigate the effect of silver nanoparticles (AgNP) on cell proliferation over a period of 12 days. Indeed, cell proliferation was inhibited by 10 μM AgNP. This effect correlated with high levels of silver being associated with the cells. The new cell line, RTgill-W1-pf, can serve as a unique representation of the gill cell–environment interface, offering novel opportunities to study nanoparticle–cell interactions without serum protein interference.

Upon contact with biota, nanoparticles can bind to proteins, which coat the nanoparticles and form a nanoparticle-protein corona. Knowledge of corona proteins is therefore important for a mechanistic understanding of how nanoparticles interact with biomolecules in cells. Here we present the first study to reveal the identity of corona proteins from silver nanoparticle (AgNPs)-exposed living vertebrate cells. The cells are from a rainbow trout (Oncorhynchus mykiss) gill cell line, RTgill-W1, representing the interface between the aquatic environment and one of its model species. Subcellular fractionation allowed AgNP-protein corona complexes to be recovered from intact subcellular compartments and proteins lysed from the AgNPs to be detected by mass spectrometry. The identified proteins mark the trail of AgNPs processing in the cells like a forensic fingerprint: the cells take up the AgNPs via endocytic processes and store the particles in endosomal/lysosomal compartments. Moreover, stress response proteins were recovered in the AgNPs protein corona. In this way, we established a list of AgNPs susceptible proteins which can be investigated further in targeted nanoparticle–protein interaction. As a proof of principle, we demonstrate that Na⁺/K⁺-ATPase, identified from the corona and a known key protein in ion regulation in gill cells, is inhibited in its activity by AgNPs, confirming previously published in vivo experiments. The developed methodology is broadly applicable to other nanoparticles and cell types, representing a valuable tool for mechanistic nanoparticle–cell interaction studies, ranging from environmental and human risk assessment to biomedicine. In this way, our research also contributes to safer particle design.

This paper reports the first successful derivation and characterization of humpback whale fibroblast cell lines. Primary fibroblasts were isolated from the dermal connective tissue of skin biopsies, cultured at 37 °C and 5% CO₂ in the standard mammalian medium DMEM/F12 supplemented with 10% fetal bovine serum (FBS). Of nine initial biopsies, two cell lines were established from two different animals and designated HuWa1 and HuWa2. The cells have a stable karyotype with 2n = 44, which has commonly been observed in other baleen whale species. Cells were verified as being fibroblasts based on their spindle-shaped morphology, adherence to plastic and positive immunoreaction to vimentin. Population doubling time was determined to be ∼41 h and cells were successfully cryopreserved and thawed. To date, HuWa1 cells have been propagated 30 times. Cells proliferate at the tested temperatures, 30, 33.5 and 37 °C, but show the highest rate of proliferation at 37 °C. Short-term exposure to para,para′-dichlorodiphenyldichloroethylene (p,p′-DDE), a priority compound accumulating in southern hemisphere humpback whales, resulted in a concentration-dependent loss of cell viability. The effective concentration which caused a 50% reduction in HuWa1 cell viability (EC₅₀ value) was approximately six times greater than the EC₅₀ value for the same chemical measured with human dermal fibroblasts. HuWa1 exposed to a natural, p,p′-DDE-containing, chemical mixture extracted from whale blubber showed distinctively higher sensitivity than to p,p′-DDE alone. Thus, we provide the first cytotoxicological data for humpback whales and with establishment of the HuWa cell lines, a unique in vitro model for the study of the whales' sensitivity and cellular response to chemicals and other environmental stressors.

The occurrence of chronic or delayed toxicity resulting from the exposure to sublethal chemical concentrations is an increasing concern in environmental risk assessment. The Fish Embryo Toxicity (FET) test with zebrafish provides a reliable prediction of acute toxicity in adult fish, but it cannot yet be applied to predict the occurrence of chronic or delayed toxicity. Identification of sublethal FET endpoints that can assist in predicting the occurrence of chronic or delayed toxicity would be advantageous. The present study characterized the occurrence of delayed toxicity in zebrafish larvae following early exposure to PCB126, previously described to cause delayed effects in the common sole. The first aim was to investigate the occurrence and temporal profiles of delayed toxicity during zebrafish larval development and compare them to those previously described for sole to evaluate the suitability of zebrafish as a model fish species for delayed toxicity assessment. The second aim was to examine the correlation between the sublethal endpoints assessed during embryonal and early larval development and the delayed effects observed during later larval development. After exposure to PCB126 (3–3000 ng/L) until 5 days post fertilization (dpf), larvae were reared in clean water until 14 or 28 dpf. Mortality and sublethal morphological and behavioural endpoints were recorded daily, and growth was assessed at 28 dpf. Early life exposure to PCB126 caused delayed mortality (300 ng/L and 3000 ng/L) as well as growth impairment and delayed development (100 ng/L) during the clean water period. Effects on swim bladder inflation and cartilaginous tissues within 5 dpf were the most promising for predicting delayed mortality and sublethal effects, such as decreased standard length, delayed metamorphosis, reduced inflation of swim bladder and column malformations. The EC50 value for swim bladder inflation at 5 dpf (169 ng/L) was similar to the LC50 value at 8 dpf (188 and 202 ng/L in two experiments). Interestingly, the patterns of delayed mortality and delayed effects on growth and development were similar between sole and zebrafish. This indicates the comparability of critical developmental stages across divergent fish species such as a cold water marine flatfish and a tropical freshwater cyprinid. Additionally, sublethal effects in early embryo-larval stages were found promising for predicting delayed lethal and sublethal effects of PCB126. Therefore, the proposed method with zebrafish is expected to provide valuable information on delayed mortality and delayed sublethal effects of chemicals and environmental samples that may be extrapolated to other species.

With the global nanotechnology market growing rapidly, nanomaterials are being increasingly released into aquatic environments, where they can undergo modifications and sedimentation, which will put benthic organisms at risk. Of particular interest is the study of nanomaterial effects on periphyton, a community of auto- and heterotrophic microorganisms embedded in an extracellular polymer matrix that covers submerged surfaces in aquatic ecosystems. Although periphyton assumes important functions in ecosystems, like primary production, little information is available on its sensitivity to nanomaterials and how these might be transferred to higher trophic levels in food webs. [...]

Avec le développement des nanotechnologies, le risque de contamination des milieux aquatiques par les nanoparticules est de plus en plus important, perturbant ainsi les communautés benthiques dans ces écosystèmes. Parmi ces communautés, le périphyton est un ensemble de microorganismes auto- et hétérotrophes, inclus dans une matrice d’exopolymères et fixés sur un support solide immergés dans l’eau. En dépit du fait que le périphyton joue un rôle essentiel dans les cours d’eau, peu d’informations sont disponibles sur sa sensibilité aux nanoparticules ainsi que sur les effets sur la chaine trophique. [...]

Silver nanoparticles (AgNP) are increasingly used as antimicrobials in consumer products. Subsequently released into aquatic environments, they are likely to come in contact with microbial communities like periphyton, which plays a key role as a primary producer in stream ecosystems. At present, however, very little is known about the effects of nanoparticles on processes mediated by periphyton communities. We assessed the effects of citrate-coated silver nanoparticles and silver ions (dosed as AgNO₃) on five functional end points reflecting community and ecosystem-level processes in periphyton: photosynthetic yield, respiration potential, and the activity of three extracellular enzymes. After 2 h of exposure in experimental microcosms, AgNP and AgNO₃ inhibited respiration and photosynthesis of periphyton and the activities of two of the three extracellular enzymes. Addition of a chelating ligand that complexes free silver ions indicated that, in most cases, toxicity of AgNP suspensions was caused by Ag(I) dissolved from the particles. However, these suspensions inhibited one of the extracellular enzymes (leucine aminopeptidase), pointing to a specific nanoparticle effect independent of the dissolved Ag(I). Thus, our results show that both silver nanoparticles and silver ions have potential to disrupt basic metabolic functions and enzymatic resource acquisition of stream periphyton.

The toxicity of silver nanoparticles (AgNP) to aquatic organisms, including zebrafish (Danio rerio), has been demonstrated, but differing opinions exist on the contribution of the physical properties of the particles themselves and the free dissolved silver ions (Ag⁺) to the observed effects. High concentrations of chloride ions (Cl⁻) in the routinely used exposure media can cause precipitation of Ag⁺ as AgCl, as well as complexation of silver in diverse soluble chlorocomplexes, thus masking the contribution of dissolved silver to AgNP toxicity. In the present study, we formulated a zebrafish exposure medium with a low chloride content and exposed zebrafish embryos to AgNO₃ or carbonate-coated AgNP. The severity of toxicity caused by both silver forms depended on the time of exposure start, with younger embryos being most sensitive. Toxicity caused by both AgNO₃ and AgNP was of the same order of magnitude when compared based on the total dissolved silver concentration and could be prevented by addition of the Ag⁺ chelator cysteine. Further, we have analyzed the data from several previous studies to evaluate the influence of interactions between Ag⁺ and Cl⁻ on silver toxicity to zebrafish embryos. Our analysis demonstrates that the acute toxicity of AgNP to zebrafish embryos is largely mediated by Ag⁺. The influence of particle size and coating can at least partially be explained by the differences in Ag⁺ dissolution. High Cl⁻ levels in the exposure medium indeed have a pivotal influence on the resulting toxicity of AgNP, appearing to significantly attenuate toxicity in several studies. This consideration should influence the choice of exposure medium to be used when evaluating and comparing AgNP toxicity.

To elucidate the effects of chemicals on populations of different species in the environment, efficient testing and modeling approaches are needed that consider multiple stressors and allow reliable extrapolation of responses across species. An adverse outcome pathway (AOP) is a concept that provides a framework for organizing knowledge about the progression of toxicity events across scales of biological organization that lead to adverse outcomes relevant for risk assessment. In this paper, we focus on exploring how the AOP concept can be used to guide research aimed at improving both our understanding of chronic toxicity, including delayed toxicity as well as epigenetic and transgenerational effects of chemicals, and our ability to predict adverse outcomes. A better understanding of the influence of subtle toxicity on individual and population fitness would support a broader integration of sublethal endpoints into risk assessment frameworks. Detailed mechanistic knowledge would facilitate the development of alternative testing methods as well as help prioritize higher tier toxicity testing. We argue that targeted development of AOPs supports both of these aspects by promoting the elucidation of molecular mechanisms and their contribution to relevant toxicity outcomes across biological scales. We further discuss information requirements and challenges in application of AOPs for chemical- and site-specific risk assessment and for extrapolation across species. We provide recommendations for potential extension of the AOP framework to incorporate information on exposure, toxicokinetics and situation-specific ecological contexts, and discuss common interfaces that can be employed to couple AOPs with computational modeling approaches and with evolutionary life history theory. The extended AOP framework can serve as a venue for integration of knowledge derived from various sources, including empirical data as well as molecular, quantitative and evolutionary-based models describing species responses to toxicants. This will allow a more efficient application of AOP knowledge for quantitative chemical- and site-specific risk assessment as well as for extrapolation across species in the future.

Adverse outcome pathways (AOPs) organize knowledge on the progression of toxicity through levels of biological organization. By determining the linkages between toxicity events at different levels, AOPs lay the foundation for mechanism-based alternative testing approaches to hazard assessment. Here, we focus on growth impairment in fish to illustrate the initial stages in the process of AOP development for chronic toxicity outcomes. Growth is an apical endpoint commonly assessed in chronic toxicity tests for which a replacement is desirable. Based on several criteria, we identified reduction in food intake to be a suitable key event for initiation of middle-out AOP development. To start exploring the upstream and downstream links of this key event, we developed three AOP case studies, for pyrethroids, selective serotonin reuptake inhibitors (SSRIs) and cadmium. Our analysis showed that the effect of pyrethroids and SSRIs on food intake is strongly linked to growth impairment, while cadmium causes a reduction in growth due to increased metabolic demands rather than changes in food intake. Locomotion impairment by pyrethroids is strongly linked to their effects on food intake and growth, while for SSRIs their direct influence on appetite may play a more important role. We further discuss which alternative tests could be used to inform on the predictive key events identified in the case studies. In conclusion, our work demonstrates how the AOP concept can be used in practice to assess critically the knowledge available for specific chronic toxicity cases and to identify existing knowledge gaps and potential alternative tests.

The impact of silver nanoparticles (AgNPs) on aquatic algae has largely been studied with model species that possess a rigid cell wall. Here, we explored the interactions of AgNPs with Euglena gracilis, a green alga having no cell wall but a pellicle. The toxicity and silver uptake upon 1–2 h of exposure to various concentrations of AgNO₃ and AgNPs, having a mean size of 47 nm measured in the exposure medium, were examined. The photosynthetic yield decreased in a concentration-dependent manner and AgNPs were less toxic than AgNO₃ based on the total silver added. The cell morphology was significantly altered by AgNPs and AgNO₃. The damaging effects of AgNPs on the photosynthesis and morphology were completely prevented by cysteine, suggesting that the toxicity of AgNPs was mediated by dissolved Ag. Indeed, the maximal quantity of cell-associated silver was higher upon exposure to AgNPs compared to that upon AgNO₃ exposure, amounting to 5.1 × 10⁻⁴ mol L_cell⁻¹ and 1.4 × 10⁻⁴ mol L_cell⁻¹ for AgNPs and AgNO₃, respectively. However, the difference was not caused by the cellular uptake of AgNPs, but by the strong sorption of AgNPs onto the pellicle.

Objectives
The goal of the project was to evaluate the treatment of a representative list of organic contaminants from the Contaminant Candidate List 3 (CCL 3) via UV- and ozone-based advanced oxidation processes (AOPs). The research focused on these chemicals because they are being explored for possible regulation by the EPA in the future. The specific objectives of the research were to:
1. Select and prioritize CCL₃ contaminants for investigation
2. Select bioassays used to evaluate biological activity
3. Apply advanced oxidation treatment of CCL₃ chemicals using UV-H₂O₂ and O₃/O₃-H₂O₂ processes
4. Monitor AOP treatment using bioassay testing
5. Engineer evaluation for AOP operating conditions that minimize post-treatment bioactivity [...]

Organic micropollutants such as pharmaceuticals, estrogens or pesticides enter the environment continuously through the effluent of municipal wastewater treatment plants (WWTPs). Enhanced treatment of wastewater (WW) by ozone (O₃) is probably one of the simplest measures for abatement of organic micropollutants to avoid their discharge to the aquatic environment. During ozonation most organic micropollutants present in treated WW are oxidized either by a direct reaction with O₃ or by secondarily formed hydroxyl radicals (·OH). However, undesired oxidation by-products from the oxidative transformation of matrix components can also be formed. A modular laboratory decision tool based on the findings of previous investigations is presented to test the feasibility of ozonation as an option to upgrade specific WWTPs. These modules consist of investigations to assess (i) the matrix effects on ozone stability, (ii) the efficiency of micropollutant removal, (iii) the oxidation by-product formation, as well as (iv) bioassays to measure specific and unspecific toxicity of the treated WWs.
Matrix effects on ozone stability (quantified as O₃ and ·OH exposures) can give first indications on the suitability of an ozonation step. Ozonation of WWs yielding O₃ and ·OH exposures and micropollutant abatement similar to reference values evoked a significant improvement of the water quality as indicated by a broad range of bioassays. Irregular behavior of the ozonation points towards unknown compounds, possibly leading to the formation of undesired degradation products. It has been observed that in such WWs ozonation partly enhanced toxicity. In summary, the presented tiered laboratory test procedure represents a relatively cheap and straight-forward methodology to evaluate the feasibility of ozonation to upgrade specific WWTPs for micropollutant removal based on chemical and biological measurements.

Environmental risk assessment of chemicals is essential but often relies on ethically controversial and expensive methods. We show that tests using cell cultures, combined with modeling of toxicological effects, can replace tests with juvenile fish. Hundreds of thousands of fish at this developmental stage are annually used to assess the influence of chemicals on growth. Juveniles are more sensitive than adult fish, and their growth can affect their chances to survive and reproduce. Thus, to reduce the number of fish used for such tests, we propose a method that can quantitatively predict chemical impact on fish growth based on in vitro data. Our model predicts reduced fish growth in two fish species in excellent agreement with measured in vivo data of two pesticides. This promising step toward alternatives to fish toxicity testing is simple, inexpensive, and fast and only requires in vitro data for model calibration.

The key findings of a workshop jointly organized by the Swiss Centre of Applied Ecotoxicity, the Swiss Centre for Applied Human Toxicology (SCAHT), and the Federal Office of Public Health (FOPH) are summarized and provide a critical analysis of the current regulatory framework for nanomaterials and a snapshot of some hot topics in nanoscience.

Eine Ozonung als zusätzliche Reinigungsstufe auf einer kommunalen Abwasserreinigungsanlage (ARA) bewirkt eine deutliche Abnahme der Mikroverunreinigungen. Bei speziellen Belastungen, z.B. aufgrund bedeutender Industrie- oder Gewerbeabwassereinleitungen, können durch die Ozonung jedoch neue Stoffe, sogenannte Transformationsprodukte, gebildet werden. Daher soll künftig im Einzelfall ein Testverfahren aus fünf Modulen prüfen, ob sich ein bestimmtes Abwasser für eine Ozonbehandlung eignet oder nicht.

Owing to their unique antimicrobial properties, silver nanoparticles (AgNP) are among the most widely used engineered nanoparticles in a variety of consumer products and medical applications. Their resulting release into the aquatic environment raises concern about potential adverse effects in aquatic organisms. While a number of studies measured AgNP toxicity on integrative toxicologi-cal outcomes in aquatic organisms, such as development or mortality, very little knowledge exists on the mechanisms of AgNP interactions with cells of such organisms. This thesis therefore focused on the interaction of AgNP with cells derived from fish gill, specifically a fish gill cell line from rain-bow trout (Oncorhynchus mykiss), in an integrative way, starting from AgNP behavior in exposure media and upon contact with cells, following the formation of a protein corona around the AgNP during trafficking in intact cells and quantifying short- and long-term impact on cell viability. [...]

Les nanoparticules d’argent (AgNP) font partie des nanoparticules synthétiques les plus utilisées dans divers produits de consommation et applications médicales, en raison de leurs propriétés an-timicrobiennes uniques. En conséquence, les apports de ces nanoparticules dans l’environnement aquatique donnent lieu à des questions concernant leurs possibles effets nocifs sur les organismes aquatiques. Tandis que de nombreuses études ont mesuré la toxicité des AgNP par rapport à des paramètres toxicologiques comme le développement ou la mortalité des organismes aquatiques, les mécanismes d’interactions des AgNP dans les cellules sont encore très peu connus. Cette thèse est donc focalisée sur les interactions des AgNP avec des cellules dérivées de branchies de poisson, en particulier une ligne de cellules des branchies de la truite arc-en-ciel (Oncorhynchus mykiss), avec une approche intégrative, qui comprend le comportement des AgNP dans les milieux d’exposition et au contact des cellules, puis l’étude de la formation d’une couronne de protéines autour des AgNP par interactions avec des cellules intactes et la quantification des effets à court et long terme sur la viabilité des cellules. [...]

In aqueous solutions, silver nanoparticle (AgNP) behavior is affected by a variety of factors which lead to altered AgNP size and toxicity. Our research aims to explore the effect of media composition on citrate-coated AgNP (cit-AgNP) behavior and toxicity to the cell line from rainbow trout (Oncorhynchus mykiss) gill, RTgill-W1. Three different exposure media (L15/ex, L15/ex w/o Cl and d-L15/ex) were used. These were characterized by varying ionic strength and chloride content, both of which had a dominant effect on the behaviour of cit-AgNP. Comparing the behaviour and toxicity of cit-AgNP in the different media, stronger agglomeration of cit-AgNP correlated with higher toxicity. Deposition of cit-AgNP on cells might explain the higher toxicity of agglomerated cit-AgNP compared to that of suspended cit-AgNP. The cit-AgNP concentration-response curves as a function of dissolved silver ions, and the limited prevention of toxicity by silver ligands, indicated that cit-AgNP elicited a particle-specific effect on the cells. Furthermore, the lysosomal membrane integrity was significantly more sensitive to cit-AgNP exposure than cellular metabolic activity or cell membrane integrity and showed the weakest protection by silver ligands. This revealed that cit-AgNP toxicity seems to particularly act on RTgill-W1 cell lysosomes. The newly developed low ionic strength medium, d-L15/ex, which can stabilize cit-AgNP and better mimic the freshwater environment, offers an excellent exposure solution to study cellular and molecular effects of NP to gill cells.

A targeted analytical method was established to determine a large number of chemicals known to interfere with the gluco- and mineralocorticoid signalling pathway. The analytes comprise 30 glucocorticoids and 9 mineralocorticoids. Ten out of these corticosteroids were primary metabolites. Additionally, 14 nonsteroids were included. These analytes represent a broader range of possible adverse modes of action than previously reported. For the simultaneous determination of these structurally diverse compounds, a single-step multimode solid-phase extraction and pre-concentration was applied. Extracts were separated by a short linear HPLC gradient (20 min) on a core shell RP column (2.7 μm particle size) and compounds identified and quantified by LC-MS/MS. The method provided excellent retention time reproducibility and detection limits in the low nanograms per litre range. Untreated hospital wastewater, wastewater treatment plant influent, treated effluent and river waters were analysed to demonstrate the applicability of the method. The results show that not all compounds were sufficiently eliminated by the wastewater treatment, resulting in the presence of several steroids (∼20 ng/L) and nonsteroids in the final effluent, some of them at high concentrations up to 200 ng/L. Most of the detected mono-hydroxylated steroidal transformation products were found at significantly higher concentrations than their parent compounds. We therefore recommend to include these potentially bioactive metabolites in environmental toxicity assessment.

The risk posed by complex chemical mixtures in the environment to wildlife and humans is increasingly debated, but has been rarely tested under environmentally relevant scenarios. To address this issue, two mixtures of 14 or 19 substances of concern (pesticides, pharmaceuticals, heavy metals, polyaromatic hydrocarbons, a surfactant, and a plasticizer), each present at its safety limit concentration imposed by the European legislation, were prepared and tested for their toxic effects. The effects of the mixtures were assessed in 35 bioassays, based on 11 organisms representing different trophic levels. A consortium of 16 laboratories was involved in performing the bioassays. The mixtures elicited quantifiable toxic effects on some of the test systems employed, including i) changes in marine microbial composition, ii) microalgae toxicity, iii) immobilization in the crustacean Daphnia magna, iv) fish embryo toxicity, v) impaired frog embryo development, and vi) increased expression on oxidative stress-linked reporter genes. Estrogenic activity close to regulatory safety limit concentrations was uncovered by receptor-binding assays. The results highlight the need of precautionary actions on the assessment of chemical mixtures even in cases where individual toxicants are present at seemingly harmless concentrations.

This study investigated the occurrence of corticosteroid signaling disruptors in wastewaters and rivers in the Czech Republic and in Switzerland. 36 target compounds were detected using HPLC-MS/MS, with up to 6.4 μg/L for azole antifungals that indirectly affect corticosteroid signaling. Glucocorticoid receptor (GR)-mediated activity was determined using the GR-CALUX bioassay with dexamethasone equivalent concentrations ranging from

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