There is an increasing need for new approach methodologies (NAMs) to generate relevant ecotoxicological data. This study demonstrates the strengths of calcein AM, a highly sensitive fluorescent stain for esterase activity, in an automated image-based multiwell plate assay for detecting sublethal effects in Daphnia magna. Sample processing and feeding conditions were optimized to ensure a uniform dye distribution. The protocol was validated using two esterase inhibitors, triphenyl phosphate and netilmicin sulfate, and subsequently applied to test the environmental contaminants methoxychlor, lindane, tributyltin chloride, pentachlorophenol, diuron, and ethofumesate. The test organisms were imaged in vivo using automated confocal microscopy, and fluorescence intensity was quantified to generate concentration–response curves. The effects of triphenyl phosphate and netilmicin sulfate were observed at concentrations 3-fold and 6-fold lower, respectively, than in the OECD 202 immobilization test. All tested contaminants also inhibited esterase activity, with concentrations resulting in no esterase activity at 48 h, correlating with mortality observed at 48 h. This method provides a new sensitive fluorescent tool for detecting sublethal chemical effects in D. magna, with the added advantage of visualizing intracellular processes in vivo.

This study demonstrated the strengths of invivo molecular staining coupled with automated imaging analysis in Daphnia magna. A multiwell plate protocol was developed to assess mitochondrial membrane potential using the JC-1 dye. The suitability of five common anesthetics was initially tested, and 5% ethanol performed best in terms of anesthetic effects and healthy recovery. The staining conditions were optimized to 30min staining with 2 μM JC-1 for best J-aggregate formation. The protocol was validated with the model compound carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and used to measure the effect of four environmental contaminants, 2,4-dinitrophenol, triclosan, n-(1,3-dimethylbutyl)-N′-phenyl-p-phenylenediamine (6PPD), and ibuprofen, on mitochondrial health. Test organisms were imaged using anautomated confocal microscope, and fluorescence intensities were automatically quantified. The effect concentrations for CCCP were lower by a factor of 30 compared with the traditional OECD 202 acute toxicity test. Mitochondrial effects were also detected at lower concentrations for all tested environmental contaminants compared to the OCED 202 test. For 2,4-dinitrophenol, mitochondria effects were detectable after 2h exposure to environmentally relevant concentrations and predicted organism death was observed after 24h. The high sensitivity and time efficiency of this novel automated imaging method make it a valuable tool for advancing ecotoxicological testing.

Nontarget analysis by liquid chromatography-high-resolutionmass spectrometry (LC-HRMS) is now widely used to detect pollutants in the environment. Shifting away from targeted methods has led to detection of previously unseen chemicals, and assessing the risk posed by these newly detected chemicals is an important challenge. Assessing exposure and toxicity of chemicals detected with nontarget HRMS is highly dependent on the knowledge of the structure of the chemical. However, the majority of features detected in nontarget screening remain unidentified and therefore the risk assessment with conventional tools is hampered. Here, we developed MS2Quant, a machine learning model that enables prediction of concentration from fragmentation(MS2) spectra of detected, but unidentified chemicals. MS2Quant is an xgbTree algorithm-based regression model developed using ionization efficiency data for 1191 unique chemicals that spans 8 orders of magnitude. The ionization efficiency values are predicted from structural fingerprints that can be computed from the SMILES notation of the identified chemicals or from MS2 spectra of unidentified chemicals using SIRIUS+CSI: FingerID software. The root mean square errors of the training and test sets were 0.55(3.5x) and 0.80 (6.3x) log-units, respectively. In comparison, ionization efficiency prediction approaches that depend on assigning an unequivocal structure typically yield errors from 2x to 6x. The MS2Quant quantification model was validated on a set of 39 environmental pollutants and resulted in a mean prediction error of 7.4x, ageometric mean of 4.5x, and a median of 4.0x. For comparison, a model based on PaDEL descriptors that depends on unequivocal structural assignment was developed using the same dataset. The latter approach yielded a comparable mean prediction error of 9.5x, a geometricmean of 5.6x, and a median of 5.2x on the validation set chemicals when the top structural assignment was used as input. This confirms that MS2Quant enables to extract exposure information for unidentified chemicals which, although detected, have thus far been disregarded due to lack of accurate tools for quantification. TheMS2Quant model is available as an R-package in GitHub for improving discovery and monitoring of potentially hazardous environmental pollutants with nontarget screening.

To achieve water quality objectives of the zero pollution action plan in Europe, rapid methods are needed to identify the presence of toxic substances in complex water samples. However, only a small fraction of chemicals detected with nontarget high-resolution mass spectrometry can be identified, and fewer have ecotoxicological data available. We hypothesized that ecotoxicological data could be predicted for unknown molecular features in data-rich high-resolution mass spectrometry (HRMS) spectra, thereby circumventing time-consuming steps of molecular identification and rapidly flagging molecules of potentially high toxicity in complex samples. Here, we present MS2Tox, a machine learning method, to predict the toxicity of unidentified chemicals based on high-resolution accurate mass tandem mass spectra (MS2). The MS2Tox model for fish toxicity was trained and tested on 647 lethal concentration (LC50) values from the CompTox database and validated for 219 chemicals and 420 MS2 spectra from MassBank. The root mean square error (RMSE) of MS2Tox predictions was below 0.89 log-mM, while the experimental repeatability of LC50 values in CompTox was 0.44 log-mM. MS2Tox allowed accurate prediction of fish LC50 values for 22 chemicals detected in water samples, and empirical evidence suggested the right directionality for another 68 chemicals. Moreover, by incorporating structural information, e.g., the presence of carbonyl-benzene, amide moieties, or hydroxyl groups, MS2Tox outperforms baseline models that use only the exact mass or logKOW. 

Green public procurement has been identified as an important instrument to achieve the aim of substituting hazardous chemicals to more benign alternatives. Nevertheless, few studies have investigated the specific challenges associated with practically applying procurement requirements for reducing the content of hazardous chemicals. This study contributes with identifying and describing the specific challenges, improvement options, and conflicting goals relating to the aim of reducing exposure to hazardous chemicals through green public procurement.

The production and use of plastic over many decades has resulted in its accumulation in the world’s oceans. Plastic debris poses a range of potential risks to the marine environment and its biota. Especially, the potential hazards of small plastic debris and chemicals associated with plastic have not been extensively studied. When buoyant plastic is exposed to ultraviolet radiation, it will slowly degrade and leach chemicals into surrounding waters. These leachates can include additives, sorbed organic pollutants, and degradation products of the plastic polymers. While most hazard assessments have focused on studying adverse effects due to the uptake of plastic, toxicity studies of the leachates of plastics are less common. To begin to address this knowledge gap, we studied the acute toxicity of leachates from diverse plastics in the harpacticoid copepod Nitocra spinipes. Our results show that leachates caused a higher toxicity after plastic was exposed to ultraviolet light compared to leaching in darkness. We observed differences in toxicity for different polymer types: polyvinyl chloride and polypropylene resulted in the most toxic leachates, while polystyrene and poly[ethylene terephthalate] were least toxic. Furthermore, we observed increased toxicity of leachates from some plastics that had been weathered in the real marine environment compared to matching new materials. Our results indicate that both weathering condition and polymer type influence the toxicity of plastic leachates.

To improve assessment of risks associated with pharmaceutical contamination of the environment, it is crucial to understand effects and mode of action of drugs in non-target species. The evidence is accumulating that species with well-conserved drug targets are prone to be at risk when exposed to pharmaceuticals. An interesting group of pharmaceuticals released into the environment is imidazoles, antifungal agents with inhibition of ergosterol synthesis as a primary mode of action in fungi. However, imidazoles have also been identified as competitive antagonists of calmodulin (CaM), a calcium-binding protein with phylogenetically conserved structure and function. Therefore, imidazoles would act as CaM inhibitors in various organisms, including those with limited capacity to synthesize sterols, such as arthropods. We hypothesized that effects observed in crustaceans exposed to imidazoles are related to the CaM inhibition and CaM-dependent nitric oxide (NO) synthesis. To test this hypothesis, we measured (i) CaM levels and its gene expression, (ii) NO accumulation and (iii) gene expression of NO synthase (NOS1 and NOS2), in the cladoceran Daphnia magna exposed to miconazole, a model imidazole drug. Whereas significantly increased CaM gene expression and its cellular allocation were observed, supporting the hypothesized mode of action, no changes occurred in either NO synthase expression or NO levels in the exposed animals. These findings suggest that CaM inhibition by miconazole leads to protein overexpression that compensates for the loss in the protein activity, with no measurable downstream effects on NO pathways. The inhibition of CaM in D. magna may have implications for effect assessment of exposure to mixtures of imidazoles in aquatic non-target species.

Chlorinated paraffins (CPs) are industrial chemicals, subdivided into three categories: short chain (SCCPs), medium chain (MCCPs), and long chain (LCCPs) chlorinated paraffins. SCCPs are currently restricted in Europe and North America. MC and LCCPs are being used as substitution products, but there is a knowledge gap concerning their bioaccumulation potential in aquatic organisms. In this work, we performed laboratory bioconcentration (passive uptake) and bioaccumulation (including dietary uptake) experiments with Daphnia magna using five different CP technical substances. All tested CP technical substances were bioaccumulative in D. magna, with log BCF and log BAF values ranging between 6.7-7.0 and 6.5-7.0 (L kg lipid(-1)), respectively. An increase in carbon chain length and an increase in chlorine content (% w/w) of the CP technical substances had significant positive effects on the log BCF and log BAF values. For the different CP technical substances, 50% depuration was achieved after 2 to 10 h when D. magna were transferred to clean media. Our results show that SC, MC, and LCCPs are (very)bioaccumulative in aquatic organisms. We believe these data can aid the ongoing policy discussion concerning the environmental risk posed by CPs.

Cellulose nanofibril (CNF)-based materials are increasingly used in industrial and commercial applications. However, the impacts of CNF on aquatic life are poorly understood, and there are concerns regarding their potential toxicity. Using a combination of standard ecotoxicological tests and feeding experiments, we assessed the effects of CNF exposure (0.206-20.6 mg/L) on the feeding (food uptake and gut residence time) and life-history traits (growth and reproduction) in the cladoceran Daphnia magna. No mortality was observed in a 48 h acute exposure at 2060 mg/L. Moreover, a 21-day exposure at low food and moderate CNF levels induced a stimulatory effect on growth, likely driven by increased filtration efficiency, and, possibly, partial assimilation of the CNF by the animals. However, at low food levels and the highest CNF concentrations, growth and reproduction were negatively affected. These responses were linked to caloric restriction caused by dilution of the food source, but not an obstruction of the alimentary canal. Finally, no apparent translocation of CNF past the alimentary canal was detected. We conclude that CNF displays a low toxic potential to filter-feeding organisms and the expected environmental risks are low.

Background: Freshwater sediments have been recognized as a long-term sink and potential source for environmental pollutants released into the aquatic ecosystems. In this study, the sediment quality of Taihu Lake, which is susceptible to anthropogenic contamination, was assessed by a combination of chemical analytical and biological end points. Specifically, the snail Bellamya aeruginosa was caged in situ at two locations representing different pollution levels for different exposure times (7, 14 and 21 days). At each of these time points, biochemical parameters, i.e., phase I biotransformation enzymes ethoxyresorufin-O-deethylase (EROD), the antioxidant enzymes superoxide dismutase and catalase, reactive oxygen species, protein carbonyl content and lipid peroxidation, were evaluated in the hepato-pancreas of snails. In addition, surface sediments were collected for analysis of contaminants of concern, including inorganic pollutants, organochlorine pesticides, polychlorinated biphenyls and polybrominated diphenyl ethers.

Results: Chemical analyses revealed that sediments from Taihu Lake were contaminated with trace elements and organic pollutants. Concentrations of trace elements (Cu, Ni and As) and organochlorinated pesticides (4,4'-DDE) exceeded their corresponding threshold effect level according to the sediment quality assessment values for freshwater ecosystems in Canada, indicating that adverse biological effects may occur. All biomarkers, except EROD activity, were induced in snails during all exposure times. The integrated biomarker response index (IBR) indicated that during the initial exposure phase (7 days), B. aeruginosa were subjected to significant environmental stress, which diminished during later sampling time points.

Conclusions: Results showed that IBR correlated well with the levels of environmental contaminants, demonstrating the applicability of this biomonitoring approach to complex environmental exposure scenarios.