Sediment quality has become a growing concern, as sediment-bound anthropogenic pollutants, particularly genotoxic compounds, may serve as secondary pollution sources, posing significant risks to aquatic ecosystems and human health. The in vitro micronucleus (MN) assay, standardized by ISO and OECD guidelines, is widely used for genotoxicity assessment; however, traditional manual MN scoring is labor-intensive, time-consuming, and susceptible to observer bias. Therefore, this study proposed a deep learning-based model for automated identification and quantification of cell nuclei and MN. Among the three trained models, the architecture incorporating hierarchical attention mechanisms, including self-attention and channel-spatial attention, was selected due to its superior segmentation performance. Compared with manual scoring, the model showed 95.63% (nuclei) and 97.38% (MN) agreement in Bland-Altman analysis, while achieving processing speeds approximately 20-fold higher per hour and 60-fold higher per day. Using the model, sediment genotoxicity from East Taihu and Yangcheng Lakes, two major freshwater systems and drinking water resources in China heavily impacted by human activities, was evaluated under both rat-S9 metabolically active and inactive conditions. Significant genotoxicity was observed, with minor discrepancies between manual and model counts, primarily in weakly genotoxic samples. Genotoxicity decreased following S9-activation, likely due to metabolic detoxification or inhibitory effects of co-existing substances. Regardless of metabolic activation, Yangcheng Lake sediments consistently exhibited higher genotoxic effects than those from East Taihu Lake. As a proof-of-concept application of deep learning in environmental genotoxicity assessment, the model architecture has been made publicly available to support high-throughput MN assay applications.

Understanding how organisms respond to chemical stress requires disentangling genetically encoded (constitutive) adaptations from environmentally induced (plastic) responses. This challenge is particularly acute for polycyclic aromatic hydrocarbons (PAHs), widespread aquatic pollutants with well-documented toxicity, where mechanisms of tolerance, including host-microbiome interactions, are unexplored. We used Daphnia magna, a keystone freshwater species with clonal reproduction and dormant egg banks to test population-specific (constitutive) responses to phenanthrene (PHE), a common PAH. Populations resurrected from contrasting historical environments were exposed to sub-lethal PHE concentrations, and both host transcriptomes and gut microbiomes were profiled to assess induced responses. Transcriptomic analysis revealed distinct, population-specific responses in detoxification, stress signalling, and endocrine regulation. Unexpectedly, the semi-pristine (pollution-naïve) population showed higher tolerance, with robust induction of cytochrome P450 and hormonal pathways, while populations historically exposed to pollution exhibited chronic stress signatures and reduced plasticity. Gut microbiome profiling revealed PHE-induced functional shifts across populations, with the pollution-naïve population showing broader stress-associated responses and historically exposed populations to pollutants exhibiting more detoxification-focused microbiome profiles. Both host and microbial datasets consistently showed enrichment in pyruvate and carbon metabolism, indicating coordinated energy mobilisation and detoxification responses. Our results show that historical exposure to chemical stress and wider pollution does not necessarily confer enhanced physiological tolerance to PHE. Instead, hydrocarbon stress elicits coordinated, functionally linked responses across the host and its associated microbiome. By leveraging Daphnia's unique ecology and evolutionary history, we disentangle constitutive from plastic responses and show that microbiome functional reconfiguration under PHE exposure is coordinated with host responses, contributing to population-specific profiles.

Anthropogenic stressors, such as pollution and climate change, are altering selective pressures on natural populations, but the evolutionary consequences of chronic exposure to complex mixtures of contaminants remain poorly understood. Addressing this knowledge gap is critical to the emerging field of evolutionary ecotoxicology, which aims to understand how long-term exposure to environmental contaminants shapes adaptive evolution and genome-wide variation. In this study, we used urban runoff sediment as complex and environmentally realistic model stressor to investigate how multigenerational exposure affects fitness and potentially drives genomic adaptation in the freshwater midge Chironomus riparius. We combined an evolutionary life-cycle test with the evolve and resequence approach, exposing replicate populations over seven generations to three treatments: a control and two concentrations of urban runoff sediment (0.5% and 10%). Key fitness traits, including mortality, mean emergence time, fertility, and population growth rate, were measured, and allele frequency changes were tracked to identify genomic signatures of selection. The results revealed distinct and nonlinear fitness responses across treatments, including transgenerational effects, recovery of performance, and evidence of life-history trade-offs. Candidate haplotypes were enriched for genes involved in membrane transport, metabolism, and gene regulation, suggesting selection on general stress-response pathways consistent with polygenic adaptation. Signals of selection were also detected in control populations, underscoring the evolutionary influence of laboratory conditions. Overall, our findings demonstrate how evolutionary ecotoxicology can reveal both the potential and the constraints of rapid adaptation to realistic environmental stressors and highlight the importance of integrating evolutionary perspectives into ecological risk assessment.

Climate change, pollution, and biodiversity loss constitute the Triple Planetary Crisis, eroding the ecological foundations of economies, public health, and human wellbeing. Yet among these threats, biodiversity remains the least understood and least integrated into policy and decision-making. Monitoring efforts are fragmented and often fail to capture how multiple pressures—chemical pollution, invasive species, habitat degradation, eutrophication, and climate extremes—interact to drive non-linear ecosystem decline. This gap leaves societies poorly equipped to anticipate and mitigate ecological risks.We outline an interdisciplinary framework that combines deep-time ecological and environmental records with emerging forecasting tools to reconstruct long-term baselines and predict complex biodiversity responses to interacting stressors. We further identify pathways to embed these diagnostics into economic and governance systems, linking biodiversity directly to risk assessment, investment decisions, and regulatory frameworks. Together, these advances point toward a decision-support platform that enables governments, businesses, and communities to anticipate risks, evaluate interventions, and design strategies that align biodiversity protection with resilience in economies and societies

6PPD-quinone (6PPD-Q), a transformation product of the tire antioxidant 6PPD, is among the most acutely toxic chemicals to certain salmonids. Since its 2020 identification, it has been implicated in coho die-offs in the U.S. Pacific Northwest and prompted regulatory responses in North America. In Europe, however, awareness, monitoring, and risk assessment remain limited despite dense road networks, urban runoff, and sensitive fish communities. This Perspective synthesizes current knowledge and key gaps in occurrence, exposure, and species-specific toxicity, and outlines a tiered response: harmonized water screening, targeted tests in native taxa, interim stormwater mitigation, and innovation toward safer tire additives.

Effect-based methods (EBMs) may be included in the European Water Framework Directive (WFD) to evaluate estrogenic substances. The European Commission's Joint Research Centre conducted an interlaboratory study to assess estrogenic EBMs and effect-based trigger (EBTs) values derived using three options: (1) linking the EBT value to environmental quality standards (EQS), (2) correlating in vitro and in vivo data, and (3) averaging bioassay-specific EBT values. Surface water samples from eight Northern-Italian sites containing estrogenic hormones and endocrine-disrupting chemicals (EDCs) were analysed by fourteen laboratories employing EBMs, while four laboratories performed chemical analysis. Chemical data indicated cumulative risk in several samples, with estrone and bisphenol A identified as main drivers. All EBMs detected estrogenic activity, but specificity differed: some bioassays responded mainly to hormones, whereas others also responded to non-hormonal EDCs. EBMs flagged estrogenic risk in a sample that showed no individual exceedances of EQS. Applying EBT option 1 yielded the highest concordance with chemical results, achieving full compliance in eight bioassays and proven to be the most protective. Indeed, option 2 reduced the risk quotient (RQ) by > 30%, leading to two bioassays in full compliance, while Option 3 resulted in RQ changes (<20%) for most EBMs, with seven bioassays in full compliance with chemical analysis. The study underscores the need to harmonise EBMs - including data evaluation - to address chemical mixtures and provides recommendations for Member States on their application in the WFD. Integrating EBMs with conventional monitoring enhances protection against cumulative estrogenic risks from both hormones and EDCs.

Organic farming, defined by rigorous adherence to ecological principles and the prohibition of synthetic inputs, has expanded rapidly in response to consumer demand for environmental sustainability and food safety. However, the integrity of this sector, here defined along four interrelated dimensions of chemical purity, certification compliance, consumer trust, and economic viability, is increasingly threatened by the recurring detection of synthetic pesticide residues in certified organic produce. This disjunction between regulatory standards and chemical reality erodes the "zero-tolerance" expectation held by consumers (a marketing and perceptual construct rather than a regulatory one) and poses a serious risk to the credibility of organic labeling. Atmospheric pesticide drift from adjacent conventional operations is identified as a major contamination pathway. Through spray-droplet transport, volatilization, and long-range particulate-bound deposition, pesticides can travel considerable distances before depositing on non-target organic crops, a process that can be quantitatively described by regulatory drift models (e.g., AgDRIFT, AGDISP) and by atmospheric boundary-layer physics. This phenomenon converts environmental contamination into a governance and equity problem, as organic producers, and especially smallholders in low-and middle-income countries, disproportionately bear the financial and reputational costs of pollution generated externally, exposing a critical deficiency in current regulatory frameworks. These dynamics create a fundamental tension around the long-term viability of organic farming in chemically intensive landscapes. This review synthesizes current research on airborne pesticides as a primary contamination pathway, systematically distinguishing drift-driven contamination from legacy soil pollution and supply-chain contamination, and examines how atmospheric conditions, landscape configuration, regulatory architecture, and global trade flows intersect to jeopardize the authenticity of the organic supply chain. It further evaluates, on the basis of quantitative evidence where available, the effectiveness, cost, and feasibility of drift-mitigation strategies.

Endocrine disrupting compounds (EDCs) are substances that can interfere with hormonal systems, posing risks to wildlife and human health. For wildlife, a substance must exhibit endocrine activity at a relevant target (receptor or enzyme) and, as a direct consequence, cause population-relevant adverse effects in the exposed organism or its offspring to be classified as an EDC. Current regulatory frameworks utilize various in vivo assays to identify potential EDCs related to estrogenic, androgenic and steroidogenesis (EAS) modalities. However, ethical concerns have generated increasing momentum to align regulatory approaches with the 3Rs principle (Refinement, Reduction, Replacement) to reduce dependency on animal-based test systems. This study explores the potential of transcriptomics in non-transgenic fish embryos as a 3R-aligned complementary screening approach and proposes a conceptual framework for the identification of endocrine activity across all three EAS modalities. We compiled an overview of known gene networks associated with EAS modalities and mapped publicly available literature data on gene expression and functionality during early embryonic development. We found that during the first 96 h post fertilization, zebrafish embryos naturally express many key genes coding for enzymes and receptors involved in sex hormone biosynthesis, estrogen and androgen receptor signaling pathways. Additionally, zebrafish embryos express genes in EAS-associated pathways, namely mineralocorticoid, glucocorticoid, progesterone and glucuronosylation pathways. While available transcriptomic and functional data suggests that fish embryos could in principle generate transcriptional responses indicative of endocrine activity, the currently available information is insufficient to identify robust, specific and mechanistically informed biomarkers associated with endocrine activity in general or specific for EAS modalities. To resolve this, we propose a systematic empirical assessment of transcriptomic responses to additional endocrine-active substances of EAS modalities.

Food contact materials may contain thousands of chemicals that can migrate into food and be absorbed by consumers. Current target analysis overlooks unknown hazardous substances without any toxicological data. Thus, non-target effect-directed strategies were developed and applied to six migrates for potentially adverse effects on human health. Four coffee-to-go cups and two meal-to-go boxes were analyzed for endocrine-active, cytotoxic, genotoxic, and dioxin-like migrants by in vitro and planar bioassays. Cytotoxic, genotoxic and endocrine-active substances migrated from the polyethylene-coated cellulose-based coffee-to-go cups, and additionally, dioxin-like, genotoxic and estrogenic substances from the cellulose-based meal-to-go boxes. Via the S9 liver enzyme system, both metabolic activation and deactivation were observed. High-performance thin-layer chromatography coupled with high-resolution mass spectrometry, followed by co-analysis of candidates, identified five prioritized compound zones to be the estrogenic 2,5-dihydroxycyclohexa-2,5-diene-1,4-dione, 4-octylphenol, and dibutyl phthalate, cytotoxic (hydroxyethyl) methacrylate, and genotoxic 1,6-hexanediol dimethacrylate. Two detected perfluorotelomers were of low concern.

Microplastics are recognised as terrestrial pollutants, but their sublethal effects on soil fauna remain poorly understood. We examined biochemical responses to three environmentally relevant microplastic types in two Collembola species, Folsomia candida and Sinella curviseta. Juveniles were exposed for 28 days in standardised soil to 0-10,000 mg kg-1 of polystyrene-HBCD, car tyre abrasion or a starch-based bioplastic blend. Analysed biomarker responses included acetylcholinesterase, carboxylesterase, catalase, and glutathione S-transferase activities; glutathione, reactive oxygen species and carbohydrate concentrations. Multivariate analysis showed that Species explained 84% of biomarker variation, whereas Type and Concentration had weak but significant effects. Car tyre particles induced the strongest glutathione S-transferase, glutathione, and reactive oxygen species responses in both species. Polystyrene-HBCD stimulated detoxification responses, while the starch blend caused weaker changes. Concentration effects were significant but non-linear and species-specific. These findings demonstrate species- and polymer-specific responses and support multivariate biomarkers as indicators of stress in soil invertebrates.

Organophosphate esters (OPEs) can be biodegraded through phase Ⅰ process in liver by cytochrome P450 enzymes and cause adverse effects to target organs. This study examined the transformation of three OPEs using electrochemistry (EC) and rat liver S9 system to compare the distinctions between the two systems concerning their transformation products (TPs) formed. The toxic effects of OPEs and the primary TPs include endocrine disruption, genotoxicity and dioxin-like potential were further investigated across a range of exposure concentrations that do not induce significant cytotoxicity. OPEs were transformed in both EC and S9 with the degradation degree followed as tris(4-isopropylphenyl) phosphate (T4IPPP)> tris(2-butoxyethyl) phosphate (TBOEP)> tris(1,3-dichloro-2-propyl) phosphate (TDCIPP). Compared with S9 system, the transformation of OPEs in EC method was relatively slower. Bis(butoxyethyl) phosphate (BBOEP), hydroxyethyl phosphate triester (BBOEHEP) and bis(2-butoxyethyl) 2-(3-hydroxybutoxy) ethyl phosphate triester (3-OH-TBOEP) were the most abundant TPs of TBOEP, while bis(1,3-dichloro-2-propyl) phosphate (BDCIPP) was the only TPs of TDCIPP detected in EC and S9 system. Concerning the toxic effects, OPEs declined the cell viability significantly in H4ⅡE, A549 and U2OS cells in time- and concentration- specific relationship. However, no significant cytotoxic effect occurred after exposure to the TPs up to 1000 μM. TDCIPP showed significant antagonistic activities against ERα and AR with IC10 value of 42.4 and 0.301 μM, respectively. BBOEHEP and BDCIPP also exhibited ERα agonistic activity and antagonistic AR activity, which was lower than that of their respective parent compounds. Furthermore, OPEs and their TPs caused significant micronucleus formation in the absence or presence of S9 fraction compared to the solvent control. Taken together, TDCIPP pose considerable risks to humans and the environment considering the limited transform capability and strong toxic effects.

Many rivers and streams are affected by chemical pollution, yet current chemical monitoring methods are limited for technical and economic reasons. Biomonitoring has been increasingly used as a means of assessing the impacts of chemical pollution and indirectly monitoring river contamination. To provide an overview of the wide variety of biomonitoring approaches, we conducted a systematic review of the literature investigating the relationship between chemical pollution and biological responses. We distinguished five main approaches for the biomonitoring of chemical pollution: (i) monitoring of native communities; (ii) laboratory bioassays; (iii) in situ bioassays; (iv) mesocosms; and (v) monitoring of wild populations. Although each of them covers a wide range of methods and endpoints, we have highlighted their main advantages and limitations. Because native communities are exposed to a wide range of stressors, isolating the effects of chemical pollution alone is often limited. Most of the existing community indices cannot depict the full extent of the impact of pollutants on communities, but rather provide information on either general degradation of water and/or sediment quality. Effect-based methods (EBMs), including ecotoxicological bioassays and biomarkers, can better isolate the effects of pollution, and, to some extent, of specific types of pollutants. The experimental design of EBMs must be adapted to the research question and the context of the study, so that the test organisms, exposure scenarios and endpoints accurately reflect the contamination. In this context, a comparison with theoretically non-stressful situations with either a dilution series of the exposure solution in the laboratory or a comparison of laboratory and field treatment is relevant. The main difficulties encountered in the approaches investigated in the present review are the comparability of sampling strategies, non-linear concentration–response relationships, extrapolation from laboratory to field exposure, the highly variable sensitivity of organisms and the geographical specificities. Overall, a combination of different EBMs can integrate the effects of exposure to specific contaminants at both spatial and temporal scales while accounting for confounding factors. The establishment of thresholds and guidelines would facilitate the integration of EBMs into regular monitoring programmes. This in turn will greatly facilitate the assessment of chemical impairment.

Outdoor workers who are exposed to traffic-derived pollutants often suffer from a range of diseases, with liver disease being particularly notable. Recently, a rubber stabilizing additive antioxidant N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and its transformed-quinone product 6PPD-quinone (6PPD-Q) attracted attention. However, their implication for human health remains inadequately elucidated. In this study, outdoor and indoor workers were recruited to analyze 6PPD and 6PPD-Q distribution in their serum and urine. Simultaneously, blood cell counts, liver function, renal function, blood glucose level, and lipid profile were evaluated by 23 physiological parameters. For the first time, we found that the concentrations of 6PPD (0.54 - 1.66 μg L-1) and 6PPD-Q (0.58 - 4.04 μg L-1) in outdoor group serum were two- and three-fold in the indoor group, respectively. Compared with indoor workers, 18 biochemical parameters, notably total bilirubin and indirect bilirubin, were elevated in outdoor workers (p < 0.05). A computed tomography scan showed liver lesions in 60% of the outdoor group, whereas only 30% of the indoor group. The statistical analysis exhibited that significant positive correlations exist between the serum 6PPD-Q and immune cell counts, total bilirubin, indirect bilirubin, and triglycerides in human beings (p < 0.05). The logistic regression implied that for each 1 μg L-1 increase of 6PPD-Q in serum, the risk of human liver lesions increased by 2.31 times. Our results suggest that outdoor exposure is associated with increased concentrations of 6PPD-Q in serum, which could potentially influence glucose and lipid metabolism, immune cell regulation, and liver health.

Thiamethoxam (THM) is one of the most widely used insecticides globally, which was designed to selectively target nicotinic acetylcholine receptors (nAChRs) in the insect nervous system and is generally considered safe for non-targeted organisms. However, increasing evidence has demonstrated its neurotoxicity in aquatic organisms, though the underlying mechanisms, especially at environmentally relevant concentrations, remain largely unclear. In this study, the swimming distance of zebrafish was significantly shortened by 14.06%-21.64% after exposure to THM at 10-1000 ng/L. This behavioral impairment may result from the damage to nervous and visual systems, as confirmed by notable apoptosis, histological analysis of the eyes, and differential expression of numerous genes. Molecular docking and biomarkers assays found that THM can bind with nAChR and multiple hormone receptors, with binding energies varying from -3.75 to -6.74 kcal/mol. Consequently, the concentrations of a neurotransmitter (acetylcholine) and related hormones (cortisol, triiodothyronine, thyroxine, and thyroid-stimulating hormone) were significantly affected. Further investigations using a weighted gene correlation network and metabolomics suggest that THM may enter the cell via endocytosis and bind with multiple hormone receptors, potentially activating the MAPK signaling pathway. This activation may disrupt purine and pyrimidine metabolism in the cell nucleus, ultimately leading to cell apoptosis and neurotoxicity. This study reveals that THM, even at environmentally relevant concentrations, poses neurological risks to zebrafish and underscore the need for urgent attention to the ecological impacts of THM in aquatic environments.

Microplastic pollution and its environmental consequences have been a research topic for decades. Alongside sewage sludge and compost, mulch films are one of the most important sources of organic matter in agricultural soils. As microplastics accumulate in these environments, they increasingly interact with other agrochemical contaminants, such as pesticides. The consequences of the combined presence of mulch film microplastic and pesticides, especially on bioavailability and toxicity, still need to be understood. We investigated the interaction of pesticides with polyethylene mulch films by choosing the neonicotinoid Thiacloprid and the fungicide Tebuconazole in their commercial formulations as plant protection products, serving as representative hydrophobic and hydrophilic substances. The impact of PE microplastic (150 mg/kg) on both pesticides' extractability and earthworm mortality was determined. The hypothesis is that microplastics reduce pesticide bioavailability in soil. Hence, reduced earthworm toxicity might be observed. Acute toxicity testing with Eisenia fetida showed a slight decrease in toxicity while MPP was present, shifting the LC50 from 110.3 to 120.6 mg/kg (Tebuconazole) and 20.3-22.6 mg/kg (Thiacloprid). The chemical analysis after two-time extraction of test soil with CaCl2 and then solvents confirmed the reduced availability of pesticide in the presence of MPP. Our research shows that microplastic at application-relevant concentrations can retain pesticides and reduce pesticide toxicity. These findings impact pesticide efficiency in soils that accumulate microplastic, potentially requiring the adaptation of agricultural practices. This study underscores the need for further research and mitigation strategies as our understanding of the interactions between pesticides and microplastics in soil evolves.

Abstract Background This article provides an overview of the iMulch joint project, which analysed the use of polyethylene (PE) and biodegradable mulch films made of a polylactide (PLA) and polybutylene adipate terephthalate (PBAT) on agricultural land as a source of microplastic. The development of a detection methodology using Raman spectroscopy and thermo-extraction desorption gas chromatography mass–spectrometry (TED-GC–MS), the adsorption behaviour, ageing in drainage water and soil, their transport behaviour in lysimeters, ecotoxicity, uptake in plants, a life cycle assessment (LCA) and upcycling were considered. Results The PE film tested showed hardly any degradation or fragmentation during the ageing tests. The biodegradable films showed incipient degradation after 8 weeks in drainage water and initial degradation after 12 weeks in soil ageing experiments. Additionally no degradation could be detected in the lysimeter test within the 24 months analysed. The biodegradable films could be metabolized in laboratory tests with some microorganisms present in the soil. This indicates that these films can be degraded in the environment if the conditions for degradation are optimal. No microorganisms or fungi that could degrade the PE film within a respective period of time were detected in the soil. Adsorption of the tested substances was not observed. Incorporated in soil, mulch film microplastic showed retention of extractable pesticides. In the ecotoxicological tests, both film types showed no acute toxic effects in the earthworm Eisenia fetida and the springtail Folsomia candida. Endocrine activity was observed in eluate samples from both films. However, aged films showed fewer effects than non-aged films. Conclusion Both types of film show no transport or degradation in the tests under real conditions, which means that they remain in the upper soil layer, where they are available to soil organisms and can lead to high concentrations in the future. As the biodegradable film could be degraded, at least under ideal conditions, we recommend its use. However, proof of degradation must first be verified under real field conditions. In addition, we recommend the use of thicker conventional mulch films to minimize the emission of plastic particles. For this purpose, a minimum lower limit for the material thickness should be defined. Graphical Abstract

Azoles control fungal growth by inhibiting sterol biosynthesis in fungi according to the fungicide resistance action committee. Furthermore, previous studies have highlighted several effects of azole fungicides in fish including endocrine disruption. In this study, we analysed the transcriptome responses of zebrafish embryos exposed to azole fungicides to identify gene expression fingerprints indicating toxic effects such as endocrine disruption induced by sterol biosynthesis inhibition. Firstly, a modified zebrafish embryo toxicity test was conducted following the OECD 236 guideline, exposing embryos to difenoconazole, epoxiconazole, and tebuconazole. After 96 h, RNA was extracted for transcriptome analysis, which revealed concentration-dependent responses for each fungicide. Additionally, overrepresentation analysis of significantly differentially expressed genes revealed biological functions related to sterol biosynthesis and endocrine disruption. A gene set with specific expression patterns was was identified as molecular signature for indicating adverse effects induced by sterol biosynthesis inhibitors in zebrafish embryos. After further validation, the gene expression fingerprints and biomarkers identified in this study may be used in the future to identify endocrine activity of substances under development in a pre-regulatory screening using the zebrafish embryo model.

Nitrogen pollution represents one of the most significant threats to European freshwater ecosystems, with nitrite (NO2-N) standing out as a highly toxic compound for aquatic organisms, particularly vertebrates. Despite its recognized toxicity, little is known about its effects on invertebrates, even as riverine ecosystems experience profound species turnover. Here, we investigated the lethal and sublethal effects of nitrite on three representative amphipod species (Gammarus fossarum, G. pulex, and G. roeselii), which occupy distinct river sections and ecological niches. These species serve as models for assessing how nitrogen pollution may shape invertebrate communities across freshwater habitats. A series of laboratory bioassays revealed that G. fossarum, a species associated with upstream sections and pristine conditions, was the most sensitive to nitrite exposure, followed by the midstream species G. pulex and the long-established downstream species G. roeselii. To contextualize these findings, we compared the nitrite vulnerability of these amphipods with that of other freshwater invertebrates, offering a comprehensive perspective on how nitrogen pollution reshapes aquatic communities. While many invertebrate groups exhibit lower vulnerability to nitrite due to their reliance on hemocyanin—an oxygen-transport molecule mostly unaffected by nitrite oxidation—our results underscore significant interspecific differences in tolerance. For sensitive insect species, lethal effects occurred already at environmentally relevant concentrations, highlighting their exceptional vulnerability. In contrast, more tolerant groups such as amphipods survived higher concentrations, yet still displayed sublethal impairments, most notably a reduced leaf litter consumption—a key process in stream nutrient cycling—and altered behavioral responses at comparable exposure levels. Molluscs exhibit the highest tolerance, whereas insects are the most sensitive. Among crustaceans, tolerance varies widely, with a relationship to chloride content of the water mitigating the toxicity of nitrite. Chloride concentrations generally rise along the course of a river, placing upstream regions with naturally low chloride levels and their species at heightened risk. These differences highlight the potential role of nitrogen pollution as a driver of species turnover, particularly in multistressor environments. By linking species-specific sensitivity to broader ecological processes, like leaf litter consumption, this study provides critical insights into cascading effects of nitrogen pollution on freshwater biodiversity and ecosystem stability.