Science of The Total Environment

Subalpine forests in the Alps are fragile ecosystems that play a crucial role in regional water resources and the local climate. These ecosystems are ecologically significant due to their unique biodiversity and vulnerability to climate change. While several components of the hydrological balance have been studied, the interplay between catchment-scale processes and plot-scale drivers such as fog presence and forest age remains insufficiently understood. To address this, we investigated the hydrological balance of a subalpine coniferous forest catchment at the Renon site in the Italian Alps, integrating observations across spatial scales. The study area includes a mosaic of mature and younger regrowth forest, where both interannual and seasonal variability in precipitation and fog presence are pronounced. At the catchment scale, we quantified above-canopy precipitation, evapotranspiration (ET, measured via eddy covariance at the ICOS tower), stream discharge, and soil moisture dynamics. Within the catchment, we characterised water partitioning using sap flow sensors for tree transpiration, throughfall and stemflow collectors with rain gauges above and below the canopy and epiphyte sampling. Mixed fog-rain events frequently coincided with higher throughfall. However, these changes had a minor effect on soil water storage and catchment discharge in the annual water balance, which was nearly closed. At the plot scale, our results show that tree transpiration was higher in the younger forest structure, while canopy interception is a dominant process in water partitioning in the older forest structure, where lichen abundance likely enhances interception. This study highlights the importance of multi-scale monitoring in temperate mountain forests, where forest age influences water partitioning, and fog presence, though not directly quantified, can still contribute to reducing evaporative processes. Such contributions may gain importance under changing climate conditions, albeit less prominently than in tropical or subtropical cloud forests.

Background Following the end of a public health emergency of international concern, divergence emerged between reported coronavirus disease 2019 (COVID-19) cases and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentrations in wastewater. Exploring viral, clinical, patient, and surveillance-related factors underlying this divergence, we developed models to predict clinically confirmed infections, hospitalizations, and severe cases. Methods In this observational study, we analyzed ~2 years of data from January 2022 in Kanagawa Prefecture, Japan, assessing associations between wastewater SARS-CoV-2 RNA concentrations and confirmed, hospitalized, and severe cases, adjusting for wave and variant effects. Findings Our models based on wastewater viral RNA concentrations showed high predictive accuracy (R^2 = 0.8199-0.9961), closely tracking confirmed, hospitalized, and severe cases. Models derived from earlier waves were applied to subsequent waves with residual correction based on prior prediction errors and maintained good predictive performance (root mean square error = 0.0665-0.2065). Divergence between wastewater viral RNA concentrations and reported cases was not explained by changes in viral shedding. Declines in patients' healthcare-seeking behavior and testing were associated with trends in confirmed cases, whereas milder clinical presentation was associated with severe case trends. The lineages XBB.1.9.2 and BA.2.86 were identified as candidates associated with reduced virulence. Interpretation By incorporating understanding of viral, clinical, and surveillance-related mechanisms, wastewater surveillance may enable prediction of case trends approximately one week earlier than official reporting and inform healthcare capacity planning.

Microplastics (MP) are ubiquitous environmental contaminants with diverse physicochemical characteristics. Many studies have shown that size, shape, and polymer type are responsible for their toxicity, but this also seems to differ among MP from the same plastic type. One parameter likely contributing to these differences is plastic chemicals, a broad class of compounds intentionally or unintentionally added to plastics during their production and manufacturing. However, knowledge on the composition of plastic chemicals and their effects remains scarce. Therefore, to elucidate the chemical aspect of MP toxicity, we exposed Daphnia magna individuals to MP (PET, PBS, and PDLLA), cellulose, extracted particles (eMP), and methanol-based extracts of these particles for 10 days. Chemicals within such extracts were analyzed via GC-MS. This study was conducted with reduced food availability to investigate plastic effects in an environmentally relevant scenario. The introduction of a high-food control suggests that a more realistic feeding regime might exacerbate the plastic effects of the selected treatments. Our results indicated that, depending on the polymer type, plastic chemicals determine MP toxicity, which varies according to the endpoint investigated (i.e., body length, reproduction, levels of ROS and LPO). Body length, in particular, was significantly impaired by PET and PDLLA extracts, whereas reproduction was affected by most treatments. The investigated biochemical parameters (ROS and LPO) were not affected by the exposure. These results suggest that MP toxicity strongly depends on their chemical composition, whereas adverse effects due to physical properties are present independently of chemical composition across all MP types. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=80 SRC="FIGDIR/small/724551v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@3c2d4forg.highwire.dtl.DTLVardef@c2ccd7org.highwire.dtl.DTLVardef@116721dorg.highwire.dtl.DTLVardef@9df888_HPS_FORMAT_FIGEXP M_FIG C_FIG

Although natural forests sequester carbon, this function may decline under chronic herbivory by abundant ungulates (hereafter overbrowsing). Specifically, overbrowsing alters stand structure, potentially impair carbon exchanges related to the vegetation. Further, overbrowsing may also accelerate soil erosion, especially in heavy-rainfall regions like Monsoon Asia. We quantified these impacts by estimating net ecosystem carbon balance (NECB; g C m-2 yr-1) by subtracting heterotrophic respiration (Rh) and lateral carbon export via erosion (Se) from net primary production (Pn) in southern Kyushu, Japan. Here, about 40-years of overbrowsing by sika deer (Cervus nippon) altered mixed broadleaf-conifer stands with presence of understory (PU) into stands with no understory (NU), then further altered into stands dominated by unpalatable shrublands (SR) or stands with canopy gaps (CG). The PU maintained a positive NECB (plot mean = 307.0 g C m-2 yr-1) because high Pn (721.9) exceeded the sum of Rh (175.4) and Se (239.5). Alteration from PU into NU converted NECB to negative (-98.2 g C m-2 yr-1). This was because the suppressed Pn (400.2 g C m-2 yr-1) could not offset the sum of Rh (170.6) and Se (327.7). Further degradation into CG caused a profound negative NECB (-894.4 g C m-2 yr-1), where Pn (71.9) offset only 7% of the sum of surging Rh (464.8) and Se (501.5). Alteration into SR showed a partially recovered NECB (97.3 g C m-2 yr-1), driven by shrub growth (Pn; 554.5, Rh; 175.4, Se; 239.5). However, this recovery is still limited given that lowered shrub biomass and prior topsoil loss via erosion. Our results validate previous findings that stand alteration from PU to SR or CG through NU leads to up to a 49% loss of ecosystem carbon stocks. Preventing stand alteration and soil erosion are key countermeasures against chronic overbrowsing and subsequent erosion. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/719128v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@167aef0org.highwire.dtl.DTLVardef@e45a6org.highwire.dtl.DTLVardef@fec89borg.highwire.dtl.DTLVardef@1246bea_HPS_FORMAT_FIGEXP M_FIG C_FIG We reported that over 40 years of sika deer overbrowsing and subsequent soil erosion severely degraded the net ecosystem carbon balance (NECB) of mountain forests in Japan. The loss of understory vegetation drove the transition of intact stands into degraded states (no-understory, shrub-dominated, or canopy gaps). Based on field measurements, we quantified that this structural alteration suppressed net primary production (Pn) while increased both heterotrophic respiration (Rh) and lateral carbon loss via soil erosion (Se). Consequently, the forest shifted from a net carbon sink (+307 g C m-{superscript 2} yr-{superscript 1}) to a source (up to -894 g C m-{superscript 2} yr-{superscript 1}). These findings provide compelling empirical evidence that increasing ungulate populations, compounded by the rising frequency of heavy rainfall, may severely undermine the carbon sequestration functions traditionally expected of natural forests.

The East Asian region, known for its high levels of human and fishery activities, experiences serious plastic pollution in the marine environment, especially in seawater and along coastlines. Wharf roaches (Ligia spp.) collected from the coast of western Japan frequently ingest expanded polystyrene (EPS), which is then excreted as microplastic through their feces. However, the impact of EPS exposure and ingestion on the gut microbiome of wharf roaches remains unclear. Thus, this study aimed to investigate the effects of EPS ingestion on the gut microbiota of wharf roaches by examining their gut microbiota and gene expression. The expression levels of more than 400 genes, including those associated with xenobiotic metabolism, and the abundance of gut microbial community were altered. Microbial analysis revealed that at least five archaeal types, two to four bacterial types, three to seven eukaryotic types, and three viral types were involved in a correlation network composed of strong associations. Among them, Haloquadratum, Halalkalicoccus, and Methanospirillum (archaea); Volvox (eukaryote); and Varicellovirus and T4-like viruses showed significantly increased abundance. Furthermore, covariance structure analysis indicated that the viruses and methanogens played key causal roles as characteristic factors related to EPS administration. In conclusion, EPS disrupts the intestinal environment of wharf roaches and serves as a potential material for viral activation and methane production. Building on our previous field study that identified wharf roaches as potential indicators of coastal EPS pollution, this study provides novel insights into the ecological impacts of EPS ingestion and consequences of plastic pollution.

The increasing discharge of treated wastewater effluent poses a growing threat to freshwater ecosystems. Although wastewater treatment plants reduce chemical pollution, they do not fully remove many biologically active compounds. Behavioural responses in aquatic organisms provide sensitive and ecologically relevant indicators of sublethal contaminant exposure, offering insight into underlying physiological disruption and potential ecological consequences. Here, we examined the behavioural and neurotoxic effects of a seven-day experimental exposure to treated wastewater effluent in the noble crayfish (Astacus astacus). We quantified four ecologically important behaviours: (1) shelter use, a key antipredator strategy, (2) food seeking, (3) the ability to detect and respond to wastewater-associated olfactory cues, and (4) locomotor activity was assessed across all behavioural contexts. Cholinesterase (ChE) activity was measured as a biomarker of neurotoxicity. Exposure to wastewater effluent significantly altered crayfish behaviour. Exposed individuals exhibited higher locomotor activity compared to controls, exposed crayfish avoided areas containing wastewater cues, spending less time near the effluent source. Similarly, ChE activity was significantly reduced in exposed crayfish, indicating neurotoxic effects. The concurrence of ChE inhibition and behavioural modification suggests that effluent-derived contaminants may interfere with neural signalling pathways underlying crayfish locomotion and habitat selection. Overall, our results demonstrate that short-term exposure to treated wastewater effluent can induce both neurochemical disruption and ecologically relevant behavioural changes in A. astacus. Such alterations may increase vulnerability to predation and influence population dynamics in effluent-receiving waters, highlighting the importance of integrating behavioural endpoints with mechanistic biomarkers in assessing sublethal impacts of wastewater contamination.

Since 1995, European fisheries of Pecten maximus have faced the presence of Pseudo-nitzschia species, which are able to produce the neurotoxin domoic acid responsible for Amnesic Shellfish Poisoning (ASP). As filter-feeders, scallops can accumulate and retain domoic acid much longer than most other bivalves, from months to years. When concentrations exceed the regulatory threshold, fisheries are closed leading to economic concern. Inter-individual variability increases the difficulty to predict the depuration dynamics. Quantifying the correlations between domoic acid depuration in P. maximus and individual physiological traits, particularly body size, could improve the understanding of contamination and depuration. We analysed toxin dynamics in organs and assessed the effects of body size and growth. This analysis was based on two datasets from an experimental and an in situ depuration monitoring of P. maximus exposed to a natural bloom of toxic P. australis. Results showed that the distribution of domoic acid shifted among organs between contamination and two months of depuration. Toxin concentrations correlated negatively with body size during contamination and after two months of depuration, but shifted to a positive correlation after 7 months of depuration. This suggested that smaller scallops both accumulate more domoic acid and depurate it more rapidly. Dilution by growth appeared to explain the inversion of the correlation between domoic acid and body size throughout depuration. These results yield useful information for modelling these mechanisms, thus providing valuable tools for scallop fishery management facing ASP. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=60 SRC="FIGDIR/small/708139v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1fd317org.highwire.dtl.DTLVardef@15b9032org.highwire.dtl.DTLVardef@57dae8org.highwire.dtl.DTLVardef@1e4c7fc_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIExperimental and in situ datasets allowed to quantify DA proportion dynamics in organs in P. maximus C_LIO_LIDA concentration and body size are negatively correlated during contamination phase, but positively after a 7-month depuration C_LIO_LIConsidering dilution by growth is important for young scallops to assess DA depuration dynamics C_LIO_LIBoth depuration rate and dilution by growth need to be considered to model DA depuration over the whole scallop size range C_LI

The prevalence of nitrogen limitation and nitrogen-phosphorus co-limitation (henceforth referred to as nitrogen-related limitation) in Norwegian lakes and their relationships with atmospheric nitrogen deposition, climate, dissolved organic matter (DOM), and catchment characteristics were assessed across space and time. Routine monitoring data from 1,529 lakes in the national Vannmiljo database were analyzed for two multi-year periods (1995-2009 and 2010-2025). Limitation was inferred using the molar NO--N/TP ratio as an indicator of dissolved inorganic nitrogen availability. Nitrogen-related limitation was widespread in both periods and exhibited strong regional structure, with highest prevalence in northern regions and lowest prevalence in southwestern Norway. Overall prevalence increased from 31% to 38% between periods, with significant increases in western regions. Regional-scale models identified climate, forest cover, DOM, agriculture, and atmospheric nitrogen deposition as predictors of limitation probability, whereas study period per se and bog/peatland cover were not significant. At the local scale, atmospheric nitrogen deposition and DOM were the only consistent predictors, with substantially lower explanatory power than at the regional scale. These results indicate that large-scale environmental gradients play a major role in shaping nutrient stoichiometry in Norwegian lakes. Because the monitoring dataset primarily represents lakes affected by human activities, the findings are particularly relevant for water management. The widespread occurrence of nitrogen-related limitation suggests that nitrogen availability may influence phytoplankton growth in many systems and that dual-nutrient management strategies addressing both nitrogen and phosphorus may be required in many regions.

The accumulation of microorganisms and macroorganisms on aquatic surfaces poses economic and ecological challenges, particularly in maritime transport. Traditional antifouling methods, such as biocidal coatings containing toxic compounds like tributyltin (TBT) and copper, are effective but harmful to the environment. This study investigates eco-friendly antifouling alternatives, focusing on nature-inspired compounds (NIAFs) GBA 26 (GBA) and DPC345DHC (DH345), derived from polyphenols and flavonoids, respectively. The ecotoxicity of these compounds was evaluated using standardized assays with various species, including embryos of Danio rerio (zebrafish) (OECD TG 236), the algae Raphidocelis subcapitata (OECD TG 201), and the bacteria Vibrio fischeri (ISO 11348-2), along with nuclear receptor transactivation assays in Mytilus galloprovincialis (Mediterranean mussel). Gallic acid derivative GBA and 24h-transformation products showed low toxicity in zebrafish embryos, while dihydrochalcone DH345 inflicted developmental toxicity in zebrafish at 1 mg/L and above. Comparatively, tralopyril, a commercial biocide, exhibited significant toxicity at lower concentrations. Transcriptomic analysis of zebrafish embryos treated with GBA revealed selective gene modulation related to stress response, ion transport, and protein synthesis. Both, GBA and DH345, were shown to inhibit algae growth at 0.1 mg/L. Vibrio fischeri assay showed no toxic effects for any of the tested compounds. Nuclear receptor transactivation assays conducted with GBA revealed no activation of PPAR or PXR receptors. These findings suggest GBA and DH345 as potential eco-friendly antifouling agents with lower environmental risks than established antifoulants such as tralopyril. However, further research is needed to evaluate their potential long-term ecological impacts, particularly chronic toxicity across various organisms. This study advances the pursuit of sustainable antifouling solutions that prioritize environmental protection.

To understand the utility of healthcare facility-level wastewater surveillance (WWS) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is important to correlate wastewater SARS-CoV-2 RNA detection with the number of clinical infections. WWS for SARS-CoV-2 was performed at three skilled nursing facilities (SNFs) over 25 weeks. Electronegative membrane filtration (enMF) and Nanotrap(R) Magnetic Virus Particles (NP) virus concentration methods were compared. Extracts were tested by droplet digital polymerase chain reaction. Spearman's correlations ({rho}) between wastewater virus RNA concentrations and infection counts were calculated. From split wastewater samples, enMF recovered higher SARS-CoV-2 RNA concentrations than NP. Combining data from all facilities, the median concentrations were 53.0 versus 38.6 gc/100 mL for enMF and NP, respectively (p=0.001). Using enMF, correlations were moderate to strong at SNF A ({rho} ranged 0.67 to 0.86, all p-values <0.001). Weak to moderate correlations can be explained by the sampled manhole not representing the entire facility (SNF B, {rho} ranged 0.47 to 0.72, p-values ranged <0.001 to 0.12) and longitudinal data gaps from summer heat and equipment maintenance (SNF C, {rho} ranged 0.14 to 0.59, p-values ranged 0.52 to <0.01). WWS can be a valuable tool for tracking dynamics of SARS-CoV-2 infections in healthcare facilities.

Chelating ion exchange resins are widely used to eliminate metal interferences in the analysis of ammonium (NH4+) in soil extraction solutions. However, their potential to co-adsorb NH4+ remains underexplored. Here, synthetic metal ion solutions containing 6-30 mg L-1 NH4+ and the metal cations Ca2+, Mg2+, Cu2+, Mn2+, and Zn2+ were treated with Amberlite IRC-748 resin. The resin efficiently removed Ca2+ (-42.2%), Mg2+ (-21.1%), Cu2+ (-99.9%), Mn2+ (-56.9%), and Zn2+ (-93.6%). However, NH4+ losses of 2.2-5.6% were observed, indicating concentration-dependent co-adsorption. While these losses may be acceptable for concentration measurements via routine assays such as photometric analysis, they may still affect the accuracy of high-precision N analyses that rely on quantitative NH4+ recovery. This highlights a methodological caveat for resin-treated samples, especially in low-NH4+ environments. We therefore recommend including recovery assessments and correction factors when using chelating resins to improve accuracy in NH4+ quantification.

While tree pollen is a major trigger of allergic respiratory conditions and different taxa exhibit varying allergenic potentials, the lack of high-resolution, taxon-specific exposure metrics have limited our ability to identify which local pollen taxa are primarily responsible for respiratory illness. Traditional pollen monitoring networks, which have an intermittent sampling schedule, are not ideal for examining the delayed effects of pollen exposure due to the missing days. In this study, we developed a modeling framework integrating atmospheric dispersion effects, taxa-specific phenology, and machine learning to predict daily counts of 13 tree taxa in the five-county Metro Atlanta area, Georgia at a 1-km resolution from 2020 to 2024. Machine learning model performance was validated with daily pollen counts collected by a multi-site monitoring network equipped with automated pollen sensors. Findings showed that Betula and Quercus pollens exhibited higher predictive performance than other taxa, with R2 values ranging from 0.69 to 0.92 and from 0.71 to 0.89, respectively. Our 1-kilometer prediction data provides gapless exposure metrics to understand the spatial and temporal variability in pollen exposure, can facilitate investigation of urban pollen hotspots and support epidemiologic studies of pollen-related respiratory outcomes.

The release and management of pheasants (Phasianus colchicus) in the UK for recreational shooting exerts a range of effects on the ecosystem into which they are released. We studied possible effect of nutrient deposition on epiphytic tree flora at 20 pheasant release sites distributed through England (18) and Wales (2) during winter and spring 2023/24. Sites were all Ancient Semi-natural Woodlands (ASNWs) and had substantial (600-8000 pheasants) in a single release pen. We measured N-sensitive and N-tolerant indicator bryophyte and lichen species on tree trunks near to the pen and then in plots along a transect 100m, 250m, 500m and 1km+ away from the pen. To achieve a gradient of pheasant use, the transects were located in the opposite direction to the game managed / shooting area. We recorded 1.9 times more coverage of N-tolerant lichens and bryophytes combined on selected tree species at the pen-edge compared to the control plots. The relationship showed a decline from the pen edge to 250m away but then stabilised. We also detected higher levels of coverage of N-sensitive tree flora at 100m and 250 m compared to the penedge plot. These measures were also higher at these mid distances compared to the 500m and 1000m plots. We suggest far plots were nearer wood edges and were affected by ambient inputs of aerial N from farmland and other external sources. The overall interpretation is that concentrations of pheasants in and around release pens for several months from late summer until early winter in ASNWs does affect the balance of N-sensitive and tolerant tree flora up to potentially 250m and this is a consideration when locating release pens in and near to sensitive woods.

BackgroundContinuous, non-invasive viral surveillance is essential to monitor emerging pathogens and guide public health responses. Most environmental surveillance studies use targeted qPCR approaches, and comparisons between wastewater and indoor air surveillance remain limited. We aimed to compare the utility of emergency department indoor air and urban wastewater for tracking circulating viruses and resolving genomic information. MethodsWe conducted a matched-pair study comparing 19 weekly indoor air samples from the central ventilation exhaust shaft of an emergency department and 19 24-hour composite municipal wastewater samples in Leuven, Belgium, from December 2024 to April 2025. Both sample sets were processed using probe-based hybrid-capture viral metagenomics targeting over 3000 viral species, using influenza A as a clinically relevant test case. FindingsWastewater captured higher overall viral diversity (233 versus 106 species) and more complete genomes compared to indoor air, showing a relatively stable composition, mainly of enteric and animal-associated viruses. Indoor air demonstrated lower overall diversity but was enriched for respiratory viruses, including influenza A, coronaviruses, metapneumovirus, and respiratory syncytial virus, and more frequently achieved high genome coverage for these pathogens. Although both sample types permitted influenza A subtype characterization, influenza A genomes from wastewater were often less well covered. When coverage thresholds were met, indoor air supported targeted antiviral resistance-site screening for influenza A and RSV-A. InterpretationWastewater and indoor air generate distinct but complementary viromes. Wastewater acts as a diverse, population-level monitor for One-Health applications, whereas indoor air serves as a targeted, human-centric sentinel system facilitating further genomic characterization for respiratory viruses. FundingMustafa Karatas is supported by a Research Foundation Flanders (FWO) fundamental research scholarship (number: 11P7I24N). C.G., L.C., E.H., S.G. and E.A. acknowledge support from the DURABLE project. The DURABLE project has been co-funded by the European Union, under the EU4Health Programme (EU4H), project no. 101102733. Research in context Evidence before this studyWe searched PubMed for studies published between Jan 2000 and March 2024 using the terms "wastewater surveillance", "metagenomics", "indoor air", and "viral metagenomics". Previous studies have shown that wastewater surveillance can detect population-level viral circulation, and more recent work has explored indoor air sampling as a method for monitoring respiratory virus transmission. However, environmental metagenomic studies have largely examined these two sample types separately. Furthermore, most studies relied on untargeted sequencing approaches, which often yield fragmented genomes in these environments. To date, no study has systematically compared indoor air and wastewater using a comprehensive hybrid-capture viral metagenomics approach for virus surveillance. Added value of this studyWe conducted a matched comparison of indoor air from a hospital emergency department and municipal wastewater collected during the same weeks in Leuven, Belgium. We analyzed both sample types using an identical hybrid-capture viral metagenomics workflow targeting more than 3000 viral species. This design enabled a direct evaluation of how the two environmental surveillance lenses differ in viral diversity, genomic recovery, and epidemiological relevance. Wastewater captured broader viral diversity and a stable background dominated by enteric and animal-associated viruses, whereas indoor air captured more respiratory viruses and more frequently yielded high genome completeness for these pathogens. When genome coverage thresholds were met, indoor air data enabled influenza subtype identification and screening for antiviral resistance markers. Implications of all the available evidenceOur findings support a layered environmental surveillance strategy in which different environmental samples provide complementary information. Wastewater offers a stable, population-level view of viral circulation and captures broad viral diversity, including human and animal-associated viruses. Indoor air sampling in human-dominated settings provides a more direct signal of respiratory virus circulation and can yield genomes suitable for subtype and mutation-level characterization. Combining these approaches could strengthen metagenomic surveillance frameworks by improving the interpretation of environmental viral signals, supporting early detection of emerging pathogens, and helping distinguish human virus circulation from environmental or animal-derived detections.

Quaternary ammonium compounds (QACs) are high production volume biocidal compounds increasingly scrutinized for their potential to promote antimicrobial resistance spread. This study compared the release of QACs, QAC resistance indicator genes (qacE/qacE{Delta}1), and QAC tolerant bacteria from livestock and human waste streams into the environment. Five livestock farms with on-farm biogas plants (BGPs), a rural and an urban municipal wastewater treatment plant (WWTP) were studied in parallel. In WWTPs, <1% of incoming QACs were discharged with treated wastewater but 10-20% were transferred to sewage sludge. QAC concentrations in sewage sludge far exceeded those in raw and digested manure. The qacE/qacE{Delta}1 genes were detected in all samples with a higher relative abundance in solid than liquid samples. Relative abundances of QAC tolerant fast growing heterotrophic bacteria cultivated under high nutrient conditions at 37{degrees}C were higher in human than livestock waste streams. Providencia and Pseudomonas dominated the cultivated QAC tolerant bacteria in both systems but showed higher QAC tolerance when originating from human waste streams. Additionally, Enterobacteriaceae with higher QAC tolerance were cultivated from human waste streams. Most QAC tolerant strains carried antibiotic resistances without strong system differences. Only few strains carried the qacE/qacE{Delta}1 gene indicating that other mechanisms must be responsible for the increased QAC tolerance. In conclusion, QACs, qacE/qacE{Delta}1, and viable QAC tolerant bacteria including potential pathogenic bacteria were released from livestock and human waste streams into the environment with highest abundances in a post-pandemic sewage sludge sample. Highlights- QACs most abundant in human waste streams, especially biosolids - Higher relative abundance of QAC tolerant bacteria in human waste streams - Pseudomonas and Providencia dominated QAC tolerant bacteria in both waste streams - Enterobacteriaceae with higher QAC tolerance abundant in human waste streams - Most QAC tolerant strains carried additional antibiotic resistances Environmental implicationMunicipal wastewater treatment plants (WWTPs) and livestock farms are hotspots for antimicrobial resistance (AMR) propagation. We compared the simultaneous occurrence of quaternary ammonium compounds (QACs), resistance genes (RGs), QAC-tolerant bacteria, and their multidrug-resistance status in livestock and human waste streams. QACs, indicators of QAC tolerance and AMR occurred in both systems but were higher in WWTPs, especially sewage sludge. Our findings highlight the need for prudent disinfectant use and enhanced waste treatments to reduce the risks of spreading micropollutants, pathogens, and AMR via organic fertilizers or treated wastewater recycled in circular agricultural practice.

IntroductionThe environmental impacts of pesticides have raised increasing concern, prompting the development of indicators to assess associated risks across ecosystems. Two main categories are generally distinguished: score-based indicators, which aggregate variables into scores, and risk-based indicators, grounded in the definition of risk as the product of hazard and exposure. Although more data-intensive and more complex to implement, risk-based indicators are recognized to better preserve proportionality with actual risk levels. ObjectivesThis study presents Phytorisque, a model based on the exposure-toxicity ratio to monitor risks associated with pesticide use in Walloon agriculture, from farm to regional scales, and to identify the most contributing active substances in support of risk-reduction policies MethodPhytorisque is a hybrid model that combines mechanistic, empirical, and statistical approaches, integrating quantities of active substances, their ecotoxicological characteristics, and their mobility, persistence, and bioaccumulation properties to generate indices specific to different environmental compartments. ResultsThe indices obtained enable comparison across substances, agricultural sectors, years, and management scenarios. The Phytorisque model provides an integrated assessment of risk across environmental compartments. It can monitor risk evolution over the years for policy impacts evaluation, diagnose the most problematic substances and prospect environmental risks associated with the use of chemical phytoproducts. ConclusionsPhytorisque provides an integrated risk assessment approach adapted to temporal monitoring, diagnosis, and forecasting. It is a relevant operational tool for supporting regional strategies aimed at reducing pesticide-related risks. The model is also transferable to other regions through the adaptation of parameters to local conditions and context.

BackgroundCase-based infectious disease surveillance is subject to ascertainment bias when testing intensity varies across time and population subgroups. We previously developed a regression-based test adjustment methodology using Standardized Testing Ratios (STRs) to correct for differential testing patterns in COVID-19 surveillance data. Wastewater-based surveillance (WWS) measures viral burden in the community independently of diagnostic testing behavior, making it a valuable external validation tool for test-adjusted case estimates. MethodsWe analyzed 111 weeks of paired wastewater and case surveillance data from Ontario, Canada (July 19, 2020 to August 28, 2022). Wastewater SARS-CoV-2 signals from 107 sewersheds across 34 public health units were normalized within sewersheds and aggregated using population-weighted averages. We compared wastewater correlations with crude reported and test-adjusted case counts using Spearman rank correlations, linear regression, and negative binomial distributed lag nonlinear models (DLNM), stratified by epidemic period. ResultsTest-adjusted cases correlated substantially more strongly with wastewater signals than crude reported cases overall (Spearman {rho} = 0.849 vs. 0.679; linear R{superscript 2} = 0.609 vs. 0.191). The advantage of test adjustment was greatest during the Omicron wave, when population-level diagnostic testing contracted sharply following PCR eligibility restrictions ({rho} = 0.924 vs. 0.604; R{superscript 2} = 0.815 vs. 0.470). DLNM incorporating the wastewater signal explained substantially more variance in test-adjusted than crude reported cases (McFadden pseudo-R{superscript 2} 0.898 vs. 0.776), despite similar lag-response structure for both outcomes. ConclusionsWastewater surveillance provides compelling independent validation of a previously described test adjustment methodology for COVID-19 case surveillance. The agreement between wastewater signals and test-adjusted cases was strongest precisely when testing scarcity was most severe, supporting the use of test adjustment to recover accurate infection dynamics from case surveillance data during periods of changing testing access and policy.

Withdrawal statementThe authors have withdrawn this manuscript as appropriate permission from the sample providers was not obtained prior to submission. This oversight was unintentional, and we sincerely apologise for the error. We are currently in the process of contacting the relevant sample providers to seek the necessary permissions and will address this matter accordingly. In the meantime, the authors request that this work not be cited as a reference. For any questions or further information, please contact the corresponding author.

The use of field water for laboratory rearing of mosquitoes could offer a better representation of the natural aquatic environment than laboratory tap or deionised water. For logistical reasons, such water may be stored in the laboratory environment for an extended period, but its stability is poorly documented. This study evaluated the influence of laboratory storage conditions on the kinetics of physicochemical parameters of breeding water collected from a field habitat. To capture within-habitat variability, water was collected from multiple spatial points from a breeding site and transferred into plastic containers for storage under laboratory conditions. Water physicochemical parameters were measured in the field to establish baseline readings, while laboratory measurements were done at 2-3-day intervals over 2 months to evaluate temporal changes. A linear mixed-effects model was fitted to evaluate the determinants of changes in physicochemical parameters under laboratory storage. Most parameters exhibited high stability; however, water temperature increased significantly by an average of [~]1.5 (p= 0.046) relative to the field. Water pH demonstrated a long-term rise over the 2-month storage period with a transient, significant dip of 0.71 units after a week of storage (p< 0.001). Overall, LMM analyses revealed that ambient relative humidity was the strongest statistical predictor of change in all water parameters except pH (p< 0.05). Ambient temperature correlated positively with water temperature and ammonium nitrogen (NH4-N) (p<0.002), and negatively with dissolved oxygen (p< 0.002). These results indicate that stored field water is highly sensitive to the laboratory microclimate. Specifically, water temperature, pH, and NH4-N serve as candidate indicators for storage-related physicochemical drift. We recommend the rigorous standardisation of insectary humidity and temperature, and monitoring of water parameters, which are likely relevant for bioassay reproducibility.

O_LICliffs are environmentally extreme yet biodiversity-rich ecosystems that harbour specialist plants, many endemic and threatened. Plant persistence in these nutrient-poor substrates may depend on tightly linked soil- and root-associated microbial communities, which remain poorly understood. These interactions may become increasingly important with the global expansion of recreational climbing. While physical climbing impacts on vegetation are documented, potential chemical effects, from the use of climbing chalk (magnesium carbonate), on soil properties and plant-associated microbiota remain unknown. C_LIO_LIWe sampled soils and roots beneath cliff-specialist and generalist plants, and unvegetated soils, across climbed and unclimbed routes in northern, central, and southern Spain. Soil physicochemical properties were quantified, fungal communities were characterized using ITS-metabarcoding, and structural equation modelling was used to disentangle direct and indirect effects. C_LIO_LIClimbing increased soil pH and altered soil chemical properties, driving shifts in fungal diversity and functional composition in soil and roots. The relative read abundance of root-associated symbiotrophic fungi declined, whereas arbuscular mycorrhizal fungi and pathogens increased in climbed cliffs. Overall effects were consistent, with cliff-specialist plants mediating nutrient and fungal shifts. C_LIO_LIur findings show that climbing can reshape cliff soil chemistry and fungal communities, with potential cascading consequences for plant functional performance, nutrient dynamics, and ecosystem resilience. C_LI