A planetary health innovation for disease, food and water challenges in Africa.

Many communities in low- and middle-income countries globally lack sustainable, cost-effective and mutually beneficial solutions for infectious disease, food, water and poverty challenges, despite their inherent interdependence1-7. Here we provide support for the hypothesis that agricultural development and fertilizer use in West Africa increase the burden of the parasitic disease schistosomiasis by fuelling the growth of submerged aquatic vegetation that chokes out water access points and serves as habitat for freshwater snails that transmit Schistosoma parasites to more than 200 million people globally8-10. In a cluster randomized controlled trial (ClinicalTrials.gov: NCT03187366) in which we removed invasive submerged vegetation from water points at 8 of 16 villages (that is, clusters), control sites had 1.46 times higher intestinal Schistosoma infection rates in schoolchildren and lower open water access than removal sites. Vegetation removal did not have any detectable long-term adverse effects on local water quality or freshwater biodiversity. In feeding trials, the removed vegetation was as effective as traditional livestock feed but 41 to 179 times cheaper and converting the vegetation to compost provided private crop production and total (public health plus crop production benefits) benefit-to-cost ratios as high as 4.0 and 8.8, respectively. Thus, the approach yielded an economic incentive-with important public health co-benefits-to maintain cleared waterways and return nutrients captured in aquatic plants back to agriculture with promise of breaking poverty-disease traps. To facilitate targeting and scaling of the intervention, we lay the foundation for using remote sensing technology to detect snail habitats. By offering a rare, profitable, win-win approach to addressing food and water access, poverty alleviation, infectious disease control and environmental sustainability, we hope to inspire the interdisciplinary search for planetary health solutions11 to the many and formidable, co-dependent global grand challenges of the twenty-first century.

Maximizing US nitrate removal through wetland protection and restoration.

Growing populations and agricultural intensification have led to raised riverine nitrogen (N) loads, widespread oxygen depletion in coastal zones (coastal hypoxia)1 and increases in the incidence of algal blooms.Although recent work has suggested that individual wetlands have the potential to improve water quality2-9, little is known about the current magnitude of wetland N removal at the landscape scale. Here we use National Wetland Inventory data and 5-kilometre grid-scale estimates of N inputs and outputs to demonstrate that current N removal by US wetlands (about 860 ± 160 kilotonnes of nitrogen per year) is limited by a spatial disconnect between high-density wetland areas and N hotspots. Our model simulations suggest that a spatially targeted increase in US wetland area by 10 per cent (5.1 million hectares) would double wetland N removal. This increase would provide an estimated 54 per cent decrease in N loading in nitrate-affected watersheds such as the Mississippi River Basin. The costs of this increase in area would be approximately 3.3 billion US dollars annually across the USA-nearly twice the cost of wetland restoration on non-agricultural, undeveloped land-but would provide approximately 40 times more N removal. These results suggest that water quality improvements, as well as other types of ecosystem services such as flood control and fish and wildlife habitat, should be considered when creating policy regarding wetland restoration and protection.

Getting off the ladder: Disentangling water quality indices to enhance the valuation of divergent ecosystem services.

Many water quality valuation studies and Federal cost-benefit analyses build from pioneering work using a "water quality ladder" or a single water quality index (WQI) to characterize both current conditions and effects of policies. When policies lead to contrasting changes in valued ecosystem services like recreational fishing and swimming, analyses using a single ladder or index might obscure important underlying service trade-offs. We test for this effect using alternative approaches that separate water quality indices and value changes in distinct ecosystem services stemming from policies with small to moderate changes in water quality. The indices we test relate to nutrient loadings in Michigan's rivers, lakes, and Great Lakes. Our split-sample experiment compares economic values for treatments with two versus three quality metrics. The key distinction is that the two-index survey, like many existing studies, aggregates subindices for water contact (for swimming and boating) and fish biomass scores (for fishing) into a single WQI, whereas the three-index survey separately utilizes both. We find that changes in our index reflecting changes in fecal bacteria and water clarity are valued differently from changes in our recreational fishing index. Aggregating changes in these two distinct recreational services using a single WQI yields consistently lower benefit estimates across a range of underlying changes in our experiment. In valuation scenarios with small changes in overall water quality, the WQI-based benefit estimates can differ substantially from benefits measured by decomposing the index and valuing the disparate subindices, differences which might change balance of benefits and costs in regulatory evaluations.

Perspectives on valuing water quality improvements using stated preference methods.

Improvements to the quality of freshwater rivers and lakes can generate a wide array of benefits, from "use values" such as recreational boating, fishing, and swimming to "nonuse values" such as improved outcomes for aquatic biodiversity. Bringing these nonmarket values into decision-making is crucial to determining appropriate levels of investment in water quality improvements. However, progress in the economic valuation of water quality benefits has lagged similar efforts to value air quality benefits, with implications for water policy. New data sources, modeling techniques, and innovation in stated preference survey methods offer notable improvements to estimates of use and nonuse benefits of improved water quality. Here, we provide a perspective on how recent applications of stated preference techniques to the valuation of the nonmarket benefits of water quality improvements have advanced the field of environmental valuation. This overview is structured around four key questions: i) What is it about water quality that we seek to value? ii) How should we design and implement the surveys which elicit individuals' stated preferences? iii) How do we assess the validity of the findings provided by such studies? and iv) What are the contributions of these valuation exercises to public policy? In answering these questions, we make reference to the contributions provided by the papers in this Symposium.

Estimating biotic integrity to capture existence value of freshwater ecosystems.

The US Environmental Protection Agency (EPA) uses a water quality index (WQI) to estimate benefits of proposed Clean Water Act regulations. The WQI is relevant to human use value, such as recreation, but may not fully capture aspects of nonuse value, such as existence value. Here, we identify an index of biological integrity to supplement the WQI in a forthcoming national stated preference survey that seeks to capture existence value of streams and lakes more accurately within the conterminous United States (CONUS). We used literature and focus group research to evaluate aquatic indices regularly reported by the EPA's National Aquatic Resource Surveys. We chose an index that quantifies loss in biodiversity as the observed-to-expected (O/E) ratio of taxonomic composition because focus group participants easily understood its meaning and the environmental changes that would result in incremental improvements. However, available datasets of this index do not provide the spatial coverage to account for how conditions near survey respondents affect their willingness to pay for its improvement. Therefore, we modeled and interpolated the values of this index from sampled sites to 1.1 million stream segments and 297,071 lakes across the CONUS to provide the required coverage. The models explained 13 to 36% of the variation in O/E scores and demonstrate how modeling can provide data at the required density for benefits estimation. We close by discussing future work to improve performance of the models and to link biological condition with water quality and habitat models that will allow us to forecast changes resulting from regulatory options.

Modeling global oceanic nitrogen deposition from food systems and its mitigation potential by reducing overuse of fertilizers.

Growing population and consumption pose unprecedented demands on food production. However, ammonia emissions mainly from food systems increase oceanic nitrogen deposition contributing to eutrophication. Here, we developed a long-term oceanic nitrogen deposition dataset (1970 to 2018) with updated ammonia emissions from food systems, evaluated the impact of ammonia emissions on oceanic nitrogen deposition patterns, and discussed the potential impact of nitrogen fertilizer overuse. Based on the chemical transport modeling approach, oceanic ammonia-related nitrogen deposition increased by 89% globally between 1970 and 2018, and now, it exceeds oxidized nitrogen deposition by over 20% in coastal regions including China Sea, India Coastal, and Northeastern Atlantic Shelves. Approximately 38% of agricultural nitrogen fertilizer was excessive, which corresponds to 15% of global oceanic ammonia-related nitrogen deposition. Policymakers and water quality managers need to pay increasingly more attention to ammonia associated with food production if the goal of reducing coastal nitrogen pollution is to be achieved for Sustainable Development Goals.

Decoding the metabolic response of Escherichia coli for sensing trace heavy metals in water.

Heavy metal contamination due to industrial and agricultural waste represents a growing threat to water supplies. Frequent and widespread monitoring for toxic metals in drinking and agricultural water sources is necessary to prevent their accumulation in humans, plants, and animals, which results in disease and environmental damage. Here, the metabolic stress response of bacteria is used to report the presence of heavy metal ions in water by transducing ions into chemical signals that can be fingerprinted using machine learning analysis of vibrational spectra. Surface-enhanced Raman scattering surfaces amplify chemical signals from bacterial lysate and rapidly generate large, reproducible datasets needed for machine learning algorithms to decode the complex spectral data. Classification and regression algorithms achieve limits of detection of 0.5 pM for As3+ and 6.8 pM for Cr6+, 100,000 times lower than the World Health Organization recommended limits, and accurately quantify concentrations of analytes across six orders of magnitude, enabling early warning of rising contaminant levels. Trained algorithms are generalizable across water samples with different impurities; water quality of tap water and wastewater was evaluated with 92% accuracy.

Widespread global increase in intense lake phytoplankton blooms since the 1980s.

Freshwater blooms of phytoplankton affect public health and ecosystem services globally1,2. Harmful effects of such blooms occur when the intensity of a bloom is too high, or when toxin-producing phytoplankton species are present. Freshwater blooms result in economic losses of more than US$4 billion annually in the United States alone, primarily from harm to aquatic food production, recreation and tourism, and drinking-water supplies3. Studies that document bloom conditions in lakes have either focused only on individual or regional subsets of lakes4-6, or have been limited by a lack of long-term observations7-9. Here we use three decades of high-resolution Landsat 5 satellite imagery to investigate long-term trends in intense summertime near-surface phytoplankton blooms for 71 large lakes globally. We find that peak summertime bloom intensity has increased in most (68 per cent) of the lakes studied, revealing a global exacerbation of bloom conditions. Lakes that have experienced a significant (P < 0.1) decrease in bloom intensity are rare (8 per cent). The reason behind the increase in phytoplankton bloom intensity remains unclear, however, as temporal trends do not track consistently with temperature, precipitation, fertilizer-use trends or other previously hypothesized drivers. We do find, however, that lakes with a decrease in bloom intensity warmed less compared to other lakes, suggesting that lake warming may already be counteracting management efforts to ameliorate eutrophication10,11. Our findings support calls for water quality management efforts to better account for the interactions between climate change and local hydrological conditions12,13.

Managing nitrogen to restore water quality in China.

The nitrogen cycle has been radically changed by human activities1. China consumes nearly one third of the world's nitrogen fertilizers. The excessive application of fertilizers2,3 and increased nitrogen discharge from livestock, domestic and industrial sources have resulted in pervasive water pollution. Quantifying a nitrogen 'boundary'4 in heterogeneous environments is important for the effective management of local water quality. Here we use a combination of water-quality observations and simulated nitrogen discharge from agricultural and other sources to estimate spatial patterns of nitrogen discharge into water bodies across China from 1955 to 2014. We find that the critical surface-water quality standard (1.0 milligrams of nitrogen per litre) was being exceeded in most provinces by the mid-1980s, and that current rates of anthropogenic nitrogen discharge (14.5 ± 3.1 megatonnes of nitrogen per year) to fresh water are about 2.7 times the estimated 'safe' nitrogen discharge threshold (5.2 ± 0.7 megatonnes of nitrogen per year). Current efforts to reduce pollution through wastewater treatment and by improving cropland nitrogen management can partially remedy this situation. Domestic wastewater treatment has helped to reduce net discharge by 0.7 ± 0.1 megatonnes in 2014, but at high monetary and energy costs. Improved cropland nitrogen management could remove another 2.3 ± 0.3 megatonnes of nitrogen per year-about 25 per cent of the excess discharge to fresh water. Successfully restoring a clean water environment in China will further require transformational changes to boost the national nutrient recycling rate from its current average of 36 per cent to about 87 per cent, which is a level typical of traditional Chinese agriculture. Although ambitious, such a high level of nitrogen recycling is technologically achievable at an estimated capital cost of approximately 100 billion US dollars and operating costs of 18-29 billion US dollars per year, and could provide co-benefits such as recycled wastewater for crop irrigation and improved environmental quality and ecosystem services.

Current water quality guidelines across North America and Europe do not protect lakes from salinization.

Human-induced salinization caused by the use of road deicing salts, agricultural practices, mining operations, and climate change is a major threat to the biodiversity and functioning of freshwater ecosystems. Yet, it is unclear if freshwater ecosystems are protected from salinization by current water quality guidelines. Leveraging an experimental network of land-based and in-lake mesocosms across North America and Europe, we tested how salinization-indicated as elevated chloride (Cl-) concentration-will affect lake food webs and if two of the lowest Cl- thresholds found globally are sufficient to protect these food webs. Our results indicated that salinization will cause substantial zooplankton mortality at the lowest Cl- thresholds established in Canada (120 mg Cl-/L) and the United States (230 mg Cl-/L) and throughout Europe where Cl- thresholds are generally higher. For instance, at 73% of our study sites, Cl- concentrations that caused a ≥50% reduction in cladoceran abundance were at or below Cl- thresholds in Canada, in the United States, and throughout Europe. Similar trends occurred for copepod and rotifer zooplankton. The loss of zooplankton triggered a cascading effect causing an increase in phytoplankton biomass at 47% of study sites. Such changes in lake food webs could alter nutrient cycling and water clarity and trigger declines in fish production. Current Cl- thresholds across North America and Europe clearly do not adequately protect lake food webs. Water quality guidelines should be developed where they do not exist, and there is an urgent need to reassess existing guidelines to protect lake ecosystems from human-induced salinization.

A hybrid empirical and parametric approach for managing ecosystem complexity: Water quality in Lake Geneva under nonstationary futures.

Severe deterioration of water quality in lakes, characterized by overabundance of algae and declining dissolved oxygen in the deep lake (DOB), was one of the ecological crises of the 20th century. Even with large reductions in phosphorus loading, termed "reoligotrophication," DOB and chlorophyll (CHL) have often not returned to their expected pre-20th-century levels. Concurrently, management of lake health has been confounded by possible consequences of climate change, particularly since the effects of climate are not neatly separable from the effects of eutrophication. Here, using Lake Geneva as an iconic example, we demonstrate a complementary alternative to parametric models for understanding and managing lake systems. This involves establishing an empirically-driven baseline that uses supervised machine learning to capture the changing interdependencies among biogeochemical variables and then combining the empirical model with a more conventional equation-based model of lake physics to predict DOB over decadal time-scales. The hybrid model not only leads to substantially better forecasts, but also to a more actionable description of the emergent rates and processes (biogeochemical, ecological, etc.) that drive water quality. Notably, the hybrid model suggests that the impact of a moderate 3°C air temperature increase on water quality would be on the same order as the eutrophication of the previous century. The study provides a template and a practical path forward to cope with shifts in ecology to manage environmental systems for non-analogue futures.

Impact of operational conditions on drinking water biofilm dynamics and coliform invasion potential.

Biofilms within drinking water distribution systems serve as a habitat for drinking water microorganisms. However, biofilms can negatively impact drinking water quality by causing water discoloration and deterioration and can be a reservoir for unwanted microorganisms. In this study, we investigated whether indicator organisms for drinking water quality, such as coliforms, can settle in mature drinking water biofilms. Therefore, a biofilm monitor consisting of glass rings was used to grow and sample drinking water biofilms. Two mature drinking water biofilms were characterized by flow cytometry, ATP measurements, confocal laser scanning microscopy, and 16S rRNA sequencing. Biofilms developed under treated chlorinated surface water supply exhibited lower cell densities in comparison with biofilms resulting from treated groundwater. Overall, the phenotypic as well as the genotypic characteristics were significantly different between both biofilms. In addition, the response of the biofilm microbiome and possible biofilm detachment after minor water quality changes were investigated. Limited changes in pH and free chlorine addition, to simulate operational changes that are relevant for practice, were evaluated. It was shown that both biofilms remained resilient. Finally, mature biofilms were prone to invasion of the coliform, Serratia fonticola. After spiking low concentrations (i.e., ±100 cells/100 mL) of the coliform to the corresponding bulk water samples, the coliforms were able to attach and get established within the mature biofilms. These outcomes emphasize the need for continued research on biofilm detachment and its implications for water contamination in distribution networks. IMPORTANCE: The revelation that even low concentrations of coliforms can infiltrate into mature drinking water biofilms highlights a potential public health concern. Nowadays, the measurement of coliform bacteria is used as an indicator for fecal contamination and to control the effectiveness of disinfection processes and the cleanliness and integrity of distribution systems. In Flanders (Belgium), 533 out of 18,840 measurements exceeded the established norm for the coliform indicator parameter in 2021; however, the source of microbial contamination is mostly unknown. Here, we showed that mature biofilms, are susceptible to invasion of Serratia fonticola. These findings emphasize the importance of understanding and managing biofilms in drinking water distribution systems, not only for their potential to influence water quality, but also for their role in harboring and potentially disseminating pathogens. Further research into biofilm detachment, long-term responses to operational changes, and pathogen persistence within biofilms is crucial to inform strategies for safeguarding drinking water quality.

A comprehensive review on the utilization of probiotics in aquaculture towards sustainable shrimp farming.

Probiotics in shrimp aquaculture have gained considerable attention as a potential solution to enhance production efficiency, disease management, and overall sustainability. Probiotics, beneficial microorganisms, have shown promising effects when administered to shrimp as dietary supplements or water additives. Their inclusion has been linked to improved gut health, nutrient absorption, and disease resistance in shrimp. Probiotics also play a crucial role in maintaining a balanced microbial community within the shrimp pond environment, enhancing water quality and reducing pathogen prevalence. This article briefly summarizes the many ways that probiotics are used in shrimp farming and the advantages that come with them. Despite the promising results, challenges such as strain selection, dosage optimization, and environmental conditions are carefully addressed for successful probiotic integration in shrimp aquaculture. The potential of probiotics as a sustainable and ecologically friendly method of promoting shrimp development and health while advancing environmentally friendly shrimp farming techniques is highlighted in this analysis. Further research is required to fully exploit probiotics' benefits and develop practical guidelines for their effective implementation in shrimp aquaculture.

Recognizing Agricultural Headwaters as Critical Ecosystems.

Agricultural headwaters are positioned at the interface between terrestrial and aquatic ecosystems and, therefore, at the margins of scientific disciplines. They are deemed devoid of biodiversity and too polluted by ecologists, overlooked by hydrologists, and are perceived as a nuisance by landowners and water authorities. While agricultural streams are widespread and represent a major habitat in terms of stream length, they remain understudied and thereby undervalued. Agricultural headwater streams are significantly modified and polluted but at the same time are the critical linkages among land, air, and water ecosystems. They exhibit the largest variation in streamflow, water quality, and greenhouse gas emission with cascading effects on the entire stream networks, yet they are underrepresented in monitoring, remediation, and restoration. Therefore, we call for more intense efforts to characterize and understand the inherent variability and sensitivity of these ecosystems to global change drivers through scientific and regulatory monitoring and to improve their ecosystem conditions and functions through purposeful and evidence-based remediation.

Sensors for Emerging Water Contaminants: Overcoming Roadblocks to Innovation.

Ensuring water quality and safety requires the effective detection of emerging contaminants, which present significant risks to both human health and the environment. Field deployable low-cost sensors provide solutions to detect contaminants at their source and enable large-scale water quality monitoring and management. Unfortunately, the availability and utilization of such sensors remain limited. This Perspective examines current sensing technologies for detecting emerging contaminants and analyzes critical barriers, such as high costs, lack of reliability, difficulties in implementation in real-world settings, and lack of stakeholder involvement in sensor design. These technical and nontechnical barriers severely hinder progression from proof-of-concepts and negatively impact user experience factors such as ease-of-use and actionability using sensing data, ultimately affecting successful translation and widespread adoption of these technologies. We provide examples of specific sensing systems and explore key strategies to address the remaining scientific challenges that must be overcome to translate these technologies into the field such as improving sensitivity, selectivity, robustness, and performance in real-world water environments. Other critical aspects such as tailoring research to meet end-users' requirements, integrating cost considerations and consumer needs into the early prototype design, establishing standardized evaluation and validation protocols, fostering academia-industry collaborations, maximizing data value by establishing data sharing initiatives, and promoting workforce development are also discussed. The Perspective describes a set of guidelines for the development, translation, and implementation of water quality sensors to swiftly and accurately detect, analyze, track, and manage contamination.

Keeping Nitrogen Use in China within the Planetary Boundary Using a Spatially Explicit Approach.

Nitrogen (N) supports food production, but its excess causes water pollution. We lack an understanding of the boundary of N for water quality while considering complex relationships between N inputs and in-stream N concentrations. Our knowledge is limited to regional reduction targets to secure food production. Here, we aim to derive a spatially explicit boundary of N inputs to rivers for surface water quality using a bottom-up approach and to explore ways to meet the derived N boundary while considering the associated impacts on both surface water quality and food production in China. We modified a multiscale nutrient modeling system simulating around 6.5 Tg of N inputs to rivers that are allowed for whole of China in 2012. Maximum allowed N inputs to rivers are higher for intensive food production regions and lower for highly urbanized regions. When fertilizer and manure use is reduced, 45-76% of the streams could meet the N water quality threshold under different scenarios. A comparison of "water quality first" and "food production first" scenarios indicates that trade-offs between water quality and food production exist in 2-8% of the streams, which may put 7-28% of crop production at stake. Our insights could support region-specific policies for improving water quality.

Environmental predictors of Escherichia coli concentration at marine beaches in Vancouver, Canada: a Bayesian mixed-effects modelling analysis.

Understanding historical environmental determinants associated with the risk of elevated marine water contamination could enhance monitoring marine beaches in a Canadian setting, which can also inform predictive marine water quality models and ongoing climate change preparedness efforts. This study aimed to assess the combination of environmental factors that best predicts Escherichia coli (E. coli) concentration at public beaches in Metro Vancouver, British Columbia, by combining the region's microbial water quality data and publicly available environmental data from 2013 to 2021. We developed a Bayesian log-normal mixed-effects regression model to evaluate predictors of geometric E. coli concentrations at 15 beaches in the Metro Vancouver Region. We identified that higher levels of geometric mean E. coli levels were predicted by higher previous sample day E. coli concentrations, higher rainfall in the preceding 48 h, and higher 24-h average air temperature at the median or higher levels of the 24-h mean ultraviolet (UV) index. In contrast, higher levels of mean salinity were predicted to result in lower levels of E. coli. Finally, we determined that the average effects of the predictors varied highly by beach. Our findings could form the basis for building real-time predictive marine water quality models to enable more timely beach management decision-making.

Improving Private Well Testing Programs: Experimental Evidence from Iowa.

Approximately 23 million U.S. households rely on private wells for drinking water. This study first summarizes drinking water behaviors and perceptions from a large-scale survey of households that rely on private wells in Iowa. Few households test as frequently as recommended by public health experts. Around 40% of households do not regularly test, treat, or avoid their drinking water, suggesting pollution exposure may be widespread among this population. Next, we utilize a randomized control trial to study how nitrate test strips and information about a free, comprehensive water quality testing program influence households' behaviors and perceptions. The intervention significantly increased testing, including high-quality follow-up testing, but had limited statistically detectable impacts on other behaviors and perceptions. Households' willingness to pay for nitrate test kits and testing information exceeds program costs, suggesting that the intervention was welfare-enhancing.

Climate-Driven Increases in Source Water Natural Organic Matter: Implications for the Sustainability of Drinking Water Treatment.

This study presents an updated analysis spanning over two decades (1999-2023) of climate, water quality, and operational data from two drinking water facilities in Atlantic Canada that previously experienced gradual increases in the natural organic matter (NOM) concentration and brownification. The goal was to assess the impact of recent extreme weather events on acute NOM concentration increases and drinking water treatment processes. In 2023, a dry spring combined with a warm and wet summer caused NOM in the water supplies to increase by >67% (as measured by color). To mitigate increased NOM concentration, the alum dose nearly doubled in 2023 compared to that in 2022. Disinfection byproducts were elevated following the event but remained within the compliance levels. From 1999 to 2023, the two plants responded to gradual climate change impacts and brownification, with alum dose increases of between 4.1 and 8.3 times. Equivalent CO2 emissions were estimated for alum usage, which increased by 3 to 7-fold in 2023 compared to when the plants were commissioned decades prior. The plants were not only adversely impacted by climate change but also contributed to the global CO2 burden. Thus, a paradigm shift toward sustainable alternatives for NOM removal is required in the water sector, and climate change adaptation and mitigation principles are urgently needed.

Water Quality Monitoring with the Multiplexed Assay MitoOxTox for Mitochondrial Toxicity, Oxidative Stress Response, and Cytotoxicity in AREc32 Cells.

Mitochondria play a key role in the energy production of cells, but their function can be disturbed by environmental toxicants. We developed a cell-based mitochondrial toxicity assay for environmental chemicals and their mixtures extracted from water samples. The reporter gene cell line AREc32, which is frequently used to quantify the cytotoxicity and oxidative stress response of water samples, was multiplexed with an endpoint of mitochondrial toxicity. The disruption of the mitochondrial membrane potential (MMP) was quantified by high-content imaging and compared to measured cytotoxicity, predicted baseline toxicity, and activation of the oxidative stress response. Mitochondrial complex I inhibitors showed highly specific effects on the MMP, with minor effects on cell viability. Uncouplers showed a wide distribution of specificity on the MMP, often accompanied by specific cytotoxicity (enhanced over baseline toxicity). Mitochondrial toxicity and the oxidative stress response were not directly associated. The multiplexed assay was applied to water samples ranging from wastewater treatment plant (WWTP) influent and effluent and surface water to drinking and bottled water from various European countries. Specific effects on MMP were observed for the WWTP influent and effluent. This new MitoOxTox assay is an important complement for existing in vitro test batteries for water quality testing and has potential for applications in human biomonitoring.