SARS-CoV-2 viral titer measurements in Ontario, Canada wastewaters throughout the COVID-19 pandemic.

During the COVID-19 pandemic, the Province of Ontario, Canada, launched a wastewater surveillance program to monitor SARS-CoV-2, inspired by the early work and successful forecasts of COVID-19 waves in the city of Ottawa, Ontario. This manuscript presents a dataset from January 1, 2021, to March 31, 2023, with RT-qPCR results for SARS-CoV-2 genes and PMMoV from 107 sites across all 34 public health units in Ontario, covering 72% of the province's and 26.2% of Canada's population. Sampling occurred 2-7 times weekly, including geographical coordinates, serviced populations, physico-chemical water characteristics, and flowrates. In doing so, this manuscript ensures data availability and metadata preservation to support future research and epidemic preparedness through detailed analyses and modeling. The dataset has been crucial for public health in tracking disease locally, especially with the rise of the Omicron variant and the decline in clinical testing, highlighting wastewater-based surveillance's role in estimating disease incidence in Ontario.

The role of catchment population size, data normalization, and chronology of public health interventions on wastewater-based COVID-19 viral trends.

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic presented the most challenging global crisis in recent times. A pandemic caused by a novel pathogen such as SARS-CoV-2 necessitated the development of innovative techniques for the monitoring and surveillance of COVID-19 infections within communities. Wastewater surveillance (WWS) is recognized as a non-invasive, cost-effective, and valuable epidemiological tool to monitor the prevalence of COVID-19 infections in communities. Seven municipal wastewater sampling sites representing distinct sewershed communities were selected for the surveillance of the SARS-CoV-2 virus in Durham Region, Ontario, Canada over 8 months from March 2021 to October 2021. Viral RNA fragments of SARS-CoV-2 and the normalization target pepper mild mottle virus (PMMoV) were concentrated from wastewater influent using the PEG/NaCl superspeed centrifugation method and quantified using RT-qPCR. Strong significant correlations (Spearman's rs = 0.749 to 0.862, P < 0.001) were observed between SARS-CoV-2 gene copies/mL of wastewater and clinical cases reported in each delineated sewershed by onset date. Although raw wastewater offered higher correlation coefficients with clinical cases by onset date compared to PMMoV normalized data, only one site had a statistically significantly higher Spearman's correlation coefficient value for raw data than normalized data. Implementation of community stay-at-home orders and vaccinations over the course of the study period in 2021 were found to strongly correspond to decreasing SARS-CoV-2 wastewater trends in the wastewater treatment plants and upstream pumping stations.

Metabolomic-Based Comparison of Daphnia magna and Japanese Medaka Responses After Exposure to Acetaminophen, Diclofenac, and Ibuprofen.

Pharmaceuticals are found in aquatic environments due to their widespread use and environmental persistence. To date, a range of impairments to aquatic organisms has been reported with exposure to pharmaceuticals; however, further comparisons of their impacts across different species on the molecular level are needed. In the present study, the crustacean Daphnia magna and the freshwater fish Japanese medaka, common model organisms in aquatic toxicity, were exposed for 48 h to the common analgesics acetaminophen (ACT), diclofenac (DCF), and ibuprofen (IBU) at sublethal concentrations. A targeted metabolomic-based approach, using liquid chromatography-tandem mass spectrometry to quantify polar metabolites from individual daphnids and fish was used. Multivariate analyses and metabolite changes identified differences in the metabolite profile for D. magna and medaka, with more metabolic perturbations for D. magna. Pathway analyses uncovered disruptions to pathways associated with protein synthesis and amino acid metabolism with D. magna exposure to all three analgesics. In contrast, medaka exposure resulted in disrupted pathways with DCF only and not ACT and IBU. Overall, the observed perturbations in the biochemistry of both organisms were different and consistent with assessments using other endpoints reporting that D. magna is more sensitive to pollutants than medaka in short-term studies. Our findings demonstrate that molecular-level responses to analgesic exposure can reflect observations of other endpoints, such as immobilization and mortality. Thus, environmental metabolomics can be a valuable tool for selecting sentinel species for the biomonitoring of freshwater ecosystems while also uncovering mechanistic information. Environ Toxicol Chem 2024;43:1339-1351. © 2024 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Waterborne exposure to the antineoplastic 5-fluorouracil alters lipid composition in larval zebrafish (Danio rerio).

Antineoplastic medications are present in aquatic environments and are measured at relatively high concentrations in hospital sewage effluent. Thus, it is important to characterize risk associated with waterborne exposures to anticancer drugs. The drug 5-fluorouracil (5-FU) is used to treat several types of cancers, acting to inhibit cell division and cellular metabolism. The objectives of this study were to determine the effects of 5-FU on developmental endpoints and lipid composition in zebrafish. 5-FU did not negatively affect development nor survival in developing zebrafish at concentrations up to 1000 µg/L. However, 5-FU increased neutral lipid content in zebrafish larvae, indicating potential for lipid dysregulation. To further discern effects on lipids, lipidomics was conducted and a total of 164 lipids belonging to 14 lipid classes were identified. Significant changes (false discovery rate < 0.05) in abundance were detected for 19 lipids including some ceramides, ether-linked phosphatidylethanolamines, and sphingomyelins among others. We also measured the expression levels of 14 lipid-related enzymes and transporters (e.g., acox3, dgat1, fads2, fasn, elovl2) using real-time PCR; however, mRNA abundance levels were not affected, suggesting transcriptional changes may not be a primary mechanism underlying lipid dysregulation. Locomotor activity was measured in zebrafish as lipids are needed for swimming activity in larvae. Exposure to 5-FU did not affect locomotor activity up to 1000 µg/L. We conclude that lipids accumulate in larval zebrafish with exposure to 5-FU, which can subsequently affect lipid composition. These data reveal potential lipid signatures of 5-FU exposure and contribute to risk assessments for antineoplastic exposure in aquatic environments.

Metabolic, neurotoxic and immunotoxic effects of PFAAs and their mixtures on the proteome of the head kidney and plasma from rainbow trout (Oncorhynchus mykiss).

PFAAs (Perfluoroalkyl acids) are a class of bioaccumulative, persistent and ubiquitous environmental contaminants which primarily occupy the hydrosphere and its sediments. Currently, a paucity of toxicological information exists for short chain PFAAs and complex mixtures. In order to address these knowledge gaps, we performed a 3-week, aqueous exposure of rainbow trout to 3 different concentrations of a PFAA mixture (50, 100 and 500 ng/L) modeled after the composition determined in Lake Ontario. We conducted an additional set of exposures to individual PFAAs (25 nM each of PFOS (12,500 ng/L), PFOA (10,300 ng/L), PFBS (7500 ng/L) or PFBA (5300 ng/L) to evaluate differences in biological response across PFAA congeners. Untargeted proteomics and phosphorylated metabolomics were conducted on the blood plasma and head kidney tissue to evaluate biological response. Plasma proteomic responses to the mixtures revealed several unexpected outcomes including Similar proteomic profiles and biological processes as the PFOS exposure regime while being orders of magnitude lower in concentration and an atypical dose response in terms of the number of significantly altered proteins (FDR < 0.1). Biological pathway analysis revealed the low mixture, medium mixture and PFOS to significantly alter (FDR < 0.05) a number of processes including those involved in lipid metabolism, oxidative stress and the nervous system. We implicate plasma increases in PPARD and PPARG as being directly related to these biological processes as they are known to be important regulators in all 3 processes. In contrast to the blood plasma, the high mixture and PFOA exposure regimes caused the greatest change to the head kidney proteome, altering many proteins being involved in lipid metabolism, oxidative stress and inflammation. Our findings support the pleiotropic effect PFAAs have on aquatic organisms at environmentally relevant doses including those on PPAR signaling, metabolic dysregulation, immunotoxicity and neurotoxicity.

How does temperature rise affect a freshwater catfish Rhamdia quelen? A proteomic approach.

Outside of scientific circles, climate change is a hotly debated topic due to all its consequences. Changes in the temperature can affect aquatic organisms and it is important to understand and to detect earlier signals. This study aimed to analyze how a Neotropical fish species responds to temperature increases, using proteomic analysis as a tool. For this, fish of the species Rhamdia quelen, male and female, were exposed to two temperatures: 25 °C and 30 °C. After 96 h, the animals were anesthetized, euthanized and the liver was collected for proteomic analysis. Using freely available online software and databases (e.g. MetaboAnalyst, Gene Ontology and UniProt), we define the altered proteins in both sexes: 42 in females and 62 in males. Data are available via ProteomeXchange with identifier PXD046475. Differences between the two temperatures were observed mainly in the amino acid metabolic pathways. The cellular process and the immune response was altered, indicating that effects at lower levels of biological organization could serve as a predictor of higher-level effects when temperature rise affects wildlife populations. Thus, we conclude that the increase in temperature is capable of altering important cellular and physiological processes in R. quelen fish, with this response being different for males and females.

Different response of females and males Neotropical catfish (Rhamdia quelen) upon short-term temperature increase.

Climate change has been one of the most discussed topics in the world. Global warming is characterized by an increase in global temperature, also in aquatic environments. The increased temperature can affect aquatic organisms with lethal and sublethal effects. Thus, it is necessary to understand how different species respond to temperature. This study aimed to evaluate how the Neotropical catfish species Rhamdia quelen responds to temperature increases. The fish were exposed to temperatures of 25 °C (control) and 30 °C after gradual temperature increase for 7 days. After 96 h in each temperature, the fish were anesthetized, blood was collected, and after euthanasia, brain, liver, posterior kidney, gills, muscle, and gonads were collected. The gonads were used for sexing, while other tissues were used for the hematological, biochemical, genotoxic, and histopathological biomarkers analysis. Hepatic proteomic analysis with a focus on energy production was also carried out. Blood parameter changes in both sexes, including an increase in glucose in males, leukopenia in females, and genotoxicity in both sexes. Hepatic proteins related to energy production were altered in both sexes, but mainly in males. Others biomarker alterations, such as histopathological, were not observed in other tissues; however, the antioxidant system was affected differently between sexes. These showed that R. quelen juveniles, at temperatures higher than its optimum temperature such as 30 °C, has several sublethal changes, such as hematological alterations, antioxidant system activation, and energetic metabolism alteration, especially in males. Thus, short-term temperature rise can affect females and males of R. quelen differently.

An alternative method for monitoring and interpreting influenza A in communities using wastewater surveillance.

Seasonal influenza is an annual public health challenge that strains healthcare systems, yet population-level prevalence remains under-reported using standard clinical surveillance methods. Wastewater surveillance (WWS) of influenza A can allow for reliable flu surveillance within a community by leveraging existing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) WWS networks regardless of the sample type (primary sludge vs. primary influent) using an RT-qPCR-based viral RNA detection method for both targets. Additionally, current influenza A outbreaks disproportionately affect the pediatric population. In this study, we show the utility of interpreting influenza A WWS data with elementary student absenteeism due to illness to selectively interpret disease spread in the pediatric population. Our results show that the highest statistically significant correlation (Rs = 0.96, p = 0.011) occurred between influenza A WWS data and elementary school absences due to illness. This correlation coefficient is notably higher than the correlations observed between influenza A WWS data and influenza A clinical case data (Rs = 0.79, p = 0.036). This method can be combined with a suite of pathogen data from wastewater to provide a robust system for determining the causative agents of diseases that are strongly symptomatic in children to infer pediatric outbreaks within communities.

Characterizing the origin of blood plasma proteins from organ tissues in rainbow trout (Oncorhynchus mykiss) using a comparative non-targeted proteomics approach.

Protein expression patterns adapt to various cues to meet the needs of an organism. The dynamicity of an organism's proteome can therefore reveal information about an organism's health. Proteome databases contain limited information regarding organisms outside of medicinal biology. The UniProt human and mouse proteomes are extensively reviewed and ∼50 % of both proteomes include tissue specificity, while >99 % of the rainbow trout proteome lacks tissue specificity. This study aimed to expand knowledge on the rainbow trout proteome with a focus on understanding the origin of blood plasma proteins. Blood, brain, heart, liver, kidney, and gills were collected from adult rainbow trout, plasma and tissue proteins were analyzed using liquid chromatography tandem mass spectrometry. Over 10,000 proteins were identified across all groups. Our data indicated that the majority of the plasma proteome is shared amongst multiple tissue types, though 4-7 % of the plasma proteome is uniquely originated from each tissue (gill > heart > liver > kidney > brain).

Phytoplankton metabolite profiles from two Lake Ontario Areas of Concern reveal differences associated with taxonomic community composition.

Water quality and phytoplankton community composition are important factors that can indicate freshwater ecosystem health. We combined water quality, phytoplankton community, and metabolomic data from algae and water sampled from two embayments in Lake Ontario, Hamilton Harbour and the Bay of Quinte, over ten weeks from August to October in the year 2020. Metabolomics was performed using liquid chromatography tandem mass spectroscopy (LC-MS/MS) to identify changes in intracellular metabolites within algae communities over time, and Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) was used to characterize putative isomers of extracellular metabolites at sub-ppb mass accuracy. Results from this study indicate that Hamilton Harbour and the Bay of Quinte are two very different ecosystems with respect to water quality, phytoplankton metabolites, and phytoplankton community composition. Community composition is strongly driven by conductivity and nitrates in Hamilton Harbour, while the opposite is true in the Bay of Quinte. Metabolites including α-aminobutyric acid and glycine were found in larger abundance within algal communities at both locations, while taurine was more predominant in algal communities from the Bay of Quinte. These metabolic profiles could reflect the different communities of phytoplankton, and be alternative indicators of algal bloom growth.

Lipidomic, metabolomic, and behavior responses of zebrafish (Danio rerio) exposed to environmental levels of the beta blocker atenolol.

Blood pressure medications are used to treat hypertension; however, low concentrations of beta-blockers in water systems can negatively impact aquatic wildlife. Here, we conducted a metabolic and behavioral study investigating atenolol, a beta-blocker frequently detected in global wastewater systems. The objectives were to determine the effects of low-level atenolol exposure on early stages of zebrafish. We measured survival, deformities, heartbeat, mitochondrial function, lipid and amino acid profiles, and locomotor activity to discern mechanisms of metabolic disruption. We hypothesized that atenolol disrupts lipid metabolism, which would negatively impact locomotor activity. Atenolol showed no overt toxicity to larval zebrafish up to 10 µg/L and deformities were infrequent (<5 %), and included cardiac edema and larvae with kinked tails. A hatch delay was observed at 2-day post-fertilization (dpf) for fish exposed to >5 µg/L atenolol. Heart rates were reduced in 2 and 3 dpf in fish treated with >500 ng/L atenolol. There was no change in oxygen consumption rates (basal and maximum respiration) of embryos when exposed to a range of atenolol concentrations, suggesting mitochondrial respiration was intact. Oil red staining for lipid content in larvae showed a global reduction in lipids with 10 µg/L exposure, prompting deeper investigation into the lipid profiles. Lipidomics quantified 86 lipids and revealed reduced abundance in Ceramide 18: 1 16:0 (Cer_NS d18:1_16:0), Ether linked Phosphatidylethanolamine 16:0 22:6 (EtherPE 16:0e_22:6), and Ether linked Phosphatidylcholine 16:0 22:6 (EtherPC 16:0e_22:6). We also quantified 12 amino acids and observed a subtle dose-dependent reduction in the levels of L-Histidine. Exposure to atenolol did not impact larval locomotor activity based on a Visual Motor Response test. Taken together, atenolol at environmentally relevant levels decreased heart rate of developing zebrafish and altered lipid content. As such, exposure to beta-blockers like atenolol may have negative consequences for developmental trajectories and growth of aquatic species.

Proteome changes in an aquatic invertebrate consumer in response to different nutritional stressors.

Nutrient imbalances in zooplankton are caused by the differences in elemental content of producers and the demand for elements in consumers, which alter the life-history traits in consumers. Changes in life-history traits are mediated through metabolic pathways that affect gene expression and the metabolome. However, less is known about proteomic changes to elemental-limitation in zooplankton. Here, we grew Daphnia pulex under high food quantity and quality (HF), low food quantity (LF), and phosphorus (P)-limited (PL) diets for six days and measured growth, elemental composition, and the proteome. Daphnids in both LF and PL diets grew less. Animals in LF diets had less carbon (C), while daphnids in PL diets had less P compared to HF fed animals. In total, we identified 1719 proteins that were used in a partial least squares regression discriminant analysis (PLS-DA). Focusing on a subset of the proteome, the PLS-DA resulted in a clear separation between animals fed HF diets and PL and LF diets. Many proteome changes in nutrient-limited diets are associated with growth, reproduction, lipid metabolism, and nutrient assimilation. Regardless of the limiting nutrient, there were less hemoglobin and small subunit processome component proteins compared to HF fed animals. Daphnids fed LF diets had less vitellogenin fused superoxide dismutase and more lipid-droplet hydrolase, whereas Daphnia fed PL diets had higher abundances of cytochrome P450 and serine protease. Our proteome results compliment other "omic" studies that could be used to study Daphnia physiology in lakes.

Detection of SARS-CoV-2 Proteins in Wastewater Samples by Mass Spectrometry.

The recent COVID-19 pandemic overwhelmed the health system worldwide, and there was a need to track outbreaks and try to use this information as an early warning system. Wastewater-based epidemiology (WBE) enabled detection of the SARS-CoV-2 virus in wastewater treatment plant influents. Until now, the most used technique for this detection has been the quantitative polymerase chain reaction (qPCR)-based quantification of SARS-CoV-2 RNA. This study proposes a mass spectrometry (MS)-based method that detected specific SARS-CoV-2 proteins in wastewater, 5 and 6 days ahead of the case data for two municipalities. We identified unique peptides of eight proteins related to the SARS-CoV-2 virus and COVID-19 infection. We detected the nonstructural protein (NSP) pp1ab (transcribed after host cell infection) most frequently in all of the samples. As a result, we suspect that in the active cases of COVID-19, the pp1ab protein is present in high abundance in the urine and feces and that this protein could be used as an alternative biomarker. These data were collected before mass vaccination occurred in the population.

Pasteurization, storage conditions and viral concentration methods influence RT-qPCR detection of SARS-CoV-2 RNA in wastewater.

The COVID-19 pandemic presents many public health challenges including the tracking of infected individuals from local to regional scales. Wastewater surveillance of viral RNA has emerged as a complementary approach to track and monitor the presence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus in a variety of communities of different land use and population size. In the present study, we investigate how five different parameters (pasteurization, storage temperature, storage time, polyethylene glycol (PEG) concentration, and pellet mass) affect the detection of the SARS-CoV-2 N gene and fecal abundance indicator pepper mild mottle virus (PMMoV) gene. Pre-treatment of 24-h composite wastewater samples (n = 14) by pasteurization at 60 °C resulted in a significant reduction of total RNA concentration and copies of the SARS-CoV-2 N gene copies/L (paired Student's t-test, P < 0.05). Comparing the wastewater samples collected from 6 wastewater treatment plants (WWTPs) for a storage period of 7 and 14 days at 4 °C, -20 °C and -80 °C, demonstrated a decrease in SARS-CoV-2 N gene copies/L when samples were stored for 14 days at -20 °C. Polyethylene glycol-NaCl for purification and concentration of viral particles from the wastewater samples demonstrated that a short PEG incubation of 2 h during centrifugation at 4 °C was sufficient for the consistent detection of the SARS-CoV-2 N gene from a 30 mL sample volume. Combined, this paper presents method recommendations for developing a reliable, accurate, sensitive, and reproducible estimation of the SARS-CoV-2 virus in diverse domestic wastewater samples.

Fish tissue accumulation and proteomic response to microcystins is species-dependent.

Cyanotoxins including microcystins are increasing globally, escalating health risks to humans and wildlife. Freshwater fish can accumulate and retain microcystins in tissues; however, uptake and depuration studies thus far have not exposed fish to microcystins in its intracellular state (i.e., cell-bound or conserved within cyanobacteria), which is a primary route of exposure in the field, nor have they investigated sublethal molecular-level effects in tissues, limiting our knowledge of proteins responsible for microcystin toxicity pathways in pre-to-postsenescent stages of a harmful algal bloom. We address these gaps with a 2-wk study (1 wk of 'uptake' exposure to intracellular microcystins (0-40 µg L-1) produced by Microcystis aeruginosa followed by 1 wk of 'depuration' in clean water) using Rainbow Trout (Oncorhynchus mykiss) and Lake Trout (Salvelinus namaycush). Liver and muscle samples were collected throughout uptake and depuration phases for targeted microcystin quantification and nontargeted proteomics. For both species, microcystins accumulated at a higher concentration in the liver than muscle, and activated cellular responses related to oxidative stress, apoptosis, DNA repair, and carcinogenicity. However, intraspecific proteomic effects between Rainbow Trout and Lake Trout differed, and interspecific accumulation and retention of microcystins in tissues within each species also differed. We demonstrate that fish do not respond the same to cyanobacterial toxicity within and among species despite being reared in the same environment and diet.

Cyanotoxins within and Outside of Microcystis aeruginosa Cause Adverse Effects in Rainbow Trout (Oncorhynchus mykiss).

The global expansion of toxic Microcystis blooms, and production of cyanotoxins including microcystins, are an increasing risk to freshwater fish. Differentiating intracellular and extracellular microcystin toxicity pathways (i.e., within and outside of cyanobacterial cells) in fish is necessary to assess the severity of risks to populations that encounter harmful algal blooms in pre-to-postsenescent stages. To address this, adult and juvenile Rainbow Trout (Oncorhynchus mykiss) were, respectively, exposed for 96 h to intracellular and extracellular microcystins (0, 20, and 100 µg L-1) produced by Microcystis aeruginosa. Fish were dissected at 24 h intervals for histopathology, targeted microcystin quantification, and nontargeted proteomics. Rainbow Trout accumulated intracellular and extracellular microcystins in all tissues within 24 h, with greater accumulation in the extracellular state. Proteomics revealed intracellular and extracellular microcystins caused sublethal toxicity by significantly dysregulating proteins linked to the cytoskeletal structure, stress responses, and DNA repair in all tissues. Pyruvate metabolism in livers, anion binding in kidneys, and myopathy in muscles were also significantly impacted. Histopathology corroborated these findings with evidence of necrosis, apoptosis, and hemorrhage at similar severity in both microcystin treatments. We demonstrate that sublethal concentrations of intracellular and extracellular microcystins cause adverse effects in Rainbow Trout after short-term exposure.

Hypoxic responses in Oncorhynchus mykiss involve angiogenesis, lipid, and lactate metabolism, which may be triggered by the cortisol stress response and epigenetic methylation.

The incidence of hypoxia in water bodies is increasing more rapidly than aquatic life can adapt. This study aimed to determine the effects of hypoxia on fish physiology, as well as protein expression through proteomics. To do this, 40 rainbow trout were divided into normoxic control (11.5 mg/L dissolved oxygen) and hypoxic treatment (5 mg/L dissolved oxygen) tanks for a period of 7 days. Fish were then anesthetized and blood was sampled. Fish were then euthanized and heart and liver samples were taken. Blood glucose, cortisol and lipid, body and liver mass, fork length, hematocrit and, blood cell counts and global heart methylation were measured. Red blood cell counts were significantly lower, while hematocrit and mean corpuscular volume were significantly higher in the hypoxic treatment. Global DNA methylation was significantly decreased in hypoxic heart tissue. Plasma cortisol and 18:1 monoacylglyerol increased, while 15:0-18:1 phosphatidylethanolamine, and 18:1 lysophosphatidylethanolamine decreased in plasma of rainbow trout under hypoxic conditions. Plasma proteomics revealed 70 significantly altered proteins (p < 0.05) in the hypoxia treatment (Data are available via ProteomeXchange with identifier PXD026589). Many of these molecular changes appear to be related to the observed increase in red blood cell volume and epigenetic modifications, as well as to angiogenesis, lipid, and glucose metabolism. This study highlights a range of cellular and molecular responses in the blood and plasma of freshwater fish that may be phenotypic adaptions to hypoxia, and that could aid in diagnosing the health status of wild fish populations using several, potential, discovered biomarkers.

An 'omics approach to investigate the growth effects of environmentally relevant concentrations of guanylurea exposure on Japanese medaka (Oryzias latipes).

Metformin is a widely prescribed pharmaceutical used in the treatment of numerous human health disorders, including Type 2 Diabetes, and as a results of its widespread use, metformin is thought to be the most prevalent pharmaceutical in the aquatic environment by weight. The removal of metformin during the water treatment process is directly related to the formation of its primary degradation product, guanylurea, generally present at higher concentrations in surface waters relative to metformin. Growth effects observed in 28-day early life stage (ELS) Japanese medaka exposed to guanylurea were found to be similar to growth effects in 28-day ELS medaka exposed to metformin; however, effect concentrations were orders of magnitude below those of metformin. The present study uses a multi-omics approach to investigate potential mechanisms by which low-level, 1 ng · L-1 nominal, guanylurea exposure may lead to altered growth in 28-day post hatch medaka via shotgun metabolomics and proteomics and qPCR. Specifically, analyses show 6 altered metabolites, 66 altered proteins and 2 altered genes. Collectively, metabolomics, proteomics, and gene expression data (using qPCR) indicate that developmental exposure to guanylurea exposure alters a number of important pathways related to the overall health of ELS fish, including biomolecule metabolism, cellular energetics, nervous system function/development, cellular communication and structure, and detoxification of reactive oxygen species, among others. To our knowledge, this is the first study to both report the molecular level effects of guanylurea on non-target aquatic organisms, and to relate molecular-level changes to whole organism effects.