Characterization of Adverse Outcomes from Legacy-Contaminated Groundwater Exposure to Early Life Stages of Fathead Minnow.

Complex mixtures of chemicals present in groundwater at legacy-contaminated industrial sites can pose significant risks to adjacent surface waters. The combination of short-term molecular and chronic apical effect assessments is a promising approach to characterize the potential hazard of such complex mixtures. The objectives of this study were to: (1) assess the apical effects (survival, growth, development, and liver histopathology) after chronic exposure of early life stages (ELSs) of fathead minnows (FHM; Pimephales promelas) to contaminated groundwater from a legacy-contaminated pesticide manufacturing and packaging plant, and (2) identify possible molecular mechanisms responsible for these effects by comparing results to mechanistic outcomes previously determined by a short-term reduced transcriptome assay (EcoToxChips). This study revealed a significant increase in mortality and prevalence of spinal curvatures, as well as a significant reduction in the length of FHMs exposed to the groundwater mixtures in a concentration-dependent manner. There was an increasing trend in the prevalence of edema in FHMs, though not significantly different from controls. Additionally, no histopathological effects were observed in the liver of FHMs exposed to the groundwater mixtures. Short-term molecular outcomes determined in a parallel study were found to be informative of chronic apical outcomes, including cardiotoxicity, spinal deformities, and liver toxicity. Overall, the results observed in this study demonstrated that short-term transcriptomics analyses could support the hazard assessment of complex contaminated sites.

A Novel Multispecies Toxicokinetic Modeling Approach in Support of Chemical Risk Assessment.

Standardized laboratory tests with a limited number of model species are a key component of chemical risk assessments. These surrogate species cannot represent the entire diversity of native species, but there are practical and ethical objections against testing chemicals in a large variety of species. In previous research, we have developed a multispecies toxicokinetic model to extrapolate chemical bioconcentration across species by combining single-species physiologically based toxicokinetic (PBTK) models. This "top-down" approach was limited, however, by the availability of fully parameterized single-species models. Here, we present a "bottom-up" multispecies PBTK model based on available data from 69 freshwater fishes found in Canada. Monte Carlo-like simulations were performed using statistical distributions of model parameters derived from these data to predict steady-state bioconcentration factors (BCFs) for a set of well-studied chemicals. The distributions of predicted BCFs for 1,4-dichlorobenzene and dichlorodiphenyltrichloroethane largely overlapped those of empirical data, although a tendency existed toward overestimation of measured values. When expressed as means, predicted BCFs for 26 of 34 chemicals (82%) deviated by less than 10-fold from measured data, indicating an accuracy similar to that of previously published single-species models. This new model potentially enables more environmentally relevant predictions of bioconcentration in support of chemical risk assessments.

Toxicokinetic Models for Bioconcentration of Organic Contaminants in Two Life Stages of White Sturgeon (Acipenser transmontanus).

The white sturgeon (Acipenser transmontanus) is an endangered ancient fish species that is known to be particularly sensitive to certain environmental contaminants, partly because of the uptake and subsequent toxicity of lipophilic pollutants prone to bioconcentration as a result of their high lipid content. To better understand the bioconcentration of organic contaminants in this species, toxicokinetic (TK) models were developed for the embryo-larval and subadult life stages. The embryo-larval model was designed as a one-compartment model and validated using whole-body measurements of benzo[a]pyrene (B[a]P) metabolites from a waterborne exposure to B[a]P. A physiologically based TK (PBTK) model was used for the subadult model. The predictive power of the subadult model was validated with an experimental data set of four chemicals. Results showed that the TK models could accurately predict the bioconcentration of organic contaminants for both life stages of white sturgeon within 1 order of magnitude of measured values. These models provide a tool to better understand the impact of environmental contaminants on the health and the survival of endangered white sturgeon populations.

Development of a Comprehensive Toxicity Pathway Model for 17α-Ethinylestradiol in Early Life Stage Fathead Minnows (Pimephales promelas).

There is increasing pressure to develop alternative ecotoxicological risk assessment approaches that do not rely on expensive, time-consuming, and ethically questionable live animal testing. This study aimed to develop a comprehensive early life stage toxicity pathway model for the exposure of fish to estrogenic chemicals that is rooted in mechanistic toxicology. Embryo-larval fathead minnows (FHM; Pimephales promelas) were exposed to graded concentrations of 17α-ethinylestradiol (water control, 0.01% DMSO, 4, 20, and 100 ng/L) for 32 days. Fish were assessed for transcriptomic and proteomic responses at 4 days post-hatch (dph), and for histological and apical end points at 28 dph. Molecular analyses revealed core responses that were indicative of observed apical outcomes, including biological processes resulting in overproduction of vitellogenin and impairment of visual development. Histological observations indicated accumulation of proteinaceous fluid in liver and kidney tissues, energy depletion, and delayed or suppressed gonad development. Additionally, fish in the 100 ng/L treatment group were smaller than controls. Integration of omics data improved the interpretation of perturbations in early life stage FHM, providing evidence of conservation of toxicity pathways across levels of biological organization. Overall, the mechanism-based embryo-larval FHM model showed promise as a replacement for standard adult live animal tests.

Developmental expression profiles and thyroidal regulation of cytokines during metamorphosis in the amphibian Xenopus laevis.

Early life-stages of amphibians rely on the innate immune system for defense against pathogens. While thyroid hormones (TH) are critical for metamorphosis and later development of the adaptive immune system, the role of TH in innate immune system development is less clear. An integral part of the innate immune response are pro-inflammatory cytokines - effector molecules that allow communication between components of the immune system. The objective of this study was to characterize the expression of key pro-inflammatory cytokines, tumor necrosis factor-α (TNFα), interleukin-1ß (IL-1ß) and interferon-γ (IFN-γ), throughout amphibian development and determine the impacts of thyroidal modulation on their expression. Xenopus laevis were sampled at various stages of development encompassing early embryogenesis to late prometamorphosis and cytokine expression was measured by real-time PCR. Expression of TNFα and IL-1ß were transient over development, increasing with developmental stage, while IFN-γ remained relatively stable. Functionally athyroid, premetamorphic tadpoles were exposed to thyroxine (0.5 and 2 µg/L) or sodium perchlorate (125 and 500 µg/L) for seven days. Tadpoles demonstrated characteristic responses of advanced development with thyroxine exposure and delayed development (although to a lesser extent) and increased thyroid gland area and follicular cell height with sodium perchlorate exposure. Exposure to thyroxine for two days resulted in decreased expression of IL-1ß in tadpole trunks. Sodium perchlorate had negligible effects on cytokine expression. Overall, these results demonstrate that cytokine transcript levels vary with stage of tadpole development but that their ontogenic regulation is not likely exclusively influenced by thyroid status. Understanding the direct and indirect effects of altered hormone status may provide insight into potential mechanisms of altered immune function during amphibian development.

In vitro assessment of endocrine disrupting potential of naphthenic Acid fractions derived from oil sands-influenced water.

Oil sands-influenced process waters have been observed to cause reproductive effects and to induced CYP1A activity in fishes; however, little progress has been made in determining causative agents. Naphthenic acids (NAs) are the predominant organic compounds in process-affected waters, but due to the complexity of the mixture, it has been difficult to examine causal linkages in fishes. The aim of this study was to use in vitro assays specific to reproductive and CYP1A mechanisms to determine if specific acid extractable fractions of NAs obtained from oil sands-influenced water are active toward reproductive processes or interact with the Ah receptor responsible for CYP1A activity. NAs were extracted from aged oil sands-influenced waters by use of acid precipitation, and the mixture was fractionated into three acidic and one neutral fraction. The four fractions were examined for Ah receptor-mediated potency by use of the H4IIE-luc bioassay, effects on production of steroid hormones by use of the H295R steroidogenesis assay, and sex steroid receptor binding activity using the yeast estrogen screen and yeast androgen screen. The mixtures were characterized by high resolution mass spectrometry, (1)H nuclear magnetic resonance, and attenuated total reflectance infrared spectroscopy. The neutral fraction elicited Ah-receptor mediated activity after 24 h but not after 48 or 72 h. None of the fractions contained measurable levels of estrogen or androgen receptor agonists nor did they cause reductions in steroidogenesis. A number of fractions showed antiestrogenic or antiandrogenicity potency, with the neutral and main acidic fractions being the most potent. Neutral aromatic compounds are likely responsible for the CYP1A activity observed. Direct estrogenic, androgenic, or steroidogenic mechanisms are unlikely for NAs based on these results, but NAs act as potent antiandrogen or antiestrogens.

Using near infrared transmittance to generate sorted fractions of Fusarium-infected wheat and the impact on broiler performance.

The objective of this study was to investigate the effects of naturally contaminated Fusarium wheat containing deoxynivalenol (DON) on growth and performance of broiler chickens from 0 to 35 d. The BoMill TriQ individual kernel sorting technology uses near infrared transmittance (NIT) spectra to separate Fusarium-damaged kernels (FDK) from healthy kernels based on individual kernel CP. Three Fusarium-contaminated wheat sources were individually sorted into 3 test fractions: outlier (10% of the source), high mycotoxin (20% of the source), and low mycotoxin (70% of the source). These fractions were reconstituted into 4 ratios-M0, M20, M40, and M60-relating to the proportion of the high mycotoxin fraction in the reconstituted diets. These 12 reconstituted wheat sources with varying levels of DON were incorporated at ∼70% (starter) or ∼75% (grower/finisher) into diets. The fractions of wheat used had FDK ranging from 0.1 to 25.8% and DON from 0.0 to 14.3 ppm. A total of 480 newly hatched Ross 308 male broilers were randomly divided into 96 cages. Each test diet was assigned to 8 replicates with 5 birds per replicate cage. At 21 d, 180 birds were transferred to 36 cages, allowing 3 replicates of 5 birds per diet until 35 d. A factorial arrangement analysis compared the 3 wheat sources and 4 ratios produced from each sorted wheat. Growth and performance were evaluated as BW (g), feed intake (FI; g/bird/day), feed conversion ratio (FCR; g:g), AME (kcal ME/kg diet), nitrogen retention (NR; %), and mortality (%) for 0 to 21 d and 21 to 35 d. Results indicate no significant difference in BW, FI, and FCR (P > 0.05). Significant differences were seen in AME and NR (P < 0.01). This study demonstrates the potential of this novel sorting technology to produce naturally contaminated diets with a large range of mycotoxin concentrations from a single wheat source to enable future investigations of mycotoxin exposure in any species.

Hazard assessment of complex legacy-contaminated groundwater mixtures using a novel approach method in adult fathead minnows.

Traditional risk assessment methods face challenges in the determination of drivers of toxicity for complex mixtures such as those present at legacy-contaminated sites. Bioassay-driven analysis across several levels of biological organization represents an approach to address these obstacles. This study aimed to apply a novel transcriptomics tool, the EcoToxChip, to characterize the effects of complex mixtures of contaminants in adult fathead minnows (FHMs) and to compare molecular response patterns to higher-level biological responses. Adult FHMs were exposed for 4 and 21 days to groundwater mixtures collected from a legacy-contaminated site. Adult FHM showed significant induction of micronuclei in erythrocytes, decrease in reproductive capacities, and some abnormal appearance of liver histology. Parallel EcoToxChip analyses showed a high proportion of upregulated genes and a few downregulated genes characteristic of compensatory responses. The three most enriched pathways included thyroid endocrine processes, transcription and translation cellular processes, and xenobiotics and reactive oxygen species metabolism. Several of the most differentially regulated genes involved in these biological pathways could be linked to the apical outcomes observed in FHMs. We concluded that molecular responses as determined by EcoToxChip analysis show promise for informing of apical outcomes and could support risk assessments of complex contaminated sites.

Time-course of oral toxicity to contaminated groundwater in male Sprague Dawley rats.

Assessing toxicity of complex mixtures of contaminants from industrial sites with historic and ongoing contamination remains a challenge for risk assessors. Groundwater from a pesticide packaging site in Canada containing a complex mixture of known and unknown contaminants was examined in male rats to determine the target organ toxicity. This study determined the time-course of toxicity (7, 14, 28, and 60 days) following ad libitum oral exposure to 0.05% v/v contaminated groundwater compared to tap water (control) in male Sprague Dawley rats (n=5 /group/time). Exposure to groundwater resulted in inflammation, indicated by a statistically significant increase in plasma lymphocyte and neutrophil counts on days 7 and 60, respectively, but a reduction in the plasma alpha 2 macroglobulin levels by day 60. Gonadotoxicity was indicated by a reduced Johnsen score (grading spermatogenesis) in all exposed groups at all time points, while seminiferous epithelial height was reduced on days 7, 14, and 28 compared to controls. Plasma testosterone was reduced in exposed groups on days 7 and 28, accompanied by elevated testicular lipid peroxidation at all time points compared to control. In contrast, lipid peroxidation in the lungs from exposed rats was elevated on days 7, 14, and 28. Plasma symmetric dimethylarginine was elevated on day 14 in the exposed group indicating renal impairment. Taken together, these results indicate that testes, kidney, immune and lung are target organs for the contaminated groundwater from this industrial site. The current study highlights the challenge in hazard assessment for complex mixtures and highlights the need for effects-directed analysis and the continued, albeit limited, use of animal models in toxicity testing.

Comparing Transcriptomic Responses to Chemicals Across Six Species Using the EcoToxChip RNASeq Database.

The EcoToxChip project includes RNA-sequencing data from experiments involving model (Japanese quail, fathead minnow, African clawed frog) and ecological (double-crested cormorant, rainbow trout, northern leopard frog) species at multiple life stages (whole embryo and adult) exposed to eight chemicals of environmental concern known to perturb a wide range of biological systems (ethinyl estradiol, hexabromocyclododecane, lead, selenomethionine, 17ß trenbolone, chlorpyrifos, fluoxetine, and benzo[a]pyrene). The objectives of this short communication were to (1) present and make available this RNA-sequencing database (i.e., 724 samples from 49 experiments) under the FAIR principles (FAIR data are data which meet principles of findability, accessibility, interoperability, and reusability), while also summarizing key meta-data attributes and (2) use ExpressAnalyst (including the Seq2Fun algorithm and EcoOmicsDB) to perform a comparative transcriptomics analysis of this database focusing on baseline and differential transcriptomic changes across species-life stage-chemical combinations. The database is available in NCBI GEO under accession number GSE239776. Across all species, the number of raw reads per sample ranged between 13 and 58 million, with 30% to 79% of clean reads mapped to the "vertebrate" subgroup database in EcoOmicsDB. Principal component analyses of the reads illustrated separation across the three taxonomic groups as well as some between tissue types. The most common differentially expressed gene was CYP1A1 followed by CTSE, FAM20CL, MYC, ST1S3, RIPK4, VTG1, and VIT2. The most common enriched pathways were metabolic pathways, biosynthesis of cofactors and biosynthesis of secondary metabolites, and chemical carcinogenesis, drug metabolism, and metabolism of xenobiotics by cytochrome P450. The RNA-sequencing database in the present study may be used by the research community for multiple purposes, including, for example, cross-species investigations, in-depth analyses of a particular test compound, and transcriptomic meta-analyses. Environ Toxicol Chem 2024;00:1-6. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Maternal Transfer and Apical and Physiological Effects of Dietary Hexabromocyclododecane Exposure in Parental Fathead Minnows (Pimephales promelas).

Hexabromocyclododecane (HBCD) is a persistent organic pollutant that has been characterized as an endocrine disruptor, undergoes maternal transfer, and hinders development and growth in oviparous organisms. The present study examined the apical effects of dietary HBCD (11.5, 36.4, 106 mg/kg, wet wt) on adult fathead minnow exposed for 49 days and the subsequent accumulation and maternal transfer kinetics in adult tissue and eggs, respectively. Exposed adults displayed a significant increase in egg production in the medium treatment group, but no other significant effects were noted. Maternal transfer of dietary HBCD had a similar egg-to-muscle ratios (EMR) in the low and medium treatment groups (1.65 and 1.27 [wet wt], respectively). However, the high treatment group deviated from other treatments with an EMR of 4.2 (wet wt), potentially due to differences in total lipid content in food and/or reaching diffusion/lipid saturation limits in adult tissue, resulting in lower accumulation in the adult muscle tissue. A positive correlation was observed between egg HBCD concentration and time of exposure, which indicates that maternal transfer of HBCD is of concern in fish, and further studies should be conducted to fully elucidate the potential adverse effects that may be observed in the early life stage of oviparous organisms. Environ Toxicol Chem 2023;42:143-153. © 2022 SETAC.

Development and Initial Testing of EcoToxChip, a Novel Toxicogenomics Tool for Environmental Management and Chemical Risk Assessment.

New approach methods (NAMs) are increasingly important to help accelerate the pace of ecological risk assessment and offer more ethical, affordable, and efficient alternatives to traditional toxicity tests. In the present study, we describe the development, technical characterization, and initial testing of a toxicogenomics tool, EcoToxChip (384-well quantitative polymerase chain reaction [qPCR] array), to support chemical management and environmental monitoring for three laboratory model species-fathead minnow (Pimephales promelas), African clawed frog (Xenopus laevis), and Japanese quail (Coturnix japonica). Chip design, including gene selection, was informed by a diverse end-user group and quality control metrics (e.g., primer assay, reverse transcription, and PCR efficiency) performed well based on a priori established criteria. Correlation with RNA sequencing (seq) data provided additional confidence in this novel toxicogenomics tool. Although the present study represents an initial testing of only 24 EcoToxChips for each of the model species, the results provide increased confidence in the robustness/reproducibility of EcoToxChips for evaluating perturbations in gene expression associated with chemical exposure and thus, this NAM, combined with early-life stage toxicity testing, could augment current efforts for chemical prioritization and environmental management. Environ Toxicol Chem 2023;42:1763-1771. © 2023 SETAC.

Characterization of molecular and apical effects of legacy-contaminated groundwater on early life stages of fathead minnows.

Mechanistic toxicology approaches represent a promising alternative to traditional live animal testing; however, the often-noted uncertainties concerning the linkages between effects observed at molecular and apical levels curtails the adoption of such approaches. The objective of this study was to apply a novel transcriptomics tool, EcoToxChips, to characterize the effects of complex mixtures of contaminants in fish and to compare molecular response patterns to higher-level biological responses including swimming behavior, deformities, and mortality. Fathead minnow (FHM) embryos were exposed for seven days to increasing concentrations of groundwater collected from moderate (MIAZ) and high (HIAZ) industrial activity zones of a legacy contaminated site. There was a concentration-dependent disruption of photo-dependent swimming responses associated with avoidance behavior patterns and spinal deformities (HIAZ and MIAZ), and an induction of pericardial edema and mortality (HIAZ-10%). Parallel EcoToxChip analyses showed a shift from a majority of upregulated genes at lower concentrations to a majority of downregulated genes at higher concentrations for both treatment conditions. Many of the significantly differentially regulated genes were involved in biological pathways including induction of oxidative stress, activating of several metabolic processes and growth, cell death, and inhibition of signal transduction signaling processes. Several contaminants present in the groundwater mixtures could have contributed to an exceedance of antioxidant system capacities that possibly led to the deformities, altered swimming behaviours, and mortality observed in FHMs. Therefore, molecular response patterns could be linked to apical outcomes observed in this study. Overall, the results observed in this study demonstrate that transcriptomics approaches such as the EcoToxChip system could be supportive of risk assessment of complex contaminated sites.

Developmental profiles and thyroid hormone regulation of brain transcripts in frogs: a species comparison with emphasis on Physalaemus pustulosus.

In amphibians, thyroid hormones (THs) are considered key regulators of brain remodeling during metamorphosis, while sex steroids (estrogens and androgens) control sexual differentiation and gonadal development. However, these two endocrine axes can interact during tadpole brain development. Previously, we demonstrated that THs affect sex steroid-related gene expression in the developing brain of Silurana tropicalis and Rana pipiens; however, the gene expression changes differed between species. We chose to study a third anuran species, Physalaemus pustulosus, to test new hypotheses about the role of THs in the regulation of brain gene expression. We first established developmental transcript profiles of TH- and sex steroid-related genes in the brain of P. pustulosus. Then, following the same protocols as in our previous studies, we investigated triiodothyronine (T3) regulation of brain transcripts in premetamorphic P. pustulosus and then compared the results with our previous two studies. In the case of TH-related genes, TH receptor beta (trbeta) and deiodinase type 3 (dio3), mRNA developmental profiles were similar in the three species and with respect to other species in the published literature. However, the profiles of TH receptor alpha (tralpha) and deiodinase type 2 (dio2) mRNA revealed differences between anuran species. Among the three anurans we have studied, the direction of the T3 regulation of TH-related genes was overall similar, but the magnitude of gene expression change differed depending on the rate of metamorphosis in a given species. For the sex steroid-related genes, each species exhibited similar developmental profiles but differed in their response to T3. In P. pustulosus, T3 reduced the expression of aromatase (cyp19) while increasing mRNA levels of androgen and estrogen receptors. These results are similar to previous research in R. pipiens but differ from data for S. tropicalis, for which we found an increase in androgen synthesis enzymes but no effect on cyp19. Together, we propose that T3 has the potential to induce the brain androgen system in anurans. This could be achieved by increasing androgen synthesis enzymes (S. tropicalis) or by decreasing estrogen synthesis (due to a decrease in cyp19 in P. pustulosus and R. pipiens). In conclusion, we demonstrated that mechanisms of hormone interactions differ between anuran species, but in all cases T3 appears to affect the balance of sex steroids in the brain, stimulating the androgen system. We have shown that the regulation of sex steroid-related genes by T3 is more similar among closely related species than species with similar reproductive and developmental characteristics.

Immunological impacts of oil sands-affected waters on rainbow trout evaluated using an in situ exposure.

Rainbow trout were exposed in situ to oil sands-affected waters for 21 d, either with or without an immune stimulation using inactivated Aeromonas salmonicida. Three aquatic systems were utilized for the experiment: a pond containing oil sands tailings capped with approximately 3 m of natural surface water, a second pond where unextracted oil sands materials were deposited in the watershed, and a reservoir receiving Athabasca River water as a reference caging location. The three systems showed a gradient of oil sands-related compounds, most notably, total naphthenic acids were highest in the system containing tailings (13 mg/L), followed by the system influenced by unextracted oil sands (4 mg/L), followed by the reference cage location (1 mg/L). Biochemical and chemical measures of exposure in rainbow trout showed the same trend, with the tailings-influenced system having the highest hepatic EROD activity and elevated bile fluorescence measured at phenanthrene wavelengths. Trout caged in the tailings-influenced location had significantly fewer leukocytes and smaller spleens as compared to the reference fish, though liver size and condition factor were unaffected. Fish in the tailings-influenced waters also demonstrated increased fin erosion, indicative of opportunistic infection. The trout exposed to tailing-influenced waters also showed a significantly decreased ability to produce antibodies to the inactivated A. salmonicida. Given the complexity of the exposure conditions, exact causative agents could not be determined, however, naphthenic acids, polycyclic aromatic hydrocarbons and pH correlate with the immunotoxic effects while elevated salinity or metals seem unlikely causes.

Correlation between in vitro toxicity of pesticides and in vivo risk guidelines in support of complex operating site risk management: A meta-analysis.

In vitro cell systems can support hazard characterization and identify mechanisms involved in toxicity; however, using in vitro data for risk assessment still is challenging. As part of an effort to develop approaches for a complex operating site used for biocide packaging and distribution, we evaluated in vitro assays that could be used in a site management format. Across 66 studies, 108 pesticides were assessed on ten human-derived cell types at four endpoints. In vitro IC50s were compared to in vivo guidelines, NOEL/NOAELs, and ADIs using Spearman correlation and linear regression models. While human neuroblastoma cells (SH-SY5Y) were the most sensitive, HepG2 was the most used cell line in evaluating the toxicity of pesticides. Amongst the ten human cell lines, the IC50s derived from SH-SY5Y cells, using MTT-24 & 48 h (the most used assay) correlated (rho = 0.56-0.79; p < 0.05) with ADIs and NOEL/NOAELs. Although in vitro cell systems have some limitations, the correlation between in vitro data derived from SH-SY5Y cells and in vivo safety guidelines can provide site investigators with a tool to survey and prioritize areas and media of concern at complex operating sites impacted by pesticide mixtures.

Characterizing toxicity pathways of fluoxetine to predict adverse outcomes in adult fathead minnows (Pimephales promelas).

Current ecotoxicity testing programs are impeded as they predominantly rely on slow and expensive animal tests measuring adverse outcomes. Therefore, new approach methodologies (NAMs) increasingly involve short-term mechanistic assays that employ molecular endpoints to predict adverse outcomes of regulatory relevance. This study aimed to elucidate the application of NAMs in adult fathead minnows using fluoxetine (FLX) as a model compound. Fish were exposed to three FLX concentrations (measured: 2.42, 10.7, and 56.7 µgL-1) and a control. After 96 h, molecular toxicity signatures were characterized using proteomics and transcriptomics analyses in livers and brains of a sub-set of fish. The remaining fish were sampled at 21 days and assessed for liver histopathology and morphometric measurements. Fecundity was monitored throughout the study. In the livers, 56.7 µgL-1 FLX caused enrichment of PPAR signaling in the proteome and fatty acid-related pathways in the transcriptome, potential upstream responses that led to lipid-type vacuolation of hepatocytes, observed via histopathology. Upregulated genes in the brain suggested alterations in serotonin-related signaling processes and reproductive behaviour, which may explain the observed significant decrease in fecundity. While the relationships between molecular responses and adverse outcomes remain complex, this research provided important insights into the mechanistic toxicity of FLX.

RNA in Municipal Wastewater Reveals Magnitudes of COVID-19 Outbreaks across Four Waves Driven by SARS-CoV-2 Variants of Concern.

There are no standardized protocols for quantifying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in wastewater to date, especially for population normalization. Here, a pipeline was developed, applied, and assessed to quantify SARS-CoV-2 and key variants of concern (VOCs) RNA in wastewater at Saskatoon, Canada. Normalization approaches using recovery ratio and extraction efficiency, wastewater parameters, or population indicators were assessed by comparing to daily numbers of new cases. Viral load was positively correlated with daily new cases reported in the sewershed. Wastewater surveillance (WS) had a lead time of approximately 7 days, which indicated surges in the number of new cases. WS revealed the variant α and δ driving the third and fourth wave, respectively. The adjustment with the recovery ratio and extraction efficiency improved the correlation between viral load and daily new cases. Normalization of viral concentration to concentrations of the artificial sweetener acesulfame K improved the trend of viral load during the Christmas and New Year holidays when populations were dynamic and variable. Acesulfame K performed better than pepper mild mottle virus, creatinine, and ammonia for population normalization. Hence, quality controls to characterize recovery ratios and extraction efficiencies and population normalization with acesulfame are promising for precise WS programs supporting decision-making in public health.

Comparative analysis of transcriptomic points-of-departure (tPODs) and apical responses in embryo-larval fathead minnows exposed to fluoxetine.

Current approaches in chemical hazard assessment face significant challenges because they rely on live animal testing, which is time-consuming, expensive, and ethically questionable. These concerns serve as an impetus to develop new approach methodologies (NAMs) that do not rely on live animal tests. This study explored a molecular benchmark dose (BMD) approach using a 7-day embryo-larval fathead minnow (FHM) assay to derive transcriptomic points-of-departure (tPODs) to predict apical BMDs of fluoxetine (FLX), a highly prescribed and potent selective serotonin reuptake inhibitor frequently detected in surface waters. Fertilized FHM embryos were exposed to graded concentrations of FLX (confirmed at < LOD, 0.19, 0.74, 3.38, 10.2, 47.5 µg/L) for 32 days. Subsets of fish were subjected to omics and locomotor analyses at 7 days post-fertilization (dpf) and to histological and biometric measurements at 32 dpf. Enrichment analyses of transcriptomics and proteomics data revealed significant perturbations in gene sets associated with serotonergic and axonal functions. BMD analysis resulted in tPOD values of 0.56 µg/L (median of the 20 most sensitive gene-level BMDs), 5.0 µg/L (tenth percentile of all gene-level BMDs), 7.51 µg/L (mode of the first peak of all gene-level BMDs), and 5.66 µg/L (pathway-level BMD). These tPODs were protective of locomotor and reduced body weight effects (LOEC of 10.2 µg/L) observed in this study and were reflective of chronic apical BMDs of FLX reported in the literature. Furthermore, the distribution of gene-level BMDs followed a bimodal pattern, revealing disruption of sensitive neurotoxic pathways at low concentrations and metabolic pathway perturbations at higher concentrations. This is one of the first studies to derive protective tPODs for FLX using a short-term embryo assay at a life stage not considered to be a live animal under current legislations.

Does habitat quality matter to soil invertebrates in metal-contaminated soils?

This study investigated the influence of habitat quality (HQ) on the reproduction and bioenergetics (energy reserve and metabolic enzyme activities) of the oribatid mite, Oppia nitens, in response to cadmium (Cd). In the baseline toxicity test, Cd elevated the carbohydrate reserve of adult mites at intermediate Cd concentrations (88 and 175 mg Cd kg-1) but without a change in lipid and protein reserve across 0-700 mg Cd kg-1. The activities of glucose metabolism enzymes, glucose-6-phosphate dehydrogenase (G6PDH) and pyruvate kinase (PK) were inhibited in the mites at 700 mg Cd kg-1. Adult mites reared in high HQ soils had higher reproduction relative to mites from low HQ soils when exposed to Cd in OECD soil, but there was no difference in bioenergetics between mites from low and high HQ soils. Hence, HQ significantly (p = 0.024) influenced the reproduction of mites (i.e., juvenile production) irrespective of the Cd concentration in the OECD soil but did not significantly affect the bioenergetics of the mites. We suggest that habitat quality's effect could be more significant than metal concentration on the biological fitness (juvenile production) of O. nitens in metal-contaminated soils.