John P. Giesy


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A wastewater-based risk index for SARS-CoV-2 infections among three cities on the Canadian Prairie
Mohsen Asadi, Femi F. Oloye, Yuwei Xie, Jenna Cantin, Jonathan K. Challis, Kerry N. McPhedran, Warsame Yusuf, David Champredon, Ximing Pu, Chantel De Lange, Seba El-Baroudy, Mark R. Servos, Paul D. Jones, John P. Giesy, Markus Brinkmann
Science of The Total Environment, Volume 876

Wastewater surveillance (WWS) is useful to better understand the spreading of coronavirus disease 2019 (COVID-19) in communities, which can help design and implement suitable mitigation measures. The main objective of this study was to develop the Wastewater Viral Load Risk Index (WWVLRI) for three Saskatchewan cities to offer a simple metric to interpret WWS. The index was developed by considering relationships between reproduction number, clinical data, daily per capita concentrations of virus particles in wastewater, and weekly viral load change rate. Trends of daily per capita concentrations of SARS-CoV-2 in wastewater for Saskatoon, Prince Albert, and North Battleford were similar during the pandemic, suggesting that per capita viral load can be useful to quantitatively compare wastewater signals among cities and develop an effective and comprehensible WWVLRI. The effective reproduction number (Rt) and the daily per capita efficiency adjusted viral load thresholds of 85 × 106 and 200 × 106 N2 gene counts (gc)/population day (pd) were determined. These values with rates of change were used to categorize the potential for COVID-19 outbreaks and subsequent declines. The weekly average was considered 'low risk' when the per capita viral load was 85 × 106 N2 gc/pd. A 'medium risk' occurs when the per capita copies were between 85 × 106 and 200 × 106 N2 gc/pd. with a rate of change <100 %. The start of an outbreak is indicated by a 'medium-high' risk classification when the week-over-week rate of change was >100 %, and the absolute magnitude of concentrations of viral particles was >85 × 106 N2 gc/pd. Lastly, a 'high risk' occurs when the viral load exceeds 200 × 106 N2 gc/pd. This methodology provides a valuable resource for decision-makers and health authorities, specifically given the limitation of COVID-19 surveillance based on clinical data.

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A comparative analysis of the partitioning behaviour of SARS-CoV-2 RNA in liquid and solid fractions of wastewater
Patrick Breadner, Hadi A. Dhiyebi, Azar Fattahi, Nivetha Srikanthan, Samina Hayat, Marc G. Aucoin, Scott Joseph Boegel, Leslie M. Bragg, Paul M. Craig, Yuwei Xie, John P. Giesy, Mark R. Servos
Science of The Total Environment, Volume 895

As fragments of SARS-CoV-2 RNA can be quantified and measured temporally in wastewater, surveillance of concentrations of SARS-CoV-2 in wastewater has become a vital resource for tracking the spread of COVID-19 in and among communities. However, the absence of standardized methods has affected the interpretation of data for public health efforts. In particular, analyzing either the liquid or solid fraction has implications for the interpretation of how viral RNA is quantified. Characterizing how SARS-CoV-2 or its RNA fragments partition in wastewater is a central part of understanding fate and behaviour in wastewater. In this study, partitioning of SARS-CoV-2 was investigated by use of centrifugation with varied durations of spin and centrifugal force, polyethylene glycol (PEG) precipitation followed by centrifugation, and ultrafiltration of wastewater. Partitioning of the endogenous pepper mild mottled virus (PMMoV), used to normalize the SARS-CoV-2 signal for fecal load in trend analysis, was also examined. Additionally, two surrogates for coronavirus, human coronavirus 229E and murine hepatitis virus, were analyzed as process controls. Even though SARS-CoV-2 has an affinity for solids, the total RNA copies of SARS-CoV-2 per wastewater sample, after centrifugation (12,000 g, 1.5 h, no brake), were partitioned evenly between the liquid and solid fractions. Centrifugation at greater speeds for longer durations resulted in a shift in partitioning for all viruses toward the solid fraction except for PMMoV, which remained mostly in the liquid fraction. The surrogates more closely reflected the partitioning of SARS-CoV-2 under high centrifugation speed and duration while PMMoV did not. Interestingly, ultrafiltration devices were inconsistent in estimating RNA copies in wastewater, which can influence the interpretation of partitioning. Developing a better understanding of the fate of SARS-CoV-2 in wastewater and creating a foundation of best practices is the key to supporting the current pandemic response and preparing for future potential infectious diseases.

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Assessment of seasonality and normalization techniques for wastewater-based surveillance in Ontario, Canada
Hadi A. Dhiyebi, Jad Farah, Heather Ikert, Nivetha Srikanthan, Samina Hayat, Leslie M. Bragg, Asim Qasim, Mark Payne, Linda Kaleis, Caitlyn Paget, Dominika Celmer‐Repin, Arianne M. Folkema, Sarah Drew, Robert Delatolla, John P. Giesy, Mark R. Servos
Frontiers in Public Health, Volume 11

Introduction Wastewater-based surveillance is at the forefront of monitoring for community prevalence of COVID-19, however, continued uncertainty exists regarding the use of fecal indicators for normalization of the SARS-CoV-2 virus in wastewater. Using three communities in Ontario, sampled from 2021–2023, the seasonality of a viral fecal indicator (pepper mild mottle virus, PMMoV) and the utility of normalization of data to improve correlations with clinical cases was examined. Methods Wastewater samples from Warden, the Humber Air Management Facility (AMF), and Kitchener were analyzed for SARS-CoV-2, PMMoV, and crAssphage. The seasonality of PMMoV and flow rates were examined and compared by Season-Trend-Loess decomposition analysis. The effects of normalization using PMMoV, crAssphage, and flow rates were analyzed by comparing the correlations to clinical cases by episode date (CBED) during 2021. Results Seasonal analysis demonstrated that PMMoV had similar trends at Humber AMF and Kitchener with peaks in January and April 2022 and low concentrations (troughs) in the summer months. Warden had similar trends but was more sporadic between the peaks and troughs for PMMoV concentrations. Flow demonstrated similar trends but was not correlated to PMMoV concentrations at Humber AMF and was very weak at Kitchener ( r = 0.12). Despite the differences among the sewersheds, unnormalized SARS-CoV-2 (raw N1–N2) concentration in wastewater ( n = 99–191) was strongly correlated to the CBED in the communities ( r = 0.620–0.854) during 2021. Additionally, normalization with PMMoV did not improve the correlations at Warden and significantly reduced the correlations at Humber AMF and Kitchener. Flow normalization ( n = 99–191) at Humber AMF and Kitchener and crAssphage normalization ( n = 29–57) correlations at all three sites were not significantly different from raw N1–N2 correlations with CBED. Discussion Differences in seasonal trends in viral biomarkers caused by differences in sewershed characteristics (flow, input, etc.) may play a role in determining how effective normalization may be for improving correlations (or not). This study highlights the importance of assessing the influence of viral fecal indicators on normalized SARS-CoV-2 or other viruses of concern. Fecal indicators used to normalize the target of interest may help or hinder establishing trends with clinical outcomes of interest in wastewater-based surveillance and needs to be considered carefully across seasons and sites.

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Omicron COVID-19 Case Estimates Based on Previous SARS-CoV-2 Wastewater Load, Regional Municipality of Peel, Ontario, Canada
Lydia Cheng, Hadi A. Dhiyebi, Monali Varia, Kyle Atanas, Nivetha Srikanthan, Samina Hayat, Heather Ikert, Meghan Fuzzen, Carly Sing-Judge, Yash Badlani, Eli Zeeb, Leslie M. Bragg, Robert Delatolla, John P. Giesy, Elaine Gilliland, Mark R. Servos
Emerging Infectious Diseases, Volume 29, Issue 8

We determined correlations between SARS-CoV-2 load in untreated water and COVID-19 cases and patient hospitalizations before the Omicron variant (September 2020-November 2021) at 2 wastewater treatment plants in the Regional Municipality of Peel, Ontario, Canada. Using pre-Omicron correlations, we estimated incident COVID-19 cases during Omicron outbreaks (November 2021-June 2022). The strongest correlation between wastewater SARS-CoV-2 load and COVID-19 cases occurred 1 day after sampling (r = 0.911). The strongest correlation between wastewater load and COVID-19 patient hospitalizations occurred 4 days after sampling (r = 0.819). At the peak of the Omicron BA.2 outbreak in April 2022, reported COVID-19 cases were underestimated 19-fold because of changes in clinical testing. Wastewater data provided information for local decision-making and are a useful component of COVID-19 surveillance systems.

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Wastewater discharges alter microbial community composition in surface waters of the canadian prairies
Milena Esser, Cameron Hoggarth, Helen M. Baulch, Jonathan K. Challis, Yuwei Xie, John P. Giesy, Markus Hecker, Markus Brinkmann
Chemosphere, Volume 334

Microbial communities are an important component of freshwater biodiversity that is threatened by anthropogenic impacts. Wastewater discharges pose a particular concern by being major sources of anthropogenic contaminants and microorganisms that may influence the composition of natural microbial communities. Nevertheless, the effects of wastewater treatment plant (WWTP) effluents on microbial communities remain largely unexplored. In this study, the effects of wastewater discharges on microbial communities from five different WWTPs in Southern Saskatchewan were investigated using rRNA gene metabarcoding. In parallel, nutrient levels and the presence of environmentally relevant organic pollutants were analyzed. Higher nutrient loads and pollutant concentrations resulted in significant changes in microbial community composition. The greatest changes were observed in Wascana Creek (Regina), which was found to be heavily polluted by wastewater discharges. Several taxa occurred in greater relative abundance in the wastewater-influenced stream segments, indicating anthropogenic pollution and eutrophication, especially taxa belonging to Proteobacteria, Bacteroidota, and Chlorophyta. Strong decreases were measured within the taxa Ciliphora, Diatomea, Dinoflagellata, Nematozoa, Ochrophyta, Protalveolata, and Rotifera. Across all sample types, a significant decline in sulfur bacteria was measured, implying changes in functional biodiversity. In addition, downstream of the Regina WWTP, an increase in cyanotoxins was detected which was correlated with a significant change in cyanobacterial community composition. Overall, these data suggest a causal relationship between anthropogenic pollution and changes in microbial communities, possibly reflecting an impairment of ecosystem health.

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An improved method for determining frequency of multiple variants of SARS-CoV-2 in wastewater using qPCR assays
Meghan Fuzzen, Nathanael B.J. Harper, Hadi A. Dhiyebi, Nivetha Srikanthan, Samina Hayat, Leslie M. Bragg, Shelley Peterson, Ivy Yang, Jianxian Sun, Elizabeth Edwards, John P. Giesy, Chand Mangat, Tyson E. Graber, Robert Delatolla, Mark R. Servos
Science of The Total Environment, Volume 881

Wastewater-based surveillance has become an effective tool around the globe for indirect monitoring of COVID-19 in communities. Variants of Concern (VOCs) have been detected in wastewater by use of reverse transcription polymerase chain reaction (RT-PCR) or whole genome sequencing (WGS). Rapid, reliable RT-PCR assays continue to be needed to determine the relative frequencies of VOCs and sub-lineages in wastewater-based surveillance programs. The presence of multiple mutations in a single region of the N-gene allowed for the design of a single amplicon, multiple probe assay, that can distinguish among several VOCs in wastewater RNA extracts. This approach which multiplexes probes designed to target mutations associated with specific VOC's along with an intra-amplicon universal probe (non-mutated region) was validated in singleplex and multiplex. The prevalence of each mutation (i.e. VOC) is estimated by comparing the abundance of the targeted mutation with a non-mutated and highly conserved region within the same amplicon. This is advantageous for the accurate and rapid estimation of variant frequencies in wastewater. The N200 assay was applied to monitor frequencies of VOCs in wastewater extracts from several communities in Ontario, Canada in near real time from November 28, 2021 to January 4, 2022. This includes the period of the rapid replacement of the Delta variant with the introduction of the Omicron variant in these Ontario communities in early December 2021. The frequency estimates using this assay were highly reflective of clinical WGS estimates for the same communities. This style of qPCR assay, which simultaneously measures signal from a non-mutated comparator probe and multiple mutation-specific probes contained within a single qPCR amplicon, can be applied to future assay development for rapid and accurate estimations of variant frequencies.

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Multiple anthropogenic stressors influence the taxonomic and functional homogenization of macroinvertebrate communities on the mainstream of an urban-agricultural river in China
Yue Ma, Zongling Yu, Shiqi Jia, Naicheng Wu, Kai Yin, Yeyao Wang, John P. Giesy, Xin Jin
Journal of Environmental Management, Volume 341

Biodiversity loss is caused by intensive human activities and threatens human well-being. However, less is known about how the combined effects of multiple stressors on the diversity of internal (alpha diversity) and multidimensional (beta diversity) communities. Here, we conducted a long-term experiment to quantify the contribution of environmental stressors (including water quality, land use, climate factors, and hydrological regimes) to macroinvertebrate communities alpha and beta diversity in the mainstream of the Songhua River, the third largest river in China, from 2012 to 2019. Our results demonstrated that the alpha and beta diversity indices showed a decline during the study period, with the dissimilarity in community composition between sites decreasing significantly, especially in the impacted river sections (upper and midstream). Despite overall improvement in water quality after management intervention, multiple human-caused stressors still have led to biotic homogenization of macroinvertebrate communities in terms of both taxonomic and functional diversities in the past decade. Our study revealed the increased human land use explained an important portion of the variation of diversities, further indirectly promoting biotic homogenization by changing the physical and chemical factors of water quality, ultimately altering assemblage ecological processes. Furthermore, the facets of diversity have distinct response mechanisms to stressors, providing complementary information from the perspective of taxonomy and function to better reflect the ecological changes of communities. Environmental filtering determined taxonomic beta diversity, and functional beta diversity was driven by the joint efforts of stressors and spatial processes. Finally, we proposed that traditional water quality monitoring alone cannot fully reveal the status of river ecological environment protection, and more importantly, we should explore the continuous changes in biodiversity over the long term. Meanwhile, our results also highlight timely control of nutrient input and unreasonable expansion of land use can better curb the ecological degradation of rivers and promote the healthy and sustainable development of floodplain ecosystems.

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Perfluoroethylcyclohexane sulphonate, an emerging perfluoroalkyl substance, disrupts mitochondrial membranes and the expression of key molecular targets in vitro
Hannah Mahoney, Jenna Cantin, Yuwei Xie, Markus Brinkmann, John P. Giesy
Aquatic Toxicology, Volume 257

Perfluoroethylcyclohexane sulphonate (PFECHS) is an emerging, replacement perfluoroalkyl substance (PFAS) with little information available on the toxic effects or potencies with which to characterize its potential impacts on aquatic environments. This study aimed to characterize effects of PFECHS using in vitro systems, including rainbow trout liver cells (RTL-W1 cell line) and lymphocytes separated from whole blood. It was determined that exposure to PFECHS caused minor acute toxic effects for most endpoints and that little PFECHS was concentrated into cells with a mean in vitro bioconcentration factor of 81 ± 25 L/kg. However, PFECHS was observed to affect the mitochondrial membrane and key molecular receptors, such as the peroxisome proliferator receptor, cytochrome p450-dependent monooxygenases, and receptors involved in oxidative stress. Also, glutathione-S-transferase was significantly down-regulated at a near environmentally relevant exposure concentration of 400 ng/L. These results are the first to report bioconcentration of PFECHS, as well as its effects on the peroxisome proliferator and glutathione-S-transferase receptors, suggesting that even with little bioconcentration, PFECHS has potential to cause adverse effects.

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Acute Exposure of Zebrafish (<i>Danio rerio</i>) to the Next-Generation Perfluoroalkyl Substance, Perfluoroethylcyclohexanesulfonate, Shows Similar Effects as Legacy Substances
Hannah Mahoney, Jenna Cantin, Josephine Rybchuk, Yuwei Xie, John P. Giesy, Markus Brinkmann
Environmental Science & Technology, Volume 57, Issue 10

Perfluoroethylcyclohexanesulfonate (PFECHS) is an emerging perfluoroalkyl substance (PFAS) that has been considered a potential replacement for perfluorooctanesulfonic acid (PFOS). However, there is little information characterizing the toxic potency of PFECHS to zebrafish embryos and its potential for effects in aquatic environments. This study assessed toxic potency of PFECHS in vivo during both acute (96-hour postfertilization) and chronic (21-day posthatch) exposures and tested concentrations of PFECHS from 500 ng/L to 2 mg/L. PFECHS was less likely to cause mortalities than PFOS for both the acute and chronic experiments based on previously published values for PFOS exposure, but exposure resulted in a similar incidence of deformities. Exposure to PFECHS also resulted in significantly increased abundance of transcripts of peroxisome proliferator activated receptor alpha (pparα), cytochrome p450 1a1 (cyp1a1), and apolipoprotein IV (apoaIV) at concentrations nearing those of environmental relevance. Overall, these results provide further insight into the safety of an emerging PFAS alternative in the aquatic environment and raise awareness that previously considered "safer" alternatives may show similar effects as legacy PFASs.

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Understanding common population markers for SARS-CoV-2 RNA normalization in wastewater – A review
Femi F. Oloye, Yuwei Xie, Jonathan K. Challis, Oluwabunmi P. Femi-Oloye, Markus Brinkmann, Kerry N. McPhedran, Paul D. Jones, Mark R. Servos, John P. Giesy
Chemosphere, Volume 333

Wastewater monitoring and epidemiology have seen renewed interest during the recent COVID-19 pandemic. As a result, there is an increasing need to normalize wastewater-derived viral loads in local populations. Chemical tracers, both exogenous and endogenous compounds, have proven to be more stable and reliable for normalization than biological indicators. However, differing instrumentation and extraction methods can make it difficult to compare results. This review examines current extraction and quantification methods for ten common population indicators: creatinine, coprostanol, nicotine, cotinine, sucralose, acesulfame, androstenedione 5-hydroindoleacetic acid (5-HIAA), caffeine, and 1,7-dimethyluric acid. Some wastewater parameters such as ammonia, total nitrogen, total phosphorus, and daily flowrate were also evaluated. The analytical methods included direct injection, dilute and shoot, liquid/liquid, and solid phase extraction (SPE). Creatine, acesulfame, nicotine, 5-HIAA and androstenedione have been analysed by direct injection into LC-MS; however, most authors prefer to include SPE steps to avoid matrix effects. Both LC-MS and GC-MS have been successfully used to quantify coprostanol in wastewater, and the other selected indicators have been quantified successfully with LC-MS. Acidification to stabilize the sample before freezing to maintain the integrity of samples has been reported to be beneficial. However, there are arguments both for and against working at acidic pHs. Wastewater parameters mentioned earlier are quick and easy to quantify, but the data does not always represent the human population effectively. A preference for population indicators originating solely from humans is apparent. This review summarises methods employed for chemical indicators in wastewater, provides a basis for choosing an appropriate extraction and analysis method, and highlights the utility of accurate chemical tracer data for wastewater-based epidemiology.

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Aqueous Geochemical Controls on the Sestonic Microbial Community in Lakes Michigan and Superior
Asha Rani, Ravi Ranjan, Solidea Bonina, Mahsa Izadmehr, John P. Giesy, An Li, Neil C. Sturchio, Karl J. Rockne
Microorganisms, Volume 11, Issue 2

Despite being the largest freshwater lake system in the world, relatively little is known about the sestonic microbial community structure in the Laurentian Great Lakes. The goal of this research was to better understand this ecosystem using high-throughput sequencing of microbial communities as a function of water depth at six locations in the westernmost Great Lakes of Superior and Michigan. The water column was characterized by gradients in temperature, dissolved oxygen (DO), pH, and other physicochemical parameters with depth. Mean nitrate concentrations were 32 μmol/L, with only slight variation within and between the lakes, and with depth. Mean available phosphorus was 0.07 μmol/L, resulting in relatively large N:P ratios (97:1) indicative of P limitation. Abundances of the phyla Actinobacteria, Bacteroidetes, Cyanobacteria, Thaumarchaeota, and Verrucomicrobia differed significantly among the Lakes. Candidatus Nitrosopumilus was present in greater abundance in Lake Superior compared to Lake Michigan, suggesting the importance of ammonia-oxidating archaea in water column N cycling in Lake Superior. The Shannon diversity index was negatively correlated with pH, temperature, and salinity, and positively correlated with DO, latitude, and N2 saturation. Results of this study suggest that DO, pH, temperature, and salinity were major drivers shaping the community composition in the Great Lakes.

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Acute exposure to microcystins affects hypothalamic-pituitary axes of male rats
Ting Shi, Linlin Xu, Liang Chen, Jun He, Yeke Wang, Feng Chen, Yang Chen, John P. Giesy, Yu-Ting Wang, Qianhui Wu, Wenli Xu, Jun Chen, Ping Xie
Environmental Pollution, Volume 318

Microcystins (MCs) produced by some cyanobacteria can cause toxicity in animals and humans. In recent years, growing evidence suggests that MCs can act as endocrine disruptors. This research systematically investigated effects of microcystin-LR (MC-LR) on endocrine organs, biosynthesis of hormones and positive/negative feedback of the endocrine system in rats. Male, Sprague-Dawley rats were acutely administrated MC-LR by a single intraperitoneal injection at doses of 45, 67.5 or 90 μg MC-LR/kg body mass (bm), and then euthanized 24 h after exposure. In exposed rats, histological damage of hypothalamus, pituitary, adrenal, testis and thyroid were observed. Serum concentrations of corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and corticosterone (CORT), expressions of genes and proteins for biosynthesis of hormones were lesser, which indicated an overall suppression of the hypothalamus-pituitary-adrenal (HPA) axis. Along the hypothalamus-pituitary-gonadal (HPG) axis, lesser concentrations of gonadotropin-releasing hormone (GnRH) and testosterone (T), but greater concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH) and estradiol (E2) were observed. Except for greater transcription of cyp19a1 in testes, transcriptions of genes and proteins for T and E2 biosynthesis along the HPG axis were lesser. As for the hypothalamus-pituitary-thyroid (HPT) axis, after MCs treatment, greater concentrations of thyroid-stimulating hormone (TSH), but lesser concentrations of free tri-iodothyronine (fT3) were observed in serum. Concentrations of free tetra-iodothyronine (fT4) were greater in rats dosed with 45 μg MCs/kg, bm, but lesser in rats dosed with 67.5 or 90 μg MCs/kg, bm. Transcripts of genes for biosynthesis of hormones and receptors along the HPT axis and expressions of proteins for biosynthesis of tetra-iodothyronine (T4) and tri-iodothyronine (T3) in thyroid were significantly altered. Cross-talk among the HPA, HPG and HPT axes probably occurred. It was concluded that MCs caused an imbalance of positive and negative feedback of hormonal regulatory axes, blocked biosynthesis of key hormones and exhibited endocrine-disrupting effects.

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Sorption behaviour and toxicity of an herbicide safener “cyprosulfamide”
Oluwabunmi P. Femi-Oloye, Femi F. Oloye, Paul D. Jones, John P. Giesy
Science of The Total Environment, Volume 859

Cyprosulfamide is a herbicide safener that works against the injurious effects of herbicides such as isoxaflutole, dicamba, nicosulfuron, tembotrione, thiencarbazone-methyl. However, its sorption behaviour in soils and toxicity to aquatic organisms are yet to be thoroughly examined. This study determined the octanol-water partition coefficient, sorption properties, acute and chronic toxic effects, and potency of cyprosulfamide to the cladoceran water flea (Daphnia magna). The influence of soil properties such as organic carbon content, cation exchange capacity, pH, and field capacity on adsorption and desorption properties were also examined. The Log Kow (0.55) of cyprosulfamide was less than that of some other safeners, such as benoxacor or furilazole, found in aquatic environments. The sorption of cyprosulfamide to the soil was driven by pH, so sorption decreased with an increase in pH. Other characteristics, such as cation exchange capacity (CEC), organic carbon content, and field capacity, do not directly correlate with the distribution coefficient. Cyprosulfamide generally has a low affinity for soil and is thus mobile and prone to transport to surrounding surface waters. No lethality was observed at the highest concentration (120 mg/L) tested for acute toxicity to D. magna; hence the LC50 will be >120 mg/L. During chronic exposures, cyprosulfamide caused adverse effects at a concentration of 120 mg/L on the number of neonates and brood size. The death rate for the chronic study was a function of concentration and increased with days of exposure. Cyprosulfamide is unlikely to cause lethality to D. magna at relevant environmental concentrations.


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RNA metabarcoding helps reveal zooplankton community response to environmental stressors
Phillip Ankley, Yuwei Xie, Sonya M. Havens, Lisa Peters, Lauren Timlick, José Luis Rodríguez‐Gil, John P. Giesy, Vince Palace
Environmental Pollution, Volume 292

DNA metabarcoding can provide a high-throughput and rapid method for characterizing responses of communities to environmental stressors. However, within bulk samples, DNA metabarcoding hardly distinguishes live from the dead organisms. Here, both DNA and RNA metabarcoding were applied and compared in experimental freshwater mesocosms conducted for assessment of ecotoxicological responses of zooplankton communities to remediation treatment until 38 days post oil-spill. Furthermore, a novel indicator of normalized vitality (NV), sequence counts of RNA metabarcoding normalized by that of DNA metabarcoding, was developed for assessment of ecological responses. DNA and RNA metabarcoding detected similar taxa richness and rank of relative abundances. Both DNA and RNA metabarcoding demonstrated slight shifts in measured α-diversities in response to treatments. NV presented relatively greater magnitudes of differential responses of community compositions to treatments compared to DNA or RNA metabarcoding. NV declined from the start of the experiment (3 days pre-spill) to the end (38 days post-spill). NV also differed between Rotifer and Arthropoda, possibly due to differential life histories and sizes of organisms. NV could be a useful indicator for characterizing ecological responses to anthropogenic influence; however, the biology of target organisms and subsequent RNA production need to be considered. • RNA normalized by DNA metabarcoding functions as normalized vitality. • Normalized vitality reflected temporal dynamics of zooplankton communities. • Normalized vitality revealed greater community differences between treatments. • Rotifer had greatest normalized vitality compared to Arthropoda.

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Multiplex RT-qPCR assay (N200) to detect and estimate prevalence of multiple SARS-CoV-2 Variants of Concern in wastewater
Meghan Fuzzen, Nathanael B.J. Harper, Hadi A. Dhiyebi, Nivetha Srikanthan, Samina Hayat, Shelley Peterson, Ivy Yang, Jianxian Sun, Elizabeth A. Edwards, John P. Giesy, Chand Mangat, Tyson E. Graber, Robert Delatolla, Mark R. Servos

Abstract Wastewater-based surveillance (WBS) has become an effective tool around the globe for indirect monitoring of COVID-19 in communities. Quantities of viral fragments of SARS-CoV-2 in wastewater are related to numbers of clinical cases of COVID-19 reported within the corresponding sewershed. Variants of Concern (VOCs) have been detected in wastewater by use of reverse transcription quantitative polymerase chain reaction (RT-qPCR) or sequencing. A multiplex RT-qPCR assay to detect and estimate the prevalence of multiple VOCs, including Omicron/Alpha, Beta, Gamma, and Delta, in wastewater RNA extracts was developed and validated. The probe-based multiplex assay, named “N200” focuses on amino acids 199-202, a region of the N gene that contains several mutations that are associated with variants of SARS- CoV-2 within a single amplicon. Each of the probes in the N200 assay are specific to the targeted mutations and worked equally well in single- and multi-plex modes. To estimate prevalence of each VOC, the abundance of the targeted mutation was compared with a non- mutated region within the same amplified region. The N200 assay was applied to monitor frequencies of VOCs in wastewater extracts from six sewersheds in Ontario, Canada collected between December 1, 2021, and January 4, 2022. Using the N200 assay, the replacement of the Delta variant along with the introduction and rapid dominance of the Omicron variant were monitored in near real-time, as they occurred nearly simultaneously at all six locations. The N200 assay is robust and efficient for wastewater surveillance can be adopted into VOC monitoring programs or replace more laborious assays currently being used to monitor SARS- CoV-2 and its VOCs.

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Absorption and elimination of per and poly-fluoroalkyl substances substitutes in salmonid species after pre-fertilization exposure
Shu Su, Paul D. Jones, Jason C. Raine, Zilin Yang, Yufeng Gong, Yuwei Xie, Jie Tang, Chao Wang, Xiaoli Zhao, John P. Giesy
Science of The Total Environment, Volume 814

Due to their relatively large production and few restrictions on uses, novel substitutes for historically used per and poly-fluoroalkyl substances (PFAS) are being used and accumulating in the environment. However, due to a lack of information on their toxicological properties their hazards and risks are hard to estimate. Before fertilization, oocytes of two salmonid species, Arctic Char (Salvelinus alpinus) and Rainbow Trout (Oncorhynchus mykiss), were exposed to three PFAS substances used as substitutes for traditional PFAS, PFBA, PFBS or GenX or two archetypical, historically used, longer-chain PFAS, PFOA and PFOS. Exposed oocytes were subsequently fertilized, incubated and were sampled during several developmental stages, until swim-up. All five PFAS were accumulated into egg yolks with similar absorption rates, and their concentrations in egg yolks were less than respective concentrations in/on egg chorions. Rapid elimination of the five PFAS was observed during the first 3 days after fertilization. Thereafter, amounts of PFOS and PFOA were stable until swim-up, while PFBA, PFBS and GenX were further eliminated during development from one month after the fertilization to swim-up. In these two salmonid species, PFBA, PFBS and GenX were eliminated faster than were PFOS or PFOA.

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Effects of aqueous fluoxetine exposure on gut microbiome of adult Pimephales promelas
Alana Weber, Yuwei Xie, Jonathan K. Challis, Abigail DeBofsky, Phillip Ankley, Markus Hecker, Paul D. Jones, John P. Giesy
Science of The Total Environment, Volume 813

The microbiome of the gut is vital for homeostasis of hosts with its ability to detoxify and activate toxicants, as well as signal to the immune and nervous systems. However, in the field of environmental toxicology, the gut microbiome has only recently been identified as a measurable indicator for exposure to environmental pollutants. Antidepressants found in effluents of wastewater treatment plants and surface waters have been shown to exhibit antibacterial-like properties in vitro, where some bacteria are known to express homologous proteins that bind antidepressants in vertebrates. Therefore, it has been hypothesized that exposure to antidepressant drugs might affect gut microbiota of aquatic organisms. In this study, the common antidepressant, fluoxetine, was investigated to determine whether it can modulate the gut microbiome of adult fathead minnows. A 28-day, sub-chronic, static renewal exposure was performed with nominal fluoxetine concentrations of 0.01, 10 or 100 μg/L. Using 16S rRNA amplicon sequencing, shifts among the gut-associated microbiota were observed in individuals exposed to the greatest concentration, with greater effects observed in females. These changes were associated with a decrease in relative proportions of commensal bacteria, which can be important for health of fish including bacteria essential for fatty acid oxidation, and an increase in relative proportions of pathogenic bacteria associated with inflammation. Results demonstrate, for the first time, how antidepressants found in some aquatic environments can influence gut microbiota of fishes.

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RNA in Municipal Wastewater Reveals Magnitudes of COVID-19 Outbreaks across Four Waves Driven by SARS-CoV-2 Variants of Concern
Yuwei Xie, Jonathan K. Challis, Femi F. Oloye, Mohsen Asadi, Jenna Cantin, Markus Brinkmann, Kerry N. McPhedran, Natacha S. Hogan, Mike Sadowski, Paul D. Jones, Chrystal Landgraff, Chand Mangat, Mark R. Servos, John P. Giesy
ACS ES&T Water, Volume 2, Issue 11

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.

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Monthly variations of unregulated brominated disinfection by-products in chlorinated water are correlated with total bromine
Christena Watts, Jianxian Sun, Paul D. Jones, Hui Peng, John P. Giesy
Eco-Environment & Health, Volume 1, Issue 3

Brominated disinfection by-products (Br-DBPs) can form during chlorination of drinking water in treatment plants (DWTP). Regulations exist for a small subset of Br-DBPs; However, hundreds of unregulated Br-DBPs have been detected and limited information exists on their occurrence, concentrations, and seasonal trends. Here, a data-independent precursor isolation and characteristic fragment (DIPIC-Frag) method was optimized to screen chlorinated waters for Br-DBPs. There were 553 Br-DBPs detected with m/z values ranging from 170.884 to 497.0278 and chromatographic retention times from 2.4 to 26.2 min. With MS 2 information, structures for 40 of the 54 most abundant Br-DBPs were predicted. The method was then applied to a year-long study in which raw, clear well, and finished water were analyzed monthly. The 54 most abundant unregulated Br-DBPs were subjected to trend analysis. Br-DBPs with higher oxygen-to-carbon (O/C) and bromine-to-carbon (Br/C) ratios increased as water moved from the clear well to the finished stage, which indicated the dynamic formation of Br-DBPs. Monthly trends of unregulated Br-DBPs were compared to raw water parameters such as natural organic matter, temperature, and total bromine, but no correlations were observed. It was found that total concentrations of bromine (TBr) in finished water (0.04–0.12 mg/L) were consistently and significantly greater than in raw water (0.013–0.038 mg/L, P < 0.001), suggesting the introduction of bromine during the disinfection process. Concentrations of TBr in treatment units, rather than raw water, were significantly correlated to 34 of the Br-DBPs at α = 0.05. This study provides the first evidence that monthly trends of unregulated Br-DBPs can be associated with the concentration of TBr in treated waters. - Ultrahigh resolution mass spectrometry was used to identify novel brominated disinfection byproducts in a Canadian water treatment facility. - Several hundred novel brominated compounds were identified of which 54 were assigned chemical structures. - Seasonal variation in the generated DBPs were assessed over 11 months of sampling. - Increases in total bromine in drinking water was noted with progress thru the treatment process.

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16S rRNA metabarcoding unearths responses of rare gut microbiome of fathead minnows exposed to benzo[a]pyrene
Abigail DeBofsky, Yuwei Xie, Jonathan K. Challis, Phillip Ankley, Markus Brinkmann, Paul D. Jones, John P. Giesy
Science of The Total Environment, Volume 807

Activities of gut microbiomes are often overlooked in assessments of ecotoxicological effects of environmental contaminants. Effects of the polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP) on active gut microbiomes of juvenile fathead minnows (Pimephales promelas) were investigated. Fish were exposed for two weeks, to concentrations of 0, 1, 10, 100, or 1000 μg BaP g-1 in the diet. The active gut microbiome was characterized using 16S rRNA metabarcoding to determine its response to dietary exposure of BaP. BaP reduced alpha-diversity at the greatest exposure concentrations. Additionally, exposure to BaP altered community composition of active microbiome and resulted in differential proportion of taxa associated with hydrocarbon degradation and fish health. Neighborhood selection networks of active microbiomes were not reduced with greater concentrations of BaP, which suggests ecological resistance and/or resilience of gut microbiota. The active gut microbiome had a similar overall biodiversity as that of the genomic gut microbiota, but had a distinct composition from that of the 16S rDNA profile. Responses of alpha- and beta-diversities of the active microbiome to BaP exposure were consistent with that of genomic microbiomes. Normalized activity of microbiome via the ratio of rRNA to rDNA abundance revealed rare taxa that became active or dormant due to exposure to BaP. These differences highlight the need to assess both 16S rDNA and rRNA metabarcoding to fully derive bacterial compositional changes resulting from exposure to contaminants.

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Impacts of wastewater effluents and seasonal trends on levels of antipsychotic pharmaceuticals in water and sediments from two cold-region rivers
Ana Sharelys Cardenas Perez, Jonathan K. Challis, Xiaowen Ji, John P. Giesy, Markus Brinkmann
Science of The Total Environment, Volume 851

Most pharmaceuticals are found at trace concentrations in aquatic systems, but their continuous release and potential accumulation can lead to adverse health effects in exposed organisms. Concentrations can vary temporally, driven by variations in discharges of receiving waters, sorption to sediments, and other biotic and abiotic exchange processes. The principal aim of this research was to better understand the occurrence, trends, and dynamics of pharmaceuticals in a cold-climate, riverine environment. To this end, a suite of seven representative antipsychotic pharmaceuticals was measured upstream and downstream of two wastewater treatment plants (WWTPs) in Saskatchewan, Canada, located in the South Saskatchewan River and Wascana Creek, respectively, across three seasons. Concentrations of analytes were in the ng/L range and generally greater downstream of both WWTPs compared to upstream. Some compounds, including the tricyclic antidepressant amitriptyline, which was the most abundant analyte in water and sediment from both sites and across seasons, reached low μg/L concentrations. Data collected from this research effort indicate contamination with antipsychotic pharmaceuticals, with the potential to adversely impact exposed organisms.

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Next generation per- and poly-fluoroalkyl substances: Status and trends, aquatic toxicity, and risk assessment
Hannah Mahoney, Yuwei Xie, Markus Brinkmann, John P. Giesy
Eco-Environment & Health, Volume 1, Issue 2

Widespread application of poly- and per-fluoroalkyl substances (PFAS) has resulted in some substances being ubiquitous in environmental matrices. That and their resistance to degradation have allowed them to accumulate in wildlife and humans with potential for toxic effects. While specific substances of concern have been phased-out or banned, other PFAS that are emerging as alternative substances are still produced and are being released into the environment. This review focuses on describing three emerging, replacement PFAS: perfluoroethylcyclohexane sulphonate (PFECHS), 6:2 chlorinated polyfluoroalkyl ether sulfonate (6:2 Cl-PFAES), and hexafluoropropylene oxide dimer acid (HFPO-DA). By summarizing their physicochemical properties, environmental fate and transport, and toxic potencies in comparison to other PFAS compounds, this review offers insight into the viabilities of these chemicals as replacement substances. Using the chemical scoring and ranking assessment model, the relative hazards, uncertainties, and data gaps for each chemical were quantified and related to perfluorooctane sulfonic acid (PFOS) and perfluorooctanoic acid (PFOA) based on their chemical and uncertainty scores. The substances were ranked PFOS > 6:2 Cl-PFAES > PFOA > HFPO-DA > PFECHS according to their potential toxicity and PFECHS > HFPO-DA > 6:2 Cl-PFAES > PFOS > PFOA according to their need for future research. Since future uses of PFAS remain uncertain in the face of governmental regulations and production bans, replacement PFAS will continue to emerge on the world market and in the environment, raising concerns about their general lack of information on mechanisms and toxic potencies.

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Effects of in situ experimental selenium exposure on finescale dace (Phoxinus neogaeus) gut microbiome
Phillip Ankley, Stephanie D. Graves, Yuwei Xie, Abigail DeBofsky, Alana Weber, Markus Brinkmann, Vince Palace, Karsten Liber, Markus Hecker, David M. Janz, John P. Giesy
Environmental Research, Volume 212

Selenium (Se) is an environmental contaminant of global concern that can cause adverse effects in fish at elevated levels. Fish gut microbiome play essential roles in gastrointestinal function and host health and can be perturbed by environmental contaminants, including metals and metalloids. Here, an in-situ Se exposure of female finescale dace (Phoxinus neogaeus) using mesocosms was conducted to determine the impacts of Se accumulation on the gut microbiome and morphometric endpoints. Prior to this study, the gut microbiome of finescale dace, a widespread Cyprinid throughout North America, had not been characterized. Exposure to Se caused a hormetic response of alpha diversity of the gut microbiome, with greater diversity at the lesser concentration of 1.6 μg Se/L, relative to that of fish exposed to the greater concentration of 5.6 μg Se/L. Select gut microbiome taxa of fish were differentially abundant between aqueous exposure concentrations and significantly correlated with liver-somatic index (LSI). The potential effects of gut microbiome dysbiosis on condition of wild fish might be a consideration when assessing adverse effects of Se in aquatic environments. More research regarding effects of Se on field-collected fish gut microbiome and the potential adverse effects or benefits on the host is warranted.

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Comparison of primary and secondary sludge carbon sources derived from hydrolysis or acidogenesis for nitrate reduction and denitrification kinetics: Organics utilization and microbial community shift
Yiding Guo, Liang Guo, Jin Chen, Yangguo Zhao, Mengchun Gao, Junyuan Ji, Zhigang She, John P. Giesy
Environmental Research, Volume 212

Seeking available and economical carbon sources for denitrification process is an intractable issue for wastewater treatment. However, no study compared different types of waste sludge as carbon source from denitrification mechanism, organics utilization and microbial community aspects. In this study, primary and secondary sludge were pretreated by thermophilic bacteria (TB), and its hydrolysis or acidogenic liquid were prepared as carbon sources for denitrification. At C/N of 8-3, the variations of NO3--N and NO2--N were profiled in typical cycles and denitrification kinetics was analyzed. Primary sludge achieved a competitive NOX-N removal efficiency with less dosage than secondary sludge. Fourier transform infrared (FTIR) spectroscopy was introduced to analyze organic composition from functional-group perspective and the utilization of organic matters in different sludge carbon sources was investigated. To further analyze the microbial community shift in different denitrification systems, high-throughput sequencing technology was applied. Results showed that denitrifier Thauera, belonging to Proteobacteria, was predominant, and primary sludge acidogenic liquid enriched Thauera most intensively with relative abundance of 47.3%.

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Rapid transition between SARS-CoV-2 variants of concern Delta and Omicron detected by monitoring municipal wastewater from three Canadian cities
Femi F. Oloye, Yuwei Xie, Mohsen Asadi, Jenna Cantin, Jonathan K. Challis, Markus Brinkmann, Kerry N. McPhedran, Kevin Kristian, Mark P. Keller, Mike Sadowski, Paul D. Jones, Chrystal Landgraff, Chand Mangat, Meghan Fuzzen, Mark R. Servos, John P. Giesy
Science of The Total Environment, Volume 841

Monitoring the communal incidence of COVID-19 is important for both government and residents of an area to make informed decisions. However, continuous reliance on one means of monitoring might not be accurate because of biases introduced by government policies or behaviours of residents. Wastewater surveillance was employed to monitor concentrations of SARS-CoV-2 RNA in raw influent wastewater from wastewater treatment plants serving three Canadian Prairie cities with different population sizes. Data obtained from wastewater are not directly influenced by government regulations or behaviours of individuals. The means of three weekly samples collected using 24 h composite auto-samplers were determined. Viral loads were determined by RT-qPCR, and whole-genome sequencing was used to charaterize variants of concern (VOC). The dominant VOCs in the three cities were the same but with different proportions of sub-lineages. Sub-lineages of Delta were AY.12, AY.25, AY.27 and AY.93 in 2021, while the major sub-lineage of Omicron was BA.1 in January 2022, and BA.2 subsequently became a trace-level sub-variant then the predominant VOC. When each VOC was first detected varied among cities; However, Saskatoon, with the largest population, was always the first to present new VOCs. Viral loads varied among cities, but there was no direct correlation with population size, possibly because of differences in flow regimes. Population is one of the factors that affects trends in onset and development of local outbreaks during the pandemic. This might be due to demography or the fact that larger populations had greater potential for inter- and intra-country migration. Hence, wastewater surveillance data from larger cities can typically be used to indicate what to expect in smaller communities.

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Health Risks of Chronic Exposure to Small Doses of Microcystins: An Integrative Metabolomic and Biochemical Study of Human Serum
Jun He, Jun Chen, Feng Chen, Liang Chen, John P. Giesy, Hyewon Lee, Liang Gao, Xuwei Deng, Wenjing Wang, Ping Xie
Environmental Science & Technology, Volume 56, Issue 10

Health risks of chronic exposure to microcystins (MCs), a family of aquatic contaminants produced mainly by cyanobacteria, are critical yet unsolved problems. Despite a few epidemiological studies, the metabolic profiles of humans exposed to MCs remain unknown, hindering the deep understanding of the molecular toxicity mechanisms. Here, sensitive nuclear magnetic resonance (NMR)- and liquid chromatography-mass spectrometry (LC-MS)-based metabolomics were applied to investigate the serum metabolic profiles of humans living near Lake Chao, where toxic cyanobacterial blooms occur annually. MCs were positively detected in 92 of 144 sera by ultra-high-pressure liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) with a median concentration of 0.016 μg/L. The estimated daily intake (0.15-0.27 μg MC-LReq/day) was less than the tolerable daily intake (TDI, 2.4 μg MC-LR for 60 kg adults) recommended by the World Health Organization (WHO). Obvious disruptions of the amino acid metabolism were confirmed and played important roles in renal impairments associated with serum MC burdens. Chronic oral exposure of mice to 30 μg MC-LR/kg body mass, which is less than the no observed adverse effect level, also led to obvious renal lesions and metabolic dysfunction. These observations provide the first evidence of metabolic disturbance of humans exposed to MCs and indicate that the WHO's TDI value determined traditionally should be lessened to protect human health effectively.

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Molecular Characterization of Estrogen Receptor Agonists during Sewage Treatment Processes Using Effect-Directed Analysis Combined with High-Resolution Full-Scan Screening
Jiyun Gwak, Junghyun Lee, Jihyun Cha, Kim Mun-Gi, Jin Hur, Jinwoo Cho, Min Sung Kim, Kyoung‐Soon Jang, John P. Giesy, Seongjin Hong, Jong Seong Khim
Environmental Science & Technology, Volume 56, Issue 18

Endocrine-disrupting potential was evaluated during the sewage treatment process using in vitro bioassays. Aryl hydrocarbon receptor (AhR)-, androgen receptor (AR)-, glucocorticoid receptor (GR)-, and estrogen receptor (ER)-mediated activities were assessed over five steps of the treatment process. Bioassays of organic extracts showed that AhR, AR, and GR potencies tended to decrease through the sewage treatment process, whereas ER potencies did not significantly decrease. Bioassays on reverse-phase high-performance liquid chromatography fractions showed that F5 (log KOW 2.5-3.0) had great ER potencies. Full-scan screening of these fractions detected two novel ER agonists, arenobufagin and loratadine, which are used pharmaceuticals. These compounds accounted for 3.3-25% of the total ER potencies and 4% of the ER potencies in the final effluent. The well-known ER agonists, estrone and 17β-estradiol, accounted for 60 and 17% of the ER potencies in F5 of the influent and primary treatment, respectively. Fourier transform ion cyclotron resonance mass spectrometry analysis showed that various molecules were generated during the treatment process, especially CHO and CHOS (C: carbon, H: hydrogen, O: oxygen, and S: sulfur). This study documented that widely used pharmaceuticals are introduced into the aquatic environments without being removed during the sewage treatment process.

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Predicting Hepatic Clearance of Psychotropic Drugs in Isolated Perfused Fish Livers Using a Combination of Two <i>In Vitro</i> Assays
Zoey M. Bourgeois, Jordan J Comfort, Matthew Schultz, Jonathan K. Challis, Jenna Cantin, Xiaowen Ji, John P. Giesy, Markus Brinkmann
Environmental Science & Technology, Volume 56, Issue 22

In vitro biotransformation assays with primary trout hepatocytes (RT-HEP) or liver subcellular fractions (RT-S9) have been proposed as valuable tools to help scientists and regulators better understand the toxicokinetics of chemicals. While both assays have been applied successfully to a diversity of neutral organic chemicals, only the RT-S9 assay has been applied to a large number of ionizable organic chemicals. Here, a combination of an in vitro biotransformation assay with RT-HEP with an active transport assay based on the permanent rainbow trout liver cell line RTL-W1 was used to qualitatively predict the potential hepatic clearance of nine psychotropic drugs with various degrees of ionization. Predictions were compared with rates of clearance measured in isolated perfused rainbow trout livers, and the importance of active transport was verified in the presence of the active transport inhibitor cyclosporin A. For the first time, it was demonstrated that a combination of biotransformation and active transport assays is powerful for the prediction of rates of hepatic clearance of ionizable chemicals. Ultimately, it is expected that this approach will allow for use of fewer animals while at the same time improving our confidence in the use of data from in vitro assays in chemical risk assessment.

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Exposure to the Tire Rubber-Derived Contaminant 6PPD-Quinone Causes Mitochondrial Dysfunction <i>In Vitro</i>
Hannah Mahoney, Francisco C. da Silva, Catherine Roberts, Matthew Schultz, Xiaowen Ji, Alper James Alcaraz, David W. Montgomery, Summer Selinger, Jonathan K. Challis, John P. Giesy, Lynn P. Weber, David M. Janz, Steve Wiseman, Markus Hecker, Markus Brinkmann
Environmental Science & Technology Letters, Volume 9, Issue 9

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Polyhalogenated Carbazoles in Sediments of Lower Laurentian Great Lakes and Regional Perspectives
An Li, Shanshan Zhou, Huan He, Junmeng Guo, Karl J. Rockne, Neil C. Sturchio, John P. Giesy
ACS ES&T Water, Volume 2, Issue 9

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Phytochemical Analysis and Antioxidant, Antibacterial, and Antifungal Effects of Essential Oil of Black Caraway (Nigella sativa L.) Seeds against Drug-Resistant Clinically Pathogenic Microorganisms
Otmane Zouirech, Abdullah A. Alyousef, Azeddin El Barnossi, Abdelfattah El Moussaoui, Mohammed Bourhia, Ahmad Mohammad Salamatullah, Lahcen Ouahmane, John P. Giesy, Mourad A. M. Aboul‐Soud, Badiâa Lyoussi, Elhoussine Derwich
BioMed Research International, Volume 2022

Nigella sativa (NS) is a plant that has long been utilized in traditional medicine as a treatment for certain diseases. The aim of this work was to valorize the essential oil (EO) of this species by phytochemical analysis and antimicrobial and antioxidant evaluation. EO was extracted by hydrodistillation from the seeds of Nigella sativa (EO-NS). Phytochemical content of EO-NS was evaluated by use of gas chromatography coupled to mass spectrometry (GC-MS/MS). Antioxidant ability was in vitro determined by use of three assays: 2.2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing power (FRAP), and total antioxidant capacity (TAC) relative to two synthetic antioxidants: BHT and quercetin. Antimicrobial effect was evaluated against four clinically important bacterial strains (Staphylococcus aureus, ATCC 6633; Escherichia coli, K12; Bacillus subtilis, DSM 6333; and Proteus mirabilis, ATCC 29906) and against four fungal strains (Candida albicans, ATCC 10231; Aspergillus niger, MTCC 282; Aspergillus flavus, MTCC 9606; and Fusarium oxysporum, MTCC 9913). Fifteen constituents that accounted for the majority of the mass of the EO-NS were identified and quantified by use of GC-MSMS. The main component was O-cymene (37.82%), followed by carvacrol (17.68%), α-pinene (10.09%), trans-sabinene hydrate (9.90%), and 4-terpineol (7.15%). EO-NS exhibited significant antioxidant activity with IC50, EC50, and total antioxidant capacity (TAC) of <math xmlns="" id="M1"> <mn>0.017</mn> <mo>±</mo> <mn>0.0002</mn> </math> , <math xmlns="" id="M2"> <mn>0.1196</mn> <mo>±</mo> <mn>0.012</mn> </math> , and <math xmlns="" id="M3"> <mn>114.059</mn> <mo>±</mo> <mn>0.97</mn> </math> mg EAA/g, respectively. Additionally, EO-NS exhibited promising antibacterial activity on all strains under investigation, especially on E. coli K12 resulting in inhibition diameter of <math xmlns="" id="M4"> <mn>38.67</mn> <mo>±</mo> <mn>0.58</mn> </math> mm and a minimum inhibitory concentration (MIC) of <math xmlns="" id="M5"> <mn>1.34</mn> <mo>±</mo> <mn>0.00</mn> </math> μg/mL. Also, EO-NS had significant antifungal efficacy, with a percentage of inhibition of <math xmlns="" id="M6"> <mn>67.45</mn> <mo>±</mo> <mn>2.31</mn> </math> % and MIC of <math xmlns="" id="M7"> <mn>2.69</mn> <mo>±</mo> <mn>0.00</mn> </math> μg/mL against F. oxysporum, MTCC 9913 and with a diameter of inhibition <math xmlns="" id="M8"> <mn>42</mn> <mo>±</mo> <mn>0.00</mn> </math> mm and MIC of <math xmlns="" id="M9"> <mn>0.67</mn> <mo>±</mo> <mn>0.00</mn> </math> μg/mL against C. albicans. To minimize development of antibiotic-resistant bacteria, EO-NS can be utilized as a natural, alternative to synthetic antibiotics and antioxidants to treat free radicals implicated in microbial infection-related inflammatory reactions.


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Using zooplankton metabarcoding to assess the efficacy of different techniques to clean-up an oil-spill in a boreal lake
Phillip Ankley, Yuwei Xie, Tyler A. Black, Abigail DeBofsky, McKenzie Perry, Michael Paterson, Mark L. Hanson, Scott N. Higgins, John P. Giesy, Vince Palace
Aquatic Toxicology, Volume 236

Abstract Regulators require adequate information to select best practices with less ecosystem impacts for remediation of freshwater ecosystems after oil spills. Zooplankton are valuable indicators of aquatic ecosystem health as they play pivotal roles in biochemical cycles while stabilizing food webs. Compared with morphological identification, metabarcoding holds promise for cost-effective, high-throughput, and benchmarkable biomonitoring of zooplankton communities. The objective of this study was to apply DNA and RNA metabarcoding of zooplankton for ecotoxicological assessment and compare it with traditional morphological identification in experimental shoreline enclosures in a boreal lake. These identification methods were also applied in context of assessing response of the zooplankton community exposed to simulated spills of diluted bitumen (dilbit), with experimental remediation practices (enhanced monitored natural recovery and shoreline cleaner application). Metabarcoding detected boreal zooplankton taxa up to the genus level, with a total of 24 shared genera, and while metabarcoding-based relative abundance served as an acceptable proxy for biomass inferred by morphological identification (ρ ≥ 0.52). Morphological identification determined zooplankton community composition changes due to treatments at 11 days post-spill (PERMANOVA, p = 0.0143) while metabarcoding methods indicated changes in zooplankton richness and communities at 38 days post-spill (T-test, p

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Life Cycle Exposure to Environmentally Relevant Concentrations of Diphenyl Phosphate (DPhP) Inhibits Growth and Energy Metabolism of Zebrafish in a Sex-Specific Manner
Qiliang Chen, Xiaolong Lian, Jie An, Ningbo Geng, Haijun Zhang, Jonathan K. Challis, Yun Luo, Yaxin Liu, Guanyong Su, Yuwei Xie, Yingwen Li, Zhihao Liu, Yanjun Shen, John P. Giesy, Yufeng Gong
Environmental Science & Technology

Due to commercial uses and environmental degradation of aryl phosphate esters, diphenyl phosphate (DPhP) is frequently detected in environmental matrices and is thus of growing concern worldwide. However, information on potential adverse effects of chronic exposure to DPhP at environmentally realistic concentrations was lacking. Here, we investigated the effects of life cycle exposure to DPhP on zebrafish at environmentally relevant concentrations of 0.8, 3.9, or 35.6 μg/L and employed a dual-omics approach (metabolomics and transcriptomics) to characterize potential modes of action. Exposure to DPhP at 35.6 μg/L for 120 days resulted in significant reductions in body mass and length of male zebrafish, but did not cause those same effects to females. Predominant toxicological mechanisms, including inhibition of oxidative phosphorylation, down-regulation of fatty acid oxidation, and up-regulation of phosphatidylcholine degradation, were revealed by integrated dual-omics analysis and successfully linked to adverse outcomes. Activity of succinate dehydrogenase and protein content of carnitine O-palmitoyltransferase 1 were significantly decreased in livers of male fish exposed to DPhP, which further confirmed the proposed toxicological mechanisms. This study is the first to demonstrate that chronic, low-level exposure to DPhP can retard growth via inhibiting energy output in male zebrafish.

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Comparison of approaches to quantify SARS-CoV-2 in wastewater using RT-qPCR: Results and implications from a collaborative inter-laboratory study in Canada
Alex H. S. Chik, Melissa B. Glier, Mark R. Servos, Chand Mangat, Xiaoli Pang, Yuanyuan Qiu, Patrick M. D’Aoust, Jean-Baptiste Burnet, Robert Delatolla, Sarah Dorner, Qiudi Geng, John P. Giesy, R. Michael L. McKay, Michael R. Mulvey, Natalie Prystajecky, Nivetha Srikanthan, Yuwei Xie, Bernadette Conant, Steve E. Hrudey
Journal of Environmental Sciences, Volume 107

Detection of SARS-CoV-2 RNA in wastewater is a promising tool for informing public health decisions during the COVID-19 pandemic. However, approaches for its analysis by use of reverse transcription quantitative polymerase chain reaction (RT-qPCR) are still far from standardized globally. To characterize inter- and intra-laboratory variability among results when using various methods deployed across Canada, aliquots from a real wastewater sample were spiked with surrogates of SARS-CoV-2 (gamma-radiation inactivated SARS-CoV-2 and human coronavirus strain 229E [HCoV-229E]) at low and high levels then provided "blind" to eight laboratories. Concentration estimates reported by individual laboratories were consistently within a 1.0-log10 range for aliquots of the same spiked condition. All laboratories distinguished between low- and high-spikes for both surrogates. As expected, greater variability was observed in the results amongst laboratories than within individual laboratories, but SARS-CoV-2 RNA concentration estimates for each spiked condition remained mostly within 1.0-log10 ranges. The no-spike wastewater aliquots provided yielded non-detects or trace levels (<20 gene copies/mL) of SARS-CoV-2 RNA. Detections appear linked to methods that included or focused on the solids fraction of the wastewater matrix and might represent in-situ SARS-CoV-2 to the wastewater sample. HCoV-229E RNA was not detected in the no-spike aliquots. Overall, all methods yielded comparable results at the conditions tested. Partitioning behavior of SARS-CoV-2 and spiked surrogates in wastewater should be considered to evaluate method effectiveness. A consistent method and laboratory to explore wastewater SARS-CoV-2 temporal trends for a given system, with appropriate quality control protocols and documented in adequate detail should succeed.

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Responses of juvenile fathead minnow (Pimephales promelas) gut microbiome to a chronic dietary exposure of benzo[a]pyrene
Abigail DeBofsky, Yuwei Xie, Jonathan K. Challis, Niteesh Jain, Markus Brinkmann, Paul D. Jones, John P. Giesy
Environmental Pollution, Volume 278

The microbiome has been described as an additional host “organ” with well-established beneficial roles. However, the effects of exposures to chemicals on both structure and function of the gut microbiome of fishes are understudied. To determine effects of benzo[ a ]pyrene (BaP), a model persistent organic pollutant, on structural shifts of gut microbiome in juvenile fathead minnows ( Pimephales promelas ), fish were exposed ad libitum in the diet to concentrations of 1, 10, 100, or 1000 μg BaP g −1 food, in addition to a vehicle control, for two weeks. To determine the link between exposure to BaP and changes in the microbial community, concentrations of metabolites of BaP were measured in fish bile and 16S rRNA amplicon sequencing was used to evaluate the microbiome. Exposure to BaP only reduced alpha-diversity at the greatest exposure concentrations. However, it did alter community composition assessed as differential abundance of taxa and reduced network complexity of the microbial community in all exposure groups. Results presented here illustrate that environmentally-relevant concentrations of BaP can alter the diversity of the gut microbiome and community network connectivity. Highlights • Dominant phyla of gut microbiome are consistent with those of other freshwater fishes. • BaP metabolites and exposure doses were consistent with those found in contaminated sites. • Dietary BaP exposure has significant, dose-dependent effects on the fish gut microbiome. • Dietary BaP exposure altered association networks of gut microbiome. Environmentally-relevant concentrations of BaP can alter the diversity of the gut microbiome and community network connectivity via dietary exposure route.

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Remodeling of Arctic char ( <i>Salvelinus alpinus</i> ) lipidome under a stimulated scenario of Arctic warming
Chao Wang, Yufeng Gong, Fuchang Deng, Enmin Ding, Jie Tang, Garry Codling, Jonathan K. Challis, Derek Green, Jing Wang, Qiliang Chen, Yuwei Xie, Shu Su, Zilin Yang, Jason C. Raine, Paul D. Jones, Song Tang, John P. Giesy
Global Change Biology, Volume 27, Issue 14

Arctic warming associated with global climate change poses a significant threat to populations of wildlife in the Arctic. Since lipids play a vital role in adaptation of organisms to variations in temperature, high-resolution mass-spectrometry-based lipidomics can provide insights into adaptive responses of organisms to a warmer environment in the Arctic and help to illustrate potential novel roles of lipids in the process of thermal adaption. In this study, we studied an ecologically and economically important species-Arctic char (Salvelinus alpinus)-with a detailed multi-tissue analysis of the lipidome in response to chronic shifts in temperature using a validated lipidomics workflow. In addition, dynamic alterations in the hepatic lipidome during the time course of shifts in temperature were also characterized. Our results showed that early life stages of Arctic char were more susceptible to variations in temperature. One-year-old Arctic char responded to chronic increases in temperature with coordinated regulation of lipids, including headgroup-specific remodeling of acyl chains in glycerophospholipids (GP) and extensive alterations in composition of lipids in membranes, such as less lyso-GPs, and more ether-GPs and sphingomyelin. Glycerolipids (e.g., triacylglycerol, TG) also participated in adaptive responses of the lipidome of Arctic char. Eight-week-old Arctic char exhibited rapid adaptive alterations of the hepatic lipidome to stepwise decreases in temperature while showing blunted responses to gradual increases in temperature, implying an inability to adapt rapidly to warmer environments. Three common phosphatidylethanolamines (PEs) (PE 36:6|PE 16:1_20:5, PE 38:7|PE 16:1_22:6, and PE 40:7|PE 18:1_22:6) were finally identified as candidate lipid biomarkers for temperature shifts via machine learning approach. Overall, this work provides additional information to a better understanding of underlying regulatory mechanisms of the lipidome of Arctic organisms in the face of near-future warming.

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Environmental DNA of preservative ethanol performed better than water samples in detecting macroinvertebrate diversity using metabarcoding
Yu Wang, Kai Chen, Jin Gao, Meng Wang, Jie Dong, Yuwei Xie, John P. Giesy, Xiaowei Jin, Beixin Wang
Diversity and Distributions, Volume 27, Issue 10

High‐throughput pipelines supported by eDNA metabarcoding have been applied in various freshwater ecosystems. Both eDNA in ethanol (EtOH) samples (ES‐eDNA) and in water samples (WS‐eDNA) can provide comprehensive classification lists with good taxonomic resolution and coverage for determining freshwater biodiversity and biomonitoring. But, the advantages of ES‐eDNA metabarcoding over WS‐eDNA metabarcoding remain unclear for routine assessments of diversity of benthic macroinvertebrates in streams.

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Prefertilization Exposure of Rainbow Trout Eggs to Per‐ and Polyfluoroalkyl Substances to Simulate Accumulation During Oogenesis
Jason C. Raine, Shu Su, Eric S. Lin, Zilin Yang, John P. Giesy, Paul D. Jones
Environmental Toxicology and Chemistry, Volume 40, Issue 11

Aqueous film–forming foams (AFFFs) are used in firefighting and are sources of per- and polyfluoroalkyl substances (PFAS) to the environment through surface runoff and groundwater contamination at defense and transportation sites. Little is known regarding the toxicity and bioaccumulation of newer AFFF formulations containing novel PFAS. To mimic maternal transfer of PFAS, prefertilization rainbow trout eggs were exposed to three PFAS using novel methodologies. Batches of unfertilized oocytes were exposed for 3 h to 0, 0.01, 0.1, 1, or 10 µg/ml separately to perfluorooctanoic acid, perfluorohexanoic acid, or perfluorooctanesulfonic acid in either coelomic fluid or Cortland's solution. After exposure, the gametes were fertilized and rinsed with dechlorinated water. Egg yolk was aspirated from a subset of fertilized eggs for PFAS quantification. Each PFAS was detected in yolks of eggs exposed to the respective PFAS, and yolk concentrations were directly proportional to concentrations in aqueous media to which they were exposed. Exposure in coelomic fluid or Cortland's solution resulted in similar concentrations of PFAS in egg yolks. Ratios of PFAS concentrations in oocytes to concentrations in exposure media (oocyte fluid ratios) were <0.99 when exposed from 0.01 to 10 µg/ml and <0.45 when exposed from 0.1 to 10 µg/ml for both media and all three PFAS, demonstrating that the water solubility of the chemicals was relatively great. Prefertilization exposure of eggs effectively introduced PFAS into unfertilized egg yolk. This method provided a means of mimicking maternal transfer to evaluate toxicity to developing embryos from an early stage. This method is more rapid and efficient than injection of individual fertilized eggs and avoids trauma from inserting needles into eggs. Environ Toxicol Chem 2021;40:3159–3165. © 2021 SETAC

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Ractopamine and Other Growth-Promoting Compounds in Beef Cattle Operations: Fate and Transport in Feedlot Pens and Adjacent Environments
Jonathan K. Challis, Srinivas Sura, Jenna Cantin, Ashley Curtis, K. M. Shade, Tim A. McAllister, Paul D. Jones, John P. Giesy, Francis J. Larney
Environmental Science & Technology, Volume 55, Issue 3

The current study represents a comprehensive investigation of the occurrence and fates of trenbolone acetate (TBA) and metabolites 17α-trenbolone (17α-TBOH), 17β-TBOH, and trendione (TBO); melengesterol acetate (MGA); and the less commonly studied β-andrenergic agonist ractopamine (RAC) in two 8 month cattle feeding trials and simulated rainfall runoff experiments. Cattle were administered TBA, MGA, or RAC, and their residues were measured in fresh feces, pen floor material, and simulated rainfall runoff from pen floor surfaces and manure-amended pasture. Concentrations of RAC ranged from 3600 ng g–1, dry weight (dw), in pen floor to 58 000 ng g–1 in fresh feces and were, on average, observed at 3–4 orders of magnitude greater than those of TBA and MGA. RAC persisted in pen floors (manure t1/2 = 18–49 days), and contamination of adjacent sites was observed, likely via transport of windblown particulates. Concentrations in runoff water from pen floors extrapolated to larger-scale commercial feedlots revealed that a single rainfall event could result in mobilization of gram quantities of RAC. This is the first report of RAC occurrence and fate in cattle feedlot environments, and will help understand the risks posed by this chemical and inform appropriate manure-management practices.

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Temporal Patterns of Bacterial and Viral Communities during Algae Blooms of a Reservoir in Macau
Dini Hu, John P. Giesy, Min Guo, Wai Kin Ung, Yijun Kong, Kai Meng Mok, Simon Ming‐Yuen Lee
Toxins, Volume 13, Issue 12

Compositions of microbial communities associated with blooms of algae in a storage reservoir in Macau, China were investigated between 2013 and 2016. Algae were enumerated by visible light microscopy. Profiles of organisms in water were examined by 16S rRNA sequences and viral metagenomics, based on next generation sequencing. Results of 16S rRNA sequencing indicated that majority of the identified organisms were bacteria closely related to Proteobacteria, Cyanobacteria, Verrucomicrobia, Bacteroidetes, and Actinobacteria. Metagenomics sequences demonstrated that the dominant virus was Phycodnavirus, accounting for 70% of the total population. Patterns of relative numbers of bacteria in the microbial community and their temporal changes were determined through alpha diversity indices, principal coordinates analysis (PCoA), relative abundance, and visualized by Venn diagrams. Ways in which the bacterial and viral communities are influenced by various water-related variables were elucidated based on redundancy analysis (RDA). Relationships of the relative numbers of bacteria with trophic status in a reservoir used for drinking water in Macau, provided insight into associations of Phycodnavirus and Proteobacteria with changes in blooms of algae.

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High Throughput Sequencing of MicroRNA in Rainbow Trout Plasma, Mucus, and Surrounding Water Following Acute Stress
Heather Ikert, Michael Lynch, Andrew C. Doxey, John P. Giesy, Mark R. Servos, Barbara A. Katzenback, Paul M. Craig
Frontiers in Physiology, Volume 11

Circulating plasma microRNAs (miRNAs) are well established as biomarkers of several diseases in humans and have recently been used as indicators of environmental exposures in fish. However, the role of plasma miRNAs in regulating acute stress responses in fish is largely unknown. Tissue and plasma miRNAs have recently been associated with excreted miRNAs; however, external miRNAs have never been measured in fish. The objective of this study was to identify the altered plasma miRNAs in response to acute stress in rainbow trout ( Oncorhynchus mykiss ), as well as altered miRNAs in fish epidermal mucus and the surrounding ambient water. Small RNA was extracted and sequenced from plasma, mucus, and water collected from rainbow trout pre- and 1 h-post a 3-min air stressor. Following small RNA-Seq and pathway analysis, we identified differentially expressed plasma miRNAs that targeted biosynthetic, degradation, and metabolic pathways. We successfully isolated miRNA from trout mucus and the surrounding water and detected differences in miRNA expression 1-h post air stress. The expressed miRNA profiles in mucus and water were different from the altered plasma miRNA profile, which indicated that the plasma miRNA response was not associated with or immediately reflected in external samples, which was further validated through qPCR. This research expands understanding of the role of plasma miRNA in the acute stress response of fish and is the first report of successful isolation and profiling of miRNA from fish mucus or samples of ambient water. Measurements of miRNA from plasma, mucus, or water can be further studied and have potential to be applied as non-lethal indicators of acute stress in fish.


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Metals and PFAS in stormwater and surface runoff in a semi-arid Canadian city subject to large variations in temperature among seasons
Garry Codling, Hongda Yuan, Paul D. Jones, John P. Giesy, Markus Hecker
Environmental Science and Pollution Research, Volume 27, Issue 15

Because compounds accumulate through dry periods and enter aquatic systems in just a few seasonal events such as snowmelt and summer storms, surface waters in semi-arid, cold regions, such as the Canadian Prairies, are particularly vulnerable to loading of contaminant from runoff events from surfaces. This study assessed concentrations of metals and selected trace organics entering a river via surface runoff from an urban region and how these semi-arid regions with large seasonal variations in temperature might differ from more temperate regions. Selected potentially harmful elements (PHEs) including, Mn with Cr, Cu, Zn, Ba and U all exceeded guideline discharge values set by the Canadian Council of the Ministers of the Environment (CCME) by as much as 16-fold. Variation among discharges during spring, summer and winter was observed. For example, across the whole city, an estimated 6 kg of zinc was discharged in a spring storm, 36 kg in a summer storm and 17 tonnes in snowmelt. The mass of Zn discharged is similar to the annual loading estimated for Stockholm, Sweden, but in Saskatoon, Saskatchewan, Canada, the bulk of runoff was during snowmelt. The mean sum of poly- and per-fluoroalkyl substances (PFAS) in stormwater was 9.0 ng L−1, which is consistent with concentrations observed in other Canadian cities (6.5–16 ng L−1). These concentrations of PFAS are likely due to dispersed sources and orders of magnitude less than thresholds for toxicity to fish and aquatic invertebrates.

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Differential responses of gut microbiota of male and female fathead minnow (Pimephales promelas) to a short-term environmentally-relevant, aqueous exposure to benzo[a]pyrene
Abigail DeBofsky, Yuwei Xie, Chelsea Grimard, Alper James Alcaraz, Markus Brinkmann, Markus Hecker, John P. Giesy
Chemosphere, Volume 252

In addition to aiding in digestion of food and uptake of nutrients, microbiota in guts of vertebrates are responsible for regulating several beneficial functions, including development of an organism and maintaining homeostasis. However, little is known about effects of exposures to chemicals on structure and function of gut microbiota of fishes. To assess effects of exposure to polycyclic aromatic hydrocarbons (PAHs) on gut microbiota, male and female fathead minnows ( Pimephales promelas ) were exposed to environmentally-relevant concentrations of the legacy PAH benzo[ a ]pyrene (BaP) in water. Measured concentrations of BaP ranged from 2.3 × 10 −3 to 1.3 μg L −1 . The community of microbiota in the gut were assessed by use of 16S rRNA metagenetics. Exposure to environmentally-relevant aqueous concentrations of BaP did not alter expression levels of mRNA for cyp1a1 , a “classic” biomarker of exposure to BaP, but resulted in shifts in relative compositions of gut microbiota in females rather than males. Results presented here illustrate that in addition to effects on more well-studied molecular endpoints, relative compositions of the microbiota in guts of fish can also quickly respond to exposure to chemicals, which can provide additional mechanisms for adverse effects on individuals. • Female and male fathead minnows exhibited significantly different gut microbiota. • Exposure to BaP altered structures in female gut microbiota, but not in males. • Exposure to BaP altered predicted functions in gut microbiota of fathead minnow. • Gut microbiome was more sensitive to a low dose BaP than host’s ahr1 and cyp1a1.

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Integrated assessment of west coast of South Korea by use of benthic bacterial community structure as determined by eDNA, concentrations of contaminants, and in vitro bioassays
Aslan Hwanhwi Lee, Jung-Hyun Lee, Seongjin Hong, Bong-Oh Kwon, Yuwei Xie, John P. Giesy, Xiaowei Zhang, Jong Seong Khim
Environment International, Volume 137

During the past few decades, contamination of sediments by persistent toxic substances (PTSs) has been observed in estuarine and coastal areas on the west coast of South Korea. The contaminants are suspected to cause toxicities in aquatic biota, but little is known about their ecological effects, particularly on benthic microbial communities. In this study, an eDNA-based assessment was applied along with classic assessments of exposure, such as chemistry and in vitro bioassays, to evaluate condition of benthic bacterial communities subjected to PTSs. Two strategies were adopted for the study. One was to conduct a comprehensive assessment in space (by comparing seawater and freshwater sites at five coastal regions) and in time (by following change over a 5-y period). Although we found that bacterial composition varied among and within years, some phyla, such as Proteobacteria (28.7%), Actinobacteria (13.1%), Firmicutes (12.7%), and Chloroflexi (12.5%) were consistently dominated across the study regions. Certain bacterial groups, such as Firmicutes and Verrucomicrobia have been linked to contamination at some sites in the study area and at specific points in time. Bacterial communities were not significantly correlated with salinity or AhR- and ER-mediated potencies, whereas concentrations of PAHs, APs, and certain metals (Cd and Hg) exhibited significant associations to the structure of bacterial communities at the phylum level. In fact, the relative abundance of microbes in the phylum Planctomycetes was significantly and negatively correlated with concentrations of PAHs and metals. Thus, the relative abundance of Planctomycetes could be used as an indicator of sedimentary contamination by PAHs and/or metals. Based on our correlation analyses, Cd and ER-mediated potencies were associated more with bacterial abundances at the class taxonomic level than were other PTSs and metals. Overall, the eDNA-based assessment was useful by augmenting more traditional measures of exposure and responses in a sediment triad approach and has potential as a more rapid screening tool.

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Concentrations of Metals in Fishes from the Athabasca and Slave Rivers of Northern Canada
Brett Tendler, Ehimai Ohiozebau, Garry Codling, John P. Giesy, Paul D. Jones
Environmental Toxicology and Chemistry, Volume 39, Issue 11

There is growing concern about possible effects of exploitation of the Alberta Oil Sands on the ambient environment, including possible effects on populations of fishes in the Athabasca River and farther downstream in Lake Athabasca and the Slave River. In the present study, concentrations of metals in dorsal muscle tissue of 5 fish species-goldeye, northern pike, walleye, whitefish, and burbot-from the Slave, Peace, and Athabasca Rivers were quantified. A suite of 25 metals including As, Hg, Se, Tl, and V was analyzed. Most metals exhibited no significant variations in concentration among locations. Concentrations of 5 metals, As, Hg, Se, Tl, and V, revealed significant variations among locations and were of sufficient magnitude to be of interest. Concentrations of Hg did not vary significantly among locations; however, because it was detected at concentrations of concern and the use of the selected fishes was a local source of food for humans and pets, it was of interest. Concentrations of As, Se, Tl, and V in dorsal muscle of certain fishes in the farthest downstream sites on the Slave River were greater than those in the same tissues and species in the farther upstream sites on the Peace and Athabasca Rivers. This phenomenon was most prevalent with Tl and to a lesser extent with As and Se. Nevertheless, concentrations were not of concern for the health of human consumers. Although metals did not appear to be increased in fish in the Alberta Oil Sands region in the present study, further research is needed to understand the potential impacts. Environ Toxicol Chem 2020;39:2180-2195. © 2020 SETAC.

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In vitro-in vivo and cross-life stage extrapolation of uptake and biotransformation of benzo[a]pyrene in the fathead minnow (Pimephales promelas)
Chelsea Grimard, Annika Mangold-Döring, Markus Schmitz, Hattan A. Alharbi, Paul D. Jones, John P. Giesy, Markus Hecker, Markus Brinkmann
Aquatic Toxicology, Volume 228

• A concentration dependent increase of B[ a ]P metabolites was observed • No induction of phase I or II activity was observed with increasing B[ a ]P exposure • Biotransformation of B[ a ]P was successfully implemented into in silico models • The models accurately predicted life stage-specific abundances of B[ a ]P metabolites Understanding internal dose metrics is integral to adequately assess effects environmental contaminants might have on aquatic wildlife, including fish. In silico toxicokinetic (TK) models are a leading approach for quantifying internal exposure metrics for fishes; however, they often do not adequately consider chemicals that are actively biotransformed and have not been validated against early-life stages (ELS) that are often considered the most sensitive to the exposure to contaminants. To address these uncertainties, TK models were parameterized for the rapidly biotransformed chemical benzo[ a ]pyrene (B[ a ]P) in embryo-larval and adult life stages of fathead minnows. Biotransformation of B[ a ]P was determined through measurements of in vitro clearance. Using in vitro-in vivo extrapolation, in vitro clearance was integrated into a multi-compartment TK model for adult fish and a one-compartment model for ELS. Model predictions were validated using measurements of B[ a ]P metabolites from in vivo flow-through exposures to graded concentrations of water-borne B[ a ]P. Significantly greater amounts of B[ a ]P metabolites were observed with exposure to greater concentrations of parent compound in both life stages. However, when assessing biotransformation capacity, no differences in phase I or phase II biotransformation were observed with greater exposures to B[ a ]P. Results of modelling suggested that biotransformation of B[ a ]P can be successfully implemented into in silico models to accurately predict life stage-specific abundances of B[ a ]P metabolites in either whole-body larvae or the bile of adult fish. Models developed increase the scope of applications in which TK models can be used to support environmental risk assessments.

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Effects of the husky oil spill on gut microbiota of native fishes in the North Saskatchewan River, Canada
Abigail DeBofsky, Yuwei Xie, Timothy D. Jardine, Janet E. Hill, Paul D. Jones, John P. Giesy
Aquatic Toxicology, Volume 229

• Concentrations of PAHs in muscle suggests continued exposure to the residual spilled oil. • Identity of the host species was the dominant driver in shaping the gut microbiome of fish. • Structures of gut microbiomes were correlated with concentrations of PAHs in muscle in walleye. In July 2016, a Husky Energy pipeline spilled 225,000 L of diluted heavy crude oil, with a portion of the oil entering the North Saskatchewan River near Maidstone, SK, Canada. This event provided a unique opportunity to assess potential effects of a crude oil constituent (namely polycyclic aromatic hydrocarbons, PAHs) on a possible sensitive indicator of freshwater ecosystem health, the gut microbiota of native fishes. In summer 2017, goldeye ( Hiodon alosoides ), walleye ( Sander vitreus ), northern pike ( Esox lucius ), and shorthead redhorse ( Moxostoma macrolepidotum ) were collected at six locations upstream and downstream of the spill. Muscle and bile were collected from individual fish for quantification of PAHs and intestinal contents were collected for characterization of the microbial community of the gut. Results suggested that host species is a significant determinant of gut microbiota, with significant differences among the species across sites. Concentrations of PAHs in dorsal muscle were significantly correlated with gut community compositions of walleye, but not of the other fishes. Concentrations of PAHs in muscle were also correlated with abundances of several families of bacteria among fishes. This study represents one of the first to investigate the response of the gut microbiome of wild fishes to chemical stressors.

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Mechanisms of pH-Dependent Uptake of Ionizable Organic Chemicals by Fish from Oil Sands Process-Affected Water (OSPW)
Markus Brinkmann, Hattan A. Alharbi, Ulyana Fuchylo, Steve Wiseman, Garrett Morandi, Hui Peng, John P. Giesy, Paul D. Jones, Markus Hecker
Environmental Science & Technology, Volume 54, Issue 15

Uptake and effects of ionizable organic chemicals (IOCs) that are weak acids in aqueous solution by fish can differ as a function of pH. While the pH-dependent behavior of select IOCs is well-understood, complex mixtures of IOCs, e.g., from oil sands process-affected water (OSPW), have not yet been studied systematically. Here, we established an in vitro screening method using the rainbow trout gill cell line, RTgill-W1, to investigate pH-dependent cytotoxicity and permeation of IOCs across cultured epithelia using ultra-high-performance liquid chromatography with high-resolution mass spectrometry (UPLC-HRMS). The assay was benchmarked using model chemicals and technical mixtures, and then used to characterize fractions and reconstituted extracts of field-collected OSPW. Significant pH-dependent cytotoxicity of individual IOCs, acidic fractions, and reconstituted extracts of OSPW was observed. In vitro data were in good agreement with data from a 96 h in vivo exposure experiment with juvenile rainbow trout. Permeation of some IOCs from OSPW was mediated by active transport, as revealed by studies in which inhibitors of these active transport mechanisms were applied. We conclude that the RTgill-W1 in vitro assay is useful for the screening of pH-dependent uptake of IOCs in fish, and has applications for in vitro-in vivo extrapolation, and prioritization of chemicals in nontarget screenings.

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Biochemical and Molecular Investigation of In Vitro Antioxidant and Anticancer Activity Spectrum of Crude Extracts of Willow Leaves Salix safsaf
Mourad A. M. Aboul‐Soud, Abdelkader E. Ashour, Jonathan K. Challis, Atallah F. Ahmed, Ashok Kumar, Amr Nassrallah, Tariq A Alahmari, Quaiser Saquib, Maqsood A. Siddiqui, Yazeed A. Al‐Sheikh, Hany A. El‐Shemy, Ahmed M. Aboul‐Enein, Khalid M. AlGhamdi, Paul D. Jones, John P. Giesy
Plants, Volume 9, Issue 10

Organic fractions and extracts of willow (Salix safsaf) leaves, produced by sequential solvent extraction as well as infusion and decoction, exhibited anticancer potencies in four cancerous cell lines, including breast (MCF-7), colorectal (HCT-116), cervical (HeLa) and liver (HepG2). Results of the MTT assay revealed that chloroform (CHCl3) and ethyl acetate (EtOAc)-soluble fractions exhibited specific anticancer activities as marginal toxicities were observed against two non-cancerous control cell lines (BJ-1 and MCF-12). Ultra-high-resolution mass spectrometry Q-Exactive™ HF Hybrid Quadrupole-Orbitrap™ coupled with liquid chromatography (UHPLC) indicated that both extracts are enriched in features belonging to major phenolic and purine derivatives. Fluorescence-activated cell sorter analysis (FACS), employing annexin V-FITC/PI double staining indicated that the observed cytotoxic potency was mediated via apoptosis. FACS analysis, monitoring the increase in fluorescence signal, associated with oxidation of DCFH to DCF, indicated that the mechanism of apoptosis is independent of reactive oxygen species (ROS). Results of immunoblotting and RT-qPCR assays showed that treatment with organic fractions under investigation resulted in significant up-regulation of pro-apoptotic protein and mRNA markers for Caspase-3, p53 and Bax, whereas it resulted in a significant reduction in amounts of both protein and mRNA of the anti-apoptotic marker Bcl-2. FACS analysis also indicated that pre-treatment and co-treatment of human amniotic epithelial (WISH) cells exposed to the ROS H2O2 with EtOAc fraction provide a cytoprotective and antioxidant capacity against generated oxidative stress. In conclusion, our findings highlight the importance of natural phenolic and flavonoid compounds with unparalleled and unique antioxidant and anticancer properties.


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Comparison of the Effects of Extraction Techniques on Mass Spectrometry Profiles of Dissolved Organic Compounds in Oil Sand Process-Affected Water
Hattan A. Alharbi, Garrett Morandi, Paul D. Jones, Steve Wiseman, John P. Giesy
Energy & Fuels, Volume 33, Issue 8

Recent advances in mass spectrometry have facilitated chemical characterization and profiling of complex environmental mixtures such as oil sand process-affected water (OSPW) and identification of previously unresolved chemicals. However, because OSPW is a complex mixture of salts, metals, suspended particulate matter, and dissolved organics, extraction techniques are required to reduce the effects of signal suppression/enhancement. In this work, Orbitrap, ultrahigh resolution mass spectrometry was used to perform a comprehensive comparison of solid phase extraction (SPE) and liquid–liquid extraction (LLE) techniques on profiling of dissolved organic chemicals in OSPW. When operated in negative ion mode, extraction of naphthenic acid (NAs–O2) was dependent on acidification of OSPW samples for C18 and LLE techniques. However, when applying a hydrophilic lipophilic balance (HLB) sorbent (ABN) SPE technique, the extractability of NAs was independent of pH. When operated in positive ion mode, for all extracti...

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Abundances and concentrations of brominated azo dyes detected in indoor dust
Birendra Dhungana, Hui Peng, Steven Kutarna, Gisela de Aragão Umbuzeiro, Sujan Shrestha, Jing Liu, Paul D. Jones, Bikram Subedi, John P. Giesy, George P. Cobb
Environmental Pollution, Volume 252

Dust samples were collected from four indoor environments, including childcare facilities, houses, hair salons, and a research facility from the USA and were analyzed for brominated compounds using full scan liquid chromatography high-resolution mass spectrometry. A total of 240 brominated compounds were detected in these dust samples, and elemental formulas were predicted for 120 more abundant ions. In addition to commonly detected brominated flame retardants (BFRs), nitrogen-containing brominated azo dyes (BADs) were among the most frequently detected and abundant. Specifically, greater abundances of BADs were detected in indoor dusts from daycares and salons compared to houses and the research facility. Using authentic standards, a quantitative method was established for two BADs (DB373: Disperse Blue 373 and DV93: Disperse Violet 93) and 2-bromo-4,6-dinitroaniline, a commonly used precursor in azo dye production, in indoor dust. Generally, greater concentrations of DB373 (≤3850 ng/g) and DV93 (≤1190 ng/g) were observed in indoor dust from daycares highlighting children as a susceptible population to potential health risk from exposure to BADs. These data are important because, to date, targeted analysis of brominated compounds in indoor environments has focused mainly on BFRs and appears to underestimate the total amount of brominated compounds.

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Temporal variation in zooplankton and phytoplankton community species composition and the affecting factors in Lake Taihu—a large freshwater lake in China
Cuicui Li, Weiying Feng, Haiyan Chen, Xiaofeng Li, Fanhao Song, Wenjing Guo, John P. Giesy, Fubao Sun
Environmental Pollution, Volume 245

Monitoring diverse components of aquatic ecosystems is vital for elucidation of diversity dynamics and processes, which alter freshwater ecosystems, but such studies are seldom conducted. Phytoplankton and zooplankton are integral components which play indispensable parts in the structure and ecological service function of water bodies. However, few studies were made on how zooplankton and phytoplankton community may respond simultaneously to change of circumstance and their mutual relationship. Therefore, we researched synchronously the phytoplankton communities as well as zooplankton communities based on monthly monitoring data from September 2011 to August 2012 in heavily polluted areas and researched their responses to variation in environmental parameters and their mutual relationship. As indicated by Time-lag analysis (TLA), the long-term dynamics of phytoplankton and zooplankton were undergoing directional variations, what's more, there exists significant seasonal variations of phytoplankton and zooplankton communities as indicated by Non-Metric Multidimensional scaling (NMDS) methods. Also, Redundancy Analysis (RDA) demonstrated that environmental indicators together accounted for 25.6% and 50.1% variance of phytoplankton and zooplankton, respectively, indicating that environmental variations affected significantly on the temporal dynamics of phytoplankton as well as zooplankton communities. What's more, variance partioning suggested that the major environmental factors influencing variation structures of zooplankton communities were water temperature, concentration of nitrogen, revealing the dominating driving mechanism which shaped the communities of zooplankton. It was also found that there was significant synchronization between zooplankton biomass and phytoplankton biomass (expressed as Chl-a concentration), which suggested that zooplankton respond to changes in dynamic structure of phytoplankton community and can initiate a decrease in phytoplankton biomass through grazing in a few months.

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Acid mine drainage affects the diversity and metal resistance gene profile of sediment bacterial community along a river
Xiaohui Zhang, Song Tang, Mao Wang, Weimin Sun, Yuwei Xie, Hui Peng, Aimin Zhong, Hongling Liu, Xiaowei Zhang, Yu H, John P. Giesy, Markus Hecker
Chemosphere, Volume 217

Acid mine drainage (AMD) is one of the most hazardous byproducts of some types of mining. However, research on how AMD affects the bacterial community structure of downstream riverine ecosystems and the distribution of metal resistance genes (MRGs) along pollution gradient is limited. Comprehensive geochemical and high-throughput next-generation sequencing analyses can be integrated to characterize spatial distributions and MRG profiles of sediment bacteria communities along the AMD-contaminated Hengshi River. We found that (1) diversities of bacterial communities significantly and gradually increased along the river with decreasing contamination, suggesting community composition reflected changes in geochemical conditions; (2) relative abundances of phyla Proteobacteria and genus Halomonas and Planococcaceae that function in metal reduction decreased along the AMD gradient; (3) low levels of sediment salinity, sulfate, aquatic lead (Pb), and cadmium (Cd) were negatively correlated with bacterial diversity despite pH was in a positive manner with diversity; and (4) arsenic (As) and copper (Cu) resistance genes corresponded to sediment concentrations of As and Cu, respectively. Altogether, our findings offer initial insight into the distribution patterns of sediment bacterial community structure, diversity and MRGs along a lotic ecosystem contaminated by AMD, and the factors that affect them.


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Perfluorobutanesulfonate Exposure Causes Durable and Transgenerational Dysbiosis of Gut Microbiota in Marine Medaka
Lianguo Chen, James C.W. Lam, Chen-Yan Hu, Mirabelle M.P. Tsui, Qi Wang, John P. Giesy, Patrick T.I. Lam
Environmental Science & Technology Letters, Volume 5, Issue 12

Environmental pollutants are known as disruptors of gut microbiota. However, it remains unexplored whether the dysbiosis of gut microbiota by pollutants is durable and transgenerational in teleost. Therefore, this study exposed eggs of marine medaka to environmentally realistic concentrations (0, 1.0, 2.9, or 9.5 μg/L) of perfluorobutanesulfonate (PFBS), a persistent organic pollutant of emerging concern, until sexual maturity. A proportion of F0 adults was dissected after exposure (F0-exposed). Remaining fish were depurated in clean seawater (F0-depurated). F1 offspring were also cultured in clean seawater for a complete life-cycle. Substantial amounts of PFBS were accumulated in F0-exposed intestines, while F1 intestines contained no PFBS. Significant alterations were observed in physiological activities of F0-exposed and F1 medaka. The gut microbial community in F0-exposed, F0-depurated, and F1 medaka were restructured in a concentration-dependent manner by PFBS exposure. Dysbiosis of gut microbiota ca...

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eDNA-based bioassessment of coastal sediments impacted by an oil spill
Yuwei Xie, Xiaowei Zhang, Jianghua Yang, Seon Jin Kim, Seongjin Hong, John P. Giesy, Un Hyuk Yim, Won Joon Shim, Yu H, Jong Seong Khim
Environmental Pollution, Volume 238

Oil spills offshore can cause long-term ecological effects on coastal marine ecosystems. Despite their important ecological roles in the cycling of energy and nutrients in food webs, effects on bacteria, protists or arthropods are often neglected. Environmental DNA (eDNA) metabarcoding was applied to characterize changes in the structure of micro- and macro-biota communities of surface sediments over a 7-year period since the occurrence of Hebei Spirit oil spill on December 7, 2007. Alterations in diversities and structures of micro- and macro-biota were observed in the contaminated area where concentrations of polycyclic aromatic hydrocarbons were greater. Successions of bacterial, protists and metazoan communities revealed long-term ecological effects of residual oil. Residual oil dominated the largest cluster of the community-environment association network. Presence of bacterial families (Aerococcaceae and Carnobacteriaceae) and the protozoan family (Platyophryidae) might have conferred sensitivity of communities to oil pollution. Hydrocarbon-degrading bacterial families (Anaerolinaceae, Desulfobacteraceae, Helicobacteraceae and Piscirickettsiaceae) and algal family (Araphid pennate) were resistant to adverse effects of spilt oil. The protistan family (Subulatomonas) and arthropod families (Folsomia, Sarcophagidae Opomyzoidea, and Anomura) appeared to be positively associated with residual oil pollution. eDNA metabarcoding can provide a powerful tool for assessing effects of anthropogenic pollution, such as oil spills on sediment communities and its long-term trends in coastal marine environments.