Venlafaxine deposition in the zygote disrupts the endocrine control of growth in juvenile zebrafish.

The antidepressant venlafaxine can be found at levels nearing µg/L in waterways receiving municipal wastewater effluent, exposing non-target organisms, such as fish, to this chemical. We showed previously that zygotic exposure to venlafaxine alters neurodevelopment and behaviour in zebrafish (Danio rerio) larvae. Here, we tested the hypothesis that the zygotic deposition of venlafaxine disrupts endocrine pathways related to growth in zebrafish. This was carried out by microinjecting embryos (1-4 cell stage) with either 0, 1, or 10 ng venlafaxine. Zygotic venlafaxine deposition reduced the growth of fish after 30 days post-fertilization. Specific growth rate was particularly impacted by 1 ng venlafaxine. This growth retardation corresponded with the disruption of endocrine pathways involved in growth and metabolism. Venlafaxine exposed embryos displayed reduced transcript abundance of key genes involved in anabolic hormone action. Early-life venlafaxine exposure also reduced whole-body insulin and glucose content in juveniles. Target-tissue glucose uptake measurements indicated that high venlafaxine deposition preferentially increased glucose uptake to the brain. Zygotic venlafaxine did not affect feed intake nor altered the transcript abundance of key feeding-related peptides. Taken together, zygotic venlafaxine deposition compromises zebrafish growth by disrupting multiple endocrine pathways, and this study has identified key markers for potential use in risk assessment.

The removal of micropollutants from treated effluent by batch-operated pilot-scale constructed wetlands.

Micropollutants (MPs), such as pharmaceuticals and antibiotics, are present in the environment at low concentrations (ng/L-µg/L). A constructed wetland (CW) is a nature-based wastewater treatment technology, which can be used to remove MPs from wastewater treatment plant effluent. This study aimed to improve MP removal of CWs by optimizing the design of batch-operated CW. Three pilot-scale CWs were built to study the effect of two design-features: the use of a support matrix (a mixture of bark and biochar) and continuous aeration. The use of bark-biochar as support matrix increased the removal of 11 of 12 studied MPs compared to the CW filled with conventional material sand. The highest improved removal by the addition of bark-biochar was more than 40% (median) for irbesartan, carbamazepine, hydrochlorothiazide and benzotriazole. Aerating the bed of the bark-biochar CW did not change MP removal. Besides, the presence of bark-biochar also enhanced the removal of total nitrogen during 10 months of operation, but no improvement was observed on the total organic carbon and total phosphorus removal. Considering the application in a batch-operated CW, MP removal can be greatly enhanced by replacing sand with bark-biochar that will act as MP adsorbing matrix.

Impact of effluent organic matter on perfluoroalkyl acid removal from wastewater effluent by granular activated carbon and alternative adsorbents.

Occurrence of perfluoroalkyl acids (PFAAs) in wastewater effluent coupled with increasingly stringent regulations has increased the need for more effective sorption-based PFAA treatment approaches. This study investigated the impact of ozone (O3)- biologically active filtration (BAF) as integral components of non-reverse osmosis (RO)-based potable reuse treatment trains and as a potential pretreatment option to improve adsorptive PFAA removal from wastewater effluent by nonselective (e.g., granular activated carbon (GAC) and selective (e.g., anionic exchange resins (AER) and surface-modified clay (SMC)) adsorbents. For nonselective GAC, O3 and BAF resulted in similar PFAA removal improvements, while BAF alone performed better than O3 for AER and SMC. O3-BAF in tandem resulted in the highest PFAA removal performance improvement among pretreatments investigated for selective and nonselective adsorbents. Side by side evaluation of the dissolved organic carbon (DOC) breakthrough curves and size exclusion chromatography (SEC) for each pretreatment scenario suggested that despite the higher affinity of selective adsorbents towards PFAAs, the competition between PFAA and effluent organic matter (EfOM) (molecular weights (MWs): 100-1000 Da) negatively impacts the performance of these adsorbents. The SEC results also demonstrated that transformation of hydrophobic EfOM to more hydrophilic molecules during O3 and biotransformation of EfOM during BAF were the dominant mechanisms responsible for alleviating the competition between PFAA and EfOM, resulting in PFAA removal improvement.

The antidepressant venlafaxine perturbs cardiac development and function in larval zebrafish.

Venlafaxine, a selective serotonin and norepinephrine reuptake inhibitor, is a highly prescribed antidepressant and is detected at µg/L concentrations in waterways receiving municipal wastewater effluents. We previously showed that early-life venlafaxine exposure disrupted the normal development of the nervous system and reduces larval activity in zebrafish (Danio rerio). However, it is unclear whether the reduced swimming activity may be associated with impaired cardiac function. Here we tested the hypothesis that zygotic exposure to venlafaxine impacts the development and function of the larval zebrafish heart. Venlafaxine (0, 1 or 10 ng) was administered by microinjection into freshly fertilized zebrafish embryos (1-4 cell stage) to assess heart development and function during early-life stages. Venlafaxine deposition in the zygote led to precocious development of the embryo heart, including the timing of the first heartbeat, increased heart size, and a higher heart rate at 24- and 48-hours post-fertilization (hpf). Also, waterborne exposure to environmental levels of this antidepressant during early development increased the heart rate at 48 hpf of zebrafish larvae mimicking the zygotic deposition. The venlafaxine-induced higher heart rate in the embryos was abolished in the presence of NAN-190, an antagonist of the 5HT1A receptor. Also, heart rate dropped below control levels in the 10 ng, but not 1 ng venlafaxine group at 72 and 96 hpf. An acute stressor reduced the venlafaxine-induced heart rate at 48 hpf but did not affect the already reduced heart rate at 72 and 96 hpf in the 10 ng venlafaxine group. Our results suggest that the higher heart rate in the venlafaxine group may be due to an enhanced serotonin stimulation of the 5HT1A receptor. Taken together, early-life venlafaxine exposure disrupts cardiac development and has the potential to compromise the cardiovascular performance of larval zebrafish.

Antidepressants as Endocrine Disrupting Compounds in Fish.

As antidepressant usage by the global population continues to increase, their persistent detection in aquatic habitats from municipal wastewater effluent release has led to concerns of possible impacts on non-target organisms, including fish. These pharmaceuticals have been marketed as mood-altering drugs, specifically targeting the monoaminergic signaling in the brain of humans. However, the monoaminergic systems are highly conserved and involved in the modulation of a multitude of endocrine functions in vertebrates. While most studies exploring possible impact of antidepressants on fish have focused on behavioural perturbations, a smaller spotlight has been placed on the endocrine functions, especially related to reproduction, growth, and the stress response. The purpose of this review is to highlight the possible role of antidepressants as endocrine disruptors in fish. While studies linking the effects of environmentally relevant levels of antidepressant on the endocrine system in fish are sparse, the emerging evidence suggests that early-life exposure to these compounds have the potential to alter the developmental programming of the endocrine system, which could persist as long-term and multigenerational effects in teleosts.

Altered microbiomes of aquatic macroinvertebrates and riparian spiders downstream of municipal wastewater effluents.

Municipal wastewater treatment plants (WWTPs) contain numerous contaminants, including antimicrobials, that could affect the composition of the beneficial bacterial communities associated with host aquatic organisms. There is also potential for these effects to transfer to terrestrial predators. Riparian spiders and five families of aquatic macroinvertebrates were collected from sites upstream and downstream of two WWTPs, Waterloo and Kitchener, discharging to the Grand River, Ontario, Canada. Whole-body microbiota were analyzed following the extraction, PCR amplification, and sequencing of bacterial DNA using the V3-V4 hypervariable regions of the 16S rRNA genetic barcode. Changes in the relative abundance of major microbiome phyla were observed in all downstream aquatic insects except Hydropsychidae caddisflies, which exhibited little site variation. Shannon alpha diversity differed among sites for Tetragnathidae spiders, Perlidae, Hydropsychidae, and Heptageniidae. Downstream of the Waterloo WWTP alpha diversity decreased in spiders, while downstream of the Kitchener WWTP this measure decreased in Perlidae and increased in spiders. Bray-Curtis beta diversity was dissimilar among sites in all invertebrate taxa; upstream sites differed from those downstream of Waterloo in spiders, Perlidae, and Hydropsychidae, and from those downstream of Kitchener in spiders, Perlidae, and Hydropsychidae. Finally, effluent-derived bacteria were found in the microbiomes of downstream spiders and aquatic insects and not upstream. Overall, results indicated that the microbiomes of invertebrates collected downstream differed from those collected upstream of WWTPs, which has implications for altered host health and transport of WWTP-derived bacteria through aquatic ecosystems.

Sex-Specific and Long-Term Impacts of Early-Life Venlafaxine Exposure in Zebrafish.

Venlafaxine, a selective serotonin and norepinephrine reuptake inhibitor, is a widely prescribed antidepressant that is detected in municipal wastewater effluents at µg/L concentrations. It has been shown to impact the early life stages of fish, including neurodevelopment and behaviour in larvae, but whether such early exposures have longer-term consequences are far from clear. Here, we sought to determine whether zygotic deposition of venlafaxine, mimicking a maternal transfer scenario, disturbs the metabolic rate and behavioural performance using zebrafish (Danio rerio). This was tested using freshly fertilized embryos (1-4 cell stage) microinjected with either 0, 1 or 10 ng of venlafaxine and raised to either juvenile (60 days post-fertilization) or adult (10-12 months post-fertilization). Zygotic venlafaxine exposure led to a reduction in the active metabolic rate and aerobic scope, but this was only observed in female fish. On the other hand, the total distance travelled in an open field assessment was greater at the highest concentration of venlafaxine only in the adult males. At the juvenile stage, behavioural assessments demonstrated that venlafaxine exposure may increase boldness-including hyperactivity, lower thigmotaxis, and a reduction in the distance to a novel object. Taken together, these results demonstrate that zygotic venlafaxine exposure may impact developmental programming in a sex-specific manner in fish.

Impact of ozone-biologically active filtration on the breakthrough of Perfluoroalkyl acids during granular activated carbon treatment of municipal wastewater effluent.

The presence of perfluoroalkyl acids (PFAAs) in municipal wastewater has highlighted the need to develop PFAA treatment approaches for wastewater effluent and potable reuse applications. Ozone (O3) and biologically active filtration (BAF) were investigated as standalone and combined pretreatment processes to improve the performance of granular activated carbon (GAC) for PFAA removal from wastewater effluent. As individual processes, ozonation at all three investigated doses (0.35, 0.75, 1.0 mg O3/mg DOC) and BAF at both tested empty bed contact times (EBCT; 15 and 20 min) led to significant improvement in PFAA removal by subsequent GAC treatment. With respect to standalone ozonation, the specific O3 dose of 0.75 mg O3/mg DOC was proven to be the optimum operating condition as further increase of the specific ozone dose to 1.0 mg O3/mg DOC did not provide considerable additional improvement. Extending the EBCT during standalone BAF from 15 to 20 minutes significantly improved the efficacy of GAC for the removal of tested PFAAs. Pretreatment with O3-BAF (0.75 mg O3/mg DOC; 20 min EBCT) in tandem outperformed both standalone ozonation and BAF for the removal of PFAA by GAC. Characterization of effluent organic matter (EfOM) by size exclusion chromatography (SEC) and Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR-MS) before and after pretreatments suggest that among multiple co-occurring phenomena, the shift towards smaller and more polar EfOM may have predominantly alleviated pore constriction/blockage without having adverse impact on direct site competition. This observation is supported by SEC and FT-ICR-MS results indicating reduced EfOM molecular size through O3 and BAF pretreatment as well as transition to more hydrophilic byproducts.

Behaviour of 27 selected emerging contaminants in vertical flow constructed wetlands as post-treatment for municipal wastewater.

Six substrates (i.e. sand enriched with activated or non-activated biochar or zeolite in different ratios) were tested in Vertical Flow Constructed Wetlands (VFCWs) planted with Phragmites australis and Iris pseudacorus for the removal of 27 emerging contaminants from municipal wastewater. The laboratory investigation under controlled conditions (spiked constant concentrations in synthetic wastewater) lasted 357 days and proved VFCWs being able to provide excellent effluent quality in terms of both macro - and micropollutant elimination. Because overall removal efficiencies exceeded 90% in most of the cases, significant differences among the substrates were not detectable. For compounds with medium elimination (i.e. AMPA) the type of substrate seemed to play a strong role and the maximum amount of active ingredient adsorbed per amount of substrate has been quantified (i.e. 0.77 µg of AMPA per g of 30% biochar mixed with sand). Three of the most promising substrates from laboratory where thus selected to be tested under real conditions (fluctuation in concentration, variable temperature). As result, VFCWs with 15% activated biochar mixed with sand proved to be effective in the removal of 18 emerging contaminants and complying with national discharge standards for 4 selected compounds.

Wild longnose dace downstream of wastewater treatment plants display an obese phenotype.

Wild fish living downstream of wastewater treatment plants (WWTPs) often have increased body condition factors or body mass indices compared to upstream fish. This observation has been largely attributed to increased nutrient loading and food availability around wastewater effluent outflows. While a higher condition factor in fish is generally considered a predictor of healthy ecosystems, the metabolic status and capacity of the animals downstream of WWTPs may be a better predictor of fitness and potential population level effects. To address this, we sampled wild longnose dace (Rhinichthys cataractae), a native species in North American waterways, from sites upstream and downstream of WWTPs. Downstream fish had higher body mass indices, which corresponded with higher nutrient (lipid, protein, and glycogen) storage in somatic tissues compared to upstream fish. Liver transcriptome analysis revealed metabolic reprogramming favoring lipid synthesis, including higher hepatic triglyceride levels and transcript abundance of targeted lipogenic genes. This suggests that effluent exposure-mediated obesity in dace is a result of changes at the transcriptional level. To determine potential ecological consequences, we subjected these fish to an acute stressor in situ to determine their stress performance. Downstream fish failed to mobilize metabolites post-stress, and showed a reduction in liver aerobic and anaerobic metabolic capacity. Taken together, fish living downstream of WWTPs exhibit a greater lipid accumulation that results in metabolic disruption and may compromise the ability of these fish to cope with subsequent environmental and/or anthropogenic stressors.

Rainbow darter (Etheostoma caeruleum) from a river impacted by municipal wastewater effluents have altered gut content microbiomes.

Municipal wastewater treatment plant (WWTP) effluent contains pharmaceuticals and personal care products known to affect fish health and reproduction. The microbiome is a community of bacteria integral in maintaining host health and is influenced by species, diet, and environment. This study investigated changes in the diversity and composition of the gut content microbiome of rainbow darter (Etheostoma caeruleum) at ten sites on the Grand River, Ontario, Canada. Gut contents were collected in fall 2018 from these fish at sites upstream and downstream of two municipal wastewater treatment plants (WWTPs; Waterloo and Kitchener). 16S rRNA genes were sequenced to determine the composition and diversity (alpha and beta) of microbial taxa present. Gut content bacterial alpha diversity increased downstream of both WWTP outfalls; dominance of bacterial amplicon sequence variants decreased compared to upstream fish. Fish collected at different sites had distinct bacterial communities, with upstream samples dominant in Proteobacteria and Firmicutes, and downstream samples increasingly abundant in Proteobacteria and Cyanobacteria. In mammals, increased abundance of Proteobacteria is indicative of microbial dysbiosis and has been linked to altered health outcomes, but this is not yet known for fish. This research indicates that the fish gut content microbiome was altered downstream of WWTP effluent outfalls and could lead to negative health outcomes.

Zygotic exposure to venlafaxine disrupts cortisol stress axis activity in multiple generations of zebrafish.

Ubiquitous use of antidepressants has resulted in increased concentrations of these pharmaceuticals in waterways receiving municipal wastewater effluent. Amongst these, venlafaxine, a selective serotonin and norepinephrine reuptake inhibitor, is commonly found at concentrations surpassing 1 ppb in surface waters. We recently showed that the deposition of venlafaxine in zebrafish (Danio rerio) embryos impacts neural development in the hypothalamus, suggesting the possibility of neuroendocrine disruptions due to this antidepressant. Here, we tested the hypothesis that early developmental exposure to venlafaxine disrupts the long-term functioning of the hypothalamus-pituitary-interrenal (HPI) axis in zebrafish. Embryos (1-4 cell stage) were injected with either 0, 1, or 10 ng venlafaxine, and the ontogeny of cortisol content, as well as changes in cortisol levels following a stressor in larvae and adults were assessed across 3 generations. Zygotic venlafaxine exposure did not affect the ontogeny of cortisol production, but there was a disruption in the cortisol response to stressor exposure, which was also evident in multiple generations. In the F0 generation, venlafaxine exposure did not affect cortisol levels in response to stressor exposure in larvae, but adult females, and not males, showed an attenuated cortisol response compared to control fish. This reduction in cortisol levels in the females was rescued by stimulation with adrenocorticotropic hormone, suggesting that the disruption was at the level of the hypothalamus-pituitary axis. Venlafaxine-mediated disruption in HPI axis functioning was also evident in the F1 and F2 generations, including impaired cortisol responses to a stressor in adult female and larval fish, respectively. Taken together, our results suggest that venlafaxine is an endocrine disruptor, and early developmental exposure to this antidepressant may have long-term and generational consequences on cortisol stress axis activity in zebrafish.

Municipal wastewater effluent exposure disrupts early development, larval behavior, and stress response in zebrafish.

While wastewater treatment standards have been progressively increasing, emerging contaminants such as pharmaceuticals can nonetheless pass through treatment and end up in our watersheds. Pharmaceuticals in the parts-per-billion range can impact fish behavior, survival, and recruitment in the wild. However, the ecological risk posed by whole municipal wastewater effluents (MWWE), a complex mixture, is not clear. This knowledge gap is particularly evident for early lifestages (ELS) of fish, and because effluent discharge events are typically short, the effects of short-term MWWE exposures to ELS fish are particularly important from an environmental perspective. Here we tested the effects of rapid 30-min exposures, and short-term 24- and 72-h exposures to MWWE on development, behaviors, and stress response in zebrafish (Danio rerio) embryos, larvae, and juveniles. We obtained 24-h composite samples of tertiary-treated MWWE that contained a mixture of chemicals with affinities for serotonergic, adrenergic, dopaminergic, and ion-channel receptors. Embryos exposed to 5%, 10%, and 50% MWWE experienced developmental delays in somitogenesis and hatching rate, although there was no effect on survival. Embryonic photomotor responses were affected following 30-min and 24-h exposures to 10% and 50% MWWE, and larval visual motor responses were reduced from 24-h exposure to 10% MWWE. Exposure to 10% MWWE dulled the juvenile cortisol and lactate response following an acute air-exposure. Compromised behavioral and stress performances demonstrate the capacity of MWWE to impact phenotypes critical to the survival of fish in the environment. Taken together, we found that zebrafish were sensitive to toxic effects of MWWE at multiple life-stages.

Environmental levels of venlafaxine impact larval behavioural performance in fathead minnows.

Venlafaxine, a selective serotonin and norepinephrine reuptake inhibitor, is one of the most abundant antidepressants in municipal wastewater effluents (MWWE). The early life stages are particularly sensitive to contaminant exposure, but few studies have examined whether persistent exposure to venlafaxine impart adverse developmental outcomes. The fathead minnow (Pimephales promelas) is a widely used model for ecotoxicological studies, and this fish is native to Alberta, Canada. We tested the hypothesis that environmental levels of venlafaxine compromises early developmental behavioural performances in fathead minnows. Embryos were exposed to waterborne venlafaxine at either 0, 0.06, 0.33, 0.66, 1.37 or 3 µg L-1 concentration for 7 days. Environmental levels of venlafaxine did not impact the survival, hatch rate or heart rate of fathead minnow embryos and larvae but reduced the growth of larvae even at concentrations as low as 0.06 µg L-1. We validated thigmotaxis as a screen for anxiolytic and anxiogenic behaviour in fathead minnow larvae by exposing them to concentrations of ethanol and caffeine, respectively. Behavioural analyses revealed that early developmental exposure to venlafaxine does not alter thigmotaxis but reduced the activity of fathead minnows. The larval behavioural assays reported here for fathead minnow have the potential to be used as screening tools for the risk assessment of neurotoxic contaminants in MWWE. Overall, we demonstrate for the first time that exposure to environmental levels of venlafaxine during the critical early developmental window does not elicit an anxiogenic response but may adversely affect the larval growth performance of fathead minnows.

Assessing the effects of environmentally relevant concentrations of antidepressant mixtures to fathead minnows exposed over a full life cycle.

Antidepressant drugs have been detected in municipal wastewater effluents (MWWEs) at ng/L to low µg/L concentrations. We exposed fathead minnow (Pimephales promelas) over a full lifecycle to a mixture of five antidepressants at concentrations similar to a MWWE (1× AntiD Mix); venlafaxine at 2400 ng/L, citalopram at 240 ng/L, fluoxetine at 90 ng/L, sertraline at 20 ng/L, and bupropion at 90 ng/L, and 10× these concentrations (i.e. 10× AntiD Mix). Mean measured concentrations of venlafaxine, citalopram, fluoxetine, sertraline, and bupropion were 2300, 160, 110, 7 ng/L, and below detection limits, respectively, for the 1× AntiD Mix, and 33,000, 2900, 1000, 210, and 100 ng/L, respectively for the 10× AntiD Mix. During the life-cycle exposure, no significant changes were observed in survival of fathead minnows. When male fish from the exposed treatments reached maturity, their weights were increased compared to control males. There were no significant differences in condition factor, gonadosomatic index, or liver-somatic index in the exposed fish. Exposed fathead minnows produced similar numbers of eggs as control fish, and there were no changes in nest-defense behaviours of male minnows. Egg quality, % fertilization, and % hatching in F1 fry were unaffected by exposure to the antidepressants. Eggs hatched 0.5 d earlier, deformities in fry were 50% lower, and there were transient decreases in length of F1 larvae at 8 days post-hatch in offspring from the treatment with the 10× AntiD Mix. Overall, exposure to the antidepressant mixture at environmentally relevant concentrations (i.e. 1× AntiD Mix) caused no adverse effects in fathead minnows. Exposure to the 10× AntiD Mix increased the weight of adult male minnows and caused subtle effects in F1 offspring. This study is the first to assess sublethal effects in fish exposed to mixtures of antidepressants over a full lifecycle. CAPSULE: No effects were observed in fathead minnow exposed for a lifecycle to antidepressant mixtures at environmentally-relevant concentrations.

Municipal wastewater effluent characterization and variability analysis in view of an ozone dose control strategy during tertiary treatment: The status in Belgium.

Ozonation is known for removing trace organic contaminants (TrOCs) from secondary wastewater effluent. However, its implementation and overall efficiency on a broad scale depends on effluent characteristics, which can differ both in time as well as between different treatment plants (nowadays referred to as water resource recovery facilities (WRRFs)). Therefore, water quality was assessed over time at 15 different Belgian sampling locations to increase the understanding of effluent variability in view of online control of the tertiary ozonation step. Conventional and surrogate parameters as well as those specifically related to tertiary ozonation (e.g. instantaneous ozone demand) were assessed. Little differences between the different locations were found for spectral measurements (e.g. UVA254 or fluorescence). The small amount of observed outliers was clearly site or event dependent. A lower variability (for spectral measurements) is advantageous in simplifying the development and application of a generic control framework based on these spectral measurements. In addition, also variations in TrOC concentration levels seemed to be small, as the concentration of most individual compounds resided within one order of magnitude over multiple sampling events at two different WRRFs. The combination of this low variability in TrOC levels in the effluent before ozonation with a control strategy using a TrOC removal efficiency set-point, allows to indicatively assess absolute TrOC levels after ozonation. In contrast, significant variations between different plants (especially smaller sized plants) were observed and could be related to the conventional water quality parameters alkalinity (correlated with the electrical conductivity) and pH which are both known to have an influence on the ozonation process. This confirms that a differential dosing control strategy (i.e. accounting for the matrix reactivity) should be applied instead of one solely based on the (organic) effluent load before ozonation.

Inactivation efficiency of Escherichia coli and autochthonous bacteria during ozonation of municipal wastewater effluents quantified with flow cytometry and adenosine tri-phosphate analyses.

Inactivation kinetics of autochthonous bacteria during ozonation of wastewater effluents were investigated using cultivation-independent flow cytometry (FCM) with total cell count (TCC) and intact cell count (ICC) and intracellular adenosine triphosphate (ATP) analysis. The principles of the methods including ozone inactivation kinetics were demonstrated with laboratory-cultured Escherichia coli spiked into filtered and sterilized wastewater effluent. Both intracellular ATP and ICC decreased with increasing ozone doses, with ICC being the more conservative parameter. The log-inactivation levels (-log(N/N0) of E. coli reached the method detection limits for FCM (∼3) and ATP (∼1.7) at specific ozone doses of ≥0.5 gO3/gDOC. During ozonation of four real wastewater effluents, the log-inactivation of autochthonous bacteria with FCM ICC was 0.3-1.0 for 0.25 gO3/gDOC and increased to 1.1-2.1 for 0.5 gO3/gDOC, but remained at a similar level of 1.5-2.8 for a further increase of the specific ozone doses to 1.0 and 1.5 gO3/gDOC. The FCM data also showed that autochthonous bacteria were composed of communities with high and low ozone reactivity. The inactivation levels measured with intracellular ATP were reasonably correlated to ICC (r(2) = 0.8). Overall, FCM and ATP measurements were demonstrated to be useful tools to monitor the inactivation of autochthonous bacteria during ozonation of municipal wastewater effluents.

Reduction of antibiotic resistance genes in municipal wastewater effluent by advanced oxidation processes.

This study investigated the reduction of antibiotic resistance genes (ARGs), intI1 and 16S rRNA genes, by advanced oxidation processes (AOPs), namely Fenton oxidation (Fe(2+)/H2O2) and UV/H2O2 process. The ARGs include sul1, tetX, and tetG from municipal wastewater effluent. The results indicated that the Fenton oxidation and UV/H2O2 process could reduce selected ARGs effectively. Oxidation by the Fenton process was slightly better than that of the UV/H2O2 method. Particularly, for the Fenton oxidation, under the optimal condition wherein Fe(2+)/H2O2 had a molar ratio of 0.1 and a H2O2 concentration of 0.01molL(-1) with a pH of 3.0 and reaction time of 2h, 2.58-3.79 logs of target genes were removed. Under the initial effluent pH condition (pH=7.0), the removal was 2.26-3.35 logs. For the UV/H2O2 process, when the pH was 3.5 with a H2O2 concentration of 0.01molL(-1) accompanied by 30min of UV irradiation, all ARGs could achieve a reduction of 2.8-3.5 logs, and 1.55-2.32 logs at a pH of 7.0. The Fenton oxidation and UV/H2O2 process followed the first-order reaction kinetic model. The removal of target genes was affected by many parameters, including initial Fe(2+)/H2O2 molar ratios, H2O2 concentration, solution pH, and reaction time. Among these factors, reagent concentrations and pH values are the most important factors during AOPs.

Concentration, composition, bioavailability, and N-nitrosodimethylamine formation potential of particulate and dissolved organic nitrogen in wastewater effluents: A comparative study.

Wastewater-derived organic nitrogen (org-N) can act as both nutrients and carcinogenic nitrogenous disinfection byproduct precursors. In this study, the concentration, composition, bioavailability, and N-nitrosodimethylamine (NDMA) formation potential of particulate organic nitrogen (PON) from three different municipal wastewater treatment plants were characterized and compared with that of effluent dissolved organic nitrogen (DON). The average effluent PON and DON concentrations ranged from 0.09 to 0.55mgN/L and from 0.91 to 1.88mgN/L, respectively. According to principal component analysis, org-N composition and characterization differed in PON and DON samples (n=20). Compared with DON, PON tended to be enriched in protein and nucleic acids, and showed a more proteinaceous character. Composition of org-N functional groups estimated from the X-ray photoelectron spectroscopy N 1s spectra indicate no significant differences in the molecular weight distribution of the protein-like materials between PON and DON. Moreover, PON exhibited a significantly higher bioavailability (61.0±13.3%) compared to DON (38.5±12.4%, p˂0.05, t-test) and a significantly higher NDMA yields (791.4±404.0ng/mg-N) compared to DON (374.8±62.5ng/mg-N, p˂0.05, t-test). Accordingly, PON contributed to approximately 12.3-41.7% of the total bioavailable org-N and 22.0-38.4% of the total NDMA precursors in wastewater effluents. Thus, the potential adverse effects of PON on wastewater discharge and reuse applications should not be overlooked, even though it only accounted for 7.4-26.8% of the total effluent org-N.

A multi-level biological approach to evaluate impacts of a major municipal effluent in wild St. Lawrence River yellow perch (Perca flavescens).

The development of integrated ecotoxicological approaches is of great interest in the investigation of global concerns such as impacts of municipal wastewater effluents on aquatic ecosystems. The objective of this study was to investigate the effects of a major wastewater municipal effluent on fish using a multi-level biological approach, from gene transcription and enzyme activities to histological changes. Yellow perch (Perca flavescens) were selected based on their wide distribution, their commercial and recreational importance, and the availability of a customized microarray. Yellow perch were sampled upstream of a major municipal wastewater treatment plant (WWTP) and 4 km and 10 km downstream from its point of discharge in the St. Lawrence River (Quebec, Canada). Concentrations of perfluoroalkyl substances (PFASs), polybrominated diphenyl ethers (PBDEs) and metals/trace elements in whole body homogenates were comparable to those from other industrialized regions of the world. Genomic results indicated that the transcription level of 177 genes was significantly different (p<0.024) between exposed and non-exposed fish. Among these genes, 38 were found to be differentially transcribed at both downstream sites. Impacted genes were associated with biological processes and molecular functions such as immunity, detoxification, lipid metabolism/energy homeostasis (e.g., peroxisome proliferation), and retinol metabolism suggesting impact of WWTP on these systems. Moreover, antioxidant enzyme activities were more elevated in perch collected at the 4 km site. Biomarkers of lipid metabolism, biosynthetic activity, and aerobic capacities were significantly lower (p<0.05) in fish residing near the outfall of the effluent. Histological examination of the liver indicated no differences between sites. Correlations between PFAS, PBDE, and metal/trace element tissue concentrations and markers of peroxisomal proliferation, oxidative stress, and retinoid metabolism were found at the gene and cellular levels. Present results suggest that relating transcriptomic analyses to phenotypic responses is important to better understand impacts of environmental contamination on wild fish populations.