Dosing the Coast: Leaking Sewage Infrastructure Delivers Large Annual Doses and Dynamic Mixtures of Pharmaceuticals to Urban Rivers.

Pharmaceuticals are commonly detected at low concentrations in surface waters, where they disrupt biological and ecological processes. Despite their ubiquity, the annual mass of pharmaceuticals exported from watersheds is rarely quantified. We used liquid chromatography-mass spectroscopy to screen for 92 pharmaceuticals in weekly samples from an urban stream network in Baltimore, MD, USA, that lacks wastewater treatment effluents. Across the network, we detected 37 unique compounds, with higher concentrations and more compounds in streams with higher population densities. We also used concentrations and stream discharge to calculate annual pharmaceutical loads at the watershed outlet, which range from less than 1 kg to ∼15 kg and are equivalent to tens of thousands of human doses. By calculating annual watershed mass balances for eight compounds, we show that ∼0.05 to ∼42% of the pharmaceuticals consumed by humans in this watershed are released to surface waters, with the importance of different pathways (leaking sewage vs treated wastewater effluent) differing among compounds. These results demonstrate the importance of developing, maintaining, and improving sewage infrastructure to protect water resources from pharmaceutical contamination.

Impacts of Oxazepam on Perch (Perca fluviatilis) Behavior: Fish Familiarized to Lake Conditions Do Not Show Predicted Anti-anxiety Response.

A current theory in environmental science states that dissolved anxiolytics (oxazepam) from wastewater effluents can reduce anti-predator behavior in fish with potentially negative impacts on prey fish populations. Here, we hypothesize that European perch (Perca fluviatilis) populations being exposed to oxazepam in situ show reduced anti-predator behavior, which has previously been observed for exposed isolated fish in laboratory studies. We tested our hypothesis by exposing a whole-lake ecosystem, containing both perch (prey) and northern pike (Esox lucius; predator), to oxazepam while tracking fish behavior before and after exposure in the exposed lake as well as in an unexposed nearby lake (control). Oxazepam concentrations in the exposed lake ranged between 11 and 24 µg L-1, which is >200 times higher than concentrations reported for European rivers. In contrast to our hypothesis, we did not observe an oxazepam-induced reduction in anti-predator behavior, inferred from perch swimming activity, distance to predators, distance to conspecifics, home-range size, and habitat use. In fact, exposure to oxazepam instead stimulated anti-predator behavior (decreased activity, decreased distance to conspecifics, and increased littoral habitat use) when using behavior in the control lake as a reference. Shoal dynamics and temperature changes may have masked modest reductions in anti-predator behavior due to oxazepam. Although we cannot fully resolve the mechanism(s) behind our observations, our results indicate that the effects of oxazepam on perch behavior in a familiar natural ecosystem are negligible in comparison to the effects of other environmental conditions.

Exposure via biotransformation: Oxazepam reaches predicted pharmacological effect levels in European perch after exposure to temazepam.

It is generally expected that biotransformation and excretion of pharmaceuticals occurs similarly in fish and mammals, despite significant physiological differences. Here, we exposed European perch (Perca fluviatilis) to the benzodiazepine drug temazepam at a nominal concentration of 2 µg L-1 for 10 days. We collected samples of blood plasma, muscle, and brain in a time-dependent manner to assess its bioconcentration, biotransformation, and elimination over another 10 days of depuration in clean water. We observed rapid pharmacokinetics of temazepam during both the exposure and depuration periods. The steady state was reached within 24 h of exposure in most individuals, as was complete elimination of temazepam from tissues during depuration. Further, the biologically active metabolite oxazepam was produced via fish biotransformation, and accumulated significantly throughout the exposure period. In contrast to human patients, where a negligible amount of oxazepam is created by temazepam biotransformation, we observed a continuous increase of oxazepam concentrations in all fish tissues throughout exposure. Indeed, oxazepam accumulated more than its parent compound, did not reach a steady state during the exposure period, and was not completely eliminated even after 10 days of depuration, highlighting the importance of considering environmental hazards posed by pharmaceutical metabolites.

Chronic Exposure to Oxazepam Pollution Produces Tolerance to Anxiolytic Effects in Zebrafish (Danio rerio).

Environmental concentrations of the anxiolytic drug oxazepam have been found to disrupt antipredator behaviors of wild fish. Most experiments exposed fish for a week, while evidence from mammals suggests that chronic exposure to therapeutic concentrations of benzodiazepines (such as oxazepam) results in the development of tolerance to the anxiolytic effects. If tolerance can also develop in response to the low concentrations found in the aquatic environment, it could mitigate the negative effects of oxazepam pollution. In the current study, we exposed wild-caught zebrafish to oxazepam (∼7 µg L-1) for 7 or 28 days and evaluated behavioral and physiological parameters at both time points. Females showed reduced diving responses to conspecific alarm pheromone after 7 days, but not after 28 days, indicating that they had developed tolerance to the anxiolytic effects of the drug. Zebrafish males were not affected by this oxazepam concentration, in line with earlier results. Serotonin turnover (ratio 5-HIAA/5-HT) was reduced in exposed females and males after 28 days, indicating that brain neurochemistry had not normalized. Post-confinement cortisol concentrations and gene expression of corticotropin-releasing hormone (CRH) were not affected by oxazepam. We did not find evidence that chronically exposed fish had altered relative expression of GABAA receptor subunits, suggesting that some other still unknown mechanism caused the developed tolerance.

Novel metabolomic method to assess the effect-based removal efficiency of advanced wastewater treatment techniques.

Unprecedented levels of chemicals of anthropogenic origin are currently released into surface waters globally. Wastewater treatment plant effluent has been identified as a major source, containing a broad mixture of pharmaceuticals and consumer chemicals. Therefore, there is a need for implementation of advanced wastewater treatment techniques, such as ozonation and adsorption methods, to reduce the contamination. However, there are conflicting findings on the toxicity of treated effluent and only limited possibilities for assessing the effect-based removal efficiency (EBRE) of different treatment techniques. Here, we describe a metabolomics approach to detect perturbations in fatty acid catabolic pathways as a proxy for biological effects. Metabolites in three fatty acid pathways were analyzed in a common damselfly larva (Coenagrion hastulatum) by liquid chromatography coupled to mass spectrometry. The larvae were exposed for one week to either conventionally treated effluent (activated sludge treatment), effluent additionally treated with ozone or effluent additionally treated with biochar filtration and results were compared with those from tap water control exposure. Five lipoxygenase-derived oxylipins (9,10,13-TriHOME, 9,12,13-TriHOME, 9-HODE, 9-HOTrE, and 13-HOTrE) decreased in response to conventionally treated effluent exposure. By using an additional treatment step, oxylipin levels were restored with exception of 9,10,13-TriHOME (ozonated effluent), and 9-HOTrE and 13-HOTrE (effluent filtered with biochar). In conclusion, exposure to wastewater effluent affected fatty acid metabolite levels in damselfly larvae, and a subset of the analyzed metabolites may serve as indicators for biological effects in biota in response to effluent exposure. To that effect, our findings suggest a new metabolomics protocol for assessing EBRE.

Slow-Release Implants for Manipulating Contaminant Exposures in Aquatic Wildlife: A New Tool for Field Ecotoxicology.

Field-based ecotoxicology studies are invaluable for uncovering the effects of contaminants of emerging concern (CECs) on aquatic organisms. However, large-scale exposures are still very rare due to prohibitive costs, the availability of replicated habitats, and the potential for exposure to cause lasting damage to the environment. Here, we evaluated the viability of internal slow-release implants as an alternative method for manipulating CEC exposures in aquatic wildlife using two fat-based carriers (coconut oil and vegetable shortening). We treated roach (Rutilus rutilus) with implants containing a high (50 µg/g), low (25 µg/g), or control (0 µg/g) concentration of the behavior-modifying pharmaceutical oxazepam. We then measured oxazepam uptake in four tissues (plasma, muscle, liver, and the brain) over 1 month. The two carriers released oxazepam differently: coconut oil was the superior implant type because it delivered a more consistent dose across time, while vegetable shortening released oxazepam rapidly at the start of the exposure period. For both carriers and treatments, the brain and liver contained the most oxazepam. Overall, the method is a promising technique for controlled manipulations of pharmaceuticals in fish, and we have provided some of the first data on the suitability and contaminant release kinetics from different implant types.

Population-level surveillance of antibiotic resistance in Escherichia coli through sewage analysis.

IntroductionThe occurrence of antibiotic resistance in faecal bacteria in sewage is likely to reflect the current local clinical resistance situation.AimThis observational study investigated the relationship between Escherichia coli resistance rates in sewage and clinical samples representing the same human populations.MethodsE. coli were isolated from eight hospital (n = 721 isolates) and six municipal (n = 531 isolates) sewage samples, over 1 year in Gothenburg, Sweden. An inexpensive broth screening method was validated against disk diffusion and applied to determine resistance against 11 antibiotics in sewage isolates. Resistance data on E. coli isolated from clinical samples from corresponding local hospital and primary care patients were collected during the same year and compared with those of the sewage isolates by linear regression.ResultsE. coli resistance rates derived from hospital sewage and hospital patients strongly correlated (r2 = 0.95 for urine and 0.89 for blood samples), as did resistance rates in E. coli from municipal sewage and primary care urine samples (r2 = 0.82). Resistance rates in hospital sewage isolates were close to those in hospital clinical isolates while resistance rates in municipal sewage isolates were about half of those measured in primary care isolates. Resistance rates in municipal sewage isolates were more stable between sampling occasions than those from hospital sewage.ConclusionOur findings provide support for development of a low-cost, sewage-based surveillance system for antibiotic resistance in E. coli, which could complement current monitoring systems and provide clinically relevant antibiotic resistance data for countries and regions where surveillance is lacking.

A Comprehensive Screening of Escherichia coli Isolates from Scandinavia's Largest Sewage Treatment Plant Indicates No Selection for Antibiotic Resistance.

There is concern that sewage treatment plants (STPs) serve as hotspots for emergence and selection of antibiotic resistant bacteria. However, field studies investigating resistance selection by comparing bacterial populations in influents and effluents have produced variable and sometimes contradictive results. Also, large taxonomic changes between influents and effluents make interpretation of studies measuring relative gene abundances ambiguous. The aim here was to investigate whether within-species selection occurs by conducting a comprehensive screening of Escherichia coli isolated from composite influent and effluent samples collected at Scandinavia's largest STP, accompanied by analyses of antibiotics residues. In total, 4028 isolates, collected on eight occasions during 18 months, were screened for resistance to seven antibiotics. Although differences in proportions of resistant E. coli between influent and effluent samples were detected for a few antibiotics on two occasions, aggregated data over time showed no such differences for any of the investigated antibiotics. Neither was there any enrichment of multiresistant or extended-spectrum beta-lactamase-producing isolates through the treatment process. Despite some antibiotics were detected at or close to concentrations predicted to provide some selective pressure, field observations of resistance profiles in E. coli do not provide support for systematic selection in the investigated STP.

Environmental relevant levels of a benzodiazepine (oxazepam) alters important behavioral traits in a common planktivorous fish, (Rutilus rutilus).

Environmental pollution by pharmaceuticals is increasingly recognized as a major threat to aquatic ecosystems worldwide. A complex mix of pharmaceuticals enters waterways via treated wastewater effluent and many remain biochemically active after the drugs reach aquatic systems. However, to date little is known regarding the ecological effects that might arise following pharmaceutical contamination of aquatic environments. One group of particular concern is behaviorally modifying pharmaceuticals as seemingly minor changes in behavior may initiate marked ecological consequences. The aim of this study was to examine the influence of a benzodiazepine anxiolytic drug (oxazepam) on key behavioral traits in wild roach (Rutilus rutilus) at concentrations similar to those encountered in effluent surface waters. Roach exposed to water with high concentrations of oxazepam (280 µg/L) exhibited increased boldness, while roach at low treatment (0.84 µg/L) became bolder and more active compared to control fish. Our results reinforce the notion that anxiolytic drugs may be affecting fish behavior in natural systems, emphasizing the need for further research on ecological impacts of pharmaceuticals in aquatic systems and development of new tools to incorporate ecologically relevant behavioral endpoints into ecotoxicological risk assessment.

Improving environmental risk assessment of human pharmaceuticals.

This paper presents 10 recommendations for improving the European Medicines Agency's guidance for environmental risk assessment of human pharmaceutical products. The recommendations are based on up-to-date, available science in combination with experiences from other chemical frameworks such as the REACH-legislation for industrial chemicals. The recommendations concern: expanding the scope of the current guideline; requirements to assess the risk for development of antibiotic resistance; jointly performed assessments; refinement of the test proposal; mixture toxicity assessments on active pharmaceutical ingredients with similar modes of action; use of all available ecotoxicity studies; mandatory reviews; increased transparency; inclusion of emission data from production; and a risk management option. We believe that implementation of our recommendations would strengthen the protection of the environment and be beneficial to society. Legislation and guidance documents need to be updated at regular intervals in order to incorporate new knowledge from the scientific community. This is particularly important for regulatory documents concerning pharmaceuticals in the environment since this is a research field that has been growing substantially in the last decades.

Fluoroquinolones and qnr genes in sediment, water, soil, and human fecal flora in an environment polluted by manufacturing discharges.

There is increasing concern that environmental antibiotic pollution promotes transfer of resistance genes to the human microbiota. Here, fluoroquinolone-polluted river sediment, well water, irrigated farmland, and human fecal flora of local villagers within a pharmaceutical industrial region in India were analyzed for quinolone resistance (qnr) genes by quantitative PCR. Similar samples from Indian villages farther away from industrial areas, as well as fecal samples from Swedish study participants and river sediment from Sweden, were included for comparison. Fluoroquinolones were detected by MS/MS in well water and soil from all villages located within three km from industrially polluted waterways. Quinolone resistance genes were detected in 42% of well water, 7% of soil samples and in 100% and 18% of Indian and Swedish river sediments, respectively. High antibiotic concentrations in Indian sediment coincided with high abundances of qnr, whereas lower fluoroquinolone levels in well water and soil did not. We could not find support for an enrichment of qnr in fecal samples from people living in the fluoroquinolone-contaminated villages. However, as qnr was detected in 91% of all Indian fecal samples (24% of the Swedish) it suggests that the spread of qnr between people is currently a dominating transmission route.

Pesticide screening of surface water and soil along the Mekong River in Cambodia.

Widespread use of pesticides globally has led to serious concerns about environmental contamination, particularly with regard to aquatic and soil ecosystems. This work involved investigating concentrations of 64 pesticides in surface-water and soil samples collected in four provinces along the Mekong River in Cambodia during the dry and rainy seasons (276 samples in total), and conducting semi-structured interviews with local farmers about pesticide use. Furthermore, an ecological risk assessment of the detected pesticides was performed. In total, 56 pesticides were detected in surface water and 43 in soil, with individual pesticides reaching maximum concentrations of 1300 ng/L in the surface-water samples (tebufenozide) and 1100 ng/g dry weight in the soil samples (bromophos-ethyl). The semi-structured interviews made it quite evident that the instructions that farmers are provided regarding the use of pesticides are rudimentary, and that overuse is common. The perceived effect of pesticides was seen as an end-point, and there was a limited process of optimally matching pesticides to pests and crops. Several pesticides were used regularly on the same crop, and the period between application and harvest varied. Risk analysis showed that bromophos-ethyl, dichlorvos, and iprobenfos presented a very high risk to aquatic organisms in both the dry and rainy seasons, with risk quotient values of 850 for both seasons, and of 67 in the dry season and 78 in the rainy season for bromophos-ethyl, and 49 in the dry season and 16 in the rainy season for dichlorvos. Overall, this work highlights the occurrence of pesticide residues in surface water and soil along the Mekong River in Cambodia, and emphasizes the urgent need for monitoring and improving pesticide practices and regulations in the region.

The synthetic progestin levonorgestrel is a potent androgen in the three-spined stickleback (Gasterosteus aculeatus).

The use of progestins has resulted in contamination of aquatic environments and some progestins have in experimental studies been shown to impair reproduction in fish and amphibians at low ng L(-1) concentrations. The mechanisms underlying their reproductive toxicity are largely unknown. Some progestins, such as levonorgestrel (LNG), exert androgenic effects in mammals by activating the androgen receptor (AR). Male three-spined stickleback (Gasterosteus aculeatus) kidneys produce spiggin, a gluelike glycoprotein used in nest building, and its production is directly governed by androgens. Spiggin is normally absent in females but its production in female kidneys can be induced by AR agonists. Spiggin serves as the best known biomarker for androgens in fish. We exposed adult female sticklebacks to LNG at 5.5, 40, and 358 ng L(-1) for 21 days. Androgenic effects were found at LNG concentrations ≥40 ng L(-1) including induction of spiggin transcription, kidney hypertrophy, and suppressed liver vitellogenin transcription. These are the first in vivo quantitative data showing that LNG is a potent androgen in fish supporting the contention that androgenic effects of certain progestins contribute to their reproductive toxicity.

Neuroactive pharmaceuticals in estuaries: Occurrence and tissue-specific bioaccumulation in multiple fish species.

Contamination of surface waters by pharmaceuticals is an emerging problem globally. This is because the increased access and use of pharmaceuticals by a growing world population lead to environmental contamination, threatening non-target species in their natural environment. Of particular concern are neuroactive pharmaceuticals, which are known to bioaccumulate in fish and impact a variety of individual processes such as fish reproduction or behaviour, which can have ecological impacts and compromise fish populations. In this work, we investigate the occurrence and bioaccumulation of 33 neuroactive pharmaceuticals in brain, muscle and liver tissues of multiple fish species collected in four different estuaries (Douro, Tejo, Sado and Mira). In total, 28 neuroactive pharmaceuticals were detected in water and 13 in fish tissues, with individual pharmaceuticals reaching maximum concentrations of 1590 ng/L and 207 ng/g ww, respectively. The neuroactive pharmaceuticals with the highest levels and highest frequency of detection in the water samples were psychostimulants, antidepressants, opioids and anxiolytics, whereas in fish tissues, antiepileptics, psychostimulants, anxiolytics and antidepressants showed highest concentrations. Bioaccumulation was ubiquitous, occurring in all seven estuarine and marine fish species. Notably, neuroactive compounds were detected in every water and fish brain samples, and in 95% of fish liver and muscle tissues. Despite variations in pharmaceutical occurrence among estuaries, bioaccumulation patterns were consistent among estuarine systems, with generally higher bioaccumulation in fish brain followed by liver and muscle. Moreover, no link between bioaccumulation and compounds' lipophilicity, species habitat use patterns or trophic levels was observed. Overall, this work highlights the occurrence of a highly diverse suite of neuroactive pharmaceuticals and their pervasiveness in waters and fish from estuarine systems with contrasting hydromorphology and urban development and emphasizes the urgent need for toxicity assessment of these compounds in natural ecosystems, linked to internalized body concentration in non-target species.

Cost-Effective Pharmaceutical Implants in Fish: Validating the Performance of Slow-Release Implants for the Antidepressant Fluoxetine.

Internal, slow-release implants can be an effective way to manipulate animal physiology or deliver a chemical exposure over long periods of time without the need for an exogenous exposure route. Slow-release implants involve dissolving a compound in a lipid-based carrier, which is inserted into the body of an organism. However, the release kinetics of the compound from the implant to body tissues also requires careful validation. We tested and validated a slow-release implant methodology for exposing fish to a pharmaceutical pollutant, fluoxetine. We tested two lipid-based carriers (coconut oil or vegetable shortening) in the common roach (Rutilus rutilus). The implants contained either a high (50 µg/g), low (25 µg/g), or control (0 µg/g) concentration of fluoxetine, and we measured tissue uptake in the brain, muscle, and plasma of implanted fish over 25 days. The two carriers released fluoxetine differently over time: coconut oil released fluoxetine in an accelerating manner (tissue uptake displayed a positive quadratic curvature), whereas vegetable shortening released fluoxetine in a decelerating manner (a negative quadratic curvature). For both carrier types, fluoxetine was measured at the highest concentration in the brain, followed by muscle and plasma. By comparing the implant exposures with waterborne exposures in the published literature, we showed that the implants delivered an internal exposure that would be similar if fish were exposed in surface waters containing effluents. Overall, we showed that slow-release internal implants are an effective method for delivering chronic exposures of fluoxetine over at least 1-month time scales. Internal exposures can be an especially powerful experimental tool when coupled with field-based study designs to assess the impacts of pharmaceutical pollutants in complex natural environments. Environ Toxicol Chem 2023;42:1326-1336. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Disrupted oogenesis in the frog Xenopus tropicalis after exposure to environmental progestin concentrations.

Levonorgestrel is a synthetic progesterone commonly used in pharmaceuticals (e.g., in contraceptives). It is found in sewage treatment plant effluents at concentrations up to 30 ng/L and was recently shown to pose a threat to egg laying in fish. Information on the susceptibility of adult amphibians to progestin toxicity is lacking. The present study aimed to 1) characterize progestogenic effects on the full cycle of oogenesis (egg development) in frogs and 2) determine female amphibians' susceptibility to reproductive impacts from progestogenic compounds in the environment. Sexually mature female Xenopus tropicalis were exposed to levonorgestrel via the surrounding water for 7 days (0, 51, or 307 ng/L) or 28 days (0, 1.3, 18, 160, or 1240 ng/L). Their ovaries were analyzed histologically with respect to frequencies of immature (in early meiotic prophase I), previtellogenic, vitellogenic, mature, and atretic oocytes. The 28-day exposure caused reduced proportions of oocytes at immature, vitellogenic, and mature stages, and increased proportions of previtellogenic oocytes compared with the control. The lowest tested concentration, 1.3 ng/L, increased the proportions of previtellogenic oocytes and reduced the proportions of vitellogenic oocytes, indicating inhibited vitellogenesis. The present study shows that progestin concentrations found in the aquatic environment impaired oogenesis in adult frogs. Our results indicate that progestogenic effects on oocyte development include interrupted germ cell progression into meiosis and inhibited vitellogenesis. Considering the crucial role of oogenesis in female fertility, our results indicate that progestogenic pollutants may pose a threat to reproduction in wild amphibian populations.

An evaluation of free water surface wetlands as tertiary sewage water treatment of micro-pollutants.

Increased attention is currently directed towards potential negative effects of pharmaceuticals and other micro-pollutants discharged into the aquatic environment via municipal sewage water. A number of additional treatment technologies, such as ozonation, have therefore been suggested as promising tools for improving the removal efficiency of pharmaceuticals in existing Sewage Treatment Plants (STPs). Constructed wetlands are also capable of removing a variety of micro-pollutants, including some pharmaceuticals, and could hence be a resource efficient complement to more advanced treatment technologies. The purpose of the present study was therefore to increase the knowledge base concerning the potential use of constructed wetlands as a treatment step to reduce emissions of organic micro-pollutants from municipal sewage effluents. Under cold winter conditions, incoming and outgoing waters from four Swedish free water surface wetlands, operated as final treatment steps of sewage effluent from municipal STPs, were sampled and analyzed for levels of a set of 92 pharmaceuticals and 22 inorganic components as well as assessed using subchronic ecotoxicity tests with a macro-alga and a crustacean. Sixty-five pharmaceuticals were detected in the range from 1 ng L(-1) to 7.6 µg L(-1) in incoming and outgoing waters from the four investigated wetlands. Although the sampling design used in the present study lacks the robustness of volume proportional to 24h composite samples, the average estimated removal rates ranged from 42% to 52%, which correlates to previous published values. The effects observed in the ecotoxicity tests with the macro-alga (EC(50)s in the range of 7.5-46%) and the crustacean (LOECs in the range of 11.25-90%) could not be assigned to either pharmaceutical residues or metals, but in general showed that these treatment facilities release water with a relatively low toxic potential, comparable to water that has been treated with advanced tertiary treatments. From the present study it can be concluded that constructed wetlands may provide a complementary sewage treatment option, especially where other treatment is lacking today. To fully remove micro-pollutants from sewage effluent, however, other more advanced treatment technologies are likely needed.

Environmentally relevant concentration of caffeine-effect on activity and circadian rhythm in wild perch.

We studied the ecological consequences of widespread caffeine contamination by conducting an experiment focused on changes in the behavioral traits of wild perch (Perca fluviatilis) after waterborne exposure to 10 µg L-1 of caffeine. We monitored fish swimming performance during both light and dark conditions to study the effect of caffeine on fish activity and circadian rhythm, using a novel three-dimensional tracking system that enabled positioning even in complete darkness. All individuals underwent three behavioral trials-before exposure, after 24 h of exposure, and after 5 days of exposure. We did not observe any effect of the given caffeine concentration on fish activity under light or dark conditions. Regardless of caffeine exposure, fish swimming performance was significantly affected by both the light-dark conditions and repeating of behavioral trials. Individuals in both treatments swam significantly more during the light condition and their activity increased with time as follows: before exposure < after 24 h of exposure < after 5 days of exposure. We confirmed that the three-dimensional automated tracking system based on infrared sensors was highly effective for conducting behavioral experiments under completely dark conditions.

Bioconcentration of neuroactive pharmaceuticals in fish: Relation to lipophilicity, experimental design and toxicity in the aquatic environment.

Uptake of contaminants is linked to their toxicity and is usually estimated through their lipophilicity (logKow). Here, we review current literature regarding bioconcentration, i.e. uptake of contaminants from the external environment only, and the effects of exposure to neuroactive pharmaceuticals in fish. We aim to determine if lipophilicity is a suitable predictor of bioconcentration of these compounds in fish, to identify major drivers of bioconcentration and explore the link between bioconcentration potential and toxicity, focusing on survival, growth, condition, behaviour and reproduction endpoints. Additionally, we compare concentrations known to elicit significant effects in fish with current environmental concentrations, identifying exposure risk in ecosystems. The majority of studies have focused on antidepressants, mainly fluoxetine, and encompasses mostly freshwater species. Few studies determined pharmaceuticals bioconcentration, and even a smaller portion combined bioconcentration with other toxicity endpoints. Results show that lipophilicity isn't a good predictor of neuroactive pharmaceuticals' bioconcentration in fish, which in turn is highly influenced by experimental parameters, including abiotic conditions, species and life-stage. The need for increased standardization of experimental settings is key towards improving accuracy of environmental risk assessments and application in future regulatory schemes. Still, increased fish lethality was linked to increased bioconcentration, yet no other correlations were observed when considering effects on growth, condition, behaviour or reproduction, likely as a result of insufficient and variable data. In the context of current environmental concentrations, several neuroactive pharmaceuticals were found to be potentially threatening, while data on occurrence is lacking for some compounds, particularly in brackish/marine systems. Specifically, nine compounds (fluoxetine, citalopram, sertraline, amitriptyline, venlafaxine, clozapine, carbamazepine, metamfetamine and oxazepam) were found at concentrations either above or critically close to minimum response concentrations, thus likely to affect fish in freshwater and brackish or marine environments, which supports further exploration in risk management strategies and monitoring programs in aquatic environments.

Phosphorus release from hydrothermally carbonized digested sewage sludge using organic acids.

Hydrothermal carbonization (HTC) is a treatment technique with great potential for sanitizing digested sewage sludge (SS) and converting it into valuable products. In particular, phosphorus (P) recovery from hydrothermally carbonized SS has attracted special attention in recent years. This work aims to examine the leaching efficiency of P and the consequent release of metals and heavy metals from SS hydrochars (at 180, 215 and 250 °C) using organic acids (oxalate and citrate) over a range of pH values (0-4) and extraction times (5 min-24 h). Both organic acids triggered P extraction efficiencies exceeding 75 % at the lowest pH, but only oxalate reached a nearly complete P release from hydrochars at pH > 0 and for all carbonization temperatures. Low HTC temperature (180 °C) and short extraction time (5 min) were the optimal conditions treatment for P recovery when reacted in oxalate solutions of maximal pH buffering capacity (pH = 1.4). However, oxalate leaching also transferred metals/heavy metals into the P-leachate, with the exception of Ca being retained in the solid residue from HTC as Ca-oxalate precipitate. Different characterization methods confirmed the presence of this precipitate, and provided information about the surface and morphological changes of the SS hydrochars following acid treatment. The results suggest that HTC not only a promising technique to sanitize and reduce the volume of SS, but also an efficient means for P recovery using oxalic acid, thus contributing to the circular economy of P.