Comprehensive profiling and semi-quantification of exogenous chemicals in human urine using HRMS-based strategies

Chemicals infiltrate our daily experiences through multiple exposure pathways. Human biomonitoring (HBM) is routinely used to comprehensively understand these chemical interactions. Historically, HBM depended on targeted screening methods limited to a relatively small set of chemicals with triple quadrupole instruments typically. However, recent advances in high-resolution mass spectrometry (HRMS) have facilitated the use of broad-scope target, suspect, and non-target strategies, enhancing chemical exposome characterization within acceptable detection limits. Despite these advancements, establishing robust and efficient sample treatment protocols is still essential for trustworthy broad-range chemical analysis. This study sought to validate a methodology leveraging HRMS-based strategies for accurate profiling of exogenous chemicals and related metabolites in urine samples. We evaluated five extraction protocols, each encompassing various chemical classes, such as pharmaceuticals, plastic additives, personal care products, and pesticides, in terms of their extraction recoveries, linearity, matrix effect, sensitivity, and reproducibility. The most effective protocol was extensively validated and subsequently applied to 10 real human urine samples using wide-scope target analysis encompassing over 2000 chemicals. We successfully identified and semi-quantified a total of 36 chemicals using an ionization efficiency-based model, affirming the methodology's robust performance. Notably, our results dismissed the need for a deconjugation step, a typically labor-intensive and time-consuming process

Insights into environmental caffeine contamination in ecotoxicological biomarkers and potential health effects of Danio rerio

Caffeine (CAF) exposures have been shown to cause several pharmacological and biological effects in target and non-target organisms. Although there are already several ecotoxicological studies with CAF in non-target organisms, they are focused on marine organisms, with relevant concentrations in these ecosystems, therefore, less ecologically relevant to freshwater ecosystems (the main ecoreceptor of this type of anthropogenic contaminant). The present study aimed to assess the chronic effects (28 days) of sub-lethal and environmentally relevant concentrations of CAF (0.16, 0.42, 1.09, 2.84, 7.40, 19.23, and 50 mu g/L) in Danio rerio. Biochemical endpoints as biomarkers of antioxidant defense, biotransformation, lipid peroxidation, energy sources, and neurotransmission were assessed. CAF exposure induced alterations in antioxidant defenses (su-peroxide dismutase and glutathione reductase activities, and glutathione content) preventing lipid peroxidation. Lactate dehydrogenase activity decreased in all the concentrations tested, while acetylcholinesterase activity was only affected by the highest concentrations tested (19.23 and 50 mu g/L). We also utilized a multi-biomarker approach (Integrated Biomarker Response version 2, IBRv2) to investigate the effects of CAF in the dispersion scope of individual biochemical responses of D. rerio. IBRv2 showed that the concentration of 50 mu g/L promotes the highest stress. However, the results showed that CAF induced disturbances in the metabolic pathways studied in D. rerio. These results demonstrated the toxic effects of CAF on freshwater fish, compromising their physiological functions and evidencing the need for monitoring the residues of CAF released into the inland aquatic environments. Furthermore, this research evidence that phylogenetically and physiologically different species may present different biological responses with concern for ecologically relevant environmental conditions. In this sense, the present study generated ecotoxicologically relevant data, that can be considered by environment regulators, since the here-endpoints evaluated showed sensitivity and consistency in the evaluation of caffeine risks in freshwater environments

Use of lignocellulosic substrate colonized by oyster mushroom (Pleurotus ostreatus) for removal of organic micropollutants from water

Efficient removal techniques are urgently needed to remove organic micropollutants (OMPs) from wastewater, in order to protect water resources. In this study, laccase activity of mushroom substrate colonized by Pleurotus ostreatus was evaluated as a novel wastewater treatment method for removal of OMPs, including diclofenac, bicalutamide, lamotrigine, and metformin at environmentally relevant concentrations. Laccase activity of the colonized mushroom substrate was found to be highest, 0.8 enzyme activity (U)/g mushroom substrate wet weight, immediately before initiation of fruiting body formation. The selected OMPs were treated for 5 min with suspensions of mushroom substrate with laccase activity of approximately 50 U/L. Removal of all OMPs was significant, with the highest removal for diclofenac of 90% compared with a control with uncolonized mushroom substrate. To our knowledge, direct use of colonized mushroom substrate in removing diclofenac from water has not been reported previously. Removal efficiency of bicalutamide, lamotrigine, and metformin was 43%, 73%, and 59%, respectively. This demonstrates potential for using mushroom substrate colonized by P. ostreatus for removal of OMPs from wastewater

Risk-based screening for prioritisation of organic micropollutants in Swedish freshwater

Concerns about environmental contamination by organic micropollutants (OMPs) are increasing, due to their potential bioaccumulative and toxic properties. This study evaluated the risk posed by OMPs to aquatic ecosystems in Swedish freshwaters. The assessment was based on measured environmental concentrations (MEC) of OMPs in surface waters upstream and downstream of Swedish wastewater treatment plants (WWTPs). A novel optimised risk quotient (RQ(f)) was used to identify potential high-risk substances in the aquatic environment. A secondary objective was to assess the impact of WWTP effluent on aquatic ecosystems using a novel impact factor (I) based on the risk quotient (RQ). Among the 126 substances investigated, four compounds (metformin, N,N-dimethyltetradecylamine, oxazepam, and venlafaxine) were identified as likely to pose a risk to aquatic ecosystems in Swedish surface waters (RQ(f)>1), and five compounds (clindamycin, gemfibrozil, sertraline, o-desmethylvenlafaxine, and diclofenac) were identified as posing a moderate risk to aquatic ecosystems (0.1<1). WWTP effluent appeared to pose an environmental risk for all recipient sites, but the impact of calculated RQ was site-specific. These results can be used by authorities to prioritise OMPs and contaminated hotspots, in order to decrease negative impacts on aquatic ecosystems.Synopsis: A novel optimised risk assessment approach for identification of high-concern organic micropollutants in aquatic environments

What is in the fish? Collaborative trial in suspect and non-target screening of organic micropollutants using LC- and GC-HRMS

A collaborative trial involving 16 participants from nine European countries was conducted within the NORMAN network in efforts to harmonise suspect and non-target screening of environmental contaminants in whole fish samples of bream (Abramis brama). Participants were provided with freeze-dried, homogenised fish samples from a contaminated and a reference site, extracts (spiked and non-spiked) and reference sample preparation protocols for liquid chromatography (LC) and gas chromatography (GC) coupled to high resolution mass spectrometry (HRMS). Participants extracted fish samples using their in-house sample preparation method and/or the protocol provided. Participants correctly identified 9–69 % of spiked compounds using LC-HRMS and 20–60 % of spiked compounds using GC-HRMS. From the contaminated site, suspect screening with participants’ own suspect lists led to putative identification of on average ~145 and ~20 unique features per participant using LC-HRMS and GC-HRMS, respectively, while non-target screening identified on average ~42 and ~56 unique features per participant using LC-HRMS and GC-HRMS, respectively. Within the same sub-group of sample preparation method, only a few features were identified by at least two participants in suspect screening (16 features using LC-HRMS, 0 features using GC-HRMS) and non-target screening (0 features using LC-HRMS, 2 features using GCHRMS). The compounds identified had log octanol/water partition coefficient (KOW) values from − 9.9 to 16 and mass-to-charge ratios (m/z) of 68 to 761 (LC-HRMS and GC-HRMS). A significant linear trend was found between log KOW and m/z for the GC-HRMS data. Overall, these findings indicate that differences in screening results are mainly due to the data analysis workflows used by different participants. Further work is needed to harmonise the results obtained when applying suspect and non-target screening approaches to environmental biota samples

Production of oyster mushroom (Pleurotus ostreatus) on sawdust supplemented with anaerobic digestate

Anaerobic digestion of organic waste results in production of biogas and a nutrient-rich digestate that has an established use as fertilizer in plant production. This study evaluated use of anaerobic digestate based on a high concentration of organic household waste as a fertilizer in sawdust-based production of oyster mushrooms (Pleurotus ostreatus). Inclusion of 0.5 L of anaerobic digestate (AD) per kg sawdust gave similar productivity in terms of biological efficiency (79.5 +/- 5.4 %), and protein concentration (24.7 +/- 2.4 % of dry weight (dw)) as standard mushroom substrate (78.1 +/- 5.3 %, and 21.9 +/- 3.0 % of dw, respectively). However, mushroom growth was impaired at the highest concentration of anaerobic digestate tested, 1 L digestate per kg dw sawdust. Comparison of the AD-fertilized substrate with a mushroom substrate with standard components (sawdust, wheat bran, calcium sulfate) and with similar C/N-ratio revealed some differences in elemental composition of the fruiting bodies, with an major increase in sodium concentration for the AD-fertilized substrate compared with the standard substrate (413.3 +/- 28.9 and 226.7 +/- 30.6 mg kg(-1) dw, respectively). This difference can be explained by high sodium concentration in the anaerobic digestate, most likely due to inclusion of food scraps from households and restaurants in the biodigester feedstock. Screening of both substrates for a total of 133 micro -pollutants revealed that total sum of micropollutants was significantly higher in the AD-fertilized substrate (258 +/- 12 ng/g dw substrate) than in the standard substrate (191 +/- 35 ng/g dw substrate). Nitrogen losses during preparation of the AD-fertilized substrate were negligible

New extraction method prior to screening of organic micropollutants in various biota matrices using liquid chromatography coupled to high-resolution time-of-flight mass spectrometry

A new extraction method with limited clean-up requirements prior to screening various matrices for organic micropollutants using liquid chromatography-high resolution mass spectrometry (LC-HRMS) for analysis was developed. First, the performance of three extraction methods (QuEChERS with SPE clean-up, ultrasonication with SPE clean-up, extraction without SPE clean-up) was tested, optimized, and compared using > 200 contaminants of emerging concern (CECs) together covering a wide range of physicochemical properties applicable for suspect and non-target screening in biota. White-tailed sea eagle (Haliaeetus albicilla) muscle tissue was used in method development and optimization. The method without SPE clean-up was then applied to European perch (Perca fluviatilis) muscle, heart, and liver tissues. The optimization and application of the method demonstrated a wide applicable domain of the novel extraction method regarding species, tissues, and chemicals. For future applications, the suitability of the method for suspect and non-target screening was tested. Overall, our extraction method appears to be sufficiently simple and broad (relatively non-discriminant) for use prior to analysis of CECs in various biota

Occurrence and mass flows of contaminants of emerging concern (CECs) in Sweden's three largest lakes and associated rivers

Contaminants of emerging concern (CECs) are a concern in aquatic environments due to possible adverse effects on the environment and humans. This study assessed the occurrence and mass flows of CECs in Sweden's three largest lakes and 24 associated rivers. The occurrence and distribution of 105 CECs was investigated, comprising 71 pharmaceuticals, 13 perfluoroalkyl substances (PFASs), eight industrial chemicals, four personal care products (PCPs), three parabens, two pesticides, and four other CECs (mostly anthropogenic markers). This is the first systematic study of CECs in Sweden's main lakes and one of the first to report environmental concentrations of the industrial chemicals tributyl citrate acetate and 2,2 '-dimorpholinyldiethyl-ether. The Sigma CEC concentration was generally higher in river water (31-5200 ng/L; median 440 ng/L) than in lake water (36-900 ng/L; median 190 ng/L). At urban lake sites, seasonal variations were observed for PCPs and parabens, and also for antihistamines, antidiabetics, antineoplastic agents, antibiotics, and fungicides. The median mass CEC load in river water was 180 g/day (range 4.0-4300 g/day), with a total mass load of 5000 g/day to Lake Vanern, 510 g/day to Lake Vattern, and 5600 g/day to Lake Malaren. All three lakes are used as drinking water reservoirs, so further investigations of the impact of CECs on the ecosystem and human health are needed

Gene co-expression network analysis reveals mechanisms underlying ozone-induced carbamazepine toxicity in zebrafish (Danio rerio) embryos

Sewage effluent ozonation can reduce concentrations of chemical pollutants including pharmaceutical residues. However, the formation of potentially toxic ozonation byproducts (OBPs) is a matter of concern. This study sought to elucidate toxicity mechanisms of ozonated carbamazepine (CBZ), an anti-epileptic drug frequently detected in sewage effluents and surface water, in zebrafish embryos (Danio rerio). Embryos were exposed to ozonated and non-ozonated CBZ from 3 h post-fertilization (hpf) until 144 hpf. Embryotoxicity endpoints (proportion of dead and malformed embryos) were assessed at 24, 48, and 144 hpf. Heart rate was recorded at 48 hpf. Exposure to ozonated CBZ gave rise to cardiovascular-related malformations and reduced heart rate. Moreover, embryo-larvae exposed to ozonated CBZ displayed a lack of swim bladder inflation. Hence, the expression patterns of CBZ target genes involved in cardiovascular and embryonal development were investigated through a stepwise gene co-expression analysis approach. Two co-expression networks and their upstream transcription regulators were identified, offering mechanistic explanations for the observed toxicity phenotypes. The study presents a novel application of gene co-expression analysis elucidating potential toxicity mechanisms of an ozonated pharmaceutical with environmental relevance. The resulting data was used to establish a putative adverse outcome pathway (AOP).Peer reviewe

Uptake of perfluoroalkyl substances, pharmaceuticals, and parabens by oyster mushrooms (Pleurotus ostreatus) and exposure risk in human consumption

Organic micropollutants (MPs) pose potential threats to environmental ecosystems and human health. This study investigated uptake of perfluoroalkyl substances (PFASs), pharmaceuticals, and paraben by edible oyster mushrooms (Pleurotus ostreatus), cultivated on spiked growth substrate. Concentrations of pharmaceuticals and paraben in substrate showed a decreasing trend over a 20-day harvesting period, whereas PFAS concentrations were variable over the harvesting period. However, only propylparaben, clarithromycin, and PFASs were detected in fruiting bodies of oyster mushroom. Uptake of PFASs by oyster mushroom fruit bodies was negatively correlated with perfluorocarbon chain length. An impact of MPs on fungal colonization was observed, with decreased respiration in treatments with the highest concentration of MPs, but production of fruiting bodies was not affected by exposure level. The potential human risk from ingestion of MPs was evaluated for oyster mushrooms exposed to the highest concentration of MPs in substrate, based on acceptable daily intake (ADI)