Tracking Aromatic Amines from Sources to Surface Waters.

This review examines the environmental occurrence and fate of aromatic amines (AAs), a group of environmental contaminants with possible carcinogenic and mutagenic effects. AAs are known to be partially responsible for the genotoxic traits of industrial wastewater (WW), and AA antioxidants are acutely toxic to some aquatic organisms. Still, there are gaps in the available data on sources, occurrence, transport, and fate in domestic WW and indoor environments, which complicate the prevention of adverse effects in aquatic ecosystems. We review key domestic sources of these compounds, including cigarette smoke and grilled protein-rich foods, and their presence indoors and in aquatic matrices. This provides a basis to evaluate the importance of nonindustrial sources to the overall environmental burden of AAs. Appropriate sampling techniques for AAs are described, including copper-phthalocyanine trisulfonate materials, XAD resins in solid-phase extraction, and solid-phase microextraction methods, which can offer insights into AA sources, transport, and fate. Further discussion is provided on potential progress in the research of AAs and their behavior in an aim to support the development of a more comprehensive understanding of their effects and potential environmental risks.

Investigation of occurrence of aromatic amines in municipal wastewaters using passive sampling.

Aromatic amines (AAs) are human-made compounds known for their mutagenic properties, entering surface waters from various sources, often originating as transformation products of dyes or pesticides. Despite their low concentrations in surface waters, AAs can exhibit mutagenicity. Our study focused on evaluating three passive samplers (PSs) for enriching these compounds from influent and effluent of a wastewater treatment plant (WWTP) in Brno, Czech Republic. The PSs tested included variants containing AttractSPE™ SDB-RPS sorbent disk, one with and one without a diffusive agarose hydrogel layer, and a modified Speedisk (Bakerbond Speedisk® H2O-Philic). PSs were deployed in wastewater (WW) for one to four weeks in various overlapping combinations, and the uptake of AAs to PSs was compared to their concentrations in 24-hour composite water samples. A targeted LC/MS analysis covered 42 amines, detecting 11 and 13 AAs in daily composite influent and effluent samples, respectively. In the influent, AAs ranged from 1.5 ng L-1 for 1-anilinonaphthalene to 1.0 µg L-1 for aniline, and the highest concentration among all measured amines was observed for cyclohexylamine at 2.9 µg L-1. In the effluent, concentrations ranged from 0.5 ng L-1 for 1-anilinonaphthalene to 88 ng L-1 for o-anisidine. PSs demonstrated comparable accumulation of amines, with integrative uptake up to 28 days in both influent and effluent and detection of up to 23 and 27 amines in influent and effluent, respectively; altogether 34 compounds were detected in the study. Sampling rates (Rs) were estimated for compounds present in at least 50 % of the samples and showing <40 % aqueous concentration variability, with robustness evaluated by comparing values for compounds in WWTP influent and effluent. Although all devices performed similarly, hydrogel-based PS exhibited superior performance in several criteria, including time integration and robustness of sampling rates, making it a suitable monitoring tool for AAs in WW.

Dataset comprising pesticides, pharmaceuticals and personal care products, and industrial chemicals detected in streams and rivers of Central Chile.

Chemical pollution caused by synthetic organic chemicals at low concentrations in the environment poses a growing threat to the ecological status of aquatic ecosystems. These chemicals are regularly released into surface waters through both treated and untreated effluents from wastewater treatment plants (WWTPs), agricultural runoff, and industrial discharges. Consequently, they accumulate in surface waters, distribute amongst environmental compartments according to their physicochemical properties, and cause adverse effects on aquatic organisms. Unfortunately, there is a lack of data regarding the occurrence of synthetic organic chemicals, henceforth micropollutants, in South American freshwater ecosystems, especially in Chile. To address this research gap, we present a comprehensive dataset comprising concentrations of 153 emerging chemicals, including pesticides, pharmaceutical and personal care products (PPCPs), surfactants, and industrial chemicals. These chemicals were found to co-occur in surface waters within Central Chile, specifically in the River Aconcagua Basin. Our sampling strategy involved collecting surface water samples from streams and rivers with diverse land uses, such as agriculture, urban areas, and natural reserves. For sample extraction, we employed an on-site large-volume solid phase extraction (LVSPE) device. The resulting environmental extracts were then subjected to wide-scope chemical target screening using gas chromatography and liquid chromatography high-resolution mass spectrometry (GC- and LC-HRMS). The dataset we present holds significant value in assessing the chemical status of water bodies. It enables comparative analysis of pollution fingerprints associated with emerging chemicals across different freshwater systems. Moreover, the data can be reused for environmental risk assessment studies. Its utilisation will contribute to a better understanding of the impact and extent of chemical pollution in aquatic ecosystems, facilitating the development of effective mitigation strategies.

Neurotoxicity in complex environmental mixtures-a case-study at River Danube in Novi Sad (Serbia) using zebrafish embryos.

Acetylcholinesterase (AChE) inhibitors are an important class of neuroactive chemicals that are often detected in aquatic and terrestrial environments. The correct functionality of the AChE enzyme is linked to many important physiological processes such as locomotion and respiration. Consequently, it is necessary to develop new analytical strategies to identify harmful AChE inhibitors in the environment. It has been shown that mixture effects and oxidative stress may jeopardize the application of in vivo assays for the identification of AChE inhibitors in the environment. To confirm that in vivo AChE assays can be successfully applied when dealing with complex mixtures, an extract from river water impacted by non-treated wastewater was bio-tested using the acute toxicity fish embryo test (FET) and AChE inhibition assay with zebrafish. The zebrafish FET showed high sensitivity for the extract (LC10 = relative extraction factor 2.8) and we observed a significant inhibition of the AChE (40%, p < 0.01) after 4-day exposure. Furthermore, the extract was chromatographically fractionated into a total of 26 fractions to dilute the mixture effect and separate compounds according to their physico-chemical properties. As expected, non-specific acute effects (i.e., mortality) disappeared or evenly spread among the fractions, while AChE inhibition was still detected in five fractions. Chemical analysis did not detect any known AChE inhibitors in these active fractions. These results confirm that the AChE assay with Danio rerio can be applied for the detection of neuroactive effects induced in complex environmental samples, but also, they highlight the need to increase analytical and identification techniques for the detection of neurotoxic substances.

Morphological and behavioral alterations in zebrafish larvae after exposure to contaminated river sediments collected in different weather conditions.

Wastewater treatment plants (WWTPs) are the primary source of micropollutants in aquatic ecosystems. Many micropollutants tend to bind to sediments and persist until remobilizion by bioturbation or flood events. Advanced effluent treatment by ozonation has been proven to eliminate most micropollutants. The present study characterizes sediments' toxic potential regarding zebrafish embryo development, which highly complex nervous system is vulnerable to exposure to neurotoxic substances. Furthermore, behavioral changes can be induced even at low pollutant concentrations and do not cause acute toxicity. The study area includes stretches of the main waterbody, the Wurm River (sampling sites W1-W5), and its tributary the Haarbach River (sampling sites H1, and H2) in North-Rhine Westphalia, Germany. Both waterbodies serve as recipients of WWTPs' effluents. The effluent entering the Haarbach River is conventionally treated, while the Wurm River receives ozonated effluent from the Aachen-Soers WWTP. Seven sampling sites up- and downstream of the WWTPs were investigated in June of two subsequent years. The first sampling campaign in 2017 was characterized by prolonged dry weather. The second sampling campaign in 2018 occurred after prolonged rain events and the release of the rainwater overflow basin. Direct exposure of zebrafish embryos to native sediments using the sediment contact test represented an ecologically realistic scenario and showed no acute sublethal effects. Exposure of the zebrafish embryo to freeze-dried sediments representing the ecotoxicological status of sediments during flood events unfolded acute sublethal toxicity. Behavioral studies with zebrafish larvae were an essential part of environmental neurotoxicity testing. Zebrafish larvae exposed to sediments' concentrations causing no acute effects led to behavioral changes signalizing neurotoxic substances in sediments. Polyaromatic hydrocarbons, polychlorinated biphenyls, and nitroaromatic compounds were identified as potential toxicity drivers, whereby the rainwater overflow basin served as a possible source of pollution. Mixture toxicity, effect-directed analysis, and further sediment monitoring are needed.

Extensive rain events have a more substantial impact than advanced effluent treatment on the endocrine-disrupting activity in an effluent-dominated small river.

Wastewater treatment plants (WWTPs) remain an important primary source of emission for endocrine-disrupting compounds in the environment. As an advanced wastewater treatment process, ozonation is known to reduce endocrine-disrupting activity. However, it remains unclear to which extend improved wastewater treatment may reduce the endocrine-disrupting activity in the receiving water body. The present study investigated possible factors for the endocrine-disrupting activity in a small receiving water body, the Wurm River (North-Rhine Westphalia, Germany), up- and downstream of a local WWTP. The cell-based reporter gene CALUX® assay was applied to identify the endocrine-disrupting activity in the water, sediment, and suspended particulate matter. The water phase and the effluent sampling were primarily driven by applying the full-scale effluent ozonation (sampling campaigns in June 2017 and March 2019). In contrast, the sediment sampling aimed to compare the particle-bound endocrine-disrupting activity during dry (June 2017) and rainy summer (June 2018) seasons. The water phase showed low to moderate estrogenic/antiandrogenic activity. Advanced effluent treatment by ozonation led to a complete reduction of the endocrine-disrupting activity according to the limit of detection of the CALUX® assays. The suspended particulate matter originated from the water phase of the second sampling campaign revealed antiandrogenic activity only. Sediments at the sampling sites along the local WWTP revealed higher estrogenic and antiandrogenic activity after extensive rain events and were not affected by the ozonated effluent. Fluctuation patterns of the endocrine-disrupting activity in sediments were in line with fluctuated concentrations of polycyclic aromatic hydrocarbons. Rainwater overflow basin release was suggested as a vector for particle-bound and dissolved endocrine-disrupting activity in the receiving water body. The present study underlined the necessity for monitoring both water and sediment phases to achieve reliable profiling of the endocrine-disrupting activity. The receptor-mediated CALUX® assays were proven to be suitable for investigating the endocrine-disrupting activity distribution in different river compartments and WWTP effluents.

Assessing the genotoxic potential of freshwater sediments after extensive rain events - Lessons learned from a case study in an effluent-dominated river in Germany.

Wastewater treatment plant effluents and releases from rainwater overflow basins can contribute to the input of genotoxic micropollutants in aquatic ecosystems. Predominantly lipophilic genotoxic compounds tend to sorb to particulate matter, making sediment a source and a sink of pollution. Therefore, the present study aims to investigate the genotoxic potential of freshwater sediments (i) during the dry period and (ii) after extensive rain events by collecting sediment samples in one small anthropogenically impacted river in Germany up- and downstream of the local wastewater treatment plant. The Micronucleus and Ames fluctuation assays with Salmonella typhimurium strains TA98, TA100, YG1041, and YG1042 were used to assess the genotoxic potential of organic sediment extracts. For evaluation of possible genotoxicity drivers, target analysis for 168 chemical compounds was performed. No clastogenic effects were observed, while the genotoxic potential was observed at all sampling sites primarily driven by polycyclic aromatic hydrocarbons, nitroarenes, aromatic amines, and polycyclic heteroarenes. Freshwater sediments' genotoxic potential increased after extensive rain events due to sediment perturbation and the rainwater overflow basin release. In the present study, the rainwater overflow basin was a significant source for particle-bound pollutants from untreated wastewater, suggesting its role as a possible source of genotoxic potential. The present study showed high sensitivity and applicability of the bacterial Salmonella typhimurium strains YG1041 and YG1042 to organic sediment extracts to assess the different classes of genotoxic compounds. A combination of effect-based methods and a chemical analysis was shown as a suitable tool for a genotoxic assessment of freshwater sediments.

Removing Disturbing Matrix Constituents from Biota Extracts from Total Extraction and Silicone-Based Passive Sampling.

Contaminant analysis in biota extracts can be hampered by matrix interferences caused by, for example, co-extracted lipids that compromise the quality of the analytical data and require frequent maintenance of the analytical instruments. In the present study, using gas chromatography coupled to high resolution mass spectrometry (GC-HRMS), we aimed to develop and validate a straightforward, robust, and reproducible cleanup method with acceptable recoveries for diverse compound classes with a wide range of physicochemical properties representative of pollutant screening in biota extracts. We compared Oasis PRiME HLB cartridges, Agilent Captiva EMR-Lipid cartridges, and "Freeze-Out" with salmon lipids spiked with 113 target chemicals. The EMR-Lipid cartridges provided extracts with low matrix effects at reproducible recoveries of the multi-class target analytes (93 ± 9% and 95 ± 7% for low and high lipid amounts, respectively). The EMR-Lipid cartridges were further tested with spiked pork lipids submitted to total extraction or silicone-based passive sampling. Reproducible recoveries were achieved and matrix residuals were largely removed as demonstrated gravimetrically for both types of extracts. Ion suppression of halogenated compounds was not as efficiently removed by the cleanup of total and silicone-based extracts of pork lipids as for the salmon lipids. However, the samples with clean up provided better instrument robustness than those without cleanup. Hence, EMR-Lipid cartridges were shown to be efficient as a cleanup method in multi-class monitoring of biota samples and open up new possibilities as a suitable cleanup method for silicone extracts in biota passive sampling studies using GC-HRMS analysis. Environ Toxicol Chem 2021;40:2693-2704. © 2021 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Bioavailable Environmental Pollutant Patterns in Sediments from Passive Equilibrium Sampling.

Sediment-associated risks depend on the bioavailable fraction of organic chemicals and cannot be comprehended by their total concentrations. The present study investigated contamination patterns of bioavailable chemicals in sediments from various sites around the globe by using passive equilibrium sampling. The extracts had been characterized previously for mixture effects by in vitro reporter gene assays and were in this study analyzed using gas chromatography-high resolution mass spectrometry for 121 chemicals including both legacy and emerging contaminants. The spatial distribution of the detected chemicals revealed distinct contamination patterns among sampling sites. We identified compounds in common at the different sites but most contaminant mixtures were site-specific. The mixture effects of the detected chemicals were predicted with a mixture toxicity model from effect concentrations of bioactive single chemicals and detected concentrations, applying a joint model for concentration addition and independent action. The predicted mixture effects were dominated by polycyclic aromatic hydrocarbons, and among the chemicals with available effect data, 17% elicited oxidative stress response and 18% activated the arylhydrocarbon receptor. Except for two sites in Sweden, where 11 and 38% of the observed oxidative stress response were explained by the detected chemicals, less than 10% of effects in both biological end points were explained. These results provide a comprehensive investigation of bioavailable contamination patterns of sediments and may serve as an example of employing passive equilibrium sampling as a monitoring technique to integrate the risk of bioavailable sediment-associated chemicals in aquatic environments.

Mixture Risk Drivers in Freshwater Sediments and Their Bioavailability Determined Using Passive Equilibrium Sampling.

The identification of mixture risk drivers is a great challenge for sediment assessment, especially when taking bioavailability into consideration. The bioavailable portion, which comprises the organic contaminants in pore water and the ones bound to organic carbon, was accessed by equilibrium partitioning to polydimethylsiloxane (PDMS). The exhaustive solvent and PDMS extracts were toxicologically characterized with a battery of in vitro reporter gene assays and chemically analyzed with liquid and gas chromatography coupled to high-resolution mass spectrometry. The bioavailable fractions of mixture effects and individual chemicals were mostly lower than 0.1, indicating that more than 90% of the substances are strongly bound and would not pose an immediate risk but could potentially be remobilized in the long term. Despite 655 organic chemicals analyzed, only 0.1-28% of the observed biological effects was explained by the detected compounds in whole sediments, while 0.009-3.3% was explained by bioavailable chemicals. The mixture effects were not only dominated by legacy pollutants (e.g., polycyclic aromatic hydrocarbons (PAHs) in the bioassay for activation of the aryl-hydrocarbon receptor (AhR) and oxidative stress response (AREc32)) but also by present-use chemicals (e.g., plastic additives for binding to the peroxisome proliferator-activated receptor γ (PPARγ)), with different fingerprints between whole sediments and bioavailable extracts. Our results highlight the necessity to involve different bioassays with diverse effect profiles and broader selection of contaminants along with bioavailability for the risk assessment of chemical mixtures in sediments.

Passive equilibrium sampling of hydrophobic organic compounds in homogenised fish tissues of low lipid content.

Passive equilibrium sampling using polymer samplers in lean tissue is one of the current challenges in assessing bioaccumulation and biomagnification due to the long time needed to reach equilibrium. Despite recent progress achieved by rolling pieces of intact fish fillet with sheets of silicone, there is still a need for a passive sampling method for homogenates that achieves equilibrium before tissue decay starts. In this work, a new approach for relocation of silicone passive samplers in homogenates of lean fish was established for three homogenates with lipid contents varying from 1.2% to 6.1%. Results showed that for 20 model hydrophobic organic compounds with log KOW between 3.9 and 7.8, equilibrium between the silicone and the tissue was achieved in less than 3 days at 4 °C. The concentrations in lipids obtained using passive equilibrium sampling and those from traditional total solvent extraction agreed well, within a factor of 1.3. This new procedure extends the use of passive samplers to homogenised fish tissues of low lipid content, which is highly relevant for environmental studies focused on bioaccumulation of contaminants.

European demonstration program on the effect-based and chemical identification and monitoring of organic pollutants in European surface waters.

Growing concern about the adverse environmental and human health effects of a wide range of micropollutants requires the development of novel tools and approaches to enable holistic monitoring of their occurrence, fate and effects in the aquatic environment. A European-wide demonstration program (EDP) for effect-based monitoring of micropollutants in surface waters was carried out within the Marie Curie Initial Training Network EDA-EMERGE. The main objectives of the EDP were to apply a simplified protocol for effect-directed analysis, to link biological effects to target compounds and to estimate their risk to aquatic biota. Onsite large volume solid phase extraction of 50 L of surface water was performed at 18 sampling sites in four European river basins. Extracts were subjected to effect-based analysis (toxicity to algae, fish embryo toxicity, neurotoxicity, (anti-)estrogenicity, (anti-)androgenicity, glucocorticoid activity and thyroid activity), to target analysis (151 organic micropollutants) and to nontarget screening. The most pronounced effects were estrogenicity, toxicity to algae and fish embryo toxicity. In most bioassays, major portions of the observed effects could not be explained by target compounds, especially in case of androgenicity, glucocorticoid activity and fish embryo toxicity. Estrone and nonylphenoxyacetic acid were identified as the strongest contributors to estrogenicity, while herbicides, with a minor contribution from other micropollutants, were linked to the observed toxicity to algae. Fipronil and nonylphenol were partially responsible for the fish embryo toxicity. Within the EDP, 21 target compounds were prioritized on the basis of their frequency and extent of exceedance of predicted no effect concentrations. The EDP priority list included 6 compounds, which are already addressed by European legislation, and 15 micropollutants that may be important for future monitoring of surface waters. The study presents a novel simplified protocol for effect-based monitoring and draws a comprehensive picture of the surface water status across Europe.

Identification of Mutagenic Aromatic Amines in River Samples with Industrial Wastewater Impact.

Aromatic amines are one of the most important classes of compounds contributing to surface water mutagenicity due to their widespread occurrence as precursors and transformation products of dyes, pharmaceuticals, agrochemicals, and other compound classes. In this study, we implemented a workflow including novel analytical and data evaluation methods aiming to identify aromatic amines in six mutagenic wastewater effluents from a chemical-industrial area in Germany, collected by the passive sampler Blue Rayon. We identified 14 amines including the two potent mutagenic aromatic amines 2,3- and 2,8-phenazinediamine, which were reported for the first time as environmental contaminants. These two isomers accounted between 4.2 and 86% of the mutagenicity of the blue rayon extracts and may be byproducts of dye production at the studied site.

Mutagenicity in Surface Waters: Synergistic Effects of Carboline Alkaloids and Aromatic Amines.

For decades, mutagenicity has been observed in many surface waters with a possible link to the presence of aromatic amines. River Rhine is a well-known example of this phenomenon but responsible compound(s) are still unknown. To identify the mutagenic compounds, we applied effect-directed analysis (EDA) utilizing novel analytical and biological approaches to a water sample extract from the lower Rhine. We could identify 21 environmental contaminants including two weakly mutagenic aromatic amines, and the known alkaloid comutagen norharman along with two related ß-carboline alkaloids, carboline, and 5-carboline, which were reported the first time in surface waters. Results of mixture tests showed a strong synergism of the identified aromatic amines not only with norharman, but also with carboline and 5-carboline. Additionally, other nitrogen-containing compounds also contributed to the mutagenicity when aromatic amines were present. Thus, comutagenicity of ß-carboline alkaloids with aromatic amines is shown to occur in surface waters. These results strongly suggest that surface water mutagenicity is highly complex and driven by synergistic mechanisms of a complex compound mixture (of which many are yet unidentified) rather than by single compounds. Therefore, mixture effects should be considered not only from mutagens alone, but also including possible comutagens and nonmutagenic compounds.

Assessment of a novel device for onsite integrative large-volume solid phase extraction of water samples to enable a comprehensive chemical and effect-based analysis.

The implementation of targeted and nontargeted chemical screening analysis in combination with in vitro and organism-level bioassays is a prerequisite for a more holistic monitoring of water quality in the future. For chemical analysis, little or no sample enrichment is often sufficient, while bioanalysis often requires larger sample volumes at a certain enrichment factor for conducting comprehensive bioassays on different endpoints or further effect-directed analysis (EDA). To avoid logistic and technical issues related to the storage and transport of large volumes of water, sampling would benefit greatly from onsite extraction. This study presents a novel onsite large volume solid phase extraction (LVSPE) device tailored to fulfill the requirements for the successful effect-based and chemical screening of water resources and complies with available international standards for automated sampling devices. Laboratory recovery experiments using 251 organic compounds in the log D range from -3.6 to 9.4 (at pH7.0) spiked into pristine water resulted in acceptable recoveries and from 60 to 123% for 159 out of 251 substances. Within a European-wide demonstration program, the LVSPE was able to enrich compounds in concentration ranges over three orders of magnitude (1ngL-1 to 2400ngL-1). It was possible to discriminate responsive samples from samples with no or only low effects in a set of six different bioassays (i.e. acetylcholinesterase and algal growth inhibition, androgenicity, estrogenicity, fish embryo toxicity, glucocorticoid activity). The LVSPE thus proved applicable for onsite extraction of sufficient amounts of water to investigate water quality thoroughly by means of chemical analysis and effect-based tools without the common limitations due to small sample volumes.

Nontargeted detection and identification of (aromatic) amines in environmental samples based on diagnostic derivatization and LC-high resolution mass spectrometry.

The presence of aromatic amines in the environment has been in the focus of research, as many of these compounds are known or suspected mutagens and carcinogens. To facilitate the detection of aromatic amines in complex environmental samples by LC-high resolution mass spectrometry, an on-line-post-column and a pre-column derivatization method to label (in an ideal case) all aromatic amines was evaluated by applying different derivatization reagents. 4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was found to be the most promising labeling reagent due to its high reactivity with both primary and secondary amines and its low signal in positive mode electrospray ionization (ESI+). Post-column on-line derivatization did not result in sufficient signal intensities of derivatives. With pre-column derivatization most of the selected aromatic amines resulted in a derivative that shows common fragments of diagnostic value. The selectivity of NBD-F was studied in depth with a data set of 220 compounds with different functional groups showing that also aliphatic amines and some thiols yield a derivative. The developed method was successfully applied to wastewater effluent samples and several derivatives were confirmed by diagnostic neutral losses.

Determination of selected natural hormones and endocrine disrupting compounds in domestic wastewater treatment plants by liquid chromatography electrospray ionization tandem mass spectrometry after solid phase extraction.

A new analytical method for the simultaneous determination of two natural hormones (progesterone and estrone) and two selected endocrine disrupter compounds (EDCs) (diltiazem and carbamazepine (Cbz)) was developed by liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) after pre-concentration with solid phase extraction (SPE). Influent and effluent samples taken from five different wastewater treatment plants throughout Turkey namely Hurma/Antalya, Lara/Antalya, Kemer-1 and Kemer-2 and METU/Ankara were analyzed for their EDCs contents under the optimum conditions. All of the parameters in the pre-concentration step were optimized and the best recoveries for all compounds of interest were achieved at pH 7 (about 100%). Progesterone was not detected in any of the treatment plants while diltiazem was found in all samples with the exception of Lara effluent.