Quantification and risk assessment of polar organic contaminants in two chalk streams in Hampshire, UK using the Chemcatcher passive sampler.

Freshwater systems are facing a number of pressures due to the inputs of polar organic contaminants from a range of sources including agriculture, domestic and industry. The River Itchen and River Test are two sensitive chalk streams in Southern England that are experiencing a decline in invertebrate communities. We used Chemcatcher passive samplers to measure time-weighted average concentrations (14 days) of polar pollutants at nine sites on the River Itchen and eight sites on the River Test over a 12-month period. Sampler extracts were analysed using a targeted LC/MS method. In total, 121 plant protection products and pharmaceutical and personal care products were quantified (range of log Kow from - 1.5 to 7). Concentrations (sub ng L-1 to >500 ng L-1) in both rivers showed spatial and temporal variations. A greater number of compounds and higher concentrations were found in the River Test. The chemical profile was dominated by inputs from wastewater treatment plants and legacy plant protection products. On the River Itchen, high concentrations (∼100 ng L-1) of caffeine were observed directly downstream of a fish farm. Using the NORMAN database, the predicted no effect concentration (PNEC) freshwater values were exceeded by only five contaminants (2-hydroxy-terbuthylazine, alprazolam, azithromycin, diclofenac and imidacloprid). In addition, venlafaxine was detected above its EU Watch List concentration. These exceedances were mainly downstream of direct inputs from treatment plants. These compounds are known to have ecotoxicological effects on a range of aquatic biota including macroinvertebrates. Of concern is the ubiquitous presence of the ectoparasiticide imidacloprid, highlighting the need to control its use. The impact of the cocktail of pollutants found in this study on the long-term effects on chalk stream ecosystems remains unknown and needs further investigation.

High enrichment factors in chemical analysis of progestins and in bioassays: insights beyond trace levels.

Concerns are growing about adverse effects of progestins on biota, even at ultra-trace concentrations. The enrichment factor (EF) from extraction of analytes in environmental samples that is needed for sample pre-concentration can affect not only performance of the analytical method but also the matrix effect. Therefore, the present study aimed to assess the influence of high sample EF on performance of the high-performance liquid chromatography with atmospheric pressure chemical ionization and photoionization coupled with high-resolution mass spectrometry (HPLC-APCI/APPI-HRMS) method for analysis of progestins in waste water treatment plant (WWTP) effluents and surface waters and analysis of (anti-)progestogenic activities measured by (anti-)PR-CALUX bioassays. The results showed that HPLC-APCI/APPI-HRMS coupled with solid-phase extraction and a high EF (33,333 Lwater/Lextract) enabled the detection of more compounds compared to samples with lower sample EF (10,000 Lwater/Lextract). The matrix effect did not increase proportionally compared to lower EFs (10,000 and 16,666 Lwater/Lextract), and lower limits of quantification were achieved in WWTP effluents and surface waters. The results of bioassays have shown that relative EF of 25 Lwater/Lbioassay appears high enough to detect progestogenic activity in treated waste water. Our study is one of the first to provide insights into sample pre-concentration in analysis of progestins and progestogenicity in aquatic environments.

Identification of hot spots and co-occurrence patterns of activities on thyroid hormone receptor and transthyretin binding in passive samplers from Czech surface waters.

One of the less studied in vitro biological activities in the aquatic environment are thyroid hormone receptor beta (TRß)-mediated agonistic and antagonistic activities and transthyretin (TTR) binding activity. They were measured mostly using active sampling methods, but rarely found. It is unclear if these activities co-occur, and the drivers of the (anti-)TRß activity are mostly unknown. Therefore, the main aim of the study was to determine (anti-)TRß activities as well as transthyretin (TTR) binding activity in passive samplers from Czech surface waters in combination with the search for the effect drivers based on liquid chromatography-high resolution mass spectrometry (LC-HRMS) analysis by applying suspect screening. Passive samplers (polar organic chemical integrative samplers, POCIS) were deployed at twenty-one sites (all ends of watersheds and other important sites in Elbe River) in the Czech rivers. The (anti-)TRß and TTR binding activity were measured using (anti-)TRß-CALUX and TTR-TRß-CALUX bioassays. Anti-TRß activity was found at eight sites, and TTR binding activity co-occurred there at six of these sites. The co-occurrence of TRß-mediated antagonistic activity and TTR binding indicate that they may have common effect drivers. No sample exhibited TRß agonistic activity. The extract from the site Bílina River, the most burdened with anti-TRß activity, was further successfully fractionated, and this activity was revealed in the fraction, where mid-polar compounds prevailed. However, the suspect LC-HRMS analysis did not reveal the chemical effect drivers. Our results showed that anti-TRß activity can be found in surface waters by employing passive sampling and frequently co-occurs with TTR binding activity. Overall, the fractionation procedure and non-target data acquisition used in this study can serve as a basis for searching the effect drivers in future research.

Identifying organic micropollutants' transformation products from the soil dissipation experiment by non-targeted high-resolution mass spectrometry approach: Can we gain more than transformation product identity?

Risk assessment of environmental hazards originating from xenobiotics extensively used worldwide (e.g., pharmaceuticals, bisphenols, or preservatives) requires a combined study of their effects, mobility, dissipation mechanisms, and subsequent transformation product identification and evaluation. We have developed an efficient accelerated solvent extraction method for a broad range of micropollutants of variable physical-chemical properties in soils to enable more accurate hazard characterisation. Micropollutant recoveries from freeze-dried soils were 60-120%, with the exception of atorvastatin, fexofenadine, and telmisartan, which had reduced recoveries (40-66%). The observed matrix effect ranged from -26% to 17% and was corrected by the matrix matching standard for quantitative analysis. The method allows sensitive and reliable determination of a wide range of analytes in soil samples and, consequently, qualitative analysis of transformation products (TP) with variable physicochemical properties. We identified TPs of five compounds (venlafaxine, telmisartan, valsartan, atorvastatin, and sertraline) by applying suspect and non-targeted data analyses. To our knowledge, the transformation product of atorvastatin was reported for the first time. All others were found in soil or other matrices. Valsartan (formed valsartan acid) and atorvastatin (transformed probably by oxidative decarboxylation of beta, delta dihydroxy heptanoic acid chain to propionic acid) were modified to a relatively large extent. All other compounds identified were only hydroxylated (sertraline and telmisartan) or demethylated (venlafaxine). We estimated the stability and presence of the identified TPs based on the constructed time trends and the ratio between TP formation and degradation rates. We demonstrated how valuable a non-targeted approach can be for complex evaluation of the fate and effect of soil pollutants.

Toxicological effects of diclofenac on signal crayfish (Pacifastacus leniusculus) as related to weakly acidic and basic water pH.

The widespread use and continuous discharge of pharmaceuticals to environmental waters can lead to potential toxicity to aquatic biota. Pharmaceuticals and their metabolites are often complex organic and environmentally persistent compounds that are bioactive at low doses. This study aimed to investigate the effects of diclofenac (DCF) on the antioxidant defence system and neurotoxicity biomarkers in signal crayfish (Pacifastacus leniusculus) under weakly acidic and basic conditions. Crayfish were exposed to 200 µg/L of DCF at pH 6 and 8 for 96 h and subsequently underwent the depuration phase for 96 h. Gills, hepatopancreas, and muscle were sampled after the exposure and depuration phases to assess the toxicological biomarker responses of DCF in crayfish by evaluating lipid peroxidation (LPO) levels, activities of antioxidant enzymes and acetylcholinesterase. After the exposure phase, the hemolymph DCF concentration was detected one order higher at pH 6 than at pH 8. The DCF was subsequently fully eliminated from the hemolymph during the depuration phase. Our results showed that DCF caused alteration in the activities of six of the seven tested biomarkers in at least one crayfish tissue. Although exposure to DCF caused imbalances in the detoxification system on multiple tissue levels, it was regenerated to a balanced state after the depuration phase. Integrated biomarker response (IBRv2) showed that the highest toxicological response to DCF exposure was elicited in the gills, whereas the hepatopancreas was the highest-responding tissue after the depuration phase. Exposure to DCF at pH 6 caused higher toxicological effects than at pH 8; however, crayfish antioxidant mechanisms recovered more quickly at pH 6 than at pH 8 after the depuration phase. Our results showed that water pH influenced the toxicological effects of DCF, an ionisable compound in crayfish.

Effects of mifepristone, a model compound with anti-progestogenic activity, on the development of African clawed frog (Xenopus laevis).

The objective of this study was to assess the effects of a model substance with anti-progestogenic activity on development of African clawed frog (Xenopus laevis) from tadpole to juvenile stage. Mifepristone, a synthetic progesterone receptor-blocking steroid hormone used in medicine as an abortifacient, was chosen as a model compound with anti-progestogenic activity. In the experiment, African clawed frog tadpoles were exposed to mifepristone at three concentrations (2, 21, and 215 ng L-1). A control group was exposed to dimethyl sulfoxide (DMSO; 0.001 %). The experiment started when tadpoles reached stages 47-48 according to Nieuwkoop and Faber (NF; 1994) and continued until stage NF 66, when metamorphosis was complete. Exposure to mifepristone had no significant effect on the rate of tadpole development, occurrence of morphological anomalies, weight, body length, or sex ratio. Mortality was within an acceptable range of 0-3.6 % throughout the test and did not differ among the groups. Histopathological examination of the gonads and thyroid gland revealed no significant changes. Therefore, we can conclude that mifepristone had no negative effect on development of the African clawed frog up to juvenile stage. Nevertheless, at the highest tested mifepristone concentration (215 ng L-1), gene expression analysis revealed up-regulation of mRNA expression of nuclear progesterone receptor (npr), membrane progesterone receptor (mpr), estrogen receptor beta (esrß), and luteinizing hormone (lh) in the brain-pituitary complex of exposed frogs at stage NF 66. Higher mRNA expression of npr was also found in frogs exposed to 22 ng L-1 mifepristone compared to the solvent control. These findings confirmed the anti-progestogenic activity of mifepristone in frogs because the up-regulation of progesterone receptors occurs if progesterone availability in the body is reduced. All the observed changes in combination may have negative consequences for reproduction and reproductive behavior later in life.

Novel nontarget LC-HRMS-based approaches for evaluation of drinking water treatment.

A conventional evaluation methodology for drinking water pollution focuses on analysing hundreds of compounds, usually by liquid chromatography-tandem mass spectrometry. High-resolution mass spectrometry allows comprehensive evaluation of all detected signals (compounds) based on their elemental composition, intensity, and numbers. We combined target analysis of 192 emerging micropollutants with nontarget (NT) full-scan/MS/MS methods to describe the impact of treatment steps in detail and assess drinking water treatment efficiency without compound identification. The removal efficiency based on target analytes ranged from - 143 to 97%, depending on the treatment section, technologies, and season. The same effect calculated for all signals detected in raw water by the NT method ranged between 19 and 65%. Ozonation increased the removal of micropollutants from the raw water but simultaneously caused the formation of new compounds. Moreover, ozonation byproducts showed higher persistence than products formed during other types of treatment. We evaluated chlorinated and brominated organics detected by specific isotopic patterns within the developed workflow. These compounds indicated anthropogenic raw water pollution but also potential treatment byproducts. We could match some of these compounds with libraries available in the software. We can conclude that passive sampling combined with nontargeted analysis shows to be a promising approach for water treatment control, especially for long-term monitoring of changes in technology lines because passive sampling dramatically reduces the number of samples and provides time-weighted average information for 2 to 4 weeks.

Rapid target screening and quantitative analysis of various environmental pollutants.

RATIONALE: The presented analytical method demonstrates a straightforward approach for environmental applications based on laser diode thermal desorption (LDTD). The study aims to examine abilities to achieve environmentally relevant outcomes for different types of pollutants with a fast method following the green chemistry principle. METHODS: Treatment of environmentally relevant sample matrix (river water) was limited to filtration with a cellulose filter. Samples fortified with analytes were spotted in a LazWell plate and dried before analysis. Samples thermally desorbed using LDTD were detected with Q Exactive hybrid high-resolution mass spectrometer operation in full-scan data-dependent acquisition mode (LDTD-FullMS-dd-MS/MS). RESULTS: LDTD-FullMS-dd-MS/MS exhibits the lowest quantification limits for anatoxin-A, atrazine, caffeine, methamphetamine, methylbenzotriazole, paracetamol, perfluorobutanoic acid, perfluorohexanoic acid, and perfluorooctanoic acid of between 0.10 and 1.0 ng mL-1 in the environmentally relevant sample matrix. CONCLUSIONS: The developed method was successfully evaluated for different environmental pollutants and radically reduced sample treatment and time requirements for analysis and sample preparation.

Assessment of potential mobility of selected micropollutants in agricultural soils of the Czech Republic using their sorption predicted from soil properties.

The sorption of organic contaminants in soils and sediment is a crucial factor affecting their mobility in the vadose zone environment. The Freundlich sorption isotherms were evaluated for eleven micropollutants and eight soils. The highest Freundlich sorption coefficients, KF, were obtained for triclosan (324 ± 153 cm3/nµg1-1/ng-1) followed by sertraline (120 ± 74), venlafaxine (74.3 ± 41.2), telmisartan (33.3 ± 13.6), atorvastatin (8.66 ± 4.78), bisphenol S (8.03 ± 4.87), lamotrigine (6.92 ± 3.02), 2-phenylbenzimidazole-5-sulfonic acid (3.77 ± 2.25), memantine (3.42 ± 1.64), 1-methyl-1H-benzotriazole (2.05 ± 0.99), and valsartan (0.88 ± 0.89). The KF values for the individual compounds were correlated with soil properties. Multiple linear regressions were used to derive equations for predicting the KF values using the soil properties. The first set of equations contained mainly properties with the strongest correlations with the KF values, e.g., a base cation saturation for positively charged compounds or a hydrolytic acidity for negatively charged compounds. The second set of equations contained properties included in the map of agricultural soils of the Czech Republic. These equations always indicated positive correlations with oxidizable organic carbon and clay content. They also included either a negative or positive correlation with pHKCl. A positive correlation with pHKCl was obtained for venlafaxine, memantine, and sertraline, which were mostly positively charged. A negative correlation with pHKCl was obtained for the remaining compounds. The second set of equations, the soil map, and the database of soil properties were used to predict the KF value distributions within the Czech agricultural soils. It resulted in similar KF distributions' patterns for valsartan, lamotrigine, atorvastatin, and telmisartan (with a positive correlation between KF and hydrolytic acidity), which considerably differed from the KF patterns for the other compounds. These maps were used to delineate areas with a leaching potential of the compounds toward groundwater that will serve as a tool for assessing a potential groundwater vulnerability.

Environmental water extracts differentially activate zebrafish and human nuclear progesterone receptors.

Many reports on anti-progestogenic activities in aquatic environments have been published in the past decade. These are monitored mainly by in vitro reporter gene bioassays based upon the human progesterone receptor (PR). However, results obtained by some human in vitro bioassays may not be relevant for aquatic animals, especially fish. The present work aimed to detect fish (anti-)PR activity in waste- and receiving surface waters. In parallel, human (anti-)PR activity was analysed to determine if there was any connection between human and fish (anti-)PR activities. Finally, (anti-)PR activities were linked to the occurrence of progestins in water samples. Human PR agonistic activity was detected in all wastewater and most receiving surface water samples. Nevertheless, zebrafish PR (zfPR) agonistic activity was found in only two influent wastewater samples (max. 117 ng/L 17α,20ß-dihydroxy-4-pregnen-3-one [DHP] equivalents). Analysed synthetic progestins and progesterone accounted for 14 % to 161 % of detected human PR (hPR) agonistic activity in water samples. Progesterone also contributed significantly to zfPR agonistic activity (up to 10 %) in raw wastewater. The anti-hPR activity was detected also in most wastewater and some surface water samples, but synthetic progestins did not trigger anti-zfPR activity in excess of LOQ values. In addition, altrenogest, dienogest, and ulipristal acetate were tested for their potency to zfPR for the first time. The activity analyses of both pure substances and environmental samples showed that human and zebrafish progesterone receptors are differentially activated. Therefore, results based on human PR in vitro bioassays could not predict fish PR activities in the environment.

Effects of the synthetic progestin levonorgestrel on some aspects of thyroid physiology in common carp (Cyprinus carpio).

The objective of the present study was to assess the effects of levonorgestrel (LNG), a synthetic progestin, on early development and the thyroid system of carp using morphological, histological, immunohistochemical, and gene expression analysis. Fish were exposed to LNG at three levels (3, 31, and 310 ng L-1) from eggs to the onset of juvenile stage (47 days). LNG had no significant effect on early development in common carp or on the occurrence of morphological anomalies. No pathological alterations of the thyroid follicles were found. Immunohistochemical examination of the thyroid follicles using antibodies against thyroxin did not show any differences in fish exposed to 310 ng L-1 LNG compared to the controls. mRNA expression of iodothyronine deiodinases (dio1, 2, 3) was differentially affected by LNG treatment during carp development. Most importantly, dio3 was markedly downregulated in fish exposed to all three LNG levels compared to the controls at the conclusion of the experiment (47 days post-fertilization). A decrease in dio1 or dio3 or an increase in dio2 transcription observed at different time points of the study may be a sign of hypothyroidism. mRNA expression of genes npr, esr1, and esr2b in the body and npr and esr2b in the head of fish exposed to 310 ng L-1 LNG was significantly upregulated compared to the solvent control group at the end of the test. Together, these results show that levonorgestrel caused parallel changes in the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-gonad axes.

Integrated biomarker response in signal crayfish Pacifastacus leniusculus exposed to diphenhydramine.

Diphenhydramine (DPH) is a pharmaceutical with multiple modes of action, primarily designed as an antihistamine therapeutic drug. Among antihistamines, DPH is a significant contaminant in the environment, frequently detected in surface waters, sediments, and tissues of aquatic biota. In the present study, signal crayfish Pacifastacus leniusculus was used as a model organism because of their prominent ecological roles in freshwater ecosystems. The biochemical effects were investigated in crayfish exposed to the environmental (low: 2 µg L-1), ten times elevated (medium: 20 µg L-1), and the sublethal (high: 200 µg L-1) nominal concentrations of DPH in water for 96 h. Lipid peroxidation, antioxidant enzyme activities, and acetylcholinesterase activity were assessed as toxicological biomarkers in crayfish hepatopancreas, gills, and muscles. Low and medium DPH exposure caused imbalances only in glutathione-like enzyme activities. Integrated biomarker response showed the absolute DPH toxicity effects on all tested tissues under high exposure. This study identified that high, short-term DPH exposure induced oxidative stress in crayfish on multiple tissue levels, with the most considerable extent in muscles.

Determination of citalopram in fish brain tissue: benefits of coupling laser diode thermal desorption with low- and high-resolution mass spectrometry.

Recent state-of-the-art methods developed for the analysis of polar xenobiotics from different types of biological matrices usually employ liquid chromatography with mass spectrometry. However, there are limitations when a small amount of sample mass is available. For example, individual benthic invertebrates or fish tissue samples often weigh less than 100 mg (e.g., brain, liver) but are necessary to understand environmental fate and bioaccumulation dynamics. We developed ultra-fast methods based on a direct sample introduction technique. This included coupling laser diode thermal desorption with atmospheric pressure chemical ionization mass spectrometry (LDTD-APCI-MS). We then quantitated a common selective serotonin reuptake inhibitor (citalopram) in brain tissues of individual juvenile fish after in vivo exposure to environmentally relevant concentration. Two mass spectrometric methods based on low (LDTD-APCI-triple quadrupole (QqQ)-MS/MS) and high (LDTD-APCI-high-resolution product scan (HRPS)) resolutions were developed and evaluated. Individual instrument conditions were optimized to achieve an accurate and robust analytical method with minimum sample preparation requirements. We achieved very good recovery (97-108%) across the range of 1-100 ng g-1 for LDTD-APCI-HRPS. LDTD-APCI-QqQ-MS/MS showed poorer performance due to interferences from the matrix at the lowest concentration level. LDTD-APCI ionization was successfully validated for analysis of non-filtered sample extracts. Evaluation of final methods was performed for a set of real fish brain samples, including comparison of LDTD-APCI-HRPS with a previously validated LC-heated electrospray ionization-HRPS method. This new LDTD-APCI-HRPS method avoids the chromatographic step and provides important benefits such as analysis of limited sample masses, lower total sample volume (typically µL), and reduction in analysis time per sample run to a few seconds. Graphical abstract.

Do progestins contribute to (anti-)androgenic activities in aquatic environments?

Unknown compounds with (anti-)androgenic activities enter the aquatic environment via municipal wastewater treatment plants (WWTPs). Progestins are well-known environmental contaminants capable of interfering with androgen receptor (AR) signaling pathway. The aim of the present study was to determine if 15 selected progestins have potential to contribute to (anti-)androgenic activities in municipal wastewaters and the respective recipient surface waters. AR-specific Chemically Activated LUciferase gene eXpression bioassay in agonistic (AR-CALUX) and antagonistic (anti-AR-CALUX) modes and liquid chromatography tandem atmospheric pressure chemical ionization/atmospheric photoionization with hybrid quadrupole/orbital trap mass spectrometry operated in high resolution product scan mode (LC-APCI/APPI-HRPS) methods were used to assess (anti-)androgenic activity and to detect the target compounds, respectively. The contribution of progestins to (anti-)androgenic activities was evaluated by means of a biologically and chemically derived toxicity equivalent approach. Androgenic (0.08-59 ng/L dihydrotestosterone equivalents - DHT EQs) and anti-androgenic (2.4-26 µg/L flutamide equivalents - FLU EQs) activities and progestins (0.19-75 ng/L) were detected in selected aquatic environments. Progestins displayed androgenic potencies (0.01-0.22 fold of dihydrotestosterone) and strong anti-androgenic potencies (9-62 fold of flutamide). Although they accounted to some extent for androgenic (0.3-29%) and anti-androgenic (4.6-27%) activities in influents, the progestins' contribution to (anti-)androgenic activities was negligible (≤2.1%) in effluents and surface waters. We also tested joint effect of equimolar mixtures of target compounds and the results indicate that compounds interact in an additive manner. Even if progestins possess relatively strong (anti-)androgenic activities, when considering their low concentrations (sub-ng/L to ng/L) it seems unlikely that they would be the drivers of (anti-)androgenic effects in Czech aquatic environments.

Development of a robust extraction procedure for the HPLC-ESI-HRPS determination of multi-residual pharmaceuticals in biota samples.

A simple, robust and effective extraction procedure for the determination of 74 pharmaceuticals in different fish tissues by ultrasensitive high performance liquid chromatography with electrospray high resolution product scan (HPLC-ESI-HRPS) was developed and validated. Different extraction solvent mixtures were tested to achieve the highest recoveries of the selected analytes, to minimize the influence of a complex matrix and to reduce the total analysis time as well as cost of analysis. A mixture of acetonitrile + isopropanol (3:1 v/v) acidified with 0.1% formic acid was the best extraction solvent among the five solvents tested for most of the tissues with the exception of plasma samples, where only acidified acetonitrile exhibited the best performance. The developed method was validated at three concentration levels (5, 20 and 50 ng g-1) in five different fish tissues (liver, kidney, brain, muscle and plasma). Most of the target analytes were extracted with a recovery between 60 and 130%. Very low limits of quantification (LOQs) were obtained for the majority of the pharmaceuticals in all of the studied matrices. The developed analytical method was successfully applied for the analysis of common carp (Cyprinus carpio) originating from the waste water effluent-dominated pond Cezarka (Czech Republic). The results confirmed the importance of multi-tissue analysis to obtain complex information on the distribution of pharmaceuticals in fish.

Two synthetic progestins and natural progesterone are responsible for most of the progestagenic activities in municipal wastewater treatment plant effluents in the Czech and Slovak republics.

Vast numbers of xenobiotics are known still to be present in treated municipal wastewater treatment plant (WWTP) effluents. Some of these possess endocrine-disrupting potency and pose risks for exposed aquatic animals. We searched for 17 potential environmental contaminants having affinity to the progesterone receptor. Relative potency values of these progesterone receptor-active chemicals were obtained. On the basis of relative potencies and measured environmental concentrations, the contribution of progestins to measured progestagenic activities was evaluated. Wastewaters (influent and effluent) and surrounding surface waters (upstream and downstream) at six municipal WWTPs were screened using instrumental chemical analysis and in vitro reporter gene bioassay. We showed the presence of target compounds and (anti-)progestagenic activities in municipal wastewater and surface water. Nine and seven progestins were identified in influent and effluent wastewaters, respectively. Only two compounds, progesterone and medroxyprogesterone were found in surface waters. Progestagenic agonistic activities in influents were partially masked by strong anti-progestagenic activities that were detected in all influents and ranged from 2.63 to 83 ng/L of mifepristone equivalents (EQs). Progestagenic activities were detected in all effluents and ranged from 0.06 to 0.47 ng/L of reference compound ORG 2058 EQs (a synthetic progestin equivalents), thus indicating incomplete removal of progestins during wastewater treatment processing. This activity poses a continuing risk for the aquatic environment. By contrast, anti-progestagenic activities showed better removal efficiency in WWTPs compared to progestagenic agonistic activities. Anti-progestagenic activities were found in only three of six effluents and ranged from 0.26 to 2.1 ng/L mifepristone EQs. We explained most of the progestagenic activity in municipal WWTP effluents by the presence of synthetic progestins and progesterone, which contributed 65-96% of such activity in samples where no antagonistic activity was found. The progestins medroxyprogesterone acetate, megestrol acetate and progesterone contributed most to the progestagenic activity detected in municipal effluents. Anti-progestagenic activities were found in some municipal effluents, but no causative agents were revealed because two analysed selective progesterone receptor modulators (SPRMs) with anti-progestagenic activities, mifepristone and ulipristal acetate, were not present in the effluents.