Dietary patterns, untargeted metabolite profiles and their association with colorectal cancer risk.

We investigated data-driven and hypothesis-driven dietary patterns and their association to plasma metabolite profiles and subsequent colorectal cancer (CRC) risk in 680 CRC cases and individually matched controls. Dietary patterns were identified from combined exploratory/confirmatory factor analysis. We assessed association to LC-MS metabolic profiles by random forest regression and to CRC risk by multivariable conditional logistic regression. Principal component analysis was used on metabolite features selected to reflect dietary exposures. Component scores were associated to CRC risk and dietary exposures using partial Spearman correlation. We identified 12 data-driven dietary patterns, of which a breakfast food pattern showed an inverse association with CRC risk (OR per standard deviation increase 0.89, 95% CI 0.80-1.00, p = 0.04). This pattern was also inversely associated with risk of distal colon cancer (0.75, 0.61-0.96, p = 0.01) and was more pronounced in women (0.69, 0.49-0.96, p = 0.03). Associations between meat, fast-food, fruit soup/rice patterns and CRC risk were modified by tumor location in women. Alcohol as well as fruit and vegetables associated with metabolite profiles (Q2 0.22 and 0.26, respectively). One metabolite reflecting alcohol intake associated with increased CRC risk, whereas three metabolites reflecting fiber, wholegrain, and fruit and vegetables associated with decreased CRC risk.

OMICs Signatures Linking Persistent Organic Pollutants to Cardiovascular Disease in the Swedish Mammography Cohort.

Cardiovascular disease (CVD) development may be linked to persistent organic pollutants (POPs), including organochlorine compounds (OCs) and perfluoroalkyl and polyfluoroalkyl substances (PFAS). To explore underlying mechanisms, we investigated metabolites, proteins, and genes linking POPs with CVD risk. We used data from a nested case-control study on myocardial infarction (MI) and stroke from the Swedish Mammography Cohort - Clinical (n = 657 subjects). OCs, PFAS, and multiomics (9511 liquid chromatography-mass spectrometry (LC-MS) metabolite features; 248 proteins; 8110 gene variants) were measured in baseline plasma. POP-related omics features were selected using random forest followed by Spearman correlation adjusted for confounders. From these, CVD-related omics features were selected using conditional logistic regression. Finally, 29 (for OCs) and 12 (for PFAS) unique features associated with POPs and CVD. One omics subpattern, driven by lipids and inflammatory proteins, associated with MI (OR = 2.03; 95% CI = 1.47; 2.79), OCs, age, and BMI, and correlated negatively with PFAS. Another subpattern, driven by carnitines, associated with stroke (OR = 1.55; 95% CI = 1.16; 2.09), OCs, and age, but not with PFAS. This may imply that OCs and PFAS associate with different omics patterns with opposite effects on CVD risk, but more research is needed to disentangle potential modifications by other factors.

Untargeted plasma metabolomics and risk of colorectal cancer-an analysis nested within a large-scale prospective cohort.

BACKGROUND: Colorectal cancer (CRC) is a leading cause of cancer-related death worldwide, but if discovered at an early stage, the survival rate is high. The aim of this study was to identify novel markers predictive of future CRC risk using untargeted metabolomics. METHODS: This study included prospectively collected plasma samples from 902 CRC cases and 902 matched cancer-free control participants from the population-based Northern Sweden Health and Disease Study (NSHDS), which were obtained up to 26 years prior to CRC diagnosis. Using reverse-phase liquid chromatography-mass spectrometry (LC-MS), data comprising 5015 metabolic features were obtained. Conditional logistic regression was applied to identify potentially important metabolic features associated with CRC risk. In addition, we investigated if previously reported metabolite biomarkers of CRC risk could be validated in this study population. RESULTS: In the univariable analysis, seven metabolic features were associated with CRC risk (using a false discovery rate cutoff of 0.25). Two of these could be annotated, one as pyroglutamic acid (odds ratio per one standard deviation increase = 0.79, 95% confidence interval, 0.70-0.89) and another as hydroxytigecycline (odds ratio per one standard deviation increase = 0.77, 95% confidence interval, 0.67-0.89). Associations with CRC risk were also found for six previously reported metabolic biomarkers of prevalent and/or incident CRC: sebacic acid (inverse association) and L-tryptophan, 3-hydroxybutyric acid, 9,12,13-TriHOME, valine, and 13-OxoODE (positive associations). CONCLUSIONS: These findings suggest that although the circulating metabolome may provide new etiological insights into the underlying causes of CRC development, its potential application for the identification of individuals at higher risk of developing CRC is limited.

IBS randomized study: FODMAPs alter bile acids, phenolic- and tryptophan metabolites, while gluten modifies lipids.

Diet is considered a culprit for symptoms in irritable bowel syndrome (IBS), although the mechanistic understanding of underlying causes is lacking. Metabolomics, i.e., the analysis of metabolites in biological samples may offer a diet-responsive fingerprint for IBS. Our aim was to explore alterations in the plasma metabolome after interventions with fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAPs) or gluten versus control in IBS, and to relate such alterations to symptoms. People with IBS (n = 110) were included in a double-blind, randomized, crossover study with 1-wk provocations of FODMAPs, gluten, or placebo. Symptoms were evaluated with the IBS severity scoring system (IBS-SSS). Untargeted metabolomics was performed on plasma samples using LC-qTOF-MS. Discovery of metabolite alterations by treatment was performed using random forest followed by linear mixed modeling. Associations were studied using Spearman correlation. The metabolome was affected by FODMAP [classification rate (CR) 0.88, P < 0.0001], but less by gluten intake CR 0.72, P = 0.01). FODMAP lowered bile acids, whereas phenolic-derived metabolites and 3-indolepropionic acid (IPA) were higher compared with placebo. IPA and some unidentified metabolites correlated weakly to abdominal pain and quality of life. Gluten affected lipid metabolism weakly, but with no interpretable relationship to IBS. FODMAP affected gut microbial-derived metabolites relating to positive health outcomes. IPA and unknown metabolites correlated weakly to IBS severity. Minor symptom worsening by FODMAP intake must be weighed against general positive health aspects of FODMAP. The gluten intervention affected lipid metabolism weakly with no interpretable association to IBS severity. Registration: www.clinicaltrials.gov as NCT03653689.NEW & NOTEWORTHY In irritable bowel syndrome (IBS), fermentable oligo-, di-, monosaccharides, and polyols (FODMAPs) affected microbial-derived metabolites relating to positive health outcomes such as reduced risk of colon cancer, inflammation, and type 2 diabetes, as shown in previous studies. The minor IBS symptom induction by FODMAP intake must be weighed against the positive health aspects of FODMAP consumption. Gluten affected lipids weakly with no association to IBS severity.

In Vivo Bioconcentration of 10 Anionic Surfactants in Rainbow Trout Explained by In Vitro Data on Partitioning and S9 Clearance.

Bioconcentration factors (BCFs) in rainbow trout were measured for 10 anionic surfactants with a range of alkyl chain lengths and different polar head groups. The BCFs ranged from 0.04 L kg-1 ww (for C10SO3) to 1370 L kg-1 ww (C16SO3). There was a strong correlation between the log BCF and log membrane lipid-water distribution ratio (DMLW, r2 = 0.96), and biotransformation was identified as the dominant elimination mechanism. The strong positive influence of DMLW on BCF was attributed to two phenomena: (i) increased partitioning from water into the epithelial membrane of the gill, leading to more rapid diffusion across this barrier and more rapid uptake, and (ii) increased sequestration of the surfactant body burden into membranes and other body tissues, resulting in lower freely dissolved concentrations available for biotransformation. Estimated whole-body in vivo biotransformation rate constants kB-BCF are within a factor three of rate constants estimated from S9 in vitro assays for six of the eight test chemicals for which kB-BCF could be determined. A model-based assessment indicated that the hepatic clearance rate of freely dissolved chemicals was similar for the studied surfactants. The dataset will be useful for evaluation of in silico and in vitro methods to assess bioaccumulation.

LongITools: Dynamic longitudinal exposome trajectories in cardiovascular and metabolic noncommunicable diseases.

The current epidemics of cardiovascular and metabolic noncommunicable diseases have emerged alongside dramatic modifications in lifestyle and living environments. These correspond to changes in our "modern" postwar societies globally characterized by rural-to-urban migration, modernization of agricultural practices, and transportation, climate change, and aging. Evidence suggests that these changes are related to each other, although the social and biological mechanisms as well as their interactions have yet to be uncovered. LongITools, as one of the 9 projects included in the European Human Exposome Network, will tackle this environmental health equation linking multidimensional environmental exposures to the occurrence of cardiovascular and metabolic noncommunicable diseases.

Toxicometabolomics and Biotransformation Product Elucidation in Single Zebrafish Embryos Exposed to Carbamazepine from Environmentally-Relevant to Morphologically Altering Doses.

Toxicometabolomics and biotransformation product (bioTP) elucidation were carried out in single zebrafish (ZF) embryos exposed to carbamazepine (CBZ). Exposures were conducted in 96-well plates containing six CBZ concentrations ranging from 0.5 µg/L to 50 mg/L (n = 12 embryos per dose). In the 50 mg/L dose group, 33% of embryos developed edema during the exposure (120 hpf), while hatching was significantly delayed in three of the lower-dose groups (0.46, 3.85, and 445 µg/L) compared to the control at 48 hpf. Toxicometabolomic analysis together with random forest modeling revealed a total of 80 significantly affected metabolites (22 identified via targeted lipidomics and 58 via nontarget analysis). The wide range of doses enabled the observation of both monotonic and nonmonotonic dose responses in the metabolome, which ultimately produced a unique and comprehensive biochemical picture that aligns with existing knowledge on the mode of action of CBZ. The combination of high dose exposures and apical endpoint assessment in single embryos also enabled hypothesis generation regarding the target organ for the morphologically altering insult. In addition, two CBZ bioTPs were identified without additional exposure experiments. Overall, this work showcases the potential of toxicometabolomics and bioTP determination in single ZF embryos for rapid and comprehensive chemical hazard assessment.

Microbiota-Dependent and -Independent Production of l-Dopa in the Gut of Daphnia magna.

Host-microbiome interactions are essential for the physiological and ecological performance of the host, yet these interactions are challenging to identify. Neurotransmitters are commonly implicated in these interactions, but we know very little about the mechanisms of their involvement, especially in invertebrates. Here, we report a peripheral catecholamine (CA) pathway involving the gut microbiome of the model species Daphnia magna. We demonstrate the following: (i) tyrosine hydroxylase and Dopa (3,4-dihydroxyphenylalanine) decarboxylase enzymes are present in the gut wall; (ii) Dopa decarboxylase gene is expressed in the gut by the host, and its expression follows the molt cycle peaking after ecdysis; (iii) biologically active l-Dopa, but not dopamine, is present in the gut lumen; (iv) gut bacteria produce l-Dopa in a concentration-dependent manner when provided l-tyrosine as a substrate. Impinging on gut bacteria involvement in host physiology and ecologically relevant traits, we suggest l-Dopa as a communication agent in the host-microbiome interactions in daphnids and, possibly, other crustaceans. IMPORTANCE Neurotransmitters are commonly implicated in host-microbiome communication, yet the molecular mechanisms of this communication remain largely elusive. We present novel evidence linking the gut microbiome to host development and growth via neurotransmitter l-Dopa in Daphnia, the established model species in ecology and evolution. We found that both Daphnia and its gut microbiome contribute to the synthesis of the l-Dopa in the gut. We also identified a peripheral pathway in the gut wall, with a molt stage-dependent dopamine synthesis, linking the gut microbiome to the daphnid development and growth. These findings suggest a central role of l-Dopa in the bidirectional communication between the animal host and its gut bacteria and translating into the ecologically important host traits suitable for subsequent testing of causality by experimental studies.

Rapid in-plate screening of biotransformation products in single zebrafish embryos.

A procedure was developed for rapid screening of xenobiotic biotransformation products (bioTPs) in single zebrafish (ZF; Danio rerio) embryos. Exposure was carried out from 0-120 hours post fertilization (hpf) to 6 different concentrations of the model compound propranolol (PPL). Following in-plate extraction and non-target instrumental analysis by high resolution mass spectrometry, suspected bioTPs were identified using custom data filtration scripts and matching to in silico structural predictions. A total of eight PPL bioTPs were identified (five at a level 1 confidence and one at a level 2-3 confidence). These findings supplement previously generated toxicometabolomic models derived from the same dataset, and were obtained without conducting additional exposure experiments. In addition to facilitating assessments of inter-individual variability in bioTP production in ZF embryos, we demonstrate that bioTPs can be elucidated using extremely small quantities of biomass (i.e. ∼200 µg). To the best of our knowledge, this is the first time bioTP elucidation has been carried out in single ZF embryos.

In-plate toxicometabolomics of single zebrafish embryos.

Toxicometabolomic studies involving zebrafish embryos have become increasingly popular for linking apical endpoints to biochemical perturbations as part of adverse outcome pathway determination. These experiments involve pooling embryos to generate sufficient biomass for metabolomic measurement, which adds both time and cost. To address this limitation, we developed a high-throughput toxicometabolomic assay involving single zebrafish embryos. Incubation, microscopy, embryo extraction, and instrumental metabolomic analysis were all performed in the same 96-well plate, following acquisition of conventional toxicological endpoints. The total time for the assay (including testing of 6 doses/n = 12 embryos per dose plus positive and negative controls, assessing conventional endpoints, instrumental analysis, data processing and multivariate statistics) is <14 days. Metabolomic perturbations at low dose were linked statistically to those observed at high dose and in the presence of an adverse effect, thereby contextualizing omic data amongst apical endpoints. Overall, this assay enables collection of high resolution metabolomic data in a high throughput manner, suitable for mode of action hypothesis generation in the context of pharmaceutical or toxicological screening.

Release of Side-Chain Fluorinated Polymer-Containing Microplastic Fibers from Functional Textiles During Washing and First Estimates of Perfluoroalkyl Acid Emissions.

The quantity and composition of fibers released from functional textiles during accelerated washing were investigated using the GyroWash method. Two fabrics [polyamide (PA) and polyester/cotton (PES/CO)] were selected and coated with perfluorohexane-based side-chain fluorinated polymers. Fibers released during washing ranged from ∼10 to 500 µ with a similar distribution for the two textile types. The PA-based fabric released considerably more fibers >20 µm in length compared to the PES/CO-based fabric (>1000/GyroWash for PA vs ∼200/GyroWash fibers for PES/CO). After one GyroWash (2-15 domestic washes), fibers that contained approximately 240 and 1300 µg total fluorine per square meter (µg F/m2) were released from the PA and PES/CO fabrics, respectively. Current understanding of the fate of microplastic fibers suggests that a large fraction of these fibers reach the environment either in effluent wastewater or sewage sludge applied to land. In the environment, the fluorinated side chains will be slowly cleaved from the backbone of the side-chain fluorinated polymers coated on the fibers and then transformed into short-chain perfluoroalkyl acids. On the European scale, emissions of up to ∼0.7 t of fluorotelomer alcohol (6:2 FTOH) per year were estimated for outdoor rain jackets treated with fluorotelomer-based side-chain fluorinated polymers.

Children's exposure to perfluoroalkyl acids - a modelling approach.

Adults are mainly exposed to per- and polyfluoroalkyl substances (PFASs) via ingestion of food, inhalation of air and ingestion of dust, whereas for children the exposure to PFASs is largely unknown. This study aimed to reconstruct the serum concentrations of perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS) and perfluorohexane sulfonic acid (PFHxS) in children after infancy up to 10.5 years of age and to test if dietary intake is the major exposure pathway for children to PFOA, PFOS and PFHxS after infancy. For this work, a dataset from a Finnish child cohort study was available, which comprised serum concentrations of the studied perfluoroalkyl acids (PFAAs) and PFAS concentration measurements in dust and air samples from the children's bedrooms. The calculated PFAA intakes were used in a pharmacokinetic model to reconstruct the PFAA serum concentrations from 1 to 10.5 years of age. The calculated PFOA and PFOS intakes were close to current regulatory intake thresholds and diet was the major exposure medium for the 10.5 year-olds. The one-compartment PK model reconstructed median PFOA and PFOS serum concentrations well compared to corresponding measured median serum concentrations, while the modelled PFHxS serum concentrations showed a constant underestimation. The results imply that children's exposure to PFOA and PFOS after breastfeeding and with increasing age resembles the exposure of adults. Further, the children in the Finnish cohort experienced a rather constant exposure to PFOA and PFOS between 1 and 10.5 years of age. The PFHxS exposure sources and respective pharmacokinetic parameter estimations need further investigation.

Neurodevelopmental and Metabolomic Responses from Prenatal Coexposure to Perfluorooctanesulfonate (PFOS) and Methylmercury (MeHg) in Sprague-Dawley Rats.

Methylmercury (MeHg) and perfluorooctanesulfonate (PFOS) are major contaminants of human blood that are both common in dietary fish, thereby raising questions about their combined impact on human development. Here, pregnant Sprague-Dawley rats ingested a daily dose, from gestational day 1 through to weaning, of either 1 mg/kg bw PFOS (PFOS-only), 1 mg/kg MeHg (MeHg-only), a mixture of 0.1 mg/kg PFOS and 1 mg/kg MeHg (Low-Mix), or of 1 mg/kg of PFOS and 1 mg/kg MeHg (High-Mix). Newborns were monitored for physical milestones and reflexive developmental responses, and in juveniles the spontaneous activity, anxiety, memory, and cognition were assessed. Targeted metabolomics of 199 analytes was applied to sectioned brain regions of juvenile offspring. Newborns in the High-Mix group had decreased weight gain as well as delayed reflexes and innate behavioral responses compared to controls and individual chemical groups indicating a toxicological interaction on early development. In juveniles, cumulative mixture effects increased in a dose-dependent manner in tests of anxiety-like behavior. However, other developmental test results suggested antagonism, as PFOS-only and MeHg-only juveniles had increased hyperactivity and thigmotaxic behavior, respectively, but fewer effects in Low-Mix and High-Mix groups. Consistent with these behavioral observations, a pattern of antagonism was also observed in neurochemicals measured in rat cortex, as PFOS-only and MeHg-only juveniles had altered concentrations of metabolites (e.g., lipids, amino acids, and biogenic amines), while no changes were evident in the combined exposures. The cortical metabolites altered in PFOS-only and MeHg-only exposed groups are involved in inhibitory and excitatory neurotransmission. These proof-of-principle findings at relatively high doses indicate the potential for toxicological interaction between PFOS and MeHg, with developmental-stage specific effects. Future mixture studies at lower doses are warranted, and prospective human birth cohorts should consider possible confounding effects from PFOS and mercury exposure on neurodevelopment.

Amitriptyline at an Environmentally Relevant Concentration Alters the Profile of Metabolites Beyond Monoamines in Gilt-Head Bream.

The antidepressant amitriptyline is a widely used selective serotonin reuptake inhibitor that is found in the aquatic environment. The present study investigates alterations in the brain and the liver metabolome of gilt-head bream (Sparus aurata) after exposure at an environmentally relevant concentration (0.2 µg/L) of amitriptyline for 7 d. Analysis of variance-simultaneous component analysis is used to identify metabolites that distinguish exposed from control animals. Overall, alterations in lipid metabolism suggest the occurrence of oxidative stress in both the brain and the liver-a common adverse effect of xenobiotics. However, alterations in the amino acid arginine are also observed. These are likely related to the nitric oxide system that is known to be associated with the mechanism of action of antidepressants. In addition, changes in asparagine and methionine levels in the brain and pantothenate, uric acid, and formylisoglutamine/N-formimino-L-glutamate levels in the liver could indicate variation of amino acid metabolism in both tissues; and the perturbation of glutamate in the liver implies that the energy metabolism is also affected. These results reveal that environmentally relevant concentrations of amitriptyline perturb a fraction of the metabolome that is not typically associated with antidepressant exposure in fish. Environ Toxicol Chem 2019;00:1-13. © 2019 SETAC.

Development, characterization and comparisons of targeted and non-targeted metabolomics methods.

The potential of a metabolomics method to detect statistically significant perturbations in the metabolome of an organism is enhanced by excellent analytical precision, unequivocal identification, and broad metabolomic coverage. While the former two metrics are usually associated with targeted metabolomics and the latter with non-targeted metabolomics, a systematic comparison of the performance of both approaches has not yet been carried out. The present work reports on the development and performance evaluation of separate targeted and non-targeted metabolomics methods. The targeted approach facilitated determination of 181 metabolites (quantitative analysis of 18 amino acids, 11 biogenic amines, 5 neurotransmitters, 5 nucleobases and semi-quantitative analysis of 50 carnitines, 83 phosphatidylcholines, and 9 sphingomyelins) using ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) and flow injection-tandem mass spectrometry (FI-MS/MS). Method accuracy and/or precision were assessed using replicate samples of NIST SRM1950 as well as fish liver and brain tissue from Gilthead Bream (Sparus aurata). The non-target approach involved UPLC-high resolution (Orbitrap) mass spectrometry (UPLC-HRMS). Testing of ionization mode and stationary phase revealed that a combination of positive electrospray ionization and HILIC chromatography produced the largest number of chromatographic features during non-target analysis. Furthermore, an evaluation of 4 different sequence drift correction algorithms, and combinations thereof, revealed that batchCorr produced the best precision in almost every test. However, even following correction of non-target data for signal drift, the precision of targeted data was better, confirming our existing assumptions about the strengths of targeted metabolomics. Finally, the accuracy of the online MS2-library mzCloud was evaluated using reference standards for 38 different metabolites. This is among the few studies that have systematically evaluated the performance of targeted and non-targeted metabolomics and provides new insight into the advantages and disadvantages of each approach.

Passive dosing of triclosan in multigeneration tests with copepods - stable exposure concentrations and effects at the low µg/L range.

Ecotoxicity testing is a crucial component of chemical risk assessment. Still, due to methodological difficulties related to controlling exposure concentrations over time, data on long-term effects of organic chemicals at low concentrations are limited. The aim of the present study was, therefore, to test the applicability of passive dosing to maintain stable concentrations of the organochlorine bacteriocide triclosan in the water phase during a 6-wk multigeneration population development test with the harpacticoid copepod Nitocra spinipes. Triclosan was loaded into silicone (1000 mg), which was used as passive dosing phase in the exposure vials. The distribution ratio for triclosan between silicone and water (Dsilicone-water ) was 10466 ± 1927. A population development test was conducted at 3 concentration levels of triclosan that were measured to be 3 µg/L to 5 µg/L, 7 µg/L to 11 µg/L and 16 µg/L to 26 µg/L. The results demonstrate that passive dosing is applicable for long-term ecotoxicity testing of organic chemicals, including during significant growth of the test organism population. Shifts in the demographic structure of the population during exposure suggest the most severe effects were exerted on juvenile development. Progressively lower development index values in the populations exposed to increasing triclosan concentrations suggest developmental retardation. The results further stress the need for chronic exposure during ecotoxicity testing in chemical risk assessment because even the most sensitive endpoint was not significant until after 7 d of exposure. Environ Toxicol Chem 2017;36:1254-1260. © 2016 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

Risks of hormonally active pharmaceuticals to amphibians: a growing concern regarding progestagens.

Most amphibians breed in water, including the terrestrial species, and may therefore be exposed to water-borne pharmaceuticals during critical phases of the reproductive cycle, i.e. sex differentiation and gamete maturation. The objectives of this paper were to (i) review available literature regarding adverse effects of hormonally active pharmaceuticals on amphibians, with special reference to environmentally relevant exposure levels and (ii) expand the knowledge on toxicity of progestagens in amphibians by determining effects of norethindrone (NET) and progesterone (P) exposure to 0, 1, 10 or 100 ng l(-1) (nominal) on oogenesis in the test species Xenopus tropicalis. Very little information was found on toxicity of environmentally relevant concentrations of pharmaceuticals on amphibians. Research has shown that environmental concentrations (1.8 ng l(-1)) of the pharmaceutical oestrogen ethinylestradiol (EE2) cause developmental reproductive toxicity involving impaired spermatogenesis in frogs. Recently, it was found that the progestagen levonorgestrel (LNG) inhibited oogenesis in frogs by interrupting the formation of vitellogenic oocytes at an environmentally relevant concentration (1.3 ng l(-1)). Results from the present study revealed that 1 ng NET l(-1) and 10 ng P l(-1) caused reduced proportions of vitellogenic oocytes and increased proportions of previtellogenic oocytes compared with the controls, thereby indicating inhibited vitellogenesis. Hence, the available literature shows that the oestrogen EE2 and the progestagens LNG, NET and P impair reproductive functions in amphibians at environmentally relevant exposure concentrations. The progestagens are of particular concern given their prevalence, the range of compounds and that several of them (LNG, NET and P) share the same target (oogenesis) at environmental exposure concentrations, indicating a risk for adverse effects on fertility in exposed wild amphibians.