Pesticide drift mitigation measures appear to reduce contamination of non-agricultural areas, but hazards to humans and the environment remain.

Pesticide drift onto non-agricultural land is a common problem in intensively farmed regions, and national action plans have been established across Europe to prevent it. Here, we analyzed official data on pesticide residues in grass samples collected over six years to determine whether implemented measures to reduce pesticide drift were effective. We used 306 samples collected between 2014 and 2020 on non-agricultural land in one of the most intensively managed apple and wine growing regions in Europe, the Autonomous Province of Bolzano-South Tyrol, Italy. Samples were analyzed for up to 314 substances by gas chromatography and mass spectrometry. Percentage of sites with multiple pesticides and number of pesticides decreased between 2014 and 2020. Fungicides were most often detected, with fluazinam found on 74 % and captan on 60 % of the contaminated sites (53 sites out of a total of 88 sites were contaminated). The most frequently found insecticide, phosmet, was detected in 49 % of the contaminated sites. Only one herbicide, oxadiazon, was detected in <1 % of the sites; glyphosate was not analyzed. The percentage of residues with human hazard properties increased significantly across years regarding reproductive toxicity (from 21 % of the detected substances in 2014 to 88 % in 2020) and specific target organ toxicity (0 % in 2014 to 21 % in 2020). Percentages of substances associated with endocrine-disruption (89 % of substances across years) or carcinogenic properties (45 % of substances across years) remained constant. The percentage of sites where concentrations in grass samples exceeded the surrogate maximum residue levels (MRLs) for lettuce also remained constant. Potential ecotoxicological hazards of detected residues regarding acute contact toxicity to honeybees remained high over the study years, while the acute and chronic toxicity to earthworms decreased. Our results suggest that while drift mitigation measures contributed some reduction in pesticide contamination, they were not sufficient to eliminate substantial risks to human health and the environment in nontarget areas.

Obesogens in the aquatic environment: an evolutionary and toxicological perspective.

The rise of obesity in humans is a major health concern of our times, affecting an increasing proportion of the population worldwide. It is now evident that this phenomenon is not only associated with the lack of exercise and a balanced diet, but also due to environmental factors, such as exposure to environmental chemicals that interfere with lipid homeostasis. These chemicals, also known as obesogens, are present in a wide range of products of our daily life, such as cosmetics, paints, plastics, food cans and pesticide-treated food, among others. A growing body of evidences indicates that their action is not limited to mammals. Obesogens also end up in the aquatic environment, potentially affecting its ecosystems. In fact, reports show that some environmental chemicals are able to alter lipid homeostasis, impacting weight, lipid profile, signaling pathways and/or protein activity, of several taxa of aquatic animals. Such perturbations may give rise to physiological disorders and disease. Although largely unexplored from a comparative perspective, the key molecular components implicated in lipid homeostasis have likely appeared early in animal evolution. Therefore, it is not surprising that the obesogen effects are found in other animal groups beyond mammals. Collectively, data indicates that suspected obesogens impact lipid metabolism across phyla that have diverged over 600 million years ago. Thus, a consistent link between environmental chemical exposure and the obesity epidemic has emerged. This review aims to summarize the available information on the effects of putative obesogens in aquatic organisms, considering the similarities and differences of lipid homeostasis pathways among metazoans, thus contributing to a better understanding of the etiology of obesity in human populations. Finally, we identify the knowledge gaps in this field and we set future research priorities.

The Mammalian "Obesogen" Tributyltin Targets Hepatic Triglyceride Accumulation and the Transcriptional Regulation of Lipid Metabolism in the Liver and Brain of Zebrafish.

Recent findings indicate that different Endocrine Disrupting Chemicals (EDCs) interfere with lipid metabolic pathways in mammals and promote fat accumulation, a previously unknown site of action for these compounds. The antifoulant and environmental pollutant tributyltin (TBT), which causes imposex in gastropod snails, induces an "obesogenic" phenotype in mammals, through the activation of the nuclear receptors retinoid X receptor (RXR) and peroxisome proliferator-activated receptor gamma (PPARγ). In teleosts, the effects of TBT on the lipid metabolism are poorly understood, particularly following exposure to low, environmental concentrations. In this context, the present work shows that exposure of zebrafish to 10 and 50 ng/L of TBT (as Sn) from pre-hatch to 9 months of age alters the body weight, condition factor, hepatosomatic index and hepatic triglycerides in a gender and dose related manner. Furthermore, TBT modulated the transcription of key lipid regulating factors and enzymes involved in adipogenesis, lipogenesis, glucocorticoid metabolism, growth and development in the brain and liver of exposed fish, revealing sexual dimorphic effects in the latter. Overall, the present study shows that the model mammalian obesogen TBT interferes with triglyceride accumulation and the transcriptional regulation of lipid metabolism in zebrafish and indentifies the brain lipogenic transcription profile of fish as a new target of this compound.

Chronic effects of clofibric acid in zebrafish (Danio rerio): a multigenerational study.

Clofibric acid (CA) is an active metabolite of the blood lipid lowering agent clofibrate, a pharmaceutical designed to work as agonist of peroxisome proliferator-activated receptor alpha (PPARa). It is the most commonly reported fibrate in aquatic environments with low degradation rate and potential environmental persistence. Previous fish exposures showed that CA may impact spermatogenesis, growth and the expression of fat binding protein genes. However, there are limited data on the effects of chronic multigenerational CA exposures. Here, we assessed chronic multigenerational effects of CA exposure using zebrafish (Danio rerio) as a teleost model. Zebrafish were exposed through the diet to CA (1 and 10mg/g) during their whole lifetime. Growth, reproduction-related parameters and embryonic development were assessed in the exposed fish (F1 generation) and their offspring (F2 generation), together with muscle triglyceride content and gonad histology. In order to study the potential underlying mechanisms, the transcription levels of genes coding for enzymes involved in lipid metabolism pathways were determined. The results show that chronic life-cycle exposure to CA induced a significant reduction in growth of F1 generation and lowered triglyceride muscle content (10mg/g group). Also, an impact in male gonad development was observed together with a decrease in the fecundity (10mg/g group) and higher frequency of embryo abnormalities in the offspring of fish exposed to the lowest CA dose. The profile of the target genes was sex- and tissue-dependent. In F1 an up-regulation of male hepatic pparaa, pparb and acox transcript levels was observed, suggesting an activation of the fatty acid metabolism (provided that transcript level change indicates also a protein level change). Interestingly, the F2 generation, raised with control diet, displayed a response pattern different from that observed in F1, showing an increase in weight in the descendants of CA exposed fish, in comparison with control animals, which points to a multigenerational effect.

Gemfibrozil modulates cytochrome P450 and peroxisome proliferation-inducible enzymes in the liver of the yellow European eel (Anguilla anguilla).

The human lipid regulator gemfibrozil (GEM) has been shown to induce peroxisome proliferation in rodents leading to hepatocarcinogenesis. Since GEM is found at biological active concentrations in the aquatic environment, the present study investigates the effects of this drug on the yellow European eel (Anguilla anguilla). Eels were injected with different concentrations of GEM (0.1 to 200 µg/g) and sampled 24- and 96-h post-injection. GEM was shown to inhibit CYP1A, CYP3A and CYP2K-like catalytic activities 24-h post-injection, but at 96-h post-injection, only CYP1A was significantly altered in fish injected with the highest GEM dose. On the contrary, GEM had little effect on the phase II enzymes examined (UDP-glucuronyltransferase and glutathione-S-transferase). Peroxisome proliferation inducible enzymes (liver peroxisomal acyl-CoA oxidase and catalase) were very weakly induced. No evidence of a significant effect on the endocrine system of eels was observed in terms of plasmatic steroid levels or testosterone esterification in the liver.

Triphenyltin alters lipid homeostasis in females of the ramshorn snail Marisa cornuarietis.

Molluscs are sensitive species to the toxic effects of organotin compounds, particularly to masculinisation. Both tributyltin (TBT) and triphenyltin (TPT) have been recently shown to bind to mollusc retinoid X receptor (RXR). If RXR is involved in lipid homeostasis, exposure to TPT would have an immediate effect on endogenous lipids. To test this hypothesis, the ramshorn snail Marisa cornuarietis was exposed to environmentally relevant concentrations of TPT (30, 125, 500 ng/L as Sn) in a semi-static water regime for 7 days. Percentage of lipids and total fatty acid content decreased significantly in TPT-exposed females while the activity of peroxisomal acyl-CoA oxidase, involved in fatty acid catabolism, increased. In addition, fatty acid profiles (carbon chain length and unsaturation degree) were significantly altered in exposed females but not in males. This work highlights the ability of TPT to disrupt lipid metabolism in M. cornuarietis at environmentally realistic concentrations and the higher susceptibility of females in comparison to males.

Comparative study on the metabolism of the androgen precursor androstenedione in two gastropod species: in vitro alterations by TBT and TPT.

A comparative study was performed to assess the metabolism of the androgen precursor androstenedione (AD) in two gastropod species from the Muricidae family: Bolinus brandaris and Hexaplex trunculus. AD was mainly converted to 5alpha-dihydrotestosterone by microsomal fractions isolated from Bolinus brandaris, whereas it was primarily metabolized to testosterone by Hexaplex trunculus. Sex differences in the metabolism of AD were only detected in Bolinus brandaris and attributed to higher 5alpha-reductase activity in males. Thereafter, the effect of the organotin compounds, tributyltin (TBT) and triphenyltin (TPT), on the metabolism of AD was investigated. A significant interference was only detected in females, and differences between the modes of action of the two compounds were observed: TPT was a strong inhibitor of 5alpha-reductase activity in B. brandaris at a concentration as low as 100 nM whereas only TBT (10 microM) altered the metabolism of AD in H. trunculus by increasing the activity 17beta-hydroxysteroid dehydrogenase (17beta-HSD). Thus, this work shows that the metabolism of the androgen precursor AD strongly differs among gastropod species, both in terms of activity and metabolic profile, and further demonstrates the ability of TBT and TPT to interfere with key enzymatic pathways involved in androgen synthesis.

Short-term exposure to the organotin compound triphenyltin modulates esterified steroid levels in females of Marisa cornuarietis.

Long-term exposures to organotin compounds have shown alterations on endogenous steroid levels in gastropods together with the development of imposex. However, information regarding short-term effects of these compounds on the endocrine system of gastropods is lacking. This work aimed at investigating those responses in the ramshorn snail Marisa cornuarietis by looking at both endogenous levels of free and esterified steroids and the metabolism of the androgen precursor androstenedione by digestive gland/gonad microsomal fractions. One-week exposure to the organotin compound triphenyltin (TPT) led to a significant increase in esterified testosterone (60-85%) and a decrease in esterified estradiol (50-84%) in females, but had no effect on males. The observed alterations in esterified steroids were not directly related to changes in P450 aromatase activity that remained unchanged in exposed females. The enzymes involved in the metabolism of the androgen precursor androstenedione, namely 17beta-hydroxysteroid dehydrogenases and 5alpha-reductases, were not significantly altered by TPT exposure, suggesting that such enzymes are not primary targets of TPT in M. cornuarietis. Additional studies are needed to fully understand the significance of the observed alterations in females and their potential relationship with the development of imposex.

Alteration of brain and interrenal StAR protein, P450scc, and Cyp11beta mRNA levels in atlantic salmon after nominal waterborne exposure to the synthetic pharmaceutical estrogen ethynylestradiol.

Pharmaceuticals are ubiquitous pollutants in the aquatic environment, where their potential effects on nontarget species like fish has only recently become subject of systematic investigations. Recently, it was shown that the documented xenoestrogen nonylphenol produced variations in brain steroidogenic acute regulatory (StAR) protein, cytochrome P-450-mediated cholesterol side-chain cleavage (P450scc), and cytochrome P-45011beta hydroxylase (CYP11beta) gene transcripts of exposed juvenile salmon (Arukwe, 2005). In the present study, experiments were undertaken to examine the effect of the synthetic pharmaceutical endocrine disruptor ethynylestradiol (EE2), given in water at 5 or 50 ng/L and sampled at d 0 (control), 3, and 7 after exposure, on these key and rate-limiting brain and interrenal steroidogenic pathways of juvenile salmon using quantitative (real-time) polymerase chain reaction (qPCR). Our data, which are based on nominal exposure concentrations, show that brain and head kidney StAR and P450scc expression were modulated by EE2 in a time- and concentration-specific manner. While the StAR protein and P450scc showed EE2 concentration-dependent transcriptional increases in the brain and head kidney at d 3 after exposure, no significant effect was observed at d 7. The EE2 induced effects at d 7 were underscored because the carrier solvent (dimethyl sulfoxide, DMSO) produced significant induction of the StAR protein and P450scc in both the brain and head kidney at d 7 compared to d 3 postexposure. CYP11beta transcript was detected in the brain and head kidney, where the expression patterns were modulated by EE2 in a concentration-and time-specific manner. In the brain, DMSO produced significant changes in the CYP11beta gene expression at d 7 compared to d 3 after exposure. These changes in the levels of StAR, P450scc, and CYP11beta mRNA levels in important steroidogenic organs suggest that the experimental animals are experiencing a time-dependent impaired steroidogenesis. Thus, the StAR protein, P450scc, and CYP11beta might represent sensitive diagnostic tools for short-term and acute exposure to endocrine disrupting chemicals. In view of the present study and high concentrations of EE2 reported in effluents and surface waters from Europe and the United States, pharmaceuticals in the environment represent potentially more serious health concern both to humans and wildlife than earlier anticipated.

Sexual dimorphism in esterified steroid levels in the gastropod Marisa cornuarietis: the effect of xenoandrogenic compounds.

Molluscs can conjugate a variety of steroids to form fatty acid esters. In this work, the freshwater ramshorn snail Marisa cornuarietis was used to investigate sex differences in endogenous levels of esterified steroids. Testosterone and estradiol were mainly found in the esterified form in the digestive gland/gonad complex of M. cornuarietis, and males had higher levels of esterified steroids than females (4-10-fold). Additionally, the ability of several xenobiotics, namely tributyltin (TBT), methyltestosterone (MT) and fenarimol (FEN) to interfere with the esterification of testosterone and estradiol was investigated. All three compounds induced imposex - appearance of male sexual characteristics in females. Exposure to TBT led to a decrease in both esterified testosterone (60-85%) and estradiol (16-53%) in females after 100 days exposure, but had no effect on males. Exposure to FEN and MT did not alter levels of esterified steroids in males or in females, although exposed females developed imposex after 150 days exposure. The decrease in esterified steroids by TBT could not be directly linked with a decrease in microsomal acyl-CoA:testosterone acyltransferase (ATAT) activity, which catalyzes the esterification of steroids. In fact, ATAT activity was marginally induced in organisms exposed to TBT for 50 days (1.3-fold), and significantly induced in males and females exposed to MT for 50 days (1.8- and 1.5-fold, respectively), whereas no effect on ATAT activity was observed after 150 days exposure.

Brain cytochrome P450 aromatase gene isoforms and activity levels in atlantic salmon after waterborne exposure to nominal environmental concentrations of the pharmaceutical ethynylestradiol and antifoulant tributyltin.

In this study, the effects of two environmental endocrine disruptors, the synthetic pharmaceutical estrogen (ethynylestradiol, EE2) and antifoulant (tributyltin, TBT) representing two different modes of action on the endocrine system, were studied on brain steroidogenic pathway of juvenile Atlantic salmon (Salmo salar). Neurosteroidogenesis was studied using brain aromatase gene isoforms and enzyme activity, in parallel with typical xenoestrogen responses, such as brain estrogen receptor (ERalpha) and plasma vitellogenin (Vtg) levels. Fish were exposed to nominal waterborne EE2 (5 and 50 ng/l) and TBT (50 and 250 ng/l) concentrations dissolved in dimethyl sulfoxide (DMSO), singly and in combination. Gene expressions were quantified using real-time PCR with gene-specific primers, aromatase activity was analyzed using the tritiated water-release assay, and plasma Vtg was analyzed using competitive ELISA. Our data show that EE2 induced a concentration-specific modulation of P450aromA, P450aromB, and aromatase activity in addition to ERalpha and plasma Vtg levels in juvenile salmon at day 3 postexposure. TBT exposure caused both the elevation and inhibition of P450aromA, P450aromB, and aromatase activity levels, depending on concentration, at day 7 postexposure. TBT elevated and inhibited ERalpha and plasma Vtg and also antagonized EE2-induced expression of the studied variables at day 7 postexposure. Interestingly, the carrier vehicle DMSO modulated the receptor-mediated and non-receptor-mediated estrogenic responses at day 7 postexposure, compared to day 3. In general, these findings suggest that the exposed animals are experiencing impaired steroidogenesis and modulations of receptor-mediated endocrine responses. Given the integral role of neurosteroids in homeostatic process, growth, metabolism, reproduction, and development of central nervous system and function, these effects may have serious impact on this endocrine pathway and potentially affect organismal reproductive performance and health. In conclusion, the regulation of steroidogenesis is a fundamental mechanism involved in the biosynthesis of important biological compounds, irrespective of organ; therefore, the search for the molecular targets of xenoestrogens, given singly and also in combination, in these pathways will increase our understanding of organismal endocrine disruption and potential consequences.