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AbstractPhthalate esters, such as di(n-butyl) phthalate, (DBP), are synthetic chemical pollutants commonly used as plasticizers in the manufacture of plastics. In the present study, we investigated the effects of DBP in the testes of adult male quails (Coturnix cortunix japonica) exposed by oral gavage to variable doses of DBP (0 [control], 1, 10, 50, 200, and 400 mg/kgbw−d), for 30 days during the prepubertal period, using histo-morphometric and ultrastructural techniques. Generally, significant decreases in seminiferous tubular diameter (STD) and epithelial height (SEH) were observed predominantly at the highest DBP doses (200 and 400 mg/kg), as compared to medium (50 mg/kg), and lowest doses (1 and 10 mg/kg) as well as the control group. Ultrastructurally, apparent dose-specific degenerative changes were observed in the Leydig cells. The lowest DBP doses (1 and 10 mg/kg) did not produce significant effects on Leydig cell ultrastructure, whereas, at the highest doses (200 and 400 mg/kg), the Leydig cells were remarkably conspicuous in the interstitium and appeared foamy. There was a preponderance of electron-lucent lipid droplets which crowded out the normal organelles of the cell, as well as increases in the number of dense bodies in the cytoplasm. The smooth endoplasmic reticulum (sER) was less obvious, compacted, and wedged between the abundant lipid droplets and mitochondria. Taken together, these findings indicate that pre-pubertal exposure of precocious quail birds to DBP, produced parameter-specific histometric tubular changes, as well as dose-dependent cyto-structural derangement of the Leydig cells; which consequently may lead to overt reproductive impairments in the adult bird in the environment.Graphical Abstract

AbstractMicroplastics (MPs) are physical anthropogenic pollutants and their ability to act as contaminant vectors in biological matrices is of serious ecosystem and human health concern. In the present study, we have, for the first time, screened and detected MPs in the stomach of a select group of commonly consumed fish species from a municipal water supply lake (Eleyele) in Nigeria. A total of 109 fish samples consisting of eight (8) species: Coptodon zillii (CZ: n = 38), Oreochromis niloticus (ON: n = 43), Sarotheron melanotheron (SM: n = 19), Chrysicthys nigrodigitatus (CN: n = 3), Lates niloticus (LN: n = 3), Paranchanna obscura (PO: n = 1), Hemichromis fasiatus (HF: n = 1), and Hepsetus odoe (HO: n = 1) were collected between February–April, 2018. Fish stomach content was screened for the presence of MPs using the density gradient separation technique (NaCl hypersaline solution) and examined using a fluorescence microscope. MPs were present in all the species screened (except H. fasciatus) with a frequency of 69.7% positive individuals in the examined species. MP prevalence was highest in ON (34%) > CZ (32%) > SM (13%) > CN (6%) and 5% each, for PO HO, and LN. On average, 1–6 MPs with sizes ranging between 124 μm and 1.53 mm were detected per individual. However, the highest number (34) of MPs was detected in the stomach of SM. Principal coordinate analysis (PCA) identified ecological variables such as habitat, feeding mode, and trophic levels as critical factors that may determine and influence MP uptake in fish population. The PCA showed stronger association between fish habitat, feeding mode, and trophic level with MP size and number in the benthopelagic species (ON CZ and SM), compared to demersal species (PO CN HO and LN). Given that MPs can act as vectors for the transfer of pathogens and environmental contaminants (both legacy and emerging), in addition to direct health risks to aquatic organisms, our findings raise concerns on the potential human/wildlife health effects of MPs in these economically and ecologically important food fishes.

Due to the heavy fuel oil (HFO) ban in Arctic maritime transport and new legislations restricting the sulphur content of fuel oils, new fuel oil types are continuously developed. However, the potential impacts of these new fuel oil types on marine ecosystems during accidental spills are largely unknown. In this study, we studied the toxicity of three marine fuel oils (two marine gas oils with low sulphur contents and a heavy fuel oil) in early life stages of cod (Gadus morhua). Embryos were exposed for 4 days to water-soluble fractions of fuel oils at concentrations ranging from 4.1 - 128.3 µg TPAH/L, followed by recovery in clean seawater until 17 days post fertilization. Exposure to all three fuel oils resulted in developmental toxicity, including severe morphological changes, deformations and cardiotoxicity. To assess underlying molecular mechanisms, we studied fuel oil-mediated activation of aryl hydrocarbon receptor (Ahr) gene battery and genes related to cardiovascular, angiogenesis and osteogenesis pathways. Overall, our results suggest comparable mechanisms of toxicity for the three fuel oils. All fuel oils caused concentration-dependant increases of cyp1a mRNA which paralleled ahrr, but not ahr1b transcript expression. On the angiogenesis and osteogenesis pathways, fuel oils produced concentration-specific transcriptional effects that were either increasing or decreasing, compared to control embryos. Based on the observed toxic responses, toxicity threshold values were estimated for individual endpoints to assess the most sensitive molecular and physiological effects, suggesting that unresolved petrogenic components may be significant contributors to the observed toxicity. ; publishedVersion

Thousands different chemicals are discharged into the environment from agriculture, industry, medical facilities, house-holds. Currently, there is an increasing concern for the environmental impact of mixture of compounds since the additive and eventual synergistic effects are unknown and could produce serious adverse effects. Recently, a document from the European Commission on combination effects of chemicals highlighted the need to ensure that risks associated with chemical mixtures are properly understood and assessed. To address this issue, a joint-effort of 16 European and associated research groups participated to an exercise to test a synthetic reference chemical mixture on the own routine bioassays to investigate the chemical mixtures effects. The reference material included class of pesticides, pharmaceuticals, industrial products, heavy metals and polyaromatic hydrocarbons. The mixures were prepared, each compound at Equivalent Quality Standard (EQS) value, the safety limit concentration allowed by the European Water Framework Directive, (WFD).The bioassays proposed by the groups could cover the entire ecosystem from bacteria to fish as well in vitro assays providing an unique scenario from ecological risk assessment perspective. The results showed that effects were observed at very low concentration on algal-bacteria composition in a marine microcosm, immobilization in crustacean, fish embryo toxicity and frog embryo development. We conclude that some precaution on the chemical mixture assessment should be taken even in case the individual compounds are present at EQS, the safety limit concentration under European legislation.

Thousands different chemicals are discharged into the environment from agriculture, industry, medical facilities, house-holds. Currently, there is an increasing concern for the environmental impact of mixture of compounds since the additive and eventual synergistic effects are unknown and could produce serious adverse effects. Recently, a document from the European Commission on combination effects of chemicals highlighted the need to ensure that risks associated with chemical mixtures are properly understood and assessed. To address this issue, a joint-effort of 16 European and associated research groups participated to an exercise to test a synthetic reference chemical mixture on the own routine bioassays to investigate the chemical mixtures effects. The reference material included class of pesticides, pharmaceuticals, industrial products, heavy metals and polyaromatic hydrocarbons. The mixures were prepared, each compound at Equivalent Quality Standard (EQS) value, the safety limit concentration allowed by the European Water Framework Directive, (WFD).The bioassays proposed by the groups could cover the entire ecosystem from bacteria to fish as well in vitro assays providing an unique scenario from ecological risk assessment perspective. The results showed that effects were observed at very low concentration on algal-bacteria composition in a marine microcosm, immobilization in crustacean, fish embryo toxicity and frog embryo development. We conclude that some precaution on the chemical mixture assessment should be taken even in case the individual compounds are present at EQS, the safety limit concentration under European legislation.

Thousands different chemicals are discharged into the environment from agriculture, industry, medical facilities, house-holds. Currently, there is an increasing concern for the environmental impact of mixture of compounds since the additive and eventual synergistic effects are unknown and could produce serious adverse effects. Recently, a document from the European Commission on combination effects of chemicals highlighted the need to ensure that risks associated with chemical mixtures are properly understood and assessed. To address this issue, a joint-effort of 16 European and associated research groups participated to an exercise to test a synthetic reference chemical mixture on the own routine bioassays to investigate the chemical mixtures effects. The reference material included class of pesticides, pharmaceuticals, industrial products, heavy metals and polyaromatic hydrocarbons. The mixures were prepared, each compound at Equivalent Quality Standard (EQS) value, the safety limit concentration allowed by the European Water Framework Directive, (WFD).The bioassays proposed by the groups could cover the entire ecosystem from bacteria to fish as well in vitro assays providing an unique scenario from ecological risk assessment perspective. The results showed that effects were observed at very low concentration on algal-bacteria composition in a marine microcosm, immobilization in crustacean, fish embryo toxicity and frog embryo development. We conclude that some precaution on the chemical mixture assessment should be taken even in case the individual compounds are present at EQS, the safety limit concentration under European legislation.

Thousands different chemicals are discharged into the environment from agriculture, industry, medical facilities, house-holds. Currently, there is an increasing concern for the environmental impact of mixture of compounds since the additive and eventual synergistic effects are unknown and could produce serious adverse effects. Recently, a document from the European Commission on combination effects of chemicals highlighted the need to ensure that risks associated with chemical mixtures are properly understood and assessed. To address this issue, a joint-effort of 16 European and associated research groups participated to an exercise to test a synthetic reference chemical mixture on the own routine bioassays to investigate the chemical mixtures effects. The reference material included class of pesticides, pharmaceuticals, industrial products, heavy metals and polyaromatic hydrocarbons. The mixures were prepared, each compound at Equivalent Quality Standard (EQS) value, the safety limit concentration allowed by the European Water Framework Directive, (WFD).The bioassays proposed by the groups could cover the entire ecosystem from bacteria to fish as well in vitro assays providing an unique scenario from ecological risk assessment perspective. The results showed that effects were observed at very low concentration on algal-bacteria composition in a marine microcosm, immobilization in crustacean, fish embryo toxicity and frog embryo development. We conclude that some precaution on the chemical mixture assessment should be taken even in case the individual compounds are present at EQS, the safety limit concentration under European legislation.

Thousands different chemicals are discharged into the environment from agriculture, industry, medical facilities, house-holds. Currently, there is an increasing concern for the environmental impact of mixture of compounds since the additive and eventual synergistic effects are unknown and could produce serious adverse effects. Recently, a document from the European Commission on combination effects of chemicals highlighted the need to ensure that risks associated with chemical mixtures are properly understood and assessed. To address this issue, a joint-effort of 16 European and associated research groups participated to an exercise to test a synthetic reference chemical mixture on the own routine bioassays to investigate the chemical mixtures effects. The reference material included class of pesticides, pharmaceuticals, industrial products, heavy metals and polyaromatic hydrocarbons. The mixures were prepared, each compound at Equivalent Quality Standard (EQS) value, the safety limit concentration allowed by the European Water Framework Directive, (WFD).The bioassays proposed by the groups could cover the entire ecosystem from bacteria to fish as well in vitro assays providing an unique scenario from ecological risk assessment perspective. The results showed that effects were observed at very low concentration on algal-bacteria composition in a marine microcosm, immobilization in crustacean, fish embryo toxicity and frog embryo development. We conclude that some precaution on the chemical mixture assessment should be taken even in case the individual compounds are present at EQS, the safety limit concentration under European legislation.

International audience ; Estrogenic compounds are widely released to surface waters and may cause adverse effects to sensitive aquatic species. Three hormones, estrone, 17β-estradiol and 17α-ethinylestradiol, are of particular concern as they are bioactive at very low concentrations. Current analytical methods are not all sensitive enough for monitoring these substances in water and do not cover mixture effects. Bioassays could complement chemical analysis since they detect the overall effect of complex mixtures. Here, four chemical mixtures and two hormone mixtures were prepared and tested as reference materials together with two environmental water samples by eight laboratories employing nine in vitro and in vivo bioassays covering different steps involved in the estrogenic response. The reference materials included priority substances under the European Water Framework Directive, hormones and other emerging pollutants. Each substance in the mixture was present at its proposed safety limit concentration (EQS) in the European legislation. The in vitro bioassays detected the estrogenic effect of chemical mixtures even when 17β-estradiol was not present but differences in responsiveness were observed. LiBERA was the most responsive, followed by LYES. The additive effect of the hormones was captured by ERα-CALUX, MELN, LYES and LiBERA. Particularly, all in vitro bioassays detected the estrogenic effects in environmental water samples (EEQ values in the range of 0.75–304 × EQS), although the concentrations of hormones were below the limit of quantification in analytical measurements. The present study confirms the applicability of reference materials for estrogenic effects' detection through bioassays and indicates possible methodological drawbacks of some of them that may lead to false negative/positive outcomes. The observed difference in responsiveness among bioassays – based on mixture composition - is probably due to biological differences between them, suggesting that panels of bioassays with different characteristics should be applied according to specific environmental pollution conditions.

Estrogenic compounds are widely released to surface waters and may cause adverse effects to sensitive aquatic species. Three hormones, estrone, 17β-estradiol and 17α-ethinylestradiol, are of particular concern as they are bioactive at very low concentrations. Current analytical methods are not all sensitive enough for monitoring these substances in water and do not cover mixture effects. Bioassays could complement chemical analysis since they detect the overall effect of complex mixtures. Here, four chemical mixtures and two hormone mixtures were prepared and tested as reference materials together with two environmental water samples by eight laboratories employing nine in vitro and in vivo bioassays covering different steps involved in the estrogenic response. The reference materials included priority substances under the European Water Framework Directive, hormones and other emerging pollutants. Each substance in the mixture was present at its proposed safety limit concentration (EQS) in the European legislation. The in vitro bioassays detected the estrogenic effect of chemical mixtures even when 17β-estradiol was not present but differences in responsiveness were observed. LiBERA was the most responsive, followed by LYES. The additive effect of the hormones was captured by ERα-CALUX, MELN, LYES and LiBERA. Particularly, all in vitro bioassays detected the estrogenic effects in environmental water samples (EEQ values in the range of 0.75–304 × EQS), although the concentrations of hormones were below the limit of quantification in analytical measurements. The present study confirms the applicability of reference materials for estrogenic effects' detection through bioassays and indicates possible methodological drawbacks of some of them that may lead to false negative/positive outcomes. The observed difference in responsiveness among bioassays – based on mixture composition - is probably due to biological differences between them, suggesting that panels of bioassays with different characteristics should be ...