Previous studies have detected numerous organic contaminants and in vitro bioactivities in surface water from the South Platte River near Denver, Colorado, USA. To evaluate the temporal and spatial distribution of selected contaminants of emerging concern, water samples were collected throughout 2018 and 2019 at 11 sites within the S. Platte River and surrounding tributaries with varying proximities to a major wastewater treatment plant (WWTP). Water samples were analyzed for pharmaceuticals, pesticides, steroid hormones, and wastewater indicators and screened for in vitro biological activities. Multiplexed, in vitro assays that simultaneously screen for agonistic activity against 24 human nuclear receptors detected estrogen receptor (ER), peroxisome proliferator activated receptor-gamma (PPARγ), and glucocorticoid receptor (GR) bioactivities in water samples near the WWTP outflow. Targeted in vitro bioassays assessing ER, GR, and PPARγ agonism corroborated bioactivities for ER (up to 55 ± 9.7 ng/L 17β-estradiol equivalents) and GR (up to 156 ± 28 ng/L dexamethasone equivalents), while PPARγ activity was not confirmed. To evaluate the potential in vivo significance of the bioactive contaminants, sexually-mature fathead minnows were caged at six locations upstream and downstream of the WWTP for 5 days after which targeted gene expression analyses were performed. Significant up-regulation of male hepatic vitellogenin was observed at sites with corresponding in vitro ER activity. No site-related differences in GR-related transcript abundance were detected in female adipose or male livers, suggesting observed environmental concentrations of GR-active contaminants do not induce a detectable in vivo response. In line with the lack of detectable targeted in vitro PPARɣ activity, there were no significant effects on PPARɣ-related gene expression. Although the chemicals responsible for GR and PPAR-mediated bioactivities are unknown, results from the present study provide insights into the significance (or lack thereof) of these bioactivities relative to short-term in situ fish exposures.

To assess the time-dependent stress evidence in dynamic allocation of physiological metabolism of Nilaparvata lugens nymphs in response to elevated CO₂, we measured the time-dependent allocation of nutrient compositions and physiological metabolism in the bodies of N. lugens at 1h, 4h and 12h under elevated CO₂. Elevated CO₂ significantly increased the contents of nutrient compositions (protein, glucose and total amino acids) and catalase (CAT) enzyme activity in the body of N. lugens at 12h relative to 1h and 4h (P < 0.05). Significantly higher genes expression levels of acetylcholinesterase (AChE), heat shock protein (HSP70) and vitellogenin gene (vg) were observed in the body of N. lugens compared with those in ambient CO₂ at 4h (P < 0.05). These results showed that there was an instantaneous reaction of N. lugens nymphs to elevated CO₂, which indicated N. lugens may enhance stress defense response to future increasing CO₂ levels.

This study aimed to investigate the effect of mono-(2-ethylhexyl) phthalate (MEHP), one of the major phthalate metabolites that are widespread in aquatic environments, on reproductive dysfunction, particularly on endocrine activity in adult male and female zebrafish. For 21 days, the zebrafish were exposed to test concentrations of MEHP (0, 2, 10, and 50 μg/mL) that were determined based on the effective concentrations (ECx) for zebrafish embryos. Exposure to 50 μg/mL MEHP in female zebrafish significantly decreased the number of ovulated eggs as well as the hepatic VTG mRNA abundance when those of the control group. Meanwhile, in female zebrafish, the biosynthetic concentrations of 17β-estradiol (E2) and the metabolic ratio of androgen to estrogen were remarkably increased in all MEHP exposed group compared with those in the control group, along with the elevated levels of cortisol. However, no significant difference was observed between these parameters in male zebrafishes. Therefore, exposure to MEHP causes reproductive dysfunction in female zebrafishes and this phenomenon can be attributed to the alteration in endocrine activities. Moreover, the reproductive dysfunction in MEHP-exposed female zebrafishes may be closely associated with stress responses, such as elevated cortisol levels. To further understand the effect of MEHP on the reproductive activities of fish, follow-up studies are required to determine the interactions between endocrine activities and stress responses. Overall, this study provides a response biomarker for assessing reproductive toxicity of endocrine disruptors that can serve as a methodological approach for an alternative to chronic toxicity testing.

Perfluorooctane sulfonate (PFOS) has been reported to be widely distributed in the environment and wildlife with persistence. PFOS has various biological toxicity, especially disturbing the endocrine system. However, few studies have systematically evaluated its effect on sexual behaviors alteration and reproduction-related genes. This study was performed to assess the effect of PFOS exposure on sexual behaviors and genes in hypothalamic–pituitary–gonadal–liver (HPGL) axis in adult zebrafish.Male adult zebrafish were exposed to PFOS (0, 2, 20, and 200 μg/L) and 5 μg/L estradiol (E₂) continuously for 21 days. Sexual behaviors were analyzed by zebrafish behavior tracking system and the mRNA levels of HPGL-related genes was detected by RT-qPCR.Body weight of the fish was increased in 2, 200 μg/L PFOS and E₂ groups, and body length was increased with exposure to 2 μg/L PFOS and E₂. The hepatic-somatic index was decreased significantly after 2 and 20 μg/L PFOS treatments. Highest PFOS (200 μg/L) and E₂ exposure impaired standard zebrafish sexual behaviors significantly such as chasing, nose-tail and tail-touching. In brains, the genes gonadotropin-releasing hormone (GnRH), gonadotropin-releasing hormone receptor (GnRHr) were down-regulated with exposure to PFOS with linear trend and E₂ exposure, and follicle-stimulating hormone and luteinizing hormone were also down-regulated with exposure to 20 and 200 μg/L PFOS. In livers, the genes vitellogenin 1 and 3 were upregulated with some concentrations of PFOS and E₂, but estrogenic receptor α, β2 were upregulated in any concentration of PFOS and E₂. In testes, the expressions of follicle-stimulating hormone receptor, luteinizing hormone receptor, and androgen receptor genes were all significantly down-regulated with any exposure concentration of PFOS and E₂.PFOS may alter the zebrafish reproductive system by disrupting endocrine activity and impairing sexual behaviors.

Climate change and pharmaceuticals contamination constitute two of the most relevant stressors on the aquatic ecosystems, however, there is a huge lack of information regarding the interactive effects of both stressors. For that, a mesocosm experiment was implemented where adult zebrafish were exposed to combined temperature and the progestin levonorgestrel (LNG) for 21 days. Considering that the liver is one of the organs where there is a greater metabolization and accumulation of toxicants, the main objective of this work was to assess the effects of both stressors on the female zebrafish hepatocytes morphology and functioning, through stereological and immunohistochemical techniques.Our results revealed an increase of coefficient of variation of the number distribution of hepatocytes volume (CVN(υ)) for individuals exposed to LNG, which denotes an increase of the hepatocytes size variability and is suggestive of functional impacts. This was corroborated by the signs of increased glycogen content with the exposure to increased LNG concentrations and temperature, indicating modified hepatocyte glycogen metabolism. Such disturbances can be considered indicators that the fish had to deal with impacts caused by the stress factors.Regarding the immunoreactivity, from the four proteins selected (catalase, CYP1A, HSP90 and Vtg), just in two of them (catalase and Vtg) were observed some responses to both stressors. For catalase there was a hormetic response, in which exposure to lower LNG concentrations caused a significant higher positive immunostaining than under higher LNG concentrations. While, for Vtg, significant effects of temperature and LNG existed, in which a decline in Vtg immunostaining was observed with exposure to higher temperature and lower LNG concentrations. These results should be seen as a warning sign about fine impacts of multiple stressors, such as temperature and progestogens, on the structure and functioning of zebrafish liver and potentially in other aquatic organisms, and on their health implications.

People living a subsistence lifestyle in the Arctic are highly exposed to persistent organic pollutants, including polychlorinated biphenyls (PCBs). Formerly Used Defense (FUD) sites are point sources of PCB pollution; the Arctic contains thousands of FUD sites, many co-located with indigenous villages. We investigated PCB profiles and biological effects in freshwater fish (Alaska blackfish [Dallia pectoralis] and ninespine stickleback [Pungitius pungitius]) living upstream and downstream of the Northeast Cape FUD site on St. Lawrence Island in the Bering Sea. Despite extensive site remediation, fish remained contaminated with PCBs. Vitellogenin concentrations in males indicated exposure to estrogenic contaminants, and some fish were hypothyroid. Downstream fish showed altered DNA methylation in gonads and altered gene expression related to DNA replication, response to DNA damage, and cell signaling. This study demonstrates that, even after site remediation, contaminants from Cold War FUD sites in remote regions of the Arctic remain a potential health threat to local residents – in this case, Yupik people who had no influence over site selection and use by the United States military.

Interactions between organic toxicants and nano-particles in the aquatic environment may modify toxicant bioavailability and consequently the toxicant's fate and toxicity. To evaluate the potential impact of nano-titanium dioxide (TiO₂) on the bio-concentration and reproductive endocrine disruption of tris(1,3-dichloro-2-propyl) phosphate (TDCIPP) in fish, a comparative bioaccumulation study was conducted on zebrafish (Danio rerio, AB strain) treated with 0, 5.74, 23.6, or 90.7 μg L⁻¹ TDCIPP alone or co-exposed to TDCIPP and 0.09 mg L⁻¹ nano-TiO₂ for 21 days. Nano-TiO₂ can absorb TDCIPP and nano-TiO₂ is taken up into zebrafish. Chemical measurements showed that TDCIPP was bio-concentrated in zebrafish, and the highest level was detected in the liver, followed by the brain and gonads. Compared with TDCIPP treatment, increased tissue burdens of both TDCIPP were observed in the liver, brain, and gonads suggesting that nano-TiO₂ adsorbed TDCIPP and acted as a carrier facilitating the uptake and translocation of TDCIPP in tissues. Higher bio-concentration in the presence of nano-TiO₂ resulted in a significant decrease in the hepatic-somatic index, gonad-somatic index and brain-somatic index in F0 females but not F0 males. Moreover, a further gender-dependent reduction in testosterone (T), estradiol (E2), follicle-stimulating hormone (FSH) and luteinizing hormone (LH), and induction of plasma vitellogenin (VTG) concentrations in adults were observed following co-exposure. Co-exposure also inhibited egg production and caused significant developmental toxicity in F1 larvae. The results obtained using this multi-marker approach suggested that nano-TiO₂ is a carrier of TDCIPP and accelerated its bio-concentration in adult zebrafish, resulting in adverse reproduction outcomes.

Leaf-cutting ants are important pests of forests and agricultural crops in the Neotropical region. Atta sexdens colonies can be composed of thousands of individuals, which form a highly complex society with a single reproductive queen. Successful control of this species is achieved only if the queen is affected. Few data are available on the lethal or sublethal effects of toxic compounds on leaf-cutting ant queens. Azadirachtin has been claimed as an effective biopesticide for insect control, but its action on leaf-cutting ants has been little explored. This study shows that azadirachtin affects oviposition in A. sexdens queens, impairing egg development by decreasing protein reserves. Azadirachtin inhibits the synthesis of vitellogenin, the major yolk protein precursor. The negative effects of azadirachtin on the reproduction of leaf-cutting ant queens suggest a potential use for the control of these insects.

Among the many factors responsible for the decline of bee populations are plant protection products such as neonicotinoids. In general, bees are exposed to not only one but mixtures of such chemicals. At environmental realistic concentrations neonicotinoids may display negative effects on the immune system, foraging activity, learning and memory formation of bees. Neonicotinoids induce alterations of gene transcripts such as nicotinic acetylcholine receptor (nAChR) subunits, vitellogenin, genes of the immune system and genes linked to memory formation. While previous studies focused on individual compounds, the effect of neonicotinoid mixtures in bees is poorly known. Here we investigated the effects of neonicotinoids acetamiprid, clothianidin, imidacloprid and thiamethoxam as single compounds, and binary mixtures thereof in honeybees. We determined transcriptional changes of nAChR subunits and vitellogenin in the brain of experimentally exposed honeybees after exposure up to 72 h. Exposure concentrations were selected on the basis of lowest effect concentrations of the single compounds. Transcriptional induction of nAChRs and vitellogenin was strongest for thiamethoxam, and weakest for acetamiprid. To a large extent, binary mixtures did not show additive transcriptional inductions but they were less than additive. Our data suggest that the joint transcriptional activity of neonicotinoids cannot be explained by concentration addition. The in vivo effects are not only governed by agonistic interaction with nAChRs alone, but are more complex as a result of interactions with other pathways as well. Further studies are needed to investigate the physiological joint effects of mixtures of neonicotinoids and other plant protection products on bees to better understand their joint effects.