Combined effects of binary mixtures of 17ß-estradiol and testosterone in western mosquitofish (Gambusia affinis) after full life-cycle exposure.

Estrogens and androgens are typical steroid hormones and often occur together in contaminated aquatic environments, but their mixed effects in aquatic organisms have been less well reported. In this study, the endocrine disrupting effects of binary mixtures of 17ß-estradiol (E2) and testosterone (T) in western mosquitofish (Gambusia affinis) were assessed by analyzing the sex ratio, secondary sex characteristics, gonadal histology, and transcriptional expression of target genes related to the hypothalamic-pituitary-gonadal (HPG) axis in G. affinis (from embryos) continuously exposed to E2 (50 ng/L), T (T1: 50 ng/L; T2: 200 ng/L), and mixtures of both (E2 + T1: 50 + 50 ng/L; E2 + T2: 50 + 200 ng/L) for 119 d. The results showed that exposure to E2 + T1 and E2 + T2 reduced the length ratio of ray 4/6 ratio in male G. affinis, suggesting feminized phenomenon in male G. affinis. Furthermore, 16.7-38.5 % of female G. affinis showed masculinized anal fins and hemal spines when exposed to T alone and in combination with E2. Importantly, the transcriptional levels of certain target genes related to the HPG axis were significantly altered in G. affinis following exposure to E2 and T alone and in combinations. Moreover, exposure to E2 and T in combinations can lead to combined effects (such as synergistic and antagonistic effects) on the transcriptional levels of some genes. These results collectively suggest that exposure to environmentally relevant concentrations of E2 and T alone and in mixtures can impact the endocrine system of G. affinis, and may pose potential risks in aquatic systems.

Interaction of 17α-ethinylestradiol and methyltestosterone in western mosquitofish (Gambusia affinis) across two generations.

The interactions between estrogen and androgen in aquatic animals remain largely unknown. In this study, two generations (F0 and F1) of western mosquitofish (Gambusia affinis) were continuously exposed to 17α-ethinylestradiol (EE2, 10 ng/L), methyltestosterone (MT, 10 ng/L (MTL); 50 ng/L (MTH)), and mixtures (EE2+MTL and EE2+MTH). Various endpoints, including sex ratio (phenotypic and genetic), secondary sex characteristics, gonadal histology, and transcriptional profile of genes, were examined. The results showed that G. affinis exposed to MTH and EE2+MTH had a > 89.7 % of phenotypic males in F1 generation, with 34.5 and 50.0 % of these males originated from genetic females, respectively. Moreover, females from F0 and F1 generations exposed to MTH and EE2+MTH exhibited masculinized anal fins and skeletons. The combined effect of MT and EE2 on most endpoints was dependent on MT. Furthermore, significant transcriptional alterations in certain target genes were observed in both the F0 and F1 generations by EE2 and MT alone and by mixtures, showing some degree of interactions. These findings that the effects of EE2+MTH were primarily on the phenotypic sex of G. affinis in offspring generation suggest that G. affinis under chronic exposure to the binary mixture contaminated water could have sex-biased populations.

Identification of Additives in Disposable Face Masks and Evaluation of Their Toxicity Using Marine Medaka (Oryzias melastigma).

The COVID-19 pandemic has resulted in huge amounts of face masks worldwide. However, there is a lack of awareness on the additives and their potential risk to aquatic ecosystems of face masks. To address this issue, the additives and their toxicity in 13 face masks (e.g., polypropylene, polyethylene, and polylactic acid) were determined using nontarget analysis and bioassays. A total of 826 organic additives including intermediates (14.8%), surfactants (9.3%), plasticizers (8.2%), and antioxidants (6.1%) were tentatively identified, with 213 compounds being assigned confidence levels of 1 and 2. Interestingly, polylactic acid masks contained more additives than most polypropylene or polyethylene masks. Among these additives, the concentration of tris(2-ethylhexyl) phosphate in masks was 9.4-978.2 ng/g with a 100% detection frequency. Furthermore, 13 metals such as zinc (up to 202.0 µg/g), copper (32.5 µg/g), and chromium (up to 5.7 µg/g) were detected in the face masks. The methanol extracts of the masks showed the developmental toxicity, swimming behavior, and/or endocrine disruption in embryos/larvae of Oryzias melastigma. The findings demonstrate that face masks contain various toxic additives to marine medaka, which deserves close attention to pollution by face masks.

Endocrine disruptor responses in the embryos of marine medaka (Oryzias melastigma) after exposure to aged plastic leachates.

The issue of the additives leached from plastics has attracted widespread attention. More crucially, endocrine disruptor status for several leached additives has been established. However, little is known about the overall endocrine disrupting effects of aged plastic leachates. Therefore, the transcriptional responses of endocrine-related genes were assessed in the embryos of marine medaka (Oryzias melastigma), which were exposed to the leachates from aged plastics that were immersed into the simulated seawater (SW) or fish digest (FD). The results revealed that there was a great difference between the SW and FD leachates in the transcripts of endocrine-related genes. With the exception of cyp1a, all target genes had their transcripts potentially down-regulated by the FD leachates. Chgl (a biomarker for estrogens), pparß (related to lipid metabolism), and cyp19a (related to sexual differentiation and reproduction) transcripts tended to be repressed by the SW leachates, while pparα, pparγ and cyp1a (mediating metabolism of xenobiotics) transcripts were stimulated. In addition, a redundancy analysis was carried out to determine the relationship between the leached additives and the transcriptional changes. However, the additives only partially explained the variation in the transcripts of endocrine-related genes (24.8%), indicating that other leached additives may have an impact on target gene transcription. This study provided molecular evidence of the aged plastic leachates' endocrine disrupting effects. Exploring the primary factors that affect the transcriptional alterations would require more research.

Toxicity and Estrogenicity of Bisphenol TMC in Oryzias melastigma via In Vivo and In Silico Studies.

Bisphenol 4-[1-(4-hydroxyphenyl)-3,3,5-trimethylcyclohexyl] phenol (BPTMC), as a substitute for bisphenol A, has been detected in environments. However, the ecotoxicological data of BPTMC are extremely scarce. Here, the lethality, developmental toxicity, locomotor behavior, and estrogenic activity of BPTMC at different concentrations (0.25-2000 µg/L) in marine medaka (Oryzias melastigma) embryos were examined. In addition, the in silico binding potentials of O. melastigma estrogen receptors (omEsrs) with BPTMC were assessed by docking study. Low-concentration BPTMC exposure (including an environmentally relevant concentration, 0.25 µg/L) resulted in stimulating effects, including hatching rate, heart rate, malformation rate, and swimming velocity. However, elevated concentrations of BPTMC led to an inflammatory response, changed heart rate and swimming velocity in the embryos and larvae. In the meantime, BPTMC (including 0.25 µg/L) altered the concentrations of estrogen receptor, vitellogenin, and endogenous 17 ß-estradiol as well as the transcriptional levels of estrogen-responsive genes in the embryos or/and larvae. Furthermore, elaborate tertiary structures of omEsrs were built by ab initio modeling, and BPTMC exerted potent binding potential with three omEsrs with -47.23, -49.23, and -50.30 kJ/mol for Esr1, Esr2a, and Esr2b, respectively. This work suggests that BPTMC has potent toxicity and estrogenic effects in O. melastigma.

Exposure to estrone disrupts the endocrine system of western mosquitofish (Gambusia affinis).

Estrone (E1) is one of the predominant natural estrogens detected in aquatic environments, yet little is known about its effects on the endocrine system in fish. In this study, the sex ratio, secondary sexual characteristics, gonadal histology, and transcriptional levels of genes closely related to sex differentiation and hypothalamic-pituitary-gonadal-liver (HPGL) axis were assessed in western mosquitofish (Gambusia affinis) after a full life-cycle exposure to E1 (0, 25.4, 143, 740, and 4300 ng/L) for 119 days. The results showed that exposure to 4300 ng/L of E1 resulted in 100% female and inhibited the growth of females. Exposure to environmentally relevant concentrations of E1 (143 and 740 ng/L) led to obvious feminization of skeletons and anal fins in males. Exposure to 740 and 4300 ng/L of E1 increased the proportion of mature spermatocytes in females, and exposure to 143 and 740 ng/L decreased the proportion of mature spermatocytes in males. Moreover, the transcripts of genes related to sex differentiation and HPGL axis were changed in the E1-exposed adult fish and embryos inside females. This study has provided valuable data on the endocrine disruption effects of E1 at environmentally relevant concentrations in G. affinis.

A comparative study on endocrine disrupting effects of leachates from virgin and aged plastics under simulated media in marine medaka larvae (Oryzias melastigma).

Marine plastic pollution has garnered substantial attention, but the potential endocrine disrupting effects of plastic leachates in marine organisms remain unclear. In this study, the larvae of marine medaka (Oryzias melastigma) were exposed to the leachates from virgin and aged plastics soaked in simulated seawater and fish digest for 3 days. The concentrations of vitellogenin (VTG), estradiol (E2), and 11-ketotestosterone (11-KT), as well as the transcripts of endocrine-related genes were measured in the larvae. The results revealed that endogenous E2 was more sensitive to plastic leachates than VTG and 11-KT, which was significantly affected by 26.7 % of all plastic leachates. Among all genes, estrogen receptor α was impacted mostly, being up-regulated by 53.3 % of leachates from aged plastics. The comparative results demonstrated that the leachates from plastics with different statuses caused a greater difference than those from plastics in different simulated media, and the leachates from aged plastics resulted in higher endocrine disrupting effects than those from virgin plastics. In addition, seven leached additives (plasticizers and flame retardants) could explain 25.6 % of the hormonal effects using redundancy analysis, indicating that other additives in the plastic leachates can also play important roles in regulating the endocrine system of O. melastigma larvae.