The effects of treatment processes on estrogenicity were evaluated by examining estradiol equivalent (EEQ) concentrations in influents and effluents of sewage treatment plants (STPs) located along Yeongsan and Seomjin rivers in Korea. The occurrence and distribution of estrogenic chemicals were also estimated for surface water in Korea and compared with seven other Asian countries including Laos, Cambodia, Vietnam, China, Indonesia, Thailand and Malaysia. Target compounds were nonylphenol (NP), octylphenol (OP), bisphenol A (BPA), estrone (E1), 17beta-estradiol (E2), 17alpha-ethynylestradiol (EE2) and genistein (Gen). Water samples were pretreated and analyzed by liquid-liquid extraction (LLE) and gas chromatography/mass spectrometry (GC/MS). The results showed that the treatment processes of Korean STPs were sufficient to reduce the estrogenic activity of municipal wastewater. The concentrations of phenolic xenoestrogens (i.e., NP, OP and BPA) in samples of Yeongsan and Seomjin rivers were smaller than those reported by previous studies in Korea. In most samples taken from the seven Asian countries, the presence of E2 and EE2 was a major contributor toward estrogenic activity. The EEQ concentrations in surface water samples of the seven Asian countries were at a higher level in comparison to that reported in European countries, America and Japan. However, further studies with more sampling frequencies and sampling areas should be carried out for better evaluation of the occurrence and distribution of estrogenic compounds in these Asian countries.
Per- and polyfluoroalkyl substances (PFAS) are gaining worldwide attention because of their toxicity, bioaccumulative and resistance to biological degradation in the environment. PFAS can be categorised into endocrine disrupting chemicals (EDCs) and identified as possible carcinogenic agents for the aquatic ecosystem and humans. Despite this, only a few studies have been conducted on the aquatic toxicity of PFAS, particularly in invertebrate species such as zooplankton. This study evaluated the acute toxicity of two main PFAS, perfluorooctanoic acid (PFOA) and perfluorooctanesulfonate (PFOS), by using freshwater cladocerans (Moina micrura) as bioindicators. This study aimed to assess the adverse effects at different levels of organisations such as organ (heart size and heart rate), individual (individual size and mortality) and population (lethal concentration, LC50). PFOA was shown to be more hazardous than PFOS, with the LC50 values (confidence interval) of 474.7 (350.4-644.5) μg L-1 and 549.6 (407.2-743.9) μg L-1, respectively. As the concentrations of PFOS and PFOA increased, there were declines in individual size and heart rate as compared to the control group. The values of PNECs acquired by using the AF method (PNECAF) for PFOA and PFOS were 0.4747 and 0.5496 μg L-1, respectively. Meanwhile, the PNEC values obtained using the SSD method (PNECSSD) were 1077.0 μg L-1 (PFOA) and 172.5 μg L-1 (PFOS). PNECAF is more protective and conservative compared to PNECSSD. The findings of this study have significant implications for PFOS and PFOA risk assessment in aquatic environments. Thus, it will aid freshwater sustainability and safeguard the human dependency on water resources.
Bisphenol A (BPA) is a well-known endocrine-disrupting compound that causes several toxic effects on human and aquatic organisms. The restriction of BPA in several applications has increased the substituted toxic chemicals such as bisphenol F (BPF) and bisphenol S (BPS). A native tropical freshwater cladoceran, Moina micrura, was used as a bioindicator to assess the adverse effects of bisphenol analogues at molecular, organ, individual and population levels. Bisphenol analogues significantly upregulated the expressions of stress-related genes, which are the haemoglobin and glutathione S-transferase genes, but the sex determination genes such as doublesex and juvenile hormone analogue genes were not significantly different. The results show that bisphenol analogues affect the heart rate and mortality rate of M. micrura. The 48-h lethal concentration (LC50) values based on acute toxicity for BPA, BPF and BPS were 611.6 µg L-1, 632.0 µg L-1 and 819.1 µg L-1, respectively. The order of toxicity based on the LC50 and predictive non-effect concentration values were as follows: BPA > BPF > BPS. Furthermore, the incorporated method combining the responses throughout the organisation levels can comprehensively interpret the toxic effects of bisphenol analogues, thus providing further understanding of the toxicity mechanisms. Moreover, the output of this study produces a comprehensive ecotoxicity assessment, which provides insights for the legislators regarding exposure management and mitigation of bisphenol analogues in riverine ecosystems.
The usage of cladocerans as non-model organisms in ecotoxicological and risk assessment studies has intensified in recent years due to their ecological importance in aquatic ecosystems. The molecular assessment such as gene expression analysis has been introduced in ecotoxicological and risk assessment to link the expression of specific genes to a biological process in the cladocerans. The validity and accuracy of gene expression analysis depends on the quantity, quality and integrity of extracted ribonucleic acid (RNA) of the sample. However, the standard methods of RNA extraction from the cladocerans are still lacking. This study evaluates the extraction of RNA from tropical freshwater cladocerans Moina micrura using two methods: the phenol-chloroform extraction method (QIAzol) and a column-based kit (Qiagen Micro Kit). Glycogen was introduced in both approaches to enhance the recovery of extracted RNA and the extracted RNA was characterised using spectrophotometric analysis (NanoDrop), capillary electrophoresis (Bioanalyzer). Then, the extracted RNA was analysed with reverse transcription polymerase chain reaction (RT-PCR) to validate the RNA extraction method towards downstream gene expression analysis. The results indicate that the column-based kit is most suitable for the extraction of RNA from M. micrura, with the quantity (RNA concentration = 26.90 ± 6.89 ng/μl), quality (A260:230 = 1.95 ± 0.15, A280:230 = 1.85 ± 0.09) and integrity (RNA integrity number, RIN = 7.20 ± 0.16). The RT-PCR analysis shows that the method successfully amplified both alpha tubulin and actin gene at 33-35 cycles (i.e. Ct = 32.64 to 33.48). The results demonstrate that the addition of glycogen is only suitable for the phenol-chloroform extraction method. RNA extraction with high and comprehensive quality control assessment will increase the accuracy and reliability of downstream gene expression, thus providing more ecotoxicological data at the molecular biological level on other freshwater zooplankton species.
Moina micrura represents a promising model species for ecological and ecotoxicological investigations in tropical freshwater ecosystems. Illumina NovaSeq™ 6000 sequencing was employed in this study to analyze M. micrura across three distinct developmental stages: juvenile, adult, and male. Current study successfully annotated 51,547 unigenes (73.11%) derived from seven (7) different databases. A total of 554 genes were found to be significantly upregulated, while 452 genes showed significant downregulation between juvenile and male. Moreover, 1001 genes were upregulated, whereas 830 genes exhibited downregulation between the adult and male. Analysis of differentially expressed genes revealed upregulation of chitin, cuticle, myosin (MYO), mitogen-activated protein kinases (MAPK), fibrillin (FBN), cytochrome (CYP), glutathione s-transferase (GST), vitellogenin (VTG), acetylcholinesterase (AChE), and transforming growth factor beta (TGFB) under unfavorable environmental conditions (male), as compared to favorable environmental conditions (juveniles and adults). These alterations in gene expression significantly impact the phenological and life-history traits of M. micrura. Furthermore, the upregulation of hemoglobin (HMB), doublesex (DSX), juvenile hormone analogs (JHA), heat shock protein (HSP), and methyltransferase (METT) genes in males initiates the sex-switching effects observed in M. micrura. These findings hold substantial value for researchers interested in determining M. micrura sequences for future investigations of gene expression and comparative reproductive genome analysis within the Moina genus and cladoceran families.