Anupama Kumar

Vitellogenin induction by 17β‐estradiol and 17α‐ethynylestradiol in male Murray rainbowfish (Melanotaenia fluviatilis)

Hepatic vitellogenin (VTG) mRNA and plasma protein assays were developed for the Murray rainbowfish (Melanotaenia fluviatilis), a native freshwater fish species in Australia. Adult male Murray rainbowfish were exposed to 17β-estradiol (E2) or 17α-ethynylestradiol (EE2) at 0, 1, 5, 10, 50, or 100 ng/L in a semistatic system for 7 d. Vitellogenin mRNA was quantified by quantitative polymerase chain reaction, and VTG protein was semiquantified in plasma using an enzyme-linked immunosorbent assay. Water concentrations were quantified by liquid chromatography coupled with tandem mass spectrometry and were on average 110% and 85% nominal concentrations for E2 and EE2, respectively. Vitellogenin transcripts and protein were upregulated in male Murray rainbowfish exposed to either E2 or EE2 in a dose-responsive manner, with calculated 10% effective concentration (EC10) values for E2 of 3.71 ng/L and 11.6 ng/L for VTG mRNA and protein, respectively, and for EE2 of 2.77 ng/L and 8.47 ng/L for VTG mRNA and protein, respectively. Comparisons of these responses with responses of commonly used test species, including zebrafish (Danio rerio), fathead minnow (Pimephales promelas), and Japanese medaka (Oryzias latipes), revealed the Murray rainbowfish to be a sensitive test species for estrogenic effects via the estrogen receptor pathway. The present study suggests that the Murray rainbowfish would be a suitable candidate for future field studies designed for assessing estrogenic effects of effluent discharges in the Australian freshwater environment.

Ecological evidence links adverse biological effects to pesticide and metal contamination in an urban Australian watershed

Summary: Aquatic ecosystems near urban areas are often ecologically impaired, but causative factors are rarely identified. Effects may be revealed by considering multiple lines of evidence at different levels of biological organization. Biological impairment is evident in the urban section of the Upper Dandenong Creek Catchment (Victoria, Australia). We assessed whether episodic sewage spills or other pollutants were the cause of poor ecological condition in the stream. The evidence evaluated included chemical and invertebrate assessments, caging studies of mudsnails Potamopyrgus antipodarum, antioxidant biomarkers and endocrine disruption-related endpoints in fish (Carassius auratus and Gambusia holbrooki) and toxicological studies with chironomids (Chironomus tepperi). A combination of metals and pesticides is likely to be affecting the aquatic fauna across all biological levels, with macroinvertebrate communities, P. antipodarum and C. tepperi populations and C. auratus individuals all ecologically impaired. Adverse alterations to aquatic fauna were consistently seen in Bungalook Creek and persisted downstream of this confluence into Dandenong Creek. In addition, chemical assessments and toxicity identification evaluation (TIEs) resulted in several point sources of both metals and pesticides being identified as origins of impairment. This contrasted with an expectation that adverse effects were likely to be associated with sewer-related pollution. As a consequence, target areas and specific pollutants were identified for remediation instead of an expensive sewer upgrade. Synthesis and applications. The results demonstrate that it is important to investigate biological effects in different taxa, in both the laboratory and field, to understand which stressors are causing adverse effects on faunal assemblages. When adverse effects are seen across multiple levels of biological organization and caused by the same pollutant from an identifiable source, there is a clear

Di-n-butyl phthalate causes estrogenic effects in adult male Murray rainbowfish (Melanotaenia fluviatilis)

Phthalic acid esters (PAEs) are a class of synthetic industrial chemicals commonly found in the aquatic environment worldwide. PAEs have been recognised as anti-androgens in male mammals but little is known about their endocrine disrupting effects in fish. This study investigated the effects of 7-day exposures to nominal (measured) concentrations of 125 (62), 250 (140), 500 (230) and 1,000 (383) μg/L of di-n-butyl phthalate (DnBP) on the biomarkers of reproduction in adult male Murray River rainbowfish (Melanotaenia fluviatilis) using molecular, biochemical and histological endpoints. None of the tested concentrations of DnBP had any effect on survival or the vital body indices of the fish. The sizes of spermatogonia, Type A and B spermatocytes and spermatids were significantly smaller relative to the controls after treatment with DnBP. This was accompanied by a significant increase in the proportion of spermatogonia in fish treated with 250-1,000 μg/L of DnBP in comparison to the unexposed fish. At the end of the exposure period, the expressions of the transcripts for the androgen receptors α and β were significantly elevated in the livers of the fish treated with 500 and 1,000 μg/L of DnBP. In addition, there was also an increase in the circulating concentrations of vitellogenin in the plasma in the higher treatment groups. An induction in the activity of aromatase was noted in the brains of 1,000 μg/L DnBP-treated fish. This was accompanied by an increase in the hepatic expression of the genes (here and later, whenever the phrase gene expression is used as a synonym for gene transcription although it is acknowledged that it is also regulated, e.g., by translation, mRNA stability and protein stability) encoding for the oestrogen receptors α and β and choriogenin L. Collectively, an increase in the proportion of spermatogonia in the testes, the upregulation of the genes for the oestrogen receptors and choriogenin in the liver, an induction in the brain aromatase activity and the increase in the circulating levels of plasma vitellogenin suggest that continuous exposures for 7 days to sub-acute concentrations of DnBP can adversely affect the reproductive health of the male Murray rainbowfish by an estrogenic mode of action.

Localisation of estrogen responsive genes in the liver and testis of Murray rainbowfish Melanotaenia fluviatilis exposed to 17β-estradiol

The localisation of estrogen receptors (ERalpha and ERbeta) and vitellogenin (VTG) transcripts were examined in the liver and testis in male rainbowfish exposed to 17beta-estradiol (E2; 0, 50 and 500 ng/L) via the water for up to 7 days. The ER transcripts were localised within the perinuclear region of the hepatocytes and were up-regulated with E2 exposure. A parallel induction of liver VTG transcripts and protein was observed within 24h, followed by a time-dependent increase in VTG protein. In the testis, both ERs were up-regulated in the germ and epithelial cells, while VTG protein was detected in the cellular space surrounding the spermatids and in association with the connective tissue of the sperm tubules. These results indicate that the ERs are positively auto-regulated in the liver and testis of male rainbowfish. The cellular localisation of VTG within the testis may suggest implication in the mediation of adverse effects of endocrine disrupting chemicals such as testicular growth inhibition, testis-ova and sex reversal.

Effects of the commercial antiandrogen flutamide on the biomarkers of reproduction in male Murray rainbowfish (Melanotaenia fluviatilis)

The endocrine responses in male Murray rainbowfish (Melanotaenia fluviatilis) were evaluated after exposures to biologically active concentrations of the nonsteroidal pharmaceutical, flutamide. Fish were exposed to nominal concentrations of 125 µg/L, 250 µg/L, 500 µg/L, and 1000 µg/L of flutamide for 7 d, after which plasma vitellogenin concentration; brain aromatase activity; and hepatic expression of the genes for vitellogenin, choriogenin, and androgen and estrogen receptors were assessed. Qualitative assessment of the testes of the fish exposed to flutamide exhibited hindrance in the transformation of spermatogonia to spermatozoa and increased testicular anomalies, such as multinucleated and pyknotic cells and interstitial fibrosis. An increase in the hepatosomatic index with respect to the controls was noted after treating the fish with flutamide at all concentrations. Vitellogenin was induced in plasma in the 1000 µg/L flutamide group. The activity of aromatase in the brain declined significantly after exposures to flutamide at all concentrations. Males exposed to 1000 µg/L of flutamide showed a downregulation in the genes encoding androgen receptors α and β. The expression of the gene for the estrogen receptor α was induced and of vitellogenin was downregulated after treatment with 250 µg/L to 1000 µg/L of flutamide. The results suggest that 7-d exposures to 125 µg/L to 1000 µg/L flutamide can impair the reproductive endocrine system in male Murray rainbowfish at multiple levels by an antiandrogenic mode of action.

Comparative study on the toxicity of pyrethroids, α-cypermethrin and deltamethrin to Ceriodaphnia dubia.

Two synthetic pyrethroid pesticides, α-cypermethrin and deltamethrin were investigated as potential toxic contaminants. The acute and chronic bioassays were conducted using Ceriodaphnia dubia. The toxicity of α-cypermethrin and deltamethrin to C. dubia increased with increasing concentrations and exposure time. C. dubia was three times more sensitive to deltamethrin than to α-cypermethrin with 48-h EC(50) of 0.06 μg/L and 0.23 μg/L, respectively. The chronic EC(50) values for α-cypermethrin and deltamethrin were 97.8 and 34.7 ng/L, respectively. Eight-day growth of Ceriodaphnia neonates during chronic exposures was the most sensitive endpoint measured in comparison to the endpoints of survival and number of neonates produced. To gain a better understanding of the link between acute and chronic toxicity, the acute-to chronic ratios (ACRs) were also calculated for survival, growth and reproduction endpoints. ACRs varied between 11 and 224 for the two pyrethroids. These results suggest that at environmentally relevant low concentrations, α-cypermethrin and deltamethrin could have significant adverse effects on the survival, reproduction and growth of C. dubia.

Oxidation of ciprofloxacin and enrofloxacin by ferrate(VI): Products identification, and toxicity evaluation

Ferrate(VI) (Fe(VI)) has been known to react with emerging organic contaminants containing electron-rich organic moieties, such as phenols, anilines, olefins, reduced sulfur and deprotonated amines. Oxidation of fluoroquinolone antibiotics, ciprofloxacin (CIP) and enrofloxacin (ENR), by Fe(VI) were investigated for their reaction products and toxicity changes as well as biodegradability of these products. Ten products were identified for both CIP and ENR reactions with Fe(VI) using a high-resolution accurate-mass Orbitrap mass analyzer. Structural changes to the CIP and ENR molecule included dealkylation, formation of alcohols and amides in piperazine ring and oxygen transfer to the double bond in quinolone structure. An enamine formation mechanism was tentatively proposed to facilitate the interpretation of CIP and ENR oxidation pathways. Toxicity evaluation using Microbial Assay for toxicity Risk Assessment (MARA) bioassay indicated that Fe(VI) oxidation products of CIP and ENR contributed negligible antibacterial potency and Fe(VI) oxidation treatment can remove the residual toxicity of CIP and ENR impacted source waters. The Fe(VI) oxidation treatment resulted in formation of relatively more biodegradable products (based on in silico assessment) than their corresponding parent compounds. The results showed that Fe(VI) has a good potential to degrade fluoroquinolone antibiotics and their antimicrobial potency in natural waters.

Nature of the clay–cation bond affects soil structure as verified by X-ray computed tomography

Non-destructive X-ray computed tomography (µCT) scanning was used to characterise changes in pore architecture as influenced by the proportion of cations (Na, K, Mg, or Ca) bonded to soil particles. These observed changes were correlated with measured saturated hydraulic conductivity, clay dispersion, and zeta potential, as well as cation ratio of structural stability (CROSS) and exchangeable cation ratio. Pore architectural parameters such as total porosity, closed porosity, and pore connectivity, as characterised from µCT scans, were influenced by the valence of the cation and the extent it dominated in the soil. Soils with a dominance of Ca or Mg exhibited a well-developed pore structure and pore interconnectedness, whereas in soil dominated by Na or K there were a large number of isolated pore clusters surrounded by solid matrix where the pores were filled with dispersed clay particles. Saturated hydraulic conductivities of cationic soils dominated by a single cation were dependent on the observed pore structural parameters, and were significantly correlated with active porosity (R2 = 0.76) and pore connectivity (R2 = 0.97). Hydraulic conductivity of cation-treated soils decreased in the order Ca > Mg > K > Na, while clay dispersion, as measured by turbidity and the negative charge of the dispersed clays from these soils, measured as zeta potential, decreased in the order Na > K > Mg > Ca. The results of the study confirm that structural changes during soil–water interaction depend on the ionicity of clay–cation bonding. All of the structural parameters studied were highly correlated with the ionicity indices of dominant cations. The degree of ionicity of an individual cation also explains the different effects caused by cations within a monovalent or divalent category. While sodium adsorption ratio as a measure of soil structural stability is only applicable to sodium-dominant soils, CROSS derived from the ionicity of clay–cation bonds is better suited to soils containing multiple cations in various proportions.

Di‐n‐butyl phthalate causes antiestrogenic effects in female murray rainbowfish (Melanotaenia fluviatilis)

Di-n-butyl phthalate (DnBP) is an industrial pollutant with antiandrogenic effects reported in male mammals and fish. Little research has been done on the endocrine effects of DnBP in female fish. The present study investigated the changes in ovarian histology and serum vitellogenin concentrations in adult Murray rainbowfish (Melanotaenia fluviatilis) after exposure to 125 µg/L, 250 µg/L, 500 µg/L, and 1000 µg/L DnBP for 7 d. Treatment at 125 µg/L to 1000 µg/L DnBP for 7 d had no significant effect on the survival, condition factor, gonadosomatic index, hepatosomatic index, and developmental stage of the fish. Based on the histological investigation, the sizes of the previtellogenic oocytes in the fish treated at 250 µg/L to 1000 µg/L were found to be significantly higher than in the corresponding control fish (p ≤ 0.05). The early vitellogenic oocytes in the fish treated at 1000 µg/L were significantly smaller relative to those in the unexposed fish (p ≤ 0.05). Histological changes like chorion folding, shrunken ooplasm, impaired yolk production, granulomatous inflammation, and interstitial fibrosis were observed in the ovaries of the fish treated with DnBP. The circulating levels of plasma vitellogenin were significantly lower in the fish exposed to 500 µg/L and 1000 µg/L DnBP (p ≤ 0.05). These data show that a continuous exposure to subacute concentrations of DnBP for 7 d can cause antiestrogenicity in female adult Murray rainbowfish.

Impact of microplastic beads and fibers on waterflea (Ceriodaphnia dubia) survival, growth and reproduction: Implications of single and mixture exposures

There is limited knowledge regarding the adverse effects of wastewater-derived microplastics, particularly fibers, on aquatic biota. In this study, we examined the acute (48 h) and chronic (8 d) effects of microplastic polyester fibers and polyethylene (PE) beads on freshwater zooplankton Ceriodaphnia dubia. We also assessed the acute response of C. dubia to a binary mixture of microplastic beads and fibers for the first time. Acute exposure to fibers and PE beads both showed a dose-dependent effect on survival. An equitoxic binary mixture of beads and fibers resulted in a toxic unit of 1.85 indicating less than additive effects. Chronic exposure to lower concentrations did not significantly affect survival of C. dubia, but a dose-dependent effect on growth and reproduction was observed. Fibers showed greater adverse effects than PE beads. While ingestion of fibers was not observed, scanning electron microscopy showed carapace and antenna deformities after exposure to fibers, with no deformities observed after exposure to PE beads. While much of the current research has focused on microplastic beads, our study shows that microplastic fibers pose a greater risk to C. dubia, with reduced reproductive output observed at concentrations within an order of magnitude of reported environmental levels.