Anu Kumar

Benchmarking Organic Micropollutants in Wastewater, Recycled Water and Drinking Water with In Vitro Bioassays

Thousands of organic micropollutants and their transformation products occur in water. Although often present at low concentrations, individual compounds contribute to mixture effects. Cell-based bioassays that target health-relevant biological endpoints may therefore complement chemical analysis for water quality assessment. The objective of this study was to evaluate cell-based bioassays for their suitability to benchmark water quality and to assess efficacy of water treatment processes. The selected bioassays cover relevant steps in the toxicity pathways including induction of xenobiotic metabolism, specific and reactive modes of toxic action, activation of adaptive stress response pathways and system responses. Twenty laboratories applied 103 unique in vitro bioassays to a common set of 10 water samples collected in Australia, including wastewater treatment plant effluent, two types of recycled water (reverse osmosis and ozonation/activated carbon filtration), stormwater, surface water, and drinking water. Sixty-five bioassays (63%) showed positive results in at least one sample, typically in wastewater treatment plant effluent, and only five (5%) were positive in the control (ultrapure water). Each water type had a characteristic bioanalytical profile with particular groups of toxicity pathways either consistently responsive or not responsive across test systems. The most responsive health-relevant endpoints were related to xenobiotic metabolism (pregnane X and aryl hydrocarbon receptors), hormone-mediated modes of action (mainly related to the estrogen, glucocorticoid, and antiandrogen activities), reactive modes of action (genotoxicity) and adaptive stress response pathway (oxidative stress response). This study has demonstrated that selected cell-based bioassays are suitable to benchmark water quality and it is recommended to use a purpose-tailored panel of bioassays for routine monitoring.

A review of the fate of potassium in the soil–plant system after land application of wastewaters

Irrigation with wastewaters from agri-industry processes such as milk factories, piggeries, wineries and abattoirs is commonplace. These wastewaters all have high levels of potassium (K). Potassium concentration in effluents from domestic wastewater sources are relatively low, reported to vary between 10 and 30 mg L(-1). Higher levels of potassium are reported for effluents from olive oil mills, 10,000-20,000 mg KL(-1), wool scouring, 4200-13,000 mg KL(-1), cheese and lactic whey and potato processing, approximately 1800 mg KL(-1), piggery effluent, 500-1000 mg KL(-1) and winery wastewaters, up to 1000 mg KL(-1). Application of wastewaters with these high potassium levels has been found to increase the overall level of soil fertility, with the exception of alkaline effluents which can dissolve soil organic carbon. Long-term application of such wastewater may cause the build-up of soil potassium and decrease the hydraulic conductivity of the receiving soils. These potential impacts are uncertain and have been inadequately researched. Regulatory limits for potassium in drinking water have been set only by the European Union with no toxicological or physiological justification. The literature shows that grasses and legume herbages accumulate high levels of potassium, up to 5% dry weight, and some grasses, such as turfgrass are particularly tolerant to high levels of potassium, even under saline conditions. This adaptation is considered useful for increasing potassium immobilization and sustainable practices of land wastewater disposal. Potassium availability is significantly affected by the cation ratios of the wastewater, the existing soil water solution and of soil exchange sites.

Occurrence and implications of estrogens and xenoestrogens in sewage effluents and receiving waters from South East Queensland.

We report a survey on the occurrence of estrogens (estrone, E1; 17beta-estradiol, E2; 17alpha-ethynylestradiol, EE2) and xenoestrogens (bisphenol-A, BPA; 4-t-octylphenol, 4-t-OP; 4-nonylphenols, 4-NP; and nonylphenol mono- and di-ethoxylates, NPE1 and NPE2) in effluents from five wastewater treatment plants and their receiving waters in South East Queensland. The total xenoestrogen concentrations in effluent ranged between 2446 ng/L and 6579 ng/L, with 4-NP and NPE1-2 having much higher concentration levels than BPA and 4-t-OP. The estrogen levels in effluent varied from 9.12 to 32.22 ng/L for E1, from 1.37 ng/L to 6.35 ng/L for E2 and from 0.11 ng/L to 1.20 ng/L for EE2. No significant differences (p<0.05) in the concentrations of the selected estrogenic compounds were found for the effluents from the five sewage treatment plants. The estrogens and xenoestrogens were also found in the receiving waters at relatively lower concentration levels due to dilution of effluents in the rivers. Based on the chemical analysis data and relative potency of the compound from in vitro and in vivo bioassays from the literature, the calculated in vitro EEQ values (estrogen equivalents) in the receiving river waters downstream of the effluent discharge points ranged from 1.32 to 11.79 ng/L, while the in vivo EEQ values (vitellogenin response in rainbow trout) ranged from 2.48 to 21.18 ng/L. The three estrogens accounted for the majority of the EEQ in the water samples. This study indicates that the rivers of South East Queensland are at potential risk.

FATE OF ESTROGENS AND XENOESTROGENS IN FOUR SEWAGE TREATMENT PLANTS WITH DIFFERENT TECHNOLOGIES

The fate and removal of the estrogens 17beta-estradiol (E(2)), estrone (E(1)), and 17alpha-ethynylestradiol (EE(2)) and of the xenoestrogens bisphenol A (BPA), 4-tert-octylphenol (4-t-OP), 4-nonylphenol (4-NP), and nonylphenol mono- and diethoxylate (NPEO1 and NPEO2, respectively) were investigated in four South Australian sewage treatment plants (STPs; plants A-D) with different treatment technologies. The concentrations in the effluent from the two-year survey were similar to those reported in other studies. In the effluent, 4-NP, NPEO1, and NPEO2 had total concentrations up to 8 microg/L, which were much higher than those of BPA and 4-t-OP. Estrone had the highest concentrations among the three estrogens, ranging between 13.3 and 39.3 ng/L, whereas the concentrations for E(2) and EE(2) varied between 1.0 and 4.2 ng/L and between 0.1 and 1.3 ng/L, respectively. The removal rates for the estrogens and xenoestrogens were variable but consistent with the plant performance parameters (biochemical oxygen demand, suspended solids, and ammonia). Considering all the estrogenic compounds analyzed in the present study, plant D, with a series of anaerobic and aerobic lagoons, was the least efficient of the four STPs in the removal of these compounds. The removal rates for 4-NP, NPEO1, and NPEO2 within the plants were 92% for plant A, with conventional activated sludge treatment; 80% for plant B, with two oxidation ditches; 70% for plant C, with three bioreactors; and 64% for plant D, with 10 lagoons in series. Comparatively, the removal of estrogens was lower, with rates ranging between 47 and 68% for E(2) at the four plants. Both E(1) and EE(2) were more persistent during treatment, especially in plants C and D.

Toxicity of copper, lead, and zinc mixtures to Ceriodaphnia dubia and Daphnia carinata.

Acute and chronic bioassays were conducted to determine the effects of copper, lead, and zinc mixtures on Ceriodaphnia dubia and Daphnia carinata. Copper, lead, and zinc combined at up to 5.2, 4.5, and 51.8 microg/L, respectively, did not cause significant mortality during acute exposures, although mixtures of 10.6, 9, and 101.1 microg/L and higher resulted in 65-100% mortality. Binary combinations of Cu+Zn (1.3+13.0 microg/L) and Cu+Pb (1.3+1.1 microg/L) and ternary combinations of Cu+Pb+Zn (1.3+1.1+13.0 microg/L) had a significant effect on reproduction of C. dubia. Toxic units and associated confidence intervals were calculated to characterize the nature of metal interactions. In most cases, and based on confidence intervals encompassing a value of 1, most of the metal interactions would be classified as additive. However, a more than additive effect was indicated by the acute tests for both species exposed to Cu+Pb, for D. carinata exposed to Cu+Zn, and for C. dubia exposed to all three metals.

Response and recovery of acetylcholinesterase activity in freshwater shrimp, Paratya australiensis (Decapoda: Atyidae) exposed to selected anti-cholinesterase insecticides

The toxicity of carbaryl, chlorpyrifos, dimethoate and profenofos to the freshwater shrimp, Paratya australiensis was assessed by measuring acetylcholinesterase (AChE) inhibition after 96h exposures. Shrimp exposed to these pesticides exhibited significant AChE inhibition, with mortality in shrimp corresponding to 70-90% AChE inhibition. The sensitivity of P. australiensis to the four pesticides based on AChE inhibition can be given as chlorpyrifos > profenofos > carbaryl > dimethoate. Recovery of AChE activity was followed in shrimp after 96 h exposures to carbaryl, chlorpyrifos and dimethoate. Recovery after exposure to the carbamate pesticide carbaryl was more rapid than for the two organophosphorus pesticides, chlorpyrifos and dimethoate. The slow recovery of depressed AChE activity may mean that affected organisms in the natural system are unable to sustain physical activities such as searching for food or eluding predators. To investigate the ecological significance of AChE inhibition, chemotaxis behaviour was assessed in shrimp exposed to profenofos for 24h. Abnormal chemotaxis behaviour in the exposed shrimp was observed at concentrations representing 30-50% AChE inhibition. A clear relationship existed between the depression of AChE activity and observed chemotaxis responses, such as approaching and grasping the chemoattractant source. These results suggest that in vivo toxicity tests based on this specific biomarker are sensitive and present advantages over conventional acute tests based on mortality. Behavioural studies of test organisms conducted in conjunction with measurement of AChE inhibition will provide data to clarify the toxic effects caused by sublethal chemical concentrations of anti-cholinesterase compounds.

Assessment of multiple hormonal activities in wastewater at different stages of treatment

Changes in the endocrine potency of municipal wastewater at 3 wastewater treatment plants (WWTPs) in Australia were investigated using a panel of in vitro receptor-driven transactivation assays. The assays were based on human estrogen receptor α, androgen receptor, progesterone receptor, glucocorticoid receptor, and peroxisome proliferator-activated receptor γ2. Total removal efficiencies for estrogenic activity in the dissolved phase were 79.8% to 99.4%. Chemical analysis of 17β-estradiol, estrone, and 17α-ethinylestradiol levels showed that they accounted for the majority of the observed in vitro estrogenic activity in the final effluents but only 18% to 70% of estrogenic activity in the influents. Removal efficiency for androgenic activity was 97.5% to 100%. Endocrine activity levels were low in the final effluent of the WWTP with the lowest catchment population, with only estrogenic activity detected. In the final effluent of the WWTP with an intermediate catchment population, estrogenic, glucocorticoid, and peroxisome proliferator activities were detected. Estrogenic, antiandrogenic, progestagenic, glucocorticoid, and peroxisome proliferator activities were detected in the final effluent of the WWTP with the highest catchment population. The present study confirms the efficacy of secondary and tertiary treatment in reducing the concentrations of endocrine-active compounds in municipal wastewater. Further work is required to determine the possible health risks to aquatic biota posed by multiple hormonal activities present at low levels.

Toxicity of selected pesticides to freshwater shrimp, Paratya australiensis (Decapoda: Atyidae): use of time series acute toxicity data to predict chronic lethality.

Toxicity of six pesticides (carbaryl, chlorpyrifos, cypermethrin, dimethoate, diuron and fenarimol) to the freshwater shrimp, Paratya australiensis was assessed after 96 h exposures. Of the six pesticides tested, alpha cypermethrin was the most toxic to the shrimp followed by chlorpyrifos, carbaryl, dimethoate, fenarimol and diuron. Regression methods for predicting chronic toxicity (lethality) from acute lethality data with shrimp were developed and compared, and it was found that the log-log model gives the most reliable predictions of the probability of death as a function of extended exposure times. Based on this model, chronic toxicity (21 days) to P. australiensis was estimated as 0.0058 microg/L for alpha cypermethrin, 4.9 microg/L for carbaryl, 0.004 microg/L for chlorpyrifos, 89 microg/L for dimethoate, 240 microg/L for diuron and 1500 microg/L for fenarimol. Acute LC(10) values were also useful predictors of the chronic lethality. The log-log model was used to derive extrapolated chronic values that were compared to measured experimental chronic values for two fish species. The predictions of chronic toxicity based on acute toxicity data were found to give credible results for both fish species. These predictions of chronic toxicity can therefore be used in ecological risk assessments to fill in gaps with reasonable confidence where no measured estimates of chronic toxicity are available.

Nortestosterone-derived synthetic progestogens do not activate the progestogen receptor of Murray–Darling rainbowfish (Melanotaenia fluviatilis) but are potent agonists of androgen receptors alpha and beta

Synthetic progestogens derived from 19-nortestosterone can elicit a number of adverse effects in fish including decreased fecundity, altered hormone levels, disruption of normal breeding cycles, expression in females of male-specific biomarkers, development of male secondary sexual characteristics in females, and changes in the expression of steroidogenic genes. A recent in vitro study showed that a number of representatives from this class of progestins were potent agonists of fathead minnow androgen receptor (AR) and only weak agonists of progesterone receptor (PR) from the same species. This confirms that synthetic progestogens derived from 19-nortestosterone function as AR agonists in otomorphs, which express a single AR subtype. However, numerous perciformes are known to express two AR subtypes. We have recently shown that ARα and ARβ from Murray-Darling rainbowfish (Melanotaenia fluviatilis) respond differently to certain androgens and anti-androgens. The goal of the present study was to determine concentration-response profiles for selected progestins in transactivation assays driven by rainbowfish ARα, ARβ and PR in order to ascertain the relative potency of progestins against these receptors. As a means of confirming the expected activity of the progestins and reference compounds used in the study against human-derived receptors, we also established concentration-response relationships using transactivation assays driven by human PR and AR. We found that all five 19-nortestosterone-derived progestins tested were highly potent agonists of rainbowfish ARα, but that only four of the five progestins were potent agonists of rainbowfish ARβ, with norgestimate exhibiting only weak activity against rainbowfish ARβ. The spironolactone-derived progestin, drospirenone, was not an agonist of rainbowfish ARα or ARβ but was a weak agonist of rainbowfish PR. None of the 19-nortestosterone-progestins activated rainbowfish PR. These findings confirm that the majority of 19-nortestosterone-derived progestins are likely to elicit strong androgenic activity in teleosts, but that PR-mediated effects would be minimal. In species that express two AR subtypes similar to rainbowfish ARα and ARβ, biological processes mediated by a specific subtype may be affected differently by progestins such as norgestimate.

Bioconcentration of triclosan and methyl-triclosan in marine mussels (Mytilus galloprovincialis) under laboratory conditions and in metropolitan waters of Gulf St Vincent, South Australia

The anti-microbial agent triclosan (TCS), and its derivative methyl-triclosan (Me-TCS), are discharged with treated effluents from wastewater treatment plants to receiving environments. We investigated the bioconcentration of TCS and Me-TCS in mussels (Mytilus galloprovincialis) exposed to TCS (100 ng L(-1)) for 30 days in seawater aquaria (19±2°C) with fresh phytoplankton as a food source. Bioconcentration increased with time reaching a steady-state around 24-30 days. The bioconcentration factor (log BCF) for TCS were 2.81 L kg(-1) (dry weight) and 4.13 L kg(-1), when lipid normalised concentrations were used. Mussels were also deployed in cages at four marine locations receiving effluents from WWTPs. The mean (±SD) TCS and Me-TCS concentrations for mussels from these sites were 9.87 (±1.34) and 6.99 (±2.44) μg kg(-1). The study showed that mussels can be a useful tool for monitoring pollution of TCS and Me-TCS in marine and estuarine environments.