Changes in estrogenicity and micropollutant concentrations across unit processes in a biological wastewater treatment system.

The behavior of 10 micropollutants, i.e. four estrogens (estrone, 17ß-estradiol, estriol, 17α-ethynylestradiol), carbamazepine (CBZ), sulfamethoxazole (SMX), triclosan, oxybenzone, 4-nonylphenol, and bisphenol A, was investigated in a typical domestic wastewater treatment plant. LC-MS and yeast estrogen screen bioassay were used to study the changes in micropollutants and estrogenicity across unit processes in the treatment system. Primary treatment via sedimentation showed that only 4-nonylphenol was removed, but led to no significant change in estrogenicity. Secondary treatment by the biological nitrification-dentrification process showed complete removal of oxybenzone and partial removal of the estrogens, which led to a decrease in estrogenic activity from 80 to 48 ng/L as estradiol equivalent (EEq). Ultraviolet treatment completely degraded the estrogens and triclosan, but failed to lower the concentrations of bisphenol A, SMX, and CBZ; a decrease in estrogenic activity from 48 to 5 ng/L EEq across the unit, a value that was only slightly larger than the observed EEq of 1 ng/L for the deionized control. Similarly, the anaerobic digestion of sludge completely degraded estrogens, oxybenzone, and SMX, but had no impact on bisphenol A, triclosan, and CBZ. The study emphasises the need to complement chemical analyses with estrogenic bioassays to evaluate the efficacy of waste water treatment plants.

In vivo metabolism of norbormide in rats and mice.

Norbormide's species-selective lethality displays 150-fold and 40-fold more sensitivity to rats than mice and guinea pigs, respectively. Our previous study revealed marked inter-species differences in rate and route of metabolism in liver preparations from different species, with hydroxylation the major route. To examine whether rapid metabolic clearance or species-dependent formation of a toxic metabolite play a role in the marked species-sensitivity, we initiated in vivo metabolic studies in rats and mice. After oral dosing, norbormide was detected in mouse but not rat blood. In contrast, liver analysis revealed that norbormide concentration was significantly higher in rat compared with mouse, and that it underwent extensive metabolism tentatively identified via hydroxylation in rat, whilst none was detected in mouse. Although an unidentified metabolite (M3) was detected in rat blood after oral dosing, no metabolites were detected 1min after intravenous dosing, which proved lethal at 0.5mg/kg. Taken together, the data indicate that the toxicity resides with the parent compound, rather species-dependent formation of a potent metabolite and that species sensitivity may be controlled at the pharmacodynamic level.

In vitro metabolism of norbormide in rat, mouse and guinea pig liver preparations.

Differences between species in response to norbormide (NRB) may arise through differential pharmacodynamic and/or pharmacokinetic properties. We hypothesise that species-selectivity is at least partly determined by differences in metabolism based on in vitro data generated in liver preparations from rats, mice and guinea pigs. HPLC separation and LC/MS identification revealed that NRB undergoes metabolism primarily to hydroxylated form that was tentatively identified in both rat and non-rat species with NADPH as the preferred cofactor. However, the metabolic profile and the rate are different between species. Gender differences are also reported in the metabolic rate in rats and we postulate that this may be responsible for different toxic sensitivities seen between sexes. Using this knowledge, we aim to develop pharmacological tool(s) for use in designing a new class of drugs that can be targeted in a tissue-selective manner. Further in vivo pharmacokinetic with receptor affinity studies are warranted.

Investigating hybridization in the parthenogenetic New Zealand stick insect Acanthoxyla (Phasmatodea) using single-copy nuclear loci.

The New Zealand stick insect genus Acanthoxyla Uvarov is extremely unusual among higher taxa of animals in that all known species are obligate parthenogens. We have used a combination of the mitochondrial DNA genes cytochrome oxidase subunits I and II, 28S nuclear ribosomal RNA, and the two single-copy nuclear genes elongation factor 1alpha and phosphoglucose isomerase to test hypotheses on the role of hybridization in the evolution of this genus. Alleles at the single-copy nuclear loci in three sampled species of Acanthoxyla were resolved by cloning the PCR products. Analysis of multilocus genotypes shows that most sampled individuals of Acanthoxyla possess three alleles at the single-copy nuclear loci, which we have interpreted to indicate triploidy. Because most of the alleles from Acanthoxyla form a monophyletic group, including sets of alleles possessed by the putative triploids, we have inferred that the extant parthenogenetic lineages formed via hybridization between species of Acanthoxyla, at least one of which must have been sexual. More recently, there have been multiple introgression events from the related species Clitarchus hookeri White, although C. hookeri does not appear to be involved with the origin of parthenogenesis in Acanthoxyla. Our study demonstrates the utility of cloning alleles from multiple single-copy nuclear genes for resolving the origins of parthenogenetic lineages.

Catalytic oxidative degradation of 17α-ethinylestradiol by FeIII-TAML/H2O2: estrogenicities of the products of partial, and extensive oxidation.

The oxidative degradation of the oral contraceptive 17α-ethinylestradiol (EE(2)) in water by a new advanced catalytic oxidation process was investigated. The oxidant employed was hydrogen peroxide in aqueous solution and the catalyst was the iron tetra-amido macrocyclic ligand (Fe(III)-TAML) complex that has been designated Na[Fe(H(2)O)(B*)] (Fe(III)-B*). EE(2) (10 µM) was oxidised rapidly by the Fe(III)-B*/H(2)O(2) (5 nM/4 mM) catalytic oxidation system at 25 °C, and for reactions at pH 8.40-11.00, no unchanged EE2 was detected in the reaction mixtures after 60 min. No oxidation of EE(2) was detected in blank reactions using either H(2)O(2) or Fe(III)-B* alone. The maximum rate of EE(2) loss occurred at pH 10.21. At this pH the half-life of EE(2) was 2.1 min and the oxidised products showed around 30% estrogenicity removal, as determined by the yeast estrogen screen (YES) bioassay. At pH 11.00, partial oxidation of EE(2) by Fe(III)-B*/H(2)O(2) (5 nM/4 mM) was studied (half-life of EE(2) was 14.5 min) and in this case the initial intermediates formed were a mixture of the epimers 17α-ethynyl-1,4-estradiene-10α,17ß-diol-3-one (1a) and 17α-ethynyl-1,4-estradiene-10ß,17ß-diol-3-one (1b) (identified by LC-ToF-MS and (1)H NMR spectroscopy). Significantly, this product mixture displayed a slightly higher estrogenicity than EE(2) itself, as determined by the YES bioassay. Upon the addition of further aliquots of Fe(III)-B* (to give a Fe(III)-B* concentration of 500 nM) and H(2)O(2) (to bring the concentration up to 4 mM assuming the final concentration had dropped to zero) to this reaction mixture the amounts of 1a and 1b slowly decreased to zero over a 60 min period as they were oxidised to unidentified products that showed no estrogenicity. Thus, partial oxidation of EE(2) gave products that have slightly increased estrogenicity, whereas more extensive oxidation by the advanced catalytic oxidation system completely removed all estrogenicity. These results underscore the importance of controlling the level of oxidation during the removal of EE(2) from water by oxidative processes.