Validation of Arxula Yeast Estrogen Screen assay for detection of estrogenic activity in water samples: Results of an international interlaboratory study.

Endocrine-active substances can adversely impact the aquatic ecosystems. A special emphasis is laid, among others, on the effects of estrogens and estrogen mimicking compounds. Effect-based screening methods like in vitro bioassays are suitable tools to detect and quantify endocrine activities of known and unknown mixtures. This study describes the validation of the Arxula-Yeast Estrogen Screen (A-YES®) assay, an effect-based method for the detection of the estrogenic potential of water and waste water. This reporter gene assay, provided in ready to use format, is based on the activation of the human estrogen receptor alpha. The user-friendly A-YES® enables inexperienced operators to rapidly become competent with the assay. Fourteen laboratories from four countries with different training levels analyzed 17ß-estradiol equivalent concentrations (EEQ) in spiked and unspiked waste water effluent and surface water samples, in waste water influent and spiked salt water samples and in a mixture of three bisphenols. The limit of detection (LOD) for untreated samples was 1.8ng/L 17ß-estradiol (E2). Relative repeatability and reproducibility standard deviation for samples with EEQ above the LOD (mean EEQ values between 6.3 and 20.4ng/L) ranged from 7.5 to 21.4% and 16.6 to 28.0%, respectively. Precision results are comparable to other frequently used analytical methods for estrogens. The A-YES® has been demonstrated to be an accurate, precise and robust bioassay. The results have been included in the ISO draft standard. The assay was shown to be applicable for testing of typical waste water influent, effluent and saline water. Other studies have shown that the assay can be used with enriched samples, which lower the LOD to the pg/L range. The validation of the A-YES® and the development of a corresponding international standard constitute a step further towards harmonized and reliable bioassays for the effect-based analysis of estrogens and estrogen-like compounds in water samples.

Ozonation of anilines: Kinetics, stoichiometry, product identification and elucidation of pathways.

Anilines as archetypes for aromatic amines, which play an important role in the production of, e.g., dyestuffs, plastics, pesticides or pharmaceuticals were investigated in their reaction with ozone. Due to their high reactivity towards ozone (1.2 × 10(5)-2.4 × 10(6) M(-1) s(-1)) the investigated aniline bearing different substituents are readily degraded in ozonation. However, around 4 to 5 molecules of ozone are needed to yield a successful transformation of aniline, most likely due to a chain reaction that decomposes ozone without compound degradation. This is inferred from OH radical scavenging experiments, in which compound transformation per ozone consumed is increased. Mechanistic considerations based on product formation indicate that addition to the aromatic ring is the preferential reaction in the case of aniline, p-chloroaniline and p-nitroaniline (high amounts of o-hydroxyaniline, p-hydroxyaniline, chloride, nitrite and nitrate, respectively were found). For aniline an addition to the nitrogen happens but to a small extent, since nitroso- and nitrobenzene were observed as well. In the case of N-methylaniline and N,N-dimethylaniline, an electron transfer reaction from nitrogen to ozone was proven due to the formation of formaldehyde. In contrast, for p-methylaniline and p-methoxyaniline the formation of formaldehyde may result from an electron transfer reaction at the aromatic ring. Additional oxidation pathways for all of the anilines under study are reactions of hydroxyl radicals formed in the electron transfer of ozone with the anilines (OH radical yields = 34-59%). These reactions may form aminyl radicals which in the case of aniline can terminate in bimolecular reactions with other compounds such as the determined o-hydroxyaniline by yielding the detected 2-amino-5-anilino-benzochinon-anil.

Transformation products and reaction kinetics of fragrances in advanced wastewater treatment with ozone.

The reaction of the fragrance compounds 4,6,6,7,8,8-hexamethyl-1,3,4,7-tetrahydrocyclopenta[g]isochromene (HHCB), 1-(3,5,5,6,8,8-hexamethyl-6,7-dihydronaphthalen-2-yl)ethanone (AHTN), 1-tert-butyl-3,5-dimethyl-2,4,6-trinitrobenzene (musk xylene/MX), 1-(4-tert-butyl-2,6-dimethyl-3,5-dinitrophenyl)ethanone (musk ketone/MK), and 1-(2,3,8,8-tetramethyl-1,3,4,5,6,7-hexahydronaphthalen-2-yl)ethanone (OTNE) with ozone in tap water as well as waste water treatment plant (WWTP) effluents is described. Several transformation products are characterized by means of gas chromatography coupled to mass spectrometry. One transformation product (HHCB-Lactone) was confirmed by means of a true standard. Musk xylene and musk ketone do not react with ozone under the conditions used in this study. AHTN and HHCB reacted slowly to a multitude of transformation products, while OTNE reacted quickly to several stable transformation products. The reaction constants and half lives are used to predict removal efficiencies for full scale reactors.