Ispitivanje estrogene aktivnosti u vodama pomoću kvasaca

The presence of endocrine-disrupting compounds in wastewater, surface water, groundwater and even drinking water has become a major concern worldwide, since they negatively affect wildlife and humans. Therefore, these substances should be effectively removed from effluents before they are discharged into surface water to prevent pollution of groundwater, which can be a source of drinking water. Furthermore, an efficient control of endocrine-disrupting compounds in wastewater based on biological and analytical techniques is required. In this study, a yeast estrogen screen (YES) bioassay has been introduced and optimized with the aim to assess potential estrogenic activity of waters. First, assay duration, concentration of added substrate to the assay medium and wavelength used to measure the absorbance of the substrate were estimated. Several compounds, such as 17-β-estradiol, 17-α-ethinylestradiol, bisphenol A, nonylphenol, genisteine, hydrocortisone, dieldrin, atrazine, methoxychlor, testosterone and progesterone were used to verify its specificity and sensitivity. The optimized YES assay was sensitive and responded specifically to the selected estrogenic and nonestrogenic compounds in aqueous samples. Potential estrogenicity of influent and effluent samples of two wastewater treatment plants was assessed after the samples had been concentrated by solid-phase extraction (SPE) procedure using Oasis® HLB cartridges and methanol as eluting solvent. Up to 90 % of relative estrogenic activity was detected in concentrated samples of influents to wastewater treatment plants and estrogenic activity was still present in the concentrated effluent samples. We found that the introduced YES assay is a suitable screening tool for monitoring the potential estrogenicity of effluents that are discharged into surface water.Prisutnost spojeva koji imaju estrogeni učinak u otpadnim, površinskim, podzemnim, a čak i pitkim vodama sve više zabrinjava stručnjake diljem svijeta zbog negativnog učinka na zdravlje ljudi i životinja. Stoga takve spojeve treba odstraniti iz otpadnih voda prije njihovog ispuštanja u površinske vode, te na taj način spriječiti zagađivanje podzemnih voda, što se koriste kao izvor pitke vode. Kemijskom analizom i biološkim testovima potrebno je učinkovito pratiti prisutnost spojeva s estrogenim učinkom u otpadnim vodama. U ovom smo radu razvili i optimirali metodu ispitivanja estrogenog učinka spojeva pomoću kvasca (test YES) radi procjene potencijalne estrogene aktivnosti u vodama. Optimirali smo trajanje testa, koncentraciju supstrata dodanog podlozi i valnu duljinu za mjerenje apsorbancije razgrađenog supstrata. Specifičnost i osjetljivost testa YES provjerili smo pomoću sljedećih spojeva: 17-β-estradiol, 17-α-etinilestradiol, bisfenol A, nonilfenol, genistein, hidrokortizon, dieldrin, atrazin, metoksiklor, testosteron i progesteron. Optimiranim testom YES mogu se u uzorcima voda detektirati odabrani spojevi s estrogenim učinkom ili bez njega. Moguća estrogenost pritoka i istoka vode iz dva postrojenja za obradu otpadnih voda određena je prethodnom ekstrakcijom uzoraka vode na čvrstoj fazi (SPE ekstrakcija) pomoću kolone Oasis® HLB i metanola kao otapala. U koncentriranim je uzorcima pritoka u postrojenjima određeno 90 % relativne estrogene aktivnosti. Isto tako je estrogena aktivnost pronađena i u koncentriranim uzorcima istoka iz postrojenja. Iz dobivenih se rezultata može zaključiti da je test YES prikladan za ispitivanje estrogene aktivnosti otpadnih voda, što naknadno dospijevaju u površinske vode

Yeast Estrogen Screen Assay as a Tool for Detecting Estrogenic Activity in Water Bodies

The presence of endocrine-disrupting compounds in wastewater, surface water, groundwater and even drinking water has become a major concern worldwide, since they negatively affect wildlife and humans. Therefore, these substances should be effectively removed from effluents before they are discharged into surface water to prevent pollution of groundwater, which can be a source of drinking water. Furthermore, an efficient control of endocrine-disrupting compounds in wastewater based on biological and analytical techniques is required. In this study, a yeast estrogen screen (YES) bioassay has been introduced and optimized with the aim to assess potential estrogenic activity of waters. First, assay duration, concentration of added substrate to the assay medium and wavelength used to measure the absorbance of the substrate were estimated. Several compounds, such as 17-β-estradiol, 17-α-ethinylestradiol, bisphenol A, nonylphenol, genisteine, hydrocortisone, dieldrin, atrazine, methoxychlor, testosterone and progesterone were used to verify its specificity and sensitivity. The optimized YES assay was sensitive and responded specifically to the selected estrogenic and nonestrogenic compounds in aqueous samples. Potential estrogenicity of influent and effluent samples of two wastewater treatment plants was assessed after the samples had been concentrated by solid-phase extraction (SPE) procedure using Oasis® HLB cartridges and methanol as eluting solvent. Up to 90 % of relative estrogenic activity was detected in concentrated samples of influents to wastewater treatment plants and estrogenic activity was still present in the concentrated effluent samples. We found that the introduced YES assay is a suitable screening tool for monitoring the potential estrogenicity of effluents that are discharged into surface water

Membrane changes associated with exposure of Pseudomonas putida to selected environmental pollutants and their possible roles in toxicity

A bacterial model system (Pseudomonas putida DSM 50026) was used in this research to assess potential effect of five selected chemically diverse environmental pollutants on cell membranes. Long chain fatty acid profiles of cultures exposed to environmentally relevant concentrations of atrazine (ATR), metolachlor (MET), pentachlorobiphenyl (PCB), hexachlorobenzene (HCB) and fluoranthene (FL), were analyzed and compared to non0exposed cultures. To assess sensitivity of membrane-based response, the impact of each toxicant on culture growth was also followed spectrophotometrically. Results revealed changes in fatty acid profiles when cells were exposed to PCB, HCB and FL in concentrations below the inhibitory levels. Moreover, the observed membrane responses were similar to the ones previously associated with adaption to some membrane-active compounds. On the other hand, exposure of cells to any of the two herbicides, ART or MET, did not induce any significant changes in fatty acid profiles. However, when combined with a commonly used fertilizer compound, NH[down]4NO[down]3, growth impairment was observed. Synergisitc effect of the two herbicides with NH[down]4NO[down]3, might be a consequence of changes in fatty acid profile increasing membrane fluidity, likely induced by NH[down]4+ ions

Methylmercury inhibits growth and induces membrane changes in Pseudomonas putida

A bacterial model system (Pseudomonas putida DSM 50026) was used in this research to assess toxicity of the environmentally relevant concentrations of mercury species (MeHg and Hg(II)) that represent important pollutants of aquatic ecosystems at sites of industrial or mining activities. In addition to direct monitoring of bacterial growth, we also analyzed fatty acid profiles of exposed and non-exposed cultures to determine possible toxic effects manifested on membrane level. The results showed that exposure of P. putida to Hg(II) in concentrations of 0.2-200 g/L did not have any significant effects on growth nor fatty acid composition of exposed bacterial culture. On the other hand, when bacteria were exposed to up to 1600-times lower concentrations of MeHg (0.12-12.5 g/L), growth inhibition as well as significant changes in fatty acid composition were detected. Observed adaptive membrane changes due to MeHg exposure were similar to those associated with responses to organic solvents and some other membrane-disrupting compounds.V raziskavi smo na bakterijskem modelu (Pseudomonas putida DSM 50026) analizirali strupenost okoljskih koncentracij anorganske (Hg(II)) in organske (MeHg) oblike živega srebra, ki predstavljata pomembna vira onesnaenja vodnih ekosistemov v bliini nekaterih industrijskih in rudarskih območij. Poleg neposrednega spremljanja bakterijske rasti smo analizirali tudi maščobnokislinske profile izpostavljenih bakterijskih kultur in jih primerjali s tistimi, ki živosrebrovima spojinama niso bili izpostavljeni. Rezultati so pokazali, da izpostavitev P. putida Hg(II) v koncentracijah med 0,2 in 200 g/L ne inhibira rasti, niti ne vpliva na maščobnokislinsko sestavo bakterijskih membran. Nasprotno pa je izpostavitev celic do 1600-krat nižjim koncentracijam MeHg povzročila tako upočasnitev rasti kot tudi prilagoditvene spremembe na membranskem nivoju. Slednje so bile podobne kot tiste, opaene ob izpostavitvi bakterij organskim topilom in nekaterim drugim spojinam, ki motijo integriteto membran

Membrane changes associated with exposure of Pseudomonas putida to selected environmental pollutants and their possible roles in toxicity

Abstract A bacterial model system (Pseudomonas putida DSM 50026) was used in this research to assess potential effect of five selected chemically diverse environmental pollutants on cell membranes. Long chain fatty acid profiles of cultures exposed to environmentally relevant concentrations of atrazine (ATR), metolachlor (MET), pentachlorobiphenyl (PCB), hexachlorobenzene (HCB) and fluoranthene (FL), were analyzed and compared to non-exposed cultures. To assess sensitivity of membrane-based responses, the impact of each toxicant on culture growth was also followed spectrophotometrically. Results revealed changes in fatty acid profiles when cells were exposed to PCB, HCB and FL in concentrations below the inhibitory levels. Moreover, the observed membrane responses were similar to the ones previously associated with adaptation to some membrane-active compounds. On the other hand, exposure of cells to any of the two herbicides, ATR or MET, did not induce any significant changes in fatty acid profiles. However, when combined with a commonly used fertilizer compound, NH 4 NO 3 growth impairment was observed. Synergistic effect of the two herbicides with NH 4 NO 3 might be a consequence of changes in fatty acid profile increasing membrane fluidity, likely induced by NH 4 + ions