Name: Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency (Video) | JoVE Journal
Uploaded: 2016-09-11T14:00:00.000+00:00
Duration: 9 min 49 s
Description: Learn analytical chemistry and ecotoxicology methods to assess wastewater treatment efficacy for removing estrogenic contaminants. This battery of tests evaluates emerging technologies to protect aquatic ecosystems.

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5 minutes

Projects presented in this paper were funded by Severn Trent Water and Brunel University London. The authors would like to thank Alan Henshaw and John Churchley for providing field and laboratory assistance. T.J.C. thanks the Heinz Endowments for support. M.R.M. thanks the Steinbrenner Institute for a Steinbrenner Doctoral Fellowship and Carnegie Mellon University for a Presidential Fellowship.

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<li>Jobling, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Predicted+exposures+to+steroid+estrogens+in+U.K.+rivers+correlate+with+widespread+sexual+disruption+in+wild+fish+populations%5BTitle%5D)+AND+%22Environ.+Health+Perspect%22%5BJournal%5D)">Predicted exposures to steroid estrogens in U.K. rivers correlate with widespread sexual disruption in wild fish populations.</a> Environ. Health Perspect. 114, Suppl 1. 32-39 (2006).</li>
<li>Baynes, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Additional+treatment+of+wastewater+reduces+endocrine+disruption+in+wild+fish+-+a+comparative+study+of+tertiary+and+advanced+treatments%5BTitle%5D)+AND+%22Environ.+Sci.+Technol%22%5BJournal%5D)">Additional treatment of wastewater reduces endocrine disruption in wild fish - a comparative study of tertiary and advanced treatments.</a> Environ. Sci. Technol. 46 (10), 5565-5573 (2012).</li>
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<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22The+determination+of+steroid+oestrogens+in+waters+using+chromatography+and+mass+spectrometry+%282008%29+Methods+for+the+Examination+of+Waters+and+Associated+Materials%22">The determination of steroid oestrogens in waters using chromatography and mass spectrometry (2008) Methods for the Examination of Waters and Associated Materials</a>. (2008), Environment Agency. Bristol, UK. (2008).</li>
<li>Van den Belt, K., Berckmans, P., Vangenechten, C., Verheyen, R., Witters, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparative+study+on+the+in+vitro%2Fin+vivo+estrogenic+potencies+of+17beta-estradiol%2C+estrone%2C+17alpha-ethynylestradiol+and+of+17beta-estradiol%2C+estrone%2C+17alpha-ethynylestradiol+and+nonylphenol%5BTitle%5D)+AND+%22Aquat.+toxicol%22%5BJournal%5D)">Comparative study on the in vitro/in vivo estrogenic potencies of 17beta-estradiol, estrone, 17alpha-ethynylestradiol and of 17beta-estradiol, estrone, 17alpha-ethynylestradiol and nonylphenol.</a> Aquat. toxicol. 66 (2), 183-195 (2004).</li>
<li>Mills, M. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Removal+of+ecotoxicity+of+17%CE%B1-ethinylestradiol+using+TAML%2Fperoxide+water+treatment%5BTitle%5D)+AND+%22Sci.+Rep%22%5BJournal%5D)">Removal of ecotoxicity of 17α-ethinylestradiol using TAML/peroxide water treatment.</a> Sci. Rep. 5, 10511(2015).</li>
<li><a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22EN+14962%3A2006+Water+quality+-+Guidance+on+the+scope+and+selection+of+fish+sampling+methods%22">EN 14962:2006 Water quality - Guidance on the scope and selection of fish sampling methods</a>. , British Standards Institute. London, UK. (2006).</li>
<li>Beresford, N., Brian, J. V., Runnalls, T. J., Sumpter, J. P., Jobling, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estrogenic+activity+of+tropical+fish+food+can+alter+baseline+vitellogenin+concentrations+in+male+fathead+minnow+%28Pimephales+promelas%29%5BTitle%5D)+AND+%22Environ.+Toxicol.+Chem%22%5BJournal%5D)">Estrogenic activity of tropical fish food can alter baseline vitellogenin concentrations in male fathead minnow (Pimephales promelas).</a> Environ. Toxicol. Chem. 30 (5), 1139-1145 (2011).</li>
<li>Nolan, M., Jobling, S., Brighty, G., Sumpter, J. P., Tyler, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+histological+description+of+intersexuality+in+the+roach%5BTitle%5D)+AND+%22J.+Fish+Biol%22%5BJournal%5D)">A histological description of intersexuality in the roach.</a> J. Fish Biol. 58 (1), 160-176 (2001).</li>
<li>Dascenzo, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fate+of+natural+estrogen+conjugates+in+municipal+sewage+transport+and+treatment+facilities%5BTitle%5D)+AND+%22Sci.+Total+Environ%22%5BJournal%5D)">Fate of natural estrogen conjugates in municipal sewage transport and treatment facilities.</a> Sci. Total Environ. 302 (1-3), 199-209 (2003).</li>
<li>Gomes, R. L., Birkett, J. W., Scrimshaw, M. D., Lester, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Simultaneous+determination+of+natural+and+synthetic+steroid+estrogens+and+their+conjugates+in+aqueous+matrices+by+liquid+chromatography%2Fmass+spectrometry%5BTitle%5D)+AND+%22Int.+J.+Environ.+Anal.+Chem%22%5BJournal%5D)">Simultaneous determination of natural and synthetic steroid estrogens and their conjugates in aqueous matrices by liquid chromatography/mass spectrometry.</a> Int. J. Environ. Anal. Chem. 85 (1), 1-14 (2005).</li>
<li>Metcalfe, C. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Estrogenic+potency+of+chemicals+detected+in+sewage+treatment+plant+effluents+as+determined+by+in+vivo+assays+with+Japanese+medaka+%28Oryzias+latipes%29%5BTitle%5D)+AND+%22Environ.+Toxicol.+Chem%22%5BJournal%5D)">Estrogenic potency of chemicals detected in sewage treatment plant effluents as determined by in vivo assays with Japanese medaka (Oryzias latipes).</a> Environ. Toxicol. Chem. 20 (2), 297-308 (2001).</li>
<li>Stalter, D., Magdeburg, A., Wagner, M., Oehlmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ozonation+and+activated+carbon+treatment+of+sewage+effluents%3A+Removal+of+endocrine+activity+and+cytotoxicity%5BTitle%5D)+AND+%22Water+Res%22%5BJournal%5D)">Ozonation and activated carbon treatment of sewage effluents: Removal of endocrine activity and cytotoxicity.</a> Water Res. 45 (3), 1015-1024 (2011).</li>
<li>Caldwell, D. J., Mastrocco, F., Anderson, P. D., Länge, R., Sumpter, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Predicted-no-effect+concentrations+for+the+steroid+estrogens+estrone%2C+17%CE%B2-estradiol%2C+estriol%2C+and+17%CE%B1-ethinylestradiol%5BTitle%5D)+AND+%22Environ.+Toxicol.+Chem%22%5BJournal%5D)">Predicted-no-effect concentrations for the steroid estrogens estrone, 17β-estradiol, estriol, and 17α-ethinylestradiol.</a> Environ. Toxicol. Chem. 31 (6), 1396-1406 (2012).</li>
<li>Gardner, M., Comber, S., Scrimshaw, M. D., Cartmell, E., Lester, J., Ellor, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+significance+of+hazardous+chemicals+in+wastewater+treatment+works+effluents%5BTitle%5D)+AND+%22Sci.+Total+Environ%22%5BJournal%5D)">The significance of hazardous chemicals in wastewater treatment works effluents.</a> Sci. Total Environ. 437, 363-372 (2012).</li>
<li>Filby, A. L., Shears, J. A., Drage, B. E., Churchley, J. H., Tyler, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+advanced+treatments+of+wastewater+effluents+on+estrogenic+and+reproductive+health+impacts+in+fish%5BTitle%5D)+AND+%22Environ.+Sci.+Technol%22%5BJournal%5D)">Effects of advanced treatments of wastewater effluents on estrogenic and reproductive health impacts in fish.</a> Environ. Sci. Technol. 44 (11), 4348-4354 (2010).</li>
<li>Magdeburg, A., Stalter, D., Oehlmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Whole+effluent+toxicity+assessment+at+a+wastewater+treatment+plant+upgraded+with+a+full-scale+post-ozonation+using+aquatic+key+species%5BTitle%5D)+AND+%22Chemosphere%22%5BJournal%5D)">Whole effluent toxicity assessment at a wastewater treatment plant upgraded with a full-scale post-ozonation using aquatic key species.</a> Chemosphere. 88 (8), 1008-1014 (2012).</li>
<li>Collins, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((TAML+oxidant+activators%3A+a+new+approach+to+the+activation+of+hydrogen+peroxide+for+environmentally+significant+problems%5BTitle%5D)+AND+%22Acc.+Chem.+Res%22%5BJournal%5D)">TAML oxidant activators: a new approach to the activation of hydrogen peroxide for environmentally significant problems.</a> Acc. Chem. Res. 35 (9), 782-790 (2002).</li>
<li>Collins, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Designing+Ligands+for+Oxidizing+Complexes%5BTitle%5D)+AND+%22Acc.+Chem.+Res%22%5BJournal%5D)">Designing Ligands for Oxidizing Complexes.</a> Acc. Chem. Res. 27 (9), 279-285 (1994).</li>
<li>Truong, L., Denardo, M. A., Kundu, S., Collins, T. J., Tanguay, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish+Assays+as+Developmental+Toxicity+Indicators+in+The+Design+of+TAML+Oxidation+Catalysts%5BTitle%5D)+AND+%22Green+Chem%22%5BJournal%5D)">Zebrafish Assays as Developmental Toxicity Indicators in The Design of TAML Oxidation Catalysts.</a> Green Chem. 15 (9), 2339-2343 (2013).</li>
<li>Murk, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+estrogenic+potency+in+wastewater+and+surface+water+with+three+in+vitro+bioassays%5BTitle%5D)+AND+%22Environ.+Toxicol.+Chem%22%5BJournal%5D)">Detection of estrogenic potency in wastewater and surface water with three in vitro bioassays.</a> Environ. Toxicol. Chem. 21 (1), 16-23 (2002).</li>
<li>Test No 455: The Stably Transfected Human Estrogen Receptor-alpha Transcriptional Activation Assay for Detection of Estrogenic Agonist-Activity of Chemicals. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22OECD+Guidelines+for+the+Testing+of+Chemicals%2C+Section+4%22">OECD Guidelines for the Testing of Chemicals, Section 4</a>. , OECD Publishing. Paris. (2009).</li>
<li>Kolle, S. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+house+validation+of+recombinant+yeast+estrogen+and+androgen+receptor+agonist+and+antagonist+screening+assays%5BTitle%5D)+AND+%22Toxicol+in+Vitro%22%5BJournal%5D)">In house validation of recombinant yeast estrogen and androgen receptor agonist and antagonist screening assays.</a> Toxicol in Vitro. 24 (7), 2030-2040 (2010).</li>
<li>Lee, O., Tyler, C. R., Kudoh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+transient+expression+assay+for+detecting+environmental+oestrogens+in+zebrafish+and+medaka+embryos%5BTitle%5D)+AND+%22BMC+Biotechnology%22%5BJournal%5D)">Development of a transient expression assay for detecting environmental oestrogens in zebrafish and medaka embryos.</a> BMC Biotechnology. 12, 32(2012).</li>
<li>Brian, J. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Accurate+prediction+of+the+response+of+freshwater+fish+to+a+mixture+of+estrogenic+chemicals%5BTitle%5D)+AND+%22Environ+Health+Perspect%22%5BJournal%5D)">Accurate prediction of the response of freshwater fish to a mixture of estrogenic chemicals.</a> Environ Health Perspect. 113 (6), 721-728 (2005).</li>
<li>Test No 229: Fish Short Term Reproduction Assay. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22OECD+Guidelines+for+the+Testing+of+Chemicals%2C+Section+2%22">OECD Guidelines for the Testing of Chemicals, Section 2</a>. , OECD Publishing. Paris. (2009).</li>
<li>Test No 230: 21-day Fish Assay: A Short-Term Screening for Oestrogenic and Androgenic Activity and Aromatase Inhibition. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=%22OECD+Guidelines+for+the+Testing+of+Chemicals%2C+Section+2%22">OECD Guidelines for the Testing of Chemicals, Section 2</a>. , OECD Publishing. Paris. (2009).</li>
</ol>

The authors have nothing to disclose.

Wastewater treatment plants are the major route of surface water contamination with EDC. An evaluation of the efficacy of removal of endocrine activity of conventional, advanced or emerging treatment processes requires the use of a variety of chemical and biological assays. Chemical analysis using non-targeted and targeted analysis provides qualitative or quantitative data on the efficacy of removal of individual components and therefore allows an assessment to be made against environmental quality standards or predicted no effect concentrations for the compounds or mixtures of compounds analyzed.
The generation of transformation products resulting from incomplete mineralization of substances following treatment and the presence of unknown biologically active components in wastewater limits the usefulness of chemical testing alone. A combination of in vivo and in vitro bioassays in combination with analytical chemistry screening provides a useful toolbox to determine the efficacy of EDC removal by emerging wastewater treatment processes. These tests, when conducted alongside traditional water quality parameters and other toxicological and microbiological end-points allow a critical evaluation of current and emerging wastewater treatment technologies.
It is important to note that Yeast based estrogen screens (e.g., YES) are not the only in vitro assays to determine estrogenic potency of chemicals and wastewaters. A number of stably transfected mammalian cell based assays have been developed too, for example, the ER-CALUX27 and hER&#945;-HeLa-990328 with human breast cancer cells or cervical tumor cells respectively. The YES has been compared to similar mammalian cell based assays and has been found to have a comparable high level of reproducibility, true positive and true negative estrogenic identification rates29, although it is sometimes considered to be slightly less sensitive27. One benefit of Yeast based reporter assays is that in labs without significant experience with mammalian cell culture the YES can be more easily adopted, as it requires less stringent bio-control measures and sterile techniques (YES can be performed on the bench top if necessary). The human cell based assays also require CO2 incubators and luminometers compared to the standard incubator and microplate readers used in the YES. Two yeast based estrogen reporter assays (YES, Saccharomyces cerevisiae and A-YES, Arxula adeninivorans) are currently undergoing inter-laboratory trails for the validation of ISO 19040 &#34;Water quality - Determination of the estrogenic potential of water and waste water&#34; highlighting the industries interest in these techniques.
There are a number of limitations of the methods described which include the potential contamination of samples during sampling, sample storage and analysis with estrogenic substances originating from the field or laboratory environment or by human contamination (e.g., plasticizers, surfactants, personal care products). This type of contamination in the YES assay (or other cell based reporter assays) will elevate the background and impact the use of the assay. Water samples or solvents stored in plastic bottles can easily cause false positives. False negatives are also of concern as both LCMS/MS and the YES assay require SPE to concentrate estrogens to detectable levels. The matrix, the choice of SPE sorbent and elution solvent can affect the extraction efficiency and the types of compounds eluted. Using C18 SPE cartridges for extraction using the conditions described in this protocol may generate a negative bias, as highly polar and basic compounds would be poorly retained by the sorbent. Furthermore, this protocol requires reconstitution of the eluted YES eluent from methanol to ethanol via evaporation to dryness funder nitrogen resulting in the loss of volatile compounds. As a result the protocol could provide underestimated estrogenic activity of tested samples. These limitations are especially important when considering the YES assay as unknown or unexpected compounds might be missed, because they have not been extracted or they are lost due to evaporation. Furthermore, the LCMS/MS technique makes use of labeled internal standards to correct for recovery; this approach cannot be used with the YES assay.
Significant limitations of in vivo testing of effluents include high cost and time required for assessment compared to in vitro methods. Currently the use of fish embryo tests to detect estrogenic activity is limited. However, there has been some success with producing estrogen responsive transgenic glowing fish embryos30, which could have future applications. Fathead minnows (used in this protocol) are a common laboratory species and VTG induction in male fish is a well-documented bio-marker of estrogenic exposure and a quantifiable measure of wastewater effluent estrogenicity22 or other estrogenic compounds or mixtures31. OECD test guidelines for endocrine disrupting chemicals have been validated using adult fathead minnow, Japanese medaka and zebrafish32,33, with VTG being a sensitive biomarker of estrogen exposure in all three species. However, VTG induction does not directly correlate to reproductive impairment and therefore the ecological consequences of wastewater exposure, as seen in severely intersex roach3. On the other hand, roach are not a classic &#39;laboratory species&#39; for ecotoxicology research due to their large size, long generation time (2-3 years to reach sexual maturity), reproductive style; group spawning (breeding) takes place once a year, and the difficulty to identify males from females (other than during the spawning season). However, this normally gonochoristic species has been very well studied in the UK, due to the discovery that downstream of estrogenic wastewater effluents, male fish exhibited perturbations to their endocrinology (e.g., presence of female-specific vitellogenin in their blood) and histopathology (ovotestes - developing eggs in the testis and/or female reproductive ducts)5,6. Therefore, as a future application of these protocols, roach (or similar species) could be a useful wild sentinel species to show if real improvements to wastewater quality (and reduced estrogenicity) are seen in rivers receiving advanced treated effluents. They can also be employed in end of pipe systems to monitor technologically improved effluents from pilot scale plants7. When considering which species to use in in vivo wastewater assessments there is a tradeoff between relatively quick and controlled testing using laboratory species compared to the longer field based, but more environmentally relevant, testing using native species. However, such in vivo assessments are high cost and should only be considered as the final set of tests following assessments using chemical analysis and in vitro assays.
Critical steps within the protocols described include the preparation and handling of samples and glassware (i.e., bottles and sampling equipment should be pre-treated with suitable surface active cleaning agent) to avoid contamination of samples from environmental contaminants including limiting contact of samples with plastics and other materials that can produce false positives. This is equally important when designing and building aquaria and fish exposure systems. Ideally aquaria (housing stocks and during exposures) should be built from materials with low adsorption32 with minimal contamination risk. Stainless steel can be used for effluent or water holding tanks. Whereas tanks of a glass construction are preferred for fish tanks (as this also provides easy observation of the fish). The use of low grade plastic pipes or tubing should be avoided32, PVC34 and ABS can be used if &#39;properly seasoned&#39;, i.e., left to leach out any contaminants in running dilution water for at least 12 hr prior to use. Medical grade silicon tubing has been employed successfully in our facility for peristaltic pump delivery of chemicals and wastewater/dilution water to tanks. As well as considering estrogenic contamination in construction and running of the aquatics system, it is also important to think about the diet of the fish; many propriety fish foods have been found to be estrogenic to fish. Therefore it is important to test any foods for activity (e.g., in the Yeast Estrogen Screen, See Beresford et al.14) prior to using them in these types of studies.
Troubleshooting of the chemical analysis or YES assay protocols described is simplified if quality assurance samples including multiple travel, laboratory and solvent blanks are analyzed alongside positive controls and real samples to eliminate false positive and false negative results. Positive (e.g., EE2) and negative (dilution water only) control should also always be used in the in vivo assays to confirm sensitivity of expected biological biomarker or endpoint (i.e., VTG or histopathology), and allow any unexpected contamination to be detected (e.g., from experimental set up, diet, or dilution waters). Any modifications in the protocol should be validated prior to conducting any study.
With stricter regulation of estrogenic compounds entering the environment via WWTP effluents it is envisage that more effective wastewater treatment technologies will need to be developed. The battery of tests described in this manuscript compliment the ecotoxicological and chemical evaluation tests normally applied to wastewater treatment plant effluent discharges. Therefore, future application of this type of holistic battery of test should enable wastewater technology developers, and plant operators, to implement the most ecologically safe designs considering the best methods to remove both specific regulated estrogenic chemicals and overall biological activity.

Concerns regarding the adverse effects of endocrine disrupting compounds on wildlife reproductive health has led the European Union to place two estrogenic substances (estradiol and ethinylestradiol) on a &#34;watch list&#34; under the Water Framework Directive (WFD). EDC encompass a variety of chemical classes, including natural and synthetic steroid estrogens, drugs, pesticides, and industrial chemicals and constituents of consumer products with known adverse effects on wildlife. Some of these compounds may potentially impact human health1.
Research has shown that effluents from WWTP are estrogenic to fish2 and as a consequence many receiving waters are also estrogenic to fish3. This was first demonstrated through national surveys in the United Kingdom that showed increased vitellogenin concentrations (a female specific yolk protein precursor4) in the blood of wild male fish and a high prevalence of intersex (developing eggs and/or female reproductive ducts in the testis of male fish) in normally gonochoristic fish species5,6.
Conventional sewage treatment is typically a three stage process consisting of a preliminary screening followed by primary and secondary treatment which removes both dissolved and suspended organic matter. The efficacy of removal of individual EDC is dependent on the physicochemical properties of the substances and on the effectiveness of the treatment process applied. For many EDC removal via adsorption and biological degradation can be significant but incomplete. Tertiary treatment, such as sand filtration, can be effective at increasing EDC removal7 whereas advanced treatment using advanced oxidation (for example ozone) or activated carbon can be effective in achieving near complete removal7.
The assessment of any new technology for wastewater treatment needs to determine the efficacy of the proposed process in EDC removal. A battery of tests, including targeted chemical analysis alongside ecotoxicology testing, using in vivo and in vitro bioassays, provides comprehensive data for this purpose. Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Bioassays additionally allow the &#34;detection&#34; of adverse effects of metabolites and treatment-generated wastewater transformation by-products that would otherwise be undetected8,9. This paper describes the use of a battery of chemical and ecotoxicity laboratory assays to assess the efficacy of a number of advanced and emerging wastewater treatment processes in removing the estrogenic potency of crude and treated sewage and receiving waters.

<table><tbody><tr><td>Wellwash Versa plate washer</td><td>Thermo Scientific</td><td>5165010</td><td></td></tr><tr><td>Plate reader</td><td>Molecular Devices</td><td>SpectraMax 340PC</td><td></td></tr><tr><td>Incubator</td><td>Memmert</td><td>INB 400</td><td>37 &amp;#176;C incubation required for carp assay</td></tr><tr><td>Fisherbrand whirlimixer</td><td>Fisher Scientific</td><td>13214789</td><td></td></tr><tr><td>Icemaker</td><td>Scotsman</td><td>AF80</td><td></td></tr><tr><td>12-Channel F1 digital multichannel pipette</td><td>Thermo Scientific Finnpipette</td><td>4661070</td><td></td></tr><tr><td>ELISA kits</td><td>Biosense Laboratories</td><td>V01018401-096 (Fathead minnow)&lt;br/&gt; V01003402-096 (Carp)</td><td></td></tr><tr><td>Microfuge tubes, 0.5 ml</td><td>Alpha labs</td><td>LW2372</td><td></td></tr><tr><td>Microfuge tubes, 1.5 ml</td><td>Alpha labs</td><td>LW2375</td><td></td></tr><tr><td>Sulphuric acid, 95-98%</td><td>Sigma-Aldrich</td><td>258105</td><td></td></tr><tr><td>&lt;strong&gt;Histology&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Tissue processor</td><td>Leica Biosystems</td><td>TP1020</td><td></td></tr><tr><td>Wax dispenser</td><td>Thermo Scientific Raymond Lamb</td><td>E66HC</td><td></td></tr><tr><td>Metal embedding mold</td><td>Leica Biosystems</td><td>Various</td><td></td></tr><tr><td>Hot plate</td><td>Thermo Scientific Shandon</td><td>3120063</td><td></td></tr><tr><td>Cold plate (EG1150 C)</td><td>Leica Biosystems</td><td>14038838037</td><td></td></tr><tr><td>Heated forceps (EG F)</td><td>Leica Biosystems</td><td>14038835824</td><td></td></tr><tr><td>Microtome</td><td>Leica Biosystems</td><td>RM2235</td><td></td></tr><tr><td>Paraffin section floatation&amp;nbsp; bath</td><td>Electrothermal</td><td>MH8517</td><td></td></tr><tr><td>Slide drying bench</td><td>Electrothermal</td><td>MH6616</td><td></td></tr><tr><td>Stainmate automated stainer</td><td>Thermo Scientific Shandon</td><td>E103/S10L</td><td></td></tr><tr><td>Cassettes, Histosette II, biopsy</td><td>Simport</td><td>M493</td><td></td></tr><tr><td>Paraffin wax</td><td>Thermo Scientific Raymond Lamb</td><td>W1</td><td></td></tr><tr><td>Histo-Clear II</td><td>National Diagnostics</td><td>HS-202</td><td></td></tr><tr><td>IMS (ethanol mix), IDA99</td><td>Tennants</td><td>ID440</td><td></td></tr><tr><td>Polysine adhesion slides</td><td>Thermo Scientific Gerhard Menzel</td><td>J2800AMNZ</td><td></td></tr><tr><td>Cover slips, 22x50 mm</td><td>VWR</td><td>631-0137</td><td></td></tr><tr><td>Histomount</td><td>National Diagnostics</td><td>HS-103</td><td></td></tr><tr><td>Haematoxylin Harris GURR</td><td>VWR</td><td>351945S</td><td></td></tr><tr><td>Eosin, 1%, aqueous</td><td>Pyramid Inovation</td><td>S20007-E</td><td></td></tr><tr><td>Fisherbrand slide boxes</td><td>Fisher Scientific</td><td>11701486</td><td></td></tr><tr><td>Microtome blades, MB35</td><td>Thermo Scientific Shandon</td><td>3050835</td><td></td></tr><tr><td>Bouin&amp;rsquo;s solution</td><td>Sigma Aldrich</td><td>HT10132-1L</td><td></td></tr><tr><td>&lt;strong&gt;Yeast screen&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Flow cabinet</td><td>Labcaire Systems Ltd</td><td>SC12R</td><td></td></tr><tr><td>Cooled incubator</td><td>LMS Cooled Incubator</td><td>303</td><td></td></tr><tr><td>Incubator</td><td>Memmert</td><td>INB 400</td><td></td></tr><tr><td>Shaker</td><td>Grant</td><td>PSU-10i</td><td></td></tr><tr><td>Fisherbrand whirlimixer</td><td>Fisher Scientific</td><td>13214789</td><td></td></tr><tr><td>Plate shaker</td><td>Heidolph Titramax 100</td><td>544-11200-00</td><td></td></tr><tr><td>12-Channel F1 digital multichannel pipette</td><td>Thermo Scientific Finnpipette</td><td>4661070</td><td></td></tr><tr><td>12-channel pipette, electronic</td><td>Sartorius</td><td>735441</td><td></td></tr><tr><td>96-well flat-bottom microplates</td><td>MP Biomedicals&lt;br/&gt; Thermo Scientific Nunc&lt;br/&gt; Sarstedt</td><td>76-232-05&lt;br/&gt; 260860&lt;br/&gt; 82.1581.001</td><td>We have found that these multiwell plates all produce low backgrounds</td></tr><tr><td>HPLC grade water</td><td>Rathburn</td><td>RH1020</td><td></td></tr><tr><td>Absolute ethanol</td><td>Hayman Kimia</td><td>F200238</td><td></td></tr><tr><td>Potassium phosphate monobasic anhydrous</td><td>Sigma-Aldrich</td><td>P-5655</td><td></td></tr><tr><td>Ammonium sulphate</td><td>Sigma-Aldrich</td><td>A-2939</td><td></td></tr><tr><td>Potassium hydroxide, pellets</td><td>Sigma-Aldrich</td><td>P-1767</td><td></td></tr><tr><td>Magnesium sulfate, anhydrous</td><td>Sigma-Aldrich</td><td>M-2643</td><td></td></tr><tr><td>Iron(III) sulfate</td><td>Sigma-Aldrich</td><td>307718</td><td></td></tr><tr><td>L-Leucine</td><td>Sigma-Aldrich</td><td>L-8912</td><td></td></tr><tr><td>L-Histidine</td><td>Sigma-Aldrich</td><td>H-6034</td><td></td></tr><tr><td>Adenine</td><td>Sigma-Aldrich</td><td>A-2786</td><td></td></tr><tr><td>L-Argenine, hydrochloride</td><td>Sigma-Aldrich</td><td>A-6969</td><td></td></tr><tr><td>L-Methionine</td><td>Sigma-Aldrich</td><td>M-5308</td><td></td></tr><tr><td>L-Tyrosine</td><td>Sigma-Aldrich</td><td>T-8566</td><td></td></tr><tr><td>L-Isoleucine</td><td>Sigma-Aldrich</td><td>I-7403</td><td></td></tr><tr><td>L-Lysine, hydrochloride</td><td>Sigma-Aldrich</td><td>L-8662</td><td></td></tr><tr><td>L-Phenylalanine</td><td>Sigma-Aldrich</td><td>P-5482</td><td></td></tr><tr><td>L-Glutamic acid</td><td>Sigma-Aldrich</td><td>G-8415</td><td></td></tr><tr><td>L-Valine</td><td>Sigma-Aldrich</td><td>V-0513</td><td></td></tr><tr><td>L-Serine</td><td>Sigma-Aldrich</td><td>S-4311</td><td></td></tr><tr><td>Thiamine, hydrochloride</td><td>Sigma-Aldrich</td><td>T-1270</td><td></td></tr><tr><td>Pyridoxine</td><td>Sigma-Aldrich</td><td>P-5669</td><td></td></tr><tr><td>D-Pantothenic acid, hemicalcium salt</td><td>Sigma-Aldrich</td><td>P-5155</td><td></td></tr><tr><td>Inositol</td><td>Sigma-Aldrich</td><td>I-5125</td><td></td></tr><tr><td>D-Biotin</td><td>Sigma-Aldrich</td><td>B-4639</td><td></td></tr><tr><td>D-(+)-Glucose anhydrous; mixed anomers</td><td>Sigma-Aldrich</td><td>G-7021</td><td></td></tr><tr><td>L-Aspartic acid</td><td>Sigma-Aldrich</td><td>A-4534</td><td></td></tr><tr><td>L-Threonine</td><td>Sigma-Aldrich</td><td>T-8441</td><td></td></tr><tr><td>Copper(II) sulfate, anhydrous</td><td>Sigma-Aldrich</td><td>C-1297</td><td></td></tr><tr><td>Chlorophenolred-&amp;#946;-D galactopyranoside (CPRG)</td><td>Sigma-Aldrich</td><td>10884308001</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G-2025</td><td></td></tr><tr><td>17 &amp;beta;-Estradiol</td><td>Sigma-Aldrich</td><td>E-8875</td><td></td></tr><tr><td>&lt;strong&gt;Steroids&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Acetone</td><td>Rathburn</td><td></td><td></td></tr><tr><td>Acetonitrile</td><td>Rathburn</td><td></td><td></td></tr><tr><td>Ammonia solution</td><td>Rathburn</td><td></td><td></td></tr><tr><td>Ethylacetate</td><td>Rathburn</td><td></td><td></td></tr><tr><td>Copper(II) nitrate</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Acetone</td><td>Rathburn</td><td></td><td></td></tr><tr><td>Dichloromethane</td><td>Rathburn</td><td></td><td></td></tr><tr><td>2,4,16,16-d&lt;sub&gt;4&lt;/sub&gt;-17&amp;#946;-estradiol</td><td>CDN Isotopes</td><td></td><td></td></tr><tr><td>2,4,16,16-d&lt;sub&gt;4&lt;/sub&gt;-estrone</td><td>CDN Isotopes</td><td></td><td></td></tr><tr><td>2,4,16,16-d&lt;sub&gt;4&lt;/sub&gt;-17&amp;#945;-ethynyl oestradiol</td><td>CDN Isotopes</td><td></td><td></td></tr><tr><td>17&amp;#946;-estradiol</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Estrone</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>17&amp;#945;-ethynyl oestradiol</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Hexane</td><td>Rathburn</td><td></td><td></td></tr><tr><td>Hydrochloric acid</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Sodium hydrogen carbonate</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>Styrene divinyl benzene cartridge (Isolute ENV+) solid phase extraction cartridge (200 mg/6 ml)</td><td>Biotage</td><td></td><td></td></tr><tr><td>Isolute aminopropyl solid phase extraction cartridge (500 mg/6 ml)</td><td>Biotage</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Fish study&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>orange-white silicon manifold tubing 0.63 bore pk 6</td><td>Watson Marlow</td><td>982.0063.000</td><td></td></tr><tr><td>straight connectors for 0.5/0.8 bore pk 20</td><td>Watson Marlow</td><td>999.2008.000</td><td></td></tr><tr><td>pumsil silicon tubing 0.8 bore 15 m</td><td>Watson Marlow</td><td>913.A008.016</td><td></td></tr><tr><td>200 series multi-channel persitaltic pump</td><td>Watson Marlow</td><td>205CA</td><td></td></tr><tr><td>Silicone tubing x 15 m (dosing tanks)</td><td>VWR</td><td>SFM1-3250</td><td></td></tr><tr><td>silicone tubing x 15 m (large for inflow/outflow)</td><td>VWR</td><td>SFM1-5450</td><td></td></tr><tr><td>2.5 L glass winchester pk 4</td><td>Fisher Scienctific</td><td>BTF-505-050B</td><td></td></tr><tr><td>magnetic stir bar 51 x 8 mm pk 10</td><td>Fisher Scienctific</td><td>FB55595&amp;nbsp;</td><td></td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfonate (MS222)</td><td>Sigma Aldrich</td><td>E10521-10G</td><td></td></tr><tr><td>17&amp;alpha;-Ethynylestradiol</td><td>Sigma Aldrich</td><td>E4876-100MG</td><td></td></tr><tr><td>Absolute ethanol</td><td>Hayman Kimia</td><td>F200238</td><td></td></tr><tr><td>&lt;strong&gt;SPE&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>1/8 inch PTFE tubes &#039;straws&#039; colour coded pk 4</td><td>Sigma Aldrich</td><td>57276</td><td></td></tr><tr><td>disposable liners for manifold</td><td>Sigma Aldrich</td><td>57059</td><td></td></tr><tr><td>filtration tubes without frits 6 ml pk 30</td><td>Sigma Aldrich</td><td>57242</td><td></td></tr><tr><td>reservior adaptors pk 12</td><td>Sigma Aldrich</td><td>57020-U</td><td></td></tr><tr><td>stainless steel weight for manifold pk 4</td><td>Sigma Aldrich</td><td>57278</td><td></td></tr><tr><td>male Luer plug for manifold pk 12</td><td>Sigma Aldrich</td><td>504351</td><td></td></tr><tr><td>SPE Vacuum Manifold</td><td>Sigma Aldrich</td><td>57265</td><td></td></tr><tr><td>stop cocks for extraction mainfold (supelco) pk 12</td><td>Waters</td><td>WAT054806</td><td></td></tr><tr><td>Sep-Pak Plus C18 cartridge box 50&amp;nbsp;</td><td>Waters</td><td>WAT020515</td><td></td></tr><tr><td>Methanol HPLC grade 2.5 L</td><td>Fisher Scientific</td><td>M/4056/17</td><td></td></tr><tr><td>7 ml glass vials with lids (58 x 17 mm) pk 399</td><td>Fisher Scientific</td><td>TUL-520-031K</td><td></td></tr><tr><td>Absolute ethanol</td><td>Hayman Kimia</td><td>F200238</td><td></td></tr><tr><td>vacuum pump, &lt;em&gt;e.g.&lt;/em&gt;, VP Series Vacuum Pump</td><td>Camlab</td><td>1136915</td><td></td></tr></tbody></table>

use of a battery of chemical and ecotoxicological methods for the assessment of the efficacy of wastewater treatment processes to remove estrogenic potency

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in a watch list of environmental pollutants under the European Water Framework Directive. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. Much of the estrogenic potency of wastewater treatment plant (WWTP) effluents can be attributed to the discharge of steroid estrogens including estradiol (E2), EE2 and E1 due to incomplete removal of these substances at the treatment plant. An evaluation of the efficacy of wastewater treatment processes requires the quantitative determination of individual substances most often undertaken using chemical analysis methods. Most frequently used methods include Gas Chromatography-Mass Spectrometry (GCMS/MS) or Liquid Chromatography-Mass Spectrometry (LCMS/MS) using multiple reaction monitoring (MRM). Although very useful for regulatory purposes, targeted chemical analysis can only provide data on the compounds (and specific metabolites) monitored. Ecotoxicology methods additionally ensure that any by-products produced or unknown estrogenic compounds present are also assessed via measurement of their biological activity. A number of in vitro bioassays including the Yeast Estrogen Screen (YES) are available to measure the estrogenic activity of wastewater samples. Chemical analysis in conjunction with in vivo and in vitro bioassays provides a useful toolbox for assessment of the efficacy and suitability of wastewater treatment processes with respect to estrogenic endocrine disrupting compounds. This paper utilizes a battery of chemical and ecotoxicology tests to assess conventional, advanced and emerging wastewater treatment processes in laboratory and field studies.

Attempts to understand the impact of improvements to wastewater treatment processes or to determine the most appropriate technology to retrofit equipment as tertiary treatment at existing WWTP with respect to the efficacy of removal of endocrine disrupting activity of discharged effluents, requires not only the measurement of key chemical components that enter the works but requires the analysis of the breakdown products which may also have endocrine disrupting activity. In domestic sewage effluents, the most estrogenic substances present are the steroid hormones, estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2)5,8. Steroid estrogens are primarily excreted from the body as a mixture of inactive conjugates16,17. These conjugated estrogens are substantially deconjugated in the sewerage system by bacterial activity and further degradation occurs in the WWTP. The deconjugated steroids are removed from the wastewater stream by adsorption to sludge or biodegraded during secondary treatment resulting in the formation, firstly of transformation byproducts and ultimately complete mineralization can occur of the initial active component. The chemical analysis of all individual compounds in the effluent stream would be difficult, time consuming and costly and would not cover unknown active components present in a sample. Furthermore, a sum of the estrogenic contribution of each component will only provide an indication of the cumulative estrogenic potency of a sample of the compounds analyzed. This is a risk where transformation processes generate unknown estrogenic substances or where the influent is of industrial origin. Combining chemical analysis with in vivo and in vitro ecotoxicology bioassays provides a solution to the presence of unknown estrogenic components in mixtures such as treated sewage. In vitro assays such as the Yeast Estrogen Screen (YES) have been used extensively to determine the estrogenic activity of sewage effluents and to help identify the active components in treated samples8,18,19. However, comparisons between in vivo and in vitro testing can be significant11 and a comprehensive assessment of new processes with respect to treatment of endocrine disrupting potency requires a battery of chemical and ecotoxicology tests.
A determination of whether individual treatment plants or processes remove active compounds from the wastewater stream can be achieved using chemical analysis which follows sample extraction, concentration and clean-up of the extract prior to analysis, most often undertaken using LCMS(/MS) or GCMS(/MS) methods. The data obtained from chemical analysis can be used to determine compliance with individual predicted no effect concentrations (PNEC)20 or environmental quality standards (EQS)21 of specific individual compounds and therefore such methods are vital for regulatory compliance data. Furthermore, targeted or non-targeted chemical analysis methods allow the identification and quantitation of individual compounds or isomers compared with biological methods, which provide a total response. Chemical analysis methods therefore allow the assessment of discrete compounds to be made to meet and to address these wastewater treatment challenges on an individual treatment plant basis. Studies have shown that conventional wastewater treatment (e.g., activated sludge plants) can be highly effective in the removal of natural steroid hormones although removal of the synthetic hormone EE2 tends to be less effective. Field studies using advanced treatment utilizing techniques such as ozone, granulated activated carbon (GAC) and membranes have demonstrated, albeit at high cost, that they can be used as an end-of-pipe solution to remove EE2 to below predicted effect levels and to below the detection limits. Figure 3 shows the removal of EE2 using GAC at a pilot scale municipal wastewater treatment plant. Studies undertaken at pilot scale at municipal wastewater treatment plants using end of pipe GAC treatment also show the reduction in estrogenic potency following GAC measured using the Yeast Estrogen Screen (YES) as seen in Figure 4.
<img alt="HPLC chromatograms; tripalmitin extraction; spectra(A-C); absorption, internal standard analysis." src="/files/ftp_upload/54243/54243fig3.jpg" data-altupdatedat="1788235205000" data-alt="Figure 3"> 
Figure 3. Example field data showing the removal of ethinylestradiol following advanced tertiary treatment. (A) Samples are collected from the WWTP following conventional (activated sludge plant) treatment following the procedures described for sample preservation. (B) Samples are extracted using solid phase extraction, cleaned-up to remove interfering substances using normal phase SPE and gel permeation chromatography. (C) The clean concentrated extract is concentrated to a low volume and analyzed using negative ion electrospray LCMS/MS in MRM mode. Results are calculated using internal standardization using isotopically labeled internal standards. In the example shown, EE2 is present in the final ASP effluent at a concentration above the predicted no effect level (PNEC) of 0.1 ng/L and is removed using GAC and ozone (O3) to an environmentally safe concentration. <a href="https://www.jove.com/files/ftp_upload/54243/54243fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Microplate assay absorbance analysis; graphs show estradiol, ethanol impact; 540nm absorbance data." src="/files/ftp_upload/54243/54243fig4.jpg" data-altupdatedat="1788235205000" data-alt="Figure 4"> 
Figure 4. Photo of a Yeast Estrogen Screen (YES) assay plate (A) showing color change from yellow to red, relating to estrogenic activity of the samples. Plots created from the YES assay plate showing corrected absorbance (540 nm) of the estradiol standard (B), activated sludge process effluent (ASP) and Granular activated carbon (GAC) treated wastewater effluent samples (C). Each sample was tested in duplicate. ASP and GAC effluents were extracted and concentrated using the SPE methods outlined in section 1. <a href="https://www.jove.com/files/ftp_upload/54243/54243fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Ozonation is also efficient in removing steroid estrogens and estrogenic activity from conventionally treated wastewater treatment plants. Ozone is able to oxidize a wide range of organic contaminants and dissolved organic matter in wastewater samples and provides disinfection properties. The effectiveness of ozonation depends on water characteristics such as pH, amount of organic matter and the applied dose of ozone. Estrogens that are poorly removed by conventional treatment can be removed from wastewater with doses between 0.8 and 2 mg O3/mg DOC. Ozone is a selective oxidizing agent, which reacts with electron rich sites (unsaturated carbon-carbon bonds, aromatic compounds including aromatic alcohols), which makes ozone applicable for the breakdown of a number of EDC. However, the elimination of individual compounds does not necessarily lead to the complete mineralization of the original compound. Organic substances following ozonation may be transformed generating intermediates or transformation oxidation by-products which include a number of low molecular weight, polar classes of compounds such as aldehydes, ketones, carboxylic acids, keto acids, and brominated compounds. Examples include, bromate, formaldehyde, acetaldehyde and carboxylic acids. Using in vivo and in vitro bioassays it has been shown that although ozone only partially oxidizes some chemical substances, the resulting major transformation products have a lower estrogenic potency and hence the application of ozone at an appropriate dose results in a high removal of estrogenic activity.
One of the major benefits of additional treatment of wastewaters is the reduction in the feminization of male fish in receiving waters; an adverse effect that can lead to reduced fertility3. In vivo studies using fish (e.g., roach or fathead minnow) exposed to wastewater show female germ cells or oocytes in the testis of male fish (e.g., as seen in Figure 5). Intersex or male VTG is absent or significantly reduced in fish following advanced treatment such as GAC7 or ozone22. These studies show that transformation products produced during ozonation are not estrogenic, however this does not address the toxicity of the effluent produced. This issue has been addressed in other studies, for example a study by Magdeburg et al.23 which shows that ozone oxidation by-products are toxic to rainbow trout but this toxicity can be removed by downstream sand filtration following ozonation.
<img alt="Histology of muscle tissue sections; microscopic images showing cellular structure and fiber arrangement." src="/files/ftp_upload/54243/54243fig5.jpg" data-altupdatedat="1788235205000" data-alt="Figure 5"> 
Figure 5. Photomicrographs of a normal male (A) and intersex (B, C) gonads from adult roach (Rutilus rutilus) exposed to wastewaters in a field based assessment. Photomicrograph-A, depicts a histological section of normal male testis. Photomicrograph-B and -C, depicts histological sections of an intersex male fish, having been exposed to activated sludge process wastewater effluent for six months. Arrows indicate oocytes present in the testicular tissue. Scale bar represents 100 µm in each photomicrograph. <a href="https://www.jove.com/files/ftp_upload/54243/54243fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The high cost of end of pipe treatment using ozone, GAC or membrane technology necessitates the development of alternative lower cost, sustainable methods for endocrine disrupting chemical (EDC) removal. Furthermore, adsorption and separation methods simply separate EDC from one phase to another rather than eliminating them via degradation. TAML activators have been developed to catalyze hydrogen peroxide oxidation of organic micropollutants in wastewater12,24-26. TAML activators with H2O2 effectively degrade EE2 and other steroid estrogens in pure laboratory water as well as in effluents from municipal wastewater treatment plants and in spiked urine samples12. Laboratory studies, shows TAML/H2O2 treatment provides high steroid estrogen removal including EE2 removal and substantially reduces estrogenic activity measured in vitro using the YES bioassay and substantially diminishes fish feminization in vivo measured using the VTG bioassay (Figure 1 and Figure 6).
<img alt="Bar graph comparing plasma VTG levels and LC-MS/MS with YES assay across treatment controls." src="/files/ftp_upload/54243/54243fig6.jpg" data-altupdatedat="1788235205000" data-alt="Figure 6"> 
Figure 6. Average EE2 concentration and estrogenic activity in treated and untreated tank waters (A) and plasma vitellogenin in baseline and exposed male fish (B). A) EE2 concentration (ng/L, dark blue bars) was measured by LCMS/MS, estrogenic activity (EE2 equivalent ng/L, dark green bars) was measured via in vitro Yeast Estrogen Screen (YES). B) Plasma vitellogenin (ng/ml, light blue bars) concentration in male fathead minnows were measured via a quantitative enzyme-linked immunosorbent assay (ELISA). EE2 chemical analysis results reported as &lt; 0.03 ng/L EE2 (i.e., lower than detection limit (LOD)) were treated as having half LOD (i.e., 0.015 ng/L EE2) for use in calculations of averages, standard error and statistical analysis. EE2 and estrogenic activity are average measured concentrations sampled over the 21 day exposure. Plasma VTG was measured prior to exposure (baseline) and after 21 days exposure. The treatment regime consisted of; negative control (dilution water only), EE2+H2O2+TAML, EE2+H2O2, and EE2-only. Error bars in graph-A represent standard error of the mean, error bars in graph-B represent standard deviation. Letters above bars in graph-B represent statistical similarity. This figure has been modified from Mills et al.12 <a href="https://www.jove.com/files/ftp_upload/54243/54243fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Endocrine Disrupting Compounds (EDC) pose a substantial risk to the aquatic environment. Municipal wastewater treatment plants are major contributors to the estrogenic potency of surface waters. The methodology provided in this paper allows for an assessment of the efficacy and suitability of wastewater treatment processes with respect to EDC removal.

Endocrine Disrupting Compounds pose a substantial risk to the aquatic environment. Ethinylestradiol (EE2) and estrone (E1) have recently been included in ...

The overall goal of the battery of tests outlined here, including analytical chemistry and in vitro and in vivo methods, is to determine the efficacy of emerging and novel wastewater treatment technologies, to remove estrogenic contaminants. These methods can help answer key questions in environmental engineering, for example, when selecting the most effective wastewater treatment processes in a moving estrogenic potency of wastewater. The main advantage of these techniques, is that they are sensitive to very low concentrations of active compounds, thus evidencing, the best technologies to protect aquatica life.Demonstrating the procedures will be postdoctoral fellow, Dr.Chris Green, graduate student, Angela Pinzon, research fellow, Dr.Alice Baynes, and senior research technician, Nicola Beresford. The SPE process requires tight attention to cleanliness and orderliness to prevent sample contamination. Pre-clean all the tubes, taps and frits with methanol.Before running samples, attach the transfer lines to the extraction manifold, and rinse the entire system with deionized water. To run the samples, first attach the disposable valve liners, then attach the styrene divinylbenzene SPE cartridges. Next, pipette five milliliters of ethyl acetate into each reservoir to condition the cartridges.During this step, make sure the SPE is sealed. Then, switch on the vacuum pump for a flow rate of 10 millimeters per minute. Before the cartridges dry out, load five milliliters of methanol, followed by five milliliters of water.It's very important that the SPE cartridges do not dry out. After the water has mostly drawn through, switch off the pump and top up each cartridge reservoir with water. Then, connect 1/8th inch PTFE tubes between the cartridge reservoirs and glass sample bottles.Now, switch the vacuum to a flow rate below 10 milliliters per minute, and allow the whole sample to pass through the cartridge. Next, thoroughly dry the SPE cartridges, until the cartridge contents change color. Use a vacuum or a gas stream.Now, load clean, dry, 10 milliliter glass collection vials into a rack and place the rack inside the extraction manifold. Check that each liner is above a vial. Load the sample reservoirs with two times four milliliters, for a total of eight milliliters of dichloromethane, switch on the vacuum pump, and let the liquid pass through the SPE and into the collection vials.Then, use a concentrator to reduce the volume in all the collection vials to one milliliter. Transfer each sample into an autosampler vial, and further concentrate them down to 100 microliters, using nitrogen blow down equipment. The next stage in the process, is to use GPC to remove macromolecular interferences.First, inject 95 microliters of the eluded sample extract, into the GPC equipped, HPLC. Concentrate the GPC extract down to 200 microliters, using a concentrator, and the nitrogen blow down apparatus, and then fill it up to two milliliters with hexane. Next, attach disposable valve liners, amino purple cartridges, and cartridge reservoirs to the SPE manifold, and through the 10 milliliter glass collection vials.Now, load two milliliters of hexane into each cartridge, to condition them and discard the solvent. Next, load the GPC sample extract into the reservoir, and suck it through. Set the extract collection aside, and replace the vial in the rack to collect the wash.Next, pass two milliliters of 30 percent volume by volume ethyl acetate and hexane through the cartridge. Then, add two more milliliters of ethyl acetate and hexane, then discard the washing solution collections and return the 10 milliliter collection vial to the rack. Now, add two milliliters of 50 percent volume by volume ethyl acetate and acetone into the reservoir, and start the pump at a low flow rate, below two milliliters per minute.After the liquid pulls through, add another two milliliters of 50 percent ethyl acetate to the reservoir. Now, use a concentrator to reduce the extract volume to one milliliter. Then, transfer the sample to a smaller glass vial, and use nitrogen blow down equipment to evaporate the extract to incipient to Add 100 microliters of methanol, mix the sample well and transfer it to an autosampler vial with a 0.3 milliliter insert.Then, perform LC-MS/MS analysis of the extract. The day before starting the assay, prepare fresh growth medium, and inoculate it with the tenax dock of the HER used strain. The following day, prepare for the assay, by making 100 microliter serial delusions of the test chemicals or effluent extracts, and then E2 standard curve in ethanol.Now, transfer 10 microliter aliquots to a labelled sterile 96 well plate. Then, leave the plate uncovered to evaporate the ethanol. Next, prepare 50 milliliters of growth medium, with 0.5 milliliters of CPRG solution.From the 24-hour yeast culture, measure the turbidity at 620 nanometers to estimate the cell density. Then, add 40 million yeast cells to the 50 milliliters of assay medium. Transfer the inoculated assay medium in a sterile trough, and use a multichannel pipette to transfer 200 microliters of medium to each well of the assay plate.Replace the assay plate lid, seal the edges with tape, and shake the assay plate vigorously for two minutes using a plate shaker. Then, incubate the plate at 32 Celsius, in a naturally ventilated heating cabinet. After three days of incubation, repeat the shake again, and then let the yeast settle for about one hour.Finally, measure the light absorbents by the samples, at 540 and 620 nanometers. Water samples from a conventional wastewater treatment plan to activate it's sludge process, were analyzed using negative ion electrospray LCMS, ethinyl estradiol was present at a concentration above the predicted no effect level. Using advanced water treatment, such as granulated activated carbon, or GAC, it was possible to substantially reduce the ethinyl estradiol levels.All wastewater extracts were analyzed with an isotopically labelled internal standard, red peak, to allow quantification. The yeast to estrogen screen assay was clearly sensitive to the standard estradiol, but chose no response to the ethanol, which is essentially the blank. The yeast screen cells were clearly responsive to the activated sludge process effluent, while the GAC treated effluent produced a diminished response, it was still more biologically active than the blank.Using this novel wastewater treatment technology, both ethanol estradiol concentration, shown in blue, and estrogenic activity, shown in green, were significantly reduced by combined hydrogen peroxide and TAML treatment. Wastewater treatment plants are the major source of surface water contamination with estrogenic substances. New ways for water treatment processes to improve these effluents require in depth testing of estrogenic activity.After watching this video, you should have a good understanding of a battery of chemical and ecotoxicology test, needed to measure estrogenic activity in water samples. It is extremely important to insure that the contamination of samples and extracts is minimized with a careful handling of samples and extracts. For example, commonly used plastics contain estrogenic compounds, therefore it is essential to include negative controls to check for chemical contamination, and positive controls to check that the extraction method has worked and that there is good recovery of spiked estrogens.

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Learn analytical chemistry and ecotoxicology methods to assess wastewater treatment efficacy for removing estrogenic contaminants. This battery of tests evaluates emerging technologies to protect aquatic ecosystems.

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency (Video) | JoVE Journal

Use of a Battery of Chemical and Ecotoxicological Methods for the Assessment of the Efficacy of Wastewater Treatment Processes to Remove Estrogenic Potency

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