Funding was provided by State Water Resources Control Board (Agreements No. 10-096-250 and 14-090-270). We thank S. Abbasi, M. Connor, S. Engelage, K. North, J. Armstrong, S. Asato, M. Dojiri, D. Schlenk, S. Snyder, S. Westerheide, B. Escher, F. Leusch, G. Pelanek, K. Bi, and J. Printen. The authors declare no conflict of interest, and reference to trade names does not imply endorsement.

1. Collect and Process Water Sample (Modified from Escher et al.9)
<ol>
	<li>Fill a clean 1 L amber glass bottle containing 1 g sodium azide and 50 mg ascorbic acid to the top with water sample of interest. Store sample at 4 &#176;C and process within 72 hr. 
	NOTE: Sodium azide is highly toxic and must be handled with caution. Use protective gear (eye/face, gloves, clothing) and weigh in a properly functioning fume-hood. Do not use a metal spatula for weighing.</li>
	<li>Pass the sample through a 1.6 &#181;M glass fiber filter and then through a preconditioned Solid Phase Extraction (SPE) cartridge at a flow rate of 5-10 ml/min. Adjust the pressure of the vacuum pump to control the flow rate.</li>
	<li>Vacuum dry the cartridge for 15 min.</li>
	<li>Elute the cartridge with 10 ml of methanol, followed by 10 mL&#160;of acetone:hexane (1:1, v/v).</li>
	<li>Concentrate eluate to ~1 ml under a gentle stream of high purity nitrogen. Solvent exchange by adding 500 &#181;l of dimethyl sulfoxide (DMSO) and evaporating the extract down to 500 &#181;l.</li>
	<li>Transfer the extract to an amber glass autosampler vial. Store at -20 &#176;C.</li>
</ol>
2. Prepare Dilutions of the Assay Specific Reference Chemical and Water Extract
<ol>
	<li>Prepare 9 dilutions for the calibration curve.
	<ol>
		<li>Make a stock solution of the assay specific reference chemical in 100% DMSO. The final concentration must be 2 &#181;M 17&#946;-estradiol for the ER assay, and 100 &#181;M dexamethasone for the GR assay. Store the stock solutions at -20 &#176;C.</li>
		<li>Add 15 &#181;l of the appropriate reference chemical stock to 285 &#181;l of assay medium in a sterile tube (tube #1). Mix the sample by pipetting up and down.</li>
		<li>Add 200 &#181;l of a solution of 5% DMSO in assay medium in 8 additional tubes (tubes #2-9).</li>
		<li>Transfer a 100 &#181;l aliquot from tube #1 to tube #2 through tube #9 to perform a 3-fold dilution series. Each diluted sample must be mixed thoroughly with a pipette before taking an aliquot and adding it to the next tube.</li>
	</ol>
	</li>
	<li>Prepare a solvent control sample by mixing 10 &#181;l of DMSO in 190 &#181;l of assay medium.</li>
	<li>Prepare four dilutions for each water extract.
	<ol>
		<li>In the first tube, add 5 &#181;L&#160;of water extract in 95 &#181;L of assay medium and mix thoroughly. Add 50 &#181;l of 5% DMSO in assay medium in three other tubes.</li>
		<li>Perform a 2-fold dilution by transferring 50 &#181;l of solution from one tube to the next.</li>
	</ol>
	</li>
</ol>
3. Prepare the Cell Suspension to Conduct the FRET Bioassay
<ol>
	<li>Prepare the assay specific medium according to the manufacturer&#39;s instructions. Assay medium must be stored at 4 &#176;C and warmed to 37 &#176;C in a water-bath before use.</li>
	<li>Take a vial of ER or GR division-arrested cells out of the cryogenic freezer and thaw the cells quickly by placing the vial in a 37 &#176;C water-bath for 2 min with gentle agitation. Decontaminate the vial with 70% ethanol and place it in a Class II biological safety cabinet.</li>
	<li>Open the vial using aseptic techniques and transfer the cells into 10 ml of assay medium.</li>
	<li>Centrifuge at 200 x g for 5 min.</li>
	<li>Aspirate the supernatant using a sterile glass pipet and resuspend the cell pellet in 6 ml of assay medium.</li>
	<li>Mix 5 &#181;l of cell suspension with 5 &#181;l of a vital stain solution and add an aliquot into a counting chamber.</li>
	<li>Count the number of live cells using a light microscope or automated cell counter and estimate the density of live cells in the cell suspension. Dilute if necessary to obtain a final density of 550,000 live cells per ml of assay medium.</li>
</ol>
4. Plate Cells in a 96-well Black Wall Clear-bottom Plate and Add Diluted Water Extract
<ol>
	<li>Create a plate layout that includes a 9-point assay specific calibration curve, QA/QC samples and multiple dilutions for each water extract. An example of a 96-well plate layout is shown in Figure 1.</li>
	<li>Add 90 &#181;l of assay medium to the replicate cell-free control wells.</li>
	<li>Pour the cell suspension in a sterile pipetting reservoir and add 90 &#181;l of cell suspension to the other wells using a multichannel pipette.</li>
	<li>Add 10 &#181;l of the diluted samples prepared during step 2 to the appropriate wells. The final concentration of DMSO per well must be 0.5% maximum.</li>
	<li>Cover the plate with a lid and place it in a 5% CO2 incubator at 37 &#176;C for 16 hr.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/54725/54725fig1.jpg" /> 
Figure 1: Example of a 96-well Plate Layout. The multi-well plate is designed to include an assay specific calibration curve, 3 types of QA/QC controls (media only, cells in clean medium and cells in DMSO spiked medium) and 4 dilutions per water extract. Each control and dilution of water extract is analyzed in triplicate wells. <a href="https://cloudfront-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54725/54725fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
5. Prepare the Loading Solution and Add to Each Well
<ol>
	<li>After incubation, allow the plate to equilibrate to RT. For this FRET bioassay, steps 5.2 through 5.6 should be conducted in the absence of direct light.</li>
	<li>Prepare a 6x loading solution according to the manufacturer&#39;s instructions.</li>
	<li>Add 20 &#181;l of the 6x loading solution to each well.</li>
	<li>Add 10 &#181;l of cell viability reagent to each well to evaluate the cytotoxicity of the diluted water extract.</li>
	<li>Seal the plate with aluminum adhesive film.</li>
	<li>Incubate the plate in the dark at RT for 2 hr.</li>
</ol>
6. Measure Cytotoxicity and Endocrine Activity Response
<ol>
	<li>Set up the microplate reader with bottom-reading capabilities following the manufacturer&#39;s instructions.</li>
	<li>For the FRET transactivation bioassay, measure fluorescence in the blue (409/460 nm, excitation (Ex)/emission (Em) wavelength) and green (409 Ex/530 Em nm) channels.</li>
	<li>For the cytotoxicity assay, measure fluorescence at 560 Ex/590 Em nm.</li>
</ol>
7. Assess QA/QC Checks to Determine the Quality of the Data
<ol>
	<li>Compare the average raw fluorescence of the cell-free (media only) and cells-only (cells in clean assay medium) controls. The media-only background fluorescence should be at least 25% lower than the response of the cells-only control.</li>
	<li>Process the raw FRET data. For both &#39;blue&#39; and &#39;green&#39; datasets, subtract the average fluorescence of the cell-free control wells from all cell containing well. Calculate the blue/green ratio for each experimental well.</li>
	<li>Compare blue/green ratio of cells-only and cells with DMSO controls. Fluorescence values of these controls should be within 15% Relative Standard Deviation (RSD).</li>
	<li>Plot the assay specific calibration curve as blue/green ratio against the sample concentration expressed as a log molecular mass (log M). Calculate the slope, R2 and log EC50 (50% Effect Concentration). Calibration parameters should be within the expected range listed in Table 1.</li>
	<li>Calculate the RSD&#160;for all triplicate wells. The variability among replicates should be less than 20%.</li>
	<li>For the cytotoxicity data (560 Ex/590 Em nm), subtract the cell-free control average (i.e. media background) from all cell-containing wells.</li>
	<li>Calculate the average background-subtracted fluorescence for each sample. Plot the resulting fluorescence data as a percent of the DMSO control. Sample dilutions should not exhibit more than 20% cell mortality compared to the cells-only and DMSO controls.</li>
</ol>
8. Data Analyses
<ol>
	<li>Calculate the assay Limit of Detection (LOD) as the minimum calibration response plus two standard deviations of the mean of that response.</li>
	<li>For samples showing a dose response, derive the EC50&#160;of the water extract using the slope of a linear regression of the calibration curve between 10 and 50% effect concentrations of the reference chemical.</li>
	<li>Calculate the Bioanalytical Equivalent Concentration (BEQ) using the formula: BEQ = EC50 of reference chemical/EC50 of water sample.</li>
</ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+tiered,+integrated+biological+and+chemical+monitoring+framework+for+contaminants+of+emerging+concern+(CECs)+in+aquatic+ecosystems.">A tiered, integrated biological and chemical monitoring framework for contaminants of emerging concern (CECs) in aquatic ecosystems.</a> Integr. Environ. Assess. Manag. , (2015).</li><li>Van der Linden, S. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+multiple+hormonal+activities+in+wastewater+effluents,+surface+water,+using+a+panel+of+steroid+receptor+CALUX+bioassays.">Detection of multiple hormonal activities in wastewater effluents, surface water, using a panel of steroid receptor CALUX bioassays.</a> Environ. Sci. Technol. 42 (15), 5814-5820 (2008).</li><li>Leusch, F. D. 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The authors have nothing to disclose.

The well documented potency of environmental estrogens, such as 17&#946;-estradiol (E2), warrants screening for these chemicals at ng/L concentrations23,24. In this study, the ER response for wastewater effluents (BEQ range: 2.3 to 17 ng E2/L) was somewhat higher than reported for secondary effluent from Australian WWTPs20, whereas the BEQs for surface water (&#60;0.5 to 4 ng E2/L) were within the range reported for surface and stormwater elsewhere (&#60;1 to 11 ng E2/L)16. Despite the low levels of ER activity measured in the surface water samples, this assay represents a relevant screening endpoint for water quality assessment. Estrogens such as bisphenol A and alkylphenol surfactants are often detected in the aquatic environment, but they can be difficult to quantify using conventional analytical chemistry. ER-cell assays may also prove useful for screening of currently and newly registered pesticides, as shown by Kojima et al25.
The GR activity exceeded the ER activity by an order of magnitude in all 4 effluent samples analyzed in this study (Table 2). This trend is consistent with previous studies on wastewater effluent14, and the range for GR-BEQs reported here is slightly higher but comparable to that reported for other secondary effluents using the same in vitro bioassay12. Using a different cell bioassay, the range for GR-BEQs for effluent from wastewater treatment plants in Japan was lower (&#60;3 to 78 ng Dex/L) than those reported for secondary effluent in the present study22. With the exception of the single surface water sample that registered a maximum GR response of 30 ng Dex/L (Table 2, sample E), the GR activity in the remaining surface water samples was consistent with the low activity reported for Dutch surface waters21. However, the environmental impact for GR active chemicals is less well characterized than for ER active chemicals26, making assessment of the potential for higher order effects a challenge.
As with conventional chemical analysis, adherence to written protocols and validation of measurements using a performance-based QA/QC approach maximizes data quality and robustness. Although validation of cell bioassays adapted for water quality assessment will continue to evolve and improve, measurement of the QA/QC parameters first outlined in Mehinto et al.14 and applied to this study showed that our results were well within prespecified guideline values (Table 1). The calibration curve parameters for these bioassays mirror those used for calibrating other analytical protocols (e.g., GC-MS), with the addition of criteria that assess the viability of the cells (e.g., cytotoxicity) representing the main difference. Another operational difference made possible using cells in a high throughput format is the inclusion of multiple sample dilutions that illustrate the concentration dependent response expected when bioactivity is measured (Figure. 2). In reviewing the sample measurements, the lower ER and GR responses for the tertiary effluent (sample D) compared to the three secondary effluent samples (A-C) provides additional evidence of the relative accuracy of our bioassay screening results in Table 2. Incorporation of control or reference samples that represent the matrix of interest, in this case water, would further enhance the ability to compare bioassay results within and especially across measuring entities.
The responses of the ER and GR transactivation cell lines utilized in this study were expressed relative to the activity of the strong agonists E2 and dexamethasone, respectively. This allows for a quantitative estimate of bioactivity expressed as a Bioassay Equivalent Concentration (BEQ). This further allows for investigation of chemicals that contribute to the bioassay response, i.e. a direct comparison of BEQs with concentrations of individual agonists determined via conventional analytical chemistry. This concept underlines the added utility of bioscreening in directing diagnostic investigation of causative agents, also known as effects-directed analysis.
Since SPE is routinely used to isolate a wide range of pharmaceuticals and other &#34;emerging contaminants&#34; from water, we employed a previously published protocol9, 20 that featured a sorbent designed to capture both hydrophilic and hydrophobic chemicals in water. Although we assume quantitative capture of all bioactive organic compounds using this technique, additional work is needed to fully standardize and validate the performance of such extraction methods. The type of cell assay employed in this study may present another limitation. These assays measure the interactions between contaminants and nuclear receptors and do not take into account other possible interactions, such as chemical-membrane receptor. Thus, it is possible that the screening responses measured here were underestimated. Like sample extraction, there are no standardized methods to analyze cell assay data and different approaches may produce different results. The linear regression used in this study to model the response for the lower half of the calibration curve may not be appropriate for all cell assays, particularly if modeled response is extended beyond the relatively narrow range specified in the present study. Additional investigation into the above issues is needed to understand the extent to which these tools can be applied as a robust monitoring tool.
Whereas much of the groundwork is being laid for ER and GR as screens for endocrine active chemicals, researchers are broadening the scope of bioanalytical tools to include other relevant mode of actions, e.g., androgenicity, genotoxicity, neurotoxicity and immunotoxicity9,27. A more complete toolbox will enhance the ability to screen for bioactive chemicals, including transformation products of known and/or unknown chemicals that can occur28, now and into the future, in various aquatic environments19.

Current water quality monitoring is predicated on the ability to accurately and precisely measure the occurrence of chemical contaminants as a proxy for exposure to wildlife and humans. However, this chemical-by-chemical monitoring and assessment paradigm cannot keep pace with the ever-changing chemical universe that we face. As we learn more about the fate and effects of synthetic and natural chemicals, we continue to search for measurement tools that address expected biological impacts, and that at the same time are immune to changes in chemical production, usage and environmental input. Such tools are especially relevant for understanding whether unknown or new chemicals, and transformation products, deserve our attention. Moreover, complex mixtures of chemicals present in water are poorly addressed by individual chemical monitoring. Thus, we face the challenge of modernizing the existing monitoring toolbox to better address these issues in surface waters that receive discharge of treated wastewater effluent and urban/stormwater runoff.
In recent years, bioanalytical techniques have shown promise as screening tools for water quality assessment. In particular, in&#39;vitro bioassays that respond to chemicals acting via known, specific modes of action1,2 are of great interest to the environmental monitoring community3. Numerous investigations have employed in vitro bioassays to quantify the endocrine activity of drinking, surface and wastewaters4 -6. Moreover, a number of bioassays target molecular initiating events (e.g., receptor activation) which can potentially be linked to deleterious effects via adverse outcome pathway analyses7,8.
The evolution of bioscreening for water quality assessment has been relatively rapid, with hundreds of different in vitro bioassay endpoints having been evaluated for their utility9,10. Currently, only a handful of bioassays have been shown to achieve good measurement precision (within laboratories) while demonstrating the ability to differentiate among water qualities5,6. For treated wastewater effluent in particular, the occurrence of estrogens and glucocorticoid steroids has been successfully accounted for using in vitro transactivation assays11,12. However, most studies to date have employed bioassays whose cell lines are proprietary (and thus not widely available), require continuous care and manipulation, or both. As a result, the ability to standardize protocols, perform inter-laboratory calibration exercises, and ultimately to transfer this screening technology to the water resources community remains hindered.
At least one supplier of in vitro bioassays vetted through the U.S. ToxCast program is commercially available13 in easy to use &#34;freeze and thaw&#34; formats. These division-arrested cell &#34;kits&#34; have been shown to be robust in measuring the activity of chemicals extracted from water representing different levels of treatment14. Although vendor protocols are available to screen the bioactivity of individual chemicals or mixtures, some of them require modification before they can be applied to water samples. Treated wastewater effluent15, stormwater runoff16, receiving waters17,18 and more recently recycled water19,20 are prime examples of aqueous media that are of interest to the water quality community.
This study presents a single, standardized protocol to measure the endocrine activity in water samples using commercially available, division-arrested in vitro transactivation bioassays. We demonstrated robustness of the protocol through a comprehensive assessment of background, dose responsivity and repeatability of response for two endpoints of particular interest Estrogen and Glucocorticoid Receptor transactivation (ER and GR, respectively). The protocol was applied to screen samples of treated wastewater effluent and surface water from freshwater systems in California.

<table border="0" cellpadding="0" cellspacing="0" width="826">
	<tbody>
		<tr height="42">
			<td height="42" style="height:43px;width:275px;">GeneBLAzer ER alpha DA assay kit</td>
			<td style="width:123px;">ThermoFisher</td>
			<td style="width:115px;">K1393</td>
			<td style="width:313px;">Kit includes ER division arrested (DA) cells and LiveBLAzer FRET loading kit.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:275px;">GeneBLAzer GR DA assay kit</td>
			<td style="width:123px;">ThermoFisher</td>
			<td style="width:115px;">K1391</td>
			<td style="width:313px;">Kit includes GR division arrested (DA) cells and LiveBLAzer FRET loading kit.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">PrestoBlue cell viability reagent&#160;</td>
			<td style="width:123px;">ThermoFisher</td>
			<td style="width:115px;">A-13261</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Trypan blue, 0.4% in PBS</td>
			<td style="width:123px;">Sigma-Aldrich&#160;</td>
			<td style="width:115px;">T8154</td>
			<td>Also available at ThermoFisher</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Corning 96 well black wall, clear-bottom plate</td>
			<td style="width:123px;">Corning</td>
			<td style="width:115px;">3603</td>
			<td style="width:313px;">Individually wrapped, sterile with lid</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Whatman glass fiber filters, GF/A, 1.6 &#181;M</td>
			<td style="width:123px;">Sigma-Aldrich&#160;</td>
			<td style="width:115px;">WHA1820025</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Microplate aluminum sealing film</td>
			<td style="width:123px;">E&#38;K Scientific</td>
			<td style="width:115px;">T592100</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Oasis HLB 6 cc cartridge, 200 mg sorbent</td>
			<td style="width:123px;">Waters</td>
			<td style="width:115px;">WAT106202</td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">17&#946; Estradiol</td>
			<td style="width:123px;">Sigma-Aldrich&#160;</td>
			<td style="width:115px;">E2758</td>
			<td style="width:313px;">CAS #50-28-2</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Ascorbic acid</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:115px;">A61-100</td>
			<td style="width:313px;">Also available at Sigma-Aldrich</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Dexamethasone&#160;</td>
			<td style="width:123px;">Sigma-Aldrich&#160;</td>
			<td style="width:115px;">D4902</td>
			<td style="width:313px;">CAS #50-02-2</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:123px;">Sigma-Aldrich&#160;</td>
			<td style="width:115px;">D8418</td>
			<td>Molecular grade</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Solvents (acetone, hexane, methanol)</td>
			<td style="width:123px;">Fisher Scientific</td>
			<td style="width:115px;"></td>
			<td style="width:313px;">HPLC grade</td>
		</tr>
		<tr height="83">
			<td height="83" style="height:83px;width:275px;">Sodium azide</td>
			<td style="width:123px;">Sigma-Aldrich&#160;</td>
			<td style="width:115px;">S2002</td>
			<td style="width:313px;">Chemical is highly toxic and must be handled with caution. Use protective clothing and weigh under a fumehood. Also available at EMD Millipore.</td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:275px;">Automated cell counter or hemocytometer</td>
			<td style="width:123px;">Various*</td>
			<td style="width:115px;"></td>
			<td style="width:313px;">Suppliers include Bio-Rad, Fisher Scientific, Sigma-Aldrich and ThermoFisher.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">Class II biological safety cabinet</td>
			<td style="width:123px;">Various*</td>
			<td style="width:115px;"></td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:23px;width:275px;">CO2 incubator</td>
			<td style="width:123px;">Various*</td>
			<td style="width:115px;"></td>
			<td style="width:313px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:275px;">Cryogenic freezer&#160;</td>
			<td style="width:123px;">Various*</td>
			<td style="width:115px;"></td>
			<td style="width:313px;">Liquid nitrogen storage dewar is recommended.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:275px;">Fluorescence microplate reader</td>
			<td style="width:123px;">Various*</td>
			<td style="width:115px;"></td>
			<td style="width:313px;">&#160;The reader must have bottom read capabilities.</td>
		</tr>
		<tr height="21">
			<td colspan="4" height="21" style="height:21px;">* No recommended source, the choice of this equipment depends on budget, frequency of use, and lab space.</td>
		</tr>
	</tbody>
</table>

screening for endocrine activity in water using commercially-available in vitro transactivation bioassays

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been hindered partly due to a lack of standardized methods and availability of robust, user-friendly technology. In this study, commercially available, division-arrested cell lines were employed to quantitatively screen for endocrine activity of chemicals present in water samples of interest to environmental quality professionals. A single, standardized protocol that included comprehensive quality assurance/quality control (QA/QC) checks was developed for Estrogen and Glucocorticoid Receptor activity (ER and GR, respectively) using a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Samples of treated municipal wastewater effluent and surface water from freshwater systems in California (USA), were extracted using solid phase extraction and analyzed for endocrine activity using the standardized protocol. Background and dose-response for endpoint-specific reference chemicals met QA/QC guidelines deemed necessary for reliable measurement. The bioassay screening response for surface water samples was largely not detectable. In contrast, effluent samples from secondary treatment plants had the highest measurable activity, with estimated bioassay equivalent concentrations (BEQs) up to 392 ng dexamethasone/L for GR and 17 ng 17&#946;-estradiol/L for ER. The bioassay response for a tertiary effluent sample was lower than that measured for secondary effluents, indicating a lower residual of endocrine active chemicals after advanced treatment. This protocol showed that in vitro transactivation bioassays that utilize commercially available, division-arrested cell &#34;kits&#34;, can be adapted to screen for endocrine activity in water.

In the present study, 4x 24 hr composite samples of treated municipal wastewater effluent, 6 grab samples of surface water from freshwater systems in southern California and a field blank consisting of ultrapure water were selected to illustrate this protocol. 3 of the 4&#160;effluent samples were from conventional activated sludge wastewater treatment plants (&#34;secondary effluent&#34;), and the fourth one from an advanced wastewater treatment plant with sand/carbon filtration added post biological treatment (&#34;tertiary effluent&#34;). Surface water samples were collected from watersheds representing different land use (open, agricultural and urban).
Dilutions of the water extracts showed no apparent cytotoxicity with less than 20% cell mortality observed compared to the DMSO controls (Table 1). Cell-free (media only) background was low, and the comparison between cells-only and cells with DMSO responses indicated that the solvent had no measurable impact on the fluorescence measurements. Parameters of the assay specific calibration curves, including slope and log EC50, were within the range of acceptable historical values (Table 1) demonstrating a good reproducibility of the calibration curves over time. An example of acceptable calibration curve for the ER assay is shown in Figure 2. The selection of 17&#946;-estradiol and dexamethasone as reference chemicals was based on the strength of assay response, likelihood of occurrence in treated wastewater and historical use by other investigators20-22. The variability in the response among triplicate samples was less than 20% RSD. Thus, all data were deemed to be of acceptable quality, and were subsequently used to estimate the ER and GR bioactivity (expressed as BEQ in ng/L) in wastewater and surface water samples.
A Relative Enrichment Factor (REF) of 5 to 10 and 4&#160;dilutions per sample appeared suitable for screening of the water samples analyzed in the present study. The majority of WWTP effluent samples showed a dose response above LOD for both ER and GR bioassays; the lowest dilution had a response within the calibration curve and the highest dilution was typically below LOD (Figure 3). However, surface water extracts did not show strong ER or GR activity. Despite an REF of 10, the fluorescent responses were often close to or below the LOD. The absence of bioassay responses for the field blank confirmed that the detections noted above LOD were not the result of sampling or procedural lab artifacts.
ER and GR activities were detected in 7 and 4 of the 10 representative water samples, respectively (Table 2), with the highest of BEQs of 17 ng E2/L and 392 ng Dex/L found in the secondary effluents. The GR activity in the sample of tertiary effluent was below the bioassay LOD. Likewise, most surface water samples exhibited no GR activity above LOD and much lower levels of ER activity than the secondary effluents.
<img alt="Figure 2" src="/files/ftp_upload/54725/54725fig2.jpg" /> 
Figure 2: Example of Calibration Curve for the ER Assay. The dose-response curve, plotted as the mean fluorescence ratio (&#177; SEM), is sigmoidal and used to determine the EC10 and EC50 values. This portion of the curve is fitted using linear regression and used to derive a bioassay equivalent concentration. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54725/54725fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54725/54725fig3.jpg" /> 
Figure 3: Mean Blue/green Fluorescence Ratio (&#177; SEM) of Water Samples Plotted Against theRrelative Enrichment Factor. Bioassay Equivalent Concentrations (BEQ) are calculated for samples whose dilution series show a concentration dependent-response with a minimum of two points above the limit of detection (LOD). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54725/54725fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td colspan="2">Estrogen Receptor (ER) Assay</td>
			<td colspan="2">Glucocorticoid Receptor (GR) Assay</td>
		</tr>
		<tr>
			<td></td>
			<td>Study results&#160;</td>
			<td>Acceptance criteria&#160;</td>
			<td>Study results&#160;</td>
			<td>Acceptance criteria&#160;</td>
		</tr>
		<tr>
			<td>Calibration parameters</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Mean log EC50 (M)</td>
			<td>-9.6</td>
			<td>-10.1 to -9.1</td>
			<td>-8.5</td>
			<td>-9.0 to -8.0</td>
		</tr>
		<tr>
			<td>Mean slope</td>
			<td>1.1</td>
			<td>0.9 to 1.5</td>
			<td>2.0</td>
			<td>1.8 to 2.4</td>
		</tr>
		<tr>
			<td>R2</td>
			<td>0.99</td>
			<td>&#62;0.95</td>
			<td>0.99</td>
			<td>&#62;0.95</td>
		</tr>
		<tr>
			<td>QA/QC controls</td>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Media background</td>
			<td>yes</td>
			<td>media only &#60;cell+media</td>
			<td>yes</td>
			<td>media only &#60;cell+media</td>
		</tr>
		<tr>
			<td>Cytotoxicity (% mortality)</td>
			<td>0%</td>
			<td>&#60;20% mortality</td>
			<td>0 to 5%</td>
			<td>&#60;20% mortality</td>
		</tr>
		<tr>
			<td>Sample precision (%RSD)</td>
			<td>2 to 18%</td>
			<td>&#60;20%</td>
			<td>4 to 13%</td>
			<td>&#60;20%</td>
		</tr>
	</tbody>
</table>
Table 1: Summary of QA/QC Results From Bioanalytical Screening of Treated Wastewater Effluent and Surface Water Samples.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Sample ID and Description</td>
			<td>ER-BEQ (ng E2/L)</td>
			<td>GR-BEQ (ng Dex/L)</td>
		</tr>
		<tr>
			<td>A &#8211; final effluent from full secondary treatment plant</td>
			<td>6.4</td>
			<td>248</td>
		</tr>
		<tr>
			<td>B &#8211; final effluent from full secondary treatment plant</td>
			<td>13</td>
			<td>392</td>
		</tr>
		<tr>
			<td>C &#8211; final effluent from full secondary treatment plant</td>
			<td>17</td>
			<td>236</td>
		</tr>
		<tr>
			<td>D &#8211; final effluent from tertiary treatment plant</td>
			<td>2.3</td>
			<td>&#60;22</td>
		</tr>
		<tr>
			<td>E &#8211; surface water from agricultural area</td>
			<td>&#60;0.5</td>
			<td>30</td>
		</tr>
		<tr>
			<td>F &#8211; surface water from agricultural area</td>
			<td>&#60;0.5</td>
			<td>&#60;22</td>
		</tr>
		<tr>
			<td>G &#8211; surface water from urban area</td>
			<td>4</td>
			<td>&#60;22</td>
		</tr>
		<tr>
			<td>H &#8211; surface water from urban area</td>
			<td>0.9</td>
			<td>&#60;22</td>
		</tr>
		<tr>
			<td>I &#8211; surface water from open field</td>
			<td>0.8</td>
			<td>&#60;22</td>
		</tr>
		<tr>
			<td>J &#8211; surface water from open field</td>
			<td>&#60;0.5</td>
			<td>&#60;22</td>
		</tr>
		<tr>
			<td>K &#8211; field blank (ultrapure water)</td>
			<td>&#60;0.5</td>
			<td>&#60;22</td>
		</tr>
	</tbody>
</table>
Table 2: Bioassay Equivalent Concentrations for Estrogen Receptor (ER-BEQ) and Glucocorticoid Receptor (GR-BEQ) activity. Three types of samples collected in California (USA) are analyzed: municipal wastewater effluent (A-D), surface water (E-J), and field blank (K).

Watch this Scientific Journal Video about Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays at JoVE.com

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

A protocol to screen for endocrine activity in organic extracts of water samples, including treated wastewater effluent and surface (receiving) water, was adapted using commercially available division-arrested ("freeze and thaw") in vitro transactivation bioassays.

Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

In vitro transactivation bioassays have shown promise as water quality monitoring tools, however their adoption and widespread application has been ...

screening-for-endocrine-activity-water-using-commercially-available

Nanyang Technological University

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JoVE Journal

Environment

7.5K Views.  Southern California Coastal Water Research Project Authority. A protocol to screen for endocrine activity in organic extracts of water samples, including treated wastewater effluent and surface (receiving) water, was adapted using commercially available division-arrested ("freeze and thaw") in vitro transactivation bioassays.

Video: Screening for Endocrine Activity in Water Using Commercially-available In Vitro Transactivation Bioassays

EPA Method 1615. Measurement of Enterovirus and Norovirus Occurrence in Water by Culture and RT-qPCR. Part III. Virus Detection by RT-qPCR

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a standardized method for use in multiple analytical laboratories during monitoring period 3 of the Unregulated Contaminant Monitoring Rule. Herein we present the protocol for extraction of viral ribonucleic acid (RNA) from water sample concentrates and for quantitatively measuring enterovirus and norovirus concentrations using reverse transcription-quantitative PCR (RT-qPCR). Virus concentrations for the molecular assay are calculated in terms of genomic copies of viral RNA per liter based upon a standard curve. The method uses a number of quality controls to increase data quality and to reduce interlaboratory and intralaboratory variation. The method has been evaluated by examining virus recovery from ground and reagent grade waters seeded with poliovirus type 3 and murine norovirus as a surrogate for human noroviruses. Mean poliovirus recoveries were 20% in groundwaters and 44% in reagent grade water. Mean murine norovirus recoveries with the RT-qPCR assay were 30% in groundwaters and 4% in reagent grade water.

Here we present a procedure to quantify enterovirus and norovirus in environmental and drinking waters using reverse transcription-quantitative PCR. Mean virus recovery from groundwater with this standardized procedure from EPA Method 1615 was 20% for poliovirus and 30% for murine norovirus.

EPA Method 1615 measures enteroviruses and noroviruses present in environmental and drinking waters. This method was developed with the goal of having a ...

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated HF183 marker. The EntHF183 duplex dPCR (referred as EntHF183 dPCR hereon) assay uses the same primer and probe sequences as its published individual quantitative PCR (qPCR) counterparts. Likewise, the same water filtration and DNA extraction procedures as performed prior to qPCR are followed prior to running dPCR. However, the duplex dPCR assay has several advantages over the qPCR assays. Most important, the dPCR assay eliminates the need for running a standard curve and hence, the associated bias and variability, by direct quantification of its targets. In addition, while duplexing (i.e. simultaneous quantification) Enterococcus and HF183 in qPCR often leads to severe underestimation of the less abundant target in a sample, dPCR provides consistent quantification of both targets, whether quantified individually or simultaneously in the same reaction. The dPCR assay is also able to tolerate PCR inhibitor concentrations that are one to two orders of magnitude higher than those tolerated by qPCR. These advantages make the EntHF183 dPCR assay particularly attractive because it simultaneously provides accurate and repeatable information on both general and human-associated fecal contamination in environmental waters without the need to run two separate qPCR assays. Despite its advantages over qPCR, the upper quantification limit of the dPCR assay with currently available instrumentation is approximately four orders of magnitude lower than that achievable by qPCR. Consequently, dilution is needed for measurement of high concentrations of target organisms such as those typically observed following sewage spills.

A Duplex Digital PCR Assay for Simultaneous Quantification of the Enterococcus spp. and the Human Fecal-associated HF183 Marker in Waters

This manuscript describes a duplex digital PCR assay that can be used to simultaneously quantify Enterococcus spp. and the HF183 genetic markers as indicators of general and human-associated fecal contamination in recreational waters.

This manuscript describes a duplex digital PCR assay (EntHF183 dPCR) for simultaneous quantification of Enterococcus spp. and the human fecal-associated ...

Characterization and Application of Passive Samplers for Monitoring of Pesticides in Water

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study provides a protocol for the passive sampler preparation, calibration, extraction method and instrumental analysis. Sampling rates (RS) and passive sampler-water partition coefficients (KPW) were calculated for silicone rubber, polar organic chemical integrative sampler POCIS-A, POCIS-B, SDB-RPS and C18 disk. The uptake of the selected compounds depended on their physicochemical properties, i.e., silicone rubber showed a better uptake for more hydrophobic compounds (log octanol-water partition coefficient (KOW) &#62; 5.3), whereas POCIS-A, POCIS-B and SDB-RPS disk were more suitable for hydrophilic compounds (log KOW &#60; 0.70).

A protocol about the characterization and application of five different passive sampling devices is presented.

Five different water passive samplers were calibrated under laboratory conditions for measurement of 124 legacy and current used pesticides. This study ...

A Simple Planting Technique for Re-establishing Trees Where Frequent Inundation Occurs

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with herbaceous plants. This protocol describes a simple, cost-effective tree planting technique needed for restoring degraded Everglade tree islands. The design is patterned after a natural Everglades process that creates floating peat islands, which allows tree survival and growth in flooded conditions and often leads to the development of tree islands. Commercially available peat bags were used as the medium for the growth and establishment of potted native tree saplings. The pop-up configuration floated initially and provided additional elevation to minimize inundation, with a single native tree species sapling and a single tree fertilizer spike. During a 3 year study involving 105 pop-ups, most plants survived (80%) and many thrived. Determining whether this technique can establish trees on a degraded tree island will require longer studies and extensive field tests.

This protocol describes a simple, cost-effective tree planting technique for restoring degraded Everglades tree islands that experience inundation. The design creates an island (pop-up) which floats initially and adds elevation to promote tree survival and growth under flooded conditions.

Many of the Everglades tree islands have lost elevation over the past century and most of their trees have died such that they are now covered with ...

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol for performing in situ juvenile exposure tests within oligotrophic river catchments over one-month and three-month periods. Two methods (in both modifications) are presented to evaluate the juvenile growth and survival rate. The methods and modifications differ in value for the locality bioindication and each has its benefits as well as limitations. The sandy cage method works with a large set of individuals, but only some of the individuals are measured and the results are evaluated in bulk. In the mesh cage method, the individuals are kept and measured separately, but a low individual number is evaluated. The open water exposure modification is relatively easy to apply; it shows the juvenile growth potential of sites and can also be effective for water toxicity testing. The within-bed exposure modification needs a high workload but is closer to the conditions of a natural juvenile environment and it is better for reporting the real suitability of localities. On the other hand, more replications are needed in this modification due to its high-hyporheic environment variability.

Bioindication Testing of Stream Environment Suitability for Young Freshwater Pearl Mussels Using In Situ Exposure Methods

In situ bioindications enable determination of the suitability of an environment for endangered mussel species. We describe two methods based on the juvenile exposure of freshwater pearl mussels in cages to oligotrophic river habitats. Both methods are implemented in variants for open water and hyporheic water environments.

Knowledge of habitat suitability for freshwater mussels is an important step in the conservation of this endangered species group. We describe a protocol ...

Optimized Procedure for Determining the Adsorption of Phosphonates onto Granular Ferric Hydroxide using a Miniaturized Phosphorus Determination Method

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide (GFH), with little effort and high reliability. The phosphonate, e.g., nitrilotrimethylphosphonic acid (NTMP), is brought into contact with the GFH in a rotator in a solution buffered by an organic acid (e.g., acetic acid) or Good buffer (e.g., 2-(N-morpholino)ethanesulfonic acid) [MES] and N-cyclohexyl-2-hydroxyl-3-aminopropanesulfonic acid [CAPSO]) in a concentration of 10 mM for a specific time in 50 mL centrifuge tubes. Subsequently, after membrane filtration (0.45 &#181;m pore size), the total phosphorus (total P) concentration is measured using a specifically developed determination method (ISOmini). This method is a modification and simplification of the ISO 6878 method: a 4 mL sample is mixed with H2SO4 and K2S2O8 in a screw cap vial, heated to 148-150 &#176;C for 1 h and then mixed with NaOH, ascorbic acid and acidified molybdate with antimony(III) (final volume of 10 mL) to produce a blue complex. The color intensity, which is linearly proportional to the phosphorus concentration, is measured spectrophotometrically (880 nm). It is demonstrated that the buffer concentration used has no significant effect on the adsorption of phosphonate between pH 4 and 12. The buffers, therefore, do not compete with the phosphonate for adsorption sites. Furthermore, the relatively high concentration of the buffer requires a higher dosage concentration of oxidizing agent (K2S2O8) for digestion than that specified in ISO 6878, which, together with the NaOH dosage, is matched to each buffer. Despite the simplification, the ISOmini method does not lose any of its accuracy compared to the standardized method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric hydroxide, with little effort and high reliability. In a buffered solution, the phosphonate is brought into contact with the adsorbent using a rotator and then analyzed via a miniaturized phosphorus determination method.

This paper introduces a procedure to investigate the adsorption of phosphonates onto iron-containing filter materials, particularly granular ferric ...

Construction of a Low-cost Mobile Incubator for Field and Laboratory Use

Incubators are essential for a range of culture-based microbial methods, such as membrane filtration followed by cultivation for assessing drinking water quality. However, commercially available incubators are often costly, difficult to transport, not flexible in terms of volume, and/or poorly adapted to local field conditions where access to electricity is unreliable. The purpose of this study was to develop an adaptable, low-cost and transportable incubator that can be constructed using readily available components. The electronic core of the incubator was first developed. These components were then tested under a range of ambient temperature conditions (3.5 &#176;C - 39 &#176;C) using three types of incubator shells (polystyrene foam box, hard cooler box, and cardboard box covered with a survival blanket). The electronic core showed comparable performance to a standard laboratory incubator in terms of the time required to reach the set temperature, inner temperature stability and spatial dispersion, power consumption, and microbial growth. The incubator set-ups were also effective at moderate and low ambient temperatures (between 3.5 &#176;C and 27 &#176;C), and at high temperatures (39 &#176;C) when the incubator set temperature was higher. This incubator prototype is low-cost (&lt; 300 USD) and adaptable to a variety of materials and volumes. Its demountable structure makes it easy to transport. It can be used in both established laboratories with grid power or in remote settings powered by solar energy or a car battery. It is particularly useful as an equipment option for field laboratories in areas with limited access to resources for water quality monitoring.

This paper describes a method for building an adaptable, low-cost and transportable incubator for microbial testing of drinking water. Our design is based on widely available materials and can operate under a range of field conditions, while still offering the advantages of higher-end laboratory-based models.

Incubators are essential for a range of culture-based microbial methods, such as membrane filtration followed by cultivation for assessing drinking water ...

Colletotrichum fioriniae Development in Water and Chloroform-based Blueberry and Cranberry Floral Extracts

To accurately monitor the phenology of the bloom period and the temporal dynamics of floral chemical cues on fungal fruit rotting pathogens, floral extraction methods and coverslip bioassays were developed utilizing Colletotrichum fioriniae. In blueberry and cranberry, this pathogen is optimally controlled by applying fungicides during the bloom period because of the role flowers play in the initial stages of infection. The protocol detailed here describes how floral extracts (FE) were obtained using water-, chloroform-, and field rainwater-based methods for later use in corresponding glass coverslip bioassays. Each FE served to provide a different set of information: response of C. fioriniae to mobilized floral chemical cues in water (water-based), pathogen response to flower and fruit surface waxes (chloroform-based), and field-based monitoring of collected floral rainwater, moving in vitro observations to an agricultural setting. The FE is broadly described as either water- or chloroform-based, with an appropriate bioassay described to compensate for the inherent differences between these two materials. Rainwater that had run off flowers was collected in unique devices for each crop, alluding to the flexibility and application of this approach for other crop systems. The bioassays are quick, inexpensive, simple, and provide the ability to generate spatiotemporal and site-specific information about the presence of stimulatory floral compounds from various sources. This information will ultimately better inform disease management strategies, as FE decrease the time needed for infection to occur, thus providing insight into changing risks for pathogen infection over the growing season.

Colletotrichum fioriniae Development in Water and Chloroform-based Blueberry and Cranberry Floral Extracts

Here, bioassays designed to monitor the development of a fungal pathogen, Colletotrichum fioriniae, in the presence of blueberry or cranberry floral extracts on glass coverslips are described. Water-, chloroform-, and field rainwater- based floral extraction techniques are detailed as well as insight into how this information can be applied.

To accurately monitor the phenology of the bloom period and the temporal dynamics of floral chemical cues on fungal fruit rotting pathogens, floral ...

Leaf Area Index Estimation Using Three Distinct Methods in Pure Deciduous Stands

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for describing the vegetation structure in the fields of ecology, forestry, and agriculture. Therefore, procedures of three commercially used methods (litter traps, needle technique, and a plant canopy analyzer) for performing LAI estimation were presented step-by-step. Specific methodological approaches were compared, and their current advantages, controversies, challenges, and future perspectives were discussed in this protocol. Litter traps are usually deemed as the reference level. Both the needle technique and the plant canopy analyzer (e.g., LAI-2000) frequently underestimate LAI values in comparison with the reference. The needle technique is easy to use in deciduous stands where the litter completely decomposes each year (e.g., oak and beech stands). However, calibration based on litter traps or direct destructive methods is necessary. The plant canopy analyzer is a commonly used device for performing LAI estimation in ecology, forestry, and agriculture, but is subject to potential error due to foliage clumping and the contribution of woody elements in the field of view (FOV) of the sensor. Eliminating these potential error sources was discussed. The plant canopy analyzer is a very suitable device for performing LAI estimations at the high spatial level, observing a seasonal LAI dynamic, and for long-term monitoring of LAI.

An accurate estimation of leaf area index (LAI) is crucial for many models of material and energy fluxes within plant ecosystems and between an ecosystem and the atmospheric boundary layer. Therefore, three methods (litter traps, needle technique, and PCA) for taking precise LAI measurements were in the presented protocol.

Accurate estimations of leaf area index (LAI), defined as half of the total leaf surface area per unit of horizontal ground surface area, are crucial for ...

Measuring Phosphorus Release in Laboratory Microcosms for Water Quality Assessment

Phosphorus (P) is a critical limiting nutrient in agroecosystems requiring careful management to reduce transport risk to aquatic environments. Routine laboratory measures of P bioavailability are based on chemical extractions performed on dried samples under oxidizing conditions. While useful, these tests are limited with respect to characterizing P release under prolonged water saturation. Labile orthophosphate bound to oxidized iron and other metals can rapidly desorb to solution in reducing environments, increasing P mobilization risk to surface runoff and groundwater. To better quantify P desorption potential and mobility during extended saturation, a laboratory microcosm method was developed based on repeated sampling of porewater and overlying floodwater over time. The method is useful for quantifying P release potential from soils and sediments varying in physicochemical properties and can improve site-specific P mitigation efforts by better characterizing P release risk in hydrologically active areas. Advantages of the method include its ability to simulate in situ dynamics, simplicity, low cost, and flexibility.

Accurate quantification of phosphorus (P) desorption potential in saturated soils and sediments is important for P modeling and transport mitigation efforts. To better account for in situ soil-water redox dynamics and P mobilization under prolonged saturation, a simple approach was developed based on repeated sampling of laboratory microcosms.

Phosphorus (P) is a critical limiting nutrient in agroecosystems requiring careful management to reduce transport risk to aquatic environments. Routine ...