The overall goal of this standardized procedure is to screen water samples for endocrine active chemicals, using commercially-available, receptor-based cell assays. This method can help answer key questions in the water quality monitoring field, such as, are endocrine active chemicals actually present in the sample? The main advantage of this technique is that it can analyze for a group of endocrine active compounds much faster than the current chemical methods.
This technique can help diagnose water quality issues that arise from the presence of endocrine disrupting chemicals. This method provides insight into contaminants in water, but it can also be applied to other environmental matrices, such as sediment and tissues. Generally, individuals new to this technique may struggle, because it requires high precision when using pipetting techniques.
To begin this procedure, prepare the water sample in stock solutions of assay-specific medium and reference chemical, as detailed in the text protocol. Store the assay-specific medium at four degrees Celsius for long-term storage. Next, add 15 microliters of the appropriate reference chemical stock to 285 microliters of assay medium in a sterile tube.
Mix the sample by pipetting up and down. Then, add 200 microliters of 5%DMSO in assay medium to eight additional tubes. Transfer a 100-microliter aliquot from tube one to tube two, to begin a three-fold dilution series.
Mix the diluted sample in tube two thoroughly with a pipette. Then, transfer a 100-microliter aliquot from tube two to tube three. Continue this process for each tube.
Then, prepare a solvent control sample by mixing 10 microliters of DMSO in 190 microliters of assay medium. Next, collect and label four new tubes. Add 95 microliters of assay medium to the first tube.
Then, add five microliters of water extract and mix thoroughly. Add 50 microliters of 5%DMSO in assay medium to the other three tubes. Then, perform a two-fold dilution by transferring 50 microliters of solution from one tube to the next.
Begin by taking a vial of ER or GR division arrested cells out of the cryogenic freezer. Thaw the cells quickly by placing the vial in a 37-degree-Celsius water bath, with gentle agitation for two minutes. Then, decontaminate the vial with 70%Ethanol.
Place it in a class two biological safety cabinet. Open the vial using aseptic techniques and transfer the cells into 10 milliliters of assay medium. Centrifuge at 200 times g for five minutes.
When centrifugation is complete, use a sterile glass pipette to aspirate the supernatant. Then, re-suspend the cell pellet in six millileters of assay medium. Mix five microliters of cell suspension with five microliters of vital stain solution.
Then, add an aliquot into a counting chamber. Count the number of live cells using a light microscope or automated cell counter. Then, estimate the density of live cells in the cell suspension.
Dilute if necessary, to obtain a final density of 550, 000 live cells per milliliter of assay medium. Begin by creating a plate layout that includes a nine point assay specific calibration curve, QA/QC samples, and multiple dilutions for each water extract. Add 90 microliters of assay medium to the replicate cell free control wells.
Pour the cell suspension into a sterile pipetting reservoir. Then, using a multi-channel pipette, add 90 microliters of cell suspension to the other wells. Add 10 microliters of the diluted samples to the appropriate wells.
Cover the plate with a lid. Then, place the plate in a 5%C02 incubator at 37 degrees Celsius for 16 hours. Once incubation is complete, allow the plate to equilibrate to room temperature.
Then next steps should be conducted in the absence of direct light. Prepare a six x loading solution according to the manufacturer's instructions. Then, add 20 microliters of the loading solution to each well.
Add 10 microliters of cell viability reagent to each well, to evaluate the cytotoxicity of the diluted water extract. Seal the plate with aluminum adhesive film. Then, incubate the plate in the dark at room temperature for two hours.
Once incubation is complete, add the plate to the microplate reader with bottom reading capabilities. Measure the fluorescence and analyze the data as detailed in the text protocol. In this study, four 24-hour composite samples of treated municipal waste water effluent, six grab samples of surface water from freshwater systems in Southern California, and a field blank consisting of ultra pure water were analyzed.
ER activity was detected in seven of the 10 representative water samples, while GR activity was detected in four. The QA/QC performance guidelines for the estrogen receptor and glucocorticoid receptor assays can be seen here. The study results for cytotoxicity and sample precision fell well within acceptance criteria, validating the water sample measurements.
The highest bioassay equivalent concentrations were found in the secondary effluents, while the GR activity of the tertiary effluent was found to be below the bio assay limit of detection. Likewise, most surface water samples exhibited no GR activity above the limit of detection and had much lower levels of ER activity than the secondary effluents. The majority of waste water treatment plant effluent samples showed a dose response above the limit of detection for both ER and GR bioassays.
The representative results for the mean blue/green fluorescence ratio of these samples, plotted against the relative enrichment factor, can be seen here. This technique can be done in 24 hours, if performed properly. While attempting it, it's important to follow all the QA/QC guidelines.
This technique paved the way for managers to explore alternative approaches for monitoring endocrine disrupting chemicals in water. Don't forget that working with sodium azide can be hazardous. Precautions should be taken, such as working in a properly functioning fume hood.
