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.
