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Investigating Long-Distance Transport of Perfluoroalkyl Acids in Wheat via a Split-Root Exposure Technique
Chapters
Summary September 28th, 2022
The present protocol describes a simple and efficient method for the long-distance transport of perfluoroalkyl acids in wheat.
Transcript
The phloem was collected directly from plants to observe the translocation of organic compounds through long distances, whereas it is difficult to acquire phloem from plant seedlings. A simple and effective method, the split-root technique, was introduced to study the translocation of PFAAs in plants, during relatively short-term exposure. This method can also disclose the long-distance transport of different compounds in plants.
The given concentration of target PFAAs must be relatively higher than their concentration in the real environment, ensuring the monitoring of the target PFAAs in wheat in unspiked solution. Begin by selecting the similar-sized Triticum aestivum, or wheat seeds, and disinfect them for 15 minutes with an 8%hydrogen peroxide solution. Thoroughly rinse the disinfected seeds with deionized water.
Place the seeds on the humid filter paper in the dark at room temperature for 5 days for germination. Select approximately 9 germinated seedlings, of uniform size, and transfer them to plastic beakers with 250 milliliters of 1/4 strength of Hoagland's solution. Before the treatment, cultivate the seedlings in growth chambers for 7 days with a cycle of 14 hours at 22 degrees Celsius, and 10 hours at 27 degrees Celsius.
For seedling cultivation, prepare 2 centrifuge tubes, A and B, each containing 50 milliliters of 1/4 strength of Hoagland's solution. Dissolve the commercial perfluorooctane acid, or PFOA, and perfluorooctanesulfonic acid, or PFOS, in methanol, and dilute it with the sterile nutrient solution as the stock solution, then add the stock solution to tube B at 100 micrograms per liter of PFOA and PFOS. Next, separate the whole roots of a wheat seedling into 2 equal parts using tweezers with the roots connected to the same shoot, then insert them into tubes A and B respectively.
Seal the 2 tubes with aluminum foil before culturing them in an incubator for 7 days with a cycle of 14 hours at 22 degrees Celsius, and 10 hours at 27 degrees Celsius. After 7 days, collect the wheat seedlings and separate them into 3 parts using scissors, such as shoots and roots cultured in the spiked solution of perfluoroakyl acids, or PFAAs, in unspiked solution. Freeze dry the plant samples in a lyophilizer at minus 55 degrees Celsius for 48 hours.
After 2 days, homogenize. Weigh the root and shoot samples. Collect the spiked and unspiked solution samples.
In a 15-milliliter polypropylene tube, add 2 milliliters of sodium carbonate buffer, 1 milliliter of tetrabutylammonium hydrogen sulfate, 5 milliliters of methyl tert-butyl ether, and the homogenized root or shoot. When done, shake the tube at 250 RPM for 20 minutes, before centrifugation at 2000 g for 10 minutes at room temperature to obtain the supernatant organic phase. After the second extraction, pool the similar treatment extracts and vaporize them to dryness in a gentle nitrogen stream, then reconstitute with 5 milliliters of methanol and vortex them, maintaining the same speed for approximately 30 seconds.
Condition the PestiCarb cartridge with 5 milliliters of 0.1%ammonium hydroxide in methanol, 5 milliliters of water and 5 milliliters of methanol. Add 5 milliliters of extracting methanol solution through the PestiCarb cartridge to remove the pigment. Elute the cartridge with 5 milliliters of methanol and collect in the sample tubes.
Evaporate the collected 10 milliliters of methanol solution to dryness and reconstitute with 200 microliters of methanol before vortexing, then centrifuge the sample at 10, 000 g for 20 minutes at room temperature. Condition the polar-enhanced polymer extraction cartridge with 5 milliliters of methanol and 5 milliliters of water to activate. Add 1 milliliter of the spiked solution or 50 milliliters of the un spiked solution samples through the cartridge.
Elude the target PFAAs with 10 milliliters of methanol. Evaporate the extract with a gentle nitrogen stream and then reconstitute the evaporated extract with 200 microliters of methanol for analysis. The split-root experiment investigated the long-distance transport of PFAAs in wheat.
It was observed that PFOA and PFOS were taken up by the wheat root with a concentration of 11.94 nanograms per gram and 30.67 nanograms per gram dry weight in spiked solution respectively, and were transferred to the shoots with a concentration of 5.01 nanograms per gram and 4.17 nanograms per gram dry weight. It was found that PFOA and PFOS were detected in wheat roots cultured in the unspiked solution with a concentration of 0.26 nanograms per gram and 0.64 nanograms per gram dry weight respectively. It suggested that PFOA and PFOS could experience long-distance transport through the phloem from the shoot to the root.
It was noted that PFOA and PFOS were also found in the unspiked nutrient solution with a concentration of 17.8 nanograms per liter and 28.5 nanograms per liter respectively, suggesting that PFOA and PFOS could pass through the root Casparian strip. Precaution must be taken to ensure that the spiked solution in tube B does not contaminate the unspiked solution in tube A.The visual demonstration of this method can provide a simple operation to disclose the long-distance transport of PFAAs in wheat.
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