Name: investigating long-distance transport of perfluoroalkyl acids in wheat via a split-root exposure technique
Uploaded: 2022-09-28T14:05:00
Description: Watch this Scientific Journal Video about Investigating Long-Distance Transport of Perfluoroalkyl Acids in Wheat via a Split-Root Exposure Technique at JoVE.com

We gratefully acknowledge financial support from the Natural Science Foundation of China (NSFC 21737003), Chinese Universities Scientific Fund (No. 2452021103), and Chinese Postdoctoral Science Foundation (No. 2021M692651, 2021M702680).

Wheat seeds, Triticum aestivum L., were procured (see Table of Materials) and used for the present study.
1. Wheat seedling germination and hydroponic culture
<ol>
	<li>Select similar-sized wheat seeds and disinfect them for 15 min with 8% (w/w) hydrogen peroxide solution.</li>
	<li>Rinse the disinfected seeds with deionized water thoroughly, and then place them on humid filter paper in the dark at room temperature to germinate for 5 days.</li>
	<li...

<ol>
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To ensure the accuracy of this method, careful operation must be taken to ensure that the spiked solution in tube B does not contaminate the unspiked solution in tube A. The given concentration of target PFAAs in the present study was relatively higher than their concentration in the real environment, ensuring to monitor target PFAAs in wheat and unspiked solution using LC-MS/MS. 
There are limitations to this method. Since only one wheat seedling was used in each treatment group and the root ...

Perfluoroalkyl acids (PFAAs) are widely utilized in various commercial and industrial products due to their excellent physicochemical properties, including surface activity and thermal and chemical stability1,2,3. Perfluorooctane sulfonic acid (PFOS) and perfluorooctane acid (PFOA) are the two most important PFAAs used worldwide4,5,6, although these compounds were listed in the international Stockholm Convention in 2009 and 20197,8, respectively. Due to their persistence and widespread use, PFOS and PFOA have been widely detected in various environmental matrices. The concentrations of PFOA and PFOS in surface water from different worldwide rivers and lakes are 0.15-52.8 ng/L and 0.09-29.7 ng/L, respectively9. Due to the use of groundwater or reclaimed water for irrigation and also using biosolids as fertilizer, PFOA and PFOS are widely present in the soil, ranging between 0.01-123 &#181;g/kg and 0.003-162 &#181;g/kg, respectively10, which could introduce a large amount of PFAAs into plants and pose potential risks to human health. The PFAA (C4-C8) concentrations in agricultural soil and grain (wheat and maize) show a positive linear correlation11. Therefore, it is imperative to investigate the accumulation and translocation of PFAAs within plants.
The translocation of PFAAs in plants firstly occurs from the roots to the aboveground tissues, and the translocation of PFAAs from the roots to the edible tissues is regarded as long-distance transport12,13. Previous studies have detected bisphenol A, nonylphenol, and natural estrogens in vegetables and fruits14, which implies that these chemicals might migrate via the phloem. Hence, uncovering the translocation of PFAAs in plants is important to assess their potential risk. However, the accumulation and translocation of PFAAs are impacted by their bioavailability in the soil, so it is not easy to evaluate the translocation ability of target PFAAs in plants. Additionally, hydroponic experiments are generally limited by several factors, making it more difficult to acquire the edible tissues of plants. Typically, the phloem was collected directly from plants to observe the translocation of organic compounds through long distances in plants, whereas it is difficult to acquire phloems from plant seedlings15. Hence, a simple and effective method, the split-root technique, was introduced to study the translocation of PFAAs in plants during relatively short-term exposure. As for the split-root investigation, the roots in one plant seedling are separated into two parts; one part is put into the nutrient solution containing target PFAAs (tube A), and the other is placed in the nutrient solution in the absence of PFAAs (tube B). After exposure for several days, the PFAAs in tube B are measured by LC-MS/MS. The concentration of PFAAs in tube B discloses the translocation potential of PFAAs through the phloem within plants16,17,18.
The split-root experiment has been reported for studying the long-distance translocation of many compounds in plants, such as CuO nanoparticles17, steroid estrogens18, and organophosphate esters16. These studies provided evidence that these compounds could transfer via the phloem to the edible parts of plants. However, whether PFAAs could aid in translocation in plants and the impact of compound properties need to be further explored. Based on these reports, the split-root experiment was conducted in the present study to disclose the long-distance transport of PFAAs in wheat.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ACQUITY UPLC BEH C18 column</td>
			<td>Waters, Milford, MA</td>
			<td></td>
			<td>Liquid chromatographic column</td>
		</tr>
		<tr>
			<td>Cleanert PEP cartridge</td>
			<td>Bonna- Angel Technologies, China</td>
			<td></td>
			<td>Solid phase extraction column</td>
		</tr>
		<tr>
			<td>Clearnert Pesticarb cartridge</td>
			<td>Bonna- Angel Technologies, China</td>
			<td></td>
			<td>Solid phase extraction column</td>
		</tr>
		<tr>
			<td>LC-MS/MS(Waters Acquity UPLC i-Class Coupled to Xevo TQ-S)</td>
			<td>Waters, Milford, MA</td>
			<td></td>
			<td>Liquid chromatography and mass spectrometry</td>
		</tr>
		<tr>
			<td>Lyophilizer&#160;</td>
			<td>Boyikang Instrument Ltd., Beijing, China</td>
			<td>FD-1A50</td>
			<td>Freeze-dried sample</td>
		</tr>
		<tr>
			<td>Masslynx</td>
			<td>Waters, Milford, MA</td>
			<td></td>
			<td>data analysis software</td>
		</tr>
		<tr>
			<td>Methyl tert-butyl ether</td>
			<td>Sigma-Aldrich Chemical Co. (St. Louis, US)</td>
			<td></td>
			<td>use for extracting target compounds from plant tissues</td>
		</tr>
		<tr>
			<td>MPFAC-MXA</td>
			<td>Wellington Laboratories (Ontario, Canada)</td>
			<td>PFACMXA0518</td>
			<td>the internal standards</td>
		</tr>
		<tr>
			<td>PFAC-MXB</td>
			<td>Wellington Laboratories (Ontario, Canada)</td>
			<td>PFACMXB0219</td>
			<td>mixture of PFAA calibration standards</td>
		</tr>
		<tr>
			<td>PFOA</td>
			<td>Sigma-Aldrich Chemical Co. (St. Louis, US)</td>
			<td>335-67-1</td>
			<td>a represent PFAAs</td>
		</tr>
		<tr>
			<td>PFOS</td>
			<td>Sigma-Aldrich Chemical Co. (St. Louis, US)</td>
			<td>2795-39-3</td>
			<td>a represent PFAAs</td>
		</tr>
		<tr>
			<td>Sodium carbonate buffer</td>
			<td>Sigma-Aldrich Chemical Co. (St. Louis, US)</td>
			<td></td>
			<td>use for extracting target compounds from plant tissues</td>
		</tr>
		<tr>
			<td>Tetrabutylammonium hydrogen sulfate</td>
			<td>Sigma-Aldrich Chemical Co. (St. Louis, US)</td>
			<td></td>
			<td>use for extracting target compounds from plant tissues</td>
		</tr>
		<tr>
			<td>Wheat seeds</td>
			<td>Chinese Academy of Agricultural Sciences (Beijing,China)&#160;</td>
			<td></td>
			<td>Triticum aestivum L.</td>
		</tr>
	</tbody>
</table>

investigating long-distance transport of perfluoroalkyl acids in wheat via a split-root exposure technique

Large amounts of perfluoroalkyl acids (PFAAs) have been introduced into the soil and accumulated by plants, posing potential risks to human health. It is imperative to investigate the accumulation and translocation of PFAAs within plants. Long-distance transport is an important pathway for PFAAs transferred from the plant leaves to the edible tissues through the phloem. However, it was previously difficult to assess the translocation potential of organic contamination in a short-term exposure period. The split-root experiment provides a solution to effectively uncover the long-distance translocation of PFAAs using a hydroponic experiment, which, in this study, was carried out in two 50 mL centrifuge tubes (A and B), of which centrifuge tube A had 50 mL of one-quarter strength Hoagland sterile nutrient solution, while centrifuge tube B had the same amount of nutrient concentration, and the target PFAAs (perfluorooctane sulfonic acid, PFOS, and perfluorooctane acid, PFOA) added at a given concentration. A whole wheat root was manually separated into two parts and inserted carefully into tubes A and B. The concentration of PFAAs in the roots, shoots of wheat, and solutions in tubes A and B were evaluated using LC-MS/MS, respectively, after being cultured in an incubator for 7 days and harvested. The results suggested that PFOA and PFOS experience a similar long-distance transport process through the phloem from the shoot to the root and could be released into the ambient environment. Thus, the split-root technique can be used to evaluate the long-distance transport of different chemicals.

The split-root experiment investigated the long-distance transport of PFAAs in wheat. As shown in Figure 2A,C, both PFOA and PFOS could be taken up by the wheat root and transferred to the shoot. PFOS and PFOA were not detected in the wheat root and solution in tube A of the blank control. It was found that PFOS and PFOA were detected in the wheat roots cultured in the unspiked solution, with a concentration of 0.26 ng/g &#177; 0.02 ng/g and 0.64 ng/g &#177; 0.05 ng/g dry we...

Watch this Scientific Journal Video about Investigating Long-Distance Transport of Perfluoroalkyl Acids in Wheat via a Split-Root Exposure Technique at JoVE.com

Investigating Long-Distance Transport of Perfluoroalkyl Acids in Wheat via a Split-Root Exposure Technique

The present protocol describes a simple and efficient method for the long-distance transport of perfluoroalkyl acids in wheat.

Investigating Long-Distance Transport of Perfluoroalkyl Acids in Wheat via a Split-Root Exposure Technique

Large amounts of perfluoroalkyl acids (PFAAs) have been introduced into the soil and accumulated by plants, posing potential risks to human health. It is ...

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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