Summary
The present protocol describes a simple and efficient method for the long-distance transport of perfluoroalkyl acids in wheat.
Abstract
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.
Introduction
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 µg/kg and 0.003-162 µg/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.
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Protocol
Wheat seeds, Triticum aestivum L., were procured (see Table of Materials) and used for the present study.
1. Wheat seedling germination and hydroponic culture
- Select similar-sized wheat seeds and disinfect them for 15 min with 8% (w/w) hydrogen peroxide solution.
- 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.
- Select approximately nine germinated seedlings of uniform size and transfer them to plastic beakers with 250 mL of nutrient solution (1/4 strength of Hoagland's solution; its chemical composition is shown in Table 1).
NOTE: Out of the nine seeds, three each were selected for blank, PFOA, and PFOS, respectively. - Cultivate the seedlings in growth chambers for 7 days before exposure with a cycle of 14 h at 22 °C and 10 h at 27 °C.
2. The root splitting experiment
- Carry out the seedling cultivation in two 50 mL centrifuge tubes (A and B).
NOTE: In centrifuge tube A, 50 mL of sterile 1/4 strength Hoagland's solution was present, and the same amount of nutrient solution was present in centrifuge tube B.- Dissolve the commercial PFOA and PFOS (see Table of Materials) in methanol and dilute them with the sterile nutrient solution as the stock solution. Then, add the stock solution to tube B at a PFOA/PFOS concentration of 100 µg/L.
- Perform the treatments in triplicates with a blank control to monitor the background contamination. A schematic diagram of the split-root exposure experiments is shown in Figure 1.
- Separate the whole roots of the wheat seedling using tweezers into two equal parts so that the roots are still hooked to the same shoot and carefully insert them into tubes A and B, respectively.
- Seal the two tubes with aluminum foil and culture them in an incubator for 7 days. Maintain the same incubation conditions as stated in step 1.4.
- Collect the wheat seedlings after 7 days of culture and separate the wheat into three parts: shoots and roots cultured in the spiked solution of PFAAs and unspiked solution, respectively, using sterilized scissors.
- Freeze-dry the plant samples in a lyophilizer at −55 °C for 48 h.
- Homogenize and weigh the root and shoot samples. Collect the spiked and unspiked solution samples.
3. Extraction of PFOA and PFOS from plant tissues
- Add 2 mL of sodium carbonate buffer (0.25 mol/L), 1 mL of tetrabutylammonium hydrogen sulfate (0.5 mol/L), and 5 mL of methyl tert-butyl ether (see Table of Materials) to a 15 mL polypropylene tube, including the homogenized root or shoot.
- Shake the tube at 250 rpm for 20 min and centrifuge at 2,000 x g for 10 min at room temperature to obtain the supernatant organic phase. Perform the extraction process twice.
- Mix the collected extracts, vaporize to dryness in a gentle nitrogen (N2) stream, and then reconstitute with 5 mL of methanol and vortex them, maintaining the same speed for approximately 30 s.
- Condition the pesticarb cartridge (see Table of Materials) with 5 mL of 0.1% NH4OH in methanol, 5 mL of water, and 5 mL of methanol.
- Add the 5 mL of extracting methanol solution through the pesticarb cartridge (500 mg/6 mL) to remove the pigment, elute the cartridge with 5 mL of methanol, and collect in the same tubes.
- Evaporate the collected 10 mL of methanol solution to almost dryness and reconstitute with 200 µL of methanol, followed by vortexing and centrifugation at 10,000 x g for 20 min at room temperature.
4. Sample preparation from the nutrient solution
- Condition with 5 mL of methanol and 5 mL of water to activate the polar enhanced polymer (PEP) extraction cartridge (60 mg/g, 3 mL) (see Table of Materials).
- Add 1 mL of the spiked solution or 50 mL of the unspiked solution samples (step 2.6) through the cartridge, respectively.
- Elute the target PFAAs with 10 mL of methanol, evaporate the extract with gentle N2, and then reconstitute with 200 µL of methanol for analysis.
5. Instrumental analysis
- Use an ultra-performance liquid chromatography UPLC coupled with tandem mass spectrometry (LC-MS/MS) for quantification of the target PFAAs in multi-reaction mode (MRM) and negative electrospray ionization (ESI-) (see Table of Materials).
- Inject 10 µL of samples and separate the target PFAAs using a C18 liquid chromatographic column (1.7 µm, 2.1 mm x 50 mm, see Table of Materials), and use 2 mM ammonium acetate in water (phase A) and methanol (phase B) as the mobile phase for UPLC, with a flow rate of 0.3 mL/min. Maintain the column temperature at 50 °C.
NOTE: The ion transitions of PFOA and PFOS are 413 to 369 and 499 to 80, respectively. The gradient elution program and the LC-MS/MS instrumental parameters for quantification of the target PFAAs are listed in Table 2. - Process the data with the data analysis software (see Table of Materials).
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Representative Results
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 ± 0.02 ng/g and 0.64 ng/g ± 0.05 ng/g dry weight (dw) (n = 3), respectively, which account for 1.5% and 1.8% of the amount of accumulation in the whole wheat plant. This result suggests that PFOS and PFOA could experience long-distance transport through the phloem from the shoot to the root. It was worth noting that PFOS and PFOA were also found in the unspiked nutrient solution with a concentration of 17.8 ng/L ± 0.28 ng/L and 28.5 ng/L ± 5.9 ng/L (n = 3), respectively, which suggests that PFOA and PFOS could pass through the root Casparian strip19,20 and be released into the ambient environment. The results from the present work provide solid evidence that long-distance transport is also an important pathway for wheat to eliminate PFAAs.
Figure 1: Schematic diagram of the split-root experiments. The whole roots of the wheat seedling were equally separated into two parts and carefully inserted into tubes (A) and (B). A hydroponic plastic root retainer with a matching sponge was used to connect the two tubes and fix the seedling. The blank group is set to the solution in A; B tubes are all unspiked. Please click here to view a larger version of this figure.
Figure 2: Distribution of PFOA and PFOS concentrations in the split-root experiment after 7 days of exposure. The spiked solution (solution containing target PFAAs), spiked root (root in PFAAs-spiked solution), and shoot of (A) PFOA and (C) PFOS. Unspiked solution (solution without PFAAs) and unspiked root (root in unspiked solution) of (B) PFOA (D) and PFOS. The error bars denote the standard deviations (n = 3). Abbreviation: dw = dry weight. Please click here to view a larger version of this figure.
| Component | Molecular weight | Conc. of stock solution (g/L) | Volume of stock solution per litre of final solution (mL) | Element | Final conc. of element in nutrient solution (ppm) |
| Macronutrients | |||||
| KNO3 | 101.1 | 101.1 | 1.25 | K | 56 |
| Ca(NO3)2.4H2O | 236.16 | 236.16 | 1 | N | 58.75 |
| NH4H2PO4 | 115.08 | 115.08 | 0.5 | Ca | 40 |
| MgSO4.7H2O | 246.48 | 246.48 | 0.25 | P | 15.5 |
| Mg | 6 | ||||
| S | 8 | ||||
| Iron (EDTA-FeNa) | |||||
| EDTA-FeNa | 367.05 | 7.342 | 0.25 | Fe | 0.28 |
| Micronutrients | |||||
| H3BO3 | 61.83 | 2.86 | B | 0.125 | |
| MnCl2.4H2O | 197.91 | 1.81 | Mn | 0.125 | |
| ZnSO4.7H2O | 287.56 | 0.22 | Zn | 0.0125 | |
| CuSO4 | 159.61 | 0.051 | Cu | 0.005 | |
| H2MoO4(85% MoO3) | 161.97 | 0.017 | Mo | 0.0025 |
Table 1: Chemical compositions of the 1/4 strength Hoagland nutrient solution. This nutrient solution represents the unspiked solution in the split-root experiment.
| Column Temperature | 50 °C | |||||
| Mobile phase | 2 mM ammonium acetate in water pH = 9 (A) and methanol (B) | |||||
| Gradient | Time (min) | Flow rate (mL/min) | A (%) | B (%) | ||
| Initial | 0.3 | 75 | 25 | |||
| 0.5 | 0.3 | 75 | 25 | |||
| 5 | 0.3 | 15 | 85 | |||
| 5.1 | 0.3 | 0 | 100 | |||
| 7 | 0.3 | 0 | 100 | |||
| 7.1 | 0.3 | 75 | 25 | |||
| 9 | 0.3 | 75 | 25 | |||
| Mass parameters | Capillary voltage: -1.5 kV | |||||
| Desolvation temperature 500 °C | ||||||
| Desolvation gas flow: 1000 L/h | ||||||
| Cone gas flow: 150 L/h | ||||||
| Multiple | Compounds | Parent Ions | Product Ions (m/z) | |||
| reaction | (m/z) | |||||
| monitoring | ||||||
| (MRM) | PFOA | 413 | 369 | |||
| transitions | PFOS | 499 | 80 | |||
Table 2: LC-MS/MS instrumental parameters for quantification of the target PFAAs.
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Discussion
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 was split in half, if the initial concentration of the spiked solution is relatively low, the less biomass obtained from the final treatment may result in the concentration of PFAAs in the roots cultured in the unspiked solution being below the limit of the detection. In addition, due to the short exposure time, the transport of PFAA from the roots to the edible parts of wheat could not be determined. The split-root experiment could only analyze the phloem transport of PFAAs with different properties within plants16.
This method is of great significance for understanding the long-distance transport12,13 of pollutants in plant tissues. According to the results, PFAAs can be taken up by roots and transported to shoots mainly through the xylem; however, it is to be noted that they could be translocated from leaves to edible tissues, as well as from shoots to roots through the phloem, which is important for the assessment of their potential risk of translocation in plants. Furthermore, the translocation of PFAAs from the aboveground tissues to roots and then release into the ambient environment provides solid evidence for the elimination pathways of PFAAs in plants.
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Disclosures
The authors have nothing to disclose.
Acknowledgments
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).
Materials
| Name | Company | Catalog Number | Comments |
| ACQUITY UPLC BEH C18 column | Waters, Milford, MA | Liquid chromatographic column | |
| Cleanert PEP cartridge | Bonna- Angel Technologies, China | Solid phase extraction column | |
| Clearnert Pesticarb cartridge | Bonna- Angel Technologies, China | Solid phase extraction column | |
| LC-MS/MS(Waters Acquity UPLC i-Class Coupled to Xevo TQ-S) | Waters, Milford, MA | Liquid chromatography and mass spectrometry | |
| Lyophilizer | Boyikang Instrument Ltd., Beijing, China | FD-1A50 | Freeze-dried sample |
| Masslynx | Waters, Milford, MA | data analysis software | |
| Methyl tert-butyl ether | Sigma-Aldrich Chemical Co. (St. Louis, US) | use for extracting target compounds from plant tissues | |
| MPFAC-MXA | Wellington Laboratories (Ontario, Canada) | PFACMXA0518 | the internal standards |
| PFAC-MXB | Wellington Laboratories (Ontario, Canada) | PFACMXB0219 | mixture of PFAA calibration standards |
| PFOA | Sigma-Aldrich Chemical Co. (St. Louis, US) | 335-67-1 | a represent PFAAs |
| PFOS | Sigma-Aldrich Chemical Co. (St. Louis, US) | 2795-39-3 | a represent PFAAs |
| Sodium carbonate buffer | Sigma-Aldrich Chemical Co. (St. Louis, US) | use for extracting target compounds from plant tissues | |
| Tetrabutylammonium hydrogen sulfate | Sigma-Aldrich Chemical Co. (St. Louis, US) | use for extracting target compounds from plant tissues | |
| Wheat seeds | Chinese Academy of Agricultural Sciences (Beijing,China) | Triticum aestivum L. |
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