GenX uptake by wheat and flooded and non-flooded rice: greenhouse experiment.

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Amnah Al Zbedy, Viktoria Müller, Andrew Kindness, Rainer Ebel, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-2889643/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 04 Dec, 2023 Read the published version in Environmental Science and Pollution Research → Version 1 posted 5 You are reading this latest preprint version Abstract GenX (hexafluoropropylene oxide dimer acid) belongs to the group of per- and poly-fluoroalkyl substance (PFAS) compounds introduced to replace perfluorooctanoic acid (PFOA), which has been phased out in industrial and consumer product formulations. While GenX has been investigated in lab animals, there is limited information available regarding its uptake and translocation in wheat and rice. This study reports on a greenhouse experiment in which wheat and rice grown under flooded and non-flooded conditions were exposed to two GenX concentrations in the soil (0.4 mg kg − 1 and 2 mg kg − 1 ). GenX was analysed in the soil, porewater and shoots using targeted liquid chromatography-tandem mass spectroscopy (LC-MS/MS) analysis. Extractable organic fluorine (EOF) was determined using high-resolution continuum source graphite furnace molecular absorption spectrometry (HR GFMAS). Results showed that different species took up different amounts of GenX. The GenX in rice shoots was found to be 2.34 (± 0.45) µg g − 1 and 4.11 (± 0.87) µg g − 1 under flooded and non-flooded conditions, respectively, at a low exposure level. At high exposure, the GenX concentrations in flooded and non-flooded rice shoots increased threefold to 10.4 (± 0.41) and 13.4 (± 0.72) µg g − 1 , respectively. Wheat shoots showed similar concentrations and increases between low- and high-level exposure. The translocation factor was significantly higher ( P = 0.013) in non-flooded rice compared to flooded rice. The GenX bioaccumulation behaviours under the same culture conditions (e.g. temperature, humidity, light, same GenX concentration in the soil) were significantly different in non-flooded and flooded rice ( P < 0.001). Non-flooded rice plants displayed a higher level of GenX bioaccumulation than flooded ones. Following exposure to GenX, flooded rice plants showed a reduction in biomass (25%) compared to the control plants ( P < 0.014). Our findings indicate that GenX is a bioaccumulative compound, the presence of which likely inhibits the growth of plants. PFAS plant uptake rice agriculture practise wheat EOF Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Hexafluoropropylene oxide dimer acid (HFPO-DA), a perfluoroalkyl ether carboxylic acid also known as GenX, has been used to aid polymerisation in the production of high-performance fluoropolymers (Bokkers et al. 2018 ). It was introduced as an alternative for perfluorooctanoic acid (PFOA), which was used from 1970 to 2012 (Rivm and Poll). Organisations such as the Stockholm Convention and the European Chemical Agency began investigating PFOA as a hazardous contaminant due to its persistence, bioaccumulation and toxicity (Brandsma et al. 2019 ), leading to its restriction in 2017 by the European Union (Brendel et al. 2018 ). This restriction gave rise to so-called replacement chemistries, including GenX, which was introduced for its lower bioaccumulation potential compared to PFOA, despite the limited toxicokinetic data available for GenX (Heydebreck et al. 2015 ; Zhang et al. 2021 ). The United States Environmental Protection Agency (US-EPA) recently published toxicity assessments for PFOA (2016) and perfluorooctanesulfonic acid PFOS (2016), showing that chronic oral reference doses (RfD) for PFOS and PFOA are higher than those for GenX. Based on the available animal toxicity data, it appears that GenX has greater hazardous potency than PFOA and PFOS (Wang et al. 2015 ; Gomis et al. 2018 ; US Environmental Protection Agency et al. 2021). GenX has been shown to have adverse implications in mice and zebrafish (Satbhai et al. 2022 ), such as inducing benign tumours, gene changes in the liver, thyroid hormone level disturbance and hepatocellular damage (Cannon et al. 2020 ). GenX has been detected in increasing amounts in drinking water and soil (Gebbink and van Leeuwen 2020 ). In 2015, GenX contamination was discovered near North Carolina’s Cape Fear River, downstream of a chemical manufacturing company (Cahoon 2019 ). GenX was also detected in plant leaves found within three kilometres of a fluoropolymer manufacturing Teflon plant in the Netherlands (Brandsma et al. 2019 ). It is unclear exactly how GenX and PFASs are absorbed by grass and foliage. The GenX concentrations seen in the grass and leaves gathered around the plant may be the consequence of air deposition, absorption from contaminated soil or both (Brandsma et al. 2019 ). It has also been found that perfluoroether chains are just as resistant to biotic and abiotic degradation as PFOA (Wang et al. 2015 ). While most studies have focused on the effects of GenX on animals, there is limited knowledge about GenX bioaccumulation and its adverse effects on plants. While research on the uptake and accumulation of GenX by plants is still limited, it is a growing area of interest and investigation. In recent research, the accumulation and toxicity of GenX and perfluorooctanoic acid (PFOA) have been studied and compared using Arabidopsis thaliana and Nicotiana benthamiana as model plants in a hydroponic system. Findings indicate that at concentrations between 20 and 200 mg L − 1 , GenX inhibited plant growth and lowered chlorophyll content and enzyme activity (Chen et al. 2020 ). Zhang et al. ( 2021 ) found that Carex comosa absorb nearly eight percent of the GenX in soil after 80 days of exposure (Zhang et al. 2021 ). Moreover, Zhi et al. ( 2022 ) studied the bioaccumulation of PFAS in spontaneous urban plants and reported that GenX had a lower bioaccumulation factor (0.66–2.5) than PFOA (3.5–10.5) in plant roots (Zhi et al. 2022 ). PFAS are typically analysed with targeted LC-MS/MS; however, only those that ionise easily using electrospray ionisation can be analysed. Extractable organic fluorine (EOF) or total fluorine analysis are broader PFAS analysis methods. Different instruments can be used for EOF and total fluorine analysis, such as combustion ion chromatography (CIC) and high-resolution graphite furnace molecular absorption spectrometry (HR-GFMAS). Although results obtained with the two different instruments are comparable (Gehrenkemper et al. 2021 ), HR-GFMAS analysis has been found to be more sensitive and less time consuming (Gehrenkemper et al. 2021 ). Mass balance analysis, meanwhile, combines the target analysis and EOF analysis to identify the fraction of PFAS that can and cannot be determined using the target method (Aro et al. 2021 ). Rice is one of the most representative foods among primary nutritional foods (Liu et al. 2018 ) and is consumed by more than fifty percent of people around the world (da Silva et al. 2018 ). Wheat is also a significant source of carbohydrates, fibre and vitamins worldwide (Shewry and Hey 2015 ). Although PFAS intake from rice and wheat consumption is a health concern, considering the uptake of legacy and replacement PFAS, few studies (Stahl et al. 2009 ; Lan et al. 2018 ; Kim et al. 2019 ) on the uptake and bioaccumulation of these chemicals in rice and wheat are available. The present research uses a mass balance approach to study the accumulation of GenX in plants grown on contaminated soil and to determine how much GenX contributes to EOF in wheat and rice. To our knowledge, this study is the first to investigate GenX uptake by wheat and rice from soil systems and the first to measure the total EOF in wheat and rice using high resolution graphite furnace molecular absorption spectrometry (HR-GFMAS) in addition to targeted LC-MS/MS. 2. Material and methods 2.1 Chemical reagents and laboratory materials The chemical 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy) propanoic acid GenX (97% purity) was obtained from SynQuest Laboratories. A PFAS standard solution (MPFAC-MXC) containing perfluorinated carboxylic acid (PFCA) and perfluoroalkanesulfonic acids PFSA was purchased from Wellington Laboratories. Ultrapure water with a resistivity of 18.2 MΩ cm was obtained from Smart2 Pure, Thermo Fisher Scientific (Loughborough, UK). Ammonium hydroxide (Merck) and HPLC-grade methanol (Honeywell Riedel-de Haen, Germany) were used for sample extraction, and Envi carb (Merck) was used for cleaning purposes. Acetonitrile (Honeywell Riedel-de Haen, Germany) was used to prepare the mobile phase for HPLC. For the HR-GFMAS analysis, BOC (Dublin, Ireland) provided 99.998% purity argon gas, and a W (Merck) standard solution was used for the graphite tube coating as a permanent modifier. Ca(NO 3 ) 2 x4H 2 O (VWR chemicals, Leicestershire, UK) was used as a forming reagent at a concentration of 1% Ca (w/v). PFOA, which was used as a calibration solution, was obtained from Sigma Aldrich (St Louis, MO, USA). 2.2 Soil properties and treatment Soil was collected from a field in Insch Aberdeenshire. Soil samples were taken from the top 10 cm of the field. The soil was air-dried for two weeks and sifted through a 2-mm sieve. Soil pH (7.29) was measured in deionised water with a soil-to-water ratio of 1:2.5 after shaking for 1 hour. The soil was then spiked with a stock solution of GenX to achieve two nominal concentrations of 0.4 mg kg − 1 (low level) and 2.0 mg kg − 1 (high level). The control soil was also spiked with methanol. Once the soils had been spiked, they were placed in a fume hood for solvent evaporation at room temperature. Once the solvent evaporated, the soil was incubated in darkness for 14 d at room temperature. Concentrations of GenX in the soil were determined after incubation but before adding plants to the soil (0 d) and at the end of the plant growth experiment (30 d). 2.3 Plant exposure in soil and sampling Plastic pots (0.9 L) were filled with 650 g of soil. Four groups of tests were conducted: 1) soil alone, spiked with water (control 1); 2) spiked soil alone, spiked with methanol (control 2); 3) and 4) spiked soil with low and high GenX concentrations, respectively. Each group contained four replicates for each plant species and water treatment. For the wheat and non-flooded rice experiments, a piece of filter paper was placed at the bottom of the pot to restrict soil loss. For the flooded rice experiment, the pots were lined with a plastic bag so the soil could be flooded without loss. Wheat (Tybalt) and rice (BJ 1) seeds were germinated for 3 d on a petri dish, with the wheat at room temperature and the rice at 25 o C. Ten seeds were transplanted into each pot. The pots were irrigated daily to maintain a moisture content at 80% water-holding capacity for the wheat and non-flooded rice and completely flooded for rice. To account for any spatial variations in light and temperature within the greenhouse, the pots were positioned at random each day. After twenty days, 2 mL of 10x Yoshida’s nutrient (goleman, daniel; boyatzis, Richard; Mckee et al. 1976) were added. This experiment was divided into two halves because the rice and wheat were grown in separate greenhouses for four weeks at the University of Aberdeen in Aberdeen, Scotland (Coordinates: 57.165°N 2.100°W). For the rice experiment, the day temperature was 28°C and the night temperature 25°C. Supplemental lights were turned on between 7 am and 7 pm if the natural light was less than 24,000 Lux. The wheat experiment was kept at 15°C. After 30 d, the shoots of wheat and rice were harvested, freeze-dried and homogenised, then stored in polypropylene (PP) vials at -20°C prior to analysis. Following the removal of the plants from each pot, all test soils were taken out and air- and freeze-dried for 48 hours at room temperature. The dried soil samples were stored in polypropylene (PP) tubes at -20°C before chemical analysis. 2.4 Measurements of plant height and porewater collection Rhizon samplers attached a 10 mL syringe were buried at a 45-degree angle in the soil to allow sampling of porewater. Samplers of the porewater was taken on first week transplanting (day 0) and at the end of experiment at day 30. Plant growth was determined by measuring plant height, dry shoot biomass and number of tillers. The measurements were performed on day 30. Shoots were cut at 3 cm above the soil level to avoid soil contamination and its dry weight biomass were determined using an analytical balance. 2.5 Analysis and extraction of PFAS 2.5.1 Extraction procedure The GenX in the plant was extracted using a previously described method (Blaine et al. 2013 ; Chen et al. 2020 ). As an extraction solvent, a mixture of 50:50 (v/v) DCM and MeOH with 1% ammonium hydroxide (v/v) was prepared. For the extraction, 1 mg of dried plant sample was placed into a 15-mL polypropylene tube, and 3 mL of the extraction solvent was added. The tube was then vortexed for 30 s, sonicated for 15 min at 30°C, then shaken on an orbital shaker for 1 hour. The supernatant was collected after centrifugation at 1,500 rpm for 10 min. This extraction process was repeated twice. The extracts were then pooled and dried under a gentle nitrogen stream. The dried extract was reconstituted with 1 mL of MeOH, then mixed with 50 mg of ENVI-Carb for 20 s for clean-up. This was followed by centrifugation at 13,000 rpm for 10 min. Then, 450 µL of the extract was transferred into HPLC vials. Finally, 50 µL of the 50 µL L − 1 isotopically mass-labelled standard was introduced to enable target analysis using LC-MS/MS. Soil samples were extracted by transferring a 0.5 g aliquot into a 50 mL polypropylene vial, to which a solution containing an isotopically labelled surrogate standard and 3 mL of 0.1% ammonium hydroxide (NH 4 OH) in methanol was added. This was then sonicated for 15 min at 30°C, as per the established protocols (Higgins et al. 2005 ; Houtz et al. 2013 ; Zhang et al. 2021 ). The solution was then shaken for 1 hour on an orbital shaker. The supernatant was collected after centrifugation at 1,500 rpm for 10 min. This extraction process was repeated twice. All extracts were combined, dried under a gentle nitrogen stream, reconstituted in 1 mL methanol and cleaned with 50 mg ENVI-Carb. Then, 450 µL was taken into HPLC vials, and 50 µL of the 50 µL L − 1 isotopically mass-labelled standard was added for target analysis using LC-MS/MS. The porewater samples were analysed directly via LC-MS/MS. Prior to injection, each vial was centrifuged for 20 min at 2,700 rpm. An aliquot (225 µL) of the supernatant was then removed and transferred to a PP microcentrifuge tube containing 250 µL of methanol. Then, 50 µL of the 50 µL L − 1 isotopically mass-labelled standard was added for target analysis using LC-MS/MS. 2.6 Instrumentation 2.6.1 Target PFAS Agilent 1200 infinity HPLC (Agilent Technologies, Germany), coupled with Agilent 6465 Triple Quadrupole MS/MS (Agilent Technologies, Germany), operated in negative electrospray ionisation (ESI) mode. PFAS separation was performed using 5 mM ammonium acetate (CH 3 COONH 4 ) in reagent water, and 100% LCMS-grade acetonitrile was used as the mobile phase of the analysis of PFAS ionic compounds. Ten µL of the extract was automatically injected onto a BrownLee SPP C18 column (2.7 µm, 3 x 100 mm, PerkinElmer, UK) at 30°C. Chromatograms were recorded using multiple reaction monitoring (MRM) mode. A list of the analytes, transitions and optimised MRM conditions and LC method used can be found in this study’s supplementary materials (Table S1 -S3). 2.6.2 Extractable organic fluorine (EOF) The EOF were analysed according to the method described by Akhdhar et al. (Akhdhar et al. 2019 ). All measurements were performed using an Analytik Jena ContrAA 700 High Resolution Continuum Source Graphite Furnace Atomic Absorption Spectrometer (HR GFAAS) with a transversely heated graphite tube atomizer. A 300W xenon short-arc lamp was used as the instrument’s continuous radiation source for wavelengths between 185 and 900 nm. A charge-coupled device (CCD) array detector with 588 pixels, 200 and a high-resolution double echelle monochromator were used for analytical purposes. The measurements were conducted using coated graphite tubes with an integrated PIN platform (Analytical Jena part No. 407-A81.025). All fluorine measurements were performed three times at a wavelength of 606.417 nm to monitor the absorption of the formed CaF. The graphite furnace platform was coated with tungsten (W), while calcium (Ca) was utilized as the forming reagent.). Table S4 details the temperature programme used. 2.6.3 Fluorine mass balance analysis Fluorine mass balance analysis was conducted by converting the concentration of PFAS analyte in the samples measured by LCMS/MS into the corresponding fluorine concentrations equivalents. This was then compared to the fluorine concentrations obtained from the EOF analysis using HR-GFMAS. 2.7 Quality control (QC) and statistical analysis 2.7.1 Targeted LC-MS/MS analysis A 4- to 5-point calibration curve in 50% (v/v) methanol, ranging from 0.05–0.1 to 5–20 g kg − 1 , was prepared prior to the run. To evaluate instrumental drift, three calibration blanks were processed with extra higher and lower QC standards at concentrations of 0.5 µg kg − 1 and 5 µg kg − 1 , respectively. The standard error of the y intercept of the linear regression line was used as the basis for calculating the limits of detection and quantification (LOD and LOQ). LOD and LOQ were calculated at 3 and 10 times the y intercept error and found to be 2.31 µg kg − 1 and 7.72 µg kg − 1 , respectively. 2.7.2 Total extractable organic fluorine (EOF) A calibration curve was prepared using PFOA in 50% (v/v) methanol ranging from 0 to 2000 µg F L − 1 . Ten blank measurements were conducted using deionized water, then the blank standard deviation (SD) was calculated, and the result was divided by the slope of the calibration curve, which was taken on the same day of blank measurement, multiplied by 3 to obtain the value of the instrumental LOD. The LOD calculated to be 0.49 µg F L − 1 . 2.7.3 Statistical analysis Data were expressed as the mean ± standard deviation (SD) on a dry weight (dw) basis. The statistical analysis included one- and two-way analysis of variance (ANOVA) approaches. All analyses were performed using Minitab 20 (Minitab LLC, USA) and Microsoft Excel (Microsoft, USA) software. The least significant differences ( P < 0.05) were determined and used to compare the statistical significance between treatments. 3. Results 3.1 Effects of GenX on the growth of rice and wheat Growth characters, including shoot biomass and the length of the wheat and rice crops, under GenX application are presented in (Fig. 1 ). The number of tillers for wheat, shoot length and dry biomass showed non-significant ( P > 0.05) results with both GenX treatments compared to the control with P = 0.451, P = 0.762 and P = 0.178 values, respectively (Table 1 ). The rice shoots’ dry biomass, however, exhibited a significant reduction of 25% ( P = 0.02) at 2 mg kg − 1 GenX concentrations compared to the control under flooded soil conditions (Fig. 1 ). Further, reductions of 9% and 7% were observed for the rice shoots’ dry biomass at 0.4 and 2 mg kg − 1 GenX concentrations, respectively, compared to the non-flooded control. The flooded and non-flooded soil conditions exhibited non-significant ( P = 0.096) effects on the rice shoots’ dry biomass at both GenX concentrations. Rice plant height however affected ( P = 0.048) under varied soil conditions. Rice shoot length showed a significant reduction with GenX and with varied soil environments (flooded ( P 0.048) and non-flooded ( P = 0.001)), indicating that the percentage of reduction increases with higher GenX concentrations. The number of rice tillers exhibited a non-significant effect with GenX exposure ( P = 0.301) and under varied soil conditions ( P = 1). The interaction between GenX treatments and soil conditions is only significant for the number of tillers, with a P value ( P = 0.017). Overall, the rice crops grown under non flooded soil exhibited a lower tolerance to GenX treatments compared to the wheat crops. 3.2 Uptake of GenX into shoots The concentration of GenX significantly varied ( P > 0.05) with the GenX treatments and under different soil conditions (Fig. 2 ). The uptake of GenX increased as the level of GenX treatment increased in both tested crops. After 30 days of GenX exposure, the wheat shoot’s uptake was 2.71 (± 0.92) and 10.5 (± 1.05) µg g − 1 at 0.4 and 2.0 mg kg − 1 of GenX application, respectively. This concentration of GenX in the plant shoot was significantly higher than that of the control group. The control soil had a GenX concentration below LOQ. The rice plants showed significantly varied GenX concentration values in their shoots according to the different soil conditions. The maximum uptake of GenX was observed in non-flooded soil conditions over flooded ( P < 0.003). At 0.4 mg kg − 1 of GenX, the rice shoots exhibited 2.34 (± 0.45) and 4.11 (± 0.87) µg g − 1 of GenX uptake in flooded and non-flooded soil environments, respectively. When exposed to 2.0 mg kg − 1 of GenX, the rice shoots showed a GenX uptake level of 10.4 (± 0.41) µg g − 1 under flooded conditions and 13.4 (± 0.72) µg g − 1 under non-flooded conditions. The uptake of GenX by the rice shoots showed a non-significant effect under the interactivity of the GenX treatment and soil condition ( P = 0.34). Overall, the rice shoots in non-flooded soil conditions exhibited more GenX uptake when exposed to higher concentrations of GenX than did the wheat shoots. The GenX levels in the rice and wheat shoots exhibited similar distribution patterns to the GenX concentrations in plant tissues (Fig S1 and Fig S2). Of all the experiments, the non-flooded rice absorbed the most GenX. Table 1 Statistical analysis of GenX concentrations in rice under varying treatment conditions. Parameters GenX treatment Growth conditions GenX growth conditions interaction Biomass P = 0.001 P = 0.096 P = 0.436 Plant height P = 0.001 P = 0.048 P = 0.091 Number of tillers P = 0.301 P = 1 P = 0.017 Shoot GenX conc. P = 0.001 P = 0.003 P = 0.349 The removal efficiency (%) of the rice and wheat was significantly ( P > 0.05) affected by the GenX application level and soil conditions. This was calculated by dividing the mass of the GenX in the plant tissues by the mass of spiked GenX (Fig. 4 ). Generally, a higher removal percentage was observed at 0.4 mg kg − 1 of GenX exposure with respect to the 2.0 mg kg − 1 treatment. The wheat plants exhibited a removal efficiency of 2% and 1% with low and high concentrations of GenX, respectively, while the rice crops exhibited a removal efficiency of 3% and 2% at lower and higher GenX levels, respectively, in both soil environments. 3.3 Distribution coefficient The distribution coefficient, K d , describes the distribution of a chemical between two media or two phases. Prior studies have used this approach to evaluate the accumulation and mobility of PFAS in solid-water systems (Milinovic et al. 2015 ). Further, its value has been used to provide information on the distribution and final course of PFAS. The K d value for the present study was determined as the ratio of the concentration of GenX measured in the soil phase and the concentration of GenX measured in the aqueous phase, as detailed below (Nguyen et al. 2020 ): $$Kd\left(\frac{L}{kg}\right)=\frac{ \text{m}\text{e}\text{a}\text{s}\text{u}\text{r}\text{e}\text{d} \text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n} \text{i}\text{n} \text{t}\text{h}\text{e} \text{s}\text{o}\text{i}\text{l} \text{p}\text{h}\text{a}\text{s}\text{e} \text{m}\text{g}/\text{k}\text{g}}{\text{m}\text{e}\text{a}\text{s}\text{u}\text{r}\text{e}\text{d} \text{i}\text{n} \text{t}\text{h}\text{e} \text{a}\text{q}\text{u}\text{e}\text{o}\text{u}\text{s} \text{p}\text{h}\text{a}\text{s}\text{e} \text{m}\text{g}/\text{L}}$$ Table 2 K d values in rice under two different treatments. Errors are represented by one standard deviation for four replicates. Treatment Flooded rice Non-flooded rice Week 1 Week 4 Week 1 Week 4 Low 0.54(± 0.02) 0.04(± 0.03) 0.35 (± 0.05) 0. 07(± 0.02) High 0.49(± 0.10) 1.08(± 0.34) 0.43(± 0.02) 0.68(± 0.23) GenX exhibits low k d , which indicates that GenX is very mobile. K d levels with low and high concentrations of GenX were higher in the flooded rice than in the non-flooded rice ( P = 0.028). Hence, GenX is significantly more mobile in the non-flooded environment. 3.4 Translocation factor (TF) The translocation factor was used to describe the ability of the rice to translocate GenX to the plant shoots. The TF values were calculated by the ratio of the concentration of GenX measured in the shoot and the concentration of GenX measured in porewater, as detailed below: $$\text{T}\text{F}=\frac{\text{m}\text{e}\text{a}\text{s}\text{u}\text{r}\text{e}\text{d} \text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n} \text{i}\text{n} \text{t}\text{h}\text{e} \text{s}\text{h}\text{o}\text{o}\text{t}}{\text{m}\text{e}\text{a}\text{s}\text{u}\text{r}\text{e}\text{d} \text{c}\text{o}\text{n}\text{c}\text{e}\text{n}\text{t}\text{r}\text{a}\text{t}\text{i}\text{o}\text{n} \text{i}\text{n} \text{t}\text{h}\text{e} \text{p}\text{o}\text{r}\text{e}\text{w}\text{a}\text{t}\text{e}\text{r}}$$ The values for the translocation factor significantly varied under application of GenX ( P = 0.001) and soil conditions (flooded and non-flooded) ( P = 0.02) (see Table 3 ). Maximum TF values were observed at 2.0 mg kg − 1 of GenX treatment, not at the 0.4 mg kg − 1 treatment. TF values of 3.08 (± 0.12) and 3.80 (± 0.35) were observed under the flooded and non-flooded conditions, respectively, at low exposure. At high exposure, the TF values were observed at 5.00 (± 0.07) and 5.33 (± 0.14) in the flooded and non-flooded environments, respectively. These higher TF values in the non-flooded environment indicate that rice plants can translocate more effectively in non-flooded conditions than in flooded conditions. Table 3 Translocation factor (TF) values in rice under two different growing conditions. Error represents one SD (n = 4). Treatment Flooded rice Non-flooded rice GenX at 0.4 mg kg − 1 3.08 (± 0.12) 3.80 (± 0.35) GenX at 2 mg kg − 1 5.00 (± 0.07) 5.33 (± 0.14) 3.5 Total extractable organic fluorine (EOF) measurement and mass balance analysis A fluorine mass balance analysis was performed to estimate the levels of unidentified organic fluorine compounds (UOF) that resulted from the degradation of GenX or another PFAS or other fluorinated compounds such as pesticides and pharmaceuticals occurring in the soil. The study determined the extractable organic fluorine using HR-GFMAS and percentage contributions of the total PFAS, including the GenX in the rice, wheat, porewater and soil (Fig. 5 – 7 ). An LC-MS/MS analysis was performed to identify the PFAS in the rice and wheat shoots, soil and porewater. No other PFAS, apart from GenX, was detected among the 35 targeted PFAS, which included PFCA and PFSA with perfluorocarbon chain lengths of C4-C12, as well as PFAS sulphonamides (table S1 ). Notably, ultra-short PFCAs were not monitored in this study due to challenges in their measurement. GenX at low and high exposure levels was found to be the major contributor to the EOF in the flooded rice shoots, contributing to 78 to 97% of the extractable organic fluorine. In the non-flooded rice shoots, it contributed to 68 to 95% at both exposure levels. The difference in mass balance between flooded and non-flooded conditions was not significant ( P = 0.133), as well as between different exposure levels ( P value = 0.103). In the wheat shoots, GenX was found to contribute to only 50–72% of the total extractable fluorine at both low and high exposure levels. Similarly, in the flooded soil, GenX contributed to 64–84% of the extractable fluorine and in non-flooded rice soil, it contributed to 21–92% at both exposure levels. While in non-flooded wheat soil contributed to 57–97% of the extractable fluorine. The difference in mass balance between flooded and non-flooded conditions was also not significant ( P value = 0.176), but significant between different exposure levels ( P value = 0.03). GenX was also found to account for 53 to 94% of the total extractable organic fluorine in non-flooded soil porewater samples, while in flooded soil porewater, it accounted for 53% and 98% at low and high exposure levels respectively. This difference in mass balance between flooded and non-flooded conditions was not significant (p value = 0.447), as well as between different exposure levels (p value = 0.480). 4. Discussion 4.1 Effects of GenX on growth and biomass of rice and wheat The 30-day exposure of GenX at the rate of 0.4 and 2.0 mg kg − 1 significantly affected the growth parameters of both tested crops, but most notably the rice. The results demonstrate that the rice shoots’ dry biomass significantly reduced under 2.0 mg g − 1 compared to the dry shoot biomass of wheat (Fig. 1 ). The growth of the rice was more inhibited in non-flooded soil than was wheat, indicating that rice is more sensitive to GenX than wheat. Similar findings have been reported by Chen et al. ( 2020 ), who found that in a hydroponic experiment, plant species A. thaliana and N. benthamiana did not exhibit any growth issues when exposed to 5 mg L − 1 GenX but when GenX dosage increased, shoot growth was affected. Both the growth and development of the shoots and roots of N. benthamiana were severely hindered when exposed to the highest level of GenX (20 mg L − 1 ). N. benthamiana showed reduced root and shoot biomass, with tolerance index values for GenX decreasing from 100–40% and 55%, respectively (Chen et al. 2020 ). A recent study that investigated the phytotoxicity of GenX in lettuce showed that the shoots’ dry biomass remained unaffected when treated with a concentration of 100 µg L − 1 of GenX. This suggests that at this concentration, GenX does not have a significant adverse effect on the growth and development of lettuce plants (Wang et al. 2023 ). These findings suggest that the effects of GenX on plant growth and development are concentration and species-specific. 4.2 Uptake of GenX While a proportional uptake of GenX was observed for both crops (rice and wheat), the amounts as measured by their biomass quantities differed. This observation further confirms that uptake and bioaccumulation does not only depend on GenX properties and chemical characteristics but also on plant species and its capacity for bioaccumulation. As Doucette et al. ( 2018 ) noted, the differences in PFAS uptake among species are generally due to varying biotransformation capacities, rates of dissipation, ease of root uptake and temperature (Doucette et al. 2018 ). Further, Zhao et al. ( 2016 ) found that wheat’s uptake of PFCA increased by 1.5- to 2.3-fold when the temperature increased from 20 to 30°C (Zhao et al. 2016 ). In our study, the rice and wheat were grown in different greenhouses with different temperatures. The rice was grown at 28°C, while the wheat was grown at 15°C. This may have impacted the uptake of GenX between the wheat and non-flooded rice. Zhao et al. ( 2016 ) found that the growth of wheat at a temperature of around 30°C can lead to nutrient diffusion rates increase, water viscosity decreases and photosynthesis is given more energy, all of which increase the uptake of pollutants (Zhao et al. 2016 ). To avoid changing the PFAS uptake, plant growth conditions must be carefully monitored. 4.3 GenX mechanism of intoxication and growth inhibition Despite the different uptake and bioaccumulation capacities, GenX was found to inhibit the growth of shoot biomass more in rice. GenX, like other PFASs, initiates a mechanism that inhibits shoot growth in plants, but this appears to be species-specific at the concentration used in this study. Our findings are in line with studies involving both human cells in vitro and plant species. Such studies have concluded that exposure to GenX induces an intracellular toxicity mechanism that leads to apoptosis and a reduction of cell viability (Yoo et al. 2021 ). Similarly, GenX uptake has been observed to have an adverse effect on lettuce even at low concentration levels (100 µg L − 1 ), which suggests that GenX could induce oxidative stress in lettuce plants (Wang et al. 2023 ). The H 2 O 2 content in lettuce tissues revealed that GenX causes stronger oxidative stress than does PFOA. Previous research also found that 100 to 200 mg L − 1 of GenX increased H 2 O 2 in Nicotiana benthamiana plants. These findings suggest that even low concentrations of GenX can cause significant oxidative stress in plants, even more than the phased-out PFOA (Chen et al. 2020 ). Both outcomes indicate that a similar mechanism of GenX inhibition affects the growth and development of different species, albeit at different intoxication levels. 4.4 Impact of soil conditions on GenX bioaccumulation Apart from the plant species and chemical properties of GenX, soil conditions also impacted the uptake and bioaccumulation of GenX in rice. In this experiment, the rice grown in non-flooded soil had higher concentrations of GenX compared to that grown in flooded soil (Fig. 2 ). This observation is in line with findings from studies that revealed that efficient transpiration facilitates the translocation of more molecules through the water gradient (Lesmeister et al. 2021 ). Similarly, it has been established that flooding reduces transpiration rate, thereby affecting the distribution of the substance located in the soil and the plant tissues. In non-flooded conditions, the rate of transpiration is generally higher than in flooded conditions, which can lead to higher rates of PFAS uptake by plants. This causes less GenX to be concentrated in the flooded plant comparing to the non-flooded one. It has been suggested that the amount of water that transpires during growth can explain the various absorption and translocation abilities among crops (Blaine et al. 2014 ). Anaerobic (flooded) and aerobic (non-flooded) soil conditions cause the soil to exhibit different biological and chemical characteristics. For example, there is less oxygen in flooded soil because the soil pores fill with water. In the absence of oxygen, the reduced chemical forms of compounds predominate. When oxygen availability is limited, bacteria opt to use other compounds as electron acceptors to maintain their metabolism, thereby reducing the elements in these compounds. The result of such microbial metabolism is the conversion of oxidised elements to their corresponding reduced forms under anaerobic conditions. In non-flooded soil, oxygen is available for bacterial metabolism, so the compounds stay in their oxidised forms. Although the degradation of such PFAS compounds as FTOH has been reported under aerobic and anaerobic conditions, the degradation of GenX has not been studied in much depth. One study on the sorption of GenX onto sediments found that within 14 days, the concentration of GenX in freshwater and estuary sediments decreased by 40 to 59%. Moreover, there were no statistically significant variations in GenX loss between the bioactive and autoclaved sediments, so the decrease could not be explained by biological degradation (Harfmann et al. 2021 ). 4.5 Translocation factor (TF) At higher GenX exposure in the present study, its concentration in both wheat and rice shoots rose as well, but not proportional. Moreover, higher transfer factor (TF) values were found at higher exposure levels, indicating that the plants were taking up more GenX with increasing exposure levels. Interestingly, the TF values for GenX in rice shoots were higher in non-flooded soil than in flooded soil conditions. This suggests that rice plants in non-flooded conditions can translocate GenX more easily from the roots to the shoots compared to rice grown in flooded conditions. This can be explained by the higher concentration of GenX in the soil porewater under non-flooded conditions compared to flooded conditions. This difference in concentration of GenX in the soil porewater can be attributed to the lower mobility of GenX in flooded soil conditions due to its strong adsorption to soil particles, which reduces its availability for plant uptake. Conversely, in non-flooded soil conditions, GenX is more mobile and has a lower K d value, which allows it to move freely in the soil water, making it more accessible for plant uptake. This would mean that when rice is grown in non-flooded conditions to safe water, it would increase the uptake of GenX, which may have implications for human health and the environment. Zhang et al. ( 2021 ) (Zhang et al. 2021 ) found that among the five ether-PFAS studied, GenX had the highest translocation factor (TF) value in wetland plants (C. comosa ) exposed to 500 ng L − 1 for 52 days, which indicates that GenX can be translocated easily to shoots of a wetland plant. Gu et al. ( 2023 ) (Wang et al. 2023 ) also reported that short-chain PFAS accumulate more easily in the edible parts of plants compared to long-chain PFAS. Overall, in both the flooded and non-flooded rice, TF was statistically different between the GenX treatments. This suggests that the increase in GenX exposure impacted the crop’s efficiency of GenX uptake as well. 4.6 Fluorine mass balance and EOF The results of the fluorine mass balance analysis show that higher exposure levels of GenX significantly contribute to the total extractable fluorine. These results suggest that a close fluorine mass balance exists at high exposure levels, and that most of the extractable fluorine can be accounted for by the presence of GenX. The high contribution of GenX to the total extractable fluorine in rice shoots, porewater and soil implies no GenX degradation in the plants. However, discrepancies between the EOF and GenX concentrations in the soil at low exposure levels suggest that GenX may degrade to other short-chain PFCAs not included in the targeted LC-MS/MS analysis. A recent study examined the effects of fluoroalkylether compounds, including GenX, on the microbial community in soil–plant systems. The results of the study showed that the structure of the community and species diversity were significantly impacted by ether-PFAS at concentrations of 500 ng L − 1 and 2,000 ng L − 1 (Jiang et al. 2021 ). Although the study did not find any evidence of biodegradation of the spiked ether-PFAS, it did reveal that the presence of these compounds seemed to stimulate the growth of certain microbes with the ability to break down hydrocarbon chemicals and contaminants. In our study it is significant that at low exposure level the mass balance between GenX and EOF shows unaccounted organofluorines, which however were not detected in the targeted LC-MS/MS analysis. The unaccounted organofluorines might however be ultra-short chain PFAS as degradation products which were not monitored. It is important to note that our study did not aim to investigate the mechanisms or pathways of GenX degradation in these plants. Therefore, further research is required to understand the course of this compound in agricultural environments comprehensively in the field. 4.7 GenX distribution coefficient In this experiment, the substance molecule distribution was determined using the distribution coefficient, K d . An earlier study assessed the behaviours of a wider selection of PFAS on soils of different properties. The study found that per- and polyfluoroalkyl ether acids, including GenX, have low K d values, which means that they are very mobile. This indicates the hydrophilic nature (hydrophilicity) of these compounds and the ease by which they may be taken up in groundwater to pollute waterways and the environment [18]. As indicated in Table 2 , the present study observed K d values for GenX in rice are influenced by treatment conditions (flooded vs. non-flooded) and exposure levels. A lower K d value for GenX suggests a higher degree of mobility, allowing it to be present in the water phase instead of adsorbing onto soil particles. In this study, it has been found that GenX had the lowest K d value in non-flooded conditions, especially at low exposure levels, which suggests that it is more likely to be taken up by rice under non flooding conditions compared to flooded conditions. In other words, the mobility of GenX is higher under non-flooded conditions, resulting in its increased presence in the water phase. This characteristic makes it more available for plant uptake by rice, resulting in a higher likelihood of accumulation. Hence, the results of this study suggest that the risk of GenX accumulation in rice is higher under non-flooded conditions, particularly at low exposure level. These findings are in line with previous evidence indicating that short-chain PFASs like GenX are more readily absorbed through a water gradient and anion channels when in adequate concentrations in the growth medium (Li et al. 2022 ). To our knowledge, the uptake mechanisms of new alternatives like GenX in plants have not been well studied. Gu et al. ( 2023 ) investigated the differences between PFOA and its alternatives, finding that PFOA and GenX were likely transported via a diffusion process. Water channels only have a limited effect on the uptake of PFOA and its alternatives, and slow anion channels rather than rapid ones were mainly responsible for the uptake of PFCAs (Gu et al. 2023 ). If this applies also to rice and wheat, the difference in GenX uptake of the plant species in our experiment can therefore not be a temperature effect but rather a plant species effect. As observed previously, despite being industrially used as a short-chain alternative to PFOA, GenX still has similar properties that make it unsafe for the environment. According to recent research, PFASs such as GenX accumulate to different degrees in crops, depending on the components of said crops (Krippner et al. 2015 ; Bizkarguenaga et al. 2016 ). PFASs have been reported to accumulate in protein-rich plant parts; as a result, GenX may accumulate in the bran and germ of rice and wheat. Although wheat and rice are the model crops for this study, other findings raise concerns about the safety of crops whose shoots are consumed directly, such as lettuces. In China, for instance, water scarcity has forced a shift from cultivating rice in traditional high-water-consuming lowlands to controlled and non-flooded irrigation approaches. Although non-flooded conditions have been tested successfully for the reduction of other contaminants of concern, such as arsenic (as rice grown in traditional flooded paddies has been found to contain arsenic levels 10 to 15 times greater than rice cultivated in non-flooded conditions (Li et al. 2019 )), here we observed higher GenX concentrations in rice shoots grown under non-flooded conditions. This may increase safety risks for crops cultivated under water stressed and rainfed areas. In contrast to the available published information on PFAS and PFOA substances, this study’s findings are crucial because they demonstrate that despite being a short-chain alternative to PFOA, GenX exhibits chemical properties and environmental safety concerns. Even more than long-chain PFAS (like PFOA), the industrial use of which has been replaced, GenX has the potential to impede plant growth. Studies in line with our findings on shoot biomass and growth length indicate that due to GenX’s high solubility in water, it can enter the environment and affect plants more easily than other PFAS (Ahrens et al. 2010 , 2011 ). Prior studies in rice paddy fields, which are consistent with the present flooded soil conditions, have determined that due to their high mobility in water, short-chain PFAS like GenX are likely to leach into groundwater systems, thereby reducing their availability for plant uptake (Eun et al. 2022 ). This explains our observation that less GenX accumulated in the shoots of rice grown in flooded soil. Based on these study findings, GenX is not better for the environment because of its higher bioaccumulation and poor degradation. Further, GenX is only poorly absorbed in flooded conditions and better taken up by the rice under non-flooded conditions, making it a concern for modern crop irrigation approaches that aim to optimise production with minimal water use. 5. Conclusions This study assessed the bioaccumulation potential of an emerging fluorinated ether, GenX, in wheat and rice in two concentrations. The results demonstrate that GenX was taken up by plant roots, translocated to shoots and accumulated in the tissues of the studied plants. Among the two investigated plant species, rice demonstrated the highest concentrations of GenX in its shoots. The accumulation of GenX in rice and wheat increased when the plants were exposed to higher levels of GenX, this means that rice and wheat growing in hot spots of GenX contamination would be far more affected as would low level exposure experiments suggest. The results of this study indicate that rice is more sensitive to GenX than wheat at relatively high exposure levels. Moreover, the existence of GenX might pose similar or even higher ecotoxicological risks to the environment than PFOA. Long-term PFAS accumulation in surface and wastewater, as well as the potential risk to organisms due to environmental persistence, must be considered seriously and dealt with effectively to reduce adverse environmental consequences. Therefore, it is important to conduct additional research to compare the accumulation and phytotoxic effects of GenX with other plant species and other PFAS under longer exposure times. Declarations Acknowledgment A.A.Z. thank the Culture Bureau of Saudi Arabia for their financial support. V.M. thank the Macaulay Development Trust for her scholarship. Ethical Approval Compliance with ethical standards Consent to Participate No consent of participate was necessary for this study. Consent to Publish All co-authors gave the consent to publish. Authors Contributions Amnah Al Zbedy: Laboratory work, HR GFMAS, greenhouse work, methodology, writing- original draft preparation. Viktoria Müller: Laboratory work, Target LC-MS/MS, help with manuscript writing Andrew Kindness: Supervision, help with manuscript writing Rainer Ebel: Supervision, help with manuscript writing. Gareth Norton: Conceptualisation, methodology, supervision, help with manuscript writing. Jörg Feldmann: Conceptualisation, methodology, supervision, help with manuscript writing. Funding Amnah Al Zbedy: financial support of her scholarship from the Cultural Bureau of Saudi Arabia, London. Viktoria Müller: financial support of her scholarship from the Macaulay Development Trust, UK. Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 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Accessed 19 Feb 2023b Supplementary Files supplementarymaterialGenXAmnahfinal.docx Cite Share Download PDF Status: Published Journal Publication published 04 Dec, 2023 Read the published version in Environmental Science and Pollution Research → Version 1 posted Reviewers agreed at journal 27 Jul, 2023 Reviewers invited by journal 21 Jul, 2023 Editor invited by journal 30 May, 2023 Editor assigned by journal 23 May, 2023 First submitted to journal 17 May, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2889643","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":220548682,"identity":"44fbd0d5-6a5c-4596-bb16-4bb80cee3fb5","order_by":0,"name":"Amnah Al Zbedy","email":"","orcid":"","institution":"University of Aberdeen","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Amnah","middleName":"Al","lastName":"Zbedy","suffix":""},{"id":220548683,"identity":"94cd6cb1-207b-41a1-aa24-4b97b572fab9","order_by":1,"name":"Viktoria Müller","email":"","orcid":"","institution":"University of Graz: Karl-Franzens-Universitat Graz","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Viktoria","middleName":"","lastName":"Müller","suffix":""},{"id":220548684,"identity":"0ad6bfde-12ab-4d01-97f9-a3ba19196a9b","order_by":2,"name":"Andrew Kindness","email":"","orcid":"","institution":"The James Hutton Institute Aberdeen","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Andrew","middleName":"","lastName":"Kindness","suffix":""},{"id":220548685,"identity":"6fa62ff6-46ed-4a7b-8869-097c542177f1","order_by":3,"name":"Rainer Ebel","email":"","orcid":"","institution":"University of Aberdeen","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Rainer","middleName":"","lastName":"Ebel","suffix":""},{"id":220548686,"identity":"e51f2e1c-56bf-4879-8542-b3a8daaf0160","order_by":4,"name":"Gareth J Norton","email":"","orcid":"","institution":"University of Aberdeen","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Gareth","middleName":"J","lastName":"Norton","suffix":""},{"id":220548687,"identity":"a76cf647-a0c6-4c90-935f-caecdde72371","order_by":5,"name":"Jörg Feldmann","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-0524-8254","institution":"University of Graz","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Jörg","middleName":"","lastName":"Feldmann","suffix":""}],"badges":[],"createdAt":"2023-05-03 11:38:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2889643/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2889643/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11356-023-31160-w","type":"published","date":"2023-12-04T15:01:43+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":40607655,"identity":"a2d77065-006e-45e1-839f-9aeb05874b2b","added_by":"auto","created_at":"2023-07-26 14:35:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":81440,"visible":true,"origin":"","legend":"\u003cp\u003eTreatment of rice (A and C) and wheat (B and D) biomass (g) and shoot length (cm). The error bars represent one standard deviation. Different letters represent significant differences among the treatments, means that don’t share a letter are significantly different.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-2889643/v1/79e9e9ef4d7c26382c9cc0ab.png"},{"id":40607651,"identity":"9c653199-2000-4950-a70d-33e36ca7177e","added_by":"auto","created_at":"2023-07-26 14:35:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":71620,"visible":true,"origin":"","legend":"\u003cp\u003eConcentration of GenX in rice (A) and wheat (B) shoots exposed to GenX at 0.4 mg kg\u003csup\u003e-1\u003c/sup\u003e (low) and 2 mg kg\u003csup\u003e-1\u003c/sup\u003e (high) after 30 days.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-2889643/v1/b59f80d10e925789dbe60c58.png"},{"id":40607650,"identity":"f16e1831-c7dd-456f-abda-2caeb14a092b","added_by":"auto","created_at":"2023-07-26 14:35:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":19389,"visible":true,"origin":"","legend":"\u003cp\u003eConcentration of GenX in porewater under flooding (black bar) and non-flooding (grey bar) condition exposed to GenX at 0.4 mg kg\u003csup\u003e-1 \u003c/sup\u003e(low) and 2 mg kg\u003csup\u003e-1 \u003c/sup\u003e(high) at the start and the end of rice growth experiment.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-2889643/v1/49f446f1fd916955aa38f2f2.png"},{"id":40609065,"identity":"6ae12d22-5352-404b-b93c-f1169473a95a","added_by":"auto","created_at":"2023-07-26 14:43:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":49368,"visible":true,"origin":"","legend":"\u003cp\u003eRemoval of GenX in soil by plant (translocation of GenX from soil to plants) after 30 days. Error bars represent standard deviations (n = 4).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-2889643/v1/a5bdec5243ec8488d1b42653.png"},{"id":40607652,"identity":"1d39f269-4a81-44d6-b912-e99c7a8f18d6","added_by":"auto","created_at":"2023-07-26 14:35:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":74795,"visible":true,"origin":"","legend":"\u003cp\u003eA. Fluorine mass balance analyses of rice A (flooded), B (non-flooded) and C (wheat) shoots. The black bars represent the contribution of GenX mg F kg\u003csup\u003e-1\u003c/sup\u003e using LCMS/MS, while grey bars represent the total extractable organic fluorine using HR-GFMAS. Error bars represent by standard deviations (n = 4).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-2889643/v1/3a2503c6e7c50cb914bf9374.png"},{"id":40607659,"identity":"632ae594-e2ce-47cc-9627-5f830836b135","added_by":"auto","created_at":"2023-07-26 14:35:13","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":90680,"visible":true,"origin":"","legend":"\u003cp\u003eFluorine mass balance analyses of soil A (flooded), B (non-flooded) and C (wheat). The black bars represent the contribution of GenX mg F Kg\u003csup\u003e-1\u003c/sup\u003e using LCMS/MS, while grey bars represent the total extractable organic fluorine using HR-GFMAS. Error bars represent standard deviations (n = 4).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-2889643/v1/3bcee4affae7266e5a9962fb.png"},{"id":40607654,"identity":"0513a948-a0a8-47ad-a43f-91f81b4db834","added_by":"auto","created_at":"2023-07-26 14:35:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":69239,"visible":true,"origin":"","legend":"\u003cp\u003eFluorine mass balance analyses of porewater A (flooded), B (non-flooded). The black bars represent the contribution of GenX mg F L\u003csup\u003e-1\u003c/sup\u003e using LCMS/MS, while grey bars represent the total extractable organic fluorine using HR-GFMAS. Error bars represent standard deviations (n = 3).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-2889643/v1/82a29688fc873f74c90fcc24.png"},{"id":47989956,"identity":"9777c7ba-3279-4325-9965-10fec2a6e104","added_by":"auto","created_at":"2023-12-11 15:11:01","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":993479,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2889643/v1/2b24740c-2eff-4afc-8d44-a8b463a25499.pdf"},{"id":40607656,"identity":"945944f8-8134-4b5d-8f1f-ebf7edc8cf9f","added_by":"auto","created_at":"2023-07-26 14:35:09","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":45448,"visible":true,"origin":"","legend":"","description":"","filename":"supplementarymaterialGenXAmnahfinal.docx","url":"https://assets-eu.researchsquare.com/files/rs-2889643/v1/8595c4c00ef06ab04dea372d.docx"}],"financialInterests":"","formattedTitle":"GenX uptake by wheat and flooded and non-flooded rice: greenhouse experiment.","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eHexafluoropropylene oxide dimer acid (HFPO-DA), a perfluoroalkyl ether carboxylic acid also known as GenX, has been used to aid polymerisation in the production of high-performance fluoropolymers (Bokkers et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). It was introduced as an alternative for perfluorooctanoic acid (PFOA), which was used from 1970 to 2012 (Rivm and Poll). Organisations such as the Stockholm Convention and the European Chemical Agency began investigating PFOA as a hazardous contaminant due to its persistence, bioaccumulation and toxicity (Brandsma et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), leading to its restriction in 2017 by the European Union (Brendel et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This restriction gave rise to so-called replacement chemistries, including GenX, which was introduced for its lower bioaccumulation potential compared to PFOA, despite the limited toxicokinetic data available for GenX (Heydebreck et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The United States Environmental Protection Agency (US-EPA) recently published toxicity assessments for PFOA (2016) and perfluorooctanesulfonic acid PFOS (2016), showing that chronic oral reference doses (RfD) for PFOS and PFOA are higher than those for GenX. Based on the available animal toxicity data, it appears that GenX has greater hazardous potency than PFOA and PFOS (Wang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Gomis et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; US Environmental Protection Agency et al. 2021). GenX has been shown to have adverse implications in mice and zebrafish (Satbhai et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), such as inducing benign tumours, gene changes in the liver, thyroid hormone level disturbance and hepatocellular damage (Cannon et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGenX has been detected in increasing amounts in drinking water and soil (Gebbink and van Leeuwen \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In 2015, GenX contamination was discovered near North Carolina\u0026rsquo;s Cape Fear River, downstream of a chemical manufacturing company (Cahoon \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). GenX was also detected in plant leaves found within three kilometres of a fluoropolymer manufacturing Teflon plant in the Netherlands (Brandsma et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It is unclear exactly how GenX and PFASs are absorbed by grass and foliage. The GenX concentrations seen in the grass and leaves gathered around the plant may be the consequence of air deposition, absorption from contaminated soil or both (Brandsma et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). It has also been found that perfluoroether chains are just as resistant to biotic and abiotic degradation as PFOA (Wang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile most studies have focused on the effects of GenX on animals, there is limited knowledge about GenX bioaccumulation and its adverse effects on plants. While research on the uptake and accumulation of GenX by plants is still limited, it is a growing area of interest and investigation. In recent research, the accumulation and toxicity of GenX and perfluorooctanoic acid (PFOA) have been studied and compared using \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eNicotiana benthamiana\u003c/em\u003e as model plants in a hydroponic system. Findings indicate that at concentrations between 20 and 200 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, GenX inhibited plant growth and lowered chlorophyll content and enzyme activity (Chen et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Zhang et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) found that \u003cem\u003eCarex comosa\u003c/em\u003e absorb nearly eight percent of the GenX in soil after 80 days of exposure (Zhang et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Moreover, Zhi et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) studied the bioaccumulation of PFAS in spontaneous urban plants and reported that GenX had a lower bioaccumulation factor (0.66\u0026ndash;2.5) than PFOA (3.5\u0026ndash;10.5) in plant roots (Zhi et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003ePFAS are typically analysed with targeted LC-MS/MS; however, only those that ionise easily using electrospray ionisation can be analysed. Extractable organic fluorine (EOF) or total fluorine analysis are broader PFAS analysis methods. Different instruments can be used for EOF and total fluorine analysis, such as combustion ion chromatography (CIC) and high-resolution graphite furnace molecular absorption spectrometry (HR-GFMAS). Although results obtained with the two different instruments are comparable (Gehrenkemper et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), HR-GFMAS analysis has been found to be more sensitive and less time consuming (Gehrenkemper et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Mass balance analysis, meanwhile, combines the target analysis and EOF analysis to identify the fraction of PFAS that can and cannot be determined using the target method (Aro et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eRice is one of the most representative foods among primary nutritional foods (Liu et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) and is consumed by more than fifty percent of people around the world (da Silva et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Wheat is also a significant source of carbohydrates, fibre and vitamins worldwide (Shewry and Hey \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Although PFAS intake from rice and wheat consumption is a health concern, considering the uptake of legacy and replacement PFAS, few studies (Stahl et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Lan et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Kim et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) on the uptake and bioaccumulation of these chemicals in rice and wheat are available.\u003c/p\u003e \u003cp\u003eThe present research uses a mass balance approach to study the accumulation of GenX in plants grown on contaminated soil and to determine how much GenX contributes to EOF in wheat and rice. To our knowledge, this study is the first to investigate GenX uptake by wheat and rice from soil systems and the first to measure the total EOF in wheat and rice using high resolution graphite furnace molecular absorption spectrometry (HR-GFMAS) in addition to targeted LC-MS/MS.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Chemical reagents and laboratory materials\u003c/h2\u003e \u003cp\u003eThe chemical 2,3,3,3-tetrafluoro-2-(heptafluoropropoxy) propanoic acid GenX (97% purity) was obtained from SynQuest Laboratories. A PFAS standard solution (MPFAC-MXC) containing perfluorinated carboxylic acid (PFCA) and perfluoroalkanesulfonic acids PFSA was purchased from Wellington Laboratories. Ultrapure water with a resistivity of 18.2 MΩ cm was obtained from Smart2 Pure, Thermo Fisher Scientific (Loughborough, UK). Ammonium hydroxide (Merck) and HPLC-grade methanol (Honeywell Riedel-de Haen, Germany) were used for sample extraction, and Envi carb (Merck) was used for cleaning purposes. Acetonitrile (Honeywell Riedel-de Haen, Germany) was used to prepare the mobile phase for HPLC. For the HR-GFMAS analysis, BOC (Dublin, Ireland) provided 99.998% purity argon gas, and a W (Merck) standard solution was used for the graphite tube coating as a permanent modifier. Ca(NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003ex4H\u003csub\u003e2\u003c/sub\u003eO (VWR chemicals, Leicestershire, UK) was used as a forming reagent at a concentration of 1% Ca (w/v). PFOA, which was used as a calibration solution, was obtained from Sigma Aldrich (St Louis, MO, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Soil properties and treatment\u003c/h2\u003e \u003cp\u003eSoil was collected from a field in Insch Aberdeenshire. Soil samples were taken from the top 10 cm of the field. The soil was air-dried for two weeks and sifted through a 2-mm sieve. Soil pH (7.29) was measured in deionised water with a soil-to-water ratio of 1:2.5 after shaking for 1 hour. The soil was then spiked with a stock solution of GenX to achieve two nominal concentrations of 0.4 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (low level) and 2.0 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (high level). The control soil was also spiked with methanol. Once the soils had been spiked, they were placed in a fume hood for solvent evaporation at room temperature. Once the solvent evaporated, the soil was incubated in darkness for 14 d at room temperature. Concentrations of GenX in the soil were determined after incubation but before adding plants to the soil (0 d) and at the end of the plant growth experiment (30 d).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Plant exposure in soil and sampling\u003c/h2\u003e \u003cp\u003ePlastic pots (0.9 L) were filled with 650 g of soil. Four groups of tests were conducted:\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e1) soil alone, spiked with water (control 1);\u003c/h3\u003e\n\n\u003ch3\u003e2) spiked soil alone, spiked with methanol (control 2);\u003c/h3\u003e\n\n\u003ch3\u003e3) and 4) spiked soil with low and high GenX concentrations, respectively.\u003c/h3\u003e\n\u003cp\u003eEach group contained four replicates for each plant species and water treatment.\u003c/p\u003e \u003cp\u003eFor the wheat and non-flooded rice experiments, a piece of filter paper was placed at the bottom of the pot to restrict soil loss. For the flooded rice experiment, the pots were lined with a plastic bag so the soil could be flooded without loss. Wheat (Tybalt) and rice (BJ 1) seeds were germinated for 3 d on a petri dish, with the wheat at room temperature and the rice at 25\u003csup\u003eo\u003c/sup\u003eC. Ten seeds were transplanted into each pot. The pots were irrigated daily to maintain a moisture content at 80% water-holding capacity for the wheat and non-flooded rice and completely flooded for rice. To account for any spatial variations in light and temperature within the greenhouse, the pots were positioned at random each day. After twenty days, 2 mL of 10x Yoshida\u0026rsquo;s nutrient (goleman, daniel; boyatzis, Richard; Mckee et al. 1976) were added. This experiment was divided into two halves because the rice and wheat were grown in separate greenhouses for four weeks at the University of Aberdeen in Aberdeen, Scotland (Coordinates: 57.165\u0026deg;N 2.100\u0026deg;W). For the rice experiment, the day temperature was 28\u0026deg;C and the night temperature 25\u0026deg;C. Supplemental lights were turned on between 7 am and 7 pm if the natural light was less than 24,000 Lux. The wheat experiment was kept at 15\u0026deg;C.\u003c/p\u003e \u003cp\u003eAfter 30 d, the shoots of wheat and rice were harvested, freeze-dried and homogenised, then stored in polypropylene (PP) vials at -20\u0026deg;C prior to analysis.\u003c/p\u003e \u003cp\u003eFollowing the removal of the plants from each pot, all test soils were taken out and air- and freeze-dried for 48 hours at room temperature. The dried soil samples were stored in polypropylene (PP) tubes at -20\u0026deg;C before chemical analysis.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Measurements of plant height and porewater collection\u003c/h2\u003e \u003cp\u003eRhizon samplers attached a 10 mL syringe were buried at a 45-degree angle in the soil to allow sampling of porewater. Samplers of the porewater was taken on first week transplanting (day 0) and at the end of experiment at day 30. Plant growth was determined by measuring plant height, dry shoot biomass and number of tillers. The measurements were performed on day 30. Shoots were cut at 3 cm above the soil level to avoid soil contamination and its dry weight biomass were determined using an analytical balance.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Analysis and extraction of PFAS\u003c/h2\u003e \u003cdiv id=\"Sec11\" class=\"Section3\"\u003e \u003ch2\u003e2.5.1 Extraction procedure\u003c/h2\u003e \u003cp\u003eThe GenX in the plant was extracted using a previously described method (Blaine et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Chen et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). As an extraction solvent, a mixture of 50:50 (v/v) DCM and MeOH with 1% ammonium hydroxide (v/v) was prepared. For the extraction, 1 mg of dried plant sample was placed into a 15-mL polypropylene tube, and 3 mL of the extraction solvent was added. The tube was then vortexed for 30 s, sonicated for 15 min at 30\u0026deg;C, then shaken on an orbital shaker for 1 hour. The supernatant was collected after centrifugation at 1,500 rpm for 10 min. This extraction process was repeated twice. The extracts were then pooled and dried under a gentle nitrogen stream. The dried extract was reconstituted with 1 mL of MeOH, then mixed with 50 mg of ENVI-Carb for 20 s for clean-up. This was followed by centrifugation at 13,000 rpm for 10 min. Then, 450 \u0026micro;L of the extract was transferred into HPLC vials. Finally, 50 \u0026micro;L of the 50 \u0026micro;L L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e isotopically mass-labelled standard was introduced to enable target analysis using LC-MS/MS.\u003c/p\u003e \u003cp\u003eSoil samples were extracted by transferring a 0.5 g aliquot into a 50 mL polypropylene vial, to which a solution containing an isotopically labelled surrogate standard and 3 mL of 0.1% ammonium hydroxide (NH\u003csub\u003e4\u003c/sub\u003eOH) in methanol was added. This was then sonicated for 15 min at 30\u0026deg;C, as per the established protocols (Higgins et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Houtz et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The solution was then shaken for 1 hour on an orbital shaker. The supernatant was collected after centrifugation at 1,500 rpm for 10 min. This extraction process was repeated twice. All extracts were combined, dried under a gentle nitrogen stream, reconstituted in 1 mL methanol and cleaned with 50 mg ENVI-Carb. Then, 450 \u0026micro;L was taken into HPLC vials, and 50 \u0026micro;L of the 50 \u0026micro;L L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e isotopically mass-labelled standard was added for target analysis using LC-MS/MS.\u003c/p\u003e \u003cp\u003eThe porewater samples were analysed directly via LC-MS/MS. Prior to injection, each vial was centrifuged for 20 min at 2,700 rpm. An aliquot (225 \u0026micro;L) of the supernatant was then removed and transferred to a PP microcentrifuge tube containing 250 \u0026micro;L of methanol. Then, 50 \u0026micro;L of the 50 \u0026micro;L L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e isotopically mass-labelled standard was added for target analysis using LC-MS/MS.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Instrumentation\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003e2.6.1 Target PFAS\u003c/h2\u003e \u003cp\u003eAgilent 1200 infinity HPLC (Agilent Technologies, Germany), coupled with Agilent 6465 Triple Quadrupole MS/MS (Agilent Technologies, Germany), operated in negative electrospray ionisation (ESI) mode. PFAS separation was performed using 5 mM ammonium acetate (CH\u003csub\u003e3\u003c/sub\u003eCOONH\u003csub\u003e4\u003c/sub\u003e) in reagent water, and 100% LCMS-grade acetonitrile was used as the mobile phase of the analysis of PFAS ionic compounds. Ten \u0026micro;L of the extract was automatically injected onto a BrownLee SPP C18 column (2.7 \u0026micro;m, 3 x 100 mm, PerkinElmer, UK) at 30\u0026deg;C. Chromatograms were recorded using multiple reaction monitoring (MRM) mode. A list of the analytes, transitions and optimised MRM conditions and LC method used can be found in this study\u0026rsquo;s supplementary materials (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e-S3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003e2.6.2 Extractable organic fluorine (EOF)\u003c/h2\u003e \u003cp\u003eThe EOF were analysed according to the method described by Akhdhar \u003cem\u003eet al.\u003c/em\u003e (Akhdhar et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). All measurements were performed using an Analytik Jena ContrAA 700 High Resolution Continuum Source Graphite Furnace Atomic Absorption Spectrometer (HR GFAAS) with a transversely heated graphite tube atomizer. A 300W xenon short-arc lamp was used as the instrument\u0026rsquo;s continuous radiation source for wavelengths between 185 and 900 nm. A charge-coupled device (CCD) array detector with 588 pixels, 200 and a high-resolution double echelle monochromator were used for analytical purposes. The measurements were conducted using coated graphite tubes with an integrated PIN platform (Analytical Jena part No. 407-A81.025). All fluorine measurements were performed three times at a wavelength of 606.417 nm to monitor the absorption of the formed CaF. The graphite furnace platform was coated with tungsten (W), while calcium (Ca) was utilized as the forming reagent.). Table S4 details the temperature programme used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section3\"\u003e \u003ch2\u003e2.6.3 Fluorine mass balance analysis\u003c/h2\u003e \u003cp\u003eFluorine mass balance analysis was conducted by converting the concentration of PFAS analyte in the samples measured by LCMS/MS into the corresponding fluorine concentrations equivalents. This was then compared to the fluorine concentrations obtained from the EOF analysis using HR-GFMAS.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Quality control (QC) and statistical analysis\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e2.7.1 Targeted LC-MS/MS analysis\u003c/h2\u003e \u003cp\u003eA 4- to 5-point calibration curve in 50% (v/v) methanol, ranging from 0.05\u0026ndash;0.1 to 5\u0026ndash;20 g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, was prepared prior to the run. To evaluate instrumental drift, three calibration blanks were processed with extra higher and lower QC standards at concentrations of 0.5 \u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 5 \u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. The standard error of the y intercept of the linear regression line was used as the basis for calculating the limits of detection and quantification (LOD and LOQ). LOD and LOQ were calculated at 3 and 10 times the y intercept error and found to be 2.31 \u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 7.72 \u0026micro;g kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e2.7.2 Total extractable organic fluorine (EOF)\u003c/h2\u003e \u003cp\u003eA calibration curve was prepared using PFOA in 50% (v/v) methanol ranging from 0 to 2000 \u0026micro;g F L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Ten blank measurements were conducted using deionized water, then the blank standard deviation (SD) was calculated, and the result was divided by the slope of the calibration curve, which was taken on the same day of blank measurement, multiplied by 3 to obtain the value of the instrumental LOD. The LOD calculated to be 0.49 \u0026micro;g F L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section3\"\u003e \u003ch2\u003e2.7.3 Statistical analysis\u003c/h2\u003e \u003cp\u003eData were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) on a dry weight (dw) basis. The statistical analysis included one- and two-way analysis of variance (ANOVA) approaches. All analyses were performed using Minitab 20 (Minitab LLC, USA) and Microsoft Excel (Microsoft, USA) software. The least significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were determined and used to compare the statistical significance between treatments.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Effects of GenX on the growth of rice and wheat\u003c/h2\u003e \u003cp\u003eGrowth characters, including shoot biomass and the length of the wheat and rice crops, under GenX application are presented in (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The number of tillers for wheat, shoot length and dry biomass showed non-significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) results with both GenX treatments compared to the control with \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.451, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.762 and \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.178 values, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The rice shoots\u0026rsquo; dry biomass, however, exhibited a significant reduction of 25% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02) at 2 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e GenX concentrations compared to the control under flooded soil conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Further, reductions of 9% and 7% were observed for the rice shoots\u0026rsquo; dry biomass at 0.4 and 2 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e GenX concentrations, respectively, compared to the non-flooded control. The flooded and non-flooded soil conditions exhibited non-significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.096) effects on the rice shoots\u0026rsquo; dry biomass at both GenX concentrations. Rice plant height however affected (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.048) under varied soil conditions. Rice shoot length showed a significant reduction with GenX and with varied soil environments (flooded (\u003cem\u003eP\u003c/em\u003e 0.048) and non-flooded (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001)), indicating that the percentage of reduction increases with higher GenX concentrations.\u003c/p\u003e \u003cp\u003eThe number of rice tillers exhibited a non-significant effect with GenX exposure (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.301) and under varied soil conditions (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1). The interaction between GenX treatments and soil conditions is only significant for the number of tillers, with a \u003cem\u003eP\u003c/em\u003e value (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.017).\u003c/p\u003e \u003cp\u003eOverall, the rice crops grown under non flooded soil exhibited a lower tolerance to GenX treatments compared to the wheat crops.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Uptake of GenX into shoots\u003c/h2\u003e \u003cp\u003eThe concentration of GenX significantly varied (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) with the GenX treatments and under different soil conditions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The uptake of GenX increased as the level of GenX treatment increased in both tested crops. After 30 days of GenX exposure, the wheat shoot\u0026rsquo;s uptake was 2.71 (\u0026plusmn;\u0026thinsp;0.92) and 10.5 (\u0026plusmn;\u0026thinsp;1.05) \u0026micro;g g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e at 0.4 and 2.0 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of GenX application, respectively. This concentration of GenX in the plant shoot was significantly higher than that of the control group. The control soil had a GenX concentration below LOQ.\u003c/p\u003e \u003cp\u003e The rice plants showed significantly varied GenX concentration values in their shoots according to the different soil conditions. The maximum uptake of GenX was observed in non-flooded soil conditions over flooded (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.003). At 0.4 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of GenX, the rice shoots exhibited 2.34 (\u0026plusmn;\u0026thinsp;0.45) and 4.11 (\u0026plusmn;\u0026thinsp;0.87) \u0026micro;g g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of GenX uptake in flooded and non-flooded soil environments, respectively. When exposed to 2.0 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of GenX, the rice shoots showed a GenX uptake level of 10.4 (\u0026plusmn;\u0026thinsp;0.41) \u0026micro;g g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e under flooded conditions and 13.4 (\u0026plusmn;\u0026thinsp;0.72) \u0026micro;g g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e under non-flooded conditions. The uptake of GenX by the rice shoots showed a non-significant effect under the interactivity of the GenX treatment and soil condition (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.34). Overall, the rice shoots in non-flooded soil conditions exhibited more GenX uptake when exposed to higher concentrations of GenX than did the wheat shoots.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe GenX levels in the rice and wheat shoots exhibited similar distribution patterns to the GenX concentrations in plant tissues (Fig \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Fig S2). Of all the experiments, the non-flooded rice absorbed the most GenX.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eStatistical analysis of GenX concentrations in rice under varying treatment conditions.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eParameters\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGenX treatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGrowth conditions\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGenX growth conditions interaction\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eBiomass\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.096\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.436\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePlant height\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.091\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNumber of tillers\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.301\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.017\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eShoot GenX conc.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.349\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe removal efficiency (%) of the rice and wheat was significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) affected by the GenX application level and soil conditions. This was calculated by dividing the mass of the GenX in the plant tissues by the mass of spiked GenX (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Generally, a higher removal percentage was observed at 0.4 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of GenX exposure with respect to the 2.0 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e treatment. The wheat plants exhibited a removal efficiency of 2% and 1% with low and high concentrations of GenX, respectively, while the rice crops exhibited a removal efficiency of 3% and 2% at lower and higher GenX levels, respectively, in both soil environments.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Distribution coefficient\u003c/h2\u003e \u003cp\u003eThe distribution coefficient, K\u003csub\u003ed\u003c/sub\u003e, describes the distribution of a chemical between two media or two phases. Prior studies have used this approach to evaluate the accumulation and mobility of PFAS in solid-water systems (Milinovic et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Further, its value has been used to provide information on the distribution and final course of PFAS. The K\u003csub\u003ed\u003c/sub\u003e value for the present study was determined as the ratio of the concentration of GenX measured in the soil phase and the concentration of GenX measured in the aqueous phase, as detailed below (Nguyen et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2020\u003c/span\u003e):\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$Kd\\left(\\frac{L}{kg}\\right)=\\frac{ \\text{m}\\text{e}\\text{a}\\text{s}\\text{u}\\text{r}\\text{e}\\text{d} \\text{c}\\text{o}\\text{n}\\text{c}\\text{e}\\text{n}\\text{t}\\text{r}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n} \\text{i}\\text{n} \\text{t}\\text{h}\\text{e} \\text{s}\\text{o}\\text{i}\\text{l} \\text{p}\\text{h}\\text{a}\\text{s}\\text{e} \\text{m}\\text{g}/\\text{k}\\text{g}}{\\text{m}\\text{e}\\text{a}\\text{s}\\text{u}\\text{r}\\text{e}\\text{d} \\text{i}\\text{n} \\text{t}\\text{h}\\text{e} \\text{a}\\text{q}\\text{u}\\text{e}\\text{o}\\text{u}\\text{s} \\text{p}\\text{h}\\text{a}\\text{s}\\text{e} \\text{m}\\text{g}/\\text{L}}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eK\u003csub\u003ed\u003c/sub\u003e values in rice under two different treatments. Errors are represented by one standard deviation for four replicates.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003eFlooded rice\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eNon-flooded rice\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWeek 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWeek 4\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eWeek 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eWeek 4\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.54(\u0026plusmn;\u0026thinsp;0.02)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e0.04(\u0026plusmn;\u0026thinsp;0.03)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.35 (\u0026plusmn;\u0026thinsp;0.05)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0. 07(\u0026plusmn;\u0026thinsp;0.02)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e0.49(\u0026plusmn;\u0026thinsp;0.10)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.08(\u0026plusmn;\u0026thinsp;0.34)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e0.43(\u0026plusmn;\u0026thinsp;0.02)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.68(\u0026plusmn;\u0026thinsp;0.23)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eGenX exhibits low k\u003csub\u003ed\u003c/sub\u003e, which indicates that GenX is very mobile. K\u003csub\u003ed\u003c/sub\u003e levels with low and high concentrations of GenX were higher in the flooded rice than in the non-flooded rice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.028). Hence, GenX is significantly more mobile in the non-flooded environment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e3.4 Translocation factor (TF)\u003c/h2\u003e \u003cp\u003eThe translocation factor was used to describe the ability of the rice to translocate GenX to the plant shoots. The TF values were calculated by the ratio of the concentration of GenX measured in the shoot and the concentration of GenX measured in porewater, as detailed below:\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\text{T}\\text{F}=\\frac{\\text{m}\\text{e}\\text{a}\\text{s}\\text{u}\\text{r}\\text{e}\\text{d} \\text{c}\\text{o}\\text{n}\\text{c}\\text{e}\\text{n}\\text{t}\\text{r}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n} \\text{i}\\text{n} \\text{t}\\text{h}\\text{e} \\text{s}\\text{h}\\text{o}\\text{o}\\text{t}}{\\text{m}\\text{e}\\text{a}\\text{s}\\text{u}\\text{r}\\text{e}\\text{d} \\text{c}\\text{o}\\text{n}\\text{c}\\text{e}\\text{n}\\text{t}\\text{r}\\text{a}\\text{t}\\text{i}\\text{o}\\text{n} \\text{i}\\text{n} \\text{t}\\text{h}\\text{e} \\text{p}\\text{o}\\text{r}\\text{e}\\text{w}\\text{a}\\text{t}\\text{e}\\text{r}}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e \u003cp\u003eThe values for the translocation factor significantly varied under application of GenX (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.001) and soil conditions (flooded and non-flooded) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.02) (see Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Maximum TF values were observed at 2.0 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of GenX treatment, not at the 0.4 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e treatment. TF values of 3.08 (\u0026plusmn;\u0026thinsp;0.12) and 3.80 (\u0026plusmn;\u0026thinsp;0.35) were observed under the flooded and non-flooded conditions, respectively, at low exposure. At high exposure, the TF values were observed at 5.00 (\u0026plusmn;\u0026thinsp;0.07) and 5.33 (\u0026plusmn;\u0026thinsp;0.14) in the flooded and non-flooded environments, respectively. These higher TF values in the non-flooded environment indicate that rice plants can translocate more effectively in non-flooded conditions than in flooded conditions.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eTranslocation factor (TF) values in rice under two different growing conditions. Error represents one SD (n\u0026thinsp;=\u0026thinsp;4).\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTreatment\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFlooded rice\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eNon-flooded rice\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenX at 0.4 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.08 (\u0026plusmn;\u0026thinsp;0.12)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e3.80 (\u0026plusmn;\u0026thinsp;0.35)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGenX at 2 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e5.00 (\u0026plusmn;\u0026thinsp;0.07)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e5.33 (\u0026plusmn;\u0026thinsp;0.14)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec25\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Total extractable organic fluorine (EOF) measurement and mass balance analysis\u003c/h2\u003e \u003cp\u003eA fluorine mass balance analysis was performed to estimate the levels of unidentified organic fluorine compounds (UOF) that resulted from the degradation of GenX or another PFAS or other fluorinated compounds such as pesticides and pharmaceuticals occurring in the soil. The study determined the extractable organic fluorine using HR-GFMAS and percentage contributions of the total PFAS, including the GenX in the rice, wheat, porewater and soil (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAn LC-MS/MS analysis was performed to identify the PFAS in the rice and wheat shoots, soil and porewater. No other PFAS, apart from GenX, was detected among the 35 targeted PFAS, which included PFCA and PFSA with perfluorocarbon chain lengths of C4-C12, as well as PFAS sulphonamides (table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Notably, ultra-short PFCAs were not monitored in this study due to challenges in their measurement.\u003c/p\u003e \u003cp\u003eGenX at low and high exposure levels was found to be the major contributor to the EOF in the flooded rice shoots, contributing to 78 to 97% of the extractable organic fluorine. In the non-flooded rice shoots, it contributed to 68 to 95% at both exposure levels. The difference in mass balance between flooded and non-flooded conditions was not significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.133), as well as between different exposure levels (\u003cem\u003eP\u003c/em\u003e value\u0026thinsp;=\u0026thinsp;0.103).\u003c/p\u003e \u003cp\u003eIn the wheat shoots, GenX was found to contribute to only 50\u0026ndash;72% of the total extractable fluorine at both low and high exposure levels. Similarly, in the flooded soil, GenX contributed to 64\u0026ndash;84% of the extractable fluorine and in non-flooded rice soil, it contributed to 21\u0026ndash;92% at both exposure levels. While in non-flooded wheat soil contributed to 57\u0026ndash;97% of the extractable fluorine. The difference in mass balance between flooded and non-flooded conditions was also not significant (\u003cem\u003eP\u003c/em\u003e value\u0026thinsp;=\u0026thinsp;0.176), but significant between different exposure levels (\u003cem\u003eP\u003c/em\u003e value\u0026thinsp;=\u0026thinsp;0.03). GenX was also found to account for 53 to 94% of the total extractable organic fluorine in non-flooded soil porewater samples, while in flooded soil porewater, it accounted for 53% and 98% at low and high exposure levels respectively. This difference in mass balance between flooded and non-flooded conditions was not significant (p value\u0026thinsp;=\u0026thinsp;0.447), as well as between different exposure levels (p value\u0026thinsp;=\u0026thinsp;0.480).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e \u003ch2\u003e4.1 Effects of GenX on growth and biomass of rice and wheat\u003c/h2\u003e \u003cp\u003eThe 30-day exposure of GenX at the rate of 0.4 and 2.0 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e significantly affected the growth parameters of both tested crops, but most notably the rice. The results demonstrate that the rice shoots\u0026rsquo; dry biomass significantly reduced under 2.0 mg g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e compared to the dry shoot biomass of wheat (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The growth of the rice was more inhibited in non-flooded soil than was wheat, indicating that rice is more sensitive to GenX than wheat. Similar findings have been reported by Chen et al. (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), who found that in a hydroponic experiment, plant species \u003cem\u003eA. thaliana\u003c/em\u003e and \u003cem\u003eN. benthamiana\u003c/em\u003e did not exhibit any growth issues when exposed to 5 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e GenX but when GenX dosage increased, shoot growth was affected. Both the growth and development of the shoots and roots of \u003cem\u003eN. benthamiana\u003c/em\u003e were severely hindered when exposed to the highest level of GenX (20 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). \u003cem\u003eN. benthamiana\u003c/em\u003e showed reduced root and shoot biomass, with tolerance index values for GenX decreasing from 100\u0026ndash;40% and 55%, respectively (Chen et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). A recent study that investigated the phytotoxicity of GenX in lettuce showed that the shoots\u0026rsquo; dry biomass remained unaffected when treated with a concentration of 100 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of GenX. This suggests that at this concentration, GenX does not have a significant adverse effect on the growth and development of lettuce plants (Wang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). These findings suggest that the effects of GenX on plant growth and development are concentration and species-specific.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003e4.2 Uptake of GenX\u003c/h2\u003e \u003cp\u003eWhile a proportional uptake of GenX was observed for both crops (rice and wheat), the amounts as measured by their biomass quantities differed. This observation further confirms that uptake and bioaccumulation does not only depend on GenX properties and chemical characteristics but also on plant species and its capacity for bioaccumulation. As Doucette et al. (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) noted, the differences in PFAS uptake among species are generally due to varying biotransformation capacities, rates of dissipation, ease of root uptake and temperature (Doucette et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Further, Zhao et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) found that wheat\u0026rsquo;s uptake of PFCA increased by 1.5- to 2.3-fold when the temperature increased from 20 to 30\u0026deg;C (Zhao et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). In our study, the rice and wheat were grown in different greenhouses with different temperatures. The rice was grown at 28\u0026deg;C, while the wheat was grown at 15\u0026deg;C. This may have impacted the uptake of GenX between the wheat and non-flooded rice. Zhao et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) found that the growth of wheat at a temperature of around 30\u0026deg;C can lead to nutrient diffusion rates increase, water viscosity decreases and photosynthesis is given more energy, all of which increase the uptake of pollutants (Zhao et al. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). To avoid changing the PFAS uptake, plant growth conditions must be carefully monitored.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec29\" class=\"Section2\"\u003e \u003ch2\u003e4.3 GenX mechanism of intoxication and growth inhibition\u003c/h2\u003e \u003cp\u003eDespite the different uptake and bioaccumulation capacities, GenX was found to inhibit the growth of shoot biomass more in rice. GenX, like other PFASs, initiates a mechanism that inhibits shoot growth in plants, but this appears to be species-specific at the concentration used in this study. Our findings are in line with studies involving both human cells \u003cem\u003ein vitro\u003c/em\u003e and plant species. Such studies have concluded that exposure to GenX induces an intracellular toxicity mechanism that leads to apoptosis and a reduction of cell viability (Yoo et al. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similarly, GenX uptake has been observed to have an adverse effect on lettuce even at low concentration levels (100 \u0026micro;g L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e), which suggests that GenX could induce oxidative stress in lettuce plants (Wang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e content in lettuce tissues revealed that GenX causes stronger oxidative stress than does PFOA. Previous research also found that 100 to 200 mg L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e of GenX increased H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e in \u003cem\u003eNicotiana benthamiana\u003c/em\u003e plants. These findings suggest that even low concentrations of GenX can cause significant oxidative stress in plants, even more than the phased-out PFOA (Chen et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Both outcomes indicate that a similar mechanism of GenX inhibition affects the growth and development of different species, albeit at different intoxication levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec30\" class=\"Section2\"\u003e \u003ch2\u003e4.4 Impact of soil conditions on GenX bioaccumulation\u003c/h2\u003e \u003cp\u003eApart from the plant species and chemical properties of GenX, soil conditions also impacted the uptake and bioaccumulation of GenX in rice. In this experiment, the rice grown in non-flooded soil had higher concentrations of GenX compared to that grown in flooded soil (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). This observation is in line with findings from studies that revealed that efficient transpiration facilitates the translocation of more molecules through the water gradient (Lesmeister et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Similarly, it has been established that flooding reduces transpiration rate, thereby affecting the distribution of the substance located in the soil and the plant tissues. In non-flooded conditions, the rate of transpiration is generally higher than in flooded conditions, which can lead to higher rates of PFAS uptake by plants. This causes less GenX to be concentrated in the flooded plant comparing to the non-flooded one. It has been suggested that the amount of water that transpires during growth can explain the various absorption and translocation abilities among crops (Blaine et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnaerobic (flooded) and aerobic (non-flooded) soil conditions cause the soil to exhibit different biological and chemical characteristics. For example, there is less oxygen in flooded soil because the soil pores fill with water. In the absence of oxygen, the reduced chemical forms of compounds predominate. When oxygen availability is limited, bacteria opt to use other compounds as electron acceptors to maintain their metabolism, thereby reducing the elements in these compounds. The result of such microbial metabolism is the conversion of oxidised elements to their corresponding reduced forms under anaerobic conditions. In non-flooded soil, oxygen is available for bacterial metabolism, so the compounds stay in their oxidised forms. Although the degradation of such PFAS compounds as FTOH has been reported under aerobic and anaerobic conditions, the degradation of GenX has not been studied in much depth. One study on the sorption of GenX onto sediments found that within 14 days, the concentration of GenX in freshwater and estuary sediments decreased by 40 to 59%. Moreover, there were no statistically significant variations in GenX loss between the bioactive and autoclaved sediments, so the decrease could not be explained by biological degradation (Harfmann et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003e4.5 Translocation factor (TF)\u003c/h2\u003e \u003cp\u003eAt higher GenX exposure in the present study, its concentration in both wheat and rice shoots rose as well, but not proportional. Moreover, higher transfer factor (TF) values were found at higher exposure levels, indicating that the plants were taking up more GenX with increasing exposure levels. Interestingly, the TF values for GenX in rice shoots were higher in non-flooded soil than in flooded soil conditions. This suggests that rice plants in non-flooded conditions can translocate GenX more easily from the roots to the shoots compared to rice grown in flooded conditions. This can be explained by the higher concentration of GenX in the soil porewater under non-flooded conditions compared to flooded conditions. This difference in concentration of GenX in the soil porewater can be attributed to the lower mobility of GenX in flooded soil conditions due to its strong adsorption to soil particles, which reduces its availability for plant uptake. Conversely, in non-flooded soil conditions, GenX is more mobile and has a lower K\u003csub\u003ed\u003c/sub\u003e value, which allows it to move freely in the soil water, making it more accessible for plant uptake. This would mean that when rice is grown in non-flooded conditions to safe water, it would increase the uptake of GenX, which may have implications for human health and the environment. Zhang et al. (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) (Zhang et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) found that among the five ether-PFAS studied, GenX had the highest translocation factor (TF) value in wetland plants \u003cem\u003e(C. comosa )\u003c/em\u003e exposed to 500 ng L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for 52 days, which indicates that GenX can be translocated easily to shoots of a wetland plant. Gu et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) (Wang et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) also reported that short-chain PFAS accumulate more easily in the edible parts of plants compared to long-chain PFAS. Overall, in both the flooded and non-flooded rice, TF was statistically different between the GenX treatments. This suggests that the increase in GenX exposure impacted the crop\u0026rsquo;s efficiency of GenX uptake as well.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec32\" class=\"Section2\"\u003e \u003ch2\u003e4.6 Fluorine mass balance and EOF\u003c/h2\u003e \u003cp\u003eThe results of the fluorine mass balance analysis show that higher exposure levels of GenX significantly contribute to the total extractable fluorine. These results suggest that a close fluorine mass balance exists at high exposure levels, and that most of the extractable fluorine can be accounted for by the presence of GenX. The high contribution of GenX to the total extractable fluorine in rice shoots, porewater and soil implies no GenX degradation in the plants. However, discrepancies between the EOF and GenX concentrations in the soil at low exposure levels suggest that GenX may degrade to other short-chain PFCAs not included in the targeted LC-MS/MS analysis. A recent study examined the effects of fluoroalkylether compounds, including GenX, on the microbial community in soil\u0026ndash;plant systems. The results of the study showed that the structure of the community and species diversity were significantly impacted by ether-PFAS at concentrations of 500 ng L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 2,000 ng L\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (Jiang et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Although the study did not find any evidence of biodegradation of the spiked ether-PFAS, it did reveal that the presence of these compounds seemed to stimulate the growth of certain microbes with the ability to break down hydrocarbon chemicals and contaminants. In our study it is significant that at low exposure level the mass balance between GenX and EOF shows unaccounted organofluorines, which however were not detected in the targeted LC-MS/MS analysis. The unaccounted organofluorines might however be ultra-short chain PFAS as degradation products which were not monitored. It is important to note that our study did not aim to investigate the mechanisms or pathways of GenX degradation in these plants. Therefore, further research is required to understand the course of this compound in agricultural environments comprehensively in the field.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec33\" class=\"Section2\"\u003e \u003ch2\u003e4.7 GenX distribution coefficient\u003c/h2\u003e \u003cp\u003eIn this experiment, the substance molecule distribution was determined using the distribution coefficient, K\u003csub\u003ed\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eAn earlier study assessed the behaviours of a wider selection of PFAS on soils of different properties. The study found that per- and polyfluoroalkyl ether acids, including GenX, have low K\u003csub\u003ed\u003c/sub\u003e values, which means that they are very mobile. This indicates the hydrophilic nature (hydrophilicity) of these compounds and the ease by which they may be taken up in groundwater to pollute waterways and the environment [18].\u003c/p\u003e \u003cp\u003eAs indicated in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the present study observed K\u003csub\u003ed\u003c/sub\u003e values for GenX in rice are influenced by treatment conditions (flooded vs. non-flooded) and exposure levels. A lower K\u003csub\u003ed\u003c/sub\u003e value for GenX suggests a higher degree of mobility, allowing it to be present in the water phase instead of adsorbing onto soil particles. In this study, it has been found that GenX had the lowest K\u003csub\u003ed\u003c/sub\u003e value in non-flooded conditions, especially at low exposure levels, which suggests that it is more likely to be taken up by rice under non flooding conditions compared to flooded conditions. In other words, the mobility of GenX is higher under non-flooded conditions, resulting in its increased presence in the water phase. This characteristic makes it more available for plant uptake by rice, resulting in a higher likelihood of accumulation. Hence, the results of this study suggest that the risk of GenX accumulation in rice is higher under non-flooded conditions, particularly at low exposure level. These findings are in line with previous evidence indicating that short-chain PFASs like GenX are more readily absorbed through a water gradient and anion channels when in adequate concentrations in the growth medium (Li et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTo our knowledge, the uptake mechanisms of new alternatives like GenX in plants have not been well studied. Gu et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) investigated the differences between PFOA and its alternatives, finding that PFOA and GenX were likely transported via a diffusion process. Water channels only have a limited effect on the uptake of PFOA and its alternatives, and slow anion channels rather than rapid ones were mainly responsible for the uptake of PFCAs (Gu et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). If this applies also to rice and wheat, the difference in GenX uptake of the plant species in our experiment can therefore not be a temperature effect but rather a plant species effect.\u003c/p\u003e \u003cp\u003eAs observed previously, despite being industrially used as a short-chain alternative to PFOA, GenX still has similar properties that make it unsafe for the environment. According to recent research, PFASs such as GenX accumulate to different degrees in crops, depending on the components of said crops (Krippner et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Bizkarguenaga et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). PFASs have been reported to accumulate in protein-rich plant parts; as a result, GenX may accumulate in the bran and germ of rice and wheat. Although wheat and rice are the model crops for this study, other findings raise concerns about the safety of crops whose shoots are consumed directly, such as lettuces. In China, for instance, water scarcity has forced a shift from cultivating rice in traditional high-water-consuming lowlands to controlled and non-flooded irrigation approaches. Although non-flooded conditions have been tested successfully for the reduction of other contaminants of concern, such as arsenic (as rice grown in traditional flooded paddies has been found to contain arsenic levels 10 to 15 times greater than rice cultivated in non-flooded conditions (Li et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2019\u003c/span\u003e)), here we observed higher GenX concentrations in rice shoots grown under non-flooded conditions. This may increase safety risks for crops cultivated under water stressed and rainfed areas.\u003c/p\u003e \u003cp\u003eIn contrast to the available published information on PFAS and PFOA substances, this study\u0026rsquo;s findings are crucial because they demonstrate that despite being a short-chain alternative to PFOA, GenX exhibits chemical properties and environmental safety concerns. Even more than long-chain PFAS (like PFOA), the industrial use of which has been replaced, GenX has the potential to impede plant growth. Studies in line with our findings on shoot biomass and growth length indicate that due to GenX\u0026rsquo;s high solubility in water, it can enter the environment and affect plants more easily than other PFAS (Ahrens et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Prior studies in rice paddy fields, which are consistent with the present flooded soil conditions, have determined that due to their high mobility in water, short-chain PFAS like GenX are likely to leach into groundwater systems, thereby reducing their availability for plant uptake (Eun et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This explains our observation that less GenX accumulated in the shoots of rice grown in flooded soil. Based on these study findings, GenX is not better for the environment because of its higher bioaccumulation and poor degradation. Further, GenX is only poorly absorbed in flooded conditions and better taken up by the rice under non-flooded conditions, making it a concern for modern crop irrigation approaches that aim to optimise production with minimal water use.\u003c/p\u003e \u003c/div\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eThis study assessed the bioaccumulation potential of an emerging fluorinated ether, GenX, in wheat and rice in two concentrations. The results demonstrate that GenX was taken up by plant roots, translocated to shoots and accumulated in the tissues of the studied plants. Among the two investigated plant species, rice demonstrated the highest concentrations of GenX in its shoots. The accumulation of GenX in rice and wheat increased when the plants were exposed to higher levels of GenX, this means that rice and wheat growing in hot spots of GenX contamination would be far more affected as would low level exposure experiments suggest. The results of this study indicate that rice is more sensitive to GenX than wheat at relatively high exposure levels. Moreover, the existence of GenX might pose similar or even higher ecotoxicological risks to the environment than PFOA. Long-term PFAS accumulation in surface and wastewater, as well as the potential risk to organisms due to environmental persistence, must be considered seriously and dealt with effectively to reduce adverse environmental consequences. Therefore, it is important to conduct additional research to compare the accumulation and phytotoxic effects of GenX with other plant species and other PFAS under longer exposure times.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.A.Z. thank the Culture Bureau of Saudi Arabia for their financial support. V.M. thank the Macaulay Development Trust for her scholarship.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCompliance with ethical standards\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo consent of participate was necessary for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to Publish\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll co-authors gave the consent to publish.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmnah Al Zbedy: Laboratory work, HR GFMAS, greenhouse work, methodology, writing- original draft preparation.\u003c/p\u003e\n\u003cp\u003eViktoria M\u0026uuml;ller: Laboratory work, Target LC-MS/MS, help with manuscript writing\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAndrew Kindness: Supervision, help with manuscript writing\u003c/p\u003e\n\u003cp\u003eRainer Ebel: Supervision, help with manuscript writing.\u003c/p\u003e\n\u003cp\u003eGareth Norton: Conceptualisation, methodology, supervision, help with manuscript writing.\u003c/p\u003e\n\u003cp\u003eJ\u0026ouml;rg Feldmann: Conceptualisation, methodology, supervision, help with manuscript writing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAmnah Al Zbedy: financial support of her scholarship from the Cultural Bureau of Saudi Arabia, London.\u003c/p\u003e\n\u003cp\u003eViktoria M\u0026uuml;ller: financial support of her scholarship from the Macaulay Development Trust, UK.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMost data are made available in the supplementary material. Additional data will be made available from the corresponding author on request.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAhrens L, Taniyasu S, Yeung LWY et al (2010) Distribution of polyfluoroalkyl compounds in water, suspended particulate matter and sediment from Tokyo Bay. Japan Chemosphere 79:266\u0026ndash;272. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.chemosphere.2010.01.045\u003c/span\u003e\u003cspan address=\"10.1016/j.chemosphere.2010.01.045\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAhrens L, Yeung LWY, Taniyasu S et al (2011) Partitioning of perfluorooctanoate (PFOA), perfluorooctane sulfonate (PFOS) and perfluorooctane sulfonamide (PFOSA) between water and sediment. 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Springerplus 5:\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhi Y, Lu H, Grieger KD et al (2022) Bioaccumulation and Translocation of 6:2 Fluorotelomer Sulfonate, GenX, and Perfluoroalkyl Acids by Urban Spontaneous Plants. ACS ES\u0026amp;T Eng 2:1169\u0026ndash;1178. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acsestengg.1c00423\u003c/span\u003e\u003cspan address=\"10.1021/acsestengg.1c00423\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCandidate List of substances of very high concern for Authorisation - ECHA. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.echa.europa.eu/candidate-list-table\u003c/span\u003e\u003cspan address=\"https://www.echa.europa.eu/candidate-list-table\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Accessed 19 Feb 2023a\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eReports, Decisions \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://chm.pops.int/TheConvention/POPsReviewCommittee/ReportsandDecisions/tabid/3309/Default.aspx\u003c/span\u003e\u003cspan address=\"http://chm.pops.int/TheConvention/POPsReviewCommittee/ReportsandDecisions/tabid/3309/Default.aspx\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. Accessed 19 Feb 2023b\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"PFAS, plant uptake, rice, agriculture practise, wheat, EOF","lastPublishedDoi":"10.21203/rs.3.rs-2889643/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2889643/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eGenX (hexafluoropropylene oxide dimer acid) belongs to the group of per- and poly-fluoroalkyl substance (PFAS) compounds introduced to replace perfluorooctanoic acid (PFOA), which has been phased out in industrial and consumer product formulations. While GenX has been investigated in lab animals, there is limited information available regarding its uptake and translocation in wheat and rice. This study reports on a greenhouse experiment in which wheat and rice grown under flooded and non-flooded conditions were exposed to two GenX concentrations in the soil (0.4 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 2 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). GenX was analysed in the soil, porewater and shoots using targeted liquid chromatography-tandem mass spectroscopy (LC-MS/MS) analysis. Extractable organic fluorine (EOF) was determined using high-resolution continuum source graphite furnace molecular absorption spectrometry (HR GFMAS). Results showed that different species took up different amounts of GenX. The GenX in rice shoots was found to be 2.34 (\u0026plusmn;\u0026thinsp;0.45) \u0026micro;g g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 4.11 (\u0026plusmn;\u0026thinsp;0.87) \u0026micro;g g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e under flooded and non-flooded conditions, respectively, at a low exposure level. At high exposure, the GenX concentrations in flooded and non-flooded rice shoots increased threefold to 10.4 (\u0026plusmn;\u0026thinsp;0.41) and 13.4 (\u0026plusmn;\u0026thinsp;0.72) \u0026micro;g g\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, respectively. Wheat shoots showed similar concentrations and increases between low- and high-level exposure.\u003c/p\u003e \u003cp\u003eThe translocation factor was significantly higher (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.013) in non-flooded rice compared to flooded rice. The GenX bioaccumulation behaviours under the same culture conditions (e.g. temperature, humidity, light, same GenX concentration in the soil) were significantly different in non-flooded and flooded rice (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Non-flooded rice plants displayed a higher level of GenX bioaccumulation than flooded ones. Following exposure to GenX, flooded rice plants showed a reduction in biomass (25%) compared to the control plants (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.014). Our findings indicate that GenX is a bioaccumulative compound, the presence of which likely inhibits the growth of plants.\u003c/p\u003e","manuscriptTitle":"GenX uptake by wheat and flooded and non-flooded rice: greenhouse experiment.","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-07-26 14:35:03","doi":"10.21203/rs.3.rs-2889643/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2023-07-27T04:19:00+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-07-21T15:11:01+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Environmental Science and Pollution Research","date":"2023-05-30T17:39:18+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-05-23T04:09:19+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Science and Pollution Research","date":"2023-05-17T04:54:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0b1c145e-175b-4df6-8d93-7064c9ab577e","owner":[],"postedDate":"July 26th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2023-12-11T15:10:46+00:00","versionOfRecord":{"articleIdentity":"rs-2889643","link":"https://doi.org/10.1007/s11356-023-31160-w","journal":{"identity":"environmental-science-and-pollution-research","isVorOnly":false,"title":"Environmental Science and Pollution Research"},"publishedOn":"2023-12-04 15:01:43","publishedOnDateReadable":"December 4th, 2023"},"versionCreatedAt":"2023-07-26 14:35:03","video":"","vorDoi":"10.1007/s11356-023-31160-w","vorDoiUrl":"https://doi.org/10.1007/s11356-023-31160-w","workflowStages":[]},"version":"v1","identity":"rs-2889643","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2889643","identity":"rs-2889643","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}