Science of the Total Environment 472 (2014) 20–26

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Uptake of perfluorinated compounds by plants grown in nutrient solution A.I. García-Valcárcel a,⁎, E. Molero a, M.C. Escorial b, M.C. Chueca b, J.L. Tadeo a a b

Department of Environmental Science, INIA, Ctra. De La Coruña Km 7, 28040 Madrid, Spain Department of Plant Protection, INIA, Ctra. De La Coruña Km 7, 28040 Madrid, Spain

H I G H L I G H T S • • • •

Levels of perfluorinated compounds (PFCs) in plants were determined by LC-MS/MS Uptake of PFCs by plants growing in nutrient solution was determined along 20 days Plant uptake of perfluorinated carboxylates was related to the carbon chain length Uptake of perfluorinated sulfonates was independent of the carbon chain length

a r t i c l e

i n f o

Article history: Received 9 September 2013 Received in revised form 9 October 2013 Accepted 16 October 2013 Available online 28 November 2013 Keywords: Perfluorinated compounds Plant Hydroponic system MSPD extraction Uptake rates

a b s t r a c t The uptake rates of three perfluorinated carboxylates and three perfluorinated sufonates by a grass (B diandrus) grown in nutrient solution at two different perfluorinated compounds (PFCs) concentrations were assessed. Grass can be ingested by grazing animals causing the PFCs to enter the food chain, which is a pathway of human exposure to these compounds. A rapid and miniaturized method was developed to determine PFCs in plants, based on a matrix solid-phase dispersion (MSPD) extraction procedure followed by quantitation by HPLC-MS/MS with an MQL in the range from 1 to 9 ng/g. An increase of PFCs levels in plant was observed along the exposure time. Differences in uptake for studied perfluorinated carboxylates were found, showing a decrease with carbon chain length (from 3027 to 1167 ng/g at the end of assay), whereas no significant differences in absorption were obtained between perfluorinated sulfonates (about 1700 ng/g). Initially, higher PFC transfer factors (ratio between concentration in plant and concentration in initial nutrient solution) were obtained for plants growing in the nutrient solution at the highest PFC concentration, but these factors became similar with time to plants exposed to the lowest concentration. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Perfluorinated compounds (PFCs) are used in a variety of consumers and industrial applications such as protective coatings in textiles, carpets, packets and leather, as well as surfactants in fire-fighting foam and insecticides among others. These compounds exhibit both hydrophobic and lipophilic properties due to the attachment of a sulfonate, carboxylate or sulfonamide group to the end of an aliphatic carbon chain fully fluorinated. These compounds are a cause for concern due to their persistence, bioaccumulation and ubiquitous presence in the environment (Prevedouros et al., 2006; Jahnke and Berger, 2009; Ahrens, 2011). Perfluorinated pollutants present in soil, water and air may enter into the food chain, being dietary intake the predominant route of human exposure to these compounds (Tittlemier et al., 2007; Vestergren et al., 2008; Picó et al, 2011), which have been also found ⁎ Corresponding author. Tel.: +34 91 347 6823; fax: +34 91 357 2293. E-mail address: [email protected] (A.I. García-Valcárcel). 0048-9697/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scitotenv.2013.10.054

in human and animal tissues (Zhao et al., 2012; Hanssen et al., 2013), where they may cause adverse health effect (Lau et al., 2007; Steenland et al., 2010; Wang et al., 2011). One way of entry of the perfluorinated compounds into human and animal populations is via plants, which constitute one of the basic diets for humans and animals. Several studies have shown that vegetable food contains PFCs (Haug et al., 2010; Domingo et al., 2012; Ullah et al., 2012; Vestergren et al., 2012). Plants can be exposed to PFCs in different ways, like when they grow on soils amended with sludge contaminated by PFCs, when they are irrigated with waste water containing these compounds or by aerial deposition. Whereas in the first two cases perfluoroalkyl sulfonates and perfluoroalkyl carboxylates are the compounds involved, in the third one volatile neutral PFCs, such as fluorotelomers and perfluoroalkyl sulfonamides, precursors of the ionic PFCs, are the contaminants involved. In soil, absorption of PFCs may occur through active or passive uptake by the roots, possibly followed by transport along the plant with the transpiration stream. Plant uptake from air occurs by wet and/or dry deposition on the

A.I. García-Valcárcel et al. / Science of the Total Environment 472 (2014) 20–26

above-ground parts and subsequent absorption and translocation over the cuticles. Plant uptake and distribution have been shown to be dependent on the physical–chemical properties of the chemicals, the soil and irrigation water characteristics, and the plant species and physiology, including properties such as lipid or water content and transpiration rates (Paterson et al., 1994). However, data available on uptake of PFCs by plants are scarce (Stahl et al., 2009, 2013; Lechner and Knapp, 2011; Yoo et al., 2011; Felizeter et al., 2012) and some of them only consider perfluooroctane sulfonate (PFOS) and perfluoroctanoic acid (PFOA) (Stahl et al., 2009, 2013; Lechner and Knapp, 2011). In these works, values of transfer factors from soil or water to plant were reported, but only at one time, generally at time of harvest, and none of them give the rate of uptake by plant at different times during the growing period, which may have interest for the determination of the time of the maximum uptake, and its correlation with the concentration in the medium. On the other hand, although there are analytical methods for the quantitation of PFCs in different matrices (soil, water, sludge, air, animal tissues…), specific methods for the determination of PFCs in green plants are scarcer. Therefore, it was necessary to develop a reliable method to quantify these compounds in plants, mainly in their green parts, because PFC accumulation takes place at higher extend in the aerial parts than in roots (Felizeter et al., 2012) or other edible parts such as storage organs in potatoes, carrots or cucumbers (Lechner and Knapp, 2011). The aim of this work was to develop a fast and efficient method to quantify PFCs in grass, specifically in Bromus diandrus, which may be ingested by grazing animals and enter the food chain, and to assess the rate and extent of PFC uptake by plants grown in a hydroponic system using the developed method. A hydroponic system was chosen to make bioavailable to plants all PFCs present in a nutrient solution, avoiding differences due to the varied adsorption of PFCs to soil.

21

The aerial part of plants was harvested and the fresh weight obtained after 1, 2, 6, 13 and 20 days growing in the hydroponic system. Five replicates for each PFC concentration and sampling time were used, including blank controls. Additionally, five vessels without plants per sampling time with the same spiked nutrient solutions (dose 0.5 and 1 μg/mL) were placed in the climate chamber to determine possible dissipation of PFCs with time. The harvested samples of the aerial parts of plants and spiked nutrient solutions were stored at −20 °C until analysis. 2.3. Extraction procedure For the extraction of PFCs from plant tissues, an MSPD method using Florisil in the homogenization step was employed. Florisil was washed several times with a mixture of acetonitrile/methanol (50:50 v/v) and air-dried for further use. Bromus diandrus, 0.5 g, was mixed with 1.5 g of the previously washed Florisil in a glass mortar using a glass pestle. The homogenized matrix was transferred to a polypropylene syringe barrel containing a cellulose frit at the bottom and finally other cellulose frit was placed on top applying a slight compression with a syringe plunger. Then, elution of analytes from the matrix was carried out with 7 mL of acetonitrile/methanol (50:50 v/v) and the eluent was collected into a polypropylene graduate tube by gravity flow applying vacuum at the end of elution. The total volume of eluate collected was taken to 5 mL. An extract aliquot (1 mL) was transferred to another polypropylene tube and 30 mg of graphitized carbon black was added, vortexed by 1 min and centrifuged at 4000 rpm during 10 min. The supernatant was filtered through a 0.22 μm polypropylene syringe filter into a polypropylene vial and analyzed by LC-MS/MS. Nutrient solutions and spiked nutrient solutions were filtered through a 0.22 μm polypropylene syringe filter before LC-MS/MS analysis. 2.4. LC-MS/MS determination

2. Materials and methods 2.1. Chemicals Perfluorobutanoic acid (PFBA), perfluorobutane sulfonic acid (PFBS), perfluorohexane sulfonic acid (PFHxS), perfluorooctanoic acid (PFOA), perfluorooctane sulfonic acid (PFOS) and perfluorodecanoic acid (PFDA) were obtained from Wellington Laboratories (Ontario, Canada). Methanol and acetonitrile (HPLC grade) were purchased from Sharlau Chemie S.A. (Barcelona, Spain). Deionized water was generated from a Q-pod water purifying system of Milipore. Florisil 60–100 mesh was obtained from Acròs Organics (New Jersey, USA), Silica gel 60 (35–70 mesh) was purchased from Merck (Darmstadt, Germany), sand (fat free quartz sand) and diatomaceous earth were obtained from Büchi (Flawil, Switzerland). Bondesil-PSA (primary–secondary amine), 40 μm particle diameter, graphitized carbon (ENVI-Carb 120– 140 mesh) and ammonium acetate were purchased from Sharlau Chemie S.A. (Barcelona, Spain), Supelco (Bellefonte, USA) and Panreac (Barcelona, Spain), respectively.

PFCs were determined by LC-MS/MS using an Agilent 1200 HPLC system coupled to an Agilent 6410 tandem triple quadrupole mass spectrometer, with electrospray ionization (ESI) interface operating in negative mode. Nitrogen was the gas used in the nebulizer and collision cell. The optimized instrument parameters were: drying gas flow 10 L/min at 300 °C, nebulizer pressure 45 psi and capillary voltage −4.0 kV. The two most sensitive MRM transitions were selected; one was used for quantitation and the other for confirmation of each target perfluorinated compound. Table 1 shows the optimized conditions for the studied PFCs by LC-MS/MS. Separation of analytes was carried out using a Kinetex-C18 (75 mm × 3 mm i.d, 2.6 μm particle size) core–shell analytical column with a C18 security guard cartridge. The mobile phase employed was 2 mM ammonium acetate (A) and methanol (B). Elution gradient started at 70% A, decreased to 10% in 5 min and to 0% in 1 additional Table 1 Optimized multiple reaction monitoring (MRM) parameters for the studied PFCsa.

2.2. Plant uptake study

Compound

Precursor ion (m/z)

Product ion (m/z)

Cone voltage (V)

Collision energy (eV)

Retention time (min)

Bromus diandrus Roth. seeds were pre-germinated for one week in Petri dishes containing two layers of sterile filter paper moistened with water. Next, five selected seedlings of uniform size were transferred to an experimental hydroponic system that consisted in a plastic grid placed into a PVC vessel, wrapped in black cardboard, containing 170 mL of Hewitt's nutrient solution or Hewitt's nutrient solution spiked with PFCs at 0.5 μg/mL or 1 μg/mL. The volume of nutrient solution was maintained at grid level during the entire assay by adding nutrient solution. The plants were grown in a growth chamber with a photoperiod of 16 h of light (250–300 μE m−2 s−1 PAR) at 22 ± 1 °C and 8 h of darkness at 16 ± 1 °C, and a relative humidity of 60%.

PFBA

213 299

PFHxS

399

PFOA

413

PFOS

499

PFDA

513

60 60 150 150 150 150 70 70 200 200 90 90

5 0 30 25 50 35 5 10 45 45 5 15

2.48

PFBS

169 213 80 99 80 99 369 169 80 99 469 269

a

Quantitation transitions are showed in bold.

6.18 7.88 7.99 8.22 8.47

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min, was maintained at 0% A for 1 min and then back to initial conditions in 1 min. A post-run time of 5 min was done before the next injection. Flow rate was set at 0.35 mL/min, column was kept at 25 °C and injection volume was 5 μL. Quantitation was performed using matrix-matched standards from 0 to 1250 ng/mL for plant samples and nutrient solutions to correct possible matrix effect and background in plants or nutrient solutions. 2.5. Method validation The method performance was investigated under the analytical conditions described above: 0.5 g of plant sample, 1.5 g of Florisil, MeOH/ACN (50/50 v/v; 5 mL) and DSPE with 30 mg/mL of graphitized carbon. Linearity was evaluated with a triplicate seven point calibration curve ranging from 0 to 1250 ng/mL for all compounds in a blank sample extract. Accuracy was determined by spiking plant samples at three different concentration levels of PFCs before extraction and the concentration was calculated with the corresponding matrix matched calibration standard. Method detection limits (MDL) for PFCs were determined by spiking a blank sample with PFCs to give a concentration of 0.5 ng/mL in nutrient solution or 0.5 ng/g in plant samples and were calculated as MDL = 3.143 × SD, where 3.143 corresponds to the Student's value for a 99% confidence level and six degrees of freedom and SD is the standard deviation of seven replicate analysis. Method quantitation limits (MQL) were established as 10 × SD. The precision of the method was calculated by determining the average coefficient of variation (CV) of the replicate analysis (n = 6) of a spiked extract, during the same day for repeatability and in different days for reproducibility. Two procedural reagent blanks were included in each batch of 5 samples, in order to determine background during sample preparation and monitor instrumental background. The average concentration obtained in the blanks was subtracted from the sample analyte concentration. In general, these background signals were close to the MQL. No detectable signal was observed for PFBS and PFHxS.

pretreatment. To improve detection limits, a clean-up using a DSPE with PSA or graphitized carbon was evaluated. Graphitized carbon removed a higher amount of pigments, producing cleaner extracts than PSA without a decrease in the recovery of analytes, and then it was chosen as the purification adsorbent. To the best of our knowledge, this is the first paper on the analysis of PFCs in plants using an MSPD method. This miniaturized procedure has a low sample and solvent consumption. In addition, homogenization and extraction are incorporated into the same step reducing manipulation of samples. This presents an advantage versus other methods used in plant sample preparation for the determination of PFCs, where there is a previous homogenization using freeze drying or grinding after drying, blending with a disperser or mixer, or grinding in a mortar using liquid nitrogen, and usually employing a higher amount of sample (Ballesteros-Gómez et al., 2010; Haug et al., 2010; Lechner and Knapp, 2011; Yoo et al., 2011; Domingo et al., 2012; Felizeter et al., 2012; Stahl et al., 2013). Satisfactory mean recoveries of the six analytes in plant at the different concentration levels, 10, 100 and 500 ng/mL, were obtained by the proposed method, with values ranging from 70.1% to 104.3% (Table 2). These recoveries are in the range of those obtained by other authors in vegetables employing different PFC extraction methods (BallesterosGómez et al., 2010; Lechner and Knapp, 2011; Felizeter et al., 2012; Stahl et al., 2013). The recovery of PFCs from nutrient solutions was not studied because they were analyzed by direct sample injection. The precision of the method expressed as reproducibility (interday) was below 8.2% and 6.1% for plant samples and nutrient solutions, respectively, and the repeatability (intraday) was below 5.5% and 3.7%, for plants and nutrient solutions, respectively, (Tables 2 and 3). For both matrices, nutrient solution and plant samples, the correlation coefficient values of matrix-matched calibration curves were higher than 0.99 for all PFCs analyzed. The MDL obtained varied between 0.01 and 0.19 ng/mL for nutrient solution and between 0.5 and 3 ng/g for B. diandrus fresh samples, with MQL between 0.04 and 0.61 ng/mL and between 1 and 9 ng/g fresh weight for nutrient solution and B. diandrus, respectively (Tables 2 and 3). 3.2. Dissipation in nutrient solution

2.6. Statistical analysis Analysis of variance (ANOVA) with the least-significant-difference procedure (LSD) for a multiple statistical comparison of groups of means and p = 0.05 as the level of significance was employed in the statistical analyses to evaluate the statistical significance of differences between samples subjected to the same experimental conditions. The statistical package used for the ANOVA analysis was Statgraphics Centurion XVI (Manugistics, Rockville, MD, USA). 3. Results and discussion 3.1. Determination of PFCs The plant extraction method used in this work is based on matrix solid-phase dispersion (MSPD), where a disruption of the structure of the vegetal is achieved and its homogeneous distribution around the sorbent particles may allow a clean-up of the extract in the same process. In the MSPD procedure, the selection of the dispersant and the elution solvent are the key factors for the efficacy of the extraction. In this study, different sorbents, such as Silica, Florisil, sand and diatomaceous earth, were tested as dispersants. Interferences were observed in the extracts coming from all dispersants, but a washing of Florisil sorbent with acetonitrile/methanol prior to use gave the best results. The elution solvent selected was acetonitrile/methanol (50:50 v/v), which ensured efficient extraction of the long and short chain PFCs used in this study, as it was reported in a previous work (García-Valcarcel and Tadeo, 2013). A pretreatment of matrix with acetic acid or sodium hydroxide was also assayed, but the extraction efficacy was similar to that obtained without

To determine the real amount of PFCs removed by plant from the spiked nutrient solution during the exposure time, a possible dissipation of target compounds in nutrient solutions must be taken into account. Then, a dissipation study was carried out in parallel to that of the uptake of PFCs by plant, in vessels with nutrient solution but without plant under the same conditions. The decline in concentration of PFCs in nutrient solution during this period (20 days) was small, between 7 and 13% of its initial value, with a significant decrease of concentration with time for PFBA, PFBS, PFHxS and PFOA (Table 4). In general, a higher rate of dissipation was found for the lowest concentration level of PFCs in nutrient solution (0.5 μg/mL versus 1 μg/mL). Nevertheless, for PFDA and PFOS no significant differences (P value N0.05, for LSD 95%) were obtained in the concentration values along the assay. This may be explained by their higher molecular weights

Table 2 Recoveries (mean ± SD), precisiona and method detection and quantitation limits of PFCs in Bromus diandrus. Recovery (%) ± SD

PFBA PFBS PFHxS PFOA PFOS PFDA a

Precision (%RSD)

10 ng/g

100 ng/g

500 ng/g

Interday Intraday

79.8 ± 2.0 104.3 ± 1.5 96.9 ± 0.4 92.1 ± 2.7 95.3 ± 5.1 71.8 ± 2.2

89.6 93.3 94.9 70.4 96.4 76.5

72.3 95.4 98.2 70.1 99.5 80.4

7.9 6.8 5.7 8.2 6.5 6.4

± ± ± ± ± ±

1.9 2.2 3.4 4.6 2.3 1.9

± ± ± ± ± ±

2.9 3.1 3.6 2.2 3.0 2.7

Determined with a matrix-matched standard of 5 ng/g.

5.5 4.1 3.1 5.4 3.1 4.3

MDL MQL (ng/g) (ng/g) 1 3 1 0.5 2 0.7

3 9 1 2 6 2

A.I. García-Valcárcel et al. / Science of the Total Environment 472 (2014) 20–26 Table 3 Precisiona and limits of detection (MDL) and quantitation (MQL) in nutrient solution. Precision (%RSD)

PFBA PFBS PFHxS PFOA PFOS PFDA a

Interday

Intraday

4.6 3.1 6.1 3.3 3.1 3.7

1.7 1.2 3.7 1.1 1.2 1.7

MDL (ng/mL)

MQL (ng/mL)

0.02 0.01 0.04 0.07 0.14 0.19

0.06 0.04 0.13 0.20 0.46 0.61

Determined with a matrix matched standard of 2 ng/mL.

and particular physical–chemical properties as well as their higher variation coefficients between replicate samples. Determination of the dissipation rate constants and half-lives for each perfluorinated compound in nutrient solution was carried out using the equation: −kt

C ¼ C0 e

Where C0 is the initial concentration of the perfluorinated compound (ng/mL), C is the concentration at time t (days) in ng/mL and k is the dissipation rate constant. Half-lives of PFCs were calculated using the k value, obtained by plotting the ln of the concentration against time, as follows: t1=2 ¼ ðln2Þ=k Except PFOS and PFDA, that did not fit well to this dissipation equation, the rest of PFCs showed low dissipation rate constants, between 0.004 and 0.007, with half-lives ranging between 99 and 173 days, depending on the initial concentration in the nutrient solution. This confirms the persistence of perfluorinated compounds, as indicated by other authors (Sáez et al., 2008; Liou et al., 2010). These dissipation rates were not taken into account in the determination of the uptake by plants, because the low dissipation observed can be considered negligible versus the amount found in plants during the assay time (about 20 days). 3.3. Vegetation study 3.3.1. PFC concentration in plant At the end of assay, differences in the fresh weight of plants grown in untreated and treated nutrient solutions were observed, being more pronounced for the high PFC dose. This is in accordance with Stahl et al. (2009), who observed a direct proportional effect of a decrease in growth with concentration for several plant species. Nevertheless, Lechner and Knapp (2011) only found this effect for cucumbers in a study with carrots, potatoes and cucumbers cultivated in soil contaminated with PFCs. Felizeter et al. (2012), found a non-remarkable effect

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in the growth of lettuce when submitted to PFC uptake from spiked nutrient solutions. Probably, this may be explained by the contamination levels to which the plants were subjected, from 0.5 to 10 μg/kg in Lechner and Knapp (2011), or from 0.01 to 10 μg/L in Felizeter et al. (2012), versus 10,000–50,000 μg/kg used by Stahl et al. (2009), in different crops or 500–1000 μg/L in the present work. Levels of PFCs in the aerial parts of plants were determined along the assay, for plants grown in nutrient solution containing PFCs at 500 or 1000 ng/mL. Fig. 1 represents the amount of the different PFCs found in plants, expressed as ng/g fresh weight as a function of exposure time. In this figure, the following trend can be observed: 1) Independently of compounds, the concentration of PFCs in plant increased, in general, with the exposure time and higher levels were observed for plants growing in the nutrient solution with the highest PFC dose, in accordance with Lechner and Knapp (2011). 2) For the same time of sampling, a decrease in concentration in plant with chain length was observed for perfluorioalkyl carboxylates (Fig. 1 A1–B1) whereas for perfluoroalkyl sulfonate acids the concentration in plant was, in general, similar irrespective of their molecule size (Fig. 1 A2–B2). 3) Higher amounts of PFBA (perfluorinated carboxylic acid) than PFBS (perfluorinated sulfonic acid), with the same carbon chain length (C4), were found in plants; nevertheless, similar concentrations of PFOA and PFOS, also with the same carbon chain length (C8), were obtained. This can be explained by the higher hydrophobicity of perfluorinated sulfonates versus perfluorinated carboxylates, when molecules with the same carbon number are compared, due to the higher molecular size of perfluorinated sulfonates. However, as the chain length increases a higher impediment for the plant uptake occurs, being this fact even more important than the hydrophobicity differences between carboxylates and sulfonates compounds, which can explain the similar uptake of PFOA and PFOS. 4) At both tested doses, the highest uptake of all PFCs assayed was for PFBA and the lowest for PFDA, being both perfluoroalkyl carboxylates compounds with the shortest and longest chain, respectively.

3.3.2. Plant uptake rates Uptake rates of PFCs from nutrient solutions to plants were calculated as the total absorbed amount per plant along the assay. Uptake rates are dependent on the perfluorinated compound type (Fig. 2). Low and similar amounts of PFCs are taken by plant during the first 6 days of the assay; after this time, an increase in uptake is observed for all the compounds with the highest rate for PFBA and the lowest for PFDA. At the end of assay, 31 μg of PFBA, 14 μg of PFBS and 12 μg of PFDA, were absorbed by plants, whereas it was about 18 μg for PFHxS, PFOA and PFOS. These values mean percentages of PFCs from the initial content in the nutrient solution with 0.5 μg/mL that range from 35.5% for PFBA to 14.5% for PFDA. At the dose of 1 μg/mL, a lower percentage of PFCs was absorbed by plants, varying between 17% for PFBA and 7.5% for PFDA. This could be explained by a reduced growth of plants subjected to a higher dose of PFCs.

Table 4 Dissipation in nutrient solution along the assay (ng/ml). PFCs

Dose

T1

T2

T3

T4

T5

T6

P value

PFBA

0.5 μg/mL 1 μg/mL 0.5 μg/mL 1 μg/mL 0.5 μg/mL 1 μg/mL 0.5 μg/mL 1 μg/mL 0.5 μg/mL 1 μg/mL 0.5 μg/mL 1 μg/mL

406.7 a 776.9 a 505.5 a 975.4 a 611.85 a 963.7 a 458.8 a 957.3 a 519.7 a 982.3 a 382.8 a 918.4 a

392.3 b 727.1 b 500.9 a 974.7 a 610.14 a 944.4 a 460.36 a 911.24 b 406.5 a 1012.7 a 283.0 a 903.4 a

383.7 c 708.4 c 492.3 b 966.1 a 592.2b 905.4 bc 459.9 a 884.8 bc 466.2 a 982.8 a 381.5 a 967.6 a

372.2 d 708.5 c 494.1 b 933.3 b 583.2 c 912.4 b 455.84 a 863.4 cd 586.5 998.8 a 428.7 a 906.7 a

361.3 e 687.6 d 459.4 c 908.5 c 561.7 d 893.2 bc 436.38 b 859.4 cd 501.6 a 969.4 a 421.9 a 915.8 a

354.8 f 692.6 d 440.9 d 885.0 d 539.8 e 888.3 c 426.04 b 857.0 d 519.7 a 1054.5 a 444.3 a 1005.0 a

0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.0000 0.1662 0.9967 0.0773 0.7323

PFBS PFHxS PFOA PFOS PFDA

Different letters in a row show a significant difference at 95% confidence level (P values lower than 0.05).

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A.I. García-Valcárcel et al. / Science of the Total Environment 472 (2014) 20–26

PFBA

4000

PFOA

PFBS

PFDA

A1

4000

ng/g

ng/g

2000

A2

2000 1000

1000

0

0 1

3

6

PFBA

13

PFOA

1

20

PFDA

3

PFBS 6000

B1

5000

5000

4000

4000

ng/g

ng/g

PFOS

3000

3000

6000

PFHxS

3000

13

PFHxS

20

PFOS

B2

3000

2000

2000

1000

1000

0

6

0 1

3

6

13

20

1

3

6

13

20

Fig. 1. Amount of perfluorinated compounds (ng/g) found in the aerial part of plants along the assay (days) at an initial dose in nutrient solution of A: 0.5 μg/mL and B: 1 μg/mL.

3.3.3. Transfer factors To evaluate the transport of each perfluorinated compound from nutrient solutions to the aerial part of plants, transfer factors (TF) were calculated. TF was calculated as the ratio of the concentration at harvest in aerial tissues (ng/g fresh plant) to that in the nutrient solution (ng/mL) (Table 5). For calculation, five independent plant samples were taken. 35.0

PFBA PFOA

30.0

PFDA PFBS

25.0

PFHxS

µg/plant

PFOS 20.0

15.0

10.0

5.0

0.0 0

5

10

15

20

25

time (days) Fig. 2. Uptake of PFCs (μg/plant) by B. diandrus with time from a nutrient solution containing 0.5 μg/mL.

An increase of the transfer factor with exposure time was observed for both concentration levels in nutrient solution, but when plant was exposed at 1000 ng PFCs/mL, the increase in the transfer factor from day 13 to 20 was not observed. In addition, though initially higher transfer factors were obtained for plant growing in the solution with the highest PFC concentration, the transfer factors become similar with time for plant growing in both PFC solutions, except for PFBA which had a higher transfer factor at low concentration. Lechner and Knapp (2011) obtained also similar TF values for PFOS in vegetables grown in soil with low and high amounts of PFOS. One possible explanation is that, although the PFC bioavailability from both concentrations in nutrient solution is similar, the transport to the plant at higher concentrations gives higher concentrations in plant but also had an effect on the plant growth. In addition, it could be that the PFC accumulation in plant reaches its maximum in 13 days for plants grown in a dose of 1 μg/mL in nutrient solution whereas 20 days is needed at a dose of 0.5 μg/mL. This is in accordance with Zhao et al. (2013), which found that PFOS uptake by wheat seedlings increased with exposure time until reaching an apparent uptake plateau. This was explained by some processes such as foliar volatilization, solute metabolism in plants, formation of bound residues and plant-growth induced dilution that can reduce the levels of PFCs in plants. These processes could, in our case, explain the apparent decrease in PFC levels from day 13 to day 20 of plants subjected to a nutrient solution with a dose of 1000 ng PFCs per mL. At the end of exposure time (20 days), statistical differences were obtained at both doses, for perfluorinated carboxylic acids, with the highest transfer factor for PFBA followed by PFOA and then by PFDA, whereas no statistical differences in TF values were obtained for the perfluorinated sulfonic acids (PFBA, PFHxS and PFOS). This is in accordance with Yoo et al. (2011), who reported that the shortest perfluorinated carboxylates had the highest transfer factor decreasing over the homologue range. However, Felizeter et al. (2012) found a decrease with increasing chain length from PFBA to PFOA and then an increase from PFOA to PFDA, they also found TF values higher for PFOS than for PFBS and PFHxS in a hydroponical assay with lettuce.

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Table 5 Transfer factors determined for the different dose application. Dose

Harvest day

PFBA

PFBS

PFHxS

PFOA

PFOS

PFDA

F value

P value

0.5 μg/mL

1 3 6 13 20 1 3 6 13 20

0.258 c 2.053 c 2.91 c 4.249 d 5.65 c 0.523 c 1.824 d 1.826 b 4.150 b 4.29 c

0.145 b 0.977 b 1.340 ab 2.596 bc 3.332 ab 0.501 c 1.783 d 1.269 a 3.283 b 3.071 ab

0.102 b 0.913 b 1.235 a 2.918 c 3.733 b 0.518 c 1.707 cd 1.452 ab 3.711 b 3.495 b

0.030 a 0.848 ab 1.298 a 2.837 bc 3.634 ab 0.292 ab 1.078 ab 1.708 b 3.354 b 3.416 b

0.092 b 0.998 b 1.698 b 2.521 b 3.425 ab 0.353 bc 1.216 bc 1.907 c 3.332 b 3.098 ab

0a 0.502 a 1.064 a 1.513 a 2.33 a 0.145 a 0.595 a 1.419 ab 2.182 a 2.036 a

33.27 22.3 32.05 45.30 8.69 5.04 6.66 17.23 4.63 5.79

0.0000 0.0000 0.0000 0.0000 0.0003 0.0051 0.0008 0.0000 0.0068 0.0023

1 μg/mL

Different letters in a row show significant difference at 95% confidence level (P values lower than 0.05).

Nevertheless, these authors also found a considerable higher transfer factor for PFBA in comparison with the others PFCs studied, which is in accordance with our results. In the present study, the mean transfer factors during 20 days of plant exposure at the two doses employed, on fresh weight basis, were 4.97 for PFBA, 3.20 for PFBS, 3.72 for PFHxS, 3.52 for PFOA, 3.26 for PFOS and 2.18 for PFDA, which are higher than those found in the literature, probably due to the higher dose of PFCs used in nutrient solutions versus the levels assayed in soil available in the literature. Nevertheless, most studies on uptake of PFCs by plant are focused solely in PFOA and PFOS and only a comparison of our results with these two compounds can be made. The TF mean values calculated from the data given by Stahl et al. (2009) for perennial wheatgrass varied between 0.274 and 4.45 for PFOA and from 0.127 to 1.43 for PFOS, depending on the previous number of cutting in plants when harvested. TF obtained by Felizeter et al. (2012) were 1.2 for PFOA and 1.1 for PFOS in lettuce grown in a hydroponically system; TF reported by Yoo et al. (2011) in grass samples grown in soil amendment with contaminated sludge were 0.25 for PFOA and 0.07 for PFOS and those reported by Lechner and Knapp (2011) in vegetative compartments of carrot, cucumbers and potatoes grown in soil varied from 0.38 to 0.99 for PFOA and from 0.12 to 0.45 for PFOS, depending on vegetable culture. Regarding these data, it can be seen that plants grown in PFC contaminated soil absorb lower amounts of PFOS than PFOA, whereas those grown in contaminant nutrient solution uptake similar amount of PFOS and PFOA. This can be due to the higher soil adsorption of PFOS in comparison with PFOA (Ahrens, 2011; Zareitalabad et al., 2013) and, therefore, to the lower bioavailability of PFOS in plants growing in soils contaminated with the same amount of PFOS and PFOA. In this work, a miniaturized method with a low sample and solvent consumption has been developed to determine levels of PFCs in plants. The results obtained show the ability of plants to uptake PFCs from solution, being the absorption of perfluorinated carboxylates dependent of the chain length, whereas the perfluorinated sulfates, with the same number of carbon in the molecule, have higher molecular weight and hydrophobicity than perfluorinated carboxylates, which may make the uptake by plant independent of the carbon chain length. Although these PFCs are absorbed by plants and can enter the food chain, the levels found in plants cultivated hydroponically are magnified in comparison to the expected levels in those cultivated in soil under field conditions, because soil can adsorb these compounds at variable amounts depending on its physico-chemical properties and the environmental conditions. Nevertheless, for vegetables hydroponically cultivated, which are commercially available, an important uptake may occur when these compounds are present in the hydroponic medium and, therefore, monitoring PFC levels in this case would be recommended. Conflict of interest We wish to confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome.

We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In doing so we confirm that we have followed the regulations of our institutions concerning intellectual property. We understand that the Corresponding Author is the sole contact for the Editorial process (including Editorial Manager and direct communications with the office). She is responsible for communicating with the other authors about progress, submissions of revisions and final approval of proofs. We confirm that we have provided a current, correct email address which is accessible by the Corresponding Author and which has been configured to accept email from [email protected]. Acknowledgment The authors gratefully acknowledge the financial support provided by the Spanish Ministry of Science and Innovation (RTA 2011-0047-00-00). References Ahrens L. Polyfluoroalkyl compounds in the aquatic environment: a review of their occurrence and fate. J Environ Monit 2011;13:20–31. Ballesteros-Gómez A, Rubio S, Van Leeuwen S. Tetrahydrofuran-water extraction, in line clean-up and selective liquid chromatography/tandem mass spectrometry for the quantitation of perfluorinated compounds in food at the low pictogram per gram level. J Chromatogr A 2010;1217:5913–21. Domingo JL, Jogsten IE, Eriksson U, Martorell I, Perelló G, Nadal M, et al. Human dietary exposure to perfluoroalkyl substances in Catalonia. Spain Food Chem 2012;135: 1575–82. Felizeter S, McLachlan MS, de Voogt P. Uptake of perfluorinated alkyl acids by hydroponically grown lettuce (Lactuca sativa). Environ Sci Technol 2012;46:11735–43. García-Valcarcel AI, Tadeo JL. Fast ultrasonic assisted extraction combined with LC–MS/MS of perfluorinated compounds in manure. J Sep Sci 2013;36:2507–13. Hanssen L, Dudarev AA, Huber S, Odland J- Ø, Nieboer E, Sandanger TM. Partition of perfluoroalkyl substances (PFASs) in whole blood and plasma, assessed in maternal and umbilical cord samples from inhabitants of arctic Russia and Uzbekistan. Sci Total Environ 2013;447:430–7. Haug LS, Salihovic S, Jogsten IE, Thomsen C, Van Bavel B, Lindström G, et al. Levels in food and beverages and daily intake of perfluorinated compounds in Norway. Chemosphere 2010;80:1137–43. Jahnke A, Berger U. Trace analysis of per- and polyfluorinated alkyl substances in various matrices-How do current methods perform? J Chromatogr A 2009;1216:410–21. Lau C, Anitole K, Hodes C, Lai D, Pfahles-Hutchens A, Seed J. Perfluoroalkyl acids: a review of monitoring and toxicological finding. Toxicol Sci 2007;99:366–94. Lechner M, Knapp H. Carryover of perfluooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) from soil to plant and distribution to the different plant compartments studied in cultures of carts (Daucus carota ssp. Sativus), potatoes (Solanum tuberosum), and cucumbers (Cucumis Sativus). J Agric Food Chem 2011;59:11011–8. Liou SC, Szostek B, DeRito CM, Madsen EL. Investigating the biodegradability of perfluorooctanoic acid. Chemosphere 2010;80:176–83. Paterson S, Mackay D, Mcfarlane C. A model of organic-chemical uptake by plants from soil and the atmosphere. Environ Sci Technol 1994;28:2259–66. Picó Y, Farré M, Llorca M, Barceló D. Perfluorinated compounds in food: a global perspective. Crit Rev Food Sci Nutr 2011;51:605–25.

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