Interaction effects on uptake and toxicity of perfluoroalkyl substances and cadmium in wheat (Triticum aestivum L.) and rapeseed (Brassica campestris L.) from co-contaminated soil

Ecotoxicology and Environmental Safety 137 (2017) 194–201

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Interaction effects on uptake and toxicity of perfluoroalkyl substances and cadmium in wheat (Triticum aestivum L.) and rapeseed (Brassica campestris L.) from co-contaminated soil

MARK

⁎

Shuyan Zhao , Ziyan Fan, Lihui Sun, Tao Zhou, Yuliang Xing, Lifen Liu Key Laboratory of Industrial Ecology and Environmental Engineering, School of Food and Environment, Dalian University of Technology, Panjin, Liaoning 124221, PR China

A R T I C L E I N F O

A B S T R A C T

Keywords: PFASs Cd Phytotoxicity Bioaccumulation Wheat Rapeseed

A vegetation study was conducted to investigate the interactive effects of perfluoroalkyl substances (PFASs), including perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS), and Cadmium (Cd) on soil enzyme activities, phytotoxicity and bioaccumulation of wheat (Triticum aestivum L.) and rapeseed (Brassica campestris L.) from co-contaminated soil. Soil urease activities were inhibited significantly but catalase activities were promoted significantly by interaction of PFASs and Cd which had few effects on sucrase activities. Joint stress with PFASs and Cd decreased the biomass of plants and chlorophyll (Chl) content in both wheat and rapeseed, and malondialdehyde (MDA) content, superoxide dismutase (SOD) and peroxidase (POD) activities were increased in wheat but inhibited in rapeseed compared with single treatments. The bioconcentration abilities of PFASs in wheat and rapeseed were decreased, and the translocation factor of PFASs was decreased in wheat but increased in rapeseed with Cd addition. The bioaccumulation and translocation abilities of Cd were increased significantly in both wheat and rapeseed with PFASs addition. These findings suggested important evidence that the co-existence of PFASs and Cd reduced the bioavailability of PFASs while enhanced the bioavailability of Cd in soil, which increased the associated environmental risk for Cd but decreased for PFASs.

1. Introduction Persistent organic pollutants (POPs) (Shrestha et al., 2009) and heavy metal (Wei and Yang, 2010) contaminations are of concern on a worldwide scale as two predominant contaminant families in soil. Perfluoroalkyl substances (PFASs) are widely used in various commercial products due to their hydrophobic and lipophobic properties in the past six decades (e.g., refrigerating fluid, film-forming foams, textiles, lubricant, cleaners, cosmetics and food packaging). During the production, application and disposal of PFASs, they are released into the environment and ubiquitous in environmental matrices such as water, sediment and soil (Paul et al., 2009). Otherwise, PFASs can be bioaccumulated and biomagnified throughout the food chain (Loi et al., 2011) and have been found to be toxicity to wildlife species (Lau et al., 2007) and humans (D'eon and Mabury, 2011). PFOS and PFOA are typical PFASs and have been paid more attention by people because of their high detection rate in the environment and biota (Zareitalabad et al., 2013). PFOS was added in the list of persistent organic pollutants (POPs) regulated by the Stockholm Convention. Although 3M company volunteered to stop the production of PFOS, its ⁎

production and usage have been continued in some countries including China (UNIDO, 2012). In recent years, the use of sewage sludge as fertilizer in agriculture causes contamination of soil with PFASs. It was reported that the concentrations of PFASs in sludge-applied soils near Decatur, Alabama, USA were in the range of 16–986 ng g−1 dry weight, PFOA (≤320 ng g−1) and PFOS (≤410 ng g−1) both at a high level (Washington et al., 2010). Sepulvado et al. (2011) found that concentration of PFOS ranged from 2 to 483 ng g−1 in biosolids-amended soils in Chicago, USA. PFASs were also detected in the soil of China, and PFOS (≤1.88 ng g−1) and PFOA (≤2.32 ng g−1) contributed most to the total PFASs concentration (Meng et al., 2015). Many previous studies have demonstrated that PFASs in soil could be taken up by plant roots and then translocated to above-ground compartments (Blaine et al., 2013, 2014a, 2014b; Lechner and Knapp, 2011; Müller et al., 2016; Stahl et al., 2009; Zhao et al., 2014). Heavy metal pollution of soil has become an important global environmental issue (Liu et al., 2009). The behaviors of heavy metals in contaminated soil have been extensively studied over many decades. The principal and significance potential for heavy metals is to enter the soil solution phase where their mobility and bioavailability increases

Corresponding author. E-mail address: [email protected] (S. Zhao).

http://dx.doi.org/10.1016/j.ecoenv.2016.12.007 Received 29 July 2016; Received in revised form 14 November 2016; Accepted 5 December 2016 0147-6513/ © 2016 Elsevier Inc. All rights reserved.

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collected from a farm of Panjin, China. The selected physicochemical properties of the soil were characterized as follows: pH 7.51, total organic carbon 1.81%, total nitrogen 0.34%; total phosphorus 0.03%; moisture content 1.04% for air-dried soil; Cd concentration was 0.063 mg kg−1. The soil was air-dried for 14 d and sifted through a 2-mm mesh and then received mineral nutrients as basal fertilizers at a rate of 100 mg P (KH2PO4), 300 mg N (NH4NO3) and 200 mg K (K2SO4) kg−1 soil. The pre-weighed soil was divided into four equal parts and amended to contain 20 mg Cd kg−1 by adding appropriate concentrations of CdCl2. To ensure even distribution of Cd, soils were thoroughly mixed while adding CdCl2 and water. The soils were incubated at room temperature (at about 20–25 °C) for 30 d allowing Cd to distribute into various fractions. During the period, soil moisture content was carefully monitored to keep 60–70% of field holding capacity (Yu et al., 2005). A small portion of pre-weighed soil was spiked with 1 mL of methanol solution containing PFOA and PFOS and mixed thoroughly and then placed in a fume hood to allow the solvent to evaporate for 24 h. The untreated soil was added to the spiked soil continuously and then mixed thoroughly until all the pre-weighed soil was mixed. The soil was spiked with PFASs compound at an original theoretical level of 300 ng g−1. They were shaken for 5 times (30 min/each time) each day for 6 d continuously, and incubated in the dark for 14 d at room temperature. The soils which were co-spiked with PFASs and Cd were firstly spiked with Cd and then PFASs as above. The concentrations of the target PFASs and Cd spiked in the soil were conducted by considering the background values of pollution levels in nature soils and other studies conducted in the laboratory. And their concentrations in the spiked soil were measured at the end of incubation but before transplanting seedlings (Table S1). The concentrations of PFOA were 285 ng g−1 (Group I) and 292 ng g−1 (Group III), PFOS were 264 ng g−1 (Group I) and 294 ng g−1(Group III), Cd were 14.7 (Group II) and 14.2 mg kg−1 (Group III). No PFAS was detected in non-spiked soil (Control).

their associated environmental risk (Ashworth and Alloway, 2007). Cadmium (Cd) is a heavy metal which is particular concern due to its high toxicity and carcinogenic to organism and humans, and it is the most widespread pollution heavy metal in agricultural soils (Wei and Yang, 2010) by the application of metal-containing sewage sludge and fertiliser, disposal of municipal and industrial wastes (Loska et al., 2004) and sewage irrigation (Liu et al., 2005). The concentrations of Cd in urban soils, urban road dusts and agricultural soils were in the range of 0.11–8.59 mg kg−1 in China (Wei and Yang, 2010) and has been reported as a major constraint on agricultural land quality in China and food safety. For a relatively high mobile metal in soil, Cd is readily taken up by plants through membrane transporters (Zheng et al., 2011). The co-occurrence of PFASs with Cd are found in sediments in China (Zheng et al., 2015). Soil contaminated with PFASs and Cd was discharged from the same sources, including sludge-applied and sewage irrigation in agriculture soil and might lead to co-existence of PFASs and Cd in soil environment. The presence of co-contaminated multiple contaminants will influence the environmental behaviors because of their interactive effects on soil processes, plant growth, and rhizosphere biota (Almeida et al., 2008). Previous studies have found the combined effect of pollution on soil enzymes between organic matters and heavy metals (Liu et al., 2008; Shen et al., 2006). The combined presence of different pollutants might influence the micro-ecological processes including physio-chemical and biological changes of soil and plant growth and microorganisms in the rhizosphere, and then affect the plant physiological and biochemical properties and the bioaccumulation of contaminants in plants (Lu et al., 2014; Wen et al., 2011; Zhang et al., 2011, 2012). PFASs are anion organic pollutant, which may interact with cationic metal potentially, such as Cd, leading to the changes of their speciation, interfacial behavior, bioaccumulation and toxicity of both PFASs and metals. However, little information has been conducted to investigate the effects of their combined contamination on the terrestrial environment. Only one recent work has studied toxicological interactions of PFOS and PFOA with heavy metals (Hg2+, Cd2+) in bioluminescent cyanobacteria and they observed the antagonistic interaction, decreasing metal toxicity (Palomares et al., 2012). The transfer of PFASs and Cd from soil to plants implies a serious risk not only to the plants but also biota and humans feeding on them. Hence, it is necessary to assess the combined effects of PFASs and Cd on terrestrial environment, including in the soil media and in plants. This study was conducted by a greenhouse pot experiment to investigate the effects of PFASs and Cd combined contamination on soil enzyme activities, plant biomass and biochemical responses, as well as evaluate the mutual impacts of the co-contaminants on uptake and translocation of PFASs and Cd in plants. The results obtained from this study are expected to supply important information about the behavior of co-contamination of PFASs and Cd in terrestrial ecological system.

2.3. Experimental design

2. Materials and methods

The experiment was conducted in plastic pots (volume was 2 L) and the plastic pots were packed with 2 kg of contaminated soil. Four groups of tests were conducted: PFASs was individually occurred in two series of pots cultured with wheat (Triticum aestivum L.) (Group IA) and rapeseed (Brassica campestris L.) (Group IB); Cd was individually spiked in pots cultured with wheat (Group IIA) and rapeseed (Group IIB); and they were co-spiked in another group of pots cultured with wheat (Group IIIA) and rapeseed (Group IIIB); soils alone, which were spiked with Cd (Group SA), PFASs (Group SB) and Cd-PFASs (Group SC) separately but without plant. Each group test was conducted with triplicates in three parallel pots and the four test groups were accompanied with control experiments using non-spiked soil, in which the soil was clean without spiking PFASs and Cd (Control).

2.1. Chemicals

2.4. Plants

The standard of perfluorooctane sulfonate (PFOS, 98%) was purchased from Shanghai Aladdin Reagent Co., Ltd. (China). Perfluorooctanoic acid (PFOA, 98%) was purchased from J & K Chemical Ltd. (Shanghai, China). Cd2+(as CdCl2, 97.5%) was purchased from Sigma-Aldrich (Shanghai, China). Methanol of highperformance liquid chromatography (HPLC) grade was obtained from Dikma Technology Inc., USA. Dichloromethane (DCM), methanol for extraction and other chemicals were bought from Dalian Bono Biochemical Reagent Ltd. (Dalian, China). All solvents, including methanol and water were HPLC grade.

Wheat (Triticum aestivum L.) and rapeseed seeds (Brassica campestris L.) were obtained from the Chinese Academy of Agricultural Sciences, Beijing, China. They were firstly surface sterilized in 3% H2O2 solution, washed with distilled water, soaked in 2.8 mmol L−1 Ca(NO3)2 solution for 4 h in darkness and germinated in a cultivation dish on moist filter paper at 22–27 °C. After 3 d of germination, uniform seedlings were sown in plastic pots (10 plants pot−1). The seedlings were grown under plant growth chamber conditions for 14 h at 25 °C (day) and for 10 h at 20 °C (night). Plants were irrigated daily with distilled water to maintain soil moisture at approximately 60–70% of water holding capacity by weight. The pots were positioned randomly and rerandomized every two days during the exposure time. After cultivation for 70 d, roots and shoots were separately harvested. They were washed thoroughly with distilled water, one part was freeze-dried for 48 h in a

2.2. Soil properties and treatment A surface agricultural soil (0–10 cm) without detectable PFASs was 195

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-1 -1 -1 -1 Surase activity(mg g-1d-1) Urease activity(mg g h ) Catalase activity(mL g h )

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lyophilizer and weighed again and the other part was used to evaluate biological response. The dried root and shoot samples were homogenized and stored in polypropylene (PP) tubes at −20 °C before chemical analysis. 2.5. Chemical extraction and analysis Urease, catalase and sucrase activities in soil were assayed following the procedure by Zhang and Wang (2006). Catalase activities were back-titrated by KMnO4, and were expressed in mL H2O2 decomposed by 1 g soil. The urease activities were expressed in mg NH3-N generated by 1 g soil at 37 °C h−l. Sucrase activities were expressed in mg glucose generated by 1 g soil at 37 °C d−1. The Chl concentration and MDA content in plant tissues were measured according to the method of Hegedüs et al. (2001). The assay of antioxidative enzymes including SOD and POD referred to the method described by Wu and Tiedemann (2002). Extractions of PFASs in plant samples followed the steps in our previous study with minor modifications (Zhao et al., 2014), and the soil samples were subjected to modification on the basis of the original method provided by Zhang et al. (2010). The details are described in the Supporting Information (SI). LC/MS analysis and quantitation are available in the SI and Table S2. Prior to analysis of Cd concentrations in soils and plants, samples were digested with a solution of 3:1 HNO3 and HClO4 (v:v) (Sahan et al., 2007). The free Cd2+ concentrations in soil solution spiked with Cd (Group SA) and Cd-PFASs (Group SC) were sampled according to Van Gestel et al. (2012), and determined by the electrochemical comprehensive tester (Princeton Parstat4000), and the details are available in the SI. The modified BCR-sequential extraction technique was applied in triplicate to obtain the fraction which was used to assess bioavailability of metal ions in the soil samples of Group SA and SC, and the method was described in detail elsewhere (Rauret et al., 1999). An inductively coupled plasma mass spectrometer (ICP-MS, Agilent 7500) was used to determine the concentrations of Cd. The MDLs of Cd were 0.25 µg kg−1 for soil and 0.23–0.38 µg kg−1 for plants. Further details about the extraction, determination and quality control are supplied in SI. 2.6. Statistical analysis Statistically significant differences between the results were evaluated on the basis of paired-samples T test and analysis of variance method (ANOVA). When the p value was < 0.05, the hypothesis was accepted as statistically significant (IBM SPSS Statistics 22).

0.5

catalase

*

0.4 0.3 0.2 0.1 0.0 6

Urease

4

*

*

2

0 7

* Surase

6 5 4 3 2 1 0

Control

PFASs

Cd

PFASs+Cd

Fig. 1. Catalase, urease and sucrase activities in soils in single and joint treatments of PFASs and Cd. Statistically significant differences from control are marked with asterisks (*p < 0.05).

2.7. Data analysis

3. Results and discussion

The root concentration factor (RCF), shoot concentration factor (SCF), bioconcentration factor (BCF) and the translocation factor (TF) from root to shoot of PFASs or Cd in plants were determined as follows:

3.1. Interactions between PFASs and Cd on soil enzyme activities

RCF =

Croot Cs

(1)

SCF =

Cshoot Cs

(2)

BCF =

TF =

Soil enzyme activities have been proposed as indicators for measuring the degree of soil pollution. A laboratory incubation experiment was carried out to analyze the changes in soil urease, catalase and sucrase activities of group S (A, B and C) under single and co-contamination of PFASs and Cd (Fig. 1). As shown in Fig. 1, the change rate of soil enzyme activities suggested a sensitivity order of catalase > urease > sucrase under joint stress (Group SC), suggesting that catalase was more sensitive to contamination of PFASs and Cd. Catalase is involved in the biological processes of soil energy and nutrient transformation, and it can facilitate decomposition of soil H2O2 which is harmful to plant growth (Zhang and Wang, 2006). Catalase activities were slightly promoted in single PFASs (by 5.9%) and single Cd (by 17.6%) treatments, but were significantly (p < 0.05) increased in co-contaminated treatments (by 141%) compared with control. The results indicated that combined effect of PFASs and Cd on catalase activities produced a stronger effect than single treatments. Similarly, Liu et al.

Cplants Cs

Cshoot Croot

(3) (4)

where Croot, Cshoot and Cplants are the concentrations of PFASs or Cd in the root, shoot and whole plants (ng g−1 dry weight) respectively, and Cs is the organic carbon-normalized concentration of PFASs or Cd in the dry soil (ng goc−1). 196

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significant difference in wheat biomass among these three treatments (Group I, II and III, p > 0.05). The total biomass of rapeseed was significantly influenced rather than wheat in joint treatments. The rapeseed biomass decreased by 13.4% and 37.3% respectively under joint stress compared with single PFASs and single Cd treatments. The effect trend of PFASs on plant growth was similar to Qu et al. (2010) who found that the low concentration (equal to and lower than 1000 ng mL−1) of PFOS could slightly stimulate the growth of wheat seedlings cultured in Hoagland's solution. Previous studies have also demonstrated that benzo[a]pyrene (B[a]P) and Cd at low levels could facilitate the growth of plants (Sun et al., 2011) and the addition of PAHs significantly increased the total biomass of Juncus subsecundus grown in Cd treatments compared without PAHs (Zhang et al., 2012).

(2008) found that catalase activities were stimulated in soil co-spiked with cypermethrin (1000 ng g−1) and Cu (100 mg kg−1). Kordybach and Smreczak (2003) also suggested that combined effect of polycyclic aromatic hydrocarbons (PAHs) and heavy metals (Zn2+, Pb2+ and Cd2+) on soil microorganisms activity can be stronger than in soils amended with heavy metals or PAH separately. Zhang et al. (2013) reported that PFOA introduced into the soil would exert an influence on the microbiota, which manifested itself in changes in enzyme activities. In the present study, the increased activities of catalase might be due to the changes of soil microorganisms in single and joint treatments of PFASs and Cd. Urease activities were significantly (p < 0.05) inhibited in single PFASs treatments (by 39.6%), single Cd treatments (by 26.1%) and joint treatments (by 89%) compared with control, and they were significantly (p < 0.05) decreased in co-contamination compared with single PFASs treatments (by 81.8%) and single Cd treatments (by 85.1%). The trend was similar to Zhang et al. (2013) who found that soil urease activities were highly inhibited (p < 0.01) by PFOA after exposure for 30 d at treatment levels of 10,000 ng g−1. Previous studies found that the interaction between Zn and benzo(a)pyrene decreased the soil urease activity from 14 to 21 d of incubation (Shen et al., 2006), the addition of phenanthrene enhanced the toxicity of Cd to soil enzymes and microorganisms which mainly affected the growth of fungi and the activity of urease (Shen et al., 2005). Urease is an important enzyme in soil nitrogen transformation, which can decompose urea into ammonia (Zhang and Wang, 2006). In the present study, the inhibition of urease activities might be due to the changes of fungi membrane structure and permeability under joint stress. There had been little change on sucrase activities both in single and joint treatments of PFASs and Cd. It might be related to tolerance and adaptation of the microorganism (Shen et al., 2006).

3.3. Biological responses of plants to single and co-contamination of PFASs and Cd Toxic effects of PFASs, Cd and PFASs-Cd on wheat and rapeseed grown in soil (Group S, I, II and III) were investigated using the developmental indexes, including chlorophyll (Chl), malondialdehyde (MDA), superoxide dismutase (SOD) and peroxidase (POD) in the shoot tissues. The contents of the developmental indexes in wheat and rapeseed are shown in Fig. 3. The biosynthesis of Chl (21.92%) and MDA (19.37%), and the activities of SOD (97.9%) and POD (93.8%) in wheat were inhibited with PFASs addition (Fig. 3). The Chl (14.61%), MDA (60.65%) and POD activities (99.71%) were enhanced, but SOD activity (19.87%) was inhibited in rapeseed with PFASs addition. As one of the visible symptoms, Chl could be used to monitor the oxidative damage level of the growth and development of plants (Song et al., 2007). The inhibition of Chl accumulation in wheat indicated that PFASs might be active in the chloroplast electron-transport system and disturb the photosynthesis of wheat after exposure to PFASs for 70 d. Antioxidant defense systems comprising of SOD and POD play important roles in scavenging reactive oxygen species (ROS) produced under oxidative stress (Wu and Tiedemann, 2002). The enhancement of POD activities in rapeseed indicated that the antioxidant system played a positive role against PFASs stress. Biological responses of wheat and rapeseed to Cd in soils were also investigated using the developmental indexes in the shoot tissue. The Chl (12.19%), MDA (19.80%), SOD (32.5%) and POD (52.0%) activities in wheat (Group II) were inhibited with Cd addition (Fig. 3). The contents of Chl (54.44%), MDA (43.53%) and POD (334.41%) activities in rapeseed (Group II) were enhanced, but SOD activities were inhibited (11.16%) with Cd addition. Previous studies have shown that Cd toxicities decreased photosynthetic pigments in many plant species such as wheat (Rizwan et al., 2012) and Brassica napus (Ehsan et al., 2014). The difference of Cd toxic effect on physiology indicated different metal tolerance ability of plants. In the case of co-contamination (Group III), the biosynthesis of Chl (18.80%) and MDA (4.73%) in wheat were inhibited, and SOD (0.78%) and POD (184%) activities were promoted compared with control. The Chl (1.11%) and MDA (9.59%) content, and the activities of SOD (29.32%) and POD (26.79%) were inhibited in rapeseed compared with control. The biosynthesis of Chl (4.04%) and MDA (18%) in wheat under joint stress of PFASs and Cd were enhanced compared with single PFASs treatments, and SOD (49.22%) and POD (479%) activities were promoted compared with single PFASs treatments. The Chl (14.0%) and MDA (43.53%) contents, the activities of SOD (14.0%) and POD (83.67%) in rapeseed were inhibited compared with single PFASs treatments. The significant reduction in wheat Chl in the single and joint PFASs and Cd treatments were observed, and the Chl content in joint stress was mainly determined by PFASs. Similarly, Chen et al. (2010) found significant inhibition of Chl biosynthesis in early developmental stages of wheat by the joint stress with polycyclic musk and Cd. In this study, the MDA content of wheat under joint stress was significantly higher than that in either PFASs or Cd treatments, which indicated that the co-existence of PFASs and Cd could increase the

3.2. Plant growth and biomass

60 50

Wheat Rapeseed *

40

*

-1

The biomass (g.pot FW) of plants

Both wheat and rapeseed survived and appeared in good health in all test groups after grown for 70 d. An increase in plant biomass suggested that plants were influenced by single and joint treatments of PFASs and Cd. (Fig. 2). The biomass of wheat and rapeseed increased (p < 0.05) by 15.2% and 31.0% respectively with PFASs addition. The addition of Cd significantly (p < 0.05) increased the biomass of wheat and rapeseed by 20.2% and 50.7% separately. The biomasses of wheat and rapeseed were increased by 16.6% and 13.5% respectively under joint stress. The effects of single Cd treatments on plant biomass were much stronger than that of single PFASs and joint treatments. Significant increase of rapeseed biomass had occurred in single treatments of PFASs and Cd than joint treatments. But there was no

30

*

*

20 10 0

Control

PFASs

Cd

PFASs+Cd

Fig. 2. The biomass of wheat and rapeseed influenced by PFASs and Cd treatments and their interactions after grown in single and co-spiked soil. Significant differences from control are indicated with asterisks (*p < 0.05).

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3.5 3.0

1.8

Wheat Rapeseed

2.0 1.5 1.0 0.5 Wheat Rapeseed

-1

1500 1000 500 0

1.2

-1

2.5

0.0

Wheat Rapeseed

1.5 MDA (ug g FW)

4.0

POD activity (U mg FW)

-1

-1 SOD activity (U mg FW) Chlorophyll content (mg g FW)

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Control

PFASs

Cd

0.9 0.6 0.3 0.0 180

Wheat Rapeseed

160 140 40 20 0

PFASs+Cd

Control

PFASs

Cd

PFASs+Cd

Fig. 3. Toxic effects of Group I (spiked with PFASs), Group II (spiked with Cd) and Group III (co-spiked) on activities of antioxidant enzyme in wheat and rapeseed.

-1

PFOA conc.(ng g DW)

damage to cell membranes of wheat. Banni et al. (2009) also found that the mixture of benzo[a]pyrene and Cd induced concentration of MDA in Hediste diversicolor compared with their single treatments. The changes of Chl and MDA contents demonstrated that PFASs and/or Cd caused a variety of physiological stresses in the plants. The increased activities of SOD and POD in wheat caused by the joint of PFASs and Cd were typical stress response to remove the peroxides and maintain the function of cells. Similarly, significant inducement of SOD and POD activities were found in early developmental stages of wheat by the joint stress with polycyclic musk and Cd (Chen et al., 2010).

PFOS conc.(ng g DW)

3.4. Interaction impacts on bioaccumulation of PFASs and Cd in plants

-1

Cd conc.(mg kg DW)

-1

No PFASs and Cd were detected in the roots and shoots of both wheat and rapeseed from the control treatments, suggesting that there was no contribution from foliar uptake from the air to concentrations in the plant. The detailed concentrations of PFASs and Cd detected in testing plants are provided in Table S3. As seen from Table S3, the concentrations of PFASs in plant roots and shoots were in the range of 332–1411 and 39.6–821 ng g−1 respectively, and concentrations of Cd were in the range of 65.3–206 and 13.0–44.0 mg kg−1 respectively. The result indicated that roots could efficiently take up PFASs and Cd from soil and then translocate to shoots through xylem in both wheat and rapeseed. The concentrations of PFOA (p < 0.05) and PFOS in plant tissues grown in co-contaminated soil, including roots (wheat 28.9% and 11.2%, rapeseed 22.2% and 18.0%), shoots (wheat 60.1% and 47.3%, rapeseed 18.3% and 5.8%) and total plants (wheat 55.2% and 34.6%, rapeseed 17.6% and 4.9%) were decreased compared with single PFASs treatments (Fig. 4A and B). To compare the bioaccumulation abilities of PFASs and Cd in plants between single and combined testing groups, RCF, SCF, BCF and TF were calculated for PFOA/PFOS and Cd using Eqs. (1)–(4). Table S4 illustrated all the concentration factors (RCFs/SCFs/BCFs) and TF values of PFASs and Cd in wheat and rapeseed in Group I, II and III. The RCFs (wheat 30.0% and 21.9%, rapeseed 24.4% and 24.2%), SCF (wheat 62.5% and 60.0%, rapeseed 19.2% and 9.1%), and BCFs (wheat 50.0% and 25.0%, rapeseed 20.7% and 20.0%) of PFOA and PFOS in both wheat and rapeseed were

2000 1600

PFASs PFASs+Cd

A

1200

*

800

*

400

*

0 2000

* PFASs PFASs+Cd

1600

B

1200 800 400 0 250 200

Cd PFASs+Cd *

C

150 100

*

50 0

*

*

Shoot (W) Root (W) Shoot (R) Root (R)

Fig. 4. Impacts of combination of PFASs and Cd on accumulation of PFOA (A), PFOS (B) and Cd (C) in different compartments (roots and shoots) of plants (wheat and rapeseed). W: wheat; R: rapeseed. Significant differences between single PFASs/Cd and PFASs-Cd treatments are represented by asterisks (*p < 0.05).

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PFASs PFASs+Cd

RCF of PFOA and PFOS

0.05 0.04 0.03 0.02 0.01 0.00 0.07

PFASs PFASs+Cd

0.06 0.05 0.04 0.03 0.02 0.01 0.00 PF

O

W A(

) PF

O

W S(

PFASs PFASs+Cd

0.12

TF of PFOA and PFOS

BCF of PFOA and PFOS

SCF of PFOA and PFOS

0.06

) O PF

R A(

) O PF

R S(

0.10 0.08 0.06 0.04 0.02 0.00 0.7

PFASs PFASs+Cd

0.6 0.5 0.4 0.3 0.2 0.1 0.0

)

P

A FO

(W

) P

S FO

(W

)

OA PF

(R

)

OS PF

(R

)

Concentration and translocation factors of Cd in wheat and rapeseed

Fig. 5. Impacts of PFASs and Cd combination on the concentration factors (SCFs/RCFs/BCFs) and translocation factors (TFs) of PFOA and PFOS in testing plants (wheat and rapeseed). W: wheat; R: rapeseed.

decreased compared with single PFASs treatments (Fig. 5). However, TFs of PFOA (45.5%) and PFOS (41.7%) were decreased in wheat but increased in rapeseed (PFOA 5.2% and PFOS 14.3%) with Cd addition. PFOA and PFOS could be taken up by roots of plants from soil mainly through pore water and then be transported to the shoots through xylem or phloemsap via transpiration (Lechner and Knapp, 2011). Müller et al. (2016) reported that accumulation of short-chain PFASs was dominated by uptake and translocation, whereas long-chain PFASs was primarily the result of sorption affinity. It was also reported that, the absorption of organic components could be described as partitioning between soil aqueous solution and plant roots (Lu et al., 2014). In this study (Table S5), the concentration of free Cd2+ in soil solution of Group SA (11.3 µg L−1) increased as a result of PFASs addition in Group SC (21.1 µg L−1), which suggested the complexation between Cd and PFASs did not occur. Qi and Chen (2010) found a positive correlation between bioavailability and the degree of desorption ability from soil of hydrophobic organic contaminant. The study of Dontsova and Bigham (2005) have demonstrated that cations will increase sorption of organic anion to sediment. Higgins and Luthy (2006) interpreted the sorption of PFOA and PFOS to sediments increased with increasing solution divalent cations (Ca2+) might be due to a reduction in the charge present on the organic matter (i.e., a reduction in the potential, ψ), and it suggested that electrostatic interactions played a role in the PFOA and PFOS sorption. Adak et al. (2005) reported that cations (Fe3+) increased the surface charge of alumina and also resulted in more anionic surfactant (sodium dodecyl sulfate; SDS) adsorption. Thus, the addition of Cd might increase the sorption of PFASs in soil and lead to the reduction of PFASs concentrations in the pore water, which could be taken up by roots of plants, and then decreased the bioaccumulation of PFASs in plants. The concentrations and bioaccumulation factors of Cd in plant tissues were significantly (p < 0.05) influenced by the addition of PFASs (Fig. 6). The RCF (wheat 25.0%, rapeseed 53.0%), SCF (wheat 64.8%, rapeseed 146%), BCF (wheat 28.3%, rapeseed 73.4%) and TF

30 25 20 15

Cd PFASs+Cd

Wheat

Cd PFASs+Cd

Rapeseed

10

1.5 1.0 0.5 0.0 8 6 4 2 0

SCF

RCF

TF

BCF

Fig. 6. Effects of PFASs and Cd combination on the concentration factors (SCFs/RCFs/ BCFs) and translocation factors (TFs) of Cd in wheat and rapeseed.

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Table 1 Concentrations (mg kg−1) of Cd in modified BCR-sequential (European Community Bureau of Reference) extraction fractions of soil samples spiked with single Cd and PFASs-Cd respectively. Fraction A: Exchangeable and weak-acid soluble (chemically labile) fraction; Fraction B: Reducible fraction; Fraction C: Oxidizable fraction. Extractions

Group SA

Group SC

Fraction A Fraction B Fraction C

1.16 ± 0.37 0.34 ± 0.11 0.32 ± 0.07

5.50 ± 0.20 1.64 ± 0.37 0.34 ± 0.09

Group SA: soil alone, spiked with single Cd; Group SC: soil alone, co-spiked with PFASs and Cd.

Appendix A. Supporting information

(wheat 2.4%, rapeseed 60.6%) were all increased compared with single Cd treatments suggesting that PFASs could enhance the uptake of Cd by roots from soil and subsequent translocation to shoots. Table S5 showed that the presence of PFASs increased free Cd2+ concentration in the soil solution by 86.7%, and significantly increased Cd concentrations in both roots and shoots of plants. The results were in agreement with the free ion activity model (FIAM), the free metal ion activity reflects its bioavailability (Morel, 1983). Heavy metals are readily taken up by plants through membrane transporters due to their relatively mobile in soil (Zhang et al., 2012). The BCR sequential extraction method was also used to evaluate plant availability of heavy metals in soils in previous studies (Sungur et al., 2014; Tokalioğlu et al., 2006). The fractionation results of Cd in the soil samples (Group SA and SC) obtained by the modified BCR sequential extraction procedure are given in Table 1. As shown in Table 1, the first step extracts of BCR sequential extraction of Cd which have high mobility and can be readily available to biota was 1.16 mg kg−1 in Group SA and 5.5 mg kg−1 in Group SC. The proportion of fraction A (soluble and exchangeable fractions) in PFASs-Cd treatment was increased by 374% relative to a single Cd treatment, suggesting that PFASs addition increased the proportion of Cd mobile forms, since the labile acid exchangeable fraction is generally considered to represent bioavailable forms of soil Cd (Degryse et al., 2006; Fleming et al., 2013).

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4. Conclusions The single and co-contamination of PFASs and Cd in soil could affect the soil enzyme activities, bioaccumulation and toxicity of PFASs and Cd in wheat and rapeseed. Soil enzyme activities indicated a sensitivity order of catalase > urease > sucrase under joint stress of PFASs and Cd, and soil urease activities were decreased but catalase activities were increased significantly in co-contaminated soil. The biomass and biosynthesis of Chl in wheat were decreased and MDA was enhanced grown in PFASs and Cd co-spiked soil, while SOD and POD activities were promoted. The Chl and MDA contents, activities of SOD and POD in rapeseed were inhibited compared with single PFASs and Cd treatments. The co-existence of PFASs and Cd in soil mutually affected their bioaccumulation and translocation abilities in plants. The bioaccumulation was decreased for PFASs but increased for Cd. The translocation abilities of PFASs were decreased in wheat but increased in rapeseed, while increased for Cd in both wheat and rapeseed. The cocontamination of PFASs and Cd decreased the bioavailability of PFASs but increased the bioavailability of Cd in soil. These results are important to predict the environmental fate and assess the environmental risk of PFASs and heavy metals in co-contaminated soil.

Acknowledgments The authors gratefully acknowledge the financial support from the Natural Science Foundation of China (NSFC 41603106) and Fundamental Research Funds for the Central Universities (Grant No. DUT16RC(4)83). 200

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