CuO nanoparticles modify bioaccumulation of perfluorooctanoic acid in radish (Raphanus sativus L.)

ABSTRACT Research on combined phytotoxicity of perfluorooctanoic acid (PFOA) and nanoparticles is very important for the remediation of PFOA contaminated soil and further assessment for the potential of nano-enhanced phytoremediation. Here, joint effects of PFOA and CuO nanoparticles (nCuO) in plants were studied by exposing radish (Raphanus sativus L.) to PFOA (4 mg/kg) and nCuO (200 and 400 mg/kg) for 30 days, and measuring for contaminant accumulation, radish biomass, photosynthesis profiles and nutrient contents. Results showed that PFOA accumulated highly in radish organs but showed limited effects on radish biomass. nCuO could increase the transfer rate of PFOA from root to shoot and reduce PFOA accumulation in edible root part, but higher nCuO lead to decreased radish biomass. Reversely, PFOA alleviated the adverse effects of nCuO on leaf photosynthesis and root metabolism of vitamins and amino acids. These results provided basics for exploring possibility of nano-enhanced phytoremediation for PFOA soil pollution.


Introduction
Owing to its neurotoxicity, genotoxicity and development toxicity to endocrine disruption in organisms, perfluorooctanoic acid (PFOA) has been shortlisted in the Stockholm Convention in 2019 [1][2][3]. However, due to its high stability and unique hydrophilicity, PFOA is still produced for domestic and international demands [4][5][6]. PFOA has been frequently detected in agricultural soil throughout the world, especially in some hot spots with concentration up to 123.6 µg/kg [2,7]. PFOA can be accumulated in plant's roots and transported to shoots or other edible parts, posing potential food exposure and threatening human health [3,[8][9][10][11].
Nanoparticles, functioning as regulators, pesticides and fertilizers, have been documented as promising soil amendments in sustainable agriculture [12]. Nanoparticles existing in the soil will alter not only the properties of soil but also the migration, transformation and toxicity of coexisting pollutants [13][14][15][16]. Hence, theoretically, it can be assumed that nanoparticles have potential effects on the carryover and bioavailability of PFOA when they coexisting in soil. In particular, metal-based nanoparticles, not only with large surface area but also releasing cations, can interact with PFOA via electrostatic interaction and ligand exchange, and thus affecting PFOA bioavailability [17]. However, there is a lack of research on the interaction between nanoparticles and PFOA in soil-plant system, which is very important for the agricultural product safety and remediation technology development for PFOA contaminated soil.
Here, we conducted an experiment to test the joint effects of PFOA and CuO nanoparticles (nCuO) on radish (Raphanus sativus L.). Radish is a traditionally and widely consumed vegetable in east Asian countries, and nCuO is largely and extensively applied in agriculture as pesticides and biocides [12,18,19]. The uptake and translocation of PFOA and nCuO in radish, and their effects on radish photosynthetic parameters, biomass and nutritional values (total sugar, starch , vitamins and amino acids) were measured.

Plant growth and exposure
Radish (Raphanus sativus L.) seeds were purchased from Wanbang Seed Corporation (Nanjing, China). 14 C-PFOA was purchased from American Radiolabeled Chemicals, Inc. (St. Louis, MO) with a specific radioactivity of 2.035 GBq/mM. Non-labeled PFOA and nCuO were purchased from Sigma-Aldrich Co., St. Louis, MO. According our previous study, nCuO has a primary size at 10-100 nm and zeta potential at −34.4 ± 0.5 mV [19].
Commercial soil (potting mix, Miracle Gro® with micromax, Marysville, OH, USA) was thoroughly mixed with PFOA solutions to contain 0 (control) and 4 mg PFOA per kg soil (dry weight). PFOA solutions were prepared by adding 14 C-PFOA and unlabeled PFOA stock solutions to ultrapure water (Milli-Q, Sigma-Aldrich) and homogenized by sonication in a water bath (180 watts × 20°C × 30 min). Subsequently, nCuO suspensions were thoroughly mixed with the spiked soil to contain 0 (control), 200 and 400 mg Cu per kg soil (dry weight). Nanoparticle suspensions were prepared in Millipore water and homogenized using ultrasonic in a 5°C water bath for 30 min. In all, there are six treatments: one for control, soil without any supplementation; one for PFOA treatment, soil amended with PFOA (4 mg kg −1 ); two for nCuO treatments, soil amended with nCuO (200 or 400 mg kg −1 ); two for PFOA+nCuO treatments, soil amended with combination of PFOA (4 mg kg −1 ) and nCuO (200 or 400 mg kg −1 ).
The spiked soil was placed in plastic pots (19 cm diameter x 18 cm height) and seeded 24 h after treatment application. Four replicates for each treatment were allocated in a completely random design. Plants were maintained in a greenhouse with day/night temperatures at 28/20°C, relative humidity at 60%, illumination at 200 µmol·m −2 ·s −1 for 14 h photoperiod.

Photosynthetic index analysis
Prior to harvest, chlorophyll contents in cucumber leaves were measured according to the method described by Lichtenthaler and Wellburn [20) at 645 and 663 nm using a spectrophotometer (UV-1800, Shimadzu, Kyoto, Japan). Net photosynthetic rate (Pn), stomata conductance (Gs), intercellular CO 2 concentration (Ci) and transpiration rate (Tr) were measured by a Li-Cor 6800 portable photosynthesis system (Li-Cor Inc., Lincoln, NE). The measurements were carried out at the aforementioned light intensity, with an air flow of 700 µmol s −1 through the sample chamber, and a CO 2 concentration of 400 µmol mol −1 in the sample chamber.

Biomass, copper and PFOA concentration analysis
Thirty days after germination, plants were removed from soil, rinsed with tap water and ultrapure water over 10 times, and separated into root and shoot parts. Plant biomass was then measured after lyophilization. For copper analysis in radish roots and shoots, 100 mg samples of oven dried tissues were digested with HNO 3 and HClO 4 (4:1, v/v) and adjusted to 10 ml with Millipore water. Element copper was analyzed using an inductively coupled plasma-optical emission spectroscopy (ICP-OES, Optima 5300 DV, Perkin-Elmer, USA). The recovery for Cu was 94-105%.
Concentrations of PFOA in plants were calculated by the initial 14 C-radioactivity and PFOA concentration in spiked soil. Briefly, 0.1 g plant samples were combusted at 800-900°C in a combustion unit (Ox500 Biological Oxidizer, Zinsser Analytic, Germany), mixed with a scintillation cocktail (Gold Star multipurpose, Meridian Biotechnologies Ltd., UK) and 14 C-activity was quantified by a liquid scintillation counter (LS6500, Beckman Coulter, Brea, CA). The recovery for 14 C-chemicals was tested [79-82%) and subsequently calibrated by radioactivity.

Nutrient contents in radish roots
Amino acid contents were analyzed using external standard method according to 21. Amino acid standards were purchased from Sigma Aldrich Co. St. Samples were ground to a powder in liquid N 2 and subsequently dissolved in ultrapure water. Derivatization was performed with an AccQ-Tag Ultra Derivatization Kit (Waters, USA). UPLC-MS analyses were performed using an ultra-performance liquid chromatography (UPLC, U3000 DGLC, Thermo Fisher, USA) containing an ACCQ-TAG TMULTRA C18 column [1.7 µm, 2.1 × 100 mm, Waters, USA].
Vitamin B and C contents were analyzed using external standard method according to 22. Radish root at 200 mg was extracted using 500 μL 80% methanol (containing 1% acetic acid) and ultrasonic for 15 min. The supernatant was centrifuged at 12,000 rpm for 10 min. Repeated extraction for 3 times, and collected the supernatant into a tube for determination. Vitamin contents were extrapolated from a standard curve by Liquid Chromatography Mass Spectrometry (LC-MS, DGLC-QE, Thermo Fisher, USA). Starch contents were analyzed using glucose standard calibration curve at 510 nm according to 23. Total sugar content was quantified from the glucose standard calibration curve at 620 nm according to 24.

Statistical analyses
The data were expressed as mean ± standard deviation (n = 4). Statistical significance of differences among treatments was evaluated by one-way analyses of variance (ANOVA) performed by SPSS (IBM Statistics 22). Reference to a significant difference between treatments was based on a probability of p < 0.05.

PFOA and Cu accumulation
PFOA accumulation in radish roots and shoots is presented in Figure 1. Contents of 14 C-activity chemicals were 1.55-2.18 mg/kg in roots and 3.46-4.44 mg/kg in shoots after PFOA exposure. PFOA in the radish shoots was approximately 2-fold higher than that in roots, hypothesizing that phytoremediation could be considered for PFOA contaminated soil [8,25]. Generally, the adsorption affinity of long-chain compounds in plant is dependent on compound hydrophobicity, so PFOA was thought more efficiently adsorbed to root but less available for translocation through transpiration stream [26,27]. However, radish roots, unlike a typical dicotyledonous root, are primarily consist of vascular tissue, lacking the Casparian strip as barrier to prevent PFOA's transport from roots to shoots [8]. Consequently, PFOA was found concentrated in radish shoots.
Notably, nCuO significantly increased the transport of PFOA to shoots by 11% and 28% for 200 mg/kg and 400 mg/kg, respectively. nCuO could remodify the protein receptor that regulate the transport of PFOA or mechanically damage the structure of membranes, promoting the PFOA translocation [12,28]. Besides, PFOA may be transferred to leaves by adsorbing on the surface of nCuO [16]. Although the dissolution of nCuO proceeded all the time, the particles of CuO still existed even after 88 days of culture [15]. nCuO could adsorb PFOA through electrostatic interaction and ligand exchange and thus affect distribution of PFOA in plants [14].
Meanwhile, PFOA also modified Cu accumulation in roots and upward transport to leaves (Figure 2). nCuO alone significantly improved Cu contents, tested 0.29-0.63 mg/kg in roots and 28-72 mg/kg in shoots, but without difference between 200 mg/kg and 400 mg/ kg. The periderm, a layer of dead cells covering the radish root, has a clear capacity to adsorb and retain a large amount of nCuO and Cu 2+ released from nCuO [29]. So, nCuO with high dissolution rate and the translocation could promote Cu accumulation in shoots [28]. Here, PFOA obviously promoted the uptake of Cu in radish roots but not changed Cu upward transfer to leaves, indicating that PFOA may increase Cu bioavailable forms' proportion via the desorption of Cu from soil, functioning as low-molecular-weight organic acids (LMWOAs) [30,31]. The dissolution and aggregation of nCuO were generally affected by numerous soil conditions such as pH, organic matter, ionic species and colloids [32]. Especially, organic acids may absorb on the surface of nCuO through electrostatic interaction or complex with free Cu 2+ ions by ligand exchange [31,32]. So, PFOA could adsorb on the nCuO surface by the carboxyl group, or form a complex with Cu through sequestration or ion exchange, decreasing the adsorption of Cu and increasing the availability of Cu in soil [14,33,34]. However, the PFOA-Cu complex may have a different transport behavior from individual Cu, so the limited adsorption sites of radish roots and the finite amount of Cu transporters may restrict the uptake and the translocation of Cu in radish [35,36].

Effects on radish photosynthetic parameters and final biomass
PFOA alone showed no obvious effect on chlorophyll contents in radish leaves, while nCuO at 400 mg/kg significantly reduced the contents of chlorophyll b ( Figure 3A). Further disturbance in chloroplast electron-transport system were reflected by changes in Pn, Tr, and Fv/Fm value as shown in Figure 3. Pn is   a reliable method for gauging the primary production of plants; Fv/Fm value is a useful way to detect disturbances in the photosynthetic system caused by various stressors [37]. PFOA caused no obvious change in such two indices, while nCuO at 400 mg/kg lead to an obvious reduce. Nanoparticles may generate reactive oxygen species (ROS) and decrease leaves' chlorophyll contents, subsequently reducing PSII reaction centers and inhibiting electron transport from the water splitting system to Q A reduction [38][39][40]. In addition, transpiration rate was thought playing an important role in the transport of perfluorinated compounds in plants [8,41]. But, here there was no change observed in transpiration rate, revealing that changed PFOA accumulation in radish shoots were not result from the disturbance of nCuO to plant transpiration. The specific reasons need further research. Still, it demonstrated that nanoparticles have the ability to redistribute organic compounds in plants [16,36,42]. Figure 4 shows the biomass of final harvest radish. nCuO alone caused a significant decrease in root and shoot biomass, while PFOA showed limited effects. Accumulation of Cu in plant could decrease the chlorophyll contents, inhibit the photosynthesis, and finally reduce plant biomass [15]. Notably, PFOA significantly alleviated such reduce in biomass by nCuO, even though PFOA increased the accumulation of Cu in radish roots. PFOA, functioning as LMWOAs, may enhance the bioavailability of metal nutrients in soil [31]. But, as a kind of organic acid, it could also hinder dissolution of nCuO via blocking the active sites of nCuO surface [13]. nCuO assumed to be retained in tissue as PFOA-Cu complex through strong COObinding group [43]. Figure 5 and Table S1 present the summary statistics for changed nutrients in harvest radish roots. PFOA alone showed limited modification in radish nutrient except for an increase in biotin (VB7) when compared with control. Biotin, as an essential cofactor related to carboxylation reactions, was increased by PFOA, indicating that PFOA caused some disturbance in carbon and energy metabolism [44]. While nCuO alone obviously disturbed metabolism of starch, amino acids, and vitamin. Especially, nCuO at 400 mg/kg decreased contents of starch, asparagine (Asn) and VC by 9%, 31% and 16%, but increased those of serine (Ser), alanine (Ala) and VB7 by 19%, 22% and 49%, respectively. It indicated that nCuO could disturbed the metabolism of carbohydrate, amino acid and vitamin, some of which are related to energy status maintenance such as glycolysis and the TCA cycle [38,45]. Decline in starch contents was consistent with the reduction in chlorophyll contents, implying that nCuO has negative effects on the metabolism of carbohydrate [46]. Higher Ala accumulating indicated that excessive accumulation of Cu led to the breakdown of protein synthesis [47,48]. Besides, changes in Ser and Asn contents indicated a reprogramming of nitrogen and sulfur metabolism may occur in radish roots to modulate carbon, nitrogen and sulfur status [49,50]. Ser, as an important precursor of cysteine (Cys) synthesis, may contribute to the biosynthesis of glutathione (GSH), an essential component of resistance systems to heavy metals [51]. Nicotinic acid (VB5) and ascorbic acid (VC) are associated with the antioxidant systems in plants, so their up-regulation indicated the generation of ROS by nCuO [52,53]. nCuO also inhibited the biosynthesis of folic acid (VB9), a cofactor consisted with one-carbon (C1) biosynthesis, implying an inhibition of cell division and consequent decrease in root biomass [54].

Changed nutrients in radish
Compared with individual nCuO (400 mg/kg) exposure, combined PFOA and nCuO (400 mg/kg) led to obvious up-regulation in Asn and Ser, and downregulation in Ala, indicated that PFOA partially alleviated the toxicity of nCuO to radish nutrients. This was contrary to the content trend of Cu in roots, indicated that PFOA might change Cu form in plant, might as PFOA-Cu complex, and thus inhibited copper toxicity [43]. Accumulation of Asn was related to a nitrogen storage pathway and reflected plant response to the abiotic stresses [55]. And recovery in Ser and Ala contents also reflected the restoration of nitrogen metabolism, sulfur metabolism, and protein synthesis [50,56]. PFOA also increased the contents of VB9, indicating the recovery of cell division process, in correspondence with the restoration of radish biomass [54]. PFOA may activate a stress protective strategy in plants or directly interact with nCuO to inhibit the negative effect of nCuO [17]. However, both nCuO (400 mg/kg) alone and its combination with PFOA significantly reduced starch contents in roots, which may ascribe to the disturbance in the starch biosynthesis. In addition, the nutrient value of radish is mainly dependent on the above bio-molecules such as starch, folic acid and ascorbic acid, which are essential for plant and human growth. As the application of nCuO at high concentration slightly inhibited the growth of plants, so it is essential to control dose of nCuO in handling PFOA risk to food safety. Besides, the antagonism between nCuO and PFOA could reduce some adverse effects of nCuO on plant growth and expand the safety concentration of nCuO application.

Conclusions
How nanoparticles will affect plant utilization and toxicity of PFOA was assessed, and results confirmed that nCuO at appropriate dose could redistribute PFOA in plant, increasing transport to aboveground part and decreasing accumulation in edible part. This work highlighted that nanoparticles have potential to regulate PFOA risk in agriculture field and enhance phytoremediation, but attention should be focused on the application concentration and mode in practice. Further studies are needed to expand more kinds of nanoparticles and explore the underlying mechanism.