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The role of different fractions of humic acid in the physiological response of amaranth treated with magnetic carbon nanotubes
Ecotoxicology and Environmental Safety 169 (2019) 848–855
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Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv
The role of different fractions of humic acid in the physiological response of amaranth treated with magnetic carbon nanotubes
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Weili Jiaa, Sheng Zhaib, Chuanxin Mac,d, Huimin Caoa, Cuiping Wanga, , Hongwen Suna, Baoshan Xingc a
Key Laboratory of Pollution Processes and Environmental Criteria, Ministry of Education, Tianjin Key Laboratory of Environmental Remediation and Pollution Control, College of Environmental Science and Engineering/Sino-Canada R&D Centre on Water and Environmental Safety, Nankai University, Tianjin 300071, China b School of Environment and Planning, Liaocheng University, Liaocheng 252059, China c Stockbridge School of Agriculture, University of Massachusetts, Amherst, MA 01003, USA d Department of Analytical Chemistry, The Connecticut Agricultural Experiment Station, New Haven, CT 06504, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Magnetic carbon nanotubes Humic acids Toxicity Amaranth
Dissolved humic acid (DHA) from soil can interact with multi-walled carbon nanotubes (MWCNTs) and magnetic-modified multi-walled carbon nanotubes (MMWCNTs), and subsequently alter the toxicity of MWCNTs and MMWCNTs to amaranth. This is the first study to compare the effects of MWCNTs and MMWCNTs under natural DHAs on their toxicity to amaranth. When DHAs were combined with 0.5 g/L MWCNTs, 1:2:1 MMWCNTs and 4:2:1 MMWCNTs nanomaterials, DHA1 and DHA4 both increased the pH of Hoagland's solutions. DHA1 more severely decreased the soluble protein levels in shoots than DHA4 in the 1:2:1 MMWCNT and 4:2:1 MMWCNT treatments. DHA1 and DHA4 both increased the chlorophyll concentrations of amaranth treated with MWCNTs, decreased the chlorophyll concentrations in the MMWCNT treatments. Co-exposure of DHAs and carbon-based CNTs caused further decreases in the anthocyanin level as compared to the respective CNT alone treatment. In the nanomaterial alone treatment, both 0.25 and 4:2:1 MMWCNTs greatly lowered the anthocyanin level as compared to the other two CNTs with the same exposure dose. Transmission electron microscopy images showed that the interaction between 4:2:1 MMWCNT and DHA4 had more serious effects on plant cells across all the treatments.
1. Introduction Carbon nanotubes (CNTs) have been investigated as a promising adsorbent for removing environmental pollutants and largely applied (Li et al., 2001; Ai et al., 2011) due to their large specific surface areas and hollow and layered structures. However, the recovery of these adsorbents may suffer from a difficult separation process, therefore resulting in their reduced reusability and increased regeneration costs. The recovery defect of CNTs has attracted many researchers to focus on combining CNTs and magnetic oxides to synthetize a promising magnetic carbon nanotube (Chen et al., 2009) that effectively recovers the CNTs via magnetic separation. Though some manufactured magnetic carbon nanotubes, which are similar to other engineered nanomaterials (ENMs), could still be released to the environment (water, air and/or soil) during their handling, disposal and effluent management during CNT synthesis (Cornelis et al., 2014; Aziz et al., 2015; Prasad et al., 2016; Tripathi et al., 2017).
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Once ENMs were released into the environment, their potential effects on living organisms and the non-living (abiotic) components of ecosystems should raise concerns (Hao et al., 2018). Plants are a vital component of ecological systems, serving as both important ecological receptors and potential routes for the transport and bioaccumulation of ENMs in the food chain at different trophic levels (Gardea-Torresdey et al., 2014). Studies performed using advanced detection techniques have demonstrated that ENMs can be readily absorbed by plants germinated or grown in nutrient solutions (Irin et al., 2012; Khodakovskaya et al., 2012) or soil (Rico et al., 2015) supplemented with nanosized materials, which can alter their morphoanatomical, physiological, biochemical and genetic compositions (Rico et al., 2015; Hao et al., 2016; Tripathi et al., 2016). However, there are currently very few studies that have been performed to characterize the effects of magnetic carbon nanotubes on plants. Many studies have also shown that the adsorption of dissolved organic matter (DOM) on the surface of ENMs can affect the dissolution,
Corresponding author. E-mail address: [email protected] (C. Wang).
https://doi.org/10.1016/j.ecoenv.2018.11.072 Received 21 June 2018; Received in revised form 15 November 2018; Accepted 17 November 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.
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(Nicolet iS50, Thermo Fisher Scientific Co., Beijing, China) (Fig. S2).
surface charge, and aggregation behavior of ENMs (Edgington et al., 2010). The interaction between DOM and ENMs may occur and subsequently alter the plant physiological response. Previous studies have demonstrated that DOM could diminish nanoparticle toxicity by reducing its bioavailability to micro crustaceans and bacteria ( Fabrega et al., 2009; Edgington et al., 2010; Chen et al., 2011; Jiang et al., 2011). Humic acids (HAs) are one of the most important components of organic matter, with the ability to affect seed germination and plant growth (Lee and Bartlett, 1976). However, current research on plant tolerance to magnetic carbon nanotubes, especially the physiological response of plants due to both dissolved HA (DHA) combined with magnetic carbon nanotube stress, is rare. In this study, we examined the role of different DHA fractions on the multi-walled carbon nanotubes and magnetic-modified multi-walled carbon nanotube (MMWCNT) in the toxicity of plants. We used soil HA as the aquatic DOM model because it is well distributed in the soil and directly interacts with any ENMs present. Amaranth was chosen as the plant model because it is one of the most widely planted vegetables in China. By analyzing their physiological parameters and subcellular structures in plants, the results could provide important information for evaluating the potential benefits of different soil HA fractions on the environment.
2.3. Plant preparation and experimental design Amaranth seedlings were prepared according to Ma et al. (2013). Twenty sterilized seeds were placed on each filter paper for germination and cultivated in half-strength Hoagland's solution for 20 days in a controlled environment cabinet (22 °C/18 °C, 14 h/8 h, day/night) (HH.BH-600, Tianjin Tianyu Experimental Equipment Co., Ltd, China). The uniform-sized amaranth seedlings (approximately 5 cm in length) were then transferred to Hoagland solutions for 5 days before exposure. In the exposure experiment, each amaranth seedling was grown in a 250 mL glass jar containing 230 mL Hoagland's solution mixed with 10 mg/L DHA and 0.1–0.5 g/L MWCNTs or MMWCNTs for 10 days, each treatment has three replicates. The experimental setup regarding the detailed information for each treatment is provided in Table S1. The seedlings after exposure were harvested and rinsed with deionized H2O to remove the attached particulates from the root surface. The plant roots and shoots were separately frozen in liquid nitrogen and stored in a −80 °C freezer (DW86L388A, Qingdao Haier Electric Appliance Co., Ltd., China) until further analysis. Parts of the roots and shoots were freeze-dried for biomass and element analysis. The rest of the samples remained at −80 °C for the antioxidant enzymes activity analysis.
2. Materials and methods 2.1. MMWCNT preparation and characterization
2.4. Soluble protein and pigment measurement Multi-walled carbon nanotubes (MWCNTs), with 95% purity, a 20–30 nm outer diameter, and 10–30 µm length, were purchased from Xianfeng Nano Material Co. Ltd., China. Analytical grade chemicals including FeCl3·6H2O, FeCl2·4H2O and NH3·H2O were obtained from the Tianjin Yingdaxigui Chemical Reagent Co. Ltd., China. The MWCNT was acidified before loading Fe elements. Briefly, raw MWCNTs were purified with 1 M acidic solution (H2SO4: HNO3; v/v = 3:1) for 24 h before sonicated for 20 min. The mixture was then heated to 100 °C for 40 min. After cooling, the solution was washed to pH was 7.0 and filtered. The precipitates were collected and dried for 12 h at 100 °C in an oven, and stored MWCNTs in a desiccator for further use. Then the magnetic carbon nanotubes were prepared according to the following procedure. 200 mg acidified MWCNTs, 200 mg FeCl2 and 400 mg FeCl3 were added to a 200 mL flask containing 80 mL deionized water. The mixture was sonicated for 30 min and then heated under magnetic stirring at 70 °C, along with a peristaltic pump slowly pumped 6% NH3·H2O (30 mL) to the mixture. The mixture was then cooled naturally to room temperature (25 °C), filtered through a 0.45 µm filter, and washed with deionized water until the pH of the filtrate was approximately 7.0. The dark precipitates were collected and dried for 24 h at 50 °C in an oven. This process yielded 1:2:1MMWCNT (FeCl2·4H2O: FeCl3·6H2O: MWCNT) nanomaterials. The synthesized 4:2:1MMWCNT samples were obtained following the above mentioned procedure and a FeCl2·4H2O: FeCl3·6H2O: MWCNT mass ratio of 4:2:1. All the samples characterized in Fig. 1 were observed using a transmission electron microscope (TEM) (JEM-2010, JEOL, Japan) operating at 20 kV. Fe(II) and Fe(III) in samples were characterized by X-ray photoelectron spectrum (XPS) (ESCALAB 250Xi, Thermo Scientific, USA) in Fig. 2.
The soluble protein was measured according to Singh and Lee (2016). The chlorophyll and anthocyanin concentrations were analyzed using a modified method (Ma et al., 2013; Wang et al., 2017). A portion of 20 mg fresh leaves was incubated in 10 mL 95% ethanol in the dark for 3 days. The absorbance of supernatant was measured at 664.2 and 648.6 nm using a UV−Vis spectrophotometer (TU-1810, Persee, China). The total chlorophyll concentrations were calculated using the following equations: Chla = 13.36A664.2 − 5.19A648.6, Chlb = 27.43A648.6 − 8.12A664.2, Total chlorophyll = Chla + Chlb. The anthocyanin concentrations were measured as follows. 100 mg amaranth leaves were ground in liquid nitrogen and then mixed with 2 mL 1% (v/v) HCl acidified methanol prior to incubate in the dark at 4 °C overnight. Then deionized H2O2 (1 mL) and chloroform (1 mL) were added and mixed. The mixture was centrifuged at 13,000 g for 2 min at 4 °C. The absorbance of the supernatant was measured at 530 and 657 nm and the final anthocyanin concentration was determined by the formula A530 − 1/4 A657. 2.5. TEM observation The cell and chloroplast morphology was observed by a transmission electron microscopy (TEM; Hitachi H-7650, Hitachi Corp., Tokyo, Japan) at 180 kV (Nhan et al., 2015). Briefly, the fresh amaranth leaves were washed with deionized water, the apex were excised and prefixed in 2.5% glutaraldehyde, dehydrated in a graded acetone series, and embedded in Spurr's resin. The fixed samples were sectioned using a microtome JEM-1230 (JEOL, Ltd., Japan) with a diamond knife for TEM analysis.
2.2. Preparation and characterization of different DHA fractions 2.6. Statistical analysis Soil was sampled and prepared as what we reported before (Li et al., 2016). Different fractions of DHAs were sequentially extracted from the soil and characterized follow the approach of Wang et al. (2017). DHAs were characterized by scanning electron microscopy (SEM; S-3500N, Hitachi Co., Tokyo, Japan) at 20.0 kv × 20 k (Fig. S1); Fourier-transform infrared spectra was recorded from the DHAs in the range of 4000–400 cm−1 with a resolution of 2 cm−1 by FTIR spectroscopy
The values in each assay were determined as the mean ± standard deviation of three replicates. All data analysis was performed using the SPSS version 18.0 software package. A one-way analysis of the variance followed by a least significant difference (LSD) test was used to determine the statistical significance (p ≤ 0.05 or p < 0.01) of each parameter among all the treatments. 849
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Fig. 1. The SEM images of unmodified MWCNTs (a), 1:2:1 MMWCNTs (b) and 4:2:1 MMWCNTs (c). Arrow: MWCNT; COM: MWCNT/iron oxide magnetic compositions. Measurements are of three replicates. (Note: 1:2:1 MMWCNT refer to the mole ratios of Fe2+: Fe3+: MWCNT= 1:2:1; 4:2:1 MMWCNT refer to the mole ratios of Fe2+: Fe3+: MWCNT= 4:2:1).
3. Results
44% Fe(II) (710.8 eV binding energy) and 56% Fe(III) (712.5 eV). The iron spectrum clearly showed 62% Fe(II) and 38% Fe(III) on the 4:2:1 MMWCNT surface satellited at 710.9 and 712.8 eV, respectively. Therefore, the results further confirmed that different Fe concentrations were loaded with MWCNTs.
3.1. Magnetic carbon nanotube characterization SEM images of the oxidized MWCNTs and two kinds of MMWCNT composites are shown in Fig. 1. An entangled network of oxidized MWCNTs, with clusters of iron oxides attached to the MWCNTs (Fig. 1B), suggests the formation of MWCNT/iron oxide magnetic composites. The SEM image of the composites (Fig. 1C) shows that a high density of composites was loaded onto the network structure of the MMWCNTs. X-ray photoelectron spectrometry (XPS) of the iron-loaded MWCNTs is shown in Fig. 2. The spectrum in the Fe 2p3/2 region indicates that the iron on the 1:2:1 MMWCNT surface is approximately
Fe(III)
3.2. pH values of the Hoagland solutions Fig. 3 shows that the addition of three nanomaterials, MWCNT, 1:2:1 MMWCNT and 4:2:1 MMWCNT, has different effects on the Hoagland solution pH values. Exposure to a 0.1 g/L three-nanomaterial dose resulted in statistically significant increases in the Hoagland solution pH values relative to the control plants (Fig. 3A), with the low-tohigh ranking of the materials being as follows: Control group (pH
a
Fe(III)
Fe(II)
b Fe(II)
Fig. 2. X-ray photoelectron spectrum of two magnetic carbon nanotubes. Measurements are of three replicates (n = 3). (a: 4:2:1MMWCNT; b: 1:2:1MMWCNT). 850
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Fig. 3. pH changes of Hoagland's solutions for amaranth growth as affected by different doses of MWCNTs and 1:2:1 MMWCNTs and 4:2:1 MMWCNTs (A) and the relationship between doses of three nanomaterials and pH values (B), and 0.5 g/L of MWCNTs and 1:2:1 MMWCNTs and 4:2:1 MMWCNTs combined with DHAs (C). Data are mean ± standard error of three replicates (n = 3). Values followed by different word are significantly different at p ≤ 0.05.
≈ 5.0) < MWCNT < 1:2:1 MWCNT < 4:2:1 MWCNT (pH ≈ 6.0). Exposure to a 0.25 g/L three-nanomaterial dose did not yield obvious changes in the Hoagland solution pH values relative to the untreated control plants (Fig. 3A), whereas exposure to a 0.5 g/L three nanomaterial dose yielded a significant decrease in the pH values of the solution, especially 4:2:1 MWCNT. The solution pH values therefore decreased with increasing exposure doses of the three nanomaterials. In this study, a decrease in the pH of the collection solutions was negatively correlated with an increase in the three-nanomaterial doses (Fig. 3B). The relationship coefficients for pH and MWCNT, 1:2:1 MWCNT, 4:2:1 MWCNT were 0.9313, 0.9998 and 1.0, respectively. The addition of different DHA fractions to the 0.5 g/L MWCNT, 1:2:1 MWCNT and 4:2:1 MWCNT-amended solution yielded pH values that were approximately 2.0 higher than the corresponding three-material treatments (Fig. 3C). The pH changes were dependent on the DHA addition, but they were virtually independent of the DHA fractions.
while the concentration was 11.92 ± 1.19 mg/g in the control root group (Fig. 4B). Therefore, the soluble proteins levels in the CNT treatment groups did not increase with the increase in MWCNT doses for the same material, while The soluble proteins levels in 1:2:1 MMWCNT and 4:2:1 MMWCNT increased as the corresponding material doses were increased. DHA1 and DHA4 combined with MWCNT did not decrease the soluble protein levels in shoots when different DHA fractions were added to the 0.5 g/L MWCNT-amended solution (Fig. 4C). However, DHA1 and DHA4 combined with 0.5 g/L 1:2:1 MWCNT decreased the soluble protein levels in shoots compared to the 1:2:1 MWCNT only treatments, with DHA1 yielding a more obvious decline in soluble protein levels relative to DHA4 (Fig. 4C). Similarly, DHA1 combined with 0.5 g/L 4:2:1 MWCNT decreased the soluble protein levels in shoots compared to the 4:2:1 MWCNT only treatments, whereas DHA4 had no obvious impact (Fig. 4C). The DHA effects on the soluble protein trends in the roots were the opposite to those in the shoots (Fig. 4D). For example, DHAs combined with 0.5 g/L three-material doses increased the soluble protein levels in the roots compared to the MWCNT, 1:2:1 MWCNT and 4:2:1 MWCNT only treatments. The order of the soluble protein levels in the roots was: DHA1 + MWCNT > DHA4 + MWCNT > Control > MWCNT, DHA1 + 4:2:1 MWCNT > DHA4 + 4:2:1 MWCNT > Control > 4:2:1 MWCNT, whereas the order in the shoots was: DHA4 + 1:2:1 MWCNT > DHA1 + 1:2:1 MWCNT > 1:2:1 MWCNT > Control. DHA1 therefore increased the 1:2:1 MWCNT and 4:2:1 MWCNT toxicities to the amaranth shoots more than DHA4 did (Fig. 4C).
3.3. Soluble proteins concentrations in shoots and roots There was a decline in the soluble protein levels in the shoots when the MWCNT, 1:2:1 MMWCNT and 4:2:1 MMWCNT concentrations increased from 0.1 to 0.5 g/L. For example, the 0.1, 0.25 and 0.5 g/L MWCNT concentrations reduced the soluble protein levels by 29.9%, 32.2% and 32.6%, whereas the 1:2:1 MWCNT concentrations reduced them by 30.5%, 22.2% and 15.1%, and the 4:2:1 MMWCNT concentrations reduced them by 39.3%, 67.1% and 12.8% relative to the control, respectively (Fig. 4A). The current order of the soluble proteins for the 0.1 and 0.25 g/L three-nanomaterial doses are as follows: Control > 1:2:1 MWCNT ≈ CNT > 4:2:1 MWCNT, whereas the order for the 0.5 g/L dose is: Control > 4:2:1 MMWCNT > 1:2:1 MMWCNT > CNT. The soluble protein levels in roots treated with 0.1, 0.25 and 0.5 g/L nanomaterials were: 9.33 ± 0.13, 8.97 ± 0.19 and 7.43 ± 0.98 mg/ g, respectively (MWCNT); 6.63 ± 1.31, 8.57 ± 0.50 and 13.22 ± 1.36 mg/g, respectively (1:2:1 MMWCNT); 6.51 ± 0.20, 6.47 ± 0.69 and 10.43 ± 1.22 mg/g, respectively (4:2:1 MMWCNT),
3.4. Chlorophyll concentrations in shoots Different doses of the same nanomaterial had varying effects on chlorophyll concentration. Amaranth exposed to 0.1, 0.25 and 0.5 g/L MWCNT yielded chlorophyll values of 2.91 ± 0.09, 5.0 ± 0.91 and 2.54 ± 0.29 mg/g, while the control concentration was 4.0 ± 0.10 mg/g. The chlorophyll concentration changes were therefore related to the MWCNT doses, with the appropriate MWCNT dose (0.25 g/L) helpful in amaranth photosynthesis. Amaranth exposed to 851
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Fig. 4. Soluble protein content in amaranth shoots (A) and roots (B) as affected by different doses of MWCNTs, 1:2:1 MMWCNTs and 4:2:1 MMWCNTs; soluble proteins in shoots (C) and roots (D) as affected by 0.5 g/L of MWCNTs and MMWCNTs combined with DHAs. Data are mean ± standard error of three replicates (n = 3). Values followed by different word are significantly different at p ≤ 0.05.
influence on the chlorophyll concentration of amaranth treated with MWCNT. When different DHA fractions were added to the 0.5 g/L 1:2:1 MWCNT-amended solution, DHA1 and DHA4 combined with 1:2:1 MWCNT or 4:2:1 MWCNT yielded obvious declines in chlorophyll concentrations compared to the 1:2:1 MWCNT or 4:2:1 MWCNT only treatments.
0.1, 0.25 and 0.5 g/L 1:2:1 MWCNT gave the highest chlorophyll values of 2.57 ± 0.24, 2.28 ± 0.14 and 4.13 ± 0.14 mg/g, respectively. The chlorophyll concentrations therefore increased with increasing 1:2:1 MWCNT doses. Amaranth exposed to 0.1, 0.25 and 0.5 g/L 4:2:1 MWCNT gave the highest chlorophyll values of 5.34 ± 0.36, 4.59 ± 0.08 and 3.09 ± 0.34 mg/g, respectively, with a decline in the chlorophyll concentrations as the 4:2:1 MWCNT doses were increased. The chlorophyll produced by amaranth was therefore influenced by the iron doses loaded with MWCNTs. Different nanomaterials at the same dosage also had varying effects on chlorophyll concentration. The resultant chlorophyll concentrations were ordered as follows when the amaranth exposure dose was 0.1 g/L: 1:2:1 MWCNT ≤ MWCNT < Control < 4:2:1 MWCNT. When the exposure dose was 0.25 g/L, the chlorophyll concentration order was: 1:2:1 MMWCNT < Control < 4:2:1 MMWCNT ≤ MWCNT. When the exposure dose was 0.5 g/L, the chlorophyll concentration order was: MWCNT < 4:2:1 MWCNT < 1:2:1 MWCNT ≤ Control. DHAs significantly (p < 0.05) altered the chlorophyll concentration of amaranth when different DHA fractions were added to the 0.5 g/ L MWCNT-amended solution (Fig. 5B). DHA1 combined with MWCNT yielded an obvious increase in chlorophyll compared to the MWCNT only treatments, whereas the presence of DHA4 had no obvious
3.5. Anthocyanin concentrations in shoots Amaranth exposed to 0.1, 0.25 and 0.5 g/L MWCNT yielded significant declines (p < 0.05) in anthocyanin compared to the control (Fig. 6A). Amaranth exposed to 0.1, 0.25 and 0.5 g/L 1:2:1 MWCNT also yielded decreased anthocyanin concentrations. Furthermore, the extent of the decreased anthocyanin concentrations in amaranth diminished with the increase in MWCNT and 1:2:1 MWCNT doses. The anthocyanin values decreased the most by increasing the 4:2:1 MWCNT doses (Fig. 6A), even though amaranth exposed to 0.1, 0.25 and 0.5 g/L 4:2:1 MWCNT yielded significant declines (p < 0.05) in anthocyanin concentrations. The anthocyanin concentrations were therefore impacted by the nanomaterial doses. Different nanomaterials at the same dosage also had varying effects on the anthocyanin concentrations. When amaranth was exposed to a Fig. 5. Total chlorophyll content of amaranth as affected by different doses of MWCNTs and 1:2:1 MMWCNTs and 4:2:1 MMWCNTs (A) and 0.5 g/L of MWCNTs and MMWCNTs combined with DHAs (B). Data are mean ± standard error of three replicates (n = 3). Values followed by different word are significantly different at p ≤ 0.05.
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Fig. 6. Anthocyanin level of amaranth as affected by different doses of MWCNTs and 1:2:1 MMWCNTs and 4:2:1 MMWCNTs (A) and 0.5 g/L of MWCNTs and MMWCNTs combined with DHAs (B). Data are mean ± standard error of three replicates (n = 3). Values followed by different word are significantly different at p ≤ 0.05.
0.1 g/L dose, the anthocyanin concentration order was: CNT ≤ 1:2:1 MWCNT ≤ 4:2:1 MWCNT < Control. When the exposure dose was 0.25 g/L, the anthocyanin concentration order was: 4:2:1 MWCNT ≤ CNT < 1:2:1 MWCNT < Control. When the exposure dose was 0.5 g/L, the anthocyanin concentration order was: 4:2:1 MWCNT < CNT < 1:2:1 MWCNT < Control. The anthocyanin produced by amaranth was therefore influenced by the nanomaterial properties. The DHAs significantly (p < 0.05) changed the anthocyanin of amaranth (Fig. 6B) when different DHA fractions were added to the 0.5 g/L MWCNT-amended solution. DHA1 or DHA4 combined with MWCNT both yielded obvious declines in the anthocyanin concentrations compared to the MWCNT only treatment, whereas there were no differences between the influence of DHA1 and DHA4 on anthocyanin in amaranth treated with MWCNT. When different DHA fractions were added to the 0.5 g/L 1:2:1 MWCNT-amended solution, DHA1 and DHA4 combined with 1:2:1 MWCNT or 4:2:1 MWCNT yielded obvious declines in the anthocyanin concentrations compared to the 1:2:1 MWCNT or 4:2:1 MWCNT only treatments, with DHA4 yielding a more obvious decline than DHA1 in the 1:2:1 MWCNT treatment. It is a remarkable fact that anthocyanin was not detected in the treatments where DHA1 and DHA4 were combined with 4:2:1 MWCNT.
A
B
3.6. Plant cell structure characterized by transmission electron microscopy (TEM) Intact amaranth shoot and root cells were observed in the control group (Fig. 7A and E). The plant cells were also intact and undeformed after the MWCNT, MWCNT combined with DHAs, and 1:2:1 MWCNT treatments (not given). Furthermore, the internal structure of the grana lamella and stroma lamella in the chloroplast was intact and undeformed. These results indicated that MWCNT, MWCNT combined with DHAs, and 1:2:1MMWCNT treatments have no obvious negative effects on the plant cells and photosynthesis. However, the chloroplast was deformed and starch grains formed in the chloroplast when 1:2:1 MMWCNT combined with DHAs were added to the solution, and the grana lamella and stoma lamella became distorted (Figs. 7B, S4B and S4E). Some black granules, which were suspected to be iron particles or MMWCNTs, were found in the intercellular spaces (Fig. 7 B and F). These results imply that the 1:2:1MMWCNTs exerted an adverse effect on the plant cell and chloroplast when they interacted with the DHAs, which was likely due to the DHAs enhancing the biotoxicity of MWCNTs when in the presence of excess iron. The 4:2:1MMWCNT alone treatments also have an effect on the plant cell and chloroplast (Fig. S4F). For example, some small starch grains formed in the chloroplast, with slight deformation of the interior chloroplast structure. The deformation of the cell and chloroplast became even more serious when 4:2:1 MWCNT interacted with the DHAs (Fig. 7 C, D and S4 C, D). More and larger starch grains formed in the chloroplast, which seriously
C
SG
D
BG
SG SL
SG BG
E
F
SG
BG G
H
BG BG BG
Fig. 7. TEM images of amaranth chloroplast and roots cells among different treatments. Figure A, B, C and D represents the chloroplast structure of amaranth leaves upon exposure to control, 1:2:1 MMWCNT+DHA4, 4:2:1 MMWCNT+DHA1, 4:2:1 MMWCNT+DHA4, respectively. Figure E, F, G and H represent cellular structure of amaranth roots upon exposure to control, 1:2:1 MMWCNT+DHA4, 4:2:1 MMWCNT+DHA1, 4:2:1 MMWCNT+DHA4, respectively. Measurements are of three replicates. SL: stroma lamella; SG: starch grain; BG: black granules. 853
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acids concentrations may destabilize CNTs over HA-bridging flocculation. 10 mg/L DHAs to 0.5 g/L iron-MWCNT ratio in our study sufficiently promoted the iron-MWCNT availability. It was very likely that dissolved iron metals act as bridging agents between DHAs and MWCNT surfaces provided a more stable suspension of iron-MWCNT.
impaired photosynthesis. Furthermore, some black granules appeared around the shoot and root cell spaces (Fig. 7 C, D, G and H), likely due to the addition of DHAs enhancing the bioavailability of 4:2:1MMWCNT transferred to the plant cell. This is also supported by the total chlorophyll in Fig. 5. For example, 4:2:1MMWCNT combined with or without DHAs, all yielded significant declines in the chlorophyll concentration. The starch grains and black granules from the DHA4 treatment were more obvious than those from the DHA1 treatment, therefore indicating that the interaction between 4:2:1 MMWCNT and DHA4 had a more serious effect on the plant cells.
4.3. MWCNTs and DHAs caused effects on pigments contents Few studies have compared the effects of MWCNT coated with different doses of iron on plants. In our study, the chlorophyll produced by amaranth was generally affected by the iron-nanomaterial properties (Fig. 5A). The change in chlorophyll concentrations highlighted a fact that the iron in MMWCNTs was capable of either inhibiting or enhancing chlorophyll synthesis mainly depends on irons concentration. Besides, different DHA fractions played different roles in chlorophyll synthesis that were also dependent on the iron concentrations of the MWCNTs/MMWCNTs (Fig. 5B). The dissolved irons may play an important role by work as bridging agents in dominating the MMWCNT stabilization and bioavailability. All doses of nanomaterials decreased the anthocyanin contents as compared to control. However, with dose increasing, the diminishing effects decreased when applied with MWCNT and 1:2:1 MWCNT, while such trend was not obvious in 4:2:1 MWCNT treatments (Fig. 6A). Besides, the nanomaterials properties also affect the anthocyanin production of amaranth because that a varying effects existed between different materials at the same dosage. Biochemical changes induced by MWCNT-based modifications in certain ions, such as Fe2+ or Fe3+, or cationic exchange in the amaranth cell wall matrix were likely to alter the antioxidant defense of the plants. Different fractions of DHA played varying roles in decreasing the anthocyanin produced by amaranth (Fig. 6B). Studies involving dissolved organic matter (DOM) have reported DOM to be an effective suspending agent for CNTs (Hyung et al., 2007). This means that MWCNTs and MMWCNTs will be suspended by DOM, resulting in the easy transport of MWCNTs and MMWCNTs to amaranth, which further enhances their toxicity to amaranth. Shen et al. (2015) found that highmolecular-weight (MW) DOM stabilizes nC60 in NaCl more effectively than its low-MW counterparts. Therefore, we conclude that DHA1 with high-MW DOM could stabilize MWCNTs and MMWCNTs in comparison to DHA4 with low-MW DOM, with DHA4 elevating the MWCNT and MMWCNT toxicities considerably more than DHA1 (Fig. 6B).
4. Discussion 4.1. The role of DHAs in the solution pH changes and potential mechanisms treated with MWCNT It has been reported that MWCNTs can penetrate the seeds of cabbage, rice, tomato, barley, soybean and maize in hydroponic conditions, with functionalized MWCNTs directly penetrating the cells (Liné et al., 2017), which further stimulates the secretion of some organic acids via the plant roots. MWCNTs loaded with more doses of iron severely weaken the root exudation of organic acids by amaranth. One report noted that the root exudation of phenolic compounds associated with the presence of Cu in the rhizosphere depended on the Cu concentration (Meier et al., 2012). The increased pH in the presence of different DHA fractions is likely due the HAs, where they not only slowed the agglomeration between nanoparticles but also increased the bioavailability of nanoparticles to the roots (Jayalath et al., 2018) and enhanced the nanomaterial toxicity toward plants and root proton release (H+). In present study, DHAs also had influence on TOC contents in Hoagland solution. When nanomaterials MWCNT or 4:2:1 MWCNT existed, adding DHA significantly decreased the TOC content in solution (Fig. S3). However, DHAs have no effect on treatments with 1:2:1 MWCNT. The results indicated DHAs could obviously change the root exudation release when treating with different nanomaterials. 4.2. MWCNTs-induced toxicity and physiochemical response of amaranth as affected by DHAs Lower doses of MWCNTs obviously decreased the soluble protein levels in both shoots and roots, while increasing nanomaterials doses increased the soluble protein levels. This could be attributed to the release of endogenous redox-active iron. Fe2O3 nanoparticles affect the physiology of Arabidopsis, which are dependent on the coating, charge, and concentration of the nanoparticles, with only the lowest of the two concentrations (1 and 3 mg/L) found to negatively affect the pollen viability, pollen tube growth, and seed production (Bombin et al., 2015). However, another study demonstrated that tested Fe3O4 NPs have an increased negative effect on root length as the concentration increases (400, 2000, and 4000 mg/L), with the highest concentration completely inhibiting root elongation (Lee et al., 2010). Plants will therefore respond to various forms of soluble proteins at different nanomaterial stresses through an alteration in the material levels. DOM will alter the physicochemical properties and colloidal behaviors of nanoparticles (Zhang et al., 2016) by means of its high electron pair donor capacity or its high polarity, which will enhance or hamper the CNT suspension (Aksenov et al., 2007; Mac Kernan and Blau, 2008). Zhang et al. (2015) found that DOM enhanced the MWCNTs-induced to green algae toxicity. However, DOM was also reported to reduce NP toxicities, such as CeO2 nanotoxicities to plants, zero valent iron to bacteria and CeO2 to algae (Zhang et al., 2016). Therefore, DOM from diverse sources may have different effects on the algal toxicity of CNTs that merit specific investigations. Schwyzer et al. (2012) reported the higher negative zeta potential in presence of humic acid, the higher electrostatic stabilization of the CNTs by humic acid, hence, low humic
4.4. TEM observation of MWCNTs in amaranth seedlings According to Fig. 7 and Fig. S4, high ratio of irons in nanomaterials (4:2:1 MWCNT) had more negative effects on amaranth cells and chloroplasts as compared to MWCNT and 1:2:1 MWCNT. Hao et al. (2018) also found MWCNTs at 50 mg/kg and 500 mg/kg caused cortical cell damage in the rice root maturation zone. However, Joshi et al. (2018) found MWCNTs increase the cells or vascular region which might enhance the capacity of plant cells and further stimulate growth and yield. The difference among studies could be due to if the MWCNT is modified (loaded with ions or radical groups) (Liné et al., 2017), dosages used in experiments, duration of exposure and plants’ tissue sensitivity. Furthermore, DHAs, especially DHA4 combined with iron-MWCNTs has more damages to amaranth cells and more black granules suspected to MWCNTs were found in subcellular structure. The results suggested that DOM accelerate the availability and stabilization of nanomaterials in solution, and DHA4 with low MW-DOM has a better dispersion of iron-MWCNTs which enhanced the plants uptake. When 4:2:1 MWCNT combined with DHA4, damages on cell and chloroplasts became even more serious. Investigations on the changes in cell structure induced by exposure to MWCNTs combined with DHAs are scarce. Until now, only Zhang et al. (2015) reported that HA could alleviate the toxicity of MWCNTs through limiting green algae cells’ internalization of 854
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MWCNTs and reducing oxidative stress. Therefore, the present study further reveal the role of humic acids in cell structure of plants and ecological toxicity of nanomaterials.
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5. Conclusions The outcomes of this work indicate that DHAs in soil alter the toxicities of nanomaterials to plants. DHAs increased the pH of Hoagland's solutions when combined with the three nanomaterials. DHAs also severely lowered the anthocyanin levels of amaranth combined with nanomaterials compared to only nanomaterials treatments. Furtherly, relative to DHA4, DHA1 play a more important role in impairing the soluble protein and chlorophyll concentrations in shoots when treated with iron-loaded MMWCNTs. However, TEM images showed that the combination of 4:2:1 MWCNT and DHA4 had most serious effects on the plant cells than the other treatments. This study therefore provides information on how different DHA fractions influence the physiological response of plants treated with nanomaterials. However, there are still limitations in the present study. The interaction mechanism between nanomaterials and DHAs, the transportation of MWCNTs in plants under the effect of DHAs need our further exploration. Acknowledgments This work was supported by the Ministry of Science and Technology of China (18ZXSZSF00110), Natural Science Foundation of China (41673104), Tianjin Science and Technology Committee (17JCZDJC39600), Science and Technology Commission of Tianjin Binhai New Area (BHXQKJXM-PT-ZJSHJ-2017002), Fundamental Research Funds for the Central Universities, and 111 program, Ministry of Education, China (T2017002), and Tianjin Agricultrual Affair Committee, China (201604010). Supporting information Additional information including experimental design and characterization were provided in the supplementary document. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ecoenv.2018.11.072. References Ai, L., Zhang, C., Liao, F., Wang, Y., Li, M., Meng, L., Jiang, J., 2011. Removal of methylene blue from aqueous solution with magnetite loaded multi-wall carbon nanotube: kinetic, isotherm and mechanism analysis. J. Hazard Mater. 198, 282–290. Aksenov, V., Avdeev, M., Kyzyma, E., Rosta, L., Korobov, M., 2007. Effect of the age of the C 60/N-methyl-2-pyrrolidone solution on the structure of clusters in the C 60/Nmethyl-2-pyrrolidone/water system according to the small-angle neutron scattering data. Crystallogr. Rep. 52, 479–482. Aziz, N., Faraz, M., Pandey, R., Shakir, M., Fatma, T., Varma, A., Barman, I., Prasad, R., 2015. Facile algae-derived route to biogenic silver nanoparticles: synthesis, antibacterial, and photocatalytic properties. Langmuir 31, 11605–11612. Bombin, S., LeFebvre, M., Sherwood, J., Xu, Y., Bao, Y., Ramonell, K.M., 2015. Developmental and reproductive effects of iron oxide nanoparticles in Arabidopsis thaliana. Int. J. Mol. Sci. 16, 24174–24193. Chen, C., Hu, J., Shao, D., Li, J., Wang, X., 2009. Adsorption behavior of multiwall carbon nanotube/iron oxide magnetic composites for Ni (II) and Sr (II). J. Hazard Mater. 164, 923–928. Chen, J., Dong, X., Xin, Y., Zhao, M., 2011. Effects of titanium dioxide nano-particles on growth and some histological parameters of zebrafish (Danio rerio) after a long-term exposure. Aquat. Toxicol. 101, 493–499. Cornelis, G., Hund-Rinke, K., Kuhlbusch, T., Van den Brink, N., Nickel, C., 2014. Fate and bioavailability of engineered nanoparticles in soils: a review. Crit. Rev. Environ. Sci. Technol. 44, 2720–2764. Edgington, A.J., Roberts, A.P., Taylor, L.M., Alloy, M.M., Reppert, J., Rao, A.M., Mao, J., Klaine, S.J., 2010. The influence of natural organic matter on the toxicity of multiwalled carbon nanotubes. Environ. Toxicol. Chem. 29, 2511–2518.
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The role of different fractions of humic acid in the physiological response of amaranth treated with magnetic carbon nanotubes
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Weili Jiaa, Sheng Zhaib, Chuanxin Mac,d, Huimin Caoa, Cuiping Wanga, , Hongwen Suna, Baoshan Xingc a
Key Laboratory of Pollution Processes and Environmental Criteria, Ministry of Education, Tianjin Key Laboratory of Environmental Remediation and Pollution Control, College of Environmental Science and Engineering/Sino-Canada R&D Centre on Water and Environmental Safety, Nankai University, Tianjin 300071, China b School of Environment and Planning, Liaocheng University, Liaocheng 252059, China c Stockbridge School of Agriculture, University of Massachusetts, Amherst, MA 01003, USA d Department of Analytical Chemistry, The Connecticut Agricultural Experiment Station, New Haven, CT 06504, USA
A R T I C LE I N FO
A B S T R A C T
Keywords: Magnetic carbon nanotubes Humic acids Toxicity Amaranth
Dissolved humic acid (DHA) from soil can interact with multi-walled carbon nanotubes (MWCNTs) and magnetic-modified multi-walled carbon nanotubes (MMWCNTs), and subsequently alter the toxicity of MWCNTs and MMWCNTs to amaranth. This is the first study to compare the effects of MWCNTs and MMWCNTs under natural DHAs on their toxicity to amaranth. When DHAs were combined with 0.5 g/L MWCNTs, 1:2:1 MMWCNTs and 4:2:1 MMWCNTs nanomaterials, DHA1 and DHA4 both increased the pH of Hoagland's solutions. DHA1 more severely decreased the soluble protein levels in shoots than DHA4 in the 1:2:1 MMWCNT and 4:2:1 MMWCNT treatments. DHA1 and DHA4 both increased the chlorophyll concentrations of amaranth treated with MWCNTs, decreased the chlorophyll concentrations in the MMWCNT treatments. Co-exposure of DHAs and carbon-based CNTs caused further decreases in the anthocyanin level as compared to the respective CNT alone treatment. In the nanomaterial alone treatment, both 0.25 and 4:2:1 MMWCNTs greatly lowered the anthocyanin level as compared to the other two CNTs with the same exposure dose. Transmission electron microscopy images showed that the interaction between 4:2:1 MMWCNT and DHA4 had more serious effects on plant cells across all the treatments.
1. Introduction Carbon nanotubes (CNTs) have been investigated as a promising adsorbent for removing environmental pollutants and largely applied (Li et al., 2001; Ai et al., 2011) due to their large specific surface areas and hollow and layered structures. However, the recovery of these adsorbents may suffer from a difficult separation process, therefore resulting in their reduced reusability and increased regeneration costs. The recovery defect of CNTs has attracted many researchers to focus on combining CNTs and magnetic oxides to synthetize a promising magnetic carbon nanotube (Chen et al., 2009) that effectively recovers the CNTs via magnetic separation. Though some manufactured magnetic carbon nanotubes, which are similar to other engineered nanomaterials (ENMs), could still be released to the environment (water, air and/or soil) during their handling, disposal and effluent management during CNT synthesis (Cornelis et al., 2014; Aziz et al., 2015; Prasad et al., 2016; Tripathi et al., 2017).
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Once ENMs were released into the environment, their potential effects on living organisms and the non-living (abiotic) components of ecosystems should raise concerns (Hao et al., 2018). Plants are a vital component of ecological systems, serving as both important ecological receptors and potential routes for the transport and bioaccumulation of ENMs in the food chain at different trophic levels (Gardea-Torresdey et al., 2014). Studies performed using advanced detection techniques have demonstrated that ENMs can be readily absorbed by plants germinated or grown in nutrient solutions (Irin et al., 2012; Khodakovskaya et al., 2012) or soil (Rico et al., 2015) supplemented with nanosized materials, which can alter their morphoanatomical, physiological, biochemical and genetic compositions (Rico et al., 2015; Hao et al., 2016; Tripathi et al., 2016). However, there are currently very few studies that have been performed to characterize the effects of magnetic carbon nanotubes on plants. Many studies have also shown that the adsorption of dissolved organic matter (DOM) on the surface of ENMs can affect the dissolution,
Corresponding author. E-mail address: [email protected] (C. Wang).
https://doi.org/10.1016/j.ecoenv.2018.11.072 Received 21 June 2018; Received in revised form 15 November 2018; Accepted 17 November 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.
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(Nicolet iS50, Thermo Fisher Scientific Co., Beijing, China) (Fig. S2).
surface charge, and aggregation behavior of ENMs (Edgington et al., 2010). The interaction between DOM and ENMs may occur and subsequently alter the plant physiological response. Previous studies have demonstrated that DOM could diminish nanoparticle toxicity by reducing its bioavailability to micro crustaceans and bacteria ( Fabrega et al., 2009; Edgington et al., 2010; Chen et al., 2011; Jiang et al., 2011). Humic acids (HAs) are one of the most important components of organic matter, with the ability to affect seed germination and plant growth (Lee and Bartlett, 1976). However, current research on plant tolerance to magnetic carbon nanotubes, especially the physiological response of plants due to both dissolved HA (DHA) combined with magnetic carbon nanotube stress, is rare. In this study, we examined the role of different DHA fractions on the multi-walled carbon nanotubes and magnetic-modified multi-walled carbon nanotube (MMWCNT) in the toxicity of plants. We used soil HA as the aquatic DOM model because it is well distributed in the soil and directly interacts with any ENMs present. Amaranth was chosen as the plant model because it is one of the most widely planted vegetables in China. By analyzing their physiological parameters and subcellular structures in plants, the results could provide important information for evaluating the potential benefits of different soil HA fractions on the environment.
2.3. Plant preparation and experimental design Amaranth seedlings were prepared according to Ma et al. (2013). Twenty sterilized seeds were placed on each filter paper for germination and cultivated in half-strength Hoagland's solution for 20 days in a controlled environment cabinet (22 °C/18 °C, 14 h/8 h, day/night) (HH.BH-600, Tianjin Tianyu Experimental Equipment Co., Ltd, China). The uniform-sized amaranth seedlings (approximately 5 cm in length) were then transferred to Hoagland solutions for 5 days before exposure. In the exposure experiment, each amaranth seedling was grown in a 250 mL glass jar containing 230 mL Hoagland's solution mixed with 10 mg/L DHA and 0.1–0.5 g/L MWCNTs or MMWCNTs for 10 days, each treatment has three replicates. The experimental setup regarding the detailed information for each treatment is provided in Table S1. The seedlings after exposure were harvested and rinsed with deionized H2O to remove the attached particulates from the root surface. The plant roots and shoots were separately frozen in liquid nitrogen and stored in a −80 °C freezer (DW86L388A, Qingdao Haier Electric Appliance Co., Ltd., China) until further analysis. Parts of the roots and shoots were freeze-dried for biomass and element analysis. The rest of the samples remained at −80 °C for the antioxidant enzymes activity analysis.
2. Materials and methods 2.1. MMWCNT preparation and characterization
2.4. Soluble protein and pigment measurement Multi-walled carbon nanotubes (MWCNTs), with 95% purity, a 20–30 nm outer diameter, and 10–30 µm length, were purchased from Xianfeng Nano Material Co. Ltd., China. Analytical grade chemicals including FeCl3·6H2O, FeCl2·4H2O and NH3·H2O were obtained from the Tianjin Yingdaxigui Chemical Reagent Co. Ltd., China. The MWCNT was acidified before loading Fe elements. Briefly, raw MWCNTs were purified with 1 M acidic solution (H2SO4: HNO3; v/v = 3:1) for 24 h before sonicated for 20 min. The mixture was then heated to 100 °C for 40 min. After cooling, the solution was washed to pH was 7.0 and filtered. The precipitates were collected and dried for 12 h at 100 °C in an oven, and stored MWCNTs in a desiccator for further use. Then the magnetic carbon nanotubes were prepared according to the following procedure. 200 mg acidified MWCNTs, 200 mg FeCl2 and 400 mg FeCl3 were added to a 200 mL flask containing 80 mL deionized water. The mixture was sonicated for 30 min and then heated under magnetic stirring at 70 °C, along with a peristaltic pump slowly pumped 6% NH3·H2O (30 mL) to the mixture. The mixture was then cooled naturally to room temperature (25 °C), filtered through a 0.45 µm filter, and washed with deionized water until the pH of the filtrate was approximately 7.0. The dark precipitates were collected and dried for 24 h at 50 °C in an oven. This process yielded 1:2:1MMWCNT (FeCl2·4H2O: FeCl3·6H2O: MWCNT) nanomaterials. The synthesized 4:2:1MMWCNT samples were obtained following the above mentioned procedure and a FeCl2·4H2O: FeCl3·6H2O: MWCNT mass ratio of 4:2:1. All the samples characterized in Fig. 1 were observed using a transmission electron microscope (TEM) (JEM-2010, JEOL, Japan) operating at 20 kV. Fe(II) and Fe(III) in samples were characterized by X-ray photoelectron spectrum (XPS) (ESCALAB 250Xi, Thermo Scientific, USA) in Fig. 2.
The soluble protein was measured according to Singh and Lee (2016). The chlorophyll and anthocyanin concentrations were analyzed using a modified method (Ma et al., 2013; Wang et al., 2017). A portion of 20 mg fresh leaves was incubated in 10 mL 95% ethanol in the dark for 3 days. The absorbance of supernatant was measured at 664.2 and 648.6 nm using a UV−Vis spectrophotometer (TU-1810, Persee, China). The total chlorophyll concentrations were calculated using the following equations: Chla = 13.36A664.2 − 5.19A648.6, Chlb = 27.43A648.6 − 8.12A664.2, Total chlorophyll = Chla + Chlb. The anthocyanin concentrations were measured as follows. 100 mg amaranth leaves were ground in liquid nitrogen and then mixed with 2 mL 1% (v/v) HCl acidified methanol prior to incubate in the dark at 4 °C overnight. Then deionized H2O2 (1 mL) and chloroform (1 mL) were added and mixed. The mixture was centrifuged at 13,000 g for 2 min at 4 °C. The absorbance of the supernatant was measured at 530 and 657 nm and the final anthocyanin concentration was determined by the formula A530 − 1/4 A657. 2.5. TEM observation The cell and chloroplast morphology was observed by a transmission electron microscopy (TEM; Hitachi H-7650, Hitachi Corp., Tokyo, Japan) at 180 kV (Nhan et al., 2015). Briefly, the fresh amaranth leaves were washed with deionized water, the apex were excised and prefixed in 2.5% glutaraldehyde, dehydrated in a graded acetone series, and embedded in Spurr's resin. The fixed samples were sectioned using a microtome JEM-1230 (JEOL, Ltd., Japan) with a diamond knife for TEM analysis.
2.2. Preparation and characterization of different DHA fractions 2.6. Statistical analysis Soil was sampled and prepared as what we reported before (Li et al., 2016). Different fractions of DHAs were sequentially extracted from the soil and characterized follow the approach of Wang et al. (2017). DHAs were characterized by scanning electron microscopy (SEM; S-3500N, Hitachi Co., Tokyo, Japan) at 20.0 kv × 20 k (Fig. S1); Fourier-transform infrared spectra was recorded from the DHAs in the range of 4000–400 cm−1 with a resolution of 2 cm−1 by FTIR spectroscopy
The values in each assay were determined as the mean ± standard deviation of three replicates. All data analysis was performed using the SPSS version 18.0 software package. A one-way analysis of the variance followed by a least significant difference (LSD) test was used to determine the statistical significance (p ≤ 0.05 or p < 0.01) of each parameter among all the treatments. 849
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Fig. 1. The SEM images of unmodified MWCNTs (a), 1:2:1 MMWCNTs (b) and 4:2:1 MMWCNTs (c). Arrow: MWCNT; COM: MWCNT/iron oxide magnetic compositions. Measurements are of three replicates. (Note: 1:2:1 MMWCNT refer to the mole ratios of Fe2+: Fe3+: MWCNT= 1:2:1; 4:2:1 MMWCNT refer to the mole ratios of Fe2+: Fe3+: MWCNT= 4:2:1).
3. Results
44% Fe(II) (710.8 eV binding energy) and 56% Fe(III) (712.5 eV). The iron spectrum clearly showed 62% Fe(II) and 38% Fe(III) on the 4:2:1 MMWCNT surface satellited at 710.9 and 712.8 eV, respectively. Therefore, the results further confirmed that different Fe concentrations were loaded with MWCNTs.
3.1. Magnetic carbon nanotube characterization SEM images of the oxidized MWCNTs and two kinds of MMWCNT composites are shown in Fig. 1. An entangled network of oxidized MWCNTs, with clusters of iron oxides attached to the MWCNTs (Fig. 1B), suggests the formation of MWCNT/iron oxide magnetic composites. The SEM image of the composites (Fig. 1C) shows that a high density of composites was loaded onto the network structure of the MMWCNTs. X-ray photoelectron spectrometry (XPS) of the iron-loaded MWCNTs is shown in Fig. 2. The spectrum in the Fe 2p3/2 region indicates that the iron on the 1:2:1 MMWCNT surface is approximately
Fe(III)
3.2. pH values of the Hoagland solutions Fig. 3 shows that the addition of three nanomaterials, MWCNT, 1:2:1 MMWCNT and 4:2:1 MMWCNT, has different effects on the Hoagland solution pH values. Exposure to a 0.1 g/L three-nanomaterial dose resulted in statistically significant increases in the Hoagland solution pH values relative to the control plants (Fig. 3A), with the low-tohigh ranking of the materials being as follows: Control group (pH
a
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b Fe(II)
Fig. 2. X-ray photoelectron spectrum of two magnetic carbon nanotubes. Measurements are of three replicates (n = 3). (a: 4:2:1MMWCNT; b: 1:2:1MMWCNT). 850
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Fig. 3. pH changes of Hoagland's solutions for amaranth growth as affected by different doses of MWCNTs and 1:2:1 MMWCNTs and 4:2:1 MMWCNTs (A) and the relationship between doses of three nanomaterials and pH values (B), and 0.5 g/L of MWCNTs and 1:2:1 MMWCNTs and 4:2:1 MMWCNTs combined with DHAs (C). Data are mean ± standard error of three replicates (n = 3). Values followed by different word are significantly different at p ≤ 0.05.
≈ 5.0) < MWCNT < 1:2:1 MWCNT < 4:2:1 MWCNT (pH ≈ 6.0). Exposure to a 0.25 g/L three-nanomaterial dose did not yield obvious changes in the Hoagland solution pH values relative to the untreated control plants (Fig. 3A), whereas exposure to a 0.5 g/L three nanomaterial dose yielded a significant decrease in the pH values of the solution, especially 4:2:1 MWCNT. The solution pH values therefore decreased with increasing exposure doses of the three nanomaterials. In this study, a decrease in the pH of the collection solutions was negatively correlated with an increase in the three-nanomaterial doses (Fig. 3B). The relationship coefficients for pH and MWCNT, 1:2:1 MWCNT, 4:2:1 MWCNT were 0.9313, 0.9998 and 1.0, respectively. The addition of different DHA fractions to the 0.5 g/L MWCNT, 1:2:1 MWCNT and 4:2:1 MWCNT-amended solution yielded pH values that were approximately 2.0 higher than the corresponding three-material treatments (Fig. 3C). The pH changes were dependent on the DHA addition, but they were virtually independent of the DHA fractions.
while the concentration was 11.92 ± 1.19 mg/g in the control root group (Fig. 4B). Therefore, the soluble proteins levels in the CNT treatment groups did not increase with the increase in MWCNT doses for the same material, while The soluble proteins levels in 1:2:1 MMWCNT and 4:2:1 MMWCNT increased as the corresponding material doses were increased. DHA1 and DHA4 combined with MWCNT did not decrease the soluble protein levels in shoots when different DHA fractions were added to the 0.5 g/L MWCNT-amended solution (Fig. 4C). However, DHA1 and DHA4 combined with 0.5 g/L 1:2:1 MWCNT decreased the soluble protein levels in shoots compared to the 1:2:1 MWCNT only treatments, with DHA1 yielding a more obvious decline in soluble protein levels relative to DHA4 (Fig. 4C). Similarly, DHA1 combined with 0.5 g/L 4:2:1 MWCNT decreased the soluble protein levels in shoots compared to the 4:2:1 MWCNT only treatments, whereas DHA4 had no obvious impact (Fig. 4C). The DHA effects on the soluble protein trends in the roots were the opposite to those in the shoots (Fig. 4D). For example, DHAs combined with 0.5 g/L three-material doses increased the soluble protein levels in the roots compared to the MWCNT, 1:2:1 MWCNT and 4:2:1 MWCNT only treatments. The order of the soluble protein levels in the roots was: DHA1 + MWCNT > DHA4 + MWCNT > Control > MWCNT, DHA1 + 4:2:1 MWCNT > DHA4 + 4:2:1 MWCNT > Control > 4:2:1 MWCNT, whereas the order in the shoots was: DHA4 + 1:2:1 MWCNT > DHA1 + 1:2:1 MWCNT > 1:2:1 MWCNT > Control. DHA1 therefore increased the 1:2:1 MWCNT and 4:2:1 MWCNT toxicities to the amaranth shoots more than DHA4 did (Fig. 4C).
3.3. Soluble proteins concentrations in shoots and roots There was a decline in the soluble protein levels in the shoots when the MWCNT, 1:2:1 MMWCNT and 4:2:1 MMWCNT concentrations increased from 0.1 to 0.5 g/L. For example, the 0.1, 0.25 and 0.5 g/L MWCNT concentrations reduced the soluble protein levels by 29.9%, 32.2% and 32.6%, whereas the 1:2:1 MWCNT concentrations reduced them by 30.5%, 22.2% and 15.1%, and the 4:2:1 MMWCNT concentrations reduced them by 39.3%, 67.1% and 12.8% relative to the control, respectively (Fig. 4A). The current order of the soluble proteins for the 0.1 and 0.25 g/L three-nanomaterial doses are as follows: Control > 1:2:1 MWCNT ≈ CNT > 4:2:1 MWCNT, whereas the order for the 0.5 g/L dose is: Control > 4:2:1 MMWCNT > 1:2:1 MMWCNT > CNT. The soluble protein levels in roots treated with 0.1, 0.25 and 0.5 g/L nanomaterials were: 9.33 ± 0.13, 8.97 ± 0.19 and 7.43 ± 0.98 mg/ g, respectively (MWCNT); 6.63 ± 1.31, 8.57 ± 0.50 and 13.22 ± 1.36 mg/g, respectively (1:2:1 MMWCNT); 6.51 ± 0.20, 6.47 ± 0.69 and 10.43 ± 1.22 mg/g, respectively (4:2:1 MMWCNT),
3.4. Chlorophyll concentrations in shoots Different doses of the same nanomaterial had varying effects on chlorophyll concentration. Amaranth exposed to 0.1, 0.25 and 0.5 g/L MWCNT yielded chlorophyll values of 2.91 ± 0.09, 5.0 ± 0.91 and 2.54 ± 0.29 mg/g, while the control concentration was 4.0 ± 0.10 mg/g. The chlorophyll concentration changes were therefore related to the MWCNT doses, with the appropriate MWCNT dose (0.25 g/L) helpful in amaranth photosynthesis. Amaranth exposed to 851
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Fig. 4. Soluble protein content in amaranth shoots (A) and roots (B) as affected by different doses of MWCNTs, 1:2:1 MMWCNTs and 4:2:1 MMWCNTs; soluble proteins in shoots (C) and roots (D) as affected by 0.5 g/L of MWCNTs and MMWCNTs combined with DHAs. Data are mean ± standard error of three replicates (n = 3). Values followed by different word are significantly different at p ≤ 0.05.
influence on the chlorophyll concentration of amaranth treated with MWCNT. When different DHA fractions were added to the 0.5 g/L 1:2:1 MWCNT-amended solution, DHA1 and DHA4 combined with 1:2:1 MWCNT or 4:2:1 MWCNT yielded obvious declines in chlorophyll concentrations compared to the 1:2:1 MWCNT or 4:2:1 MWCNT only treatments.
0.1, 0.25 and 0.5 g/L 1:2:1 MWCNT gave the highest chlorophyll values of 2.57 ± 0.24, 2.28 ± 0.14 and 4.13 ± 0.14 mg/g, respectively. The chlorophyll concentrations therefore increased with increasing 1:2:1 MWCNT doses. Amaranth exposed to 0.1, 0.25 and 0.5 g/L 4:2:1 MWCNT gave the highest chlorophyll values of 5.34 ± 0.36, 4.59 ± 0.08 and 3.09 ± 0.34 mg/g, respectively, with a decline in the chlorophyll concentrations as the 4:2:1 MWCNT doses were increased. The chlorophyll produced by amaranth was therefore influenced by the iron doses loaded with MWCNTs. Different nanomaterials at the same dosage also had varying effects on chlorophyll concentration. The resultant chlorophyll concentrations were ordered as follows when the amaranth exposure dose was 0.1 g/L: 1:2:1 MWCNT ≤ MWCNT < Control < 4:2:1 MWCNT. When the exposure dose was 0.25 g/L, the chlorophyll concentration order was: 1:2:1 MMWCNT < Control < 4:2:1 MMWCNT ≤ MWCNT. When the exposure dose was 0.5 g/L, the chlorophyll concentration order was: MWCNT < 4:2:1 MWCNT < 1:2:1 MWCNT ≤ Control. DHAs significantly (p < 0.05) altered the chlorophyll concentration of amaranth when different DHA fractions were added to the 0.5 g/ L MWCNT-amended solution (Fig. 5B). DHA1 combined with MWCNT yielded an obvious increase in chlorophyll compared to the MWCNT only treatments, whereas the presence of DHA4 had no obvious
3.5. Anthocyanin concentrations in shoots Amaranth exposed to 0.1, 0.25 and 0.5 g/L MWCNT yielded significant declines (p < 0.05) in anthocyanin compared to the control (Fig. 6A). Amaranth exposed to 0.1, 0.25 and 0.5 g/L 1:2:1 MWCNT also yielded decreased anthocyanin concentrations. Furthermore, the extent of the decreased anthocyanin concentrations in amaranth diminished with the increase in MWCNT and 1:2:1 MWCNT doses. The anthocyanin values decreased the most by increasing the 4:2:1 MWCNT doses (Fig. 6A), even though amaranth exposed to 0.1, 0.25 and 0.5 g/L 4:2:1 MWCNT yielded significant declines (p < 0.05) in anthocyanin concentrations. The anthocyanin concentrations were therefore impacted by the nanomaterial doses. Different nanomaterials at the same dosage also had varying effects on the anthocyanin concentrations. When amaranth was exposed to a Fig. 5. Total chlorophyll content of amaranth as affected by different doses of MWCNTs and 1:2:1 MMWCNTs and 4:2:1 MMWCNTs (A) and 0.5 g/L of MWCNTs and MMWCNTs combined with DHAs (B). Data are mean ± standard error of three replicates (n = 3). Values followed by different word are significantly different at p ≤ 0.05.
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Fig. 6. Anthocyanin level of amaranth as affected by different doses of MWCNTs and 1:2:1 MMWCNTs and 4:2:1 MMWCNTs (A) and 0.5 g/L of MWCNTs and MMWCNTs combined with DHAs (B). Data are mean ± standard error of three replicates (n = 3). Values followed by different word are significantly different at p ≤ 0.05.
0.1 g/L dose, the anthocyanin concentration order was: CNT ≤ 1:2:1 MWCNT ≤ 4:2:1 MWCNT < Control. When the exposure dose was 0.25 g/L, the anthocyanin concentration order was: 4:2:1 MWCNT ≤ CNT < 1:2:1 MWCNT < Control. When the exposure dose was 0.5 g/L, the anthocyanin concentration order was: 4:2:1 MWCNT < CNT < 1:2:1 MWCNT < Control. The anthocyanin produced by amaranth was therefore influenced by the nanomaterial properties. The DHAs significantly (p < 0.05) changed the anthocyanin of amaranth (Fig. 6B) when different DHA fractions were added to the 0.5 g/L MWCNT-amended solution. DHA1 or DHA4 combined with MWCNT both yielded obvious declines in the anthocyanin concentrations compared to the MWCNT only treatment, whereas there were no differences between the influence of DHA1 and DHA4 on anthocyanin in amaranth treated with MWCNT. When different DHA fractions were added to the 0.5 g/L 1:2:1 MWCNT-amended solution, DHA1 and DHA4 combined with 1:2:1 MWCNT or 4:2:1 MWCNT yielded obvious declines in the anthocyanin concentrations compared to the 1:2:1 MWCNT or 4:2:1 MWCNT only treatments, with DHA4 yielding a more obvious decline than DHA1 in the 1:2:1 MWCNT treatment. It is a remarkable fact that anthocyanin was not detected in the treatments where DHA1 and DHA4 were combined with 4:2:1 MWCNT.
A
B
3.6. Plant cell structure characterized by transmission electron microscopy (TEM) Intact amaranth shoot and root cells were observed in the control group (Fig. 7A and E). The plant cells were also intact and undeformed after the MWCNT, MWCNT combined with DHAs, and 1:2:1 MWCNT treatments (not given). Furthermore, the internal structure of the grana lamella and stroma lamella in the chloroplast was intact and undeformed. These results indicated that MWCNT, MWCNT combined with DHAs, and 1:2:1MMWCNT treatments have no obvious negative effects on the plant cells and photosynthesis. However, the chloroplast was deformed and starch grains formed in the chloroplast when 1:2:1 MMWCNT combined with DHAs were added to the solution, and the grana lamella and stoma lamella became distorted (Figs. 7B, S4B and S4E). Some black granules, which were suspected to be iron particles or MMWCNTs, were found in the intercellular spaces (Fig. 7 B and F). These results imply that the 1:2:1MMWCNTs exerted an adverse effect on the plant cell and chloroplast when they interacted with the DHAs, which was likely due to the DHAs enhancing the biotoxicity of MWCNTs when in the presence of excess iron. The 4:2:1MMWCNT alone treatments also have an effect on the plant cell and chloroplast (Fig. S4F). For example, some small starch grains formed in the chloroplast, with slight deformation of the interior chloroplast structure. The deformation of the cell and chloroplast became even more serious when 4:2:1 MWCNT interacted with the DHAs (Fig. 7 C, D and S4 C, D). More and larger starch grains formed in the chloroplast, which seriously
C
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D
BG
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SG BG
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H
BG BG BG
Fig. 7. TEM images of amaranth chloroplast and roots cells among different treatments. Figure A, B, C and D represents the chloroplast structure of amaranth leaves upon exposure to control, 1:2:1 MMWCNT+DHA4, 4:2:1 MMWCNT+DHA1, 4:2:1 MMWCNT+DHA4, respectively. Figure E, F, G and H represent cellular structure of amaranth roots upon exposure to control, 1:2:1 MMWCNT+DHA4, 4:2:1 MMWCNT+DHA1, 4:2:1 MMWCNT+DHA4, respectively. Measurements are of three replicates. SL: stroma lamella; SG: starch grain; BG: black granules. 853
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acids concentrations may destabilize CNTs over HA-bridging flocculation. 10 mg/L DHAs to 0.5 g/L iron-MWCNT ratio in our study sufficiently promoted the iron-MWCNT availability. It was very likely that dissolved iron metals act as bridging agents between DHAs and MWCNT surfaces provided a more stable suspension of iron-MWCNT.
impaired photosynthesis. Furthermore, some black granules appeared around the shoot and root cell spaces (Fig. 7 C, D, G and H), likely due to the addition of DHAs enhancing the bioavailability of 4:2:1MMWCNT transferred to the plant cell. This is also supported by the total chlorophyll in Fig. 5. For example, 4:2:1MMWCNT combined with or without DHAs, all yielded significant declines in the chlorophyll concentration. The starch grains and black granules from the DHA4 treatment were more obvious than those from the DHA1 treatment, therefore indicating that the interaction between 4:2:1 MMWCNT and DHA4 had a more serious effect on the plant cells.
4.3. MWCNTs and DHAs caused effects on pigments contents Few studies have compared the effects of MWCNT coated with different doses of iron on plants. In our study, the chlorophyll produced by amaranth was generally affected by the iron-nanomaterial properties (Fig. 5A). The change in chlorophyll concentrations highlighted a fact that the iron in MMWCNTs was capable of either inhibiting or enhancing chlorophyll synthesis mainly depends on irons concentration. Besides, different DHA fractions played different roles in chlorophyll synthesis that were also dependent on the iron concentrations of the MWCNTs/MMWCNTs (Fig. 5B). The dissolved irons may play an important role by work as bridging agents in dominating the MMWCNT stabilization and bioavailability. All doses of nanomaterials decreased the anthocyanin contents as compared to control. However, with dose increasing, the diminishing effects decreased when applied with MWCNT and 1:2:1 MWCNT, while such trend was not obvious in 4:2:1 MWCNT treatments (Fig. 6A). Besides, the nanomaterials properties also affect the anthocyanin production of amaranth because that a varying effects existed between different materials at the same dosage. Biochemical changes induced by MWCNT-based modifications in certain ions, such as Fe2+ or Fe3+, or cationic exchange in the amaranth cell wall matrix were likely to alter the antioxidant defense of the plants. Different fractions of DHA played varying roles in decreasing the anthocyanin produced by amaranth (Fig. 6B). Studies involving dissolved organic matter (DOM) have reported DOM to be an effective suspending agent for CNTs (Hyung et al., 2007). This means that MWCNTs and MMWCNTs will be suspended by DOM, resulting in the easy transport of MWCNTs and MMWCNTs to amaranth, which further enhances their toxicity to amaranth. Shen et al. (2015) found that highmolecular-weight (MW) DOM stabilizes nC60 in NaCl more effectively than its low-MW counterparts. Therefore, we conclude that DHA1 with high-MW DOM could stabilize MWCNTs and MMWCNTs in comparison to DHA4 with low-MW DOM, with DHA4 elevating the MWCNT and MMWCNT toxicities considerably more than DHA1 (Fig. 6B).
4. Discussion 4.1. The role of DHAs in the solution pH changes and potential mechanisms treated with MWCNT It has been reported that MWCNTs can penetrate the seeds of cabbage, rice, tomato, barley, soybean and maize in hydroponic conditions, with functionalized MWCNTs directly penetrating the cells (Liné et al., 2017), which further stimulates the secretion of some organic acids via the plant roots. MWCNTs loaded with more doses of iron severely weaken the root exudation of organic acids by amaranth. One report noted that the root exudation of phenolic compounds associated with the presence of Cu in the rhizosphere depended on the Cu concentration (Meier et al., 2012). The increased pH in the presence of different DHA fractions is likely due the HAs, where they not only slowed the agglomeration between nanoparticles but also increased the bioavailability of nanoparticles to the roots (Jayalath et al., 2018) and enhanced the nanomaterial toxicity toward plants and root proton release (H+). In present study, DHAs also had influence on TOC contents in Hoagland solution. When nanomaterials MWCNT or 4:2:1 MWCNT existed, adding DHA significantly decreased the TOC content in solution (Fig. S3). However, DHAs have no effect on treatments with 1:2:1 MWCNT. The results indicated DHAs could obviously change the root exudation release when treating with different nanomaterials. 4.2. MWCNTs-induced toxicity and physiochemical response of amaranth as affected by DHAs Lower doses of MWCNTs obviously decreased the soluble protein levels in both shoots and roots, while increasing nanomaterials doses increased the soluble protein levels. This could be attributed to the release of endogenous redox-active iron. Fe2O3 nanoparticles affect the physiology of Arabidopsis, which are dependent on the coating, charge, and concentration of the nanoparticles, with only the lowest of the two concentrations (1 and 3 mg/L) found to negatively affect the pollen viability, pollen tube growth, and seed production (Bombin et al., 2015). However, another study demonstrated that tested Fe3O4 NPs have an increased negative effect on root length as the concentration increases (400, 2000, and 4000 mg/L), with the highest concentration completely inhibiting root elongation (Lee et al., 2010). Plants will therefore respond to various forms of soluble proteins at different nanomaterial stresses through an alteration in the material levels. DOM will alter the physicochemical properties and colloidal behaviors of nanoparticles (Zhang et al., 2016) by means of its high electron pair donor capacity or its high polarity, which will enhance or hamper the CNT suspension (Aksenov et al., 2007; Mac Kernan and Blau, 2008). Zhang et al. (2015) found that DOM enhanced the MWCNTs-induced to green algae toxicity. However, DOM was also reported to reduce NP toxicities, such as CeO2 nanotoxicities to plants, zero valent iron to bacteria and CeO2 to algae (Zhang et al., 2016). Therefore, DOM from diverse sources may have different effects on the algal toxicity of CNTs that merit specific investigations. Schwyzer et al. (2012) reported the higher negative zeta potential in presence of humic acid, the higher electrostatic stabilization of the CNTs by humic acid, hence, low humic
4.4. TEM observation of MWCNTs in amaranth seedlings According to Fig. 7 and Fig. S4, high ratio of irons in nanomaterials (4:2:1 MWCNT) had more negative effects on amaranth cells and chloroplasts as compared to MWCNT and 1:2:1 MWCNT. Hao et al. (2018) also found MWCNTs at 50 mg/kg and 500 mg/kg caused cortical cell damage in the rice root maturation zone. However, Joshi et al. (2018) found MWCNTs increase the cells or vascular region which might enhance the capacity of plant cells and further stimulate growth and yield. The difference among studies could be due to if the MWCNT is modified (loaded with ions or radical groups) (Liné et al., 2017), dosages used in experiments, duration of exposure and plants’ tissue sensitivity. Furthermore, DHAs, especially DHA4 combined with iron-MWCNTs has more damages to amaranth cells and more black granules suspected to MWCNTs were found in subcellular structure. The results suggested that DOM accelerate the availability and stabilization of nanomaterials in solution, and DHA4 with low MW-DOM has a better dispersion of iron-MWCNTs which enhanced the plants uptake. When 4:2:1 MWCNT combined with DHA4, damages on cell and chloroplasts became even more serious. Investigations on the changes in cell structure induced by exposure to MWCNTs combined with DHAs are scarce. Until now, only Zhang et al. (2015) reported that HA could alleviate the toxicity of MWCNTs through limiting green algae cells’ internalization of 854
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MWCNTs and reducing oxidative stress. Therefore, the present study further reveal the role of humic acids in cell structure of plants and ecological toxicity of nanomaterials.
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5. Conclusions The outcomes of this work indicate that DHAs in soil alter the toxicities of nanomaterials to plants. DHAs increased the pH of Hoagland's solutions when combined with the three nanomaterials. DHAs also severely lowered the anthocyanin levels of amaranth combined with nanomaterials compared to only nanomaterials treatments. Furtherly, relative to DHA4, DHA1 play a more important role in impairing the soluble protein and chlorophyll concentrations in shoots when treated with iron-loaded MMWCNTs. However, TEM images showed that the combination of 4:2:1 MWCNT and DHA4 had most serious effects on the plant cells than the other treatments. This study therefore provides information on how different DHA fractions influence the physiological response of plants treated with nanomaterials. However, there are still limitations in the present study. The interaction mechanism between nanomaterials and DHAs, the transportation of MWCNTs in plants under the effect of DHAs need our further exploration. Acknowledgments This work was supported by the Ministry of Science and Technology of China (18ZXSZSF00110), Natural Science Foundation of China (41673104), Tianjin Science and Technology Committee (17JCZDJC39600), Science and Technology Commission of Tianjin Binhai New Area (BHXQKJXM-PT-ZJSHJ-2017002), Fundamental Research Funds for the Central Universities, and 111 program, Ministry of Education, China (T2017002), and Tianjin Agricultrual Affair Committee, China (201604010). Supporting information Additional information including experimental design and characterization were provided in the supplementary document. Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ecoenv.2018.11.072. References Ai, L., Zhang, C., Liao, F., Wang, Y., Li, M., Meng, L., Jiang, J., 2011. Removal of methylene blue from aqueous solution with magnetite loaded multi-wall carbon nanotube: kinetic, isotherm and mechanism analysis. J. Hazard Mater. 198, 282–290. Aksenov, V., Avdeev, M., Kyzyma, E., Rosta, L., Korobov, M., 2007. Effect of the age of the C 60/N-methyl-2-pyrrolidone solution on the structure of clusters in the C 60/Nmethyl-2-pyrrolidone/water system according to the small-angle neutron scattering data. Crystallogr. Rep. 52, 479–482. Aziz, N., Faraz, M., Pandey, R., Shakir, M., Fatma, T., Varma, A., Barman, I., Prasad, R., 2015. Facile algae-derived route to biogenic silver nanoparticles: synthesis, antibacterial, and photocatalytic properties. Langmuir 31, 11605–11612. Bombin, S., LeFebvre, M., Sherwood, J., Xu, Y., Bao, Y., Ramonell, K.M., 2015. Developmental and reproductive effects of iron oxide nanoparticles in Arabidopsis thaliana. Int. J. Mol. Sci. 16, 24174–24193. Chen, C., Hu, J., Shao, D., Li, J., Wang, X., 2009. Adsorption behavior of multiwall carbon nanotube/iron oxide magnetic composites for Ni (II) and Sr (II). J. Hazard Mater. 164, 923–928. Chen, J., Dong, X., Xin, Y., Zhao, M., 2011. Effects of titanium dioxide nano-particles on growth and some histological parameters of zebrafish (Danio rerio) after a long-term exposure. Aquat. Toxicol. 101, 493–499. Cornelis, G., Hund-Rinke, K., Kuhlbusch, T., Van den Brink, N., Nickel, C., 2014. Fate and bioavailability of engineered nanoparticles in soils: a review. Crit. Rev. Environ. Sci. Technol. 44, 2720–2764. Edgington, A.J., Roberts, A.P., Taylor, L.M., Alloy, M.M., Reppert, J., Rao, A.M., Mao, J., Klaine, S.J., 2010. The influence of natural organic matter on the toxicity of multiwalled carbon nanotubes. Environ. Toxicol. Chem. 29, 2511–2518.
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