Amelioration of arsenic toxicity in rice: Comparative effect of inoculation of Chlorella vulgaris and Nannochloropsis sp. on growth, biochemical changes and arsenic uptake

Ecotoxicology and Environmental Safety 124 (2016) 68–73

Contents lists available at ScienceDirect

Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv

Amelioration of arsenic toxicity in rice: Comparative effect of inoculation of Chlorella vulgaris and Nannochloropsis sp. on growth, biochemical changes and arsenic uptake A.K. Upadhyay a, N.K. Singh b, R. Singh a, U.N. Rai a,n a b

Plant Ecology and Environmental Science Division, CSIR-National Botanical Research Institute, Lucknow 226001, India Institute of Environment and Sustainable Development, Banaras Hindu University, Varanasi 221005, India

art ic l e i nf o

a b s t r a c t

Article history: Received 9 May 2015 Received in revised form 30 September 2015 Accepted 4 October 2015

The present study was conducted to assess the responses of rice (Oryza sativa L. var. Triguna) by inoculating alga; Chlorella vulgaris and Nannochlropsis sp. supplemented with As(III) (50 mM) under hydroponics condition. Results showed that reduced growth variables and protein content in rice plant caused by As toxicity were restored in the algae inoculated plants after 7 d of treatment. The rice plant inoculated with Nannochloropsis sp. exhibited a better response in terms of increased root, shoot length and biomass than C. vulgaris under As(III) treatment. A significant reduction in cellular toxicity (thiobarbituric acid reactive substances) and antioxidant enzyme (SOD, APX and GR) activities were observed in algae inoculated rice plant under As(III) treatment in comparison to uninoculated rice. In addition, rice treated with As(III), accumulated 35.05 mg kg
1 dw arsenic in the root and 29.96 mg kg
1 dw in the shoot. However, lower accumulation was observed in As(III) treated rice inoculated with C. vulgaris (24.09 mg kg
1 dw) and Nannochloropsis sp. (20.66 mg kg
1 dw) in the roots, while in shoot, it was 20.10 mg kg
1 dw and 11.67 mg kg
1 dw, respectively. Results demonstrated that application of these algal inoculum ameliorates toxicity and improved tolerance in rice through reduced As uptake and modulating antioxidant enzymes. Thus, application of algae could provide a low-cost and eco-friendly mitigation approach to reduce accumulation of arsenic in edible part of rice as well as higher yield in the As contaminated agricultural field. & 2015 Elsevier Inc. All rights reserved.

Keywords: Rice As(III) Chlorella vulgaris Nannochloropsis sp. Antioxidant enzyme

1. Introduction Inorganic arsenic (iAs) is a toxic groundwater contaminant of geogenic origin, particularly in large deltas and along major rivers in the deprived regions of South and East-Asia (Sharma et al., 2014). Arsenic is present predominantly as arsenate (As(V)) in the aerobic environment and as arsenite (As(III)) under anaerobic or waterlogged conditions (Lomax et al., 2011). However, different organic species such as methylarsenate [MAs(V)], dimethylarsenate [DMAs(V) or cacodylate] and trimethylarsine oxide [TMAsO (V)] of As have been also reported (Yin et al., 2011). Besides, use of organic As compound like ROX (4-hydroxyl-3-nitrophenylarsenic acid) and p ASA (4-aminophenylarsonic acid) in agricultural application poses toxicity and risk to human health (Zheng et al., 2014). Recent studies have shown that contamination of arsenic (As) in paddy soils is a widespread problem due to irrigation of Asladen groundwater and mining activities or uses of arsenical n

Corresponding author. Fax: þ91 522 2205836/39. E-mail address: [email protected] (U.N. Rai).

http://dx.doi.org/10.1016/j.ecoenv.2015.10.002 0147-6513/& 2015 Elsevier Inc. All rights reserved.

agrochemicals in other regions of the world (Williams et al., 2007; Zhu et al., 2008; Brammer, 2009). Although the main source of As exposures are drinking water and the food supply (Guha Mazumder et al., 2014), concern is growing over the human exposure of As through dietary consumption of rice (Meharg, 2004; Smith et al., 2009). Rice, being particularly efficient in assimilating arsenic from paddy soils becomes part of the food chain and develops arrays of diseases such as keratosis, cancer, cerebrovascular disease, diabetes mellitus, and kidney disease resulting into slow and painful death (Ma et al., 2008; Jomova et al., 2011; Rossman and Klein, 2011). In addition, the toxicity associated with organoarsenic compounds depends on associated organic functional groups and biological or environmental induced biotransformation (Zheng et al., 2014). The accumulation of As in rice may be attributed to two main factors: the reductive mobilization of As(III) in anaerobic paddy soils (Xu et al., 2008) and uptake of arsenite via the silicic acid pathway in rice (Ma et al., 2008). Plants exposed to As, develop reactive oxygen species (ROS) leading to photosynthetic pigment degradation, lipid peroxidation, electrolyte leakage and DNA damage (Rai et al., 2011). Malondialdehyde, a common oxidation product of polyunsaturated fatty acids, refers

A.K. Upadhyay et al. / Ecotoxicology and Environmental Safety 124 (2016) 68–73

as TBARS has been considered as a marker of oxidative damage and commonly used to determine the stress level in the plants. To overcome the ROS toxicity, plants are equipped with various enzymatic and non-enzymatic antioxidant defense systems to reduce the stress level (Upadhyay et al., 2014). Thus, rice being an important dietary component for many people in the world, its quality needs to be assured. Algae are an important component of aquatic environments and soil and play major role in bio-geochemical cycle (Ye et al., 2012). Recently, algae have received much attention due to their ability to absorption, sequestration and capacity to synthesize phytochelatins and metallothioneins that can form complexes of heavy metals and translocate them into vacuoles (Suresh and Ravishankar, 2004). Uptake of toxic elements by algae is basically dependent on the process of adsorption and metabolism-dependent active uptake (Lomax et al., 2011). Competence of green algae for metal accumulation is considered as a more attractive and potential technique for the restoration of metal contaminated water and soil. Chlorella vulgaris is widely distributed microalgae found mostly in fresh, stagnant and differently contaminated water and waste water, having a short life cycle and easier to handle in the laboratory (Rai et al., 2013). Nannochloropsis sp. is photoautotrophic, spherical, fast-growing and comparatively smaller than C. vulgaris (Sukenik et al., 2009; Kilian et al., 2011). However, to the extent of our knowledge, no work has been done about how algalization assists in As amelioration in rice. In this study, work is focused on comparative As tolerance and uptake in the rice plant inoculated with the C. vulgaris and Nannochloropsis sp. to assess their potential for amelioration of As toxicity.

69

Hewitt nutrient medium served as control. The experiments were conducted in aseptic laboratory conditions. The harvesting of rice was done after 7 d of treatment. All the experiments were carried out under controlled laboratory conditions, i.e., 14 h light and 10 h dark, light intensity of approximately 280 mmol m
2 s
1, 25 72 °C temperature and 60% relative humidity in triplicate and repeated twice. Harvested rice plants were blotted to remove moisture content and measured the root, shoot length (cm) and fresh weight (g) with the help of metric scale and weighing balance. 2.4. Biochemical analysis 2.4.1. Photosynthetic pigments The blotted dry rice leaves were crushed in 3 ml 80% acetone with the help of pestle and mortar under dark cold conditions and centrifuged at 8000g for 10 min. Supernatants were used for the estimation of chlorophyll content in treated and control plants following the method of Arnon (1949). Carotenoids content was calculated at the wavelength of 480 and 510 nm using the formula given by Duxbury and Yentsch (1956). 2.4.2. Protein content Protein content was estimated by the method of Lowry et al. (1951) using bovine serum albumin (Sigma) as standard. Plant material (100 mg) was crushed in 5 ml 10% chilled trichloroacetic acid (TCA) and centrifuged at 10,000g for 10 min. After decanting the supernatant, pellets were washed and resuspended in 5 ml of 0.1 N NaOH and again centrifuged at 10,000g for 10 min. Supernatant was mixed with alkaline Cu solution and Folin-phenolciocalteau reagent and leave for 30 min. The absorbance was recorded at 650 nm.

2. Material and methods 2.1. Collection and cultivation of algae Microalgae C. vulgaris and Nannochloropsis sp. were collected and isolated from As contaminated area of West Bengal, India and grown in the BG-11 medium. The medium consists of the following components; NaNO3 (1.5 g/L), K2HPO4 (0.04 g/L), MgSO4  7H2O (0.075 g/L), CaCl2  2H2O (0.036 g/L), citric acid (0.01 g/L), ferric ammonium citrate (0.006 g/L), Na2  EDTA (0.001 g/L), Na2CO3 (0.02 mg/L) and trace metal solution. The trace metal solution contains H3BO3 (61.0 mg/L), MnSO4  H2O (169.0 mg/L), ZnSO4  7H2O (287.0 mg/L), CuSO4  5H2O (2.5 mg/L), and (NH4)6MoO4  4H2O (12.5 mg/L). 2.2. Germination of rice seed Rice (Oryza sativa L.) seedlings were obtained by growing rice seed var. triguna in a seed germinator. Rice seeds were washed with 70% ethanol for 30 s and sterilize in 2.5% sodium hypochlorite solution for 15 min. Then rinsed with Milli-Q water 5 times, and incubated for 2–3 d covered with soaked blotting paper in petridish. 2.3. Experimental setup Rice seedlings (7 d old) approx. same size, weight and numbers (15–20) were transferred into a plastic tray containing 24 PVC cups for the anchoring and proper growth of plants in hydroponic condition and grown in 4 l modified Hewitt's nutrient medium (Liu et al., 2004). The pH of the media was maintained between 5.5 and 6.0 with the help of 0.1% KOH and HCl. After 7 d of growth and acclimatization, plants were inoculated with the thick culture (10%) of algae and left for 7 d for colonization, and then treated with 50 mM As(III) in a total 4 l nutrient solution. Rice grown in

2.4.3. TBARS content TBARS content was estimated according to the method of Heath and Packer (1968). 250 mg of plant leaves was crushed in 5 ml of 0.1% TCA and centrifuged at 10,000g for 10 min. 1 ml aliquot of the supernatant was mixed with 4 ml of 20% trichloroacetic acid (TCA) containing 0.5% of thiobarbituric acid and subjected to heat at 95 °C for 30 min followed by cooling in an ice bath and centrifuged at 10,000g for 10 min. The absorbance of the supernatant was taken at 532 nm and 600 nm using a spectrophotometer. 2.5. Antioxidant enzyme assay 2.5.1. Preparation of enzyme extract Enzyme extracts were prepared by homogenizing 200 mg plant leaves in 2 ml of 100 mM potassium phosphate buffer (pH 7.5), containing 1 mM of EDTA and a pinch of polyvinyl polypyrrolidone (PVP). The homogenate was centrifuged at 12,000g for 15 min at 4 °C. All the steps in the preparation of enzyme extract were carried out at 4 °C. The extract was used to measure the activities of antioxidant enzymes. 2.5.2. Superoxide dismutase assay The activity of superoxide dismutase (SOD) was measured by the method of Nishikimi and Rao (1972), using the enzyme extracts. SOD activity was assayed by its ability to inhibit the photochemical reduction of nitroblue tetrazolium (NBT). Inhibition of 50% shows the expression of 1 Unit (1 U) enzyme. A system devoid of enzyme served as control. 2.5.3. Ascorbate peroxidase assay Ascorbate peroxidase (APX) activity was measured in the leaves of the rice plant by the method of Nakano and Asada (1981). The reaction mixture was prepared by adding 50 mM pentose

A.K. Upadhyay et al. / Ecotoxicology and Environmental Safety 124 (2016) 68–73

2.6. Estimation of As For the estimation of total arsenic in rice roots and shoots, dried samples (100 mg) were weighed and soaked in 3 ml of HNO3. After standing overnight at room temperature, the samples were digested at 120 °C for 6 h on a heating plate. The digested samples were diluted to 15 ml with Milli-Q water filtered and stored at 4 °C until the estimation. Arsenic in the diluted sample was measured by inductively coupled plasma mass spectrometry (ICP-MS, 7500; Agilent Technologies, USA). The standard reference material of As (Agilent, Part 8500-6940) was used for the calibration and quality assurance for each analytical batch. The detection limit of As was 1 mg L
1. 2.7. Statistical analysis All determinations were carried out in three replicates in each case. To confirm validity of the data an analysis of variance (ANOVA, p o0.01) was performed and significant differences in various parameters were verified by Duncan's multiple range tests (DMRT, p o0.05).

3. Results and discussion 3.1. Growth response of rice plant The rice plant treated with As(III) showed reduced growth in terms of root length, shoot length and biomass (Fig. 1). After 7 d of treatment, root length, shoot length and biomass decreased from 7.54 cm to 5.9 cm, 20.17 cm to 14.83 cm and 2.29 g to 1.70 g, respectively, as compared with control plants. In contrast, rice inoculated with the algae Nannochloropsis sp. and C. vulgaris showed an increasing trend in root length, shoot length and biomass, and it was comparatively higher in the case of Nannochloropsis sp. inoculated rice. Reduction in root length was due to the fact that plant roots were the first point of contact for this toxic arsenic in the growth media (Liu et al., 2005). Compartmentalization of As (III) into root vacuole and poor translocation to the shoot was also an important factor for reduction in root length of rice plants (Upadhyaya et al., 2014). There are several reports on the loss of fresh and dry biomass of roots as well as shoots, yield, fruit production and morphological changes in the plants grown in Astreated soils (Whitaker et al., 2001; Mokgalaka-Matlala et al., 2008; Shaibur et al., 2008). However, increase in the root shoot length and biomass in rice inoculated with algae may be ascribed to reduced accumulation of As(III), exudates of organic acids by

Root length

shoot length

Biomass

3.5 3

20

2 1.5

10

Biomass (g)

2.5 15

1

5

0.5 Rice +As(III)+ Nanno. sp.

Rice +As(III)+C. vulgaris

Rice +As(III)

0 Rice+Nanno. sp.

0 Rice+C. vulgaris

2.5.4. Glutathione reductase assay Activity of glutathione reductase (GR) was assayed following the method of Smith et al. (1988). The reaction mixture included: 1.0 ml of 0.2 M potassium phosphate buffer (pH, 7.5), 1 mM EDTA, 0.5 ml 3 mM 5, 5′-dithiobis (2-nitrobenzoic acid) in 0.01 M phosphate buffer (pH, 7.5), 0.25 ml H2O, 0.1 ml 2 mM NADPH, 0.05 ml enzyme extract and 0.1 ml 20 mM GSSG. The components were added in the order as mentioned above directly in cuvette and the reaction was started by the addition of GSSG. The increase in absorbance was monitored for 3 min at definite intervals at 412 nm wavelength. The rate of enzyme activity was calculated using standard curve prepared by known amounts of GR (Sigma, USA).

25

Control rice

phosphate buffer (pH, 7.0), 0.5 mM ascorbate, 0.25 mM H2O2 and 50 ml of the plant extracts in a total volume of 1.075 ml. The hydrogen peroxide-dependent oxidation of ascorbate was followed by a decrease in the absorbance at 290 nm. APX activity was expressed as mmol ascorbate oxidized min
1 g
1 fw.

Length (cm)

70

Treatments Fig. 1. Growth characteristics of As(III) treated rice inoculated with algae C. vulgaris and Nannochlropsis sp. All values are mean 7S.D.

algae resulting in enhanced nutrient and mineral, which may positively affect the photosynthesis and thus growth of the plants (Dwivedi et al., 2007). 3.2. Effect on photosynthetic pigment and protein content The chlorophyll a concentration in rice inoculated with the alga C. vulgaris decreased to 2.56 mg g
1 fw with respect to control (2.91 mg g
1 fw), while in case of Nannochloropsis sp., it was recorded 2.66 mg g
1 fw (Table 1). Reduction in chlorophyll content may be due to the nutrient deficiency generated by the algae and chloroplast deformities. Rice plant showed a significant decrease in chlorophyll a concentration (2.19 mg g
1 fw) under As(III) treatment (pr 0.05). Reduced photosynthetic rate, chlorophyll concentration and growth are considered as one of the most damaging effects of this metalloid (Miteva and Merakchiyska, 2002; Stoeva et al., 2005). Miteva and Merakchiyska (2002) reported that arsenic exposure led to an alternation of the chloroplast shape, concaving membrane, bending and partial destruction as well as changes in the accumulation and flow of assimilates resulting from the decrease of chlorophyll content in rice leaves. Increased chlorophyll content in rice leaves inoculated with C. vulgaris and Nannochloropsis sp. might be due to lower accumulation, adsorption of As on the root surface, pH change and chelation of metal by acids secreted by both plant and algae (Hinsinger et al., 2006; Gadd, 2007). Carotenoids present as accessory pigment and serve as antioxidants by scavenging free radicals and diminish the cellular damage, destruction of chloroplast membrane and its main genetic composition induced by heavy metals and metalloids (Czerpak and Piotrowska, 2006). The carotenoids concentration was decreased in rice plant inoculated with algae C. vulgaris (0.216 mg g
1 fw) and Nannochloropsis sp. (0.206 mg g
1 fw) in comparison to control (0.251 mg g
1 fw) (Table 1). However, an increased carotenoids level in rice inoculated with C. vulgaris and Nannochlropsis sp. was observed under As(III) treatment. Increased carotenoids content in rice showed its scavenging potential. In contrast, decrease in carotenoids content is due to low toxicity responses exhibited by the plants. Upadhyay et al. (2014) also reported decreased carotenoids content under heavy metal stress in hydroponically grown aquatic plants. Protein content in the As(III) treated rice plant was decreased sharply (8.47 mg g
1 fw) in comparison to control (11.03 mg g
1 fw). In contrast, algae inoculated rice plant showed better tolerance and high protein content under As(III) treatment. Protein content was increased from 8.47 to 12.91 and

A.K. Upadhyay et al. / Ecotoxicology and Environmental Safety 124 (2016) 68–73

71

Table 1 Concentration of chlorophyll (mg g
1 fw), carotenoids (mg g
1 fw) and protein (mg g
1 fw) in hydroponically grown rice leaves after 7 d of treatment. All values are mean 7 SD. (n¼ 3), ANOVA significant at p r 0.01. Identical superscripts denote no significant difference between means in column according to DMRT (p r0.05). Treatments

Chlorophyll a

Chlorophyll b

Carotenoids

Protein

Control Rice þ C. vulgaris Rice þ Nanno. sp. Rice þ As(III) Rice þ As(III)þ C. vulgaris Rice þ As(III)þ Nanno. sp.

2.91 7 0. 61b 2.56 7 0.08ab 2.69 7 0.13ab 2.10 7 0.05a 2.55 7 0.4ab

1.187 0.62ab 1.067 0.07ab 1.147 0.42ab 0.85 7 0.12a 1.067 0.27ab

0.25 7 0.02ab 0.217 0.01a 0.20 7 0.01ab 0.28 7 0.08b 0.23 7 0.01ab

11.03 70.38b 10.89 70.57b 11.83 71.1c 8.47 71.3a 12.91 71.9d

2.777 0.15b

1.707 0.07b

0.20 7 0.01ab

13.99 70.76e

Nanno. sp. ¼Nannochloropsis sp.
1

13.99 mg g fw in C. vulgaris and Nannochloropsis sp. inoculated rice plants under As(III) treatments, respectively. Increase in protein content in rice inoculated algae may be due to high biomass, more nutrient availability and reduced toxicity of As (Dheeba et al., 2014).

reports by various authors under metal stress (Singh et al., 2006; Maheshwari and Dubey, 2009). Reduction in TBARS content in rice inoculated with both the algae in comparison to As treated plant reveals its detoxification potential. 3.4. Effect on antioxidant enzyme activity

3.3. Effect on TBARS content The TBARS concentration in rice enhanced significantly (1508.06 mmol g
1 fw) as comparison to control (841.40 mmol g
1 fw) under As(III) treatment (Fig. 2A, p r0.05). In contrast, the concentration of TBARS was reduced in As treated rice plant inoculated with algae C. vulgaris and Nannochloropsis sp., which was 1399.19 mmol g
1 fw and 1241.70 mmol g
1 fw, respectively. However, no significant change was observed in the rice inoculated with both the algae as compared to control. Increased TBARS suggests that excess ROS is produced leading to membrane damage due to peroxidation of poly unsaturated lipid (Singh et al., 2007) generating malondialdehyde as one of the byproducts of lipid peroxidation. This is in accordance with earlier

Rice inoculated with algae C. vulgaris and Nannochloropsis sp. showed significant reduction in SOD activity (163.79 and 191.95 Unit g m
1 fw, respectively) (p r0.05) under As stress in comparison to uninoculated rice plant (224.71 Unit g m
1 fw). This suggested their detoxification response, which was more pronounced in Nannochloropsis sp. inoculated rice in comparison to rice inoculated with C. vulgaris (Fig. 2B). Plants are equipped with an efficient biochemical and scavenging machinery (enzymatic and non-enzymatic antioxidants) such as SOD, CAT, APX and GR to manage ROS levels as possible to avoid the toxicity under different environmental stress and thus play an important role in plant protection and detoxification. SODs are the major O
2 scavengers, providing the first line of defense against the cell injury from

250 d

1600

c

SOD (U g-1 fw)

TBARS ( mol g-1 fw)

2000 b

1200 a

a

a

800 400

150

a

a

b

a

100 50

d

e

c

0.9

b

a

0.3 0

GR (U g-1 fw)

10 8 6

c bc

bc a

bc

ab

4 2 0

III

Ri ce

Ri ce

+A

S(

Ri

ce +

As (II +A )+C I) .v s(I II) ulga +N ri an s no .s p

.

APX (mmol min-1g-1 fw)

f

1.5

0.6

bc

200

0

0

1.2

c

.

II) aris . sp lg nno vu a + ce +C. )+N i R II) I I I s(I As( +A ce+ e c Ri Ri (I As

Fig. 2. Thiobarbituric acid reactive substances (TBARS) content and antioxidant enzyme activity in As(III) treated rice inoculated with algae C. vulgaris and Nannochlropsis sp. All values are mean7 S.D. One-way ANOVA was performed and significant differences in different parameters were tested by DMRT. Identical superscripts denote no significant difference between means according to DMRT (p r 0.05).

A.K. Upadhyay et al. / Ecotoxicology and Environmental Safety 124 (2016) 68–73

As accumulation (mg kg-1 dw)

72

50

Root

adsorption and accumulation of As by these algae (Hinsinger et al., 2006). Algae mediated transformation of As also contribute for the lower accumulation of As, as evident by various authors (Meng et al., 2011; Yin et al., 2011; Bahar et al., 2013; Wang et al., 2013). In addition, algalization around the root surface of rice make less accessible to plant and thus reduces uptake of arsenic. Further, Tripathi et al. (2007) reported that iron plaque formation in rice and the precipitation of iron oxide on the root surface decreased As concentration.

Shoot

40 30 20 10 0 Rice+As(III)

Rice+C. vulgaris+As(III)

Rice+Nanno. sp.+As(III)

4. Conclusions

Treatments Fig. 3. As accumulation in root and shoot of rice plant inoculated with algae C. vulgaris and Nannochlropsis sp. in As(III) treated rice. All values are mean 7 S.D.

stress (Sandalio et al., 2009). Reduction in SOD activity was possibly due to binding of metals with algae either its surface or compartmentalization inside the cell and enhanced bio-geochemical and nutrient cycle within the rhizosphere of plants (Smith and Read, 2008) and by providing a minimum area for As accumulation. As induced increased ROS production and oxidative damage have been reported in various plants (Singh et al., 2006; Lin et al., 2008; Mokgalaka-Matlala et al., 2008) in which over expression of the SOD and APX protects against As toxicity. An increased APX activity was observed in the uninoculated rice plant under As(III) stress as compared to control plant after 7 d of treatment. However, reduction in APX activity was observed in rice plant inoculated with algae C. vulgaris and Nannochloropsis sp. reflecting the role of algae in toxicity amelioration (Fig. 2C). APX scavenges H2O2 using ascorbate for reduction and is inevitable to protect photosynthetic machinery and other cell constituent from damage, thus fend the accumulation of H2O2 in the cell (Upadhyay et al., 2014). Increased APX activity under metal stress indicates its role in constant detoxification of H2O2 (Singh et al., 2006; Bajguz, 2010). GR activity in rice inoculated with algae C. vulgaris and Nannochloropsis sp. was decreased significantly (p r0.05) in comparison to control (Fig. 2D). Rice plant treated with As(III) showed an increased GR activity. In the case of As treated rice inoculated with both the algae, GR activity decreased significantly. However, no significant difference was observed in GR activity in relation to the both algae (p r0.05). Glutathione reductase is a key enzyme in the ASC–GSH cycle participates in removing H2O2 by maintaining the intracellular pool of GSH/GSSG (Shri et al., 2009; Kumar et al., 2014). Increased APX and GR activity under arsenic stress was also reported by Neto et al. (2006). Reduced activity of GR in rice inoculated with algae suggests a decreased GSH turnover rate, which clearly indicates low As stress in plants (Shri et al., 2009). The lack of responsiveness of GR to As stress is in agreement on the results of Norton et al. (2008) in rice plants. 3.5. As accumulation Rice treated with arsenic accumulated appreciable amount of As in the roots (35.02 mg kg
1 dw) followed by shoots (29.96 mg kg
1 dw) (Fig. 3). However, rice inoculated with C. vulgaris and Nannochloropsis sp., showed lower accumulation in the roots, i.e., 24.09 and 20.66 mg kg
1 dw and in shoots 20.10 and 11.67 mg kg
1 dw, respectively. Roots are the primary site of accumulation of toxic elements (Czerpak and Piotrowska, 2006). Results showed that the As accumulation was in the order of root4shoot. Higher retention of arsenic in the roots might be attributed to its compartmentalization in root vacuoles. Reduction in metal accumulation in rice inoculated algae may be ascribed to

Present study concludes that the algae C. vulgaris and Nannochloropsis sp. ameliorates As induced oxidative stress through the detoxification processes in rice, which was high in the case of rice inoculated with Nannochloropsis sp. Cellular toxicity decreased significantly by the application of Nannochloropsis sp. Increased root, shoot length, biomass and protein content in algae inoculated rice and reduced enzymatic activity of SOD, APX and GR signify detoxification capacity. Further, reduced uptake of As in shoot mitigates the acute toxicity induced by As in rice plant. Therefore, inoculation of rice seedlings with algae C. vulgaris and Nannochloropsis sp. may be adapted for cultivating rice in As contaminated agricultural field. However, field trials are required before their application at a large scale.

Acknowledgments The authors are thankful to Dr. C.S. Nautiyal, Director, CSIRNational Botanical Research Institute, Lucknow, for providing the required facilities. The author NKS is thankful to Science and Engineering Research Board, New Delhi for award of Young Scientist.

References Arnon, D.I., 1949. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1–15. Bahar, M.M., Megharaj, M., Naidu, R., 2013. Toxicity transformation and accumulation of inorganic arsenic species in microalga Scendesmus sp. isolated from soil. J. Appl. Phycol. 25, 913–917. Bajguz, A., 2010. An enhancing effect of exogenous brassinolide on the growth and antioxidant activity in Chlorella vulgaris cultures under heavy metals stress. Environ. Exp. Bot. 68, 175–179. Brammer, H., 2009. Mitigation of arsenic contamination in irrigated paddy soils in south and south-east Asia. Environ. Int. 35, 856–863. Czerpak, R., Piotrowska, A., 2006. Jasmonic acid affects changes in the growth and some components content in Chlorella vulgaris. Acta Physiol. Plant. 28, 95–203. Dheeba, B., Sampathkumar, P., Kannan, K., 2014. Growth, chromium accumulation potential and biochemical changes of Vigna radiate and Zea mays grown with effective microbes. Proc. Natl. Acad. Sci. India Sect. B: Biol. Sci. 84, 381–387. Duxbury, A.C., Yentsch, C.S., 1956. Plankton pigment monograph. J. Mar. Res. 15, 93–101. Dwivedi, S., Tripathi, R.D., Srivastava, S., Mishra, S., Shukla, M.K., Tiwari, K.K., Rai, U. N., 2007. Growth performance and biochemical responses of three rice (Oryza sativa L.) cultivars grown in fly-ash amended soil. Chemosphere 67 (1), 140–151. Gadd, G.M., 2007. Geomycology: biogeochemical transformations of rocks, minerals, metals and radionuclides by fungi, bioweathering and bioremediation. Mycol. Res. 111, 3–49. Guha Mazumder, D.N., Deb, D., Biswas, A., Saha, C., Nandy, A., Das, A., Ghose, A., Bhattacharya, K., Mazumdar, K.K., 2014. Dietary arsenic exposure with low level of arsenic in drinking water and biomarker: a study in West Bengal. J. Environ. Sci. Health Part A 49, 555–564. Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts kinetic and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125, 189–198. Hinsinger, P., Plassard, C., Jaillard, B., 2006. Rhizosphere: a new frontier for soil biogeochemistry. J. Geochem. Explor. 88, 210–213. Jomova, K., Jenisova, Z., Feszterova, M., Baros, S., Liska, J., Hudecova, D., Rhodes, C.J., Valko, M., 2011. Arsenic: toxicity, oxidative stress and human disease. J. Appl. Toxicol. 31, 95–107. Kilian, O., Benemann, C.S.E., Niyogi, K.K., Vick, B., 2011. High-efficiency homologous

A.K. Upadhyay et al. / Ecotoxicology and Environmental Safety 124 (2016) 68–73

recombination in the oil-producing alga Nannochloropsis sp. Proc. Natl. Acad. Sci. USA 108, 21265–21269. Kumar, A., Singh, R.P., Singh, P.K., Awasthi, S., Chakrabarty, D., Trivedi, P.K., Tripathi, R.D., 2014. Selenium ameliorates arsenic induced oxidative stress through modulation of antioxidant enzymes and thiols in rice (Oryza sativa L.). Ecotoxicology 23, 1153–1163. Lin, A., Zhang, X., Zhu, Y.G., Zhao, F.J., 2008. Arsenate-induced toxicity: effects on antioxidative enzymes and DNA damage in Vicia faba. Environ. Toxicol. Chem. 27, 413–419. Liu, W.J., Zhu, Y.G., Smith, F.A., Smith, S.E., 2004. Do phosphorus nutrition and iron plaque alter arsenate uptake by rice seedlings in hydroponic culture. New Phytol. 162, 481–488. Liu, X., Zhang, S., Shan, X., Zhu, Y.G., 2005. Toxicity of arsenate and arsenite on germination, seedling growth and amylolytic activity of wheat. Chemosphere 61, 293–301. Lomax, C., Liu, W.J., Wu, L., Xue, K., Xiong, J., Zhou, J., McGrath, S.P., Meharg, A.A., Miller, A.J., Zhao, F.J., 2011. Methylated arsenic species in plants originate from soil microorganisms. New Phytol. 193, 665–672. Lowry, O.H., Rosenbrough, N.M., Farr, A.L., Randall, R.J., 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265–275. Ma, J.F., Yamaji, N., Mitani, N., Xu, X.Y., Su, Y.H., McGrath, S.P., Zhao, F.J., 2008. Transporters of arsenite in rice and their role in arsenic accumulation in rice grain. Proc. Natl. Acad. Sci. USA 105, 9931–9935. Maheshwari, R., Dubey, R.S., 2009. Nickel induced oxidative stress and role of antioxidant defense in rice seedlings. Plant Growth Regul. 59, 37–49. Meharg, A.A., 2004. Arsenic in rice-understanding a new disaster for South-East Asia. Trends Plant Sci. 9, 415–417. Meng, X.Y., Qin, J., Wang, L.H., Duan, G.L., Sun, G.X., Wu, H.L., Chu, C.C., Ling, H.Q., Rosen, B.P., Zhu, Y.G., 2011. Arsenic biotransformation and volatilization in transgenic rice. New Phytol. 191, 49–56. Miteva, E., Merakchiyska, M., 2002. Response of chloroplasts and photosynthetic mechanism of bean plants to excess arsenic in soil. Bulg. J. Agric. Sci. 8, 151–156. Mokgalaka-Matlala, N.S., Flores-Tavizón, E., Castillo-Michel, H., Peralta-Videa, J.R., Gardea-Torresdey, J.L., 2008. Toxicity of arsenic (III) and (V) on plant growth, element uptake, and total amylolytic activity of mesquite (Prosopis Juliflora x P. Velutina). Int. J. Phytoremediation 10, 47–60. Nakano, Y., Asada, K., 1981. Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol. 22, 867–880. Neto, A.D.d.A., Prisco, J.T., Filho, J.E., Abreu, C.E.B.d., Filho, E.G., 2006. Effect of salt stress on antioxidative enzymes and lipid peroxidation in leaves and roots of salt-tolerant and salt-sensitive maize genotypes. Environ. Exp. Bot. 56, 87–94. Nishikimi, M., Rao, N.A., 1972. The occurrence of superoxide anion in the reaction of reduced phenazine methosulphate and molecular oxygen. Biochem. Biophys. Res. Commun. 48, 849–854. Norton, G.J., Lou-Hing, D.E., Meharg, A.A., Price, A.H., 2008. Rice arsenate interactions in hydroponics: whole genome transcriptional analysis. J. Exp. Bot. 59, 2267–2276. Rai, R., Pandey, S., Rai, S.P., 2011. Arsenic-induced changes in morphological, physiological, and biochemical attributes and artemisinin biosynthesis in Artemisia annua, an antimalarial plant. Ecotoxicology 20, 1900–1913. Rai, U.N., Singh, N.K., Upadhyay, A.K., Verma, S., 2013. Chromate tolerance and accumulation in Chlorella vulgaris L.: role of antioxidant enzymes and biochemical changes in detoxification of metals. Bioresour. Technol. 136, 604–609. Rossman, T.G., Klein, C.B., 2011. Genetic and epigenetic effects of environmental arsenicals. Metallomics 3, 1135–1141. Sandalio, L.M., Rodríguez-Serrano, M., del Río, L.A., Romero-puertas, M.C., 2009. Reactive oxygen species and signalling in cadmium toxicity. In: del Río, L.A., Puppo, A. (Eds.), Reactive Oxygen Species in Plant Signalling. Springer, Heidelberg, pp. 175–190. Shaibur, M.R., Kitajima, N., Sugawara, R., Kondo, T., Huq, S.M.I., Kawai, S., 2008. Physiological and mineralogical properties of arsenic-induced chlorosis in rice seedlings grown hydroponically. Soil Sci. Plant Nutr. 52, 691–700. Sharma, A.K., Tjell, J.C., Sloth, J.J., Holm, P.E., 2014. Review of arsenic contamination,

73

exposure through water and food and low cost mitigation options for rural areas. Appl. Geochem. 41, 11–33. Shri, M., Kumar, S., Chakrabarty, D., Trivedi, P.K., Mallick, S., Mishra, P., Shukla, D., Mishra, S., Srivastava, S., Tripathi, R.D., Tuli, R., 2009. Effect of arsenic on growth, oxidative stress and antioxidant system in rice seedlings. Ecotoxicol. Environ. Saf. 72, 1102–1110. Singh, N., Ma, L.Q., Shrivastava, M., Rathinasapathi, B., 2006. Metabolic adaptation to arsenic-induced oxidative stress in Pteris vittata L. and Pteris ensiformis L. Plant Sci. 170, 274–282. Smith, E., Kempson, I., Juhasz, A.L., Weber, J., Skinner, W.M., Gräfe, M., 2009. Localization and speciation of arsenic and trace elements in rice tissues. Chemosphere 76, 529–535. Singh, H.P., Batish, D.R., Kohli, R.K., Arora, K., 2007. Arsenic-induced root growth inhibition in mung bean (Phaseolus aureus Roxb.) is due to oxidative stress resulting from enhanced lipid peroxidation. Plant Growth Regul. 53 (1), 65–73. Smith, I.K., Vierheller, T.L., Thorne, C.A., 1988. Assay of glutathione reductase in crude tissue homogenates using 5,5′-dithiobis (2-nitrobenzoic acid). Anal. Biochem. 175, 408–413. Smith, S.E., Read, D.J., 2008. Mycorrhizal Symbiosis. Elsevier Academic Press, London. Stoeva, N., Berova, M., Zlatev, Z., 2005. Effect of arsenic on some physiological parameters in bean plants. Biol. Plant. 49, 293–296. Sukenik, A., Beardall, J., Kromkamp, J.C., Kopeck, J., Masojídek, J., Bergeijk, S.V., Gabai, S., Shaham, E., Yamshon, A., 2009. Photosynthetic performance of outdoor Nannochloropsis mass cultures under a wide range of environmental conditions. Aquat. Microb. Ecol. 56, 297–308. Suresh, B., Ravishankar, G.A., 2004. Phytoremediation: a novel and promising approach for environmental cleanup. Crit. Rev. Biotechnol. 24, 97–124. Tripathi, R.D., Srivastava, S., Mishra, S., Singh, N., Tuli, R., Gupta, D.K., Maathuis, F.J. M., 2007. Arsenic hazards: strategies for tolerance and remediation by plants. Trends Biotechnol. 25, 158–165. Upadhyay, A.K., Singh, N.K., Rai, U.N., 2014. Comparative metal accumulation potential of Potamogeton pectinatus L. and Potamogeton crispus L.: role of enzymatic and non-enzymatic antioxidants in tolerance and detoxification of metals. Aquat. Bot. 117, 27–32. Upadhyaya, H., Shome, S., Roy, D., Bhattacharya, M.K., 2014. Arsenic induced changes in growth and physiological responses in Vigna radiata seedling: effect of curcumin interaction. Am. J. Plant Sci. 5 (24), 3609. Wang, N.X., Li, Y., Deng, X.H., Miao, A.J., Ji, R., Yang, L.Y., 2013. Toxicity and bioaccumulation kinetics of arsenate in two freshwater green algae under different phosphate regimes. Water Res. 47, 2497–2506. Whitaker, J.H., Ainsworth, G., Meharg, A.A., 2001. Copper and arsenate-induced oxidative stress in Holcus lanatus L. clones with differential sensitivity. Plant Cell Environ. 24, 713–722. Williams, P.N., Villada, A., Deacon, C., Raab, A., Figuerola, J., Green, A.J., Feldmann, J., Meharg, A.A., 2007. Greatly enhanced arsenic shoot assimilation in rice leads to elevated grain levels compared to wheat and barley. Environ. Sci. Technol. 41, 6854–6859. Xu, X.Y., McGrath, S.P., Meharg, A.A., Zhao, F.J., 2008. Growing rice aerobically markedly decreases arsenic accumulation. Environ. Sci. Technol. 42, 5574–5579. Ye, J., Rensing, C., Rosen, B.P., Zhu, Y.G., 2012. Arsenic biomethylation by photosynthetic organisms. Trends Plant Sci. 17, 155–162. Yin, X.X., Chen, J., Qin, J., Sun, G.X., Rosen, B.P., Zhu, Y.G., 2011. Biotransformation and volatilization of arsenic by three photosynthetic cyanobacteria. Plant Physiol. 156, 1631–1638. Zhu, Y.G., Sun, G.X., Lei, M., Teng, M., Liu, Y.X., Chen, N.C., Wang, L.H., Carey, A.M., Deacon, C., Raab, A., Meharg, A.A., Williams, P.N., 2008. High percentage inorganic arsenic content of mining impacted and non impacted Chinese rice. Environ. Sci. Technol. 42, 5008–5013. Zheng, S., Jiang, W., Cai, Y., Dionysiou, D.D., O’Shea, K.E., 2014. Adsorption and photocatalytic degradation of aromatic organoarsenic compounds in TiO2 suspension. Catal. Today 224, 83–88.