Ecotoxicological effects of alpha-cypermethrin on freshwater alga Chlorella sp.: Growth inhibition and oxidative stress studies

Journal Pre-proof Ecotoxicological effects of alpha-cypermethrin on freshwater alga Chlorella sp: growth inhibition and oxidative stress studies Prithu Baruah, Neha Chaurasia

PII:

S1382-6689(20)30023-5

DOI:

https://doi.org/10.1016/j.etap.2020.103347

Reference:

ENVTOX 103347

To appear in:

Environmental Toxicology and Pharmacology

Received Date:

15 March 2019

Revised Date:

15 October 2019

Accepted Date:

29 January 2020

Please cite this article as: Baruah P, Chaurasia N, Ecotoxicological effects of alpha-cypermethrin on freshwater alga Chlorella sp: growth inhibition and oxidative stress studies, Environmental Toxicology and Pharmacology (2020), doi: https://doi.org/10.1016/j.etap.2020.103347

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Title: Ecotoxicological effects of alpha-cypermethrin on freshwater alga Chlorella sp: growth inhibition and oxidative stress studies

Prithu Baruah1 and Neha Chaurasia 1*

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Environmental Biotechnology laboratory, Department of Biotechnology and Bioinformatics,

North-Eastern Hill University, Shillong, India-793022 Tel: +91-364-2722424; Fax No: +91-

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364-2550108; E-mail address: [email protected], [email protected]. Name and Full address of corresponding author: Neha Chaurasia

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Environmental Biotechnology laboratory, Department of Biotechnology and Bioinformatics,

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North-Eastern Hill University, Shillong, India-793022 Tel: +91-364-2722424; Fax No: +91-

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Graphical Abstract

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364-2550108; E-mail address: [email protected], [email protected]

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Highlights



High dose of alpha-cypermethrin (ACy) inhibited the growth of Chlorella sp.



The 96 h EC50 of ACy was estimated to be 11.00 mg L-1.



ACy induced enhanced generation of ROS and intracellular lipid accumulation.



ACy altered photosynthetic pigment content and antioxidant enzyme activity.

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 ACy induced lipid peroxidation in Chlorella sp.

Abstract

Alpha-cypermethrin (ACy) is a synthetic pyrethroid insecticide commonly used in

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agricultural practices for controlling a broad range of insect pests particularly belonging to

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the order Lepidoptera and Coleoptera. The present study aims to evaluate the toxic effect of ACy on microalgae by studying its influence on Chlorella sp. According to our knowledge,

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this is the first detailed study of ACy toxicity on microalgae.Significant growth inhibition of Chlorella sp. was observed at high ACy concentration (6-48 mg L-1) during the entire 96 h

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bioassay. The 96 h effective concentration (EC50) of ACy was estimated to be 11.00 mg L-1. Flow cytometry analysis showed an enhanced generation of reactive oxygen species (ROS)

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and intracellular lipid accumulation after 96 h exposure to 11.00 mg L-1 of ACy. Further, the same ACy concentration showed a significant decrease in photosynthetic pigment content

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and an increase in antioxidant enzyme activity and malondialdehyde (MDA) content in Chlorella sp.

Keywords: alpha-cypermethrin, microalgae, toxicity, oxidative stress, antioxidant enzymes

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1. Introduction Pesticides find widespread application in modern day’s agricultural practices and have successfully revolutionized the entire pest control system. According to reports, the annual sale of active ingredients is estimated to be 2.7 billion Kg across the world (Centner, 2018). Pyrethroids constitute about 30% of globally used insecticide and have become a potential alternative to its organophosphate and organochlorine counterparts (Gao et al., 2016). They are synthesized artificially based on the chemical structure of pyrethrin, produced naturally

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by Chrysanthemum cineraraefolum. Pyrethroids are extensively used in agriculture, food storage and are a popular household insecticide (Yao et al., 2017). Spray drift, accidental spillage or watershed drainage leads to

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its spread in water bodies and subsequent deterioration of quality of the aquatic environment.

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This may cause hazardous effects on non-target micro-organisms like algae and cyanobacteria and as well as to human beings (Fatma et al., 2007; Rodger et al., 1994).

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In the aquatic ecosystem, microalgae are important primary producers providing food for diverse invertebrates and fish species (Mofeed and Mosleh, 2003). In addition, they play a

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major role in nitrogen and phosphorus cycling (Kallquist and Svenson, 2003; Sabater and Carrasco, 2001). Thus any adverse effect on the microalgal community can result in

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structural changes in the entire ecosystem (Martinez et al., 2015; Villem, 2011). Considering these facts, evaluation of toxicity of pyrethroids on ecologically relevant organism like

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microalgae becomes important. Characteristic features of microalgae such as short generation time and rapid response to environmental changes give credence to their use as an ideal ecological testing organism (Chen et al., 2016; Eguchi et al., 2004). Moreover, microalgaebased tests are rapid as well as cost-effective (Sosak-swiderska et al., 1998).

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Alpha-cypermethrin (ACy) is a racemic mixture of alpha C isomers of the pyrethroid insecticide, cypermethrin, with two to three folds more toxicity than cypermethrin (Hartnik et al., 2008). High toxicity of pyrethroid insecticides can be attributed to its lipophilic nature which makes them readily absorbed into biomembranes and tissues (Gao et al., 2016; Oros et al., 2005). In paddy fields, pyrethroids such as ACy are used for controlling snout moth’s larvae and during spraying time the compound may reach a high concentration (Yao et al., 2017). Marino & Ronco (2005) reported a cypermethrin concentration as high as 194 µg L-1

sub-surface water concentration of 0.6 µg L-1

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in surface water due to agricultural activitiy. Garforth & Woodbridge (1984) demonstrated a shortly after application of ACy. Higher

concentration of pyrethroid can be expected in surface water depending on intensity of

2012).

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rainfall and quantity of water leaving the agricultural fields through run off (Sáenz et al., The primary producers of the aquatic ecosystem such as microalgae are easily

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exposed to these contaminants. ACy has been reported to have exceedingly high toxicity to

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aquatic organisms making its use questionable in agricultural practices (Hartnik et al., 2008; Sololmon et al., 2001). The high toxicity of ACy towards non-target organisms is reflected by the median lethal concentration (LC50) of 0.3 and 2.8 µg L-1 for Daphnia magna and rainbow

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trout respectively (European Commission, 2004). Moreover, reports on ACy induced DNA damage in human peripheral blood lymphocytes reveals its genotoxic potential (Kocaman

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and Topaktas, 2009). Due to the high toxicity of ACy, studies on its environmental behaviour

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have received sufficient attention (Yao et al., 2019). Although reports on toxic effects of ACy on non-target organisms are available, to the best of our knowledge there is no detailed study on its impact on microalgae. Thus, the present study aims to evaluate the toxic effects of ACy using Chlorella sp. as a test organism. In this work, toxic effects of ACy were analysed by studying the growth of Chlorella sp. under different concentration of ACy. Furthermore, the impact of median effective dose (EC50) of ACy on reactive oxygen species (ROS) formation,

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intracellular lipid accumulation, photosynthetic pigment content, antioxidant enzyme activity and lipid peroxidation was evaluated for underpinning its toxicity mechanism. The reason behind choosing Chlorella sp. in our study is its wide distribution in aquatic ecosystem and its sensitivity towards environmental contaminants (Ramandass et al., 2016). The findings of the present study will be helpful in understanding the impact of ACy on microalgae and will add new dimensions to the toxicological perspective of pyrethroids.

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2. Materials and Methods 2.1. Chemicals

Commercial formulation of alpha-cypermethrin (10% emulsifiable concentrate) was

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purchased from local market of Meghalaya, India. All other chemicals were of analytical

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grade and purchased from SigmaAldrich (USA) and HiMedia (India).

2.2. Experimental design: In the present study Chlorella sp. NC-MKM (accession number

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KY048406) was used as a test organism. For evaluating the ecotoxicological effects of ACy on Chlorella sp., at first algal growth inhibition test was performed and 96 h median effective

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concentration (EC50) of ACy was estimated. Following this, the estimated EC50 of ACy was tested on Chlorella sp. cultures for 96 h to evaluate its effect on ROS generation, intracellular

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lipid accumulation, photosynthetic pigment content, antioxidant enzyme activity and lipid

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peroxidation.

2.3. Experimental conditions and algal growth inhibition test: Cultures of unicellular microalgae Chlorella sp. NC-MKM were obtained from Environmental Biotechnology Laboratory, North Eastern Hill University, Meghalaya, India. The stock cultures were prepared by growing the strain photoautotrophically in sterile Bold’s basal medium (BBM, pH=7.4) which is a commonly used media in ecotoxicological studies 5

(Xiong et al., 2017b; Xiong et al., 2017c;Tayemeh et al.,2020). Microalga strain was inoculated in 500 mL

Erlenmeyer flasks containing 250 mL of BBM media at 10%

concentration (Vinoculum/Vmedia) (Bischoff and Bold 1963, Kumar et al., 2016). The cultures were incubated in a shaker incubator (REMI, India) at 150 rpm under 14/10-h light/dark cycle(lights were switched on at 7:00 pm and switched off at 9:00 am based on Indian Standard Time) using fluorescent illumination of 45–50 μmol photon m-2 s-1 at 25±2°C. In order to reduce the probable spatial variations in illumination and temperature, all the flask

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were repositioned two times daily within the shaker incubator (Fu et al.,2017). Algal growth inhibition test was performed by exposing the test alga to varying concentrations of a commercial alpha-cypermethrin formulation (10 % emulsifiable

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concentrate). In agricultural practices it is usually the commercial formulation which is used

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and not the technical grade chemical. Thus, primary producers in aquatic ecosystem like algae are most likely to be exposed to the commercial formulation. Therefore in order to

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obtain more physiologically relevant data we preferred a commercial formulation in this study (Feckler et al., 2018; Smythers et al., 2019). Diluted stock solution of ACy was added

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to 100 mL sterile Bold’s basal medium contained in 250 mL Erlenmeyer flask in order to obtain concentrations 0.75, 1.5, 3, 6, 12, 24 and 48 mg L-1 of active ingredient (a.i). These

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concentrations were selected on the basis of our preliminary range finding experiment and concentration were arranged in a geometric progression of factor two (Duan et al. 2017). The

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concentrations were analytically validated by high-performance liquid chromatography (HPLC, Agilent/1260 Infinity, USA) using alpha-cypermethrin as external standard (purchased from SigmaAldrich, USA, 98.9% purity). HPLC detection was carried out at 25°C with a C18 column and DAD detector set at 235 nm. The mobile phase used was acetonitrile:water in a ratio of 98:2 at a flow rate of 1 mg L-1. Quantification was done using the following equation as obtained from the standard plot of concentration vs peak area: 6

Concentration (mg L-1) = 2.36×10-5 × peak area+1.18(R2 = 0.996) The deviation (%) of actual concentration of ACy with respect to the nominal concentration was calculated as: 100 ̶ (actual ACy concentration×100/nominal concentration) (Ferreira et al., 2016). In the present study, deviation (%) of actual concentration of ACy from the nominal concentration was less than 20% and therefore nominal concentration was used for statistical

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analyses ( Fang et al., 2018). To each dilution exponentially growing cells of Chlorella sp. were added to obtain an initial concentration of 1.33x104 cells m L-1. Sterile BBM medium inoculated with the test organism

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without ACy treatment served as control. The microalga was cultured in aforementioned conditions for 96h. For estimating the microalgal growth, 1 mL culture was withdrawn

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regularly after every 24h and counted directly in a Neubauer chamber during the entire 96 h

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bioassay.

The specific growth rate (µ, day-1) was calculated using the following equation (Fogg, 1975):

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µ=

𝑙𝑛𝑁1 − 𝑙𝑛𝑁0 △𝑡

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Where, △t (t1-t0) is the exposure time, N1 is the cell density after time t1, N0 is the initial cell density and µ is the specific growth rate per day. The percent inhibition of specific growth

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rate was calculated using the following equation (USEPA, 1989):

%Inhibition =

C−T X100 T

Where T is the specific growth rate (µ) for the treatment replicate and C is the mean value for specific growth rate in the control.

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EC50 was determined by linear regression of specific growth rate inhibition vs. logarithmic concentrations (Yuan et al., 2013). All glassware used in this study were cleaned with phosphate free detergent (Vetroclean, Borosil, India).The glassware were soaked in 10% v/v HCl and neutralized with saturated sodium biocarbonate solution. Finally they were rinsed in deionized double distilled water (Chen et al., 1997).

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2.4. Extraction and quantification of photosynthetic pigments Pigments were extracted from the algal sample using dimethylsulphoxide (DMSO) as described by Griffiths et al. (2011). For this, 2 mL culture was withdrawn and centrifuged at

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10,000×g for 5 min. The supernatant was discarded and 2 mL hot (60 °C) DMSO was added to the cell pellet. The sample was vortexed and incubated at 60 °C in a water bath for 10 min.

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The absorbance of the supernatant obtained after centrifugation was measured at 649, 665

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and 480 nm using a UV–vis spectrophotometer (Eppendorf Biospectrometer, Germany). Based on the resolution of the spectrophotometer used, the pigment content was determined

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using the equations below (Wellburn, 1994):

Chlorophyll a (Chl-a) (µ mL−1) = 12.19(A665) − 3.45 (A649)

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Chlorophyll b (Chl-b) (µ mL−1) = 21.99(A649) – 5.32 (A665)

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Carotenoids (µ mL−1) = [1000(A480) − 2.14(Chl-a) − 70.16(Chl-b)]/220 Where, A665, A649 and A480 represent absorbance at 665 nm, 649 nm and 480 nm respectively. 2.5. Flow cytometry analysis of ROS production ROS was detected using cell permeable probe 2ʹ, 7ʹ - dichlorodihydrofluorescein diacetate (H2DCFDA) as described previously by Knauert and Knauer (2008). Inside the microalgal 8

cell, H2DCFDA is hydrolyzed to non-fluorescent 2ʹ, 7ʹ - dichlorodihydrofluorescein (H2DCF) by cellular esterase which is further changed to fluorescent 2ʹ, 7ʹ- dichlorofluorescein (DCF) in the presence of intracellular ROS. For ROS detection, 10 mL algal culture was collected at 96 h and centrifuged at 10000×g for 10 min. Following this, 10 mM freshly prepared H2DCFDA was added to the cell pellet under dim light. Samples were then incubated in a water bath at 37°C for 30 min in the dark. Flow cytometry (FCM) analysis of the samples was done using Becton Dickinson FACS Calibur (Becton Dickinson Instruments, Franklin

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Lakes, NJ) at excitation and an emission wavelength of 485 nm and 530 nm respectively. Green fluorescent emission of DCF was collected in FL1 channel. Results were expressed as the percentage of cells with positive DCF fluorescence (DCF +). FCM analyses were done in

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duplicate.

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2.6. Flow cytometric detection of intracellular lipid accumulation

For detecting the accumulation of intracellular lipid, the fluorescent dye Nile Red (NR) was

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used. Inside the cell, NR binds with intracellular lipid droplets and gives yellow-gold fluorescence (Alemán-Nava et al., 2016). For this, 10 mL algal cultures were harvested at 96

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h by centrifugation at 10000xg for 10 min. The cell pellets obtained after centrifugation were stained with 3.0 x 10-3 μM NR dye and incubated at 37°C for 15min. Flow cytometry

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analysis of the samples were done using Becton Dickinson FACSCalibur (Becton Dickinson Instruments, Franklin Lakes, NJ) at excitation and emission wavelength of 488 nm and

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586/42 nm respectively. The fluorescent emission of NR was collected in FL2 channel and results were expressed as the percentage of cells with positive NR fluorescence (NR+). FCM analysis was carried out in duplicate.

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2.7. Analysis of antioxidant enzyme activity and lipid peroxidation For estimation of superoxide dismutase activity (SOD), 10 mL culture was withdrawn at 96 h and centrifuged at 5000×g for 10 min. The pellets were washed two times with double distilled water and homogenized in 2 mL 0.1 M phosphate buffer (pH 7.5). This was followed by centrifugation of the homogenate at 15000×g at 4°C for 10 min. The supernatant obtained after centrifugation was used for enzyme assay. SOD activity was assayed by monitoring the inhibition of reduction of nitroblue tetrazolium chloride (NBT) photochemically. The

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following reaction mixture was used: 1 M Na2CO3, 200 mM methionine, 2.25 mM NBT, 3 mM EDTA, 60 μM riboflavin and 0.1 M phosphate buffer (pH 7.8). The absorbance was measured at 560 nm (Dhindsa et al., 1981). One unit of SOD activity is the amount of

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enzyme required to inhibit photochemical reduction of NBT by 50%.

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For estimation of catalase activity, algal cell pellets (pellets were obtained in the same way as described in SOD assay) were homogenized in 2 mL 0.5 M phosphate buffer (pH 7.8) and

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centrifuged at 15000×g at 4°C for 10 min. The supernatant obtained after the process was used for catalase assay. In a test tube, 1.6 mL phosphate buffer, 0.2 mL 0.3% H2O2 and 3 mM

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EDTA were added to 100 μL of enzyme extract and the reaction was allowed to run for 3 min. One unit of catalase is the amount of enzyme required to decompose 1 μL of H2O2 per

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minute at 25 °C (Kumar et al., 2016). The absorbance was measured at 240 nm (Aebi, 1984).

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The amount of malondialdehyde (MDA) in the cell was taken as a measurement of lipid peroxidation (Janero, 1990). MDA content was determined as previously described by Health and Packer (1968). 10 mL algal culture was collected at 96 h and centrifuged at 12,000×g for 10 min at 4 °C. The pellet obtained after centrifugation was homogenized with 2 mL 10% (w/v) trichloroacetic acid (TCA). This was followed by centrifugation of the homogenate at 12,000×g for 10 min at 4°C. The supernatant was used for estimation of MDA content. 2 mL

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of the supernatant and 2 mL of thiobarbituric acid (TBA) reagent (0.25% in 10% TCA) were mixed properly and heated at 95°C for 30 min. The mixture was immediately cooled and centrifuged at 15,000×g for 10 min. The absorbance was measured at 532 nm and nonspecific turbidity was corrected using absorbance at 600 nm. MDA content was calculated by using the extinction coefficient of 155 mM-1 cm-1 (Health and Packer 1968). The protein content was measured according to the method described by Bradford (1976). The results of SOD and CAT activity were expressed as U mg-1 protein and MDA content as

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nmol mg-1 protein. 2.8. Statistical analyses

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All the experiments were carried out in triplicate and the data is presented as mean and standard deviations. One-way analysis of variance (ANOVA) was employed to evaluate

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whether there was any statistical difference between microalgal growth (cell density and

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specific growth rate) at different treatment concentration. Before performing one-way ANOVA, Kolmogorov–Smirnov and Levene tests were used for checking the normality and

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homogeneity of variances respectively in experimental data. When statistical differences were found in one way ANOVA, post hoc (Dunnett) test was employed. The statistical difference between control and treatment group at 96 h for ROS generation, intracellular lipid

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accumulation, pigment content, antioxidant enzyme activity and MDA content was analysed

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by student’s t-test. All analysis was done using GraphPrism version 5.01 and Origin 7.0 software package. Values with p<0.05 was taken as statistically significant. 3. Results and discussion 3.1. Effect of ACy on growth behaviour of Chlorella sp. Phytotoxicity tests based on growth inhibition of microalgae is a broadly used technique to evaluate the toxicity of a compound. In these tests, cell density is a popular parameter for 11

toxicity assessment (Xiong et al., 2016). In order to evaluate the ecotoxicological effects of ACy, Chlorella sp. was exposed to different concentrations of ACy (0, 0.75, 1.5, 3, 6, 12, 24 and 48 mg L-1) for 96 h. The effects of ACy on cell density of Chlorella sp. is shown in Fig 1(a). Cell density of Chlorella sp. was negligibly effected (p>0.05) at low ACy concentration (0.75 - 3 mg L-1), but significant decrease (p<0.05) in cell density was observed at high ACy concentration (6-48 mg L-1) during the entire 96 h bioassay. At the end of cultivation, 6, 12, 24 and 48 mg L-1 of ACy inhibited the growth of Chlorella sp. cultures by 34.36±2.17%,

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45.97±1.73%, 59.82±1.32% & 60.32±2.51% respectively. The specific growth rate (SGR) of Chlorella sp. decreased by 36.90±4.89%, 53.76±0.70%, 72.95±2.89%, 80.88±1.23% after exposure to 6, 12, 24 and 48 mg L-1 of ACy concentrations for 96 h [Fig.1 (b)]. Growth

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inhibition of microalgae by environmental contaminants has been reported by several researchers earlier. It has been demonstrated that pyrethroid insecticide such as cypermethrin

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and cyfluthrin caused significant growth inhibition in Scenedesmus obliquus, S. quadricauda,

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S. acutus, Chlorella vulgaris & Pseudokirchneriella subcapitata ( Li et al., 2005; Sáenz et al., 2012). Adverse effects on growth is also reported in Ankistrodesmus gracilis, C. pyrenoidosa, Merismopedia sp. & C. vulgaris exposed to organophosphorus insecticides (Asselborn et al.,

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2015, Chen et al., 2016; Kurade et al., 2016). Prolonged exposure to organic pollutants causes severe damage to the cellular membrane and facilitates their interaction with the cellular

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structures (Kurade et al., 2016). Growth inhibition of Chlorella sp. by ACy can be attributed

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to its severe impact on cellular membranes of the microalgae which resulted in its toxicity. Median effective concentration (EC50) is a popular index used in toxicological assessment of compounds (Huang et al., 2012). In our study, EC50 was calculated based on specific growth rate inhibition (Kabra et al., 2014). 96 h EC50 was estimated to be 11.00 mg L-1 (Table 1). EC50 can be used for classifying matter into different toxic classes as per the EU-Directive 93/67/EEC: EC50-values <1 mg L-1 (very toxic to aquatic organisms); 1–10 mg L-1 (toxic to 12

aquatic organisms); 10–100 mg L-1 (harmful to aquatic organisms); >100 mg L-1 (not be classified) (Commission of the European Communities, 1996; Chen et al., 2016). From our study, ACy could be classified as harmful to Chorella sp. (EC50=11.00 mg L-1) according to this standard. EC50 value obtained in our study is similar to 72 h EC50 (11.49 mg L-1) of ACy for the green alga P. subcapitata (Satyavani et al., 2012). Marked variation in sensitivity between microalgae to pyrethroids has been reported earlier. For example, Li et al., 2005 demonstrated 96 h EC50 value as high as 112.45 mg L-1 for cypermethrin on S. obliquus.

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While the 96 h EC50 for S. quadricauda, S.acutus, C. vulgaris and P. subcapitata on exposure to cypermethrin was 0.48 mg L-1, 0.94 mg L-1, 0.23 mg L-1 & 0.20 mg L-1 respectively (Sáenz et al., 2012). Differential toxicity of the same contaminant to different microalgal strains

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might be due to the species specific diversity in biological characteristics (morphology,

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cytology, physiology etc.) of the organism (Xiong et al., 2017b).

The EC50 obtained in our study is higher than concentration of ACy found in the

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environment. But high concentration of pesticide is expected in water bodies near agricultural areas (Martínez-Ruiz and Martínez-Jerónimo, 2017). In addition to this, lipophilic pesticides

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might bioaccumulate in algae and reach a concentration several folds higher than the environmental concentration (DeLorenzo et al., 2001). For instance, Rao and Lal (1987)

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demonstrated rapid accumulation of lipophilic agricultural pesticide in microalgae, with levels reaching 700 folds of the exposed concentration within 48 h. Our study is consistent

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with previous studies where higher EC50 of contaminant compared to environmental concentration has been demonstrated. For example, Andrieu et al., 2015 demonstrated that EC50 of enrofloxacin was as high as 111.00 mg L-1 for Chlorella sp. while the measured environmental concentration of the same contaminant was only 0.68 µg L-1.

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Table 1:

Equation

R2

EC50 (mg L-1)

96 h

ya = 55.96xb - 8.42

0.907

11.00

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Cultivation period

ya - SGR inhibition (%) xb - log transformed ACy concentration

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Median effective concentration (EC50) of ACy for Chlorella sp. at 96 h

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3.2. Effect of ACy on photosynthetic pigment content

Photosynthetic organisms transform light energy into chemical energy by the process of

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photosynthesis. Pigments such as chlorophylls are an integral part of this vital physiological process (Xiong et al., 2017a). Meanwhile, chlorophyll-a(Chl-a) is admitted to be a potential

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biomarker indicating the light harvesting capabilities of plants exposed to contaminants (Chen et al., 2016; Kurade et al., 2016; Oguntimehin et al., 2008). Chlorophyll-a content of

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Chlorella sp. was significantly reduced by 53.50% (p < 0.01) after 96 h exposure to 11.00 mg L-1 of ACy (Fig 2). Chlorophyll-b (Chl-b) is an accessory pigment that trap light energy from

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the sun and transmit it to Chl-a. Reduction of Chl-b content can adversely affect the photosynthesis process (Fang et al., 2018). In the present study, Chl-b content of Chlorella sp. was significantly reduced by 48.00% (p < 0.01) after 96 h exposure to 11.00 mg L-1 of ACy (Fig 2). These results are in agreement with previous reports where cypermethrin exposure greatly reduced Chl-a and Chl-b content in S. obliquus (Li et al, 2005). A decrease in chlorophyll-a content was evident when Chlamydomonas reinhardtii was exposed to a 14

sublethal dose of atrazine and C. mexicana cells exposed to 15 mg L-1 of acephate and imidacloprid for 12 days (Esperanza et al., 2016; Kumar et al., 2016). Carotenoids are accessory photosynthetic pigments which play a crucial role in light harvesting during photosynthesis. They harvest the light which the chlorophyll fails to absorb proficiently and thereby contribute to the smooth functioning of the photosynthesis process (Xiong et al., 2017b). Carotenoid content of Chlorella sp. was significantly reduced (p<0.01) by 66.28% upon exposure to 11.00 mgL-1 of ACy for 96 h (Fig 2). A similar reduction in

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carotenoid content was reported in C. reinhardtii exposed to atrazine and S. obliquus exposed to insecticide cypermethrin (Esperanza et al., 2016; Li et al., 2005).

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The reason for reduction of photosynthetic pigment content in the present study can be explained as follows. Firstly, the increased production of ROS evoked by ACy exposure

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might have directly damaged the pigment structure (Huang et al., 2012). Secondly, ROS might have induced peroxidation of chloroplast membranes resulting in impaired pigment

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synthesis (Fang et al., 2018). This alteration in photosynthetic pigment content of Chlorella sp. by ACy reflects the sensitivity of the photosynthetic apparatus of the microalgae to

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environmental contaminants.

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3.3. Effect of ACy on ROS generation Plants respond to environmental stresses by enhanced generation of reactive oxygen species

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(ROS), which includes free radicals such as superoxide radical (O2.), hydroxyl radical (OH.) and non-radicals such as hydrogen peroxide (H2O2), singlet oxygen (1O2) (Khare et al., 2015; Lesser et al., 2006). In this study, ROS generation upon ACy exposure was studied by FCM analysis of Chlorella sp. using the dye 2ʹ, 7ʹ- dichlorodihydrofluorescein diacetate (H2DCFHDA). As illustrated in Fig. 3(a,b), ACy resulted in a profound increase in intracellular ROS levels depicted by an increase in the percentage of cells with positive DCF fluorescence 15

(DCF +). After 96 h exposure to ACy, the percentage of DCF (+) microalgal cells increased by 2.77 folds compared to control (p<0.05). Enhanced generation of ROS in microalgae on exposure to pesticide has been reported in many studies. Recently, enhanced generation of ROS by pesticide Rac - and S - metolachlor have been reported in S. obiquus (Liu et al., 2017). Electron Transport Activities in chloroplast and plasma membrane are the major site of ROS generation in microalgae under diverse environmental stresses such as salinity, temperature, drought, heavy metals, organic

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pollutants etc. (Das and Roychoudhury, 2014). A significant enhancement of intracellular ROS level was observed in algae such as S. obliquus, C. pyrenoidosa, C. reinhardtii and Closterium ehrenbergii exposed to dibutyl phthalate (plasticizer), aclonifen (biocide),

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chlorine (disinfectant) (Almeida et al., 2017; Gu et al., 2017; Sathasivam et al., 2016).

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Increased generation of ROS can lead to severe cellular consequences such as peroxidation of cellular membranes and degradation of important biomolecules such as pigments, protein,

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carbohydrate, DNA (Kärkönen and Kuchitsu, 2015; Das and Roychoudhury , 2014).

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3.4. Effect of ACy on intracellular lipid accumulation In the present study, intracellular lipid accumulation has been investigated by Flow cytometry analysis in association with Nile Red (NR) dye. There was a significant increase in the

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percentage of cells (p<0.05) with positive NR fluorescence in ACy treated cells compared to

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untreated cells at the end of 96 h (Fig. 3 c,d). In the untreated cultures, only 12.56 % cells showed positive (+) NR fluorescence while in ACy treated cells 75.35% cells showed (+) NR fluorescence. In other words, the percentage of cells with NR fluorescence was 5.99 times higher with respect to untreated cultures after 96 h exposure to 11.00 mgL-1 of ACy. Intracellular lipid accumulation demonstrated in the present study can be attributed to increased production of ROS in Chlorella sp. cells by ACy exposure. ROS produced under 16

stress condition has been admitted to be a mediator of intracellular lipid accumulation in organisms like algae (Shi et al., 2017). Microalgae might use these accumulated lipids to absorb the lipophilic xenobiotic as a detoxification mechanism for minimizing their bioavailability (Yang et al., 2002). These microalgae with accumulated pesticide can be consumed by organisms of the successive trophic resulting in biomagnifications which is extremely dangerous for the ecosystem (Guanzon et al., 1996). Increase in lipid accumulation was also reported in A. gracilis exposed to the pesticide chlorpyrifos (Asselborn et al., 2015).

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In addition to this, the presence of large lipid bodies, as an indication of lipid accumulation, was observed in S. microspina exposed to diesel fuel oil (Tukaj et al., 1998) and Anabaena variabilis and A. flos-aquae exposed to zinc (Rachlin et al., 1985). Recently, Duan et al.

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(2017) demonstrated an increase in the number of lipid droplets in marine microalgae

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exposed sub lethal dose of phenol.

3.5. Effect of ACy on antioxidant enzyme activity and lipid peroxidation

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Overproduction of ROS in microalgae on exposure to environmental contaminants is a well known phenomenon. In order to combat the deleterious effects of ROS, plants have

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developed a well defined system comprising antioxidant molecules and enzymes. Both SOD and CAT are important antioxidant enzymes involved in ROS scavenging and a potential

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biomarker of environmental pollution (Obermeier et al., 2015). In our study, ACy exposure resulted in significant increase of both SOD and CAT activity at the end of 96 h (Fig. 4 a, b).

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SOD activity was increased by 4.78 times while CAT by 4.34 times relative to the control. SOD catalyzes the production of H2O2 and O2 by disproportionation of superoxide anions O2.- which is further dismutated into H2O and O2 by the catalytic activity of CAT. A coordinated function of SOD-CAT redox system plays a significant role in ROS scavenging (Ballesteros et al., 2009; Ken et al., 2005). Li et al. (2005) demonstrated a significant stimulation in SOD activity by the pyrethroid cypermethrin in S. obliquus and recommended 17

it as a biomarker of environmental pollution. Increase in SOD activity in our study is an indirect evidence of overproduction of superoxide anion, O2.-. Increase in CAT activity in the present study indicates initiation of cellular tolerance to oxidative stress in order to cope with the excessive production of H2O2 (Kurade et al., 2016). However, CAT may not be able to completely catalyze the dismutation of H2O2 and the residual H2O2 might lead to oxidative damage of the cellular organelles (e.g. chloroplast) and important biomolecules such as carotenoid. Sáenz et al. (2012) noted an increased CAT

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activity in C. vulgaris on exposure to pyrethroid Cyfluthrin which is consistent with our results. Chen et al. (2016) also demonstrated a notable acceleration in SOD and CAT activity of the freshwater microalgae C. pyrenoidosa and Merismopedia sp. exposed to chlorpyrifos.

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A significant enhancement in SOD and CAT activity was also observed in C. mexicana cells

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exposed to 15mg L-1 of acephate and imidacloprid for 12 days (Kumar et al., 2016). ROS generated under chemical stress attacks the polyunsaturated fatty acids present in

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cellular membranes resulting in lipid peroxidation (Chen et al., 2006). Malondialdehyde (MDA) is a product of lipid peroxidation and a biomarker of this ROS induced damage

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(Wang et al., 2012). In the present study, exposure of Chlorella sp. to 11.00 mg L-1 of ACy for 96 h significantly increased (p< 0.001) MDA content in the microalgal cells (Fig. 4 c).

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The MDA was noted to increase by 7.36 folds with respect to the control at the end of the cultivation period. This result indicates that ACy might have caused peroxidation of

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membrane lipids in Chlorella sp. Peroxidation results in an increase of membrane permeability and easier penetration of contaminants into the cell (Tanq et al., 2016, WongEkkabut et al., 2007). Lipid peroxidation demonstrated in the present study might be due to increased production of ROS by ACy exposure. Wang et al., (2012) reported an increased MDA content in microalgal cells on exposure to cypermethrin which is consistent with our results. A profound increase in MDA content was also observed in microalgal cells exposed 18

to dibutylphthalate and ciprofloxacin (Gu et al., 2017; Xiong et al., 2017a). Increased MDA content recorded in the present study indicates ACy induced instability in the cellular membranes of the microalgae and thereby adversely affecting its physiological features. 3.7 Relationship between various end points evaluated in our study Based on our findings a tentative relationship between the evaluated endpoints is shown in Fig .5. Exposure of ACy to the microalgae resulted in an enhanced generation of ROS which

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might have created oxidative stress in the organism resulting in peroxidation of membrane lipids and increase in accumulation of MDA. Peroxidation of thylakoid lipids by ROS can be a probable reason for decrease in photosynthetic pigment content. The activity of antioxidant

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enzymes (SOD, CAT) was increased probably to scavenge the excess ROS produced and maintaining ROS homeostasis. H2O2 produced due to disproportionation of superoxide anions

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(O2.- ) by SOD may further be dismutated into H2O and O2 by CAT. However, CAT may not be able to completely catalyze the dismutation of H2O2 and the residual H2O2 might have

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led to oxidative damage of the cellular organelles (e.g. chloroplast) and important biomolecules such as chlorophyll and carotenoid. Further, increased ROS production induced

microalgae.

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4. Conclusion

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by ACy exposure might have acted as a mediator for accumulation of intracellular lipid in the

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In the present study, ACy was found to be harmful to Chlorella sp. ACy exposure provoked increased ROS production, altered antioxidant enzymatic response, reduced pigment content, and induced lipid peroxidation and intracellular lipid accumulation in Chlorella sp. Although concentration of ACy reported in natural environment is low, continuous application of pesticides in crops, accidental spillage, spray drift and unsafe disposal of expired pesticides can result in build-up of pesticide in the environment. Thus, microalgae such as Chlorella sp. 19

may be exposed to ACy concentration higher than those reported in natural waters. Such exposure to high ACy concentration can result in growth inhibition, oxidative stress and impaired photosynthesis in microalgae. Further, intracellular lipid accumulation induced by ACy may act as site for lipophilic pollutants to bioaccumulate and may contribute to biomagnification which is dangerous for the ecosystem. Thus the results of the present investigation effectively illustrated the ecotoxicological effects of ACy on microalgae which will be helpful in understanding the ecological risk of ACy to the primary producers in the

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aquatic ecosystems. Acknowledgements

The authors thankfully acknowledge Department of Science and Technology (Government of

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India) - Fund for Improvement of S&T Infrastructure in Higher Educational Institutions

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(DST-FIST) [SR/FST/LSI-666/2016(C)] and University Grants Commission - Special Assistance Programme (UGC-SAP) [F.4-7/2016/DRS-1 (SAP-II)] for financial support and

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the Head of department, Biotechnology and Bioinformatics, North-Eastern Hill University for providing the necessary facilities. Authors thank Guwahati Biotech Park, Assam, India for

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support in HPLC analysis. Also, the authors thank Dr. Anna Aksmann, University of Gdansk, Poland and Dr. Krishnappa Rangappa, ICAR Research Complex for NER Region,

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Meghalaya, India for critically reviewing and editing the manuscript.

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References

Aebi, H., 1984. Catalase in vitro. Methods. Enzymol. 105, 121-126. Alemán-Nava, G.S., Casiano-Flores, V.H., Cárdenas-Chávez, D.L., Díaz-Chavez, R., Scarlat, N., Mahlknecht, J., Dallemand, J.F., Parra, R., 2014. Renewable energy research progress in Mexico: a review. Renew. Sust. Energ. Rev. 32, 140-153.

20

Almeida, A.C., Gomes, T., Langford, K., Thomas, K.V., Tollefsen, K.E., 2017. Oxidative stress in the algae Chlamydomonas reinhardtii exposed to biocides. Aquat. Toxicol.189, 50-59. Andrieu, M., Rico, A., Phu, T.M., Huong, D. T. T , Phuong, N.T, Van den Brink, P.J.2015. Ecological risk assessment of the antibiotic enrofloxacin applied to Pangasius catfish farms in the Mekong Delta, Vietnam. Chemosphere.119, 407-414. Asselborn, V., Fernandez, C., Zalocar, Y., Parodi, E.R., 2015. Effects of chlorpyrifos on the growth and ultrastructure of green algae, Ankistrodesmus gracilis. Ecotoxicol. Environ. Saf. 120, 334-341.

ro of

Ballesteros, M.L., Wunderlin, D.A., Bistoni, M.A., 2009. Oxidative stress responses in different organs of Jenynsia multidentata exposed to endosulfan. Ecotoxicol.Environ. Saf. 72, 199-205. Bischoff, H.W., Bold, H.C., 1963. Phycological Studies IV. In: Some soil algae from enchanted rock and related algal species. University of Texas Publication, Austin, 195.

re

-p

Bradford, M.M., 1976. A rapid and sensitive method for the quantification of microgram quantity of proteins utilising the principle of protein dye binding. Anal. Biochem.72, 248–254.

lP

Centner, T.J., 2018. Canceling pesticide registrations and revoking tolerances: The case of chlorpyrifos. Environ. Toxicol. Phar. 57, 53-61.

na

Chen, C. Y., Lin, K. C., & Yang, D. T., 1997. Comparison of the relative toxicity relationships based on batch and continuous algal toxicity tests. Chemosphere, 35(9), 1959-1965.

ur

Chen, H.L., Hsu, C.H., Hung, D.Z., Hu, M.L., 2006. Lipid peroxidation and antioxidant status in workers exposed to PCDD/Fs ofmetal recovery plants. Sci Total Environ. 372,12-19.

Jo

Chen, S., Chen, M., Wang, Z., Qiu, W., Wang, J., Shen, Y., Wang, Y., 2016. Toxicological effects of chlorpyrifos on growth, enzyme activity and chlorophyll a synthesis of freshwater microalgae. Environ. Toxicol. Pharmacol. 45, 179-186. Commission of the European Communities, 1996. Technical Guidance Document in Support of Commission Directive 93/67/EEC on Risk Assessment for New Notified Substances and Commission Regulation (EC) No 1488/94 on Risk Assessment. Office for Existing Substances. Part II; Environmental Risk Assessment. Office for Official Publications of the European Communities, Luxembourg. Das, K., Roychoudhury, A., 2014. Reactive oxygen species (ROS) and response of antioxidants as ROS-scavengers during environmental stress in plants. 2, 53.

21

Delorenzo, M.E., Scott, G.I., E. Ross.P.E 2001. Toxicity of pesticides to aquatic microorganisms: a review.Environ. Toxicol. Chem.20 (1), 84-98. Dhindsa, R.S., Dhindsa, P.P., Thorpe, T.A., 1981. Leaf senescence: correlated with increased level of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. J. Exp. Bot. 32, 93-101. Duan, W., Meng, F., Lin, Y., Wang, G., 2017. Toxicological effects of phenol on four marine microalgae. Environ. Toxicol. Phar. 52, 170-176. Eguchi, K., Nagase, H., Ozawa, M., Endoh, Y.S., Goto, K., Hirata, K., Miyamoto, K., Yoshimura, H., 2004. Evaluation of antimicrobial agents for veterinary use in the ecotoxicity test using microalgae. Chemosphere. 57, 1733-1738.

ro of

Esperanza, M., Seoane, M., Rioboo, C., Herrero, C., Cid, A., 2016. Early alterations on photosynthesis-related parameters in Chlamydomonas reinhardtii cells exposed to atrazine: A multiple approach study. Sci. Total. Environ. 554-555, 237-245.

-p

European Commission, 2004. Review report for the active substance alpha-cypermethrin Annex B, B.8 Ecotoxicology. SANCO/4335/2000 Final Report. Health and Consumer Protection Directorate, Brussels, Belgium.

lP

re

Fang, B., Shi, J., Qin, L., Feng,M., Cheng,D., Wang,T., Zhang,X.,2018. Toxicity evaluation of 4,4′ di-CDPS and 4,4′-di-CDE on green algae Scenedesmus obliquus: growth inhibition, change in pigment content, and oxidative stress. Environ. Sci. Pollut. Res. 25,15630–15640. Fatma, T., Khan, M.A., Choudhary, M., 2007. Impact of environmental pollution on cyanobacterial proline content. J. Appl. Phycol.19, 625-629.

ur

na

Feckler, A., Rakovic, J., Kahlert, M., Tröger, R., Bundschuh, M. 2018. Blinded by the light: Increased chlorophyll fluorescence of herbicide-exposed periphyton masks unfavourable structural responses during exposure and recovery. Aquat. Toxicol. 203: 187 – 193.

Jo

Ferreira, P., Fonte, E., Soares, M.E., Carvalho, F., Guilhermino, L., 2016. Effects of multistressors on juveniles of the marine fish Pomatoschistus microps: gold nanoparticles, microplastics and temperature. Aquat. Toxicol. 170, 89–103. Fogg, G.E., 1975. Algal Cultures and Phytoplankton Ecology. The University of Wisconsin Press, USA. Fu, L., Huang, T., Wang, S., Wang, X., Su, L., Li, C., & Zhao, Y., 2017. Toxicity of 13 different antibiotics towards freshwater green algae Pseudokirchneriella subcapitata and their modes of action. Chemosphere, 168, 217-222. Gao, Y., Lim, T.K., Lin, Q., Li, S.F.Y., 2016. Identification of cypermethrin induced protein changes in green algae by iTRAQ quantitative proteomics. J .Proteomics. 139, 67-76. 22

Garforth, B.M & Woodbridge, A.P., 1984. Spray drift from an aerial application of Fastac; fate and biological effects in an adjacent freshwater ditch. Sittingbourne, Shell Research (SBGR 84.055). Griffiths, M.J., Garcia, C., Hille, R.P., Harrison, S.T.L., 2011. Interference by pigment in the estimation of microalgal biomass concentration by optical density. J. Microbiol. Methods. 85, 119-123 Gu, S., Zheng, H., Xu, Q., Sun, C., Shi, M., Wang, Z., Li, F., 2017. Comparative toxicity of the plasticizer dibutyl phthalate to two freshwater algae. Aquat. Toxicol. 191, 122130.

ro of

Guanzon, N.G., Fukuda, M., Nakahara, H., 1996. Accumulation of agricultural pesticides by three freshwater microalgae. Fish.Sci. 62, 690-697. Hartnik, T., Sverdrup, L.E., Jensen J., 2008.Toxicity of the pesticide alpha-cypermethrin to four soil non-target invertebrates and implications for risk assessment. Environ. Toxicol. Chem. 27, 1408-1415.

-p

Health, R.L., Packer, G., 1968. Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125, 189-198.

re

Huang, L.D., Lu, D.H., Diao, J.L., Zhou, Z.Q. 2012. Enantioselective toxic effects and biodegradation of benalaxyl in Scenedesmus obliquus. Chemosphere. 87, 7-11.

lP

Huang, L.D., Lu, D.H., Zhang, P., Diao, J.L., Zhou, Z.Q., 2012. Enantioselective toxic effects of hexaconazole enantiomers against in Scenedesmus obliquus. Chirality. 24,610-614.

na

Janero, D.R., 1990. Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free. Radical. Biol. Med. 9, 515540.

ur

Kabra, A.N., Ji, M.K., Choi, J., Kim, J.R., Govindwar, S.P., Jeon, B.H., 2014. Toxicity of atrazine and its bioaccumulation and biodegradation in a green microalga, Chlamydomonas mexicana. Environ. Sci. Pollut. Res. 21, 12270-12278.

Jo

Källqvist, T., Svenson, A., 2003. Assessment of ammonia toxicity in test with the microalga, Nephroselmis pyriformis, Chlorophyta. Water Res. 37, 477-484. Kärkönen, A., Kuchitsu, K., 2015. Reactive oxygen species in cell wall metabolism and development in plants. Phytochemistry .112, 22-32. Ken, C.F., Hsiung, T.M., Huang, Z.X., Juang, R.H., Lin, C.T., 2005. Characterization of the Fe/Mn-superoxide dismutase from diatom (Thallassiosira weissflogii); cloning, expression, and property. J. Agric. Food Chem. 9, 1470-1474.

23

Khare, T., Kumar, V., Kishor, P.B.K., 2015. Na+ and Cl- ions show additive effects under NaCl stress on induction of oxidative stress and the responsive antioxidative defense in rice. Protoplasma .252 (4), 1149-1165. Knauert, S., Knauer, K., 2008. The role of reactive oxygen species in copper toxicity to two freshwater green algae. J. Phycol. 44, 311-319. Kocaman, A.Y., Topaktas, M., 2009. The in vitro genotoxic effects of a commercial formulation of α-cypermethrin in human peripheral blood lymphocytes. Environ. Mol. Mutagen. 50, 27-36.

ro of

Kumar, M.S., Aabra, A.N., Min, B., Ei-Dalatony, M.M., Xiong, J., Thajuddin, N., Lee, D. S., Jeon, B.H., (2016). Insecticide induced biochemical changes in freshwater microalga Chlamydomonas mexicana. Environ. Sci. Pollut. Res. 23, 1091-1099. Kurade, M.B., Kim, J.R., Govindwar, S.P., Jeon, B.H. 2016. Insights into microalgae mediated biodegradation of diazinon by Chlorella vulgaris: Microalgal tolerance to xenobiotic pollutants and metabolism. Algal Res. 20, 126-134.

-p

Lesser, M.P., 2006. Oxidative stress in marine environments: biochemistry and physiological ecology. Annu. Rev. Physiol. 68, 253-278.

re

Li, X., Ping, X., Sheng, X.M., Wu, Z.B., Xie, L.Q., 2005. Toxicity of cypermethrin on growth, pigments, and superoxide dismutase of Scenedesmus obliquus. Ecotoxicol. Environ. Saf. 60, 188-192.

lP

Liu, H., Xia, Y., Cai, W., Zhang, Y., Zhang, X., Du, S., 2017. Enantioselective oxidative stress and oxidative damage caused by Rac- and S-metolachlor to Scenedesmus obliquus. Chemosphere. 173, 22-30.

na

Marino, D., Ronco, A., 2005. Cypermethrin and chlorpyrifos concentration levels in surface water bodies of Pampa Ondulada, Argentina. Bull. Environ. Contam. Toxicol.74, 820826.

ur

Martinez, R.S., DiMarzio, W.D., Saénz, M.E., 2015. Genotoxic effects of commercial formulations of Chlorpyrifos and Tebuconazole on green algae. Ecotoxicology. 24, 45–54.

Jo

Martínez-Ruiz, E.B., Martínez-Jerónimo, F., 2017. Exposure to the herbicide 2, 4-D produces different toxic effects in two different phytoplankters: A green microalga (Ankistrodesmus falcatus) and a toxigenic cyanobacterium (Microcystis aeruginosa). Sci. Total. Environ. 619-620, 1566-1578. Mofeed, J., Mosleh, Y.Y., 2013. Toxic responses and antioxidative enzymes activity of Scenedesmus obliquus exposed to fenhexamid and atrazine, alone and in mixture. Ecotoxicol. Environ. Saf. 95, 234-240.

24

Obermeier, M., Schroder, C.A., Helmreich, B., Schroder, P., 2015. The enzymatic and antioxidative stress response of Lemna minor to copper and a chloroacetamide herbicide. Environ. Sci. Pollut. R. 22 (23), 18495-18507. Oguntimehin, I., Nakatani, N., Sakugawa, H., 2008. Phytotoxicities of fluoranthene and phenanthrene deposited on needle surfaces of the evergreen conifer, Japanese red pine (Pinus densifloraSieb et Zucc.). Environ. Pollut. 154, 264-271. Oros, D.R., Werner, I., 2005. Pyrethroid Insecticides: An Analysis of Use Patterns, Distributions, Potential Toxicity and Fate in the Sacramento-San Joaquin Delta and Central Valley, San Francisco Estuary Institute Oakland, CA.

ro of

Rachlin, J.W., Jensen, T.E., Warkentine, B.E., 1985. Morphometric analysis of the response of Anabaena flos-aquae and Anabaena variabilis (Cyanophyceae) to selected concentrations of zinc. Arch.Environ.Contam.Toxicol. 14, 395–402. Ramadass, K., Megharaj, M., Venkateswarlu, K., Naidu, R., 2016. Sensitivity and antioxidant response of Chlorella sp. MM3 to used engine oil and its water accommodated fraction. Bull. Environ. Contam. Toxicol. 97, 71-77.

-p

Rao, V.V.S.N., Lal, R., 1987. Uptake and metabolism of insecticides by blue-green algae Anabaena and Aulosira fertilissima. Microbios. Lett. 36,143-147.

lP

re

Rodger, P.A., Simpson, I., Official, R., Ardales, S., Jimenez, R., 1994. Effects of pesticides on soil and water microflora and mesofauna in wetland rice fields: a summary of current knowledge and extrapolation to temperate environments. Aust. J. Exp. Agric. 34, 1057-1068.

na

Sabater, C., Carrasco, J.M., 2001. Effects of pyridaphenthion on growth of five freshwater species of phytoplankton: a laboratory study. Chemosphere. 44, 1775-1781.

ur

Sáenz, M.E., Marzio, W.D.D, Alberdi, J.L., 2012. Effects of a commercial formulation of cypermethrin used in biotech soybean crops on growth and antioxidant enzymes of freshwater algae. IJEP.2 (1), 15-22.

Jo

Sáenz, M.E., Marzio, W.D.D., Alberdi, J.L. Assessment of Cyfluthrin commercial formulation on growth, photosynthesis and catalase activity of green algae. Pesticide. Biochem. Physiol. 104, 50-57. Sathasivam, R., Ebenezer, V., Guo, R., Ki, J.S., 2016. Physiological and biochemical responses of the freshwater green algae Closterium ehrenbergii to the common disinfectant chlorine. Ecotox. Environ. Safe. 133, 501-508. Satyavani, G., Chandrasehar, G., Varma, K.K., Goparaju, A., Ayyappan S., Reddy, P.N., Murthy, P.B., 2012. Toxicity Assessment of Expired Pesticides to Green Algae Pseudokirchneriella subcapitata. ISRN. Toxicol. 2012, 10.

25

Shi, K., Gao, Z., Shi, T.Q, Song, P., Ren, L.J, Huang, H., Ji, X.J, 2017. Reactive oxygen species-mediated cellular stress response and lipid accumulation in oleaginous microorganisms: the state of the art and future perspectives. Front. Microbiol. 8(793), 1-9. Smythers, A.L., Garmany, A., Perry, N.L., Higginbotham, E.L., Adkins, P.E., Kolling, D.R.J., 2019. Characterizing the effect of Poast on Chlorella vulgaris, a non target organism. Chemosphere. 219, 704-712. Solomon, K.R., Giddings, J.M., Maund, S.J., 2001. Probabilistic risk assessment of cotton pyrethroids: I. Distributional analyses of laboratory aquatic toxicity data. Environ. Toxicol. Chem. 20, 652-659.

ro of

Sosak-Swiderska, B., Tyrawska, D., Maslikowska, B., 1998. Microalgal ecotoxicity test with 3, 4-dichloroaniline. Chemosphere. 37, 2975-2982. Tang, S., Yin, H., Chen, S., Peng, H., Chang, J., Liu, Z., Dang, Z., 2016. Aerobic degradationof BDE-209 by Enterococcus casseliflavus: isolation, identification and cell changes during degradation process. J. Hazard. Mater. 308, 335-342.

re

-p

Tayemeh, M. B., Esmailbeigi, M., Shirdel, I., Joo, H. S., Johari, S. A., Banan, A., ... & Tabarrok, M., 2020. Perturbation of fatty acid composition, pigments, and growth indices of Chlorella vulgaris in response to silver ions and nanoparticles: A new holistic understanding of hidden ecotoxicological aspect of pollutants. Chemosphere. 238, 124576.

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Tukaj, Z., Bohdanowicz, J., Aksmann, A., 1998. A morphometric and stereological analysis of ultrastructural changes in two Scenedesmus (Chlorococcales, Chlorophyta) strains subjected to diesel fuel oil pollution. Phycologia. 37, 388-393.

ur

na

USEPA (U.S.Environmental Protection Agency), 1989. In short-term methods for estimating the chronic toxicity of effluents and receiving water to freshwater organisms. Environmental Monitoring and Support Laboratory Office of Research and Development, Cincinnati, Ohio.

Jo

Villem, A., 2011.Algae Pseudokirchneriella subcapitata in environmental hazard evaluation of chemicals and synthetic nanoparticles. Ph.D. Thesis. Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences. Wang, Z.H., Nie, X.P., Yue, W.J., Li, X., 2012. Physiological responses of three marine microalgae exposed to cypermethrin.Environ.Toxicol. 27(10), 563-572. Wellburn, A.R., 1994. The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. J. Plant Physiol. 144, 307-313.

26

Wong-ekkabut, J., Xu, Z., Triampo, W., Tang, I.M., Tieleman, D.P., Monticelli, L., 2007. Effect of lipid peroxidation on the properties of lipid bilayers: a molecular dynamics study. Biophys. J. 93, 4225- 4236. Xiong, J. Q., Kurade, M. B., & Jeon, B. H., 2017c. Biodegradation of levofloxacin by an acclimated freshwater microalga, Chlorella vulgaris. Chem. Eng. J. 313, 1251-1257. Xiong, J.Q., Kurade, M.B., Abou-Shanab, R.A.I., Ji, M.K., Choi, J., Kim, J.O., Jeon, B.H., 2016. Biodegradation of carbamazepine using freshwater microalgae Chlamydomonas mexicana and Scenedesmus obliquus and the determination of its metabolic fate, Bioresour. Technol. 205,183-190.

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Xiong, J.Q., Kurade, M.B., Jeon, B.H., 2017b . Ecotoxicological effects of enrofloxacin and its removal by monoculture of microalgal species and their consortium. Environ. Pollut. 226, 486-493. Xiong, J.Q., Kurade, M.B., Kim, J.R., Roh, H.S., Jeon, B.H., 2017a. Ciprofloxacin toxicity and its co-metabolic removal by a freshwater microalga Chlamydomonas mexicana. J. Hazard. Mater.323, 212-219.

re

-p

Yang, S., Wu, R.S.S.B., Kong, R.Y.C., 2002. Physiological and cytological responses of themarine diatom Skeletonema costatum to 2, 4-dichlorophenol. Aquat. Toxicol. 60, 33-41.

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Yao, G., Gao, J., Zhang, C., Jiang, W., Wang,P., Liu,X., Liu D., Zhou, Z., 2019. Enantioselective degradation of the chiral alpha-cypermethrin and detection of its metabolites in five plants. Environ. Sci. Pollut. Res. 26, 1558-1564.

na

Yao, G., Jing, X., Liu. C., Wang, P., Liu, X., Hou, Y., Zhou, Z., Liu, D., 2017. Enantioselective degradation of alpha-cypermethrin and detection of its metabolites in bullfrog (rana catesbeiana). Ecotox. Environ. Safe. 141, 93-97.

Jo

ur

Yuan, N.N., Chen, S.N., Zhai, L.X., Schnabel, G., Yin, L.F., Luo, C.X., 2013. Baseline sensitivity of Monilia yunnanensis to the DMI fungicides tebuconazole and triadimefon. Eur. J. Plant. Pathol. 136, 651-655.

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Figure 1: Effects of ACy concentrations on growth of Chlorella sp. in terms of cell density

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during 0-96 h of cultivation (a) and specific growth rate at end of 96 h cultivation (b). Error bars represent standard deviation (n=3). Asterisks (*) indicates significant differences relative

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to control (* ̶ p < 0.05, ** ̶ p< 0.01, *** ̶ p< 0.001).

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Figure 2: Chlorophyll-a, Chlorophyll-b and carotenoid content in Chlorella sp. cells in control cultures (0 mg L-1 ACy) and treated cultures (11.00 mg L-1 ACy) after 96 h exposure. Error bars represent standard deviation (n=3). Asterisks (*) indicates significant differences

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relative to control (** ̶ p< 0.01).

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Figure 3: Flow Cytogram showing: ROS production in Chlorella sp. (a) Control cells without ACy treatment (b) Cells treated with ACy (11.00 mgL-1), Intracellular lipid accumulation in (c) Control cells without ACy treatment (d) Cells treated with ACy (11.00 mg L-1).

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Figure 4: Effect of ACy on (a) SOD activity, (b) CAT activity and (c) MDA in Chlorella sp. at 11.00 mg L-1 after 96 h exposure. Error bars represent standard deviation (n=3). (** ̶ p<

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0.01, *** ̶ p< 0.001).

Figure 5: A conceptual framework showing relationship between various endpoints evaluated in the present study. 30