S C I E N C E O F T H E T O T A L E N V I R O N M E N T 4 0 3 ( 2 0 0 8 ) 1 3 0 – 1 3 8

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Endosulfan induced biochemical changes innitrogen-fixing cyanobacteria

Satyendra Kumar, Khalid Habib, Tasneem Fatma⁎

Cyanobacterial Biotechnology and Environmental Biology Laboratory, Department of Biosciences, Jamia Millia Islamia (Central University),New Delhi –110025, India

A R T I C L E I N F O

⁎ Corresponding author. Tel.: +91 (011) 269219E-mail addresses: satya3369@gmail.com, sa

fatmacbl@gmail.com (T. Fatma).

0048-9697/$ – see front matter © 2008 Elsevidoi:10.1016/j.scitotenv.2008.05.026

A B S T R A C T

Article history:Received 3 January 2008Received in revised form 2 May 2008Accepted 19 May 2008Available online 26 June 2008

Pesticide contamination in aquatic ecosystem including paddy fields is a serious globalenvironmental concern. Cyanobacteria are also affected by pesticides as non- target organism.For better exploitation of cyanobacteria as biofertiliser, it is indispensable to select tolerantstrains along with understanding of their tolerance. Three cyanobacterial strains viz. Aulosirafertilissima, Anabaena variabilis and Nostoc muscorum were studied for their stress responses toan organochlorine pesticide ‘endosulfan’ with special reference to oxidative stress, role ofproline and antioxidant enzymes in endosulfan induced free radical detoxification. Reductionin growth, photosynthetic pigments and carbohydrate of the test microorganisms wereaccompanied with increase in their total protein, proline, malondialdehye (MDA), superoxidedismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) in higher endosulfan doses.Increased amount of MDA is indicative of formation of free radicals, while increased level ofCAT,APX, SOD andproline indicated their involvement in free radical scavengingmechanism.In lower concentrations, test pesticide showed increase in photosynthetic pigments. Order oftolerance was Nostoc muscorumNAnabaena variabilisNAulosira fertilissima.

© 2008 Elsevier B.V. All rights reserved.

Keywords:CyanobacteriaInsecticideGrowthphotosynthetic pigmentsCarbohydrateProteinProlineFree radicalsAntioxidant

1. Introduction

The increasing use of pesticides in agriculture demandsinvestigation to examine the effect of pesticides on the non-target soil micro-organisms including nitrogen fixing cyano-bacteria. Cyanobacteria have been applied in rice fields as abiofertilizer for better yield of paddy (Relwani, 1963). Weedi-cides, fungicides and insecticides used for plant protection inrice fields affect adversely on the cyanobacterial population(Anand, 1980; Stratton, 1987; Kolte and Goyal, 1990).

Parathion-methyl, thiobencarb, paraquat reduce growth,biochemical production (pigment, carbohydrate, protein etc.),heterocyst differentiation and nitrogen fixation in cyanobac-teria (Padhy, 1985; Ahluwalia, 1988).

08; fax: +91 (011) 2698022t@iitk.ac.in (S. Kumar), kh

er B.V. All rights reserved

Endosulfan is most popular amongst the organochlorineinsecticides. It is being extensively used in crops field due to itsbroadspectrumofactivity and relatively lowcost.Application rateof 35 EC endosulfan is 560ml in 100 litres of water per acre in ricefield (ICAR).Dependingon the typeof cropand thearea inwhich itis grown, application rates usually range between 0.45 kg ai and1.4 kg/ha, but both smaller and larger doses have occasionallybeen used (Hoechst, 1977). It is often repeatedly used on crops inone crop period leading to build upof its residues both in crop andsoil (Kwon and Penner, 1995). It has been reported that extensiveusage of endosulfan in various tropical and subtropical countriesfor control of the insect population, is accompanied withreduction in non-targetmicrobial population including cyanobac-teria (Satish and Tiwari, 2000; Shetty et al., 2000).

9; mobile: +91 9891408366, +91 9868301050.alid_habib7@yahoo.com (K. Habib), fatma_cbl@yahoo.com,

.

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Most environmental stresses are affecting on the productionof active oxygen species in plants, causing oxidative stress(Smirnoff, 1993; Hendry, 1994; Bartosz, 1997). The balancebetween the production of activated oxygen species and thequenchingactivity of antioxidant isupset,whichoften results inoxidative damage (Del-Rio et al., 1991; Del Vos et al., 1992;Smirnoff, 1993). Among the four major active oxygen species(superoxide radical O-

2, hydrogen peroxide H2O2, hydroxylradical OH and singlet oxygen 1O2) H2O2 and the hydroxylradical are most active, toxic and destructive (Smirnoff, 1993).Under normal circumstances, concentration of oxygen radicalsremain low because of the activity of protective enzymes,including superoxide dismutase, catalase and ascorbate perox-idase (Asada, 1984) but under stress conditions imposed byphysical, chemical and biological pollutants this balance mayget disturbed, causing enhancement of detrimental processes.Photosynthetic cells are prone to oxidative stress because theycontain an array of photosensitizing pigments and they bothproduce and consume oxygen. The photosynthetic electrontransport system is themajor source of active oxygen species inplant tissues (Asada, 1997), have the potential to generatesinglet oxygen 1O2 and superoxide O-

2. The main cellularcomponents susceptible to damage by free radicals are lipids(peroxidation of unsaturated fatty acids in membranes), pro-teins (denaturation), carbohydrates and nucleic acids (Olgaetal., 2003).The formationof ROS ispreventedbyanantioxidantsystem: low molecular mass antioxidants (ascorbic acid,glutathione, and tocopherols), enzymes regenerating thereduced forms of antioxidants, and ROS-interacting enzymessuch as SOD, peroxidases and catalases. Though considerablework has been done on pesticide induced inhibitory effect ongrowth, photosynthetic pigments, photosynthesis and N2 fixa-tion but, to the best of our knowledge there is no report oninsecticide, particularly endosulfan induced ROS generationand their detoxification in resistant heterocystous cyanobac-teria. Only non heterocystous cyanobacteria Plectonema borya-num has been studied recently with reference to endosulfaninduced free radical generation and their detoxification (Prasadet al., 2005). From our lab heterocystous cyanobacteria Westiel-lopsis prolifica–Janet strain–NCCU331 has been reported todevelop resistance against pyrethroid pesticide with the helpof proline (Fatma et al., 2007).

We hypothesize that in non target cyanobacterial hetero-cystous nitrogen fixers, common paddy crop pesticide-endo-sulfan impart detrimental effect through free radicalmediatedoxidative stress and the universal osmoprotectant prolineplays significant role in resistance development againstendosulfan in addition to SOD,APX, CATantioxidant enzymes.

2. Materials and Methods

2.1. Pesticide

For evaluating pesticide toxicity, endosulfan (35 % E.C.) wasseparately added to the fresh medium in calculated amountsto obtain final concentration of 2.5, 5, 7.5, 10, 12.5 and 15 µg/ml.For ‘control’ sets test microorganism were grown withoutadding the pesticide. Commercial grade ‘endosulfan’ (35% E.C)was procured from Excel Industries Limited, Mumbai (India).

2.2. Test strains

Three cyanobacterial strains viz. Aulosira fertilissima, Anabaenavariabilis and Nostoc muscorum were procured from NationalCenter for Conservation and Utilization of Blue Green Algae,Indian Agricultural Research Institute, New Delhi, India. Thetest strains were raised in BG-11 medium (pH 7.3) withoutSodium nitrate (Stainer et al., 1971). The flask and media weresterilized in anautoclave (Yorco, India)maintaining 15 lb/in2 orKg/ cm2 pressure for 15 minutes. 50 ml inoculums weresuspended in 500 ml sterile medium taken in 1000 ml EMflask (three sets) maintaining (O.D-0.4±0.1) after dilution at560 nm. Culture were allowed to grow for 20 days at 30±2 °Cunder light intensity of 2000±200 lux provided by 20 Wfluorescent tubes following a 16: 8 h light/dark regime.Repeated shaking was done at regular intervals. The biomasswas harvested by filtration through fine nylon cloth, washedtwice with distilled water to remove the remaining pesticide.

2.3. Growth measurement

The growth of the test cyanobacterial strains was determinedover a period of 20 days by recording optical density of the 5mlalgal suspension at 560 nm, on a UV–VIS Spectrophotometer –SPECORD 200 at 4 days interval. The growth absorbance datawas supported by biomass data (dry weight).

2.4. Biochemical analysis

Biochemical analysis of 20 days old harvested bio-mass of testorganismsunder stress and control conditionswas carried out intriplicate for evaluating chlorophyll, carotenoid, phycobilipro-tein, carbohydrate, total protein, proline, MDA, SOD, APX, CAT.

2.5. Chlorophyll (Mackinney, 1941)

Extraction was made using 5 mg dry weight in 10 ml 95%methanol in the test tube that was placed in a water bath at65 °C for 30 minutes. The pellet was discarded and theabsorbance of the supernatant was observed at 650 nm and665 nm against 95% methanol as blank.

2.6. Carotenoid (Hellebust and Craige, 1978)

Using 5 mg dry biomass in 10 ml 85% acetone carotenoid wasextracted. Repeated freezing and thawing was done for celldisruption. The absorbance of the supernatant was observedat 450 nm against 85 % acetone as blank.

2.7. Phycobiliprotein (Siegelman and Kygia, 1978)

Repeated freezing and thawing 5 mg of biomass in 10 ml 0.1 Mphosphate buffer was done for phycobiliprotein extract. Thesuspension was centrifuged at 3000-5000 rpm for 5 minutes.The absorbance of the blue supernatant was observed at562 nm, 615 nm and 652 nm.

2.8. Carbohydrate (Spiro, 1966)

The algal sample (1 mg) was taken in a test tube and 1.25 mldoubledistilledwaterwasaddedto it.To1.25mlblank /standard /

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sample solution added 4.0ml of anthrone reagent and placed in aboiling water bath for 8 -10 minutes. The absorbance of thesupernatant was observed at 620 nm against blank. Thecarbohydrate content was evaluated from concentration of theglucose solution known from the standard curve.

2.9. Protein (Lowry et al., 1951)

The algal mass (1 mg) was taken in a test tube and 1 ml 1 NNaOH was added to it. The test tube was placed in a boilingwater bath for 10 minutes. The blank / sample solution weretaken and added 5 ml of Reagent A (prepared by adding 1 mlfreshly prepared l% Na-K tartarate solution containing 0.5%CuSO4 into 50 ml 2% Na2CO3 solution) and incubated at roomtemperature for 10 minutes. Then added 0.5 ml reagent B(Folin reagent) and again incubated at room temperature for15 minutes. The absorbance of the supernatant was observedat λ650 nm. Protein content was evaluated from the concen-tration of BSA solution known from standard curve.

2.10. Proline (Bates et al. (1973)

Cells were suspended in 10 ml of 3% Sulphosalicylic acid andcentrifuged at 5000 g for 10 min to remove cell debris. To 2 mlof supernatant, 2 ml of ninhydrin was added, followed byaddition of 2 ml glacial acetic acid and incubated at boilingtemperature for one hour. The mixture was extracted withtoluene. Proline was quantified spectrophotometrically at520 nm from organic phase.

2.11. MDA (Heath and Packer, 1968)

Harvested cyanobacterium cells (50 mg) were homogenized in1% Trichloroacetic acid (TCA) (2.5 ml) and then centrifuged at10,000 rpm for 10 minutes at room temperature. Equalvolumes of supernatant and 0.5% Thiobarbituric acid (TBA)in 20% TCA solutions (freshly prepared) were added into a newtest tube and incubated at 95 °C for 30 minutes in water bath.The supernatant were transferred into ice bath and thencentrifuged at 10,000 rpm for 5minutes. The absorbance of thesupernatant was recorded at 532 nm and corrected or non-specific turbidity by subtracting the absorbance at 600 nm,0.5% TBA in 20% TCAwas used as the blankMDA contents wasdetermined using the coefficient of 155 mM cm-1.

2.12. SOD (Dhindsa et al., 1981)

Cyanobacterial cells were harvested by centrifugation and then50 mg dry biomass was homogenized in 2 ml 0.5 M phosphatebuffer (pH 7.5). Supernatant obtained after centrifugation of thehomogenate at 15,000 rpm at 4 °Cwas used for the enzyme assay.SOD activity was assayed by monitoring the inhibition ofphotochemical reduction of nitroblue tetrazolium chloride (NBT),using a reaction mixture consisting of 1 M Na2CO3, 200 mMmethionine, 2.25mMNBT, 3mMEDTA, 60μMRiboflavinand0.1Mphosphate buffer (pH 7.8). Absorbance was read at 560 nm.

2.13. Catalase (Aebi, 1984)

To estimate the antioxidant enzyme (catalase) in desiredcyanobacteria, known amount of algal biomass (50 mg) was

taken and homogenized with 2 ml of extraction buffer (0.5 Mphosphate buffer, pH 7.5). The homogenate was centrifuged at12,000 rpm for 20 min and the supernatant (enzyme extract)was separated for assay. To 100 μl of enzyme extract, 1.6 mlphosphate buffer, 0.2 ml 0.3% H2O2 and 3mM EDTAwas addedin a test tube. The reaction was allowed to run for 3 min.Enzyme activity was calculated by using extinction coefficient0.036 per mM/cm and was expressed in enzyme (unit/mgprotein). One unit of enzyme is the amount necessary todecompose 1 μl of H2O2 per minute at 25 °C. The absorbance ofthe supernatant was observed at 240 nm against blank.

2.14. Ascorbate peroxidase (Nakano and Asada, 1981)

To estimate ascorbate peroxidase in the cyanobacteria ofinterest, we used the same procedure for extracting enzymefrom the sample as in catalase (stated as above). Using thesame enzyme extract of catalase, ascorbate peroxidase wasestimated as follow. The reaction mixture containing 0.5 mMNa-phosphate buffer (pH 7.5), 0.5 mM ascorbate, 3.0 mM EDTA,1.2 mM H2O2, and 0.1 ml enzyme extract in a final assayvolume of 1 ml. Ascorbate oxidation was read at 290 nm. Theconcentration of oxidized ascorbate was calculated usingextinction coefficient (2.8 mM/cm). One unit of APX may bedefined as nmol/mg ascorbate oxidized per minute.

2.15. Statistical analysis

Data were statistically analyzed and the results wereexpressed as means (±SE) of 3 independent replicates.

3. Results and Discussion

3.1. Growth behavior

Exogenous addition of different concentrations (2.5, 5, 7.5, 10,12.5 and 15 µg/ml) of pesticide (endosulfan) showed varyingtoxicity to the test cyanobacterial strains. The extent of toxicityincreased with increasing concentration of the pesticide (Fig. 1(a-f)). The order of toxicity of the test organisms is AulosirafertilissimaNAnabaena variabilisNNostoc muscorum. The order oftolerance is based on 10 µg/ml tested concentration where thepercentage increase in growth (absorbance) were 36.44, 36.17and 28.24 % in Nostoc muscorum, Anabaena variabilis and Aulosirafertilissima respectively. After that their growth showed manyfold inhibition from first day to 20th day. Thismay be attributedto the differential permeability of the pesticide across the cellmembrane of test experimental micro-organism. The relativelyhigher toleranceofN.muscorummaybepartiallydue topresenceof consistent mucilaginous envelope as compared to the thinand diffluent mucilage in Anabaena variabilis and weak sheathcovering in Aulosira fertilissima (Ahmed and Venkataraman,1973). Visually yellowing and reduction in aggregate formationrepresented the adverse effect of endosulfan of the three teststrains. Maximum yellowing and least aggregation wereobserved in Aulosira fertilissima and were followed by Anabaenavariabilis and Nostoc muscorum.

The pattern of growth in these organisms was almostsimilar. In low concentration of endosulfan (2.5, 5, 7.5 µg/ml),

Fig. 1 – (a-f) Effect of endosulfan on growth of Nostoc muscorum (NM), Anabaena variabilis (AV), Aulosira fertilissima (AF) asabsorbance and biomass. Values are means±S.E with n=3.

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there was a gradual increase in growth reaching peak on 16th /20th day. But in higher concentrations (10, 12.5, 15 µg/ml)growthwas usually less than the control till 12th / 16th day butlater it increased though at a very slow rate. This could beexplained on the basis of the cellular degradation of endo-sulfan or due to the adaptability of cyanobacteria to thepesticide. Growth response of cyanobacterial strains in pre-sence of endosulfan was found to be inhibitory but extent ofinhibition was more damaging beyond 7.5 µg/ml endosulfan.Similar findings have also been reported with propanil inAnabaena cylindrica and Anabaena variabilis (Wright et al., 1977),with benthiocarb and butachlor in Anabaena sp. (Zarger andDar, 1990) and with endosulfan in Nostoc linkia (Satish and

Tiwari, 2000). In present study Aulosira fertilissima, Anabaenavariabilis and Nostoc muscorum tolerated 10 μg/ml endosulfan.Almost similar range of tolerance has been reported in othercyanobacteria with other pesticide. Anabaena doliolum, Aulosirafertilissima and Nostoc sp. have been shown to tolerate 9, 15,10 μg/ml of lindane respectively (Sharma and Gaur, 1981). Thereduction in the growth rate of cyanobacteria in presence ofpesticidemay be due to a decrease in photosynthesis speciallychlorophyll-a (Padhy, 1985; Abou–waly et al., 1991) whichsubsequently leads to several secondary effects. BHC, carbo-furan, phorate and malathion inhibitory effect on growth hasbeen correlated with the inhibition in chlorophyll-a synthesis,photosynthesis activity and nitrogen fixation in Oscillatoria,

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Hapalosiphon sp. and Calothrix brauni ARM 367 (Kaushik andVenkataraman, 1993; Torres and 'Flaeerty, 1976). The site ofaction of pesticides inhibiting electron transport is closelyassociated with PSII such as non – cyclic electron acceptor, getinhibited. Some pesticides have also been shown to inhibitboth electron flow and ATP formation in coupled system(Moreland, 1980).

3.2. Biochemical analysis

3.2.1. Photosynthetic pigmentsIn order to find out tolerance potential of cyanobacteria forendosulfan the three test strains were grown under controland stressed conditions for 20 days and then their biomass

Fig. 2 – (a-f) Effect of endosulfan on Chlorophyll, Carotenoid, Phycmuscorum (NM), Anabaena variabilis (AV), Aulosira fertilissima (AF

were washed, harvested and stored in deep freezer forbiochemical analysis. In low endosulfan doses (2.5 µg/ml)chlorophyll, carotenoid and phycobiliprotein contentincreased in comparision to respective control in all teststrains (Fig. 2a, b and c). Chlorophyll at different pesticideconcentrations in studied strains was almost same. Theirmaximum amounts found in presence of 2.5 µg/ml endosul-fan. But beyond this chlorophyll amount decreased graduallywith increasing pesticide concentrations. Carotenoid andphycobiliprotein content increase were more noticeable inAulosira fertilissima. Carotenoid content increased only at2.5 µg/ml endosulfan in Aulosira fertilissima while extendedupto 7.5 µg/ml and 10 µg/ml endosulfan in Nostoc muscorumand Anabaena variabilis respectively. Maximum carotenoid

obiliprotein, Carbohydrate, Protein and Proline of Nostoc). Values are means±S.E with n=3.

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increase was noted in Anabaena variabilis at 2.5 µg/mlendosulfan. Phycobiliprotein was more than control upto7.5 µg/ml in Aulosira fertilissima where as till 5 µg/mlendosulfan in Nostoc muscorum and Anabaena variabilis. Max-imum phycobiliprotein was decteded in Aulosira fertilissima in5 µg/ml endosulfan. Inhibitory effect of endosulfan (5 µg/ml)has been reported as chlorophyll, carotenoid, phycocyanincontent of Plectonema boryanum (Prasad et al., 2005). We havefound 5 µg/ml endosulfan inhibitory effects only on chlor-ophyll of test strains, whereas carotenoids and phycobilipro-teins were stimulated at this concentration. Earlier we havealso reported endosulfan toxicity on Spirulina platensis andAnabaena sp. (Kumar et al., 2004).

3.2.2. CarbohydrateThe carbohydrate content wasmore than control upto 5 μg/mlendosulfan exposures in Nostoc muscorum, Anabaena variabiliswhile till 7.5 μg/ml endosulfan in Aulosira fertilissima, but thereafter it decreased gradually (Fig. 2d). Similar observations havebeen reported for Nostoc kihlmani and Anabaena oscillariodeswhere at lower concentration of thiobencarb showed increasein the contents of reducing sugar, sucrose, polysaccharidesand total sugars but higher concentration of the pesticideshowed significant decrease (Mansour et al., 1994).

Fig. 3 – (a-d) Effect of endosulfan on MDA, SOD, APX and CAT of Nfertilissima (AF). MDA content in control was 752.3±11.4, 631.7±1content in control was 14.4±0.02, 15.0±0.34, 18.4±0.71 (unit/mgcontrol was 249.2±17.2, 252.7±27.0, 336.0±4.9 (nmol/mg proteinwas 6.58±0.46, 9.61±0.39, 7.95±0.46 (unit/mg protein/min) in NM

3.2.3. ProteinThe total protein contents were more than control till 7.5 μg/mlpesticide concentrations in all test strains (Fig. 2e). The max-imum protein enhancement was observed in Aulosira fertilissima(43%) at 7.5 µg/ml pesticide concentration. At lower concentra-tions of pesticide, increase in the protein content suggests thatlower concentrations of pesticide stimulate synthesis of stressretarding proteins. Increase in protein content of Anabaenasphaerica due to the effect of 25 µg/ml molinate (Yan et al.,1997), 2-6 µg/ml benthiocarb (Bhunia et al., 1991) and 50 µg/mlbavistin1µg/mlnimbicidin (Rajendranetal., 2007)havealsobeendemonstrated. Where as in case of Anabaena sp. (0.5–2 µg/ml)showed decrease in protein content (Babu et al., 2001). In ourstudy such decrease in protein content was observed beyond7.5 µg/ml endosulfan. The decrease in protein content may alsobedue topresenceof pesticidebeyond their tolerance range. Thisdecrease in protein contentmay also be due to increased level ofROS (Leitao et al., 2003) or increased protease activity. It resultedretarded growthanddecreased carbonandnitrogenassimilationunder Lindane stress (Babu et al., 2001).

3.2.4. ProlineThe proline content increased drastically under pesticidestress conditions (Fig. 2.f). It was maximum in presence of

ostoc muscorum (NM), Anabaena variabilis (AV), Aulosira9.5, 717.9±23.9 (nmol/g) in NM, AV and AF respectively. SODprotein/h) in NM, AV and AF respectively. APX content in/min) in NM, AV and AF respectively. CAT content in control, AV and AF respectively. Values are means±S.E with n=3.

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2.5 µg/ml endosulfan in Nostoc muscorum and then started todecrease gradually. In studied strains, proline content washigher than control in Nostoc muscorum (29.2%), Anabaenavariabilis (2.3%) and Aulosira fertilissima (0.08%) at 15 µg/ml.Very high accumulation of cellular proline (upto 80% of thetotal amino acid pool under stress as compared to just 5%under the normal condition) has been reported earlier inmanyhigher plant species due to increased synthesis and decreaseddegradation under variety of stress conditions such as water,salt, drought and heavy metal (Bates et al., 1973; Bohnert andJensen, 1996; Delauney and Verma, 1993; Kavi kishor et al.,2005). Although the actual reason behind the accumulation ofproline (presumably byway of synthesis from glutamic acid) isyet to be known, in plants or plant parts exposed to stress, itcould probably be due to a decrease in the activity of electrontransport system (Venekemp, 1989).

3.2.5. Oxidative damageIn present study MDA content increased with increasingconcentrations of pesticide suggesting formation of freeradicals eliciting endosulfan toxicity (Fig. 3a). Our results arein consonance with finding on Plectonema boryanum withendosulfan (Prasad et al., 2005) it has been suggested thatfree radical formation occur due to strong inhibition of PSII.Several herbicides have been found to generate active oxygenspecies, either by direct involvement in radical production orby inhibition of biosynthetic pathways. The generation of thehydrocarbon gas ethane, the production of malonaldehydeand changes in electrolytic conductivity has frequently beenused as sensitive markers for herbicide action in plants(Kunert et al., 1985; Peleg et al., 2001). Compounds such asparaquat (also known as methyl viologen) induce lightdependent oxidative damage in plants (Dodge, 1971). The PSImediated reduction of the paraquat di-cation results in theformation of a mono-cation radical which then reacts withmolecular oxygen to produce O-

2 with the subsequent pro-duction of other toxic species, such as H2O2 and OH (Elstneret al., 1988). These compounds cause severe toxicologicalproblems and results in peroxidation of membrane lipids andgeneral cellular oxidation. Increase in both proline and MDAcontents with increasing pesticide concentration are indica-tive of a correlation between free radical generation andproline accumulation (Figs. 2f and 3a). This is also in agree-ment with the earlier reports of our lab on Spirulina platensisand Westellopis prolifica (Choudhary et al., 2007; Fatma et al.,2007).

3.2.6. AntioxidantPhotosynthetic organisms counteract the toxicity of pesti-cide induced free radicals by increasing their antioxidativedefense mechanisms that include enzymes such as super-oxide dismutase (SOD), catalase (CAT), ascorbate peroxidase(APX) and low molecular weight compounds such asascorbate, glutathione, flavonoids, tocopherols, and carote-noids. Ascorbate peroxidase (APX) utilizes the reducingpower of ascorbic acid to eliminate potentially harmfulH2O2. SOD neutralizes the highly reactive superoxide radicalgenerated in the cell especially under stress condition(Elstner et al., 1988). The experimental concentrations ofendosulfan accelerated the activities of defense enzymesSOD, APX and CAT in test cyanobacterial strains (Fig. 3b-d).

The increase in SOD and CAT continued till the 15 µg/ml inall test organisms while APX increased till the 12.5 µg/ml inA. variabilis and 10 µg/ml in N. muscorum and A. fertilissima.We have noted 85.9%, 85.7% SOD increase in N. muscorumand A. variabilis respectively and only 3.79% observed in A.fertilissima at 10 µg/ml while 41% SOD increase is beingreported in Plectonema boryanum at 10 µg/ml endosulfan(Prasad et al., 2005). Maximum SOD, APX and CAT wereobserved in N. muscorum. It is suggesting that SOD and CATare playing greater role in ROS detoxification.

4. Conclusions

N. muscorum, A. variabilis, and A. fertilissima have varyingtolerance potential to endosulfan and the order of toleranceis Nostoc muscorumNAnabaena variabilisNAulosira fertilissima.This observation is based on 10 µg/ml tested concentrationwhere the percentage increases in absorbance growth curve(36.44, 36.17 and 28.24 %). After that their growth showedmany fold inhibition from first day to 20th day. In otherobservations in the case of proline 65, 23.61 and 3.1% andSOD 85.9, 58.7, 3.79% increase with respect to control(untreated sample) at 10 µg/ml concentrations in Nostocmuscorum, Anabaena variabilis and Aulosira fertilissima respec-tively. At lower tested concentration (2.5 µg/ml) showedstimulatory response in all tested strains. It is due to stressresponse and assimilation of uptake carbon source ofpesticide by the organisms during the processes of degrada-tion of pesticide. Therefore we conclude that up to 2.5 µg/mlendosulfan concentrations does not affect adversely oncyanobacterial population. During present investigation wehave successfully attempted our proposed hypothesis andachieved expected results. The common chlorinated pesti-cide of paddy fields the endosulfan was found to be toxic tothe non-target organism, the heterocystous N2 fixingcyanobacteria frequently used as biofertilisers at higherconcentrations. The observations suggested that the endo-sulfan exert its toxic effect through free radical mediatedoxidative stress and resistance is being imparted throughantioxidant enzymes as well as proline. Proline has beenproduced during the stress condition so it might be helpfulin degradation of pesticide and cope up with adversecondition. For the first time we showed the osmoprotectant(proline) role in detoxification of chlorinated pesticide(endosulfan).

Acknowledgments

The authors are thankful to NCCU - BGA, I.A.R.I., New Delhi,for providing the test strains and to Indian Council of MedicalResearch for providing financial assistance to SatyendraKumar as SRF.

R E F E R E N C E S

Abou-waly H, Abou-Setta MM, Nigg HN, Mallory L. Growthresponse of freshwater algae, Anabaena flos- aquaeand Selenastrum capricornutum, to atrazine and hexazinoneherbicides. Bull Environ Contam Toxicol1991;46:223–9.

137S C I E N C E O F T H E T O T A L E N V I R O N M E N T 4 0 3 ( 2 0 0 8 ) 1 3 0 – 1 3 8

Aebi H. Catalase in vitro. Methods Enzymol 1984;05:121–6.Ahluwalia AS. Influence of satum and knockweed on the growth

and heterocyst formation in the nitrogen fixing blue greenalgae. Pesticide 1988;22:43–94.

Ahmed MH, Venkataraman GS. Tolerance of Aulosira fertilissimato pesticides. Curr Sci 1973:42–108.

Anand N. BGA populations of rice field. In: Seshadri CV, Thomas S,Jeeji Bai N, editors. Proc. Nat. Work, Algal System Indian SocBiotech IIT/New Delhi; 1980. p. 51–5.

AsadaK. The role of ascorbate peroxidase andmonodehydrpascorbatereductase in H2O2 scavenging in plants. In: Scandolios JG, editor.Oxidative stress and themolecular biology of antioxidant defenses.Plainview: Cold Spring Harbor Laboratory Press; 1997. p. 715–35.

Asada K. Chloroplasts: formation of active oxygen and itsscavenging. Methods Enzymol 1984;10:422–9.

Babu GS, Hans RK, Singh J, Viswanathan PN, Joshi PC. Effect oflindane on the growth and metabolic activities ofcyanobacteria. Ecotoxicol Environ Saf 2001;48:219–21.

Bartosz G. Oxidative stress in plants. Acta Physiol Plant1997;19:47–64.

Bates LS, Wadern RP, Teare ID. Rapid estimation of free proline forwater stress determination. Plant Soil 1973;39:205–7.

Bhunia AK, Basu NK, Roy D, Chakrabarti A, Banerjee SK. Growth,chlorophyll a content, nitrogen fixing ability and certainmetabolic activities of N. muscorum: Effect of methylparathionand benthiocarb. Bull Environ Contam Toxicol 1991;47:43–50.

Bohnert HJ, Jensen RG. Strategies for engineering water stresstolerance in plants. Trends Biotechnol 1996;4:89–97.

Choudhary M, Jetely UK, Khan A, Zutshi S, Fatma T. Effect of heavymetal stress on proline and malondialdehyde andsuperoxidedismutase activity in the cyanobacterium Spirulinaplatensis S-5. Ecotoxicol Environ Saf 2007;66:204–9.

Del Vos CH, VonkMJ, Schat H. Glutathione depletion due to copperinduced phytochelatin synthesis causes oxidative stress inSilene cucubalus. Plant Physiol. 1992;98:853–8.

Delauney AJ, Verma DPS. Proline biosynthesis and osmoregulationin plants. J Plant 1993;4:215–23.

Del-Rio LA, Sevilla F, Sandalio LM, Palma JM. Nutritional effect andexpression of superoxide dismutase; induction and geneexpression, diagnostics, prospective protection against oxygentoxicity. Free Radic Res Commun 1991;12(13):819–28.

Dhindsa RS, Dhindsa PP, Thorpe TA. Leaf senescence: correlatedwith increased level of membrane permeability and lipidperoxidation, and decreased levels of superoxide dismutaseand catalase. J Exp Bot 1981;32:93–101.

Dodge AD. The mode of action of the bipyridylium herbicides,paraquat and diqual. Endeavour 1971;30:135–40.

Elstner EF, Wagner GA, Schutz W. Activated oxygen in greenplants in relation to stress situation. Curr Top Plant BiochemPhysiol 1988;7:159–87.

Fatma T, KhanMA, ChoudharyM. Impact of environmental pollutionon cyanobacterial proline content. J Appl Phycol 2007;19:625–9.

Heath RL, Packer L. Photoperoxidation on in isolated Chloroplasts,I. Stoichiometry of fatty acid peroxidation. Arch BiochemBiophys 1968;125:189–98.

Hellebust JA, Craige JS. Handbook of physiological and biochemicalmethods. Cambridge: Cambridge university press; 1978. p. 64–70.

Hendry GAF. Oxygen and environmental stress in plants: anevolutionary context. Proc R Soc Edinb 1994;102B:155–65.

Hoechst AG. Thiodan: the versatile but selective insecticide.Instructions for its use, Frankfurt, West Germany HoechstAktiengesellschaft; 1977.

Indian Council for Agriculture Research (ICAR). www.iffco.nic.in/applications/Agriinfo.nsf.

Kaushik BD, Venkataraman GS. Response of cyanobacterialnitrogen fixation to insecticides. Curr Sci 1993;52:321–3.

Kavi Kishor PB, Sangam S, Amrutha RN, Sri laxami P, Naidu KR,Rao KRSS, Rao S, Reddy KJ, Theriappan P, Sreenivasulu N.Regulation of proline biosynthesis, degradation, uptake and

transport in higher plants: Its implications in plant growth andabiotic stress tolerance. Cur Sci 2005;88(3):424–38.

Kolte SO, Goyal SK. Inhibition of growth and nitrogen fixation inCalothrix marchia by herbicide in vitro. In: Kaushik BD, editor.Proc National Symposium on Cyanobacterial NitrogenFixation. New Delhi: Pub Associated Co.; 1990. p. 507–10.

Kumar S, Jetley UK, Fatma T. Tolerance of Spirulina platensis-S5and Anabaena sp. to Endosulfan an organochlorine pesticide.Ann Plant Physiol 2004;18(2):103–7.

Kunert KJ, Homrighausen C, Bohme H, Boger P. Oxyfluorfen andlipid peroxidation: Protein damage as a phototoxicconsequence. Weed Sci 1985;33:766–79.

Kwon CS, Penner D. The interaction of insecticides with herbicideactivity. Weed technol 1995;9:119–24.

Leitao M, das A, Cardozo KHM, Pinto E, Colepicolo P. PCB inducedoxidative stress in the unicellular marine dinoflagellatesLingulodinium polyedrum. Arch Environ Comtam Toxicol2003;45:59–65.

Lowry OH, RosebroughNJ, Farr AL, Randall RJ. Proteinmeasurementwith the Follin. phenol reagent. J Biol Chem 1951;193(1):265–75.

Mackinney G. Absorption of light by chlorophyll solution. J BiolChem 1941;140:315–22.

Mansour FA, SolimanARI, Shaaban -Desouki SA,HusseinMH. Effectof herbicides on cyanobacteria. I. Changes in carbohydratecontent, Pmase and GOT activities in Nostoc kihlmani andAnabaena oscillarioides. Phykos 1994;33:153–62.

Moreland DE. Mechanism of action of herbicides. Ann Rev PlantPhysiol 1980;315:97–638.

Nakano Y, Asada K. Hydrogen peroxide is scavenged byascorbate-specific peroxidase in spinach chloroplasts. PlantCell Physiol 1981;22:867–80.

Olga B, Eija V, Kurt VF. Antioxidants, oxidative damage and oxygendeprivation stress: a review. Ann Bot 2003;91:179–94.

Padhy RN. Cyanobacteria and pesticides. Res Rev 1985:941–4.Peleg L, Zer H, Chevion M. Paraquat toxicity in Pisum sativum.

Effects on phosphorus-31 nuclear magnetic resonance studies.Plant Physiol 2001;125:912–925.100.

Prasad SM, Kumar D, Zeeshan M. Growth, photosynthesis, activeoxygen species and antioxidants responses of paddy fieldcynobacterium Plectonema boryanum to endosulfan stress.J Gen Appl Microbiol 2005;51:115–23.

Rajendran UM, Elango K, Anand N. Effects of a fungicide, aninsecticide, and a biopesticdie on Tolypothrix scytonemoides.Pestic Biochem Physiol 2007;87:164–71.

Relwani LL. Role of blue green algae on paddy field. Curr Sci1963;32:417–8.

Satish N, Tiwari GL. Pesticide tolerance in Nostoc linckia inrelation to the growth and nitrogen fixation. Proc Natl Acad SciIndia 2000;70:319–23.

SharmaVK, GaurYS.Nitrogen fixation by pesticide adopted strainsof paddy field cyanophyte. Int J Ecol Env Sci 1981;7:117–22.

Shetty PK, Mitra J, Murthy NBK, Namitha KK, Savitha KN,Raghu K. Biodegradation of cyclodiene insecticideendosulfan by Mucor thermohyalospora MTCC 1384. Curr Sci2000;79:1381–3.

Siegelman HW, Kygia JH. Handbook of phycological methods.Cambridge University Presss; 1978. p. 73–9.

Smirnoff N. The role of active oxygen in the response of plants towater deficit and desiccation. New Phytol 1993;125:27–58.

Spiro RG. Analysis of sugars found in glycoproteins. Methodsenzymol 1966;8:3–26.

Stainer RY, Kunisawa R, Mandel M, Cohin-Bazire G. Purificationand properties of unicellular blue green algae (orderChrococcales). Bacteriol Rev 1971;35:171–205.

Stratton GW. The effects of pesticides and heavy metals onphototrophic micro-organisms. Rev Environ Toxicol1987;3:71–112.

Torres AMR, 'Flaeerty LMO. Influence of pesticides on Chlorella,Chlorococcum, Stigeoclonium (Chlorophyceae) Tribonema,

138 S C I E N C E O F T H E T O T A L E N V I R O N M E N T 4 0 3 ( 2 0 0 8 ) 1 3 0 – 1 3 8

Vaucheria (Xanthophyceae) and Oscillatoria (Cyanophyceae).Phycologia 1976;15:25–36.

Venekemp JH. Regulation of cytosolic acidity in plants undercondition of drought. Plant Physiol 1989;76:112–7.

Wright SJL, Stantharpe AF, Downs JD. Interaction of herbicidepropanil and a metabolite, 3, 4-dichloromiline with blue greenalgae. Acta Pathol Acad Hung 1977;12:51–7.

Yan GA, Yan X,WuW. Effect of herbicide Molinate onmixotrophicgrowth, photosynthetic pigments and protein Content ofAnabaena sphaerica under different light conditions.Ecotoxicol Environ Saf 1997;38:144–9.

Zarger MY, Dar GH. Effect of benthiocarb and butachlor on growthand nitrogen fixation by cyanobacteria. Bull Environ ContamToxicol 1990;45:232–4.