SC IE N CE OF T H E TOT AL E N V I RO N ME N T 4 0 3 ( 2 00 8 ) 1 3 0–1 38
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / s c i t o t e n v
Endosulfan induced biochemical changes in
nitrogen-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
AR TIC LE I N FO
ABS TR ACT
Article history:
Pesticide contamination in aquatic ecosystem including paddy fields is a serious global
Received 3 January 2008
environmental concern. Cyanobacteria are also affected by pesticides as non- target organism.
Received in revised form 2 May 2008
For better exploitation of cyanobacteria as biofertiliser, it is indispensable to select tolerant
Accepted 19 May 2008
strains along with understanding of their tolerance. Three cyanobacterial strains viz. Aulosira
Available online 26 June 2008
fertilissima, Anabaena variabilis and Nostoc muscorum were studied for their stress responses to
an organochlorine pesticide ‘endosulfan’ with special reference to oxidative stress, role of
Keywords:
proline and antioxidant enzymes in endosulfan induced free radical detoxification. Reduction
Cyanobacteria
in growth, photosynthetic pigments and carbohydrate of the test microorganisms were
Insecticide
accompanied with increase in their total protein, proline, malondialdehye (MDA), superoxide
Growth
dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) in higher endosulfan doses.
photosynthetic pigments
Increased amount of MDA is indicative of formation of free radicals, while increased level of
Carbohydrate
CAT, APX, SOD and proline indicated their involvement in free radical scavenging mechanism.
Protein
In lower concentrations, test pesticide showed increase in photosynthetic pigments. Order of
Proline
tolerance was Nostoc muscorum N Anabaena variabilisN Aulosira fertilissima.
Free radicals
© 2008 Elsevier B.V. All rights reserved.
Antioxidant
1.
Introduction
The increasing use of pesticides in agriculture demands
investigation to examine the effect of pesticides on the nontarget soil micro-organisms including nitrogen fixing cyanobacteria. Cyanobacteria have been applied in rice fields as a
biofertilizer for better yield of paddy (Relwani, 1963). Weedicides, fungicides and insecticides used for plant protection in
rice 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 cyanobacteria (Padhy, 1985; Ahluwalia, 1988).
Endosulfan is most popular amongst the organochlorine
insecticides. It is being extensively used in crops field due to its
broad spectrum of activity and relatively low cost. Application rate
of 35 EC endosulfan is 560 ml in 100 litres of water per acre in rice
field (ICAR). Depending on the type of crop and the area in which it
is grown, application rates usually range between 0.45 kg ai and
1.4 kg/ha, but both smaller and larger doses have occasionally
been used (Hoechst, 1977). It is often repeatedly used on crops in
one crop period leading to build up of its residues both in crop and
soil (Kwon and Penner, 1995). It has been reported that extensive
usage of endosulfan in various tropical and subtropical countries
for control of the insect population, is accompanied with
reduction in non-target microbial population including cyanobacteria (Satish and Tiwari, 2000; Shetty et al., 2000).
⁎ Corresponding author. Tel.: +91 (011) 26921908; fax: +91 (011) 26980229; mobile: +91 9891408366, +91 9868301050.
E-mail addresses: [email protected], [email protected] (S. Kumar), [email protected] (K. Habib), [email protected],
[email protected] (T. Fatma).
0048-9697/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.scitotenv.2008.05.026
S CIE N CE OF T H E TOT AL E N V I RO N ME N T 4 0 3 ( 2 00 8 ) 1 3 0–1 38
Most environmental stresses are affecting on the production
of active oxygen species in plants, causing oxidative stress
(Smirnoff, 1993; Hendry, 1994; Bartosz, 1997). The balance
between the production of activated oxygen species and the
quenching activity of antioxidant is upset, which often results in
oxidative 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, hydroxyl
radical OH and singlet oxygen 1O2) H2O2 and the hydroxyl
radical are most active, toxic and destructive (Smirnoff, 1993).
Under normal circumstances, concentration of oxygen radicals
remain low because of the activity of protective enzymes,
including superoxide dismutase, catalase and ascorbate peroxidase (Asada, 1984) but under stress conditions imposed by
physical, chemical and biological pollutants this balance may
get disturbed, causing enhancement of detrimental processes.
Photosynthetic cells are prone to oxidative stress because they
contain an array of photosensitizing pigments and they both
produce and consume oxygen. The photosynthetic electron
transport system is the major source of active oxygen species in
plant tissues (Asada, 1997), have the potential to generate
singlet oxygen 1O2 and superoxide O-2. The main cellular
components susceptible to damage by free radicals are lipids
(peroxidation of unsaturated fatty acids in membranes), proteins (denaturation), carbohydrates and nucleic acids (Olga
et al., 2003). The formation of ROS is prevented by an antioxidant
system: low molecular mass antioxidants (ascorbic acid,
glutathione, and tocopherols), enzymes regenerating the
reduced forms of antioxidants, and ROS-interacting enzymes
such as SOD, peroxidases and catalases. Though considerable
work has been done on pesticide induced inhibitory effect on
growth, photosynthetic pigments, photosynthesis and N2 fixation but, to the best of our knowledge there is no report on
insecticide, particularly endosulfan induced ROS generation
and their detoxification in resistant heterocystous cyanobacteria. Only non heterocystous cyanobacteria Plectonema boryanum has been studied recently with reference to endosulfan
induced free radical generation and their detoxification (Prasad
et al., 2005). From our lab heterocystous cyanobacteria Westiellopsis prolifica–Janet strain–NCCU331 has been reported to
develop resistance against pyrethroid pesticide with the help
of proline (Fatma et al., 2007).
We hypothesize that in non target cyanobacterial heterocystous nitrogen fixers, common paddy crop pesticide-endosulfan impart detrimental effect through free radical mediated
oxidative stress and the universal osmoprotectant proline
plays significant role in resistance development against
endosulfan in addition to SOD, APX, CAT antioxidant enzymes.
2.2.
131
Test strains
Three cyanobacterial strains viz. Aulosira fertilissima, Anabaena
variabilis and Nostoc muscorum were procured from National
Center for Conservation and Utilization of Blue Green Algae,
Indian Agricultural Research Institute, New Delhi, India. The
test strains were raised in BG-11 medium (pH 7.3) without
Sodium nitrate (Stainer et al., 1971). The flask and media were
sterilized in an autoclave (Yorco, India) maintaining 15 lb/in2 or
Kg/ cm2 pressure for 15 minutes. 50 ml inoculums were
suspended in 500 ml sterile medium taken in 1000 ml EM
flask (three sets) maintaining (O.D-0.4 ± 0.1) after dilution at
560 nm. Culture were allowed to grow for 20 days at 30 ± 2 °C
under light intensity of 2000 ± 200 lux provided by 20 W
fluorescent tubes following a 16: 8 h light/dark regime.
Repeated shaking was done at regular intervals. The biomass
was harvested by filtration through fine nylon cloth, washed
twice with distilled water to remove the remaining pesticide.
2.3.
Growth measurement
The growth of the test cyanobacterial strains was determined
over a period of 20 days by recording optical density of the 5 ml
algal suspension at 560 nm, on a UV–VIS Spectrophotometer –
SPECORD 200 at 4 days interval. The growth absorbance data
was supported by biomass data (dry weight).
2.4.
Biochemical analysis
Biochemical analysis of 20 days old harvested bio-mass of test
organisms under stress and control conditions was carried out in
triplicate for evaluating chlorophyll, carotenoid, phycobiliprotein, 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 at
65 °C for 30 minutes. The pellet was discarded and the
absorbance of the supernatant was observed at 650 nm and
665 nm against 95% methanol as blank.
2.6. Carotenoid (Hellebust and Craige, 1978)
Using 5 mg dry biomass in 10 ml 85% acetone carotenoid was
extracted. Repeated freezing and thawing was done for cell
disruption. The absorbance of the supernatant was observed
at 450 nm against 85 % acetone as blank.
2.7. Phycobiliprotein (Siegelman and Kygia, 1978)
2.
Materials and Methods
2.1.
Pesticide
For evaluating pesticide toxicity, endosulfan (35 % E.C.) was
separately added to the fresh medium in calculated amounts
to obtain final concentration of 2.5, 5, 7.5, 10, 12.5 and 15 µg/ml.
For ‘control’ sets test microorganism were grown without
adding the pesticide. Commercial grade ‘endosulfan’ (35% E.C)
was procured from Excel Industries Limited, Mumbai (India).
Repeated freezing and thawing 5 mg of biomass in 10 ml 0.1 M
phosphate buffer was done for phycobiliprotein extract. The
suspension was centrifuged at 3000-5000 rpm for 5 minutes.
The absorbance of the blue supernatant was observed at
562 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 ml
double distilled water was added to it. To 1.25 ml blank / standard /
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sample solution added 4.0 ml of anthrone reagent and placed in a
boiling water bath for 8 -10 minutes. The absorbance of the
supernatant was observed at 620 nm against blank. The
carbohydrate content was evaluated from concentration of the
glucose 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 N
NaOH was added to it. The test tube was placed in a boiling
water bath for 10 minutes. The blank / sample solution were
taken and added 5 ml of Reagent A (prepared by adding 1 ml
freshly prepared l% Na-K tartarate solution containing 0.5%
CuSO4 into 50 ml 2% Na2CO3 solution) and incubated at room
temperature for 10 minutes. Then added 0.5 ml reagent B
(Folin reagent) and again incubated at room temperature for
15 minutes. The absorbance of the supernatant was observed
at λ650 nm. Protein content was evaluated from the concentration of BSA solution known from standard curve.
2.10.
Proline (Bates et al. (1973)
Cells were suspended in 10 ml of 3% Sulphosalicylic acid and
centrifuged at 5000 g for 10 min to remove cell debris. To 2 ml
of supernatant, 2 ml of ninhydrin was added, followed by
addition of 2 ml glacial acetic acid and incubated at boiling
temperature for one hour. The mixture was extracted with
toluene. Proline was quantified spectrophotometrically at
520 nm from organic phase.
2.11.
MDA (Heath and Packer, 1968)
Harvested cyanobacterium cells (50 mg) were homogenized in
1% Trichloroacetic acid (TCA) (2.5 ml) and then centrifuged at
10,000 rpm for 10 minutes at room temperature. Equal
volumes of supernatant and 0.5% Thiobarbituric acid (TBA)
in 20% TCA solutions (freshly prepared) were added into a new
test tube and incubated at 95 °C for 30 minutes in water bath.
The supernatant were transferred into ice bath and then
centrifuged at 10,000 rpm for 5 minutes. The absorbance of the
supernatant was recorded at 532 nm and corrected or nonspecific turbidity by subtracting the absorbance at 600 nm,
0.5% TBA in 20% TCA was used as the blank MDA contents was
determined using the coefficient of 155 mM cm- 1.
2.12.
SOD (Dhindsa et al., 1981)
Cyanobacterial cells were harvested by centrifugation and then
50 mg dry biomass was homogenized in 2 ml 0.5 M phosphate
buffer (pH 7.5). Supernatant obtained after centrifugation of the
homogenate at 15,000 rpm at 4 °C was used for the enzyme assay.
SOD activity was assayed by monitoring the inhibition of
photochemical reduction of nitroblue tetrazolium chloride (NBT),
using a reaction mixture consisting of 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). Absorbance was read at 560 nm.
2.13.
Catalase (Aebi, 1984)
To estimate the antioxidant enzyme (catalase) in desired
cyanobacteria, known amount of algal biomass (50 mg) was
taken and homogenized with 2 ml of extraction buffer (0.5 M
phosphate buffer, pH 7.5). The homogenate was centrifuged at
12,000 rpm for 20 min and the supernatant (enzyme extract)
was separated for assay. To 100 μl of enzyme extract, 1.6 ml
phosphate buffer, 0.2 ml 0.3% H2O2 and 3 mM EDTA was added
in a test tube. The reaction was allowed to run for 3 min.
Enzyme activity was calculated by using extinction coefficient
0.036 per mM/cm and was expressed in enzyme (unit/mg
protein). One unit of enzyme is the amount necessary to
decompose 1 μl of H2O2 per minute at 25 °C. The absorbance of
the supernatant was observed at 240 nm against blank.
2.14.
Ascorbate peroxidase (Nakano and Asada, 1981)
To estimate ascorbate peroxidase in the cyanobacteria of
interest, we used the same procedure for extracting enzyme
from the sample as in catalase (stated as above). Using the
same enzyme extract of catalase, ascorbate peroxidase was
estimated as follow. The reaction mixture containing 0.5 mM
Na-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 assay
volume of 1 ml. Ascorbate oxidation was read at 290 nm. The
concentration of oxidized ascorbate was calculated using
extinction coefficient (2.8 mM/cm). One unit of APX may be
defined as nmol/mg ascorbate oxidized per minute.
2.15.
Statistical analysis
Data were statistically analyzed and the results were
expressed 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 varying
toxicity to the test cyanobacterial strains. The extent of toxicity
increased with increasing concentration of the pesticide (Fig. 1
(a-f)). The order of toxicity of the test organisms is Aulosira
fertilissima N Anabaena variabilis N Nostoc muscorum. The order of
tolerance is based on 10 µg/ml tested concentration where the
percentage increase in growth (absorbance) were 36.44, 36.17
and 28.24 % in Nostoc muscorum, Anabaena variabilis and Aulosira
fertilissima respectively. After that their growth showed many
fold inhibition from first day to 20th day. This may be attributed
to the differential permeability of the pesticide across the cell
membrane of test experimental micro-organism. The relatively
higher tolerance of N. muscorum may be partially due to presence
of consistent mucilaginous envelope as compared to the thin
and diffluent mucilage in Anabaena variabilis and weak sheath
covering in Aulosira fertilissima (Ahmed and Venkataraman,
1973). Visually yellowing and reduction in aggregate formation
represented the adverse effect of endosulfan of the three test
strains. Maximum yellowing and least aggregation were
observed in Aulosira fertilissima and were followed by Anabaena
variabilis and Nostoc muscorum.
The pattern of growth in these organisms was almost
similar. In low concentration of endosulfan (2.5, 5, 7.5 µg/ml),
S CIE N CE OF T H E TOT AL E N V I RO N ME N T 4 0 3 ( 2 00 8 ) 1 3 0–1 38
133
Fig. 1 – (a-f) Effect of endosulfan on growth of Nostoc muscorum (NM), Anabaena variabilis (AV), Aulosira fertilissima (AF) as
absorbance and biomass. Values are means ± S.E with n = 3.
there was a gradual increase in growth reaching peak on 16th /
20th day. But in higher concentrations (10, 12.5, 15 µg/ml)
growth was usually less than the control till 12th / 16th day but
later it increased though at a very slow rate. This could be
explained on the basis of the cellular degradation of endosulfan or due to the adaptability of cyanobacteria to the
pesticide. Growth response of cyanobacterial strains in presence of endosulfan was found to be inhibitory but extent of
inhibition was more damaging beyond 7.5 µg/ml endosulfan.
Similar findings have also been reported with propanil in
Anabaena cylindrica and Anabaena variabilis (Wright et al., 1977),
with benthiocarb and butachlor in Anabaena sp. (Zarger and
Dar, 1990) and with endosulfan in Nostoc linkia (Satish and
Tiwari, 2000). In present study Aulosira fertilissima, Anabaena
variabilis and Nostoc muscorum tolerated 10 μg/ml endosulfan.
Almost similar range of tolerance has been reported in other
cyanobacteria with other pesticide. Anabaena doliolum, Aulosira
fertilissima and Nostoc sp. have been shown to tolerate 9, 15,
10 μg/ml of lindane respectively (Sharma and Gaur, 1981). The
reduction in the growth rate of cyanobacteria in presence of
pesticide may be due to a decrease in photosynthesis specially
chlorophyll-a (Padhy, 1985; Abou–waly et al., 1991) which
subsequently leads to several secondary effects. BHC, carbofuran, phorate and malathion inhibitory effect on growth has
been 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 and
Venkataraman, 1993; Torres and 'Flaeerty, 1976). The site of
action of pesticides inhibiting electron transport is closely
associated with PSII such as non – cyclic electron acceptor, get
inhibited. Some pesticides have also been shown to inhibit
both electron flow and ATP formation in coupled system
(Moreland, 1980).
3.2.
Biochemical analysis
3.2.1.
Photosynthetic pigments
In order to find out tolerance potential of cyanobacteria for
endosulfan the three test strains were grown under control
and stressed conditions for 20 days and then their biomass
were washed, harvested and stored in deep freezer for
biochemical analysis. In low endosulfan doses (2.5 µg/ml)
chlorophyll, carotenoid and phycobiliprotein content
increased in comparision to respective control in all test
strains (Fig. 2a, b and c). Chlorophyll at different pesticide
concentrations in studied strains was almost same. Their
maximum amounts found in presence of 2.5 µg/ml endosulfan. But beyond this chlorophyll amount decreased gradually
with increasing pesticide concentrations. Carotenoid and
phycobiliprotein content increase were more noticeable in
Aulosira fertilissima. Carotenoid content increased only at
2.5 µg/ml endosulfan in Aulosira fertilissima while extended
upto 7.5 µg/ml and 10 µg/ml endosulfan in Nostoc muscorum
and Anabaena variabilis respectively. Maximum carotenoid
Fig. 2 – (a-f) Effect of endosulfan on Chlorophyll, Carotenoid, Phycobiliprotein, Carbohydrate, Protein and Proline of Nostoc
muscorum (NM), Anabaena variabilis (AV), Aulosira fertilissima (AF). Values are means ± S.E with n = 3.
S CIE N CE OF T H E TOT AL E N V I RO N ME N T 4 0 3 ( 2 00 8 ) 1 3 0–1 38
increase was noted in Anabaena variabilis at 2.5 µg/ml
endosulfan. Phycobiliprotein was more than control upto
7.5 µg/ml in Aulosira fertilissima where as till 5 µg/ml
endosulfan in Nostoc muscorum and Anabaena variabilis. Maximum phycobiliprotein was decteded in Aulosira fertilissima in
5 µg/ml endosulfan. Inhibitory effect of endosulfan (5 µg/ml)
has been reported as chlorophyll, carotenoid, phycocyanin
content of Plectonema boryanum (Prasad et al., 2005). We have
found 5 µg/ml endosulfan inhibitory effects only on chlorophyll of test strains, whereas carotenoids and phycobiliproteins were stimulated at this concentration. Earlier we have
also reported endosulfan toxicity on Spirulina platensis and
Anabaena sp. (Kumar et al., 2004).
3.2.2.
Carbohydrate
The carbohydrate content was more than control upto 5 μg/ml
endosulfan exposures in Nostoc muscorum, Anabaena variabilis
while till 7.5 μg/ml endosulfan in Aulosira fertilissima, but there
after it decreased gradually (Fig. 2d). Similar observations have
been reported for Nostoc kihlmani and Anabaena oscillariodes
where at lower concentration of thiobencarb showed increase
in the contents of reducing sugar, sucrose, polysaccharides
and total sugars but higher concentration of the pesticide
showed significant decrease (Mansour et al., 1994).
3.2.3.
135
Protein
The total protein contents were more than control till 7.5 μg/ml
pesticide concentrations in all test strains (Fig. 2e). The maximum protein enhancement was observed in Aulosira fertilissima
(43%) at 7.5 µg/ml pesticide concentration. At lower concentrations of pesticide, increase in the protein content suggests that
lower concentrations of pesticide stimulate synthesis of stress
retarding proteins. Increase in protein content of Anabaena
sphaerica 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/ml
bavistin 1 µg/ml nimbicidin (Rajendran et al., 2007) have also been
demonstrated. Where as in case of Anabaena sp. (0.5–2 µg/ml)
showed decrease in protein content (Babu et al., 2001). In our
study such decrease in protein content was observed beyond
7.5 µg/ml endosulfan. The decrease in protein content may also
be due to presence of pesticide beyond their tolerance range. This
decrease in protein content may also be due to increased level of
ROS (Leitao et al., 2003) or increased protease activity. It resulted
retarded growth and decreased carbon and nitrogen assimilation
under Lindane stress (Babu et al., 2001).
3.2.4.
Proline
The proline content increased drastically under pesticide
stress conditions (Fig. 2.f). It was maximum in presence of
Fig. 3 – (a-d) Effect of endosulfan on MDA, SOD, APX and CAT of Nostoc muscorum (NM), Anabaena variabilis (AV), Aulosira
fertilissima (AF). MDA content in control was 752.3 ± 11.4, 631.7 ± 19.5, 717.9 ± 23.9 (nmol/g) in NM, AV and AF respectively. SOD
content in control was 14.4 ± 0.02, 15.0 ± 0.34, 18.4 ± 0.71 (unit/mg protein/h) in NM, AV and AF respectively. APX content in
control was 249.2 ± 17.2, 252.7 ± 27.0, 336.0 ± 4.9 (nmol/mg protein/min) in NM, AV and AF respectively. CAT content in control
was 6.58 ± 0.46, 9.61 ± 0.39, 7.95 ± 0.46 (unit/mg protein/min) in NM, AV and AF respectively. Values are means ± S.E with n = 3.
136
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2.5 µg/ml endosulfan in Nostoc muscorum and then started to
decrease gradually. In studied strains, proline content was
higher than control in Nostoc muscorum (29.2%), Anabaena
variabilis (2.3%) and Aulosira fertilissima (0.08%) at 15 µg/ml.
Very high accumulation of cellular proline (upto 80% of the
total amino acid pool under stress as compared to just 5%
under the normal condition) has been reported earlier in many
higher plant species due to increased synthesis and decreased
degradation under variety of stress conditions such as water,
salt, drought and heavy metal (Bates et al., 1973; Bohnert and
Jensen, 1996; Delauney and Verma, 1993; Kavi kishor et al.,
2005). Although the actual reason behind the accumulation of
proline (presumably by way of synthesis from glutamic acid) is
yet to be known, in plants or plant parts exposed to stress, it
could probably be due to a decrease in the activity of electron
transport system (Venekemp, 1989).
3.2.5.
Oxidative damage
In present study MDA content increased with increasing
concentrations of pesticide suggesting formation of free
radicals eliciting endosulfan toxicity (Fig. 3a). Our results are
in consonance with finding on Plectonema boryanum with
endosulfan (Prasad et al., 2005) it has been suggested that
free radical formation occur due to strong inhibition of PSII.
Several herbicides have been found to generate active oxygen
species, either by direct involvement in radical production or
by inhibition of biosynthetic pathways. The generation of the
hydrocarbon gas ethane, the production of malonaldehyde
and changes in electrolytic conductivity has frequently been
used as sensitive markers for herbicide action in plants
(Kunert et al., 1985; Peleg et al., 2001). Compounds such as
paraquat (also known as methyl viologen) induce light
dependent oxidative damage in plants (Dodge, 1971). The PSI
mediated reduction of the paraquat di-cation results in the
formation of a mono-cation radical which then reacts with
molecular oxygen to produce O-2 with the subsequent production of other toxic species, such as H2O2 and OH (Elstner
et al., 1988). These compounds cause severe toxicological
problems and results in peroxidation of membrane lipids and
general cellular oxidation. Increase in both proline and MDA
contents with increasing pesticide concentration are indicative of a correlation between free radical generation and
proline accumulation (Figs. 2f and 3a). This is also in agreement with the earlier reports of our lab on Spirulina platensis
and Westellopis prolifica (Choudhary et al., 2007; Fatma et al.,
2007).
3.2.6.
Antioxidant
Photosynthetic organisms counteract the toxicity of pesticide induced free radicals by increasing their antioxidative
defense mechanisms that include enzymes such as superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase
(APX) and low molecular weight compounds such as
ascorbate, glutathione, flavonoids, tocopherols, and carotenoids. Ascorbate peroxidase (APX) utilizes the reducing
power of ascorbic acid to eliminate potentially harmful
H2O2. SOD neutralizes the highly reactive superoxide radical
generated in the cell especially under stress condition
(Elstner et al., 1988). The experimental concentrations of
endosulfan accelerated the activities of defense enzymes
SOD, APX and CAT in test cyanobacterial strains (Fig. 3b-d).
The increase in SOD and CAT continued till the 15 µg/ml in
all test organisms while APX increased till the 12.5 µg/ml in
A. variabilis and 10 µg/ml in N. muscorum and A. fertilissima.
We have noted 85.9%, 85.7% SOD increase in N. muscorum
and A. variabilis respectively and only 3.79% observed in A.
fertilissima at 10 µg/ml while 41% SOD increase is being
reported in Plectonema boryanum at 10 µg/ml endosulfan
(Prasad et al., 2005). Maximum SOD, APX and CAT were
observed in N. muscorum. It is suggesting that SOD and CAT
are playing greater role in ROS detoxification.
4.
Conclusions
N. muscorum, A. variabilis, and A. fertilissima have varying
tolerance potential to endosulfan and the order of tolerance
is Nostoc muscorum N Anabaena variabilis N Aulosira fertilissima.
This observation is based on 10 µg/ml tested concentration
where the percentage increases in absorbance growth curve
(36.44, 36.17 and 28.24 %). After that their growth showed
many fold inhibition from first day to 20th day. In other
observations in the case of proline 65, 23.61 and 3.1% and
SOD 85.9, 58.7, 3.79% increase with respect to control
(untreated sample) at 10 µg/ml concentrations in Nostoc
muscorum, Anabaena variabilis and Aulosira fertilissima respectively. At lower tested concentration (2.5 µg/ml) showed
stimulatory response in all tested strains. It is due to stress
response and assimilation of uptake carbon source of
pesticide by the organisms during the processes of degradation of pesticide. Therefore we conclude that up to 2.5 µg/ml
endosulfan concentrations does not affect adversely on
cyanobacterial population. During present investigation we
have successfully attempted our proposed hypothesis and
achieved expected results. The common chlorinated pesticide of paddy fields the endosulfan was found to be toxic to
the non-target organism, the heterocystous N 2 fixing
cyanobacteria frequently used as biofertilisers at higher
concentrations. The observations suggested that the endosulfan exert its toxic effect through free radical mediated
oxidative stress and resistance is being imparted through
antioxidant enzymes as well as proline. Proline has been
produced during the stress condition so it might be helpful
in degradation of pesticide and cope up with adverse
condition. 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 Medical
Research for providing financial assistance to Satyendra
Kumar as SRF.
REFERENCES
Abou-waly H, Abou-Setta MM, Nigg HN, Mallory L. Growth
response of freshwater algae, Anabaena flos- aquae
and Selenastrum capricornutum, to atrazine and hexazinone
herbicides. Bull Environ Contam Toxicol
1991;46:223–9.
S CIE N CE OF T H E TOT AL E N V I RO N ME N T 4 0 3 ( 2 00 8 ) 1 3 0–1 38
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 green
algae. Pesticide 1988;22:43–94.
Ahmed MH, Venkataraman GS. Tolerance of Aulosira fertilissima
to 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 Soc
Biotech IIT/New Delhi; 1980. p. 51–5.
Asada K. The role of ascorbate peroxidase and monodehydrpascorbate
reductase in H2O2 scavenging in plants. In: Scandolios JG, editor.
Oxidative stress and the molecular biology of antioxidant defenses.
Plainview: Cold Spring Harbor Laboratory Press; 1997. p. 715–35.
Asada K. Chloroplasts: formation of active oxygen and its
scavenging. Methods Enzymol 1984;10:422–9.
Babu GS, Hans RK, Singh J, Viswanathan PN, Joshi PC. Effect of
lindane on the growth and metabolic activities of
cyanobacteria. Ecotoxicol Environ Saf 2001;48:219–21.
Bartosz G. Oxidative stress in plants. Acta Physiol Plant
1997;19:47–64.
Bates LS, Wadern RP, Teare ID. Rapid estimation of free proline for
water 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 certain
metabolic activities of N. muscorum: Effect of methylparathion
and benthiocarb. Bull Environ Contam Toxicol 1991;47:43–50.
Bohnert HJ, Jensen RG. Strategies for engineering water stress
tolerance in plants. Trends Biotechnol 1996;4:89–97.
Choudhary M, Jetely UK, Khan A, Zutshi S, Fatma T. Effect of heavy
metal stress on proline and malondialdehyde and
superoxidedismutase activity in the cyanobacterium Spirulina
platensis S-5. Ecotoxicol Environ Saf 2007;66:204–9.
Del Vos CH, Vonk MJ, Schat H. Glutathione depletion due to copper
induced phytochelatin synthesis causes oxidative stress in
Silene cucubalus. Plant Physiol. 1992;98:853–8.
Delauney AJ, Verma DPS. Proline biosynthesis and osmoregulation
in plants. J Plant 1993;4:215–23.
Del-Rio LA, Sevilla F, Sandalio LM, Palma JM. Nutritional effect and
expression of superoxide dismutase; induction and gene
expression, diagnostics, prospective protection against oxygen
toxicity. Free Radic Res Commun 1991;12(13):819–28.
Dhindsa RS, Dhindsa PP, Thorpe TA. Leaf senescence: correlated
with increased level of membrane permeability and lipid
peroxidation, and decreased levels of superoxide dismutase
and 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 green
plants in relation to stress situation. Curr Top Plant Biochem
Physiol 1988;7:159–87.
Fatma T, Khan MA, Choudhary M. Impact of environmental pollution
on 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 Biochem
Biophys 1968;125:189–98.
Hellebust JA, Craige JS. Handbook of physiological and biochemical
methods. Cambridge: Cambridge university press; 1978. p. 64–70.
Hendry GAF. Oxygen and environmental stress in plants: an
evolutionary context. Proc R Soc Edinb 1994;102B:155–65.
Hoechst AG. Thiodan: the versatile but selective insecticide.
Instructions for its use, Frankfurt, West Germany Hoechst
Aktiengesellschaft; 1977.
Indian Council for Agriculture Research (ICAR). www.iffco.nic.in/
applications/Agriinfo.nsf.
Kaushik BD, Venkataraman GS. Response of cyanobacterial
nitrogen 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
137
transport in higher plants: Its implications in plant growth and
abiotic stress tolerance. Cur Sci 2005;88(3):424–38.
Kolte SO, Goyal SK. Inhibition of growth and nitrogen fixation in
Calothrix marchia by herbicide in vitro. In: Kaushik BD, editor.
Proc National Symposium on Cyanobacterial Nitrogen
Fixation. New Delhi: Pub Associated Co.; 1990. p. 507–10.
Kumar S, Jetley UK, Fatma T. Tolerance of Spirulina platensis-S5
and Anabaena sp. to Endosulfan an organochlorine pesticide.
Ann Plant Physiol 2004;18(2):103–7.
Kunert KJ, Homrighausen C, Bohme H, Boger P. Oxyfluorfen and
lipid peroxidation: Protein damage as a phototoxic
consequence. Weed Sci 1985;33:766–79.
Kwon CS, Penner D. The interaction of insecticides with herbicide
activity. Weed technol 1995;9:119–24.
Leitao M, das A, Cardozo KHM, Pinto E, Colepicolo P. PCB induced
oxidative stress in the unicellular marine dinoflagellates
Lingulodinium polyedrum. Arch Environ Comtam Toxicol
2003;45:59–65.
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement
with the Follin. phenol reagent. J Biol Chem 1951;193(1):265–75.
Mackinney G. Absorption of light by chlorophyll solution. J Biol
Chem 1941;140:315–22.
Mansour FA, Soliman ARI, Shaaban - Desouki SA, Hussein MH. Effect
of herbicides on cyanobacteria. I. Changes in carbohydrate
content, Pmase and GOT activities in Nostoc kihlmani and
Anabaena oscillarioides. Phykos 1994;33:153–62.
Moreland DE. Mechanism of action of herbicides. Ann Rev Plant
Physiol 1980;315:97–638.
Nakano Y, Asada K. Hydrogen peroxide is scavenged by
ascorbate-specific peroxidase in spinach chloroplasts. Plant
Cell Physiol 1981;22:867–80.
Olga B, Eija V, Kurt VF. Antioxidants, oxidative damage and oxygen
deprivation 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, active
oxygen species and antioxidants responses of paddy field
cynobacterium Plectonema boryanum to endosulfan stress.
J Gen Appl Microbiol 2005;51:115–23.
Rajendran UM, Elango K, Anand N. Effects of a fungicide, an
insecticide, and a biopesticdie on Tolypothrix scytonemoides.
Pestic Biochem Physiol 2007;87:164–71.
Relwani LL. Role of blue green algae on paddy field. Curr Sci
1963;32:417–8.
Satish N, Tiwari GL. Pesticide tolerance in Nostoc linckia in
relation to the growth and nitrogen fixation. Proc Natl Acad Sci
India 2000;70:319–23.
Sharma VK, Gaur YS. Nitrogen fixation by pesticide adopted strains
of 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 insecticide
endosulfan by Mucor thermohyalospora MTCC 1384. Curr Sci
2000;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 to
water deficit and desiccation. New Phytol 1993;125:27–58.
Spiro RG. Analysis of sugars found in glycoproteins. Methods
enzymol 1966;8:3–26.
Stainer RY, Kunisawa R, Mandel M, Cohin-Bazire G. Purification
and properties of unicellular blue green algae (order
Chrococcales). Bacteriol Rev 1971;35:171–205.
Stratton GW. The effects of pesticides and heavy metals on
phototrophic micro-organisms. Rev Environ Toxicol
1987;3:71–112.
Torres AMR, 'Flaeerty LMO. Influence of pesticides on Chlorella,
Chlorococcum, Stigeoclonium (Chlorophyceae) Tribonema,
138
SC IE N CE OF T H E TOT AL E N V I RO N ME N T 4 0 3 ( 2 00 8 ) 1 3 0–1 38
Vaucheria (Xanthophyceae) and Oscillatoria (Cyanophyceae).
Phycologia 1976;15:25–36.
Venekemp JH. Regulation of cytosolic acidity in plants under
condition of drought. Plant Physiol 1989;76:112–7.
Wright SJL, Stantharpe AF, Downs JD. Interaction of herbicide
propanil and a metabolite, 3, 4-dichloromiline with blue green
algae. Acta Pathol Acad Hung 1977;12:51–7.
Yan GA, Yan X, Wu W. Effect of herbicide Molinate on mixotrophic
growth, photosynthetic pigments and protein Content of
Anabaena sphaerica under different light conditions.
Ecotoxicol Environ Saf 1997;38:144–9.
Zarger MY, Dar GH. Effect of benthiocarb and butachlor on growth
and nitrogen fixation by cyanobacteria. Bull Environ Contam
Toxicol 1990;45:232–4.