Differential physiological and biochemical responses of two cyanobacteria Nostoc muscorum and Phormidium foveolarum against oxyfluorfen and UV-B radiation.

Abstract

In the present study, degree of tolerance and tolerance strategies of two paddy field cyanobacteria viz. Nostoc muscorum and Phormidium foveolarum against oxyfluorfen (10 and 20 μg ml(-1)) and UV-B (7.2 kJ m(-2)d(-1)) stress were investigated. Oxyfluorfen and UV-B decreased growth, photosynthesis, nutrient uptake, nitrate reductase, acid and alkaline phosphatase activities, which accompanied with the increase in the level of oxidative stress. However, growth was more affected in N. muscorum than P. foveolarum. Antioxidants exhibited differential responses against oxyfluorfen and UV-B stress. Ascorbate and proline levels were higher in P. foveolarum. A protein of 66 kDa was expressed in N. muscorum, however, it was absent in P. foveolarum than those of N. muscorum. Besides this, a protein of 29 kDa appeared in P. foveolarum under all the treatments, but it was present only in control cells of N. muscorum cells. Overall results indicated resistant nature of P. foveolarum against oxyfluorfen and UV-B stress in comparison to N. muscorum.

Full text

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Differential physiological and biochemical responses of two cyanobacteria
Nostoc muscorum and Phormidium foveolarum against oxyfluorfen
and UV-B radiation
Sheeba, Vijay Pratap Singh, Prabhat Kumar Srivastava, Sheo Mohan Prasadn
Ranjan Plant Physiology and Biochemistry Laboratory, Department of Botany, University of Allahabad (A Central University of India), Allahabad (UP) 211002, India
a r t i c l e i n f o
Article history:
Received 16 December 2010
Received in revised form
6 July 2011
Accepted 11 July 2011
Available online 28 July 2011
Keywords:
Cyanobacteria
Differential sensitivity
Oxidative stress
Oxyfluorfen
Photosynthesis
UV-B radiation
a b s t r a c t
In the present study, degree of tolerance and tolerance strategies of two paddy field cyanobacteria viz.
Nostoc muscorum and Phormidium foveolarum against oxyfluorfen (10 and 20 mg ml?1) and UV-B
(7.2 kJ m?2d?1) stress were investigated. Oxyfluorfen and UV-B decreased growth, photosynthesis,
nutrient uptake, nitrate reductase, acid and alkaline phosphatase activities, which accompanied with
the increase in the level of oxidative stress. However, growth was more affected in N. muscorum than
P. foveolarum. Antioxidants exhibited differential responses against oxyfluorfen and UV-B stress.
Ascorbate and proline levels were higher in P. foveolarum. A protein of 66 kDa was expressed in
N. muscorum, however, it was absent in P. foveolarum than those of N. muscorum. Besides this, a protein
of 29 kDa appeared in P. foveolarum under all the treatments, but it was present only in control cells of
N. muscorum cells. Overall results indicated resistant nature of P. foveolarum against oxyfluorfen and
UV-B stress in comparison to N. muscorum.
& 2011 Elsevier Inc. All rights reserved.
1. Introduction
Soil and water pollution due to pesticides has become a
common concern among environmentalists. Use of pesticides
became indispensible and an integral part of modern agriculture
and their use under Integrated Pest Management Programme to
save the crop losses becomes quite decisive in countries like India
in the wake of second green revolution likely to be experienced in
next few years. We cannot rule out the use of pesticides in our
agricultural fields because of steady but continuous rise in
population and lesser availability of agricultural fields. But exces-
sive and imprudent use of pesticides will definitely lead to
environmental degradation in several ways. Pesticides that are
used in agricultural practices are transported to water bodies
through run-off, drift and leaching, and increase the risk of
exposure to non-target organisms (Chen et al., 2007). Oxyfluor-
fen(2-chloro-1-(3-ethoxy-4-nitrophenyl)-4-trifluoromethyl)ben-
zene) is an ortho-substituted diphenylether, which is a selective
herbicide and used to control pre- and post-emergent broad leaf
and grassy weeds in agricultural fields (Ahrens, 1979). Studies
have demonstrated that diphenyl ethers inhibit photosynthesis
by affecting electron transport activities in chloroplasts of higher
plants (Moreland et al., 1970; Bugg et al., 1980; Pritchard et al.,
1980). Bugg et al. (1980) suggested that the site of inhibition in
electron transport by diphenyl ethers is in the region of plasto-
quinone and cytochrome f. However, Moreland et al. (1970)
suggested that interference with ATP generation could be one of
the mechanisms of diphenyl ether phytotoxicity. Besides this,
oxyfluorfen has the ability to decrease growth indirectly by
generating reactive oxygen species (ROS) such as singlet oxygen
(1O2), superoxide radical (O2
hydroxyl radicals (dOH) (Geoffroy et al., 2002). Oxyfluorfen has
long residence time (half life 72–160 days) in natural conditions;
it persists in fertile layer of soil and harms non-target organisms
of soil including cyanobacteria leading to considerable losses in
crop yield (Baruah and Mishra, 1986; Yen et al., 2003). Several
studies demonstrated impact of oxyfluorfen on various metabolic
activities of higher plants; however, impact of oxyfluorfen on
metabolic activities of cyanobacteria is less explored despite their
ecological as well as economic importance.
Besides this, incoming solar UV-B radiation may also adversely
affect cyanobacteria. Solar UV-B radiation comes on the earth
surface due to depletion of stratospheric ozone layer. It is
reported that chlorofluorocarbons, which can deplete the ozone
layer, can remain in the upper atmosphere for 40–150 years,
hence, the global UV-B radiation will not recover to the levels of
the pre-industrialization era by 2050, even if all the nations
d?), hydrogen peroxide (H2O2) and
Contents lists available at ScienceDirect
journal homepage: www.elsevier.com/locate/ecoenv
Ecotoxicology and Environmental Safety
0147-6513/$-see front matter & 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.ecoenv.2011.07.006
nCorresponding author. Mobile: þ919451373143.
E-mail addresses: vijaypratap.au@gmail.com (V.P. Singh),
prabhatsrivastava.au@gmail.com (P.K. Srivastava),
profsmprasad@gmail.com (S.M. Prasad).
Ecotoxicology and Environmental Safety 74 (2011) 1981–1993

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implement
Organization, 2007; Mohammed and Tarpley, 2011). Ultraviolet-
B radiation affects plants including cyanobacteria from the
molecular level to the ecosystem level (He and H¨ ader, 2002). It
has been reported that UV-B induces decrease in growth and
photosynthesis by enhancing damage to lipids, proteins and
nucleic acids (He and H¨ ader, 2002).
Cyanobacteria, in general, are exposed to harsh conditions
such as extremes of high and low light, UV radiations, heavy
metals, salinity, drought, temperature, xenobiotics, etc. and
respond by generating ROS (Gadkari, 1988; Geoffroy et al.,
2002; He and H¨ ader, 2002; Prasad and Zeeshan, 2005; Wang
et al., 2008). ROS are the inevitable product of oxygen metabo-
lism. They may originate in various electron transport chains—
photosynthetic or respiratory (B¨ oger and Sandmann, 1998). Their
least amount is necessary in cell signaling but when they are
present in greater amount than needed, they harm various
components of cells—a situation termed as oxidative stress. Both
the stresses—UV-B and oxyfluorfen may also generate ROS in
plants often more than required (Geoffroy et al., 2002; He and
H¨ ader, 2002). These ROS may readily peroxidise membrane lipids
and proteins (He and H¨ ader, 2002; Prasad and Zeeshan, 2005). So
their concentration ought to be controlled. In order to control
their amount, a full-fledged antioxidant system has evolved in
cellular system.
It is known that cyanobacteria occupy the lowest trophic level
within food webs. Therefore, changes in their community may
have indirect but significant effects on the rest of the fresh water
communities, nitrogen status and organic matter accumulation
(Li et al., 2010). It is also known that organisms differ in their
sensitivity to different stress factors. In modern agricultural
technological programs cyanobacteria are utilized to increase
the availability of nitrogen and organic matter in soil for better
production of rice in rice producing countries. Nostoc muscorum, a
heterocystous cyanobacterium, fixes molecular nitrogen under
aerobic condition while Phormidium foveolarum, a non-heterocys-
tous strain, fixes nitrogen in darkness. Herbicide and UV-B have
been reported to inhibit nitrogenase activity (Gadkari, 1988;
Sinha et al., 1996). Therefore, for better exploitation of cyano-
bacteria as biofertilizer we must know about their degree of
tolerance and tolerance strategies to various stresses. Therefore,
in the present study, an attempt has been made to investigate
the degree of tolerance of two paddy field cyanobacteria viz.
N. muscorum and P. foveolarum against oxyfluorfen and UV-B by
analyzing different physiological and biochemical parameters.
the MontrealProtocol(World Meteorological
2.Materials and methods
2.1. Reagents
Oxyfluorfen (trade name ‘‘Oxyflour’’ (EC 23.5%), active ingredient oxyfluorfen
(2-chloro-1-(3-ethoxy-4-nitrophenyl)-4-trifluoromethyl)benzene)
present study was purchased from Divine Tree Limited, Gujarat, India. All
chemicals used in present work were of analytical grade and purchased from
different companies. Bovine serum albumin, 3-(3, 4-dichloro diphenyl)-1,
1-dimethyl urea, methyl viologen, p-benzoquinone and diphenyl carbazide were
purchased from Sigma Aldrich, USA. Methanol, acid ninhydrin, sucrose, hydro-
xylamine, sulphuric acid, toluene, trichloroacetic acid, thiobarbituric acid and
hydrogen peroxide were purchased from Merck, India. Sodium nitrate, K2HPO4,
MgCl2, sodium azide, manganese chloride, potassium nitrate, succinate, Tris–HCl
buffer, potassium nitrite, sulfanilamide, naphthylethylene diamine dihydrochlor-
ide, sodium nitrite, methionine, sulfosalicylic, sodium molybdate, sodium dodecyl
sulphate and polyacrylamide were purchased from Himedia, India. HEPES, NaOH
and sodium carbonate were purchased from Qualigens Fine Chemicals, India. 2,6-
dichlorophenol indophenols and nitroblue tetrazolium chloride were obtained
from LOBA Chemie, Mumbai, India. p-nitrophenyl phosphate was obtained from
CDH, India, riboflavin from Hoffmann-La Roche, Inc., Nutley, New Jersey, USA, and
pyrogallol from S.D. fine-CHEM Limited, India.
used inthe
2.2. Organisms and culture conditions
The cyanobacteria
N. muscorum
(filamentous and heterocystous) and
P. foveolarum (filamentous non-heterocystous) were isolated in pure forms from
rice fields of Allahabad city using standard microbiological techniques. Their pure
stocks were maintained in our laboratory. Homogenous cultures of N. muscorum
and P. foveolarum were grown in BG 11 medium without and with nitrogen
(NaNO3 as nitrogen source), respectively, at 2572 1C in culture room under
the photosynthetically active radiation (PAR) of 70 mM photons m?2s?1with a
14:10 h light:dark period. All experiments were performed with 7 days old
exponential phase of culture.
2.3.Oxyfluorfen and UV-B treatments
After series of screening experiments with various doses of oxyfluorfen,
effective doses of 10 and 20 mg ml?1of oxyfluorfen were selected for the present
study. These two concentrations were outcome of number of screening experi-
ments ranging from 1 to 100 mg ml?1, toxic to the test cyanobacteria at different
levels. The exponentially grown cyanobacterial cells were harvested by centrifu-
gation at 4000g for 5 min and washed twice with distilled water and then the
pellets were resuspended in BG 11 medium containing 0, 10 and 20 mg ml?1of
oxyfluorfen. Oxyfluorfen treated and untreated cultures were kept in Petri dishes
under 70 mM photons m?2s?1of PAR. Petri dishes were kept on a magnetic
shaker to reduce the cell aggregation and sedimentation and then cultures were
irradiated with artificial UV-B radiation equivalent to 7.2 kJ m?2d?1, which is
biologically effective. Ultraviolet-B radiation was provided by UV-lamp (TL 40 W/12
Philips, Holland) along with 70 mM photons m?2s?1of PAR. The irradiance was
measured with the help of power meter (Spectra physics, USA Model 407, A–2). The
artificial UV-B radiation was equivalent to 15% stratospheric ozone depletion
at Varanasi, an adjoining city of Allahabad, and having fluence rate of 0.4 W m?2.
To filter out UV-C radiation (o280 nm), the lamp was wrapped with 0.127 mm
cellulose acetate film (Johnston Industrial Plastics, Toronto, Canada). After 72 h of
experiment, different parameters were analyzed by harvesting the cyanobacterial
cells. Cyanobacterial cells, which were not treated with oxyfluorfen and UV-B, were
regarded as ‘control’.
2.4.Measurement of growth and extraction of photosynthetic pigments
Growth was measured by estimating dry mass after 72 h of experiment.
Treated and untreated cells (80 ml) were harvested by centrifugation at 4000g for
10 min. The cells were washed thrice with 1 mM of ethylene diaminetetra-acetic
acid (EDTA) to remove surface bound metals. Cells of each sample were oven dried
at 80 1C for 48 h then weighted. For chlorophyll a (Chl a) and carotenoids
estimations, 40 ml of culture [equivalent to 66 mg dry mass (ml culture)?1and
33 mg dry mass (ml culture)?1, respectively, in unstressed N. muscorum and
P. foveolarum cells] from each sample was centrifuged at 4000g for 10 min, and
pigments from cells were extracted in 100% methanol. The concentrations of Chl a
and carotenoids were quantified by the method of Porra et al. (1989) and Goodwin
(1954), respectively. For phycocyanin determination, cells of each sample were
treated with toluene and extracted with 2.5 mM potassium phosphate buffer
(pH 7.0) after repeated freezing and thawing, and the absorbance of the super-
natant was recorded at 620 nm. The amount of phycocyanin was determined by
the method of Blumwald and Tel-Or (1982).
2.5. Measurements of whole cell photosynthetic O2-evolution and respiration
Photosynthetic activity in terms of whole cell O2-evolution of treated and
untreated cyanobacterial cells was estimated by measuring O2-evolution for 5 min
with a Clark type O2-electrode (Rank Brothers, UK) in a temperature controlled
airtight reaction vessel at 25 1C. The cyanobacterial cells were illuminated by a
projector lamp providing 360 mM photons m?2s?1PAR at the surface of vessel.
Respiration rate of treated and untreated cyanobacterial cells was estimated by
measuring the O2-consumption in darkness with the Clark type oxygen electrode
at 25 1C for 5 min.
2.6.Measurements of photosynthetic electron transport activities
Spheroplasts of N. muscorum and P. foveolarum were prepared according to the
method of Spiller (1980) and resuspended in the HEPES-NaOH buffer (pH 6.9)
containing 0.5 M sucrose, 10 mM HEPES-NaOH, 5 mM K2HPO4, 10 mM MgCl2and
2% (w:v) bovine serum albumin (BSA). The rates of photosystem I (PS I) and whole
photosynthetic chain reaction were measured in terms of O2-consumption.
The 6 ml reaction mixture consisted of buffer (pH 6.9), 10 mM [3-(3,4-dichloro
diphenyl)-1,1-dimethyl urea)] (DCMU), 1 mM sodium ascorbate, 50 mM 2,
6-dichlorophenol indophenol (DCPIP), 50 mM sodium azide (NaN3), 100 mM
methyl viologen (MV) and spheroplasts, which were used for measuring PS I
activity, while for whole photosynthetic chain reaction activity the mixture
Sheeba et al. / Ecotoxicology and Environmental Safety 74 (2011) 1981–1993
1982

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consisted of buffer (pH 6.9), 50 mM NaN3, 100 mM MV and spheroplasts. Photo-
system II (PS II) electron transport activity was measured in terms of O2-evolution.
The 6 ml reaction mixture contained buffer (pH 6.9), p-benzoquinone (p-BQ;
1 mM) and spheroplasts. Spectrophotometric assay of PS II activity in terms of
DCPIP photoreduction was also monitored by measuring changes in absorption at
600 nm in the presence of various exogenous electron donors, i.e. hydroxylamine
(NH2OH), diphenyl carbazide (DPC) and manganese chloride (MnCl2).
2.7. Nutrient uptake
The uptake of nitrate (NO3
the external medium following the method of Nicholas and Nason (1957). Cells
from each sample (40 ml) were harvested by centrifugation at 4000g for 10 min,
washed twice with double distilled water and resuspended in growth medium
(20 ml) containing 1 mM KNO3. NO3
75 mM photons m?2s?1PAR at 25 1C. Cell suspension (10 ml) from each sample
was withdrawn after 4 h of incubation and centrifuged to obtain cell free super-
natant (5 ml). To this, 2 ml of brucine reagent was added and then samples were
kept in ice bath and 5 ml of H2SO4was added slowly drop by drop. Later on, these
were placed in a water bath at 90 1C for 10 min until the chloroform evaporates.
The color developed was read spectrophotometrically at 410 nm and NO3
in the sample was calculated using the standard curve.
The uptake of PO4
the external medium. For PO4
harvested by centrifugation at 4000g for 10 min, and resuspended in fresh BG 11
medium containing 100 mM phosphate and incubated at 25 1C under a PAR
of 75 mM photons m?2s?1. For PO4
3–4 times with double distilled water to avoid any possibility of presence of PO4
Samples were withdrawn after 4 h of incubation, immediately centrifuged and the
cell-free supernatants were analyzed for residual PO4
660 nm following the method of Ames and Dubin (1960).
?) was determined by measuring its depletion from
?uptake experiments were carried out under
?present
3?was determined by measuring the depletion of PO4
3?uptake, cells (40 ml) from each sample were
3?from
3?uptake experiments, glassware were rinsed
3?.
3?spectrophotometrically at
2.8. Measurements of acid and alkaline phosphatase and nitrate reductase activities
Acid phosphatase activity (ACP, EC 3.1.3.2) was determined by measuring
formation of p-nitrophenol from substrate p-nitrophenyl phosphate (p-NPP)
according to the method of Torriani (1960). A definite volume (40 ml) of each
sample was harvested, washed and resuspended in 4 ml of succinate buffer
(pH 6.0) and then treated with toluene (1%, v/v) for 30 min to permeabilize the
cells. The reaction was started by the addition of 0.2 ml of p-NPP (final
concentration of 5.2 mM) at 37 1C and terminated by the addition of 1 ml of 1 N
NaOH after 30 min of incubation. Samples were centrifuged and absorbance of
supernatant was recorded at 410 nm and the amount of p-nitrophenol in each
sample was calculated with the help of standard curve.
Alkaline phosphatase activity (ALP, EC 3.1.3.1) was assayed by the determina-
tion of p-nitrophenol formed from the substrate p-nitrophenyl phosphate (p-NPP)
following the method of Ihlenfeldt and Gibson (1975). A known volume (40 ml) of
treated and untreated cells was harvested, washed and resuspended in 4 ml of
0.2 M Tris–HCl buffer (pH 8.5) and then treated with toluene (1%, v/v) for 30 min.
The reaction was initiated by the addition of 0.2 ml of p-NPP (final concentration
of 5.2 mM) at 37 1C and terminated after 30 min of incubation by the addition of
0.2 ml of 1 M K2HPO4. Samples were centrifuged and absorbance of supernatant
was read at 420 nm. The amount of p-nitrophenol formed was calculated with the
help of standard curve.
Nitrate reductase (NR, EC 1.6.6.1) activity was measured by the method of
Camm and Stein (1974). Measurement of NR activity was based on total nitrite
(NO2
with double distilled water and incubated in growth medium containing 5 mM
KNO3. Samples were withdrawn at desired time and formation of NO2
measured by diazo-coupling method of Lowe and Evans (1964). The absorbance of
pink color was read at 540 nm.
?) formed. Cyanobacterial suspension (40 ml) was centrifuged, washed thrice
?was
2.9.Measurements of superoxide radicals (SOR; O2
d?) and hydrogen peroxide (H2O2)
O2
d?in each sample was measured by the method of Elstner and Heupel
(1976). This assay is based on the formation of NO2
presence of O2
homogenized in 65 mM potassium phosphate buffer (pH 7.8) and centrifuged at
10,000g for 10 min at 4 1C. The reaction mixture consisted of 65 mM potassium
phosphate buffer (pH 7.8), 10 mM hydroxylamine hydrochloride and cell extract
was incubated for 20 min at 25 1C. After this, 17 mM sulfanilamide and 7 mM
naphthylethylene diamine dihydrochloride (NEDD) were mixed to the incubated
reaction mixture. After 15 min of incubation, diethyl ether was mixed to the same
reaction mixture gently and centrifuged at 2000 g for 5 min. The absorbance of the
colored aqueous phase was recorded at 530 nm. Production of O2
with the help of standard curve prepared with NaNO2
each sample (40 ml) was centrifuged at 4000g for 10 min and the cells were
homogenized in 3.5 ml of 5% (w/v) trichloroacetic acid (TCA) and centrifuged at
?from hydroxylamine in the
d?. Cells obtained from 40 ml culture of each sample were
d?was calculated
?. For H2O2measurement,
10,000g for 15 min. The supernatant obtained was used to analyze total peroxide
by the method of Sagisaka (1976).
2.10.
electrolyte leakage
Measurements of lipid peroxidation (as malondialdehyde; MDA content) and
Lipid peroxidation was determined in terms of MDA (malondialdehyde)
content, a product of unsaturated fatty acid peroxidation known as 2-thiobarbi-
turic acid (TBA) reactive metabolite. MDA concentration was estimated by the
method of Heath and Packer (1968). Culture suspension (40 ml) from each sample
was harvested by centrifugation and washed twice in 50 mM phosphate buffer
(pH 7.0). Cells were homogenized in 5% (w/v) TCA. The resulting homogenate was
centrifuged at 10,000g for 10 min. To 0.5 ml of the aliquot of the supernatant, 2 ml
of 20% TCA containing 0.5% (w/v) TBA was added. The mixture was heated at 90 1C
for 20 min and then quickly cooled in ice bath followed by centrifugation.
Absorbance of the supernatant was read at 532 and 600 nm. The value for non-
specific absorption of each sample at 600 nm was subtracted from absorption
recorded at 532 nm. The MDA concentration was calculated using the extinction
coefficient 155 mM?1cm?1.
The intactness of plasma membrane in cells of each sample was estimated in
terms of electrolyte leakage by the method of Gong et al. (1998). Each sample
(40 ml) was centrifuged at 4000g for 10 min and the cells were washed thoroughly
with double distilled water and placed in test tubes containing 30 ml double
distilled water. The tubes were incubated in a water bath at 30 1C for 2 h and
the initial electrical conductivity (EC1) of supernatant was measured by Con-
ductivity Meter. One of the samples from control set was boiled at 100 1C for
15 min to release all electrolytes, cooled and the final electrical conductivity (EC2)
was measured. The percentage of electrolyte leakage was calculated using the
formula:
EC ¼EC1
EC2? 100ð%Þ:
2.11.Assay of antioxidant enzymes
In vivo catalase (CAT, EC 1.11.1.6) activity was determined polarographically
by the method of Egashira et al. (1989). Cyanobacterial cells (40 ml) were
harvested from different test samples by centrifugation, washed twice with
double distilled water and finally suspended in 50 mM of phosphate buffer
(pH 7.0). In each sample, CAT activity was determined by recording O2-evolution
for 1 min in darkness after the addition of 5 ml of 50 mM phosphate buffer
(pH 7.0) containing 50 mM H2O2. To this, 1 ml of cell suspension was added
in darkness and O2-evolution was monitored by Clark type O2-electrode
(Rank Brothers, UK). Temperature around the vessel was maintained at 25 1C.
One unit of CAT is the amount of the enzyme producing 1 nM O2min?1.
Superoxide dismutase (SOD, EC 1.15.1.1) activity was assayed by the method
of Giannopolitis and Ries (1977). Cells collected from each sample (40 ml) by
centrifugation were washed twice with 100 mM EDTA–phosphate buffer (pH 7.8).
Cells were homogenized in an ice cold 100 mM EDTA–phosphate buffer (pH 7.8).
The homogenate was centrifuged for 20 min at 10,000g. The supernatant fraction
was used as the enzyme source. The 3 ml reaction mixture contained 1.3 mM
riboflavin, 13 mM L-methionine, 0.05 M Na2CO3, (pH 10.2), 63 mM p-nitroblue
tetrazolium chloride (NBT) and 0.1 ml of enzyme extract. Reaction was carried
out for 15 min in similar test tubes at 25 1C under an illumination of
75 mM photons m?2s?1. The initial rate of reaction as measured by the difference
in the increase in absorbance at 560 nm in the presence and absence of extract
was proportional to the amount of enzyme. The unit of SOD activity is defined as
the amount of enzyme, which caused a 50% inhibition of the reaction observed in
the absence of enzyme. For the blank, the reaction was run in darkness.
Peroxidase (POD, EC 1.11.1.7) activity was determined by the method of
Gahagen et al. (1968). Cells obtained from 40 ml culture suspension of each
sample were homogenized in 2 ml 100 mM phosphate buffer (pH 7.0) at 5 1C. The
homogenate was centrifuged at 10,000g for 30 min. The supernatant was con-
sidered as the enzyme extract. The 3 ml reaction mixture consisted of 1 ml 25 mM
H2O2, 1 ml 100 mM pyrogallol and 1 ml enzyme extract. After thorough mixing
of the reaction mixture, change in optical density was monitored at 430 nm for
2–3 min.
2.12.Measurement of ascorbate and proline
Ascorbate content in cells of each sample was estimated by the method of
Oser (1979). Cells (40 ml) were homogenized in 2 ml of 5% (w/v) sulfosalicylic
acid, and then the homogenate was centrifuged at 10,000g for 10 min. The
reaction mixture consisted of 2 ml 2% (w/v) sodium molybdate, 2 ml 0.15 N
H2SO4, 1 ml 1.5 mM K2HPO4and 1 ml crude extract. The reaction mixture was
incubated at 60 1C in water bath for 40 min, cooled, centrifuged at 8000g for
10 min and the absorbance was recorded at 660 nm. The amount of ascorbate was
calculated by comparing absorbance with standard curve.
Sheeba et al. / Ecotoxicology and Environmental Safety 74 (2011) 1981–1993
1983

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Proline content was estimated according to the method of Bates et al. (1973).
Cells obtained from 40 ml culture suspension of each sample were crushed in 3%
(w/v) sulfosalicylic acid, centrifuged at 10000g and 1 ml of this extract was mixed
with 3% (v/v) glacial acetic acid and acid ninhydrin solution. Samples were heated
for 1 h in a water bath maintained at 95 1C, cooled and extracted with 4 ml toluene
by vortexing for 5 min with a test tube mixer. The toluene layer was taken for
recording the absorbance at 520 nm using toluene as blank. The proline content in
each sample was calculated with the help of standard curve.
2.13.SDS-PAGE analysis of proteins
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of
both the cyanobacterial proteins was done on 12% polyacrylamide resolving
gel and the separation of individual protein was carried out in a vertical gel
electrophoresis system (Perfit, India) with gels of 160?120 mm?2and 1 mm
thick, according to the method of Laemmli (1970).
2.14. Statistical analysis
All the experiments were conducted in triplicate and repeated at least thrice to
confirm the reproducibility of the results. A one-way ANOVA test was applied to
confirm the significance of data. Comparison between control and treatment’s
means was carried out using Duncan’s multiple range test (DMRT) at Po0.05
significance level and different parameters were correlated by regression analysis
in which growth (dry mass) was regarded as independent variable. SPSS-10
software was used for DMRT and regression analysis.
3. Results and discussion
3.1. Growth, pigments, photosynthesis and respiration
Oxyfluorfen showed (differential) inhibitory effects on both the
cyanobacteria as indicated by decreased biomass accumulation,
photosynthetic pigments and photosynthetic activities (Tables 1–5;
Fig. 1). Exposure of 10 and 20mg ml?1oxyfluorfen caused reduction
in dry masses by 41% and 50% in N. muscorum and only by 6% and
15% in P. foveolarum, respectively, in comparison to their respective
controls (Fig. 1). These reductions were site manifestation of various
pigments (Chl a, carotenoids, phycocyanin) and physiological
changes (Tables 1–8, Figs. 1–3 and 6). Chlorophyll biosynthesis is
catalyzed by several enzymes in multiple steps. Out of these, in the
last step protoporphyrinogen IX is converted to protoporphyrin IX,
which is catalyzed by protoporphyrinogen IX oxidase (EC 1.3.3.4)
(Jacobs and Jacobs, 1987; Matringe et al., 1992; B¨ oger and
Sandmann, 1998; Geoffroy et al., 2003). Proper activity of this
protoporphyrinogen IX oxidase is necessary for chlorophyll biosynth-
esis (Jung, 2011). Oxyfluorfen (chemically diphenyl ether) is a
peroxidizing herbicide, which has been reported to alter the activity
of enzyme protoporphyrinogen oxidase (Duke et al., 1991). The
inhibition in pigment synthesis due to alteration in protoporphyr-
inogen oxidase activity may disturb the function of light harvesting
complex and energy transfer within photosystems (Karapetyan et al.,
1983), which ultimately results in alterations in photosynthetic
electron transport reactions as indicated by inhibition in PS II, PS I
and whole photosynthetic electron transport chain reactions
(Table 3). Further, the alteration in protoporphyrinogen IX oxidase
activity will cause an accumulation of protoporphyrinogen IX, which
is oxidized to protoporphyrin IX by non-specific peroxidases (Aizawa
and Brown, 1999). Protoporphyrin IX has strong photosensitizing
property and is responsible for generation of ROS and causing
oxidative stress (B¨ oger and Sandmann, 1998; Aizawa and Brown,
1999). Besides protoporphyrin IX-mediated generation of ROS, over-
reduction of photosynthetic electron transport chain due to herbicide
may also generate ROS by spilling out electrons and giving it to
Table 1
Effect of oxyfluorfen and UV-B alone and together on photosynthetic pigments in N. muscorum and P. foveolarum.
Treatments Photosynthetic pigments (mg (ml culture)?1)
N. muscorumP. foveolarum
Chlorophyll a
Carotenoids PhycocyaninChlorophyll a
Carotenoids Phycocyanin
Oxyfluorfen (mg/ml)
0
10
20
1.1470.051a
0.71470.032 (?37)c
0.59570.030(?48)d
0.39670.020a
0.27670.014 (?30)c
0.24070.011 (?39)d
7.3070.33a
3.8070.17 (?48)c
3.2770.15 (?55)d
0.53670.027a
0.40870.019 (?24)c
0.36870.018 (?31)d
0.25670.012a
0.20870.009 (?19)c
0.20070.010 (?22)cd
4.3570.20a
2.8670.14 (?34)c
2.5870.13 (?41)d
Oxyfluorfen (mg/ml)þ7.2 kJ m?2d?1(UV-B)
0 0.91870.037 (?19)b
100.32370.013 (?72)e
200.22170.011 (?81)f
Data are means7SE of three independent experiments. Values with different letters within the same column are significantly different (Po0.05) according to the
Duncan’s multiple range test. Values in parentheses indicate percent decrease (?) over control value.
0.34270.016 (?14)b
0.17470.009 (?56)e
0.13870.006 (?65)f
5.2370.24 (?28)b
1.2070.064 (?84)e
0.87270.040 (?88)ef
0.51270.026 (?4)ab
0.40070.020 (?25)c
0.34470.018 (?36)e
0.22470.012 (?13)b
0.20870.010 (?19)c
0.17270.009 (?33)e
4.0170.18 (?8)b
2.7270.14 (?37)c
2.4570.13 (?44)de
Table 2
Effect of oxyfluorfen and UV?B alone and together on photosynthetic activity and respiration in N. muscorum and P. foveolarum.
Treatments
N. muscorumP. foveolarum
Photosynthetic activity (mM O2
evolved (mg protein)?1h?1)
Respiration (mM O2
consumed (mg protein)?1h?1)
Photosynthetic activity
(mM O2evolved (mg protein)?1h?1)
Respiration (mM O2
consumed (mg protein)?1h?1)
Oxyfluorfen (mg/ml)
0
10
20
18.570.85a
9.970.51 (?46)c
8.370.40 (?55)d
2.770.12ab
3.070.14 (þ11)ab
3.170.13 (þ15)ab
14.170.68a
10.070.52 (?29)c
9.170.41 (?35)cd
1.670.072a
1.870.091 (þ13)a
1.970.087 (þ19)a
Oxyfluorfen (mg/ml)þ7.2 kJ m?2d?1(UV-B)
0 13.970.71 (?25)b
106.670.30 (?64)e
205.570.29 (?70)f
Data are means7SE of three independent experiments. Values with different letters within same column are significantly different (Po0.05) according to the Duncan’s
multiple range test. Values in parentheses indicate percent decrease (?) or increase (þ) over control value.
3.070.16 (þ11)ab
3.270.17 (þ19)a
3.570.17 (þ30)a
11.670.63 (?18)b
8.570.43 (?40)d
7.970.41 (?44)de
1.870.094 (þ13)a
1.870.077 (þ13)a
2.070.098 (þ25)a
Sheeba et al. / Ecotoxicology and Environmental Safety 74 (2011) 1981–1993
1984

Page 5

molecular oxygen (O2) instead of NADPþ(B¨ oger and Sandmann,
1998). The formation of ROS may further trigger lipid peroxidation,
which then alters the structure of thylakoid membrane and its
fluidity, which may again reduce photosystem-mediated electron
transport efficiency as we report in our study. Alteration in mem-
brane fluidity due to oxyfluorfen treatment is supported by the data
of electrolyte leakage (Table 9). Pritchard et al. (1980) have also
reported oxyfluorfen-mediated electrolyte leakage and was corre-
lated with one of the sites of oxyfluorfen action. Our results also
showed that oxyfluorfen significantly increased O2
production, which was accompanied by a significant increase in lipid
peroxidation and electrolyte leakage (Tables 8 and 9). Thus, results
d?
and H2O2
showed that oxyfluorfen directly affects growth of both the cyano-
bacteria by inhibiting chlorophyll synthesis and electron transport
activities, and indirectly by generating ROS and oxidative damage.
Other herbicide 2,4-dichlorophynoxyacetic acid (2,4-D) has also been
reported to decrease growth of cyanobacterium Anabaena fertilissima
by decreasing the level of photosynthetic pigments and activities of
nitrate reductase (Kumar et al., 2010). Furthermore, Li et al. (2010)
have reported that insecticide acetamiprid reduces photosynthetic
performance of the cyanobacterium Synechocystis sp. by inhibiting
electron transport and inactivating reaction centers. Lesser negative
impact of oxyfluorfen on P. foveolarum than N. muscorum may be
attributed to lesser damage on pigments and photosynthetic activity
Table 4
Restoration of photosystem II activity (PS II; mmol DCPIP reduced (mg Chl a)?1h?1) in spheroplasts prepared from N. muscorum exposed to oxyfluorfen and UV-B
radiation.
Treatments PS II activity (mM DCPIP reduced (mg Chl a)?1h?1) with donors
Without donorDPCNH2OHMnCl2
Oxyfluorfen (mg/ml)
0
10
20
90.073.8a
77.474.0 (?14)b
71.073.7 (?21)c
110.075.2a
106.775.0 (?3)ab
103.475.0 (?6)b
104.075.0a
102.074.9 (?2)a
99.875.2 (?4)ab
95.074.4a
90.274.7 (?5)b
85.574.1 (?10)c
Oxyfluorfen (mg/ml)þ7.2 kJ m?2d?1(UV-B)
0
10
20
78.373.6 (?13)b
65.773.0 (?27)d
62.173.2 (?31)de
101.274.7 (?8)b
94.674.8 (?14)c
91.374.7 (?17)c
97.875.1 (?6)b
94.674.7 (?9)bc
92.674.3 (?11)c
91.274.2 (?4)b
80.773.9 (?15)d
76.974.0 (?19)de
All the values are means7SE of three independent experiments. Values with different letters within same column are significantly different (Po0.05) according to the
Duncan’s multiple range test. Values in parentheses indicate percent decrease (?) over control value.
Table 5
Restoration of photosystem II activity (PS II; mmol DCPIP reduced (mg Chl a)?1h?1) in spheroplasts prepared from P. foveolarum exposed to oxyfluorfen and UV-B
radiation.
TreatmentsPS II activity (mM DCPIP reduced (mg Chl a)?1h?1) with donors
Without donorDPC NH2OHMnCl2
Oxyfluorfen (mg/ml)
0
10
20
80.073.4a
72.873.5 (?9)b
68.073.3 (?15)c
95.074.4a
93.174.3 (?2)ab
87.474.5 (?8)c
89.074.1a
87.273.9 (?2)ab
83.774.0 (?6)c
84.074.7a
80.674.2 (?4)ab
75.673.7 (?10)b
Oxyfluorfen (mg/ml)þ7.2 kJ m?2d?1(UV-B)
0
10
20
73.673.5 (?8)b
66.473.1 (?17)c
59.272.9 (?26)d
93.174.8 (?2)ab
82.674.0 (?13)d
79.674.3 (?16)de
87.274.2 (?2)ab
80.174.0 (?10)cd
78.373.8 (?12)cd
80.673.2 (?4)ab
70.573.4 (?16)c
68.973.4 (?18)cd
All the values are means7SE of three independent experiments. Values with different letters within same column are significantly different (Po0.05) according to the
Duncan’s multiple range test. Values in parentheses indicate percent decrease (?) over control value.
Table 3
Effect of oxyfluorfen and UV?B radiation alone and together on photosynthetic electron transport activities in N. muscorum and P. foveolarum.
TreatmentsPhotosynthetic electron transport activities
N. muscorumP. foveolarum
PS I PS II Whole chainPS I PS II Whole chain
Oxyfluorfen (mg/ml)
0
10
20
620727.9a
607725.5 (?2)ab
601727.0 (?3)ab
360716.2a
306715.6 (?15)b
288712.4 (?20)c
205710.7a
16677.6 (?19)b
15577.4 (?24)bc
540723.2a
529725.4 (?2)ab
523727.7 (?3)ab
320713.8a
288715.6 (?10)b
268711.3 (?16)c
19078.7a
16177.4 (?15)b
15277.0 (?20)bc
Oxyfluorfen (mg/ml)þ7.2 kJ m?2d?1(UV-B)
0 607728.5 (?2)ab
10 595730.9 (?4)ab
20 582729.7 (?6)b
All the values are means7SE of three independent experiments. Values with different letters within the same column are significantly different (Po0.05) according to the
Duncan’s multiple range test. Values in parentheses indicate percent decrease (?) over control value. Photosystem I activity (PS I; mM O2consumed (mg Chl a)?1h?1),
photosystem II activity (PS II; mM O2evolved (mg Chl a)?1h?1) and whole chain electron transport activity [mM O2consumed (mg Chl a)?1h?1).
306714.4 (?15)b
270712.4 (?25)cd
255713.3 (?29)d
16478.4 (? 20)b
14377.4 (?30)c
13375.6 (?35)d
529724.3 (?2)ab
518724.9 (?4)ab
507723.3 (?6)b
291713.1 (?9)b
256713.3 (?20)cd
243712.6 (?24)d
15277.3 (?20)bc
14276.5 (?25)cd
13375.6 (?30)d
Sheeba et al. / Ecotoxicology and Environmental Safety 74 (2011) 1981–1993
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Page 6

due to the lesser accumulation of ROS, hence lesser oxidative damage
(MDA and electrolyte leakage) as shown by their respective regres-
sion curves (Figs. 4 and 5).
UV-B also caused drastic decrease in biomass accumulation,
pigments and photosynthesis (Fig. 1; Tables 1–3). Exposure of
UV-B caused a reduction in dry mass by 15% in N. muscorum and
by 6% in P. foveolarum in comparison to control (Fig. 1). Reduction
in dry mass has further increased when the two cyanobacteria
were given the combined treatments of oxyfluorfen and UV-B.
Treatment of 20 mg ml?1oxyfluorfenþUV-B led to a dry mass
reduction of 77% in N. muscorum while it was able to cause only
24% reduction in P. foveolarum in comparison to control, proving
P. foveolarum more tolerant against these two stresses in compar-
ison to N. muscorum (Fig. 1). It is well known that increased UV-B
radiation can directly decrease growth and survival and cause
photo-bleaching of photosynthetic pigments (He and H¨ ader,
2002; Gao et al., 2007). UV-B radiation is reported to decrease
photosynthetic parameters such as oxygen evolution, photosyn-
thetic activity and14CO2fixation (Sinha and H¨ ader, 2008). The
decrease in these processes has been linked with UV-B-mediated
alterations in light harvesting complex and damage to oxygen
evolving complex, and D1 and D2 polypeptides of PS II reaction
center (Greenberg et al., 1989). UV-B can also indirectly affect
various physiological and biochemical processes by producing
excess ROS resulting into oxidative stress (He and H¨ ader, 2002;
Wang et al., 2008). UV-B can induce lipid peroxidation and
protein oxidation of biological membranes, DNA damage and
hormone inactivation (He and H¨ ader, 2002; Wang et al., 2008).
Prasad and Zeeshan (2004) have also reported that UV-B induces
reduction in biomass to which they have associated the reduction
in chlorophyll. Mitchell and Karentz (1993) have showed that
UV-B radiation caused damage in growth, which was due to
irreversible damage to DNA. Thus, UV-B-mediated reduction in
growth of both the cyanobactria may be correlated with
decreased photosynthetic pigments and thereby decreased photo-
synthetic activities (Fig. 1; Tables 1–5).
Results related to photosynthetic pigments in N. muscorum
and P. foveolarum are shown in Table 1. Oxyfluorfen and UV-B
induced the reduction in pigments in both the cyanobacteria,
however, pigments were severely affected in N. muscroum than
P. foveolarum. Comparing the effects of oxyfluorfen and UV-B on
photosynthetic pigments, phycocyanin was the most affected and
carotenoids were the least affected (Table 1). It is known that
photosynthetic pigments such as Chl a and carotenoids are
integrated in the membrane, while phycocyanin is found attached
to the outer surface of the thylakoid membrane. Because of this
reason, phycocyanin appeared to be highly sensitive to oxyfluor-
fen and UV-B. Phycocyanins are major light harvesting pigments
Growth [µg dry mass (ml culture)-1]
0
20
40
60
80
0
9
18
27
36
a
b
c
d
e
f
a
b
b
c
cd
d
- UV-B
0 µg/ml
10 µg/ml
20 µg/ml
+ UV-B
Fig. 1. Effects of oxyfluorfen and UV-B radiation alone and together on growth of
N. muscorum (A) and P. foveolarum (B). Data are means7SE of three independent
experiments. Mean values followed by different letters on bars are significantly
different (Po0.05) according to the Duncan’s multiple range test.
Table 6
Effect of oxyfluorfen and UV-B alone and together on NO3
P. foveolarum.
?uptake [mM NO3(g dry mass)?1h?1) and PO4
?3uptake (mM PO4
3(g dry mass)?1h?1) in N. muscorum and
Treatments
N. muscorum P. foveolarum
NO3
?
PO4
3?
NO3
?
PO4
3?
Oxyfluorfen (mg/ml)
0
10
20
223.0710.7a
103.075.2 (?54)c
92.074.2 (?59)cd
158.077.4a
135.076.5 (?15)b
117.075.5 (?26)c
184.078.3a
160.078.3 (?13)c
113.075.9 (?39)e
148.077.5a
107.075.1 (?28)c
95.074.6 (?36)d
Oxyfluorfen (mg/ml)þ7.2 kJ m?2d?1(UV-B)
0
10
20
188.079.8 (?16)b
83.074.4 (?63)e
67.072.7 (?70)f
129.076.2 (?18)b
84.074.0 (?47)d
49.072.5 (?69)e
199.079.2 (?8)b
137.076.3 (?26)d
97.075.0 (?47)f
139.077.2 (?6)b
94.074.9 (?36)d
92.074.3 (?38)d
Data are means7SE of three independent experiments. Values with different letters within same column are significantly different (Po0.05) according to the Duncan’s
multiple range test. Values in parentheses indicate percent decrease (?) over control value.
Sheeba et al. / Ecotoxicology and Environmental Safety 74 (2011) 1981–1993
1986

Page 7

and reserves of nitrogen in cyanobacteria (Cohen-Bazire and
Bryant, 1982). Thus, the steeper decline in phycocyanin in
N. muscorum suggests severe destruction in growth in comparison
to P. foveolarum as justified by data of dry mass (Fig. 1; Table 1).
The essential function of carotenoids in protecting the photo-
synthetic system from photo-oxidative damage is well documen-
ted and the carotenoids of the xanthophyll cycle play a major role
in photoprotection (Carletti et al., 2003). Carotenoids were
observed to be least affected pigment in comparison to Chl a
and phycocyanin for the similar treatments (except in UV-B alone
exposed P. foveolarum cells). Lesser reduction in carotenoids
(in comparison to Chl a, phycocyanin and dry mass) is suggesting
their protective role against oxyfluorfen and UV-B stress. Carotenoids
plays major role in directly quenching singlet oxygen (1O2), since no
antioxidant has been reported to directly quench1O2(Siefermann-
Harms, 1987).
Results pertaining to photosynthetic activities are shown in
Tables 2–5. We report that exposure of both the cyanobacteria
to oxyfluorfen and UV-B led to a reduction in whole cell
O2-evolution and photosynthetic electron transport activities (PS
II, PS I and whole chain) in comparison to control (Tables 2 and 3).
Again, these processes were more affected in N. muscorum in
comparison to P. foveolarum. Under oxyfluorfen and UV-B treat-
ments decrease in whole cell O2-evolution can directly be
correlated with decreased photosynthetic electron transport
activities (Tables 2 and 3). Among photosynthetic electron trans-
port activities, whole chain electron transport activity was com-
paratively more affected followed by PS II and PS I under
oxyfluorfen and UV-B stress (Table 3). Oxyfluorfen and UV-B
mediated decrease in photosynthetic electron transport activities
can indirectly be correlated with increased level of ROS, lipid
peroxidation and electrolyte leakage (Tables 8 and 9). As shown in
earlier studies that ROS cause damaging effects on photosynthetic
pigments associated with PS II, I and also on water oxidation side
and reaction center complexes (He and H¨ ader, 2002; Prasad and
Zeeshan, 2004). It is known that oxyfluorfen inhibits the photo-
synthetic electron transport system by interacting with electron
carriers of photosynthetic chain reaction (Bugg et al., 1980).
Recently, it has been reported that insecticide acetamiprid caused
toxicity in the cyanobacterium Synechocystis sp. by arresting
electron transfer at acceptor side of PS II (Li et al., 2010).
Ultraviolet-B affects photosynthetic electron transport activities
by damaging the integrity of membrane system, thereby inter-
rupting the electron transport across the thylakoid membrane
(Renger et al., 1989). Under oxyfluorfen and UV-B stress, com-
paratively more decrease in whole chain electron transport
activity suggests that these two stresses also damaged other
components of photosynthetic electron transport chain in addi-
tion to PS II.
In order to find out possible site of damage in photosynthetic
electron transport chain caused by oxyfluorfen and UV-B, the
DCPIP photoreduction was measured in the presence of various
exogenous electron donors—DPC, NH2OH, MnCl2and compared
with DCPIP photoreduction in PS II without donor (Tables 4 and 5).
Results indicated that all the three exogenous electron donors
successfully restored PS II activity but DPC was comparatively
more effective. This shows that the site of damage within PS II lies
in between oxygen evolving complex (OEC) and PS II.
Respiration rate showed increasing trend under oxyfluorfen
and UV-B treatments (Table 2). Similar results were reported in
Table 7
Effect of oxyfluorfen and UV-B alone and together on activities of acid phosphatase (ACP; nM p-NP formed (mg protein)?1min?1), alkaline phosphatase (ALP; nM p-NP
formed (mg protein)?1min?1] and nitrate reductase (NR; mM NO2
?produced (g protein)?1h?1) in N. muscorum and P. foveolarum.
TreatmentsEnzyme activities
N. muscorum P. foveolarum
ACP ALPNRACP ALPNR
Oxyfluorfen (mg/ml)
0
10
20
4.6470.22a
3.0470.15 (?34)c
2.8070.14 (?40)d
8.8070.35a
5.7270.27 (?35)c
5.1070.27 (?42)cd
35.071.6b
30.871.6 (?12)c
29.571.4 (?16)c
3.1070.16a
2.2170.11 (?29)c
2.1270.10 (?32)cd
11.270.58a
9.4470.46 (?16)c
8.6470.39 (?23)d
59.773.2c
70.473.6 (þ18)a
71.573.2 (þ20)a
Oxyfluorfen (mg/ml) þ 7.2 kJ m?2d?1(UV-B)
0 3.6870.19 (?21)b
10 2.1670.11 (?53)e
201.7670.089 (?62)f
All the values are means7SE of three independent experiments. Values with different letters within same column are significantly different (Po0.05) according to the
Duncan’s multiple range test. Values in parentheses indicate percent decrease (?) or increase (þ) over control value.
7.7070.35 (?13)b
2.9170.14 (?67)e
2.5370.14 (?71)ef
44.072.3 (þ26)a
27.571.3 (?21)cd
14.070.64 (?60)e
2.4470.13 (?21)b
2.0270.084 (?35)d
1.7470.083 (?44)e
10.170.53 (?10)b
8.3270.42 (?26)de
7.3670.33 (?34)e
69.373.3 (þ16)a
65.173.5 (þ9)b
40.572.2 (?32)d
Table 8
Effect of oxyfluorfen and UV-B alone and together on level of superoxide radicals (SOR; nM (mg dry mass)?1) and hydrogen peroxide (H2O2; mM (g dry mss)?1) in
N. muscorum and P. foveolarum.
Treatments
N. muscorumP. foveolarum
SORH2O2
SORH2O2
Oxyfluorfen (mg/ml)
0
10
20
11.970.57f
14.370.74 (þ20)d
15.370.64 (þ29)c
28.071.3d
36.471.7 (þ30)c
40.072.1 (þ43)b
12.470.56cd
16.770.77 (þ35)b
17.670.84 (þ42)ab
39.272.1e
43.972.1 (þ12)d
48.572.0 (þ24)c
Oxyfluorfen (mg/ml)þ7.2 kJ m?2d?1(UV-B)
0
10
20
12.770.58 (þ7)e
17.370.90 (þ45)b
18.170.85 (þ52)a
36.871.9 (þ31)c
40.072.2 (þ43)b
46.471.9 (þ66)a
13.070.67 (þ5)c
17.370.83 (þ40)b
18.170.85 (þ46)a
44.872.2 (þ14)d
51.372.7 (þ31)b
58.373.1 (þ49)a
All the values are means7SE of three independent experiments. Values with different letters within same column are significantly different (Po0.05) according to the
Duncan’s multiple range test. Values in parentheses indicate percent increase (þ) over control value.
Sheeba et al. / Ecotoxicology and Environmental Safety 74 (2011) 1981–1993
1987

Page 8

the cyanobacterium Plectonema boryanum by Prasad and Zeeshan
(2005). Our results indicated that exposure of both the cyano-
bacteria to oxyfluorfen and UV-B inhibited photosynthetic elec-
tron transport activities (Table 3), thereby reducing the supply of
ATP. Thus, under oxyfluorfen and UV-B stress, increased respira-
tory rate is to meet out the demand of ATP for carrying out the
basic life processes in the cell.
3.2. NO3
?and PO4
3?uptake
NO3
?and PO4
3?uptake, both decreased after oxyfluorfen and
UV-B treatments, however, these declines were more with
N. muscorum (Table 6). Inhibition in nutrient uptake by oxyfluor-
fen and UV-B stress may be due to alterations in the membrane
permeability as well as cellular homeostasis as evidenced by
increasedROS, lipid peroxidation
(Tables 8 and 9). It has been reported that pesticides and UV-B
decreased nutrient uptake and probable cause was decreased
ATP pool as a consequence of damaged photosynthetic electron
transport chain, which led to reduced photosynthesis (Tables 2
and 3), thus limiting the availability of assimilatory powers
andelectrolyteleakage
(NADPH and ATP) (Prasad et al., 2008). It is well known that
availability of phosphate and nitrate to cyanobacteria is critical
for their growth. Our results also prove this interlink (Fig. 1;
Table 6).
3.3. ACP, ALP and NR activities
Results pertaining to ACP and ALP activities have been pre-
sented in Table 7. Exposure of both the cyanobacteria to oxy-
fluorfen and UV-B resulted into decreased activities in ACP and
ALP. However, decrease in these enzyme activities were lower in
P. foveolarum in comparison to N. muscorum. Between the two
enzymes, ACP was more affected in P. foveolarum while ALP was
more affected in N. muscorum (Table 7). Reduced activities of
ACP and ALP activities were reported by Prasad et al. (2008) in
P. boryanum under endosulfan toxicity. Acid and alkaline phos-
phatase activities are used to detect pesticides in the environment
(Dyk and Pletschke, 2010). Besides this, ACP and ALP are regarded
as the principal enzymes responsible for hydrolysis of polypho-
sphates and are shown to be localized around the polyphosphate
bodies (Dyk and Pletschke, 2010) and their inhibition may starve
- UV-B
POD activity [units (mg protein)-1 min-1]
0.0
0.1
0.2
0.3
a
ab
c
cd
d
e
POD activity [ units (mg protein)-1 min-1]
0.0
0.1
0.2
0.3
a
a
ab
b
b
c
CAT activity [units (mg protein)-1 min-1]
0.0
2.5
5.0
7.5
10.0
a
b
b
c
c
CAT activity [units (mg protein)-1 min-1]
0.0
2.5
5.0
7.5
10.0
a
ab
b
b
b
c
SOD activity [units (mg protein)-1 min-1]
0
4
8
12
16
20
b
a
a
c
c
d
0
4
8
12
16
20
d
c
a
b
cdd
SOD activity-1 min-1] [units (mg protein)
0 µg/ml
10 µg/ml 20 µg/ml
+ UV-B
Fig. 2. Effects of oxyfluorfen and UV-B radiation alone and together on enzymatic antioxidants in N. muscorum (A–C) and P. foveolarum (D–F). Data are means7SE of three
independent experiments. Mean values followed by different letters on bars are significantly different (Po0.05) according to the Duncan’s multiple range test.
Sheeba et al. / Ecotoxicology and Environmental Safety 74 (2011) 1981–1993
1988

Page 9

the cyanobacteria from inorganic phosphorus and thus may cause
reduction in growth as observed in the present study (Fig. 1;
Table 7).
Our results showed that oxyfluorfen alone and together with
UV-B drastically decreased NR activity in N. muscorum (except
UV-B alone), however it was increased in P. foveolarum (except
20 mg ml?1þUV-B) (Table 7). There has been report that carba-
mate (an insecticide) reduced growth, survival and nitrogen
fixation in Anabaena sp. and Westiellopsis prolifica and these
adverse effects were associated with a fall in the nitrogen fixing
ability of both the organisms (Adhikary et al., 1984). Therefore,
under oxyfluorfen and UV-B stress, high activity of NR in
P. foveolarum suggests that this cyanobacterium efficiently man-
aged sequestration of nitrogen for the synthesis of protective
compounds (e.g. glutamate) in comparison to N. muscorum
and thus exhibiting lesser reduction in growth performance
(Fig.1; Table 7).
3.4. ROS, lipid peroxidation and electrolyte leakage
ROS produced under normal conditions are used generally as
signaling molecules for various processes. However, under stress
conditions their generation increase several folds and cause
oxidative stress and damage (He and H¨ ader, 2002; Lukaszewicz-
Hussain, 2010). Exposure of both the cyanobacteria to oxyfluorfen
and UV-B increased the level of O2
of both the ROS further got accelerated when these two stresses
were applied in combination (Table 8). However, the inherent
level of H2O2was more in P. foveolarum but the percent increase
was greater in N. muscorum in comparison to their respective
controls. As stated earlier, oxyfluorfen are known to generate
ROS, either by direct involvement in radical production or by
inhibition of biosynthetic pathways (B¨ oger and Sandmann, 1998;
d?and H2O2and the generation
Geoffroy et al., 2002), similarly, UV-B radiation is also known to
generate ROS by interfering with different electron carriers of
photosynthetic pathway (He and H¨ ader, 2002; Prasad and
Zeeshan, 2005). Excessive accumulation of ROS is very damaging,
since they can attack lipids, proteins and DNA to induce oxidation,
which cause membrane damage, protein modification and DNA
damage (He and H¨ ader, 2002). Under oxyfluorfen and UV-B
treatments, lipid peroxidation, i.e. MDA formation and electrolyte
leakage also increased as the result of different ROS formations
(Table 9). However, level of lipid peroxidation and electrolyte
leakage was higher in case of N. muscorum in comparison to
P. foveolarum. Our results show that lipid peroxidation and
electrolyte leakage is associated with oxyfluorfen and UV-B
induced ROS generation, which can further be correlated with
inhibited growth and photosynthetic electron transport activities
(Fig. 1; Tables 2, 3, 8 and 9).
3.5. Antioxidant system
The activities of antioxidant enzymes in both the cyanobac-
teria are depicted in Fig. 2. The enzymatic antioxidants studied,
i.e. SOD, CAT and POD showed differential responses in both the
cyanobacteria when treated with 10 and 20 mg ml?1of oxyfluor-
fen with or without UV-B. In N. muscorum cells, SOD and CAT
activities increased with 10 mg ml?1of oxyfluorfen alone and
when oxyfluorfen combines with UV-B the activities of these
enzymatic antioxidants decreased in comparison to control
(Fig. 2A–C). However in case of P. foveolarum, only 20 mg ml?1
oxyfluorfen could cause decrease in CAT activity and 20 mg ml?1
oxyfluorfenþUV-B could cause decrease in POD activity (Fig. 2D–F).
These results suggested that P. foveolarum is better equipped with
enzymatic antioxidants (SOD, CAT and POD) to counteract the
Proline [nM (mg dry mass)-1]
Ascorbate [nM (mg dry mass)-1]
0
200
400
600
800
a
b
c
c
cdcd
0 µg/ml
10 µg/ml
20 µg/ml
Ascorbate [nM (mg dry mass)-1]
0
12
200
400
600
800
b
b
c
a
d
d
0
3
6
9
12
c
b
a
d
e
- UV-B
b
Proline [nM (mg dry mass)-1]
0
3
6
9
b
c
a
cd
d
b
- UV-B
+ UV-B
+ UV-B
Fig. 3. Effects of oxyfluorfen and UV-B radiation alone and together on non-enzymatic antioxidants in N. muscorum (A–B) and P. foveolarum (C–D). Data are means7SE of
three independent experiments. Mean values followed by different letters on bars are significantly different (Po0.05) according to the Duncan’s multiple range test.
Sheeba et al. / Ecotoxicology and Environmental Safety 74 (2011) 1981–1993
1989

Page 10

Chlorophyll a
706050 403020 10
1.2
1.0
0.8
0.6
0.4
0.2
r = 0.993
Carotenoids
706050 40 30 20 10
0.5
0.4
0.3
0.2
0.1
r = 0.997
Phycocyanin
70 60 50403020 10
8
7
6
5
4
3
2
1
0
r = 0.988
Photosynthetic oxygen yield
70 60 5040 30 2010
20
18
16
14
12
10
8
6
4
r = 0.982
Respiration
7060 50 4030 2010
3.6
3.4
3.2
3.0
2.8
2.6
r = -0.923
Photosystem I
70 60 50 4030 20 10
630
620
610
600
590
580
r= 0.935
Photosystem II
7060 50 403020 10
380
360
340
320
300
280
260
240
r= 0.942
Whole Chain
706050 4030 2010
220
200
180
160
140
120
r= 0.922
Nitrate uptake
7060 504030 2010
300
200
100
0
r= 0.966
Phosphate uptake
70605040 30 2010
180
160
140
120
100
80
60
40
r= 0.907
Acid phosphatase
7060 5040 3020 10
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
r = 0.990
Alkaline phosphatase
70 6050 403020 10
10
9
8
7
6
5
4
3
2
r = 0.994
Nitrate reductase
70 6050 40 3020 10
50
40
30
20
10
r = 0.834
Superoxide radical
7060 50 4030 2010
19
18
17
16
15
14
13
12
11
r = -0.988
Hydrogen peroxide
70 6050 4030 2010
50
40
30
20
r = -0.910
Lipid peroxidation
GROWTH
70 6050 4030 2010
2.0
1.8
1.6
1.4
1.2
1.0
0.8
r = -0.946
Electrolyte leakage
7060 50 4030 2010
40
30
20
10
0
r = -0.969
Superoxide dismutase
7060 5040 30 2010
16
14
12
10
8
6
r = 0.702
Catalase
70 60 5040 302010
13
12
11
10
9
8
r = 0.247
Peroxidase
70 6050 403020 10
11
10
9
8
7
6
5
r = 0.590
Ascorbate
70 6050 4030 2010
500
400
300
200
100
r= 0.971
Proline
70 6050 40
r= 0.554
30 2010
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
Fig. 4. Regression curves for N. muscorum. X-axis represents dry mass, which is an independent variable in all curves, while Y-axis represents different parameters, which
are dependent variables in N. muscorum.
Table 9
Effect of oxyfluorfen and UV-B alone and together on level of lipid peroxidation (LPO; nM MDA (mg dry mass)?1) and electrolyte leakage (EL; %) in N. muscorum and
P. foveolarum.
Treatments
N. muscorumP. foveolarum
LPOEL LPO EL
Oxyfluorfen (mg/ml)
0
10
20
0.8770.042d
1.1370.054 (þ30)c
1.6770.081 (þ92)ab
6.070.27f
17.570.81 (þ192)d
25.571.3 (þ325)bc
0.7470.038cd
0.8270.043 (þ11)c
0.8770.047 (þ18)b
4.070.21e
10.370.47 (þ158)c
14.070.71 (þ250)b
Oxyfluorfen (mg/ml)þ7.2 kJ m?2d?1(UV-B)
0
10
20
1.1070.053 (þ26)c
1.7770.092 (þ103)ab
1.9770.083 (þ126)a
11.070.57 (þ83)e
24.071.2 (þ300)b
33.071.7 (þ450)a
0.8970.045 (þ20)b
1.0070.052 (þ35)a
1.1670.049 (þ57)a
7.370.33 (þ83)d
15.070.78 (þ275)b
18.771.0 (þ368)a
All the values are means7SE of three independent experiments. Values with different letters within same column are significantly different (Po0.05) according to the
Duncan’s multiple range test. Values in parentheses indicate percent increase (þ) over control value.
Sheeba et al. / Ecotoxicology and Environmental Safety 74 (2011) 1981–1993
1990

Page 11

adverse effects of oxyfluorfen and UV-B induced ROS and thus has
greater tolerance in comparison to N. muscorum.
The level of ascorbate drastically decreased in N. muscorum
when cells exposed to single and combined doses of oxyfluorfen
and UV-B (Fig. 3A). However, in the case of P. foveolarum, the
ascorbate level is less affected under oxyfluorfen and UV-B
treatments in comparison to N. muscorum (Fig. 3C). Ascorbate is
one of the most studied and powerful non-enzymatic antioxi-
dants. It can directly scavenge O2
via ascorbate peroxidase reaction (Noctor and Foyer, 1998).
Ascorbate regenerates tocopherol from tocopheroxyl radical pro-
viding membrane protection (Noctor and Foyer, 1998). Higher
amount of ascorbate in cells is expected to provide greater
protection to sulphydryl groups—a functional integrity of protein
molecules. These results indicated that ascorbate level is higher
and less affected in P. foveolarum than N. muscorum and can be
correlated with greater tolerance of P. foveolarum. Regression
curves for ascorbate also show that decrease in growth is
positively correlated in both the cyanobacteria but ‘r’ value is
d?,dOH and reduce H2O2to water
much high in case of N. muscorum (r¼0.971) in comparison to
P. foveolarum (r¼0.395) (Figs. 4 and 5).
Apart from ascorbate, the accumulation of compounds like
proline in stressed cells is well documented (Alia and Pardha
Saradhi, 1993; Matysik et al., 2002). Proline provides less than 5%
of the total pool of free amino acids in plants under stress free
condition, whereas the concentration increased up to 80% during
stress (Matysik et al., 2002). Results pertaining to proline ( Fig. 3B
and D) show that in N. muscorum, under oxyfluorfen and UV-B
treatments alone, accumulation of proline increased while on
combining these stresses, proline content decreased indicating
the severity of the stress (Fig. 3B). In contrast to this, proline
content showed a continuous increase in P. foveolarum under
oxyfluorfen and UV-B treatments suggesting its protective role
during stress. Regression curves for proline also show that
decrease in growth is positively correlated in N. muscorum
(r¼0.554) while it is negatively correlated with proline in
P. foveolarum (r¼–0.920) (Figs. 4 and 5). Continuous accumula-
tion of proline in P. foveolarum further confirms its greater
Chlorophyll a
34323028
r = 0.856
26 24
Carotenoids
34 323028
r = 0.853
26 24
0.26
0.24
0.22
0.20
0.18
0.16
Phycocyanin
34 3230 28
r = 0.838
26 24
4.5
4.0
3.5
3.0
2.5
2.0
Photosynthetic oxygen yield
34 323028
r = 0.916
2624
Respiration
34 32 3028
r= -0.810
2624
2.1
2.0
1.9
1.8
1.7
1.6
1.5
Photosystem I
34323028
r = 0.967
26 24
Photosystem II
343230 28
r = 0.981
26 24
340
320
300
280
260
240
Whole chain
3432 30 28
r = 0.889
2624
200
190
180
170
160
150
140
130
Nitrate uptake
34 32 3028
r = 0.888
26 24
Phosphate uptake
34 3230 28
r = 0.877
2624
150
140
130
120
110
100
90
80
Acid phosphatase
34 3230 28
r = 0.887
2624
Alkaline phosphatase
3432 30 28
r = 0.964
26 24
12
11
10
9
8
7
Nitrate reductase
34 3230 28
r = 0.586
2624
80
70
60
50
40
Superoxide radical
34323028
r = -0.844
26 24
Hydrogen peroxide
34 3230 28
r = -0.969
26 24
60
50
40
30
Lipid peroxidation
3432 3028
r = -0.953
Ascorbate
2624
Electrolyte leakage
34 3230 28
r= -0.969
Proline
2624
20
18
16
14
12
10
8
6
4
2
Superoxide dismutase
3432 3028 2624
6.5
6.0
5.5
5.0
4.5
4.0
r = 0.567
Catalase
343230 28
r = 0.911
2624
15
14
13
12
11
10
9
8
7
200
180
160
140
120
100
80
19
18
17
16
15
14
13
12
0.4
0.3
0.2
0.1
Peroxidase
3432 3028
r = 0.277
2624
0.21
0.20
0.19
0.18
0.17
0.16
0.15
34 3230 28
r = 0.395
26 24
0.6
0.5
0.4
0.3
550
540
530
520
510
500
3.2
3.0
2.8
2.6
2.4
2.2
2.0
1.8
1.6
1.2
1.1
1.0
0.9
0.8
0.7
800
700
600
500
400
300
34 32 3028
r = -0.920
26 24
12
11
10
9
8
7
6
Fig. 5. Regression curves for P. foveolarum. X-axis represents dry mass, which is an independent variable in all curves, while Y-axis represents different parameters, which
are dependent variables in P. foveolarum.
Sheeba et al. / Ecotoxicology and Environmental Safety 74 (2011) 1981–1993
1991

Page 12

resistance against oxyfluorfen and UV-B stress in comparison to
N. muscorum.
3.6. SDS-PAGE analysis
Sodium dodecyl sulphate polyacrylamide gel electrophoresis
(SDS-PAGE) analysis showed differential expression of proteins in
both the cyanobacteria under oxyfluorfen and UV-B treatments
(Fig. 6). A protein of 29 kDa was detected in P. foveolarum under
all treatments of oxyfluorfen and UV-B, which is not present at all
in N. muscorum except control cells, suggesting its important role
in normal functioning of physiological processes. Brown et al.
(1998) have shown that in plants and animals glutathione-
S-transferases (GSTs) have molecular weights in the range of
24–29 kDa. Therefore, in the present study, this protein of 29 kDa
may be ascribed as GST. Glutathione-S-transferases are enzymes
that remove xenobiotics including pesticides and decrease the
level of oxidative stress in plants as well as in animals (Brown
et al., 1998). On the other hand, a protein of 66 kDa appeared in N.
muscorum cells treated with oxyfluorfen and UV-B, which was
completely absent in P. foveolarum under oxyfluorfen and UV-B
treatments. Ferreira et al. (1996) reported that 66 kDa protein is a
photomodified form of the large subunit of ribulose-1,5-bispho-
sphate carboxylase/oxygenase (RUBISCO) enzyme. Thus under
oxyfluorfen and UV-B treatments, enormous accumulation of
66 kDa protein suggests the modification of RUBISCO enzyme
that might have led to a weakened photosynthesis in N. muscorum
in comparison to P. foveolarum (Fig. 6)
4. Conclusion
Our study shows the inhibitory effect of pesticide oxyfluorfen
and UV-B on both the cyanobacteria and effect of one stress is
aggravated by other stress and vice-versa. Oxyfluorfen decreased
growth of both the cyanobacteria by directly affecting chlorophyll
biosynthesis, other pigments and photosynthetic electron trans-
port activities and indirectly by increasing ROS and oxidative
damage (MDA and electrolyte leakage). UV-B stress also followed
similar mechanisms. Further, greater tolerance of P. foveolarum
against oxyfluorfen and UV-B stress in comparison to the
N. muscorum may be attributed to strong antioxidant defense
system (particularly ascorbate and proline), low level of oxidative
stress and absence of 66 kDa protein in comparison to N. muscorum.
Besides this, presence of 29 kDa protein in P. foveolarum further
makes it more tolerant against oxyfluorfen and UV-B stress
in comparison to N. muscorum. Our study indicates that use of
P. foveolarum may be a better option over N. muscorum to sustain
fertility and productivity of soils under such stressed conditions.
Acknowledgments
The authors are thankful to The Head, Department of Botany,
University of Allahabad, Allahabad, for providing necessary
laboratory facilities. The authors are also thankful to UGC, New
Delhi, for providing financial support to carry out this work.
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