International Journal of Latest Research in Science and Technology ISSN (Online):2278-5299

Volume 3, Issue 4: Page No.165-170. July-August 2014

http://www.mnkjournals.com/ijlrst.htm

EFFECT OF TANNERY EFFLUENT TOXICITY ON

SEED GERMINATION -AMYLASE ACTIVITY
AND EARLY SEEDLING GROWTH OF MUNG
BEAN (VIGNA RADIATA) SEEDS
1
Vineeta Kumari,1Sharad Kumar, 1Izharul Haq, 1Ashutosh Yadav,2Vinay Kumar Singh,2Zulfiqar Ali,1*Abhay Raj
1
Environmental Microbiology Section, CSIR-Indian Institute of Toxicology Research, M.G. Marg,
Post Box No. 80, Lucknow-226 001 (U.P.), India
2
Department of Chemistry, Integral University, Kursi Road, Lucknow, (U.P.), India

Abstract- From the Common effluent treatment plant (CETP), treated tannery effluent was analyzed for physico-chemical characteristics
and its effect on seed germination, -amylase activity and early seedling growth of mung bean seeds (Vigna radiata (L.) Wilczek).The
effluent had alkaline pH (8.5), high TDS (19700 mg/L), EC (671 mS/cm), BOD (650 mg/L) and COD (1280 mg/L). Germination of seed
was inhibited by 50% and prevented by 75 and 100% of the tannery effluent. No -amylase activity was recorded in seeds treated with 75%
and 100% of the effluent, suggested -amylase enzyme dependent inhibition of seed germination. No seedling growth was found in 50%
effluent even after germination. Total seedling length and root length showed an increase at 10% over the control and then there was
decreased from 25% of the effluent onwards. In contrast, compared to the control, shoot length, leaf length, -amylase activity (IU/g) and
fresh weight biomass (g/plant) of seedling found to decrease as the effluent concentration increased. The result of the present study,
revealed that CETP, Unnao treated wastewater is not suitable for irrigation purposes, even at lower concentration.
Keywords-CETP Tannery effluent, physico-chemical parameters, mung bean, seed germination, -amylase activity, zymogram

1. INTRODUCTION an activated sludge process-based plant which receives 1.9

million liters per day (MLD) of tannery wastewater from a
Tannery effluents are ranked as the highest pollutants
cluster of 21 tanneries processing approximately 47.5 tons
among all industrial wastes. India is the third largest producer
average daily raw hides. The treated effluent from CETP is
of leather in the world having about 3000 tanneries with
finally discharged in the Ganga River through drain. This
annual processing capacity of 0.7 million tonnes of hides and
wastewater is used for irrigation purposes in agricultural field
skin. The tannery industry effluent is highly toxic to flora and
on both side of drain before entering in the Ganga River.
fauna due to the presence of excess amount of dissolved
Ramteke et al. [10] assessed the efficiency of CETP and found
solids, chlorides, sulphides, chromium with a very high BOD,
a significant reduction in COD and BOD levels during the
COD and conductivity in the effluent.[1-2] Chromium
course of treatment in CETP. In a previous study, inhibition
(trivalent, Cr III and hexavalent, Cr VI) present in effluent is
of seed germination, plant growth and pigments content in
one of the most toxic pollutants. Cr VI is more toxic and
Phaseolus mungo Roxb with increasing concentration of
carcinogen than the Cr III due to its highly solubility in
CETP-treated effluent have been reported. [11] Although -
water, rapid permeability through biological membrane, and
amylase has decisive role during seed germination and
subsequent interaction with intracellular proteins and nucleic
seedling growth, information on the correspondence between
acid.[3] Tannery effluent has been reported to inhibit seed
-amylase activity and seed germination under tannery
germination, root growth and biochemical activity of crop
effluent stress is lacking. Therefore, the aim of this study was
plants.[4-7] Besides, tannery effluent induced various
to investigate the effect of diluted and undiluted tannery
chromosomal abnormalities in plant cells thereby severely
effluent on seed germination, -amylase activity and early
reducing mitotic index and root growth.[8-9]
seedling growth of mung bean seedling.
Unnao district (U.P.), India is one of the major industrial
2. MATERIAL AND METHODS
areas especially for leather tannery industry. As most of the
tanneries in this area are in the small scale and cannot be 2.1 Wastewater Samples
afford expensive treatment plant on their own. Therefore, to
Tannery effluents were collected from outlet point of
mitigate the pollution from tanneries, a common effluent
CETP-Unnao (26.48N, 80.43), U.P. state, India during the
treatment plant (CETP) was established in 1996 having
month of February, 2013. Effluents were taken in five litres
treating capacity of 2.15 million liter per day (MLD). This is
of sterilized Jerry cane and transported to laboratory for
further analysis. The effluents were filtered through

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Whtaman filter paper No. 1 to remove solids and stored at
4C.
2.2 Physico-chemical Parameters Analysis
The effluents were analyzed for various physico-chemical
parameters viz., pH, total dissolved solids (TDS), electrical
conductivity (EC), biological oxygen demand (BOD),
chemical oxygen demand (COD) and total chromium. TDS,
BOD and COD in effluent were determined following
standard method described in APHA. [12] Effluent pH was
measured with digital pH meter (Metrohm, USA). EC was
determined by conductivity meter (Thermo Orion, model-
162A, USA). Total chromium was analysed with atomic
absorption spectrometer model A. analyst-300 (Perkin Elmer)
after digestion of samples (100 mL) in digestion mixture of
(5:1) of nitric-perchloric acid. [12]
2.3 Seed Germination Bioassay
The toxicity of tannery effluent was conducted using seed
germination bioassay. [13-15] Five test solutions (10, 25, 50, 75
and 100% v/v) prepared by diluting tannery effluent with
distilled water were used to investigate the effect of tannery
effluent on germination of mung bean seeds. Mung bean
(Vigna radiata L. var. K-851) purchased from local certified
shop was chosen for the test. Seeds were surface-sterilized
with 0.1% HgCl2 and then washed thrice to remove all the
traces of mercury. Seed germination and seedling growth test
on filter paper was carried out in glass Petri dishes (20 mm
120 mm) with two layer of filter paper (125 mm in diameter,
Whatman No.1) followed by a layer of cotton bed on the
bottom. Each dish contained 10 mL of test solutions or tap
water (control), and ten seeds. The Petri dishes were covered
by lid, and incubated at 28C in dark conditions for 5 days.
The seed germination percentage, sprout length and -
amylase activity were observed in each test solution on 24
and 48 h intervals. The growth parameters for three plants,
such as root, shoot, leaf length and fresh weight of seedling
were recorded on 5th day. The -amylase activity in seedling
grew in control and different concentration of effluent was
also measured and presented in IU/g fresh weight.
2.4 Extraction and Assay of -Amylase
The control and effluent treated seeds after 48 h of
germination were homogenized in a cold mortar with 3 mL of
50 mM sodium acetate buffer (pH 4.8) containing 20 mM
CaCl2, 60 mM NaCl and 1 mM of phenylmethylsulfonyl
fluoride (PMSF) as protease inhibitors.[16] The homogenates
was centrifuged at 12,000 x g for 10 min in a refrigerated
centrifuge at 4C (Sigma 3K-30, UK) and supernatant was
heated at 70C for 15 min to inactivate -amylase,
debranching enzyme, and -glucosidase. [17] The heat-treated
supernatant was used as the crude enzyme source. The -
amylase was assayed by quantifying the reducing sugars
(maltose equivalent) liberated from soluble starch using
method. [18] The reaction mixtures consisting of 500 L crude
enzyme and 500 L of 2% starch solution in 50 mM sodium
acetate buffer (pH 4.8) were incubated at 30C for 10 min.
The reaction was terminated by adding 1 mL of DNSA
reagent (3, 5-dinitrosalicylic acid), followed by boiling in
water bath for 5 min. Then, it was cooled and diluted with 2
mL of distilled water. The color formation was monitored at
540nm with spectrophotometer (UV-visible 2300
ISSN:2278-5299
spectrophotometer, Techcomp, Korea). The release of
reducing sugars formed by enzymatic reaction was calculated
using standard curve of D-maltose. One unit of enzyme
activity was defined as the amount of enzyme that produced 1
mol of reducing sugar/min under the condition defined. All
measurements were performed in triplicates and the values
are reported as mean standard deviation (n=3). All the
chemicals used in this study were of analytical grade. 3, 5-
dinitrosalicylic acid was purchased from Sigma (St. Louis,
MO, USA).
2.5 Zymogram Analysis
Activity of -amylase was also detected on native
polyacrylamide gel electrophoresis (native PAGE) zymogram
using 10% polyacrylamide in gel.[19] Protein samples were
electrophoresed on native PAGE at a constant current of 20
mA at room temperature in a Mini-Gel Electrophoresis unit
(Microkin, Techno Source, Mumbai, India). After
electrophoresis, the gel was immersed in 2% starch solution
(prepared in 50 mM sodium acetate buffer, pH 4.8) for 60
min. After washing with distilled water, the gel was stained
with 10 mM iodine in 14 mM potassium iodide for 5 min.
Excess iodine was washed off with cold distilled water and
the gel was soaked in 1% acetic acid after visualization of
activity bands. The molecular weight of -amylase protein
was compared with native PAGE proteins marker (Bangalore
Genei, India) after Coomassie brilliant blue R-250 staining.
2.6 Water Uptake Test
Water uptake (%) by control and effluent treated seeds was
determined in separately conducted experiments. The initial
weight (W1) of seeds was taken and they were kept on wet
filter paper in petridish moisten with tap water (control)
different concentrations of tannery effluent (10, 25, 50, 75
and 100%) at 28C in dark conditions. Final weight (W2)
was recorded after 8 h of imbibition and water uptake
percentage was calculated:
Water uptake (%) = (W2-W1)/ W1*100
3. RESULTS
Table 1 shows the CETP treated tannery effluent was
alkaline (pH 8.5). It contained higher EC (671 mS/cm), TDS
(19700 mg/L) BOD (650 mg/L), COD (1280 mg/L) and total
chromium (2.41 mg/L). The value of TDS, COD and BOD
values in the effluent was found to be 9.4, 5.1 and 21.7 times
higher than the permissible limits respectively. [20] Further,
the values of EC, TDS, COD and BOD showed an increasing
trend with increasing concentrations of the effluent. There
was no significant variation in the values of pH at different
concentrations. Control (tap water) had pH=7.71, TDS 481
mg/L and EC 7.2 mS/cm and BOD and COD values were nil
(data not shown).
Germination was 100% in control seeds within 24 h,
whereas 80% and 30% germination was observed in 24 h in
seeds treated with 10 and 25% effluent (Table 2). However,
100% germination was observed in 48 h in both 10 and 25%
effluent treated seeds, respectively showing a delayed
germination. At 50% effluent concentration, the percentage
of seed germination was 50%. Germination was prevented in
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seeds treated with 75 and 100% effluent concentration
throughout the experiment.
The sprout length of 48 h seeds has been showed a
concentration dependent reduction (Table 2). It was 2.0 cm in
control, 1.5 cm in 10%, 1.2 cm in 25% and 0.5 cm in 50%
effluent treated seeds, respectively. At the same, no sign of
sprout development in both 75 and 100% treated effluent
seeds were observed till the end of the experiment.
The -amylase activity in the germinated seeds after 48 h
(Table 2) showed a concentration dependent inhibition. It was
6.6 IU/g fresh weight in control germinated seeds and 6.0, 5.2
and 4.2 IU/g fresh weight in 10, 25 and 50% effluent treated
seeds respectively, showed 9.0%, 21.2% and 36.4%
inhibition over the control. No -amylase activity was
observed in 75 and 100% effluent treated seeds. This result
suggested a -amylase enzyme dependent germination of
mung bean seed. Similar pattern was also detected on native
PAGE zymogram (Fig. 1), which again showed no -amylase
activity band in the seeds treated with 75 and 100% effluent.
Further, zymogram analysis showed presence of a single -
amylase with estimated molecular weight of 67 kDa based on
its relative mobility on native PAGE in comparison with
standard proteins marker.
The amount of water uptake by seeds through imbibition
process (absorption of water by seed coat during
germination) showed a decreasing trend of water uptake with
increasing concentration of the effluent (Fig. 2). The control
seeds showed 87% water uptake over its dry weight and
seeds treated with effluent of different concentrations are
67%, 64%, 45%, 44% and 39% at 10, 25, 50, 75 and 100%
concentrations over their dry weights, respectively.
The pattern of seedling growth in control and different
concentrations of effluent after 5 days is given in Fig. 3. At
10% effluent concentration, whole seedling length was
slightly increased by 17.7 cm as compared to 17.0 cm at
control. However, it decreased to 12.8 cm at 25% effluent.
Whereas at 50% effluent, the seedling could not established
even after germination and remained undeveloped till the end
of experiment.
The length of root, shoot and leaf for seedling grew in control
and 10%, 25% effluent concentrations are presented in Table
3. The root length of seedling grew in control and 10%, 25%
effluent concentration was 3.0 and 5.03, 5.0 cm respectively
and showed an increase in root growth up to 67.7 and 66.7%
at 10%, 25% concentration over the control. Conversely, the
shoot length of the seedling under similar treatment
conditions gradually decreased as compared to control. It
was 14.0, cm in control, 12.67 cm in 10% and 7.83 cm in
25% effluent concentration respectively showing percentage
of reduction up to 9.5 and 44.1%. A similar trend was also
observed in case of the leaf length which was 1.67 cm in
control and 1.50, 0.93 cm in 10%, 25% effluent
concentrations respectively. Further, the seedling grew in
10% and 25% effluent concentration had less fresh weight
than that of seedling grew in control as 0.21 and 0.19 g fresh
weight in 10% and 25% treated effluent respectively. The -
amylase activity in 5 days old seedling was also recorded in
similar pattern, with 3.12% and 31.06% reduction in 10% and
25% effluent concentration respectively. -amylase activity
ISSN:2278-5299
was also detected in 50% effluent treated germinating seeds
which were 70% less than the control.
4. DISCUSSION
The basic tool for the evaluation of complex industrial
waste toxicity is the physico-chemical analysis and
toxicological bioassay. The CETP-Unnao treated effluent
contained much higher amount of EC, TDS, BOD and COD
due to extensive use of salts mainly sodium chloride, sodium
sulphate, chromium and etc. during transformation of raw
hide/skin into leather. The results of effluent analysis is
comparable with the previous reports.[21-22] The EC which
bear direct relation with salinity affects seed germination and
plant growth if it is higher than 200 mS/cm.[23] The high TDS
is due to use of large quantities of salts. Salts are very much
soluble in water and chemically stable under aquatic
conditions, making it effectively impossible to remove them
easily from effluent. The higher TDS is toxic to aquatic lives
by causing osmotic stress and affecting the osmoregulatory
function of the organisms.[24] High BOD indicates poor
dissolved oxygen (DO) in the effluent. On the other hand
high COD is due to high amount of organic compounds in the
effluent which is not affected by the bacterial decomposition.
Although, the chromium concentration in the effluent was
2.41 mg/L, but continuous discharge of chromium in effluent
even at low concentration can cause toxic effect to aquatic
life by disrupting food chain.[7, 25]
Due to presence of various chemicals in industrial
wastewater, chemical and physical tests alone are not
sufficient when assessing the potential effect of wastewater in
aquatic and terrestrial biota. Environmental toxicity
assessment of industrial wastewater was conducted with a
range of test models encompassing bacteria, algae, plants,
and fishes can be used.[12] However, the use of plant seed
germination test to evaluate toxicity of industrial effluents
has been considered as one of the simplest and short-term
methods.[13-14] During seed germination sufficient amount of
water uptake is needed for triggering enzymatic activity.
Among these enzymes, -amylase induced under the
influence of signals transduced by gibberellins, plays an
important role during seed germination through hydrolysis of
stored starch into soluble one required for energy creation.[26]
Any inhibitory substance in the growing environment can
disturbs seed germination process.
In present study, complete inhibition of seed germination
above 50% effluent concentrations (Table 1) was correlated
with less water uptake (Fig. 2) which was insufficient for
triggering -amylase enzyme in seeds (Fig. 1). All these
physiological and biochemical changes in the seeds was
occurred due to the cumulative effect of excess amount of
TDS, EC, BOD, COD and chromium in the effluent. This
observation has the conformity with the result obtained by
Karunyal et al. [27], as they reported that the germination of
Oryza sativa, Acaia holosericca, and Leucaena leucocephala
was totally inhibited at 75% and 100% tannery effluent.
Similar observation was observed by Calheiros et al. [28] for
seed germination in Trifolium pretense at 50% effluent.
Therefore, it is derived that type of seed is not important for
this type of study and any seed can be used.
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 International Journal of Latest Research in Science and Technology.
The inhibition of seed germination may be due to high
level of dissolved solids, which enrich the salinity, and
conductivity of the absorbed solute by seed before
germination. [29] The increased salinity decrease the osmotic
potential to such an extent, which retard or prevent the uptake
of water necessary for germination or by toxic effects of ions
on embryo viability. [30] The salt content outside the seed is
also known to act as limiting factor and causes less absoption
of water by osmosis and inhibit the germination of seeds.[31] It
has been also reported that salts stress either alter metabolic
pattern or does not permit the synthesis of enzymes required
for seed germination.[32] Other possibility of inhibition in seed
germination at higher concentration may be due to the
presence of 2.41 mg/L chromium in the effluent which
concomitantly affected -amylase activity in germinating
seeds, as documented by previous reports in various
plants.[33-34]
The increase in root length of seedling treated with 10%
and 25% effluent concentration over the control was
attributed due to decrease in concentration of various
chemicals present in the diluted effluent. These observations
are in conformity with previous report of effect of tannery
effluent on groundnut. [35] A gradual decrease in shoot length,
leaf length and fresh weight of biomass of seedling treated
with 10% and 25% may be due to excess amount of salts
which affected plant growth. These findings are similar to
result of Nath et al.[33] who have reported that different
concentration of tannery effluent inhibited seedling growth
and pigments of Phaseolus mungo Roxb. with increase in
concentration. The complete inhibition of plant growth at
50% effluent even after germination may be due to presence
of TDS which interfered and inhibited the uptake of other
elements. [36] Rao and Kumar [4] have reported that under
higher effluent concentration, germinated seeds get low
oxygen, which restrict the energy supply, and thus retard the
growth and development of seedling. The root which
continuously remains in direct contact with the effluent hence
the higher concentrations of the effluent could affect cell
multiplication or the growth. [37]
Stimulatory as well as inhibitory effects of tannery
effluent at lower and higher concentration on plant growth
have been reported. [5, 11, 38] But in this study, tannery effluent
even at lower concentration (10%) has adverse effect on
seedling growth except root length. It may be due to
variations in the chemical constituents from one industry to
another. It was noteworthy to mention that CETP-Unnao
received not only effluent from tannery industries, but also
from many other industries as listed by Report of the
committee on pollution caused by leather tanning industry to
the water bodies/ground water in Unnao district of Uttar
Pradesh.[21]
5. CONCLUSIONS
This study showed that treated tannery effluent prevented
germination of V. radiata L. seeds at 75% concentration due
to presence of variety of toxic substances. This study also
demonstrated -amylase dependent inhibition in seed
germination under tannery effluent stress. Since, CETP,
Unnao treated tannery effluent carries higher amount of salts
and chromium it may interfere with the metabolic activities
of the plants irrigated with it. There could also be some
ISSN:2278-5299
changes in soil characteristics when the tannery effluent used
for irrigation. Thus, it is needed that tannery effluents should
be properly treated to bring down their adverse effects within
tolerable limits.
ACKNOWLEDGEMENTS
We gratefully acknowledge the facilities provided by Director,
CSIR-Indian Institute of Toxicology Research, Lucknow.
Financial support for this study was provided by CSIR under
INDEPTH project (BSC0111).
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Table 1 Physico-chemical characteristics of CETP, Unnao treated tannery effluent at different concentrations. The values are
mean of triplicate samples

Effluent concentration (%) pH TDS EC BOD COD Chromium

(mg/L) (mS/cm) (mg/L) (mg/L) (mg/L)
10 8.39 2670 91 125 227 0.20
25 8.42 4410 149 235 427 0.52
50 8.46 11300 379 300 619 1.10
75 8.48 19600 669 390 820 1.68
100 8.48 19700 671 650 1280 2.41
ISI standard* 5.5-9.0 2100 NS 30 250 2.0
*ISI standards No. 2490 (1974); NS=not specified.

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 International Journal of Latest Research in Science and Technology.

Fig. 2 Effect of different concentrations of tannery effluent on water uptake Fig. 3 Effect of different concentrations of tannery effluent on total length of
mung bean seedling. The values are as mean SD (n=3) of triplicate
samples.

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