12 Chapter 4

Results and Discussion

Effect of tannery effluent on water and soil profile, plant growth and human health

51

RREESSUULLTTSS AANNDD DDIISSCCUUSSSSIIOONN

In developing as well as underdeveloped countries, the industrial

effluents are released directly or indirectly into natural water resources, mostly

without proper treatment, thus posing a serious threat to the environment

(Altug and Balkis, 2009). Environmental pollution is an extremely important

issue today, affecting all of us in one way or the other. Due to rapid increase in

human population and industrialization, the demand for natural raw materials

and source of energy are increasing day by day (Abhay and Rajput, 2009).

Many rivers of the world receive flux of sewage, domestic waste, industrial

effluents and agricultural waste which contain substances varying from simple

nutrients to highly toxic chemicals (Benazir et al., 2010). Tannery industry

contributes significantly towards exports, employment generation and occupies

an important role in Indian economy. Heavy metals released from tanneries are

kept under environment pollutant category due to their toxic effects on plants,

animals and human beings. They interfere with physiological activities of plants

such as photosynthesis, gaseous exchange and nutrient absorption and cause

reduction in plant growth, dry matter accumulation and yield (Sharma and

Agrawal, 2005). They cause direct toxicity, both to human and other living

beings due to their presence beyond specified limits. Heavy metal pollution of

soil and waste water is a significant environmental problem and has a negative

impact on human health and agriculture (Michalak, 2006).

The reuse of waste waters and industrial effluents for irrigation to

crop plants after proper dilution is an useful technique (Rehman et al., 2007).

Tannery effluent can be diluted and reused for agriculture purpose which can

also act as a good fertilizer (Mariappan and Rajan, 2002).

In the selected area of our study the continuous discharge of tannery

waste water has polluted the water and lands of the nearby villages namely

Chinnalapatti, Begampur, Kottapatti and many other places. The potable water

4



52

of the residential area was found to be salty and polluted and soil properties of

the cultivable land of the nearby area was also affected. The negligence of the

safe disposal of tannery wastes had led to deleterious effects on the biosphere

as a whole. Hence, in the present study, the impact of the tannery effluent on

water, soil, plant and human beings were assessed. The results of the study

entitled “Effect of tannery effluent on water and soil profile, plant growth

and human health” are presented and discussed as follows:

PHASE I

4.1 Characterization of the tannery effluent, target area water and soil samples

4.1.1 Characterization of tannery industry effluent

4.1.2 Biochemical profile of the target area water samples

4.1.3 Physicochemical characteristics of target area soil samples

PHASE II

4.2 Growth studies of selected plants using diluted tannery effluent

4.2.1 Biometric observations

4.2.2. Biochemical parameters

4.2.3 .Histochemical observations of root samples of the selected plants

4.2.4. Yield parameters

PHASE III

4.3 Health profile of the tannery industry workers

4.3.1 Hematological parameters

4.3.2 Assessment of the liver function

4.3.3 Assessment of the renal function

4.3.4 Assessment of metal contents



53

PHASE I

4.1 Characterization of tannery effluent, target area water and soil samples

4.1.1 Characterization of tannery industry effluent

Tannery industry contains several organic and inorganic chemicals,

which are toxic metals and they cause soil and ground water pollution. These

chemicals cause adverse effect on plant growth and the health of animals and

people living in that area. Processing of hides and skin to leather uses many

chemicals at various stages and hence releases many toxic substances at each

stage. The overall tanning process performed in drums can be characterized by

a high consumption of water and chemicals with collagen. Chemicals are added

in excess and are only partly taken up by the leather and the remaining is

released in the effluent (Scholz and Lucas, 2003).

Heavy metals can pose health hazards if their concentrations exceed

allowable limits. Even when the concentration of metals does not exceed these

limits there is still a potential for long-term contamination, and heavy metals are

known to accumulate within biological system (Altaf et al., 2008). Hence the

effluent released is expected to have a higher amount of chemicals and toxic

metals.

In every step of tanning process a considerable amount of waste water is

released. The waste water was found to contain salts, fat, protein and

preservatives for soaking, lime, ammonia and sulphides for fleshing, trimming

and bating, chromium salts and polyphenolic compounds for tanning and

dye and solvent chemicals with metals for wet finishing. Hence tanneries

that perform the complete tanning process produces a complete tanning

mixed waste water. In this view, in the present study, the combined tannery

effluent was collected and characterized for certain physicochemical

parameters. The results of the physical parameters of the raw effluent

are presented in Table 6.



54

TABLE 6

PHYSICAL PARAMETERS ASSESSED IN RAW TANNERY EFFLUENT

PARAMETERS RAW

EFFLUENT BIS LIMITS

IS 2490-2009

Colour Brown -

Odour Offensive -

Turbidity Turbid -

pH 10.5 5.5 - 9.0

Total hardness (mg/l) 5400 100

Electrical conductivity (mg/l) 24,500 NM

Total suspended solids (mg/l) 650 100

Total dissolved solids (mg/l) 17,150 2100

Values are mean of triplicates

NM - Not mentioned

BIS - Tolerance limits for industrial effluent discharged into inland surface waters prescribed by the Bureau of Indian Standards (2009)

The effluent released from tannery industry was brown in colour and had

an offensive odour. The colour of the effluent might be due to the presence of

biodegradable and nonobiodegradable high molecular weight organic

compounds and high amount of chemicals used during the processing and the

odour may be due to the processing of skin and hides by soaking and liming.

The yellowish brown colour might be hindering the penetration of sunlight

causing depletion in the rate of oxidation process (Ravibabu et al., 2007) and

this colour might be due to physico chemical treatments (Zahid et al ., 2006).

Turbidity is an expression of the optical property that causes light to

be scattered and absorbed rather than transmitted with no change in direction

or flux level through the sample (APHA, 2005). The turbidity of the effluent

might be due to the discharge of high concentrations of carbonate,

bicarbonate, chloride, calcium, magnesium and sodium used in tanning industry

(Chakrapani, 2005).



55

pH was recorded as 10.5, which was above the tolerance limit of

5.5 - 9.0 prescribed by the Bureau of Indian Standards (2009). The higher pH of

the effluent indicates the basic nature of the effluent. The pH of waste water

could vary due to the presence of various tanning and colouring materials.

According to Fadali et al.(2005), the pH of the tannery effluent assessed by

them was found to be 10.0, which was above the permissible limits and they

suggested that this alkaline pH of the effluent could affect the biological property

of the receiving water body.

Total hardness of the tannery effluent in the present study was found to

be 5400 mg/l. Hardness is an indication of calcium and magnesium found in

higher concentration in effluent.

The higher electrical conductivity value of the effluent indicates that

the discharge of chemicals as cations and anions were higher in the waste

water. The higher conductivity alters the chelating properties of water

bodies and creates an imbalance of free metal availability for flora and

fauna (Akan et al., 2008). Venkatesh et al. (2009) recorded that the electrical

conductivity, pH, chloride, sulfides, biological oxygen demand and chemical

oxygen demand in tannery effluent were much higher than the tolerance limits

for industrial effluent discharged into land surface.

Levels of total suspended solids (TSS) found in the effluent (650mg/l)

were greater than that of the permissible limit (100 mg/l). Somnath (2003)

reported that larger solid particulate matter remains suspended as a result of

charges on the surface of small particles in the effluent.

The effluent showed a higher level of total dissolved solids (TDS)

(17,150 mg/l) . The value was much greater than the tolerance limits (2100 mg/l)

prescribed by Bureau of Indian Standards. Total dissolved solids are mainly

due to carbonates, bicarbonates, chlorides, sulphates, phosphates, nitrates,

nitrogen, calcium, sodium, potassium and iron (Kannan et al., 2009). In the



56

liming section of tanning process, protein, hair, skin and emulsified fats are

removed from the hides, which are released in the effluent and therefore

increase the total solids (Bhalli and Khan, 2006).

Table 7 indicates the chemical characteristics studied in tannery effluent.

TABLE 7

CHEMICAL CHARACTERISTICS OF RAW TANNERY EFFLUENT

PARAMETERS RAW

EFFLUENT BIS LIMITS

IS 2490-2009

Chemical Oxygen Demand 3180 250

Biological Oxygen Demand 1300 30

Carbonate 9850 600

Bicarbonate 10423 NM

Calcium 1440 200

Magnesium 432 30-100

Chloride 5100 1000

Sodium 2300 NM

Potassium mg/l 600 NM

Fluoride 1.0 2.0

Nitrate 440 100

Nitrite 32 10

Sulphate 1080 1000

Chromium 19.3 2.0

Nickel 5.5 3.0

Zinc 10.8 1.0

Cadmium 4.2 2.0


NM - Not mentioned

BIS - Tolerance limits for industrial effluent discharged into inland surface waters prescribed by the Bureau of Indian Standards (2009)



57

Chemical oxygen demand (COD) is the test used to measure pollution

of domestic and industrial waste (Chavan and Wagh, 2005). It is the amount of

oxygen required for the oxidation of inorganic matter using a strong chemical

oxidant. COD is tested to determine the degree of pollution in the effluent

samples (Bhalli and Khan, 2006). COD values in the effluent were found to be

12 times higher than that of the tolerance limits.

Biological oxygen demand (BOD) is the parameter which is widely

used to determine the pollution load of waste water (Chavan and Wagh, 2005).

It is the amount of organic matter in the water and the amount of oxygen

required by the micro organisms to stabilize the biologically decomposable

organic matter in wastes under aerobic conditions (Bhalli and Khan, 2006).

BOD of tannery effluent was found to be 1300 mg / l which was higher than that

of the BIS limits (30 mg/l).

Akan et al. (2007) also reported higher levels of BOD, COD and TSS in

tannery effluent. Industrial effluents and municipal sewage carry organic and

inorganic substances which utilize dissolved oxygen resulting in oxygen

depletion (Ravindran and vasudevan, 2008). Nearly 70% of the emission loads

of biochemical oxygen demand (BOD), chemical oxygen demand (COD)

and total dissolved solids (TDS) emanate from the pretanning operations

(Calherios et al., 2008b).

High carbonate and bicarbonate contribute to the total alkalinity of the

sample (Balakrishnan and Karruppusamy, 2005). Carbonate and bicarbonate of

the effluent were found to be 9850 mg/l and 10423 mg/l respectively. Usage of

sodium bicarbonate during the process of pickling in tannery industry might

have caused the excessive amounts of these in the effluent.

The cations calcium and magnesium present in the effluent were found

to be at higher levels (1440 mg/l and 432 mg/l respectively) compared to BIS

limits. Calcium and magnesium contribute to the hardness of the water and it



58

imparts unpleasant odour, when present in higher levels (More et al., 2002). The

tannery effluent contains fairly large amount of calcium and magnesium

because lime is used for loosening the hair.

Chloride is an indicator of pollution when present in higher

concentrations (Singh et al., 2009). Sodium chloride used as a dehydrating and

antiseptic agent is the source of chloride (Mehdi, 2005). The level of chloride in

the effluent (5100 mg/l) was 5 fold higher than that prescribed by BIS (2009).

The presence of very high amounts of chloride and sulphate is responsible

for high hardness and further it increases the degree of eutrophication

(Kannan et al., 2005).

The level of sodium and potassium in the effluent were 2300 mg/l and

600 mg/l respectively. Sodium sulphide is used in the liming process of hide and

skin. The residual sulphide in the range of 100 – 200 mg/l goes in the discharge

and causes serious environmental problem (Ram and Roger, 2004). The high

concentrations of sodium and chloride in the effluent were mainly due to the use

of huge salts in different stages of tanning process (Zahid et al., 2006).

Fluoride levels in the effluent were lesser (1.0 mg/l) than that of Bureau

of Indian Standards (2.0 mg/l). Very high nitrate content (440 mg/l) was present

in tannery effluent than the standard value. Nitrite content in tannery effluent

(32 mg/l) was also above the permissible limits (10 mg/l). Waste generated from

tanning generally contains much higher concentration of total dissolved solids

(TDS), total suspended solids (TSS), phenols, chromium, chlorides, nitrates,

nitrites, ammonia and heavy metals. Nitrate is the highest oxidized form of

nitrogen and causes blue baby disease when consumed in excess (Das et al.,

2010)

Sulphate is one of the important anions present in natural water and

produces laxative effect when exceeds the limit (Kasthuri et al., 2005). Sulphate

levels in the effluent were found to be 1080 mg/l. The tolerance limits prescribed

by BIS (2009) for sulphate was 1000 mg/l which was lesser than the amount



59

present in effluent. In a similar study done by Zahid et al. (2006), extremely

high concentrations of sodium, magnesium, chloride, calcium, sulphate and

bicarbonate were reported in tannery effluent. It was also reported by Sheela

and Peethambaram (2007) that excess amount of sulphate, nitrogen and

dissolved solids were present in the tannery effluent.

Chromium level in raw effluent was found to be 19.3 mg/l which was 10

times higher than the amount prescribed by BIS (2.0 mg/l). Chromium is the

major chemical used in tanning process and hence its discharge in the effluent

was found to be high. Continuous discharge of chromium in low concentration

has been reported to be toxic to aquatic life and has been shown to disrupt the

aquatic food chain (Fent, 2004). Extremely high concentrations of chromium,

sodium, magnesium, calcium and ammonia were detected in the tannery

effluent in a study conducted by Krantz and Kifferstein (2002).

Nickel, zinc and cadmium in the effluent were 5.5 mg/l, 10.8 mg/l and

4.2 mg/l respectively. All these metals were present in higher concentrations

compared to the prescribed limits of BIS (2009) (3.0 mg/l, 1.0 mg/l and 2.0 mg/l

respectively). The tannery wastewater is being contaminated with higher levels

of metals (iron, nickel, chromium, zinc, cadmium, manganese and copper) and

their irrigation contaminates the soil vegetables and crops, which when

consumed causes serious health hazards to the consumer (Mohanta et al.,

2010). The presence of cadmium and other heavy metals in the environment

has become a major threat to plant, animal and human life due to their toxic

effect and therefore must be removed from industrial effluent before discharge

(Vinod and Anirudhan, 2001). Deepali and Gangwar (2010) reported that the

soil and ground water samples showed the presence of high level metal

contamination due to the receipt of industrial effluent from tannery industry.

The above results indicate that the raw tannery effluent released into the

atmosphere was found to contain chemicals which were above the permissible

limit prescribed by BIS (2009). The impact of raw effluent on potable water in

the target areas was studied in the next stage.



60

4.1.2. Biochemical profile of the target area water samples Ground water is the principal source of drinking water in urban and rural

areas of our country. The quality of drinking water in Indian cities has been

detoxified in the recent years mainly due to increase in population and improper

disposal of waste water from industries. These factors have influenced the

surface and ground water (Venkatasubramani et al., 2007). The groundwater in

industrial areas across the country has undergone severe contamination by

industrial waste, effluents and emissions which are discharged indiscriminately

without any regulatory system (Viswanatham et al., 2007). Industrial and

household waste discharged directly or indirectly, through leakages in the

sewage system enters into water sources. The changes in the properties of

water affect the biological lives in that area (Singh, 2006).

The major source of water pollution is industrial and the waste water

generated from various industries which is being discharged into common

drainage. The effluents pollute not only nearby soil but also pollute and alter the

biochemical nature of drinking water. This may be due to the presence of

chemicals in the industrial effluents which pollute and alter the biochemical

nature of drinking water (Bernal et al., 2006).

The organic waste in water may spread pathogenic diseases like

diarrhea and cholera. Along with these diseases, some long term effects can

also occur in some cases (Sathe and Kulkarni, 2001).Tannery effluent when

discharged untreated damages the normal life of receiving stream and if allowed

to percolate into the ground for a prolonged period , seriously affects the

ground water table of that locality ( Mondel et al., 2003; Prasad, 2007).

Quality of water is an important consideration in any appraisal of salinity

or alkalinity conditions in an irrigated area (Acharya et al., 2008). The water

used for drinking purpose should be free from toxic elements and excessive

amount of minerals that may be harmful to health. Keeping this in focus, to

assess the extent of ground water deterioration, a detailed analysis of ground



61

water quality has been carried out. Biochemical characteristics of ground water

near tannery industry area and water collected from area 15 km away from the

effluent discharge site were analysed and shown in Table 8.

TABLE 8

BIOCHEMICAL PROFILE OF TARGET AREA WATER SAMPLE

S.No Parameters Control water

Target area water sample

1 pH 7.21 8.14

2 Electrical conductivity (mho/cm) 0.65 2.88

3 Turbidity 0.05 1.28

4 Total alkalinity 600 914

5 Total suspended solids 15 418

6 Total dissolved solids 500 2467

7 Total hardness 200 830

8 Calcium hardness 75 454

9 Magnesium hardness (mg/l) 50 405

10 Fluoride 1.5 0.70

11 Chloride 600 1178

12 Chromium 0.05 10.40

13 Nickel 0.6 3.90

14 Zinc 4.5 7.00

15 Cadmium 1.9 3.40


Control water- Collected from the area 15 kilometers away from the effluent discharge site

Target area water sample - Water collected within 1 kilometer radius of the effluent discharge site.

pH and Electrical conductivity (EC)

The pH of the target area water sample was 8.14 and EC was 2.88 in the

present study. The higher pH value indicates the alkaline nature of the effluent.



62

The higher values of EC could be the result of extreme concentrations of soluble

salts from the tannery effluent discharged into the low lands and surface water

bodies of the area. Electrical conductivity and total dissolved solids are

proportional to each other (Gupta et al., 2010). The negative impact of the

effluent on the water quality includes increase in turbidity, colour, nutrient load

and presence of toxic and persistent compounds (Sharief et al., 2005).

Turbidity and Total alkalinity

Turbidity and total alkalinity were found to be increased in the target

area water sample. The total alkalinity of the water is mainly caused by the

contents of calcium, magnesium, sodium, potassium, ammonia and iron,

combined either with carbonates and/or bicarbonates or occasionally by

hydroxide (Jhingran, 2003).

Total suspended solids and Total dissolved solids Total suspended solids were found to be high in target area water sample

compared to the non contaminated water. Total dissolved solid (TDS) was one

of the often neglected parameter, eventhough it has a tremendous effect on the

overall quality of water. High TDS levels are indicative of the presence of high

levels of both inorganic and organic compounds present in the given water

sample. The total dissolved solids of the target area water sample was high

compared to the BIS value for dissolved solids which is upto 500 mg/l and the

maximum permissible quantity is 2000 mg/l (WHO, 2005). The present study

showed a level of 2467 mg/l of dissolved solids.

The high amount of dissolved solids might be due to the presence

of inorganic salts and small amounts of organic matter dissolved in

water (Lofrano et al., 2008). Jayaseelan et al. (2008) reported that the higher

concentration of total dissolved solids in surface water could be due to the

industrial activities such as tanneries, where huge amount of soluble salts are

used for processing of leather.



63

Total hardness, calcium and magnesium hardness Hardness is one of the very important properties of ground water

from utility point of view for different purposes. It is a well known fact

that hardness is not caused by a single substance but by a variety of dissolved

polyvalent metallic ions, predominantly calcium and magnesium cations,

although cations like barium, iron, manganese, strontium and zinc also

contribute (Chaudhary et al., 2005). Calcium and magnesium are the principal

ions of hardness.

In the present study, total hardness, calcium and magnesium hardness

were found to be at a higher level in the target area water sample compared to

the control water. Calcium and magnesium causes by far the greatest portion of

the hardness occurring in the natural waters (Acharya et al., 2008). Tanning

process uses these chemicals at greater amounts in liming, pickling and bating

processes which are let out along with the effluent.

Fluoride

Fluoride enters the body through food, water, industrial exposure, drugs

and cosmetics. Presence of fluoride in water may affect the photosynthesis,

respiration and protein synthesis and enzyme activities of higher plants (Sarala

and Rao, 1993). The fluoride content in target area water sample was found to

be lesser than the control water.

Chloride

The chloride content of target area water sample recorded a value of

1178 mg/l which was twofold higher than the chloride content (600mg/l) of the

control water sample. The addition of excess sodium chloride during the tanning

process might be responsible for this.

Chromium, nickel, zinc and cadmium Chromium, nickel, zinc and cadmium were found to be high in the

target area water sample than the control water. According to World Health



64

Organization (2005), the metals of most immediate concern are aluminum,

chromium, magnesium, manganese, iron, cobalt, copper, nickel, zinc, cadmium,

mercury and lead (Ulmanu et al., 2003) which are discharged into water bodies

that can be toxic to aquatic life and may cause natural water unsuitable as

potable water sources (Cooman et al., 2007). According to Oliveria et al. (2007),

the presence of heavy metals in industrial and urban wastewater is one of the

main causes of water and soil pollution. Accumulation of these elements in

waste water depends on a number of local factors such as the type of industries

in the region, lifestyle and awareness of the impact to the environment by

careless disposal of water (Nordberg et al., 2007).

The results of the study showed that due to unsafe disposal of tannery

waste water on the bare land, the organic and inorganic chemical compounds

present in the effluent have leached and found their way into the ground water.

Hence the potable water in the industrial area was significantly contaminated

with cadmium, chromium, nickel and zinc which were used in the wet finishing

process of tannery process and released along with the effluent.

4.1.3. Physiochemical Characteristics of the target area soil samples

Among various environmental hazards, pollution of soil and water caused

by various effluents has become a serious problem. The tannery industries

discharge large quantities of common salt during the process of tanning.

Deposition of these salts into the soil takes place when the effluent comes in

contact with the soil. Besides chlorides, toxic substances like chromium, sodium

sulphide, sodium carbonate and ammonium sulphate are present in the

discharged effluent which manifolds the soil pollution (Rajan and Arias, 2007).

Heavy metals emitted either from anthropogenic or natural activities can

disperse in the environment and may ultimately get deposited in the soil.

Plants growing in such areas may absorb heavy metals in to their body.

Although metals like iron, molybdenum, manganese, zinc, copper, magnesium,

copper, selenium and nickel have a major role in the growth and development of

plants, they may be toxic beyond a certain level. Long term and indiscriminate



65

application of raw sewage effluent results in the accumulation of these nutrients

into surface and subsurface soils (Vaani and Kumar, 2008).

The dissolved constituents of the industrial waste reacts with clay

complex of soil leading to accumulation of salts and an increase in the amount

of exchangeable sodium and other nutrients. A study on physical and chemical

properties of soil is a basic and practical utility in agriculture. It aims at providing

a tool for proper management of soil (Altieri and Nichollas, 2003).

The tannery waste water being alkaline in nature, leads to the

deterioration of the concrete, metallic pipe through which the effluent passes

and thus causes seepage of the waste water into the land. Free disposal of the

sludge and effluent on the land affects the soil property and fertility of the soil in

the long run.

The selected industry discharges the effluent directly on land. Two soil

types namely red soil and black soil are commonly found in the areas in and

around Dindigul district. Hence, in the present study, the impact of the effluent

on both the soil types were studied and compared with controls of the respective

types.

Physicochemical profile of red soil and black soil

It has to be taken into consideration that sand generally allows the

contaminant to pass into deeper zones. Hence there is a possibility of

contamination of the deeper zones of groundwater in the future if the soil and

groundwater environment are not protected from the tannery wastes. The

presence of heavy metals even in low concentration is an indication of industrial

activity in that area (Zahid et al., 2006). According to Cao and Zhu (1999), red

soil is an important resource for the exploitation and utilization of agriculture and

forestry. Black soil is rich in organic matter in the form of humus. It has large

quantities of nutrients, excellent structure and good water holding capacity,

making it very suitable for agriculture.



66

In the present study, red soil and black soil samples were collected from

the effluent discharge area (Target area soil samples). Red and Black soil

samples collected from the area 15 kms away from the target area served as

control samples.

Table 9 indicates the profile of the selected soil samples.

TABLE 9

PHYSICOCHEMICAL PROFILE OF RED SOIL AND BLACK SOIL

Parameters Red soil (control)

Target area red

soil

Black soil (control)

Target area black soil

pH 7.5 8.7 7.15 8.8

EC mho/cm 0.32 0.43 0.19 0.87

Sodium 122 52 32.0 24.82

Nitrogen 95 98 66 73

Phosphorous 8.57 14.06 4.25 8.21

Potassium 474 650 149 378

Calcium 1.25 2.38 0.45 0.63

Magnesium mg/kg 175 140 164 138

Copper 15.86 62 4.86 19.64

Iron 63.0 86.0 20.9 52.9

Chromium 22.01 93 15.52 48.72

Nickel 2.0 8.7 2.6 9.6

Zinc 18.22 29.05 8.72 11.83

Cadmium 1.5 7.4 1.8 9.2


Control soil- Soil collected from 15 kilometers away from the effluent discharge site

Target area soil sample - Soil collected within one kilometer radius of the effluent discharge site.

Soil pH is one of the most influential parameters controlling the

conversion of metals from immobile solid phase forms to more mobile form

(Abou et al., 2008). pH of the target area black soil was found to be 8.8 and that



67

of red soil was found to be 8.7. Zahid et al., (2006) reported that the pH varies

between 7.5 and 8.5 in the top soil, where carbonates generally precipitate

and many trace metals co precipitate with them and make the soil alkaline.

Sinha et al.(2006) suggested that soil becomes alkaline due to the alkalinity of

the tannery effluent discharged.

Electrical conductivity of the target area red soil (0.43 mho/cm) was

greater compared to the control red soil (0.32 mho/cm). The conductivity of the

target area black soil was found to be 0.87 which was higher compared to that

of control black soil (0.19). High conductivity of the soil indicates the presence of

higher levels of anions and cations in the soil. Soil discharged with effluents

from cotton ginning mills and paper mills showed higher electrical conductivity

(Medhi, 2005). The addition of tannery effluent to the soil affects the physical

properties of the soil. The effluent is rich in salts, particularly sodium chloride,

which on continuous irrigation increased the concentration in soil and reflected

in increased electrical conductivity (Thangavel et al., 2003).

Levels of sodium in the target area red soil were found to be 52 mg/kg

which was less than the control red soil (122 mg/kg). 24.82 mg/kg of sodium

was found to be present in target area black soil whereas 32 mg/kg of sodium

was found in control black soil (Figure 2). Sodium content in the red soil was

higher than that of the black soil. Krishna and Govil (2008) reported that the

level of cations (sodium and potassium) in the soil irrigated with tannery waste

water varied differently from control sites.

Generally nitrogen is the growth limiting nutrient, which is needed in the

highest concentration. Nitrogen content in the target area black soil (73 mg/kg)

was found to be higher than that of the control black soil (66 mg/kg). The target

area red soil contained 98 mg/kg nitrogen (Figure 2) which was on par with the

control red soil (95 mg/kg). According to Kabdali et al. (2003), the high nitrogen

content in the target area soil might favour plant growth.



68

FIGURE 2

SODIUM AND NITROGEN CONTENT IN THE TARGET AREA SOILS

CR - Control red soil TR - Target area red soil

CB - Control black soil TB - Target area black soil

The phosphorus content of the target area red soil was found to be

14.06 mg/kg, which was greater than the control red soil (8.57 mg/kg).

Phosphorus content in the target area black soil was found to be 8.21 mg/kg

while that of control soil was found to be 4.25 mg/kg. The results of the study

conducted by Chonkar et al., (2003) revealed that the phosphorus content in soil

increased significantly due to application of industrial sludge.

Potassium levels in the target area red soil (650 mg/kg) were found to

be higher than the control red soil (474 mg/kg). Target area black soil was found

to contain 378 mg/kg of potassium and control black soil without effluent was

found to contain 149 mg/kg (Figure 3). Nitrogen, phosphorus and potassium

contents in both the soils were found to be higher in target area soil compared

to that of the control soil.

Nitrogen, phosphorus, potassium and calcium contents were increased in

soils after disposal of sewage wastes (Girisha et al., 2006). Soil analysis in the

0

20

40

60

80

100

120

140

mg

/kg

Sodium Nitrogen

CR TR

CB TB



69

study done by Sheela and Peethambaram (2007) revealed that NPK content of

the soil was slightly increased by effluent treatment as the nutrients nitrogen,

phosphorus and potassium present in diluted effluent played a role in promoting

plant growth at lower concentration. Irrigation with sewage and pulp paper cult

effluent was reported to enrich the soil, mainly with respect to nitrogen,

phosphorus and potassium, and enhanced the crop yields considerably (Nan

and Chung, 2001).

FIGURE 3

POTASSIUM AND MAGNESIUM CONTENT IN THE TARGET AREA SOILS



Calcium levels of red soil in target area with tannery effluent were higher

than the control red soil (2.38 mg/kg and 1.25 mg/kg respectively). Calcium

content of the target area black soil and control black soil were 0.63 mg/kg and

0.45 mg/kg respectively. Red soil had a higher concentration of calcium

compared to that of black soil.

The red soil collected from the target area was found to contain a low

level of magnesium (140 mg/kg) compared to the control red soil which had

175 mg/kg of magnesium (Figure 3). Magnesium content in the black soil of

0

100

200

300

400

500

600

700

mg

/kg

Potassium Magnesium

CR TR

CB TB



70

target area was lesser than that of the control black soil (138 mg/kg and 164

mg/kg respectively). Target area red soil was found to contain a higher level of

magnesium compared to the target area black soil.

A threefold increase in copper content was recorded in target area red

soil compared to that of the control red soil. The content of copper in target area

black soil was found to be 19.62 mg/kg while 4.86 mg/kg of copper was found to

be present in control black soil (Figure 4). Though copper is a micronutrient of

prime importance in agricultural production, it may cause environmental problem

when accumulated in soils. Adsorption of copper depends upon soil properties

like pH, organic matter, clay and cation exchange capacity (Anuradha, 2005).

Copper is an essential element and good for health in very small quantities but

at excessive dose it is toxic.

FIGURE 4

CHROMIUM, ZINC AND COPPER CONTENT IN THE TARGET AREA SOILS



Iron content was higher in target area red soil (86.0 mg/kg) than control

red soil. In black soil 52.9 mg/kg of iron was present in target area sample which

0

10

20

30

40

50

60

70

80

90

100

mg

/kg

Chromium Zinc Copper

CR TR

CB TB



71

was higher than the control black soil. Increased amount of iron may be

contributed by the weathering of rocks and also by the discharge of effluent and

other wastes on surface that percolated into the ground water (Jain et al., 2000).

Higher values of iron were noted in tannery effluent contaminated soil

(Alverz-Bernal et al., 2006). As suggested by Xiong et al. (2001) untreated

industrial effluent contains higher amounts of cadmium, lead, zinc, copper,

manganese and iron which would have enhanced the concentration of heavy

metals in irrigated soil. From the composition of the heavy metals in the soils,

it was obvious that tannery industries were responsible for not only the increase

in chromium content in the soil inherent to the tanning process but also

cause an increase in iron, zinc, manganese, copper and sulphate contents

(Zahid et al., 2006).

Rajkumar et al. (2005) observed that chromium is a transition metal that

is discharged into the environment through the disposal of wastes from

industries like leather tanning and metallurgical, leading to contamination of soil.

Chromium is the main tanning agent and most hazardous chemical used in

chrome tanning process. The excessive use of this chemical leads to higher

concentration in the effluent (Bhalli and Khan, 2006). Chromium levels in the

target area red soil was found to be increased three fold than the control red soil

(93 mg/kg and 22.01 mg/kg respectively). The target area black soil was found

to contain 48.72 mg/kg of chromium while that of control black soil was

15.52 mg/kg (Fig 4). It is the major chemical present in the effluent, which, when

released into the soil, percolates the layers of soil.

Krishna and Govil (2008) reported that the level of chromium was found

to be high in soils receiving treated tannery waste water for irrigation than

control soil. Inadequate disposal of waste containing chromium at industrial site

had contaminated both the ground and water. High chromium levels have been

well documented to have negative impact on plant growth.

The results show that the target area red soil had 8.7mg/kg and control

red soil had 2.0 mg/kg of Nickel. Nickel level in target area black soil was found



72

to be 9.6 mg/kg compared to that of control black soil which was 2.6 mg/kg

(Figure 5). Presence of chromium and nickel in soil disturbs the pattern of

nutrient uptake by increasing or decreasing the nutrient content in plants

due to the heavy metal and nutrient metal interactions (Zupansic et al., 2004;

Harikrishnan and Kumar, 2009).

FIGURE 5

NICKEL AND CADMIUM CONTENT IN THE TARGET AREA SOILS



The metals, zinc and cadmium, were found to be 29.05 mg/kg and

7.4 mg/kg respectively in target area red soil which were higher compared to the

control red soil (18.22 mg/kg and 1.5 mg/kg respectively). Zinc and cadmium

content in the target area black soil were found to be 11.83 mg/kg and

9.2 mg/kg respectively while those of control black soil were 8.72 mg/kg and

1.8 mg/kg respectively (Figure 4 and 5). Higher amounts of heavy metals like

copper, zinc, iron and manganese were recorded in irrigated soil near industrial

complex as reported by Barman et al.(2001).

Excessive accumulation of heavy metals such as cadmium, lead,

chromium and nickel in the soil due to effluent discharge and the resultant

phytotoxicity was reported by Peralta et al. (2001) and Tsakou et al.(2001).

0

2

4

6

8

10

12

mg

/kg

Nickel Cadmium

CR TR CB TB



73

Cadmium dispersed in the environment persists in soils and sediments

for decades and taken up by plants which accumulates in the biosphere

(Bernard, 2008).

According to Sinha et al. (2006), chromium and other metals were found

to be high in effluent contaminated soils. Significant increases in soil metal

content were found in areas of high industrial activity where accumulation may

be several times higher than that of the average content in non contaminated

areas (Krishna and Govil, 2008).

From the results of the study it was observed that control red soil was

found to be rich in minerals and nutrients compared to the control black soil. The

contents of sodium, nitrogen, phosphorus, potassium, calcium and magnesium

were found to be at a higher level in target area red soil compared to the target

area black soil.

PHASE II

4.2 Growth studies of selected plants using diluted tannery effluent

The use of plants to improve water quality in municipal and more recently

industrial waste water treatment system is of great use and an emerging

technology of phytoremediation (Suseela et al., 2002). Girisha et al. (2006) in

their study acclaimed that tannery and textile industrial waste water contains

appreciable amount of plant nutrients such as nitrogen, phosphorus, potassium,

calcium, magnesium and sulphur. These nutrients could be used for plant

growth after proper treatment.

Direct discharge of effluents changes the physico chemical and biological

characteristics of soils and was responsible for the reduction in the rate of

germination of seeds but studies have proved that properly diluted effluents can

be used for irrigation (Sheela and Soumya, 2004).

Favorable effect of diluted effluents on seedling growth have been

investigated and well documented (Chandra et al., 2009; Shreshtha and

Niroula, 2003).



74

Plants Vigna radiata and Vigna mungo belong to the family leguminosae.

They have been recognized for their economic value and are rich in protein,

calcium, phosphorus and vitamins (Simanova et al., 2007). These leguminous

plants have a property of impermeability of seed coat and also reported to show

resistance against salinity. Since it has been well documented that raw effluent

discharged from the tannery industry has a detrimental effect on the plant

growth, in the present study, various dilutions of the effluent were used for the

measurement of growth of the selected plants Vigna radiata and Vigna mungo.

Nutrients added through the effluent were retained to some extent; at the same

time the high salt concentration of the effluent was reduced by dilution. Several

growth parameters such as percentage of germination, seedling survival,

seedling height, yield parameters and biochemical parameters have been taken

as criteria to assess plant responses to a specific pollutant.

In the present study, plants Vigna radiata and Vigna mungo were grown

in effluent contaminated red soil and black soil. The growth was continued with

25% and 50% tannery effluent. Plants were grown in red soil (CR), red soil +

25% effluent (T1R), red soil+50% effluent (T2R), black soil (C2B), black soil +

25% effluent (T1B), black soil + 50% effluent (T2B) for 90 days till the seeds

were produced. Germination percentage and vigour index were observed on

8th day after sowing. Selected biometric observations (root length, shoot length,

fresh weight, dry weight, number of leaves and number of roots), selected

biochemical parameters such as chlorophyll, carotenoid, carbohydrate and

protein in the leaf samples, total phenol, carotenoid, carbohydrate and protein

contents in the seed samples were analysed. Leaves and fresh seeds of all the

plants were analysed for their enzymic and non enzymic antioxidant status.

Metals like chromium, nickel, zinc and cadmium were analysed in roots,

shoots, leaves and grains of both the plants grown in red and black soil using

both the effluent concentrations. Histochemical staining of root samples were

observed for the accumulation of metals and yield parameters such as number

of nodules, flowering time, pod weight, pod length, number of pods per plant,



75

number of seeds per plant, number of seeds per pod, 100 seed weight and total

seed weight per plant were studied.

4.2.1 Biometric observations of the plant samples

Plant growth can be best monitored by observing the biometric

observations of the plants. Tannery effluent has a higher concentration of total

nitrogen, sulphide, dissolved and suspended solids and due to excess of these

nutrients plant growth is inhibited as it affects the water absorption and other

metabolic processes of the plant (Sheela and Peethambaram, 2007). Panda

and Choudry (2005) and Yongpisanphop et al. (2005) observed that raw effluent

irrigation adversely affects the plant growth and development but use of diluted

effluent would enhance plant growth.

4.2.1.1 Germination percentage and vigour index

Plant growth and development are essential processes of life and

propagation of the species. They are continuous and mainly depend on external

resource present in soil and air. Growth is chiefly expressed as a function of

genotype and environment, which consists of external and internal growth

factors. Presence of contaminants in the external environment leads to change

in the growth and development pattern of the plant.

Among the growth process, seed germination and seedling growth have

been considered critical for raising a successful agricultural crop. The process of

germination and growth of young seedlings are susceptible to toxic materials

in water. The ability of a crop to germinate and establish under stress by

environmental contaminants is an early indicator of tolerance of the plant. Seed

germination was the first physiological process affected by the heavy metals

when present in soil and water (Peralta et al., 2001).

Table 10 and Figure 6 and 7 show the percent germination and vigour

index of the plants Vigna radiata and Vigna mungo grown in red soil and black

soil using 25% and 50% tannery effluent. These parameters were observed on

the 8th day after sowing.



76

TABLE 10

GERMINATION PERCENTAGE AND VIGOUR INDEX OF Vigna radiata AND Vigna mungo GROWN IN RED SOIL AND BLACK SOIL USING

DILUTED TANNERY EFFLUENT

Germination percentage Vigour index Soil

Groups Vigna radiata

Vigna mungo

Vigna radiata

Vigna mungo

CR 98.0 ± 1.63 96.0 ± 3.27 392 ± 2.43 364 ± 5.20

T1R 94.0 ± 3.01 90.7 ± 6.94 329 ± 1.58 300 ± 4.90 Red soil

T2R 91.0 ± 3.15 86.0 ± 4.20 273 ± 2.45 249 ± 2.05

CB 96.0 ± 4.90 94.0 ± 3.17 393 ± 2.42 366 ± 7.06

T1B 93.0 ± 2.35 91.0 ± 4.18 344 ± 3.24 327 ± 2.43 Black soil

T2B 81.0 ± 3.64 84.0 ± 3.16 259 ± 3.30 260 ± 4.05

CD ( 0.05) 8.11 6.36

Values are mean ± SD of triplicates

CR-Control red soil, T1R-Red soil with 25% effluent, T2R-Red soil with 50% effluent, CB-Control black soil, T1B-Black soil with 25% effluent, T2B- Black soil with 50% effluent.

FIGURE 6

GERMINATION PERCENTAGE OF Vigna radiata AND Vigna mungo

CR-Control red soil, T1R-Red soil with 25% effluent, T2R-Red soil with 50% effluent, CB-Control black soil, T1B-Black soil with 25% effluent, T2B- Black soil with 50% effluent.

0

10

20

30

40

50

60

70

80

90

100

Vigna radiata Vigna mungo

Germ

inati

on

perc

en

tag

e

CR T1R T2R CB T1B T2B



77

FIGURE 7

VIGOUR INDEX OF Vigna radiata AND Vigna mungo

Seeds of Vigna mungo grown with 50% effluent showed a significant

reduction in germination compared to the control plants. Both the plants grown

in black soil with 50% tannery effluent showed a significant reduction in

germination percentage compared to the control plants of black soil.

A significant reduction (p< 0.05) in vigour index was observed in both the

plants grown in both the soil types using 25% and 50% effluent compared to the

control plants.

According to Malla and Mohanty (2005), there was significant decrease in

percentage germination, root length and shoot length with increase in the

effluent concentration. In their work with mung seeds, they reported that

percentage germination and the concentration of effluent treatment were

negatively correlated. Pandey et al. (2008) reported that supply of the undiluted

distillery effluent produced significant inhibition in seed germination

4.2.1.2 Root length

Table 11 shows the root length of the plants Vigna radiata and Vigna

mungo grown in red and black soils using 25% and 50% tannery effluent.

0

50

100

150

200

250

300

350

400

450


Vig

ou

r in

dex

CR T1R T2R CB T1B T2B



78

T

AB

LE

1

1

RO

OT

LE

NG

TH

(c

m)

OF

Vigna radiata

AN

D Vigna mungo

GR

OW

N I

N R

ED

AN

D B

LA

CK

SO

ILS

US

ING

DIL

UT

ED

TA

NN

ER

Y E

FF

LU

EN

T O

N 3

0, 6

0 A

ND

90

DA

YS

AF

TE

R S

OW

ING

Vigna radiata

Vigna mungo

Da

ys

aft

er

so

win

g (

DA

S)

S

oil

Gro

up

s

30

60

90

3

0

6

0

9

0

CR

4

.00 ±

1.6

3

6.0

0 ±

3.2

7

9.0

0 ±

2.4

5

6.0

0 ±

2.4

3

7.0

0 ±

2.3

0

10

.00

± 4

.08

T1

R

3.4

0 ±

0.3

4

.67 ±

0.9

4

7.0

0 ±

2.2

0

5.0

0 ±

2.3

8

6.2

0 ±

0.1

6

8.0

0 ±

1.6

3

Re

d s

oil

T2

R

3.1

0 ±

0.1

6

3.0

0 ±

0.8

2

6.0

0 ±

2.3

5

4.5

0 ±

0.4

1

5.0

0 ±

3.2

7

7.5

0 ±

0.4

1

CB

4

.20 ±

0.1

1

6.0

0 ±

1.5

3

8.4

0 ±

0.1

3

5.3

0 ±

0.2

4

7.8

0 ±

0.1

4

8.9

0 ±

0.0

8

T1

B

3.4

0 ±

0.3

3

5.7

0 ±

0.1

0

7.8

0 ±

0.1

7

4.7

0 ±

0.2

1

6.8

0 ±

0.1

3

8.6

0 ±

0.2

1

Bla

ck

s

oil

T

2B

2

.90 ±

0.1

2

4.7

0 ±

0.2

0

6.8

0 ±

0.1

9

3.2

7 ±

0.3

9

5.8

0 ±

0.1

8

7.3

0 ±

0.2

4

CD

( 0

.05

) 2

.92

3

.43

V

alu

es a

re m

ean ±

SD

of

trip

lica

tes

CR

- C

ontr

ol re

d s

oil,

T1R

- R

ed s

oil

with 2

5%

eff

luent,

T2R

- R

ed s

oil

with 5

0%

eff

luent, C

B-

Contr

ol b

lack s

oil,

T1B

- B

lack s

oil

with 2

5%

eff

luent,

T

2B

- B

lack s

oil

with 5

0%

eff

luent.

78



79

Plant biomass, root length and shoot length are used as indices of growth

performance. Tolerance to toxic elements and biotic stress depends on well

branched and extensive root systems. In our study, Vigna radiata and Vigna

mungo highlighted significant differences in their responses to various

concentrations of tannery effluent.

It was observed that both the plants recorded no significant difference in

the root length when grown with 25% effluent in both the soils. A significant

reduction in root length was observed on 90th day only when Vigna radiata was

grown with 50% effluent. The reduction might be due to the metals present in

the tannery effluent.

According to Pandey et al. (2008) supply of untreated effluent produced

significant inhibition in seed germination and seedling growth parameters in both

maize and rice. According to them a significant inhibition in root length was

observed with 50% effluent.

Studies on Allicum cepa (Palacio et al., 2005), Zea mays (Akbar et al.,

2009) and Arachis hypogea (Nagajyoti et al., 2009) showed significant decrease

in root length with higher chromium concentration in soil. The reduction in root

growth could be due to the direct contact of seedling roots with pollutants

causing a collapse and subsequent inability of the root to absorb water.

4.2.1.3. Shoot length Shoot length is considered as an important morphological parameter

related to growth and development of the whole plant.

Table 12 shows the shoot length of the plants Vigna radiata and Vigna

mungo grown in red soil and black soil using 25% and 50% tannery effluent on

30th, 60th and 90th days after sowing.

Both the plants recorded significant reduction in shoot length when grown

with 50% effluent in red soil. In black soil Vigna mungo recorded significant

reduction on the 60th day of growth.



80

TA

BL

E 1

2

S

HO

OT

LE

NG

TH

(cm

) O

F Vigna radiata

AN

D Vigna mungo

GR

OW

N I

N R

ED

AN

D B

LA

CK

SO

ILS

US

ING

DIL

UT

ED

TA

NN

ER

Y E

FF

LU

EN

T O

N 3

0, 6

0 A

ND

90

DA

YS

AF

TE

R S

OW

ING

Vigna radiata

Vigna mungo

Da

ys

aft

er

so

win

g (

DA

S)

So

il

Gro

up

s

30

60

90

30

60

9

0

CR

2

3.0

± 2

.45

4

0.0

± 2

.42

6

8.0

± 1

.63

2

2.0

± 1

.58

3

7.0

± 2

.41

5

5.0

± 4

.08

T1

R

20

.0 ±

2.3

2

37

.0 ±

3.1

7

63

.0 ±

2.3

5

19

.0 ±

0.7

2

34

.0 ±

3.2

7

49

.0 ±

0.8

2

Re

d s

oil

T2

R

16

.0 ±

2.4

7

35

.33

± 2

.87

5

8.0

± 1

.73

1

4.0

± 3

.47

3

2.0

± 1

.43

4

7.0

± 2

.15

CB

2

1.0

± 2

.34

3

8.0

± 2

.40

5

7.0

± 2

.42

2

0.0

± 3

.24

3

7.0

± 2

.25

5

5.0

± 2

.31

T1

B

17

.0 ±

2.3

0

35

.0 ±

4.0

8

54

.0 ±

3.0

6

17

.0 ±

1.4

5

36

.0 ±

2.2

7

52

.0 ±

1.4

3

Bla

ck

s

oil

T2

B

16

.0 ±

2.1

9

34

.0 ±

3.3

2

53

.0 ±

2.6

5

16

.0 ±

1.7

4

32

.0 ±

1.6

2

51

.0 ±

1.6

0

CD

( 0

.05

) 5

.36

4

.95

V

alu

es a

re m

ean ±

SD

of

trip

lica

tes

CR

- C

ontr

ol re

d s

oil,

T1R

- R

ed s

oil

with 2

5%

eff

luent,

T2R

- R

ed s

oil

with 5

0%

eff

luent, C

B-

Contr

ol b

lack s

oil,

T1B

- B

lack s

oil

with 2

5%

eff

luent,

T

2B

- B

lack s

oil

with 5

0%

eff

luent.

80



81

Purohit et al. (2003) reported that the shoot length of tomato plant

decreased when the concentration of the tannery effluent was increased. Babu

and Vishnuvardhan (2006) reported that the shoot length and root length of

Vigna mungo was retarded and inhibited at higher effluent concentration.

According to Rajula and Padmadevi (2000), the germination percentage

and morphological characters like shoot length and root length decreased

gradually with increase in effluent concentration. A gradual decrease in

germination percentage, root length, shoot length, fresh weight and dry weight

of black gram seedlings with increase in chromium concentrations was

observed by Chidambaram et al., (2009).

4.2.1.4 Fresh weight

Table 13 shows the fresh weight of Vigna radiata and Vigna mungo

plants grown in red soil and black soil using 25% and 50% tannery effluent.

Vigna radiata grown with black soil using 50% effluent showed significant

reduction of fresh weight on 90th day compared to that grown with red soil.

Vigna mungo recorded significant reduction (p<0.05) of fresh weight on 90th day

with 50% effluent using red soil compared to that with black soil.

Chidarambam et al., (2009) in their work on black gram (Vigna mungo)

reported a gradual decrease of root length, shoot length, fresh weight and dry

weight with increase in chromium concentrations.

Decrease in the fresh weight may be the outcome of a decreased water

uptake or enhanced water loss, both of which may occur following membrane

damage since plant cell membranes are generally considered as the primary

sites of metal injury (Diwan et al., 2010b).



82

T

AB

LE

13

FR

ES

H W

EIG

HT

(g

) O

F Vigna radiata

AN

D Vigna mungo

GR

OW

N I

N R

ED

AN

D B

LA

CK

SO

ILS

US

ING

DIL

UT

ED

TA

NN

ER

Y E

FF

LU

EN

T O

N 3

0, 6

0 A

ND

90

DA

YS

AF

TE

R S

OW

ING

Vigna radiata

Vigna mungo

Da

ys

aft

er

so

win

g (

DA

S)

So

il

Gro

up

s

30

60

90

30

60

90

CR

2

1.0

± 3

.27

2

9.0

± 0

.82

6

3.0

± 2

.45

1

8.0

± 1

.63

2

9.0

± 0

.92

8

4.0

± 3

.65

T1

R

19

.0 ±

0.9

1

28

.0 ±

1.5

3

61

.0 ±

2.2

5

18

.0 ±

2.4

3

25

.0 ±

4.0

8

53

.0 ±

1.2

3

Re

d s

oil

T2

R

16

.0 ±

3.2

3

26

.0 ±

2.1

1

59

.0 ±

0.8

2

17

.0 ±

2.4

1

22

.7 ±

3.7

2

51

.0 ±

3.3

7

CB

1

7.0

± 2

.34

2

7.0

± 2

.41

5

8.0

± 1

.73

1

6.0

± 3

.19

2

5.0

± 3

.56

5

3.0

± 2

.02

T1

B

16

.0 ±

1.4

3

26

.0 ±

3.4

3

56

.0 ±

3.0

4

15

.0 ±

4.0

8

24

.0 ±

3.1

7

52

.0 ±

2.3

8

Bla

ck

s

oil

T2

B

16

.0 ±

0.7

2

25

.0 ±

4.1

8

53

.0 ±

2.3

5

15

.0 ±

2.4

9

23

.0 ±

2.6

1

49

.0 ±

0.2

2

CD

( 0

.05

) 4

.76

6

.53

Valu

es a

re m

ean ±

SD

of

trip

licate

s

C

R-

Con

trol re

d s

oil,

T1R

- R

ed s

oil

with 2

5%

eff

luent,

T2R

- R

ed s

oil

with 5

0%

eff

lue

nt, C

B-

Con

trol bla

ck s

oil,

T1B

- B

lack s

oil

with 2

5%

eff

luent,

T

2B

- B

lack s

oil

with 5

0%

eff

luent.

82



83

4.2.1.5. Dry weight

Table 14 shows the dry weight of the plants Vigna radiata (Figure14)

and Vigna mungo (Figure 15) grown in red soil and black soil using 25% and

50% tannery effluent on 30th, 60th and 90th days after sowing.

Vigna radiata plants grown in red soil did not show any differnece in their

dry weights on comparision with control plants. While on growth with black soil

no significance difference was obtained on 30th and 60th day. But on 90th day a

significant reduction was observed in 50% effluent.

Vigna mungo plants grown in red soil was found to have no significant

difference in dry weight when grown in 25% effluent. But with 50% effluent a

signficant difference was noticed on 30th day plant. Plants grown with black soil

showed a significant reduction on 90th day.

This indicated that plants showed a better growth with 25% effluent which

is more diluted compared to 50% effluent. In a study conducted by Diwan et al.,

(2010), significant reduction in dry weight was observed due to increased

effluent concentration.

4.2.1.6. Number of leaves and Number of roots

Table 15 and 16 show the number of leaves and roots of the plants Vigna

radiata and Vigna mungo grown in red soil and black soil using 25% and 50%

tannery effluent on 30th, 60th and 90th day after sowing.

Vigna raidiata and Vigna mungo plants grown in 25% and 50% effluent

had no significant difference in their number of leaves when grown in red soil

and black soil.

No. of roots of Vigna radiata and Vigna mungo plants showed a gradual

increase from 30th to 90th day of growth. Plants grown in red soil and black soil

followed the same trend and found to be on par with the control plants. Similarly

with effect of different dilutions of the effluent (25% and 50%) was not found to

be significant on the number of roots.



84

T

AB

LE

14

DR

Y W

EIG

HT

(g

) O

F Vigna radiata

AN

D Vigna mungo

GR

OW

N I

N R

ED

AN

D B

LA

CK

SO

ILS

US

ING

D

ILU

TE

D

T

AN

NE

RY

EF

FL

UE

NT

ON

30

, 6

0 A

ND

90

DA

YS

AF

TE

R S

OW

ING

Vigna radiata

Vigna mungo

Da

ys

aft

er

so

win

g (

DA

S)

So

il

Gro

up

s

30

6

0

90

3

0

60

9

0

CR

2

.0 ±

0.4

1

2.7

0 ±

0.1

3

3.4

6 ±

0.0

3

2.6

0 ±

0.3

3

2.6

0 ±

0.2

8

4.0

1 ±

0.0

1

T1

R

1.9

0 ±

0.0

8

2.3

0 ±

0.2

4

3.1

6 ±

0.0

5

2.0

3 ±

0.6

9

2.3

0 ±

0.2

4

3.8

6 ±

0.0

2

Re

d s

oil

T2

R

1.7

0 ±

0.7

4

2.1

3 ±

0.2

9

3.0

1 ±

0.0

3

1.5

0 ±

0.3

1

2.0

± 0

.16

3

.71 ±

0.2

3

CB

1

.80 ±

0.1

4

2.5

0 ±

0.4

4

3.5

7 ±

0.0

6

1.8

0 ±

0.2

6

2.6

0 ±

0.2

3

4.0

± 0

.42

T1

B

1.7

0 ±

0.3

4

2.2

0 ±

0.1

2

3.2

1 ±

0.0

4

1.5

0 ±

0.3

7

2.5

0 ±

0.4

5

3.8

0 ±

0.1

9

Bla

ck

s

oil

T

2B

1

.60 ±

0.2

1

2.2

0 ±

0.2

4

2.9

1 ±

0.0

1

1.7

0 ±

0.0

8

2.1

0 ±

0.1

6

3.2

0

± 0

.13

CD

( 0

.05

) 0

.47

0

.60

V

alu

es a

re m

ean ±

SD

of

trip

lica

tes

CR

- C

ontr

ol re

d s

oil,

T1R

- R

ed s

oil

with 2

5%

eff

luent,

T2R

- R

ed s

oil

with 5

0%

eff

luent, C

B-

Contr

ol b

lack s

oil,

T1B

- B

lack s

oil

with 2

5%

eff

luent,

T

2B

- B

lack s

oil

with 5

0%

eff

luent.

84



85

T

AB

LE

15

N

UM

BE

R O

F L

EA

VE

S O

F Vigna radiata

AN

D Vigna mungo

GR

OW

N I

N R

ED

AN

D B

LA

CK

SO

ILS

US

ING

DIL

UT

ED

TA

NN

ER

Y E

FF

LU

EN

T O

N 3

0, 6

0 A

ND

90

DA

YS

AF

TE

R S

OW

ING

Vigna radiata

Vigna mungo

Da

ys

aft

er

so

win

g (

DA

S)

So

il

Gro

up

s

30

6

0

90

3

0

60

9

0

CR

1

3.0

± 2

.45

2

4.0

± 3

.27

3

5.0

± 4

.08

1

0.0

± 4

.05

2

1.0

± 0

.82

3

2.0

± 1

.63

T1

R

12

.0 ±

1.6

1

21

.0 ±

1.5

8

34

.0 ±

3.2

7

9.0

± 0

.72

1

9.0

± 0

.61

3

1.0

± 3

.11

R

ed

so

il

T2

R

11

.33

± 2

.87

2

0.0

± 4

.02

3

2.0

± 1

.43

7

.0 ±

2.2

5

18

.0 ±

1.4

3

29

.0 ±

2.5

1

CB

1

3.0

± 2

.65

2

3.0

± 2

.38

3

4.0

± 3

.17

1

0.0

± 2

.45

2

1.0

± 1

.69

3

1.0

± 0

.34

T1

B

11

.0 ±

1.5

3

20

.0 ±

4.0

6

32

.0 ±

0.3

2

9.0

± 0

.62

2

0.0

± 1

.51

2

7.0

± 0

.80

B

lac

k

so

il

T2

B

10

.0 ±

4.1

8

19

.0 ±

1.2

3

29

.0 ±

0.8

2

6.0

± 3

.46

1

7.0

± 2

.32

2

9.0

± 0

.81

CD

( 0

.05

) 5

.64

4

.23

V

alu

es a

re m

ean ±

SD

of

trip

licate

s

C

R-

Co

ntr

ol re

d s

oil,

T1R

- R

ed s

oil

with

25%

eff

luent,

T2R

- R

ed s

oil

with 5

0%

eff

luent, C

B-

Co

ntr

ol b

lack s

oil,

T1B

- B

lack s

oil

with

25%

eff

luent,

T

2B

- B

lack s

oil

with 5

0%

eff

luent.

85



86

T

AB

LE

16

NU

MB

ER

OF

RO

OT

S O

F Vigna radiata

AN

D Vigna mungo

GR

OW

N I

N R

ED

AN

D B

LA

CK

SO

ILS

US

ING

DIL

UT

ED

T

AN

NE

RY

EF

FL

UE

NT

ON

30

, 6

0 A

ND

90

DA

YS

AF

TE

R S

OW

ING

Vigna radiata

Vigna mungo

Da

ys

aft

er

so

win

g (

DA

S)

So

il

Gro

up

s

30

6

0

90

3

0

60

9

0

CR

9

.0 ±

0.8

2

12

.0 ±

1.6

3

14

.0 ±

1.2

7

10

.0 ±

1.0

8

13

.0 ±

1.4

5

15

.0 ±

1.0

6

T1

R

8.0

± 1

.66

1

0.0

±1

.1

8

12

.0 ±

0.8

3

9.3

3 ±

1.2

5

10

.0 ±

4.0

4

12

.0 ±

1.6

2

Re

d

so

il

T2

R

7.0

± 2

.43

9

.0 ±

0.5

2

13

.0 ±

2.4

4

8.3

3 ±

1.3

5

11

.0 ±

0.7

2

14

.0 ±

3.0

7

CB

9

.0 ±

1.4

3

13

.0 ±

2.6

5

13

.0 ±

0.4

2

9.3

3 ±

2.0

5

12

.0 ±

1.3

3

13

.0 ±

2.7

5

T1

B

8.0

± 1

.67

1

1.0

± 2

.49

1

2.0

± 1

.69

8

.0 ±

2.4

1

10

.0 ±

4.0

7

11

.0 ±

1.6

5

Bla

ck

s

oil

T2

B

6.0

± 1

.68

1

2.0

± 1

.61

1

0.0

± 0

.80

7

.0 ±

2.4

7

11

.0 ±

0.8

0

12

.0 ±

1.6

2

CD

( 0

.05

) 4

.05

5

.29

Valu

es a

re m

ean ±

SD

of

trip

licate

s

C

R-

Con

trol re

d s

oil,

T1R

- R

ed s

oil

with 2

5%

eff

luent,

T2R

- R

ed s

oil

with 5

0%

eff

lue

nt, C

B-

Con

trol bla

ck s

oil,

T1B

- B

lack s

oil

with 2

5%

eff

luent,

T

2B

- B

lack s

oil

with 5

0%

eff

luent.

86



87

4.2.2. Biochemical parameters

Morphological or physiological changes in plants will not be visible unless

there is certain metabolic change in cellular activities such as in photosynthesis.

Essential heavy metals like iron, manganese, zinc and copper for all higher

plants are absorbed and accumulated in plant tissues based on their solubility,

concentration and availability of different ions in the soils. Excess accumulation

of heavy metals causes either deficiency or enrichments of other essential

micronutrients which will a have direct effect on the concentration of chlorophyll

(Sharma and Chettri, 2008).

The toxic chemicals and pollutants especially heavy metals present in the

tannery effluent may affect the biochemical characteristics such as chlorophyll,

carotenoids, carbohydrate, protein and enzymes such as glucose 6 phosphate

dehydrogenase, peroxidase and superoxide dismutase (Singh and Tiwari, 2003;

Sharma and Agrawal, 2005). The subsequent biochemical changes of the plant

samples due to waste water irrigation were studied by Gupta et al., (2010).

Hence in the present study an attempt was made to study the changes

in biochemical parameters after irrigating the selected plants with various

concentrations of tannery effluent. Biochemical parameters were analysed in

roots, shoots, leaves and seeds of both the plants grown in 25% and 50%

tannery effluent.

4.2.2.1. Biochemical parameters in the leaf samples

Biochemical parameters such as chlorophyll, carotenoid, carbohydrate

and protein were analysed in the leaves of both the plants grown with 25% and

50% tannery effluent. Estimation was done on the leaves of plants collected on

30, 60 and 90 days after sowing.



88

T

AB

LE

17

CH

LO

RO

PH

YL

L C

ON

TE

NT

(m

g/g

) IN

TH

E L

EA

VE

S O

F Vigna radiata A

nd

Vigna mungo G

RO

WN

IN

RE

D A

ND

B

LA

CK

SO

IL U

sin

g D

ILU

TE

D T

AN

NE

RY

EF

FL

UE

NT

Vigna radiata

Vigna mungo

Da

ys

aft

er

so

win

g (

DA

S)

So

il

Gro

up

s

30

6

0

90

3

0

60

9

0

C1

0

.48 ±

0.0

4

0.3

71

± 0

.03

0

.22 ±

0.0

3

0.7

1 ±

0.0

2

0.6

3 ±

0.0

5

0.4

9 ±

0.0

4

T1

0

.41 ±

0.0

4

0.3

1 ±

0.0

3

0.1

9 ±

0.0

4

0.5

7 ±

0.0

5

0.4

6 ±

0.0

4

0.3

4 ±

0.0

3

Re

d s

oil

T2

0

.32 ±

0.0

3

0.2

1 ±

0.0

2

0.1

0 ±

0.0

2

0.4

6 ±

0.0

4

0.3

6 ±

0.0

3

0.2

7 ±

0.0

2

C2

0

.40 ±

0.0

4

0.3

1 ±

0.0

3

0.2

0 ±

0.0

3

0.4

2 ±

0.0

3

0.3

5 ±

0.0

2

0.1

9 ±

0.0

1

T3

0

.39 ±

0.0

3

0.2

9 ±

0.0

5

0.1

6 ±

0.0

4

0.2

5 ±

0.0

2

0.2

2 ±

0.0

5

0.1

7 ±

0.0

2

Bla

ck

s

oil

T

4

0.2

9 ±

0.0

2

0.2

0 ±

0.0

4

0.0

9 ±

0.0

2

0.1

8 ±

0.0

1

0.1

4 ±

0.0

4

0.1

1 ±

0.0

1

CD

(0

.05

) 0

.004

0

.002

V

alu

es a

re m

ean ±

SD

of

trip

lica

tes

C

R-

Co

ntr

ol

with r

ed s

oil,

T1R

- R

ed s

oil

with 2

5%

eff

luent, T

2R

- R

ed s

oil

with 5

0%

eff

luent, C

B-

Co

ntr

ol

bla

ck s

oil,

T1B

- B

lack s

oil

with 2

5%

eff

luent, T

2B

- B

lack s

oil

with 5

0%

eff

luent

88



89

a) Chlorophyll Chlorophylls are the most important pigments in plants because of their

photosynthetic process. A quantitative analysis of these pigments is important in

elucidating the photosynthetic productivity of plants. Synthesis of chlorophyll

is controlled by the interaction of a number of environmental factors such as

light, temperature, oxygen, moisture content, metallic ions and nutrients.

Table 17 shows the chlorophyll content in the leaves of Vigna radiata

(Figure 8) and Vigna mungo (Figure 9) grown in red and black soil using 25%

and 50% tannery effluent.

FIGURE 8

CHLOROPHYLL CONTENT IN Vigna radiata

Chlorophyll content in the leaves of both the plants in both the soils using

25% and 50% tannery effluent were found to be significantly reduced than that

of the plants grown without effluent. A decrease in the chlorophyll content

suggests pollution injury.

Mishra et al. (2008), in their study demonstrated that the levels of

chlorophyll and protein decreased in Eichornea crassipies when the metal levels

increased. According to Sharma and Chettri (2008) excess heavy metals

0

0.1

0.2

0.3

0.4

0.5

0.6

30 DAS 60 DAS 90 DAS

Ch

loro

ph

yll c

on

ten

t (m

g/g

)

CR T1R T2R

CB T1B T2B



90

accumulated in different plant tissues may influence the disturbances in plant

nutrition. Thus, excess accumulation of heavy metals causes either deficiency

or enrichment of other nutrients which has a direct effect on chlorophyll.

Magnesium of chlorophyll molecule is substituted by heavy metals (copper,

cadmium, nickel, lead) resulting in heavy metal substituted chlorophylls.

FIGURE 9

CHLOROPHYLL CONTENT IN Vigna mungo

Accumulation of copper, zinc and cadmium would have inhibited the

chlorophyll biosynthesis due to prevention of photoreactive protochlorophyll

reductase complex formation and amino levulinic acid synthase as opined

by Singh et al. (2003). Reduction in chlorophyll content may be attributed to

impaired uptake of essential elements, damage of photosynthetic apparatus or

due to chlorophyll degradation by increased chlorophyllase activity (Sharma and

Dubey, 2005). The chemicals released from tannery effluent might be the

reason for the reduction in chlorophyll content in the experimental plants.

b) Carotenoid

Table 18 shows the carotenoid content in the leaves of the plants Vigna

radiata and Vigna mungo grown in red soil and black soil grown using 25% and

50% effluent.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8


Ch

loro

ph

yll c

on

ten

t (m

g/g

)

CR T1R T2R

CB T1B T2B



91

TA

BL

E 1

8

CA

RO

TE

NO

ID C

ON

TE

NT

(m

g/g

) IN

TH

E L

EA

VE

S O

F Vigna radiata A

ND

Vigna mungo G

RO

WN

IN

RE

D A

ND

BL

AC

K S

OIL

US

ING

DIL

UT

ED

TA

NN

ER

Y E

FF

LU

EN

T

Vigna radiata

Vigna mungo

S

oil

Gro

up

s

Da

ys

aft

er

so

win

g (

DA

S)

30

6

0

90

3

0

60

9

0

C1

0

.04 ±

0.0

1

0.1

2 ±

0.0

2

1.0

3 ±

0.1

0

.07 ±

0.0

5

0.2

0 ±

0.0

2

1.0

3 ±

0.1

T1

0

.04 ±

0.0

2

0.1

2 ±

0.0

3

1.0

3 ±

0.3

0

.06 ±

0.5

0

.12 ±

0.0

2

1.0

2 ±

0.1

R

ed

so

il

T2

0

.03 ±

0.0

1

0.1

1 ±

0.0

1

1.0

2 ±

0.2

0

.05 ±

0.0

2

0.1

0 ±

0.1

0

.08 ±

0.5

C2

0

.04 ±

0.0

2

1.1

0 ±

0.1

1

.02 ±

0.3

0

.05 ±

0.0

2

1.1

1 ±

0.1

1

.02 ±

0.5

T3

0

.04 ±

0.0

5

0.8

± 0

.01

1

.02 ±

0.3

0

.05 ±

0.0

2

0.2

± 0

.02

1

.1 ±

0.3

B

lac

k

so

il

T4

0

.03 ±

0.0

2

0.2

± 0

.02

1

.01 ±

0.4

0

.04 ±

0.0

5

0.1

± 0

.01

1

.0 ±

0.2

CD

(0

.05

) 0

.06

0

.01

V

alu

es a

re m

ean o

f tr

iplic

ate

s

C

R-

Co

ntr

ol

with r

ed s

oil,

T1R

- R

ed s

oil

with 2

5%

eff

luent, T

2R

- R

ed

soil

with 5

0%

eff

luent,

CB

- C

ontr

ol

bla

ck s

oil,

T1B

- B

lack s

oil

with 2

5%

eff

luent, T

2B

- B

lack s

oil

with 5

0%

eff

luent

91



92

Carotenoid content in the leaves of Vigna radiata showed significant

reduction with black soil grown in 50% tannery effluent only on 60th day of

growth whereas Vigna mungo recorded significant reduction of carotenoid

content in both the soils grown with 25% and 50% effluents from 60th day to 90th

day.

Earlier reports suggested that effects of heavy metals on carotenoid

content were plant and metal specific. Chromium induced degradation of

carotenoid has been reported in V.spiralis by Vajpayee et al. (2001).

c) Carbohydrate

Table 19 shows the carbohydrate content in the leaves of Vigna radiata

(Figure 10) and Vigna mungo (Figure 11) plants grown in red soil and black soil

using 25% and 50% tannery effluent.

The carbohydrate content in the leaves of Vigna radiata and Vigna

mungo (control) plants increased with increase in time of growth and showed a

maximum in the 90th day plant. The carbohydrate content in the leaves of the

plants grown with 25% effluent showed a value of 4.18 and with 50% effluent

it was 3.16 mg/g. Similarly the carbohydrate content of the leaves of Vigna

radiata plants grown in black soil with 25% and 50% effluent were 3.78 and

2.03 mg/g respectively on 30th day. A significant decrease was observed in the

carbohydrate content of the leaves of T1R, T2R, T1B and T2B plants compared

to control plants.

When the concentration of pollutants in the effluent exceeds the

detoxifying capacity of the tissue through their normal metabolism, there is a

decrease in the biochemical parameters such as chlorophyll, protein, amino

acid, carbohydrate and nucleic acid (Hamid and Jawaid, 2009; Adekunle et al.,

2010).

Changes in pigment concentration by effluent treatment affected the

carbohydrate content in a study conducted by Malla and Mohanty (2005) which

supports our observation.



93

TA

BL

E 1

9

CA

RB

OH

YD

RA

TE

CO

NT

EN

T (

mg

/g)

IN T

HE

LE

AV

ES

OF

Vigna radiata A

ND

Vigna MUNGO G

RO

WN

IN

R

ED

AN

D B

LA

CK

SO

IL U

SIN

G D

ILU

TE

D T

AN

NE

RY

EF

FL

UE

NT

Vigna radiata

Vigna mungo

Da

ys

aft

er

so

win

g (

DA

S)

So

il

Gro

up

s

30

6

0

90

3

0

60

9

0

CR

4

.86 ±

0.5

4

5.3

2 ±

0.2

5

6.2

1 ±

0.2

6

4.9

6 ±

0.8

5

5.7

3 ±

0.6

8

6.8

1 ±

0.8

6

T1

R

4.1

8 ±

0.5

5

4.9

2 ±

0.2

8

5.7

0 ±

0.3

3

4.8

2 ±

0.6

9

5.3

6 ±

0.6

2

5.8

6 ±

0.9

5

Re

d s

oil

T2

R

3.1

6 ±

0.3

6

3.7

0 ±

0.6

5

4.4

1 ±

0.4

6

3.4

5 ±

0.4

8

4.8

4 ±

0.3

4

5.1

4 ±

0.4

8

CB

4

.18 ±

0.4

2

4.9

0 ±

0.5

6

5.1

5 ±

0.9

5

3.9

0 ±

0.4

5

4.4

4 ±

0.3

4

5.8

7 ±

97

T1

B

3.7

8 ±

0.5

4

.07 ±

0.2

5

4.9

7 ±

0.8

8

3.7

1 ±

0.7

3

4.2

5 ±

0.8

5

5.6

5 ±

0.9

5

Bla

ck

s

oil

T2

B

2.0

3 ±

0.4

2

.95 ±

0.3

5

3.2

7 ±

0.2

8

2.9

3 ±

0.8

2

4.0

2 ±

0.2

9

5.3

5 ±

0.3

8

CD

(0

.05

)

0.0

1

0.0

1

V

alu

es a

re m

ean ±

SD

of

trip

licate

s

C

R-

Co

ntr

ol

with r

ed s

oil,

T1R

- R

ed s

oil

with 2

5%

eff

luent, T

2R

- R

ed

soil

with 5

0%

eff

luent,

CB

- C

ontr

ol

bla

ck s

oil,

T1B

- B

lack s

oil

with 2

5%

eff

luent, T

2B

- B

lack s

oil

with 5

0%

eff

luent

93



94

FIGURE 10

CARBOHYDRATE CONTENT IN THE LEAVES OF Vigna radiata

FIGURE 11

CARBOHYDRATE CONTENT IN THE LEAVES OF Vigna mungo

0

1

2

3

4

5

6

7


Carb

oh

yd

rate

co

nte

nt

(mg

/g)

CR T1R T2R

CB T1B T2B

0

1

2

3

4

5

6

7

8

9


Carb

oh

yd

rate

co

nte

nt

(mg

/g)

CR T1R T2R

CB T1B T2B



95

d) Protein

Table 20 shows the protein content in the leaves of Vigna radiata

(Figure 12) and Vigna mungo (Figure 13) grown in red and black soil using 25%


FIGURE 12

PROTEIN CONTENT IN THE LEAVES OF Vigna radiata

FIGURE 13

PROTEIN CONTENT IN THE LEAVES OF Vigna mungo

0

1

2

3

4

5

6


Pro

tein

co

nte

nt

(mg

/g)

CR T1R T2R

CB T1B T2B

0

1

2

3

4

5

6

7

8


Pro

tein

co

nte

nt

(mg

/g)

CR T1R T2R

CB T1B T2B



96

TA

BL

E 2

0

P

RO

TE

IN C

ON

TE

NT

(m

g/g

) IN

TH

E L

EA

VE

S O

F Vigna radiata A

ND

Vigna mungo G

RO

WN

IN

RE

D A

ND

B

LA

CK

SO

IL U

SIN

G D

ILU

TE

D T

AN

NE

RY

EF

FL

UE

NT

Vigna radiata

Vigna mungo

Da

ys

aft

er

so

win

g (

DA

S)

So

il

Gro

up

s

30

6

0

90

3

0

60

9

0

CR

3

.89 ±

0.3

5

4.7

5 ±

0.7

0

5.2

6 ±

0.3

8

4.9

6 ±

0.6

7

5.7

2 ±

0.6

5

6.8

2 ±

0.3

7

T1

R

3.1

1 ±

0.4

1

4.1

0 ±

0.6

0

4.8

5 ±

0.2

5

4.8

5 ±

0.8

9

5.6

4 ±

0.3

7

5.8

6 ±

0.6

7

Re

d s

oil

T2

R

2.0

6 ±

0.2

1

2.9

3 ±

0.6

5

3.2

4 ±

0.6

4

3.7

0 ±

0.2

5

5.2

0 ±

0.6

4

5.1

7 ±

0.4

7

CB

3

.15 ±

0.3

4

4.2

5 ±

0.5

3

4.9

1 ±

0.6

3

3.6

5 ±

0.6

8

4.6

2 ±

0.3

5

5.3

0 ±

0.2

8

T1

B

2.1

2 ±

0.8

8

3.8

8 ±

0.3

5

4.0

6 ±

0.3

4

3.4

8 ±

0.7

5

4.3

2 ±

0.6

2

5.2

1 ±

0.6

8

Bla

ck

s

oil

T2

B

1.8

2 ±

0.7

6

2.2

2 ±

0.4

4

2.9

4 ±

0.3

8

2.7

2 ±

0.2

2

3.9

0 ±

0.7

7

5.9

0 ±

0.9

7

CD

(0

.05

)

0.1

2

0.1

1

V

alu

es a

re m

ean ±

SD

of

trip

licate

s

CR

- C

on

trol

with

red s

oil,

T1R

- R

ed s

oil

with 2

5%

eff

luent, T

2R

- R

ed s

oil

with 5

0%

eff

luent, C

B-

Co

ntr

ol

bla

ck s

oil,

T1B

- B

lack s

oil

with

25%

eff

luent, T

2B

- B

lack s

oil

with 5

0%

eff

luent

96



97

Protein content in the leaves recorded significant reduction (p<0.05)

compared to the control plants. Similar results were obtained by Malla and

Mohanty (2005), who stated that there was a negative correlation between the

effluent treatment and biochemical parameters such as chlorophyll, protein and

sugar. Vajpayee et al. (2001) observed a reduction in protein content and an

inhibition of nitrogenase activity in Vallisneria spiralis plant due to the presence

of chromium in the effluent.

Leaf part of R.sativus and B.nigra showed lower protein content in waste

water irrigated samples. Guo et al. (2007) reported that stress induced a decline

in soluble protein contents in plants but increase in soluble sugar contents.

There was a considerable reduction in the level of protein, lipid and

carbohydrate content in the leaves of plant treated with various concentrations

of effluent in a study conducted by Azmat et al. (2010). Gill and Sago (2010)

reported reduction of carbohydrate and protein content in turnip plants due to

higher concentration of cadmium, chromium and lead.

Reduction in protein content may be due to degradation by proteases.

High salinity was found to decrease protein content. The tannery effluent rich in

chromium might have caused the stress and thereby reduced the protein

content in the leaves.

4.2.2.2. Biochemical parameters in the seeds of Vigna radiata and Vigna

mungo Total phenol, carotenoid, carbohydrate and protein contents were

analysed in the seeds of both the plants grown with the effluent.

1) Total phenol and carotenoid

Table 21 shows the total phenol (Figure 14) and carotenoid (Figure 15)

contents in the seeds of Vigna radiata and Vigna mungo grown in red and black

soil using 25% and 50% tannery effluent.



98

TA

BL

E 2

1

TO

TA

L P

HE

NO

L A

ND

CA

RO

TE

NO

ID C

ON

TE

NT

(m

g/g

) IN

TH

E S

EE

DS

OF

Vigna radiata A

ND

Vigna mungo

GR

OW

N I

N R

ED

AN

D B

LA

CK

SO

IL U

SIN

G D

ILU

TE

D T

AN

NE

RY

EF

FL

UE

NT

To

tal

ph

en

ol

Ca

rote

no

id

So

il

Gro

up

s

Vigna radiata

Vigna mungo

Vigna radiata

Vigna mungo

CR

1

2.9

5 ±

0.2

1

3.8

1 ±

0.4

0

.95 ±

0.0

1

1.1

3 ±

0.0

2

T1

R

12

.0 ±

0.1

1

3.2

2 ±

0.3

0

.90 ±

0.0

2

1.1

1 ±

0.0

2

Re

d s

oil

T2

R

11

.12

± 0

.1

12

.13

± 0

.2

0.8

8 ±

0.0

1

0.0

9 ±

0.0

1

CB

1

3.0

1 ±

0.3

1

3.9

2 ±

0.4

0

.88 ±

0.0

1

1.1

0 ±

0.0

2

T1

B

12

.33

± 0

.2

13

.33

± 0

.3

0.8

5 ±

0.0

2

1.0

8 ±

0.0

2

Bla

ck

so

il

T2

B

11

.83

± 0

.1

12

.23

± 0

.2

0.8

0 ±

0.0

1

1.0

5 ±

0.0

2

CD

( 0

.05

) 0

.27

0

.52

V

alu

es a

re m

ean ±

SD

of

trip

licate

s

C

R-

Co

ntr

ol

with r

ed s

oil,

T1R

- R

ed s

oil

with 2

5%

eff

luent, T

2R

- R

ed s

oil

with 5

0%

eff

luent,

CB

- C

ontr

ol

bla

ck s

oil,

T1B

- B

lack s

oil

with 2

5%

eff

luent, T

2B

- B

lack s

oil

with 5

0%

eff

luent

98



99

FIGURE 14

TOTAL PHENOL CONTENT IN THE SEEDS OF Vigna radiata AND Vigna mungo

FIGURE 15

CAROTENOID CONTENT THE IN SEEDS OF Vigna radiata AND Vigna mungo

0

2

4

6

8

10

12

14

16

18


To

tal p

hen

ol co

nte

nt

(mg

/g)

CR T1R T2R

CB T1B T2B

0

0.2

0.4

0.6

0.8

1

1.2

1.4


Caro

ten

oid

co

nte

nt

(mg

/g)

CR T1R T2R

CB T1B T2B



100

Total phenol content of the seeds of T1R plants of Vigna radiata and

Vigna mungo were 12 and 13.22 mg/g respectively. The T2R plants had

11.2 and 12.13 mg/g in Vigna radiata and Vigna mungo respectively. Thus there

was a significant decrease in total phenol content in seeds at (p<0.05) level.

Similar trend was observed in both the plants when grown with black soil.

A decrease in phenol content was observed in rice seedlings on

treatment with sewage sludge amendments in a study conducted by Singh and

Agrawal (2010).

The carotenoid content in the seeds of T1R of Vigna radiata and Vigna

mungo plants were found to be 0.90 and 1.11 mg/g respectively. T2R recorded

a value of 0.88 and 0.09 mg/g when grown in red soil whereas in black soil the

T1B and was found to be 0.85 and 1.08 mg/g and that of T2B was recorded

as 0.80 and 1.05 mg/g in Vigna radiata and Vigna mungo respectively. The

carotenoid contents in the seeds of Vigna radiata and Vigna mungo showed no

significant reduction when compared with control seeds.

According to the results of the study done by Okamoto et al. (2001), the

exposure to metals increase the activity of SOD and peroxidase but no

significant changes were detected in carotenoid contents of G. Polyedra which

was in agreement with our results.

b) Carbohydrate and protein

Table 22 shows the carbohydarate (Figure 16) and protein (Figure 17)

contents in the seeds of Vigna radiata and Vigna mungo grown in red and black

soil using 25% and 50% tannery effluent.

Carbohydrate and protein contents in the seeds of Vigna radiata and

Vigna mungo showed no significant reduction when grown in 25% and 50%

effluent.



101

T

AB

LE

22

CA

RB

OH

YD

AR

AT

E A

ND

PR

OT

EIN

CO

NT

EN

T (

mg

/g)

IN T

HE

SE

ED

S O

F Vigna radiata A

ND

Vigna mungo

GR

OW

N I

N R

ED

AN

D B

LA

CK

SO

IL U

SIN

G D

ILU

TE

D T

AN

NE

RY

EF

FL

UE

NT

Ca

rbo

hyd

rate

Pro

tein

So

il

Gro

up

s

Vigna radiata

Vigna mungo

Vigna radiata

Vigna mungo

CR

3

30

±1

2.5

7

32

1± 1

1.2

2

44

± 6

.45

2

34

± 5

.74

T1

R

32

6 ±1

0.3

3

15

± 1

0.3

2

24

0± 5

.05

2

31

± 6

.13

R

ed

so

il

T2

R

32

1 ±

9.6

5

31

0± 9

.48

2

37

± 6

.43

2

30

± 5

.39

CB

3

28

± 1

1.2

9

30

5± 1

0.6

6

24

1± 4

.26

2

33

± 6

.23

T1

B

32

0 ±

8.4

7

30

2± 8

.73

2

37

± 5

.68

2

30

± 4

.69

B

lac

k s

oil

T2

B

31

9 ±

9.6

3

30

1± 8

.86

2

35

± 5

.82

2

24

± 5

.0

CD

( 0

.05

) 1

4.3

4

10

.59

Valu

es a

re m

ean ±

SD

of

trip

lica

tes

C

R-

Contr

ol w

ith r

ed

so

il, T

1R

- R

ed s

oil

with 2

5%

eff

luent, T

2R

- R

ed

soil

with

50%

eff

luent, C

B-

Contr

ol bla

ck s

oil,

T1B

- B

lack s

oil

with

25%

eff

luent, T

2B

- B

lack s

oil

with 5

0%

eff

luent

101



102

FIGURE 16

CARBOHYDRATE CONTENT IN THE SEEDS OF Vigna radiata AND Vigna mungo

FIGURE 17

PROTEIN CONTENT IN THE SEEDS OF Vigna radiata AND Vigna mungo

290

300

310

320

330

340

350


Carb

oh

yd

rate

co

nte

nt

(mg

/g)

CR T1R T2R

CB T1B T2B

200

205

210

215

220

225

230

235

240

245

250

255


Pro

tein

co

nte

nt

(mg

/g)

CR T1R T2R

CB T1B T2B



103

The highest amount of protein content in Vigna mungo was recorded at

10% sago effluent concentration and the lowest content was recorded at 75%

(Sivaraman and Tamizhiniyan, 2005). The same trend was observed in paddy

seedling under tannery effluent treatment by Lakshmi and Sundaramurthy,

(2001).The increase in protein content at lower concentration of effluents could

be due to adsorption of most of the nitrogen by plants. The diluted effluent

reduces the salinity and enhances the plant growth.

c) Seed protein profile of Vigna radiata and Vigna mungo

Legume seeds have a high protein content compared to other

sources of plant proteins (Abou-El-Enain, 2002) and are highly stable and

unaffected by environmental conditions. Therefore electrphoretic technique for

analysis of total seed storage protein have been recognized as a valid method

(Emre et al., 2006; Tamilselvi, 2010).

Plates 5 and 6 show the polyacrylamide gel electrophoresis banding

pattern of the seed proteins of Vigna radiata and Vigna mungo respectively. The

banding patterns of seeds collected from control plants and the plants irrigated

with 25% and 50% effluent and grown in red soil and black soil are given in

lanes 2-7. The banding of protein marker of range 14.3 to 97.3 KDa is shown

in lane 1.

The storage protein profile of seeds of Vigna radiata (CR and CB)

showed 11 of bands with molecular weights 16.5 to 84.8 KDa, having 2 dense,

5 medium and 4 light bands. With respect to Vigna mungo (CR and CB) the total

bands were 12 with molecular weight ranging from 16.8 to 84.3 having 3 dense

5 medium and 4 light bands. The seeds of Vigna radiata experimental plants

namely T1R, T2R, T1B and T2B showed 10, 9, 8 and 7 bands respectively. The

molecular weight of T1R ranged from 16.5 to 84.8 KDa, while that of T2R

ranged from 19.3 to 69.1KDa. Similarly seeds of T1B ranged from 19.3 to 78.8

and T2B ranged from 29.2 to 69.1 KDa , which shows that the plants grown

with 50% effluent was found to have a stress on protein pattern.



104

PLATE 5

SEED PROTEIN PROFILE OF Vigna radiata

Lane 7 6 5 4 3 2 1

Lane 1 - Marker (M)

Lane 2 - Control red soil (CR)

Lane 3 - Red soil with 25% effluent (T1R)


Lane 5 - Control black soil (CB)

Lane 6 - Black soil with 25% effluent (T1B)




105

PLATE 6

SEED PROTEIN PROFILE OF Vigna mungo

Lane 7 6 5 4 3 2 1

Lane 1 - Marker (M)

Lane 2 - Control red soil (CR)



Lane 5 - Control black soil (CB)





106

The seeds of Vigna mungo experimental plants namely T1R, T2R, T1B

and T2B showed 12, 7, 10 and 7 bands respectively. The molecular weight of

T1R ranged from 16.8 to 84.3, while that of T2R ranged from 20.1 to 84.3 KDa .

Similarly seeds of T1B ranged from 18.3 to 84.3 KDa and T2B ranged from

29.8 to 84.3 KDa, which shows that the plants grown with 50% effluent was

found to have a stress on protein pattern. But the seeds of both the plants

grown with 25% effluent were not affected by the translocation of chemicals and

metals into the plants.

From the study it was found that the protein banding pattern was not

affected by growth with 25 % diluted tannery effluent however a differential

banding pattern was observed in the seeds of the plants grown with 50%

effluent.

According to Danimihardja and Lester (1974) it is genetically determined

that development of seeds always get priority over all physiological processes in

any plant. Hence, Vigna radiata and Vigna mungo plants were found to be

tolerant to contaminations, and the seed proteins were also found to be

unaltered in our study.

It could be derived from our study that the seeds of the plants grown with

25% tannery effluent did not show any modification and those grown with 50%

effluent showed a slight alteration in their molecular weights compared to the

control plants.

4.2.2.3 Enzymic and non enzymic antioxidants in the leaves and seeds of

Vigna radiata and Vigna mungo

Plants are rich sources of natural antioxidants that play a vital role in the

prevention or progression of the degenerative diseases. The consumption of

fruits, vegetables and herbs rich in antioxidants is associated with a decline in

the incidence of degenerative diseases and cancer (Harish et al., 2005).



107

Plants possess two very efficient antioxidant defense systems:

the enzymic which includes catalase, peroxidase, superoxide dismutase,

polyphenol oxidase, glutathione reductase and the non enzymic antioxidant

defense systems such as ascorbic acid, glutathione, tocopherol and

carotenoids. Both allow scavenging of reactive oxygen species leading to

protection of plant cells from oxidative damage (Blokhina et al., 2003;

Gratao et al., 2005). Indeed, activities of antioxidant enzymes have been

detected in various cellular organelles of various plant species. These

antioxidant enzymes were found in various compartments of the plant leaf cell,

eg: superoxide dismutase (SOD) found in chloroplasts (Mittova et al., 2000). It is

found that people who eat fruits and vegetables rich in polyphenols

and anthocyanins have a lower risk of cancer, heart disease and some

neurological diseases (Stanner et al., 2004). Antioxidants can cancel out the cell

damaging effects of free radicals. These compounds might prevent conditions

such as macular degeneration, suppressed immunity to poor nutrition and

neurodegeneration which is caused by oxidative stress (Wang et al., 2005).

The term antioxidant originally was used to refer to a chemical that

prevented the consumption of oxygen. Research into how vitamin E prevents

the process of lipid peroxidation led to the identification of antioxidants as

reducing agents that prevent oxidative reactions, often by scavenging reactive

oxygen species before they can damage cells (Wolf, 2005).

Heavy metals induce oxidative stress by generating free radicals and

toxic reactive oxygen species. These species react with lipids, proteins,

pigments and nucleic acids and cause lipid peroxidation, membrane damage

and inactivation of enzymes, thus affecting the cell viability. The deleterious

effects resulting from the cellular oxidative state may be alleviated by the

enzymatic and non enzymatic antioxidant machinery of the plant (Mittler 2002;

Sharma and Agrawal, 2005).

In the present study certain enzymic and non enzymic antioxidants were

analysed in the leaves and seeds of both the plants grown with the effluent.



108

Enzymic antioxidants

The enzymic antioxidants and free radical scavengers may provide a

defensive mechanism against the deleterious actions of reactive oxygen species

(ROS). Some of the antioxidant enzymes that are found to provide protection

against the ROS are superoxide dismutase, catalase, peroxidase, glutathione

reductase and ascorbate oxidase (Rani et al.,2004). Free radicals are implicated

in several degenerative diseases such as arteriosclerosis, diabetes, arthritis,

cancer and aging. The harmful effects of free radicals on living systems could

be attenuated by antioxidants that scavenge the free radicals. Uptake of any

excess heavy metal induces a deficiency of essential nutrients affecting the

cationic balance at subcellular level altering certain enzymes (Sharma and

Chettri, 2008).

The deleterious effects from the cellular oxidative state may be alleviated

by the enzymic and non enzymic antioxidant machinery of the plant. The

antioxidants of legume leaves and seeds have been examined in considerable

detail (Matamoras et al., 2003; Palma et al., 2006). The enzymic antioxidants

analysed in the seeds and leaves of Vigna radiata and Vigna mungo plants

grown in red and black soils using 25% and 50% tannery effluent were catalase,

peroxidase, super oxide dismutase and glutathione reductase.

a.) Catalase and Peroxidase

Catalases are enzymes that catalyze the conversion of hydrogen

peroxide to water and oxygen using either iron or manganese as cofactor

(Chelikani et al., 2004) and an enzymic antioxidant which protects the tissue

from highly reactive hydroxyl radical (Himer et al., 2002; Dash et al., 2007)

Peroxidases are widely distributed in plant tissues and are of immense

physiological interest because of their association with numerous catalytic

functions. Numerous functions have been proposed and the most important

among these are the ability to oxidize indole-3-acetic acid, ethylene

biosynthesis, hydroxylation of proline, lignifications and disease resistance

(Hiraga et al., 2001).



109

TA

BL

E 2

3

CA

TA

LA

SE

AN

D P

ER

OX

IDA

SE

AC

TIV

ITIE

S I

N T

HE

LE

AV

ES

AN

D S

EE

DS

OF

Vigna radiata A

ND

Vigna mungo

GR

OW

N I

N R

ED

AN

D B

LA

CK

SO

IL U

SIN

G D

ILU

TE

D T

AN

NE

RY

EF

FL

UE

NT

Ca

tala

se

(U

nit

s/g

) P

ero

xid

as

e (

Un

its

/g

)

Le

ave

s

Se

ed

s

Le

ave

s

Se

ed

s

G

rou

ps

Vigna

radiata

Vigna

mungo

Vigna

radiata

Vigna

mungo

Vigna

radiata

Vigna

mungo

Vigna

radiata

Vigna

mungo

CR

2

.01 ±

0.0

5

1.9

8 ±

0.0

8

1.3

2 ±

0.0

8

1.1

8 ±

0.0

8

0.1

5±0

.03

0

.06

±0

.01

0

.13

±0

.02

0

.11

±0

.03

T1

R

1.9

2 ±

0.0

3

1.7

8 ±

0.0

5

1.2

2 ±

0.1

1

.08 ±

0.6

0

.19

±0

.01

0

.07

±0

.01

0

.15

±0

.05

0

.16

±0

.04

T2

R

1.7

8 ±

0.0

4

0.8

2 ±

0.0

3

0.0

7 ±

0.0

1

0.0

9 ±

0.0

2

0.2

1±0

.02

0

.08

±0

.02

0

.17

±0

.03

0

.17

±0

.05

CB

2

.16 ±

0.0

9

2.0

1 ±

0.0

7

1.4

8 ±

0.0

1

1.3

2 ±

0.0

6

0.2

2±0

.03

0

.11

±0

.02

0

.13

±0

.02

0

.12

±0

.04

T1

B

2.0

1 ±

0.1

0

1.9

4 ±

0.1

1

1.3

2 ±

0.1

4

0.2

1 ±

0.0

3

0.2

8±0

.04

0

.12

±0

.02

0

.17

±0

.01

0

.16

±0

.03

T2

B

1.8

1 ±

0.2

1

1.0

4 ±

0.0

8

1.0

2 ±

0.5

0

.98 ±

0.2

3

0.3

1±0

.02

0

.13

±0

.05

0

.18

±0

.01

0

.17

±0

.01

CD

( 0

.05

) 0

.21

0

.29

0

.06

0

.04

V

alu

es a

re m

ean ±

SD

of

trip

lica

tes

Cata

lase u

nits -

am

ount of

en

zym

e r

eq

uire

d to

decre

ase the o

ptical density b

y 0

.05 u

nits.

Pero

xid

ase u

nits –

cha

nge in a

bsorb

ance a

t 450

nm

/ m

in.

CR

- C

ontr

ol

with r

ed s

oil,

T1R

- R

ed s

oil

with 2

5%

eff

luent, T

2R

- R

ed s

oil

with 5

0%

eff

luent, C

B-

Con

trol

bla

ck s

oil,

T1B

- B

lack s

oil

with 2

5%

eff

luent, T

2B

- B

lack s

oil

with 5

0%

eff

luent

109



110

Table 23 shows the catalase (Figure 18) and peroxidase (Figure 19)

activities in the leaves and seeds of Vigna radiata and Vigna mungo grown in

red and black soil using 25% and 50% tannery effluent.

FIGURE 18

CATALASE ACTIVITIES IN THE LEAVES AND SEEDS OF Vigna radiata AND Vigna mungo

FIGURE 19

PEROXIDASE ACTIVITIES IN THE LEAVES AND SEEDS OF Vigna radiata AND Vigna mungo

0

0.5

1

1.5

2

2.5

Vigna radiata Vigna mungo Vigna radiata Vigna mungo

Leaves Seeds

Un

its / g

CR T1R T2R

CB T1B T2B

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4


Leaves Seeds

Un

its

/ g

CR T1R T2R

CB T1B T2B



111

The catalase activity was significantly reduced in the leaves of Vigna

radiata and Vigna mungo when grown with 50% effluent. Seeds also recorded

significant reduction in the catalase activity when both the plants were grown

with 50% effluent.

Peroxidase activity of the leaves and seeds of Vigna radiata showed

significant increase on growth with both the types of effluents. Vigna mungo

plants were found to exhibit increased activities of peroxidase in both leaves

and seeds compared to the control plants.

Singh and Agrawal (2007) observed an increase in the activity of

peroxidase in root, stem and leaf of Beta vulgaris plants grown in sewage

sludge amended pots. Toxic doses of zinc, uranium and cadmium inhibited

shoot growth but increased the glucose 6 phosohate dehydrogenase activity

and peroxidase activity in the leaves of dwarf beans (Vandenhove et al., 2006).

Many authors have shown a concomitant increase in the peroxidase

activity in plants after prolonged exposure to high cadmium concentration

(Tamas et al., 2003; Chaoui et al., 2004; Saffar et al., 2009).

b) Superoxide dismutase and Glutathione reductase Superoxide dismutase (SOD) enzymes are present in all aerobic cells

and in extra cellular fluids (Johnson and Giulivi, 2005). This contains metal ion

cofactors like zinc, manganese or iron. In plants SOD isozymes are present in

the cytosol and mitochondria, with an iron SOD found in chloroplast which is not

present in vertebrates and yeast. SOD catalyzes the dismutation of superoxide

into oxygen and hydrogen peroxide (Alscher et al., 2003).

Glutathione reductase is ubiquitous NADPH dependant enzyme which

catalyses the reduction of oxidized glutathione present in cells. It has been well

documented that the activity of glutathione reductase altered due to metal stress

(Schutzendubel and Polle, 2002; Meloni et al., 2003).



112

TA

BL

E 2

4

SU

PE

RO

XID

E D

ISM

UT

AS

E A

ND

GL

UT

AT

HIO

NE

RE

DU

CT

AS

E A

CT

IVIT

IES

IN

TH

E L

EA

VE

S A

ND

SE

ED

S O

F

Vigna radiata A

ND

Vigna mungo G

RO

WN

IN

RE

D A

ND

BL

AC

K S

OIL

US

ING

D

ILU

TE

D T

AN

NE

RY

EF

FL

UE

NT

Su

pe

rox

ide d

ism

uta

se

(U

nit

s/g

) G

luta

thio

ne

re

du

cta

se

(U

nit

s /

g)

Le

ave

s

Se

ed

s

Le

ave

s

Se

ed

s

Gro

up

s

Vigna

radiata

Vigna

mungo

Vigna

radiata

Vigna

mungo

Vigna

radiata

Vigna

mungo

Vigna

radiata

Vigna

mungo

CR

7

.2 ±

0.1

2

6.0

1 ±

0.1

5

7.8

± 0

.19

6

.51 ±

0.2

1

1.8

±0

.29

1

.0 ±

0.2

7

1.3

±0

.55

1

.5 ±0

.30

T1

R

8.8

±0

.08

7

.62 ±

0.1

4

8.5

±0

.18

7

.01 ±

0.1

9

2.3

±0

.28

2

.21 ±

0.3

6

2.2

±0

.21

2

.0 ±

0.5

1

T2

R

9.0

2 ±

0.8

2

8.7

1 ±

0.1

6

9.7

1 ±

0.1

1

8.2

1 ±

0.1

8

3.0

±0

.26

2

.9 ±

0.2

2

2.9

±0

.36

2

.4 ±

1.6

1

CB

5

.41 ±

0.2

3

5.2

2 ±

0.1

3

5.8

±0

.13

5

.73 ±

0.2

0

1.0

±0

.35

0

.92 ±

0.2

6

1.2

±0

.34

1

.4 ±

1.5

2

T1

B

6.1

1 ±

0.2

4

6.8

±0

.11

6

.5 ±

0.1

7

6.2

±0

.20

2

.82 ±

0.3

8

1.1

3 ±

0.2

1

2.0

±0

.23

1

.9 ±

1.2

4

T2

B

7.2

2 ±

0.5

6

7.2

±0

.10

7

.91 ±

0.0

9

7.5

±0

.23

3

.2 ±

0.2

8

2.1

±0

.23

2

.8 ±

1.1

0

2.5

±1

.16

CD

( 0

.05

) 1

.08

1

.12

1

.4

0.9

8

V

alu

es a

re m

ean ±

SD

of

trip

licate

s

S

OD

- U

nit-

the a

mount th

at causes 5

0%

red

uctio

n in

the e

xte

nt

of

NB

T o

xid

atio

n.

G

luta

thio

ne r

ed

ucta

se-

Un

it-

µm

ole

s o

f glu

tath

ion

e c

onsum

ed /m

in/g

of

the s

am

ple

C

R-

Contr

ol

with r

ed s

oil,

T1R

- R

ed s

oil

with 2

5%

eff

luent, T

2R

- R

ed s

oil

with 5

0%

eff

luent, C

B-

Co

ntr

ol

bla

ck s

oil,

T1B

- B

lack s

oil

with 2

5%

eff

luent, T

2B

- B

lack s

oil

with 5

0%

eff

luent

112



113

Table 24 shows the superoxide dismutase (Figure 20) and glutathione

reductase (Figure 21) activities in the leaves and seeds of Vigna radiata and

Vigna mungo grown in red and black soil using 25% and 50% tannery effluent.

FIGURE 20

SUPEROXIDE DISMUTASE ACTIVITIES IN THE LEAVES AND SEEDS OF Vigna radiata AND Vigna mungo

FIGURE 21

GLUTATHIONE REDUCTASE ACTIVITIES IN THE LEAVES AND SEEDS OF Vigna radiata AND Vigna mungo

0

2

4

6

8

10

12


Leaves Seeds

Un

its / g

CR T1R T2R

CB T1B T2B

0

0.5

1

1.5

2

2.5

3

3.5

4


Leaves Seeds

Un

its / g

CR T1R T2R

CB T1B T2B



114

There was significant increase in the SOD activities in the leaves of

Vigna radiata and Vigna mungo plants on treatment with effluents. Seeds of

both the plants showed a significant increase in SOD activities on treatment with

50% effluent.

The high SOD activity indicates that this plant can efficiently detoxify toxic

superoxide radicals produced by the accumulated chromium and it has been

associated with stress tolerance in plants because it neutralizes the reactivity of

oxygen. It has been well documented that SOD activity has a protective role in

heavy metal accumulated plants (Gratao et al., 2005 ; Labra et al., 2006).

Glutathione reductase activity in the leaves of Vigna radiata and Vigna

mungo showed a significant increase when the concentration of the effluent was

increased correspondingly. Seeds of plants grown with 50% effluent showed

significant increase of glutathione reductase activity compared to that of control

plants. Increase in glutathione reductase activity might be due to the result of

reaction of metal with sulfhydral groups.

A similar study conducted by Diwan et al. (2010a) reported that SOD

activity of Vigna radiata was increased 7- folds on chromium exposure and

glutathione reductase increased significantly over untreated plants.

Non enzymic antioxidants

The non enzymic antioxidants analysed in the leaves and seeds of Vigna

radiata and Vigna mungo grown in red and black soils using 25% and 50%

tannery effluent were ascorbic acid, riboflavin, tocopherol and flavonoids.

a) Ascorbic acid and riboflavin

Ascorbic acid has antioxidant activity and it reduces oxidizing

substances such as hydrogen peroxide (Duarte and Lunec, 2005). It can also

reduce metal ions which lead to the generation of free radicals through the

Fenton reaction (Valko et al., 2005).



115

T

AB

LE

25

AS

CO

RB

IC A

CID

AN

D R

IBO

FL

AV

IN C

ON

TE

NT

(m

g/g

) IN

TH

E L

EA

VE

S A

ND

SE

ED

S O

F Vigna radiata

AN

D Vigna mungo G

RO

WN

IN

RE

D A

ND

BL

AC

K S

OIL

US

ING

DIL

UT

ED

TA

NN

ER

Y E

FF

LU

EN

T

As

co

rbic

ac

id

Rib

ofl

avin

co

nte

nts

Le

ave

s

Se

ed

s

Le

ave

s

Se

ed

s

G

rou

ps

Vigna

radiata

Vigna

mungo

Vigna

radiata

Vigna

mungo

Vigna

radiata

Vigna

mungo

Vigna

radiata

Vigna

mungo

CR

7

.2 ±

0.2

1

6.0

1 ±

0.3

7

.8 ±

0.1

6

.51 ±

0.2

1

.8 ± 0

.05

1

.0 ±

0.0

9

1.3

± 0

.03

1

.5 ± 1

.03

T1

R

8.8

± 0

.23

7

.62 ±

0.2

8

.5 ±

0.2

7

.01 ±

0.3

2

.3 ±

0.0

6

2.2

1 ±

0.0

5

2.2

± 0

.06

2

.0 ±

1.0

5

T2

R

9.0

2 ±

0.1

3

8.7

1 ±

0.4

9

.71 ±

0.3

8

.21 ±

0.4

3

.0 ±

0.0

7

2.9

± 0

.04

2

.9 ±

0.0

5

2.4

± 1

.05

CB

5

.41 ±

0.3

0

5.2

2 ±

0.6

5

.8 ±

0.6

5

.73 ±

0.4

1

.0 ±

0.0

3

0.9

2 ±

0.0

2

1.2

± 0

.02

1

.4 ±

1.0

4

T1

B

6.1

1 ±

0.5

1

6.8

± 0

.7

6.5

± 0

.7

6.2

± 0

.6

2.8

2 ±

0.0

2

1.1

3 ±

0.0

7

2.0

± 0

.05

1

.9 ±

1.0

8

T2

B

7.2

2 ±

0.7

4

7.2

± 0

.8

7.9

1 ±

0.4

7

.5 ±

0.3

3

.2 ±

0.0

3

2.1

± 0

.06

2

.8 ±

1.0

4

2.5

± 1

.09

CD

( 0

.05

) 2

.1

2.2

4

0.4

0

.32

V

alu

es a

re m

ean ±

SD

of

trip

lica

tes

CR

- C

ontr

ol

with r

ed s

oil,

T1R

- R

ed s

oil

with 2

5%

eff

luent, T

2R

- R

ed s

oil

with 5

0%

eff

luent, C

B-

Con

trol

bla

ck s

oil,

T1B

- B

lack s

oil

with 2

5%

eff

luent, T

2B

- B

lack s

oil

with 5

0%

eff

luent

115



116

In cells ascorbic acid is maintained in its reduced form by reaction with

glutathione, which can be catalyzed by protein disulphide isomerase and

glutaredoxins. Ascorbic acid is a reducing agent that can reduce and thereby

neutralize oxygen species such as hydrogen peroxide (Padayatty et al., 2003).

In addition to its direct antioxidant effects, ascorbic acid is also a substrate for

the antioxidant enzyme ascorbate peroxidase, a function that is particularly

important in stress resistance in plants (Shigeoka et al., 2002).

Table 25 shows the ascorbic acid (Figure 22) and riboflavin (Figure 23)

contents in the leaves and seeds of Vigna radiata and Vigna mungo grown in

red and black soil using 25 % and 50 % tannery effluent.

Ascorbic acid content in the leaves of both the plants reported a

significant increase when grown with the effluent whereas in seeds,

the significant increase was noted only with 50% effluent. According to

Guo et al. (2005) ascorbic acid was found to increase in all waste water

irrigated plants than control.

FIGURE 22

ASCORBIC ACID CONTENT IN THE LEAVES AND SEEDS OF Vigna radiata AND Vigna mungo

0

2

4

6

8

10

12


Leaves Seeds

Asc

orb

ic a

cid

(m

g/g

)

CR T1R T2R

CB T1B T2B



117

FIGURE 23

RIBOFLAVIN CONTENT IN THE LEAVES AND SEEDS OF Vigna radiata AND Vigna mungo

Gupta et al. (2010) reported that heavy metal accumulation showed a

varied response in plant species and also increases the ascorbic acid contents.

In a study conducted by Singh and Agrawal (2009), application of sewage

sludge increased the ascorbic acid contents in Abelmochus esculentus plants.

The riboflavin content in the leaves and seeds Vigna radiata showed

a significant increase in plants grown with 25 and 50% effluent. Similar

observation was noticed in the leaves and seeds of Vigna mungo plants.

b) Flavonoid and tocopherol

Table 26 shows the flavonoid (Figure 24) and tocopherol (Figure 25)

contents in the leaves and seeds of Vigna radiata and Vigna mungo grown in

red and black soil using 25 % and 50 % tannery effluent.

0

0.5

1

1.5

2

2.5

3

3.5


Leaves Seeds

Rib

ofl

avin

(m

g/g

)

CR T1R T2R

CB T1B T2B



118

TA

BL

E 2

6

FL

AV

ON

OID

(µ

g/g

) A

ND

TO

CO

PH

ER

OL

(m

g/g

) C

ON

TE

NT

IN

TH

E L

EA

VE

S A

ND

SE

ED

S O

F Vigna radiata A

ND

Vigna mungo GROWN

IN

RE

D A

ND

BL

AC

K S

OIL

US

ING

DIL

UT

ED

TA

NN

ER

Y E

FF

LU

EN

T

Fla

vo

no

id

To

co

ph

ero

l

Le

ave

s

Se

ed

s

Le

ave

s

Se

ed

s

G

rou

ps

Vigna

radiata

Vigna

mungo

Vigna

radiata

Vigna

mungo

Vigna

radiata

Vigna

mungo

Vigna

radiata

Vigna

mungo

CR

1

62

± 2

.1

12

0 ±

1.9

1

48

± 2

.6

10

5 ±

2.9

1

.9 ±

0.0

8

1.7

± 0

.08

0

.73 ±

0.0

4

0.6

3 ±

0.0

8

T1

R

19

2 ±

2.8

1

49

± 1

.8

16

2 ±

2.8

1

32

± 2

.15

1

.93 ±

0.0

2

1.9

1 ±

0.0

9

1.9

8 ±

0.0

5

0.8

5 ±

0.0

5

T2

R

20

9 ±

3.1

1

72

± 1

.4

19

4 ±

2.7

1

66

± 2

.16

2

.2 ±

0.0

4

2.0

± 0

.06

2

.14 ±

0.0

6

1.9

4 ±

0.0

6

CB

1

70

± 2

.9

13

5 ±

1.6

1

55

± 2

.8

11

4 ±

2.3

2

1.9

8 ±

0.0

3

1.8

4 ±

0.0

5

0.8

4 ±

0.0

7

0.7

2 ±

0.0

9

T1

B

19

9 ±

2.7

1

56

± 1

.7

17

6 ±

3.1

1

38

± 2

.17

2

.11 ±

0.0

3

2.0

3 ±

0.0

4

1.0

2 ±

0.0

9

1.9

2 ±

0.0

3

T2

B

21

3 ±

3.0

1

83

± 1

.6

19

9 ±

3.6

1

75

± 2

.32

2

.4 ±

0.0

4

2.1

2 ±

0.0

6

2.3

2 ±

0.0

8

2.0

4 ±

0.0

2

CD

( 0

.05

) 4

.8

4.9

8

1.7

1

.1

V

alu

es a

re m

ean ±

SD

of

trip

licate

s

C

R-

Contr

ol

with r

ed s

oil,

T1R

- R

ed s

oil

with 2

5%

eff

luent, T

2R

- R

ed s

oil

with 5

0%

eff

luent, C

B-

Co

ntr

ol

bla

ck s

oil,

T1B

- B

lack s

oil

with 2

5%

eff

luent, T

2B

- B

lack s

oil

with 5

0%

eff

luent

118



119

FIGURE 24

FLAVONOID CONTENT IN THE LEAVES AND SEEDS OF Vigna radiata AND Vigna mungo

FIGURE 25

TOCOPHEROL CONTENT IN THE LEAVES AND SEEDS OF Vigna radiata AND Vigna mungo

0

50

100

150

200

250


Leaves Seeds

Fla

vo

no

id (µµ µµ

g/g

)

CR T1R T2R

CB T1B T2B

0

0.5

1

1.5

2

2.5

3


Leaves Seeds

To

co

ph

ero

l (m

g/g

)

CR T1R T2R

CB T1B T2B



120

Dietary flavonoid makes an important contribution to health and

cardiovascular system (Lazarus et al., 2000). Flavonoids are secondary

metabolites and functions as chelators for metals (Korkina, 2007). It has a vast

array of functions in plants including antioxidant activity (Havsteen, 2002).

Tocopherol is the most important lipid soluble antioxidant and it protects

membranes from oxidation by reacting with lipid radicals produced in the lipid

peroxidation chain reaction (Traber and Atkinson, 2007). This removes the free

radicals intermediates and prevents the propagation reaction. The oxidized

α–tocopherol radicals produced in this process may be recycled back to the

active reduced form through reduction by ascorbate, retinol and ubiquinol

(Wang and Quinn, 1999).

Flavonoid content in the leaves and seeds of both the plants had a

significant increase (p<0.05) when grown with both effluent concentrations than

that of control plants. Keilig and Muller (2009) observed an increase in flavonoid

content in Arabidopsis thaliana on growth with different concentrations of

cadmium and zinc.

The tocopherol content of the T1R, T2R, T1B and T2B plants showed no

significant difference on comparison with control plants. Tocopherol content of

the seeds of T2R and T2B plants recorded a significant increase.

According to Gajewska and Sklodowska (2007), the tocopherol content

increased in shoots of wheat seedlings on treatment with nickel.

Menach et al. (2004), reported that leguminous plants are rich sources of

flavonoids. Flavonoids are postulated to play a pivotal role in adaptation of

producer legumes to their biological environments both as defensive

compounds and chemical signals in symbiotic nitrogen fixation with rhizobia

(Aoki et al., 2000)



121

4.2.2.4. Metal contents in various parts of Vigna radiata and Vigna mungo plants

The use of industrial effluents carrying a high load of heavy metals such

as zinc, chromium and nickel for irrigation of crops produces adverse effect on

plant growth (Pandey et al., 2008). Essential heavy metals like iron, manganese,

zinc and copper in all higher plants were found to be absorbed and accumulated

in plant tissues based on their solubility, concentration and availability of

different ions in the soil (Sharma and Chettri, 2008).

Accumulation and exclusion are two basic strategies by which plants

respond to elevated concentration of heavy metals. It is known that some plants

can survive quite well under elevated metal conditions without the hyper

accumulation characteristic. These plants appear to tolerate metals in the

environment by using a variety of mechanism including exudation of compounds

that complex metals in the contaminated environment, thereby preventing their

entry into the root (Vogel-Mikus et al., 2005).

Chromium

Chromium is known to be highly toxic to biological systems. The

morphological growth parameters like germination percentage, root length,

shoot length, fresh weight and dry weight of black gram seedling were

decreased when chromium concentration was increased (Chidambaram et al.,

2009).

Tables 27 and 28 show accumulation of chromium in roots, shoots,

leaves and seeds of the plants Vigna radiata (Figure 26) and Vigna mungo

(Figure 27) grown in red soil and black soil using 25% and 50% tannery effluent.

In 25 % effluent, the chromium content in the roots of both the plants

were found to be 4.8 mg/g and 4.6 mg/g respectively. The chromium content

was decreased to 3.84 mg/g and 2.1 mg/g in shoots which indicates the upward

translocation of chromium to shoots.



122

T

AB

LE

27

CH

RO

MIU

M C

ON

TE

NT

(m

g/g

) IN

VA

RIO

US

PA

RT

S O

F Vigna radiata G

RO

WN

IN

RE

D S

OIL

AN

D

BL

AC

K S

OIL

US

ING

DIL

UT

ED

EF

FL

UE

NT

RO

OT

S

SH

OO

TS

L

EA

VE

S

SO

IL

C

T1

T

2

C

T1

T

2

C

T1

T

2

Re

d s

oil

0

.3 ±

0.0

1

4.8

± 0

.89

5

.4 ±

1.0

4

0.2

± 0

.01

3

.84 ±

0.5

2 4

.93 ±

0.7

5

0.1

± 0

.01

2

.53 ±

0.3

5

3.2

1 ±

0.3

9

Bla

ck

s

oil

0

.4 ±

0.0

1

4.9

± 0

.92

5

.9 ±

0.9

7

0.2

± 0

.01

3

.97 ±

0.6

3 5

.35 ±

0.9

8

0.2

± 0

.01

2

.86 ±

0.4

1

3.9

2 ±

0.4

7

CD

(0

.05

) 0

.063

0

.046

0

.53

Valu

es a

re m

ean ±

SD

of

trip

lica

tes

C-

Contr

ol soil

, T

1-

Soil

with 2

5%

eff

luent, T

2 -

So

il w

ith 5

0%

eff

luent.

See

ds : N

ot

dete

cta

ble

122



123

TA

BL

E 2

8

C

HR

OM

IUM

CO

NT

EN

T (

mg

/g)

IN V

AR

IOU

S P

AR

TS

OF

Vigna mungo G

RO

WN

IN

RE

D S

OIL

AN

D B

LA

CK

SO

IL U

SIN

G D

ILU

TE

D E

FF

LU

EN

T

RO

OT

S

SH

OO

TS

L

EA

VE

S

C

T

1

T2

C

T

1

T2

C

T

1

T2

Re

d s

oil

0

.6 ±

0.0

3

4.6

± 0

.8

5.3

± 0

.7

0.4

± 0

.03

2

.1 ±

0.0

3

2.9

± 0

.4

0.2

± 0

.03

1

.43 ±

0.0

2

1.5

3 ±

0.2

Bla

ck

s

oil

0

.9 ±

0.0

3

4.5

± 0

.6

6.3

2 ±

0.7

0

.5 ±

0.0

2

2.5

4 ±

0.3

4

.1 ±

0.4

0

.2 ±

0.0

3

2.2

± 0

.5

3.1

2 ±

0.4

CD

(0

.05

) 0

.063

0

.046

0

.53

Valu

es a

re m

ean ±

SD

of

trip

lica

tes

C-

Contr

ol soil

, T

1-

Soil

with 2

5%

eff

luent, T

2 -

So

il w

ith 5

0%

eff

luent.

S

ee

ds : N

ot

dete

cta

ble

123



124

FIGURE 26

CHROMIUM CONTENT IN VARIOUS PARTS OF Vigna radiata

FIGURE 27

CHROMIUM CONTENT IN VARIOUS PARTS OF Vigna mungo

0

1

2

3

4

5

6

7

C T1 T2 C T1 T2

mg

/ g

Root Shoot Leaf

0

1

2

3

4

5

6

7

8

C T1 T2 C T1 T2

mg / g

Root Shoot Leaf

Red soil Black soil

Red soil Black soil



125

Leaves of both plants recorded a value of 2.53 mg/g and 1.43 mg/g

respectively whereas chromium was not detected in grains. The same trend was

noted when the plants were grown with 50% effluent except that seed contained

negligible amounts of chromium. Calheiros et al. (2008a) reported the effect of

tannery waste water in the development of P.australis as the accumulation

of the metal in the plant was found to be in the following decreasing order

root > shoot> leaf. The concentration of chromium in the leaves, shoots and

roots increased with the concentration applied. According to Weis and Weis

(2004) the degree of upward translocation depends upon the plant species,

metal and several environmental conditions.

Several workers have documented high levels of chromium accumulation

in roots than in the top portions of the plant namely pod, leaves and stem which

was in agreement with our result (Ghosh and Singh, 2005 ; Yang et al., 2003).

Nickel

It is the essential heavy metal for plant growth and development. Under

normal conditions plants take up small quantities of nickel from soils. It can be

toxic to plants when its concentration in the soil is high. In plants under nickel

stress, the absorption of nutrients, root development and metabolism are

strongly retarded. Along with the toxicity symptoms in plants that develop later,

plant tissues are known to inhibit photosynthesis and transpiration (Zarkovic and

Blagojevic, 2009).

Tables 29 and 30 show the accumulation of nickel in roots, shoots,

leaves and seeds of the plants Vigna radiata and Vigna mungo grown in red soil

and black soil using 25% and 50% tannery effluent.

In Vigna radiata plants grown using 25 and 50% effluent, the nickel

content was found to be higher in roots compared to other parts of the plant The

value in grains was negligible. Similar trend was followed in Vigna mungo plants

also.



126

T

AB

LE

29

NIC

KE

L C

ON

TE

NT

(m

g/g

) I

N V

AR

IOU

S P

AR

TS

OF

Vigna radiata G

RO

WN

IN

RE

D S

OIL

AN

D

BL

AC

K S

OIL

US

ING

DIL

UT

ED

EF

FL

UE

NT

RO

OT

S

SH

OO

TS

L

EA

VE

S

So

il

C

T1

T

2

C

T1

T

2

C

T1

T

2

Re

d s

oil

0

.6 ±

0.0

2 3

.01 ±

0.4

5

3.7

± 0

.56

0

.32 ±

0.0

1

2.1

± 0

.44

2

.9 ±

0.5

3

0.1

4 ±

0.0

4 1

.17 ±

0.2

5

2.5

8 ±

0.4

1

Bla

ck

s

oil

0

.9 ±

0.0

3

3.3

± 0

.61

3

.9 ±

0.7

2

0.0

7 ±

0.0

3

2.2

± 0

.34

3

.1 ±

0.3

9

0.3

± 0

.02

1

.65 ±

0.2

4

2.6

8 ±

0.2

9

CD

(0

.05

) 0

.85

0

.76

0

.37

Valu

es a

re m

ean ±

SD

of

trip

lica

tes

C-

Contr

ol soil

, T

1-

Soil

with 2

5%

eff

luent, T

2 -

So

il w

ith 5

0%

eff

luent.

S

ee

ds : N

ot

dete

cta

ble

126



127

T

AB

LE

30

NIC

KE

L C

ON

TE

NT

(m

g/g

) IN

VA

RIO

US

PA

RT

S O

F Vigna mungo G

RO

WN

IN

RE

D S

OIL

AN

D

BL

AC

K S

OIL

US

ING

DIL

UT

ED

EF

FL

UE

NT

RO

OT

S

SH

OO

TS

L

EA

VE

S

So

il

C

T1

T

2

C

T1

T

2

C

T1

T

2

Re

d s

oil

0

.5 ±

0.0

3

3.2

± 0

.24

3

.5 ±

0.2

7

0.3

± 0

.02

2

.9 ±

0.1

8

2.7

± 0

.21

0

.1 ±

0.0

3

1.5

4 ±

0.2

1

.68 ±

0.3

Bla

ck

s

oil

0

.8 ±

0.0

4

2.5

± 0

.21

3

.0 ±

0.2

3

0.6

± 0

.02

1

.9 ±

0.1

5

2.4

± 0

.22

0

.5 ±

0.1

1

1.4

8 ±

0.3

0

1.9

5 ±

0.4

CD

(0

.05

) 0

.34

0

.27

0

.20

Valu

es a

re m

ean ±

SD

of

trip

lica

tes

C-

Contr

ol soil

, T

1-

Soil

with 2

5%

eff

luent, T

2 -

So

il w

ith 5

0%

eff

luent.

See

ds : N

ot

dete

cta

ble

127



128

A similar observation was reported by Zarkovic and Blagojevic, (2009) in

their study on uptake if nickel by maize plants, where the concentration of nickel

was 8 times higher in roots than in shoots

Metals in tannery waste water occur in a complex form and vary in their

availability to the plant parts (Gupta and Sinha, 2007).

Zinc

Zinc is an essential micronutrient for all organisms and form active site for

various metalloenzymes. Excessive intake of zinc may lead to vomiting,

dehydration and other adverse effects. Industrial waste water rich in zinc

cadmium, chromium and other heavy metals pose a major threat to the

agricultural fields.

The plants irrigated with diluted form of industrial effluent do not face

much hazards as with the raw effluent which contains the heavy metals at a

higher concentration.

Tables 31 and 32 show the accumulation of zinc in roots, shoots, leaves

and seeds of the plants Vigna radiata and Vigna mungo grown in red soil and

black soil using 25% and 50% tannery effluent

Zinc contents of the plant Vigna radiata and Vigna mungo in various parts

were found to be higher than that of the control plants. Zinc content was higher

in roots but negligible in grain. The accumulation of zinc followed a decreasing

order from roots, shoots, leaves and grain.

Khilji and Barbeen (2008) observed the percentage reduction in the

amount of metals in different concentrations of tannery sludge after growing

H. Umbellata for 90 days. Akinola and Ekiyoyo (2006) studied that accumulation

of cadmium, lead and chromium in crop plants were high in roots compared to

other parts.



129

T

AB

LE

31

ZIN

C C

ON

TE

NT

(m

g/g

) IN

VA

RIO

US

PA

RT

S O

F Vigna radiata G

RO

WN

IN

RE

D S

OIL

AN

D

BL

AC

K S

OIL

US

ING

DIL

UT

ED

EF

FL

UE

NT

RO

OT

S

SH

OO

TS

L

EA

VE

S

So

il

C

T1

T

2

C

T1

T

2

C

T1

T

2

Re

d s

oil

0

.7 ±

0.0

2

4.2

± 0

.45

5

.1 ±

0.6

3

0.6

± 0

.02

3

.4 ±

0.2

5

2.2

5 ±

0.0

2 0

.48 ±

0.0

3

1.3

7 ±

0.1

4

1.0

± 0

.21

Bla

ck

s

oil

0

.8 ±

0.0

2

4.5

± 0

.71

4

.9 ±

0.8

2

0.6

± 0

.03

3

.6 ±

0.2

9

3.9

± 0

.32

1

.54 ±

0.1

2

1.3

± 0

.13

2

.9 ±

0.1

9

CD

(0

.05

) 0

.91

0

.58

0

.20

Valu

es a

re m

ean ±

SD

of

trip

lica

tes

C-

Contr

ol soil

, T

1-

Soil

with 2

5%

eff

luent, T

2 -

So

il w

ith 5

0%

eff

luent.

See

ds : N

ot

dete

cta

ble

129



130

T

AB

LE

32

ZIN

C C

ON

TE

NT

(m

g/g

) IN

VA

RIO

US

PA

RT

S O

F Vigna mungo

GR

OW

N I

N R

ED

SO

IL A

ND

BL

AC

K S

OIL

US

ING

DIL

UT

ED

EF

FL

UE

NT

RO

OT

S

SH

OO

TS

L

EA

VE

S

So

il

C

T1

T

2

C

T1

T

2

C

T1

T

2

Re

d s

oil

0

.8 ±

0.0

3

4.9

± 0

.53

5

.6 ±

1.7

3

0.7

± 0

.04

3

.9 ±

0.3

2

4.8

± 0

.41

0

.56 ±

0.0

4

1.3

3 ±

0.1

5

1.7

2 ±

0.1

6

Bla

ck

s

oil

0

.9 ±

0.0

4

4.7

± 0

.62

5

.1 ±

1.8

1

0.7

± 0

.03

3

.8 ±

0.2

9

4.1

± 0

.22

0

.57

± 0

.12

2

.10 ±

0.1

9

2.1

8 ±

0.2

0

CD

(0

.05

) 0

.89

0

.61

0

.18

Valu

es a

re m

ean ±

SD

of

trip

lica

tes

C-

Contr

ol soil

, T

1-

Soil

with 2

5%

eff

luent, T

2 -

So

il w

ith 5

0%

eff

luent.

S

ee

ds : N

ot

dete

cta

ble

130



131

Cadmium

Cadmium is one of the most toxic heavy metals and is recognized for its

negative effect on the environment where it accumulates throughout the food

chain posing a serious threat to human health (Xiaomei et al., 2004).The uptake

of cadmium by roots and transport to the upper parts takes place in the upward

translocation mechanism (Huttova et al., 2006).

Cadmium has been recognised to have a negative impact on the

environment in high concentration. The presence of excessive amount of

cadmium in soil causes reduction in root growth, disturbance in mineral nutrients

and carbohytrate metabolism. They were found to reduce biomass production

due to the direct consequence of the inhibition of chlorophyll synthesis and

photosysnthesis. The stress caused by cadmium and zinc in the tannery effluent

could be reduced by diluting the effluent before application on the vegetative

crops.

Table 33 and 34 show the accumulation of cadmium in roots,

shoots, leaves and seeds of the plants Vigna radiata and Vigna mungo grown in

red soil and black soil using 25% and 50% tannery effluent.

Cadmium content in various plant parts of Vigna radiata in 50% effluent

in the present study was found to have the following order of accumulation as

3.1 mg/g, 2.5 mg/g and 2.25 mg/g in root, shoot and leaves respectively. But it

was not detectable in grains. The same trend was noticed in Vigna mungo

plants.

According to Sharma and Chettri (2008) cadmium and lead were found

to have accumulated in plant tissues. Although cadmium adversely affects plant

growth, root growth is severely affected and results in faster reduction of root

biomass compared to the shoot resulting in an increased shoot root biomass

ratio (Chandra et al., 2010).



132

TA

BL

E 3

3

CA

DM

IUM

CO

NT

EN

T (

mg

/g)

IN V

AR

IOU

S P

AR

TS

OF

Vigna radiata G

RO

WN

IN

RE

D S

OIL

AN

D B

LA

CK

SO

IL U

SIN

G D

ILU

TE

D E

FF

LU

EN

T

RO

OT

S

SH

OO

TS

L

EA

VE

S

So

il

C

T1

T

2

C

T1

T

2

C

T1

T

2

Re

d s

oil

1

.05 ±

0.1

1

2.8

± 0

.19

3

.1 ±

0.2

1

0.8

2 ±

0.0

7 2

.27 ±

0.1

6

2.5

± 0

.24

0

.24 ±

0.0

5

1.9

± 0

.14

2

.25 ±

0.2

5

Bla

ck

s

oil

1

.09 ±

0.1

4

2.9

± 0

.16

3

.9 ±

0.2

7

0.7

± 0

.05

2

.8 ±

0.1

9

3.1

± 0

.29

0

.31

±0

.12

1

.8 ±

0.1

5

2.9

0 ±

0.2

3

CD

(0

.05

) 0

.25

0

.31

0

.26

Valu

es a

re m

ean ±

SD

of

trip

lica

tes

C-

Contr

ol soil,

T1-

So

il w

ith 2

5%

eff

luent, T

2 -

So

il w

ith 5

0%

eff

luent.

S

ee

ds : N

ot

dete

cta

ble

132



133

T

AB

LE

34

CA

DM

IUM

CO

NT

EN

T (

mg

/g)

IN V

AR

IOU

S P

AR

TS

OF

Vigna mungo G

RO

WN

IN

RE

D S

OIL

AN

D

BL

AC

K S

OIL

US

ING

DIL

UT

ED

EF

FL

UE

NT

RO

OT

S

SH

OO

TS

L

EA

VE

S

So

il

C

T1

T

2

C

T1

T

2

C

T1

T

2

Re

d s

oil

1

.04 ±

0.0

7 2

.35 ±

0.1

2

3.8

± 0

.32

0

.71 ±

0.1

1 2

.2 ±

0.0

9

2.7

± 0

.18

0

.03 ±

0.9

6

1.3

1 ±

0.2

4

2.1

2 ±

0.4

1

Bla

ck

s

oil

1

.0 ±

0.0

3

3.0

± 0

.24

3

.6 ±

0.3

5

0.8

± 0

.10

2

.85 ±

0.1

2

3.0

± 0

.20

0

.64 ±

0.0

9

2.1

3 ±

0.2

9

2.6

2 ±

0.3

6

CD

(0

.05

) 0

.44

0

.23

0

.45

Valu

es a

re m

ean ±

SD

of

trip

lica

tes

C-

Contr

ol soil,

T1-

So

il w

ith 2

5%

eff

luent, T

2-

So

il w

ith 5

0%

eff

luent.

See

ds : N

ot

dete

cta

ble

133



134

Stolt et al. (2006) opined that the rate of absorption and translocation of

cadmium varies from plant to plant and genetic variation exists in the

accumulation rate of cadmium in different parts of the plant.

Low grain cadmium accumulation was observed in many studies. The

mechanism of cadmium uptake, translocation and grain accumulation depends

on lower cadmium pools in leaves. Much of the cadmium was found to be

retained in the root cell walls during the growth with 25% effluent

The gradient of accumulation of heavy metals was found to be highest in

roots followed by stem, branches, leaves and then in grains or fruits as reported

by Sharma and Agrawal (2005). Fritioff and Gregor (2006) in their study on

distribution of heavy metals such as zinc, copper, lead and cadmium by leaves,

stems and roots of Potamogeton natans, found the highest accumulation in

roots than other parts in conformity with the present study. Angelova and Ivanov

(2008), in the work on distribution of heavy metals in Brassica Nigera, reported

that the metal content of the seeds were lower in comparision to other parts. It

follows the order roots> stems> leaves>fruit shells> seeds. Fruit shells act as a

barrier on their way towards the seeds.

In accordance with these observations the concentration of metals in our

study were found to be higher in roots followed by shoots and leaves, with least

concentrations in grain. The seed coat of the grains might have acted as a

barrier in Vigna radiata and Vigna mungo in condtions of soil contamination.

4.2.3. Histochemical observations of root sections of the selected plants

Plate 7 show the cross sections of root of Vigna radiata and Plate 8 show

that of Vigna mungo plants grown in 25% and 50% tannery effluent.



135

PLATE 7

HISTOCHEMICAL OBSERVATION OF ROOTS OF Vigna radiata

CR- Control with red soil, T1R- Red soil with 25% effluent, T2R- Red soil with 50% effluent, CB- Control black soil, T1B- Black soil with 25% effluent, T2B- Black soil with 50% effluent

CB

T1B

T2B

CR

T1R

T2R



136

PLATE 8

HISTOCHEMICAL OBSERVATION OF ROOTS OF Vigna mungo


C1R C1B

T1R T1B

T2R T2B



137

Localization of metals in various plant tissues and the concerned

anatomical variations in effluent cultivated plants was done by histochemical

staining method.

Both the plants grown with 25% effluent (T1R and T1B) did not show any

accumulation in their root tissues. Roots of T2R and T2B Vigna radiata plants

exhibited accumulation of heavy metal. Similarly the Vigna mungo plants grown

with 50% effluent (T2R and T2B) were found to have accumulations of metals in

the cross sectional study of roots.

It was observed that there were many sites of accumulation in the roots

of Vigna mungo plant compared to the roots of Vigna radiata plants indicating

that metals are fast absorbed in Vigna mungo plants than Vigna radiata plants.

An important reason for enhanced accumulation of chromium in the root may be

due to presence of organic acids in the root exudates which form complexes

with chromium, thereby making them available for the uptake by the root.

Histological changes in the root showed highly distorted piliferous layer

and cortex. Thickened cell walls of vessels and pith were noted. The distortion

of cells of various tissues may be the result of interferences with the cell division

or with cell elongation.

Though metal concentrations were found in leaves and shoots of both the

plants, the histochemical sections did not show any accumulation. As Shankar

et al. (2005) suggested, translocation of chromium from root to shoot is slow.

Pulford et al. (2001) in a study with temperate trees confirmed that chromium is

poorly taken up into the aerial parts and is predominantly held in the roots.

In our study, in both the species of Vigna chromium accumulation was

high in the roots compared to the stems, leaves and grains which was in

concomitment with the results obtained by Chandra et al. (2010).



138

4.2.4. Yield parameters

a. Number of nodules In nodulating plants, nitrogen fixation is the primary route of nitrogen

nutrition and hence what happens to the nodules at the stimulated growth

conditions will have a profound effect on the overall growth of the plants

themselves. Leguminous plants exhibit differential response in nodulation to

heavy metal toxicity (Veliappan et al., 2002).

Table 35 and 36 show the number of nodules formed in plants Vigna


effluent concentrations respectively.

TABLE 35

NUMBER OF NODULES OF Vigna radiata GROWN IN RED SOIL

AND BLACK SOIL USING DILUTED EFFLUENT

Vigna radiata

Days after sowing (DAS) Soil Groups

30 60 90

C1 10.0 ± 4.08 19.0 ± 0.82 21.0 ± 0.80

T1 9.0 ± 0.82 18.0 ± 0.81 20.0 ± 0.82 Red soil

T2 8.0 ± 1.63 17.0 ± 0.82 19.0 ± 0.80

C2 11.0 ± 0.82 18.0 ± 1.63 19.0 ± 1.60

T3 10.0 ± 0.82 16.0 ± 6.24 16.0 ± 1.63 Black soil

T4 9.0 ± 0.82 13.3 ± 2.45 16.0 ± 0.82

CD ( 0.05) 4.2



Number of nodules were increased with increase in the time point and

reached a maximum level on 60th day and sustained till 90th day in both the

plants. According to Geetha et al. (2008), the nodule formation in soybean

reached its peak during flowering stage and senescence of nodules occurred as

the plant matured.



139

TABLE 36

NUMBER OF NODULES OF Vigna mungo GROWN IN RED SOIL

AND BLACK SOIL USING DILUTED EFFLUENT

Vigna radiata

Days after sowing (DAS) Soil Groups

30 60 90

C1 11.0 ± 1.63 15.0 ± 4.08 16.0 ± 1.63

T1 9.0 ± 0.79 13.0 ± 2.45 14.0 ± 3.27 Red soil

T2 8.0 ± 0.82 12.0 ± 1.63 13.0 ± 2.45

C2 12.0 ± 1.61 16.0 ± 1.63 16.0 ± 0.82

T3 11.0 ± 4.08 15.0 ± 3.27 14.0 ± 3.25 Black soil

T4 10.0 ± 0.80 14.0 ± 4.08 13.0 ± 2.45

CD ( 0.05) 5.1



b. Flowering time

Table 37 shows the day of first flowering of the plants Vigna radiata and

Vigna mungo grown in red soil and black soil using 25 % and 50% tannery

effluent.

TABLE 37

FLOWERING TIME OF PLANTS Vigna radiata AND Vigna mungo GROWN IN RED SOIL AND BLACK SOIL USING DILUTED EFFLUENT

Flowering time Soil Groups


CR 45 ±4.08 45 ± 4.04

T1R 47 ±0.82 47 ± 1.63 Red soil

T2R 49 ±0.80 49 ± 0.86

CB 44 ±3.27 45 ± 4.05

T1B 47± 0.85 46 ± 0.82 Black soil

T2B 49 ±0.81 48 ± 1.62

CD ( 0.05) 4.9





140

There was no significant delay in flowering time of Vigna radiata and

Vigna mungo plants grown using both the effluents in both the soils.

c. Pod length, Pod weight, Number of Pods/plant Table 38 shows the pod length and pod weight of the plant of Vigna


tannery effluent.

Vigna radiata recorded significant (p<0.05) reduction in pod length grown

with black soil and 50% effluent. The pod weight of Vigna mungo was

decreased significantly with 50% effluent.

TABLE 38

POD LENGTH, POD WEIGHT OF Vigna radiata AND Vigna mungo GROWN IN RED AND BLACK SOILS USING DILUTED

TANNERY EFFLUENT

Pod length (cm) Pod weight(g) Soil Groups Vigna

radiata Vigna mungo

Vigna radiata

Vigna mungo

CR 4.70 ± 0.16 1.42 ± 0.02 1.07 ± 0.01 19.0 ± 0.82

T1R 4.50 ± 0.41 1.30 ± 0.04 1.02 ± 0.01 18.0 ± 1.62

Red soil

T2R 4.40 ± 0.31 1.23 ± 0.03 0.99 ± 0.02 16.0 ± 0.81

CB 4.50 ± 0.43 1.31 ± 0.01 1.02 ± 0.03 18.0 ± 0.80

T1B 4.10 ± 0.09 1.28 ± 0.02 0.99 ± 0.02 17.0 ± 0.84 Black soil

T2B 3.90 ± 0.05 0.99 ± 0.01 0.81 ± 0.03 15.0 ± 0.86

CD ( 0.05) 0.5 2.6



Table 39 shows number of pods/ plant of Vigna radiata and Vigna mungo

grown in red soil and black soil using 25% and 50% tannery effluent.

A significant decrease was not observed in number of pods/plant in both the

plants and soils.



141

TABLE 39

NUMBER OF PODS / PLANT OF Vigna radiata AND Vigna mungo GROWN IN RED AND BLACK SOILS USING DILUTED

TANNERY EFFLUENT

Pod length (cm) Soil Groups


CR 4.70 ± 0.16 1.42 ± 0.02

T1R 4.50 ± 0.41 1.30 ± 0.04 Red soil

T2R 4.40 ± 0.31 1.23 ± 0.03

CB 4.50 ± 0.43 1.31 ± 0.01

T1B 4.10 ± 0.09 1.28 ± 0.02 Black soil

T2B 3.90 ± 0.05 0.99 ± 0.01

CD ( 0.05) 0.5



d. Number of seeds / plant and Number of seeds /pod Table 40 shows the number of seeds /plant and number of seeds /pod of

Vigna radiata and Vigna mungo plants grown in red soil and black soil using

25% and 50% tannery effluent.

The decrease in the number of seeds/ plant and number of seeds / pod in

effluent treated Vigna radiata and Vigna mungo plants grown in red and

black soils was not found to be significant compared to the control plants.

Sinha et al. (2008) demonstrated that V.radiata exhibited a significant increase

in growth parameters when grown on lower amendments of sludge.



142

TABLE 40

NUMBER OF SEEDS /PLANT AND NUMBER OF SEEDS /POD OF Vigna radiata AND Vigna mungo GROWN IN RED AND BLACK SOILS

USING DILUTED TANNERY EFFLUENT

Number of seeds /plant Number of seeds /pod Soil Groups Vigna

radiata Vigna mungo

Vigna radiata

Vigna mungo

CR 219 ± 0.82 113 ± 2.43 13.0 ± 2.45 8.0 ± 0.81

T1R 213 ± 2.46 109 ± 0.83 12.0 ± 1.63 7.0 ± 0.80

Red soil

T2R 202 ± 1.62 106 ± 0.85 11.0 ± 0.87 6.0 ± 0.88

CB 201 ± 0.86 82 ± 1.53 11.0 ± 1.65 7.0 ± 1.66

T1B 193 ± 4.45 79 ± 0.52 10.0 ± 0.72 5.0 ± 0.89 Black soil

T2B 185 ± 4.08 72 ± 1.61 10.0 ± 1.67 6.0 ± 0.81

CD ( 0.05) 2.02 2.75


CR- Control with red soil, T1R- Red soil with 25% effluent, T2R- Red soil with 50% effluent, CB- Control black soil, T1B- Black soil with 25% effluent, T2B- Black soil with 50% effluent.

e. Total seed weight / plant and 100 seed weight

Table 41 shows the total seeds weight / plant and 100 seeds weight of

Vigna radiata and Vigna mungo plants grown in red soil and black soil in 25%


Total seeds weight / plant of Vigna radiata and Vigna mungo showed no

significant reduction when grown in 25% and 50% effluent. This indicates that

the diluted tannery effluent might be used for plant growth. 100 seed weight of

both the plants were found to be significantly decreased with 50% effluent in

both the soils.

Results of yield parameters of both the plants grown in red soil and black

soil in both the effluent concentrations indicated that plants grow better in 25%

tannery effluent and diluted effluent could be a better choice for plant growth in

industrialized area.



143

TABLE 41

TOTAL SEED WEIGHT/ PLANT AND 100 SEED WEIGHT OF Vigna radiata

AND Vigna mungo GROWN IN RED AND BLACK SOILS USING

DILUTED TANNERY EFFLUENT

Total seed weight/ plant (g) 100 seed weight (g) Soil Groups Vigna

radiata Vigna mungo

Vigna radiata

Vigna mungo

CR 16.40 ± 0.33 45.0 ± 4.08 7.60 ± 0.33 4.51 ± 0.01

T1R 15.70 ± 0.06 47.0 ± 0.82 7.40 ± 0.28 4.21 ± 0.03

Red soil

T2R 14.30 ± 0.24 49.0 ± 0.80 7.10 ± 0.16 3.80 ± 0.17

CB 15.80 ± 0.18 44.0 ± 3.27 7.90 ± 0.08 4.60 ± 0.15

T1B 14.20 ± 0.16 47.20 ± 0.85 7.40 ± 0.35 4.20 ± 0.18 Black soil

T2B 13.70 ± 0.13 49.0 ± 0.81 6.90 ± 0.07 4.0 ± 0.41

CD ( 0.05) 2.02 2.75



Hence, from the results of this phase it was found that the biometric

observations and biochemical parameters such as chlorophyll, carbohydrate,

carotenoid, ascorbic acid and yield parameters of the plants grown in red soil

were at higher levels compared to those grown in black soil. Vigna radiata

showed a better response with respect to biometric observations and certain

biochemical parameters compared to Vigna mungo. Plants grown with 25%

effluent exhibited a better growth compared to those grown with 50% effluent.

PHASE III

4.3. Health profile of Tannery workers Any effort to evaluate occupational health risks includes assessing the

health of individual workers with the goal of keeping the worker healthy and

reducing the overall risks in the work environment.



144

A worker is at risk if he/she has a greater chance of developing disease

than a non exposed worker. It is very important to identify all harmful

substances and to monitor and control exposures in order to manage the risk

(Hall, 2001).

Leather production includes many operations with different exposures,

which can be harmful for the health of the workers and particularly be

carcinogenic (Issever et al., 2007). Certain chemicals such as benzene based

dyes and formaldehyde are considered to be carcinogenic (Budhwar, 2005).

Besides these, scores of other chemicals and organic solvents such as

chromate and bichromate salts, aniline, butyl acetate, ethanol, benzene,

toluene, suplhuric acid and ammonium hydrogen sulfide are used in the

tannery industry. An important health risk factor for the tannery workers is

occupational exposure to chromium mainly in organic form or in protein bound

form caused by leather dust (Mikoczy and Hagmar, 2005). Chromium may

enter the body by inhalation, ingestion and by direct cutaneous contact.

Professional exposure to chromium increases the risk of dermatitis, ulcers and

perforation of the nasal septum and respiratory illness as well as increased lung

and nasal cancers. Chromium specific health hazards like carcinoma of the

larynx and lung parenchyma and paranasal sinusis have also been reported

(Rastogi et al., 2007).

According to Bulletin of the WHO (2005), 58% of the tannery workers

were found to suffer from gastrointestinal diseases, 31% from dermatological

diseases, 12% from hypertension and 0.9 % from jaundice.

Working conditions, nature of work, vocational and professional status

and geographical location of industries and employment have a profound

impact on the social status and social well being of the workers (Babalola and

Babajide, 2009)

Exposure is the contact of toxic substance by the body. It may be acute

(immediate) or chronic (long term). During leather tanning, the workers are



145

exposed to chemicals such as sodium chloride during soaking stage, sulphide

and lime in fleshing and trimming stage, acid and ammonium salts in bating

stage, fungicides and bactericides in picking stage, chromium salts during

tanning stage, nickel, arsenic, zinc, cadmium, copper, dye, and solvents

during wet finishing stage. The chemicals used in the process of tanning have

been proved to be toxic.People involved in producing leather have a

significant risk of presenting clinical conditions attributed to chromium exposure

(Cuberos et al., 2009).

The use of sulphides and hydrosulphides in dehairing operations may

carry a risk of skin contact for the operator. A similar risk is possible in the use of

caustic materials such as sodium hydroxide or calcium hydroxide causing skin

burns. Formaldehyde, glutaraldehyde and hydrogen peroxide used in tanning

processes causes irritation of body tissues even in if minute quantities are

inhaled. A variety of dyestuffs and fungicides are used which are quite serious if

inhaled or injested (Taylor et al., 2006).

Continuous exposure to these chemicals results in entry into the body.

Workers who have been dealing with these chemicals and who do not follow

any safety measures in preventing the entry of the chemicals into the body,

were found to acquire many forms of ill effects. Eating in contaminated area,

using the chemicals with bare hand, breathing without using mask in the

workplace are some of the reasons of the entry of these hazardous chemicals.

Hence in this phase of the present study an attempt was made to analyze

the biochemical parameters associated with hepatic, renal and skin disorders

caused, if any in the selected group tannery workers who have been exposed to

these chemicals for many years and who do not follow any safety measures

while using the chemicals.

Tannery workers with one to five years of experience (Group II: n=20)

and workers with five to ten years of experience (Group III : n= 20), were



146

selected for the study. A reference group of 20 subjects belonging to the similar

age group who never had any occupational exposure in the tanneries served as

control (Groiup I). Each group comprised of 60% men and 40% women workers

who belong to the same socio economic group and dietary pattern. They

belonged to an age group of 20-50 years and they were under cumulative

exposure to numerous pollutants in the workplace. They had no history of

defect, infections or metabolic disorders. These workers were not engaged in

any other occupation and hence not exposed to other types of pollutants.

Hematological parameters (hemoglobin, total count and

immunoglobulin E), assessment of liver function (alanine transaminase,

aspartate transamiase, alkaline phosphatase, acid phosphatase and lactate

dehydrogenase), assessment of renal function (urea, uric acid and creatinine)

and mineral status (chromium, nickel, zinc and cadmium) of the selected

tannery workers were performed and their results are discussed as follows.

4.3.1 Hematological parameters of blood sample

Hematological parameters are related to the changing environmental

conditions and therefore can be used to screen the health state of organisms

exposed to a particular toxicant (Tripathi et al., 2002).

4.3.1.1. Hemoglobin and total leukocyte count

The mean values of hemoglobin and total leukocyte count estimated in

control and experimental groups are presented Table 42 and Figure 28 and

Figure 29.

The normal hemoglobin level is 12-14 g/dl. Group I participants

(control) had hemoglobin contents within the normal limits, whereas group II and

group III recorded significantly lower levels than the control. The total count of

the experimental groups was found to be increased than that of the control

groups.



147

0

2000

4000

6000

8000

10000

12000

14000

Group I Group II Group III

To

tal

leu

co

cy

te c

ou

nt

(mm

3)

0

2

4

6

8

10

12

14

16


Hem

og

lob

ulin

(g

/100m

l)

TABLE 42

HEMOGLOBIN CONTENT AND TOTAL LEUKOCYTE COUNT OF THE TANNERY WORKERS

Groups Hemoglobin (g/100 ml)

Total leukocyte count (mm3)

Group I

Control 12.33 ± 0.08 10,100 ± 105.7

Group II

1-5 years 9.60 ± 0.7 11,000 ± 104.9

Group III

5-10 years 7.87 ± 0.9 12,320 ± 96.2

CD (0.05) 0.19 112.57

Values are presented as mean± SD (n = 20 in each group)

The reduction

of hemoglobin content might

be due to the effect of

pollutants on hematopoietic

system which leads to

anemic condition in

human (Mathivanan, 2004).

Hematological values such

as white blood cell count and

red blood cell count of population residing in industrial area exposed to toxic

pollutants showed an

increased trend while

the hemoglobin content

decreased in exposed

population compared to

the non exposed

population (Ahsan, 2003).

According to Benova et al.

(2002), chromium is rapidly

FIG. 28 HEMOGLOBIN CONTENT

FIG. 29 TOTAL LEUKOCYTE COUNT



148

absorbed by the lungs into the blood and easily penetrates the cellular

membranes and binds to the hemoglobin in the red blood cells, thereby affecting

the oxygen carrying capacity and impairing the lung function status.

Elevated leucocyte count might be due to increase in the population

of neutrophils, acidophils and basophils (Joshi et al., 2002). Cases of

hematological effects have been reported in humans after the ingestion of lethal

or sublethel doses of chromium. Decreased hemoglobin (anemia) content and

increased total white blood cell count (thrombocytopenia) were noted by

Parveen and Rawat (2010). Alterations in blood hemoglobin, total cell count

and erythrocyte sedimentation have provided a useful means of detecting

and assessing the severity of anemia due to exposure to toxic pollutants

(Sharma et al., 2004).

4.3.1.2 Immunoglobulin E levels

Table 43 and Figure 30 shows the Immunoglobulin E levels in blood

sample of the participants.

TABLE 43

Ig E LEVELS OF THE WORKERS

Groups IgE (IU/L)

Group I 98.6± 1.3

Group II 732± 1.9

Group III 800± 2.3

CD ( 0.05) 4.8


The results show that both the experimental groups had significantly

increased levels of Immunoglobin E compared to the Group I (control)

participants. For any allergic reactions, elevated Ig E is an important

determinant. Occupational exposure and elevated serum Ig E levels were well

correlated in the study conducted by Kim et al., (2010).



149

0

100

200

300

400

500

600

700

800

900


Imm

un

og

lob

ulin

(IU

/ L

)

Sensitizers are agents

that may cause allergic or

allergic-like responses to

occur. After an initial

exposure to a substance an

individual may become

sensitized to that substance.

Subsequent exposures to

the same substance, often at

a much lower concentration than before, produce an allergic response. This

response may be a skin rash (dermatitis) or an asthmatic-like attack, depending

on the route of exposure (Shahzad et al., 2008).

Leather tanning is principally chemical preservation of raw hide by the

process in which binding of various chemicals (mainly chromium salts as

potassium dichromate) to proteins in raw hide takes place. Chromium has

potential to bind with skin proteins of tannery workers to produce complex

antigens which lead to hypersensitivity. The resulting contact dermatitis could be

preliminary condition to the development of bronchial asthma (Lockman, 2002).

Tannery workers are thus potentially exposed to harmful agents,

rendering them vulnerable to health problems especially those of skin and

gastrointestinal problems. Due to this exposure, health hazards namely eye

irritation and repiratory tract irritation would be caused. Skin ulcer might develop

if chromium compounds come into contact with an abrasion, a scratch of

laceration of the skin (Shahzad et al., 2006).

In the present study, 12 out of 20 persons of groups III who had more

years of exposure were found to possess skin rashes and scales indicating

allergic response. All the respondents were associated with chrome tanning

process. They handle lead chromate and nickel chromate every day. And their

contact with these chemicals occurs for more than 6 hours a day. The route of

FIG. 30 Ig E LEVELS OF THE WORKERS



150

entry was mainly absorption by skin contact since they seldom wore gloves

while operating the chemicals. Other possible routes of entry were inhalation of

chrome dust and ingestion since they have lunch in the work place itself without

proper handwash. Repeated contact with toxic metals might be the cause for

allergic contact dermatitis.

Plate 9 shows a person affected by dermatitis. He was associated with all

the processes of tanning since he has 18 years of experience in tannery

industry. His Ig E levels were found to be increased than the normal limits and

had skin allergy. This person was associated with all the process of tanning. He

handled chemicals namely chrome, formate and potassium dichromate daily.

PLATE 9

DERMATITIS LIKE SYMPTOM

In a study conducted by Rastogi et al. (2008), the leather tanners who

had a mean exposure of 8 years in the tanneries were found to have

dermatological diseases such as rashes and papules along with complaints of

itching. The burning sensation was also reported by 15 subjects in the exposed

workers. Chromium toxicity can produce penetrating lesions known as chrome

hole or chrome ulcers particularly in areas where a break in the epidermis is



151

already present. These commonly occur on the fingers, knuckles, and fore

arms. The characteristic chrome sore begins as a papule forming an ulcer with

raised hard edges. Ulcers can penetrate deep into soft tissue or become the

sites of secondary infection (Meditext, 2005). Chrome ulceration is a specific

skin lesion caused as a result of direct contact with trivalent or hexavalent

chromium compounds and is especially observed among chrome tanners.

The affected workman has painless, multiple ulcers of holes on the skin

of the exposed parts of the body, especially hands and feet. In a study

conducted in North India, the prevalence of ulcers of fingers and toes among

chrome tanners was found to be 10.6% (Raidas, 2007).

The most viable condition that aggravates the risk of developing

dermatitis is the constant wetting of the skin. Persons who are engaged in

soaking operations were found to be maximum affected (10.46%) with

dermatitis.

4.3.2. Assessment of liver function

Clinical laboratories use the measurement of alanine transaminase

(ALT), aspartate transaminase (AST), alkaline phosphatase (ALP) , acid

phosphatase (ACP) and lactate dehydrogenase (LDH) in serum and tissue for

assessing liver damage (Karthikeyan et al., 2004).

Table 44, Figures 31 and 32 shows the activities of the liver enzymes

alanine transaminase, aspartate transaminase, alkaline phosphatase, acid

phosphatase and lactate dehydrogenase of tannery workers

The normal value of ALT and AST were up to 40 IU/L and 38 IU/L

respectively. The control group recorded a value within the normal range,

whereas ALT of group III workers showed a significant increase and AST

activity of group II and group III workers showed a significant increase.



152

TABLE 44

ALANINE TRANSAMINASE (ALT), ASPARTATE TRANSAMINASE (AST),

ALKALINE PHOSPHATASE (ALP), ACID PHOSPHATASE (ACP)

AND LACTATE DEHYDROGENASE (LDH) ACTIVITIES

OF THE TANNERY WORKERS

GROUPS ALT (IU/L) AST (IU/L) ALP (IU/L) ACP (IU/L) LDH (IU/L)

Group I 38.20±0.78 34.15 ±0.01 32.9±0.9 4.78±0.01 162±0.54

Group II 41.20±0.17 38.58±0.98 56.8±0.5 3.63±0.06 241±0.62

Group III 42.36±0.42 39.43±0.84 83.2±0.4 3.87±0.08 286±0.71

CD (0.05) 1.10 0.16 0.16 0.11 1.99


ALT - Activity of enzymes that transforms 1 µmol of the L.alanine in 1 min to pyruvate

AST - Activity of enzymes that transforms 1 µmol of the aspartate in 1 min to pyruvate

ALP - Activity of enzyme that converts 1 mg phenol in 1 minute in 100 ml of serum

ACP - Moles of p-nitrophenol released/ min/mg protein

LDH - The activity which produces a change in extinction of 0.001/ min

FIGURE 31

ALANINE TRANSAMINASE AND ASPARTATE TRANSAMINASE ACTIVITIES OF THE TANNERY WORKERS

0

5

10

15

20

25

30

35

40

45

Alanine transaminase Aspartate transaminase

IU / L



153

FIGURE 32

ALKALINE PHOSPHATASE, ACID PHOSPHATASE AND LACTATE DEHYDROGENASE ACTIVITIES OF THE

TANNERY WORKERS

0

10

20

30

40

50

60

70

80

90


AL

P (

IU /

L)

0

1

2

3

4

5

6


AC

P (

IU /

L)

0

50

100

150

200

250

300

350


LD

H (

IU / L

)

ALKALINE PHOSPHATASE

ACID PHOSPHATASE

LACTATE DEHYDROGENASE



154

Group II and group III participants showed a significant increase in

alkaline phosphatase and lactate dehydrogenase activities whereas acid

phosphatase activity of group II and group III participants decreased significantly

compared to the group I participants.

Increase in serum aminotransferase levels characterizes cirrhosis,

cholestatic liver disease, fatty liver and hepatic neoplasms (Das and

Vasudevan, 2005). Gupta et al. (2005) and Johri et al.(2004) reported that rise

in aspartate aminotransferase and alanine aminotransferase is indicative of

leakage of enzymes from liver resulting in alterations in the cell permeability.

Chromium has been reported to cause severe hepatic effects, elevated liver

enzyme levels, hepatomegaly and hepatic failure in four of five workers exposed

to chromium in leather tanning industry (Munagala et al., 2003). The present

study falls in line with the work of Yun et al. (2008) who reported a significant

increase in serum LDH level in tannery industry workers when compared to

control subjects.

LDH serves as a sensitive marker of epidermal toxicity of stress induced

by pathological conditions and environmental conditions (Sharma et al., 2010)

Its level is increased in persons with anemia, leukemia, urinary tract infection

and pulmonary embolism reflecting its diagnostic utility in specific clinical cases

(Nussinovitch et al., 2009; Shahi et al., 2009).

4.3.3. Assessment of renal function

In order to see the long term effects of pollutants on kidney different renal

indices were measured in both exposed and non exposed groups and the

results are presented.

Tables 45, 46 and Figure 33 indicate the urea, uric acid and creatinine

contents of the serum sample and urine sample of the participants respectively.

The normal blood urea level is 15-45 mg/dl, uric acid is up to 4.8 mg/dl and

creatinine is 0.7 to 1.4 mg/dl. In our study, all the three groups recorded normal

values of urea, uric acid and creatinine contents in the blood sample.



155

TABLE 45

UREA, URIC ACID AND CREATININE LEVELS IN THE BLOOD SAMPLE OF THE TANNERY WORKERS

Groups Urea

(mg/dl) Uric acid (mg/dl)

Creatinine (mg/dl)

Group I 26.39 ± 0.26 3.29 ± 0.09 0.94 ± 0.02

Group II 30.35 ± 0.29 3.97 ± 0.08 1.10 ± 0.06

Group III 37.70 ± 0.31 4.26 ± 0.05 1.29 ± 0.07

CD (0.05) 0.19 0.18 0.02


TABLE 46

UREA, URIC ACID AND CREATININE LEVELS IN THE URINE SAMPLE OF THE TANNERY WORKERS

Groups Urea

(g/day) Uric acid (g/day)

Creatinine (g/day)

Group I 12.20±1.4 0.41±0.05 2.52±0.11

Group II 14.20±1.1 0.48±0.02 2.71±0.28

Group III 15.10±1.02 0.53±0.06 3.01±0.19

CD (0.05) 0.18 0.02 0.21




156

FIGURE 33

UREA, URIC ACID AND CREATININE CONTENTS IN THE BLOOD AND URINE SAMPLES OF THE TANNERY WORKERS

BLOOD SAMPLE URINE SAMPLE

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6


Cre

ati

nin

e (

mg

/dl)

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5


Uri

c a

cid

(m

g/d

l)

0

5

10

15

20

25

30

35

40


Ure

a

(mg

/dl)

UREA

URIC ACID

CREATININE

0

2

4

6

8

10

12

14

16

18


Ure

a (

g/d

ay)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7


Uri

c a

cid

(g

/da

y)

0

0.5

1

1.5

2

2.5

3

3.5

Group I Group II Group II

Cre

ati

nin

e (

g/d

ay

)

UREA

URIC ACID

CREATININE



157

Similarly, urea, uric acid and creatinine contents in urine were well within the

normal limits indicating that exposure to pollutants had no ill effect on the renal

function. This was supported by the findings of Orisakwe et al.(2007), who found

no detectable differences in the renal indices like urea, uric acid and creatinine

contents between exposed and non exposed groups.

3.5. Metal contents in the tannery workers

Heavy metals namely zinc, cadmium and lead are considered to be the

frequent pollutants of natural environments causing serious health hazards

(Mathad et al., 2004). They are generally deposited in liver, muscle, kidney,

spleen, skin, bone and soft tissues of human being. USEPA (United States

Environmental Protection Agency) considers eight trace metals as high priority

critical metals namely arsenic, cadmium, copper, chromium, lead, mercury,

nickel and zinc (Sarker and Gupta, 2003).

Environment pollution by compounds of heavy metals is increasing with

extensive industrial developments. Large amounts of chromium were introduced

into the environment through tannery, textile, chemical manufacture, metal

plating and many other industrial effluents thus making them available to

plants, animals and humans (Raj and Raghavan, 2002). More than 170,000

tons of chromium wastes are discharged to the environment annually as a

consequence of industrial and manufacturing activities (Kamaludeen et al.,

2003). Zinc as a trace element, is regarded as an essential nutrient for human

beings and has also been found to be protective in some kind of liver injury.

Alcoholic cirrhosis may be associated with a state of zinc deficiency (Dhawan

et al., 2005). It is an important component of several enzymes and nutrients.

The water having more than 5mg/L of zinc gives an undesirable astringent taste

and is unsuitable for drinking and cooking purposes (Rani and Reddy, 2003).

The various production processes in tanning industry pose many hazards

to the health of its employees. Toxic chemicals such as hydrogen sulphide,

chromium, bleaching agent, disinfectants, dyes and physical and biological



158

agents are a few to mention. Their effects vary from minor irritation while

working to serious and disabling diseases (Raidas, 2007).

In the present study, to find out the toxicological aspects of the exposed

metals to human beings blood and urine samples were analyzed for the metal

contents namely chromium, nickel, zinc and cadmium.

Table 47 and Figure 34 indicate the chromium, nickel, zinc and cadmium

contents of blood samples and Table 48 indicates that of urine samples of the

participants respectively.

TABLE 47

CHROMIUM, NICKEL, ZINC AND CADMIUM LEVELS IN THE BLOOD SAMPLE OF THE PARTICIPANTS

Chromium Nickel Zinc Cadmium Groups

mg/l

Group I 0.010±0.001 0.007±0.00 0.016±0.001 0.016±0.003

Group II 0.019±0.002 0.019±0.001 0.016±0.002 0.019±0.002

Group III 0.032±0.002 0.019±0.001 0.026±0.002 0.026±0.001

CD ( 0.05) 0.002 0.001 0.006 0.003


TABLE 48

CHROMIUM, NICKEL ZINC AND CADMIUM LEVELS IN THE URINE SAMPLE OF THE PARTICIPANTS

Chromium Nickel Zinc Cadmium Groups

mg/l

Group I 0.01±0.001 ND ND ND

Group II 0.01±0.001 ND ND ND

Group III 0.02±0.001 ND ND ND

CD ( 0.05) 0.002 - - -


ND- Not detectable



159

FIGURE 34

CHROMIUM, NICKEL, ZINC AND CADMIUM CONTENTS OF THE BLOOD SAMPLE

Chromium levels in the blood of the control group were found to be

0.01 mg/l which was significantly increased (0.019 and 0.032 mg/l) in group II

and group III participants. Chromium level in urine was found to be 0.01 mg/l.

The value was significantly increased in group III participants. Chromium is toxic

and mutagenic to most organisms and is known to cause irritation, corrosion of

the skin and respiratory tract; it also causes lung carcinoma in humans (Ganguli

and Tripathi 2002). Rastogi et al. (2008) reported that the urinary and blood

concentrations of chromium were found to be significantly raised among the

leather tanners thereby reflecting the body’s burden of chromium in the exposed

workers as a result of a high concentration of environmental chromium at the

work place.

An important health risk factor for the tannery workers is occupational

exposure to chromium which is used as a basic tanning pigment. The workers

on exposure to leather dust which contains chromium in the protein bound form

exhibited a higher mean concentration of urinary and blood chromium. The

lungs, intestinal tract, the liver and the kidney are the target organs for

0

0.005

0.01

0.015

0.02

0.025

0.03

0.035

0.04

Chromium Nickel Zinc Cadmium

mg / l




160

chromate toxicity (Rom, 2007). Higher values of blood and urinary chromium

concentration were observed in the tannery workers when compared with

healthy normal adult values as reported by Kornhauser et al. (2002).

Nickel content in the blood sample was found to be 0.007 mg/l in control

group which increased to 0.019 in both the group. There was no detectable

nickel in the urine sample. Nickel is the potential carcinogen for lung and may

cause allergies, lung fibrosis and cancer of respiratory tract in occupationally

exposed populations (Kazprazak et al., 2003). Nickel used as nickel carbonate

in wet finishing and pickling processes of leather making might be the cause of

increased nickel concentration in the blood sample of tannery workers.

The zinc content of the control and group II sample remained the same

ie. 0.016 mg/l which was increased to 0.026 mg/l in group III. Zinc was not

detectable in the urine sample. Acute zinc toxicity in humans includes vomiting,

dehydration, drowsiness, lethargy, nausea, lack of muscular co-ordination, and

renal failure. Workers exposed to zinc fumes from smelting or welding have

suffered from a short term illness called mental fern-fever (Leghouchi et al.,

2008).

Zinc hydrosulfate and zinc sulfite were used in the process of tanning

instead of wet blue which was banned after 1990. Workers exposed to hides

treated with zinc chloride, zinc hydroxide and zinc sulfite were found to have a

higher zinc content in blood sample due to their frequent handling.

Cadmium content of the blood sample of group I participants was

0.016 mg/l whereas group II and group III showed a value of 0.019 and

0.026 mg/l. the content was not detectable in urine sample. Prolonged exposure

to cadmium can cause yellow stain that gradually appears on necks and teeth

(Meena et al., 2004). Cadmium induced adverse health effects in humans were

reported by Pizent et al. (2003).

Heavy metals affect individuals at sublethal concentration by changing

the activities of key biomolecules. A chronic dose of zinc, lead, copper,



161

cadmium, nickel and chromium increases the risk of developing anemia,

damage to pancreas, lowers down HDL cholesterol levels and raises

LDL cholesterol levels and possibly enhances the symptoms of the Alzhemiers

disease, hepatic and renal damage (Sharma and Agrawal, 2005). The

excessive intake of metal by man leads to severe mucosal irritation, widespread

capillary damage, hepatic and renal damage, central nervous problems followed

by depression, gastrointestinal irritation and possible necrotic changes in the

liver and kidney (Kalavathy et al., 2005).

Hence, the clinical study indicates that the workers with greater years of

exposure had a risk of dermatitis like symptom due to unsafe handling of

chemicals.

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