Allium Cepa And Lemna Minor Biology Essay

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Tannery effluent is one of the major pollutant which contributes significantly to the environmental pollution. Tannery effluent from Ranipet, Vellore was used to evaluate the toxicity. In this study, the phytotoxic and cytotoxic effects of tannery effluent were performed on Allium cepa and Lemna minor by measuring root growth inhibition, estimating EC50 value, induction of chlorosis in fronds, estimation of protein and chlorophyll content. Allium cepa was exposed to various concentrations of tannery effluent (5, 10,15,20, 25, 30, 35,40,45,50 and 55 %) for 96 hours. Root growth was enhanced with low concentrations of tannery effluent, but beyond a concentration of 20% the root growth was inhibited. The EC50 value of Allium cepa exposed to various dilutions of tannery effluent was estimated as 12%. On the other hand, Lemna minor was exposed to concentrations of tannery effluent (5, 10,15 and 20%) for 96 hours. The number of fronds, protein content and chlorophyll content significantly reduced with high percentage of tannery effluent. So, the tannery effluent of Ranipet, Vellore is highly toxic pollutant which can result in loss of vegetation and soil fertility.

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Keywords : Allium cepa, Tannery effluents, Lemna minor, fronds, chlorosis, phytotoxic, cytotoxic, EC50 .

Introduction

Indiscriminate discharge of untreated waste water directly or indirectly into aquatic bodies may render water resources hazardous to man and other living systems. The environment is under increasing pressure from solid and liquid wastes emanating from the leather industry. These are inevitable by-products of the leather manufacturing process and cause significant pollution unless treated in some way prior to discharge. A discharge of liquid waste, as from a factory or nuclear plants is known as Tannery effluents. Tannery effluents are ranked as the highest pollutants among all industrial wastes. India is the third largest producer of leather in the world having about 3000 tanneries with annual processing capacity of 0.7 million tonnes of hides and skin. The metals that might be present in tannery effluents (viz. chromium, aluminium and perhaps zirconium) are all classified as having a 'high'/'moderately acute' or 'chronic' toxic effect upon organic life and being 'accumulative'.Treated wastewater discharged from tanning industries contains high level of BOD,COD, electrical conductivity and heavy metals especially Cr above permissible making it potentially toxic. Usually tanning industries discharge their wastewater into nearby rivers and indirectly is being used for irrigation of crops and vegetables. This practice has ultimately led to movement of potentially toxic metals from water to plant system and ultimately to human beings. It is well known that Cr (VI) is a potent carcinogen to humans and animals as it enters cells via surface transport system and gets reduced to Cr (III) inducing genotoxicity. Thus, Cr loaded effluent used for irrigation disrupts several physiological and cytological processes in cells leading to reduced root growth, biomass, seed germination, early seedling development, and induces chlorosis, photosynthetic impairment and finally leading to plant death . Previous studies have shown tannery effluent and Cr induced various chromosomal abnormalities in plant cells thereby severely reducing mitotic index and root growth.

We have chosen onion (Allium cepa) as our model , as it is good genetic models for the assessment of environmental pollutants. Due to their sensitivity in evaluation of the genotoxicity of dangerous /harmful chemicals, but also due to potential of assessing numerous genetic end points, varying from point mutation to chromosome aberrations and micronucleus formation in cells. Second model used to evaluate effluent toxicity was Duckweed, Lemna minor L., as it is a suitable plant model for toxicity evaluation of many contaminants due to its small size and rapid growth and possess the ability to accumulate huge quantities of heavy metals. The effluent also results in reduction of root growth, decoloration of the frond etc. Because of the potential environmental and human health impact connected with the heavy use of Cr, the aim of the present study was to investigate the physico-chemical properties of tannery effluent and evaluate the phytotoxic, cytotoxic and clastogenic effects of tannery effluent and using Allium cepa bioassay.

Materials and Methods:

Cultivation Of Allium cepa : Effluent samples were collected in plastic container of 5-liter capacity from the point of discharge, close to the effluent treatment plant (E.T.P.), of tannery industry situated Ranipet, vellore tamilnadu.Onions were purchased and washed with running water to remove contamination of outer covering coat .A known volume of different concentration of tannery effluent (5, 10,15,20, 25, 30, 35,40,45,50 and 55 percent) was poured into different containers. All the onions, germinated and grown in distilled water served as control. Each treatment including control was performed in triplicate and for every container one onion was used. Length of root, was measured with the help of meter scale and minimum root inhibition was calculated as EC 50.

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Cultivation of Lemna minor : Fresh Duck weeds (Lemna minor L.,) were collected from Nursery at VIT University. Tannery effluent was collected from Ranipet, Tamil Nadu. Fronds were separated and taken in different beaker. The ranges of concentration used in the experiment with Duck weeds were 5%, 10%, 15% and 25% for 96 hrs. There were two replicates for each concentration. After, the completions of incubation period leaves were dead when color changes from green to white.

Protein Extraction: Fronds from each test concentration were crushed in pestle and mortar with 5ml of Potassium Phosphate buffer and collected in 15ml centrifuge tubes. And the tubes were centrifuged at 12,000rpm for 20mins which removes turbidity. The clear supernatant was collected.

Estimation of Protein by Lowry's Method: For preparation of analytical reagents for Lowry's estimation, 50 ml of 2% sodium carbonate was mixed with the 50 ml of 0.1 N sodium hydroxide solutions to make Reagent A. Further, 10 ml of 1.56% copper sulphate solution mixed with 10 ml of 2.37% sodium potassium tartarate solution to make Reagent B. To prepare the Reagent C for the test 1 ml of reagent B was mixed with 50 ml of Reagent A. 0.2 ml of different protein samples of Lemna minor was taken in separate test tubes. 2 ml of Reagent C was added in each test tube and the mixture was incubated for 10 minutes at room temperature. After 10 minutes incubation, 1 ml of commercially prepared Folin-Ciocalteau solution was added in each test tube and was again kept for incubation in dark for 30 minutes. Finally the absorbance of the protein solution was measured at 660 nm (L. Li and X.Z.H. Jiao, 1999).

Determination of photosynthetic pigments and soluble protein: Approximately 150 mg of Lemna fronds of different concentrations were homogenized on ice with mortar and pestle in 3 ml of 66 mM phosphate buffer, pH 7.2 with 10 mM KCl sequentially. The homogenate was extracted with 80% of cold acetone and control was stored for standardization. The absorbance of pigment extract was measured at wavelength of 470,537, 647, 663 and 730 nm with spectrophotometer (Wenhua et al., 2007). The contents of chlorophyll a, chlorophyll b, and carotenoid were calculated in accordance with experimental equations as described by Lichtenthaler (Lichtenthaler, 1987).

RESULTS:

Root growth inhibition:

It was observed that the tannery effluent at low concentrations enhanced the growth of root in Allium cepa . But it started inhibiting the root growth beyond a concentration of 20% and at the concentration of 45 % it totally inhibited the growth of root of A.cepa . No root growth of Allium cepa were observed from a concentration of 45 % of tannery effluent. The root growth of Allium cepa started from 1.5cm in 5% of tannery effluent and gradually increased to 2.1cm, 2.5cm and 2.8cm with the higher concentration of tannery effluent, that is, 10%, 15 % and 20% respectively. But the root growth started decreasing from 2.2 cm, 1.8cm, 0.4cm, 0.2cm to no growth in a concentration of tannery effluent ranging from 25 % to 55 %. The EC50 (the concentration of effluent causing 50 % of total damage ) of Allium cepa exposed to tannery effluent was found to be 12 % as shown in figure 2.

Table 1: Root growth inhibition Allium cepa exposed to various concentration of tannery effluent

Concentration of tannery effluent (%)

Root length (cm)

Root Growth (%)

0

5.5

100

5

1.5

27.2

10

2.1

38.1

15

2.5

45.4

20

2.8

50.9

25

2.2

40

30

1.8

32.7

35

0.4

7.2

40

0.2

3.6

45

0

0

50

0

0

55

0

0

Figure 1: Onion root growth inhibition due to tannery effluent exposure

Figure 2: Graph showing the value of EC50

Effect of tannery effluent on the number of fronds of Lemna minor (duckweeds)

Table 2: Decrease in number of fronds of Lemna minor due to tannery effluent exposure

Concentration of tannery effluent (%)

Number of fronds

0 hour

0

42

5

42

10

42

15

42

20

42

The growth of Lemna minor was drastically effected by the tannery effluent. In the initials around 42 fronds were taken for each dilution and the control . After 96 hours, the number of leaves in control increased to 56 which showed enhanced growth in the presence of tape water. But in other dilutions of tannery effluent: 5 %, 10 % , 15 % and 20 % , the number of fronds of Lemna minor decreased gradually from 42 fronds to 38,33, 22 and 18 respectively. The number of fronds decreased with the increase in concentration of tannery effluent. The lowest dilution of tannery effluent showed the highest inhibition in the growth of Lemna minor . Sometimes fronds of of Lemna minor exposed to various dilutions of tannery effluent were found to be whitish in colour which indicated the death of the fronds and in some chlorosis were also observed.

Figure 3: Decrease in number of fronds of Lemna minor due to tannery effluent exposure

Estimation of protein content of Lemna minor exposed to various concentration of tannery effluent:

Table 3: Reduction in protein content of Lemna minor

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Concentration of tannery effluent (%)

Protein concentration (µg/ml)

0

60

5

52

10

50

15

38

20

12

Exposure of tannery effluent to Lemna minor resulted in the rapid decrease in concentration of the protein content . The protein concentration of the control was found to be 60 µg/ml and it gradually decreased with increase in the concentration of tannery effluent. The concentration of protein content reduced to 52 µg/ml, 50 µg/ml, 38 µg/ml and 12 µg/ml with increase in the concentration of tannery effluent. The reduction in protein content was maximum in the highest concentration of tannery effluent.

Figure 4: Reduction in protein content of Lemna minor

Determination of photosynthetic pigments of Lemna minor exposed to various concentration of tannery effluent:

Table 4 : Reduction of photosynthetic pigments of Lemna minor due to exposure of tannery effluent:

Concentration of tannery effluent (%)

Chlorophyll a concentration (µg/ml)

Chlorophyll b concentration (µg/ml)

Carotenoids concentration (µg/ml)

0

6.687

1.00

3.474

5

5.79

0.916

2.95

10

0.907

0.811

1.946

15

0.738

0.800

1.044

20

0.636

0.640

0.360

Exposure of tannery effluent significantly affected photosynthetic pigments of Lemna minor. The chlorophyll a content decreased by only 13.5 % in 5% of tannery effluent. But a sharp fall in concentration were observed in subsequent concentrations of tannery effluent. The chlorophyll a content reduced to 13 %, 11 % and 0.09 % in the increasing concentration of tannery effluent of 10%, 15 % and 20 %.

The chlorophyll b content showed narrow decrease with increase in concentration of tannery effluent. In concentration of 5 % and 10 %of tannery effluent, the pigment reduced by only 8.4 % and 19 % whereas in the other two dilutions of tannery effluent the pigment content decreased to 80 % and 64%.

The Carotenoids contents also showed sharp decrease in the concentration when exposed to tannery effluent. The concentration of the carotenoid of the control was estimated to be 3.474 µg/ml. The pigment content decreased to 85% in a concentration of 5 % of tannery effluent, but the reduction continued by around 56 %, 30 % and 10 % with gradual increase in the concentration of tannery effluent.

Figure 5: Reduction of photosynthetic pigments of Lemna minor due to exposure of tannery effluent

Discussion:

The leather industry mainly causes high influx of Chromium into the biosphere which contributes 40 % of the total industrial use (Barnhart, 1997). In India, the annual outcome of elemental Cr from tanning industries into the biosphere is about 2000-32,000 tons. The entry of chromium into the food chain occurs mainly through the food chain starting from the consumption of plant materials. High concentration of Chromium in tannery effluent can result in loss of vegetation and barren of the land (Dube et al., 2003).

The main effect of chromium on the plant root growth can be observed by decrease in root length and dry weight, increase in root diameter and root hairs. The estimated EC50 value of of Allium cepa exposed to tannery effluent was 12 % which showed similarity with the findings of Gupta et al (2011). His findings stated that the EC50 value of Allium cepa exposed to tannery effluent was 12.5 %. Lower concentration of tannery effluent enhanced the root growth of Allium cepa exposed to it. This result was also in agreement with the findings by Olusegun B. Samuel (2009). Golovatyj et al. (1999) findings showed that root contained the maximum amount of the element contaminant and vegetative and reproductive organs held minimum amount of the contaminant. This high accumulation of chromium in the root is due to the immobilization of chromium in the vacuole of root cells. The inhibition of root growth may be due to inhibition of root cell division/ root elongation or to the extension of cell cycle in the roots.

When exposed to higher concentration of tannery effluent containing chromium; it can also lead to phyto-genetic toxicity in plant. The main symptoms of the toxicity by chromium includes reduction in root length , leaf chlorosis, inhibition of seed germination and depressed biomass (Sharma et al., 1995). Certain iron containing enzymes such as catalase, peroxidase, a cytochrome oxidase can also be affected by chromium. Major toxicity of chromium in plant can observed by with respect to photosynthetic pigment, photosynthesis and protein content of some algae (Rai et al., 1992). It can also result in chlorosis, necrosis and change in the concentration of essential minerals in plant. Earlier also several workers have reported inhibition of chlorophyll biosynthesis by metal in higher plants (Baszinsky et al., 1980, Prasad and Prasad, 1987) and in algae (Defil ippis and Pal aghy, 1976 and Hamp and Ziegler, 1981). Reduction in photosynthesis may result from the stomatal closure and alteration in ultra structure of chroloplast. High concentration of cobalt, chromium and copper greatly affect the concentration of iron, chlorophyll "a" and "b", protein and catalase activity in plant.

The results showed that the protein concentration of Lemna minor of the control was 60 µg/ml and it gradually decreased with increase in the concentration of tannery effluent. The concentration of protein content reduced to 52 µg/ml, 50 µg/ml, 38 µg/ml and 12 µg/ml with increase in the concentration of tannery effluent. The reduction in protein content of Lemna minor was maximum in the highest concentration of tannery effluent. Almost similar results were found by Lal et al(2009). Al. His results stated that the free amino acid content of control was 0.90 mg/g. free amino acid content decreased to 0.55 mg/g with increase in the concentration of tannery effluent. Chromium toxicity can reduce the size of the peripheral part of the antenna complex in chloroplast which can ultimately decrease the chlorophyll a content. It can also lead to destabilization and degradation of the proteins of the peripheral part of antenna reducing the chlorophyll b content of the plant. Under the Chromium stress , there can be inactivation of enzymes which are involved in the biosynthesis pathway of chlorophyll ; ultimately reducing the chlorophyll content as a whole.

Conclusion:

From the result obtained, it can be concluded that the tannery effluent from Ranipet, Vellore is highly toxic to both Allium cepa and Lemna minor as it drastically affected the root growth, protein and chlorophyll content respectively. Since the experimental conditions were carried out using diluted form of effluent , it can be interpreted that in reality the agricultural crops when directly exposed to the effluent can be more toxic. It can also act as major water and soil pollutant in that area which may result in loss of crop yield and can turn the land barren.

Acknowledgement:

It is our great pleasure to acknowledge the assistance and contributions of those who have played an important part in the successful completion of this project.

We express our heartfelt gratitude to the Director of SBST, Dr. Anilkumar Gopinathan and the management of VIT , for permitting us to use the facilities in the laboratory for experimental work.