Changes in pigment content (chlorophyll and carotenoid ......Carotenoid content of fresh leaf sample...

International Journal of Scientific & Engineering Research, Volume 4, Issue 6, June-2013 2706 ISSN 2229-5518

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Changes in pigment content (chlorophyll and carotenoid), enzyme activities (catalase and peroxidase), biomass and yield of rice plant (Oriza sativa.L) following irrigation of rice mill wastewater under pot culture conditions

Abanti Pradhan , Sanjat K Sahu , Aditya K Dash ABSTRACT A work was undertaken to study the changes in pigment (chlorophyll and carotenoid) content, enzyme activities (catalase and peroxidase), biomass and yield of rice plant (Oriza sativa. L) following irrigation of rice mill wastewater under pot culture conditions. Prior to the experiment, the physic-chemical characteristics of the rice mill wastewater were measured. The wastewater revealed an alkaline pH (8.0) with low concentration of DO (0.9 mg/ l), moderate concentration of COD (630 mg/l), TDS (670 mg/ l) and high concentration of total suspended solids (530 mg/ l) and BOD ( 450 mg/l). Moreover the wastewater was rich in sodium (235 mg/l), total phenols (35 / l) as well as silica (58 mg /l). The experiment was carried out during the month of winter (December- February) when prevailing atmospheric temperature was between 10- 300C. Twenty cemented pots of 40 cm (l) X 40 (b) cm X 80(h) cm, each filled with 10kg of soil, were used for the pot experiment. Different concentrations (0.25, 50, 75 and 100%) of rice mill wastewater were irrigated to the pots before plantation of rice saplings. Twenty days old Jaya T 90 variety of rice saplings were planted in the pots with three saplings in each pot. Biochemical analysis (chlorophyll, carotenoid, catalase and peroxidase) of plant leaves was carried out at 15 days interval until the rice plants started drying (i.e. up to 90 days). At the time of harvest, above ground biomass (stem, leaf and grain) and below ground biomass and many yield parameters like the length of the panicle, number of panicle/plant, filled grains / panicle, weight of panicle, grain yield/ plant, harvest index, shoot / root ratio and root/shoot ratio were recorded. The values of four replicates were averaged for each parameter. The results showed that pigment content (Chlorophyll-a, b and total carotenoid) of the 75 and 100% effluent irrigated rice plants was remarkably inhibited with significant increase in catalase and peroxidase activity at higher concentrations (75 and 100%) of rice mill wastewater. The significant decrease in grain and straw yield of rice plant irrigated with higher concentrations of rice mill wastewater (75 and 100%) was also observed. However, a marked increase in pigment content, biomass and yield parameters was seen up to 50% effluent treatment. The difference in responsiveness of rice plant may be due to the fact that dilution of effluent brings down the toxicity and favours the productivity. Therefore, from the above study it may be concluded that the rice mill waste water may be diluted to 50% before use for cultivation of rice. Keywords: biomass, carotenoid, catalase, chlorophyll, peroxidase, rice mill, wastewater, yield 1. INTRODUCTION Rapid increase in human population in developing countries, more particularly in India, during last few decades has put a tremendous pressure on existing natural resources. For example, increased per capita water consumption and increased water requirements for industry and irrigation have resulted in considerable decrease of usable water resources [1]. Industrialization has its inevitable effect on pollution of air, water and soil based on the type of industry, nature of raw material used and the manufacturing processes, involved [2], [3], [4], [5], [6], [7], [8], [9] . Industrial effluents are being used for irrigation in dry areas. These effluents not only contain nutrients that

enhance the growth of crop plants but also have other toxic materials [8], [10]. Therefore, it is essential that the implications of the use of industrial effluents in the crop field should be assessed before they are recommended for use in irrigation. Keeping the above facts in view a work was undertaken to study the suitability of rice mill waste water for cultivation of rice (Oriza sativa. L) under pot culture conditions. The parameters selected for the study are pigment (chlorophyll and carotenoid) content, enzyme activities (catalase and peroxidase), biomass and yield.

———————————————— • Dr. Abanti Pradhan is currently working as Assistant Professor in Environmental Engineering in Institute of Technical Education and Research, under Siksha ‘O’

Anusandhan University, Odisha, India. E-mail: [email protected] • Prof. Sanjat K Sahu is currently working as Professor, PG Department of Environmental Sciences, in Sambalpur University, Odisha, India. E-mail:

[email protected] • Dr. Aditya K Dash is currently working as Assistant Professor in Environmental Engineering in Institute of Technical Education and Research, under Siksha ‘O’

Anusandhan University, Odisha, India. E-mail: [email protected]

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2. MATERIALS AND METHODS The effluent of a nearby rice mill having milling capacity 10 MT/day has been used for the experiment. The

physicochemical characteristics of the effluent (Table 1) were analyzed following the procedures recommended by APHA [11] and values have been given in table-1.

Table1. Physico-chemical characteristics of the effluent of a rice mill at Sambalpur, Orissa with their maximum permissible

limits as recommended by Indian Standard Institution.

Parameters Range Mean± SD ISI limit for discharge of industrial effluents

on land for irrigation (ISI,1977).

into inland surface waters (ISI, 1974)

Colour Brown - - Odour Unpleasant - - Temperature (0 C) 35.0-48.0 38.0±5.09 - 40 Conductivity (m mho cm-1) 0.46-0.86 0.66± 0.15 - - pH 7.2 - 8.8 8.0±0.54 5.5-9.0 5.5-9 Total solids (mg l-1) 998.1-1459.1 1200.0±189.48 - - TSS (mg l-1) 432.5-576.0 530.0±53.00 100 100 TDS (mg l-1) 522.1-883.1 670.0±149.2 2100 2100 Dissolved oxygen (mg l-1) 0.2-1.6 0.9±0.52 - - BOD at 20 0C (mg l-1) 312.1-540.1 450.0±76. 61 100 30 COD(mg l-1) 400.2-892.1 630.0±183.03 - - Total Alkalinity (mg l-1) 180.7-340.1 272.0±58.29 - - Total hardness (mg l-1) 98.3-256.4 182.0±59.84 - - Ca hardness (mg l-1) 38.4-98.3 78.0±22.22 - - Mg Hardness (mg l-1) 14.1-24.3 21.0±3.68 - - Chloride (mg l-1) 95.1-170.3 140.0±28.06 600 1000 Sulphate (mg l-1) 28.4-70.1 40.0±15.66 1000 1000 Phosphate (mg l-1) 10.1-35.2 21.0±11.11 - - Nitrate (mg l-1) 0.3-0.8 0.5±0.15 - - Sodium (mg l-1) 213.4-263.7 235.0±20.34 60% - Potassium (mg l-1) 14.1-32.1 20.0±7.12 - - Phenols (mg l-1) 13.3-50.4 35.0±13.98 - 1.0 SiO2 (mg l-1) 35.4-75.1 58.0±15.5 - -

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2.1 Experimental design for pot culture study. Twenty cemented pots of 40 cm (l) X 40 (b) cm X 80(h) cm were used for the pot experiment. Pots were filled with 10kg of soil each, collected from the upland non-irrigated paddy field, where field experiment was initiated. Different concentrations (0, 25, 50, 75 and 100%) of rice mill wastewater were prepared in dilution with de-chlorinated tap water supplied from river the Mahanadi and irrigated to the pots before plantation of rice saplings. For each concentration four replicate were kept. Twenty days old Jaya T 90 variety of rice saplings, were planted in the pots with three saplings in each pot. A standing water of 5 cm using different concentrations of rice mill effluent was maintained through out the cropping period except planting and pre-harvesting. The experiment was carried out during the month of winter (December- February) when prevailing atmospheric temperature was between 10- 300C. Biochemical analysis of plant leaves was carried out at 15 days interval until the rice plants started drying (i.e. up to 90 days). The values of four replicates were averaged for each parameter at each sampling occasion. 2.1.1 Pigment Content --- The Chl-a, Chl-b and Carotenoid content of fresh leaf sample was measured by extracting the pigment with 80% chilled acetone. The absorbance at 475,645 and 663 nm were read in a Spectrophotometer (Systronics, 106 model) and the calculation was made as per the following equation of Arnon [12] .

a. Chl –a (mg/ g fresh tissue) 12.7 A (663)-- 2 .69 A (645) x V/ (1000 x W)

b. Chl- b (mg g fresh tissue-1) 22.9 A (645) – 4.68 A (663) x V/ (1000 x W)

c. Carotenoid (mg g fresh tissue -1) A (475) 10 x V / (2500 x W)

Where A - Absorbance at specific wavelength V -Final volume of the extract W - Fresh weight of leaves taken for extraction

2.1.2 Ezymes activities. The fresh weighted leaf sample was homogenized in chilled 0.1 M Sodium phosphate buffer (pH-7) and centrifuged at 40C for 10 minutes at 7000 rpm. The supernatant was used as the enzyme extract for the assay of both Catalase and Peroxidase [13] . 2.1.2.1 Catalase : One ml of enzyme extract was added to the reaction mixture containing 1ml of 0.01 M hydrogen peroxide and 3 ml of 0.01 M Sodium phosphate buffer (pH 7). After incubating at 200C for 10 minutes the reaction was stopped by adding 10 ml of 2 % Sulphuric acid (H2SO4). The acidified reaction mixture with or without the enzyme extract was titrated against 0.005 N KMn04 to determine the quantity of hydrogen peroxide utilized by the enzyme. The catalase activity was calculated in milli moles of hydrogen peroxide utilized per gram fresh weight per minute. 2.1.2.2 Peroxidase: One ml of enzyme extract was added to the reaction mixture containing 2 ml of 0.1M sodium phosphate buffer (pH 7), 1ml of 0.01M Pyrogallol and 1 ml of 0.01M H2O2. After incubating at 200C for five minutes the reaction was stopped by adding 5% Sulphuric acid (H2SO4). The concentration of Purpurosgallin formed was determined spectrophotometrically at 430nm. The enzyme activity was calculated as absorbance per gram fresh weight per minute. 2.1.3 Biomass and yield : At the time of harvest, above ground biomass (stem/leaf and grain) and below ground biomass (root) were separated and dry weight of each part was determined (by oven drying at 850C for 48 hours) and the values were expressed in gm dry wt /plant. The length of the panicle (cm), number of panicle/plant, filled grains / panicle, weight of panicle (g dry wt), grain yield/ plant (g dry wt), Harvest index, Shoot/root ratio and Root/shoot ratio were calculated.

3. RESULTS 3.1 Pigment Content 3.1.1 Chlorophyll – a . The chlorophyll-a content of 20 days old rice plants at the time at plantation was 1.015 ± 0.103 mg/g fresh weight (Table 2). The chlorophyll-a content increased with increases in the age of the plant up to 60 days at all the concentrations of wastewater treated soil and decreased thereafter. When comparison was

made with their counterpart in control condition at different day’s intervals, the chlorophyll a content showed maximum increased (32.6%) up to 50% rice mill wastewater treated soil. At higher concentration of treatment (75 and 100%) the chlorophyll-a content decreased and maximum decrease of 33.7% was noticed at 100% effluent treated pots (Figure 1).

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Percentage increase/decrease in chlorophyll-a content of rice plant (Oryza sativa )

-50

-30

-10

10

30

50

0 15 30 45 60 75 90

Days

% c

hang

e ov

er c

ontro

l

25%

50%

75%

100%

Figure-1

Statistical analysis of the data by two-way ANOVA showed significant difference between days as well as treatments (F1=22.45, F2= 20.17, P < 0.05), whereas, one-way ANOVA test showed significant difference between treatments from 30 days onwards till the end of the

experiment (F ≥ 10.99, P< 0.05), (Table 2). LSD test however reveal that the chlorophyll-content was significantly higher from 45 days onwards in 25 and 50% effluent treated pots and significantly lower from 45 days onwards in 75 and 100% effluent treated soil pots.

Table 2. Chlorophyll-a content (mg/ g fresh wt.) of rice plant (O. sativa L) in different percent of effluent treated pots under laboratory conditions

Days

Control

Concentration of effluent (%) One-way ANOVA (F)

LSD (P



Percentage increase/decrease in chlorophyll-b content of rice plant (Oryza sativa )

-50

-30

-10

10

30

50

0 15 30 45 60 75 90

Days

% c

hang

e ov

er c

ontro

l

25%

50%

75%

100%

Figure-2

Two-way ANOVA test showed significant difference in chl.-b content of rice plant between days as well as concentrations (F1= 17.13, F2 = 34.08, P < 0.05). However, one-way ANOVA test also showed significant difference between different treatments after 30, 45, 60, 75 and 90

days (F≥7.52, p



Percentage increase/decrease in carotenoid content of rice plant (Oryza sativa )

-50

-30

-10

10

30

50

0 15 30 45 60 75 90

Days

% c

hang

e ov

er c

ontro

l

25%

50%

75%

100%

Figure-3

Statistical analysis of data by two-way ANOVA test showed significant difference in the carotenoid content of rice plants with respect to different days as well as concentrations of wastewater treatment (F1 = 15.15, F2 =

54.44, p < 0.05). However significant difference in carotenoid content of rice plant were also observed between different treatments (F≥ 6.69, P< 0.05) from 30 days onwards and was continued up to 90 days (Table 4).

Table 4. Carotenoid (mg/g fresh wt) of rice plant (O. sativa L) in different percent of effluent treated pots under laboratory conditions

Days

Control

Concentration of effluent (%) One-way ANOVA (F)

LSD (P



100% effluent treated pots after 90 days, indicating a maximum increase in higher concentration (75 and 100%)

of effluent treated soil (Fig.4).

Figure-4

Statistical analysis of peroxidase activity by two-way ANOVA test showed significant differences between days and treatments (F1 = 21.96, F2 = 250.69, P < 0.05). One-way ANOVA test however showed significant differences from 45 days onwards (F≥ 4.51, P.0.05). But

comparison of LSD, values between mean peroxidase activity of control and experimental plants showed significant difference from 45 days onwards only in case of 75 and 100% effluent treated pots (Table 5).

Table 5. Effect of rice mill waste water on peroxidase activities (change in OD/g fresh tissue / min. x 10-3) of rice plant (O. sativa. L) under laboratory condition

Days

Control Concentration of effluent (%) LSD (P



after 90 days and during the same period the increase in 25 and 50% effluent treated pot was 7.09 and 10.6% over

control respectively (Figure 5).

Percentase increase/decrease in catalase activity of rice plant (Oryza sativa )

-5

5

15

25

35

0 15 30 45 60 75 90Days

% c

hang

e ov

er c

ontro

l

25%

50%

75%

100%

Figure-5

Two-way ANOVA test of catalase activity showed a significant differences with respect to days and concentrations (F1 =38.54, F2 = 125.43, P



3.3 Biomass and Yield. The effect of rice mill effluent on total plant biomass and yield or rice plant at the time of

harvest under pot culture has been given in Table 7.

Table 7. Effect of rice mill effluent on total biomass and yield (g dry wt. /plant) of rice plants (O. sativa.L ) at the time of harvest under pot culture

Sl. No

.

Parameter studied Control Concentration of effluent (%) One-way ANOVA (F)

LSD (P



and 100% effluent irrigated pots respectively. Weight of unfilled grain was found to be less in all the effluent treated pots but significantly less in 50% effluent treated pots. Average weight and grain content of single main panicle was respectively increased by 24.98% and 24.9% in 50% effluent treated pots and respectively decreased by 20.5 and 39.27 % in 100 % wastewater treated pots. One-way ANOVA test showed significant differences (P



Murumkar and Chavan [36] studied the activity of these enzymes in the gram treated with wastewater and observed that an increase peroxidase activity might be contributing to growth suppression at high dose. 4.3 Biomass and Yield. From the field experiment results, it was observed that the effect of rice mill wastewater irrigation was not so much pronounced at the initial stages. But as the irrigation continued, the effect was remarkably visible particularly between tillering and maturity. Since the plant growth and yield is directly related with pigment content, any decrease in its amount clearly reflects the hazardous nature of the effluent “[37], [8], [24]” . The reduction in growth, biomass and yield of rice plant in experimental plots may thus be due to influence of alkaline pH and accumulation of silica, organic matter, sodium and phenolic compounds. The significant decrease in grain and straw yield of rice plant irrigated with higher concentrations of rice mill wastewater (75 and 100%) under pot conditions may be due to the fact that stressed plants in hostile environment require more energy towards survival than what could have been ordinarily available for growth and yield process. This corroborates our findings with rice in effluent irrigated plots under field conditions. The data for above ground, belowground biomass / plant, weight of filled grain/ plant, nos of panicle/ plant, weight of filled grain / panicle, nos of filled grain/ panicle etc. under pot conditions further indicate that the values of these parameters were maximal in plants irrigated with 50% effluent. The difference in responsiveness of rice plant may be due to the fact that dilution of effluent brings down the toxicity and favours the productivity. 5. CONCLUSION Industrial effluents are being used for irrigation in dry areas. These effluents not only contain nutrients that enhance the growth of crop plants but also have toxic materials. In the present investigation, it is found that the application of diluted form of rice mill wastewater was encouraging the growth of rice plant. Therefore, it is recommended that, rice mill wastewater which contain different nutrient can be used for agricultural practices after careful dilution. REFERENCES [1] Nawaz, S.; Ali, S. M.; Yasmin, A., “Effects of

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Changes in pigment content (chlorophyll and carotenoid), enzyme activities (catalase and peroxidase), biomass and yield of rice plant (Oriza sativa.L) following irrigation of rice mill wastewater under pot culture conditionsAbanti Pradhan , Sanjat K Sahu , Aditya K DashABSTRACT1. INTRODUCTION

Keeping the above facts in view a work was undertaken to study the suitability of rice mill waste water for cultivation of rice (Oriza sativa. L) under pot culture conditions. The parameters selected for the study are pigment (chlorophyll and caroteno...2. MATERIALS AND METHODS

2.1 Experimental design for pot culture study.Twenty cemented pots of 40 cm (l) X 40 (b) cm X 80(h) cm were used for the pot experiment. Pots were filled with 10kg of soil each, collected from the upland non-irrigated paddy field, where field experiment was initiated. Different concentrations (0,...2.1.2 Ezymes activities.3. RESULTS3.1 Pigment Content3.2 Enzymes Activities3.3 Biomass and Yield. The effect of rice mill effluent on total plant biomass and yield or rice plant at the time of harvest under pot culture has been given in Table 7.From the table it is evident that the above ground biomass was increased by 19.2% and decreased by 34.43% in 50 and 100% effluent treated pots respectively. Similarly the total plant biomass was also increased by 20.4% and decreased by 32.8% in 50% an...

4. DISCUSSION

Changes in pigment content (chlorophyll and carotenoid ......Carotenoid content of fresh leaf sample...

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