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The effect of paper industry effluent on growth, pigments, carbohydrates and proteins of rice seedlings
Environmental Pollution 72 (1991) 159 167 Y~': ~:. :7'
The Effect of Paper Industry Effluent on Growth, Pigments, Carbohydrates and Proteins of Rice Seedlings R. N. Misra Department of Botany, Government Science College, Chatrapur 761020, Orissa, India
& P. K. Behera Plant Physiology Laboratory, Botany Department, Berhampur University, Berhampur 760007, India (Received 3 April 1990; revised version received 3 December 1990: accepted 6 December 19901
ABSTRACT The effect t~f paper industry effluent on the growth and content of certain macromoleeules of seedlings of rice (Oryza sativa L. cv. Kesari-82K ) has been examined. The effects were investigated in relation to both concentration ~/" effluent and time of exposure to the effluent. Percentage of germination, wtaer imbibing capacity, growth, pigment, carbohydrate and protein content showed a decreasing trend with increase in effluent concentration and time. Protein content was the most sensitive macromolecule affected by effluent. Measurement of protein and protein enzymes might therefore provide a use/hi criterion fi~r the evaluation of the phytotoxici O, of'effluent released from the pulp and paper industries.
INTRODUCTION It is well known that living in the neighbourhood of a paper mill gives moderately increased environmental risks (Olin et al., 1987). Pollution arises from the pulp and paper industries, with the discharge of untreated effluents 159 Environ. Pollut. 0269-7491/91/$03"50 ,© 1991 Elsevier Science Publishers Ltd, England. Printed in Great Britain
160
R. N. Misra, P. K. Behera
contaminating the environment to some degree (Freedman & Jaggi, 1986). Physico-chemical characteristics have been estimated in paper mill effluent, and the impact of this pollutant on river water has also been established (Reddy & Venkateswarulu, 1986). Moreover, with regard to the impact of industrial effluents on living organisms, studies on physiological effects usefully estimate the phytotoxicity of the effluent. 'Physiological effects', defined as the effect of sublethal concentrations of toxicants on plant metabolism and plant composition, should be more extensively investigated for industrial effluents. Considerable change in the 'physiological effect' is observed in crops grown in soils contaminated with even moderate levels of some industrial effluents. For example, it has been reported that rice seedlings grown in sugar factory effluent show retarded shoot and root growth, inhibition of root primordia and coleoptile emergence (Behera & Misra, 1982) and decreased synthesis of various cellular macromolecules (Behera & Misra, 1983). The physiotoxicity and cytotoxicity effects of the released effluent of a papermill in a modified Allium test have also been reported (Misra & Sahu, 1983). In order to obtain a better understanding of the basis of the 'physiological effect', in the present paper we report the effects of paper industry effluent on growth, chlorophyll, carbohydrate and protein content of seedlings of rice (Oryza sativa L. cv. Kesari 82K).
MATERIALS A N D METHODS The paper industry (J. K. Papermill, Rayagada, Koraput), situated in the Eastern Ghats of Orissa about 210km from Berhampur University (83 ° 27' E longitude and 19° 09' N latitude), releases its effluents into the river 'Nagabali'. The effluent was sampled from time to time during the experiments from the existing release point, before it falls to the river stream. Seeds of rice (Oryza sativa L. cv. Kesari-82K) were soaked in distilled water at 2 5 _ 2°C for 12 h and placed in sterilised petri dishes which contained soaked filter papers. One hundred seeds were evenly placed in each petri dish. A measured quantity (10ml) of distilled water (Control) and of test solutions (different concentrations of the effluent, i.e. 25, 50, 75 and 100% v/v) were added to each set and were exposed for 6, 12, 18 and 24h. Accordingly, samples were taken from the treated dishes at 6 h intervals and the seedlings then grown in distilled water in a growth chamber under fluorescent illumination of 2000 lux at 28 _+2°C until 7 days old. A measured quantity (5 ml) of distilled water was added to each dish to moisten the filter paper when necessary. The pH was maintained between 4-5 and 5"0 during the growth period (Yoshida et al., 1972). Each treatment and control was
Effect of paper industry effluent on rice seedlings
161
replicated five times. Two sets of seedlings were tested in each experiment. In the first set, the following measurements of the seedlings were made on the 7th day, random samples being taken from each treatment: (1) percentage of germination, (2) percentage of water imbibing capacity, (3) shoot and root length and (4) tolerance index (TI). The tolerance index (TI) of each seedling population against each of the effluent concentrations was calculated according to the tolerance testing of rice species by the procedure of Wilkins (1978). In the second set, chlorophyll, carbohydrate and protein controls were measured.
Chlorophyll estimation Pigments of the chloroplasts were extracted with 80% acetone and the amount of total chlorophyll was estimated by the procedure of Arnon (1949).
Carbohydrate estimation Carbohydrate contents of shoot and root were measured according to the method of Yemm & Willis (1954), using glucose as a standard.
Protein estimation The TCA insoluble protein of shoot and root was measured by the procedure described by Lowry et al. (1951), using Bovin serum albumin as a standard.
Statistical analysis The data obtained of different measurements and analyses were subjected to different statistical analyses (Gomez & Gomez, 1984). The coefficients of correlation (r) between the concentrations of effluents and macromolecule content of plant-parts against each exposure time were calculated by regression analysis. Levels of significance (P) were determined with d f = 9.
RESULTS A N D DISCUSSION The results of the effect of four concentrations of papermill effluents, i.e. 25, 50, 75 and 100% (v/v), on percentage of germination, water imbibing capacity, root and shoot length and tolerance index of the root in seedlings of Oryza sativa are given in Table 1. The data indicate that exposure to
Number of seeds
1 000
1 000
1000
1 000
1 000
Concentration of eJfluent
Control
25%
50%
75%
100%
6 12 18 24
6 12 18 24
6 12 18 24
6 12 18 24
6 12 18 24
Period of treatment (h)
16-5 _____1-3 13"2+1"2 8.7 +- I-1 5"3 -+_0'7
21-5 _+ 1-5 19.8_+1.4 17"3-+ 1'3 14"6 +- 1"2
28"3+1.9 25"6+ 1"4 22.1 +- 1'3 20"3-+1'2
36"6 ± 2"1 27"8+ 1"9 24-2+1.6 22.8_+ 1"4
53.7 ± 1.2 60.6+ 1.3 65-3 ___2.7 74-4+4-7
Percentage of germination
42-3 ± 1"4 35.2_+1-2 21'3 ± 1"2 12.2 +- i'I
62"6 _+ 3"3 51"2__+2-4 42"7+-2"3 38"4 ± 2"3
73"3+3"2 68"6-+ 1-3 52"4-+3"1 49-3+-4"3
78"2 -+ 1'0 70"2+___2'4 61"5+1"9 59-6+2"6
88'0 -t- 1.4 88-1 + 1.3 88.2 + 1-3 88-3+1.3
Water imbibing capacity (%)
0-64 + 0'0 0"51±0-1 0"35 + 0"1 0"28 +- 0-0
0"91 _+0"1 0"75+0"0 0"62_+0-0 0"83 ± 0-0
1"08+0-0 0"91 - + 0 " 1 0"90+0-1 1"03+0"0
1"38 + 0'1 1'21 -+_0"I 1"11 +0"1 1"21 +0-1
1.63 + 0"2 1.62+0-1 1-64 _ 0"2 1.59+0-1
Length of root (cm)
1-88 ± 0"0 1"31_+0-0 1"17 _+0"0 0'86 +- 0-0
2"36 _+0"1 2"11__+0-0 2"01 _+0'1 2"16 ± 0-1
2"99+0-1 2"90+-0"1 2"51 -+0-1 2"14-+0'1
3"55 + 0'1 3"24+0-2 3"17+0"1 2"98+0-2
4"88 -+ 0-2 4.89+0.1 4-87 + 0.1 4.79+0-1
Length of shoot (cm)
50-9 _+ 1"5 39'5 ± 1-1 32-1 _+ 1-0 17.7 _+ 1"0
63"2 _+ 1"! 56'2__+3'4 47'2+ 1"1 39"2 _+2-0
74-2+3-1 66-7 +2"1 57-2-t- 1'6 57"0_+2-3
90"0 __+2"1 85"2_____2"0 76"1__+1"0 70"3-+2"8
ToleranceIndex (TI) of root
TABLE 1 Percentage of Seed Germination, Water Imbibing Capacity, Root and Shoot Length and Tolerance Index of Root of 7-day-old Seedlings of Oryza sativa L. cv. Kesari-82K under Different Concentrations of Paper Industry Effluent. Values are Mean + SD of 100 Samples
.~
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.~
t,9
5O-
r U
_o
e,i
'-
O u
10-
20-
30-
~¢- 40-
E
01
60-
Fig. 1.
18hr
r= - 0 . 7 4 1 p
1
T i m e of e x p o s u r e t o e f f l u e n t
12hr
p <0.06
r= - 0 - 6 6 6
r= - 0 . 8 3 1
24 hr
I
/
effluent
100°/o
./25°1o effluent ~. j S O °/o e f f l u e n t ~75°/o effluent
.Control
p < 0.01
Effect of effluent on total chlorophyll content of rice seedlings.
6hr
r = - 0-632 p
2"
&
O"
10-
20-
30-
O-
10-
~- 20-
u
L_
0
~ 4o-
r 0 U
E v
O1
"':
30-
40 ¸
Fig. 2.
6hr
1
18hr
r= -0-834 <0"01
18hr
r = - 0.941 p <0.001
Time of exposure to effluent
12hr
r =-0-727 p
12hr
1
r= -0-762 )<(}01
24 hr
100Olo effluent
25 ~/o e f f l u e n t 50 /°effluent 5°1o e f f l u e n t
Control
r= -0.937 < 0-001
24 hr
1O0 °1° effluent
50°/o e f f l u e n t 5°1° e f f l u e n l
< (}001
r = -0.973
Effect o f effluent o n total c a r b o h y d r a t e c o n t e n t o f rice seedlings.
r = -0.721 p <0.02
6hr
r = -0.731 < 0.02
4"
Effect o f paper industry effluent on rice seedlings
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effluent, both in concentration and time-dimensions, retarded growth of the rice seedlings. Decrease in seedling growth is associated with increase in effluent concentration. The above results are in agreement with the earlier results of Rajannan and Oblisamai (1979) and Behera and Misra (1982), but contradictory to those of Choudhury et al. (1989). The contents of pigments, protein and carbohydrate of the leaf extracts of rice seedlings grown in several concentrations of effluent are illustrated in Figs 1-3. Compared with the control, the effluent showed adverse effects at all concentrations on chlorophyll, protein and carbohydrate contents. These results are similar to the earlier results of Behera and Misra (1983), but do not agree with those obtained by Choudhury et al. (1989). The longer the exposure of the rice seedlings to the effluent the lower is the level of these cellular constituents. A gradual decline with time of exposure to effluent and with concentration of effluent in the protein and carbohydrate contents seems to be correlated with the loss of chlorophyll. Of the two studied macromolecular components of rice seedlings, protein seemed to be more susceptible to effluent stress (Figs 1 and 3). Our data on the degradation of cellular macromolecules and growth of seedlings suggest that the papermill effluent has greatest effect at prolonged exposure and could lead to decreased growth of rice. The adverse effects may be because the papermill effluent consists of three different types of suspended solids (silicon, cellulose and hemicellulose) together with 18 different types of dissolved chemicals, as reported by Nestmann et al. (1980). At present, a firm conclusion cannot be drawn in these pilot experiments regarding the significance of the decline in protein to a longer duration of exposure. However, the results suggest that in an evaluation ofphytotoxicity and pollution risk, protein, and enzymes involved in protein synthesis, can act as bio-indicators at a particular time of exposure to the effluent.
REFERENCES Arnon, D. I. (1949). Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol., 24, 1-15. Behera, B. K. & Misra, B. N. (1982). Analysis of the effect of industrial effluent on growth and development of rice seedlings. Environ. Res., 28, 10-20. Behera, B.~K. & Misra, B. N. (1983). Analysis of effect of industrial effluent on pigments, proteins, nucleic acids and DCPIP Hill reaction of rice seedlings. Environ. Res., 31, 381-9. Choudhury, S. K., Jha, A. N. & Srivastava, D. K. (1989). Impact of paper mill effluent on germination, seedling growth and pigment content of Hordeum vulgare. Env. Eco., 7(1), 193-5.
Effect of paper industry effluent on rice seedlings
167
Freedman, Martin & Bikki Jaggi (1986). Pollution performance of firms from pulp and paper industries. Environ. Manage., 10(3), 359-66. Gomez, K. A. & Gomez, A. A. (1984). Statistical Procedures for Agricultural Research. (2nd edn) Wiley-Interscience Publication, p. 680. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with Folin phenol reagent. J. Biol. Chem., 193, 265-75. Misra, R. N. & Sahu, R. K. (1983). Studies on physiotoxicity and cytotoxicity of some dyes released from papermills in a modified Allium test. Abst. 6th bzd. Bot. Con£ J. Incl. Bot. Soc. Supp. 62, 66. Nestmann, E. R., Lee, E. G. H., Metula, T. I., Douglas, G. R. & Muller, J. C. (19801. Mutagenicity of constituents identified in pulp and paper mill effluents using the Salmonella/mammalian microsome assay. Mutation Res., 79, 203 12. Olin, Robert G., Anders, Ahlbom., Ingen Hindberg Navier, Stuffan E. Norell & Bo Spannare (1987). Occupational factors associated with astro cytomas: A c~secontrol study. Am. J. Ind. Med., 11(6), 615-26. Rajannan, G. & Oblisamai, G. (1979). Effect of paper factory effluents on soil and crop plants. Intl. J. Environ. 21(2), 120 30. Reddy, P. Manikya & Venkateswarulu, V. (1986). Ecology of algae in the paper mill effluents and their impact on the river Tungabhadra (India). J. Environ. Biol., 7(4), 215-24. Wilkins, D. A. (1978). The measurement of tolerance to edaphic factors by means of root growth. New Phytol., 80, 623 33. Yemm, E. W. & Willis, A. J. (1954). The estimation of carbohydrate in plant extracts by anthrone, Biochem. J., 57, 509---13. Yoshida, S., Forno, D. A., Cock, J. H. & Gomez, K. A. (1972). Laboratoo' Manual Jor Physiological Studies of Rice (2nd edn). The Philippines, The International Rice Research Institute, p. 70.
The Effect of Paper Industry Effluent on Growth, Pigments, Carbohydrates and Proteins of Rice Seedlings R. N. Misra Department of Botany, Government Science College, Chatrapur 761020, Orissa, India
& P. K. Behera Plant Physiology Laboratory, Botany Department, Berhampur University, Berhampur 760007, India (Received 3 April 1990; revised version received 3 December 1990: accepted 6 December 19901
ABSTRACT The effect t~f paper industry effluent on the growth and content of certain macromoleeules of seedlings of rice (Oryza sativa L. cv. Kesari-82K ) has been examined. The effects were investigated in relation to both concentration ~/" effluent and time of exposure to the effluent. Percentage of germination, wtaer imbibing capacity, growth, pigment, carbohydrate and protein content showed a decreasing trend with increase in effluent concentration and time. Protein content was the most sensitive macromolecule affected by effluent. Measurement of protein and protein enzymes might therefore provide a use/hi criterion fi~r the evaluation of the phytotoxici O, of'effluent released from the pulp and paper industries.
INTRODUCTION It is well known that living in the neighbourhood of a paper mill gives moderately increased environmental risks (Olin et al., 1987). Pollution arises from the pulp and paper industries, with the discharge of untreated effluents 159 Environ. Pollut. 0269-7491/91/$03"50 ,© 1991 Elsevier Science Publishers Ltd, England. Printed in Great Britain
160
R. N. Misra, P. K. Behera
contaminating the environment to some degree (Freedman & Jaggi, 1986). Physico-chemical characteristics have been estimated in paper mill effluent, and the impact of this pollutant on river water has also been established (Reddy & Venkateswarulu, 1986). Moreover, with regard to the impact of industrial effluents on living organisms, studies on physiological effects usefully estimate the phytotoxicity of the effluent. 'Physiological effects', defined as the effect of sublethal concentrations of toxicants on plant metabolism and plant composition, should be more extensively investigated for industrial effluents. Considerable change in the 'physiological effect' is observed in crops grown in soils contaminated with even moderate levels of some industrial effluents. For example, it has been reported that rice seedlings grown in sugar factory effluent show retarded shoot and root growth, inhibition of root primordia and coleoptile emergence (Behera & Misra, 1982) and decreased synthesis of various cellular macromolecules (Behera & Misra, 1983). The physiotoxicity and cytotoxicity effects of the released effluent of a papermill in a modified Allium test have also been reported (Misra & Sahu, 1983). In order to obtain a better understanding of the basis of the 'physiological effect', in the present paper we report the effects of paper industry effluent on growth, chlorophyll, carbohydrate and protein content of seedlings of rice (Oryza sativa L. cv. Kesari 82K).
MATERIALS A N D METHODS The paper industry (J. K. Papermill, Rayagada, Koraput), situated in the Eastern Ghats of Orissa about 210km from Berhampur University (83 ° 27' E longitude and 19° 09' N latitude), releases its effluents into the river 'Nagabali'. The effluent was sampled from time to time during the experiments from the existing release point, before it falls to the river stream. Seeds of rice (Oryza sativa L. cv. Kesari-82K) were soaked in distilled water at 2 5 _ 2°C for 12 h and placed in sterilised petri dishes which contained soaked filter papers. One hundred seeds were evenly placed in each petri dish. A measured quantity (10ml) of distilled water (Control) and of test solutions (different concentrations of the effluent, i.e. 25, 50, 75 and 100% v/v) were added to each set and were exposed for 6, 12, 18 and 24h. Accordingly, samples were taken from the treated dishes at 6 h intervals and the seedlings then grown in distilled water in a growth chamber under fluorescent illumination of 2000 lux at 28 _+2°C until 7 days old. A measured quantity (5 ml) of distilled water was added to each dish to moisten the filter paper when necessary. The pH was maintained between 4-5 and 5"0 during the growth period (Yoshida et al., 1972). Each treatment and control was
Effect of paper industry effluent on rice seedlings
161
replicated five times. Two sets of seedlings were tested in each experiment. In the first set, the following measurements of the seedlings were made on the 7th day, random samples being taken from each treatment: (1) percentage of germination, (2) percentage of water imbibing capacity, (3) shoot and root length and (4) tolerance index (TI). The tolerance index (TI) of each seedling population against each of the effluent concentrations was calculated according to the tolerance testing of rice species by the procedure of Wilkins (1978). In the second set, chlorophyll, carbohydrate and protein controls were measured.
Chlorophyll estimation Pigments of the chloroplasts were extracted with 80% acetone and the amount of total chlorophyll was estimated by the procedure of Arnon (1949).
Carbohydrate estimation Carbohydrate contents of shoot and root were measured according to the method of Yemm & Willis (1954), using glucose as a standard.
Protein estimation The TCA insoluble protein of shoot and root was measured by the procedure described by Lowry et al. (1951), using Bovin serum albumin as a standard.
Statistical analysis The data obtained of different measurements and analyses were subjected to different statistical analyses (Gomez & Gomez, 1984). The coefficients of correlation (r) between the concentrations of effluents and macromolecule content of plant-parts against each exposure time were calculated by regression analysis. Levels of significance (P) were determined with d f = 9.
RESULTS A N D DISCUSSION The results of the effect of four concentrations of papermill effluents, i.e. 25, 50, 75 and 100% (v/v), on percentage of germination, water imbibing capacity, root and shoot length and tolerance index of the root in seedlings of Oryza sativa are given in Table 1. The data indicate that exposure to
Number of seeds
1 000
1 000
1000
1 000
1 000
Concentration of eJfluent
Control
25%
50%
75%
100%
6 12 18 24
6 12 18 24
6 12 18 24
6 12 18 24
6 12 18 24
Period of treatment (h)
16-5 _____1-3 13"2+1"2 8.7 +- I-1 5"3 -+_0'7
21-5 _+ 1-5 19.8_+1.4 17"3-+ 1'3 14"6 +- 1"2
28"3+1.9 25"6+ 1"4 22.1 +- 1'3 20"3-+1'2
36"6 ± 2"1 27"8+ 1"9 24-2+1.6 22.8_+ 1"4
53.7 ± 1.2 60.6+ 1.3 65-3 ___2.7 74-4+4-7
Percentage of germination
42-3 ± 1"4 35.2_+1-2 21'3 ± 1"2 12.2 +- i'I
62"6 _+ 3"3 51"2__+2-4 42"7+-2"3 38"4 ± 2"3
73"3+3"2 68"6-+ 1-3 52"4-+3"1 49-3+-4"3
78"2 -+ 1'0 70"2+___2'4 61"5+1"9 59-6+2"6
88'0 -t- 1.4 88-1 + 1.3 88.2 + 1-3 88-3+1.3
Water imbibing capacity (%)
0-64 + 0'0 0"51±0-1 0"35 + 0"1 0"28 +- 0-0
0"91 _+0"1 0"75+0"0 0"62_+0-0 0"83 ± 0-0
1"08+0-0 0"91 - + 0 " 1 0"90+0-1 1"03+0"0
1"38 + 0'1 1'21 -+_0"I 1"11 +0"1 1"21 +0-1
1.63 + 0"2 1.62+0-1 1-64 _ 0"2 1.59+0-1
Length of root (cm)
1-88 ± 0"0 1"31_+0-0 1"17 _+0"0 0'86 +- 0-0
2"36 _+0"1 2"11__+0-0 2"01 _+0'1 2"16 ± 0-1
2"99+0-1 2"90+-0"1 2"51 -+0-1 2"14-+0'1
3"55 + 0'1 3"24+0-2 3"17+0"1 2"98+0-2
4"88 -+ 0-2 4.89+0.1 4-87 + 0.1 4.79+0-1
Length of shoot (cm)
50-9 _+ 1"5 39'5 ± 1-1 32-1 _+ 1-0 17.7 _+ 1"0
63"2 _+ 1"! 56'2__+3'4 47'2+ 1"1 39"2 _+2-0
74-2+3-1 66-7 +2"1 57-2-t- 1'6 57"0_+2-3
90"0 __+2"1 85"2_____2"0 76"1__+1"0 70"3-+2"8
ToleranceIndex (TI) of root
TABLE 1 Percentage of Seed Germination, Water Imbibing Capacity, Root and Shoot Length and Tolerance Index of Root of 7-day-old Seedlings of Oryza sativa L. cv. Kesari-82K under Different Concentrations of Paper Industry Effluent. Values are Mean + SD of 100 Samples
.~
~"
.~
t,9
5O-
r U
_o
e,i
'-
O u
10-
20-
30-
~¢- 40-
E
01
60-
Fig. 1.
18hr
r= - 0 . 7 4 1 p
1
T i m e of e x p o s u r e t o e f f l u e n t
12hr
p <0.06
r= - 0 - 6 6 6
r= - 0 . 8 3 1
24 hr
I
/
effluent
100°/o
./25°1o effluent ~. j S O °/o e f f l u e n t ~75°/o effluent
.Control
p < 0.01
Effect of effluent on total chlorophyll content of rice seedlings.
6hr
r = - 0-632 p
2"
&
O"
10-
20-
30-
O-
10-
~- 20-
u
L_
0
~ 4o-
r 0 U
E v
O1
"':
30-
40 ¸
Fig. 2.
6hr
1
18hr
r= -0-834 <0"01
18hr
r = - 0.941 p <0.001
Time of exposure to effluent
12hr
r =-0-727 p
12hr
1
r= -0-762 )<(}01
24 hr
100Olo effluent
25 ~/o e f f l u e n t 50 /°effluent 5°1o e f f l u e n t
Control
r= -0.937 < 0-001
24 hr
1O0 °1° effluent
50°/o e f f l u e n t 5°1° e f f l u e n l
< (}001
r = -0.973
Effect o f effluent o n total c a r b o h y d r a t e c o n t e n t o f rice seedlings.
r = -0.721 p <0.02
6hr
r = -0.731 < 0.02
4"
Effect o f paper industry effluent on rice seedlings
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R. N. Misra, P. K. Behera
effluent, both in concentration and time-dimensions, retarded growth of the rice seedlings. Decrease in seedling growth is associated with increase in effluent concentration. The above results are in agreement with the earlier results of Rajannan and Oblisamai (1979) and Behera and Misra (1982), but contradictory to those of Choudhury et al. (1989). The contents of pigments, protein and carbohydrate of the leaf extracts of rice seedlings grown in several concentrations of effluent are illustrated in Figs 1-3. Compared with the control, the effluent showed adverse effects at all concentrations on chlorophyll, protein and carbohydrate contents. These results are similar to the earlier results of Behera and Misra (1983), but do not agree with those obtained by Choudhury et al. (1989). The longer the exposure of the rice seedlings to the effluent the lower is the level of these cellular constituents. A gradual decline with time of exposure to effluent and with concentration of effluent in the protein and carbohydrate contents seems to be correlated with the loss of chlorophyll. Of the two studied macromolecular components of rice seedlings, protein seemed to be more susceptible to effluent stress (Figs 1 and 3). Our data on the degradation of cellular macromolecules and growth of seedlings suggest that the papermill effluent has greatest effect at prolonged exposure and could lead to decreased growth of rice. The adverse effects may be because the papermill effluent consists of three different types of suspended solids (silicon, cellulose and hemicellulose) together with 18 different types of dissolved chemicals, as reported by Nestmann et al. (1980). At present, a firm conclusion cannot be drawn in these pilot experiments regarding the significance of the decline in protein to a longer duration of exposure. However, the results suggest that in an evaluation ofphytotoxicity and pollution risk, protein, and enzymes involved in protein synthesis, can act as bio-indicators at a particular time of exposure to the effluent.
REFERENCES Arnon, D. I. (1949). Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol., 24, 1-15. Behera, B. K. & Misra, B. N. (1982). Analysis of the effect of industrial effluent on growth and development of rice seedlings. Environ. Res., 28, 10-20. Behera, B.~K. & Misra, B. N. (1983). Analysis of effect of industrial effluent on pigments, proteins, nucleic acids and DCPIP Hill reaction of rice seedlings. Environ. Res., 31, 381-9. Choudhury, S. K., Jha, A. N. & Srivastava, D. K. (1989). Impact of paper mill effluent on germination, seedling growth and pigment content of Hordeum vulgare. Env. Eco., 7(1), 193-5.
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Freedman, Martin & Bikki Jaggi (1986). Pollution performance of firms from pulp and paper industries. Environ. Manage., 10(3), 359-66. Gomez, K. A. & Gomez, A. A. (1984). Statistical Procedures for Agricultural Research. (2nd edn) Wiley-Interscience Publication, p. 680. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). Protein measurement with Folin phenol reagent. J. Biol. Chem., 193, 265-75. Misra, R. N. & Sahu, R. K. (1983). Studies on physiotoxicity and cytotoxicity of some dyes released from papermills in a modified Allium test. Abst. 6th bzd. Bot. Con£ J. Incl. Bot. Soc. Supp. 62, 66. Nestmann, E. R., Lee, E. G. H., Metula, T. I., Douglas, G. R. & Muller, J. C. (19801. Mutagenicity of constituents identified in pulp and paper mill effluents using the Salmonella/mammalian microsome assay. Mutation Res., 79, 203 12. Olin, Robert G., Anders, Ahlbom., Ingen Hindberg Navier, Stuffan E. Norell & Bo Spannare (1987). Occupational factors associated with astro cytomas: A c~secontrol study. Am. J. Ind. Med., 11(6), 615-26. Rajannan, G. & Oblisamai, G. (1979). Effect of paper factory effluents on soil and crop plants. Intl. J. Environ. 21(2), 120 30. Reddy, P. Manikya & Venkateswarulu, V. (1986). Ecology of algae in the paper mill effluents and their impact on the river Tungabhadra (India). J. Environ. Biol., 7(4), 215-24. Wilkins, D. A. (1978). The measurement of tolerance to edaphic factors by means of root growth. New Phytol., 80, 623 33. Yemm, E. W. & Willis, A. J. (1954). The estimation of carbohydrate in plant extracts by anthrone, Biochem. J., 57, 509---13. Yoshida, S., Forno, D. A., Cock, J. H. & Gomez, K. A. (1972). Laboratoo' Manual Jor Physiological Studies of Rice (2nd edn). The Philippines, The International Rice Research Institute, p. 70.