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Effect of sugar mill effluent on oxygen uptake and carbon dioxide output of rice (Oryza sativa L.c.v. Mushoori) seedlings
ENVIRONMENTAL RESEARCH 43, 135-141 (1987)
Effect of Sugar Mill Effluent on Oxygen Uptake and Carbon Dioxide Output of Rice (Oryza sativa L.c.v. Mushoori) Seedlings B . K . B E H E R A AND SHABBIR A . SAYEED*
Department of Bio-Sciences, M,D. University, Rohtak, Haryana, Pin-124001, India, and *Bioenergetic Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, Pin-110067, India Received February 7, 1986 The effects of a sugar mill effluent on respiration of rice (Oryza sativa L.c.v. Mushoori) seedlings have been investigated, Each experiment consisted of two parts, (i) the effect of various concentrations of the effluent and (ii) the time-dependent changes induced by the undiluted effluent. A marked increase in the rate of respiration was noticed upon treatment with various concentrations of the effluent. The time-dependent changes in the respiratory rate were about threefold with the 12 hr of treatment, and thereafter a rapid loss was noticed. Additionally, the respiratory quotient was studied to elucidate the nature of the metabolism of seedlings as influenced by the effluent. © 1987AcademicPress,Inc.
INTRODUCTION
Effluent from sugar mill distilleries contains pollutants such as organic and inorganic compounds, i.e., protein, free ammonia, sodium, potassium, calcium, and chlorine (Behera et al., 1980; Behera and Misra, 1982). The wastewater, locally known as " s p e n d w a s h , " resulting from sugar mill distilleries in India amounts to more than 10,000 million liters per day. Disposal of liquid wastes from distilleries has presented one of the most urgent and serious environmental problems in most of the tropical countries like India. In the present study, effluent from a distillery of a sugar factory is taken and its impact on rice seedlings is investigated. As reported earlier, the rice seedlings grown in the effluent show morphological (Behera and Misra, 1982) and biochemical disorders such as loss of macromolecules and photosynthetic pigments (Behera and Misra, 1983), and reduction in the activities of various enzymes that participate in the tricarboxylic acid cycle enzymes may provide additional substrate to the respiratory metabolism and may bring changes in the rate of respiration. So, in the present investigation an attempt is made to elucidate the oxygen uptake and carbon dioxide output by the rice seedlings under effluent stress. MATERIALS AND METHODS Plant materials. Seeds of rice (O. sativa) were soaked for 12 hr in distilled water at a temperature of 25 _+ 2°C and placed in sterilized petri dishes containing soaked filter papers. Fifty seeds were spaced evenly in each petri dish and each treatment was replicated five times, including the control. The petri dishes were illuminated from above by two fluorescent tube lights (60 W) providing a light intensity of 3000 lux at the level of the seeds. The dishes were kept inside a 135 0013-9351/87 $3.00 Copyright© 1987by AcademicPress,Inc. All rightsof reproductionin any formreserved.
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BEHERA AND SAYEED
temperature-controlled culture room at 25°C in order to maintain a steady atmosphere and also to check evaporation. However, a measured quantity (5 ml) of distilled water or test solution and several concentrations of effluent (v/v) were added to the dishes to moisten the filter paper as necessary. The pH of treatment set varied between 4.5 and 5.0 during the growth period and the control set was adjusted to the same pH. Two sets of seedlings were tested in each experiment. One was a study of the effect of various concentrations (v/v) of the effluent, and the other was a study of the time-dependent changes induced by the 100% effluent. The effluent from sugar mill distilleries was collected directly from the main source of discharge. At this point the temperature of the effluent was 80 to 90°C. The high temperature of the effluent was brought to ambient temperature by keeping it exposed to laboratory atmosphere. Then, the effluent was sealed airtight in Coming glass bottles and was stored frozen at 5°C for further use. Samples of 7-day-old rice seedings, at steady state of growth, were taken at random from the first set. Similarly, samples were taken from the second set at 6-hr intervals. Measurement of respiration. The oxygen uptake and carbon dioxide output were measured using Warburg's apparatus with reaction flask of 25-ml capacity. The reaction temperature was maintained at 30 -+ 0.1°C throughout the experiments. Two sets of reaction flasks were maintained in each experiment. In one series the control well of the flask contained 0.2 ml of 15% KOH solution while in the other it had the same amount of distilled water. The former set gave the oxygen uptake while the latter indicated the difference in the O2 uptake and carbon dioxide output. At each observation, samples of from 5 to 10 seedlings were selected and kept in the flask solution (different concentration of effluent) as necessary. Duplicate manometers were maintained together with adequate thermobarometers. An initial shaking of the flask for 15 rain for stabilizing the temperature was followed by two successive 15-min experimental shakings. The manometers were shaken 60 oscillations per minute. The oxygen uptake (QO2) and carbon dioxide liberated (QCO2) in microliters by 1 g fresh wt of plant in 1 hr, together with respiratory quotient, were calculated according to the method given by Umbreit et al. (1949). RESULTS
Uptake of Oxygen The results on the effluent-induced changes in uptake of oxygen of rice seedlings are given in Fig. 1A. The results show that low concentrations of effluent (2.5%, v/v) stimulated both shoot and root respiration (QO2). However, at this concentration, the increase in respiration rate of shoot and root was relatively slow compared with that of controls. But, with the increase in effluent concentration a rapid increase in oxygen uptake was noticed to be flattened after 20% (v/v) concentration of effluent. The highest increase, nearly threefold oxygen uptake of shoot and root, was found with 50% (v/v) effluent treatment. The time-dependent changes in oxygen uptake of rice seedlings treated with
137
INDUSTRIAL E F F L U E N T AND RICE SEEDLINGS B
A 300
~
200
z 0 o u. O
~
Root
,-.
100
0 0
J I0
I 20
EFFLUENT (% V/V )
1/5 ~0
i 12
i ]8
J 24
TIME ( h )
FIG. 1. (A) Changes in the rates of respiration (QO2) of 7-day-old rice seedlings grown in several concentrations of effluent. The initial (control) values for shoot (350 U liter/g/fresh wt/hr) and root (340 ~ liter/g/fresh wt/hr) were set equal to 100%. Each point represents an average of three experiments. Vertical bars represent SD. (B) Changes in the rate of respiration of 7-day-old rice seedlings (distilled water grown) treated with 100% effluent. The initial (zero-time) values were control and set equal to 100%. Each point represents an average for three experiments. Vertical bars represent SD.
100% effluent are shown in Fig. lB. A rapid increase in shoot and root oxygen uptake was noticed during effluent treatment. The highest increase in both shoot and root oxygen uptake, nearly threefold, was noticed with 12 hr of treatment. After this, a rapid decline in the oxygen uptake of shoot and root system was noticed.
Output of Carbon Dioxide The results on effluent-induced changes in the output of carbon dioxide of shoot and root of rice seedlings are presented in Fig. 2A. Effluent 2.5% (v/v) stimulated both shoot and root carbon dioxide output. But at this concentration, the increase in respiration (QCO2) was slow as compared with that of controls. With the increase in effluent concentration, a rapid rise in carbon dioxide output was marked. The curves of QCO2 of shoot and root were observed to be somewhat flattened after 20% (v/v) effluent. The highest increase, nearly threefold carbon dioxide output of shoot and root, was found with 50% (v/v) effluent treatment. The time-dependent changes in QCO 2 of rice seedlings treated with 100% (v/v) effluent are shown in Fig. 2B. A rapid increase in shoot and root Q C O 2 was noticed during effluent treatment. The highest increase in both shoot and root CO2, nearly threefold, was noticed at the 12th treatment. After this, a rapid decline in Q C O 2 of shoot and root system was noticed.
138
BEHERA
AND SAYEED
300
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~
o~
ROOT
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20
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24
FIG. 2. (A) Changes in the rates of respiration (QCO2) of 7-day-old rice seedlings grown in several concentrations of effluent. The initial (control) values for shoot (245 ix liter/g/fresh wt/hr) and root (225 tx liter/g/fresh wt/hr) were set equal to 100%. Each point represents an average of three experiments. Vertical bars represent SD. (B) Changes in the rate of respiration of 7-day-old rice seedlings (distilled water grown treated with 100% effluent. The initial (zero-time) values were control and set equal to 100%. Each point represents an average for three experiments. Vertical bars represent SD.
Respiratory Quotient (RQ) Results on respiratory quotient of shoot and root system of seedlings grown in several concentrations of effluent are shown in Figs. 3A and B. The curves of RQ of shoot and root systems declined gradually from control till 20% (v/v) effluent. With a further increase in effluent concentration, the declined curve of RQ appeared flattened. The time-dependent changes in RQ of rice seedlings treated with 100% (v/v) effluent are shown in Fig. 3B. Relative to the control, the decline in RQ values of shoot and root was noticed during the treatment period. The decline curves of RQ were noticed to be flattened after 12 hr of treatment.
DISCUSSION The uptake of oxygen and output of carbon dioxide of shoot and root systems increased substantially over those of the control when rice seedlings were subjected to effluent stress. Due to the presence of many c o m p o n e n t s (Behera et al., 1980; Behera and Misra, 1982) in the effluent, it is difficult to specify which of their c o m p o n e n t s are responsible for the increase in QCO2. However, results could be analyzed, along with the findings of earlier workers, as follows: (1) The presence of high amounts of N a + , K + , N H + , Ca 2+ , Mg 2÷ , Fe 3+ , C I - , PO-4, etc., in effluent (Behera and Misra, 1982) in general suggests a close similarity between effluent stress (Figs. 1-3) and the salt stress-induced changes (Siew and Klein, 1968; Pokrovaskaia, 1958; Livne and Levin, 1967; M a k s i m o v a
139
I N D U S T R I A L E F F L U E N T A N D RICE S E E D L I N G S
,.5[. A
o--o
SHOOT
H
ROOT
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- - R L
~I
0.5
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' I0
' 20
EFFLUENT(% V//V )
//s'0o
~
,~ TIME(h)
I
,8
I
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FIG. 3. (A) Changes in the RQ of 7-day-old rice seedlingsgrown in several concentrations of effluent. The control (distilled water grown) values for shoot (0.97) and root (0.97) are an average of three experiments. Vertical bars represent SD. (B) Changes in the RQ of 7-day-old rice seedlings (distilled water grown) treated with 100%effluent.The initial(zero-time) values were controls. Each point represents an average of three experiments. Vertical bars represent SD.
and Metukhin, 1965; Goris, 1969; Matsumoto et al., 1971; Wakiuchi et al., 1971, 1972). (2) Generally, high amounts of salt (s) caused inhibition in respiratory metabolism (Boyer, 1965; Munda, 1969; Sarin and Rao, 1958; Bhardwaj and Rao, 1960). But in our present findings it is interesting to note the stimulatory affect of effluent (0 to 20%, v/v) on oxygen uptake and carbon dioxide output. The presence of high amounts of ammonia (800-1000 mg liter -1) might have been uncoupling (Wakiuchi et al., 1971) and resulted in increases in QO 2 and QCO 2 during effluent treatment of rice seedlings. (3) In an earlier study (Behera and Misra, 1985) it was noticed that some respiratory enzyme activities increased during effluent treatment. This increase in respiratory enzymes might have served as an additional substrate(s) to respiratory metabolism (Hackett, 1956; Hackett et al., 1959) and, as a result, increased in QO 2 and CO2 were noticed during effluent treatment. (4) Some information is available on the effect of salt stress on respiratory quotient (Bhardwaj and Rao, 1960; Sarin, 1961). The changes in RQ might have resulted from some anabolic process involving fixation of oxygen or carbon dioxide (James, 1953; James and James, 1940; Fritz et al., 1958; Brown, 1943). However, in the present study the substrate-induced changes in RQ values cannot be ignored. Loss of photosynthetic activities (Behera and Misra, 1983) and [3-amylase (Behera and Misra, 1985) might have created starvation conditions in vegetable tissue. Use of cellular proteins during starvation could have been responsible for protoplasmic respiration (Deleano, 1912; Yema, 1935). As proteins have lower a amount of oxygen as compared with carbonhydrates, more oxygen obviously is needed for its complete oxidation into carbon dioxide and water. Thus, in our present results, RQ values under effluent stress appeared as less than a unit.
140
BEHERA AND SAYEED
ACKNOWLEDGMENTS The authors are gratefully indebted to Professor E Mohanty, School of Life Sciences, J.N. University, New Delhi, and Professor B. N. Misra, Department of Botany, Berhampur University, Berhampur, for providing necessary facilities. The authors also thank Professor H. S. Srivastav, Department of Biosciences, M.D. University, for help while preparing the manuscript.
REFERENCES Behera, B. K., Misra, B. N. (1982). Analysis of effect of industrial effluent on growth development of rice seedlings. Environ. Res. 28, 10-20. Behera, B. K., and Misra, B. N. (1983). Analysis of effect of industrial effluent on pigments, proteins, nucleic acids and Hill reaction of rice seedlings. Environ. Res. 31, 381-389. Behera, B. K., and Misra, B. N. (1985). The effect of sugar mill effluent on enzyme activities of rice seedlings. Environ. Res. 37, 390-398. Behera, B. K., Misra, B. N., and Patnaik, H. (1980). Nutrient value of distillery effluent. Geobios 7, 316-318. Bhardwaj, S. N., and Rao, I. M. (1960). Physiological studies on salt tolerance in crop plants. IX. Effect of sodium chloride and sodium carbonate on seedling respiration and growth of wheat and gram. Indian J. Plant Physiol. 3, 56-71. Brown, R. N. (1943). Studies in germination and seedling growth, gaseous exchange and dry weight of attached and isolated embryos of barley. Ann. Bot. 25, 43-114. Boyer, J. S. (1965). Effect of osmotic water stress on metabolic rates of cotton plants with open stomata. Plant Physiol. 40, 229-234. Deleano, M. T. (1912). Studien uber den Atmungsstoff-wechsel obgesihnittenen Laubhlatter. Jahrb. wiss. Bot. 51, 541-592. Fritz, G. J., Miller, W. G., Burris, R. H., and Anderson, L. (1958). Direct incorporation of molecule oxygen into organic material by repairing corn seedlings. Plant Physiol. 33, 159-161. Goris, I. Ya. (1969). Respiratory metabolism of seeds germinating under salinization conditions. Sel'skokhoz Biol. 4, 246-251. Hackett, D. E (1956). Pathways of oxidation in cell free potato fractions. Plant Physiol. 31, 111-118. Hackett, D. E, Hass, D. W., Grifflrths, S. K., and Niederpruem, D. J. (1959). Studies on development of cyanide resistant respiration in potato tuber slices. Plant Physiol. 35, 8-19. James, W. O. (1953). "Plant Respiration." Clarendon Press, Oxford. James, W., and James, A. L. (1940). Respirations of barley germinating in darks. New Phytol. 39, 149-176. Livne, A , and Levin, N. (1967). Tissue respiration and mitochondrial oxidative phosphorylation of NaCl-treated pea seedlings. Plant Physiol. 42, 407-417. Maksimova, E. G., and Matukhin, G. R. (1965). Effect of soil salinization on the respiration rate and terminal oxidase activity of millet leaves. Fiziol. Rast. 12, 540-542. Matsumoto, H., Wakiuchi, N., and Takahashi, E. (1971). Changes of some mitochondrial enzyme activities of cucumber leaves during ammonium toxicity. Physiol. Plant. 25, 353-357. Munda, I. (1969). The effect of salinity on the respiration and photosynthesis on the brown alga Ascophyllurn nodosum. Biol. Vestn. 11, 3-13. Pokrovaskaia, E. I. (1958). Salt hardiness and various metabolic pathways and glycophytes. Fiziol. Rast. 5,250-266. Sarin, M. N. (1961). Physiological studies on salt tolerance in crop plants. XIV. Further studies on the effect of sodium sulphate on respiration of wheat and gram seedlings. Indian J. Plant Physiol. 4, 38-46. Sarin, M. N., and Rao, I. M. (1958). Physiological studies on salt tolerance in crop plants. III. Influence of sodium sulphate on seedling respiration in wheat and gram. Indian J. Plant Physiol. 4, 38-46. Siew, D., and Klein, S. (1968). The effect of sodium chloride on some metabolic and fine structural changes during greening of etiolated (bean) leaves. J. Cell Biol. 37, 590-595.
INDUSTRIAL E F F L U E N T AND RICE SEEDLINGS
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Umbreit, W. W., Burries, R. H., and Statner, J. R (1949). "Manometric Techniques and Tissue Metabolism." Burgess, Minneapolis, MN. Wakiuchi, N., Matsumoto, H., Kondo, S., and Takahashi, E. (1972). Changes in nicotinamide nucleotide coenzymes in cucumber leaves during ammonium toxicity. Physiol. Plant 26, 230-232. Wakiuchi, N., Matsumoto, H., and Takahashi, E. (1971). Changes of some enzyme activities of cucumber during ammonium toxicity. Physiol. Plant 24, 248-253. Yema, E. W. (1935). The respiration of barley plant. II. Carbohydrate concentrations and carbon dioxide production in starting leaves. Proc. R. Soc. B 117, 504-525.
Effect of Sugar Mill Effluent on Oxygen Uptake and Carbon Dioxide Output of Rice (Oryza sativa L.c.v. Mushoori) Seedlings B . K . B E H E R A AND SHABBIR A . SAYEED*
Department of Bio-Sciences, M,D. University, Rohtak, Haryana, Pin-124001, India, and *Bioenergetic Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi, Pin-110067, India Received February 7, 1986 The effects of a sugar mill effluent on respiration of rice (Oryza sativa L.c.v. Mushoori) seedlings have been investigated, Each experiment consisted of two parts, (i) the effect of various concentrations of the effluent and (ii) the time-dependent changes induced by the undiluted effluent. A marked increase in the rate of respiration was noticed upon treatment with various concentrations of the effluent. The time-dependent changes in the respiratory rate were about threefold with the 12 hr of treatment, and thereafter a rapid loss was noticed. Additionally, the respiratory quotient was studied to elucidate the nature of the metabolism of seedlings as influenced by the effluent. © 1987AcademicPress,Inc.
INTRODUCTION
Effluent from sugar mill distilleries contains pollutants such as organic and inorganic compounds, i.e., protein, free ammonia, sodium, potassium, calcium, and chlorine (Behera et al., 1980; Behera and Misra, 1982). The wastewater, locally known as " s p e n d w a s h , " resulting from sugar mill distilleries in India amounts to more than 10,000 million liters per day. Disposal of liquid wastes from distilleries has presented one of the most urgent and serious environmental problems in most of the tropical countries like India. In the present study, effluent from a distillery of a sugar factory is taken and its impact on rice seedlings is investigated. As reported earlier, the rice seedlings grown in the effluent show morphological (Behera and Misra, 1982) and biochemical disorders such as loss of macromolecules and photosynthetic pigments (Behera and Misra, 1983), and reduction in the activities of various enzymes that participate in the tricarboxylic acid cycle enzymes may provide additional substrate to the respiratory metabolism and may bring changes in the rate of respiration. So, in the present investigation an attempt is made to elucidate the oxygen uptake and carbon dioxide output by the rice seedlings under effluent stress. MATERIALS AND METHODS Plant materials. Seeds of rice (O. sativa) were soaked for 12 hr in distilled water at a temperature of 25 _+ 2°C and placed in sterilized petri dishes containing soaked filter papers. Fifty seeds were spaced evenly in each petri dish and each treatment was replicated five times, including the control. The petri dishes were illuminated from above by two fluorescent tube lights (60 W) providing a light intensity of 3000 lux at the level of the seeds. The dishes were kept inside a 135 0013-9351/87 $3.00 Copyright© 1987by AcademicPress,Inc. All rightsof reproductionin any formreserved.
136
BEHERA AND SAYEED
temperature-controlled culture room at 25°C in order to maintain a steady atmosphere and also to check evaporation. However, a measured quantity (5 ml) of distilled water or test solution and several concentrations of effluent (v/v) were added to the dishes to moisten the filter paper as necessary. The pH of treatment set varied between 4.5 and 5.0 during the growth period and the control set was adjusted to the same pH. Two sets of seedlings were tested in each experiment. One was a study of the effect of various concentrations (v/v) of the effluent, and the other was a study of the time-dependent changes induced by the 100% effluent. The effluent from sugar mill distilleries was collected directly from the main source of discharge. At this point the temperature of the effluent was 80 to 90°C. The high temperature of the effluent was brought to ambient temperature by keeping it exposed to laboratory atmosphere. Then, the effluent was sealed airtight in Coming glass bottles and was stored frozen at 5°C for further use. Samples of 7-day-old rice seedings, at steady state of growth, were taken at random from the first set. Similarly, samples were taken from the second set at 6-hr intervals. Measurement of respiration. The oxygen uptake and carbon dioxide output were measured using Warburg's apparatus with reaction flask of 25-ml capacity. The reaction temperature was maintained at 30 -+ 0.1°C throughout the experiments. Two sets of reaction flasks were maintained in each experiment. In one series the control well of the flask contained 0.2 ml of 15% KOH solution while in the other it had the same amount of distilled water. The former set gave the oxygen uptake while the latter indicated the difference in the O2 uptake and carbon dioxide output. At each observation, samples of from 5 to 10 seedlings were selected and kept in the flask solution (different concentration of effluent) as necessary. Duplicate manometers were maintained together with adequate thermobarometers. An initial shaking of the flask for 15 rain for stabilizing the temperature was followed by two successive 15-min experimental shakings. The manometers were shaken 60 oscillations per minute. The oxygen uptake (QO2) and carbon dioxide liberated (QCO2) in microliters by 1 g fresh wt of plant in 1 hr, together with respiratory quotient, were calculated according to the method given by Umbreit et al. (1949). RESULTS
Uptake of Oxygen The results on the effluent-induced changes in uptake of oxygen of rice seedlings are given in Fig. 1A. The results show that low concentrations of effluent (2.5%, v/v) stimulated both shoot and root respiration (QO2). However, at this concentration, the increase in respiration rate of shoot and root was relatively slow compared with that of controls. But, with the increase in effluent concentration a rapid increase in oxygen uptake was noticed to be flattened after 20% (v/v) concentration of effluent. The highest increase, nearly threefold oxygen uptake of shoot and root, was found with 50% (v/v) effluent treatment. The time-dependent changes in oxygen uptake of rice seedlings treated with
137
INDUSTRIAL E F F L U E N T AND RICE SEEDLINGS B
A 300
~
200
z 0 o u. O
~
Root
,-.
100
0 0
J I0
I 20
EFFLUENT (% V/V )
1/5 ~0
i 12
i ]8
J 24
TIME ( h )
FIG. 1. (A) Changes in the rates of respiration (QO2) of 7-day-old rice seedlings grown in several concentrations of effluent. The initial (control) values for shoot (350 U liter/g/fresh wt/hr) and root (340 ~ liter/g/fresh wt/hr) were set equal to 100%. Each point represents an average of three experiments. Vertical bars represent SD. (B) Changes in the rate of respiration of 7-day-old rice seedlings (distilled water grown) treated with 100% effluent. The initial (zero-time) values were control and set equal to 100%. Each point represents an average for three experiments. Vertical bars represent SD.
100% effluent are shown in Fig. lB. A rapid increase in shoot and root oxygen uptake was noticed during effluent treatment. The highest increase in both shoot and root oxygen uptake, nearly threefold, was noticed with 12 hr of treatment. After this, a rapid decline in the oxygen uptake of shoot and root system was noticed.
Output of Carbon Dioxide The results on effluent-induced changes in the output of carbon dioxide of shoot and root of rice seedlings are presented in Fig. 2A. Effluent 2.5% (v/v) stimulated both shoot and root carbon dioxide output. But at this concentration, the increase in respiration (QCO2) was slow as compared with that of controls. With the increase in effluent concentration, a rapid rise in carbon dioxide output was marked. The curves of QCO2 of shoot and root were observed to be somewhat flattened after 20% (v/v) effluent. The highest increase, nearly threefold carbon dioxide output of shoot and root, was found with 50% (v/v) effluent treatment. The time-dependent changes in QCO 2 of rice seedlings treated with 100% (v/v) effluent are shown in Fig. 2B. A rapid increase in shoot and root Q C O 2 was noticed during effluent treatment. The highest increase in both shoot and root CO2, nearly threefold, was noticed at the 12th treatment. After this, a rapid decline in Q C O 2 of shoot and root system was noticed.
138
BEHERA
AND SAYEED
300
j 200 0 z o (..> o
1oo
~
o~
ROOT
~9 O
I
I0 EFFLUENT
I
III 50
20
(°/o
V/V )
6
12 18 TIME ( h )
24
FIG. 2. (A) Changes in the rates of respiration (QCO2) of 7-day-old rice seedlings grown in several concentrations of effluent. The initial (control) values for shoot (245 ix liter/g/fresh wt/hr) and root (225 tx liter/g/fresh wt/hr) were set equal to 100%. Each point represents an average of three experiments. Vertical bars represent SD. (B) Changes in the rate of respiration of 7-day-old rice seedlings (distilled water grown treated with 100% effluent. The initial (zero-time) values were control and set equal to 100%. Each point represents an average for three experiments. Vertical bars represent SD.
Respiratory Quotient (RQ) Results on respiratory quotient of shoot and root system of seedlings grown in several concentrations of effluent are shown in Figs. 3A and B. The curves of RQ of shoot and root systems declined gradually from control till 20% (v/v) effluent. With a further increase in effluent concentration, the declined curve of RQ appeared flattened. The time-dependent changes in RQ of rice seedlings treated with 100% (v/v) effluent are shown in Fig. 3B. Relative to the control, the decline in RQ values of shoot and root was noticed during the treatment period. The decline curves of RQ were noticed to be flattened after 12 hr of treatment.
DISCUSSION The uptake of oxygen and output of carbon dioxide of shoot and root systems increased substantially over those of the control when rice seedlings were subjected to effluent stress. Due to the presence of many c o m p o n e n t s (Behera et al., 1980; Behera and Misra, 1982) in the effluent, it is difficult to specify which of their c o m p o n e n t s are responsible for the increase in QCO2. However, results could be analyzed, along with the findings of earlier workers, as follows: (1) The presence of high amounts of N a + , K + , N H + , Ca 2+ , Mg 2÷ , Fe 3+ , C I - , PO-4, etc., in effluent (Behera and Misra, 1982) in general suggests a close similarity between effluent stress (Figs. 1-3) and the salt stress-induced changes (Siew and Klein, 1968; Pokrovaskaia, 1958; Livne and Levin, 1967; M a k s i m o v a
139
I N D U S T R I A L E F F L U E N T A N D RICE S E E D L I N G S
,.5[. A
o--o
SHOOT
H
ROOT
1,0
- - R L
~I
0.5
O:
' I0
' 20
EFFLUENT(% V//V )
//s'0o
~
,~ TIME(h)
I
,8
I
2,
FIG. 3. (A) Changes in the RQ of 7-day-old rice seedlingsgrown in several concentrations of effluent. The control (distilled water grown) values for shoot (0.97) and root (0.97) are an average of three experiments. Vertical bars represent SD. (B) Changes in the RQ of 7-day-old rice seedlings (distilled water grown) treated with 100%effluent.The initial(zero-time) values were controls. Each point represents an average of three experiments. Vertical bars represent SD.
and Metukhin, 1965; Goris, 1969; Matsumoto et al., 1971; Wakiuchi et al., 1971, 1972). (2) Generally, high amounts of salt (s) caused inhibition in respiratory metabolism (Boyer, 1965; Munda, 1969; Sarin and Rao, 1958; Bhardwaj and Rao, 1960). But in our present findings it is interesting to note the stimulatory affect of effluent (0 to 20%, v/v) on oxygen uptake and carbon dioxide output. The presence of high amounts of ammonia (800-1000 mg liter -1) might have been uncoupling (Wakiuchi et al., 1971) and resulted in increases in QO 2 and QCO 2 during effluent treatment of rice seedlings. (3) In an earlier study (Behera and Misra, 1985) it was noticed that some respiratory enzyme activities increased during effluent treatment. This increase in respiratory enzymes might have served as an additional substrate(s) to respiratory metabolism (Hackett, 1956; Hackett et al., 1959) and, as a result, increased in QO 2 and CO2 were noticed during effluent treatment. (4) Some information is available on the effect of salt stress on respiratory quotient (Bhardwaj and Rao, 1960; Sarin, 1961). The changes in RQ might have resulted from some anabolic process involving fixation of oxygen or carbon dioxide (James, 1953; James and James, 1940; Fritz et al., 1958; Brown, 1943). However, in the present study the substrate-induced changes in RQ values cannot be ignored. Loss of photosynthetic activities (Behera and Misra, 1983) and [3-amylase (Behera and Misra, 1985) might have created starvation conditions in vegetable tissue. Use of cellular proteins during starvation could have been responsible for protoplasmic respiration (Deleano, 1912; Yema, 1935). As proteins have lower a amount of oxygen as compared with carbonhydrates, more oxygen obviously is needed for its complete oxidation into carbon dioxide and water. Thus, in our present results, RQ values under effluent stress appeared as less than a unit.
140
BEHERA AND SAYEED
ACKNOWLEDGMENTS The authors are gratefully indebted to Professor E Mohanty, School of Life Sciences, J.N. University, New Delhi, and Professor B. N. Misra, Department of Botany, Berhampur University, Berhampur, for providing necessary facilities. The authors also thank Professor H. S. Srivastav, Department of Biosciences, M.D. University, for help while preparing the manuscript.
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