ECOTOXICOLOGY
AND
ENVIRONMENTAL
SAFETY
19,135- 142 (1990)
Evaluation of Hematotoxic Effects of Two Commonly Used Fertilizers, Diammonium Phosphate and Urea, on Fish C/arias batrachus S. P. TRIVEDI,
P. SINGH, N. SETHI,* AND R. K. SINGH*
Department of Zoology, University oflucknow, Lucknow 226007, India; and *Division Central Drug Research Institute, Lucknow 226001, India
of Toxicolofl,
Received December 15, 1988 The effects of two commonly used fertilizers, diammonium phosphate and urea, on hematological parameters (hemoglobin, red blood cell count, hematocrit, and total leucocyte count) of fresh water fish Clarias batruchus were studied. The toxic effect of diammonium phosphate was more pronounced than that of urea. The toxic effect of diammonium phosphate resulted in a sudden fall of hematological parameters-Hb, RBC count, Hct-at higher concentrations, and at lower concentrations gradual decreases were seen over comparatively longer durations. In urea intoxication, slight decreases in the three parameters were seen at lower concentrations during shorter intervals, while at higher concentrations, significant decreases during shorter intervals were observed. Total leucocyte count (TLC) increased during toxicity with both fertilizers, but higher elevations in TLC were produced by diammonium phosphate than by urea. Q 1990 Academic
Press, Inc.
INTRODUCTION Under natural ecological conditions and in the experimentally created aquatic environment polluted with contaminants under controlled laboratory conditions, hematological parameters have been studied as indicators or monitors of prevailing aquatic pollution (Joshi, 1978; Vet-ma et al., 1979; Sharma and Gupta, 1982; Singh, 1982; Gopal et al., 1982; Goel et al., 1982; Naqvi, 1983; Mishra and Srivastava, 1983; Goel and Maya, 1986). In this laboratory, the effects of commonly used fertilizers are being studied (Sethi et al., 1986, 1987; Trivedi et al., 1989a, 1989b). In this report, the effects of two commonly used fertilizers, diammonium phosphate (nitrogen 18%, phosphoric acid 46%) and urea (nitrogen 46.4%), on hematological parameters (hemoglobin, red blood cell count, hematocrit, and total leucocyte count) of the fish Clurius butruchus exposed for 24 to 144 hr to six different concentrations were studied, and the results are presented. MATERIALS
AND
METHODS
The fish were collected from the river Gomati of Lucknow with the help of local fishermen using hand nets. They were brought to the experimental laboratory in large plastic containers in natural water, avoiding injuries and stresses of all kinds. The fish were washed five times in tap water, then treated with 2% KMn04 to remove external infections. Normal, uninfected, and apparently healthy fish selected for the experiment were transferred to large glass aquaria and acclimated for 120 hr. Concentrations of DAP and urea lethal (90-100%) to C. butruchus at 24,48, 72, 96, 120, and 144 hr have been recorded earlier (Singh, 1982; Naqvi, 1983). 135
0147-6513/90$3.00 Copyright
0 1990 by Academic
Press, Inc.
aControlvalues.
1.10
1.01
0.97
0.92
0.86
0.81
0.00"
00" 144” 00 144 00 120 00 96 00 72 00 48 00 24
30.5” 30.0” 30.5 33.0 30.5 29.8 30.5 28.4 29.5 29.7 30.5 30.4 30.5 30.2
24.0“ 23.6” 24.8 25.2 24.3 25.1 24.0 24.8 23.1 24.5 23.0 24.3 22.2 23.1
Fertilizer Exposure Room Aquaria concentration time temp temp (g/liter) (W (“Cl (“Cl
7.05a 7.15” 7.10 7.20 7.25 7.45 7.27 7.41 7.34 7.47 7.39 7.50 7.44 7.57
PH
Parameters
PHOSPHATE FERTILIZER AT DIFFERENT INTERVALS ANDCONCENTRATIONS
184” 163“ 180 148 162 150 154 142 150 140 144 136 133 112
Nil” Nil” Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil 184” 163” 180 148 162 150 154 142 150 140 144 136 133 112
Nil” Nil“ Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil
260” 290“ 450 405 465 430 480 465 495 455 515 570 530 585
7.10 7.60 6.90 7.80 7.00 7.50 6.80
7.70 7.30 7.90
8.00” 7.10” 7.90 7.20
Hardness Hardness Alkalinity Alkalinity Dissolved Hardness (temporary (permanent (phenophthaline (methyl orange oxygen (total ppm) mm) wm) mm) mm) (ppm)
WATER CHARACTERISTICS AFTER DISSOLVING DIAMMONIUM
TABLE 1
F
3
2 ? B
EFFECTS
OF
FERTILIZERS
ON
137
C. batrachus
TABLE 2 EFFECT
OF FERTILIZERS DIAMMONIUM OF FRESH WATER
PHOSPHATE AND UREA ON HEMOGLOBIN TELEOST FISH Clarias batrachus
I. Effect of diammonium
Fertilizer concentration (g/liter)”
0.8 1 0.86 0.92 0.97 1.01 1.10
LEVELS
phosphate
Hemoglobin (g%) mean + SD Exposure time (hr) 24
6.57 f 0.22 5.87 f 0.49 4.20 + 0.57 2.57 f 0.22 1.32kO.12 0.95 f 0.05
72
48 Control:
8.50 + 0.52
5.12+0.17 4.05 f 0.60 3.12 f 0.26 2.62 + 0.22 0.97 +- 0.08
3.95 3.42 1.42 1.04
f f f *
0.23 0.33 0.30 0.09
96
120
144
3.52 f 0.38 1.95 -to.12 0.97 f 0. I 1
1.19 kO.09 1.08 + 0.06
0.82 f 0.11
4.57 k 0.22 4.72 f 0.17 1.82kO.17
3.00 f 0.21 2.62 f 0.43
2.90 f 0.08
II. Effect of urea
7.50 11.50 16.25 20.30 24.70 31.35 * Ten observations
6.45 T 0.36 5.45 f 0.36 3.72k0.17 2.60 +- 0.18 2.50 f 0.29 1.75 kO.12
Control:
8.80 f 0.18
6.00 + 0.2 1 5.30k0.18 3.55 + 0.31 2.27 +0.17 1.95 kO.12
5.07 5.25 2.17 2.00
f + f f
0.17 0.40 0.45 0.29
were made in each case.
Water characteristics were analysed throughout the study before and after dissolving an amount of fertilizer, using the standard procedure (APHA, 1976). Proper oxygen supply was maintained throughout the experiment. Fish were tested at measured intervals and exposures to particular concentrations. Blood was collected in vials by puncturing the caudal vein and was mixed with 1% EDTA (ethylenediaminetetraacetic acid). Hematological parameters-hemoglobin (g%), RBC count (million/cm*), Hct (%), and TLC (cells/cm*)-were analyzed using a semiautomatic blood cell counter (Boehringer-Mannheim Diagnostics, HC-555). OBSERVATIONS A. Effect of DAP on Hernatological
AND
RESULTS
Parameters
1. Water characteristics. pH increased slightly at the termination of the experiment (Table I), while other water characteristics -hardness, temperature, dissolved oxygen, etc.-analyzed before and after dissolving different amounts of fertilizer, were almost the same. 2. Hemoglobin. Hemoglobin content decreased at all concentrations and exposures. However, at higher concentrations (0.97, 1.O1, 1.10 g/liter) a sudden fall in hemoglobin was observed, while at lower concentrations (0.8 1,0.86 g/liter) hemoglobin decreased gradually and was minimum at highest exposures (Table 2), which proved lethal to the fish.
138
TRIVEDI
ET
TABLE EFFECT
OF FERTILIZERS
AL.
3
DIAMMONIUM PHOSPHATE AND UREA ON RBC WATER TELEOST FISH Clariasbatrachus 1. Effect of Diammonium
24
0.81 0.86 0.92 0.97 1.01 1.10
1.05 + 0.55 0.87 + 0.09 1.14+0.11 0.92 f 0.05 0.9 1 f 0.03 0.80 f 0.10
OF FRESH
phosphate
RBC Count (million/cm2) mean Exposure time (hr)
Fertilizer concentration (g/liter) a
COUNT
+ SD
48
72
96
120
144
Control: 1.25 + 0.02 1.29 +0.15 1.16kO.08 0.89 + 0.06 0.67 f 0.12
3.56 f 0.14 1.08 f 0.06 1.30*0.14 0.95 + 0.04 0.67 + 0.04
0.89 k 0.12 1.03kO.13 0.69 + 0.02
0.84 + 0.09 0.78kO.06
0.62 + 0.06
2.66~0.11 2.38 f 0.03 2.17 kO.13
2.30 + 0.11 2.18 f 0.09
2.13 +0.13
II. Effect of Urea 7.50 11.50 16.50 20.30 24.70 31.35
3.28 f 0.10 2.94 + 0.07 2.73 -+ 0.07 2.57kO.10 2.63 kO.17 2.15kO.11
a Ten observations
Control: 2.95 + 0.05 2.80 + 0.05 2.66kO.12 2.58k0.16 2.27 f 0.11
3.43 + 0.06 2.76 f 0.05 2.72 + 0.06 2.24kO.11 2.13+0.06
were made in each case.
TABLE
4
EFFECT OF FERTILIZERS DIAMMONIUM PHOSPHATE AND UREA ON HEMATOCRIT WATER TELEOST FISH Clariasbatrachus I. Effect of Diammonium
OF FRESH
phosphate
Hematocrit (%) mean + SD Exposure time (hr)
Fertilizer concentration (g/liter)”
24
0.81 0.86 0.92 0.97 1.01 1.10
2.42 2.72 2.40 2.65 2.55 3.20
+ 0.33 kO.17 + 0.3 I + 0.68 k 0.26 f 1.00
48
72
96
Control: 2.47kO.12 3.35 + 0.26 2.30 IL 0.18 2.57 + 0.17 2.62 + 0.26
22.35 + 1.10 3.20~0.21 4.40 + 0.40 2.50 + 0.29 2.47-cO.17
4.22 + 0.25 4.12 f 0.49 2.45 + 0.34
120
3.87 f 0.29 2.55 + 0.3 1
144
2.80 f 0.25
II. Effect of Urea 7.20 11.50 16.25 20.30 24.70 31.35
23.32+ 21.17 15.52 12.85+ 10.67 9.40
a Ten observations
1.26 +- 0.74 + 0.60 1.06 + 0.38 + 0.42
Control: 24.45 f 0.62 22.82k0.38 18.32& 1.05 18.62 f 1.08 17.87 f 0.84 15.05 + 0.88 13.95 + 0.58 11.80+ 1.15 9.00k0.35 8.85 f 0.3 1
were made in each case.
17.15k2.17 14.35 + 0.64 9.95 + 0.36
15.272 I.26 10.42 + 0.65
10.82kO.97
EFFECTS
OF
FERTILIZERS
ON
C. batruchus
139
3. RBC count. At all concentrations, the RBC count was lowest at the termination of the experiment. Smaller decreases were seen for initial exposures at lower concentrations, and at higher concentrations the cell count decreased suddenly (Table 3). 4. Hct. The hematocrit content, during shorter intervals and at lower concentrations, was almost unaffected, but decreased over longer durations (Table 4). 5. TLC. At all concentrations, TLC increased gradually with exposure time (Table 5) and was maximum at the end, which proved lethal to experimental fish. B. Effect of Urea on Hematological
Parameters
1. Water characteristics. Variable changes in water characteristics were observed (Table 6) after dissolving the fertilizer; pH of the medium changed slightly while the other characteristics remained almost unaffected. 2. Hb, RBCcount, and Hct. These three parameters decreased at all concentrations and exposures (Tables 2, 3, and 4). Maximum lowerings of 80.11% for Hb content, 37.90% for RBC count, and 15.60% for Hct were recorded. 3. TLC. During urea intoxication, TLC increased but the increase was not pronounced as in DAP toxicity.
DISCUSSION The significant alterations in hematological parameters due to toxicity by various pollutants have been observed in fish (Van Vuren and Hattingh, 1978; Srivastava and Agarwal, 198 1; Raizada and Gupta, 1982). Phosphorus is one of the important constituents of the animal body and is present in soils as tri-, di-, and monophosphate. Phosphates are transported to and from water as adsorbed materials on soil particles and as part of microorganisms. Solid residues, water effluents, and air emissions from phosphate fertilizer-producing industries cause the pollution of water, air, and land. In our experiment diammonium phosphate toxicity caused a sharp fall in RBC count, hemoglobin content, and hematocrit. A decrease in RBC number and hemoglobin was observed in the fish Trichogasterfasciatus after exposure to the fungicide RH-216 (Raizada and Gupta, 1982). In urea toxicity, the three parameters, RBC count, hemoglobin, and hematocrit, decreased at all concentrations and exposures, but these decreases were greater in diammonium phosphate toxicity. A sublethal dose of 10 ppm of the organophosphorus pesticide Rogor resulted in an increase in leukocytes in C. batrachus from 25,000-55,000/cm2 to 32,000/cm2 in 96 hr (Goel and Maya, 1986). CCL, (injected at 0.03 and 0.06 ml/100 g body weight at intervals of 3 days) caused considerable lymphocytosis; i.e., within 6 days, lymphocytes increased from 33 to 72% (Sharma and Gupta, 1982). A IO-ppm dose of Rogor resulted in a fall of total RBC count in C. batrachus from 3,850,OOO to 2,210,000/ cm2 in 96 hr (Goel and Maya, 1986), while CCL, injections caused a decrease in total RBC count from 5,050,OOO to 2,700,000/cm2 in 6 days (Sharma and Gupta, 1982). The RBC count fell from 2,9 10,000 to 1,730,000/cm2 in 10 days in endosulfon poisoning (Gopal et al., 1982). In Heteropneustes fossilis, the pesticide malathion resulted in a decrease in RBC count from 6,400,OOO to 3,460,00O/cm* in 96 hr at 7.6 ppm (Mishra and Srivastava, 1983). In C. batrachus, hemoglobin content decreased from 15.34 to 10.30 g% in 96 hr with 10 ppm of Rogor (Goel and Maya, 1986) and from 10.03 to 8.60 g% in 6 days
25075 f 26925 + 30825 k 36125 + 402OOk 38750 +
7.50 11.50 16.25 20.30 24.70 31.35
16393 4832 2392 1314 535 3500
1206 3287 580 2321 2034 2449
3920 1873 2458 247 1 2 160
1709 4832 1150 1541 1685
Control: 31425 -t 24000 + 30875 + 37225 + 42825 f
48
I. Effect
PHOSPHATE
Control: 56475 + 43775 + 55550 f 57950 + 68000 +
DIAMMONIUM
aTen observationswere madein eachcase.
46625 + 37725 + 40450+ 45825 + 60600 2 74000 +
24
OF FERTILIZERS
0.81 0.86 0.92 0.97 1.01 1.10
Fertilizer concentration (g/liter)*
EFFECT
OF FRESH
phosphate
TLC WATER
22850 54000 46225 66575 63875
-I f + f +
72 1195 2535 1209 1150 2808
20175 26500 22750 33850 38825
+ + + f f
1027 3415 1424 1975 3009
29525 + 1014 23150 + 1554 33700 + 1061
64575 + 994
2465Ok 23425k
911 613
54725 + 3387 55500 k 2434
120
TELEOSTFISH Clarias batruchus
47100 + 1471 49625 + 1941
96
TLC (cells/cm*) mean + SD Exposure time (hr)
Diammonium
ON
II. Effect of Urea
of
AND UREA
TABLE 5
23400 + 1329
50475 k 1828
144
F
2
28
$
z 0
"Control values.
31.35
24.70
20.30
16.25
11.50
7.50
0.00”
00” 144” 00 144 00 120 00 96 00 72 00 48 00 24
35.5” 35.0” 35.5 35.0 35.5 34.8 35.5 34.4 34.5 34.6 35.5 35.2 35.5 35.0
28.0” 27.6“ 27.8 28.2 27.3 28.1 27.0 27.8 26.1 27.5 26.0 21.3 24.2 25.7
Fertilizer Exposure Room Aquaria concentration time temp temp (g/liter) (W 03 (“C)
7.05” 7.65” 7.95 8.23 8.05 9.00 8.10 8.52 8.12 8.38 8.16 8.80 8.22 8.90
pH 242” 252” 239 226 236 224 230 219 226 207 216 192 207 185
Nil” Nil“ Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil
242” 252” 239 226 236 224 230 219 226 207 216 192 207 185
15.0” 10.0” 18.0 151.5 24.0 144.0 32.0 122.0 78.0 39.0 95.5 58.0 122.0 70.0
240.0” 255.0” 301.0 408.0 312.0 392.0 351.5 418.0 368.0 421.5 392.0 422.5 412.5 446.5
9.10” 8.80” 9.00 8.15 9.15 8.45 9.10 8.60 9.20 8.65 9.05 8.70 8.90 8.20
Alkalinity Hardness Hardness Alkalinity Hardness (temporary (permanent (phenophthaline (methyl orange Alkalinity (total ppm) pm) ppm) mm) pv-4 (oxygenppm)
Water characteristics
WATER CHARACTERISTICSAFTERDISSOLVINGUREAFERTILIZERATDIFFERENTINTERVALSANDCONCENTRATIONS
TABLE 6
142
TRIVEDI
ET AL.
with CC& injections (Sharma and Gupta, 1982) while in H. fossilis 7.6 ppm of malathion in the medium resulted in a fall in hemoglobin content from 12.35 to 11.42 g% (Mishra and Srivastava, 1983). CONCLUSION These data lead to the conclusion that lowering of RBC count and hemoglobin levels and leukocytosis in the blood of fresh water fish C. batruchus and H. fossilis are common effects of the pollutants diammonium phosphate and urea fertilizers in the aquatic environment. ACKNOWLEDGMENTS We express our sincere thanks to Professor Bhoomitra Dev, Head of the Department ofZoology, University of Lucknow, Lucknow, India, for providing laboratory facilities and constructive criticism throughout the work. Mr. S. P. Trivedi thanks UGC, Government of India, for the award of Junior Research Fellowship and for financial assistance in carrying out the present study.
REFERENCES APHA (American Public Health Association) (1976). Standard Methods fir the Examination of Water and Waste Waters, 14th ed. American Public Health Assoc., New York. GOEL, K. A., AND MAYA (1986). Haematological anomalies in Clarius butrachus under the stressof Rogor. Adv. Biosci. 5, 187-192. GOEL, K. A., TYAGI, S. K., AND AWASTHI, A. K. (1982). Effect of malathion on some haematological values in Heteropneustesfossilis. Comp. Physiol. Ecol. 7,259-26 1. GOPAL, K., KHANNA, R. N., ANAND, M., AND GUPTA, G. S. D. (1982). Haematological changes in fresh water fish exposed to endosulfon. Indust. Health 20, 157- 159. JOSHI, B. D. (1978). Studies on Blood of Fresh Water Fishes of India. Ph.D. thesis, Lucknow University, Lucknow. MISHRA, J., AND SRIVASTAVA, A. K. (1983). Malathion-induced haematological and biochemical changes in the Indian catfish, Heteropneustesfossilis. Environ. Res. 30,393-398. NAQVI, M. S. (1983). Effect of Environment Pollution on Physiology of Fresh Water Fishes, Ph.D. thesis, Lucknow University, Lucknow. RAIZADA, M. N., AND GUPTA, A. (1982). Toxic effect of Rh-2 16 (a systemic fungicide) from total etythrocyte counts (RBC) and haemoglobin (Hb) content of Trichogasterfasciatus. Comp. Physiol. Ecol. 7( 1), 29-30. SETHI, N., SINGH, R. K., AND DAYAL, R. (1986). Toxic effects of diammonium phosphate on serum aminotransferases activity of teleost, Heteropneustes fossilis. Ind. J. Bio. Res. 2(3), 247-254. SETHI, N., AND SINGH, R. K. (1987). Serum inorganic phosphorus levels of fresh water mud-eel, Amphipnous cuchia, under intoxication of pesticide sumithion. Adv. Biosci. 2, 175- 178. SHARMA, R. C., AND GLJPTA, NEELAM (1982). Carbon tetrachloride-induced haematological alterations in Clark batrachus. J. Environ. Biol. 3, 127-129. SINGH, R. K. (1982). Ecophysiological Studies on Some Fresh Water Fishes. Ph.D. thesis, Lucknow University, Lucknow. SRIVASTAVA, A. K., AND AGARWAL, V. (198 1). Seasonal and diurual variations of blood cell types in a fresh water teleost Colisca fasciatus. Camp. Physiol. Ecol. 6( I), 19. TRIVEDI, S. P., SETHI, N., SINGH, R. K., AND SINGH, P. (1989a). Effect of urea fertilizer on haematological parameters ofteleost fish, Heteropneustesfossilis. Biol. Mem. 15( 1), 54-58. TRIVEDI, S. P., SETHI, N., SINGH, R. K., AND SINGH, P. (1989b). Haematological profile of fish Heteropneustes fossilis in the toxicity of fertilizer diammonium phosphate. Biol. Mem. 15(3), 166-l 76. VAN VUREN, J. H. J., AND HA~TINGH, J. (1978). Seasonal changes in the haemoglobins of fresh water fish in their natural environment. Camp. Biochem. Physiol. 60,265-268. VERMA, S. R., BANSAL, S. K., GUPTA, A. K., AND DALELA, R. C. ( 1979). Pesticide induced haematological alterations in a fresh water fish, Saccobranchusfossilis. Bull. Environ. Contam. Toxicol. 22,467-474.
AND
ENVIRONMENTAL
SAFETY
19,135- 142 (1990)
Evaluation of Hematotoxic Effects of Two Commonly Used Fertilizers, Diammonium Phosphate and Urea, on Fish C/arias batrachus S. P. TRIVEDI,
P. SINGH, N. SETHI,* AND R. K. SINGH*
Department of Zoology, University oflucknow, Lucknow 226007, India; and *Division Central Drug Research Institute, Lucknow 226001, India
of Toxicolofl,
Received December 15, 1988 The effects of two commonly used fertilizers, diammonium phosphate and urea, on hematological parameters (hemoglobin, red blood cell count, hematocrit, and total leucocyte count) of fresh water fish Clarias batruchus were studied. The toxic effect of diammonium phosphate was more pronounced than that of urea. The toxic effect of diammonium phosphate resulted in a sudden fall of hematological parameters-Hb, RBC count, Hct-at higher concentrations, and at lower concentrations gradual decreases were seen over comparatively longer durations. In urea intoxication, slight decreases in the three parameters were seen at lower concentrations during shorter intervals, while at higher concentrations, significant decreases during shorter intervals were observed. Total leucocyte count (TLC) increased during toxicity with both fertilizers, but higher elevations in TLC were produced by diammonium phosphate than by urea. Q 1990 Academic
Press, Inc.
INTRODUCTION Under natural ecological conditions and in the experimentally created aquatic environment polluted with contaminants under controlled laboratory conditions, hematological parameters have been studied as indicators or monitors of prevailing aquatic pollution (Joshi, 1978; Vet-ma et al., 1979; Sharma and Gupta, 1982; Singh, 1982; Gopal et al., 1982; Goel et al., 1982; Naqvi, 1983; Mishra and Srivastava, 1983; Goel and Maya, 1986). In this laboratory, the effects of commonly used fertilizers are being studied (Sethi et al., 1986, 1987; Trivedi et al., 1989a, 1989b). In this report, the effects of two commonly used fertilizers, diammonium phosphate (nitrogen 18%, phosphoric acid 46%) and urea (nitrogen 46.4%), on hematological parameters (hemoglobin, red blood cell count, hematocrit, and total leucocyte count) of the fish Clurius butruchus exposed for 24 to 144 hr to six different concentrations were studied, and the results are presented. MATERIALS
AND
METHODS
The fish were collected from the river Gomati of Lucknow with the help of local fishermen using hand nets. They were brought to the experimental laboratory in large plastic containers in natural water, avoiding injuries and stresses of all kinds. The fish were washed five times in tap water, then treated with 2% KMn04 to remove external infections. Normal, uninfected, and apparently healthy fish selected for the experiment were transferred to large glass aquaria and acclimated for 120 hr. Concentrations of DAP and urea lethal (90-100%) to C. butruchus at 24,48, 72, 96, 120, and 144 hr have been recorded earlier (Singh, 1982; Naqvi, 1983). 135
0147-6513/90$3.00 Copyright
0 1990 by Academic
Press, Inc.
aControlvalues.
1.10
1.01
0.97
0.92
0.86
0.81
0.00"
00" 144” 00 144 00 120 00 96 00 72 00 48 00 24
30.5” 30.0” 30.5 33.0 30.5 29.8 30.5 28.4 29.5 29.7 30.5 30.4 30.5 30.2
24.0“ 23.6” 24.8 25.2 24.3 25.1 24.0 24.8 23.1 24.5 23.0 24.3 22.2 23.1
Fertilizer Exposure Room Aquaria concentration time temp temp (g/liter) (W (“Cl (“Cl
7.05a 7.15” 7.10 7.20 7.25 7.45 7.27 7.41 7.34 7.47 7.39 7.50 7.44 7.57
PH
Parameters
PHOSPHATE FERTILIZER AT DIFFERENT INTERVALS ANDCONCENTRATIONS
184” 163“ 180 148 162 150 154 142 150 140 144 136 133 112
Nil” Nil” Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil 184” 163” 180 148 162 150 154 142 150 140 144 136 133 112
Nil” Nil“ Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil
260” 290“ 450 405 465 430 480 465 495 455 515 570 530 585
7.10 7.60 6.90 7.80 7.00 7.50 6.80
7.70 7.30 7.90
8.00” 7.10” 7.90 7.20
Hardness Hardness Alkalinity Alkalinity Dissolved Hardness (temporary (permanent (phenophthaline (methyl orange oxygen (total ppm) mm) wm) mm) mm) (ppm)
WATER CHARACTERISTICS AFTER DISSOLVING DIAMMONIUM
TABLE 1
F
3
2 ? B
EFFECTS
OF
FERTILIZERS
ON
137
C. batrachus
TABLE 2 EFFECT
OF FERTILIZERS DIAMMONIUM OF FRESH WATER
PHOSPHATE AND UREA ON HEMOGLOBIN TELEOST FISH Clarias batrachus
I. Effect of diammonium
Fertilizer concentration (g/liter)”
0.8 1 0.86 0.92 0.97 1.01 1.10
LEVELS
phosphate
Hemoglobin (g%) mean + SD Exposure time (hr) 24
6.57 f 0.22 5.87 f 0.49 4.20 + 0.57 2.57 f 0.22 1.32kO.12 0.95 f 0.05
72
48 Control:
8.50 + 0.52
5.12+0.17 4.05 f 0.60 3.12 f 0.26 2.62 + 0.22 0.97 +- 0.08
3.95 3.42 1.42 1.04
f f f *
0.23 0.33 0.30 0.09
96
120
144
3.52 f 0.38 1.95 -to.12 0.97 f 0. I 1
1.19 kO.09 1.08 + 0.06
0.82 f 0.11
4.57 k 0.22 4.72 f 0.17 1.82kO.17
3.00 f 0.21 2.62 f 0.43
2.90 f 0.08
II. Effect of urea
7.50 11.50 16.25 20.30 24.70 31.35 * Ten observations
6.45 T 0.36 5.45 f 0.36 3.72k0.17 2.60 +- 0.18 2.50 f 0.29 1.75 kO.12
Control:
8.80 f 0.18
6.00 + 0.2 1 5.30k0.18 3.55 + 0.31 2.27 +0.17 1.95 kO.12
5.07 5.25 2.17 2.00
f + f f
0.17 0.40 0.45 0.29
were made in each case.
Water characteristics were analysed throughout the study before and after dissolving an amount of fertilizer, using the standard procedure (APHA, 1976). Proper oxygen supply was maintained throughout the experiment. Fish were tested at measured intervals and exposures to particular concentrations. Blood was collected in vials by puncturing the caudal vein and was mixed with 1% EDTA (ethylenediaminetetraacetic acid). Hematological parameters-hemoglobin (g%), RBC count (million/cm*), Hct (%), and TLC (cells/cm*)-were analyzed using a semiautomatic blood cell counter (Boehringer-Mannheim Diagnostics, HC-555). OBSERVATIONS A. Effect of DAP on Hernatological
AND
RESULTS
Parameters
1. Water characteristics. pH increased slightly at the termination of the experiment (Table I), while other water characteristics -hardness, temperature, dissolved oxygen, etc.-analyzed before and after dissolving different amounts of fertilizer, were almost the same. 2. Hemoglobin. Hemoglobin content decreased at all concentrations and exposures. However, at higher concentrations (0.97, 1.O1, 1.10 g/liter) a sudden fall in hemoglobin was observed, while at lower concentrations (0.8 1,0.86 g/liter) hemoglobin decreased gradually and was minimum at highest exposures (Table 2), which proved lethal to the fish.
138
TRIVEDI
ET
TABLE EFFECT
OF FERTILIZERS
AL.
3
DIAMMONIUM PHOSPHATE AND UREA ON RBC WATER TELEOST FISH Clariasbatrachus 1. Effect of Diammonium
24
0.81 0.86 0.92 0.97 1.01 1.10
1.05 + 0.55 0.87 + 0.09 1.14+0.11 0.92 f 0.05 0.9 1 f 0.03 0.80 f 0.10
OF FRESH
phosphate
RBC Count (million/cm2) mean Exposure time (hr)
Fertilizer concentration (g/liter) a
COUNT
+ SD
48
72
96
120
144
Control: 1.25 + 0.02 1.29 +0.15 1.16kO.08 0.89 + 0.06 0.67 f 0.12
3.56 f 0.14 1.08 f 0.06 1.30*0.14 0.95 + 0.04 0.67 + 0.04
0.89 k 0.12 1.03kO.13 0.69 + 0.02
0.84 + 0.09 0.78kO.06
0.62 + 0.06
2.66~0.11 2.38 f 0.03 2.17 kO.13
2.30 + 0.11 2.18 f 0.09
2.13 +0.13
II. Effect of Urea 7.50 11.50 16.50 20.30 24.70 31.35
3.28 f 0.10 2.94 + 0.07 2.73 -+ 0.07 2.57kO.10 2.63 kO.17 2.15kO.11
a Ten observations
Control: 2.95 + 0.05 2.80 + 0.05 2.66kO.12 2.58k0.16 2.27 f 0.11
3.43 + 0.06 2.76 f 0.05 2.72 + 0.06 2.24kO.11 2.13+0.06
were made in each case.
TABLE
4
EFFECT OF FERTILIZERS DIAMMONIUM PHOSPHATE AND UREA ON HEMATOCRIT WATER TELEOST FISH Clariasbatrachus I. Effect of Diammonium
OF FRESH
phosphate
Hematocrit (%) mean + SD Exposure time (hr)
Fertilizer concentration (g/liter)”
24
0.81 0.86 0.92 0.97 1.01 1.10
2.42 2.72 2.40 2.65 2.55 3.20
+ 0.33 kO.17 + 0.3 I + 0.68 k 0.26 f 1.00
48
72
96
Control: 2.47kO.12 3.35 + 0.26 2.30 IL 0.18 2.57 + 0.17 2.62 + 0.26
22.35 + 1.10 3.20~0.21 4.40 + 0.40 2.50 + 0.29 2.47-cO.17
4.22 + 0.25 4.12 f 0.49 2.45 + 0.34
120
3.87 f 0.29 2.55 + 0.3 1
144
2.80 f 0.25
II. Effect of Urea 7.20 11.50 16.25 20.30 24.70 31.35
23.32+ 21.17 15.52 12.85+ 10.67 9.40
a Ten observations
1.26 +- 0.74 + 0.60 1.06 + 0.38 + 0.42
Control: 24.45 f 0.62 22.82k0.38 18.32& 1.05 18.62 f 1.08 17.87 f 0.84 15.05 + 0.88 13.95 + 0.58 11.80+ 1.15 9.00k0.35 8.85 f 0.3 1
were made in each case.
17.15k2.17 14.35 + 0.64 9.95 + 0.36
15.272 I.26 10.42 + 0.65
10.82kO.97
EFFECTS
OF
FERTILIZERS
ON
C. batruchus
139
3. RBC count. At all concentrations, the RBC count was lowest at the termination of the experiment. Smaller decreases were seen for initial exposures at lower concentrations, and at higher concentrations the cell count decreased suddenly (Table 3). 4. Hct. The hematocrit content, during shorter intervals and at lower concentrations, was almost unaffected, but decreased over longer durations (Table 4). 5. TLC. At all concentrations, TLC increased gradually with exposure time (Table 5) and was maximum at the end, which proved lethal to experimental fish. B. Effect of Urea on Hematological
Parameters
1. Water characteristics. Variable changes in water characteristics were observed (Table 6) after dissolving the fertilizer; pH of the medium changed slightly while the other characteristics remained almost unaffected. 2. Hb, RBCcount, and Hct. These three parameters decreased at all concentrations and exposures (Tables 2, 3, and 4). Maximum lowerings of 80.11% for Hb content, 37.90% for RBC count, and 15.60% for Hct were recorded. 3. TLC. During urea intoxication, TLC increased but the increase was not pronounced as in DAP toxicity.
DISCUSSION The significant alterations in hematological parameters due to toxicity by various pollutants have been observed in fish (Van Vuren and Hattingh, 1978; Srivastava and Agarwal, 198 1; Raizada and Gupta, 1982). Phosphorus is one of the important constituents of the animal body and is present in soils as tri-, di-, and monophosphate. Phosphates are transported to and from water as adsorbed materials on soil particles and as part of microorganisms. Solid residues, water effluents, and air emissions from phosphate fertilizer-producing industries cause the pollution of water, air, and land. In our experiment diammonium phosphate toxicity caused a sharp fall in RBC count, hemoglobin content, and hematocrit. A decrease in RBC number and hemoglobin was observed in the fish Trichogasterfasciatus after exposure to the fungicide RH-216 (Raizada and Gupta, 1982). In urea toxicity, the three parameters, RBC count, hemoglobin, and hematocrit, decreased at all concentrations and exposures, but these decreases were greater in diammonium phosphate toxicity. A sublethal dose of 10 ppm of the organophosphorus pesticide Rogor resulted in an increase in leukocytes in C. batrachus from 25,000-55,000/cm2 to 32,000/cm2 in 96 hr (Goel and Maya, 1986). CCL, (injected at 0.03 and 0.06 ml/100 g body weight at intervals of 3 days) caused considerable lymphocytosis; i.e., within 6 days, lymphocytes increased from 33 to 72% (Sharma and Gupta, 1982). A IO-ppm dose of Rogor resulted in a fall of total RBC count in C. batrachus from 3,850,OOO to 2,210,000/ cm2 in 96 hr (Goel and Maya, 1986), while CCL, injections caused a decrease in total RBC count from 5,050,OOO to 2,700,000/cm2 in 6 days (Sharma and Gupta, 1982). The RBC count fell from 2,9 10,000 to 1,730,000/cm2 in 10 days in endosulfon poisoning (Gopal et al., 1982). In Heteropneustes fossilis, the pesticide malathion resulted in a decrease in RBC count from 6,400,OOO to 3,460,00O/cm* in 96 hr at 7.6 ppm (Mishra and Srivastava, 1983). In C. batrachus, hemoglobin content decreased from 15.34 to 10.30 g% in 96 hr with 10 ppm of Rogor (Goel and Maya, 1986) and from 10.03 to 8.60 g% in 6 days
25075 f 26925 + 30825 k 36125 + 402OOk 38750 +
7.50 11.50 16.25 20.30 24.70 31.35
16393 4832 2392 1314 535 3500
1206 3287 580 2321 2034 2449
3920 1873 2458 247 1 2 160
1709 4832 1150 1541 1685
Control: 31425 -t 24000 + 30875 + 37225 + 42825 f
48
I. Effect
PHOSPHATE
Control: 56475 + 43775 + 55550 f 57950 + 68000 +
DIAMMONIUM
aTen observationswere madein eachcase.
46625 + 37725 + 40450+ 45825 + 60600 2 74000 +
24
OF FERTILIZERS
0.81 0.86 0.92 0.97 1.01 1.10
Fertilizer concentration (g/liter)*
EFFECT
OF FRESH
phosphate
TLC WATER
22850 54000 46225 66575 63875
-I f + f +
72 1195 2535 1209 1150 2808
20175 26500 22750 33850 38825
+ + + f f
1027 3415 1424 1975 3009
29525 + 1014 23150 + 1554 33700 + 1061
64575 + 994
2465Ok 23425k
911 613
54725 + 3387 55500 k 2434
120
TELEOSTFISH Clarias batruchus
47100 + 1471 49625 + 1941
96
TLC (cells/cm*) mean + SD Exposure time (hr)
Diammonium
ON
II. Effect of Urea
of
AND UREA
TABLE 5
23400 + 1329
50475 k 1828
144
F
2
28
$
z 0
"Control values.
31.35
24.70
20.30
16.25
11.50
7.50
0.00”
00” 144” 00 144 00 120 00 96 00 72 00 48 00 24
35.5” 35.0” 35.5 35.0 35.5 34.8 35.5 34.4 34.5 34.6 35.5 35.2 35.5 35.0
28.0” 27.6“ 27.8 28.2 27.3 28.1 27.0 27.8 26.1 27.5 26.0 21.3 24.2 25.7
Fertilizer Exposure Room Aquaria concentration time temp temp (g/liter) (W 03 (“C)
7.05” 7.65” 7.95 8.23 8.05 9.00 8.10 8.52 8.12 8.38 8.16 8.80 8.22 8.90
pH 242” 252” 239 226 236 224 230 219 226 207 216 192 207 185
Nil” Nil“ Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil Nil
242” 252” 239 226 236 224 230 219 226 207 216 192 207 185
15.0” 10.0” 18.0 151.5 24.0 144.0 32.0 122.0 78.0 39.0 95.5 58.0 122.0 70.0
240.0” 255.0” 301.0 408.0 312.0 392.0 351.5 418.0 368.0 421.5 392.0 422.5 412.5 446.5
9.10” 8.80” 9.00 8.15 9.15 8.45 9.10 8.60 9.20 8.65 9.05 8.70 8.90 8.20
Alkalinity Hardness Hardness Alkalinity Hardness (temporary (permanent (phenophthaline (methyl orange Alkalinity (total ppm) pm) ppm) mm) pv-4 (oxygenppm)
Water characteristics
WATER CHARACTERISTICSAFTERDISSOLVINGUREAFERTILIZERATDIFFERENTINTERVALSANDCONCENTRATIONS
TABLE 6
142
TRIVEDI
ET AL.
with CC& injections (Sharma and Gupta, 1982) while in H. fossilis 7.6 ppm of malathion in the medium resulted in a fall in hemoglobin content from 12.35 to 11.42 g% (Mishra and Srivastava, 1983). CONCLUSION These data lead to the conclusion that lowering of RBC count and hemoglobin levels and leukocytosis in the blood of fresh water fish C. batruchus and H. fossilis are common effects of the pollutants diammonium phosphate and urea fertilizers in the aquatic environment. ACKNOWLEDGMENTS We express our sincere thanks to Professor Bhoomitra Dev, Head of the Department ofZoology, University of Lucknow, Lucknow, India, for providing laboratory facilities and constructive criticism throughout the work. Mr. S. P. Trivedi thanks UGC, Government of India, for the award of Junior Research Fellowship and for financial assistance in carrying out the present study.
REFERENCES APHA (American Public Health Association) (1976). Standard Methods fir the Examination of Water and Waste Waters, 14th ed. American Public Health Assoc., New York. GOEL, K. A., AND MAYA (1986). Haematological anomalies in Clarius butrachus under the stressof Rogor. Adv. Biosci. 5, 187-192. GOEL, K. A., TYAGI, S. K., AND AWASTHI, A. K. (1982). Effect of malathion on some haematological values in Heteropneustesfossilis. Comp. Physiol. Ecol. 7,259-26 1. GOPAL, K., KHANNA, R. N., ANAND, M., AND GUPTA, G. S. D. (1982). Haematological changes in fresh water fish exposed to endosulfon. Indust. Health 20, 157- 159. JOSHI, B. D. (1978). Studies on Blood of Fresh Water Fishes of India. Ph.D. thesis, Lucknow University, Lucknow. MISHRA, J., AND SRIVASTAVA, A. K. (1983). Malathion-induced haematological and biochemical changes in the Indian catfish, Heteropneustesfossilis. Environ. Res. 30,393-398. NAQVI, M. S. (1983). Effect of Environment Pollution on Physiology of Fresh Water Fishes, Ph.D. thesis, Lucknow University, Lucknow. RAIZADA, M. N., AND GUPTA, A. (1982). Toxic effect of Rh-2 16 (a systemic fungicide) from total etythrocyte counts (RBC) and haemoglobin (Hb) content of Trichogasterfasciatus. Comp. Physiol. Ecol. 7( 1), 29-30. SETHI, N., SINGH, R. K., AND DAYAL, R. (1986). Toxic effects of diammonium phosphate on serum aminotransferases activity of teleost, Heteropneustes fossilis. Ind. J. Bio. Res. 2(3), 247-254. SETHI, N., AND SINGH, R. K. (1987). Serum inorganic phosphorus levels of fresh water mud-eel, Amphipnous cuchia, under intoxication of pesticide sumithion. Adv. Biosci. 2, 175- 178. SHARMA, R. C., AND GLJPTA, NEELAM (1982). Carbon tetrachloride-induced haematological alterations in Clark batrachus. J. Environ. Biol. 3, 127-129. SINGH, R. K. (1982). Ecophysiological Studies on Some Fresh Water Fishes. Ph.D. thesis, Lucknow University, Lucknow. SRIVASTAVA, A. K., AND AGARWAL, V. (198 1). Seasonal and diurual variations of blood cell types in a fresh water teleost Colisca fasciatus. Camp. Physiol. Ecol. 6( I), 19. TRIVEDI, S. P., SETHI, N., SINGH, R. K., AND SINGH, P. (1989a). Effect of urea fertilizer on haematological parameters ofteleost fish, Heteropneustesfossilis. Biol. Mem. 15( 1), 54-58. TRIVEDI, S. P., SETHI, N., SINGH, R. K., AND SINGH, P. (1989b). Haematological profile of fish Heteropneustes fossilis in the toxicity of fertilizer diammonium phosphate. Biol. Mem. 15(3), 166-l 76. VAN VUREN, J. H. J., AND HA~TINGH, J. (1978). Seasonal changes in the haemoglobins of fresh water fish in their natural environment. Camp. Biochem. Physiol. 60,265-268. VERMA, S. R., BANSAL, S. K., GUPTA, A. K., AND DALELA, R. C. ( 1979). Pesticide induced haematological alterations in a fresh water fish, Saccobranchusfossilis. Bull. Environ. Contam. Toxicol. 22,467-474.
