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Sublethal effects of an in situ oil shale retort water on rainbow trout
TOXICOLOGY
AND APPLIED
Sublethal
PHARMACOLOGY
54, 462-468
(1980)
Effects of an in Situ Oil Shale Retort Water on Rainbow M. E. LEBSACK,
A. DUANE
ANDERSON, KENNETH AND DAVID S. FARRIER*
Trout’
F. NELSON,
School of Pharmacy, University of Wyoming, Laramie, Wyoming 82071, and *Laramie Energy Technology Center, U.S. Department of Energy, Laramie, Wyoming 82071
Received September 18, 1979; accepted March 20, 1980 Sublethal Effects of an in Situ Oil Shale Retort Water on Rainbow Trout. LEBSACK, M. E., A. D.,NELsoN,K. F., AND FARRIER, D. S. (1980). Toxicol. AppLPharmacol. 54, 462-468. An in situ oil shale process water, designated Omega-9 water, was used in sublethal concentrations in flow-through bioassays with rainbow trout. Exposure for 96 hr to 0.3% of Omega-9 water (approximately 70% of the LCSO) decreased blood packed cell volume (PCV) and hemoglobin concentration. This concentration of Omega-9 water also decreased plasma alkaline phosphatase (AP) activity and protein concentration and caused threefold increases in plasma ammonia levels. Exposure to sublethal concentration of a solution of the 13 major inorganic constituents of Omega-9 water also decreased PCV and plasma AP activity and increased plasma ammonia levels. This study also found that no sublethal effects on blood hemoglobin, protein and alkaline phosphatase occurred at a toxic level less than a fifth of the level of the LCSO. ANDERSON,
Significant quantities of waste waters are produced during in situ combustion retorting of shale to produce shale oil. The initial water produced along with shale oil is referred to as retort water and the minimal amount produced is estimated to at least equal the amount of oil produced (Farrier et al., 1978). Retort waters are heavily contaminated with both inorganic and organic constituents (Jackson et al., 1975; Fox et al., 1978). In an earlier study we showed that one in situ retort water, Omega9 water (described below), is toxic to aquatic species in acute and embryo-larval bioassays (Anderson et al., 1979). This Omega-9 water, which was available in sufficient quantities as a relatively homogeneous solution, was used in the present studies to determine the effects of exposure ’ This work was presented in part at the August 1979 meeting of the American Society for Pharmacology and Experimental Therapeutics. 0041-008X/80/090462-07$02.00/0 Copyright All rights
0 1980 by Academic Press. Inc. of reproduction in any form reserved.
462
to sublethal concentrations on rainbow trout. One purpose of these studies was to obtain data about the mechanism of toxicity of this retort water. An attempt was also made to develop biochemical screens for detrimental effects of Omega-9 water. METHODS Retort water was acquired from the U.S. Department of Energy’s Laramie Energy Technology Center (LETC) site 9 experimental in situ oil shale retorting facility located near Rock Springs, Wyoming. The water was homogenized and pressure filtered through nominal 0.4~pm filters as described by Farrier et al. (1977) and designated “Omega-9 water.” Organic analyses of Omega-9 have been conducted (Stuber and Leenheer 1977) as have those for trace elements, water quality parameters and inorganics (Fox et al., 1978). The average concentrations of the 13 major inorganic ions are presented in Table 1. Rainbow trout, Salmo gairdneri, were obtained from the Wyoming Game and Fish Department or from commercial sources. Fish were maintained in
SUBLETHAL TABLE
EFFECTS
1
CONCENTRATION OF MAJOR INORGANIC CONSTITUENTS OF OMEGA-~ WATER USED -r0 PREPARE THE ART~FEIAL INORGANIC MIXTURE Ion Bicarbonate Sodium Ammonium Thiosulfate Sulfate Chloride Carbonate Tetrathionate Thiocyanate Fluoride Potassium Magnesium Calcium
Concentration (mg/liter) 15,940 4,263 3,470 2,740 1,690 764 500 280 136 62 46 19 11
Note. Other characteristics of “Omega-9” water are as follows: a pH of 8.6, 1003 mg/liter total organic carbon, and 30,300 mg/liter total dissolved solids (Stuber and Leenheer, 1977). dechlorinated, aerated tap water at 10 to 15°C and fed commercial trout pellets. Ten fish, 15 to 22 cm in length, were placed in Xl-liter aquariums provided with continuously floating water. Fish were not fed during the 96-hr test periods and tanks were cleaned daily by siphoning. Fish were never exposed to more than one concentration of contaminated water. Omega-9 water or a solution containing its major inorganic constituents was pumped into a mixing tank above each aquarium and mixed with the flowing water. The actual concentration of the process water delivered to each test tank was monitored spectrophotometrically at 222 nm by comparison to standard Omega-9 water dilutions. Blood for packed cell volume (PCV) and hemoglobin concentration determination was obtained after severing caudal peduncles. (Davidsohn and Nelson, 1974). The remaining pooled blood was assayed for the content of various serum electrolytes, enzyme activities, and other clinical chemistry determinants with a Hycel Autoanalyzer. An adaptation of the method of Lied et al. (1975) was used to obtain sufficient blood from juvenile rainbow trout. Immediately after the fish lost equilibrium (less than 2 min of exposure to a l:lO,OOO dilution of ethyl-m-aminobenzoatemethanesulfonic acid) blood was withdrawn from the vasculature beneath the gills with a tuberculin syringe (23-gauge needle) and rinsed with a 10,000 USP unit/ml solution of
OF OIL
SHALE
WATER
463
sodium heparin. Blood was collected by syringe with caudal amputation and placed in capillary tubes for PCV determinations. The excess blood in the syringe was collected and utilized in protein, ammonia, and alkaline phosphatase (AP) activity determinations. Plasma AP activity was determined kinetically at 26°C as described by Massod et al. (1970). Plasma samples of 25 ~1 were added to 1.5-ml volumes of 0.3 M 2-amino-2-methyl-1,3-propanediol buffer, pH 10.3, containing 0.2 mM MgC& and 6.5 mM pnitrophenol phosphate. The increase in absorbance at 405 nm as p-nitrophenol phosphate was converted to p-nitrophenol was measured with a recording spectrophotometer and reaction rates were linear for at least 10 min. Rates were determined using the extinction coefficient of 1.8 x 104 M-’ cm-’ for p-nitrophenol. Plasma protein concentrations were determined by the biuret method (Coma11 er al., 1949). The concentration of un-ionized ammonia in plasma was measured from the reductive amination of a-ketoglutarate by L-glutamate dehydrogenase as described by Dewan (1938). The oxidation of the coenzyme NADH to NAD was measured spectrophotometrically at 340 nm. Data from trout exposed to Omega-9 water was analyzed with a one-way analysis of variance using a computer program from the Statistical Package for the Social Sciences (Nie, 1975). Treatment means were declared significantly different when p < 0.05 using Duncan’s (1955) mean range test. Means from fish exposed to the inorganic mixture were compared to control means using Student’s t test (Schefler, 1969).
RESULTS Paradoxically, the serum of trout exposed for 96 hr to 0.36% Omega-9 water had lower CPK and AP activities when compared to control serum. After exposure to this concentration of retort water, serum LDH, GOT, GPT, and electrolytes, including sodium, potassium, chloride, calcium, and inorganic phosphorus, were unchanged. Retort water exposure led to a decrease in serum glucose, cholesterol, total protein, globulin, urea nitrogen, bilirubin, and creatinine. Since the sera of many fish had to be pooled to obtain enough serum for these tests, only one control and three samples from exposed fish could be run. Therefore, statistical analysis of these data was impossible. These data indicate that further
464
LEBSACK
ET AL.
measurements should be run to determine the effect on these biochemical indicators. The PCV of blood of individual fish could be measured since the procedure for its determination requires only small volumes of blood. As shown in Table 2, 96 hr of exposure to Omega-9 water decreased PCVs in a concentration-dependent manner regardless of the method by which blood was obtained. Hemoglobin concentrations (Table 2) also significantly decreased in fish exposed to 0.3% Omega-9 water for 96 hr. Table 3 illustrates the changes in plasma parameters resulting from 96 hr of exposure to Omega-9 concentrations of 0.08 to 0.3%. Plasma ammonia levels were increased nearly threefold by exposure to 0.3% Omega-9 water. Exposure to a concentration of 0.15, but not 0.08%, significantly decreased plasma protein concentrations. Plasma AP activity was significantly decreased by 96 hr of exposure to 0.1% Omega-9 water. Exposure to 0.2 or 0.3% of this process water decreased plasma AP activity to approximately 60% of control levels. Based on our previous determinations of the 96-hr LCSOs of Omega-9 water and the artificial mixture of its major inorganic constituents (Anderson et al., 1979), 0.3%
Omega-9 water and 0.4% of the inorganic solution are both approximately 70% of their respective LCSO concentrations. Plasma ammonia was increased as much by 96 hr of exposure to 0.4% of the inorganic solution as to 0.3% Omega-9 water (Table 4), but the resulting decreases in PCV and plasma AP activity, although significant, were less pronounced and plasma protein content was unaffected. DISCUSSION Exposure of animals, including fish, to sublethal concentrations of toxic substances often elevates serum or plasma enzyme activity and other biochemical measurements and these increases usually correlate with tissue damage. Plasma enzyme activities, including LDH, GOT, and GPT, are increased in rainbow trout injected with carbon tetrachloride (Racicot et al., 1975). Brook trout exposed for 6 days to sublethal concentrations of copper have elevated plasma GOT activities as well as increased PCVs hemoglobin concentrations and plasma protein concentrations (McKim et al., 1970). Eight-day exposure of pike to phenol elevates plasma LDH, GOT, and GPT activities (Kristoffersson et al., 1974).
TABLE PACKED
CELL
VOLUMES
OF RAINBOW
2
TROUT
EXPOSED
TO OMEGA-~
WATER
Blood sampling method-PCV Omega-9 water concentration (%o) 0 0.8 0.15 0.30
Caudal amputation 55.7 50.8 46.4 41.8
2 k k k
1.2 1.7 2.1 1.9
(10) (10) (lO)O (lO)a*b
Syringe 50.7 ” 1.9 (16) 42.8 + 1.4 (18)a 38.9 + 1.2 (18)a
Hemoglobin (g/l~ ml) concentrations 10.0 9.72 9.02 7.98
‘2 + +
0.3 0.26 0.33 0.54
(10) (10) (10) (10)
Note. Fish were exposed to Omega-9 water for 96 hr in a flow-through system. PCV values are means + SE expressed in percentage with the number of animals in each group in parentheses. a Significantly (p < 0.05) different from control mean. b Significantly (p < 0.05) different from 0.08% mean. c Significantly different from control and 0.08% means (p < 0.05).
SUBLETHAL
EFFECTS OF OIL SHALE TABLE
PLASMA
VALUES
WATER
465
3
AFTEREXPOSURETOOMEGA-~WATER
Plasma parameter Omega-9 water concentration (“ro) 0 0.08 0.10 0.15 0.20 0.30
Protein (g/100 ml) 3.38 + 0.16 3.05 k 0.22 2.71 ? 0.14 2.76 + 0.11
Alkaline phosphatase (pmol/min/liter)
Ammonia (raW
(15) (3)
6.17 k 1.03 (6) 9.49 k 1.37 (8) 16.9 ? 2.5 (lO)c(,p
(11)” (20)”
124 rfr 5.5 106 -r- 6.1 98.4 + 8.4 76.2 + 6.4 78.3 + 7.9
(63) (40)” (17)” (14)“,b (15)a,b
Note. Rainbow trout were exposed for 96 hr to Omega-9 water in flow-through experiments. Blood was obtained from behind the gills with a syringe. Values are means 2 SE and the number of animals in each group is given in parentheses. u Significantly (p < 0.05) different from control mean. b Significantly (p < 0.05) different from 0.1% mean. c Significantly (p < 0.05) different from 0.15% mean.
Factors other than exposure to toxicant can also increase plasma values in fish. Wedemeyer (1972) reported that the stress resulting from the handling of the fish increased blood glucose and plasma cholesterol, chloride, and calcium levels in Salmo gairdneri. Hemorrhagic stress increases the activity of LDH and CPK in the plasma of rainbow trout (Cairns and Christian, 1978). In our preliminary screen of the effects of exposure to Omega-9 water, serum parameters were unchanged or decreased in TABLE EFFECTOFTHE
rainbow trout. Upon further study we found that blood PCV and hemoglobin concentration as well as plasma AP activity and protein concentration were decreased in a concentration dependent manner when fish were exposed to Omega-9 water. Decreases, unlike increases, in plasma enzyme activities do not pinpoint damage to specific organs. Since none of the blood parameters were increased after exposure to Omega-9 water, the lowered values might have resulted from overhydration or some other 4
MAIORINORGANICCONSTITUENTSOFOMEGA-~WATER ON BLOODVALUESOFRAINBOWTROUT
Ionic solution concentration 0 (Control) Packed Plasma Plasma Plasma
cell volume (%) proteins (g/100 ml) ammonia (&ml) alkaline phosphatase activity (~mol/min/liter)
Note. Values exposed for 96 hr a Significantly b Significantly
47.5 3.60 4.34 132
t k + +
1.7 0.08 0.16 13
0.4% (19) (16) (10) (20)
42.5 3.38 13.1 101
t 2 * +
1.6 0.12 0.5 8
(17)” (20) (14)b (25)”
are means 2 SE. The number of animals in each group is given in parentheses. Fish were in a flow-through system. different from control, p < 0.05. different from control, p < 0.001.
466
LEBSACK
mechanism resulting in dilution of the blood. Parameters such as sodium, potassium and chloride levels that are unchanged after 96 hr of exposure of rainbow trout to Omega-9 water may be elements that are quickly equilibrated with intracellular elements to maintain relatively constant blood levels. These elements are also contained in the Omega-9 water and, therefore, are available for the maintenance of normal blood levels. The biochemical changes resulting from Omega-9 exposure resemble the sublethal effects of ammonia. Lloyd and Orr (1968) postulate that ammonia increases the permeability of fish to water since this compound greatly increases the urinary output of rainbow trout without changing body weight. Omega-9 water contains substantial quantities of a number of ions including a total ammonia (NH: + NH,) concentration of approximately 3.5 g/liter (Table 1). Un-ionized ammonia (NH,) is considered the toxic form of this chemical (Downing and Merkens, 1955). Using the table of percentage un-ionized ammonia from Trussell (1972) based on the water pH and temperature of our studies, a 0.3% dilution of Omega-9 water would contain about 0.15 mg/liter NHB. The European Inland Fisheries Advisory Commission (1973) reports that adverse effects of NH, are absent only at concentrations lower than 0.03 mg/liter. The data of Lloyd and Orr (1969) indicate that concentrations of NH, lower than 0.05 mg/liter, 12% of the lethal threshold concentration may be without toxic effect. An NH3 level of 0.05 mg/liter corresponds to approximately 0.1% of Omega-9 water which was the lowest retort water concentration at which any blood parameter was decreased. Exposure for 96 hr to 0.3% Omega-9 water causes almost threefold increases in plasma levels of total ammonia (Table 3). Some of the increases in plasma ammonia could be due to increased protein catabolism as rainbow trout excrete nitrogen in the form of ammonia. It is likely,
ET AL.
however, that much of the elevated plasma ammonia level results from increased movement of exogenous ammonia into the animal. Time-course studies of the development of the changes in ammonia could provide additional insight into the mechanism of this change. Exposure to a dilute solution of the 13 major inorganic constituents of Omega-9 water caused qualitatively the same changes as those induced by Omega-9 water itself. A 0.4% concentration of the synthetic inorganic ion solution (approximately 70% of the 96-hr LC50) caused approximately the same threefold increase in plasma ammonia levels that resulted from exposure to 0.3% Omega-9 water (also 70% of the 96-hr LC50). However, the 0.4% inorganic ion solution was not nearly as effective as the 0.3% Omega-9 water in lowering PCV, plasma AP activity or plasma protein concentration. These findings suggest that some of the Omega-9 water induced decreased in blood values are due to components other than the 13 major inorganic constituents. Injection of petroleum ether or gasoline ip has been reported to decrease serum AP activity in rats (Kala et al., 1978) and exposure to sodium lauryl sulfate decreased liver and kidney AP activity in some teleosts (Verma et al., 1979). It is therefore possible that some of the decrease in AP activity in rainbow trout plasma may be due to organic constituents of Omega-9 water. Tucker and Leitzke (1979) concluded that no sublethal effect tends to occur at a level much less than a quarter or a sixth of the LC50 concentration. Our study is consistent with their observations. At 0.3% concentration (70% of LC50 concentration) blood hemoglobin, protein, and alkaline phosphatase levels were reduced 80, 82, and 63%, respectively, when compared to control values (See Tables 2 and 3). Exposure to 0.08% concentration lowered hemoglobin and protein levels 10 and 9%, respectively. This exposure (0.08/0.3 x 70% of the LC50) equals approximately one-
SUBLETHAL
EFFECTS
fifth of the LCSO concentration. Alkaline phosphatase activity was lowered 15% at 0.1% Omega-9 water (O.UO.3 x 70% of the LC50) which equals approximately one quarter of the LCSO concentration. This information is consistent with Tucker and Leitzke’s (1979) observation. We have studied a number of parameters that can be used as indicators of Omega-9 water toxicity at sublethal levels. Blood can be obtained relatively easily without permanent damage to rainbow trout. Significant changes in PCV, plasma protein and plasma AP activity occurred after 96 hr of exposure to 0.15% dilutions of Omega-9 water. Exposure to 0.1% Omega-9 water (less than 20% of the 96-hr LC50) significantly decreased plasma AP activity. This dilution of Omega-9 water is near the 0.16% dilution that was the lowest concentration that decreased the size of rainbow trout fry hatched in this Omega-9 water (Anderson et al., 1979). The biochemical changes caused by Omega-9 water substantiate the suggestion that levels of this process water may cause adverse effects if allowed to exceed 0.1%. Omega-9 water may not be representative of all fossil fuel process waters but it provided a stable, homogenous sample for initial testing purposes. Extrapolation of biochemical perturbations to other waters or their toxic components will require a comparative study of a variety of waters. ACKNOWLEDGMENTS This work was performed pursuant to an Interagency Agreement between the U.S. Department of Energy and the U.S. Environmental Protection Agency under Contract DE-AS20-79 LC 01761 to the Rocky Mountain Institute of Energy and Environment, University of Wyoming. We would like to thank Cynthia A. Jones for her fine technical and editorial assistance.
REFERENCES ANDERSON, A. D., LEBSACK, M. E., DEGRAEVE, G. M., FARRIER, D. S., AND BERGMAN, H. L. (1979).
OF OIL
SHALE
467
WATER
Toxicity of an in situ oil shale process water to rainbow trout and fathead minnows. Arch. Environ. Contam. Toxicol., in press. CAIRNS, M. A., and CHRISTIAN, A. R. (1978). Effects of hemorrhagic stress on several blood parameters in adult rainbow trout (Salmo gairdneri). Trans. Amer. Fish.
Sot.
107, 334-340.
DAVIDSHON, I., AND NELSON, D. A. (1974). The blood. In Clinical Diagnosis by Laboratory Methods. (I. Davidsohn and J. B. Henry, eds.), 15th ed., p. 106. Saunders, Philadelphia. DEWAN, J. G. (1938). The L(+) glutamic dehydrogenase of animal tissues. Biochem. J. 32, 1378. DOWNING, K. M.. AND MERKENS, J. C. (1955). The influence of dissolved-oxygen concentration on the toxicity of un-ionized ammonia to rainbow trout (Salmo gairdneri Richardson). Ann. Appl. Biol. 43, 243-246. DUNCAN, D. B. (1955). Multiple range and multiple F tests. Biometrics 11, l-42. European Inland Fisheries Advisory Commission (1973). Water quality criteria for fresh water fish. Report on ammonia and inland fisheries. Water Res. 7,1011-1122. FARRIER, D. S., POULSON, R. E., SKINNER, Q. D., ADAMS, J. C., AND BOWER, J. B. (1977). Acquisition, processing and storage for environmental research of aqueous effluents from in situ oil shale processing. In Proceedings Engineering
of the Congress,
Second
Pacific
Chemical
Denver, Cola., Vol. 2, pp.
1031-1045. FARRIER, D. S., VIRGONA, J. E., PHILLIPS, T. E., AND POULSON, R. E. (1978). Environmental research for in situ oil shale processing. In Proceedings of the 1 I th Annual Mines,
Oil Shale
Symposium,
Colorado
School
of
Golden Colo. Fox, J. P., FARRIER, D. S., AND POULSON, R. E. (1978). Chemical Characterization and Analytical Considerations for an in Situ Oil Shale Process Water. Laramie Energy Technology Center Report
N. LETCIRI-7817, Technical U.S. Department of Energy. GORNALL,
A. G., BARDAWILL,
Information
C. J., AND DAVID,
Center, M. M.
(1949). Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177, 751-766. JACKSON, L. P., POULSON, R. E., SPEDDING, T. J., PHILLIPS, T. E., AND JENSEN, H. B. (1975). Char-
acteristics and possible roles of various waters significant to in situ oil shale processing. Colorado School Mines Quart. 70, 105-134. KALA, A., RAO, G. S., AND PANDYA, K. P. (1978). Effects of petroleum hydrocarbon solvents on alkaline phosphatase of rats. Bull. Environ. Contam. Toxicol. 19, 287-294. KRISTOFFERSSON, R., BROBERG, S., OIKARI, A., AND PEKKARINEN, M. (1974). Effect of a sublethal con-
centration of phenol on some blood plasma enzyme
468
LEBSACK
activities in the pike (Esox Lucius L.) in brackish water. Ann. Zool. Fennici 11, 220-223. LIED, E., GJERDE, J., AND BRAEKKAN, 0. R. (1975). Simple and rapid technique for repeated blood sampling in rainbow trout (S&no gairdneri). J. Fish. Res. Bd. Canada 32, 699-701. LLOYD, R., AND ORR, L. D. (1969). The diuretic response by rainbow trout to sublethal concentrations of ammonia. Water Res. 3, 335-344. MASSOD, J. F., WERNER, K. R., AND MCGUIRE, S. L. (1970). Kinetic determination of serum alkaline phosphatase activity. Amer. J. Clin. Pathol. 54, 1lo117. MCKIM, J. M., CHRISTENSEN, G. M., AND HUNT, E. P. (1970). Changes in the blood of brook trout (Salvelinus fontinalis) after short-term and long-term exposure to copper. J. Fish. Res. Bd. Canada 27, 1883- 1889. NIE, N. H. (1975). SPSS-Statistical Package for the Social Sciences. McGraw-Hill, New York. RACICOT, J. G., GAUDET, H., AND LERAY, C. (1975). Blood and liver enzymes in rainbow trout (S&no gairdneri Rich.) with emphasis on their diagnostic use: Study of CC& toxicity and a case of Aeromonas infection. J. Fish Biol. 7, 825-835.
ET AL. SCHEFLER, W. C. (1969). Statistics for the Biological Sciences. Addison-Wesley, Reading, Mass. STUBER, H. A., AND LEENHEER, J. A. (1977). Fractionation of Organic Solutes in Oil Shale Retort Waters for Sorption Studies, Preprints American Chemical Society, Division of Fuel Chemistry. 23, pp. 165- 174. TUCKER, R. K., AND LEITZKE, J. S. (1979). Comparative toxicology of insecticides for vertebrate wildlife and fish. Pharm. Ther. 6, 167-220. TRUSSELL, R. P. (1972). The percent unionized ammonia in aqueous ammonia solutions at different pH levels and temperatures. J. Fish. Res. Bd. Canada 19, 150.5-1507. VERMA, S. R.. PAL, N., TYAGI, A. K., AND DALELA, R. C. (1979). Toxicity of SwascolR 1P (SLS) to Chnnna puncfutus and Cirrhina mrigala: Biochemical alterations. Bull. Environ. Contam. Toxico[. 21,711-718. WEDEMEYER, G. (1972). Some physiological consequences of handling stress in the juvenile coho salmon (Oncorhyndhus Kisutch) and steelhead trout (Salmo gairdneri). J. Fish. Res. Bd. Canada 29, 1780- 1783.
AND APPLIED
Sublethal
PHARMACOLOGY
54, 462-468
(1980)
Effects of an in Situ Oil Shale Retort Water on Rainbow M. E. LEBSACK,
A. DUANE
ANDERSON, KENNETH AND DAVID S. FARRIER*
Trout’
F. NELSON,
School of Pharmacy, University of Wyoming, Laramie, Wyoming 82071, and *Laramie Energy Technology Center, U.S. Department of Energy, Laramie, Wyoming 82071
Received September 18, 1979; accepted March 20, 1980 Sublethal Effects of an in Situ Oil Shale Retort Water on Rainbow Trout. LEBSACK, M. E., A. D.,NELsoN,K. F., AND FARRIER, D. S. (1980). Toxicol. AppLPharmacol. 54, 462-468. An in situ oil shale process water, designated Omega-9 water, was used in sublethal concentrations in flow-through bioassays with rainbow trout. Exposure for 96 hr to 0.3% of Omega-9 water (approximately 70% of the LCSO) decreased blood packed cell volume (PCV) and hemoglobin concentration. This concentration of Omega-9 water also decreased plasma alkaline phosphatase (AP) activity and protein concentration and caused threefold increases in plasma ammonia levels. Exposure to sublethal concentration of a solution of the 13 major inorganic constituents of Omega-9 water also decreased PCV and plasma AP activity and increased plasma ammonia levels. This study also found that no sublethal effects on blood hemoglobin, protein and alkaline phosphatase occurred at a toxic level less than a fifth of the level of the LCSO. ANDERSON,
Significant quantities of waste waters are produced during in situ combustion retorting of shale to produce shale oil. The initial water produced along with shale oil is referred to as retort water and the minimal amount produced is estimated to at least equal the amount of oil produced (Farrier et al., 1978). Retort waters are heavily contaminated with both inorganic and organic constituents (Jackson et al., 1975; Fox et al., 1978). In an earlier study we showed that one in situ retort water, Omega9 water (described below), is toxic to aquatic species in acute and embryo-larval bioassays (Anderson et al., 1979). This Omega-9 water, which was available in sufficient quantities as a relatively homogeneous solution, was used in the present studies to determine the effects of exposure ’ This work was presented in part at the August 1979 meeting of the American Society for Pharmacology and Experimental Therapeutics. 0041-008X/80/090462-07$02.00/0 Copyright All rights
0 1980 by Academic Press. Inc. of reproduction in any form reserved.
462
to sublethal concentrations on rainbow trout. One purpose of these studies was to obtain data about the mechanism of toxicity of this retort water. An attempt was also made to develop biochemical screens for detrimental effects of Omega-9 water. METHODS Retort water was acquired from the U.S. Department of Energy’s Laramie Energy Technology Center (LETC) site 9 experimental in situ oil shale retorting facility located near Rock Springs, Wyoming. The water was homogenized and pressure filtered through nominal 0.4~pm filters as described by Farrier et al. (1977) and designated “Omega-9 water.” Organic analyses of Omega-9 have been conducted (Stuber and Leenheer 1977) as have those for trace elements, water quality parameters and inorganics (Fox et al., 1978). The average concentrations of the 13 major inorganic ions are presented in Table 1. Rainbow trout, Salmo gairdneri, were obtained from the Wyoming Game and Fish Department or from commercial sources. Fish were maintained in
SUBLETHAL TABLE
EFFECTS
1
CONCENTRATION OF MAJOR INORGANIC CONSTITUENTS OF OMEGA-~ WATER USED -r0 PREPARE THE ART~FEIAL INORGANIC MIXTURE Ion Bicarbonate Sodium Ammonium Thiosulfate Sulfate Chloride Carbonate Tetrathionate Thiocyanate Fluoride Potassium Magnesium Calcium
Concentration (mg/liter) 15,940 4,263 3,470 2,740 1,690 764 500 280 136 62 46 19 11
Note. Other characteristics of “Omega-9” water are as follows: a pH of 8.6, 1003 mg/liter total organic carbon, and 30,300 mg/liter total dissolved solids (Stuber and Leenheer, 1977). dechlorinated, aerated tap water at 10 to 15°C and fed commercial trout pellets. Ten fish, 15 to 22 cm in length, were placed in Xl-liter aquariums provided with continuously floating water. Fish were not fed during the 96-hr test periods and tanks were cleaned daily by siphoning. Fish were never exposed to more than one concentration of contaminated water. Omega-9 water or a solution containing its major inorganic constituents was pumped into a mixing tank above each aquarium and mixed with the flowing water. The actual concentration of the process water delivered to each test tank was monitored spectrophotometrically at 222 nm by comparison to standard Omega-9 water dilutions. Blood for packed cell volume (PCV) and hemoglobin concentration determination was obtained after severing caudal peduncles. (Davidsohn and Nelson, 1974). The remaining pooled blood was assayed for the content of various serum electrolytes, enzyme activities, and other clinical chemistry determinants with a Hycel Autoanalyzer. An adaptation of the method of Lied et al. (1975) was used to obtain sufficient blood from juvenile rainbow trout. Immediately after the fish lost equilibrium (less than 2 min of exposure to a l:lO,OOO dilution of ethyl-m-aminobenzoatemethanesulfonic acid) blood was withdrawn from the vasculature beneath the gills with a tuberculin syringe (23-gauge needle) and rinsed with a 10,000 USP unit/ml solution of
OF OIL
SHALE
WATER
463
sodium heparin. Blood was collected by syringe with caudal amputation and placed in capillary tubes for PCV determinations. The excess blood in the syringe was collected and utilized in protein, ammonia, and alkaline phosphatase (AP) activity determinations. Plasma AP activity was determined kinetically at 26°C as described by Massod et al. (1970). Plasma samples of 25 ~1 were added to 1.5-ml volumes of 0.3 M 2-amino-2-methyl-1,3-propanediol buffer, pH 10.3, containing 0.2 mM MgC& and 6.5 mM pnitrophenol phosphate. The increase in absorbance at 405 nm as p-nitrophenol phosphate was converted to p-nitrophenol was measured with a recording spectrophotometer and reaction rates were linear for at least 10 min. Rates were determined using the extinction coefficient of 1.8 x 104 M-’ cm-’ for p-nitrophenol. Plasma protein concentrations were determined by the biuret method (Coma11 er al., 1949). The concentration of un-ionized ammonia in plasma was measured from the reductive amination of a-ketoglutarate by L-glutamate dehydrogenase as described by Dewan (1938). The oxidation of the coenzyme NADH to NAD was measured spectrophotometrically at 340 nm. Data from trout exposed to Omega-9 water was analyzed with a one-way analysis of variance using a computer program from the Statistical Package for the Social Sciences (Nie, 1975). Treatment means were declared significantly different when p < 0.05 using Duncan’s (1955) mean range test. Means from fish exposed to the inorganic mixture were compared to control means using Student’s t test (Schefler, 1969).
RESULTS Paradoxically, the serum of trout exposed for 96 hr to 0.36% Omega-9 water had lower CPK and AP activities when compared to control serum. After exposure to this concentration of retort water, serum LDH, GOT, GPT, and electrolytes, including sodium, potassium, chloride, calcium, and inorganic phosphorus, were unchanged. Retort water exposure led to a decrease in serum glucose, cholesterol, total protein, globulin, urea nitrogen, bilirubin, and creatinine. Since the sera of many fish had to be pooled to obtain enough serum for these tests, only one control and three samples from exposed fish could be run. Therefore, statistical analysis of these data was impossible. These data indicate that further
464
LEBSACK
ET AL.
measurements should be run to determine the effect on these biochemical indicators. The PCV of blood of individual fish could be measured since the procedure for its determination requires only small volumes of blood. As shown in Table 2, 96 hr of exposure to Omega-9 water decreased PCVs in a concentration-dependent manner regardless of the method by which blood was obtained. Hemoglobin concentrations (Table 2) also significantly decreased in fish exposed to 0.3% Omega-9 water for 96 hr. Table 3 illustrates the changes in plasma parameters resulting from 96 hr of exposure to Omega-9 concentrations of 0.08 to 0.3%. Plasma ammonia levels were increased nearly threefold by exposure to 0.3% Omega-9 water. Exposure to a concentration of 0.15, but not 0.08%, significantly decreased plasma protein concentrations. Plasma AP activity was significantly decreased by 96 hr of exposure to 0.1% Omega-9 water. Exposure to 0.2 or 0.3% of this process water decreased plasma AP activity to approximately 60% of control levels. Based on our previous determinations of the 96-hr LCSOs of Omega-9 water and the artificial mixture of its major inorganic constituents (Anderson et al., 1979), 0.3%
Omega-9 water and 0.4% of the inorganic solution are both approximately 70% of their respective LCSO concentrations. Plasma ammonia was increased as much by 96 hr of exposure to 0.4% of the inorganic solution as to 0.3% Omega-9 water (Table 4), but the resulting decreases in PCV and plasma AP activity, although significant, were less pronounced and plasma protein content was unaffected. DISCUSSION Exposure of animals, including fish, to sublethal concentrations of toxic substances often elevates serum or plasma enzyme activity and other biochemical measurements and these increases usually correlate with tissue damage. Plasma enzyme activities, including LDH, GOT, and GPT, are increased in rainbow trout injected with carbon tetrachloride (Racicot et al., 1975). Brook trout exposed for 6 days to sublethal concentrations of copper have elevated plasma GOT activities as well as increased PCVs hemoglobin concentrations and plasma protein concentrations (McKim et al., 1970). Eight-day exposure of pike to phenol elevates plasma LDH, GOT, and GPT activities (Kristoffersson et al., 1974).
TABLE PACKED
CELL
VOLUMES
OF RAINBOW
2
TROUT
EXPOSED
TO OMEGA-~
WATER
Blood sampling method-PCV Omega-9 water concentration (%o) 0 0.8 0.15 0.30
Caudal amputation 55.7 50.8 46.4 41.8
2 k k k
1.2 1.7 2.1 1.9
(10) (10) (lO)O (lO)a*b
Syringe 50.7 ” 1.9 (16) 42.8 + 1.4 (18)a 38.9 + 1.2 (18)a
Hemoglobin (g/l~ ml) concentrations 10.0 9.72 9.02 7.98
‘2 + +
0.3 0.26 0.33 0.54
(10) (10) (10) (10)
Note. Fish were exposed to Omega-9 water for 96 hr in a flow-through system. PCV values are means + SE expressed in percentage with the number of animals in each group in parentheses. a Significantly (p < 0.05) different from control mean. b Significantly (p < 0.05) different from 0.08% mean. c Significantly different from control and 0.08% means (p < 0.05).
SUBLETHAL
EFFECTS OF OIL SHALE TABLE
PLASMA
VALUES
WATER
465
3
AFTEREXPOSURETOOMEGA-~WATER
Plasma parameter Omega-9 water concentration (“ro) 0 0.08 0.10 0.15 0.20 0.30
Protein (g/100 ml) 3.38 + 0.16 3.05 k 0.22 2.71 ? 0.14 2.76 + 0.11
Alkaline phosphatase (pmol/min/liter)
Ammonia (raW
(15) (3)
6.17 k 1.03 (6) 9.49 k 1.37 (8) 16.9 ? 2.5 (lO)c(,p
(11)” (20)”
124 rfr 5.5 106 -r- 6.1 98.4 + 8.4 76.2 + 6.4 78.3 + 7.9
(63) (40)” (17)” (14)“,b (15)a,b
Note. Rainbow trout were exposed for 96 hr to Omega-9 water in flow-through experiments. Blood was obtained from behind the gills with a syringe. Values are means 2 SE and the number of animals in each group is given in parentheses. u Significantly (p < 0.05) different from control mean. b Significantly (p < 0.05) different from 0.1% mean. c Significantly (p < 0.05) different from 0.15% mean.
Factors other than exposure to toxicant can also increase plasma values in fish. Wedemeyer (1972) reported that the stress resulting from the handling of the fish increased blood glucose and plasma cholesterol, chloride, and calcium levels in Salmo gairdneri. Hemorrhagic stress increases the activity of LDH and CPK in the plasma of rainbow trout (Cairns and Christian, 1978). In our preliminary screen of the effects of exposure to Omega-9 water, serum parameters were unchanged or decreased in TABLE EFFECTOFTHE
rainbow trout. Upon further study we found that blood PCV and hemoglobin concentration as well as plasma AP activity and protein concentration were decreased in a concentration dependent manner when fish were exposed to Omega-9 water. Decreases, unlike increases, in plasma enzyme activities do not pinpoint damage to specific organs. Since none of the blood parameters were increased after exposure to Omega-9 water, the lowered values might have resulted from overhydration or some other 4
MAIORINORGANICCONSTITUENTSOFOMEGA-~WATER ON BLOODVALUESOFRAINBOWTROUT
Ionic solution concentration 0 (Control) Packed Plasma Plasma Plasma
cell volume (%) proteins (g/100 ml) ammonia (&ml) alkaline phosphatase activity (~mol/min/liter)
Note. Values exposed for 96 hr a Significantly b Significantly
47.5 3.60 4.34 132
t k + +
1.7 0.08 0.16 13
0.4% (19) (16) (10) (20)
42.5 3.38 13.1 101
t 2 * +
1.6 0.12 0.5 8
(17)” (20) (14)b (25)”
are means 2 SE. The number of animals in each group is given in parentheses. Fish were in a flow-through system. different from control, p < 0.05. different from control, p < 0.001.
466
LEBSACK
mechanism resulting in dilution of the blood. Parameters such as sodium, potassium and chloride levels that are unchanged after 96 hr of exposure of rainbow trout to Omega-9 water may be elements that are quickly equilibrated with intracellular elements to maintain relatively constant blood levels. These elements are also contained in the Omega-9 water and, therefore, are available for the maintenance of normal blood levels. The biochemical changes resulting from Omega-9 exposure resemble the sublethal effects of ammonia. Lloyd and Orr (1968) postulate that ammonia increases the permeability of fish to water since this compound greatly increases the urinary output of rainbow trout without changing body weight. Omega-9 water contains substantial quantities of a number of ions including a total ammonia (NH: + NH,) concentration of approximately 3.5 g/liter (Table 1). Un-ionized ammonia (NH,) is considered the toxic form of this chemical (Downing and Merkens, 1955). Using the table of percentage un-ionized ammonia from Trussell (1972) based on the water pH and temperature of our studies, a 0.3% dilution of Omega-9 water would contain about 0.15 mg/liter NHB. The European Inland Fisheries Advisory Commission (1973) reports that adverse effects of NH, are absent only at concentrations lower than 0.03 mg/liter. The data of Lloyd and Orr (1969) indicate that concentrations of NH, lower than 0.05 mg/liter, 12% of the lethal threshold concentration may be without toxic effect. An NH3 level of 0.05 mg/liter corresponds to approximately 0.1% of Omega-9 water which was the lowest retort water concentration at which any blood parameter was decreased. Exposure for 96 hr to 0.3% Omega-9 water causes almost threefold increases in plasma levels of total ammonia (Table 3). Some of the increases in plasma ammonia could be due to increased protein catabolism as rainbow trout excrete nitrogen in the form of ammonia. It is likely,
ET AL.
however, that much of the elevated plasma ammonia level results from increased movement of exogenous ammonia into the animal. Time-course studies of the development of the changes in ammonia could provide additional insight into the mechanism of this change. Exposure to a dilute solution of the 13 major inorganic constituents of Omega-9 water caused qualitatively the same changes as those induced by Omega-9 water itself. A 0.4% concentration of the synthetic inorganic ion solution (approximately 70% of the 96-hr LC50) caused approximately the same threefold increase in plasma ammonia levels that resulted from exposure to 0.3% Omega-9 water (also 70% of the 96-hr LC50). However, the 0.4% inorganic ion solution was not nearly as effective as the 0.3% Omega-9 water in lowering PCV, plasma AP activity or plasma protein concentration. These findings suggest that some of the Omega-9 water induced decreased in blood values are due to components other than the 13 major inorganic constituents. Injection of petroleum ether or gasoline ip has been reported to decrease serum AP activity in rats (Kala et al., 1978) and exposure to sodium lauryl sulfate decreased liver and kidney AP activity in some teleosts (Verma et al., 1979). It is therefore possible that some of the decrease in AP activity in rainbow trout plasma may be due to organic constituents of Omega-9 water. Tucker and Leitzke (1979) concluded that no sublethal effect tends to occur at a level much less than a quarter or a sixth of the LC50 concentration. Our study is consistent with their observations. At 0.3% concentration (70% of LC50 concentration) blood hemoglobin, protein, and alkaline phosphatase levels were reduced 80, 82, and 63%, respectively, when compared to control values (See Tables 2 and 3). Exposure to 0.08% concentration lowered hemoglobin and protein levels 10 and 9%, respectively. This exposure (0.08/0.3 x 70% of the LC50) equals approximately one-
SUBLETHAL
EFFECTS
fifth of the LCSO concentration. Alkaline phosphatase activity was lowered 15% at 0.1% Omega-9 water (O.UO.3 x 70% of the LC50) which equals approximately one quarter of the LCSO concentration. This information is consistent with Tucker and Leitzke’s (1979) observation. We have studied a number of parameters that can be used as indicators of Omega-9 water toxicity at sublethal levels. Blood can be obtained relatively easily without permanent damage to rainbow trout. Significant changes in PCV, plasma protein and plasma AP activity occurred after 96 hr of exposure to 0.15% dilutions of Omega-9 water. Exposure to 0.1% Omega-9 water (less than 20% of the 96-hr LC50) significantly decreased plasma AP activity. This dilution of Omega-9 water is near the 0.16% dilution that was the lowest concentration that decreased the size of rainbow trout fry hatched in this Omega-9 water (Anderson et al., 1979). The biochemical changes caused by Omega-9 water substantiate the suggestion that levels of this process water may cause adverse effects if allowed to exceed 0.1%. Omega-9 water may not be representative of all fossil fuel process waters but it provided a stable, homogenous sample for initial testing purposes. Extrapolation of biochemical perturbations to other waters or their toxic components will require a comparative study of a variety of waters. ACKNOWLEDGMENTS This work was performed pursuant to an Interagency Agreement between the U.S. Department of Energy and the U.S. Environmental Protection Agency under Contract DE-AS20-79 LC 01761 to the Rocky Mountain Institute of Energy and Environment, University of Wyoming. We would like to thank Cynthia A. Jones for her fine technical and editorial assistance.
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467
WATER
Toxicity of an in situ oil shale process water to rainbow trout and fathead minnows. Arch. Environ. Contam. Toxicol., in press. CAIRNS, M. A., and CHRISTIAN, A. R. (1978). Effects of hemorrhagic stress on several blood parameters in adult rainbow trout (Salmo gairdneri). Trans. Amer. Fish.
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DAVIDSHON, I., AND NELSON, D. A. (1974). The blood. In Clinical Diagnosis by Laboratory Methods. (I. Davidsohn and J. B. Henry, eds.), 15th ed., p. 106. Saunders, Philadelphia. DEWAN, J. G. (1938). The L(+) glutamic dehydrogenase of animal tissues. Biochem. J. 32, 1378. DOWNING, K. M.. AND MERKENS, J. C. (1955). The influence of dissolved-oxygen concentration on the toxicity of un-ionized ammonia to rainbow trout (Salmo gairdneri Richardson). Ann. Appl. Biol. 43, 243-246. DUNCAN, D. B. (1955). Multiple range and multiple F tests. Biometrics 11, l-42. European Inland Fisheries Advisory Commission (1973). Water quality criteria for fresh water fish. Report on ammonia and inland fisheries. Water Res. 7,1011-1122. FARRIER, D. S., POULSON, R. E., SKINNER, Q. D., ADAMS, J. C., AND BOWER, J. B. (1977). Acquisition, processing and storage for environmental research of aqueous effluents from in situ oil shale processing. In Proceedings Engineering
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(1949). Determination of serum proteins by means of the biuret reaction. J. Biol. Chem. 177, 751-766. JACKSON, L. P., POULSON, R. E., SPEDDING, T. J., PHILLIPS, T. E., AND JENSEN, H. B. (1975). Char-
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centration of phenol on some blood plasma enzyme
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activities in the pike (Esox Lucius L.) in brackish water. Ann. Zool. Fennici 11, 220-223. LIED, E., GJERDE, J., AND BRAEKKAN, 0. R. (1975). Simple and rapid technique for repeated blood sampling in rainbow trout (S&no gairdneri). J. Fish. Res. Bd. Canada 32, 699-701. LLOYD, R., AND ORR, L. D. (1969). The diuretic response by rainbow trout to sublethal concentrations of ammonia. Water Res. 3, 335-344. MASSOD, J. F., WERNER, K. R., AND MCGUIRE, S. L. (1970). Kinetic determination of serum alkaline phosphatase activity. Amer. J. Clin. Pathol. 54, 1lo117. MCKIM, J. M., CHRISTENSEN, G. M., AND HUNT, E. P. (1970). Changes in the blood of brook trout (Salvelinus fontinalis) after short-term and long-term exposure to copper. J. Fish. Res. Bd. Canada 27, 1883- 1889. NIE, N. H. (1975). SPSS-Statistical Package for the Social Sciences. McGraw-Hill, New York. RACICOT, J. G., GAUDET, H., AND LERAY, C. (1975). Blood and liver enzymes in rainbow trout (S&no gairdneri Rich.) with emphasis on their diagnostic use: Study of CC& toxicity and a case of Aeromonas infection. J. Fish Biol. 7, 825-835.
ET AL. SCHEFLER, W. C. (1969). Statistics for the Biological Sciences. Addison-Wesley, Reading, Mass. STUBER, H. A., AND LEENHEER, J. A. (1977). Fractionation of Organic Solutes in Oil Shale Retort Waters for Sorption Studies, Preprints American Chemical Society, Division of Fuel Chemistry. 23, pp. 165- 174. TUCKER, R. K., AND LEITZKE, J. S. (1979). Comparative toxicology of insecticides for vertebrate wildlife and fish. Pharm. Ther. 6, 167-220. TRUSSELL, R. P. (1972). The percent unionized ammonia in aqueous ammonia solutions at different pH levels and temperatures. J. Fish. Res. Bd. Canada 19, 150.5-1507. VERMA, S. R.. PAL, N., TYAGI, A. K., AND DALELA, R. C. (1979). Toxicity of SwascolR 1P (SLS) to Chnnna puncfutus and Cirrhina mrigala: Biochemical alterations. Bull. Environ. Contam. Toxico[. 21,711-718. WEDEMEYER, G. (1972). Some physiological consequences of handling stress in the juvenile coho salmon (Oncorhyndhus Kisutch) and steelhead trout (Salmo gairdneri). J. Fish. Res. Bd. Canada 29, 1780- 1783.