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Short-term toxicity of fluoride ion (F-) in soft water to rainbow trout and brown trout
Chemosphere, Voi.22, Nos.5-6, Printed in Great Britain
p? 605-611,
!991
0045-6535/91 $3.00 + 0.00 Pergamon Press plc
SHORT-TERM TOXICITY OF FLUORIDE ION (F-) IN SOFT WATER TO RAINBOW TROUT AND BROWN TROUT
Julio A. Camargo* and Jose V. Tarazona Department of Animal Health, CIT-INIA, Embajadores 68, 28012 Madrid, Spain and (present address) Department of Biology, Colorado State University, Fort Collins, CO 80523, USA*
ABSTRACT Short-term static bioassays were conducted in duplicate to determine the toxicity of fluoride ion (F-) in soft water (hardness average value of 22 ppm CaCO3) to fingerlings (two months old) of Oncorhynchus mykiss Richardson and Salmo truttafario Linnaeus. Animals were exposed to five different concentrations of sodium fluoride (NaF) for 8 days. They were also exposed to a high concentration of sodium chloride (NaC1) (800 ppm for O. mykiss and 1,000 ppm for S. truttafario) as sodium and conductivity toxicity controls. No significant effect was obseved during NaC1 tests, indicating that toxic effects generated by NaF were fundamentally due to fluoride ion. Fish in fluoride aquaria showed hypoexcitability, darkened backs and a decrease in respiration before their death. The 120, 144, 168 and 192 hour LCs0 values were respectively 92.4, 85.1, 73.4 and 64.1 ppm F- for rainbow trout and 135.6, 118.5, 105.1 and 97.5 ppm F- for brown trout. O. mykiss appears significantly (P <0.05) more sensitive to fluoride ion than S. truttafario. INTRODUCTION Fluorine is the most electronegative of all elements. It does not occur naturally as a free element and only appears with a valence o f - 1 . The fluoride concentration in sea waters normally ranges from 1.2 to 1.4 ppm (Dobbs, 1974). In contrast, most fresh waters contain less than 0.2 ppm, although total concentrations can be considerably higher if the fluoride is bound to small suspended particles (Dave, 1984). Nevertheless, the fluoride ion concentration in many surface waters is significantly increasing as a result of industrial pollution (Martin and Salvadori, 1983). In this sense, McClurg (1984) has indicated that fresh water organisms may be far more sensitive to fluoride pollution than those living in sea waters, because the toxicity of fluoride is decreased by the formation of innocuous complexes with one or more ions of sea water (Oliveira et al., 1978).
605
606
Toxic effects of fluoride compounds have been described in aquatic invertebrates such as Daphnia magna (Dave, 1984; LeBlanc, 1980), Artemia salina (Pankhurst et al., 1980), Penaeus indicus (McClurg, 1984; Hemens and Warwick, 1972) and Hydropsyche spp. (Camargo and Tarazona, 1990), and the species tested in soft water appear more sensitive to fluoride than those tested in hard or sea water. In fish, the fluoride toxicity may be influenced not only by the usual factors such as size (Hemens et al., 1975), species (Milhaud et al., 1981), and physiological state (Pillai and Mane, 1984), but also by the physico-chemical characteristics of the water. Thus, the tolerance of fish to fluoride is increased by low temperatures (Angelovic et al., 1961) and high levels of calcium hardness (Herbert and Shurben, 1964). Pimentel and Bulkley (1983) found in Oncorhynchus mykiss that 96 hour LC50 values increased from 51 to 193 ppm F- as water hardness levels rose from 17 to 385 ppm CaCO3, and Smith etal. (1985) concluded that LCs0 values in Gasterosteus aculeatus and Pimephalespromelas varied with the initial water hardness due to the precipitation of calcium and magnesium salts. Nevertheless, up to the present, the chemical basis of fluoride toxicity to aquatic organisms is not well understood. In this connection, Chitra et al. (1983) have suggested that the toxic action of fluoride on fish may be mainly due to an inhibition of enzymatic activity. The main purposes of this study were to determine the short-term (8 days) toxicity of fluoride ion (F-) in soft water to fingerlings of Oncorhynchus mykiss Richardson and Salmo truttafario Linnaeus, common fish species in cool-water streams from the Iberian Peninsula, and to determine significant differences between these trout species in relation to their respective sensitivity to fluoride during short-term exposures. Prior to this toxicological study, Camargo (1989) estimated 96 hour LC50 values in soft water of 107.5 and 164.5 ppm F- for O. mykiss and
S. trutta fario, respectively. MATERIALS AND METHODS Fingerlings (about two months old) of rainbow trout (Oncorhynchus mykiss Richardson) and brown trout (Salmo truttafario Linnaeus) were obtained from a Spanish ICONA trout hatchery and were certified disease free at CIT-INIA laboratories. No fish died during transportation. In the laboratory, fishes were randomly distributed into test aquaria and acclimatized for four days prior to fluoride toxicity tests. They were not fed during their acclimatization nor during the bioassays. Static fluoride toxicity bioassays were conducted in duplicate for 8 days using glass aquaria, each with a volume of 20 L dechlorinated Madrid tap water. Water oxygenation and turbulence were produced by air pumps. Natural photoperiod was utilized and water temperature was maintained by a cooling unit with thermostat. A control (fluoride ion concentration of 0.08 ppm) and five different fluoride ion concentrations were used per bioassay. Test fluoride solutions were made from sodium fluoride (NaF pro analysi, Merck), geometrically increasing with an approximate factor of 1.6. NaF was added to aquaria at the start of each experiment. Trout species were tested separately using ten fishes per aquarium. Dead animals were removed every day during fluoride toxicity bioassays. Sublethal effects were daily checked by comparing fish in fluoride aquaria with those in the control aquarium. The respiration rate was measured according to the method described by Tarazona et al. (1987). Hardness, alkalinity, chloride, sodium, potassium, ammonia, nitrite, pH, water temperature, dissolved oxygen and conductivity were analysed at the start and at the end of each toxicity bioassay, using analytical methods described by APHA (1980) and Rodier (198 I). Fluoride concentrations were monitored daily using an Orion-USA model 94-09 specific ion electrode and an Orion-USA model 90-02 calomel reference electrode. Water samples
607
were analysed at pH 5.5 after adding total ionic strength adjustment buffer (TISAB-III) with cyclohexanediamine tetra acetic acid (CDTA) as complexing agent for total fluoride ion analysis. The 120, 144, 168 and 192 hour LCs0 values, their 95% confidence limits and ~ values were estimated by the method of Litchfield and Wilcoxon (1949), using mortalities and mean assayed F- concentrations obtained in duplicate for each trout species. Death was defined as the fish floating upside down and not operculating. In addition, the formula of factors (Litchfield and Wilcoxon, 1949; APHA, 1980) was applied for estimating significant (P < 0.05) differences between test species. In order to verify whether the toxicity of sodium fluoride was due to fluoride ion fundamentally, sodium and conductivity toxicity controls were conducted parallel to fluoride toxicity bioassays, using sodium chloride (NaC1 pro analysi, Merck). For that, 10 fingerlings of rainbow trout and brown trout were exposed in soft water (23 ppm CaCO3) for 196 hours to high sodium concentrations (300-380 ppm Na+) and conductivities (710-1200 ~tmhos/cm) respectively. These toxicity controls were performed in duplicated. The possible mortality and sublethal effects were checked every day. RESULTS No mortality occurred during 196 hours of exposure to NaC1. Fingerlings showed symptoms of hyperexcitability and hyperventilation at first, returning to their normal state after about 10 hours. Sublethal effects such as hypoexcitability, darkened backs and a decrease in respiration were not observed during these toxicity controls. Values of water quality parameters analysed during short-term fluoride toxicity bioassays are presented in Table 1. All values are within water quality criteria for aquatic organisms (USEPA, 1986). Ammonia and nitrite were not detected at the start, and no precipitation of fluoride salts was observed during these bioassays. The mean dry weight (60 *C for 24 hours) of each trout species after fluoride toxicity tests was 118.9-&14.9 mg for O. mykiss and 123.6+20.9 mg for S. truttafario. Mortality percentages, and fluoride, sodium and conductivity mean values estimated in each aquarium during duplicate fluoride bioassays are presented in Table 2. Physicochemical standard deviations were lower than 10% of their respective mean values. No mortality occurred in control aquaria. The mortality increased as fluoride concentrations and exposure times increased. Fingerlings in fluoride aquaria showed hyperexcitability and hyperventilation at first and, at alternate times during rest, hypoexcitability, darkened backs and a decrease in respiration before their death. However, these sublethal symptoms were not observed in fingerlings of rainbow trout exposed to a mean concentration of 22.3 ppm F- after 8 days. Respiratory alterations, one of the first symptoms in fish stress, have been reported in freshwater fish exposed to other toxic pollutants including surfactants (Maki, 1979) and ammonia (Tarazona et al., 1987). The 120, 144, 168 and 192 hour LCs0 values, their 95% confidence limits and )(2 values calculated for each trout species are presented in Table 3. All X2 values were lower than those for P = 0.05, indicating that data are not significantly heterogeneous (Litchfield and Wilcoxon, 1949). From a simple comparison of median lethal concentrations (Table 3), we can see that rainbow trout appears to be a more sensitive species to fluoride ion during short-term exposures because its LCs0 values are smallest. In addition, the 95% confidence limits of rainbow trout for exposures of 120, 144 and 192 hours do not overlap significantly (P < 0.05) with the 95% confidence limits of brown trout.
608
Table 1. Mean values and standard deviations (n=24) of water quality parameters analysed during fluoride toxicity bioassays.
Oncorhynchus mykiss
Salrno trutta fario
Water temperature (*C)
15.3 + 0.22
16.1 + 0.13
Alkalinity (ppm CaCO3)
37.5 + 2.09
32.2 + 1.92
Hardness (ppm CaCO3)
22.4 + 1.79
21.2 + 2.55
Chloride (ppm CI-)
10.0 + 1.22
10.8 + 0.43
Dissolved oxygen (ppm 02)
10.1 + 0.28
10.1 + 0.20
Total ammonia (ppm N)
0.15 + 0.16
0.12 + 0.12
Nitrite (ppm N)
0.01 _+0.01
0.01 + 0.01
Potassium (ppm K÷)
0.08 + 0.02
0.14 + 0.02
pH
7.58 + 0.18
7.63 + 0.19
Table 2. Mortality percentages after exposures of 120, 144, 168 and 192 hours to NaF solutions, and mean values (n=2; n*=8) of conductivity (~mhos/cm), sodium (ppm Na÷) and fluoride (ppm F-) analysed in each aquarium during duplicated (a,b) fluoride toxicity bioassays. C = control aquaria; 1, 2, 3, 4 and 5 = fluoride aquaria.
Oncorhynchus mykiss 120
144
168
192
Ca
0
0
0
0
Cb
0
0
0
0
Ia
0
0
0
0
Cond 32.5 30.0 142
Salmo trutta fario Sod
Fluor* 120
4.8
0.08
0
144
168
192
Cond
0
0
0
42.5
Sod
Fluor*
7.3
0.08
5.1
0.09
0
0
0
0
40.0
8.3
0.08
26.1
22.3
0
0
0
10
218
° 48.2
33.9
lb
0
0
0
0
145
26.4
22.3
0
0
10
10
213
48.8
35.0
2a
10
10
20
20
202
46.3
34.4
10
10
10
20
318
68.2
55.4
2b
10
10
10
10
202
44.1
34.2
0
10
10
10
315
69.3
53.8
3a
20
20
20
20
325
69.7
57.6
20
30
30
40
485
111
3b
30
30
30
40
335
71.1
57.3
30
30
40
40
495
113
4a
50
60
60
70
485
114
91.4
50
60
60
60
650
189
4b
40
40
60
70
495
113
91.0
30
40
50
50
665
190
150
5a
70
70
90
90
635
190
144
80
80
100
100
1 0 7 5 293
236
5b
80
90
90
100
640
190
146
90
90
90
100
1 0 2 5 291
228
90.3 90.6 146
609
Table 3. LC50 values (ppm F-), their 95% confidence limits (ppm F-) and X2 values calculated for each test species after exposures of 120, 144, 168 and 192 hours to fluoride solutions.
Oncorhynchus mykiss
120 hours
Salmo trutta fario
LC50
95%
X2
LC50
95%
X2
92.4
73.6-116.0
1.47
135.6
114.0-161.2
5.40
144 hours
85.1
68.0-106.5
2.88
118.5
94.1-149.2
4.79
168 hours
73.4
55.9-96.4
2.90
105.1
81.8-134.9
6.63
192 hours
64.1
50.0-82.2
3.15
97.5
76.8-123.8
6.90
DISCUSSION Although it has already been indicated that sodium ion (Na÷) is the metal ion with the lowest toxicity for aquatic organisms (Hellawell, 1986), this present study has demonstrated the toxicity generated by sodium fluoride on trout species is due fundamentally to fluoride ion (F-). Because ages and weights of all animals were very similar during this study, the lower sensitivity ofS. truttafario to fluoride could be due to a higher physiological ability of this trout species to inhibit the toxic action of fluoride on groups of enzymes within cells, by removing or immobilizingfluoride ions in a more suitable way. On the other hand, rainbow trout and brown trout appear more resistant to fluoride than freshwater benthic macroinvertebrates. Camargo and Tarazona (1990) have estimated 96 hour LCs0 values in soft water of 26.3, 26.5, 38.5, 48.2 and 44.9 ppm F- for Hydropsyche bulbifera, H. exocellata, H. pellucidula, H. lobata and
Chimarra marginata larvae, respectively. This lower sensitivity of trout species to fluoride could be explained because fluoride ions may form stable complexes with calcium in blood and bone of fish (Sigler and Neuhold, 1972), whereas stable complexes could not be formed in freshwater insect larvae. However, marine invertebrates exposed to fluoride compounds tend to accumulate fluoride in their exoskeleton during chronic exposures (Wright and Davidson, 1975). Maximum safe criteria of fluoride ion for fish in natural ecosystems have not yet been determined (USEPA, 1986) because a range of widely LCs0 values has been reported (Smith et al., 1985). However, it is evident that freshwater fish may resist higher fluoride concentrations in hard water than in soft water (Herbert and Shurben, 1964; Sigler and Neuhold, 1972; Pimentel and Bulkley, 1983; Smith et al., 1985). In this connection, Pimentel and Bulkley (1983) have suggested that a reservoir of calcium in the water surrounding fish tends to compensate for the loss of calcium and thereby delays toxic effects of fluoride on the organisms. Studies on fluoride toxicity to freshwater fish should be conducted in water quality conditions of highest toxicity (e.g., soft water) for determining safe criteria of fluoride ion for fish, and chronic toxicity bioassays should be performed to improve these fluoride quality criteria. In this sense, the data offered in this paper may provide a suitable background for future long-term toxicity studies.
610
ACKNOWLEDGEMENTS The authors thank the Department of Animal Health (CIT-INIA) for its logistical support during laboratory studies. Funds for this toxicological research were provided by a grant from the National Institute of Agrarian Researches in Spain.
REFERENCES AngeIovic J.W., Sigler W.F. and Neuhold J.M. (1961) Temperature and fluorosis in rainbow trout. J. Water
Pollut. Control Fed. 33, 371-381. A.P.H.A. (1980) Standard Methods for the Examination of Water and Wastewater, 15th edition. APHA-AWWAWPCF, Washington, DC. Buikema A.L., Niederlehner B.R. and Cairns J.Jr. (1982) Biological monitoring Part IV- Toxicity testing. Water
Res. 16, 239-262. Camargo J.A. (1989) Estudio Ecotoxicol6gico del impacto ambiental generado por una regulaci6n de candales y un vertido de flfior, sobre las comunidades de animales acmiticos del Rfo Durat6n. PhD Dissertation, Madrid Autonomous University, Madrid (Spain). Camargo J.A. and Tarazona J.V. (1990) Acute toxicity to freshwater benthic macroinvertebrates of fluoride ion (F-) in soft water. Bull. Environ. Contain. Toxicol. 45, 883-887. Chitra T., Reddy M.M. and Ramana Rao J.V. (1983) Levels of muscle and liver tissue enzymes in Charma
punctatus exposed to NaF. Fluoride 16, 48-51. Dave G. (1984) Effects of fluoride on growth, reproduction and survival in Daphnia Magna. Comp. Biochem.
Physiol. 78C, 425-431. Dobbs C.G. (1974) Fluoride and the Environment. Fluoride 7, 123-135. Hellawell J.M. (1986) Biological Indicators of Freshwater Pollution and Environmental Management. Elsevier Applied Science Publishers, London and New York. Hemens J. and Warwick R.J. (1972) The effects of fluoride on estuarine organisms. Water Res. 6, 1301-1308. Hemens J., Warwick R.J. and Oliff W.D. (1975) Effect of extended exposure to low fluoride concentration on estuarine fish and crustacea. Progr. in Water Tech. 7, 579-585. Herbert D.W.N. and Shurben D.S. (1964) The toxicity of fluoride to rainbow trout. Water and Waste Treatment 10, 141-142. LeBlanc G.A. (1980) Acute toxicity of priority pollutants to water flea Daphnia magna. Bull. Environ.
Contam.Toxicol. 24, 684-691. Litchfield J.T. and Wilcoxon F. (1949) A simplified method of evaluating dose-effect experiments. J. Pharrnacol. Exp. Ther. 96, 99-113. Maki A.W. (1979) Respiratory activity of fish as a predictor of chronic fish toxicity values for surfactants. In
Aquatic Toxicology. Edited by L.L. Marking & R.A. Kimerle. pp. 77-95. American Society for Testing and Materials, New York. Martin J.M. and Salvadori F. (1983) Fluoride pollution in French rivers and estuaries. Estuarine, Coastal and
Shelf Science 17, 231-242.
611
McClurg T.P. (1984) Effects of fluoride, cadmium and mercury on the estuarine prawn Penaeus indicus. Water
SA 10, 39-45. Milhaud L, Bahri E. and Dridi A. (1981) The effects of fluoride on fish in Gabes Gulf. Fluoride 14, 161-168. Oliveira L., Antia N.J. and Bisalputra T. (1978) Culture studies on the effects from fluoride pollution on the growth of marine phytoplankters. J. Fish. Res. Board Can. 35, 1500-1504. Pankhurst N.W., Boyden C.R. and Wilson J.B. (1980) The effect of a fluoride effluent on marine organisms.
Environmental Pollution 23, 299-312. Pillai K.S. and Mane U.H. (1984) Effect of F- effluent on some metabolites and minerals in fry of Catla carla.
Fluoride 17, 224-233. Pimentel R. and Bulkley R.V. (1983) Influence of water hardness on fluoride toxicity to rainbow trout.
Environmental Toxicology and Chemistry 2, 381-386. Rodier J. (1981) Analisis de las Aguas. Omega Ed., Barcelona. Sigler W.F. and Neuhold J.M. (1972) Fluoride intoxication in fish: A review. J. Wildl. Dis. 8, 252-254. Smith L.R., Holsen T.M., Ibay N.C., Block R.M. and De Leon A.B. (1985) Studies on the acute toxicity of fluoride ion to stickleback, fathead minnow and rainbow trout. Chemosphere 14, 1383-1389. Tarazona J.V., Mufioz M.J., Ortiz J.A., Nufiez M.O. and Camargo J.A. (1987) Fish mortality due to ammonia exposure. Aquaculture and Fisheries Management 18, 167-172. U.S.E.P.A. (1975) Quality Criteria for Water. U.S. Environmental Protection Agency, 440/5-86-001, Washington, DC. Wright D.A. and Davidson A.W. (1975) The accumulation of fluoride by marine and intertidal animals. Envir. Pollut. 8, 1-13. (Received
in USA 2 October
1990;
accepted
16 February
1991)
p? 605-611,
!991
0045-6535/91 $3.00 + 0.00 Pergamon Press plc
SHORT-TERM TOXICITY OF FLUORIDE ION (F-) IN SOFT WATER TO RAINBOW TROUT AND BROWN TROUT
Julio A. Camargo* and Jose V. Tarazona Department of Animal Health, CIT-INIA, Embajadores 68, 28012 Madrid, Spain and (present address) Department of Biology, Colorado State University, Fort Collins, CO 80523, USA*
ABSTRACT Short-term static bioassays were conducted in duplicate to determine the toxicity of fluoride ion (F-) in soft water (hardness average value of 22 ppm CaCO3) to fingerlings (two months old) of Oncorhynchus mykiss Richardson and Salmo truttafario Linnaeus. Animals were exposed to five different concentrations of sodium fluoride (NaF) for 8 days. They were also exposed to a high concentration of sodium chloride (NaC1) (800 ppm for O. mykiss and 1,000 ppm for S. truttafario) as sodium and conductivity toxicity controls. No significant effect was obseved during NaC1 tests, indicating that toxic effects generated by NaF were fundamentally due to fluoride ion. Fish in fluoride aquaria showed hypoexcitability, darkened backs and a decrease in respiration before their death. The 120, 144, 168 and 192 hour LCs0 values were respectively 92.4, 85.1, 73.4 and 64.1 ppm F- for rainbow trout and 135.6, 118.5, 105.1 and 97.5 ppm F- for brown trout. O. mykiss appears significantly (P <0.05) more sensitive to fluoride ion than S. truttafario. INTRODUCTION Fluorine is the most electronegative of all elements. It does not occur naturally as a free element and only appears with a valence o f - 1 . The fluoride concentration in sea waters normally ranges from 1.2 to 1.4 ppm (Dobbs, 1974). In contrast, most fresh waters contain less than 0.2 ppm, although total concentrations can be considerably higher if the fluoride is bound to small suspended particles (Dave, 1984). Nevertheless, the fluoride ion concentration in many surface waters is significantly increasing as a result of industrial pollution (Martin and Salvadori, 1983). In this sense, McClurg (1984) has indicated that fresh water organisms may be far more sensitive to fluoride pollution than those living in sea waters, because the toxicity of fluoride is decreased by the formation of innocuous complexes with one or more ions of sea water (Oliveira et al., 1978).
605
606
Toxic effects of fluoride compounds have been described in aquatic invertebrates such as Daphnia magna (Dave, 1984; LeBlanc, 1980), Artemia salina (Pankhurst et al., 1980), Penaeus indicus (McClurg, 1984; Hemens and Warwick, 1972) and Hydropsyche spp. (Camargo and Tarazona, 1990), and the species tested in soft water appear more sensitive to fluoride than those tested in hard or sea water. In fish, the fluoride toxicity may be influenced not only by the usual factors such as size (Hemens et al., 1975), species (Milhaud et al., 1981), and physiological state (Pillai and Mane, 1984), but also by the physico-chemical characteristics of the water. Thus, the tolerance of fish to fluoride is increased by low temperatures (Angelovic et al., 1961) and high levels of calcium hardness (Herbert and Shurben, 1964). Pimentel and Bulkley (1983) found in Oncorhynchus mykiss that 96 hour LC50 values increased from 51 to 193 ppm F- as water hardness levels rose from 17 to 385 ppm CaCO3, and Smith etal. (1985) concluded that LCs0 values in Gasterosteus aculeatus and Pimephalespromelas varied with the initial water hardness due to the precipitation of calcium and magnesium salts. Nevertheless, up to the present, the chemical basis of fluoride toxicity to aquatic organisms is not well understood. In this connection, Chitra et al. (1983) have suggested that the toxic action of fluoride on fish may be mainly due to an inhibition of enzymatic activity. The main purposes of this study were to determine the short-term (8 days) toxicity of fluoride ion (F-) in soft water to fingerlings of Oncorhynchus mykiss Richardson and Salmo truttafario Linnaeus, common fish species in cool-water streams from the Iberian Peninsula, and to determine significant differences between these trout species in relation to their respective sensitivity to fluoride during short-term exposures. Prior to this toxicological study, Camargo (1989) estimated 96 hour LC50 values in soft water of 107.5 and 164.5 ppm F- for O. mykiss and
S. trutta fario, respectively. MATERIALS AND METHODS Fingerlings (about two months old) of rainbow trout (Oncorhynchus mykiss Richardson) and brown trout (Salmo truttafario Linnaeus) were obtained from a Spanish ICONA trout hatchery and were certified disease free at CIT-INIA laboratories. No fish died during transportation. In the laboratory, fishes were randomly distributed into test aquaria and acclimatized for four days prior to fluoride toxicity tests. They were not fed during their acclimatization nor during the bioassays. Static fluoride toxicity bioassays were conducted in duplicate for 8 days using glass aquaria, each with a volume of 20 L dechlorinated Madrid tap water. Water oxygenation and turbulence were produced by air pumps. Natural photoperiod was utilized and water temperature was maintained by a cooling unit with thermostat. A control (fluoride ion concentration of 0.08 ppm) and five different fluoride ion concentrations were used per bioassay. Test fluoride solutions were made from sodium fluoride (NaF pro analysi, Merck), geometrically increasing with an approximate factor of 1.6. NaF was added to aquaria at the start of each experiment. Trout species were tested separately using ten fishes per aquarium. Dead animals were removed every day during fluoride toxicity bioassays. Sublethal effects were daily checked by comparing fish in fluoride aquaria with those in the control aquarium. The respiration rate was measured according to the method described by Tarazona et al. (1987). Hardness, alkalinity, chloride, sodium, potassium, ammonia, nitrite, pH, water temperature, dissolved oxygen and conductivity were analysed at the start and at the end of each toxicity bioassay, using analytical methods described by APHA (1980) and Rodier (198 I). Fluoride concentrations were monitored daily using an Orion-USA model 94-09 specific ion electrode and an Orion-USA model 90-02 calomel reference electrode. Water samples
607
were analysed at pH 5.5 after adding total ionic strength adjustment buffer (TISAB-III) with cyclohexanediamine tetra acetic acid (CDTA) as complexing agent for total fluoride ion analysis. The 120, 144, 168 and 192 hour LCs0 values, their 95% confidence limits and ~ values were estimated by the method of Litchfield and Wilcoxon (1949), using mortalities and mean assayed F- concentrations obtained in duplicate for each trout species. Death was defined as the fish floating upside down and not operculating. In addition, the formula of factors (Litchfield and Wilcoxon, 1949; APHA, 1980) was applied for estimating significant (P < 0.05) differences between test species. In order to verify whether the toxicity of sodium fluoride was due to fluoride ion fundamentally, sodium and conductivity toxicity controls were conducted parallel to fluoride toxicity bioassays, using sodium chloride (NaC1 pro analysi, Merck). For that, 10 fingerlings of rainbow trout and brown trout were exposed in soft water (23 ppm CaCO3) for 196 hours to high sodium concentrations (300-380 ppm Na+) and conductivities (710-1200 ~tmhos/cm) respectively. These toxicity controls were performed in duplicated. The possible mortality and sublethal effects were checked every day. RESULTS No mortality occurred during 196 hours of exposure to NaC1. Fingerlings showed symptoms of hyperexcitability and hyperventilation at first, returning to their normal state after about 10 hours. Sublethal effects such as hypoexcitability, darkened backs and a decrease in respiration were not observed during these toxicity controls. Values of water quality parameters analysed during short-term fluoride toxicity bioassays are presented in Table 1. All values are within water quality criteria for aquatic organisms (USEPA, 1986). Ammonia and nitrite were not detected at the start, and no precipitation of fluoride salts was observed during these bioassays. The mean dry weight (60 *C for 24 hours) of each trout species after fluoride toxicity tests was 118.9-&14.9 mg for O. mykiss and 123.6+20.9 mg for S. truttafario. Mortality percentages, and fluoride, sodium and conductivity mean values estimated in each aquarium during duplicate fluoride bioassays are presented in Table 2. Physicochemical standard deviations were lower than 10% of their respective mean values. No mortality occurred in control aquaria. The mortality increased as fluoride concentrations and exposure times increased. Fingerlings in fluoride aquaria showed hyperexcitability and hyperventilation at first and, at alternate times during rest, hypoexcitability, darkened backs and a decrease in respiration before their death. However, these sublethal symptoms were not observed in fingerlings of rainbow trout exposed to a mean concentration of 22.3 ppm F- after 8 days. Respiratory alterations, one of the first symptoms in fish stress, have been reported in freshwater fish exposed to other toxic pollutants including surfactants (Maki, 1979) and ammonia (Tarazona et al., 1987). The 120, 144, 168 and 192 hour LCs0 values, their 95% confidence limits and )(2 values calculated for each trout species are presented in Table 3. All X2 values were lower than those for P = 0.05, indicating that data are not significantly heterogeneous (Litchfield and Wilcoxon, 1949). From a simple comparison of median lethal concentrations (Table 3), we can see that rainbow trout appears to be a more sensitive species to fluoride ion during short-term exposures because its LCs0 values are smallest. In addition, the 95% confidence limits of rainbow trout for exposures of 120, 144 and 192 hours do not overlap significantly (P < 0.05) with the 95% confidence limits of brown trout.
608
Table 1. Mean values and standard deviations (n=24) of water quality parameters analysed during fluoride toxicity bioassays.
Oncorhynchus mykiss
Salrno trutta fario
Water temperature (*C)
15.3 + 0.22
16.1 + 0.13
Alkalinity (ppm CaCO3)
37.5 + 2.09
32.2 + 1.92
Hardness (ppm CaCO3)
22.4 + 1.79
21.2 + 2.55
Chloride (ppm CI-)
10.0 + 1.22
10.8 + 0.43
Dissolved oxygen (ppm 02)
10.1 + 0.28
10.1 + 0.20
Total ammonia (ppm N)
0.15 + 0.16
0.12 + 0.12
Nitrite (ppm N)
0.01 _+0.01
0.01 + 0.01
Potassium (ppm K÷)
0.08 + 0.02
0.14 + 0.02
pH
7.58 + 0.18
7.63 + 0.19
Table 2. Mortality percentages after exposures of 120, 144, 168 and 192 hours to NaF solutions, and mean values (n=2; n*=8) of conductivity (~mhos/cm), sodium (ppm Na÷) and fluoride (ppm F-) analysed in each aquarium during duplicated (a,b) fluoride toxicity bioassays. C = control aquaria; 1, 2, 3, 4 and 5 = fluoride aquaria.
Oncorhynchus mykiss 120
144
168
192
Ca
0
0
0
0
Cb
0
0
0
0
Ia
0
0
0
0
Cond 32.5 30.0 142
Salmo trutta fario Sod
Fluor* 120
4.8
0.08
0
144
168
192
Cond
0
0
0
42.5
Sod
Fluor*
7.3
0.08
5.1
0.09
0
0
0
0
40.0
8.3
0.08
26.1
22.3
0
0
0
10
218
° 48.2
33.9
lb
0
0
0
0
145
26.4
22.3
0
0
10
10
213
48.8
35.0
2a
10
10
20
20
202
46.3
34.4
10
10
10
20
318
68.2
55.4
2b
10
10
10
10
202
44.1
34.2
0
10
10
10
315
69.3
53.8
3a
20
20
20
20
325
69.7
57.6
20
30
30
40
485
111
3b
30
30
30
40
335
71.1
57.3
30
30
40
40
495
113
4a
50
60
60
70
485
114
91.4
50
60
60
60
650
189
4b
40
40
60
70
495
113
91.0
30
40
50
50
665
190
150
5a
70
70
90
90
635
190
144
80
80
100
100
1 0 7 5 293
236
5b
80
90
90
100
640
190
146
90
90
90
100
1 0 2 5 291
228
90.3 90.6 146
609
Table 3. LC50 values (ppm F-), their 95% confidence limits (ppm F-) and X2 values calculated for each test species after exposures of 120, 144, 168 and 192 hours to fluoride solutions.
Oncorhynchus mykiss
120 hours
Salmo trutta fario
LC50
95%
X2
LC50
95%
X2
92.4
73.6-116.0
1.47
135.6
114.0-161.2
5.40
144 hours
85.1
68.0-106.5
2.88
118.5
94.1-149.2
4.79
168 hours
73.4
55.9-96.4
2.90
105.1
81.8-134.9
6.63
192 hours
64.1
50.0-82.2
3.15
97.5
76.8-123.8
6.90
DISCUSSION Although it has already been indicated that sodium ion (Na÷) is the metal ion with the lowest toxicity for aquatic organisms (Hellawell, 1986), this present study has demonstrated the toxicity generated by sodium fluoride on trout species is due fundamentally to fluoride ion (F-). Because ages and weights of all animals were very similar during this study, the lower sensitivity ofS. truttafario to fluoride could be due to a higher physiological ability of this trout species to inhibit the toxic action of fluoride on groups of enzymes within cells, by removing or immobilizingfluoride ions in a more suitable way. On the other hand, rainbow trout and brown trout appear more resistant to fluoride than freshwater benthic macroinvertebrates. Camargo and Tarazona (1990) have estimated 96 hour LCs0 values in soft water of 26.3, 26.5, 38.5, 48.2 and 44.9 ppm F- for Hydropsyche bulbifera, H. exocellata, H. pellucidula, H. lobata and
Chimarra marginata larvae, respectively. This lower sensitivity of trout species to fluoride could be explained because fluoride ions may form stable complexes with calcium in blood and bone of fish (Sigler and Neuhold, 1972), whereas stable complexes could not be formed in freshwater insect larvae. However, marine invertebrates exposed to fluoride compounds tend to accumulate fluoride in their exoskeleton during chronic exposures (Wright and Davidson, 1975). Maximum safe criteria of fluoride ion for fish in natural ecosystems have not yet been determined (USEPA, 1986) because a range of widely LCs0 values has been reported (Smith et al., 1985). However, it is evident that freshwater fish may resist higher fluoride concentrations in hard water than in soft water (Herbert and Shurben, 1964; Sigler and Neuhold, 1972; Pimentel and Bulkley, 1983; Smith et al., 1985). In this connection, Pimentel and Bulkley (1983) have suggested that a reservoir of calcium in the water surrounding fish tends to compensate for the loss of calcium and thereby delays toxic effects of fluoride on the organisms. Studies on fluoride toxicity to freshwater fish should be conducted in water quality conditions of highest toxicity (e.g., soft water) for determining safe criteria of fluoride ion for fish, and chronic toxicity bioassays should be performed to improve these fluoride quality criteria. In this sense, the data offered in this paper may provide a suitable background for future long-term toxicity studies.
610
ACKNOWLEDGEMENTS The authors thank the Department of Animal Health (CIT-INIA) for its logistical support during laboratory studies. Funds for this toxicological research were provided by a grant from the National Institute of Agrarian Researches in Spain.
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in USA 2 October
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accepted
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1991)