TOXIC AND SUBLETHAL EFFECT OF FORMALIN ON FRESHWATER FISHES

Supranee Chinabut
Chalor Limsuwan
Kamonporn Tonguthai
Temdoung Pungkachonboon

Network of Aquaculture Centres in Asia

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TOXIC AND SUBLETHAL EFFECT OF FORMALIN ON FRESHWATER FISHES

ABSTRACT

Toxicity of formalin to three freshwater fish species was determined by using static bioassay. The 96-hour LC₅₀ of formalin on silver barb (Puntius gonionotus Bleeker), common carp (Cyprinus carpio Linn.) and snakehead fish (Channa striatus Flower) were 67–80, 106–128 and 147–166 ppm, respectively. Toxicity of formalin was most obvious during the first 24 hours of exposure. Water pH and hardness had no effect on the toxicity. There were significant differences in percentage weight gain among the control common carp fry and fish treated with 25, 50 and 75 ppm formalin for 8 weeks. Fish exposed to 75 ppm formalin had the lowest growth throughout the 8-week period. However, no histological changes were observed in gills, liver, kidney, spleen digestive tract and muscle of common carp fry exposed to formalin during the 8-week period. No histological changes were found in common carp and snakehead fry that survived from acute toxicity test but hyperplasia of gill lamellae and fatty degeneration in the liver were observed in silver barb.

INTRODUCTION

Formalin (CH₂(OH)₂ and HO. (CH₂O) n. H) is a 37–50 percent aqueous solution of dissolved formaldehyde CH₂O. Formaldehyde was described by Alexander M. Butleroo in 1854 (Walker, 1964). It was first used in fish culture by Le'ger in 1909 to control Costia on trout. Formalin was brought into common usage by Fish (1940) to control every ectoparasitic disease of hatchery trout and salmon.

Formalin is one of the most effective and widely used compounds in fish culture for therapeutic and prophylactic treatment of fungal infection and external parasites of fish and fish eggs. Uses of formalin in fish culture were reviewed by Schnick (1974). Toxicities of formalin to fishes in waters of different quality were studied by Phelps (1975) and Bill et al. (1977).

In Thailand, formalin is the most commonly used chemical in fish culture for therapeutic treatment of external parasites. However, there is no published information on the toxicity or pathology of formalin to the key farmed species in Thailand. This study investigates the toxicity and toxic pathology of formalin to three important farmed fish species in Thailand, namely, common carp (Cyprinus carpio), silver barb (Puntius gonionotus) and snakehead (Channa striatus). The specific objectives of the study are as follows:

1. To determine the 96 hour LC₅₀ of formalin for common carp, silver barb and snakehead fish under standard test conditions at various pH and hardness;

2. To determine the chronic pathological effects, if any of formalin at therapeutic levels on common carp for 8 weeks;

3. To determine the site of action of formalin by pathological examination of survival fish at LC₅₀ levels;

4. To determine the chronic pathological effects of formalin at higher than therapeutic levels on common carp for 8 weeks and;

5. To evaluate the effect of formalin on growth rate of common carp for 8 weeks.

MATERIALS AND METHODS

The experiments were conducted in the laboratory of the National Inland Fisheries Institute. Common carp fry were obtained from private hatchery, silver barb fry from the aquaculture section of the Institute (NIFI) and snakehead fry were collected from the natural water bodies on the Kasetsart University campus.

1. Acute Toxicity

The static bioassay was used to determine the 96-hour LC₅₀ of formalin for common carp, silver barb and snakehead fish fry.

1.1 Test fish and acclimation

Average weights for common carp, silver barb and snakehead fry were 0.8, 0.27 and 0.17 g, respectively. Fish were acclimated in aquaria containing well water and fed twice daily during the acclimation period. Before the test, fish were acclimated in 150-litre aquaria containing 120 liters of water for 24 hours. They were not fed during the 24 hours acclimation to the aquaria before the test or during the test period.

1.2 Dilution water

Distilled water was reconstituted by adding to it reagent-grade chemicals (Table 1). The water was aerated at least 1 hour in the aquarium before and after chemicals were added. Water quality was checked before the fish were placed in the test chamber. Soft reconsituted water with alkalinity between 30 to 35 mg/L as CaCO₃, hardness 40 to 48 mg/L as CaCO₃ and pH 7.2 +- 0.1 (Table 1) was used for the preparation of different pH water. For the pH test (Table 2), chemicals were added to the aerated soft reconstituted water, mixed with a glass rod, and then the pH was checked and adjusted by adding 1N NaOH and 0.5 M H₃BO₃. Water hardness was adjusted by the addition of reagent-grade chemicals to distilled water (Table 3). Water analysis was conducted according to procedure outlined in Standard Methods for the Examination of Water and Wastewater (American Public Health Association, 1975).

1.3 Chemicals and test procedure

Analytical grade formalin (37% formaldehyde) was used in this study. Stock solution was prepared in distilled water (liquid formulation was measured volumetrically and diluted with distilled water). Ten fish were moved into each of the 15-liter jar containing 10 liters of reconstituted water for 24 hours before the test. Formalin was added to the aquaria and stirred with a glass rod. Formalin concentrations ranged from that causing no deaths to that causing 100% mortality. Each test had a control jar that contained reconstituted water and fish but no formalin. No aeration was used during the 96-hour experimental period.

1.4 Observations

Fish responses to formalin were recorded for several hours and daily thereafter throughout the 96-hour bioassay. Dead fish were recorded and removed. Fish were regarded as dead when all opercular movements ceased. Fish which survived the 96-hour bioassay were used for histological examination.

1.5 Statistical analysis

Data were analyzed statistically by the Litchfield and Wilcoxon method (1949). Data were plotted on logarithmic-probability paper. Results were analyzed statistically to determine the LC₅₀ with 95% confidence interval and the slope function. Chi-square tests were used to test the goodness of fit of the log dose-percent effect curves. The parallelism of dose-percent effect curves were tested, using the slope function ratio, as well as the potency ratio.

2. Growth Study

A feeding experiment was conducted to study growth rate of common carp fry continuously exposed to therapeutic levels of formalin 25 and 50 mg/l, and higher than therapeutic levels at 75 mg/l. The experiment was conducted in 150-liter glass aquaria for an 8-week feeding period. Well water was used during the experimental period. Fry averaging 1.2 g were acclimated 2 weeks in the laboratory before stocking in the experimental aquaria. They were fed a nutritionally complete practical diet (NIFI 12) during this holding period.

Twenty fish were stocked into each experimental aquarium. Formalin was added into duplicate aquaria for making each concentration at 0, 25, 50 and 75 mg/l. Water was changed every two days and new formalin solutions were added into aquaria. Aeration was supplied through airstone during the 8-week experimental period. The fish were fed with NIFI-12 diet twice daily at a rate of 3% (dry matter) their body weight per day. Fish were weighed every two weeks and the feed allowance adjusted accordingly. Analysis of variance and Duncan's new multiple range test (Stell and Torrie, 1960) were used to compute statistical differences among sample means.

3. Histopathological Study

The experiment was conducted to study the effect of formalin on histology of common carp. Twenty fry averaging 1.2 g were stocked in each of the aquarium containing formalin 0, 25, 50 and 75 mg/l. Fish were fed and water was changed in the same manner as described in the growth study. At the end of 2, 4, 6 and 8 weeks, three fish were randomly collected from each aquarium for histopathological examination.

Fish samples for histological study were fixed in 10% buffered formalin, dehydrated in a series of alcohols and embedded in paraplast. Embedded tissues were sectioned to thickness of 5–6 microns which were mounted on glass microscope slides and stained with hematoxylin and eosin (Humason, 1979).

RESULTS AND DISCUSSION

Acute Toxicity

Percentage mortalities of fish during the 96-hour exposure to formalin at different water hardness and pH are given in Table 4–9 and the 24-, 48-and 96-hour LC₅₀ are given in Table 10 and 11. The 96-hour LC₅₀ was similar to the 24-hour LC₅₀. The low number of fish deaths after 24 hours may have resulted from a decreased concentration of formalin by absorption and metabolism in the fish, natural degradation of formalin in solution, or greater activity of the chemical in fish during the early hours of exposure.

There were no significant differences in toxicity of formalin to the three test species of different water hardness and pH. Similar results were reported by Marking et al (1972) and Piper and Smith (1973). In contrast, Phelps (1975) found that formalin was more toxic to channel catfish in soft water than in hard water. Bills et al (1977) reported that water hardness did not affect the toxicity of formalin to fishes but in soft water with pH 9.5 formalin was more toxic than at pH 6.5 and 8.5. In this study, pH 6.5, 7.5 and 8.5 with hardness 40–50 mg/l as CaCO₃ had no effect on the toxicity of formalin. Silver barb was the most sensitive to formalin probably due to species differences.

Observation of behavioral changes in fish exposed to formalin indicated that at high concentration there was an increase in opercular beat but slower swimming than the controls. After 18 hours of exposure fish swam near the surface. Moribund fish swam up and down rapidly and frequently gulping at the surface. Fish showed uncoordinated movements, finally lay on the bottom, and within 12–18 hours. Lanzing (1963) and Wedemeyer and Yasutake (1974) reported that fish exposed to formalin exhibit hyperventilation because formalin decreases the dissolved oxygen. However, Wedemeyer (9171) stated that rainbow trout (Salmo gairdneri) and coho salmon (Oncorhynchus Kisutch) exposed to formalin had a 10–15 percent decreased in respiration rate.

Histopathological Effects

1. Survived fish from acute toxicity test

No histological changes were observed in gills, livers, spleens, kidneys and muscles of common carp and snakehead fry that survived from 96-hour acute toxicity test. However, silver barb fry that survived from exposure to formalin at the concentration of 83.0 mg/l had hyperplasia of secondary gill lamellae (Fig. 1) and fatty degeneration in the liver (Fig. 2). Wedemeyer and Yasutake (1974) found that steelhead trout (S. gairdneri) and chinook salmon (O. tshawytscha) exposed to 200 mg/l formalin for one hour developed hypertrophy at the gills and the longer exposure to formalin resulted in degeneration and necrosis of the lamellae. Williams and Wootten (1981) found cytoplasmic degeneration in the liver of rainbow trout exposed to 200 mg/l formalin for 72 hours.

2. Common carp continuously exposed to formalin for 8 weeks

There were no histological changes in gills, liver, kidney, spleen, intestine, muscle and skin of common carp fry exposed to formalin at the levels of 25, 50 and 75 mg/l for 8 weeks. However, exposure to formalin at these concentrations for 8 weeks reduced the growth of common carp fry. Wedemeyer (1971) found the reduction of chloride; calcium ions and vitamin C concentration in interregnal tissue of fish exposed to formalin. Smith and Piper (1972) reported that rainbow trout treated with formalin had an elevation of hematocrit and immature erythrocytes.

Effect on Growth

Average weights of common carp fry during 8 weeks of exposure to formalin at the concentrations of 25, 50 and 75 mg/l are shown in Table 12. Percentage weight gain every two weeks are given in Table 13. There were no significant differences in weight gain among fish exposed to three concentrations of formalin at the end of the second week. After four weeks fish exposed to 4 mg/l formalin had significant by lower percentage weight gain than the other groups. At the end of 8 week feeding period, fish exposed to formalin 25, 50 and 75 mg/l had lower percentage weight gain than the control; fish in the 75 mg/l formalin group had the lowest weight gain.

Results of this study indicate that formalin at the therapeutic levels of 25–50 mg/l should be safe for fish unless continuously exposed more than 4 weeks. Formalin at higher than therapeutic level of 75 mg/l slightly affected the growth of common carp after an exposure of more than 2 weeks.

Fig 1. Hyperplasia of the epithelial cells (arrow) lining the secondary gill lamellae of silver barb fry exposed to formalin at the concentration of 83.0 mg/l for 96 hours. H & E a = × 290 b = × 305 c= × 590

Fig 2. Hepatocytic fatty degeneration (D) in the liver of silver barb fry exposed to formalin at the concentration of 83.0 mg/l for 96 hours.

H & E a = × 290 b and c = × 570

Table 1. Quantities of salts and characteristics of reconstituted water used for preparation of water with different pH

Table 2. Quantities of chemicals added to soft reconstituted water for buffering pH

Table 3. Quantities of salts and characteristics of reconstituted water used for hardness test

Table 4. Cumulative percentage mortality of silver barb to formalin at various water hardness

Table 5. Accumulation percentage mortality of silver barb to formalin at various pH

Table 6. Cumulative percentage mortality of common carp to formalin at various water hardness

Table 7. Cumulative percentage mortality of common carp to formalin at various pH.

Table 8. Cumulative percentage mortality of snakehead fish to formalin at various water hardness.

Table 9. Cumulative percentage mortality of snakehead fish to formalin at various pH.

Table 10. LC₅₀ with 95 percent confidence interval of formalin on silver barb, common carp and snakehead fish at various water hardness

Table 11. LC₅₀ with 95 percent confidence interval of formalin on silver barb, common carp and snakehead fish at various pH.

Table 13. Percentage weight gain of common carp during 8 weeks exposure to various concentrations of formalin*

* mean with the same letter within a column are not significantly different

Table 12. Average weight of common carp during 8 weeks exposure to various concentration of formalin.

RECOMMENDATION

Future investigations should include the degradation rate of formalin in water of different temperature. Toxicity of formalin to bacteria, phytoplanktons and zooplanktons should be studied.

REFERENCES

American Public Health Association. 1975. Standard Methods for the Examination of Water and Wastewater, 14th ed. American Public Health Association, Washington, D.C. 1193 p.

Bills, T.D., L.L, Marking and J.H. Chandler, Jr. 1977. Formalin: Its toxicity to nontarget aquatic organisms, persistence, and counteraction. U.S. Fish Wildl. Sero. Invest. Fish Control. 73: 1–7.

Fish, F.F. 1940. Formalin for external protozoa parasites, Prog. Fish Cult. 48: 1–10.

Humason, G.L. 1979. Animal Tissue Technique. W.H. Freeman and Company, San Francisco. 641 p.

Lanzing, W.J.R. 1963. Observation on malachite green in relation to its application to fish disease. Hydrobiologica 25: 426–441.

Le'ger, L. 1909. La costiase et son traitment chezles jeuned alevins de truete. Comtes rendus 48: 1284–1286.

Litchfield, J.T. and F. Wilcoxon. 1949. A simplified method of evaluating dose-effect experiments. J. Pharmacol. Exp. Ther. 96 : 99–113.

Marking, L.L., E.L. King, Tr., and J.H. Chandler, Jr. 1972. Section of toxicology, pp. 3–8 In Quarterly report of progress for July-September 1972. Fish Control Laboratory, La Crosse, Wisconcin.

Phelps, R.P. 1975. Toxicity and efficacy of five chemotherapeutics used in aquaculture when applied to waters of different quality. Ph.D. thesis, Auburn University, Auburn, Alabama. 103 p.

Piper, R.G. and C.E. Smith. 1973. Factors influencing formalin toxicity in trout. Prog. Fish-Cult. 35(2) : 78–81.

Schnick, R.A. 1974. Formalin as a therapeutant in fish culture. U.S. Fish Wildl. Serv. Lit. Rev. 74–09. Natl. Tech. Inf. Serv. No. PB-235 448/AS. 145 p.

Smith, C.E. and R.G. Piper. 1972. Pathological effect in formalin-treated rainbow trout (Salmo gairdneri). J. Fish Res. Board Can. 29: 328–329.

Steel, R.G.D. and J.H. Torrie. 1960. Principle and Procedures in Statistics. McGraw-Hill, New York. 481 p.

Walker, J.F. 1964. Formaldehyde. Am. Chem. Soc. monograph seried, 3rd ed. Rainhold Publishing Corp. New York.

Wedemeyer, G. 1971. The stress of formalin treatments in rainbow trout (Salmon gairdneri) and coho salmon (Oncorhynchus kisutch). J. fish Res. Board Can. 28: 1899–1904.

Wedemeyer, G. and W.T. Yasutake. 1974. Stress of formalin treatment in juveniles spring chinook salmon (Oncorhynchus tshawytsha) and steelhead trout (Salmo gairdneri). J. Fish. Res. Board. Can. 31: 179– 184.

Williams, H.A. and R. Wootten. 1981. Some effect of therapeutic levels of formalin and copper sulphate on blood parameters in rainbow trout. Aquaculture 24: 341–353.