A BIOASSAY ON THE EFFECTS OF FORMALIN ON THE BEHAVIOR AND

SURVIVAL OF TILAPIA (Oreochromis nilotica) FRY

INTRODUCTION

Biological assay, or bioassay, is a type of experiment which is conducted to measure the

effects of substances or a specific chemical on a living organism. Bioassays are used alongside
with inorganic tests in determining the effects of certain compounds. While inorganic tests can
tell scientists the molecules present in a substance, living organisms are needed in order to take
into considerations the effect of a substance on the behavior or physiology of the species being
studied.

In aquaculture, it is usually used in testing the tolerance of a fish to a certain chemicals

which are then used as disinfectant or treatment for a certain disease caused by fungi,
ectoparasites, protozoans or bacteria. Some disinfectants used which are being tested through
bioassay include iodophores, salts, organo-chloride compounds, aldehydes, hydrogen peroxide,
quaternary ammonium compounds, and antiseptic dyes.

Formalin is a 37–50% 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).

Formalin is efficient to treat white spot disease (ichthyophthiriasis) caused by

Ichthyophthiriusmultifiliis (Kinnunen et al. 2005), and effective for the control of ectoparasitic
diseases in Oreochromis niloticus (Badran et al. 1996). However, the formaldehyde is a mutagen
and carcinogen even at low concentrations and presented toxicity in a variety of organisms. In
aquatic organisms, deleterious effects were observed on the concentration of 1 mg/L for fish and
5 mg/L for micro crustaceans and algae (Park, K., and G. Heo 2008, Srivastava et al. 2009). The
effects of these products in fish can be manifested at various levels of biological organization,
including physiological dysfunction, structural changes in organs and tissues and behavioral
changes that lead to impaired growth and reproduction (Santos et al. 2012). Further studies on
the toxicities of formalin to fishes were done by Phelps (1975) and Bill et al. (1977).
 This experiment aims to investigate the acute toxicity of formalin to tilapia fry to
formalin at different concentrations (control, 200 ppm, 500 ppm, 1000 ppm). Specifically,

1. To determine the LC50 of formalin for tilapia fry;

2. To determine the effects on swimming behavior and physical appearance of fish

MATERIALS AND METHODS

Materials

Tilapia fry Forceps

8 jars Petri plate
Formalin Thermometer
Chlorinated freshwater Aeration set-up
Analytical balance Beaker
Pipette Aspirator

Experimental Fish

Oreochromis niloticus fry were collected from Freshwater Aquaculture Station of the
University of the Philippines Visayas, Miagao, Iloilo. Larvae (ave. 1.1 cm, 0.05 g) were
acclimatized for one hour prior to exposure.

Experimental Set-up and Water Parameters

Four treatments were used (control, 200 ppm, 500 ppm, 1000 ppm) and two replicates
run for each treatment. Totally 8 aquaria each filled with 2 L water were used for the tests. Ten
tilapia fry were stocked into each jars and exposed to different concentrations for 24 hours. Each
treatment was provided with constant aeration and temperature was maintained at 28oC.

24 Hour Observation

The fish were observed based on its physical appearance, behavior and mortality. On the
first hour, observations were done in every 15 minutes. During the 2nd to the 6th hours, the fish
were observed in every 60 minutes. The next observation was done in the 12th hour and lastly in
the 24th hour.
LC50 determination

The LC50 was determined using the Reed-Muench method of estimating LC50. The following
computations were used:

Accumulated dead
% Cumulative mortality = × 100
Total

50−M below 50%

LC50 = log conc. below 50% M + × log conc above 50% − log conc below 50%
M above 50%− M below 50%

RESULTS

The toxicity of formalin on Nile tilapia was determined by computing the LC50 which
indicates the toxic concentration at which 50% of fish number died after 5 hours. Mortality after
six hours of exposure was not computed for LC50 because there was no data for the succeeding
treatments aside from the control.
No mortality was observed in the control group during the trial. Fish showed behavioral
changes after formalin exposure, including: avoidance behavior (fish gathered in a corner of the
tanks) and gasping at surface at the highest concentration, while other treatments appeared
normal. As the exposure period progresses, these symptoms were accompanied by loss of
equilibrium, imbalanced or upside down swimming, coming to surface or going to bottom of the
chamber in the form of upside down or laying on one side and finally death, at higher
concentrations. Table 1 and figure 1 shows the percent mortality of tilapia fry at different time
intervals.

Table 1: Percentage death of fish exposed to different formalin concentrations in different time
period.
Time (h)
Concentration
(mg/L) 0.15 0.30 0.45 1 2 3 4 5 6 18 24
Control 0 0 0 0 0 0 0 0 0 0 5
0.2 0 0 0 0 0 0 5 10 15 70 100
0.5 0 0 0 0 0 0 10 45 55 100 ND
1.0 0 0 10 20 20 60 60 100 ND ND ND
 120

100

80
Mortality (%)
60

40

20

0
0.15 0.30 0.45 1 2 3 4 5 6 18 24
Time exposed (h)

Control A B C

Figure 1. Percentage mortality of tilapia fry exposed at different concentrations of formalin

Table 2. Data for computing LC50 using the Reed-Muench Method

Accumulated Cumulati
Conc. Log conc Number Number
N Total ve %
(ppm) (ln) Dead Alive
Dead Alive Mortality
0 0 10 0 10 0 24.5 24.5 0
250 5.52 10 1 9 1 14.5 15.5 6.45
500 6.21 10 4.5 5.5 5.5 5.5 11 50
1000 6.91 10 10 0 10.5 0 10.5 100

The LC50 of tilapia fry exposed to various concentration of formalin after five hours was
500 ppm. During exposure, the test fish were darker compared to the control and produces
excessive amounts of mucus. The most pronounced reaction to formalin was observed at the
highest concentrations, with abrupt increase in mortality starting from the 3rd hour of exposure.

DISCUSSION

Mucus accumulation was observed on the body surfaces and gill filament of dead fish.
This is in similar with the findings of Annune, et al. (1994) and Buchmann et al. (2004) where
mucus accumulation results from increase in the activity of mucus cells due to subsequent
exposure to pollutants. This results in an increase in the production and accumulation of mucus
over the body surface and gills of the fish which reduced respiratory activities of fish as reported
by Konar (1970).

The mortality of the fish is formalin-induced rather than gill-damage as observed in long
term exposures. Lanzing (1963) and Wedemeyer and Yasutake (1974) reported that fish exposed
to formalin exhibit hyperventilation because formalin decreases the dissolved oxygen. This
behavior was observed to fishes at early hours of exposure and nearing to moribund stage. Fishes
exposed to formalin also exhibited weak swimming compared to the control in order for them to
compensate the energy loss due to exposure to the chemical.

CONCLUSION

The LC50 of formalin to tilapia fry is 500 ppm. There was 100% mortality of fish at 5, 18
and 24 hours of exposure at 1000 ppm, 500 ppm and 200 ppm of formalin concentrations
respectively. It is therefore suggested to use formalin at much lower levels because this could
affect the survival of the fish. Appropriate management practices must be implemented in using
formalin especially at high concentrations.

References
Annune, P.A., Ebele, S.O. and Oladimeji, A.A. (1994). Acute toxicity of Cadmium to juveniles
of Clarias gariepinus (Teugels) and Oreochromis nilolicus (Trewavas) Journal of Environmental
Science and Health 29(7):1357-1361.
Badran, A., S. Aly, A. Abdel-Aal, 1996.Studies on skin parasitic diseases of hybrid tilapia.
Assiut Vet. Med. J., l35(70):163-175.

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.
Buchman, K. Bresciani, J. and Jape, C. (2004). Effects of formalin treatment on Epithelial
strucuture and mucous cell densities in rainbow trout, oncorhynchus mykiss (wallbaum), skin,
journal of fish diseases 27 (2):99.
Fish, F.F. 1940. Formalin for external protozoa parasites, Prog. Fish Cult. 48: 1–10.
Kinnunen, P., M.Rahkonen, H. Mykrä and E. Valtonen,2005. Treatment of ichthyophthiriasis
after malachite green. II. Earth ponds at salmonid farms. Dis. Aquat. Org., 66: 15–20.

Konar, S.K. (1970). Nicotine as a fish poison. Progressive Fish Culturist. 32:103-104.
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.
Park, K., and G. Heo, 2008.Acute and Subacute Toxicity of Copper Sulfate Pentahydrate
(CuSO45·H2O) in the Guppy (Poeciliareticulata) J. Vet. Med. Sci., 71(3): 333–336.

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.
Reed, L.J. and H. Muench. 1938. A simple method of estimating Fifty percent end points. Amer.
J. Hygiene. 27:493-497.
Santos, R., H. Dias and R. Fujimoto,2012.Acute toxicity and histopathology in ornamentalfish
amazon bluespottedcorydora (Corydorasmelanistius) exposed to formalin. An. Acad.
Bras.Cienc., 84 (4):1001-1007.

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.
Srivastava, S., A.Gupta, S.Chaturvedi, A. Abhinavand R. Singh, 2009. Effects of short-term
therapeutic bath of malachite green and formalin on certain hematological parameters of
freshwater catfish, (Heteropneustes fossilis) (Bloch).Journal of Experimental Zoology,
India.12(1):79-83.
Walker, J.F. 1964. Formaldehyde. Am. Chem. Soc. monograph seried, 3rd ed. Rainhold
Publishing Corp. New York.
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.