Toxicity of formalin on behaviour and respiration in Danio rerio

INTERNATIONAL JOURNAL OF ENVIRONMENTAL SCIENCES Volume 2, No 4, 2012
© Copyright 2010 All rights reserved Integrated Publishing Association
Research article ISSN 0976 – 4402
<strong>Toxicity</strong> <strong>of</strong> <strong>formalin</strong> on behaviour and respiration in Danio rerio
Varusai Naina Mohammed. S, Asrar Sheriff. M, Sultan Mohideen. A.K, Md. Azmathullah.N
P.G & Research Department <strong>of</strong> Zoology, The New College, Chennai – 600 014.
azmathullah0109@yahoo.co.in
doi:10.6088/ijes.00202030073
ABSTRACT
Formalin is an effective chemotherapeutic agent for combating several infectious conditions
affecting fish <strong>of</strong> aquacultural importance and ornamental fish. It is applied as bath, flush or
flowing treatment method and has proved to be effective against a variety <strong>of</strong> fish parasites
and microbes. The toxicity <strong>of</strong> <strong>formalin</strong> on the behavior and respiration <strong>of</strong> the popular
aquarium Zebra fish Danio rerio was evaluated 96 hour LC50 value was found to be 30ppm.
Abnormal behavior and swimming movements, reduced opercular beats, increased mucus
secretion and mortality were recorded due to <strong>formalin</strong> toxicity on zebrafish. Oxygen
consumption <strong>of</strong> zebrafish exposed to sub-lethal concentration <strong>of</strong> <strong>formalin</strong> showed a time
dependent decrease with 62.4% reduction at the end <strong>of</strong> 96 hours. The need for judicious use
<strong>of</strong> fish chemotherapeutants like <strong>formalin</strong> and their impact on fish life are discussed.
Key words: Formalin, <strong>Toxicity</strong>, Behaviour, Oxygen consumption, Zebrafish.
1. Introduction
Fishes are a rich source <strong>of</strong> protein available in a digestible and convertible form. Besides
being a nutritional delicacy, fishes have been valued for many years as excellent bioindicators
<strong>of</strong> water quality bio-control agents <strong>of</strong> mosquito larvae ( Jhingran, 1977) and
have also been attracting attention for their ornamental nature and as game fishes
(angling).
In aquaculture, factors such as unfavorable water conditions, overcrowding, frequent
administration <strong>of</strong> medicines, handling and transportation stress (Schaperclaus, 1991)
have resulted in defective growth in fishes, mass attack by parasites and episodes <strong>of</strong>
mass mortality. For the prophylaxis and treatment <strong>of</strong> fish diseases, a large number <strong>of</strong>
disinfectants, anaesthetics, antibiotics and other drugs are in use for a long time
(Schaperclaus, 1991). According to Roberts (1978), therapy can be applied to fishes in three
ways external treatment involving baths, dips, etc., systemic treatment via the diet and
parenteral treatment involving injection <strong>of</strong> drugs.
Despite the fact that effects <strong>of</strong> several chemicals employed in fish disease therapy have
been analysed by various workers, there seems to be little attention given to <strong>formalin</strong>
(Razia & Radhakrishnan, 1987). Formalin (37-40% formaldehyde) is an effective
chemotherapeutic agent successfully used against several infectious conditions, particularly
those affecting the body surface and skin <strong>of</strong> fishes (Roberts & Shepherd, 1979).
Formalin can be applied by bath, flush or flowing treatment methods and has proved effective
against a variety <strong>of</strong> parasites and micro-organisms (Bauer et. al 1969; Schaperclaus, 1991;
Shuzo Egusa, 1991). There has been very little work on the toxicity <strong>of</strong> <strong>formalin</strong> on fishes.
Received on March 2012 Published on May 2012 1904

<strong>Toxicity</strong> <strong>of</strong> <strong>formalin</strong> on behaviour and respiration in Danio rerio
Smith & Piper (1972), Williams & Wootten (1981) and Razia & Radhakrishnan (1987) had
cautioned on the need for close monitoring <strong>of</strong> oxygen levels following <strong>formalin</strong> treatment as
it is a reducing agent.
The Zebrafish, Danio rerio, is a tropical freshwater fish belonging to the family Cyprinidae.
It is a popular aquarium fish, frequently sold under the trade name Zebra Danio, and is an
important model organism in scientific research.
Danio rerio, is a common and useful model organism for studies on vertebrate development
and gene function (Lieschke & Currie, 2007). Its greatest advantages for use as a model
system include:
Fully sequenced genetic code; well understood, easily observable and testable developmental
behaviors; Availability <strong>of</strong> well-characterized mutants. Rapid embryonic development. Large,
robust and transparent embryos that develop outside the mother and ability to regenerate fins,
skin, heart and the brain (in larval stages).
2. Materials and methods
Fish response to <strong>formalin</strong> was monitored and recorded daily throughout the 96 hours bioassay.
Dead fish were counted & removed. Fish were regarded as dead when all opercular
movements ceased. Regular visual observations <strong>of</strong> the fish (maintained in different
concentration <strong>of</strong> <strong>formalin</strong>) was made to record the general behaviour, opercular movements,
swimming orientation etc. during the entire exposure period.
Using the mortality record, LC 50 for 96 hours for <strong>formalin</strong> was determined graphically
following the method <strong>of</strong> Varma & Srivastava (1984)).
Oxygen consumption <strong>of</strong> the control and treated fish was determined following Strickland and
Parsons (1968).
3. Results
In the control fish, no apparent abnormalities in the behaviour pattern were observed. In the
case <strong>of</strong> <strong>formalin</strong> exposed fish, there was not much change in the behavioral patterns at
concentrations <strong>of</strong> 10ppm and 20ppm. At 40ppm, the fingerlings showed increased swimming
movements and decreased rate <strong>of</strong> opercular movements in comparison to the control
fingerlings. At 80ppm, no fingerlings survived at the end <strong>of</strong> 96 hours exposure to <strong>formalin</strong>.
The fingerlings showed initial restlessness followed by abnormal movements such a leaping
to the surface, decreased respiratory frequency (opercular movement) and secretion <strong>of</strong> mucus.
At 40 ppm concentration <strong>of</strong> <strong>formalin</strong>, the fingerlings showed an avoidance response by
exhibiting increased swimming and opercular movements immediately after exposure. Later
within a few hours <strong>of</strong> exposure, the fingerlings showed loss <strong>of</strong> equilibrium, secretion <strong>of</strong>
mucus, swimming at upside down position, followed by death usually within 24 hours.
The mortality record for 96 hours exposure at various concentration <strong>of</strong> <strong>formalin</strong> is given in
Table 1. It is evident that at concentrations <strong>of</strong> 10 ppm and 20 ppm the % mortality is less than
50%, at 40 ppm it is 70% and shows a marked increase at 80 ppm (100%). Graphical
determination <strong>of</strong> 96 hours LC 50 value was found to be 30 ppm by the straight line graphical
method.
Varusai Naina Mohammed. S, Asrar Sheriff. M, Sultan Mohideen. A.K, Md. Azmathullah. N
International Journal <strong>of</strong> Environmental Sciences Volume 2 No.4, 2012
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<strong>Toxicity</strong> <strong>of</strong> <strong>formalin</strong> on behaviour and respiration in Danio rerio
A change in the respiration rate is one <strong>of</strong> the common physiological responses to toxicants
and is easily detectable through changes in oxygen consumption rate, which is frequently
used to evaluate the changes in metabolism under environmental deterioration. It is evident
from the study that <strong>formalin</strong> affected the oxygen consumption <strong>of</strong> Zebrafish Danio rerio sub –
lethal concentration <strong>of</strong> the rate <strong>of</strong> O2 consumption in treated fish showed a progressive
decrement compared to the control. At 24 hours, oxygen consumption in the treated fish was
1.48 ml/g/hr, followed by1.01, 0.08 and 0.65 ml/g/hr at 48, 72 and 96 hours respectively.
The % decrease in O2 consumption was 10.6, 19.8, 37.5 and 62.4 during the study period and
this was statistically significant at P< 0.01.
4. Discussion
Chemotherapy is widely practiced for treating various infectious disease <strong>of</strong> fishes. According
to Schaperclaus (1991), there are a large number <strong>of</strong> drugs, antiparasitics, disinfectants,
vitamins, hormones etc. at our disposal whose scientific application could give satisfactory
results. Among the various chemotherapeutic agents in use, <strong>formalin</strong> and KMnO4 are
commonly used as antiparasitics against a variety <strong>of</strong> pathogenic organisms. According to
Strelkow (1983), the usage <strong>of</strong> these agents depends essentially on the working spectrum,
toxicity, metabolism and residue formation, technological and economical aspect <strong>of</strong>
applicability.
Formalin is one <strong>of</strong> the most effective and widely used compounds in fish culture for
therapeutic and prophylactic treatment <strong>of</strong> fungal infection and external parasites <strong>of</strong> fish and
fish eggs (Roberts, 1978). Shuzo Egusa (1991) recommended the use <strong>of</strong> <strong>formalin</strong> at 25 ppm
for the white spot disease caused by Ichthyopthirius multifilis and at 15 – 25 ppm in ponds for
alternate days for 5 – 7 days to control trematodes. According to Kabata (1970) treatment for
1 hour with 250 ppm <strong>formalin</strong> destroyed the larvae <strong>of</strong> the copepod Salmincola sp.
The mortality rate and behavior <strong>of</strong> Zebra fish during experimentation was found to be
dependent on duration <strong>of</strong> exposure and concentration <strong>of</strong> <strong>formalin</strong> which is in accordance with
similar results obtained for other toxicants (Vutukuru, 2005). Behavioural toxicology is an
important tool for hazardous assessment <strong>of</strong> water pollution and helps in understanding the
manifestations <strong>of</strong> environmental stress on fish (Rao and Rao, 1987). The abnormal
behavioural changes exhibited by zebra fish on exposure to <strong>formalin</strong> such as erratic
swimming behavior, increased secretion <strong>of</strong> mucous, reduced opercular movements have also
been reported in fishes exposed to various toxicants like lead, cadmium, chromium and
fertilizers (Konar and Sarkar, 1983; Olaifa et al., 2003; Tawari-Fufeyin et al., 2008). The
behavioural changes seen in zebra fish in the present study may be an indicative <strong>of</strong> internal
disturbances <strong>of</strong> body functions such as inhibition <strong>of</strong> enzymes, impairment <strong>of</strong> neural
transmission, disturbances in metabolic pathways and respiratory distress (Shah and Altindag,
2005).
The rate <strong>of</strong> O2 consumption <strong>of</strong> a fish is considered a reflection <strong>of</strong> total metabolism and hence
the metabolic state <strong>of</strong> the fish. The present study clearly indicates that fingerlings <strong>of</strong> Zebra
fish exposed to various concentrations <strong>of</strong> <strong>formalin</strong> showed toxicity & mortality. This could
partly be attributed to the role <strong>of</strong> <strong>formalin</strong> as a reducing agent resulting in reduction <strong>of</strong>
oxygen levels in the water (Roberts, 1978). Reduced oxygen tension along with other
environmental stressors like high ammonia levels, suspended solids will impose stress and
predispose the fishes to various secondary microbial infections (Shepherd and Poupard,
1975 ). Another reason for the reduction in the rate <strong>of</strong> oxygen consumption by the fingerlings
Varusai Naina Mohammed. S, Asrar Sheriff. M, Sultan Mohideen. A.K, Md. Azmathullah. N
International Journal <strong>of</strong> Environmental Sciences Volume 2 No.4, 2012
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<strong>Toxicity</strong> <strong>of</strong> <strong>formalin</strong> on behaviour and respiration in Danio rerio
may be due to damage caused to the gill epithelia by exposure to <strong>formalin</strong> (Smith and Piper,
1972).
Hence, judicious use <strong>of</strong> the chemotherapeutant with proper monitoring is essential to
understand its toxicity on fish and thereby help to achieve the aim <strong>of</strong> preventing the spread <strong>of</strong>
fish infections and infestations.
Table 1: Mortality <strong>of</strong> Zebra fish fingerlings at different concentration <strong>of</strong> <strong>formalin</strong>
S. NO FORMALIN CONCENTRATION %
1
(ppm)
CONTROL
MORTALITY
0
2 10 20
3 20 50
4 40 70
5 80 100
Table 2: O2 consumption in Danio rerio exposed to sub lethal concentration <strong>of</strong> <strong>formalin</strong>
S.
NO
TIME
(HOUR)
OXYGEN
CONSUMPTION IN
CONTROL (ml/g/hr)
OXYGEN
CONSUMPTION IN
TREATED FISH
(ml/g/hr)
1 24 1.4 1.48 +10.6
2 48 1.01 -19.8
3 72 0.080 -37.5
4 96 0.065 -62.4
(Significant at P< 0.01)
5. References
%
INCREASE OR
DECREASE
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