Abstract: The toxicity studies were conducted on the fish Channa punctata (Bloch) by employing static and continuous flow through systems, for the toxicant butachlor (technical grade + ) and its commercial formulation + (machete 50% E

K S Tilak

K Veeraiah

M S Butchiram

P Bhaskara

Thathaji

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Journal of Environmental Biology  April, 2007
Toxicity studies of butachlor to the freshwater fish Channa punctata (Bloch)
K.S. Tilak*, K. Veeraiah, P. Bhaskara Thathaji and M.S. Butchiram
Department of Zoology, Acharya Nagarjuna University, Nagarjunanagar-522 510, India
(Received: May 17, 2005 ; Revised received: December 18, 2005 ; Accepted: January 14, 2006)
Abstract: The toxicity studies were conducted on the fish Channa punctata (Bloch) by employing static and continuous flow through systems, for the
toxicant butachlor (technical grade+) and its commercial formulation+  (machete 50% EC). The LC
50
 values are 297.89 ppb and 247.46 ppb for 24 hr
and 48 hr in static for technical and 636.45 and 546.09 for machete. In continuous flow through the values are 270.05, 233.52 to the technical and
567.85 and 481.49 respectively for machete. The tissues show qualitative accumulation and were quantitatively analysed by gas liquid chromatography
(GLC).
Key words: Toxicity, Channa punctata, Butachlor, Machete, Gas liquid chromatography
Introduction
Butachlor was the first rice herbicide to be introduced  in
India. It is chemically 2-chloro 2, 6 diethyl N, butoxymethyl
acetanilide (Fig. 1).
For many years, bioassays have remained simple tools
to assess acute and chronic toxicity. Even in sublethal
concentrations butachlor effects are multifold ranging from simple
respiratory distress, accumulation, effecting the biochemical
pathway at cellular neurological levels and finally culminating in
inhibit ion/decrease of  the neuromotor enzyme acetyl
cholinesterase. In the present study, an attempt has, therefore,
been made to assess the toxicity of acute nature and the residue
study of qualitative and quantitative nature of the toxicant
butachlor after exposure of the fish Channa punctata (Bloch) to
sublethal doses of the herbicide.
Materials and Methods
The fish Channa punctata (Bloch) of 8 to 10 cm length
and 6 to 9 g of weight were brought from the river Krishna. Toxicity
studies were conducted using the machete (50% EC) by
employing static and continuous flow through systems as per
the recommendations of APHA (1998). For flowthrough system,
test solutions of desired concentrations were prepared once every
five hours in glass reservoirs and delivered into the test containers
*Corresponding author: E-mail: kstilak@yahoo.com, Tel.: 0091-863-2594208 (O), 0091-866-2451169 (R), 94403-32474 (Cell)
through thin walled polyethelene tubes. The flow rate was
adjusted with regulators such that 4 liter of water passed through
containers in one hour. The conditions of the test medium were:
temperature 28oC ± 2 oC, oxygen 6-8 mg l-1, hardness 80 mg l-1,
alkalinity 425 mg l-1 and pH 8.3. Pilot experiments were conducted
to determine the concentrations causing 10-90% mortality of test
fish. For each concentration, 10 fish were tested and the
experiment was repeated thrice. Probit analysis (Finney, 1971)
as recommended by Roberts and Boyce (1972) was followed to
calculate the LC
50
 values.
For the residue analysis, the fish were acclimatized at
room temperature (28oC ± 2) in the laboratory conditions for 7
days in well aerated water and these acclimatized fish were
exposed for 10 days to sublethal concentration (1/
5
th of static LC
50
)
of butachlor. After the exposure, the residues were extracted from
the tissues by the method of Seleh et al. (1986), but the brain
tissue was extracted in acetonitrile, in the presence of anhydrous
sodium sulphate, the residue was finally washed in 4 ml of hexane.
Clean up: The extracts of the tissues viz., gill, liver, kidney, muscle
and brain were cleaned up by column chromatography using silica
gel as adsorbent covered with a layer of anhydrous sodium
sulphate, packed with hexane. The butachlor was eluted in a
mixture of hexane and acetone (9 : 1). A 100 ml portion of the
elutant was collected and evaporated to a final volume of 1 ml
according to Goughan et al. (1978).
Gas liquid chromatographic (GLC) analysis: The quantitative
analysis by gas liquid chromatography was performed according
to Bradbury and Coats method (1982). Residues of butachlor
were determined by using GC-17A. GC 17A is an autocontrol
system of Shimadzu, Japan equipped with electron capture
detector and coiled glass column (S.E-30) of 25 metres and 0.31
mm inner diameter with 0.17 micro meters film thickness. Helium
was used as carrier gas through 1.5 ml/min. The standard curves
Fig. 1: Chemical formula of machete
Cl CH
2 C
N CH
2 O
CH
2
CH
3
CH
2
CH
2
CH
2
CH
3
CH
3
H
2
C
O
Journal of Environmental Biology April 2007, 28(2) 485-487 (2007)
©Triveni Enterprises, Lucknow (India) For personal use only
Free paper downloaded from: www. jeb.co.in Commercial distribution of this copy is illegal
Journal of Environmental Biology  April, 2007
Tilak et al.
based on the 8 cm of peak heights were used for quantification.
The amount of butachlor residue was calculated against known
standard values by employing the following formula.
   Area of sample
Concentration  =  ——————————  x  Amount of standard
 Area of standard
The residue analysis by GLC is being done by Indian
Institute of Chemical Technology (IICT), a CSIR Laboratory,
Hyderabad-500 007, India.
Results and Discussion
The LC
50
 values of machete for 24 hr and 48 hr to the fish,
Channa punctata (Bloch) are given in Table 1. Farah et al. (2004)
reported  the LC
50
 values and stress behaviour in the fish and
mosquito larvae under exposure to three widely used xenobiotic
compounds like pentachlorophenol (PCP), 2,4-D and butachlor.
Mosquito larvae were generally more resistant than the fish.
Junghans et al.  (2003) studied the effects of
chloroacetanilides (acetachlor, alachlor, butachlor, dimethachlor,
metazachlor, pretilachlor and propachlor) individually and in
combination on the reproduction of the green algae, Scenedesmus
vaculatus. Individually, chloroacetanilides impaired algal
reproduction with EC
50
 values ranging from 3 to 232 mg l-1. The
differences in EC
50
 values were strongly correlated with the
lipophilicites of the compound synergistically. The acute toxicity
of butachlor on earthworm Drawide willsi was determined by
Smeeth and Sanjat (2002). The reported 96 hr LC
50
 values were
7.72 to 10.22 mg/kg for juveniles as well as for adults respectively.
Tantawy (2002) studied the effect of two herbicides on biological
and biochemical parameters of Biomphalaria alexandrina and
reported that after 6 hr of exposure to 6.5 mg l-1 concentration of
butachlor, the mortality rate was increased. Suseela (2001)
reported that the herbicide, as toxicant, did not have any adverse
effects but it accelerated the nitrogen fixation. Hashimoto and
Nishituchi (1983) studied the toxic effects of butachlor not only in
fish but also in Daphnia and other aquatic organisms and
concluded that it is toxic. Wang et al. (1991) reported that the
residues of butachlor in paddy field, even though lower than the
safe concentration, caused toxic effect to Cyprinus carpio.
Table - 1: The LC
50
 values and their confidence limits of butachlor technical grade and machete (50% EC) formulation to fish, Channa punctata
(Bloch) for 24 and 48 hr static and continuous flow (CF) through system
              Fish                24 hr                  48 hr
Toxicant
Length Weight Static C.F. Static C.F.
   cm     g  ppb ppb   ppb ppb
Technical grade 8 - 10 6 - 9 297.89 270.05 247.46 233.52
of butachlor (4.56-4.38) (4.51-4.35) (4.49-4.28) (4.49-4.24)
Machete 50% E C 8 – 10 6 - 9 636.45 567.85 546.09 481.49
(4.86-4.74) (4.91-4.60) (4.78-4.68) (4.73-4.63)
CF = Continuous flow through,   ppb = parts per billion
Alachlor, closely related to butchlor is also toxic to fish.
The LC
50
 value for rainbow trout is 2.4 mg l-1, bluegill fish is 4.3
mg l-1, catfish is 6.5 mg l-1 and carp is 4.6 mg l-1 after exposure of
96 hr (Kidd and James, 1991; Usnlm, 1995). Hill et al. (1997)
studied the most widely used herbicides of chloroacetalnilides,
alachlor and butachlor and made the hypothesis that they exert
genotoxic effect in cultured lymphocytes.
Hard et al. (1995) studied the stomach tumours that were
induced in Sprague Dawley rats during two chronic bioassays
with acetanilide herbicide, butachlor at a dietery concentration of
3000 mg l-1, the results in the experimental animals indicated
that butachlor has low mammalian toxicity following acute oral,
dermal and inhalation exposure. Some earlier reports revealed
that commercial formulations are more toxic than technical grade
pesticides, but in the present study the technical grade herbicide
i.e., butachlor was found more toxic than commercial formulations
i.e., machete 50% E.C. The reason is that the ingredients mixed
in the formulations are not causing toxicity, an observation which
is contrary to the earlier reports of Bradbury et al. (1987) and
Tilak et al. (2003).
Generally aquatic organisms are chemically affected by
three different classes of pesticides viz., organochlorine,
organophosphates and carbamates (Edwards, 1973; Brown,
1978; Rand and Sam, 1985). Among them, organochlorines act
as contact poison which effect the central nervous system of the
organisms, whereas organophosphates and carbamates inhibit
the enzyme acetylcholinesterase. The acetanilide group of
herbicides, as per the earlier reports, proved to have different
toxic values for different aquatic organisms and the mechanism
of action is of cumulative nature.
The results of tissue analysis of Channa punctata (Bloch)
exposed to sublethal concentration are given in Table 2 and the
residues are in the following order:
Liver > Kidney > Muscle > Gill > Brain
The variations in the residue analysis are due to factors
like difference in uptake rate, lipid content of respective animal
tissue, chemical structure, solubility, and metabolic pattern. (Zitko
486
Journal of Environmental Biology  April, 2007
Toxicity studies of Butachlor to Channa punctata
et al., 1977; Mulla et al., 1978; Coats and O’ Donnel Jaffery, 1979;
Zitko et al., 1979; Bradbury et al., 1987; Haya, 1989; Tripathi,
1992; Tilak 1982; Tilak, et al., 1980, 2003 and 2004). The
accumulation of residue is a factor responsible for changes either
in biochemical or pathological disturbance of overall biochemical
cyclic reactions which are cumulative, causing lethality even at
the sublethal concentrations.
The results of present study revealed that prolonged
exposure to sublethal concentrations led to increase in the
accumulation of residue.  Several chemicals are absorbed and
metabolised.  Radio labelled butachlor absorption was measured
in human skin and the unchanged compound and metabolites
were quantified by high pressure liquid chromatography (HPLC)
and qualified by thin layer chromatography (TLC) (Ademola et
al. 1993).  The final action of toxicity is due to metabolism and
accumulation of the residues in fish tissues as reported by Susan
et al. (1999a and b).  The residues are accumulated in different
tissues, causing toxicity to the fish which ultimately results in bio-
magnification through the food chain.
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487


Toxicity studies of butachlor to the freshwater fish, Channa punctatus (Bloch).

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Toxicity studies of butachlor to the freshwater fish, Channa punctatus (Bloch).

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