MEASUREMENTS OF THE ACUTE TOXICITY OF CYPERMETHRIN TO NILE TILAPIA ( Oreochrotnie niloticue ) , USING A STATIC AND A CONTINUOUS . FLOW SYSTEM

Experiments on the toxicity of the insecticide cypermethrin, a synthetic pyrethroid, to nile tilapia (Oreoehromia niloticue) were conducted, using static and continuous flow systems.


INTRODUCTION
Modern agriculture relies heavily on pesti- cides to control pests and to increase yields of many crops.Pesticides are also increasingly used to aid the control of many serious vector- borne diseases, such as bilharzia in Africa and malaria in Asia, as well as diseases in cattle.Unfortunately, despite the many benefits of pesticides there are considerable problems associated with their use (Willis and McDowell, 1982).For example many pesticides are now recognized as serious pollutants in aquatic environments, with deleterious effects on many aquatic organisms 197

l).
The new synthetic pyrethroid insecticides have been found to be highlv active against insects and larvae of several species of mos- quitoes.Th'e chemical penetrates the cuticle of the insect and then transported in the haemolymph to the central nervous system, where toxic effects are manifested (Burt and   Goodchild, 1974).
It has been reported, however, that at least some of the synthetic pyrethroids have a high level of toxicity to fish (Mulla et al., 1978;Stephenson, 1982;Kumaraguru and Beamish, 1981;Shires, l98B).Decamethrin, for example, was found to be the most toxic pyrethroid compound to all the fish tested by Mulla et al. (7978), causing g0% mortality at concentration levels of l-2 ppb (parts per billion).
The heavy usage of agricultural chemicals and their associated effects on fish represent considerable problems for fish culture in many parts of the world, particularly where agriculture and fish culture activities exist in the same agrosystem.The danger of pesti- cides is probably greatest where fish culture is practised in irrigated rice fields, a tech- nique which is extensively conducted in Indonesia and many other Asian countries.Fish mortalities are commonly seen directly or shortly after insecticide application, depend- ing on the toxicity of the chemical.Insecti' cides may persist in the rice field environ' ment depending on their chemical structure (Edwards, 1977).High levels of insecticide usage can cause downstream contamination, associated with serious deleterious effects to fisheries (Gorbach et al., l97la, 1971b).The use of less toxic and less persistent insecti' cides in irrigated rice fields should be encour- aged.Since many new insecticides that appear on the market pose a threat to the envi- ronment.there is a need for an assessment of the potential impact of insecticides, such as synthetic pyrethroids, on fish culture in rice fields.
This paper aimed to evaluate the acute toxicity of cypermethrin, a synthetic pyre' throid insecticide, to the nile tilapia, based on the results of static and continuous-flow toxicity tests.

Ineecticide Sample
Cypermethrin is the active ingredient of widely used formulated insecticides.The chemical name of cypermethrin is (R,S)-a- cyano-3-pheno-xy benzy I ( IR,IS,cis,trans)-3 - ( 2, 2 -dichlorouiny l) -2, 2 -dimethy I cyclop ropane - carboxylate.For the purpose of the experi- ments a commercial insecticide product con- taining 1009 of cypermethrin per litre, was used.The material was a yellowish liquid, which was miscible in water, producing a whitish suspension.
A solution of cypermethrin in water was used for the experiments.This solution was prepared by mixing 1 mL of the formulated insecticide product in 100 mL of Analar ace- tone, to give I g cypermethrin per litre of acetone, as suggested by Stephenson (1982).
The solution was then made up to 1 L with distilled water, producing a stock of 1000 ppm of the commercial insecticide product.Subse- quent dilutions of this stock solution were prepared for the toxicity tests.

Teet Fish
Fingerlings of nile tilapia, Oreochrontis niloticus, with average weigh[of 1g, obtained from a local hatchery unit, were used in the tests.The fishes were reared in fibre glass tanks, supplied with water from a recycling water system which produced an average water flow of 0.72Usec., at a water temperature of 25"-27"C.The fishes were fed with commercial fish pellets, containing 40o/o pro- tein.Daily feeding rate of 5o/o body weight was given.
No mortality was recorded in the frsh stock population, and no sign of any health problem was observed during the two weeks before the experiment.Two days before each experi- ment, fish were transferred to the test con- tainers, and were starved to prevent fouling the test solutions.

Dilution Water
The average physical and chemical characteristics of the dilution water were as follows: Temperature ("C) pH Total hardness (as ppm CaCO) N-NH3 (ppm) Dissolved oxygen (ppm) The above parameters of water quality, except ammonia, were monitored and recorded daily during the course of the experi- ment.

Static Test
The main objective of the static toxicity test was to determine the acute or lethal toxicity of the insecticide to fish.The acute toxicity was defined as the Median Lethal Concentration or LC50, i.e the concentration which produces frfty percent mortality to test fish in a certain period of time (usually 24, 48 or 96 hours), under specified test conditions.
The methodology of fish toxicity testing has been described by many investigators and research institutions (Duodoroff et aL,l95I: Alabaster and Abrams, 1965;Sprague, 1969;APHA et al., 1974;EIFAC/FAO, 1975 andBuikema et al., 1982), and the basic proce' dures postulated in these standard methods were adopted in the tests.
Two tests were carried out in the static system.The tests were conducted in six test containers made of transparent plastic, each with dimensions of 40 x 18 x 20 cm (length, width and depth).
Ten litres of dilution water were added to each test container.Twenty fish were selected at random from the stock populations, and introduced into the test containers, allowing an average loading ratio of 2 g per I L of dilution water.In order to maintain the dissolved oxygen concentration in each test container, ventilation was provided by means of a compressor and air stones.
Aliquots of 1000 ppm cypermethin stock solution were added to each container to make certain concentration at the start of each experiment.To find out the lethal range of cypermethrin for the nile tilapia, an explor- atory test was carried out using 0.01, 0.1 and 1.0 ppm of the commercial insecticide pro- duct, and five fishes per concentration.Full scale tests were conducted using five concentrations based on progressive bisection of intervals on a logarithmic scale (Duodoroff el al., 1951and APHA et al., 1974).These test concentrations were: 0.37ppm, 0.05ppm, 0.87ppm, 1.15ppm and 1.55ppm (formulated product of cypermethrin), The control fish were exposed to the highest concentrations of the solvent in which fish in the test solutions were exposed (i.e.155ppm acetone which is non lethal to fish).
The test fish were observed and mortality recorded at the following time intervals: (a) 15, 30, 60 minutes, (b) 2, 4, 8, 16 and 24 hours, and (c) 2, 3, and 4 days.Test iish were considered dead when any movement of the operculum had ceased.Dead fish were imme- diately removed from the test containers to prevent fouling of the test solutions.
Cumulative fish mortality at24, 48,72 and, 96 hours exposure periods, was recorded and plotted against concentration on logarithm- probability graph papers.The data were then IFR Journal Vol.II No.l, 1996   analysed according to the statistical method described by Litchfield and Wilcoxon (1949), to determine the values of the Median Lethal Concentrations (LC50s), the slope functions of the dose-response lines, and their g5-percent confident limit intervals.
Testing for significant differences between LC50s (P = 0.05) was carried out by calculating the potency ratio of the values, as de- scribed by Litchfield & Wilcoxon (1949).

Continuous-Flow Test
The objective of this test was to determine the acute toxicity of the insecticide to fish by means of a continuous-flow system.This allows a continous supply of new test solution through the tank.
The system consisted of: The system allowed a rate of test solution flow of l.2Ug of fish/day, and a replacement time of test solution of 90o/o per 20 -24 hours.
More reasonable test solution rates of [2][3] Uglday and replacement time of 9O% in 8-12 hours, as recommended by Sprague (1969), could not be applied here due to limited sup- ply of dilution water.
Except for the incorporation of the continuous-flow apparatus, the general procedures of the tests were the same as those described in the static test.
High pH value was maintained by adding 20ppm of calcium hydroxide to the test solu- tions.
The test concentrations used in the above experiments were based on the formulated product of cypermethrin as indicated in Table 1.RESULTS

Static Teete
The two acute static tests showed that the insecticide is highly toxic to nile tilapia, with mean 24-hour LC50 concentrations of 0.90 ppm (0.090 mg/L active ingredient of cyper- methrin) (   The LC50s in both static tests were not signifcantly different (P <0.05), and there was no significant change in LC50 values after 24 hours (Table 3).This was indicated by the relative potency ratio of the action of the IFR Journal Vol.II No.L I996 insecticide against nile tilapia between 24 hours and subsequent exposure periods, which is estimated after testing the parallel- ism oftheir dose-effect lines, as described by' Litchfield and Wilcoxon (1949) (see Table 3).
Table 3.The potency ratio of cypermethrin formulation (100 g/L) to nile tilapia between 24,48 and 72 hours exposure period in the Static Test I and 2.
Test No.
Potency Ratio (PR)  The physico-chemical characteristics of the test solutions measured were according to the recommended guidelines of APHA et aI. (1974).No significant fluctuations was noted in the values of the parameter measured (Table 4).Median Lethal Concentration (LC50), and 95% confrdence limit intervals (ppm) Slope Function and g5% con- fidence limit intervals 24 48 72 96 0.90 (0.69-1.17) 0.55 (0.44.0.69) 0.36 (0.27-0.48) < 0.35 2.32 (1.76-3.69).88)ity in the test frsh after 96 hours in the lowest concentration, and thus the value of 96-hour LC50 could not be calculated, but was clearly less than 0.35 ppm.However, the potency ratios clearly showed that continuous expo- sure is more toxic over a longer period of time than a single dose of the insecticide under the present experimental conditions (Table 6).7. The results show, similar to those in the previous experiment, that there was an increasing mortality with longer exposure period.There was no significant difference (F>0.05) between the LCbOs in both acidic experiments.

-Alkaline conditions
The values of LC50 obtained from the tests in alkaline water are presented in Table 8.
The comparison of the results between the toxicity tests in acidic and alkaline media suggests that cypermethrin is more toxic in acidic conditions (P<0.05) (Table 9).There was no significant difference between the toxicity of the insecticide in static and continuous-flow test within 48 hours exlosure time (F>0.05) (Table 10).However, the difference was significantly pronounced after 22 hours of exposure.
The physico-chemical characteristics of the water used in the tests did not show any significant fluctuations, except in pH.The hardness of the water was higher than those measured in the static test, as shown in Table 11.
It has been pointed by Stephenson (1982) that synthethic pyrethroids have a very low solubility in water (5 x 10.3 to l0 x 10'3 mg/L) and a strong tendency to be adsorbed onto t6 surfaces.The cypermethrin may have been adsorbed onto the surfacee of the test containers or adsorbed onto organic matter during the couree of the test, resulting in a reduction of its toxic effects on fish.The lower toxicity in static teets may also have been due to the removal and metabolism of the test material by the tieh.However, specific information about this effect with regard to cypermethrin is not available.It was also observed during the experiment that fish mortality waa more rapid in the continuous- flow system relative to the static system in the same level of concentration, perhaps due to the constant availability of the insecticide in this system to enter the fish vascular system via the gills.This result is in accordance with similar experiments by Lincer et al. (1970) in determining the toxicity of DDT and Endrin.These investigators stated that toxicity of DDT and Endrin to fish are greater in a continuous'flow system then a the static system.
The results of the above toxicity tests of cypermethrin strongly suggest the loss of the bioactivity of this material or the reduction of its initial concentration in the test solutions.It has been pointed by Stephenson (1982) that synthethic pyrethroids have a very low solubility in water (5 x 10'3 to lO x 103 mg/) and a strong tendency to be adsorbed onto surfaces.The cypermethrin may have been adsorbed onto the surfaces of the test containers or adsorbed onto organic matter during the course of the test, resulting in a reduction of its toxic effects on fish.The lower toxicity in static tests may also have been due to the removal and metabolism of the test material by the fish.However, specific information about this effect with regard to cypermethrin is not available.It was also observed during the experiment that fish mortality was more rapid in the continuousflow system relative to the static system in the same level of concentration, perhapa due to the constant availability of the insecticide in this system to enter the fish vascular system via the gills.This result is in accordance with similar experiments by Lincer et al. (7970) in determining the toxicity of DDT and Endrin.These investigators stated that toxicity of DDT and Endrin to fish are greater in the continuous-flow system relative to the static sYstem.
Stephenson (1982) obtained a 96-hour value of O.OO22 ppm for technical cyper- methrin containing more than 95%o cypet' methrin, ttsing Oreochromis niloticus as a test fish in a continuous-flow system.This value is IFR Journol VoI.II No.L t996 about 15 times lower than the value obtained from the present test, ifcalculated based on the active ingredient of cypermethrin in the test material.This large difference in results may be due to the different teet material used or experimental method.According to Alabaster (1969) it is impossible in most instances to deduce LC50 values from concentrations of the active constituent, as the other components may themselves be toxic, or may reduce the toxicity of the pesticides.Variation of the results may also have been due to different testing conditions.One of the test conditions which was shown to influence toxicity was water pH, with toxicity being greater in acidic conditions.This result may be due to fish in acid water being stressed by the relatively low pH.It is well known that additional stress on a fieh which is already stressed can cause mortality (Wedemeyer et a1.,1984).It is also possible that pH may influence the toxicity of pesticide itself (Muirhead-Thomson, 1972, andAlabaster, 1969).The results of the experi' ment suggest, however, that water quality in the field will play an important rule in the toxicity of cypermethrin to fish.

CONCLUSION
The results of these experiments showed that the insecticide cypermethrin is highly toxic to the nile tilapia.The experiments also confirmed that substantial improvements in determining more accurately the toxicity of cypermethrin could be made by employing a continuous-flow test system, relative to a static system.Accurate information from toxicity tests can be used in (a) prediction of environmental effects of pesticides, O) comparison of potential hazard of pesticidee to aquatic organisms, and (c) regulation of the usage of pesticides.
It is therefore advantageous to obtained toxicological informations of all new pesti' cides, before the materials are to be recom' mended for their use in agricultural pest control, especially in rice plants' (a) a constant head water reservoir which supplied dilution water to the system; (b) a peristaltic pump (Watson-Marlow HR Flow Inducer Type MHRE), which delivered insecticide solutions to the system by means of five tubings with different diameters and a pump speed of 20 rpm; (c) mixing chambers which were attached to each test container, to receive the dilution water and the insecticide solu- tion, respectively from the constant head water tank and the stock solution tank, and subsequently mix and deliver the new test solutions into the test containers.

Table 2 ) . Table 1 .
Concentration of cypermethrin formulation (100 g/L) used in the Continuous Flow

Table 2 .
The acute toxicity of cypermethrin formulation (100 etL) to nile tilapia in test using a

Table 4 .
The physico-chemical characteristics of test solutions measured during the test in the Static System (Mean t standard deviation, N = 5).
The data indicate a decrease in the median lethal concentrations through out the period of the test.There was more than 50% mortal- the the

Table 5 .
The acute toxicity of cypermethrin formulation (100 g/L) to nile tilapia in acidic conditions (pH: 3.6-3.9),using a Continuous-Flow Test System.

Table 6 .
The potency ratio of cypermethrin formulation (100 g/L) to nile tilapia between 24,4A and 72 hours exposure periods, in the Continuous-Flow Test System.

Table 9 .
The potency ratio of cypermethrin formulation (100 g/l) to nile tilapia between acidic, slightly acidic and alkaline condition, in the Continuous-Flow Test System.

Table 10 .
The potency ratio of cypermethrin formulation (lm g/L) to nile tilapia between the Static Test and the Continuous Flow Test conditione.The potency ratios were based on the average values of 24 and 48 hours LC50s obtained from two static tests and three continuous flow tests.

Table 11 .
The physico-chemical characterietics of test eolutions measured during the test in the Continuous Flow System (Means t etandard deviation, N = 5).