Aquaculture International(2005)13:451–458óSpringer2005 DOI10.1007/s10499-005-0615-5
Acute toxicity of synthetic pyrethroid cypermethrin
on the common carp(Cyprinus carpio L.)embryos
and larvae
RAHM_I AYDIN1,*,KENAN KO PRU CU2,MUSTAFA DO RU CU2,
S_IBEL S_IMS EK KO PRU CU2and MURAT PALA3
1Firat University,College of Bingol,Fisheries Programme,Bingol,Turkey;2Firat University,Fish-eries Faculty,23119Elazig,Turkey;3The State Hydraulic Works,Elazig,Turkey;*Author for correspondence(e-mail:raydin@?https://www.doczj.com/doc/1313940125.html,.tr)
Key words:Acute toxicity,Common carp,Cypermethrin,Cyprinus carpio,Embryo,Hatching success,Larvae,Synthetic pyrethroid
Abstract.In this study,the toxic e?ects on the embryos and larvae of the common carp were used as a model to investigate the synthetic pyrethroid pesticide,cypermethrin,which contaminates aquatic ecosystems.Data obtained from the cypermethrin acute toxicity tests were evaluated using the Probit Analysis Statistical Method.The control and eight test experiments were repeated?ve times.The number of dead embryos signi?cantly increased in response to cypermethrin concen-trations0.0001,0.001,0.01,0.1,1,2,4and8l g là1(p<0.05for each case).The48h LC50value (with95%con?dence limits)of cypermethrin for common carp embryos was estimated at0.909 (0.256–5.074)l g là1.Dose–response decreases in hatching success were recorded as87.4,85.0,80.2, 71.4,56.3,48.6,38.8and23.5%,respectively.The lowest concentration of cypermethrin (0.0001l g là1)produced a signi?cant increase in the number of dead larvae compared to the control group(p<0.05).The number of dead larvae signi?cantly increased with increasing cy-permethrin concentrations exposed for1–96h(p<0.05).The highest concentration(8l g là1) showed the highest larvae mortality.The96h LC50value(with95%con?dence limits)of cyper-methrin for common carp larvae was estimated at0.809(0.530–1.308)l g là1.The results of the study suggest that low levels of cypermethrin in the aquatic environment may have a signi?cant e?ect on the reproduction and development of carp.
Introduction
Recently,many new broad-spectrum pesticides have been developed that have the potential for widespread use in the environment.In large-scale applications of these pesticides by methods such as crop dusting,orchard and forest spraying or mosquito control,some inevitably enter the aquatic environment (Environment Agency1997).
Synthetic pyrethroid insecticides are extensively used in place of organo-chlorine,organophosphorus insecticides and carbamates to control pests. These insecticides are more likely to be toxic to?sh and other aquatic organisms(Elliot1977;Casida et al.1983;Smith and Stratton1986;Moore and Waring2001;Polat et al.2002;Ko pru cu and Aydin2004).Synergistic interactions between the active ingredient and other components of the for-mulation should be taken into consideration when evaluating toxicity.Several
452
larvicides and adulticides,including resmethrin and permethrin,were evaluated for toxicity to measure the e?ects of mosquito control pesticides on non-target organisms(Milam et al.2000).Many products containing cypermethrin are classi?ed as‘restricted use pesticides’by the United States Environmental Protection Agency due to cypermethrin’s toxicity to?sh.Cypermethrin is classi?ed as a toxicity class II(moderately toxic)chemical,whilst others are designated as toxicity class III(slightly toxic)(US EPA1989).
Non-target organisms such as aquatic invertebrates and?sh are extremely sensitive to the neurotoxic e?ects of these insecticides(Reddy et al.1991; Philip et al.1995).Toxic e?ects of pyrethroids on non-target organisms have been reviewed and reported to be in the l g là1toxicity range(Smith and Stratton1986).In both the laboratory and?eld,absorption of pyrethroids substantially reduces toxicity.Cypermethrin has been classi?ed‘immobile’by the United States Environmental Protection Agency(US EPA1989),there-fore,in the?eld most of the a?ected organisms show rapid recovery.The environmental fate and e?ects of synthetic pyrethroid insecticides have been summarized in Hill(1989).In mammals and insects,pyrethroids have been shown to be more toxic at low temperatures than at high temperatures (Moore and Waring2001).In addition,Kumaragura and Beamish(1981) reported that acute toxicity of synthetic pyrethroids in?sh was negatively correlated to temperature.
Pyrethroids have been shown to be up to1000times more toxic to?sh than to mammals and birds at comparable concentrations(Edwards et al.1986; Bradbury and Coats1989).The hypersensitivity of?sh to pyrethroid intoxi-cation is due partly to species speci?c di?erences in pyrethroid metabolism,but principally to the increased sensitivity of the piscine nervous system to these pesticides(Moore and Waring2001).One such type II pyrethroid,cyper-methrin,is being increasingly used in the world as the active ingredient in many applications.According to environmental quality standards,the maximum allowable concentration is1ng là1(Environment Agency1997).
This study was conducted to determine acute toxicity of cypermethrin,a synthetic pyrethroid pesticide,to the embryos and larvae of common carp, Cyprinus carpio.The common carp is widespread and presently cultured all over Asia,in most parts of Europe,and on a small scale in some countries of Africa and Latin America.Therefore,common carp was selected for the bio-assay experiments.
Materials and methods
Adult?sh and chemical supply
Four to?ve year old male and female common carp samples weighing3–5kg, total length50–68cm,were obtained from a?sh hatchery unit of the State Hydraulic Works,Elazig,Turkey.The brood?sh were kept at24±1°C
453 (Rothbard and Yaron1996),in500l?berglass tanks and in natural light conditions.
Polytrin200EC,with the active molecule cypermethrin[(R,S)-alphacyano-3-phenoxybenzyl-(1RS)-cis/trans-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane-carboxylate],purity20%(dissolved in80%acetone)was purchased from Novartis.
Collection of gametes,arti?cial fertilization and incubation of eggs
Carp gametes were obtained through the hormonal induction of ovulation and spermiation by the intramuscular injection of carp pituitary powder (Chaudhuri1976)suspended in a0.9%NaCl solution.The suspended carp pituitary was administered at a dose of0.5mg là1of?sh for males and 1.0mg là1of?sh for females.The‘dry method’of fertilization was used in this study(Rothbard and Yaron1996).Twenty-four hours after injection,the gametes of both sexes were stripped into separate bowls,by applying gentle pressure to the in?ated belly.Before the stripping,brood?sh were anaesthe-tized with benzocaine dissolved in water at the concentration of1000ppm for 15m(Ferreira et al.1984).
The stripped eggs and milt were then mixed in a bowl.Fertilization and degumming of eggs was performed using the modi?ed Woynarovich method. Eggs were activated with a solution of4g NaCl and3g urea là1and5m later they were transferred into a solution of4g NaCl and20g urea là1 (Woynarovich and Woynarovich1980).Fertilised eggs were washed by the addition of water(24°C)for5m.
The fertilised eggs were rinsed and transported from the hatchery unit to the reproduction laboratory of the Fisheries Faculty of Firat University within 30m.These eggs were immediately placed in experimental units.Each exper-imental unit had?ve incubation chambers(each containing approximately200 eggs)containing2.5l of medium.The levels of medium in both aquaria and chambers were kept in the same position in order to maintain the same tem-perature in each chamber.The experimental medium from the reservoir was pumped through the chambers in order to keep the eggs suspended.The water temperature was kept at24±1°C and a12L:12D photoperiod was provided for the experimental units.
Experimental treatment
The experimental water was kept in the tank for24h before cypermethrin was added.Water quality characteristics of the reservoir in the experimental media were determined according to APHA(1985).The mean values for test water qualities were as follows:temperature24±1°C,pH7.3±0.3,dissolved oxy-gen7.8±0.2mg là1and total hardness120.5±3.4mg là1.Eight di?erent
454
concentrations(0.0001,0.001,0.01,0.1,1,2,4and8l g là1)for embryonal and larval stages of C.carpio were obtained by the addition of cypermethrin,fol-lowing preparation from a stock solution.The stock solutions were renewed every12h.The control group received acetone at the maximum acetone vol-ume used in the dilution of the dosing concentrations.
Embryonal Stage:The temperature of all the experimental media was kept at 24±1°C.After the fertilization procedure,unfertilized eggs were removed and the number of fertilized eggs per experimental unit(200fertilized eggs)was assessed.Every24h,until the end of the experiment,dead and mouldy eggs and later on dead larvae were counted and removed.
Larval Stage:At the beginning of the larval stage,200larvae were placed into each chamber with?ve replicates from the stock aquarium.After1,24,47, 72and96h exposure periods to eight di?erent concentrations of cypermethrin, dead larvae in experimental and control groups were counted.
Statistical analyses
For statistical analyses,the statistical software package SPSS(Version10.0, SPSS Inc.Chicago,Illinois,USA)was used.Data obtained from the delta-methrin acute toxicity tests were evaluated using the Probit Analysis Statistical Method.The LC50values(with95%con?dence limits)were calculated.The signi?cance level between the LC50values obtained and the di?erent exposure times were analysed using a chi-square test.
Results
In the embryonal stage,the number of dead embryos signi?cantly increased in response to cypermethrin concentrations0.0001,0.001,0.01,0.1,1,2,4and 8l g là1(p<0.05for each case,Table1).The incubation times of fertilised common carp eggs were determined as48h in the control and experimental groups.The48h LC50value(95%con?dence limits)of cypermethrin for common carp embryos was found to be0.909(0.256–5.074)l g là1.Cyper-methrin exposure resulted in a decrease in hatching success as follows,cyper-methrin concentrations0.0001,0.001,0.01,0.1,1,2,4and8l g là1led to hatching success values of87.4,85.0,80.2,71.4,56.3,48.6,38.8and23.5%, respectively.
In the larval stage,the number of dead larvae at certain cypermethrin doses was examined in relation to the duration(1,24,48,72and96h)of exposure (Table1).The lowest concentration of cypermethrin(0.0001l g là1)produced a signi?cant increase in the number of dead larvae compared with the control group(p<0.05).The number of dead larvae signi?cantly increased with increasing concentrations exposed for1to96h(p<0.05).There were signi?cant di?erences in number of dead larvae between the exposure times
T a b l e 1.A c u t e t o x i c i t y o f c y p e r m e t h r i n o n c o m m o n c a r p e m b r y o s a n d l a r v a e (n =1000f o r i n i t i a l e g g s a n d l a r v a e i n ?v e r e p l i c a t e s ).
C o n c e n t r a t i o n s (l g l à1)
E m b r y o n a l s t a g e
L a r v a l s t a g e
N u m b e r o f d e a d e m b r y o s
H a t c h i n g s u c c e s s (%)
N u m b e r o f d e a d l a r v a e
1h
24h 48h 72h
96h
0.000112687.47088971171860.00115085.0921121381952550.0119880.21391682052693270.128671.4211244290356413143756.3332358400453501251448.6390415459502547461238.84864915156121000876523.55755856301000N D C o n t r o l 4995.1
37556686119C h i -s q u a r e v a l u e 172.1665.3251.2541.4120.9120.50p <0.05<0.05<0.05<0.05<0.05<0.05L C 50v a l u e w i t h 95%c o n ?d e n c e l i m i t s *0.909(0.256–5.074)
7.813a
(2.829–33.652)6.196b
(2.481–22.897)2.940c
(1.327–8.125)1.304d
(0.612–3.389)0.809e
(0.530–1.308)
*
L C 50v a l u e s w i t h t h e d i ?e r e n t s u p e r s c r i p t a r e s i g n i ?c a n t l y d i ?e r e n t (p <0.05).N D :N o d a t a b e c a u s e 100%m o r t a l i t y .
455
456
1–96h in each concentration(p<0.05).The highest concentration8l g là1 showed the highest larvae mortality.The1,24,48,and96h LC50values(with 95%con?dence limits)of cypermethrin for common carp larvae were esti-mated as7.813(2.829–33.652), 6.196(2.481–22.897), 2.940(1.327–8.125), 1.304(0.612–3.389)and0.809(0.530–1.308)l g là1,respectively.There were signi?cant di?erences in the LC50values obtained at di?erent exposure times (p<0.05).
In addition,the yolk-sac absorption time of larvae was determined as48h after hatching in the control and experimental groups.In the larval stage, feeding was initiated immediately after yolk sack absorption in the control and experimental groups.
Discussion
It has been observed that increasing cypermethrin concentrations had signi?-cant e?ects on hatching success.For example,when the embryos of C.carpio were exposed to0.0001l g là1cypermethrin,87.4%hatched whereas only 23.5%hatched when exposed to the8l g là1cypermethrin concentration.In addition,the96h LC50value of cypermethrin for common carp larvae were found to be0.809l g là1.
USDA National Agricultural Pesticide Impact Assessment Program’s document reports that cypermethrin causes acute toxicity in?sh in laboratory tests at an average range of1.8–8.2l g là1(US EPA1989).Shires(1985) exposed Salmo gairdneri to an emulsion concentrate formulation of cyper-methrin(WL85871),and2–5l g là1was found to be toxic to rainbow trout. Edwards et al.(1987)reported that0.25and0.17l g là1levels of cis-and trans-cypermethrin,respectively,were toxic signs for trout.They attributed the high toxicity of pyrethroids in?sh to a combination of three factors:a sensitive central nervous system,rather slow hydrolytic detoxi?cation,and the route of exposure(direct absorption via the gills into the bloodstream). Smith and Stratton(1986)compiled a list of toxic e?ects of cypermethrin on di?erent?sh species as follows(LC50):Salmo salar96h2l g là1;S.gairdneri 96h6l g là1;Gambussia a?nis24h9l g là1,and Cyprinodon macularius24h 10l g là1and48h6l g là1.Bradbury and Coats(1989)reported96h cyper-methrin toxicity(LC50)to C.carpio0.9–1.1l g là1;Salmo trutta1.2l g là1; S.gairdneri0.5l g là1;Scardinius erythropthalmus0.4l g là1,Tilapia nilotica 2.2l g là1.Reddy et al.(1991)calculated24h LC50value of cypermethrin for Tilapia mossambica as0.2l g là1.Polat et al.(2002)estimated a48h LC50 value for Poecilia reticulata at21.4l g là1.Examining cypermethrin toxicity in other aquatic organisms,the work of Clark et al.(1987)reported a96h LC50 value of cypermethrin for grass shrimp,Palaemonetes pugio at0.0016l g là1. The author concluded that exposure of aqueous organisms to pyrethroids may also secondarily induce an osmotic imbalance that contributes to their toxicity. Bradbury and Coats(1989)determined the cypermethrin’s acute toxicity96h
457 LC50value for Daphnia magna,a small freshwater crustacean,at0.0002l g là1. Our results are in agreement with the reports of Bradbury and Coats(1989)for C.carpio.
United States Environmental Protection Agency states cypermethrin’s bio-concentration factor was444·for whole?sh using14C-cyclopropyl labeled cypermethrin and468·using14C-benzyl labeled cypermethrin(US EPA1989). Although under?eld conditions cypermethrin is considered to pose less risk due to its high adsorption to soil,these data should be considered when assessing potential ecosystem risks.In addition,Kumaragura and Beamish (1981)reported that acute toxicity of synthetic pyrethroids to?sh was nega-tively correlated to temperature.Therefore,the presence of pyrethroids in the aquatic environment when water temperatures are decreasing during coldwater ?sh spawning season,may increase the toxic impact on reproduction(Moore and Waring2001).
Physiological processes including neural control of hatching remain unclear. However,exposure of teleost embryos to neurotransmitter agonists and antagonists has suggested such a role.Schoots et al.(1983)reported that dopaminergic agonists increase the time of hatch and antagonists cause a decrease,whereas DiMichele and Taylor(1981)reported that epinephrine decreased average hatch times.In addition,they demonstrated that atropine,a muscarinic receptor antagonist(Katzung1992),inhibited hatching in Fundulus heteroclitus.More work is needed to understand the normal biology of the hatching process and how cypermethrin interferes with the development of the hatching gland.While hatching success was decreased by cypermethrin expo-sure,e?ects on the time to hatch were not part of our study.
We can conclude that cypermethrin contamination is dangerous to aquatic ecosystems,and this fact should be taken into consideration when this insec-ticide is used in agriculture or in the control of mosquito populations.In addition,potential risk from cypermethrin metabolities should be investigated to get a more complete picture in terms of toxicity.
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