Pesticide Biochemistry and Physiology 82 (2005) 220225 www.elsevier.

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Acute toxicity of diazinon on the common carp (Cyprinus carpio L.) embryos and larvae
Rahmi Aydn a,, Kenan Kprc b
a

University of Firat, College of Bingol, Fisheries Programme, Bingol, Turkey b University of Firat, Faculty of Fisheries, 23119 Elazig, Turkey Received 22 December 2004; accepted 14 March 2005 Available online 22 April 2005

Abstract In the present study, the toxic eVects on the embryos and larvae of the common carp were used as a model to investigate the organophosphorus pesticide, diazinon, which contaminates aquatic ecosystems. Data obtained from the diazinon acute toxicity tests were evaluated using the Probit Analysis Statistical Method. The control and six test experiments were repeated Wve times. The number of dead embryos signiWcantly increased in response to diazinon concentrations 0.25, 0.5, 1, 2, 4, and 8 mg L1 (p < 0.05 for each case). The 48 h LC50 value (with 95% conWdence limits) of diazinon for common carp embryos was estimated at 0.999 (0.6981.427) mg L1. Doseresponse decreases in hatching success were recorded as 84.60, 75.2, 54.1, 31.0, 6.0, and 0.0%, respectively (p < 0.05). The number of dead larvae signiWcantly increased with increasing diazinon concentrations exposed for 2496 h (p < 0.05). The 24, 48, 72, and 96 h LC50 values (with 95% conWdence limits) of diazinon for common carp larvae were estimated at 3.688 (2.4648.495), 2.903 (2.0195.433), 2.358 (1.6724.005), and 1.530 (1.0093.948) mg L1, respectively. There were signiWcant diVerences in the LC50 values obtained at diVerent exposure times (p < 0.05). The results of the study suggest that low levels (0.25 mg L1) of diazinon in the aquatic environment may have a signiWcant eVect on the reproduction and development of carp. 2005 Elsevier Inc. All rights reserved.
Keywords: Common carp; Embryo; Larvae; Acute toxicity; Hatching success; Organophosphorus pesticide; Diazinon

1. Introduction During the last decades, signiWcant amounts of pesticides belonging to the classes of organophos*

Corresponding author. E-mail address: raydin@Wrat.edu.tr (R. Aydn).

phates have been released into the environment. The organophosphorus pesticides were developed by chemical manipulation of nerve gases and further modiWcations have resulted in chemicals with greater species selectivity. Organophosphate compounds are useful as pesticides due to their ability to inhibit acetylcholinesterase, an enzyme

0048-3575/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2005.03.001

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responsible for inactivating the neurotransmitter acetylcholine [1,2]. Diazinon is a contact organophosphate pesticide and extensively used, both in agriculture and households to control insects in soil, plants, fruit and vegetable crops. Agricultural spray contains 8590% diazinon [3]. After its application on crops and plants, diazinon is easily washed into surface waters and enters the ground water. Eventually, it enters the aquatic environment in large quantities [49]. Diazinon degrades rapidly, but under conditions of low temperature, low moisture, high alkalinity, and lack of suitable microbiological degraders, it may remain biologically active in soils for six months or longer, Because of its aquatic distribution, diazinon aVects a wide range of non-target organisms, like invertebrates, mammals, birds, and Wshes, especially those inhabiting aquatic environment [7,10,11]. In Wshes, exposure to diazinon in sublethal doses is known to aVect the nervous system by inhibition of acetylcholinesterase activity [12]. Sublethal doses may lead to reduced growth and reproduction in aquatic invertebrates [10]. Acute toxicity tests of adult Wsh using diazinon have shown that 96 h sublethal values vary by several orders of magnitude between species [1315]. While experience shows that early life stages of Wshes are often the most sensitive to toxic eVects, little is known about the toxicity of diazinon to Wsh during these developmental stages. This is important since during development sensitivity may change with some compounds showing higher sensitivity in embryos [7,16] whereas others are more toxic to larvae [1719]. Takimoto et al. [20] reported that timing of exposure of killiWsh (Oryzias latipes) embryos to sublethal concentrations of the organophosphate fenitrothion resulted in signiWcantly diVerent degrees of mortality and hatching success. The present study was performed to determine acute toxicity of diazinon on the embryos and larvae of common carp (Cyprinus carpio). The common carp was selected for the bioassay experiments because of it 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.

2. Materials and methods 2.1. Adult Wsh and chemical supply Four- to Wve-year-old male and female common carp samples weighing 1.85.2 kg, total length 45.465.2 cm, were obtained from a Wsh hatchery unit of the State Hydraulic Works, Elazig, Turkey. The broodWsh were kept at 24 1 C [21], in 500 L Wberglass tanks and in natural light conditions. Basudin 60 EM, with the active molecule diazinon [0,0-diethyl-0-(2-isopropyl-6-methyl-4-pyrimidyl) phosphorothioate], purity 63% (dissolved in 80% acetone) was purchased from Chem Services (Elazig, Turkey). 2.2. Collection of gametes, artiWcial 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 [22] suspended in a 0.9% NaCl solution. The suspended carp pituitary was administered at a dose of 0.5 mg L1 of Wsh for males and 1.0 mg L1 of Wsh for females. The dry method of fertilization was used in this study [21]. Twenty-four hours after injection the gametes of both sexes were stripped into separate bowls, by applying gentle pressure on the inXated belly. Before the stripping, broodWsh were anaesthetized with benzocaine dissolved in water at the concentration of 1000 ppm for 15 min [23]. The stripped eggs and milt were then mixed in a bowl. Fertilization and degumming of eggs was performed using the modiWed Woynarovich method. Eggs were activated with a solution of 4 g NaCl and 3 g urea L1 and 5 min later they were transferred into a solution of 4 g NaCl and 20 g urea L1 [24]. Fertilised eggs were washed by the addition of water (24 C) for 5 min. The fertilised eggs were rinsed and transported from the hatchery unit to the reproduction laboratory of the Fisheries Faculty of Firat University within 30 min. These eggs were immediately placed in experimental units. Each experimental unit had Wve incubation chambers (each containing approximately 200 eggs) containing 2.5 L of medium. The levels of medium in both aquaria

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and chambers were kept in the same position to maintain the same temperature in each chamber. The experimental medium from the reservoir was pumped through the chambers to keep the eggs suspended. The water temperature was kept at 24 1 C and a 12L:12D photoperiod was provided for the experimental units. 2.3. Experimental treatment The experimental water was kept in the tank for 24 h before diazinon was added. Water quality characteristics of the reservoir in the experimental media were determined according to APHA [25]. The mean values for test water qualities were as follows: temperature 24 1 C, pH 7.1 0.2, dissolved oxygen 7.4 0.1 mg L1 and total hardness 125.1 2.2 mg L1. Six diVerent concentrations (0.25, 0.5, 1, 2, 4, and 8 mg L1) for embryonal and larval stages of common carp were obtained by the addition of diazinon, following preparation from a stock solution. The stock solutions were renewed every 12 h. The control group received acetone at the maximum acetone volume 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 (200 fertilized eggs) was assessed. Every 24 h, 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, 200 larvae were placed into each chamber with Wve replicates from the stock aquarium. After 24, 48, 72, and 96 h exposure periods to six diVerent concentrations of diazinon, dead larvae in experimental and control groups were counted. 2.4. Statistical analyses For statistical analyses, the statistical software package SPSS (Version 10.0, SPSS Chicago, Illinois, USA) was used. Data obtained from the deltamethrin acute toxicity tests were evaluated using the Probit Analysis Statistical Method. The LC50 values (with 95% conWdence limits) were calculated. The signiWcance level between the LC50 values obtained and the diVerent exposure times was analysed using a 2 test.

3. Results In the embryonal stage, the number of dead embryos signiWcantly increased in response to diazinon concentrations 0.25, 0.5, 1, 2, 4, and 8 mg L1 (p < 0.05 for each case, Table 1). The

Table 1 Acute toxicity of diazinon on common carp embryos and larvae (n D 1000 for initial eggs and larvae in Wve replicates) Concentrations (mg L1) Embryonal stage Number of dead embryos 0.25 0.5 1 2 4 8 Control
2 value p LC50 value with 95% conWdence limits

Larval stage Hatching success (%) 84.6 75.2 54.1 31.0 6.0 00.0 96.5 Number of dead larvae 24 h 21 38 94 232 586 1000 12 61.53 <0.05 3.688a (2.4648.495) 48 h 34 59 135 324 667 ND 24 57.65 <0.05 2.903b (2.0195.433) 72 h 46 91 185 389 738 ND 32 56.80 <0.05 2.358c (1.6724.005) 96 h 78 149 307 631 1000 ND 45 26.34 <0.05 1.530d (1.0093.948)

154 248 459 690 940 1000 35 62.38 <0.05 0.999 (0.6981.427)

ND, no data because of 100% mortality. LC50 values with diVerent superscripts are signiWcantly diVerent (p < 0.05).

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incubation times of fertilised common carp eggs were determined as 48 h in the control and experimental groups. The 48 h LC50 value (95% conWdence limits) of diazinon for common carp embryos was found to be 0.999 (0.698 1.427) mg L1. Diazinon exposure resulted in a decrease in hatching success, as follows diazinon concentrations 0.25, 0.5, 1, 2, 4, and 8 mg L1 lead to hatching success values as 84.6, 75.2, 54.1, 31.0, 6.0, and 00.0%, respectively. In the larval stage, the number of dead larvae at certain diazinon doses was examined in relation to the duration (24, 48, 72, and 96 h) of exposure (Table 1). The number of dead larvae signiWcantly increased with increasing concentrations exposed for 2496 h (p < 0.05). There were signiWcant diVerences in number of dead larvae between the exposure times 2496 h in each concentration (p < 0.05). The highest concentration of 8 mg L1 showed the highest larvae mortality. The 24, 48, 72, and 96 h LC50 values (with 95% conWdence limits) of diazinon for common carp larvae were estimated as 3.688 (2.4648.495), 2.903 (2.0195.433), 2.358 (1.6724.005), and 1.530 (1.0093.948) mg L1, respectively. There were signiWcant diVerences in the LC50 values obtained at diVerent exposure times (p < 0.05). In addition, the yolk-sac absorption time of larvae was determined as 48 h 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.

4. Discussion It has been observed that increasing diazinon concentrations had signiWcant eVects on hatching success. For example, when common carp (C. carpio) embryos were exposed to 0.25 mg L1 diazinon, 84.6% hatched whereas only 6.0% hatched when exposed to the 4 mg L1 diazinon concentration. In addition, the 48 h LC50 value of diazinon for common carp embryos were found to be 0.999 mg L1. Marty et al. [16] exposed three age groups of medaka (O. latipes) embryos to a 2 h pulse of Nnitroso-N-methylurea to deWne age dependent tox-

icity and reported increased hatching success when embryos were exposed at early organogenesis rather than at later embryonic stages. Hamm and Hinton [7] reported that early embryonic exposure in medaka was the greatest eVect on hatching success. For example, when embryos were exposed to 26 mg L1 diazinon from days 1 to 5, only 16% hatched whereas 100% hatched when exposed to the same concentration from days 5 to 9. Takimoto et al. [20] demonstrated that pulse exposure of medaka embryos to fenitrothion between days 0 and 3 was associated with a decrease in hatch which was quantitatively greater than that occurring in exposures conducted from days 3 to 6 or days 5 to 8. The question which now arises concerns the mechanism whereby diazinon exposure early in development can lead to decreased hatch. An important element in understanding the toxicity of diazinon is its requirement for bioactivation, the question of where and by which steps this reaction occurs is particularly important. Cantrell et al. [26] reported that piperonyl butoxide, a P450 inhibitor, blocked both DNA degradation and embryotoxicity. Here, it will be necessary to have detailed data on the time of appearance of cytochrome P450 and/or to determine whether the parent compound itself is interacting to inhibit hatching success. Moore and Wairing [27] and Wall [28] reported signiWcantly reduced levels of the reproductive steroids in zebraWsh (Danio rerio) and Atlantic salmon (Salmo salar) after sublethal doses of diazinon. Hatchability was observed to be 30 and 50% in eggs obtained from the mother Wsh exposed chronically to 2.6 and 1.3 mg L1 for 30 days, respectively [29]. Embryolarval toxicity assays with diazinon were performed on various Wsh species: Japanese Xounder, Paralichthys olivacens [30], rainbow trout, Oncorhynchus mykiss [31] and medaka, O. latipes [7]. Decreased total length of the larval body was a highly sensitive indicator of the diazinon eVect in the course of embryolarval development. 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. [32] reported that dopaminergic agonists increase the time of hatch and antagonists cause a

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decrease, whereas DiMichele and Taylor [33] reported that epinephrine decreased average time to hatch. In addition, DiMichele and Taylor [33] demonstrated that atropine, a muscarinic receptor antagonist [34], inhibited hatch in Fundulus heteroclitus. More work is needed to understand the normal biology of the hatching process and how diazinon interferes with the development of the hatching gland. While hatch success was decreased by diazinon exposure, eVects on the time to hatch were not seen. In this study, with increasing diazinon concentrations, the larvae exposed duration 2496 h signiWcantly increased the number of dead larvae (see Table 1). The diVerences in number of dead larvae between the durations 24 and 96 h in each concentration were found signiWcant (p < 0.05). In the present work, the 24, 48, 72, and 96 h LC50 values of diazinon for common carp, larvae were found as 3.688, 2.903, 2.358, and 1.530 mg L1, respectively and here we report diazinon to be highly toxic to common carp larvae. During development sensitivity may change with some compounds showing higher sensitivity in embryos whereas others are more toxic to larvae [17,18]. Marty et al. [16] found that early life stage of O. latipes was the most sensitive to toxic eVects. Takimoto et al. [20] also reported that timing of exposure of O. latipes embryos to sublethal concentrations of the organophosphate fenitrothion resulted in signiWcantly diVerent degrees of mortality and hatching success. Our results support earlier Wndings by Marty et al. [16] and Takimoto et al. [20]. There are diVerences in the acute toxicity of diazinon for various Wsh species. The 96 h LC50 values range in tenths to several tens of milligram per liters [30,35]. In European eel (Anguilla anguilla), the 96 h LC50 values range even in hundredths of milligrams per liters [36]. The 96 h LC50 values of diazinon was reported as 0.10.5 mg L1 for fry bluegill (Lepomis macrochirus), 0.88 mg L1 for American oyster (Crassostrea virginica), 1.47 mg L1 for sheepshead minnow (Cyprinodon variegates), 1.65 mg L1 for fry rainbow trout (O. mykiss), and 7.80 mg L1 for fathead minnow, Pimephales minnow [15]. The diVerent toxicity of diazinon may be demonstrated on the example of two Wsh species used for ecotoxicological assessment of

chemical substances. The 96 h LC50 values of diazinon for guppy (Poecilia reticulate) was found to be 0.8 mg L1 but for zebraWsh (Brachydanio rerio) it was found to be 8 mg L1 [37]. Oh et al. [38] present three factors causing the selective toxicity of diazinon for various Wsh species: diVerent inhibition of acetylcholinesterase, diVerent detoxiWcation and absorption. The results indicate that low levels of diazinon (0.25 mg L1) in the aquatic environment may have a signiWcant eVect on common carp populations. In addition, data presented herein on developmental sensitivity and toxicological endpoints may guide the development of experimental protocols employing species of concern.

References
[1] D.J. Ecobichon, R.M. Joy, Organophosphorus Ester Insecticides in Pesticides and Neurological Diseases, second ed., CRC Press, Boca Raton, FL, 1994 pp. 171249, Chapter 4. [2] D. Pesando, P. Huitorel, V. Dolcini, C. Angelini, P. Guidetti, C. Falugi, Biological targets of neurotoxic pesticides analysed by alteration of developmental events in the Mediterranean sea urchin, Paracentrotus lividus, Marine Environ. Res. 55 (2003) 3957. [3] URL 1. Available from: <http://www.atstr.ede.gov/ tfacts86.html>. [4] B.A. Ansari, K. Kumar, Diazinon toxicity on protein and nucleic acid metabolism in the liver of zebra Wsh, Brachydaniorerio (Cyprinidae), Sci. Total Environ. 76 (1988) 6368. [5] K.M. Kuivila, C.G. Foe, Concentrations, transport and biological eVects of dormant spray pesticides in the San Francisco Estuary, California, Environ. Toxicol. Chem. 14 (1995) 11411150. [6] M.J. Ferrari, S.W. Ator, J.D. Blomquist, J.E. Dysart, Pesticides in surface water of the Mid-Atlantic Region, U.S. Geological Survey Water-Resources Investigations Report 97974280, 1997, p. 20. [7] J.T. Hamm, D.E. Hinton, The role of development and duration of exposure to the embryotoxicity of diazinon, Aquat. Toxicol. 48 (2000) 403418. [8] N., Saglam, Aquaculture Legislation (in Turkish), Nobel Publication, Ankara, 2003, p. 281. [9] H.M. Dutta, H.J.M. Meijer, Sublethal eVects of diazinon on the structure of the testis of bluegill, Lepomis macrochirus: a microscopic analysis, Environ. Pollut. 125 (2003) 355360. [10] R. Eisler, Diazinon hazards to Wsh, wildlife, and invertebrates: a synoptic review. U.S. Fish and Wildlife Service, U.S. Dep. Int. Washington D.C. 85 (19) (1986) 138. [11] D.E. Burkepile, M.T. Moore, M.M. Holland, The susceptibility of Wve nontarget organisms to aqueous diazinon

R. Ayd n, K. Kprc / Pesticide Biochemistry and Physiology 82 (2005) 220225 exposure, Bull. Environ. Contam. Toxicol. 64 (2000) 114 121. H.M. Dutta, D. Arends, EVects of endosulfan on brain acetylcholinesterase activity in juvenile bluegill sunWsh, Environ. Res. 91 (2003) 157162. M.D. Ferrando, E. Sancho, E. Andreu-Moliner, Comparative acute toxicities of selected pesticides to Anguilla anguilla, J. Environ. Sci. Health B 26 (1991) 491498. M.K. Alam, O.E. Maughan, Acute toxicity of selected organophosphorus pesticides to Cyprinus carpio and Barillus vagra, J. Environ. Sci. Health B 28 (1993) 8189. OYce of Pesticide Programs, Pesticide Ecotoxicity Database (Formerly: Environmental EVects Database), Environmental Fate and EVects Division, U.S. EPA, Washington, DC, 2000. G.D. Marty, J.M. Nunez, D.J. Lauren, D.E. Hinton., Agedependent changes in toxicity to Japanese medaka (Oryzias latipes) embriyos, Aquat. Toxicol. 17 (1990) 4562. K. Fent, W. Meier, EVects of triphenyltin on Wsh early life stages, Arch. Environ. Contam. Toxicol. 27 (1994) 224231. M.P. Gaikowski, S.J. Hamilton, K.J. Buhl, S.F. McDonald, C.H. Summers, Acute toxicity of Wre Wghting chemical formulations to four life stages of fathead minnow, Ecotoxicol. Environ. Saf. 34 (1996) 252263. K. Kprc, R. Aydn, The toxic eVects of pyrethroid deltamethrin on the common carp (Cyprinus carpio L.) embryos and larvae, Pestic. Biochem. Physiol. 80 (1) (2004) 4753. Y. Takimoto, S. Hagino, H. Yamada, J. Miyamoto, The acute toxicity of fenitrothion to killiWsh (Oryzias latipes) at twelve diVerent stage of its life history, J. Pestic. Sci. 9 (1984) 463470. S. Rothbard, Z. Yaron, Carps cyprinidae, in: N.R. Bromage, R.J. Roberts (Eds.), Broodstock Management and Egg Larval Quality Blackwell Science, Printed and bound in Great Britain by the University Press, Cambridge, 1996, pp. 321352. H. Chaudhuri, Use of hormones in induced spawning of carps, J. Fish Res. Bd. Can. 33 (1976) 940947. J.T. Ferreira, H.J. Schoonbee, G.L. Smit, The uptake of the anaesthetic benzocaine-hydrochloride by the gills and skin of three freshwater Wsh species, J. Fish Biol. 25 (1984) 35 41. E. Woynarovich, A. Woynarovich, ModiWed technology for elimination of stickiness of common carp (Cyprinus carpio) eggs, Aquac. Hungaria 2 (1980) 1921.

225

[12]

[13]

[14]

[15]

[16]

[17] [18]

[19]

[20]

[21]

[22] [23]

[24]

[25] APHA, American Public Health Association, Standard Methods for the Examination of Water and Wastewater, ed. 16th., Washington, DC, 1985, p. 1268. [26] S.M. Cantrell, J. Joy-Schlezinger, J.J. Stegeman, D.E. Tillitt, M. Hannink, Correlation of 2,3,7,8-tetra-chlorodibenzo-pdioxin-induced apoptotic cell death in the embryonic vasculature with embryotoxicity, Toxicol. Appl. Pharmacol. 148 (1998) 2434. [27] A. Moore, C.P. Wairing, Sublethal eVects of the pesticide diazinon on olfactory function in mature male Atlantic salmon parr, J. Fish Biol. 48 (1996) 758775. [28] S.B. Wall, Sublethal eVects of cadmium and diazinon on reproduction and larval behaviour in zebraWsh, Diss. Abst. Inter. B. Sci. Eng. 60 (2000) 3829. [29] J. Iqbal, S.A. Mufti, EVect of diazinon on egg hatchability in a freshwater teleost, Colisa fasciata, Proc. Pak. Congress Zool. 11 (1992) 231238. [30] M.S. Menendez, A. Ishimatsu, Pesticide toxicity in the larvae of Japanese Xounder, Paralichthys olivaceus, Bull. Fac. Fish. Nagasaki Univ. Chodai Suikenpo. 74/75 (1993) 3136. [31] M. Kikuchi, T. Miyagaki, M. Wakabayashi, Evaluation of pesticides used in golf links by acute toxicity in test on rainbow trout, Nippon Suisan Gakkaishi. 62 (1996) 414419. [32] A.F.M. Schoots, R.J. Meijer, J.M. Denuce, Dopaminergic regulation of hatching in Wsh embryos, Dev. Biol. 100 (1983) 5963. [33] L. DiMichele, M.H. Taylor, The mechanism of hatching in fundulus heteroclitus: development and physiology, J. Exp. Zool. 217 (1981) 7379. [34] B.G. Katzung, Basic and Clinical Pharmacology, Wfth ed., Appleton and Lange, Connecticut, 1992 p. 1017. [35] J.M. Giddings, R.C. Biever, M.F. Annunziato, A.J. Hosmer, EVects of diazinon on large outdoor pond microcosmes, Environ. Toxicol. Chem. 15 (1996) 618629. [36] E. Sancho, M.D. Ferrando, E. Andreau, M. Gamon, Bioconcentration and excretion of diazinon by eel, Bull. Environ. Contam. Toxicol. 50 (1993) 578585. [37] J. Keizer, G. DeAgostino, I. Vittozzi, The importance of biotransformation in the toxicity of xenobiotics to Wsh. 1: toxicity and bioaccumulation of diazinon in guppy (Poecilia reticulata) and zebra Wsh (Brachydanio rerio), Aquat. Toxicol. 21 (1991) 239254. [38] H.S. Oh, S.K. Lee, Y.H. Kim, J.K. Roh, Mechanism of selective toxicity of diazinon to killiWsh (Oryzias latipes) and loach (Misgurnus anguillicaudatus), Aquat. Toxicol. Risk Assess. 14 (1991) 343353.