Effects of diazinon on female Chalcalburnus chalcoides Cyprinidae) serum estradiol levels

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LC-50 values for DZN vary widely and depend upon organism age, weight, gender, and climatic conditions. Sub-lethal doses may lead to (1) reduced growth and reproduction in aquatic invertebrates, (2) decreased emergence in stream insects, (3) interference with algae–invertebrate interactions, (4) reduced egg-production, (5) decreased food-intake and body weight loss in birds, (6) offspring with altered brain pathology, (7) delayed sexual maturity and (8) adverse behavioral modifications in mammals [3]. In fish, exposure to DZN in sub-lethal doses is known to affect the nervous system by inhibition of AChE activity [5,6], to produce gill abnormalities [7] and diminished olfactory functioning [8]. Further DZN was found to alter reproductive behavior [8] and disrupt ovarian structure [9] in fish. DZN is readily hydrolyzed and converted into tetraethyl monothiopyrophosphate in water and this metabolite is more toxic [10]. Lab and field studies provided evidence to suggest that reproductive hazards due to pesticide exposure are affecting wildlife survival [11]. Recent studies confirmed that a large number of xenobiotic chemicals intreract and modify the vertebrate endocrine system [12–14]. Data showed that these compounds act in several ways, but most seem to act as estrogens or antiandrogens [12,13,2]. These contaminants are capable of acting as hormonal mimics and may modify the development of the reproductive system permanently [15]. Several studies demonstrated reproductive and developmental abnormalities in various taxa of organisms such as birds, reptiles, fish, and invertebrates which raised concerns for susceptibility of wildlife to endocrine disruptions in or via the aquatic environment [16]. Therefore, studies were conducted to determine the influence of DZN on blood estradiol levels in order to establish whether this compound exerts an estrogenic action on fish.

Materials and methods Fish characteristics Adult female white anchovy Chalcalburnus chalcoides (Cyprinidae), were obtained from Anzali Pond near the Caspian Sea, Iran in spring, using beach seine and identified using available references. White anchovy is distributed in Asia and southeastern Europe. Fish mature in 2–3 years, and the egg laying date is based on water temperature from April to June. Fish with an average weight of 30 g and an average length of 15 cm were maintained in 200-gallon fiberglass aquaria with dechlorinated city water under lab conditions for a period of 1 week. Vitality experience and lethality Commercial DZN produced by Golsam (Gorgan) was obtained for use in this study. DZN lethality for white anchovy was taken as 60 mg L
1 based on OECD (1992) [17] TCR guidelines. Determination of water quality Water quality characteristics were determined. Alkalinity and total water hardness were measured with a digital titrator and a Hach’s water analysis kit. The mean values for test water qualities were as follows: temperature 19.5  1 C, pH 7.9  0.18, dissolved oxygen 8.3 mg L
1 and total hardness was 326 mg L
1.

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Effect of diazinon on estradiol Fish exposure to DZN

After a 1 week acclimation period 20 fish per group were exposed to five treatments as follows: 1 (control), and DZN at 10, 20, 40 and 60 mg L
1. Each treatment was repeated for 24, 48, 72, 96 and 168 h (1 week) [17].

Bleeding and radioimmunoassay (RIA) testing All treated and control fish were bled, blood was allowed to clot at room temperature and subsequently serum was obtained after centrifugation. Serum estradiol levels were measured using a radioimmunoassay (RIA) testing (Spectria Estradiol RIA Kit).

Data analysis Data were analyzed using the SPSS for windows release 10.0 Statistical Package for Social Scientists consisting of means and standard deviations (M  SD). One way ANOVA was followed by Tukey’s test for significant differences (P50.05).

Results Data in Table I show the estradiol levels in fish following exposure to varying amounts of DZN for various periods. Treatment with DZN at 10 mg L
1 significantly decreased serum estradiol levels at all time periods studied. As the concentration of DZN was increased the reduction in serum estradiol levels was greater with maximal decreases noted at 60 mg L
1. However at 60 mg L
1 time was not a factor as the fall in estradiol levels at 24 h was similar to the changes noted at 168 h. Hence exposure duration did not seem to be a factor in DZN-induced estradiol level reduction in fish. Figure 1(a–e) shows serum estradiol data plotted in response of female C. chalcoides to five different DZN concentrations. Blood serum estradiol levels were highest in the control groups. For all fish exposures means were less than control. Results also showed that there were significant differences between groups for serum estradiol levels in female C. chalcoides to five different DZN concentrations. The greatest effects noted were at 60 mg L
1 at 24 or 168 h. Table I. Influence of DZN on serum estradiol levels. Ng mL
1 fish blood estradiol level under different exposure period (h) 24

48

72

96

168

Diazinon (mg L
1)

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Control 0 10 20 40 60

97.4 72.3* 55.3* 52.0* 16.0*

1.11 7.74 1.01 0.77 0.75

92.3 68.6* 81.8* 71* 32.9*

1.19 1.02 4.64 10.02 0.7

101.1 84.5* 49.0* 35.1* 53.1*

0.7 1.68 3.16 14.92 2.12

97.7 70.9* 21.3* 20.9* 34.5*

0.7 5.94 1.18 5.94 3.72

88.2 59.0* 56.6* 18.5* 15.0*

0.74 0.77 6.66 3.51 4.01

Data are given as mean þ SD. *Significant from control p50.05.

M. Malek-Hoseini et al.

(a) 120

100

(b)

100 5

90 1 80

80 T48

T24

70 60

60 50

40 40 20

30 20

0 Control

C10

C20

C40

Control

C60

C10

C20

C40

C60

C

C (c) 120

(d)

120

100

100

80

80 T96

T72

60

60 40

40

20

20

0

0

Control Control

C10

C20

C40

C10

C60

C20

C40

C60

C

C (e) 100

80

60 tweek

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40

20

0 Control

C10

C20

C40

C60

C

Figure 1. (a–e) Blood estradiol levels in female C. chalcoides exposed to five different DZN concentrations for either, (a) 24, (b) 48, (c) 72, (d) 96 and (e) 168 h (horizontal axis is DZN concentration and vertical axis is ng mL
1 fish blood estradiol level).

Disscussion Results showed that using a sublethal dose of DZN (60 mg L
1) altered serum estradiol levels in C. chalcoides. Similar to the effect of other organophosphates [18], it is conceivable

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Effect of diazinon on estradiol

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that the production of gonadotropin I (GtH I) and/or gonadotropin II (GtH II) may have been affected by DZN in C. chalcoides through a disruption of the hypothalamicpituitary-gonadal axis. GtH I is involved in the process of gametogenesis and GtH II is involved in the final follicular maturation process. Histopathological studies of [9] showed damage occurred at an early stage of follicular development. In this study DZN exerted an effect as early as 24 h, thus inhibiting production of estrogen needed for completion of gametogenesis process. In Table I, means serum estradiol levels following 24 h DZN exposure were less than at 48 h of exposure for groups. This may be attributed to an initial stress insult followed by a recovery period or adaptation. It is also possible that a few healthy follicles within the ovary were available to produce more estrogen to compensate for the damaged follicles. Following 72 h exposure, estradiol levels continued to decline in 20 and 40 DZN groups, which can be correlated to ovarian structural damage (Figure 1(c)). Estradiol levels continued to decline at 96 h at all DZN concentrations (Figure 1(d)) which may be associated with breakdown of the ovigerous lamellae. Destruction of the ovigerous lamellae prevents adequate distribution of estradiol into the blood stream, and thus, a decrease in serum estradiol levels is expected. It is possible that both damaged follicular structures and inadquate blood supply are responsible for the inability of the ovarian follicles to fully recover from DZN-induced exposure. After 1 week of DZN exposure, an increase in serum estradiol level occurred in 20 mg L
1 group, but all other groups had reductions (Table I), which cannot be explained. This study showed that DZN altered the production of estradiol. The occurrence of endocrine disruption as evidenced by the level of estradiol production and damaged ovarian structures, may result in decreased fertility and egg production in female white anchovy C. chalcoides.

Acknowledgments We wish to thank Dr Khanipoor (Head of Guilan Fishery Research Center). We would also like to thank of Dr Mohajerani and Mr Maleki for their help in the Pathology Lab.

References 1. Dutta HM, Meijer HJM. Sublethal effects of Diazinon on the structure of the testis of bluegill, Lepomis macrochirus: A microscopic analysis. Environ. Pollut. 2003;125:355–360. 2. Maxwell BL, Dutta HM. Diazinon-induced endocrine disruption in bluegill sunfish, Lepomis macrochris. Exotoxicol. Environ. Safety 2005;60:21–27. 3. Eisler R. Diazinon hazards to fish, wildlife and invertebrates: A synoptic report, USDOI Fish and Wildlife Service. U. S. Dep. Int. Was. DC. Biological Report 85 (1.9) 1986;1–38. 4. Burkepile DE, Moore MT, Holland MM. The susceptibility of five nontarget organisms to aqueous diazinon exposure. Bull. Environ. Contam. Toxicol. 2000;64:114–121. 5. Pan G, Dutta HM. The inhibition of brain acetylcholinesterase activity of juvenile largemouth bass Micropterus salmoides by sublethal concentrations of diazinon. Environ. Res. A 1998;79:133–137. 6. Dutta HM, Arends D. Effects of endosulfan on brain acetylcholinesterase activity in juvenile bluegill sunfish. Environ. Res. 2003;91:157–162. 7. Dutta HM, Qadri N, Ojha J, Singh NK, Adhikari S, Datta Munshi JS, Roy PK. Effect of diazinon on macrophages of bluegill sunfish, Lepomi macrochrius: A cytochemical evaluation. Bull. Environ. Contam. Toxicol. 1997;58:134–141. 8. Moore A, Waring CP. Sublethal effects of the pesticide diazinon on olfactory function in mature male Atlantic salmon parr. J. Fish. Biol. 1996;48:758–775. 9. Dutta HM, Maxwell L. Histological examination of sublethal effects of diazinon on ovary of bluegill, Lepomis macrochirus. Environ. Pollut. 2003;121:95–102.

Downloaded By: [Agricultural Research and Education Organization (AREO)] At: 11:27 26 July 2008

112

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10. ATSDR. ToxFAQS: Diazinon. Available from http://www.atstr.edc.gov/tfacts86.html. 2002. 11. Moore A, Waring CP. The effects of synthetic pyrethroid pesticide on some aspects of reproduction in Atlantic salmon. Aquat. Toxicol. 2001;52:1–12. 12. DeRosa C, Richter P, Pohl H, Jones DE. Environmental exposures that affect the endocrine system: Public health implications. J. Toxicol. Environ. Health B 1998;1:3–26. 13. Roy D, Palangat M, Chen C-W, Thomas RD, Colerangle J, Atkinson A, Yan Z-J. Biochemical and molecular changes at the cellular level in response to exposure to environmental estrogen-like chemicals. J. Toxicol. Environ. Health 1997;50:1–29. 14. Choi SM, Yoo SD, Lee BM. Toxicological characteristics of endocrine-disrupting chemicals: Developmental toxicity, carcinogenicity and mutagenicity. J. Toxicol. Environ. Health 2004;7:1–24. 15. Guillette L, Guillette E. Environmental contaminants and reproductive abnormalities in wildlife implications for public health? Toxicol. Ind. Health 1996;12:537–550. 16. Campbell P, Hutchinson T. Wildlife and endocrine disruptors: Requirements for hazard identification. Environ. Toxicol. Chem. 1998;17:127–135. 17. OECD (Organization Economic Cooperation and Development), TCR, Guidelines for testing chemicals, No. 203 and 204. Paris: OECD; 1992. 18. Khan IA, Thomas P. Disruption of neuroendocrine control of luteinizing hormone secretion by Aroclar 1254 involves inhibition of hypothalamic tryptophan hydroxylase activity. Biol. Reprod. 2001;64:955–964.