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Toxicology Letters 138 (2003) 253 /260 www.elsevier.

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Assessment of reproductive and fertility effects of amitraz pesticide in male mice


Rawdah K. Al-Thani a,*, Aisha S. Al-Thani a, Ahmed Elbetieha b,1, Homa Darmani b
b

Department of Biological Science, University of Qatar, P.O. Box 2713, Doha, Qatar Department of Applied Biology, Faculty of Science, Jordan University of Science and Technology, Irbid, Jordan Received 1 July 2002; received in revised form 30 October 2002; accepted 4 November 2002

Abstract Forty adult male Swiss mice were exposed to tap water containing 0, 40, 80, or 160 ppm amitraz for 12 weeks. Based on fluid consumption the mice received an average of 0, 5.429/0.47, 10.569/0.97, and 20.399/2.17 mg/kg/day amitraz, respectively. The average body weights gains and fluid consumption were significantly decreased in males exposed to amitraz pesticide. Fertility was significantly reduced in male mice ingesting 10.569/0.97 or 20.399/2.17 mg/kg/day amitraz in that the number of females impregnated by them was significantly reduced. The number of viable fetuses was significantly reduced in females mated with males that ingested 10.569/0.97 or 20.399/2.17 mg/kg/day amitraz. A significant increase in the total number of resorptions and the number of females with resorptions was observed in females impregnated with the exposed males. Absolute testis weight was significantly decreased at 10.569/0.97 mg/kg concentration. The weight of the epididymis was decreased in test males ingested 20.399/2.17 mg/kg amitraz. The seminal vesicles weights were significantly increased in male mice ingested 10.569/0.97 or 20.399/2.17 mg/kg/day amitraz. Similarly, the preputial gland weights were increased in males that ingested 5.429/0.47 or 10.569/0.97 mg/kg and decreased in males ingested 20.399/2.17 mg/kg amitraz. Testicular sperm counts and daily sperm production were significantly decreased in males that ingested 10.569/0.97 or 20.399/2.17 mg/kg/day amitraz. Epididymal sperm counts were significantly decreased in exposed males at 10.569/0.97 or 20.399/2.17 mg/kg amitraz. These results strongly suggest that exposure to amitraz pesticide have an adverse effect on the fertility and reproductive system of male mice. # 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Amitraz; Fertility; Male mice

1. Introduction
* Corresponding author. Tel.: '/974-485-2709; fax: '/9744800-786. E-mail address: dr_althani99@hotmail.com (R.K. AlThani). 1 Present address: Department of Biological Science, University of Qatar, P.O. Box 2713, Doha, Qatar (Sabbatical Leave).

Amitraz is a trade name of triazapentadiene compound (N ,N ?-[(methylimino) dimethylidyne]di-2,4-xylidine), a member of the formamidine class chemical family (The Agrochemicals Handbook, 1994). It is an insecticide and acaricide used

0378-4274/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 3 7 8 - 4 2 7 4 ( 0 2 ) 0 0 4 1 8 - 6

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to control red spider mites, leaf miners, scale insects and aphids. On cotton it is used to control bollworms, white fly and leaf worms. On animals it is used to control ticks, mites, lice and other animal pests (Thomson, 1993; Budavari, 1989). The US Environmental Protection Agency classifies amitraz as class III-slightly toxic (U.S. EPA, 1987). The oral LD50 for mice is greater than 1600 mg/kg. Long term feeding studies show that amitraz is not carcinogenic in rats. However, it can cause tumors in female mice (Hayes and Laws, 1991). At high doses, amitraz can reduce the function of the hypothalamus (U.S. EPA, 1987). Animal studies have shown that amitraz may have serious effects on fertility and reproduction. Female mice treated orally for 5 days with 50 mg/kg/ day of amitraz and then mated showed a slight increase in loss of fetuses and a decrease in the number of living offspring (Hayes and Laws, 1991). In another study female mice who received 400 mg/kg/day of amitraz in their diet for up to 30 weeks, showed a significant increase in the time they were sexually receptive (Hayes and Laws, 1991). Following acute exposure in female rats, both amitraz and chlordimeform (CDF), another formamidine, have been found to block the luteinizing hormone (LH) surge and thus delay or block ovulation (Goldman et al., 1991; Cooper et al., 1999), most likely as a consequence of their activity as a-noradrenergic antagonists. Furthermore, in the male mice CDF was observed to decrease circulating LH and testosterone concentrations and to dramatically suppress the release in vitro of gonadotropin releasing hormone from hypothalamic tissue (Goldman et al., 1990). However the effects of acute dosing didnt persist, and serum hormonal levels in the male returned to normal within 4 days. In addition yohimbine, an a 2-adrenergic antagonist, has been found to attenuate the delayed lethality induced by amitraz (Moser and Macphail, 1985). Furthermore, females rabbit that received 25 mg/kg/day of amitraz from days 6 /18 of pregnancy had fewer and smaller litters (Meister, 1994). Amitraz has been shown to be teratogenic in laboratory animals. In one study, rats treated with 12 mg/kg/day of amitraz from days 8/20 of pregnancy, the offspring were heavier but had

less bone development than the offspring of untreated rats (Hayes and Laws, 1991). However, an EPA study indicates that the highest dose at which amitraz has no observable effect on test rats offspring (teratogenic NOEL) is 12 mg/kg/day. Recently, the teratogenic effect of amitraz has been demonstrated in the developing frog Xenopus laevis , embryos (Osano et al., 2002). Amitraz is broken down rapidly in soil containing oxygen. The half-life in soil is less than 1 day. Degradation occurs more rapidly in acidic soils than in alkaline or neutral soils (Kidd and James, 1991). Available data suggest that amitraz, following absorption into the blood, is not readily absorbed into tissues, and is mostly excreted unchanged via the urine (Kidd and James, 1991; U.S. EPA, 1987; Hayes and Laws, 1991). Although pesticides like amitraz may be valuable in agriculture, many pesticides or their breakdown products can be found in trace amounts or higher levels in air, soil and water. Environmental exposure to these agents may cause serious health risks including infertility. The objective of this study is to further evaluate the effect of amitraz pesticide on fertility of male mice.

2. Materials and methods 2.1. Animals Forty adult male Swiss mice, at day 60 of age, weighing approximately 32 g were used in this study. They were raised in the animal house unit in the Faculty of Medicine at Jordan University of Science and Technology under controlled temperature of 219/1 8C in 12 h light, 12 h darkness schedule (lights on 06.00 /18.00 h). Food (manufactured by the Faculty of Veterinary Medicine at Jordan University of Science and Technology, Irbid, Jordan, according to standard recipes) and water were offered ad libitum. 2.2. Administration of amitraz pesticide Amitraz pesticide in the form of emulsifiable concentrates (200 g/l) was purchased from the

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Veterinary and Agricultural Products Co. Ltd (VAPCO), (Amman, Jordan) and was diluted with tap water on daily bases to the appropriate concentrations of 40 ppm (1/40 of the LD50), 80 ppm (1/20 of the LD50) and 160 ppm (1/10 of the LD50). Male mice were randomly assigned into groups of 10 animals each and allowed ad libitum access to tap water containing amitraz. Control male mice were given tap water only. Based on fluid consumption the mice exposed to 0, 40, 80, and 160 ppm amitraz received an average dose of 0, 5.429/0.47, 10.569/0.97, and 20.399/2.17 mg/kg/ day amitraz, respectively. Mice oral LD50 for amitraz is around 1600 mg/kg (Meister, 1994).

Procedures employed in the use and sacrifice of mice were in accordance to the NIH Guide for the Care and Use of Laboratory Animals. 2.4. Evaluation of reproductive organs weights Amitraz exposed and control males were sacrificed after 12 weeks of amitraz ingestion and the 10 days period of mating. The following organs were excised and weighed: paired testes, seminal vesicles (stripped of seminal fluid), epididymis and preputial glands. 2.5. Testicular and epididymal sperm counts

2.3. Fertility test Animals were observed daily from the first day of exposure to amitraz for clinical signs of toxicity. Their water consumption was measured every day and the body weights every week. Fertility was estimated in adult male mice exposed to 0, 40, 80 and 160 ppm amitraz for 12 weeks. After 12 weeks of amitraz ingestion, dosing was discontinued and each treated male was placed in an individual cage with two virgin untreated females of the same strain. They were left together for 10 days during which two estrus cycles should have elapsed (Rugh, 1968). Adult male mice ingested amitraz as well as the control males, were then removed and sacrificed for further evaluations. Ten days later, the mated females were killed by cervical dislocation under light ether anesthesia and the following measurements were recorded: number of pregnant females, number of implantation sites, number of viable fetuses, number of resorptions, and number of females with resorptions. It is noteworthy to point out that 3 animals out of 10 and 1 out of 10 were died during the third week of exposure to 160 and 80 ppm amitraz, respectively, most likely due to amitraz toxicity. Other sign of amitraz toxicity observed in this study was an increase in aggressive behavior, as evidenced by fighting and resultant cutaneous lesions, among males treated at 160 ppm. Food consumption did not differ significantly between the control and treated groups (data are not shown).

The excised left testis and epididymis were weighed. Testis from each mouse was placed in 10 ml of normal saline (0.9% sodium chloride) and refrigerated for later homogenization for spermatid count. Epididymis was placed in 10 ml of normal saline (0.9% sodium chloride) and refrigerated for later homogenization for epididymal sperm count. Sperm count was performed according to the method of Amman and Lambiase (1969). Briefly, the excised left testis or epididymis from each mouse was sectioned by a disposable blade in 4 ml of normal saline in a conical glass petri dish, and then minced using a manual glass homogenizer. The homogenate was mixed using a vortex mixer and the number of sperm measured using a hemocytometer. Epididymal sperm counts were expressed as number of sperms per mg of epididymis. Testicular spermatid counts were expressed as the number of spermatids per mg of testis. The estimate of daily sperm production (DSP) per testis per day was calculated based on a factor of 4.84 as described by Vom Saal et al. (1998). 2.6. Statistical analysis Data are expressed as mean9/S.D. Differences between control and test groups were analyzed using either Students t-test or Fishers exact test. A P -value less than 0.05 was considered significant.

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3. Results 3.1. Effect of amitraz on body weight gain and uid consumption Table 1 presents the effects on body weight and fluid consumption of male mice exposed to 0, 40, 80, and 160 ppm amitraz. Body weight gain was reduced in males that ingested an average of 10.569/0.97 (P B/0.05) or 20.399/2.17 mg/kg (P B/0.001) amitraz. Fluid consumption decreased in males that ingested 5.429/0.47 (P B/0.001) and 10.569/0.97 (P B/0.0001) or 20.399/2.17 mg/kg amitraz. 3.2. Effect of amitraz on fertility The results presented in Table 2 demonstrate the adverse effect of amitraz on male mice fertility. The number of pregnancies in females mated with the test males was reduced in males that ingested 10.569/0.97 (P B/0.01) or 20.399/2.17 mg/kg amitraz. There were no significant differences in the number of implantations in females impregnated with either control or test male mice. However, the number of viable fetuses was reduced in females mated with males that ingested 10.569/0.97 (P B/ 0.05) or 20.399/2.17 mg/kg (P B/0.005) amitraz. Likewise, the total number of resorptions per group increased in females exposed to all three concentrations of amitraz (P B/0.005). The number of females with resorptions increased when mated with males that ingested 5.429/0.47 or 20.399/2.17 mg/kg amitraz (P B/0.01).

3.3. Effects of amitraz on weights of reproductive organs The data presented in Table 3 show the effect of exposure to amitraz on weights of some male reproductive organs. Absolute testis weight was decreased in males that ingested 10.569/0.97 mg/ kg amitraz (P B/0.01). Epididymal weights were decreased in males that ingested 20.399/2.17 mg/ kg amitraz (P B/0.005). Absolute weight of seminal vesicles was increased in males that ingested 10.569/0.97 (P B/0.005) or 20.399/2.17 mg/kg (P B/0.05) amitraz. The relative weights of seminal vesicles were also increased in males that ingested 10.569/0.97 or 20.399/2.17 mg/kg amitraz (P B/0. 005). Absolute preputial gland weight was decreased in males that ingested 20.399/2.17 mg/kg amitraz (P B/0.05). However, the relative weights of preputial glands were increased in males that ingested 5.429/0.47 (P B/0.01) or 10.569/0.97 mg/ kg (P B/0.005) amitraz. 3.4. Effect of amitraz on testicular and epididymal sperm counts Table 4 presents testicular and epididymal sperm counts and DSP of male mice in the control and the test groups. Testicular sperm counts were decreased in males that ingested 20.399/2.17 mg/ kg (P B/0.01) amitraz. Likewise, Epididymal sperm counts were decreased in males that ingested any of the two higher concentrations of amitraz (P B/0.01). The DSP was decreased in males that ingested 10.569/0.97 (P B/0.005) or 20.399/2.17 mg/kg (P B/0.0001) amitraz.

Table 1 Effect of 12 weeks ingestion of amitraz via drinking water on body weight gain and average water consumption of adult male mice Treatment Control (tap water) Amitraz (40 ppm) Amitraz (80 ppm) Amitraz (160 ppm)
a

Body weight (g)a 35.359/2.33 33.479/2.14 33.269/1.46** 31.969/1.70*

Fluid consumption (ml)a 5.659/0.49 4.509/0.38* 4.349/0.42*** 4.009/0.46***

Actual dose consumption (mg/kg/d)a / 5.429/0.47 10.569/0.97 20.399/2.17

Results are expressed as mean9/S.D. * P B/0.001. ** P B/0.05. *** P B/0.0001 (Students t -test).

R.K. Al-Thani et al. / Toxicology Letters 138 (2003) 253 /260 Total no. of resorptions/total no. of No. (%) of animals with implantation site resorptions

257

4. Discussion The present study was conducted to evaluate any adverse effects of amitraz pesticide on fertility and reproductive system of male mice. Amitraz pesticide was chosen because it is widely used in the state of Qatar as well as in many parts of the world. The animal model used in this study was used previously to assess the adverse effects of pesticides on fertility and reproduction in laboratory animals (Costa et al., 1989). The results presented in this study clearly demonstrate that ingestion of amitraz for 12 weeks caused a significant decrease in the average body weight gain in the test males (Table 1). This reduction in body weight gain is a clear indication of general toxicity. This magnitude of toxicity might have affected the animals indirectly rather than having any specific effect on fertility. However, it has been shown that the reproductive system of male mice was relatively resistant to body weight decrease down to even 70% of the body weight of control animals (Chapin et al., 1993). Several reproductive parameters were adversely affected after ingestion of amitraz by adult male mice. The pregnancy rate was significantly reduced in females impregnated by the test males, which could be related to the significant reduction in sperm count and/or to poor quality of the semen. Furthermore, it cannot be excluded that the pesticide could have acted directly on the testes and influenced the androgen biosynthesis pathway. In addition, an agent acting directly on the brain, hypothalamus, or anterior pituitary gland will indirectly affect the testes and possibly affect sexual activity (Amman, 1982). There is increasing evidence suggesting that the effects of amitraz in mammals are mediated by its interaction with a 2adrenoceptors (Costa et al., 1989), and of neurotoxicity due to inhibition of monoamine oxidase (Moser and Macphail, 1985). These mechanisms might relate to inhibition of LH (Goldman et al., 1991). Thus, if amitraz is inhibiting spermatogenesis, and doing so by inhibiting LH, then it would be expected that testosterone would be lower. However, if this is the case, it is hard to explain the increase in the seminal vesicle weights, which is testosterone dependent. In contrast to our results,

9/16*** (56.3)

13/126**

2/150

No. (%) of pregnant No. of implantation No. of viable females sitesa fetusesa

Table 2 Effect of 12 weeks ingestion of amitraz via drinking water on fertility of adult male mice

7.509/1.67

8.069/2.26

6.339/1.61 12/18*** (61.1)

16/20 (80.0)

8/14*** (57.1)

20/20 (100.0)

6.509/1.51

5.389/1.19' '

5.589/1.93'

7.409/1.70

6.819/2.54

9/52****

9/76**

5/8*** (62.5)

2/20 (10.0)

5/12 (41.7)

Treatment No. of males

Control 10 (tap water) Amitraz 10 (40 ppm) Amitraz 9 (80 ppm) Amitraz 7 (160 ppm)

Results are expressed as mean9/S.D. P B/0.05 (Students t -test). '' P B/0.005 (Students t -test). ** P B/0.005 (Fishers exact test). *** P B/0.01 (Fishers exact test).
' /
/ /

No. of mated females

20

20

18

14

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Table 3 Effect of 12 weeks ingestion of amitraz via drinking water on weights of reproductive organs of adult male mice Treatment group Control (tap water) Amitraz (40 ppm) Amitraz (80 ppm) Amitraz (160 ppm)
a b

No. of males 10 10 9 7

Absolute testes weight (g)a (mg/10 g B.wt.)b 0.209/0.01 (54.249/4.26) 0.199/0.01 (55.669/3.39) 0.189/0.01** (53.969/3.46) 0.209/0.04 (61.479/9.95)

Epididymis weight (mg)a 32.319/1.66 31.889/1.46 31.759/1.45 29.809/0.93***

Absolute seminal vesicles weight (g)a (mg/10 g B.wt.)b 0.139/0.02 (35.129/5.59) 0.139/0.02 (37.509/5.29) 0.229/0.02*** (67.609/7.50)*** 0.169/0.02* (49.369/7.61)***

Absolute preputial gland weight (g)a (mg/10 g B.wt.)b 0.109/0.01 (27.799/1.66) 0.119/0.01 (31.799/3.71)** 0.119/0.01 (34.069/4.45)*** 0.079/0.03* (22.519/6.45)

Results are expressed as mean9/S.D. Relative weights. * P B/0.05. ** P B/0.01.

it has been found that single or multiple doses of CDF in the male rats had minimal effects on gonadal function. Following two injections of CDF (50 mg/kg), serum LH and testosterone were significantly lower than vehicle treated controls. (Goldman et al., 1990). After 4 days of repeated CDF exposure at the same concentration. Both hormones returned to control values (Laws et al., 1991). The significant increase in the number of resorptions in females mated with test males may be attributed to an increase in periimplantation mortality of unhealthy fertilized ova due to alterations in sperm quality */a matter that needs further investigation. The increase in the number of resorptions in females impregnated with the test males observed in this study is in agreement with the results obtained by Hayes and Laws (1991).

They showed that when male mice were given 50 mg/kg/day of amitraz orally for 5 days and then mated, the resulting embryos were significantly less likely to grow in the mothers uterus. In contrast, Rehnberg and Cooper (1993) exposed male rats to CDF for 1, 7, 14, or 21 days. At each test interval, sexual activity was impaired (i.e., longer latency to first mount, first mount with intromission, and firs ejaculation) when measured within 1 h of treatment. However this treatment was transient, since the CDF-treated males all produced sperm-positive smears when housed with a proestrous female overnight. Furthermore, the cohabited females all became pregnant, and there were no differences in liter size following any of the CDF exposure periods. In addition, evaluation of sperm number, morphology, and motility revealed no noticeable effect of CDF on any of

Table 4 Effect of 12 weeks ingestion of amitraz via drinking water on testicular and epididymal sperm counts and DSP in adult male mice Treatment Control (tap water) Amitraz (40 ppm) Amitraz (80 ppm) Amitraz (160 ppm)
a

Sperm/mg testisa (No. )/103) 44.929/2.70 47.019/8.18 41.799/4.55 33.699/6.53**

Sperm/mg epididymisa (No.)/103) 187.949/15.25 169.729/15.48 146.919/21.58** 143.959/15.02**

DSPa (No. )/105) 10.319/0.62 9.349/1.33 8.159/0.51*** 6.149/0.97****

Results are expressed as mean9/S.D. ** P B/0.01 (Students t -test). *** P B/0.005 (Students t -test). **** P B/0.0001 (Students t -test).

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these parameters. The discrepancy between our results and their results might be due to the period of exposure of the test males, which in our case extended over 12 weeks. Furthermore, species differences cannot be excluded. The results presented in this work also show that ingestion of amitraz pesticide by the test males caused a significant disturbance in the weights of the testes and other reproductive accessory glands. The decrease in the weight of the testes and the epididymis could be attributed to the significant decreases in the sperm counts observed in these organs. Such reduction in sperm counts may be resulted from the direct effect of the pesticide on testicular Leydig and Seroli cells causing a decrease in testosterone production. The data presented in this work indicate that the weight of the preputial gland was significantly increased in test males ingested the pesticide at the lower two concentrations. However, at high concentration the weight of these glands was significantly decreased. It has been noted that the size and activity of the preputial gland in rodents are clearly influenced by a variety of steroid hormones (Elbing, 1963). Preputial glands also produce behavior-modulating pheromones that alter fighting and other behaviors in rodents (Brain et al., 1983). Similar studies have indicated a strong link between male infertility and exposure to more than 50 pesticides (Cox, 1996). A significant increase in the production of dead or abnormal sperm in mice was reported after exposure to the pesticide cypermethrin (Bunya and Pati, 1988) or deltamethrin (Bunya and Pati, 1990). Recently, we have shown that exposure to the insecticide cypermethrin, a synthetic pyrthroid, caused a significant reduction in fertility of male Sprague/Dawley rats (Elbetieha et al., 2001). Taken together, the data presented in this work suggest that exposure to amitraz pesticide could have adverse effects on fertility and reproduction in adult male mice. However, the dosage levels of amitraz employed in this investigation are relatively high and it is likely that human exposures are very much less under normal circumstances.

Acknowledgements This work was supported by the Scientic and Applied Research Center (SARC) at the University of Qatar (grant No. 1/7/2001).

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