UDC 615.285.7.032.015.4:612.64

Experimental Study of the Embryotropic Effects of Methylmercaptophos Following Inhalation Exposure

V. A. Gofmekler, Candidate of Medical Sciences, and B. B. Khuriev

A. N. Sysin Institute of General and Municipal Hygiene, Academy of Medical Sciences of the USSR, Moscow, and Tashkent Regional Sanitary and Epidemiological Station, Uzbek SSR

Methylmercaptophos is widely used in Uzbekistan as an insecticide against numerous cotton pests. As our studies and those of B. K. Akhmedov, V. B. Danilov, S. Yu. Mukhamedova, and others have shown, after fields are treated by aircraft or tractor, organophosphorus compounds persist in ambient air for up to 7–10 days, and sometimes longer, at concentrations not exceeding the maximum permissible limits, and disperse over distances of up to 1.5 km.

The literature indicates that pesticides exert embryotropic as well as general toxic effects. The effects of pesticides on the first generation of rats have been investigated by R. A. Ryazanova and by Mestit-

zova and Beno. In the first case, the effects of ziram and zineb added to the food of experimental animals were studied; in the second, the effects of heptachlor, also administered orally, were examined. In both cases, the findings indicated changes in the reproductive function of rats. According to Kalow and Marton, malathion reduced the survival of rat offspring and caused abnormal development in those that survived. E. A. Goloma and N. D. Mukhtarov observed accidental poisoning of pregnant women with Granosan through consumption of flour contaminated with the compound, which affected the duration of pregnancy and fetal viability. The infants of 10 of these women died shortly after birth, 3 pregnancies ended in miscarriage, and the children born showed marked delays in mental and physical development.

In view of these findings, as well as the considerable vulnerability of women and children, we decided to study the embryotropic effects of methylmercaptophos in animal experiments, with a view to subsequently comparing the results with data on the health of women exposed to methylmercaptophos during its application to cotton fields.

The studies were conducted on sexually mature, 3-month-old outbred female white rats weighing 170–220 g. After mating, groups of 15–20 females were placed for the entire duration of pregnancy in exposure chambers in which specified concentrations of methylmercaptophos had been established beforehand. The first day of pregnancy was determined by the presence of spermatozoa in vaginal smears.

Four concentrations of methylmercaptophos were studied in the experiment (\(0.5\pm0.0075\), \(0.105\pm0.0064\), \(0.00068\pm0.000015\), and \(0.00020\pm0.000011\ \mathrm{mg/m^3}\)); one group served as a control. The lowest concentration studied was close to the maximum permissible single-exposure concentration of methylmercaptophos in ambient air recommended by B. B. Khuriev (\(0.0003\ \mathrm{mg/m^3}\)). In the experiment, the compound was measured spectrophotometrically at a wavelength of \(220\ \mathrm{nm}\). Air samples were collected in 96% alcohol.

We studied female fertility by counting live embryos; death of ova before implantation and of embryos after implantation by counting ovarian corpora lutea and dead and live embryos; the sex distribution of embryos by counting males and females; weight measures, including embryonic weight and the relative weights of the thymus, heart, liver, lung, kidneys, and adrenal glands; embryonic length; morphological and histochemical changes in embryonic organs; visible external and internal developmental abnormalities of the organs, using Wilson’s method of sagittal sectioning of embryos fixed in Bouin’s fluid, and of the embryonic skeleton, using tissue clearing and alizarin staining; and the ascorbic and nucleic acid content of the maternal and embryonic brain and liver and of the placenta.

A total of 88 females and 855 embryos were studied. The experimental data were analyzed statistically, with significance assessed by the range method (R. N. Biryukov).

Group fertility of the females is determined by the number of live embryos and the number of nonpregnant females in the group. Following exposure to relatively high concentrations of methylmercaptophos (\(0.1\) and \(0.5\ \mathrm{mg/m^3}\)), one quarter of all females were found not to be pregnant. The number of embryos per pregnant female was also reduced; these concentrations substantially decreased the overall fertility of the females compared with the other groups (Table 1).

The sex distribution changed markedly following exposure to methylmercaptophos at \(0.5\ \mathrm{mg/m^3}\): the male-to-female ratio was 1.3–1.6 in all groups except the last (\(0.5\ \mathrm{mg/m^3}\)), in which the ratio was reversed (0.65).

Exposure to methylmercaptophos at concentrations of \(0.0007\), \(0.1\), and \(0.5\ \mathrm{mg/m^3}\) sharply increased embryonic mortality during early development

Table 1

Summary data on female fertility and embryonic mortality following exposure to methylmercaptophos

Notes. 1. Confidence levels: a—95%; b—99%; c—99.9%.
2. Mortality was calculated as follows: before implantation, \(В-(A+Б)\); after implantation, it equals the number of dead embryos.

…of the ovum before its implantation in the uterus. This is also supported by the increase in the number of nonpregnant females in the last two groups. Given that nonpregnant females generally lack corpora lutea (only 2 of 12 such females had them), it may be assumed that the agent acts on the female at the very earliest stage after mating, disrupting fertilization of the ovum and thereby increasing preimplantation

Fig. 1. Dead embryo with fused placentas (476).

mortality of ova. In 2 females, this mortality apparently occurred during the 3 days in which the fertilized ovum travels to the uterus.

Although postimplantation embryonic mortality increases with increasing methylmercaptophos concentration, the increase is not significant. A tendency toward an increase in the number of dead (resorbed) embryos was observed in females exposed to 0.0007, 0.1, and 0.5 mg/m³. Thus, in rat No. 16 (0.1 mg/m³), 5 of 12 embryos were dead, one of which was in the initial stage of regression, probably in connection with an abnormality of the placenta—a triple, fused placenta of irregular shape (Fig. 1). The vast majority of resorbed embryos were extremely small…

Fig. 2. Underdeveloped (758 and 861) and normal (K) embryos.

…in size (\(2 \times 3\) mm in diameter), indicating that they had died at an early stage. Large resorbed embryos were encountered only sporadically. Along with the increase in the number of dead embryos, general underdevelopment of the living embryos was observed, manifested in a disproportion of body parts visible to the naked eye and in reduced length and weight (Fig. 2, Table 2).

The data presented in Table 2 show significant changes in embryo length and weight on the 20th and 21st days of pregnancy at all concentrations of methylmercaptophos studied. Against the background of reduced overall embryo weight, a relative increase in the weight of individual organs (liver, adrenal glands, lungs, and heart) was observed, which should evidently be regarded as a compensatory response to the adverse exposure.

Developmental abnormalities caused by methylmercaptophos included changes in the size of the cerebral ventricles, hemorrhages into the thoracic and abdominal cavities, incomplete ossification of the skeleton, and so on (Table 3).

Table 2

Selected weight measurements and embryo size following exposure of pregnant females to methylmercaptophos

Note. Confidence level: a—95%, b—99%, c—99.9%.

Following exposure to methylmercaptophos at a concentration of 0.5 mg/m³, marked enlargement of the cavities of the cerebral ventricles was observed in 20 of 38 cases (53%) (Fig. 3), and extensive hemorrhage into the abdominal cavity in 5.3%. In embryos from this group, the general picture of underdevelopment is corroborated by incomplete ossification of the small bones of the limbs and curvature of the spine (Fig. 4).

Table 3

Visible internal and external developmental abnormalities in embryos following exposure of pregnant females to methylmercaptophos

Values are numbers of cases, in percent.

Fig. 3. Dilated III (806) and normal (763) brain ventricles.

Fig. 4. Curved spine of an embryo (2137).

At lower concentrations (0.1, 0.0007, and 0.0002 mg/m³), abnormalities in embryonic development consisted primarily of incomplete ossification of the skeleton and curvature of the spine. Such developmental anomalies were either entirely absent or very rare in the embryos of control animals.

Thus, the experiment described shows that all the methylmercaptophos concentrations studied, when introduced into the body through the respiratory tract, had adverse effects on female white rats and their first offspring.

References

Akhmedov, B. K. Gigiena i sanitariya, 1968, no. 10, p. 10. — Biryukova, R. N. Ibid., 1962, no. 7, p. 43. — Goloma, E. A. Ibid., no. 9, p. 101. — Danilov, V. B. In: Proceedings of the 2nd Republican Conference of Pathologists of Uzbekistan. Tashkent, 1966, p. 106. — Mukhamedova, S. Yu. Gigiena i sanitariya, 1968, no. 12, p. 8. — Mukhtarov, N. D. In: Collected Scientific Papers of the Department of Nervous Diseases, Kirghiz Medical Institute. Frunze, 1963, p. 77. — Ryazanova, R. A. Gigiena i sanitariya, 1967, no. 2, p. 26. — Kalow, Marton. Cited in: S. N. Golikov and V. I. Rozengart. Cholinesterases and Anticholinesterase Agents. Leningrad, 1964, p. 277. — Mestitzova, M., Beno, M. Pracow. Lek., 1966, vol. 18, p. 153.

Received February 19, 1969.

An Experimental Study of the Embryotropic Action of Methylmerkaptophose Introduced by the Respiratory Route

V. A. Hofmekler, B. B. Khuriev

In order to define the embryotropic action of the investigated substance the following indices were studied: the weight of embryos and their organs, the fertility of females, the mortality of embryos before and after implantation, visible external and internal anomalies of development. Introduction by the respiratory route of methylmerkaptophose at concentrations of 0.5, 0.1, 0.0007 and 0.0002 mg/m³ into the body of female animals in the course of the gestation period proved noxious both to the pregnant animals and their first generation.