UDC 613.155.3:615.778.3

Experimental Substantiation of the Maximum Permissible Concentration of Bi-58 (Rogor, Phosphamide) in Ambient Air

F. Kaloyanova, L. Ivanova, G. Dimov, M. Mukhtarova

Organophosphorus insecticides are widely used not only in agriculture but also in the home to combat flies and other insects, and to spray trees in parks and along streets. These agents include Bi-58 (O,O-dimethyl-S-N-methylcarbamidomethyl dithiophosphate), a white crystalline powder with an extremely unpleasant mercaptan odor. It is moderately soluble in water at 20° (up to 3%) and readily soluble in organic solvents and oils. Bi-58 is used as an insecticide in the form of a 0.05–0.1% solution.

Published data on the toxicity of Bi-58 are limited. Studies by T. N. Panshina and by T. N. Panshina and M. A. Klisenko on the Soviet preparation phosphamide have been reported. Phosphamide does not irritate the skin when applied to it, but rapidly penetrates it and produces systemic toxic effects. The oral \(\mathrm{LD}_{50}\) of phosphamide in various animal species (T. N. Panshina; Giring) ranges from 100 to 250 mg/kg, whereas the \(\mathrm{LD}_{50}\) following skin application ranges from 1,000 to 1,500 mg/kg.

According to T. N. Panshina, cholinesterase activity is strongly inhibited after a single toxic dose of phosphamide and recovers after 8–10 days.

The threshold concentration is 10 mg/m³ for a single exposure and 1–5 mg/m³ for chronic exposure. These data indicate that phosphamide at low concentrations has a weakly expressed

cumulative effect. In determining the threshold effect of a substance, specific inhibition of cholinesterase activity is a more sensitive indicator than changes in conditioned reflex activity. Similar findings have also been obtained for chlorthion (Kalojanova). Thus, cholinesterase activity should be adopted as the principal indicator for establishing hygienic standards for organophosphorus compounds.

During the agricultural use of phosphamide, ambient air concentrations were found to range from 0.7 to 4 mg/m³ and, in some cases, to reach 9 mg/m³. In the room where solutions were prepared, concentrations ranged from 0.4 to 1 mg/m³. Examination of workers under these conditions revealed no abnormalities in cholinesterase activity, the autonomic nervous system, or the morphological composition of the blood.

The study to establish the maximum permissible concentration of Bi-58 in ambient air was conducted according to the procedure adopted in the USSR (Ryazanov). To determine the maximum permissible single-exposure concentration of the substance, 12 people under 30 years of age with no abnormalities of the upper respiratory tract were examined. The odor threshold of the preparation for the most sensitive individuals was 0.01 mg/m³.

Bi-58 in air was determined chemically by a method developed by G. Bobev and M. Mukhtarova. The sensitivity of the method was 2 µg per 5 ml sample.

The threshold for reflex effects was determined by measuring adaptation in four subjects with normal vision and olfaction. The electrical sensitivity of the eyes was studied as follows. After 5 minutes of adaptation to standard light, the subject was fitted with black goggles that completely excluded light, and ocular rheobase was measured in darkness for 40 minutes in a well sound-insulated room. Electrical sensitivity was determined every 5 minutes. This method was developed in detail by M. Lukanov and N. Tsaneva.

Most subjects could clearly detect the preparation by smell at a concentration of 0.025 mg/m³. A concentration of 0.1 mg/m³ caused negative emotions because of the substance’s strong odor. Bi-58 was administered for 5 minutes at the 15th and 30th minutes of the study. The effect of each concentration was tested four times in each subject. During the experiment, administration of the test substance was periodically alternated with clean air.

In determining the baseline adaptation curve, we sought to extinguish the response to the delivery of air by repeating the experiment over 4–5 days, but did not succeed.

The delivery of clean air and of Bi-58 at a concentration of 0.025 mg/m³ produced a substantial stimulatory effect on the central nervous system, manifested by increased electrical sensitivity of the visual analyzer. A decrease in electrical excitability was observed at this concentration in only 4 of 40 cases. The same was observed in isolated cases during the delivery of clean air. At a concentration of 0.1 mg/m³, the opposite effect was obtained: the electrical excitability of the visual analyzer decreased significantly in 26 of 40 cases. This is clearly illustrated in the figure, which shows changes in the electrical excitability of the eye (in %). The ordinate represents electrical sensitivity, with the baseline taken as 100. The values above the bars indicate decreases in electrical excitability, and those below the bars indicate increases (in %).

The ocular rheobase studies were repeated under somewhat modified conditions. Adaptation was measured while air was supplied continuously.

From the 20th to the 25th minute, the substance was supplied, after which air was supplied again. The purpose of this experiment was to identify a concentration that would increase the excitability of the visual analyzer, taking into account the findings of some authors (S. V. Kravkov; M. Lukanov and N. Tsanev, 1957) that low concentrations have a stimulating effect and high concentrations an inhibitory effect. We hypothesized that, in the first experimental setup, the air itself acted as a stimulus, masking the effects of low concentrations of the preparation.

A statistically significant increase in the excitability and electrical sensitivity of the eye was observed at a Bi-58 concentration of 0.025 mg/m³. The lower concentration tested (0.005 mg/m³) caused no deviation. A concentration of 0.04 mg/m³ produced an increase in excitability that was not statistically significant. Interestingly, a higher concentration of the substance (0.09 mg/m³) did not produce the statistically significant decrease in excitability that we had expected. This may be attributed to a slight inhibition of electrical excitability at this concentration, superimposed on the natural decline in excitability during dark adaptation. A decrease in excitability was observed only at a higher Bi-58 concentration (0.1 mg/m³).

Changes in the electrical excitability of the eye (%), under the influence of different concentrations of Bi-58.
1 — clean air; 2 — 0.025 mg/m³; 3 — 0.1 mg/m³.
Difference in changes (%): at 20 min, 1: +8%, 2: +3%, 3: −20%; at 35 min, 1: +9%, 2: +5%, 3: −1.3%.

To establish the maximum permissible daily average concentration of Bi-58 in ambient air, studies were conducted on three groups of animals exposed by inhalation to three different concentrations of the substance. In the first experiment, the effects of a concentration of 4.5 mg/m³ were tested on 10 rats and 5 guinea pigs. There were equal numbers of control animals. The experiment lasted 3 months, with an 8-hour daily exposure. To assess the effects of Bi-48 on the animals, their behavior and weight were monitored, and the following were examined: blood cell composition; cholinesterase activity in blood serum, erythrocytes, whole blood, and brain; liver glycogen; glucose-6-phosphate isomerase activity; C-reactive protein; and amino acid nitrogen in urine. At the end of the experiment, the rats were subjected to physical exertion by swimming in a pool at a water temperature of 32°, after which their urine was examined for mucoproteins.

Statistically significant deviations were observed only in cholinesterase activity, total leukocyte count, and urinary nitrogen content. Reduced cholinesterase activity was observed throughout the exposure period in both rats and guinea pigs. The increase in leukocyte count was statistically significant in the third month of exposure. By the end of the experiment, the level of amino acid nitrogen in urine had decreased: it was \(0.11 \pm 0.009\) mg/mL in the experimental rats and \(0.13 \pm 0.06\) mg/mL in the controls, with \(P = 0.037\).

The second experiment was conducted only on rats; a Bi-58 aerosol concentration of 0.05 mg/m³ was tested. The animals were exposed for 14 hours a day over 3 months. During the exposure period, no …

…no substantial deviations were observed in most of the parameters studied, but cholinesterase activity was significantly reduced, particularly during the first month after exposure began. In the following days, cholinesterase activity returned to normal. Examination of liver and brain cholinesterase revealed a significant reduction in its activity. Two experimental animals tested positive for C-reactive protein. The precipitates formed by binding of the antiserum to C-reactive protein were barely visible.

Morphological examination of the animals’ brains revealed mild circulatory disturbances, perivascular edema, and tigrolysis of individual neurons. Silver impregnation and the reaction for cholinesterase activity revealed no clear differences between the changes in control and experimental animals. Histoenzymatic reactions for oxidative enzymes (succinate dehydrogenase and triphosphopyridine nucleotide and diphosphopyridine nucleotide diaphorases) showed a slight reduction in enzyme activity in the brain tissue of experimental animals. A more pronounced reduction in enzymatic activity was found in the parenchymal cells of the liver.

In the third experiment, rats were exposed to Bi-58 aerosol at a concentration of \(0.01\ \text{mg/m}^3\). As in the second experiment, exposure lasted 14 hours per day; the experiment continued for 3 months. No changes were detected in the parameters studied. Cholinesterase activity was depressed in only one observation, at the very beginning of exposure, and subsequently returned to normal. Histological examination of the organs revealed no changes.

Thus, the principal evidence for establishing the maximum single-exposure permissible concentration of the substance comes from the results concerning its reflex action and odor threshold. A concentration below the odor threshold (\(0.005\ \text{mg/m}^3\)) caused no deviations in the adaptation curve of the visual analyzer; it therefore has no reflex action. The electrical excitability of the eye increased at a concentration (\(0.25\ \text{mg/m}^3\)) higher than the odor threshold (\(0.01\ \text{mg/m}^3\)).

These changes were statistically significant.

Our results support a proposed maximum single-exposure permissible concentration of Bi-58 in ambient air of \(0.005\ \text{mg/m}^3\); it is not detectable by smell even by the most sensitive individuals and has no reflex action.

The experimental studies indicate that the threshold for chronic effects does not differ substantially from the threshold for reflex effects in humans. The chronic experiment established that a concentration of \(0.05\ \text{mg/m}^3\) produces a minimal toxic effect, whereas a concentration of \(0.01\ \text{mg/m}^3\) has no effect. This provides grounds for setting the maximum single-exposure permissible concentration of Bi-58 at the same level as the average daily permissible concentration.

References

Lukanov M., Tsaneva N. Ocular rheobase as a method for determining changes in the excitability of the visual analyzer. Tr. NITKh, 1954, vol. 11, p. 201. — The same authors. Changes in ocular rheobase under the influence of caffeine and bromine. Tr. NITKh, 1957, vol. 4, p. 115. — Panshina T. N., Klisenko M. A. In: Industrial Toxicology and Clinical Aspects of Occupational Diseases of Chemical Etiology. Moscow, 1962, p. 181. — Panshina T. N. Gig. i San., 1963, no. 3, p. 21. — The same author. Farmakol. i Toksikol., 1963, no. 4, p. 476. — Giring. In: New Insectofungicides and Herbicides, edited by N. N. Melnikov. Moscow, 1960, p. 24. — Kalojanova F., Activ. nerv. sup. Praha, 1961, vol. 3, p. 284. — Ryazanov V. A., Arch. environm. Hlth., 1962, vol. 5, p. 480.

Received January 28, 1967.

Experimental Substantiation of the Maximum Permissible Concentration of Bi-58 (Rogor, Phosphamide) in the Atmosphere

F. Kaloyanova, L. Ivanova, G. Dimov, M. Mukhtarova

The findings showed that Bi-58 at a concentration of 0.005 mg/m³ had no effect on human reflex reactions. In a chronic exposure experiment, the subthreshold concentration of the same substance was 0.01 mg/m³. The maximum one-time and daily average concentrations of the compound in the atmosphere are proposed to be 0.005 mg/m³.