Author(s): <p>Muna Salem M Solyman, Fatma Hassan Boshahma, Huda Mohamed, Fawzeia H Elmhalli Abdalla I Mohamed and Abeer H Amer*</p>
Approximately two thirds of deaths and millions of nonfatal cases are attributed to organophosphorus pesticides (OPP) annually. Diazinon, a prominent member of the OP pesticides, was introduced for both agriculture and public health usages during. For several decades, the compound was among the top pesticides in use in many countries, including Libya. The toxicity of this compound is based on its ability to inhibiting the acetylcholinesterase enzyme (AchE), which is vitally important for normal nerve impulse transmission. This study evaluated the acute toxicity of diazinon through acute exposure testing on female Swiss albino mice. In the acute toxicity test, 72 mice were subcutaneously injected with doses of diazinon ranging from 320 to 480 μL/ kg. Mortality and symptoms were recorded over four days, with the median lethal dose found to be 403.5 μL/kg. Based on the findings, the present study suggests that stringent controls and surveillance must be placed on diazinon and similar chemicals.
Approximately 34% of pesticides manufactured and distributed for use in agriculture globally are composed of organophosphorus [1]. During the 1940’s, the organophosphorus compounds appeared to represent an extremely important class of organic pesticides. They are widely used in agricultural practices to control pests on vegetable crops, fruit trees, grains, cotton, and cabbage. However, the uncontrolled usage of organophosphorus compounds causes pollution to the environment and negatively effects on plants, animals, and humans [2].
Diazinon is probably one of the most important OP pesticides that was introduced by the Geigy Company in 1952 and by the 1970s. It has a relatively low mammalian toxicity and a wide margin of safety [3]. Moreover, it was described as selective against adult and juvenile forms of insects and with moderate activity against acarina. The common spectrum of diazinon activity includes, flying insects and crawling insects; therefore, it is used as a pesticide and as a veterinary drug to control ectoparasites.
Also, it has garnered attention due to its efficacy and associated health and environmental issues [4,5]. Its mode of action causes long-lasting ecological effects by endangering water systems and wildlife in addition to agricultural laborers [6-9]. WHO classified diazinon as a moderately hazardous class II OP pesticide.
The continuous use of diazinon causes it to accumulate in the environment, food chain, and animal tissues and then eventually stimulate, cause, or be a major contributing factor in a number of serious human diseases, including immune system and neurological disorders, sterility, various cancers (breast, lung, cervix, and prostate), heart diseases, Alzheimer’s, and Parkinsonism[9-11].
Furthermore, the sources of human exposures are accidental, occupational, or through diet [12]. On the other hand, there are several ways that pesticides, and their harmful effects can come into contact with people [13]. The two possible routes of exposure are oral consumption (food and drink) or absorption via skin by exposure to the pesticide-containing dust [12,14]. After being absorbed, organophosphorus chemicals cause excessive cholinergic stimulation by primarily blocking the enzyme acetylcholinesterase (AChE), which interferes with normal nervous system function in both target and non-target organisms [15-17]. According to the Centers for Disease Control and Prevention the amount of organophosphorus pesticides found in the urine and blood serum of humans varies according to the extent of their exposure to these pesticides [18-20].
Symptoms of acute toxicity manifest as headaches, dizziness, nausea, respiratory distress, and potentially severe outcomes like seizures and coma [12]. Furthermore, it leads to decreased MCHC and MCV levels along with increased leukocyte and erythrocyte values [21]. Importantly, even low-level exposures can pose long-term health risks [22]. Regulatory agencies have set safety standards to reduce risks associated with diazinon usage; however, reports of acute poisoning incidents continue to highlight the need for caution [23].
Ongoing research into the mechanisms of diazinon toxicity and its overall environmental impact is essential for enhancing safety protocols and public health guidelines. This introduction establishes a foundation for understanding the acute toxicity of diazinon and emphasizes the necessity for further investigation. Therefore, this work was designed to evaluate the available formulation of Neocidol in the local market on vertebrate animal models. Neocidol, a common widely used diazinon formulation, was chosen to be evaluated in terms of its acute toxicity to the Swiss albino female mice as a vertebrate animal model. Acute toxicity was expressed as LD50 value within 24, 48 or 72 hours.
The total number of animals used in the experiments were 72 female Swiss albino mice Mus musculus produced from several parents that were obtained from the animal house of the Faculty of Medicine University of Benghazi. The animals were kept under laboratory conditions, where the temperature was 7±2°C and the relative humidity was 71±11% . The electrical current regulator was set to regulate the laboratory photoperiod at 14 hours of light and 10 hours of dark throughout animal rearing and experiment duration. The animals were housed in plastic cages; their sizes were 50 x 30 x 19cm, 4 to 6 animals per cage. A little wooden sawdust was placed at the bottom of each cage before putting the mice in for additional care. The animals were supplied with the diet two to three times daily to ensure their feeding demands. Clean bottles approximately 350 cc size were used for the watering. Cages were washed with soap and water, every other day, and fresh wooden sawdust was layered.
The main chemical is the organophosphorus pesticide, diazinon, manufactured by Novartis Inc Basle, Switzerland under the trade name Neocidol. This commercial preparation was designated as “60” emulsifiable concentrate and was obtained from a local pesticide market. The other chemicals used in this study were of a technical grade with known structures and functions.
Five concentrations of Neocidol were selected for this study, these were 320, 360, 400, 440, and 480 µl/kg (ppm) body weight. These concentrations, however, were based on a preliminary test, where two and four mice were tested by way of subcutaneous injection with an aqueous diluted emulsion of the Neocidol and on the information’s concern with toxicity found in the literature for the determination of the mortality range of the dosages. A digital laboratory balance, (Mettler PM 3000) was used to measure the weight of the animals and the other chemicals.
Neocidol concentrations were prepared using distilled water in a conical flask. Twelve female mice with approximate age and weight in three replicates (four per replicate) were selected randomly for each concentration.
Injection was subcutaneously performed by an ultrafine sterile and pyrogen free disposable syringe, where the calculated dose (ul/kg body weight) was delivered in volumes ranging from 0.1 to 0.2 ml/ mice according to weight in the inner upper side of the hind leg of each animal. Every care was taken to bring the temperature of the injected suspension close to the body temperature before the time of the injection. Twelve animals (three replicates of four animals each) were injected similarly, but with distilled water alone to serve as control. All treated animals, along with the control, were then kept in their rearing cages under the laboratory conditions, (temperature, humidity, and light) and supplied with the rearing diet and water. Ad libtium observations were made for the treated animal behavior while mortalities were recorded on an hourly basis, whereas postmortem examinations were conducted for the dead animals. The median lethal dose (LD50) was calculated for the compound, according to Finney, whereas the LT50 was calculated for selected dosages.
The probit mortality curve (LD50) for female Swiss albino mice subcutaneously injected with the organophosphorus insecticide Neocidol (diazinon) is presented in (Figure 1,2,3). The median 72h- LD50 was found at 403.5 µl Neocidol per kg of mice body weight. The lower and upper limit values and the correlation between used dosages and mortalities are shown in Figure 1. Neocidol at 280 µl/kg, the lowest tried dose in this study did not reveal any mortality, although signs of poisoning were observed during the early period of injection. Diazinon dosages and corresponding mortalities, clearly showed a dose-dependent relationship. The higher the dose, the greater the mortality. Furthermore, as diazinon dosage increased, a shorter time was required for 50% mortality (LT50) of the tested females. The severity of symptoms and time of death varied with the used dose. Thus, the median lethal time LT50 for 400 µl/kg was 33.84 hours post injection (LT50= 33.48), with a range of 12.18 to 48.99 hours (Figure 2). As the dose increased to 440 µl/kg, a shorter time of LT50 32.6 hours (range 21.5 to 41.94) was noted (Figure 3).
Also, low mortality of 8% was observed at 320 µl/kg with more pronounced ill symptoms, when Neocidol dose was increased to 360 µl/kg. An increase in mortality was also observed, where, 16% of female mice were found dead.
Besides, mortalities were then suddenly increased to 50% at Neocidol 400 µl/kg and up to 91% at 440 µl /kg. The highest mortality up to 100% was recorded for the highest used dose, 480 µl /kg mice body- weight.
On the other hand, the most obvious symptoms observed in female mice treated with Neocidol were excessive sweating, constriction of pupils, “depression” exhaustion, slow movement, reduction of food and water intake, failure of response, and paralysis at the final stages prior to death. These symptoms, however, were mainly in those mice exposed to the higher dosages, which often died. At the lower dosages, most treated mice suffered sweating, depression, and slow movement with initial reduced food and water intake. However, the majority of affected females recovered gradually to normal after 48 to 72 hours’ post-treatment.
Figure 1: Probit Mortality of Female Mice Treated with Neocidol 320,360,400,440 and 480 µl /l (g (ppm) 72h - LD50 = 403.5 (range 381.5-426.6 correlation= 0.953).
Figure 2: Lethal time (LT50) of female mice treated with Neocidol LT50 (400 ppm)= 33.84 hours (range 12.18 - 48.99 hours) (LT50 - mean time required for 400 ppm to kill 50% of tested animals)
Figure 3: Lethal Time LT50 of 440 ppm Neocidol Treated Female Mice
LT50 =32.6 hours (range 21.5 -41.94 hours). Note, the higher concentration, the shorter period required to kill 50% of treated mice.
The results of this experiment found clear support for the negative impact of diazinon on female Swiss albino mice, which at low doses of diazinon, poisoning signs were observed and the mortality increased with increase its dose. Diazinon, likes all other organophosphates, is a cholinesterase inhibitor. The clinical signs of diazinon toxicity are the results of acetylcholine accumulation which eventually causes an over-stimulation of the parasympathetic and central nervous systems and skeletal muscles [24-27]. Inactivation of AChE causes a blockage of the cholinergic transfer of nerve signal, paralysis, and death due to asphyxia [27].
In line with previous studies reported that diazinon produces depression effects on the respiratory centers and leads to impairment of respiratory muscle efficiency [28]. Acute toxicity of diazinon varies among different animals, and among different formulations. The acute oral 72hour LD50 of the commercial Neocidol reported in this test was found to be 403 5 µl/kg female body weight. This value confirms the ranking of diazinon among the moderately toxic compounds listed by Matsumura and Loomis and Hays [29,30]. Overall, these findings are in accordance with findings reported by WHO, which it that confirms the diazinon classification was being among the moderately hazardous pesticides WHO [31].
In line with previous studies, acute diazinon exposure induced oxidative stress indicators in a time-dependent manner that matched the effects on the pattern of hepatic antioxidant gene expression. Also, it had an influence on the gene expression of antioxidant enzymes even at non-lethal doses [32].
Other confirming reports of diazinon acute toxicity include that of Howard who reported repeated single dose LD50 range from 2.75mg/kg to nearly 450 mg/kg/day for rats, and that of the National Library of Medicine, who reported repeated single dose LD50, as high as 720 mg/kg/day [33]. Acute toxicity in animals other than mammals was also reported. The LD50 values for birds range from 2.75 mg/kg/day to 40.8 mg/kg, and for the rainbow trout the LC50 values range from 2.6 to 3.2 mg/L [34]. As it can be seen, both birds and fish (wildlife) were found quite susceptible to diazinon poisoning when compared to most mammals, including humans. The toxicity of diazinon to all animal species, however, is dose-dependent, the higher the dose, the higher the mortality rate accompanied by shorter mortality time. The results reported here, showed that diazinon at 440 µl/kg mice body weight inflicted 50% mortality among the tested animals within 32.6 hours (LT50=32.6h) which was a shorter time than that reported for diazinon 400µl/ kg with 33.84 hours (LT50 33.84 hours). The symptoms of acute toxicity of diazinon treated female mice reported in this study were clearly indicating OP-poisoning. These symptoms included excessive sweating, depression, slow movement, and exhaustion, these were followed by failure to response, and then paralysis, then coma before death.
And this is coming in agreement with the present study which found that the most noticeable signs seen in female mice given Neocidol were increased perspiration, narrowed pupils, “depression” fatigue, sluggishness, decreased consumption of food and liquids, inability to react, and paralysis in the final moments before death. However, the mice exposed to the larger dosages exhibited these symptoms primarily, and these mice frequently died. These symptoms confirm the previously reported symptoms of OP including diazinon by Matsumura, Hodgson and Levi and Ganie who stated that OP compounds induce a broad spectrum of clinical effects that are indicative of over stimulating the cholinergic system [29,35,36]. The toxicity of diazinon as an OP compound was described by Hodgson and levi [35]. These compounds are toxic because of their inhibition of the enzyme acetylcholinesterase found in the nervous system. This enzyme inhibition results in the accumulation of acetylcholine in nerve tissue and effector organs, primarily at muscarinic and nicotinic synapses in the periphery. This leads to cholinergic hyperstimulation, which is characterized by bradycardia, bronchospasm, diarrhea, hypersalivation, loss of bladder control, and pupillary miosis and lacrimation.
Hyperstimulation of nicotinic receptors causes hypercontraction and spasms in the muscles, which eventually result in paralysis. Confusion, nausea, and dizziness, as well as a possible coma, seizures, and centrally mediated cardiac and respiratory arrest, are additional impacts of the central nervous system [37].
Diazinon, one of the most prominent OP compounds, was proved to be moderately toxic to female Swiss Albino mice with an LD50 of 403.5 ppm. The observed trend of toxicity is similar to other toxic compounds, where, a positive correlation between the dose and percent mortality was very evident. Vital organs (lung and heart) did not reveal weight differences between control and treated groups. However, these organs were relatively less in weight in the treated females. No gross histological abnormalities were observed in any of the vital organs studied, however, minor to moderate histopathological effects were evident in these organs. Such effects included distorted bronchioles and disrupted alveolar septa in the treated lung. No sign of histopathological abnormalities, however, was observed in the treated heart as compared to that control group. Even though the results of this investigation do not offer adequate evidence to conclusively establish the detrimental effects of diazinon independently, given the precautionary principle, the authors advocate for immediate control and surveillance on the importation, sale, and distribution of diazinon and other pesticides that could harm human health or the environment [38-40].
No conflict of interest between the authors
Animal experiments were designed and carried out in compliance with the guidelines set by the institutional animal ethics committee.
