The residential and industrial use of carbamate and organophosphate pesticides is widespread in the United States. According to the US Environmental Protection Agency in 1997, over 40 organophosphate pesticides and 22 carbamate pesticides were included in the list of 900 pesticides that posed the highest risks to human health and were registered for use in the United States (3). Both organophosphate and carbamate pesticides primarily target the nervous system of insects. Exhibiting many structural similarities with naturally occurring compounds, organophosphates and carbamates interfere with the conduction of signals and cholinergic reactions in the nervous system of insects by inhibiting the release of the enzyme acetylcholinesterase (AChE) at the synaptic junction. Eserine, parathion, and malathion are examples of cholinesterase inhibitors responsible for the hydrolysis of body choline esters, including acetylcholine, at the cholinergic synapses (3, 18).
Basically, organophosphates and carbamates are neurotoxicants, whether directly or indirectly, and several vital organs are affected; these chemicals produce a variety of toxicological effects on the central nervous, peripheral nervous, cardiovascular, pulmonary, ocular, neurobehavioral, immunological, reproductive, placental, cutaneous, and other body systems. In addition, these insecticides cause neurodegeneration, oxidative stress, endocrine disruption, and many other toxic effects (3).
Carbofuran (furadan) is still causing intoxication in animals even after almost a decade of being banned (10). Illegal poisoning of wildlife and domestic animals is a worldwide issue (28). There are high numbers of carbofuran poisoning incidents in birds. Novotny et al. (28) found sporadic cases of small carnivore intoxication, and martens and foxes are thought to be the main object of poisoners. Additionally, domesticated animals, such as pets, mainly dogs, and livestock, are at risk of being poisoned with carbofuran (40). The clinical signs of accidental or intentional carbamate poisoning are nonspecific, reflecting a combination of muscarinic and nicotinic hyperstimulation (18).
Carbamates are reversible AChE inhibitors derived from carbamic acid. Carbamate causes inhibition of the activity of AChE, which is an enzyme responsible for the hydrolysis of the neurotransmitter acetylcholine in two separate components: choline and acetic acid (16, 21). This results in an excess of acetylcholine in the synaptic cleft and prolonged binding to postsynaptic receptors (32). AChE inhibition causes hyperstimulation of cholinergic receptors, followed by muscarinic, nicotinic and central nervous signs. AChE inhibitors may also impair endothelial function due to their toxicity to endothelial cells (20, 46) and the vascular wall (46). The overstimulation of the somatic nervous system usually results in tremors, muscle twitches, and piloerection, as well as ataxia and paresis. Cholinergic tracts are also important to both the parasympathetic and sympathetic autonomic nervous systems, but especially to the former. They conduct impulses from the neural ganglia to a multitude of organs, such as the heart, endocrine glands, and digestive system (27).
Systemic effects may occur within 30-60 minutes, generally occur after 6 hours, and rarely occur after 12 hours. Muscarinic symptoms are usually associated with salivation, lacrimation, urination, diarrhea, and gastroenteritis (SLUDGE) in addition to bradycardia, dyspnea, and miosis. Local effects usually occur because of direct contact with the product. Symptoms can be observed after a few minutes or can be delayed several days in the case of cutaneous exposure (30).
Intoxication with a cholinesterase inhibitor may lead to apparently opposite clinical signs, such as either constriction or dilation of the pupils or a speeding up or slowing down of the heartbeat.
The autonomic nervous system is subjected to constant adjustment through feedback mechanisms, and because of this, each individual may react differently to various levels of cholinergic stimulation. Death usually occurs due to respiratory failure and cardiac arrest (19).
The 7 cases described here are a clear example of carbofuran used for the intentional poisoning of dogs. In most of these confirmed cases, the results were used by authorities in legal investigations. According to police report information, there are common reasons for killing both dogs and cats, many of which are related to domestic or social violence (1, 2, 11).
The investigation of cases of intentional animal poisoning is as serious as that in human cases (23, 26), yet it is a very challenging and difficult process (8).
The investigation of an incident that involves the death of wildlife generally consists of a field inquiry, a postmortem examination and, when necessary, chemical analysis to determine whether a poison might be responsible (5). For pesticide detection, multistage mass spectrometry (MS/MS) is considered a very useful tool to detect low levels of an analyte when coupled with chromatographic techniques (22). For our toxicological analysis, we used a triple quadrupole mass spectrometry analyzer operated in the selective reaction monitoring mode, which significantly improved both the sensitivity and selectivity of the analytical determination, similar to Luzardo et al. (22), who developed a method for the identification of 117 pesticides. The main differences were the type (they used liver) and the weight (2 g of sample) of the samples, as well as the dilutions and the quantity of the solvent. Similarly, the use of sonication should be mentioned, which improves the extraction efficiency and recovery rate of certain key pesticides, such as carbofuran. Therefore, Luzardo et al. (22) added a 5-min sonication to the extraction protocol; in our cases, sonication was performed for 15 minutes. Another method using 2 g of homogenized liver samples is based on a new analytical multiclass method named the Quick, Easy, Cheap, Rugged and Safe (QuEChERS) technique (35), developed by Sell et al. and validated according to the requirements of SANCO/12571/2013 (34).
In our study, pathological examinations revealed predominant pulmonary lesions. Thus, carbofuran poisoning induced respiratory and cardiac depression, which led to the death of the dogs. Hyperstimulation affects vascular tone and cell permeability and tissue perfusion (9), which could cause interstitial blood pooling (congestion) and edema. Similar to Motas-Gusman et al. (24), we found acute pulmonary congestion, pulmonary edema, and emphysema but without constriction or bronchial rupture. Pulmonary hemorrhage is typically described, especially in acute intoxication cases (18), and these lesions were also present in our study. Novotny et al. (28) reported dried saliva around the oral cavity, congestion of the organs and hemorrhagic necrosis of the small gut. In our study, we found only one dog with foamy salivation; four dogs presented epistaxis and four had staining by a pink-colored foreign substance (interpreted as being the consumed carbofuran) around the oral cavity. In 6 cases, we observed ocular changes consisting of conjunctival hemorrhages or congestion and unilateral or bilateral hyphema (Tab. 1).