Residential and industrial use of carbamate and organophosphate pesticides was widespread in the United States. According to the 1997 findings of the US Environmental Protection Agency, over 40 organophosphate pesticides and 22 carbamate pesticides were listed among 900 pesticides which pose the highest risks to human health were registered for use in the United States (3). Both organophosphate and carbamate pesticides primarily target of action in the nervous system of insects. Exhibiting many structural similarities with naturally occurring compounds, organophosphate and carbamate interfere with the conduction of signals and cholinergic reactions in the nervous system of insects via inhibiting the release of the enzyme acetyl cholinesterase at the synaptic junction. Eserine, parathion, and malathion are further examples of cholinesterase inhibitors responsible for the hydrolysis of body choline esters, including acetylcholine at cholinergic synapses (3, 18).
Basically, organophosphates and carbamates are neurotoxicants, but directly or indirectly several vital organs are affected, as these chemicals produce a variety of toxicological effects on the central nervous system, peripheral nervous system, 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) after almost a decade of banning is still causing intoxications amongst animals (10). Illegal poisoning of wildlife and domestic animals is a worldwide issue (28).There are high numbers of carbofuran poisonings in birds, Novotny et al (28) found sporadic cases of small carnivore intoxication, although martens and foxes are thought to be the main object of poisoners. Also, domesticated animals such as pets, mainly dogs, and livestock are at risk of being poisoned with carbofuran (40). The clinical signs in accidental or intentional carbamates poisoning are unspecific reflecting a combination of muscarinic and nicotinic hyperstimulation.(18)
Carbamates are reversible acetylcholinesterase inhibitors, derived from carbamic acid. Carbamate causes inhibition of the activity of acetylcholinesterase, which is an enzyme responsible for the hydrolysis of the neurotransmitter acetylcholine in two separated components: choline and acetic acid (16, 21). This results in an excess of acetylcholine in the synaptic cleft and prolonged binding to the post-synaptic receptors (32). AChE inhibition is causing hyperstimulation of cholinergic receptors, followed by muscarinic, nicotinic and central nervous signs. AChE inhibitors may also impair the endothelial function, due to their toxicity to endothelial cells (20, 46) and to 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, the endocrine glands, and the digestive system (27).
Systemic effects may occur in less than 30-60 minutes, generally after 6 hours, and rare after 12 hours. Muscarinic symptoms are associated usually with SLUDGE symptoms (salivation, lacrimation, urination, diarrhea, gastroenteritis) 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 lid too apparently opposite clinical signs like individuals who may show either constriction or dilation of the pupils, or a speeding up or slowing down of the heartbeat.
The autonomic nervous system is a constant subject of 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).
The7 cases described here are a clear example of carbofuran used for intentional poisoning in dogs. In most of these confirmed-cases, the results were further used by authorities for legal investigations. However, based on the information of the police reports, there are common reasons to kill both dogs and cats, and these can be related to domestic or social violence (1, 2, 11).
The investigation in cases of intentional animal poisoning is as serious as those in human cases (23, 26), yet it is a very challenging and difficult activity (8).
The investigation of an incident that involves the death of wildlife specimens 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 improved significantly both the sensitivity and selectivity of the analytical determination, as the Luzardo et al (22) developed method for the identification of 117 Pesticides. The differences consist in the type (they used liver) and the weight (2 g sample) of the sample and there are some differences referring to the dilutions and to the quantity of the solvent. As a similarity the use of sonication should be mentioned, which improves the extraction efficiency and improve the recoveries of certain key pesticides, such as carbofuran. Therefore Luzardo et al (22) added a 5-min sonication to the extraction protocol, which in our cases lasted 15-min. Another method using homogenized 2 g liver samples is based on a new analytical multiclass method named QuEChERS technique (35), developed by Sell et al. and validated according to the requirements of SANCO/12571/2013 (34).
In our study, the pathological exam revealed predominant pulmonary lesions. Thus, the carbofuran poisoning induced respiratory and cardiac depression, which led to the death of the dogs. The hyperstimulation affects the vascular tone and cell permeability and also the tissue perfusion (9) that could cause interstitial blood pooling (congestions) and edema. Like Motas-Gusman et al (24) we found acute pulmonary congestion, pulmonary edema, and emphysema, but without constrictions and bronchial rupture. Pulmonary haemorrhage is typically described especially in acute intoxication cases (18), lesions which were present in our study too. Novotny et al (28) revealed dried saliva around the oral cavity, congestion of the organs and haemorrhagic necrosis of the small gut. In our study we found only one dog with foamy salivation, four presented epistaxis and four were stained around the oral cavity with pink colored foreign substance (interpreted as being the consumed carbofuran).In 6 cases, we observed ocular changes consisting of conjunctival haemorrhages or congestion and unilateral or bilateral hyphema)(table 1).