Organophosphates and carbamates are commonly used as pesticides in the household environment, in agriculture, in industry and as parasiticides in veterinary medicine. The two groups of compounds share a common mechanism of action and have similar toxic effects on animals. Both groups include compounds with relatively low toxicity and others that are extremely toxic or lethal (nerve gases).
The first organophosphate was synthesised in 1854 but the greatest period of development occurred around the Second World War, when numerous compounds, some of which were extremely toxic and non-selective (e.g. the nerve gases), were produced for various purposes. Starting after the war, the development programme focused on the synthesis of more selective substances that were toxic to insects but less toxic to mammals, and for about 50 years these compounds have been the main insecticides used throughout the world. There are currently at least a hundred organophosphates used for a variety of purposes.
The first carbamate, physostigmine (alkaloid of eserine), was isolated in the 1880s. Neostigmine, an aromatic ester of carbamic acid, was synthesised at the beginning of the 1900s, but the real development of compounds used as insecticides began in the 1960s and 1970s. As for the organophosphates, numerous compounds were synthesised, but only a very small number of these are actually used as insecticides.
CHEMICAL STRUCTURE
The class of organophosphorus compounds, often called organophosphates, comprises at least 13 types of compounds. The organophosphates are essentially esters of phosphoric acid with various combinations of bonds with oxygen, carbon, sulphur and/or nitrogen. The resulting compounds are fairly complex, but all contain a phosphate group (P=O) or a thiosulphate group (P=S). Some organophosphates inhibit acetylcholinesterase directly (e.g., dichlorvos, trichlorfon) (see Mechanism of action), while others (those with a P=S bond) have only minimal or no direct inhibitory effect on the enzyme, but must undergo a process of oxidation (transformation of the P=S group of thiosulphate to the characteristic P=O group of a phosphate) to produce an oxon analogue of the molecule, which has direct anticholinesterase activity (e.g. parathion becomes the oxygenated analogue, paraoxon).
Carbamates are esters of carbamic acid and are structurally simpler molecules than the organophosphates.
SOURCES
Animals can become intoxicated following exposure to organophosphate compounds used as insecticides, which can be distributed on crops, animals, the ground, house plants, etc.
Most cases of poisoning of pets are accidental (e.g. accidental exposure to household insecticide sprays), although deliberate poisoning (through ingestion of bait) must be considered, as must the possibility of iatrogenic intoxication (overdose or accidental intake of pharmaceutical products, use of products intended for other species, use of insecticides in debilitated animals). According to data collected in Italy for the years 2004 and 2010, organophosphate and carbamate insecticides were, along with rat poisons, among the most common causes of intoxication of dogs and cats. In the dog, the most frequent carbamate products responsible for poisoning are methomyl and carbaryl, while the most frequently implicated organophosphates are diazinone and malathion.
Organophosphates, once absorbed through the oral, cutaneous or pulmonary route, are transported to all the tissues of the body, without particular selective tropism, and are rapidly metabolised in the liver before being quickly excreted through the kidneys.
MECHANISM OF ACTION
Organophosphates and carbamates have the same mechanism of action, which is inhibition of acetylchoinesterase (AChE, also called true acetylcholinesterase), an enzyme that breaks down acetylcholine (ACh), and is present mainly in the central nervous system (CNS) and red blood cells. Since the cholinergic system is widely distributed throughout the central and peripheral nervous systems, all substances able to inhibit AChE produce a vast range of well-known and well-characterized signs (see Clinical signs). Both classes of compounds act on AChE and also on pseudocholinesterases; these latter are normally present in the plasma, liver, pancreas and nervous tissue and can be hydrolysed by a variety of esterases.
- ACh is a neurotransmitter in:
- pre- and post-ganglionic neurones of the parasympathetic and sympathetic nervous syste
- post-ganglionic parasympathetic fibres, smooth muscle and exocrine glands;
- neuromuscular junctions
- cholinergic synapses in the CNS.
In physiological situations, AChE binds the terminal groups of ACh and hydrolyses the compound, leading to the release of choline and acetate. Since the acetate is released after the choline, the enzyme is “aceylated” for a fraction of time. The turnover of ACh occurs very rapidly, within about 150 µs. When a carbamate binds AChE, the insecticide is hydrolysed and the enzyme is temporarily “carbamylated” and, therefore, inhibited. This type of inhibition is usually reversible, but can last for long enough to produce the characteristic clinical signs up to the point of death (time of hydrolysis = 15-30 minutes). After binding to an organophosphate, the enzyme is “phosphorylated” and, therefore, inhibited, but since the period of hydrolysis is very long (days), the bond and the type of inhibition it causes are considered irreversible. In some cases, following subsequent processes of dealkylation, the bond between the enzyme and the phosphorus group becomes stable (a phenomenon known as ageing). Once this process has occurred, the inhibition is definitely irreversible and even treatment with an oxime is ineffective.
The result of phosphorylation or carbamylation of AChE is cessation of the normal activity of the enzyme and a consequent increase in the concentration of ACh in the neuro-effector sites, which translates into continuous stimulation of neural, glandular and muscular receptors.
The simultaneous or temporally close administration of some other substances can potentially increase the susceptibility of animals to the toxic effects of organophosphates and carbamates. Such substances include:
- Drugs that cause neuromuscular blockade or that compete for binding with the esterases: phenothiazine, procaine, inhaled anaesthetics, neuromuscular blocking agents such as succinylcholine and decamethonium; some antibiotics: aminoglycosides (streptomycin, dihydrostreptomycin, neomycin, kanamicin, gentamicin), polypeptides (polymixin A and B, colistin), clindamycin and lincomycin can have neuromuscular blocking ef
- CNS depressants (phenothiazine tranquillisers, benzodiazepines, reserpine and barbiturates) can potentiate the effect of organophosphates and carbamates through direct or indirect synergistic pharmacodynamic activities, or though pharmacological effects (e.g. respiratory depression).
Given these potential interactions between drugs and insecticides, it may be necessary to postpone surgical interventions and/or use alternative therapeutic protocols if an animal has recently been intoxicated by organophosphates or carbamates.
Furthermore, the concomitant use of drugs with anticholinesterase effects, albeit at low doses, can induce intoxication because of the progressive inhibition of the esterases.
CLINICAL SIGNS
Most cases of poisoning in animals are acute. The first clinical signs usually occur within 15 minutes to 1 hour and these are rapidly followed by more severe signs. The time course of signs does, however, depend on the species of animal involved, the compound and the dose. In the case of some particularly liposoluble organophosphates, the appearance of the clinical signs can be delayed for some days and then last for several weeks. Given the great variability of the individual responses and the large number of compounds that can cause intoxication, not all poisoned animals present with the same signs and not all cholinesterase inhibitors cause the same syndrome.
The clinical signs can be grouped into three categories:
- Muscarinic;
- Nicotinic;
- CNS effects.
The earliest signs are usually muscarinic and can include:
- Hypersalivation;
- Lacrimation;
- Urination;
- Gastrointestinal hypermotility and defecation, diarrhoea;
- Increase in respiratory sounds because of bronchoconstriction and/or excessive bronchial secretions;
- Bradycardia;
- Miosis.
The muscarinic signs can be attenuated or completely masked by sympathetic stimulation (caused by stress or ganglionic stimulation through inhibition of AChE) which induces opposite effects (mydriasis, tachycardia, etc.)
Subsequently, the nicotinic-type signs (stimulation of skeletal muscles) appear:
- Muscle rigidity;
- Fasciculation, tremors;
- Muscle weakness;
- Paralysis.
The CNS symptoms are due to the accumulation of ACh and vary according to the species:
- Agitation;
- Anxiety;
- Hyperactivity;
- Convulsions (more probable in dogs);
- Subsequent depression.
Death due to poisoning by organophosphates or carbamates can occur as a result of one or more of the following causes:
- Hypoxia due to an increase of secretions in the respiratory tree and bronchiolar constriction, worsened by bradycardia;
- Respiratory depression as a result of paralysis due to nicotinic stimulation;
- Respiratory paralysis due to effects on the CNS.
DIAGNOSIS
The diagnosis of organophosphate or carbamate intoxication is based on the history, known contact with the substance, and a decrease in cholinesterases in whole blood (not in the serum). This last test, although not conclusive, should be carried out on whole blood taken from the living animal or cerebral tissue in the case of an already dead animal (in this circumstance, the tissue should be collected as soon as possible and frozen immediately). The assay is considered positive and, therefore, to indicate exposure and intoxication if there is a greater than 70% reduction in the activity of cholinesterases compared with normal levels of activity. It is, however, necessary to consider that there is great inter-species variability of normal values of AChE activity.
In most species, the most sensitive test for determining exposure to organophosphates is an assay of plasma pseudocholinesterases rather than whole blood cholinesterases; a fall in these latter does, therefore, more probably indicate severe intoxication.
The prognostic value of this test is difficult to determine, particularly in cats, because pseudocholinesterases are very sensitive and represent a large proportion of the total cholinesterase activity in the blood. Consequently the cat is very sensitive to a total decrease in enzymatic activity, even after exposure to amounts of organophosphates well below those able to induce evident toxic effects.
An unofficial, but useful test for evaluating the possibility of poisoning by a cholinesterase inhibitor is the atropine test: a dose of atropine 0.02-0.04 mg/kg is administered intravenously and if signs of atropinisation (tachycardia, dry mouth, mydriasis) occur, the probability that a cholinesterase inhibitor is responsible for the poisoning of the animal undergoing the test is very low.
The search for and identification of residues of compounds or their metabolites is the ideal diagnostic approach, although neither easy nor commonly performed. Tissue concentrations of carbamates and organophosphates decrease rather quickly after an animal’s exposure to the product. Samples collected before death (vomit, gastric contents, fur potentially contaminated by topical exposure) or post-mortem (liver, body fat, skin) must be frozen immediately and maintained in this state until analysis in the laboratory. The suspected source of exposure (container, food, water, etc.) should also be conserved in order to identify the substance responsible for the poisoning.
TREATMENT
Atropine sulphateis the antidote to poisoning by both carbamates and organophosphates. Before starting treatment with the antidote it is advisable to carry out a gastric lavage or induce vomiting (if the ingestion was recent) or wash the animal thoroughly, taking care to wear gloves, if the exposure was cutaneous. The administration of activated charcoal is useful in all species in order to limit further absorption of the poison. Intravenous fluid therapy should be given to compensate for the loss of fluids.
Atropine sulphate is administered at a dose of 0.2 mg/kg (of which one-quarter intravenously and the remainder intramuscularly or subcutaneously). The treatment can be repeated (at intervals of 3 to 6 hours) according to the reappearance and severity of the respiratory signs (laboured breathing, dyspnoea, etc.), cyanosis, and heart rate. Signs such as miosis and salivation, which are not risk indicators for survival, are not used as therapeutic indicators following the administration of atropine.
Treatment with an oxime (pralidoxime chloride, 2-PAM) reactivates phosphorylated AChE (from the phosphoric ester) by cleaving the phosphorus group from the esterase site of the enzyme. The dose is 20 mg/kg i.v. or i.m. Pralidoxime is ineffective in the case of carbamate poisoning and is sometimes contraindicated. However, if the substance responsible for the intoxication is not known, it is worthwhile administering pralidoxime (in addition to atropine); if no response is seen after three or four administrations, the efficacy of the drug is minimal and it is, therefore, better to interrupt the treatment.
The administration of pralidoxime should be started within 24 hours of exposure to the toxic substance. In the case of “ageing” (see Mechanism of action), oximes are ineffective. The absorption of organophosphate insecticides through the skin is slow and can take several days; oximes can, therefore, be useful in the case of transdermal intoxication even when some days have passed since the exposure. The toxicity of pralidoxime is generally low; cases of overdosage are manifested as tachycardia and cardiac arrhythmias.
The use of diphenhydramine is controversial. This drug acts in the dog by counteracting nicotinic effects and could be a valid additional treatment in poisoned animals who do not respond to treatment with atropine and oximes. The dose in the dog must be adapted to the individual case, but usually varies from 1 to 4 mg/kg per os every 6 to 8 hours. Care must be taken when using this drug to avoid CNS depression, particularly when high doses of atropine have already been used.
ORGANOPHOSPHATE-INDUCED DELAYED POLYNEUROPATHY - Besides acute toxicity, some organophosphates can cause a polyneuropathy characterized by distal and retrograde degeneration of axons and myelin sheaths of motor and sensory neurones of both peripheral nerves and nerves of the spinal cord. This organophosphate-induced delayed polyneuropathy (OPIDP) is not due to inhibition of AChE, but rather to the binding of the organophosphates with esterases present in neurones, named “neuropathy target esterases” (NTE), and subsequent “ageing” of the complex thus formed, after phosphorylation and successive dealklyation. This syndrome can appear following a single exposure or repeated exposures and the clinical signs develop insidiously 1 to 2 weeks after exposure in many animals (in humans from 2 to 4 weeks after). Various degrees of posterior paralysis, weakness and ascending ataxia and paralysis, usually flaccid, can be observed. There are signs of reduced motor neurone activity and muscle atrophy. Death occurs as a result of respiratory paralysis. The animals most frequently affected are cattle, swine, poultry and cats.
Suggested readings
- Amorena M, Caloni F, Mengozzi G. Epidemiology of intoxications in Italy. Vet Res Commun 2004; 28: 89-95.
- Baynes E. Ecoparasiticides. In: Veterinary Pharmacology and Therapeutics. Riviere JE, Papich MG (eds). 9th edn. 2009 Wiley-Blackwell, Ames, Iowa USA.
- Berny P, Caloni F, Croubels S, et al. Animal poisoning in Europe. Part 2: Companion animals. Vet J 2010; 183: 255-9.
- Costa LG. Current issues in organophosphate toxicology. Clin Chim Acta 2006; 366: 1-13.
- Gfeller RW, Messonnier SP. Organofosfati e carbamati: avvelenamento acuto. In: Tossicologia ed Avvelenamenti nei Piccoli Animali. 2005 Poletto Editore
- Giuliano A, Nebbia C. Incidence of poisonings in domestic carnivores in Italy. Vet Res Commun 2004; 28: 83-8.
- Gupta CG. Organophosphates and carbamates. In: Veterinary Toxicology. Basic and Clinical Principles. Gupta CG (ed) 2007 1st edn. Academic Press, Elsevier, USA
- Gupta CG. (Special issue on anticholinesterases). Toxicol Appl Pharmacol 2007; 219: 95-246.
- Lotti M, Moretto A. Organophosphate-induced delayed polyneuropathy. Toxicol Rev 2005; 24: 37-49.
Useful links
Chemicals Regulation Directorate Website
Merck manual