TOXICOLOGICAL INVESTIGATION AND ITS SIGNIFICANCE IN ANIMAL

HEALTH DIAGNOSIS
Diagnosis of a poisoning condition in animals is based on history, clinical signs, laboratory
examinations and test results, post-mortem lesions and response to therapy. Diagnosis of poisoning
could be tentative, presumptive and confirmative. Accurate and rapid diagnosis of intoxication is
challenging, as no single laboratory test detects all possible toxicants. A systematic approach to
collecting all evidence, proper sampling techniques, and good communication between clinician,
technicians, client and laboratory are critical for successful toxicological investigations. Various
evidences may be utilized to successfully investigate a possible case of poisoning, viz: historical,
clinical, circumstantial, pathological and analytical evidences.
Historical evidences:
Detailed history about any change in feed /climate, any animal introduced newly in and around
the habitat.
The number of animals which exhibited toxic symptoms.
The clinical signs that were observed, treatment given.
Use of pesticides/herbicides etc around the near the animal habitat, etc. should be recorded.
Answers for many of the relevant questions to be obtained from the owners/ caretakers
Clinical evidences:
If the animal is already dead, the source of clinical evidence is the caretaker/ranger, animal
attendant or others who have seen the animal at the time of death.
The symptoms noticed differ from animal to animal due to the differences in individual
response at a given dose level, the amount of poison consumed, number of doses consumed
and the interval between them, whether the animal has eaten recently or not, whether the diet
contained factors influencing the solubility and absorption of the poison, general state of
health of the animal, age and breed of the animal.
Sometimes the animals show atypical symptoms. In addition to the clinical signs,
diagnostically helpful information can be gathered from the odour of the breath or colour of
urine or faeces of the affected animal.
The odour of breath will be
phenolic after ingestion of phenol or materials such as creosote
bitter almonds after cyanide
garlic after phosphorous
The colour of the urine will be dark green after ingestion of phenolic compounds and red after
phenothiazine ingestion
The colour of the faeces
dark green due to the formation of a copper-chlorophyll complex in animals suffering
from acute copper poisoning.
Colour of blood and mucous membrane will be.
cherry red in carbon monoxide
bright red in cyanide
chocolate brown in nitrite and chlorate

Circumstantial evidence:
Undertaking thorough search of the premises involved whenever possible bearing in mind the
sources of poison may include fabric and fittings, covering and atmosphere within the
accommodation frequented by the animal, discarded domestic or industrial waste, pesticides,
food and water and any medicaments or mineral supplements provided.
Any changes related to these, which preceded the suspected poisoning incidence, like change of
feed/fodder, spray of green fodder with agrochemicals, urea dressing of crops may provide
valuable information regarding the source and nature of the poison involved.
Looking for any factories, industries in the neighbouring areas, as water and soil contaminated
with these wastes and gas released from these factories can also add to toxic effects
Pathological evidence:
Gross changes observed during post mortem or on histopathological examination of tissue
samples may provide evidence. More characteristic lesions are found in chronic poisoning.
Toxicants cause a variety of lesions ranging from no lesions to specific changes in tissue
structure. No lesions in per-acute poisoning/neurotoxicosis/residue cases are commonly
encountered and produce great confusion in interpretations and judgement.
Presence of specific lesions associated with well defined etiological agents should be correlated
with laboratory diagnosis. In addition to the pattern and type of lesions, the chronicity of those
lesions can also yield clues about the nature of the toxin and exposure pattern.
Presence of non specific lesions for example, hemorrhagic gastroenteritis (for example in
arsenic or even salt poisoning) will also divert accuracy of diagnosis towards false positive of
negative.
The odour, nature and colour of gastrointestinal contents also provide valuable information.
The colour of the gastrointestinal tract contents of interest include
greenish blue for copper sulphate,
yellow to orange green colour for chromium salts,
yellow for nitric and picric acids
black for corrosive acids like sulphuric acid.
Hepatic lesions are produced by such diverse materials as antimony, arsenic, boric acid, iron,
lead, phosphorus, selenium, thallium, chloroform, carbon tetrachloride etc.
Renal lesions are produced by irritant or otherwise nephrotoxic agents like mercury, lead,
ochrotoxin, citrinin, sulphonamides etc.
Analytical evidence:
Toxicological analysis should be considered only when there is sound evidence that the animal
has been poisoned and where at least the group of compounds involved has been established. A written
history and description of signs and lesions should be included with the samples to help the laboratory
diagnostician to provide the best advice regarding selection of tests and interpretation of findings.
It is best to collect as many samples as possible for toxicology testing at the time of necropsy.
Samples can be held frozen (tissues, serum, urine, and milk) or refrigerated (blood) until results of

other tests (eg, histopathology, bacteriology, and virology) are completed before proceeding to specific
toxicological analysis.
Generally, the most useful postmortem specimens for toxicology testing include GI contents,
liver, kidney, urine, brain, and ocular fluid. For most tissue analysis, 5 g of specimen is sufficient for
toxicology testing. Suitable samples for toxicology testing from affected, live animals include
gastrointestinal (GI) contents (rumen lavage fluid, fecal material), urine, whole blood, serum and milk.

Fresh frozen samples, not formalin fixed samples, must be collected for toxicology testing.
To avoid any dilution effect, samples should not be pooled and should be packed separately
in good quality, sterile polythene covers.
Samples of major organs should be placed in 10% buffered formalin for histopathological
examination. The formalin to tissue sample ratio should be 9:1, and the thickness of the
collected specimen should not exceed 0.5 cm.
For most analyses performed on serum, blood, urine, or milk, a 1 mL volume of sample is
sufficient. If excessive testing is expected, volumes of 5 mL are desired
Tissue biopsies, such as liver collection for copper analysis, or fat collection for persistent
organic pollutant analysis, can be useful for certain investigations.
Samples should be stored in separate containers and frozen. Whole blood can be refrigerated
at 4C.
Serum must be separated from the clot as soon as possible before freezing. Some analytes,
such as potassium, magnesium, phosphorus, and zinc, may be altered by time spent in
contact with the clot, because red cells continue to metabolize them or release additional
material.
Toxic plants
During the investigation of suspect plant intoxications, mixed feed from bunks or stalls as fed,
unusual pasture plants, all feed ingredients going into a ration, feed supplements, tags, and labels
should be collected.
In addition, information on recent feed changes (with dates), feed quality (visual),
preparation of mixed feed, feeding practices, feed supplements (recent changes, lot
numbers, use level), lot numbers, storage facilities and conditions, pasture changes, and
weather changes must be obtained.
Diagnosing a plant poisoning can be difficult. In many cases, clinical signs are nonspecific
(such as diarrhea), and postmortem lesions are not characteristic.
In many cases, the best way to support a diagnosis of a plant poisoning is to confirm the
presence of a toxic plant in the animal's environment (this will require positive identification
of the suspect plant), to confirm that the plant has been ingested (noting that the candidate
plants have been chewed and/or finding plant fragments in vomitus or GI tract samples), and
to correlate clinical findings, where possible, with those known to be associated with the
suspect plant..
Toxic residues occurring in extremely low quantities, are analysed quantitatively by the
various laboratory methods viz: Gas chromatography, High-performance liquid chromatography,

Electrophoresis and Electrochromatography, Atomic Absorption Spectrometry, Ultra Violet and

visible spectrum Spectrophotometry, Infrared and Ramans spectrophotometry and Nuclear magnetic
resonance (NMR) spectrometry etc.
SAMPLE SPECIMENS FOR ANALYTICAL TOXICANT DETECTION
Sample

Quantity

Condition

Possible toxicants detectable

ANIMAL SAMPLES
Whole blood

2-3 mL

EDTA
anticoagulant,
heparin for some analyses
(ie, some lead analyses)
refrigerated

Lead, cholinesterase activity, selenium, arsenic,

mercury, cyanide, some organic chemicals,
anticoagulant rodenticides, ammonia

Serum

1-3 mL

Spin and remove clot,

avoid rubber contact for
zinc testing, frozen

Copper, zinc, iron, magnesium, calcium,

sodium,potassium,
nitrate/nitrite,
alkaloids,
oleandrin,
drugs,gossypol,
anticoagulant
rodenticides, amanitins, some
pesticides,
perchlorate, sodium chloride, ammonia

Urine

10 mL

Plastic vial, frozen

Alkaloids, drugs, some metals, cantharidin (blister

beetle), fluoride, paraquat, oleandrin, amanitins,
some pesticides., arsenic

Milk

50-100
mL

Plastic vial, frozen

Antibiotic residues, plant toxins, aflatoxin ,

organochlorine insecticides, some pesticides,
PolyChlorinated Biphenyls, iodide, perchlorate

GI contents
(vomitus,
Ingesta) (live
animal, feces;,
do not pool
samples

500
Good quality sterile
g(each)) polythene covers, frozen,
stomach, small intestine,
and large intestine
contents; keep separated
(in 1% mercuric chloride
for cyanide)

Plant identification, seed identification, cardiac

glycosides ,grayanotoxins, alkaloids, tannins,
insecticides,
drugs,
cyanide,
ammonia,
cantharidin, petroleum hydrocarbons, heavy
metals, ionophores, microcystins, anatoxin-a,
ethylene glycol, 4-aminopyridine, anticoagulant
rodenticides, herbicides, nitrate/nitrite, urea, Zinc
phosphide

Liver

5-10 g

Good quality sterile

polythene covers, frozen

Heavy metals, minerals, oleandrin, insecticides,

organic chemicals, some pesticides, cyanide,
aflatoxin
B1,
anticoagulant
rodenticides,
amanitins,
Zinc
phosphide,
arsenic,
Organochlorines, copper

Kidney (cortex)

5-10 g

Good quality, sterile

polythene covers, frozen

Heavy metals, some plant toxins, ethylene glycol,

oxalates, amanitins, sodium fluoroacetate, Zinc
phosphide , arsenic, Organochlorine insecticides,
copper

Brain

Half of
brain

Saggital section (for ChE

activity), otherwise 5-10,
Good quality, sterile
polythene covers,
frozen

Cholinesterase activity, sodium, macrolide

endectocides, organochlorine insecticides

Ocular fluid

entire eyeball Good quality, sterile

polythene covers, frozen

Nitrate, calcium, magnesium, potassium

Fat

30 g

Good quality, sterile

polythene covers, frozen

Organochlorine insecticides, PCBs

Lung

10 g

Good quality, sterile

polythene covers, frozen

paraquat

Muscle

10 g

Good quality, sterile

polythene covers, frozen

Hair

5g

Selenium (chronic exposure)

CSF

1ml

sodium chloride

Bone

100 g or 1 long bone

Cyanide, selenium

fluorides

ENVIRONMENAL SAMPLES
Baits/source

200 ml or g

Entire suspect bait and label should be submitted if available

Feed

500 g

Multiple representative samples should be taken and then either combined

and mixed for a composite sample or retained as individual samples (to
detect variability in the source), or both .

Forages (pasture, 5 kg
hay, silage)

Samples should be taken from multiple locations in a pasture or storage

facility, using a forage (core) sampler for baled hay or stacks. Silage
should be frozen to prevent mold and deterioration.

Plants Entire plant or representative portion (including root)

Water

1 liter

Fresh; pressed and dry; or frozen

Detection of nitrates, sulphates, total solids, metals, algae, and pesticides.

Legal/ Forensic toxicological cases: In veterolegal cases, with the involvement of police, lawyers, or
insurance companies, the nature of the investigation may change substantially. Specific legal
requirements must be considered, such as chain of custody of samples, proper documentation, with
contacting the various authorities before proceeding .
General therapeutic approach in poisoning condition:
If feed-mixing errors have occurred, the feed should be withheld and good quality of roughage
should be provided to the animals. The ingestion of toxic materials should be immediately
followed with decontamination.
Administration of emetics (hydrogen peroxide, xylazine etc) in monogsatric animals within 2-4
hours of ingestion of toxicant.
Surgical ruminal evacuation (rumenotomy) can be performed if the patient is stable and
without cardiovascular compromise and before the substance is completely absorbed or
transitioned into the intestine. However, the procedure is time consuming and not practical or
economic if numerous cattle are affected.
For herd outbreaks, activated charcoal (AC) has been successfully used as an orally
administered adsorbent of toxicants. It is contraindicated to administer AC with mineral oil,
as the adsorptive properties of charcoal diminish. Dairy calves should be fed at least 3 hours

apart from charcoal administration as feeding interferes with adsorptive properties. Toxicants,
such as metals, are not adsorbed by activated charcoal.
Administration of a purgative agent: magnesium sulfate (Epsom salt) or sodium sulfate
(Glauber's salt) decrease the GI transit time and subsequently time for absorption. If affected
cattle already have diarrhea, there is no need to administer a cathartic agent.
Mineral oil (liquid paraffin) is a lubricant laxative, commonly used in large animals for the
treatment of GI constipation and fecal impaction. Its use is discouraged in intoxications
because of questionable efficiency as adsorbent for toxicants .
Depending on electrolyte and hydration status of the animal, intravenous fluid therapy may be
warranted. If a specific diagnosis has been made, possible antidotes should be administered as
soon as possible.
Intravenous lipid emulsions (ILE)
Also known as intravenous fat emulsions (IFE), they have been used in human and veterinary
medicine as part of total or partial parenteral nutrition for several decades. ILE also has been used as
a vehicle f or drug delivery for emulsions (eg, propofol) and, more recently, it has been recommended
as a potential antidote in the management of compounds that are lipophilic or cardiotoxic. The precise
mechanism of action through which ILE increases the rate of recovery and reduces the severity of
clinical signs in lipophilic drug toxicosis is unknown. ILE therapy is generally considered relatively
safe; however, its use for treatment of toxicities is considered extra-label, and rare side effects can
occur, including fat-overload syndrome, cholesterol deposits into the cornea, and coagulopathy.
ILEs formulations come in 3 concentrations: 10%, 20% and 30%; Toxicity should be
treated with 20% formulations only.ILE has a long storage half-life, and can be stored at
room temperature until it is opened. Once opened, aseptic handling and refrigeration is
necessary; the remaining product should be discarded after 24 hours.
The recommended dose is: to give a bolus of 1.5mL/kg over 5-15 minute minute , IV,
then starting a continuous rate infusion at 0.25mL/kg per minute for 30-120 minutes
In veterinary medicine, ILE is generally initiated earlier in the course of treatment, and is
recommended
For lipophilic toxins when standard therapies fail or when life threatening cardiovascular
events are imminent.
For toxicities associated with lipid- soluble compounds in which a high morbidity has been
reported.
As an adjunct therapy that can lessen hospitalization time and treatment costs.
In most cases the suitability of a compound for treatment with intravenous lipid therapy is
determined by two factors: its lipophilicity and half-life. Lipid infusion is suitable for lipophilic
compounds with short to moderate half-lives (generally less than 24 hours); it is not suitable for
lipophilic compounds with long lives such as vitamin D compounds (e.g. calciferol,
calcipotriol) and anticoagulant rodenticides (e.g. brodifacoum, bromadiolone)
Toxicants That Have Been Successfully Treated with ILE Therapy include:

Pyrethrins (eg: permethrin in cats), macrocyclic lactones (Avermectins: ivermectin,

moxidectin in dogs), calcium channel blockers(eg: amlodipine), beta blockers(eg: metoprolol),
cholecalciferol, ibuprofen, muscle relaxants (eg: methocarbamol, baclofen), local anesthetics
(lidocaine, bupivacaine) in cats. Other toxicants for which ILE may be used include: cocaine,
tricyclic antidepressants and organophosphates.
DIFFERENTIAL DIAGNOSIS OF TOXICANTS BASED ON IMPORTANT CLINICAL SIGNS

Clinical sign
Abdominal pain

Possible toxicants to be differentiated

Acids, alkalis, ammonium salts, arsenic, carbamates, chlorate, chlorophenoxy
herbicides, copper (sheep), fluoroacetate, lead, mercury (inorganic), nicotine, nitrate/
nitrite, organophosphorus insecticides, paraquat, phenol, phenothiazines, phosphorus,
selenium, thallium, urea, zinc, zinc phosphide
Anaemia
Cadmium, copper (sheep), lead, molybdenum, phenothiazines
Blindness
Arsanilic acid, benzoic acid (cat), carbon monoxide, lead, mercury (inorganic and
organic), molybdenum (lambs), phenothiazines (poultry), selenium, sodium chloride
(pigs and cattle)
Constipation
Calciferol, lead, phenothiazine (horse), sodium chloride (pig)
Convulsions
Alphachloralose, benzoic acid (cat), caffeine, carbamates, carbon tetrachloride,
chlorophenoxy herbicides, copper (sheep), crimidine, cyanide, DNOC, ethylene glycol,
fluoroacetate, garbage (staphylococcal enterotoxin; dog), hydrogen sulphide, lead,
metaldehyde, narcotics, nitrate/nitrite, organochlorine insecticides, organophosphorus
insecticides, phenol, phosphorus, red squill, sodium chloride (pig), strychnine, thallium
Cyanosis
Acrolein, alphnaphtyl thio urea (ANTU), carbamates, chlorate, hydrogen sulphide,
metaldehyde, organochlorine insecticides, organophosphorus insecticides, paraquat
Diarrhoea
Acids, alkalis, ammonium salts, anticoagulant rodenticides, antu, arsenic, cadmium,
calciferol, carbamates, carbon tetrachloride, chlorate, chlorophenoxy herbicides,
chromate, detergents, garbage (staphylococcal enterotoxin; dog), lead, mercury
(inorganic), molybdenum, nicotine, nitrate/nitrite, organophosphorus insecticides,
phenothiazine (horse), red squill, reserpine, thallium,, urea, urea herbicides, zinc
Dilated pupils
Barbiturates, cyanide, metaldehyde, strychnine,snake venom.
Dyspnoea
Acrolein, ammonium salts, anticoagulant rodenticides, ANTU, carbamates, carbon
monoxide, chlorate, chromate, cyanide, DNOC, hydrogen sulphate, lead (horse),
nitrate/nitrite, organophosphorus insecticides, kerosene (paraffin), paraquat,
phenothiazine, selenium, sulphur, thallium, TOCP, triazine herbicides, urea
Excitement
Alphachloralose, benzoic acid (cat), caffeine, cannabis, cyanide, ethylene glycol,
fluoroacetate, lead, metaldehyde, narcotics (cat, horse), nicotine, organochlorine
insecticides, strychnine
Haematuria
Anticoagulant rodenticides, chlorate, mercury (inorganic and organic)
Haemoglobinuria
Chlorate, copper (sheep), phenothiazines
Incoordination
Alphachloralose, ammonium salts, ANTU, arsenic, barbiturates, benzoic acid (cat),
caffeine, cannabis, carbamates, carbon monoxide, carbon tetrachloride, chloral
hydrate, chlorate, chlorophenoxy herbicides, chlorpromazine, ethylene glycol, garbage
(staplyococcal enterotoxin; dog), mercury (inorganic and organic), metaldehyde,
molybdenum, nicotine, nitrate/nitrite, organochlorine insecticides, organophosphorus
insecticides, paraquat, phenol, phenothiazines (pig), red squill, salicylates (cat),
selenium, sodium chloride (pig), triazine herbicides, urea, urea herbicides
Jaundice
Arsenic, carbon tetrachloride, copper (sheep), paraquat, phenol, phenothiazines,
phosphorus
Lameness
Anticoagulant rodenticides, fluoride, molybdenum, selenium
Muscle tremors Carbamates, cyanide, lead, mercury,organochlorine insecticides, organophosphorus
insecticides, phenol, sodium chloride (pig), strychnine, triazine herbicides.
Paralysis
Carbon monoxide (pig), copper (sheep), cyanide, nicotine, organophosphorus
insecticides, phenothiazines, phosphorus, selenium, sodium chloride (pig
Salivation
Alphachloralose, ANTU, arsenic, benzalkonium chloride, benzoic acid (cat),
carbamates, copper, cyanide, detergents, lead, mercury (inorganic),nicotine,
organochlorine insecticides, organophosphorus insecticides, phosphorus, sodium
chloride (pig), strychnine, triazine herbicides

Thirst
Vomiting

Arsenic, calciferol, chlorate, chromate, , sodium chloride

Acids, alkalis, anticoagulant rodenticides, ANTU, arsenic, benzalkonium chloride,
carbamates, chlorophenoxy herbicides, copper, ethylene glycol, fluoroacetate, garbage
(staphylococcal enterotoxin; dog), lead, narcotics (dog), nicotine, organochlorine
insecticides, organophosphorus insecticides, kerosene (paraffin), paraquat, phosphorus,
red squill, reserpine, rotenone, salicylates (cat), thallium, zinc

ANTIDOTAL THERAPY FOR COMMON POISONINGS IN LARGE ANIMALS

Poison/toxicity
Heavy metals
(Arsenic,antimony,
mercury,cadmium,
chromium, lead,
nickel etc)
Lead

Warfarine
Bromodiolone
Pyrethroids
(Deltamethrin,

Antidote/ treatment : Dosage and method

British anti-Lewisite(BAL)(Dimercaprol)
BAL: 3mg/kg as 5% mixture of 10% benzyl benzoate in mineral oil. Give deep i.m
injection every 4hr. on first two days, every 6hr. on third day and then b.i.d for next 10
days. In cattle and horse sodium thiosulfate can also be used @ 8-10gram in the form of
10-20%solution (i.v) or 20-30gram per-orally in 300ml water.
Calcium disodium EDTA 6.6% solution @ 73 mg/kg,i.v (repeat or two treatment daily
for 3 days if required). Combined thrapy with thiamine HCl @2-4mg/kg/day, s.c is more
effective, Anticonvulsants, if required
Vitamin- K1: 300-500mg, S.C,every 4-6hr.Blood transfusion transfusion @ 20ml/kg or
plasma @ 9ml/kg body wt; Sedatives/tranquilizer
Diazepam HCl + Atropine SO4 ; Diazepam: 0.5-2 mg/kg, i.v Atropine SO4 as required
Activated charcoal (1-2kg), P.O Fluid therapy

Cypermethrin etc.)
Carbamate (carbaryl, propoxur,
aldicarb etc) insecticides

Atropine sulphate : 0.25mg-0.5mg/kg, intravenously and remaining

by intra-muscular or sub-cutaneous route
Organophosphates Atropine sulphate + 2-PAM
Or DAM(diacetyl -monoxime)
(malathion,
Atropinization: 0.25mg-0.5mg/kg,give i.v,remaining by i.m or s.c for every 3-6
parathion,
hours. Observe for papillary dilatation and recovery symptoms and continue treatment as
chlorpyriphos,
required.
echothiopate etc)
2-PAM: 20%solution @ 25-50mg/kg,i.v or intra-muscualrly, Activated charcoal,P.O
Fluid therapy and supportive care
Organochorines
Pentobarbitone sodium
30mg/kg,i.v Or chloral hydrate
(D.D.T,gamma
benzene
hexa 5g/45 kg ,i.v and supportive care
chloride(BHC)/lindane. endosulphan etc.,)
Dinitro-herbicides In ruminants only: Treat for methaemoglobinemia with Methylene blue:
(Dinitro-orthocresol 2-4%,8-10mg/kg, i.v every 8hr.
Or Ascorbic acid: 5-10mg/kg,i.v; every8hr. for
-DNOC
& first 24-48hr. Note: Antipyretics are contraindicated. Saline purgatives are administered.
Dinitrophenol-DNP)

*****