Toxicology Laboratory Manual

TOXICOLOGY LABORATORY
MANUAL
[For Students of Toxicology (Pharmacology) of Veterinary, Medical and Pharmacy Sciences]
By
DR. GOVIND PANDEY

Professor / Principal Scientist & Sectional Head,
Department of Pharmacology & Toxicology, College of Veterinary Science &
Animal Husbandry, Rewa (The Nanaji Deshmukh Veterinary Science University,
Jabalpur), MP, India
and
DR. YASH PAL SAHNI

Director of Research Services,
The Nanaji Deshmukh Veterinary Science University, Jabalpur, MP, India
2013
International E – Publication
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International E - Publication
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© Copyright Reserved
2013
All rights reserved. No part of this publication may be reproduced, stored, in
a retrieval system or transmitted, in any form or by any means, electronic,
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permission of the publisher.
ISBN: 978-93-83520-03-9
International Science Congress Association

iii
PREFACE
“Toxicology” involves the absorption, distribution, metabolism and excretion of a

‘poison/toxicant’. ‘Toxicosis’, ‘toxicity’, ‘poisoning’ and ‘intoxication’ are synonyms for
the disease caused by a poison. Toxicity (sometimes used instead of poisoning) refers to
the amount of a toxicant which is necessary to produce a detrimental effect. ‘Toxic effect’
is the response to drug, which is harmful to the health or life of the individual. All toxic
effects are dose dependent. A dose may cause undetectable, therapeutic, toxic or lethal
effects. With respect to the chemicals which are capable of causing harm, the ‘hazard’ is
about equivalent in meaning to the toxicity. ‘Median lethal dose’ (LD50) is the dose
which is lethal to 50% of a test sample. Poisoning potential is usually determined more
by the multitude of related factors than by the actual toxicity of poison. In deed, there are
a huge number of substances which can cause toxicity or poisoning in both humans and
animals. Majority of substances are drugs, chemicals (especially metals and nonmetals)
and plants (or plant products). Diagnosis of a poisoning, as with any disease, is based on
the history, clinical signs, lesions, laboratory examinations, and in some cases, analytical
procedures. Poisoning therapy principles include: prevention of further absorption of
poison, administration of specific antidotes and supportive/symptomatic therapy.
Hence, to describe the above aspects experimentally, this “Toxicology Laboratory

Manual” has been putforth to enable the students to have a worthy knowledge of
practical veterinary toxicology. This toxicology manual will certainly fulfil the aims of
the concerned students, teachers, as well as the field veterinarians. The experiments
included in the manual are: toxicology and poisoning : an overview; poisoning by
metals, nonmetals and plants/weeds; determination of median effective and median lethal
doses; collection and dispatching of samples for toxicological tests; preparation of
toxicological kit for treatment of poisoning; and detection of metals, lead, mercury,
antimony, fluoride, copper, zinc, cyanide, nitrate/nitrite, urea, chloral hydrate,
phenobarbitone/barbiturates, sulphonamides, acetyl salicylic acid, insecticides, alkaloids,
strychnine, glycosides, digitalis and tannins.
Lastly, thanks are due to all those who have helped us in writing this manual. Our
sincere thanks and deep regards are devoted to Dr. A.B. Shrivastav, Director, Centre for
Wildlife Forensic & Health, The Nanaji Deshmukh Veterinary Science University
(NDVSU), Jabalpur, MP for the “Foreword” for this manual. We thankfully acknowledge
to the ‘Laboratory Manual: Toxicology’ (compiled by Dr. Neetu Rajput, Dr. Vidhi
Gautam and Dr. Y.P. Sahni) and ‘The Merck Veterinary Manual : Toxicology’ (Merck
Sharp & Dohme Corp., a subsidiary of Merck & Co., Inc., Whitehouse Station, NJ,
USA), besides several authors/publishers/books/websites, from where the matters and
photographs have been taken and included in the manual.
26th July, 2013 Dr. Govind Pandey

Dr. Y.P. Sahni

iv
CONTENTS
S. No. Description Page

- Foreword iii
- Preface iv
- Contents v
1 Toxicology and Poisoning : An Overview 1
Objective 1
Certain Terms of Toxicology 1
Absorption, Distribution, Metabolism and Excretion of 3
Poisons
Factors Affecting Poisoning 5
Diagnosis of Poisoning 7
Poisoning Therapy Principles 8
2 Poisoning by Metals and Nonmetals 10
Objective 10
Toxic Metals and Nonmetals 10
Metals Poisoning/Toxicity 11
3 Poisoning by Plants/Weeds 14
Objective 14
Plants Poisoning in Animals 14
Factors Influencing Plants Poisoning in Animals 14
Poisoning by Plant Poisons 15
Other Important Poisonous Plants 23
Other Kinds of Poisoning/Injury by Plants 26
4 Determination of Median Effective and Median Lethal Doses 32
Objective 32
Definition of ED50 and LD50 32
Method 33
5 Collection and Dispatching of Samples for Toxicological Tests 39
Objective 39
Principle 39
Required Samples for Diagnosis of Poisoning 39
Containers Required for Collection of Samples 39
Tissues Required for Analysis 41
Preservation of Test Samples 41
Dispatching/Submission of Test Samples 42
Examinations/Tests for Diagnosis of Poisoning 43
Precautions 48
6 Preparation of Toxicological Kit for Treatment of Poisoning 49
Objective 49
Measures Taken in Suspected Poisoning 49
Toxicological Kit 49
Gastric Lavages 50
Adsorbents 51

v
Emetics 51
Purgatives (Cathartics) 52
Diuretics 52
Antidotes 52
Miscellaneous Drugs in Toxicological Kit 53
7 Detection of Metals Poisoning 55
Objective 55
Toxicity of Metals in Animals 55
Detection of Metals 55
8 Detection of Lead Poisoning 57
Objective 57
Toxicity of Lead in Animals 57
Detection of Lead 57
9 Detection of Mercury Poisoning 59
Objective 59
Toxicity of Mercury in Animals 59
Detection of Mercury 59
10 Detection of Antimony Poisoning 62
Objective 62
Toxicity of Antimony in Animals 62
Detection of Antimony 62
11 Detection of Fluoride Poisoning 64
Objective 64
Toxicity of Fluoride in Animals 64
Detection of Fluoride 64
12 Detection of Copper Poisoning 65
Objective 65
Toxicity of Copper in Animals 65
Detection of Copper 65
13 Detection of Zinc Poisoning 67
Objective 67
Toxicity of Zinc in Animals 67
Detection of Zinc 67
14 Detection of Cyanide Poisoning and its Treatment Test 69
Objective 69
Toxicity of Cyanide in Animals 69
Mechanism of Action of Cyanide 69
Clinical Signs of Cyanide Toxicity 69
Detection of Cyanide 70
Treatment Test of Cyanide Toxicity 70
15 Detection of Nitrate/Nitrite Poisoning and its Treatment Test 72
Objective 72
Toxicity of Nitrate/Nitrite in Animals 72
Mechanism of Action of Nitrite 72
Clinical Signs of Nitrite Toxicity 73

vi
Detection of Nitrate/Nitrite 73
Treatment Test of Nitrite Toxicity 74
16 Detection of Urea Poisoning 76
Objective 76
Toxicity of Urea in Animals 76
Detection of Urea 76
17 Detection of Chloral Hydrate Poisoning 78
Objective 78
Toxicity of Drugs in Animals 78
Toxicity of Chloral Hydrate in Animals 78
Detection of Chloral Hydrate 78
18 Detection of Phenobarbitone/Barbiturates Poisoning 80
Objective 80
Toxicity of Phenobarbitone or Barbiturates in Animals 80
Detection of Phenobarbitone or Barbiturates 80
19 Detection of Sulphonamides Poisoning 82
Objective 82
Toxicity of Sulphonamides in Animals 82
Detection of Sulphonamides 82
20 Detection of Acetyl Salicylic Acid Poisoning 84
Objective 84
Toxicity of Acetyl Salicylic Acid in Animals 84
Detection of Acetyl Salicylic Acid 84
21 Detection of Insecticides Poisoning and its Treatment Test 86
Objective 86
Toxicity of Organophosphate Insecticides in Animals 86
Mechanism of Action of Organophosphate Insecticides 86
Symptoms of Organophosphate Insecticides Toxicity 86
Treatment Test of Malathion Toxicity 87
22 Detection of Alkaloids Poisoning 89
Objective 89
Toxicity of Alkaloids in Animals 89
Detection of Alkaloids 89
23 Detection of Strychnine Poisoning and its Treatment Test 92
Objective 92
Toxicity of Strychnine in Animals 92
Mechanism of Action of Strychnine 92
Clinical Signs of Strychnine Toxicity 92
Detection of Strychnine 92
Treatment Test of Strychnine Toxicity 93
24 Detection of Glycosides Poisoning 95
Objective 95
Toxicity of Glycosides in Animals 95
Detection of Glycosides 95
25 Detection of Digitalis Poisoning 97

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Objective 97
Toxicity of Digitalis in Animals 97
Detection of Digitalis 97
26 Detection of Tannins Poisoning 99
Objective 99
Toxicity of Tannins in Animals 99
Detection of Tannins 99
- About the Authors 100

viii
1
TOXICOLOGY AND POISONING : AN OVERVIEW
OBJECTIVE
To discuss general consideration of toxicology and poisoning in animals.
CERTAIN TERMS OF TOXICOLOGY

“Toxicology” is the evaluation of ‘toxicosis/toxicity’ and deficiencies, identification
and characterization of ‘toxins’, determination of their fate in the body, and treatment of
‘toxicosis/toxicity’. Veterinary toxicology can be challenging because of the low
frequency of cases noticed in a practice setting. When a ‘toxicosis’ occurs, it usually
involves a huge number of animals, and may also involve litigation.
‘Toxic agent’ is a ‘toxicant’ or ‘poison’. The term ‘toxin’ refers to ‘poison’ produced
from the biological sources (e.g., venom, plant toxin, etc.); the redundant term ‘biotoxin’
is occasionally used. The terms ‘toxicosis’, ‘poisoning’ and ‘intoxication’ are synonyms
for the disease caused by a ‘toxicant’. ‘Toxicity’ (sometimes incorrectly used instead of
the poisoning) refers to the amount of a ‘toxicant’ which is necessary to produce a
detrimental effect. On the other hand, the term ‘toxicity’ is the state or quality of being
poisonous or capable of causing harm to the exposed humans or animals. Thus, ‘toxicity’
is the degree to which a substance is poisonous, or can cause harm to the exposed humans
or animals. ‘Toxicity’ occurs when a person/animal has accumulated too much of a drug
in the bloodstream, leading to the adverse effects within the body. ‘Drug toxicity’ can
occur when the dose given is too high, or the liver or kidney is unable to remove the drug
from the bloodstream, allowing the drug to accumulate in the body. ‘Acute toxicosis’
means the effects during first 24 hr. The effects produced by prolonged exposure are
called as the ‘chronic toxicosis’. The terms, ‘subacute’ and ‘subchronic’ are used to
cover the large gap between the acute and chronic.
‘Toxic effect’ is the response to drug, which is harmful to the health or life of the

1
individual. The toxic effects may be idiosyncratic or allergic in nature, or pharmacologic

side effects, or may be an extension of therapeutic effect produced by the over dosage.
All ‘toxic effects’ are dose dependent. A dose may cause undetectable, therapeutic, toxic
or lethal effects. ‘Hazard’ is the potential for causing harm, or it is a potential cause of
harm. With respect to the chemicals which are capable of causing harm, the ‘hazard’ is
about equivalent in meaning to the ‘toxicity’. Measuring the ‘hazard’ or ‘toxicity’ of a
chemical is to measure its potency in producing the harm: the lower the dose requires to
produce the harm, the greater the ‘hazard’ or ‘toxicity’, the more hazardous or toxic is the
substance. ‘Side effect’ is the drug effect which is not desirable, or is not the part of a
therapeutic effect; the effect other than those intended.
‘Dose’ is expressed as the amount of compound per unit of body weight and toxicant
concentration as part per million or part per billion. These quantitative expressions are
also applicable for feedstuff, water and air, as well as the tissue level.
‘Median lethal dose’ (LD50) is the dose which is lethal to 50% of a test sample. It is
an estimator of ‘lethality’ and the most common expression used to rate the potency of
‘toxicants’. Other terms used for prediction of illness or ‘lethality’ are- ‘no observed
effect level’ (NOEL), ‘maximum nontoxic dose’ (MNTD) and ‘maximum tolerated dose’
or ‘minimum toxic dose’ (MTD). ‘Therapeutic index’ or ‘therapeutic ratio’ is a
comparison of the amount of a therapeutic agent that causes the therapeutic effect to the
amount which causes ‘death’ (in animal studies) or ‘toxicity’ (in human studies). In other
words, the ‘therapeutic index’ is the ratio given by the ‘lethal dose or ‘toxic dose’ divided
by the ‘therapeutic dose’. In animal studies, the ‘therapeutic index’ is the lethal dose of a
drug for 50% of the population (LD50) divided by the minimum effective dose for 50% of
the population (ED50). ‘Lethality’ is not determined in human clinical trials; instead, the
dose which produces a toxicity in 50% of the population (TD50), is used to calculate the
‘therapeutic index’. While the ‘lethal dose’ is important to determine in animal studies,
there are usually severe toxicities which occur at ‘sublethal dose’ in humans, and these
toxicities often limit the maximum dose of a drug. A higher ‘therapeutic index’ is
preferable to a lower one: a patient would have to take a much higher dose of such a drug
to reach the lethal/toxic threshold than the dose taken to elicit the therapeutic effect.

2
Thus, the ‘therapeutic index’ for animals is obtained from the LD50 divided by ED50 (i.e.,
LD50 / ED50); and for humans, the ‘therapeutic index’ is obtained from the TD50 divided
by ED50 (i.e., TD50 / ED50).
‘Agonist’ is a ligand which binds to a ‘receptor’ and alters the receptor state,
resulting in a biological response. The agonist may be: full, partial or inverse agonist. On
the contrary, an ‘antagonist’ acts against the ‘agonist’, that is to say, the effect of
‘agonist’ is diminished by the ‘antagonist’. Similary, an ‘antidote’ is an agent which
antagonizes the effect of ‘toxicant’. The ‘antidote’ may be of two types: universal and
specific antidotes.
ABSORPTION, DISTRIBUTION, METABOLISM AND EXCRETION OF

POISONS
“Toxicology” involves the absorption, distribution, metabolism and excretion of a
poison/toxicant/toxic agent.
I. Absorption:
‘Absorption’ may occur through the alimentary tract, skin, lungs, via the eye,
mammary gland or uterus, as well as from the sites of injection. The toxic effects may be
local, but the toxicant must be dissolved and absorbed to some extent to affect the cell.
‘Solubility’ is the primary factor affecting the absorption. The insoluble salts and ionized
compounds are poorly absorbed, while the lipid-soluble substances are usually readily
absorbed even through the intact skin; e.g., barium (Ba) is toxic, but barium sulphate can
be used for intestinal contrast radiography because of its low absorption.
II. Distribution:
‘Distribution’ (or ‘translocation’) of a toxicant occurs via bloodstream to reactive
sites, including storage depots. Liver receives portal circulation, and is mostly involved
with ‘intoxication’ (and ‘detoxification’). The selective deposit of foreign chemicals in
various tissues depends on the receptor sites. The ease of chemical translocation depends
largely on its water solubility. Polar- or aqueous-soluble agents tend to be excreted by the

3
kidney; lipid-soluble chemicals are more likely to be excreted via the bile and accumulate
in fat depots. The highest concentration of a toxin within an animal is not necessarily
present in the organ or tissue (‘target organ’) on which it exerts its maximal effect. Lead
(Pb) may be found in highest concentrations in bone, which is neither a site for toxic
effects nor a reliable tissue for toxicologic interpretation. The knowledge of distribution
characteristics of toxicants is essential for proper selection of organs for analysis.
III. Metabolism:
‘Metabolism’ (or ‘biotransformation’) of toxicant by the body is an ‘attempt to
detoxify’. In some cases, the metabolized xenobiotic agents are more toxic than the
original compound. This is called ‘lethal synthesis’. The biotransformation of many
organophosphorous insecticides (OPIs) produces metabolites more toxic than the initial
(or parent) compounds (e.g., parathion to paroxan). There are two phases of
biotransformation: ‘Phase I’ includes oxidation, reduction and hydrolysis. These
reactions, catalyzed by hepatic enzymes, usually convert the foreign compounds to
derivatives for ‘Phase II’ reactions. The products of ‘Phase I’, however, may be excreted
as such, if polar solubility permits the distribution. ‘Phase II’ mainly involves the
conjugation or synthesis reactions. The common conjugates are glucuronides, acetylation
products and combinations with glycine. The metabolism of xenobiotic agents seldom
follows a single pathway. Normally, a fraction is excreted unchanged and the rest is
excreted or stored as metabolites. Significant differences in metabolic mechanisms exist
between species; e.g., because the cats lack forms of glucuronyl transferase, their ability
to conjugate compounds like morphine and phenol is compromised. The increased
tolerance to subsequent exposures of a toxicant, in some cases, is due to the enzyme
induction initiated by the previous exposure.
IV. Excretion:
‘Excretion’ of most toxicants and their metabolites is by way of the kidney. Some
excretion occurs in the digestive tract and some via milk. Many polar and high molecular
weight compounds are excreted by way of the bile. An enterohepatic cycle occurs when

4
these products are excreted from the liver via bile, reabsorbed from the intestine and
returned to the liver. Milk is also an excretion pathway for some toxicants. The excretion
rate may be of primary concern because some toxicants can cause violative residues in
food-producing animals. The route of administration, dose and condition of the animal
may have a profound effect on the excretion rates. The toxicants are removed in the
kidney by glomerular filtration, tubular excretion by passive diffusion and active tubular
secretion. The damage to kidney from the excretion of xenobiotics is specific to the
anatomic location where the excretion occurs. The excretion sites are proximal tubules,
glomeruli, medulla, papilla and loop of Henle. The proximal convoluted tubule is the
most common site of toxicant induced injury.
The important ‘Phase I’ enzymes present in the kidney are cytochrome P450,
prostaglandin synthase and prostaglandin reductase. Cytochrome P450 is present in the
kidney at 10% of the level of the liver. The important ‘Phase II’ enzymes present in the
kidney are UDP-glucuronosyltransferases (UGT), sulphotransferases and glutathione-S-
transferase (GST). Medulla and papilla are the target sites for phenylbutazone and the
tubules for many plant toxins. The loop of Henle is the target site for fluoride and the
glomeruli for immune complexes.
The elimination or disappearance (by metabolic change) of a chemical from an organ
or the body is expressed in terms of ‘half-life’ (t½), defined as the amount of time
required for the disappearance of half of the compound. The rate of elimination is
normally dependent on the concentration of the compound. A constant fraction (e.g., ½)
eliminated per unit of time is called the ‘first-order kinetics’. A metabolic reaction may
dictate the rate of elimination. A constant amount eliminated per unit of time is called the
‘zero-order kinetics’. Different body compartments will likely have different elimination
rates. A ‘two-compartment system’ indicates the elimination, which is initially rapid (e.g.,
from the central or plasma component) and subsequently slower from the peripheral
component (e.g., liver, kidney or fat).
FACTORS AFFECTING POISONING

Poisoning (toxicity or toxicosis) potential is usually determined more by the

5
multitude of related factors than by the actual toxicity of poison (toxicant or toxic agent).
Exposure-related, biologic or chemical factors regulate the absorption, metabolism and
elimination, and thus, influence the observed clinical consequences. Hence, the following
factors can affect the action of any poison:
I. Exposure-related Factors:
For these factors, dose is primarily concerned; however, the exact intake of poison is
seldom known. Duration and frequency of exposure are important. Route of exposure
affects the absorption, distribution and perhaps metabolic pathways. Exposure of a poison
relative to periods of stress or food intake may also be a factor. Following ingestion of
some poisons, emesis may occur if the stomach is empty, but if partly filled, the poison is
retained and poisoning can occur. Environmental factors like temperature, humidity and
barometric pressure, affect the rates of consumption and even occurrence of some toxic
agents. Several mycotoxins and poisonous plants are correlated with seasonal or climatic
changes. For example, the ischemic effects of ergot toxicosis are more often observed
during the winter cold, and plant nitrate levels are affected by the amounts of rainfall.
II. Biologic Factors:

Different species and strains within species react differently to a particular poison
because of variations in the absorption, metabolism or elimination. The functional
differences in species may also affect the likelihood of toxicity, e.g., species unable to
vomit can be intoxicated with a lower dose of some agents. The age and size of animal
are primary factors in poisoning. The distribution and metabolism of xenobiotic agents
are compromised by the underdeveloped microsomal enzyme system in young animals.
The membrane permeability, and hepatic and renal clearance capability vary with age,
species and health. Toxicant amount required to develop pathogenesis is usually
correlated to body weight, but with greater body weight, a disproportionate increase in
toxicity (per unit body weight) of a compound often occurs. The body surface area may
correlate more closely with toxic dose. No measurement parameter is consistent for every
situation. Nutritional and dietary factors, hormonal and health status, organ pathology,

6
stress, and sex, all affect toxicity. Nutritional factors may directly affect the poison (i.e.,
by altering the absorption), or indirectly affect the metabolic processes or availability of
receptor sites. The copper-molybdenum-sulphate interaction is an example of both these.
III. Chemical Factors:

Chemical nature of a poison determines the solubility, which in turn influences the
absorption. The Non-polar or lipid-soluble substances tend to be more readily absorbed
than the polar or ionized substances. The vehicle or carrier of the toxic compound also
affects its availability for absorption. The isomers, including optical isomers, vary in
toxicity. For example, the γ isomer of hexachlorocyclohexane (lindane) is more toxic
than other isomers. The adjuvants are formulation factors used to alter the toxicologic
effect of the active constituent (e.g., piperonyl butoxide enhances the insecticidal activity
of pyrethrins). The binding agents, enteric coating and sustained-release preparations
influence the absorption of active constituent. As the absorption is delayed, the toxicity of
poison decreases. The flavoring agents affect the palatability, and thus the excess amount
of toxic drugs is ingested.
DIAGNOSIS OF POISONING
Diagnosis of a poisoning, as with any disease, is based on the history, clinical signs,
lesions, laboratory examinations, and in some cases, analytical procedures. The
circumstantial evidence is valuable and should be noted, but does not replace a thorough
clinical and postmortem (P.M.) examination. History from the animal’s owner can stress
obvious factors and omit subtle, important details. ‘Sudden death’ is often actually ‘tardy
observation’.
The concerned data and samples should be submitted to the diagnostic laboratory. A
complete history is necessary for developing the scheme of laboratory investigation and
may be valuable in case of litigation. The information should be detailed. For example, a
notation of central nervous system (CNS) signs is insufficient; most animals exhibit some
types of CNS signs prior to death. The exact actions and signs should be described.
Examples of concerned information include the following:

7
a) Number of animals exposed/sick/dead, age, weight, and a chronology of

morbidity and mortality.
b) Clinical signs and course of the disease.
c) Any prior disease conditions.
d) Lesions observed at necropsy, with careful examination of ingesta.
e) Response to treatment (medication should be listed to avoid analytic confusion).
f) Related events, e.g., feed change, water source, other medications, feed additives
pesticide applications, etc.
g) Description of facilities (a drawing or digital photograph may be helpful), access
to refuse, machinery, etc.
h) Recent past locations and when moved.
The diagnostic laboratory should be contacted if there are questions regarding the
appropriate sample, amount, or container.
POISONING THERAPY PRINCIPLES

At initial examination, certain immediate, life-saving measures are necessary.
Besides this, the treatment for poisoning includes the following three basic principles:
A. Prevention of Further Absorption:

Topically applied poisons normally can be removed by thorough washing with soap
and water; clipping of hair or wool is essential. Emesis is of value in dogs, cats and pigs,
if done within few hours of ingestion of poison. The emesis is contraindicated when the
swallowing reflex is absent; the animal is convulsing; corrosive agents, volatile
hydrocarbons or petroleum distillates are involved; or risk of aspiration pneumonia is
imminent. Oral emetics include syrup of ipecac (10-20 ml, p.o. in dogs) and hydrogen
peroxide (2 ml/kg, p.o.). Apomorphine is used in dogs parenterally @ 0.05 to 0.1 mg/kg.
Gastric lavage (using an endotracheal tube and largest bore stomach tube) is done on the
unconscious or anesthetized animal. Head is lowered to a 30° angle and 10 ml of lavage
fluid (water or saline) per kg is gently flushed into the stomach and then removed. This
process is repeated until returned fluid is clear. Cathartics and laxatives may be given in

8
some cases for more rapid elimination of poison from the gastrointestinal tract (GIT). A
gastrotomy or rumenotomy may be necessary when lavage techniques are insufficient (or
too slow in ruminants). When the poison can not be physically removed, certain agents
administered orally can adsorb it, and prevent its absorption from the alimentary tract.
Activated charcoal (1-2 g/kg) is effective in adsorbing a wide variety of compounds, and
is usually the adsorbent and detoxicant of choice when toxicity is suspected.
B. Administration of Specific Antidotes:

Antidotes are listed for each toxicant. Some complex with the toxicants (e.g., oximes
bind with OPIs, and EDTA chelates Pb). Others block or compete for receptor sites (e.g.,
vitamin K competes with the receptor for coumarin anticoagulant). A few affect the
metabolism of poison (e.g., nitrite and thiosulphate ions release and bind cyanide).
C. Supportive/Symptomatic Therapy:
The supportive or symptomatic therapy is generally needed until the toxicant can be
metabolized and eliminated. The type of support needed depends on the animal’s clinical
condition. The supportive efforts may be: control of convulsive seizures, control of
cardiac dysfunction, alleviation of pain, maintenance of respiration, treatment for shock,
and correction of electrolyte imbalance and fluid loss.

9
2
POISONING BY METALS AND NONMETALS
OBJECTIVE
To demonstrate poisoning caused by poisonous (toxic) metals and nonmetals.
TOXIC METALS AND NONMETALS
A. Toxic metals- e.g., antimony (Sb, a metalloid), arsenic (As, a metalloid),

cadmium (Cd), lead (Pb), mercury (Hg), barium (Ba), beryllium, osmium,
thallium, vanadium, and radioactive metals (viz., actinium, thorium, uranium,
radium, transuraniums- plutonium and americium, polonium, and radioactive
isotopes of metallic elements not otherwise strongly toxic- cobalt (Co)-60 and
strontium-90), etc. Aluminium (Al) has no known biological role and its
classification into ‘toxic metals’ is controversial. Its significant toxic effects and
accumulation to tissues are found in impaired renal patients. However, the
individuals with healthy kidneys can be exposed to large amounts of Al with no ill
effects. Thus, Al is not dangerous to persons with normal elimination capacity.
Vanadium poisoning is notable as it is an anticorrosive component of automotive
steel, fragments of which can be left in passengers during automobile accident.
B. Toxic trace elements- e.g., chromium (Cr) as hexavalent Cr(VI), nickel (Ni, its
salts are carcinogenic), copper (Cu), zinc (Zn) and iron (Fe), etc.
C. Toxic nonmetals- Some heavy nonmetals may be erroneously called ‘metals’, as

they have some metallic properties, e.g., selenium (Se) and tellurium, etc.
‘Metals’ are ubiquitous in nature and essential for body functions. ‘Toxic metals’
sometimes imitate the action of essential elements in body, interfering with metabolic

10
process to cause illness. Many metals, particularly ‘heavy metals’ are toxic but some are
essential and some, such as bismuth (Bi) produces low toxic effect. In most of the cases,
Cd, Pb, Hg and radioactive metals cause severe toxicity. ‘Metalloids’, e.g., As and
polonium, may also be toxic. ‘Radioactive metals’ have both radiological toxicity and
chemical toxicity. The metals in an oxidation state abnormal to the body may also
become toxic, e.g., Cr(III) is an ‘essential trace element’, but Cr(VI) is a carcinogen.
Decontamination for toxic metals is different from organic toxins because toxic
metals are elements; they can not be destroyed. Toxic metals may be made insoluble or
collected, possibly by the aid of ‘chelating agents’. They can also be diluted into
sufficiently large reservoirs like sea, because toxicity is function of concentration rather
than amount. However, the bioaccumulation has potential to reverse this. The toxic
metals can bioaccumulate in the body and food chain. Thus, a common characteristic of
toxic metals is the chronic nature of their toxicity. This is particularly notable with
‘radioactive heavy metals’ like radium, which imitates calcium (Ca) to the point of being
incorporated into human bone, although similar health implications are found in Pb or Hg
poisoning. Exceptions to this are Ba and Al, which can be removed efficiently by kidney.
METALS POISONING/TOXICITY
“Toxicity” is a function of solubility. The insoluble compounds as well as the
metallic forms often exhibit negligible toxicity. Toxicity of any metal depends on its
ligands: organo-metallic forms like methylmercury and tetraethyl lead, can be extremely
toxic; while organo-metallic derivatives are less toxic, e.g., cobaltocenium cation.
‘Metal toxicity’ is toxic effect of some metals in certain forms and doses. Some
metals are toxic when they form poisonous soluble compounds. Some metals have no
role, i.e., they are not essential minerals, or they are toxic in a certain form. In case of Pb,
any measurable amount may have ill health effect. Often ‘heavy metals’ are thought as
synonymous, but the ‘lighter metals’ may also be toxic in certain circumstances, like
beryllium; and not all heavy metals are particularly toxic, and some are essential, like Fe.
‘Trace elements’ become poisonous when taken in abnormally high, toxic doses.
The heavy metals like Fe, Cu, manganese (Mn) and Zn in small quantities are

11
essential for good health. The heavy metals, like Pb are also good industrial ingredients,
e.g., used in car batteries. However, the heavy metals become toxic when they do not get
metabolized by the body and end up accumulating in the soft tissues. Ingestion is the
most common route of exposure to heavy metals. In plants, uptake of heavy metals
depends on plant species and bioavailability of metal in the soils. Since most of the
ingestions of heavy metals occur from consumption of plants, then addressing how plants
acquire heavy metals can aid in controlling heavy metal toxicity.
If someone happens to ingest the heavy metals, that alone is not enough to cause
toxicity. In laboratory animals, absorption of toxic metals may occur as a result of the
chronic deficiencies of Ca and magnesium (Mg) in the body and in other cases, excess
levels of Al mobilizes Ca and heavy metals to move from bone to the central neural
tissue. Of the many heavy metals, Pb and As have been found to be higher than federally
set levels in most soils studied, i.e., soils closed to, or near former smelters and tailings
from metal ore mines and those close to fuel-fired electrical plants. It should be noted that
all heavy metals exist naturally in the soils largely in complex forms with other minerals.
Studies involving animals of three species (dairy cattle, growing swine and laying
chickens) indicated that the residues of Pb and Cd metals do not increase appreciably in
major food products obtained from the animals during long-term exposure to subtoxic
dietary concentrations of these heavy metals. Human risk would not be expected by the
consumption of milk, meat or eggs from the animals similarly exposed. Both Pb and Cd
accumulate in the liver and kidney, and Pb accumulates in the bone. A moderate intake of
liver and kidney from Pb-exposed animals appears to exhibit little or no health hazard.
The utilization of liver and kidney from Cd-exposed animals, however, should be
avoided. Besides the poisoning by Pb, other heavy metals are also important to cause
poisoning, which include Cd, Sb, Cr, Hg and As, since these are also of environmental
concern as they can cause illness in both humans and animals. Dogs, cats and cattle are
most likely to be affected by Pb poisoning.
The As is the most common cause of acute heavy metal poisoning in adults (but the
source is not from soils). This heavy metal is released into the environment by the
smelting process of Cu, Zn and Pb, and from the manufacture of chemicals and glasses.

12
The Pb, on the other hand, is the leading cause of heavy metal poisoning with major
source coming from the soils. Excess levels of Pb in soils greater than 400 ppm result
from prior use of Pb paint around houses, Pb-arsenate sprays for pest control, use of
leaded gasoline, locations close to former smelters and tailings from metal ore mines, and
proximity to fossil fuel-fired electrical plants. Therefore, the culprit to look for when
looking at heavy metals in soils is the Pb toxicity.
Some toxic metals/elements and their compounds are shown in Figures 1 to 9.
Fig. 1 Fig. 2 Fig. 3

Some Toxic Metals/Elements and their Compounds- Fig. 1: Lead Chloride; Fig. 2: Arsenic;
Fig. 3: Mercuric Chloride; Fig. 4: Cadmium; Fig. 5: Antimony Trioxide; Fig. 6: Sodium
Fluoride; Fig. 7: Copper Sulphate; Fig. 8: Iron (II)/Ferrous Sulphide; Fig. 9: Zinc Sulphate
[Source of figures: Different websites which are gratefully acknowledged]

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3
POISONING BY PLANTS/WEEDS
OBJECTIVE
To demonstrate poisoning caused by poisonous (toxic) plants/weeds.
PLANTS POISONING IN ANIMALS

Several plants contain chemicals, or accumulate the chemicals which are poisonous
to the livestock. The poisoning can range from minor irritations and slightly lowered
animal performance to severe cases, where the animal is in a great deal of distress and
may die. This means that the plants can be poisonous to the livestock. Some plants
mechanically injure the animals, or may cause irritation of skin on contact.
In this context, the plants can be classified into two groups:
A. Poisonous plants
B. Non-poisonous plants
FACTORS INFLUENCING PLANTS POISONING IN ANIMALS

There are many plant factors which contribute to the toxic principles in plants. The
individual plant species and varieties may differ in their poisonous contents from early
growth to maturity. With some plants, there is an increase in their ability to poison with
advanced stages of growth; whereas with others, the danger lessens. The state of the plant
when eaten may also be important. In some cases, damage to the plant or wilting may
produce poisonous chemicals in the plant which were not present in the fresh material. In
other cases, e.g., with buttercups, the poison is contained in the fresh plants but not dried
ones. Certain parts of a plant may be poisonous and other parts may not be. Rhubarb is a
good example; its leaf stalk is eatable, while the leaves are very poisonous.
Animal factors also influence the ability of plants to be toxic. Different animal
species are susceptible to different poisonous plants. Age of animal is also important; the

14
youngs are often more susceptible than the older ones, but it is not always true. Animals
may build up resistance to certain poisons by being exposed to small quantities at first. If
a large quantity is consumed, they become resistant because their metabolism has already
adjusted to handle the poison. A hungry animal, or an animal with certain dietary
deficiencies can eat more toxic quantities of a poisonous plant than a well fed animal.
POISONING BY PLANT POISONS

Variety of toxic substances are associated with plant poisonings, but for many plant
species, the nature of toxic substances is still unknown. However, most poisonous plants
contain toxic substances/metabolites from one or more of the following groups:
A. Alkaloid:
Alkaloids are complex, alkaline, nitrogenous compounds mostly isolated from the
plants. They have a marked physiological action when administered to animals. Majority
of alkaloids are derivatives of heterocyclic basic compounds such as pyrole, pyridine,
quinoline and isoquinoline. They contain at least one nitrogen atom in a heterocyclic ring.
Thus, the alkaloids are organic basic substances with a bitter taste, e.g., morphine (from
seedpod of opium or poppy- Papaver somniferum, Fig. 10), atropine (from leaf of deadly
nightshade- Atropa belladonna, Fig. 11), nicotine (from nightshades family of plants,
e.g., Nicotiana tabacum, Fig. 12; species of Solanum, Datura, Mandragora; and A.
belladonna), quinine (from flower of Cinchona pubescens, Fig. 13) and strychnine (Fig.
14, from fruit and seed of Strychnos nux-vomica, Fig. 15).
The alkaloids are colourless, crystalline compounds usually in powder form, but
some may be liquid. They are soluble in organic solutions. They are mostly stored in
stems or barks, and are regarded as the by-products of plants.
The alkaloids are highly poisonous, but are used medicinally in very small quantities,
such as quinine, morphine, cocaine (from leaf and fruit of Erythroxylum coca, Fig. 16 and
E. novogranatense), piperine (from fruit of black pepper- Piper nigrum, Fig. 17 and long
pepper- P. longum), ephedrine (from Ephedra plant- Ephedra sinica, Fig. 18), arecholine
(from nut or fruit of areca nut or betel tree- Areca catechu, Fig. 19) and berberine (from

15
fruits of Berberis aristata, Fig. 20 and other Berberis species). The alkaloids usually exist
as salts of organic or inorganic acids, and some lie in free state. Rarely, the alkaloids exist
in combination with sugars (e.g., solanine from fruits of Makoi- Solanum nigrum, Fig. 21
and other species of Solanum), as amides (e.g., piperine), or as esters (e.g., atropine).
The toxic alkaloids are found in plants like swamp and death cama, lupine (lupin-
Lupinus perennis, Fig. 22), buttercup, marsh marigold (kingcup- Caltha palustris, Fig.
23), larkspur, nightshade, squirrel corn and Dutchman’s breeches (Dicentra cucullaria).
In general, the alkaloids are irritating to the gastrointestinal tract (GIT) producing
nausea, colic and diarrhoea, and also act on the CNS to produce blindness, muscular
weakness, convulsion and death.
B. Glycoside:
Glycosides are natural plant products which contain the sugar glucose. They can be
subdivided into three main groups:
1. Cyanogenic (cyanogenetic) glycosides- They are not themselves poisonous but

in the presence of certain enzymes, they are hydrolyzed and produce ‘cyanide’
(Fig. 24) or hydrocyanic acid (hydrogen cyanide, HCN) which is highly toxic.
The HCN interferes with oxygen exchange from lungs to the body tissues so that
various tissues, including brain are starved for oxygen, and are consequently
injured. The symptoms are muscle tremor, rapid respiration and convulsion. Often
these are not seen as the death occurs within minutes. Many factors influence the
amount of cyanogenic glycosides in plants. Some plant species normally have
high levels, the highest levels occurring in early growth stages and decreasing as
the plants mature. Climatic conditions, soil factors, shade and other factors which
slow the plant growth and development increase the cyanogenic glycoside
content. Low soil moisture, high nitrogen and low phosphorus, all cause the HCN
production. Wilting, frost and other physical damages to plants may induce a
rapid increase in the HCN content. These glycosides occur in species of Sorghum
(e.g., Sorghum bicolor- jowar, Fig. 25; S. bicolor subspecies drummondii- sudan

16
grass, Fig. 26), millet, corn, linseed (Linum usitatissimum- flax, Fig. 27), lotus,
species of Acacia (e.g., A. nilotica- gum arabic tree or babul, Fig. 28), species of
Eucalyptus (e.g., E. tereticornis- eucalypts, Fig. 29), apricot, peach, apple, velvet
grass (Yorkshire fog- Holcus lanatus, Fig. 30), marsh-arrow grass, wild cherry
(Prunus avium, Fig. 31), wild clover, etc.
2. Saponin glycosides- They produce a violent gastroenteritis with vomiting,

diarrhoea and colic. If the saponin glycosides are absorbed into the bloodstream,
they cause a breakdown of red blood cells (RBCs) and injury to the CNS,
producing convulsion and paralysis. Saponin glycosides are found in purple
cockle (Agrostemma githago- corn cockle, Fig. 32), cow cockle, bouncing bet and
poke weed (Phytolacca acinosa, Fig. 33).
3. Mustard oil glycosides- They are found in plants belonging to the species of
mustard (e.g., Brassica campestris- sarson, Fig. 34; Br. nigra- black mustard; Br.
Napus- rape or colza; Br. juncea- oriental mustard; etc.). These glycosides
produce severe gastroenteritis, severe colic and purging.
The examples of some important glycosides are: digitalis (viz., digoxin, digitoxin
and digitalin), isolated from Digitalis purpurea (Fig. 35); amygdalin, isolated from the
species of Prunus, e.g., P. amygdalus (almond, Fig. 36; synonymous species are- P.
dulcis, Amygdalus communis and A. dulcis), P. pursica, P. domestica, P. laurocerasus, P.
armeniaca and P. serotina; linamarin (cyanogenic glycoside), isolated from the plants
such as Cassava utilissima (cassava, Fig. 37; or Manihot esculenta), lima beans and flax
(linseed, Fig. 27); oubain, isolated from Strophanthus gratus (Fig. 38); and gynocardin,
isolated from Gynocardia odorata (Fig. 39).
C. Tannin:
Tannins are present in the young leaves and buds of the species of Quercus (e.g., Q.
robur- oak, Fig. 40), which is toxic to grazing animals. The toxic principles in oak are
gallotanins or their metabolites, which mainly produce the nephrotoxicity.

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D. Nitrate/Nitrite:
Nitrate (Fig. 41) poisoning in animals is actually nitrite poisoning occurring when
nitrate is reduced to nitrite in the gastrointestinal tract (GIT). The nitrite is absorbed into
the bloodstream, where it reacts with haemoglobin (Hb) to form methaemoglobin. This
compound (brown in colour) is incapable of releasing oxygen. In acute cases of poisoning
in cattle, 60 to 80% of total Hb is comprised of methaemoglobin. Sheep generally do not
develop as much methaemoglobin, and are therefore, more resistant to nitrite poisoning.
Common plant species (crops and weeds) which are involved in nitrate/nitrite
poisoning are mentioned in Table 1.
Table 1: Certain Plant Species Involved in Nitrate/Nitrite Poisoning
Crop Weed
Barley Annual sow thistle
Broccoli Canada thistle
Celery Lamb’s quarters
Corn Milk thistle
Cucumber Perennial sow thistle
Kale Poison hemlock
Mangel Prickly lettuce
Oat Prostrate pigweed
Rape Rough pigweed
Rutabaga Russian thistle
Rye Spotted spurge
Sorghum Tumbling pigweed
Squash Wild morning glory
Sudan grass Witch grass
Sugar beet
Turnip
Wheat
Some plant species are naturally good accumulators of nitrates. The legume and
grass species which are used for pasture, or hay crops are not considered good nitrate
accumulators, but the right conditions can accumulate the concentrations of nitrate that
are potentially hazardous. There is a direct response in plant nitrate concentration to
increasing levels of nitrogen fertilization. The nitrate accumulation is greater when nitrate
fertilizers are used than when either urea or ammonium sulphate is the nitrogen source. A

18
number of environmental conditions can influence the accumulation of nitrates in plants

by altering mineral metabolism in the plant. The drought, uneven distribution of rainfall
and low light intensity have been identified as climatic factors, which bring about an
accumulation of nitrates and nitrites in the stems and leaves of plants.
The symptoms of acute nitrite poisoning are trembling, staggering, rapid breathing,
and death. Chronic poisoning may result in poor growth, poor milk production and
abortion. In cattle, there is evidence that vitamin A storage is affected.
E. Urea:
Urea (or carbamide, Fig. 42) is an organic compound with the chemical formula
CO(NH2)2. Urea serves an important role in the metabolism of nitrogen-containing
compounds by animals, and is the main nitrogen-containing substance in the urine of
mammals. It is a colourless, odourless, solid, highly soluble in water and practically non-
toxic. Its LD50 is 15 g/kg for rat. When urea is dissolved in water, it is neither acidic nor
alkaline. The body uses it in many processes, the most notable one being nitrogen
excretion. The urea is widely used in fertilizers as a convenient source of nitrogen. It is
also an important raw material for the chemical industry.
More than 90% of world industrial production of urea is destined for use as a
nitrogen-release fertilizer. Urea has the highest nitrogen content of all solid nitrogenous
fertilizers in common use. Hence, it has the lowest transportation costs per unit of
nitrogen nutrient. Many soil bacteria possess the enzyme urease, which catalyzes the
conversion of urea molecule to two ammonia (NH3) molecules and one carbon dioxide
(CO2) molecule. Thus, the urea fertilizers are very rapidly transformed to the ammonium
(NH4) form in soils. Among soil bacteria known to carry urease, some ammonia-
oxidizing bacteria (AOB) like species of Nitrosomonas are also able to assimilate the
CO2 released by the reaction to make biomass via the ‘Calvin cycle’, and harvest energy
by oxidizing NH3 (the other product of urease) to nitrite, a process termed nitrification.
Nitrite-oxidizing bacteria, especially Nitrobacter, oxidize nitrite to nitrate, which is
extremely mobile in soils and is a major cause of water pollution from agriculture. Nitrate
and NH3 are readily absorbed by plants, and are the dominant sources of nitrogen for
plant growth. Urea is also used in many multi-component solid fertilizer formulations.

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The most common impurity of synthetic urea is biuret, which impairs the plant
growth. The aqueous solution of urea is sprayed, or applied through irrigation systems. In
grain and cotton crops, urea is often applied at the time of last cultivation before planting.
In high rainfall areas and on sandy soils (where nitrogen can be lost through leaching)
and where good in-season rainfall is expected, urea can be side- or top-dressed during
growing season. Top-dressing is also popular on pasture and forage crops. In cultivating
sugarcane, urea is side-dressed after planting and applied to each ratoon crop. In irrigated
crops, dry urea can be used to soil, or dissolved and applied through the irrigation water.
Urea absorbs moisture from the atmosphere and, therefore, is typically stored either
in closed/sealed bags on pallets or, if stored in bulk, under cover with a tarpaulin. As with
most solid fertilizers, the storage of urea in a cool, dry, well-ventilated area is
recommended.
Urea poisoning is an acute, rapidly progressing and highly fatal condition. The
mature ruminants are most commonly affected. The death rate for urea poisoning is high.
The animals exhibit severe abdominal pain, shivering, drunken gait, bloat, salivation,
rapid breathing, violent struggling and bellowing. Some of the causes of urea poisoning
in animals are:
(a) An insufficient diet mix, which could allow pockets of high urea concentration;
the granular urea may also settle out of the dry diets.
(b) An excess of urea in the diet due to miscalculation.
(c) Cattle drinking puddles with high urea content (as the urea, being highly soluble,
washes out of the diet with rain).
To prevent the urea poisoning in animals, avoid poor mixing and keep urea-fortified
diets dry in feeding troughs. Do not feed greater than 1% urea on a dry matter basis.
F. Copper:
Cu may also accumulate in the plants, causing severe toxic effects, particularly when
the soils are rich in Cu or deficient in molybdenum (Mo). The clovers are good
accumulators of Cu, and are generally associated with copper poisoning.

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G. Selenium:
Se is a highly toxic element when taken in larger quantities. In many plants, the level
of Se is related to the level in the soils. The symptoms of Se poisoning include dullness,
stiffness of joints, lameness, hoof deformities and loss of hair from mane or tail. The
acute form of poisoning is often called ‘blind staggers’.
H. Molybdenum:
Mo poisoning can occur when there are abnormally high quantities of Mo in the soil.
The animals pasturing on areas which meet this condition are often subject to acute
scouring. The animals become emaciated, produce less milk, and their coats become
rough and often faded. The legumes, especially red and alsike clovers are usually
associated with the poisoning of Mo. To counteract the effect of Mo, it is essential to add
Cu to the diet of animals. A veterinarian should be consulted first before feeding the Cu.
I. Ergot:
Ergot, a fungal disease of cereal grasses, especially rye (Br. campestris), is caused
by the Claviceps purpurea (ascomycete fungus). Ergot fungus (Fig. 43) infests the
grasses, and if eaten in sufficient quantities, is poisonous due to the production of a
‘mycotoxin’. The presence of ergot is observed by the hard, dark-coloured masses in
flowering grass heads. These purplish or dark-brown masses are usually 2 to 5 times
larger than the grass seed, and are called ‘ergot bodies’. Ergot develops in the barley, oat,
poverty oat grass, foxtail, wheat, rye, red top, bent grass, meadow foxtail, brome grass,
orchard grass, reed canary, timothy fescue, blue grass and quack grass.
The active toxin, ‘ergotoxine’, stimulates the nerve centres which cause contraction
of the small blood vessels supplying the different parts of the body. The result of ergot
poisoning depends largely upon the amount of the fungus consumed. When only small
quantities have been ingested, the recovery without any serious symptoms occurs. When
large quantities have been consumed, dry gangrene in the extremities, possible abortion
in pregnant animals and death may result.
The common symptoms of ergot are lack of appetite, dullness, abdominal pain and
subnormal temperature. However, two distinct symptoms may occur in severe cases:

21
1. Nervous symptoms- Dullness, depression, muscular trembling, convulsion,

contraction of legs and delirium may be seen. Animal suffers from gastrointestinal
catarrh, refuses food and gradually develops a wasting condition. Sometimes, a
very rapid type in which animal dies in spasm or convulsion, is seen.
2. Gangrenous or general symptoms- Blood stoppage due to contraction of small

blood vessels causes necrosis (death) of extremities, particularly of foot, tail, or
ear tips. The affected part is cool and dries up; a small furrow or line of separation
appears and completely surrounds the limb, dividing the living tissue from the
dead. There is little or no loss of blood, and seldom any pus present. Death may
also occur due to the invasion of bacterial organisms, ‘secondary invaders’, as
well as from the gangrene. The cases that do recover may be crippled for life.
J. Mycotoxin:
Besides ergotoxine, other mycotoxins are produced by some fungi that infect the
corns and cereals. Mycotoxins are produced only if the right environments are met, and
these conditions vary depending on the fungus. It is possible for mycotoxin production to
take place while the crop is still standing in the field or after it is harvested and in storage.
Two most common forms of mycotoxins are ‘vomitoxin’ and ‘zearalenone’. The
vomitoxin affects the animal that eats the contaminated feed to vomit. Generally,
however, the animals refuse to eat the feed. Zearalenone is a type of oestrogen. Due to
this, swine (pig) is normally affected. The female pigs show the signs of irregular heat,
immature gilts with a marked swelling, inflammation of the external genital organs and
reduced litter sizes. The male pigs may lose libido.
K. Coumarin:
Coumarin, a chemical found in sweet clover (Melilot- Melilotus officinalis, Fig. 44).
It reduces the palatability of sweet clover, and is associated with a reduced blood clotting
ability in the animals, that eat sweet clover. Coumarin itself, however, does not cause this
latter problem. The coumarin is converted by fungi (including Penicillium, Aspergillus,

22
Fusarium, and Mucor) into a poisonous anticoagulant, called ‘dicoumarol’ (a chemical

derived from coumarin during heating or spoilage of sweet clover hay or silage). This
compound was the historical cause of so-called ‘sweet clover disease’, recognized in
cattle since the 1920s. In fact, the ‘dicoumarol’ produces the bad effects of coumarin. If
the clotting ability of the blood is lowered, it is possible that animals may bleed to death
from the slight wound, dehorning, castrating or from the internal haemorrhage.
OTHER IMPORTANT POISONOUS PLANTS

Calotropis gigantea (Fig. 45) is called ‘Ak or Akanda’ in Hindi and ‘milk weed or
Crown flower’ in English. It grows on waste land all over India. It has white or purple
flowers. When crushed, its leaves and stalks produce milky juice. This ‘milky juice’ is
very toxic and often used to cause the malicious poisoning in humans or animals. Its toxic
principles are calotoxin, calactin and gigantin. These phytotoxins are irritant to skin and
mucous membrane, and produce acute gastroenteritis and cardiotoxicity.
Lantana camara is commonly named as ‘Ghaneri’ in Hindi. L. camara (Fig. 46) is
widely distributed all over India. It has clusters of flowers of different colours. Red
flowered variety is considered toxic. Its toxic principles are lantadene A and B. These
phytotoxins are hepatotoxic, and cause secondary photosensitization.
Datura stramonium (Fig. 47) is named as ‘Datura’ in Hindi and ‘jimson weed or
thorn apple’ in English. It grows commonly on waste places all over the country. It has
bell shaped flowers and spherical fruits, which are covered with sharp spinous
projections. Its seeds are yellowish-brown in colour. Its toxic principles are hyoscine,
hyoscyamine, daturine and traces of atropine. Daturine is a mixture of atropine and
hyoscyamine. Toxins are present in the entire plant, but are more concentrated in seeds.
These hytotoxins elicit muscarinic receptor blocker action.
Ricinus communis is commonly called ‘Arandi’ in Hindi and ‘castor bean’ in
English. R. communis (Fig. 48) is an ornamental plant, and widely distributed all over
India. All parts of the plant are toxic, but the seeds are particularly rich in toxin. Its toxic
principles are ricin and ricinine. These phytotoxins causes gastrointestinal toxicity. Ricin
(a highly toxic protein) may produce severe abdominal pain, drooling, vomiting,

23
diarrhoea, excessive thirst, weakness and loss of appetite. Severe cases of poisoning can
result in dehydration, muscle twitching, tremor, seizure, coma and death.
Nerium odorum (N. oleander/N. indicum) is commonly called ‘Safed or sweet
scented Kaner’ in Hindi and ‘oleander’ in English. N. odorum (Fig. 49) is widely
distributed all over India. It has lanceolate leaves and white or purple flowers. Its toxic
principle is nerin, which is cardio toxic. All parts of N. oleander are considered to be
toxic, as they contain cardiac glycosides that have the potential to cause serious effects,
including GIT irritation, abnormal heart function, hypothermia and even death.
Cerbera thevetia is commonly called ‘Pila Kaner’ in Hindi. C. thevetia (Fig. 50) is
widely distributed all over India. It has lanceolate leaves and large, yellow, bell shaped
flowers. This plant is highly poisonous. Its toxic principles are thevetin and cerberin,
which are cardio toxic.
Argemone mexicana is commonly called as ‘Mexican poppy, prickly poppy or yellow
poppy’ in English. A. mexicana (Fig. 51) grows widely through out India in waste lands
and along road sides. The oil ‘argemone’ from this is used as an adulterant in mustard oil
industry. Its toxic principle is sanguinarine. Argemone oil produces dropsy in humans.
Parthenium hysterophorus is called ‘Gajar Ghas’ in Hindi and ‘whitetop weed’ in
English. P. hysterophorus weed (Fig. 52) is remarkably adaptable and grows abundantly
with crops and fodder fields, and in waste land throughout the year. Its toxic principle is
parthenin, which causes primary photosensitization, skin reactions and liver damage.
Ipomea carnea is commonly known as ‘Beshram’ in Hindi and ‘pink morning glory’
in English. I. carnea (Fig. 53) is found all over India. It has heart shaped leaves with
trumpet shaped white, pink, blue or violet flowers. I. carnea contains saponins as toxic
principles, which cause haemolysis of red blood cells (RBCs) leading to anaemia and
hypotension (fall in blood pressure).
Ingestion of Cannabis sativa (Marijuana) by companion animals can result in the
depression of CNS and incoordination, as well as vomiting, diarrhoea, drooling,
increased heart rate, and even seizure and coma. Species of Lilium (Lily) are considered
to be highly toxic to cats. While the poisonous component has not yet been identified, it
is clear that with even ingestion of very small amounts of this plant, severe kidney
damage could result. Spathiphyllum (Peace lily) contains calcium oxalate crystals that can

24
cause oral irritation, excessive drooling, vomiting, difficulty in swallowing, intense

burning, and irritation of mouth, lip and tongue in the dog who ingests. All parts of Cycas
revoluta (Sago palm) are poisonous, but the seeds or nuts contain the largest amount of
toxin. The ingestion of just one or two seeds can result in very serious effects, which
include vomiting, diarrhoea, depression, seizure and liver failure. The bulbs
of Tulipa/Narcissus species (Tulip) contain toxins, which can cause intense
gastrointestinal irritation, drooling, loss of appetite, depression of CNS, convulsion and
cardiac abnormality. Species of Rhododendron (Azalea) contain substances called
‘grayantoxins’, which can produce vomiting, drooling, diarrhoea, weakness and
depression of the CNS in animals. Severe azalea poisoning could ultimately lead to coma
and death from the cardiovascular collapse. Taxus (Yew) species contain toxic principle,
taxine, which causes CNS effects like trembling, incoordination and difficult breathing. It
can also cause gastrointestinal irritation and cardiac failure, which can result in death.
Cylamen species contain cyclamine, but the highest concentration of this toxic
component is typically located in the root of the plant. If consumed, Cylamen can
produce significant gastrointestinal irritation, including intense vomiting. Fatalities have
also been reported in some cases. Amaryllis is a common garden plant, popular around
the Easter. The Amaryllis species contain toxins, which may cause vomiting, depression,
diarrhoea, abdominal pain, hypersalivation, anorexia and tremor. Species of both
Scindapsus and Epipremnum (Pothos, the popular household plant), if chewed or
ingested, can cause significant mechanical irritation, and swelling of the oral tissues and
other parts of GIT. Schefflera and Brassaia actinophylla contain calcium oxalate crystals
that can cause oral irritation, excessive drooling, vomiting, difficulty in swallowing,
intense burning and irritation of the mouth, lip and tongue in the pet who ingests. Hedera
helix (Ivy, common ivy, English ivy, European ivy, branching ivy, glacier ivy,
needlepoint ivy, sweetheart ivy or California ivy) contains triterpenoid saponins, which
can cause vomiting, abdominal pain, hypersalivation and diarrhoea if pet ingests the
plant. Species of Kalanchoe (Kalanchoe plant) contain the constituents, that can produce
gastrointestinal irritation, as well as those that are toxic to the heart, and can seriously
affect the cardiac rhythm and rate. The popular blooms of Chrysanthemums species
(Chrysanthemums, mums or chrysanths) contain pyrethrins, which may produce

25
gastrointestinal upset including drooling, vomiting and diarrhoea, if eaten. In certain

cases, depression and loss of coordination may also develop if enough of any part of the
plant is consumed. Ingestion of Colchicum autumnale (Autumn crocus, meadow saffron
or naked lady) by the dogs can result in oral irritation, bloody vomiting, diarrhoea, shock,
multi-organ damage and bone marrow suppression.
OTHER KINDS OF POISONING/INJURY BY PLANTS
A. Photosensitization:
Some plants contain toxic substances which, when eaten, render the animal sensitive
to strong sunlight. The injury which results may range from sun-burning and swelling of
the sensitive areas to the formation of ulcer and gangrene. The animals may also become
blind. This process is known as ‘photosensitization’. The ‘phototoxic plants’ are
classified into two categories:
1. Primary phototoxic plants- They have toxins, which directly photosensitize the
skin either through the contact or by infestation. When these plants are eaten, the
toxins are absorbed and circulated in blood to the skin, where they are activated
by the rays of sun. The unpigmented (white) skin is affected. Saint John’s-wort,
spring parsley and buckwheat cause primary photosensitization.
2. Hepatogenic phototoxic plants- These plants do not directly cause the

‘photosensitization’. These plants have toxins which damage the liver. This
damage prevents a breakdown product of chlorophyll (phylloerythrin) from being
removed in the bile fluid. The phylloerythrin is circulated to the capillaries of
skin, where it is activated by the sun and produces symptoms similar to those with
primary photosensitization. It is important with hepatogenic cases to treat the
damaged liver. The blue-green algae cause hepatogenic photosensitization.

26
B. Water with Algae:

Water can have blue-green algae, which can poison the livestock. This type of algae
is usually present in the stagnant or slow-moving water during July and August. The long
periods of warm weather and a high content of organic matter in the water favour its
growth. In general, the symptoms develop very rapidly and resemble an allergic reaction.
The animals may be found dead at the water’s edge or after having walked a few metres.
The convulsions may occur, but more frequently the animal sinks to the ground and dies
without struggling. Smaller amounts of poison cause weakness and staggering, followed
by recovery. Sometimes, apparent recovery from an attack is followed in a few days or
weeks by evidence of photosensitization. There may be innammation of the muzzle, skin
of the ear, udder, or other parts of the body. Jaundice is often seen, and constipation is a
common symptom. Such cases normally recover under the good care.
C. Mechanical Injury by Plants:

Certain plants cause physical or mechanical injury to the animals. This injury may be
external or internal. When the injury occurs, there is also the danger of infection of these
injuries, which may prove to be even more serious. The barbs or awns of foxtail barley,
downy brome and wild rye are often troublesome in the mouth and throat of animals that
have fed on these plants. The small, backward-pointing spines cause the awns to stick in
the mouth or throat, and they are difficult to dislodge. The spines of the fruits of sandbur
are quite stiff, and an animal grazing may injure its muzzle while cropping, or if burs get
into its mouth, they may cause a painful injury. The burs of cocklebur and burdock are
also a source of annoyance. When the burs are eaten, they form an indigestible ball in the
stomach. The spines injure the wall of the digestive tract and may cause the secondary
infection. The sap from some plants, e.g., spurges and buttercups, is a source of irritation
to the animal skin. After contact with plant juices, the skin becomes inflamed and painful
blisters may form. This injury to mouth reduces the animal’s desire or ability to eat.
D. Milk and its Production Affected by Plants:
Some plants decrease milk production. They can also make the milk or milk products
unpalatable and unsuitable for human consumption. Some of these plants are: white
snakeroot, chicory, curled dock, broad-leaved dock, wild onion, wild garlic, garlic

27
mustard, wild mustard, rape, hedge mustard, turnip, spurge, buttercup, marsh marigold,
lupine, Saint John’s-wort, wild carrot, stinkweed, jimson weed (Datura stramonium),
burdock, false flax, flaxseed, buckthorn, yarrow, wormwood, absinth, stinking mayweed,
ox-eye daisy, ragweed and tansy, etc.
Fig. 10: Papaver somniferum Fig. 11: Atropa belladonna Fig. 12: Nicotiana tabacum
Fig. 13: Cinchona pubescens Fig. 14: Strychnine Fig. 15: Strychnos nux-vomica
Fig. 16: Erythroxylum coca Fig. 17: Piper nigrum Fig. 18: Ephedra sinica

28
Fig. 19: Areca catechu Fig. 20: Berberis aristata Fig. 21: Solanum nigrum
Fig. 22: Lupinus perennis Fig. 23: Caltha palustris Fig. 24: Cyanide
Fig. 25: Sorghum bicolor Fig. 26: S. bicolor var. drummondii Fig. 27: Linum usitatissimum
Fig. 28: Acacia nilotica Fig. 29: Eucalyptus tereticornis Fig. 30: Holcus lanatus

29
Fig. 31: Prunus avium Fig. 32: Agrostemma githago Fig. 33: Phytolacca acinosa
Fig. 34: Brassica campestris Fig. 35: Digitalis purpurea Fig. 36: Prunus amygdalus
Fig. 37: Cassava utilissima Fig. 38: Strophanthus gratus Fig. 39: Gynocardia odorata
Fig. 40: Quercus robur Fig. 41: Nitrate Fig. 42: Urea

30
Fig. 43: Ergot Fig. 44: Melilotus officinalis
Fig. 45: Calotropis gigantea Fig. 46: Lantana camara Fig. 47: Datura stramonium
Fig. 48: Ricinus communis Fig. 49: Nerium odorum Fig. 50: Cerbera thevetia
Fig. 51: Argemone mexicana Fig. 52: Parthenium hysterophorus Fig. 53: Ipomea carnea

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4
DETERMINATION OF MEDIAN EFFECTIVE
AND MEDIAN LETHAL DOSES
OBJECTIVE
To determine median effective dose (ED50) and median lethal dose (LD50).
DEFINITION OF ED50 AND LD50

“ED50” is the dose of a drug predicted (by statistical techniques) to produce a
characteristic effect in 50% of the subjects to whom the dose is given. Similarly, the
“LD50” (lethal dose, 50%) or “median lethal concentration” (LC50 or lethal concentration,
50%) of a drug/toxin/radiation/pathogen is the dose required to kill half the members of a
tested population after a specified test duration. On the other hand, the LD50/LC50 is the
lethal dose for 50% of the animals in a population. The LD50/LC50 is used as a general
indicator of the acute toxicity of a drug.
ED50 or LD50 is usually expressed as the mass of substance administered per unit
mass of the test subject, as ‘milligrams of substance per kilogram of body mass’ (mg/kg
body weight), but is stated as nanograms (suitable for botulinum, etc.), micrograms,
milligrams or grams (suitable for paracetamol, etc.) per kilogram as toxicity decreases.
Stating it this way allows the relative toxicity of different substances to be compared, and
normalizes for the variation in the size of the animals exposed (although toxicity does not
always scale simply with body mass).
The LD50 is usually determined by the tests on animals like laboratory rats and mice.
In 2011, the US Food and Drug Administration (FDA) approved the alternative methods
to LD50 for testing the cosmetic drugs. The determination of ED50 value helps in
determining the potency of a drug. The units of ED50 and LD50 are those of the dose
(mg/kg). When the ‘all-or-none’ response (quantal response) is death, the ED50 becomes

32
the LD50 value. The ED50 and LD50 of a given drug are different in different species of
animals. Likewise, the ED50 and LD50 of a given drug in a species are different for
different routes of administration. Both the ED50 and LD50 are important for knowing the
safety of a drug. The ratio between the LD50 and ED50 (i.e., LD50 / ED50) represents the
‘therapeutic index’.
METHOD
1. Take atleast 10 overnight fasted mice in each group.
2. Administer the given drug by the said route (oral, im, ip, etc.) and observe the
animals for death due to toxicity.
3. Find out the least tolerated dose (the dose producing 100% mortality) and most
tolerated dose (the dose producing no mortality) by ‘hit and trial’ method.
4. Once the two doses, i.e., LD0 and LD100 are determined, select atleast five doses in
between, and observe the mortality due to these doses.
5. Finally, find out the LD50 value by one of the following four methods:
A. Arithmetical Method of Karber (1931);
B. Arithmetical Method of Reed and Muench (1938);
C. Graphical Method of Miller and Tainter (1944);
D. Graphical Method of Litchfield and Wilcoxon (1949).
A. Arithmetical Method of Karber:

This method uses the interval mean of the number of animals died in each group, and
the difference in doses for the same interval is used. The results from the dose larger than
the ‘least tolerated dose’ (100% mortality, LD100) and the dose smaller than ‘most
tolerated dose’ (0% mortality, LD0) are not used. The method is performed as under:
i. Determine the number of animals dead in each group.
ii. Calculate the mean of maximum number of dead animals in two adjacent doses
(groups), i.e., the interval means of the two maximum numbers of dead animals.
iii. Calculate the product of mean and the dose difference.
iv. Take sum of the products.

33
v. Divide the sum of the products by the number of animals in a group, and substract
the resulting quotient from the ‘least tolerated dose’ to obtain the LD50.
Example- Endosulphan (an organphosphorous insecticide) is given in rat, orally in order
to determine its LD50. Total 10 groups (having 10 rats each) are used and the result is
shown in Table 2, below-
Table 2: Determination of LD50 of Endosulphan
Group No. of Dose Dose Difference No. of Animal Mean Product

Animals (f) (mg/kg) (a) (Rat) Died (b) (c) (d)
1 10 22 - 10 - -
2 10 20 2 10 - -
3 10 18 2 8 9 18
4 10 16 2 6 7 14
5 10 15 1 4 5 5
6 10 14 1 2 3 3
7 10 13 1 0 1 1
8 10 12 1 0 - -
Sum of Products (e) 41
Calculation from the above Table 2-

a) Mean (c) = Interval mean of two maximum number of dead animals (b) / 2 = (10
+ 8) / 2 = 9 (max. numbers of dead animals in two adjacent doses are 10 and 8).
b) Product (d) = c x maximum dose difference (a) = c x a = 9 x 2 = 18.
c) Sum of products (e) = 41.
d) Number of animals in each group (f) = 10.
e) Least tolerated dose (100% mortality) = 20 (because this is minimum dose with
which the maximum 10 animals are died).
f) LD50 = Least tolerated dose - (Sum of products / number of animals in each
group) = 20 - (41 / 10) = 20 - 4.1 = 15.9.
Therefore, the LD50 of endosulphan in rat is 15.9 mg/kg body weight (as per the
above example).
B. Arithmetical Method of Reed and Muench:

This method employs the cumulative values. It is assumed that an animal killed by a

34
particular dose of a chemical would have been killed by any higher dose, and that a
surviving animal would have survived after using the smaller dose. The method is
performed as under:
i. Note total number of animals dead and survived in each group (a and b, Table 3).
ii. Eliminate the doses larger than the ‘least tolerated dose’ (100% lethal dose) and
smaller than the ‘most tolerated dose’ (0% lethal dose).
iii. Record the cumulative dead animals by adding the successive entries (c, Table 3).
iv. Similarly, take the cumulative survived animals (d, Table 3).
v. Take the sum of cumulative dead and cumulative survived animals in each dose
(e, Table 3).
vi. Count the per cent (%) survival in two doses adjacent to the LD50 (f, Table 3), and
calculate the LD50 of the given drug as per the following example.
Example- Endosulphan is given in rat, orally in order to determine its LD50. Total 10
groups (having 10 rats each) are used and the result is shown in Table 3, below-
Table 3: Determination of LD50 of Endosulphan
Group No. of Dose Animals Animals Cumulative %

Animals (mg/kg) Died (a) Survived Dead Survived Total Survival
(b) (c) (d) (e) (f)
1 10 22 10 0 - - - -
2 10 20 10 0 10 30 40 -
3 10 18 8 2 18 30 48 -
4 10 16 6 4 24 28 52 53.8
5 10 15 4 6 28 24 52 46.2
6 10 14 2 8 30 18 48 -
7 10 13 0 10 30 10 40 -
8 10 12 0 10 - - - -

a) Group 1 is neglected, since the doses larger than the least dose killing all animals,
have no significance. Similarly, group 6 is neglected, since the dose smaller than
the greatest dose allowing all animals to survive, have no significance.
b) The % survival for both doses adjacent to the LD50 is computed.
c) Then the proportionate distance from 50% is computed.
Thus: (50.0 - 46.2) / (53.8 - 46.2) = 3.8 / 7.6 = 0.5 -------- (1)

35
(50.0 is the average of % survival for both doses adjacent to the LD50, i.e., f =
53.8 and 46.2 in groups 4 and 5, respectively. So, 53.8 + 46.2 = 100.0 / 2 = 50.0).
d) Divide the higher adjacent dose by the lower adjacent dose.
In the above Table (example), the higher adjacent dose for LD50 is 16 and the
lower adjacent dose is 15.
Thus: higher adjacent dose / lower adjacent dose = 16 / 15 = 1.1 -------- (2)
Take logarithm of (2), i.e., 1.1 = 0.0414 -------- (3)
e) Multiply (1) and (3).
Thus: 0.5 x 0.0414 = 0.0207 -------- (4)
f) Then the value of (4) is added to the logarithm of smaller adjacent dose to form
the logarithm of LD50.
Thus: smaller adjacent dose = 15 mg/kg; log of 15 = 1.1761 -------- (5)
Log LD50 = (4) + (5) = 0.0207 + 1.1761 = 1.1968 or 1.197 -------- (6)
g) Take antilog of log LD50 (6) to get LD50 value in mg/kg.
Thus: antilog of 1.197 = 15.73 -------- (7)
So, from the example, LD50 of endosulphan in rat is 15.73 mg/kg body weight.
C. Graphical Method of Miller and Tainter:

In this method, the values are converted into probits and plotted against log-dose. A
special co-ordinate paper, i.e., logarithm-probit paper is also used. In probit
transformation, the sigmoid curve becomes a straight line. This method is more reliable
than the other methods. The method is performed as under:
i. Note total number of animals dead in each group and calculate the % dead (a and
b, Table 4).
ii. Correct the data for 0% and 100% dead (c, Table 4) according to the formula. 0%
dead = 100 (0.25 / n) and 100% dead = 100 (n - 0.25 / n); where, n = number of
animals in a group.
iii. Determine the probit values of corrected % values from the table (d, Table 4) or
from the normal distribution-probit transformation table (Table 5).
iv. Plot the probit values against the log doses (e).

36
v. Find out the LD50 value corresponding to the 50%, or a probit of 5 from the
plotted curve (f).
vi. To compute the standard deviation (SD) of the mean LD50, determine the doses
for probits 4 and 6 from the plotted graph and calculate their difference “S” (g).
vii. Calculate the SD from the formula given below-
______
viii. SD = S / √ 2 x 2n -------- (h)
Example- Calculate the LD50 of ethion from the following data (Table 4)-
Table 4: Determination of LD50 of Ethion
Group No. of Dose Animals Dead % Corrected % Probit

Animals (mg/kg) Died (a) (b) (c) (d)
1 10 14.0 10 - - -
2 10 12.0 10 100.0 97.5 6.96
3 10 10.0 8 80.0 80.0 5.84
4 10 08.0 7 70.0 70.0 5.52
5 10 06.0 6 60.0 60.0 5.25
6 10 05.0 4 40.0 40.0 4.75
7 10 04.0 4 40.0 40.0 4.75
8 10 03.0 1 10.0 10.0 3.72
9 10 02.0 0 0.0 2.5 3.04
10 10 01.0 0 - - -

a) Corrected % for 0 and 100% dead-
From formula, 0% dead = 100 (0.25 / n) and 100% dead = 100 (n - 0.25 / n);
0% dead = 100 (0.25 / 10) = 2.5;
100% dead = 100 (10 - 0.25 / 10) = 97.5.
b) Probit values are plotted against their corresponding log doses and graph is
obtained.
c) LD50 from plotted graph (corresponding to probit 5) = 6.15 or 6.2 mg/kg.
d) Doses for probits 4 and 6 from the graph are-
Dose for probit 4 = 3.6 mg/kg;
Dose for probit 6 = 10.8 mg/kg;
S = 10.8 - 3.6 = 7.2; where, S = difference in doses for probits 4 and 6.
e) Standard deviation (SD) according to the formula (h)-

37
______ _______
SD = S / √ 2 x 2n = 7.2 / √ 2 x 2 x 10
= 7.2 / 6.32 = 1.14
Therefore, the LD50 of ethion with standard deviation is 6.2 ± 1.14 mg/kg (as per the
above example).
Table 5: Normal Distribution-Probit Transformation Table
Corrected Probit Value

% 0 1 2 3 4 5 6 7 8 9
0 - 2.67 2.95 3.12 3.25 3.36 3.45 3.52 3.59 3.66
10 3.72 3.77 3.82 3.87 3.92 3.96 4.01 4.05 4.08 4.12
20 4.16 4.19 4.23 4.26 4.29 4.33 4.36 4.39 4.42 4.45
30 4.48 4.50 4.53 4.56 4.59 4.61 4.64 4.67 4.69 4.72
40 4.75 4.77 4.80 4.82 4.85 4.87 4.90 4.92 4.95 4.97
50 5.00 5.03 5.05 5.08 5.10 5.13 5.15 5.18 5.20 5.23
60 5.25 5.28 5.31 5.33 5.36 5.39 5.41 5.44 5.47 5.50
70 5.52 5.55 5.58 5.61 5.64 5.67 5.71 5.74 5.77 5.81
80 5.84 5.88 5.92 5.95 5.99 6.04 6.08 6.13 6.18 6.23
90 6.28 6.34 6.41 6.48 6.55 6.64 6.75 6.88 7.05 7.33

38
5
COLLECTION AND DISPATCHING OF
SAMPLES FOR TOXICOLOGICAL TESTS
OBJECTIVE
To collect, preserve and dispatch/submit suspected samples for the toxicological
examination.
PRINCIPLE
The suspected samples for toxicological examination are taken during the P.M.
examination. In small animals (viz., small dogs, cats, piglets, poultry and small wild
animals), the whole carcass should be sent unopened. In large dogs, sheep and pigs, the
tied off stomach and tied off parts of the intestine are required. In large animals (viz.,
horses and cattle), the parts of the digestive organs are sent.
REQUIRED SAMPLES FOR DIAGNOSIS OF POISONING

The samples/specimens/materials to be submitted for the toxicological examination
in suspected case of poisoning are described in Table 6.
CONTAINERS REQUIRED FOR COLLECTION OF SAMPLES

a) The best container for collection of sample is screw-cap polyurethane jar.
b) The glass jar or pickle bottle, thoroughly cleaned, dried and fitted with air tight
seal is satisfactory but should be carefully packed to avoid any leakage.
c) Place glass container in rigid box (carton) well padded with wood sawing, hay or
saw-dust.
d) Do not use rubber closer ring as it contains certain contaminants.
e) Test the polythene bag, if used, to make sure that it does not leak; and pack the
dry material which is not liable to damp in new, strong, unused plastic bag.

39
Table 6: Required Samples for Diagnosis of Poisoning/Toxicity
Suspected Poison/Toxicant Required Samples/Specimens/Materials

Lead (Pb- Acute poisoning) Blood (no EDTA; heparinized blood), kidney, liver, urine
Lead (Pb- Chronic poisoning) Hair, liver, kidney, bone, faeces, urine
Arsenic (As- Acute
Liver, kidney, ingesta or GIT contents (stomach and intestine), urine, feed
poisoning)
Arsenic (As- Chronic
Hair, liver, urine, spleen, altered organs
poisoning)
Blood (no EDTA; heparinized blood preferred), liver, kidney, GIT contents,
Mercury (Hg)
muscle, brain, faeces, feed
Cadmium (Cd) Kidney, liver, hair
Cyanide/hydrogen cyanide GIT contents, liver, muscle, oxalated blood, brain (all these should be frozen in
(HCN) different airtight containers), feed/forage
Copper (Cu) Liver, kidney, blood, faeces, urine
Iron (Fe- ferrous or ferric) Kidney, liver, blood, serum, faeces, feed
Selenium (Se) Whole blood (heparinized), altered organs, liver, hair, feed
Nickel (Ni) Blood (no EDTA; heparinized blood), serum, kidney, liver, faeces, feed
Cobalt (Co) Kidney, liver, blood, serum, faeces, feed
Chromium (Cr) Kidney, liver, blood, serum, faeces, feed
Molybdenum (Mo) Liver, kidney, altered bone, hair, blood (no EDTA; heparinized blood), feed
Zinc (Zn) Kidney, liver, serum (use ‘trace minerals’ tubes)
Zinc phosphide Liver, stomach contents (both should be frozen)
Sodium (Na)/sodium chloride
Brain (other half of each should fixed in formalin), serum, CSF, feed
(NaCl)
Fluoride Altered bones, tooth, urine, stomach contents, liver, kidney, feed/forage, water
GIT contents, whole blood, urine, body fluids such as aqueous humor (all these
Nitrite (NO3)/nitrate (NO2)
should be refrigerated), feed/forage, water
Sulphate (SO4) Brain (should be fixed in formalin), water
Chlorate Stomach contents (should be frozen, in airtight container), urine, feed
Oxalate Kidney (do not macerate, but freeze or fix in formalin), fresh forage
Phosphide GIT contents, feed
Phosphorous Oxalated blood, GIT contents, liver, lung, vomitus, faeces
Triaryl phosphate Ingesta, feed
Alkaloid Liver, urine, brain, GIT contents
Ammonia (NH3) Blood, urine, rumen contents (all these should be frozen; in rumen contents, 1-2
drops of saturated mercuric chloride can be added if not frozen), serum, feed
Urea As for ammonia
Barbiturates Blood, brain, liver, fat
Strychnine (some other
GIT contents, urine, liver, kidney, brain
convulsants like bromethalin)
Anticoagulants (warfarin and Whole blood (add EDTA or heparin), liver, stomach contents (all these should be
related compounds) refrigerated), feed
Cholinesterase Cerebrum (should be frozen), serum
Dicoumarol Liver, forage
Ionophore Rumen contents, heart, skeletal muscle, feed
Phenol Gastric or rumen contents (in airtight container)
Ethylene glycol Serum, urine, kidney
Polychlorinated biphenyls Fat, cerebrum, feed

40
(PCBs; and polybrominated

biphenyls)
Chlorinated hydrocarbons Cerebrum, ingesta, body fat, liver, kidney (use only glass containers; avoid
contamination; all these should be refrigerated or frozen)
Organophosphate Oxalated blood or whole blood, liver, ingesta or GIT contents, urine, feed
insecticides (OPIs) and
carbamates
Organochlorine insecticides Fat, liver, stomach contents, brain
(OCIs, CHIs)
Herbicides Liver or kidney, ingesta, urine, treated weeds
Rodenticides Stomach contents, liver, kidney, urine
Mycotoxins Liver, kidney, grain, forage
Rumen pH Ingesta (should be frozen)
Total dissolved solids (TDS) Water
Vitamins A, D and E Liver (should be frozen), serum
Vitamin D3 Kidney
TISSUES REQUIRED FOR ANALYSIS

The required tissues for the toxicological test are mentioned in Table 7.
Table 7: Required Tissues for Toxicological Test
Tissue Amount Remark

Blood 100 ml Heart blood or peripheral blood is preferred
Milk 50 ml In case of poison excreted in milk
Urine 500 ml Urine contains poisons and their metabolites in most cases
Vomitus and faeces Small quantity In case of poison excreted in vomitus/faeces
Liver 100 g Important for most types of poisons
Lung 100 g Useful for inhaled poisons
Kidney One kidney Tissue of choice for metals and sulphonamides
Stomach Intact Full stomach along with contents after ligating both ends
Intestine Part of intestine Part of intestine along with its contents after tying both ends
Gall bladder Intact -
Spleen 100 g -
Urinary bladder Intact Send bladder if no urine is available
Brain 50 g Useful for volatile and lipid soluble poisons/alcohol
Bone 100 g or part of Important for pesticides and metal poisoning
altered bone
Fat 50 g Useful for pesticides and lipid soluble drugs
Hair and Nail Intact In suspected chronic metal poisoning, e.g., arsenic
PRESERVATION OF TEST SAMPLES

i. After collection and sealing, store the sample in a refrigerator or deep freeze till
the dispatch to laboratory.
ii. As far as possible, the preservative should be avoided.
41
iii. Prefer to send the sample with the ice.

iv. The sample should be dispatched to laboratory as soon as possible.
v. If necessary, it is best to use the alcohol (95% ethanol) as preservative (1 ml/g of
sample) since alcohol is rarely met with a poison in veterinary toxicology.
vi. Always send a sample of preservative separately in a small bottle along with the
thest sample.
vii. Formalin is undesirable as it interferes with many tests, but place the sample
intended for histological examination immediately in a 1:9 solution of neutral
formalin (1% of 40% formaldehyde + 9 parts of water) and dispatch it.
DISPATCHING/SUBMISSION OF TEST SAMPLES

The veterinary practitioner and veterinary diagnostic laboratory staff must maintain
the good communication in order to complete their diagnostic efforts efficiently and
provide optimal service to client. The practitioners must be specific and clear in their test
requests. The laboratory staff can provide guidance when there are questions regarding
sample collection and handling, as well as offering assistance in interpretation of test
results. Most diagnostic laboratories publish user guidelines with preferred protocols for
sample collection and dispatching/submission, but the following broad recommendations
are fairly standard.
Regardless of the type of dispatching or submission, a detailed case history should be
included with the samples to assist the laboratory personnel in determining a diagnosis.
The information should include: owner, species, breed, sex, age, animal identification,
clinical signs, gross appearance (including size and location) of the lesion(s), previous
treatment (if any), time of recurrence from any previous treatment and morbidity/
mortality in the group. If a zoonotic disease is suspected, this should also be clearly
indicated on the submission sheet to alert the lab personnel. The submission form should
be placed in a waterproof bag to protect it from any fluids which might be present in the
packaged materials. The waterproof marker should be used when labeling the specimen
bags and containers. Henceforth:

42
1) Each organ should be sent in a separate container after the proper labeling with
date, name and address of the sender; particulars of organ and species; and details
of the preservative if used in the sample.
2) A full report of the clinical and P.M. findings and also for the suspected poisons
should accompany the sample.
3) Always treat the parcel as infectious material and mark it with a ‘black cross’ to
indicate it.
4) Parcel should be properly sealed to prevent it from being tampered on route. It
should be labeled- ‘Urgent’, ‘Handle with care’ and ‘Keep away from food stuffs’.
5) An application for toxicological examination should be enclosed, including
history, clinical and P.M. findings, and a list of samples/specimens/materials sent.
EXAMINATIONS/TESTS FOR DIAGNOSIS OF POISONING

The following studies are to be done for the diagnosis of poisoning/toxicity:
I. Toxicology:
If a known poison is suspected, a specific analysis should always be requested-
laboratories can not just ‘check for poisoning’. A complete description of clinical and
epidemiologic findings may help to differentiate the poisoning from infectious diseases
which can simulate the poisoning. The appropriate samples to be sent for analysis of
different poisonings are listed in Tables 6 and 7. The most critical samples to be collected
are GIT contents, liver, kidney, whole blood, plasma/serum and urine; but in exceptional
cases, the cerebral tissue for cholinesterase analysis should be sent. Sometimes, analysis
of feed or water is also done. If there is any doubt, the laboratory can be consulted.
II. Hematology:
The routine studies require anticoagulated whole blood and several blood smears.
The blood smears should be prepared immediately after the sample has been collected to
minimize the cell deterioration. The anticoagulated blood should be kept refrigerated;
blood smears should not. Ethylenediamine tetra acetic acid (EDTA) is the anticoagulant

43
of choice, because it best preserves the cellular components of the blood and prevents the
platelet aggregation. The blood for coagulation testing should be collected into a blue top
tube, which contains sodium citrate. After mixing, the sample should be centrifuged for 5
minutes, and then plasma should be removed and transferred to a clean tube without
anticoagulant. The plasma should be kept frozen until the time of analysis. The whole
blood should not be frozen because this causes cell lysis and gross haemolysis, which
interfere with the testing.
III. Microbiology:
Any specific agents which are of interest in the diagnostic investigation should be
mentioned on the submission form; some agents have requirements (e.g., anaerobic
culture, special media, etc.) that would not be used in most laboratories unless the
pathogen was cited as a differential diagnosis. The laboratory techniques and capabilities
for microbiologic examination vary; available tests include bacteriologic culture, fungal
culture, virus isolation, in situ hybridization, a variety of PCR methods, fluorescent
antibody tests, latex agglutination tests, Western blotting, ELISA and many others. Most
tests, including the newer molecular biology techniques, rely on either the growth/
visualization of intact viable organisms or the detection of nucleic acids and proteins of
these pathogens. Thus, the unfixed specimens (tissue, fluid, etc.) should be collected
aseptically and shipped promptly to avoid the degradation. If PCR testing is to be done, it
is particularly important to avoid the cross contamination between the multiple animals in
a submission; this applies to tissues, fluids and even dissection instruments. Further, the
swabs destined for PCR analysis should not be placed in agar or charcoal based transport
media. Calcium alginate swabs should be avoided; the cotton or dacron swabs should be
shipped in a tube with few drops of sterile saline or viral transport media.
Some test protocols may permit the pooling of organ specimens from an individual,
but for the vast majority, it is preferable that each tissue be collected into separate sterile,
clearly labeled bags or tubes for shipping. Gut samples must never be pooled in a
container with other tissue samples. Tissues and fluids for most microbiologic assays
may be frozen before shipment, but generally freezing is undesirable if samples can be

44
chilled and delivered directly to the laboratory within 24 hr. Exceptions to this rule
include analysis for certain toxins like those of Clostridium perfringens and C. botulinum,
in which degradation of the toxin must be prevented by prompt freezing after collection.
Adequate refrigerant should be provided, so that the samples remain chilled (or frozen).
IV. Clinical Chemistry:

Many tests of clinical chemistry need the serum, but an occasional test may require
the plasma. The anticoagulants present in the plasma may interfere with the tests; thus,
the serum should always be submitted unless the plasma is specifically asked. Because
lipemia can interfere with a number of chemistry tests, the dogs and cats should be fasted
for 12 hr before the samples are collected.
For serum samples, the blood should be drawn into a red top tube or a separator tube.
The sample should be held at room temperature for 20 to 30 minutes to allow the
complete clot formation and retraction. The incomplete clot formation may cause the
serum to gel due to latent fibrin formation. The clot should be separated from the glass by
gently running an applicator stick around the tube walls (‘rimming’). The sample should
then be centrifuged at high speed (∼1,000 g; 2,200 rpm) for 10 minutes. Rough handling
of the sample or incomplete separation of erythrocytes (RBCs) from serum may promote
haemolysis, which can interfere with certain tests. If the sample has been collected into a
serum separator tube, the centrifugation will cause a layer of silicone gel to lodge
between the packed cells and serum. The gel layer should be inspected to ensure the
integrity of the barrier, and recentrifugation is recommended, if there is a visible crack in
this layer. If a red top tube has been used, the serum should be removed and transferred to
a clean tube. The serum should be refrigerated or frozen until analyzed.
V. Serology:
Normally, serology requires the serum, but the plasma is often satisfactory. The
samples should be collected as described for clinical chemistry tests, and should always
be free of haemolysis. In some cases, the paired samples may be required for an adequate
diagnosis. The acute sample should be collected early in the course of the disease and

45
frozen. The convalescent sample should be collected 10 to 14 days later, and both
samples should be forwarded to the laboratory at the same time.
VI. Fluid Analysis:

This analysis of effusions includes the determination of protein content, total cell
count and cytologic examination. Other tests may be performed depending on the source
(e.g., synovial fluid) or appearance (e.g., chylous fluid) of the effusion. A sample of
effusion should be collected into an EDTA (purple top) tube for routine analysis. A
second sample should be collected into a serum (red top) tube, if any biochemical
analyses (e.g., triglyceride, cholesterol, lipase) are to be done, or if a bacterial culture is
desired. Smears for cytologic examination should be prepared immediately after the
sample has been collected to minimize the cell deterioration and other in vitro artifacts.
VII. Genetic Analysis:

The tests based on the detection of specific genetic features range from karyotype
analysis to identification of specific genes. The required samples range from hair to skin
or blood. Many blood-based analyses require the collection into yellow-topped acid-
citrate-dextrose tubes and overnight shipment of the chilled tubes to the laboratory.
Tissue samples for genetic analysis should be unfixed and shipped immediately after
collection. As with most molecular techniques, aseptic collection and the prevention of
cross contamination between the samples is critical.
VIII. Histology:
Histology is relatively rapid and inexpensive diagnostic technique which can often
result in substantial savings in time, money and animal life. The increasing number of
immunohistochemical (IHC) tests that can be applied to formalin-fixed tissue has further
reinforced the utility of this diagnostic technique. Autolyzed tissues are normally useless
for histopathologic examination; prompt necropsy examination and organ sampling are
critical. Tissue should not be frozen before the fixation. Other than CNS tissues, the
samples collected for histology should never be >1 cm thick (preferably 5-7 mm) and

46
must be placed immediately into ≥10 times their volume of phosphate-buffered 10%
formalin to ensure the adequate fixation. The tissues collected for histologic examination
should be representative of any lesions present and, in the case of cutaneous punch
biopsies and biopsies obtained via endoscopic collection, should be centered directly on
the grossly visible lesions. Wedge biopsies or tissue samples collected at necropsy should
include some of the apparently normal surrounding tissue; the interface between normal
and abnormal may provide key information. Excisional biopsies of small tumors (<1.5
cm) may be cut in half. Larger tumours may be sliced like bread, so that formalin can
penetrate to the face of each slice. Alternatively, several representative samples (7 mm
wide, including the interface of normal and abnormal) may be collected. The tissues
should remain in fixative for ≥24 hr. Fixed tissues should be avoided to be freezed.
Because the GI mucosa decomposes rapidly, short sections of gut collected at
necropsy must be opened lengthwise to allow the adequate fixation. If spinal cord is to be
submitted, the dura mater should be carefully incised lengthwise to permit more rapid
penetration to the spinal cord. Fixing the brain poses a special dilemma, especially if a
neuroanatomic location of the lesion(s) within the organ could not be determined
antemortem. In fact, a whole, intact fixed brain is required for complete histopathologic
analysis. Immersion of the brain for many days in a very large volume of formalin is
required to adequately fix such a specimen, so the brain is commonly transported in an
only partially fixed state. If the specimen can be shipped by overnight delivery, it may be
acceptable to send a chilled, carefully packaged, unfixed brain, which can then be
processed at the diagnostic laboratory. Often, the brain is halved longitudinally and one-
half sent unfixed (fresh), properly refrigerated, for microbiologic tests, while the other
half partially fixes in transit. This method can prove unsatisfactory if a solitary unilateral
lesion is involved. Slicing the brain into widths suitable for rapid fixation introduces
considerable fixation artifact and should be avoided if possible. It is preferable to fix the
intact/halved brain in a large volume of formalin for >24 hr.
IX. Cytology:
For cytological studies, the air-dried smears are generally required. Rapid air drying

47
of smears minimizes cell distortion, thereby enhancing the diagnostic quality. However,
depending on the method of staining used, some laboratories prefer alcohol-fixed smears.
The samples can be obtained by fine-needle aspiration or by scraping. Imprints (touch
preparations) of external lesions can also be used, although these tend to have a greater
degree of contamination. The aspirated material should always be smeared before air
drying. The fluid smears can be prepared using a traditional blood smearing technique.
Highly cellular fluids may be smeared directly, while the fluids of low cellularity should
be centrifuged to concentrate the cells. Thick material or viscous fluid is more readily
smeared using a squash technique in which a second glass slide is placed over the
aspirated material, and then slid rapidly and smoothly down the length of lower slide. The
blood or cytologic smears should never be mailed to laboratory in the same package with
formalin-fixed tissues, because the formalin vapors will produce artifacts in the
specimen. Many laboratories now offer immunocytochemical testing. Generally, the air-
dried, unfixed smears is sufficient, but sometimes the shipping of samples in tubes
containing a transport media is needed.
PRECAUTIONS
For toxicological examination of the suspected samples, the following precautions
must be taken into account:
1. The organs to be analyzed should be protected from the contamination/infection
as far as possible.
2. The sample should not be washed.
3. The sample should be sent fresh (not in decayed condition).
4. No preservative should be added in the sample, as far as possible.
5. If any therapeutic agent is given to the animal before death, it should be clearly
written.
6. The analyst or in-chagre of the laboratory should be warned of the legal action
likely to arise regarding the case/suspected sample.

48
6
PREPARATION OF TOXICOLOGICAL KIT
FOR TREATMENT OF POISONING
OBJECTIVE
To prepare toxicological kit for the treatment of poisoning.
MEASURES TAKEN IN SUSPECTED POISONING

The following measures should be taken without delay, when a case of poisoning is
suspected:
a) Prevent more absorption of the poison, which is being taken.
b) Promote more excretion of the poison, which has already been taken.
c) Give a specific antidote, if any.
d) Start the effective symptomatic treatment.
TOXICOLOGICAL KIT
An emergency box/bag containing all the essential articles, which may be necessary
to start the treatment of poisoning immediately is called as the ‘toxicological kit’. A
physician or veterinarian should always carry this toxicological kit when he/she goes to
attend the case of acute poisoning.
The ‘toxicological kit’ should contain different instruments/appliances as well as
various drugs/chemicals, which are required for the treatment of poisoning. Therefore,
the following instruments/appliances and drugs/chemicals may be kept in the
‘toxicological kit’:
I. Instruments and Appliances:

1. Scalpels (Fig. 54)
2. Scissors (Fig. 55)

49
3. Hypodermic syringes and needles (Fig. 56)

4. Tourniquet (Fig. 57)
5. Hot water bag (Fig. 58)
6. Mouth gag (Fig. 59)
7. Stomach tube (Fig. 60)
8. Endotracheal tube (Fig. 61)
9. Rubber catheter (Fig. 62)
II. Drugs/Chemicals:
1. Gastric lavage
2. Emetics
3. Adsorbent
4. Purgatives
5. Diuretics
6. Specific antidotes
7. Miscellaneous drugs
GASTRIC LAVAGES
‘Gastric lavaging’ is the process of washing of ingesta and intestine. In this, the
solution is drenched by stomach tube/pipe and pumped out again by the suction pressure.
Some of the important gastric lavages are:
1. Normal saline
2. Tannic acid
3. Potassium permanganate (KMnO4- 1:2000)
4. Tincture iodine (1:250 of 5% solution)
5. Sodium bicarbonate solution
The above compounds/agents are used as 1% solution, and are drenched at the rate of
10 ml/kg body weight for washing of the intestine and removal of ingesta of stomach.
The gastric lavage should be performed in the unconscious/anaesthetized animals.
ADSORBENTS

50
‘Adsorption’ is the physical binding of a toxicant to an unabsorbable carrier, which is

eliminated in the faeces. For example, the activated charcoal is given as an adsorbent at
the dose rate of 2.5 g/kg, orally. However, it should be remembered that for prevention of
constipation to be occurred after the activated charcoal administration, a purgative can be
administered to the suffering individual.
EMETICS
‘Emetics’ are the drugs that cause vomition in the monogastric animals (viz., dog,
cat, pig, etc.). In these animals, the vomition may be induced to empty the stomach. The
emetics are of two kinds:
A. Centrally acting emetics
B. Peripherally acting emetics
A. Centrally Acting Emetics:

These drugs stimulate the dopaminergic receptors in ‘chemoreceptor trigger zone’
(CTZ) and produce ‘emesis’ (vomition). The examples are:
1. Apomorphine hydrochloride- By this, the vomition (emesis) occurs within 2 to 10
minutes. In dog (contraindicated in cat and pig), the dose rates are 6 mg/kg, oral;
3 mg/kg, sc; 0.07 mg/kg, im; 0.04 mg/kg, iv; and 0.02 mg/kg, subconjunctival.
2. Xylazine- It induces vomition within 10 to 20 minutes in dog and cat. It is given
@ 1 mg/kg, im.
B. Peripherally Acting Emetics:

These drugs irritate the epithelium of pharynx, larynx and stomach, and thereby
produce emesis. The examples are:
1. Copper sulphate (CuSo4)- Its dose is 50 ml of 1% solution, orally.
2. Zinc sulphate (ZnSo4)- Its dose is 50 ml of 1% solution, orally.
3. Sodium chloride (NaCl)- It is administered @ ½ t.s.f. crystals on the back of
tongue, or 1 to 2 t.s.f. crystals with half cup of lukewarm water, orally.
4. Mustard powder- It is given @ ½ to 1 t.s.f. with half cup of lukewarm water,
orally.

51
5. Hydrogen peroxide (3%)- It is given @ 1 to 5 ml/kg, orally.
PURGATIVES (CATHARTICS)
‘Purgatives’ are used to increase the elimination of unabsorbed toxicants from the
GIT. The examples are:
1. Sodium sulphate (Glauber’s salt)- Its dose is 250 mg/kg, orally.
2. Magnesium sulphate (MgSO4)- Its dose is 1 g/kg.
3. Sorbitol (70%)- Its dose is 3 ml/kg, orally.
4. Liquid paraffin- It is given @ 5 to 15 ml in dog; 0.5 to 1 L in cattle and horse; and
250 to 500 ml in sheep and goat.
It should be noted that the oil based purgatives must not be used in the poisoning
cases as they may increase the absorption of toxicants.
DIURETICS
‘Diuretics’ can be used to increase the rapid renal filtration of the absorbed toxicants
by enhancing the frequency of urination. The examples are:
1. Mannitol (5-25%)- It is given at the dose of 1 g/kg, iv.
2. Furosemide- It is given at the dose of 2 to 4 mg/kg, iv or im, twice daily.
ANTIDOTES
‘Antidote’ is an agent which antagonizes the effect of toxicant. There are two types
of antidotes:
A. Universal antidote
B. Specific antidote
A. Universal Antidote:
‘Universal antidote’ can be used non-specifically in the cases where the exact cause
of poisoning is not known. The universal antidote contains the following compounds-
Charcoal - 2 Parts (causes adsorption)
Magnesium oxide - 1 Part (causes catharsis)
Tannic acid - 1 Part (causes precipitation)

52
B. Specific Antidote:
‘Specific antidote’ can be used specifically in the cases where the exact cause of
poisoning is known. The important specific antidotes used against different poisonings
are mentioned in Table 8.
Table 8: Specific Antidotes for Various Poisonings
Poisoning Antidote Action of Antidote

Lead, mercury, arsenic BAL/dimercaprol Chelation
Lead, zinc Calcium disodium EDTA Chelation
Copper D-penicillamine Chelation
Fluoride, oxalate Calcium gluconate Complex formation
Nitrate, nitrite, chlorate Methylene blue Oxidative reduction
Silver nitrate Sodium chloride Complex formation
Iron Desferrioxamine Complex formation
Cyanide Sodium nitrite, sodium thiosulphate Chemical neutralization
Carbon monoxide Oxygen Antagonism
Alkaloid Tannic acid Precipitation
Warfarin (coumarin anticoagulant) Vitamin K Antagonism
Strychnine, nicotine Potassium permagnate Complex formation
Curare Neostigmine Receptor antagonism
Organophosphate insecticide Atropine Receptor antagonism
Carbamate insecticide 2-PAM Receptor antagonism
Morphine Nalorphine, naloxone Receptor antagonism
Barbirurate Bemegride Receptor antagonism
Benzodiazepine Flumazenil Receptor antagonism
Venom Anti-venom Complex formation
MISCELLANEOUS DRUGS IN TOXICOLOGICAL KIT

1. Mannitol/Glucose (10%)
2. Calcium borogluconate
3. Crocin
4. Coramine (Nikethamide)
5. Dopram (Doxapram)
6. Adrenaline hydrochloride
7. Pentazocine (Fortwin)
8. Atropine sulphate

53
Fig. 54: Scalpels Fig. 55: Scissors Fig. 56: Syringes with Needles
Fig. 57: Tourniquet Fig. 58: Hot Water Bag Fig. 59: Mouth Gag
Fig. 60: Stomach Tube Fig. 61: Endotracheal Tube Fig. 62: Rubber Catheter

54
7
DETECTION OF METAL POISONING
OBJECTIVE
To detect metal poisoning by the spot test in suspected sample.
TOXICITY OF METALS IN ANIMALS

In excessive amount, many metals, elements or chemical compounds can induce the
harmful effects in the body. The common sources of metal poisoning/toxicity in the
animals are the soil rich in a particular metal, minerals accumulated in the plants and
water containing the excess minerals. The potential for metallic poisoning is enormously
increasing due to the industrialization. Many metals are common ingredients in a variety
of commercial and household products, which make easy access of metals to the majority
of animals.
DETECTION OF METALS
The detection (analysis) of suspected materials like plants, water, feed, animal tissues
and P.M. specimens for the presence of poisonous metal(s) is required to aid in the
diagnosis of metal poisoning. The presence of metal(s) in a suspected sample is detected
by means of the ‘spot test’.
Reagents Required:
1. Ammonium carbonate (10%)
2. Ammonium sulphide (20%)
3. Potassium iodide (1%)
Standard Chart for Spot Test:

The ‘standard chart for spot test of poisonous metals’ has been illustrated in Table 9.

55
Table 9: Standard Chart for Spot Test of Poisonous Metals
Metal Colour Reaction of Solution by Adding Reagent

Ammonium Ammonium Potassium
Carbonate (10%) Sulphide (20%) Iodide (1%)
Lead (Pb) White Dark brown Yellow
Mercury Mercurous (Hg++) White Greenish-black Black
(Hg) Mercuric (Hg+++) White Yellow Red
Copper (Cu) Blue Dark brown Brown
Iron Ferrous (Fe++) Dirty green Black No reaction
(Fe) Ferric (Fe+++) Reddish-brown Black Brown
Cobalt (Co) Purple Black No reaction
Zinc (Zn) White White No reaction
Procedure:
1. A small amount of suspected material is triturated with 10 ml of distilled water.
2. Three drops of each reagent (viz., 10% ammonium carbonate, 20% ammonium
sulphide and 1% potassium iodide) are put on three glass slides, separately.
3. Then, 2 drops of the filtrate are added on each reagent, separately.
4. Each such solution is mixed properly and observed for the colour formed.
5. Finally, the colour of each solution is matched with the standard chart (Table 9) to
find the metal present in the sample.
Observation and Inference:

The tests are to be performed with the standard solutions of different metals, distilled
water and unknown sample to differentiate between the positive (+ve) and negative (-ve)
tests. The observations of different samples should be made on the basis of the‘standard
chart’ described in the above table. Finally, the interpretation should be written as “the
provided unknown sample contains the ……………..”.

56
8
DETECTION OF LEAD POISONING
OBJECTIVE
To detect lead poisoning in suspected sample.
TOXICITY OF LEAD IN ANIMALS

Lead (Pb) is one of the most frequently diagnosed poisoning in veterinary practice,
and has been reported in all domestic and several zoo animal species. The Pb poisoning
mainly results from the ingestion of lead-based paints, used oils, grease, linoleum,
asphalt, discarded batteries and lead arsenate in white lotion. The grass growing near
highways contains toxic amount of Pb from auto exhaust. The fumes or waste from Pb
smelting and plumbing works also accumulate the Pb in plants.
DETECTION OF LEAD
Detection of Pb in liver, renal cortex, blood, urine, faeces or fodder is done to
diagnose the Pb toxicity.
From the organic matter, Pb may be recovered by incineration. The ash is treated
with sulphuric acid (H2SO4), and again incinerated to yield the lead sulphate. The lead
sulphate and lead sulphide are, however, boiled with ammonium carbonate solution. The
resulting lead carbonate is dissolved in acetic acid and the solutions are tested for Pb. The
inorganic Pb compounds are dissolved by boiling with nitric acid (HNO3).
Procedure:
Test 1-
1. To 3 ml of sample, add a few drops of aqueous potassium dichromate solution.
2. Then, add dilute acetic acid solution until medium becomes acidic to litmus paper.
3. If Pb is present, a yellow precipitate of lead chromate will be formed.
57
Test 2-
1. Add hydrochloric acid (highly pungent solution of hydrogen chloride, HCl) to the
sample. A white precipitate of Pb will be formed.
2. This precipitate is soluble in boiling water and crystallizes on cooling.
Test 3-
1. Add few drops of potassium iodide (KI) solution to the sample. Bright yellow
precipitate will be formed.
2. This precipitate is soluble in boiling water and its crystallization to golden yellow
on cooling indicates the presence of Pb.
Test 4-
1. In 2 ml of sample, add few drops of tea infusion.
2. Formation of brown precipitate will indicate the presence of Pb.

The tests are to be performed with the standard solution of Pb, distilled water and
unknown sample to differentiate the positive (+ve) test from the negative (-ve) test.
Thereafter, the observations of different samples should be done, and the interpretation
should be written as “the provided unknown sample contains the ……………..”.

58
9
DETECTION OF MERCURY POISONING
OBJECTIVE
To detect mercury poisoning in suspected sample.
TOXICITY OF MERCURY IN ANIMALS

Mercury (Hg) exists in organic and inorganic forms. Sources of inorganic mercurial
poisoning are elemental Hg and salts, e.g., mercuric chloride, mercurous chloride, etc.
Main sources of organic mercurial toxicity are fungicides, and methyl mercuric
dicyanidiamide and methoxyethyl mercuric silicate used as seed dressing agents in
agriculture. Antiseptics and diuretics also cause toxicity. Secondary poisoning may be
due to ingestion of flesh of animals fed on mercurial fungicide. Acute and chronic
toxicities in animals are uncommon due to limited availability of toxic amounts of Hg.
DETECTION OF MERCURY
Analysis of blood, urine or suspected feed is done to detect Hg. The Hg is separated
from organic matter, and the analysis is done for mercurous or mercuric salts.
I. Non-Specific Test for Mercurous and Mercuric Salts:

1. Dissolve the sample in 1 ml concentrated H2SO4 and 3 ml concentrated HCl.
2. Add few drops of aqueous stannous chloride.
3. Formation of gray to black colour denotes the presence of Hg.
II. Specific Test for Mercurous Salts:
Test 1-
Add HCl to the suspected sample. White precipitation is formed which is insoluble
in acid, and is blackened by ammonia indicating the presence of mercurous salts.
59
Test 2-
Add KI to the suspected sample. Yellowish-green precipitate which turns grayish-
black in excess of reagent and on heating it indicates mercurous salts.
Test 3-
Mercurous salts give black precipitate with potassium hydroxide solution which is
insoluble in excess of potassium hydroxide solution.
Test 4-
Potassium dichromate solution gives brick red precipitate with mercurous salts.
Test 5-
On adding stannous chloride to the suspected sample, a white precipitate changing to
gray indicates the presence of mercurous salts.
Test 6-
Put a drop of sodium nitrite and a drop of silver nitrate on a filter paper to give the
white precipitate of silver nitrite. Add the suspected sample to this precipitate which turns
black if mercurous salt is present.
III. Specific Test for Mercuric Salts:
Test 1-
Add aqueous potassium hydroxide to the suspected sample. Yellow precipitate
denotes the presence of mercuric salts.
Test 2-
Mercuric salts give white precipitate on adding ammonia (NH3) solution.

60
Test 3-
Add dilute KI solution, drop by drop to the sample. A scarlet precipitate of mercuric
iodide, soluble in excess of reagent is shown by the mercuric salts.
Test 4-
A piece of Cu wire if introduced into the solution acidified with a few drops of HCl,
gives a silver coating of Hg on the wire.

The tests are to be performed with the standard solution of Hg, distilled water and

61
10
DETECTION OF ANTIMONY POISONING
OBJECTIVE
To detect antimony poisoning in suspected sample.
TOXICITY OF ANTIMONY IN ANIMALS

Antimony (Sb) is a silvery white metal used in alloys foils, platting, batteries and
ceramics. Some Sb preparations, e.g., stibenyl, stibamine and stibophen are used as
antiprotozoal drugs. The Sb compounds like antimony tartarate, trioxide and trichloride
are important from toxicological point of view. Toxicity of Sb may occur by overdose of
drugs, or by inhalation of vapours. The Sb is rapidly eliminated from body and may be
detected in urine. It does not disappear from the tissues after decomposition.
DETECTION OF ANTIMONY
The chemical tests for Sb do not work unless the organic matter is destroyed using
‘Strzyzowski method’ as under:
1. Gently heat 20 g of sample in 10 ml of aqueous saturated magnesium nitrite.
2. Make it alkaline with magnesium oxide. When the mass softens and chars, heat
more strongly to obtain the gray ash of magnesium pyroantimonate.
3. Soak the ash in 25 ml water and filter. Use filtrate for following chemical tests:
Test 1-
Add HCl to the filtrate. White precipitation is formed, which is soluble in excess of
solution indicating the presence of Sb.

62
Test 2-
Add little amount of sulphurated hydrogen to the filtrate. An orange precipitate of
antimony sulphide is formed, which is soluble in ammonium sulphide.

The tests are to be performed with the standard solution of Sb, distilled water and

63
11
DETECTION OF FLUORIDE POISONING
OBJECTIVE
To detect fluoride poisoning in suspected sample.
TOXICITY OF FLUORIDE IN ANIMALS

Acute fluoride poisoning is rare in animals and may occur due to the ingestion of
inorganic fluoride compounds, such as sodium fluoride (used as vermifuge) and sodium
fluorosilicate (used as rodenticide). Chronic poisoning of fluoride is more common
among animals and usually occurs after continuous ingestion of water, forages and feed
supplements higher in fluoride content. The fluoride is a normal constituent of forages
(especially legumes) and plants growing in fluoride rich soils. The pasture can be
contaminated with the fluoride fallout from nearby industries. The absorbed fluoride is
deposited in skeletal tissues causing pathological alteration in bones and teeth.
DETECTION OF FLUORIDE
The fluoride assay of feed, water, blood, urine, bone, teeth and faeces is an important
tool in the diagnosis of fluoride toxicity. The assay can be performed as under:
1. Take suspected material. Mix it with sodium hydroxide (NaOH) and incinerate it.
2. Place the resulting ash on a small shallow container and add small amounts of
concentrated H2SO4. Cover the container with glass and heat it gently.
3. The vapours of hydrofluoric acid etch the glass.

64
Standard sodium fluoride solution, distilled water and unknown sample should be
used to know the +ve and -ve tests. As per the observations of samples, the interpretation
should be written as “the provided unknown sample contains the …………..”.
12
DETECTION OF COPPER POISONING
OBJECTIVE
To detect copper poisoning in suspected sample.
TOXICITY OF COPPER IN ANIMALS

Copper (Cu) is an essential component of the animal system, and plays an important
role in various physiological functions. The Cu has complex interrelationship with some
elements like molybdenum (Mo) and sulphur (S). The sources may include the
contaminated forages in the vicinity of mines or smelters, water from Cu pipes, copper
sulphate (CuSO4) spills, foot baths containing CuSO4 and ponds treated with CuSO4 for
its algaecidal property. In animals, the acute Cu toxicosis is not common. In chronic
toxicity, the ingestion of Cu can be chronic but onset of toxicity is acute because of
sudden release of Cu from the liver.
DETECTION OF COPPER
The analysis of suspected materials such as liver, kidney, blood, urine and faeces is
done to detect the presence of Cu.
Test 1-
1. Take 2 ml of suspected sample in a test tube.
2. Add few drops of NH3 solution.
3. Formation of bluish-violet precipitate indicates the presence of Cu.

65
Test 2-
2. Add few drops of potassium ferrocyanide solution.
3. Formation of dark brown precipitate indicates the presence of Cu.
Test 3-
2. Add few drops of KI solution.
3. Formation of brown precipitate indicates the presence of Cu.

The tests are to be performed with the standard solution of Cu, distilled water and

66
13
DETECTION OF ZINC POISONING
OBJECTIVE
To detect zinc poisoning in suspected sample.
TOXICITY OF ZINC IN ANIMALS

Zinc (Zn) is an essential component of many metallo enzymes, and is required for
growth, skeletal development, collagen formation, feathering, dermal health and
reproductive performance in mammals and birds. Poisoning by the Zn compounds is rare
in animals. However, the poisoning may occur by inhalation of industrial fumes,
consumption of feed mixtures that contain excessive amounts of Zn salts, chewing on
galvanized bars, pipes, coins, wires and licking of Zn coated containers.
DETECTION OF ZINC
The analysis of materials such as plants, water, feed, animal tissues (e.g., liver,
kidney, bone, pancreas, hair, faeces, serum, etc.) is done to detect the presence of Zn.
Test 1-
2. Add few drops of potassium hydroxide (KOH) solution.
3. Formation of white precipitate which dissolve in excess of reagent indicates the
presence of Zn in the sample.
Test 2-
2. Add egg albumin in the sample
3. Formation of white precipitate indicates the presence of Zn in the sample.
67
Test 3-
1. To 2 ml of suspected sample, add few drops of potassium ferrocyanide solution.
2. White precipitate of zinc ferrocyanide is formed, which is insoluble in HCl.
3. Add few drops bromine water to the precipitate which produces greenish-yellow
or yellow colour, which on boiling forms a green or bluish-green precipitate
indicative of the presence of Zn in the sample.

The tests are to be performed with the standard solution of Zn, distilled water and

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14
DETECTION OF CYANIDE POISONING
AND ITS TREATMENT TEST
OBJECTIVE
To detect cyanide poisoning in suspected sample and perform its treatment test.
TOXICITY OF CYANIDE IN ANIMALS

Domestic animals are frequently affected by cyanide. The cyanides are present in
plants, fumigants, fertilizers and rodenticides. The most frequent cause of cyanide
toxicity to animals is the ingestion of cyanogenetic plants, containing cyanogenetic
glycosides initiated by the degradation of enzymes released after damage to plant cells. In
the alimentary tract (GIT) of animals, the cyanogenetic glycosides are hydrolyzed by
microbial or acid hydrolysis to liberate the hydrogen cyanide (HCN). Main cyanogenetic
plants are Sorghum sp. (e.g., jowar, sudan grass, etc.), millet, corn, linseed, lotus, Acacia
sp., Eucalyptus sp., apricot, peach, apple, wild cherry, wild clover, velvet grass, etc. The
plant materials containing more than 20 mg of HCN per 100 g may be toxic to animals.
MECHANISM OF ACTION OF CYANIDE

The HCN is a volatile gas, which is irritant to the mucous membranes. The ‘cyanide
radical’ forms complex with the ‘ferric’ ion of cytochrome oxidase, thus inhibiting the
electron transport system, thereby the cells die due to lack of usable oxygen. Death
occurs due to acute tissue anoxia in the brain.
CLINICAL SIGNS OF CYANIDE TOXICITY

Initially, there is excitement, rapid respiration and tachycardia; followed by
dyspnoea, salivation, lacrimation, frequent urination, vomition and colic. Tremors,

69
muscle spasms, clonic convulsions, staggering gait and collapse occur before death. The
mucous membranes are bright red which become cyanotic, terminally. Death occurs
during severe asphyxial convulsions.
DETECTION OF CYANIDE
The analysis of materials like feed, stomach contents, blood, liver and muscle is done
for the presence of HCN. The samples in dry form are triturated in a small amount of
distilled water. The triturated material is then filtered and the filtrate is used for analysis.
Preparation of Picric Acid Strips:

The strips of filter paper are dipped in saturated aqueous solution of picric acid and
dried at room temperature. These strips are soaked in 10% solution of sodium carbonate
or NaOH and again dried at room temperature.
Procedure:
After doing above, the following procedures are performed for detection of HCN:
1. One to two ml of suspected material is taken in a test tube and boiled.
2. Freshly prepared ‘picric acid strips’ are exposed to the vapours. Change of the
colour of these strips to red indicates the presence of HCN in the vapours. On the
basis of this, the suspected material is confirmed as containing the ‘cyanide’.

The tests are to be performed with the standard solution of HCN, distilled water and
TREATMENT TEST OF CYANIDE TOXICITY

Immediate treatment is necessary. The major objective is to remove the cyanide
radical from the ‘ferric’ ion of cytochrome oxidase. For this, 10% solution of nitrite is

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given @ 20 mg/kg, iv. Sodium nitrite (specific antidote of cyanide/HCN) converts some
of the Hb into the methaemoglobin, which competes with cytochrome oxidase for
‘cyanide radical’ and forms cyanmethhaemoglobin, making the cytochrome oxidase free.
Next, replace the available thiosulphate in the bloodstream. For this, 20% sodium
thiosulphate (specific antidote of cyanide/HCN) solution is injected intravenously @ 500
mg/kg. Sodium thiosulphate reacts with cyanide in the bloodstream, or with
cynomethaemoglobin and forms thiocyanate which is then excreted. This therapy is
replaced at every 2 to 4 hr by reducing the dose of sodium nitrite to 10 mg/kg. In
addition, the large dose of cyanocobalamine may be given. The cobalt in this preparation
forms the complex with additional cyanide in the circulation.
For experimentation, the items required are: rats, sodium cyanide (0.1%), sodium
nitrite (1%) and sodium thiosulphate (25%). The following procedures are to be adopted:
1. Weight of rats is recorded and total dose of chemicals to be given is calculated.
2. Sodium cyanide is injected at the dose of 5 mg/kg, ip.
3. Observations are made for the onset, nature and duration of symptoms.
4. Treatment is given immediately on the arrival of symptoms.
5. Calculations of total dose and volume of chemicals to be given for cyanide
toxicity and its treatment in rats should be written as per the Table 10.
6. Observations for toxicity and its treatment should be written as per the Table 11.
In conclusion, the sodium cyanide produces acute toxicity in rats. This toxicity is
treated by sodium nitrite (1% solution) and sodium thiosulphate (25% solution).
Table 10: Calculations of Dose and Volume of Chemicals for Toxicity/Treatment
Chemical Dose Rate Total Dose % of Total

Solution Volume (ml)
Sodium cyanide 5 mg/kg, ip Calculate as per kg body weight 0.1% Calculate
Sodium nitrite 20 mg/kg, ip Calculate as per kg body weight 1% Calculate
Sodium thiosulphate 0.5 mg/kg, ip Calculate as per kg body weight 25% Calculate
Table 11: Observations of Cyanide Toxicity Symptoms and their Recovery after Treatment
Symptom Time of Onset of Duration of Time of Recovery

Symptom Symptom after Treatment

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15
DETECTION OF NITRATE/NITRITE POISONING
OBJECTIVE
To detect nitrate/nitrite poisoning in suspected sample and perform its treatment test.
TOXICITY OF NITRATE/NITRITE IN ANIMALS

Nitrate and nitrite poisonings are common in animals because these are widely used
in fertilizers, and are naturally present in the soils, ground waters, forages, silages, crops,
weeds, animal tissues and excreta. The main hazard to livestock is the consumption of
plants growing on nitrate rich soils. The crops that concentrate nitrate are oat, millet, rye,
corn, sunflower and Sorghum sp. The plants containing more than 1% nitrate on dry
mater basis are considered to be toxic. When the animals ingest these plants, the nitrates
are converted by microbes to nitrite, which is the main cause of poisoning.
Nitrite is approximately 10 times more toxic than nitrate. The domestic animals are
highly susceptible to nitrite poisoning. This poisoning is more common in cattle, sheep
and pig. The acute oral lethal dose of nitrite is 70 mg/kg in pig and 500 mg/kg in cattle.
MECHANISM OF ACTION OF NITRITE

The acute toxicity results by two actions of nitrite:
1. Direct relaxation of vascular smooth muscle- The vasodilation leads to
hypotension, decreased cardiac output and tissue oxygen starvation. The nitrite
ions also alert certain metabolic enzymes.
2. One molecule of nitrite interacts with two molecules of Hb and oxidizes it to
methaemoglobin, which is incapable of oxygen transport leading to death from
the tissue anoxia.

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CLINICAL SIGNS OF NITRITE TOXICITY

The signs and symptoms during the nitrite toxicity appear suddenly due to the tissue
hypoxia. The initial sings are low blood pressure, rapid weak heart beat, subnormal
temperature, muscular tremor, weakness, and appearance of brown and cyanotic mucous
membrane. The rapid difficult breathing, anxiety and frequent urination are commonly
seen in the affected animals. The monogastric animals exhibit salivation, vomition,
diarrhoea, abdominal pain and gastric haemorrhage. The affected animals may die
suddenly within one hour in the terminal anoxic convulsion phase.
DETECTION OF NITRATE/NITRITE
The presence of nitrate or nitrite in the suspected sample may be detected by simple
field tests. If the sample is plant or dried material, then it is triturated, filtered and the
filtrate is used for analysis.
I. Non-Specific Test for Detection of Nitrate/Nitrite:
Test 1-
Prepare 1% solution of diphenylamine in concentrated H2SO4. Then, do the
followings-
1. One to two drops of suspected material is taken in a glass slide.
2. To this, 1 to 2 drops of diphenylamine solution is added.
3. Conversion to deep blue colour denotes the presence of nitrate or nitrite.
Test 2-
Prepare 50% solution of salicylic acid in concentrated H2SO4. Then, do the
followings-
1. One ml suspected material is taken in a test tube.
2. To this, 1 ml of salicylic acid solution is added.
3. Development of yellow colour changing to brown denotes the presence of nitrate
or nitrite.

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II. Differential Test for Nitrite:
Principle-
Sulphanilamide reacts with nitrite to form the diazo compounds. Nephthyl ethylene
diamine dihydrochloride (NEDD) forms complex with the diazo compounds to give rise
the colour.
Reagents-
1% solution of sulphanilamide prepared in 1.5N HCl and 0.02% solution of n-1
NEDD.
Procedure-
1. One to two drops of suspected material is taken in a glass tube.
2. To this, 1 to 2 drops of sulphanilamide is added and mixed.
3. Then, 1 to 2 drops of NEDD is added and mixed. Development of violet colour
indicates the presence of nitrite.

The tests are to be performed in the standard solutions of sodium nitrate and sodium
nitrite, distilled water, and unknown sample to differentiate the positive (+ve) test from
the negative (-ve) test. Thereafter, the observations of different samples should be done,
and the interpretation should be written as “the provided unknown sample contains the
……………..”.
TREATMENT TEST OF NITRITE TOXICITY

The primary aim of treatment is to convert the methaemoglobin into oxyhaemoglobin
by administering a suitable reducing agent, e.g., methylene blue (specific antidote of
nitrate/nitrite poisoning) or ascorbic acid. 1% solution of methylene blue in isotonic
saline is given by slow iv route. The methylene blue injection @ 9 mg/kg in cattle and
sheep, and 4.4 mg/kg in other species is administered. The dose is repeated within 15 to
30 minutes, if needed. Ascorbic acid is given at the dose rate of 5 to 20 mg/kg, iv. Rumen

74
lavage with cold water and oral antibiotics are given to reduce the microbial conversion
of nitrate to nitrite. Fluid therapy is provided by slow iv infusion of normal saline at the
dose of 10 to 15 ml/kg in large animals and 20 to 25 ml/kg in small animals.
For experimentation, the items required are: rats, sodium nitrite (1% solution),
methylene blue (1% solution in normal saline). The following procedures are to be
adopted:
1. Weight of each rat is recorded.
2. Sodium nitrite and methylene blue are administered to the rats, and their doses
and volumes are calculated @ 70 mg/kg, oral and 9 mg/kg, ip, respectively. These
should be written as per the Table 12.
3. The observations are made for the onset, nature and duration of symptoms, and
for time of recovery after treatment. These should be written as per the Table 13.
In conclusion, the sodium nitrite produces acute toxicity in rats. This toxicity is
treated by methylene blue.
Table 12: Calculations of Dose and Volume of Chemicals for Toxicity/Treatment

Sodium nitrite 70 mg/kg, orally Calculate as per kg body weight 1% Calculate
Methylene blue 9 mg/kg, ip Calculate as per kg body weight 1% Calculate
Table 13: Observations of Nitrite Toxicity Symptoms and their Recovery after Treatment


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16
DETECTION OF UREA POISONING
OBJECTIVE
To detect urea poisoning in suspected sample.
TOXICITY OF UREA IN ANIMALS

The toxicity of urea is an acute, rapidly progressing and highly fatal condition caused
by the excess ingestion of urea. The mature ruminants are most commonly affected. The
animals may get poisoned by accidental ingestion of solid or liquid form of urea due to
improper storage, spillage, feeding of large quantity of non-protein nitrogen (NPN) urea
molasses feeds to unaccustomed animals, improper mixed feed, etc. In the rumen, urea is
hydrolyzed by the enzyme urease to release NH3, which is absorbed from the GIT and
produces toxicity. The death in urea poisoning is very high. The animals show severe
abdominal pain, shivering, drunken gait, bloat, salivation, rapid breathing, and violent
struggling and bellowing.
DETECTION OF UREA
Required Reagent:
The reagent is prepared by dissolving 1.6 g p-dimethyl aminobenzaldehyde (p-
DMAB) in 90 ml alcohol and 10 ml concentrated HCl. This reagent is useful for the
detection of urea in the suspected sample.
Procedure:
1. Mix the suspected sample and reagent in equal quantities.
2. Formation of yellow colour indicates the presence of urea in a given suspected
sample.

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The tests are to be performed in the standard solution of urea, distilled water and
unknown sample for positive (+ve) and negative (-ve) tests. As per the observations, the
interpretation should be written as “the provided unknown sample contains the ………..”.

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17
DETECTION OF CHLORAL HYDRATE
POISONING
OBJECTIVE
To detect chloral hydrate poisoning in suspected sample.
TOXICITY OF DRUGS IN ANIMALS

The main determinant of drug toxicity is the dose of a drug/chemical compound. The
drug used injudiciously or at high doses may produce non-lethal adverse effects in the
animals. Drugs/chemical compounds having narrow margin of safety may even produce
death at the slight overdose. The young animals are particularly vulnerable to certain
types of drug toxicity. Moreover, no specific antidotes are known for most of the drugs.
TOXICITY OF CHLORAL HYDRATE IN ANIMALS

Chloral hydrate is used as a hypnotic, sedative and anesthetic agent. Its poisoning
may frequently occur due to the overdose. It has a pungent odour and bitter taste.
DETECTION OF CHLORAL HYDRATE

Different body tissues are chemically analyzed for the presence of drugs and their
metabolites to facilitate the diagnosis of drug toxicity.
As far as the anslysis (detection) of chloral hydrate is concerned, the suspected
tissues are finely minced and then distilled with steam in 20% solution of phosphoric
acid. Thereafter, the following tests should be done with the ‘distillate’:
Test 1-
1. Add a few drops of Nesseler’s reagent to the distillate.

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2. Yellowish to reddish brown precipitate is produced which changes to green or

black, indicating the presence of chloral hydrate.
Test 2-
1. Add 0.1 g of resorcinol to 2 to 3 ml of distillate.
2. Then, add 1 ml of 15% NaOH solution and boil.
3. A yellowish-red to red colour denotes the presence of chloral hydrate.
Test 3-
1. Take 2 ml of distillate in a test tube.
2. Add 1 ml 5% alcoholic solution of KOH solution and 1 ml of aniline.
3. Gently heat the mixture with shaking.
4. The offensive odour of phenyl isocyanide indicates the presence of chloral
hydrate, chloroform or other organic halogen compounds like iodoform.

The tests are to be performed in the standard solution of chloral hydrate, distilled
water and unknown sample to differentiate the positive (+ve) test from the negative (-ve)
test. Thereafter, the observations of different samples should be done, and the
interpretation should be written as “the provided unknown sample contains the
……………..”.

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18
DETECTION OF PHENOBARBITONE/
BARBITURATES POISONING
OBJECTIVE
To detect phenobarbitone (a barbiturate) poisoning in suspected sample.
TOXICITY OF PHENOBARBITONE OR BARBITURATES IN ANIMALS

Barbiturates are commonly used as sedatives, hypnotics and for induction of
anesthesia. The poisoning of barbiturates (e.g., phenobarbitone) usually occurs in animals
due to the over dosage.
DETECTION OF PHENOBARBITONE OR BARBITURATES

Presence of phenobarbitone/barbiturates can be detected by doing the following tests:
Test 1-
1. Boil the suspected sample with 8% aqueous sodium bicarbonate.
2. Liberation of NH3 indicates the presence of phenobarbitone or barbiturate.
Test 2-
1. Dissolve the suspected sample in glacial acetic acid.
2. Add few drops of 15% aqueous mercuric chloride and 4% NaOH.
3. A white precipitate indicates the presence of phenobarbitone or barbiturate.
Test 3-
1. Put a few drops of Millon’s reagent in small quantity of warm water and add to
the suspected sample.
2. A white gelatinous precipitate is formed which is insoluble in excess of reagent.

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The tests are to be performed in the standard solution of phenobarbitone or any other
barbiturate, distilled water and unknown sample to differentiate the positive (+ve) test
from the negative (-ve) test. Thereafter, the observations of different samples should be
done, and the interpretation should be written as “the provided unknown sample contains
the ……………..”.

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19
DETECTION OF SULPHONAMIDES POISONING
OBJECTIVE
To detect sulphonamides poisoning in suspected sample.
TOXICITY OF SULPHONAMIDES IN ANIMALS

Sulphonamides (sulpha drugs) are commonly used as antimicrobials in animals. In
excess amounts and chronic use, the sulphonamides cause poisoning in animals.
DETECTION OF SULPHONAMIDES
Sulphonamides are extracted from the neutral aqueous solution in acetone by “Stas-
Otto’s process”. The residues are dissolved in water, heated and filtered. The filtrate is
saturated with sodium chloride (NaCl) and treated with acetone. On evaporation to
dryness, sulphonamide with some NaCl is obtained as a residue which can be
distinguished by the following tests:
Test 1-
1. Add few drops of p-dimethylamino-benzaldehyde solution (prepared by
dissolving in water acidified by strong H2SO4) to a small amount of residue or its
solution.
2. A yellow or orange precipitate indicates the presence of sulpha drug.
Test 2-
1. Dissolve the residue in warm dilute HCl.
2. Cool and mix with 2 ml of 1% sodium nitrite solution.
3. Add 2 ml of distilled water and 1 ml of β-naphthol solution.
4. Orange solution or precipitate is formed by the sulpha drug (sulphonamide).

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Test 3-
1. Add one drop of concentrated HCl to one drop of suspected sample on a paper.
2. Orange colour indicates the presence of sulpha drug (sulphonamide).

The tests are to be performed in the standard solution of any sulphonamide, distilled
water and unknown sample to differentiate the positive (+ve) test from the negative (-ve)
test. Thereafter, the observations of different samples should be done, and the
interpretation should be written as “the provided unknown sample contains the
……………..”.

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20
DETECTION OF ACETYL SALICYLIC ACID
POISONING
OBJECTIVE
To detect acetyl salicylic acid (aspirin) poisoning in suspected sample.
TOXICITY OF ACETYL SALICYLIC ACID IN ANIMALS

Acetyl salicylic acid (aspirin) is commonly used as an analgesic, antiinflammatory
and antipyretic agent. Its excess amounts and long use may cause poisoning in animals.
DETECTION OF ACETYL SALICYLIC ACID

Aspirin can be easily extracted with water. The aqueous solution is shaken out with
ether and the ether extract is evaporated. The residue contains aspirin, which can be
detected by the following tests:
Test 1-
1. Add few drops of dilute ferric chloride solution to the residue/suspected sample.
2. A yellowish-brown colour indicates the presence of aspirin.
Test 2-
1. Add few drops of 1% ammonium molybdate solution (in H2SO4) to the residue/
suspected sample.
2. The presence of aspirin shows a blue colour changing to violet.

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Test 3-
1. Boil 0.2 to 0.4 g of residue/suspected sample with 2 ml of Millon’s reagent (5 g
mercury + 5 ml fuming nitric acid + 10 ml distilled water) for 30 seconds.
2. The presence of aspirin shows a blue colour changing to violet.

The tests are to be performed in the standard solution of aspirin, distilled water and

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21
DETECTION OF INSECTICIDES POISONING
OBJECTIVE
To detect organophosphate insecticides poisoning in suspected sample and perform
its treatment test.
TOXICITY OF ORGANOPHOSPHATE INSECTICIDES IN ANIMALS

Malathion is an indirectly acting organophosphate insecticide (OPI) which causes
toxicity after conversion to malaoxon in the body. The OPI compounds are widely used
in agriculture as pesticides. The poisoning of insecticides to animals occurs after the
consumption of plants sprayed with insecticides, or by accidental ingestion.
MECHANISM OF ACTION OF ORGANOPHOSPHATE INSECTICIDES

OPIs cause toxicity by inhibiting the acetylcholinesterase (AchE) enzyme, which is
essential for the hydrolysis of acetylcholine (Ach). This inhibition leads to the
accumulation of Ach, which produces the symptoms of parasympathomimetic
stimulation. The OPIs bind to both the anionic and esteratic sites of AchE enzyme.
SYMPTOMS OF ORGANOPHOSPHATE INSECTICIDES TOXICITY

Inhibition of AchE results into four different types of symptoms in animals:
A. Muscarinic Symptoms:

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‘Muscarinic symptoms’ result from the stimulation of muscarinic receptors in the

smooth muscles, heart and exocrine glands. The symptoms included are increased
bronchial secretion, bronchoconstriction, increased salivation and lacrimation, excessive
sweating, increased gastrointestinal motility, frequent and involuntary urination, difficult
breathing, hypotension, bradycardia, and papillary constriction.
B. Nicotinic Symptoms:
‘Nicotinic symptoms’ result from the accumulation of Ach at the skeletal motor nerve
endings and autonomic ganglia. The muscular effects included are: muscular weakness,
twitching, fasciculation, cramp, tremor and atrophy.
C. Central Nervous System Symptoms:

The symptoms of CNS include: restlessness, convulsion, cardiac arrest, respiratory
arrest and collapse. Death may result from the asphyxia due to respiratory failure.
D. Delayed Neuropathy:
‘Delayed neuropathy’ means the functional disturbances occurring in the distal part
of hind limb which progresses to increased weakness and flaccidity.
TREATMENT TEST OF MALATHION TOXICITY

Treatment for OPIs (e.g., malathion) toxicity is given to animals by two ways:
i. To prevent over stimulation of muscarinic receptors- For this, atropine sulphate
(0.5%) @ 0.2 to 0.5 mg/kg is administered with physiological saline. One-fourth
of this dose is injected iv, and three-fourth dose is injected im or sc.
ii. To regenerate inhibited cholinesterase enzyme- For this, oxime reactivator, e.g.,
DAM (6%) @ 30 mg/kg, iv or im is administered with physiological saline.
For experimentation, the items required are: rats, malathion (5%), atropine sulphate
(0.5% solution) in physiological (normal) saline and DAM (6% solution) in physiological
saline. The following procedures are to be adopted:

87
2. Total dose and volume of malathion are calculated, using the dose of 107 mg/kg
(lethal dose). This is mixed with 100 ml tap water. Similarly, the total dose and
volume of atropine and DAM are calculated. All these drugs are injected to the
rats of respective group. The data should be written as per the Table 14.
4. Atropine and DAM treatments are given at the peak of toxic symptoms.
In conclusion, malathion produces acute toxicity in animals. This toxicity is treated
by atropine and DAM, which are the antidotes of malathion.
Table 14: Calculations of Dose and Volume of Drugs for Toxicity/Treatment

Malathion 107 mg/kg, iv or im Calculate as per kg body weight 5% Calculate
Atropine 0.5 mg/kg, iv or im Calculate as per kg body weight 0.5% Calculate
sulphate
DAM 30 mg/kg, iv or im Calculate as per kg body weight 6% Calculate
Table 15: Observations of Malathion/OPIs Toxicity Symptoms and their Recovery after
Treatment


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22
DETECTION OF ALKALOIDS POISONING
OBJECTIVE
To detect alkaloids poisoning in suspected sample.
TOXICITY OF ALKALOIDS IN ANIMALS

Alkaloids are colourless, crystalline compounds usually in powder form, but some
may be liquid. They are soluble in organic solutions. They usually exist as salts of
organic or inorganic acids, and some lie in free state. Rarely, the alkaloids exist in
combination with sugars, as amides, or as esters.
The alkaloids are highly poisonous, but are used medicinally in very small quantities,
e.g., quinine, cocaine, morphine, piperine, ephedrine, arecholine and berberine.
Normally, alkaloids are irritating to GIT, producing nausea, colic and diarrhoea. They
also act on the CNS, producing blindness, muscular weakness, convulsion and death.
DETECTION OF ALKALOIDS
Firstly, the extraction of plant material is done by drying it at 60oC in an oven (40oC
for volatile alkaloids). The dried material is finely powdered and stored in dark. The
alkaloids are then extracted in ethanol (ethyl alcohol) solvent.
Thereafter, individual alkaloid is separated from a mixture of closely related
alkaloids by the process of fractional crystallization.
Then, the detection of alkaloid is accomplished with different reagents, which give
the precipitate. So, the following tests are to be carried out:
A. Hayer’s Reagent:
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Prepare ‘solution A’ by dissolving 1.36 g of mercuric chloride (HgCl2) in 16 ml of

distilled water. Then, prepare ‘solution B’ by dissolving 5 g of potassium iodide (KI) in
10 ml of distilled water. Both these solutions are mixed and diluted to 100 ml in distilled
water. Then, perform the test as described below:
2. Add few drops of Hayer’s reagent.
3. This reagent forms precipitate with hydrochloride of alkaloid.
B. Wagner’s Reagent:
Dissolve 1.27 g of iodine and 2.0 g of KI in 5 ml of distilled water, and the solution
is diluted to 100 ml. This is called the Wagner’s reagent. Then, perform the test as
described below:
2. Add few drops of Wagner’s reagent.
3. This reagent gives brown flocculant precipitate with alkaloid.
C. Dragendroff’s Reagent:
Prepare ‘solution A’ by dissolving 8 g of bismuth subnitrate in 20 ml of HNO3.
Then, prepare ‘solution B’ by dissolving 21.2 g of KI in 50 ml of distilled water. These
two solutions are mixed and allowed to stand. The supernatant is decanted and diluted to
100 ml of distilled water. The Dragendroff’s reagent gives the precipitate with alkaloid.
D. Tannic Acid:
Freshly prepared 1% aqueous solution of tannic acid gives the precipitate with
alkaloid.
E. Picric Acid:
Freshly prepared 1% aqueous solution of picric acid gives yellowish-white
precipitate with alkaloid.

90
The tests are to be performed in the standard solution of any alkaloid (e.g., atropine,
pilocarpine, strychnine, etc.), distilled water and unknown sample for positive (+ve) and
negative (-ve) tests. As per the observations, the interpretation should be written as “the
provided unknown sample contains the ………..”.

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23
DETECTION OF STRYCHNINE POISONING
OBJECTIVE
To detect strychnine poisoning in suspected sample and perform its treatment test.
TOXICITY OF STRYCHNINE IN ANIMALS

Strychnine is an alkaloid, obtained from nux-vomica (Strychnos nux-vomica) plant. It
is a potent convulsant, and is also commonly used as a rodenticide. In small doses, it is
used as tonic and stimulates the appetite. The preparation of strychnine are used in skin
infections, pruritus, mange and dermatitis. Strychnine poisoning occurs mostly in dogs
and cats either due to accidental ingestion of rodenticides or through the killed rodents.
The LD50 of strychnine in dog is 0.75 mg per kg body weight.
MECHANISM OF ACTION OF STRYCHNINE

Strychnine is a CNS stimulant. It acts as an antagonist of glycine, which is an
inhibitory neurotransmitter in CNS.
CLINICAL SIGNS OF STRYCHNINE TOXICITY

The clinical signs of acute poisoning of strychnine include: hyperexcitement,
muscular tremor, increased heart and respiration rates, convulsion, and paralysis. Death
occurs due to the respiratory failure.
DETECTION OF STRYCHNINE
The analysis of stomach contents, blood, liver, lung, urine and suspected plant is
performed to detect the presence of strychnine. The dried material or tissue is triturated in

92
a little amount of distilled water and then filtered. The filtrate is taken to detect the
strychnine with the help of following tests:
A. Tannic Acid Test:

1. Freshly prepared solution (0.1%) of tannic acid is taken as reagent.
2. One ml of suspected material (filtrate) is taken in a test tube and equal quantity of
tannic acid solution is added to it.
3. Formation of white precipitate indicates the presence of strychnine in the sample.
B. Potassium Permanganate Test:

1. Freshly prepared solution (1%) of potassium permanganate (KMnO4) is taken.
2. One ml of suspected material (filtrate) is taken in a test tube and 0.5 ml of KMnO4
reagent is added to it.
3. Discolouration of the KMnO4 solution indicates the presence of strychnine.

The tests are to be performed in the standard solution of strychnine sulphate, distilled
water and unknown sample for positive (+ve) and negative (-ve) tests. As per the
observations, the interpretation should be written as “the provided unknown sample
contains the ………..”.
TREATMENT TEST OF STRYCHNINE TOXICITY

The symptomatic treatment is given to control the nervous symptoms of strychnine
poisoning. Thus, the following symptomatic treatments can be given:
i. A CNS depressant, e.g., pentobarbitone sodium @ 50 mg/kg, iv.
ii. 2% aqueous solution of tannic acid, orally to neutralize the poison in gut.
iii. Isotonic glucose or saline, iv to accelerate the urine formation and elimination of
strychnine from the bloodstream.
iv. A large dose of cyanocobalamine; cobalt in this preparation forms the complex
with additional cyanide in the circulation.

93
For experimentation, the items required are: rats, strychnine sulphate (0.1% solution)
and pentobarbitone sodium (1% solution). Then, the following should be done:
2. Total dose and volume of strychnine sulphate are calculated, using the dose of @
10 mg/kg, ip. Similarly, the total dose and volume of pentobarbitone sodium are
calculated, using the dose of @ 50 mg/kg, ip. The data should be written as per
the Table 16.
6. As soon as the symptoms of strychnine toxicity appear, the pentobarbitone
treatment is given.
Conclusively, strychnine produces acute CNS toxicity in rats. This toxicity is treated
by administering the CNS depressants.
Table 16: Calculations of Dose and Volume of Drugs for Toxicity/Treatment

Strychnine sulphate 10 mg/kg, ip Calculate as per kg body weight 0.1% Calculate
Pentobarbitone 50 mg/kg, ip Calculate as per kg body weight 1.0% Calculate
sodium
Table 17: Observations of Strychnine Toxicity Symptoms and their Recovery after
Treatment


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24
DETECTION OF GLYCOSIDES POISONING
OBJECTIVE
To detect glycosides poisoning in suspected sample.
TOXICITY OF GLYCOSIDES IN ANIMALS

Glycosides are organic compounds which on hydrolysis with inorganic acids or
enzymes yield a sugar and one or more non-sugar products called ‘aglycone’. The
aglycone may be an alcohol or phenol. Certain examples of glycoside are digitalis (viz.,
digoxin, digitoxin and digitalin), amygdalin, linamarin, gynocardin, oubain, saponin
glycosides, mustard glycosides, etc.
Cyanogenic glycosides occur in plant species of Sorghum, Acacia and Eucalyptus,
millet, corn, linseed, lotus, apricot, peach, apple, velvet grass, marsh-arrow grass, wild
cherry, wild clover, etc. These glycosides (possessing HCN) interfere with the oxygen
exchange from the lungs to the body tissues so that various tissues, including brain are
starved for oxygen, and are consequently injured. Muscle tremor, difficult rapid
respiration and convulsion occur. Often these symptoms are not seen because death
occurs within minutes. Saponin glycosides are found in purple cockle (Agrostemma
githago), cow cockle, bouncing bet and poke weed (Phytolacca acinosa). When the
saponin glycosides are absorbed into the bloodstream, they cause a breakdown of RBCs
and injury to the CNS, producing convulsion and paralysis. Mustard oil glucosides are
found in plants belonging to the mustard family (e.g., Brassica campestris and Br. nigra)
They produce severe gastroenteritis, severe colic and purging.
DETECTION OF GLYCOSIDES
Glycosides are hydrolyzed with 6% mineral acids (H2SO4 or HCl), and are extracted
with ether.

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Procedure:
For detection of glycosides, ‘paper chromatography’ is performed. Thus, the
following solvents/reagents are required:
Solvent A = Chloroform:Tetrahydrofuran:Formamidine (50:50:6.5)
Solvent B = n-butanol:Acetic acid:Water (4:1:5)
Spraying agent: Dilute periodate solution and benzidine.
With dilute periodate solution, the glycol groups of sugar moiety are split and
periodate is reduced to iodate.

The paper chromatography is to be performed with the standard solution of any
glycoside and the distance travelled by the glycoside is recorded. Similarly, the paper
chromatography to be performed with the test (unknown) sample and the distance moved
by the test sample is noted. By comparing with the standard glycoside, the unknown
glycoside is detected in the test sample. As per the observations, the interpretation should
be written as “the provided unknown sample contains the ………..”.

96
25
DETECTION OF DIGITALIS POISONING
OBJECTIVE
To detect digitalis glycoside poisoning in suspected sample.
TOXICITY OF DIGITALIS IN ANIMALS

Digitalis is used as cardiac stimulant in congestive heart failure. It is obtained from
the leaves of Digitalis purpurea. It contains several glycosides, e.g., digoxin, digitoxin
and digitalin as active principles. Digitalis has a very narrow margin of safety. Its
poisoning may result even from the slight overdose or by the ingestion of its leaves.
DETECTION OF DIGITALIS
The digitalis glycosides are extracted from the acidified organic material with
chloroform. The following two tests are to be performed for the detection of different
digitalis glycosides:
Test 1-
1. Treat the sample with concentrated H2SO4 and add bromine water.
2. Green colour not decolourized by the bromine indicates the presence of digitoxin.
3. Orange-yellow colour rapidly changing to red or violet with bromine indicates the
presence of digitalin.
4. Red colour intensified with bromine indicates the presence of digitonin.
5. Emerald-green turning brown indicates the presence of strophanthin glycoside.
Test 2-
1. Heat the unknown sample with a few drops of the mixture containing equal parts
of strong H2SO4 and alcohol.

97
2. The digitalis turns to yellowish-brown, which changes to bluish-green on adding a

drop of dilute ferric chloride (FeCl3) solution.

The tests are to be performed in the standard solution of any digitalis, distilled water
and unknown sample to differentiate the positive test (+ve) from the negative (-ve) test.
As per the observations, the interpretation should be written as “the provided unknown
sample contains the ………..”.

98
26
DETECTION OF TANNINS POISONING
OBJECTIVE
To detect tannins poisoning in suspected sample.
TOXICITY OF TANNINS IN ANIMALS

Tannins are present in the young leaves and buds of oak, which is toxic to grazing
cattle and sheep. The toxic principles in oak are gallotanins or their metabolites, which
mainly cause the nephrotoxicity in animals.
DETECTION OF TANNINS
Analysis of the stomach contents, blood, urine, animal tissues and suspected plant is
done to detect the presence of tannins. The dried material or tissue is triturated in small
amount of distilled water and then filtered. The filtrate is taken to detect the tannins by
the ‘strychnine test’ described below:
Strychnine Test:
1. Strychnine sulphate (1% solution) is taken as reagent.
2. One ml of suspected material (filtrate) is taken on a test tube and equal quantity of
strychnine sulphate solution is added to it.
3. Formation of white precipitate indicates the presence of tannic acid in the sample.

The tests are to be performed in the standard solution of any tannic acid, distilled
water and unknown sample to differentiate the positive test (+ve) from the negative (-ve)
test. As per the observations, the interpretation should be written as “the provided
unknown sample contains the ………..”.

99
ABOUT THE AUTHORS
DR. GOVIND PANDEY:

Dr. Govind Pandey, “Professor/Principal Scientist” of
Pharmacology & Toxicology, possesses about 33 yr. of experience
in ‘Research/Teaching/Extension/Administration’. He is an able
academician, scientist, veterinarian and administrator; a Hindi
literalist and eloquent speaker endowed with strong writing flair.
Dr. Pandey is probably “only Person in Madhya Pradesh and alone
Veterinarian in India with Maximum Academic Qualifications”
(20 Degrees/Diplomas/Certificates). He obtained PhD (Hons.) in
Veterinary Pharmacology & Toxicology from the Jawaharlal
Nehru Krishi Vishwa Vidyalaya (JNKVV), Jabalpur in 1990. Presently, he is doing DSc.
His “Biography” is included in the famous directory/book of the world, “Who’s Who in
the World 2011” (28th edition, America). Dr. Pandey is honoured with 3 prestigious
‘Fellowship Titles’, viz., “FASAW, FSLSc and FISCA”.
Dr. Pandey started his career as “Veterinary Assistant Surgeon/Lecturer” on 7th
August, 1980 at Artificial Insemination Training Institute, Mandla; followed with
“Veterinary Surgeon/Senior Veterinary Surgeon” in different offices at Jabalpur,
including “Officer-In-Charge cum Drawing Disbursing Officer (DDO)” of Rinder Pest,
Jabalpur Division, Jabalpur under the Animal Husbandry Department, Government of
MP. During this tenure, he also served as “Chief Executive Officer/Block Development
Officer cum DDO” of some Janapad Panchayats under the Panchayat & Rural
Development Department, Govt. of MP; and as “Assistant Professor & Head, and
Professor/Principal Scientist & Head” of Pharmacology in Pharmacy colleges. On 20th
April, 2012, he joined as “Deputy Director of Research/Associate Professor/Senior
Scientist” at the Directorate of Research Services, Nanaji Deshmukh Veterinary Science
University (NDVSU), Jabalpur, MP, India. On 26th November, 2012, he has resumed the
post of “Professor/Principal Scientist & Sectional Head”, Department of Pharmacology &
Toxicology, College of Veterinary Science & AH, Rewa (NDVSU, Jabalpur).
He is working in different areas of Life Sciences, including Pharmacology &
Toxicology and Fishery Science. He has also made a good contribution in Hindi
literature, Human Resource Management, Political Science, Sociology, Public
Administration, Law and Astrology. Dr. Pandey has investigated some “Antihepatotoxic
and Anticancer Herbal Drugs”, and produced experimental ‘Hepatotoxic and Cancer
Models’ in animals. He has published more than 225 scientific papers and delivered
many speeches in conferences/seminars/radio stations/governmental or public
programmes. He has “Supervised/Guided/Co-Guided” many PhD/PG/UG research
scholars for their ‘Thesis/Dissertations/Project Reports’. He has also carried out some
‘Research Projects’. In science, his “1 e-Book and 1 e-Manual” have been recently
published by the International E - Publication, ISCA (2013). His “10 scientific Books/
Manuals” are likely to be published soon. He has received “30 Awards/Fellowships/
Sponsorships/Honours/Recognitions” (including “ICAR Senior Research Fellowship”
and “Sri Ram Lal Agrawal National Award”) in science, research and Hindi literature. In
100
Hindi literature, he has published “5 Books”, released “2 Audiocassettes” of own lyrics

and edited “1 Book”. His several poems, lyrics, dramas or stories have been published/
broadcasted through various media. He is the “Life Member” of 25 scientific,
professional, literary and cultural associations/societies/journals. He has chaired as the
“Chairperson/Chief Guest/Judge/Expert” in many conferences/projects/committees/
programmes. He has also acted as the “Editor/Mentor/Editorial Board Member/
Reviewer” of some books/journals/magazines. Dr. Pandey is “Ex-Captain of Badminton”,
“Ex-Sergeant of NCC”, “Ex-Literary Secretary” and “Ex-Hostel Prefect”. He has passed
“NCC C Certificate”; and “2 years’ Course of National Service Scheme” (NSS).
DR. YASH PAL SAHNI:

Dr. Yash Pal Sahni, “Director of Research Services”, NDVSU,
Jabalpur, has more than 29 yr. of experience in ‘Teaching/Research/
Extension/Administration’. He obtained PhD (Hons.) in Veterinary
Pharmacology & Toxicology from the JNKVV, Jabalpur in 1990. He
is a good academician, scientist and administrator.
Dr. Sahni joined on 18th January, 1984 in the Department of
Veterinary Pharmacology & Toxicology, College of Veterinary Science
& Animal Husbandry, JNKVV (now NDVSU), Jabalpur, and he worked as “Assistant
Professor/Scientist” till 26th May, 1996. From 27th May, 1996 to 4th July, 2008, he served
as “Associate Professor/Senior Scientist”; and thereafter up to 30th December, 2011, he
resumed the post of “Professor/Principal Scientist & Head”. Before occupying the post of
“Director Research”, Dr. Sahni also acted as the “In-charge of Dean Students’ Welfare”
from 8th October, 2010 in the NDVSU, Jabalpur.
Dr. Sahni is working in different areas of Life Sciences, and has also made a good
contribution in “Indigenous Pharmacology & Toxicology”. He has published more than 150
papers in various journals of national and international repute. Under his able guidance,
more than 20 MVSc & AH students and 1 PhD scholar have completed their research theses
in Veterinary Pharmacology & Toxicology. As “Principal Investigator”, he has successfully
completed 11 ‘Research Projects’. He is the recipient of 7 “Awards/Honours”. Dr. Sahni is
the “Life Member” of many scientific associations/societies/journals. He has also acted as
the “Chairperson/Judge” in many conferences/projects/committees/programmes.

101

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TOXICOLOGY LABORATORY

DR. GOVIND PANDEY

DR. YASH PAL SAHNI

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“Toxicology” involves the absorption, distribution, metabolism and excretion of a

Hence, to describe the above aspects experimentally, this “Toxicology Laboratory

26th July, 2013 Dr. Govind Pandey

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S. No. Description Page

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CERTAIN TERMS OF TOXICOLOGY

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individual. The toxic effects may be idiosyncratic or allergic in nature, or pharmacologic

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ABSORPTION, DISTRIBUTION, METABOLISM AND EXCRETION OF

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FACTORS AFFECTING POISONING

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II. Biologic Factors:

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III. Chemical Factors:

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a) Number of animals exposed/sick/dead, age, weight, and a chronology of

POISONING THERAPY PRINCIPLES

A. Prevention of Further Absorption:

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B. Administration of Specific Antidotes:

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TOXIC METALS AND NONMETALS

A. Toxic metals- e.g., antimony (Sb, a metalloid), arsenic (As, a metalloid),

C. Toxic nonmetals- Some heavy nonmetals may be erroneously called ‘metals’, as

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Fig. 1 Fig. 2 Fig. 3

Fig. 4 Fig. 5 Fig. 6

Fig. 7 Fig. 8 Fig. 9

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PLANTS POISONING IN ANIMALS

FACTORS INFLUENCING PLANTS POISONING IN ANIMALS

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POISONING BY PLANT POISONS

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1. Cyanogenic (cyanogenetic) glycosides- They are not themselves poisonous but

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2. Saponin glycosides- They produce a violent gastroenteritis with vomiting,

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Table 1: Certain Plant Species Involved in Nitrate/Nitrite Poisoning

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number of environmental conditions can influence the accumulation of nitrates in plants

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1. Nervous symptoms- Dullness, depression, muscular trembling, convulsion,

2. Gangrenous or general symptoms- Blood stoppage due to contraction of small

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Fusarium, and Mucor) into a poisonous anticoagulant, called ‘dicoumarol’ (a chemical

OTHER IMPORTANT POISONOUS PLANTS

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