Re-revised manuscript of toxicology manualisca.co.in/ANI_VAT_AND_FISHRY/lab_manual/Toxicology... ·...

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

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permission of the publisher.

ISBN: 978-93-83520-03-9

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International Science Congress Association

iii




iv

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




v

CONTENTS

S. No. Description Page

- Foreword iii

- Preface iv

- Contents v

1 Toxicology and Poisoning : An Overview

Objective

Certain Terms of Toxicology

Absorption, Distribution, Metabolism and Excretion of

Poisons

Factors Affecting Poisoning

Diagnosis of Poisoning

Poisoning Therapy Principles

1

1

1

3

5

7

8

2 Poisoning by Metals and Nonmetals

Objective

Toxic Metals and Nonmetals

Metals Poisoning/Toxicity

10

10

10

11

3 Poisoning by Plants/Weeds

Objective

Plants Poisoning in Animals

Factors Influencing Plants Poisoning in Animals

Poisoning by Plant Poisons

Other Important Poisonous Plants

Other Kinds of Poisoning/Injury by Plants

14

14

14

14

15

23

26

4 Determination of Median Effective and Median Lethal Doses

Objective

Definition of ED50 and LD50

Method

32

32

32

33

5 Collection and Dispatching of Samples for Toxicological Tests

Objective

Principle

Required Samples for Diagnosis of Poisoning

Containers Required for Collection of Samples

Tissues Required for Analysis

Preservation of Test Samples

Dispatching/Submission of Test Samples

Examinations/Tests for Diagnosis of Poisoning

Precautions

39

39

39

39

39

41

41

42

43

48

6 Preparation of Toxicological Kit for Treatment of Poisoning

Objective

Measures Taken in Suspected Poisoning

Toxicological Kit

Gastric Lavages

Adsorbents

49

49

49

49

50

51




vi

Emetics

Purgatives (Cathartics)

Diuretics

Antidotes

Miscellaneous Drugs in Toxicological Kit

51

52

52

52

53

7 Detection of Metals Poisoning

Objective

Toxicity of Metals in Animals

Detection of Metals

55

55

55

55

8 Detection of Lead Poisoning

Objective

Toxicity of Lead in Animals

Detection of Lead

57

57

57

57

9 Detection of Mercury Poisoning

Objective

Toxicity of Mercury in Animals

Detection of Mercury

59

59

59

59

10 Detection of Antimony Poisoning

Objective

Toxicity of Antimony in Animals

Detection of Antimony

62

62

62

62

11 Detection of Fluoride Poisoning Objective

Toxicity of Fluoride in Animals

Detection of Fluoride

64 64

64

64

12 Detection of Copper Poisoning

Objective

Toxicity of Copper in Animals

Detection of Copper

65

65

65

65

13 Detection of Zinc Poisoning

Objective

Toxicity of Zinc in Animals

Detection of Zinc

67

67

67

67

14 Detection of Cyanide Poisoning and its Treatment Test

Objective

Toxicity of Cyanide in Animals

Mechanism of Action of Cyanide

Clinical Signs of Cyanide Toxicity

Detection of Cyanide

Treatment Test of Cyanide Toxicity

69

69

69

69

69

70

70

15 Detection of Nitrate/Nitrite Poisoning and its Treatment Test

Objective

Toxicity of Nitrate/Nitrite in Animals

Mechanism of Action of Nitrite

Clinical Signs of Nitrite Toxicity

72

72

72

72

73




vii

Detection of Nitrate/Nitrite

Treatment Test of Nitrite Toxicity

73

74

16 Detection of Urea Poisoning

Objective

Toxicity of Urea in Animals

Detection of Urea

76

76

76

76

17 Detection of Chloral Hydrate Poisoning

Objective

Toxicity of Drugs in Animals

Toxicity of Chloral Hydrate in Animals

Detection of Chloral Hydrate

78

78

78

78

78

18 Detection of Phenobarbitone/Barbiturates Poisoning

Objective

Toxicity of Phenobarbitone or Barbiturates in Animals

Detection of Phenobarbitone or Barbiturates

80

80

80

80

19 Detection of Sulphonamides Poisoning

Objective

Toxicity of Sulphonamides in Animals

Detection of Sulphonamides

82

82

82

82

20 Detection of Acetyl Salicylic Acid Poisoning

Objective

Toxicity of Acetyl Salicylic Acid in Animals

Detection of Acetyl Salicylic Acid

84

84

84

84

21 Detection of Insecticides Poisoning and its Treatment Test

Objective

Toxicity of Organophosphate Insecticides in Animals

Mechanism of Action of Organophosphate Insecticides

Symptoms of Organophosphate Insecticides Toxicity

Treatment Test of Malathion Toxicity

86

86

86

86

86

87

22 Detection of Alkaloids Poisoning

Objective

Toxicity of Alkaloids in Animals

Detection of Alkaloids

89

89

89

89

23 Detection of Strychnine Poisoning and its Treatment Test

Objective

Toxicity of Strychnine in Animals

Mechanism of Action of Strychnine

Clinical Signs of Strychnine Toxicity

Detection of Strychnine

Treatment Test of Strychnine Toxicity

92

92

92

92

92

92

93

24 Detection of Glycosides Poisoning

Objective

Toxicity of Glycosides in Animals

Detection of Glycosides

95

95

95

95

25 Detection of Digitalis Poisoning

97




viii

Objective

Toxicity of Digitalis in Animals

Detection of Digitalis

97

97

97

26 Detection of Tannins Poisoning

Objective

Toxicity of Tannins in Animals

Detection of Tannins

99

99

99

99

- About the Authors 100




1

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




2

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.




3

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




4

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




5

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




6

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,




7

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:




8

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




9

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.




10

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




11

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




12

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.




13

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]




14

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




15

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




16

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




17

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.




18

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




19

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.




20

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.




21

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:




22

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,




23

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,




24

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




25

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




26

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.




27

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




28

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




29

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




30

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




31

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





32

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




33

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.




34

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

Animals (f)

Dose

(mg/kg)

Dose Difference

(a)

No. of Animal

(Rat) Died (b)

Mean

(c)

Product

(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




35

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

Animals

Dose

(mg/kg)

Animals

Died (a)

Animals

Survived

(b)

Cumulative %

Survival

(f) Dead

(c)

Survived

(d)

Total

(e)

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)




36

(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).




37

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

Animals

Dose

(mg/kg)

Animals

Died (a)

Dead %

(b)

Corrected %

(c)

Probit

(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)-




38

______ _______

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




39

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.




40

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

poisoning) Liver, kidney, ingesta or GIT contents (stomach and intestine), urine, feed

Arsenic (As- Chronic

poisoning) Hair, liver, urine, spleen, altered organs

Mercury (Hg) Blood (no EDTA; heparinized blood preferred), liver, kidney, GIT contents,

muscle, brain, faeces, feed

Cadmium (Cd) Kidney, liver, hair

Cyanide/hydrogen cyanide

(HCN)

GIT contents, liver, muscle, oxalated blood, brain (all these should be frozen in

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

(NaCl) Brain (other half of each should fixed in formalin), serum, CSF, feed

Fluoride Altered bones, tooth, urine, stomach contents, liver, kidney, feed/forage, water

Nitrite (NO3)/nitrate (NO2) GIT contents, whole blood, urine, body fluids such as aqueous humor (all these

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

convulsants like bromethalin) GIT contents, urine, liver, kidney, brain

Anticoagulants (warfarin and

related compounds)

Whole blood (add EDTA or heparin), liver, stomach contents (all these should be

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




41

(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

insecticides (OPIs) and

carbamates

Oxalated blood or whole blood, liver, ingesta or GIT contents, urine, feed

Organochlorine insecticides

(OCIs, CHIs)

Fat, liver, stomach contents, brain

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

altered bone

Important for pesticides and metal poisoning

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.




42

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:




43

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




44

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




45

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




46

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




47

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




48

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.




49

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)




50

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




51

‘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.




52

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)




53

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




54

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





55

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.




56

Table 9: Standard Chart for Spot Test of Poisonous Metals

Metal Colour Reaction of Solution by Adding Reagent

Ammonium

Carbonate (10%)

Ammonium

Sulphide (20%)

Potassium

Iodide (1%)

Lead (Pb) White Dark brown Yellow

Mercury

(Hg)

Mercurous (Hg++

) White Greenish-black Black

Mercuric (Hg+++

) White Yellow Red

Copper (Cu) Blue Dark brown Brown

Iron

(Fe)

Ferrous (Fe++) Dirty green Black No reaction

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 ……………..”.




57

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.




58

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 ……………..”.




59

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.




60

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.




61

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







62

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.




63

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







64

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.





65

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.




66

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







67

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.




68

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







69

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,




70

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




71

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

Solution

Total

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

Symptom

Duration of

Symptom

Time of Recovery

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.




73

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




75

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


Solution

Total

Volume (ml)

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


Symptom

Duration of

Symptom

Time of 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.




77


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.




79

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.




81


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).




83

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.




85

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:




87

‘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:





88

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


Solution

Total

Volume (ml)

Malathion 107 mg/kg, iv or im Calculate as per kg body weight 5% Calculate

Atropine

sulphate

0.5 mg/kg, iv or im Calculate as per kg body weight 0.5% Calculate

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


Symptom

Duration of

Symptom

Time of Recovery

after Treatment




89

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 (40

oC

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:




90

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.





91

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




93

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.




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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


Solution

Total

Volume (ml)

Strychnine sulphate 10 mg/kg, ip Calculate as per kg body weight 0.1% Calculate

Pentobarbitone

sodium

50 mg/kg, ip Calculate as per kg body weight 1.0% Calculate

Table 17: Observations of Strychnine Toxicity Symptoms and their Recovery after

Treatment


Symptom

Duration of

Symptom

Time of 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.




96

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 ………..”.




97

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.




98

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 ………..”.




99

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 ………..”.




100

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




101

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.

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