Chapter 31: General Principles of Forensic Toxicology

31.1: Introduction

• Toxicology is the branch of medicine that deals with properties, action, toxicity, and lethal dose, estimation of, treatment, and autopsy findings of poisons.
• Forensic toxicology — deals with medical and legal aspects of the harmful effects of chemicals on the human body.
• Clinical toxicology — refers to human diseases caused by or associated with abnormal exposure to chemical substances.
• Toxinology — refers to toxins produced by living organisms, which are dangerous to man, e.g. venom of snakes, spiders and bees, bacterial and fungal toxins, poisonous plants, etc.
• Drugs — natural or synthetic substances, which are used to exert a physiological or psychological effects on the consumer.
• Poison — any substance which when administered by any route can cause disease, deformity or death.
• Overdose — an intentional toxic exposure.

31.2: Actions of Poison

• Poisons usually act by three ways: locally, remotely, and both locally and remotely.
  • Locally acting: These act only at the site of application such as skin/mucosa, e.g. corrosive poisons.
  • Remotely acting: These act only after being absorbed into the circulatory system, e.g. narcotic poisons, cardiac poisons, etc.
  • Both locally and remotely acting: These act by local and remote actions, synergistically, e.g. carbolic acid, etc.

Factors Influencing the Action of Poison

• Route of Administration: A poison could be administered orally, hypodermically, intramuscularly, intravenously, endemically (rubbing), per rectally, per vaginally, per vesically (urinary bladder), by inhalation, etc.
• Idiosyncrasy
  • It means unexpected allergy or intolerance.
  • It brings about untoward effects or ill health or death, e.g. allergy to certain drugs like penicillin, certain foodstuffs like eggs, shellfish, etc.
• Age: It has got considerable relationship to dosage for any poisonous substance, e.g. dosage required for children is usually half that in an adult.
• Addiction: Taking small quantity of a poison or a drug for a long duration can lead to its diminished effect.
• Dose: Depending on dosage, a poison could be either useful or harmful, e.g. aspirin, morphine, etc. which with a proper dose can act as an analgesic, but with higher dose can induce fatality.
• Health of Individual: A healthy, normal individual can withstand a poison ingested for a longer duration than an unhealthy and debilitated one.
• Concentration of Poison: This factor is highly responsible for the development of classical effects of any poison or drug, e.g. sulfuric acid burns are developed only with concentrated form of the acid.
• Chemical State of Poison: These factors are responsible to derive the rate of solubility, absorption, etc. for a poison to get into the various systems of the living body.
• Physical State of Poison
  • This means the state of existence of a poison, i.e. gas, liquid or solid state.
  • For gas, inhalation is the best route for rapid onset of action.
  • For liquids, onset of action is more rapid than solids when administered orally.
  • Among solids, fine powder acts faster than coarse powder.

Lethal Dose (Fatal Dose)

• Lethal dose is usually difficult to fix due to various factors that can affect the action of a poison.
  • This renders the lethal dose for a given poison only in an “Approximately Fatal Dose” (AFD), which is essentially to help the treating physician to assess the prognosis of a case.
• The UFD is based on minimum lethal dose (MLD), which is usually indicative of the lethal dose that is fatal to 50 percent of animals (LD 50).

31.3: Epidemiology of Poisoning

• Epidemiology — the study of the distribution and determinants of health-related states and events in populations and the control of health problems, the study of epidemic disease.
• It also refers to the field of medicine concerned with the determination of causes, incidence, and characteristic behavior of disease outbreaks affecting human populations and includes the interrelationships of host, agent, and environment as related to the distribution and control of disease.
• Toxic epidemiology — the study of the distribution and determinants of health related states and events in populations, and the control of poisoning related health problems.

Characteristics of Ideal Suicidal and Homicidal Poisons

31.4: Occupational and Environmental Toxicology

• Occupational toxicology — deals with the chemicals found in the workplace.
  • Industrial workers may be exposed to these agents during the synthesis, manufacturing, or packaging of these substances or through their use in an occupational setting.
  • Agricultural workers may be exposed to harmful amounts of pesticides during their application in the field.
  • The major emphasis of occupational toxicology is to identify the agent of concern, define the conditions leading to their safe use, and prevent absorption of harmful amounts.
• Environmental toxicology — deals with the potentially deleterious impact of chemicals, present as pollutants of the environment, to living organisms.
  • It is defined as the science that deals with effects of pollutants on environment and wildlife.
  • Environment — includes all the surroundings of an individual organism, but particularly the air, soil, and water.
  • Pollutant — a substance that occurs in the environment, at least in part as a result of human activity, and which has a deleterious effect on living organisms.
  • Air pollution — a product of industrialization, technological and biological development, increased urbanization and indoor sources.
• Ecotoxicology — a specialized area of environmental toxicology dealing with effects of pollutants on population dynamics in an ecosystem.
  • It is concerned with the toxic effects of chemical and physical agents on living organisms, especially in populations and communities within defined ecosystems; it includes the transfer pathways of those agents and their interactions with the environment.

Toxicity, Hazard and Risk

• Toxicity — the ability of a chemical agent to cause injury. It is a qualitative term. Whether these injuries occur depends on the amount of chemical absorbed.
• Hazard — the likelihood that injury will occur in a given situation or setting, the conditions of use and exposure are primary considerations.
• Risk — the expected frequency of the occurrence of an undesirable effect arising from extrapolation from the observed relationships to the expected responses at doses occurring in actual exposure situations.

Classification of Occupational/Environmental Poisons

1. Air pollutants
   • The major substances that account for about 98% of air pollution are carbon monoxide, sulfur dioxides, nitrogen dioxide and ozone.
     • Sources of these chemicals include transportation, industry, generation of electric power, space heating, and refuse disposal.
   • Carbon monoxide (CO) — a colorless, tasteless, odorless, and nonirritating gas, a by product of incomplete combustion.
     • The average concentration of CO in the atmosphere is about 0.1 ppm; in heavy traffic, the concentration may exceed 100 ppm.
   • Sulfur dioxide (SO2) — a colorless, irritant gas generated primarily by the combustion of sulfur containing fossil fuels.
   • Nitrogen dioxide (NO2) — a brownish irritant gas sometimes associated with fires. It is also formed from fresh silage; exposure of farmers to NO2 in the confines of a silo can lead to “silo filler’s disease.”
   • Ozone (O3) — a bluish irritant gas that occurs normally in the earth’s atmosphere, where it is an important absorbent of ultraviolet light.
2. Solvents
   • These agents find wide use as industrial solvents, degreasing agents, and clearing agents.
     • The substances include carbon tetrachloride, chloroform, trichloroethylene, tetrachloroethylene (perchloroethylene), 1,1,1–trichloroethane methyl chloroform.
   • Benzene is widely used for its solvent properties and as an intermediate in the synthesis of other chemicals.
   • Toluene (methylbenzene) does neither possess the myelotoxic properties of benzene, nor has it been associated with leukemia.
3. Insecticides
   • Chlorinated Hydrocarbon Insecticides:
     • These agents are usually classified into four groups: chlorophenothane, benzene hexachlorides, cyclodienes, and toxaphenes.
     • They are aryl, carbocyclic, or heterocyclic compounds containing chlorine substituents.
     • The individual compounds differ widely in their biotransformation, and capacity for storage; toxicity and storage is not always correlated. They can be absorbed through the skin as well as by inhalation or oral ingestion.
     • The chlorinated hydrocarbon insecticides are considered “persistent” chemicals.
   • Organophosphorus Insecticides:
     • These agents are utilized to combat a large variety of pests.
     • They are useful pesticides when in direct contact with insects or when used as ‘plant systemics’, where the agent is trans-located within the plant and exerts its effects on insects that feed on the plant.
   • Carbamate Insecticides:
     • These compounds inhibit acetylcholinesterase by carbamylation of the esteratic site.
     • They possess the toxic properties associated with inhibition of this enzyme, as described for the organophosphorus insecticides.
   • Botanical Insecticides:
     • These insecticides derived from natural sources include nicotine, rotenone, and pyrethrum.
     • Nicotine — obtained from the dried leaves of Nicotiana tobacum and Nicotiana rustica plants.
     • Rotenone — obtained from Derris elliptica, Derris malaccensis, Lonchocarpus utilis, and Lonchocarpus urucu. The oral ingestion of rotenone produces gastrointestinal irritation.
     • Pyrethrum — consists of six known insecticidal esters: pyrethrin I, pyrethrin II, cinerin I, cinerin II, jasmolin I, and jasmolin II. Pyrethrum may be absorbed after inhalation or ingestion; absorption from the skin is not significant.
4. Herbicides
   • Chlorophenoxy Herbicides:
     • Dichlorophenoxyacetic acid and their salts and esters are the major compounds of interest as herbicides used for the destruction of weeds.
   • Bipyridyl Herbicides
     • Paraquat is the most important agent of this class. It has been given a toxicity rating of 4, which places the probable human lethal dosage at 50 to 500 mg/kg.
5. Environmental Pollutants
   • The polychlorinated biphenyls (PCBs) have been used in a large variety of applications as dielectric and heat transfer fluids, plasticizers, wax extenders, and flame-retardants.
     • These chemicals are highly stable and highly lipophilic, poorly metabolized, and very resistant to environmental degradation, therefore they bioaccumulate in food chains.

31.5: Clinical Management of Poisoning

Diagnosis of Poisoning

• A standard clinical examination is carried out in every poisoned patient:
  • Record the vitals — This includes recording of pulse, respiratory rate, blood pressure, body temperature and pupillary manifestations.
  • Note the odor — A number of toxins have a characteristic odor and may help in detection of poison consumed.
  • Note the color of urine — Observing the color of urine passed, may also give vital clue towards the diagnosis of type of poison.

Characteristic of Odor Toxins

Urine Color Caused by Certain Toxins

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Certain Condition Arousing Suspicion of Poisoning

Duties of a Doctor in a Poisoning Case

General Measures

• Dealing with Unconscious Patient:
  • First ensure that the airway is clear and the patient is breathing adequately, circulation is not compromised and there is no depression of CNS.
  • If the patient is stable then proceed to take history and assessing the depth of unconsciousness.
  • In a poisoned patient, this is important and is preferably done as per the Reeds Classification/ Edinburgh Method.
  • The Glasgow Coma Scale is the most frequently used in assessment of the degree of impaired consciousness, though remarkably it has never been validated for use in poisoned patients.
  • When patient is unconscious and no history is available, the diagnosis of poisoning depends on exclusion of other causes of coma and consideration of circumstantial evidence.
• Dealing with Conscious Patients:
  • Take a brief history. In the vast majority of cases, the diagnosis of acute poisoning is obtained from the history given by the patient.
  • Doctors need to be aware that patients may not always furnish them with the correct answer. This may be because they do not always know what they have taken, not least because they may have been under the influence of alcohol or the drug itself at the time of ingestion or while narrating the history.
  • Full details of how many and what type of substance that has been taken must be recorded, as well as timing of ingestion or exposure.
  • Ask the patient why the overdose, if any, was taken and take time to listen to the explanation.
  • Often reasons include relationship difficulties, work or school related commitments, problems of addiction, psychiatric illness or bereavement.
  • Beware of those claiming ‘accidental overdose’. Whilst this clearly can also occur.

The Reed’s Classification of Comatose Patient

Specific Measures

Decontamination

• Decontamination refers to skin/eye decontamination, gut evacuation and administration of activated charcoal.

Gastric Lavage

• Gastric lavage (stomach washout) should only be undertaken if the patient has ingested a potentially life-threatening amount of a poison and presents within 1 hour of the ingestion. If performed later than this the amount of poison removed is insignificant and lavage can actually make the situation worse by pushing unabsorbed poison into the small intestine.
• Complications:
  • Rupture of Esophagus/Stomach
  • Aspiration pneumonitis
  • Lipoid pneumonia
  • Hypoxia, Cardiac Arrhythmias
• Contradictions:
  • Corrosive poisoning.
  • Convulsive poisoning.
  • Comatose patients.

Emesis

• Emesis or vomiting may be induced by several methods, which are tickling the throat, administering the oral emetics and parenteral emetics.

Oral Emetics

• Common salt — 1tbsp.
• Mustard — 2tsp.
• Tincture iodine — 20 drops.
• Zinc sulfate — 250 mg
• Ammonium carbonate — 1 gm
• Tartar emetics — 100 mg
• Powder ipecac — 1 gm
• Syrup ipecac — 30 ml

Activated Charcoal

• Activated charcoal is a black slurry material, which owing to its large surface area is highly effective at adsorbing many toxins.

• It is highly effective at adsorbing most poisons, with a few exceptions, owing to its large surface area and porous structure.

• It should be given to all patients who present within 1 hour of ingestion of a potentially toxic amount of poison, which would binds to charcoal.

• Activated charcoal is given in 50 gm doses for adults and in the dose of 1 gm/kg for children.

• Certain agents cannot be adsorbed by activated charcoal and they are:

  • Metal salts, e.g. iron, lithium, potassium.
  • Alcohols, e.g. ethanol, ethylene glycol, methanol.
  • Other agents, e.g. cyanide, hydrocarbons, solvents, acids, alkalies, fluoride.

• Available formulations of activated charcoal vary from one country to another:

  1. Medicoal® is an effervescent preparation containing sodium citrate and povidone and must be mixed with water before use.
  2. Carbomix® comes in ready to use containers, to which water must be added.
  3. Liquichar® and Actidose aqua® come as ready to use prepared mixtures.

Catharsis

• Catharsis is known to reduce the transit time of drugs in the gastrointestinal tract.
• Ionic/ Saline Cathartics: These cathartics alter the physicochemical forces within the intestinal lumen leading to osmotic retention of fluid which activates motility reflexes and enhances expulsion.
• Saccharides cathartics: This consists of giving sorbitol (D-glucitol), which is the cathartic of choice in an adult because of its better efficacy than saline cathartics.

Whole Bowel Irrigation

• Whole bowel irrigation involves administration of non-absorbable polyethylene glycol solution to cause a liquid stool and reduce drug absorption by physically forcing gastrointestinal contents.
  • This means washing the gut rapidly.
• It is a newer method of gut decontamination that is indicated for a limited number of poisons such as large ingestion of iron, lithium, sustained release or enteric coated drugs.
• Complications:
  • Hemodynamic compromise — Do not use in patient with hemodynamic compromise from bleeding, e.g. severe iron poisoning.
  • Obstruction — Do not use in patients with ileus.

Methods of Enhancing Elimination of Toxins

1. Urinary Alkalization
   • This is also known as alkaline diuresis and is indicated for serious poisoning with:
     • Chlorpropamide
     • Mecoprop
     • Phenobarbitone
     • Phenoxyacetate herbicides
     • Salicylates including aspirin
2. Extracorporeal Techniques
   • Hemodialysis, charcoal hemoperfusion, hemofiltration are some of the extracorporeal techniques.
   • There are a limited number of poisonings in which one of these procedures may be indicated.
   • Before deciding whether or not to undertake one of these procedures in a poisoned patient, the case should be discussed with a clinical toxicologist.
3. Diaphoresis
   • It means inducing excessive perspiration, and poison is excreted through sweat.
   • Methods adopted include application of heat by fomenting with hot water bottles or covering the body with thick blankets, administering hot beverages like hot tea, coffee or milk, hot lemonade, etc can induce increased perspiration.
   • Profuse perspiration can be induced by giving subcutaneous injection of 5 mg of pilocarpine nitrate.
   • Giving alcohol, salicylates or antipyretics are also used to produce less marked perspiration.

Administration of Antidotes

• Antidotes — substances, which, on administration counteract or neutralize the effect of a poison.

Classification According to Action

1. Physical/ Mechanical Antidotes: These neutralize the poison by their mechanical action.
2. Chemical Antidotes: These acts by forming new compounds with the poison, which will be either nontoxic or less active or insoluble.
3. Universal Antidote: It is an antidote, which is a combination of physical and chemical antidote. It can be administered, when the exact nature of poison taken is not known or when one or more poison is/are taken.
4. Physiological/Pharmacological Antidotes: These act by producing exactly the opposite action to those produced by the poison.
5. Chelating Agents: These are true physiological agents which act by forming stable and soluble complexes by the inner ring structure which can combine with the poison molecules easily.
   • British Anti-Lewisite (BAL) — a colorless liquid in peanut oil, given deep intramuscularly.
     • It is administered in a dose of 2.5 mg/kg body weight, four hourly for 2 days followed by six hourly for 2 days and 12 hourly for next subsequent 10 days or till improvement.
   • Ethylene diamine tetra-acetic acid (EDTA) — it is rarely used in heavy metallic poisoning, as it is prone to remove blood and bone calcium also producing fatal hypocalcaemia.
     • It is administered in a dose of 50-70 mg/kg body weight (1 gm in 500 ml. of 5 percent glucose or normal saline, by slow intravenous drip, twice a day for 3 to 5 days).
   • Penicillamine — it is obtained by hydrolytic dehydration of penicillin. It is well absorbed from the gastrointestinal tract.
     • It is administered in a dose of 30 mg/ kg body weight per day orally.
   • Deferoxamine — a water soluble compound with great affinity for ferric iron.
     • It is administered in a dose of 8 gm in 50 to 200 ml of water given orally or by nasogastric tube, 1-2 gm intravenously or intramuscularly 4 hourly for 2 days.

31.6: Diagnosis of Poisoning in the Dead

• Certain Specific Changes in the Cadaver: If the dead body is closely observed there will be several changes noticed which help in detection of poison causing death**.**
• Analytical toxicology: It is to help not only in diagnosis of the poisoning in a living case in a hospital, but also facilitate poison identification in a dead body in the mortuaries.
• Histopathological examination (HPE): HPE of tissues could give clues regarding degenerative change due to certain poisons. It is valuable, as it could furnish corroborative evidence in poisoning death.
• Circumstantial evidence: It is obtained from the belongings of the deceased such as a suicide note in the pocket of the garments worn by the deceased, an empty container (bottle) in the hand of the dead with a label of poisonous material consumed.

31.7: Viscera and Chemical Examination

Preservation of Viscera

• Saturated solution of common salt is prepared by adding salt to water till it remains undissolved on vigorous stirring. It is indicated in all poisoning cases.
• Rectified spirit is indicated for most of the poisons, exceptions being alcohol, kerosene, phosphorus, paraldehyde, carbolic acid, acetic acid, etc.

Preservation of Blood and Urine

• For 25 ml blood, add 125 mg sodium fluoride as preservative and 75 mg potassium oxalate as anticoagulant. Refrigeration of the sample may be helpful in alcohol poisoning cases. In case of carbon monoxide poisoning, pour 2-3 cm thick layer of liquid paraffin over the blood sample.
• For urine, add thymol as preservative. Other alternatives are saturated solution of sodium chloride or 5 ml of concentrated hydrochloric acid for every 250/500 ml.
• Specimen of bones does not require preservative.

Notes

• Always send a sample of the preservative used to chemical examiner in separate bottle.
• Never use formalin as a preservative for sending the viscera to chemical examiner as it hardens (fixes) the tissue, renders difficulty to extract poison (formalin is used as a fixative for histopathological examination of tissues).

Sending Viscera to Chemical Examiner

• Write a requisition
• Send the specimen packed ideally in separate containers, which are closed and sealed from outside, through a police constable (note his PC No)
• Always demand and collect a receipt for delivering the specimen to the police constable.

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31.8: Analytical Toxicology

Apparatus and Reagents

• Analytical toxicology services can be provided in clinical biochemistry laboratories that serve a local hospital or accident and emergency unit.

Steps to be undertaken in an analytical toxicological investigation

• Pre-analytical phase:

  • Step 1: Obtain details of current admission, including any circumstantial evidence of poisoning and results of biochemical and blood investigations.
  • Step 2: Obtain patient’s medical history, if available, ensure access to the appropriate sample(s), and decide priorities for the analysis.

• Analytical phase: Step 3: Perform the agreed analysis

• Post-Analytical Phase:

  • Step 4: Interpret the results and discuss them with the clinician looking after the patient.
  • Step 5: Perform additional analysis, if indicated, on the original samples/ on further samples from the patient.

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Wet Chemistry

• Traditional wet chemistry: Refers to the wet chemical methods of analysis.
  • This comprises a painstaking testing and retesting of unknown material using different reagents, until the composite behavior patterns give a clue to the identity of the material/ poison.
• Wet chemistry: Refers to the analytical techniques in which various chemical reagents, such as acids, bases and salts are applied to a sample, identified on the basis of its reactions with the reagents.
• Qualitative Tests: This includes certain biochemical tests wherein the nature of the poison is identified using the biological material samples from the victim of poisoning are in common practice.
  • Color Test
• Quantitative Analytical Methods: This comprises methods where in the tests are done not only to exactly identify the poison but also to estimate the concentration of the poison in the body of the poisoned victim.
  • Chromatography
  • Ultraviolet spectrophotometry
  • Mass spectrometry

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Recommended Qualitative Color Tests

1. Trinder’s Test — Salicylic acid & Acetyl salicylic acid.
   • Add 100 ml of Tinder’s reagent to 2 ml of urine and mix for 5 seconds.
   • A violet color indicates the presence of salicylate.
   • If only stomach contents or scene residue are available, first hydrolyze the same by heating with 0.5 mol/L hydrochloric acid on a boiling water bath for 2 minutes, and neutralize with 0.5 mol/L sodium hydroxide before performing the test.
   • If a positive result is obtained in this test, carry out a qualitative assay on plasma/serum.
2. FPN Test — Phenothiazines
   • Add 1 ml of FPN reagent to 1 ml of of sample and mix for 5 seconds.
   • Colors ranging from pink to red, orange, violet or blue suggest the presence of phenothiazines.
   • TLC should confirm positive results.
3. Forrest test — Imipramine and related compounds
   • Add 1 ml of Forrest reagent, 25 ml of aqueous perchloric acid, and 25 ml of aqueous nitric acid (500 ml/ L) to 0.5 ml of sample and mix for 5 seconds.
   • A yellow-green color deepening through dark green to blue indicates the presence of imipramine or related compounds.
   • TLC should confirm positive results.
4. Fujiwara test — Trichloro compounds, including chloral hydrate, chloroform, dichloral phenazone and Trichloroethylene.
   • To three 10 ml tubes labeled A, B and C, add respectively 1 ml portion of – sample, purified; water; and aqueous Trichloroacetic acid.
   • Add 1 ml of sodium hydroxide solution and 1 ml of pyridine to each test-tube.
   • Mix carefully and heat in a boiling water bath for 2 minutes.
   • An intense red/purple color in the top (pyridine) layer of tube A as in tube C indicates the presence of Trichloro compounds; tube B should show no coloration.
5. O-cresol/ammonia test — Paracetamol, Phenacetin
   • Add 0.5 ml of concentrated hydrochloric acid to 0.5 ml of sample, heat in a boiling waterbath for 10 minutes and cool.
   • Add 1 ml of aqueous Ocresol solution (10 gm/L) to 0.2 ml of hydrolysate, add 2 ml of ammonium hydroxide solution (4 mol/L), and mix for 5 seconds.
   • A strong blue to blue-black color, which forms immediately, indicates the presence of Paracetamol or Phenacetin.
   • If a positive test result is obtained in this test, carry out a quantitative assay for Paracetamol on plasma or serum.
6. Dithionite test — Paraquat, diquat
   • Add 0.5 ml of aqueous ammonium hydroxide (2 mol/L) to 1 ml of test solution, mix for 5 seconds and add about 20 mg of solid sodium dithionite.
   • A strong blue to blue-black color indicates Paraquat; diquat gives a yellow-green color, but this is insignificant in the presence of Paraquat.
   • If the color fades on continued agitation in air and is restored by adding further sodium dithionite, Paraquat or diquat is confirmed.
7. Dichromate test — Ethanol and other volatile reducing agents
   • Apply 50 ml of potassium dichromate to strip of glass fiber filter paper in the neck of the test-tube containing 1 ml urine.
   • Lightly stop the tube and place in a boiling water-bath for 3 minutes.
   • A change in color from orange to green indicates the presence of volatile substances.
   • If a positive result is obtained in this test, carry out a quantitative assay for ethanol on blood.
8. Diphenylamine test — Chlorates and other oxidizing agents
   • Carefully add 0.5 ml of diphenylamine to 0.5 ml of filtered stomach contents or scene residue.
   • A strong blue color, which develops rapidly, indicates the presence of oxidizing agents.
9. Ferricyanide/ ferrocyanide test — Ferrous and Ferric Iron
   • To 50 ml of filtered stomach contents or scene residue, add 100 ml of aqueous hydrochloric acid and 50 ml of aqueous potassium ferricyanide solution.
   • To a further 50 ml of sample add 100 ml of hydrochloric acid and 50 ml potassium ferrocyanide solution.
   • A deep blue precipitate with potassium ferricyanide or ferrocyanide indicates the presence of ferrous or ferric iron.
   • If a positive result is obtained in this test, carry out a quantitative assay for iron on serum.
   1. Meixner test — Poisonous mushroom
   • This test is done with stool sample.
   • To a stool sample add methanol and centrifuge and filter it.
   • Place one or two drops of this on a piece of filter paper and then add a few drops of hydrochloric acid to them.
   • A bluish coloration indicates the presence of anatoxins, which is present in most of the poisonous mushrooms.
   1. Reinsch test — Arsenic, antimony, bismuth and mercury
   • Applicable to urine, stomach contents and scene residues.
   • Take a copper foil or mesh (5×10) or wire (2-3 cm), which is cleaned in nitric acid until the copper acquires a bright surface.
   • Rinse the same with purified water and add 10 ml of concentrated hydrochloric acid and 20 ml of test solution in a 100 ml conical flask.
   • Heat on a boiling water bath in a fume cupboard for 1 hour.
   • Maintain the volume of the solution by adding dilute hydrochloric acid as necessary.
   • Color staining on the copper can be interpreted as follows:
     • Purple black: antimony.
     • Dull black: arsenic.
     • Shiny black: bismuth.
     • Silvery: mercury.
   1. Marquis’ reagent test — Morphine and other opium
   • Applicable to scene residue, stomach contents.
   • Place a drop of Marquis’ reagent on the suspected scene residue, or add a few drops to stomach contents.
   • A purple coloration which gradually turns into violet and finally to blue color indicates the presence of opium and its derivatives.

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