SUBJECT FORENSIC SCIENCE

Paper No. and Title PAPER No.10: Forensic Toxicology

Module No. and Title MODULE No.8: Organic Poisons- II: Animal Poisons

Module Tag FSC_P10_M8

FORENSIC SCIENCE PAPER No.10: Forensic Toxicology MODULE No.8: Organic Poisons: Animal Poisons

TABLE OF CONTENTS

1. Learning Outcome 2. Introduction to Animal Poisoning 3. Forensic Issues 4. Categorization of Venomous Animals 4.1 Vertebrates 4.1.1 Reptiles 4.1.2 Fishes 4.1.3 Amphibians 4.1.4 Birds 4.2 Non- Vertebrates 4.2.1 Arthropods 4.2.2 Arachnids 4.2.3 Cnidaria (or Coelenterates) 4.2.4 Molluscs 5. Summary

FORENSIC SCIENCE PAPER No.10: Forensic Toxicology MODULE No.8: Organic Poisons: Animal Poisons

1. Learning Outcomes

After studying this module, you shall be able to know about –  The toxins of Animal origin.  General introduction of certain animal toxins  Forensic significance of animal poisoning

2. Introduction to Animal Poisoning Largely it talks about the poisons originated from living organisms which are obviously animals. While there is so much diversity in the animal kingdom, the methods of predation and self-protection is varies from species to species. Similarly, some animals use poisons as a weapon of predation or self-defence or both. Snakes are the most popular among these type creatures besides other known venomous organisms like, fishes, insects, etc. In India, deaths as a result of snake-bite are mainly accidental in nature. Venoms used in an offensive posture are generally associated with the oral pole, as in the snakes and spiders, while those used in a defensive function are usually associated with the aboral pole or with spines, as in the stingrays and scorpion fishes. In the snake, the venom provides a food-getting mechanism. Its secondary function is its defensive status. In contrast, in venomous spiders, toxin is used to paralyze the prey before the extraction of hemolymph and body fluids. The venom is not primarily designed to kill the prey, but it is only to immobilize the organism for feeding. The same can be said for scorpions, although they do use their venom in defense. In fishes, such as the scorpion fishes and stone fishes, and in elasmobranches, such as the stingray, the venom apparatus is generally used in the animal’s defense. Poisonous animals, on the other hand, usually derive their toxins through the food chain. As such, poison is often a metabolite produced by microorganisms, plants, or animals.

3. Forensic Issues Practically almost all cases of animal bites reported from around the world are accidental in nature, and the vast majority are due to unintentional or deliberate provocation of the animal by humans itself. Likewise Snakes, rarely if ever, attack human beings on their own. A number of occupations are associated with increased risk of snake bites like grass-cutting, working in rubber, coconut, areca- nut, and tea & coffee plantations. Perhaps, the only recorded case of suicide accomplished with the help of a venomous snake is that of Queen Cleopatra of Egypt (69 – 30 BC), who is said to have deliberately prompted a snake (an adder) to bite her. While homicides can be accomplished by using a venomous snake as an instrument but actual instances of murder committed by such an unusual method are rare. Deaths due to snakebites are regarded as medicolegal in nature, and a forensic autopsy is mandatory. Unfortunately, precised characters of envenomation may be lacking in such cases, and even fang marks may not always be visible.

FORENSIC SCIENCE PAPER No.10: Forensic Toxicology MODULE No.8: Organic Poisons: Animal Poisons

Today, immuno-diagnosis with the help of, ELISA makes it possible to conclusively diagnose death due to snakebite by analysing tissues around bite site, or blister fluid, or even body fluids such as blood and urine for venom antigens.

4. Categorization of Venomous Animals Several species of animals, with or without backbones, produce venoms or contain certain toxins that may be dangerous to humans, either externally by stinging, biting or by ingestion. Both the categories are discussed as following:

4.1 VERTEBRATES

4.1.1 REPTILES

4.1.1.1 SNAKES

Snakes belong to the class Reptilia, order squamata, and sub-order Serpentes. Snakes are found all over the world except New Zealand, Ireland and in Arctic lands. Of the 2500-3000 species of snakes distributed world-wide, about 500 are poisonous to Humans. The three main families of poisonous snakes exists are the Elapidae (cobras), the Viperidae (vipers) and the Crotalidae (pit vipers). The elapids cover around half the world’s species of venomous snakes and contain the Cobras and the Mambas. Genera of the elapid family are found in Asia, the Pacific, the Americas and Africa. The existent vipers, Viperidae, of which the common viper is the best known, inhabit Europe, Asia and Africa. The Pit Vipers, Crotalidae, are largely found in North, Central and South America and include the Rattlesnake genera Crotalus and Sistrurus. There are 238 species of snakes in India out of which only 52 are poisonous. Of these 52 poisonous species majority of bites and consequential mortality is attributed to 5 species viz. Ophiophagus hannah (King Cobra), Naja naja (Common Cobra), Daboia rusellii (Russell’s Viper), Bungarus caeruleus (Krait) and Echis carinatae (Saw- Scaled viper).

Venom

The snake venom is the secretion of the racemose salivary glands that is a modification of the parotid salivary gland of other vertebrates, and is usually situated on each side of the head below and behind the eye, invested in a muscular sheath. It is provided with large alveoli in which the venom is stored before being conveyed by a duct to the base of the channelled or tubular fang through which it is ejected. Poison is a clear limpid fluid of a pale straw or amber colour, more rarely greenish, sometimes with a certain amount of suspended matter, is exhausted after several bites, and the glands have to recuperate. The venom retains its poisonous properties for several years in dried state. The Cobra venom is slightly viscous and when exposed to sun it becomes a little turbid.

FORENSIC SCIENCE PAPER No.10: Forensic Toxicology MODULE No.8: Organic Poisons: Animal Poisons

The venom of Russell’s viper is usually white or yellow. Venoms of different species of poisonous snakes vary in the toxicity, composition and antigenic structure. It is basically a mixture of one or more of the toxic substances, toxalbumins and enzymes in varying proportions such as:

(1) Proteolysins: It causes liberation of histamine from the damaged muscular endothelium leading to dissolution of walls of blood vessels with extravasation of blood in the tissue space, also causes digestion of tissue proteins and peptides and produces marked tissue destruction.

(2) Fibrinolysins: It enhances the coagulation process.

(3) Neurotoxins: It is mainly found in elapidae venom producing curare like effect causing paralysis especially of respiratory centre.

(4) Cholinestrase: It is also found in elapidae venom causing hydrolysis of acetylcholine to choline and acetic acid thus causes impairment of neuromuscular transmission.

(5) Haemolysin: It is found chiefly in viper venom causing widespread haemolysis in presence of lecithinase.

(6) Cytolysin: It is mainly found in viprine venom and causes lysis of cell structures of blood and tissues.

(7) Agglutinins: It causes agglutination of Red Blood Corpuscles.

(8) Phosphatidases: It produces haemolysis and toxic effects on heart and circulation with haemorrhage from lungs.

(9) Proteinases: It has trypsin like action, causing tissue damage. It produces anticoagulant effect from destruction of fibrinogen. It also catalyses the conversion of prothrombin to thrombin thus producing coagulant effect.

(10) Phospholipase: It acts as a catalyst in hydrolysis of lipids. By destroying phospholipids in nervous tissues, it alters neuromuscular conduction. It also helps in penetration of neurotoxin in to nervous tissue.

(11) Hyaluronidase: It helps in rapid spreading of the venom from the local site of bite and thereby quick absorption. It is present in all snake venoms.

(12) Ribonuclease and deoxyribonuclease: It helps in rapid spreading of the venom from the local site of bite and thereby quick absorption. It is present in all venoms.

FORENSIC SCIENCE PAPER No.10: Forensic Toxicology MODULE No.8: Organic Poisons: Animal Poisons

(13) Protease: It causes dissolution of vascular wall and help in spreading of red blood cells and serum in the tissues.

(14) Lecithinases: It acts on the lipid layer of the cells causing increasing fragility and permeability leading to cell destruction.

The cobra venom in Neurotoxic and produces muscular paralysis involving firstly the muscles of the mouth, throat and lastly muscles of respiration, seats the action of venom being upon the motor nerve cells, the action resembling curare. It consists of: (i) Neurotoxin (ii) Cholinesterase (iii) Proteases (iv) Phosphatidases (v) Hyaluronidase (vi) Ribonuclease (vii) Thromboplastin (viii) Fibrinolysin (ix) Proteolysin (x) Cardiotoxin (xi) Phospholipase- A. Cobra venom produces convulsions and paralysis, while krait venom produces only muscular paralysis.

The viperine venom is predominantly haemolytic and hemotoxic. It causes lysis of red cells and other tissue cells and coagulation disorders bringing about coagulation of the blood and clotting of the pulmonary arteries. There will be haemorrhage from the site of bite, associated with the necrosis of renal tubules, convulsions from intra-cerebral haemorrhage. It consists of: (i) Proteases (ii) Hyaluronidase (iii) Haemorrhagin (iv) Haemolysin (v) Leucolysin (vi) Lecithinase (vii) Cytolysin (viii) Thromboplastin (ix) Phospholipase- A (x) Proteinases.

Fatal doses of various bites are: (i) Dried cobra venom : 12 - 15 mg (ii) Dried viper venom : 15 - 20 mg (iii) Dried krait venom : 5 - 6 mg (iv) Dried Saw Scaled viper venom : 8 mg

Fatal Period: The death may occur instantaneously from neurogenic shock resulting from fright otherwise the fatal period is: (i) In colubrine bite : ½ - 24 hours (ii) In viperine bite : 2 - 4 days

Antivenoms

Antivenoms are prepared by immunizing horses with venom from poisonous snakes and extracting the serum and purifying it. Antivenoms or antivenins may be species specific (monovalent) or effective against several species (polyvalent). Monovalent antivenom is ideal, but the cost and non-availability, besides the difficulty of accurately identifying the offending species makes its use less common.

FORENSIC SCIENCE PAPER No.10: Forensic Toxicology MODULE No.8: Organic Poisons: Animal Poisons

4.1.1.2 GILA MONSTERS

These poisonous lizards belong to the genus Helodermatidae and are indigenous to the south- western USA and Mexico. They have poison glands on the mandible and deliver the venom along corrugated teeth into a bite. The venom contains hyaluronidase and proteases, in addition to gilatoxin. The bite can lead to anaphylactoid syndrome.

4.1.2 FISHES

The sea is host to a wide variety of venomous creatures which stings or bites, and is a special threat to swimmers and divers. Marine envenomations have risen sharply in incidence over the last few decades owing to an increase in popularity of recreational diving and other water related sports. Approximately 225 species of marine fish are known to be venomous. These include the stingray, scorpionfish, lion or zebra fish, stonefish, weeverfish, toadfish, stargazer, and certain catfish, sharks, ratfish, and surgeonfish.

Venom

Poisoning by the consumption of fish meat (ichthyosarcotoxism) usually occurs in warm climates, but is also observed in moderate climate zones when hygiene measures are ignored. Tetrodotoxic and ciguatoxic fish poisoning are caused by ingestion of fish that accumulate toxin-producing organisms, such as bacteria or protozoa, without being affected themselves.

Ciguatoxin

Scombroid poisoning is an example of a toxin produced by improper storage after death, and other fish produce poisonous stings.

FORENSIC SCIENCE PAPER No.10: Forensic Toxicology MODULE No.8: Organic Poisons: Animal Poisons

Tetrodotoxin

4.1.3 AMPHIBIANS

Of the amphibians, the toads are of foremost interest to the toxicologist because a number of species produce noxious substances in their dermal glands. These compounds include amines, peptides, proteins, steroids and both water-soluble and lipid-soluble alkaloids. With the exception of the last, these substances are produced by the toad itself rather than bioaccumulated. The genus Bufo emanates the alkaloid bufotenine (N, N-dimethyl-5- hydroxytryptamine).

Bufotenine 4.1.4 BIRDS

Certain birds possess poisons, although they cannot produce it. Some Pitohui bird’s skin and feathers contains powerful neurotoxic alkaloids of the batrachotoxin group. These are supposed to aid the birds as a chemical defence, either against ectoparasites or against predators such as snakes, raptors or humans. The birds perhaps do not produce batrachotoxin themselves. The toxins most likely come from the beetle genus Choresine which is a part of the birds' diets

FORENSIC SCIENCE PAPER No.10: Forensic Toxicology MODULE No.8: Organic Poisons: Animal Poisons

Batrachotoxin

4.2 INVERTEBRATES

4.2.1 ARTHROPODS

4.2.1.1 CANTHARIDES (SPANISH FLY)

The dried beetle Cantharis vesicatoria, known as ‘Spanish fly’ contains a vesicant, ‘cantharidin’. Contact of either with the live or the dried beetle with the skin results in immediate burning with vesication. Besides this, other beetles causing similar representations includes Epicauta cinerea (blister beetle) and members of genus Paederus.

Venom

Cantharidin is a powerful vesicant and may be administered in the form of powdered beetles or the tincture or active principle. It is also used as an aphrodisiac or an abortificient or a counter irritant to the skin in the blistering plaster or a promoter in the growth of hair. Cantharidin is a nephrotoxic or kidney poison and is readily absorbed from all surfaces including the skin. 10 mg. of Cantharidin or 1.5 gm of powder cantharides is found to be a fatal dose.

4.2.1.2 INSECTS (BEES, WASPS & ANTS)

While snakebites are more common in tropical countries such as India, anaphylactic reaction to hymenoptera stings are much more common in temperate countries. This is despite the gross under-reporting of such stings. Hymenoptera stings are unvaryingly caused by the Honeybee (Apis mellifera), Paper Wasp (Polistes annularis; Ropalidia gregaria), European Wasp (Vespula germanica), Hornets (Vespa & Dolichovespula sp.), and Yellow Jackets (Vespula pensylvanica). A few incidents result from stings of fire ants (Solenopsis invicta) and rarely Jumper Ants (Myrmecia pilosula).

FORENSIC SCIENCE PAPER No.10: Forensic Toxicology MODULE No.8: Organic Poisons: Animal Poisons

They produce a painful local reaction, which recedes with time. An allergic phenomenon produced by them may kill the patient because of prior sensitization. The number of stings may be so great or the patient is so young as to kill with primary toxicity.

Insect Venom

Hymenoptera venom is a mixture of biogenic amines (Histamine, 5-hydroxytryptamine and Acetylcholine), enzymes (Phospholipase- A and Hyaluronidase), and toxic peptides (Kinins in wasps; apamin, melittin, and mast cell degranulating peptides in bees).

4.2.2 ARACHNIDS

4.2.2.1 SCORPIONS

Scorpions are poisonous arthropods that have a fleshy segment body with four pair of legs, a pair of claws and a tail. The posterior segment is long and bulbous and contains the venom gland and a hollow sting at the end of the tail in its last joint. The sting communicates with the venom gland by means of a duct. The stings inject venom in the injury produced by it. The adult scorpion are 2 - 20 cm long and big, black coloured are more dangerous that the small, brown ones. The scorpion’s bites are always accidental as they live in cracks and holes in the houses.

Venom

The main toxins include phospholipase, acetylcholinesterase, hyaluronidase, serotonin, and neurotoxins. The venom of Buthus species of India contains Phospholipase- A, which causes gastrointestinal and pulmonary haemorrhages, and disseminated intravascular coagulation. Scorpion venom is clear and colourless toxalbumins that is more toxic than the snake venom.

4.2.2.2 SPIDERS

All spiders, with the exception of two small groups, are venomous. There are over 100,000 species of spiders. However, only about 20 species cause serious envenoming in humans, while about 150 to 180 can cause significant toxicity. The common Indian species that cause serious envenomation include Brown Recluse, Black Widow, Wolf Spider, and Tarantula. Other spiders such as Orb Weaver, Running Spider, Hackled-band Spider, Giant Crab Spider, Lynx Spider, Jumping Spider, and Tangleweb Weaver, which are also encountered in India, do not cause significant envenomation. Funnel Web Spider which can cause significant envenomation is found only in the Australian continent.

FORENSIC SCIENCE PAPER No.10: Forensic Toxicology MODULE No.8: Organic Poisons: Animal Poisons

Venom

The venom of Brown Recluse is cytotoxic and consists of several toxic components including hyaluronidase, ribonuclease, deoxyribonuclease, alkaline phosphatase, lipase, and Sphingomyelinase - D. The last mentioned is the main constituent which is responsible for tissue destruction. The venom of Black Widow is neurotoxic and contains six active components of molecular weight ranging from 5000 – 130,000D. The main component is alpha latrotoxin which binds avidly to a specific presynaptic receptor.

4.2.2.3 MITES & TICKS

Gastrointestinal illness has been reported in children who handled the mite Holothyrus coccinella and then placed their fingers in their mouths. Skin reactions and irritation in humans is seen from a number of different mites including Dermanyssus gallinae (Chicken Mite), Ornithonyssus sylviarum (Northern Fowl Mite), Ornithonyssus bacoti (Tropical Rat Mite), Allodermanyssus sanguineus (Rodent Mite), and Rickettsia akari. Respiratory allergies due to house dust mites are fairly common. Rhinitis and extrinsic asthma are caused by house dust mites (Dermatophagoides species). The scabies mite is antigenic and stimulates autoantibodies leading to a pemphigoid-like reaction. Scabies may also mimic other skin disorders like contact dermatitis, scalp dermatitis, and generalised urticaria.

4.2.2.4 CENTIPEDES

Centipedes can inflict painful bites characterised by immediate local burning pain, erythema, swelling, inflammation, superficial necrosis, lymphadenopathy, and lymphangitis. The oedema may last for several hours. Local pain may be excruciating and the wound may bleed profusely. The commonest genus encountered in India is Scolopendra. Occasionally, systemic features are seen: anxiety, dizziness, vomiting, headache, convulsions, irregular pulse, and cardiac arrhythmias. Rhabdomyolysis and renal failure have been reported with the Giant Desert Centipede (Scolopendra sp.).

4.2.2.5 MILLEPEDES

Millipedes do not bite, and are not venomous, but have a glandular system that produces a foul-smelling, disagreeable fluid containing phenols and hydrocyanic acid. Some millipedes secrete or squirt these irritant liquids for defensive purposes, which can cause brown or purple skin lesions that blister after a few days and then peel. Sometimes these lesions take a long time to heal. Contact with the eye can result in severe conjunctivitis, corneal ulceration, and even blindness.

FORENSIC SCIENCE PAPER No.10: Forensic Toxicology MODULE No.8: Organic Poisons: Animal Poisons

4.2.3 CNIDARIANS

4.2.3.1 BOX JELLY FISH

The Box Jellyfish, Fire Medusa, or Sea Wasp (Chironex fleckeri) is the most venomous of all stinging marine creatures. Box Jelly fish has a transparent box-like bell with four pedalia (feet). Each pedalia may have up to 15 tentacles attached. Because of its transparency, the Box Jellyfish is virtually invisible under natural conditions, including clear, bright seawater.

Venom

The venom is a complex mixture of serotonin, histamine, bradykinin, haemolysin, prostaglandins, Hyaluronidase, phosphodiesterases, Fibrinolysin, RNAase, DNAase, adenosine triphosphatase, alkaline and acid proteases, as well as alkaline and acid phosphatases. Each box jellyfish carries enough venom to kill several adults. Features include profound muscle spasm, hypotension, acute respiratory distress, respiratory paralysis, cyanosis, haemolysis, arrhythmias, and cardiac arrest. Severe parasympathetic dysfunction (abdominal distension, urinary retention, dry eyes) is common. Death can occur in less than a minute.

4.2.4 MOLLUSCS

Mollusc poisoning or ‘shellfish poisoning’ is caused by the consumption of bivalve molluscs that accumulate toxins of protozoal or algal origin. Toxins from algal sources are also referred to as phycotoxins, analogous to the mycotoxins from fungal sources. Mollusc poisoning caused by algal toxins is usually classified according to the symptoms they cause in humans:  Paralytic Shellfish Poisoning (Saxitoxins)  Diarrhoetic Shellfish Poisoning (Okadaic acid)  Neurotoxic Shellfish Poisoning (Brevetoxin)  Amnestic Shellfish Poisoning (Domoic acid)  Intoxication with venoms (the Conotoxins) from snails belonging to the genus Conus is another form of mollusc poisoning.

Paralytic Shellfish Poisoning (PSP) is caused by Saxitoxins (STX) produced by marine ‘red tide’ dinoflagellates and freshwater blue-green algae such as Alexandrium spp., Gymnodinium catenatu and Pyrodinium bahamense. STX are potent agents that can block sodium channels in nerves and muscles at the extracellular side of the channel, which leads to conductivity disturbances and paralysis. In severe cases, the neurological symptoms spread to the extremities and respiratory muscles and, without ventilatory support, patients may die between 2 and 12 hours after ingestion.

FORENSIC SCIENCE PAPER No.10: Forensic Toxicology MODULE No.8: Organic Poisons: Animal Poisons

Saxitoxin Domoic Acid

The major causative agent of Diarrhoetic Shellfish Poisoning (DSP) is Okadaic acid, which is produced primarily by ‘red tide’ dinoflagellates belonging to the genera Dinophysis and Prorocentrum. DSP toxins are lipophilic and accumulate in the digestive gland of mussels. Okadaic acid is a potent inhibitor of protein phosphatases- 1 and 2A. In humans, consumption of contaminated molluscs leads almost exclusively to gastrointestinal symptoms: diarrhoea, nausea, vomiting and abdominal pain, which appear between 30 minutes and a few hours after the meal and can be caused by as little as 40 μg of toxin.

Okadaic Acid

Neurotoxic Shellfish Poisoning (NSP) is caused by a toxin produced by another ‘red tide’ dinoflagellate, Gymnodinium breve. The active principle is the lipid soluble polyether Brevetoxin, one of the most potent neurotoxins known. In humans, ingestion of brevetoxin- contaminated shellfish can result in gastroenteritis with neurological symptoms. Within 3 hours, nausea and vomiting, paraesthesias, reversal of hot/cold sensation, throat tightness and ataxia may occur. There is no paralysis. There is complete recovery from these symptoms within 2 days without specific treatment. No human deaths have been reported with brevetoxin poisoning. Domoic acid is a neurotoxic agent. Acute symptoms include vomiting and diarrhoea and, in some cases, are followed by confusion, memory loss, disorientation, coma or death.

FORENSIC SCIENCE PAPER No.10: Forensic Toxicology MODULE No.8: Organic Poisons: Animal Poisons

Brevetoxin

5. Summary

• The animal kingdom comprises of more than 100,000 species spread through major phyla including arthropods, molluscs, chordates, etc. Venomous animals are proficient of producing a poison in a highly developed exocrine gland or group of cells. • Venomous or poisonous animals and insects are capable to deliver toxins during biting or stinging. They produce the toxins in highly developed secretory glands or group of cells or the by-product of metabolism. • Every Animal toxin varies considerably to each other in their chemistry and toxicology. Venoms, for instance, may be composed of proteins of both large and small molecular weight including polypeptides and enzymes. They may be amines, lipids, steroids, amino polysaccharides, quinones, 5-HT glycosides or other substances. • Venoms are very complex, containing polypeptides, high- and low-molecular-weight proteins, amines, lipids, steroids, aminopolysaccharides, quinones, glucosides, and free amino acids, as well as serotonin, histamine, and other substances. Some venoms are known to consists of more than a hundred proteins. • The bioavailability of venom is determined by its composition, molecular size, amount or concentration gradient, solubility, degree of ionization, and the rate of blood flow into that tissue, as well as the properties of the engulfing surface itself. • Among all of poisonous insect and animals, cantharides, snakes and scorpions are of medico-legal importance. • Poisoning resulting from fish and other marine creatures is referred to as Ichthyism, which may result either from envenomation by stinging or biting, or from ingestion of toxic or decomposing fish.

FORENSIC SCIENCE PAPER No.10: Forensic Toxicology MODULE No.8: Organic Poisons: Animal Poisons