Relative Toxicity

Snake venoms are like soups. Most of them contain many different components and some of these are toxic to man. For instance, in the common tiger snake venom, Notechis scutatus, there have been at least 6 neurotoxins described1,2. There are several haemotoxins 3,4,5.6, 2 interfering with activation of the clotting factors and some weak haemorrhagins. Some of the neurotoxins also cause muscle damage. There are also toxins which can cause a drop in blood pressure in this venom3. Most venoms have many components, some of which are known to be toxic to man. There are also many components in most venoms that are still uncharacterised. It is the combined effect of all the components in snake venoms that gives it its overall toxicity. In the past, and to a lesser extent today, toxicity was measured by injecting test animals with venoms7. Many different types of animals have been used. In the 1960's and 1970's, mice became the universally accepted test animal and comparative toxicity was expressed as an LD50. This is usually measured by testing the crude venom in mice at varying doses and when a calculated concentration causes 50% of the test animals to die, it is referred to as the LD50. Today, these types of animal experiments are not performed as much because of animal welfare concerns, however they were invaluable in understanding the toxicity of both crude venoms and purified single toxins and are still the best way of measuring overall toxicity in living organism. There are other specific tests that look at the effect of single toxins. These can be clotting assays to measure the effect of coagulants8, nerve-muscle preparations to measure neurotoxic activity9, phosphilipase and phosphodiesterase assays which measure these respective agents in the venoms. Even muscle or myoblast preparations to measure the myotoxic activity of venoms have been used. These tests are useful to investigate the relative toxicity of purified toxins but there is sometimes a poor correlation between these results and the in vivo effects of the toxin. There is still no alternative that surpasses the in vivo LD50 for both purified toxins and crude venoms. In 1979, the Commonwealth Serum Laboratories in Australia conducted an extensive comparative study of the toxicity of most of the Australian medically significant snake venoms and some venoms from non-Australian snakes10. Here they used mice which were subcutaneously injected with the crude venoms. In the early 1980's, Richard Davis and I published the cobra scale11, which compared venoms with that of the well known Indian cobra venom. Cobra venom is assigned the value of 1 and the toxicity of other venoms are compared to this venom. Table 1 is an expanded version of this comparison.

Australian snakes
		Relative Toxicity
Inland taipan	Oxyuranus microlepidotus	50.0
Common brown snake	Pseudonaja textilis	12.5
Taipan	Oxyuranus scutellatus	7.8
Reevesby Is. tiger snake	Notechis ater niger	5.1
Common tiger snake	Notechis scutatus	4.2
Western tiger snake	Notechis ater occidentalis	4.0
Beaked sea snake	Enhydrina schistosa	2.9
Chappell Is. tiger snake	Notechis ater serventyi	1.8
Common death adder	Acanthophis antarcticus	1.5
Western brown snake	Pseudonaja nuchalis	1.5
Copperhead	Austrelaps superbus	1.0
Dugite	Pseudonaja affinis	0.9
Stephens banded snake	Hoplocephalus stephensi	0.4
Rough scaled snake	Tropidechis carinatus	0.5
Spotted black snake	Pseudechis guttatus	0.3
King brown snake	Pseudechis australis	0.3
Colletts snake	Pseudechis colletti	0.2
Red bellied black snake	Pseudechis porphyriacus	0.2
Small-eyed snake	Cryptophis nigrescens	0.2
Whip snake	Demansia olivacea	< 0.1
Non-Australian
Indian cobra	Naja naja	1.0
Papuan black snake	Pseudechis papuanus	0.4
King cobra	Ophiophagus hannah	0.3
Eastern diamond-back rattlesnake	Crotalus adamanteus	<< 0.1
Brazillian viper (Barba amarilla)	Bothrops atrox	<< 0.1

Table 1 Relative toxicity compared to the Indian cobra.

This type of comparison still relies on LD50 results being available, but provides a simple comparison of venoms for the lay person. It is important when using this type of comparison, to only use LD50 figures that quoted in the same form using the same test animal, same route of injection and if possible from the same data set. Table 2 shows how toxicity can differ from one data set to another and also with route of injection. Generally, intravenous (i.v.) injections will produce lower LD50's than intraperitoneal (i.p.) injections and both usually produce lower results than subcutaneous (s.c.) injections.

	Route of injection
	LD50 mg/kg (Minton & Minton 1969)			LD50 mg/kg (Broad et al 1979)
Species	i.p.	i.v.	s.c.	s.c.
Oxyuranus microlepidotus				0.03
Acanthophis antarcticus		0.25	0.50	0.40
Notechis scutatus	0.04		0.18	0.12
Pseudonaja textilis		0.01	0.25	0.05
Pseudechis porphyriacus		0.50	2.00	2.52
Dendroaspis polyepis		0.25	0.28
Enhydrina schistosa	0.12	0.13	0.15	0.17
Daboia (Vipera) russelli		0.08	4.75
Ophiophagus hannah			1.7	1.8
Bothrops atrox	3.80	4.25	22	> 27.8

Table 2. Comparison of different toxicity data and route of injection (mice).

Venoms can vary for many reasons so it is important, to specify the relevant parameters when reporting results. Some of the more important parameters that can affect venom toxicity and cause variable results are as follows:
Geography
Sometimes, venoms from the same species can differ in their components, and therefore toxicity, when compared from different geographical areas. For instance the tiger snake, Notechis scutatus, venom from both Mount Gambier and Melbourne differ in their component composition and toxicity12. In these 2 populations and also another population at Lake Alexandrina in South Australia, the neurotoxic components vary considerably. For instance, notexin and scutoxin are 2 very potent presynaptic neurotoxins in this venom, and vary in their relative proportions. They are shown in table 3:

	Notexin	Scutoxin
Notechis scutatus (Lake Alexandrina, SA)	+++	+
Notechis scutatus (Mt. Gambier, SA)	++	+
Notechis scutatus (Melbourne, Victoria)	++	++

Table 3 Variation of individual neurotoxins in different populations of tiger snakes. (This information was kindly supplied by Professor Ivan Kaiser, Laramie Wyoming USA).

Also the notexin and Notechis II V components varied in proportion between Mt Gambier and Melbourne. The Melbourne population lacked Notechis II V in their venoms but was rich in notexin whereas the notexin was less in proportion in the Mt Gambier population but Notechis II V existed12. Daltry et al (1996)13, showed there was considerable venom variation of the Malayan pit viper Calloselasma rhodostoma venom from different geographical areas due to prey differences13, however Williams et al (1988)20, explained differences they found in Notechis ater niger venoms by the length of time in separation of their habitats.

Seasons
Seasonal variation in venom toxicity may affect results of some venoms. Preliminary studies by Vaughan Williams at the Adelaide Woman's and Children's Hospital and Venom Supplies Pty Ltd, have shown that there is little or no seasonal variation with tiger snake venom from Lake Alexandrina but in a separate study, Vaughan Williams , using venom supplied by Ted Mertens showed measurable differences occurred in a single brown snake venom samples when compared over 1 year.

Snake age
As snakes age, venom components can vary. Furtado et al (1991)17 compared the age difference of venoms in 9 species of the Bothrops genus and found significant variation. Likewise Meier (1986)18 and Minton and Weinstein (1986)19, compared venoms from Bothrops atrox and Crotalus atrox respectively and also found differences in venoms from adults and juveniles. In studies we shared with Dr Nget-Hong Tan and his co-workers from the University of Malaysia, we found there were very little differences in the toxicity and other specific venom characters in Notechis scutatus, Oxyuranus scutellatus and Oxyuranus microlepidotus venoms when juvenile venoms were compared to adult venoms of these species14,15,16. This is probably due to less prey difference in juveniles to adults in the 3 Australian species studied.

References
1. Sutherland,S.K. (1983). Australian Animal Toxins. Oxford University Press. Melbourne. p54.
2. Francis, B., John, T.R., Seebart, C. and Kaiser, I.I. (1996). New toxins from the venom of the common tiger snake (Notechis scuatatus scutatus). Toxicon 29(1) 85-96.
3. Francis, B., Williams, E. S., Seebart, C., Kaiser, I. I. (1993). Proteins isolated from the venom of the common tiger snake ( Notechis scutatus scutatus ) promote hypotension and haemorrhage. Toxicon 31(4). 447-458.
4. Tans, G., Govers-Rienslag, J.W.P., van Rijn, J.M.L. and Rosing, J. (1985). Purification and properties of a prothrombin activator from the venom of Notechis scutatus scutatus. The Journal of Biological Chem. 260(16). 9366-9372.
5. Jobin, F. and Esnouf, M.P. (1966). Coagulant activity of tiger snake (Notechis scutatus scutatus) venom. Nature 211(5051). 873-875.
6. Picciuto, R., Marshall, L.R.(1994). Unique anticoagulant activity of tiger snake venoms (Notechis species). In press
7. Minton, S and Minton, M.R. (1969). Venomous Reptiles. Scribners New York.
8. Nahas, L., Denson, K.W.E. and Macfarlane, R.G. (1964). A study of the coagulant action of eight snake venoms. Thromb. Diath. Treat. 12 355-367.
9. Thesleff, S. (1979). Reptile toxins and neurotransmitter release.. Eds. I.W. Chubb and L.B. Geffen. Neurotoxins. Fundamental & Clinical Advances. Centre for Neurosciences. Flinders University. 19-25
10. Broad, A. J., Sutherland, S. K., Coulter, A.R. (1979). The lethality in mice of dangerous Australian and other snake venoms. Toxicon (17). 664-667. 11. Mirtschin,P.J. and Davis, R. (1982). Dangerous Snakes of Australia. Rigby. Adelaide.
12. Yang, C.C., Chang, L.S. and Wu, F.S. (1991). Venom constituents of Notechis scutatus scutatus (Australian tiger snake) from differing geographic regions. Toxicon 29(11) 1337-1344.
13. Daltry, J. C., WÜster, W., Thorpe, R. S. (1996). Diet and snake venom evolution. Nature 379 537-540
14. Tan, N., Ponnudurai, G., Mirtschin, P. J. (1993). A comparative study on the biological properties of venoms from juvenile and adult Common Tiger Snake (Notechis scutatus ) venoms. Comp. Biochem. Physiol. 106B (3) 651-654
15. Tan, N., Ponnudurai, G., Mirtschin, P. J. (1993). A comparative study of the biological properties of venoms from juvenile and adult Inland Taipan ( Oxyuranus microlepidotus ) snake venoms. Toxicon 31 (3). 363-367. 16. Tan, N., Armugam, A., Mirtschin, P. J. (1992). The biological properties of venoms from juvenile and adult Taipan ( Oxyuranus scutellatus ) snakes. Comp. Biochem. Physiol. 103B (3). 585-588
17. Furtado, M. F. D., Maryuyama, M., Kamiguti, A. S. and Antonio L.C. (1991). Comparative study of nine Bothrops snake venoms from adult female snakes and their offspring. Toxicon 29 (2). 219-236.
18. Meier, J. (1986). Individual and age-dependent variations in the venom of the Fer-de-lance (Bothrops atrox). Toxicon 24 (1) 41-45.
19. Minton, S. A. and Weinstein, S. A. (1986). Geographic and Ontogenetic variation in venom of the western diamondback rattlesnake (Crotalus atrox). Toxicon 24 (1) 71-80.
20. Williams, V., White, J., Schwaner, T. D., Sparrow, A. (1988). Variation in venom proteins from isolated populations of tiger snakes (Notechis ater Niger, N. scutatus ) in South Australia Toxicon 26(11). 1067-1075.