The Art and Science of Poisons

Stella.

The Deadliest Poison

Olen R. Brown

Abstract

Beauty is said to be in the eyes of the beholder. Likewise, the deadliness of a poison depends on subjective criteria. Is more weight to be given for quickness of action, stealth, whether an antidote is available, or how little is required? Most accounts declare botulinum A, the toxin produced by a species of anaerobic bacteria, to win the contest based on its LD50 (the amount that kills half of those exposed). Its toxicity, measured this way, is greater than any known substance. I propose a new way of ranking poisons, the LD50*, based on the number of molecules in a deadly dose. This is more equitable because poisons differ greatly in their molecular weights – some are very small and some are very large molecules, and poisons kill molecule-by-molecule. Several snake venoms are deadly and the most toxic is that of the inland taipan, although the coral snake and cobras have very toxic venoms, and rattlesnake, because of the volume injected and the multiplicity of toxic ingredients, deserve mention. Only two species of scorpions (the death stalker and man killer; neither found in the United States) have stings that are life threatening for humans. Spider venoms don’t quite make it to our most deadly list. Radiation exposure is a different kind of poisoning and Polonium-210 makes our list because of the small amount required and its intense radiation based on its very short half-life. The most deadly, quick-acting toxins affect the nervous system and cessation of respiration or heart function stops the supply of oxygen to tissues to cause death. It is thought-provoking to consider that all things are poisonous, and that only the dose makes the difference (to paraphrase toxicologists). In this context, life-giving pure water becomes deadly when several liters are consumed rapidly. Why poisons exist has a scientific answer, but perhaps not a satisfactory philosophical answer.

All things are poison and nothing is without poison; only the dose makes a thing not a poison.¹

Keywords: Acetylcholinesterase, Anaphylaxis, Antidote, Antitoxin, Antivenin, Avogadro’s Number, Bane, Biological Warfare, Botulinum A, Clostridium Botulinum, Coral Snake LD50, LD50*, Myoneural Junction, Nerve Gas, Paracelcus, Poison, Polonium-210, Potion, Sarin, Skull and Crossbones, Taipoxin, Toxin, Venom, VX Agent.

INTRODUCTION

The word poison, to me, conjures up thoughts of death, skull and crossbones, chemicals, harm by intrigue and stealth, serpents, object of aversion, and when

thinking technically, an inhibitor of a chemical reaction, especially an enzyme. Although we may not usually consider medicines (including the drugs known as statins that are used to treat elevated cholesterol) as toxic, many actually poison enzymes in our bodies. Merriam-Webster [1] gives several meanings for poison including that it is … a substance that through its chemical action usually kills, injures or impairs an organism. This dictionary states that the origin of the word is from Middle English (derived from Anglo-French meaning poisun drink, potion, poison); or from the Latin for potio. Synonyms are bane, toxic, toxin, and venom. Bane is seldom used now but usually means a cause of great distress or annoyance, but can refer to a deadly poison, and conveys other meanings: killer, slayer, death, destruction and probably dates back to the 12th century. These meanings will define the scope of this book: stories of discovery, intrigue, murder, venomous animals, toxic bacteria, beautiful but deadly plants, and warfare by mankind.

POISONS MOST VILE

Paracelsus (1493-1541), credited with originating the discipline known as Toxicology (the study of poisons), is famous for having penned what is most often paraphrased simply as the dose makes the poison. This provides very wide latitude for the subject of this chapter and I will take into consideration much more than how little of a substance is required to kill a human. Thus, we shall approach the question of the most toxic substance from a variety of perspectives.

Candidates of poison which kill a human with the least amount of substance traditionally have included botulinum [2] the protein produced by the microbe that causes botulism [3], the radioactive element polonium-210 [4], the venom of poisonous snakes including the taipan, cobra, mamba, saw-scaled viper, and others [5] and the nerve agents which have been used as weapons of war [6]. Use of poisons is often considered insidious and secretive; some are deadly but slow to act and some do their deadly work quickly. Some poisons always, or most usually, are encountered accidentally and others mostly by evil design.

Poisons are connected with murder by stealth and poisoning has been considered a cowardly act. John Fletcher (1579- 1625) is quoted to have used the words: The coward’s weapon, poison [7]. Fletcher was a dramatist and wrote and collaborated to write comedies and tragedies. Unfortunately he died in the London plague of 1625 and it has been said that this resulted because he lingered in the city to be measured for a suit of clothes instead of quickly fleeing to the country as did many [8].

The universal symbol for a poisonous substance is the skull and crossbones (Fig. 1). The origin of this is lost in history. However, I found the following information (with original attributions listed) [9]. I quote from this site as follows:

… it was first seen on the tomb of Tutankhamen… It is widely believed that the skull and crossbones was first used by the Knights Templar… in the Middle Ages… According to Masonic legend, the skull and crossbones are the bones of Jackes de Molay… Toward the end of the Roman Empire and into the Middle Ages, Christians frequently used the Skull and Crossbones to symbolize death… Originally, pirates used a red flag on the top of their ships’ mast… many pirates soon changed their flags from red to black, and began to weave the skull and crossbones into them… Yale University formed a secret society called Skull and Crossbones"… The Death’s Head or Totenkopf was a symbol of the Nazi Schultzstaffel (SS: literally defense echelon, especially Hitler’s body guard)… In 1829, New York State Law was changed to require that all containers of poisonous substances be labeled… The skull and crossbones first illustrated those labels in 1850."

Fig. (1))

Universal symbol of a toxic substance.

Ranking of Poisons by Lethal Dose

It has become customary to compare the lethal amounts of poisons based on weight (mass). By this measure it is generally agreed that botulinum toxin has the smallest lethal dose of all poisons [2, 10-12]. Taipoxin (from the inland taipan snake), is second and it is the most potent of all venoms [13]. Third is polonium-210, which is extremely unstable (radioactive), and it is often cited as the most dangerous radioactive element [4, 14, 15]. Fourth in toxicity by required amount is the chemically-synthesized nerve poison VX agent (developed for warfare). It is the most toxic man-made chemical with a lethal dose that is difficult to accurately assess. Ranking poisons is problematic and will be a struggle faced throughout this book. Based on the sources cited directly above, my judgment is that the toxicities (per kg of body weight), of these agents are: type A botulinum toxin: 1.0 nanogram, polonium-210: 11.1 nanograms, VX-agent: 143 micrograms, and taipoxin: 2 milligrams. Note the difference in units: nanograms, micrograms, and milligrams; each is a thousand times smaller than the next, respectively. Some comments are necessary. The toxicity for taipoxin (the best source is [13]) is in the range of 0.01 mg/kg to 0.025 mg/kg for the complete venom but 2 mg/kg for taipoxin which is the presynaptic neurotoxin (surely the most toxic component). The difference in toxicity is in the range of 80-fold to 200-fold! Thus, if the data are correctly reported, the answer to this quandary may lie in the complex interaction of the venom molecules. A sophisticated study of the venom by Laura Cendron and coworkers provided data supporting several relevant conclusions [16]. Venom of the Australian taipan snake contains taipoxin, a neurotoxin with phospholipase A2 activity that is a potent inducer of paralysis through the specific disruption of the neuromuscular junction pre-synaptic membrane. They state: Although no correlation has been reported between neurotoxicity and enzymatic activity, toxicity increases with structural complexity and phospholipase A2 oligomers show 10-fold lower LD50 values compared to their monomeric counterparts. This strongly indicates a correlation between structural complexity of the toxin and its neurotoxicity. They further state that their research revealed … there are two isoforms of the taipoxin β subunit which show no neurotoxic activity but enhance the activity of the other subunits in the complex. In the introduction to their paper, they state: Taipoxin is composed of three homologous non-covalently bound class I secretory PLA2 subunits… We report the structural characterization of two isoforms of the taipoxin subunit β, which retains no enzymatic activity but is crucial for the elevated neurotoxicity of taipoxin. Moreover, we propose a novel model for the quaternary structure of a trimeric PLA2neurotoxin that has been determined under physiological conditions.

Thus, it appears that this toxin is more than the sum of its parts. Indeed, study of toxins at the molecular level is aided by considering their toxicities on a molecular basis and the LD50* that we propose appears to be a helpful approach.

Scientific assessment of toxicity for humans is complex for many reasons which I will address near the end of this chapter. For now, let us compare, for four poisons that I calculate are the most toxic, the lethal amount, time required to kill, and availability and effectiveness of an antidote (Table 1). Note especially that I have calculated the lethal amount in two ways. The traditional way to assess toxic potency is according to dose by weight. I propose a comparison, based not on weight but on the number of molecules of the poison in a lethal dose.

I propose that it is better to compare the lethal dose in terms of the number of molecules (atoms for polonium-210) because poisons exert their effects molecule- by-molecule. Note that plonium-210 is an atom and not a molecule. The time to death and availability of an antidote also are attributes that I propose (Table 1). They are arguably arbitrary but all are justifiably quite important.

For clarity of comparison, the LD50 of botulinum can be equated to one with the LD50 of the other agents calculated proportionately with the following results: botulinum = 1; polonium = 11.1; taipoxin = 2 x 10⁶; and VX agent = 0.143 x10⁶. Comparisons of LD50* (based on number of molecules) are: botulinum = 1; taipoxin = 6.5 x 10⁶; polonium = 8,000; and VX agent = 8 x 10⁷. It is remarkable that botulinum retains its first place rank in both assessments in spite of the fact that it is by far the largest molecule among the toxins and therefore there are fewer molecules in a lethal dose (Table 1). The size comparisons for these poisons are as follows (in Dalton units): botulinum = 150,000; Taipoxin = 45,600; VX agent = 267; and polonium-210 = 209. If we equate polonium-210 to 1 the comparison is easier to comprehend: VX agent is 1.28 times as large, Taipoxin is 218 times as large, and botulinum is 718 times as large as polonium-210.

When assessment is based on the time to death from a lethal dose, granting a great deal of uncertainty in reported values, the order is VX agent, taipoxin, botulism, and polonium (Table 1).

Toxin from Clostridium Botulinum

Botulinum is the toxin in spoiled food that is produced anaerobically at alkaline pH by Clostridium botulinum, the microbe that causes botulism. There are seven known types (A-G) of the toxin which have slightly different molecular structures but which can be identified by laboratory tests. Botulism in humans is usually caused by types identified as A, B, E (and rarely by type F). The toxins are composed of amino acids linked together in a protein with a large molecular weight of approximately 150,000 Daltons for type-A toxin (Fig. 2). This is huge, approximately 150,000 times the weight of one hydrogen atom [17]. Type-A toxin has 1,259 amino acids linked together by peptide bonds [18]. This large arrangement of atoms is poisonous by two chemical actions. One poisonous action results from a smaller part that is approximately 50,000 Daltons in size. A larger part (approximately 100,000 Daltons) of the molecule (Fig. 3) is the component that actually binds to nerves and blocks impulse transmission to muscle fibers [19]. The estimated lethal dose for a human is 0.091 microgram. This is equivalent to only 365 billion molecules. This may appear to be a huge number; however, there are 602 billion trillion molecules in a quantity of the toxin equal to its molecular weight expressed in grams. A molecule of botulinum toxin has a weight of 150,000 Daltons (molecular weight expressed in grams). For comparison, the smallest known protein has only 20 amino acids, and weighs only 2,171 Da; it is found in the saliva of the Gila monster [20].

Fig. (2))

Botulinum toxin, depicted as a ribbon diagram to show the complex 3-D structure of the protein.

Fig. (3))

Botulinum toxin, diagrammatically, showing two polypeptide chains linked by a disulfide bridge. The gap between the light and heavy chains (green and lavender) is the point of cleavage of the toxin at its site of cellular action into portions of 100,000 and 50,000 Daltons (see text).

The lethal quantity of botulinum toxin is a microscopic amount, far too small to be visible by the unaided, human eye. On a weight basis, nothing has been described to be more toxic for humans. This results directly from the fact that the toxin binds to nerves at junctions (synapses) and prevents nerve transmission. With a lethal dose, a person without treatment likely will die from paralysis of the breathing muscles. Paralysis develops first in muscles in the head area and descends. More specifically, botulinum toxin binds to receptors at myoneural junctions (connections between nerves and muscles) and prevents nerve impulse from activating muscle which causes flaccid paralysis. However, an antiserum is available as an antidote and if administered after rapid diagnosis, and with proper supportive care, the death rate is reduced to approximately 3% to 5% for clinical cases [10]. Breathing can be maintained by mechanical means if medical care is available and over time the body can replace the connections of nerves to muscles and patients can recover. However, this may require several months or even longer. A heptavalent botulism antitoxin (active against all seven known botulinum nerve toxin serotypes of Clostridium botulinum) has been developed but it is an effective antidote only when a proper diagnosis is made and treatment begun soon after ingesting the poison [21]. Damage that has already resulted from this toxin to nerves is not directly reversed by the antitoxin. After a lethal dose of botulinum death is unlikely before one day [10] and if the person seeks medical treatment, is properly diagnosed and given antitoxin (or mechanical ventilation and oxygen is available) the person likely will survive.

Nerve Gas

VX agent is the most deadly of the nerve gasses that have been synthesized in the laboratory for use as a terror weapon and in warfare (Fig. 4). The estimated lethal dose by skin contact is 10,000 micrograms (about 1/100th of a milliliter by volume) [22]. Calculated in terms of the number of molecules, the estimated lethal dose of VX agent for a human (70 kg) by skin contact is approximately 22 billion billion molecules (compared to 280 billion for botulinum). Although the dose of VX agent is a large number of molecules, it is a very small amount. An ordinary drop is 1/20th of a milliliter; thus, divide an ordinary drop of liquid into 5 drops and one of these smaller drops is the amount of VX agent that kills by contact with the skin. Because the mechanism of poisoning is known and can be negated by an antidote which soldiers carry when there is threat of chemical warfare, and because sensors to detect VX agent are available, my assessment of the toxicity rank of this poison is decreased somewhat. This agent has a molecular weight of approximately 267 grams. This means, as is the case for all chemicals, the weight in grams is calculated based on the chemical composition of the substance. Small molecules are composed of fewer atoms and weigh less as a general characteristic. VX agent, therefore, is a comparatively small molecule. For comparison, water has a molecular weight in Daltons (molecular weight expressed in grams) of approximately 18; potassium cyanide is 65; aspirin 180, VX agent 267, table sugar 360, and botulinum 150,000.

The number of molecules in one gram molecular weight is approximately 6.2x10²³ (Avogadro’s number). This number is written as a power of 10 because it is so large. Written this way, the number of zeros is indicated by the exponent. It can be translated into more familiar terms as 6.2 hundred billion trillion. This is a number larger than the number of stars calculated to be in the entire known universe. Therefore, when we calculate the number of molecules present in the

Fig. (4))

VX agent shown as a ball and stick model. White is hydrogen, black is carbon, red is oxygen, orange is phosphorous, and blue is nitrogen.

lethal dose of a poison, even when a very small amount of the poison is lethal, the number is very large. Poisons vary greatly in molecular size. The action of poisons occurs at the molecular level, so calculations based on the number of molecules is more appropriate, in my view, than calculations based on weight (mass) of the poison in the lethal dose. Polonium-210 exists as atoms so using the atomic weight is equivalent to using molecular weights for the other poisons.

The mechanism by which VX agent kills is the same as for other, similar nerve agents known as taubin (GA), sarin (GB), soman (GD), and cyclosarin (GF). All these agents poison the enzyme acetylcholinesterase [22]. Several organo-phosporous compounds act similarly but are far less toxic and some affect insects, not humans. Nerve agents are extremely toxic and act very rapidly. VX agent is designed to be less volatile and to be persistent on materials and the terrain for long periods of time. Human uptake is primarily via skin contact, but it can also be inhaled. The route of body entry affects the time to symptoms which include death. Effects occur more quickly when entry is by the respiratory route compared to absorption through the skin. At the biochemical level, VX agent and similar compounds are toxic because they poison an enzyme known as acetylcholinesterase (Fig. 5). Acetylcholinesterase functions in transmission of nerve impulses. For nerve impulses to activate muscle movement, the electrical impulse must cross a barrier called the myoneural junction. This is an actual, physical gap that the impulse must cross. To do so, a chemical signal is produced by the impulse on the side of the junction nearest the origin of the impulse (CNS side). This chemical signal is acetylcholine. Acetylcholine diffuses across the synapse and stimulates receptors on the muscle innervations site of the junction. The nerve impulse causes muscle contraction. To stop the effect of acetylcholine molecules, they are chemically cleaved by an enzyme called acetyl cholinesterase.

Fig. (5))

The myoneural junction site of action of VX; see text for explanation.

The cleavage products are recycled. The important fact is that cholinesterase erases the agent that carries the nerve impulse across the synapse to effect muscle contraction. When cholinesterase is poisoned by VX agent, the acetylcholine remains and the nerve continues to function across the synapse and muscle contraction is sustained inappropriately.

Deadly Snake Venom, Taipoxin

Taipoxin is the primary neurotoxin in the venom of a particular Taipan snake, Oxyuranus microlepidotus. The average yield of snake venom (when milked) is 44 mg, and the maximum is said to be 110 mg [13]. Taipan snake venom is a complex mixture. Another species, Oxyuranus scutellatus, has even more venom: 120 mg average and 400 mg maximum. It is said that the average venom injected at first bite (defensive strike) by O. Microlepidotus is 17.3 mg with a range of 0.7 to 45.6 mg. O. scutellatus has a neurotrophic venom very similar to O. microlepidotus. Both neurotoxins work similarly to botulinum toxin and they block signal sent to muscles and paralysis of the diaphragm impairs breathing and death can occur. More specifically, taipoxin binds to neuromuscular post-synaptic junction sites and prevents nerve impulses from activating muscles which results in a flaccid paralysis. Toxicity of the venom of Oxyuranus microlepidotus, reported as toxicity for the whole toxin, is LD50 = 0.025 mg/kg in mice (18-20 gram) with subcutaneous injection (a referenced source is given) [13]. This site gives several clinical cases including circumstances of envenomation, course of treatment, and outcome; there are 97 total references.

Each snake species has venom that is a complex mixture of proteins and non-proteins that act as neurotoxins, procoagulants, and myolysins. Neurotoxins are present that act both pre-synaptically (taipoxin) and post-synaptically. Coagulants principally are analogues of blood factors involved in coagulation but they act independently of control by cofactors including calcium, and they convert prothrombin to thrombin (to form a clot). Myolysins perform an additional toxic action at presynaptic junctions (this is an additional toxic action of taipoxin). There is some controversy about various snakes that are called taipans, and some claim the inland taipan is the most venomous. A particular snake bite will deliver a variable amount of venom depending on when the snake last used its venom, and the snake is also capable of controlling the amount of venom delivered. Apparently, this is a mechanism that allows the snake to conserve its poison. Some snake bites are described as dry to indicate essentially no poison was delivered. It is reported that early symptoms are usually seen in the first six hours, anti-coagulation effects leading to hemorrhaging may develop within 30 minutes; however, systemic collapse, unconsciousness and convulsions may occur (especially in children) occasionally as rapidly as 15 minutes after the bite [13]. For severe envenomation: … in some cases, paralysis may be sufficiently advanced at a cellular level that antivenom cannot prevent severe paralysis… overall, up to 75% of all taipan bites will prove fatal if no antivenom treatment is used [13]. An antidote (antitoxin or antivenom; the terms are used interchangeably) is available and with prompt, effective medical treatment, survival is probable. However, antitoxin does not reverse damage already done to nerves, and antivenom therapy may be too late to prevent paralysis and death [13]. The antitoxin is an antibody, usually developed in the horse.

Radioactive Poison

Polonium-210 is an element that is radioactive [23]. An international symbol of radiation hazard has been adopted (Fig. 6). Emission of radiation is a physical not a chemical action. Does this technically disqualify polonium-210? Did the creator of the definition of poison (see page 1) carefully consider the nuance implied by chemical action, and does this exclude radioactivity? After all, scientists talk about radiation poisoning. Think about it. True enough, the emission of an alpha particle is an event involving the atomic nucleus and radiation emission is physics and not defined as chemistry. However, the effect of the alpha particle on a living cell immediately involves chemistry. The toxic event can be a chemical change in a DNA molecule to lead to a fixed mutation, or it can be inactivation of function by physical destruction of cells. So, there is a gray area here that includes an immediate involvement of biochemistry (and thus, chemistry) in the toxic mechanism. I vote to include polonium as a poison.

Fig. (6))

International symbol of radiation hazard.

There are, however, special complexities encountered when considering radiation. All chemical poisons must be absorbed into the body, and transported and distributed to the site of action where further chemistry must occur. A poison that is toxic by chemical action, if it is in a container or otherwise outside the body, is not a hazard to human life. A molecule of chemical poison, even though ingested, has no deadly effect if it is purged before absorption. A molecule of chemical poison though ingested or injected has no deadly effect if it is blocked (inactivated) before it reaches its cellular site of action. A molecule of chemical poison on the skin is ineffective if it is removed before penetration and absorption. Radioactive atoms obey these rules to an extent. However, radioactivity implies generation of particles or rays which can have a variety of properties in terms of: penetrability, rate of production, and interaction with biological tissue (Fig. 7). Both polonium-210 and radioactive carbon (C-14), for example, emit alpha particles and they are not a significant hazard unless consumed. Radioactive compounds that emit beta particles or gamma rays (which possess different energy levels) can be deadly from sources outside the body.

Fig. (7))

Alpha particles, emitted by polonium-210, are blocked by a simple sheet of paper; beta particles and gamma rays are much more penetrating and some gamma rays pass through a considerable thickness of lead.

The atomic weight of polonium-210 (relevant for this comparison to the molecular weights of the other poisons) is 267 grams per gram atomic weight and its lethal dose is stated to be 0.89 micrograms for an 80 kilogram person [4]. This is equivalent to 0.78 micrograms for a 70 kilogram person which is the reference weight used in comparative toxicology. Compared to botulinum, it takes 11.1 times as much to be lethal (Table 1). Because this element’s toxicity is caused by its radioactivity, its ability to poison people has some unusual characteristics. The radiation emitted is primarily alpha particles and these particles penetrate poorly, including that they are blocked by a sheet of ordinary paper and also by the skin (Fig. 7). Thus, polonium-210 must be ingested or injected to be deadly. Because radiation poisons the body by mechanisms that are quite different from the actions of the other chemicals in Table 1, the calculation of its toxicity is complicated and details are provided near the end of the chapter. Radiation poisoning with the minimum lethal dose of polonium-210 is a slow process; there is no effective antidote and death from ingesting a lethal dose of radiation is practically certain.

Table 1 The four most toxic substances, assessed by four relevant factors.

a The traditional ranking of the toxicities of poisons is by the LD50, the amount in mg of toxin per kilogram of body weight of the test subject that causes death in half the subjects. Further information is provided in the text.

b Poisons, in the opinion of the author, should be ranked based on the number of molecules that cause death. There are 6.02 x 10²³ molecules in one gram molecular weight of a substance (one gram atomic weight for polonium). Note the exponents. Further explanation is provided in the text.

c Time to death is very difficult to assess and will be discussed in the text.

d Antidotes are available for these poisons except for polonium-210. However, except under certain battlefield conditions, described in the text, there is insufficient time (because death occurs rapidly) to administer the antidote for VX agent.

REEVALUATING THE MOST TOXIC POISONS

Certainly, by the traditional meaning of poisons, botulinum toxin wins as the world’s most toxic substance. This is valid based on the amount required to kill. However, it is also possible to consider the time required to kill and whether one can be saved by an antidote (Table 1). There is, however, another way to evaluate what is the deadliest poison. Suppose we keep the dictionary definition of poison (a substance that by chemical action usually kills, injures or impairs an organism) [1], but extend our thinking to include things that are themselves alive, or at least have the ability to replicate (increase in number) when introduced into the human body. Perhaps you protest: these are called infectious agents, not poisons. Think of botulinum toxin. It is only a complex chemical produced by a germ during its growth. The poison is produced (usually), in a jar or can of improperly canned food; or in modern times, in a biological laboratory devoted to germ warfare. The toxin accumulates in the food and the disease is called a food intoxication to distinguish it from a food infection. The germ, itself is not the poison; however, the germ is the necessary agent for the poison.

If we consider the germ as the poison, we can see how this affects our calculation. Indeed, is it possible for the botulism germ to grow inside a person and poison that person? Indeed, adult intestinal, toxemia botulism and wound infection botulism are known [10]. How does this affect our evaluation? How much does one botulinum cell weigh? We also have to assume that a single cell could be infectious. My evaluation is that a single cell of Clostridium botulinum weighs approximately 1 picogram (1 x 10-12 gram), and this is in general agreement with other data [24]. The somewhat astonishing calculated result is that the lethal dose, if we assume it to arise from a single cell, is 4 x 10⁴ (40,000) times the weight of a single botulinum cell. This implies that the lethal dose of toxin is produced by the action of many botulism cells. It is also consistent with the fact that the infectious dose is not one cell, and that the lethal dose of toxin is produced over time. To calculate the time required for one cell to produce the lethal dose of toxin, we must know the rate of toxin production. Since the botulinum cells are multiplying during this interval of toxin production, the doubling time of the cells is also a relevant factor.

There is information about rate of production of botulinum toxin. In a study published in 1979 [25] in a fermenter under controlled conditions, the maximum concentration of type A botulinum produced was 6.3 x 10⁵ mouse medium lethal doses per