Wednesday December 19, 2007 - Vol. VI Issue 8[Download PDF for Printing]
Biotoxins are toxic
chemicals produced in nature by living things.
The living things may be bacteria in the animal gut, certain plants
which produce potent toxins such as ricin, spore-forming bacteria producing anthrax
toxin, red algae producing saxitoxin, certain animals and insects producing
toxic venom, etc.; the list is long. A
terrorist or a nation state can potentially harvest and purify a toxin to be
used against an enemy, perhaps by poisoning a water supply or by releasing a
dust/aerosol in an airplane passenger compartment or inserting a fine powder in
a letter. However, most poisonings
occur accidentally when people eat contaminated food or from contact with
Two of the natural
biotoxins are classified as “Schedule 1 Chemical Warfare Agents” under the
United Nations agreement on biological weapons, e.g. the Chemical Weapons
Convention in 1993 and earlier agreements.
These are saxitoxin and ricin.
These are not the only potent biotoxins. We will look at saxitoxin and ricin in this newsletter.
On June 12, 2002,
President George W. Bush signed into law the Public Health and Safety Act of
2002 (PL 107-188), which requires that the Department of Health and Human
Services maintain a list of biological agents and toxins, which pose a severe
treat to public safety. The list of
biotoxins, as it appears in the August 23, 2002 Federal Resister, (see also 42
CFR Part 72, Appendix A) is as follows:
perfringens epsilon toxin
Saxitoxin is a potent
toxin which if ingested (or injected) works as a selective sodium channel
blocker in the nervous system resulting in rapid, incurable paralysis and
death. A dose of 0.2 milligram is fatal
to an average-weight human being.
Saxitoxin is produced by “red-tide” algae, one of the algae species “Alexandrium
This algae species is
found in marine waters throughout the world.
The usual mode in which humans ingest this toxin is from eating
contaminated shellfish (clams, mussels, oysters), which in turn have ingested
(“filtered”) the algae at some previous time in their life, even though the
“red tide” may have past and the waters look clean. Cooking the shellfish does not destroy the toxin.
Red tide off California coast, Noctiluca sp.,
photo by PJS Franks, from Woods Hole Oceanographic Institution photo gallery
Red tide off New Zealand coast, photo by M. Godfrey, from
NIWA Science website
Another photo of “red
tide” is in the May 2008 month of “Toxic by Nature” calendar issued jointly by
Drug Discovery & Development and Gilson Inc. More photos are at http://www.whoi.edu/redtide/rtphotos/rtphotos.html
Alexandrium sp. cyst, from Woods
Hole Oceanographic Institution
Chemist’s representation of Saxitoxin., photo from
University of Sussex at Brighton, UK.
In the 1950’s the CIA
began experimenting with the biotoxin.
Saxitoxin was produced and stockpiled in the United States as part of a
chemical weapons program at Fort Detrick, MD using the code name Agent TZ. It is not clear what the CIA involvement
was, but Internet sources suggest development of dart guns and manufacture of
suicide tables for their agents if captured.
Agent TZ is soluble in water and can be ingested or inhaled. If an intended victim is jabbed with a dart
tip, death occurs quickly.
President Nixon in 1969
ordered the CIA to destroy its entire stock collected over the years and not
engage in additional covert research with saxitoxin (see http://www.aarclibrary.org/publib/church/reports/vol1/pdf/ChurchV1_8_Exhibits.pdf
for details). In 1975, the CIA director
revealed to Congress that they still possessed 10 grams of saxitoxin, in
violation of the 1969 presidential order, which was then evenly distributed to
scientists and medical researchers under the auspices of the National Institute
of Health. Saxitoxin is useful in the
study of nerve disorders because it selectively blocks only the sodium channels
but does not affect potassium or calcium channels or the chloride ion count or
In 1977, a paper was
published in the open literature (Kishi et al, Journal of the American
, 1977, vol 99, page 2818) on the chemical synthesis of
saxitoxin. The synthesis was modified
in another paper published in 1984 (Jacobi et al., Journal of the American
, 1984, vol 106, page 5594) to yield an optically pure
product mimicking what is produced in nature.
The Jacobi synthesis was carried at Wesleyan University in Connecticut.
Saxitoxin is said to be
1000 times more toxic than the nerve gas Sarin. The 0.2 milligram fatal dose for an average weight human is based
on LD50 mice studies (the lethal dose resulting in the death of 50% of the test
animals in 24 hours). The LD50 for mice
is 8 microgram/kg, but humans are 4 times more sensitive to than mice to oral
doses of saxitoxin because the human digestive track is longer and more
saxitoxin is absorbed. The lethal oral
dose for humans is 1 to 4 mg depending upon age and physical condition. Children are apparently more sensitive than
is not the only algae species that produce
biotoxins. The causative algal species
mostly belong in the general classification of dinoflagellates, which include Alexandrium
tamarense, Alexandrium circinalis
(a fresh water species), A. minutum,
A. ostenfeldi, A. catenella (Pacific coast), A. fundyense
U.S. and Canadian coast), Gymnodinium catenatum, Karenia brevis
Gulf of Mexico), various Dinophysis sp.
(Europe, Asia, Japan), and Pyrodinium
(Philippines and elsewhere).
The affected waters may be red, grey, brown or other colors, e.g. “brown
tide”, or the waters may not be noticeably colored. At least 12 different biotoxins are produced from various
dinoflagellate species and have also been studied. The biotoxins fall under the general chemical classification of
tetrahydropurines, of which saxitoxin is the first studied and best known. There are also other biotoxins besides
tetrahydropurines produced from algae blooms.
No human deaths have been
directly attributed to direct contact to “red tide” algae, but people may
experience respiratory irritation when winds blow aerosol onshore from waters
containing “red tide” algae. Skin
irritation and burning is possible when swimming in areas affected by “red
tide” algae. Deaths of people and
animals (including birds, fish, turtles, etc.) occur because of consumption of
mollusks and other creatures, which feed on the algae; the mollusks concentrate
the biotoxin in their flesh as the result of their filter feeding. The biotoxins have resulted in deaths of
fish, marine animals, sea turtles, and birds.
Generally, fish and shrimp caught in “red tide” waters are safe to eat
at least in smaller quantities (check with the local health
department/authorities to be sure).
However, the 1987 deaths of 14 humpback whales off Massachusetts in Cape
Cod Bay were traced to the whales eating mackerel, which in turn had eaten
smaller fish and zooplankton, which had consumed large amounts of Alexandrium
The saxitoxin was
concentrated in the food chain. The
1987 death of 700 bluenose dolphins was also similarly traced to
bioaccumulation of neurotoxins in their fish diet.
The method used by the
CIA at the chemical weapons program at Fort Detrick, MD involved harvesting a
certain species of clam grown in “red tide” waters, and extracting the
saxitoxin from the clams.
Four types of human
shellfish poisoning have been identified:
Table 1. Example Biotoxins from Eating Shellfish
Alexandrium sp. and certain other
0.2 mg for average weight human. 100% recovery from non-fatal doses without
(There are several toxins)
Gymnodinium breve (Gulf
of Mexico and Caribbean), Karenia sp. and other dinoflagellites
Deaths rare, but patients may suffer dementia. Rat and human studies show brain damage.
Nitzchi pungens (a diatom) implemented
in 1987 Canadian outbreak. Also Pseudonitzschia
sp. (east and west coast of U.S. and Gulf of Mexico)
Rat LD50 (injected subcutaneously) 0.33 mg/kg. In a 1987 Canadian outbreak, of 145
patients studied, the three patients who died were elderly and showed severe
damage to the hippocampus and other parts of the brain. For 10 cases, from mussel analysis, a 4.2
mg/kg oral dose of toxin resulted in severe neurological effects.
Dinophysis sp.; also Prorocentrum
Deaths rare, but elderly patients experience memory
loss. Large dose of okadaic acid from
contaminated mussels results in permanent neurological sequelae. LC50 (mice, injected subcutaneously) 0.192
: The onset
of symptoms occurs within 5 to 30 minutes after ingestion of contaminated
shellfish. Initially there is a slight
tingling progressing to numbness around the mouth, neck, and face. In severe cases, these symptoms spread to
the extremities in coordination and respiratory difficulty. There may be difficulty swallowing, sense of
throat constriction, speech incoherence, headache, dizziness, nausea, possible
vomiting, and reduced eye pupil size.
In severe cases, within 2 to 12 hours, there is complete paralysis and
death from respiratory failure in the absence of ventilatory support. Without artificial respiration, up to 75% of
severely affected patients, die within 12 hours. Gastric lavage and administration of activated charcoal or dilute
bicarbonate solution is also recommended.
Benzedrine is effective in aiding artificial respiration. The PSP symptoms mimic acute
organophosphorous pesticide or nerve gas Sarin poisoning, but the use of
anticholinesterase agents is not recommended for PSP patients and may do more
harm than good. After about 12 hours,
if death has not occurred, patients start to recover gradually and are without
residual symptoms after a few days.
Cooking the shellfish
does not destroy the biotoxin.
It is imperative to
obtain samples of the shellfish tissue and their source so that diagnosis can
be made. The mouse bioassay of the food
extract is the usual diagnostic method.
Radioimmunoassay and indirect enzyme-linked immunoabsorbent assay have
been developed for saxitoxin, but not all of the biotoxins, which cause PSP. HPLC analysis methods have been developed
for all of the PSP toxins.
: There are
several different kinds of brevetoxin (given names such as brevetoxin A,
brevetoxin B, and other names).
Brevetoxins act by disrupting the flow of sodium ions in nerve
cells. They bind to the sites near the
nerve cells allowing an unchecked flow of sodium ions in and out of the cells
(in contrast to saxitoxin which binds different sites and blocks sodium ions
from passing through the sodium channel).
Brevetoxin poisoning rarely results in death, and patients recover
within a few days, but permanent nerve damage and dementia can occur. Symptoms include false temperature
sensations, muscular aches, dizziness, and anxiety. These are usually accompanied by vomiting, diarrhea, and
abdominal pain. Cooking the shellfish
does not destroy the biotoxin.
: Much of
what is known is the result of investigation of 153 cases of acute
intoxication, which were reported in 1987 as the result of individuals eating
mussels harvested from Prince Edward Island, Canada. The onset of symptoms varied between 15 minutes to 38 hours after
eating. Many of the patients were
elderly and suffered gastrointestinal distress and also neurological effects
that included memory loss and dementia.
Younger patients seemed to have more digestive problems. Twenty-three (23) patients required
intensive care because of seizures, coma, profuse respiratory secretions, or
unstable blood pressure. Three patients
died. The cause of death was coma,
encephalopathy, convulsions, and cardiovascular collapse. In the mouse bioassay, mice were given
intraperitioneal injections of extracts from the mussels. The mice soon exhibited an uncontrolled
scratching of both shoulders with their hind legs, and most of the mice died
within 3.5 hours after injection.
Further research demonstrated that the toxin was domoic acid, an amino
acid with a molecular weight of 311.
The involved mussels contained between 31 and 128 mg of domoic acid per
100 grams of mussel tissue. The toxin
was produced by an algae (diatom) called Nitzschi pungen
, which in turn
was ingested by the mussels during their normal filter feeding. In ten patients studied, there was a clear
dose response between amount of domoic acid in mussels consumed and
neurological effects. Further tests
using monkeys using mussel extracts gave similar dose response to the ten
patients studied. Later research
demonstrated the presence of domoic acid in anchovies in California, and razor
clams and crabs off British Columbia.
Domoic acid caused the death of large numbers of Brown pelicans and
cormorants in 1991 and over 400 sea lions in 1998, both incidents off the
California coast. The anchovies in
California had been eating the diatom, Pseudonitzschia australis
produced the domoic acid; the seabirds that died in 1991 in turn ate the
In the brain, domoic acid
damages the hippocampus and amygdaloid nucleus. It damages neurons by activating AMPAS and kainatic receptor
causing an influx of calcium. The uncontrolled
increase of calcium causes the nerve cells to degenerate. When the hippocampus is damaged, long-term
memory loss occurs. There is no
antidote for domoic acid.
Cooking the mussels does
not destroy domoic acid.
high-dose exposure to okadaic acid, which accumulates in certain clams and some
crabs, is the underlying cause of human DSP.
Symptoms occur between 30 minutes to 12 hours after eating contaminated
shellfish. Symptoms include diarrhea,
nausea, vomiting, abdominal cramps, and chills. Gastrointestinal bleeding and hiccups have occurred. Full recovery is usually experienced within
a few days, but death can occur especially in elderly patients due to coma,
seizures, and/or pulmonary edema. DSP
mostly occurs in Europe, Japan, and South America. Over 5000 people experienced DSP in Spain in 1981. Okadaic acid (and/or its esters) primarily
affects the cells lining the intestinal gut; the exact mechanism is not clear,
but the chemical is thought to stimulate phosphorylation that controls sodium
secretion by intestinal cells and also affects calcium ion transport in general
across cell membranes in the body.
Patients over 50 years old may also experience neurological effects
including memory loss, severe anterograde amnesia, and motor or sensorimotor
Cooking the shellfish
does not destroy the biotoxin.
: Most of
the information presented here on shellfish poisoning came from (1) a paper
prepared by Charles Baier, University of Idaho, December 2000, “Red Tide and
Shellfish Poisoning: Toxic Products of
Marine Algae” which is available at
http://www.agls.uidaho.edu/etox/resources/case_studies/REDTIDE2.PDF and (2)
from the United States National Library of Medicine TOXNET at
Ricin is a toxin
extracted from the castor bean. The
(adult) human fatal dose for ricin is 0.2 milligrams if ingested, and about
0.05 milligrams if injected. It is also
fatal by inhalation. Children are more
sensitive than adults. Ricin acts by
inhabitation of protein synthesis.
Symptoms appear within a few hours after ingestion. The initial symptoms are abdominal pain,
vomiting, and diarrhea (sometimes bloody).
Within several days there is severe dehydration, urine decrease, and
drop in blood pressure. If death has
not occurred within five days, the victim usually recovers but suffers
long-term organ damage. A castor bean
might pass through the gut unscratched if its surface is not broken (as in
chewing), but eight chewed castor beans contain enough ricin to kill an adult
and one chewed castor bean can kill a child.
There is no specific antidote.
The castor bean plant (Ricinus
) originally is a native of tropical Africa. It is now grown worldwide, sometimes as an
ornamental in gardens or as a houseplant, and also grows as a weed in tropical
and semitropical areas. The growing of
the plant is not illegal, but in the United States (see Title 18, United States
Code, part 175) a person caught manufacturing or possessing ricin may be
sentenced up to 10 years, or life for possession with intent to use as a weapon
or to provide to a foreign government.
Photographs and descriptions of the castor bean plant and
its seed are readily available from the Internet making recognition easy. In tropical areas the plant can grow to
almost tree-like size with up to 20-inch leaves. The leaves are usually eight-lobed as shown in the photograph
with slightly serrated edges and prominent central veins.
Univ. of Illinois
Source: NPR website, photo Ketzel Levine
Wikipedia as used in USA Today website
Ornamental varieties grown in gardens may be purplish with
narrower leaf lobes and smaller like the photo at the left. The flowers may be green or they may be pink
or red as shown above (center). The soft
flower capsules mature into soft spiked seed capsules each containing several
mottled castor beans about 0.4 inch long.
More than one million tons of castor beans are processed
each year to make useful products such as castor oil. Besides its use as a laxative, castor oil when dehydrated is used
extensively in paints and varnishes, and is said to have qualities superior to
linseed oil. Its water resistant
qualities make it ideal for coating fabrics and for protective coverings. Other use is in the production of sebacic
acid, which is the basic ingredient in the production of nylon. Castor oil has an ability to cling to very
hot moving parts making it an outstanding lubricant for high performance
Ricin does not partition into the castor oil product because
the ricin is water-soluble. The ricin
stays behind in the seed-pulp left over in oil extraction. The residual seed pulp (also called oil
cake, or pomace) may contain up to 5% ricin.
The seed cake does make an excellent fertilizer. There are also numerous documented cases of
ricin poisoning of horses or other livestock when they accidentally ate castor
bean seeds or meal. Castor bean meal
mixed in with bait is highly toxic to rodents and insects.
Procedures for extraction of ricin from castor seeds or seed
pulp are said to be available on the Internet, and this author has not
attempted to research these procedures.
The trick is to separate the ricin from the other complex proteins,
which form part of the castor bean, and a simple extraction using water (or lye
or acetone) plus filtration does not accomplish this. Consequently homegrown recipes are likely to produce a mixture of
plant proteins, and the ricin produced might even be partly denatured
(inactivated). Another toxin from castor
beans called “Ricinus communis agglutinin” is a powerful hemagglutinin (clumps
red blood cells) but does not penetrate the intestinal wall. U.S. Patent 3060165 (granted in 1962,
withdrawn in 2004) may be the basis of some procedures. As mentioned before, manufacture of ricin is
Mass spectrometry can be used as a method of forensic
identification of ricin including ricin in castor bean extracts. The procedure is described by Sten-Åke
Fredriksson et al, “Forensic Identification of Neat Ricin and of Ricin from
Crude Castor Bean Extracts by Mass Spectrometry”, 2005, Anal. Chem.
(An American Chemical Society publication), 77
(6), pp 1545-1555.
Ricin is toxic by inhalation, injection, and ingestion. It is also absorbed by mucous membranes and
the eyes. There is no known antidote.
There have been attempts to weaponize ricin by governments,
but because the material is difficult to deploy and is easily deactivated,
historically it has not been used as a mass casualty weapon. Historically, specific individuals have been
targeted. The potential for use as a
mass casualty weapon is there. Al Qaeda
has reportedly experimented with ricin (according to an interview with German
magazine Der Spiegel, cited in wikipedia).
There are also a few incidents where powdered ricin has been sent in
first class letters in the United States, including one sent to U.S. Senate
Majority Leader Bill Frist in November 2003.