Experimental methods of

measuring toxins

Presented by,
SRUTHY G S
Roll No 16,
1st MTECH,
UKFCET
● Toxicity - effect of a substance on an

organism, a tissue or a cell.

● Factors - gender, age and body weight.

● Population-level measure of toxicity is often

used.
Lethal dose (LD50 )


! LD50 is a general indicator of a substance’s

toxicity within a short space of time.

! It is a measure of acute toxicity.
! LD50 - dose required to kill half the members
of a specific animal population when entering
the animal’s body by a particular route.
! The information for an LD50 must include the

substance, the route of entry and the animal

species.

! Table salt has an oral LD50 of 3 gm/kg in rats.

! Paracetamol has an oral LD50 of 1.944 gm/kg

in rats.
 Lethal Concentration LC50 

! Concentration of a chemical in air or concentration
of a chemical in water (environmental studies).
! Testing of Chemicals - traditional experiment -
groups of animals exposed to a concentration for a
set period of time (usually 4 hours). The animals
are clinically observed for up to 14 days.
! The concentrations of the chemical in air that kills
50% of the test animals during the observation
period is the LC50 value.
Fixed-dose procedure


! In 1992, the fixed-dose procedure (FDP) was proposed as

an alternative test to LD50.

! It uses fewer animals, and there is less pain and suffering.

! In this procedure, the test substance is given at one of four
fixed-dose levels (5, 50, 500 and 2000 milligrams per
kilogram) to five male and five female rats.
! When a dose produces clear signs of toxicity but no death
is identified, the chemical is then classified at that level.
Parts per million


! Some chemicals can cause toxicity at very

low doses.
! ppm, ppb & ppt - most commonly used
terms to describe very small amounts of
substances.
! Measuring a toxic chemical in a lake or air
(greenhouse gases).
Mouse bioassay for shellfish


! Standard method to test for toxins and toxicity levels in shellfish.

! Injecting extracts of shellfish into mice.

! If two out of three mice die within 24 hours, the sale of that
particular shellfish is prohibited.

! Tests are then conducted systematically at least once a week

during the period of risk for toxins in that particular shellfish.

! The ban is only lifted after two consecutive tests turn up negative.
! There is no indication which toxins are present in the shellfish or
the level of toxicity.
Liquid chromatography-mass spectrometry


! Cawthron Institute in Nelson (2000) designed a way

to test for toxicity levels in shellfish using LC-MS.
! Using this technique, the actual toxin can be
identified as well as the level of its toxicity.
! Toxins can be detected and monitored using
chemical procedures instead of mice.
! Mice would only be needed to detect a new toxin –
which would be rare.
High-performance liquid
chromatography (HPLC)
❑ Technique to separate, identify, and quantify each
component in a mixture.
❑ Pressurised liquid solvent containing the sample mixture is
passed through a column filled with a solid adsorbent
material.
❑ Each component in the sample interacts slightly
differently with the adsorbent material, causing different
flow rates for the different components and leading to the
separation of the components as they flow out the
column.
! The sampler brings the sample mixture into the mobile
phase stream which carries it into the column.
! The pumps deliver the desired flow and composition of the
mobile phase through the column.
! The detector generates a signal proportional to the amount
of sample component emerging from the column, hence
allowing for quantitative analysis of the sample components.
! A digital microprocessor and user software control the
HPLC instrument and provide data analysis.
! Most HPLC instruments also have a column oven that
allows for adjusting the temperature at which the separation
is performed.
Gas chromatography (GC)
" GC is a common type of chromatography used
for separating and analyzing compounds that
can be vaporized without decomposition.
! Typical uses of GC include testing the purity
of a particular substance, or separating the
different components of a mixture, help in
identifying a compound.
● Mobile phase - carrier gas, usually an inert gas such
as helium or an unreactive gas such as nitrogen.

● Stationary phase - microscopic layer of liquid or

polymer supported on inert solid, inside a piece of
glass or metal tubing called a column.
! The gaseous compounds being analyzed interact with
the walls of the column, which is coated with a
stationary phase.
! Each compound elute at a different time, known as the
retention time of the compound.