Measuring toxicity
Toxicity can be measured by the effect the substance has on an organism, a tissue or a cell. We know that individuals will respond differently to the same dose of a substance because of a number of factors including their gender, age and body weight. Therefore a population-level measure of toxicity is often used. The probability of an outcome for a population is then related to a given individual in a population.
LD50
Paul McNabb explains LD50 – a lethal dose given to animals to determine the toxicity of a toxin. The graph in the clip shows the cumulative population response to increasing doses of a toxin.
Lethal dose (LD50)
One such population-level measure is the median lethal dose, LD50 (lethal dose, 50%). This is defined as the dose required to kill half the members of a specific animal population when entering the animal’s body by a particular route. LD50 is a general indicator of a substance’s toxicity within a short space of time. It is a measure of acute toxicity.
Most users of a substance will want to know the toxicity of that substance. The information for an LD50 must include the substance, the route of entry and the animal species. For example, table salt has an oral LD50 of 3 gm/kg in rats. Paracetamol has an oral LD50 of 1.944 gm/kg in rats.
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 2,000 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, so it is important to understand how low doses compare to one another. Parts per million (ppm), parts per billion (ppb) and parts per trillion (ppt) are the most commonly used terms to describe very small amounts of substances.
A ppm of a chemical in water means that, in a million units of water, there would only be one unit of the chemical. They are measures of concentration – the amount of one substance in a larger amount of another substance. Scientists often use these measurements when measuring a toxic chemical in a lake or toxins in the air such as greenhouse gases.
Mouse bioassay for shellfish
The standard method to test for toxins and toxicity levels in shellfish has been the mouse bioassay. This consists of injecting extracts of shellfish into mice to determine the presence of substances toxic to humans. After injection, 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.
Mouse bioassays
Paul McNabb of the Cawthron Institute in Nelson explains what a mouse bioassay is. He describes the shortcomings of mouse bioassays and explains the need to develop other ways to detect toxins.
The problem with this bioassay is that there is no indication which toxins are present in the shellfish or the level of toxicity. Sometimes, the mice die for reasons other than toxins in the shellfish, and an industry is shut down when in fact the shellfish are fine. Also, some people object to using animals in this way.
Using liquid chromatography-mass spectrometry
Monitoring shellfish safety
Paul McNabb of the Cawthron Institute in Nelson describes the system they use to monitor shellfish safety. He explains how toxins are detected in shellfish using liquid chromatography-mass spectrometry (LC-MS). Cawthron has become a world leader in developing such methods to detect toxins in shellfish. These methods are of international interest because they replace the need to use mice to detect toxins.
Jargon alert: Liquid chromatography-mass spectrometry (LC-MS) is a process using specialised equipment to detect toxins in substances. The process can accurately determine the molecular mass of different toxins, giving scientists the clue to work out what toxin it is.
In 2000, the Cawthron Institute in Nelson designed a way to test for toxicity levels in shellfish using liquid chromatography-mass spectrometry (LC-MS). Using LC-MS, the actual toxin can be identified (unless it’s a new toxin that hasn’t been identified before) as well as the level of its toxicity. This test means that toxins can be detected and monitored using chemical procedures instead of mice. As a result of this method, mice would only be needed to detect a new toxin – which would be rare. Legislation has been passed that, from 2012, all countries in the European Union use LC-MS to detect known toxins and measure toxicity levels in shellfish.
Scientist Paul McNabb using LC-MS
Scientist Paul McNabb testing for toxins using liquid chromatography-mass spectrometry.
Nature of science
Science knowledge and methodology change over time. It is only recently that chemical testing for toxins and toxicity levels has developed in favour of mouse testing. The Cawthron Institute in Nelson has been instrumental in developing a method using liquid chromatography-mass spectrometry (LC-MS) to monitor toxins in seafood.
Activity ideas
Use these activites with your students to explore measuring toxicity further:
Detecting toxins – in this activity, students explore the processes scientists used to analyse and identify the toxic substance responsible for dog deaths on Auckland beaches.
Exploring small doses – students explore small doses in the order of parts per million. They dilute food colouring to help them understand how small one part per million actually is.
Ethics, mice and toxins – in this activity, students consider the use of mice for bioassays and in establishing the lethal dose when researching/testing toxins. They consider the rights and responsibilities of all those affected by these methods.
Having a go at chromatography – during this hands-on activity, students use paper chromatography to separate the dye pigments in coloured sweets. This introduces students to chromatography and helps them to understand how scientists find toxins in substances.
