Test design and test species
Considering the large number of chemicals to be tested it is necessary to develop high throughput, rapid assays that can handle many chemicals simultaneously. Toxicity assessment is the determination of the potential to act as a poison, the conditions under which this potential will be realized and the characterization of its action. Risk assessment is a quantitative assessment of the probability of deleterious effects under given exposure conditions.
Based on physicochemical properties of the test compound, different test designs may be used when studying the aquatic environment:
- Static: the test solution is not replaced and no flow occurs, commonly used for stable and non-volatile compounds.
- Semi-static: the test solution is replaced at regular intervals.
- Flow-through: a constant flow rate and the concentration rate of the test solution is maintained during the whole toxicity test.
Studying the effects of chemicals in the environment requires the use of indicator species that may be representative of a particular trophic level organization or that play an important role in the maintenance of the ecosystem sustainability.
When selecting a test species, different factors may be taken into consideration: laboratory capacity for its maintenance, availability, ecological or commercial relevance, easy handling, sensitivity, database of background information, etc. Thus, the results of both acute and chronic tests should not be considered absolute as contaminants may elicit toxic effects in some species but not in others, even from the same taxa.
The OECD test guidelines provide the full details for acute, short-term and long-term tests for different test species of freshwater, marine water and soil. EPA has also different guidelines for testing chemicals.
For more detailed information consult:
OECD Guidelines: http://www.oecd.org/env/ehs/testing/TG_List_EN_Jul_2013.pdf
EPA Guidelines: http://www.epa.gov/ocspp/pubs/frs/home/guidelin.htm
These tests are frequently required for different regulatory agencies purposes:
- toxicity assessment of new chemicals being introduced into the market;
- the granting of permits and discharge consents;
- products classification and labeling;
- environmental risk assessment of anthropic activities;
- establishment of protective standards for the environment;
- legislative and judicial.
3.1 Acute toxicity testing
Acute toxicity can be defined as toxicity elicited immediately following a short time period of exposure to a single dose of a chemical. Considering this definition, two components comprise acute toxicity: acute exposure and acute effect. In fact chronic exposure may result in later acute effects at specific life-stages. For example, chronic exposure to a persistent, lipophilic chemical may result in sequestration of significant levels of the chemical in adipose tissue of the organisms without causing adverse effects. However, later mobilization of fatty stores and release into the bloodstream during the reproductive phase may induce mortality. Conversely, acute exposure at a specific development stage may result in later chronic toxicity, namely reproductive abnormalities once the organism has attained reproductive maturity.
Acute toxicity tests are undertaken to investigate the potential adverse effects arising from exposure to a given chemical over a short period of time. This approach can help identifying the mode of action of a compound and may provide information on dose-response and target-organ toxicity. From the different types of acute effects, lethality (or completely immobilization) is the most accepted and used endpoint. In fact, in some test species it is relatively easy to see whether or not they are dead. However, with other smaller test species it may be more difficult and immobilization is the endpoint used. Usually, immobilization measure is an EC value, the effect concentration that produces immobilization in a specific percentage of the exposed population. Properly conducted, acute toxicity tests provide also information about nonlethal effects (morphological effects, organs affected, behaviour...), mode of action and metabolic pathway of detoxification. Moreover, these tests are a useful first approximation of the risk assessment of new or emergent chemicals and can be used to support the subsequent subchronic studies design.
When death is the measured endpoint, the term EC50 should be referred as LD50, and describes the acute oral or dermal median lethal dose, i.e., the single lethal dose which will induce 50%-mortality of the test population in a specific period of time, usually expressed in milligram or gram of the test compound per kilogram of animal weight (mg kg-1). It is widely recognized that the LD50 is of marginal value as a measure of hazard, although this information can be used as a rough measure of relative toxicity and provide some insight into any systemic toxicity arising from that particular exposure route (see Figure 1).
The lower the LD50 value the more toxic is the test compound. However both EC50/LD50 are not a biological constant and many factors may influence toxicity and thus may alter the estimation of this measure in any particular study. The value obtained in acute tests will depend on the selected test species, age and weight, type of feed, caging, pretrial fasting time, method of administration, volume and type of suspension medium, duration... Moreover, toxicity tests can also be influenced by abiotic factors (temperature, photoperiod, pH, O2, test medium, solvent) or biotic factors (genetic variances, test species health). The following LC50 categories are used by the Organization for Economic Cooperation and Development (OECD):
- Less than 5 mg/kg: very toxic
- More than 5 mg/kg and less than 50 mg/kg: toxic
- More than 50 mg/kg and less than 500 mg/kg: harmful
- More than 500 mg/kg: no label required
Normal distribution
Only few animals responded to the
lowest dose and the highest dose. Larger numbers of animals responded
to intermediate doses and the maximum response and the maximum
frequency of response occurred in the middle portion of the dose range.
The reason for this normal distribution is that there are differences
in susceptibility to chemicals among individuals, known as biological
variation.
3.2 Short-term toxicity testing
Subacute or subchronic studies are designed to investigate the adverse effects resulting from repeated exposures to lower chemical levels over part of the organism life cycle. Short-term and long-term studies do not use lethality as the principal endpoint and attempt to find the lowest observable effect concentration (LOEC) and the no observable effect concentration (NOEC) for the selected endpoints. Both LOEC and NOEC for a given chemical vary with the route and duration of exposure and the nature of the selected adverse effect. The NOEC for the most sensitive test species and the most sensitive indicator of toxicity is usually employed for regulatory purposes.
Short-term studies can help to record
potential target-organ effects. Moreover for chemicals with very low
acute effects, these studies allow to determine whether there is any
possible accumulative effect that may interfere with critical body
functions and produce some "delayed toxicity". Appropriate dose level
for longer-term studies can be determined considering the results from
such tests.
3.3 Chronic toxicity testing
Chronic toxicity is characterized by prolonged exposure over significant part of the life cycle of the test species. Frequently, chronic exposure results in sublethal effects elicited through mechanisms that are distinct from those that cause acute toxicity. Hence, from a chronic toxicity point of view, acute toxicity testing may be seen as an initial approach and is a gross indicator for chronic toxicity testing, whereby it is not recommended to predict the potential adverse health effects arising from chronic exposure to a chemical based on acute toxicity test results. Different sublethal endpoints are generally associated to chronic exposure including reproductive, immune, endocrine and developmental function. For this reason, once selected the endpoints to be assessed in a chronic toxicity test, different conclusions may emerge, therefore the choice of the endpoints is of extremely importance.
Theoretically, all chemicals elicit acute toxicity at a sufficiently high dose, while some chemicals do not elicit chronic exposures. Raising the dose of a chemical does not ensure that chronic toxicity will ultimately occur. Moreover, since chronic toxicity typically occurs at dosages below those that elicit acute toxicity, toxicity observed at higher dosage may be a consequence of acute rather than chronic toxicity.
Different mechanisms may be involved in the toxic effects resulting from single or repeated exposures to large or small amounts of the same chemical. Interactions among abiotic and biotic components of the environment may contribute to the chronic toxicity of the chemicals, while such interactions may not occur in laboratory conditions. Also, since chronic toxicity studies take place over the whole or most of the lifetime of the test species, the aging process itself as well related spontaneous diseases may influence the final outcome.
