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The behavioural tests have been used frequently in pharmacological evaluations of drugs for several years.
These tests have only recently been recognized as an essential part of toxicity screening because of belief that behavioural responses of animals have been selected as a measure of toxicity of xenobiotics because of the following reasons:
(i) The behaviour of the animals is the end- point of functional integration of nervous system encompassing sensory, motor and cognitive aspects.
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(ii) The functional capacity of central nervous systems cannot be determined by histological and even physiological studies independent of behavioural analyses.
(iii) The morphological and chemical alterations cannot be detected at time when behaviour is profoundly disturbed.
(iv) The behavioural tests may prove to be more sensitive than other tests in tracing central nervous system toxicosis.
(v) Neurotoxicants may be used as a tool to understand the behavioural phenomena.
Criteria of Behavioural Tests:
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The main target of behavioural toxicity screening is to obtain information that can be used to protect useful life and human health.
The following criteria must be considered while selecting a behavioural response for toxicity testing:
(i) The selected behavioural pattern should be amenable to laboratory or field controlled investigations. In other words, much of the background on the behaviour should be already known.
(ii) The behavioural patterns that depend on diverse sensory and/or motor mechanisms should be examined.
(iii) The behavioural response chosen should be ecologically relevant.
(iv) The behavioural pattern should be closely examined in order to identify the effects of major biotic and abiotic variables.
(v) The behavioural test procedure should be easy to conduct in a routine way. It should be sensitive to measure even subtle changes in behaviour. The technique should be able to measure such behavioural changes in a wide range of animals in relation to a variety of toxicants.
U.S. National Academy of Science, National Research Council and Environmental Protection Agency in 1975, have jointly prepared a report for behavioural tests.
As per their report, the behavioural protocol consists of two types of behavioural tests:
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(i) Elementary behavioural tests, and
(ii) Sensitive behavioural tests.
(i) Elementary Behavioural Tests:
The elementary behavioural tests for acute toxicity studies are:
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(a) Activity Responses:
Locomotion of a standard distance in standard time, gross movements, excitement or depression in spontaneous exploratory locomotion, abnormal locomotion such as circling etc.
(b) Objective Signs:
Tremors, lacrimation, defecation, convulsions, abnormal postural changes etc.
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(c) Reflex Responses:
Hypo or hyper reflexia such as corneal and pinna righting, placing etc.
(d) Elicited Responses:
Grasping response to nose or tail pinch, handling or noise induced convulsion, orientation etc.
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(e) Body Weight Changes:
Immediate and brief weight losses and failure to gain weight as compared to normal growth curves.
(ii) Sensitive Behavioural Tests:
The sensitive behavioural tests include:
(a) Sensory Function:
Vision, audition and olfaction.
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(b) Motor Control:
Rotarod, tremor and force transducer.
(c) Complex Processes:
Operant behaviour.
(d) Phylogenetically Determined Performances:
Sex or parental behaviour, aggressive behaviour, thermoregulatory behaviour, circadian rhythms and ingestive behaviour.
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(e) Tolerance and dependence.
However, Tilson et al (1979) rectified aforementioned behavioural screening proposals and cited following measures in the primary test protocols:
(a) Motor Tests:
Motor tests include spontaneous motor activity, forelimb and hind limb grip strength and tremor.
(b) Sensory Tests:
These tests include the behavioural responses such as visual orientation/localization, auditory localization, tactile stimulation, orientation, negative geotaxis and pain.
(c) Tests for Arousal Deficits:
These tests include emergence and startle response such as, air-puff, auditory cue etc.
(d) Tests for Cognitive Functions:
These tests cover rapid escape/avoidance, conditioning test, retention and passive avoidance.
(e) Tests for Physiological and Consumatory Responses:
These tests include body- weight, rectal temperature, autonomic signs and respiration.
Some common behaviours of aquatic organisms which are used as a measure of toxicity of toxicants in aquatic systems are:
(a) Feeding behaviour,
(b) Locomotory behaviour including directed and undirected behaviour,
(c) Predatory behaviour — such as predator- prey interaction,
(d) Reproductive behaviour,
(e) Social interactions — such as territoriality, dominance and aggregation.
(f) Breathing mode.
Behavioural Testing Procedures:
Direct visual observations of behaviour — both in the laboratory and field — are tedious, time- consuming, often difficult to evaluate and quantify and sometimes too subjective. Recent technological advances — such as acoustic and radiotelemetry, advanced photography, sonar and closed-circuit television, mini-computers, microprocessors and other electronic tools have greatly enhanced our ability to collect and analyse behavioural toxicity data.
(a) Methods of Exposure:
If the goal of behavioural test is to collect information concerning the protection of human health, the selection of route of administration is important. The route of entry of toxicants should be similar to that encountered in the environment. The toxicants show maximum effect when administered via the intravenous route.
The most common routes of exposure in the environment for human beings are by inhalation, topical application and oral ingestion. Inhalation exposure is often not practical because of expensive and complex facilities required to control and monitor the exposure. Therefore, oral route is often used as a substitute.
(b) Test Animals:
Keeping in view the moral and ethical considerations, human being cannot be used as a suitable subject for behavioural toxicity screening. Therefore, animal models are generally used for behavioural toxicity studies and the data obtained from such studies are employed to extrapolate for human beings.
The behaviour is generally species specific. The primates are generally the subjects of choice. Generally, rats and mice are used as animal models. Apart from rats and mice, other animals such as rabbits, cats, dogs, squirrels and monkeys are generally used.
In aquatic toxicology, aquatic species such as fathead, minnows, perches, daphnids, mysids, sheep-head minnows etc. have been used for acute early stage or chronic toxicity studies.
But, there is little background data on their general behaviour and biology, so that their use in behavioural tests are limited. For these reasons, a wealth of information on behavioural effects of drugs/ toxicants is available on fishes like Betta spendens, Colisa fascintus, Clarius batrachus, Carassius auratus, Cyprinus carpio, Miystus species, Channna species etc.
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(c) Test Design:
Because of many unanswered questions in behavioural toxicology, it is difficult to describe a specific test design for a behavioural study.
A simple generalized design for behavioural study is given here:
i. Test animals after 15 days acclimatization may be first exposed to toxicant and then monitored in experimental situation to determine the behavioural responses of exposure.
ii. The behavioural responses should be studied following exposure to a range of 3-5 sublethal doses/concentrations of toxicants.
iii. Concurrent control groups (untreated animals) should also be tested much in the same way as toxicant exposed groups for comparative considerations.
iv. The duration of exposure is also important in behavioural testing because certain cyclic phenomena (e.g., daily, tidal and lunar) may make the animals more susceptible to environmental stimuli including toxicants.
v. The duration of exposure is dependent on test animals and nature of toxicants. For example, for animals having short life- cycles, animals selected from sea-shores and toxicants having short half-life (e.g., organophosphate insecticides), and exposure should be of short duration.
vi. Varieties of tests with different behavioural parameters should be included. The tests should be conducted in stepwise manner. The simple responses should be studied first and complex behaviours later on.
vii. Samples of test animals from overall population should be selected randomly for study. The size of each group must be large enough to apply statistical procedures viz., student ‘t’ test to determine the significance of data.
The behavioural responses are especially susceptible to endogenous and environmental variations. The responses of animals are highly variable — both with the same species and within same animal — at different times depending upon the biological status and environmental conditions. It is, therefore, important to adhere to proper experimental procedures such as large number of animals, controlled experimental environment and statistical analysis of results.
Toxicological Importance of Behavioural Tests:
Behavioural tests have been extensively used in assessing the neurotoxicity of solvents, heavy metals (especially lead, cadmium and mercury); metalloids viz., arsenic; pesticides, and certain CNS drugs like alcohol and amphetamines.
The behavioural effects are especially susceptible to endogenous and environmental variations. For example, Norton (1980) reported data to indicate a large variability of results both between animals of the same species and within the variations in the same animal at different times. It is, therefore, important to adhere to proper experimental procedures, e.g., sufficiently large number of animals rigorously controlled experimental environment and statistical analysis of results.
To facilitate proper interpretation of behavioural effects of neurotoxic agents, Tilson et al (1979) recommended validation with agents that are known to produce specific effects. For example, they recommended the use of triethyltin, methyl mercury and inorganic lead in studies involving mixed central and peripheral neuropathies.
The significance of a neurotoxic effect depends on its reversibility. In general, irreversible effects are more serious than reversible ones. The site of the effect also plays an important role. There are areas in the nervous system more critical to physiologic function than others. Flarov (1974) pointed out that the toxicological nature of the surrounding can be assessed with the study of behavioural responses of the animals.
