Assessment of Herbicide Toxicity and Herbicide Residue by Bioassay Method
1. Introduction:
In spite of rapid developments in analytical methods. Bioassay remains a major tool for qualitative and quantitative determination of herbicides. A major advantage of the bioassay is the assurance that the phototoxic activity of the herbicide molecule is being measured. A secondary advantage is that it is not generally necessary to exact the herbicides form the substrate. Bioassay procedures are usually more economical, less difficult to perform and do not require as much expensive equipments as in chemical analytical methods.
There are various procedures for conducting the bioassay studies. The choice of a particular procedure will depends upon the herbicide and its phytotoxicity to bioassay species. At the same time the bioassay must also satisfy certain requirements. In an effective bioassay,, the indicator species should be sufficiently sensitive to detect even small amounts of herbicides and should express the response with increasing herbicide concentrations. The rest should be rapid and the time required for the conduct should be minimal.
2. Indicator Species:
Various organisms can serve as an indicator for a given herbicide and conversely, many herbicides though belong o different chemical groups, can be tested by the same organism. Microorganisms also have been used in some bioassays (Hess, 1980). The indicator species should be sensitive to minute amounts of the chemical and should respond by clear, easily observable and measurable symptoms. Herbicide bioassays are usually conducted with sensitive plant species referred as indicator / test species. Cucumber, sorghum mustard soybean, oat and minor millets are some commonly used indicator plants in herbicide bioassays.
3. Assessment Parameter:
The response of indicator plants to herbicides can be evaluated in various ways.
A) Germination Tests:
Many herbicides strongly inhibit the germination of sensitive species but few tests have been based simply on germination sumbers. Sublethal concentrations frequently produce an inhibition of radical of radical or shoot elongation which is dose related in a range sufficiently large to allow valid measurements. Typically, the root or shoot elongation is observed after a period of 24 – 96 hours. Cucumber, sorghum and oat are the main species used in germination tests butthey are not sensitive to photosynthetic inhibitors (Kratky and Warren 1971).
B) Assessment of Plants:
Determination of dry weight is most common assessment used in bioassay. Generally, the tops only are weighed since the separation of roots from soil is laborious. Observation based on fresh weight and dry weights generally give good estimations.
For many photosynthesis inhibitors shoot growth is reduced before the appearance of the injury symptoms. Measurement of plant height or leaf length provide an assessment of herbicidal activity which may replace weighing. While leaf length is easily measured on monocots, other measurement may be considered on the other species. For instance, the petiole of the first trifoliate leaf of legumes gives dependable estimation.
Observation of plant height generally give similar results. However, with herbicides inducing deformation in the shoot, the plant height values should be used with care, Koren et al. (1968) found that with thiocarbamates the height reduction was greater than weight reduction. Recording both the parameters is useful in experiments including different type of herbicides (Horowitz and Blumenfeld, 1973). Visual estimation of relative development or of injury intensity is often use (Scifres et al. 1972).
C) Physiological and Morphological Effects:
The physiological, bio-chemical and structural modifications of plants induced by herbicides can be used for bioassay assessment. Reduction of photosynthetic activity by photosynthesis inhibitors has been measured or leaf discus (De Silva et al,1976). Chlorosis may be evaluated by determining chlorophyll content through spectrophotometer (Horowitz 1970). Duffy (1972) estimation the decrease in viability of root tissue with a modified tetrazoltum test.
D) Symptoms:
Symptoms which are typical of a certain group of herbicides or of a given compound can be used for qualitative assay and if the intensity of symptoms are dose related, it can also be used for quantitative determinations. Ex. Epinasty of cotton have been used to measure the effect of 2, 4 – D.
4. Application of Bioassay:
Bioassay procedures have been used to investigate many practical aspects of herbicide behaviors in the environment. The different methods and approaches in herbicide research based on bioassay are outlined below:
A) Soil Effects:
The effect of soil factors on herbicidal activity may be determined by bioassay and correlate phytotoxicity with relevant soil physical and chemical characteristics.
B) Dissipation from Soil Surface:
Bioassays have been used in experiments dealing with the process of dissipation from soil surface especially volatilization and photodecomposition.
In volatility studies, a source at a sink for herbicide vapors may be set up in a closed container or treated soil may be exposed to an air flow. The bioassay, measures the concentration of herbicide remaining in the soil and from this the amount of herbicide volatilized is computed (Talbert et al. 1971).
Photodecomposition can be induced experimentally by irradiation herbicides in soil solution or on artificial surfaces or on soil with UV lamps or natural sunlight. The herbicidal activity is measured by bioassay after certain period of exposure and compared with non ex. Posed treatments (Parochetti and Hein. 1973).
C) Movement in Soil:
Various bioassay methods have been developed for studying the movement of herbicides in soil. In field experiments soil samples may be taken from different depths and assayed. In laboratory leaching studies using soil columns, various methods have been used to assess the movement of co pounds. Nishimoto and Warren (1971) also developed bioassay techniques to examine upward and lateral movement of herbicides.
D) Degradation and Persistence:
Soil samples at different depths may be collected from the herbicide applied plots and assayed in the laboratory. A similar procedure has been used to follow dissipation of herbicides in water (Hiltribran, 1962).
E) Residual Effect:
The residual effect of herbicides on succeeding crop can be assessed using bioassays.
5. Biossay Procedure:
The procedure developed by Crafts (1935) is followed by many researchers with little modifications. Soils treated with the herbicide at different concentrations are sown with an indicator plant and after measuring the germination and response (plant height and dry weight) of the indicator plants standard curves are drawn. By comparing these parameters obtained from the field sample where the said herbicide has been applied with that of standard the residua is quantities. Jayakumar (1987) standardized the bioassay technique for a number of herbicides different soil types and brought out quadratic models for easy assessment.