**5. Herbicide metabolism in resistant weeds**

Usually, the translocation studies are conducted right after the absorption studies, although they demand more work and time. Differently from the absorption, which occurs within hours after the treatment, the translocation of herbicides may take up to days after the treatment. Due to this reason, in order to evaluate the translocation, the previous knowledge must be considered in order to determine the times after the treatment in which this variable should be evaluated. The biological combustion is the most used procedure to quantify the translocation of herbicides on plants. However, care must be taken when stating that the detection of the radioactivity on other parts of the plant, outside the treated leaf, means that the herbicide is on its parental form. It might have been converted into a non-phytotoxic metabolite. In order to state this, one must investigate the potential for the herbicide to have been metabolized by the

**Figure 5.** Absorption of 14C-quinclorac by propanil- and quinclorac-resistant and susceptible barnyardgrass (*Echinochloa crus-galli*) biotypes over time. No differences were detected between biotypes at any time. LSD (0.05) bar to make

To study the movement of herbicides on weeds, the qualitative techniques involving autoradiography or phosphorus blade images have been used for over 50 years [20]. While the biological combustion offers a quantitative estimation of the herbicide on the treated weed, autoradiography (**Figure 6**), or the phosphorus blade image provides a qualitative measurement of the movement of the herbicide on the weed, in addition to the location where it

For the exposition of the treated and untreated plants, the use of phosphorus blade images is safer in comparison to the use of autoradiography, since it does not require handling chemical compounds that are harmful to the health. Despite more expensive, the technique is also quicker. A single day of exposition of a plant on a phosphorus blade resulted on images with

Therefore, in order to study the translocation, the plants treated as on the absorption study must be exposed on phosphorus blade for 72 h, in order to scan the image for qualitative

studied weed, through the information available in the literature.

comparisons between biotypes at a particular time interval. Source: Lovelace et al. [23].

166 Herbicide Resistance in Weeds and Crops

superior quality than the exposition for 3 weeks with the X-ray film [24].

occurs.

The use of radiolabeled herbicides to investigate whether the herbicide is being metabolized in the resistant weed is an efficient method, and it is the most indicated method to diagnose the resistance related to other phenomena that are not related to the change on the action site of the herbicide [26]. The analytical method aiming at studying the metabolism of herbicides in plants comprehends three fundamental steps: preparation of the plants and application of treatments; extraction and separation; and identification of the herbicide and its metabolites, if any.

The steps to conduct the study on the metabolism of herbicides in plants are described as follows. The preparation of plants and application of the treatments must be conducted as described for the absorption and translocation study. In case the fresh samples of plants are not adequate for processing after the collection, the ideal is to store them at −20°C to assure the stability of the active substances and metabolites. The techniques employed on studies on the metabolism of herbicides in plants are thin layer chromatography (TLC), high-performance liquid chromatography (HPLC), and gas chromatography (GC), depending on the herbicide molecule.

For the extraction, the adequate system of solvents for the studies herbicide must be known. The treated leaf must be rinsed with non-polar solvent (usually ethanol or methanol). Then, the plant must be dried with an absorbing paper, immediately frozen in liquid nitrogen and stored at −80°C up to its use. The plant tissue must be macerated in crucibles that must be previously cooled with N2 , and homogenized with the specific cold solvent at a concentration of 80% (v v−1). A stainless steel homogenizer may also be used. The solution must be centrifuged; the supernatant decanted; and the residue must go through re-extraction with the chosen cold solvent at 80%, followed by extraction with the same cold solvent at 50% (v v−1). The supernatants must be mixed, and the radioactivity must be determined by LSS, in order to know the mass balance, which is expressed as the rate between the radioactivity applied at the beginning of the experiment and the total radioactivity measured (originated from rinsing all parts of the plant). The mass balance may be also referred to as the radioactivity recovery percentage. Approximately 7 mL of the supernatant must be evaporated, resuspended in 300 mL of the solvent at 50%, and centrifuged. The final sample may be analyzed by any previously described technique, usually TLC or HPLC, with the respective solvent system [4].
