**4. Herbicide absorption and translocation in resistant weeds**

Studies on the absorption and translocation of herbicides in plants are usually conducted to evaluate the behavior of a new herbicide on a certain plant species, comparing two or more herbicides, specific formulations, additives, or the effect of environmental standards. The growing problem regarding the resistance of weeds to herbicides promoted the studies on the absorption, translocation, and metabolism of herbicides as the methodology to elucidate the resistance mechanisms [20]. So these procedures need to be better explained to researchers, as will be described in this chapter.

The studies on the absorption of herbicides use a destructive sampling of treated plants on several post-treatment periods, which allows the characterization of the absorption standard on the plant, considering the planning and adequate statistical analyses [22].

On the adequate phenological stage for each species, susceptible and resistant weeds must be adequately identified by treatment. The leaves that have been predetermined to receive the radiolabeled herbicide must be covered with plastic film, aluminum paper, or small paper envelopes. Then, the "cold" herbicide is applied to the plants (without the radioisotope) at the dose recommended by the manufacturer, as a solution with adjuvant (when indicated) and water, followed by the immediate removal of the protective plastic film of the applied leaf.

The radiolabeled herbicide solution must be prepared on a solution containing its commercial formulation at the recommended dose for the considered phenological stage. After applying the "cold" product, its radiolabeled version is applied. It is important for the radiolabeled herbicide to be applied with at least 170 Bq of specific activity, in the case of studies with most of the annual weeds [20].

Resistant weed samples are not readily soluble on scintillation cocktails. Due to this reason, such samples go through biological combustion on oxidizer. The combustion of the sample creates an atmosphere that is rich in hydrogen, which is oxidized by the water, while the

is trapped in a 2 M NaOH solution and subsequently mixed in an adequate scintillating cock-

Coughtrey et al. [19] described a wet oxidation technique using potassium dichromate and concentrated sulfuric and phosphoric acid, which can be done in a modified filter flask. This technique can accommodate up to 0.3 g of dry resistant weed. Recovery of 14C is consistent between batches, with an average recovery of 97.2% over 15 standards. These authors reported that technique described does not involve large capital expenditure and is relatively rapid.

The expression of the resistant weed sample's activity in becquerel (Bq) of 14C per kg of carbon also requires measuring its elementary carbon content, generally by gas chromatography. According Nandula and Vencil [20], the commonly accepted unit of measurement of radioactivity is the Bq, derived from the International System of Units. It is defined as follows:

 1 becquerel (Bq) = 1 disintegration/s (dps) = 60 disintegrations/min (dpm) (1) A description of the research procedures and the methodology related for detection of resistant weeds using 14C-herbicide absorption, translocation, and metabolism compared with susceptible weeds will be described below, based on Nandula and Vencil [20] and Mendes et al. [21].

Studies on the absorption and translocation of herbicides in plants are usually conducted to evaluate the behavior of a new herbicide on a certain plant species, comparing two or more herbicides, specific formulations, additives, or the effect of environmental standards. The growing problem regarding the resistance of weeds to herbicides promoted the studies on the absorption, translocation, and metabolism of herbicides as the methodology to elucidate the resistance mechanisms [20]. So these procedures need to be better explained to researchers, as

The studies on the absorption of herbicides use a destructive sampling of treated plants on several post-treatment periods, which allows the characterization of the absorption standard

On the adequate phenological stage for each species, susceptible and resistant weeds must be adequately identified by treatment. The leaves that have been predetermined to receive the radiolabeled herbicide must be covered with plastic film, aluminum paper, or small paper envelopes. Then, the "cold" herbicide is applied to the plants (without the radioisotope) at the dose recommended by the manufacturer, as a solution with adjuvant (when indicated) and water, followed by the immediate removal of the protective plastic film of the applied leaf.

The radiolabeled herbicide solution must be prepared on a solution containing its commercial formulation at the recommended dose for the considered phenological stage. After applying

**4. Herbicide absorption and translocation in resistant weeds**

on the plant, considering the planning and adequate statistical analyses [22].

). Evolved 14CO<sup>2</sup>

entire carbon content is oxidized by the carbon dioxide containing 14C (14CO<sup>2</sup>

tail for ß counting on a LSS [18].

164 Herbicide Resistance in Weeds and Crops

will be described in this chapter.

The radiolabeled product is applied using a micro-syringe, by applying a 1 μL droplet (the total radiolabeled product applied depends on the molecule and the radioactivity of the radiolabeled molecule), on the leaf blade of the upper part of the expanded leaf of each plant (**Figure 4**). The choice for the leaf on which the application will occur depends on the studied species. Each plant (or part of the plant) must be collected according to the pre-established times for each situation. However, it is suggested that at least six collection times are used, in addition to time zero (immediately after the application), and that the untreated plants are included as control. For each collection, the treated leaf from each plant must be rinsed with the adequate solvent. The concentration (v v−1) of the solvent must be established on preliminary tests with the studied molecule. Then, the radioactivity during the rinsing must be quantified by LSS in order to determine the non-absorbed radioactivity. The leaf absorption is calculated by the difference between the applied and the non-absorbed radioactivity. The plants must be dried with an absorbing paper, pressed, and dried on an air circulation oven at 70°C for 48 h.

In the preparation of the absorption studies, we must select resistant and susceptible weeds of the same age and/or growth stage. According Nandula and Vencill [20] to plot, the figure is necessary use at least six time points in addition to a 0 time point of tissue harvest, as illustrated in **Figure 5**. However, under conditions of limited resources, it is better to increase the number of time points and reduce the number of replications (*n* ≥ 2). Include non-treated weeds as a blank or control is very important for research. Then, the steps to evaluate the translocation are conducted.

**Figure 4.** Application of 14C-glyphosate with a micro-syringe on glyphosate tolerant *Spermacoce verticillata* leaves at the Laboratory of Ecotoxicology of CENA/USP.

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

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 studied weed, through the information available in the literature.

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 occurs.

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

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

**Figure 6.** Autoradiography of glyphosate tolerant (a and b) and susceptible (c and d) *Richardia brasiliensis* with application of the leaf of 14C-glyphosate at 48 HAT (hours after the treatment). Photograph weed to the right (a and c) and autoradiography of the weed translocation to the left (b and d) at the Laboratory of Ecotoxicology of CENA/USP. Arrows indicate the sites of application.

analysis. The usual procedure to quantify the translocation of herbicides on plants is the biological combustion, in which dry samples of each part of the plant (both the treated leaf and the part above and below it, as well as the roots) are oxidized by the presence of O<sup>2</sup> , and the resulting CO<sup>2</sup> is captured on a special solvent. Then, the radioactivity must be measured on the scintillation counter.

The quantitative analysis of the translocation may also be conducted through the volume analysis, offered by the software provided together with the image scanner, as of its purchase. The volume is the total signal intensity of the radioactivity within defined limits of the image. The translocation is then expressed as the rate between the percentage of signal intensity on the applied zone, as well as above and below it, and the total signal intensity on a defined image containing 14C [25].
