**3. Preparation of resistant weed samples by oxidizer**

the herbicide into plant cells, possibly via a phosphate transporter, and operates at low concentrations. Other may be a passive mass flow system which is gradient dependent (**Figure 1**). The exact mechanism that promotes the reduction of cellular absorption and translocation of glyphosate in resistant weeds is not clear yet. Shaner [10] described four potential mechanisms that may cause the restriction of glyphosate movement (**Figure 2**): (1) alteration in a putative phosphate transporter responsible for the active cellular absorption of glyphosate, in a way that the transporter is no longer present or no longer recognizes glyphosate, resulting in reduced absorption and translocation; (2) evolution of a new transporter that pumps glyphosate into the vacuole, thus sequestering the herbicide and preventing it from reaching either the chloroplast or the phloem; (3) evolution of a new transporter that actively pumps glyphosate out of the cell into the apoplast; or (4) evolution of a transporter at the chloroplast envelope that pumps glyphosate out of the chloroplast, preventing the herbicide from reach-

In order to study glyphosate resistance, 31P nuclear magnetic resonance (NMR) spectroscopy studies were employed to track glyphosate movement and metabolism in resistant and susceptible biotypes of horseweed (*Conyza canadensis*), and the results showed that the rate of vacuole accumulation of this herbicide is faster and occurs to a greater extent in the resistant

These results have been confirmed in different glyphosate-resistant *Lolium* spp. biotypes collected on three different continents [16], pointing to vacuolar glyphosate sequestration as the

**Figure 1.** Proposed mechanisms of glyphosate absorption into plant cells. G, glyphosate (the size of the letter indicates relative size of glyphosate pool). (1) Active absorption of glyphosate into cell. (2) Passive diffusion of glyphosate into the cell. Arrows indicate direction of movement of glyphosate pools into and out of the cell, chloroplast, and vacuole.

ing its target site.

162 Herbicide Resistance in Weeds and Crops

Source: Shaner [10].

biotype rather than in the susceptible [15].

primary mechanism of resistance in these biotypes.

According IRSN [17], the 14C contained in the resistant weed (test portion) is transformed to <sup>14</sup>CO<sup>2</sup> from which a sample is prepared for measurement by liquid scintillation spectrometry (LSS), and combustion by oxidizer (**Figure 3**) is main method used.

**Figure 3.** Oxidizer OX500 (R.J. Harvey Instrument Corporation) (a) and liquid scintillation equipment, Tri-Carb 2910 TR LSA counter (PerkinElmer) (b) from the Laboratory of Ecotoxicology of CENA/USP.

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 entire carbon content is oxidized by the carbon dioxide containing 14C (14CO<sup>2</sup> ). Evolved 14CO<sup>2</sup> is trapped in a 2 M NaOH solution and subsequently mixed in an adequate scintillating cocktail for ß counting on a LSS [18].

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:

$$1\text{becquerel (Bq)} = 1\text{ disintegration/s (dps)} = 60\text{ disintegrations/min (dpm)}\tag{1}$$

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].
