3.2. Aminocyclopyrachlor Mineralization to 14CO2
Low mineralization of herbicide in the soil increases the bound residue formation and bioavailability of extractable residue. The 14
produced directly from 14
C-aminocyclopyrachlor mineralization was evaluated weekly for 126 days, and results are shown in Figure 1
During the first 28 days incubation, the amount of herbicide mineralized was very low, with values of 0.9%, 1.2%, and 1.4% of the initially applied aminocyclopyrachlor recorded in the loamy sand, sandy clay, and clay soil samples, respectively (Figure 1
). These results indicated the occurrence of a similar lag phase in all three soils, suggesting that time was needed for the acclimatization of the microbial degraders of aminocyclopyrachlor [18
]. As the soils used in the present study were never treated with aminocyclopyrachlor prior to the experiment, we assumed that all three soils did not contain any natural microorganisms able to mineralize this particular herbicide. However, Boivin et al. [15
] reported that soils never treated with the herbicide 2,4-d
had been known to host significant but low populations of microbial decomposers, and the same principle likely applies here. Assessing the size of the microbial biomass and composition of the microbial community originally present in the tested soils would thus represent a valuable avenue for future investigation.
In all three soils, the added aminocyclopyrachlor underwent slow mineralization, with total accumulated 14
following the order of clay (9.48%) > loamy sand (7.46%) > sandy clay (6.15%) soil (p <
0.05), as shown in Table 3
. These data agree with those reported by Durkin [6
], who found a maximum percentage of CO2
evolution of 23% at 360 days aminocyclopyrachlor incubation. In the present study, the clay soil had the highest clay (74.1%) and FC content (31.05%), with intermediate OC content (1.7%) and pH (4.7), as shown in Table 2
, which could have contributed to the observed higher levels of aminocyclopyrachlor mineralization. According to Oliveira and Brighenti [25
], soils with higher OC content present increased biological activity, and therefore higher mineralization rates should be expected in such soils. On the other hand, soils with high OC content can also adsorb herbicides more intensively, promoting the formation of bound residues and thus inhibiting herbicide mineralization. However, the mineralization rate of an herbicide in a soil is a function of multiple factors, such as the density of the microbial population, the physico-chemical properties of the soil and herbicide, soil depth, molecule bioavailability, and climatic conditions [26
]. Moreover, further research is required regarding the fate of aminocyclopyrachlor in other soil types with different physico-chemical properties and environmental conditions.
Indeed, mineralization appeared not to be the main aminocyclopyrachlor dissipation process during incubation in the three tropical soils analyzed in the present study, with the first-order kinetics of the herbicide characterized by a declining mineralization rate over time. As shown in Table 3
, the mineralization rate (k) was very low throughout the incubation period in all soils, with values ranging from 0.00050% to 0.00079% 14
. MT50 values of aminocyclopyrachlor were higher in sandy clay (1376 days), intermediate in loamy sand (1128 days), and lower in clay soil (877 days) (p <
0.05). The data regarding aminocyclopyrachlor mineralization recorded in this study indicate that the herbicide has a relatively long persistence in all soils and thus could provide prolonged weed control in drought years and this herbicide has high leaching potential, particularly in wet years, since Francisco et al. reported that aminocyclopyrachlor was detected at all depths (0–30 cm) in the agricultural soils and can cause damage to sensitive plants, such as tomatoes [28
], soybean, and beet [29
All aminocyclopyrachlor not mineralized by microorganisms remained in the three soils as residues, in either extractable or non-extractable form, as described in the following sections.
3.3. Dissipation of 14C-Extractable Residue
High dissipation would imply that the herbicide is subject to leaching and groundwater contamination problems, consequently decrease efficacy of the product. The extractable residues of an herbicide are formed by the parent product and its metabolites. Total 14
C-extractable residue of aminocyclopyrachlor decreased slowly with incubation time in the three tested soil types in the present study, with values ranging from 90.9% to 59.0%, 94.2% to 60.0%, and 88.9% to 44.9% in loamy sand, sandy clay and clay soil, respectively (Figure 1
). At the end of the study after 126 days, the reduction in extractable residue was significantly higher in the clay soil (~50%), followed by the sandy clay (~36%) and the loamy sand (~31%) (p <
0.05). This pattern is likely due to the high clay content (74.1%) of the clay soil compared to that of the other two soils (Table 1
Francisco et al. [5
] also found that aminocyclopyrachlor not only exhibited low sorption capacity (Kf
= 0.37–1.34 µmol (1−1/n)
) and high desorption capacity (Kf
= 3.62–5.36 µmol (1−1/n)
) in the same three tropical soils studied in the present study, but also showed a significant positive correlation with soil clay content. Since this herbicide remains weakly sorbed, it can be easily extracted from the soil using solvents, as proven here.
During the first 28 days of incubation, the fraction of total extractable residues from all extraction steps in the three tested soils was more than 72% of the initially applied 14
C-aminocyclopyrachlor (Figure 1
). However, after 28 days the amount of extractable herbicide residues gradually decreased. Such a decrease in recovery rate through solvent extraction indicates the tendency of aminocyclopyrachlor to form bound residues in the soil, as detailed below.
3.4. 14C-Bound Residue Formation
High bound residue formation of an herbicide contributes to long persistence of this herbicide in soils. The formation of bound (non-extractable) residues of aminocyclopyrachlor increased slowly with incubation time in all three soils (Figure 1
). Total 14
C-bound aminocyclopyrachlor residue levels were slightly higher in the clay soil over the incubation period, followed by the reverse order recorded for extractable residues: clay > sandy clay = loamy sand soil (p <
0.05). At the end of the study after 126 days, high levels of bound residues (41.4%) were detected in the clay soil compared to the loamy sand and sandy clay soils (32.4% and 22.5%, respectively), as already justified based on their respective physico-chemical properties.
Bound residues were detected immediately after the application of aminocyclopyrachlor at 6.5%, 3.0%, and 9.6% in the loamy sand, sandy clay and clay soils, respectively, and by the end of the study at 126 days had increased ~5.0-, 7.5-, and 4.3-fold in the same soils (Figure 1
). We assume that the formation of bound residues of aminocyclopyrachlor is not related to its low sorption and high desorption rates in the soils studied, because most of this herbicide is bioavailable in the soil solution, according to Francisco et al. [5
]. Indeed, Gevao et al. [12
] reported that bound residue formation is not equivalent to the strong sorption of compounds because those sorptive processes that are reversible by traditional chemical extraction techniques do not lead to bound residue formation.
A similarly large bound residue fraction was also reported by Wang et al. [19
] who found that pyribambenz propyl herbicide residue was consistently non-extractable, accounting for up to 57% in some soil samples, indicating the importance of considering this form of residue in the comprehensive risk assessment of new herbicides.
Barriuso et al. [14
] reported that the main issue with this kind of approach is the lack of information regarding the exact nature of the bound residues, which is required to convert 14
C-bound residues into 14
C-parent herbicide or 14
C-metabolites. It should also be noted that bound residues are constituted by the sum of the original compound and its fractions immobilized in the soil colloids, which cannot return to the soil solution and thus become unavailable to plants and soil microorganisms [12
]. Consequently, the absorption, mineralization and degradation rates of aminocyclopyrachlor are reduced, leading to the greater persistence of the herbicide in the environment and more available herbicide in soil solution.