Soil collection

Twenty-seven orchards in the Central Valley (Tulare or Fresno Counties) and Ventura County with different simazine use histories were identified and soil samples were collected. The targeted simazine use histories included: (1) annual simazine use, (2) recent simazine use only, (3) a short break since the last simazine application, and (4) no recent simazine use. In this context, annual use was defined as at least 15 consecutive years of at least one simazine application; recent use only means less than 7 years of annual use; short break since last means 2-5 years since last simazine use, and no recent use means at least 15 years since the last simazine application. Soil was collected in October 2006 from the top 15 cm within the tree row in each orchard, placed in a plastic bag, and stored in a sealed 20-L plastic container until further analysis. Based on herbicide use information from the growers nine of the 27 sites were classified as annual use, five as short recent use only, seven as short break since last use, and six as no recent use (Table 1).

Rapid Laboratory Dissipation assay

One hundred g subsamples of each soil were weighed into wide-mouth 250-mL Wheaton jars with Teflon-lined lids. Soil was treated with 15 mL of 66.67 µg mL^-1 formulated simazine (Princep 4L, Syngenta Crop Protection, Greensboro, NC) in water and manually homogenized to achieve a nominal simazine concentration of 10 µg g^-1 soil, a level that approximates initial simazine residues following applications in California orchards. Samples were taken at 0, 1, 2, 3, 4, 6, 9, 14, 21, 28, and 35 d after treatment (DAT), and simazine was extracted using a water-based procedure described by Shaner et al.¹² At each sampling time, soil samples were physically stirred and a 5 g subsample of moist soil was weighed into a 50 mL centrifuge tube. An equivalent amount (w/w) of distilled water was added to each tube and samples were mixed on a reciprocating shaker for 1 hour then centrifuged at 2000 x g for 20 min at 20 ^°C. One-half to 1-mL aliquots of the supernatant were transferred to microfuge tubes (Millipore Corporation, Bedford, MA) with 0.22-µM Teflon filter inserts and centrifuged at 10,000 x g for 10 min. The filtrates were analyzed using a high-performance liquid chromatograph (HPLC) (Agilent Technologies, Wilmington DE) equipped with a multiple wavelength detector and a 4.6-by 250-mm C18 column (Agilent Technologies, Wilmington DE). The mobile phase was acetonitrile:water:phosphoric acid (35:65:0.05 vol/vol/vol) and was run isocratically at 1 mL min^-1 at 40 ^°C. The injection volume was 100 µL, and simazine was detected at 223 nm. A series of simazine standards were included with each sample run to determine herbicide concentration and retention. Simazine retention time under these conditions was between 5.5 and 6.5 min and extraction efficiency in these soils was 69.5% ± 1.5% in preliminary experiments.

Table 1

Location, simazine use history, and soil classification of 27 California citrus orchard soils collected in 2006.

Microbial Activity determination

From the initial 27 orchards, eight soils (one of each simazine use class from the two regions) were selected for more detailed simazine dissipation analyses. Soil samples 10 and 26 (annual simazine use), 5 and 24 (recent simazine use only), 12 and 20 (short break since last), and 6 and 21 (no simazine use) were selected for this study (Tables 1 and 2). Approximately 20 kg soil (dry weight equivalent) from each sample was divided into two; half was triple autoclaved to eliminate existing microbial populations and the other half was left unsterilized. Autoclaved (sterile) or non-autoclaved (live) soil was passed through a 4-mm sieve and soil moisture was measured. Three 100 g replicate subsamples of each soil were weighed into wide-mouth 250-mL Wheaton jars with Teflon-lined lids. Soil was treated with either 15 mL water or 15 mL of 66.67 µg mL^-1 simazine in water and manually homogenized resulting in a nominal final simazine concentration of 10 µg g^-1 soil (w/w). Samples were taken at 0, 1, 2, 3, 7, 14, 21, 28, and 35 d after treatment (DAT), and simazine was extracted using the previously described water-based extraction procedure. Data were analyzed as a completely randomized experimental design with three replications and the experiment was repeated.

Table 2

Location, simazine use history, and physical properties of California citrus orchard soils used for microbial activity and mineralization assays in 2006.

Mineralization assay

Mineralization of ¹⁴C-ring-labed simazine in eight representative soils (Table 2) was evaluated in biometer flasks as described by Krutz et al.¹³ Thirty g soil (dry weight equivalent) was mixed with ring-labeled ¹⁴C-simazine (≥95% radiological purity with specific activity of 9.9 mCi mmol^-1) and analytical grade simazine (99% purity) for an initial concentration of 1 µg simazine g^-1 soil and radioactivity of 58.6 Bq g^-1 soil. The experiment was arranged as a completely randomized design with three replications and was repeated. Final soil moisture content in each flask was adjusted to 30% (w/w) by addition of deionized water, and biometers were incubated in the dark at 25 ^°C ± 2 ^°C. Evolved ¹⁴C-CO₂ was collected in sodium hydroxide traps and quantified by liquid scintillation spectroscopy (LSS) using Hionic-Fluor (Perkin Elmer, Shelton, CT). The sodium hydroxide solution in the traps was replaced after each sampling. Samples were collected at 0, 1, 7, 11, 14, 18, 21, 25, 28, 32, and 35 DAT. Soil was destructively sampled 35 d after herbicide application. Air-dried soil was manually crushed into uniform particle size, and duplicate samples (0.30 g) were weighed onto Whatman 1 qualitative filter paper (Whatman Inc., Florham Park, NJ). Samples were combusted in a Packard model 306 oxidizer (Packard Instruments, Chicago, IL), and evolved ¹⁴CO₂ was trapped in scintillation vials containing Carbo-Sorb and Permafluor (20 mL; 1 + 1 by volume) (Perkin Elmer, Meridian, CT). Radioactivity was determined by LSS. The amount of ¹⁴C-CO₂ recovered from the combusted samples was added to the cumulative ¹⁴C-CO₂ evolved to determine the ¹⁴C mass balance.

Rapid Dissipation assay

Dissipation data were converted to percent simazine extracted 1 hr after equilibration, ie, time zero, and were grouped together on the basis of simazine history. Percent simazine extracted data were described using a first order kinetics model using Sigma Plot 11:

Microbial Activity determination

Data on simazine dissipation was analyzed using mixed procedure in Statistical Analysis System (SAS version 9.1; SAS Institute Inc., Cary, NC) with sampling time as fixed effects and replications and experimental runs as random effects. Sampling time means for simazine dissipation were then separated using LSMEANS at the 5% level of significance. The relationship between dissipation data to sampling time for sterile soils was described using a first order kinetics model. The live soils dissipation data were described using the three-parameter, sigmoidal logistic function:

Mineralization assay

Analysis of variance and mean separation of mineralization assay data were performed using Proc Mixed at P ≤ 0.05. When interactions with the sampling time main effect were noted, the relationship between time and simazine mineralization was described by the Gompertz growth model in Sigma Plot 11. The general form of the Gompertz model utilized is as follows:

Rapid Dissipation assay

When the 27 citrus orchard soils were grouped according to broad simazine use classes, simazine dissipation was more rapid in soils with a history of simazine use compared to soils with no recent use (Fig. 1). In soils with simazine use history, 90% or greater was dissipated by 14 days in the 35 day laboratory assay. In contrast, in soils with no use history (for at least 15 years), simazine was at least 40% higher at 35 days after treatment. Soil from orchards treated recently with simazine dissipated the herbicide the fastest which suggests that enhanced degradation can develop after relatively few years of simazine treatment. Others have also reported that enhanced atrazine degradation can occur following a single atrazine application.¹⁴ Moreover, a break of a few years between simazine treatments appears to slow the degradation rate but does not fully reset degradation to the level in non-adapted fields.

Microbial Activity determination

More detailed degradation assays conducted with a subset of the orchard soils indicated that microbial activity is the primary contributor to simazine degradation. In autoclaved soil, simazine dissipation was reduced four-fold compared to non-sterilized soil (Fig. 2A and B, dashed line) indicating that simazine persistence in these orchard soils is principally governed by biotic activity.^11,14

In soils from Tulare County, simazine was quickly degraded below analytical detection limits if the orchard had a history of simazine use (Fig. 2A). The fitted logistic curve revealed that the simazine half-life in soils with simazine use histories were 2-fold (recent use only) and 3-fold (annual use and short break after last use) faster compared with soil with no recent simazine use history. Similar degradation rates between adapted and non-adapted soils were reported in a related vineyard experiment.¹⁵ In this experiment, reduced simazine persistence in soils with long use history and short break after last use were expected. However, it is interesting to note that soils with only recent simazine use also exhibited enhanced degradation of the herbicide which suggests that the microbial population was already present and can build up quickly after one or two applications of simazine. In the Ventura County soils, fitted logistic curves indicated that the half-life of all soils regardless of simazine use history was 11 d or shorter (Fig. 2B). These half-life values are similar to soils with long simazine use and short break after last use in the Tulare County soils which suggests that the microbial population that can quickly degrade simazine is widely spread in these orchards regardless of recent simazine use. A similar phenomenon has been reported in western and southern U.S. corn production regions.¹⁶

Figure 1.

Simazine soil dissipation in a laboratory assay using soil from citrus orchards with various simazine use histories.

Mineralization

Cumulative mineralization of ¹⁴C ring-labeled s-triazine herbicides can be used as a diagnostic test to confirm enhanced degradation although the critical threshold may vary among specific pesticides and environments. Krutz et al¹⁶ suggested that soils that mineralize available s-triazine herbicide more than 50% within 30 d could be considered adapted. In Tulare County, soils with a short break after use, annual use, and recent use only had cumulative simazine mineralization that exceeded 50% in less than 15 days indicating that these soils are adapted (Fig. 3A). Simazine mineralization in soils with long annual use and short break after long use peaked at 15 days then leveled off. Simazine mineralization in soils recently treated with simazine plateaued after 19 days of incubation in the laboratory. Conversely, cumulative mineralization in soil with no simazine history did not exceed 30% after 30 days of incubation, which is typical of non-adapted soils. In Ventura County, all soils had cumulative mineralization of 14C-simazine that exceeded 50% before 30 d of incubation indicating that these soils are adapted to simazine regardless of simazine use history in the last 15 years (Fig. 3B).

At each location, the cumulative mineralization and degradation data were in agreement. In Tulare county, adapted soils had shorter half-life and exceeded the critical cumulative mineralization threshold level faster than the non-adapted soil. No distinct differences in cumulative mineralization and simazine degradation were observed among soils with different simazine use histories in the Ventura County soils. All soils appear to have shorter half-lives and exceeded the critical cumulative mineralization threshold level before 30 days. Although available records indicated that no simazine use had been used for at least 15 years in the orchard classified as no recent simazine use, the similarity of cumulative mineralization and simazine degradation among the soils in Ventura County suggest that all four orchards have some level of enhanced degradation. Because of the long history of orchard crops and simazine availability in this area, it is fairly likely that the soil we classified as non-adapted had actually been treated with an s-triazine herbicide at some time in the past. The triazine-degrading microbial population may have been previously selected but had decreased to a low level following at least 15 years of no s-triazine treatments. However, the re-application of simazine to these soils may have re-activated the remaining degraders in the soil thus field dissipation kinetics was generally similar on all soils. Krutz et al¹³ reported that once a soil is adapted to s-triazine herbicides, the s-triazine degrader population will persist at some level in the soil. In the absence of continued s-triazine use, the population declines to a low but static level but can quickly rebound following a subsequent application of an s-triazine herbicide. It should be noted, however, that not all soils that received s-triazine treatments mineralized the herbicides despite a history of application.

Figure 2.

Effects of soil sterilization and simazine use history on simazine dissipation in California citrus orchard soils.

Figure 3.

Cumulative mineralization of ¹⁴C-simazine in citrus orchard soils with different simazine use histories.

The biological degradation of s-triazine in the soil depends on the presence and activity of indigenous microorganisms that possess enzymatic mechanism to transform and degrade the s-triazine molecule.^8,17,18 Several factors are thought to influence these microorganisms in soils including microbial composition (diversity and biomass) or soil conditions (nutrient limitations and specificity, moisture requirement, pH, temperature, soil texture and porosity, and organic matter content).^19–202122 Depending on the microbial population and the factor(s) affecting these microorganisms, simazine half-life may vary from few weeks to months.

Moreover, applying simazine in orchards before weeds emerge reduces its efficacy owing increased herbicide dissipation due to microbial or chemical degradation, photolysis, or leaching through the target zone.^23–242526 To reduce early winter losses, citrus producers could delay simazine applications until late winter, although a tank mix partner would likely be required to control emerged weeds. This practice may also minimize the potential for off-site transport of the herbicide because fewer and smaller precipitation events would occur following the applications.