**3. Results**

#### *3.1. Climatic and Edaphic Conditions*

Our analyses confirmed earlier data published by other authors [32–35] that the average annual temperatures in Tovarnik (Table 1) are higher than in Lukaˇc. Precipitation varied from place to place in one of the two years of investigation and confirmed earlier published data [32–35] that when comparing Lukaˇc (west) and Tovarnik (east), temperatures increased while precipitation decreased in the eastern part.

In both years the mean air and soil temperatures in the area of Lukaˇc were significantly lower compared to Tovarnik, and the precipitation was significantly higher in 2015 in the same place, while in 2016 the differences were not significant. Between the years studied (2015 vs. 2016) there were no significant differences between the climatic conditions at both locations.


**Table 1.** Characteristics of the weather conditions prevailing at the two locations where the field investigations were carried out and the corresponding ANOVA results.

\* Values followed by the same lowercase letters are not significantly different (*p* > 0.05; HSD test), 1, small letters refer to no differences among locations; 2, small letters refer to no differences among years within same location; ns, letters refer to no differences.

The edaphic conditions di ffered between the locations. The soil in Tovarnik has a higher content of soil organic matter than the soil in Lukaˇc (Table 2). In addition, both soils are classified as silty clay according to the soil particle size fractions. A detailed description of the regional physical and chemical soil properties is given in Table 2.


**Table 2.** Physical and chemical soil properties in Lukaˇc and Tovarnik, 2016.

#### *3.2. Degradation in Soil*

Table 3 shows that there were no residues of neonicotinoids above LOQ in Lukaˇc. Tovarnik showed concentrations of imidacloprid residues above LOQ and slightly increased thiamethoxam, while higher residues were found in the greenhouse.

**Table 3.** Residues of neonicotinoids (mg/kg) in soil samples taken from field sites at the end of the growing season 2016 (i.e., 180 days' post planting), Croatia.


#### *3.3. Degradation Dynamics in Plants*

Figure 1 shows a degradation dynamic of imidacloprid in sugar beet plants.

**Figure 1.** Degradation dynamics of imidacloprid during the growing seasons 2015 (**a**) and 2016 (**b**) in sugar beet plants in Lukac, Tovarnik and in greenhouse trials, in compliance with the maximum permitted residue level of 0.5 mg/kg; LOQ— limit of quantification; MRL—maximum residue level.

The maximum residue level (MRL) for imidacloprid in sugar beet roots is 0.5 mg/kg (EU No 491/2014) [36]. Concentrations of imidacloprid in whole plants collected in field trials (Lukaˇc and Tovarnik) fell below the MRL of 0.5 mg/kg (EU No. 491/2014) 40–55 days after sowing in both years under investigation [36] (Figure 1). After that, residues in the leaves of sugar beets grown under field conditions were almost no longer detectable. Root samples were taken 60 days after sowing, and from the first sample onwards the residue level in the roots was below the MRL. At the time of harvesting the roots (180 days after planting), no residues above LOQ were detected. In the greenhouse trial (Zagreb), degradation was much slower because no regular water rinsing was possible. Residues of imidacloprid in leaves from greenhouse trials fell below the MRLs ten days later compared to field conditions (i.e., 60 days after sowing). A slightly faster degradation of imidacloprid residues in roots of sugar beet grown in greenhouse trials was observed in 2016 compared to 2015. In general, the residue level of imidacloprid in roots was below the MRL 80 days after sowing. At the time of harvest, the residue level in roots was quite low, 0.08 mg/kg in 2015 and <0.01 mg/kg in 2016.

The results of the statistical analysis are presented in Tables 4–7. Residue levels were significantly affected by treatment with imidacloprid at almost all sampling times, except for two final samples where degradation was completed in both years of the study. Residue levels in plants from treated seeds were significantly higher compared to those in untreated plants throughout the vegetation until harvest where degradation was completed. In 2015, residues of imidacloprid were significantly influenced by location (i.e., agroclimatic conditions) in almost all but two of the last samples taken (Tables 4 and 5). In 2016, residues were significantly site-dependent (i.e., agroclimatic conditions) in only one sampling (76–85 days after sowing) when residues were significantly higher under greenhouse conditions in Zagreb (Tables 6 and 7). The third factor (plant part) was observed in three samples. In 2015, residues of imidacloprid were significantly a ffected in two out of three samples (Table 5), while in 2016 residues of plant parts were not a ffected at all (Table 7), confirming the good systemic translocation of imidacloprid.


**Table 4.** Imidacloprid residues in the whole sugar beet plants during the first three observing periods and for roots at harvesting in 2015.

Analysis of variance for imidacloprid residues in the whole sugar beet plants and root. \*\* significant at *p* = 0.01.


**Table 5.** Imidacloprid residues in di fferent plant parts during the vegetation period in 2015.

Analysis of variance for imidacloprid residues in different plant parts. \* significant at *p* = 0.05, \*\* significant at *p* = 0.01.

**Table 6.** Imidacloprid residues in the whole plants during the first two observing periods and for roots at harvesting in 2016.


Analysis of variance for imidacloprid residues in the whole sugar beet plants and root. \* significant at *p* = 0.05, \*\* significant at *p* = 0.01.


**Table 7.** Imidacloprid residues in the di fferent plant parts during the vegetation period in 2016.

Analysis of variance for imidacloprid residues in different plant parts. \* significant at *p* = 0.05, \*\* significant at *p* = 0.01.

The significant interaction between all three factors (location × insecticide treatment × plant part) for the imidacloprid residue level was present at the first sampling when plant parts were sampled separately (i.e., 66–68 days after sowing in 2015 and 76–85 days after sowing in 2016). A significant insecticide "treatment × location" interaction for imidacloprid residues was not observed in the first and the last two samples in 2015 (Tables 4 and 5), while in 2016 the significant interaction was only observed when samples were taken 76 to 85 days after sowing (Tables 6 and 7). For all other sampling data, the significant interaction "insecticide treatment × location" did not exist for imidacloprid residues. Significant interactions between "location × plant part" and "insecticide application × plant part" for imidacloprid residues existed only occasionally in both years of the study.

Figure 2 shows a degradation dynamic of thiamethoxam (expressed as sum of thiamethoxam and clothianidin) in sugar beet plants.

The maximum residue level (MRL) for thiamethoxam and clothianidin has been reduced in Europe from 0.05 mg/kg to 0.02 mg/kg in 2017 (EU 2017/671) [37]. For sugar beets grown under field conditions, the residue content of thiamethoxam in the leaves and roots of sugar beets dropped below the MRL between 70 and 80 days after sowing, depending on the year and location (Figure 2). No residues were found in sugar beet roots in open field cultivation at the time of harvest.

Similar to imidacloprid, the degradation of thiamethoxam was much slower in greenhouse trials. The residues of thiamethoxam in sugar beet roots in greenhouse cultivation were above the MRL (i.e., 0.053 mg/kg) at harvest time in 2015 (Figure 2), while in 2016, 100 days after sowing, the residues fell below the MRL of 0.02 mg/kg in 2016.

The results of the statistical analysis are presented in Tables 8–11. Residue levels were significantly affected by thiamethoxam treatment at all sampling dates including the last sampling in 2015, indicating that degradation at harvest is not complete in all trials. At the time of harvest in 2015, residues (0.053 mg/kg) were confirmed in beet roots grown in greenhouses (see Figure 2).

**Figure 2.** Degradation dynamics of thiamethoxam (expressed as sum of thiamethoxam and clothianidin) during the growing seasons 2015 (**a**) and 2016 (**b**) in sugar beet plants in Lukac, Tovarnik and in greenhouse trials, in compliance with the maximum permitted residue level of 0.02 mg/kg; LOQ— limit of quantification; MRL—maximum residue level.


**Table 8.** Thiamethoxam (including chlothianidin) residues in the whole plants during the first three observing periods and for roots at harvesting in 2015.

Analysis of variance for thiamethoxam residues in the whole sugar beet plants and root. \* significant at *p* = 0.05, \*\* significant at *p* = 0.01.


**Table 9.** Thiamethoxam (including chlothianidin) residues in different plant parts during the vegetation period in 2015.

Analysis of variance for thiamethoxam residues in different plant parts \* significant at *p* = 0.05, \*\* significant at *p* = 0.01.

**Table 10.** Thiamethoxam (including chlothianidin) residues in the whole plants during the first three observing periods and for roots at harvesting in 2016.


Analysis of variance for thiamethoxam residues in the whole sugar beet plants and root. \* significant at *p* = 0.05, \*\* significant at *p* = 0.01.


**Table 11.** Thiamethoxam (including chlothianidin) residues in the different plant parts during the vegetation period in 2016.

Analysis of variance for thiamethoxam residues in different plant parts. \* significant at *p* = 0.05, \*\* significant at *p* = 0.01.

In 2016, residue levels were significantly affected by thiamethoxam treatment on all but the last sampling dates, indicating that degradation at harvest was complete under all conditions studied, including greenhouse trials. In 2015, residues of thiamethoxam were significantly influenced by location (i.e., agroclimatic conditions) at almost all sampling dates except the first sampling (Table 8). In 2016, residues were significantly influenced by the location (Tables 10 and 11) on only two samples (76–85 and 92–97 days after sowing), when residues were significantly higher under greenhouse conditions in Zagreb (Figure 2). The third factor (plant part) was observed in three samples. In 2015

the residues of thiacloprid were significantly influenced by plant parts in three samples (Table 9), whereas in 2016 the residues were not influenced by plant parts at all (Table 11). A significant insecticide "treatment × location" interaction for thiamethoxam residues was observed in 2015 in all samples (Tables 8 and 9), while in 2016 the significant interaction was observed in only two samples taken after 76–85 days and 92–97 days after sowing (Table 11). Significant interactions between "location × plant part" and "insecticide application × plant part" for thiacloprid residues were complete in 2015. In 2016, these interactions only existed on a single sampling date for the "location × plant part" interaction. The significant interaction between all three factors (location × insecticide treatment × plant part) for thiacloprid residue level existed in 2015 for all three samples and in 2016 for only one sample when plant parts were sampled separately (i.e., 76–85 days after sowing in 2016).
