Amitrole

Biodegradation in soil

Description of key information

Two studies similar to OECD 307 are available for the biodegradation of amitrole in soil.

The objective of the key study (Hall, 1994) was to investigate the route and rate of degradation of [14C]-amitrole in soil under aerobic conditions at nominal temperatures at 7 and 22°C. The degradation half-life calculated at 22°C using aqueous ethanol extracts profiled by TLC was 20.5 days; the equivalent value by HPLC was 22.5 days. Incorporation of Soxhlet data resulted in half-life (TLC) of 25.9 days.

The supporting study (Hawkins, 1982) confirmed these results and showed that 14C-aminotriazole was rapidly and almost completely degraded when applied to aerobic soil stored at 25°C in the dark

Key value for chemical safety assessment

Half-life in soil:

25.9 d

at the temperature of:

22 °C

Additional information

Two studies similar to OECD 307 are available for the biodegradation of amitrole in soil:

The objective of the key study (Hall, 1994) was to investigate the route and rate of degradation of [14C]-amitrole in soil under aerobic conditions at nominal temperatures at 7 and 22°C. Either [14C]-amitrole or non-labelled amitrole was applied to samples of a borderline sandy-loamy sand (100 g oven dry weight equivalent) at an application rate of 0.8 mg per sample (representing a field application rate of 8 kg/ha). The treated soil samples were divided into study groups identified as A-F. Non-specific radiolabelled volatiles were maintained at a moisture level equivalent to ca 75% of moisture holding capacity (at 0.33 bar). The nature of the extractable radioactivity from samples in study groups A, C and D was investigated by TLC and/or HPLC.

Study Group A (viable soil, incubation temperature ca 22°C)

Overall mean recoveries of applied radioactivity ranged from 90.22% to 99.97% over the 52 week incubation period. The recovery of radioactivity from ethanolamine traps, as 14CO2, increased steadily throughout incubation reaching a maximum mean value of 58.39% at 52 weeks. The evolution of other non-specific radiolabelled volatiles was negligible (=<0.13%) throughout the incubation period.

The mean percentage of applied radioactivity extracted by aqueous ethanol was relatively constant at 74.22 -79.03% during the initial 3 days of incubation but subsequently declined to a minium value of 1.86% at week 26; thereafter extractability remained relatively constant.

The levels of non-extractable (by aqueous ethanol) radioactivity also remained relatively constant (mean range 11.02% to 15.40%) during the initial 3 days of incubation before gradually increasing to a mean of 43.11% after 13 weeks. Further incubation had relatively little effect on the magnitude of the residue. A further mean of 7.43% to 13.94% of the applied radioactivity was extracted by ethanolic Soxhlet from those residues remaining from samples up to Day 30. After this time Soxhlet extractability declined markedly to a minimum mean value of 0.66% at week 52. Extraction of a zero-time (flask 2) and 7 day (flask 12) sample with aqueous ethanol (pH 2.5) and methanol:acetone: water (1:1:1 by volume, pH 2.5) prior to Soxhlet residue of flask 12 with 0.01M potassium bromide ina queous methanol demonstrated the effectiveness of the Soxhlet extraction procedure.

Degradate profiling using thin layer chromatography (TLC) indicated that amitrole and a minimum of 2 radiolabelled degradates were presnet in aqueous ethanol extracts. Amitrole was the sole/main component present in these extracts during the earlier phases of incubation accounting for a mean of >=94.94% of the extractable radioactivity (>= 35.49% of the applied) in samples incubated for up to 21 days. thereafter the relative contribution of amitrole decreased ultimately accounting <1% of the applied radioactivity from weeks 26 onwards. A radiolabelled degradate, designated B, was present in extracts of those samples incubated for 7 days or longer. By the end of 52 week incubation period, this degradate comprised a mean 90.78% of the extractable radioactivity (2.01% of the applied). TLC examination of filtered concentrated extracts showed Degradate B to be comprised of a solubilized degradate and particulate bound radioactivity. only one refernce compound, urazole, possessed similar chromatographic properties to Degradate B.

TLC analysis also indicated the presence of a minor radiolabelled degradate, designated C, in week 8 and 13 extracts.The mean contribution of degradate C did not exceed 0.3% of the applied dose.

Soxhlet extracts of study Group A samples were shown by TLC to contain amitrole and Degradate B. For the majority of samples incubated fro 21 days or less, amitrole comprised >90% of the Soxhlet extractable radioactivity. By week 52, the mean proportion of Soxhlet extract attributable to amitrole was 39.54% (0.26% of applied). The mean contribution of degradate B remained relatively low initially before gradually increasing to account for 37.89% of the Soxhlet extractable radioactovity by week 52. At no time did degradate B comprise greater than a mean of 0.59% of the applied radioactivity.

Degradate profiling by high liquid chromatography (HPLC) indicated that amitrole and a minimum of 3 radiolabelled degradation products may be present in aqueous ethanol extracts. Amitrole was the only radiolabelled component present in extracts incubated for a period up to 10 days. Over this time period extractability declined and amitrole comprised between means of 52.04% and 69.87% of the applied radioactivity. Levels of amitrole declined to a mean of 35.84% of the extractable radioactivity (0.82% of applied) by week 52. Only one radiolabelled degradate was detectable by on-line techniques.Designated Degradte D, this component first appeared in day 14 samples and thereafter gradually increased throughout incubation accounting for 36.61% of the extractable radioactivity (0.81% of teh applied dose) by the end of teh 52 week incubation period. In terms of teh applied dose, the overall contribution of Degradate D remained relatively constant throughout the incubation period.

Two minor degradates, E and F, were detected by LSC in HPLC eluates of extracts from week 13 onwards. The levels of radioactivity associated with these components were very low, the majority of the constituent count rates being below 30 d.p.m., the limit of the reliable determination.

The degradation half-life calculated using aqueous ethanol extracts profiled by TLC was 20.5 days; the equivalent value by HPLC was 22.5 days. Incorporation of Soxhlet data resulted in half-life (TLC) of 25.9 days.

Study Group B (Viable Soil; 22°C)

The use of sodium hydroxide to collect evolved radiolabelled volatiles confirmed that 14CO2 was the sole radiolabelled component present in effluent gases from soil samples treated with [14C]-amitrole.

Study Group C (Sterile Soil; 22°C)

Total Viable Bacteria (TVB) analysis showed that, with 2 limited exceptions, sterile incubation conditios had been maintained for this study group.

Overall mean recoveries of applied radioactivity ranged from 85.77% to 91.73% over the 52 week incubation period. the evolution of 14CO2 was negligible (ca 1% at 52 weeks). No other radiolabelled volatiles were collected.

The mean percentage of applied radioactivity extracted by aqueous ethanol decreased from 55.24% at 30 days (the first sampling interval) to 19.16% at week 52. Decreases to extractability were accompanied by a concomitant increase in the level of non-extractable residue; ethanolic Soxhlet extraction removed a further 9.94%-18.42% of the applied radioactivity from the residue.

TLC-radioanalysis indicated that amitrole together witha minimum of 2 radiolabelled degradates were present in aqueous ethanol extracts of Study Group C samples. The contribution of amitrole ranged from a mean of 97.23% of the extractable radioactivity (50.20% of applied) at day 30 to 62.23% (9.98%) at week 52. The relative contribution of Degradate B increased throughout incubation eventually accounting for a mean of 34.39% of the extractable radioactivity (5.50% applied) by week 52.Minor quantities of Degradte C appeared during the latter stages on incubation. Ethanolic Soxhlet extracts contained primarily amitrole with only samall quantities of Degradate B appearing as incubation progressed.

Study Group D (Viable Soil; 7°C)

Total recoveries of apllied radioactivity were in the range 89.30% to 95.74%. The evolution of 14CO2 increased steadily during incubation ultimatelyaccounting for 44.17% of the applied radioactivity by 52 weeks. The evolution of non-specific radiolabelled volatiles was low, accounting for only 0.91% of the applied radioactivity at the end of the incubation period.

Aquaeous ethanol extractability was relatively constant during the initial 14 days of incubation (68.35 -79.27% of the applied radioactivity). Subsequent to this period, extractability gradually decreased to 4.69% by week 52. Non-extractable radioactivity accounted for between 12.28% and 15.01% applied radioactivity during the initial 3 days of incubation. Thereafter residue levels increased to a peak value of 51.86% at week 26 before decreasing slightly to 44.48% at Weeek 52.

TLC- and HPLC- radioanalysis indicated that amitrole and at least one radiolabelled degradte were present in aqueous ethanol extracts. TLC showed amitrole to account for => 9.33% of the extractable radioactivity in samples up to 13 weeks. Due to the decrease in extractability, only 31.15% of the applied radioactivity was accounted for by amitrole at week 13.By Week 52 amitrole accounted for 36.93% of teh extractable radioactivity (1.30% of teh applied). degradate B was presnet at low levels up to 13 weeks after which the levels, relative to amitrole, increased such that 60.19% of the extractable radioactivity (2.12% of the apllied) was attribuable to Degradtes B at week 52.

HPLC data were similar to those generated y TLC. Amitrole was the sole/main extractable component ins amples up to 26 weeks(=>91.76% of the extractable radioactivity). During this period, the extractable amitrole accounted for between 8.49% and 75.59% of the applied radioactivity. By week 52 amitrole comprised 50.66% of the extractable radioactivity, equivalent to 1.78% of that applied.

Degradte D apeared in samples from week 13 onwards. By week 52, Degradates D accounted for 33.62% of the extractable radioactivity (1.18% of the applied).

TLC and HPLC analysis of aqueous ethanol extracts provided similar data with respect to the calculation of the degradation half-life for amitrole. By TLC a value of 63.9 days was calculated; the corresponding value calculated from HPLC data was 68.8 days.

Study Groups E (Viable; 22°C) and F (Viable; 7°C)

TVB analysis of samples fom both these study groups demonstrated that soil samples at both study temperatures had remained viable throughout incubation.

The supporting study (Hawkins, 1982) showed that 14C-aminotriazole was rapidly and almost completely degraded when applied to aerobic soil stored at 25°C in the dark. In the case of german standard soil 2.2 about 70-75% of the applied radioactivity appeared as 14CO2 within 28 days. the remaining radioactivity became strongly bound to soil, possibly being incorporated into organic matter as single carbon fragments, and disappeared much more slowly. In the case of English loam soil the initial rapid production of 14CO2 accounted for 40-50% of the applied radioactivity with a correspondingly  higher proportion becoming bound to soil. Some minor radioactive components detected in extracts of soil may have been intermediates in the degradation pathway of 14C-aminotriazole, but these were also rapidly degraded  and never accounted for a significant proportion of soil radioactivity. Neither urea nor cyanamide, which are compounds which could conceivably arise from the breakdown of aminotriazole, were detected in soil extracts, altough this does not exclude them from the degradation pathway. A separate experiment showed that 14C-urea was almost completely converted to volatile products within 7 days of application to soil. urea could therefore  have been an intermediate degradation product of aminotriazole, but a separate pathway would have to exist to account for the bound radioactivity resulting from 14C-aminotriazole. Production of 14CO2 from soil was similar when either [3,5-14C]- or [5-14C]-aminotriazole was used showing that 14CO2 resulted from both carbon atomes in approximately equal proportions. This could suggest the existence of symmetrical compound in the degradation pathway of aminotriazole. However, urazole, a possible candidate, was not detected in soil extracts.