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Toxicity to soil microorganisms

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Reference
Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February-April 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
Soil Preparation
The soil moisture was determined about four days before initiation of the test, the soil was filled into incubation flasks and equilibrated at 20 ± 2°C in the dark. The soil moisture content was adjusted to 42% of the maximum water holding capacity (MWC) after the application.
Furthermore, all samples used for the determination of the nitrification process were amended with lucerne meal as a source of organic nitrogen. The lucerne meal contained approximately 3% nitrogen.

Incubation
After application, the test item, control, and dinoseb acetate treated samples were adjusted to 42% of the soil MWC (i.e 15.6 g water per 100 g dry soil). The samples were then incubated in the dark at 20 ± 2 °C. The incubation temperature was monitored continuously. The moisture content of the samples was monitored on a weekly basis and moisture loss was compensated by the addition of purified water. Thereafter, the soil samples were thoroughly mixed.

Application of Test Item
For the preparation of the high dose application solution, 7.97 mg of 1,2,4 Triazole was diluted with 150 ml purified water, resulting in a final concentration of 0.053 mg1,2,4 Triazole/ml. For the low dose application solution, a 2,5 ml aliquot of the high dose solution was transferred to a 25 ml flask and made up to the volume with purified water. This resulted in a final concentration of 0.0053 mg 1,2,4 Triazole/ml.

Before application, an aliquot of 1.5 g sand was added to the samples and the resulting mixture was mixed homogeneously. The application solutions were added dropwise to 150 g of soil (dry weight) with a Hamilton syringe. During application, the soil was thoroughly mixed. For the nitrification experiments, the soil samples were amended with 0.75 g lucerne meal (nitrogen content approximately 3%) after application. Finally, the soil moisture content of all samples was adjusted to 42% of the respective MWC by adding purified water.
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20 +/-2°C
Moisture:
42% of the maximum water holding capacity
Organic carbon content (% dry weight):
0.71
Nitrogen content (% dry weight):
0.09
Nominal and measured concentrations:
Preparation of the Application Solution
The test item taking into account the maximum formation from parent, was tested at a field rate concentration of 25 g 1,2,4 Triazole/ha and at 10 times this rate corresponding to 250 g 1,2,4 Triazole/ha.
The treatment dose per sample was calculated assuming uniform distribution of the test item in the top 5 cm of the soil and a bulk density of 1.5 g soil dry weight/cm3 using equation (1).

Low Dose
The soil was treated with the test item at the maximum recommended field rate i.e. 25 g 1,2,4 Triazole/ha. This rate corresponds to 0.033 mg test item/kg dry soil. The actual dose applied was 0.035 mg test item/kg dry soil.

High Dose
The soil was treated with the test item at the maximum recommended field rate i.e. 250 g 1,2,4 Triazole/ha. This rate corresponds to 0.333 mg test item/kg dry soil. The actual dose applied was 0.353 mg test item/kg dry soil.

Control
The control soil samples were adjusted to 42% of its respective MWC with purified water.

Addition of test item.
The application volume was one millilitre per 150 g dry soil.

Treatment Rate of Disoseb Acetate
Soil samples for the nitrification test were treated with 3.75 mg dinoseb acetate/150 g dry soil, correspond to 25 mg dinoseb acetate/kg dry soil.
Reference substance (positive control):
yes
Remarks:
Dinoseb Acetate
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
0.353 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Key result
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
0.353 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
respiration rate
Details on results:
see section any other information.

Results and Discussion

Microbial Biomass and Optimum Glucose Amendment

The maximum rate of initial CO2 evolution from 1 kg dry soil equivalents was 5.01 ml/h. By applying the formula of Anderson and Domsch (1978), the microbial biomass, expressed ad microbial carbon per kg of dry weight soil (microbial C/kg dry weight soil) was calculated to be 203 mg microbial carbon (C).

Amendments with different amounts of glucose/kg dry soil resulted in a maximum and constant respiration response (Figure 1). The lowest amount of glucose resulting in a maximum CO2 production, i.e. 1.16 g/kg dry soil was used for the short-term respiration experiment.

Glucose induced Short-Term Respiration

Respiration can be regarded as a measure of the general turn-over of organic matter in soil. Respiration levels were determined by monitoring glucose-induced evolution of CO2 resulting from microbial activity during short-term experiments (for approximately 24 hours each). The respiration rates during 12 consecutive hours, for treated and control soil, were compared in order to determine possible side effects of the test item on microorganisms. In the present study, the influence of 1,2,4 Triazole on glucose-induced soil respiration was monitored in short-term experiments over an incubation period of 28 days (35 days for dinoseb acetate and a control sample). The results are given in Table 2 and in Figures 2 to 5.

The rate of respiration (Table 2 and figure 2) at test initiation was 5.14 ml CO2/h per kg dry soil for the control, 5.38 ml CO2/h for the low dose and 5.43 ml CO2/h for the high dose treated samples (Table 2). In comparison to the control, increases in respiration of 4.7% and 5.7% were observed for the low and high doses rates, respectively, in comparison to the control. By day 14 (Figure 4) mean respiration was 7.7 % and 8.7% above the mean control respiration in the low and high dose samples, respectively (Table 2 and Figure 5).

After 28 days of incubation, the rate of respiration was 4.11 ml CO2/h for the control and 4.34 ml CO2/h and 4.45 CO2/h for the low and high dose treated samples, respectively. This resulted in a deviation of low and high dose treated samples from the control of 5.5% and 8.3% respectively. Thus, no significant influence of 1,2,4 Triazole on microbial respiration in soil was observed.

The dinoseb acetate treated samples showed an increasing effect when compared to the non-treated samples. The deviation from the control decreased from -39.6% on day 0 to 58.1% (day 7), 61.1% (day 14), 62.1% (day 35), cf. Table 2 and Figures 2 to 5. Thus, the reference item showed an important inhibitory effect.

According to Malkomes scheme, rates up to ten times the maximum field application rate of 1,2,4 Triazole result in "negligible" effects on soil respiration. Dinoseb acetate results in tolerable to critical effects on soil respiration, the effect on microorganisms increases.

Nitrification of Lucerne Meal

Initially, no nitrite (NO2--N) and 7.71 mg nitrate (NO3--N) per kg dry soil were detected in untreated and unamended soil (Table 1).

On day 0 for the control, low dose and high dose treated samples, the NO2--N concentration was 0.1 mg nitrite per kg dry soil. On days 7, 14 and 28 the NO2--N concentrations for these samples was below the limit of determination (<0.1 mg/kg dry soil). Thus, the treatment with 1,2,4 Triazole had no influence on the nitrite formation and transformation (Tables 3 and 4). In the dinoseb acetate treated samples the amount of NO2--N per kg dry soil reached 0.1 mg, 1.3 mg, 11.4 mg and 0.1 mg on days 0, 7, 14 and 28 respectively.

The mean initial concentration of nitrate-nitrogen was 8.2 mg for the control and 7.4 mg and 7.3 mg for the low and high dose treated samples, respectively. Mean nitrate levels decreased until day 7 and thereafter increased in all samples types, reaching 13.5 mg, 12.8 mg and 13.3 mg, respectively, at day 28.The calculated deviation to control was -5.2% and -1.5%, respectively.

The nitrate concentration at day 28 for the dinoseb acetate treated samples was 25.3 mg NO3--N/kg dry soil (Table 3). This resulted in a deviation of 87.4% to control (Table 4). Thus, the reference item dinoseb acetate showed a very strong stimulating effect.

In the Malkomes scheme the results for 1,2,4 Triazole range in the area of a negligible effect for day 28 (cf. figure 7). The results for dinoseb acetate after 28 days range in the area of an intolerable effect. A graphical presentation of NO3--N values are presented in figure 8.

Validity criteria fulfilled:
yes
Conclusions:
1,2,4 Triazole showed no effect on the soil respiration as measured by CO2 evolution. An impairment of the carbon cycle in the soil is not to be expected after application of 1,2,4 Triazole up to a concentration in the soil corresponding to ten times the maximum expected dose rate.
1,2,4 Triazole also had no influence on the nitrification process in soil. It may be conducted that no adverse effects of the test item on organic matter turnover, and hence on soil fertility, are to be expected from its use according to the label recommendations.
The reference item dinoseb acetate had an important influence on the microflora thereby showing the sensitivity of the test system and validity of the experimental design.
Executive summary:

The influence of 1,2,4 Triazole on soil microorganisms was determined in control and treated soil samples by measuring the microbial CO2 evolved during short-term respiration experiments after glucose amendments. Furthermore, its influence on the nitrification of lucerne meal, was investigated.

In this study, one fresh agricultural soil, a sandy loam, was moistened to 42% of its maximum water-holding capacity and incubated in the dark at 20 ±2°C following treatment with the test item.

The two application rates are equivalent to doses of 0.033 mg and 0.333 mg 1,2,4 Triazole/kg dry soil, respectively.

On the basis of the results obtained, it can be concluded that 1,2,4 Triazole will cause no adverse effect on organic matter turnover, and hence on soil fertility, even at rates up to ten times the recommended field rate.

Description of key information

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:
0.353 mg/kg soil dw

Additional information

1,2,4 Triazole showed no effect on the soil respiration as measured by CO2 evolution. An impairment of the carbon cycle in the soil is not to be expected after application of 1,2,4 Triazole up to a concentration in the soil corresponding to ten times the maximum expected dose rate.

1,2,4 Triazole also had no influence on the nitrification process in soil. It may be conducted that no adverse effects of the test item on organic matter turnover, and hence on soil fertility, are to be expected from its use according to the label recommendations.

The reference item diseb acetate had an important influence on the microflora thereby showing the sensitivity of the test system and validity of the experimental design.