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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
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:
yes
Remarks:
Prior to test initiation, the soil was preincubated at room temperature at 39% of its WHCmax for at least two days in plastic containers (protocol limit is 40 % to 60 %). However, this deviation has no impact the integrity of the study.
Qualifier:
according to
Guideline:
OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)
Deviations:
yes
Remarks:
Prior to test initiation, the soil was preincubated at room temperature at 39% of its WHCmax for at least two days in plastic containers (protocol limit is 40 % to 60 %). However, this deviation has no impact the integrity of the study.
GLP compliance:
yes
Analytical monitoring:
no
Vehicle:
yes
Details on preparation and application of test substrate:
AMENDMENT OF SOIL
- Type of organic substrate: Lucerne meal: 5 g/kg soil dry weight (nitrogen determination); Glucose: 2 g/kg soil wet weight (short-term respiration study)

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: 260 mg of the test item were dissolved in 25 ml acetone. Appropriate amounts of this stock solution were applied onto quartz sand and the solvent was allowed to evaporate overnight. The treated quartz sand was then mixed thoroughly into the soil. Pure water was added to the soil to reach the desired range of soil moisture, resulting in a final water content of 48% to 51% of the WHCmax. After the addition of lucerne meal (for nitrogen transformation) and the test substance to the soil, the soil samples were thoroughly mixed by a mixer and divided in 3 replicates each.

VEHICLE:
- Chemical name of vehicle: Acetone and fine quartz sand
- Evaporation of vehicle before use: yes
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20 ± 2°C
Moisture:
Nitrogen transformation test: 48% to 51% of the WHCmax
Carbon transformation test: 47% to 49% of the WHCmax
Details on test conditions:
TEST SYSTEM
- Testing facility: ibacon GmbH, Arheilger Weg 17, 64380 Rossdorf, Germany
- Test container: Nitrogen transformation test: 500 mL plastic boxes with perforated plastic lids containing approximately 400 g soil dry weight; Respiration test: 1000 mL plastic boxes, with perforated plastic lids containing approximately 800 grams of soil dry weight
- No. of replicates per concentration: 3
- No. of replicates per control: 3

SOIL INCUBATION
- Method: After treating the soil, three equally sized soil sub-samples from each treatment group were placed in appropriate plastic boxes. The boxes were covered by perforated lids to enable a slight, but sufficient air exchange and weighed for the determination of the starting weight. The plastic boxes were incubated at 20 ± 2°C in the dark.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site: longitude 8° 44' 38.70'' E; latitude 49° 51' 59.59'' N
- History of site: For at least four years prior to test initiation, no pesticides had been used on the soil. No organic or mineral fertilizer had been applied to the soils for two years prior to study initiation, respectively.
- Vegetation cover: One single, common, agricultural soil type was used
- Depth of sampling: 0.05 to 0.2 m
- Soil texture: Silty sand
- % sand: 63.2
- % silt: 29.3
- % clay: 7.5
- pH: 7.0
- Maximum water holding capacity (in % dry weigth): 37.4
- Cation exchange capacity (mmol/kg): 43
- Pretreatment of soil: For the nitrogen transformation the soil was thoroughly mixed with the ground lucerne meal before application. The final concentration of the dried lucerne meal was 0.5% of the soil dry weight. For the short-term respiration the amount of glucose needed to obtain maximum shortterm rates of respiration in the test soil was determined periodically in the soil batch used for the reference test. A concentration of 2 g glucose/kg soil wet weight was found to be the optimum and was used in the test.
- Storage (condition, duration): 20 ± 2°C
- Initial microbial biomass as % of total organic C: 2.24

DETAILS OF PREINCUBATION OF SOIL: The sieved soil was pre-incubated at 39% of its maximum water hold capacity in ventilated boxes at about 20°C before used in the experiment

EFFECT PARAMETERS MEASURED: Samples were collected for determination of nitrogen transformation and soil respiration at days 0, 7, 14, and 28 following application of the test item.

VEHICLE CONTROL PERFORMED: yes
Nominal and measured concentrations:
Nominal: 1.94 (Test Rate 1) and 19.37 mg/kg soil dry weight (Test Rate 2)
Reference substance (positive control):
yes
Remarks:
Sodium chloride
Key result
Basis for effect:
nitrate formation rate
Remarks on result:
other: Deviations in nitrate levels/nitrate formation rates and rates of short-term respiration at day 28 (soil treated with ≥19.37 mg test item/kg soil dw were low (< 25% compared with control) indicating no effect occurred in carbon or nitrogen transformation.
Details on results:
Effects on the nitrate content of the test substance at concentrations of 1.94 mg and 19.37 mg test item/kg soil dry weight were significant when compared to the controls for the high test item concentration at day 28. At the end of the nitrate study (day 28), the deviations of the test item compared to the control soil were -2.03%, and 8.53% and therefore below the threshold of 25% specified in the OECD guideline 216.
Both nitrate formation rates were below the threshold of 25% according to the OECD guideline 216 at the end of the study. On day 28, the deviations to control were -2.93% and 9.99% for the cumulative nitrate formation rate (statistically significant when compared to the control for the high rate) and 6.68% and 13.92% for the incremental nitrate formation rate (statistically significant when compared to the control for the high rate) for the test concentrations of 1.94 mg/kg soil dry weight and 19.37 mg/kg soil dry weight, respectively.
At the end of the soil respiration study (day 28), deviations of the test item concentration compared to the control were within the 25% range specified by the OECD guidelines up to and including 19.37 mg test item/kg soil (dry weight equivalent). At day 28, the short-term respiration rates in soil treated with the test substance was not statistically significantly different from the control for both test rates and differed from the control by 10.26% and 1.31% in the 1.94 mg/kg soil dry weight and 19.37 mg/kg soil dry weight treatments.
Results with reference substance (positive control):
- Results with reference substance valid: Yes
- Other: the reference item sodium chloride (tested at least once a year) had a retarding or stimulating effect of more than ± 25% compared to the control at day 28 after application; the validity criterion according to the OECD test guidelines was met.
Reported statistics and error estimates:
The results of the nitrogen transformation (nitrate levels) and the short-term respiration measurements were submitted to a statistical analysis procedure for comparison of samples using the pair wise comparison-t-test for significant and non-significant differences between the control and the study groups.

Summary of Effects of the test substance on Nitrate Formation and Short-term Respiration in Soil 

Test substance Concentrationa

NO3-N levels

(day 28)

Nitrate formation

rate

(day 14 to 28)

Nitrate formation

rate

(day 0 to 28)

Respiration rate

(day 28)

mg/kg

sdw

% Dev.

from

controlb,c

mg/kg

sdw/d

% Dev.

from

controlb,c

mg/kg

sdw/d

% Dev.

from

controlb,c

mg CO2

/h/sdw

% Dev.

from

controlb,c

Control

42.380

---

1.257

---

1.261

---

15.009

---

1.94 mg/kg sdw

(Test Rate 1)

41.519

-2.03

1.341

6.68

1.224

-2.93

16.549

10.26

19.37 mg/kg sdw

(Test Rate 2)

45.994*

8.53

1.432*

13.92

1.387*

9.99

15.205

1.31

aTest item concentrations correspond to 1 time and 10 time the application rate (single maximum application rate is 250 g ai/ha)

bNegative value =% inhibition, positive value =% stimulation

cStatistical evaluation (Student t-test, two sided, a = 0.05): *significant differences from the control sdw: soil dry weight

Validity criteria fulfilled:
yes
Conclusions:
The test substance has no effect on nitrogen transformation or respiration at concentrations up to and including 19.37 mg/kg soil dry weight (corresponding to 10 times the application rate)
Executive summary:

The effects of the test substance on soil microflora were assessed in a test that measured nitrogen transformation and short-term respiration following an application of the test substance to soil. The test was conducted in accordance with the OECD-Guideline for the Testing of Chemicals, Soil Microorganisms: Nitrogen Transformation Test, Guideline 216 and OECD-Guideline for the Testing of Chemicals, Soil Microorganisms: Carbon Transformation Test, Guideline 217, dated 21 January 2000.

The test substance was incubated in a silty sand soil over a period of 28 days (carbon transformation and nitrogen transformation) at concentrations of 1.94 mg and 19.37 mg/kg soil dry weight (Test Rate 1 and Test Rate 2, respectively). Deviations in nitrate levels and nitrate formation rates at day 28 and rates of short-term respiration at day 28 in soil treated with up to and including 19.37 mg test item/kg soil dry weight were low (< 25% compared with control) indicating no effect occurred in carbon or nitrogen transformation.

Effects on nitrogen transformation and respiration at all test concentrations of the test substance were below the effects threshold specified by the OECD guidelines at day 28(deviations from control were <25%). Therefore the test substance has no effect on nitrogen transformation or respiration at concentrations up to and including 19.37 mg/kg soil dry weight (corresponding to 10 times the application rate).

Description of key information

The test substance has no effect on nitrogen transformation or respiration at concentrations up to and including 19.37 mg/kg soil dry weight (corresponding to 10 times the application rate); OECD 216, OECD 217; Reliability = 1

Key value for chemical safety assessment

Additional information