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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:
Q4 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP OECD 222 study with all validity criteria fulfilled and no deviation.
Qualifier:
according to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable.
Analytical monitoring:
yes
Details on sampling:
On day 0 and on day 28, the water content and the nitrate content of each replicate was determined. From each replicate, a sample containing 12.5 g wet soil was taken for nitrate analysis.
Vehicle:
no
Details on preparation and application of test substrate:
The test system was delivered by LUFA Speyer, Obere Langgasse 40, 67346 Speyer. Major components were sand (around 58%) and silt (around 35%). Water holding capacity (WHC) was 35.4%. The soil was not treated with plant protection products within the last year and no organic fertilizer was applied for at least 6 months. No inorganic fertilizer was applied for at least 3 month as required in the guideline.
The soil used was freshly collected and stored in the test facility for 1 week under aerobic conditions at 18.8 - 21.1 °C. A mixture of 3 parts of whole wheat flour and 1 part of soya flour with a C/N ratio of 12.49/1 (determined by elemental analysis performed at Mikroanalytisches Labor Pascher, Rema-gen, Germany) was used as a nitrogen source for the test.
The soil was divided into six portions of 900 g. The required amount of 4.3 g organic substrate was given to each portion and the soil samples were homogenised. To each portion the required amount of test item was added using 87 mL deionised water which was also used for adjustment of the soil moisture and then thoroughly mixed. Each portion was divided into 3 replicates à 300 g and incubated in glass jars. The amount of organic substrate corresponded to the recommended value in the guideline (5 g substrate per 1000 g dry weight of used test soil)
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
17.9°C - 21.8°C min - max
Moisture:
approx. 41% of WHC.
Details on test conditions:
TEST SYSTEM
- Testing facility:
- Test container (type, material, size): glass jar of 1 L
- Amount of soil: 300 g
- No. of replicates per concentration: 3
- No. of replicates per control: 3

SOIL INCUBATION
- Method: The replicates were left to stand for 28 days. During this period, the replicates were manually homogenised twice a week. Each week, the water content of each test vessel was adjusted to maintain the water content of the start of the test.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Offenbach, Germany
- History of site: Site not used in the past two years
- Vegetation cover: Uncultivated
- Treatments with pesticides or fertilizers: None for at least one year
- Accidental contamination: None known
- Depth of sampling: 20 cm
- Soil texture
- % sand: 54.7 - 60.5%
- % silt: 32.7 - 39.1%
- % clay: 4.9 - 8.3%
- Soil taxonomic classification: Silty sand
- pH (in water): 5.7
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight):
- Maximum water holding capacity (in % dry weigth): 35.4%
- Cation exchange capacity (meq/100g): 7.5 ± 0.9
- Storage (condition, duration): The soil used was freshly collected and stored in the test facility for 1 week under aerobic conditions at 18.8 - 21.1 °C
- Initial microbial biomass as % of total organic C: 1.90 - 2.08 % of organic carbon

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : NO3 at D0 and D28
Nominal and measured concentrations:
0 / 10 / 32 / 100 / 320 / 1000 mg/kg dry soil (nominal concentrations). Concentrations were not measured (not requested by the guideline)
Reference substance (positive control):
no
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
320 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
nitrate formation rate
Details on results:
The quantities of nitrate in mg nitrate/kg dry weight soil formed in the treated samples after 28 days were compared to that formed in the control. From these data, the % inhibition values for each test concentration were calculated. “Nitrate formed” means “nitrate measured at D28” minus “nitrate measured at D0”. The % inhibition values were expressed in % of the nitrate formed in the control group.
Results with reference substance (positive control):
None.
Reported statistics and error estimates:
Calculation of results was performed with the help of validated software. The estimation of the biological results was accomplished using the software ToxRat® Professional, version 3.2.

Table 1: Nitrate production in % compared to blank control

Concentration in mg/kg
dry matter

Inhibition of Nitrate formation in % compared to blank control

10 mg/kg

-11.2

32 mg/kg

-19.3

100 mg/kg

-60.2

320 mg/kg

-95.8

1000 mg/kg

168.0

Negative inhibition implies a boost of nitrate production compared to the blank control.

Validity criteria fulfilled:
yes
Conclusions:
The study was performed using 5 treatments of 10 / 32 / 100 / 320 / 1000 mg/kg nominal concentration. Sieved soil was exposed to the test item for 28 days. After 0 and 28 days, the nitrate content of the soil of each replicate was determined. A blank control was tested. Only the highest concentration showed significant inhibition of nitrate production compared to the control. NOEC was determined as 320 mg/kg soil dry weight.
Executive summary:

The study was performed according to the OECD 216 guideline. The experimental design was in triplicate, using 5 treatments of 10 / 32 / 100 / 320 / 1000 mg/kg soil dry weight nominal concentration and a control. A sieved natural soil was used. Each replicate was exposed to a relevant amount of test item for 28 days. After 0 and 28 days, the nitrate content of the soil of each replicate was determined. A blank control was tested. Only the highest concentration showed significant inhibition of nitrate production compared to the control. NOEC was determined as 320 mg/kg soil dry weight.

Description of key information

The study was performed using 5 treatments of 10 / 32 / 100 / 320 / 1000 mg/kg nominal concentration. Sieved soil was exposed to the test item for 28 days. After 0 and 28 days, the nitrate content of the soil of each replicate was determined. A blank control was tested. Only the highest concentration showed significant inhibition of nitrate production compared to the control. NOEC was determined as 320 mg/kg soil dry weight.

Key value for chemical safety assessment

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

Additional information

The study was performed according to the OECD 216 guideline. The experimental design was in triplicate, using 5 treatments of 10 / 32 / 100 / 320 / 1000 mg/kg soil dry weight nominal concentration and a control. A sieved natural soil was used. Each replicate was exposed to a relevant amount of test item for 28 days. After 0 and 28 days, the nitrate content of the soil of each replicate was determined. A blank control was tested. Only the highest concentration showed significant inhibition of nitrate production compared to the control. NOEC was determined as 320 mg/kg soil dry weight.