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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:
28th of February 2019 to 19th of July 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Version / remarks:
OECD, 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Details on sampling:
Analysis for the determination of the concentration of nitrate after 0 and 28 days of incubation:
- Concentrations: 1.2, 3.7, 11, 33, and 100 mg/kg dry weight soil.
- Sampling method: At each sampling interval, a subsample was taken from each test sample and transferred to a Nalgene bottle. The subsamples were extracted with potassium chloride solution on a shaker table set. Following the extraction, the samples were centrifuged and the supernatant was decanted and each sample was additionally diluted with purified reagent water. An aliquot of the diluted extract was centrifuged and transferred to an HPLC vial. These vials were analyzed using a validated HPLC-UV method to quantify nitrate concentration against a nitrate standard calibration curve.
- Sample storage conditions before analysis: not specified
Vehicle:
no
Details on preparation and application of test substrate:
AMENDMENT OF SOIL
- Type of organic substrate: Speyer 2.3 (sandy loam)
Sand content: 68%
pH in water: 6.5
Organic carbon content: 0.60%
Microbial biomass: 2.2% of total organic carbon

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: Due to the limited water solubility of the test substance and because the test substance is a liquid at ambient conditions, the test substance was applied directly to soil without a carrier.
Six 1.0-kg (dry weight equivalent) batches of soil were weighed out into beakers. Starting with the control, each batch of soil was dosed and mixed as follows. Approximately half of the soil batch was transferred to a 4-L jar. An appropriate amount of the test substance was dosed dropwise across the soil surface using a pipette. The remaining half of the soil batch was then added to the 4-L jar. The soil was mixed by rolling the capped jar for approximately 3 hours at approximately 15 rpm on a rolling mill. The fortified soil was transferred to the bowl of a food mixer and mechanically mixed for 10 minutes at low speed. During this mechanical mixing, the soil was amended with 5.0 g of lucerne meal (i.e. alfalfa). Also during this mechanical mixing a volume of purified reagent water was added to the soil in order to bring the moisture content to 45 ± 5% of the soil’s maximum water holding capacity. After dosing and mixing, each batch of soil was divided equally into three replicate test samples in 1-L glass bottle test vessels.
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20 ± 2 °C
Moisture:
45 ± 5%
Details on test conditions:
TEST SYSTEM
- Testing facility: Smithers Viscient
- Test container: Jar, no more details
- Amount of soil: 1.0 kg of dry weight equivalent batches of soil
- No. of replicates per concentration: 3
- No. of replicates per control: 3
- No. of replicates per vehicle control: n/a

SOIL INCUBATION
- Method: Series of individual subsamples occured after 0 and 28 days of incubation.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site: Offenbach, Rheinland-Pfalz, Germany “Rechts der Landauer Str.”, No. 826/7
- History of site: Not specified
- Vegetation cover: not specified
- Treatments with pesticides or fertilizers: None used within the last 4 years
- Accidental contamination: No
- Other:
- Depth of sampling: 0-20 cm
- Soil texture
- % sand: 68
- % silt: 26
- % clay: 6
- Soil taxonomic classification: sandy loam
- Soil classification system: not specified
- pH (in water): 6.5
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 93.9 mg/kg
- Maximum water holding capacity (in % dry weight): 26.4
- Cation exchange capacity (mmol/kg): 57
- Pretreatment of soil: not specified
- Storage: The test substance was determined to be stable for 66 months under ambient storage conditions
- Initial microbial biomass as % of total organic C: 2.2

DETAILS OF PREINCUBATION OF SOIL: n/a

EFFECT PARAMETERS MEASURED: n/a

VEHICLE CONTROL PERFORMED: n/a

RANGE-FINDING STUDY
- Test concentrations: n/a
- Results used to determine the conditions for the definitive study: n/a
Nominal and measured concentrations:
Nominal concentrations were 0, 1.2, 3.7, 11, 33, and 100 mg/kg dry weight soil.
Reference substance (positive control):
no
Key result
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
> 100 mg/kg soil dw
Nominal / measured:
nominal
Basis for effect:
nitrate formation rate
Remarks on result:
other: No long-term effects on the nitrogen transformation rates of soil microflora at environmentally-relevant concentrations were observed.
Reported statistics and error estimates:
Relative standard deviation in nitrate concentration and formation rate for the untreated control samples ranged from 0.4 to 1.5%, meeting the 15% validity criteria.
The nitrate formation rates were input into CETIS (Ives, 2019). Differences between the untreated control and the treated soils were tested for significance using the ANOVA and Dunnett’s Multiple Comparison Test statistical programs. All statistical conclusions were made at the 95% level of certainty. The nitrate formation rates were analyzed using the linear interpolation (ICPIN) statistical program in CETIS to determine EC10, EC25, and EC50 values. The results were investigated for outliers using the Grubbs Extreme Value Test statistical program in CETIS.

Table 1 Summarised nitrate concentrations and formation rates

Test System

Mean Day 0 Nitrate

Concentration

(mg/kg)

Mean Day 28 Nitrate

Concentration

(mg/kg)

Mean Nitrate

Formation Rate

(mg/kg/day)

Rate Difference

from Untreated

Control (%)

 Untreated control

 93.9

 286

 6.87

 NA *

 1.2 mg/kg

 94.6

 268

 6.21

 -9.63

 3.7 mg/kg

 93.8

 266

 6.14

 -10.7

 11 mg/kg

 94.0

 280

 6.62

 -3.6

 33 mg/kg

 93.4

 272

 6.39

 -6.96

 100 mg/kg

 94.8

 272

 6.33

 -7.95

* NA = Not Applicable

Table 2 - Nitrate Concentrations and Formation Rates

Test System

Sample ID F0319-

Day 0 Nitrate

Concentration

(mg/kg)

Day 28 Nitrate

Concentration

(mg/kg)

Nitrate Formation

Rate

(mg/kg/day)

Untreated Control

110

93.9

288

6.91

Untreated Control

111

93.4

282

6.73

Untreated Control

112

94.3

289

6.97

Untreated Control

Mean:

93.9

286

6.87

Untreated Control

SD:

0.37

3.18

0.10

Untreated Control

RSD:

0.39

1.11

1.47

1.2 mg/kg

113

95.1

277

6.51

1.2 mg/kg

114

94.2

271

6.31

1.2 mg/kg

115

94.5

257

5.81

1.2 mg/kg

Mean:

94.6

268

6.21

1.2 mg/kg

SD:

0.37

8.44

0.30

1.2 mg/kg

RSD:

0.40

3.14

4.75

3.7 mg/kg

116

94.1

263

6.03

3.7 mg/kg

117

92.6

266

6.18

3.7 mg/kg

118

94.7

269

6.21

3.7 mg/kg

Mean:

93.8

266

6.14

3.7 mg/kg

SD:

0.88

2.25

0.08

3.7 mg/kg

RSD:

0.94

0.85

1.25

11 mg/kg

119

94.6

280

6.64

11 mg/kg

120

93.8

277

6.54

11 mg/kg

121

93.7

281

6.70

11 mg/kg

Mean:

94.0

280

6.62

11 mg/kg

SD:

0.40

1.87

0.06

11 mg/kg

RSD:

0.43

0.67

0.98

33 mg/kg

122

94.1

263

6.03

33 mg/kg

123

93.0

275

6.49

33 mg/kg

124

93.2

280

6.66

33 mg/kg

Mean:

93.4

272

6.39

33 mg/kg

SD:

0.48

7.02

0.27

33 mg/kg

RSD:

0.51

2.58

4.16

100 mg/kg

125

93.3

280

6.67

100 mg/kg

126

95.8

283

6.68

100 mg/kg

127

95.2

253

5.63

100 mg/kg

Mean:

94.8

272

6.33

100 mg/kg

SD:

1.07

13.60

0.50

100 mg/kg

RSD:

1.12

5.00

7.83

Ta

Validity criteria fulfilled:
yes
Conclusions:
In a soil microorganism toxicity study, conducted according to the appropriate OECD TG 216 and in compliance with GLP, the reported EC10 value was empirically estimated to be >100 mg/kg dw based on the nitrogen transformation rate of soil microflora.
Executive summary:

In a soil microorganism toxicity study, conducted according to the appropriate OECD TG 216 and in compliance with GLP, the reported EC10 value was empirically estimated to be >100 mg/kg dw of the nitrogen transformation rate of soil microflora. This was based on no observed effects at highest concentration. Furthermore, it is expected that the test substance does not have any long-term effects on the nitrogen transformation rates of soil microflora at environmentally-relevant concentrations.

Description of key information

28-day EC10 >100 mg/kg dry weight in accordance with OECD TG 216 based on nitrogen transformation rate.

Key value for chemical safety assessment

Additional information

A soil microorganism toxicity test in accordance with OECD TG 216 nitrogen transformation test has been conducted with D5.

No effects at the highest test concentration were observed. A 28-day EC10 value of >100 mg/kg dry weight based on nitrogen transformation rate was determined.

Soil stability tests have been carried out with the test substance and other similarly volatile siloxane substances. The test soil preparation method used has been shown to minimise volatilisation losses. The test organisms are therefore likely to have been exposed to the test substance.