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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:
2018-10-09 to 2018-11-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
yes
Remarks:
Quartz sand
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
nominal: 20 +/- 2 °C
measured: 21.2-22.7 °C
Moisture:
Maximum water holding capacity: 34.9 ± 1.8 g/100 g DW
The soil was adjusted to approx. 45% of MWHC.
Soil moisture content in terms of percent MWHC before application: 41.6%
Moisture on day 0: 43.1-43.8%
Moisture on day 28: 42.9-433.6%
Organic carbon content (% dry weight):
0.465
Details on test conditions:
TEST SYSTEM
- Testing facility: DR.U.NOACK-LABORATORIEN
- Test container (type, material, size): Plastic boxes (volume 1.0 L, food grade) with perforated tops to enable gas exchange
- Amount of soil: 400 g soil DW
- No. of replicates per concentration: 3
- No. of replicates per control: 3
- Control: Untreated soil with 1% quartz sand (tested under the same conditions as the test replicates)


SOIL INCUBATION
- Method: bulk approach


SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Soil: LUFA soil 2.3
- Geographical reference of sampling site (latitude, longitude):Offenbach, “rechts der Landauer Str.“ Nr. 826/7, Rheinland-Pfalz, Germany
- History of site: Cultures: 2014 - 2018: uncultivated
Fertilisation: 2015 - 2018: none, no organic fertilisation.
- Vegetation cover: Not specified
- Treatments with pesticides or fertilizers: No crop protection products applied during sampling year and 4 preceding years.
- Accidental contamination: No
- Depth of sampling: ca. 20 cm
- Soil texture
Sand:
2.0 - 0.63 mm: 2.7 ± 0.5 %
0.63 - 0.2 mm: 29.4 ± 1.2 %
0.2 - 0.063 mm: 25.2 ± 1.4 %
Silt:
0.063 - 0.02 mm: 18.7 ± 1.3 %
0.02 - 0.006 mm: 11.0 ± 0.7 %
0.006 - 0.002 mm: 5.3 ± 0.8 %
Clay:
< 0.002 mm: 7.6 ± 0.4 %
- Soil taxonomic classification: silty sand
- Soil classification system: acc. to German DIN classification
- pH (in water): 5.9+/- 0.5
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 7.18
- Maximum water holding capacity (in % dry weigth): 34.9+/- 1.8
- Cation exchange capacity: 7.3 +/- 1.1 meq/100 g

LUFA Speyer:
The soil was manually cleared of large objects and then sieved to a particle size of up to 2 mm (carried out by LUFA Speyer).
The maximal water holding capacity and the pH-value were determined.
Test facility:
The soil moisture content was determined.
The soil was adjusted to about 45 % of its maximal water holding capacity with demineralised water. Drying out of the soil was prevented by moistening with demineralised water as necessary.
The soil was checked for a detectable microbial biomass (result in terms of percentage of total organic carbon) and the amount of total inorganic nitrogen.
The soil amounts were amended with powdered lucerne-green-grass-meal (0.5 % of soil dry weight).

At experimental starting the soil was adjusted to approximately 42 % of its maximal water holding capacity. All replicates were checked once per week for water losses by evaporation (recommended maximum 5 %, actual 0.5 - 1.0 %). Compensation with demineralised water was done. Replicates were weighed for this procedure.

- Storage (condition, duration): The soil was stored for 63 days (2018-07-18 to 2018-09-19) in the dark at 4 ± 2 °C in a climatic room (TE1200, VIESSMANN).
- Initial microbial biomass as % of total organic C: 3.30

DETAILS OF PREINCUBATION OF SOIL (if any): Subsequently, the soil was pre-incubated at room temperature (ca. 20 °C) for 19 days (2018-09-19 to 2018-10-09) before experimental starting to guarantee a temperature adaptation of the micro-organisms.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
Measurements of inorganic nitrate were carried out after 0, 7, 14 and 28 days. The pH values and water contents were determined after 0 and 28 days.The room temperature was measured and recorded continuously by a datalogger.

Determination of nitrate concentrations:
Nitrate was extracted from soil with a mineral salt solution. For the elimination of coloured organic matter in the extraction solution a cleaning step with solid phase extraction (SPE) cartridges was carried out. Thereafter, photometric determination took place.



VEHICLE CONTROL PERFORMED: No vehicle used

RANGE-FINDING STUDY: Yes, with 10, 100 and 1000 mg/kg soil dw. Based on the results the concentrations for the definitive study were selected.
Nominal and measured concentrations:
Nominal test concentrations: 1000 - 316 - 100 - 31.6 - 10.0 mg/kg soil dry weight (factor 3.16).
Reference substance (positive control):
yes
Remarks:
Cyanoguanidine, conducted from 2018-05-18 to 2018-06-15 with 50 mg/kg soil dry weight and resulted in 33% compared to control on the basis of Nitrate-N.
Key result
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
19.8 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Duration:
28 d
Dose descriptor:
EC25
Effect conc.:
82 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
274 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Results with reference substance (positive control):
- Results with reference substance valid? Yes,
- Relevant effect levels: difference to control > 25 %

Nitrate-N Contents -Day 0

 

Test concentration
[mg/kg soil dry weight]

Nitrate-N

Repl.

MV±SD

CV

[mg NO3-N/kg soil dry weight]

[%]

Control

26.5

26.5 ± 0.10

0

26.6

26.4

10.0

27.8

27.2 ± 0.60

2

26.6

27.2

31.6

26.6

27.3 ± 0.82

3

27.1

28.2

100

28.5 

27.8 ± 0.96

3

28.2

26.7

316

29.1

29.1 ± 2.05

7

31.2 

27.1 

1000

26.7

27.4 ± 1.33

5

28.9

26.5

Nitrate-N Contents -Day 7

Test concentration
[mg/kg soil dry weight]

Nitrate-N

Nitrate-N Formation Rate

Repl.

MV±SD

CV

Repl.

MV±SD

CV

[mg NO3-N/kg soil dry weight]

[%]

[mg NO3-N×(kg soil dry weight×d)-1]

Control

34.0

35.3 ± 2.25

6

1.07

1.26 ± 0.32

26

37.9

1.63

34.0

1.07

10.0

37.6

35.6 ± 2.16

6

1.49

1.20 ± 3.11

26

35.8

1.23

33.3

0.87

31.6

34.5

36.0 ± 1.31

4

1.03

1.24 ± 0.19

15

37

1.39

36.4

1.30

100

36.3

35.4 ± 0.78

2

1.21

1.08 ± 0.11

10

35.0

1.03

34.9

1.01

316

37.3

36.9 ± 0.55

1

1.17

1.12 ± 0.08

7

36.3

1.03

37.2

1.16

1000

35.4

36.6 ± 1.11

3

1.14

1.31 ± 0.16

12

 37.6  1.46
 36.8

 1.34

Nitrate-N Contents -Day 14

Test concentration
[mg/kg soil dry weight]

Nitrate-N

Nitrate-N Formation Rate

Repl.

MV±SD

CV

Repl.

MV±SD

CV

[mg NO3-N/kg soil dry weight]

[%]

[mg NO3-N×(kg soil dry weight×d)-1]

Control

42.8

44.3 ± 1.97

4

1.16

1.27 ± 0.14

11

43.5

1.21

46.5

1.43

10.0

47.7

45.2 ± 1.35

3

1.39

1.29 ± 0.09

7

44.1

1.21

44.8

1.26

31.6

44.7

44.2 ± 1.61

4

1.24

1.21 ± 0.11

9

42.4

1.08

45.5

1.30

100

40.6

40.9 ± 1.82

4

0.91

0.94 ± 0.13

14

42.9

1.08

39.3

0.82

316

35.6

34.6 ± 0.91

3

0.46

0.40 ± 0.07

16

33.8

0.34

34.5

0.39

1000

28.3

29.8 ± 1.41

5

0.06

0.17 ± 0.10

59

30.0 0.19
 31.1

 0.26

Nitrate-N Contents -Day 28

Test concentration
[mg/kg soil dry weight]

Nitrate-N

Nitrate-N Formation Rate

Repl.

MV±SD

CV

Repl.

MV±SD

CV

[mg NO3-N/kg soil dry weight]

[%]

[mg NO3-N×(kg soil dry weight×d)-1]

Control

57.6

57.9 ± 0.31

6

1.11

1.12 ± 0.01

26

58.0

1.13

58.2

1.13

10.0

57.1

57.1 ± 1.65

6

1.07

1.07 ± 0.06

26

58.8

1.13

55.5

1.01

31.6

54.4

54.9 ± 0.92

4

0.97

0.99 ± 0.004

15

56.0

1.03

54.4

0.97

100

46.7

48.1 ± 2.42

2

0.68

0.73 ± 0.009

10

46.7

0.68

50.9

0.83

316

51.0

45.0 ± 5.23

1

0.78

0.57 ± 0.19

7

42.6

0.48

41.4

0.44

1000

17.9

16.9 ± 1.18

3

n.d.

-

-

 15.6  n.d.
17.2

 n.d.

Repl.: Replicate MV: Mean value

SD: Standard deviation CV: Coefficient of variation

n.d. not determinable, due to negative Nitrate-N Formations rates

Validity criteria fulfilled:
yes
Conclusions:
Based on results of an OECD 216 compliant study with the test item an EC10 (based on the Nitrate-N formation rate) of 19.8 mg/kg soil dw was determined as key value.
Executive summary:

The effects of the test item on the metabolic activity of soil micro-organisms were determined according to OECD Guideline 216 (2000) over 28 days. The test item was applied via quartz sand with the test concentrations 1000 - 316 - 100 - 31.6 - 10 mg/kg soil dry weight. Untreated soil was tested as control under the same test conditions as the test item concentrations. Standard soil, moistened to nominally 45 % of its maximal water holding capacity, was used. Plastic boxes (volume 1.0 L) with perforated tops to enable gas exchange and filled with 400 g soil dry weight were used. The effects of the test item on the metabolic activity of the nitrogen-N formation rate (nitrate) were measured on the day of treatment (day 0) and after 7, 14 and 28 days.

After 7 days of exposure, no significant inhibition of the microbial nitrate transformation was observed for all test item concentrations up to 1000 mg/kg soil dry weight. After 14 days of exposure, a significant inhibition of the microbial nitrate transformation was observed for test item concentrations 100 mg/kg soil dry weight. After 28 days of exposure a significant inhibition of the microbial nitrate transformation was observed for test item concentrations > 100 mg/kg soil dry weight. For the test item concentration 1000 mg/kg soil dry weight, nitrate formation rates were negative, due to low nitrate contents. On day 28 the NOEC for nitrogen transformation in soil measured as nitrate was 31.6 mg/kg soil dry weight, the EC10 for nitrogen transformation in soil measured as nitrate was 19.8 mg/kg soil dry weight.

Description of key information

Based on results of an OECD 216 compliant study with the test item an EC10 (based on the Nitrate-N formation rate) of 19.8 mg/kg soil dw was determined as key value for the risk assessment.

Key value for chemical safety assessment

Short-term EC50 for soil microorganisms:
274 mg/kg soil dw
Long-term EC10 or NOEC for soil microorganisms:
19.8 mg/kg soil dw

Additional information

The effects of the test item on the metabolic activity of soil micro-organisms were determined according to OECD Guideline 216 (2000) over 28 days. The test item was applied via quartz sand with the test concentrations 1000 - 316 - 100 - 31.6 - 10 mg/kg soil dry weight. Untreated soil was tested as control under the same test conditions as the test item concentrations. Standard soil, moistened to nominally 45 % of its maximal water holding capacity, was used. Plastic boxes (volume 1.0 L) with perforated tops to enable gas exchange and filled with 400 g soil dry weight were used. The effects of the test item on the metabolic activity of the nitrogen-N formation rate (nitrate) were measured on the day of treatment (day 0) and after 7, 14 and 28 days.

After 7 days of exposure,no significant inhibition of the microbial nitrate transformation was observed for all test item concentrations up to 1000 mg/kg soil dry weight. After 14 days of exposure,a significant inhibition of the microbial nitrate transformation was observed for test item concentrations 100 mg/kg soil dry weight. After 28 days of exposurea significant inhibition of the microbial nitrate transformation was observed for test item concentrations > 100 mg/kg soil dry weight. For the test item concentration 1000 mg/kg soil dry weight, nitrate formation rates were negative, due to low nitrate contents.On day 28 the NOEC for nitrogen transformation in soil measured as nitrate was 31.6 mg/kg soil dry weight, the EC10 for nitrogen transformation in soil measured as nitrate was 19.8 mg/kg soil dry weight.