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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:
15 Mar - 23 Apr 2018
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:
January 2000
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)
Version / remarks:
January 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The Department of Health of the Government of the United Kingdom
Analytical monitoring:
no
Vehicle:
no
Details on preparation and application of test substrate:
APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: The test substance, a non-agrochemical, was tested at five treatment concentrations (10, 32, 100, 320 and 1000 mg/kg dry weight soil).
Test item was incorporated into test soil by mixing test item with sand (at a rate of 10 g sand per kg dry weight soil + test item) and then distributed evenly in several aliquots over the surface of each soil replicate (spread as a thin layer of approximately 2.5 cm).
Nitrogen transformation test: Test item was mixed with soil and lucerne meal as nitrogen source.
Carbon transformation: No addition of lucerne meal
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20 °C ± 2 °C
Moisture:
45% of WHC
Details on test conditions:
TEST SYSTEM
- Test container: 5 L polypropylene containers and perforated lids fitted to limit water loss and allow gas exchange
- Amount of soil: Nitrogen transformation: 20 g; Carbon transformation: 100 g
- No. of replicates per concentration: 3
- No. of replicates per control: 3

SOIL INCUBATION
- Method: . Bulk soils were thoroughly mixed prior to sub-sampling at each time point. The soil samples were extracted on the same day of sampling and sample extracts were stored at approx. -20 °C.

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Leamington Spa, Warwickshire, England, Landlook (Midlands) soil series/number: Bromsgrove 317, CEMAS Ltd. study number CEMS-8056
- History of site: Fallow agricultural site
- Vegetation cover: Grass cover
- Treatments with pesticides or fertilizers: The site had received no pesticides or fertilisers for 5 years prior to sampling.
- Depth of sampling: 5 - 20 cm of top soil
Soil texture
- % sand: 71 % w/w
- % silt: 17 % w/w
- % clay: 12 % w/w
- Soil taxonomic classification: sandy loam
- Soil classification system: USDA pipette method
- pH (in water): 6.5
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): <0.003 g N/g dry weight soil (<0.3%), 61.86 mg NO3/kg dry weight soil
- Maximum water holding capacity (in % dry weigth): 41.3% w/w
- Cation exchange capacity (mmol/kg): 10.4 meq/100 g
- Pretreatment of soil: Test soil (Bromsgrove 317/30.01.18) was received at test facility and stored at 2 – 8 °C in the dark and acclimatised at 20 ± 2 °C for 16 days prior to testing.
- Storage (condition, duration): ambient temperature prior to delivery to test facility
- Initial microbial biomass as % of total organic C: 4.24 % (Microbial biomass) and Total biomass (BC) mg C/kg soil: 606

EFFECT PARAMETERS MEASURED:
Nitrogen Transformation: Analyses of nitrogen transformation were performed within 6 hours of treatment on Day 0 and 28 days after preparation.
Carbon transformation: Analyses of carbon transformation were performed at each hourly interval over 12 hours at Day 0 and at Day 28.

RANGE-FINDING STUDY: no range-finding test was performed
Nominal and measured concentrations:
nominal: Control, 10, 32, 100, 320 and 1000 mg/kg dry weight soil
Reference substance (positive control):
no
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
320 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
respiration rate
Details on results:
No statistically significant effects were observed for carbon transformation so the NOEC was determined to be greater or equal the highest test item concentration of 1000 mg/kg dry weight soil. In case of nitrogen transformation effects were observed at the highest test item concentration of 1000 mg/kg dry weight soil, therefore the NOEC was determiend to be at a level of 320 mg/kg dry weight soil.
Reported statistics and error estimates:
Data were received in an Excel file and were analyzed using SAS 9.4 (SAS Institute Inc. 2002) and Quasar 1.5 (Quasar 1.5 2017).

Table 1: Nitrate concentrations in soil† - group mean values

Day

 

Group

 

Group 2:

Group 1

% deviation

Group 3:

Group 1

% deviation

 

1

Non-treated

control soil

2

10 mg Test item/kg

dry weight soil

3

32 mg Test item

/kg dry weight soil

0

61.86

56.13

53.12

-9.26

-14.13

28

200.16

204.20

203.26

2.02

1.55

 

Day

 

Group

 

Group 4:

Group 1

% deviation

Group 5:

Group 1

% deviation

Group 6:

Group 1

% deviation

 

4

100 mg
Test item

/kg dry weight soil

5

320 mg
Test item

/kg dry weight soil

6

1000 mg
Test item

/kg dry weight soil

0

62.61

62.52

59.61

1.21

1.07

-3.64

28

198.47

229.58

253.84**

-0.84

14.70

26.82

† Nitrate concentrations in soil expressed as mg NO3/kg dry weight soil; each value is a mean figure derived from 3 replicate analyses

** p<0.01

Table 2: Nitrate concentrations in soil* - individual values

Day

Group 1

Non-treated

control soil

Group 2

10 mg Test item/kg dry weight soil

Group 3

32 mg Test item/kg dry weight soil

1A

1B

1C

2A

2B

2C

3A

3B

3C

0

64.02

58.95

62.61

49.92

56.69

61.77

68.25

48.51

42.59

28

198.56

209.28

192.64

209.84

192.07

210.69

197.15

199.97

212.66

 

Day

Group 4

100 mg Test item/kg dry weight soil

Group 5

320 mg Test item/kg dry weight soil

Group 6

1000 mg Test item/kg dry weight soil

4A

4B

4C

5A

5B

5C

6A

6B

6C

0

55.56

62.61

69.66

54.72

62.33

70.51

58.38

64.02

56.41

28

182.76

227.33

185.30

214.35

228.17

246.22

233.81

259.48

268.22

*Nitrate concentrations in soil expressed as mg NO3/kg dry weight soil

  

Table 3: Carbon transformation in soil† - Day 0 group mean values

Day 0

 

Group

 

 

 

Hourly interval after treatment

1

Non-treated control soil

2

10 mg Test item

/kg dry weight soil

3

32 mg Test item

/kg dry weight soil

Group 2 : Group 1
% deviation

Group 3 : Group 1
% deviation

1

8.95

8.50

8.63

-5.0

-3.6

2

8.53

8.11

8.24

-4.9

-3.4

3

8.55

8.15

8.22

-4.7

-3.9

4

8.80

8.38

8.56

-4.8

-2.8

5

9.18

8.78

8.95

-4.4

-2.5

6

9.59

9.17

9.36

-4.4

-2.5

7

10.16

9.78

9.96

-3.7

-1.9

8

11.03

10.58

10.79

-4.0

-2.2

9

11.90

11.50

11.78

-3.4

-1.0

10

12.96

12.54

12.89

-3.2

-0.5

11

14.41

13.90

14.30

-3.5

-0.7

12

16.27

15.71

16.18

-3.5

-0.5

Mean % deviation from Gp 1 control

-

-

-

-4.1

-2.1

 

Day 0

 

Group

 

 

 

 

Hourly interval after treatment

4

100 mg Test item

/kg dry weight soil

5

320 mg Test item

/kg dry weight soil

6

1000 mg Test item

/kg dry weight soil

Gp 4 : Gp 1
% dev

Gp 5 : Gp 1
% dev

Gp 6 : Gp 1
% dev

1

8.71

8.86

8.80

-2.7

-1.0

-1.7

2

8.42

8.45

8.46

-1.2

-0.9

-0.8

3

8.64

8.64

8.66

1.1

1.1

1.2

4

9.18

9.10

9.10

4.4

3.4

3.4

5

9.56

9.54

9.58

4.1

3.9

4.4

6

10.00

10.01

10.03

4.2

4.3

4.5

7

10.66

10.62

10.60

5.0

4.6

4.4

8

11.52

11.42

11.48

4.5

3.6

4.1

9

12.50

12.45

12.48

5.0

4.6

4.8

10

13.63

13.52

13.59

5.2

4.3

4.8

11

15.05

14.88

14.93

4.4

3.3

3.6

12

16.91

16.59

16.74

3.9

2.0

2.9

Mean % deviation from Gp 1 control

-

-

-

3.2

2.8

3.0

†         Values expressed as mg C as CO2 per kg dry weight soil/hour; each value is a mean figure derived from 3 replicate analyses

 

Table 4: Carbon transformation in soil† - Day 28 group mean values

Day 28

 

Group

 

 

 

Hourly interval after treatment

1

Non-treated control soil

2

10 mg Test item

/kg dry weight soil

3

32 mg Test item

/kg dry weight soil

Group 2 : Group 1
% deviation

Group 3 : Group 1
% deviation

1

6.39

6.27

6.46

-1.9

1.1

2

6.24

6.15

6.24

-1.4

0.0

3

6.23

6.13

6.23

-1.6

0.1

4

6.33

6.34

6.44

0.2

1.8

5

6.59

6.59

6.71

0.0

1.8

6

6.60

6.59

6.74

-0.2

2.1

7

7.10

7.09

7.24

-0.1

1.9

8

7.42

7.42

7.54

0.1

1.6

9

8.04

8.03

8.29

-0.1

3.0

10

8.49

8.50

8.75

0.2

3.1

11

9.48

9.50

9.75

0.2

2.9

12

10.49

10.54

10.89

0.5

3.8

Mean % deviation from Gp 1 control

-

-

-

-0.3

1.9

 

Day 28

 

Group

 

 

 

 

Hourly interval after treatment

4

100 mg Test item/kg

dry weight soil

5

320 mg Test item/kg

dry weight soil

6

1000 mg Test item

/kg dry weight soil

Gp 4 : Gp 1
% dev

Gp 5 : Gp 1
% dev

Gp 6 : Gp 1
% dev

1

7.36

7.12

6.90

15.1

11.4

7.9

2

6.95

6.79

6.64

11.5

8.8

6.5

3

6.78

6.65

6.50

9.0

6.9

4.4

4

6.87

6.78

6.59

8.6

7.2

4.2

5

7.12

7.02

6.85

8.2

6.6

4.0

6

7.41

7.30

7.14

12.2

10.6

8.1

7

7.72

7.59

7.41

8.8

6.9

4.3

8

8.12

7.99

7.78

9.5

7.7

4.9

9

8.72

8.65

8.42

8.4

7.5

4.7

10

9.44

9.36

9.14

11.2

10.3

7.7

11

10.31

10.19

9.97

8.8

7.5

5.2

12

11.36

11.26

10.93

8.3

7.4

4.2

Mean % deviation from Gp 1 control

-

-

-

10.0

8.2

5.5

†         Values expressed as mg C as CO2 per kg dry weight soil/hour; each value is a mean figure derived from 3 replicate analyses

 

Table 5: Validity criteria.

Criterion from the guideline 216

Outcome

Validity criterion fulfilled

The variation between replicate control samples should be less than ± 15%.

4.21%

Yes

Criterion from the guideline 217

Outcome

Validity criterion fulfilled

The variation between replicate control samples should be less than ± 15%.

1.13% to 3.68%

Yes

 

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.
Conclusions:
In conclusion, assessment of the effects of test item on nitrogen transformation in soil in this study demonstrated a NOEC level of 320 mg test item /kg dry weight soil. Assessment of the effects of test item on carbon transformation in soil in this study demonstrated a NOEC level of >=1000 mg test item/kg dry weight soil. However there was an insufficient dose-response relationship across the treatment groups to enable estimation of EC values for both nitrogen and carbon transformation. This demonstrated that test item is not anticipated to have any adverse influence on nitrogen transformation at concentrations up to 320 mg/kg dry weight soil or on carbon transformation at concentrations up to 1000 mg/kg dry weight soil.

Description of key information

NOEC (28 d) 320 mg /kg soil (OECD 216, N-transformation); increase of N-transformation

NOEC (28 d) ≥ 1000 mg /kg soil (OECD 217, C-transformation)

Key value for chemical safety assessment

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

Additional information

One experimental study is available investigating the potential effects of the test item Reaction mass of 1,1'-(isopropylidene)bis[3,5-dibromo-4-(2,3-dibromo-2-methylpropoxy)benzene] and 1,3-dibromo-2-(2,3-dibromo-2-methylpropoxy)-5-{2-[3,5-dibromo-4-(2,3,3-tribromo-2-methylpropoxy)phenyl]propan-2-yl}benzene (EC 944-461-4) on nitrogen and carbon transformation of soil microorganisms. The study was performed according to OECD guideline 216 and 217 under GLP conditions. For the nitrogen as well as for the carbon transformations sandy loam soil was exposed for 28 d to the nominal concentrations of 10, 32, 100, 320 and 1000 mg/kg dry weight soil. In case of nitrogen transformation, Lucerne-grass-green meal was added to the soil (4.07 g/kg dry weight soil) to stimulate nitrogen transformation. For nitrogen transformation statistically significant effects were observed at 1000 mg /kg dry weight soil, therefore the NOEC was considered to be 320 mg /kg dry weight soil. However, this effect is an increase in N-transformation compared to the control. Thus, it can be assumed that the N-cycle in soil is not affected by the test item and this effect is not considered to be relevant. Concerning the carbon transformation no statistically significant effects were observed up to the test item concentration of 1000 mg /kg dry weight soil. Therefore the NOEC was determined to be ≥1000 mg /kg dry weight soil.