Reaction mass of 1-[(1R*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol and 1-[(1S*,6S*)-2,2,6-trimethylcyclohexyl]hexan-3-ol

Toxicity to soil microorganisms

Link to relevant study record(s)

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.