C16-(branched), C20-(branched) and C24-(branched)-alkanes

Toxicity to soil microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-11-11 to 2008-12-09

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Reliable guideline study with acceptable restrictions. High test substance concentrations were tested, exceeding the solubility in test medium. The test substance has been weight into the substrate, homogen distribution has not been determined. Therefore, physical effects cannot be excluded.

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Water solubility (under test conditions): Insoluble
- Density: 0.81 g/cm3 (20 °C)

Analytical monitoring:

no

Details on sampling:

No analytical monitoring

Vehicle:

yes

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- Type of organic substrate: Standardized loamy sand soil (LUFA 2.3) amended with 0.5 % (dry weight) Lucerne grass-green meal.

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: The respective test item concentrations were weighed out and mixed thoroughly with quartz sand in a glass vessel. Afterwards the test item quartz sand mix, the soil and the amount of demineralised water to adjust the soil to 50 % of the maximum water holding capacity, were mixed thoroughly with a long handled blender for at least three minutes. Bulk samples were prepared and divided into three replicates per concentration.

VEHICLE:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Quartz sand
- Concentration of vehicle in test medium (stock solution and final test solution): The amount of quartz sand corresponds to 1 % of the bulk sample (10 g quartz sand/kg soil DW)
- Evaporation of vehicle before use: Not necessary

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

nominal: 20 +/- 2 °C, actual: 20 - 22 °C

Moisture:

Application rate Soil Dry Weights [%] Moisture content as [%] of the Maximal Water Holding Capacity
[mg/kg DW] day 0 day 28 day 0 day 28
Control 86.1 86.0 46.8 47.3
125 86.2 86.1 46.4 46.8
250 85.8 85.7 48.1 48.4
500 85.7 85.8 48.4 48.3
1000 86.0 85.7 47.5 48.4
2000 85.9 85.5 47.6 49.5

Details on test conditions:

TEST SYSTEM
- Testing facility: DR.U.NOACK-LABORATORIEN
- Test container (type, material, size): Plastic boxes (volume 0.5 L, food grade) with perforated tops.
- Amount of soil: 450 g soil DW
- No. of replicates per concentration: 3
- No. of replicates per vehicle control: 3


SOIL INCUBATION
- Method: bulk approach


SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Offenbach, Im Bildgarten Nr. 570, Rheinland-Pfalz, Germany
- History of site:
Cultures:
2008: uncultivated
2007: uncultivated
2006: pumpkin
2005: pumpkin
2004: pumpkin
Fertilisation:
No organic fertilisation.
2008: none
2007: none
2006: none
2005: 300 kg/ha Nitrophoska Spezial (12% N, 12% P2O5, 17% K2O)
2004: 400 kg/ha Nitrophoska Spezial (12% N, 12% P2O5, 17% K2O)

- Vegetation cover: Not specified
- Treatments with pesticides or fertilizers: No crop protection products applied during sampling year and 4 former years
- Accidental contamination: No
- Depth of sampling: 20 cm
- Soil texture
- % sand:
2.0 - 063 mm 2.5 ± 0.8
0.63 - 0.2 mm 26.9 ± 0.7
0.2 - 0.063 mm 29.3 ± 3.4
- % silt:
0.063 - 0.02 mm 18.6 ± 2.3
0.02 - 0.006 mm 9.1 ± 0.5
0.006 - 0.002mm 4.2 ± 0.8
- % clay:
< 0.002 mm 9.4 ± 0.9
- Soil taxonomic classification: loamy sand
- Soil classification system: acc. to German DIN classification
- pH (in water): 6.4 +/- 0.6
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 16.07
- Maximum water holding capacity (in % dry weigth): 34.4 +/- 2.0
- Cation exchange capacity: 8 +/- 2 mVal/100 g
- Pretreatment of soil: - The soil was manually cleared of large objects and then sieved to a particle size of 2 mm
- The maximal water holding capacity and the pH-value were
determined by LUFA SPEYER.
- The soil moisture content was determined.
- The soil was adjusted to 45 % of its maximal water holding
capacity with demineralised water.
- The soil was checked for a detectable microbial biomass
(result in terms of percentage of total organic carbon).

- Storage (condition, duration): The soil was stored for 56 days (2008-09-05 to 2008-10-31) in the dark at 6 ± 2 °C in a climatic room (TE1200, VIESSMANN).
- Initial microbial biomass as % of total organic C: 1.11


DETAILS OF PREINCUBATION OF SOIL (if any): The soil was preincubated at room temperature (ca. 20 °C) for 11 days before starting to the definitive study to guarantee a temperature adaptation of the micro-organisms.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Measurements of nitrate were carried out after 0, 7, 14 and 28 days. The pH-values and moisture contents were determined
on day 0 and 28.
Nitrate was extracted from the soil with a mineral salt solution.
For the elimination of coloured organic matter in the extraction solutions a cleaning step with solid phase extraction (SPE) cartridges was carried out according to ISO 14238. Thereafter, nitrate was determined photometrically.



VEHICLE CONTROL PERFORMED: yes


RANGE-FINDING STUDY
A range-finding study was carried out with the test concentrations: 100 - 1000 mg/kg soil DW

Nominal and measured concentrations:

nominal test concentrations: 125 - 250 - 500 - 1000 - 2000 mg/kg soil dry weight (DW)

Reference substance (positive control):

yes

Remarks:

Cyanoguanidin

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

743 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: 95% confidence interval 705 - 784 mg/kg dw.

Duration:

28 d

Dose descriptor:

EC25

Effect conc.:

358 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other:

Remarks:

95% confidence interval 269 to 411 mg/kg dw.

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

< 125 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Details on results:

- Effect concentrations exceeding solubility of substance in test medium: Yes

Results with reference substance (positive control):

Cyanoguanidin is tested once per year as toxic reference with 50 and 100 mg/kg DW.

Mean Nitrate-N Content of the Reference Item [mg N/kg DW]
Cyanoguanidin
[mg/kg DW] Mean Nitrate-N Content [mg N/kg DW]
0 d 7d 14 d 28 d
Control 11.5 16.4 23.0 32.8
50 11.1 13.0 14.9 28.0
100 11.3 11.2 14.4 14.0



Nitrate-N Formation Rates of Reference Item Soils Compared to the Control Soils [%]
Cyanoguanidin
[mg/kg DW] Nitrate-N Formation Rates [%]
7 d 14 d 28 d
50 61 66 20
100 103 73 88
Positive values = indicate inhibition

Reported statistics and error estimates:

One Way Analysis of Variance (ANOVA) and DUNNETT’s test were carried out for the determination of statistically significant differences compared to control replicates. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The a-value for ANOVA and DUNNETT’s test (acceptable probability of incorrectly concluding that there is a difference) is a = 0.05.

EC25 and EC50 values were calculated by sigmoidal dose-response regression. Calculations of the confidence intervals for EC25 and EC50 were carried out using standard procedures.

Validity criteria fulfilled:

yes

Conclusions:

The EC50-value (d 0 - 7) with 95 % confidence intervals for inhibition of the nitrate-N formation rate on day 7 was 565 (478 - 670) mg/kg DW. The EC25-value with 95 % confidence intervals for inhibition on day 7 days was 219 (125 - 301) mg/kg DW. The NOEC on day 7 was < 125 mg/kg DW.

The EC50-values (d 0 - 14) with 95 % confidence intervals for inhibition of the nitrate-N formation rate on day 14 was 552 (503 - 611) mg/kg DW. The EC25-value with 95 % confidence intervals for inhibition on day 14 days was 354 (263 - 407) mg/kg DW. The NOEC on day 14 was 125 mg/kg DW.

The EC50-value (d 0 - 28) with 95 % confidence intervals for inhibition of the nitrate-N formation rate on day 28 was 743 (705 - 784) mg/kg DW. The EC25-value with 95 % confidence intervals for inhibition on day 28 days was 358 (269 - 411) mg/kg DW. The NOEC on day 28 was < 125 mg/kg DW.

Executive summary:

The effects of Tetrabutane on the metabolic activity of soil micro-organisms were determined according to OECD Guideline 216 (January 2000) and 68/548/EEC, Annex V, Method C.21 (2004). The test item was applied via quartz sand with the test concentrations 2000 - 1000 - 500 - 250 - 125 mg/kg soil dry weight (DW). Untreated soil was tested as control. Standard soil type 2.3 (Batch-No. F2.33508; loamy sand) of the LUFA Speyer was used. Plastic boxes (volume 0.5 L) with perforated tops to enable gas exchange and filled with 450 g soil dry weight (DW), moistened to nominally 50 % of its maximal water holding capacity were used.

The treated soils were incubated for 28 days at 46.4 - 49.5 % (measured) of maximum water holding capacity and a pH-value between 7.03 and 7.22. The effects of the test item on the metabolic activity of the nitrogen transformation were measured on the day of treatment (day 0) and after 7, 14 and 28 days and compared to the untreated control.

Nitrate-N Formation Rate: NOEC and EC- Values with Confidence Intervals

	Day 7	Day 14	Day 28
	[mg/kg DW]
EC50	565	552	743
95 % confidence interval	478 - 670	503 - 611	705 - 784
EC25	219	354	358
95 % confidence interval	125 - 301	263 - 407	269 - 411
NOEC	125	125	125

Reference 2

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-11-11 to 2008-12-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: test peformed under GLP conditions at an GLP accredited test facility

Qualifier:

according to guideline

Guideline:

OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Water solubility (under test conditions): Insoluble
- Density: 0.81 g/cm3 (20 °C)

Analytical monitoring:

no

Details on sampling:

No analytical monitoring

Vehicle:

yes

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- Type of organic substrate: Standardized loamy sand soil (LUFA 2.3)



APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: The respective test item concentrations were weighed out and mixed thoroughly with quartz sand in a glass vessel. Afterwards the test item quartz sand mix and the soil were mixed thoroughly with a long handled blender for at least three minutes. Bulk samples were prepared and divided into three replicates per concentration.


VEHICLE:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Quartz sand
- Concentration of vehicle in test medium (stock solution and final test solution): The amount of quartz sand corresponds to 1 % of the bulk sample (10 g quartz sand/kg soil DW)
- Evaporation of vehicle before use: Not necessary

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

nominal: 20 +/- 2 °C; measured: 18 - 22 °C

Moisture:

Soil Dry Weight, Moisture Content and pH-Values of the Soil after 0 and 28 Days
Test concentration Soil Dry Weights [%] Moisture content as [%] of Maximal Water Holding Capacity pH-Values
[mg/kg DW] day 0 day 28 day 0 day 28 day 0 day 28
Control 85.6 85.6 48.9 48.8 6.50 6.42
4.401 85.7 85.3 48.5 50.2 6.77 6.76
22.0 86.0 85.4 47.5 49.8 6.85 6.89

Details on test conditions:

TEST SYSTEM
- Testing facility: Dr.U.NOACK-LABORATORIEN
- Test container (type, material, size): Plastic boxes, volume 6.25 L with perforated tops to enable gas exchange
- Amount of soil: 2 kg soil dw
- No. of replicates per concentration: 3
- No. of replicates per vehicle control: 3


SOIL INCUBATION
- Method: bulk approach


SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Offenbach, Im Bildgarten Nr. 570, Rheinland-Pfalz, Germany
- History of site:
Cultures:
2008: uncultivated
2007: uncultivated
2006: pumpkin
2005: pumpkin
2004: pumpkin
Fertilisation:
No organic fertilisation.
2008: none
2007: none
2006: none
2005: 300 kg/ha Nitrophoska Spezial (12% N, 12% P2O5, 17% K2O)
2004: 400 kg/ha Nitrophoska Spezial (12% N, 12% P2O5, 17% K2O)

- Vegetation cover: Not specified
- Treatments with pesticides or fertilizers: No crop protection products applied during sampling year and 4 former years
- Accidental contamination: No
- Depth of sampling: 20 cm
- Soil texture
- % sand:
2.0 - 063 mm 2.5 ± 0.8
0.63 - 0.2 mm 26.9 ± 0.7
0.2 - 0.063 mm 29.3 ± 3.4
- % silt:
0.063 - 0.02 mm 18.6 ± 2.3
0.02 - 0.006 mm 9.1 ± 0.5
0.006 - 0.002mm 4.2 ± 0.8
- % clay:
< 0.002 mm 9.4 ± 0.9
- Soil taxonomic classification: loamy sand
- Soil classification system: acc. to German DIN classification
- pH (in water): 6.4 +/- 0.6
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 16.07
- Maximum water holding capacity (in % dry weigth): 34.4 +/- 2.0
- Cation exchange capacity: 8 +/- 2 mVal/100 g
- Pretreatment of soil: - The soil was manually cleared of large objects and then sieved to a particle size of 2 mm
- The maximal water holding capacity and the pH-value were
determined by LUFA SPEYER.
- The soil moisture content was determined.
- The soil was adjusted to 45 % of its maximal water holding
capacity with demineralised water.
- The soil was checked for a detectable microbial biomass
(result in terms of percentage of total organic carbon).

- Storage (condition, duration): The soil was stored for 56 days (2008-09-05 to 2008-10-31) in the dark at 6 ± 2 °C in a climatic room (TE1200, VIESSMANN).
- Initial microbial biomass as % of total organic C: 1.11


DETAILS OF PREINCUBATION OF SOIL (if any): The soil was preincubated at room temperature (ca. 20 °C) for 11 days before the experimental start to guarantee a temperature adaptation of the micro-organisms.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The measurements of respiration rates were carried out after 0, 7, 14 and 28 days. The pH-values and water contents were
determined on days 0 and 28.
Soil samples of each replicate were mixed with a sufficient induced respiration rates amount of glucose (4000 mg/kg) to produce an immediate maximum respiratory response. Measurements started within 2 hours after glucose supplement. 200 g soil was filled into 500 mL glass flasks and closed with Oxitop® sensors. Incubation took place for 24 h in the dark at 20 ± 2 °C. The pressure was measured 360 times in 24 h. CO2 was adsorbed by sodium lime deposited in the headspace of Oxitop® sensors. Due to the adsorption of CO2 and the oxygen uptake by the soil the pressure in the glass flasks was reduced and measured. Based on the
change of pressure the evolved CO2 and thus the consumed O2 were calculated.




VEHICLE CONTROL PERFORMED: yes


RANGE-FINDING STUDY
A range-finding study was carried out with the test concentrations: 20 - 100 - 1000 mg/kg soil DW

Nominal and measured concentrations:

nominal concentrations: 500 - 1000 mg/kg soil DW

Reference substance (positive control):

yes

Remarks:

Dinoterb

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

respiration rate

Details on results:

- Effect concentrations exceeding solubility of substance in test medium: Yes

Results with reference substance (positive control):

Dinoterb is tested yearly as toxic reference item with a concentration of 13.75 kg/ha, corresponding to 18.3 mg/kg DW.

Mean Respiration Rates of the Reference Item Dinoterb
[kg/ha] Mean Respiration Rate [mg O2 x (kg DW x h)-1]
0 d 7d 14 d 28 d 42 d
Control 9.17 9.42 8.49 9.67 10.7
13.75 10.5 12.8 10.3 8.17 6.37


Respiration Rate of the Reference Item in Comparison to the Untreated Control Dinoterb
[kg/ha] Respiration Rate [%]
0 d 7d 14 d 28 d 42 d
18.3 mg/kg DW
(13.75 kg/ha) -14 -36 -22 16 40
positive values = indicate inhibition negative values = indicate increase

Reported statistics and error estimates:

One Way Analysis of Variance (ANOVA) was carried out for the determination of statistically significant differences compared to control replicates. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The a-value for ANOVA and DUNNETT’s test (acceptable probability of incorrectly concluding that there is a difference) is a = 0.05.
EC25 and EC50 values were calculated by sigmoidal dose-response regression. Calculations of the confidence intervals for EC25 and EC50 were carried out using standard procedures.

Respiration Rate in Comparison to the Untreated Control

	Respiration Rate [%]
Day	0	7	14	28
Tetrabutane 500 mg/kg DW	0	4	0	4
Tetrabutane 1000 mg/kg DW	-2	6	2	11

Respiration Rate at Day 0

Test concentration	Repl.	Respiration Rate                  Mean Value	Respiration Rate MV ± Std.Dev.	Coefficient of Variation
[mg/kg DW]		[mg O2 × (kg DW × h)^-1]	[mg O2 × (kg DW × h)^-1]	[%]
	1	10.8
Control	2	11.2	10.9 ± 0.32	3
	3	10.6
	1	10.8
500	2	10.9	10.9 ± 0.14	1
	3	11.0
	1	11.5
1000	2	10.7	11.1 ± 0.41	4
	3	11.2

Respiration Rate at Day 7

Test concentration	Repl.	Respiration Rate                  Mean Value	Respiration Rate MV ± Std.Dev.	Coefficient of Variation
[mg/kg DW]		[mg O2 × (kg DW × h)^-1]	[mg O2 × (kg DW × h)^-1]	[%]
	1	-*
Control	2	10.4	10.6 ± 0.30	3
	3	10.9
	1	10.1
500	2	10.2	10.2 ± 0.10	1
	3	10.3
	1	10.3
1000	2	9.96	9.95 ± 0.34	3
	3	9.61

Respiration Rate at Day 14

Test concentration	Repl.	Respiration Rate                  Mean Value	Respiration Rate MV ± Std.Dev.	Coefficient of Variation
[mg/kg DW]		[mg O2 × (kg DW × h)^-1]	[mg O2 × (kg DW × h)^-1]	[%]
	1	9.75
Control	2	9.83	9.63 ± 0.29	3
	3	9.30
	1	9.92
500	2	9.65	9.59 ± 0.36	4
	3	9.20
	1	9.20
1000	2	9.20	9.45 ± 0.43	5
	3	9.94

Respiration Rate at Day 28

Test concentration	Repl.	Respiration Rate                  Mean Value	Respiration Rate MV ± Std.Dev.	Coefficient of Variation
[mg/kg DW]		[mg O2 × (kg DW × h)^-1]	[mg O2 × (kg DW × h)^-1]	[%]
	1	10.8
Control	2	9.82	10.3 ± 0.47	5
	3	10.3
	1	9.49
500	2	9.53	9.85 ± 0.60	6
	3	10.5
	1	9.40
1000	2	9.13	9.15 ± 0.24	3
	3	8.93

Validity criteria fulfilled:

yes

Conclusions:

Tetrabutane caused no effects on the soil microflora when applied at the test concentrations 500 and 1000 mg/kg DW. The study was terminated after 28 days. No inhibition or stimulation was observed and therefore Tetrabutane is not expected to lead to any long detrimental effects on carbon turnover in soil under normal conditions.

Executive summary:

The effects of Tetrabutane on the metabolic activity of soil micro-organisms were determined according to OECD Guideline 217 (January 2000). The test item was applied with the test concentrations 1000 - 500 mg/kg soil dry weight (DW). Untreated soil was tested as control. Triplicates were set up. Standard soil type 2.3 (Batch-No. F2.33508; loamy sand) of the LUFA Speyerwas used. Plastic boxes (volume 6.25 L) with perforated tops were used to enable gas exchange and filled with 3 kg soil dry weight (DW), moistened to nominally 50 % of its maximal water capacity.

The treated soils were incubated for 28 days at 47.5 - 50.2 % (measured) of maximum water holding capacity and a pH-value between 6.42 and 6.89. The effects of the test item on the metabolic activity of the microbial biomass (glucose induced respiration) were measured on the day of treatment (day 0) and after 7, 14 and 28 days. On each occasion, aliquots of soil were amended with glucose (4000 mg/kg). Oxygen consumption was measured over the subsequent 24 h period and compared to that of the untreated control.

Tetrabutanecaused no effects on the soil microflora when applied with the test concentrations 1000 and 500 mg/kg DW. The study was terminated after 28 days. No inhibition or stimulation was observed and therefore Tetrabutaneis not expected to lead to any long detrimental effects on carbon turnover in soil under normal conditions.

Respiration Rate: NOEC and EC- Values with Confidence Intervals

	Day 7	Day 14	Day 28
	[mg/kg DW]
EC50	> 1000	> 1000	> 1000
95 % confidence interval	–	–	–
EC25	> 1000	> 1000	> 1000
95 % confidence interval	–	–	–
NOEC	1000	1000	1000

Description of key information

Tetrabutane has no effect on carbon transformation by soil micro-organisms. NOEC (28-days) ≥ 1000 mg/kg soil dw. (OECD 217). The EC50 value for nitrogen transformation by soil micro-organisms has been determined to be 743 mg/Kg dw.

Additional information

The effect of Tetrabutane on the metabolic activity of soil micro-organisms was determined in a carbon transformation test according to the guideline OECD 217. The test was conducted at concentrations of 500 and1000 mg/kg soil dw, over a duration of 28 days. The effect of the test substance on glucose induced respiration of the microbial biomass was determined on the day of treatment (day 0) and after 7, 14 and 28 days. On each occasion glucose was added to aliquots of soil (4000 mg/kg soil) and oxygen consumption measured over a subsequent 24 hour period. Tetrabutane caused no effect on the soil micro-organisms at 500 or 1000 mg/kg soil dw. No inhibition of stimulation was observed and therefore Tetrabutane is not expected to lead to any long-term detrimental effects on the carbon transformation in soil under normal conditions. The NOEC was determined to be≥1000 mg/kg soil dw.

In an additional test, the metabolic activity of soil micro-organisms were determined according to OECD Guideline 216 (January 2000) and 68/548/EEC, Annex V, Method C.21 (2004) with Tetrabutane. The test item was applied via quartz sand with the test concentrations 2000 - 1000 - 500 - 250 - 125 mg/kg soil dry weight (DW). After incubation for 28 days, effects on the nitrogen transformation were observed and resulted in an EC50 value (28 days) of 743 mg/kg dw and a NOEC <125 mg/kg dw. However, high test substance concentrations were used in the study, which are above the limit of solubility. Therefore, physical effects in this study cannot be excluded.