Quaternary ammonium compounds, C20-22-alkyltrimethyl, chlorides

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:

2010-01-25 to 2010-02-23

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:

yes

Remarks:

The soil was stored at 6 +/- 2 °C instead of 4 +/- 2 °C due to organisational reasons.

GLP compliance:

yes

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: A silty sand soil (uS acc. to German DIN classification, field fresh sampled) and amended with powdered Lucerne-grass-green meal (0.5 % of dry weight) was used.



APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: The test item was weighed out on quartz sand, pulverized and mixed homogeneously. Afterwards the test item quartz sand mix, the soil and the amount of demineralised water to adjust the soil to 55 % 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: 19 - 22 °C

Moisture:

Dry weight before application: 85.9 g/100 g soil

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:Cultures:
2009: uncultivated
2008: uncultivated
2007: uncultivated
2006: uncultivated
2005: pumpkin
Fertilisation:
No organic fertilisation.
2009: none
2008: none
2007: none
2006: None
2005: 300 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 ± 0.7
0.63 - 0.2 mm 27.6 ± 1.8
0.2 - 0.063 mm 29.5 ± 3.6
- % silt:
0.063 - 0.02 mm 18.1 ± 2.7
0.02 - 0.006 mm 9.0 ± 0.6
0.006 - 0.002mm 4.3 ± 0.7
- % clay:
< 0.002 mm 8.7 ± 1.6
- Soil taxonomic classification: silty sand (uS)
- Soil classification system: acc. to German DIN classification
- pH (in water): 6.6 +/- 0.5
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 4.25
- Maximum water holding capacity (in % dry weigth): 35.1 +/- 2.0
- Cation exchange capacity: 9 +/- 3 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 55 % 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 79 days (2009-10-21 to 2010-01-07) in the dark at 6 ± 2 °C in a climatic room (TE1200, VIESSMANN).
- Initial microbial biomass as % of total organic C: 1.30


DETAILS OF PREINCUBATION OF SOIL (if any): The soil was preincubated at room temperature (ca. 20 °C) for 18 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
Inhibition of Nitrate-N Formation Rate Compared to the Control
Nominal test item concentration
[mg/kg soil dry weight] Day after application
7 14 28
10 mg/kg soil dry weight 110 21 -4
100 mg/kg soil dry weight 93 43 46
1000 mg/kg soil dry weight90 86 113

Nominal and measured concentrations:

nominal test concentrations: 500 - 200 - 80 - 32 - 12.8 mg/kg soil dry weight (DW)

Reference substance (positive control):

yes

Remarks:

Cyanoguanidin

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

76 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: (66.0 - 84.3)

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

ca. 15 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: ca. 12-38

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

ca. 10 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: n.a.

Details on results:

- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

Cyanoguanidin is tested once per year as toxic reference with 50 and 100 mg/kg DW.
Changes of the Nitrate-N Formation Rates of Reference Item Soils Compared to the
Control Soils [%]

[%] compared to Untreated Control
Day 7 14 28
Cyanoguanidine 50 mg/kg soil dry weight 60 70 72
Cyanoguanidine 100 mg/kg soil dry weight 79 75 90

Reported statistics and error estimates:

One Way Analysis of Variance (ANOVA) with following 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.

ECx values were calculated by sigmoidal dose- response regression. Calculations of the confidence intervals for EC12.5, EC25 and EC50 were carried out using standard procedures.

Inhibition of Nitrate-N Contents

Test concentration	[%] compared to Untreated Control
[mg/kg soil dry weight]	0 d	7 d	14 d	28 d
12.8	-2	1	4	7
32	-3	9	11	9
80	3	15	15	36
200	5	18	27	43
500	11	19	31	54

Inhibition of Nitrate-N Formation Rates Soil [%]

Test concentration	[%] compared to Untreated Control
[mg/kg soil dry weight]	7 d	14 d	28 d
12.8	6	11	12*
32	34*	28*	15*
80	40*	30*	53*
200	47*	56*	63*
500	37*	57*	75*

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

	Day 7	Day 14	Day 28
	[mg/kg soil dry weight]
EC50	> 500	176	76.0
95 % confidence interval	–	93.8 - > 500	66.0 - 84.3
EC25	22.0	32	49.5
95 % confidence interval	15.0 - 34.1	–	43.0 - 57.7
EC12.5			16.6
95 % confidence interval			12.8 - 38.2
LOEC	32	32	12.8
NOEC	12.8	12.8	12.8

Validity criteria fulfilled:

yes

Conclusions:

After 7 days of exposure the EC25-value for inhibition of the nitrate-N formation rate in soil was 22.0 mg/kg soil dry weight. After 7 days the inhibition of nitrate transformation was below 50 % at all test item concentrations resulting in an EC50-value > 500 mg/kg soil dry weight. The NOEC for nitrate transformation in soil on day 7 was determined to be 12.8 mg/kg soil dry weight.

After 14 days of exposure the EC25-value for inhibition of the nitrate-N formation rate in soil was < 32 mg/kg soil dry weight. The EC50 for inhibition of the nitrate transformation was determined to be at 176 mg/kg soil dry weight. The NOEC for nitrate transformation in soil on day 14 was set at 12.8 mg/kg soil dry weight.

After 28 days of exposure the following EC-values for inhibition of the nitrate-N formation rate were determined: EC12.5: 16.6 mg/kg soil dry weight, EC25: 49.5 mg/kg soil dry weight, EC50: 76.0 mg/kg soil dry weight. The NOEC for nitrate transformation in soil on day 28 was set < 12.8 mg/kg soil dry weight.

Executive summary:

After 7 days of exposure the EC₂₅-value for inhibition of the nitrate-N formation rate in soil was 22.0 mg/kg soil dry weight. After 7 days the inhibition of nitrate transformation was below 50 % at all test item concentrations resulting in an EC₅₀-value > 500 mg/kg soil dry weight. The NOEC for nitrate transformation in soil on day 7 was determined to be 12.8 mg/kg soil dry weight.

After 14 days of exposure the EC₂₅-value for inhibition of the nitrate-N formation rate in soil was 32 mg/kg soil dry weight. The EC₅₀ for inhibition of the nitrate transformation was determined to be at 176 mg/kg soil dry weight. The NOEC for nitrate transformation in soil on day 14 was set at 12.8 mg/kg soil dry weight.

After 28 days of exposure the following EC-values for inhibition of the nitrate-N formation rate were determined: EC_12.5: 16.6 mg/kg soil dry weight, EC₂₅: 49.5 mg/kg soil dry weight, EC₅₀: 76.0 mg/kg soil dry weight. The NOEC for nitrate transformation in soil on day 28 was set 12.8 mg/kg soil dry weight.

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

	Day 7	Day 14	Day 28
	[mg/kg soil dry weight]
EC50	> 500	176	76.0
95 % confidence interval	–	93.8 - > 500	66.0 - 84.3
EC25	22.0	32	49.5
95 % confidence interval	15.0 - 34.1	–	43.0 - 57.7
EC12.5			16.6
95 % confidence interval			12.8 - 38.2
LOEC	32	32	12.8
NOEC	12.8	12.8	<12.8

Description of key information

After 7 days of exposure the EC25-value for inhibition of the nitrate-N formation rate in soil was 22.0 mg/kg soil dry weight. After 7 days the inhibition of nitrate transformation was below 50 % at all test item concentrations resulting in an EC50-value > 500 mg/kg soil dry weight. The NOEC for nitrate transformation in soil on day 7 was determined to be 12.8 mg/kg soil dry weight.
After 14 days of exposure the EC25-value for inhibition of the nitrate-N formation rate in soil was < 32 mg/kg soil dry weight. The EC50 for inhibition of the nitrate transformation was determined to be at 176 mg/kg soil dry weight. The NOEC for nitrate transformation in soil on day 14 was set at 12.8 mg/kg soil dry weight.
After 28 days of exposure the following EC-values for inhibition of the nitrate-N formation rate were determined: EC12.5: 16.6 mg/kg soil dry weight, EC25: 49.5 mg/kg soil dry weight, EC50: 76.0 mg/kg soil dry weight. The NOEC for nitrate transformation in soil on day 28 was set < 12.8 mg/kg soil dry weight.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

10 mg/kg soil dw