Quaternary ammonium compounds, C12-14-alkyl(hydroxyethyl)dimethyl, chlorides

Toxicity to soil microorganisms

Administrative data

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-09-30 to 2009-10-28

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

[see concordance under 1.1. of the CSR]

IUPAC Name:

[see concordance under 1.1. of the CSR]

Details on test material:

- Name of test material (as cited in study report): HYEQS
- Chemical name: Quaternary ammonium compounds, C12-18-alkyl (hydroxyethyl) dimethyl, chlorides
- Physical state: Solid, white granules
- Analytical purity: 98.8 %
- Purity test date: 2009-06-10
- Lot/batch No.: DEG4052477
- Expiration date of the lot/batch: 2011-03-01
- Storage condition of test material: Room temperature, protected from moisture and light

Sampling and analysis

Analytical monitoring:

no

Details on sampling:

No analytical monitoring

Test substrate

Vehicle:

no

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- Type of organic substrate: A loamy sand soil (lS, acc. to German DIN classification, field fresh sampled) was used in the study.

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: The test item was suspended in demineralised water and applied to the surface of the soil. The soil was mixed and care was taken to ensure a homogeneous distribution of the test item in the soil. Afterwards the soil was distributed to the replicates.
Application Conditions of the Soil
Dry Weight (DW) before application [g/100 g soil] 86.2
Maximal water holding capacity (MWHC) [g/100 g soil dry weight] 35.1
Soil moisture content in terms of percent MWHC before application [%] 45.7
Water amount to adjust to 50 % MWHC [g/100 g soil dry weight] 1.52
Water amount / test group [mL] 20.6

Test organisms

Test organisms (inoculum):

soil

Study design

Total exposure duration:

28 d

Test conditions

Test temperature:

19 - 20 °C

Moisture:

Dry Weights, Maximal Water Holding Capacity and pH-Values of the Soil after 0 and 28 Days
Application rate Soil Dry Weights [%] Moisture content as [%] of the Maximal Water Holding Capacity pH-Values
[mg/kg soil dry weight] day 0 day 28 day 0 day 28 day 0 day 28
Control 85.9 84.8 46.9 51.2 6.82 7.03
25.6 85.8 85.5 47.2 48.3 6.86 7.04
64 86.0 84.3 46.4 53.2 6.90 7.04
160 85.9 85.1 46.7 49.8 6.92 6.98
400 86.5 86.1 44.7 46.2 6.94 6.93
1000 86.4 84.5 44.7 52.3 6.92 6.83

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. Incubation was performed in bulk and sub samples were taken
- Amount of soil: 450 g dry weight per vessel
- No. of replicates per concentration: 3
- No. of replicates per control: 3


SOIL INCUBATION
- Method: bulk


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:
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)
No crop protection products applied during sampling year (2008) and the four previous years.
Sampling date: 2009-07-06
- Depth of sampling: ca. 20 cm
- Soil texture
Particle size distribution acc. to DIN*
Sand:
0.63 – 2.0 mm % DW 2.5 ± 0.7
0.2 – 0.63 mm % DW 27.9 ± 1.8
0.063 – 0.2 mm % DW 29.5 ± 3.6
Silt:
0.02 – 0.063 mm % DW 18.1 ± 2.7
0.006 – 0.02 mm % DW 9.0 ± 0.6
0.002 – 0.006 mm % DW 4.3 ± 0.7
Clay:
< 0.002 mm % DW 8.7 ± 1.6
- Soil taxonomic classification: loamy sand (lS)
- Soil classification system: German DIN classification
- pH (in water): 6.6 +/- 0.5
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 11.1
- Maximum water holding capacity (in % dry weigth): 35.1 +/- 2.0
- Cation exchange capacity (mmol/kg): 9.0 +/- 3.0 (mVal/100g)
- Pretreatment of soil: The soil was pre-incubated at room temperature (ca. 20 °C) for 24 days before experimental starting to guarantee a temperature adaptation of the micro-organisms.
- Storage (condition, duration): 61 d, in the dark at 6 +/- 2 °C
- Initial microbial biomass as % of total organic C: 2.0


DETAILS OF PREINCUBATION OF SOIL (if any):
The soil was pre-incubated at room temperature (ca. 20 °C) for 24 days before experimental starting 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 content were determined on day 0 and 28. The room temperature was measured and recorded continuously by a thermohygrograph.

VEHICLE CONTROL PERFORMED: no


RANGE-FINDING STUDY
- Test concentrations: 10 - 100 - 1000 mg/kg soil dry weight
- Results used to determine the conditions for the definitive study:
Nitrate-N Formation Rates in Comparison to the Untreated Control
Differences of the Nitrate-N Formation Rates
Day after application 7 14 28
10 mg/kg soil dry weight -11 3 2
100 mg/kg soil dry weight -28 -8 -8
1000 mg/kg soil dry weight 32 38 -39

Nominal and measured concentrations:

1000 - 400 - 160 - 64 - 25.6 mg/kg soil dry weight

Reference substance (positive control):

yes

Remarks:

Cyanoguanidin

Results and discussion

Effect concentrations

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:

nitrate formation rate

Details on results:

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

Results with reference substance (positive control):

Mean Nitrate-N Content of the Reference Item [mg N/kg Soil Dry Weight]
Test concentrations
[mg/kg soil dry weight] Mean Nitrate-N Content [mg N/kg soil dry weight]
0 d 7d 14 d 28 d
Control 8.70 14.7 17.7 27.6
50 8.21 10.6 10.9 13.7
100 8.00 9.26 10.2 10.1

Changes of the Nitrate-N Formation Rates of Reference Item Soils Compared to the
Control Soils [%]

Formation Rates
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.
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.

Any other information on results incl. tables

Nitrate-N Contents in Comparison to the Untreated Soil [%]

Test concentration	Nitrate-N Contents [%]
[mg/kg soil dry weight]	0 d	7 d	14 d	28 d
25.6	-2	-2	-5	4
64	6	4	-2	2
160	1	6	-5	2
400	4	1	10	6
1000	5	16	24	8

Nitrate-N Formation Rates of Test Item Soils Compared to the Untreated Soil [%]

Test concentration [mg/kg soil dry weight]	Nitrate-N Formation Rates [%]
	7 d	14 d	28 d
25.6	-4	-10	9
64	2	-15	-2
160	16	-14	5
400	-5	-18	9
1000	36*	51*	11

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

	Day 7	Day 14	Day 28
	[mg/kg soil dry weight]
EC50	> 1000	908	> 1000
95 % confidence interval	–	738 - > 1000	–
EC25	782	562	> 1000
95 % confidence interval	657 - 955	465 - 688	–
LOEC	1000	1000	> 1000
NOEC	400	400	1000

Nitrate-N Contents - Day 0

Test concentration [mg/kg soil dry weight]	Nitrate-N
	Repl.	MV ± Std. Dev.	CV [%]
	[mg N/kg soil dry weight]
Control	14.2	14.6 ± 0.67	5
	15.4
	14.3
25.6	13.2	14.9 ± 1.51	10
	16.1
	15.4
64	13.7	13.7 ± 0.06	0
	13.7
	13.6
160	13.6	14.5 ± 0.85	6
	14.6
	15.3
400	14.1	14.0 ± 0.40	3
	13.6
	14.4
1000	13.6	13.9 ± 0.55	4
	13.5
	14.5

Nitrate-N Contents and Nitrate-N Formation Rates - Day 7

Test concentration [mg/kg soil dry weight]	Nitrate-N			Nitrate-N Formation Rate
	Repl.	MV ± Std. Dev.	CV [%]	Repl.	MV ± Std. Dev.	CV [%]
	[mg N/kg soil dry weight]			[mg NO3-N × (kg soil dry weight × d)^-1]
Control	22.7	22.6 ± 1.11	5	1.16	1.14 ± 0.16	14
	21.4			0.97
	23.6			1.29
25.6	22.5	23.1 ± 0.85	4	1.09	1.18 ± 0.12	10
	24.1			1.31
	22.8			1.13
64	22.0	21.6 ± 0.40	2	1.19	1.12 ± 0.06	5
	21.2			1.07
	21.5			1.11
160	21.3	21.2 ± 1.30	6	0.97	0.96 ± 0.19	20
	22.5			1.14
	19.9			0.77
400	22.8	22.4 ± 0.32	1	1.26	1.20 ± 0.05	4
	22.3			1.19
	22.2			1.17
1000	19.1	19.0 ± 0.56	3	0.74	0.73 ± 0.08	11
	18.4			0.64
	19.5			0.80

Nitrate-N Contents and Nitrate-N Formation Rates - Day 14

Test concentration [mg/kg soil dry weight]	Nitrate-N			Nitrate-N Formation Rate
	Repl.	MV ± Std. Dev.	CV [%]	Repl.	MV ± Std. Dev.	CV [%]
	[mg N/kg soil dry weight]			[mg NO3-N × (kg soil dry weight × d)^-1]
Control	23.7	24.7 ± 1.00	4	0.65	0.72 ± 0.07	10
	24.6			0.71
	25.7			0.79
25.6	25.5	25.9 ± 0.67	3	0.76	0.79 ± 0.05	6
	26.7			0.84
	25.6			0.76
64	26.9	25.3 ± 1.42	6	0.94	0.83 ± 0.10	12
	24.4			0.76
	24.5			0.77
160	25.7	26.0 ± 0.74	3	0.80	0.82 ± 0.05	6
	25.4			0.78
	26.8			0.88
400	22.7	22.3 ± 0.93	4	0.62	0.59 ± 0.07	12
	22.9			0.64
	21.2			0.51
1000	18.6	18.8 ± 0.32	2	0.34	0.35 ± 0.02	6
	18.7			0.34
	19.2			0.38

Nitrate-N Contents and Nitrate-N Formation Rates - Day 28

Test concentration [mg/kg soil dry weight]	Nitrate-N			Nitrate-N Formation Rate
	Repl.	MV ± Std. Dev.	CV [%]	Repl.	MV ± Std. Dev.	CV [%]
	[mg N/kg soil dry weight]			[mg NO3-N × (kg soil dry weight × d)^-1]
Control	34.2	33.0 ± 1.07	3	0.70	0.66 ± 0.04	6
	32.4			0.64
	32.3			0.63
25.6	30.6	31.8 ± 1.53	5	0.56	0.60 ± 0.05	8
	31.2			0.58
	33.5			0.66
64	32.8	32.5 ± 1.04	3	0.68	0.67 ± 0.04	6
	33.3			0.70
	31.3			0.63
160	34.7	32.2 ± 2.37	7	0.72	0.63 ± 0.08	13
	31.8			0.62
	30.0			0.55
400	32.6	30.9 ± 1.60	5	0.66	0.60 ± 0.06	10
	29.4			0.55
	30.8			0.60
1000	33.5	30.4 ± 2.84	9	0.70	0.59 ± 0.10	17
	27.9			0.50
	29.9			0.57

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The NOEC for nitrogen transformation in soil measured as nitrate on day 7 was 400 mg/kg soil dry weight. The EC50-value (d 0 - 7) for inhibition of the nitrate-N formation rate on day 7 was > 1000 mg/kg soil dry weight. The EC25-value with 95 % confidence intervals for inhibition on day 7 days was 782 (657 - 955) mg/kg soil dry weight.

The NOEC for nitrogen transformation in soil measured as nitrate on day 14 was 400 mg/kg soil dry weight. The EC50-values (d 0 - 14) with 95 % confidence intervals for inhibition of the nitrate-N formation rate on day 14 was 908 (738 - > 1000) mg/kg soil dry weight. The EC25-value with 95 % confidence intervals for inhibition on day 14 days was 562 (465 - 688) mg/kg soil dry weight.

The NOEC for nitrogen transformation in soil measured as nitrate on day 28 was 1000 mg/kg soil dry weight. The EC25 - and the EC50-value (d 0 - 28) for inhibition of the nitrate-N formation rate on day 28 was > 1000 mg/kg soil dry weight.

Executive summary:

The effects of HYEQS(Batch no. DEG4052477) on the metabolic activity of soil micro-organisms were determined according to OECD Guideline 216 (2000) at Dr.U.Noack-Laboratorien, 31157 , , from September 30^thto October 28^th, 2009. The test item was applied via demineralised water with the test concentrations 1000 - 400 - 160 - 64 - 25.6 mg/kg soil dry weight. Untreated soil was tested as control. Standard loamy sand soil was used. Plastic boxes with perforated tops to enable gas exchange and filled with 450 g soil dry weight, moistened to nominally 50 % of its maximal water holding capacity, were used.

The treated soils were incubated for 28 days at 44.7 - 52.3 % (measured) of the maximum water holding capacity and a pH-value between 6.82 and 7.04. 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 as amount of nitrate and compared to the untreated control.

The NOEC for nitrogen transformation in soil measured as nitrate on day 7 was determined to be 400 mg/kg soil dry weight. The EC₅₀-value (d 0 - 7) for inhibition of the nitrate-N formation rate on day 7 was > 1000 mg/kg soil dry weight. The EC₂₅-value with 95 % confidence intervals for inhibition on day 7 days was 782 (657 - 955) mg/kg soil dry weight.

The NOEC for nitrogen transformation in soil measured as nitrate on day 14 was set at 400 mg/kg soil dry weight. The EC₅₀-values (d 0 - 14) with 95 % confidence intervals for inhibition of the nitrate-N formation rate on day 14 was 908 (738 - > 1000) mg/kg soil dry weight. The EC₂₅-value with 95 % confidence intervals for inhibition on day 14 days was 562 (465 - 688) mg/kg soil dry weight.

The NOEC for nitrogen transformation in soil measured as nitrate on day 28 was determined to be at 1000 mg/kg soil dry weight. The EC₂₅ - and the EC₅₀-value (d 0 - 28) for inhibition of the nitrate-N formation rate on day 28 was > 1000 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	> 1000	908	> 1000
95 % confidence interval	–	738 - > 1000	–
EC25	782	562	> 1000
95 % confidence interval	657 - 955	465 - 688	–
LOEC	1000	1000	> 1000
NOEC	400	400	1000