Registration Dossier

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:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
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
Organic carbon content (% dry weight):
1.37
Nitrogen content (% dry weight):
11.1 (Total inorganic Nitrogen)
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 30thto October 28th, 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