D-Glucitol, 1-deoxy-1-(methylamino)-, N-[C18-C18 (unsaturated) acyl] derivs.

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:

2014-06-19 to 2014-07-17

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP studie, no deficiencies

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

yes

Remarks:

see "Principle of methods if other than guideline"

Principles of method if other than guideline:

The soil was stored at 6  2 °C instead of 4 ± 2 °C due to organisational reasons.
The temperature was under 18° C for 39 hours in total, due to technical reasons.

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

Soil handling
The soil moisture content was determined.
The soil was adjusted to about 42% of its maximum 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). The C/N ratio was between 12/1 and 16/1. A ratio of of 5 g Lucerne per kg of soil (dry weight was used.
Content of total inorganic nitrogen: 2.8% (dry weight)
Content of total organic carbon: 42.4% (dry weight)
C/N-ratio: 15.31
Particle size: 0.0288 - 1.008 mm
Origin: Alfalis, la maîtrise des luzernes, DESIALIS, Société par actions simplifiée BP 124, F-51007 Chalons en Champagne, France

Application
The respective test item amounts were weighed out for each test item concentration, demineralised water was added and stirred at 50 °C for 1 hour. Afterwards the test item solution was mixed carefully into the soil with a mixer to ensure a homogeneous distribution of the test item in the soil. Thereafter the soil was distributed to the replicates.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

Nominal: 20 +/- 2 °C
Actual: 16.4 22.0°C
The temperature was < 18 °C for 39 hours in total.

Moisture:

Dry weight before application: 87.3 g/100 g soil
Maximal water holding capacity: 35.6 +/- 1.7 g/100 g soil DW

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 as stated below (see type and frequency of measurements and observations).
- Amount of soil: 400 g soil dry weight per replicate
- No. of replicates per concentration: Triplicates
- No. of replicates per control: Trplicates

SOIL INCUBATION
- Method: bulk approach

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude):
Offenbach, “rechts der Landauer Str.“ Nr. 826/7, Rheinland-Pfalz, Germany
Gauß-Krüger-Coordinates: R-439683; H-544955
- History of site:
Cultures:
2010 - 2014: uncultivated
Fertilisation:
No organic fertilisation.
2010 - 2014: none
Pesticides:
No crop protection products applied during sampling year and 4 former years.
- Accidental contamination: No
- Depth of sampling: ca. 20 cm
- Soil texture
Particle size distribution acc. to DIN*
Sand:
2.0 - 0.63 mm 2.5 ± 0.9 % dry weight
0.63 - 0.2 mm 30.4 ± 0.6 % dry weight
0.2 - 0.063 mm 24.7 ± 1.8 % dry weight
Silt:
0.063 - 0.02 mm 19.5 ± 1.9 % dry weight
0.02 - 0.006 mm 11.5 ± 0.7 % dry weight
0.006 - 0.002 mm 5.0 ± 0.2 % dry weight
Clay:
< 0.002 mm 6.4 ± 2.0 % dry weight
- Soil taxonomic classification: Silty sand
- Soil classification system: acc. to German DIN classification
- pH (in water): 5.8 +/- 0.7
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 4.91
- Maximum water holding capacity (in % dry weigth): 35.6 +/- 1.7
- Cation exchange capacity (mmol/kg): 7.3
- Pretreatment of soil:
LUFA Speyer:
The soil was manually cleared of large objects and then sieved to a particle size of up to 2 mm.
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 42% of its maximum 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). The C/N ratio was between 12/1 and 16/1. A ratio of of 5 g Lucerne per kg of soil (dry weight was used).
- Storage (condition, duration):
The soil was stored for 16 days (2014-05-12 to 2014-05-28) in the dark at 6 ± 2 °C in a climatic room (TE1200, VIESSMANN).
- Initial microbial biomass as % of total organic C: 2.03
DETAILS OF PREINCUBATION OF SOIL (if any):
Subsequently, the soil was pre-incubated at room temperature (ca.20 °C) for 22 days (2014-05-28 to 2014-06-19) 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 on day 0 and 28. The room temperature was measured and recorded continuously by a data logger.
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.
Wave length 588 nm for nitrate
Solutions Extraction solution: Potassium chloride, 1 M and 2 M, respectively
Reagent: 2 g/L 4-Ethylresorcin in 2-Propanol
Standard Potassium nitrate > 99%
Preparation of standard solutions
A stock solution of 100 mg nitrate/L was prepared in demineralised water. 6 concentrations were prepared by dilution with 1 M potassium chloride and used for calibration.
Calibration was performed prior to experimental starting.
Working steps:
15 g soil of each replicate were weighed into shaking flasks. 60 mL extraction solution were added. Shaking was carried out for 1 h with 150 - 200 rpm. Filtration was carried out thereafter. The first 20 mL of filtrate were rejected.
Sample cleaning:
10 mL of the filtrated extract was cleaned via C18-SPE-cartridges to remove dissolved and coloured organic matter which would have influence on photometric determination. Conditioning of the cartridges was done with 2 x 2.5 mL methanol and thereafter with 2 x 2.5 mL demineralised water. Dryness of the cartridges was avoided. After conditioning, the sample was applied. The first 2.5 mL of each cleaned extract were rejected. The following volumes were stored in reagent tubes.
Nitrate determination:
1 mL of the cleaned extract was diluted with 1 mL demineralised water. A cuvette was filled with 1.8 mL sulphuric acid (86 %). 0.5 mL sample were added. The cuvette was closed and shaken gently. After 15 min 0.3 mL of the reagent solution were added. After 45 min photometric determination at 588 nm was carried out. Extraction solution was used as ground signal. On day 28 samples were diluted 1:4 before measurements.
Method validation:
The nitrate method was evaluated on linearity and limit of quantification (LOQ). In the range of calibration standards the second lowest standard was chosen as limit of quantification.

VEHICLE CONTROL PERFORMED: no

RANGE-FINDING STUDY
- Test concentrations: 1000 - 100 - 10 mg/kg soil dry weight
- Results used to determine the conditions for the definitive study:
Inhibition of Nitrate-N Content
Test concentration Inhibition [%] compared to untreated Control
[mg/kg soil DW] 0 d 7 d 14 d 28 d
10 12 5 -4 -3
100 2 8 3 -7
1000 5 < LOQ 37 -11

Inhibition of Nitrate-N Formation Rates
Test concentration Inhibition [%] compared to untreated Control
[mg/kg soil DW] 7 d 14 d 28 d
10 -8 -19 -11
100 20 5 -12
1000 n.d. 66 -20
DW = soil dry weight negative value = promotion of nitrate-N Content / formation rates
LOQ = Limit of quantification n.d.= not determinable, Nitrate content

Nominal and measured concentrations:

Nominal test concentrations: 1000 - 500 - 250 - 125 - 62.5 mg/kg soil dry weight (factor 2)

Reference substance (positive control):

yes

Remarks:

Cyanoguanidine

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:

nitrate formation rate

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

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

221 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: 214 - 226

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

587 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Remarks on result:

other: 538 - 644

Results with reference substance (positive control):

Mean Nitrate-N Content in the Reference Item Test
Reference Item Concentration
[mg/kg soil dry weight] Mean ± SD of Nitrate-N Content [mg NO3-N/kg soil dry weight]
0 d 7 d 14 d 28 d
Control 16.1 ± 0.93 24.5 ± 0.46 33.6 ± 1.67 43.8 ± 1.66
50 16.0 ± 0.21 18.7 ± 1.67 18.3 ± 0.27 19.9 ± 0.65
100 17.8 ± 0.46 18.1 ± 0.21 18.5 ± 1.08 19.0 ± 0.96

Inhibition of Nitrate-N Formation Rates of the Reference Item Test
Reference Item Concentration Nitrate-N Formation Rate
[%] compared to Untreated Control
[mg/kg soil dry weight] 7 d 14 d 28 d
50 68* 87* 86*
100 97* 96* 96*
*) difference to control ≥ 25 %

Reported statistics and error estimates:

NOEC
One Way Analysis of Variances (ANOVA) and DUNNETT’S test, if possible, were carried out for the determination of statistically significant differences compared to control replicates. When running a One Way Analysis of Variances a Normality test and an Equal Variance test was done first. P-values for both Normality and Equal Variances test are 0.05. The α-value for ANOVA and DUNNETT’S test (acceptable probability of incorrectly concluding that there is a difference) is α = 0.05.
EC-values and confidence intervals
EC25 and EC50 values were calculated by sigmoidal dose- response regression (day 7 and 14). Calculations of the confidence intervals for EC25 and EC50 were carried out using standard procedures.

Inhibition of Inorganic-N Contents

Nitrate-N Content
Test concentration	Inhibition [%] compared to control
[mg/kg soil dry weight]	0 d	7 d	14 d	28 d
62.5	6	-6	5	-2
125	9	-2	7	2
250	6	29*	11	7
500	15	68*	22	4
1000	9	< LOQ	54*	9

positive values = inhibition           negative values = increase              *) Difference to control ≥ 25%

LOQ = Limit of quantification

 Inhibition of Inorganic-N Formation Rates

Nitrate-N Formation Rate
Test concentration	Inhibition [%] compared to control
[mg/kg soil dry weight]	7 d	14 d	28 d
62.5	-32*	3	-5
125	-25*	5	-1
250	83*	15	7
500	189*	29*	-1
1000	n.d.	98*	9

*) Difference to control ≥ 25% and statistically significant difference

positive values = inhibition               negative values = increase

Validity criteria fulfilled:

yes

Conclusions:

After 7 days of exposure C18/C18 unsatd.-Glucamide stimulated the microbial nitrate transformation to 32 % and 25 % compared to the control when applied at the test item concentrations 62.5 and 125 mg/kg soil dry weight and inhibited the microbial nitrate transformation to 83 % and 189 % at the test item concentrations 250 and 500 mg/kg soil dry weight. For the test item concentration 1000 mg/kg soil dry weight no nitrate-N formation rates could be determined, because the nitrate-N contents were under the limit of quantification. After 14 days of exposure no statistically significant effects on the nitrate transformation were determined at the test item concentrations 62.5 to 250 mg/kg soil dry weight. At the test item concentrations 500 and 1000 mg/kg soil dry weight the nitrate transformation was inhibited to 29 % and 98 %, respectively. After 28 days of exposure no statistically significant effects on the nitrate transformation were determined for all tested test item concentrations.
After 7 days of exposure the EC25-value for inhibition of the nitrate-N formation rate in soil was determined to be 198 mg/kg soil dry weight and the EC50-value at 221 mg/kg soil dry weight. The NOEC for nitrate transformation in soil on day 7 was determined to be < 62.5 mg/kg soil dry weight.
After 14 days of exposure the EC25-value for inhibition of the nitrate-N formation rate in soil was 483 mg/kg soil dry weight. The EC50 for inhibition of the nitrate transformation was determined to be 587 mg/kg soil dry weight. The NOEC for nitrate transformation in soil on day 14 was set at 250 mg/kg soil dry weight.
After 28 days of exposure no EC-values of the nitrate-N formation rate were determined, due to effects < 25 %. The NOEC for nitrate transformation in soil on day 28 was determined to be 1000 mg/kg soil dry weight.
The test item is rapidly biodegraded in soil with a half-live of < 5d (see IUCLID Chapter 5. 2.3 Biodegradation in soil). This means that at the test end after 28d nearly all of the test item is degraded. Although effects were observed at Day 7 and 14, no effects were measured at the highest tested concentration of 1000 mg/kg dw,at the test end (Day 28) which can be explained by the rapid biodegradation of the test item. It can also be concluded that the soil microorganism population was not damaged during the test

Executive summary:

The effects of C18/C18 unsatd.-Glucamide (batch no. SN 119/13/3 -8) on the metabolic activity of soil micro-organisms were determined according to OECD Guideline 216 (2000) at Dr.U.Noack-Laboratorien, D-31157 Sarstedt, Germany from 2014-06-19 to 2014-07-17. The test item was applied via demineralised water with the test item concentrations 1000 - 500 - 250 - 125 - 62.5 mg/kg soil dry weight. Untreated soil was tested as control under the same test conditions as the test item replicates. Standard soil, moistened to nominally 46 % of its maximum 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 applied.

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 C18/C18 unsatd.-Glucamide stimulatedthe microbial nitrate transformation to 32 % and 25 % compared to the control when applied at the test item concentrations 62.5 and 125 mg/kg soil dry weight and inhibited the microbial nitrate transformation to 83 % and 189 % at the test item concentrations 250 and 500 mg/kg soil dry weight. For the test item concentration 1000 mg/kg soil dry weight no nitrate-N formation rates could be determined, because the nitrate-N contents were under the limit of quantification. After 14 days of exposure no statistically significant effects on the nitrate transformation were determined at the test item concentrations 62.5 to 250 mg/kg soil dry weight. At the test item concentrations 500 and 1000 mg/kg soil dry weight the nitrate transformation was inhibited to 29 % and 98 %, respectively. After 28 days of exposure no statistically significant effects on the nitrate transformation were determined for all tested test item concentrations.

After 7 days of exposure the EC₂₅-value for inhibition of the nitrate-N formation rate in soil was determined to be 198 mg/kg soil dry weight and the EC₅₀-value at 221 mg/kg soil dry weight. The NOEC for nitrate transformation in soil on day 7 was determined to be < 62.5 mg/kg soil dry weight.

After 14 days of exposure the EC₂₅-value for inhibition of the nitrate-N formation rate in soil was 483 mg/kg soil dry weight. The EC₅₀for inhibition of the nitrate transformation was determined to be 587 mg/kg soil dry weight. The NOEC for nitrate transformation in soil on day 14 was set at 250 mg/kg soil dry weight .

After 28 days of exposure no EC-values of the nitrate-N formation rate were determined, due to effects < 25 %. The NOEC for nitrate transformation in soil on day 28 was determined to be 1000 mg/kg soil dry weight.

The test item is rapidly biodegraded in soil with a half-live of < 5d (see IUCLID Chapter 5. 2.3 Biodegradation in soil). This means that at the test end after 28d nearly all of the test item is degraded. Although effects were observed at Day 7 and 14, no effects were measured at the highest tested concentration of 1000 mg/kg dw,at the test end (Day 28) which can be explained by the rapid biodegradation of the test item. It can also be concluded that the soil microorganism population was not damaged during the test.

Description of key information

Details see IUCLID Chapter 6.3.4 and Discussion below

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

1 000 mg/kg soil dw

Additional information

The test item is rapidly biodegraded in soil with a half-live of < 5d (see IUCLID Chapter 5. 2.3 Biodegradation in soil). This means that at the test end after 28d nearly all of the test item is degraded. Although effects were observed at Day 7 and 14, no effects were measured at the highest tested concentration of 1000 mg/kg dw,at the test end (Day 28) which can be explained by the rapid biodegradation of the test item. It can also be concluded that the soil microorganism population was not damaged during the test.