Hexanamide, N-(2-ethylhexyl)-3,5,5-trimethyl-

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:

2015-11-06 to 2015-12-07

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.

Principles of method if other than guideline:

The soil was stored at 6  2 °C instead of 4 ± 2 °C due to organisational 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: 18 - 20°C

Moisture:

Dry weight before application: 87.2 g/100 g soil
Maximal water holding capacity: 35.4 +/- 1.5 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-5449554
- History of site:
Cultures:
2011 - 2015: uncultivated
Fertilisation:
2015: none
2014: 3500 kg/ha CaO (05.06.2014 and 24.09.2014)
3500 kg/ha CaO; 1463 kg/ha MgO (15.12.2014)
2013: none
2012: none
2011: 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 2.7 ± 0.8 % dry weight
0.63 - 0.2 mm 30.3 ± 0.6 % dry weight
0.2 - 0.063 mm 24.6 ± 1.5 % dry weight
Silt:
0.063 - 0.02 mm 19.0 ± 1.8 % dry weight
0.02 - 0.006 mm 11.5 ± 0.6 % dry weight
0.006 - 0.002 mm 5.4 ± 0.8 % dry weight
Clay:
< 0.002 mm 6.6 ± 1.7 % dry weight
- Soil taxonomic classification: Silty sand
- Soil classification system: acc. to German DIN classification
- pH (in water): 5.7 +/- 0.6
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 15.8
- Maximum water holding capacity (in % dry weigth): 35.4 +/- 1.5
- Cation exchange capacity (mmol/kg): 7.5 +/- 0.9
- 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).
The soil amounts were amended with powdered lucerne-green-grass-meal (0.5% of soil dry weight). Content of total inorganic nitrogen: 28.3 g/kg Content of total organic carbon: 42.8 % C/N-ratio: 15.1

- Storage (condition, duration):
The soil was stored for 8 days (2015-10-07 to 2015-10-15) in the dark at 6 ± 2 °C in a climatic room (TE1200, VIESSMANN).
- Initial microbial biomass as % of total organic C: 2.37

DETAILS OF PREINCUBATION OF SOIL (if any):
Subsequently, the soil was pre-incubated at room temperature (ca.20 °C) for 22 days (2015-10-15 to 2015-11-06) 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 0 and 7 all samples were diluted 1:2 and on day 14 and day 28 all 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 - 1 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
1 2 -8 -3 -2
10 0 -7 -4 -5
100 1 -7 -5 1
1000 0 -16 -6 1

 
Inhibition of Nitrate-N Formation Rates
Test concentration Inhibition [%] compared to untreated Control
[mg/kg soil DW] 7 d 14 d 28 d
1 -23 -7 -5
10 -19 -7 -7
100 -20 -9 0
1000 -39 -12 1
DW = soil dry weight negative value = promotion of nitrate-N formation rate

Nominal and measured concentrations:

Nominal test concentrations: 1000 -and 500 mg/kg soil dry weight

Reference substance (positive control):

yes

Remarks:

Cyanoguanidine

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

Results with reference substance (positive control):

Cyanoguanidine a well-known inhibitor of nitrification is tested once per year as toxic reference with 50 and 100 mg/kg soil dry weight in LUFA soil of the same origin as that used for studies with test items. The results are given in Table 14 and Table 15.

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 10.9 ± 0.27 23.5 ± 0.35 29.0 ± 1.35 38.1 ± 0.60
50 11.3 ± 0.67 13.1 ± 0.52 14.0 ± 0.06 26.5 ± 0.44
100 11.0 ± 0.27 12.9 ± 0.56 13.1 ± 1.04 11.6 ± 0.12

 
Inhibition of Nitrate-N Formation Rates of the Reference Item Test
Nitrate-N Formation Rate
Test concentration [%] compared to Untreated Control
[mg/kg soil dry weight] 7 d 14 d 28 d
50 86* 85* 44*
100 85* 88* 98*
*) difference to control ≥ 25 %

The observed effects caused by the reference item Cyanoguanidine prove the sensitivity of the test system thereby showing its suitability for this kind of study.

Reported statistics and error estimates:

Software
The data for the tables in the report were computer generated and have been rounded for presentation from the fully derived data. Consequently, if calculated manually based on the given data minor variations may occur from these figures.

Calculations were carried out using software
- Excel, MICROSOFT CORPORATION
- SigmaPlot, SPSS INC.

Changes of Nitrate-N Contents

Nitrate-N Content
AntimussolZJ Test concentration [mg/kg soil DW]	Changes [%] compared to control
	0 d	7 d	14 d	28 d
500	-1	-16	-7	5
1000	-2	-18	-13	1

positive values = inhibition           negative values = increase

Changes of Nitrate-N Formation Rates

Nitrate-N Formation Rate
AntimussolZJ Test concentration [mg/kg soil DW]	Changes [%] compared to control
	7 d	14 d	28 d
500	-47	-14	9
1000	-49	-25	3

positive values = inhibition                              negative values = increase

Validity criteria fulfilled:

yes

Conclusions:

The effects of the test item on the metabolic activity of the nitrogen-N (nitrate) formation rate were measured on the day of treatment (day 0) and subsequently after 7, 14 and 28 days.

Antimussol ZJ did not inhibit microbial nitrate formation rates at concentrations of 500 and 1000 mg/kg soil dry weight, throughout 28 days of exposure. On day 7 the nitrate-N formation rate was significantly stimulated in both test item concentrations. On Day 28 no inhibition nor stimulation (differences ≥ 25 %) of the nitrogen-N nitrate formation rate was observed and thus Antimussol ZJ is not expected to cause any long term detrimental effects on nitrate transformation in soil under normal conditions.

Executive summary:

The effects of Antimussol ZJ (batch no.ESD0017844) on the metabolic activity of soil micro-organisms were determined according to OECD Guideline 216 (2000) at Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany, from 2015-11-06 to 2015-12-07, with a definitive exposure phase from 2015-11-06 to 2015-12-04. The test item was applied via quartz sand at concentrations of 500 and 1000 mg/kg soil dry weight. Untreated standard silty soil was tested as control.

 

The effects of the test item on the metabolic activity of the nitrogen-N (nitrate) formation rate were measured on the day of treatment (day 0) and subsequently after 7, 14 and 28 days.

 

Antimussol ZJ did not inhibit microbial nitrate formation rates at concentrations of 500 and 1000 mg/kg soil dry weight, throughout 28 days of exposure. On day 7 the nitrate-N formation rate was significantly stimulated in both test item concentrations. On Day 28 no inhibition nor stimulation (differences ≥ 25 %) of the nitrogen-N nitrate formation rate was observed and thus Antimussol ZJ is not expected to cause any long term detrimental effects on nitrate transformation in soil under normal conditions.

 

Changes of Nitrate-N Formation Rates

Nitrate-N Formation Rate
AntimussolZJ Test concentration [mg/kg SDW]	Changes [%] compared to control
	7 d	14 d	28 d
500 mg/kg SDW	-47	-14	9
1000 mg/kg SDW	-49	-25	3

positive values = inhibition                              negative values = increase 

Description of key information

The effects of the test item on the metabolic activity of the nitrogen-N (nitrate) formation rate were measured on the day of treatment (day 0) and subsequently after 7, 14 and 28 days.

Antimussol ZJ did not inhibit microbial nitrate formation rates at concentrations of 500 and 1000 mg/kg soil dry weight, throughout 28 days of exposure. On day 7 the nitrate-N formation rate was significantly stimulated in both test item concentrations. On Day 28 no inhibition nor stimulation (differences ≥ 25 %) of the nitrogen-N nitrate formation rate was observed and thus Antimussol ZJ is not expected to cause any long term detrimental effects on nitrate transformation in soil under normal conditions.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

1 000 mg/kg soil dw