2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine

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:

2015-07-03 to 2015-07-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: The respective test item amounts were weighed out for each test item concentration and dissolved in demineralised water. Afterwards the test item solutions were mixed carefully into the soil with a mixer to ensure a homogeneous distribution of the test item in the soil. Subsequently the soil was distributed to the replicates.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20 ± 2 °C (nominal)

Moisture:

45 % of its maximum water holding capacity.

Details on test conditions:

TEST SYSTEM
- Testing facility:
- 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
- Amount of soil: 400 g soil dry weight
- No. of replicates per concentration: 3
- No. of replicates per control: 3

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): R-439683; H-5449554
- History of site: 2011 - 2014: uncultivated
- Treatments with pesticides or fertilizers: Fertilizers: 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; No crop protection products applied during the year of sampling and 4 preceding years.
- Sampling date: 2015-03-24
- Depth of sampling: ca. 20 cm
- Soil texture: field fresh, silty sand soil
- % sand: 50 – 75 %
- % silt: 35.9%
- % clay: 6.6 +/- 1.7%
- Organic carbon content 0.5 - 1.5 %
- Microbial biomass (expressed as carbon content): at least 1 % of the total soil organic carbon.
- Soil taxonomic classification: silty sand (uS)
- Soil classification system: according to German DIN
- pH (in water): 5.5 - 7.5
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): 10.5 mg/kg dw
- Maximum water holding capacity (in % dry weigth): 35.4 ± 1.5
- Cation exchange capacity (meq / 100 g): 7.5 ± 0.9
- Pretreatment of soil: The soil was manually cleared of large objects and then sieved to a particle size of up to 2 mm (LUFA SPEYER, Obere Langgasse 40, D-67346 Speyer, Germany); The soil was checked for a detectable microbial biomass (result expressed 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 5 g Lucerne per kg of soil (dry weight was used.
- Storage (condition, duration): for 63 days (2015-04-08 to 2015-06-10) in the dark at 6 ± 2 °C in a climatic room (TE1200, VIESSMANN).

DETAILS OF PREINCUBATION OF SOIL (if any):
Subsequently, the soil was pre-incubated at room temperature (ca.20 °C) for 23 days (2015-06-10 to 2015-07-03) to adapt the micro-organisms to test conditions before the start of the study

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- nitrate concentrations (at d 0, d 7, d 14, d 28)

Nominal and measured concentrations:

Nominal: 1000 - 316 – 100 – 31.6 – 10 mg /kg soil dry weight (factor 2.5)

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

Details on results:

After 7 days of exposure to the test item, a significant stimulation of the microbial nitrate transformation was observed for the test item concentrations 100 and 316 mg/kg soil dry weight. No significant difference was determined compared to the control at all test item concentrations after 14 days of exposure. After 28 days of exposure a statistical significant stimulation of the nitrate transformation was determined for the test item concentrations 10 and 1000 mg/kg soil dry weight (Figure 1). These stimulation were below 25 % and showed no dose-response relationship.

Results with reference substance (positive control):

Performance once every twelve months at concentrations of 50 and 100 mg/kg soil dry weight (laboratory ID: TBN1505, duration: 2015-05-18 to 2015-06-15). 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.

Validity criteria fulfilled:

yes

Conclusions:

There was no evidence that exposure to the test item inhibited the nitrogen transformation activity of soil microflora at the range of concentrations applied. The EC50 based on inhibition of nitrogen transformation processes could therefore not be determined reliably, but it exceeded 1000 mg/kg soil dry weight, the highest concentration applied in the definitive test. The corresponding no-observed effect concentration (NOEC) was 1000 mg/kg soil dry weight.

Executive summary:

The effects of the test item 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. The test item was applied via demineralised water at concentrations of 10, 31.6, 100, 316 and 1000 mg/kg soil dry weight. Untreated field soil, adjusted to a moisture content corresponding to nominally 45% of its maximum water holding capacity and mixed with a source of organic nitrogen (lucerne meal), was tested as the control under the same test conditions as the test item replicates. The effects of the test item on nitrogen transformation by soil microflora were determined by comparing rates of formation of the terminal transformation product, nitrate-nitrogen (NO₃^--N), in soil treated with the test substance against the rate observed in the untreated control. After 28 days of exposure a statistical significant stimulation of the nitrate transformation was determined for the test item concentrations 10 and 1000 mg/kg soil dry weight (Figure1). These stimulation were below 25 % and showed no dose-response relationship. The corresponding no-observed effect concentration (NOEC) was 1000 mg/kg soil dry weight.

Description of key information

The effects of the test item 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. The test item was applied via demineralised water at concentrations of 10, 31.6, 100, 316 and 1000 mg/kg soil dry weight. Untreated field soil, adjusted to a moisture content corresponding to nominally 45% of its maximum water holding capacity and mixed with a source of organic nitrogen (lucerne meal), was tested as the control under the same test conditions as the test item replicates. The effects of the test item on nitrogen transformation by soil microflora were determined by comparing rates of formation of the terminal transformation product, nitrate-nitrogen (NO3--N), in soil treated with the test substance against the rate observed in the untreated control. After 28 days of exposure a statisticaly significant stimulation of the nitrate transformation was determined for the test item concentrations 10 and 1000 mg/kg soil dry weight (Figure1). These stimulations were below 25 % and showed no dose-response relationship. The corresponding no-observed effect concentration (NOEC) was 1000 mg/kg soil dry weight.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

1 000 mg/kg soil dw