2,6-di-tert-butylphenol

Toxicity to soil microorganisms

Administrative data

Endpoint:

toxicity to soil microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-03 to 2017-06-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

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

Test material

Test material information

1

Reference substance name:

2,6-di-tert-butylphenol

EC Number:

204-884-0

EC Name:

2,6-di-tert-butylphenol

Cas Number:

128-39-2

Molecular formula:

C14H22O

IUPAC Name:

2,6-di-tert-butylphenol

Test material form:

solid

Details on test material:

- State of aggregation: White to yellow, solid
- Water solubility: The water solubility of 2,6-di-tert-butylphenol is 4.11 mg/L at 25 °C at pH 7 determined with a method equivalent or similar to OECD Guideline 105 (Water Solubility)

Sampling and analysis

Analytical monitoring:

no

Test substrate

Vehicle:

yes

Remarks:

Quartz sand

Details on preparation and application of test substrate:

Application
The respective test item amount for each test item concentration were treated with a mortar, weight out on quartz sand (1 % of the entire soil amount per concentration), mixed thoroughly and applied to the surface of the soil. Additional demineralized water was added to adjust the humidity of the artificial soil. Afterwards the soil was mixed to ensure a homogeneous distribution of the test item in the soil and portioned into replicates (three replicates each per concentration and control).

Test organisms

Test organisms (inoculum):

soil

Study design

Total exposure duration:

28 d

Remarks:

For the test item concentration 1000 mg/kg soil dry weight inconclusive results were determined on day 28, so the measurements were repeated on day 29.

Test conditions

Test temperature:

Nominal: 20 +/- 2 °C
Actual: 21 - 22 °C

Moisture:

Dry weight before application: 87.5 g/100 g soil
Maximal water holding capacity: 35.6 +/- 1.4 g/100 g soil DW

Details on test conditions:

TEST SYSTEM
- Testing facility: Noack Laboratorien GmbH
- 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: 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:
2012 - 2016: uncultivated
Fertilisation:
2016: none
2015: none
2014: 3500 kg/ha CaO (2014-06-05 and 2014-09-24)
3500 kg/ha CaO; 1463 kg/ha MgO (2014-12-15)
2013: none
2012: none
Pesticides
No crop protection products applied during the year of sampling and 4 preceding years.

Soil Parameters
Parameter LUFA-soil 2.3
Batch-No. F2.30217
sampling depth* ca. 20 cm
pH value* 5.9 ± 0.6
Dry weight (DW) before application
[g/100 g soil] 87.5
Maximum water holding capacity*
[g/100 g DW] 35.6 ± 1.4
Particle size distribution acc. to DIN*
Sand:
2.0 - 0.63 mm % dry weight 2.7 ± 0.7
0.63 - 0.2 mm % dry weight 30.1 ± 0.6
0.2 - 0.063 mm % dry weight 24.7 ± 1.4
Silt:
0.063 - 0.02 mm % dry weight 18.8 ± 1.6
0.02 - 0.006 mm % dry weight 11.5 ± 0.5
0.006 - 0.002 mm % dry weight 5.3 ± 0.7
Clay:
< 0.002 mm % dry weight 6.9 ± 1.6
Organic carbon content [%]1 0.631
Microbial biomass [%] of total organic carbon 3.16
Nitrate-N [mg NO3-N/kg DW] 9.31
Ammonium-N [mg NH4-N/kg DW] 0.484
Total inorganic Nitrogen [mg/kg DW] 9.79
Cation exchange capacity [meq / 100 g]* 7.6 ± 0.8
Soil texture* silty sand (uS)#
*) data provided by LUFA Speyer
#) acc. to German DIN classification
1) determined by AGROLAB Agrar und Umwelt GmbH, based on dry weight (non GLP)

Soil handling
LUFA Speyer:
The soil was manually cleared of large objects and then sieved to a particle size of up to 2 mm.
The maximum water holding capacity and the pH value were determined.
 
Soil preparation
Test facility:
The soil moisture content was determined.
Prior to final moistening at test start, 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 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).

Soil storage
The soil was stored for 75 days (2017-01-25 to 2017-04-11) in the dark at 6 ± 2 °C in a climatic room (TE1200, Viessmann). Subsequently, the soil was pre-incubated at room temperature (ca.20 °C) for 22 days (2017-04-11 to 2017-05-03) to adapt the micro-organisms to test conditions before the start of the study.
The storage temperature was higher as stated in the guideline (4 ± 2 °C) due to technical reasons, but did not influence the activity of the micro-organisms in the soil.
For soil parameter at test start (microbial biomass and amount of total inorganic nitrogen) please refer to section 10.

Extraction and Determination of Nitrate
Determination of nitrate concentrations
Nitrate was extracted from soil with a mineral salt solution. A cleaning step with solid phase extraction (SPE) cartridges was carried out to eliminate potential interference from coloured organic matter in the extraction solution. Nitrate-nitrogen concentrations were then determined photometrically.

Wave length 588 nm for nitrate
Solutions Extraction solution: Potassium chloride, 1 M and 2 M
Reagent solution: 2 g/L 4-Ethylresorcinol in 2-Propanol
Standard Potassium nitrate > 99%

Preparation of standard solutions
A stock solution of 100 mg nitrate/L was prepared in demineralized water. 6 concentrations were prepared by dilution with 1 M potassium chloride and used for calibration.
Calibration
Calibration was performed at experimental starting.
Working steps
15 g soil of each replicate were weighed into shaking flasks and shaken with 60 mL extraction solution for 1 h at 150 - 200 rpm. Mixtures were then filtered. The first 20 mL of each filtrate was rejected and the remainder collected for SPE clean-up and photometric analysis.
Sample clean-up
10 mL of each filtered extract was cleaned via C18-SPE-cartridges to remove dissolved and coloured organic matter which could interfere with the photometric determination. Conditioning of the cartridges was done with 2 x 2.5 mL methanol and thereafter with 2 x 2.5 mL demineralized 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. On day 14 and 28/29 all samples were diluted 1:4.
Method validation
The linearity and limit of quantification (LOQ) of the nitrate method were evaluated. In the range of calibration standards the second lowest standard was chosen as the limit of quantification.

VEHICLE CONTROL PERFORMED: no

Preliminary results (Non-GLP)

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 3 -3 1 4
100 4 -2 -1 -2
1000 -2 -8 -5 0

Inhibition of Nitrate-N Formation Rates
Test concentration Inhibition [%] compared to untreated Control
[mg/kg soil DW] 7 d 14 d 28 d
10 -30 -8 -6
100 -31 -22 -16
1000 -31 -18 5
DW = dry weight
negative value = promotion of nitrate-N content / nitrate-N formation rate

Nominal and measured concentrations:

Nominal test concentrations:
1000 - 316 - 100 - 31.6 - 10 - 3.16 mg /kg soil dry weight (factor √10)

Reference substance (positive control):

yes

Remarks:

Cyanoguanidine

Results and discussion

Effect concentrations

Effect concentrations 1

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

Effect concentrations 2

Duration:

28 d

Dose descriptor:

other: LOEC

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Effect concentrations 3

Duration:

28 d

Dose descriptor:

EC25

Effect conc.:

> 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

nitrate formation rate

Effect concentrations 4

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

Results with reference substance (positive control):

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

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 41.0 ± 1.19 49.5 ± 2.00 55.4 ± 1.35 52.4 ± 1.52
50 41.7 ± 1.33 38.9 ± 1.10 38.0 ± 1.70 44.6 ± 2.11


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 n.d. n.d. 75*
*) difference to control ≥ 25 %
n.d.= not determinable, due to negative nitrate formation rates.

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.

Any other information on results incl. tables

Reduction of Nitrate-N Content in soil containing2,6-DI-TERT-BUTYLPHENOL HIGH PURITY, relative to the untreated control

Nitrate-N Content
Test concentration	Inhibition [%] compared to control
[mg/kg soil dry weight]	0 d	7 d	14 d	28/29 d
3.16	2	-2	3	-3
10	3	-4	3	1
31.6	-2	-11	5	-2
100	-1	-16	4	-5
316	-3	-22	5	4
1000	7	-6	5	1

negative values = increase

 Inhibition of Nitrate-N Formation Rates in soil containing2,6-DI-TERT-BUTYLPHENOL HIGH PURITY, relative to the untreated control

Nitrate-N Formation Rate
Test concentration	Inhibition [%] compared to control
[mg/kg soil dry weight]	7 d	14 d	28 / 29 d
3.16	-11	5	-8
10	-20	4	-1
31.6	-34*	12	-2
100	-54*	8	-8
316	-76*	10	8
1000	-39*	3	-4

*) Statistically significant difference compared to control (ANOVA, p ≤ 0.05)

  negative values = increase

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

After 7 days of exposure to 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY a significant increase of the nitrate-N formation rate was determined for the test item concentrations 31.6, 100, 316 and 1000 mg/kg soil dry weight.
After 14 and finally after 28 (29) days of exposure to 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY, no significant inhibition or increase was observed for the test item concentrations up to 1000 mg/kg soil dry weight.
For the test item concentration 1000 mg/kg soil dry weight inconclusive results were determined on day 28, so the measurements were repeated on day 29
Thus 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY is not expected to cause any long term detrimental effects towards nitrate transformation in soil under normal conditions.
Since no inhibition ≥ 25 % of the nitrate-N formation rate was observed, no ECx-values were determined. 
The corresponding no-observed effect concentration (NOEC) was 1000 mg 2,6-DI-TERT-BUTYLPHENOL HIGH PURITY/kg soil dry weight on day 28/29. The LOEC was determined to be > 1000 mg/kg soil dry weight on day 28/29.

Executive summary:

The effects of 2,6-DI-TERT-BUTYLPHENOLHIGHPURITY (Batch no. 222428901) on the metabolic activity of soil micro-organisms were determined according to OECD Guideline 216 (2000) at Noack Laboratorien GmbH, D-31157 Sarstedt, Germany from 2017-05-03 to 2017-06-01. The test guideline presents alternative testing procedures that apply to agrochemical and non-agrochemical substances. The approach for non-agrochemical substances, which entails a dose-response test design and focuses primarily on inhibitory effects was adopted in this study performed with 2,6-DI-TERT-BUTYLPHENOLHIGHPURITY.

The test item was applied via quartz sand at concentrations 3.16, 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. Plastic boxes (volume 1.0 L) with perforated tops to enable gas exchange and filled with 400 g soil dry weight were used as test vessels.

In the presence of a viable community of soil microflora, organic nitrogen undergoes conversion to mineralised nitrogen and a sequence of microbially mediated transformations that result in the formation of a series of inorganic nitrogen species: ammonification (product = NH₄⁺‑N), followed by Stage 1 nitrification (product = NO₂^-‑N) and finally Stage 2 nitrification (product = NO₃^--N). 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 with2,6-DI-TERT-BUTYLPHENOLHIGHPURITYagainst the rate observed in the untreated control. Measurements of nitrate-nitrogen concentrations in soil were made in all treatments and the control on the day of treatment application (day 0) and after 7, 14 and 28 days.

After 7 days of exposure to 2,6-DI-TERT-BUTYLPHENOLHIGHPURITY a significant increase of the nitrate-N formation rate was determined for the test item concentrations 31.6, 100, 316 and 1000 mg/kg soil dry weight.

After 14 and finally after 28 (29) days of exposure to 2,6-DI-TERT-BUTYLPHENOLHIGHPURITY, no significant inhibition or increase was observed for the test item concentrations up to 1000 mg/kg soil dry weight.

For the test item concentration 1000 mg/kg soil dry weight inconclusive results were determined on day 28, so the measurements were repeated on day 29.

Thus2,6-DI-TERT-BUTYLPHENOLHIGH PURITY is not expected to cause any long term detrimental effects towards nitrate transformation in soil under normal conditions.

Since no inhibition ≥25 % of the nitrate-N formation rate was observed, no EC_x-values were determined.

The corresponding no-observed effect concentration (NOEC) was 1000 mg 2,6-DI-TERT-BUTYLPHENOLHIGHPURITY/kg soil dry weight on day 28/29. The LOEC was determined to be > 1000 mg/kg soil dry weight on day 28/29.

Nitrate-N Formation Rate: NOEC-, LOEC- and EC_x-Values

		Day 28/29
		[mg/kg soil dry weight]
NOEC		1000
LOEC		> 1000
EC25		> 1000
95 % confidence interval		–
EC50		> 1000
95 % confidence interval		–