2,4-di-tert-butylphenol

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study following OECD guideline 216

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Lot-no.: 1437
Purity: 99.95%

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 1000 µg/g, 49.4 µ/g, 2.49 µg/g, 0.125 µg/g, 0.00619 µg/g, 0.0 µg/g (control)
- Sampling method: Soil samples were extracted using 100 mL of 0.1 M KCl. The soil and 0.1 M KCl mixtures were shaken on a platform shaker for 60 minutes. The mixture was then centrifuged for 20 minutes at approximately 3,700 × g. The supernatant was transferred to a graduated cylinder and was adjusted to 100 mL with 0.1 M KCl. The supernatants were stored frozen when not in use.
- Sampling intervals: Day 0, Day 7, Day 28

Vehicle:

yes

Details on preparation and application of test substrate:

AMENDMENT OF SOIL
- Type of organic substrate: Alfalfa meal which had a C/N ratio of 15/1 at an alfalfa-soil ratio of 5 g of alfalfa per kilogram of soil (dry weight).


APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: Approximately 2 gram aliquots of sand were weighed into separate 20 mL scintillation vials. Then, 0.5 mL of each dose solution was added according to the table below to the respective sand vials. Once the sand aliquots were dosed, solvent was allowed to evaporate on the benchtop with periodic swirling of sand. Once dried, the samples were stored refrigerated until ready for use.

VEHICLE:
- Chemical name of vehicle: Acetone
- Method: A 400 mg/mL stock solution of the test substance was prepared by weighing 799 mg of test substance into a 2-mL volumetric flask, correcting for the active ingredient content (100%), and bringing to volume with acetone. This 400 mg/mL stock solution was diluted to prepare 20.0, 1.00, 0.0500, and 0.00250 mg/mL dose solutions in acetone.
- Evaporation of vehicle before use: Yes.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20 ± 2°C

Moisture:

40-60% ±5%

Details on test conditions:

TEST SYSTEM
- Testing facility: ABC Laboratories, Columbia, Missouri, US
- Test container (type, material, size): 250-mL Nalgene bottles
- Amount of soil: 20 g (dry weight) per test vessel
- No. of replicates per concentration: 9
- No. of replicates per control: 9 (as vehicle control)

SOIL INCUBATION
- Method: Series of individual subsamples; The soil was added to test vessels and acclimated to the test temperature for a period of 13 days (at 19.61 to 20.33°C). The soil was amended with alfalfa meal, which had a C/N ratio of 15/1 at an alfalfa-soil ratio of 5 g of alfalfa per kilogram of soil (dry weight).

SOURCE AND PROPERTIES OF SUBSTRATE (if soil)
- Geographical reference of sampling site (latitude, longitude): Offenbach, Rheinland-Pfalz (E: 439713 N:5449850)
- History of site: Uncultivated.
- Treatments with pesticides or fertilizers: No pesticide use for current or 4 previous years, no fertilizer for current year.
- Accidental contamination: None.
- Depth of sampling: 20 cm
- Soil texture
- % sand: 66
- % silt: 24
- % clay: 10
- Soil taxonomic classification: Sandy loam
- pH (in water): 6.4
- Initial nitrate concentration for nitrogen transformation test (mg nitrate/kg dry weight): ~ 141 mg/kg
- Maximum water holding capacity (in % dry weigth): > 50%
- Cation exchange capacity (mmol/kg): 6.4 (meq/100 g)
- Pretreatment of soil: Sieving (2-mm sieve)
- Storage: In refrigerator at 4°C
- Initial microbial biomass as % of total organic C: 483.9 μg/g Dry Weight

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Nitrate concentration in soil (Days 0, 7, and 28).

VEHICLE CONTROL PERFORMED:
Yes.

RANGE-FINDING STUDY
No.

Nominal and measured concentrations:

Nominal: 0.0, 1000, 49.4, 2.49, 0.125, 0.00619 µg/g

Reference substance (positive control):

yes

Remarks:

Nitrate Standard for IC

Key result

Duration:

28 d

Dose descriptor:

NOEC

Effect conc.:

49.4 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of nitrate formation

Duration:

28 d

Dose descriptor:

EC25

Effect conc.:

ca. 1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of nitrate formation

Key result

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:

other: Inhibition of nitrate formation

Details on results:

- Effect concentrations exceeding solubility of substance in test medium: Not applicable.
- Analytics: Quality control spike recoveries for nitrate ranged from 108.4 to 113.8% throughout the
study.
- Temperature of the environmental chamber ranged from 19.61 to 20.33°C during incubation of the soils before dosing and through Day 28 of the test.

Results with reference substance (positive control):

Not applicable.

Reported statistics and error estimates:

Percent Inhibition Calculations
The mean nitrate concentration of the triplicate samples collected from each control, reference, and test substance treatment were calculated. Percent difference from the mean control nitrate concentration was calculated at each sampling time as follows:

Percent Difference = ( (Nc-Nt)/Nc) x 100%

where:
NT = Mean concentration of nitrate (μg/g) in test or reference treatment
NC = Mean concentration of nitrate (μg/g) in control treatment

Microbial evaluation:

The results of bacterial plate counts showed that the soils in the control treatment contained 1.27 x 10^8 CFU/g at initiation (Day 0) and 1.19 x 10^7 CFU/g at termination (Day 28). The bacterial plate count for test substance 1,000, 49.4, 2.49, 0.125, and 0.00619 μg/g treatments were 2.8 x 10^7, 9.9 x 10^7, 2.84 x 10^7, 6.0 x 10^7, and 5.5 x 10^7 at initiation and 1.53 x 10^7, 1.29 x 10^7, 1.40 x 10^7, 1.71 x 10^7, and 1.65 x 10^7 CFU/g at termination, respectively. The microbial evaluation data indicate that the microbial populations in each treatment remained active and viable during the course of the study.

Validity criteria fulfilled:

yes

Conclusions:

The percent inhibition on Day 28, based on nitrate concentration, was 25.9, -1.2, -1.2, -2.4, and -0.4% in the 1,000, 49.4, 2.49, 0.125, and 0.00619 μg/g treatments, respectively. The derived effect values EC50, EC25, and NOEC are >1000µg/g, ~1000µg/g and 49.4 µg/g, respectively.

Executive summary:

The purpose of this test was to investigate the effects of the test substance on the process of nitrogen transformation in aerobic surface soils. The study was conducted according to the study protocol entitled “Determination of the Effect of 2,4-di-tert-butylphenol on the Nitrogen Transformation Activity of Soil Microorganisms”, following OECD Guideline 216.

Nominal Concentrations of 0 (control); 1,000; 49.4; 2.49; 0.125; and 0.00619 μg/g soil of the test Substance 2,4 -di-tert-butylphenol were applied to the soil and exposure was followed over the 28 -day test period. The nitrate concentration was determined on Day 0, 7 and 28 and inhibition of nitrate formation was calculated as effect basis. The test soil was received from Lufa, Speyer, Germany, classified as sandy loam. Soil characterization was perfomed by Agvise Laboratories, North Dakota. The test temperature was within the accepted range of 20 ± 2°C throughout the study and all validity criteria were fulfilled.

The percent inhibition on Day 28, based on nitrate concentration, was 25.9, -1.2, -1.2, -2.4, and -0.4% in the 1,000, 49.4, 2.49, 0.125, and 0.00619 μg/g treatments, respectively.

The EC values for the effect of the test substance on the nitrogen transformation in soil were estimated based on the percent inhibition values, and are presented below.

Day	EC Type	EC Value (μg a.i./g)
28	EC50	>1,000
28	EC25	~1,000
28	NOEC	49.4

Description of key information

The percent inhibition on Day 28, based on nitrate concentration, was 25.9, -1.2, -1.2, -2.4, and -0.4% in the 1,000, 49.4, 2.49, 0.125, and 0.00619 μg/g treatments, respectively. The derived effect values EC50, EC25, and NOEC are >1000µg/g, ~1000µg/g and 49.4 µg/g, respectively.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

49.4 mg/kg soil dw

Additional information

The effects of 2,4-DTBP were tested on the microbial community according to OECD test guideline 216. Effects were determined as the percent inhibition on Day 28, based on nitrate concentration, 25.9, -1.2, -1.2, -2.4, and -0.4% in the 1,000, 49.4, 2.49, 0.125, and 0.00619 μg/g treatments, respectively. The derived effect values EC50, EC25, and NOEC are >1000µg/g, ~1000µg/g and 49.4 µg/g, respectively.