Tris(2-ethylhexyl) phosphate

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:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)

Qualifier:

according to guideline

Guideline:

OECD Guideline 217 (Soil Microorganisms: Carbon Transformation Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Sampling scheme carbon transformation: CO2-production was determined over a period of up to 24 hours at different sampling intervals
Sampling scheme nitrogen transformation: 0 and 28 days after treatment

Vehicle:

no

Remarks:

As the test item turned out to be not totally soluble in acetone in the range-finder test for the highest test item concentrations, the test item will be directly applied onto quartz sand for the maintest for all test item concentrations.

Details on preparation and application of test substrate:

The test item was directly applied onto quartz sand for the main test for all test item concentrations. As the controls should be treated in the same way, quartz sand was added to the controls.
Throughout the application the soil was ventilated and the soil water content for the test item treatments were adjusted to 49% and 50% of WHC with ultrapure water.
The soil water content of the control was adjusted to 49% of WHC.
For the nitrogen transformation test, additionally 0.5% lucerne meal (related to soil dry weight) was added to the soil.

Application Scheme: The test item was applied to the total soil amount for each treatment and soil from each treatment was divided into three replicates after application. The applications were conducted in ascending order, according to the guidelines.

Test organisms (inoculum):

soil

Total exposure duration:

28 d

Test temperature:

20+-2 °C

Moisture:

Carbon transformation: 49 - 51 %
Nitrogen transformation: 48 - 51 %

Organic carbon content (% dry weight):

0.89

Nitrogen content (% dry weight):

0.11

Details on test conditions:

Light Regime: In the dark
Replicates: 3 replicates per treatment
Exposure Time: 28 days

Carbon Transformation
For the carbon transformation test, the glucose induced respiration rate was determined in each sample of treated and control soils after 0 (within 6 hours) and 28 days. The soil samples (100 g) were taken and mixed with 3 g/kg (moist soil) of glucose (1.2 mL of a solution of 250 g glucose/L ultrapure water), corresponding to a glucose concentration of 3.6 g/kg soil dry weight. The amount of glucose was determined to give the highest respiration rates (periodically determined in the soil used for the reference test).
The glucose amended soil samples were incubated at 20°C ± 2°C. The pressure decrease in the reaction vessels was measured up to 24 consecutive hours using the BSB-Sensomat System®. The oxygen consumption and the carbon dioxide release were calculated there from.
For calculation of short term respiration, usually the linear parts of the respiration curve after 2 hours up to 14 hours were used.

Nitrate Determination:
For the determination of nitrogen content, soil samples were taken within 6 hours after application and at day 28. The nitrogen content was determined in each sample of treated and control soils.
For extraction, 24 g to 25 g soil were suspended in 100 mL 0.1 M KCl-solution and agitated for one hour. The suspension was centrifuged (Multifuge 3s+, 4350 rpm) and the extracts were stored deep frozen (day 0) or analysed immediately (day 28).
Amounts of 14.8 mg and 72.2 mg sodium nitrite and potassium nitrate, respectively, were diluted in 200 mL (sodium nitrite) and 100 mL (potassium nitrate) 0.1 M KCl to prepare the standard stock solutions for nitrite-N and nitrate-N + nitrite-N determination. Appropriate aliquots of the stock solutions were automatically diluted by the dilution unity with 0.1 M KCl to prepare 6 standard solutions at a range of 0.5 mg/L to 3.0 mg/L for nitrite-N and 7 standard solutions at a range of 1.0 mg/L to 12.0 mg/L for nitrate-N +
nitrite-N determination. Before photometric determination, frozen soil extracts were thawed. For determination, undiluted extracts were used. To get the nitrate-N concentrations, the nitrite-N values were subtracted from the nitrate-N + nitrite-N values.

Nominal and measured concentrations:

Control,
nominal: 95.26 mg/kg soil dry weight, 171.5 mg/kg soil dry weight, 380.6 mg/kg soil dry weight, 555.6 mg/kg soil dry weight, 1000 mg/kg soil dry weight

Reference substance (positive control):

yes

Remarks:

sodium chloride Effects of sodium chloride were determined at a rate of 16 g/kg dry soil in a separate study (ibacon study code: 116524080) within one year before start of the experimental phase of this study

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:

respiration 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

Results with reference substance (positive control):

The reference item had a retarding effect of more than ± 25% compared to the control at days 28 and 98 after application.
Results: Deviation from control
day 28 day 98
Soil respiration rates -68.71 -68.68%
Soil nitrate content -42.60% -68.42%
Soil nitrate formation rate -111.81% -103.86%

Reported statistics and error estimates:

Normality and homogeneity of variances were tested using the Shapiro-Wilk`s-Test (α = 0.01) and Levene´s test (α = 0.01), respectively and onesided smaller comparisons of treated and control values according to Student-t-test (α = 0.05) were conducted.
For the threshold concentrations (NOEC) determination at day 28 and the interval day 0-28 (nitrogen transformation only), the Shapiro-Wilk`s Test on normal distribution (α = 0.01) and Levene´s test (α = 0.01) for variance homogeneity were performed, following by a trend analysis by contrasts (monotonicity of concentrations/response) and the Dunnett`s multiple sequential t-test procedure (α = 0.05, one-sided smaller).
The software used to conduct the statistical analysis was ToxRat Professional®, Version 3.3.0, ToxRat Solutions GmbH.

Validity criteria fulfilled:

yes

Remarks:

variation between the replicate control samples was less than 15% throughout the study; reference item had a retarding or stimulating effect of more than ± 25% compared to the control at day 28 after application

Conclusions:

The test item had neither a stimulating nor inhibiting impact on carbon transformation (soil respiration) and nitrogen transformation (nitrate-N content, nitrate-N formation rate) of soil microorganisms when applied at test item concentrations up to and including 1000 mg/kg soil dry weight treatment. The statistical analysis revealed that the NOEC for inhibition of the carbon and nitrogen transformation in soil was 1000 mg Tris(2-ethylhexyl)phosphate /kg soil dry weight.

Executive summary:

A study was conducted to assess the effects of the test item on the activity (carbon and nitrogen transformation) of soil microorganisms in the laboratory after 28 d exposure in the dark.

The soil microorganisms were exposed to 5 concentrations of the test item: 95.26 mg/kg soil dry weight, 171.5 mg/kg soil dry weight, 380.6 mg/kg soil dry weight, 555.6 mg/kg soil dry weight, 1000 mg/kg soil dry weight. The test item was directly applied onto quartz sand for the main test for all test item concentrations. A control was conducted additionally. Per Treatment and concentration three replicates were conducted. The carbon transformation (soil respiration) in soil was determined after addition of glucose. A BSB-Sensomat System® was used to determine the CO2-production over a period of up to 24 hours at different sampling intervals.The nitrogen transformation was determined in soil enriched with lucerne meal. NO3-nitrogen formed in the nitrification process was determined by continuous flow analysis.

The effects on O2-consumption (soil carbon transformation) correlating to CO2-production and on NO3-nitrogen production (soil nitrogen transformation) after 28 days exposure were determined.

The test item had neither a stimulating nor inhibiting impact on carbon transformation (soil respiration) and nitrogen transformation (nitrate-N content, nitrate-N formation rate) of soil microorganisms when applied at test item concentrations up to and including 1000 mg/kg soil dry weight treatment. Therefore, no ECx for inhibiting effects of Tris(2-ethylhexyl)phosphate could be calculated. The statistical analysis revealed that the NOEC for inhibition of the carbon and nitrogen transformation in soil was 1000 mg Tris(2-ethylhexyl)phosphate /kg soil dry weight.

Description of key information

A study was conducted to assess the effects of the test item on the activity (carbon and nitrogen transformation) of soil microorganisms in the laboratory after 28 d exposure in the dark.

The soil microorganisms were exposed to 5 concentrations of the test item: 95.26 mg/kg soil dry weight, 171.5 mg/kg soil dry weight, 380.6 mg/kg soil dry weight, 555.6 mg/kg soil dry weight, 1000 mg/kg soil dry weight. A control was conducted additionally. Per Treatment and concentration three replicates were conducted. The carbon transformation (soil respiration) in soil was determined after addition of glucose. A BSB-Sensomat System® was used to determine the CO2-production over a period of up to 24 hours at different sampling intervals.The nitrogen transformation was determined in soil enriched with lucerne meal. NO3-nitrogen formed in the nitrification process was determined by continuous flow analysis.

The effects on O2-consumption (soil carbon transformation) correlating to CO2-production and on NO3-nitrogen production (soil nitrogen transformation) after 28 days exposure were determined.

The test item had neither a stimulating nor inhibiting impact on carbon transformation (soil respiration) and nitrogen transformation (nitrate-N content, nitrate-N formation rate) of soil microorganisms when applied at test item concentrations up to and including 1000 mg/kg soil dry weight treatment. Therefore, no ECx for inhibiting effects of Tris(2-ethylhexyl)phosphate could be calculated. The statistical analysis revealed that the NOEC for inhibition of the carbon and nitrogen transformation in soil was 1000 mg Tris(2-ethylhexyl)phosphate /kg soil dry weight.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:

1 000 mg/kg soil dw