Tris(2-ethylhexyl) phosphate

Toxicity to soil macroorganisms except arthropods

Link to relevant study record(s)

Reference

Endpoint:

toxicity to soil macroorganisms except arthropods: long-term

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 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Vehicle:

yes

Remarks:

acetone

Details on preparation and application of test substrate:

A stock solution was prepared by dissolving 10500.1 mg of Tris(2-ethylhexyl)phosphate in 50 mL acetone. A sequential dilution series was prepared.
Then 10.0 mL of the corresponding dilutions were added to 50 g fine quartz sand. The treated sand was left for three hours in a fume hood until the solvent had evaporated and was mixed with a spoon. Then artificial soil equivalent to 2050 g dry weight was added to test item blended sand.
There were no significant deviations to the nominal target concentration (< 5%). The control was treated with the same amount of acetone and sand as the test item groups. While mixing the artificial soil in a laboratory mixer for approximately 5 minutes the soil of each treatment group was moistened with deionised water. Each group was treated in one batch (two in the control) which was then split into the replicates.

Test organisms (species):

Eisenia andrei

Animal group:

annelids

Details on test organisms:

- Taxonomic Group: Earthworms (Annelida: Oligochaeta)
- Species: Eisenia andrei
- Life Stage: Adults
- Age: Approximately 7 months, with well-developed clitellum, age range between test individuals not differing by more than 4 weeks
- Body Weight: 302 mg to 595 mg
- Origin: Bred under standardised conditions at ibacon laboratories in a breeding medium of cattle manure, peat, sand, calcium carbonate and straw, fed with cattle manure, stored at room temperature.
- Acclimatisation: 1 day, in artificial soil, under test conditions

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

8 wk

Test temperature:

18 - 22 °C

pH:

5.8 - 6.3

Moisture:

49.4 - 58.1 % of maximum water holding capacity (WHC)

Details on test conditions:

Test conditions
- Test Environment: Controlled environment room, in a ventilated area
- Temperature: Within the range of 18 °C to 22 °C
- Light Intensity: Within the range of 400 lux to 800 lux
- Light Regime: 16 h light : 8 h dark
- Water Content Maintenance: Once a week the water content of the soil was checked by weighing each container and evaporated water was replenished. In this way, it was ensured that the difference in water content between experimental start and end was less than 10%.
- pH: pH was determined at the start and end of the test for each treatment group, according to ISO 10390 (CaCl2).
pH range at experimental start: 5.8, and thus did not deviate from the values given by the guidelines (6.0 ± 0.5).
pH range at experimental end: 6.2 to 6.3.
- Water Content: Water content was determined at the start and end of the experiment for each test concentration, according to ISO 11465. Therefore, one soil sample was taken from one replicate of each test item concentration. For the control soil samples from two replicates were taken and the mean of the two samples is reported.
At experimental start: 26.2% to 27.5% (49.4% to 51.9% of the maximum water holding capacity, i.e. within the recommended range 40-60 % of the total water holding capacity).
At experimental end: 27.1% to 30.8% (51.0% to 58.1% of the maximum water holding capacity.
- Test Substrate: Artificial Soil: Based on OECD 222 but with reduced organic matter content:
• 5% Sphagnum-peat, air-dried and finely ground (<2 mm, with no visible plant remains); (Floragard, Vertriebs GmbH für Gartenbau, 26138 Oldenburg, Germany)
• 20% Kaolin clay (Erbslöh, 65558 Lohrheim, Germany)
• 74.8% fine quartz-sand (F34) containing more than 50% by mass of particle size 0.05 mm to 0.2 mm; (Quarzwerke Frechen, Postfach 1780, 50207 Frechen, Germany)
• 0.2% Calcium carbonate (CaCO3) was added to adjust pH to 6.0 ± 0.5 (Merck, 64293 Darmstadt, Germany).
According to OECD 222 and EPPO (2003), 5% peat was used in the artificial soil considering a potential influence of the test item on bioavailability.
The artificial soil was moistened to approximately half of the final water content 1 day before the application. The additional water required to achieve the final water content was added when applying the test item.

Food
- Feeding: Air dried and finely ground cattle manure was used as food. 5 g/container was scattered on the soil surface at day 1 after application and was moistened with 5 g deionised water; 5 g/container (moistened with 2 g deionised water) was added each week for the first 4 weeks of the experiment, when the food of the previous week had almost been consumed. If the food was not quite fully consumed, the added amount of food was adjusted to replace the visually estimated consumption. Four weeks after application, the food was mixed into the substrate following removal of the adult earthworms.
- Food Source: Cattle manure is collected at an organic farm, the cows are not treated with antibiotics or worm treatments.

Course of the Test
- No. of Replicates: 8 per control, 4 per test item treated group
- No. of Individuals: 10 per replicate, i.e. 80 individuals per control, 40 individuals per test item treated group
Introduction of Individuals: All earthworms were rinsed with tap water, dried with dry paper towels, weighed individually and randomly assigned to batches of 10 earthworms. The different batches were sorted into four classes on the basis of the total weight and one batch of each weight class was assigned to each treatment group (two batches for the control) to ensure weights were homogeneous. The earthworms were placed on the surface of the artificial soil after application.
- Test Duration: 8 weeks
- Exposure Time of Adult Earthworms: After 4 weeks, the artificial soil was transferred to a tray and adult earthworms were counted, removed and weighed per replicate after being rinsed under tap water and dried on paper towels. The remaining soil (without the adult earthworms) was then returned to the respective test containers.
- Exposure Time of Offspring: Additional 28 days
- Removal of Juveniles: Juveniles were removed by placing the test units in a water bath at 50 - 60 °C and counting all emerging earthworms. In addition, the soil of each container was emptied out onto a tray and checked visually for any remaining juvenile earthworms.

Test Parameters
- Mortality: Number of dead adult earthworms at day 28 after application (including any missing ones)
- Behavioural and Morphological Abnormalities: Number of affected adult earthworms (e.g. lack of movement, rigidity) at day 28 after application
- Food Given: Cumulative amount of food added to each test container during the test period
- Mean Body Weight: Body weights were determined at start (day 0) and 28 days after application
- Reproduction: Number of juveniles 56 days after application

Nominal and measured concentrations:

Nominal concentrations: 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg test item/kg soil

Reference substance (positive control):

yes

Remarks:

Carbendazim; the effects of the reference item were investigated in a separate GLP study

Duration:

56 d

Dose descriptor:

NOEC

Effect conc.:

1 000 mg/kg soil dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Results with reference substance (positive control):

In the most recent test with the reference item Carbendazim (performed under ibacon Study No. 105685022 from May to July 2019), there were statistically significant effects on reproduction at a concentration of 0.695 mg a.i./kg soil and above, which is in line with the guideline OECD 222 (significant effects should be observed between 1 and 5 mg a.i./kg soil). The EC50 for reproduction was calculated as 0.92 mg a.i./kg soil.

Reported statistics and error estimates:

The mortality data were not statistically analysed as no mortality was observed in any treatment group.
The body weight change and reproduction data were tested for normal distribution and homogeneity of variance (α = 0.01) using the Shapiro-Wilk´s test and the Levene´s test, respectively. Since the data for body weight changes were normally distributed and homogeneous and followed a monotonicity trend (contrast trend), the Williams t-test (multiple comparison, α = 0.05, one-sided greater) was used to compare treatment and control values.
Since the reproduction data were normally distributed and homogeneous but did not follow a monotonicity trend (contrast trend), the Dunnett’s t-test was used to compare treatment and control values (multiple comparison, α = 0.05, one-sided smaller).
Due to the lack of a concentration-response relationship no reliable ECx-calculation is possible.
The software used to perform the statistical analysis was ToxRat Professional, Version 3.3.0, ToxRat® Solutions GmbH.

Validity criteria fulfilled:

yes

Remarks:

Control Mortality: Should not exceed 10% over initial 4-week test period; Reproduction of Control: Should be ≥ 30 earthworms per replicate container; Coefficient of Variation of Reproduction in Control: Should not exceed 30%

Conclusions:

In an earthworm reproduction and growth study with Tris(2-ethylhexyl)phosphate the No Observed Effect Concentration (NOEC) for mortality, growth and reproduction of the earthworm Eisenia andrei was determined to be 1000 mg test item/kg soil, i.e. the highest concentration tested.
Due to the lack of a concentration-response relationship no reliable ECx-calculation is possible but the EC50 is estimated to be >1000 mg test item/kg soil.

Executive summary:

A study was conducted to assess the toxicity of Tris(2ethylhexyl)phosphate towards the reproduction and growth of earthworms according to OECD 222 for a 56-day period in treated artificial soil.

Adult earthworms (Eisenia andrei), approximately 7 months old, were exposed to 8 different concentrations of the test item incorporated into the soil (control, 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg Tris(2-ethylhexyl)phosphate/kg soil). 4 replicates were prepared for the test item treatments and 8 replicates for the control with 10 earthworms each. The earthworms were fed wit cattle manure from an organic farm.

An assessment of adult earthworm mortality, behavioural effects and biomass development was carried out after 28 days exposure of adult earthworms in treated artificial soil. Reproduction rate (number of offspring) was assessed after additional 28 days (assessed 56 days after application).

No mortality was observed in any treatment group.

The body weight changes of the earthworms after 4 weeks exposure to Tris(2-ethylhexyl)phosphate were not statistically significantly different compared to the control up to and including the highest test concentration of 1000 mg test item/kg soil (Williams t-test, α = 0.05, one-sided greater).

The reproduction rates were not statistically significantly different compared to the control up to and including the highest test concentration of 1000 mg test item/kg soil (Dunnett´s t-test, α = 0.05, one-sided smaller). No behavioural abnormalities were observed in any of the treatment groups. The feeding activity in all treated groups was comparable to the control.

The No Observed Effect Concentration (NOEC) for mortality, growth and reproduction of the earthworm Eisenia andrei was determined to be 1000 mg test item/kg soil, i.e. the highest concentration tested.

Due to the lack of a concentration-response relationship no reliable ECx-calculation is possible but the EC50 is estimated to be >1000 mg test item/kg soil.

Description of key information

A study was conducted to assess the toxicity of Tris(2ethylhexyl)phosphate towards the reproduction and growth of earthworms according to OECD 222 for a 56-day period in treated artificial soil.

Adult earthworms (Eisenia andrei), approximately 7 months old, were exposed to 8 different concentrations of the test item incorporated into the soil (control, 16.3, 29.4, 52.9, 95.3, 171.5, 308.6, 555.6 and 1000 mg Tris(2-ethylhexyl)phosphate/kg soil). 4 replicates were prepared for the test item treatments and 8 replicates for the control with 10 earthworms each. The earthworms were fed wit cattle manure from an organic farm.

An assessment of adult earthworm mortality, behavioural effects and biomass development was carried out after 28 days exposure of adult earthworms in treated artificial soil. Reproduction rate (number of offspring) was assessed after additional 28 days (assessed 56 days after application).

The No Observed Effect Concentration (NOEC) for mortality, growth and reproduction of the earthworm Eisenia andrei was determined to be 1000 mg test item/kg soil, i.e. the highest concentration tested.

Due to the lack of a concentration-response relationship no reliable ECx-calculation is possible but the EC50 is estimated to be >1000 mg test item/kg soil.

Key value for chemical safety assessment

Short-term EC50 or LC50 for soil macroorganisms:

1 000 mg/kg soil dw

Long-term EC10, LC10 or NOEC for soil macroorganisms:

1 000 mg/kg soil dw

Additional information

Should read as "EC50 > 1000 mg/kg soil dw" and "NOEC = 1000 mg/kg soil dw"