Tris(2,4-ditert-butylphenyl) phosphite

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))

Version / remarks:

(2004)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on preparation and application of test substrate:

The test substance is a solid material. The water solubility is not enough to prepare a sufficient stock solution with deionized water. Therefore, required amounts of test substance were weighed to 25 g quartz sand and thoroughly mixed and afterwards added to 2500 g (dry weight) prepared test substrate.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

- Common name: earthworm- Origin: Agricultural Center Limburgerhof of BASF SE, D-67056 Ludwigshafen, Germany- Specifics: Only adult worms (with clitellum) with a fresh weight between 250 and 600 mg were used. - Breeding: The worms inserted to the test were bred in horse manure and had an age from about 10 month. - Acclimatization: A transport box filled up with approx. 1.5 kg wet test substrate was provided to the supplier. A sufficient number of adult worms were sorted out from the breeding boxes into the transport box. The worms selected for the test, were acclimatized in the test substrate in the transport box under test conditions overnight under permanent light without feeding before starting the test.

Study type:

laboratory study

Substrate type:

artificial soil

Remarks:

73.7 % quartz sand; 21.0 % kaolin clay; 5.1 % sphagnum peat; 0.2 % CaCO3.

Limit test:

no

Total exposure duration:

56 d

Remarks:

The adult worms were sampled out of the test containers.

Test temperature:

20.6 - 21.6° C

pH:

- Start: 5.7 - 5.8- End: 6.5 - 6.6

Moisture:

- Start: 41.5 - 41.9% WHCmax- End: 45.8 - 55.3% WHCmax

Details on test conditions:

TEST SYSTEM- Test substrate: Total mass of the test substrate: 45.18 kg; water holding capacity: 46.3 g/100g d.w. (mean value).- Test units: Bellaplast plastic container were used as test vessels. The containers consist of inert and non-toxic plastic. The test vessels have a bottom area of 11 × 15.5 cm and a height of 6 cm. The test vessels were covered with perforated transparent lids.- Test substrate per test container: Approx. 678 g artificial soil (wet weight)- Number of animals / test container: 10- Worm weight at start of exposure: In a range of 250 - 600 mg- Number of replicates: 8 replicates for control and 4 replicates for each test substance concentration- Number of test substance concentrations: 5- Determination of water content of the dry test substrate: 0.7 % (mean of 2 single values; 0.7%/0.7%)- Illumination: Light/day cycle of 16:8 hours, over the exposure period- Light intensity: 535 lux- Feeding: The adult worms were fed with finely ground cow manure (free of growth promoters, nematicides or similar veterinary pharmaceuticals, provided by Jesus Bruderschaft e.V. Gnadenthal, D-65597 Hünfelden). After the start of the experiment, food was first provided one day after application of the test substance and introduction of the adult worms. Thereafter, the adult worms were fed weekly during the first 4 weeks of the test. An amount of 5 g moistened with about 5-6 mL deionized water was spread on the test substrate surface of each test container. If food remained unconsumed, the ration was reduced on demand. After removing the adults on day 28 further 5 g of food moistened with about 5-6 mL deionized water was mixed with the test substrate of each test vessel.TEST MIXTURES- Preparation: The required amounts of artificial soil were weighed into a mixing bowl. The test substance / quartz sand mixtures were added and thoroughly mixed with the soil. Amounts of deionized water were dosed in order to adjust the water content to about 50% WHCmax. and mixed once again. The test mixtures were distributed to the test units. Due to the high number of replicates the test assay of control was prepared in two portions.- Distribution: Each replicate container was filled with about 678 g of the prepared test mixture (corresponding to about 550 g Dw artificial soil) to a layer of about 4 cm and a corresponding surface area of about 170 cm².- Insertion of earthworms: The acclimatized worms were washed in a fine meshed sieve and gently dried on a paper towel. Ten worms were randomly selected and placed on the surface of the test substrate in each test container. The body weight of the worms was determined per animal before insertion of the worms.EFFECT PARAMETERS MEASUREDNumber and biomass [mg] of the adult earthworms determined at start of exposure and after 28 days of the test. Reproduction, mean number of juveniles per test vessel 56 days after application.EXPERIMENTAL PROCEDUREIn the control group eight test units and in each further treatment group four test units were used, which were closed with perforated lids, and placed in a temperature controlled incubation room and a photoperiod of 16 h light 8 h dark.- Day 0 (test preparation): For each test concentration, required aliquots of the test substance were mixed with 25 g quartz sand. The quartz/substance mixtures were blended with approx. 2500 g artificial soil. After adjustment of the water content to approx. 50±10 % WHCmax, the test mixtures were distributed to the test units. The test mixture of the control contained only quartz sand without test substance. Eight replicates were prepared for the control assay and four replicates were prepared for each test substance concentration. Determination of the pH value and the moisture of artificial soil once for each concentration (samples are taken from the mixing bowl).- Day 0 (Introduction of the worms): The acclimatized worms were weighed and distributed into 28 test units, each unit 10 individuals. The animals were put onto the soil surface of each test unit.- Day 1 of the test: Moistening: The weight of the test containers was verified. The loss of water was compensated by addition of deionized water. Feeding: About 5g dried cow manure was spread on the soil surface of each test container. The food was moistened with 5-6 mL deionized water per test container. The total weights of the test containers were determined which were used on day 7 for comparison with actual weights. - Day 7 of the test: Moistening: The weight of the test containers was verified. The loss of water was compensated by addition of deionized water. Feeding: The consumption of food was recorded for each test container. About 5g dried cow manure was spread on the soil surface of each test container. The food was moistened with 5-6 mL deionized water per test container. The total weights of the test containers were determined which were used on day 14 for comparison with actual weights.- Day 14 of the test: Moistening: The weight of the test containers was verified. The loss of water was compensated by addition of deionized water. Feeding: The consumption of food was recorded for each test container. About 5g dried cow manure was spread on the soil surface of each test container. The food was moistened with 5-6 mL deionized water per test container. The total weights of the test containers were determined which were used on day 21 for comparison with actual weights.- Day 21 of the test: Moistening: The weight of the test containers was verified. The loss of water was compensated by addition of deionized water. Feeding: The consumption of food was recorded for each test container. About 5g dried cow manure was spread on the soil surface of each test container. The food was moistened with 5-6 mL deionized water per test container. The total weights of the test containers were determined which were used on day 28 for comparison with actual weights.- Day 28 of the Test: Moistening: Determination of the weight of the test containers. The loss of water was compensated by addition of deionized water. The adult worms were sampled out of the test containers after the feed consumption has been recorded for each test container. During this process, no morphological and behavioral changes were observed. The number of surviving worms in each test container was recorded. Worms were classified as dead when they did not respond to a gentle mechanical stimulus to the front end. Mixing in of dried, feed material (5 g per test container separately) into the artificial soil. The artificial soil (including cocoons) was put back into the test containers. The test containers were closed again. For further 28 days they were investigated under the same conditions as described above. The new initial weight of the test vessels for the comparison of the actual weight was determined.- Day 35 of the Test: Moistening: Verification of the weight of the test containers. Water losses were compensated by addition of deionized water.- Day 42 of the Test: Moistening: Verification of the weight of the test containers. Water losses were compensated by addition of deionized water.- Day 49 of the Test: Moistening: Determination of the weight of the test containers. Water losses were compensated by addition of deionized water.- Day 56 of the Test: The juvenile worms were sampled out of the test containers. Extraction was done using the water bath method (at a setting of approx. 59 °C). During this process morphological and behavioral changes and the number of juveniles in each test container were recorded. No morphological and behavioral changes of the juveniles were observed. Determination of the pH-value and the moisture of the artificial soil once for each concentration (samples were taken from one test vessel).

Nominal and measured concentrations:

- Nominal concentrations: 0 (control), 62.5, 125, 250, 500 and 1000 mg/kg soil d.w.- Measured concentrations: no analysis performed.

Reference substance (positive control):

yes

Remarks:

BAS 346 F (Carbendazim) (CAS# 10605-21-7)

Key result

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:

other: mortality and biomass

Key result

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

Details on results:

No significant effects were noted at any of the tested concentrations.

Results with reference substance (positive control):

EC10: 1.30 mg/kg d.w. (LCL: 1.17 mg/kg d.w.; UCL: 1.46 mg/kg d.w.)EC50: 1.93 mg/kg d.w. (LCL: 1.82 mg/kg d.w.; UCL: 2.05 mg/kg d.w.)

Reported statistics and error estimates:

- For the changes in body weight of live adults (% of initial weight) after the first four weeks the statistical evaluation was carried out using Williams trend test. If this test is not significant the Dunnett’s test for a simultaneous comparison of several dose groups with the control was carried out. The statistical unit was the test container. The tests were performed one-sided.- For the number of juveniles (offsprings) the Jonckheere-Terpstra trend test was performed. If this test is not significant, a pair-wise comparison of each dose group with the control was carried out using Wilcoxon-test. The statistical unit was the test container. The tests were performed one-sided. - No ECx calculations were performed, because there was no dose response relationship.

Validity criteria fulfilled:

yes

Description of key information

The toxicity to terrestrial macroorganisms was determined in a study according to OECD guideline No 222 and in compliance with GLP criteria (BASF, 2016). In this study, groups of 10 earthworms (E. fetida) were exposed to nominal test substance concentrations of 0 (control), 62.5, 125, 250, 500 and 1000 mg/kg soil d.w. Artificila soil was used consisting of 73.7 % quartz sand, 21.0 % kaolin clay, 5.1 % sphagnum peat and 0.2 % CaCO3. No test substance analysis was performed. Number and biomass [mg] of the adult earthworms was determined at the start of exposure and after 28 days of the test. Reproduction, as mean number of juveniles per test vessel, was determined after 56 days exposure. The results in this study are consistent with all validity criteria and the test is valid according to the test guideline of this study.

No significant effects on mortality, biomass or reproduction were noted at any of the tested concentrations. Based on these findings the NOEC for all these effect parameters was determined at 1000 mg/kg soil d.w.

Key value for chemical safety assessment

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

1 000 mg/kg soil dw