Ethylenebis(oxyethylene) bis[3-(5-tert-butyl-4-hydroxy-m-tolyl)propionate]

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

Study period:

2018

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))

Deviations:

no

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 was not enough to prepare a sufficient stock solution with deionized water. Therefore, the test substance was mixed with quartz sand for the application. For this, the test substance was finely ground using a mortar and pestle. Afterwards, required amounts of test substance were weighed to 25 g quartz sand, thoroughly mixed and later added to 2500 g (dry weight) of prepared test substrate.

The required amounts of artificial soil were weighed into a mixing tray. The test substance / quartz sand mixtures were added and thoroughly mixed with the soil. Thereafter, the water content of the soil was adjusted to about 50% WHCmax using deionized water and was mixed once again. The test mixtures were distributed to the test units.

Test organisms (species):

Eisenia fetida

Animal group:

annelids

Details on test organisms:

Species: Eisenia fetida
Reason for selection of species: Recommended by the test guideline
Breeding facility: BASF SE, Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany

Only adult worms (with clitellum) with a fresh weight between 300 and 600 mg were used. The worms that were introduced into the test were bred in horse manure and had an age from about 11 months.

Acclimatization:
Before starting the test, the worms selected were acclimatized in the test substrate in a plastic box under test conditions through overnight under permanent light and without feeding. For this, a plastic box was filled up with approx. 750 g test substrate (Dw) and added with a sufficient number of adult worms that were sorted out from the breeding boxes.

Study type:

laboratory study

Substrate type:

artificial soil

Limit test:

no

Total exposure duration:

56 d

Test temperature:

20.6 °C – 21.5 °C (exception on 8th -10th days of exposure with ≤ 0.5 °C increase in measurement due to technical reasons; with 22.5 ° C on 8th day and 22.4 °C on 10th day of exposure)

pH:

pH value of the dry test substrate: 6.4 (mean value of 2 single values; 6.4/6.4)

Moisture:

Water content in the test: 41.3% WHCmax (mean value)

Details on test conditions:

Test conditions and procedure
Test temperature: 20.6° C – 21.5 °C (exception on 8th -10th days of exposure with ≤ 0.5 °C increase in measurement due to technical reasons; with 22.5 ° C on 8th day and 22.4 °C on 10th day of exposure)
Measurement of the temperature: In a separate vessel, filled with about 750 g test substrate (dry weight), two times per week during the exposure time with an electronic thermometer. The water content of this vessel was adjusted initially (on day 0) to the water content of test vessels.
Test container: Plastic dishes with a volume of about 1.0 L, dimensions 11×15.5×6 [cm] with transparent and punctured lid
Test substrate per test container: Approx. 664 g artificial soil (wet weight)
Number of animals / test container: 10
Worm weight at start of exposure: Body weight of all added worms in the required range of 300-600 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: 0.5% (mean of 2 single values: 0.5%/0.5%)
Illumination: Light/dark cycle of 16:8 hours, over the exposure period
Measurement of light intensity, once at start of exposure: 528 Lux, (mean of 5 single measurements: 783, 497, 447, 478 and 434 [Lux])
Measurement of the temperature at adaptation: 20.9° C
Measurement of light intensity, once at start of adaption: 742Lux
pH value of the dry test substrate: 6.4 (mean value of 2 single values; 6.4/6.4)
Water content in the test: 41.3% WHCmax (mean value)

Test Units
Bellaplast plastic containers 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 and were covered with perforated transparent lids.

Test Substrate
The earthworms were tested in an artificial soil. The composition of this soil was based on OECD Guideline No. 222 (the percentages refer to the dry weight). 18.5 kg quartz sand (74% of total substrate mass) Supplier: Gebrüder Willersinn GmbH, 67071 Ludwigshafen, Germany 5.3 kg kaolin clay (21% of total substrate mass) Supplier: Co. Bassermann minerals GmbH, 68169 Mannheim, Germany 1.3 kg sphagnum peat (5% of total substrate mass), finely grounded. Supplier: Raiffeisen Mannheim eG, 68307 Mannheim, Germany 70.4 g CaCO3, p.A. (0.3 % of total substrate mass) Supplier: Merck Total mass of the test substrate: 25.17 kg Water holding capacity: 41.3 g/100g Dw (mean value) The availability of lipophilic test item (ingredients) in soil and therefore their toxicity to earthworms depends on the organic matter content of the soil. For chemicals with log Kow >2, adsorption is expected to be linearly related to soil organic matter content. With respect to the properties of the test item (log Kow >2), 5% of peat instead of 10% were used, considering the influence on bioavailability (OECD 222 and EPPO 2003).

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). The experiment began with the application of the test substance as well as the introduction of the adult worms. Initial food supply was given one day after the introduction of worms into test substrate with the test substance. Thereafter, the adult worms were fed weekly during the initial 4 weeks of the test. An amount of about 5 g finely ground cow manure moistened with about 5-6 mL deionized water was sprayed 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.

Distribution of the test mixtures with test substance
Each replicate container was filled with about 664 g of the prepared test substrate-test substance 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.

Introduction of the earthworms
The acclimatized worms were washed with tap water 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 introduction of the worms. Only worms without morphological defects were used in the test.


Experimental Procedure
In 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 and 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 (Dw) of artificial soil. After adjusting the water content to 50 ±10% WHCmax (mean value), the test mixtures were distributed to the test units. The test mixture of the control had only quartz sand without test substance. Eight replicates were prepared for the control assay and four replicates were prepared for each test substance concentration. The pH-values and the moisture of the test assays were checked once for each concentration (samples were taken from the mixing container).
Day 0 (Introduction of the worms)
The acclimatized worms were weighed and distributed into 28 test units, each unit having 10 individuals. The animals were put onto the soil surface of each test unit. No abnormalities or behavioural changes were observed
Day 1 of the Test:
Feeding: About 5 g dried mixture of cow manure were sprayed on to the soil surface of each test container. The food was moistened with about 5-6 mL deionized water per test container. Determination of the initial weight of the test vessels were carried out for later comparison with its actual initial weights. No abnormalities or behavioural changes were observed.
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:
No abnormalities were observed.
Moistening: The weight of each test container was compared with the weight of the previous feeding days. To compensate the loss of water (in comparison with the initial weight of the test container), deionized water was added, and the test containers were weighed again.
Feeding: The consumption of food was recorded for each test container. About 5 g 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 weight of each test container was determined afterwards. No abnormalities or behavioural changes were seen.
Day 22 of the Test:
Moistening: The weight of each test container was compared with the weight of the previous feeding day, i.e., day 14 and the water loss was compensated with the addition of deionized water. Later, the new weight was recorded.
Feeding: The consumption of food was recorded for each test container. About 5 g 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 weight of each test container was determined afterwards. No abnormalities or behavioural changes were seen.
Instead of 21st day as mentioned in study plan, physiological observations were made on 22nd day.
Day 28 of the Test:
Moistening: The weight of each test container was compared with the previous feeding day. The loss of water was compensated by addition of deionized water. The adult worms were recovered from each of the test containers and during this process, no morphological or behavioral changes were observed. The number of surviving adult worms in each test container was documented. Worms were classified as dead when they did not respond to a gentle mechanical stimulus to the front end. The individual weight of the surviving adult worms from each replicate was recorded.
Feeding: The supplied food on day 22 was completely consumed in all test containers. Dried food material (5 g per test container separately) was gently blended into the test substrate and the test substrate (including cocoons) was placed back into the test containers. The test containers were closed again. For another 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 noted.
Day 35 of the Test:
Moistening: The weight of each test container was determined, and the loss of water was compensated by the addition of deionized water. No abnormal changes were observed.
Day 42 and 49 of the Test:
Moistening: The weight of each test container was determined, and the loss of water was compensated by the addition of deionized water. No abnormal changes were observed.
Day 56 of the Test: The juvenile worms were brought to the surface of the test substrate by using the water bath method (at a setting of 60.2 °C). During this process morphological and behavioral changes and the number of juveniles in each test container were recorded. No morphological and behavioral changes were observed. The pH-value and the moisture content of the test substrate of one representative sample from the replicates of each test concentration were also determined as single determinations.

Nominal and measured concentrations:

nominal: 0 (control), 62.5, 125, 250, 500, 1000 mg/kg soil dw.

Reference substance (positive control):

yes

Remarks:

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:

mortality

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: other

Remarks:

biomass change

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:

Test Item

Adult worms:
No mortality of adult worms was observed in the controls and in all replicates with test substance
NOECMortality: ≥ 1000 mg test substance/kg soil (dw)
LOECMortality: > 1000 mg test substance /kg soil (dw).
NOECBiomass change: ≥ 1000 mg test substance/kg soil (dw)
LOECBiomass change: > 1000 mg test substance /kg soil (dw)

Statistical analysis shows no significant difference (Williams Test one sided and Dunnett test one sided p ≤0.05) concerning the body weight change of individual adults over 28 days between the control and all replicates of the tested test substance concentrations.

Juvenile worms:
Reproduction by count of juvenile worms:
NOECReproduction: ≥ 1000 mg test substance/kg soil (dw)
LOECReproduction: > 1000 mg test substance/kg soil (dw)
No ECx calculations were performed, because there was no dose response
relationship.

Results with reference substance (positive control):

Reference item (= reference substance)

Reference Item: BAS 346 F (Carbendazim)
Substance No.: 00/0902-8
CAS-No: 10605-21-7
Batch No.: 5385000
Expiry date: 30 Sep. 2020

The reference substance BAS 346 F (Carbendazim) was tested by a non-GLP project 68E0902/00G026 (start of exposure 16 January 2018). The raw data and the report are archived. The result of the reference substance is presented below.

ECx values [mg/kg soil (dw)], reproduction, conf. limits 95%
EC10: 0.80 LCL: 0.68 UCL: 0.95
EC50: 1.19 LCL: 1.03 UCL: 1.37
These observed effects show a high sensitivity of the test system and the derived EC50 value is within the expected range from the test guideline (1-5 mg/kg carbendazim/kg soil (dw)) and hence acceptable sensitivity of the test system is assured.

Reported statistics and error estimates:

The statistical calculations to determine the NOEC/LOEC were carried out using SAS System computer software. For the changes in body weight of live adults (% of initial weight) after the first 4 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. Dunnett’s test was performed, because the Williams trend test was not significant. For the number of juveniles (offsprings) the Jonkheere-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. The Wilcoxon-test was performed, because the Jonkheere-Terpstra trend test was not significant. The trend tests showed no significant result. No ECx calculations were performed, because there was no dose response relationship. A significant effect was demonstrated at 250 mg/kg of test substance concentration in the number of offspring in Wilcoxon test (one sided; p ≤0.01). However, this effect can be disregarded as the highest concentrations above this treatment (1000 mg and 500mg test substance/kg soil (dw)) showed no inhibition. In this treatment, the fourth replicate had a very low number of juveniles compared to other replicates. Due to this one replicate, the mean value deviated significantly from the range of reproduction in control assays. Hence, we can conclude that this observation is an outlier.

Validity criteria fulfilled:

yes

Conclusions:

Mean mortality in the control should be ≤ 10 %. The reproduction in the controls should be at least 30 juveniles per container and the coefficient of variance of reproduction in the control should be ≤ 30 %.

All validity criteria were met. After 28 days, no adult worm in the control assay was dead. A number > 250 juvenile (mean value) were counted in the test units of the control assay. The mortality of the adult worms in the control was 0 % and the coefficient of variation concerning to the offspring juvenile worms in the pooled control assay was 12.7 %.

Description of key information

No toxic effects have been observed towards soil macroorganisms.

Additional information

The toxicity to soil macroorganisms was investigated in an earthworm reproduction test according to OECD TG 222 under consideration of GLP. The test was conducted with artificial soil with nominal test concentrations of 62.5, 125, 250, 500, and 1000 mg/kg soil dw. After 28d the mortality and biomass change of the adult worms was measured and after 56d the