Reaction mass of 2-(1,1-dimethylethyl)-4-{[5-(1,1-dimethylethyl)-4-hydroxy-2-methylphenyl]thio}-5-methylphenyl 3-(dodecylthio)propionate and thiobis[2-(1,1-dimethylethyl)-5-methyl-4,1-phenylene] bis[3-(dodecylthio)propionate]

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

yes

Details on test solutions:

The method of preparation used during the definitive test was based on the results of formulation trials, a range finding test and a main test. The test substance (500, 227.5, 103.5 or 47.0 mg) was added to 4 L dilution medium in an aspirator (5 L), and content mixed before being made up to volume. These preparations were stirred for approximately 24 hours in the dark, then left to stand for approximately 70 hours in the dark. An aliquot (2 L) was then syphoned from a mid-vessel location of each preparation vessel, of which the first litre was discarded, to give 1 L aliquots of Water Accommodated Fractions (WAF) with nominal loading rates of 100, 45.5, 20.7 and 9.39 mg/L as AO-26. The above procedure was replicated using 42.7 mg of the test substance, which was added to 9 L dilution medium in a 10 L aspirator, to give a WAF with a nominal loading rate of 4.27 mg/L as AO-26. Each aliquot of WAF was centrifuged for 30 minutes at 4000 rpm. These centrifuged media was then used as the test media providing nominal loading rates of 100, 45.5, 20.7, 9.39 and 4.27 mg/L.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (Straus)
- Source: National Institute for Applied Chemical Research (IRCHA), France.
- Age of parental stock (mean and range, SD):
- Feeding during test
- Food type: suspension of the unicellular green algae, Pseudokirchneriella subcapitata
- Amount: 0.1 to 0.2 mg
- Frequency: per day, except during the initial three days when a slightly lower ration was given.

ACCLIMATION
- Acclimation period:
- Acclimation conditions (same as test or not):
- Type and amount of food:
- Feeding frequency:
- Health during acclimation (any mortality observed):

QUARANTINE (wild caught)
- Duration:
- Health/mortality:

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:

Test type:

static

Water media type:

not specified

Total exposure duration:

48 h

Post exposure observation period:

Observations of the Daphnia in each control and test vessel made after 24 and 48 hours.

Hardness:

The total hardness Elendt M4 medium used from two batches were 256 and 247 mg/L as CaCO3, respectively.

Test temperature:

During the definitive test, continuous monitoring of the medium in an additional vessel indicated that temperatures ranged between 20.7 and 21.4°C, confirming that the test area was maintained to within ±1°C.

pH:

7-8

Dissolved oxygen:

99-102 (%ASV)

Nominal and measured concentrations:

The range finding test employed WAFs with nominal loading rates of 1, 10 and 100 mg/L as AO-26. After 48 hours, 15% immobilisation had occurred at 100 mg/L, no immobilisation occurred in other groups. The main test (originally designed as the definitive test) employed WAFs with nominal loading rates of 4.27, 9.39, 20.7, 45.5 and 100 mg/L as AO-26.

Details on test conditions:

During the definitive test, continuous monitoring of the medium in an additional vessel indicated that temperatures ranged between 20.7 and 21.4°C, confirming that the test area was maintained to within ±1°C. A photoperiod of 16 hours light : 8 hours dark was maintained, with periods of subdued lighting at the beginning and end of each light phase. No supplementary aeration was employed and no feed was given during the exposure period. The temperature, pH and dissolved oxygen levels of control and test media were recorded at the start and at the end of the study. The total hardness and alkalinity of the dilution medium was measured before use.

Reference substance (positive control):

yes

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

>= 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

The measured concentrations of AO-26 in samples of freshly prepared WAFs media were less than 1% of nominal. After 48 hours, in samples of expired media, the measured concentrations had decreased to between 30 and 59% of their starting values excluding the interim level at 20.7 mg/L where the measured level was below the limit of quantification (1 μg/L). Based on a geometric mean, the overall measured levels of AO-26 were 2.14, 5.25, 0.29, 10.3 and 26.9 μg/L. These results were not unexpected as information provided by the Sponsor indicated that AO-26 was poorly soluble in water (<0.1 mg/L). Study end-points are based on nominal loading rates. There was no significant immobilisation in any test groups.

Validity criteria fulfilled:

yes

Conclusions:

Daphnia magna were exposed for 48 hours to AO-26 as Water Accommodated Fractions at six loading rates up to 100 mg/L. No significant immobilisation was observed, therefore the median effect loading rate, (EL50) could not be determined but must be greater than 100 mg/L WAF and the “no observed effect loading rate” (NOELR) was 100 mg/L WAF (nominal).

Executive summary:

The acute toxicity of AO-26 to Daphnia magna was assessed under static exposure conditions.

The study was conducted in accordance with the following guidelines:

1. Procedure 202 of the “Guidelines for Testing of Chemicals" of the Organisation for Economic Co-operation and Development: Daphnia sp., Acute Immobilisation Test of the Guidelines for Testing of Chemicals' of the OECD (2004);
2. Part C2 (Acute Toxicity for Daphnia) of the EC Directive 440/2008.

Groups of twenty Daphnia, less than 24 hours old, were exposed to water accommodated fractions (WAFs) of AO-26, prepared from aqueous mixtures with initial nominal loading rates of 4.27, 9.39, 20.7, 45.5 and 100 mg/L. The test media were individually prepared by the direct addition of the test substance to the dilution medium (Elendt M4 medium). The aqueous mixtures were stirred for approximately 24 hours in the dark and then left to stand for approximately 70 hours in the dark before the aqueous phases (WAFs) were removed and centrifuged (4000 rpm for 30 minutes) to provide the test media

The test concentrations of AO-26 were measured using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS). The measured concentrations of AO-26 in samples of freshly prepared WAFs media were less than 1% of nominal. After 48 hours, in samples of expired media, the measured concentrations had decreased to between 30 and 59% of their starting values excluding the interim level at 20.7 mg/L where the measured level was below the limit of quantification (1 μg/L). Based on a geometric mean, the overall measured levels of AO-26 were 2.14, 5.25, 0.29, 10.3 and 26.9 μg/L. These results were not unexpected as information provided by the Sponsor indicated that AO-26 was poorly soluble in water (<0.1 mg/L). Study end-points are based on nominal loading rates.

Observations of the Daphnia in each control and test vessel were made after approximately 24 and 48 hours. There was no significant immobilisation in all groups. The following study end-points were therefore estimated in terms of nominal loading rates:

48-hour EL50 value: > 100 mg/L.

No observed effect loading rate (NOELR): ≥ 100 mg/L.

Description of key information

Daphnia magna were exposed for 48 hours to AO-26 as Water Accommodated Fractions at six loading rates up to 100 mg/L. No significant immobilisation was observed, therefore the median effect loading rate, (EL50) could not be determined but must be greater than 100 mg/L WAF and the “no observed effect loading rate” (NOELR) was 100 mg/L WAF (nominal).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L