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]

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Deviations:

yes

Remarks:

Further details are provided in the materials and methods section.

Principles of method if other than guideline:

The following deviation from protocol occurred:
The pH and temperature of the medium at each vessel (excluding those prepared solely for chemical analysis) should have been measured at the end of the test, after the removal of samples for cell counting. This was not undertaken in error but pooled
sample temperature and pH was recorded instead.

This deviation was considered not to affect the integrity or validity of the study.

GLP compliance:

yes (incl. QA statement)

Vehicle:

yes

Remarks:

The test substance (499.4, 156.1, 49.1 or 15.4 mg) was added to 4 L dilution medium in an aspirator (5 L) and the contents mixed before being made up to volume.

Details on test solutions:

The test substance (499.4, 156.1, 49.1 or 15.4 mg) was added to 4 L dilution medium in an aspirator (5 L) and the contents mixed before being made up to volume. These preparations were stirred for approximately 24 hours and then left to stand for approximately 24 hours. A portion (ca. 2L) was siphoned from a mid-vessel location of each vessel of which the first ca. 1 L was discarded, to give water accommodated fractions (WAFs) with nominal loading rates of 100, 31.3, 9.77, 3.05 and 0.954 mg/L. Each WAF was then centrifuged at 4000 rpm for at least 30 minutes and an aliquot of the supernatant was removed and used as test media.

The above procedure was replicated using 10.1 mg of the test substance, which was added to 9 L dilution medium in a 10 L aspirator, to give a centrifuged WAF with a nominal loading rate of 0.954 mg/L as AO-26.

An aliquot (4.1 mL) of the secondary algal inoculum was added to a portion (400 mL) of the test media at 3.05, 9.77, 31.3 and 100 mg/L WAF, to give an initial cell density of ca. 1 x 104 cells/mL. At 0.954 mg/L WAF a 3.6 mL aliquot was added to 350 mL test medium to give the same initial cell density.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata,
- Strain: CCAP 278/4.
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd.,

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.0 and 23.2°C

pH:

7-9

Nominal and measured concentrations:

The definitive test employed WAFs with nominal loading rates of 0.954, 3.05, 9.77, 31.3 and 100 mg/L as AO-26

Details on test conditions:

A range finding test was followed by a definitive test with five test concentrations, plus an algal nutrient medium control group.

Six flasks were established and incubated for the control group and three flasks for each test group, plus an additional flask at nominal concentrations of 0.954 and 100 mg/L WAF, which contained test medium but no algal cells. The media remaining in the preparation flasks were used for water quality measurements at the start.

Before the start of the test, the required number of empty test vessels (250 mL conical flasks),were loosely stoppered with foam bungs, covered with aluminium foil that was secured by autoclave tape and sterilised by autoclaving (121°C for at least 15 minutes). After the addition of the inoculated test medium (100 mL), each flask was then loosely plugged with a foam bung. The control cultures were prepared as for the test medium except that no test substance was added and a larger volume (700 mL) of medium was made, requiring 7.2 mL of inoculum.

The range finding test employed WAFs with nominal loading rates of 1, 10 and 100 mg/L as AO-26. After 72 hours, there was 45% inhibition in algal growth at 100 mg/L WAF, 25% inhibition at 10 mg/L WAF, and no significant inhibition at 1 mg/L WAF. Based on these findings, the definitive test employed WAFs with nominal loading rates of 0.954, 3.05, 9.77, 31.3 and 100 mg/L as AO-26.

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Yield

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

At the start of the test, the measured concentrations of AO-26 in samples of the test cultures were less than 1% of nominal, ranging between 2.18 and 25.4 μg/L, with no marked dose related trend in concentration. These measurements demonstrated that at the start of the test quantities of AO-26 in the test media were detectable. After 72 hours, the measured levels at all test concentrations had decreased to below the limit of detection or quantitation. These results were not unexpected as information provided by the sponsor indicated that AO-26 was poorly soluble in water (<0.1 mg/L).

After 72 hours, analysis of samples of media containing AO-26 at nominal loading rates of 0.954 and 100 mg/L WAF without algal cells gave similar results to test media incubated in the presence of algal cells.

There was no significant inhibition of growth at any test concentrations. Based on nominal loading rates, the following study end-points were therefore estimated for average specific growth rate and yield:
72-hour ErL50 and EyL50: > 100 mg/L.
No observed effect loading rate (NOELR): ≥ 100 mg/L.
The mean coefficient of variation (CoV) for section by section daily growth rates in the control cultures was 11.7 during the definitive test and the CoV for the average specific growth rates of the control culture was 1.45 during the 72 hour exposure period.

Low levels of swollen or irregular shaped cells were observed at a nominal loading rate of 100 mg/L WAF from Day 1; otherwise, no microscopic abnormalities of the cells were detected.

Validity criteria fulfilled:

yes

Conclusions:

The effect of AO-26 (tested as a centrifuged WAF) on the growth of the unicellular green alga Pseudokirchneriella subcapitata was assessed over a 72 hour period.
Based on nominal loading rates, the 72-hour EC50 for growth rate and yield was greater than 100 mg/L, the highest concentration tested. The ‘no-observed effect loading rate’ for growth rate and yield was 100 mg/L.

Executive summary:

The effect of AO-26 on the growth of the unicellular green alga Pseudokirchneriella subcapitata was assessed under non-axenic conditions.

The study was conducted in accordance with the following guidelines: Procedure 201 (Freshwater Algae and Cyanobacteria, Growth Inhibition Test) of the

"Guidelines for Testing of Chemicals" of the Organisation for Economic Co-operation and Development" (adopted 23 March 2006; corrected 28 July 2011) and Part C3 (Freshwater Algae and Cyanobacteria, Growth Inhibition Test) of Commission Regulation (EC) No 761/2009: Annex IV, adopted 24 August 2009.

Triplicate algal cultures, with an initial cell density of ca. 1 x 104/mL, were exposed to water accommodated fractions (WAF) of AO-26 prepared from aqueous mixtures with nominal loading rates of 0.954, 3.05, 9.77, 31.3 and 100 mg/L. Aliquots of the test substance were added to OECD medium; these mixtures were stirred for a minimum of 24 hours in the dark and then left to stand for a minimum of 24 hours in the dark before the aqueous phase (WAFs) were removed and centrifuged (4000 rpm for 30 minutes) to provide the test media. The cultures were incubated in an orbital incubator under continuous illumination with the algal cultures at temperatures ranging from 23.0 to 23.2°C for 72 hours.

The test concentrations of AO-26 were measured using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS). At the start of the test, the measured levels of AO-26 in samples of the test cultures were less than 1% of nominal, ranging between 2.18 and 25.4 μg/L with no marked dose related increase in concentration. After 72 hours, the measured levels at all test concentrations had decreased to below the limit of detection or quantitation. These results were not unexpected as information provided by the sponsor indicated that AO-26 was poorly soluble in water (<0.1 mg/L).

Cell numbers were counted daily to monitor growth. There was no significant inhibition of growth at any of the test levels. Based on nominal loading rates, the following study endpoints were estimated for average specific growth rate and yield:

72-hour EL50 and EL50: > 100 mg/L.

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

Description of key information

Based on nominal loading rates, the 72-hour EC50 for growth rate and yield was greater than 100 mg/L, the highest concentration tested. The ‘no-observed effect loading rate’ for growth

rate and yield was 100 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L