Tetramethylcyclotetrasiloxane substituted dipropylether bisphenol A

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental dates: 04 December 2020 to 26 August 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2022

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

2009

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of (Tetramethylcyclotetrasiloxane propylether) prop-1-enylether bisphenol A and Di(tetramethylcyclotetrasiloxane propylether) bisphenol A

Cas Number:

203874-34-4

Molecular formula:

(OSiHxCH3)4-CH2-CH2 -CH2- O-C6H4-C(CH3)2-C6H4-0-CH2-CH2-CH2-(SiHxCH3O)4

IUPAC Name:

Reaction mass of (Tetramethylcyclotetrasiloxane propylether) prop-1-enylether bisphenol A and Di(tetramethylcyclotetrasiloxane propylether) bisphenol A

Test material form:

liquid

Details on test material:

Appearance: Pale yellow liquid

Specific details on test material used for the study:

Storage Conditions: Room temperature in the dark under nitrogen

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Samples were taken from the control and each test group from the uninoculated bulk test preparation at 0 hours and from an additional uninoculated sample ran alongside the test at 72 hours for TOC analysis. All samples were stored in the fridge prior to analysis. Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a WAF of the test item. Preliminary investigational work indicated that there was no significant increase in the measured amount of test item when the preparation period was extended for longer than 24 hours. Therefore, for the purpose of testing the WAF was prepared using a stirring period of 23 hours followed by a 1-Hour settlement period.

Based on the results of the range-finding test the following loading rates were assigned to the definitive test: 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L. Nominal amounts of test item (10, 32, 20, 64 and 100 mg) were each separately added to the surface of 10, 10, 2, 2 and 2 L of culture medium to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates respectively. After the addition of the test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1-Hour. The aqueous phase or WAF was removed by mid-depth siphoning (the first ~100 mL discarded) to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAFs. Microscopic inspection of the WAFs showed no micro-dispersions of test item to be present. The 1.0 mg/L loading rate WAF was further diluted to give a further test concentration of 0.10 mg/L loading rate WAF and the 3.2 mg/L loading rate WAF was further diluted to give a further test concentration of 0.32 mg/L loading rate WAF.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source: Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland.

ACCLIMATION
- Culturing media and conditions: The culture medium was prepared using reverse osmosis purified deionized water and the pH adjusted to 7.5 ± 0.1 with 0.1N NaOH or HCl. The culture medium used for the validation of mixing period trial, initial experiment, range-finding test and definitive test was the same as that used to maintain the stock culture.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

24±1°C

pH:

7.3 - 8.5

Nominal and measured concentrations:

Nominal: 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L

Measured: Total Organic Carbon analysis of the WAFs containing no algal cells was conducted at 0 and 72 hours. Measured carbon concentrations of less than the LOQ, considered to be 1.0 mg C/L were obtained from all samples. This does not infer that no test item was present and as such no carbon was in solution, just that any carbon present was at a level below the quantifiable limit.
Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Details on test conditions:

Test system: Approximately 3 to 4 days before the start of the test, inoculum cultures of algae were set up at an initial cell density of approximately 10^3 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (approximately 150 rpm) and constant illumination at 24 ±1 °C until the algal cell density was approximately 10^5 to 10^6 cells/mL.

Reference substance (positive control):

yes

Remarks:

A positive control used potassium dichromate as the reference item at concentrations of 0.125, 0.25, 0.50, 1.0 and 2.0 mg/L.

Results and discussion

Effect concentrations

Effect concentrations 1

Key result

Duration:

72 h

Dose descriptor:

other: EyL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

yield

Effect concentrations 2

Key result

Duration:

72 h

Dose descriptor:

other: ErL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

All test and control cultures were inspected microscopically at 72 hours. After 72 hours there were no abnormalities detected in the control or test cultures 0.10, 0.32, 1.0, 3.2, 10 and 32 mg/L loading rate WAFs, however swollen cells were observed to be present in the test cultures at 100 mg/L loading rate WAF.

The pH value of the control cultures was observed to increase from pH 7.6 at 0 hours to pH 8.3 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was within the limits given in the Test Guidelines. Temperature was maintained at 24±1ºC throughout the test.

At the start of the mixing period all loading rate WAFs were observed to have formed clear colorless media columns with test item visible at the surface. After 23 hours stirring the WAFs were observed to have formed clear colorless media columns with test item at the surface and fine material in suspension. After a 1-Hour standing period all loading rate WAFs were observed to be clear colorless media columns with an oily slick of test item at the surface. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present and as such the aqueous phase was removed by mid-depth siphon.
At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control 0.10, 0.32, 1.0, 3.2 and 10 mg/L loading rate WAF test cultures were observed to be green dispersions whilst the 32 and 100 mg/L loading rate WAF test cultures were observed to be pale green dispersions.

Results Total Organic Carbon Analysis
Measured carbon concentrations of less than the LOQ, considered to be 1.0 mg C/L were obtained from all samples. This does not infer that no test item was present and as such no carbon was in solution, just that any carbon present was at a level below the quantifiable limit.
Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Results with reference substance (positive control):

Exposure of Raphidocelis subcapitata (CCAP 278/4) to the reference item gave the following results:
ErC50 (0 to 72 hour) : 1.3 mg/L; 95% confidence limits 1.2 to 1.5 mg/L
EyC50 (0 to 72 hour) : 0.44 mg/L; 95% confidence limits 0.37 to 0.52 mg/L
No Observed Effect Concentration based on growth rate: 0.125 mg/L
No Observed Effect Concentration based on yield: 0.125 mg/L
Lowest Observed Effect Concentration based on growth rate: 0.25 mg/L
Lowest Observed Effect Concentration based on yield: 0.25 mg/L

The results from the positive control with potassium dichromate were within the normal ranges for this reference item.

Reported statistics and error estimates:

Statistical analysis of the growth rate data was carried out for the control and all loading rates using Williams Multiple Sequential t-test Procedure. There were no statistically significant differences between the control and the 0.10, 0.32, 1.0, 3.2 and 10 mg/L loading rate WAFs (P=0.05); however, all other loading rates were significantly different (P<0.05). Inspection of the data showed that in the 32 and 100 mg/L loading rate WAF test groups, 6% and 9% inhibition of growth rate occurred; respectively which was considered not to be significant. As such the NOEL based on growth rate was considered to be 100 mg/L loading rate WAF.

There were no statistically significant differences between the control and the 0.10, 0.32, 1.0, 3.2 and 10 mg/L loading rate WAFs (P=0.05); however, all other loading rates were significantly different (P<0.05) and, therefore the NOEL based on yield was 10 mg/L loading rate WAF. Correspondingly, the LOEL based on yield was 32 mg/L loading rate WAF.

Any other information on results incl. tables

Range-finding test results:

Growth was observed to be reduced at 1.0, 10 and 100 mg/L loading rate WAF. Based on this information loading rates of 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.
Chemical analysis of the test preparations at 0 hours showed measured test concentrations to range from less than the limit of detection (LOD) to 0.047 mg/L. Analysis of the corresponding aged preparations at 72 hours showed measured test concentrations of less than LOD for all test samples indicating that the test item was stable under test conditions.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Raphidocelis subcapitata has been investigated over a 72-Hour period according to OECD guideline 201 and GLP principles. The results indicated a NOELs for Growth Rate and Yield of 100 mg/L (loading rate).

Executive summary:

A study was performed to assess the effect of the test item on the growth of the green alga Raphidocelis subcapitata (formerly known as Pseudokirchneriella subcapitata) according to  OECD Guideline 201 and GLP principles.

Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.
Following a preliminary range-finding test, Raphidocelis subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 0.10, 0.32, 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24±1°C.
Samples of the algal populations were removed daily and cell concentrations were determined for each control and treatment group.

Total Organic Carbon (TOC) analysis of the WAFs containing no algal cells was conducted at 0 and 72 hours. Measured carbon concentrations of less than the LOQ, considered to be 1.0 mg C/L were obtained from all samples. This does not infer that no test item was present and as such no carbon was in solution, just that any carbon present was at a level below the quantifiable limit. Given that toxicity cannot be attributed to a single component or mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

Exposure of Raphidocelis subcapitata to the test item gave the following results:

Growth Rate - EL50: >100 mg/L - NOEL: 100 mg/L - LOEL: Not determined
Yield - EL50: >100 mg/L - NOEL: 10 mg/L - LOEL: 32 mg/L