2,2-bis[[(mercaptoacetyl)oxy]methyl]-1,3-propanediyl bis(mercaptoacetate)

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

Study period:

2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

The samples were taken from the biological phase of the study. Collecting, storage and handing over of the samples were the Study Director’s responsibility. The information concerning the samples was provided by the Study Director.
One sample from the freshly prepared filtrated of the stock solution and duplicate samples from the freshly prepared test media (without algae) of all test concentrations and from the controls were taken at the start of the test.
For the determination of the stability of the test item under the test conditions and of the maintenance of the test item concentrations during the test period, duplicate samples from the test media of all test concentrations and the controls (containing algae) were taken after 24 and 48 hours test duration and at the end of the test (after the 72 hours test period by pouring together the contents of the test beakers of each treatment).
All samples were diluted by a factor of two with acetonitrile.
Additional samples of the untreated controls and of the sample dilution solvent were taken at each sampling without any sample treatment.
Storage of test samples:
All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed. Afterwards the samples were again stored deep frozen and will be kept stored up to the date of the final report.
Storage of standard solutions and final extracts:
All samples were analysed directly after sample preparation without further storage.
Analyses:
The concentrations of the test item PETMA were analysed in the duplicate test media samples from all test concentrations and in the duplicate control samples from all sampling times (0, 24, 48 and 72 hours) and in the additional control and solvent samples from 0 and 72 hours.

Vehicle:

no

Details on test solutions:

Pre-Experiments:
Pre-experiments were performed to determine a suitable concentration range and to establish suitable methods to prepare the test solutions.
The pre-experiments were not performed in compliance with the GLP-Regulations and are excluded from the Statement of Compliance in the final report, but the raw data of these tests will be archived under the study number of the present study.
Test Concentrations:
Due to the low limit of water solubility of the test item, dilutions of 1:5, 1:15, 1:45, 1:135, 1:405 and 1:1215 (spacing factor 3) of a filtrate of nominal 100 mg test item/L were tested, corresponding to loading rates of 20, 6.7, 2.2, 0.74, 0.25 and 0.082 mg test item/L (spacing factor 3).
Control:
In the control, test water was used without addition of the test item.

Test organisms (species):

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

Details on test organisms:

Pseudokirchneriella subcapitata (KORSHIKOV), Strain No. 61.81 SAG, formerly known as Selenastrum capricornutum, and recently renamed as Raphidocelis subcapitata (KORSHIKOV).
The algae were originally supplied by the "Sammlung von Algenkulturen, Albrecht-von-Haller-Institut für Pflanzenwissenschaften, Universität Göttingen", 37073 Göttingen, Germany.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3.

Test temperature:

In a range of 21 - 24 °C, controlled at ± 2 °C.
The temperature will be measured daily in an Erlenmeyer flask filled with water and incubated under the same conditions as the test flasks.

pH:

The pH of the untreated test water should be pH 8.1 ± 0.1 at test start.
If necessary, the pH will be adjusted with e.g. 1 M HCl or 1 M NaOH.

Nominal and measured concentrations:

Due to the low limit of water solubility of the test item, dilutions of 1:5, 1:15, 1:45, 1:135, 1:405 and 1:1215 (spacing factor 3) of a filtrate of nominal 100 mg test item/L were tested, corresponding to loading rates of 20, 6.7, 2.2, 0.74, 0.25 and 0.082 mg test item/L (spacing factor 3).

Details on test conditions:

Water temperature: 22.4 to 22.6 °C;
pH value in the control at test start: 8.0,
pH value in the control at test end: 8.0;
pH values in the test item treatments at test start: 8.0,
pH values in the test item treatments at test end: 7.9 to 8.0;
continuous illumination; mean light intensity: 4897 lux (4530 to 5160 lux).


Reconstituted Water (OECD Medium):
Analytical grade salts were added at the following nominal concentrations in deionised water:
Macro-nutrients:
NaHCO3 50.0 mg/L CaCl2 × 2 H2O 18.0 mg/L NH4Cl 15.0 mg/L MgSO4 × 7 H2O 15.0 mg/L MgCl2 × 6 H2O 12.0 mg/L KH2PO4 1.6 mg/L
Trace Elements:
Na2EDTA × 2 H2O 100.0 μg/L FeCl3 × 6 H2O 64.0 μg/L MnCl2 × 4 H2O 415.0 μg/L H3BO3 185.0 μg/L Na2MoO4 × 2 H2O 7.0 μg/L ZnCl2 3.0 μg/L CoCl2 × 6 H2O 1.5 μg/L CuCl2 × 2 H2O 0.01 μg/L
Calculated water hardness of the test water: 0.24 mmol/L (= 24 mg/L) as CaCO3.
The culture medium was prepared 6 days before test start to allow pH to stabilise. The pH of untreated test water was checked before test start and was in range of 8.1 ± 0.1.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.74 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

7.67 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

The 72-hour EyC50 was calculated to be 1.72 mg test item/L and the ErC50 7.67 mg test item/L. The 72-hour EyC10 was calculated to be 0.696 mg test item/L and the ErC10 0.816 mg test item/L. The 72-hour NOEyC was determined to be 0.74 mg test item/L and the associated 72-hour LOEyC of 2.2 mg test item/L. The 72-hour NOErC was determined to be 0.74 mg test item/L and the associated 72-hour LOErC is 2.2 mg test item/L.

Table 3. Influence of PETMA on the Growth of Pseudokirchneriella subcapitata Parameter	Yield [mg test item/L]	Growth rate [mg test item/L]
72-hour EC50	1.72	7.67
95 % conf. interval	1.51 - 1.96	6.48 - 9.19
72-hour EC20	0.950	1.76
95 % conf. interval	0.728 - 1.13	1.31 - 2.22
72-hour EC10	0.696	0.816
95 % conf. interval	0.484 - 0.872	0.533 - 1.12
72-hour NOEC	0.74	0.74
72-hour LOEC	2.2	2.2
Values refer to loading rates.

Validity criteria fulfilled:

yes

Conclusions:

The influence of PETMA on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 1.72 mg test item/L and the 72-hour ErC50 value was calculated to be 7.67 mg test item/L. The 72-hour NOEyC was determined to be 0.74 mg test item/L and the associated 72-hour LOEyC was 2.2 mg test item/L. The 72-hour NOErC was determined to be 0.74 mg test item/L and the associated 72-hour LOErC was 2.2 mg test item/L.
The initial concentrations and the maintenance of the exposure concentrations during the test were examined in the analytical part. All reported results refer to nominal loading levels.

Description of key information

The influence of PETMA on the growth of the freshwater green algae Pseudokirchneriella subcapitata was assessed in a static concentration-response test. The 72-hour EyC50 was calculated to be 1.72 mg test item/L and the 72-hour ErC50 value was calculated to be 7.67 mg test item/L. The 72-hour NOEyC was determined to be 0.74 mg test item/L and the associated 72-hour LOEyC was 2.2 mg test item/L. The 72-hour NOErC was determined to be 0.74 mg test item/L and the associated 72-hour LOErC was 2.2 mg test item/L.
The initial concentrations and the maintenance of the exposure concentrations during the test were examined in the analytical part. All reported results refer to nominal loading levels.

Key value for chemical safety assessment

EC50 for freshwater algae:

7.67 mg/L