Butanedioic acid, sulfo-, C-[2-[(1-oxododecyl)amino]ethyl] ester, monosodium salt

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:

July 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

Sampling time:
0, 24, 48 and 72 h after study initiation

Vehicle:

yes

Remarks:

test medium (composition according to OECD 201)

Details on test solutions:

Growth and test medium:
the following OECD medium (according to OECD 201) was used for culturing and in the test:

- Macronutrients
NH4Cl: 15.0 mg/L
MgCl2 x 6 H2O: 12.0 mg/L
CaCl2 x 2 H2O: 18.0 mg/L
MgSO4 x 7 H2O:15.0 mg/L
KH2PO4: 1.6 mg/L
NaHCO3: 50.0 mg/L

- Trace elements
H3BO3: 185.0 μg/L
MnCl2 x 4 H2O: 415.0 μg/L
ZnCl2: 3.0 μg/L
CoCl2 x 6 H2O: 1.5 μg/L
CuCl2 x 2 H2O: 0.01 μg/L
Na2MoO4 x 2 H2O: 7.0 μg/L
FeCl3 x 6 H2O 64.0 μg/L
Na2EDTA x 2 H2O: 100.0 μg/L
(reagents of analytical grade)

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: freshwater green alga
- Strain: Desmodesmus subspicatus HILSE, 86.81 SAG
- Source (laboratory, culture collection): MBM ScienceBridge GmbH, Hans-Adolf-Krebs-Weg 1, 37077 Göttingen, Germany
- Age of inoculum (at test initiation): axenic stock cultures grew in culturing vessels for 4 days prior to test initiation
- Method of cultivation: cultivation was performed in glass flasks with the medium described above; algae were kept at the same temperature and light conditions as in the test itself

ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions: same as test
Temperature (exposure phase): 22.8 – 22.9 °C (recorded continuously in a vessel containing test medium)
Illumination: continuous cold white fluorescent light; light intensity was measured once before test start: light intensity: on average 76 μE·m-2·s-1 measured at 400-700 nm differences from the selected light intensity over the test area did not exceed the range ± 15%

- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.8 – 22.9°C

pH:

8.14-.8.19

Nominal and measured concentrations:

Nominal:
0, 26.0, 36.4, 51.0, 71,4 and 100.0 mg/L
Measured (mean over 72 h):
0, 23.9 (92%), 34.13 (94%), 47.73 (94%), 66.87 (94%) and 100.62 mg/L (101%)

Reference substance (positive control):

yes

Remarks:

The reference item potassium dichromate was tested in a separate study to verify the sensitivity of the test system.

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

66.9 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

Analytical determination
Samples of the test solution were taken from the bulk solution at the test start and spent test solutions were sampled as composite specimens per treatment group. Specimens were stabilised with methanol (v:v, 1:1) and were stored deep frozen (≤ -18°C) until they were analysed. Concentrations of the test item CS-2021-939 in test solutions at test start (0 h), at 24 and 48 hours after test start and at test end (72 hours) were analysed. The method of the analytical determination of the test item and the results are described in the related analytical phase report.
Measured concentrations of the test item in test solutions were within a range of 93 to 97% of nominal values at test start and after 24, 48 and 72 hours the concentrations ranged from 84 to 108% in spent test solutions.
Therefore, the calculated endpoints are based on the nominal and mean measured concentrations for test item.

Validity criteria
All validity criteria for this type of study were met:
- The biomass in the control cultures increased exponentially by a factor of at least 16 within the 72-hour test period (being 70.9).
- The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures did not exceed 35% (being 19.6%).
- The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures did not exceed 7% (being 1.4%).

NOEC and LOEC calculation (growth rate)
A NOEC (No observed effect concentration) of ≥ 100.0 mg/L test item and a LOEC (Lowest observed effect concentration) of > 100.0 mg/L test item was determined for 24 hours for growth rate based on nominal concentrations.
A NOEC (No observed effect concentration) of 71.4 mg/L test item and a LOEC (Lowest observed effect concentration) of 100.0 mg/L test item was determined for 48 and 72 hours for growth rate based on nominal concentrations.

The pH-value of the control medium did not increase by more than 1.5 units during the test.
Temperature:
During the test period the following water temperature was measured in the vessel containing test medium: 22.8 – 22.9°C.
Additional observations:
No differences such as sedimentation of test solution, cell aggregation or colour differences of algae cells were noticed between the control vessels and the test vessels containing the test item during the test.

Results with reference substance (positive control):

To verify the sensitivity of the test system the reference item potassium dichromate is routinely tested at concentrations of 0.125, 0.250, 0.500, 1.00 and 2.00 mg/L in a separate study.

The reference toxicity test was valid because the cell density increased by a factor of more than 16 within the test period of 72 hours (actually 81.7). This increase corresponds to a growth rate of 1.467 d-1. The variation coefficient of the growth rate was < 7 % (actually 0.7 %). The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures did not exceed 35% (actually 12.8 %).

Please refer to attachment

Validity criteria fulfilled:

yes

Conclusions:

A growth inhibition test was performed to assess the effects of the test item CS-2021-939 to green algae (Desmodesmus subspicatus) during 72 hours of exposure.
Significant effects on growth rate were found using Williams t-test (alpha = 0.05) at the nominal test concentration of 100.0 mg/L test item. As a result, a LOEC of 100.0 mg/L test item was determined. The corresponding NOEC was 71.4 mg/L test item.
Effect concentrations of ErC10, ErC20 and ErC50 were higher than 100.0 mg/L test item for growth rate based on nominal concentrations and higher than 100.6 mg/L test item based on mean measured concentrations.

Executive summary:

In the present study the toxicity of the test substance to Desmodesmus subspicatus was determined in a static 72-Hour Algal Growth Inhibition Test. The test was performed according to OECD 201 and EU Method C.3.

The nominal concentrations of 26.0, 36.4, 51.0, 71.4 and 100 mg test item/L (corresponding to 23.9, 34.13, 47.73, 66.87 and 100.2 mg/L measured) were tested in parallel with a control. Dose verification analysis was performed. The mean measured concentrations of the test item in the test media were between 92 to 101% of the nominal values. Thus, the correct dosing of the test item was confirmed. During the test period of 72 hours, no decrease of test item concentration in the test media occurred.

Measured concentrations of the test item in test solutions were within a range of 93 to 97% of nominal values at test start and after 24, 48 and 72 hours the concentrations ranged from 84 to 108% in spent test solutions. Therefore, the calculated endpoints are based on the nominal and mean measured concentrations for test item. The cell density was determined at 0, 24, 48 and 72 hours. The growth of the control cultures fulfilled the validity criteria from OECD 201 (2006). The 72 -hour ErC50 is > 100.6 mg/L.

Description of key information

72 h ErC50: > 100.6 mg/L (mean measured)

Key value for chemical safety assessment

EC50 for freshwater algae:

100.6 mg/L

EC10 or NOEC for freshwater algae:

100.6 mg/L

Additional information

In the Klimisch 1 GLP study the toxicity of the test substance to Desmodesmus subspicatus was determined in a static 72-Hour Algal Growth Inhibition Test. The test was performed according to OECD 201 and EU Method C.3.

The nominal concentrations of 26.0, 36.4, 51.0, 71.4 and 100 mg test item/L (corresponding to 23.9, 34.13, 47.73, 66.87 and 100.2 mg/L measured) were tested in parallel with a control. Dose verification analysis was performed. The mean measured concentrations of the test item in the test media were between 92 to 101% of the nominal values. Thus, the correct dosing of the test item was confirmed. During the test period of 72 hours, no decrease of test item concentration in the test media occurred.

Measured concentrations of the test item in test solutions were within a range of 93 to 97% of nominal values at test start and after 24, 48 and 72 hours the concentrations ranged from 84 to 108% in spent test solutions. Therefore, the calculated endpoints are based on the nominal and mean measured concentrations for test item. The cell density was determined at 0, 24, 48 and 72 hours. The growth of the control cultures fulfilled the validity criteria from OECD 201 (2006). The 72 -hour ErC50 is > 100.6 mg/L.