Reaction mass of C18 (unsatd.) fatty acid amides/esters of diethanolamine, C16-18 (even-numbered) fatty amine ethoxylates, castor oil ethoxylates and sulfosuccinates of C18 (unsatd.) fatty acid diethanolamide, sodium 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:

From October 12, 2015 to November 11, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

Analytical Determinations:
Analytical samples were taken from each test substance concentration and control at 0 h (initial value) and after 72 h from aged test solutions. All analytical samples taken were analysed by TOC analysis. These samples were taken from a separate vessel without algae.

Vehicle:

no

Details on test solutions:

A stock solution (Stock A) was prepared by directly weighing 200 mg in 1,000 mL deionised water. This stock solution was homogenized by shaking. Afterwards the solution was clear and transparent. The Stock A solution was diluted with deionised water 1 to 3.2 to lead to a final volume of 1,000 mL (= Stock B). The stock solutions Stock C - E were made by diluting the appropriate stock solutions.
The medium used for the test was AAP-Medium, as per Annex 3 of OECD Guideline 201.

Test organisms (species):

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

Details on test organisms:

- Test species: Pseudokirchneriella subcapitata Hindák, strain SAG 61.81.
- Source: MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany.
The algae were grown semi-continuously in sterile cultures under permanent illumination in the laboratory. Old medium was periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Stock cultures were ordered regularly from the culture collection.
Culture conditions were as follows:
- Illumination: from the top by light tubes, 60 – 120 μEm-2s-1 at cell culture level
- Temperature: 21 – 24°C
- Culture flasks: 100 mL Erlenmeyer flasks
- Visually healthy cells were used for the test. CO2 supply by shaking on a rotating shaker, approximately 105 rpm. Cells from this semi-continuous liquid stock culture were used for the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

Standard OECD medium

Test temperature:

21.9 – 22.7°C

pH:

7.68 – 8.40

Dissolved oxygen:

-

Salinity:

Standard OECD medium

Nominal and measured concentrations:

Nominal: 0, 0.954, 3.05, 9.77, 31.3 and 100 mg/L.

Details on test conditions:

- Test procedure: Static
- Duration: 3 d (72 h)
- Initial cell density: 0.5 × 10000 cells/mL
- Temperature: 21.9 – 22.7°C
- pH of control: 7.68 – 8.40
- Illumination: Continuously from the side, 70.9 – 82.1 μEm-2s-1
- Culture flasks: 100 mL Erlenmeyer flasks with aluminium caps
- CO2 supplied: By continuously shaking

Reference substance (positive control):

yes

Remarks:

Potassium dichromate, 99.95% w/w purity, batch no. 112403J

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

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth and growth rate

Results with reference substance (positive control):

Potassium dichromate is tested as the toxic reference substance in a separate study twice a year to confirm the sensitivity of the test organism against compounds with known effects under the test conditions. From results, an ErC50 and EyC50 (0 - 72 h) of potassium dichromate of 1.77 and 0.749 mg/L, respectively, was reported. The overall LOEC and NOEC were determined to be at 0.320 mg/mL and 0.128 mg/mL, respectively. All the validity criteria were fulfilled. The ECx values calculated in this reference test were considered to be within an acceptable range therefore it can be considered that the algae cells are sensitive.

Reported statistics and error estimates:

The measured cell numbers (Nn) in the test cultures and controls were tabulated together with the concentrations of the test substance and the times of measurement (tn). The mean value of the cell numbers for each test substance concentration and the control was plotted versus time to produce growth curves.

Analytical Results

The test substance is a complex mixture of various organic ingredient groups having substantially different chemical structures and the toxicity cannot be assigned to a special ingredient. Consequently, no appropriate analytical method for an accurate analysis was available. However, the content (approximately) of test substance in aqueous media as the content of total organic carbon (TOC) was measured. In addition, the TOC content of the test substance as a bulk material was determined. The content of TOC in the test substance was analysed to be 55.7%. Since no effect was observed up to and including 100 mg/L only the analytical results of the highest test substance concentration was reported. TOC analysis showed that the test substance was stable throughout the period of the test. The initial measured concentration of TOC was 156% of the nominal concentration. The measured concentration of TOC in the aged samples was 155%. The toxicological endpoints were evaluated using the nominal concentrations.

All validity criteria were fulfilled: ·

- Cell numbers, measured in the controls between 0 and 72 h, were found to increase by a factor of 58.0 for the control, which exceeds the threshold of 16. It corresponds to a growth rate of 1.34997 d^-1, respectively.

- The coefficient of variation of average growth in replicate control cultures was 5.3% for the control and did not exceed 7% for the whole test period.

- The mean coefficient of variation for the section-by-section specific growth rates (h 24 - 0, 48 - 24 and 72 - 48) in the control cultures was 32% for the control and did not exceed 35%.

Validity criteria fulfilled:

yes

Conclusions:

The ErC50 and EyC50 (0 -72 h) of test substance for Pseudokirchneriella subcapitata were >100 mg/L (nominal). The NOEC for effect on algal growth and growth rate was determined to be 100 mg/L (nominal).

Executive summary:

A study was conducted to evaluate the toxicity of the test substance to single cell green algae Pseudokirchneriella subcapitata according to OECD Guideline 201, in compliance with GLP. A static main test, wherein the organisms were exposed to the substance at 0, 0.954, 3.05, 9.77, 31.3 and 100 mg/L was performed. After 24, 48 and 72 h, cell growth was measured by fluorescence detection. The mean value of the cell concentration was plotted versus time to produce growth curves for each concentration. EC values (EC10, EC20, EC50, NOEC, LOEC) were calculated where possible for growth rate (ErC) and yield (EyC).Analytical samples were taken from each test item concentration and control at0 h (initial value) and after 72 h from aged test solutions. All analytical samples taken were analysed by TOC analysis. These samples were taken from a separate vessel without algae. All the validity criteria were met in this test. Stimulating effects towards the algae were observed at concentrations up to and including 100 mg/L after 72 h for growth rate and for yield. Under the study conditions, the ErC50, ErC20, ErC10, EyC50, EyC20, EyC10 were > 100 mg/L (nominal). The overall NOEC was estimated to be 100 mg/L (nominal) (Falk S, 2015).

Key value for chemical safety assessment

EC50 for freshwater algae:

100 mg/L

EC10 or NOEC for freshwater algae:

100 mg/L

Additional information

A study was conducted to evaluate the toxicity of the test substance to single cell green algae Pseudokirchneriella subcapitata according to OECD Guideline 201, in compliance with GLP. A static main test, wherein the organisms were exposed to the substance at 0, 0.954, 3.05, 9.77, 31.3 and 100 mg/L was performed. After 24, 48 and 72 h, cell growth was measured by fluorescence detection. The mean value of the cell concentration was plotted versus time to produce growth curves for each concentration. EC values (EC10, EC20, EC50, NOEC, LOEC) were calculated where possible for growth rate (ErC) and yield (EyC).Analytical samples were taken from each test item concentration and control at0 h (initial value) and after 72 h from aged test solutions. All analytical samples taken were analysed by TOC analysis. These samples were taken from a separate vessel without algae. All the validity criteria were met in this test. Stimulating effects towards the algae were observed at concentrations up to and including 100 mg/L after 72 h for growth rate and for yield. Under the study conditions, the ErC50, ErC20, ErC10, EyC50, EyC20, EyC10 were > 100 mg/L (nominal). The overall NOEC was estimated to be 100 mg/L (nominal) (Falk S, 2015).