Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid

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:

January 22 - 25, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Remarks:

No confirmatory analysis of the test solutions was performed due to lack of an analytical method.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A primary stock concentrate of Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid with a nominal concentration of 10 mg/L was prepared by weighing a nominal 0.010 g of test substance (actual measured: 0.01005 g) into a 1000 mL volumetric flask and made up to volume with AAP culture medium. The stock was then sonicated for 60 minutes. This stock was used to prepare the test solutions by the direct addition of the appropriate amount of concentrate to dilution water in a volumetric flask.
- Controls: Culture medium control.
- Evidence of undissolved material: The resultant stock was observed to be a slightly cloudy, colourless homogenous dispersion with very fine particles visible.

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 cultures maintained under axenic conditions.
- Age of inoculum: A 3-day old culture of the alga in the exponential growth phase was used as inoculum for the test.
- Method of cultivation: The culture was grown in the medium (AAP-medium), and under the environmental conditions, described for the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

The daily temperature measurements remained within 22 ± 2 °C.

pH:

At the start of the test the pH ranged from 7.22 to 7.30 and at the end of the test the range was from 7.80 to 7.92.

Nominal and measured concentrations:

Nominal exposure concentrations of 0.095, 0.31 0.98, 3.1, and 10 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass conical flasks.
- Type: Closed with foam bungs.
- Material, size, headspace, fill volume: 250 mL.
- Type of flow-through: Static.
- Initial cells density: Each replicate test vessel was inoculated with 0.474 mL of the inoculum culture to give a nominal cell density of 0.5 × 10^4 cells/mL.
- Control end cells density: Culture medium control had a 72-hour cell density of 35.2 x 10^4 cells/mL.
- No. of vessels per concentration (replicates): Triplicates of each concentration of the test substance were employed.
- No. of vessels per control (replicates): Six replicates of the culture medium control.

GROWTH MEDIUM
- Standard medium used: AAP-medium.

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: The culture was grown in the medium, and under the environmental conditions, described for the test.

OTHER TEST CONDITIONS
- Light intensity and quality: The mean light intensity calculated from a range of representative positions, measured once during the study, was 6438 lux (by cosine receptor), within a range of ± 15 %.

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: The algal cell densities of the inoculum and test cultures were determined by electronic particle counting, using a Coulter counter and counting between a lower and upper threshold equivalent spherical diameter of approximately 2.3 and 5.0 µm respectively.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: A spacing factor between test concentrations of 3.2 was selected based on the toxicity and solubility observed in a non-GLP range-finding test.
- Range finding study
- Test concentrations: A non-GLP range-finding test was conducted prior to study initiation. The range-finding test was run with a culture medium control, 0.01, 0.1, 1.0, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

no

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.98 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.98 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.:

> 0.98 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.68 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.63 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.44 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

For both growth rate and the yield, the nominal 3.1 and 10 mg/L test concentrations were excluded from the calculation of the ECx values due to high background blank counts. The microscopic observations, made at the end of the test, showed that compared to the control the algal cells sampled from the nominal 0.095, 0.31 and 0.98 mg/L test concentrations appeared normal. The cells from the nominal 3.1 mg/L test solution appeared to be stuck together in clumps of approximately hundreds. No cells were visible in the nominal 10 mg/L test solution.

Validity criteria fulfilled:

yes

Conclusions:

Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid was exposed to Pseudokirchneriella subcapitata in a 72-hour test for aquatic toxicity. Based on growth rate, a NOEC and LOEC of 0.31 and 0.98 mg/L, respectively, was determined. EC10 was 0.44 and EC50 was >0.98 after 72 hours. Based on yield, NOEC = 0.31 mg/L; LOEC = 0.98 mg/L; EC10 = 0.31 mg/L; EC50 = 0.68 mg/L. According to CLP Regulation (EC) No 1272/2008, Annex I, Table 4.1.0 an ErC50 of >0.98, which is less than the 1 mg/L threshold, suggests that a classification as acute aquatic hazard may be required.

Executive summary:

A green alga, Pseudokirchneriella subcapitata, was exposed to Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid for a 72-hour period in order to determine its potential to induce acute aquatic toxicity. The experiment was performed without deviation according to Good Laboratory Practise (GLP) and OECD Guideline 201 (Alga, Growth Inhibition Test). A 10 mg/L test concentration was selected as the highest concentration and a spacing factor between test concentrations of 3.2 was selected based on toxicity and solubility observed in an initial non-GLP range-finding test. Test concentrations consisted of a media control and nominal concentrations of 0.095, 0.31, 0.98, 3.1 and 10 mg/L. The culture medium used for the experiment was AAP-medium. Cell counts were made at 24, 48, and 72 hours. The cell density count was used to calculate growth rate and yield.

The validity criteria of the test were satisfied. For both growth rate and the yield, the nominal 3.1 and 10 mg/L test concentrations were excluded from the calculation of the ECx values due to high background blank counts. Based on growth rate, the following results were obtained; NOEC = 0.31 mg/L; LOEC = 0.98 mg/L; EC50 = >0.98 mg/L; EC20 = 0.63 mg/L; EC10 = 0.44 mg/L. Based on yield, the following results were obtained; NOEC = 0.31 mg/L; LOEC = 0.98 mg/L; EC50 = 0.68 mg/L; EC20 = 0.40 mg/L; EC10 = 0.31 mg/L. As the derived ErC50 value >0.98 mg/L is slightly below the concentration threshold considered to be hazardous to the aquatic environment, Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid may require classification as Acute Category 1 (CLP Regulation (EC) No 1272/2008).

Description of key information

A 72-hour EC10 and EC50 of 0.44 and >0.98 mg/L (based on growth rate), respectively, was obtained in an experiment for aquatic toxicity in algae for Reaction products of stearic acid, diethylenetriamine and N-aminoethylethanolamine and acetic acid. NOEC and LOEC were 0.31 and 0.98 mg/L, respectively. Based on algal yield, NOEC = 0.31 mg/L; LOEC = 0.98 mg/L; EC10 = 0.31 mg/L; EC50 = 0.68 mg/L. According to the obtained ErC50 of >0.98, the registered substance may possess properties hazardous to aquatic algae and cyanobacteria, which would require classification as Acute Category 1 (CLP Regulation (EC) No 1272/2008).