Amides, C12-18 and C18-unsatd., N,N-bis(hydroxyethyl)

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 24, 1986 to October 27, 1986

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, short report, few details but follows scientific principles of OECD 201 guideline

Remarks:

No analytical dose verification

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
First a stock solution of 10 g of the test material/L was prepared. From this solution appropriate aliquots were pipetted into the test vessels and filled up to final volumes of 100 mL with mineral nutrient. At last the solutions were inoculated with algae and incubated at 23 ±2°C

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Source (laboratory, culture collection): Pflanzenphysiol. Institut der Universitat Gottingen

ACCLIMATION
- Acclimation period: 3-4 d
- Culturing media and conditions (same as test or not): Yes, without test material

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.5-24.0°C

pH:

7.6-9.6

Nominal and measured concentrations:

0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, 100, 300 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 300mL conical flasks
- Material, size, headspace, fill volume: 100 mL liquid volume
- Initial cell/biomass concentration: 10(4) cells/mL
- No. of vessels per concentration (replicates): Three

GROWTH MEDIUM
- Standard medium used: Yes

OTHER TEST CONDITIONS
- Light intensity and quality: Continuous uniform illumination with approximately 2000 lux (daylight-spectrum)
- Incubation temperature: 23 ±2°C

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]
- Chlorophyll measurement:
- Other:

TEST CONCENTRATIONS
- Spacing factor for test concentrations:
- Justification for using less concentrations than requested by guideline:
- Range finding study: No

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 3.9 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 2.1 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.3 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

- For details on biodegradation results, please refer to 'Table 1' in the ‘Any other information on results incl. tables’.

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

The NOEC (No Observed Effect Concentration or EbC0) could be determined using a t-Test statistical method (P = 95%).

Table 1: Algal cell Concentrations [1/mL]

Substance concentrations [mg product/L]	Algae cell concentrations (1/mL)
	Incubation time [h]
	24	48	72
0	4.40E+04	4.10E+05	2.30E+06
0.01	4.50E+05	4.60E+05	2.60E+06
0.03	4.70E+04	4.40E+05	2.60E+06
0.1	4.80E+04	3.60E+05	2.40E+06
0.3	6.10E+04	5.20E+05	2.20E+06
1	5.00E+04	3.80E+05	1.80E+06
3	3.10E+04	7.10E+04	5.30E+05
10	1.70E+04	2.10E+04	1.70E+04
30	1.30E+04	1.00E+04	1.30E+04
100	3.30E+03	1.00E+04	1.30E+04
300	1.00E+04	6.70E+03	1.30E+04
1000	1.00E+04	6.70E+03	3.30E+03

Validity criteria fulfilled:

not specified

Conclusions:

Under the study conditions, the nominal 72 h ErC50 and EbC50 were estimated to be 3.9 and 2.1 mg/L, respectively. The 72 h NOEC was 0.3 mg/L.

Executive summary:

A study was performed to determine the toxicity of the test substance, C12-18 and C18-unsatd. DEA, to Selenastrum subspicatus according to OECD Guideline 201, in compliance with GLP. The algae were exposed to the test substance (directly dissolved in test medium) at concentrations of 0.01 to 1000 mg /L for 72 h under static conditions. Growth inhibition was measured at 24, 48 and 72 h. Both algae biomass growth (EbC50) and algal growth rate (ErC50) were used to calculate the (inhibition) effect-concentrations (EC50) by the probit method. No analytical dose verification was conducted. Under the study conditions, the nominal 72 h ErC50 and EbC50 were estimated to be 3.9 and 2.1 mg/L, respectively. The 72 h NOEC was 0.3 mg/L (Gode, 1991).

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From May 08, 1995 to May 11, 1995

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:

- Concentrations: Single samples of 100 mg/L stock solution and duplicate samples of 0.32, 3.2 and 32 mg/L test solutions and control at the start of the test; duplicate samples of 0.32, 3.2 and 32 mg/L test solutions and the control at the end of the test (after 72 h test period) for stability testing
- Sample storage conditions before analysis: : Samples were treated with 8% (v/v) of a Formalin solution (37%), to preserve the samples until analysis

Vehicle:

yes

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test substance was homogenized by intense stirring. Then an adequate amount of the homogenized test substance was weighed into sterile test water to prepare for the preparation of a concentrated stock solution of 100 mg/L concentration. Adequate volumes of this intensively stirred stock solution were added to test water to prepare the test media of the following nominal test concentrations: 0.32, 1.0, 3.2, 10 and 32 mg/L.
- Controls: Deionised water
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No turbidity or precipitation was observed in the test media after addition of the test substance and during the test period up to and including the test concentration of nominal 10 mg/L. At the highest concentration of nominal 32 mg/L the test medium was slightly turbid due to the high test substance concentration.

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: 86.81 SAG
- Source (laboratory, culture collection): Sammlung von Algenkulturen, Pflanzenphysiologisches lnstitut der Universitat Gottingen, D-37073 Gottingen
- Method of cultivation: The algae were grown in the laboratories of RCC under standardized conditions according to the test guideline.

ACCLIMATION
- Acclimation period: 72 h before the test
- Culturing media and conditions (same as test or not): Yes

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

0.24 mmol/L (= 24 mg/L) as CaCO3

Test temperature:

23.4-23.8ºC

pH:

7.9-10.3

Nominal and measured concentrations:

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

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks
- Type (delete if not applicable): Closed with glass dishes
- Material, size, headspace, fill volume: 50 mL erlenmeyer flasks containing 50 mL algal suspension continuously stirred by magnetic stirrers
- Renewal rate of test solution (frequency/flow rate):
- Initial cells density: 10,000 algal cells/mL test solution
- Control end cells density: 69.17 X 104 cells/mL (initial 1x104 cells/mL)
- No. of vessels per concentration (replicates): Three
- No. of vessels per particle control (replicates): Two
- No. of vessels per vehicle control (replicates): Six

GROWTH MEDIUM
- Standard medium used: yes/no
- Detailed composition: Refer to the attached document for details

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionised water (MilliQ-water) Refer to the attached document for details on the composition of the test water
- Conductivity: 0.1 µS cm-1

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: The pH-values of the test media were measured in samples from all test concentrations and the control at the start and at the end of the test
- Photoperiod: Continuously illuminated
- Light intensity and quality: approximately 7780 Lux (range: 7100 to 8300 Lux) using fluorescent tubes (universal white L 25, 36 W), installed above
the water bath in a distance of about 35 cm from the test flasks

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Algal cell densities were counted using electronic particle counter (AL CELLCOUNTER, Model 871, AL-Systeme, D76149 Karlsmhe) (three measurements/sample)
- Shape of algal cells: A sample was taken from the control and from a test concentration with reduced algal growth (i.e., nominal 3.2 mg/L) after the test period of 72 h and the shape of these treated algal cells was microscopically examined and compared with the cells in the control.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.1-3.2
- Range finding study
- Test concentrations: Not reported
- Results used to determine the conditions for the definitive study: Yes

DETERMINATION OF PARTICLE DENSITY OF TEST SUBSTANCE:
- Two replicates per test concentration were additionally prepared with test media without algae for corrections at the algal cell density determinations, since at least in the two highest test concentrations a significant test substance particle density had been determined.
- For the verification of the cell countings by the electronic particle counter, the algal cell densities in the two highest test concentrations were counted also microscopically in a counter chamber.

Reference substance (positive control):

no

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 7.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 1.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

ca. 0.32 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.:

ca. 2.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

ca. 0.35 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

- In the control, the cell density was increased from nominal N = 1x104 cells/mL at the start of the test (0 h) to N = 69.17 X 104 cells/mL (mean value) after 72 h i.e., by a factor of approximately 69 (refer to Table 2). Thus, the algal growth in the control was sufficiently high under the test conditions.
- The microscopic counting of the algal cell densities at the test concentration of nominal 10 mg/L with a significant test substance particle density showed an algal cell density of approximately 5x104 cells/mL after 72 h, and thus a value in the same magnitude as determined by the electronic palticle counter after the test substance particle correction.
- At the highest test concentration of nominal 32 mg/L, the algal cell density was too low for the microscopic counting despite an enrichment of the algae by a factor of 60.
- At the microscopic examination of the shape of the algal cells after 72 h test period no difference was observed between the algae growing in the test concentration of nominal 3.2 mg/L and the algal cells in the control. Thus, the shape of the algal cells growing at least up to this test concentration was obviously not affected.

Refer to Tables 2-6 of the appended word document under 'attached background material for further details on results

Reported statistics and error estimates:

Dunnett test

Analytical determinations:

- The analytically determined test substance concentration in the sample from the freshly prepared stock solution amounted to 112% of the nominal concentration.

- The analytically determined test substance concentrations in the samples from the analysed test media ranged from 80% to 103% of the nominal values.

- In the test media, incubated under the test conditions but without algae, the test substance was sufficiently stable during the test period of 72 h.

- The mean measured values of all measurements per test concentration ranged from 81% to 101% of the respective nominal value. Therefore, all biological results are based on the nominal concentrations of the test substance.

Validity criteria fulfilled:

not specified

Conclusions:

Under the study conditions, the nominal 72 h ErC50 and EbC50 were estimated to be 7.4 and 2.2 mg/L, respectively. The nominal 72 h NOEC was 0.32 mg/L.

Executive summary:

A study was performed to evaluate the toxicity of the test substance, C12-18 and C18-unsatd. DEA, to the green algae Selenastrum subspicatus according to OECD Guideline 201, in compliance with GLP. The algae were exposed to the test substance (dissolved in test medium by intense stirring) at concentrations ranging from 0.32 to 32 mg/L for 72 h under static conditions. The growth inhibition concentrations in terms of algae biomass growth (EbC50) and algal growth rate (ErC50) were determined at 24, 48 and 72 h. Calculations were performed by the Probit method. Samples were collected for analytical dose verification by HPLC-UV at test start and end. Measured values ranged from 80 to 103% of nominal values, so that the results were expressed based on nominal concentrations. Under the study conditions, the nominal 72 h ErC50 and EbC50 were estimated to be 7.4 and 2.2 mg/L, respectively. The nominal 72 h NOEC was 0.32 mg/L (Memmert, 1995).

Key value for chemical safety assessment

EC50 for freshwater algae:

3.9 mg/L

EC10 or NOEC for freshwater algae:

0.3 mg/L

Additional information

A study was performed to determine the toxicity of the test substance, C12-18 and C18-unsatd. DEA, to Selenastrum subspicatus according to OECD Guideline 201, in compliance with GLP. The algae were exposed to the test substance (directly dissolved in test medium) at concentrations of 0.01 to 1000 mg /L for 72 h under static conditions. Growth inhibition was measured at 24, 48 and 72 h. Both algae biomass growth (EbC50) and algal growth rate (ErC50) were used to calculate the (inhibition) effect-concentrations (EC50) by the probit method. No analytical dose verification was conducted. Under the study conditions, the nominal 72 h ErC50 and EbC50 were estimated to be 3.9 and 2.1 mg/L, respectively. The 72 h NOEC was 0.3 mg/L (Gode, 1991).

A study was performed to evaluate the toxicity of the test substance, C12-18 and C18-unsatd. DEA, to the green algae Selenastrum subspicatus according to OECD Guideline 201, in compliance with GLP. The algae were exposed to the test substance (dissolved in test medium by intense stirring) at concentrations ranging from 0.32 to 32 mg/L for 72 h under static conditions. The growth inhibition concentrations in terms of algae biomass growth (EbC50) and algal growth rate (ErC50) were determined at 24, 48 and 72 h. Calculations were performed by the Probit method. Samples were collected for analytical dose verification by HPLC-UV at test start and end. Measured values ranged from 80 to 103% of nominal values, so that the results were expressed based on nominal concentrations. Under the study conditions, the nominal 72 h ErC50 and EbC50 were estimated to be 7.4 and 2.2 mg/L, respectively. The nominal 72 h NOEC was 0.32 mg/L (Memmert, 1995).