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Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2003-09-23 to 2003-09-26
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
No analytical measurements.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
1984. Validity, reliability, and deviations evaluated against OECD 201 (2006).
Deviations:
yes
Remarks:
(No measurements of test concentrations Nominal only)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Analytical monitoring:
no
Details on sampling:
NOTE: samples were taken, preserved, and sent to the sponsor for measurment. The analytical results and details are not available in this study report.
- Concentrations: At 0 h, the sampling volume was 15 ml for the highest and 20 ml for the other concentrations. At 72 h, the sampling volume was 20 ml for all the test concentrations. Samples with algae at 72 h were fillered with a 0.45 um syring filter.
- Sampling method: Per 20 ml sample, 660 ul of a 35% active formaldehyde was added to preserve the samples. For the 15 ml samples, 495 ul of a 35% active formaldehyde was added.
- Sample storage conditions before analysis: All samples were kept in the freezer until they were sent to the sponsor (under frozen condition).
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 1.005 g/L test item was prepared by dissolving 0.1005 g GTS02902 in a certified volumetric flask. After solubilization in an appropriate volume of demineralized water, the solution was made up to the mark by carefully adding demineralized water. 8 concentrations were prepared in a geometric series at a concentration ratio of 2.2, by appropriate dilution of this stock solution with algal medium.
- Eluate: Not applicable
- Differential loading: Not applicable
- Controls: Algal medium without test item was used as a control.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None
Test organisms (species):
Chlorella vulgaris
Details on test organisms:
TEST ORGANISM
- Common name: Chlorella vulgaris
- Strain: CCAP 211/11D
- Source (laboratory, culture collection): LISEC-lab
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: The culture was incubated for 4 days before use.


ACCLIMATION
- Acclimation period: 4 days
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: Not available
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not applicable
Hardness:
Not available, Therefore calculated 45 mg/L as CaCO3
Test temperature:
Temperature measured at 0, 24, 48 and 72 h. Temperature ranged as follows:
Control, 0.05, 0.23, 0.51, 2.49 and 12.06 mg/L: range 22.9-23.2 degree C
0.11 mg/L: range 22.8-23.1 degree C
1.14 mg/L: range 22.9-23.1 degree C
5.48 mg/L: range 22.8-23.2 dgree C
pH:
pH measured at 0 and 72 h. pH ranged as follows:
Control: range 7.74-8.32
0.05 mg/L: range 7.70-8.38
0.11 mg/L: range 7.70-8.37
0.23 mg/L: range 7.68-8.46
0.51 mg/L: range 7.68-8.49
1.14 mg/L: range 7.70-8.12
2.49 mg/L: range 7.71-831
5.48 mg/L: rannge 7.69-7.82
12.06 mg/L: 7.68
Dissolved oxygen:
Not available
Salinity:
Not aaplicable
Nominal and measured concentrations:
Nominal exposure concentrations based on 31.20% active were 0, 0.05, 0.11, 0.23, 0.51, 1.14, 2.49, 5.48 and 12.06 mg/L. No measurements of test concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open / closed: Closed
- Material, size, headspace, fill volume: 250 ml glass flask containing 100 ml of test solution
- Aeration: Not available
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- Initial cells density: 5X10(+3) - 1X10(+4) cells/ml
- Control end cells density: 2.09X10(+5) - 4.18x10(+5) cells/ml. This represents a 42x increase in cell density.
- No. of organisms per vessel: 5X10(+3)-1X10(+4) cells/ml
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): Not applicable


GROWTH MEDIUM
- Standard medium used: yes/no: Yes, Medium prepared according to OECD 201 guideline
- Detailed composition if non-standard medium was used: Not applicable


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The algal medium was prepared by appropriate dilution of the chemicals in demineralized water, The test solution were prepared with 10 times concentrated algal medium.
- Total organic carbon: Not available
- Particulate matter: Not available
- Metals: MgCl2.6H2O: 12 mg/L, CaCl2.2H2O: 18 mg/L, MgSO4.7H2O: 15 mg/L, FeCl3. 6H2O: 0.08 mg/L, H3BO3: 0.185 mg/L, MnCl2.4H2O:0.415 mg/L, CuCl2.2H2O: 0.00001 mg/L, ZnCl2: 0.003 mg/L, CoCl2.6H2O: 0.0015 mg/L, Na2MoO4.2H20: 0.007 mg/L
- Pesticides: Not available
- Chlorine: Not available
- Alkalinity: Not available
- Ca/mg ratio: 18 mg/L CaCl2.2H2O/ 12 mg/LMgCl2.6H2O
- Conductivity: Not available
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature was recorded daily in one replicate of the each test concentration. The pH was measured in one replica of each test concentration at the start and end of the test.


OTHER TEST CONDITIONS
- Sterile test conditions: yes/no: Yes
- Adjustment of pH: Not applicable
- Photoperiod: Continuous uniform illumination.
- Light intensity and quality: 6000 lux intensity was provided by Philips TL-33 lamps at the surface of the test solution.
- Salinity (for marine algae): Not applicable


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: Spectrophotometer (UNICAM UV2, 720 nm, 5cm light path cuvette.
- Chlorophyll measurement: Not applicable
- Other: None


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.2
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study: No
- Test concentrations: Not applicable
- Results used to determine the conditions for the definitive study: Not applicable
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.14 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 95% CL for EbC50: 1.06-1.22 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
0.77 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 95% CL for EbC20: 0.71-0.85 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.63 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 95% CL for EbC10: 0.56-0.71 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.016 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: (-0.053)-0.019 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.7 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL for ErC50: 1.69-1.72 mg/L
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
1.55 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL for ErC20: 1.51-1.59 mg/L
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
1.47 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL for ErC10: 1.47-1.47 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.16 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: Not available
Details on results:
- Exponential growth in the control (for algal test): yes/no: Yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: None
- Colour differences: None
- Flocculation: None
- Adherence to test vessels: None
- Aggregation of algal cells: None
- Other: None
- Any stimulation of growth found in any treatment: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: None
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
The concentration- effect curve fittings and EC50 were based on the model of Bruce and Versteeg (1992). The model was fitted to date using SAS procedure NLIN. The model was also used to calculate the EC20 and EC10- values. The NOEC for growth was calculated via the Dunnett's T-test. The NOEC for growth rate calculated via the Welch T-test.
Validity criteria fulfilled:
yes
Conclusions:
The nominal 72 h EC50 for C12/14 alkyl dimethyl amine oxide to Chlorella vulgaris was 1.14 mg a.i./L (based on biomass), and 1.70 mg a.i./L (based on growth rate).
Executive summary:

In a 72 -hour algae growth inhibition study, Chlorella vulgaris was exposed to alkyl dimethyl amine oxide at nominal concentrations of 0, 0.05, 0.11, 0.23, 0.51, 1.14, 2.49, 5.58 and 12.06 mg a.i./L in accordance with the OECD 201 guideline. The NOEC and EC50 values based on cell density were 0.16 mg/L and 1.14 mg/L, respectively. The % growth inhibition in the treated algal cultures as compared to the control ranged from 1 to 99%.

There were no compound related phytochemical effects.

This toxicity study is classified as acceptable and satisfies the guideline requirements for the OECD 201 freshwater algae and cyanobacteria toxicity study.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
supporting study
Study period:
13-11-2000 to 16-11-2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
No analytical measurement of test material concentration
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
DOC analysis was carried out only at the beginning of the test in accordance with the sponsor's wish.
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
not specified
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
The stock solution (100 mg/L) and control were analytically verified via analysis of disolved organic carbon (DOC, according to DIN guideline 38409 part 3) after 0 h.
Vehicle:
not specified
Test organisms (species):
Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)
Details on test organisms:
TEST ORGANISM
- Common name:
- Strain: CHODAT SAG 86.81
- Source: Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universitat Gottinghen.
- Age of inoculum (at test initiation): Three-day old subculture.
- Method of cultivation: Fresh stocks were prepared every month on Z agar. Light intensity amounted to 35 - 70 μE/m2/s for 24 h per day.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.1 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.25
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.02 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test substance to unicellular green alga was determined according to OECD guideline 201 and EC method C.3 under static conditions over a duration of 72 h. Microscopic evaluation revealed that the cells at nominal concentration levels of 0.5 to 1 mg/L were larger and agglutinated cells were observed. The EbC50 of the test substance was 0.34 mg/L with confidence limits of 0.29 - 0.38 mg/L. The ErC50 of the test substance was 0.82 mg/L with 95 % confidence limits of 0.66 - 1.00 mg/L, with a NOEC of 0.0624 mg/L. The EbC50 of the active ingredient was therefore 0.1 mg/L. The ErC50 of the active ingredient was 0.25 mg/L with a NOEC of 0.02 mg/L.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
no analytical validification of the test concentrations.
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock solution of test material prepared in deionised water (1 g/L)
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Method of cultivation: 2.5E04 exponentially growing algal cells/mL as test innoculum. Algal preculture set up two to four days before the start of the test.

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: not specified
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Test temperature:
20 - 22°C
Details on test conditions:
TEST SYSTEM
- Test vessel: 100 mL Erlenmeyers
- Material, size, headspace, fill volume: 40 mL
- Initial cells density: 2.5E04 cells/mL
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes with two deviations; 100 mg NaHCO3 instead of 50 mg/L and 161.6 KH2CO3 mg/L instead of 1.6 mg/L
- Detailed composition if non-standard medium was used:

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: deionised water
- Total organic carbon: < 2 mg/L
- Metals: < 0.01 mg Cu/L
- Conductivity: < 5µS/cm
- Culture medium different from test medium: No


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No
- Photoperiod: continuous illumination
- Light intensity and quality: 400 - 700 nm (fluorescent lamps)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: spectrophotometer (4 cm cell, 436 nm) at 0, 24, 48 and 72 hours.
- Chlorophyll measurement: No

TEST CONCENTRATIONS
- Range finding study: 0.001, 0.01, 0.1, 1 and 10 mg/L
- Test concentrations: 0.1, 0.2, 0.4, 0.8 and 1.6 mg/L
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.81 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL = 0.69 - 0.97 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.19 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.4 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: 95% CL = 0.35 - 0.46 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.095 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Details on results:
No NOEC value derived.

Table 1: % inhibition – definitive test

Concentration (mg/L)

% inhibition growth

% inhibition growth rate

0.1

5.5

3.3

0.2

23.7

9.5

0.4

58.1

29.6

0.8

78.7

55.8

1.6

84.6

67.3

Validity criteria fulfilled:
not specified
Conclusions:
The EbC50 (72h) amounted to 0.40 mg/L with 95% confidence limits of 0.35 - 0.46 mg/L and the ErC50 (72h) amounted to 0.81 mg/L with 95% confidence limits of 0.69 - 0.97 mg/L.
If it is assumed that the technical product consists of 100% active ingredient and that the observed toxic effect is related to the active ingredient only, the EbC50 (72h) amounts to 0.095 mg/L and the ErC50(72h) to 0.19 mg/L.
Executive summary:

In a 72-hour algal growth inhibition study performed according to OECD 201, Pseudokirchnerella subcapitata were exposed to C14 AO at nominal test concentrations of 0, 0.024, 0.047, 0.095, 0.190 or 0.379 mg AO/L. The 72-h EbC50 was 0.095 mg AO/L. The 72-h ErC50 was 0.19 mg AO/L. No NOEC value was derived.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
no analysis of test concentrations during the study, some other deficiencies in reporting
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
no
Analytical monitoring:
no
Details on sampling:
not applicable
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
A stock solution containing 1 g of test substance/L of deionised water was prepared. The test solutions were prepared by dilution of the respective amounts of stock solution with test medium.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: green algae
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
not applicable
Hardness:
no data
Test temperature:
20-22 deg C
pH:
No pH measurements were performed during the test
Dissolved oxygen:
no data
Salinity:
not measured
Nominal and measured concentrations:
Nominal concentrations:
Range-finding test: 0.001, 0.01, 0.1, 1 or 10 mg/L (equivalent to 0.000237, 0.00237, 0.0237, 0.237 or 2.37 mg AO/L)
Definitive test: 0.06, 0.12, 0.24, 0.48 or 0.96 mg/L (equivalent to 0.014, 0.028, 0.057, 0.114 or 0.228 mg AO/L)
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Material, size, headspace, fill volume: glass, 100 mL capacity, filled to 40 mL
- Aeration: no data
- Initial cells density: 2.5E04 cells/mL
- No. of vessels per concentration (replicates):range-finding - 2; definitive test 3
- No. of vessels per control (replicates): range-finding and definitive test - 6

GROWTH MEDIUM
- Standard medium used: no
- Detailed composition if non-standard medium was used: Deviation from standard: 100 mg/L NaHCO3 used instead of 50 mg/L and 161.6 mg/L KH2CO3 used instead of 1.6 mg/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Deionized water
- Total organic carbon: < 2 mg/L
- Metals: < 0.01 mg Cu/L
- Conductivity: < 5 uS/cm
- Culture medium different from test medium: No


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: fluorescent lamps in the spectral range 400-700 nm


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Extinction in each test vessel was measured after 0, 24, 48 & 72 hours
- Determination of cell concentrations: UV spectrophotometer at 436 nm


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study
- Test concentrations: 0.001, 0.01, 0.1, 1 or 10 mg/L (equivalent to 0.000237, 0.00237, 0.0237, 0.237 or 2.37 mg AO/L)
- Results used to determine the conditions for the definitive study: see Table 1 below
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.28 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.07 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.86 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.2 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
The EC50 values were computed from the best fitted line (least squares method) through the points given by the probit of the percentage inhibition and the logarithm of the concentration fo the test substance. The EC50 value calculated with teh area under the growth curve is termed the EbC50 (0-72h) whereas the EC50 value calculated with the specific growth rate is termed ErC50. It is defeined as the lowest concentration tested at which growth and growth confidence limits were computed on the basis of Fieller's theorem. All computations were performed using the Akzo programme 'Algal' version 3.0 made in SAS.

Table 1: Range-finding test

Concentration (mg/L)

% inhibition (growth)

% inhibition (growth rate)

0.001

-6.3

-3.2

0.01

-20.8

-6.0

0.1

-2.6

-1.2

1

82.6

61.1

10

89.4

74.7

 

Table 2: Definitive test

Concentration (mg/L)

% inhibition (growth)

% inhibition (growth rate)

0.06

14.3

4.9

0.12

29.6

11.1

0.24

47.9

21.9

0.48

65.4

36.2

0.96

76.7

52.4

Executive summary:

In a 72-hour algal growth inhibition study performed according to OECD 201, Pseudokirchnerella subcapitatawere exposed to C12 AO at nominal test concentrations of 0, 0.014, 0.028, 0.057, 0.114 or 0.228 mg AO/L. The 72-h EbC50 was 0.07 mg AO/L. The 72-h ErC50 was 0.20 mg AO/L. No NOEC value was derived.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
No analytical monitoring of test material concentration.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
Concentrations of KH2PO4 and NACO3 in the test medium were increased to 160 and 100 mg/L respectively to maintain a constant pH
GLP compliance:
yes
Analytical monitoring:
no
Details on sampling:
No data
Vehicle:
no
Details on test solutions:
Deionized water had a conductivity of less than 5 uS/cm and contained less than 0.01 mg/L of copper.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchnerella subcapitata
- Strain: ATCC 22662

Algae maintenance and pre-culture

The strain was maintained on mineral salts agar slants in the light at room temperature. The mineral salts medium of the slants contained the following quantities per litre of dionized water: 1.25 g KNO3; 0.01 g CaCl2.2H2O; 0.3 g MgSO4.7H2O; 0.01 g Fe(III)EDTA; 0.29 g K2HPO4; 0.19 g KH2PO4; 0.014 g H3BO3; 0.009 g MnCl2.4H2O; 0.001 g ZnSO4.7H2O; 0.0001 g (NH4)2MoO4; 0.0004 g CuSO4.5H2O and 0.025 g Co(NO3)2.6H2O. The test was inoculated with algae taken from exponentially growing pre-culture.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No data
Hardness:
No data
Test temperature:
The temperature varied from 22.5 degrees Centigrade to 24 degrees Centigrade during the definitive test
pH:
The pH was determined using a microcomputer pH meter (Consort P207). The values obtained are reported in Table IV (attached) as 6.9 for all test concentrations and associated controls at the start of the test. The pH values varied between 7.6 and 7.9 after 72 hours.
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
Nominal concentrations for the range finding test: 1, 10, 100 and 1000 mg/L
Nominal concentrations for the definitive test: 0.063, 0.125, 0.25, 0.5 and 1.0 mg/L
Details on test conditions:
The test was performed in 100 mL erlenmeyers containing 40 mL of mineral salts medium.

The culturing apparatus was a cabinet in which the temperature was maintained between 22 and 24 degrees Centigrade and continuous uniform illumination was provided in the spectral range of 400 to 700 nm. The light intensity was in the range 6,000 to 10,000 lux, which was obtained by using seven 30 W fluorescent lamps (colour temperature of approximately 4,300 K) at a distance of 0.35 m from the algal culture. The culture flasks were rotated continuously at 100 rpm to prevent sedimentation of the algae.

Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.266 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95 % confidence limits 0.214 to 0.446 mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.205 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 95% confidence limits 0.140 to 0.267 mg/L
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.078 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Details on results:
In the range finding test, growth of S. capricornutum was inhibited at 1, 10, 100 and 1000 mg/L of test substance (see Table I, attached).

The results of the extinction measurements of the definitive test are presented in Table II (attached). The growth curve and the specific growth rates calculated from the mean extinctions at various test concentrations are shown in Figures 1 and 2 (attached). The growth of S.capricornutum is stimulated at low concentrations and those concentrations were not considered when determining the concentration/effect relationship.

The EC50 (0-72 h) based on growth rate and EC50 (0-72 h) based on biomass were calculated from information shown in Figures 3 and 4 (attached).
Results with reference substance (positive control):
No data
Reported statistics and error estimates:
The method used to evaluate data is shown in section 2.11 of the study report and that portion of the report is attached.

The extinction of the controls increased by a factor of 37 within 72 hours, which met the validity criteria of at least a 16 fold increase.

The extinctions of potassium dichromate are shown in Table III (attached). From those extinctions an EC50 (72h) of 0.56 mg/L was calculated based on growth rate and 0.69 mg/L based on biomass (see Figures 5 and 6 (attached)). Those values were in agreement with expected results and show that the test organisms were not changed significantly and that the slight modification of the mineral salts medium did not influence the result.

The pH measurements in Table IV (attached) show a limited increase. However, the measurements did not deviate by more than 1.5 units. It was also noted that, although the test material is readily biodegradable, it was not likely that the test compound was biodegraded within three days.

Conclusions:
An EC50 (72 h) of 0.86 mg/L was calculated based on growth rate. The equivalent EC50 (72 h) based on biomass was calculated as 0.66 mg/L. The NOEC of the test substance was reported as 0.25 mg/L.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1995-09-05 to 1996-01-23
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
No analytical measurements of test concentrations.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
Reliability, deviations, and validity evaluated against OECD 201 (2006).
Deviations:
yes
Remarks:
(No measurement of test concentrations (Nominal only), 2 replicates of each test concentrations instead of 3)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Analytical monitoring:
no
Details on sampling:
- Concentrations: Not applicable
- Sampling method: Not available
- Sample storage conditions before analysis: Not available
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: For range finding test: an amount of 52 mg was dissolved in 250 ml of medium (pH 8), from which dilutions in medium were prepared so as to yield a concentration series of 0, 0.003, 0.3, 0.3, 3.2, 31.8 mg/L.
For growth inhibition test: an amount of 52.8 mg was dissolved in 250 ml of medium (pH 8), from which dilutions in medium were prepared, so as to yield a concentration series of 0, 0.0032, 0.01, 0.018, 0.032, 0.058, 0.1, 0.18 and 0.32 mg/. 100 ml of the stock solution 52.1 mg/250ml was kept in a freezer (max -20 degree C)
- Eluate: Not applicable
- Differential loading: Not applicable
- Controls: Four controls containing algae only, and a single background series containing test substance without algae.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green alga
- Strain: ATCC 22662
- Source (laboratory, culture collection): The culture was supplied by the 'American Type Culture Collection', c/o Sales Department, 12301 Parklawn Drive, Rockville, Maryland 20852, USA.
- Age of inoculum (at test initiation): Not available
- Method of cultivation: A pre-culture of algae in the exponential growth phase was prepared as datailed in OECD guideline no. 201.


ACCLIMATION
- Acclimation period: 3-4 days.
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: Cells were normal.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not applicable
Hardness:
Not available, Therefore calculated as 10.78 mg/L as CaCO3
Test temperature:
23+/-1 degree C.
pH:
pH was ranged from 7.9 at the begenning of the test to 8.9 at the end of the test .
Dissolved oxygen:
Not available
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal exposure concentrations based on 30.61% active were 0, 0.0032, 0.01, 0.018, 0.032, 0.058, 0.1, 0.18 and 0.32 mg/L. No measurement of test concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open / closed: Not available
- Material, size, headspace, fill volume: 200 ml conical flask containing 100 ml of the test solution.
- Aeration: Not available
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Not applicable
- Initial cells density: 0.9X10(+4) cell/ml
- Control end cells density: 117.1X10(+4) cell/ml
- No. of organisms per vessel: 0.9X10(+4) cell/ml
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): Not applicable


GROWTH MEDIUM
- Standard medium used: yes/no: Yes. According to OECD guideline 201 (1984) with 2 exceptions. One, 150 mg/L NaHCO3 (not 50 mg/L) to improve buffering. Two, Fe-citrate was included (in absence of complexed iron, the algae growth would be erratic).
- Detailed composition if non-standard medium was used: Not applicable


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The dilution water was algae growth medium. The medium was prepared from concentrated stock solutions in Milli-Q-filtered water. It was sterillized by micropore filteration
- Total organic carbon: Not available
- Particulate matter: Not available
- Metals: MgSO4. 7H2O: 15mg/L, Fe-citrate.3H2O: 80ug/L, Na2EDTA.2H2O: 100ug/L, H3BO3: 185ug/L, MnCl2.4H2O: 415ug/L, ZnSO4.7H2O: 6.3 ug/L, CoCl2.6H2O: 1.5ug/L, CuSO4.5H2O: 0.015 ug/L, Na2MoO4.2H2O: 7ug/L
- Pesticides: Not available
- Chlorine: Not available
- Alkalinity: Not available
- Ca/mg ratio: 18 mg/L CaCl2.2H2O/12 mg/L MgCl2.6H2O
- Conductivity: Not available
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH was measured at the start and after 71.5 h in in selected cultures.


OTHER TEST CONDITIONS
- Sterile test conditions: yes/no: Yes
- Adjustment of pH: Not applicable
- Photoperiod: Continuous illumination
- Light intensity and quality: 60-120 uE.m(-2).s(-1)
- Salinity (for marine algae): Not applicable


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter]: Electronic particle counter (Coulter model TAII).
- Chlorophyll measurement: Not applicable
- Other: None


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8 except for factor between first (0.0032 mg/L) and second (0.010 mg/L) concentration was 3.13
- Justification for using less concentrations than requested by guideline: Not applicable
- Range finding study
- Test concentrations: 0, 0.003, 0.3, 0.3, 3.2, 31.8 mg/L
- Results used to determine the conditions for the definitive study: The range finding test revealed thet inhibiting effects could be expected at test substance concentrations higher then 0.003 mg a.i./L.
Reference substance (positive control):
yes
Remarks:
K2Cr2O7 and/or 3,5-dichlorophenol
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.082 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.063 - 0.106
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.015 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 0.0106 - 0.0224
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.003 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
not specified
Details on results:
- Exponential growth in the control (for algal test): yes/no: Yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: Morphologically abnormal cells were found at test substance concentrations of 0.010 mg a.i./L and higher.
- Colour differences: Not available
- Flocculation: Not availabe
- Adherence to test vessels: Not available
- Aggregation of algal cells: Not available
- Other: None
- Any stimulation of growth found in any treatment: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: None
Results with reference substance (positive control):
Not available
Reported statistics and error estimates:
The EC values with respect to the growth rate and exponential growth (ErC values), were calculated by means of a parametric model developed by Kooijman et.al.
The EC values with respect to the area under curve (EbC values), were calculated by the method given in the OECD guideline Alga, Growth Inhibition Test, Paris (1984). The values were calculated by linear interpolation of a plot of the percentage reductionin growth (IA) against the log concentration of the test substance.
Validity criteria fulfilled:
yes
Remarks:
All three validity criteria of OECD 201 (2006) were fulfilled.
Conclusions:
The 72 h toxicity of C12/C14 Amine Oxide (CAS#70592-80-2) to Selenastrum capricornutum was 0.0154 mg/L (EbC50) and 0.082 mg/L (ErC50).
Executive summary:

In a 72h algae growth inhibition study, the cultures of Selenastrum capricornutum were exposed to C12/C14 Amine oxide at nominal concentrations of 0 (Control), 0.0032, 0.010, 0.018, 0.032, 0.058, 0.10, 0.18 and 0.32 mg a.i./L in accordance with the OECD 201 guideline.

The ErC50 value (based on growth rate) was 0.082 mg a.i./L with 95%CL from 0.063 to 0.106 mg a.i./L. The EbC50 value (based on biomass) was 0.0154 mg a.i./L with 95%CL from 0.0106 to 0.0224 mg a.i./L. The % growth inhibition in the treated algal cultures, as compared to the control, ranged from 3% to 92%.

At the end of the test morphologically abnormal cells were found at test substance concentrations of 0.010 mg a.i./L and higher.

This toxicity study is classified as acceptable and satisfies the guideline requirements for the OECD 201 (2006) freshwater algae and cyanobacteria toxicity study.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1996-09-02 to 1996-09-05
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
No analytical measurement of test substance concentrations.
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Version / remarks:
Deviations, reliability, and validity evaluated against the current OECD 201 guideline (2006).
Deviations:
yes
Remarks:
(No measurements of test concentrations (Nominal only), 2 replicates of test concentrations were taken instead of 3 replicates)
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Details on sampling:
- Note: samples were taken for analysis by the sponsor. Those results (if any) are not available in this study report.
- Concentrations: At the start of the growth inhibition test, and during the test, single 50 ml samples were taken from the 0, 0.01, 0.11, and 1.09 mg test substance/L.
- Sampling method: Samples were placed in the polycarbonate sample beakers.
- Sample storage conditions before analysis: Immediately after sampling, samples were frozen and stored at -80 degree C, pending transfer to the sponsor for chemical analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: An amount of 54.4 g was dissolved in 250 ml of medium (pH 8) from which dilutions in medium were prepared so as to yield a test substance concentration series of 0, 0.02, 0.07, 0.12, 0.22, 0.39, 0.70, 1.22 and 2.18 mg/L. 50 ml of the stock solution of 218 mg/L was kept in an ultra low freezer (-80 degree C).
- Controls: Four controls containing algae only, and a single background series containing test substance without algae.
- Chemical name of vehicle: none used
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green alga
- Strain: ATCC 22662
- Source (laboratory, culture collection): The culture was supplied by the 'American Type Culture Collection', c/o Sales Departement, 12301 Parklawn Drive, Rockville, Maryland 20852, USA.
- Age of inoculum (at test initiation): Not available
- Method of cultivation: A pre-culture of algae in the exponential growth phase was prepared as detailed in OECD guideline 201.

ACCLIMATION
- Acclimation period: A preculture of algae in the experimental growth phase was prepared as detailed in OECD 201 guideline, using the same medium as used in the test.
- Culturing media and conditions: Same as test
- Any deformed or abnormal cells observed: Cells were normal.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
71 h
Post exposure observation period:
Not available
Hardness:
Not available. Calculated to be 12.2 mg/L as CaCO3, based on composition of growth medium (18 mg/L CaCl2*2H2O).
Test temperature:
All flasks were incubated at 23+/-2 degree C.
pH:
pH ranged from 8.0 initially to 8.6 at the end of the test.
Dissolved oxygen:
Not available
Nominal and measured concentrations:
Nominal exposure concentrations were 0, 0.01, 0.03, 0.06, 0.11, 0.20, 0.35, 0.61 and 1.09 mg/L (based on the test substance, E5138.01). No measurements of test concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: open
- Material, size, fill volume: 200 ml conical flask with 100 ml test media
- Aeration: No
- Initial cells density: 1.0 X 10 (+4) cells/ml
- Control end cells density: 51.05 X 10 (+4) cells/ml
- No. of organisms per vessel: 1.0 X 10 (+4) cells/ml
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 4

GROWTH MEDIUM
- Standard medium used: Yes, the OECD 201 media, with the exception that it contained 150 mg/L NaHCO3 (not 50 mg/L) to improve the buffer capacity of the medium. The media also contained ferric citrate (not ferric chloride) because the growth of the algae would be erratic without complexed iron. See study report for details.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: The medium was prepared from concentrated stock solutions in Milli-Q-filtered water. It was sterilized by micropore filteration and contained 150 mg/L NaHCO3 (not 50 mg/L) to improve buffer capacity of the medium.The medium contained Fe-citrate.
- Total organic carbon: Not available
- Particulate matter: Not available
- Metals: see composition of test medium
- Pesticides: Not available
- Chlorine: Not available
- Alkalinity: Not available
- Ca/mg ratio: 18 mg/L CaCl2.2H2O/12 mg/L MgCl2.6H2O
- Conductivity: Not available
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH was measured at the start (medium without alga) and after 71 h in the culture.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: Not applicable
- Photoperiod: Not available
- Light intensity and quality: Light intensity radiated by the fluorescent lamps was within the standard range of 60-120 umol/Sec/m(2)

EFFECT PARAMETERS MEASURED:
- Determination of cell concentrations: Electronic particle counter (Coulter Counter model TAII).
- Chlorophyll measurement: Not available

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.74-3
- Range finding study: No
Reference substance (positive control):
yes
Remarks:
The test system is checked with reference substances (K2Cr2O7 and/or 3,5-dichlorophenol) on a yearly basis (data not included in this study report). The results of these reference tests demonstrate a constant quality of the test system for 15 years.
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.007 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 0.003 - 0.014
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.12 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 0.009 - 0.153
Duration:
72 h
Dose descriptor:
EC90
Effect conc.:
2 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 1.19 - 3.37
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
0.002 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 0.001 - 0.003
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.024 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 0.018 - 0.032
Duration:
72 h
Dose descriptor:
EC90
Effect conc.:
0.328 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 0.254 - 0.398
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.003 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: Growth and biomass
Details on results:
- Exponential growth in the control (for algal test): yes/no: Yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: At the end of the test morphological abnormal algal cells were found at test substance concentrations of 0.03 mg/L and higher. At concentrations higher than 0.06 mg/L, all cells had a deviating morphology.
- Colour differences: None
- Flocculation: None
- Adherence to test vessels: None
- Aggregation of algal cells: None
- Other: None
- Any stimulation of growth found in any treatment: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None
- Effect concentrations exceeding solubility of substance in test medium: None
Results with reference substance (positive control):
Not available
Reported statistics and error estimates:
EC10, EC50, and EC90 values were derived using the ECx program compiled in SAS v. 9.1.3 based on Bruce & Versteeg (1992).
Validity criteria fulfilled:
yes
Remarks:
All three validity criteria of OECD 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test) were fulfilled.
Conclusions:
The 72 h toxicity of C12/C14 amine oxide (E-5138.01) to Selenastrum capricornutum was 0.12 mg a.i./L (ErC50), 0.024 mg a.i./L (EbC50), and 0.003 mg a.i./L (NOEC).
Executive summary:

In a 71h algal toxicity study, cultures of green algae (Selenastrum capricornutum) were exposed to the test substance E5138.01 (30.61% C12/C14 Amine oxide) at nominal concentrations of 0 (Control), 0.01, 0.03, 0.06, 0.11, 0.20, 0.35, 0.61 and 1.09 mg/L in accordance with OECD 201.

The key results (based on active ingredient) were:

ErC50       0.12 mg a.i./L (0.009 -0.153)

EbC50      0.024 mg a.i./L (0.018 -0.032)

The NOEC was 0.003 mg a.i./L.

This toxicity study is classified as acceptable and satisfies the guideline requirements for the OECD 201 freshwater algae and cyanobacteria toxicity study.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2010-05-25 To 2010-05-28
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Not to GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
no
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Samples of each test solution were taken for analytical analysis at 0, 24, 48, and 72 hrs.
- Sampling method: Each test solution was preserved in 50% Methanol (1 mL test solution + 1 mL MeOH), placed in VWR 1-dram vials with Teflon-lined caps.
- Sample storage conditions before analysis: Samples were stored at 4 degree C for a maximum of 3 days.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The stock solution was prepared based on % active of the test material at a concentration of 100 mg/L by adding 154.2 mg of amine oxide to 500 mL water. The dosing solutions were then individually prepared in sterile 100 mL glass volumetric flasks in fresh 1XAAP media. This test medium was prepared from concentrated stock solutions in Milli-Q filtered water, adjusted to pH 7.51 and sterilized by micropore filtration
- Controls: Prepared using algal medium
- Evidence of undissolved material: None
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Green algae
- Strain: Not available
- Source: Carolina Biological Supply Company, Unialgal Cultures, 2700 York St. Burlington, NC, 27215
- Age of inoculum (at test initiation): 4 days
- Method of cultivation:
Culture Volume: 100 mL in 250 mL flasks
Culture Density: Approximately 4.25x10(+6) cells/mL
Culture Waters: 1XAAP Media (prepared in high quality water (HQW)
Duration: Continuous subculture, Transferred weekly


ACCLIMATION
- Acclimation period: Cultured and tested under same conditions
- Culturing media and conditions: Same as test
- Any deformed or abnormal cells observed: None
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
Not applicable
Hardness:
Not available. Test medium was according to OECD 201 (1XAAP media).
Test temperature:
Test temperature was registered continuously and remained within the required limits 24 +/- 2 degree C.
0 (control): ranged 22- 24.9 degree C
0.0125mg/L: ranged 22- 24.9 degree C
0.025mg/L: ranged 22- 24.9 degree C
0.05mg/L: ranged 22- 24.9 degree C
0.1mg/L: ranged 21.8- 24.9 degree C
0.2mg/L: ranged 21.8- 24.9 degree C
0.4mg/L: ranged 21.8- 24.9 degree C
pH:
pH was measured at the beginning of the test and after 72 h in each test concentration and the control. Overall, pH ranged 7.21- 8.66.
0 (control): ranged 7.48- 8.66
0.0125mg/L: ranged 7.31- 8.59
0.025mg/L: ranged 7.22- 8.57
0.05mg/L: ranged 7.24- 8.14
0.1mg/L: ranged 7.22- 7.93
0.2mg/L: ranged 7.21- 8.35
0.4mg/L: ranged 7.21- 7.79
Dissolved oxygen:
Not available
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal test concentrations based on 32% active ingredient were: 0 (control), 0.012, 0.025, 0.05, 0.1, 0.2, 0.4 mg/L.
Mean measured concentrations (geometric mean) were 0.010mg/L, 0.017mg/L, 0.033mg/L, 0.076mg/L, 0.141mg/L, 0.281mg/L.
Mean measured concentrations were 66-87% of nominal. Results are based on mean measured concentrations. Details on analytical results are in "any other information on materials and methods" below.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: open
- Material: Sterile 6- well Falcon polystyrene plates containing 10 mL of test solution
- Aeration: No
- Initial cells density: 1x10(+4) cells/mL
- Control end cells density: 83x10(+4) cells/ml (83-fold increase)
- No. of organisms per vessel: 1x10(+4) cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

GROWTH MEDIUM
- Standard medium used: Yes (AAP media)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 1XAAP Media
- Total organic carbon: Not available
- Particulate matter: Not available
- Metals: Not available
- Pesticides: Not available
- Chlorine: Not available
- Alkalinity: Not available
- Ca/mg ratio: 4.4 mg/L CaCl2.2H2O / 12.2 mg/L MgCl2.6H2O (based on composition of AAP media)
- Conductivity: Not available
- Culture medium different from test medium: No
- Intervals of water quality measurement: Temperature was registered continuously. pH was measured at the beginning of the test and after 72 h.

OTHER TEST CONDITIONS
- Sterile test conditions: Yes
- Adjustment of pH: No
- Photoperiod: continuous
- Light intensity and quality: fluorescent lighting (~86µE/m2)

EFFECT PARAMETERS MEASURED: cell count, daily
- Determination of cell concentrations: Fluorimeter
- Chlorophyll measurement: Yes (by fluorimetry)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: No
Reference substance (positive control):
no
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.076 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.159 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Remarks on result:
other: 95% CL: 0.139-0.182mg/L
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.076 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
biomass
Remarks on result:
other: 95% CL: 0.068- 0.085mg/L
Details on results:
- Exponential growth in the control: Yes (83-fold increase)
- Observation of abnormalities (for algal test): N/A
- Unusual cell shape: N/A
- Colour differences: N/A
- Flocculation: N/A
- Adherence to test vessels: N/A
- Aggregation of algal cells: N/A
- Any stimulation of growth found in any treatment: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Growth inhibition information was summarized using the ECx program compiled in SAS v. 9.1.3 based on Bruce and Versteeg (1992) to generate 72-hr ErC10, ErC50, EbC10 and EbC50 statistics.
Validity criteria fulfilled:
yes
Remarks:
All the validity criteria of OECD 201 (2006) were fulfilled.
Conclusions:
The effect of C12/14-alkyl dimethylamine oxide to Pseudokirchneriella subcapitata was 0.076 mg a.i./L (EbC50), 0.159 mg a.i./L (ErC50), and 0.076 mg/L (NOECr), based on mean measured concentrations.
Executive summary:

In a 72 h algal toxicity test, the cultures of green algae (Pseudokirchneriella subcapitata) were exposed to C12 -14 alkyldimethylamine oxide at nominal test concentrations of 0, 0.0125, 0.025, 0.05, 0.1, 0.2, and 0.4 mg/L in accordance with OECD Guideline 201. Mean measured concentrations were 0, 0.010, 0.017, 0.033, 0.076, 0.141, and 0.281mg/L. Mean measured concentrations were 66 -87% of nominal, and results are based on mean measured concentrations.

The results of this algal growth inhibition test may be summarized as follows:

72h EbC50: 0.076 mg a.i./L (0.068 - 0.085)

72h ErC50: 0.159 mg a.i./L (0.139 - 0.182)

72h NOErC: 0.076

There were no compound related phytotoxic effects.

This toxicity study is classified as acceptable and satisfies the guideline requirements for the OECD Guideline 201 (Alga, Growth Inhibition Test) freshwater algae and cyanobacteria toxicity study.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996-05-24 to 1998-02-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
no guideline available
Principles of method if other than guideline:
The objective of this study was to determine the effect of a cationic surfactant (amine oxide) on algae in a much more realistic test than the standard lab algae growth inhibition test. Substrates (tiles, cobbles) were naturally colonized by periphyton in high quality natural streams in Ohio. After 5 weeks of in-stream colonization, the substrates were recovered, brought to the lab, and carefully exposed to the test compound for 28 days in a flow-through system. Effects on algal population and community level structural indices were used to quantify effects of the test chemical.
GLP compliance:
yes
Remarks:
The test was not technically a GLP study. However, the study was conducted in accordance with GLP principles, and was QA-audited internally. The analytical measurement of test material concentration was GLP.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Melting point: Not Applicable
- Boiling point: Not Applicable
- Vapour pressure: Not Applicable
- Water solubility (under test conditions): Not Applicable
- Henry's law constant: Not Applicable
- log Pow: Not Applicable
- pKa: Not Applicable
- Stability in water: Not Applicable
- Stability in light: Not Applicable
- pH dependance on stability: Not Applicable


OTHER PROPERTIES (if relevant for this endpoint)
- Results of test for ready biodegradability: Not Applicable
- Other: Not Applicable
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test concentrations (0, 6.25, 12.5, 25, 50, 100 ug/L) were sampled on days 0, 7, 14, 21, and 28.
- Sampling method: Water column samples for amine oxide were taken in the middle of the test chamber, immediately freeze dried and shipped to BCQ Analytical Services (Breda, Netherlands) for quantification of the surfactant. Stock solutions were evaluated on four occasions, whereas test chamber samples were sampled on 5 occasions (Days 0, 7, 14,21, and 28 of the study) in each of the three replicates at each concentration. Day 7 samples and part of Day 0 were lost at BCO. Other than Day 0 when 2 or all 3 replicate chambers were assessed, samples at all other times involved only one replicate.
- Sample storage conditions before analysis: Samples were immediately freeze dried.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of the test material (290 µg/L) was prepared every other day.
- Eluate: No Data
- Differential loading: No Data
- Controls: The control was the dilution water without test chemical added.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not Applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Not Applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No Data
Test organisms (species):
other: Periphyton community. Two substrates (cobble, tile) were naturally colonized by periphyton in two high quality streams in Ohio (Little Miami River, Big Darby Creek). A total of 110 taxa were encountered.
Details on test organisms:
TEST ORGANISM
- Common name: periphyton community
- Strain: Natural strains from source river.
- Source (laboratory, culture collection): Naturally colonized from sites from upstream reaches of both the Little Miami River (LMR), north of Xenia, Ohio, and Big Darby Creek (BD), north of Darbyville, Ohio. Both locations are characterized by high water quality by the state of Ohio based on biological and chemical assessments. In addition on 31 May 1996, tile substrates were placed in the Maumee River in northern Ohio where water quality is somewhat compromised by agricultural nutrient and pesticide runoffs. Vandalism of this site negated this portion of the study from being completed.
- Age of inoculum (at test initiation): Not Applicable (cobbles & tiles were naturally colonized for 5 weeks in the streams)
- Method of cultivation: Colonization of tile substrates began on 24 June 1996 in the LMR and BD. Tiles (2514 square mm, approx. 5cm x 5 cm) were placed in each location using the colonization methods described by Belanger et al. (1996). Tiles were colonized for approximately five weeks in each stream, until 31 July 1996. They were retrieved from the field, held in a large (~50 L) cooler, and directly transported to the periphyton bioassay dilutor at Ivorydale Technical Center. Summer storms resulted in the loss of some substrates at the BD site. Because Maumee tiles were lost, cobble was collected from the LMR and substituted as a new treatment. Tiles and cobble were immediately dosed when they arrived at the lab and assigned to chambers. LMRT (Little Miami River Tile), LMRC (Little Miami River Cobble), and BDT (Big Darby Tile) were sampled and transferred to the lab on the same day.


ACCLIMATION
- Acclimation period: Not possible. The cobbles/tiles were naturally colonized in river water, and then immediately (within 4-5 hours) placed in the lab test, which was conducted with ESD blended water.
- Culturing media and conditions (same as test or not): No. Periphyton colonized in natural waters, tested in ESD blended water (lab water).
- Any deformed or abnormal cells observed: No Data
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
28 d
Remarks on exposure duration:
Lab testing immediately followed the 5-week colonization period in the 3 streams.
Post exposure observation period:
Not Applicable
Hardness:
No Data in the study report. ESD blended water typically has a hardness of ~150 mg/L as CaCO3.
Test temperature:
22 - 23 deg C
pH:
No Data
Dissolved oxygen:
No Data. DO is expected to be adequate in a flow-through test.
Salinity:
No Data
Nominal and measured concentrations:
0, 6.25, 12.5, 25, 50, 100 µg/L (nominal).
4.3 ± 5.7, 8.6 ± 5.8, 9.2 ± 5.6, 16.1 ± 8.4, 33.1 ± 17.1, 67.1 ± 23.4 µg/L (average measured active concentrations). See Table in "any other information on materials and methods" below.
Details on test conditions:
The experiment involved two substrates and two sources of colonization for periphyton, resulting in three different experimental treatments. The experimental design consisted of Little Miami River tile, Little Miaimi River cobble, and Big Darby tile chambers for each concentration tested (See attached Figure 1). Colonization sites were from upstream reaches of both the Little Miami River and Big Darby Creek. Both locations are characterized by high water quality by the State of Ohio in biological and chemical assessments.

TEST SYSTEM
- Test vessel: Substrates were placed into exposure chambers in what would be considered a pseudo-replicated design. In other words, replicate samples from each of the groups (LMRT, LMRC, BDT) were removed from the same chamber on each day. Six treatment levels of amine oxide (AO) were used. Typically chambers would be replicated for each dose. Here we have used each group of 3 chambers at the same dose to house all the tiles for a substrate location group.
- Type (delete if not applicable): Open
- Material, size, headspace, fill volume: Chamber dimensions were 325 mm longx 128 mm wide x 10.2 mm high
- Aeration: Naturally aerated waters
- Type of flow-through (e.g. peristaltic or proportional diluter): proportional diluter
- Renewal rate of test solution (frequency/flow rate): Turnover 15.3 times daily
- Initial cells density: Not Applicable
- Control end cells density: Not Applicable
- No. of organisms per vessel: Not Applicable
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- No. of vessels per vehicle control (replicates): Not Applicable

GROWTH MEDIUM
- Standard medium used: Natural waters at the 3 stream colonization sites
- Detailed composition if non-standard medium was used: No Data


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ESD blended water (a mixture of well water and deionized water)
- Total organic carbon: No data
- Particulate matter: No data
- Metals: No data
- Pesticides: No data
- Chlorine: No data
- Alkalinity: No data
- Ca/mg ratio: No data
- Conductivity: No data
- Culture medium different from test medium: Yes
- Intervals of water quality measurement: Weekly


OTHER TEST CONDITIONS
- Sterile test conditions: No Data
- Adjustment of pH: No Data
- Photoperiod: 15 hours on / 9 hours off
- Light intensity and quality: 530 ± 60 micro-Einsteins/sq.meter/sec
- Salinity (for marine algae): Not applicable


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Palmer-Maloney nannoplankton counting chamber
- Chlorophyll measurement: Not Applicable
- Other: No Data


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Justification for using less concentrations than requested by guideline: Not Applicable
- Range finding study: No
- Test concentrations: Not applicable.
- Results used to determine the conditions for the definitive study: Not applicable

For additional in-laboratory periphyton community bioassay test conditions see attached Table 1.
Reference substance (positive control):
no
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 67 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Population abundances based on cell and biovolume density, community cell and biovolume density, taxonomic richness, and Shannon Weiner Diversity.
Details on results:
- Exponential growth in the control (for algal test): Not applicable
- Observation of abnormalities (for algal test): No Data
- Unusual cell shape: No data
- Colour differences: No Data
- Flocculation: No Data
- Adherence to test vessels: No data
- Aggregation of algal cells: No Data
- Other: No data
- Any stimulation of growth found in any treatment: No Data
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No Data
- Effect concentrations exceeding solubility of substance in test medium: No
Results with reference substance (positive control):
- Results with reference substance valid? Not Applicable
- EC50: Not Applicable
- Other: Not Applicable
Reported statistics and error estimates:
Dominant populations and community metric data were subjected to one-way ANOVA for each day and for each group to determine no-observed-effect-concentrations (NOEC). Analyses were performed parametrically as assumptions allowed, otherwise the data were subjected to non parametric Kruskal-Wallis. Two way ANOVAs were performed to address questions of whether location had an effect for the same substrate and whether the substrate had an effect for the same location. Data were managed and analyzed using SAS for Windows (v 6.12).

A total of 110 taxa were identified (87 diatom, 12 green, 7 blue-green, 2 euglenoid, 1 chrysophyte, and 1 red taxon were present). SIMI is Stander's Similarity Index (Stander 1970) and can be viewed as a correlation coefficient based on proportional abundance of each taxon in the community. A value near 1, perfect correlation, would indicate all taxa in the same proportions were found in both communities. A value near 0, no correlation, would mean no taxa would be in common. SIMI values for cell density were between 0.73-0.82, and values for biovolume density were between 0.092-0.14 (See attached Table 2). The communities were sufficiently different, based on biovolume abundance, and these will be considered as separately tested communities. A variety of dominant taxa were present. A total of 18 different taxa were dominant in some capacity. Cell and biovolume density dominants were distinctly different. However, taxa richness was similar among the 3 communities (see attached Figure 2).

To evaluate the effect of location on sensitivity of algal communities to amine oxide, tile substrates were used. A 3-way ANOVA with all potential interaction terms was used where main terms included river, exposure concentration, and day. Interaction terms were often significant. In general, strong statistically significant effects of location were present (p<0.001), although in many cases the magnitude of location effects varied by concentration and day. Similarly, location effects were frequently present, 61% of the time overall, when the data were analyzed on each study day separately using a 2-way ANOVA model that included effects for river, concentration, and their interaction. The predominant main effect was the river in these models (See attached Table 3).

In a similar manner, the effects of substrate were evaluated from the Little Miami River. The initial 3-way ANOVA model with interaction terms was complicated by the significance of interactions, especially those involving substrate. There were highly significant substrate effects overall. When 2-way ANOVA models were run on each sample day a very frequent statistical difference for substrate effects occurred as 57% of the time the ANOVA was Significant.

To evaluate exposure effects only, NOECs were determined for each unique test group and each parameter measured. One-way ANOVA was followed by Dunnett's multiple comparison procedure. A total of 280 possible statistical tests per concentration were available. No adverse effects were indicated for any substrate-river combination. A pattern of increases above the control was stronger than for decreases (See attached Table 4). This was most clear for Little Miami River tile at the highest concentration tested (100 ug/L), where there were significant increases for 8 of 35 endpoints, compared to the control. At the next highest test concentration (50 ug/L), 4 of those 8 endpoints were also significantly greater than the control.

Conclusions:
The No-Observed Effect Concentration (NOEC) of amine oxide to periphyton communities was ≥67 µg/L (mean measured concentration). This NOEC is based on periphyton colonized on 2 substrates in 3 natural streams, and exposed for 28 days in a laboratory flow-through test. The NOEC is based on the mean measured concentration of the test substance.
Executive summary:

The toxicity of amine oxide to algae was evaluated in a periphyton microcosm test. Two substrates (tiles, cobbles) were naturally colonized for 5 weeks in high quality flowing streams/rivers in Ohio. The periphyton-colonized tiles & cobbles were then brought into the laboratory, and placed in a 28 -day flow-through test of amine oxide. Periphyton communities were exposed to 0, 6.25, 12.5, 25, 50, 100 µg/L (nominal) amine oxide concentrations for 28 days. Mean measured concentrations were 4.3 (control), 8.6, 9.2, 16.1, 33.1, and 67.1 µg/L. Results are based on mean measured concentrations.

There were clear location (i.e. colonization stream) and substrate effects in this study. Tile and cobble were substantially different, as were the Little Miami River and Big Darby Creek communities. Adverse responses were absent by day 28 in all three communities. There were significant increases in some endpoints at the 2 highest test concentrations (50, 100 µg/L, nominal), but there were no significant decreases (adverse effects) for any endpoint.

The No-Observed Effect Concentration (NOEC) of amine oxide to the periphyton community was determined to be >67 µg/L, the highest test concentration evaluated (mean measured concentration).

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Both the target and source substances contain identical functional groups and differ only by the C-chain length and hence will have similar ecotoxicological properties.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source: Amines, C12-14 (even numbered) -alkyldimethyl, N-oxides (EC 931-292-6) C12 71.0%, C14 24.6%, C16 4.4%.
Target: dodecyldimethylamine oxide (EC 216-700-6; CAS 1643-20-5)

3. ANALOGUE APPROACH JUSTIFICATION
The target and source substances contain identical functional groups. The only difference between the two substances is the alkyl chain length (C12 in the target and mainly C12 (70.1%) in the source along with C14 (24.6%) and minor amounts of other analogues). The results of the acute toxicity tests on Pseudokirchnerella subcapitata performed using C12, C12-14 and C14 AO showed that there was no effect on acute toxicity to algae due to differences in alkyl chain length over the range of chain lengths considered. It is expected that this finding is also applicable to the periphyton study and hence the result of this study may be read across to C12 AO.

4. DATA MATRIX
Target: 72-h ErC50 = 0.20 mg AO/L (Pseudokirchnerella subcapitata).
Source: ErC50 (72 h) = 0.266 mg AO/L (Pseudokirchnerella subcapitata), ErC50 (72 h) = 0.159 mg AO/L (Pseudokirchnerella subcapitata), ErC50 (72 h) = 0.12 mg AO/L (Pseudokirchnerella subcapitata), ErC50 (72 h) = 0.082 mg AO/L (Pseudokirchnerella subcapitata), ErC50 (72 h) = 1.14 mg AO/L (Chlorella vulgaris), ErC50 (72 h) = 0.25 mg AO/L ( Desmodesmus subspicatus). NOEC (28 d) = 0.067 mg AO/L (periphyton microcosm study).
Reason / purpose for cross-reference:
read-across source
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
>= 67 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: Population abundances based on cell and biovolume density, community cell and biovolume density, taxonomic richness, and Shannon Weiner Diversity.

Description of key information

The 72-h ErC50 for C12 AO is 0.20 mg/L. The results of the acute toxicity tests on Pseudokirchnerella subcapitata performed using C12, C12-14 and C14 AO showed that there was no effect on acute toxicity to algae due to differences in alkyl chain length over the range of chain lengths considered. It is expected that this finding is also applicable to the periphyton study and hence the result of this study may be read across to C12 AO. The 28-d NOEC of 0.067 mg AO/L is derived from a periphyton microcosm study in which more than 110 taxa of algae were exposed to C12-14 AO.

Key value for chemical safety assessment

EC50 for freshwater algae:
0.2 mg/L
EC10 or NOEC for freshwater algae:
0.067 mg/L

Additional information

In a 72-hour algal growth inhibition study performed according to OECD 201 Pseudokirchnerella subcapitata were exposed to C12 AO at nominal test concentrations of 0, 0.014, 0.028, 0.057, 0.114 or 0.228 mg AO/L [Mark UE & Arends ICM (1992c)]. The 72-h EbC50 was 0.07 mg AO/L. The 72-h ErC50 was 0.20 mg AO/L. No NOEC value was derived.

One reliable study is available for C14 AO. In this study, performed according to OECD 201, Pseudokirchnerella subcapitata were exposed to C14 AO for 72 hours at nominal test concentrations of 0, 0.024, 0.047, 0.095, 0.190 or 0.379 mg AO/L [Mark UE (1992d)]. The 72-h EbC50 was 0.095 mg AO/L. The 72-h ErC50 was 0.19 mg AO/L. No NOEC value was derived.

Six reliable studies are available for C12-14 AO. In a study performed according to OECD TG 201 under GLP [Ginkel & Kroon (1990)] Pseudokirchnerella subcapitata were exposed to C12-14 AO under static conditions for 72 hours at nominal concentrations of 0, 0.020, 0.039, 0.078, 0.155 or 0.31 mg AO/L. The ErC50 (72 h) was 0.266 mg AO/L. In three supporting studies performed according to OECD TG 201 Pseudokirchnerella subcapitata were exposed to C12-14 AO for 72 hours under static conditions. ErC50 values of 0.159 mg AO/L [Brill J (2010)], 0.12 mg AO/L [Hanstveit & Oldersma (1997)] and 0.082 mg AO/L [Hanstveit & Oldersma (1997)] were reported. Exposure of Chlorella vulgaris to C12-14 AO for 72 hours under static conditions in accordance with OECD TG 201 resulted in an ErC50 of 1.14 mg AO/L [Vreys (2003)], whilst exposure of Desmodesmus subspicatus to C12-14 AO for 72 hours under static conditions in accordance with OECD TG 201 resulted in an ErC50 of 0.25 mg AO/L [Scheerbaum (2000)].

From the available studies for C12-14 AO it appears that Pseudokirchnerella subcapitata is the most sensitive of the species tested. It is noted that the ErC50 values from studies performed using this species are consistent over the range of, C12 AO (0.20 mg AO/L), C12-14 AO (0.082, 0.12, 0.159 & 0.266 mg/L) and C14 AO (0.19 mg AO/L).

The toxicity of C12-14 AO to algae was evaluated in a 28 -day freshwater periphyton microcosm assay [Belanger SE (1999)]. The microcosm was composed of a complex consortia of bacterial, cyanobacterial, algal, and fungal species, and included 110 taxa of algae (notably 87 diatom, 12 green, 7 blue-green, 2 euglenoid, 1 chrysophyte and 1 red). Two substrates (tiles, cobbles) were naturally colonised for 5 weeks in high quality flowing streams/rivers in. The periphyton-colonised tiles & cobbles were then brought into the laboratory and placed in a 28 day flow through test of the C12-14 AO. Periphyton communities were exposed to 0, 6.25, 12.5, 25, 50 or 100 µg AO/L (nominal) for 28 days. Mean measured concentrations were 4.3 (control), 8.6, 9.2, 16.1, 33.1 or 67.1 µg AO/L. There were clear location (i.e. colonisation stream) and substrate effects in this study. Tile and cobble were substantially different, as were the Little Miami River and Big Derby Creek communities. Adverse responses were absent by day 28 in all three communities. There were significant increases in some endpoints at the 2 highest test concentrations (50, 100 µg/L, nominal), but there were no significant decreases (adverse effects) for any endpoint. The No-Observed Effect Concentration (NOEC) of the substance to the periphyton community was determined to be >67 µg AO/L, the highest test concentration evaluated (mean measured concentration).

The results of the acute toxicity tests on Pseudokirchnerella subcapitata performed with C12 AO, C12-14 AO and C14 AO showed that there was no effect on acute toxicity to algae due to differences in alkyl chain length over the range of chain lengths considered. It is expected that this finding is also applicable to the periphyton study and hence the result of this study may be read across to C12 AO.