1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-C8-10 (even numbered) acyl derivs., hydroxides, inner salts

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:

weight of evidence

Study period:

07.09.1992-11.09.1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

without analytical monitoring

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

GLP compliance:

not specified

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 0.38 g of Coco AAPB was weighed out and dissolved in 100 mL of distilled water to give a nominal 1000 mg a.i./L stock solution. The test solutions were obtained by dilution with ISO algal medium.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle used
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not reported

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga (Selenastrum capricornutum)
- Strain: not reported
- Source (laboratory, culture collection): laboratory culture
- Age of inoculum (at test initiation): 3 d
- Method of cultivation: the algae were cultivated in ISO medium

ACCLIMATION
- Acclimation period: 3 d
- Culturing media and conditions (same as test or not): not reported
- Any deformed or abnormal cells observed: not reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

23°C

pH:

initial: 6.8-7.4
at the end: 7.0-7.3

Nominal and measured concentrations:

0 (control), 0.56, 1.0, 1.8, 3.2, 5.6, and 10.0 mg a.i./L nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: not reported
- Type (delete if not applicable): test vessels were loosely stoppered
- Material, size, headspace, fill volume: not reported
- Aeration: not reported
- Type of flow-through (e.g. peristaltic or proportional diluter): test conducted under static conditions
- Initial cells density: 10E4 cells/mL
- Control end cells density: 4.42 x 10E6 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (ISO medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH was measured at time 0 and after 96 h

OTHER TEST CONDITIONS
- Sterile test conditions: not reported
- Adjustment of pH: no
- Photoperiod: continuous lighting
- Light intensity and quality: not measured

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : growth rate, biomass
- Determination of cell concentrations: Coulter counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 1.8
- Range finding study: not reported

Reference substance (positive control):

not specified

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: biomass and growth rate

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

ca. 8 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Validity criteria fulfilled:

yes

Conclusions:

In a study conducted according to OECD Guideline 201, EU Method C.3 and ISO 8692, the 96 h EbC50, ErC50 and NOEC were found to be ca. 8 mg a.i./L, >10 mg a.i./L and 3.2 mg a.i./L (nominal each).

Executive summary:

The toxicity of Coco AAPB to Selenastrum capricornutum was investigated in a study conducted according to OECD Guideline 201 (Alga, Growth Inhibition Test), EU Method C.3 (Algal Inhibition test) and ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum). Selenastrum capricornutum was exposed to nominal concentrations of 0 (control), 0.56, 1.0, 1.8, 3.2, 5.6, and 10.0 mg a.i./L for 96 h under static conditions. The 96 h ErC50, EbC50 and NOEC were determined to be >10 (growth rate) and ca. 8 (biomass) and 3.2 mg a.i./L nominal (growth rate and biomass), respectively.

This study is regarded as reliable with restrictions.

Results Synopsis
Test Organism: Scenedesmus subspicatus (Desmodesmus subspicatus)

Test Type: Static
Growth rate:
96 hr ErC50: >10 mg a.i./L
96 hr NOEC: 3.2 mg a.i./L

Biomass:
96 hr EbC50: ca. 8 mg a.i./L
96.hr NOEC: 3.2 mg a.i./L
(all values refer to nominal concentrations)

Endpoint(s) Effected: growth rate, biomass

Reference 2

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

22.07.1991-25.07.1991

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

without analytical monitoring

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

GLP compliance:

not specified

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 0.38 g of Coco AAPB was weighed out and dissolved in 100 mL of distilled water to give a nominal 1000 mg a.i./L stock solution. The test solutions were obtained by dilution with ISO algal medium.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle used
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not reported

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: green alga (Scenedesmus subspicatus)
- Strain: 86.81
- Source (laboratory, culture collection): laboratory culture
- Age of inoculum (at test initiation): 3 d
- Method of cultivation: the algae were cultivated in ISO medium

ACCLIMATION
- Acclimation period: 3 d
- Culturing media and conditions (same as test or not): not reported
- Any deformed or abnormal cells observed: not reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23-24.5°C

pH:

initial: 7.4-7.6
at the end: 7.0-8.6

Nominal and measured concentrations:

0 (control), 1.0, 1.8, 3.2, 5.6, and 10.0 mg a.i./L nominal

Details on test conditions:

TEST SYSTEM
- Test vessel: glass
- Type (delete if not applicable): test vessels were loosely stoppered
- Material, size, headspace, fill volume: glass/-/-/150 mL
- Aeration: not reported
- Type of flow-through (e.g. peristaltic or proportional diluter): test conducted under static conditions
- Initial cells density: 10E4 cells/mL
- Control end cells density: 2.17 x 10E6 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes (ISO medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH was measured at time 0 and after 72 h

OTHER TEST CONDITIONS
- Sterile test conditions: not reported
- Adjustment of pH: no
- Photoperiod: continuous lighting
- Light intensity and quality: 2600-3700 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : growth rate, biomass
- Determination of cell concentrations: Coulter counter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 1.8
- Range finding study: not reported

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.8 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: biomass and growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

4.66 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.6 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Reported statistics and error estimates:

The EC50 based on biomass and growth rate was calculated using the SAS analysis program on the Vax computer system.

Validity criteria fulfilled:

yes

Conclusions:

In a study conducted according to OECD Guideline 201, EU Method C.3 and ISO 8692, the 72 h EbC50, ErC50 and NOEC were found to be 4.66 mg a.i./L, 5.6 mg a.i./L and 1.8 mg a.i./L (nominal each).

Executive summary:

The toxicity of Coco AAPB to Scenedesmus subspicatus was investigated in a study conducted according to OECD Guideline 201 (Alga, Growth Inhibition Test), EU Method C.3 (Algal Inhibition test) and ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum). Scenedesmus subspicatus was exposed to nominal concentrations of 0 (control), 1.0, 1.8, 3.2, 5.6, and 10.0 mg a.i./L for 72 h under static conditions. The 72 h EC50 and NOEC based on biomass and growth rate were determined to be 5.6 (growth rate) and 4.66 (biomass) and 1.8 mg a.i./L nominal (growth rate and biomass), respectively .

This study is regarded as reliable with restrictions.

Results Synopsis
Test Organism: Scendesmus subspicatus (Desmodesmus subspicatus)

Test Type: Static
Growth rate:
72 hr ErC50: 5.6 mg a.i./L
72 hr NOEC: 1.8 mg a.i./L

Biomass:
72 hr EbC50: 4.66 mg a.i./L
72.hr NOEC: 1.8 mg a.i./L
(all values refer to nominal concentrations)

Endpoint(s) Effected: growth rate, biomass

Reference 3

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2007-04-23 to 2007-05-02

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Lauramidopropyl betaine and control were analytically verified at the beginning and the end of the test in all test vessels. Separate replicates (all concentrations without algae) were prepared and analysed at 0 h and at the end of the test.
- Sample storage conditions before analysis: All samples were stored at room temperature until start of analysis if necessary

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 100 mg C12 AAPB/L stock solution was freshly prepared and dispersed by agitation

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus
- Strain: Chodat SAG 86.81
- Source (laboratory, culture collection): Collection of algal Cultures (SAG), Inst. Plant Physiology, University of Göttingen (Germany)
- Age of inoculum (at test initiation): 3 days old preculture
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar (nutrient medium acc. to Lüttge et al. 1994) at the testing facility. Light intensity was 35 - 70 µE/ m² x s for 24h/d

ACCLIMATION
- Acclimation period: no (preculture was directly pipetted to the test and control replicates)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Algae from the test vessels of 3.2 - 32 mg test item/L and the controls were transferred into fresh untreated medium (0.5 - 1 mL algae suspension from each replicate were filled up with dilution water to 10 mL) and allowed to grow for further 4 - 6 d to determine whether the effect was reversible.

Hardness:

0.24 mmol Ca+Mg/L (nominal)

Test temperature:

21 - 23°C

pH:

7.67 - 8.05

Nominal and measured concentrations:

0.10, 0.32, 1.0, 3.2, 10, 32 mg/L test material (nominal)
corresponding to
0.03, 0.096, 0.3, 0.96, 3, 9.6 mg/L mg a.i. (nominal)
0.091, 0.31, 0.93, 3.27, 9.70, 33.36 mg/L test material (measured)
corresponding to
0.027, 0.093, 0.28, 0.98, 2.91, 10.01 mg/L a.i. (measured)

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL sterile Erlenmeyer flasks
- Type: sealed with cotton wool plugs
- Material, size, headspace, fill volume: test volume 100 mL
- Initial cells density: 2 - 5 x10E3 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24 h/day light
- Light intensity and quality: 60 - 120 µE/m² x s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Chlorophyll-a-fluorescence, excitation at 435 nm, emission at 685 nm
- Chlorophyll measurement: Each replicate was measured 6-fold, values (flash) no. 3 - 6 were used for calculations
Chlorophyll was measured at the beginning and every 24 h
- at start and at the end cells were checked for unusual cell shape, colour differences, differences in chloroplast morphology, flocculation, adherence of algae to test containers and aggregation of alga cells

TEST CONCENTRATIONS
- Spacing factor for test concentrations: square root of 10
- Range finding study
- Test concentrations: 0.1, 1, 10, 100 mg/L (nominal)
- Results used to determine the conditions for the definitive study:
Yield inhibition: -7, 20, 95, 100 %
Specific Growth Rate Inhibition: -1, 4, 54, 100 %

Reference substance (positive control):

yes

Remarks:

potassium dichromate (CAS 7778-50-9

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.15 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 2.82 - 3.51 mg act. ingr./L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

1.31 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 1.11 - 1.48 mg act. ingr./L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.825 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 0.663 - 1.01 mg act. ingr./L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.3 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.96 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.01 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: yield inhibition

Remarks on result:

other: 0.801 - 1.29 mg act. ingr./L

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.558 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: yield inhibition

Remarks on result:

other: 0.396 . 0.603 mg act. ingr./L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.426 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: yield inhibition

Remarks on result:

other: 0.287 - 0.603 mg act. ingr./L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.3 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: yield inhibition

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.96 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

other: yield inhibition

Details on results:

Rate-related inhibition (inhibition of growth rate):
ErC50 (95% confidence interval): 10.5 mg test mat./L (9.40 – 11.7 mg test mat./L)
ErC20 (95% confidence interval): 4.35 mg test mat./L (3.70 – 4.94 mg test mat./L)
ErC10 (95% confidence interval): 2.75 mg test mat./L (2.21 – 3.37 mg test mat./L)
NOEC: 1.0 mg test mat. /L (0.3 mg act. ingr./L)
LOEC: 3.2 mg test mat. /L (0.96 mg act. ingr./L)

Yield inhibition
EyC50 (95% confidence interval): 3.38 mg test mat./L (2.67 – 4.29 mg test mat./L)
EyC20 (95% confidence interval): 1.86 mg test mat./L (1.32 – 2.43 mg test mat./L)
EyC10 (95% confidence interval): 1.42 mg test mat./L (0.955 – 2.01 mg test mat./L)
NOEC: 1.0 mg test mat. /L (0.3 mg act. ingr./L)
LOEC: 3.2 mg test mat. /L (0.96 mg act. ingr./L)

- Observation of abnormalities: no morphological abnormalities were observed by microscopic evaluation at start and at the end of the test
- Any stimulation of growth found in any treatment: A slight stimulation was observed at test concentration 0.32 mg test mat./L

Post exposure observation
The test item effects were observed to be reversible at these test concentrations. There is potential for recovery following exposure up to the highest test concentration of 32 mg/L

Results with reference substance (positive control):

- The effect value (rate related inhibition) is in accordance with the range of data determined in a ring test (ISO 8692)
Rate-related inhibition:
- EC50: 0.92 mg/L (nominal), 95% confidence interval: 0.87 - 0.97 mg/L
Yield inhibition:
- EC50: 0.38 mg/L (nominal), 95% confidence interval: 0.36 - 0.39 mg/L

Reported statistics and error estimates:

EC10, EC20 and EC50 values of growth rate and yield inhibition after 72 h were calculated by sigmiodal dose-response regression. Confidence intervals were calculated using standard procedures according to Clopper and Pearson (1934)

Table 1: Evaluation ofter 72 h

Nominal concentration  [mg test mat. /L]	Mean yield [N/ml]	Mean inhibition of yield [%]	Mean growth rate [d^-1]	Mean rate-related inhibition [%]
32	10437	98.50	0.346	78.47
10	63074	90.93	0.835	48.04
3.2	375795	45.96	1.401	12.82
1.0	659085	5.22	1.592	0.96
0.32	851865	-22.50	1.676	-4.29
0.10	684705	1.54	1.605	0.17
Control	695415		1.607

Negative inhibitions = increase of growth

Table 2: Mean cell densities

Nominal concentration  [mg test mat. /L]	0 h	24 h	48 h	72 h
32	5601	7439	13623	16038
10	5601	15030	34508	68675
3.2	5601	27945	119369	381396
1.0	5601	38561	156990	664686
0.32	5601	43055	204051	857466
0.10	5601	46005	197436	690306
Control	5601	38955	193131	701016

No morphological abnormalities were observed by microscopic evaluatuion at start and at the end of the test

A slight stimulation was observed at test concentration 0.32 mg test mat./L

Algae from the test vessels of 3.2 - 32 mg test item/L and the controls were transferred into untreated medium and allowed to grow for further 4 - 6 d to determine whether the effect was reversible. The test item effects were observed to be reversible at these test concentrations. There is potential for recovery following exposure up to the highest test concentration of 32 mg/L.

The study meets the validity criteria of the guideline:

The cell growth increased 125-fold after 72 h in the control (required 16-fold)

The temperature was in the range of 21 -24°C

The difference of the temperature from the mean values was within±2°C

The pH value in the control replicates increased not higher than 1.5 units

The mean coefficient of variation of daily growth rates was 20.45 (required < 35%)

The mean coefficient of variation of average growth during the whole test period in replicate cultures was 2.76 (required <7%)

Validity criteria fulfilled:

yes

Conclusions:

In a 72 hour acute toxicity study, the cultures of Desmodesmus subspicatus were exposed to C12 AAPB under static conditions in accordance with the OECD Guideline 201 (2006).  The 72 h ErC50 value based on inhibition of specific growth rate was 3.15 mg a.i./L, the 72 h EyC50 value based on yield inhibition was 1.01 mg a.i./L, respectively. The 72 h NOEC based on growth rate and yield was determined to be 0.3 mg a.i./L nominal. No abnormalities were noted. The test item effects were observed to be reversible at the test concentrations 0.32 - 32 mg test mat. /L. (0.096 - 9.6 mg a.i./L).

Executive summary:

In a 72 hour acute toxicity study, the cultures of Desmodesmus subspicatus were exposed to C12 AAPB at nominal concentrations of 0.03, 0.096, 0.3, 0.96, 3, 9.6 mg a.i./L / under static conditions in accordance with the OECD Guideline 201 (2006).  The 72 h ErC50 value based on inhibition of specific growth rate was 3.15 mg a.i./L, the 72 h EyC50 value based on yield inhibition was 1.01 mg a.i./L, respectively. The 72 h NOEC based on growth rate and yield was determined to be 0.3 mg a.i./L nominal. No abnormalities were noted. The test item effects were observed to be reversible at the test concentrations 0.32 - 32 mg test mat. /L. (0.096 - 9.6 mg a.i./L).

This toxicity study is classified as reliable without restriction and satisfies the requirements of the OECD Guideline 201 Alga, Growth inhibition test (2006).

Results Synopsis
Test Organism: Desmodesmus subspicatus
Test Type: Static
72 h ErC50:  3.15mg a.i./L                95% C.I.:  2.82 to 3.51 mg a.i./L
72 h EyC50:  1.01 mg a.i./L                95% C.I.:  0.801 to 1.29 mg a.i./L
72 h NOEC: 0.3 mg a.i./L (all values refer to nominal concentrations) Endpoint(s) Effected: Rate-related inhibition; Yield inhibition

Reference 4

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28.02.2006-03.03.2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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:

yes (incl. QA statement)

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution (200 mg/L) was freshly prepared with dilution water. The test concentrations were obtained by dilution
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle used
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not reported

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus CHODAT (current scientific name)
- Strain: SAG 86.81
- Source (laboratory, culture collection): Culture Collection of Algae (SAG), University Göttingen
- Age of inoculum (at test initiation): 4 d
- Method of cultivation: fresh stocks were prepared every month on Z-Agar (light intensity: 35-70 µE/m² x s for 24 h per day; nutrient medium Z according to Lüttge et al. 1994)

ACCLIMATION
- Acclimation period: not reported
- Culturing media and conditions (same as test or not): yes
- Any deformed or abnormal cells observed: no

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

After 72 h, algae from test concentrations 25 - 100 mg/l and control were transferred into fresh untreated water (0.5 ml algae suspension + dilution water to 10 ml)) and allowed to grow for 3-7 days to determine whether the effect is reversible.

Hardness:

nominal: 0.24 mmol Ca+Mg/l

Test temperature:

min: 22°C
max: 23°C

pH:

start: 8.08 - 8.2
end: 7.46 - 8.85

Nominal and measured concentrations:

Concentrations:
3.13, 6.25, 12.5, 25, 50, 100 mg product/l
0.97, 1.93, 3.86, 7.72, 15.45, 30.9 mg active substance/l

Details on test conditions:

TEST SYSTEM
- Test vessel: sterile Erlenmeyer flasks (250 ml)
- Type (delete if not applicable): not reported
- Material, size, headspace, fill volume: glass/250 mL/-/100 mL
- Aeration: not reported
- Type of flow-through (e.g. peristaltic or proportional diluter): test conducted under static conditions
- Initial cells density: 8869 cells/mL (mean)
- Control end cells density: 865156 cells/mL (mean)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH was measured at time 0 and after 72 h. Temperature was measured continuously. Light intensity was measured prior to test start

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: 24 h/d light (continuous lighting)
- Light intensity and quality: 65.2 - 76.4 µE/m² x s; mean: 71.6 µE/m² x s

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : inhibition of biomass growth, rate-related inhibition
- Chlorophyll measurement: Chlorophyll fluorescence was determined at 0, 24, 48 and 72h, Fluorescence values were related to cell density values according to a calibration curve.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 0.1, 1, 10, and 100 mg product/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.86 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.86 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% c.i. 9.33 - 10.4 mg/L

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

7.73 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

5.31 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Remarks on result:

other: 95% c.i. 5.06 - 5.53 mg/L

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.93 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

3.86 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

RESULTS: EXPOSED
- Nominal/measured concentrations: nominal

72h NOEC growth rate = 12.5 mg product/l
72h LOEC growth rate = 25 mg product/l
72h ErC50 = 31.9 mg product/l

72h NOEC biomass = 6.25 mg product/l
72h LOEC biomass = 12.5 mg product/l
72h EbC50 = 17.2 mg product/l

Assuming a content of 30.9% active substance:
72h NOEC = 3.86 mg active substance/l
72h LOEC = 7.73 mg active substance/l
72h ErC50 = 9.86 mg active substance/l

72h NOEC biomass = 1.93 mg active substance/l
72h LOEC biomass = 3.86 mg active substance/l
72h EbC50 = 5.31 mg active substance/l)

The test item effect was observed to be reversible up to the highest concentration tested (100 mg product/l = 30.9 mg active substance/l).

Results with reference substance (positive control):

REFERENCE SUBSTANCE
- potassium dichromate
- Concentrations: 0.1, 0.2, 0.4, 0.8, 1,6 and 3.2 mg/l

RESULTS: TEST WITH REFERENCE SUBSTANCE
- Date: 13.-16.9.2005
- Results: ErC50 = 0.56 mg/l (EbC50 = 0.31 mg/l)

Reported statistics and error estimates:

EC values were calculated by sigmoidal dose-response regression. The calculation of confidence intervals was carried out using standard procedures according to Clopper and Pearson (1934). The NOEC/LOEC values were calculated using ANOVA and Dunnett's-test (95%).

 Effect of Coco AAPB on the cell density [cells/ml], growth rate and biomass integral of Desmodesmus subspicatus
(mean values of three replicates (6 replicates in control))

Nominal   / Measured*  /    0h    /24h/48h/72h
 [mg/l]     [mg/l]
  0      /   0        /   8869  /  44571  /  242465  /  865156
  3.13   /   0.190    /   9450  /  45382  /  246812  /  915270
  6.25   /   1.80     /   9086  /  49177  /  239057  /  837485
 12.5    /   3.55     /   9262  /  53043  /  166689  /  649837
 25      /   7.1      /  10332  /  41516  /   50752  /  222302
 50      /  14.2      /  10285  /  39401  /   18322  /   28744
100      /  28.4      /  10990  /  12917  /   2671   /  ≤ 1000


Effect of Coco AAPB on the growth rate of Desmodesmus subspicatus
(mean values of three replicates (6 replicates in control))

Nominal  / Measured*  /  Growth rate  /  Inhibition of Growth rate
 [mg/l]     [mg/l]         [d-1]              [%]
  0      /   0        /      1.53     /      -
  3.13   /   0.190    /  (-) 1.53     /     0.27
  6.25   /   1.80     /  (-) 1.51     /     1.39
 12.5    /   3.55     /  (-) 1.42     /     7.34
 25      /   7.1      /  (+) 1.03     /    33.0
 50      /  14.2      /  (+) 0.34     /    77.98
 100     /  28.4      /      n.a.     /   100
 negative inhibitions = increase of growth


Effect of Coco AAPB on the biomass integral of Desmodesmus subspicatus
(mean values of three replicates (6 replicates in control))

Nominal  /Measured*    /  Biomass integral    / Inhibition of biomass
 [mg/l]     [mg/l]                                    [%]
  0      /   0        /        697442         /       -
  3.13   /   0.190    /    (-) 726204         /    - 4.12
  6.25   /   1.80     /    (-) 684262         /      1.39
 12.5    /   3.55     /    (+) 521495         /     25.23
 25      /   7.1      /    (+) 177590         /     74.54
 50      /  14.2      /    (+) 46383          /     93.35
 100     /  28.4      /    (+) – 13342        /    100

Validity criteria fulfilled:

yes

Conclusions:

In a 72 hour toxicity study conducted according to OECD Guideline 201 and EU Method C.3, the cultures of Desmodesmus subspicatus were exposed to Coco AAPB under static conditions. The 72 h NOEC, LOEC and EC50 values based on growth rate were 3.86, 7.73 and 9.86 mg a.i./L nominal, respectively. The 72 h NOEC, LOEC and EC50 values based on biomass were 1.93, 3.86 and 5.31 mg a.i./L nominal, respectively.

Executive summary:

In a 72 hour toxicity study conducted according to OECD Guideline 201 (Alga, Growth Inhibition Test) and EU Method C.3 (Algal Inhibition test), the cultures of Desmodesmus subspicatus were exposed to Coco AAPB (30.9 % active matter) at nominal concentrations of 0.97, 1.93, 3.86, 7.72, 15.45, 30.9 mg active substance/L under static conditions. The 72 h NOEC, LOEC and EC50 values based on growth rate were 3.86, 7.73 and 9.86 mg a.i./L nominal, respectively. The 72 h NOEC, LOEC and EC50 values based on biomass were 1.93, 3.86 and 5.31 mg a.i./L nominal, respectively. There were no compound related phytotoxic effects.
This toxicity study is regarded as reliable without restriction and satisfies the requirements of the guideline.

Results Synopsis
Test Organism: Desmodesmus subspicatus

Test Type: Static

Growth rate:
72 hr ErC50:  9.86 mg a.i./L; 95% C.I.:  9.33 to 10.4 mg a.i./L
72 hr NOEC:  3.86 mg a.i./L
72 hr LOEC: 7.73 mg a.i./L
Biomass:
72 hr EC50:  5.31 mg a.i./L; 95% C.I.:  5.06 to 5.53 mg a.i./L
72 hr NOEC:  1.93 mg a.i./L

72 hr LOEC:  3.86 mg a.i./L

(all values refer to nominal concentrations)

Endpoint(s) Effected: growth rate, biomass

Reference 5

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

19.10.1992-23.10.1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

without analytical monitoring

Qualifier:

according to guideline

Guideline:

other: DIN 38412, part 9: Bestimmung der Hemmwirkung von Wasserinhaltsstoffen auf Grünalgen

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution (10 g/L) was prepared. Furthermore a diluted solution of 1 mg/L was prepared. The corresponding test concentrations were obtained by pipetting an appropriate amount of the aforementioned solutions into the test vessels.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle used
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not reported

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus (reported as Scenedesmus subspicatus)
- Strain: SAG 86.81
- Source (laboratory, culture collection): origin: Pflanzenphysiologisches Institut der Universität Göttingen
- Age of inoculum (at test initiation): 3 d
- Method of cultivation: not reported

ACCLIMATION
- Acclimation period: 3 d
- Culturing media and conditions (same as test or not): not reported
- Any deformed or abnormal cells observed: not reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Test temperature:

23-24°C

pH:

8.0-10.0

Nominal and measured concentrations:

0.01, 0.03, 0.1, 0.3, 1.0, 3.0, and 10 mg product/L (nominal)
0.003, 0.009, 0.03, 0.09, 0.3, 0.9, 3.0 mg a.i./L (nominal)

Details on test conditions:

TEST SYSTEM
- Test vessel: 300 mL Erlenmeyer flask
- Type (delete if not applicable): not reported
- Material, size, headspace, fill volume: glass/300 mL/-/100 mL
- Aeration: not reported
- Type of flow-through (e.g. peristaltic or proportional diluter): test conducted under static conditions
- Initial cells density: 1 x 10E4 cells/mL
- Control end cells density: 1.6 x 10E6 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: test culture medium: Algenn 10.10 (not further specified)
- Culture medium different from test medium: not reported
- Intervals of water quality measurement: pH and temperature were measured daily

OTHER TEST CONDITIONS
- Sterile test conditions: not reported
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 2000 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : biomass, growth rate
- Determination of cell concentrations: coulter counter; Thoma chamber

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 3
- Range finding study: not reported

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 1.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

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

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 0.57 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Reported statistics and error estimates:

Originally, the data were evaluated according to the test guideline (DIN 38412, Part 9) using the EDV-program of Mr. Holtmann (Henkel KGaA). Based on today knowledge, this calculation model is no longer valid or only valid with restriction. Therefore, a recalculation of the data was performed taking into account the OECD principles.

RESULTS: EXPOSED
- Nominal/measured concentrations: nominal

re-evaluated values (Cognis, 27.5.2004)
growth rate
72h ErC50 = 5.0 mg product/L

biomass
72 h EbC50 = 1.9 mg product/L
72 h NOEC = 1.0 mg product/L

Assuming a content of 30% active substance:
growth rate
72h ErC50 = 1.5  mg active substance/L

biomass
72h EbC50 = 0.57 mg active substance/L
72 h NOEC = 0.3 mg active substance/L

Validity criteria fulfilled:

yes

Conclusions:

The toxicity of Coco AAPB to Desmodesmus subspicatus was investigated in a study conducted according to DIN 38412, part 9 (comparable to OECD Guideline 201). Originally, the data were evaluated according to the test guideline (DIN 38412, Part 9) using the EDV-program of Mr. Holtmann (Henkel KGaA). Based on today knowledge, this calculation model is no longer valid or only valid with restriction. Therefore, a recalculation of the data was performed taking into account the OECD principles. The 72 h ErC50, EbC50, and NOEC were found to be 1.5, 0.57, and 0.3 mg a.i./L nominal, respectively.

Executive summary:

In a test conducted according to DIN 38412, part 9 (comparable to OECD Guideline 201), the cultures of Desmodesmus subspicatus were exposed to Coco AAPB at nominal concentrations of 0.003, 0.009, 0.03, 0.09, 0.3, 0.9, 3.0 mg a.i./L under static conditions for 72 h. Originally, the data were evaluated according to the test guideline (DIN 38412, Part 9) using the EDV-program of Mr. Holtmann (Henkel KGaA). Based on today knowledge, this calculation model is no longer valid or only valid with restriction. Therefore, a recalculation of the 72 h EC50 values was performed taking into account the OECD principles. The 72 h ErC50, EbC50, and NOEC were found to be 1.5, 0.57, and 0.3 mg a.i./L nominal, respectively.

Results Synopsis
Test Organism: Desmodesmus subspicatus
Test Type: Static
Growth rate:
72 hr ErC50: 1.5 mg a.i./L  
Biomass:
72 hr EbC50: 0.57  mg a.i./L

72 hr NOEC: 0.3 mg a.i./L

(all values refer to nominal concentations)

Endpoint(s) Effected: growth rate, biomass

Reference 6

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

06/02/96 - 09/02/96

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Analytical monitoring via TOC, not via specific analysis.

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Version / remarks:

cited as Directive 92/69/EEC, C.3

GLP compliance:

yes

Analytical monitoring:

yes

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution (2.86 g/L) was prepared. The test concentrations were obtained by dilution.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle used
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not reported

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Scenedesmus subspicatus CHODAT
- Strain: 86.81
- Source (laboratory, culture collection): WaBoLu
- Age of inoculum (at test initiation): 3 d
- Method of cultivation: not reported

ACCLIMATION
- Acclimation period: 3 d
- Culturing media and conditions (same as test or not): not reported
- Any deformed or abnormal cells observed: not reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23.2-23.4°C

pH:

start: 7.9-9.1
end: 7.7-9.6

Nominal and measured concentrations:

0 (control), 4, 10, 25, 63, 158, and 395 mg dry residue/L

Details on test conditions:

TEST SYSTEM
- Test vessel: not reported
- Aeration: not reported
- Type of flow-through (e.g. peristaltic or proportional diluter): test conducted under static conditions
- Initial cells density: 2 x 10³ cells/mL
- Control end cells density: 104 x 10³ cells/mL
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 8

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH was measured at test start and test end; temperature was continuously measured

OTHER TEST CONDITIONS
- Sterile test conditions: not reported
- Adjustment of pH: no
- Photoperiod: continuous lighting
- Light intensity and quality: ca. 8000 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : growth rate, biomass
- Determination of cell concentrations: spectrophotometer

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study: not reported

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 334 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

37.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

10 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

RESULTS: EXPOSED
- Nominal/measured concentrations: nominal

72h ErC10 = 44 mg dry residue/l
72h ErC50 > 395 mg dry residue/l
(72h NOEC biomass = 10 mg dry residue/l
 72h EbC10 = 26 mg dry residue/l
 72h EbC50 = 167 mg dry residue/l)

Assuming a content of 35% dry residue:
72h ErC10 = 126 mg product/l
72h ErC50 > 1129 mg product/l
(72h NOEC biomass = 28.6 mg product/l
 72h EbC10 = 74.3 mg product/l
 72h EbC50 = 477 mg product/l)

Assuming a content of 29.6% active substance:
72h ErC10  = 37.2 mg active substance/l
72h ErC50 > 334  mg active substance/l
(72h NOEC biomass = 8.46 mg product/l
 72h EbC10 = 22 mg product/l
 72h EbC50 = 141 mg active substance/l)

RESULTS: Analysis of test substance concentration
- at 0h: >80%
- after 72h: >80%

Validity criteria fulfilled:

yes

Conclusions:

In a test conducted according to OECD Guideline 201, the cultures of Desmodesmus subspicatus were exposed to Coco AAPB for 72 h under static conditions. The 72 h ErC50 and EbC50 were determined to be >334 mg a.i./L and 37.2 mg a.i./L nominal.

Executive summary:

In a test conducted according to OECD Guideline 201, the cultures of Desmodesmus subspicatus were exposed to Coco AAPB at nominal concentrations of 0 (control), 4, 10, 25, 63, 158, and 395 mg dry residue/L for 72 h under static conditions. The stability of the test item was measured via TOC measurements. The recovery rate was >80%. After 72 h, the measured values differed from freshly prepared solutions by < 20%. The 72 h EC50 were determined to be 37.2 (biomass) and >334 mg a.i./L nominal (growth rate), respectively.

This study is regarded as reliable with restrictions.

Results Synopsis
Test Organism: Desmodesmus subspicatus (reported as Scenedesmus subspicatus)

Test Type: Static
Growth rate:
72 hr ErC50:  >334 mg a.i./L

Biomass:
72 hr EbC50:  37.2 mg a.i./L

72 hr NOEC:  8.46 mg a.i./L

(all values refer to nominal concentations)

Endpoint(s) Effected: growth rate, biomass

Reference 7

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

07.12.1993-10.12.1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

without analytical monitoring

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution (200 mg/L) was prepared. The test concentrations were obtained by dilution
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): no vehicle used
- Evidence of undissolved material (e.g. precipitate, surface film, etc): not reported

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus CHODAT (current scientific name)
- Strain: SAG 86.81
- Source (laboratory, culture collection): own breeding (origin: Sammlung von Algenkulturen, Uni Göttingen)
- Age of inoculum (at test initiation): 4 d
- Method of cultivation: The algae were cultivated in sterile flasks with ca. 2 L culture medium according to Kuhl (1962) at room temperature (light intensity: 35-70 µE/m² x s; continuous lighting)

ACCLIMATION
- Acclimation period: 4 d
- Culturing media and conditions (same as test or not): no
- Any deformed or abnormal cells observed:not reported

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

23 +/- 2°C

pH:

7.79-8.13 (start), 8.03-8.19 (end)

Nominal and measured concentrations:

0.32; 1; 3.2; 10; 32; 100 mg product/l
corresponding to 0.098, 0.31, 0.98, 3.1, 9.8, 31 mg active substance/l

Details on test conditions:

TEST SYSTEM
- Test vessel: plastic cuvettes ( cross section dimension=50 mm, clear, with cover, 20 ml nominal volume)
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: plastic/20 mL/10 mL/10 mL
- Aeration: not reported
- Type of flow-through (e.g. peristaltic or proportional diluter): test conducted under static conditions
- Initial cells density: 7 x 10³ cells/mL
- Control end cells density: 110 x 10³ cells/mL
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Culture medium different from test medium: yes
- Intervals of water quality measurement: pH was measured at the beginning and at the end of the test; temperature was continuously measured

OTHER TEST CONDITIONS
- Sterile test conditions: not reported
- Adjustment of pH: no
- Photoperiod: continuous light
- Light intensity and quality: 35-70 µE/m² x sec (acc. to OECD: 120 µE/m² x sec)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : biomass, growth rate
- Chlorophyll measurement: Algal cells density was determined by measuring chlorophyll a-fluorescence via chlorophyll-fluorometrie. Chlorophyll fluorescence was determined at 0; 24; 48 and 72h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 3
- Range finding study: not reported

Reference substance (positive control):

not specified

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 14.7 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

ca. 9.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

biomass

Details on results:

RESULTS: EXPOSED
- Nominal/measured concentrations: nominal

EC0 was obtained from concentration-response relation and not calculated;
EC10 and EC50 were determined according to DIN 38412 L9 (graphically from a log concentration/biomass or growth rate inhibition plot)

Results, based on nominal concentration:

ErC0 : 3.2 mg product/l
ErC10 : 7.0 mg product/l
ErC50 : 48 mg product/l

Results for the endpoint biomass inhibition after 72 h:
EbC0 : 3.2 mg product/l
EbC10 : 4.9 mg product/l
EbC50 : 30 mg product/l


Assuming a content of 30.7% active substance:
Results for the endpoint growth rate inhibition after 72 h:
ErC0 : 0.98 mg active substance/l
ErC10 : 2.1 mg active substance/l
ErC50 : 14.7 mg active substance/l

Results for the endpoint biomass inhibition after 72 h:
EbC0 : 0.98 mg active substance/l
EbC10 : 1.5 mg active substance/l
EbC50 : 9.2 mg active substance/l

Reported statistics and error estimates:

SigmaPlot, Jandel Scientific Version 5.0 (1992)

 Number of cells/ml:
Test item  |   0h  |  24h  |  48h  |  72h  |
[mg/l]     |       |       |       |       |
--------------------------------------------
           |   number of cell [*10E3]
control    |   7   |   13  |   45  |  110  |
0.32       |   8   |   14  |   39  |  103  |
1.0        |   8   |   16  |   43  |  107  |
3.2        |   7   |   10  |   43  |  123  |
10.0       |   8   |   13  |   32  |   93  |
32.0       |   7   |    9  |   24  |   64  |
100        |   9   |    4  |    4  |    5  |


Inhibition of growth rate and biomass integral after 72h:
Test item  |   growth  |  inhibition |  inhibition |
[mg/l]     |   grate   |   biomass   |  growth rate|  
           |           |    [%]      |     [%]     |
---------------------------------------------------   
control    |    0.94   |      ---    |     ---     |
0.32       |    0.86   |     12.3    |       9.2   |
1.0        |    0.87   |      4.2    |       8.0   |
3.2        |    0.94   |      0.8    |       0.7   |
10.0       |    0.81   |     26.7    |      14.1   |
32.0       |    0.72   |     51.7    |      23.8   |
100        |    0.00   |    100.0    |     100.0   |

Validity criteria fulfilled:

yes

Conclusions:

In a 72 hour toxicity study conducted according to OECD Guideline 201 (Alga, Growth Inhibition Test), the cultures of Desmodesmus subspicatus were exposed to C8-18 AAPB under static conditions. The 72 h ErC50 and 72 h EbC50 were 14.7 and 9.2 mg active substance/l nominal, respectively. 

Executive summary:

In a 72 hour toxicity study conducted according to OECD Guideline 201 (Alga, Growth Inhibition Test), the cultures of Desmodesmus subspicatus were exposed to C8 -18 AAPB at nominal concentrations of 0, 0.098, 0.31, 0.98, 3.1, 9.8, and 31 mg active substance/L under static conditions. The 72 h ErC50 and 72 h EbC50 were 14.7 and 9.2 mg active substance/L nominal, respectively.  There were no compound related phytotoxic effects.

This study is regarded as reliable with restrictions and satisfies the guideline requirements for a toxicity study with freshwater algae.

Results Synopsis
Test Organism: Desmodesmus subspicatus
Test Type: Static
Growth rate:
72 hr ErC50: 14.7  mg a.i./L  
Biomass:
72 hr EbC50: 9.2  mg a.i./L

(all values refer to nominal concentations)

Endpoint(s) Effected: growth rate, biomass

Description of key information

72 h ErC50 = 1.5 mg a.i./L, 72 h NOEC = 0.3 mg a.i./L, DIN 38412, Part 9 (comparable to OECD TG 201), Desmodesmus subspicatus, GLP, RL2; Read-across: C8-18 and C18 unsatd. AAPB

Key value for chemical safety assessment

EC50 for freshwater algae:

1.5 mg/L

EC10 or NOEC for freshwater algae:

0.3 mg/L

Additional information

No experimental data are available for the target substance C8-10 Alkylamidopropyl betaine. However, toxicity studies with algae are available for the source substances Formamidopropylbetaine, C8-18 AAPB and C8-18 and C18 unsatd. AAPB. A justification for read-across is given in the Endpoint summary “Aquatic Toxicity”.

 

The toxicity of C8-18 AAPB and C8-18 and C18 unsatd. AAPB to fresh water algae was investigated in 6 valid studies with Desmodesmus subspicatus  and Pseudokirchnerella subcapitata  (72 hErC50 1.5 - > 334 mg/L a.i.). A further supporting study is available for Formamidopropylbetaine.

 

Summary 

In a weight of evidence approach the acute aquatic toxicity to algae was found to be in the range of >1 to ≤ 10 mg/L. For practical reasons (to name a value for the algae effect concentration) the algae study with the lowest effect result within this range is used for the assessment of algae toxicity: 72 h ErC50 = 1.5 mg a.i./L, 72 h NOEC = 0.3 mg a.i./L.

 

Studies in detail - data included in the weight of evidence approach

1. In a test conducted according to DIN 38412, part 9 (comparable to OECD Guideline 201), the cultures of Desmodesmus subspicatus were exposed to C8-18 and C18 unsatd. AAPB at nominal concentrations of 0.003, 0.009, 0.03, 0.09, 0.3, 0.9, 3.0 mg a.i./L under static conditions for 72 h. Originally, the data were evaluated according to the test guideline (DIN 38412, Part 9) using the EDV-program of Mr. Holtmann (Henkel KGaA). Based on today knowledge, this calculation model is no longer valid or only valid with restriction. Therefore, a recalculation of the 72 h EC50 values was performed taking into account the OECD principles. The 72 h ErC50, EbC50, and NOEC were found to be 1.5, 0.57, and 0.3 mg a.i./L nominal, respectively.

 

2. In a 72 hour toxicity study conducted according to OECD Guideline 201 (Alga, Growth Inhibition Test) and EU Method C.3 (Algal Inhibition test), the cultures of Desmodesmus subspicatus  were exposed to C8-18 and C18 unsatd. AAPB (30.9 % active matter) at nominal concentrations of 0.97, 1.93, 3.86, 7.72, 15.45, 30.9 mg active substance/L under static conditions. The 72 h NOEC, LOEC and EC50 values based on growth rate were 3.86, 7.73 and 9.86 mg a.i./L nominal, respectively. The 72 h NOEC, LOEC and EC50 values based on biomass were 1.93, 3.86 and 5.31 mg a.i./L nominal, respectively. There were no compound related phytotoxic effects. This toxicity study is regarded as reliable without restriction and satisfies the requirements of the guideline.

 

3. In a 72 hour toxicity study conducted according to OECD Guideline 201 (Alga, Growth Inhibition Test), the cultures of Desmodesmus subspicatus  were exposed to C8 -18 AAPB at nominal concentrations of 0, 0.098, 0.31, 0.98, 3.1, 9.8, and 31 mg active substance/L under static conditions. The 72 h ErC50 and 72 h EbC50 were 14.7 and 9.2 mg active substance/L nominal, respectively.  There were no compound related phytotoxic effects. This study is regarded as reliable with restrictions and satisfies the guideline requirements for a toxicity study with freshwater algae.

 

4. The toxicity of C8-18 and C18 unsatd. AAPB to Scenedesmus subspicatus  was investigated in a study conducted according to OECD Guideline 201 (Alga, Growth Inhibition Test), EU Method C.3 (Algal Inhibition test) and ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum). Scenedesmus subspicatus  was exposed to nominal concentrations of 0 (control), 1.0, 1.8, 3.2, 5.6, and 10.0 mg a.i./L for 72 h under static conditions. The 72 h EC50 and NOEC based on biomass and growth rate were determined to be 5.6 (growth rate) and 4.66 (biomass) and 1.8 mg a.i./L nominal (growth rate and biomass), respectively. This study is regarded as reliable with restrictions.

 

5. The toxicity of C8-18 and C18 unsatd. AAPB to Selenastrum capricornutum  was investigated in a study conducted according to OECD Guideline 201 (Alga, Growth Inhibition Test), EU Method C.3 (Algal Inhibition test) and ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum). Selenastrum capricornutum  was exposed to nominal concentrations of 0 (control), 0.56, 1.0, 1.8, 3.2, 5.6, and 10.0 mg a.i./L for 96 h under static conditions. The 96 h ErC50, EbC50 and NOEC were determined to be >10 (growth rate) and ca. 8 (biomass) and 3.2 mg a.i./L nominal (growth rate and biomass), respectively. This study is regarded as reliable with restrictions.

 

6. In a test conducted according to OECD Guideline 201, the cultures of Desmodesmus subspicatus  were exposed to C8-18 and C18 unsatd. AAPB at nominal concentrations of 0 (control), 4, 10, 25, 63, 158, and 395 mg dry residue/L for 72 h under static conditions. The stability of the test item was measured via TOC measurements. The recovery rate was >80%. After 72 h, the measured values differed from freshly prepared solutions by < 20%. The 72 h EC50 were determined to be 37.2 (biomass) and >334 mg a.i./L nominal (growth rate), respectively. This study is regarded as reliable with restrictions.

 

Supporting data:

In a study according to OECD Guideline 201 cultures of Selenastrum capricornutum were exposed to Formamidopropylbetaine at the regulatory concentration limit concentration of 100 mg/L. No inhibition of cell growth or reduction of growth rate was recorded. Therefore, the NOEC for both cell growth inhibition and growth rate reduction was 100 mg/L. The EC50 for both cell growth inhibition (EbC50: 0-72h) and growth rate reduction (ERC50: 0-72h) was beyond 100 mg/L.