Registration Dossier

Ecotoxicological information

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2008-03-06 to 2008-06-06
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study is GLP compliant and follows OECD 201 guideline (Freshwater Alga and Cyanobacteria, Growth Inhibition Test). Full sample characterisation is provided. The analytical chemistry supporting this study was performed to GLP and confirms the identity of the test item but, the separate chemistry report has not been audited at the current time (04 November 2010).

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): SLI (76) stripped
- Molecular formula (if other than submission substance): not applicable (mixture)
- Molecular weight (if other than submission substance): not applicable (mixture)
- Smiles notation (if other than submission substance): not applicable (mixture)
- InChl (if other than submission substance): not known
- Structural formula attached as image file (if other than submission substance): not applicable (mixture)
- Substance type:
- Physical state: cream coloured granular powder
- Analytical purity: 96%
- Impurities (identity and concentrations):
- Composition of test material, percentage of components:
- Isomers composition: not applicable (mixture)
- Purity test date:
- Lot/batch No.: S2849001
- Expiration date of the lot/batch:
- Stability under test conditions:
- Storage condition of test material:
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations (nominal): 0, 3, 10, 30, 100, 300 mg/L
- Concentrations (measured): 0.11, 0.37, 0.79, 7.86 and 135 mg/L
- Sampling method: Test concentrations at 0, 24, 48 and 72 h were determined by LC/MS.
- Sample storage conditions before analysis: Chemical analysis conducted immediately after sampling.

Test solutions

Vehicle:
yes
Details on test solutions:
A stock solution with a nominal concentration of 1000 mg/L was prepared by dissolving 1000 mg of test item in 1000 mL of purified water. The solution was heated to approximately 40 -50 oC whilst stirring continuously. Test media was prepared by dosing the appropriate volume of warm stock and ISO concentrate into purified water to give nominal concentrations of 0, 3, 10, 30, 100 and 300 mg/L.

Test organisms

Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Algae
- Strain: Pseudokirchneriella subcapitata
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa, SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, OBAN, Argyll, PA37 1QA, Scotland.
- Age of inoculum (at test initiation): No data - study conducted according to OECD 201 guideline
- Method of cultivation: No data - study conducted according to OECD 201 guideline


ACCLIMATION
- Acclimation period: No data - study conducted according to OECD 201 guideline
- Culturing media and conditions (same as test or not): ISO media (Reference: ISO 8692:1989 (E) Water Quality-Freshwater algal growth inhibition test with Selenastrum capricornutum and Scenedesmus subspicatus).
- Any deformed or abnormal cells observed: No data

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
No data

Test conditions

Hardness:
No data
Test temperature:
22-25 degrees C
pH:
In the control the pH at 0 and 72 h were 6.9 and 7.7, respectively.
In test item (nominal 3.0 mg/L) the pH at 0 and 72 h were 7.4 and 7.4, respectively.
In test item (nominal 10.0 mg/L) the pH at 0 and 72 h were 7.5 and 7.2, respectively.
In test item (nominal 30.0 mg/L) the pH at 0 and 72 h were 7.5 and 6.8, respectively.
In test item (nominal 100.0 mg/L) the pH at 0 and 72 h were 7.6 and 6.7, respectively.
In test item (nominal 300.0 mg/L) the pH at 0 and 72 h were 7.6 and 6.6, respectively.
Dissolved oxygen:
No data
Salinity:
No data
Nominal and measured concentrations:
Nominal: 0, 3, 10, 30, 100, 300 mg/L
Total Measured: 0, 0.11, 0.37, 0.79, 7.86 and 135 mg/L
Dissolved measured: 0, 0.10, 0.31, 0.68, 2.00, 102 mg/L
Details on test conditions:
Ask Jayne
For the definitive study a series of test solutions were prepared by diluting a 1000 mg/L stock solution with ISO media to give final test media concentrations of 0, 3, 10, 30, 100 and 300 mg/L. A calculated aliquot of Pseudokirchneriella subcapitata inoculum was transferred into each test solution to give a nominal algal density of 10E4 cells/mL. Cell concentration was determined at 48 and 72 hours by haemocytometer. Test concentrations were determined every 24 hours by Liquid Chromatography/Mass Spectroscopy (LC/MS).

Blank flasks were prepared by removing an aliquot of test media from each volumetric flask before the Pseudokirchneriella subcapitata inoculum was added. Glassware was not conditioned prior to the start of the test. Test flasks were placed in illuminated orbital incubators according to the randomised distribution numbers generated by the Random.bas computer software. Incubator temperatures were recorded at 24-hour intervals for the duration of the test. Light intensity was not recorded.

A 10 mL aliquot of each solution was provided to Chemical Analysis at 0, 24, 48 and 72 hours for determination of test item concentration. Test concentrations were determined by LC/MS by the Chemical Analysis.

The algal concentrations in the test media were determined at 0, 48 and 72 hours using a haemocytometer.

Incubator temperatures ranged between 22 and 25 oC throughout the experimental period. The pH of each test solution was measured at 0 hours. The pH of the remaining test media in the first replicate flask of each test concentration was measured at 72 hours.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
>= 1.87 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
>= 7.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
(total concentration)
Basis for effect:
growth rate
Remarks on result:
other: 95% fiducial interval (5.64, 11.56)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
>= 2.88 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
(total concentration)
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
>= 4.8 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
growth rate
Remarks on result:
other: 95% fiducial interval (3.66, 6.50)
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.37 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
(total concentration)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.37 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Remarks:
(total concentration)
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.31 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.31 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
dissolved
Basis for effect:
biomass
Details on results:
- Exponential growth in the control (for algal test): Yes, 110-fold increase in the cell density of control cultures over 72 h
- Observation of abnormalities (for algal test):
- No data
- Other obsevations:
The test item has limited solubility in water and ISO media. There are significant losses of the test item over a 72 hr period. At 0 hours the measured concentration of each chain length dissolved in solution is close to the expected nominal concentration taking into account the chain length distribution of the test item. The concentrations of C8, C10 and C12 remain stable over the test period however there is a significant decrease in the concentration of chain lengths ≥ C14 in solution. Comparisons between solutions with and without algae reveal a significant decrease in the total concentratio remaining at 72 hours when algae is present (30% of the 100 mg/L solution remaining compared to 65% for the same concentration without algae).
Results with reference substance (positive control):
No reference substance used
Reported statistics and error estimates:
95% fiducial intervals given for 72-h EC50 (measured total and dissolved concentrations) - see results

Any other information on results incl. tables

Table 1: Cell densitites (cells/mL)

Flask code

Mean measured conc (mg/L)

Time (hours)

Total

Dissolved

0

48

72

A1

0.0

0.0

10000

272000

1111500

 

A2

0.0

0.0

10000

212000

760500

 

A3

0.0

0.0

10000

214000

934500

 

A4

0.0

0.0

10000

251000

1272000

 

A5

0.0

0.0

10000

279500

1165500

 

A6

0.0

0.0

10000

346500

1362000

 

B1

0.11

0.10

10000

283500

1122000

 

B2

0.11

0.10

10000

279500

1009500

 

B3

0.11

0.10

10000

318000

1296000

 

C1

0.37

0.31

10000

338500

1266000

 

C2

0.37

0.31

10000

209500

835500

 

C3

0.37

0.31

10000

326500

1161000

 

D1

0.79

0.68

10000

192500

482500

 

D2

0.79

0.68

10000

93500

393500

 

D3

0.79

0.68

10000

239000

473000

 

E1

7.86

2.00

10000

57000

157500

 

E2

7.86

2.00

10000

54000

177500

 

E3

7.86

2.00

10000

45500

153500

 

F1

135

102

10000

10000

11000

 

F2

135

102

10000

10000

14500

 

F3

135

102

10000

13000

11500

 

Table 2: 72 -h mean percentage inhibition of biomass and growth rates

Mean measured concentration (mg/L)

 

 Biomass

 Growth rate

 Total

 Dissolved

 

 

 0.11

 0.10

 -7.30

 -1.48

 0.37

0.31

-3.98

 -0.27

 0.79

 0.68

 49.44

 18.14

 7.86

 2.00

 84.89

 41.78

 135

 102

 99.71

 95.92

Table 3: Summary of SLI(76) stripped concentrations during 72 -h study

 Nominal concentration (mg/L)

Measured concentration (mg/L)

Mean measured concentration (mg/L)*

0 hours

24 hrs

48 hrs

72 hrs

Total

Dissolved

Total

Dissolved

Total

Dissolved

Total

Dissolved

Total

Dissolved

3

2.35

2.02

<0.18

<0.19

<0.05

<0.05

<0.05

<0.05

0.11

0.10

10

10.1

7.91

3.04

1.95

<0.05

<0.05

<0.05

<0.05

0.37

0.31

30

36.2

29.2

17.3

11.6

<0.05

<0.05

<0.05

<0.05

0.79

0.68

100

114

89.8

71.1

42.1

18.8

<0.10

<0.05

<0.17

7.86

2.00

300

344

272

263

158

103

59.0

36.0

42.5

135

102

Table 4: Total concentrations of SLI(76) stripped in solution during definitive study according to chain length distribution.

Nominal concentration (mg/L)

 

Measured concentration of chainlengths (mg/L)

Total Measured Concentration (mg/L)

Time (hrs)

C8

C10

C12

C14

C16

C18

3

0

0.12

0.13

0.69

0.29

0.51

0.61

2.35

24

0.09

0.05

<0.01

<0.01

<0.01

<0.01

<0.18

48

<0.01

0.01

<0.01

<0.01

<0.01

<0.01

<0.05

72

<0.01

<0.01

<0.01

<0.01

<0.01

<0.01

<0.05

10

0

0.43

0.45

2.57

1.20

2.80

2.66

10.1

24

0.41

0.37

0.92

0.04

0.47

0.82

3.04

48

<0.01

0.01

<0.01

<0.01

<0.01

<0.01

<0.05

72

<0.01

<0.01

<0.01

<0.01

<0.01

<0.01

<0.05

30

0

1.43

1.48

8.84

4.28

10.32

9.83

36.2

24

1.33

1.33

5.08

0.96

4.02

4.54

17.3

48

<0.01

0.01

<0.01

<0.01

<0.01

<0.01

<0.05

72

<0.01

<0.01

<0.01

<0.01

<0.01

<0.01

<0.05

100

0

4.40

4.51

28.2

13.4

32.5

31.2

114

24

4.18

4.27

20.3

6.43

17.9

18.0

71.1

48

<0.01

0.02

0.04

0.95

5.98

11.8

18.8

72

<0.01

<0.01

<0.01

<0.01

<0.01

<0.01

<0.05

300

0

13.1

13.5

84.7

41.9

97.2

93.1

344

24

13.0

13.4

71.8

30.5

70.9

63.7

263

48

12.0

9.27

9.77

3.61

16.0

52.0

103

72

6.24

1.22

<0.02

0.14

1.44

27.0

36.0

Table 5: Dissolved concentrations of SLI(76) stripped in solution during definitive study according to chain length distribution.

Nominal concentration (mg/L)

 

Measured concentration of chainlengths (mg/L)

Total Measured Concentration (mg/L)

Time (hrs)

C8

C10

C12

C14

C16

C18

3

0

0.12

0.13

0.68

0.29

0.31

0.49

2.02

24

0.10

0.05

<0.01

<0.01

<0.01

<0.01

<0.19

48

<0.01

0.01

<0.01

<0.01

<0.01

<0.01

<0.05

72

<0.01

<0.01

<0.01

<0.01

<0.01

<0.01

<0.05

10

0

0.44

0.45

2.62

1.04

1.72

1.64

7.91

24

0.43

0.41

0.97

0.01

<0.01

0.18

1.95

48

<0.01

0.01

<0.01

<0.01

<0.01

<0.01

<0.05

72

<0.01

0.01

<0.01

<0.01

<0.01

<0.01

<0.05

30

0

1.45

1.48

8.88

3.62

6.87

6.78

29.2

24

1.38

1.39

5.28

0.59

0.97

2.01

11.6

48

<0.01

0.01

<0.01

<0.01

<0.01

<0.01

<0.05

72

<0.01

0.01

<0.01

<0.01

<0.01

<0.01

<0.05

100

0

4.46

4.56

28.2

11.1

21.1

20.4

89.8

24

4.12

4.28

20.1

3.30

4.15

6.10

42.1

48

<0.02

0.02

<0.02

<0.01

<0.02

<0.33

<0.10

72

<0.01

<0.01

<0.01

<0.01

0.16

1.02

<1.17

300

0

13.2

13.4

84.1

37.3

63.6

60.5

272

24

13.3

13.8

72.5

24.9

19.2

14.0

158

48

12.7

9.66

8.58

1.15

5.27

21.6

59.0

72

11.4

3.17

<0.01

0.14

1.50

26.3

42.5

Applicant's summary and conclusion

Conclusions:
In this study the effects of the test item SLI(76) stripped on the growth of the freshwater green alga Pseudokirchneriella subcapitata were tested. The EC50-values for specific growth rate inhibition (ErC50) and inhibition of yield (EyC50) after 72 h were >= 1.87 mg/L (dissolved measured concentration). The NOEC values were >= 0.31 mg/L (dissolved measured concentration).
Executive summary:

This study was used to determine the 72-hour EC50 and NOEC (No Observed Effect Concentration) of SLI (76) stripped toPseudokirchneriella subcapitata.

 

SLI (76), provided by Unilever Mannheim was stripped of its fatty acid content via Soxhlet extraction at the Materials Science Laboratory, Trumbull, USA. The resulting material, SLI (76) stripped was used for this study.

 

Prior to testing the solubility of the test item and its individual actives were determined by Liquid Chromatography/Mass Spectroscopy (LC/MS) by the Chemical Analysis.

 

The test item has limited solubility in water and ISO media. There are significant losses of the test item over a 72 hr period. At 0 hours the measured concentration of each chain length dissolved in solution is close to the expected nominal concentration taking into account the chain length distribution of the test item. The concentrations of C8, C10and C12remain stable over the test period however there is a significant decrease in the concentration of chain lengths ≥ C14in solution. Comparisons between solutions with and without algae reveal a significant decrease in the total concentration remaining at 72 hours when algae is present (30% of the 100 mg/L solution remaining compared to 65% for the same concentration without algae).

 

For the definitive study a series of test solutions were preparedby diluting a 1000 mg/L stock solutionwith ISO media to give final test media concentrations of 0, 3, 10, 30, 100 and 300 mg/L. A calculated aliquot ofPseudokirchneriella subcapitatainoculum was transferred into each test solution to give a nominal algal density of 104cells/mL. Cell concentration was determined at 48 and 72 hours by haemocytometer. Test concentrations were determined every 24 hours by Liquid Chromatography/Mass Spectroscopy (LC/MS).

In this study the effects of the test item SLI(76) stripped on the growth of the freshwater green alga Pseudokirchneriella subcapitata were tested. The EC50-values for specific growth rate inhibition (ErC50) and inhibition of yield (EyC50) after 72 h were >= 1.87 mg/L (dissolved measured concentrations). The NOEC values were >= 0.31 mg/L (dissolved measured concentrations).