Fatty acids, C12-18 and C18-unsatd., 2-sulfoethyl esters, sodium salts

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

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 concentrations

Effect concentrations 1

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

Effect concentrations 2

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)

Effect concentrations 3

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

Effect concentrations 4

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)

Effect concentrations 5

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

Effect concentrations 6

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

Effect concentrations 7

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

Effect concentrations 8

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).