Alcohols, C10-16, ethoxylated, sulfosuccinates, disodium 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:

02 Jan - 26 Feb 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)

Version / remarks:

2006, corrected 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesanstalt für Umwelt, Messungen und Naturschutz Baden-Württemberg, Karlsruhe, Germany

Test material

Test material information

Constituent 1

Reference substance name:

Alcohols, C10-16, ethoxylated, sulfosuccinates, disodium salts

EC Number:

500-232-7

EC Name:

Alcohols, C10-16, ethoxylated, sulfosuccinates, disodium salts

Cas Number:

68815-56-5

Molecular formula:

not applicable to UVCB

IUPAC Name:

Alcohols, C10-16, ethoxylated, sulfosuccinates, disodium salts

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Analytical samples were taken and analysed in the main test from control and all test item concentrations at 0 hours (initial value) from fresh test solutions and after 24 hours and 72 hours from aged test solutions.
- Sample storage conditions before analysis: All samples were stored deep frozen until they were transferred to the analytical laboratory.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The necessary amount of test item for preparing the stock solution was weighed on a weighing scoop and transferred to a volumetric flask. Test medium was added up to the bench mark and the solution was homogenised by 5 minutes of ultrasonication treatment. Lower test solutions were prepared by dilution of the stock solution with test medium. Algae were added to each solution individually targeting nominal cell densities of 0.5 × 10E+04 cells per mL in each solution.
- Controls: blank control without test material
- Eluate: no
- Differential loading: no
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): After homogenisation by ultrasonic treatment, the solution was clear and transparent, foam formation was observed.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: freshwater green algae
- Strain: SAG 61.81
- Source: MBM Sciencebridge GmbH, Hans-Adolf-Krebs-Weg 1, D-37077 Göttingen, Germany
- Age of inoculum (at test initiation): 3-4 days
- Method of cultivation: The algae are grown semi-continuously in sterile cultures in the laboratory. Old medium is periodically replaced by fresh mineral solution in order to keep the algae in an exponential growth state. Conditions of cultivation: Illumination: continuously (approx. 4440 - 8880 lux at cell culture level or 60 - 120 µEm-2s-1); Temperature: 21 - 24 °C; Culture flasks: 100 mL Erlenmeyer flasks; CO2 supply by shaking on a rotating shaker, approximately 105 rpm

ACCLIMATION
- Acclimation period: 3-4 days
- Culturing media and conditions: same as test

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Test temperature:

22.2 – 23.3 °C

pH:

control: 7.35-7.93
treatment: 7.29-7.96

Nominal and measured concentrations:

nominal concentrations: 100, 31.3, 9.77, 3.05, 0.954 mg/L and control
measured concentrations: 88.6-89.9, 23.2-26.9, 5.73-7.74, 0.781-1.95, 0.205-0.703 mg/L and < LOD for the control

Details on test conditions:

TEST SYSTEM
- Test vessel: Erlenmeyer flasks (100 mL) with aluminium caps were filled up with ~ 50 mL test solution
- Initial cells density: 0.5 × 10E+04 (targeted)
- Control end cells density: 52.57 × 10E+04
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: AAP-Medium (according to Annex 3 of OECD 201)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Intervals of water quality measurement: Measurements of pH-value were performed at t = 0 h and t = 72 h, the temperature was measured continuously and recorded at hours 0, 24, 48 and 72 h.

OTHER TEST CONDITIONS
- Adjustment of pH: pH was adjusted to 7.5 ± 0.1 with NaOH or HCl, if necessary
- Photoperiod: continuous light
- Light intensity and quality: 88.8 µEm-2s-1 (mean)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: fluorescence measurement (fluorescence microplate reader (infinite 200Pro) with an emission wavelength of 670 nm and evaluated with Tecan i-control (Software for Tecan Readers Tecan i-control, 1.11.1.0)). Additionally, the morphological appearance of the algae cells was observed microscopically at the end of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: yes
- Test concentrations in range finding study: Control, 100, 10.0, 1.00, 0.100 and 0.0100 mg/L
- Results used to determine the conditions for the definitive study: After 72 h at termination of the test a concentration response relation was observed for the inhibition of growth rate and yield from nominal test item concentrations of 1.00 to 100 mg/L. The inhibition of growth rate and the inhibition of yield peaked in >100 % at a nominal test item concentration of 100 mg/L.

Reference substance (positive control):

yes

Remarks:

Potassium Dichromate; tested regularly

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.38 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 2.92-3.92 mg/L

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 1.18-1.80 mg/L

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.76 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 1.57-2.09 mg/L

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.992 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: yield

Remarks on result:

other: 95% CI: 0.787-1.13 mg/L

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.462 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Effect concentrations 6

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

1.4 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): normal for the control and up to and including a nominal test item concentration of 0.954 mg/L. No cells were observed at 3.05 mg/L nominal test item concentration and above.

For further details on biological results, please refer to section "Any other information on results incl. tables".

Results with reference substance (positive control):

EC50 (growth rate) = 0.971 mg/L
EC50 (yield) = 0.622 mg/L

Reported statistics and error estimates:

The statistical evaluation for the 72 hours period was performed for growth rate and yield using SAS® (2002–2010). A test for normality of the data was performed by calculating the Shapiro-Wilk statistic and the homogeneity of variance of the data was evaluated by calculating the Levene Test. The NOEC and LOEC were determined by using a multiple comparison method (Dunnets-ttest, left sided, for growth rate and for yield). The EC10, 20, 50-values for growth rate and yield were determined by probit analysis following normal and logistic distributions, respectively. Due to statistical reasons any inhibition-values above 100 % were set to 100 and values below 0 % were set to zero. Only concentrations within a clear concentration response relation were used for calculations.

Any other information on results incl. tables

Table 1: Average cell numbers for each sampling time and concentration

Nominal conc.	Average cell numbers [10^4/mL]
[mg/L]	0 h	24 h	48 h	72 h
Control	0.57	1.65	10.40	52.57
0.954	0.57	2.05	15.16	74.60
3.05	0.57	1.36	5.68	36.71
9.77	0.57	0.43	0.32	1.10
31.3	0.57	0.00	0.00	0.00
100	0.57	0.00	0.00	0.00

Table 2: Percentage inhibition of growth rate

Nominal conc.	% Inhibition of growth rate
[mg/L]	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	0.0	0.0	0.0
0.954	-21.7	-13.2	-7.9^2)
3.05	18.7	20.7	8.6 *
9.77	126.7	119.8	85.5 *
31.3	≥ 100^1)	≥ 100^1)	≥ 100 *^1), 3)
100	≥ 100^1)	≥ 100^1)	≥ 100 *^1), 4)

¹⁾Due tocell numbers of zerothe value was not calculable. Since the cell number did not increase during the test period the inhibition of growth rate was equal to or above 100.

²⁾Value was set to zero for EC_{10, 20, 50}-calculation

³⁾Value was set to 100 for EC_{10, 20, 50}-calculation

⁴⁾Value was omitted for EC_{10, 20, 50}-calculation

* Statistically significant different to the control

Table 3: Percentage inhibition of yield

Nominal conc.	% Inhibition of yield
[mg/L]	0 h – 24 h	0 h – 48 h	0 h – 72 h
Control	0.0	0.0	0.0
0.954	-37.0	-48.4	-42.4^1)
3.05	26.9	48.0	30.5 *
9.77	113.0	102.5	99.0 *
31.3	152.8	105.8	101.1 *^2)
100	152.8	105.8	101.1 *^3)

¹⁾Value was set to zero for EC_{10, 20, 50}-calculation

²⁾Value was set to 100 for EC_{10, 20, 50}-calculation

³⁾Value was omitted for EC_{10, 20, 50}-calculation

* Statistically significant different to the control

Table 4: Main test: Individual cell numbers

Nominal conc.	Cell numbers [10^4/mL]				Yield
[mg/L]	0 h	24 h	48 h	72 h	0 h – 72 h
	0.50	1.99	10.82	54.14	53.64
	0.59	1.65	10.33	53.61	53.02
Control	0.62	1.47	9.12	41.38	40.76
	0.57	1.82	12.09	60.48	59.91
	0.60	1.72	12.03	60.86	60.26
	0.54	1.22	7.99	44.92	44.38
Mean	0.57^1)	1.65	10.40	52.57	52.00
	0.57	1.99	16.40	72.74	72.17
0.954	0.57	2.10	15.81	77.49	76.92
	0.57	2.05	13.27	73.57	73.00
Mean	0.57	2.05	15.16	74.60	74.03
	0.57	1.71	6.25	50.99	50.42
3.05	0.57	1.19	5.36	28.93	28.36
	0.57	1.18	5.44	30.21	29.64
Mean	0.57	1.36	5.68	36.71	36.14
	0.57	0.41	0.28	1.17	0.60
9.77	0.57	0.50	0.32	1.05	0.48
	0.57	0.39	0.37	1.07	0.50
Mean	0.57	0.43	0.32	1.10	0.53
	0.57	0.00	0.00	0.00	-0.57
31.3	0.57	0.00	0.00	0.00	-0.57
	0.57	0.00	0.00	0.00	-0.57
Mean	0.57	0.00	0.00	0.00	-0.57
	0.57	0.00	0.00	0.00	-0.57
100	0.57	0.00	0.00	0.00	-0.57
	0.57	0.00	0.00	0.00	-0.57
Mean	0.57	0.00	0.00	0.00	-0.57

¹⁾The mean cell density of all control replicates is used as initial cell density for all treatment groups

Table 5: Validity criteria

Criterion from the guideline	Outcome	Validity criterion fulfilled
The biomass in the control cultures should have increased exponentially by a factor of at least 16 within the 72-hour test period.	factor of 92.23	yes
The mean coefficient of variation for section-by-section specific growth rates (days 0-1, 1-2 and 2-3, for 72-hour tests) in the control cultures must not exceed 35%	28%	yes
The coefficient of variation of average specific growth rates during the whole test period in replicate control cultures must not exceed 7% in tests with Pseudokirchneriella subcapitata and Desmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	4.1%; did not exceed 7% for the whole test period	yes

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For further details please refer to “Any other information on results incl. tables”.