Alcohols, C16-18 and C18-unsatd., ethoxylated, sulfates, sodium 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:

supporting study

Study period:

01 - 11 Dec 2015

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: separate replicates for the test item analysis after 0 and 72 h were prepared without algae and incubated under the same test conditions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: test container was heated at 35 - 40 °C and homogenized by gently shaking before use. Stock solution of 100 mg/L was freshly prepared with dilution water and gently agitated until the solution was clear. Test solutions were prepared by dilution.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: HINDAK, SAG 61.81
- Source (laboratory, culture collection): Sammlung von Algenkulturen (SAG) Pflanzenphysiologisches Institut der Universität Göttingen, Germany
- Age of inoculum (at test initiation): 4 d
- Method of cultivation: fresh stocks are prepared every month of Z-Agar. Light intensity amounted to 35 - 70 µE x m^-2 x s^-1 for 24 h/day. Culture medium: nutrient medium Z according to Lüttge et al. 1994, Botanica Acta, Journal of the German Botanical Society, No. 3 Volume 107 page 111-186, Thieme Verlag

ACCLIMATION
- Acclimation period: 4 d
- Culturing media and conditions (same as test or not): same as test

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

22.3 CaCO3 mg/L

Test temperature:

21 - 22.5 °C

pH:

7.5 - 8.86 (in vessels with test item)
8.04 - 9.00 (in control)

Nominal and measured concentrations:

Nominal concentrations: 0 (control), 0.625, 1.25, 2.50, 5, 10, 20 and 40 mg/L test item; corresponding to 34.3% a.i.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL sterile Erlenmeyer flasks with cotton wool plugs
- Fill volume: 100 mL
- Initial cells density: 6153 cells/mL actual (5 x 10^3 - 10^4 nominal cells/mL)
- Control end cells density: 1986581 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline
- Total organic carbon: 18.4% (at test initiation)
- Active ingredient: 34.3% non - aqueous components
- Alkalinity: 0.6 mmol/L
- Conductivity: 158 µS/cm
- Culture medium different from test medium: yes (for more details see details on test organism)
- Intervals of water quality measurement: pH was measured in one additional replicate of each item concentration and the control at the beginning of the test. At the end of the test pH was measured in a pooled sample per test item concentration and the control. Temperature was measured continuously while light intensity was measured prior to the start of the test.

OTHER TEST CONDITIONS
- Photoperiod: 24 h day light
- Light intensity and quality: 60 - 120 µE x m^-2 x s^-1, light source: fluorescent tubes OSRAM L 36 W/11-865, cool daylight
- Agitation: 70 rpm on a rotary shaker

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: microscopically, at the beginning and at the end of the test
- Chlorophyll a measurement: cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm (dilution water used as background signal). No auto fluorescence was observed in any test concentration at test initiation.

TEST CONCENTRATIONS
- First definitive test: 0.625, 1.25, 2.50, 5, 10 mg/L (nominal test item concentrations) caused 0, 1, 6, 18, 24% growth rate inhibition, respectively. Test was followed by a range finding test as the tested concentrations were too low for the calculation of EC50 value.
- Range finding study: yes, after a first definitive test with too low concentrations
- Test concentrations: 0 (control), 1, 10, 100 mg/L (corresponding to <2, <2, <2, 11.8 mg C/L at test initiation and <2, <2, 2.46 and 14.7 mg C/L at test end). LOQ = 2 mg/L
- Results used to determine the conditions for the definitive study: 1, 10 and 100 mg/L test item concentrations resulted to -4, 100 and 100% growth rate inhibition, respectively.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.606 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 0.467 - 0.762 mg/L

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

1.77 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 1.36 - 2.22 mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

3.19 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 2.48 - n.a. mg/L

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

9.31 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 7.24 - n.a. mg/L

Details on results:

- Exponential growth in the control (for algal test): yes, 323-fold
- Flocculation: the substance has surface active properties and it is assumed that at concentrations over 10 mg/L the test item forms micelles and is not completely dissolved.

Results with reference substance (positive control):

- Results with reference substance valid: yes
- EC50: 0.564 (95% CI: 0.546 - 0.582 mg/L)

Reported statistics and error estimates:

Since test item is an UVCB all effect values are based on nominal concentrations.
Effect values were calculated by sigmoidal dose-response regression. 95% confidence limits were calculated from the standard error and the t-distribution. The two highest test concentrations of 20 and 40 mg/L were omitted for the calculations of ECx-values in order to obtain a better fit.

ErC20 (72 h) = 0.966 mg/L (nominal, act. ingr., 95% CI: 0.822 - 1.11 mg/L)

ErC20 (72 h) = 2.82 mg/L (nominal, test mat., 95% CI: 2.40 - 3.23 mg/L)

Table 1: % of growth rate inhibition at the different concentrations.

Nominal test item concentration [mg/L]	Replicate	Growth rate inhibition [%]
40	1	51
	2	48
	3	47
	mean	48
20	1	46
	2	44
	3	45
	mean	45
10	1	58
	2	46
	3	48
	mean	51
5	1	37
	2	39
	3	37
	mean	38
2.5	1	15
	2	15
	3	20
	mean	16
1.25	1	10
	2	5
	3	7
	mean	7
0.625	1	3
	2	0
	3	5
	mean	3

In the lower concentrations the background from the TOC values in the control did not allow a reliable calculation of the concentration of the test item (below the LOQ). TOC of the test item could be determined with sufficient reliability only in the 3 highest test concentrations at the start of the exposure and in the 2 highest concentrations at the end of the exposure.

Table 2: TOC content of the test item at the start and at the end of the exposure.

Nominal test item conc. [mg/L]	Nominal TOC conc. [mg C/L]	TOC [mg C/L]
		Test start (without algae)			Test end (without algae)
		Measured TOC	Calculated TOC	% of nominal	Measured TOC	Calculated TOC	% of nominal
40	7.36	7.06	6.18	84	6.02	4.97	67
20	3.68	4.12	3.24	88	3.79	2.74	74
10	1.84	2.89	2.01	109	2.38*	1.33	n.a.
5	0.92	2.17	1.29	n.a.	1.96*	0.91	n.a.
2.5	0.46	1.7	0.822	n.a.	1.95*	0.9	n.a.
1.25	0.23	2.05	1.17	n.a.	2.19*	1.14	n.a.
0.625	0.115	1.44	0.562	n.a.	1.67*	0.62	n.a.
Control	-	0.0878	-	-	1.05*	-	-

* =< LOQ

Table 3: Validity criteria for OECD 201 (2006)

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.	323	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%	13.4%	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 withPseudokirchneriella subcapitataandDesmodesmus subspicatus. For other less frequently tested species, the value should not exceed 10%.	1.82%	yes

 

Validity criteria fulfilled:

yes

Remarks:

Please refer to table 3 at "any other information on results incl. tables.

Reference 2

Endpoint:

effects on growth of green algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 - 29 June 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

adopted 23 March 2006, Annex 5 corrected 28 July 2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentrations including the control
- Sampling method: Samples (5 mL) were taken from the control and test concentrations at 0 (non-inoculated fresh media), 24, 48 and 72 (old media) for analysis. Inoculated samples were taken from pooled replicates and non-inoculated samples were taken from individual replicates. The samples were taken using an air displacement pipette fitted with a plastic pipette tip into 20 mL glass scintillation vials containing 5 mL of methanol.
- Sample storage conditions before analysis: no, samples were analysed on the day of receipt

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A 10 mg a.i./L solution was prepared by adding 29.94 mg of test substance to 1000 mL of EC medium and then mixed well. The test substance was mixed well before use. The stock solution was checked for the tyndall effect using a Hach 2100 N turbidity meter. The test solutions were prepared by dilution of the stock solution.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: 278/4
- Source (laboratory, culture collection): cultured at testing facility, originally obtained from the Culture Collection of Algae and Protozoa (CCAP)
- Age of inoculum (at test initiation): Prior to testing, duplicate starter cultures were prepared and incubated under test conditions to obtain sufficient algal cells in exponential growth and to achieve a starting alga cell density of 1 x 10^04 cells/mL.

ACCLIMATION
- Culturing media and conditions (same as test or not): same

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.4 - 23.3°C

pH:

6.89 – 7.90

Nominal and measured concentrations:

Nominal test concentrations: 0.05, 0.1, 0.2, 0.4 and 1 mg a.i./L
Calculated exposure concentrations: 0.0196, 0.0393, 0.0655, 0.181, 0.849 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: conical flasks
- Type (delete if not applicable): capped with foam bungs
- Material, size, headspace, fill volume: glass, nominal 250 mL, fill volume 100 mL
- Aeration: no
- Initial cells density: 10000 algae cells/mL
- Control end cells density: 793840 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes, OECD TG 201 medium according to Annex 3

TEST MEDIUM / WATER PARAMETERS
- Intervals of water quality measurement: 0, 24, 48 and 72 h

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: constant light
- Light intensity and quality: 4410 - 6620 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: electronic particle counter, every 24 h

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: , 0.1, 1 and 10 mg a.i./L
- Results used to determine the conditions for the definitive study: ErC50 and EyC50: > 0.1 - < 1 mg a.i./L.

Reference substance (positive control):

yes

Remarks:

3,5 Dichlorophenol

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.386 mg/L

95% CI:

<= 0.601

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

based on nine representative constituents

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.099 mg/L

95% CI:

>= 0.07 - <= 0.163

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

based on nine representative constituents

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.066 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Remarks:

based on nine representative constituents

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Unusual cell shape: After 72 h green, sickle shaped cells were observed at 0.0393 mg/L, pale green, few sickle shaped cells were observed at 0.181 mg/L and colourless, very few sickle shaped cells. Few swollen and few small cells at 0.849 mg/L
- Colour of test medium: After 72 h the colour of the test medium was green at 0.0393 mg/L, at 0.181 mg/L the test medium was Pale green, Colourless/clear, Green in replicates R1, R2 and R3 respectively. At 0.849 mg/L the test medium was colourless

Results with reference substance (positive control):

- Results with reference substance valid? yes
- ErC50: 2.08 mg/L
- Other: experimental test dates: 20-23 Dec 2021

Reported statistics and error estimates:

The average specific growth rate (μ) at 48 and 72 hours and the final yield over 72 hours were analysed using Jonckheere-Terpstra Step-Down test to determine the no observed effect concentration (NOEC). A Jonckheere-Terpstra Step-Down test was used because the data was continuous, monotonic and non-parametric. The average specific growth rate (μ) at 24, 24-48 and 48-72 hours were analysed using Williams Multiple Comparison test to determine the no observed effect concentration (NOEC). A Williams Multiple Comparison test was used because the data was continuous, monotonic and parametric.
Linear interpolation analysis was performed in order to estimate EC10, EC20 and EC50 values. Where possible, 95 % confidence limits were calculated for the EC10, EC20 and EC50 values.
The data was assessed for outliers using grubbs extreme value method. No consistent outliers were found.

Table 1: Growth rate of Raphidocelis subcapitata cultures exposed to Alcohols, C16-18 and C18-unsatd., ethoxylated, sulfates, sodium salts during the Definitive Test

Nominal Concentration [mg a.i./L]		Calculated exposure concentrations [mg a.i./L]	Average Specific Growth Rate [µ] [µ10^-2/hr]				Reduction in Average Specific Growth Rates Relative to the Control [%]
			0-24 hours		0-48 hours	0-72 hours		0-24 hours		0-48 hours		0-72 hours
Control		Control	4.383	5.879		6.086	NA		NA		NA
0.05		0.0196	4.412	5.923		6.093	-0.66		-0.75		-0.12
0.1		0.0393	4.221	5.882		6.076	3.70		-0.05		0.16
0.2		0.0655	4.613	5.891		6.071	-5.25		-0.20		0.25
0.4		0.181	3.439	3.839		4.080	21.54		34.70		32.96
1		0.849	2.272	1.763		1.180	48.16		70.01		80.61

NA: Not Applicable
Negative percentage inhibition values relative to the control represent an increase in growth relative to the control

Table 2: Geometric mean measured concentrations and Calculated Concentrations during the definitive test

Nominal Concentration (mg a.i./L)	Summed Concentration of Geomeans of Measured Components (mg/L)	Calculated Test Substance Concentration (mg a.i./L)1	Percentage of Nominal (%)
0.05	0.0126	0.0196	39.2
0.1	0.0253	0.0393	39.3
0.2	0.0421	0.0655	32.8
0.4	0.117	0.181	45.3
1.0	0.546	0.849	84.9

 

Table 3: Validity criteria for OECD 201 (2006)

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.	79	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%	27%	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%.	2.6 %	Yes

Validity criteria fulfilled:

yes

Remarks:

Please refer to table 3 at "Any other information on results incl. tables"

Description of key information

ErC50 (72h): 0.386 mg a.i./L (meas. geom. mean, OECD 201)

ErC10 (72h): 0.0985 mg a.i./L (meas. geom. mean, OECD 201)

Additional information

The assessment is based on the data currently available. New studies, based on the category review and the final decisions issued for some of the category substances, which are also relevant for this assessment, are currently being conducted. The hazard assessment with respect to aquatic toxicity will be updated once all ongoing studies have been finalised.

The toxicity of AES - Alcohols, C16-18 and C18-unsatd., ethoxylated, sulfates, sodium salts to aquatic algae was investigated in an OECD 201 guideline study (GLP). The green algae Raphidocelis subcapitata was exposed to nominal test substance concentrations of 0.05, 0.1, 0.2, 0.4 and 1 mg a.i./L. The test concentrations were analytically monitored by LC-TQMS. Nine constituents of the UVCB substance were targeted in the analysis. The geom. mean test concentrations were 0.0196, 0.0393, 0.0655, 0.181, 0.849 mg/L. The determined ErC50 (72h) was 0.386 mg a.i./L (meas. geom. mean). The ErC10 (72h) was 0.0985 mg a.i./L (meas. geom. mean).

A supporting GLP study was performed according to OECD guideline 201 with Pseudokirchnerella subcapitata as test species. Seven test item concentrations were tested in a geometric series with a dilution factor of 2 (0.625, 1.25, 2.5, 5, 10, 20 and 40 mg/L test item; corresponding to 34.3% a.i.). The test concentrations were verified by TOC measurement. The test item concentration could be determined with sufficient reliability only in the three highest test concentrations at the start of the exposure and in the 2 highest concentrations at the end of the exposure. For these concentrations, all values determined were within the range of ± 20% of the nominal TOC concentrations at the start of exposure and decreased to values of 68 to 74% of the nominal value at the end of exposure. The test item was found to inhibit the growth of the green algae over the period of 72 hours. Based on growth inhibition the ErC50 (72h) was 3.19 mg/L a.i. (nominal) and the ErC10 (72h) was determined to be 0.606 mg/L a.i. (nominal).