C16-18 (evennumbered, C18 unsaturated) alkyl bis(2-hydroxyethyl) amine oxide

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2018-01-08 to 2018-01-11 at the test facility.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

All concentration levels and the control were analytically verified via LC-MS/MS at the start (0 hours) and at the end of the exposure (72 hours) with algae. The samples were analysed with a LC-MS/MS method, implemented under non-GLP and documented in the GLP raw data. The method was validated.

Vehicle:

no

Details on test solutions:

Stock solution, media preparation
The test item was warmed up to 50 °C before used for preparation of the stock solution. A stock solution with a nominal test item concentration of 1000 mg/L was freshly prepared with demineralized water and treated with ultrasound for 30 minutes at 50 °C.

Test concentrations Out of the stock solution five concentration levels were tested in a
(nominal) geometrical series with a dilution factor of 2: 0.0625 - 0.125 - 0.250 - 0.500 - 1.00 mg/L. The concentration levels are based on the results of a preliminary range finding test.

Control Six replicates (without test item) were exposed under the same conditions as the test loadings.

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata HINDÁK
- Strain: SAG 61.81
- Source (laboratory, culture collection): SAG, Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, D-37073 Göttingen
- Age of inoculum (at test initiation): A three day old preculture, prepared in dilution water, was used as inoculum.
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 2567 – 5130 lux corresponding to 35 - 70 µE ∙ m-2 ∙ s-1 for 24 h per day.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

No

Hardness:

Not specified

Test temperature:

min.: 22.0 max.: 22.5 mean value: 22.3

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
1.00 7.71 8.01
0.500 7.74 8.18
0.250 7.80 8.71
0.125 7.87 8.96
0.0625 7.96 9.09
Control 8.01 9.14
Room temperature [°C]: min.: 22.0 max.: 22.5 mean value: 22.3

Light intensity
[lux] n = 20 mean value: 4779
range of the measured values: 4517 to 5032
equalling -5.48 to 5.29% CV [%]: 3.37

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 5 x 103 - 104 cells/mL. Actual: 6839 cells/mL

- Control end cells density: Mean 2842582 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:
Composition of Dilution Water
according to the guideline
Component Concentration [mg/L]
NH4Cl 15
MgCl2 x 6 H2O 12
CaCl2 x 2 H2O 18
MgSO4 x 7 H2O 15
KH2PO4 1.6
FeCl3 x 6 H2O 0.064
a2EDTA x 2 H2O 0.1
H3BO3 0.185
MnCl2 x 4 H2O 0.415
ZnCl2 3 x 10^-3
Na2MoO4 x 2 H2O 7 x 10^-3
CoCl2 x 6 H2O 1.5 x 10^-3
CuCl2 x 2 H2O 1 x 10^-5
NaHCO3 50
pH 8.1 ± 0.2

This medium had a nominal hardness of 0.24 mmol Ca+Mg/L.


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: Approximately 4440 to 8880 lux, corresponding to 60 to 120
µE ∙ m-2 ∙ s-1, mean value: 4779 lux,



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Chlorophyll a-fluorescence
The cell density was measured daily via Chlorophyll a-fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as a background signal. Self-fluorescence was observed in the saturated solution. A separate replicate of each test item concentration were prepared without algae and treated like the other test replicates. The self-fluorescence of each measuring point was taken into account in the evaluation.

Reference substance (positive control):

yes

Remarks:

(Potassium dichromate)

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.111 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: CI (0.0977 - 0.127)

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

0.039 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: CI (0.0249 - 0.0511)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

0.038 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.074 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.058 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: CI (0.0482 - 0.0693)

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.014 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield Inhibition

Duration:

72 h

Dose descriptor:

LOEC

Effect conc.:

0.014 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield Inhibition

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 0.014 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield Inhibition

Duration:

72 h

Dose descriptor:

EC20

Effect conc.:

0.023 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield Inhibition

Remarks on result:

other: CI (< 0.0143 - 0.0357)

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.047 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: Yield Inhibition

Remarks on result:

other: CI (0.0389 - 0.0567)

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: All test solutions were visually clear throughout the exposure period, any effects directly related to algae growth not taken into account.
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

The toxicity of potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0 %, CAS RN 7778-50-9) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined over a period of 72 hours from 2017-10-10 to 2017-10-13. The reference item toxicity is in the valid range following test facility SOPS.

EC50-Values of the Reference Item
based on nominal concentrations mg/L, (0-72 hours)
Current Study Valid Range (average ± 3 x SD)
Growth Rate inhibition
ErC50 0.535 0.745 ± 0.524
95 % confidence interval 0.523 – 0.547
Yield inhibition
EyC50 0.320 0.398 ± 0.291
95 % confidence interval 0.249 – 0.339
SD = Standard deviation

Reported statistics and error estimates:

EC-values and statistical analyses
EC10-, EC20- and EC50-values with confidence intervals of growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression.

NOEC- and LOEC-values The NOEC / LOEC was determined by calculation of statistical significance of biomass integrals, growth rate and yield.
The following statistical tests were conducted:

Shapiro-Wilk’s test on normal distribution was done with a significance level 0.01 for growth rate and yield.

Levene’s test on variance homogeneity was done with a significance level 0.01 for growth rate and yield.

Welsh-t-test after Bonferroni-Holm was done with a significance level 0.05 for growth rate and yield.



Software The data for the tables in this report were computer-generated and have been rounded for presentation from the fully derived data. Consequently, if calculated manually based on the given data minor deviations may occur from these figures.
Calculations were carried out using software
- Excel, MICROSOFT CORPORATION
- SigmaPlot, SPSS INC.
- ToxRat, ToxRat Solutions GmbH
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.

Cell Densities

Nominal test item concentration	Replicate	Cell density [cells/mL]
[mg/L]	No.	0 hours	24 hours	48 hours	72 hours
1.00	1	6839	11137	14965	8796
	2	6839	16395	14385	10002
	3	6839	16670	22519	14953
	Mean	6839	14734	17290	11250
0.500	1	6839	23573	37337	31438
	2	6839	26411	49535	48725
	3	6839	24734	48660	43827
	Mean	6839	24906	45177	41330
0.250	1	6839	28333	87240	251628
	2	6839	31848	100091	380665
	3	6839	34703	151904	928470
	Mean	6839	31628	113078	520254
0.125	1	6839	32954	198843	1413192
	2	6839	34741	238602	2468541
	3	6839	36968	337734	1580208
	Mean	6839	34888	258393	1820647
0.0625	1	6839	42864	357854	2207191
	2	6839	41199	351580	2432844
	3	6839	43300	395658	2422779
	Mean	6839	42454	368364	2354271
Control	1	6839	42612	432947	3006750
	2	6839	44040	354939	2876192
	3	6839	49664	432104	2693024
	4	6839	47247	441561	2815623
	5	6839	41015	456144	2874553
	6	6839	43148	393598	2789348
	Mean	6839	44621	418549	2842582

Evaluation after 72 hours

Statistically significant differences of growth rates and yield compared to control values are marked (+), not significant differences are marked (-). 

Nominal test item concentration	Replicate	Growth rate		Inhibition of growth rate	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
1.00	1		0.084	96		1957	100
	2		0.127	94		3163	100
	3		0.261	87		8114	100
	Mean	(+)	0.157	92	(+)	4411	100
0.500	1		0.508	75		24599	99
	2		0.655	67		41886	99
	3		0.619	69		36988	99
	Mean	(+)	0.594	70	(+)	34491	99
0.250	1		1.20	40		244789	91
	2		1.34	33		373826	87
	3		1.64	19		921631	68
	Mean	(+)	1.39	31	(+)	513415	82
0.125	1		1.78	12		1406353	50
	2		1.96	2		2461702	13
	3		1.81	10		1573369	45
	Mean	(-)	1.85	8	(+)	1813808	36
0.0625	1		1.93	4		2200352	22
	2		1.96	3		2426005	14
	3		1.96	3		2415940	15
	Mean	(+)	1.95	3	(+)	2347432	17
Control	1		2.03			2999911
	2		2.01			2869353
	3		1.99			2686185
	4		2.01			2808784
	5		2.01			2867714
	6		2.00			2782509
	Mean		2.01			2835743

Section-by-Section and Average Specific Growth Ratesof the Control Group (0 - 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 hours)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.83	2.32	1.94	2.03	0.257	12.7	10.7
	2	1.86	2.09	2.09	2.01	0.131	6.5
	3	1.98	2.16	1.83	1.99	0.167	8.4
	4	1.93	2.24	1.85	2.01	0.202	10.0
	5	1.79	2.41	1.84	2.01	0.343	17.0
	6	1.84	2.21	1.96	2.00	0.189	9.4
				Mean	2.01
				SD ±	0.01
				CV [%]	0.61

SD = Standard deviation CV = Coefficient of variation

Measured Exposure Concentrations during the Definitive Test

 

The concentrations of three components ofTallowBis(2-hydroxyethyl) amineoxide CASno. 61791-46-6were determined in fresh media (0 hours) and old media (72 hours) of all tested concentration levels and the control via LC-MS/MS.

 

The measured concentrations of the of three components were between 47% and 67% of the nominal value at the start of the exposure (0 hours) and between 35% and 89% of the nominal value at the end of the exposure (72 hours). The geometric mean measured concentration of total active substance was calculated.

 

Measured Concentrations and Recovery Rates of Test Item for Component 346 Da in Fresh Medium (0 hours) and Old Medium (72 hours) with Algae

Sampling date		0 hours		72 hours
Nominalconcentrationof the		Tallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6 (346 Da - C16:0)
test item   [mg/L]	a.s.   [mg/L]	Meas. conc. a.s. [mg/L]	[%]	Meas. conc. a.s. [mg/L]	[%]	Geometric Mean Measured Concentration of the active substance [mg/L]
1.00	0.151	0.102	67	0.131	87	0.116
0.500	0.0755	0.0461	61	0.0669	89	0.0555
0.250	0.0378	0.0228	60	0.0261	69	0.0244
0.125	0.0189	0.0107	56	0.0145	77	0.0125
0.0625	0.00944	0.00463	49	0.00481	51	0.00472
Control		< LOQ		< LOQ

Meas. Conc.   = measured concentration of the active substance (dilution and weighing factor taken into account)

a.s.                  = active substance

%                     = percentage of the nominal concentration of the active substance

LOQ           = limit of quantification of the analytical method (15 µg test item /L, corresponding to 2.27 µg a.s./L)

Validity criteria fulfilled:

yes

Conclusions:

In this study, Tallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6 was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on geometric mean measured concentrations): The EC50-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 0.111 (0.0977 – 0.127) mg/L and 0.0473 (0.0389 – 0.0567) mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were 0.0383 mg/L and < 0.0143 mg/L, respectively.
All effect levels presented here are based on active ingredient concentrations.

Executive summary:

The toxicity of Tallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6 (Batch no.:1548134) to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 andCouncil Regulation (EC) No. 266/2016/Method C.3from 2018-01-08 to 2018-01-11 at the test facility.The aim of the study was the determination of NOEC, LOEC, EC₁₀- , EC₂₀- and EC₅₀-values of growth rate and yield over a period of 72 hours.

 

The test item Tallow Bis (2-hydroxyethyl) amineoxide CAS no. 61791-46-6is an light yellow paste UVCB substance, which is dispersible in water.The study was conducted under static conditions with an initial cell density of 6839 cells/mL. The test item was warmed up to 50 °C before used for preparation of the stock solution. A stock solution of 1000 mg/L was freshly prepared with demineralized water and treated with ultrasound for 30 minutes at 50 °C. Five concentrations were tested in a geometrical series with a dilution factor of 2:0.0625 - 0.125 - 0.250 - 0.500 - 1.00 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditionswere within the acceptable limits.

 

All concentration levels and the control were analytically verified by LC-MS/MS at the start and the end of the exposure.The concentration levels of the test item Tallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6 at the start of the exposure (0 hours) were in the range of 47 to 67% of the nominal values.The measured concentrations ofthe three compoundsof the test item at the end of the exposure (72 hours) were in the range of 35 to 89% of the nominal value.

 

Since the test item is labeled as an UVCB and complex mixture, all effect values given are based on the nominal test item concentrations of Tallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6 and on the geometric mean measured concentrations of the three compounds of the test item.

NOEC, LOEC, EC-values and 95% Confidence Intervals of Tallow Bis (2-hydroxyethyl) amineoxide CAS no. 61791-46-6(0 - 72 hours)

                  based on geometric mean measured concentrations of the total active substance and the nominal test item concentrations [mg/L]

	Growth Rate Inhibition geometric mean measured concentration of the total active substance [mg/L]	Growth Rate Inhibition Nominal test item concentration [mg/L]
NOEC	0.0383	0.125
LOEC	0.0744	0.250
ErC10	0.0394 (0.0249 – 0.0511)	0.138 (0.0904 – 0.176)
ErC20	0.0584 (0.0482 – 0.0693)	0.198 (0.165 – 0.232)
ErC50	0.111 (0.0977 – 0.127)	0.350 (0.314 – 0.393)
	Yield Inhibition geometric mean measured concentration of the total active substance [mg/L]	Yield Inhibition Nominal test item concentration [mg/L]
NOEC	< 0.0143	< 0.0625
LOEC	0.0143	0.0625
EyC10	< 0.0143	< 0.0625
EyC20	0.0226 (< 0.0143 – 0.0357)	0.0783 (< 0.0625 – 0.116)
EyC50	0.0473 (0.0389 – 0.0567)	0.155 (0.127 – 0.187)

Description of key information

The toxicity of C16 -18 (evennumbered, C18 unsaturated) alkyl bis(2 -hydroxyethyl) amine oxide to algae was observed to be ErC50 (72h) of 0.111 mg a.i./L and ErC10 (72h) of 0.0394 mg a.i./L based on Geometric mean measured concentrations.

Key value for chemical safety assessment

EC50 for freshwater algae:

0.11 mg/L

EC10 or NOEC for freshwater algae:

0.039 mg/L

Additional information

Two algae studies have been performed with Tallow Bis(2-hydroxyethyl) amineoxide

The first study is a repetition of the second study as this second study from 1990 was performed at a time when no suitable analytical method was available. The results of the first study are used for risk assessment and C&L as this study result has a higher reliability.

In the study from Klix (2018), the toxicity of Tallow Bis(2 -hydroxyethyl) amineoxide to Pseudokirchneriella subcapitata was determined according to OECD 201 and EU Method C.3 in compliance with GLP. 

In this test triplicate flasks with five concentrations were tested in a geometrical series with a dilution factor of 2: 0.0625 - 0.125 - 0.250 - 0.500 - 1.00 mg/L nominal test concentrations of the test substance for 72 h under static conditions. Biomass and growth rate were determined after 0, 24, 48 and 72 h.  

All concentration levels and the control were analytically verified by LC-MS/MS at the start and the end of the exposure. The concentration levels of Tallow Bis(2-hydroxyethyl) amineoxide at the start of the exposure (0 hours) were in the range of 47 to 67% of the nominal values. The test substance is a UVCB substance and during chemical analysis the three main constituents representing 94.9% were quantified. The effect concentrations were corrected to 100% active ingredient for the calculation of the endpoints. The measured concentrations of the three main constituents at the end of the exposure (72 hours) were in the range of 35 to 89% of the nominal value.

Tallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6 was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on geometric mean measured concentrations): The EC50-values with 95% confidence intervals for inhibition of growth rate (ErC50) and yield (EyC50) after 72 hours were 0.111 (0.0977 – 0.127) mg/L and 0.0473 (0.0389 – 0.0567) mg/L, respectively. The NOEC-values for inhibition of growth rate and yield after 72 hours were 0.0383 mg/L and < 0.0143 mg/L, respectively. All effect levels presented here are based on active ingredient concentrations.

 

In the study from Kroon (1990) the toxicity of C16-18 (evennumbered, C18 unsaturated) alkyl bis(2-hydroxyethyl) amine oxide to the freshwater algae Pseudokirchneriella subcapitata (formally called Selenastrum Capricornutum) was determined according to OECD Guideline 201, EU Method C.3 and ISO Method 8692, in compliance with GLP. In this study, triplicate Erlenmeyers containing the freshwater algae were exposed to 0.01, 0.02, 0.04, 0.08 and 0.16 mg/L nominal concentrations of the test substance for 72 h under static conditions. Biomass and growth rate were determined after 0, 24, 48 and 72 h. Quantification of the applied concentrations was not possible due to the absence of a suitable analytical method. Considering the high water solubility and stability of the test substance, it was considered reasonable to assume that the actual concentrations would be identical to the nominal concentrations during the entire test. All the validity criteria were considered to be fulfilled. Under the study conditions, the 72 h EbC50 and ErC50, based on nominal concentrations were 0.55 and 0.41 mg/L test substance, respectively or 0.28 and 0.21 mg a.i./L. The NOEC of 0.2 mg/L test substance or 0.1 mg a.i./L can be used as long term endpoint (Kroon, 1990).