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

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2017-11-30 to 2018-03-14, with the definitive exposure phase from 2018-02-13 to 2018-03-07

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All concentration levels and the control were analyzed via LC-MS/MS 3 times during the test (e.g. once within a period of 7 days) in fresh media at the start of an exposure interval (0 hours) and in old media at the end of an exposure interval (48 or 72 hours). The analytical method was implemented under non-GLP and documented finally in the GLP raw data.

Sampling for the analytical monitoring
At the start of the exposure intervals, samples of the fresh media were taken after preparation of all test item concentrations and analyzed.
At the end of the exposure intervals (48 or 72 hours), samples of the old media were taken directly from the test vessels.
For the longest exposure interval of 72 hours samples were taken at the start (0 hours) and at the end of the exposure interval (72 hours) once within the test period. In concentration levels showing an effect of 100% mortality during the test, the respective concentration levels were not analyzed thereafter

Criteria for the analytical monitoring (target)
Recoveries of the test item should be within ± 20% of the nominal or initially measured concentrations.

Vehicle:

not specified

Details on test solutions:

Pre-treatment
For homogenization prior to use of the test item, the test item was heated in a water bath at approximately 35 °C until the test item was a clear liquid.

Preparation of the stock solution
A stock solution of 10.0 mg/L of the test item was prepared with test medium one day prior to each exposure-renewal interval as follows:
20 mg of the test item was weighed out and transferred with 2000 mL of the test medium into a brown glass flask.
The dispersion was shaken for 24 hours with 20 rpm at room temperature. After completion of shaking and following a separation phase of at least 1 hour of standing at room temperature, the aqueous phase was removed by siphoning from the approximate center of the water body.

Test item concentrations Five concentrations of the test item were prepared by dilution of the stock solution of 10.0 mg/L with the test medium and tested at follows:
0.100 - 0.316 - 1.00 - 3.16 - 10.0 mg/L (separation factor √10)

Control 10 replicates of dilution water without test item

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system Daphnia magna STRAUS (Clone 5)

Reason for the selectionof the test system
Daphnia magna is the preferred species in accordance with the
test guideline and is bred at the test facility.
The Daphnia magna culture of Clone 5 has shown to meet the validity criteria for the reproduction test of a mean of ≥ 60 living juveniles per survived parent animal when cultured under the described conditions.

Origin
Institut Dr. Nowak, Mayenbrook 1, 28870 Ottersberg, Germany

Breeder
Noack Laboratorien GmbH,
Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany

Culture
In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2 °C, in an incubator, 16 hours illumination; light intensity of max. 1500 lx

Culture medium
Elendt M4, according to OECD 202, Annex 3 (2004) is used. The composition of the culture medium is presented below.

Composition of the Culture Medium Elendt M4
according to OECD 202, Annex 3 (2004)
Component Concentration [mg/L]
CaCl2 x 2 H2O 294
MgSO4 x 7 H2O 123
KCl 5.80
NaHCO3 64.8
Na2SiO3 x 5 H2O 7.47
NaNO3 0.274
KH2PO4 0.143
K2HPO4 0.184
Na2EDTA x 2 H2O 2.50
FeSO4 x 7 H2O 0.996
H3BO3 2.86
MnCl2 x 4 H2O 0.361
LiCl 0.306
SrCl2 x 6 H2O 0.152
RbCl 0.0710
NaBr 0.0160
Na2MoO4 x 2 H2O 0.0615
CuCl x 2 H2O 0.0168
ZnCl2 0.0130
CoCl2 x 6 H2O 0.0100
KI 0.00325
Na2SeO3 0.00219
NH4VO3 0.000575
Thiaminhydrochloride 0.075
Cyanocobalamin 0.0010
Biotin 0.00075
pH 8.2  0.8



Culture feeding
The culture daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus, with an algae cell density of > 106 cells/mL. The algae are cultured at the test facility.

Origin of the food algae
Sammlung von Algenkulturen (SAG), Pflanzenphysiologisches Institut der Universität Göttingen, Nikolausberger Weg 18, 37073 Göttingen, Germany

Test type:

semi-static

Water media type:

other: Same composition as the culture medium

Limit test:

no

Total exposure duration:

21 d

Hardness:

Nominal test item concentrations
[mg/L] fresh old fresh old fresh old
Day 0 Day 3 Day 10 Day 12 Day 17 Day 19
10.0 249 247 - - - -
3.16 - - 178 235 - -
1.00 - - - - 183 199
Control 253 253 172 249 183 150

The total hardness was above 140 mg/L as CaCO3.

Test temperature:

Nominal test item concentrations
[mg/L] fresh old fresh old fresh old
Day 0 Day 3 Day 10 Day 12 Day 17 Day 19
10.0 20.4 20.7 - - - -
3.16 - - 20.2 20.3 - -
1.00 - - - - 20.4 20.1
Control 20.4 20.7 20.2 20.3 20.7 20.1


The temperature was within the range of 20 ± 1 °C.

pH:

Nominal test item concentrations
[mg/L] fresh old fresh old fresh old
Day 0 Day 3 Day 10 Day 12 Day 17 Day 19
10.0 8.13 8.08 - - - -
3.16 - - 7.92 8.04 - -
1.00 - - - - 7.41 7.21
Control 8.03 8.12 7.84 7.96 7.55 6.95

The pH-values should be in the range of 6 – 9. Significant deviations ≥ 1.5 units between the initial and final pH-values were not observed.

Dissolved oxygen:

Nominal test item concentrations
[mg/L] fresh old fresh old fresh old
Day 0 Day 3 Day 10 Day 12 Day 17 Day 19
10.0 8.23 8.12 - - - -
3.16 - - 9.28 8.29 - -
1.00 - - - - 9.43 8.14
Control 8.59 8.43 9.14 8.45 9.45 7.30


The dissolved oxygen concentration was above 3 mg/L.

Details on test conditions:

Control 10 replicates of dilution water without test item

Test method The study was performed with a semi-static test design.

Renewal of the test solutions
The test solutions were renewed 3 times per week (after 48 or 72 hours). For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred with a pipette (see ‘Application’).

Test duration 21 days

Test vessels Glass beakers (5 (ID) x 8 (H) cm), 100 mL capacity, loosely covered with watch glasses

Test volume 50 mL

Test medium Same composition as the culture medium


Number of daphnids and replicates
10 daphnids in 10 replicates were used for all concentration levels and the control with one daphnid per replicate, which was held individually.

Age of the daphnids at the start of the exposure
Less than 24 hours old daphnids from a healthy stock were used for the study. Juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within this period of max. 24 hours preceding the exposure were used for the test. No first brood progeny was used for the test.

Acclimatization Acclimatization was not necessary, because the composition of the dilution water was equivalent to the culture medium.

Renewal of the test solutions
The test solutions were renewed 3 times per week (after 48 or 72 hours). For this purpose, a second set of test vessels was filled with the freshly prepared test solutions and the daphnids were transferred by pipette (see ‘Application’).

Application 50 g test solution per replicate was weighed out into each test vessel. This corresponds to 50 mL. The daphnids were transferred in a small amount of dilution water or test solution by pipette.

Feeding
Daily; A suspension of Pseudokirchneriella subcapitata and Desmodesmus subspicatus was provided as food corresponding to 0.2 mg C per Daphnia and day.

Test temperature (target) 20 ± 1 °C

Light intensity (target) Max. 1500 lx

Photoperiod 16/8 hours light/dark cycle

Aeration Test vessels were not aerated during the test

Reference substance (positive control):

yes

Key result

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.018 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: CI (0.00880 - 0.0362)

Key result

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.034 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: CI (0.0154 - 0.0787)

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.046 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.015 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

other: LC20

Effect conc.:

0.053 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Adult mortality

Remarks on result:

other: CI (0.0179 - 0.0955)

Key result

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

0.129 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Adult mortality

Remarks on result:

other: CI (0.0679 - 0.257)

Duration:

21 d

Dose descriptor:

other: LC100

Effect conc.:

1.13 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Adult mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.209 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Adult mortality

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.046 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

test mat.

Basis for effect:

other: Adult mortality

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

0.11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: CI (0.0593 - 0.205)

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

0.232 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: CI (0.114 - 0.480)

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

0.316 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

other: LC20

Effect conc.:

0.318 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Adult mortality

Remarks on result:

other: Confidence intervals not determinable

Duration:

21 d

Dose descriptor:

LC50

Effect conc.:

0.667 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Adult mortality

Remarks on result:

other: Confidence intervals not determinable

Duration:

21 d

Dose descriptor:

other: LC100

Effect conc.:

3.16 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Adult mortality

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Adult mortality

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.316 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Adult mortality

Details on results:

- Behavioural abnormalities: Accidental and inadvertent mortality as defined in the OECD test guideline and abnormalities were not observed.
- Observations on body length and weight:see "any other information on results incl. tables"
- Other biological observations: No
- Mortality of control: The average number of living juveniles at the end of the test after 21 days per survived parental daphnid was 93.4 in the control group and thus the validity criterion for the reproduction of a mean of ≥ 60 living offspring per survived parent animal in the control was met.
- Other adverse effects control:No parental daphnids were excluded due to accidental or inadvertent mortality from the evaluation of the reproductive output.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:No
- Effect concentrations exceeding solubility of substance in test medium:No

Results with reference substance (positive control):

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2018-02-27 to 2018-02-28. For results of the most recent of the monthly performed reference tests, see the table below.

EC50-Value (with 95% Confidence Limits) of the Reference Item Potassium dichromate
based on nominal concentrations mg/L, (0 - 24 hours)
Current Study Valid Range
EC50 1.24 mg/L
0.6 - 2.4 mg/L, acc. to AQS P 9/2 (02/2000); clone 5
0.6 - 2.1 mg/L, acc. to OECD 202 (2004); clone A
95% confidence limits < 1.00 - 2.00 mg/L

Reported statistics and error estimates:

Statistical evaluation
The NOEC and LOEC for the reproduction and the adult mortality were determined with the software ToxRat Professional as specified below. Significant deviations of reproduction were determined in comparison to the control using statistical standard procedures as normality test (Shapiro-Wilk’s test), variance homogeneity test (Levene’s test) and a multiple test (Multiple Sequentially-rejective Welsh-t-test after Bonferroni-Holm).
Prior to running the multiple test, a normality test and a variance homogeneity test were performed. P values of the normality and the variance homogeneity test were 0.01. The -value (acceptable probability of incorrectly concluding that there is a difference) was  = 0.01 for the normality test and the variance homogeneity test.
For the effects on reproduction, the juveniles per introduced parent at the start of the test excluding accidental and inadvertent mortality (reproductive output) were calculated. Since no accidental and inadvertent mortality was observed, no parental daphnids were excluded from the evaluation of the reproductive output. The effects on reproduction per survived parental animal at the end of the exposure were also calculated.
For the determination of significant deviations of the adult mortality, the Step-down Cochran-Armitage test was used.
The coefficients of variation around the mean number of living offspring produced per introduced parent and per survived parent in the control and the test groups were evaluated.
All effect levels (NOEC, LOEC, EC10 / 50 and LC20 / 50 / 100) given are based on the nominal and time weighted average test item concentrations.


 

Determination of the EC-/LC-values The EC10 / 50-values for the reproductive output and their 95% confidence limits were calculated by point estimates from the 3 parametric normal CDF with the software ToxRat Professional. The LC20 / 50-values for the adult mortality were calculated by probit analysis with the software ToxRat Professional. The 95% confidence intervals were determined if possible. The LC100 was empirically derived from the observed data. The EC50-value for the reference item was calculated by linear dose-response regression. The respective 95% confidence limits for the EC50-value of the reference item were calculated from the standard error and the t-distribution. These calculations were carried out from the best-fit values with the software GraphPad Prism.

Calculation of the intrinsic rate The intrinsic rate of natural increase (IR) was calculated for the survived parental daphnids with the following EULER-LOTKA-equation with the ToxRat Professional.  (e -A  IR) * Sx * Jx = 1 A= Age IR= Intrinsic rate of natural increase Sx = Survival rate on day x Jx = Reproduction rate on day x The intrinsic rate of natural increase is a measure of population growth which integrates reproductive output and age-specific mortality. In steady state populations, it will be zero. For growing populations, it will be positive and for shrinking populations, it will be negative. Clearly, the latter is not sustainable and ultimately will lead to extinction.

Software

All data are computer-processed and rounded for presentation. Consequently, minor variations may occur from the original figures if manual calculations based on the original figures are made subsequently. Calculations were made using the following software: - Excel, MICROSOFT CORPORATION - GraphPad Prism, GraphPad Software, Inc. - ToxRat Professional, TOXRAT SOLUTIONS GMBH

Reproductive Output 

The number of juveniles in all replicates of the control and the test groups was counted and recorded every day. Results concerning the number of living juveniles of all concentration levels and the control for every test day are presented in the tables below.

The average number of living juveniles at the end of the test after 21 days per survived parental daphnid was 93.4 in the control group and thus thevalidity criterion for the reproduction of a mean of ≥ 60 living offspring per survived parent animalin the control was met.

No parental daphnids were excluded due to accidental or inadvertent mortality from the evaluation of the reproductive output. Since the Cochran-Armitage test procedure revealed a significant increasing trend (p≤0.05) in adult mortality, the evaluation was done based on the cumulative offspring per introduced parent. A statistically significant reduction of the reproductive output in comparison to the reproductive output in the control was determined at the concentration levels from 0.100 to 10.0 mg/L nominal (Levenes test on Variance Homogeneity,α = 0.05). The determination of the EC₁₀- and the EC₅₀-value and their 95% confidence limits was carried out by point estimates from the 3‑parametric normal CDF.

The EC₁₀for the test itemTallowbis(2-hydroxyethyl) amineoxide (CAS no. 61791-46-6)was 0.110 mg/L nominal (95% confidence limits: 0.0593 – 0.205 mg/L).The EC₅₀was 0.232 mg/L nominal (95% confidence limits: 0.114 – 0.480 mg/L).The LOEC was 0.316 mg/L nominal. The NOEC was 0.100 mg/L nominal.

 

Effects on Reproduction for all Introduced and for Surviving Parents

(based on the nominaltest item concentrations)

Nominal test item concentration [mg/L]	Mean number of offspring per introduced parental daphnid			Mean number of offspring per survived parental daphnid
	Mean	SD	CV	Mean	SD	CV
10.0	0	--		0	--
3.16	0	--		0	--
1.00	11.2	14.9	133	22.7	19.1	84.2
0.316	27.0	18.8	69.4	27.0	18.8	69.4
0.100	89.1	30.1	33.8	90.3	32.0	35.4
Control	93.4	17.1	18.3	93.4	17.1	18.3

--   = not applicable, due to the mortality of the parental daphnid(s)

The parameter living offspring per survived parental animal at the end of exposure was also evaluated. Nevertheless, the evaluation based on the cumulative offspring per introduced parent is the ecologically most relevant response variable.

 

Adult Mortality

 

Accidental and inadvertent mortality as defined in the OECD test guideline and abnormalities were not observed. The test item induced statistically significant adult mortality of 100% after 21 days in the nominal concentration levels 10.0 – 3.16 – 1.00 mg/L (Step‑down Cochran-Armitage Test Procedure,α = 0.05). In the concentration levels of 0.100 mg/L a mortality rate of 20% of the introduced adults was observed, which is not statistically significant. In the concentration level of 0.316 mg/L and in the control all parental daphnids survived until the end of the test (21 days). The Cochran-Armitage test procedure revealed a significant increasing trend (p≤0.05) in adult mortality. Therefore, the LC₂₀- and LC₅₀-values were calculated by probit analysis.

 

Mortality [%] of the Adult Daphnids after 7, 14 and 21 Days of Exposure

                (n = 10)

Nominal test item concentration [mg/L]	Adult Mortality [%]
	7 days	14 days	21 days
10.0	100	100	100^1)
3.16	90	100	100^1)
1.00	30	50	70^1)
0.316	0	0	0
0.100	10	10	20
Control	0	0	0

¹⁾= statistically significant (Step‑down Cochran-Armitage Test Procedure,α = 0.05)

 

Intrinsic Rates of Natural Increase (IR)

The intrinsic rates of natural increase (IR)of the survived parental daphnids accounting for generation time and number of offspring were used for calculation of population growth and maintenance.

 

Intrinsic Rates of Natural Increase

Nominal test item concentration [mg/L]	Intrinsic rate of natural increase in replicate no.										Mean IR	CV
	1	2	3	4	5	6	7	8	9	10	MV±SD	[%]
10.0	---	---	---	---	---	---	---	---	---	---	---
3.16	---	---	---	---	---	---	---	---	---	---	---
1.00	---	0.173	---	---	0.172	---	---	---	0.167	---	0.171±0.0031	1.8
0.316	0.175	0.133	0.161	0.191	0.178	0.172	0.205	0.353	0.240	0.148	0.195±0.0628	32.2
0.100	0.293	---	0.283	0.277	0.356	0.271	0.280	0.268	0.249	---	0.284±0.0316	11.1
Control	0.320	0.275	0.281	0.264	0.281	0.285	0.283	0.287	0.292	0.275	0.284±0.0148	5.2

--   = not applicable, due to the mortality of the parental daphnid(s)

 

First Appearance of living Juveniles

 

Thefirst appearance ofliving juveniles was observed between days 9 and 13 at the introduced parental daphnids of the control. For the nominal test item concentrations 0.100 - 0,316 – 1.00 mg/L first appearance ofliving juveniles was observed between days 9 and 16 at the introduced parental daphnids, while 1 parental daphnid for the 0.100 mg/L concentration level and 5 parental daphnids at the 1.00 mg/L concentration level died before producing juveniles. No juveniles were observed in the two highest concentration levels 3.16 – 10.0 mg/L.

 

First Appearance of Living Juveniles in the Individual Groups

Nominal test item concentration [mg/L]	Day of first appearance of living juveniles at the introduced parental daphnids in replicate no.										First
											appearance
	1	2	3	4	5	6	7	8	9	10	mean day
10.0	No juveniles after 21 days										-
3.16	No juveniles after 21 days										-
1.00	9	10	9	--	11	--	--	--	10	--	9.8
0.316	15	15	16	11	12	13	12	10	12	12	12.9
0.100	12	10	10	9	10	12	12	13	--	12	11.1
Control	10	12	12	11	12	11	10	12	10	13	10.3

--   = not applicable, due to the mortality of the parental daphnid(s)

Stillborn Juveniles and Aborted Eggs

No stillborn juveniles or aborted eggs were observed in the control or the concentration levels during the exposure period of 21 days.

 

Stillborn Juveniles and Aborted Eggs produced by the Survived Parental Daphnids after 21 Days

Nominal test item concentration [mg/L]	Number of		Total no.   å	Number of survived adults N
	stillborn juveniles	aborted eggs
10.0	0	0	0	0
3.16	0	0	0	0
1.00	0	0	0	3
0.316	0	0	0	10
0.100	0	0	0	8
Control	0	0	0	10

N   = number of the survived parental daphnids

Growth (Total Body Length and Mean Dry Weight) of the Survived Parental Daphnids

 

The mean values of the body length (excluding the anal spine) of the survived parental daphnids in the tested nominal concentration levels 0.100 to 1.00 mg/L were in the range of 3.95 to 4.33 mm per daphnid and 4.48 mm per daphnid in the control group. At the two highest test concentration levels 3.16 – 10.0 mg/L no parent animals survived until the end of the exposure.

The mean dry weight of the survived parental daphnids was in the range of 0.49 to 0.63 mg per daphnid in the nominal concentration levels of 0.100 to 1.00 mg/L and 0.68 mg per daphnid in the control.

 

Total Body Length and Dry Weight of the Survived Parental Daphnids

Nominal test item concentration [mg/L]	Total length of the survived parent animals [mm]										MV	N	Dry weight [mg]
	Replicate no.
	1	2	3	4	5	6	7	8	9	10	[mm]		S	MV
10.0	---	---	---	---	---	---	---	---	---	---	---	0	---	---
3.16	---	---	---	---	---	---	---	---	---	---	---	0	---	---
1.00	---	4.50	---	---	4.00	---	---	---	4.50	---	4.33	3	1.50	0.50
0.316	4.00	3.75	4.25	3.50	4.00	4.50	3.75	4.00	4.25	3.50	3.95	10	4.90	0.49
0.100	4.50	3.75	4.25	5.00	4.50	4.00	3.75	4.25	---	4.50	4.28	9	5.70	0.63
Control	4.00	4.25	5.00	4.25	4.50	4.75	4.50	4.50	4.50	4.50	4.48	10	6.80	0.68

N  = number of the survived parental daphnids

--   = not applicable, due to the mortality of the parental daphnid(s)

 

Presence of Males 

No males were observed in the control or in the test groups during the test.

 

Occurrence of Ephippia (Winter Eggs) 

No ephippia were observed in the control or in the test groups during the test.

Water Quality Parameters 

The measured water quality parameters (i.e. pH-value, dissolved oxygen concentration, total water hardness and water temperature) were within the acceptable limits. The incubator temperature was in the range of 20 to 21 °C.

 

Validity criteria fulfilled:

yes

Conclusions:

The effects on reproduction were evaluated based on the reproduction per introduced parent animal, since this parameter is the ecologically most relevant response variable and is required by the OECD test guideline 211 (2012), when a significant trend in mortality is detected.
A summary of the assessed effect levels based on the nominal and time weighted average concentrations of the test item Tallow bis(2-hydroxyethyl) amineoxide (CAS no. 61791-46-6) is given in the table below.
The effect threshold for effects on reproduction of the test item under the test conditions was 0.0146 mg/L (NOEC) and 0.0436 mg/L (LOEC) based on the time weighted average concentrations of the test item Tallow bis(2-hydroxyethyl) amineoxide (CAS no. 61791-46-6).


Endpoints for Reproduction and Mortality
(based on the nominal and time weighted average concentrations of the test item)
Effect values Tallow bis(2-hydroxyethyl) amineoxide (CAS no. 61791-46-6)

Nominal concentration of the test item
[mg/L] Time weighted average concentration of the test item
[mg/L]
EC10 Reproduction
(with 95% confidence limits) 0.110 (0.0593 – 0.205) 0.0178 (0.00880 – 0.0362)
EC50 Reproduction
(with 95% confidence limits 0.232 (0.114 – 0.480) 0.0343 (0.0154 – 0.0787)
LOEC Reproduction 0.316 0.0456
NOEC Reproduction 0.100 0.0147
LC20 Adult mortality after 21 days (with confidence limits) 0.318 (n.d.) 0.0530 (0.0179 – 0.0955)
LC50 Adult mortality after 21 days
(with confidence limits) 0.667 (n.d.) 0.129 (0.0679 – 0.257)
LC100 Adult mortality after 21 days 3.16 1.13
LOEC Adult mortality after 21 days 1.00 0.209
NOEC Adult mortality after 21 days 0.316 0.0456
n.d. = Confidence limits not determinable

Executive summary:

A Daphnia magna reproduction test (semi-static, 21 d) of the test item Tallow bis(2-hydroxyethyl) amineoxide (CAS no. 61791-46-6)(batch number: 1548134) was conducted at the test facility according to OECD 211 (2012) from 2017-11-30 to 2018-03-14, with the definitive exposure phase from 2018-02-13 to 2018-03-07.

Test species was Daphnia magna STRAUS (Clone 5). Ten daphnids, held individually, were used per concentration level and control. At test start the daphnids were 2 to 24 hours old. The study was carried out undersemi-static conditions with a medium renewal three times per week (i.e. on Monday, Wednesday and Friday). Aim of the test was to assess the effects on the reproduction capacity and other test item-related effects or parameters such as the intrinsic rate of the natural increase, first appearance of living juveniles, occurrence of aborted eggs and stillborn juveniles, adult mortality, body length and dry weight of the parental daphnids.

The test item Tallow bis(2-hydroxyethyl) amineoxide (CAS no. 61791-46-6) is a light yellow paste (liquid), which is dispersible in water. Five concentration levels of the test item Tallow bis(2-hydroxyethyl) amineoxide (CAS no. 61791-46-6) were selected based on the results of previous tests under GLP conditions as follows:0.100 - 0.316 - 1.00 - 3.16 - 10.0 mg/L as a dilution from a stock solution.

A stock solution with a nominal concentration of 10.0 mg/L was prepared with dilution water and was used as the highest concentration level and for the preparation of further four concentration levels in the range of 0.100 - 0.316 - 1.00 - 3.16 - 10.0 mg test item /L.

The test item concentrations of Tallow bis(2-hydroxyethyl) amineoxide (CAS no. 61791-46-6) wereanalytically verified via LC-MS/MS in fresh media at the start of the exposure intervals on study days 0, 9 and 14 (0 hours) and in old media at the end of the exposure intervals on study days 2, 12 and 16 (48 or 72 hours, respectively) in all concentration levels and the control.

Since the test item is labelled as an UVCB substance, the three constituents C16:0 (346 Da), C18:1 (372 Da) und C18:0 (374 Da) were used for analytical verification. The proportion of the constituents in the test item was given by the sponsor.

The time weighted average concentrations were calculated from the analytical recoveries of the three mentioned constituents and used for evaluation of the effect-concentrations together with the nominal concentrations. The time weighted average concentrations are: 0.0147 – 0.0456 – 0.209 – 1.13 – 3.21 mg/L.

The environmental conditionswere within the acceptable limits.The validity criteria of the test guideline were met .

The effects on reproduction were evaluated based on the reproduction per introduced parent animal, since this parameter is the ecologically most relevant response variable and is required by the OECD test guideline 211 (2012), when a significant trend in mortality is detected. A summary of all endpoints based on the nominaland time weighted average concentrations of the test item Tallow bis(2-hydroxyethyl) amineoxide (CAS no. 61791-46-6) is given in the table below.

 

Endpoints for Reproduction and Mortality

(based on the nominal and time weighted average concentrations of the test item)

Effect values	Tallow bis(2-hydroxyethyl) amineoxide (CAS no. 61791-46-6)
	Nominal concentration of the test item [mg/L]	Time weighted average concentration of the test item [mg/L]
EC10 Reproduction (with 95%confidence limits)	0.110 (0.0593 – 0.205)	0.0178 (0.00880 – 0.0362)
EC50 Reproduction (with 95%confidence limits	0.232 (0.114 – 0.480)	0.0343 (0.0154 – 0.0787)
LOECReproduction	0.316	0.0456
NOECReproduction	0.100	0.0147
LC20 Adult mortality after 21 days(withconfidence limits)	0.318 (n.d.)	0.0530 (0.0179 – 0.0955)
LC50 Adult mortality after 21 days (withconfidence limits)	0.667 (n.d.)	0.129 (0.0679 – 0.257)
LC100 Adult mortality after 21 days	3.16	1.13
LOECAdult mortality after 21 days	1.00	0.209
NOECAdult mortality after 21 days	0.316	0.0456

               n.d. = Confidence limits not determinable

Description of key information

A Daphnia magna reproduction test (semi-static, 21 d) with Tallow bis(2-hydroxyethyl) amineoxide was conducted according to OECD 211 (2012). Ten daphnids (daphnia magna Straus), held individually, were used per concentration level and control. At test start the daphnids were 2 to 24 hours old. The study was carried out under semi-static conditions with a medium renewal three times per week (i.e. on Monday, Wednesday and Friday).

Nominal test concentrations were: 0.100 - 0.316 - 1.00 - 3.16 - 10.0 mg/L prepared as a dilution from a stock solution. The time weighted average concentrations were: 0.0147 – 0.0456 – 0.209 – 1.13 – 3.21 mg/L.

The EC10 and EC50 reproduction were: 0.0178 and 0.0343 mg/L based on TWA concentrations respectively.

The EC50 for adult mortality was 0.129 mg/L TWA.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

0.018 mg/L

Marine water invertebrates

Marine water invertebrates

Effect concentration:

0.034 mg/L