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

Sediment toxicity

Link to relevant study record(s)

Reference

Endpoint:

sediment toxicity: long-term

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-16 to 2017-09-14

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

other: Sediment-Water Lumbriculus Toxicity Test Using Spiked Sediment (October 2007)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

All concentration levels and the control were analytically verified by UPLC-MS/MS. The concentrations of the test item were analysed on the day of worm insertion (day 0) as well as on day 7 and at test end (day 28) in the sediment layer, aqueous phase and pore water. Three additional replicates per treatment were set up for the analytical verification on day 0, 7 and 28.

Vehicle:

no

Details on sediment and application:

PREPARATION OF SPIKED SEDIMENT
- Details of spiking:
The test item was heated up to approx. 60 °C. The respective test item amount (actual: 2250.2 mg) was weighed out for a stock solution (500 mL) and dissolved in demineralised water (approx. 80 °C). The stock solution was stored at room temperature under stirring conditions (300 rpm) for three days until application.
On application day the stock solution was further diluted with demineralised water to receive the respective application solutions. The application solutions were thoroughly mixed with a sub-quantity of the artificial sediment. Subsequently, the treated portion of artificial sediment was thoroughly mixed with the remaining sediment. Additional demineralised water was added to the artificial soil to adjust the humidity of the artificial soil to a moisture of 30 %.
Subsequently, the test medium was thoroughly mixed to ensure a homogenous distribution and about 45 g soil dry weight were filled into the test vessels, each.

- Equilibration time:
2 days prior to insertion of test organisms
- Equilibration conditions:
Same as test conditions
- Controls:
Six replicates with untreated artificial sediment

PREPARATION OF SPIKED WATER
- Details of spiking:
- Controls:
- Chemical name of vehicle (organic solvent, emulsifier or dispersant):
- Concentration of vehicle in final test solution:

Test organisms (species):

Lumbriculus variegatus

Details on test organisms:

TEST ORGANISM
- Common name:
Lumbriculus variegatus (Müller), Lumbriculidae, Oligochaeta
- Source:
BUNDESANSTALT FÜR GEWÄSSERKUNDE (BfG), Am Mainzer Tor 1, 56068 Koblenz, Germany
- Age of parental stock (mean and range, SD):
Synchronized from cultured organisms 14 days before insertion
- Breeding conditions:
20 +/- °C, diffuse light (100 - 1000 lx, 16 h photoperiod), dissolved oxygen concentration > 60 % of the air saturation value
- Handling of egg masses and larvae:
13 days before insertion into the test, the worms were synchronized. Therefore, a sufficient amount of worms was taken from the breeding and cutted into half. After regenerationof heads, the worms were inserted into the test.
- Age of animals at beginning of exposure:
13 days after synchronization, completely regenerated head region. Synchronization was done to minimize uncontrolled regeneration and reproduction during the test.
- Feeding during test
- Food type:
Powder of Urtica dioica (heinrich Klein GmbH & Co. KG, 97525 Schwebheim, Germany)
- Amount:
0.4 % Urtica powder
- Frequency:
Once at test start

ACCLIMATION
- Acclimation period:
13 days (after synchrinization)
- Acclimation conditions (same as test or not):
Same as test
- Type and amount of food:
Same as test
- Feeding frequency:
Same as test (once)
- Health during acclimation (any mortality observed):
No mortality observed

Study type:

laboratory study

Test type:

static

Water media type:

freshwater

Type of sediment:

artificial sediment

Limit test:

no

Duration:

28 d

Exposure phase:

total exposure duration

Post exposure observation period:

No

Hardness:

Day Parameter Control 31.25 62.5 125 250 500 1000
mg test item/kg sediment dry weight

0
(before
insertion of worms)
Total hardness [mg/L CaCO3] 288 319 319 344 303 320 342
28 Total hardness [mg/L CaCO3] 288 246 295 267 255 263 299

Test temperature:

Day Parameter Control 31.25l 62.5 125 250 500 1000
mg test item/kg sediment dry weight
0
(before
insertion of worms) Temperature [°C] 20.6 20.5 20.3 20.4 20.4 20.3 20.4
7 Temperature [°C] 20.8 20.9 20.8 20.7 20.8 20.6 20.8
14 Temperature [°C] 20.9 20.9 21.1 21.1 21.1 20.8 21.0
21 Temperature [°C] 21.2 21.1 21.0 21.1 21.1 21.1 21.0
28 Temperature [°C] 21.1 20.9 20.3 20.6 20.7 20.9 20.3

pH:

Day Parameter Control 31.25 62.5 125 250 500 1000
mg test item/kg sediment dry weight
0
(before
insertion of worms)
pH 8.33 8.12 7.85 7.77 8.02 7.85 7.77
7 pH 8.23 8.30 8.12 8.11 8.02 8.18 7.86
14 pH 8.29 8.37 8.36 8.36 8.35 8.34 8.34
21 pH 8.09 8.06 8.22 8.23 8.13 8.25 8.03
28 pH 7.76 7.87 7.92 8.09 8.07 8.11 8.16

Dissolved oxygen:

Day Parameter Control 31.25l 62.5 125 250 500 1000
mg test item/kg sediment dry weight
0
(before
insertion of worms)
O2 [mg/L]* 8.81 8.11 7.61 7.71 8.73 8.16 7.62
7 O2 [mg/L]* 8.43 8.53 8.31 8.45 7.58 8.68 7.30
14 O2 [mg/L]* 8.29 8.37 8.36 8.36 8.35 8.34 8.34
21 O2 [mg/L]* 8.09 8.06 8.22 8.23 8.13 8.25 8.03
28 O2 [mg/L]* 8.40 8.43 8.59 8.60 8.35 8.35 8.48
*) 70 % of the air saturation value corresponds to 6.2 mg O2/L.

Salinity:

Not measured, fresh water conditions

Ammonia:

Day after worm insertion Control 31.25 62.5 125 250 500 1000
mg test item/kg sediment dry weight
Ammonium-N [mg/L]
0 0.30 0.38 0.26 0.22 0.22 0.12 0.36
2 0.88 1.38 0.57 0.26 1.13 0.18 0.88
5 0.19 0.04 0.22 0.07 0.07 0.21 0.05
7 0.12 0.06 0.14 0.09 0.07 0.24 0.11
9 0.08 0.06 0.14 0.07 0.07 0.16 0.14
12 0.09 0.09 0.14 0.18 0.09 0.14 0.13
14 0.17 0.15 0.14 0.13 0.12 0.26 0.13
16 0.23 0.11 0.19 0.12 0.11 0.18 0.12
19 0.20 0.14 0.21 0.12 0.07 0.17 0.07
21 0.12 0.15 0.20 0.11 0.06 0.19 0.06
23 0.11 0.18 0.22 0.16 0.08 0.30 0.07
26 0.25 0.27 0.24 0.19 0.11 0.31 0.10
28 0.19 0.35 0.34 0.32 0.17 0.34 0.08

Nominal and measured concentrations:

Measured Concentrations and Recovery Rates of Test Item for Component 346 Da
(Aqueous Layer, Pore Water and Sediment) - Day 0
Sampling date: 2017-08-16
Concentration test item [mg/kg DW] 1000 500 250 125 62.5 31.25 Control
Amount per test vessel [mg]1) 45.0 22.5 11.3 5.63 2.81 1.41
Aqueous Layer Meas. amount
[mg] 0.0526 0.0263 0.00762 < LOQ < LOQ < LOQ < LOQ
% 0 0 0
Pore
Water Meas. amount
[mg] 0.00282 0.000929 0.000310 < LOQ < LOQ < LOQ < LOQ
% 0 0 0
Sediment Meas. amount
[mg] 39.3 19.0 8.78 4.28 2.12 0.910 < LOQ
% 87 85 78 76 75 65
Total
Amount [mg] 39.3 19.1 8.79 4.28 2.12 0.910 < LOQ
% 87 85 78 76 75 65

Measured Concentrations and Recovery Rates of Test Item for Component 372 Da
(Aqueous Layer, Pore Water and Sediment) - Day 0
Sampling date: 2017-08-16
Concentration test item [mg/kg DW] 1000 500 250 125 62.5 31.25 Control
Amount per test vessel [mg]1) 45.0 22.5 11.3 5.63 2.81 1.41
Aqueous Layer Meas. amount
[mg] 0.0348 0.0151 0.00526 < LOQ < LOQ < LOQ < LOQ
% 0 0 0
Pore
Water Meas. amount
[mg] 0.00235 0.000506 0.000156 < LOQ < LOQ < LOQ < LOQ
% 0 0 0
Sediment Meas. amount
[mg] 39.7 19.2 9.04 4.40 2. 08 0.844 < LOQ
% 88 85 80 78 74 60
Total
Amount [mg] 39.7 19.2 9.05 4.40 2. 08 0.844 < LOQ
% 88 85 80 78 74 60
1) = Based on 45 g sediment (DW) and 150 mL dilution water
 
Measured Concentrations and Recovery Rates of Test Item for Component 374 Da
(Aqueous Layer, Pore Water and Sediment) - Day 0
Sampling date: 2017-08-16
Concentration test item [mg/kg DW] 1000 500 250 125 62.5 31.25 Control
Amount per test vessel [mg]1) 45.0 22.5 11.3 5.63 2.81 1.41
Aqueous Layer Meas. amount
[mg] 0.0399 0.0315 0.00965 < LOQ < LOQ < LOQ < LOQ
% 0 0 0
Pore
Water Meas. amount
[mg] 0.00110 0.000378 0.000235 < LOQ < LOQ < LOQ < LOQ
% 0 0 0
Sediment Meas. amount
[mg] 42.7 20.4 9.40 4.61 2.09 0.909 < LOQ
% 95 91 84 82 74 65
Total
Amount [mg] 42.7 20.4 9.41 4.61 2. 09 0.909 < LOQ
% 95 91 84 82 74 65

Details on test conditions:

TEST SYSTEM
- Test container (material, size): 250 mL glass beakers (6 cm diameter)
- Sediment volume: 45 g dry weight per vessel / ca. 1.0 - 3.0 cm from the ground
- Overlying water volume: 150 mL
- Aeration: yes
- Aeration frequency and intensity: Gentle aeration via Pasteur pipettes. Aeration was checked each working day.
- Replacement of evaporated test water, if any: Water levels were topped up with demineralised water each working day during the study to compensate losses due to evaporation.

EXPOSURE REGIME
- No. of organisms per container (treatment): 10 synchronized worms per replicate (60 per treatment)
- No. of replicates per treatment group: 6 replicates per treatment group
- No. of replicates per control / vehicle control: 6 replicates per control
- Feeding regime: Once at test start
- Type and preparation of food: Powder of Urtica dioica (stinging nettle) was mixed into the sediment.
- Amount of food: 0.4 % of thge sediment dry weight

OVERLYING WATER CHARACTERISTCS
- Type of water (e.g. deionized, ground water, sea water, Elendt medium acc. to OECD 219): Reconstituted water acc. to OECD 203

Day Parameter Control 31.25 62.5 125 250 500 1000
mg test item/kg sediment dry weight
0
(before
insertion of worms) Temperature [°C] 20.6 20.5 20.3 20.4 20.4 20.3 20.4
pH 8.33 8.12 7.85 7.77 8.02 7.85 7.77
O2 [mg/L]* 8.81 8.11 7.61 7.71 8.73 8.16 7.62
Total hardness [mg/L CaCO3] 288 319 319 344 303 320 342


CHARACTERIZATION OF (ARTIFICIAL; delete if not applicable) SEDIMENT
- 5 % peat, air-dried and finely ground
- 20 % kaolin, with a kaolinite content > 30 %
- 75 % air-dried quartz sand (sand with > 50 % particle size of 0.05 - 0.2 mm)
- 0.35 % calcium carbonate (CaCO3) to achieve a pH of 6.58

Sediment Properties at Test Start
Total Organic Carbon [%] pH-value Moisture [%]
Sediment at test start 2.43* 6.58 30
*) external measurement, non-GLP

OTHER TEST CONDITIONS
Environmental Conditions
Room temperature [°C] Photoperiod [h light] Light intensity [lx]
Nominal 20 ± 2 16 100 - 500
Actual 18 - 22 16 225 ± 74

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Visual observations of behavioural differences to the controls were recorded three times per week. The endpoints of the study as determined after day 28 were the number of surviving worms per replicate and the total dry biomass of worms per replicate. Worms were considered dead if showing no response to a mechanical stimulus, signs of decomposition or by absence. Living worms were assigned as adult (large complete worms) or offspring (small complete worms with regenerated body regions or incomplete worms), if possible.

VEHICLE CONTROL PERFORMED: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study
- Test concentrations: 1000 - 100 - 10 - 1 mg test item/kf sediment dry weight
- Results used to determine the conditions for the definitive study:
Preliminary Range Finding Test (28 d): Mortality, Reproduction and Biomass (non-GLP)
(Application with acetone, 3 replicates, each with 10 synchronized worms)
Nominal Test Item Concentration Mortality
of adult worms Mean number of worms per replicate Mean dry weight
per replicate
[mg test item/kg sediment dry weight] [%] [n] [mg]
Control 0.33 36.3 37.3

1 0 42.0 34.7
10 0 42.3 35.6
100 0 33.3 32.7
1000 0 20.0 17.4

Reference substance (positive control):

yes

Remarks:

Potassium chloride

Key result

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

169.7

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks on result:

other: Nominal test concentrations used because of >80% recovery at start of the test.

Duration:

28 d

Dose descriptor:

EC10

Effect conc.:

335 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 50.66% active ingredient

Key result

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 506.6 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

reproduction

Remarks on result:

other: Nominal used because of the >80% recovery at the start of the test.

Duration:

28 d

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/kg sediment dw

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Remarks on result:

other: 50.66% active ingredient

Details on results:

- Changes in body weight of live adults (% of initial weight) at end of exposure period:
At test start the worms had a body weight of 8.75 mg dry weight/10 worms (mean weight of two determinations). At test end, the biomass dry weight of the worms was not statistically significantly different at the test item concentrations 31.25 to 250 mg/kg sediment dry weight compared to the control. However, at the test item concentrations 500 and 1000 mg/kg sediment dry weight the biomass gain was statistically significantly reduced
- No. of offspring produced:
The total number of worms and thus the reproduction did not statistically significantly differ compared to the control in the test item concentrations 31.25 to 250 mg/kg sediment dry weight. However, at the test item concentrations 500 and 1000 mg/kg sediment dry weight the total number of worms and thus the reproduction was statistically significantly reduced.
- Morphological abnormalities: None
- Behavioural abnormalities:
No evident changes in the worm behaviour or pathological symptoms were observed in the control as well as in all test item concentrations during the 28 days of exposure

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels:
Summary of all Effects and resulting NOEC, LOEC of the Reference Item Study
Effects Potassium Chloride
[g/kg sediment dry weight]
NOEC
Total worm number, Biomass 2
LOEC
Total worm number, Biomass 4
EC50 Total worm number
(CI 95%) 3.36
(2.34 – 4.89)
EC50 Biomass
(CI 95%) 2.79
(2.21 – 3.55)
CI = Confidence Interval

Reported statistics and error estimates:

NOEC/LOEC-values and Multiple Sequentially-rejective Welsh-t-test After Bonferroni-Holm
statistical analyses and Williams Multiple Sequential t-test Procedure was carried out for LOEC and NOEC calculations for reproduction and biomass, respectively.
When running the tests, Shapiro-Wilk’s Test on Normal Distribution and Levene’s Test on Variance Homogeneity (with Residuals) were done first. Additionally, a trend analysis by Contrasts (Monotonicity of Concentration/Response) was set up for biomass. The -value (acceptable probability of incorrectly concluding that there is a difference) is  = 0.05.
EC-values and statistical analyses
ECx-values for reproduction and biomass and confidence intervals were calculated by 3-parameter normal CDF regression analysis.

Mortality after 28 Days of Exposure

Test item concentration [mg/kg sediment dry weight]	Number of Dead or Missing						MV	SD ±	Mortality[%]
	1	2	3	4	5	6
Control	0	0	0	0	0	0	0	0	0
31.25	0	0	0	0	-		0	0	0
62.5	0	0	0	0			0	0	0
125	0	0	0	0			0	0	0
250	0	0	0	0			0	0	0
500	0	0	0	1			0.25	0.5	2.5
1000	0	0	0	0			0	0	0

MV = MeanValue                SD = Standard Deviation

 

 

 Total Worm Number after 28 Days of Exposure

Test item concentration [mg/kg sediment dry weight]	Number of Surviving						MV	SD ±	Inhibition to control[%]	Sign.*
	1	2	3	4	5	6
Control	49	42	44	40	35	36	41.0	5.22	-	-
31.25	39	31	33	35	-		34.5	3.42	15.9	No
62.5	35	47	45	35			40.5	6.40	1.22	No
125	40	43	37	40			40.0	2.45	2.44	No
250	35	36	33	35			34.8	1.26	15.2	No
500	35	32	34	30			32.8	2.22	20.1	Yes
1000	22	27	25	22			24.0	2.45	41.5	Yes

*) = Statistically significantly different compared to control (Multiple Sequentially-rejective Welsh-t-test After

      Bonferroni-Holm, alpha = 0.05)

 

 

 

 Reproduction after 28 days of Exposure

Test item concentration [mg/kg sediment dry weight]	Number of Reproduced Worms						MV	SD ±	Inhibition to control[%]
	1	2	3	4	5	6
Control	39	32	34	30	25	26	31.0	5.22	-
31.25	29	21	23	25	-		24.5	3.42	21.0
62.5	25	37	35	25			30.5	6.40	1.61
125	30	33	27	30			30.0	2.45	3.23
250	25	26	23	25			24.8	1.26	20.2
500	25	22	24	20			22.8	2.22	26.6
1000	12	17	15	12			14.0	2.45	54.8

MV = Mean = Standard Deviation    

Validity criteria fulfilled:

yes

Conclusions:

After 28 days of exposure, Tallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6 induced no evident mortality (< 10%) in all test item concentrations.
The total number of worms and thus the reproduction did not statistically significantly differ compared to the control in the test item concentrations 31.25 to 250 mg/kg sediment dry weight. However, at the test item concentrations 500 and 1000 mg/kg sediment dry weight the total number of worms was statistically significantly reduced.
As well compared to the control, the total biomass per replicate was not statistically significantly different in the test item concentrations 31.25 to 250 mg/kg sediment dry weight but statistically significantly decreased in the test item concentrations 500 and 1000 mg/kg sediment dry weight.

Executive summary:

The effects of the test itemTallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6(batch no.1548134) on the oligochaeteLumbriculus variegatusin a water-sediment system were determined at the test facility in Sarstedt, Germany, from 2017-08-14 to 2017-09-14, with the definitive exposure phase from 2017-08-16 to 2017-09-13. The study was carried out according to OECD Guideline 225 (2007). The test duration was 28 days from the insertion of the test organisms.The study was performed by spiking artificial sediment with the test item concentrations 31.25 – 62.5 – 125 – 250 – 500 - 1000 mg/kg sediment dry weight.Six replicates per control and four replicates per test item concentration were set up for biological investigations. Water quality parameters (temperature, pH-value, O₂-concentration, ammonium and total hardness) were determined throughout the study using an additional replicate for the control and the test item concentrations, each. Three additional replicates for chemical analysis on day 0, day 7 and day 28 were set up for the control and each test item concentration.

 

Analytical evaluation for all concentrations of three components of the test itemTallow Bis           (2-hydroxyethyl) amineoxide CAS no. 61791-46-6and control was carried out via UPLC-MS/MS for sediment layer, aqueous phase and pore water on day 0, day 7 and day 28. Details of the analytical method are presented in part15.

The measured concentrations of the three components of the test item in the sediment were in the ranges from 60% to 95% at day 0, from < LOQ to 5% at day 7 and from < LOQ to 0% of the nominal concentrations at day 28. The recoveries below 80 %at day 0may be caused by the formation of Non-Extractable Residues (NER’s) during the time between spiking and extraction period. This possibility was not monitored during analytical method development. As the measured concentrations of the decisive test item concentrations (250 to 1000 mg/kg) were between 78 and 95 % for all the three test item components on day 0, results were given based on nominal values. No recoveries of the concentrations of the three components in theaqueous phase and pore waterwere determined. Control samples were < LOQ.Details of the results are presented in part9.2.4.

 

After 28 days of exposure,Tallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6induced no mortality in all test item concentrations.

The total number of worms and thus the reproduction did not statistically significantly differ compared to the control in the test item concentrations 31.25 to 250 mg/kg sediment dry weight. However, at the test item concentrations 500 and 1000 mg/kg sediment dry weight the total number of worms was statistically significantly reduced.
Compared to the control, the total biomass per replicate was not statistically significantly different in the test item concentrations 31.25 to 250 mg/kg sediment dry weight but statistically significantly decreased in the test item concentrations 500 and 1000 mg/kg sediment dry weight. For further endpoints, please refer to Table 1.

All validity criteria were fulfilled.

                 Summary of all Effects

Effects	Tallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6
	[mg/kg sediment dry weight]
NOEC (Total number of worms/Reproduction and biomass)	250
LOEC (Total number of worms/Reproduction and biomass)	500
ECTotal number of worms/Reproduction (Confidence Intervall)	EC10: 335 (87.7 - > 1000) EC20: 549 (194 - > 1000) EC50: > 1000
ECTotal Biomass per replicate (Confidence Intervall)	EC10: 435 (227 - > 1000) EC20: 623 (316 – > 1000) EC50: > 1000

Description of key information

The benthic PNEC for C16-18 (evennumbered, C18 unsaturated) alkyl bis(2-hydroxyethyl) amine oxide is calculated from the available EC10 of 169.7 mg a.i./kg dw obtained from a long term lumbriculus test according to OECD 225.

The result as can be obtained using the EPM is considered less reliable than the result of one long term sediment reworker test because the test substance is a surfactant and EPM can according to REACh guidance R.7B (Feb, 2016) only be applied to neutral organic chemicals.

This is because the EPM is based on sorption to organic matter. Therefore, it cannot be used for some classes of substances, e.g. when binding behaviour is not driven by lipophilicity (e.g. aromatic amines forming covalent bonds to sediment components, ionisable substances, surface active substances).”

Key value for chemical safety assessment

EC50 or LC50 for freshwater sediment:

506.6 mg/kg sediment dw

EC10, LC10 or NOEC for freshwater sediment:

169.7 mg/kg sediment dw

Additional information

The effects of C16-18 (evennumbered, C18 unsaturated) alkyl bis(2-hydroxyethyl) amine oxide CAS no. 61791-46-6 on the oligochaete Lumbriculus variegatus in a water-sediment system were determined according to OECD Guideline 225 (2007). The test duration was 28 days from the insertion of the test organisms.The study was performed by spiking artificial sediment with the test item concentrations 31.25 – 62.5 – 125 – 250 – 500 - 1000 mg/kg sediment dry weight. Six replicates per control and four replicates per test item concentration were set up for biological investigations. Water quality parameters (temperature, pH-value, O₂-concentration, ammonium and total hardness) were determined throughout the study using an additional replicate for the control and the test item concentrations, each. Three additional replicates for chemical analysis on day 0, day 7 and day 28 were set up for the control and each test item concentration.

 

Analytical evaluation for all concentrations of three components of C16-18 (evennumbered, C18 unsaturated) alkyl bis(2-hydroxyethyl) amine oxide and control was carried out via UPLC-MS/MS for sediment layer, aqueous phase and pore water on day 0, day 7 and day 28.

The measured concentrations of the three main components of the test item in the sediment were in the ranges from 60% to 95% at day 0, from < LOQ to 5% at day 7 and from < LOQ to 0% of the nominal concentrations at day 28. The recoveries below 80 % at day 0 may be caused by the formation of Non-Extractable Residues (NER’s) during the time between spiking and extraction period. This possibility was not monitored during analytical method development. As the measured concentrations of the decisive test item concentrations (250 to 1000 mg/kg) were between 78 and 95 % for all the three test item components on day 0, results were given based on nominal values. No recoveries of the concentrations of the three components in the aqueous phase and pore water were determined. Control samples were < LOQ.

 

After 28 days of exposure C16-18 (evennumbered, C18 unsaturated) alkyl bis(2-hydroxyethyl) amine oxide induced no mortality in all test item concentrations.

The total number of worms and thus the reproduction did not statistically significantly differ compared to the control in the test item concentrations 31.25 to 250 mg/kg sediment dry weight. However, at the test item concentrations 500 and 1000 mg/kg sediment dry weight the total number of worms was statistically significantly reduced.
Compared to the control, the total biomass per replicate was not statistically significantly different in the test item concentrations 31.25 to 250 mg/kg sediment dry weight but statistically significantly decreased in the test item concentrations 500 and 1000 mg/kg sediment dry weight.

All validity criteria were fulfilled.

Effects	C16-18 (evennumbered, C18 unsaturated) alkyl bis(2-hydroxyethyl) amine oxide CAS no. 61791-46-6
	[mg a.i./Kg sediment dry weight]
NOEC (Total number of worms/Reproduction and biomass)	126.65
LOEC (Total number of worms/Reproduction and biomass)	253.3
ECTotal number of worms/Reproduction (Confidence Intervall)	EC10: 169.71 (44.4 - > 506.6) EC20: 278.1 (98.3 - > 506.6) EC50: > 506.6
ECTotal Biomass per replicate (Confidence Intervall)	EC10: 220.4 (115 - > 506.6) EC20: 315.6 (160.1 – > 506.6) EC50: > 506.6