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

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-03 - 2017-08-04

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Product name AROMOX T/12
Batch number 1548134
CAS No. 61791-46-6
Active ingredient Tallow bis(2-hydroxyethyl)amineoxide
Purity (certified) 50.66% (UVCB)
Water solubility dispersible
Appearance light yellow paste (liquid)
Expiry date 2018-03-27
Recommended storage Store container tightly closed in a dry, well-ventilated place

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 1 - 5.6 - 32 - 180 - 1000 mg a.i./L (corresponding to 1.97 – 11.1 – 63.2 – 355 – 1974 mg test item/L) were weighed out on object slides and were transferred to laboratory bottles.
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loadings: 1 - 5.6 - 32 - 180 - 1000 mg a.i./L (corresponding to 1.97 – 11.1 – 63.2 – 355 – 1974 mg test item/L), 5 replicates
- Controls: Test medium without test or reference item. Triplicates at the beginning of the test + triplicates at the end of the test
-Reference item: three concentrations within the concentration range 58 - 180 mg/L and a dilution factor of 1.8, Triplicates

Test organisms (species):

activated sludge

Details on inoculum:

- Preparation of inoculum for exposure: Non-adapted activated sludge from the sewage plant at Hildesheim (Municipal sewage treatment plant of 31137 Hildesheim, Germany)
- Receipt: 2017-08-03
- Pretreatment: The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The sludge was used within 24 h after sampling.
- Dry sludge concentration: 2.70 g/L, corresponding to 1.35 g/L in the test vessel

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

Not determined

Test temperature:

20.2 °C

pH:

pH-value of the activated sludge 7.54
pH-value of the synthetic waste water 7.28

Salinity:

Not determined

Details on test conditions:

TEST SYSTEM
- Test vessel: 5
- 1000 mL laboratory bottles, ISO 4796
- Aeration: Shaking of the laboratory bottles at 150 rpm to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.

- Pretreatment: None

- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6 (Triplicates at the beginning of the test + triplicates at the end of the test.)
-No. of vessels per reference item (replicates): 9 (three concentrations within the range 58 - 180 mg/L and a dilution factor 1.8, at the end of the test)
- Composition of test medium:

Test item concentration [mg/L] 1.97 11.1 63.2 355 1974
Active ingredient concentration (a.i.) [mg/L] 1 5.6 32 180 1000
Active ingredient [mg] 0.5 2.8 16 90 500
Test item [mg] 0.987 5.53 31.6 178 987
Synthetic waste water [mL] 16
Demineralised water [mL] Filled up to 250 mL
Inoculum [mL] 250

Dilution Table for the Reference Item
Reference item concentration [mg/L] 58 100 180
Stock solution [µL]* 96.7 166.7 300
Synthetic waste water [mL] 16
Demineralised water [mL] Filled up to 250 mL
Inoculum [mL] 250
* Stock solution with 300g/L


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter every 5 seconds after transferring the solution to the measuring chamber and the oxygen depletion was recorded for at least 5 minutes with the software PreSens Measurement Studio 2.

TEST CONCENTRATIONS

A preliminary test (non GLP) was carried out with the concentrations of the test item Tallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6 of 10 – 100 - 1000 mg/L with two replicates each. The pH was measured in one replicate with 1000 mg/L test item. The flasks were aerated for 3 hours.

In the preliminary test, additional replicates were prepared to determine the inhibition of nitrification. Two replicates 1000 mg/L were measured with addition of Allylthiourea ATU.

No significant difference in inhibition with or without ATU was observed in the preliminary test with 507 mg (a.i.) Tallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6. This means that the inhibition of both nitrification and heterotrophic respiration show comparable values. Therefore, nitrification inhibition was not evaluated separately in the definitive study.



Inhibitions in the Preliminary Tests

Nominal Test Item Concentration
[mg/L] Active ingredient concentration (a.i.)
[mg/L] pH

(test item in medium) Inhibition


[%]
10 5.1 - 5
- 5
100 50.7 - 30
- 31
1000 507 7.59 67
- 56


Inhibitions in the Preliminary Test (with / without ATU)

Nominal Test Item Concentration
[mg/L] Active ingredient concentration (a.i.)
[mg/L] Inhibition of total respiration (without ATU)
[%] Inhibition of heterotrophic respiration (with ATU)
[%] Inhibition of nitrification
[%]
1000 507 67 58 74
56 60 52


Based on these results the definitive study was carried out with 5 concentrations within the range 1 - 1000 mg a.i./L test item in a geometrical series and with a dilution factor of 5.6. No pH adjustment was made.
All concentrations were weighed out of object slides and were transferred to laboratory bottles and stirred overnight.
Triplicates of the control without test item were included at the beginning and at the end of the test.
The reference item was tested with three concentrations within the concentration range 58 - 180 mg/L and a dilution factor of 1.8.
A fresh sample of activated sludge taken from the sewage plant Hildesheim was used for the test procedure. It was washed twice and adjusted with chlorine free tap water if necessary to a dry sludge concentration of 3.0 g/L ± 10 %.
The oxygen uptake rates of all samples were determined following an incubation period of 3 h.

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

32 other: mg a.i./L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

34.3 other: mg a.i./L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

51.9 other: mg a.i./L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

195 other: mg a.i./L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Results with reference substance (positive control):

- Results with reference substance valid? Yes

Reported statistics and error estimates:

The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. One Way Analysis of Variance (ANOVA) was used for NOEC calculation. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-value for both tests was 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

The EC-value of the test item and reference item was calculated by sigmoidal response and linear regression using software GraphPad Prism. Calculation of the confidence intervals for the EC50-value was carried out using standard procedures. The coefficient of variation of the oxygen uptake rates of the control replicates was calculated with standard methods using Excel.

From the oxygen depletion the oxygen uptake rates were calculated in mg O₂/L×h, additionally the specific respiration rates based on the dry sludge concentration were calculated in mg O₂/g×h. The inhibition of the oxygen uptake rates of the test and reference item concentrations in % was calculated in comparison to the oxygen uptake rates of the control.

Oxygen Uptake Rates and Specific Respiration Rates of the Control Replicates

	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Specific Respiration Rate Rs
		[mg O2/L]	[mg O2/(L×h)]	[mg O2/g×h]
Control	1	6.25	23.8	17.6
	2	6.44	26.3	19.5
	3	5.94	22.6	16.7
	4	6.84	25.6	19.0
	5	7.36	25.9	19.2
	6	6.61	24.1	17.9
	Mean value		24.7	18.3
	Standard deviation		1.32
	CV [%]		5.34

*) determined at start of the measurement

Repl. = Replicate

CV = Coefficient of variation

 

Oxygen Uptake Rates and Inhibition of the Respiration of the Test Item Concentrations

Test Item Concentration	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Inhibition	Mean Inhibition
[mg a.i./L]		[mg O2/L]	[mg O2/(L×h)]	[%]	[%]
1	1	6.06	26.7	-8	1
	2	6.01	26.9	-9
	3	5.42	24.1	2
	4	6.17	21.1	15
	5	5.20	23.7	4
5.6	1	7.49	28.7	-16	-9
	2	7.00	28.4	-15
	3	6.05	25.3	-2
	4	7.27	27.7	-12
	5	5.86	24.3	2
32	1	6.22	23.2	6	9
	2	5.64	23.7	4
	3	5.06	21.6	13
	4	5.18	22.2	10
	5	4.84	21.4	13
180	1	7.27	12.3	50	49
	2	7.18	13.1	47
	3	6.47	12.5	49
	4	6.98	13.2	47
	5	6.45	12.3	50
1000	1	7.39	9.3	62	57
	2	7.46	12.0	51
	3	6.69	11.0	55
	4	7.15	9.8	60
	5	6.46	10.9	56

*) determined at start of the measurement

Repl. = Replicate

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of Tallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6 is 32 mg a.i./L.
The EC10 is 34.3 mg a.i./L and the EC50 is 195 mg a.i./L.

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item Tallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6 (batch number: 1548134) from 2017-08-03 to 2017-08-04, with the definitive exposure phase on 2017-08-04 at the test facility. The test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the test item concentrations 1 - 5.6 - 32 - 180 - 1000 mg a.i./L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The mean inhibition of respiration for the test item replicates ranged from -9 % to 57 %. In the preliminary test, no significant difference in inhibition with or without ATU was observed with 507 mg (a.i.) Tallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6. This means that the inhibition of both nitrification and heterotrophic respiration show comparable values. Therefore, nitrification inhibition was not evaluated separately in the definitive study.

 

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC₅₀-value for the reference item was 111 mg/L.

 

NOEC and EC-Values with Confidence Interval of Tallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6 after 3 h exposure time, Inhibition of Total Respiration

		EC-values [mg a.i./L]		Confidence interval P = 95 [%] [mg a.i./L]
NOEC*		32		̶
EC10		34.3		24.6 – 48.7
EC20		51.9		38.9 – 73.3
EC50		195		111 – 467
EC80		> 1000		-

*) No statistically significant inhibition (P < 0.001), ANOVA, Dunnett`s Method

                        

 

The NOEC of Tallow Bis(2-hydroxyethyl) amineoxide CAS no. 61791-46-6 is 32 mg a.i./L. The EC10 is 34.3 mg a.i./L and the EC50 is 195 mg a.i./L.

Description of key information

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with C16-18 (evennumbered, C18 unsaturated) alkyl bis(2-hydroxyethyl) amine oxide CAS no. 61791-46-6. The 3h EC10 and EC50 observed are 34.3 and 195 mg a.i./L respectively.

Key value for chemical safety assessment

EC50 for microorganisms:

195 mg/L

EC10 or NOEC for microorganisms:

34.3 mg/L

Additional information

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with C16-18 (evennumbered, C18 unsaturated) alkyl bis(2-hydroxyethyl) amine oxide CAS no. 61791-46-6. The test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the test item concentrations 1 - 5.6 - 32 - 180 - 1000 mg a.i./L. The respiration rates of the control, reference and test item replicates were measured after a contact time of three hours, and the inhibitory effects of the test and reference item were determined in comparison to the control respiration rates. The mean inhibition of respiration for the test item replicates ranged from -9 % to 57 %. In the preliminary test, no significant difference in inhibition with or without ATU was observed with 507 mg (a.i.) C16-18 (evennumbered, C18 unsaturated) alkyl bis(2-hydroxyethyl) amine oxide. This means that the inhibition of both nitrification and heterotrophic respiration show comparable values. Therefore, nitrification inhibition was not evaluated separately in the definitive study. The 3h EC10 and EC50 observed are 34.3 and 195 mg a.i./L respectively.

 

In order to check the activity of the test system and the test conditions a reference test was carried out with copper (II) sulphate pentahydrate as reference item and the reference toxicity was determined. The EC₅₀-value for the reference item was 111 mg/L.