Tributylethylammonium ethyl sulphate

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:

22 March 2017 - 02 June 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation))

Version / remarks:

22 July 2010

Deviations:

no

GLP compliance:

yes

Specific details on test material used for the study:

Soluble in water: yes
Stability in water: stable

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method:
First test: 10.0 and 2.0 g of the test item was added to 500 and 250 mL Milli-RO water, respectively. Magnetic stirring for 27 to 42 minutes was applied to accelerate dissolution and to ensure homogeneity. Volumes of the slightly cloudy stock solution (pH 6.0) corresponding to the test concentration were then added to the test media (16 mL synthetic medium and made up to 250 mL with Milli-RO). Thereafter, 250 mL of activated sludge was added resulting in the required concentrations.

Second and third test: 5.0 g of the test item was added to 500 mL of Milli-RO water. In the second full test the test item was grounded with a mortar before weighing. In the second full test magnetic stirring for 16 minutes was applied to accelerate dissolution and to ensure homogeneity. In the third full test magnetic stirring for 30 minutes (T1) to a maximum of 105 minutes (T6) was applied to accelerate dissolution and to ensure homogeneity. Volumes of the clear and colourless stock solution (pH 5.9 - 6.0) corresponding to the test concentration were then added to the test media (16 mL synthetic medium and made up to 250 mL with Milli-RO). Thereafter, 250 mL of activated sludge was added resulting in the required concentrations.

Optimal contact between the test item and test organisms was ensured in all tests by applying continuous aeration and stirring.

- Controls:
Blank control: medium with sludge and without test item
Abiotic control: medium without sludge and with the test item

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Name and location of sewage treatment plant where inoculum was collected: Municipal sewage treatment plant: 'Waterschap Aa en Maas', 's- Hertogenbosch, The Netherlands, receiving predominantly domestic sewage.
- Preparation of inoculum for exposure: The sludge was coarsely sieved (1 mm) and allowed to settle. The supernatant was removed and ISO-medium was added. A small amount of the sludge was weighed and dried overnight at ca. 105°C to determine the amount of suspended solids (3.0 g/L of sludge, as used for the test).
- Pretreatment: The batch of sludge was used one day after collection; therefore 50 mL of synthetic medium (=sewage feed) was added per litre of activated sludge at the end of the collection day. The sludge was kept aerated at test temperature until use.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

First test: 18 - 21°C
Second test: 20 - 22°C
Third test: 20 - 21°C

pH:

First test: start: 7.3-7.4; end: 7.4-8.2
Second test: start: 7.5-7.6; end: 7.7-8.4
Third test: start: 7.5-7.6; end: 7.6-8.2

Dissolved oxygen:

60-70% saturation at test start

Nominal and measured concentrations:

Nominal concentrations:
First test: 200, 360, 640, 1120 and 2000 mg/L
Second test: 22, 46, 100, 220, 460 and 1000 mg/L
Third test:3.2, 10, 32, 100, 320 and 1000 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: all glass open bottles/vessels, fill volume: 500 mL
- Aeration: yes, the aeration was adjusted in such a way that the dissolved oxygen concentration at the start was above 60-70% saturation (60% of air saturation is > 5 mg/L at 20°C) and to maintain the sludge flocs in suspension.
- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6
- No. of vessels per abiotic control (replicates): 1
- Sludge concentration (weight of dry solids per volume): 3.0 g/L after collection; 1.5 g/L in the final test solutions
- Nutrients provided for bacteria: synthetic sewage feed

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Milli-RO water (tap water purified by reverse osmosis)

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED: inhibition of respiration at t=3 h

TEST CONCENTRATIONS
- Range finding study: 10, 100 and 1000 mg/L
- Results used to determined concentratins for the definitive test: yes, no significant respiration was observed at 10 and 100 mg/L and 46% inhibition of respiration was observed at 1000 mg/L. Based on these results, the EC50 was expected to be between 100 and 1000 mg/L.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: third test

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

11.8 mg/L

Nominal / measured:

nominal

Conc. based on:

other: anion

Basis for effect:

inhibition of total respiration

Remarks on result:

other: third test

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

20.2 mg/L

Nominal / measured:

nominal

Conc. based on:

other: cation

Basis for effect:

inhibition of total respiration

Remarks on result:

other: third test

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

772 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: third test

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

685 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: second test

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

612 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: first test

Details on results:

- The abiotic control used ini the combined limit/range-finding test showed that there was no oxygen uptake from abiotic processes.
- Since in the first and second test inhibition of respiration was observed at all concentrations, no NOEC could be determined and follow up tests were performed. For detailed results in individual tests, see 'any other information on results'.
- It should be noted that a statistically significant reduction of respiration was observed at a concentration of 22 mg/L in the second test, which was below the NOEC of the third test. However, a clear dose-response relation was observed in the first and last test, while the dose response in the second test was less clear and monotonous. As the effects in the first and last test were furthermore comparable, it is considered justified to conclude that the NOEC was 32 mg/L.
- The reference substance showed that the used batch of sludge had normal sensitivity.

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Concentrations tested (nominal): 1.0, 3.2, 10 and 32 mg/L
- Relevant effect levels:
First test: EC50: 2.74 mg/L (95% CI: 0.89-4.71)
Second test: EC50: 4.90 mg/L (95% CI: 2.24-7.63)
Third test: EC50: 3.10 mg/L (95% CI: 0.78-5.40)
- Other: result was within the accepted range of 2 to 25 mg/L for total respiration.

Reported statistics and error estimates:

EC50 determination:
For the reference item calculation of the EC50 value was based on a 3-parameter logistic cumulative distribution function (CDF) using a non-linear regression analysis with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the reference item.
For the test item calculation of ECx values were based on probit analysis using linear maximum likelihood regression with the percentages of respiration inhibition versus the logarithms of the corresponding concentrations of the test item.

NOEC determination:
An effect was considered to be significant if statistical analysis of the data obtained for the test concentrations compared with those obtained in the control revealed significant inhibition of the respiration rate (Two-sample t-test Procedure, α=0.05, one-sided, smaller).

All calculations were performed with ToxRat Professional v. 3.2.1. (ToxRat Solutions® GmbH, Germany).

Table 1 Results of the first test

Treatment	Concentration (mg/L)	Mean respiration rate		% Inhibition of the respiration rate (mean value)
		(mg O2/L h)	(mg O2/g h)¹
Control		46.33	30.89
T1	200	32.24	21.49*	30.42
T2	360	28.28	18.86*	38.95
T3	640	22.31	14.87*	51.85
T4	1120	17.97	11.98*	61.20
T5	2000	13.57	9.05*	70.71

¹) The amount of suspended solids in the final test mixture was 1.5 g/L.

* Statistically significantly different compared to control.

Table 2 Results of the second test

Treatment	Concentration (mg/L)	Mean respiration rate		% Inhibition of the respiration rate (mean value)
		(mg O2/L h)	(mg O2/g h)¹
Control		49.84	33.22
T1	22	41.01	27.34*	17.71
T2	46	44.94	29.96*	9.83
T3	100	38.62	25.74*	22.51
T4	220	34.48	22.98*	30.82
T5	460	28.38	18.92*	43.05
T6	1000	20.67	13.78*	58.52

¹) The amount of suspended solids in the final test mixture was 1.5 g/L.

* Statistically significantly different compared to control.

Table 3 Results of the third test

Treatment	Concentration (mg/L)	Mean respiration rate		% Inhibition of the respiration rate (mean value)
		(mg O2/L h)	(mg O2/g h)¹
Control		36.65	24.44
T1	3.2	29.32	19.55	20.01
T2	10	36.99	24.66	-0.92
T3	32	36.38	24.25	0.75
T4	100	30.08	20.05*	17.93
T5	320	25.10	16.74*	31.51
T6	1000	16.27	10.85*	55.60

¹) The amount of suspended solids in the final test mixture was 1.5 g/L.

* Statistically significantly different compared to control.

Table 4 Overview of effect parameters

Parameter	TBEAES DRY Concentration (mg/L)
	First Full Test	Second Full Test	Third Full Test
NOEC	n.d.	n.d.	32
EC10	n.d.	24 (13 – 36)	58 (32 - 87)
EC20	102 (81 - 122)	76 (53 – 99)	141 (96 - 185)
EC50	612 (566 - 662)	685 (540 - 926)	772 (612 - 1043)

n.d. not determined

Validity criteria fulfilled:

yes

Remarks:

see 'overall remarks' section for details

Conclusions:

Based on the results of three respiration inhibition tests, performed according to OECD 209 and GLP principles, the EC50 for TBEAES DRY was between 612 and 772 mg/L. The NOEC was determined to be 32 mg/L.

Executive summary:

A respiration inhibition test was performed, according to OECD 209 and GLP principles, to assess whether TBEAES DRY was toxic to wastewater bacteria. The test was repeated three times since no NOEC could be determined in the first and second test. After a limit/dose-range finding test, the test substance was tested at loading rates of 200, 360, 640, 1120 and 2000 mg/L in the first test, 22, 46, 100, 220, 460 and 1000 mg/L in the second test and 3.2, 10, 32, 100, 320 and 1000 mg/L in the third test. The exposure period was 3 hours. For each concentration, five replicates were tested next to six blank controls and one abiotic control in the limit/dose-range finding test. The results of the limit/dose-range finding test showed that there was no oxygen uptake from abiotic processes. In the first and second test, inhibition of respiration was observed at all tested concentrations. In the third test, a NOEC of 32 mg/L was determined. Since in the second test no clear dose-response relation was observed, while this was the case for the first and third test, the reduction of respiration in the second test was not taken into account for determination of the NOEC. The EC50 values were 612, 685 and 772 mg/L for the first, second and third test, respectively. Testing with 3,5-dichlorophenol as reference item showed the test system had normal sensitivity. All validity criteria were met and the study was considered to be valid.

In conclusion, TBEAES DRY was not toxic to wastewater bacteria at or below a concentration of 32 mg/L.

Description of key information

A respiration inhibition test was performed, according to OECD 209 and GLP principles, to assess whether TBEAES DRY was toxic to wastewater bacteria. The test was repeated three times since no NOEC could be determined in the first and second test. After a limit/dose-range finding test, the test substance was tested at loading rates of 200, 360, 640, 1120 and 2000 mg/L in the first test, 22, 46, 100, 220, 460 and 1000 mg/L in the second test and 3.2, 10, 32, 100, 320 and 1000 mg/L in the third test. The exposure period was 3 hours. For each concentration, five replicates were tested next to six blank controls and one abiotic control in the limit/dose-range finding test. The results of the limit/dose-range finding test showed that there was no oxygen uptake from abiotic processes. In the first and second test, inhibition of respiration was observed at all tested concentrations. In the third test, a NOEC of 32 mg/L was determined. Since in the second test no clear dose-response relation was observed, while this was the case for the first and third test, the reduction of respiration in the second test was not taken into account for determination of the NOEC. The EC50 values were 612, 685 and 772 mg/L for the first, second and third test, respectively. Testing with 3,5-dichlorophenol as reference item showed the test system had normal sensitivity. All validity criteria were met and the study was considered to be valid. In conclusion, the results of the third test are used for the CSA and therefore TBEAES DRY was determined to be not toxic to wastewater bacteria at or below a concentration of 32 mg/L (3h-NOEC). Corresponding to 11.8 and 20.2 mg/L for the anion and cation, respectively.