(2-hydroxy-1,1-dimethylethyl)ammonium toluene-4-sulphonate

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:

2020-06-09 to 2020-10-20, with the definitive exposure phase on 2020-10-20

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:

OECD 209 for Testing of Chemicals (2010)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

Test concentration:
Out of a stock solution the concentrations 500 - 707 - 1000 - 1414 - 2000 mg/L were tested, to determine the NOEC/LOEC and EC10. The concentrations are based on the results of the preliminary range finding test (non-GLP) and the first definite study.
Stock solution preparation:
5000 mg/L test item were freshly prepared with ultrapure water. The dispersion was agitated until the test item was visually diluted.
Out of a stock solution the concentrations 500 - 707 - 1000 - 1414 - 2000 mg/L were tested, to determine the NOEC/LOEC and EC10. The concentrations are based on the results of the preliminary range finding test (non-GLP) and the first definite study.
5000 mg/L test item were freshly prepared with ultrapure water. The dispersion was agitated until the test item was visually diluted.
5 Replicates
No vehicle, no pre-treatment

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Origin:
Municipal sewage treatment plant of 31137 Hildesheim, Germany

Reason for the selection:
Non-adapted activated sludge from the sewage plant at Hildesheim is well suited as it receives predominantly municipal sewage and hardly any industrial chemical waste.

Pretreatment:
The sludge was washed twice and adjusted with chlorine free tap water if necessary. The sludge was used within 24 h after sampling.

Date of receipt
2020-10-19

Dry sludge concentration
3.13 g/L, corresponding to 1.57 g/L in the test vessels for replicates

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

Nominal: 18 - 22 °C, measured: 20.1 °C

pH:

pH-value of the activated sludge: 7.54
pH-value of the synthetic waste water: 7.19

Dissolved oxygen:

Permanent aermanent, to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.

Nominal and measured concentrations:

Based on the results of the preliminary test (1 – 10 – 100 – 1000 mg test item/L) and the first definitive study (Limit test: NOEC < 1000 mg/L; 11% inhibition at 100 mg/L) this study was carried out with 5 concentrations within the range of 500 to 2000 mg/L.

Details on test conditions:

Based on the results of the preliminary test (see Annex I for details) and the first definitive study this study was carried out with 5 concentrations within the range of 500 to 2000 mg/L.
All concentrations were transferred out of a stock solution with a measuring flask and pipette into Erlenmeyer flasks.
Triplicates of the control without test item were included at the beginning and at the end of the replicates.
The reference item Copper (II)-sulfate pentahydrate was tested with three concentrations within the concentration range 58 - 180 mg/L and a dilution factor of approximately 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 to a dry sludge concentration of 3.0 g/L ± 10 %.

Reference substance (positive control):

yes

Remarks:

Reference Item: Copper (II) sulphate pentahydrate p.a. (MERCK) CAS No. :7758-99-8 Lot: AM0934890 Purity: 99.7% Expiry date: 2020-11-30 Stock solution: 300 g/L Test concentrations 58 - 100 - 180 mg/L Triplicates Range of validity (EC50): 53 - 155mg/L

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 2 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

2 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

1 414 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

In the reference test the EC50-value for copper (II) sulphate pentahydrate was
103 (95.4 – 111) mg/L. This is in the recommended range of validity of 53 - 155 mg/L.

Reported statistics and error estimates:

Shapiro-Wilk´s Test on Normal Distribution Normality check failed (p <= 0.01).
The Levene test indicates variance homogeneity (p > 0.010).
Variance homogeneity check was passed (p > 0.01).
Normal distribution is poore, but variance homogeneity requirements may be seen as fulfilled.
A parametric multiple test is yet possible.
To justify the use of the Step-down Jonckheere-Terpstra test at first a non-parametric trend analysis by contrasts is performed.
The linear trend is significant (p <= 0.05) The quadratic trend is not significant (p > 0.05)
The analysis of contrasts revealed a linear trend, thus the selected SD Jonckheere-Terpstra test was performed.

A NOEC of 1414.000 mg/L is suggested by the program.

Oxygen Uptake Rates and Percentage Inhibition of the Test Item Concentration of the First Definitive Study

Test Item Concentration	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Inhibition	Mean Inhibition
[mg/L]		[mg O2/L]	[mg O2/(L×h)]	[%]	[%]
1000	1	8.13	30.0	13	11
	2	7.87	32.1	7
	3	7.69	30.2	12

*) determined at start of the measurement

 

Second definitive study:

Oxygen Uptake Rates and Specific Respiration Rates of the Control

	Repl.	Oxygen Concentration*	Oxygen Uptake Rate RT	Specific Respiration Rate Rs
		[mg O2/L]	[mg O2/(L×h)]	[mg O2/g×h]
Control	1	7.55	30.6	19.5
	2	6.32	29.9	19.0
	3	6.69	29.9	19.0
	4	6.46	28.4	18.1
	5	7.30	29.0	18.5
	6	7.46	29.2	18.6
	Mean value		29.5	18.8
	Standard deviation		0.7
	CV [%]		2.4

*) determined at start of the measurement

Repl. = Replicate ; CV = Coefficient of variation

 

Oxygen Uptake Rates and Inhibition of the Test Item Concentrations

 

Test item Concentration	Rep	Oxygen Concentration*	Oxygen Uptake Rate RT	Inhibition	Mean Inhibition
[mg/L]		[mg O2/L]	[mg O2/(L×h)]	[%]	[%]
500	1	7.32	31.4	-6	-1
	2	7.16	30.9	-5
	3	7.70	30.1	-2
	4	7.80	30.4	-3
	5	7.41	26.6	10
707	1	7.56	28.8	2	2
	2	7.46	30.9	-5
	3	6.98	30.4	-3
	4	7.11	30.3	-3
	5	6.69	24.2	18
1000	1	7.55	30.0	-2	1
	2	7.78	30.4	-3
	3	7.61	29.4	0
	4	7.08	30.6	-4
	5	7.59	25.3	14
1414	1	7.91	29.7	-1	2
	2	6.83	29.9	-1
	3	7.06	28.2	4
	4	7.02	28.2	4
	5	7.11	28.8	2
2000	1	7.40	27.7	6	10
	2	7.82	27.7	6
	3	7.75	26.8	9
	4	7.67	26.6	10
	5	8.11	23.8	19

              *) determined at start of the measurement

              Repl. = Replicate

 

 

Validity Criteria

 

Validity Criterion	Required	First and Second Study
Mean specific oxygen uptake rate of the control replicates	³ 20 mg O2/g×h	23.4 mg O2/g×h (first) 18.8 mg O2/g×h*
Coefficient of variation of the oxygen uptake rates in the control replicates	< 30 %	2.9 % (first) 2.4 %
EC50 of the reference item	53 - 155 mg/L	101 mg/L (first) 103 mg/L

*The oxygen uptake of activated sludge from municipal treatment plants is influenced by seasonal fluctuations and fluctuations in sewage flow etc. The lower oxygen uptake rate reflects the range of common uptake rates of activated sludge from municipal treatment plants and was considered to have no impact on quality and integrity of the study. Despite the lower respiration, the EC₅₀-value of the reference item was in the required range. Therefore, no impact on the results of the test item can be expected through lower respiration.

 

 

Validity criteria fulfilled:

yes

Remarks:

The oxygen uptake of activated sludge is influenced by seasonal fluctuations etc. The lower oxygen uptake rate (18.8 mgO2/g*h) reflects the range of common fluctuations and was considered to have no impact on quality, integrity of the test.

Conclusions:

The NOEC of Propan-1-ol, 2-amino-2-methyl-, 4-methylbenzenesulphonate is 1414 mg/L.
The EC10 is 2000 mg/L and the EC50 is > 2000 mg/L.

Executive summary:

A respiration inhibition test with activated sludge according to OECD Guideline No. 209 was carried out with the test item Propan-1-ol, 2-amino-2-methyl-, 4-methylbenzenesulphonate. 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 500 - 707 - 1000 - 1414 - 2000 mg/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 total respiration for the test item replicates ranged from -1 % to 10 %.

The NOEC of Propan-1-ol, 2-amino-2-methyl-, 4-methylbenzenesulphonate is 1414 mg/L.
The EC10 is 2000 mg/L and the EC50 is > 2000 mg/L.

Description of key information

A respiration inhibition test with activated sludge according to OECD Guideline No. 209 was carried out with the test item Propan-1-ol, 2-amino-2-methyl-, 4-methylbenzenesulphonate. The NOEC of Propan-1-ol, 2-amino-2-methyl-, 4-methylbenzenesulphonate is 1414 mg/L.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 414 mg/L