[No public or meaningful name is available]

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:

11-12-2019 to 27-01-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Version / remarks:

The effect of the test substance on the inhibition of respiration rate of aerobic wastewater microorganisms of activated sludge was investigated. The test organisms were exposed to a series of test concentrations of the test substance for 3 hours and the effects on oxygen up take were measured. Results are reported relative to the reference standard controls: copper (II) sulphate pentahydrate and/or N-Methylaniline (with and without ATU).

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: July 2016 ; signature: January 2017

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct addition by pipette. Corresponding volumes were pipetted directly in laboratory bottles, for concentrations: 0 (control), 10, 32, 100, 320, 1000 mg/L test item (with and without ATU) in a geometrical series and with a dilution factor of 3.2. No pH adjustment was made. The ultrapure water, synthetic waste water and activated sludge were placed first in the test vessels. All concentrations were pipetted directly in the test solution in laboratory bottles. The bottles were closed immediately.
Triplicates of the control (without ATU) without test item were included at the beginning and at the end of the replicates without ATU. The vessels were 1000 mL laboratory bottles with screw cap, ISO 4796 with a 500 mL fill volume test medium and/or for specific inhibition of nitrification : 2.32 g/L stock solution of N-allylthiourea (ATU) in demineralized water. 2.5 mL of the stock solution correspond to 11.6 mg ATU/L in the test solutions. Full details of the dilution tables for the test vessels (with and without ATU) are provided in the full study report. Optimal contact between the test item and test organisms was ensured applying continuous aeration and shaking : at 150 rpm to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension. The flasks were closed with the vessel screw caps.
- Eluate: Not applicable.
- Differential loading: Not applicable.
- Controls: For positive control - reference item 1: Copper (II) sulphate pentahydrate was tested with three concentrations within the concentration range: 58, 100, 180 mg/L
and a dilution factor of approximately 1.8 (without ATU). For positive control - reference item 2: N-Methylaniline was tested with three concentrations within the concentration range 0.1, 1.8, 32 mg/L and a dilution factor of 18 (with and without ATU). A negative/blank control without test item or reference item was also included.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Not applicable.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None reported.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: aerobic activated sludge from 30982 Pattensen, Germany municipal sewage treatment plant
- Method of cultivation: See below.
- Preparation of inoculum for exposure: The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %.
- Pretreatment: The sludge was used within 24 h after sampling. The dry sludge concentration was 2.74 g/L, corresponding to 1.37 g/L in the test vessel for replicates
with and without ATU. The test item was not pretreated. The reference item 1 [Copper (II) sulphate pentahydrate] was ultrasonicated, whereas reference item 2 [N-Methylaniline] for nitrification was not pretreated.
- Other: Synthetic waste water was prepared according to OECD TG 209. The pH value of the activated sludge was determined prior to test start. The pH value of the synthetic waste water was determined prior to use. Adjustment to 7.5 ± 0.5 was not necessary.
- Initial biomass concentration: For ‘Total Respiration inhibition’ suspended solids 1.37 g per litre (aerobic sludge per litre water); for ‘Inhibition of Heterotrophic Respiration’: suspended solids 1.37 g per litre (aerobic sludge per litre water) and for ‘Measured Inhibition of Nitrification’: suspended solids 1.37 g per litre (aerobic sludge per litre water).

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

3 h

Remarks on exposure duration:

In accordance with the OECD TG 209 guideline.

Test temperature:

nominal 18 - 22 or 20 ± 2°C: actual Definitive test : 20.9 °C

pH:

The pH value of the activated sludge was determined prior to test start. The pH value of the synthetic waste water was determined prior to use. Adjustment to 7.5 ± 0.5 was not necessary.
In the definitive test : ‘Total Respiration inhibition’: pH value of the activated sludge was pH = 7.40 and the pH value of the synthetic waste water was pH = 7.21 ; ‘Measured Inhibition of Nitrification’: pH value of the activated sludge without ATU was pH = 7.40 and with ATU was pH = 7.40. The pH value of the synthetic waste water was pH = 7.21

Dissolved oxygen:

In the definitive test : Aeration was employed by shaking of flasks at 150 rpm to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension. The flasks were closed with screw caps.

Nominal and measured concentrations:

Preliminary test (non-GLP range finder) was conducted: 10, 100 and 1000 mg/L nominal (test item); with and without ATU at 1000 mg/L nominal (test item).
Based on this preliminary test the definitive test was conducted with five concentrations at up to 1000 mg/L with a dilution factor of 3.2.
For definitive test : ‘Total Respiration inhibition’ and : The nominal concentrations tested were: 0 (control), 10, 32, 100, 320, 1000 mg/L (in five replicates per concentration). The nominal concentrations of reference inhibitor copper (II) sulphate pentahydrate were 58, 100 and 180 mg/L (triplicate per concentration without ATU) ; and ‘Measured Inhibition of Nitrification’: The nominal concentrations tested were: 0 (control), 10, 32, 100, 320, 1000 mg/L (in five replicates per concentration without and with ATU). The nominal concentrations of reference inhibitor N-Methylaniline were 0.1, 1.8 and 32 mg/L (triplicate per concentration with and without ATU)

Details on test conditions:

TEST SYSTEM
- Test vessel: 1000 mL laboratory bottles
- Type (delete if not applicable): Open, vessel continuously aerated with seal.
- Material, size, headspace, fill volume: glass, 500 mL fill volume. See table 1 and 3.
- Aeration: Continuously aerated with compressed air.
- Type of flow-through (e.g. peristaltic or proportional diluter): Not reported.
- Renewal rate of test solution (frequency/flow rate): None.
- No. of vessels per concentration (replicates): Five
- No. of vessels per control (replicates): Six (negative) and three (reference items)
- Nitrification inhibitor used (delete if not applicable): N-allylthiourea (ATU)
- Biomass loading rate: See table 1 and 3.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Synthetic sewage water was in accordance with OECD TG 209
- Culture medium different from test medium: Yes, chlorine free tap water used for culture medium; synthetic wastewater and demineralised water used for test medium. Replaced during inoculum pretreatment stage and media preparation.
- Intervals of water quality measurement: pH and temperature were determined in all test media and controls ; prior to and at the end of the 3 hour incubation period. Dissolved oxygen values was determined in the 100 mg/L test item vessels only and/or within preliminary test.

OTHER TEST CONDITIONS
- Adjustment of pH: No.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Monitor the oxygen consumed by the test and control mixtures following a 3-hour exposure phase. The oximeter instrument measured the oxygen depletion for at least 5 minutes.

TEST CONCENTRATIONS
- Test concentrations: See table 1 and 3.
- Range finding study
- Test concentrations: 10, 100 and 1000 mg/L nominal (test item); with inhibitions with and without ATU at 1000 mg/L nominal (test item).
- Results used to determine the conditions for the definitive study: Yes.

Reference substance (positive control):

yes

Remarks:

copper (II) sulphate pentahydrate

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 10 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% C.I.: - mg/L

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% C.I.: - mg/L

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks on result:

other: 95% C.I.: - mg/L

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks on result:

other: 95% C.I.: - mg/L

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Remarks on result:

other: 95% C.I.: - mg/L

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

> 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Remarks on result:

other: 95% C.I.: - mg/L

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

>= 1 000 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Remarks on result:

other: No statistically significant inhibition at ≥ 1000 mg/L (P < or = 0.01) for the inhibition of total respiration and inhibition of the heterotrophic respiration and inhibition nitrification

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None reported.
- Effect concentrations exceeding solubility of substance in test medium: No effects observed at up to 1000 mg/L nominal test item.
- Adsorption (e.g. of test material to the walls of the test container): None reported.
- Blank controls oxygen uptake rate: See tables. Mean specific oxygen uptake rate of control replicates : 18.4 mg O2/g h; this was less than 20 mg O2/g h, whereas the oxygen uptake of activated sludge from municipal treatment plants is affected 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. For further information on positive controls, see below.
- Coefficient of variation of oxygen uptake rate in control replicates: See tables. Coefficient of variation of the oxygen uptake rates in the control replicates : 6.9% this was less than 30%.
- Other: All validity criteria were considered to be met.

Results with reference substance (positive control):

- Results with reference substance valid?: Yes.
- Relevant effect levels: for copper (II) sulphate: Definitive Test (total respiration inhibition): EC50 88.8 (95% C.I. 86.4 – 91.2) mg/L ; N-Methylaniline was additionally tested (with and without ATU) at three different concentrations: 0.1, 1.8 and 32 mg/L and a dilution factor of 18. Full information is provided in the full study report. For N-Methylaniline Definitive Test (Inhibition of Nitrification): EC50 1.04 (95% C.I. 0.429 – 2.24) mg/L.
- Other: Yes the reference substance results were valid, the EC50 for copper (II) sulphate within the expected range: 53 to 155 mg/L

Reported statistics and error estimates:

The NOEC was determined by calculation of statistical significance of the inhibition of respiration in comparison to the control. The NOEC for the oxygen uptake rate was determined with the software ToxRat Professional. The NOEC was determined by calculation of statistical significance of the oxygen uptake rate in comparison to the control using statistical standard procedure as normality test (Shapiro-Wilk), variance homogeneity test (Levene’s test) and a multiple test (Williams Multiple Sequential t-test) along with One Way Analysis of Variance (ANOVA), as applicable. Prior to running the multiple tests, a normality test and a variance homogeneity test were performed. The P-value for all tests was 0.01. The alpha-value (acceptable probability of incorrectly concluding that there is a difference) is alpha = 0.05. The EC-values of the test item and the reference item were calculated by biphasic regression and linear regression, respectively, using software GraphPadPrism. Full details of the statistical analysis are provided in the full study report.

No statistically significant inhibition at the highest tested concentration (P < or = 0.01) for the inhibition of total respiration and inhibition of the heterotrophic respiration and inhibition nitrification was observed.

Table 4. Definitive Test - ‘Total Respiration inhibition’: Oxygen uptake rates and Inhibition of the respiration of the control and test item

Test Item Concentration	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Inhibition	Mean Inhibition
[mg/L]		[mg O2/L]	[mg O2/(L×h)]	[%]	[%]
10	1	6.52	27.5	-9	-7
	2	6.22	27.8	-10
	3	5.76	26.4	-5
	4	7.41	25.5	-1
	5	6.54	28.0	-11
32	1	6.19	26.5	-5	-8
	2	5.63	27.3	-8
	3	5.57	26.8	-6
	4	6.54	28.1	-12
	5	5.32	26.9	-7
100	1	5.81	28.0	-11	-10
	2	5.67	27.7	-10
	3	6.06	27.5	-9
	4	6.96	28.5	-19
	5	5.80	27.3	-8
320	1	6.28	27.3	-8	-10
	2	6.07	27.9	-11
	3	6.26	27.2	-8
	4	7.07	28.1	-12
	5	5.89	27.4	-9
1000	1	6.09	27.9	-11	-11
	2	5.86	27.1	-8
	3	5.34	27.5	-9
	4	9.34	28.8	-14
	5	5.71	28.0	-11

*) determined at start of the measurement

Repl. = Replicate;

Coefficient of variation of oxygen uptake rates of the control: 6.9%: Suspended solids in the test vessel: 1.37 g/L

 

Table 5. Definitive Test : Specific respiration rates of the control replicates

Test Item Concentration	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Specific Respiration Rate Rs
[mg/L]		[mg O2/L]	[mg O2/(L×h)]	[mg O2/g×h]
0 (Control)	1	7.51	27.2	19.9
	2	7.18	26.7	19.5
	3	6.67	26.2	19.1
	4	7.57	23.6	17.2
	5	7.56	24.9	18.2
	6	6.86	22.3	16.3
	Mean value		25.2	18.4
	Standard deviation		1.7
	CV [%]		6.9

*) determined at start of the measurement

Repl. = Replicate;

Coefficient of variation of oxygen uptake rates of the control: 6.9%: Suspended solids in the test vessel: 1.37 g/L

 

Table 6. Definitive Test : ‘Inhibition of the Heterotrophic Respiration’ and ‘Inhibition of Nitrification’: Oxygen uptake rates and Inhibition of the respiration of the control and test item

	Inhibition of the Heterotrophic Respiration Note: Suspended solids in the test vessel: 1.37 g/L
	Test Concentration [mg/L]	Repl.	Oxygen Concentration* [mg O2/L]	Oxygen Uptake Rate R [mg O2/L×h]		Inhibition [%]	Mean Inhibition [%]
	10 + ATU *1	1	7.31	13.0		2	-3
		2	7.42	13.4		-2
		3	7.07	13.6		-3
		4	7.74	14.1		-7
		5	7.14	14.0		-6
	32 + ATU	1	7.06	13.6		-3	-7
		2	7.05	14.2		-8
		3	7.05	14.0		-6
		4	7.49	14.2		-8
		5	7.02	14.3		-8
	100 + ATU	1	6.74	14.4		-9	-11
		2	7.05	14.7		-11
		3	6.95	14.5		-10
		4	7.45	14.8		-12
		5	6.82	14.9		-13
	320 + ATU	1	7.11	15.3		-16	-16
		2	6.99	15.4		-17
		3	7.16	15.3		-16
		4	7.48	15.4		-17
		5	7.15	15.0		-14
	1000 + ATU	1	7.09	16.2		-23	-24
		2	6.91	16.2		-23
		3	6.65	16.8		-27
		4	7.38	16.4		-24
		5	6.95	16.3		-23
	Inhibition of Nitrification Note: Suspended solids in the test vessel: 1.37 g/L
	Test Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]		Inhibition [%]		Mean Inhibition [%]
	10 **2	1	13.9		-16		-12
		2	14.2		-18
		3	12.8		-6
		4	11.9		1
		5	14.4		-20
	32	1	12.4		-4		-9
		2	13.2		-10
		3	12.7		-6
		4	14.0		-17
		5	12.8		-7
	100	1	13.3		-11		-9
		2	13.0		-9
		3	18.8		-7
		4	13.8		-15
		5	12.6		-5
	320	1	12.0		0		-2
		2	12.6		-5
		3	11.9		1
		4	12.8		-7
		5	12.1		-1
	1000	1	11.5		4		4
		2	10.7		11
		3	11.1		7
		4	12.4		-4
		5	11.6		3

*) determined at start of the measurement

Repl. = Replicate

**1: Coefficient of variation of oxygen uptake rates of the control: 1.5%

**2: Coefficient of variation of oxygen uptake rates of the control: 15.1%

 

Table 7. Definitive Test - Specific respiration rates of the Heterotrophic Respiration control replicates

	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R
		[mg O2/L]	[mg O2/(L×h)]
Control	1	7.95	13.1
	2	7.67	13.4
	3	7.59	12.9
	4	7.87	13.3
	5	7.60	13.5
	6	7.78	13.2
	Mean value		13.2
	Standard deviation		0.2
	CV [%]		1.5

*) determined at start of the measurement

 

Table 8. Definitive Test - Specific respiration rates of the Nitrification control replicates

	Repl.	Oxygen Uptake Rate R
		[mg O2/(L×h)]
Control	1	14.1
	2	13.3
	3	13.3
	4	10.3
	5	11.4
	6	9.1
Mean value		11.9
Standard deviation		1.8
CV [%]		15.1

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 3-hour EC50 for total inhibition was > 1000 mg/L. The equivalent EC10 was > 1000 mg/L. The 3-hour EC50 for heterotrophic respiration inhibition was > 1000 mg/L and EC10 was > 1000 mg/L. The 3-hour EC50 nitrification inhibition was > 1000 mg/L and EC10 was > 1000 mg/L. The NOEC was ≥ 1000 mg/L. All effect levels were based on nominal test item concentrations.

Executive summary:

The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 in accordance with GLP. A definitive test was carried out under static conditions with the test item concentrations 10, 32, 100, 320 and 1000 mg/L with and without ATU. 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. Samples of activated sludge (suspended solids 1.37 g/L for total respiration inhibition and inhibition of nitrification, respectively) were exposed to dilutions of the test substance for three hours under static conditions with the concentrations: 10, 32, 100, 320 and 1000 mg/L with and without ATU inhibitor. Their rates of oxygen consumption were determined using an oximeter and were compared with controls that contained activated sludge alone. Five replicates were prepared for each concentration and six for the control. To check the activity of the test system and the test conditions single triplicates of the reference inhibitor copper (II) sulphate were used at 58, 100 and 180 mg/L. Furthermore, to confirm the activity of the test system and the test conditions a further reference test was carried out with N-Methylaniline (with and without ATU) and the reference toxicity was subsequently determined. The mean specific respiration rate (Rs) of the control cultures incubated alongside the test mixtures was 18.4 mg O2/g h. The oxygen uptake of activated sludge from municipal treatment plants is affected 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. The oxygen consumption rates of the two negative (blank; inoculum) controls differed by 6.9%. Therefore, meeting the guideline validity criteria (< 30%). The reference substance results were valid, the EC50 for copper (II) sulphate was: 88.8 mg/L. This was within the expected range: 53 to 155 mg/L. The EC50 value for the reference item N-Methylaniline was 1.04 mg/L. These results show that the sample of activated sludge employed was sensitive to inhibition and that the test was valid. The mean inhibition of total respiration for the test item replicates ranged from -11 % to -7 %. The mean inhibition of the heterotrophic respiration for the test item replicates ranged from -24 % to -3 %. The mean inhibition of nitrification for the test item replicates ranged from -12 % to 4 %.Under the conditions of the study, the 3-hour EC50 for total inhibition was > 1000 mg/L. The equivalent EC10 was > 1000 mg/L. The 3-hour EC50 for heterotrophic respiration inhibition was > 1000 mg/L and EC10 was > 1000 mg/L. The 3-hour EC50 nitrification inhibition was > 1000 mg/L and EC10 was > 1000 mg/L. The NOEC was ≥ 1000mg/L. All effect levels were based on nominal test item concentrations.

Description of key information

Total Respiration Inhibition:

3h-EC50 = > 1000 mg/L (nominal), 20 °C

3h-EC10 = > 1000 mg/L (nominal), 20°C

NOEC = ≥ 1000mg/L(nominal), 20 °C, OECD TG 209, 2020

 

Heterotrophic Respiration Inhibition:

3h-EC50 = > 1000 mg/L (nominal), 20 °C

3h-EC10 = > 1000 mg/L (nominal), 20 °C

NOEC = ≥ 1000mg/L(nominal), 20 °C, OECD TG 209, 2020

 

Nitrification Inhibition:

3h-EC50 = > 1000 mg/L (nominal), 20 °C

3h-EC10 = > 1000 mg/L (nominal), 20 °C

NOEC = ≥ 1000mg/L(nominal), 20 °C, OECD TG 209, 2020

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

1 000 mg/L

Additional information

Key study : OECD TG 209, 2020 : The effect on respiration rate of activate sludge was examined using a method according to OECD TG 209 in accordance with GLP. A definitive test was carried out under static conditions with the test item concentrations 10, 32, 100, 320 and 1000 mg/L with and without ATU. 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. Samples of activated sludge (suspended solids 1.37 g/L for total respiration inhibition and inhibition of nitrification, respectively) were exposed to dilutions of the test substance for three hours under static conditions with the concentrations: 10, 32, 100, 320 and 1000 mg/L with and without ATU inhibitor. Their rates of oxygen consumption were determined using an oximeter and were compared with controls that contained activated sludge alone. Five replicates were prepared for each concentration and six for the control. To check the activity of the test system and the test conditions single triplicates of the reference inhibitor copper (II) sulphate were used at 58, 100 and 180 mg/L. Furthermore, to confirm the activity of the test system and the test conditions a further reference test was carried out with N-Methylaniline (with and without ATU) and the reference toxicity was subsequently determined. The mean specific respiration rate (Rs) of the control cultures incubated alongside the test mixtures was 18.4 mg O2/g h. The oxygen uptake of activated sludge from municipal treatment plants is affected 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. The oxygen consumption rates of the two negative (blank; inoculum) controls differed by 6.9%. Therefore, meeting the guideline validity criteria (< 30%). The reference substance results were valid, the EC50 for copper (II) sulphate was: 88.8 mg/L. This was within the expected range: 53 to 155 mg/L. The EC50 value for the reference item N-Methylaniline was 1.04 mg/L. These results show that the sample of activated sludge employed was sensitive to inhibition and that the test was valid. The mean inhibition of total respiration for the test item replicates ranged from -11 % to -7 %. The mean inhibition of the heterotrophic respiration for the test item replicates ranged from -24 % to -3 %. The mean inhibition of nitrification for the test item replicates ranged from -12 % to 4 %.Under the conditions of the study, the 3-hour EC50 for total inhibition was > 1000 mg/L. The equivalent EC10 was > 1000 mg/L. The 3-hour EC50 for heterotrophic respiration inhibition was > 1000 mg/L and EC10 was > 1000 mg/L. The 3-hour EC50 nitrification inhibition was > 1000 mg/L and EC10 was > 1000 mg/L. The NOEC was ≥ 1000mg/L. All effect levels were based on nominal test item concentrations.