dec-1-en-4-yne

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:

09-08-2017 to 27-11-2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

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.

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

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: aerobic activated sludge from 31137 Hildesheim, 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 2.90 g/L, corresponding to 1.45 g/L in the test vessel. The test item was not pretreated. The reference item was ultrasonicated.
- 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 1.45 g per litre (aerobic sludge per litre water); for ‘Inhibition of Heterotrophic Respiration’: suspended solids 1.45 g per litre (aerobic sludge per litre water) and for ‘Measured Inhibition of Nitrification’: suspended solids 1.45 g per litre (aerobic sludge per litre water)

Test type:

static

Water media type:

freshwater

Limit test:

no

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 : 21.0 °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 non-GLP range finder: pH value measured during the study was pH = 7.59at final (test vessel at 1000 mg/L nominal test item)
In the definitive test : ‘Total Respiration inhibition’: pH value of the activated sludge was pH = 7.50 and the pH value of the synthetic waste water was pH = 7.28 ; ‘Measured Inhibition of Nitrification’: pH value of the activated sludge without ATU was pH = 7.50 and with ATU was pH = 7.62. The pH value of the synthetic waste water was pH = 7.28

Dissolved oxygen:

In the non-GLP range finder: Two replicates were directly sampled after stirring for 10 minutes and two replicates were stirred for 10 minutes and aerated by shaking at 150 rpm for 3 h. Then the flasks were sampled afterwards. Dissolved oxygen concentrations: 100 mg/L nominal test item: 2.93 – 2.14 mg O2 /L before incubation and aeration and 7.68 – 5.29 mg O2/L after 3 hours incubation and aeration.
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): 10, 100 and 1000 mg/L nominal (test item); with and without ATU at 1000 mg/L nominal (test item).
For definitive test : ‘Total Respiration inhibition’ and : The nominal concentrations tested were: 0 (control), 1.6, 8.0, 40, 200, 900 and 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), 1.6, 8.0, 40, 200, 900 and 1000 mg/L (in five replicates per concentration without and with ATU). The nominal concentrations of reference inhibitor N-Methyllaniline were 0.1, 1.8 and 32 mg/L (triplicate per concentration with and without ATU)
Note: One additional concentration of 900 mg/L (with and without ATU) was measured in order to better describe the course of the inhibition of heterotrophic respiration.

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): Five (negative) and three (reference item)
- 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.:

38.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% C.I.: 33.1 - 45.4 mg/L

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

11.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% C.I.: 8.94 – 14.0 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.:

17.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks on result:

other: 95% C.I.: 11.7 – 24.8 mg/L

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

22.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Remarks on result:

other: 95% C.I.: 18.6 – 26.0 mg/L

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

9.64 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Remarks on result:

other: 95% C.I.: 7.69 – 12.5 mg/L

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

32 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 8 mg/L (P < 0.001, ANOVA, Dunnett`s Method) for the inhibition of total respiration and inhibition of the heterotrophic respiration and inhibition nitrification

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 103 (95% C.I. 96.3- 110) 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 1.73 (95% C.I. 1.47 – 2.04) 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. 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 α-value (acceptable probability of incorrectly concluding that there is a difference) is α =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 lowest tested concentration (P < 0.001, ANOVA, Dunnett`s Method) for the inhibition of total respiration and inhibition of the heterotrophic respiration and inhibition nitrification was observed.

Table 5. 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)]	[%]	[%]
1.6	1	6.61	25.4	-14	-5
	2	6.97	21.6	3
	3	5.66	22.4	0
	4	6.72	24.7	-11
	5	6.38	23.0	-3
8	1	7.29	23.9	-7	3
	2	7.16	21.0	6
	3	6.58	20.7	7
	4	7.34	22.2	0
	5	6.93	19.8	11
40	1	6.63	11.5	48	51
	2	6.40	11.7	48
	3	6.01	10.7	52
	4	6.39	11.3	49
	5	5.89	9.3	58
200	1	7.14	8.0	64	66
	2	7.17	7.4	67
	3	6.43	7.3	67
	4	6.81	7.9	65
	5	6.24	7.3	67
900	1	7.27	7.2	68	69
	2	7.55	7.4	67
	3	6.63	6.0	73
	4	7.12	7.2	68
	5	6.46	6.7	70
1000	1	7.36	7.4	67	68
	2	7.31	7.5	66
	3	6.67	7.0	69
	4	7.06	7.3	67
	5	6.58	7.0	69

*) determined at start of the measurement

Repl. = Replicate;

Coefficient of variation of oxygen uptake rates of the control: 7.8 %: Suspended solids in the test vessel: 1.45 g/L

 

Table 6. Definitive Test : 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	6.12	23.9	16.5
	2	6.16	25.1	17.3
	3	5.25	21.9	15.1
	4	6.33	219	15.1
	5	5.82	21.5	14.8
	6	5.80	19.7	13.6
	Mean value		22.3	15.4
	Standard deviation		1.74
	CV [%]		7.8

 

Table 7. 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: 1.45 g/L
	Test Concentration [mg/L]	Repl.	Oxygen Concentration* [mg O2/L]	Oxygen Uptake Rate R [mg O2/L×h]		Inhibition [%]	Mean Inhibition [%]
	1.6 + ATU *1	1	5.78	12.0		-15	-5
		2	5.75	11.1		-7
		3	5.30	10.1		3
		4	5.98	11.0		-6
		5	5.33	10.2		2
	8 + ATU	1	6.59	11.0		-6	1
		2	6.58	10.7		-3
		3	6.04	9.9		5
		4	6.58	10.0		4
		5	5.96	9.7		7
	40 + ATU	1	6.75	8.9		14	21
		2	6.76	8.7		16
		3	6.22	7.7		26
		4	6.60	8.1		22
		5	6.04	7.6		27
	200 + ATU	1	6.98	7.3		30	33
		2	6.89	7.4		29
		3	6.24	6.9		34
		4	6.50	6.9		34
		5	6.17	6.5		38
	900 + ATU	1	7.13	6.6		37	39
		2	7.04	6.9		34
		3	6.36	6.1		41
		4	6.86	6.1		41
		5	6.29	6.0		42
	1000 + ATU	1	7.12	6.9		34	37
		2	7.12	7.0		33
		3	6.48	6.3		39
		4	6.70	6.5		38
		5	6.20	6.2		40
	Inhibition of Nitrification Note: Suspended solids in the test vessel: 1.45 g/L
	Test Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]		Inhibition [%]		Mean Inhibition [%]
	1.6 **2	1	13.4		-13		-5
		2	10.5		12
		3	12.3		-3
		4	13.7		-15
		5	12.8		-8
	8	1	12.9		-8		5
		2	10.3		13
		3	10.8		9
		4	12.2		-3
		5	10.1		15
	40	1	2.6		78		77
		2	3.0		75
		3	3.0		75
		4	3.2		73
		5	1.7		86
	200	1	0.7		94		95
		2	0.0		100
		3	0.4		97
		4	1.0		92
		5	0.8		93
	900	1	0.6		95		95
		2	0.5		96
		3	-0.1		101
		4	1.1		91
		5	0.7		94
	1000	1	0.5		96		94
		2	0.5		96
		3	0.7		94
		4	0.8		93
		5	0.8		93

Repl. = Replicate

**1: Coefficient of variation of oxygen uptake rates of the control: 7.97 %

**2: Coefficient of variation of oxygen uptake rates of the control: 9.93 %

 

Table 8. 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	6.41	10.9
	2	6.36	11.0
	3	5.80	10.0
	4	7.15	11.0
	5	7.15	10.7
	6	6.66	8.7
	Mean value		10.4
	Standard deviation		0.827
	CV [%]		7.97

 

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

	Repl.	Oxygen Uptake Rate R
		[mg O2/(L×h)]
Control	1	10.9
	2	11.0
	3	10.0
	4	11.0
	5	10.7
	6	8.7
Mean value		11.9
Standard deviation		1.18
CV [%]		9.93

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the study, the 3 hour EC50 for total inhibition was 38.7 (C.I. 33.1 – 45.4) mg/L. The equivalent EC10 was 11.1 (C.I. 8.94 – 14.0) mg/L. The 3 hour EC50 for heterotrophic respiration inhibition was > 1000 (C.I. – ) mg/L and EC10 was 17.7 (C.I. 11.7 – 24.8) mg/L. The 3 hour EC50 nitrification inhibition was 22.2 (C.I. 18.6 – 26.0) mg/L and EC10 was 9.64 (C.I. 7.69 – 12.5) mg/L. The NOEC was 8 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 1.6, 8.0, 40, 200, 900 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 1.45 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: 1.6, 8.0, 40, 200, 900 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 22.3 mg O2/g h. The three-hour 50% effect concentration (EC50) for copper (II) sulphate was 103 mg/L.The EC50 value for the reference item N-Methylaniline was 1.73 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 - 5 % to 69 %. The mean inhibition of heterotrophic respiration for the test item replicates ranged from -5% to 39%. The mean inhibition of nitrification for the test item replicates ranged from- 5% to 95 %. Under the conditions of the study, the 3 hour EC50 for total inhibition was 38.7 (C.I. 33.1 – 45.4) mg/L. The equivalent EC10 was 11.1 (C.I. 8.94 – 14.0) mg/L. The 3 hour EC50 for heterotrophic respiration inhibition was > 1000 (C.I. – ) mg/L and EC10 was 17.7 (C.I. 11.7 – 24.8) mg/L. The 3 hour EC50 nitrification inhibition was 22.2 (C.I. 18.6 – 26.0) mg/L and EC10 was 9.64 (C.I. 7.69 – 12.5) mg/L. The NOEC was 8 mg/L. All effect levels were based on nominal test item concentrations.

Description of key information

Total Respiration Inhibition:

3h-EC50 = 38.7 (C.I. 33.1 – 45.4) mg/L (nominal), 20 °C

3h-EC10 = 11.1 (C.I. 8.94 – 14.0) mg/L (nominal), 20°C

NOEC = 8 mg/L (nominal), 20 °C, OECD TG 209, 2017

 

Heterotrophic Respiration Inhibition:

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

3h-EC10 = 17.7 (C.I. 11.7 – 24.8) mg/L (nominal), 20°C

NOEC = 8 mg/L (nominal), 20 °C, OECD TG 209, 2017

 

Nitrification Inhibition:

3h-EC50 = 22.2 (C.I. 18.6 – 26.0) mg/L (nominal)

3h-EC10 = 9.64 (C.I. 7.69 – 12.5) mg mg/L (nominal)

NOEC = 8 mg/L (nominal), 20 °C, OECD TG 209, 2017

 

The nitrification inhibition effect values are suggested to be used for subsequent environmental risk assessment, as the most conservative effect levels.

Key value for chemical safety assessment

EC50 for microorganisms:

22.2 mg/L

EC10 or NOEC for microorganisms:

8 mg/L

Additional information

Key study : OECD TG 209, 2017 : 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 1.6, 8.0, 40, 200, 900 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 1.45 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: 1.6, 8.0, 40, 200, 900 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 22.3 mg O2/g h. The three-hour 50% effect concentration (EC50) for copper (II) sulphate was 103 mg/L.The EC50 value for the reference item N-Methylaniline was 1.73 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 - 5 % to 69 %. The mean inhibition of heterotrophic respiration for the test item replicates ranged from -5% to 39%. The mean inhibition of nitrification for the test item replicates ranged from- 5% to 95 %. Under the conditions of the study, the 3 hour EC50 for total inhibition was 38.7 (C.I. 33.1 – 45.4) mg/L. The equivalent EC10 was 11.1 (C.I. 8.94 – 14.0) mg/L. The 3 hour EC50 for heterotrophic respiration inhibition was > 1000 (C.I. – ) mg/L and EC10 was 17.7 (C.I. 11.7 – 24.8) mg/L. The 3 hour EC50 nitrification inhibition was 22.2 (C.I. 18.6 – 26.0) mg/L and EC10 was 9.64 (C.I. 7.69 – 12.5) mg/L. The NOEC was 8 mg/L. All effect levels were based on nominal test item concentrations.