Undecanal

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:

2018-12-19 to 2018-12-21

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:

adopted July 2010

Deviations:

yes

Remarks:

specific inhibitor of nitrification used was 1-Octyne instead of ATU, because of chemical reaction of ATU with test item

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

The test item was directly added into test media.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Test System Activated sludge

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.

Pre-treatment The sludge was washed twice and was used within 24 h after sampling.

Date of receipt 2018-12-20

Dry sludge concentration 3.29 g/L, corresponding to 1.65 g/L in the test vessel for replicates without OCT and 3.30 g/L, corresponding to 1.65 g/L in the test vessel for replicates with OCT.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

no

Hardness:

See IUCLID section "Any other information on materials and methods incl. tables" below.

Test temperature:

18 - 22 °C, actual: 20.8 °C

pH:

The pH value of the activated sludge was determined prior to test start (7.38-7.43). The pH value of the synthetic waste water was determined
prior to use (pH 7.26). Adjustment to 7.5 ± 0.5 was not necessary.

Dissolved oxygen:

See IUCLID section "Any other information on results incl. tables" below.

Salinity:

Not applicable

Conductivity:

Not applicable

Nominal and measured concentrations:

Nominal test item concentrations:
1.7, 8.4, 41, 200, 600, 1000 mg/L

Details on test conditions:

See IUCLID section "Any other information on materials and methods incl. tables" below.

Reference substance (positive control):

yes

Remarks:

copper (II) sulphate pentahydrate for total respiration; N-Methylaniline (with and without OCT) for inhibition of nitrification

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

24.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Remarks on result:

other: 95% CI: 19.9 - 30.2 mg/L

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

51.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% CI: 44.9 – 58.7

Remarks:

mg/L

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

93.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks on result:

other: 95% CI: 75.2 – 121 mg/L

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

85.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of nitrification rate

Remarks on result:

other: 95% CI: 74.3 – 99.2 mg/L

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

128 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks on result:

other: 95% CI: 117 – 140 mg/L

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

193 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of heterotrophic respiration

Remarks on result:

other: 95% CI: 148 – 233 mg/L

Details on results:

For details on results, please see IUCLID section "Any other information on results incl. tables".

Results with reference substance (positive control):

In the reference test the EC50-value for copper (II) sulphate pentahydrate was determined by linear regression with:
EC50 = 100 mg/L
95% Confidence Interval = 96.0 – 105 mg/L

This is in the recommended range of validity of 53 - 155 mg/L.

In the reference test on inhibition of nitrification, the EC50-value for N- Methylaniline was determined by linear regression with:
EC50 = 1.70 mg/L
95% Confidence Interval = 1.23 – 2.35 mg/L

Reported statistics and error estimates:

For details on statistical evaluation, please see IUCLID section "Overall remarks, attachments".
For statistical evaluation, the following software was used:
Software Excel, MICROSOFT CORPORATION
GraphPadPrism, GRAPHPAD SOFTWARE, INC.
SigmaPlot (windows), SPSS INCORPORATION
PreSens Measurement Studio 2, PRESENS GMBH

Biotic and Abiotic Factors in the Definitive Study

 

The pH-values of the activated sludge and synthetic waste water, the dry sludge concentration and the test temperature are documented in the following table:

Biotic and Abiotic Factors

pH-value of the activated sludge for test without OCT	7.43
pH-value of the activated sludge for test with OCT	7.38
pH-value of the synthetic waste water	7.26
Dry solids concentration [g/L] of sludge for test without OCT	3.29
Dry solid concentration [g/L] in test solution for test without OCT	1.65
Dry solids concentration [g/L] of sludge for test with OCT	3.30
Dry solid concentration [g/L] in test solution for test with OCT	1.65
Test temperature [°C]	20.8

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration

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. The results are listed in the following tables:

 

Oxygen Uptake Rates and Specific Respiration Rates of the total Respiration of the Control (without OCT)

	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Specific Respiration Rate Rs
		[mg O2/L]	[mg O2/(L×h)]	[mg O2/g×h]
Control	1	6.64	35.0	21.3
	2	6.25	31.2	18.9
	3	5.78	31.3	19.0
	4	5.44	30.1	18.3
	5	5.84	29.4	17.9
	6	5.84	28.9	17.5
	Mean value		31.0	18.8
	Standard deviation		2.0
	CV [%]		6.4

*) determined at start of the measurement

Repl. = Replicate ; CV = Coefficient of variation

 

Oxygen Uptake Rates and Inhibition of the total Respiration of the Test Item Concentrations (without OCT)

Test Item Concentration	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Inhibition	Mean Inhibition
[mg/L]		[mg O2/L]	[mg O2/(L×h)]	[%]	[%]
1.7	1	6.53	33.2	-7	-7
	2	6.41	31.5	-2
	3	6.89	32.7	-5
	4	6.85	34.7	-12
	5	7.03	33.4	-8
8.4	1	5.08	31.7	-2	-3
	2	5.34	31.5	-2
	3	5.58	30.7	1
	4	5.90	32.8	-6
	5	6.06	32.6	-5
41	1	5.40	29.3	5	5
	2	5.53	27.4	12
	3	5.99	28.1	9
	4	5.98	32.2	-4
	5	5.75	30.8	1
200	1	6.87	10.1	67	67
	2	6.92	9.9	68
	3	7.16	10.1	67
	4	7.13	10.3	67
	5	7.22	10.2	67
600	1	7.30	5.5	82	81
	2	7.21	5.6	82
	3	7.55	5.8	81
	4	7.37	5.8	81
	5	8.02	5.8	81
1000	1	7.51	5.6	82	82
	2	7.36	5.5	82
	3	7.40	5.4	83
	4	7.53	5.6	82
	5	7.54	5.5	82

*) determined at start of the measurement

 

Oxygen Uptake Rates of the heterotrophic Respiration of the Control Replicates (with OCT)

	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R
		[mg O2/L]	[mg O2/(L×h)]
Control	1	7.74	13.1
	2	7.32	12.6
	3	7.50	12.5
	4	7.41	13.2
	5	7.32	12.6
	6	7.34	13.2
	Mean value		12.9
	Standard deviation		0.3
	CV [%]		2.4

*) determined at start of the measurement

Repl. = Replicate

CV = Coefficient of variation

Oxygen Uptake Rates and Inhibition of the heterotrophic Respiration of the Test Item Concentrations (with OCT)

Test Item Concentration [mg/L]	Repl.	Oxygen Concentration* [mg O2/L]	Oxygen Uptake Rate R [mg O2/L×h]	Inhibition [%]	Mean Inhibition [%]
1.7 + OCT	1	7.45	13.0	-1	-6
	2	7.17	12.7	2
	3	7.51	12.5	3
	4	7.18	16.0	-24
	5	7.22	14.0	-9
8.4 + OCT	1	7.30	13.5	-5	-5
	2	7.13	13.4	-4
	3	7.29	13.3	-3
	4	7.33	14.0	-9
	5	7.36	13.7	-6
41 + OCT	1	7.88	15.8	-22	-21
	2	7.67	15.3	-19
	3	7.84	14.5	-12
	4	7.97	16.7	-29
	5	7.35	15.7	-22
200 + OCT	1	7.64	6.4	50	54
	2	7.56	6.1	53
	3	7.67	5.6	57
	4	7.68	5.4	58
	5	7.44	6.5	50
600+ OCT	1	7.64	4.0	69	69
	2	7.70	3.7	71
	3	7.95	3.5	73
	4	7.68	4.5	65
	5	7.65	4.1	68
1000+ OCT	1	7.75	3.6	72	67
	2	7.67	3.5	73
	3	7.85	3.8	71
	4	6.81	2.5	81
	5	7.30	7.7	40

*) determined at start of the measurement

CalculatedOxygen Uptake Rates of the Nitrification of the Control

	Repl.	Oxygen Uptake Rate R [mg O2/(L×h)]
Control	1	21.9
	2	18.6
	3	18.8
	4	16.9
	5	16.8
	6	15.7
Mean value		18.1
Standard deviation		2.0
CV [%]		11.1

*) determined at start of the measurement

Repl. = Replicate

CV = Coefficient of variation

Calculated Oxygen Uptake Rates and Inhibition of theNitrificationof the Test Item Concentrations

Test Item Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Inhibition [%]	Mean Inhibition [%]
1.7	1	19.6	-8	-8
	2	17.9	1
	3	19.1	-5
	4	21.1	-16
	5	19.8	-9
8.4	1	18.1	0	-1
	2	17.9	1
	3	17.1	5
	4	19.2	-6
	5	19.0	-5
41	1	13.7	24	23
	2	11.8	35
	3	12.5	31
	4	16.6	8
	5	15.2	16
200	1	4.1	77	77
	2	3.9	78
	3	4.1	77
	4	4.3	76
	5	4.2	77
600	1	1.5	91	90
	2	1.6	91
	3	1.8	90
	4	1.8	90
	5	1.8	90
1000	1	1.4	92	93
	2	1.3	93
	3	1.2	93
	4	1.4	92
	5	1.3	93

Repl. = Replicate

Preliminary testing (non-GLP)

From preliminary testing (range-finding tests) it became obvious that acid formation from aldehyde during stirring overnight as well as reaction of the test item or its product(s) with ATU was the reason for contradictory results.

Therefore, stirring overnight was omitted and 1-Octyne, which specifically and irreversibly inhibits ammonia monooxygenases, was used as an alternative to ATU.

To validate inhibition of nitrification, inhibition of nitrate formation as well as dose-dependent inhibition of total respiration was assessed. Results are given in the following tables:

Inhibition of Nitrate formation (mg/L NO3 -nitrogen) in the third Preliminary Test (with / without 1-Octyne)

	Start	3 h
Control	2.97	10.2
1-Octyne (4.2 mg/L)	3.03	3.56

 

Nitrate formation of the control replicates is very well inhibited by 1-Octyne at 4.2 mg/L.

Inhibition of total respiration by 1-Octyne (dose-response relationship)

Concentration	% Inhibition total respiration
2.1  mg/L	0
4.2 mg/L	45
8.4 mg/L	43

At the concentration of 4.2 mg/L, the plateau was reached. To be sure, a test concentration of 5 mg/L 1-Octyne was chosen for the final test.

Validity criteria fulfilled:

yes

Remarks:

For details, see IUCLID section "Overall remarks, attachments".

Conclusions:

In a reliable and valid Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) performed compliant with GLP according to OECD 209 (2010), the test item n-undecanal was observed to moderately stimulate heterotrophic respiration up to a concentration of 41 mg/L (21% stimulation), but to inhibit nitrification (23% inhibition at 41 mg/L), resulting in a NOEC for total respiration of 41 mg/L.
Overall, the following (no)effect concentrations for inhibition of respiration were determined for the test item after an exposure period of 3 hours:

NOEC (3h) [mg/L]
total respiration: 41
heterotrophic respiration: 41
nitrification: 8.4

EC10 (3h) [mg/L]
total respiration: 51.1 (95% CI: 44.9 – 58.7)
heterotrophic respiration: 93.1 (95% CI: 75.2 – 121)
nitrification: 24.7 (95% CI: 19.9 – 30.2)

EC50 (3h) [mg/L]
total respiration: 128 (95% CI: 117 – 140)
heterotrophic respiration: 193 (95% CI: 148 – 233)
nitrification: 85.3 (95% CI: 74.3 – 99.2)

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item n-Undecanal from 2018-12-19 to 2018-12-21, with the definitive exposure phase on 2018-12-21 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 1.7 - 8.4 - 41 - 200 - 600 - 1000 mg/L with and without 1 -Octyne, a specific inhibitor of nitrification. 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 -7 % to 82 %. The mean inhibition of the heterotrophic respiration for the test item replicates ranged from -21 % to 69 %. The mean inhibition of nitrification for the test item replicates ranged from -8 % to 93 %. The test item n-undecanal was observed to moderately stimulate heterotrophic respiration up to a concentration of 41 mg/L (21% stimulation), but to inhibit nitrification (23% inhibition at 41 mg/L), resulting in a NOEC for total respiration of 41 mg/L.

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 (without OCT) for total respiration and N-Methylaniline (with and without OCT) for inhibition of nitrification as reference items and the reference toxicity was determined. The EC₅₀-value for the reference item copper (II) sulphate pentahydrate was 100 mg/L. The EC₅₀-value for the reference item N-Methylaniline for inhibition of nitrification was 1.70 mg/L.

The following (no)effect concentrations for inhibition of respiration were determined for the test item after an exposure period of 3 hours:

	Total Respiration (without OCT)		Heterotrophic Respiration (with OCT)		Nitrification
	EC-values [mg/L]	Confidence interval P = 95 [%] [mg/L]	EC-values [mg/L]	Confidence interval P = 95 [%] [mg/L]	EC-values [mg./L]	Confidence interval P = 95 [%] [mg/L]
NOEC*	41	–	41	–	8.4	–
EC10	51.1	44.9 – 58.7	93.1	75.2 – 121	24.7	19.9 – 30.2
EC20	68.3	60.8 – 76.5	113	90.8 – 142	36.8	31.1 – 42.9
EC50	128	117 – 140	193	148 – 233	85.3	74.3 – 99.2
EC80	456	361 – 946	˃ 1000	–	236	188 – 293

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

Description of key information

In a reliable and valid Activated Sludge, Respiration Inhibition Test (Carbon and Ammonium Oxidation) performed compliant with GLP according to OECD 209 (2010), the test item n-undecanal was observed to moderately stimulate heterotrophic respiration up to a concentration of 41 mg/L (21% stimulation), but to inhibit nitrification (23% inhibition at 41 mg/L), resulting in a NOEC for total respiration of 41 mg/L.

Overall, the following (no)effect concentrations for inhibition of respiration were determined for the test item after an exposure period of 3 hours:

NOEC (3h) [mg/L]

total respiration: 41

heterotrophic respiration: 41

nitrification: 8.4

EC10 (3h) [mg/L]

total respiration: 51.1 (95% CI: 44.9 – 58.7)

heterotrophic respiration: 93.1 (95% CI: 75.2 – 121)

nitrification: 24.7 (95% CI: 19.9 – 30.2)

EC50 (3h) [mg/L]

total respiration: 128 (95% CI: 117 – 140)

heterotrophic respiration: 193 (95% CI: 148 – 233)

nitrification: 85.3 (95% CI: 74.3 – 99.2)

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

24.7 mg/L

Additional information

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test itemn-Undecanal from 2018-12-19 to 2018-12-21, with the definitive exposure phase on 2018-12-21at 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 concentrations1.7 - 8.4 - 41 - 200 - 600 - 1000 mg/L with and without 1 -Octyne, a specific inhibitor of nitrification. 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 -7 % to 82 %. The mean inhibition of the heterotrophic respiration for the test item replicates ranged from-21 % to 69 %. The mean inhibition of nitrification for the test item replicates ranged from -8 % to 93 %. The test item n-undecanal was observed to moderately stimulate heterotrophic respiration up to a concentration of 41 mg/L (21% stimulation), but to inhibit nitrification (23% inhibition at 41 mg/L), resulting in a NOEC for total respiration of 41 mg/L.

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 (without OCT) for total respiration and N-Methylaniline (with and without OCT) for inhibition of nitrification as reference items and the reference toxicity was determined. The EC₅₀-value for the reference item copper (II) sulphate pentahydrate was 100 mg/L. The EC₅₀-value for the reference item N-Methylaniline for inhibition of nitrification was 1.70 mg/L.

The following (no)effect concentrations for inhibition of respiration were determined for the test item after an exposure period of 3 hours:

	Total Respiration (without OCT)		Heterotrophic Respiration (with OCT)		Nitrification
	EC-values [mg/L]	Confidence interval P = 95 [%] [mg/L]	EC-values [mg/L]	Confidence interval P = 95 [%] [mg/L]	EC-values [mg./L]	Confidence interval P = 95 [%] [mg/L]
NOEC*	41	–	41	–	8.4	–
EC10	51.1	44.9 – 58.7	93.1	75.2 – 121	24.7	19.9 – 30.2
EC20	68.3	60.8 – 76.5	113	90.8 – 142	36.8	31.1 – 42.9
EC50	128	117 – 140	193	148 – 233	85.3	74.3 – 99.2
EC80	456	361 – 946	˃ 1000	–	236	188 – 293

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