Oleic acid, compound with (Z)-N-octadec-9-enylpropane-1,3-diamine (2:1)

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:

2012-03-12 to 2012-06-12

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

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Directly weighing in an Erlenmeyer flask, stirred overnight
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loading: 560 - 750 - 1000 mg/L
- Controls: Triplicates at the beginning of the test + triplicates at the end of the test

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Preparation of inoculum for exposure: Non adapted activated sludge from the sewage plant at Hildesheim (Municipal sewage treatment plant of 31137 Hildesheim, Germany)
- Pretreatment: Washed twice with autoclaved tap water and an adjustment to a dry weight concentration of 3.0 g/L +/- 10 % was not necessary. The sludge was used within 24 h after sampling
- Dry sludge concentration: 2.72 g/L

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

Not determined

Test temperature:

21.7 °C

pH:

pH-value of the activated sludge 7.72
pH-value of the synthetic waste water 7.44

Salinity:

Not determined

Details on test conditions:

TEST SYSTEM
- Test vessel: 500 mL Erlenmeyer flask, DIN 12380
- Aeration: Permanent
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3
- Composition of test medium:
250 mL inoculum
16 mL synthetic waste water
234 mL demineralised water

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
After 3 h incubation, the oxygen concentration of each control, reference item and test item replicate was determined by means of oximeter immediately after transferring the solution to the measuring cell and the oxygen depletion was recorded for at least 3 minutes on a flatbed recorder.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: None, limit test concentration
- Range finding study
Inhibition of Respiration (Preliminary Test)
Nominal Test Item
Concentration mg/L Pretreatment Inhibition [%]
10 Stirring overnight 2
100 -7
1000 -9

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

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 total respiration

Remarks:

respiration rate

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

993.2 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels: EC50: 99.1 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) and DUNNETT’s test was used for NOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. P-values for both Normality and Equal Variance test are 0.05. The ¿-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.

The EC50-value of the reference item by linear regression using software GraphPad Prism. Calculation of the confidence intervals for the EC-values were carried out using standard procedures.

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 with the oxygen uptake rates of the control.

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration
of the Control and Test Item Concentrations

Test Item Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Specific Respiration Rate Rs [mg O2/g×h]	Inhibition [%]	Mean Inhibition [%]
Control	1	20.4	15.0	–	–
	2	20.4	15.0
	3	18.4	13.5
	4	18.4	13.5
	5	18.8	13.8
	6	18.8	13.8
560	1	20.0		-4	-2
	2	20.4		-6
	3	18.4		4
750	1	15.6		19	18
	2	15.2		21
	3	16.4		15
1000	1	17.6		8	8
	2	19.6		-2
	3	16.0		17

Oxygen Uptake Rates, Specific Respiration Rates and Inhibition of the Respiration
of the Control and Reference Item Concentrations

Reference Item Concentration [mg/L]	Repl.	Oxygen Uptake Rate R [mg O2/L×h]	Specific Respiration Rate Rs [mg O2/g×h]	Inhibition [%]	Mean Inhibition [%]
Control	1	20.0	14.7	–	–
	2	18.8	13.8
	3	17.6	12.9
	4	18.4	13.5
	5	17.2	12.6
	6	16.0	11.8
58	1	12.8		29	24
	2	14.0		22
	3	14.0		22
100	1	9.2		49	50
	2	8.4		53
	3	9.2		49
180	1	3.6		80	79
	2	4.0		78
	3	4.0		78

NOEC-Value of N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate]

	Test Item Concentration [mg/L]
NOEC	1000

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of the test item N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate] is 1000 mg/L, the EC50 of the test item is > 1000 mg/L.
The test item is not toxic up to the concentration of 1000 mg/L, to activated sludge of a municipal sewage treatment plant.

Executive summary:

A respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate] from 2012-03-12 to 2012-06-12, with the definitive experimental phase on 2012-06-12 at the Dr.U.Noack-Laboratorien, 31157 Sarstedt, Germany. Test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the substance concentrations of 560 - 750 - 1000 mg/L in a geometrical series with a dilution factor of 1.33. 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. No adverse effect were observed on activated sludge up to the highest tested concentration (1000 mg/L). No hazard was identified for activated sludge.

Description of key information

The 3h-NOEC and 3h-EC50 are 993.2 and >993.2 mg/L respectively in a OECD 209 test with activated sludge under GLP (reliability 1).

Additional information

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item N-[(9Z)-octadec-9-en-1-yl]propane-1,3-diaminium di[(9Z)-octadec-9-enoate] .Test system was activated sludge of the municipal treatment plant of 31137 Hildesheim, Germany. The test was carried out under static conditions with the substance concentrations of 560 - 750 - 1000 mg/L in a geometrical series with a dilution factor of 1.33. Based on the concentration of active ingredient, the 3h-NOEC and 3h-EC50 were found to be 993.2 and >993.2 mg/L respectively.