Nonanamide, N,N-dimethyl-

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:

2017-06-01

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

Specific details on test material used for the study:

Batch number 112-2016-11 D
CAS No. 6225-08-7
EC No. 612-975-5
Active ingredient (certified) 98.3%
Sum formula C11H23NO
Molecular weight 185.31 g/mol
Purity 98.3%
Density 0.8827 g/cm3 (20 °C)
Water solubility approx. 340 mg/L (20 °C)
Appearance Yellow clear liquid
Expiry date 2018-05-31
Recommended storage Cool and dry (+2-+8 °C)

Analytical monitoring:

no

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 10 - 32 - 100 - 320 - 1000 mg/L were pipetted directly in Erlenmeyer flasks.
- Eluate: Synthetic waste water acc. to OECD 209
- Differential loadings: 10 - 32 - 100 - 320 - 1000 mg/L, 5 replicates
- Controls: Test medium without test or reference item. Triplicates at the beginning of the test + triplicates at the end of the test
-Reference item: three concentrations within the concentration range 58 - 180 mg/L and a dilution factor of 1.8., Triplicates

Test organisms (species):

activated sludge

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)
- Receipt: 2017-05-31
- Pretreatment: The sludge was washed twice with chlorine free tap water and adjusted to a dry sludge concentration of 3.0 g/L ± 10 %. The sludge was used within 24 h after sampling.
- Dry sludge concentration: 2.71 g/L, corresponding to 1.36 g/L in the test vessel

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Hardness:

Not determined

Test temperature:

20.8 °C

pH:

pH-value of the activated sludge 7.45
pH-value of the synthetic waste water 7.34

Salinity:

Not determined

Details on test conditions:

TEST SYSTEM
- Test vessel: 5
- 500 mL Erlenmeyer flasks, DIN 12380
- Aeration: Permanent, to keep the dissolved oxygen concentration above 60 - 70 % saturation and to maintain the sludge flakes in suspension.

- Pretreatment: None

- No. of vessels per concentration (replicates): 5
- No. of vessels per control (replicates): 6 (Triplicates at the beginning of the test + triplicates at the end of the test.)
-No. of vessels per reference item (replicates): 9 (three concentrations within the range 58 - 180 mg/L and a dilution factor 1.8, at the end of the test)
- Composition of test medium:

Test item concentration [mg/L] 10 32 100 320 1000
Test item [mg] 5 16 50 160 500
Test item [µl]* 5.7 18.1 56.6 181 567
Synthetic waste water [mL] 16
Demineralised water [mL] Filled up to 250 mL
Inoculum [mL] 250
*) The required volumes were calculated with the density of 0.8827 g/cm3. The respective volumes were pipetted directly into the Erlenmeyer flasks.

Dilution Table for the Reference Item
Reference item concentration [mg/L] 58 100 180
Stock solution [µL]* 96.7 166.7 300
Synthetic waste water [mL] 16
Demineralised water [mL] Filled up to 250 mL
Inoculum [mL] 250
* Stock solution with 300g/L


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 every 5 seconds after transferring the solution to the measuring chamber and the oxygen depletion was recorded for at least 5 minutes with the software PreSens Measurement Studio 2.

TEST CONCENTRATIONS

A preliminary test (non GLP) was carried out with the concentrations of the test item N,N-Dimethylnonanamide of 10 – 100 - 1000 mg/L with two replicates each. The pH was measured in one replicate with 1000 mg/L test item. The flasks were aerated for 3 hours.

In the preliminary test, additional replicates were prepared to determine the inhibition of nitrification. Two replicates 1000 mg/L were measured with addition of Allylthiourea ATU.

No indication for the requirement to measure the inhibition of nitrification in the definitive study was observed.


Inhibitions in the Preliminary Tests

Nominal Test Item Concentration
[mg/L] pH

(test item in medium) Inhibition


[%]
1st Preliminary test
10 - 0
- -2
100 - 23
- 20
1000 7.52 95
- 95

Inhibitions in the Preliminary Test (with / without ATU)

Nominal Test Item Concentration
[mg/L] Inhibition of total respiration (without ATU)
[%] Inhibition of heterotrophic respiration (with ATU)
[%] Inhibition of nitrification
[%]
1000 95 87 102
95 84 104



Based on these results the definitive study was carried out with 5 concentrations within the range 10 - 1000 mg/L test item in a geometrical series and with a dilution factor of 3.2. No pH adjustment was made. All concentrations were pipetted directly in the Erlenmeyer Flasks.

Triplicates of the control without test item were included at the beginning and at the end of the test.

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 if necessary to a dry sludge concentration of 3.0 g/L ± 10 %.

The reference item was tested with three concentrations within the concentration range 58 - 180 mg/L and a dilution factor of 1.8.

Reference substance (positive control):

yes

Remarks:

Copper (II) sulphate pentahydrate p.a.

Key result

Duration:

3 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

66.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

other: EC20

Effect conc.:

99.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

214 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Key result

Duration:

3 h

Dose descriptor:

other: EC80

Effect conc.:

440 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Results with reference substance (positive control):

- Results with reference substance valid? Yes

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 -value (acceptable probability of incorrectly concluding that there is a difference) is =0.05.

The EC-value of the reference item was calculated by linear regression using software GraphPad Prism. Calculation of the confidence intervals for the EC50-value was carried out using standard procedures. The coefficient of variation of the oxygen uptake rates of the control replicates was calculated with standard methods using Excel.

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.

Oxygen Uptake Rates and 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	7.88	32.8	24.2
	2	7.93	33.4	24.6
	3	7.24	32.6	24.0
	4	8.06	34.4	25.4
	5	8.67	33.8	24.9
	6	8.41	32.8	24.2
	Mean value		33.3	24.6
	Standard deviation		0.640
	CV [%]		1.92

*) determined at start of the measurement

Repl. = Replicate

CV = Coefficient of variation .

 

Oxygen Uptake Rates and Inhibition of the Respiration of the Test Item Concentrations

Test Item Concentration	Repl.	Oxygen Concentration*	Oxygen Uptake Rate R	Inhibition	Mean Inhibition
[mg/L]		[mg O2/L]	[mg O2/(L×h)]	[%]	[%]
10	1	7.90	34.3	-3	-1
	2	7.09	35.7	-7
	3	7.47	31.5	5
	4	7.25	35.5	-7
	5	6.96	31.7	5
32	1	6.11	34.4	-3	-2
	2	5.57	34.6	-4
	3	7.25	33.8	-2
	4	7.00	35.0	-5
	5	6.35	31.8	5
100	1	7.82	27.1	19	21
	2	7.62	26.1	22
	3	6.93	26.7	20
	4	7.60	27.9	16
	5	6.28	23.7	29
320	1	8.19	11.5	65	67
	2	8.76	11.4	66
	3	8.04	10.7	68
	4	9.02	10.7	68
	5	7.91	10.1	70
1000	1	9.79	0.0	100	99
	2	9.82	0.0	100
	3	9.38	0.2	99
	4	9.73	0.4	99
	5	8.88	0.2	99

*) determined at start of the measurement

Repl. = Replicate

Validity criteria fulfilled:

yes

Conclusions:

The NOEC of N,N-Dimethylnonanamide is 32 mg /L. The EC10 is 66.5 mg/L and the EC50 is 214 mg/L.

Executive summary:

A Respiration Inhibition Test with activated sludge according to OECD Guideline No. 209 was carried out with the test item N,N-Dimethylnonanamide on 2017-06-01 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 10 - 32 - 100 - 320 - 1000 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 respiration for the test item replicates ranged from -1% to 99%.

 

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 as reference item and the reference toxicity was determined. The EC₅₀-value for the reference item was 93.1 mg/L.

 

NOEC and EC-Values with Confidence Interval of N,N-Dimethylnonanamide after 3 h exposure time, Inhibition of Total Respiration

		EC-values [mg/L]		Confidence interval P = 95 [%] [mg/L]
NOEC*		32		̶
EC10		66.5		56.4 – 78.1
EC20		99.6		87.7 – 112
EC50		214		197 – 232
EC80		440		395 – 490

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

                        

 

The NOEC of N,N-Dimethylnonanamide is 32 mg /L. The EC10 is 66.5 mg /L and the EC50 is 214 mg /L.

Description of key information

The NOEC of N,N-Dimethylnonanamide is 32 mg /L. The EC10 is 66.5 mg/L and the EC50 is 214 mg/L.

 

Key value for chemical safety assessment

EC50 for microorganisms:

214 mg/L

EC10 or NOEC for microorganisms:

66.5 mg/L