N-[3-(dimethylamino)propyl] C6-9 alkyl amides

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:

28-11-2018 to29-11-2018

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 (incl. QA statement)

Analytical monitoring:

not required

Vehicle:

no

Details on test solutions:

The test item was added to the incubation vessels using a 1 and 2 g/L stock solution. The 1 g/L stock solution of test substance was prepared by dissolving 0.83 g in approximately 700 g deionized water, adjusting the pH from 10.0 to ~5.6 with 1 M HCl and NaOH, and finally diluting the suspension further to a total mass of 830 g with deionized water. The 2 g/L stock suspension of the test item was prepared by dissolving 1.85 g in approximately 774 g deionized water. The pH of this solution was approximately 10.4 – 10.7 and was adjusted to ~5.6 with 1 M HCl and NaOH, and finally diluted further to a total mass of 925 g with deionized water. 3,5-Dichlorophenol was added from a 1 g/L stock solution. The stock solution of 3,5-dichlorophenol was prepared by dissolving 0.1032 g 3,5-dichlorophenol in approximately 40 mL of deionized water with the addition of a few drops of 1 M NaOH. Subsequently the pH was checked and the solution was diluted further to 100 mL with deionized water.

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

Unadapted secondary activated sludge was obtained (28-11-2018) from the WWTP Nieuwgraaf in Duiven. The WWTP Duiven is an activated sludge plant treating predominantly domestic wastewater. Prior to use the activated sludge was homogenized with a syringe. The measured dry weight of the homogenized activated sludge was 3.5 g/L and was concentrated by settlement to 6.3 g/L dry weight. Subsequently, the concentrated activated sludge was diluted in the incubation vessels to an activated sludge dry weight concentration of 1.5 g/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Test temperature:

Temperature varied from 20.7 to 20.8°C.

pH:

The pH of the reaction mixtures after the incubation period ranged from 7.2 to 7.4

Dissolved oxygen:

aerobic during test

Nominal and measured concentrations:

Concentrations
Nominal: 0, 20, 40, 80, 160, 320, 640 and 1280 mg/L

Details on test conditions:

The test was performed in 300 and 1000 mL Erlenmeyer flasks with a total working volume of 50 and 200 mL, respectively. The homogenized activated sludge was incubated in a shaking water bath (100 rpm, 20°C) for 3 hours with various concentrations of the test compound and synthetic sewage.
-Synthetic sewage was added to the activated sludge (1.6 mL on a total volume of 50 mL).
Synthetic sewage contained per liter: 16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl
0.4 g CaCl2.2H2O
0.2 g MgSO4.7H2O
2.8 g K2HPO4
-Different concentrations (spaced by a factor two) of test compound and reference compound where added using stock solutions
-Tap-water was used to fill up the total test volume to 50 mL.
- The dry weight concentration of the activated sludge in the test was 1.5 g/L.
-The tested concentrations of the reference compound 3,5-dichlorophenol where: 2.5 mg/L, 5.0 mg/L,10 mg/L, 19.9 mg/L and 39.9 mg/L
-The tested concentrations of sodium cocopropylenediamine propionate where: 20 mg/L, 40 mg/L, 80 mg/L, 160 mg/L, 320 mg/L, 640 mg/L and 1280 mg/L.
-Controls without test substance (or reference compound) are measured at the beginning and at the end of the test
-The inhibitory effect of the test substance/reference compound is expressed as a percentage of the mean of the two control respiration rates.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol (p.a. quality 99.7% Aldrich Lot no MKBP6894V)

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

449 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:

EC50

Effect conc.:

1 154 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: 95% CL:979 - 1472 mg/L

Details on results:

Test conditions
The pH of the reaction mixtures after the incubation period ranged from 7.2 to 7.4. The temperature of the BOM thermostated vessel measured at the start and end of the test was 20.0 °C. The temperature of the water bath, used for the incubation of the activated sludge, at the beginning and end of the test was 21.4 °C and 21.5 °C, respectively. Temperatures measured in the Erlenmeyer flasks at the end of the incubation period varied from 20.7 to 20.8 °C. These conditions allow respiration of the activated sludge used.

Validity of the test
The validity of the test is demonstrated although only two out of three validity criteria were met according the guideline. First, the prescribed oxygen uptake rate of >20 mg O2/g dry weight/hour for the control measurements was not met. The inoculum used was obtained from a treatment plant which is operated at a low organic sludge load and as a consequence has a low oxygen uptake rate. Operating at low organic sludge loads is nowadays very common. Nonetheless the average oxygen uptake rate for the control replicates was 19 mg O2/g dry weight/hour, which is slightly lower than the prescribed rate. This does not influence the sensitivity of the activated sludge as shown by comparable EC50 values of the reference substance 3,5-dichlorophenol at oxygen uptake rates lower and higher than the prescribed rate (AkzoNobel 2012). The lower average oxygen uptake rate does therefore not compromise the test outcome in terms of robustness of the endpoints obtained for the test and reference substance and does not invalidate the test. Second, the coefficient of variation of the replicates of the control oxygen uptake rates was 12%, which shows a high precision and fulfills the maximum prescribed variation of <30%. Third, the EC50 of the reference compound for activated sludge after 3 hours contact time is 4 mg/L, which is within the prescribed range of 2 to 25 mg/L (Table I and Figure 1).

Activated sludge respiration inhibition test
The calculated respiration rates and inhibition percentages of the test item are presented in Table II and the results of the graphical probit analysis are shown in Figure 2. The inhibitory effect of the test item at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC50 of the test item for activated sludge after 3 hours contact time is 1154 mg/L with 95% confidence limits of 979 and 1472 mg/L. The EC10, EC20 and EC80 values were 449, 621 and 2145 mg/L, respectively. The test item is therefore considered not harmful to activated sludge.

Results with reference substance (positive control):

The EC50 of the reference compound for activated sludge after 3 hours contact time is 4 mg/L, which is within the prescribed range of 2 to 25 mg/L

Validity criteria fulfilled:

yes

Remarks:

See validity of test in "details on results"

Conclusions:

Test performed under GLP according guidelines meeting the validity criteria of the OECD guideline.
The EC50 of the test item for activated sludge after 3 hours contact time is 1154 mg/L. The substance is therefore considered not harmful to activated sludge.

Executive summary:

In order to predict effects of chemicals in the environment and in biological waste water treatment plants, an activated sludge respiration inhibition test was performed. The toxicity to activated sludge was determined in accordance with OECD Test Guideline 209, and in compliance with the OECD principles of Good Laboratory Practice.

The test is valid as shown by the EC₅₀ of the reference compound 3,5-dichlorophenol (4 mg/L) and the coefficient of variation of the replicates of the control oxygen uptake rates (12%). The average oxygen uptake rate for the control replicates is lower (18 mg O2/g dry weight/hour) than the prescribed rate (>20 mg O2/g dry weight /hour). This does not influence the sensitivity of the activated sludge as shown by comparable EC50 values of the reference substance 3,5-dichlorophenol at oxygen uptake rates lower and higher than the prescribed rate (AkzoNobel 2012). The lower average oxygen uptake rate does therefore not compromise the test outcome in terms of robustness of the endpoints obtained for the test and reference substance and does not invalidate the test.

The toxicity of the test item to activated sludge was determined at a contact time of 3 hours, using various concentrations of the test substance. The inhibitory effect at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC₅₀ for activated sludge after 3 hours contact time is 1154 mg/L. The substance is therefore considered not harmful to activated sludge.

Description of key information

In a test according to OECD Test Guideline 209 the toxicity of the test item to activated sludge was determined. The EC50 for activated sludge after 3 hours contact time is 1154 mg/L. The substance is therefore considered not harmful to activated.

Key value for chemical safety assessment

EC50 for microorganisms:

1 154 mg/L

EC10 or NOEC for microorganisms:

449 mg/L