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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
From 23 April, 2012 to 26 April, 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to the Quaternary ammonium salts (QAS) category or section 13 of IUCLID for details on the category justification.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Hydrogenated (tallow alkyl) trimethyl ammonium chloride was added to the incubation vessels using a stock solution of approximately 1.0 g/L. The stock solution of hydrogenated (tallow alkyl) trimethyl ammonium chloride was prepared by dissolving 0.69 g hydrogenated (tallow alkyl) trimethyl ammonium chloride in 690 mL deionized water.

pH stock solution hydrogenated (tallow alkyl) trimethyl ammonium chloride = 6.2
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Unadapted secondary activated sludge was obtained (24-04-2012) 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 dry weight of the homogenized activated sludge was determined and subsequently concentrated by settlement to the required dry weight concentration. The dry weight of the activated sludge in the incubation vessels was 1.5 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
Temperature varied from 19.8 to 20.5°C.
pH:
The pH of the reaction mixtures after the incubation period ranged from 7.2 to 7.4.
Dissolved oxygen:
aerobic during test
Details on test conditions:
Test conditions
The test was performed in 300 mL Erlenmeyer flasks with a total working volume of 50 mL. 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 feed and stocks
The synthetic sewage feed was made by dissolving the following amounts of substances in 1 liter of deionized water: 16 g peptone, 11 g meat extract, 3 g urea, 0.7 g NaCl, 0.3 g CaCl2, 0.2 g MgSO4.7H2O, 2.8 g K2HPO4. In the incubation vessels 1.6 mL of the synthetic sewage feed is added in a total volume of 50 mL. Hydrogenated (tallow alkyl) trimethyl ammonium chloride and 3,5-dichlorophenol were added to the incubation vessels using stock solutions both of approximately 1.0 g/L. The stock solution of hydrogenated (tallow alkyl) trimethyl ammonium chloride was prepared by dissolving 0.69 g hydrogenated (tallow alkyl) trimethyl ammonium chloride in 690 mL deionized water. The stock solution of 3,5-dichlorophenol was prepared by dissolving 0.1030 g 3,5- dichlorophenol in approximately 40 mL of deionized water with the addition of a few drops of 1 M NaOH. Finally the pH was corrected with 2M H2SO4 to approximately pH 7 and the solution was diluted further to 100 mL with deionized water.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: equivalent to 9.46 mg a.i./L
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
42 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: equivalent to 30.57 mg a.i./L (with 95% confidence limits of 15 and 133 mg/L, equivalent to 10.92 and 96.80 mg a.i./L)
Details on results:
Test conditions
The pH of the reaction mixtures after the incubation period ranged from 7.2 to 7.4. Temperature varied from 19.8 to 20.5°C. These conditions allow
respiration of the activated sludge used.

Validity of the test
The validity of the test is demonstrated by three criteria. First, the coefficient of variation of the replicates of the control oxygen uptake rates was 10%, which fulfils the maximum prescribed variation of <30%. Secondly, the prescribed average oxygen uptake rate of > 20 mg O2/g dry weight/h for the control measurements was achieved (30 mg O2/g dry weight/hour). The third criterion was met as shown by the EC50 of the reference compound of 15 mg/L, which is within the prescribed range of 2 to 25 mg/L.

Activated sludge respiration inhibition test
The calculated respiration rates and inhibition percentages of hydrogenated (tallow alkyl) trimethyl ammonium chloride were presented and the results of the graphical probit analysis were shown. The inhibitory effect of hydrogenated (tallow alkyl) trimethyl ammonium chloride at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC50 of hydrogenated (tallow alkyl) trimethyl ammonium chloride for activated sludge after 3 h contact time is 42 mg a.i./L with 95% confidence limits of 15 and 133 mg/L.
The EC10, EC20 and EC80 are 13, 20 and 91 mg a.i./L, respectively.
Results with reference substance (positive control):
The EC50 of the reference compound was 15 mg/L at a contact time of 3 h, respectively. These EC50 values is within the prescribed range of
2 to 25 mg/L.

Result tables

Table I Respiration rates of the activated sludge, inhibition percentages, pH and temperature at various concentrations of 3,5-dichlorophenol after 3 hours contact time.

Concentration (mg/L)

Activity (mgO2/g/min)

Inhibition (%)

pH at start

test

pHatend

test

Temperature at end test

(°C)

Control

0.5222

-

7.0

7.2

19.8

Control

0.5222

-

7.1

7.2

20.0

Control*

0.4556

-

7.1

7.2

20.2

Control*

0.4556

-

7.1

7.3

20.4

2.5

0.5000

4

7.1

7.3

20.1

5.0*

0.3778

17

7.1

7.2

20.0

10.0

0.3444

34

7.1

7.3

20.0

20.0

0.1500

71

7.0

7.3

20.2

40.0

0.1143

78

7.0

7.3

20.4

Concentrationstock solution3,5-dichlorophenol = 999 mg/L pHstock solution3,5-dichlorophenol =7.1

* = corresponding control and reference concentration

 

Table II Respiration rates of the activated sludge, inhibition percentages, pH and temperature at various concentrations of hydrogenated (tallow alkyl) trimethyl ammonium chloride after 3 hours contact time

Concentration (mg/L)

Activity (mgO2/g/min)

Inhibition (%)

pH at start

test

pHatend

test

Temperature end test

(°C)

Control

0.4952

-

7.1

7.3

20.4

Control

0.5278

-

7.1

7.4

20.5

10

0.5000

2

7.1

7.3

20.3

20

0.3667

28

7.1

7.3

20.0

40

0.2333

54

7.2

7.4

20.1

80

0.1133

78

7.2

7.4

20.3

160

0.0500

90

7.2

7.4

20.2

Concentration stock solution hydrogenated (tallow alkyl) trimethyl ammonium chloride = 1 g/L

pH stock solution of hydrogenated (tallow alkyl) trimethyl ammonium chloride = 6.2

Validity criteria fulfilled:
yes
Remarks:
see "details on results"
Conclusions:
Based on the results of the read across study, the 3 h EC50, EC10, EC20 and EC80 values for the test substance are considered to be 42, 13, 20 and 91 mg/L, respectively (equivalent to 30.57, 9.46, 14.56 and 66.22 mg/L, respectively).
Executive summary:

 

An activated sludge respiration inhibition test was performed with read across substance, C16 -18 TMAC (72.78% active) according to the OECD Guideline 209, in compliance with GLP. The toxicity of the read across substance to activated sludge was determined at a contact time of 3 h, using various concentrations of the read across substance. The respiration rate was calculated from the linear part of the respiration curve as mg O2/L/min. Divided by the activated sludge dry weight concentration in the incubation vessels a respiration rate (activity) in mg O2/g/min was calculated. To calculate the inhibitory effect of a read across substance at a particular concentration, the respiration rate was expressed as a percentage of the mean of two controls of the respiration rate. The EC values were computed from the best-fitted line (least square method) through the points given by the probit of the percentage inhibition and the logarithm of the concentration of the substance.The validity of the test is demonstrated by three criteria. First, the coefficient of variation of the replicates of the control oxygen uptake rates was 10%, which fulfils the maximum prescribed variation of <30%. Secondly, the prescribed average oxygen uptake rate of > 20 mg O2/g dry weight/hour for the control measurements was achieved (30 mg O2/g dry weight/hour). The third criterion was met as shown by the EC50 of the reference compound of 15 mg/L, which is within the prescribed range of 2 to 25 mg/L. The inhibitory effect of read across substance at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC50 of read across substance for activated sludge after 3 hours contact time is 42 mg/L with 95% confidence limits of 15 and 133 mg/L. The EC10, EC20 and EC80 are 13, 20 and 91 mg/L, respectively. The read across substance is therefore considered harmful to activated sludge. Under the study conditions, the read across substance 3 h EC50, EC10, EC20 and EC80 were determined to be 42, 13, 20 and 91 mg/L, respectively (equivalent to 30.57, 9.46, 14.56 and 66.22 mg/L, respectively) (Geerts, 2012). Based on the results of the read across study, similar effect levels are expected for the test substance.

Description of key information

 Based on the results of the read across study, the 3 h EC50, EC10, EC20 and EC80 values for the test substance are considered to be 42, 13, 20 and 91 mg/L, respectively (nominal; i.e., equivalent to 30.57, 9.46, 14.56 and 66.22 mg/L, respectively). 

Key value for chemical safety assessment

EC50 for microorganisms:
42 mg/L
EC10 or NOEC for microorganisms:
13 mg/L

Additional information

An activated sludge respiration inhibition test was performed with read across substance, C16 -18 TMAC (72.78% active) according to the OECD Guideline 209, in compliance with GLP. The toxicity of the read across substance to activated sludge was determined at a contact time of 3 h, using various concentrations of the read across substance. The respiration rate was calculated from the linear part of the respiration curve as mg O2/L/min. Divided by the activated sludge dry weight concentration in the incubation vessels a respiration rate (activity) in mg O2/g/min was calculated. To calculate the inhibitory effect of a read across substance at a particular concentration, the respiration rate was expressed as a percentage of the mean of two controls of the respiration rate. The EC values were computed from the best-fitted line (least square method) through the points given by the probit of the percentage inhibition and the logarithm of the concentration of the substance.The validity of the test is demonstrated by three criteria. First, the coefficient of variation of the replicates of the control oxygen uptake rates was 10%, which fulfils the maximum prescribed variation of <30%. Secondly, the prescribed average oxygen uptake rate of > 20 mg O2/g dry weight/hour for the control measurements was achieved (30 mg O2/g dry weight/hour). The third criterion was met as shown by the EC50 of the reference compound of 15 mg/L, which is within the prescribed range of 2 to 25 mg/L. The inhibitory effect of read across substance at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC50 of read across substance for activated sludge after 3 hours contact time is 42 mg/L with 95% confidence limits of 15 and 133 mg/L. The EC10, EC20 and EC80 are 13, 20 and 91 mg/L, respectively. The read across substance is therefore considered harmful to activated sludge. Under the study conditions, the read across substance 3 h EC50, EC10, EC20 and EC80 were determined to be 42, 13, 20 and 91 mg/L, respectively (nominal; i.e., equivalent to 30.57, 9.46, 14.56 and 66.22 mg/L, respectively) (Geerts, 2012). Based on the results of the read across study, similar effect levels are expected for the test substance.