Quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides

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:

From February 22, 2010 to February 24, 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

No chemical analyses of the test concentrations were performed during the test and all concentrations mentioned are nominal concentrations of the test substance.

Qualifier:

according to guideline

Guideline:

OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

not required

Details on sampling:

No data

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The stock solution was prepared by stirring 0.46 g quaternary ammonium compounds, di-C12-18-alkyldimethyl, chlorides in 460 mL of deionized water. The stock solution was a stable homogenous suspension (very light turbidity).
- pH stock solution = 5.0

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: Unadapted secondary activated sludge was obtained (22-02-2010) from the WWTP Nieuwgraaf in Duiven. The WWTP Duiven is an activated sludge plant treating predominantly domestic wastewater.
- Method of cultivation: 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.
- Preparation of inoculum for exposure: During the test period the activated sludge was kept in a thermostated 2 L cylindrical glass vessel to maintain the temperature at 20 ± 2 °C. A magnetic stirrer bar at the bottom and a glass frit were used to agitate and aerate the activated sludge. The dry weight of the activated sludge in the incubation vessels was 1.7 g/L.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

3 h

Post exposure observation period:

No data

Hardness:

No data

Test temperature:

20.2- 21.0 °C

pH:

6.9- 7.2

Dissolved oxygen:

No data

Salinity:

No data

Nominal and measured concentrations:

Nominal: 0, 10, 20, 40, 80, 160 and 320 mg/L

Details on test conditions:

Test conditions
The test was performed in 0.25 to 0.30 liter 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. Aeration of the activated sludge during this period was achieved by agitation.

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. The test substance and 3,5-dichlorophenol were added to the incubation vessels using stock solutions of approximately 1.0 and 0.5 g/L respectively. The stock solution of 3,5- dichlorophenol was prepared by dissolving 0.0508 g 3,5-dichlorophenol in 1 mL of 1 M NaOH. Then diluting this to approximately 30 mL with deionized water, adding under stirring 0.5 M H2SO4 to the point of incipient precipitation and finally diluting the mixture to 100 milliliter with deionized water. The test substance stock solution was prepared by stirring 0.46 g test substance in 460 mL of deionized water. The test substance stock solution was a stable homogenous suspension (very light turbidity).

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC50

Effect conc.:

ca. 68 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: with 95% confidence limits of 15 and 858 mg/L.

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

12 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

The inhibitory effect of test substance at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC50 of test substance for activated sludge after 3 hours contact time was 68 mg/L with 95% confidence limits of 15 and 858 mg/L. The EC10, EC20 and EC80 were 12, 22 and 215 mg/L, respectively. The test substance is therefore considered harmful to activated sludge. The validity of the test is demonstrated by two criteria. First, the control respiration rates were within 15% of each other and secondly, the EC50 of the reference compound was 8 mg/L which is within the prescribed range of 5 to 30 mg/L.

Results with reference substance (positive control):

The EC50 of the reference compound was 8 mg/L which is within the prescribed range of 5 to 30 mg/L

Results

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

Concentration mg/L	Activity mg O2/g.min	Inhibition%	pH start test	pH end test	Temperature end test (°C)
Control	0.30	-	6.8	7.0	20.4
Control	0.30	-	6.8	7.0	20.6
Control*	0.28	-	6.7	6.9	20.8
Control*	0.26	-	6.7	6.9	20.8
1.3*	0.24	10	6.7	7.0	20.9
2.5	0.23	24	6.8	7.2	20.2
5.1	0.20	34	6.8	7.1	20.7
10.2	0.13	55	6.8	7.1	20.3
20.3	0.06	79	6.8	7.1	20.4
40.6	0.03	88	6.8	7.1	20.3

Concentration stock solution 3,5-dichlorophenol = 508 mg/L pH stock solution 3,5-dichlorophenol = 7.0

* = corresponding control and reference concentration

 

Table II Respiration rates of the activated sludge, inhibition percentages and pH at various concentrations of  test substance after 3 hours contact time

Concentration mg/L	Activity mg O2/g.min	Inhibition%	pH start test	pH end test	Temperature end test (°C)
Control	0.45	-	6.9	7.1	20.5
Control	0.45	-	6.9	7.1	20.7
Control*	0.42	-	6.8	7.1	21.0
Control*	0.42	-	6.8	7.0	20.5
10	0.41	10	6.9	7.1	20.5
20	0.37	17	6.9	7.1	20.5
40	0.30	33	6.9	7.1	20.5
80	0.23	50	6.9	7.1	20.6
160	0.12	74	6.9	7.1	20.5
320*	0.05	89	6.8	7.0	20.7

Concentration stock solutiontest substance= 1 g/L

pH stock solution test substance = 5.0

 * = corresponding control and reference concentration

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 3 h EC50 and EC10 of the test substance for activated sludge was determined to be 68 and 12 mg/L respectively.

Executive summary:

A study was conducted to determine an activated sludge respiration inhibition potential of the test substance, C12-18 DAQ (99.6% active), according to OECD Guideline 209, in compliance with GLP. The toxicity of the test substance to activated sludge was determined at a contact time of 3 h, using the test substance at concentrations of 10, 20, 40, 80 and 160 mg/L. The inhibitory effect of the test substance at a particular concentration is expressed as a percentage of the two controls.No chemical analyses of the test concentrations were performed during the test and all concentrations mentioned are nominal concentrations of the test substance.In the study, the EC50, EC10, EC20 and EC80 of the test substance for activated sludge after 3 h contact time were determined to be 68 mg/L (95% CL: 15 and 858 mg/L), 12, 22 and 215 mg/L, respectively. The control respiration rates were within 15% of each other and the EC50 of the reference compound was 8 mg/L which is within the prescribed range of 5 to 30 mg/L. Therefore, the validity of the study was demonstrated. Under the study conditions, the 3 h EC50 and EC10 of the test substance for activated sludge were determined to be 68 and 12 mg/L (nominal) respectively (Geerts, 2010).

Description of key information

Based on the study results, the 3 h EC50 and EC10 of the test substance for activated sludge was determined to be 68 and 12 mg/L (nominal) respectively.

Key value for chemical safety assessment

EC50 for microorganisms:

68 mg/L

EC10 or NOEC for microorganisms:

12 mg/L

Additional information

A study was conducted to determine an activated sludge respiration inhibition potential of the test substance, C12-18 DAQ (99.6% active), according to OECD Guideline 209, in compliance with GLP. The toxicity of the test substance to activated sludge was determined at a contact time of 3 h, using the test substance at concentrations of 10, 20, 40, 80 and 160 mg/L. The inhibitory effect of the test substance at a particular concentration is expressed as a percentage of the two controls. No chemical analyses of the test concentrations were performed during the test and all concentrations mentioned are nominal concentrations of the test substance. In the study, the EC50, EC10, EC20 and EC80 of the test substance for activated sludge after 3 h contact time were determined to be 68 mg/L (95% CL: 15 and 858 mg/L), 12, 22 and 215 mg/L, respectively. The control respiration rates were within 15% of each other and the EC50 of the reference compound was 8 mg/L which is within the prescribed range of 5 to 30 mg/L. Therefore, the validity of the study was demonstrated. Under the study conditions, the 3 h EC50 and EC10 of the test substance for activated sludge were determined to be 68 and 12 mg/L (nominal) respectively (Geerts, 2010).