Quaternary ammonium compounds, benzyl-C16-18-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 23 April, 2012 to 26 April, 2012

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:

no

Vehicle:

no

Details on test solutions:

The test substance was added to the incubation vessels using a stock solution of approximately 1.0 g/L. The stock solution of of the test substance was prepared by dissolving 0.80 g in 800 mL deionized water.

pH stock solution of the test substance = 5.0

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Activated sludge
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 20.4°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:

- Deionized water
Deionized water containing no more than 0.01 mg/L Cu and 1 mg/L non- purgeable organic carbon was prepared in a water purification system.

- 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. The test substance and and 3,5-dichlorophenol were added to the incubation vessels using stock solutions both of approximately 1.0 g/L. The stock solution of the test substance was prepared by dissolving 0.80 g the test substance in 800 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.

-Test procedures
The activated sludge respiration inhibition test was performed according to the study plan. The study plan was developed from an OECD Test Guideline (OECD, 2010). In this test the inhibition of oxygen uptake by micro-organisms oxidizing organic carbon was not separately expressed from the respiration by micro- organisms oxidizing ammonium. The total respiration rate of activated sludge fed with a standard amount of synthetic sewage was measured. The respiration rate of the same activated sludge in the presence of various concentrations of the test substance under otherwise identical conditions was also measured. The inhibitory effect of the test substance at a particular concentration was expressed as a percentage of the mean respiration rates of the controls. Some aspects of the test are listed below:
• Prior to use the activated sludge was homogenized with a syringe.
• The activated sludge with test substances was incubated in a shaking bath (100 rpm/min) for 3 hours.
• The test substance and the reference compound (3,5-dichlorophenol) concentrations were spaced by a factor two.
• An abiotic control was not performed because neither reduction of oxygen by the test substance nor formation of oxygen due to chemical breakdown of the test substance is expected.
• No specific analyses were performed.

- Determination of respiration rates, pH, dry weight and temperature
The respiration rates of the activated sludge were measured in a Biological Oxygen Monitor (BOM) YSI 5300. The BOM consisted of a thermostated vessel with a magnetic stirrer and an oxygen electrode. The electrode to measure the oxygen depletion tightly closed the vessel. The volume of the vessel was 10 mL and the temperature in the vessel was 20°C. The pH was measured using an EUTECH pH meter. The temperature was measured with a Keithley 871A digital thermometer. The dry weight of the inoculum was determined by filtrating 50 mL of the activated sludge over a preweighed 12 m cellulose nitrate filter. This filter was dried for minimal 1.5 hours at 104 ± 5 °C and weighed after cooling. Dry weight was calculated by subtracting the weight of the filters and dividing the difference by the filtered volume.

- Calculation of test results
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.
In order to calculate the inhibitory effect of a test substance at a particular concentration, the respiration rate was expressed as a percentage of the mean of two controls of the respiration rate:
(1 – 2Rs / (Rc1 + Rc2)) x 100 = percentage inhibition

Rs = oxygen-consumption rate at tested concentration of test substance.
Rc = oxygen-consumption rate of controls.

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. All computations were performed with the program TOXCALCTM(Tidepool Scientific Software 1994- 1999).

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Key result

Duration:

3 h

Dose descriptor:

EC10

Effect conc.:

8 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:

EC50

Effect conc.:

35 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Remarks on result:

other: with 95% confidence limits of 2 and 163 mg/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 20.4°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/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.

Activated sludge respiration inhibition test
The inhibitory effect of the 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 the test substance for activated sludge after 3 hours contact time is 35 mg/L with 95% confidence limits of 2 and 163 mg/L. The EC10, EC20 and EC80 are 8, 14 and 88 mg/L, respectively. The test substance therefore considered harmful to activated sludge.

Please refer to tables below.

Results with reference substance (positive control):

The EC50 of the reference compound was 15 mg/L at a contact time of 3 hours, respectively. These EC50 values is within the prescribed range of 2 to 25 mg/L. Refer to the attachment for figure 1

TableI Respiration rates of theactivated 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= 999mg/L

pHstock solution3,5-dichlorophenol=7.1

* = corresponding control and reference concentration

TableII Respiration rates of theactivated sludge, inhibition percentages, pHand temperature at various concentrations of the test substance after 3hours contact time.

Concentration (mg/L)	Activity(mgO2/g/min)	Inhibition (%)	pH at start test	pHatend test	Temperature end test (°C)
Control	0.4556	-	7.1	7.3	20.0
Control	0.6133	-	7.1	7.3	20.2
10	0.4810	10	7.1	7.3	20.2
20	0.3500	35	7.1	7.2	20.3
40	0.2296	57	7.1	7.3	20.1
80	0.1037	81	7.1	7.4	20.0
160	0.0556	90	7.1	7.4	20.1

Concentration stock solution the test substance = 1g/L;

pH stock solution of the test substance =5.0

Validity criteria fulfilled:

yes

Conclusions:

Under the study conditions, the 3 h EC50 of the test substance was determined to be 35 mg/L and the EC10, EC20 and EC80 were 8, 14 and 88 mg/L, respectively. The test substance is therefore considered harmful to activated sludge.

Executive summary:

A study was conducted to determine the effect of the test substance, C16-18 ADBAC (98.2% active), on the respiration of activated sludge according to OECD 209 Guideline, in compliance with GLP. The toxicity to activated sludge was determined at a contact time of 3 h, using various concentrations (i.e., 2.5, 5, 10, 20 and 40 mg/L) of the test substance. No analytical determination was performed for the test substance. The total respiration rate of activated sludge fed with a standard amount of synthetic sewage was measured. The inhibitory effect of the test substance at a particular concentration was expressed as a percentage of the mean respiration rates of the controls. From the results EC values were calculated. Under the study conditions, the 3-h EC50 of the test substance for activated sludge was determined to be 35 mg/L with 95% confidence limits of 2 and 163 mg/L. The EC10, EC20 and EC80 were 8, 14 and 88 mg/L (nominal), respectively. The test substance is therefore considered harmful to activated sludge (Geerts, 2012).

Description of key information

The EC50 and EC10 values of the test substance for toxicity to microorganisms were determined to be 35 and 8 mg/L (nominal).

Key value for chemical safety assessment

EC50 for microorganisms:

35 mg/L

EC10 or NOEC for microorganisms:

8 mg/L

Additional information

A study was conducted to determine the effect of the test substance, C16-18 ADBAC (98.2% active), on the respiration of activated sludge according to OECD 209 Guideline, in compliance with GLP. The toxicity to activated sludge was determined at a contact time of 3 h, using various concentrations (i.e., 2.5, 5, 10, 20 and 40 mg/L) of the test substance. No analytical determination was performed for the test substance. The total respiration rate of activated sludge fed with a standard amount of synthetic sewage was measured. The inhibitory effect of the test substance at a particular concentration was expressed as a percentage of the mean respiration rates of the controls. From the results EC values were calculated. Under the study conditions, the 3-h EC50 of the test substance for activated sludge was determined to be 35 mg/L with 95% confidence limits of 2 and 163 mg/L. The EC10, EC20 and EC80 were 8, 14 and 88 mg/L (nominal), respectively. The test substance is therefore considered harmful to activated sludge (Geerts, 2012).