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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
23 April 2012 to 22 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:
Quaternary ammonium compounds, (C16-18 and C18-unsatd. alkyl) trimethyl, chlorides and 3,5-dichlorophenol was added to the incubation vessels using a stock solution of approximately 1.0 g/L. The stock solution of quaternary ammonium compounds, (C16-18 and C18-unsatd. alkyl) trimethyl, chlorides was prepared by dissolving 0.68 g quaternary ammonium compounds, (C16-18 and C18-unsatd. alkyl) trimethyl, chlorides in 680 mL deionized water.

pH stock solution quaternary ammonium compounds, (C16-18 and C18-unsatd. alkyl) trimethyl, chlorides = 5.8
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 20.6°C.
pH:
The pH of the reaction mixtures after the incubation period ranged from 7.2 to 7.5.
Dissolved oxygen:
aerobic during test
Details on test conditions:
-The test was performed in 300 mL Erlenmeyers.
-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
0.3 g CaCl2
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 both of
1.0 g/L
-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,10.0 mg/L, 20.0 mg/L and 40.0 mg/L
-The tested concentrations of hydrogenated (tallow alkyl) trimethyl ammonium chloride where : 10 mg/L, 20 mg/L, 40 mg/L, 80 mg/L and 160 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.
-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
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
11 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.:
34 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 9 and 86 mg/L
Details on results:
Test conditions
The pH of the reaction mixtures after the incubation period ranged from 7.2 to 7.5. Temperature varied from 19.8 to 20.6°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.
The test is valid as shown by the EC50 of the reference compound 3,5- dichlorophenol (15 mg/L), the coefficient of variation of the replicates of the control oxygen uptake rates (10%) and the average oxygen uptake rate for the control replicates (30 mg O2/g dry weight/hour).

Activated sludge respiration inhibition test
The inhibitory effect of quaternary ammonium compounds, (C16-18 and C18-unsatd. alkyl) trimethyl, chlorides at a particular concentration is expressed as a percentage of the two controls. From the results EC values were calculated. The EC50 of quaternary ammonium compounds, (C16-18 and C18-unsatd. alkyl) trimethyl, chlorides for activated sludge after 3 hours contact time is 34 mg/L with 95% confidence limits of 9 and 86 mg/L.
The EC10, EC20 and EC80 are 11, 16 and 73 mg/L, respectively. Quaternary ammonium compounds, (C16-18 and C18-unsatd. alkyl) trimethyl, chlorides is therefore considered harmful to activated sludge.

See below table for further details.
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.

Details on results:

TableI: Respiration rates of the activated sludge, inhibition percentages,pHand temperature at various concentrations of 3,5-dichlorophenol after 3 hours contacttime.

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

Concentration stock solution3,5-dichlorophenol= 999mg/LpHstock solution3,5-dichlorophenol=7.1

* = corresponding control and reference concentration

TableII: Respiration ratesof the activated sludge, inhibition percentages, pHand temperature at various concentrations of 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

0.5333

-

7.1

7.3

20.6

Control

0.5278

-

7.1

7.4

20.3

10

0.5048

5

7.1

7.3

20.5

20

0.3524

34

7.2

7.3

20.2

40

0.1933

64

7.2

7.4

20.0

80

0.0800

85

7.2

7.5

20.2

160

0.0333

94

7.2

7.5

20.1

Concentration stock solution quaternary ammonium compounds, (C16-18 and C18-unsatd. alkyl) trimethyl, chlorides = 1 g/L

pH stock solution ofquaternaryammoniumcompounds,(C16-18andC18-unsatd.alkyl)trimethyl, chlorides=5.8

Validity criteria fulfilled:
yes
Remarks:
see "details on results"
Conclusions:
Under the study conditions, the test substance is therefore considered harmful to activated sludge with an EC50 and EC10 values at 34 and 11 mg/L (nominal) respectively. 
Executive summary:

A study was conducted to determine the toxicity of the test substance, C16-18 and C18-unsatd. TMAC (96.9% active), to the microorganism 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 various concentrations ranging from 2.5 to 40 mg/L of the test substance. No chemical analyses was conducted. The EC50 of the test substance for activated sludge after 3 h contact time is 34 mg/L with 95% confidence limits of 9 and 86 mg/L. The EC10, EC20 and EC80 are 11, 16 and 73 mg/L, respectively. The validity of the test was shown by the EC50 of the reference compound 3,5-dichlorophenol (15 mg/L), the coefficient of variation of the replicates of the control oxygen uptake rates (10%) and the average oxygen uptake rate for the control replicates (30 mg O2/g dry weight/hour). Under the study conditions, the test substance is therefore considered harmful to activated sludge with an EC50 and EC10 values at 34 and 11 mg/L (nominal) respectively.                   

Description of key information

The test substance is considered harmful to activated sludge with an EC50 and EC10 values at 34 and 11 mg/L (nominal) respectively. 

Key value for chemical safety assessment

EC50 for microorganisms:
34 mg/L
EC10 or NOEC for microorganisms:
11 mg/L

Additional information

A study was conducted to determine the toxicity of the test substance, C16-18 and C18-unsatd. TMAC (96.9% active), to the microorganism 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 various concentrations ranging from 2.5 to 40 mg/L of the test substance. No chemical analyses was conducted. The EC50 of the test substance for activated sludge after 3 h contact time is 34 mg/L with 95% confidence limits of 9 and 86 mg/L. The EC10, EC20 and EC80 are 11, 16 and 73 mg/L, respectively. The validity of the test was shown by the EC50 of the reference compound 3,5-dichlorophenol (15 mg/L), the coefficient of variation of the replicates of the control oxygen uptake rates (10%) and the average oxygen uptake rate for the control replicates (30 mg O2/g dry weight/hour). Under the study conditions, the test substance is therefore considered harmful to activated sludge with an EC50 and EC10 values at 34 and 11 mg/L (nominal) respectively.