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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From February 08, 2010 to February 09, 2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA study
Justification for type of information:
Refer to section 13 for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
not required
Vehicle:
no
Details on test solutions:
The N,N,N’N’,N’’-pentamethyl-N-tallowalkyl-1,3-propanediammonium chloride stock solution was prepared by stirring 0.46 g N,N,N’N’,N’’-pentamethyl-N-tallowalkyl-1,3-propanediammonium chloride in 460 mL of deionized water. The N,N,N’N’,N’’-pentamethyl-N-tallowalkyl-1,3-propane-
diammonium chloride stock solution was a clear solution.
pH stock solution = 4.7

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Unadapted secondary activated sludge was obtained (08-02-2010) 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 1.7 g/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Test temperature:
Temperature varied from 20.1 to 20.9°C.
pH:
The pH of the reaction mixtures after the incubation period ranged from 7.4 to 7.6.
Dissolved oxygen:
aerobic during test
Details on test conditions:
-The test was performed in 250 to 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
0.4 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 of respectively
1.0 g/l and 0.5 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 1.7 g/l
-The tested concentrations of the reference compound 3,5-dichlorophenol where: 2.5 mg/l, 5.1 mg/l,10.2 mg/l, 20.3 mg/l and 40.6 mg/l
-The tested concentrations of N,N,N’N’,N’’-pentamethyl-N-tallowalkyl-1,3-propanediammonium chloride where: 5 mg/l, 10 mg/l, 20 mg/l, 40 mg/l, 80 mg/l, 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.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
32.1 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
94.5%
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
34 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.:
63 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 43 and 107 mg/L
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
59.535 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
94.5%
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Test conditions
The pH of the reaction mixtures after the incubation period ranged from 7.4 to 7.6. Temperature varied from 20.1 to 20.9°C. These conditions allowed respiration of the activated sludge used.

Validity of the test
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 9 mg/L which is within the prescribed range of 5 to 30 mg/L .

Activated sludge respiration inhibition test
The inhibitory effect of N,N,N’N’,N’’-pentamethyl-N-tallowalkyl-1,3-propanediammonium chloride at a particular concentration is expressed as a
percentage of the two controls. From the results EC values were calculated.
The EC50 of N,N,N’N’,N’’-pentamethyl-N-tallowalkyl-1,3-propanediammonium chloride for activated sludge after 3 hours contact time is 63 mg/L
with 95% confidence limits of 43 and 107 mg/L. The EC10, EC20 and EC80 are 34, 42 and 94 mg/L, respectively.
Results with reference substance (positive control):
The EC50 of the reference compound was 9 mg/L at a contact time of 3 hours, respectively. These EC50 values is within the prescribed range of 5 to 30 mg/L.
Validity criteria fulfilled:
yes
Remarks:
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 9 mg/L which is within the prescribed range of 5 to 30 mg/L .
Conclusions:
Under the study conditions, EC50 of test substance for activated sludge after 3 h contact time is 63 mg/L (i.e., equivalent to 59.535 mg a.i./L) The EC10, EC20 and EC80 are 34, 42 and 94 mg/L (i.e., equivalent to 32.13, 39.69 and 88.78 mg a.i./L), respectively.
Executive summary:

A study was conducted to determine the toxicity of the read across substance N,N,N',N',N''-Pentamethyl-N-C16-18 (even numbered) and C18 unsat.-alkyl-1,3-propanediammonium chloride to microorganisms, according to OECD Guideline 209, in compliance with GLP. The respiration inhibition test was performed on activated sludge. The toxicity of test substance to activated sludge was determined at a contact time of 3 h, using various concentrations of the test substance. The inhibitory effect of test substance at a particular concentration is expressed as a percentage of the two controls. The test is valid as shown by the EC50 of the reference substance, 3,5 -dichlorophenol (9 mg/L) and the control respiration rates which are within 15% of each other. Under the study conditions, the 3 h EC50 of the test substance was determined to be 63 mg/L (equivalent to 59.535 mg a.i./L) The EC10, EC20 and EC80 were determined to be 34, 42 and 94 mg/L (equivalent to 32.13, 39.69 and 88.78 mg a.i./L), respectively (Geerts, 2010).

Description of key information

Key value for chemical safety assessment

EC50 for microorganisms:
59.53 mg/L
EC10 or NOEC for microorganisms:
32.13 mg/L

Additional information

A study was conducted to determine the toxicity of the read across substance N,N,N',N',N''-Pentamethyl-N-C16-18 (even numbered) and C18 unsat.-alkyl-1,3-propanediammonium chloride to microorganisms, according to OECD Guideline 209, in compliance with GLP. The respiration inhibition test was performed on activated sludge. The toxicity of test substance to activated sludge was determined at a contact time of 3 h, using various concentrations of the test substance. The inhibitory effect of test substance at a particular concentration is expressed as a percentage of the two controls. The test is valid as shown by the EC50 of the reference substance, 3,5 -dichlorophenol (9 mg/L) and the control respiration rates which are within 15% of each other. Under the study conditions, the 3 h EC50 of the test substance was determined to be 63 mg/L (equivalent to 59.535 mg a.i./L) The EC10, EC20 and EC80 were determined to be 34, 42 and 94 mg/L (equivalent to 32.13, 39.69 and 88.78 mg a.i./L), respectively (Geerts, 2010).