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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:
09-03-2009 - 14-05-2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test performed under GLP according guidelines, all quality criteria were met
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
not required
Vehicle:
no
Details on test solutions:
The tallowtripropylenetetramine stock solution was prepared by stirring 0.1 g tallowtripropylenetetramine in approximately 50 mL of deionized water and finally diluting it to 100 mL with deionized water. The tallowtripropylenetetramine stock solution is a homogenous suspension
pH stock solution = 9.2

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Secondary activated sludge was obtained (1st test: 10-03-2009 and 2nd test: 12-05-2009) (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 activated sludge in the incubation vessels was 1.6 g/L (1st test) and 1.7 g/L (2nd test).

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Remarks on exposure duration:
30 minutes exposure was tested first
Test temperature:
Temperature varied from 20.0 to 20.9°C.
pH:
The pH of the reaction mixtures after the incubation period ranged from 6.6 to 7.8.
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 (The first test was performed withan incubation period of 30 minutes, because the test substance has a low solubility in water the incubation period used should be 3 hours = second test)
-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 1.6 g/l-1.7 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 tallow tripropylenetetramine where: 20.4 mg/l, 40.8 mg/l, 81.5 mg/l, 163.0mg/l, 326.1 mg/l, 652.2 (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
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
26 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
289 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
921 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
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 6.6 to 7.8. Temperature varied from 20.0 to 20.9°C. These conditions allowed respiration of the activated sludge used.
The EC50 of tallowtripropylenetetramine for activated sludge after 30 minutes contact time is 921 mg/L. The EC10, EC20 and EC80 are 138, 264 and 3205 mg/L, respectively.
The EC50 of tallowtripropylenetetramine for activated sludge after 3 hours contact time is 289 mg/L. The EC10, EC20 and EC80 are 26, 59 and 1420 mg/L, respectively.


Results with reference substance (positive control):
The EC50 of the reference compound were 8 and 11 mg/L at a contact time of 30 minutes and 3 hours, respectively. These EC50 values are 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 second¬ly, the EC50 of the reference compound were 8 and 11 mg/L at a contact time of 30 minutes and 3 hours, respectively.
Conclusions:
Test performed under GLP according guidelines. The test is valid as shown by the EC50of the reference compound, 3,5-dichlorophenol (8 mg/L and 11 mg/L contact time 30 minutes and 3 hours respectively) and the control respiration rates which are within 15% of each other.
The EC50 of tallowtripropylenetetramine for activated sludge after 30 minutes contact time is 921 mg/L. The EC10, EC20 and EC80 are 138, 264 and 3205 mg/L, respectively.
The EC50 of tallowtripropylenetetramine for activated sludge after 3 hours contact time is 289 mg/L. The EC10, EC20 and EC80 are 26, 59 and 1420 mg/L, respectively.
Tallowtripropylenetetramine is therefore considered not harmful to activated sludge.

The results of the 30 minutes incubation period should only be used as additional info. An incubation period of 3 hours is required to determine the toxicity of tallow tripropylenetetramine to activated slugde, because of the low solubility of the test substance in water.
Executive summary:

In order to predict effects of chemicals in the environment and in biological waste water treatment plants, an activated sludge respiration inhibition test was performed. The toxicity to activated sludge was determined in accordance with OECD Test Guideline 209, and in compliance with the OECD principles of Good Laboratory Practice.

The test is valid as shown by the EC50of the reference compound, 3,5-dichlorophenol (8 mg/L and 11 mg/L contact time 30 minutes and 3 hours respectively) and the control respiration rates which are within 15% of each other.

The toxicity of tallowtripropylenetetramine to activated sludge was determined at contact times i.e. 0.5 and 3 hours, using various concentrations of the test substance. The inhibitory effect of tallowtripropylenetetramine at a particular concentration is expressed as a percentage of the two controls. The EC50of tallowtripropylenetetramine for activated sludge after 30 minutes contact time is 921 mg/L. The EC10, EC20and EC80are 138, 264 and 3205 mg/L, respectively.

The EC50of tallowtripropylenetetramine for activated sludge after 3 hours contact time is 289 mg/L. The EC10, EC20and EC80are 26, 59 and 1420 mg/L, respectively.

Tallowtripropylenetetramine is therefore considered not harmful to activated sludge.

Description of key information

Key value for chemical safety assessment

EC50 for microorganisms:
289 mg/L
EC10 or NOEC for microorganisms:
26 mg/L

Additional information

30 minutes EC50 = 921 mg/L