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Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09-03-2009 - 13-05-2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test performed under GLP according guidelines, all quality criteria were met

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Reference substance name:
Reference substance 001
Cas Number:
1219458-14-6
Details on test material:
- Chemial name: Triamine C16-18, C18-unsaturated
- EC Number: 628-863-4

To the best of knowledge, the sample used is representative to the boundary composition share and agreed by each registrant.

Sampling and analysis

Analytical monitoring:
not required

Test solutions

Vehicle:
no
Details on test solutions:
The tallow dipropylene triamine stock solution was prepared by stirring 0.1 g tallow dipropylene triamine in approximately 50 mL of deionized water and finally diluting it to 100 mL with deionized water. The tallow dipropylene triamine stock solution is a homogenous suspension.
pH stock solution = 9.4

Test organisms

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.

Study design

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 conditions

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
-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 dipropylene triamine where: 20.3 mg/l, 40.6 mg/l, 81.2 mg/l, 162.4mg/l, 324.8 mg/l, 649.6 (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

Results and discussion

Effect concentrationsopen allclose all
Duration:
3 h
Dose descriptor:
EC10
Effect conc.:
40 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.:
197 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% confidence limits of 74 and 2869 mg/l
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
595 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 tallow dipropylene triamine for activated sludge after 30 minutes contact time is 595 mg/L. The EC10, EC20 and EC80 are 96, 180 and 1969 mg/L, respectively.
The EC50 of tallow dipropylene triamine for activated sludge after 3 hours contact time is 197 mg/L with 95% confidence limits of 74 and 2869 mg/L The EC10, EC20 and EC80 are 40, 69 and 564 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.

Applicant's summary and conclusion

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 tallow dipropylene triamine for activated sludge after 30 minutes contact time is 595 mg/L. The EC10, EC20 and EC80 are 96, 180 and 1969 mg/L, respectively.
The EC50 of tallow dipropylene triamine for activated sludge after 3 hours contact time is 197 mg/L with 95% confidence limits of 74 and 2869 mg/L The EC10, EC20 and EC80 are 40, 69 and 564 mg/L, respectively.
Tallow dipropylene triamine 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 dipropylene triamine 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 tallow dipropylene triamine 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 tallow dipropylene triamine at a particular concentration is expressed as a percentage of the two controls. The EC50of tallow dipropylene triamine for activated sludge after 30 minutes contact time is 595 mg/L. The EC10, EC20and EC80are 96, 180 and 1969 mg/L, respectively.

The EC50of tallow dipropylene triamine for activated sludge after 3 hours contact time is 197 mg/L with 95% confidence limits of 74 and 2869 mg/L The EC10, EC20and EC80are 40, 69 and 564 mg/L, respectively.

Tallow dipropylene triamine is therefore considered not harmful to activated sludge.

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