Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: As all validation criteria are fulfilled the present study is valid without restrictions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Qualifier:
according to
Guideline:
other: In addition the instructions of the OECD series on testing and assessment, no. 23: Guidance document on aquatic toxicity testing of difficult substances and mixtures" (ENV/JM/MONO(2000)6) were followed.
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Linkwell TBzTD -Tetrakis(phenylmethyl)thioperoxydi(carbothioamid)
- Substance type: mono-constituent substance
- Physical state: solid, powder
- Analytical purity: > 99%
- Lot/batch No.: 08060902
- Expiration date of the lot/batch: 2009-06
- Storage condition of test material: room temperature

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no

Test organisms

Test organisms (species):
activated sludge
Details on inoculum:
- Preparation of inoculum for exposure:
A fresh sample of activated sludge from the aeration basin of a municipal sewage treatment plant (Moers, Germany) was collected one day before carrying out the Activated Sludge Respiration Inhibition Test and aerated overnight until usage for the test.
After determination of the suspended solids (dry weight), 5.859 l activated sludge with a dry matter content of 2.048 g/l (which is equivalent to 12 g dry substance I 3 l) was filtered by means of a water jet pump through a filter paper (Schleicher & Schuell, black ribbon 589(1 )).
The sludge was washed twice with mineral medium. Afterwards, 4 g/l activated sludge (dry matter) was suspended in mineral medium, 50 mill synthetic sewage were added and the whole mixture was stirred and aerated (0.5 - 1.0 litre per minute) overnight at 20 ± 2°C. It was not necessary to buffer the pH of the inoculum suspension before use with sodium hydrogencarbonate solution, as, with a measured pH value of 7.87, it was within the required range of 6.0 to 8.0

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h

Test conditions

Test temperature:
20 ± 2 °C
pH:
inoculum suspension 7.87
others: see Table 1
Details on test conditions:
TEST SYSTEM
- Test vessel: 1 l beakers (3-hour contact period) and 300 ml Karlsruher flasks (O2 measurement)
- Aeration: synthetic air, flow meter



OTHER TEST CONDITIONS
- Adjustment of pH:


EFFECT PARAMETERS MEASURED:
Oxygen consumption was followed by continous measurement of the oxygen concentration with an oxygen electrode for up to 10 minutes.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Test concentrations: 10, 32, 100, 320, 1000 mg/l (nominal)
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrations
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values:
As the test substance is not soluble in water, each test concentration was prepared individually, stirred for 24 h and the remaining substance particles were removed by filtration.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: EC50 (3 h) = 14.9 mg/l is in the acepted range 5 to 30 mg/l

Any other information on results incl. tables

Table 2: Respiration rates and inhibition of respiration

 

Concentration [mg/l]

Respiration rate
[mg O2/l/h]

Inhibition
[%]

control 1

0

23.4

-

control 2

0

22.8

-

mean control 1 + 2

0

23.1

-

3,5-dichlorophenol

3.2

17.4

 

3,5-dichlorophenol

10

16.2

 

3,5-dichlorophenol

32

6.0

 

Linkwell TBzTD

10

26.5

0

Linkwell TBzTD

32

23.4

0

Linkwell TBzTD

100

27.7

0

Linkwell TBzTD

320

24.0

0

Linkwell TBzTD

1000

20.9

9.5

Applicant's summary and conclusion

Executive summary:

Linkwell TBzTD - Tetrakis(phenylmethyl)thioperoxydi(carbothioamid) was tested in an activated sludge respiration inhibition test after a 3-hour contact period according to the OECD Guideline for Testing of Chemicals, No. 209, to determine whether the test substance is toxic to the microorganisms of activated sludge.

As the test substance is not soluble in water, the test was performed with each test concentration of Linkwell TBzTD - Tetrakis(phenylmethyl)thioperoxydi(carbothioamid) prepared individually, stirred for 24 hand undissolved particles were removed by filtration.

The concentrations tested were 10, 32, 100, 320 and 1000 mg/L. Additionally two inoculum controls, one at the start and one at the end of the experiment, and 3,5-dichlorophenol in concentrations of 3.2, 10 and 32 mg/L as reference substance were tested.

The respiration rate of the activated sludge in the test vessels containing up to 1000 mg/L Linkwell TBzTD - Tetrakis(phenylmethyl)thioperoxydi(carbothioamid) (as test substance preparation) was not inhibited in comparison to the mean of the inoculum controls. Only at 1000 mg/L an inhibition rate of 9,5 % was observed, thus demonstrating that the EC50 is

above the highest tested concentration value.

EC50 > 1000 mg/L (test substance preparation)

The 3-hour EC50 for the reference substance 3,5-dichlorophenol was within the tolerable range of 5 - 30 mg/L. The deviation between the two inoculum controls was 1.8 % and therefore below the maximum permissible deviation of 15 %.