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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Draft Report. Experiment conducted under GLP, and following OECD guidelines.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Principles of method if other than guideline:
not applicable.
GLP compliance:
yes (incl. certificate)
Remarks:
2011-01-11
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
no data
Analytical monitoring:
no
Details on sampling:
- Concentrations: 1000 mg/L
- Sampling method: For measurement of the respiration rate a well-mixed sample of each test medium was poured into a BOD-flask after three hours incubation time, and was not further aerated. Then the dissolved oxygen concentration was measured with an oxygen electrode (WTW TriOxmatic® 300 and an oxygen meter WTW Oxi 539, Wissenschaftlich-Technische Werkstaetten WTW, Weilheim/Germany), and was continuously recorded. During measurement, the samples were continuously stirred on a magnetic stirrer. The oxygen consumption rate (in mg O2 L-1 minute-1) was determined from the linear part of the respiration curve in the range 6.5–2.5 mg O2/L. In case of very rapid oxygen consumption, the range used was below the limits indicated above but always within the linear part of the respiration curve. In case of low oxygen consumption the rate was determined over a period of at least ten minutes.
- Sample storage conditions before analysis: no data.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Based on the information provided by the Sponsor, no stock solution could be prepared due to the low water solubility of the test item.
Therefore, 500.2 mg of the test item was weighed by means of an analytical balance and transferred to the designated test vessel with 284 mL tap water. The test item was mixed into the tap water by intense stirring for three hours at room temperature in the dark to dissolve a maximum amount of the test item and/or disperse it as homogeneously as possible. No emulsifiers or solvents were used.
After stirring, the appearance of the test medium was evaluated, and the test concentration was clearly above the water solubility limit of Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide under the test conditions. Then, 16 mL synthetic sewage feed and 200 mL activated sludge inoculum were added.
- Eluate: tap water
- Differential loading: no
- Controls: Two controls containing only tap water, synthetic sewage feed and inoculum were tested in parallel to single test concentration of the test item under identical test conditions.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no data.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: Wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland) treating predominantly domestic wastewater.
- Method of cultivation: The sludge was washed twice with tap water by centrifugation and the supernatant liquid phase was decanted. A homogenized aliquot of the final sludge suspension was weighed, thereafter dried and the ratio of wet to dry weight was calculated.
- Preparation of inoculum for exposure: An aliquot of washed sludge was suspended in tap water to obtain a concentration equivalent to 3 g dry material per liter. During the holding period of two days prior to use, the sludge was fed daily with 50 mL synthetic sewage feed* per liter and was kept at room temperature under continuous aeration until use. Before use, the dry weight of the activated sludge was measured again in the inoculum used for the test. The pH of the activated sludge inoculum was 7.2.
- Pretreatment: none
- Initial biomass concentration: no data
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
no data
Hardness:
no data
Test temperature:
19-20°C
pH:
7.3-8.6
Dissolved oxygen:
7.9-8.4
Salinity:
not applicable
Nominal and measured concentrations:
nominal conc. 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beakers
- Type (delete if not applicable): no data
- Material, size, headspace, fill volume: 2000 mL
- Aeration: During the incubation period of 3 hours the single test medium and the controls were continuously aerated by intense stirring on magnetic stirrers to avoid possible foaming and/or stripping of the test item.
- No. of organisms per vessel: no data
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Biomass loading rate: no data

OTHER TEST CONDITIONS
- Adjustment of pH: no data
- Photoperiod: no data
- Light intensity: no data
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 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.:
> 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:
no data
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: The 3 hour EC50 of the reference item 3,5 dichlorophenol (positive control) was calculated to be 9 mg/L (the 95% confidence limits could not be calculated due to mathematical reasons). The 3 hour EC50 is within the guideline-recommended range of 5–30 mg/L, confirming suitability of the activated sludge used.
- Other: None.
Reported statistics and error estimates:
no data

Table 1: Influence of Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide (Test Item) and 3,5‑Dichlorophenol (Reference Item) on the Oxygen Consumption of Activated Sludge

 

 

 

 

 

 

 

 

Vessel no.

Nominal concentration of test chemical

Oxygen consumption rate

Inhibition

pH values

Oxygen concentration

(mg O2/L)

 

(mg/L)

(mg O2L-1min-1)

(%)

start*

end*

start*

end*

 

Control

 

 

 

 

 

 

A

0

1.033

 

7.3

8.5

8.1

7.9

B

0

1.078

 

7.5

8.4

8.0

8.0

Mean

 

1.056

 

 

 

 

 

Deviation (%)

4.3

 

 

 

 

 

 

Test item

 

 

 

 

 

 

6

1000

1.004

4.9

7.6

8.4

8.4

7.9

 

Reference item

 

 

 

 

 

 

1

5

0.821

22.2

7.6

8.6

8.1

7.6

2

16

0.240

77.3

7.5

8.6

8.2

8.6

3

50

0.089

91.6

7.5

8.6

8.1

8.6

Validity criteria fulfilled:
yes
Conclusions:
The 3‑hour NOEC (EC10) of the test item to activated sludge microorganisms was at least 1000 mg/L (i.e. the saturation concentration of the test item). This value might even be higher but concentrations above 1000 mg/L were not tested. The 3‑hour EC20, EC50, and EC80could not be calculated but were clearly higher than 1000 mg/L.
Executive summary:

The inhibitory effect of the test item Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3‑hour respiration inhibition test according to the EU Commission Directive 88/302/, Part C.11, Commission Regulation (EC) No. 440/2008, Part C.11 and the OECD Guideline for Testing of Chemicals, No. 209.

 

Based on the EU testing guidelines, a limit test with a nominal test concentration of 1000 mg/L was performed.

 

In addition, two controls and three different concentrations of the reference item 3,5‑dichlorophenol (5, 16, and 50 mg/L) were tested in parallel. The results of these treatments confirmed suitability of the activated sludge and the method used.

 

At the test item concentration of 1000 mg/L, the test item was not completely dissolved in the test medium. Thus, the test concentration was clearly above the water solubility limit of Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide under the test conditions.

 

The test item had no inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours at a loading rate of 1000 mg/L. The saturation concentration (water solubility limit of Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide under the test conditions) was reached and no inhibitory effect was determined after three hours contact time.

 

Thus, the 3‑hour NOEC (EC10) of the test item to activated sludge microorganisms was at least 1000 mg/L (i.e. the saturation concentration of the test item). This value might even be higher but concentrations above 1000 mg/L were not tested. The 3‑hour EC20, EC50, and EC80could not be calculated but were clearly higher than 1000 mg/L.

 

Description of key information

The 3‑hour NOEC (EC10) of the test item to activated sludge microorganisms was at least 1000 mg/L (i.e. the saturation concentration of the test item). This value might even be higher but concentrations above 1000 mg/L were not tested. The 3‑hour EC20, EC50, and EC80could not be calculated but were clearly higher than 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L
EC10 or NOEC for microorganisms:
1 000 mg/L

Additional information

The inhibitory effect of the test item Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3‑hour respiration inhibition test according to the EU Commission Directive 88/302/, Part C.11, Commission Regulation (EC) No. 440/2008, Part C.11 and the OECD Guideline for Testing of Chemicals, No. 209.

 

Based on the EU testing guidelines, a limit test with a nominal test concentration of 1000 mg/L was performed.

 

In addition, two controls and three different concentrations of the reference item 3,5‑dichlorophenol (5, 16, and 50 mg/L) were tested in parallel. The results of these treatments confirmed suitability of the activated sludge and the method used.

 

At the test item concentration of 1000 mg/L, the test item was not completely dissolved in the test medium. Thus, the test concentration was clearly above the water solubility limit of Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide under the test conditions.

 

The test item had no inhibitory effect on the respiration rate of activated sludge after the incubation period of three hours at a loading rate of 1000 mg/L. The saturation concentration (water solubility limit of Di-tert-butyl 1,1,4,4-tetramethyltetramethylene diperoxide under the test conditions) was reached and no inhibitory effect was determined after three hours contact time.

 

Thus, the 3‑hour NOEC (EC10) of the test item to activated sludge microorganisms was at least 1000 mg/L (i.e. the saturation concentration of the test item). This value might even be higher but concentrations above 1000 mg/L were not tested. The 3‑hour EC20, EC50, and EC80could not be calculated but were clearly higher than 1000 mg/L.