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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:
12 apr 2010 - 15 apr 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results. Conclusive valid guideline study under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No. 440/2008, Part C.11, 30 May 2008
Deviations:
no
Principles of method if other than guideline:
A limit test was performed based on the EU testing guidelines to demonstrate that the test item has no toxic effect on activated sludge up to the nominal test concentration of 1000 mg/L.
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
500.9 mg of the test item was weighed by means of an analytical balance onto a glass slide and transferred into the designated test vessel. Then, 284 mL tap water was added to each vessel.

The test item was mixed into the tap water by ultrasonic treatment for 30 minutes and intense stirring for 3 hours and 30 minutes 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 3,3,5-trimethylcyclohexylidene diperoxide under the test conditions. Then, 16 mL synthetic sewage feed and 200 mL activated sludge inoculum were added.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
The study was performed with aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland) treating predominantly domestic wastewater. 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.

Based on this ratio, 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 three 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 7.6.

* Synthetic sewage feed:

16 g peptone
11 g meat extract
3 g urea
0.7 g NaCl filled up to a final volume of 1 liter
0.4 g CaCl2 × 2H2O with deionized water
0.2 g MgSO4 × 7H2O
2.8 g K2HPO4
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Test temperature:
20 °C
pH:
7.4 - 8.3
Dissolved oxygen:
7.9 - 9.0 mg/L
Nominal and measured concentrations:
1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type: open
- Material, size, headspace, fill volume: glass, 2000 mL, open, 500 mL
- Aeration: continuously aerated by intense stirring on magnetic stirrers

- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 2
- Biomass loading rate: The inoculum had a sludge concentration of 2.7 g/L dry weight (corresponding to about 1.1 g dry material per liter test medium).


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: local tap water


OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Respiration rate (= oxygen consumption rate); measured continually.
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. 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. During measurement, the samples were continuously stirred on a magnetic stirrer.


TEST CONCENTRATIONS
- Limittest with 1000 mg/L

- Justification for using less concentrations than requested by guideline: A limit test was performed based on the EU testing guidelines to demonstrate that the test item has no toxic effect on activated sludge up to the nominal test concentration of 1000 mg/L.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol
Duration:
3 h
Dose descriptor:
EC10
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:
other: EC20
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
Duration:
3 h
Dose descriptor:
other: EC80
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:
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 3,3,5-trimethylcyclohexylidene diperoxide under the test conditions.

The test item di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide had no significant inhibitory effect (≤10%) on the respiration rate of activated sludge after the incubation period of 3 hours at the loading rate of 1000 mg/L. Based on the physical appearance of the test suspension, the saturation concentration (water solubility limit of the test item under the test conditions) was <1000 mg/L and resulted in less than 10% inhibition after three hours contact time.

The oxygen consumption rates of the two controls (run at the start and at the end of the test) differed by 5% (guideline-recommendation: the two control respiration rates should be within 15% of each other).
Results with reference substance (positive control):
- Results with reference substance valid? yes

The 3 hour EC50 of the reference item 3,5 dichlorophenol (positive control) was calculated to be 17 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.
Reported statistics and error estimates:
The 3 hour EC20, EC50 and EC80 values of the test item and their 95% confidence limits could not be calculated because of the absence of a toxic effect.

The 3 hour EC50 of the reference item was calculated by Probit Analysis.

All test results were based on nominal concentrations of the test and reference item.

 

 

 

 

 

 

 

 

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

1.443

 

7.5

8.2

8.3

8.7

B

0

1.520

 

7.5

8.2

8.3

8.2

Mean

 

1.481

 

 

 

 

 

Deviation (%)

5.4

 

 

 

 

 

 

Test item

 

 

 

 

 

 

6

1000

1.620

-9.4

7.4

8.0

7.9

8.0

 

Reference item

 

 

 

 

 

 

1

5

1.547

-4.4

7.4

8.1

8.2

8.7

2

16

0.722

51.3

7.4

8.1

8.5

9.0

3

50

0.165

88.9

7.4

8.3

8.5

8.7

*                        Start and end of the 3-hour incubation period

- % inhibition: increased oxygen consumption rate relative to control

Validity criteria fulfilled:
yes
Conclusions:
The test item di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide had no significant inhibitory effect (≤10%) on the respiration rate of activated sludge after the incubation period of 3 hours at the loading rate of 1000 mg/L. Based on the physical appearance of the test suspension, the saturation concentration (water solubility limit of the test item under the test conditions) was <1000 mg/L and resulted in less than 10% inhibition after three hours contact time.

Thus, the 3 hour NOEC (EC10) of di-tert-butyl 3,3,5-trimethylcyclohexylidene diperoxide to activated sludge microorganisms was at least 1000 mg/L. This value might even be higher but concentrations above 1000 mg/L were not tested. The 3 hour EC20, EC50, and EC80 could not be calculated but were clearly higher than 1000 mg/L.
Executive summary:

The inhibitory effect of the test item di-tert-butyl 3,3,5-trimethylcyclohexylidene 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/EEC, 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 3,3,5-trimethylcyclohexylidene 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 3,3,5-trimethylcyclohexylidene 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

Based on the available study results, 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 EC80 could not be calculated but were clearly higher than 1000 mg/L. Thus, the substance seems not to be potentially toxic to microorganisms.

Key value for chemical safety assessment

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

Additional information

One study was available to assess the toxicity of the substance on microorganisms (Eisner 2010). The inhibitory effect of the test item di-tert-butyl 3,3,5-trimethylcyclohexylidene 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/EEC, Part C.11, Commission Regulation (EC) No. 440/2008, Part C.11 and the OECD Guideline for Testing of Chemicals, No. 209.

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 3,3,5-trimethylcyclohexylidene 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.

In conclusion, the substance seems not to be potentially toxic to microorganisms.