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Ecotoxicological information

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:
from 1989-10-19 to 1989-11-02
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
All validity criteria have been met, GLP and guideline study. Some deviations from guideline but these do not impair integrity. However the exposure regime cannot be confirmed as complete diffusion of the test subtance adsorbed on silica gel cannot be assumed and part of substance might have been lost due to its high volatility.
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
vehicle and different reference substance
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
not specified
Principles of method if other than guideline:
vehicle: silica gel
reference substance: 2,4,5 trichlorophenol
No confirmation of test concentration
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not soluble in water at test concentrations
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
yes
Details on test solutions:
The test substance was not soluble at 1000 mg/l in water and therefore the test substance was first dissolved in dichloromethane. The test substance in dichloromethane was added to silica gel (100-200 mesh) weighed in a petri-dish. The solvent was allowed to evaporate by placing the petri-dish in a hood for 3 hours, and the entire contents were then transferred to the test erlenmeyers. This method allowed a good contact between the activated sludge and the test substance. Although no toxicity of the silica gel was expected, a control with silica gel was carried out as well.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Secondary activated sludge was collected from the RZWI Nieuwgraaf in Duiven (1989.10.27). This activated sludge plant predominantly treats domestic wastewater. Prior to use, the activated sludge was diluted with tap water (1.5x) and homogenized for two minutes in a lab blender 400. The activated sludge concentration in the incubation vessels was 0,5 g dry weight/litre.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
30 min
Post exposure observation period:
None
Hardness:
No data
Test temperature:
20 °C
pH:
7.5-7.9
Dissolved oxygen:
Parameter of interest in this test
Salinity:
Not applicable
Nominal and measured concentrations:
1000 mg/L nominal asorbed on silica gel.
Details on test conditions:
The homogenized activated sludge was incubated in a shaking water bath (100 HZ, 20°C) for 30 minutes with various concentrations of the test compound and synthetic sewage.
Reference substance (positive control):
yes
Remarks:
2,4,5-trichlorophenol
Duration:
30 min
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:
The test substance was adsorbed on silica gel for 30mn and this is considered sufficient given the water solubility of 171 mg/L. However, due to the high volatility of the test substance (vapour pressure is 42 hPa at 20°C) some may have evaporated when placed in the hood during the preparation of the silica gel.
Nevertheless it can be assume that the substance was tested at its water solubility level (171mg/l).
Results with reference substance (positive control):
EC50 of 10-20 mg/L, within limits according to guideline: 5-30 mg/L
Reported statistics and error estimates:
Not applicable.

Concentration mg/L

pH start

Respiration rate (mg O2/L min

Inhibition%

Control (RC1)

7.9

0.37

--

Control (RC2)

7.9

0.37

--

1000

7.5

0.37

0

Validity criteria fulfilled:
yes
Conclusions:
All validity criteria have been met, GLP and guideline study. Some deviations from guideline but these do not impair integrity. However the exposure regime cannot be confirmed as the microorganisms were exposed to the poorly soluble test subtance adsorbed on silica gel only for 30mn. Moreover, according to the high volatility of the test substance (vapour pressure is 42 hPa at 20°C) some may have evaporate when placed in the hood during the preparation of the silica gel. The EC50 of activated sludge for di-tert-butyl peroxide is concluded to be > 171 mg/L based on its water solubilty.
Executive summary:

In order to predict effects of di-tert-butyl peroxide in biological wastewater treatment plants, an activated sludge respiration inhibition test was performed, according to OECD 209 guideline and EU Method C.11.

The inoculum was an activated sludge, at 0.5 g dry weight/L, collected from the RZWI Nieuwgraaf in Duiven plant which predominantly treats domestic wastewater. Prior to use, the activated sludge was diluted with tap water (1.5x) and homogenized for two minutes.

The test substance was not soluble in water at the nominal concentration of 1000 mg/l and therefore it was first dissolved in dichloromethane and adsorbed onto silica gel in petri-dish for 30 min. The solvent was allowed to evaporate by placing the petri-dish in a hood for 3 hours, and the entire contents were then transferred to the test erlenmeyers. This method allowed a good contact between the activated sludge and the test substance. Although no toxicity of the silica gel was expected, a control with silica gel was carried out as well. The diffusion of the test material during the 30 mn of exposure is uncertain and according to the high volatility of the test substance (vapour pressure is 42 hPa at 20°C) some may have evaporate when placed in the hood during the preparation of the silica gel. The temperature was 20°C and the pH ranged from 7.5 to 7.9. 2,4,5-trichlorophenol was used as a reference substance during the test and show an EC50 of 10-20 mg/L which is within limits according to guideline (5 -30 mg/L). With the test material, no effect was recorded, but as the exposure concentration cannot be confirmed it is assumed that the highest concentration tested is equivalent at the water solubility concentration of di-tert-butyl peroxide. The EC50 therefore is >171 mg/L based on water solubility of the test material.

Description of key information

The toxicity of the substance to activated sludge was tested in a reliable GLP study following OECD 209 and guidance on the testing of difficult substances. A 30 minute EC50 >1000 mg/L, which is above the water solubility, was observed. No effect up to the highest concentration tested of 1000 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

Two studies were available for this endpoint. Granditschet al.(1979) tested toxicity of the substance on activated sludge following three different methods but after 4 hours of exposure only and assessing the concentration which killed 100% of the bacterial population. Ginkel and Stroo (1989) assessed the toxicity of the substance to activated sludge in a reliable GLP study following OECD 209 and guidance on the testing of difficult substances. They calculated a 30 min EC50 nominal as >1000 mg/L