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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:
27-10-1989
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. No confirmation that test substance did not evaporate when placed in the hood (vapour pressure?), but considered unlikely on basis of molecular properties. The exposure regime can be considered worse case as compared to non-vehicle use, therefore the outcome will be overestimates. When data on vapour pressure known, the data can be used for risk assessment.
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
a different reference compound was used, a vehicle was used
Principles of method if other than guideline:
instead of 3,5-dichlorophenol, 2,4,5-trichlorophenol was used as reference substance. Silica gel was used as vehicle.
No confirmation of actual test concentrations
GLP compliance:
yes
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
PHYSICO-CHEMICAL PROPERTIES
- Water solubility (under test conditions): not soluble at test concentrations
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
the test substance is a poorly soluble substance 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
Test temperature:
20 C
pH:
7.9 in controls
7.2 in 1000 mg/L
Dissolved oxygen:
Is parameter of interest in this test
Nominal and measured concentrations:
nominal: 1000 mg/L
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. The volume of the test vessel was 10 mL.
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:
A slight inhibition (24%) of the respiration rate occurred at 1000 mg/litre of dilauroyl peroxide absorbed onto silica gel.
Results with reference substance (positive control):
EC50: 10-20 mg/L and thus within the guideline criteria : 5-30 mg/L

Table 1: Respiration rates of the activated sludge and inhibition calculated at 1 g/L of the test compound

Concentration (mg/L)

pH start

Respiration rate (mg O2/litre min)

Inhibition (%)

Control (RC1)

7.9

0.37

--

Control (RC2)

7.9

0.37

--

1000

7.2

0.28

24

 

Table 2: respiration rates of the activated sludge and inhibition percentages calculated at various concentrations of the reference substance

Concentration (mg/L)

pH start

Respiration rate (mg O2/litre min)

Inhibition (%)

Control

7.9

0.37

--

Control

7.9

0.37

--

5

7.9

0.31

16

10

7.9

0.20

46

20

7.9

0.13

66

40

7.9

0.05

86

 Table 3: Respiration rates of the activated sludge and the inhibition percentage calculated for silicagel

Concentration (mg/L)

pH start

Respiration rate (mg O2/litre min)

Inhibition (%)

Control (RC1)

7.9

0.37

--

Control (RC2)

7.9

0.36

--

Silica gel

7.5

0.38

 0

Validity criteria fulfilled:
yes
Conclusions:
The test was performed according to guideline and GLP. Although not indicated and vapour pressure is not given, it is assumed that evaporation of the test compound in the hood had not occurred, confirmation of presence of test substance absent, but when vapour pressure known (evaporation unlikely), the data can be used. To improve exposure of the sludge, the substance was introduced via a vehicle (silica gel). This makes exposure worst case compared to the non vehicle, normal situation. The EC50 outcome will therefore be an overestimate of the actual EC50. The EC50 of dilauroyl peroxide to activated sludge was determined to be >1000 mg/L based on nominal values.
Executive summary:

In order to predict effects of chemicals in the environment and in biological wastewater treatment plants, an activated sludge respiration inhibition test was performed with dilauroyl peroxide, according to the OECD 209 guideline. The test substance was not soluble in water and therefore the activated sludge was exposed to dilauroyl peroxide adsorbed onto silica gel at a concentration of 1 g/litre. By this exposure an effect was recorded indicating that the obtained distribution of the test substance in the water system did inhibit the activated sludge slightly (24%). The

EC50 was determined to be > 1000 mg/L based on nominal concentration, when absorbed onto silica gel. A NOEC was not obtained.

Description of key information

EC50 was determined to be > 1000 mg/L based on nominal concentration, when absorbed onto silica gel. A NOEC was not obtained.

Key value for chemical safety assessment

EC50 for microorganisms:
1 000 mg/L

Additional information

An activated sludge respiration inhibition test was performed with dilauroyl peroxide, according to the OECD 209 guideline. The test substance was not soluble in water and therefore the activated sludge was exposed to dilauroyl peroxide adsorbed onto silica gel at a concentration of 1 g/litre. By this exposure an effect was recorded indicating that the obtained distribution of the test substance in the water system did inhibit the activated sludge slightly (24%). The EC50 was determined to be > 1000 mg/L based on nominal concentration, when absorbed onto silica gel. A NOEC was not obtained.