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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Version / remarks:
1984
Deviations:
yes
Remarks:
special test design
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Version / remarks:
1988
Deviations:
yes
Remarks:
special test design
Principles of method if other than guideline:
NA
GLP compliance:
yes
Analytical monitoring:
no
Details on sampling:
NA
Vehicle:
yes
Details on test solutions:
The test substance was adsorbed onto the silica gel using a stock solution of 10 g/L in dichloromethane.
Test organisms (species):
other: Secondary activated sludge
Details on inoculum:
Secondary activated sludge was collected from the RZWT Nieuwgraaf in Duiven. This activated sludge plant predominantly treats domestic wastewater. Prior to use, the activated sludge was diluted with tap water and homogenized for two minutes in a lab blender. The activated sludge concentration in the incubation vessels was 0.5 g dry weight/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
30 min
Remarks on exposure duration:
No remarks
Post exposure observation period:
NA
Hardness:
No data
Test temperature:
20 °C
pH:
pH at the beginning of the test: between 7.5 and 7.9
Dissolved oxygen:
No data
Salinity:
NA
Nominal and measured concentrations:
nominal concentrations: 31.3, 62.5, 125.0, 250.0, 500 mg/L
Details on test conditions:
TEST SYSTEM
The respiration rates of the activated sludge were measured in a Biological Oxygen Monitor YSI 5300 (Yellow Springs Instruments, Ohio, USA). The BOM consisted of a temperature controlled vessel with a magnetic stirrer and an oxygen electrode. The vessel is tightly closed by the electrode to measure the oxygen consumed. The volume of the vessel was 10 mL and the temperature in the vessel was 20 °C.
Reference substance (positive control):
not specified
Key result
Duration:
30 min
Dose descriptor:
EC50
Effect conc.:
64 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL: 15.7 and 117 mg/L
Key result
Duration:
30 min
Dose descriptor:
EC10
Effect conc.:
0.01 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: No remarks
Duration:
30 min
Dose descriptor:
other: EC20
Effect conc.:
0.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: No remarks
Duration:
30 min
Dose descriptor:
other: EC80
Effect conc.:
17 327 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: No remarks
Details on results:
The 0.5-h EC50 of TBPEH for activated sludge was 64 mg/L with 95% confidence limits of 15.7 and 117.0 mg/L. The EC10, EC20 and EC80 were 0.01, 0.2 and 17327 mg/L, respectively.
Results with reference substance (positive control):
No positive control
Reported statistics and error estimates:
NA

Respiration rates of the activated sludge and inhibition percentage calculated at various concentrations of the test compound absorbed onto silica gel:

concentration (mg/L)

pH start

respiration rate (mg O2/L min)

inhibition (%)

control (Rc1)

7.9

0.37

-

control (Rc1)

7.9

0.36

-

31.3

7.6

0.21

42

62.5

7.5

0.17

53

125.0

7.5

0.16

56

250.0

7.5

0.16

56

500.0

7.5

0.14

62

Validity criteria fulfilled:
yes
Conclusions:
The 0.5-h EC50 of tert.-Butylperoxy-2-ethylhexanoat for activated sludge was 64 mg/L with 95% confidence limits of 15.7 and 117.0 mg/L. The EC10, EC20 and EC80 were 0.01, 0.2 and 17327 mg/L, respectively.
Executive summary:

In order to predict effects of TBPEH in the environment and in biological wastewater treatment plants, an activated sludge respiration inhibition test was performed. In this toxicity test, the respiration rate of activated sludge collected from a domestic sewage treatment plant fed with synthetic sewage was measured after an incubation period of 30 minutes. For this test, a new technique was applied, in which the test substance was first adsorbed onto silica gel. The bacterial suspension was exposed to the silica gel during 30 minutes in order to establish a good equilibrium in the distribution of the test substance between the water phase an the silica gel. Thus, the measured effects are caused by exposure to silica-adsorbed amounts in water. The respiration rate of the same activated sludge in the presence of various concentrations of the test substance (31.3, 62.5, 125.0, 250.0, 500 mg/L) under otherwise identical conditions was also measured. Using these data a concentration effect relationship was obtained. The 0.5-h EC50 of TBPEH for activated sludge was 64 mg/L with 95% confidence limits of 15.7 and 117.0 mg/L. The EC10, EC20 and EC80 were0.2 and 17327 mg/L, respectively.

Description of key information

Tert.-Butylperoxy-2-ethylhexanoat was assessed in an activated sludge respiration inhibition test, according to the EU method C.11 and OECD guideline 209. The EC50 was determined at 64 mg/L and the EC10 determined at 0.01 mg/L.

Key value for chemical safety assessment

EC50 for microorganisms:
64 mg/L
EC10 or NOEC for microorganisms:
0.01 mg/L

Additional information

In order to predict effects of TBPEH in the environment and in biological wastewater treatment plants, an activated sludge respiration inhibition test was performed. In this toxicity test, the respiration rate of activated sludge collected from a domestic sewage treatment plant fed with synthetic sewage was measured after an incubation period of 30 minutes. For this test, a new technique was applied, in which the test substance was first adsorbed onto silica gel. The bacterial suspension was exposed to the silica gel during 30 minutes in order to establish a good equilibrium in the distribution of the test substance between the water phase an the silica gel. Thus, the measured effects are caused by exposure to silica-adsorbed amounts in water. The respiration rate of the same activated sludge in the presence of various concentrations of the test substance (31.3, 62.5, 125.0, 250.0, 500 mg/L) under otherwise identical conditions was also measured. Using these data a concentration effect relationship was obtained. The 0.5-h EC50 of TBPEH for activated sludge was 64 mg/L with 95% confidence limits of 15.7 and 117.0 mg/L. The EC10, EC20 and EC80 were0.2 and 17327 mg/L, respectively.