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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:
1989.10.19 to 1989.11.02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted in accordance with a recognized scientific procedure for biodegradation and is based on OECD 209 EEC Part C, Methods for the determination of ecotoxicity, biodegradation, activated sludge respiration inhibition test.
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:
yes
Remarks:
See "Any other information on materials and methods incl. tables"
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
No surrogate or test material was used.
Analytical monitoring:
not specified
Details on sampling:
No data.
Vehicle:
yes
Details on test solutions:
Stock solution
The test substance was adsorbed onto silica gel using a stock solution of 10 g/litre in dichloromethane.
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:
No post exposure observation period was realised.
Hardness:
No data
Test temperature:
20°C
pH:
The pH of the reaction mixture before the respiration measurement varies from 7.5 to 7.9.
Dissolved oxygen:
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 thermostated vessel with a magnetic stirrer and an oxygen electrode. The vessel is tightly closed by the electrode to measure the oxygen depletion. The volume of the vessel was 10 ml and the temperature in the vessel was 20°C.
Salinity:
The test was performed in freshwater.
Nominal and measured concentrations:
No data. Results are expressed as nominal concentrations.
Details on test conditions:
Test procedure
The activated sludge respiration inhibition test was performed according to EEC (4.1) and OECD test guidelines (4.2). In this test, the respiration rate of activated sludge fed with a standard amount of synthetic sewage is measured after a contact time of 30 minutes. The respiration rate of the same activated sludge in the presence of various concentrations of the test substance under otherwise identical conditions is also measured. The inhibitory effect of the test substance at a particular concentration is expressed as a percentage of the mean respiration rates of the controls. One deviation introduced and some aspects of the test are presented in "Any other information on materials and methods incl. tables".

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:
instead of 3,5-dichiorophenol, 2,4,5-trichlorophenol was used as a reference compound because 3,5-dichiorophenol was not commercially available. 2,4,5-Trichlorophenol was chosen for its structural resemblance and is expected to give the same result
Duration:
30 min
Dose descriptor:
NOEC
Effect conc.:
> 1 000 mg/L
Remarks on result:
other: IUCLID4 note: "m" (measured/nominal)
Duration:
30 min
Dose descriptor:
EC0
Effect conc.:
> 1 000 mg/L
Remarks on result:
other: IUCLID4 note: "m" (measured/nominal)
Details on results:
The results are presented in the Table in "Remarks on results including tables and figures". No inhibition of the respiration rate occurred at 1000 mg/litre of bis (tert-butyl peroxy isopropyl)benzene adsorbed onto silica gel. This result shows that [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide, at the obtained distribution, is not toxic for activated sludge.
Results with reference substance (positive control):
See table in "Remarks on results including tables and figures"
Reported statistics and error estimates:
No data.

Respiration rates of the activated sludge(R5) and inhibition percentaqes calculated at 1 q/litre of the test compound

concentration

pH

respiration rate

inhibition

(mg/litre)

start

(mg02/litre min)

(%)

control (Rci)

7.9

0.37

-‑

control (Rc2)

7.9

0.36

-‑

1000

7.5

0.36

1

Respiration rates of the activated sludqe and inhibition percentages calculated at various concentrations of 2,4,5-trichlorophenol (reference substance)

concentration

pH

respiration rate

inhibition

(mg/litre)

start

(mg02/litre min)

(%)

control

7.9

0.37

-‑

control

7.9

0.37

-‑

5

7.8

0.30

16

10

7.9

0.20

46

20

7.9

0.13

66

40

7.9

0.05

86

Respiration rates of the activated sludqe and the inhibition percentage calculated for silica gel

concentration

pH

respiration rate

inhibition

(mg/litre)

start

(mg 02/litre min)

(%)

control (Rc1)

7.9

0.37

-‑

control (Rc2)

7.9

0.36

-‑

silica gel

7.5

0.38

0

Validity criteria fulfilled:
yes
Remarks:
See Conclusions
Conclusions:
The validity of the test is shown by two criteria. Firstly, the control respiration rates are within 0% of each other and secondly, the EC50 of the reference compound is within the described range (5-30 mg/litre). Furthermore, the silica gel used is not toxic for the activated sludge. The pH of the reaction mixture before the respiration measurement varies from 7.5 to 7.9. The test substance was not soluble in water and therefore the activated sludge was exposed to [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide adsorbed onto silica gel at a concentration of 1 g/litre. By this exposure no effect was recorded indicating that the obtained distribution of the [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide in the water system did not inhibit the activated sludge, EC 50 > 1000 mg/l.
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 according to OECD 209 guideline and GLP requirements. The test substance was not soluble in water and therefore the activated sludge was exposed to [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide adsorbed onto silica gel at a concentration of 1 g/litre. By this exposure no effect was recorded indicating that the obtained distribution of [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide in the water system did not inhibit the activated sludge. Validity criteria were fulfilled.

Description of key information

In order to predict effects of [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide in the environment and in biological wastewater treatment plants, an activated sludge respiration inhibition test was performed according to OECD 209 guideline and GLP requirements. The test substance was not soluble in water and therefore the activated sludge was exposed to [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide adsorbed onto silica gel at a concentration of 1 g/litre. By this exposure no effect was recorded indicating that the obtained distribution of [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide in the water system did not inhibit the activated sludge and EC50 > 1000 mg/l.

Key value for chemical safety assessment

Additional information

In order to predict effects of chemicals in the environment and in biological wastewater treatment plants, an activated sludge respiration inhibition test was performed according to OECD 209 guideline and GLP requirements. The test substance was not soluble in water and therefore the activated sludge was exposed to [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide adsorbed onto silica gel at a concentration of 1 g/litre. By this exposure no effect was recorded indicating that the obtained distribution of [1,3-phenylenebis(1-methylethylidene)]bis[tert-butyl] peroxide in the water system did not inhibit the activated sludge. Validity criteria were fulfilled.