Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 September–15 November 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
EU Method C.11 (Biodegradation: Activated Sludge Respiration Inhibition Test)
Deviations:
no
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Chemical name: Fumaric acid bis-(2-ethylhexyl) ester
Trade name: Dioctylfumarat, DOF
Molecular formula: C20H36O4
CAS No. 141-02-6
Lot No. LEDF1B1004
Appearance: Colourless liquid
Purity: 99.5%
Density: 0.942 g/cm3
Melting point: -48 °C
Boiling point: 208 °C
Conditions of storage: Room temperature
Stability at conditions of storage: Stable
Expiry date: 31 January 2011

Test solutions

Details on test solutions:
The test substance was not enough soluble in water to prepare a stock solution. Therefore suspensions of the test substance were prepared by stirring the appropriate amounts of test substance in the test vessels with 100 mL tap water for 2 days prior to the start of the test. For the actual amounts of each sample see Table 1. The stock suspensions were used without filtration.
A stock solution of 3,5-dichlorophenol (nominally 0.5 g/L) was prepared by dissolving 50.3 mg of the substance in 100 mL of deionised water. Three concentrations, nominally from 5 to 30 mg/L, were prepared as described in Table 1. No NaOH was used to increase the solubility of dichlorophenol, therefore no adjustment of the pH was necessary.
All incubation mixtures were prepared by adding 200 mL of microbial inoculum and 200 mL of synthetic sewage solution (see 4.7.2) to 100 mL of tap water and/or the appropriate amount of the test or control substance in glass beakers with a nominal volume of 1000 mL (see Table 1).
The samples consisted of two negative controls (one before and one after all the test substance samples), six concentrations of dioctyl fumarate, and three concentrations of the positive control (3,5-dichlorophenol). Two blank values containing the two highest test substance concentrations and tap water instead of microbial inoculum were incubated to reveal a possible oxygen uptake by the test substance. Additionally, two duplicate incubation mixtures containing the two highest test substance concentrations were incubated.
After mixing, the samples were aerated at a flow rate of 0.9 L/min using oil free compressed air and a Pasteur-pipette as aeration device.
The incubation of the individual samples was started at intervals of 12-13 min and each sample was aerated for 3 hours at 18 °C.
At the end of the incubation time the pH was determined and aliquots of the samples were transferred to measuring bottles and oxygen consumption was recorded (see 5.1).
Substance target-concentrations in the incubation mixtures:
Dioctyl fumarate: 4.2, 12.3, 37.0, 111.1, 333.3, 1000.0 mg/L
3,5-dichlorophenol: 5, 12.2 and 30 mg/L

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
For the preparation of the microbial inoculum activated sludge was used. The sludge was collected from one of the return sludge channels of the sewage treatment plant in Baden near Vienna (Zubringerstraße, A-2500 Baden). Baden is a spa with only a small amount of industry and the waste water of the sewage treatment plant is predominantly domestic.

The amount of dry substance in the standardised activated sludge was 4 g/L.
A sample of activated sludge was collected on the day before the test. The container for transportation was only maximally 2/3 filled in order to maintain the contact with air. At arrival in the laboratory (less than 1 hour after sample collection), the supernatant over the sludge was decanted, the sludge was diluted with tap water and aerated by means of compressed air. 50 mL of synthetic sewage concentrate were added per litre. On the day of the test the sludge was allowed to sediment and the supernatant was decanted. The concentration of suspended solids was determined by filtering 3 mL of the sample through a pre-dried and pre-weighed glass microfibre filter. After drying at 110 °C and reweighing the amount of solids was calculated.
On the basis of this calculation the concentration of suspended solids was adjusted to 4 g/L with tap water. This suspension was used as inoculum for the samples. The inoculum was aerated until use.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h

Test conditions

Test temperature:
The test was performed in a temperature controlled room with automatic temperature registration. The temperature inside the samples was measured with a mercury thermometer.
The incubation of the individual samples was started at intervals of 12-13 min and each sample was aerated for 3 hours at 18 °C.
pH:
pH was determined with a pH-meter in the standardised microbial inoculum before the test and in each sample at the end of the incubation and aeration.
The pH of the standardised activated sludge was 7.9.
Dissolved oxygen:
The oxygen concentration was determined with a DO-meter "OXI Level 2" and an oxygen electrode (Wissenschaftlich-technische Werkstätten G.m.b.H., Dr. rer.nat. K. Slevogt, D-82362 Weilheim i. OB, Trifthofstr. 57a). The readout was set to 100 % in water-saturated air.
Respiration rates (in mg O2.L-1.h-1 ) were determined in aliquots of the incubation mixtures. Bottles with narrow neck and a volume of about 100 mL were filled with the sample and positioned on a magnetic stirrer. Then the oxygen electrode (equipped with a magnetic stirring wheel) was inserted in a way that no air remained in the bottle and that the electrode sealed the bottle neck to avoid contact of the sample with the atmosphere.
The decline of oxygen concentration was measured by recording the concentrations of oxygen at various times.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrations
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
Details on results:
As no significant inhibition occurred in any sample, no useful regression line can be calculated.
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was in the accepted range of 5 to 30 mg/L.
The actual values of the EC10, EC20, EC50 and EC80 for 3,5-dichlorophenol were:
EC10= 4.4 mg/L
EC20 = 5.9 mg/L
EC50 = 15.0 mg/L
EC80 = 37.6 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
EC50 > 1 000mg/L
The respiration rates of all samples with the test substance were in the range of 99.5 to 105.3 % of the control value.
The respiration rate of the activated sludge was not inhibited by Di-octyl-fumarate.
Executive summary:

Objective of the study

The study was performed to estimate possible effects of dioctyl fumarate on aerobic microbial sewage treatment plants. The test was performed according to the EU-COUNCIL REGULATION (EC) No 440/2008, using activated sludge from a sewage treatment plant treating predominantly domestic sewage.

Test design

Six concentrations of the test substance (4.0, 12.0, 37.0, 110.4, 329.4, and 1009.2 mg/L) were tested versus two negative controls (tap water). As the test substance was not enough soluble in water to prepare a stock solution, suspensions of the test substance in water were prepared and stirred for 49 h. As positive control substance 3,5-dichlorophenol was used and tested in three concentrations (5.0, 12.3 and 30.2 mg/L). The microbial inoculum was a preparation of activated sludge collected on the day before the test. 50 mL of synthetic sewage concentrate were added per litre per day and the sludge was kept aerated. On the day of the experiment, the concentration of the microbial inoculum was adjusted to 4 g of dry weight per litre which results in a final concentration of 1.6 g dry weight per litre in the samples. After mixing the inoculum with synthetic sewage solution and appropriate dilutions of control or test substance solutions the samples were aerated for a contact time of 3 hours at 18 °C. Negative controls were run before the first and after the last test substance sample. After incubation the respiration rates were determined in closed bottles using an oxygen sensitive electrode. The inhibition of respiration was calculated from the respiration rates using the mean value of the negative controls as 100 %.

Results

The test results fulfilled the criteria for validity:

The respiration rates of the two negative control samples were within 15 % of each other.

Actual values: ± 3.7 %.

The EC50 of 3,5-dichlorophenol was in the accepted range of 5 to 30 mg/L.

Actual EC50: 15.0 mg/L.

The pH of the incubation mixtures with the test substance after the 3 hour incubation was between 8.3 and 8.4, the pH of the negative control samples was 8.3 and 8.4.

The respiration rates of all samples with the test substance were in the range of 99.5 to 105.3 % of the control value.

The following EC-values for "DIOCTYLFUMARAT" were obtained (values rounded off to two significant figures):

EC-value confidence limits

EC10 > 1 000mg/L no confidence limits can be calculated

EC20 > 1 000mg/L no confidence limits can be calculated

EC50 > 1 000mg/L no confidence limits can be calculated

EC80 > 1 000mg/L no confidence limits can be calculated