Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Between 3 and 4 March 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Recently conducted GLP compliant study using the most recent test methods

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
yes
Remarks:
In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg 02/l and 2.5 mg 02/l respectively). This was considered to have had no adverse effect on the results of the study
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): JKY-214
- Substance type: Organic
- Physical state: white solid

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Direct dispersion in water

Test organisms

Test organisms (species):
activated sludge
Details on inoculum:
- Laboratory culture: yes, maintained on continuous aeration at approximately 21C
- Preparation of inoculum for exposure:
The pH of the sample was 7.5 measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter. Determination of the suspended solids level of the activated sewage sludge was carried out by filtering a sample (100 ml) of the activated sewage sludge by suction through a pre-weighed GF/A filter paper using a Buchner funnel which was then rinsed 3 times with 10 ml of deionised reverse osmosis water and filtration continued for 3 minutes. The filter paper was then dried in an oven at approximately 105°C for at least 1 hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 4.0 g/l prior to use.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
3 h
Post exposure observation period:
No information

Test conditions

Hardness:
No information
Test temperature:
21+/- 1 C
pH:
7.5-8.1
Dissolved oxygen:
No information
Salinity:
No information
Nominal and measured concentrations:
10, 32, 100, 320 and 1000 mg/l
Details on test conditions:
At time "0" 16 ml of synthetic sewage was diluted to 300 ml with water and 200 ml of inoculum added in a 500 ml conical flask (first control). The mixture was aerated with clean, oil-free compressed air via narrow bore glass tubes at a rate of approximately 0.5 - 1 litre per minute. Thereafter, at 15 minute intervals the procedure was repeated with appropriate amounts of the reference material being added.
As each vessel reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 ml darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Springs dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessel were stirred constantly by magnetic stirrer. The rate of respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 6.5 mg 02/l and 2.5 mg 02/l). In the case of a rapid oxygen consumption, measurements may have been outside this range but the oxygen consumption was always within the linear portion of the respiration curve. In the case of low oxygen consumption, the rate was determined over an approximate 10 minute period.
The test was conducted under normal laboratory lighting in a temperature controlled room at 21±1°C.
Observations were made on the test preparations throughout the test period. Observations of the test material vessels at 0 hours were made prior to addition of activated sewage sludge and synthetic sewage. The pH of the control, reference material and test material preparations were measured using a WTW pH/Oxi 340I pH and dissolved oxygen meter at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time.
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:
growth inhibition
Details on results:
In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guidelines (6.5 mg 02/l and 2.5 mg 02/l respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption rate was determined over the linear portion of the oxygen consumption trace.

Observations made at 0 hours prior to the addition of activated sewage sludge and synthetic sewage showed that the test concentrations of 10 and 32 mg/l contained slightly cloudy dispersions with fine particles of test material dispersed throughout. The test concentration of 100 mg/l contained a cloudy dispersion with fine particles of test material dispersed throughout and the test concentrations of 320 and 1000 mg/l contained cloudy dispersions with particles of test material dispersed throughout.
Observations made after 30 minutes and 3 hours contact time showed that the test concentrations of 10, 32 and 100 mg/l contained dark brown dispersions with fine particles of test material dispersed throughout. The test concentration of 320 mg/l contained a dark brown dispersion with particles of test material dispersed throughout and the test concentration of 1000 mg/l contained a cloudy brown dispersion with particles of test material dispersed throughout.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels: 3hr EC50 = 7.0 mg/l 95% confidence limits 5.4 to 9.1 mg/l
- Other:

Any other information on results incl. tables

 

Test substance

3,5-dichlorophenol

ECX(3 Hours) (mg/l)

95% Confidence Limits (mg/l)

ECX(3 Hours) (mg/l)

95% Confidence Limits (mg/l)

EC20

>1000

-

2.1

-

EC50

>1000

-

7.0

5.4-9.1

EC80

>1000

-

23

-

NOEC

1000

-

1.8

-

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test material on the respiration of activated sewage sludge micro-organisms gave a 3-Hour EC50 of greater than 1000 mg/l. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/l.
Executive summary:

The test substance was assessed to determine toxicity to microorganisms by means of the growth inhibition of sewage microorganisms. The test substance was determined to not inhibit the growth of activated sewage sludge microorganisms upto and including a maximum dose of 1000 mg/l and the substance is considered to be not toxic with any effect being greater than this concentration. A positive control of 3,5-dichlorophenol was used to determine the validity of the test system.