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Diss Factsheets

Administrative data

activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From2011-08-02 to 2012-03-15
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with international standard guidelines under GLP conditions. The study report was well documented with all mandatory information included.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Octylphosphonic acid
EC Number:
EC Name:
Octylphosphonic acid
Cas Number:
Molecular formula:
octylphosphonic acid
Test material form:
other: cream, waxy, granular solid
Details on test material:
- Name of test material (as cited in study report): Octylphosphonic Acid (OPA)

Sampling and analysis

Analytical monitoring:

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
An amount of test item (2500 mg) was dissolved in water with the aid of ultrasonication for 60 minutes and the volume adjusted to 1 liter to give a 2500 mg/L stock solution from which dilutions were made to give 250 and 25 mg/L stock solutions.
For each concentration, an aliquot of stock solution (200ml) was dispersed with synthetic sewage (16ml), activated sludge (250 ml) and water, to a final volume of 500ml, to give required concentrations (10, 100 and 100 mg/L)
The pH of the test item stock solution were measured and adjusted to between pH 7.0 to pH 8.0 if necessary
- Controls: Four controls containing only water, synthetic sewage and inoculum were tested in parallel to the three concentrations of the test item under identical test conditions

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: no, aerobic activated sludge came from the aeration stage of the Severn Water Plc sewage treatment plant (Loughborough, Leceistershire, UK)).
- Method of cultivation: The activated sewage sludge sample was maintained on continuous aeration in the laboratory at the temperature of approximately 21°C and was used on the day of collection.
- Preparation of inoculum for exposure: 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 (rinsed three times with 20 ml deionized reverse osmosis water prior to drying in a oven) using a Buchner funnel which was rinsed with 10 ml of deionized 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 one hour and allowed to cool before weighing. This process was repeated until a constant weight was attained. The suspended solids concentration was equal to 3.0 g/L prior to use.

Study design

Test type:
Water media type:
Total exposure duration:
3 h
Post exposure observation period:

Test conditions

no data
Test temperature:
21°C +/- 1°C
At the beginning and at the end of the 3-hour incubation, the pH value was in the range of 7.5 to 8.4.(See table 1 and 3)
Dissolved oxygen:
At the beginning and at the end of the 3-hour incubation, the oxygen concentration value in control group was in the range of 3.9 to 4.4 mg O2/L. (See table 2)
Not applicable
Nominal and measured concentrations:
nominal concentrations: 10, , 100, and 1000 mg/L
Details on test conditions:
- Test vessel:
- Type: no data
- Material, size, headspace, fill volume: 500ml conical flask
- Aeration: During the incubation period of 3 hours all test media and the controls were continuously aerated with clean, oil-free compressed air via narrow bore glass tube at a rate of 0.5-1liter per minute.
- No. of vessels per concentration (replicates): 1 for 10mg/kg and 100 mg/kg, 3 for 1000 mg/kg
- No. of vessels per control (replicates): 1
- Biomass loading rate: sludge concentration = 3.0 g dry material / L

- Source/preparation of dilution water: deionized reverse osmosis water containing less than 1 mg/l Dissolved Organic Carbon (DOC)
- Total organic carbon, Particulate matter, Metals, Pesticides, Chlorine, Alkalinity, Ca/mg ratio, Conductivity: no data
- Culture medium different from test medium: no
- Intervals of water quality measurement:
The pH values were measured at 0 hour (prior addition of activated sewage sludge) and after three hours of incubation.
Dissolved oxygen concentrations were measured at 0 hour (prior addition of activated sewage sludge) in all vessels, after 30 minutes contact time and at the end of the 3-hour incubation period. The appearance of the test media was recorded at the same time

- Adjustment of pH: yes for the Test Item stock solution
- Photoperiod: no data
- Light intensity: normal laboratory lighting

As each vessels reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessel (250 ml darkness 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 vessels were stirred constantly by magnetic stirrer. The rate of respiration measured over linear portion of the oxygen consumption trace (where possible between approximately 7.0 mg O2/L and 2.0 mg O2/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 on approximate 10 minutes period.

- Range finding study: 1, 10, 100 mg/kg
Reference substance (positive control):
3,5-dichlorophenol tested at 3.2, 10 and 32 mg/L

Results and discussion

Effect concentrationsopen allclose all
3 h
Dose descriptor:
Effect conc.:
> 1 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
3 h
Dose descriptor:
Effect conc.:
> 1 000 mg/L
Nominal / measured:
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
respiration rate
Details on results:
No statistically significantly toxic effects were shown at all test concentrations employed. It was therefore considered justifiable not to perform a definitive test.

The oxygen consumption rates of the two controls (run at the start and at the end of the test) differed by 9% (guideline-recommendation: the two control respiration rates should be within 15% of each other).
Results with reference substance (positive control):
The 3-hour EC50 of the reference item 3,5-dichlorophenol (positive control) was calculated to be 9.5 mg/L with 95% confidence limits from 7.5 to 12 mg/L. The 3-hour EC50 is within the guideline-recommended range of 5–30 mg/L, confirming suitability of the activated sludge used.

Applicant's summary and conclusion

Validity criteria fulfilled:
The EC50 of the reference item is in the acceptance range and the oxygen consumption rates of the 4 controls differed by less than 15%.
The effect of the test item on the respiration of activated sewage sludge micro-organisms gave a 3-hour EC50 value of greater than 1000 mg/L. The No Observed Concentration (NOEC) after 3 hours exposure was 1000mg/L
Executive summary:

In a study (Harlan, 2012), the inhibitory effect of the substance Octylphosphonic Acid (OPA) on the respiration rate of aerobic wastewater microorganisms of activated sludge was investigated in a 3-hour respiration inhibition test according to OECD guideline for Testing of Chemicals, n°209 (1984).

The nominal concentrations tested were 10, 100, and 1000 mg/L.

In addition, four controls and three different concentrations of the reference item 3,5-dichlorophenol (3.2, 10 and 32 mg/L) were tested in parallel. The results of these reference treatments (EC50 = 9.5 mg/L within the guideline-recommended range of 5 - 30 mg/L) confirmed suitability of the activated sludge.

EC50 >1000 mg/L

NOEC = 1000 mg/L

This study is classified as acceptable and satisfies the guideline requirements for toxicity study to activated sludge in a respiration inhibition test.