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Toxicity to microorganisms

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activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-08-02 to 2012-05-04
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:
Triethyl phosphonoacetate
EC Number:
EC Name:
Triethyl phosphonoacetate
Cas Number:
Molecular formula:
ethyl 2-(diethoxyphosphoryl)acetate
Constituent 2
Reference substance name:
Triethyl phosphono acetate
Triethyl phosphono acetate
Details on test material:
- Name of test material (as cited in study report): TEPA (Triethyl phosphono acetate)

Sampling and analysis

Analytical monitoring:

Test solutions

Details on test solutions:
An amount of test item (2500 mg) was dissolved in water and the volume adjusted to 1 liter to give a 2500mg/L stock solution from which dilutions were made to give 250 and 25 mg/L stock solutions. An aliquot (200 ml) of the 25 mg/L stock solution was dispersed with synthetic sewage (16 ml), activated sewage sludge (250 ml) and water, to a final volume of 500 mL, to give the required concentration of 10 mg/L. Similar aliquots (200 mL) of the 250 mg/L and 2500 mg/L were used to prepared the test concentrations of 100 and 1000 mg/L. the 1000 mg/L test concentration was prepared in triplicate. The volumetric flask containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.

The pH of the test item stock solution were measured (See Table 1) and adjusted to between pH 7.0 to pH 8.0 if necessary

The control group was maintained under identical conditions but not exposed to the test item.

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- A mixed population of activated sewage sludge micro-organism was obtained on 3 January 2012 from the aeration stage of the severn Trent Water Plc sewage treatment plant at Loughborough, Leceistershire, UK which treats predominantly domestic sewage

-The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of approximately 21°C and was used the day of collection.
-The pH of the sample was 8.0 measured using a WTWpH/Oxi 340i pH and dissolved 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 rinsed 3 times 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 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 3.0 g/L prior to use.

Study design

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

Test conditions

No data
Test temperature:
Approximately 20 °C ± 2°C
7.2 to 7.7 after adjustment (See Table 1)
Dissolved oxygen:
See Table 5
Not applicable
Nominal and measured concentrations:
10, 100 and 1000 mg/L (Nominal concention)
Details on test conditions:
At time "0", 16 mL of synthetic sewage was diluted to 250 mL of innoculum added in a 500mL conical flask (first control). the mixture was aerated with clean oil-free compressed air via narrow bore glass tube at a rate of approximately 0.5-1 litter per minute. Thereafter, at 15 minute interval, the procedure was repeated for the others vessels.
- No. of vessels per concentration (replicates): 1 for 10 and 100 mg/L and 3 for 1000 mg/L
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates):3
- Biomass loading rate: 3 g/L

- The test water used for the test was deionized reverses osmosis water containing less than 1 mg/L Dissolved Organic Carbon (DOC)
- Metals, pesticides, chlorine, alkalinity, Ca/mg ratio,conductivity: No data

- Adjustment of pH: Yes (result in table 1)
- Photoperiod: normal lighting during the test
- Light intensity: No data

As each vessels reached 3 hours contact time an aliquot was removed from the conical flask and poured into the measuring vessels (250 mL darkened glass Biological Oxygen Demand (BOD) bottle) and the rate of respiration measured using a Yellow Spring dissolved oxygen meter fitted with a BOD probe. The contents of the measuring vessels were stirred constantly by magnetic stirrer. The rate of the respiration for each flask was measured over the linear portion of the oxygen consumption trace (where possible between approximately 7.0 mg O2/L to 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 oxygen consumption, the rate was determined over an approximate 10 minute period.
Observation were made on the test preparation throughout the test period. Observation of the test item vessels at 0hours were made prior to addition of activated sewage sludge.
The pH of the control, reference item and test item preparations was measured at 0 hours and prior to measurement of the oxygen consumption rate after 3 hours contact time. The oxygen concentration in all vessels were measured after 30 minutes contact time.

- Spacing factor for test concentrations: 10
- Justification for using less concentrations than requested by guideline:
- Range finding study: Yes
- Test concentrations: 10, 100 and 1000 mg/L
- Results used to determine the conditions for the definitive study: O2 consumption rate
Reference substance (positive control):
3,5 dichlorophenol

Results and discussion

Effect concentrations
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:
Oxygen consumption rate and percentage inhibition value for the control, test and reference items are given in table 2. The pH values of the test preparations at the start and end of the exposure period are given in table 3. The dissolved oxygen concentration in all vessels after 30 minutes contact time are given in table 5
No statistically significantly toxic effects were shown at all of the test concentrations employed. It was therefore considered justifiable not to perform a definitive test.
Percentage inhibition is plotted against concentration for the reference item.
It was considered unnecessary and unrealistic to test at concentration in excess of 1000 mg/L.
The coefficient of variation of oxygen uptake in the control vessels was 9.2% and the specific respiration rate of the controls was 22.46 mg oxygen per gram dry weight of sludge per hour. The validation criteria has therefore been satisfied.
The validation criteria for the reference item EC50 values were also satisfied.

In some instances, the initial and final dissolved oxygen concentrations were below those recommended in the test guideline (7.0 mg O2/L and 2.0 mg 02/L respectively). This was considered to have had no adverse effect on the results of the study given that in all cases oxygen consumption rate was determined over the linear portion of the consumption trace.
Results with reference substance (positive control):
The reference item gave a 3-hour EC50 value of 9.2 mg/L, 95% confidence limits 7.2-12 mg/L

Applicant's summary and conclusion

Validity criteria fulfilled:
The effect of the test item on the respiration of activated sewage sludge micro-organism gave a 3-hour EC50 value on greater than 1000 mg/L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was 1000 mg/L.
Executive summary:

In a study (Harlan, 2012), the inhibitory effect of the substance TEPA 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 (2010).

The nominal concentrations tested was 1000 mg/L in a definitive study (limit test) after a range finding test at 10, 100 and 1000 mg/L (nominal concentration)

In addition, two 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= 9.2 mg/L within the guideline-recommended range of 5 - 30 mg/L confidence limits 7.2 -12 mg/L) confirmed suitability of the activated sludge.

EC50 >1000 mg/L

NOEC = 1000 mg/L

Based on the results of this study, TEPA would be not classified.

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