Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Type: activated sludge collected from a water treatment works containing effluent from a predominantly domestic origin.
Origin: the water treatment plant of Evreux (France).
Preparation: After sampling, the inoculum was left to settle and the main part of the supernatant was rejected. It was washed once with dechlorinated water and then filtered (to remove the biggest particles). Four samples of 50 mL were taken to determine the content in suspended matter (dry weight/v). The concentration of sludge (5.11 g/L) was then adjusted to 4 g/L by addition of the appropriate volume of dechlorinated water.

The inoculum was sampled one day before the test and maintained under agitation and aeration before use. As the sludge was not used immediately, 50 mL of sewage feed were added, per liter, just before agitation and aeration.
Synthetic sewage feed was prepared using dechlorinated water and analytical grade reagents following OECD recommendations.

Temperature: between 22°C and 23°C. The temperature in the culture room was recorded continuously and records retained.
illumination: the stock vessel was kept away from light by wrapping it in opaque material.
Aeration: clean air was passed through the inoculum at the rate of 0.5 to 1 L/min until use.
pH: was checked before use. The pH of inoculum remained within acceptable limits (7.98) and therefore was not adjusted before the beginning of the test.

Study design

Limit test:
no

Test conditions

Hardness:
280 mg/L as CaCO3
Test temperature:
24 °C
pH:
was checked before use. The pH of inoculum remained within acceptable limits (7.98) and therefore was not adjusted before the beginning of the test.
Nominal and measured concentrations:
10, 31.6, 100, 316 and 1 000 mg/L
Details on test conditions:
Temperature: 24°C throughout the test.
illumination: no special precautions were taken.
Dilution water: dechlorinated tap water was used, with a hardness of 280 mg/L as CaCO3.
Duration of test: 3 hours.
Aeration: air was bubbled through each suspension at the rate of 0.5 to 1 L/min until the start of oxygen concentration measurements.

Groups of test and reference suspensions and controls were set up as follows:
. two control replicates containing inoculum at 1.6 g/L,
. five test concentrations containing the test item 2-Ethylhexyl nitrate at 10, 31.6, 100, 316 and 1000 mg/L and inoculum at 1.6 g/L, with one replicate per concentration,
. three reference concentrations containing the reference item 3,5-dichlorophenol at 4, 12 and 36 mg/L and inoculum at 1.6 g/L, with one replicate per concentration.

The two control replicates were used to determine the background respiration rate of the sludge and were not exposed to the test or reference items.

The five test item concentrations were prepared by direct addition of the test item into the test flask (see §2.1.2 "Preparation of the test and reference suspensions").
The pH of the test suspension at the highest concentration (1000 mg/L) after addition of inoculum was 7.96.

Test and reference suspensions and controls were prepared in test flasks and then immediately aerated for 3 hours before being transferred to an oxygen measuring apparatus.

Measurement of oxygen concentration of the above suspensions was determined using an oxygen probe placed into an opaque BOD (Biological Oxygen Demand) flask filled with the test or reference suspension which was agitated with a magnetic stirrer. The oxygen probe was connected to a meter equipped with a chart recorder.

Each suspension was prepared by adding 16 mL of sewage feed to each of the flasks which were made up to 300 mL with dechlorinated water only in the case of the controls or a nominal mixture of dilution water and reference item or dilution water and direct addition of the required amount of the test item in the case of the other test suspensions (see § "Preparation of the test and reference suspensions" for details). Each mixture was then made up to 500 mL by adding 200 mL of inoculum. As 200 mL of inoculum were added in a final volume of 500 mL, the organic material concentration from inoculum was 1.6 g/L (± 10%).
The suspensions were aerated at between 0.5 to 1 liter of air per minute using a Pasteur-pipette connected with a flexible tube to an air pump as an aeration device.

Suspensions were prepared approximately every 15 minutes for test times of 3 hours.
In order to verify the quality of the inoculum, the controls were prepared at the beginning (first control) and the end (second control) of the test.

After 3 hours of aeration/contact time, the content of the test flask was poured into the measuring apparatus and the oxygen concentration was determined for a period of approximately 10 minutes.
Reference substance (positive control):
yes

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:
As the test item was poorly soluble all the results was based on the loading rate i.e. including the dissolved and undissolved fractions of the item.

The respiration rate of the test item suspension at the highest loading rate (1000 mg/L) was slightly lower than the respiration rate of the first control (i.e. these rates were not within 15% of each other). Therefore, the oxygen consumption rate of the four other test item suspensions (10 to 316 mg/L) was determined.

No significant inhibition (> 15% relative to mean of the controls) of the respiration rate was noted in the test item suspensions at 10, 31.6, 100 and 1000 mg/L relative to mean of the controls.
However inhibition of the respiration rate in the test item suspensions at 316 mg/L was 26.4%, relative to mean of the controls.
Hence, a significant inhibition of the respiration rate (> 15% relative to mean of the controls) was noted only at 316 mg/L.

It was supposed that the difference of a concentration-effect relationship between 316 and 1000 mg/L came from the low water solubility of the test item and the volatility of the test item.

Consequently, the highest test item loading rate without significant effect (difference ≤ 15%) on the respiration rate, relative to mean of the controls, was ≥ 100 mg/L.
The 3-hour EC50 was not calculated since inhibition at the all the concentration was lower than 50% at the corresponding time (maximum at 26.4%).
Results with reference substance (positive control):
As the difference in the respiration rate between the two controls was below 15% and the EC50 of 3,5-dichlorophenol was 15.2 mg/L i.e. between 5 and 30 mg/L, the test was therefore considered valid.

Any other information on results incl. tables

EC50s, based on nominal loading rates, were as follows:

  3-hour EC50 (mg/L)  95% confidence limits (mg/L)
Test item   > 1000  not applicable
Reference item   15.2  13.1 - 17.8

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under experimental conditions, the 3-hour EC50 of the test item 2-Ethylhexyl nitrate for activated sludge respiration inhibition was > 1000 mg/L (including the dissolved and undissolved fractions of the item).

The highest test item loading rate (including the dissolved and undissolved fractions of the item) without significant effect on the respiration was ≥ 100 mg/L.

The test item should be considered as non-toxic for the micro-organisms of a water treatment plant.