Registration Dossier

Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 01 March 2010 to 13 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: compliant to GLP and testing guideline (deviations were not considered to have compromised the validity of the study) ; adequate coherence between data, comments and conclusions

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
Deviations:
yes
Remarks:
the temperature in the culture room exceeded slightly 22°C during the culture period of the inoculum and during the test
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): Perchlorate d'Ammonium
- Substance type: monoconstituent
- Physical state: white powder
- Analytical purity: 99.9%
- Purity test date: 15 March 2010
- Lot/batch No.: A001/10
- Expiration date of the lot/batch: January 2015
- Storage condition of test material: at room temperature and protected from humidity

Sampling and analysis

Analytical monitoring:
not required

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION AND REFERENCE SOLUTIONS
The test item was dissolved at 2000 mg/L in dilution water. This stock solution was agitated during 5 minutes. The pH of this solution was 7.38 after agitation.
Test suspensions were prepared by further dilution of the stock solution with appropriate volumes of dilution water and inoculum (i.e. activated sludge) to provide a geometric series of concentrations (10, 31.6, 100, 316 and 1000 mg/L).

A reference stock solution was prepared by dissolving 500 mg of the reference item, 3,5 dichlorophenol, in 10 mL of NaOH at 1 N. The obtained preparation was then diluted to 30 mL with deionized water. Aliquots of H2SO4 at 1 N were added while stirring until the point of incipient precipitation was obtained (approximately 8 mL of H2SO4 at 1 N were required). The solution was made up to 1000 mL using deionized water.
The reference stock solution was agitated for 18 hours and 30 min. The pH of this solution was 7.91 after agitation.
Reference item suspensions were prepared by further dilution of the stock solution with appropriate volumes of dechlorinated water and inoculum (i.e. activated sludge)

Test organisms

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
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 was not adjusted because the quantity of suspended matter was 4 g/L +/- 10% (3.87 g/L).

Conditions of culture
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.

Study design

Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h

Test conditions

Hardness:
280 +/- 20 mg/L as CaCO3
Test temperature:
23°C
pH:
The pH of the test suspension at the highest concentration (1000 mg/L) after addition of inoculum was 7.87.
Salinity:
Not measured
Nominal and measured concentrations:
Five test concentrations containing the test item Perchlorate d’ammonium 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.
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenol. 97%

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:
The respiration rate of the test item suspension at the highest concentration (1000 mg/L) was equivalent to the respiration rate of the first control (i.e. these rates were within 15% of each other).
Nevertheless as the respiration rate was very close to 15% (14.4%) the oxygen consumption rate of the four other test item suspensions (10 to 316 mg/L) was determined in order to confirm this results.

No significant inhibition (> 15%) of the respiration rate was noted in the test item suspensions at 1000 mg/L relative to mean of the controls since the inhibition of the respiration rate was 14.3%.
Furthermore no significant inhibition (> 15%) of the respiration rate was also noted in the test item suspensions at 10, 31.6, 100 and 316 mg/L relative to mean of the controls.
Results with reference substance (positive control):
EC50 of 3,5-dichlorophenol was 13.9 mg/L i.e. between 5 and 30 mg/L

Any other information on results incl. tables

EC50s, based on nominal concentrations, were as follows:

               Item                                3-hour EC50 (mg/L)             95% confidence limits (mg/L)

           Test item                                       > 1000                                          NA

       Reference item                                    13.9                                       11.7 - 16.5

NA: not applicable

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
the difference between the respiration rates of the two controls was below 15% and the EC50 of 3,5-dichlorophenol was 13.9 mg/L i.e. between 5 and 30 mg/L
Conclusions:
Under the experimental conditions of the study, the 3-hour EC50 of the test item Perchlorate d’ammonium for activated sludge respiration inhibition was > 1000 mg/L.
The highest test item concentration without significant effect on the respiration was >= 1000 mg/L.
The test item should be considered as non-toxic for the micro-organisms of a water treatment plant.
Executive summary:

Methods

The inhibition potential of Perchlorate d’ammonium on the respiration rate of an activated sludge at 1.6 g/L in dechlorinated water was investigated using the following suspensions:

.           two control suspensions (activated sludge alone),

.           five test item suspensions: activated sludge and Perchlorate d’ammonium at a concentration of 10, 31.6, 100, 316 and 1000 mg/L,

.           three reference item (3,5-dichlorophenol) suspensions: activated sludge and the reference item at a concentration of 4, 12 and 36 mg/L.

 

The five test item concentrations were prepared by dilution of a stock solution at 2000 mg/L.

The five test item concentrations were tested without pH adjustment since the pH of the stock solution was 7.38.

The oxygen consumption (i.e.respiration rate) of the test item, reference item and control suspensions was measured for approximately 10 minutes, with a chart recorder, after an aeration of 3 hours.

The EC50 (concentration expected to cause 50% inhibition of the respiration rate) of the test item and that of the reference item were determined considering the mean oxygen consumption of the controls as 100%.

Results

As the validity criteria were met (the difference between the respiration rates of the two controls was below 15% and the EC50 of 3,5-dichlorophenol was 13.9 mg/L i.e.between 5 and 30 mg/L), the test was considered valid.

 

The respiration rate of the test item suspension at the highestconcentration(1000 mg/L) was equivalent to the respiration rate of the first control (i.e.these rates were within 15% of each other).

Nevertheless as the respiration rate was very close to 15% (14.4%) the oxygen consumption rate of the four other test item suspensions (10 to 316 mg/L) was determined in order to confirm this results.

No significant inhibition (> 15%)of the respiration rate was noted in the test item suspensions at 1000 mg/L relative to mean of the controls since the inhibition of the respiration rate was 14.3%.

Furthermore no significant inhibition (> 15%) of the respiration rate was also noted in the test item suspensions at 10, 31.6, 100 and 316 mg/L relative to mean of the controls.

Consequently, the highest test item concentration without significant effect (difference =< 15%) on the respiration rate, relative to mean of the controls, was >= 1000 mg/L.

EC50s, based on nominal concentrations, were as follows:

               Item                                3-hour EC50 (mg/L)             95% confidence limits (mg/L)

           Test item                                       > 1000                                          NA

       Reference item                                    13.9                                       11.7 - 16.5

 NA: not applicable

Conclusion

Under our experimental conditions, the 3-hour EC50 of the test item Perchlorate d’ammonium for activated sludge respiration inhibition was > 1000 mg/L.

The highest test item concentration without significant effect on the respiration was >= 1000 mg/L.

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