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Administrative data

Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 26 April 2010 to 13 august 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: compliant to GLP and testing guideline; 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
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Methylhydrazine
EC Number:
200-471-4
EC Name:
Methylhydrazine
Cas Number:
60-34-4
Molecular formula:
CH6N2
IUPAC Name:
methylhydrazine
Details on test material:
- Name of test material (as cited in study report): Mono Methyl Hydrazine
- Substance type: monoconstituent
- Physical state: slightly yellow liquid
- Analytical purity: 99.3%
- Impurities (identity and concentrations): 0.5% of monomethylamine and 0.3% of water
- Purity test date: 09 December 2009
- Lot/batch No.: 09TL120001
- Expiration date of the lot/batch: 01 January 2013
- Storage condition of test material: at room temperature and protected air (under nitrogen gaz)

Sampling and analysis

Analytical monitoring:
not required

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST AND REFERENCE SOLUTIONS
The test item was dissolved at 2000 mg/L in dilution water. The stock solution was agitated during 5 minutes. After agitation the pH of the stock solution was 9.34.
Test suspensions were prepared by further dilution of the stock solution without pH adjustment 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).
The pH of the test suspension at 1000 mg/L after addition of inoculum was 8.91 when prepared from the stock solution without pH adjustment.
So the pH of one part of this stock solution was adjusted to a value included in the range +/- 0.5 unit of that of the dilution water using HCl at 1N.
The pH of the test suspension at 1000 mg/L after addition of inoculum was 7.86 when prepared from the stock solution with pH adjustment.

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 17 hours and 45 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% (3.98 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 1000 mg/L after addition of inoculum was 8.91 when prepared from the stock solution without pH adjustment.
The pH of the test suspension at 1000 mg/L after addition of inoculum was 7.86 when prepared from the stock solution with pH adjustment.
Salinity:
Not measured
Nominal and measured concentrations:
Five test concentrations containing the test item Mono Methyl Hydrazine at 10, 31.6, 100, 316 and 1000 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.:
< 10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Inhibitions of the respiration rate in the test item suspensions at 10, 31.6, 100, 316 and 1000 mg/L, without pH adjustment, were 84.1%, 85.5%, 78.6%, 80.5% and 54.8%, respectively, relative to mean of the controls. Hence, a significant inhibition of the respiration rate (> 15% relative to mean of the controls) was noted from 10 mg/L.

The inhibition of the respiration rate of the test item suspension at the highest concentration (1000 mg/L) with pH adjustment (of the stock solution) was higher than the same suspension without pH adjustment 54.8% and 68.8% respectively.

So it was assumed that the toxicity of the test item should be dependant of pH of the test suspension. Furthermore it was observed that the toxicity of the test was not dose-related since all values of inhibition of respiration rate were around the same range of value (around 80% of inhibition of the respiration rate).

In all cases (i.e. without or with pH adjustment and at all the concentrations) the test item was found to be toxic (i.e. with a inhibition of the respiration rate > 50%)

The 3-hour EC50 was not calculated since immobilization at the lowest concentration (10 mg/L) was higher than 50% at the corresponding time (84.1%).
Results with reference substance (positive control):
The EC50 of 3,5-dichlorophenol was 13.6 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                                         < 10                                            NA

       Reference item                                    13.6                                       10.9 - 17.2

NA: not applicable

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
The difference in the respiration rate between the two controls was below 15% and the EC50 of 3,5-dichlorophenol was 13.6 mg/L i.e. between 5 and 30 mg/L
Conclusions:
Under our experimental conditions, the 3-hour EC50 of the test item Mono Methyl Hydrazine for activated sludge respiration inhibition was < 10 mg/L.
The highest test item concentration without significant effect on the respiration was < 10 mg/L.
The test item should be considered as toxic for the micro-organisms of a water treatment plant.
Executive summary:

Methods

The inhibition potential of Mono Methyl Hydrazine 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 Mono Methyl Hydrazine 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 concentrations containing the test item were prepared by dilution of a stock solution at 2000 mg/L (pH: 9.34). These test item suspensions were tested without pH adjustment of the stock solution, except for the 1000 mg/L suspension which was tested with pH adjustment (final pH after addition of activated sludge: 8.91) and without pH adjustment of the stock solution (final pH after addition of activated sludge: 7.96).

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 13.6 mg/L i.e. between 5 and 30 mg/L), the test was considered valid.

Inhibitions of the respiration rate in the test item suspensions at10, 31.6, 100, 316 and 1000 mg/L, without pH adjustment, were 84.1%, 85.5%, 78.6%, 80.5% and 54.8%, respectively, relative to mean of the controls. Hence, a significant inhibition of the respiration rate (> 15% relative to mean of the controls) was noted from 10 mg/L.

The inhibition of the respiration rate of the test item suspension at the highest concentration (1000 mg/L) with pH adjustment (of the stock solution) was higher than the same suspension without pH adjustment 54.8% and 68.8%, respectively.

 

So it was assumed that the toxicity of the test item should be dependant of pH of the test suspension. Furthermore it was observed that the toxicity of the test was not dose-related since all values of inhibition of respiration rate were around the same range of value (around 80% of inhibition of the respiration rate).

In all cases (i.e.without or with pH adjustment and at all the concentrations) the test item was found to be toxic (i.e.with an inhibition of the respiration rate > 50%).

The 3-hour EC50 was not calculated since immobilization at the lowest concentration (10 mg/L) was higher than 50% at the corresponding time (84.1%).

EC50s, based on nominal concentrations, were as follows:

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

           Test item                                         < 10                                            NA

       Reference item                                    13.6                                       10.9 - 17.2

NA: not applicable

Conclusion

Under our experimental conditions, the 3-hour EC50 of the test item Mono Methyl Hydrazine for activated sludge respiration inhibition was < 10 mg/L.

The highest test item concentration without significant effect on the respiration was < 10 mg/L.

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