Methylhydrazine

Biodegradation in water: screening tests

Administrative data

Endpoint:

biodegradation in water: ready biodegradability

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 09 June 2010 to 07 September 2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: compliant to GLP and testing guideline (the deviation was 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:

2020

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 301 D (Ready Biodegradability: Closed Bottle Test)

Deviations:

yes

Remarks:

pH of mineral medium was not recorded at the beginning of the test

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.4-E (Determination of the "Ready" Biodegradability - Closed Bottle Test)

Deviations:

yes

Remarks:

as above

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

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): monomethylamaine : 0.5%, water: 0.3%
- 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)

Study design

Oxygen conditions:

aerobic

Inoculum or test system:

activated sludge, domestic, non-adapted

Details on inoculum:

- Source of inoculum/activated sludge (e.g. location, sampling depth, contamination history, procedure): sampled from a water treatment plant receiving sewage from a predominantly domestic origin (collected from: the water treatment plant of Evreux (France).
- Storage conditions: 22°C +/- 2°C
- Preparation of inoculum for exposure: the inoculum contained approximately 11 x 104 bacteria per mL (determination with a Malassez cell counter). In order to obtain a final concentration of approximately 104 bacteria per liter in all test suspensions, inoculum was diluted 20-fold with mineral medium and 1.81 mL/L of (diluted) inoculum was added to each test vessel.
- Pretreatment: the inoculum was prepared by initially removing the biggest particles and seiving accross a filter paper (porosity 4-7 µm). In order to wash out the dissolved organic carbon (DOC) and to lower the carbon organic content, the inoculum was preconditioned for 5 days before use for the study. Air was bubbled through the inoculum during this preconditioning period. The inoculum was seived again accross a filter paper after the acclimation period.
- Concentration of sludge: 104 bacteria per liter in all test suspensions
- Initial cell/biomass concentration:
- Water filtered: yes/no

Duration of test (contact time):

28 d

Initial test substance concentration

Initial test substance concentration 1

Initial conc.:

0 mg/L

Based on:

test mat.

Initial test substance concentration 2

Initial conc.:

2 mg/L

Based on:

test mat.

Initial test substance concentration 3

Initial conc.:

4 mg/L

Based on:

test mat.

Parameter followed for biodegradation estimation

Parameter followed for biodegradation estimation:

O2 consumption

Details on study design:

TEST CONDITIONS
- Composition of medium: reconstituted water (OECD and EEC recommended) was prepared using deionized water and analytical grade reagents (see section "any other information on matherials and methods")
- Test temperature: between 20°C and 24°C
- pH: was not recorded at the beginning of the test
- Aeration of dilution water: at least 20 minutes and standing at test temperature between 20 and 24 hours before the beginning of the test
- Continuous darkness: yes

SAMPLING
- no samples were taken at day 1 and day 4 for chemical analysis (see datails on analytical methods)

CONTROL AND BLANK SYSTEM
- Inoculum blank: one group containing the inoculum
- Toxicity control: one group containing the test item (at 2 mg/L), the reference item (sodium acetate at 3 mg/L) and inoculum, and one group containing the test item (at 4 mg/L), the reference item (sodium acetate at 3 mg/L) and inoculum.
- Other: procedure control: one group containing the reference item (sodium acetate at 3 mg/L) and inoculum.

Reference substance

Reference substance:

acetic acid, sodium salt

Remarks:

at 3 mg/L

Results and discussion

% Degradation

% Degradation 1

Parameter:

% degradation (O2 consumption)

Value:

0

Sampling time:

1 d

% Degradation 2

Parameter:

% degradation (O2 consumption)

Value:

6.2

Sampling time:

4 d

% Degradation 3

Parameter:

% degradation (O2 consumption)

Value:

12

Sampling time:

8 d

% Degradation 4

Parameter:

% degradation (O2 consumption)

Value:

23

Sampling time:

16 d

Remarks on result:

other: test substance at 4 mg/L

% Degradation 5

Parameter:

% degradation (O2 consumption)

Value:

44.9

Sampling time:

28 d

Remarks on result:

other: test substance at 4 mg/L

Details on results:

For the test suspension at 2 mg/L:
No significant inhibition of the inoculum due to toxicity of the test item was noted since the toxicity control reached 45.4% of the ThOD after 14 days.
The 10-day window (the 10 days immediately following the attainment of 10% biodegradation) of the test item started on the 5th day. Biodegradation totalled 32% (mean of the two replicates) at the end of this 10-day window on the 15th day.
However for this test suspension at 2 mg/L biodegradtion values were considered like not relevant since the difference between the values of two replicates was too important and results at the end of the test day 25 and 29 were not relevant either.
Therefore results of this test concentration of 2 mg/L are not taking into account to assess the readily biodegradation of the test item. (However values are presented in the results of this report because results showed tendency of the test item to be not readily biodegradable).

For the test suspension at 4 mg/L:
No significant inhibition of the inoculum due to toxicity of the test item was noted since the toxicity control reached 39.7% of the ThOD after 14 days.
The 10-day window (the 10 days immediately following the attainment of 10% biodegradation) of the test item started on the 6th day. Biodegradation totalled 23% and 44.9% over the test period (mean of the two replicates) at the end of this 10-day window on the 16th day. Biodegradation in this control totalled 77.8% after 28 days.

BOD5 / COD results

Results with reference substance:

Day 1 : 0% biodegradation
Day 4: 52.1% biodegradation
Day 8: 66.5% biodegradation
Day 18: 71.6% biodegradation
Day 29: 77.4% biodegradation

Any other information on results incl. tables

All validity criteria were respected for the test concentration at 4 mg/L:

.           dissolved oxygen depletion in the inoculum blank did not exceed 1.5 mg/L after 28 days,

.           dissolved oxygen was >= 0.5 mg/L in all test suspension replicates during the test,

.           biodegradation values of test item replicates deviated by less than 20% at the end of the test,

.           biodegradation in the reference test was 71.6% after 14 days then it was at least 60% within this period,

.           biodegradation in the toxicity control was 39.7% (based on ThOD) after 14 days then it was at least 25% within this period.

 

All validity criteria concerning inoculum blank, reference test and the toxicity control were respected as indicated above. However, for the test concentration at 2 mg/L:

.       dissolved oxygen was < 0.5 mg/L in the test suspension replicates at day 29,

.    biodegradation values of test item replicates at the end of the test deviated by more than 20%.

Such difference at the end of the test can occurred when the concentration of test item was low because the precision of the method is lesser in these conditions.

It was assumed that the invalidated criteria for the test concentration at 2 mg/L were not sufficient to question this study since all validity criteria concerning inoculum blank and reference test and the test concentration at 4 mg/L were respected.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Interpretation of results:

other: not readily biodegradable, but biodegradable to some extent

Conclusions:

The biodegradation of Mono Methyl Hydrazine reached 23% at the end of the 10-day window (day 16) and 44.9% at the end of the test (day 28).
Under the experimental conditions of the study, the test item Mono Methyl Hydrazine was therefore not readily biodegradable.

Executive summary:

Methods

Six groups (two replicates per group) were used to determine the Biological Oxygen Demand (BOD) evolved by the degradation of the test item:

.           one group containing the inoculum (inoculum blank),

.           one group containing the test item (at 2 mg/L) and inoculum (test suspension),

.           one group containing the test item (at 4 mg/L) and inoculum (test suspension),

.           one group containing the reference item (sodium acetate at 3 mg/L) and inoculum (procedure control),

.           one group containing the test item (at 2 mg/L), the reference item (sodium acetate at 3 mg/L) and inoculum (toxicity control),

.           one group containing the test item (at 4 mg/L), the reference item (sodium acetate at 3 mg/L) and inoculum (toxicity control).

 

The test and reference items were dissolved in reconstituted water (OECD mineral medium) prepared from deionized water with a conductivity < 10 µS/cm.The mineral medium was prepared the day before the beginning of the study. It was maintained under aeration for at least 20 minutes and standing at test temperature between 20 and 24 hours before the beginning of the test.

The inoculum consisted of secondary effluent sampled from a sewage treatment plant and then aeratedfor 5 days. Inoculum was added to the test suspensions in order to obtain approximately 10⁴bacteria per liter in each group.

BOD flasks were filled to the brim and closed until O₂ measurement.

 

The dissolved oxygen was measured in duplicate (two replicates per group) at the beginning of the test, every 3 or 4 days thereafter and at the end of the test, for each group.

 

Chemical analysis

As the test item was not found to be readily biodegradable in the worst case,i.e.taking onto account theThOD NH₃, chemical analysis was not carried out in order to verify that the O₂consumption was not due to nitrification.

Results

Validity criteria

All validity criteria were respected for the test concentration at 4 mg/L:

.           dissolved oxygen depletion in the inoculum blank did not exceed 1.5 mg/L after 28 days,

.           dissolved oxygen was >= 0.5 mg/L in all test suspension replicates during the test,

.           biodegradation values of test item replicates deviated by less than 20% at the end of the test,

.           biodegradation in the reference test was 71.6% after 14 days then it was at least 60% within this period,

.           biodegradation in the toxicity control was 39.7% (based on ThOD) after 14 days then it was at least 25% within this period.

 

All validity criteria concerning inoculum blank, reference test and the toxicity control were respected as indicated above. However, for the test concentration at 2 mg/L:

.       dissolved oxygen was < 0.5 mg/L in the test suspension replicates at day 29,

.    biodegradation values of test item replicates at the end of the deviated by more than 20%.

Such difference at the end of the test can occurred when the concentration of test item was low because the precision of the method is lesser in these conditions.

It was assumed that the invalidated criteria for the test concentration at 2 mg/L were not sufficient to question this study since all validity criteria concerning inoculum blank and reference test and the test concentration at 4 mg/L were respected.

 

Test item biodegradation

The reference item degraded normally under the test conditions.

 

For the test suspension at 2 mg/L

No significant inhibition of the inoculum due to toxicity of the test item was noted since the toxicity control reached 45.4% of the ThOD after 14 days.

The 10-day window (the 10 days immediately following the attainment of 10% biodegradation) of the test item started on the 5th day. Biodegradation totalled 32% (mean of the two replicates and graphically determined) at the end of this 10-day window on the 15th day.

However for this test suspension at 2 mg/L biodegradation values were considered like not relevant since the difference between the values of two replicates was too important and results at the end of the test day 25 and 29 were not relevant either.

Therefore results of this test concentration of 2 mg/mL are not taking into account to assess the readily biodegradation of the test item. (However values are presented in the results of this report because results showed tendency of the test item to be not readily biodegradable)

 

For the test suspension at 4 mg/L

No significant inhibition of the inoculum due to toxicity of the test item was noted since the toxicity control reached 39.7% of the ThOD after 14 days.

The 10-day window (the 10 days immediately following the attainment of 10% biodegradation) of the test item started on the 6th day. Biodegradation totalled 23% and 44.9% over the test period (mean of the two replicates and graphically determined) at the end of this 10-day window on the 16th day. Biodegradation in this control totalled 77.8% after 28 days.

Conclusion

The biodegradation of Mono Methyl Hydrazine reached 23% at the end of the 10-day window (the 10 days immediately following the attainment of 10% biodegradation) on the 16 day and 44.9% at the end of the test.

Under the experimental conditions of the study, the test item Mono Methyl Hydrazine was therefore not readily biodegradable in the 28-day closed bottle test.