Methylhydrazine

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 July 2010 to 17 september 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

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

yes

Remarks:

temperature varied by more than ± 2°C during the combined range-finding and limit test and the definitive test; pH varied by more than 1.5 units in the control during the combined range-finding and limit test and the definitive test

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

as above

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OECD Series on testing and assessment, Guidance document on aquatic toxicity testing of difficult substances and mixtures, Number 23, December 2000

Deviations:

not applicable

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): Monomethyl Hydrazine
- Substance type: monoconstituent
- Physical state: slightly yellow liquid
- Analytical purity: 99.3%
- Impurities (identity and concentrations): 0.5% of monomethylamine, 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:

yes

Details on sampling:

Test solutions analysis:
Before analysis, all samples containing algae were centrifuged and the supernatant was analyzed separately to verify the test item concentration in solution throughout the test.
All samples were taken in order to avoid headspace in the sampling tube.

- Combined range-finding and limit test
At T0 hour, duplicate samples of 5 mL were taken from the limit test solution (100 mg/L nominal) just before distribution in the test vessel.
At T24, T48 and T72 hours, samples were taken from each limit test solution replicate, and then pooled in order to obtain two samples of 5 mL.
Further samples were taken at T24, T48 and T72 hours from the limit test solution which contained no algae and had been run alongside the test to determine the influence of adsorption (at the surface of algae cells) and/or bioaccumulation on the possible decrease in test item concentration throughout the test.
Although samples were taken during the test, no chemical analysis was performed on these samples because the test item was found to be toxic.

- Definitive test
Two samples of 10 mL were taken from each test solution except for the control before the start of the test, just before the distribution to the test vessels. Samples were taken from each replicate with algae each 24 hours (T24, T48 and T72 hours) and then pooled in order to obtain two samples of 10 mL.
Two samples of 10 mL were taken at T24, T48 and T72 hours from the solutions which contained no algae and had been run alongside the test to determine the influence of adsorption (at the surface of algae cells) and/or bioaccumulation on the possible decrease in test item concentration throughout the test.

Chemical analysis was only performed on samples taken at the beginning T0 hour, at T24 hours and sampling was stopped after T48 hours since measured concentrations with and without algae at T0 and T24 hours were below the limit of detection.

Test solutions

Vehicle:

no

Details on test solutions:

The stock solutions, for the combined range-finding and limit test and definitive test, were prepared by dissolving the test item directly in OECD medium.
The conditions of preparation of the stock solutions are reported in the following table:

Quantity of test item Volume of OECD medium Concentration of the test item Duration of the agitation
(mg) (mL) (mg/L) (min)
Combined range
finding and limit test 100 1000 100 5

Definitive test 100 1000 100 5

After agitation, the stock solutions were used to prepare the test solutions.

Test solutions were prepared by further dilution of the stock solution with OECD medium to provide a geometric series of concentrations:
- 0.01, 0.1, 1, 10, 100 and 100 mg/L for the combined range finding and limit test,
- 0.15, 0.32, 0.68, 1.43 and 3.00 mg/L for the definitive test.

The pH range considered as appropriate for a normal life of test organisms is circa 6 to 9.
The pH of all test solutions remained within the range 6.0 +/- 0.2 to 9.0 +/- 0.2 after preparation and there was therefore no adjustment of pH before addition of the algae.

Glass test vessels (250 mL Erlenmeyer flasks) were filled directly from the test solution containers after preparation and algae were then added to these vessels.
Test solutions remained unchanged throughout the study.

For the limit test and definitive tests, a further group of vessels was prepared for chemical analysis by adding test solutions (except for the control) but no algae.

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, OBAN, Argyll PA37 1QA, Scotland
- Method of cultivation: the algae are cultured under sterile conditions and maintained at exponential growth rate

ACCLIMATION
- Culturing media and conditions (same as test or not): LC medium. Reconstituted water prepared using deionized water with a conductivity less than 10 µS/cm. The LC medium after autoclaving has a pH of 7.5 ± 0.3.
The temperature was between 21°C and 24°C in water (temperature was checked daily).
Illumination: 24 hours per day. Constant illumination was provided by white type fluorescent lights in the spectral range 40 to 700 nm with a light intensity of approximately 2000 lux.
Aeration: the cultures were constantly aerated with air filtered using a 0.22 µm porosity filter to maximize the CO2 availability (and thereby the cell growth) while maintaining the sterility
- Any deformed or abnormal cells observed: yes

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

32.1mg/L as Ca CO3

Test temperature:

between 24.8°C and 28°C

pH:

between 6.95 and 11.14

Dissolved oxygen:

No data

Salinity:

Not measured

Nominal and measured concentrations:

Measured concentrations in all definitive test solutions were below 80% of the corresponding nominal values at T0 hours and T24 hours (Table A).
In spite of the dispositions undertaken to limit the loss of test item from the test solutions by volatilization (e.g. sealed test vessels and headspace kept to a minimum) all the analytical results were below of the limit of detection LOD (i.e.0.08 mg/L).
In addition, as all the analytical results were below the LOD at T0 and T24 hours, sampling was stopped after T48 hours and samples taken at T48 hours were not analyzed. It was assumed that these results were also below the LOD.

All toxicity conclusions are therefore based on the measured concentrations in test solutions.

Details on test conditions:

TEST SYSTEM
- Type (delete if not applicable): closed
- Renewal rate of test solution (frequency/flow rate): no renewal
- Initial cells density: 1 x 104 cells/mL
- No. of vessels per concentration (replicates): limit test: 6 (with algae) and 1 (without algae) replicates/concentration, range-finding test: 2 replicates/concentration, definitive test: 3 (with algae) and 1 (without algae) replicates/concentration
- No. of vessels per control (replicates): 6 replicates (with algae)/concentration

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: yes. Test medium was OECD medium = reconstituted water OECD medium prepared using sterile water with a conductivity of 10 µS/cm. Before use OECD medium after equilibration its pH was verified at 8.1 ± 0.1 or adjusted until this pH was obtained. It was prepared according to the OECD Guideline for testing of chemicals, No. 201, March 2006.
- Intervals of water quality measurement: The pH was measured in the control and in the limit test concentration and all test concentrations of the definitive test. It was measured at the beginning of the test, in the preparation test container, before distribution in test vessels and at the end of the test in one replicate. The temperature was measured between 0 and 2 hours after the beginning of the test in a flask containing OECD medium but no algae run alongside the test. The maximun and minimum temperatures were also recorded in this flask 24, 48 and 72 hours after the start of the test.
Hardness was measured once in OECD medium before the start of the test.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: test medium was adjusted at 6.95
- Photoperiod: continuous illumination
- Light intensity and quality: a set of 15 and 30 W Osram Fluora® fluorescent tubes between "white" and "daylight" type (spectral range 400 to 700 nm) were set approximately 40 cm above the cultures. These emit light measured using a lux meter equipped with a spherical collector at a level corresponding to half the height of the cultures in their conical flasks (corresponding to an irradiance of 252 mW/m2 for 15 W and 476 mW/m2 for 30 W tubes x no. of bulbs = 3724 mW/m2). The light intensity at each position to be occupied by a culture flask was measured regularly (approximately 7000 lux). The color temperature of the fluorescent lamps was between 4400 K and 5000 K.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: by a Malassez cell counter
- Chlorophyll measurement: no

TEST CONCENTRATIONS
- Spacing factor for test concentrations: For the combined range-finding and limit test the ratio between two consecutive concentrations was a constant factor not exceeding 10. For the definitive test, a geometric series of concentrations with a ratio between two consecutive concentrations ≤ 2.2 and not exceeding 3.2 was used.
- Range finding study: yes. Concentrations were 0.01, 0.1, 1 and 10 mg/L
- Test concentrations: nominal concentrations of 0.15, 0.32, 0.68, 1.43 and 3 mg/L

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

< 0.08 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

< 0.08 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

< 0.08 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

< 0.08 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 0.08 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

< 0.08 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: yield

Details on results:

. Limit test concentration (100 mg/L nominal)
Inhibitions of the average specific growth rate and yield at 100 mg/L nominal were 100% and 100%, respectively, relative to the control, after 72 hours

. Range-finding and limit test
Average specific growth rate inhibitions at 0.01, 0.1, 1, 10 and 10 mg/L nominal were 0.33, 0.13, 66.4, 100%, respectively, relative to the control, after 72 hours.
Yield inhibitions at 0.01, 0.1, 1, 10 and 10 mg/L nominal were 1.77, 0.76, 97.2 and 100%, respectively, relative to the control, after 72 hours.

As a significant inhibition of the algal growth (statistic analysis on raw data) was observed at 100 mg/L a definitive was performed.

Results with reference substance (positive control):

The result and conclusion of the last reference test (relative to the experimental completion date of this study) are presented below:
- Dates of the test: April 2010
- Reference substance: Potassium dichromate
- 72-hour ErC50: 1.22 mg/L of potassium dichromate
- Conclusion: ErC50 accepted.

Any other information on results incl. tables

The results of the definitive test based on the nominal concentrations are summarized in the following tables: 

Group	Control	Monomethyl Hydrazine
	Nominal concentration (mg/L)
	0	0.15	0.32	0.68	1.43	3.00
	Inhibition (%)
Growth rate - 24 h	NA	-7.13	0.30	58.8	89.1	100
Growth rate - 48 h	NA	-3.81	1.04	46.9	96.8	100
Growth rate - 72 h	NA	1.46	1.76	34.2	99.4	100
Yield - 72 h	NA	6.80	9.27	81.85	100	100

 

 

	Endpoints (mg/L)(1)
	24 hours	48 hours	72 hours
Growth rate
ErC50	0.70 (0.64 – 0.77)	0.71 (0.65 – 0.77)	0.73 (0.67 – 0.79 )
ErC10	ND	ND	0.43
Yield
EyC50	ND	ND	0.46 (0.42 – 0.51 )
EyC10	ND	ND	0.24

ND  : not determined

(1)    : EC50: Median (50%) Effective Concentration (numbers in brackets correspond to the 95% confidence limits),ExC10 is the test concentration where effect is reduced by 10%.

 

The results showed a clear dose-response relationshipwhich is why the ErC(s) and EyC(s) values were calculated from the nominal concentrations by statistical analysis. (The72-hoursErC(s) and 72-hours EyC(s)calculations are reported in Appendix 5 for information).

However all measured concentrations were below the Limit Of Detection (LOD). Therefore according to the analytical results it was assumed that the nominal concentrations were lower and not representative of the concentrations actually tested.

In addition, further testing with lower nominal concentrations was not performed since the nominal concentrations tested would have been below of the limit of quantification of 0.3 mg/L and therefore could not have been analyzed.

The conclusions of the definitive test are summarized in the following table: 

	Endpoints (mg/L)(1)
	24 hours	48 hours	72 hours
Growth rate
ErC50	< 0.08	< 0.08	< 0.08
ErC10	ND	ND	< 0.08
Yield
EyC50	ND	ND	< 0.08
EyC10	ND	ND	< 0.08

ND  : not determined

(1)    : EC50: Median (50%) Effective Concentration (numbers in brackets correspond to the 95% confidence limits),ExC10 is the test concentration where effect is reduced by 10%.

 

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In a 72-hour algal growth inhibition test with Pseudokirchneriella subcapitata, the EC50 of Monomethyl Hydrazine was < 0.08 mg/L based on average specific growth rate (ErC50) and < 0.08 mg/L based on yield (EyC50).

Executive summary:

Methods

A preliminary test (combined range-finding and limit test conducted simultaneously) was performed to determine the concentrations to be tested in the definitive test.

 

Combined range-finding and limit test

.           Limit test: two groups of six replicate algal suspensions were exposedto the nominal concentrations of 0 (control) and 100 mg/L for 72 hours. Cell growth data were recorded at 24, 48 and 72 hours,

.           Range-finding test: four groups of two replicate algal suspensions were exposed to nominal concentrations of 0.01, 0.1, 1 and 10 mg/L of the test item under the same experimental conditions.

 

Definitive test

In the definitive test, algal suspensions were exposed to the nominal concentrations of 0.15, 0.32, 0.68, 1.43 and 3 mg/L for 72 hours.

Three replicate algal suspensions were exposed to each test item concentration and six replicates to test medium only (control) for 72 hours. Cell growth data were recorded at 24, 48 and 72 hours.

 

Chemical analysis

Samples were taken daily to measure the actual concentration of the test item in all solutions, except for the control, during the exposure period.

Results

Validity criteria

All study validity criteria were respected.

 

Chemical analysis

Measured concentrations in all definitive test solutionswere below 80% of the corresponding nominal values at T0 hours and T24 hours.

Inspite of the provisions undertaken to limit the loss of test item from the test solutions by volatilization (e.g. sealed test vessels and headspace kept to a minimum) all the analytical results were below of the limit of detection LOD (i.e. 0.08 mg/L).

 

Inhibition of cell growth

The24,48and72-hour ErC (x)s and the72-hour EyCx were estimated from the % inhibition of average specific growth rate (ErC(x)s) and yield (EyCx).

They were calculated by statistical analysis as the spread of inhibition across the concentrations was sufficient to undertake the calculation.

The results of the definitive test based on the nominal concentrations are summarized in the following tables: 

Group	Control	Monomethyl Hydrazine
	Nominal concentration (mg/L)
	0	0.15	0.32	0.68	1.43	3.00
	Inhibition (%)
Growth rate - 24 h	NA	-7.13	0.30	58.8	89.1	100
Growth rate - 48 h	NA	-3.81	1.04	46.9	96.8	100
Growth rate - 72 h	NA	1.46	1.76	34.2	99.4	100
Yield - 72 h	NA	6.80	9.27	81.85	100	100

 

	Endpoints (mg/L)(1)
	24 hours	48 hours	72 hours
Growth rate
ErC50	0.70 (0.64 – 0.77)	0.71 (0.65 – 0.77)	0.73 (0.67 – 0.79 )
ErC10	ND	ND	0.43
Yield
EyC50	ND	ND	0.46 (0.42 – 0.51 )
EyC10	ND	ND	0.24

ND  : not determined

(1)    : EC50: Median (50%) Effective Concentration (numbers in brackets correspond to the 95% confidence limits),ExC10 is the test concentration where effect is reduced by 10%.

 

The results showed a clear dose-response relationship. However, all measured concentrations were below the Limit Of Detection (LOD), it was assumed that the nominal concentrations were not representative of the concentrations actually tested.

In addition, further testing with lower nominal concentrations was not performed since the nominal concentrations tested would have been below of the limit of quantification of 0.3 mg/L.

Therefore the conclusions of the definitive test (below) are based on the measured concentrations.

The conclusions of the definitive test are summarized in the following table:

	Endpoints (mg/L)(1)
	24 hours	48 hours	72 hours
Growth rate
ErC50	< 0.08	< 0.08	< 0.08
ErC10	ND	ND	< 0.08
Yield
EyC50	ND	ND	< 0.08
EyC10	ND	ND	< 0.08

ND  : not determined

(1)    : EC50: Median (50%) Effective Concentration (numbers in brackets correspond to the 95% confidence limits),ExC10 is the test concentration where effect is reduced by 10%.

 

Conclusion

In a 72-hour algal growth inhibition test with Pseudokirchneriella subcapitata, theEC50 of Monomethyl Hydrazine was < 0.08 mg/L based on average specific growth rate (ErC50) and < 0.08 mg/L based on yield (EyC50).