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Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 15 July 2010 to 16 september 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: compliant to GLP and testing guideline (reported deviations were not considered to have compromised the validity of the study and results); 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
Temperature varied by more than ± 1°C and water hardness in the control was higher than 250 mg/L as CaCO3 during/throughout the combined range-finding and limit test and during the definitive test.
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
as above
Qualifier:
according to
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. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 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:
yes
Details on sampling:
Samples were taken in the fresh solutions, i.e. at the start of the test and at the solution change, just before distribution in the tests vessels from the preparation containers.
Samples were also taken in the old solutions, i.e. at the solution change and at the end of the test, in each replicate directly from the test vessels and then pooled by concentration.

Combined range-finding and limit test:
Duplicate samples of 10 mL (no headspace in the sampling tube) were taken from the limit test concentration (100 mg/L nominal) solution container.
As the test item was found to be toxic at this limit test concentration, samples already taken were not analyzed.

Definitive test:
Duplicate samples of 5 mL (no headspace in the sampling tube) were taken in each group containing the test item and in the stock solution if its concentration is higher than the highest concentration tested.

Test solutions

Vehicle:
no
Details on test solutions:
The stock solutions, for the combined range-finding and limit test and definitive tests, were prepared by dissolving the test item directly in M4 medium.

The conditions of preparation of the stock solutions are reported in the following table:

Quantity of test Volume of M4 Concentration of the Duration of the agitation
item (mg) medium(mL) test item (mg/L) (minutes)

Combined range-finding
and limit test 100 1000 100 5

Definitive test
T0 hours 100 1000 100 5
T24 hours 1000 1000 1000 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 M4 medium to provide a geometric series of concentrations:
6 0.01, 0.1, 1, 10, 100 and 100 mg/L for the combined range-finding and limit test,
- 0.13, 0.22, 0.36, 0.60 and 1 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 incorporation of the animals.
Daphnids were placed in test vessels which were gently filled with the test solutions immediately after their preparation. The test solutions were changed after at T24 hours.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM

- Strain: STRAUS-clone 5

- Source: LIEBE - CNRS, UMR 7146, Université Paul Verlaine, 57070 Metz, France

- Age at study initiation (mean and range, SD): between 6 and 24 hours old

- Method of breeding: Breeding medium= M4 medium.= reconstituted water prepared according to the OECD guideline for testing of chemicals, N°202, April 2004. The pH of this solution was 8.0 ± 0.5; the sum of Ca and Mg ions in the solution was 2.5 mmol/L (molar ratio Ca:Mg was approximately 4:1 and Na:K approximately 10:1).
Temperature: between 18°C and 22°C. Temperature was recorded continuously and checked generally twice a week.
Loading: tanks were loaded at one animal/20 mL of culture water.
Feeding: animals were fed with a diet of 0.15 mg of carbon per Daphnia per day, in the form of the algal strain Scenedesmus subspicatus which was cultured at CIT and had previously been measured for its total organic carbon content.
Light/dark cycle: 16/8 hours.
Forced aeration: none. Oxygen concentrations remain above 80% of the saturation value at the selected temperature.
The medium was replaced generally each Monday, Wednesday and Friday. On each of these occasions, neonates were separated from the adults and either removed or, once monthly, used to start a new culture.
During breeding, pH, water hardness and dissolved oxygen concentrations were checked in the "new" and "old" media when solutions were replaced generally twice a week.

- Feeding during test: none


ACCLIMATION
- Acclimation period: none

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h

Test conditions

Hardness:
between 140 to 250 mg/L as Ca CO3
Test temperature:
set between 18°C and 22°C and not varying by more than ± 1°C throughout the test, measured using a max-min thermometer
pH:
between 6 and 9 and did not vary by more than 1 unit in the control
Dissolved oxygen:
> 3 mg/L at that temperature
Salinity:
Not measured
Nominal and measured concentrations:
Measured concentrations in all definitive test solutions were below 80% of the corresponding nominal values during the study.
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 the limit of quantification LOQ (0.3 mg/L) or the limit of detection LOD (0.08 mg/L).
In addition, no results were available at T23.9 or T24 hours since the analytical sequence failed. However it was assumed that these results were also below the LOQ or the LOD.
All toxicity conclusions are therefore based on the measured concentrations in test solutions.
Nominal concentrations of definitive test: 0.13, 0.22, 0.36, 0.60 and 1 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Aeration: none
- Renewal rate of test solution (frequency/flow rate): test solutions were changed 24 hours after the beginning of the assay in each group where at least one daphnid is not immobilized
- No. of organisms per vessel: 20 animals per group divided into four replicate groups of 5 animals
- Biomass loading rate: 50 mL (10 mL per animal).

OTHER TEST CONDITIONS
- Adjustment of pH: none
- Light/dark cycle: 16/8 hours

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Observations were carried out at 24 and 48 hours in order to determine the number of immobilized daphnids in each test solution. Immobilization was defined as the inability of a daphnia to swim within 15 seconds after gentle agitation of the test vessel.
Any other possible adverse effects (including abnormal behavior) were also noted

TEST CONCENTRATIONS
- Range finding study
- Test concentrations: 0.01, 0.1, 1 and 10 mg/L
- Results used to determine the conditions for the definitive study: During the range-finding study, at 24 hours, immobilizations were 0% at 0.01 and 0.1 mg/L nominal, 85% at 1 mg/L nominal and 100% at 10 and 100 mg/L nominal, respectively.
At 48 hours, immobilizations were 0% at 0.01 and 0.1 mg/L nominal and 100% at 1, 10 and 1 and 100 mg/L nominal, respectively.
Therefore, the 48-hour EC50 of the test item was estimated to be between 0.1 and 1 mg/L nominal under the experimental conditions.
Reference substance (positive control):
yes
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
> 0.3 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: immobilization
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
< 0.3 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: immobilization
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
< 0.08 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: immobilization
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
< 0.08 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: immobilization
Duration:
24 h
Dose descriptor:
EC0
Effect conc.:
< 0.08 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: immobilization
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
< 0.08 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Basis for effect:
other: immobilization
Details on results:
No difficulty was experienced in discerning the daphnids during the observation periods.
Apart from immobilization, no other adverse effects were observed in the control and treated daphnids.
The results showed a clear dose-response relationship which is why the 24 and 48-hours EC50 values were calculated from the nominal concentrations by statistical analysis. (The 24h and 48h EC50 calculations are reported in Appendix 4 for information).

However all measured concentrations were below the limit of quantification (LOQ) or 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.
Therefore the conclusions of the definitive test are based on the measured concentrations.

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

Concentration (mg/Lnominal)

0

0.13

0.22

0.36

0.60

1.00

 

 

 

Immobilization (%)

24 hours

0

0

0

0

30

90

48 hours

0

5

45

70

95

100

 

 

Endpoints (mg/L) (1)

 

24 hours

48 hours

EC100

>1.00

1.00

EC50

0.70
(0.61 – 0.80 )

0.26
( 0.22 – 0.31 )

EC0/NOEC

0.36

<0.13

(1) EC100: lowest concentration with 100% immobilization; EC50: Median (50%) Effective Concentration (numbers in brackets correspond to the 95% confidence limits); EC0: highest concentration without immobilization; NOEC: No Observed Effect Concentration.

The conclusions (based on measured concentrations) of the definitive test are summarized in the following table:

 

Endpoints (mg/L)(1)

 

24 hours

48 hours

EC100

>0.3

<0.3

EC50

<0.08

<0.08

EC0/NOEC

<0.08

<0.08

(1) EC100: lowest concentration with 100% immobilization; EC50: Median (50%) Effective Concentration (numbers in brackets correspond to the 95% confidence limits); EC0: highest concentration without immobilization; NOEC: No Observed Effect Concentration.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In a 48-hour semi-static acute toxicity test with Daphnia magna, the EC50 of Monomethyl Hydrazine was <0.08 mg/L.
The No Observed Effect Concentration NOEC was <0.08 mg/L.
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

In the limit test,2 groups of 20 daphnids (in 4 replicates of 5) were exposed to the test item dissolved in test medium at nominal concentration of 0 and 100 mg/L for 48 hours.

In the range-finding test,4 groups of 20 daphnids (in 4 replicates of 5) were subjected to the test item dissolved in test medium at nominal concentration of 0.01, 0.1, 1 and 10 mg/L for 48 hours.

 

Definitive test

In the definitive test, 20 daphnids (in 4 replicates of 5) were exposed to the nominal concentrations of 0, 0.13, 0.22, 0.36, 0.60 and 1 mg/L for 48 hours.

 

As the tests were performed in semi-static conditions (according to the physico-chemical properties of the test item) the solutions were changed after 24 hours.

Immobilization at each concentration was recorded at 24 and 48 hours.

 

Chemical analysis

Samples weretaken to measure the concentration of the test item in each test solution of the definitive test, except for the control.

Results

Validity criteria

All study validity criteria were respectedfor the definitive test.

 

Chemical analysis

Measured concentrations in all definitive test solutionswere below 80% of the corresponding nominal values during the study.

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 the limit of quantification LOQ (0.3 mg/L) or the limit of detection LOD (0.08 mg/L).

Immobilization

No difficulty was experienced in discerning the daphnids during the observation periods.

Apart from immobilization, no other adverse effects were observed in the control and treated daphnids.

 

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

Group

Control

Monomethyl Hydrazine

Concentration (mg/Lnominal)

0

0.13

0.22

0.36

0.60

1.00

 

 

 

Immobilization (%)

24 hours

0

0

0

0

30

90

48 hours

0

5

45

70

95

100

 

 

Endpoints (mg/L) (1)

 

24 hours

48 hours

EC100

>1.00

1.00

EC50

0.70
(0.61 - 0.80)

0.26
(0.22 - 0.31)

EC0/NOEC

0.36

<0.13

(1) EC100: lowest concentration with 100% immobilization; EC50: Median (50%) Effective Concentration (numbers in brackets correspond to the 95% confidence limits); EC0: highest concentration without immobilization; NOEC: No Observed Effect Concentration.

 

The results showed a clear dose-response relationship. However, all measured concentrations were below the limit of quantification (LOQ) or below the limit of detection (LOD), 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 0.03 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

EC100

>0.3

<0.3

EC50

<0.08

<0.08

EC0/NOEC

<0.08

<0.08

(1) EC100: lowest concentration with 100% immobilization; EC50: Median (50%) Effective Concentration (numbers in brackets correspond to the 95% confidence limits); EC0: highest concentration without immobilization; NOEC: No Observed Effect Concentration.

 

Conclusion

In a 48-hour semi-static acute toxicity test with Daphnia magna, the EC50 of Monomethyl Hydrazine was <0.08 mg/L.

The No Observed Effect Concentration NOEC was <0.08 mg/L.