N-nitro-N-(3-methyl-3,6-dihydro-2H-1,3,5-oxadiazin-4-yl)amine

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:

1995-12-14 until 1996-08-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

431-060-1

EC Name:

-

Cas Number:

153719-38-1

Molecular formula:

Hill formula: C4H8N4O3 CAS formula: C4H8N4O3

IUPAC Name:

3-methyl-N-nitro-3,6-dihydro-2H-1,3,5-oxadiazin-4-amine

Details on test material:

- Name of test material (as cited in study report): CA 2343 A (Intermediate of CGA 293343)
- Substance type: mono-constituent organic substance
- Physical state: solid, fine, white powder
- Analytical purity: 96.7 %
- Impurities (identity and concentrations): no data
- Purity test date: no data
- Lot/batch No.: P.503005
- Expiration date of the lot/batch: Date of reanalysis: December 1996
- Stability under test conditions: not reported
- Storage condition of test material: 0 - 5 °C

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 10, 18, 32, 58 and 100 mg test substance/l
- Sampling method:
Water samples of each concentration were taken at 0 and after 48 hour exposure and analyzed for the concentrations of both active ingredients of the formulation. The 0 hour samples of each test concentration (2 x 150 ml volume each) were taken from one additional beaker (600 ml volume), which equilibrated in parallel to the original test beakers of each concentration during a time of 24 hours before test initiation. Additonally one sample was taken from the freshly prepared stock solution (50 ml). At test termination (48 hour) samples of about 80 ml were taken from three of the four replicate test vessels of each treatment group. At least one out of three samples in each treatment group was analyzed separately.
- Sample storage conditions before analysis: frozen at -18 - -20 °C

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Preparation of Stock Solution and final test solutions: 200.1 mg test substance were mixed by shaking with 2000 ml reconstituted water (M4-medium, see Appendix D in original study report). Calculated amounts of this stock solution were transferred to 1000 ml testmedium and homogeneously distributed to produce the desired test concentrations. Before test initiation 4 x 100 ml of each test solution were transferred into the test vessels and
equilibrated for about 24 hours.
- Eluate: not applicable
- Differential loading: no data
- Controls: The controls were held solely in M4 medium.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no observations reported

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water fleas
- Strain: D. magna Straus Clon 5
- Source: In-house cultures
- Age at study initiation (mean and range, SD): < 24 h
- Weight at study initiation (mean and range, SD): not applicable
- Length at study initiation (length definition, mean, range and SD): not applicable
- Valve height at study initiation, for shell deposition study (mean and range, SD): not applicable
- Peripheral shell growth removed prior to test initiation: not applicable
- Method of breeding:
Daphnids were maintained in polypropylene vessels containing approximately 2.5 l of M4 medium. About one third of this culture medium was renewed three times a week. The daphnids were fed daily with the exception of the weekend with a suspension of green algae (1.0 ml/L of a Scenedesmus subspicatus suspension with a density of approximately 10^8 cells/ml). Culturing was performed in a constant environment room at 20°C ± 2 °C and a photo period of 16 hour light and 8 hours dark, with a transition period of 30 minutes. Three times a week the young daphnids were separated from the original culture to maintain a homogeneous stock of animals within the same age.
- Feeding during test: No feeding was performed during the 48 h test.



ACCLIMATION
- Acclimation period: see below
- Acclimation conditions (same as test or not): About 24 hours before test initiation (start of exposure) young daphnids were separated from reproductive specimens by sieving all individuals through a 1500 µm sieve. This procedure was repeated before the start of exposure. The young daphnids received were retained for the test.
- Type and amount of food: no feeding
- Feeding frequency:
- Health during acclimation (any mortality observed):


QUARANTINE (wild caught) not applicable
- Duration:
- Health/mortality:

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

none reported

Test conditions

Hardness:

CA 257 mg CaCO3/L

Test temperature:

between 21 and 22 °C

pH:

The pH decreased slightly from a value of 8.1 at test initiation to a value of 8.0 at test termination in all test concentrations.

Dissolved oxygen:

98 and 99% at test initiation

Salinity:

The conductivity was 635 µS/cm

Nominal and measured concentrations:

Nominal concentrations: 10, 18, 32, 58, 100 mg/l
Measured: The converted values corresponded to 9.9, 18.2, 32.0, 57.4 and 102.0 mg/land 10.2, 18.4,32.7, 59.0 and 104.5 mg/L of CA 2343 A at the beginning and the end of exposure, respectively.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers containing 100 ml of test medium or the blank culture medium; 4 test vessels for each concentration and the control.
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: size 150 ml, fill volume 100 ml, head space 50 ml
- Aeration: none
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: no data


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water (M4-medium acc. to Elendt, Appendix D of study report)
- Total organic carbon: no data
- Particulate matter: no data
- Metals: detailed list on macro and micro nutirents given in Appendix D of the original study report
- Pesticides: none
- Chlorine: none
- Alkalinity:
- Ca/Mg ratio: 2:1
- Conductivity: 635 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: not reported


OTHER TEST CONDITIONS
- Adjustment of pH: not applicable
- Photoperiod: 16 hour light and 8 hours dark, with a transition period of 30 minutes.
- Light intensity: fluorescent light


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Immobilization or other behavioral changes of the daphnids were recorded after 24 and 48 hours. Those animals not able to swim within 15 seconds after gentle agitation of the test container were considered to be immobile.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: 0.01, 0.1, 1, 10 and 100 mg/L.
- Results used to determine the conditions for the definitive study: Test concentrations of 10, 18, 32, 58 and 100 mg test substanceL were selected based on the results of the pre-test.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Effect concentrations 3

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: none reported
- Observations on body length and weight: not applicable
- Other biological observations: none reported
- Mortality of control: none reported
- Other adverse effects control: none reported
- Abnormal responses: none reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none reported
- Effect concentrations exceeding solubility of substance in test medium: not applicable

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

As in this test, no immobilization of daphnids was observed at any test concentration, no statistical evaluation was done. The effect concentration EC50 was thus determined to be higher than the highest concentration tested.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

For validity of the test the following criteria had to be fulfilled for the untreated control: Not more than 10 percent of the daphnids in the controls should be immobilized at the end of exposure.

Conclusions:

The test results showed that CA 2343 A (Intermediate of CGA 293343) had no effects on Daphnids up to concentrations of 100 mg/l. The EC 50 was estimated to be >100 mg/l. Using the classification range given in Annex VI of directive 67/548/EEC and the OECD-GHS classification system, CA 2343 A is "non-toxic to Daphnids" based on the inherent toxicity of CA 2343 A in this test alone.

Executive summary:

The objective of this study was to evaluate the acute toxicity (estimation of the EC50) of CA 2343 A (oxadiazinamine, Intermediate of CGA 293343) to the representative cladoceran species Daphnia magna STRAUS during a 48-hour exposure period under static conditions. The EC50 was defined as the concentration of test substance in dilution water which caused an immobilization of 50% among the exposed daphnids after 48 hours of exposure. The EC50 is often used as a relative indicator of potential acute hazards resulting from the release of the test substance into aquatic environments.

The test results showed that CA 2343 A (Intermediate of CGA 293343) had no effects on Daphnids up to concentrations of 100 mg/l. The EC 50 was estimated to be >100 mg/l. Using the classification range given in Annex VI of directive 67/548/EEC and the OECD-GHS classification system, CA 2343 A is "non-toxic to Daphnids" based on the inherent toxicity of CA 2343 A in this test alone.