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

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:

1995-11-03 until 1996-03-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations.

Cross-reference

Reason / purpose for cross-reference:

reference to other study

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 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

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: Nominal: 0.2, 0.4,0.8, 1.6,3.2,6.4 and 12.8 mg test substance/l
- Sampling method:
Composite samples (about 150 ml per concentration and control) of each test concentration were drawn by mixing identical volumes of the test solutions taken from the test vessels. Samples of test solutions were taken immediately before exposure and after 72 hours exposure. A sample of the
stock solution was taken 24 hours before exposure.
- Sample storage conditions before analysis: All samples were kept at - 18°C to - 22°C until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: preparation of stock solutions: 50.2 mg test substance were mixed and made up to 1000 ml with reconstituted water by ultrasonic for 1 minute.
- Eluate: not applicable
- Differential loading: no data
- Controls: blank: reconstituted water
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable
- Evidence of undissolved material (e.g. precipitate, surface film, etc): no observations reported

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green algae
- Strain: Selenastrum capricornutum ATCC 22662 = Pseudokirch. subcapitata SAG 61.81)
- Source (laboratory, culture collection): Collection of algae cultures, Pflanzenphysiologisches Institut, University Gottingen, Germany
- Age of inoculum (at test initiation): Preculture: 3 days under test conditions
- Method of cultivation: Initial cell density: 10 100 cells/ml. Culture according to the guideline cited. The parent stock is cultured under conditions similar to the test (e.g. light, temperature, medium). All operations for culturing were carried out under sterile conditions in order to avoid contamination with bacteria and other algae.


ACCLIMATION
- Acclimation period: Preculture: 3 days under test conditions
- Culturing media and conditions (same as test or not): same
- Any deformed or abnormal cells observed: none reported

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

no data

Test conditions

Hardness:

25 mg CaC03/l

Test temperature:

24 ± 1°C

pH:

The pH of the test solutions was 8.0 - 8.1 and 8.0 - 8.2 at the start and end of exposure, respectively.

Dissolved oxygen:

no data

Salinity:

Conductivety: 150 µS/cm

Nominal and measured concentrations:

- Nominal test concentrations: 0.2, 0.4, 0.8, 1.6, 3.2, 6.4 and 12.8 mg test substance/l
- Measured: 0.19, 0.38, 0.76, 1.6, 2.9, 6.3, 12.7 mg/l and 0.18, 0.36, 0.71, 1.5, 2.9, 5.9, 12.0 mg/l at the start and the end of exposure, respectively.
The measured concentrations were within 100 ± 20% the nominal concentrations.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: 100 ml Erlenmeyer flasks (total volume 125 ml), cotton stoppers, 50 ml test solution per flask
- Aeration: Labshaker, 150 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- Initial cells density: 10100 cells / ml
- Control end cells density: 990000 cells / ml
- No. of organisms per vessel: 505000
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not applicable


GROWTH MEDIUM
- Standard medium used: yes
- Detailed composition if non-standard medium was used:


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted Water, composition according to the guideline.
- Total organic carbon: no data
- Particulate matter: no data
- Metals: no data
- Pesticides: no data
- Chlorine: no data
- Alkalinity: no data
- Ca/mg ratio: no data
- Conductivity: 150 µS/cm
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH was measured at Oh and 72h exposure


OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: not reported
- Photoperiod: Continuous illumination
- Light intensity and quality: cold white fluorescent light, 120 µE/cm² sec ± 20 % (approx. 8000 lux)
- Salinity (for marine algae): not applicable


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: Cell densities were measured at 24, 48 and 72 hours exposure on a "TOA" cell counter. Test results are reported as daily cell concentration (cells/ml), area under the growth curve, which is an estimate of biomass, and average specific growth rate in each test culture.
- Chlorophyll measurement: no
- Other:


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: 0.01, 0.1, 1.0, 10 and 100 mg/l.
- Results used to determine the conditions for the definitive study: A range-finding test was conducted to determine a concentration range of CA 2343 A to use in the definitive test. An initial cell density of 10500 cells/ml was exposed for 72 hours to each of the concentrations. The measured cell concentration at the end of exposure showed an inhibition at test concentration 10 mg/l. Based on the results of the preliminary test the concentrations were selected for the definitive test.

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

16.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 15.2 - 18.1 mg/l; slope 2.38

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

8.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 7.3 - 10.2 mg/l; slope 2.69

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: significant on 5% level

Effect concentrations 4

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: significant on 5% level

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities reported
- Unusual cell shape:
- Colour differences:
- Flocculation:
- Adherence to test vessels:
- Aggregation of algal cells:
- Other:
- Any stimulation of growth found in any treatment: 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):

no reference substance applied

Reported statistics and error estimates:

The EC-50 values were calculated according to the maximun likelihood method, logit model (Mc Cullagh, P., Nelder, J.A., 1983: Generalized linear models, Chapman & Hall, London). The modified Dunnett's Multible Comparison Test was used for calculation of NOErC and NOEbC.

Any other information on results incl. tables

Table 1: Inhibition, areas under growth curves (indicator for biomass)

Conc. nominal, mg/l	Mean areas under Growth Curves (A) * 10000	Inhibition IA (0 -72h) in %
blank	1737	0
0.2	2189	0
0.4	2000	0
0.8	2061	0
1.6	1959	0
3.2	2016	0
6.4	1249	28.1
12.8	562	67.7

Table 2: Inhibion, growth rates

Conc. nominal, mg/l	Mean growth rates (µ) * 0.001	Inhibition Iµ (0 -72h)
blank	64	0
0.2	67	0
0.4	66	0
0.8	66	0
1.6	66	0
3.2	67	0
6.4	58	8.6
12.8	42	33.4

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

ErC 50 (0-72h) of CA 2343 A was determined to be 16.4 mg/l based on nominal concentrations. EbC 50 (0-72h) of CA 2343 A was determined to be 8.6 mg/l based on nominal concentrations. The NOErC (0-7%) and NOEbC (0-72h) were both 3.2 mg/l based on the nominal concentrations of CA 2343 A.

Executive summary:

The purpose of this study was to evaluate the acute toxic effects of test substance CA2343 (oxadiazinamine) to the growth of aquatic green algae Selenastrum capricornutum. The study followed the OECD-Guideline No. 201 and Guideline 92/69/EEC C.3. The test was conducted in a static system, the test duration was 72 hours.

ErC 50 (0-72h) of CA 2343 A was determined to be 16.4 mg/l based on nominal concentrations. EbC 50 (0-72h) of CA 2343 A was determined to be 8.6 mg/l based on nominal concentrations. The NOErC (0-72 h) and NOEbC (0-72h) were both 3.2 mg/l based on the nominal concentrations of CA 2343 A.

These results have some consequences on the classification and labelling of substance CA 2343, see discussion in endpoint summary 6.1 "Aquatic Toxicity".