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

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-06-11 until 2001-07-02

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:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction 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 (3,6-dihydro-3-methyl-N-nitro-(2HI ),3-, 5-oxadiazin-4-amine)
- 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: test substance concentrations 10, 18, 32, 56 and 120 mg/l
- Sampling method: Samples were taken from the centre of the test solutions. On day 0, 7, 14 and 18 samples of each new test solution were analysed for the concentration of CA2343A. Samples from the corresponding old solutions were analysed on the following renewal day.
- 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: A primary stock solution of 120 mg/l was prepared on each new test solution preparation day by dissolving 0.12 g of CA2343A per 1000 ml of dilution water, with 3 minutes of ultra-sonic treatment. On each test solution renewal day, the test solutions were prepared by the addition of
aliquots of the primary stock solution to dilution water. All the test solutions were observed to be clear and colourless
- Eluate: not applicable
- Differential loading: not reported
- Controls: dilution water control
- 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):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water fleas
- Strain/clone: Daphnia magna
- Justification for species other than prescribed by test guideline: not applicable
- Source: Daphnia were obtained from continuous laboratory in-house stock cultures, maintained in a reconstituted water medium. Culture conditions were such that the Dnphnia reproduction was by diploid parthenogenesis.
- Age of parental stock (mean and range, SD): The parent animals were 27 ± 1 days old and had been maintained with a twice weekly renewal of reconstituted water medium, since birth.
- Feeding during test
- Food type: The Dapllnia cultures were fed a defined diet of algae Chlorella vulgaris, strain CCAP 211/12 and a commercially available microencapsulated diet "Frippak Booster@".
- Amount: The Daphnia cultures were fed daily ad libitum depending on age and density of the culture.
- Frequency: see above


ACCLIMATION: not reported
- Acclimation period:
- Acclimation conditions (same as test or not):
- Type and amount of food:
- Feeding frequency:
- Health during acclimation (any mortality observed):


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


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Daphnia <24 hours old (first instar), obtained from a single culture vessel, were used for testing. The parent animals were 27 ± 1 days old and had been maintained with a twice weekly renewal of reconstituted water medium, since birth. The test organisms and the culture from which they were obtained showed no evidence of disease before the test period.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

not applicable

Test conditions

Hardness:

New solutions: mean 246 mg/l CaCO3 (SD 9.8)
Old solutions: mean 250 mg/l CaCO3 (SD 3.2)

Test temperature:

20 - 20.6 °C

pH:

New solution: 7.9 - 8.1
Old solution: 7.9 - 8.2

Dissolved oxygen:

New solutions: 9.2 to 9.4 mg/I
Old solutions: 8.6 to 9.2 mg/I

Salinity:

Conductivity data:
New solutions: mean 673 µS/cm (SD 7.0)
Old solutions: mean 674 µS/cm (SD 8.6)

Nominal and measured concentrations:

Nominal CA2343A concentrations: 10, 18, 32, 56 and 120 mg/I
Measured concentrations: The mean measured concentrations in the new test solutions ranged from 102 to 109% of the nominal values. The mean measured concentrations in the old test solutions ranged from 100 to 102% of the nominal values. On the basis of the analytical data the nominal concentrations were used for the calculation and reporting of all results.

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Borosilicate glass beakers were used as test vessels, each containing 80 ml of test solution providing a depth of 47 mm. The beakers were covered with loose fitting lids.
- Aeration: The dilution water was aerated for >2 hours before use. No aeration during test.
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): renewal of test medium on Mondays, Wednesdays, and Fridays
- No. of organisms per vessel: one Daphnia per vessel at test start
- No. of vessels per concentration (replicates): 10 replicates per exposure concentration
- No. of vessels per control (replicates): 10
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: not reported


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
- Total organic carbon: 2.5 mg/l
- Particulate matter: not reported
- Metals: Dilution water acc. to Elendt was used. Detailed analytical data of numerous metals are given in the original study report. All metal concentrations by far within the acceptability levels.
- Pesticides: Dilution water acc. to Elendt was used. Detailed analytical data of numerous pesticides are given in the original study report. All pesticides concentrations by far within the acceptability levels.
- Chlorine:
- Alkalinity: New solution: mean 246 mg/l CaCO3 (SD 9.8); Old solution: mean 250 mg/l CaCO3 (SD 3.2)
- Ca/Mg ratio: 6.6 : 1
- Conductivity: New solution: mean 673 µS/cm (SD 7.0); Old solution: mean 674 µS/cm (SD 8.6)
- Salinity: see above
- Culture medium different from test medium:
- Intervals of water quality measurement: The pH, conductivity, hardness, alkalinity and total organic carbon were determined for the dilution water batch used in this study. Batches of dilution water are also analysed periodically for a range of trace metals and pesticides.


OTHER TEST CONDITIONS
- Adjustment of pH: not reprted
- Photoperiod: 16 hours light:8 hours dark with 20 minute dawn and dusk transition periods
- Light intensity: average intensity of 8.9 µ Einsteins / m² s


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
At the start of the test, one Daphnia was randomly selected and added to each test vessel in sequence across the treatments. These Daphnia were termed the P0 generation. Mortality of the P0 generation was recorded daily for each test vessel. Mortality was defined as absence of any movement by the organism, when examined by eye, for a period of 15 seconds (after gentle agitation of the container if necessary). Observations were made at the same time for any other symptoms of toxicity. Observations were made daily for the presence of live and dead offspring (termed F1 generation) in each vessel.

VEHICLE CONTROL PERFORMED: not appliacble


RANGE-FINDING STUDY: not reported
- Test concentrations:
- Results used to determine the conditions for the definitive study:

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 2

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 3

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

length

Effect concentrations 4

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth

Remarks:

length

Effect concentrations 5

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

56 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: overall effect

Effect concentrations 6

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

120 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: overall effect

Details on results:

- Mortality of parent animals:
There were no mortalities, or other observed symptoms of toxicity, in the dilution water control, or the 56 mg/I test concentration of CA2343A after 21 days. One P0 Daphnia died in each of the 10, 32, and 120 mg/I test concentrations, and two in the 18 mg/l test concentration. Prior to their deaths, these Daphnia appeared to be healthy and producing offspring normally. The guideline allows mortality of 20% of the parent animals in the controls at the end of the test without affecting the validity of the test. The mortalities observed in the test were within this limit.
- No. of offspring produced per day per female: see table in free text below
- Body length and weight of parent animals: see table in free text below
- Type and number of morphological abnormalities: none reported
- Type and number of behavioural abnormalities: none reported
- Number of males and females (parental) parthenogenesis
- Time to first brood release or time to hatch: With the exception of one of the dilution water control Daphnia, and six of the surviving 120 mg/I animals, all P0 Daphnia had released their first offspring on Day 8.
- Egg development time: In all treatments, all P0 Daphnia, surviving to the end of the test, had completed at least four broods by the end of the study.
- Brood size: see table in free text below
- Time to sexual maturity: not reported
- Type and magnitude of biochemical changes: not reported
- Other biological observations: not 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:

The reproduction and length data for each individual P0 generation daphnid were entered into electronic data files and analysed using statistical procedures. The data were tested for normality, and homogeneity of variance. If the data had a normal distribution and the variance was homogeneous then the data were analysed using a parametric statistical procedure such as analysis of variance followed by a multiple comparison procedure, such as Dunnett's T-test. If the data were not normal or failed Bartlett's test for homogeneity of variance, then the data were analysed using a non parametric statistical procedure such as Wilcoxon's Rank Sum Test.

Any other information on results incl. tables

Tab. 1: Reproduction and length data

	CA 2343 concentration in mg/l
	control	10	18	32	56	120
Mean offspring per parent	124	144	135	145	136	104
Coefficient of variation for mean offspring per parent	15.2	7.2	9.8	6.5	9.7	25.3
Total offspring	1237	1372	1144	1372	1358	940
% offspring mortality	0	0	0	0	0	0
mean length in mm	4.5	4.5	4.5	4.5	4.4	4.3

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

The guideline allows mortality of 20% of the parent animals in the controls at the end of the test without affecting the validity of the test. The mortalities observed in the test were within this limit.

Conclusions:

The NOEC of CA2343A (based on nominal concentrations) for reproduction was 120 mg/l and the EC-50 for reproduction was > 120 mg/l. The NOEC of CA2343A for length was 56 mg/I, the LOEC was 120 mg/l. The overall NOEC of CA2343A was 56 mg/l and the overall LOEC was 120 mg/l. Since the NOECs from this chronic aquatic toxicity test are > 1 mg/l, the test substance is not classified as chronic toxic / hazardous to the aquatic environment.

Executive summary:

The purpose of this study was to evaluate the chronic aquatic toxicity of test item CA 2343 A (oxadiazinamine) to aquatic invertebrates, i.e. Daphnia magna. The study protocol followed OECD Guideline 211. The test duration was 21 days.

The NOEC of CA2343A (based on nominal concentrations) for reproduction was 120 mg/l and the EC-50 for reproduction was > 120 mg/l. The NOEC of CA2343A for length was 56 mg/I, the LOEC was 120 mg/l. The overall NOEC of CA2343A was 56 mg/l and the overall LOEC was 120 mg/l. Since the NOECs from this chronic aquatic toxicity test are > 1 mg/l, the test substance is not classified as chronic toxic / hazardous to the aquatic environment.