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

Toxicity to microorganisms

Administrative data

Endpoint:

activated sludge respiration inhibition testing

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996-01-03 until 1996-04-18

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 209 (Activated Sludge, Respiration Inhibition Test

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.11 (Biodegradation: Activated Sludge Respiration 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:

not required

Details on sampling:

- Concentrations:
- Sampling method: direct measurement of oxygen consumption per hour in mg/liter with a ORION - Electrode Type 97-08, ORION Microprocessor Ionalizer 901 and dotted on a recorder.
- Sample storage conditions before analysis: not applicable

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The preparation was carried out according to the method described in the guideline.
- Eluate: not applicable
- Differential loading: not applicable
- Controls: blank nutrient solution with inoculum (=100% respiratory activity)
- 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): none reported

Test organisms

Test organisms (species):

activated sludge of a predominantly domestic sewage

Details on inoculum:

- Laboratory culture: not applicable
- Method of cultivation: On return to the laboratory, the sludge will be washed three times by settling for a short period, e.g. 15-30 minutes, decanting the upper layer and adding the same amount of water as the decanted water. The sludge is kept aerated for use during the day. Before use the pH is
checked and adjusted.
- Preparation of inoculum for exposure: The preparation was carried out according to the method described in the guideline. The sludge was separated from the aqueous layer by settling instead of centrifugation. The pH of the sludge before use was 7.4.
- Pretreatment: If the sludge cannot be used on the day of collection. 100 ml synthetic sewage is added to each litre of the activated sludge prepared as described above; this is then aerated overnight at 20 ± 2 °C.
- Initial biomass concentration: The sludge concentration in the test bottles was 1.59 g/l (dry weight).

Study design

Test type:

static

Water media type:

freshwater

Total exposure duration:

3 h

Post exposure observation period:

none reported

Test conditions

Hardness:

not reported, however detailed composition of nutrient solution in dechlorinated drinking water is stated in the study report

Test temperature:

20 ± 2 °C

pH:

7.4

Dissolved oxygen:

not reported

Salinity:

not reported, however detailed composition of nutrient solution in dechlorinated drinking water is stated in the study report

Nominal and measured concentrations:

- nominal test item concentrations: 1, 3.2, 10, 32, and 100 mg/l
- nominal reference substance concentrations: 3.2, 10, and 32 mg/l

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): BOD flasks with gas inlet
- Material, size, headspace, fill volume: glass, size 250 ml, headspace 50 ml, fill volume 200 ml
- Aeration: stirring
- Type of flow-through (e.g. peristaltic or proportional diluter): no flow-through design applied
- Renewal rate of test solution (frequency/flow rate): not applicable
- No. of organisms per vessel: not applicable
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 2
- No. of vessels per vehicle control (replicates): 2
- Biomass loading rate: 1.59 g/l


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated drinkîng water
- Total organic carbon: no data (nutrient solution with 16 g/l peptone and 11 g/l meat extract)
- Particulate matter: activated sludge loading 1.59 g/l
- Metals: Na, K, Mg, Ca
- Pesticides: not reported
- Chlorine: dechlorinated drinking water used
- Alkalinity: not reported, but detailed composition of nutirent solution in drinking water stated in the study report
- Ca/Mg ratio: not reported for the drinking water; nutrient solution composition Ca:Mg = 3.4:1
- Conductivity: not reported
- Culture medium different from test medium: no
- Intervals of water quality measurement: at start and at end of test


OTHER TEST CONDITIONS
- Adjustment of pH: yes, from 7.8 to 7.4
- Photoperiod: not applicable
- Light intensity: not applicable


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : oxygen consumption continuously measured via electrode


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.3
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study
- Test concentrations: not applicable
- Results used to determine the conditions for the definitive study: not applicable

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorphenol

Results and discussion

Effect concentrations

Duration:

3 h

Dose descriptor:

IC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

inhibition of total respiration

Remarks:

respiration rate

Details on results:

- 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: none reported

Results with reference substance (positive control):

- Results with reference substance valid? yes
- Relevant effect levels: If the respiration rates of the two controls are not within 15 per cent of each other or the reference substance is not in the accepted range (5 to 30 mg/l), the test is invalid and must be repeated.
- Other:

Reported statistics and error estimates:

The inhibitory values were calculated on the basis of the measured time depend oxygen consumption of a Blank and Test solution. The Results ( EC50 Values ) were determined after calculation the linear regression (formula given in the study report).

Any other information on results incl. tables

Table 1: Consumption rate and inhibitory effects of the test substance and reference

Sample	Conc. (mg/l)	Consumption rate (mg/l/h)	Inhibition (%)
Blank 1	0	69.8	-2
Blank 2	0	66.7	2
Reference 1	32	10.7	84
Reference 2	10	40	41
Reference 3	3.2	60	12
Test conc. 1	100	56.6	17
Test conc. 2	32	58.8	14
Test conc. 3	10	57.7	15
Test conc. 4	3.2	57.7	15
Test conc. 5	1	57.7	15

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Based on the result EC 50 (3h) > l00 mg/l, acc. to the criteria set in 67/548/ECC and OECD-GHS, test item CA 2343 A is not toxic to bacteria.

Executive summary:

This report describes the experimental techniques and the results obtained in this study to determine the inhibitory concentration of the test substance CA 2343 A on the respiratory activity of aerobic waste-water bacteria. The method acc. to OECD Guideline 209 was applied. Based on the result EC 50 (3h) > l00 mg/l, acc. to the criteria set in 67/548/ECC and OECD-GHS, test item CA 2343 A is not toxic to bacteria.