2,3-Quinoxalinedione, 1,4-dihydro-6-methoxy-7-nitro-

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:

2011-08-02 to 2011-08-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well perormed OECD study under GLP

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

1,4-Dihydro-6-methoxy-7-nitroquinoxaline-2,3-dione

EC Number:

614-960-9

Cas Number:

69399-57-1

Molecular formula:

C9H7N3O5

IUPAC Name:

1,4-Dihydro-6-methoxy-7-nitroquinoxaline-2,3-dione

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): Nitro-C-Dion (NMQD)
- Physical state: Solid, yellow powder
- Analytical purity: 91 % (w/w) (7.3 % water, 1.7 % impurities)
- Purity test date: 2011-07-04
- Stability under test conditions: Not applicable
- Storage condition of test material: Room temperature, cool, dry
- Sum formula: C9H7N3O5
- Molecular weight: 237.17 g/mol

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The test item Nitro-C-Dion (NMQD) was analytically verified by HPLC-DAD in the fresh media (0 h) and old media (48 h) of the saturated solution and the control.
- Sampling method: For the old media, separate replicates without test organisms were prepared and incubated under test conditions. Recoveries of the test item should be within ± 20 % of the initially measured value.
- Sample storage conditions before analysis:All samples were stored at 6 ± 2 °C until sample preparation,
if necessary.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution (100 mg/L) was prepared with dilution water one day prior to application. The stock solution was shaken with 20 rpm for 24 hours (rotating shaker 3040, GFL). Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL).
- Eluate: Dilution water
- Differential loading: Saturated solution as limit concentration
- Controls: Dilution water without test item tested under the same conditions as the test groups.

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna (STRAUS)
- Strain: Clone 5
- Source: Institut für Wasser-, Boden- Lufthygiene (WaBoLu), further own breeding
- Age at study initiation (mean and range, SD): 2 to 24 h
- Method of breeding: In 2-3 L glass vessels with appr. 1.8 L culture medium, at 20 +/- 2 °C, in an incubator, 16 h illumination, illumination strength max. 20 µExm-2 x s-1
- Feeding during test: No feeding

ACCLIMATION
- Acclimation period: 2 h
- Acclimation conditions (same as test or not): Same as test
- Type and amount of food: No feeding
- Health during acclimation (any mortality observed): Healthy, no mortality observed

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Post exposure observation period:

No

Test conditions

Hardness:

Dilution water at test start: 244 (mg CaCO3/L)

Test temperature:

21.8 °C

pH:

Water Quality Parameters at Test Start (0 h)
(measured in one additional replicate of the saturated solution and the control)
pH-Value

Saturated solution 7.97
Control 7.99

Water Quality Parameters at the Test End (48 h)
(measured in all replicates)
pH-Values
Replicates
1 2 3 4
Saturated solution 7.77 7.59 7.73 7.77
Control 7.83 7.89 7.87 7.68

Dissolved oxygen:

Water Quality Parameters at Test Start (0 h)
(measured in one additional replicate of the saturated solution and the control)
Dissolved
O2-Concentration

[mg/L]
Saturated solution 6.16
Control 8.51

Water Quality Parameters at the Test End (48 h)
(measured in all replicates)
Dissolved O2 –Concentration [mg/L]
Replicates
1 2 3 4
Saturated solution 8.70 8.69 8.71 8.68
Control 8.63 8.60 8.63 8.66

Salinity:

Freshwater

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods"

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers, 50 mL
- Type (delete if not applicable): closed with parafilm
- Material, size, headspace, fill volume: Glass beakers (4 cm ID x 7 cm H), 50 mL
- Aeration: No
- Renewal rate of test solution (frequency/flow rate): One application at test start, static test
- 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): None


TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Acc. to Directive 92/69/EEC Method C.2, Annex 1
- Conductivity: Dilution water at test start: 640 µS/cm
- Culture medium different from test medium: Elendt M4 acc. to ELENDT (1990), modified to a total hardness of 160 to 180 mg CaCO3/L
- Intervals of water quality measurement: At test start and test end


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16:8 light:dark cycle
- Light intensity: Diffuse light, illumination strength max. 20 µE x m-2 x s-1


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :


TEST CONCENTRATIONS
- Spacing factor for test concentrations: None, limit test
- Range finding study
Immobilization Rates of the Preliminary Range Finding Test
(n = 20, divided into 2 replicates with 10 daphnids each)
Nominal
Test Item
Concentration
[mg/L] IMMOBILIZATION [%]
24 h 48 h
Replicates Replicates
1 2 MV 1 2 MV
100 0 0 0 0 0 0
10 0 0 0 0 0 0
1 0 0 0 0 0 0
Control 0 0 0 0 0 0

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 2.2 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

- Behavioural abnormalities: None
- Mortality of control: 0%
- Other adverse effects control: None
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50/LC50: 1.77 (CI 1.63 - 1.96) mg/L

Reported statistics and error estimates:

The effects of the limit concentration were evaluated directly from the observation data without mathematical calculation.

Any other information on results incl. tables

Immobilization Rates of the Definitive Test after 24 and 48 h of Exposure

                  (n = 20, divided into 4 replicates with 5 daphnids each)

Nitro-C-Dion (NMQD)	IMMOBILIZATION [%]
Initially measured test item concentration [mg/L]	24 h					48 h
	Replicates					Replicates
	1.	2.	3.	4.	MV	1.	2.	3.	4.	MV
2.20 (Saturated solution)	0	0	0	0	0	0	0	0	0	0
Control	0	0	0	0	0	0	0	0	0	0

There was no effect observed, either in the saturated solution or in the control group.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

At the saturated solution of the test item Nitro-C-Dion (NMQD), corresponding to the initially measured concentration of 2.20 mg/L, no effect was observed.

Executive summary:

In the acute immobilization test to Daphnia magna (STRAUS) the effects of the saturated solution* of the test item Nitro-C-Dion (NMQD) (batch number: 01/05) with a nominal loading of 100 mg/L were determined according to OECD 202 (2004). The limit test was conducted under static conditions over a period of 48 h from 2011-08-02 to 2011-08-04 at Dr. U. Noack-Laboratorien, Käthe-Paulus-Str. 1, D-31157 Sarstedt, .

Twenty daphnids were exposed to the saturated solution and the control. The saturated solution and the control were analysed by HPLC-DAD after 0 h (fresh media) and 48 h (old media).

The initially measured concentration (fresh media, 0 h) of the test item in the freshly prepared saturated solution was 2.20 mg/L, representing the maximum solubility of the test item under test conditions. The recovery rate of the test item in the old media (48 h) was 100 % of the initially measured concentration, demonstrating that concentrations of Nitro-C-Dion (NMQD) were successfully maintained for the duration of the test. All effect values given are based on the initially measured test item concentration (Table 1).

Water quality parameters (pH-value and dissolved oxygen concentration), measured in the fresh (0 h) an old media (48 h), were determined to be within the acceptable limits. The validity criteria of the test guideline were fulfilled.

EC-Values, NOEC and LOEC

(based on the initially measured test item concentration)

	Test Duration [h]	Nitro-C-Dion (NMQD) Initially Measured Test Item Concentration [mg/L]	Confidence Interval p = 95% [mg/L]
EC10 / EC50 / EC100	24	> 2.20	not applicable
	48	> 2.20	not applicable
NOEC	48	2.20	---
LOEC	48	>2.20	---

At the saturated* solution of the test item Nitro-C-Dion (NMQD),

corresponding to the initially measured concentration of 2.20 mg/L,

no effect was observed.