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

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:

2011-07-01 to 2011-07-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed 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 201 (Alga, Growth Inhibition 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

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 limit test item concentration and control were analytically verified via HPLC-DAD after 0 hours (test start) and 72 hours (test end).
- Sampling method: Samples at test start were taken from additional replicates without algae. After 72 hours samples were taken directly from pooled test replicates.
- 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 of 100 mg/L was freshly prepared with dilution water. 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 corresponding to an initially measured test item concentration of 2.12 mg/L
- Controls: Six replicates (without test item) were tested under the same test conditions as the test replicates.

Test organisms

Test organisms (species):

Desmodesmus subspicatus (previous name: Scenedesmus subspicatus)

Details on test organisms:

TEST ORGANISM
- Common name: Desmodesmus subspicatus Chodat
- Strain: SAG 86.81
- Source (laboratory, culture collection): SAG Pflanzenphysiologisches Institut der Universitaet Goettingen, Nikolausberger Weg 18, D-37073 Goettingen, Germany
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: Fresh stocks were prepared every month on Z-Agar. Light intensity amounted to 35 - 70 µE x m-2 x s-1 for 24 h per day.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

72 h

Post exposure observation period:

No

Test conditions

Hardness:

Dilution water: 0.24 mmol Ca+mg/L.

Test temperature:

min.: 22, max.: 23, mean value: 22.5

pH:

Initially measured test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
2.12 7.87 8.38
Control 7.96 8.27

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater conditions

Nominal and measured concentrations:

Please refer to "Any other information on materials and methods incl. tables"

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL Erlenmeyer flasks
- Type (delete if not applicable): open, cotton wool plugs
- Material, size, headspace, fill volume: sterile 250 mL Erlenmeyer flasks, test volume 100 mL
- Aeration: Test containers were placed on a rotary shaker and oscillated at appr. 70 rpm
- Type of flow-through (e.g. peristaltic or proportional diluter): None
- Renewal rate of test solution (frequency/flow rate): One application at test start
- Initial cells density: Nominal: approximately 2 - 5 x 103 cells/mL, Actual: mean 5248 cells/mL
- Control end cells density: Mean 834888 cells/mL
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6



GROWTH MEDIUM
- Standard medium used: yes



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water, composition acc. to the guideline
- Hardness: 0.24 mmol Ca+Mg/L
- pH-value: 8.1 +/- 0.2
- Culture medium: Nutrient Medium Z acc. to LÜTTGE et al. (1994)


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: 24 h
- Light intensity and quality: 60 - 120 µE x m-2 x s-1, mean value: 69.2



EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] fluorimeter, Cell density and self fluorescence was measured daily via Chlorophyll-a- fluorescence, excitation at 436 nm, emission at 685 nm. Dilution water was used as background signal.



TEST CONCENTRATIONS
- Range finding study: Yes
Results of the Range Finding Test (0-72 h)
Results of the Range Finding Test (0-72 hours)

Nominal
test item concentration [mg/L] Specific Growth Rate Inhibition [%] Yield Inhibition [%]
100 1 6
10 - 1 - 8
1 - 1 - 4
0.1 - 1 - 4

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

2.12 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test):
- Unusual cell shape: No
- Colour differences: No
- Flocculation: No
- Adherence to test vessels: No
- Aggregation of algal cells: No
- Other: No
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

- Results with reference substance valid? Yes
EC50 Values of the Reference Item
based on nominal concentrations (mg/L), (0-72 hours)

Current Study Valid Range (average ± 3 x SD)
Rate-related inhibition
ErC50 0.560 0.720 ± 0.445
95 % confidence interval 0.531 – 0.593
Yield inhibition
EyC50 0.260 0.321 ± 0.152
95 % confidence interval 0.234 – 0.289

Reported statistics and error estimates:

NOEC/LOEC was determined by calculation of statistical
significance of growth rates and yield. As standard One Way Analysis of Variance (ANOVA) and DUNNETT’s Test were used for NOEC/LOEC calculations. When running a One Way Analysis of Variance a Normality test and an Equal Variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. The Levene median test was used for equal variance. P-values for both Normality and Equal Variance test were 0.05. The a-value (acceptable probability of incorrectly concluding that there is a difference) is a=0.05.
EC-values of the growth rate and yield inhibition were estimated empirically based on the results of the only treatment level (Limit test design).

Any other information on results incl. tables

NOEC, LOEC, EC - values of Nitro-C-Dion (NMQD) (0-72 hours)

                   based on initially measured test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	2.12
LOEC	> 2.12
ErC10	> 2.12
ErC20	> 2.12
ErC50	> 2.12
	Inhibition of Yield
NOEC	2.12
LOEC	> 2.12
EyC10	> 2.12
EyC20	> 2.12
EyC50	> 2.12

Cell Densities

	Initially measured test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours	48 hours	72 hours
2.12		1		5248		20931	141758	925927
		2		5248		26712	151636	944477
		3		5248		22315	185757	1018981
		4		5248		21899	176488	1019116
		5		5248		23341	187195	1054050
		6		5248		27928	187659	1147036
		Mean		5248		23854	171749	1018265
Control		1		5248		27782	148037	797839
		2		5248		18507	139226	860292
		3		5248		23270	131573	820688
		4		5248		23618	138441	844518
		5		5248		22765	141856	850340
		6		5248		21029	155847	835649
		Mean		5248		22829	142497	834888

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

                   control values are marked (+), not significant differences are marked (-).

Initially measured test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
2.12	1		1.72	-2.05		920679	-11.0
	2		1.73	-2.44		939229	-13.2
	3		1.76	-3.94		1013733	-22.2
	4		1.76	-3.94		1013868	-22.2
	5		1.77	-4.60		1048802	-26.4
	6		1.80	-6.27		1141788	-37.6
	Mean	(+)*	1.76	-3.87	(+)*	1013017	-22.1
Control	1		1.68			792591
	2		1.70			855044
	3		1.68			815440
	4		1.69			839270
	5		1.70			845092
	6		1.69			830401
	Mean		1.69			829640

Section-by-Section and Average Specific Growth Rates of the Control Group
                   (0 – 72 hours)

	Replicate No.	Specific growth rate [d^-1]			Mean (0 - 72 h)	SD ±	CV [%]	Mean CV [%]
		section-by-section
		0 - 24 hours	24 - 48 hours	48 - 72 hours
Control	1	1.67	1.67	1.68	1.68	0.009	0.541	12.3
	2	1.26	2.02	1.82	1.70	0.393	23.1
	3	1.49	1.73	1.83	1.68	0.176	10.4
	4	1.50	1.77	1.81	1.69	0.165	9.76
	5	1.47	1.83	1.79	1.70	0.199	11.7
	6	1.39	2.00	1.68	1.69	0.308	18.2
				Mean	1.69
				SD ±	0.01
				CV [%]	0.54

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study no inhibition on growth of the freshwater green alga Desmodesmus subspicatus was found after 72 hours exposure to the test item Nitro-C-Dion (NMQD). The NOEC for inhibition of specific growth rate and yield after 72 hours was 2.12 mg/L. Nitro-C-Dion (NMQD) caused a slight stimulation of alga growth.
All effect levels are given based on the initially measured test item concentration of Nitro-C-Dion (NMQD) which has been analytically verified by HPLC-DAD.

Executive summary:

The toxicity of Nitro-C-Dion (NMQD) (batch number 01/05)to the unicellular freshwater green alga Desmodesmus subspicatus was determined according to the principles of OECD 201 at Dr.U.Noack-Laboratorienin 31157 Sarstedt, Germany from July 01 to 05, 2011 with the definitive exposure phase from July 02 to 05, 2011. The aim of the study was to assess the effects on growth rate and yield over a period of 72 hours.

The study was conducted under static conditions with an initial cell density of 5 x 10³ cells/mL. Based on a preliminary range finding the saturated solution was tested. Six replicates were tested for the limit concentration and the control. Environmental conditions were determined to be within the acceptable limits.

The saturated solution* and the control were analysed by HPLC-DAD after 0 h (fresh media) and 72 h (old media).

The initially measured concentration (fresh media, 0 h) of the test item in the freshly prepared saturated solution* was 2.12 mg/L, representing the maximum solubility of the test item under test conditions. The recovery rate of the test item in the old media (72 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

NOEC, LOEC, EC - values of Nitro-C-Dion (NMQD) (0-72 hours)

                   based on initially measured test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	2.12
LOEC	> 2.12
ErC10	> 2.12
ErC20	> 2.12
ErC50	> 2.12
	Inhibition of Yield
NOEC	2.12
LOEC	> 2.12
EyC10	> 2.12
EyC20	> 2.12
EyC50	> 2.12