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

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:

2011-09-13 to 2011-09-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: well performed OECD study with 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:

6-amino-7-methoxy-1,2,3,4-tetrahydroquinoxaline-2,3-dione

EC Number:

615-027-9

Cas Number:

69904-10-5

Molecular formula:

C9H9N3O3

IUPAC Name:

6-amino-7-methoxy-1,2,3,4-tetrahydroquinoxaline-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): Amino-C-Dion trocken
- Molecular formula: C9H9N3O3
- Molecular weight: 207.19 g/mol
- Physical state: Solid, yellow powder
- Expiration date of the lot/batch: 2013-05-04
- Stability under test conditions: Not applicable
- Storage condition of test material: Room temperature, protected from light, in original container

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentrations of the test item were analytically determined from all test concentrations and the control after 0 hours (test start) and 72 hours (test end).
- Sampling method: Samples at test start were taken from an additional replicate without algae. At end of the test samples were taken directly from the pooled test replicates.
- Sample storage conditions before analysis: All original samples were stored at 6 ± 2 °C until sample preparation, if necessary. Prepared samples were stored at room temperature until analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A stock solution of 160 mg/L was freshly prepared with dilution water. The stock solution was treated with ultrasound for 15 min and stirred with approximately 1100 rpm for 1 h at room temperature. Thereafter the stock solution was treated with ultrasound for 15 min again.
- Eluate: Dilution water
- Differential loading: 10.0 - 20.0 - 40.0 - 80.0 - 160 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 from Z-Agar. Light intensity amounted 35 - 70 µE x m-2 x s-1 for 24 h per day.

ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test
- Any deformed or abnormal cells observed: No

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

Not measured

Test temperature:

Mean: 21.5 °C, Min: 21 °C, Max: 22 °C

pH:

Nominal test item concentration pH-value
[mg/L] Start; 0 hours End; 72 hours
160 7.65 7.79
80.0 7.64 7.86
40.0 7.69 8.02
20.0 7.72 8.11
10.0 7.71 8.20
Control 7.93 8.30

Dissolved oxygen:

Not measured

Salinity:

Not measured, freshwater

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
- 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: 4910 cells/mL
- Control end cells density: Mean 992333 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): None

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

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [counting chamber; electronic particle counter; fluorimeter; spectrophotometer; colorimeter] Fluorimeter, via Chlorophyll-a fluorescence (excitation at 436 nm, emission at 685 nm), cell concentrations were related to all cell density values acc. to a calibration curve.

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 33 80
10 - 1 - 5
1 3 12
0.1 2 11
0.01 3 13

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

89.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: (81.2 - 98.9)

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

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
Rate-related inhibition: 0.488 (CI 0.466 - 0.512) mg/L
Yield Inhibition: 0.291 (CI 0.274 - 0.328) mg/L

Reported statistics and error estimates:

EC10-, EC20- and EC50-values of the growth rate and yield inhibition after 72 hours were calculated by sigmoidal dose-response regression. Calculation of the confidence intervals. EC10-, EC20- and EC50-values were carried out using standard procedures.

Any other information on results incl. tables

NOEC, LOEC, EC - values and 95 % Confidence Intervals of Amino-C-Dion trocken

                   (0-72 hours)

                   based on nominal test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	20.0
LOEC	40.0
ErC10	23.8 (19.8 – 28.5)
ErC20	37.9 (33.0 – 42.6)
ErC50	89.4 (81.2 – 98.9)
	Inhibition of Yield
NOEC	10.0
LOEC	20.0
EyC10	12.8 ( 10.0 – 16.9)
EyC20	17.5 (14.0 – 21.4)
EyC50	29.5 (25.5 – 33.9)

Cell Densities

	Nominal test item concentration		Replicate		Cell density [cells/mL]
[mg/L]		No.		0 hours		24 hours	48 hours	72 hours
160		1		4910		7736	10803	16419
		2		4910		9872	11923	21364
		3		4910		9181	12539	19079
		Mean		4910		8930	11755	18954
80.0		1		4910		14780	31966	90286
		2		4910		18013	41400	96416
		3		4910		17590	38614	88353
		Mean		4910		16794	37327	91685
40.0		1		4910		15609	59144	223371
		2		4910		17184	87707	389723
		3		4910		22038	87439	296500
		Mean		4910		18277	78097	303198
20.0		1		4910		16544	91840	610706
		2		4910		19042	112224	790517
		3		4910		21168	114008	812926
		Mean		4910		18918	106024	738050
10.0		1		4910		22901	110931	830460
		2		4910		20860	150630	1050925
		3		4910		21879	119810	919046
		Mean		4910		21880	127124	933477
Control		1		4910		19181	138708	959696
		2		4910		21279	143112	974522
		3		4910		27914	147797	991718
		4		4910		19898	134569	984610
		5		4910		24224	163883	1073635
		6		4910		21530	132155	969817
		Mean		4910		22338	143371	992333

Evaluation after 72 hours

                   Statistically significant differences of growth rates and yield compared to

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

Nominal test item concentration	Replicate	Growth rate		Rate-related inhibition	Yield		Inhibition of yield
[mg/L]	No.	[d^-1]		[%]	[cells/mL]		[%]
160	1		0.402	77.3		11509	98.8
	2		0.490	72.3		16454	98.3
	3		0.452	74.4		14169	98.6
	Mean	(+)	0.448	74.7	(+)	14044	98.6
80.0	1		0.971	45.2		85376	91.4
	2		0.992	43.9		91506	90.7
	3		0.963	45.6		83443	91.6
	Mean	(+)	0.975	44.9	(+)	86775	91.2
40.0	1		1.27	28.1		218461	77.9
	2		1.46	17.6		384813	61.0
	3		1.37	22.7		291590	70.5
	Mean	(+)	1.37	22.8	(+)	298288	69.8
20.0	1		1.61	9.13		605796	38.7
	2		1.69	4.27		785607	20.4
	3		1.70	3.74		808016	18.2
	Mean	(+)*	1.67	5.71	(+)	733140	25.8
10.0	1		1.71	3.34		825550	16.4
	2		1.79	-1.09		1046015	-5.93
	3		1.74	1.43		914136	7.42
	Mean	(-)	1.75	1.23	(-)	928567	5.96
Control	1		1.76			954786
	2		1.76			969612
	3		1.77			986808
	4		1.77			979700
	5		1.80			1068725
	6		1.76			964907
	Mean		1.77			987423

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	1.36	1.98	1.93	1.76	0.344	19.5	13.9
	2	1.47	1.91	1.92	1.76	0.257	14.6
	3	1.74	1.67	1.90	1.77	0.122	6.87
	4	1.40	1.91	1.99	1.77	0.321	18.2
	5	1.60	1.91	1.88	1.80	0.174	9.68
	6	1.48	1.82	1.99	1.76	0.262	14.8
				Mean	1.77
				SD ±	0.01
				CV [%]	0.76

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study Amino-C-Dion trocken was found to inhibit the growth of the freshwater green alga Desmodesmus subspicatus after 72 hours. The NOEC-values for inhibition of specific growth rate and inhibition of yield after 72 hours were 20.0 mg/L and 10.0 mg/L, respectively. The LOEC-values for inhibition of specific growth rate and inhibition of yield after 72 hours were 40.0 mg/L and 20.0 mg/L, respectively. The EC50-values for inhibition of specific growth rate (ErC50) and yield (EyC50) with 95 % Confidence Intervals after 72 hours were 89.4 (81.2 – 98.9) and 29.5 (25.5 – 33.9) mg/L, respectively.
All effect levels are given based on nominal concentrations of Amino-C-Dion trocken.

Executive summary:

The toxicity of Amino-C-Dion trocken (batch number: 241)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 September 13 to 16, 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 test 5 concentration levels were tested in a geometrical series with a dilution factor of 2, nominal: 10.0 - 20.0 - 40.0 - 80.0 - 160 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. Environmental conditions were determined to be within the acceptable limits.

The concentrations of Amino-C-Dion trocken were analytically verified by HPLC-DAD at test start and test end of all test concentrations and the control. Details of the analytical method are presented in part 9. The analytical results are presented in Table 8.
The measured concentrations of Amino-C-Dion trocken at test start were in the range of 98 to 106 % and after 72 hours in the range of 80 to 106 % of the nominal values. Therefore, all effect values given are based on the nominal concentrations of the test item Amino-C-Dion trocken.

NOEC, LOEC, EC - values and 95 % Confidence Intervals of Amino-C-Dion trocken

                   (0-72 hours)

                   based on nominal test item concentrations [mg/L]

                               

	Rate-related Inhibition
NOEC	20.0
LOEC	40.0
ErC10	23.8 (19.8 – 28.5)
ErC20	37.9 (33.0 – 42.6)
ErC50	89.4 (81.2 – 98.9)
	Inhibition of Yield
NOEC	10.0
LOEC	20.0
EyC10	12.8 ( 10.0 – 16.9)
EyC20	17.5 (14.0 – 21.4)
EyC50	29.5 (25.5 – 33.9)