Reaction mass of isomers 4-[[2-[(Aminocarbonyl)amino]-4-[(2,6-difluoro-4-pyrimidyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt and 4-[[2-[(Aminocarbonyl)amino]-4-[(4,6-difluoro-2-pyrimidyl)-amino]phenyl]azo]-1,3-benzenedisulfonic acid, sodium salt

Toxicity to aquatic plants other than algae

Administrative data

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-04-22 to 2013-05-02

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

not specified

IUPAC Name:

not specified

Details on test material:

- Name of test material (as cited in study report): Reactive Golden Yellow HF-RN 1331
- Molecular formula: C17H11F2Na2O7S2 (as sodium salt) C17H13F2N7O7S2 (as free acid)
- Physical state: Solid, red to orange powder
- Analytical purity: 100 %
- Purity test date: 2012-10-08
- Lot/batch No.: Kilo 10 & 12
- Expiration date of the lot/batch: 2014-01-17
- Stability under test conditions: Not specified
- Storage condition of test material: Room temperature, protected from light, in original container; since 2013-03-01 additionally under nitrogen

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Limit test item concentration of 100 mg/L
- Sampling method: The limit concentration of the test item Reactive Golden Yellow HF-RN 1331 and the control were analytically verified via HPLC-DAD at the start and end of the exposure. Freshly prepared and old media were analysed. For analysis of the old media the samples were taken directly from the test vessels.
- Sample storage conditions before analysis: All original samples were stored at 6 ± 2 °C until sample preparation. Prepared samples were stored at room temperature until analysis.

Test solutions

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 on test start and agitated.
- Eluate: Dilution water
- Differential loading: None, limit test
- Controls: 6 replicates (without test item); growth conditions as for the replicates treated with test item

Test organisms

Test organisms (species):

Lemna minor

Details on test organisms:

TEST ORGANISM
- Common name: Duckweed, Lemna minor
- Source (laboratory, culture collection): Federal Environmental Agency (UBA), Schichauweg 58, 12307 Berlin, Germany
- Age of inoculum (at test initiation): ca. 7 days
- Method of cultivation:
The species is bred at the test facility. Density is kept low to prevent conglomerates of plants on the surface. At least once per week, plants are transferred to freshly prepared growth medium. Growth media and breeding vessels are autoclaved before use to enable the breeding of axenic cultures. Culture medium is Swedish Standard Medium (SIS)


ACCLIMATION
- Acclimation period: at least 7 days
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: None observed

Study design

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

7 d

Test conditions

Test temperature:

Min: 22 °C, MAx: 24 °C

pH:

Nominal
test item concentration
[mg/L] pH-value
0 days
(Fresh medium) 7 days
(Old medium)
100 6.57 6.86
Control 6.53 6.96

Nominal and measured concentrations:

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

Details on test conditions:

TEST SYSTEM
- Incubation chamber used: no
- Test vessel:
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: Crystallisation dish, 500 mL capacity, covered with glass lids and filled with 200 mL test solution.
- Type of cover: glass lids
- Aeration: no active aeration
- Agitation: no
- Renewal rate of test solution (frequency/flow rate): static test
- Control end frond number: 171
- No. of colonies per vessel: 4
- No. of fronds per colony: 3
- No. of vessels per concentration (replicates): 6
- No. of vessels per control (replicates): 6
- No. of vessels per vehicle control (replicates): not necessary


GROWTH MEDIUM
- Standard medium used: yes






TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water:
Swedish Standard (SIS) Medium according to the guideline
Composition of Dilution Water

Component Concentration [mg/L]
NaNO3 85
KH2PO4 13.4
MgSO4  7 H2O 75
CaCl2  2 H2O 36
Na2CO3 20
H3BO3 1.0
MnCl2  4 H2O 0.2
NaMoO4  2 H2O 0.01
ZnSO4  7 H2O 0.05
CuSO4  5 H2O 0.005
Co(No3)2  6 H2O 0.01
FeCl3  6 H2O 0.85*
Na2-EDTA  2 H2O 1.40
MOPS (buffer) 490
pH-value 6.5  0.2


OTHER TEST CONDITIONS
- Sterile test conditions: no
- Adjustment of pH: no
- Photoperiod: Continuous fluorescent light, 6500 - 10000 lx (corresponding to 85 - 135 µE•m-2•s-1) on the surface of the test medium. Difference of light intensity at any measured incubation place was less than 15 % from the mean value.
- Light intensity and quality:
Light intensity [µEm-2s-1]: (n = 24) 103 CV: 6.23 %


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Biological data:
The amounts of plants were determined on start and end of the test and every 2 - 3 d. Every frond that were visibly projected beyond the edge of a parent frond were counted as a separate frond. Fronds that loose their pigmentation were not counted.

Observations of change in colour, break-up of plants and destructions of roots were made on every determination day and end of the test.

After 7 d determination of dry weight was carried out. Colonies from each test vessel were collected, rinsed with deionised water and then dried at 60 °C to a constant weight. Any root fragments were included. The starting biomass dry weight was determined based on a sample of colonies (same number of fronds as in the test vessels) taken from the same batch used to inoculate the test vessels.

Physicochemical data:
pH-values were measured on start and end of the test.
The room temperature was measured and recorded continuously by a thermohygrograph.
Light intensity was determined before start of the test.



RANGE-FINDING STUDY
Results of the static Range Finding Test after 7 days
(mean values, based on nominal concentrations, 2 replicates per dosage,4 control replicates)

Nominal Test Concentration Specific Growth Rate Inhibition Yield Inhibition

[mg/L] [%] [%]
100 -3 -7
10 9 19
1 1 2
0.1 2 5

Reference substance (positive control):

yes

Remarks:

3,5-Dichlorophenol

Results and discussion

Effect concentrations

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Details on results:

- Any visual signs of phytotoxicity (abnormalities):
Please refer to the respective table

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- Relevant effect levels:
EC50-Values of the Reference Item
based on the nominal concentrations [mg/L], (0-7d)
Current Study Valid Range (average ± 3 x SD)
Growth rate inhibition (frond number)
ErC50 2.66 2.24 ± 0.908
95% confidence interval 2.60 - 2.71
Yield inhibition (frond number)
EyC50 2.14 1.88 ± 0.711
95% confidence interval 2.06 - 2.23
Growth rate inhibition (dry weight)
ErdwC50 2.33 -
95% confidence interval 2.07 - 2.61
Yield inhibition (dry weight)
EydwC50 1.64 -
95% confidence interval 1.31 - 2.03

Reported statistics and error estimates:

NOEC/LOEC were determined by calculation of statistical
significance of inhibition of yield and growth rates. A t-test was used for NOEC/LOEC calculations. A normality test and an equal variance test were done first. The KOLGOMOROV-SMIRNOV-Test was used to test for normally distributed populations. P-values for both Normality and Equal Variance test are 0.05. The a-value (acceptable probability of incorrectly concluding that there was a difference) was a=0.05.
EC10-, EC20- and EC50-values of the growth rate, yield inhibition and dry weight reduction and corresponding confidence intervals after 7 d were calculated by sigmoidal dose-response regression if applicable. Calculation of the confidence intervals for EC-values were carried out using standard procedures.

Any other information on results incl. tables

Frond Numbers

Nominal test item concentration [mg/L]	Repl. No.	Frond number
		0 days*	2 days	4 days	7 days
100	1	12	21	55	149
	2	12	22	56	178
	3	12	24	56	191
	4	12	24	56	181
	5	12	22	53	151
	6	12	24	62	186
	Mean	12	23	56	173
Control	1	12	21	49	154
	2	12	20	59	177
	3	12	20	46	179
	4	12	23	51	168
	5	12	25	51	176
	6	12	21	48	171
	Mean	12	22	51	171

Evaluation of Frond Numbers after 7 days

                                Biologically and statistically significant differences of specific growth rates and yield

                                compared to control values are marked (+) and not significant differences are

                                marked (-).

                                               

Nominal test item concentration [mg/L]	Repl. No.	Average specific growth rate [d^-1]		Inhibition of average specific growth rate [%]	Yield [fronds]		Inhibition of yield [%]	Doubling time [d]
100	1		0.360	5		137	14	1.93
	2		0.385	-2		166	-5	1.80
	3		0.395	-4		179	-13	1.75
	4		0.388	-2		169	-6	1.79
	5		0.362	5		139	12	1.92
	6		0.392	-3		174	-10	1.77
	Mean	(-)	0.380	0	(-)	161	-1	1.83
Control	1		0.365			142		1.90
	2		0.384			165		1.80
	3		0.386			167		1.80
	4		0.377			156		1.84
	5		0.384			164		1.81
	6		0.380			159		1.83
	Mean		0.379			159		1.83

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

At the limit concentration of the test item Reactive Golden Yellow HF-RN 1331 with a nominal loading of 100 mg/L, an inhibiting effect on growth of dry weight of Lemna minor could be observed. The inhibition of growth rate of dry weight is considered as not biological significant as being below 10 %. The inhibition of yield of dry weight is as well statistical as biological relevant but is the only effect that could be observed.
For further values (NOEC/LOEC) of the test item Reactive Golden Yellow HF-RN 1331 please refer to Table 1. All effect values are given based on nominal test item concentrations.

Executive summary:

The effects of test item Reactive Golden Yellow HF-RN 1331 (batch no.: Kilo10&12) on the growth of the monocotyledon species Lemna minor was determined according to the principles of OECD guideline 221 at Dr.U.Noack-Laboratorien in 31157 Sarstedt, Germany from 2013-04-22 to 2013-05-02 with the definitive exposure phase from 2013-04-22 to 2013-04-29.

A static exposure over 7 days was carried out. Based on a preliminary test a limit concentration of 100 mg/L was tested. Six replicates were investigated for the limit concentration and for the control. Frond numbers were assessed on days 0, 2, 4 and 7. Environmental parameters (light intensity, room temperature and pH-value) were within the acceptable limits. The validity criteria of the test guideline were fulfilled.

The concentrations of the test item Reactive Golden Yellow HF-RN 1331 of the limit concentration and control were analytically verfied via HPLC-DAD at the start (0 d) and end (7 d) of the exposure. The measured concentration of the test item at the start of the exposure was 104 % of the nominal value. The measured concentration at the end of the exposure was 102 % of the nominal value.

All effect values are given based on nominal test item concentrations (Table 1).

Table 1:    NOEC-, LOEC- and EC-Values of Reactive Golden Yellow HF-RN 1331after 7 d

based on nominal test item concentrations [mg/L]

	Frond number		Dry weight
Growth rate Inhibition [mg/L]
NOEC	100	NOEC	100
LOEC	> 100	LOEC	100
ErC10 ErC20 ErC50	> 100	ErdwC10 ErdwC20 ErdwC50	> 100
Inhibition of Yield [mg/L]
NOEC	100	NOEC	100
LOEC	> 100	LOEC	100
EyC10 EyC20 EyC50	> 100	EydwC10 EydwC20 EydwC50	100
			> 100
			> 100