Trisodium 4-[[4-[[(2,3-dichloro-6-quinoxalinyl)carbonyl]amino]-2-sulphonatophenyl]azo]-4,5-dihydro-5-oxo-1-(4-sulphonatophenyl)-1H-pyrazole-3-carboxylate

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:

from 2017-02-14 to 2017-02-23, with the definitive exposure phase from 2017-02-15 to 2017-02-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

p-[4,5-dihydro-4-[[2-methoxy-5-methyl-4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulphonic acid, sodium salt

EC Number:

263-417-9

EC Name:

p-[4,5-dihydro-4-[[2-methoxy-5-methyl-4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-3-methyl-5-oxo-1H-pyrazol-1-yl]benzenesulphonic acid, sodium salt

Cas Number:

62121-75-9

Molecular formula:

C20H22N4O11S3.xNa C20H(22-x)N4NaxO11S3; x<=2

IUPAC Name:

sodium 4-[4-(2-{2-methoxy-5-methyl-4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}diazen-1-yl)-3-methyl-5-oxo-4,5-dihydro-1H-pyrazol-1-yl]benzene-1-sulfonate

Test material form:

solid: particulate/powder

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Determination of the test item
All test item concentrations and the control were analytically verified via HPLC-DAD at the start (0 day, fresh medium) and at the end of the exposure (7 days, old medium).

Test solutions

Vehicle:

no

Details on test solutions:

Test item
Reactive Yellow 15

Preparation of the Test item solution
A test item solution of 100 mg test item/L was prepared once 24 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out. The test item was applied onto a glass slide. The glass slide with the test item was inserted into a glass bottle with an appropriate amount of dilution water. The test item solution was stirred for 24 ± 1 hours (1100 rpm, room temperature) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the test item solution, was used as a test item solution in the test. During filtration, the filter was always be kept covered.
The test item solution was checked via laser beam (Tyndall effect) for undissolved test item, which was slightly positive.

Test concentrations
Based on the results of a preliminary range finding test. 5 concentrations were tested with a dilution factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution.

Control
Six replicates (without test item) were tested under the same test conditions as the test vessels.

Test organisms

Test organisms (species):

Lemna gibba

Details on test organisms:

Test organism
Duckweed, Lemna gibba, Lemnaceae, Arales, Arecidae, Monocotyledonae
Young, rapidly growing plants without visible lesions or discolouration (chlorosis) were used for the test.

Reason for the selection of the test organism
According to the guideline, Lemna gibba is a suitable species because it is a representative of temperate areas commonly used for toxicity tests.

Origin
EUROFINS-GAB GMBH, Eutinger Str. 24, 75223 Niefern-Öschelbronn, Germany

Date of receipt
2008-02-26

Cultivation at test facility
The species is cultured in 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 culturing vessels are autoclaved before use to enable the breeding of axenic cultures.

Breeding vessels
Crystallisation dishes of glass, vol. 900 mL, filled with ca. 500 mL growth medium, covered with glass tops

Medium
20X-AAP-medium (Algal Assay Procedure medium),
pH-value 7.5 ± 0.1, see dilution water

Temperature 24 ± 2 °C

Light regime
Continuous fluorescent light, 1100 – 4440 lux

Acclimatization of the test system
The test system (the test organism) was held for 7 days under test conditions to acclimatize. These acclimatized plants were used in the test.

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test conditions

Hardness:

not measured

Test temperature:

see any other information on materials and methods

pH:

see any other information on materials and methods

Dissolved oxygen:

not measured

Conductivity:

not measured

Nominal and measured concentrations:

Nominal test item concentrations: 6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution

Details on test conditions:

Test method
Static procedure

Test duration
7 days

Replicates
3 replicates per concentration level, 6 for the control

Test vessels/test volumes
Crystallisation dishes with a volume of 500 mL, covered with glass tops and filled with 200 mL test solution were used in the test. The test vessels were placed on a black non-reflective surface to avoid stray light.
 
Dilution water
20X-AAP-medium according to the guideline.

Composition of Dilution water
Component Concentration in stock solution [g/L] Concentration in prepared medium [mg/L]
NaNO3 26 510
MgCl2  6 H2O 12 240
CaCl2  2 H2O 4.4 90
MgSO4  7 H2O 15 290
K2HPO4 · 3 H2O 1.4 30
NaHCO3 15 300
H3BO3 0.19 3.7
MnCl2  4 H2O 0.42 8.3
FeCl3  6 H2O 0.16 3.2
Na2-EDTA · 2 H2O 0.30 6.0
ZnCl2 3.3 mg/L 66 µg/L
CoCl2  6 H2O 1.4 mg/L 29 µg/L
Na2MoO4  2 H2O 7.3 mg/L 145 µg/L
CuCl2  2 H2O 0.012 mg/L 0.24 µg/L
pH-value 7.5 ± 0.1
The pH of the test medium had to be 7.5  0.1 and was adjusted prior to testing with the addition of 1 N NaOH and HCl.

Application
Static with application of the test item at test start. At the start of the exposure, 4 uniform, healthy plants (colonies of 3 fronds each), were introduced into each test vessel containing the test media. The initial frond number per test vessel was 12. The initial numbers of colonies and fronds were the same in each test vessel.

Temperature (Target)
24 ± 2 °C

Light regime (Target)
Continuous, fluorescent light, 6500 to 10000 lux on the surface of the test medium (difference of light intensity at any measured incubation place < 15 % from the mean value)

Placement of the test vessels
A randomised placement of the test vessels was carried out.

Type and frequency of measurements
The numbers of plants and fronds were determined at the start and the end of the exposure. The number of fronds was determined every 2 - 3 days from each replicate of the control and the test concentrations. Every frond that visibly projected beyond the edge of a parent frond was counted as a separate frond. Fronds that lost their pigmentation were not counted.
Observations of frond size, appearance, indication of necrosis, chlorosis or gibbosity, colony break-up or loss of buoyancy, of root length and appearance, as well as of change in colour and destruction of roots, were made on every determination day and at the end of the exposure.
After 7 days, the determination of dry weight was carried out from 3 replicates per test concentration and 6 control replicates. 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 dry weight was expressed to an accuracy of
0.1 mg.
The dry weight of the starting biomass was determined based on a sample of fronds (same number of fronds as in the test vessels) taken from the same batch used to inoculate the test vessels.

Physico-chemical Parameters
The pH-values were measured in the freshly prepared solutions before distribution into the replicates. The pH-values of the aged solution were measured from pooled replicates per concentration and control. The temperature of the medium in a surrogate vessel held under the same conditions in the growth room was recorded daily. The light intensity was measured prior to the start of the exposure at positions which had the same distance from the light source as the Lemna fronds.

Reference substance (positive control):

yes

Remarks:

3,5-dichlorophenol

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 other: % of the saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: frond number growth rate

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % of the saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: frond number growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

6.25 other: % of the saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Frond number yield

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % of the saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: frond number yield

Effect concentrations 5

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 other: % of the saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dry weight growth rate

Effect concentrations 6

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % of the saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dry weight growth rate

Effect concentrations 7

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

12.5 other: % of the saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dry weight yield

Effect concentrations 8

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: % of the saturated solution

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dry weight yield

Details on results:

The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits.

Results with reference substance (positive control):

The acute toxicity of 3,5-Dichlorophenol (SIGMA, batch number SZBD168XV, purity 99.4%, CAS RN 591-35-5) to the monocotyledonous aquatic plant Lemna gibba was determined over a period of 7 days from 2016-10-12 to 2016-10-19 according to OECD Guideline 221. The plants used in the reference test were taken from the same laboratory culture as was used to determine the effects of the test item.

EC50-Values of the Reference Item based on the nominal concentrations [mg/L], (0-7 days)
Current Study Valid Range (average ± 3 x SD)
Growth rate inhibition (number of fronds)
ErC50 6.71 5.56 ± 2.83
95% confidence interval 5.42 – 8.00
Yield inhibition (number of fronds)
EyC50 4.50 4.59 ± 3.03
95% confidence interval 4.04 – 5.05
Growth rate inhibition (dry weight)
ErdwC50 6.76 5.50 ± 2.85
95% confidence interval 5.29 – 7.43
Yield inhibition (dry weight)
EydwC50 5.53 4.63 ± 2.47
95% confidence interval 4.56 – 6.50
SD = standard deviation

The observed responses to the reference item were within the valid range, confirming the normal sensitivity of the test system used in the study with the test item.

Reported statistics and error estimates:

Sample size for statistics
For the determination of NOEC, LOEC and EC-values, three replicates were included for the test concentrations and six replicates for the control.

NOEC and LOEC values
NOEC/LOEC was determined by calculation of the statistical significance of inhibition of growth rates and yield in comparison to the control: One Way Analysis of Variance (ANOVA) and DUNNETT’s test were used as a standard. A normality test and an equal variance test were done first. The SHAPIRO-WILK-Test was used to test for normally distributed populations. P-values for both normality and equal variance test were 0.05. The -value (acceptable probability of incorrectly concluding that there is a difference) was =0.05.

EC-values and statistical analyses
EC10-, EC20- and EC50-values (0 - 7 d) of the growth rate and yield (frond number and dry weight) inhibition were calculated by sigmoidal dose-response regression. Calculations of the confidence intervals of EC10-, EC20- and EC50-values were carried out from the best fit values, the standard error and the t-distribution with the software GraphPad Prism.

Software
The data for the tables in this report were computer-generated and rounded for presentation from the fully derived data. Consequently, if calculated manually based on the given data, minor deviations may occur from these figures.
Calculations were carried out using the following software:
- Excel, MICROSOFT CORPORATION
- SigmaPlot, SPSS INC.
- GraphPad Prism, GRAPHPAD SOFTWARE, INC.
- ToxRat Version 3.2.1, ToxRat Solutions GmbH

Any other information on results incl. tables

Frond Numbers

Nominal test item concentration [% of the saturated solution]	Geometric mean measured test item concentration [mg/L]	Repl. No.	Frond numbers per study day
			0 days*	2 days	5 days	7 days
100	15.7	1	12	19	32	50
		2	12	20	34	54
		3	12	19	34	46
		Mean	12	19	33	50
50.0	5.04	1	12	18	37	61
		2	12	21	38	58
		3	12	21	40	65
		Mean	12	20	38	61
25.0	3.31	1	12	21	42	70
		2	12	20	36	61
		3	12	18	36	60
		Mean	12	20	38	64
12.5	1.66	1	12	20	43	74
		2	12	21	42	75
		3	12	19	38	76
		Mean	12	20	41	75
6.25	0.707	1	12	18	40	78
		2	12	19	43	89
		3	12	19	44	87
		Mean	12	19	42	85
Control		1	12	20	47	89
		2	12	18	42	88
		3	12	19	45	91
		4	12	18	42	83
		5	12	19	39	80
		6	12	18	40	80
		Mean	12	19	43	85

* = 4 colonies with 3 fronds each per replicate were inoculated at start of the exposure

Repl. No. = replicate number

 Growth Rate and Yield Inhibition based on Fronds after 7 d

               Statistically significant differences of growth rates and yield

               compared to control values are marked (+) and non-significant differences are marked (-).

                                               

Nominal test item concentration [% of the saturated solution]		Geometric mean measured test item concentration [mg/L]		Repl. No.		Average growth rate [d^-1]			Inhibition of average growth rate [%]		Yield [fronds]			Inhibition of yield [%]		Doubling time [d]
100		15.7		1				0.204		27				38		48		3.40
			2				0.215		23				42		43		3.23
			3				0.192		31				34		54		3.61
			Mean		(+)		0.204		27		(+)		38		48		3.41
50.0		5.04		1				0.232		17				49		33		2.98
			2				0.225		20				46		37		3.08
			3				0.241		14				53		28		2.87
			Mean		(+)		0.233		17		(+)		49		33		2.98
25.0		3.31		1				0.252		10				58		21		2.75
			2				0.232		17				49		33		2.98
			3				0.230		18				48		34		3.01
			Mean		(+)		0.238		15		(+)		52		29		2.92
12.5		1.66		1				0.260		7				62		15		2.67
			2				0.262		7				63		14		2.65
			3				0.264		6				64		13		2.63
			Mean		(+)		0.262		7		(+)		63		14		2.65
6.25		0.707		1				0.267		5				66		10		2.59
			2				0.286		-2				77		-5		2.42
			3				0.283		-1				75		-2		2.45
			Mean		(-)		0.279		0		(-)		73		1		2.49
Control			1				0.286						77				2.42
			2				0.285						76				2.44
			3				0.289						79				2.39
			4				0.276						71				2.51
			5				0.271						68				2.56
			6				0.271						68				2.56
			Mean				0.280						73				2.48

Repl. No. = replicate number

 

 

Specific Growth Rate and Yield Inhibition of Dry Weight after 7 d

                               Statistically significant differences of specific growth rates and yield

                               compared to control values are marked (+) and non-significant differences are marked (-).

 

Nominal test item concentration [% of the saturated solution]	Geometric mean measured test item concentration [mg/L]	Repl. No.	Dry weight [mg]	Specific dry weight growth rate [d^-1]		Inhibition of specific dry weight growth rate [%]	Yield of dry weight [mg]		Inhibition of yield dry weight   [%]
100	15.7	1	9.50		0.274	16		8.10	34
		2	9.90		0.279	14		8.50	31
		3	10.0		0.281	14		8.60	30
		Mean	9.80	(+)	0.278	15	(+)	8.40	32
50.0	5.04	1	11.0		0.294	10		9.60	22
		2	10.9		0.293	10		9.50	23
		3	11.2		0.297	9		9.80	20
		Mean	11.0	(+)	0.295	10	(+)	9.60	22
25.0	3.31	1	13.6		0.325	0		12.2	1
		2	11.4		0.300	8		10.0	19
		3	11.1		0.296	9		9.70	21
		Mean	12.0	(+)	0.307	6	(+)	10.6	14
12.5	1.66	1	11.8		0.305	7		10.4	15
		2	13.4		0.323	1		12.0	2
		3	12.9		0.317	3		11.5	7
		Mean	12.7	(-)	0.315	3	(+)	11.3	8
6.25	0.707	1	12.2		0.309	5		10.8	12
		2	13.2		0.321	2		11.8	4
		3	13.2		0.321	2		11.8	4
		Mean	12.9	(-)	0.317	3	(-)	11.5	7
Control		1	14.5		0.334			13.1
		2	13.3		0.322			11.9
		3	14.5		0.334			13.1
		4	13.1		0.319			11.7
		5	13.7		0.326			12.3
		6	13.2		0.321			11.8
		Mean	13.7		0.326			12.3

The initial biomass dry weight was 1.4 mg per replicate.

Repl. No. = replicate number

 

Colony Number (Plants) on Days 0 and 7

 

Nominal test item concentration [% of the saturated solution]		Geometric mean measured test item concentration [mg/L]		Replicate No.		Colony number
			Day 0			Day 7
100		15.7		1		4		4
			2		4		4
			3		4		4
			Mean		4		4
50.0		5.04		1		4		5
			2		4		5
			3		4		5
			Mean		4		5
25.0		3.31		1		4		5
			2		4		4
			3		4		4
			Mean		4		4
12.5		1.66		1		4		5
			2		4		4
			3		4		5
			Mean		4		5
6.25		0.707		1		4		4
			2		4		5
			3		4		5
			Mean		4		5
Control			1		4		6
			2		4		5
			3		4		4
			4		4		4
			5		4		4
			6		4		5
			Mean		4		5

Further Observations on Days 2, 5 and 7

Nominal test item concentration [% of the saturated solution]		Geometric mean measured test item concentration [mg/L]		Observations on day
			2		5		7
100		15.7		1		2.1 + 2.3 + 3.3 +		2.1 + 2.3 + 3.3 ++
50.0		5.04		1		2.1 + 3.3 +		2.1 + 3.3 +
25.0		3.31		1		2.1 +		2.1 +
12.5		1.66		1		1		1
6.25		0.707		1		1		1
Control			1		1		1

Observations were made compared to the appearance of control colonies (plants) and test media

 

1      = no observedeffects

2.1   = chlorosis

2.3   = gibbosity

3.3   = discoloration of roots

+      = slight effects

++    = medium effects

+++  = strong effects

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Reactive Yellow 15 was found to inhibit the growth of the monocotyledonous aquatic plant Lemna gibba after 7-day exposure under static conditions, with the following effect values (nominal test item concentrations): The EC50-values for both inhibition of the specific growth rate and yield of fronds (ErC50, EydwC50) and dry weight (ErdwC50, EyC50) were > 100% of the saturated solution, respectively.
In this study, Reactive Yellow 15 was found to inhibit the growth of the monocotyledonous aquatic plant Lemna gibba after 7-day exposure under static conditions, with the following effect values (geometric mean measured test item concentrations): The EC50-values for both inhibition of the specific growth rate and yield of fronds (ErC50, EydwC50) and dry weight (ErdwC50, EyC50) were > 15.7 mg/L, respectively. Since the test item hydrolyses over time and neither parent compound nor hydrolysis products caused any acute effects, apparently, the EC50 based on nominal concentrations is used for the hazard assessment.

Executive summary:

The effects of the test item Reactive Yellow 15 on the growth of the monocotyledonous aquatic plant species Lemna gibba was determined according to the principles of OECD 221 at the test facility from2017-02-14 to 2017-02-23, with the definitive exposure phase from2017-02-15 to 2017-02-22.

Lemna gibba was exposed to the test item for 7 days under static conditions. Based on a preliminary test, 5 nominal test item concentration levels were tested in a geometrical series with a dilution factor of 2:6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution, corresponding to the geometric mean measured test item concentrations: 0.707 – 1.66 – 3.31 – 5.04 – 15.7 mg/L. Three replicates were investigated for each test concentration and six for the control. Frond numbers were assessed on days 0, 2, 5 and 7. Environmental parameters (light, pH and temperature) were within the acceptable limits.The validity criteria of the test guideline were fulfilled.

The concentrations ofthe test item Reactive Yellow 15 and the control were analysed via HPLC-DAD at the beginning and end of the exposure.

At the start of the exposure the initial measured concentrations of Reactive Yellow 15 were 2.00 – 3.85 – 7.53 – 15.6 – 30.9 mg/L. At the end of the exposure the measured concentrations were between < LOQ and 26% of the nominal concentrations. All effect values are given based on the nominal and geometric mean measured test item concentrations(Table 1).

 

Table1:   NOEC-, LOEC-, EC-Values and 95 % Confidence Intervals ofReactive Yellow15
after 7 Days of Exposure

                  (based on the nominal test item concentration [% of the saturated solution])

Frond number		Dry weight
Growth Rate Inhibition [% of the saturated solution]
NOEC	12.5	NOEC	12.5
LOEC	25.0	LOEC	25.0
ErC10	17.7 (13.9 – 21.8)	ErdwC10	52.9 (35.3 – 81.2)
ErC20	52.1 (41.3 – 70.4)	ErdwC20	> 100
ErC50	> 100	ErdwC50	> 100
Inhibition of Yield[% of the saturated solution]
NOEC	6.25	NOEC	12.5
LOEC	12.5	LOEC	25.0
EyC10	10.0 (7.23 – 12.8)	EydwC10	16.5 (< 6.25 – 32.2)
EyC20	18.5 (14.7 – 22.6)	EydwC20	44.0 (26.1 – 73.3)
EyC50	> 100	EydwC50	> 100

NOEC-, LOEC-, EC-Values and 95 % Confidence Intervals of Reactive Yellow 15
after 7 Days of Exposure

                  (based on the geometric mean measured concentration of the test item [mg/L])

Frond number		Dry weight
Growth Rate Inhibition [mg/L]
NOEC	1.66	NOEC	1.66
LOEC	3.31	LOEC	3.31
ErC10	2.18 (1.74 – 2.67)	ErdwC10	5.32 (3.96 – 9.49)
ErC20	6.82 (5.46 – 9.01)	ErdwC20	> 15.7
ErC50	> 15.7	ErdwC50	> 15.7
Inhibition of Yield [mg/L]
NOEC	0.707	NOEC	1.66
LOEC	1.66	LOEC	3.31
EyC10	1.20 (0.867 – 1.53)	EydwC10	2.45 (< 0.707 – 3.84)
EyC20	2.28 (1.82 – 2.80)	EydwC20	4.65 (3.11 – 8.12)
EyC50	> 15.7	EydwC50	> 15.7