Disodium 1-amino-4-[[3-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-2-methyl-5-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

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 2017-09-06 to 2017-09-18, with the definitive exposure phase from 2017-09-06 to 2017-09-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

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:

Disodium 1-amino-4-[[3-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-2-methyl-5-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

EC Number:

276-374-6

EC Name:

Disodium 1-amino-4-[[3-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-2-methyl-5-sulphonatophenyl]amino]-9,10-dihydro-9,10-dioxoanthracene-2-sulphonate

Cas Number:

72139-17-4

Molecular formula:

C25H16ClF2N5O8S2.2Na

IUPAC Name:

disodium 1-amino-4-({3-[(5-chloro-2,6-difluoropyrimidin-4-yl)amino]-2-methyl-5-sulfophenyl}amino)-9,10-dioxo-9,10-dihydroanthracene-2-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:

Preparation of the Test item solution
A stock solution with a nominal concentration of 100 mg test item/L was freshly prepared with dilution water and agitated until the solution was visually clear.

Test concentrations
The stock solution and further five dilution levels out of the stock solution were prepared in a geometrical series with a separation factor of 4 and tested as follows: 0.0977 - 0.391 – 1.56 – 6.25 – 25.0 – 100 mg/L, corresponding to the geometric mean measured test item concentration of: 0.0591 - 0.265 – 1.16 – 4.50 – 21.3 – 98.5 mg/L.

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

Salinity:

not measured

Conductivity:

not measured

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, 3 uniform, healthy plants (colonies of 4 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

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

< 0.059 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate and yield (frond number and dry weight)

Effect concentrations 2

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 98.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: growth rate (frond number and dry weight)

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

0.482 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: frond number yield

Remarks on result:

other: CI: 0.341 - 0.739

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

14 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: dry weight yield

Remarks on result:

other: CI: 2.32 - > 98.5

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 MKBZ0947V, purity 100.0 area %, CAS RN 591-35-5) to the monocotyledonous aquatic plant Lemna gibba was determined over a period of 7 days from 2017-10-06 to 2017-10-13 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 Acid Red 337.

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 6.76 5.71 ± 2.95
95% confidence interval 4.62 – 7.87
Yield inhibition (number of fronds)
EyC50 5.80 4.65 ± 2.96
95% confidence interval 4.50 – 7.05
Growth rate inhibition (dry weight)
ErdwC50 6.36 5.61 ± 2.76
95% confidence interval 5.14 – 7.12
Yield inhibition (dry weight)
EydwC50 5.39 4.67 ± 2.37
95% confidence interval 4.86 – 6.07
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, LOEC and statistical analyses
The NOEC and LOEC were determined by calculation of statistically significant differences of growth rates and yield.
The following statistical tests were conducted:
Shapiro-Wilk’s test on normal distribution was done with a significance level of 0.01.
Levene’s test on variance homogeneity was done with a significance level of 0.01.
Monotonicity of concentration/response was done by trend analysis by contrasts (significance level 0.05).
William’s multiple sequential t-test was carried out with a significance level of 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. If calculation of confidence intervals resulted in values below or above the concentration range of the study, these values were derived empirically.

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

Geometric mean measured test item concentration [mg/L]	Repl. No.	Frond numbers per study day
		0 days*	2 days	5 days	7 days
98.5	1	12	19	34	35
	2	12	19	27	33
	3	12	21	28	35
	Mean	12	20	30	34
21.3	1	12	18	26	29
	2	12	17	27	33
	3	12	20	26	35
	Mean	12	18	26	32
4.50	1	12	19	28	37
	2	12	20	27	36
	3	12	19	32	35
	Mean	12	19	29	36
1.16	1	12	18	33	39
	2	12	19	31	38
	3	12	19	32	43
	Mean	12	19	32	40
0.265	1	12	19	37	50
	2	12	22	39	61
	3	12	20	36	60
	Mean	12	20	37	57
0.0591	1	12	20	43	75
	2	12	22	40	68
	3	12	21	53	83
	Mean	12	21	45	75
Control	1	12	23	54	95
	2	12	21	52	90
	3	12	21	54	91
	4	12	20	36	68
	5	12	23	50	84
	6	12	22	46	85
	Mean	12	22	49	86

* = 3 colonies with 4 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 (-).

                                               

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]
98.5		1				0.153		45				23		69		4.53
		2				0.145		48				21		71		4.80
		3				0.153		45				23		69		4.53
		Mean		(+)		0.150		46		(+)		22		70		4.62
21.3		1				0.126		55				17		77		5.50
		2				0.145		48				21		71		4.80
		3				0.153		45				23		69		4.53
		Mean		(+)		0.141		50		(+)		20		72		4.94
4.50		1				0.161		43				25		66		4.31
		2				0.157		44				24		67		4.42
		3				0.153		45				23		69		4.53
		Mean		(+)		0.157		44		(+)		24		67		4.42
1.16		1				0.168		40				27		63		4.12
		2				0.165		41				26		65		4.21
		3				0.182		35				31		58		3.80
		Mean		(+)		0.172		39		(+)		28		62		4.04
0.265		1				0.204		27				38		48		3.40
		2				0.232		17				49		33		2.98
		3				0.230		18				48		35		3.01
		Mean		(+)		0.222		21		(+)		45		39		3.13
0.0591		1				0.262		7				63		14		2.65
		2				0.248		12				56		24		2.80
		3				0.276		1				71		3		2.51
		Mean		(+)		0.262		6		(+)		63		14		2.65
Control		1				0.296						83				2.35
		2				0.288						78				2.41
		3				0.289						79				2.39
		4				0.248						56				2.80
		5				0.278						72				2.49
		6				0.280						73				2.48
		Mean				0.280						74				2.49

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 (-).

 

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   [%]
98.5	1	5.6		0.140	34		3.5	51
	2	5.5		0.138	35		3.4	53
	3	5.7		0.143	33		3.6	50
	Mean	5.6	(+)	0.140	34	(+)	3.5	51
21.3	1	5.0		0.124	42		2.9	60
	2	5.5		0.138	35		3.4	53
	3	5.8		0.145	32		3.7	49
	Mean	5.4	(+)	0.136	36	(+)	3.3	54
4.50	1	5.8		0.145	32		3.7	49
	2	6.3		0.157	26		4.2	42
	3	6.1		0.152	28		4.0	44
	Mean	6.1	(+)	0.151	29	(+)	4.0	45
1.16	1	6.6		0.164	23		4.5	38
	2	6.1		0.152	28		4.0	44
	3	6.5		0.161	24		4.4	39
	Mean	6.4	(+)	0.159	25	(+)	4.3	40
0.265	1	6.3		0.157	26		4.2	42
	2	7.9		0.189	11		5.8	19
	3	7.4		0.180	15		5.3	26
	Mean	7.2	(+)	0.175	17	(+)	5.1	29
0.0591	1	7.5		0.182	14		5.4	25
	2	8.2		0.195	8		6.1	15
	3	9.0		0.208	2		6.9	4
	Mean	8.2	(+)	0.195	8	(+)	6.1	15
Control	1	10.3		0.227			8.2
	2	9.6		0.217			7.5
	3	9.9		0.222			7.8
	4	7.9		0.189			5.8
	5	9.2		0.211			7.1
	6	8.7		0.203			6.6
	Mean	9.3		0.212			7.2

The initial biomass dry weight was 2.1 mg per replicate.

Repl. No. = replicate number

 

Colony Number (Plants) on Days 0 and 7

 

Geometric mean measured test item concentration [mg/L]		Replicate No.		Colony number
			Day 0		Day 7
98.5		1		3		5
		2		3		4
		3		3		4
		Mean		3		4
21.3		1		3		4
		2		3		4
		3		3		4
		Mean		3		4
4.50		1		3		4
		2		3		5
		3		3		4
		Mean		3		4
1.16		1		3		4
		2		3		4
		3		3		4
		Mean		3		4
0.265		1		3		6
		2		3		8
		3		3		6
		Mean		3		7
0.0591		1		3		6
		2		3		6
		3		3		9
		Mean		3		7
Control		1		3		9
		2		3		9
		3		3		10
		4		3		6
		5		3		7
		6		3		8
		Mean		3		8

Further Observations on Days 2, 5 and 7

Geometric mean measured test item concentration [mg/L]		Observations on day
		2		5		7
98.5		1		1		2.1 + 2.3 +
21.3		1		1		2.1 + 2.3 +
4.50		1		1		1
1.16		1		1		1
0.265		1		1		1
0.0591		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

+      = slight effects

++    = medium effects

+++  = strong effects

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Reactive Blue 114 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 of fronds (ErC50) and dry weight (ErdwC50) were > 98.5 mg/L, respectively. The EC50-values for inhibition of yield (fronds, EyC50) and dry weight inhibition of yield (EydwC50) with 95% confidence intervals were 0.482 (0.341 – 0.739) mg/L and 14.0 (2.33 - > 98.5) mg/L, respectively.

Executive summary:

The effects of the test item Reactive Blue 114 on the growth of the monocotyledonous aquatic plant species Lemna gibba was determined according to the principles of OECD 221 and Council Regulation No. 761/2009 Method C.26 at the test facility from 2017-09-06 to 2017-09-18, with the definitive exposure phase from 2017-09-06 to 2017-09-13.

 

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 4: 0.0977 - 0.391 - 1.56 - 6.25 - 25.0 - 100 mg/L, corresponding to the geometric mean measured test item concentrations: 0.0591 – 0.265 – 1.16 – 4.50 - 21.3 – 98.5 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 of the test item Reactive Blue114 and the control were analysed via HPLC-DAD at the beginning and end of the exposure.

The measured concentrations at the start of the exposure of Reactive Blue114 were in the range of 88 and 98% of the nominal values. At the end of the exposure the measured concentrations were between < LOQ and 100% of the nominal concentrations. The test item solutions were clear and concentration related blue coloured. All effect values are given based on the geometric mean measured test item concentrations.

 

NOEC-, LOEC-, EC-Values and 95 % Confidence Intervals of Reactive Blue114 after 7 Days of Exposure

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

Frond number		Dry weight
Growth Rate Inhibition [mg/L]
NOEC	< 0.0591	NOEC	< 0.0591
LOEC	0.0591	LOEC	0.0591
ErC10	0.0986 (< 0.0591 – 0.160)	ErdwC10	0.0812 (< 0.591 – 0.204)
ErC20	0.248 (0.167 – 0.352)	ErdwC20	0.451 (0.180 – 0.977)
ErC50	> 98.5	ErdwC50	> 98.5
Inhibition of Yield [mg/L]
NOEC	< 0.0591	NOEC	< 0.0591
LOEC	0.0591	LOEC	0.0591
EyC10	< 0.0591	EydwC10	< 0.0591
EyC20	0.0949 (< 0.0591 – 0.150)	EydwC20	0.101 (< 0.591 – 0.236)
EyC50	0.482 (0.341 – 0.739)	EydwC50	14.0 (2.32 - > 98.5)