7-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-4-hydroxy-3-[(4-methoxy-2-sulphophenyl)azo]naphthalene-2-sulphonic 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:

from 2018-04-18 to 2018-04-26, with the definitive exposure phase from 2018-04-18 to 2018-04-25

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: Council Regulation (EC) No. 761/2009 Method C.26

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

7-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-4-hydroxy-3-[(4-methoxy-2-sulphophenyl)azo]naphthalene-2-sulphonic acid, sodium salt

EC Number:

286-839-5

EC Name:

7-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-4-hydroxy-3-[(4-methoxy-2-sulphophenyl)azo]naphthalene-2-sulphonic acid, sodium salt

Cas Number:

85391-83-9

Molecular formula:

C21H14ClF2N5O8S2.xNa

IUPAC Name:

7-[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]-4-hydroxy-3-[(4-methoxy-2-sulphophenyl)azo]naphthalene-2-sulphonic acid, sodium salt

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 first interval. The control, the lowest and the highest test item concentration were analytically verified at the end of the exposure (7 days) and on every renewal day. Freshly prepared and old media were analyzed. The samples were analyzed with an HPLC-DAD method. The method was implemented under non-GLP but documented in the raw data and validated. The method validation was not part of this GLP study.

Test solutions

Vehicle:

no

Details on test solutions:

Preparation of the Test item solution
A stock solution with a nominal test item concentration of 100 mg/L was freshly prepared with dilution water (see Table 2) and agitated until the solution was visually clear. The test item solution was red coloured.

Test concentrations
Based on the results of a preliminary range finding test (see section 18) 5 concentrations were tested with a dilution factor of 4: 0.391 - 1.56 - 6.25 - 25.0 - 100 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 G3, 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

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

Composition of Dilution water
Component Concentration in stock solution [g/L] Concentration in prepared medium [mg/L]
NaNO3 26 510
MgCl2 x 6 H2O 12 240
CaCl2 x 2 H2O 4.4 90
MgSO4 x 7 H2O 15 290
K2HPO4 · 3 H2O 1.4 30
NaHCO3 15 300
H3BO3 0.19 3.7
MnCl2 x 4 H2O 0.42 8.3
FeCl3 x 6 H2O 0.16 3.2
Na2-EDTA · 2 H2O 0.30 6.0
ZnCl2 3.3 mg/L 66 µg/L
CoCl2 x 6 H2O 1.4 mg/L 29 µg/L
Na2MoO4 x 2 H2O 7.3 mg/L 145 µg/L
CuCl2 x 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.

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:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Test conditions

Hardness:

not measured

Test temperature:

Room temperature [°C]: min.: 23.3 max.: 24.9 mean value: 23.9

pH:

Nominal test item concentration
[mg/L] pH-value
0 h (Fresh medium) 2 d (Old medium) 2 d (Fresh medium) 5 d (Old medium) 5 d (Fresh medium) 7 d (Old medium)
100 7.53 8.32 7.43 8.18 7.59 8.54
25.0 7.53 8.32 7.43 8.13 7.48 8.51
6.25 7.53 8.32 7.44 8.07 7.46 8.50
1.56 7.53 8.32 7.44 7.96 7.46 8.49
0.391 7.54 8.31 7.47 7.94 7.47 8.52
Control 7.55 8.32 7.57 7.97 7.53 8.46

Dissolved oxygen:

not measured

Salinity:

not measured

Conductivity:

not measured

Nominal and measured concentrations:

Nominal: 0.391 - 1.56 - 6.25 - 25.0 - 100 mg/L

Details on test conditions:

Test method
Semi-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.

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:

7 d

Dose descriptor:

LOEC

Effect conc.:

25 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Frond number and Dry weight

Remarks on result:

other: Growth Rate and Inhibition of Yield

Effect concentrations 2

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Frond number and Dry weight

Remarks on result:

other: Growth Rate and Inhibition of Yield

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Frond number and Dry weight

Remarks on result:

other: Growth Rate and Yield Inhibition

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-ALDRICH, 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 2018-04-04 to 2018-04-12, with the definitive exposure phase from 2018-04-04 to 2018-04-11 according to OECD Guideline 221.

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 7.68 5.82 ± 3.18
95% confidence interval 7.31 – > 8.00
Yield inhibition (number of fronds)
EyC50 4.93 4.67 ± 2.87
95% confidence interval 4.42 – 5.53
Growth rate inhibition (dry weight)
ErdwC50 > 8.00 5.61 ± 2.76
95% confidence interval -
Yield inhibition (dry weight)
EydwC50 5.95 4.75 ± 2.49
95% confidence interval 5.18 – 6.75
SD = standard deviation

Reported statistics and error estimates:

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.
Step-down Jonckheere-Terpstra Test Procedure was done 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.

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

 Biological Data

 

 Frond Numbers

Nominal test item concentration [mg/L]		Repl. No.		Frond numbers per study day
			0 days*		3 days		5 days		7 days
100		1		12		24		117		221
		2		12		25		117		222
		3		12		23		111		244
		Mean		12		24		115		229
25.0		1		12		25		118		251
		2		12		21		106		230
		3		12		25		143		333
		Mean		12		24		122		271
6.25		1		12		22		102		258
		2		12		21		112		307
		3		12		23		107		303
		Mean		12		22		107		289
1.56		1		12		23		112		333
		2		12		23		112		323
		3		12		22		123		370
		Mean		12		23		116		342
0.391		1		12		22		117		297
		2		12		22		93		278
		3		12		24		126		365
		Mean		12		23		112		313
Control		1		12		22		103		322
		2		12		23		96		276
		3		12		24		126		306
		4		12		23		98		275
		5		12		23		122		311
		6		12		22		107		297
		Mean		12		23		109		298

* = 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 (-).

                                               

Nominal 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		1				0.416		9				209		27		1.67
		2				0.417		9				210		27		1.66
		3				0.430		6				232		19		1.61
		Mean		(+)		0.421		8		(+)		217		24		1.65
25.0		1				0.434		5				239		16		1.60
		2				0.422		8				218		24		1.64
		3				0.475		-3				321		-12		1.46
		Mean		(-)		0.444		3		(-)		259		9		1.57
6.25		1				0.438		5				246		14		1.58
		2				0.463		-1				295		-3		1.50
		3				0.461		0				291		-2		1.50
		Mean		(-)		0.454		1		(-)		277		3		1.53
1.56		1				0.475		-3				321		-12		1.46
		2				0.470		-2				311		-9		1.47
		3				0.490		-7				358		-25		1.42
		Mean		(-)		0.478		-4		(-)		330		-15		1.45
0.391		1				0.458		0				285		0		1.51
		2				0.449		2				266		7		1.54
		3				0.488		-6				353		-24		1.42
		Mean		(-)		0.465		-1		(-)		301		-5		1.49
Control		1				0.470						310				1.47
		2				0.448						264				1.55
		3				0.463						294				1.50
		4				0.447						263				1.55
		5				0.465						299				1.49
		6				0.458						285				1.51
		Mean				0.459						286				1.51

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 [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		1		40.3				0.461		7				38.7		22
		2		37.1				0.449		9				35.5		28
		3		41.4				0.465		6				39.8		19
		Mean		39.6		(+)		0.458		7		(+)		38.0		23
25.0		1		45.4				0.478		3				43.8		11
		2		35.0				0.441		11				33.4		32
		3		55.6				0.507		-3				54.0		-9
		Mean		45.3		(-)		0.475		4		(-)		43.7		11
6.25		1		39.1				0.457		8				37.5		24
		2		46.8				0.482		2				45.2		9
		3		46.2				0.480		3				44.6		10
		Mean		44.0		(-)		0.473		4		(-)		42.4		14
1.56		1		54.3				0.504		-2				52.7		-7
		2		58.7				0.515		-4				57.1		-16
		3		58.3				0.514		-4				56.7		-15
		Mean		57.1		(-)		0.511		-3		(-)		55.5		-12
0.391		1		44.9				0.476		4				43.3		12
		2		48.3				0.487		1				46.7		5
		3		52.2				0.498		-1				50.6		-2
		Mean		48.5		(-)		0.487		1		(-)		46.9		5
Control		1		48.8				0.488						47.2
		2		46.6				0.482						45.0
		3		52.2				0.498						50.6
		4		50.1				0.492						48.5
		5		56.1				0.508						54.5
		6		52.4				0.498						50.8
		Mean		51.0				0.494						49.4

The initial biomass dry weight was 1.6 mg per replicate.

Repl. No. = replicate number

 

Colony Number (Plants) on Days 0 and 7

 

Nominal test item concentration [mg/L]		Replicate No.		Colony number
			Day 0		Day 7
100		1		3		39
		2		3		40
		3		3		43
		Mean		3		41
25.0		1		3		53
		2		3		37
		3		3		54
		Mean		3		48
6.25		1		3		34
		2		3		44
		3		3		40
		Mean		3		39
1.56		1		3		38
		2		3		32
		3		3		44
		Mean		3		38
0.391		1		3		48
		2		3		38
		3		3		54
		Mean		3		47
Control		1		3		45
		2		3		40
		3		3		29
		4		3		32
		5		3		29
		6		3		41
		Mean		3		36

 

 

Further Observations on Days 2, 5 and 7

Nominal test item concentration [mg/L]	Observations on day
	2	5	7
100	3.3 +	3.3 +	3.3 +
25.0	1	1	1
6.25	1	1	1
1.56	1	1	1
0.391	1	1	1
Control	1	1	1

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

 

1       = no observedeffects

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 Red 123 was found to inhibit the growth of the monocotyledonous aquatic plant Lemna gibba after 7-day exposure under semi-static conditions, with the following effect values (nominal test item concentrations): The EC50-values for both inhibition of the specific growth rate (fronds and dry weight) (ErC50, ErdwC50) were > 100 and yield (fronds and dry weight) (EyC50, EydwC50) were > 100.

Executive summary:

The effects of the test item Reactive Red123 on the growth of the monocotyledonous aquatic plant species Lemna gibba was determined according to the principles of OECD 221 and Council Regulation (EC) No. 761/2009 Method C. 26 at the test facility from 2018-04-18 to 2018-04-26, with the definitive exposure phase from 2018-04-18 to 2018-04-25.

Lemna gibba was exposed to the test item for 7 days under semi-static conditions. Based on a preliminary test, 5 nominal test item concentration levels were tested in a geometrical series with a dilution factor of 4:0.391 - 1.56 - 6.25 - 25.0 - 100 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. All test solutions were concentration-related red coloured throughout the exposure.The validity criteria of the test guideline were fulfilled.

The concentrations of the test item Reactive Red 123 and the control were analysed via HPLC-DAD at the first interval. The control, the lowest and the highest test item concentration were analytically verified at the end of the exposure and on every renewal day.

At the start of the first interval the measured concentrations of Reactive Red 123 were in the range of 96 and 102% of the nominal values. At the end of the first interval the measured concentrations were between 92 and 102% of the nominal values. All effect values are given based on the nominal test item concentrations.

 

NOEC-, LOEC-, EC-Values and 95% Confidence Intervals of Reactive Red 123 after 7 Days of Exposure

                  (based on the nominal test item concentration [mg/L])

Frond number		Dry weight
Growth Rate Inhibition [mg/L]
NOEC	25.0	25.0
LOEC	100	100
ErC10	> 100	> 100
ErC20	> 100	> 100
ErC50	> 100	> 100
Inhibition of Yield [mg/L]
NOEC	25.0	25.0
LOEC	100	100
EyC10	22.5 (4.02 - > 100)	5.63 (1.52 - > 100)
EyC20	62.1 (15.0 - > 100)	> 100
EyC50	> 100	> 100