4-(benzoylamino)-6-[[5-[[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]methyl]-1-sulpho-2-naphthyl]azo]-5-hydroxynaphthalene-1,7-disulphonic acid, lithium 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:

2018-01-17 to 2018-03-01, with the definitive exposure phase from 2018-02-21 to 2018-02-28

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:

4-(benzoylamino)-6-[[5-[[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]methyl]-1-sulpho-2-naphthyl]azo]-5-hydroxynaphthalene-1,7-disulphonic acid, lithium sodium salt

EC Number:

280-426-3

EC Name:

4-(benzoylamino)-6-[[5-[[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]methyl]-1-sulpho-2-naphthyl]azo]-5-hydroxynaphthalene-1,7-disulphonic acid, lithium sodium salt

Cas Number:

83400-11-7

Molecular formula:

C32 H21 Cl F2 N6 O11 S3 . x Li . x Na C32H(21-x-y)ClF2Li(x)N6Na(y)O11S3

IUPAC Name:

4-(benzoylamino)-6-[[5-[[(5-chloro-2,6-difluoro-4-pyrimidinyl)amino]methyl]-1-sulpho-2-naphthyl]azo]-5-hydroxynaphthalene-1,7-disulphonic acid, lithium 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 start (0 day, fresh medium) and at the end of the exposure (7 days, old medium). The samples were analysed with an HPLC-DAD method. The method was implemented under non-GLP but documented in the raw data and validated.

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 clear and red coloured.

Test concentrations
Based on the results of a limit test 5 concentrations were tested with a dilution factor of √10: 1.00 - 3.16 - 10.0 - 31.6 - 100mg/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 T
he 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  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.

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:

Room temperature [°C]: min.: 22.5 max.: 25.7 mean value: 24.4

pH:

Nominal test item concentration
[mg/L] pH-value
0 h
(Fresh medium) 7 d
(Old medium)
100 7.81 8.44
31.6 7.82 8.43
10.0 7.82 8.42
3.16 7.83 8.41
1.00 7.84 8.42
Control 7.39 8.42

Dissolved oxygen:

not measured

Salinity:

not measured

Conductivity:

not measured

Nominal and measured concentrations:

At the start of the exposure the measured concentrations of Reactive Red 158 were in the range of 98 and 100% of the nominal values. At the end of the exposure the measured concentrations were between 95 and 102% of the nominal values. All effect values are given based on the nominal test item concentrations.

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.
 
Dilution water 20X-AAP-medium according to the guideline.

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.

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.

Physical 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:

7 d

Dose descriptor:

NOEC

Effect conc.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Frond number and Dry weight

Effect concentrations 2

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Frond number and Dry Weight

Effect concentrations 3

Key result

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

1.09 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Frond number

Remarks:

CI <1.00 - 3.16; extrapolated

Effect concentrations 4

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

1.21 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Dry Weight

Remarks:

CI < 1.00 - 2.02

Effect concentrations 5

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

3.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Frond number

Remarks:

CI 2.07 - 4.45

Effect concentrations 6

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

4.43 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Dry weight

Remarks:

CI 2.53 - 7.04

Effect concentrations 7

Key result

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Frond number and Dry weight

Effect concentrations 8

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: Frond number and Dry weight

Effect concentrations 9

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: Frond number and Dry weight

Effect concentrations 10

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: Frond number and Dry weight

Effect concentrations 11

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

< 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: Frond number and dry weight

Effect concentrations 12

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

6.57 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: Frond number

Remarks:

CI 4.50 - 10.5

Effect concentrations 13

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

5.71 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: Dry weight

Remarks:

CI 3.57 - 8.97

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 with exposure phase 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 Reactive Red 158.

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:

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 alpha-value (acceptable probability of incorrectly concluding that there is a difference) was alpha=0.05.

EC-values and statistical analysis
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 and third order polynomial 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.

Any other information on results incl. tables

Frond Numbers

Nominal test item concentration [mg/L]		Repl. No.		Frond numbers per study day
			0 days*		2 days		5 days		7 days
100		1		12		19		32		40
		2		12		19		32		45
		3		12		17		27		39
		Mean		12		18		30		41
31.6		1		12		19		34		43
		2		12		17		30		42
		3		12		20		29		43
		Mean		12		19		31		43
10.0		1		12		19		36		52
		2		12		19		35		51
		3		12		21		34		49
		Mean		12		20		35		51
3.16		1		12		20		47		64
		2		12		19		45		70
		3		12		20		36		56
		Mean		12		20		43		63
1.00		1		12		18		48		79
		2		12		19		41		71
		3		12		22		58		82
		Mean		12		20		49		77
Control		1		12		20		54		100
		2		12		20		55		95
		3		12		20		54		99
		4		12		21		57		104
		5		12		18		47		92
		6		12		20		50		91
		Mean		12		20		53		97

* = 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 Days

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				0.172		42				28		67		4.03
		2				0.189		37				33		61		3.67
		3				0.168		44				27		68		4.12
		Mean		(+)		0.176		41		(+)		29		65		3.94
31.6		1				0.182		39				31		63		3.80
		2				0.179		40				30		65		3.87
		3				0.182		39				31		63		3.80
		Mean		(+)		0.181		39		(+)		31		64		3.83
10.0		1				0.209		30				40		53		3.31
		2				0.207		31				39		54		3.35
		3				0.201		33				37		56		3.45
		Mean		(+)		0.206		31		(+)		39		54		3.37
3.16		1				0.239		20				52		39		2.90
		2				0.252		15				58		32		2.75
		3				0.220		26				44		48		3.15
		Mean		(+)		0.237		20		(+)		51		39		2.93
1.00		1				0.269		10				67		21		2.57
		2				0.254		15				59		30		2.73
		3				0.275		8				70		17		2.52
		Mean		(+)		0.266		11		(+)		65		23		2.61
Control		1				0.303						88				2.29
		2				0.296						83				2.35
		3				0.301						87				2.30
		4				0.308						92				2.25
		5				0.291						80				2.38
		6				0.289						79				2.39
		Mean				0.298						85				2.33

Repl. No. = replicate number

 

 

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

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		7.7				0.292		32				6.7		65
		2		8.6				0.307		29				7.6		61
		3		7.1				0.280		35				6.1		68
		Mean		7.8		(+)		0.293		32		(+)		6.8		65
31.6		1		8.3				0.302		30				7.3		62
		2		7.2				0.282		34				6.2		68
		3		8.0				0.297		31				7.0		64
		Mean		7.8		(+)		0.294		32		(+)		6.8		65
10.0		1		10.2				0.332		23				9.2		52
		2		10.3				0.333		23				9.3		52
		3		9.6				0.323		25				8.6		55
		Mean		10.0		(+)		0.329		23		(+)		9.0		53
3.16		1		12.6				0.362		16				11.6		40
		2		12.8				0.364		15				11.8		39
		3		9.9				0.328		24				8.9		54
		Mean		11.8		(+)		0.351		18		(+)		10.8		44
1.00		1		15.8				0.394		8				14.8		23
		2		14.7				0.384		11				13.7		29
		3		16.9				0.404		6				15.9		18
		Mean		15.8		(+)		0.394		8		(+)		14.8		23
Control		1		20.1				0.429						19.1
		2		19.0				0.421						18.0
		3		20.2				0.429						19.2
		4		22.5				0.445						21.5
		5		20.3				0.430						19.3
		6		19.6				0.425						18.6
		Mean		20.3				0.430						19.3

The initial biomass dry weight was 1.0 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		5
		2		3		5
		3		3		3
		Mean		3		4
31.6		1		3		3
		2		3		4
		3		3		7
		Mean		3		5
10.0		1		3		5
		2		3		4
		3		3		3
		Mean		3		4
3.16		1		3		4
		2		3		3
		3		3		5
		Mean		3		4
1.00		1		3		5
		2		3		5
		3		3		7
		Mean		3		6
Control		1		3		6
		2		3		7
		3		3		7
		4		3		7
		5		3		7
		6		3		6
		Mean		3		7

 

 

Further Observations on Days 2, 5 and 7

Nominal test item concentration [mg/L]	Observations on day
	2	5	7
100	1	1	(3.3) ++
31.6	1	1	(3.3) ++
10.0	1	1	(3.3) +
3.16	1	1	1
1.00	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 158 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 (fronds and dry weight) (ErC50, ErdwC50) were > 100 mg/L and yield (fronds and dry weight) (EyC50, EydwC50) were 6.57 (4.50 – 10.5) mg/L and 5.71 (3.57 – 8.97) mg/L, respectively. The extrapolated ErC10 (frond number) was calculated at 1.09 mg/L.

Executive summary:

The effects of the test item Reactive Red 158 on the growth of the monocotyledonous aquatic plant speciesLemna gibba was determined according to the principles of OECD 221 at the test facility from2018 -01 -17 to 2018 -03 -01, with the definitive exposure phase from 2018-02-21 to 2018-02-28.

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 √10:1.00 - 3.16 - 10.0 - 31.6 - 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 clear and concentration-related red coloured throughout the exposure.The validity criteria of the test guideline were fulfilled.

The concentrations of the test item Reactive Red 158 and the control were analysed via HPLC-DAD at the start and end of the exposure.

At the start of the exposure the measured concentrations of Reactive Red 158 were in the range of 98 and 100% of the nominal values. At the end of the exposure the measured concentrations were between 95 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 158
after 7 Days of Exposure

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

Frond number		Dry weight
Growth Rate Inhibition [mg/L]
NOEC	< 1.00		< 1.00
LOEC	1.00		1.00
ErC10	< 1.00 1.09 (<1.00 – 3.16)*		1.21 (< 1.00 – 2.02)
ErC20	3.10 (2.07 – 4.45)		4.43 (2.53 – 7.04)
ErC50	> 100		> 100
Inhibition of Yield [mg/L]
NOEC	< 1.00		< 1.00
LOEC	1.00		1.00
EyC10	< 1.00		< 1.00
EyC20	< 1.00		< 1.00
EyC50	6.57 (4.50 – 10.5)		5.71 (3.57 – 8.97)

           * = extrapolated values. Confidence interval extrapolated from tested concentrations.