Cuprate(6-), [2-[[[[3-[[4-chloro-6-[[4-[[4-chloro-6-[(3-sulfophenyl)amino]-1,3,5-triazin-2-yl]amino]-2,5-disulfophenyl]amino]-1,3,5-triazin-2-yl]amino]-2-hydroxy-5-sulfophenyl]azo]phenylmethyl]azo]-5-sulfobenzoato(8-)]-, pentasodium hydrogen, (SP-4-3)-

Toxicity to aquatic plants other than algae

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic plants other than algae

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2017-05-05 to 2017-06-19, with the definitive exposure phase from 2017-06-09 to 2017-06-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 221 (Lemna sp. Growth Inhibition Test)

Version / remarks:

2006

Deviations:

no

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

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

Vehicle:

no

Details on test solutions:

Preparation of the Test item solution
A stock solution of 100 mg test item/L was freshly prepared with dilution water and agitated until the solution was visually clear. An appropriate amount of the test item was weighed out and inserted into a glass bottle with an appropriate amount of dilution water.

Test concentrations
Out of the stock solution the test item solutions were prepared in a geometrical series with a separation factor of 4 and tested as follows: 0.391 – 1.56 – 6.25 – 25.0 – 100 mg/L, corresponding to the measured initial test item concentration of: 0.375* – 1.32 – 5.82 – 23.8 – 96.4 mg/L.

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

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.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

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

Nominal and measured concentrations:

nominal test item concentration: 0.391 – 1.56 – 6.25 – 25.0 – 100 mg/L
measured initial test item concentration: 0.375 – 1.32 – 5.82 – 23.8 – 96.4 mg/L.

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

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.375 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: frond number growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 96.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: frond number growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.375 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: Frond number yield

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

6.86 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: frond number yield

Remarks on result:

other: CI: 3.52 - 16.7

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.375 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: dry weight growth rate

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 96.4 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: dry weight growth rate

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

0.375 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: dry weight yield

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

30.3 mg/L

Nominal / measured:

meas. (initial)

Conc. based on:

test mat.

Basis for effect:

other: dry weight yield

Remarks on result:

other: CI: 20.3 - 50.1

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 2017-04-19 to 2017-06-13, with the definitive exposure phase from 2017-06-02 to 2017-06-09 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 Blue 160.

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 6.95 5.65 ± 2.93
95% confidence interval 5.75 – 8.00
Yield inhibition (number of fronds)
EyC50 4.35 4.57 ± 2.91
95% confidence interval 3.88 – 4.91
Growth rate inhibition (dry weight)
ErdwC50 6.16 5.55 ± 2.79
95% confidence interval 5.62 – 6.64
Yield inhibition (dry weight)
EydwC50 4.31 4.61 ± 2.38
95% confidence interval 3.86 – 4.85
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:

For statistical analysis and determination of NOEC, LOEC and EC-values, all analytical recoveries in the test concentrations determined to be
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.

Frond Numbers

Measured initial test item concentration [mg/L]		Repl. No.		Frond numbers per study day
			0 days*		3 days		5 days		7 days
96.4		1		12		22		27		36
		2		12		24		29		37
		3		12		21		32		36
		Mean		12		22		29		36
23.8		1		12		25		37		40
		2		12		24		35		41
		3		12		25		36		44
		Mean		12		25		36		42
5.82		1		12		25		40		47
		2		12		26		39		56
		3		12		27		38		54
		Mean		12		26		39		52
1.32		1		12		26		44		68
		2		12		29		41		62
		3		12		32		43		60
		Mean		12		29		43		63
0.375		1		12		32		47		78
		2		12		30		45		70
		3		12		33		52		90
		Mean		12		32		48		79
Control		1		12		33		53		86
		2		12		34		54		85
		3		12		30		47		81
		4		12		33		54		103
		5		12		31		49		95
		6		12		23		43		78
		Mean		12		31		50		88

* = 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 (-).

                                               

Measured initial 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]
96.4		1				0.157		45				24		68		4.42
		2				0.161		43				25		67		4.31
		3				0.157		45				24		68		4.42
		Mean		(+)		0.158		44		(+)		24		68		4.38
23.8		1				0.172		39				28		63		4.03
		2				0.176		38				29		62		3.95
		3				0.186		35				32		58		3.73
		Mean		(+)		0.178		37		(+)		30		61		3.90
5.82		1				0.195		31				35		54		3.55
		2				0.220		23				44		42		3.15
		3				0.215		24				42		45		3.23
		Mean		(+)		0.210		26		(+)		40		47		3.31
1.32		1				0.248		13				56		26		2.80
		2				0.235		17				50		34		2.95
		3				0.230		19				48		37		3.01
		Mean		(+)		0.237		16		(+)		51		32		2.92
0.375		1				0.267		6				66		13		2.59
		2				0.252		11				58		24		2.75
		3				0.288		-1				78		-3		2.41
		Mean		(-)		0.269		5		(-)		67		11		2.58
Control		1				0.281						74				2.46
		2				0.280						73				2.48
		3				0.273						69				2.54
		4				0.307						91				2.26
		5				0.296						83				2.35
		6				0.267						66				2.59
		Mean				0.284						76				2.45

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 (-).

 

Measured initial 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   [%]
96.4	1	6.2		0.276	33		5.3	64
	2	6.6		0.285	30		5.7	62
	3	7.3		0.299	27		6.4	57
	Mean	6.7	(+)	0.286	30	(+)	5.8	61
23.8	1	9.5		0.337	18		8.6	42
	2	8.1		0.314	23		7.2	52
	3	9.5		0.337	18		8.6	42
	Mean	9.0	(+)	0.329	20	(+)	8.1	45
5.82	1	8.8		0.326	20		7.9	47
	2	11.4		0.363	11		10.5	30
	3	10.0		0.344	16		9.1	39
	Mean	10.1	(+)	0.344	16	(+)	9.2	38
1.32	1	13.0		0.381	7		12.1	19
	2	12.2		0.372	9		11.3	24
	3	13.8		0.390	5		12.9	13
	Mean	13.0	(+)	0.381	7	(+)	12.1	19
0.375	1	14.7		0.399	2		13.8	7
	2	13.6		0.388	5		12.7	15
	3	15.0		0.402	2		14.1	5
	Mean	14.4	(-)	0.396	3	(-)	13.5	9
Control	1	15.4		0.406			14.5
	2	17.2		0.421			16.3
	3	15.0		0.402			14.1
	4	16.9		0.419			16.0
	5	15.4		0.406			14.5
	6	15.0		0.402			14.1
	Mean	15.8		0.409			14.9

The initial biomass dry weight was 0.9 mg per replicate.

Repl. No. = replicate number

 

Colony Number (Plants) on Days 0 and 7

 

Measured initial test item concentration [mg/L]		Replicate No.		Colony number
			Day 0		Day 7
96.4		1		3		7
		2		3		6
		3		3		6
		Mean		3		6
23.8		1		3		6
		2		3		6
		3		3		6
		Mean		3		6
5.82		1		3		4
		2		3		6
		3		3		4
		Mean		3		5
1.32		1		3		5
		2		3		6
		3		3		5
		Mean		3		5
0.375		1		3		6
		2		3		6
		3		3		6
		Mean		3		6
Control		1		3		5
		2		3		5
		3		3		5
		4		3		8
		5		3		8
		6		3		5
		Mean		3		6

Further Observations on Days 3, 5 and 7

Measured initial test item concentration [mg/L]		Observations on day
		3		5		7
96.4		1		4.1 +		2.5 + 3.3 ++ 4.1 +
23.8		1		4.1 +		2.5 + 3.3 +
5.82		1		1		1
1.32		1		1		1
0.375		1		1		1
Control		1		1		1

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

 

1      = no observedeffects

2.5  = fronds are smaller, compared to the control

3.3  =roots colored

4.1   = break up of plants

+      = slight effects

++    = medium effects

+++  = strong effects

Validity criteria fulfilled:

yes

Conclusions:

In this study, Reactive Blue 160 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 (measured initial test item concentrations): The EC50-values for both inhibition of the specific growth rate of fronds (ErC50) and dry weight (ErdwC50) were > 96.4 mg/L, respectively. The EC50-values for inhibition of yield (fronds, EyC50) and dry weight inhibition of yield (EydwC50) with 95% confidence intervals were 6.86 (3.52 – 16.7) mg/L and 30.3 (20.3 – 50.1) mg/L, respectively.

Executive summary:

The effects of the test item Reactive Blue160 on the growth of the monocotyledonous aquatic plant speciesLemna gibba was determined according to the principles of OECD 221 and Council Regulation No. 761/2009 Method C.26 atthe test facility from2017-05-05 to 2017-06-19, with the definitive exposure phase from 2017-06-09 to 2017-06-16.

 

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 4:0.391 - 1.56 - 6.25 - 25.0 - 100 mg/L, corresponding to the measured initial test item concentrations: 0.375* – 1.32 – 5.82 – 23.8 – 96.4 mg/L. Three replicates were investigated for each test concentration and six for the control. Frond numbers were assessed on days 0, 3, 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 Blue160 and the control were analysed via photometric analysis at the beginning and end of the exposure. The measured concentrations at the start of the exposure of Reactive Blue160 were in the range of < LCL and 96% of the nominal values.

All effect values are given based on the measured initial test item concentrations.

 

NOEC-, LOEC-, EC-Values and 95 % Confidence Intervals ofReactive Blue160
after 7 Days of Exposure

                  (based on the measured initial item concentration [mg/L])

Frond number		Dry weight
Growth Rate Inhibition [mg/L]
NOEC	0.375*	NOEC	0.375*
LOEC	1.32	LOEC	1.32
ErC10	0.656 (< 0.375 – 1.13)	ErdwC10	2.01 (1.27 – 2.96)
ErC20	2.29 (1.31 – 3.69)	ErdwC20	16.4 (11.2 – 26.0)
ErC50	> 96.4	ErdwC50	> 96.4
Inhibition of Yield [mg/L]
NOEC	0.375*	NOEC	0.375*
LOEC	1.32	LOEC	1.32
EyC10	< 0.375	EydwC10	0.417 (< 0.375 – 0.686)
EyC20	0.611 (< 0.375 – 0.985)	EydwC20	1.22 (0.746 – 1.80)
EyC50	6.86 (3.52 – 16.7)	EydwC50	30.3 (20.3 – 50.1)

 

* = Measured recoveries <LOQ. Estimated as ½ LOQ.

Description of key information

In an OECD 221 guideline study, Reactive Blue 160 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 (measured initial test item concentrations): The EC50-value for inhibition of the specific growth rate of fronds (ErC50) was >96.4 mg/L. The 7-d NOErC was determined at 0.375 mg/L.

Key value for chemical safety assessment

EC50 for freshwater plants:

96.4 mg/L

EC10 or NOEC for freshwater plants:

0.375 mg/L