Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[ethyltoluenesulphonate]

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:

2017-07-12 to 2017-07-19

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

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

Version / remarks:

2009

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[ethyltoluenesulphonate]

EC Number:

280-163-4

EC Name:

Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)diimino]bis[ethyltoluenesulphonate]

Cas Number:

83027-61-6

Molecular formula:

C32H28N2Na2O8S2

IUPAC Name:

Reaction mass of disodium 2-ethyl-3-({4-[(2-ethyl-6-methyl-3-sulfonatophenyl)amino]-9,10-dioxo-9,10-dihydroanthracen-1-yl}amino)-4-methylbenzene-1-sulfonate, disodium 4-ethyl-3-({4-[(2-ethyl-6-methyl-3-sulfonatophenyl)amino]-9,10-dioxo-9,10-dihydroanthracen-1-yl}amino)-2-methylbenzene-1-sulfonate and disodium 4-ethyl-3-({4-[(6-ethyl-2-methyl-3-sulfonatophenyl)amino]-9,10-dioxo-9,10-dihydroanthracen-1-yl}amino)-2-methylbenzene-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:

Preparation of the Test item solution
Five test item solutions were freshly prepared with dilution water and agitated until the solutions were visually clear. For each test item solution an appropriate amount of the test item was weighed out and inserted into a glass bottle with an appropriate amount of dilution water excluding the test concentration 0.391 mg/L (see below). The concentrations are based on the results of a preliminary range finding test.

Test concentration
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 test item/L. Due to the low amount of test item needed, the lowest concentration of 0.391 mg/L was prepared by dilution of the test concentration 1.56 mg/L with dilution water.

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, 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

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

1.56 mg/L

Nominal / measured:

nominal

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.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: frond number growth rate

Effect concentrations 3

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

10.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: frond number yield

Remarks on result:

other: CI: 6.64 - 19.2

Effect concentrations 4

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dry weight growth rate

Effect concentrations 5

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

27.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: dry weight yield

Remarks on result:

other: CI: 18.8 - 40.6

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 2017-06-02 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 Acid Blue 204.

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:

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 (frond number and dry weight) 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. The Normality test failed for calculation of yield (dry weight). Therefore, the data were transformed (Y=Y(Log)).

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.

Any other information on results incl. tables

Frond Numbers

Nominal item concentration [mg/L]	Repl. No.	Frond numbers per study day
		0 days*	2 days	5 days	7 days
100	1	12	16	32	39
	2	12	15	29	35
	3	12	19	31	32
	Mean	12	17	31	35
25.0	1	12	19	36	39
	2	12	17	32	35
	3	12	16	31	38
	Mean	12	17	33	37
6.25	1	12	22	48	67
	2	12	19	40	53
	3	12	21	41	55
	Mean	12	21	43	58
1.56	1	12	24	53	84
	2	12	19	39	61
	3	12	20	47	75
	Mean	12	21	46	73
0.391	1	12	19	42	78
	2	12	18	41	80
	3	12	21	46	77
	Mean	12	19	43	78
Control	1	12	19	46	90
	2	12	19	40	80
	3	12	21	59	100
	4	12	20	38	83
	5	12	20	46	80
	6	12	19	46	82
	Mean	12	20	46	86

* = 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 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				0.168		40				27		63		4.12
		2				0.153		46				23		69		4.53
		3				0.140		50				20		73		4.95
		Mean		(+)		0.154		45		(+)		23		68		4.53
25.0		1				0.168		40				27		63		4.12
		2				0.153		46				23		69		4.53
		3				0.165		41				26		65		4.21
		Mean		(+)		0.162		42		(+)		25		66		4.29
6.25		1				0.246		13				55		25		2.82
		2				0.212		24				41		44		3.27
		3				0.217		23				43		42		3.19
		Mean		(+)		0.225		20		(+)		46		37		3.09
1.56		1				0.278		1				72		2		2.49
		2				0.232		17				49		34		2.98
		3				0.262		7				63		15		2.65
		Mean		(-)		0.257		8		(-)		61		17		2.71
0.391		1				0.267		5				66		11		2.59
		2				0.271		4				68		8		2.56
		3				0.266		6				65		12		2.61
		Mean		(-)		0.268		5		(-)		66		10		2.59
Control		1				0.288						78				2.41
		2				0.271						68				2.56
		3				0.303						88				2.29
		4				0.276						71				2.51
		5				0.271						68				2.56
		6				0.275						70				2.52
		Mean				0.281						74				2.47

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 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	6.4		0.166	40		4.4	64
	2	5.8		0.152	45		3.8	69
	3	6.3		0.164	41		4.3	64
	Mean	6.2	(+)	0.161	42	(+)	4.2	66
25.0	1	8.8		0.212	24		6.8	44
	2	7.5		0.189	32		5.5	55
	3	8.5		0.207	26		6.5	46
	Mean	8.3	(+)	0.202	27	(+)	6.3	48
6.25	1	12.5		0.262	6		10.5	13
	2	11.5		0.250	10		9.5	21
	3	11.3		0.247	11		9.3	23
	Mean	11.8	(+)	0.253	9	(+)	9.8	19
1.56	1	14.9		0.287	-3		12.9	-7
	2	12.2		0.258	7		10.2	16
	3	13.5		0.273	2		11.5	5
	Mean	13.5	(-)	0.273	2	(-)	11.5	5
0.391	1	14.1		0.279	0		12.1	0
	2	13.2		0.270	3		11.2	7
	3	13.6		0.274	1		11.6	4
	Mean	13.6	(-)	0.274	1	(-)	11.6	4
Control	1	14.1		0.279			12.1
	2	13.3		0.271			11.3
	3	17.0		0.306			15.0
	4	13.0		0.267			11.0
	5	13.9		0.277			11.9
	6	13.0		0.267			11.0
	Mean	14.1		0.278			12.1

The initial biomass dry weight was 2.0 mg per replicate.

Repl. No. = replicate number

 

Colony Number (Plants) on Days 0 and 7

 

Nominal item concentration [mg/L]		Replicate No.		Colony number
			Day 0		Day 7
100		1		4		5
		2		4		4
		3		4		4
		Mean		4		4
25.0		1		4		4
		2		4		4
		3		4		5
		Mean		4		4
6.25		1		4		6
		2		4		5
		3		4		4
		Mean		4		5
1.56		1		4		6
		2		4		5
		3		4		5
		Mean		4		5
0.391		1		4		5
		2		4		5
		3		4		4
		Mean		4		5
Control		1		4		7
		2		4		5
		3		4		6
		4		4		5
		5		4		4
		6		4		6
		Mean		4		6

 Further Observations on Days 2, 5 and 7

Nominal item concentration [mg/L]		Observations on day
		2		5		7
100		1		2.1 + 2.5 + 3.3 +		2.1 + 2.5 ++ 3.3 +
25.0		1		1		2.1 + 2.5 +
6.25		1		1		1
1.56		1		1		1
0.391		1		1		1
Control		1		1		1

 

1      = no observedeffects

2.1   = chlorosis

2.5  = fronds are smaller, compared to the control

3.3  =roots colored

+      = slight effects

++    = medium effects

+++  = strong effects

 

Observations were made compared to the appearance of control colonies (plants) and test media. The test solutions were concentration related blue throughout the exposure.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Acid Blue 204 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 of fronds (ErC50) and dry weight (ErdwC50) were > 100 mg/L, respectively. The EC50-values for inhibition of yield (fronds, EyC50) and dry weight inhibition of yield (EydwC50) with 95% confidence intervals were 10.1 (6.64 – 19.3) mg/L and 27.1 (18.8 – 40.6) mg/L, respectively.

Executive summary:

The effects of the test item Acid Blue204 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 from2017-07-12 to 2017-07-19.

 

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. 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 Acid Blue 204 were determined at the start of the exposure in the fresh media (0 h) and at the end of the exposure in the old media (7 d) of all tested concentration levels and the control via HPLC-DAD.

The measured concentrations of Acid Blue204 in fresh media were in the range of < LOQ to 100% of the nominal values and < LOQ to 100% in the old media. The measured test item concentrations were within±20% of the nominal concentrations, except the lowest test item concentration where values were <LOQ at all sampling points. Since biological effects were only found >1.56 mg/L, this is considered to have no effect on validity or evaluation of the study.

NOEC-, LOEC-, EC-Values and 95 % Confidence Intervals ofAcid Blue204after 7 Days of Exposure

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

Frond number		Dry weight
Growth Rate Inhibition [mg/L]
NOEC	1.56	NOEC	1.56
LOEC	6.25	LOEC	6.25
ErC10	2.86 (0.391 – 5.08)	ErdwC10	7.09 (4.49 – 10.3)
ErC20	6.16 (3.98 – 9.29)	ErdwC20	15.6 (11.7 – 20.3)
ErC50	> 100	ErdwC50	> 100
Inhibition of Yield [mg/L]
NOEC	1.56	NOEC	1.56
LOEC	6.25	LOEC	6.25
EyC10	< 0.391	EydwC10	3.12 (1.19 – 5.69)
EyC20	2.57 (< 0.391 – 4.75)	EydwC20	6.68 (4.24 – 9.69)
EyC50	10.1 (6.64 – 19.2)	EydwC50	27.1 (18.8 – 40.6)