Reaction mass of disodium 2-({4-methyl-2-[(4-{[5-methyl-1-(2-sulfonatophenyl)hexan-3-yl]amino}-9,10-dioxo-9,10-dihydroanthracen-1-yl)methyl]pentyl}amino)benzene-1-sulfonate,disodium 4-{5-methyl-3-[(4-{[5-methyl-1-(4-sulfonatophenyl)hexan-3-yl]amino}-9,10-dioxo-9,10-dihydroanthracen-1-yl)amino]hexyl}benzene-1-sulfonate anddisodium 4-{5-methyl-3-[(4-{[5-methyl-1-(2-sulfonatophenyl)hexan-3-yl]amino}-9,10-dioxo-9,10-dihydroanthracen-1-yl)amino]hexyl}benzene-1-sulfonate

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:

2017-10-25 to 2017-12-21, with the definitive exposure phase from 2017-12-13 to 2017-12-20

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:

Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)bis[imino[3-isopropylpropane-1,3-diyl]]]bis(benzenesulphonate)

EC Number:

276-822-0

EC Name:

Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)bis[imino[3-isopropylpropane-1,3-diyl]]]bis(benzenesulphonate)

Cas Number:

72749-90-7

Molecular formula:

C40H46N2O8S2.2Na

IUPAC Name:

Disodium [(9,10-dihydro-9,10-dioxo-1,4-anthrylene)bis[imino[3-isopropylpropane-1,3-diyl]]]bis(benzenesulphonate)

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 and agitated until the solution was visually clear. The test item solution was clear and blue colored.

Test concentrations
Based on the results of a preliminary range finding 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. As the inhibition values were higher than expected, the study was repeated with 6 nominal test item concentration levels in a geometrical series with a dilution factor of 4: 0.0977 - 0.391 - 1.56 - 6.25 - 25.0 - 100 mg/L. The geometric mean measured test item concentrations were calculated to be 0.500* – 0.500* – 1.22 – 5.29 – 17.2 – 79.6 mg/L (*1/2 LOQ).

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

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

Test temperature:

see any other information on materials and methods

pH:

see any other information on materials and methods

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. Random repositioning of the test vessels when observation were
made 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:

NOEC

Effect conc.:

< 0.5 mg/L

Nominal / measured:

meas. (geom. mean)

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.:

0.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Frond number and Dry Weight

Effect concentrations 3

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.652 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Frond number

Remarks:

CI (< 0.500 - 0.969)

Effect concentrations 4

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

1.67 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Dry Weight

Remarks:

CI (0.970 - 3.04)

Effect concentrations 5

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

> 79.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: Frond number and Dry weight

Effect concentrations 6

Duration:

7 d

Dose descriptor:

NOEC

Effect conc.:

< 0.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: Frond number and Dry weight

Effect concentrations 7

Duration:

7 d

Dose descriptor:

LOEC

Effect conc.:

0.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: Frond number and Dry weight

Effect concentrations 8

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

0.346 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: Frond number

Remarks:

CI (< 0.500 - 0.504)

Effect concentrations 9

Duration:

7 d

Dose descriptor:

EC10

Effect conc.:

< 0.5 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: Dry weight

Effect concentrations 10

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

0.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: Frond number

Remarks:

CI (< 0.500 - 0.919)

Effect concentrations 11

Duration:

7 d

Dose descriptor:

EC20

Effect conc.:

1.13 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: Dry weight

Remarks:

CI (< 0.500 - 2.42)

Effect concentrations 12

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

2.72 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: Frond number

Remarks:

CI (1.66 - 5.16)

Effect concentrations 13

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

10.6 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

other: Inhibition of Yield

Remarks on result:

other: Dry weight

Remarks:

CI (6.24 - 21.4)

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 Blue 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 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 -value (acceptable probability of incorrectly concluding that there is a difference) was =0.05. Normality test failed for calculation of yield (fronds). 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 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 in the second Definitive Test

Nominal test item concentration [mg/L]	Geometric mean measured test item concentration [mg/L]	Repl. No.	Frond numbers per study day
			0 days*	2 days	5 days	7 days
100	79.6	1	12	18	36	41
		2	12	20	31	35
		3	12	18	34	37
		Mean	12	19	34	38
25.0	17.2	1	12	21	36	42
		2	12	19	29	41
		3	12	20	31	40
		Mean	12	20	32	41
6.25	5.29	1	12	18	38	45
		2	12	19	38	47
		3	12	19	39	48
		Mean	12	19	38	47
1.56	1.22	1	12	20	47	65
		2	12	19	42	63
		3	12	20	48	74
		Mean	12	20	46	67
0.391	0.500	1	12	19	45	74
		2	12	20	45	70
		3	12	21	53	83
		Mean	12	20	48	76
0.0977	0.500	1	12	20	57	94
		2	12	21	59	94
		3	12	20	49	82
		Mean	12	20	55	90
Control		1	12	21	48	103
		2	12	21	55	92
		3	12	22	64	109
		4	12	19	55	86
		5	12	20	41	83
		6	12	20	67	108
		Mean	12	21	55	97

* = 3 colonies with 4 fronds each per replicate were inoculated at start of the exposure

Growth Rate and Yield Inhibition based on Fronds after 7 d in the second Definitive Test

               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]	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]
100	79.6	1		0.176	41		29	66	3.95
		2		0.153	49		23	73	4.53
		3		0.161	46		25	71	4.31
		Mean	(+)	0.163	45	(+)	26	70	4.26
25.0	17.2	1		0.179	40		30	65	3.87
		2		0.176	41		29	66	3.95
		3		0.172	42		28	67	4.03
		Mean	(+)	0.175	41	(+)	29	66	3.95
6.25	5.29	1		0.189	36		33	61	3.67
		2		0.195	34		35	59	3.55
		3		0.198	33		36	58	3.50
		Mean	(+)	0.194	35	(+)	35	59	3.57
1.56	1.22	1		0.241	19		53	38	2.87
		2		0.237	20		51	40	2.93
		3		0.260	13		62	27	2.67
		Mean	(+)	0.246	17	(+)	55	35	2.82
0.391	0.500	1		0.260	13		62	27	2.67
		2		0.252	15		58	32	2.75
		3		0.276	7		71	16	2.51
		Mean	(+)	0.263	12	(+)	64	25	2.64
0.0977	0.500	1		0.294	1		82	3	2.36
		2		0.294	1		82	3	2.36
		3		0.275	8		70	17	2.52
		Mean	(-)	0.288	3	(-)	78	8	2.41
Control		1		0.307			91		2.26
		2		0.291			80		2.38
		3		0.315			97		2.20
		4		0.281			74		2.46
		5		0.276			71		2.51
		6		0.314			96		2.21
		Mean		0.297			85		2.34

                                           

Repl. No. = replicate number

 

Specific Growth Rate and Yield Inhibition of Dry Weight after 7 d in the second Definitive Test

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]	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   [%]
100	79.6	1	5.8		0.251	32		4.8	61
		2	5.4		0.241	35		4.4	64
		3	5.5		0.244	34		4.5	63
		Mean	5.6	(+)	0.245	34	(+)	4.6	63
25.0	17.2	1	6.9		0.276	25		5.9	52
		2	7.0		0.278	25		6.0	51
		3	6.2		0.261	29		5.2	58
		Mean	6.7	(+)	0.272	26	(+)	5.7	54
6.25	5.29	1	8.8		0.311	16		7.8	37
		2	7.7		0.292	21		6.7	46
		3	8.0		0.297	20		7.0	43
		Mean	8.2	(+)	0.300	19	(+)	7.2	42
1.56	1.22	1	10.8		0.340	8		9.8	20
		2	10.2		0.332	10		9.2	25
		3	11.7		0.351	5		10.7	13
		Mean	10.9	(+)	0.341	8	(+)	9.9	20
0.391	0.500	1	10.5		0.336	9		9.5	23
		2	9.9		0.328	11		8.9	28
		3	11.9		0.354	4		10.9	11
		Mean	10.8	(+)	0.339	8	(+)	9.8	21
0.0977	0.500	1	12.9		0.365	1		11.9	3
		2	12.7		0.363	2		11.7	5
		3	10.9		0.341	8		9.9	20
		Mean	12.2	(-)	0.357	3	(-)	11.2	9
Control		1	13.9		0.376			12.9
		2	13.6		0.373			12.6
		3	13.9		0.376			12.9
		4	11.9		0.354			10.9
		5	11.5		0.349			10.5
		6	15.1		0.388			14.1
		Mean	13.3		0.369			12.3

The mean initial biomass dry weight was 1.0 mg per replicate.

Repl. No. = replicate number

Colony Number (Plants) on Days 0 and 7 in the second Definitive Test

 

Nominal test item concentration [mg/L]	Geometric mean measured test item concentration [mg/L]	Replicate No.	Colony number
			Day 0	Day 7
100	79.6	1	3	6
		2	3	5
		3	3	6
		Mean	3	6
25.0	17.2	1	3	6
		2	3	6
		3	3	4
		Mean	3	5
6.25	5.29	1	3	6
		2	3	7
		3	3	5
		Mean	3	6
1.56	1.22	1	3	8
		2	3	8
		3	3	7
		Mean	3	8
0.391	0.500	1	3	7
		2	3	8
		3	3	7
		Mean	3	7
0.0977	0.500	1	3	8
		2	3	9
		3	3	9
		Mean	3	9
Control		1	3	12
		2	3	11
		3	3	14
		4	3	8
		5	3	10
		6	3	13
		Mean	3	11

 

 

Further Observations on Days 2, 5 and 7 in the second Definitive Test

Nominal test item concentration [mg/L]	Geometric mean measured test item concentration [mg/L]	Observations on day
		2	5	7
100	79.6	3.3 ++	3.3 ++	2.3 + 2.5 + 3.3 ++
25.0	17.2	3.3 +	3.3 +	2.5 + 3.3 ++
6.25	5.29	1	1	3.3 +
1.56	1.22	1	1	1
0.391	0.500	1	1	1
0.0977	0.500	1	1	1
Control		1	1	1

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

 

1      = no observedeffects

2.3   = gibbosity

2.5  = fronds are smaller, compared to the control

3.3   = discoloration of roots

+      = slight effects

++    = medium effects

+++         = strong effects

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

In this study, Acid Blue 221 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 inhibition of the specific growth rate (fronds and dry weight) (ErC50, ErdwC50) were > 100 mg/L (geometric mean measured: 79.6 mg/L) and yield (fronds and dry weight) (EyC50, EydwC50) were 3.60 (2.31 – 6.24) mg/L and 15.2 (8.36 – 30.2) mg/L (geometric mean measured: 2.72 (1.66 – 5.16) mg/L and 10.6 (6.24 – 21.4) mg/L), respectively.

Executive summary:

The effects of the test item Acid Blue 221 on the growth of the monocotyledonous aquatic plant species Lemna gibba were determined according to the principles of OECD 221 at the test facility from2017-10-25 to 2017-12-21, with the definitive exposure phase from 2017-12-13 to 2017-12-20.

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. As the inhibition values were higher than expected, the study was repeated with 6 nominal test item concentration levels in a geometrical series with a dilution factor of 4:0.0977 - 0.391 - 1.56 - 6.25 - 25.0 - 100 mg/L. The second study is considered to be the main study, but as the first study is also valid, its results will be reported, too. 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 blue coloured throughout the exposure.The validity criteria of the test guideline were fulfilled.

The concentrations ofthe test item Acid Blue 221 and the control were analysed via HPLC-DAD at the beginning and end of the exposure.

At the start of the exposure the measured concentrations of Acid Blue 221 were in the range of < LOQ and 90% of the nominal values. At the end of the exposure the measured concentrations were between < LOQ and 80% of the nominal values. The geometric mean measured test item concentrations were calculated to be 0.500* – 0.500* – 1.22 – 5.29 – 17.2 – 79.6 mg/L (*1/2 LOQ). . All effect values are given based on the nominal and geometric mean measured test item concentrations.

 

 

NOEC-, LOEC-, EC-Values and 95% Confidence Intervals of Acid Blue221
after 7 Days of Exposure

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

Frond number		Dry weight
Growth Rate Inhibition [mg/L]
NOEC	0.0977	NOEC	0.0977
LOEC	0.391	LOEC	0.391
ErC10	0.470 (0.246 – 0.817)	ErdwC10	1.70 (0.817 – 3.13)
ErC20	1.64 (1.09 – 2.34)	ErdwC20	8.89 (5.86 – 14.2)
ErC50	> 100	ErdwC50	> 100
Inhibition of Yield[mg/L]
NOEC	0.0977	NOEC	0.0977
LOEC	0.391	LOEC	0.391
EyC10	0.108 (< 0.0977 – 0.222)	EydwC10	< 0.0977
EyC20	0.348 (0.197 – 0.601)	EydwC20	0.987 (0.352 – 2.17)
EyC50	3.60 (2.31 – 6.24)	EydwC50	15.2 (8.36 – 30.2)

 

 

NOEC-, LOEC-, EC-Values and 95% Confidence Intervals ofAcid Blue221
after 7 Days of Exposure

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

Frond number		Dry weight
Growth Rate Inhibition [mg/L]
NOEC	< 0.500		< 0.500
LOEC	0.500		0.500
ErC10	0.652 (< 0.500 – 0.969)		1.67 (0.970 – 3.04)
ErC20	1.50 (1.03 – 2.20)		6.75 (4.40 – 10.2)
ErC50	>79.6		>79.6
Inhibition of Yield [mg/L]
NOEC	< 0.500		< 0.500
LOEC	0.500		0.500
EyC10	0.346 (< 0.500 – 0.504)		< 0.500
EyC20	0.600 (< 0.500 – 0.919)		1.13 (< 0.500 – 2.42)
EyC50	2.72 (1.66 – 5.16)		10.6 (6.24 – 21.4)