Registration Dossier

Ecotoxicological information

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 2017-10-19 to 2017-10-30, with the definitive exposure phase from 2017-10-20 to 2017-10-27
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
Qualifier:
according to guideline
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium 1-amino-9,10-dihydro-4-[[4-[[methyl[(4-methylphenyl)sulphonyl]amino]methyl]phenyl]amino]-9,10-dioxoanthracene-2-sulphonate
EC Number:
276-889-6
EC Name:
Sodium 1-amino-9,10-dihydro-4-[[4-[[methyl[(4-methylphenyl)sulphonyl]amino]methyl]phenyl]amino]-9,10-dioxoanthracene-2-sulphonate
Cas Number:
72828-82-1
Molecular formula:
C29H25N3O7S2.Na
IUPAC Name:
sodium 1-amino-4-{[4-({methyl[(4-methylphenyl)sulfonyl]amino}methyl)phenyl]amino}-9,10-dioxo-9,10-dihydroanthracene-2-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). The samples were analysed 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

Details on test solutions:
Preparation of the Test item solution
A test item solution of 100 mg test item/L was prepared once 24 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out. The test item was applied onto a glass slide. The glass slide with the test item was inserted into a glass bottle with an appropriate amount of dilution water. The test item solution was stirred for about 24 ± 1 hours (1100 rpm, room temperature) with a magnetic stirrer. Undissolved particles were removed by membrane filtration (membrane filter 0.45 µm, RC, MACHEREY-NAGEL). The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the test item solution, was used as a test item solution in the test. During filtration, the filter was always be kept covered.
The test item solution was checked via laser beam (Tyndall effect) for undissolved test item, which was negative. The test item solution was clear and slightly blue colored.

Test concentrations
Based on the results of a preliminary range finding test (see section 18). 5 concentrations were tested with a dilution factor of √10: 1.00 - 3.16 - 10.0 - 31.6 - 100% of the saturated solution, corresponding to the geometric mean measured test item concentrations: 0.0750 – 0.0750 – 0.0886 – 0.224 – 0.754 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, 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, 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 concentrationsopen allclose all
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
< 1 other: % of the saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate frond number
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
< 1 other: % of the saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: yield frond number
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 1 other: % saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate and yield (dry weight)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: % of the saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: growth rate (frond number and dry weight)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
6.91 other: % of the saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: frond number yield
Remarks on result:
other: CI: 3.38 - 18.5
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: % of the saturated solution
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: dry weight yield
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 Red 337.

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:
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, 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.
Welsh-t-test after Bonferroni-Holm 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. Since lower confidence intervals to the EC10-value for Inhibition of growth rate for frond numbers and dry weight and to the EC20-value for Inhibition of yield for frond numbers and dry weight were below the lowest tested concentration, the value was given as an estimation.

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
[% of the saturated solution]

Geometric mean measured test item concentration
[mg/L]

Repl.

No.

Frond numbers per study day

0 days*

3 days

5 days

7 days

100

0.754

1

12

22

36

40

2

12

22

31

39

3

12

24

35

41

Mean

12

23

34

40

 31.6

0.224

1

12

23

33

36

2

12

24

34

42

3

12

25

36

46

Mean

12

24

34

41

 10.0

0.0886

1

12

26

35

49

2

12

23

40

44

3

12

22

32

49

Mean

12

24

36

47

  3.16

0.0750

1

12

26

40

60

2

12

27

41

61

3

12

26

39

52

Mean

12

26

40

58

  1.00

0.0750

1

12

28

50

80

2

12

23

39

60

3

12

29

45

81

Mean

12

27

45

74

Control

1

12

27

53

91

2

12

27

44

85

3

12

26

45

84

4

12

28

52

93

5

12

26

46

92

6

12

29

42

80

Mean

12

27

47

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 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
[% of the saturated solution]

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

0.754

1

 

0.172

39

 

28

63

4.03

2

 

0.168

41

 

27

64

4.12

3

 

0.176

38

 

29

62

3.95

Mean

(+)

0.172

39

(+)

28

63

4.03

 31.6

0.224

1

 

0.157

45

 

24

68

4.42

2

 

0.179

37

 

30

60

3.87

3

 

0.192

32

 

34

55

3.61

Mean

(+)

0.176

38

(+)

29

61

3.97

 10.0

0.0886

1

 

0.201

29

 

37

51

3.45

2

 

0.186

35

 

32

58

3.73

3

 

0.201

29

 

37

51

3.45

Mean

(+)

0.196

31

(+)

35

53

3.54

  3.16

0.0750

1

 

0.230

19

 

48

36

3.01

2

 

0.232

18

 

49

35

2.98

3

 

0.209

26

 

40

47

3.31

Mean

(+)

0.224

21

(+)

46

40

3.10

  1.00

0.0750

1

 

0.271

5

 

68

10

2.56

2

 

0.230

19

 

48

36

3.01

3

 

0.273

4

 

69

9

2.54

Mean

(+)

0.258

9

(-)

62

18

2.70

Control

1

 

0.289

 

 

79

 

2.39

2

 

0.280

 

 

73

 

2.48

3

 

0.278

 

 

72

 

2.49

4

 

0.293

 

 

81

 

2.37

5

 

0.291

 

 

80

 

2.38

6

 

0.271

 

 

68

 

2.56

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 (-).

 

Nominal test item concentration
[% of the saturated solution]

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

0.754

1

6.6

 

0.369

21

 

6.1

52

2

7.3

 

0.383

18

 

6.8

46

3

7.2

 

0.381

19

 

6.7

47

Mean

7.0

(+)

0.378

19

(+)

6.5

49

 31.6

0.224

1

7.6

 

0.389

17

 

7.1

44

2

7.2

 

0.381

19

 

6.7

47

3

8.2

 

0.400

15

 

7.7

39

Mean

7.7

(+)

0.390

17

(+)

7.2

44

 10.0

0.0886

1

9.1

 

0.414

11

 

8.6

32

2

8.3

 

0.401

14

 

7.8

39

3

7.6

 

0.389

17

 

7.1

44

Mean

8.3

(+)

0.402

14

(+)

7.8

38

  3.16

0.0750

1

8.9

 

0.411

12

 

8.4

34

2

8.5

 

0.405

14

 

8.0

37

3

8.3

 

0.401

14

 

7.8

39

Mean

8.6

(+)

0.406

13

(+)

8.1

36

  1.00

0.0750

1

13.0

 

0.465

1

 

12.5

2

2

8.8

 

0.410

12

 

8.3

35

3

11.0

 

0.442

6

 

10.5

17

Mean

10.9

(+)

0.439

6

(+)

10.4

18

Control

1

11.8

 

0.452

 

 

11.3

 

2

13.9

 

0.475

 

 

13.4

 

3

12.2

 

0.456

 

 

11.7

 

4

14.4

 

0.480

 

 

13.9

 

5

14.1

 

0.477

 

 

13.6

 

6

13.1

 

0.467

 

 

12.6

 

Mean

13.2

 

0.468

 

 

12.7

 

The initial biomass dry weight was 0.5 mg per replicate.

Repl. No. = replicate number

 

Colony Number (Plants) on Days 0 and 7

 

Nominal test item concentration
[% of the saturated solution]

Geometric mean measured test item concentration
[mg/L]

Replicate

No.


Colony number

Day 0

Day 7

100

0.754

1

3

4

2

3

4

3

3

6

Mean

3

5

 31.6

0.224

1

3

4

2

3

5

3

3

5

Mean

3

5

 10.0

0.0886

1

3

4

2

3

5

3

3

3

Mean

3

4

  3.16

0.0750

1

3

6

2

3

5

3

3

6

Mean

3

6

  1.00

0.0750

1

3

7

2

3

4

3

3

7

Mean

3

6

Control

1

3

10

2

3

7

3

3

7

4

3

8

5

3

10

6

3

6

Mean

3

8


Further Observations on Days 3, 5 and 7

Nominal test item concentration
[% of the saturated solution]

Geometric mean measured test item concentration
[mg/L]

Observations on day

3

5

7

100

0.754

3.3 +

3.3 ++

3.3 ++

 31.6

0.224

1

1

1

 10.0

0.0886

1

1

1

  3.16

0.0750

1

1

1

  1.00

0.0750

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, Acid Blue 264 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) (ErC50) and growth rate and yield (dry weight) (ErdwC50, EydwC50) were > 100% of the saturated solution, respectively.
The effect values based on geometric mean measured test item concentrations are: The EC50-values for inhibition of the specific growth rate (fronds) (ErC50) and growth rate and yield (dry weight) (ErdwC50, EydwC50) were > 0.754 mg/L, respectively.
Executive summary:

The effects of the test item Acid Blue264 on the growth of the monocotyledonous aquatic plant species Lemna gibba was determined according to the principles of OECD 221 at the test facility from2017-10-19 to 2017-10-30, with the definitive exposure phase from 2017-10-20 to 2017-10-27.

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% of the saturated solution, corresponding to the geometric mean measured test item concentrations: 0.0750* – 0.0750* – 0.0886 – 0.224 – 0.754 mg/L

(* ½ LOQ). 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. 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 itemAcid Blue264 and the control were analysed via HPLC-DAD at the beginning and end of the exposure.

At the start of the exposure the initial measured concentrations of Acid Blue 264 were 1.15 – 0.350 – 0.105 – < LOQ – < LOQ mg/L. At the end of the exposure the measured concentrations were between < LOQ and 43% of the initial concentrations. 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 Blue264 after 7 Days of Exposure

                  (based on the nominal test item concentration [% of the saturated solution])

Frond number

Dry weight

Growth Rate Inhibition [% of the saturated solution]

NOEC

< 1.00

NOEC

< 1.00

LOEC

  1.00

LOEC

  1.00

ErC10

1.09 (< 1.00 – 2.12)

ErdwC10

2.16 (< 1.00 – 4.60)

ErC20

2.89 (1.62 – 4.83)

ErdwC20

> 100

ErC50

> 100

ErdwC50

> 100

Inhibition of Yield [% of the saturated solution]

NOEC

1.00

NOEC

< 1.00

LOEC

3.16

LOEC

  1.00

EyC10

< 1.00

EydwC10

< 1.00

EyC20

1.09 (< 1.00 – 2.06)

EydwC20

1.09 (< 1.00 – 1.70)

EyC50

6.91 (3.38 – 18.5)

EydwC50

> 100