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 2018-06-06 to 2018-06-18, with the definitive exposure phase from 2018-06-06 to 2018-06-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
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

Constituent 1
Chemical structure
Reference substance name:
1-ethyl-2-[1,2,3,4-tetrahydro-1-(2-hydroxyethyl)-2,2,4-trimethyl-6-quinolyl]benz[cd]indolium chloride
EC Number:
264-497-8
EC Name:
1-ethyl-2-[1,2,3,4-tetrahydro-1-(2-hydroxyethyl)-2,2,4-trimethyl-6-quinolyl]benz[cd]indolium chloride
Cas Number:
63817-45-8
Molecular formula:
C27H31N2O.Cl
IUPAC Name:
2-ethyl-3-[1-(2-hydroxyethyl)-2,2,4-trimethyl-1,2,3,4-tetrahydroquinolin-6-yl]-2-azatricyclo[6.3.1.0^{4,12}]dodeca-1(11),2,4(12),5,7,9-hexaen-2-ium chloride
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 first interval. The control, the lowest and the highest test item concentration were analytically verified at the end of the exposure (7 days) and on every renewal day. Freshly prepared and old media were analyzed. The samples were analyzed 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

Vehicle:
no
Details on test solutions:
Preparation of the Test item solution
A stock solution with a nominal test item concentration of 10.0 mg/L was freshly prepared prior to test start and every media renewal with dilution water and treated with ultrasound for 10 minutes at room temperature. The test item solution was checked via laser beam (Tyndall effect) for undissolved test item. Neither Tyndall effect nor presence of undissolved test item during the test nor was observed. The concentrations are based on the results of a preliminary range finding test.

Test concentration
Out of the stock solution five dilution levels were prepared with dilution water in a geometrical series with a separation factor of √10 and tested as follows: 0.0100 - 0.0316 - 0.100 - 0.316 - 1.00 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 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
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

Composition of Dilution water
Component Concentration in stock solution [g/L] Concentration in prepared medium [mg/L]
NaNO3 26 510
MgCl2 x 6 H2O 12 240
CaCl2 x 2 H2O 4.4 90
MgSO4 x 7 H2O 15 290
K2HPO4 · 3 H2O 1.4 30
NaHCO3 15 300
H3BO3 0.19 3.7
MnCl2 x 4 H2O 0.42 8.3
FeCl3 x 6 H2O 0.16 3.2
Na2-EDTA · 2 H2O 0.30 6.0
ZnCl2 3.3 mg/L 66 µg/L
CoCl2 x 6 H2O 1.4 mg/L 29 µg/L
Na2MoO4 x 2 H2O 7.3 mg/L 145 µg/L
CuCl2 x 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:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d

Test conditions

Hardness:
not measured
Test temperature:
Room temperature [°C]: min.: 23.2 max.: 24.7 mean value: 24.1
pH:
Geometric mean measured test item concentration
[µg/L] pH-value
0 h (Fresh medium) 7 d (Old medium)
839 7.59 8.20
263 7.59 8.25
74.5 7.58 8.29
21.5 7.59 8.38
8.91 7.59 8.45
Control 7.60 8.33
Dissolved oxygen:
not measured
Salinity:
not measured
Conductivity:
not measured
Nominal and measured concentrations:
Nominal: 0.0100 - 0.0316 - 0.100 - 0.316 - 1.00 mg/L
geometric mean measured test item concentrations: 8.91 – 21.5 – 74.5 – 263 – 839 µg/L
Details on test conditions:
Test method
Semi-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.

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:
LOEC
Effect conc.:
31.6 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Frond number and Dry weight
Remarks on result:
other: Growth Rate and Inhibition of Yield
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
10 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Frond number and dry weight
Remarks on result:
other: Growth Rate and Inhibition of Yield
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
296 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Growth Rate Inhibition frond number
Remarks on result:
other: CI: 235 - 384
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
51 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry weight Inhibition of yield
Remarks on result:
other: CI: 43.8 - 59.8
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
53.6 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: frond number ihibition of yield
Remarks on result:
other: CI: 42.0 - 71.4
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
207 µg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry weight Growth Rate Inhibition
Remarks on result:
other: CI: 176 - 246
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 2018-04-04 to 2018-04-11 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 Basic Blue 147 Chloride.

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 7.68 5.82 ± 3.18
95% confidence interval 7.31 to > 8.00
Yield inhibition (number of fronds)
EyC50 4.93 4.67 ± 2.87
95% confidence interval 4.42 to 5.53
Growth rate inhibition (dry weight)
ErdwC50 > 8.00 5.61 ± 2.76
95% confidence interval Not applicable
Yield inhibition (dry weight)
EydwC50 5.95 4.75 ± 2.49
95% confidence interval 5.18 to 6.75
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 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 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 straight line and sigmoidal dose-response regression, respectively. 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

Geometric mean measured test item concentration
[µg/L]

Repl.

No.

Frond numbers per study day

0 days*

2 days

5 days

7 days

839

1

12

22

25

32

2

12

23

26

32

3

12

24

23

34

Mean

12

23

25

33

263

1

12

27

54

82

2

12

24

50

59

3

12

24

50

68

Mean

12

25

51

70

  74.5

1

12

29

68

80

2

12

28

71

107

3

12

26

76

109

Mean

12

28

72

99

  21.5

1

12

27

94

246

2

12

31

117

283

3

12

32

115

270

Mean

12

30

109

266

    8.91

1

12

32

159

382

2

12

32

122

291

3

12

32

148

408

Mean

12

32

143

360

Control

1

12

29

125

339

2

12

30

141

378

3

12

31

153

386

4

12

32

134

353

5

12

26

117

316

6

12

25

135

344

Mean

12

29

134

353

* = 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 (-).

                                               

Geometric mean measured test item concentration
[µg/L]

Repl.

No.

Average growth rate

[d-1]

Inhibition of average growth rate
[%]

Yield


[fronds]

Inhibition of yield

[%]

Doubling time

[d]

839

1

 

0.140

70.99

 

20

94.13

4.95

2

 

0.140

70.99

 

20

94.13

4.95

3

 

0.149

69.20

 

22

93.54

4.66

Mean

(+)

0.143

70.39

(+)

21

93.93

4.85

263

1

 

0.275

43.16

 

70

79.45

2.52

2

 

0.228

52.89

 

47

86.20

3.05

3

 

0.248

48.70

 

56

83.56

2.80

Mean

(+)

0.250

48.25

(+)

58

83.07

2.79

  74.5

1

 

0.271

43.89

 

68

80.04

2.56

2

 

0.313

35.29

 

95

72.12

2.22

3

 

0.315

34.74

 

97

71.53

2.20

Mean

(+)

0.300

37.97

(+)

87

74.56

2.32

  21.5

1

 

0.431

10.66

 

234

31.32

1.61

2

 

0.452

6.52

 

271

20.46

1.54

3

 

0.445

7.91

 

258

24.27

1.56

Mean

(+)

0.443

8.36

(+)

254

25.35

1.57

    8.91

1

 

0.494

-2.35

 

370

-8.60

1.40

2

 

0.455

5.70

 

279

18.11

1.52

3

 

0.504

-4.30

 

396

-16.23

1.38

Mean

(-)

0.485

-0.32

(-)

348

-2.24

1.43

Control

1

 

0.477

 

 

327

 

1.45

2

 

0.493

 

 

366

 

1.41

3

 

0.496

 

 

374

 

1.40

4

 

0.483

 

 

341

 

1.43

5

 

0.467

 

 

304

 

1.48

6

 

0.479

 

 

332

 

1.45

Mean

 

0.483

 

 

341

 

1.44

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 (-).

 

Geometric mean measured test item concentration
[µg/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
[%]

839

1

4.2

 

0.106

77.26

 

2.2

95.64

2

3.4

 

0.076

83.73

 

1.4

97.23

3

3.4

 

0.076

83.73

 

1.4

97.23

Mean

3.7

(+)

0.086

81.57

(+)

1.7

96.70

263

1

9.5

 

0.223

52.23

 

7.5

85.15

2

8.4

 

0.205

56.01

 

6.4

87.33

3

9.1

 

0.216

53.55

 

7.1

85.94

Mean

9.0

(+)

0.215

53.93

(+)

7.0

86.14

  74.5

1

14.2

 

0.280

39.91

 

12.2

75.84

2

14.9

 

0.287

38.44

 

12.9

74.46

3

14.4

 

0.282

39.48

 

12.4

75.45

Mean

14.5

(+)

0.283

39.28

(+)

12.5

75.25

  21.5

1

32.8

 

0.400

14.25

 

30.8

39.01

2

41.2

 

0.432

7.26

 

39.2

22.38

3

39.2

 

0.425

8.78

 

37.2

26.34

Mean

37.7

(+)

0.419

10.10

(+)

35.7

29.24

    8.91

1

55.1

 

0.474

-1.66

 

53.1

-5.15

2

49.8

 

0.459

1.44

 

47.8

5.35

3

55.1

 

0.474

-1.66

 

53.1

-5.15

Mean

53.3

(-)

0.469

-0.63

(-)

51.3

-1.65

Control

1

48.4

 

0.455

 

 

46.4

 

2

56.1

 

0.476

 

 

54.1

 

3

56.6

 

0.478

 

 

54.6

 

4

55.4

 

0.474

 

 

53.4

 

5

46.9

 

0.451

 

 

44.9

 

6

51.6

 

0.464

 

 

49.6

 

Mean

52.5

 

0.466

 

 

50.5

 

The initial biomass dry weight was 1.6 mg per replicate.

Repl. No. = replicate number

 


Colony Number (Plants) on Days 0 and 7

 

Geometric mean measured test item concentration
[µg/L]

Replicate

No.


Colony number

Day 0

Day 7

839

1

3

12

2

3

11

3

3

12

Mean

3

12

263

1

3

8

2

3

6

3

3

7

Mean

3

7

  74.5

1

3

11

2

3

14

3

3

13

Mean

3

13

  21.5

1

3

28

2

3

27

3

3

28

Mean

3

28

    8.91

1

3

43

2

3

30

3

3

52

Mean

3

42

Control

1

3

42

2

3

43

3

3

45

4

3

30

5

3

30

6

3

46

Mean

3

39

 


 

Further Observations on Days 2, 5 and 7

Geometric mean measured test item concentration
[µg/L]

Observations on day

2

5

7

839

2.3++
2.4+
3.1+++
3.2+++
4.1+

2.1++
2.2++
2.3+++
2.4++
3.1+++
3.3+++
4.1++

2.1++
2.2++
2.3+++
2.4++
2.5+++

3.1+++
3.3+++
4.1+++

263

2.3++
3.1++
3.2++

2.1++
2.3++
2.4+
2.5+
3.1+++
3.3++

2.1++
2.3++
2.4+
2.5+
3.1+++
3.3++

    74.5

3.1+
3.2+ 

2.1+
2.3++
3.1+
3.2+ 

2.1+
2.3++
3.1+
3.2+ 

    21.5

1

2.1+

2.1+

       8.91

1

1

1

Control

1

1

1

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

 

1       = no observedeffects

2.1    = chlorosis
2.2
    = necrosis

2.3   = gibbosity of the fronds

2.4   = discoloration of the fronds

2.5   = smaller fronds

3.1   = shortened roots

3.2  = losses of roots

3.3   = discoloration of roots

4.1   = Break up of plants

+      = slight effects

++    = medium effects

+++  = strong effects

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study, Basic Blue 147 Chloride 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 (geometric mean measured test item concentrations):
The EC50-values for inhibition of the specific growth rate (fronds and dry weight) (ErC50, ErdwC50) were 236 (191 – 301) µg/L and 165 (144 – 191) µg/L and yield (fronds and dry weight) (EyC50, EydwC50) were 37.4 (28.7 – 50.8) mg/L and 35.3 (30.0 – 41.8) µg/L, respectively.
Executive summary:

The effects of the test item Basic Blue147 Chloride on the growth of the monocotyledonous aquatic plant species Lemna gibba was determined according to the principles of OECD 221 at the test facility from 2018-06-06 to 2018-06-18, with the definitive exposure phase from 2018-06-06 to 2018-06-13.

Lemna gibba was exposed to the test item for 7 days under semi-static conditions. Based on a preliminary test, 5 nominal test item concentration levels were tested in a geometrical series with a dilution factor of √10:0.0100 - 0.0316 - 0.100 - 0.316 - 1.00 mg/L, corresponding to the geometric mean measured test item concentrations: 8.91 – 21.5 – 74.5 – 263 – 839 µg/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 blue coloured throughout the exposure.The validity criteria of the test guideline were fulfilled.

The concentrations ofthe test item Basic Blue147 Chloride and the control were analysed via HPLC-DAD at the first interval. The control, the lowest and the highest test item concentration were analytically verified at the end of the exposure and on every renewal day.

At the start of the first interval the measured concentrations of Basic Blue 147 Chloride were in the range of <LOQ to 104% of the nominal values. At the end of the first interval the measured concentrations were between <LOQ to 73% of the nominal values. All effect values are given based on the nominal and the geometric mean measured test item concentrations.

Since test solutions were prepared by dilution of a stock solution and the recoveries followed a fixed course in terms of concentration and decline over time, concentration levels with recoveries <LOQ were extrapolated for evaluation.

 

NOEC-, LOEC-, EC-Values and 95% Confidence Intervals ofBasic Blue 147Chlorideafter 7 Days of Exposure

                  (based on thenominaltest item concentrations [µg/L])

Frond number

Dry weight

Growth Rate Inhibition [µg/L]

NOEC

10.0

10.0

LOEC

31.6

31.6

ErC10

23.3 (17.3 – 30.0)

22.7 (18.2 – 27.5)

ErC20

44.0 (34.6 – 54.6)

39.4 (32.8 – 46.6)

ErC50

296 (235 – 384)

207 (176 – 246)

Inhibition of Yield [µg/L]

NOEC

10.0

10.0

LOEC

31.6

31.6

EyC10

20.2 (12.0 – 28.3)

17.8 (14.1 – 22.1)

EyC20

27.3 (20.1 – 35.9)

24.4 (20.1 – 29.3)

EyC50

53.6 (42.0 – 71.4)

51.0 (43.8 – 59.8)


 

NOEC-, LOEC-, EC-Values and 95% Confidence Intervals ofBasic Blue 147 Chlorideafter7 Days of Exposure

                  (based on thegeometric mean measured*test item concentrations [µg/L])

Frond number

Dry weight

Growth Rate Inhibition [µg/L]

NOEC

8.91

8.91

LOEC

21.5

21.5

ErC10

18.3 (13.9 – 23.2)

17.8 (14.8 – 21.1)

ErC20

34.7 (27.8 – 42.5)

31.1 (26.6 – 35.9)

ErC50

236 (191 – 301)

165 (144 – 191)

Inhibition of Yield [µg/L]

NOEC

8.91

8.91

LOEC

21.5

21.5

EyC10

14.2 (9.81 – 19.2)

12.9 (11.0 – 15.3)

EyC20

18.6 (14.3 – 24.5)

16.8 (14.4 – 19.9)

EyC50

37.4 (28.7 – 50.8)

35.3 (30.0 – 41.8)

* = For calculation of geometric mean measured test item concentrations, measured recoveries <LOQ were

used after confirmation on stringency with the overall analytical data. For details see section8.1.3.