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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 2018-05-29 to 2018-06-07, with the definitive exposure phase from 2018-05-30 to 2018-06-06.
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:
Chromate(2-), [2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)][2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-5-sulfobenzoato(3-)]-, lithium sodium
EC Number:
288-678-6
EC Name:
Chromate(2-), [2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)][2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-5-sulfobenzoato(3-)]-, lithium sodium
Cas Number:
85865-79-8
Molecular formula:
C34H23CrLiN8NaO9S
IUPAC Name:
Chromate(2-), [2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)][2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-5-sulfobenzoato(3-)]-, lithium sodium
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 test item solution of 100 mg test item/L was prepared 24 ± 1 hour prior to the start of the exposure and medium renewal. 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 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, were used as a test item solution in the test. During filtration, the filter was always kept covered. The test item solution was checked via laser beam (Tyndall effect) for undissolved test item. Tyndall effect was positive. Presence of undissolved test item during the test was visually not observed. The concentrations are based on the results of a preliminary range finding test.

Test concentrations
Five dilution levels were prepared out of the test item solution with dilution water in a geometrical series with a separation factor of 4 and tested as follows: 0.273 - 1.09 - 4.38 - 17.5 - 70.0 % of the test item solution.

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.: 23.9 max.: 24.8 mean value: 24.3
pH:
Geometric mean measured test item concentration
[mg/L] pH-value
0 h (Fresh medium) 7 d (Old medium)
48.4 7.51 7.96
9.92 7.53 8.04
2.50 7.52 8.13
0.554 7.51 8.21
0.375 7.53 8.25
Control 7.57 8.25
Dissolved oxygen:
not measured
Salinity:
not measured
Conductivity:
not measured
Nominal and measured concentrations:
Nominal: 0.273 - 1.09 - 4.38 - 17.5 - 70.0 % of the test item solution
geometric mean measured test item concentrations: 0.375 – 0.554 – 2.50 – 9.92 – 48.4 mg/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.:
9.92 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Frond number
Remarks on result:
other: Growth Rate and Inhibition of Yield
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
2.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weight growth rate inhibition and inhibition of yield
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
2.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Frond number
Remarks on result:
other: Growth Rate and Inhibition of Yield
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
0.554 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weigt growth rate inhibition and inhibition of yield
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 48.4 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Frond number and Dry weight
Remarks on result:
other: Growth Rate Inhibition
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
14.7 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Dry weight Inhibition of yield
Remarks on result:
other: CI: 8.54 – 26.6
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
10.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: frond number ihibition of yield
Remarks on result:
other: CI: 6.18 - 20.0
Details on results:
The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits. The test medium was visually clear and concentration related yellow colored.
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 Acid Yellow 232 (Li/Na).

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. Normality test failed for calculation of yield (fronds). Therefore, the data were transformed (Y=log(Y)).

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

Geometric mean measured test item concentration
[mg/L]

Repl.

No.

Frond numbers per study day

0 days*

2 days

5 days

7 days

48.4

1

12

23

74

110

2

12

22

82

97

3

12

22

78

92

Mean

12

22

78

100

  9.92

1

12

26

111

162

2

12

21

109

186

3

12

22

90

184

Mean

12

23

103

177

  2.50

1

12

23

110

260

2

12

25

133

264

3

12

24

126

275

Mean

12

24

123

266

  0.554

1

12

24

125

263

2

12

24

143

357

3

12

26

137

345

Mean

12

25

135

322

  0.375

1

12

25

136

308

2

12

24

119

314

3

12

25

129

391

Mean

12

25

128

338

Control

1

12

26

162

438

2

12

26

143

329

3

12

24

118

312

4

12

25

149

311

5

12

23

102

309

6

12

23

129

339

Mean

12

25

134

340

* = 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
[mg/L]

Repl.

No.

Average growth rate

[d-1]

Inhibition of average growth rate
[%]

Yield


[fronds]

Inhibition of yield

[%]

Doubling time

[d]

48.4

1

 

0.317

33.51

 

98

70.09

2.19

2

 

0.299

37.28

 

85

74.06

2.32

3

 

0.291

38.87

 

80

75.59

2.38

Mean

(+)

0.302

36.55

(+)

88

73.25

2.30

  9.92

1

 

0.372

21.89

 

150

54.23

1.86

2

 

0.392

17.74

 

174

46.90

1.77

3

 

0.390

18.07

 

172

47.51

1.78

Mean

(+)

0.384

19.23

(+)

165

49.55

1.80

  2.50

1

 

0.439

7.69

 

248

24.32

1.58

2

 

0.442

7.23

 

252

23.10

1.57

3

 

0.447

6.01

 

263

19.74

1.55

Mean

(-)

0.443

6.98

(-)

254

22.39

1.57

  0.554

1

 

0.441

7.35

 

251

23.41

1.57

2

 

0.485

-1.83

 

345

-5.28

1.43

3

 

0.480

-0.80

 

333

-1.62

1.44

Mean

(-)

0.469

1.57

(-)

310

5.50

1.48

  0.375

1

 

0.464

2.61

 

296

9.67

1.50

2

 

0.466

2.03

 

302

7.84

1.49

3

 

0.498

-4.56

 

379

-15.65

1.39

Mean

(-)

0.476

0.03

(-)

326

0.62

1.46

Control

1

 

0.514

 

 

426

 

1.35

2

 

0.473

 

 

317

 

1.47

3

 

0.465

 

 

300

 

1.49

4

 

0.465

 

 

299

 

1.49

5

 

0.464

 

 

297

 

1.49

6

 

0.477

 

 

327

 

1.45

Mean

 

0.476

 

 

328

 

1.46

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
[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
 
[%]

48.4

1

18.3

 

0.316

34.11

 

16.3

70.89

2

20.6

 

0.333

30.59

 

18.6

66.79

3

20.2

 

0.330

31.17

 

18.2

67.50

Mean

19.7

(+)

0.327

31.96

(+)

17.7

68.39

  9.92

1

36.5

 

0.415

13.57

 

34.5

38.39

2

31.7

 

0.395

17.76

 

29.7

46.96

3

31.2

 

0.392

18.24

 

29.2

47.86

Mean

33.1

(+)

0.401

16.52

(+)

31.1

44.40

  2.50

1

42.5

 

0.437

9.04

 

40.5

27.68

2

45.7

 

0.447

6.88

 

43.7

21.96

3

45.8

 

0.447

6.81

 

43.8

21.79

Mean

44.7

(+)

0.444

7.58

(+)

42.7

23.81

  0.554

1

47.7

 

0.453

5.60

 

45.7

18.39

2

52.9

 

0.468

2.52

 

50.9

9.11

3

57.0

 

0.479

0.30

 

55.0

1.79

Mean

52.5

(-)

0.467

2.81

(-)

50.5

9.76

  0.375

1

53.8

 

0.470

2.02

 

51.8

7.50

2

51.8

 

0.465

3.15

 

49.8

11.07

3

64.3

 

0.496

-3.29

 

62.3

-11.25

Mean

56.6

(-)

0.477

0.63

(-)

54.6

2.44

Control

1

69.6

 

0.507

 

 

67.6

 

2

54.7

 

0.473

 

 

52.7

 

3

55.9

 

0.476

 

 

53.9

 

4

54.0

 

0.471

 

 

52.0

 

5

52.4

 

0.467

 

 

50.4

 

6

61.7

 

0.490

 

 

59.7

 

Mean

58.0

 

0.480

 

 

56.0

 

The initial biomass dry weight was 2.0 mg per replicate.

Repl. No. = replicate number

 


Colony Number (Plants) on Days 0 and 7

 

Geometric mean measured test item concentration
[mg/L]

Replicate

No.


Colony number

Day 0

Day 7

48.4

1

3

16

2

3

14

3

3

16

Mean

3

15

  9.92

1

3

21

2

3

17

3

3

16

Mean

3

18

  2.50

1

3

20

2

3

26

3

3

27

Mean

3

24

  0.554

1

3

22

2

3

39

3

3

36

Mean

3

32

  0.375

1

3

32

2

3

27

3

3

42

Mean

3

34

Control

1

3

42

2

3

34

3

3

27

4

3

34

5

3

25

6

3

26

Mean

3

31

 


 

Further Observations on Days 2, 5 and 7

Geometric mean measured test item concentration
[mg/L]

Observations on day

2

5

7

48.4

1

2.1+

2.1+

    9.92

1

1

1

    2.50

1

1

1

    0.554

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

2.1    = chlorosis

+      = slight effects

++    = medium effects

+++  = strong effects

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study, Acid Yellow 232 (Li/Na) was found to inhibit the growth of the monocotyledonous aquatic plant Lemna gibba after 7-day exposure under semi-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 both > 48.4 mg/L and yield (fronds and dry weight) (EyC50, EydwC50) were 10.3 (6.18 to – 20.0) mg/L and 14.7 (8.54 to 26.6) mg/L, respectively.
Executive summary:

The effects of the test item Acid Yellow 232 (Li/Na) on the growth of the monocotyledonous aquatic plant species Lemna gibba was determined according to the principles of OECD 221 and Council Regulation (EC) No. 761/2009 Method C. 26 at the test facility from 2018-05-29 to 2018-06-07, with the definitive exposure phase from 2018-05-30 to 2018-06-06.

 

Lemna gibba was exposed to the test item for 7 days under semi-static conditions. Based on a preliminary test, 5 nominal test item dilution levels were tested in a geometrical series with a dilution factor of 4:0.273 - 1.09 - 4.38 - 17.5 - 70.0 % of the test item solution, corresponding to the geometric mean measured test item concentrations: 0.375 – 0.554 – 2.50 – 9.92 – 48.4 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. All test solutions were clear and concentration-related yellow throughout the exposure.The validity criteria of the test guideline were fulfilled.

 

The concentrations of the test item Acid Yellow 232 (Li/Na) and the controlwere 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 Acid Yellow 232(Li/Na) were in the range of 0.375 to 55.5 mg/L. At the end of the first interval the measured concentrations were between <LOQ and 76% of the initial measured concentrations. All effect values are given based on the geometric mean measured test item concentrations.

 

 NOEC-, LOEC-, EC-Values and 95% Confidence Intervals ofAcid Yellow 232(Li/Na)after 7 Days of Exposure

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

Frond number

Dry weight

Growth Rate Inhibition [mg/L]

NOEC

2.50

0.554

LOEC

9.92

2.50

ErC10

3.78 (2.13 – 5.61)

3.77 (2.12 – 5.61)

ErC20

10.7 (7.71 – 17.0)

15.3 (10.5 – 24.2)

ErC50

> 48.4

> 48.4

Inhibition of Yield [mg/L]

NOEC

2.50

0.554

LOEC

9.90

2.50

EyC10

1.02 (< 0.375 – 2.32)

0.703 (< 0.375 – 1.56)

EyC20

2.11 (1.04 – 3.77)

1.72 (0.883 – 3.12)

EyC50

10.3 (6.18 – 20.0)

14.7 (8.54 – 26.6)