Registration Dossier

Ecotoxicological information

Toxicity to aquatic plants other than algae

Currently viewing:

Administrative data

Endpoint:
toxicity to aquatic plants other than algae
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 2018-03-16 to 2018-03-26, with the definitive exposure phase from 2018-03-16 to 2018-03-23
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
Qualifier:
according to
Guideline:
other: Council Regulation (EC) No. 761/2009 Method C.26
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
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 . 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 100 mg/L was freshly prepared with dilution water (see Table 2) and agitated until the solution was visually clear. The test item solution was clear and purple coloured.

Test concentrations
The stock solution and four dilution levels were prepared with dilution water in a geometrical series with a separation factor of 4 and tested as follows: 0.391 - 1.56 - 6.25 - 25.0 - 100 mg/L, corresponding to the geometric mean measured test item concentrations: 0.138 - 0.276 – 0.545 – 2.15 – 8.56 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 9 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.: 24.3 max.: 25.2 mean value: 24.9
pH:
Nominal test item concentration
[mg/L] Geometric mean measured test item concentration
[mg/L] pH-value
0 h (Fresh medium) 3 d (Old medium) 3 d (Fresh medium) 5 d (Old medium) 5 d (Fresh medium) 7 d (Old medium)
100 8.56 7.44 8.34 7.37 8.29 7.49 8.39
25.0 2.15 7.42 8.34 7.36 8.28 7.49 8.40
6.25 0.545 7.39 8.34 7.36 8.29 7.49 8.40
1.56 0.276 7.41 8.35 7.37 8.27 7.51 8.39
0.391 0.138 7.54 8.29 7.38 8.27 7.53 8.39
Control 7.57 8.26 7.42 8.27 7.55 8.42
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.

Application
Semi-static, with renewal of the test solutions on day 3 and 5 (i.e. exposure to freshly prepared test and control solutions on two occasions during the test). 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.:
0.138 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.:
0.276 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:
EC50
Effect conc.:
> 8.56 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.:
0.385 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Frond number Inhibition of yield
Remarks on result:
other: CI: 0.292 - 0.640
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
0.409 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: 0.289 – 0.728
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
0.138 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weigt growth rate inhibition
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
0.276 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weight growth rate inhibition
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
< 0.138 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weight inhibition of yield
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
0.138 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dryweight inhibition of 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-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 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 Reactive Violet 5 (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 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, 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.
Step-down Jonckheere-Terpstra test 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.

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.
- ToxRat Version 3.2.1, ToxRat Solutions GmbH

Any other information on results incl. tables

Frond Numbers

Nominal test item concentration
[mg/L]

Geometric mean measured test item concentration
[mg/L]

Repl.

No.

Frond numbers per study day

0 days*

3 days

5 days

7 days

100

8.56

1

12

21

33

47

2

12

18

28

39

3

12

19

28

38

Mean

12

19

30

41

 25.0

2.15

1

12

21

32

47

2

12

19

29

45

3

12

19

30

45

Mean

12

20

30

46

  6.25

0.545

1

12

19

32

49

2

12

20

31

42

3

12

19

33

51

Mean

12

19

32

47

  1.56

0.276

1

12

20

39

63

2

12

22

37

63

3

12

21

38

72

Mean

12

21

38

66

  0.391

0.138

1

12

21

48

100

2

12

20

40

71

3

12

25

50

98

Mean

12

22

46

90

Control

1

12

24

52

97

2

12

21

46

96

3

12

22

41

92

4

12

25

50

108

5

12

24

43

95

6

12

22

44

89

Mean

12

23

46

96

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

Repl. No. = replicate number

Table5:      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
[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

8.56

1

 

0.195

34

 

35

58

3.55

2

 

0.168

43

 

27

68

4.12

3

 

0.165

45

 

26

69

4.21

Mean

(+)

0.176

41

(+)

29

65

3.96

 25.0

2.15

1

 

0.195

34

 

35

58

3.55

2

 

0.189

36

 

33

61

3.67

3

 

0.189

36

 

33

61

3.67

Mean

(+)

0.191

36

(+)

34

60

3.63

  6.25

0.545

1

 

0.201

32

 

37

56

3.45

2

 

0.179

40

 

30

64

3.87

3

 

0.207

30

 

39

54

3.35

Mean

(+)

0.196

34

(+)

35

58

3.56

  1.56

0.276

1

 

0.237

20

 

51

39

2.93

2

 

0.237

20

 

51

39

2.93

3

 

0.256

14

 

60

29

2.71

Mean

(+)

0.243

18

(+)

54

36

2.85

  0.391

0.138

1

 

0.303

-2

 

88

-5

2.29

2

 

0.254

14

 

59

30

2.73

3

 

0.300

-1

 

86

-2

2.31

Mean

(-)

0.286

4

(-)

78

8

2.44

Control

1

 

0.299

 

 

85

 

2.32

2

 

0.297

 

 

84

 

2.33

3

 

0.291

 

 

80

 

2.38

4

 

0.314

 

 

96

 

2.21

5

 

0.296

 

 

83

 

2.35

6

 

0.286

 

 

77

 

2.42

Mean

 

0.297

 

 

84

 

2.34

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
[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

8.56

1

5.5

 

0.230

36

 

4.4

66

2

4.6

 

0.204

44

 

3.5

73

3

4.8

 

0.210

42

 

3.7

71

Mean

5.0

(+)

0.215

41

(+)

3.9

70

 25.0

2.15

1

6.2

 

0.247

32

 

5.1

60

2

5.5

 

0.230

36

 

4.4

66

3

5.7

 

0.235

35

 

4.6

64

Mean

5.8

(+)

0.237

34

(+)

4.7

63

  6.25

0.545

1

6.5

 

0.254

30

 

5.4

58

2

6.2

 

0.247

32

 

5.1

60

3

7.1

 

0.266

26

 

6.0

53

Mean

6.6

(+)

0.256

29

(+)

5.5

57

  1.56

0.276

1

8.7

 

0.295

18

 

7.6

41

2

8.5

 

0.292

19

 

7.4

42

3

9.6

 

0.309

15

 

8.5

34

Mean

8.9

(+)

0.299

17

(+)

7.8

39

  0.391

0.138

1

12.1

 

0.343

5

 

11.0

14

2

10.1

 

0.317

13

 

9.0

30

3

14.1

 

0.364

-1

 

13.0

-2

Mean

12.1

(-)

0.341

6

(+)

11.0

14

Control

1

14.1

 

0.364

 

 

13.0

 

2

13.4

 

0.357

 

 

12.3

 

3

14.5

 

0.368

 

 

13.4

 

4

14.8

 

0.371

 

 

13.7

 

5

13.0

 

0.353

 

 

11.9

 

6

13.4

 

0.357

 

 

12.3

 

Mean

13.9

 

0.362

 

 

12.8

 

The initial biomass dry weight was 1.1 mg per replicate.

Repl. No. = replicate number


Colony Number (Plants) on Days 0 and 7

 

Nominal test item concentration
[mg/L]

Geometric mean measured test item concentration
[mg/L]

Replicate

No.


Colony number

Day 0

Day 7

100

8.56

1

3

9

2

3

9

3

3

7

Mean

3

8

 25.0

2.15

1

3

9

2

3

7

3

3

8

Mean

3

8

  6.25

0.545

1

3

8

2

3

7

3

3

6

Mean

3

7

  1.56

0.276

1

3

6

2

3

9

3

3

9

Mean

3

8

  0.391

0.138

1

3

12

2

3

6

3

3

10

Mean

3

9

Control

1

3

10

2

3

11

3

3

10

4

3

13

5

3

13

6

3

8

Mean

3

11


Further Observations on Days 2, 5 and 7

Nominal test item concentration
[mg/L]

Geometric mean measured test item concentration
[mg/L]

Observations on day

3

5

7

100

8.56

1

2.3 ++
3.3 ++

2.3 ++
3.3 ++

 25.0

2.15

1

2.3 +
3.3 ++

2.3 +
3.3 ++

  6.25

0.545

1

3.3+

2.3 +
3.3+

  1.56

0.276

1

1

1

  0.391

0.138

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 of the fronds

3.3   = discoloration of the roots

+      = slight effects

++    = moderate effects

+++  = strong effects

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study, Reactive Violet 5 (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 (nominal and geometric mean measured test item concentrations): The EC50-values for both inhibition of the specific growth rate of fronds (ErC50) and dry weight (ErdwC50) were > 0.391 mg/L [> 8.56 mg/L]. The EC50-values for inhibition of yield (fronds, EyC50) and dry weight inhibition of yield (EydwC50) with 95% confidence intervals were 3.25 (< 0.391 – 1.01) mg/L [0.385 (0.292 – 0.640) mg/L] and 3.50 (1.80 – 8.03) mg/L [0.409 (0.289 – 0.728) mg/L], respectively.
Executive summary:

The effects of the test item Reactive Violet 5 (Na) on the growth of the monocotyledonous aquatic plant speciesLemna gibba was determined according to the principles of OECD 221 at the test facility from 2018-03-16 to 2018-03-26, with the definitive exposure phase from 2018-03-16 to 2018-03-23.

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 4:0.391 - 1.56 - 6.25 - 25.0 - 100 mg/L, corresponding to the geometric mean measured test item concentrations: 0.138 - 0.276 – 0.545 – 2.15 – 8.56 mg/L. 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 purple coloured throughout the exposure.The validity criteria of the test guideline were fulfilled.

The concentrations ofthe test item Reactive Violet 5 (Na) 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 Reactive Violet 5 (Na) were in the range of 94 and 98% of the nominal values. At the end of the first interval the measured concentrations were between < LOQ and 1% of the nominal values. All effect values are given based on the nominal and geometric mean measured test item concentrations.

 

NOEC-, LOEC-, EC-Values and 95% Confidence Intervals ofReactive Violet 5 (Na)after 7 Days of Exposure

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

Frond number

Dry weight

Concentration of the test item

Growth Rate Inhibition [mg/L]

NOEC

0.138

NOEC

0.138

LOEC

0.276

LOEC

0.276

ErC10

0.205 (< 0.138 – 0.269)

ErdwC10

0.170 (< 0.138 – 0.221)

ErC20

0.293 (0.223 – 0.384)

ErdwC20

0.302 (0.223 – 0.401)

ErC50

> 8.56

ErdwC50

> 8.56

Inhibition of Yield [mg/L]

NOEC

0.138

NOEC

< 0.138

LOEC

0.276

LOEC

  0.138

EyC10

0.151 (< 0.138 – 0.229)

EydwC10

0.125 (< 0.138 – 0.157)

EyC20

0.200 (0.141 – 0.262)

EydwC20

0.161 (< 0.138 – 0.218)

EyC50

0.385 (0.292 – 0.640)

EydwC50

0.409 (0.289 – 0.728)