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 2018-01-17 to 2018-01-25, with the definitive exposure phase from 2018-01-17 to 2018-01-24
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 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 (see Table 1) and agitated until the solution was visually clear. The test item solution was clear and yellow-orange coloured.

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 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:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
7 d

Test conditions

Hardness:
not measured
Test temperature:
Room temperature [°C]: min.: 23.1 max.: 24.3 mean value: 23.7
pH:
Nominal test item concentration
[mg/L] pH-value
0 h
(Fresh medium) 7 d
(Old medium)
100 7.56 8.32
31.6 7.53 8.26
10.0 7.49 8.26
3.16 7.47 8.23
1.00 7.46 8.16
Control 7.54 8.19
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
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
Remarks:
potassium dichromate

Results and discussion

Effect concentrationsopen allclose all
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
3.16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks on result:
other: Growth Rate and Inhibition of Yield
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks on result:
other: Growth Rate and Inhibition of Yield
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
11 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Frond number
Remarks:
CI (2.80 - >100)
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
3.21 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Inhibition of Yield
Remarks on result:
other: Frond number
Remarks:
CI (1.56 - 9.75)
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks on result:
other: Frond number
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
8.05 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Inhibition of Yield
Remarks on result:
other: Frond number
Remarks:
CI (2.45 - > 100)
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks on result:
other: Growth Rate and Inhibition of Yield
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry weight
Remarks on result:
other: Growth Rate and Inhibition of Yield
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry Weight
Remarks on result:
other: Growth Rate and Inhibition of Yield
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry Weight
Remarks on result:
other: Growth Rate
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
2.99 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry Weight
Remarks on result:
other: Inhibition of Yield
Remarks:
CI (1.27 - > 100)
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry Weight
Remarks on result:
other: Grwoth Rate and Inhibition of YIeld
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
other: Dry Weight
Remarks on result:
other: Growth Rate and 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, 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 Reactive Orange 12.

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.


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]

Repl.

No.

Frond numbers per study day

0 days*

2 days

5 days

7 days

100

1

12

27

60

79

2

12

26

66

85

3

12

28

48

62

Mean

12

27

58

75

 31.6

1

12

29

53

74

2

12

29

65

72

3

12

27

66

96

Mean

12

28

61

81

 10.0

1

12

24

63

88

2

12

24

60

78

3

12

23

61

90

Mean

12

24

61

85

   3.16

1

12

24

63

93

2

12

24

57

95

3

12

26

69

105

Mean

12

25

63

97

   1.00

1

12

22

69

140

2

12

21

56

100

3

12

23

70

116

Mean

12

22

65

119

Control

1

12

27

58

90

2

12

23

58

112

3

12

24

72

118

4

12

25

67

116

5

12

24

60

95

6

12

25

61

106

Mean

12

25

63

106

* = 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
[mg/L]

Repl.

No.

Average growth rate

[d-1]

Inhibition of average growth rate
[%]

Yield


[fronds]

Inhibition of yield

[%]

Doubling time

[d]

 

100

1

 

0.269

13

 

67

29

2.57

2

 

0.280

10

 

73

23

2.48

3

 

0.235

25

 

50

47

2.95

Mean

(+)

0.261

16

(+)

63

33

2.67

 

 31.6

1

 

0.260

16

 

62

34

2.67

2

 

0.256

18

 

60

36

2.71

3

 

0.297

4

 

84

11

2.33

Mean

(+)

0.271

13

(+)

69

27

2.57

 

 10.0

1

 

0.285

8

 

76

19

2.44

2

 

0.267

14

 

66

30

2.59

3

 

0.288

7

 

78

17

2.41

Mean

(+)

0.280

10

(+)

73

22

2.48

 

   3.16

1

 

0.291

6

 

80

15

2.38

2

 

0.296

5

 

83

12

2.35

3

 

0.310

0

 

93

1

2.24

Mean

(-)

0.299

4

(-)

85

9

2.32

   1.00

1

 

0.351

-13

 

128

-36

1.97

2

 

0.303

3

 

88

7

2.29

3

 

0.324

-4

 

104

-10

2.14

Mean

(-)

0.326

-5

(-)

107

-13

2.13

Control

1

 

0.288

 

 

78

 

2.41

2

 

0.319

 

 

100

 

2.17

3

 

0.327

 

 

106

 

2.12

4

 

0.324

 

 

104

 

2.14

5

 

0.296

 

 

83

 

2.35

6

 

0.311

 

 

94

 

2.23

Mean

 

0.311

 

 

94

 

2.24

    

Repl. No. = replicate number

 

 Specific Growth Rate and Yield Inhibition of Dry Weight after 7 days

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]

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

1

12.6

 

0.286

6

 

10.9

14

2

15.0

 

0.311

-2

 

13.3

-5

3

10.3

 

0.257

15

 

8.6

32

Mean

12.6

(-)

0.285

6

(-)

10.9

14

 

 31.6

1

13.3

 

0.294

3

 

11.6

9

2

13.0

 

0.291

4

 

11.3

11

3

15.3

 

0.314

-3

 

13.6

-7

Mean

13.9

(-)

0.299

1

(-)

12.2

4

 

 10.0

1

12.6

 

0.286

6

 

10.9

14

2

12.4

 

0.284

7

 

10.7

16

3

13.1

 

0.292

4

 

11.4

10

Mean

12.7

(-)

0.287

6

(-)

11.0

13

 

   3.16

1

13.1

 

0.292

4

 

11.4

10

2

12.3

 

0.283

7

 

10.6

17

3

14.5

 

0.306

-1

 

12.8

-1

Mean

13.3

(-)

0.294

3

(-)

11.6

9

   1.00

1

19.1

 

0.346

-14

 

17.4

-37

2

12.9

 

0.290

5

 

11.2

12

3

15.6

 

0.317

-4

 

13.9

-9

Mean

15.9

(-)

0.317

-4

(-)

14.2

-12

Control

1

12.6

 

0.286

 

 

10.9

 

2

13.2

 

0.293

 

 

11.5

 

3

17.5

 

0.333

 

 

15.8

 

4

15.6

 

0.317

 

 

13.9

 

5

15.4

 

0.315

 

 

13.7

 

6

12.3

 

0.283

 

 

10.6

 

Mean

14.4

 

0.304

 

 

12.7

 

 

The mean initial biomass dry weight was 1.5 mg per replicate.

Repl. No. = replicate number


Colony Number (Plants) on Days 0 and 7

 

Nominal test item concentration
[mg/L]

Replicate

No.


Colony number

Day 0

Day 7

100

1

3

12

2

3

16

3

3

8

Mean

3

12

 31.6

1

3

7

2

3

13

3

3

16

Mean

3

12

 10.0

1

3

12

2

3

14

3

3

16

Mean

3

14

   3.16

1

3

15

2

3

16

3

3

15

Mean

3

15

   1.00

1

3

18

2

3

13

3

3

16

Mean

3

16

Control

1

3

12

2

3

15

3

3

14

4

3

14

5

3

13

6

3

12

Mean

3

13

 

Further Observations on Days 2, 5 and 7

Nominal test item concentration
[mg/L]

Observations on day

2

5

7

100

1

1

1

 31.6

1

1

1

 10.0

1

1

1

     3.16

1

1

1

    1.00

1

1

1

Control

1

1

1

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

 

1      = no observedeffects

+      = slight effects

++    = medium effects

+++         = strong effects

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
In this study, Reactive Orange 12 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 both inhibition of the specific growth rate of fronds (ErC50) and dry weight (ErdwC50) were > 100 mg/L and yield (fronds, EyC50) and dry weight (EydwC50) were > 100 mg/L.
Executive summary:

The effects of the test item Reactive Orange 12 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 2018-01-17 to 2018-01-25, with the definitive exposure phase from2018-01-17 to 2018-01-24.

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. 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.The validity criteria of the test guideline were fulfilled. The test item solutions were clear and concentration related yellow-orange coloured throughout the exposure period. The validity criteria of the test guideline were fulfilled.

The concentrations ofthe test item Reactive Orange 12 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 Reactive Orange 12 were in the range of 96 and 101 % of the nominal values. At the end of the exposure the measured concentrations were between 99 and 120 % of the nominal values. All effect values are given based on the nominal test item concentrations.

 

NOEC-, LOEC-, EC-Values and 95% Confidence Intervals of Reactive Orange 12
after 7 Days of Exposure

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

Frond number

Dry weight

Growth Rate Inhibition [mg/L]

NOEC

3.16

NOEC

≥ 100

LOEC

10.0

LOEC

> 100

ErC10

11.0 (2.80 - >100)

ErdwC10

> 100

ErC20

> 100

ErdwC20

> 100

ErC50

> 100

ErdwC50

> 100

Inhibition of Yield [mg/L]

NOEC

3.16

NOEC

≥ 100

LOEC

10.0

LOEC

> 100

EyC10

3.21 (1.56 – 9.75)

EydwC10

2.99 (1.27 - >100)

EyC20

8.05 (2.45 – > 100)

EydwC20

> 100

EyC50

> 100

EydwC50

> 100