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:
2017
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:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
GLP compliance:
yes

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).

Test solutions

Details on test solutions:
Preparation of the Saturated solution
A saturated solution with a nominal loading of 100 mg test item/L was prepared once 48 ± 1 hours 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 demineralized water. The saturated solution was stirred for 48 ± 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 for adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. After filtration the components of the dilution water were added to the saturated solution. The following solution, i.e. the saturated solution, was used in the test. During filtration, the filter was always kept covered. The saturated solution was clear yellow. The concentrations are based on the results of a preliminary range finding test.

Test concentrations
The saturated solution and further four dilution levels out of the saturated solution were prepared with dilution water (see Table 2) in a geometrical series with a separation factor of √10 and tested as follows: 0.500 - 1.58 - 5.00 - 15.8 - 50.0% of the saturated 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, 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:
semi-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
Nominal and measured concentrations:
Nominal test item concentrations: 0.500 - 1.58 - 5.00 - 15.8 - 50.0% of the saturated solution
Geometric mean measured test item concnetrations: 0.100 – 0.277 – 0.850 – 2.64 – 8.69 mg/L
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, 4 uniform, healthy plants (colonies of 3 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:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 0.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: frond number growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.17 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: frond number growth rate
Remarks on result:
other: CI: 1.07 - 1.30
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 0.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Frond number yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.791 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: frond number yield
Remarks on result:
other: 0.736 - 0.836
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weight growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.32 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weight growth rate
Remarks on result:
other: CI: 1.03 - 1.86
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
< 0.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weight yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.537 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weight yield
Remarks on result:
other: CI: 0.450 - 0.636)
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 SZBD168XV, purity 99.4%, CAS RN 591-35-5) to the monocotyledonous aquatic plant Lemna gibba was determined over a period of 7 days from 2016-10-12 to 2016-10-19 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 Red 18.1 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 6.71 5.56 ± 2.83
95% confidence interval 5.42 – 8.00
Yield inhibition (number of fronds)
EyC50 4.50 4.59 ± 3.03
95% confidence interval 4.04 – 5.05
Growth rate inhibition (dry weight)
ErdwC50 6.76 5.50 ± 2.85
95% confidence interval 5.29 – 7.43
Yield inhibition (dry weight)
EydwC50 5.53 4.63 ± 2.47
95% confidence interval 4.56 – 6.50
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 tested by trend analysis by contrasts (significance level 0.05) for yield.
Multiple sequentially-rejective Welsh-t-test after Bonferroni-Holm was carried out with a significance level of 0.05 for growth rate.
William’s multiple sequential t-test was carried out with a significance level of 0.05 for yield.

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

Geometric mean measured test item concentration
[mg/L]

Repl.

No.

Frond numbers per study day

0 days*

2 days

5 days

7 days

8.69

1

12

6

3

2

2

12

8

3

3

3

12

5

5

3

Mean

12

6

4

3

2.64

1

12

16

16

16

2

12

12

13

12

3

12

13

14

14

Mean

12

14

14

14

0.850

1

12

17

33

47

2

12

16

30

44

3

12

15

33

46

Mean

12

16

32

46

0.277

1

12

17

43

65

2

12

18

42

61

3

12

18

41

64

Mean

12

18

42

63

0.100

1

12

17

39

62

2

12

18

42

66

3

12

20

44

65

Mean

12

18

42

64

Control

1

12

18

47

82

2

12

18

42

83

3

12

18

45

85

4

12

17

47

88

5

12

19

46

86

6

12

20

44

85

Mean

12

18

45

85

* = 4 colonies with 3 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]

8.69

1

 

-0.256

100

 

-10

100

-2.71

2

 

-0.198

100

 

-9

100

-3.50

3

 

-0.198

100

 

-9

100

-3.50

Mean

(+)

-0.217

100

(+)

-9

100

-3.24

2.64

1

 

0.041

85

 

4

95

16.9

2

 

0.000

100

 

0

100

n.a.

3

 

0.022

92

 

2

97

31.5

Mean

(+)

0.021

92

(+)

2

97

n.a.

0.850

1

 

0.195

30

 

35

52

3.55

2

 

0.186

33

 

32

56

3.73

3

 

0.192

31

 

34

53

3.61

Mean

(+)

0.191

32

(+)

34

54

3.63

0.277

1

 

0.241

13

 

53

27

2.87

2

 

0.232

17

 

49

33

2.98

3

 

0.239

14

 

52

29

2.90

Mean

(+)

0.238

15

(+)

51

29

2.92

0.100

1

 

0.235

16

 

50

31

2.95

2

 

0.244

13

 

54

26

2.85

3

 

0.241

13

 

53

27

2.87

Mean

(+)

0.240

14

(+)

52

28

2.89

Control

1

 

0.275

 

 

70

 

2.52

2

 

0.276

 

 

71

 

2.51

3

 

0.280

 

 

73

 

2.48

4

 

0.285

 

 

76

 

2.44

5

 

0.281

 

 

74

 

2.46

6

 

0.280

 

 

73

 

2.48

Mean

 

0.279

 

 

73

 

2.48

Repl. No. = replicate number

n.a. = not applicable

 


  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
 
[%]

8.69

1

1.8

 

0.116

70

 

1.0

91

2

2.3

 

0.151

60

 

1.5

86

3

2.2

 

0.145

62

 

1.4

87

Mean

2.1

(+)

0.137

64

(+)

1.3

88

2.64

1

2.0

 

0.131

66

 

1.2

89

2

3.0

 

0.189

50

 

2.2

79

3

1.8

 

0.116

70

 

1.0

91

Mean

2.3

(+)

0.145

62

(+)

1.5

86

0.850

1

4.3

 

0.240

37

 

3.5

67

2

4.5

 

0.247

35

 

3.7

65

3

4.1

 

0.233

39

 

3.3

69

Mean

4.3

(+)

0.240

37

(+)

3.5

67

0.277

1

9.2

 

0.349

8

 

8.4

21

2

7.9

 

0.327

14

 

7.1

33

3

9.0

 

0.346

9

 

8.2

23

Mean

8.7

(+)

0.341

10

(+)

7.9

25

0.100

1

9.8

 

0.358

6

 

9.0

15

2

10.0

 

0.361

5

 

9.2

13

3

11.4

 

0.380

0

 

10.6

0

Mean

10.4

(-)

0.366

4

(+)

9.6

9

Control

1

12.4

 

0.392

 

 

11.6

 

2

11.6

 

0.382

 

 

10.8

 

3

10.6

 

0.369

 

 

9.8

 

4

10.8

 

0.372

 

 

10.0

 

5

10.8

 

0.372

 

 

10.0

 

6

12.4

 

0.392

 

 

11.6

 

Mean

11.4

 

0.380

 

 

10.6

 

The initial biomass dry weight was 0.8 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

8.69

1

4

2

2

4

2

3

4

3

Mean

4

2

2.64

1

4

14

2

4

11

3

4

10

Mean

4

12

0.850

1

4

7

2

4

5

3

4

8

Mean

4

7

0.277

1

4

4

2

4

6

3

4

5

Mean

4

5

0.100

1

4

4

2

4

4

3

4

4

Mean

4

4

Control

1

4

5

2

4

6

3

4

6

4

4

8

5

4

5

6

4

7

Mean

4

6


Further Observations on Days 2, 5 and 7

Geometric mean measured test item concentration
[mg/L]

Observations on day

2

5

7

8.69

2.1 +++
2.2 ++
3.2 +++
4.1 +++

2.1 +++
2.2 +++
3.2 +++
4.1 +++

2.1 +++
2.2 +++
3.2 +++
4.1 +++

2.64

2.1 +
3.2 ++
4.1 +++

2.1 ++
3.2 ++
4.1 +++

2.1 ++
3.2 ++
4.1 +++

0.850

4.1 +

2.5 ++
4.1 +

2.3 ++
2.5 ++
3.2 +
4.1 +

0.277

1

2.5 +

2.5 +

0.100

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

2.5  = fronds are smaller, compared to the control

3.2   = loses 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, the test item 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 concentrations): The EC50-values for inhibition of the specific growth rate of fronds (ErC50) and dry weight (ErdwC50) were 1.17 (1.07 – 1.30) mg/L and 1.32 (1.03 – 1.86) mg/L, respectively. The EC50-values for inhibition of yield (fronds, EyC50) and dry weight inhibition of yield (EydwC50) with 95% confidence intervals were 0.791 (0.736 – 0.836) mg/L and 0.536 (0.449 – 0.635) mg/L, respectively.
Executive summary:

The effects of the test item on the growth of the monocotyledonous aquatic plant species Lemna gibba were determined according to the principles of OECD 221 and EC C.26 .

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 √10:0.500 - 1.58 - 5.00 - 15.8 - 50.0 % of the saturated solution, corresponding to the geometric mean measured test item concentrations: 0.100 – 0.277 – 0.850 – 2.64 – 8.69 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 concentrations of the test item and the control were analysed via HPLC-DAD at the beginning and end of the exposure.

The measured initial concentrations of were < LOQ – 0.303 – 0.940 – 2.89 – 9.01 mg/L and reflect the dilution factor of √10. The measured concentrations in the old media were in the range of 82 to 93% of the initially measured values. All effect values are given based on the geometric mean measured test item concentrations.

 

NOEC-, LOEC-, EC-Values and 95 % Confidence Intervals of the test item
after 7 Days of Exposure

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

Frond number

Dry weight

Growth Rate Inhibition [mg/L]

NOEC

< 0.100

NOEC

0.100

LOEC

0.100

LOEC

0.277

ErC10

< 0.100

ErdwC10

0.286 (< 0.100 – 0.451)

ErC20

0.579 (0.468 – 0.676)

ErdwC20

0.484 (0.345 – 0.626)

ErC50

1.17 (1.07 – 1.30)

ErdwC50

1.32 (1.03 – 1.86)

Inhibition of Yield [mg/L]

NOEC

< 0.100

NOEC

< 0.100

LOEC

0.100

LOEC

0.100

EyC10

< 0.100

EydwC10

0.108 (< 0.100 – 0.176)

EyC20

< 0.100

EydwC20

0.226 (0.171 – 0.292)

EyC50

0.791 (0.736 – 0.836)

EydwC50

0.537 (0.450 – 0.636)