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 2018-04-24 to 2018-05-03, with the definitive exposure phase from 2018-04-25 to 2018-05-02.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
according to OECD and GLP guidelines

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent

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 beginning and at the end of the exposure and at 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.

Test solutions

Details on test solutions:
Preparation of the test item solution
A stock solution with a nominal test item concentration of 150 mg/L
was freshly prepared with demineralized water one day prior to the start of exposure (0 hours) and prior to the renewal of the test solutions (24 hours). The solution was stirred for 24 hours at approx. 1100 rpm at room temperature. Prior to testing, the nutrient salts for the medium were added to obtain the final stock solution.


Test concentrations
The stock solution and four dilution levels were prepared with dilution water in a geometrical series with a separation factor of 2 and tested as follows: 9.38 - 18.8 - 37.5 - 75.0 - 150 mg/L, corresponding to the geometric mean measured test item concentrations of 5.82 – 13.1 – 28.0 – 62.9 – 140 mg/L.
The test concentrations were based on the results of a preliminary range finding test.

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.: 24.3 max.: 25.7 mean value: 24.7
pH:
Geometric mean measured test item concentration
[mg/L] pH-value
0 h
(Fresh medium) 2 d
(Old medium) 2 d
(Fresh medium) 5 d
(Old medium) 5 d
(Fresh medium) 7 d
(Old medium)
140 7.64 8.46 7.65 8.42 7.66 8.52
62.9 7.65 8.46 7.65 8.42 7.65 8.49
28.0 7.63 8.45 7.61 8.41 7.66 8.50
13.1 7.62 8.42 7.60 8.42 7.67 8.56
5.82 7.61 8.37 7.54 8.41 7.68 8.61
Control 7.59 8.30 7.57 8.35 7.64 8.84
Dissolved oxygen:
not measured
Salinity:
not measured
Conductivity:
not measured
Details on test conditions:
Test method Semi-static procedure with media renewal every 2-3 days

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.
Acclimatization of The test system (the test organism) was held for 7 days under
the test system test conditions to acclimatize. These acclimatized plants were used in the test.

Application
Semi-static, with renewal of the test solutions on day 2 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.Random repositioning of the test vessels when observation were made was carried out.

Biological Parameters

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.:
< 5.82 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 and Inhibition of Yield
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
5.82 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 and Inhibition of Yield
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
< 5.82 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 and Inhibition of Yield
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
< 5.82 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 and Inhibition of Yield
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 140 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Frond number
Remarks on result:
other: Growth Rate Inhibition
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
85.3
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Dry weight
Remarks on result:
other: Growth Rate Inhibition
Remarks:
CI (62.0 - > 140)
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
< 5.82 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: Inhibition of Yield
Details on results:
The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits. The test media showed a concentration-related black colouring..
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 from 2018-04-04 to 2018-04-12, with the definitive exposure phase from 2018-04-04 to 2018-04-11 according to OECD Guideline 221.

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 – > 8.00
Yield inhibition (number of fronds)
EyC50 4.93 4.67 ± 2.87
95% confidence interval 4.42 – 5.53
Growth rate inhibition (dry weight)
ErdwC50 > 8.00 5.61 ± 2.76
95% confidence interval -
Yield inhibition (dry weight)
EydwC50 5.95 4.75 ± 2.49
95% confidence interval 5.18 – 6.75
SD = standard deviation

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) inhibition and dry weight yield as well as the EC10- and EC20-values of dry weight growth rate were calculated by empirical analysis of the inhibition values. The EC50 of fry weight growth was calculated by sigmoidal dose-response regression. Calculations of the confidence intervals 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

Biological Data

 

Frond numbers, evaluations of average specific growth rate inhibition, yield inhibition and dry weight inhibition are given in the tables below. Colony number and further observations are listed in the tables below.All effect values are given based on the geometric mean measured test itemconcentrationof the test item.

 

Frond Numbers

Geometric mean measured test item concentration
[mg/L]

Repl.

No.

Frond numbers per study day

0 days*

3 days

5 days

7 days

140

1

12

24

37

59

2

12

21

36

56

3

12

21

37

55

Mean

12

22

37

57

 62.9

1

12

24

45

65

2

12

25

43

61

3

12

24

40

61

Mean

12

24

43

62

 28.0

1

12

25

44

73

2

12

24

46

80

3

12

21

45

68

Mean

12

23

45

74

 13.1

1

12

27

55

113

2

12

24

54

100

3

12

26

57

112

Mean

12

26

55

108

  5.82

1

12

27

68

123

2

12

23

69

134

3

12

24

57

115

Mean

12

25

65

124

Control

1

12

22

99

228

2

12

28

106

272

3

12

29

106

262

4

12

29

110

248

5

12

29

122

320

6

12

31

95

236

Mean

12

28

106

261

* = 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]

140

1

 

0.228

48

 

47

81

3.05

2

 

0.220

50

 

44

82

3.15

3

 

0.217

50

 

43

83

3.19

Mean

(+)

0.222

50

(+)

45

82

3.13

 62.9

1

 

0.241

45

 

53

79

2.87

2

 

0.232

47

 

49

80

2.98

3

 

0.232

47

 

49

80

2.98

Mean

(+)

0.235

46

(+)

50

80

2.95

 28.0

1

 

0.258

41

 

61

76

2.69

2

 

0.271

38

 

68

73

2.56

3

 

0.248

44

 

56

78

2.80

Mean

(+)

0.259

41

(+)

62

75

2.68

 13.1

1

 

0.320

27

 

101

59

2.16

2

 

0.303

31

 

88

65

2.29

3

 

0.319

27

 

100

60

2.17

Mean

(+)

0.314

28

(+)

96

61

2.21

  5.82

1

 

0.332

24

 

111

55

2.08

2

 

0.345

21

 

122

51

2.01

3

 

0.323

26

 

103

59

2.15

Mean

(+)

0.333

24

(+)

112

55

2.08

Control

1

 

0.421

 

 

216

 

1.65

2

 

0.446

 

 

260

 

1.55

3

 

0.440

 

 

250

 

1.57

4

 

0.433

 

 

236

 

1.60

5

 

0.469

 

 

308

 

1.48

6

 

0.426

 

 

224

 

1.63

Mean

 

0.439

 

 

249

 

1.58

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
 
[%]

140

1

8.2

 

0.202

51

 

6.2

82

2

7.6

 

0.191

54

 

5.6

84

3

8.4

 

0.205

50

 

6.4

81

Mean

8.1

(+)

0.199

52

(+)

6.1

82

 62.9

1

8.9

 

0.213

48

 

6.9

80

2

9.4

 

0.221

46

 

7.4

78

3

8.9

 

0.213

48

 

6.9

80

Mean

9.1

(+)

0.216

48

(+)

7.1

79

 28.0

1

10.7

 

0.240

42

 

8.7

75

2

10.7

 

0.240

42

 

8.7

75

3

10.1

 

0.231

44

 

8.1

76

Mean

10.5

(+)

0.237

43

(+)

8.5

75

 13.1

1

14.0

 

0.278

33

 

12.0

65

2

13.5

 

0.273

34

 

11.5

66

3

14.1

 

0.279

32

 

12.1

65

Mean

13.9

(+)

0.277

33

(+)

11.9

65

  5.82

1

15.8

 

0.295

29

 

13.8

60

2

17.2

 

0.307

26

 

15.2

56

3

15.5

 

0.293

29

 

13.5

61

Mean

16.2

(+)

0.298

28

(+)

14.2

59

Control

1

29.5

 

0.384

 

 

27.5

 

2

38.9

 

0.424

 

 

36.9

 

3

36.6

 

0.415

 

 

34.6

 

4

37.1

 

0.417

 

 

35.1

 

5

43.8

 

0.441

 

 

41.8

 

6

31.4

 

0.393

 

 

29.4

 

Mean

36.2

 

0.413

 

 

34.2

 

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

140

1

3

6

2

3

8

3

3

8

Mean

3

7

 62.9

1

3

8

2

3

7

3

3

8

Mean

3

8

 28.0

1

3

7

2

3

6

3

3

6

Mean

3

6

 13.1

1

3

8

2

3

6

3

3

9

Mean

3

8

  5.82

1

3

10

2

3

11

3

3

10

Mean

3

10

Control

1

3

28

2

3

30

3

3

29

4

3

35

5

3

43

6

3

30

Mean

3

33

 


 

Further Observations on Days 2, 5 and 7

Geometric mean measured test item concentration
[mg/L]

Observations on day

2

5

7

140

3.3 +++
5.2 ++

2.4 +
3.3 +++
5.2 ++

2.4 ++
3.3 +++
5.2 ++

 62.9

3.3 +++
5.2 ++

2.4 +
3.3 +++
5.2 ++

2.4 ++
3.3 +++
5.2 ++

 28.0

2.4 +
3.3 +++
5.2 ++

2.4 +
3.3 +++
5.2 ++

2.4 +
3.3 +++
5.2 ++

 13.1

3.3 +++
5.2 ++

2.4 +
3.3 +++
5.2 ++

2.4 +
3.3 +++
5.2 ++

  5.82

3.3 +++
5.2 ++

3.3 +++
5.2 ++

3.3 +++
5.2 ++

Control

1

1

1

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

 

1       = no observedeffects

2.4    = discoloration of the fronds

3.3   = discoloration of roots

5.2    = sedimentation of the test item

+      = 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 test item concentrations): The EC50-values for inhibition of the specific growth rate (fronds and dry weight) (ErC50, ErdwC50) were > 140 mg/L and 85.3 (62.0 - > 140) mg/L, respectively.
Executive summary:

The effects of the test item on the growth of the monocotyledonous aquatic plant species Lemna gibba was determined according to the principles of OECD 221 atthe test facility from 2018-04-24 to 2018-05-03, with the definitive exposure phase from 2018-04-25 to 2018-05-02.

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 2:9.38 - 18.8 - 37.5 - 75.0 - 150 mg/L, corresponding to the geometric mean measured test item concentrations of 5.82 – 13.1 – 28.0 – 62.9 – 140 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 homogenous and concentration-related black colored throughout the exposure.The validity criteria of the test guideline were fulfilled.

The concentrations of the test item and the controlwere analysed via HPLC-DAD at the beginning and end of the exposure and at every renewal day. Freshly prepared and old media were analysed.

The measured concentrations of the test item in the fresh and old test media were in the range of 83 to 106% and 38 to 92% of the nominal values, respectively. 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 test item concentration [mg/L])

Frond number

Dry weight

Growth Rate Inhibition [mg/L]

NOEC

< 5.82

< 5.82

LOEC

  5.82

  5.82

ErC10

< 5.82

< 5.82

ErC20

< 5.82

< 5.82

ErC50

> 140

85.3 (62.0 - > 140)

Inhibition of Yield [mg/L]

NOEC

< 5.82

< 5.82

LOEC

  5.82

  5.82

EyC10

< 5.82

< 5.82

EyC20

< 5.82

< 5.82

EyC50

< 5.82

< 5.82