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Diss Factsheets

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 2019-01-09 to 2019-01-18, with the definitive exposure phase from 2019-01-09 to 2019-01-16.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
according to OECD and GLP guideline

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

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

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-)
EC Number:
233-357-8
EC Name:
Sodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-)
Cas Number:
10127-27-2
Molecular formula:
C16H11CrN5O8S.Na
IUPAC Name:
sodium [3-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]-2-hydroxy-5-nitrobenzenesulphonato(3-)]hydroxychromate(1-)

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Determination of the test item
Samples were analyzed with an HPLC-DAD method, implemented under non-GLP and documented finally in the GLP raw data. The method was validated .

Sampling schedule
All concentration levels and the control were analytically verified via HPLC-DAD at the start (0 hours) and at the end of the exposure (72 hours).
Freshly prepared and aged media were analyzed.

Sampling and pre-treatment
From freshly prepared media, samples were taken from the stock solutions and from the aged media samples were taken from pooled replicates.

Criteria for the analytical monitoring (target)
Recoveries of the test item should be within ± 20% of the nominal or initially measured concentrations.

Test solutions

Details on test solutions:
Stock solution, media preparation
114 mg test item/L was freshly prepared with dilution water. The dispersion was stirred with a magnetic stirrer for 1 hour (1100 rpm, room temperature).

Test concentrations
The stock solution and four dilution levels were prepared with dilution water in a geometrical series with a separation factor of 1.778 and tested as follows:
11.4 - 20.3 - 36.1 - 64.1 - 114 mg/L, corresponding to the geometric mean measured test item concentration of 12.6 – 22.9 – 43.0 – 77.3 – 134 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 Concentration in prepared medium
[g/L] [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:
Dilution water
Total hardness: Not determined
Test temperature:
Room temperature [°C]: min.: 25.0 max.: 26.2 mean value: 25.7
pH:
Geometric mean measured pH-value pH-value
test item concentration 0 h (Fresh medium) 7 d (Old medium)
[mg/L]
134 7.78 8.36
77.3 7.78 8.39
43.0 7.78 8.38
22.9 7.79 8.38
12.6 7.79 8.37
Control 7.51 8.37
Dissolved oxygen:
not measured
Salinity:
not measured
Conductivity:
Dilution water Conductivity [µS/cm]: Not determined
Nominal and measured concentrations:
Nominal test item concentration: 11.4 - 20.3 - 36.1 - 64.1 - 114 mg/L
Geometric mean measured test item concentration: 12.6 – 22.9 – 43.0 – 77.3 – 134 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.

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
Key result
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
134 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
Key result
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
134 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weight
Remarks on result:
other: inhibition of yield
Key result
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
12.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: frond number
Remarks on result:
other: inhibition of yield
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
77.3 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
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
< 12.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: Frond number
Remarks on result:
other: Inhibition of yield
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
77.3 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weight
Remarks on result:
other: inhibition of yield
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
123 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: frond number growth rate inhibition
Remarks on result:
other: 95% Confidence limits: 75.7 - > 134 mg/L
Key result
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
> 134 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: frond number growth rate inhibition
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 134 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: frond number growth rate inhibition
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
> 134 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weight growth inhibition
Key result
Duration:
7 d
Dose descriptor:
EC20
Effect conc.:
> 134 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weight growth inhibition
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 134 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weight growth inhibition
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
< 12.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: frond number inhibition of yield
Key result
Duration:
7 d
Dose descriptor:
EC20
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: frond number inhibition of yield
Remarks on result:
not determinable
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 134 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: frond number inhibition of yield
Key result
Duration:
7 d
Dose descriptor:
EC10
Effect conc.:
< 12.6 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weight inhibition of yield
Key result
Duration:
7 d
Dose descriptor:
EC20
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weight inhibition of yield
Remarks on result:
not determinable
Key result
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 134 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weight inhibition of yield
Key result
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
134 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weight
Remarks on result:
other: growth rate inhibition
Key result
Duration:
7 d
Dose descriptor:
LOEC
Effect conc.:
> 134 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: dry weight
Remarks on result:
other: growth rate inhibition
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 from 2018-11-07 to 2018-11-15, with the definitive exposure phase from 2018-11-07 to 2018-11-14 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 7.03 5.88 ± 3.20
95% confidence interval 6.53 – 7.53
Yield inhibition (number of fronds)
EyC50 4.62 4.67 ± 2.78
95% confidence interval 4.02 – 5.35
Growth rate inhibition (dry weight)
ErdwC50 6.48 5.66 ± 2.74
95% confidence interval 6.07 – 6.83
Yield inhibition (dry weight)
EydwC50 4.56 4.74 ± 2.41
95% confidence interval 4.13 – 5.27

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 / LOEC was determined by calculation of statistically significant differences of growth rate and yield using Dunnett’s Method. A Shapiro-Wilk’s Normality test and a Brown-Forsythe’s Equal Variance test were done first. P-values for both Normality and Equal Variance tests are 0.05. The @-value (acceptable probability of incorrectly concluding that there is a difference) is @ = 0.01.


EC-values and statistical analyses
EC10-, EC20- and EC50-values (after 7 d) of the growth rate and yield (frond number and dry weight) inhibition were calculated by sigmoidal dose-response. 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

134

1

12

22

75

173

2

12

22

81

179

3

12

21

65

157

Mean

12

22

74

170

 77.3

1

12

23

87

187

2

12

24

87

187

3

12

24

84

185

Mean

12

24

86

186

 43.0

1

12

23

86

179

2

12

23

87

192

3

12

24

87

204

Mean

12

23

87

192

 22.9

1

12

24

101

222

2

12

23

85

192

3

12

24

84

188

Mean

12

24

90

201

 12.6

1

12

22

84

179

2

12

24

87

191

3

12

25

95

204

Mean

12

24

89

191

Control

1

12

24

104

246

2

12

24

101

247

3

12

22

85

209

4

12

25

93

223

5

12

24

112

241

6

12

23

91

225

Mean

12

24

98

232

* = 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

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]

134

1

 

0.381

10

 

161

27

1.82

2

 

0.386

9

 

167

24

1.80

3

 

0.367

13

 

145

34

1.89

Mean

(+)

0.378

11

(+)

158

28

1.83

 77.3

1

 

0.392

7

 

175

20

1.77

2

 

0.392

7

 

175

20

1.77

3

 

0.391

8

 

173

21

1.77

Mean

(+)*

0.392

7

(+)

174

21

1.77

 43.0

1

 

0.386

9

 

167

24

1.80

2

 

0.396

6

 

180

18

1.75

3

 

0.405

4

 

192

13

1.71

Mean

(+)*

0.396

6

(+)

180

18

1.75

 22.9

1

 

0.417

1

 

210

4

1.66

2

 

0.396

6

 

180

18

1.75

3

 

0.393

7

 

176

20

1.76

Mean

(+)*

0.402

5

(+)

189

14

1.73

 12.6

1

 

0.386

9

 

167

24

1.80

2

 

0.395

7

 

179

19

1.75

3

 

0.405

4

 

192

13

1.71

Mean

(+)*

0.395

7

(+)

179

18

1.75

Control

1

 

0.431

 

 

234

 

1.61

2

 

0.432

 

 

235

 

1.60

3

 

0.408

 

 

197

 

1.70

4

 

0.417

 

 

211

 

1.66

5

 

0.429

 

 

229

 

1.62

6

 

0.419

 

 

213

 

1.66

Mean

 

0.423

 

 

220

 

1.64

Repl. No. = replicate number

 * =Since inihibtion values were< 10% and showed no concentration dependent correlation, growth rate inhibition is considered to be not significant.

 

 

 


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
 
[%]

134

1

25.7

 

0.426

6

 

24.4

19

2

27.2

 

0.434

5

 

25.9

14

3

23.4

 

0.413

9

 

22.1

27

Mean

25.4

(+)*

0.425

7

(+)

24.1

20

 77.3

1

28.2

 

0.440

3

 

26.9

11

2

27.1

 

0.434

5

 

25.8

14

3

28.7

 

0.442

3

 

27.4

9

Mean

28.0

(-)

0.439

4

(-)

26.7

11

 43.0

1

26.0

 

0.428

6

 

24.7

18

2

27.2

 

0.434

5

 

25.9

14

3

31.2

 

0.454

0

 

29.9

1

Mean

28.1

(-)

0.439

4

(-)

26.8

11

 22.9

1

29.9

 

0.448

2

 

28.6

5

2

23.0

 

0.410

10

 

21.7

28

3

24.1

 

0.417

8

 

22.8

24

Mean

25.7

(+)*

0.425

7

(+)1

24.4

19

 12.6

1

21.7

 

0.402

12

 

20.4

32

2

25.3

 

0.424

7

 

24.0

20

3

27.8

 

0.438

4

 

26.5

12

Mean

24.9

(+)*

0.421

7

(+)1

23.6

21

Control

1

33.3

 

0.463

 

 

32.0

 

2

32.8

 

0.461

 

 

31.5

 

3

28.2

 

0.440

 

 

26.9

 

4

29.9

 

0.448

 

 

28.6

 

5

34.2

 

0.467

 

 

32.9

 

6

30.2

 

0.449

 

 

28.9

 

Mean

31.4

 

0.455

 

 

30.1

 

The initial biomass dry weight was 1.3 mg per replicate.

Repl. No. = replicate number

* =Since inihibtion values were< 10% and showed no concentration dependent correlation, growth rate inhibition is considered to be not significant.

1) =statistically significant, but with a higher concentration level showing no harmful effect/statistical significance compared to control (as definition of NOEC/LOEC).

 

 

 


Colony Number (Plants) on Days 0 and 7

 

Geometric mean measured test item concentration
[mg/L]

Replicate

No.


Colony number

Day 0

Day 7

134

1

3

32

2

3

28

3

3

22

Mean

3

27

 77.3

1

3

38

2

3

36

3

3

37

Mean

3

37

 43.0

1

3

33

2

3

37

3

3

35

Mean

3

35

 22.9

1

3

39

2

3

34

3

3

30

Mean

3

34

 12.6

1

3

27

2

3

28

3

3

29

Mean

3

28

Control

1

3

33

2

3

37

3

3

29

4

3

27

5

3

33

6

3

36

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

134

5.2 ++

3.3 +
5.2 +++

2.1 +
3.3 +
5.2 +++

 77.3

5.2 ++

3.3 +
5.2 ++

3.3 +
5.2 ++

 43.0

5.2 ++

3.3 +
5.2 ++

3.3 +
5.2 ++

 22.9

5.2 +

3.3 +
5.2 +

3.3 +
5.2 +

 12.6

5.2 +

5.2 +

5.2 +

Control

1

1

1

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

 1       = no observedeffects

2.1    = chlorosis

3.3   = discoloration of roots

5.2    = sedimentation of the test item

+      = slight effects

++    = medium effects

+++  = strong effects


 

 Measured Exposure Concentrations

 

The concentrations of the test item were analytically verified via HPLC-DAD in fresh media at the start (0 days) and in old media at the end of the exposure (7 days) in all concentration levels and in the control.

The measured concentrations of the test item were in the range of 110 to 130% of the nominal test item concentrations at 0 days and at 7 days in the range of 112 to 121% of the nominal test item concentrations.

The measured test item concentrations were all over 100% of the nominal concentrations, the geometric mean measured concentrations were calculated.

Measured Concentrations of the Test Item during the Definitive Test

Sampling date

Fresh Media,
0 days

Old Media,
7 days

 

Nominal test item concentration

[mg/L]

test item

 

Meas. conc.

[mg/L]

%

Meas. conc.

[mg/L]

%

Geometric mean measured
concentration
[mg/L]

114

129

113

138

121

134

64.1

83.0

130

71.9

112

 77.3

36.1

45.0

125

41.1

114

 43.0

20.3

23.1

114

22.7

112

 22.9

11.4

12.5

110

12.8

112

 12.6

Control 1

< LOQ

< LOQ

 

Meas. conc.= measured concentration of the test item

%                  = percent of the nominal measured concentration of the test item

LOQ             = limit of quantification of the analytical method (2 mg/L of the test item)


 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The EC50-values for both inhibition of the specific growth rate (fronds and dry weight) (ErC50, ErdwC50) were > 134 mg/L. The EC50-values for both inhibition of yield (fronds and dry weight) (EyC50, EydwC50) were > 134 mg/L (geometric mean measured test item concentrations) .
Executive summary:

The effects of the test item (batch no.: CHM8004219) on the growth of the monocotyledonous aquatic plant species Lemna gibba was determined according to the principles of OECD 221and Council Regulation (EC) No. 266/2016 Method C. 26 at the test facility from 2019-01-09 to 2019-01-18, with the definitive exposure phase from 2019-01-09 to 2019-01-16.

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 1.778:11.4 - 20.3 - 36.1 - 64.1 - 114 mg/L, corresponding to the geometric mean measured test item concentration of 12.6 – 22.9 – 43.0 – 77.3 – 134 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 orange colored throughout the exposure. The validity criteria of the test guideline were fulfilled.

The concentrations of the test item were analytically verified via HPLC-DAD in fresh media at the start (0 days) and in old media at the end of the exposure (7 days) in all concentration levels and in the control. The measured concentrations of the test item were in the range of 110 to 130% of the nominal test item concentrations at 0 days and at 7 days in the range of 112 to 126% of the nominal test item concentrations. 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 thegeometric mean measuredtest item concentration [mg/L])

Frond number

Dry weight

Growth Rate Inhibition [mg/L]

NOEC

77.3

  134

LOEC

134

> 134

ErC10

123 (75.7 - > 134)

> 134

ErC20

> 134

> 134

ErC50

> 134

> 134

Inhibition of Yield [mg/L]

NOEC

< 12.6

77.3

LOEC

  12.6

134

EyC10

< 12.6

< 12.6

EyC20

   n.a.

   n.a.

EyC50

> 134

> 134

n.a. = not applicable

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