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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:
10 - 19 Jan 2018
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: Commission Regulation (EC) No 761/2009, Annex, Part C, C.26: "Lemna sp. Growth Inhibition Test"
Version / remarks:
2009
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, Klimaschutz, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test item concentrations and the control
- Sampling method: Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at test start, at each test medium renewal (day 2 and day 5). Samples from aged test solution and control were sampled at each test medium renewal and at the end of the test.
- Sample storage conditions before analysis: All samples were stored in a freezer (≤ - 20 °C), protected from light until analysis was performed.

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test medium of the highest test concentration of nominal 100 mg test item/L was prepared at test start and the two water renewal days by dissolving 112.2, 98.1 and 105.7 mg test item into 1122, 981 and 1057 mL test water, respectively by intense stirring for 20 to 40 min. Adequate volumes of this test medium were diluted with test water to prepare the test media of the other desired test concentrations.
- Controls: Yes (test medium only)
- Observations: The test medium showed blue coloration in all concentrations.

Test organisms

Test organisms (species):
Lemna gibba
Details on test organisms:
TEST ORGANISM
- Common name: Duckweed
- Strain: G 3
- Source: In-house laboratory culture
- Pre-culture: For at least 7 days under test conditions with weekly media exchange.
- Method of cultivation: Under standardised conditions according to the test guidelines.

ACCLIMATION
- Culturing media and conditions (same as test or not): Same as test.
- Any deformed or abnormal cells observed: No deformation was observed.

Study design

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

Test conditions

Test temperature:
23.4 - 24.3 °C
pH:
7.4 - 8.8 (Control)
7.5 - 8.7 (Test item concentration)
Nominal and measured concentrations:
Control, 1.0, 3.2, 10, 32 and 100 mg/L (nominal)
Details on test conditions:
TEST SYSTEM
- Incubation chamber used: Yes, controlled environment room
Test vessel:
- Type: Open (covered with glass dishes)
- Material, size, headspace, fill volume: 250 mL glass vessels with approximately 200 mL test medium; 50 mL headspace
- Type of cover: Glass dishes
- Aeration: No aeration
- Agitation: No but the test vessels were randomized daily during the exposure period.
- Control end cells density: 255 fronds at test end (mean growth rate 0.437)
- No. of colonies per vessel: 12 fronds per vessel
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

GROWTH MEDIUM
- Standard medium used: Yes (20 x APP medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 20 x AAP medium
- Conductivity: < 5 μS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: The pH-values were measured in all freshly prepared and aged test media; the temperature was measured daily in a test vessel filled with water and incubated under the same conditions as the test vessels.

OTHER TEST CONDITIONS
- Adjustment of pH: Adjusted with 1 M NaOH to 7.4
- Photoperiod: Continuous illumination
- Light intensity and quality: 6917 lux (mean value) with a range of 6590 to 7270 lux.

EFFECT PARAMETERS MEASURED:
- Determination of frond number: At test start frond and colony numbers were recorded. At days 2, 5 and 7 frond numbers were counted and appearance of colonies was observed.
- Determination of biomass: At test start the dry weight of a sample of fronds similar to those used to inoculate the test vessels was determined. At the end of the test the dry weight of all plants from each vessel was determined. Accordingly, the plants were collected at the end of the test.

RANGE-FINDING STUDY: non GLP pre-test
- Results used to determine the conditions for the definitive study: Based on th results of a non-GLP pre-experiment the test item concentration range of the main test were determined.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (tested at least twice a year)

Results and discussion

Effect concentrationsopen allclose all
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Frond number
Remarks on result:
other: 95% confidential limits
Remarks:
n.d.
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Frond number
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Dry weight
Remarks on result:
other: 95% confidential limits
Remarks:
n.d.
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
3.2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Remarks:
Dry weight
Details on results:
- Any visual signs of phytotoxicity (abnormalities): The shape of fronds and colonies after the test period of 7 days was not different to those in the control up to and including the nominal test concentration of 10 mg test item/L. At the higher test item concentrations of 32 and 100 mg test item/L the fronds showed more gibbous growth than the control replicates after 7 days.

Any other information on results incl. tables

Table 1: Summary of Analytical Results

Sample Description [mg test item/L]

% of nominal 1)

RSD [%]

 n

Control

n.a.

n.a.

12

1

98

8

12

3.2

103

3

12

10

104

1

12

32

100

2

12

100

105

2

12

1): mean value of all measured samples per treatment group RSD: relative standard deviation per treatment group n: number of analysed samples n.a.: not applicable

Table 2: Biological Results: Frond Numbers of Lemna gibba during the Test Period of 7 days

Nominal Concentration

Flask

Frond number after

[mg test item/L]

No

2 days

5 days

7days

Control

1

32

130

248

 

2

29

112

259

 

3

29

121

258

 

M

30.0

121.0

255.0

 

s

1.73

9.00

6.08

1.0

1

31

129

277

 

2

29

128

282

 

3

31

120

271

 

m

30.3

125.7

276.7

 

s

1.15

4.93

5.51

3.2

1

31

118

247

 

2

32

103

245

 

3

29

125

250

 

m

30.7

115.3

247.3

 

s

1.53

11.24

2.52

10

1

29

103

210

 

2

30

93

193

 

3

29

86

153

 

M

29.3

94.0

185.3

 

S

0.58

8.54

29.26

32

1

27

83

148

 

2

27

80

141

 

3

28

87

155

 

M

27.3

83.3

148.0

 

S

0.58

3.51

7.00

100

1

25

85

138

 

2

20

60

108

 

3

29

97

191

 

M

24.7

80.7

145.7

 

S

4.51

18.88

42.03

m: mean value; s: standard deviation starting number of fronds: 12 fronds per test vessel

Table 3: Dry Weights, Growth Rates μ (Based on Dry Weights) and Yields y (Based on Dry Weights) of Lemna gibba after 7 days of Exposure

Nominal Concentration

 

[mg test item/L]

Flask

 

 

No.

Dry weights (after 7 days)

 

[mg]

Yields y (dry weight) (after 7 days)

 [mg]

Growth rates μ (dry weight)

0 - 7 days

[1/day]

Control

1

31.7

29.9

0.410

 

2

33.2

31.4

0.416

 

3

34.9

33.1

0.424

 

M

33.3

31.5

0.417

 

S

1.60

1.6

0.007

1.0

1

35.1

33.3

0.424

 

2

37.3

35.5

0.433

 

3

33.4

31.6

0.417

 

M

35.3

33.5

0.425

 

S

1.96

2.0

0.008

3.2

1

32.0

30.2

0.411

 

2

29.5

27.7

0.400

 

3

33.5

31.7

0.418

 

M

31.7

29.9

0.409

 

S

2.02

2.0

0.009

10

1

25.8

24.0

0.380

 

2

23.2

21.4

0.365

 

3

22.3

20.5

0.360

 

M

23.8

22.0

0.368

 

S

1.82

1.8

0.011

32

1

20.2

18.4

0.345

 

2

19.2

17.4

0.338

 

3

20.9

19.1

0.350

 

M

20.1

18.3

0.345

 

S

0.85

0.9

0.006

100

1

18.5

16.7

0.333

 

2

13.9

12.1

0.292

 

3

23.8

22.0

0.369

 

M

18.7

16.9

0.331

 

S

4.95

5.0

0.038

m: mean value; s: standard deviation

initial dry weight at test start: 1.8 mg dry weight per test vessel

Table 4: Biological Results: Yields y (Based on Frond Numbers) and Percentage Inhibition of y (Based on Frond Numbers) during the Test Period of 7 days

Nominal Concentration

Yields y (frond number) and % inhibition of y

 

0-2 days

0-5 days

0-7 days

[mg test item/L]

Y

%

Y

%

Y

%

Control

18.0

-

109.0

-

243.0

-

1.0

18.3

-1.9

113.7

-4.3

264.7

-8.9

3.2

18.7

-3.7

103.3

5.2

235.3

3.2

10

17.3

3.7

82.0

24.8*

173.3

28.7*

32

15.3

14.8

71.3

34.6*

136.0

44.0*

100

12.7

29.6*

68.7

37.0*

133.7

45.0*

- % inhibition: increase in growth relative to that of the control * mean value significantly different from the control (tested with Williams t-test, α = 0.05, one-sided)

 

Table 5: Biological Results: Growth Rates μ (Based on Frond Numbers) and Percentage Inhibition of μ (Based on Frond Numbers) during the Test Period of 7 days

Nominal Concentration

Growth rates μ (frond number) [1/day] and % inhibition of μ

[mg test item/L]

0-2 days

0-5 days

0-7 days

 

µ

%

µ

%

µ

%

Control

0.458

-

0.462

-

0.437

-

1.0

0.463

-1.3

0.470

-1.7

0.448

-2.7

3.2

0.469

-2.4

0.452

2.1

0.432

1.0

10

0.447

2.4

0.411

11.0*

0.390

10.7*

32

0.412

10.1

0.387

16.1*

0.359

17.8*

100

0.355

22.5*

0.377

18.3*

0.353

19.2*

- % inhibition: increase in growth relative to that of the control * mean value significantly different from the control (tested with Williams t-test, α = 0.05, one-sided)

   

Table 6: Validity criteria

Criterion from the guideline

Outcome

Validity criterion fulfilled

The doubling time of frond number in the control must be less than 2.5 days (60 h), corresponding to approximately a seven-fold increase in seven days and an average specific growth rate of 0.275 d-1.

1.6 days

Yes

 

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
See Table 6 in section "Any other information on results incl tables"