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Ecotoxicological information

Toxicity to aquatic plants other than algae

Administrative data

Endpoint:
toxicity to aquatic plants other than algae
Remarks:
lemna
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 January 2018 to 08 February 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 guideline
Qualifier:
according to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Analytical samples were taken at 0 hours (initial value) from fresh test solutions, after 2 and 5 days from fresh and aged test solutions and after 7 days from aged test solutions from all test item concentrations and control.
All samples were stored deep frozen until they were transferred to the analytical laboratory. The samples were thawed to ambient temperature, mixed with 50 mL acetonitrile and shaken on a horizontal flatbed shaker for 15 min. If necessary, the samples were further diluted with acetonitrile/test medium (1:1, v/v) prior to analysis by HPLC-MS/MS.

Test solutions

Vehicle:
no
Remarks:
dissolved in test medium
Details on test solutions:
A stock solution was prepared in test medium and further diluted to achieve the required concentrations

Test organisms

Test organisms (species):
Lemna gibba
Details on test organisms:
TEST ORGANISM
- Common name: duck weed
- Strain: Lemna gibba G3 (Alismatales: Araceae)
- Source: inhouse clone from primary culture by Dr. Janet Slovin, Horticulture Crops Quality Laboratory, U.S. Department of Agriculture. BARC-West, Bldg. 050 HH-4, Beltsville, MD 20705, U.S.A.
- Method of cultivation: cultured in appropriate glass beakers under continuous illumination with OSRAM light tubes (L30/32-930 and L30/W72-965), at 6500 - 10000 lux. The temperature was 24 ± 2 °C.

Before the test was started, plants were cultured under the same illumination and temperature conditions as those to be used for the toxicity test. Young, light-green plants of similar size and comprising 2 - 4 fronds were transferred onto fresh medium and cultured for 7-10 days prior to testing, with two further transfers onto fresh medium before initiating the test.

Study design

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

Test conditions

Hardness:
no data
Test temperature:
23.5 – 23.9 °C (measured in separate vessel held under the same conditions as the test vessels)
pH:
fresh solutions: 7.40-7.61
aged solutions: 8.51-9.49 ( 100 ug/L 8.55-8.96)
Nominal and measured concentrations:
Nominal: 0, 0.160, 0.800, 4.00, 20.0 and 100 ug/L
Measured (fresh): 74-133% of nominal
Measure (aged): geometric mean concentrations: 0, 0.0496, 0.192, 1.04, 7.20 and 52 ug/L




Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass bottles each containing ~150 mL test medium covered with glass plate
- Aeration: no
- Renewal rate of test solution: on day 2 and 5
- No. of fronds per colony: 2-4 (total 12)
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM:no data

TEST MEDIUM / WATER PARAMETERS: 20xAAP test medium according to the guideline

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: no
- Photoperiod: continuously
- Light intensity and quality: T8-fluorescent tubes, length 900 mm, LT30W/865 daylight; 780 0 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number: manual counting on day 0, 2, 5 and 7 (inludes observation of appearance)
- Determination of biomass: dry weight on day 7 (for t=0 based on 6X12 fronds from the culture); dried at 60°C for about 72 hours
- Determination of frond area: no
- Other: plant development, frond size, necrosis and additional observations

RANGE-FINDING STUDY
- Test concentrations: 0, 0.010, 0.100, 1.00 and 10.0 mg/L
- Results used to determine the conditions for the definitive study: inhibition of yield and growth rate based on frond number at 0.01 mg/L and above
On day 3 shortened roots and yellow fronds were observed from 0.100 to 10.0 mg/L. Additionally at 0.100 mg/L fronds were tightly assembled and gibbosity was observed. At 1.00 and 10.0 mg/L fronds were partially singularized. Also, at 1.00 mg/L, the edge of the fronds was coloured red.
On day 5 at 0.0100 mg/L shortened roots and partially small offshoots were found. Small offshoots were also observed at 0.100 mg/L. Shortened roots, as well as yellow and partially singularized fronds were observed at test item concentrations 0.100 and 10.0 mg/L. At 1.00 mg/L shortened roots, and singualised, yellow fronds with red coloured edge were found.
On day 7 at 0.0100 mg/L shortened roots, small offshoots, tightly assembled fronds and gibbosity were found. Small offshoots and gibbosity were also observed at 0.100 mg/L. Shortened roots, as well as yellow and partially singularized fronds were observed at test item concentrations 0.100 and 10.0 mg/L. At 1.00 mg/L shortened roots, and singualised, yellow fronds were found.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
0.192 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks on result:
other: based on yield and growth rate
Duration:
7 d
Dose descriptor:
NOEC
Effect conc.:
1.04 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: based on yield and growth rate
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
5.5 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks on result:
other: based on yield
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
33.3 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
frond number
Remarks on result:
other: based on growth rate
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
14.6 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: based on yield
Duration:
7 d
Dose descriptor:
EC50
Effect conc.:
27.1 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: based on growth rate
Details on results:
On day 2 shortened roots and tightly assembled fronds were observed at 100 µg/L.
On day 5 shortened roots and gibbosity were observed at concentrations of 4.00 to 100 µg/L. Additionally at concentrations of 20.0 and 100 µg/L small offshoots and tightly assembled fronds were found. At 100 µg/L additionally, fronds were brown.
On day 7 shortened roots and gibbosity were observed at concentrations of 4.00 to 100 µg/L. Additionally at concentrations of 20.0 and 100 µg/L small offshoots and tightly assembled fronds were found. At 100 µg/L additionally, fronds were brown and partially singularized.
Results with reference substance (positive control):
EC50 growth rate 3,5-dichlorophenol = 6.69 mg/L (frond numbers), 11.0 mg/L (dry weight)

Any other information on results incl. tables

Frond numbers:yield

 

Mean of frond numbers

Yield based on mean frond numbers

Inhibition (%)

Days

 

 

Nominal conc.

[µg/L]

0

2

5

7

7-0

7-0 days

Control

12

24

91

242

230

0.0

0.160

12

24

91

258

246

-7.01)

0.800

12

25

93

256

244

-6.12)

4.00

12

23

79

191

179*

22.2

20.0

12

23

60

108

96*

58.3

100

12

19

35

42

30*

87.0

* Statistically significant different to the control (Dunnetts-t-test, left-sided)

1)Value was omitted for EC10, 20, 50-calculation

2)Value was set to zero for EC10, 20, 50-calculation

 

Frond numbers: growth rates

Days

Days

Nominal conc.

[µg/L]

2

5

7

2

5

7

Mean growth rate µ [d-1]

Inhibition of growth rat (%)

Control

0.3387

0.4039

0.4290

0.0

0.0

0.0

0.160

0.3389

0.4051

0.4383

-0.1

-0.3

-2.21)

0.800

0.3602

0.4102

0.4371

-6.3

-1.6

-1.92)

4.00

0.3230

0.3772

0.3952*

4.6

6.6

7.9

20.0

0.3240

0.3229

0.3129*

4.3

20.1

27.1

100

0.2198

0.2155

0.1798*

35.1

46.6

58.1

* Statistically significant different to the control (Dunnetts-t-test, left-sided)

1)Value was omitted for EC10, 20, 50-calculation

2)Value was set to zero for EC10, 20, 50-calculation

 

Frond numbers: mean doubling times (Td)

Days

Nominal conc.

[µg/L]

2

5

7

7

Td[days]

Td[hours]

Control

2.046

1.716

1.616

38.8

0.160

2.045

1.711

1.581

37.9

0.800

1.924

1.690

1.586

38.1

4.00

2.146

1.838

1.754

42.1

20.0

2.139

2.147

2.215

53.2

100

3.154

3.216

3.855

92.5

Dry weight:

 

Mean dry weight
[g]

Yield based on mean dry weight [g]

Inhibition [%]

Nominal conc.

[µg/L]

Days

0

7

0.0

7-0

Control

0.0106

0.0435

-11.61)

0.0329

0.160

0.0473

-10.02)

0.0367

0.800

0.0468

10.0

0.0362

4.00

0.0402

38.9

0.0296

20.0

0.0307

81.2

0.0201*

100

0.0168

 

0.0062*

* Statistically significant different to the control (Bonferroni-Holms corrected Welch test, left-sided)

1)Value was omitted for EC10, 20, 50-calculation

2)Value was set to zero for EC10, 20, 50-calculation

 

 

 

 

 

 

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The EC50 based on growth rate is 27.1 ug/L based on biomass, The NOEC based on growth rate is 0.192 ug/L based on biomass
Executive summary:

In a study with a semi-static design (renewal on day 2 and 5) the vegative growth of Lemna gibba G3 was observed for a test period of 7 days by measuring the increase of frond numbers and dry weight. Lemna was exposed at nominal concentrations of 0, 0.160, 0.800, 4.00, 20.0 and 100 ug/L (mean measured concentrations 0, 0.0496, 0.192, 1.04,  7.20 and 52 ug/L).

Significant inhibitory effects were determined for yield and growth rate of frond numbers and biomass at test item concentrations of 1.04 ug/L (measured) and above. The EC50-value for yield of frond numbers was determined to be 5.50 ug/L (measured), the EC50-value for growth rate of frond numbers was determined to be 33.3 ug/L (measured). The EC50-value for yield of dry weight was determined to be 14.6 ug/L (measured), the EC50-value for growth rate of dry weight was determined to be 27.1 ug/L (measured).