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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid
Details on test material:
Appearance
- color: yellow to orange
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: The test substance was stored at ambient temperature.

Sampling and analysis

Analytical monitoring:
no

Test solutions

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
The test substance is a poorly water soluble mixture; therefore a water accommodated fraction (WAF) was prepared following general guidance provided in OECD 23. The stock solutions (1 mg/L) were prepared by directly adding 2 mg of test substance to 2L of test medium. The mixtures were stirred for about 2 days. Undissolved test substance was removed by centrifugation (approximately 20 min at about 17700 G). After centrifugation, no undissolved test substance was observed but the Tyndall effect was positive. The centrifuged test solution appeared yellow and clear. Then filtration with a membrane filter (pore width 0.2 µm) was done. The first 50-100 mL of filtered solution was discarded (used to condition the filter).
After filtration, the Tyndall effect was negative. The stock solution appeared yellow and clear. The aqueous fraction of the test solution, after separation of the undissolved material, is considered the water saturated fraction (WSF) in test media. Due to the very low loading rates required, it was hot possible to prepare each test solution separately. Therefore the lower test concentrations were prepared by diluting the water accomodatet fraction (WAF) into test medium according to guidance in OECD 23.
Fresh test solutions were prepared for renewal on day 2 to maximize the exposure to all soluble components of the test substance.
For preparation and dilution scheme see below.
The control and the lower test concentration up to 0.1 mg/L were visibly clear over the exposure period. The higher test concentrations (0.32 – 1 mg/L) were yellowish and clear. In the aged test solutions after 2, 5 and 7 days undissolved test particulate material (yellow crumbs) were visible at the bottom of the test vessels.
The control solutions were treated in the same way.

Test organisms

Test organisms (species):
Lemna gibba
Details on test organisms:
TEST ORGANISM
- Common name: Fat duckweed
- Strain: Clone G3
- Source (laboratory, culture collection): The stock culture was obtained from BASF SE, Crop Protection Ecology and Environmental Analytics, Speyerer Strasse 2, 67117 Limburgerhof, Germany.
- Age of inoculum (at test initiation): An inoculum culture of Lemna gibba (7-10 days old at 24 °C 2°) is used to initiate the test (study day 0).
- Method of cultivation: A culture is maintained continuously at the test facility. Before the exposure an inoculum culture is prepared with sufficient colonies of Lemna transferred aseptically into fresh sterile 20X AAP media. The inoculum culture is incubated under test conditions (see section 3.7) for 9 days prior to test initiation.

Study design

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

Test conditions

Test temperature:
24 ± 2°C
pH:
7.7 - 9.1
Nominal and measured concentrations:
0 (control), 0.01, 0.032, 0.1, 0.32, 1.0 mg/L as loading rate based on test substance mass without a correction for purity or composition
Details on test conditions:
TEST SYSTEM
- Incubation chamber used: yes (Infors HT Multitron Pro controlled climate cabinet)
- Test vessel: Glass petri dishes covered with glass plates (nominal volume 300 mL; colorless glass with a minimum water depth of 20 mm)
- Test volume: 160 mL
- Type of cover:
- Aeration:
- Agitation: yes/no
- Renewal rate of test solution (frequency/flow rate): Semi-static exposure via the test medium; in order to insure constant exposure conditions this study was conducted as a static-renewal exposure. The renewal was done at day 2
- Control end cells density:
- Initial colonies: 11 fronds/ test vessel at the start of exposure
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 20X AAP media

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Artificial light, type universal white (OSRAM L 25), permanent illumination. To minimize the potential effect of slight variations in illumination.
- Light intensity and quality: 8526 lux (±15%) at a wave length of 400 - 700 nm
- Randomization: The test vessels were placed in the test chamber according to a randomization plan prepared by using a program of the laboratory data evaluation group of the testing facility. the test vessels were rearranged at start of exposure and each time of counting (d0, d2 and d5).

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The frond number in the test vessels were counted at the start of exposure, after day 2, day 5 and at test termination.
Dry weight was determined at the start of exposure in a comparable colony and at the end of exposure in all replicate.
Colonies consisting of 2 to 4 visible fronds were transferred from the inoculum culture and randomly assigned to the test vessels under aseptic conditions. Each test vessel should contain a total of 9 to 12 fronds. The number of fronds and colonies were the same in each test vessel. The test vessels were impartially distributed in an incubator and continuous illumination. Vessel positions were altered after each assessment. The temperature was monitored within the incubator and in a separate vessel filled with deionized water. At the end of the test, the pH was measured in all replicates of the control and treatment groups; the pH at test initiation was measured in the respective bulk solutions.
The TOC values of all the test solutions were measured at the start and end of one renewal interval as an additional water quality parameter.

Frond number
The frond number in the test vessels were counted at the start of the test, after day 2, day 4 and after end of exposure the total number of fronds was counted in each replicate. Every frond visibly projecting beyond the edge of the parent frond was counted. Observations on the appearance of the fronds included necrosis, chlorosis, changes in plant size or shape and root growth were documented.

Dry weight
The biomass based on dry weight was determined at the start of exposure from a sample of the inoculum culture representative of what was used to begin the test, and at the end of the test with the plant material from each treatment and control vessel. All colonies were collected from each of the test vessels and rinsed with deionized water. They were blotted to remove excess water and then dried at 60°C for 6 days to a constant weight. Any root fragments were included.

RANGE-FINDING STUDY
The test concentrations were selected on the basis of range finding tests (experimental conduct in accordance with GLP but without a GLP Status). The results of the 7 day range finding tests (as nominal concentrations based on frond number) can be found below.
Reference substance (positive control):
yes
Remarks:
3,5 Dichlorphenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
7 d
Dose descriptor:
EL10
Effect conc.:
0.332 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
growth rate
Remarks on result:
other: Average specific growth rate based on frond number
Duration:
7 d
Dose descriptor:
EL20
Effect conc.:
0.53 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
growth rate
Remarks on result:
other: Average specific growth rate based on frond number
Key result
Duration:
7 d
Dose descriptor:
EL50
Effect conc.:
1.3 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
growth rate
Remarks on result:
other: Average specific growth rate based on frond number
Duration:
7 d
Dose descriptor:
EL10
Effect conc.:
0.445 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
growth rate
Remarks on result:
other: Average specific growth rate based on dry weight
Duration:
7 d
Dose descriptor:
EL20
Effect conc.:
0.729 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
growth rate
Remarks on result:
other: Average specific growth rate based on dry weight
Duration:
7 d
Dose descriptor:
EL50
Effect conc.:
1.87 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
growth rate
Remarks on result:
other: Average specific growth rate based on dry weight
Duration:
7 d
Dose descriptor:
EL10
Effect conc.:
0.171 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
biomass
Remarks on result:
other: Yield based on frond number
Duration:
7 d
Dose descriptor:
EL20
Effect conc.:
0.25 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
biomass
Remarks on result:
other: Yield based on frond number
Duration:
7 d
Dose descriptor:
EL50
Effect conc.:
0.521 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
biomass
Remarks on result:
other: Yield based on frond number
Duration:
7 d
Dose descriptor:
EL10
Effect conc.:
0.23 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
biomass
Remarks on result:
other: Yield based on dry weight
Duration:
7 d
Dose descriptor:
EL20
Effect conc.:
0.341 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
biomass
Remarks on result:
other: Yield based on dry weight
Duration:
7 d
Dose descriptor:
EL50
Effect conc.:
0.725 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
biomass
Remarks on result:
other: Yield based on dry weight
Details on results:
VALIDITY CRITERIA
This test was fully compliant with all the following validity criteria required by the corresponding test guidelines and is considered valid.
• The doubling time of frond number in the control was 1.4 days (<2.5 days).
Results with reference substance (positive control):
In order to verify that the Lemna cultures are responding normally to toxic stress, tests with a reference substance (3,5-Dichlorphenol) are conducted. Reference substance tests are conducted according to OECD 221 guidelines and in accordance with GLP, but without a GLP status. The results from the reference substance test are compared to 3,5-Dichlorphenol EC50 values published in OECD Guideline, which represent the typical response range for the Lemna species tested.

The EC50(7d) of the reference substance 3,5-Dichlorphenol was 11.8 mg/L (experiment date: 03 Apr 2017, project number: 63E0308/02E014).
This result is within the range of 5.9 – 13.3 mg/L and indicates that the culture of Lemna gibbqa used in this study is responding normally to toxic stress.

Any other information on results incl. tables

Lemna morphology and visual signs of phytotoxicity

Signs of phytotoxicity were observed in the treatment groups 3.2 mg/L and higher on day 3 and in treatment groups 1.0 mg/L and higher on days 5 and 7.

Loading rate

[mg/L]

Vessel No.

Morphological changes, signs of phytotoxicity

Day 2

Day 5

Day 7

0 (control)

1 - 6

0

0

0

0.01

7 - 9

0

0

0

0.032

10 - 12

0

0

9

0.1

13 - 15

0

0

9

0.32

16 - 18

0

0

8(+++), 1(+)

1.0

19 - 21

0

1(+), 3, 4, 5, 6, 8

1(+++),4, 5, 6

Explanation of abbreviations:

0:            No findings

1:            frond or root yellowing (chlorosis)

2:            fronds humped or swollen (gibbosity)

3:            small fronds

4:            shortened roots

5:            no roots

6:            plants fragmented (fronds and/or roots)

7:            dead white plant tissue (necrosis)

 8:             plants colored by test substance (fronds and/or roots)

9:            other (plants are bright, yellowish)

(+):          slight effects

(++):       medium effects

(+++):     strong effects

 

 

 

WATER quality

Test medium

Physical and chemical characteristics of the test medium used.

Date of preparation

pH value

pH value after adjustment

Date of measurement

17 Apr 2017

8.2

7.6

17 Apr 2017

18 Apr 2017

8.3

7.6

18 Apr 2017

  

pH values

pH values in the test solutions after 0 and 2 days1st Interval

Loading rate

[mg/L]

pH value
after 0 daysa

pH value after 2 days

Replicate 1

Replicate 2

Replicate 3

Replicate 4

Replicate 5

Replicate 6

0 (control)

7.7

8.7

8.7

8.7

8.7

8.7

8.7

0.01

7.8

8.7

8.7

8.7

 

 

 

0.032

7.8

8.7

8.7

8.7

 

 

 

0.1

7.8

8.7

8.7

8.7

 

 

 

0.32

7.8

8.7

8.7

8.7

 

 

 

1.0

8.1

8.7

8.7

8.7

 

 

 

awithout Lemna

 

pH values in the test solutions after 2 and 7 days2nd Interval

Loading rate

[mg/L]

pH value
after 2 daysa

pH value after 7 days

Replicate 1

Replicate 2

Replicate 3

Replicate 4

Replicate 5

Replicate 6

0 (control)

7.8

9.0

9.1

9.1

9.1

9.0

9.0

0.01

7.7

9.2

9.0

9.1

 

 

 

0.032

7.7

9.0

9.0

9.0

 

 

 

0.1

7.7

8.9

9.0

9.0

 

 

 

0.32

7.8

8.9

8.9

8.9

 

 

 

1.0

7.8

8.7

8.7

8.7

 

 

 

awithout Lemna

 

TOC values

TOC values were measured over one renewal interval as an additional water quality measure.

Loading rate
[mg/L]

day 0 (fresh solution)

day 2 (aged solution)

0 (control)

2.2

2.1

0.1

2.1

2.1

0.32

2.2

2.2

32

2.4

2.2

 

Dissolution behavior

The following observations were recorded for the dissolution behavior of the test substance in the test water: 

Loading rate

[mg/L]

day 0

fresh

day 3

day 5

aged

day 7 aged

Aged test solution

Fresh test solution

0 (control)

0

0

0

0

0

0.01

0

0

0

0

0

0.032

0

0

0

0

0

0.1

0

0

0

0

0

0.32

1

1, 2

1

1, 2

1, 2

1.0

1

1, 2

1

1, 2

1, 2

Explanation of abbreviations:

0:          no remarkable observations, clear test medium

1:          coloration caused by the test substance

2:           precipitation, undissolved test material at the bottom of the test vessel (yellow crumbs)

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The test substance is acutely toxic to aquatic plants.