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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
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
Guideline:
OECD Guideline 221 (Lemna sp. Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of Amines, C12-14-tert-alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-)
EC Number:
916-868-7
Molecular formula:
C32H22CoN10O8.C10-14H21-29NH2 and C32H22CoN10O8.Na
IUPAC Name:
Reaction mass of Amines, C12-14-tert-alkyl, bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-) and sodium bis[2,4-dihydro-4-[(2-hydroxy-5-nitrophenyl)azo]-5-methyl-2-phenyl-3H-pyrazol-3-onato(2-)]cobaltate(1-)
Test material form:
solid
Details on test material:
- Physical state / Appearance: Solid / 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. Each test solution was prepared separately (differential loading) by directly adding test substance to test medium according to the loading rate described in the table below and stirring for about 2 days. Undissolved test substance was removed by centrifugation (approximately 20 min at about 17700 G). After centrifugation, undissolved test substance was still observed and the Tyndall effect was positive. 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). The aqueous fraction of the test solution, after separation of the undissolved material, is considered the water saturated fraction (WSF) in test media. In the filtered solution the Tyndall effect was negative.
The stock solutions for day 2 were not centrifuged, only filtration was used to separated undissolved test substance.

Fresh test solutions were prepared and renewed on day 2 to maximize the exposure to all soluble components of the test substance.

Test organisms

Test organisms (species):
Lemna gibba
Details on test organisms:
TEST ORGANISM
- Common name: 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.
- 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 °C ± 2
pH:
7.8 - 9
Nominal and measured concentrations:
0 (control), 0.32, 1, 3.2, 10, 32, 100 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: Vötsch Industrietechnik GmbH Bioline (VB1014) controlled climate cabinet.
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
- No. of colonies per vessel: 11 fronds/ test vessel at the start of exposure
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6
- 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 d4).

GROWTH MEDIUM
- Standard medium used: yes; 20X AAP media

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

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: permanent illumination
- Light intensity and quality: Artificial light, type universal white (OSRAM L 25); 8548 lux (±15%) at a wave length of 400 - 700 nm

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
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.
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 3 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 were (as nominal concentrations based on frond number):
% inhibition at test concentrations (mg/L):
1 10 100
-0.79% 62% 80%
ErL50 = approx. 1-10 mg/L
Reference substance (positive control):
yes
Remarks:
3,5 Dichlorphenol

Results and discussion

Effect concentrationsopen allclose all
Duration:
7 d
Dose descriptor:
EL50
Effect conc.:
25.7 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.:
1.04 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:
EL50
Effect conc.:
44.4 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.:
1.07 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.:
3.66 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.:
0.697 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.:
4.03 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:
EL10
Effect conc.:
0.427 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:
ANALYTICAL CONCENTRATION CONTROL
Concentration control analysis was not performed since the analytical detection limit was above the water solubility of the test substance because a reliable method for analyses in the required concentration range could not be developed. However, all reasonable efforts were taken to produce a water saturated fraction (WSF) of the test substance in test media, following the guidance in OECD 23. The test organisms were exposed to a water saturated fraction (WSF) of the test substance at the given loading rate and the effect concentrations should be expressed based on the loading rate as recommended in OECD 23.
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 on day 2, day 5 and 7.

Loading rate

[mg/L]

Vessel No.

Morphological changes, signs of phytotoxicity

Day 2

Day 4

Day 7

Control

1 - 6

0

0

0

0.32

7 - 9

0

0

0

1

10 - 12

0

0

3, 8, 9 (plants inelastic)

3.2

13 - 15

0

3, 4, 6

3, 4, 6

10

16 - 18

6, 8

1, 6, 8

1, 6, 7, 8 (+++)

32

19 - 21

5, 6, 8

1, 5, 6, 8

1, 8(+++)

100

22 - 24

5, 6, 8

1, 5, 6, 8

1, 5, 6, 8, 9 (plants dissolves) (+++)

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 substanz (fronds and/or roots)

9:            other:

(+):          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

04 Aug 2017

8.0

7.4

04 Aug 2017

04 Aug 2017

8.1

7.5

07 Aug 2017


 

ph values

pH values in the test solutions after 0 and 2 days1stInterval

 

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)

7.8

8.7

8.7

8.7

8.7

8.7

8.7

0.32

7.9

8.7

8.7

8.7

 

 

 

1

7.8

8.7

8.7

8.7

 

 

 

3.2

7.9

8.7

8.7

8.7

 

 

 

10

7.9

8.7

8.7

8.7

 

 

 

32

7.9

8.7

8.7

8.7

 

 

 

100

7.9

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

9.0

8.9

8.9

9.0

8.9

0.32

7.8

9.1

9.0

9.0

 

 

 

1

7.8

9.0

8.8

8.9

 

 

 

3.2

7.8

8.6

8.6

8.7

 

 

 

10

7.8

8.6

8.6

8.7

 

 

 

32

7.8

8.7

8.6

8.6

 

 

 

100

7.8

8.6

8.6

8.7

 

 

 

awithout Lemna

 TOCvalues

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

2.2

0.32

2.3

2.2

1

2.3

2.3

3.2

2.5

2.3

10

2.9

2.5

32

3.2

2.9

100

3.6

3.4

 

TOC could not be used as concentration control due to the low solubility of the test substance. However, TOC increased monotonially in the fresh test solutions. The higher TOC in the test solutions 10-100 mg/L most likely originates from dissolved components of the test substance.

 

 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

day 2

day 5

day 7

aged test solution

fresh test solution

0 (control)

0

1, 4

0

1

1

0.32

0

1, 4

0

1

1

1

1

1, 2, 4

1

1, 2

1, 2

3.2

1

1, 2, 4

1

1, 2

1, 2

10

1

1, 5

1

1, 5

1, 5

32

1

1, 5

1

1, 5

1, 5

100

1

1, 5

1

1, 5

1, 5

Explanation of abbreviations:

0:          no remarkable observations, clear test medium

1:          coloration caused by the test substance (light yellow up to yellow, depends on the concentration)

2:          coloration caused by the test substance (red)

4:           crumb at the bottom of the test vessel

5:          dust at the bottom of the test vessel

 

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
The test substance is acutely harmful for aquatic plants.