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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

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:
Amines, C10-14-branched and linear alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)
EC Number:
285-083-3
EC Name:
Amines, C10-14-branched and linear alkyl, bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)
Cas Number:
85029-58-9
Molecular formula:
C34H24CrN8O6.C10-14H21-29NH2
IUPAC Name:
reaction mass of: branched and linear(C10-C14)ammonium , bis[2-[(4,5-dihydro-3-methyl-5-oxo-1-phenyl-1H-pyrazol-4-yl)azo]benzoato(2-)]chromate(1-)
Test material form:
solid
Details on test material:
Appearance
- physical state: solid
- color: brown
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 poorly water soluble and a complex mixture. Therefore, the test solutions were prepared separately for each test concentration following general guidance provided in OECD 23. Each test solution was prepared by directly adding the required mass of test substance to test medium and stirring for approximately 2 days.

Test organisms

Test organisms (species):
Lemna gibba
Details on test organisms:
TEST ORGANISM
- Strain: Lemna gibba 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 (9 days old at 24 °C ±2°) was 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 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 °C
pH:
7.5 - 9
Nominal and measured concentrations:
0 (control), 0.32, 1, 3.2, 10, 32 mg/L as loading rates based on test substance mass without correction for purity
Details on test conditions:
TEST SYSTEM
- Incubation chamber used: 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 flow-through (e.g. peristaltic or proportional diluter):
- Renewal rate of test solution (frequency/flow rate): In order to insure constant exposure conditions this study was conducted as a static-renewal exposure. The renewal was done at day 3
- No. of fronds per colony: 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 (20X AAP medium according to OECD guideline 221)
Growth medium intended for testing was prepared at least 2 days before use to allow the pH to stabilize. The pH of growth medium was checked prior to use and readjusted if necessary by the addition of 0.1 or 1 M NaOH or HCl.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: 20X AAP medium according to OECD guideline 221

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: Continuous (Artificial light, type universal white (OSRAM L 25), permanent illumination. To minimize the potential effect of slight variations in illumination.)
- Light intensity and quality: 9020 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, d3 and d5).

TEST INITIATION AND MAINTAINANCE
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.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of frond number: The frond number in the test vessels were counted at the start of the test, after day 3, day 5 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.
- Determination of biomass: 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 at least 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). Results see 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.:
0.69 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
frond number
Remarks on result:
other: growth rate
Duration:
7 d
Dose descriptor:
EL50
Effect conc.:
14 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
frond number
Remarks on result:
other: growth rate
Duration:
7 d
Dose descriptor:
EL10
Effect conc.:
0.577 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
other: dry weight
Remarks on result:
other: growth rate
Duration:
7 d
Dose descriptor:
EL50
Effect conc.:
6.74 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
other: dry weight
Remarks on result:
other: growth rate
Duration:
7 d
Dose descriptor:
EL50
Effect conc.:
1.63 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
frond number
Remarks on result:
other: yield
Duration:
7 d
Dose descriptor:
EL50
Effect conc.:
1.21 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate
Basis for effect:
other: dry weight
Remarks on result:
other: yield
Details on results:
GROWTH INHIBITION
The duckweed population in the control vessels showed exponential growth, increasing from 11 fronds per vessel to an average of 295.3 fronds per vessel in the control after 7 days (corresponding to an average specific growth rate of 0.470 d-1). The dry weight increased from 1.5 mg (sample of the inoculum culture at start of exposure) to an average of 32.5 mg per vessel in the control at test termination.

The OECD 221 notes that toxicity values calculated by using the response variables averagespecific growth rate and yield are not comparable. The effect values based on average specific growth rate (ErCx) will be generally higher than those based on yield (EyCx). However, the differences are due to the mathematical basis of the calculations and should not be interpreted as a difference in sensitivity between the two variables. The results based on yield are influenced by several factors including the Lemna species and clone, control growth rate as well as the slope of the concentration response curve. Results calculated based on average specific growth rate are independent of these variables and are the scientifically preferred basis to estimate toxicity. The preferred endpoint for this test is the lowest average specific growth rate ErC(L)50 value.

ANALYTICAL CONCENTRATION CONTROL
Concentration control analysis was not performed because a sufficiently sensitive method foranalyses in the required concentration range was not available. However, all reasonable effortswere taken to produce a saturated solution of all soluble components of the test substance in test media. Since the test substance is a multi-constituent material, the test solution is considered a water accommodated fraction (WAF). The term “loading rate” is advocated to express exposure to a WAF and is considered analogous to the nominal concentration.
According to OECD 23, for tests with substances that cannot be quantified by analytical methods at the concentrations causing effects, the effect concentration can be expressed based on loading rate (for mixtures).

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 1.5 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 ErC50 for mean growth rate based on frond number was 7.1 mg/L (experiment date: 28 Oct 2016, project number: 63E0308/02E014).
This result is within the range of 5.9 – 13.3 mg/L and indicates that the culture of Lemna gibba 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 3

Day 5

Day 7

Control

1 - 6

0

0

0

0.32

7 - 10

0

0

0

1

11 - 14

0

3, 4, 6

3, 4, 6

3.2

15 - 18

4

3, 4, 6

1,3, 4, 6

10

19 - 22

4

1,3, 4, 6

1,3, 4, 6

32

23 - 26

3, 4

1, 3, 5

1, 3, 5

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)

(+):          slight effects

(++):       medium effects

(+++):     strong effects

Dissolution behavior:

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

Test Group
 [mg/L]

day 0

day 3

day 5

day 7

0 (control)

0

0

0

0

0.32

0

0

0

0

1

1

1

1

1

3.2

1

1

1

1

10

1

1

1

1

32

1

1

1

1

Explanation of abbreviations:

0:          no remarkable observations, clear test medium

1:          coloration caused by the test chemical (yellowish)

Applicant's summary and conclusion

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