Xanthylium, 3,6-bis(diethylamino)-9-[2-(ethoxycarbonyl)phenyl]-, molybdatetungstatephosphate

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 February 2018 - 03 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 0245N/041020
- Expiration date of the lot/batch: 16 March 2005
- Purity: 100%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Stock solutions were prepared in acetone at a concentration of a factor of 10,000 higher than the final test solutions.
- Preliminary purification step (if any):
- Final dilution of a dissolved solid, stock liquid or gel: The appearance of the test solutions ranged from colourless to pink, with increasing concentration.

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Range-finding test - 10 and 100 mg/L
2nd range-finding test - 0.010, 0.10 and 1.0 mg/L
Definitive test - 0.10, 0.32, 1.0, 3.2 and 10 mg/L
- Sampling method: The concentration and stability of the test item in the test preparations were verified by chemical analysis on Day 0 (fresh media), Days 3 and 5 (fresh and old media), and Day 7 (old media).
At the start of the test six replicate samples of fronds identical to those used to inoculate the test vessels were taken and the dry weight determined. At the end of the test the dry weight of colonies from each control and test vessel was determined by drying at 60 °C.
- Sample storage conditions before analysis: frozen

Vehicle:

yes

Remarks:

Stock solutions were prepared in acetone

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stirring and ultasonic waves treatment
- Controls: The solvent control solution contained the same amount of solvent as the treated solutions (0.1 ml/I).
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The test solution prepared at 100 mg/I still contained test substance particles.

Test organisms (species):

other: Lemna Gibba

Details on test organisms:

TEST ORGANISM
- Common name: Lemna gibba
- Strain: not specified
- Source (laboratory, culture collection): Bayer CropScience AG, 40789 Monheim
- Age of inoculum (at test initiation): 7 days
- method of cultivation: not specified

ACCLIMATION
- Acclimation period: 7 days
- Culturing media and conditions: same as test
- Any deformed or abnormal cells observed: none observed

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

7 d

Remarks on exposure duration:

A semi-static testing regime was employed with media renewal on Days 3 and 5.

Post exposure observation period:

not specified

Hardness:

not specified

Test temperature:

between 22 and 23 °C

pH:

Deifnitive test:
Day 1 pH 5.4 - 5.6
Day 3 pH 5.4 - 5.9 (also media change)
Day 5 pH 5.5 - 6.1 (also media change)
Day 7 pH 6.0 - 6.3

Dissolved oxygen:

not specified

Salinity:

not specified

Conductivity:

not specified

Nominal and measured concentrations:

Nominal concentrations tested were 0.1, 1, 10 and 100 mg/I.

Details on test conditions:

TEST SYSTEM
500 mL glass conical flasks were used. Three flasks each containing 250 mL of solution were prepared for the control and each treatment group.
The control group was maintained under identical conditions but not exposed to the test item.
Each control and test flask was inoculated with 3 colonies of Lemna gibba (total 12 fronds). The flasks were then incubated at 24 ± 1 ºC under constant illumination (intensity approximately 7000 lux) on a black, non-reflective surface for 7 days.
On Days 3 and 5 the test solutions were renewed.

GROWTH MEDIUM
- Standard medium used: yes
The culture medium was prepared in reverse osmosis purified water (Elga Optima 15+ or Elga Purlab Option R-15 BP). The pH of the prepared culture medium was adjusted, if necessary, to 5.5 ± 0.2 with either 1M HCl or NaOH.

OTHER TEST CONDITIONS
- Sterile test conditions: not specified
- Adjustment of pH: yes

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The number of fronds present in each test and control culture was recorded on Days 0, 3, 5 and 7 along with observations on frond size, appearance, root length and number of colonies present.
In addition the dry weight of the fronds in each control and treatment group was determined on Day 7. At the start of the test six replicate samples of fronds identical to those used to inoculate the test vessels were taken and the dry weight determined. At the end of the test the dry weight of colonies from each control and test vessel was determined by drying at 60 °C.


TEST CONCENTRATIONS
- Spacing factor for test concentrations: n/a
- Justification for using less concentrations than requested by guideline: n/a
- Range finding study: 10 and 100 mg/L
- Test concentrations: 0.10, 0.32, 1.0, 3.2 and 10 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Key result

Duration:

7 d

Dose descriptor:

EL50

Remarks:

Frond number

Effect conc.:

5.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

EL50

Remarks:

Dry weight

Effect conc.:

6.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

EL50

Remarks:

Frond number

Effect conc.:

1.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Yield

Key result

Duration:

7 d

Dose descriptor:

EL50

Remarks:

Dry weight

Effect conc.:

1.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Yield

Key result

Duration:

7 d

Dose descriptor:

NOELR

Remarks:

frond number

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

NOELR

Remarks:

Dry weight

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

NOELR

Remarks:

Frond number and dry weight

Effect conc.:

0.32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Yield

Key result

Duration:

7 d

Dose descriptor:

LOELR

Remarks:

Frond number

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

LOELR

Remarks:

Dry weight

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Key result

Duration:

7 d

Dose descriptor:

LOELR

Remarks:

Frond number & dry weight

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks:

Yield

Details on results:

- Exponential growth in the control (for algal test): n/a
- Observation of abnormalities (for algal test): none
- Unusual cell shape: slight gibbosity above 0.32 mg/L
- Colour differences: some colourosis above 3..2 mg/L

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50:
8.7 mg/L Frond number for growth rate
8.9 mg/L Dry weight for growth rate
6.4 mg/L Frond number for yield
6.0 mg/L Dry weight for yield

NOEC:
1.0 mg/L Frond number for growth rate
4.0 mg/L Dry weight for growth rate
1.0 mg/L Frond number for yield
1.0 mg/L Dry weight for yield

LOEC:
2.0 mg/L Frond number for growth rate
8.0 mg/L Dry weight for growth rate
2.0 mg/L Frond number for yield
2.0 mg/L Dry weight for yield

Reported statistics and error estimates:

Frond number

Average Specific Growth Rate: There were no statistically significant differences between the control, 0.10 and 0.32 mg/L loading rate WAFs (P≥0.05), however all other loading rates were significantly different (P<0.05) and therefore the NOEL in terms of inhibition of average specific growth rates calculated from frond numbers was 0.32 mg/L loading rate WAF. Correspondingly the LOEL was 1.0 mg/L loading rate WAF.

Yield: There were no statistically significant differences between the control, 0.10 and 0.32 mg/L loading rate WAFs (P≥0.05), however all other loading rates were significantly different (P<0.05) and therefore the NOEL in terms of inhibition of average specific growth rates calculated from yield was 0.32 mg/L loading rate WAF. Correspondingly the LOEL was 1.0 mg/L loading rate WAF.

Dry weight

Average Specific Growth Rate: There were no statistically significant differences between the control, 0.10, 0.32 and 1.0 mg/L loading rate WAFs (P≥0.05), however all other loading rates were significantly different (P<0.05) and therefore the NOEL in terms of inhibition of average specific growth rate calculated from dry weight was 1.0 mg/L loading rate WAF. Correspondingly the LOEL was determined to be 3.2 mg/L loading rate WAF.

Yield: There were no statistically significant differences between the control, 0.10, 0.32 and 1.0 mg/L loading rate WAFs (P≥0.05), however all other loading rates were significantly different (P<0.05). Visual inspection of the data however showed that 32% inhibition of yield occurred at 1.0 mg/L loading rate WAF which was considered to be significantly different. Therefore the NOEL in terms of inhibition of yield calculated from dry weight was considered to be 0.32 mg/L loading rate WAF. Correspondingly the LOEL was determined to be 1.0 mg/L loading rate WAF.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Lemna gibba has been investigated over a 7-Day period and gave the following results.
- EL50:
5.1 mg/L Frond number for growth rate
6.6 mg/L Dry weight for growth rate
1.8 mg/L Frond number for yield
1.7 mg/L Dry weight for yield
NOEL:
0.32 mg/L Frond number for growth rate
1.0 mg/L Dry weight for growth rate
0.32 mg/L Frond number for yield
0.32 mg/L Dry weight for yield
LOEL:
1.0 mg/L Frond number for growth rate
3.2 mg/L Dry weight for growth rate
1.0 mg/L Frond number for yield
1.0 mg/L Dry weight for yield

Key value for chemical safety assessment

EC50 for freshwater algae:

5.1 mg/L

EC10 or NOEC for freshwater algae:

1 mg/L