2,3,4,4'-tetrakis(6-diazo-5,6-dihydro-5-oxo-1-naphthylsulfonato)benzophenone

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:

2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Version / remarks:

2011

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Remarks:

With regard to the very low water solubility of the test item, no analytical verification of test item concentrations was performed. No analytical method with a sufficiently high sensitivity could be developed.

Details on test solutions:

A saturated solution with a nominal loading rate of 100 mg test item/L was prepared once 24 ± 1 hour prior to the start of the exposure.
The undiluted saturated solution and a further of four dilution levels were tested in a geometric series with a dilution factor of 2: 6.25 – 12.5 – 25.0 – 50 – 100% of the saturated solution, corresponding to the nominal test item loading rates of 6.25 – 12.5 – 25.0 – 50.0 – 100 mg/L. The dilution levels are based on the results of a preliminary range finding test (non-GLP).
Control: Six replicates (without test item) were exposed under the same conditions as the test loadings. The control solution was prepared without test item following the same method as specified for the saturated solution.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

Test organism Pseudokirchneriella subcapitata HINDÁK CCAP 278/4 (axenic)
Synonyms Selenastrum capricornutum; Ankistrodesmus subcapitata; Raphidocelis subcapitata; Ankistrodesmus bibraianus (Experimental Phycology and Culture Collection of Algae at the University of Goettingen 2014)
Reason for the selection: Pseudokirchneriella subcapitata is a suitable green alga species of the test organism according to the guideline.
Origin Culture: Collection of Algae and Protozoa (CCAP) SAMS Research Services Ltd, Dunstaffnage Marine Laboratory, Dunbeg, OBAN; Argyll PA37 1QA; Scotland, UK
Cultivation at test facility: Fresh stocks are prepared every month on Z-Agar. Light intensity amounts 2567 – 5130 lux for 24 hours per day.
Culture medium: Nutrient medium Z according to LÜTTGE et al. (1994)

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Hardness:

33 mg/L CaCO3

Test temperature:

21 - 24 °C, controlled at ± 2 °C

pH:

7.99-9.56

Conductivity:

139 μS/cm

Nominal and measured concentrations:

nominal test item loading rates of 6.25 – 12.5 – 25.0 – 50.0 – 100 mg/L, no measured concentrations

Details on test conditions:

TEST SYSTEM
- Test vessel: Sterile Erlenmeyer flasks, volume: 250 mL, sealed with cotton wool plugs, fill volume 100 mL
- Initial cells density: Nominal: approximately 5 x 10^3 – 10^4 cells/mL, Actual: 5375 cells/mL
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 6

GROWTH MEDIUM
- Standard medium used: yes

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Photoperiod: 24 hours/day light
- Light intensity and quality: Approximately 4440 to 8880 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Chlorophyll measurement: The cell density was measured daily via Chlorophyll afluorescence, excitation at 436 nm, emission at 685 nm.
- Other: The algae cells were evaluated microscopically at the start and the end of the incubation period

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 0.46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: Effect concentration refers to water solubilty.

Key result

Duration:

72 h

Dose descriptor:

EC10

Effect conc.:

> 0.46 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Remarks on result:

other: Effect concentration refers to water solubilty.

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

53.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

12.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

growth rate

Microscopic evaluation of the cells at the start and the end of exposure revealed no morphological abnormalities. The environmental conditions (pH-value, room temperature, light intensity) were determined to be within the acceptable limits. The test media were clear throughout exposure period with exception of the highest test concentration which was slightly brown colored (possible turbidity related to algae growth not taken into account). The reference item toxicity is in the valid range which was established by calculation of the average of the historic reference data since 2006, and the limits were set using the threefold standard deviation of these values.

 

Validity criteria fulfilled:

yes

Conclusions:

No adverse effects on algae were observed up to the limit of water solubility.

Executive summary:

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3. The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 5375 cells/mL. A saturated solution with a nominal loading rate of 100 mg test item/L was prepared once 24 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out. The test item was transferred into a glass flask with an appropriate amount of dilution water The saturated solution was stirred for 24 ± 1 hours (1100 rpm, room temperature) with a magnetic stirrer. After completion of stirring, undissolved particles were removed by membrane filtration (membrane filter 0.45 μm, RC, MACHEREY-NAGEL) to achieve the saturated solution. The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the saturated solution, was used in the test. During filtration, the filter was always kept covered. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was positive. Five concentrations were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution, corresponding to the nominal test item loading rates of 6.25 – 12.5 – 25.0 – 50.0 – 100 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The test media were visually clear and the highest test item loading rate was slightly brownish colored throughout the test period. With regard to the very low water solubility of the test item, no analytical verification of test item concentrations was performed. No analytical method with a sufficiently high sensitivity could be developed. The test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on the nominal test item loadings): The EL50-values for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were 53.7 (49.0 – 70.4) mg/L and 35.1 (30.8 – 39.9) mg/L, respectively. The NOELvalues for inhibition of growth rate and yield after 72 hours were 12.5 mg/L. It can thus be conclude that the EC50 and EC10 values for growth rate are higher than the water solubility of 0.46 mg/L.

Description of key information

No adverse effects on algae were observed up to the limit of water solubility.

Additional information

The toxicity of the test item to the unicellular freshwater green alga Pseudokirchneriella subcapitata was determined according to the principles of OECD 201 and Council Regulation (EC) No. 266/2016/Method C.3 from 2020-08-24 to 2020-08-28, with the definitive exposure phase from 2020-08-25 to 2020-08-28 at the test facility. The aim of the study was the determination of the effects on growth rate and yield over a period of 72 hours. The study was conducted under static conditions with an initial cell density of 5375 cells/mL. A saturated solution with a nominal loading rate of 100 mg test item/L wasprepared once 24 ± 1 hour prior to the start of the exposure. An appropriate amount of the test item was weighed out. The test item was transferred into a glass flask with an appropriate amount of dilution water The saturated solution was stirred for 24 ± 1 hours (1100 rpm, room temperature) with a magnetic stirrer. After completion of stirring, undissolved particles were removed by membrane filtration (membrane filter 0.45 μm, RC, MACHEREY-NAGEL) to achieve the saturated solution. The filter was saturated in order to avoid adsorption during the filtration. The first 25 mL of the filtrate were discarded. The filtration was interrupted for 15 minutes to allow adsorption and saturation of the filter material with dissolved test item. Thereafter, the filtration was continued. The next 25 mL were discarded. The following filtrate, i.e. the saturated solution, was used in the test. During filtration, the filter was always kept covered. The saturated solution was checked via laser beam (Tyndall effect) for undissolved test item. The Tyndall effect was positive. Five concentrations were tested in a geometrical series with a dilution factor of 2: 6.25 - 12.5 - 25.0 - 50.0 - 100% of the saturated solution, corresponding to the nominal test item loading rates of 6.25 – 12.5 – 25.0 – 50.0 – 100 mg/L. Three replicates were tested for each test item concentration and six replicates for the control. The environmental conditions were within the acceptable limits. The test media were visually clear and the highest test item loading rate was slightly brownish colored throughout the test period. With regard to the very low water solubility of the test item, no analytical verification of test item concentrations was performed. No analytical method with a sufficiently high sensitivity could be developed. The test item was found to inhibit the growth of the freshwater green alga Pseudokirchneriella subcapitata after 72 hours with the following effect values (based on the nominal test item loadings): The EL50-values for inhibition of growth rate (ErL50) and yield (EyL50) after 72 hours were 53.7 (49.0 – 70.4) mg/L and 35.1 (30.8 – 39.9) mg/L, respectively. The NOELvalues for inhibition of growth rate and yield after 72 hours were 12.5 mg/L. It can thus be conclude that the EC50 and EC10 values for growth rate are higher than the water solubility of 0.46 mg/L.