9,10-Anthracenedione, 1,4,5,8-tetrakis[(4-butylphenyl)amino]-

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to OECD TG 201, EPA OPPTS 850.5400, EPA OTS 797.1050, EPA OPP 122-2 and 123-2 and EC Council Directive 67/548/EEC and in accordance with the Principles of Good laboratory Practice (GLP).

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 122-2 (Algal Toxicity, Tiers I and II)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPP 123-3 (Algal Toxicity, Tiers I and II)

Test guideline 4

Qualifier:

according to guideline

Guideline:

EPA OPPTS 850.5400 (Algal Toxicity, Tiers I and II) (January 2012)

Test guideline 5

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

445-710-5

EC Name:

-

Cas Number:

108313-21-9

Molecular formula:

C54H60N4O2

IUPAC Name:

1,4,5,8-tetrakis[(4-butylphenyl)amino]-9,10-dihydroanthracene-9,10-dione

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ)
- Substance type: dark green powder
- Physical state: solid
- Analytical purity: ~ 97% active ingredient
- Lot/batch No.: Lot #020801; TD No. 02-045
- Stability under test conditions: expected to be stable for the duration of testing
- Storage condition of test material: room temperature

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

Based on these results and the limited solubility of 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ), a nominal concentration range of 0 (control), 3.1,6.3,13,25,50, and 100% water soluble fraction was selected for the definitive test. One hundred milliliter samples were collected from each treatment at test initiation and again at 72 and 96 hours. At test initiation, samples were collected from the parent solutions. At 72 hours samples were collected from replicate D and at 96 hours from a composite of replicates A, B, and C in each treatment. Quality control fortifications were prepared at each time period for the test. C 18 solid phase extraction cartridges were conditioned by rinsing with approximately two column volumes of methanol, followed by two column volumes of tetrahydrofuran (THF). The columns were then allowed to dry under vacuum, followed by the addition of one column volume of reagent water. At 72 and 96 hours, the samples were filtered through a 0.45 pm disc prior to extraction. Samples were applied to the cartridges, and percolated through at a rate of approximately 2-3 drops per second. Once the entire sample had passed through the cartridge, the cartridge was allowed to go dry. The residues were eluted from the cartridges using two volumes of approximately 5 mL THF, which was collected in a pre-calibrated culture tube. Samples were concentrated under a gentle stream of nitrogen gas, using no heat. Samples were then reconstituted in the appropriate volume with THF, followed by an equivalent volume of acetonitrile. The sampled were then mixed and analyzed by HPLC.

Test solutions

Vehicle:

no

Details on test solutions:

For the definitive test, a water soluble fraction of 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) was prepared at a loading rate of 100 mg a.i./L by adding 0.4131 g of 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) to 4000 mL of freshwater algal nutrient medium (FWAM). This solution was then stirred overnight with a teflon-coated stir bar for approximately 22.5 hours. Once stirring was terminated, the solution was allowed to settle for approximately 1.5 hours prior to draining the aqueous phase from the bottom of the carboy. Test substance treatments were prepared by diluting appropriate aliquots of the aqueous phase of the water soluble fraction to a volume of 1.0 L with algal media. One hundred milliliter aliquots of the resulting solutions were transferred to the exposure flasks.

Test organisms

Test organisms (species):

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

Details on test organisms:

TEST ORGANISM
- Common name: green alga
- Strain: capricornutum
- Source (laboratory, culture collection): Department of Botany, Culture Collection of Algae, University of Texas at Austin
- Age of inoculum (at test initiation): at least 3 days old
- Method of cultivation: The prepared cultures were maintained in a temperature-controlled environmental chamber under continuous light. Periodically, new Selenastrum cultures were cloned from an existing culture derived from the parent stock. All cultures were maintained under the same conditions as those use for testing.
- The test medium was freshwater algal nutrient medium (AAP) prepared in reagent water. After preparation, the medium was pH-adjusted to 7.5 + 0.1 using 0.1 N NaOH and filtered through a 0.45 µm Millipore filter

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

not applicable

Test conditions

Hardness:

not applicable

Test temperature:

Test solution temperature, measured at 0, 72 and 96 hours, ranged from 22.3 to 24.9 °C

pH:

Test solution pH ranged from 7.5 to 8.5 during the 96 hours.

Dissolved oxygen:

not applicable

Salinity:

not applicable

Nominal and measured concentrations:

Analytical confirmation of 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) exposure concentrations was performed at 0, 72, and 96 hours. Measured concentrations of 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) concentrations at 0-hour were All test solutions appeared clear throughout the test with no visible precipitates, surface films, or undissolved test substance.

Details on test conditions:

Erlenmeyer flasks were labeled with study number, treatment, replicate, and grid position. The flasks were randomly positioned using a computer generated random number table and incubated for 96 hours at a temperature of 23.2 to 24.0°C. Continuous lighting was provided at an average light intensity of 4495 ± 160 lux. The flasks were swirled on an orbital shaker table at approximately 100 rpm throughout the test. Each flask was inoculated with 1.0 mL of an algal concentrate containing approximately 1.0 x 10(6) cells/mL, resulting in a final density of approximately 1.0 x 10(4) cells/mL for each flask. At 24, 48, 72, and 96 (± 1) hours, cell density was measured in replicates A, B, and C in each treatment by direct microscopic counting with a hemacytometer.
At test initiation, temperature and pH were measured in all parent solutions prior to distribution to exposure flasks. At 72- and 96 hours, temperature and pH were measured in replicates D and A of the control and all test substance treatments, respectively. Temperature and pH were measured with a Denver Instruments pH meter. A continuous temperature recording of one uninoculated blank flask in the environmental chamber was made using an electronic datalogger with thermistor probe. Light intensity was measured daily with a LI-COR Model LI-189 light meter equipped with a LI-COR photometric sensor.

Reference substance (positive control):

not required

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 27.7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

cell number

Remarks on result:

other: tested at the functional solubility of the test substance

Effect concentrations 2

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 27.7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: same as above

Effect concentrations 3

Duration:

96 h

Dose descriptor:

NOEC

Effect conc.:

>= 27.7 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

act. ingr.

Basis for effect:

other: cell density, area under the growth curve, and growth rate

Remarks on result:

other: same as above

Details on results:

After 96 hours of exposure, mean cell density in the control was 114 x 10(4) cells/mL, or 114 times the initial inoculum. The coefficient of variation was 7% for the control. The mean cell density in the 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) treatments ranged from a low of 99 x 10(4) cells/mL at a concentration of 1.86 µg a.i./L to a high of 132 x 10(4) cells/mL at a concentration of 6.24 µg a.i./L. Percent inhibition in algal growth ranged from -13% at a concentration of 1.86 µg a.i./L to +16% at a concentration of 6.24 µg a.i./L. After 72 and 96 hours of exposure, no statistically significant reduction in cell density, growth curve and growth rate was observed at any test substance concentration.

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

Standard statistical methods were employed

Any other information on results incl. tables

None

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

No statistically significant adverse effects on the unicellular green alga, Selenastrum capricornutum were observed up to the functional solubility of the test substance. The 24-, 48-, 72- and 96-hour EC50 values, based on cell density, were estimated to be >27.7 µg a.i./L, the functional solubility of the test substance. The 24-, 48-, 72- and 96-hour EC50 values, based on area under the growth curve (EbC50)w, ere estimated to be >27.7 µg a.i./L. The 24-, 48-, 72-, and 96-hour EC50 values, based on growth rate (ErC50)w, ere estimated to be >27.7 µg a.i./L. The 24-, 48-, 72-, and 96-hour no-observed-effect concentration (NOEC) for cell density, area under the growth curve, and growth rate was 27.7 µg a.i./L.

Executive summary:

A toxicity test was conducted to evaluate the potential toxicity of 1, 4, 5, 8-Tetra (4'—n-butylphenylamino) Anthraquinone (TBPAAQ) to the unicellular green alga,Selenastrum capricornutum.Algal cells were exposed for 96 hours under static conditions to nominal concentrations of 0 (control), 3.1, 6.3, 13, 25, 50, and 100% water soluble fraction of 1,4,5,8- Tetra (4'-nbutylphenylamino) Anthraquinone (TBPAAQ).

 

Analytical confirmation of 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) exposure concentrations was conducted at 0, 72, and 96 hours. Zero-hour measured concentrations of 1,4,5,8 -Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) were <MQL (control), <MQL, 1.86, 3.60, 6.24, 15.1, and 27.7 μg a.i./L. Measurements at 72 and 96 hours indicated that the test substance was not soluble in the algal media and was filtered out when the algal cells were removed by filtration. EC50 estimates were based on 0-hour measured concentrations. All test solutions appeared clear throughout the test with no visible precipitate. Water quality characteristics of temperature and pH were within acceptable limits throughout the exposure.

 

No statistically significant adverse effects on the unicellular green alga,Selenastrumcapricornutum, were observed up to the functional solubility of the test substance. The 24-, 48-, 72- and 96-hour EC50 values, based on cell density, were estimated to be >27.7 μg a.i./L, the functional solubility of the test substance. The 24-, 48-, 72- and 96-hour EC50 values, based on area under the growth curve (EbC50), were estimated to be >27.7 μg a.i./L. The 24-, 48-, 72-, and 96-hour EC50 values, based on growth rate (ErC50), w ere estimated to be >27.7 μg a.i./L. The 24- 48-, 72-, and 96-hour no-observed-effect concentration (NOEC) for cell density, area under the growth curve, and growth rate was 27.7 μg a.i./L.