Registration Dossier

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
short-term toxicity to aquatic invertebrates
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 202, EPA OPPTS 850.1010, EPA TSCA 797.1300, EPA FIFRA 72-2, EC Directive 67/548/EEC and in accordance with the Principles of Good Laboratory Practice (GLP).
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Remarks:
No expiration date was provided upon receipt of the test substance
Qualifier:
according to
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Deviations:
no
Remarks:
same as above
Qualifier:
according to
Guideline:
EPA OTS 797.1300 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
Deviations:
no
Remarks:
same as above
Qualifier:
according to
Guideline:
EPA OPP 72-2 (Aquatic Invertebrate Acute Toxicity Test)
Deviations:
no
Remarks:
same as above
Qualifier:
equivalent or similar to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Remarks:
same as above
Principles of method if other than guideline:
not applicable
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
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:
- Concentrations: Based on the results of the range-finding test and the limited solubility of the test substance, nominal concentrations selected for the definitive exposure were 0 (control), 3.1,6.3, 13,25,50, and 100% water soluble fraction One hundred milliliter samples were collected from each treatment at test initiation and again at 48 hours. At test initiation, samples were collected from the parent solutions and transferred to separatory funnels. At 48 hours samples were collected from a composite of replicates A and B in each treatment. Quality control fortifications were prepared at each time period for the test. C18 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. The samples were then applied to the cartridge, and percolated through the cartridge at a flow 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 sampe was eluted from the cartridge using two volumes of approximately 5 mL THF, which was collected in a pre-calibrated culture tube. Samples were then concentrated under a gentle stream of nitrogen gas, using no heat. Samples were reconstituted in the appropriate volume with THF, then an equivalent volume of acetonitrile was added. The sampled were then shaken to mix. The samples were then analyzed by HPLC.

The dilution water was a moderately hard freshwater prepared by blending naturally hard well water with well water that was demineralized by reverse osmosis (RO). The well water and RO water was blended to yield a total hardness of 130 to 160 mg/L as CaC03 and the blended water was biologically aged (stored in a tank containing aquatic organisms). Prior to use, the dilution water was passed through a sediment filter and UV sterilizer.

Test solutions

Vehicle:
not specified
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 10.3096 g of 1,4,5,8-Tetra (4'-nbutylphenylamino) Anthraquinone (TBPAAQ) to 100 L of aged-blended freshwater. This solution was stirred overnight with an overhead stirrer for approximately 23 hours. Once the overhead stirrer was turned off, the solution was allowed to settle for approximately 1.5 hours prior to draining the aqueous phase from the bottom of the barrel. Test substance treatments were prepared by diluting appropriate aliquots of the aqueous phase of the water soluble fraction to a volume of 32 L with aged-blended freshwater. Two hundred milliliter aliquots of the resulting solutions were transferred to the exposure beakers.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: water flea
- Strain: magna
- Source: in-house cultured
- Age at study initiation (mean and range, SD): first instar (< 24 hours old, approximately 13 days old)
- Feeding during test - no

- First-instar (<24 hours old) Daphnia magna neonates were obtained from an in-house culture. The adult daphnids were cultured in a temperature-controlled waterbath at approximately 20°C. The culture was maintained on a 16-hour daylight photoperiod with 30-minute dawn/dusk transitions periods. During the holding period, the adults in the culture were fed a suspension of Selenastrum capricornutum and Ankistrodesmus falcatus at least once a day, supplemented by an artificial diet prepared. The adults that produced neonates for the test were approximately 13 days old and exhibited no signs of stress (i.e., no ephippia) or physical damage. One day prior to test initiation, the adults were transferred to a fresh culture/food suspension.

Study design

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
not applicable

Test conditions

Hardness:
Alkalinity and hardness, measured at test initiation in a sample of the dilution water, were 164 and 160 mg/L as CaC03, respectively.
Test temperature:
Test solution temperature ranged from 19.4 to 19.7°C in all solutions during the test. The continuous temperature recording indicated a periodic average temperature of 19.3 ± 0.1 °C ranging from 19.0 to 19.6 °C.
pH:
Test solution pH values ranged from 8.3 to 8.7 in all solutions during the test.
Dissolved oxygen:
Dissolved oxygen concentrations ranged from 8.1 to 9.5 mg/L (93 to 109% of saturation) over the course of the test.
Salinity:
not applicable
Nominal and measured concentrations:
Day 0 measured concentrations of 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) were
The undrained water-soluble fraction solution was clear and colorless with undissolved test substance present on the surface and bottom of the barrel. All test solutions appeared clear and colorless with no visible precipitates or surface films at test initiation and a precipitate was present at 24 and 48 hours in all test substance treatments. The control was clear and colorless with no visible precipitates or surface films throughout the exposure.
Details on test conditions:
Test chambers consisted of duplicate 250-mL glass beakers each containing approximately 200 mL of test solution. The beakers were labeled with study number, treatment, and replicate. Daphnids were randomly assigned to treatments using a computer generated random number table and observations for immobility and sublethal responses were made at approximately 24 and 48 hours. The beakers were positioned in a waterbath and maintained at a temperature of 20 * 2°C. Fluorescent lighting was maintained on a 16-hour daylight photoperiod with 30-minute simulated dawn and dusk periods. Light intensity, measured with a Li-Cor Model LI- 189 light meter equipped with a photometric sensor, was 589 lux.
Temperature, pH, and dissolved oxygen concentration were measured in each beaker at test initiation and termination. In addition, a continuous record of the temperature in a surrogate beaker in the waterbath was also maintained. Temperature and pH were measured with a Denver Instruments pH meter. Dissolved oxygen concentrations were measured with a WTW Oxi 330 dissolved oxygen meter. Alkalinity, hardness, conductivity, and total organic carbon were measured in a sample of the dilution water at test initiation.
Reference substance (positive control):
not required

Results and discussion

Effect concentrationsopen allclose all
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
> 66 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mobility
Remarks on result:
other: not calculated
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
>= 66 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mobility
Details on results:
After 48 hours, no immobility (mortality) or sublethal effects were observed in the control, 2.00, 4.10,8.20,14.2,38.6, and 66.0 µg a.i./L treatments. No sublethal effects were observed in any treatment throughout the exposure. No adverse effects were observed up to the functional limit of solubility. The 48-hour ECso for Daphnia magna exposed to 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) was estimated to be > 66.0 µg a.i./L (95% confidence limits could not be calculated). The 48-hour no-observed-effect concentration (NOEC) was 66.0 µg a.i./L, based on the lack of significant immobility and sublethal effects at this and all lower concentrations.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
No statistical analysis was performed due to insufficient immobility.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, the 48-hour EC50 for Daphnia magna exposed to 1,4,5,8-Tetra (4'-nbutylphenylamino) Anthraquinone (TBPAAQ) was estimated to be > 66.0 g a.i./L (95% confidence limits could not be calculated) and ehe 48-hour NOEC was 66.0 µg a.i./L, based on the lack of significant immobility and sublethal effects at this and all lower concentrations.
Executive summary:

A test was conducted to estimate the potential acute toxicity of 1, 4, 5, 8-Tetra (4'-nbutylphenylamino) Anthraquinone (TBPAAQ) to the water flea, Daphnia magna. Daphnids were exposed for 48 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'-n-butylphenylamino) Anthraquinone (TBPAAQ) (Lot No. 020801, -97.0% a.i.).

Mean measured concentrations of 1, 4, 5, 8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) were <MQL (control), 2.00, 4.10, 8.20, 14.2,38.6 and 66.0 μg a.i./L. Water quality characteristics of dissolved oxygen concentration, temperature, and pH, measured at test initiation and termination, remained within acceptable limits throughout the exposure. All test solutions appeared clear and colorless with no visible precipitates or surface films at test initiation. A precipitate was present at 24 and 48 hours in all test substance treatments. The control was clear and colorless with no visible precipitates or surface films throughout the exposure.

After 48 hours, no immobility (mortality) or sublethal effects were observed in the control, 2.00, 4.10, 8.20, 14.2, 38.6, and 66.0 μg a.i./L treatments. No adverse effects, including sublethal effects, were observed in Daphnia magna exposed to 1, 4, 5, 8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) up to the functional limit of solubility. The 48-hour EC50 for Daphnia magna exposed to 1,4,5,8-Tetra (4'-n-butylphenylarnino) Anthraquinone (TBPAAQ) is estimated to be >66.0 μg a.i./L (95% confidence limits could not be calculated). The 48-hour no observed- effect concentration (NOEC) was 66.0 μg a.i./L, based on the lack of significant immobility and sublethal effects at this and all lower concentrations. The slope of the 48-hour concentration-response line could not be calculated.