Registration Dossier

Administrative data

Endpoint:
short-term toxicity to fish
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 203, EPA OPPTS 850.1075, EPA OTS 797.1400, EPA FIFRA 72-1 and EC Council 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 203 (Fish, Acute Toxicity Test)
Deviations:
no
Remarks:
An exception - expiration of the test substance was not provided upon receipt
Qualifier:
according to
Guideline:
EPA OPP 72-1 (Fish Acute Toxicity Test)
Deviations:
no
Remarks:
same as above
Qualifier:
according to
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Deviations:
no
Remarks:
same as above
Qualifier:
according to
Guideline:
EPA OTS 797.1400 (Fish Acute Toxicity Test)
Deviations:
no
Remarks:
same as above
Qualifier:
equivalent or similar to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
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 96 hours. At test initiation, samples were collected from the parent solutions and transferred to separatory funnels. At 96 hours composite samples were collected from replicates A and B in each treatment. Quality control fortifications were prepared at each time period. 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 precalibrated 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:
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 10.3096 g of 1,4,5,8-Tetra (4'-nbutylphenylarnino) 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. Fifteen liter aliquots of the resulting solutions were transferred to the exposure jars.

Test organisms

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: rainbow trout
- Strain: mykiss
- Source: Trout Lodge, Sumner, WA
- Length at study initiation (length definition, mean, range and SD): 35 to 45 mm in standard length (mean = 39 ± 27 mm)
- Weight at study initiation (mean and range, SD): 0.500 to 1.037 g in blotted wet weight (mean = 0.692 ± 0.137 g)

ACCLIMATION
- Acclimation period: 14-day period prior to use in the test.
- Acclimation conditions (same as test or not): yes
- Following hatch and swim-up, fish were fed salmon starter andlor brine shrimp nauplii daily. Three days prior to test initiation a sublot of individuals was removed and transferred to a separate tank for acclimation to the test temperature. Food was withheld and no mortality was observed.
- Health during acclimation (any mortality observed): none

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:
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 11.4 to 12.7OC in all solutions during the test. The continuous temperature recording indicated a periodic average temperature of 11.4 ± 0.1 °C ranging from 11.1 to 12.3 °C.
pH:
Test solution pH values ranged from 7.7 to 8.7 in all solutions during the test.
Dissolved oxygen:
Dissolved oxygen concentrations ranged from 5.3 to 10.1 mg/L (51 to 100% 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. After siphoning, all treatments were clear and colorless with no visible precipitates or surface films throughout the test.
Details on test conditions:
Test chambers consisted of duplicate 20-L glass jars each containing approximately 15 L of test solution. The jars were labeled with study number, treatment, and replicate. Fish were impartially assigned to treatments by adding one fish per chamber proceeding from controls, low to high test substance treatments, and repeating steps as necessary until 10 fish had been added to each test chamber. Observations for mortality and sublethal responses were made at approximately 24,48,72, and 96 hours (kl hour). The jars were positioned in a waterbath and maintained at a temperature of 12 ± 1 °C. Fluorescent lighting was maintained on a 16-hr light: 8-hr dark 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 910 lux.
Temperature, pH, and dissolved oxygen concentration were measured in each jar on a daily basis. In addition, a continuous record of the temperature in one replicate 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:
96 h
Dose descriptor:
LC50
Effect conc.:
> 70.3 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Remarks on result:
other: 95% CL not calculated
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 70.3 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Details on results:
After 96 hours, no mortality or sublethal effects were observed in any treatment tested. No adverse effects were observed up to the functional limit of solubility. The 24-, 48-, 72-, and 96-hour LC50 for rainbow trout exposed to 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) was estimated to be >70.3 µg a.i./L (95% confidence limits could not be calculated). The 24-, 48-, 72-, and 96-hour no-observed-effect concentration (NOEC) was 70.3 µg a.i./L, based on the lack of mortality and sublethal effects at this and all lower concentrations. The 96-hour slope of the concentrations-response line could not be calculated.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
Statistical analysis was not performed due to the lack of mortality at any test substance concentration.

Any other information on results incl. tables

None

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the study, no adverse effects, including sublethal effects, were observed in Rainbow Trout, Oncorhynchus mykiss, exposed to 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) up to the functional limit of solubility. The 96-hour LC50 for rainbow trout exposed to 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) was estimated to be >70.3 µg a.i./L (95% confidence limits could not be calculated). The 96-hour NOEC was 70.3 µg a.i./L, based on the lack of mortality and sublethal effects at this and all lower concentrations. The slope of the 96-hour concentration-response line could not be calculated.
Executive summary:

A test was conducted to estimate the potential acute toxicity of 1, 4, 5, 8-Tetra (4'-nbutylphenylarnino) Anthraquinone (TBPAAQ) to the rainbow trout, Oncorhynchus mykiss. Trout 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'-n-butylphenylamino) Anthraquinone (TBPAAQ) (97.0% a.i.).

Mean measured concentrations of 1,4,5,8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) were <MQL (control), 3.16,4.45,9.14, 18.6,42.4, and 70.3 μg a.i./L. Water quality characteristics of temperature, dissolved oxygen concentration, and pH were measured daily during the test, remained within acceptable limits throughout the exposure. All treatments were clear and colorless with no visible precipitates or surface films throughout the test.

After 96 hours, no mortality or sublethal effects were observed in any concentration tested. No adverse effects, including sublethal effects, were observed in Rainbow Trout, Oncorhynchus mykiss, exposed to 1, 4, 5, 8-Tetra (4'-n-butylphenylamino) Anthraquinone (TBPAAQ) up to the functional limit of solubility. The 96-hour LC50 for rainbow trout exposed to 1, 4, 5, 8-Tetra (4' -nbutylphenylamino) Anthraquinone (TBPAAQ) was estimated to be > 70.3 μg a.i./L (95% confidence limits could not be estimated). The 96 -hour no-observed-effect concentration (NOEC) was 70.3 μg a.i./L, based on the lack of mortality and sublethal effects at this and all lower concentrations. The slope of the concentration response line could not be calculated.