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Short-term toxicity to fish

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short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
96 hours
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
equivalent or similar to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
EC Guideline L383A-C.1
GLP compliance:

Test material

Constituent 1
Chemical structure
Reference substance name:
1-isopropyl-2,2-dimethyltrimethylene diisobutyrate
EC Number:
EC Name:
1-isopropyl-2,2-dimethyltrimethylene diisobutyrate
Cas Number:
Molecular formula:
Details on test material:
- Name of test material (as cited in study report): TXIB Plasticizer
- Physical state: liquid
- Analytical purity: 99.0%
- Lot/batch No.: EOC 22998 #10
- Expiration date of the lot/batch: 10/99

Sampling and analysis

Analytical monitoring:
Details on sampling:
- Concentrations: 0.65, 1.1, 1.8, 3.0, 5.0 mg active ingredient/L
- Sampling method: Duplicate measures of each test concentration at 0 and 96 hours

Test solutions

Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: 0.0375 mL/minute of 50 mg a.i./mL stock solution introduced into mixing system with 0.0375 L/minute dilution water to achieve
highest nominal test concentration of 5.0 mg a.i./L. Highest concentration proportionally diluted (60% ) to produce remaining exposure
concentrations (3.0, 1.8, 1.1, 0.65 mg a.i./L)
- Controls: dilution water and solvent controls
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): Stock solution prepared by diluting 7.583 g
(7.500 g as active ingredient) of test substance with acetone vehicle to a volume of 150 mL to achieve 50 mg a.i./L solution. 0.4 mL/mL solvent
control stock solution prepared by diluting 120 mL of acetone with distilled water to a volume of 300 mL. Acetone concentration in solvent control
and highest test concentration was 0.10 mL/L. Remaining exposure concentrations proportionally diluted (60%).
- Evidence of undissolved material (e.g. precipitate, surface film, etc): No visible sign of undissolved test substance in stock solution or highest
exposure concentration

Test organisms

Test organisms (species):
Lepomis macrochirus
Details on test organisms:
- Common name: Bluegill sunfish
- Source: Bybrook Hatchery, Ashford, Connecticut
- Length at study initiation (length definition, mean, range and SD): Representative sample (n=30) mean length 42 mm, range 35-50 mm
- Weight at study initiation (mean and range, SD): Representative sample (n=30) mean weight 1.1 g, range 0.51-1.8 g
- Feeding during test: No food 24 hours prior to, or during test

- Acclimation period: 14 days
- Acclimation conditions (same as test or not): same
- Type and amount of food: commercial pelleted food, ad libitum
- Feeding frequency: daily
- Health during acclimation (any mortality observed): no mortality observed during 48 hour period prior to test

Study design

Test type:
Water media type:
Limit test:
Total exposure duration:
96 h

Test conditions

Measured at test initiation. Units mg/L CaCO3
Control = 32
Solvent Control = 36
0.65 = 36
1.1 = 38
1.8 = 38
3.0 = 40
5.0 = 40
Test temperature:
Temperature was measure at 0, 24, 48, 72, and 96 hours in all test concentrations using a Brooklyn alcohol thermometer. The temperature was
reported to be 22C at all measurements. A continuous temperature monitor of the solvent control solution indicated a range of 21 to 23C during
the exposure period.
pH was measured at 0, 24, 48, 72, and 96 hours in all test concentrations. Values below are ranges.
Control = 7.5-7.7
Solvent Control = 7.5-7.7
0.65 = 7.4-7.6
1.1 = 7.4-7.6
1.8 = 7.4-7.6
3.0 = 7.4-7.5
5.0 = 7.4-7.5
Dissolved oxygen:
Dissolved oxygen was measured at 0, 24, 48, 72, and 96 hours in all test concentrations. Values below are ranges in mg/L (% saturation).
Control = 8.0-8.5 (92-97)
Solvent Control = 8.0-8.3 (92-95)
0.65 = 7.7-8.2 (88-94)
1.1 = 7.0-8.1 (80-93)
1.8 = 7.3-7.9 (84-91)
3.0 = 7.5-8.1 (86-93)
5.0 = 7.1-8.1 (81-93)
Nominal and measured concentrations:
See Table 1 below.
Details on test conditions:
- Test vessel: Aquaria
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 49.5 x 25.5 x 29 cm, 14 cm standpipe, 18 L volume
- Aeration: no aeration
- Type of flow-through (e.g. peristaltic or proportional diluter): proportional diluter
- Renewal rate of test solution (frequency/flow rate): 6 volume replacements/day
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: maximum of 0.10 g biomass/L of flowing test solution/day

- Source/preparation of dilution water: Well water
- Total organic carbon: 1.0 mg/L
- Metals: Analyzed periodically. Not detected in toxic concentrations per ASTM (1996) standard practice.
- Pesticides: Analyzed periodically. Not detected in toxic concentrations per ASTM (1996) standard practice.
- Alkalinity: 32-35 mg/L
- Conductivity: 125-135 umhos/cm specific conductance
- Intervals of water quality measurement: DO, temperature and pH measured once daily in each treatment level and controls

- Adjustment of pH: no adjustment
- Photoperiod: 16 hours light and 8 hours darkness/day with transistions from light to dark and vice versa
- Light intensity: Measured but value not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Biological observations (lethality) as well as sublethal effects (erratic
swimming behavior, lethargy) made and recorded at 0, 3, 6, 24, 48, 72 and 96 hours of exposure.

- Spacing factor for test concentrations: 60%
- Range finding study
- Test concentrations: 5, 10 and 20 mg/L nominal concentrations used to evaulate solubility of test substance. Organisms in 5 mg/L solution only
- Results used to determine the conditions for the definitive study: 10 and 20 mg/L solutions exhibited undissolved test substance (surface film). 5.0 mg/L nominal determined to be highest concentration achievable without exceeding solubility.

Reference substance (positive control):
not required

Results and discussion

Effect concentrations
96 h
Dose descriptor:
Effect conc.:
>= 6 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
as active ingredient
Basis for effect:
mortality (fish)
with no sublethal effects observed
Details on results:
- Behavioural abnormalities: none observed
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: not observed
- Effect concentrations exceeding solubility of substance in test medium: none
Reported statistics and error estimates:
No adverse effects observed in any of the treatments or controls. Endpoint determined empirically.

Applicant's summary and conclusion

Validity criteria fulfilled:
Well conducted GLP study demonstrating no observable adverse lethal or sub-lethal effects at 96 hours in all concentration levels utilizing a flow
through proportional diluter system. Highest concentration tested approximated the water solubility limit of the test substance (i.e., no effect at