Registration Dossier

Administrative data

Endpoint:
fish early-life stage toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
This study was conducted for required USEPA TSCA regulatory purposes and therefore a test proposal for the study was not submitted to ECHA.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study conducted under GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 210 (Fish, Early-Life Stage Toxicity Test)
Qualifier:
according to
Guideline:
EPA OPPTS 850.1400 (Fish Early-life Stage Toxicity Test)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Water Quality Measurements
Dissolved oxygen concentration, pH and temperature measurements were taken in all vessels on day 0 and daily thereafter. Total hardness, total alkalinity and specific conductance were measured weekly in the control, low and high test concentrations.

Analytical Measurements
Samples were collected prior to the start of the definitive exposure. Samples from one replicate of each treatment level and the controls were removed for the first sampling interval. For the second sampling interval, samples were removed from the alternate replicate of the low, mid and high concentrations and the dilution water control solutions. All samples were analyzed for dibutyl terephthalate. Results of the pretest analyses were used to judge whether sufficient quantities of dibutyl terephthalate were being delivered and maintained in the exposure aquaria to initiate the early life-stage exposure. During the in-life phase of the definitive study, water samples were removed from alternating replicates (A and B) of each treatment level and control on test days 0, 4, 10, 14, 21 and 32. All test samples were removed from the approximate midpoint of each aquarium using a pipette. In
addition, a sample of the diluter stock solution was analyzed at each sampling interval, with the exception of day 10. Three quality control (QC) samples were prepared at each sampling interval at nominal concentrations approximating the test concentration range and remained with the exposure
solution samples throughout the analytical process. Results of the analyses of the QC samples were used to judge the precision and the quality control maintained during the analysis of exposure solution samples.

Test solutions

Vehicle:
yes
Details on test solutions:
The dilution water used during this study was unadulterated water from a 100-meter bedrock well which was pumped into an epoxy-coated concrete reservoir where it was aerated prior to use. Selection of nominal dibutyl terephthalate concentrations for the definitive toxicity test with fathead minnow embryos and larvae was based on information obtained through preliminary testing at Springborn Smithers Laboratories. Based on these results and consultation with the Study Sponsor, nominal dibutyl terephthalate concentrations of 9.4, 19, 38, 75 and 150 μg a.i./L were selected for the definitive exposure. An 18.2 mg a.i./mL diluter stock solution was prepared prior to test initiation and as needed throughout the definitive exposure by placing, for example, approximately 0.4643 g (0.4550 g as active ingredient) of test substance in a 25-mL volumetric flask and bringing it to volume with
acetone (CAS No. 67-64-1). The resultant solution was observed to be clear and colorless following preparation. A 109 μL/mL solvent stock solution was prepared by placing 109 mL of acetone in a 1000-mL volumetric flask and bringing it to volume with deionized water. The resultant solution was
observed to be clear and colorless following preparation. Prior to test initiation, a Harvard Apparatus syringe pump in conjunction with a 20-mL Glenco gas-tight syringe was calibrated to deliver 0.016 mL/cycle of the diluter stock solution (18.2 mg a.i./mL) into the diluter's chemical mixing chamber, which received 1.94 L of dilution water per cycle. The mixing chamber was positioned over a magnetic stir plate and partially submerged within an ultrasonic water bath which aided in mixing the stock solution with the dilution water. The solution contained in the mixing chamber constituted the highest nominal test concentration (150 μg a.i./L) and was subsequently proportionally diluted (50%) to provide the remaining nominal exposure concentrations (75, 38, 19 and 9.4 μg a.i./L). An FMI Pump was calibrated to deliver 0.40 mL/cycle of the solvent stock solution (109 μL/mL) to 5.338 L of
dilution water per cycle which was subsequently delivered to the solvent control and treatment vessels. The acetone concentration in the solvent control and the treatment levels was 8.2 μL/L, which was equal to that of the high test concentration. A set of control vessels was also established which contained the same dilution water and was maintained under the same conditions as the treatment level vessels, but contained no solvent or dibutyl terephthalate.

Test organisms

Test organisms (species):
Pimephales promelas
Details on test organisms:
Fathead minnow eggs were obtained from brood stock maintained at Springborn Smithers for more than 25 years. The brood stock originated from the U.S. Environmental Research Laboratory, Duluth, Minnesota. Fathead minnow from reputable commercial suppliers have been periodically added to the Springborn Smithers gene pool. No outbreak of disease has occurred and no medications have been used to treat the brood stock in the last fifteen years. The brood stock used for this exposure was approximately 7 to 8 months old. No mortality was observed in the brood stock 48 hours prior to testing. The water flowing to the culture unit was from the same source as the dilution water used during the early life-stage exposure. The exposure of fathead minnow embryos and larvae to dibutyl terephthalate was initiated when the embryo incubation cups were distributed to each of the test aquaria. The eggs were impartially distributed to the egg incubation cups in the following manner: Fourteen labeled incubation egg cups were impartially placed in a water bath containing control water maintained at 25 ºC. A dish containing the eggs in dilution water was placed in a separate water bath, also maintained at 25 ºC. The eggs were impartially placed in the egg cups five at a time until each cup contained five eggs. This process was continued until all egg cups contained 60 eggs. To initiate the study, the incubation cups, each containing 60 eggs, were then placed in the respective exposure aquaria (one cup per replicate vessel). At study initiation, the embryos were approximately 8 hours old.

Study design

Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
32 d
Remarks on exposure duration:
28 days post hatch

Test conditions

Hardness:
Weekly characterization of the high, low and control solutions established total hardness as 52 to 80 mg/L as CaCO3.
Test temperature:
Continuous temperature monitoring in replicate A of the control demonstrated that the temperature ranged from 24 to 25 ºC throughout the exposure. Daily temperature monitoring established a range of 24 to 26 ºC.
pH:
Measurements of pH ranged from 6.6 to 7.8.
Dissolved oxygen:
During the study, daily measurements of the test solutions established a dissolved oxygen concentration range of 6.8 to 9.6 mg/L.
Nominal and measured concentrations:
NOMINAL TEST CONCENTRATIONS: 9.4, 19, 38, 75 and 150 μg a.i./L
MEAN MEASURED CONCENTRATIONS: 5.1, 11, 24, 58 and 100 μg a.i./L
Details on test conditions:
The test was conducted using an exposure system consisting of an intermittent-flow proportional diluter, a temperature-controlled water bath and a set of 14 exposure aquaria. Flow-splitting cells were employed to equally distribute the solutions to the two replicate vessels for each concentration or control group at a rate of 500 mL of test solution per vessel per cycle. Flow splitting accuracy of the diluter cells was within ±5% of the nominal value. The exposure system was designed to provide five concentrations of the test substance, a solvent control and a dilution water control to duplicate test aquaria. The diluter system and exposure aquaria were fabricated of glass and silicone sealant. Each test aquarium measured 39 x 20 x 25 cm with a 19.5-cm high side drain that maintained a constant exposure solution volume of approximately 15 L. Embryo incubation cups were round glass jars (5 cm O.D., 8 cm high) with Nitex® screen bottoms with 475-μm screen openings. A rocker arm apparatus was used to gently oscillate the incubation cups in the test solutions. The diluter delivered the control and test solutions to the exposure aquaria at a rate sufficient to provide approximately 6 to 10 aquarium volumes per 24-hour period, with a 90% replacement time of approximately 6 to 8 hours. The exposure was initiated with a lower delivery rate (i.e., approximately 6 aquarium volume replacements per 24 hours, 90% replacement time of approximately 8 hours) and the rate was increased (i.e., approximately 10 aquarium volume replacements per 24 hours, 90% replacement time of approximately 6 hours) on test day 9 in an effort to maintain exposure levels in the tanks. The aquaria were impartially positioned in a water bath containing circulating water designed to maintain the test solution temperatures at 25 ± 2 ºC. Illumination was provided by Sylvania Oktron fluorescent bulbs centrally located above the test aquaria. Sixteen hours of light at 90 to 120 footcandles (970 to 1300 lux) and 8 hours of darkness at the exposure solution surface was provided each day. Sudden
transitions from light to dark and vice versa were avoided with a simulated 30-minute dusk-dawn period. Dead and live embryos were counted daily until hatching was complete; dead embryos were removed when observed. Day of hatch was considered to be exposure day 4, when all viable embryos in the control and all treatment level incubation cups were observed to be hatched. Calculations of hatching success of organisms were based on the number of live, dead or deformed larvae per incubation cup after hatching was complete (day 4) compared to the number of embryos per cup on test day 0. During the definitive test, beginning on day 4 (day 0 post-hatch), the larvae were fed live brine shrimp nauplii (Artemia salina) three times daily. At each feeding, larvae were fed ad libitum such that all larvae were afforded equal access to food. Larvae were not fed during the 24 hours prior to study termination. The 28-day post-hatch larval exposure was initiated on day of hatch (test day 4). On test day 4, the surviving larvae present in each incubation cup were thinned to 20 organisms per replicate/40 organisms per treatment level or control and placed into each respective exposure
aquarium. During the post-hatch exposure period, dead larvae were removed when observed and behavior and appearance of the larval fish were observed and recorded daily. Larval survival was estimated daily. At 28 days post-hatch exposure (test termination), the percentage of larval survival was determined. The surviving larvae in each replicate aquarium were euthanized with MS-222 (tricaine methanesulfonate) and measured and weighed individually to determine the total length and dry weight (dried in an oven at 99 ºC for approximately 16 hours). The larvae were measured and weighed to the nearest 0.01 mm and 0.1 mg, respectively. The mean and standard deviation were calculated using individual measurements (total length and dry weight) for each replicate aquaria.
Reference substance (positive control):
not required

Results and discussion

Effect concentrationsopen allclose all
Duration:
4 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
number hatched
Duration:
4 d
Dose descriptor:
NOEC
Effect conc.:
>= 100 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
larval development
Remarks:
(normal fry)
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
>= 24 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
mortality
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
>= 58 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
length
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
>= 58 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
weight
Details on results:
At the completion of hatch (day 4), hatching success in the control and solvent control averaged 93 and 88%, respectively (pooled control = 91%). Embryo hatching success in the 5.1, 11, 24, 58 and 100 μg a.i./L treatment levels averaged 89, 91, 93, 89 and 88%, respectively. At the completion of the hatching period (day 4), the percent of normal fry in the control and solvent control both averaged 100%. The percent of normal fry in the 5.1, 11, 24, 58 and 100 μg a.i./L treatment levels all averaged 100%. Following 28-days post-hatch exposure (day 32), larval survival in the control and solvent control averaged 83 and 90%, respectively (pooled control = 86%). Larval survival in the 5.1, 11, 24, 58 and 100 μg a.i./L treatment levels averaged 83, 88, 83, 63 and 70%, respectively. At test termination, total length of larvae exposed to the control and solvent control both averaged 25.1 mm. The mean total length of larvae exposed to the 5.1, 11, 24, 58 and 100 μg a.i./L treatment levels was 26.2, 25.0, 26.1, 25.8 and 22.7 mm, respectively. Dry weight of larvae in the control and solvent control averaged 0.0338 and 0.0309 g, respectively (pooled control = 0.0323 g). Dry weight of larvae exposed to the 5.1, 11, 24, 58 and 100 μg a.i./L treatment levels averaged 0.0365, 0.0310, 0.0360, 0.0368 and 0.0271 g, respectively.
Reported statistics and error estimates:
A t-Test was conducted to statistically compare control to the solvent control data. If no significant difference was determined, control and solvent control data were pooled for comparison to treatment data. If a difference was determined between the control and solvent control data, the solvent control was used for comparison with the treatment data. For this study, no significant difference was detected between the control and solvent control for any endpoint. Therefore, all treatment data were compared to pooled control data to establish treatment effects. Statistical analysis of percentage hatching success, percent normal larvae at hatch and percentage larval survival was performed following arcsine square-root percentage transformation of the data. Shapiro-Wilks' Test for normality was used to compare the observed sample distribution with a normal distribution for all endpoints. For this study, all data with the exception of percent normal larvae at hatch were normally distributed when treatment data were evaluated. As a check on the assumption of homogeneity of variance, data for each endpoint were analyzed using Bartlett's Test or Levene’s Test. For this study, all data met the assumption of homogeneity of variance when treatment data were evaluated. For this study, all data with the exception of percent normal larvae at hatch met the assumptions for normal distribution and homogeneity of variance. All data with the exception of percent normal larvae at hatch were evaluated using Williams' Test, a parametric procedure, to establish treatment effects. Percent normal larvae at hatch data did not meet the assumption for normal distribution, and was evaluated using Kruskal-Wallis’ Test.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Executive summary:

An early life-stage toxicity test of dibuty terephthalate using fathead minnows (Pimephales promelas) was conducted following OECD Guideline #210 and OPPTS Draft Guideline 850.1400. Biological observations were evalauted for embryo hatching success, percent normal larvae at hatch and larval survival and growth (total body length and dry weight). No adverse effects were observed for embryo hatching sucess and percent normal larvae thoughout the exposure concentrations (NOEC 100 ug/L). Effects were observed at the highest test concentration for length and dry weight (NOEC ≥ 58 ug/L). Survival was the most sensitive indicator of toxicity, the NOEC was determined to be 24 μg a.i./L. The solubility of dibutyl terephthalate in deionized water has been determined to be 4.51 ug/L at 24 ± 2C. In this aquatic toxicity study it is believed that exposure concentrations greater than the water solubility were achieved by use of a carrier solvent, and that the results of this study demonstrate no chronic toxicity at the limit of water solubility.