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Ecotoxicological information

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:
long-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
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 211 (Daphnia magna Reproduction Test)
Qualifier:
according to
Guideline:
EPA OPPTS 850.1300 (Daphnid Chronic Toxicity Test)
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): dibutyl terephthalate
- Substance type: production sample
- Physical state: viscous liquid
- Analytical purity: 98.0%
- Lot/batch No.: TD-9011254
- Storage condition of test material:room temperature in the original container in a dark, ventilated cabinet

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Water Quality Measurements
The dissolved oxygen concentrations, pH and temperature were measured and recorded in each test vessel at test initiation and weekly thereafter until test termination (day 21). In addition, the pH, dissolved oxygen concentration and temperature were measured daily in one vessel of each test concentration and the controls. Measurements taken from one replicate vessel were alternated among the four replicate test vessels. The temperature was also continuously monitored in replicate B of the 19 μg a.i./L nominal treatment level throughout the study using a VWR minimum/maximum thermometer. Total hardness, alkalinity and specific conductivity were monitored at test initiation and weekly thereafter in one replicate of the highest treatment level tested and the dilution water control during the exposure.

Analytical Measurements
Prior to the start of the definitive exposure, samples from two replicate vessels were removed from the low, mid and high treatment levels and the control and analyzed for dibutyl terephthalate concentration. Results of the pretest analyses were used to judge whether sufficient quantities of dibutyl terephthalate were being delivered to the test vessels, and whether the appropriate test concentrations were being maintained in order to initiate the definitive exposure. During the in-life phase, samples from two replicate vessels were removed from each treatment level and the controls and analyzed for dibutyl terephthalate concentration on test days 0, 7, 8, 14 and 21. Samples were removed from alternating replicates (A and B, C and D) at each sampling intervals. All samples were removed from the approximate mid-point of the appropriate vessels by pipette. 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:
Selection of nominal concentrations for the chronic toxicity test with daphnids was based on the results of a 21-day preliminary flow-through exposure conducted at Springborn Smithers and consultation with the Study Sponsor. Based on the results of this test, nominal concentrations of 9.4, 19, 38, 75 and 150 μg a.i./L were chosen for the definitive exposure. A 15 mg a.i./mL primary stock solution was prepared prior to test initiation by placing 1.4972 g (1.4673 g as active ingredient) of test substance in a 100-mL volumetric flask and bringing it to volume with acetone (CAS No. 67-64-1). The resulting stock solution was observed to be clear and colorless with no visible undissolved test substance following preparation. The 15 mg a.i./mL primary stock solution was diluted to prepare the following five secondary stock solutions: 1500, 750, 380, 190, and 94 ug a.i./mL. A solvent control stock solution was prepared prior to the definitive exposure which consisted of undiluted acetone and was observed to be clear and colorless. The concentration of acetone in the solvent control vessels (0.10 mL/L) was equivalent to the concentration of carrier solvent present in the highest treatment level solution. Prior to test initiation, a Harvard Apparatus pump with six 20-mL Glenco gas-tight syringes was calibrated to deliver 0.020 mL of each diluter stock solution (1500, 750, 380, 190 and 94 μg a.i./mL) and the solvent stock solution into the diluter’s chemical mixing chambers during each cycle, which also received 0.200 L of dilution water per chamber per cycle. The contents of the mixing chambers were continuously stirred, using a magnetic stirrer and Teflon-coated stir bar, to aid in the solubilization of the test substance.

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
The Daphnia magna used in this toxicity test were obtained from laboratory cultures maintained at Springborn Smithers. The adult daphnids used to produce offspring for this test were derived from a healthy culture stock (e.g., showing no signs of stress such as mortality, presence of males and ephippia). The daphnid culture area received a regulated photoperiod of 16 hours of light and 8 hours of darkness. Light intensity of 53 to 79 footcandles (570 to 850 lux) at the surface of the culture solution was provided by fluorescent bulbs. Culture vessels were maintained in a temperaturecontrolled water bath designed to maintain the culture solution temperature at 20 ± 2 oC. In culture, daphnids were fed increasing amounts of algal suspension based on their age. The algal suspension (Ankistrodesmus falcaltus) contained 4 x 107 cells/mL. Daphnids that were 0 to 6 days old were fed 1.0 mL of algae and 0.5 mL of YCT suspension per vessel per day. Daphnids that were 7 to 10 days old were fed 1.5 mL of algae and 0.5 mL of YCT suspension per vessel per day. Daphnids that were > 10 days old were fed 2.0 mL of algae and 0.5 mL of YCT suspension per vessel per day. The test was initiated when daphnids, < 24 hours old, were impartially distributed to each of the four replicates for each nominal concentration and the controls. Daphnids were impartially added to an intermediate beaker by adding no more than two daphnids to each beaker until all beakers contained two daphnids. This procedure was repeated until each intermediate beaker contained ten daphnids. The test was initiated by transferring one intermediate beaker of ten daphnids to each test vessel. The order of transfer was impartial for all treatment levels and the controls. During the exposure, the food was introduced at a rate of 3.0 mL of algal suspension (Ankistrodesmus falcatus, 4 x 107 cells/mL), and 1.0 mL of a yeast, cereal leaves and digested flaked fish food (YCT) suspension, three times daily. This quantity of algal suspension is equivalent to approximately 0.33 mg carbon/daphnid/day.

Study design

Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d

Test conditions

Hardness:
Total hardness measured in the 150 μg a.i./L (nominal) treatment level and the control were 180 to 190 mg/L as CaCO3.
Test temperature:
The daily temperature measured in all replicate vessels of each treatment and the control solutions ranged from 19 to 21 ºC. Continuous temperature monitoring of replicate B of the 19 μg a.i./L nominal treatment level established a minimum/maximum temperature range of 19 to 23 ºC during the exposure period.
pH:
Measurements of pH ranged from 7.7 to 8.3 in all replicate vessels of each treatment and the control solutions.
Dissolved oxygen:
The dissolved oxygen concentrations in all replicate vessels of each treatment and the controls ranged from 6.7 to 9.4 mg/L and were all safely above
the minimum criteria of 3 mg/L (33% saturation at 20 ºC).
Nominal and measured concentrations:
Nominal: 9.4, 19, 38, 75 and 150 μg a.i./L
Measured (as time-weighted averages): 5.2, 13, 29, 50 and 98 μg a.i./L
Details on test conditions:
Culture and test dilution water were prepared in 1900-L batches by fortifying well water according to the formula for hard water (U.S. EPA, 1975) and filtering it through an Amberlite XAD-7 resin column to remove any potential organic contaminants. The toxicity test was conducted using an exposure system consisting of an intermittent-flow proportional diluter (Mount and Brungs, 1967) with a 50% dilution factor, a temperature controlled water bath and a set of 28 exposure vessels. The exposure system was designed to provide five concentrations of the test substance, a dilution water control, and a solvent control. Test vessels were impartially placed in a water bath designed to maintain the test solution at a temperature of 20 ± 1C. The test area was illuminated with fluorescent bulbs at an intensity of 5.8 to 10 μE·m-2·s-1. The photoperiod during the test was 16 hours light and 8 hours dark. Sudden transitions from light to dark and vice versa were avoided. The diluter system was calibrated prior to test initiation and confirmed at test termination by measuring delivery volumes of the stock solutions (i.e., test substance), the dilution water, and the solvent. The function of the diluter system (e.g., flow rates, stock solution consumption) was monitored daily and a visual check of the system's operation was performed twice each day. The system was in proper operation for two days prior to test initiation. A flow-splitting chamber was used between the diluter cells and the replicate test vessels to promote mixing of the toxicant solution and the diluent water and to equally split the test solution between the test vessels. Four separate glass capillary tubes exited each splitter cell and entered individual delivery tubes which transferred the test solution to each replicate vessel. This tubing served to restrict the flow of the test solutions and minimized potentially stressful turbulence in the exposure vessels. Test vessels were glass battery jars having a total volume capacity of 1.6 L. Exposure solutions drained from each vessel through two 2-cm holes, approximately 15 cm from the bottom of the jars, which maintained the test solution volume at 1.4 L. The drain holes were covered with a Nitex® 40-mesh screen to prevent loss of the daphnids. Four replicates were maintained for all treatments and the controls and each vessel was labeled with the test concentration and replicate. Test solutions were delivered to the exposure vessels at an approximate rate of 9 test vessel volumes per 24-hour period in order to provide a 90% solution replacement rate of approximately six hours. The number of immobilized adult daphnids and observations of abnormal behavior were recorded daily. Immobilization is defined as the inability to swim within 15 seconds of gentle agitation. Assessments of offspring released were determined on day 9 and three times per week through day 21. Offspring were removed, counted and discarded at each observation interval. In addition, the number of immobilized offspring and the time to first brood release were recorded for each replicate vessel. At test termination (day 21), the total body length of each surviving adult daphnid was measured. Daphnids were measured (to the nearest 0.05 mm) from the apex of the head to the base of the shell spine using an Olympus SZ40 dissecting scope in combination with a calibrated ocular.
Reference substance (positive control):
not required

Results and discussion

Effect concentrationsopen allclose all
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 98 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 98 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
immobilisation
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 50 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 98 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 50 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
growth
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 98 µg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
growth
Details on results:
After 21 days of exposure, survival among the dilution water control and solvent control organisms averaged 88 and 93%, respectively. The cumulative number of offspring released by each female organism of the dilution water control and solvent control organisms during the 21-day test averaged 125 and 155 offspring per female, respectively. The coefficients of variation for daphnid reproduction in the dilution water control and solvent control were 4 and 18%, respectively. During water quality measurements on day 12 of the exposure, one daphnid from the 50 μg a.i./L concentration (replicate C) was inadvertently transferred by the dissolved oxygen probe to the 98 μg a.i./L concentration (replicate C). As a result, calculation of the percent survival and offspring per female for replicate C of the 50 μg a.i./L treatment level was based on the remaining nine daphnids from day 12 to day 21. Replicate C of the 98 μg a.i./L treatment level was excluded from the statistical analysis for reproduction and survival. At termination of the test, mean percent survival of 95, 90, 95, 90 and 90% was observed among daphnids exposed to the 5.2, 13, 29, 50 and 98 μg a.i./L treatment levels, respectively. Following 21 days of exposure, the organisms exposed to the 5.2, 13, 29, 50 and 98 μg a.i./L treatment levels had released a mean cumulative offspring per female of 171, 146, 164, 153 and 84, respectively. Mean total body length at test termination among daphnids exposed to the control and solvent control averaged 4.7 and 4.8 mm per daphnid, respectively. Mean total body length among daphnids exposed to the 5.2, 13, 29, 50 and 98 μg a.i./L treatment levels averaged 4.9, 4.8, 4.8, 4.7 and 4.0 mm, respectively.
Reported statistics and error estimates:
Bonferroni’s t-Test was used to determine the NOEC for immobilization. Since no concentration tested resulted in ≥ 50% immobilization, the 21-day EC50 for immobilization was empirically estimated to be greater than the highest mean measured concentration tested. Williams’ Test was used to determine the NOEC for reproduction and growth. Since no concentration tested resulted in ≥ 50% reduction of reproductive output or growth, the 21-day EC50 for those endpoints were empirically estimated to be greater than the highest mean measured concentration tested.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Executive summary:

A full life-cycle toxicity test utilizing Daphnia magna was conducted on dibutyl terephthalate under flow-through conditions following OECD Guideline #211 and OPPTS Draft Guideline 850.1300. Based on the most sensitive indicators of toxicity (reproduction and growth measured as length), the 21-day NOEC for dibutyl terephthalate and D. magna was determined to be 50 μg a.i./L. The 21-day LOEC was determined to be 98 μg a.i./L. The MATC for this exposure was determined to be 70 μg a.i./L. Since no concentration tested resulted in ≥ 50% reduction in survival, growth or reproductive output, the EC50 was empirically estimated to be > 98 μg a.i./L, the highest mean measured concentration tested. 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.