Registration Dossier

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
Study period:
2002-09-30 to 2002-10-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study (OECD)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report Date:
2002

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 211 (Daphnia magna Reproduction Test)
Deviations:
yes
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
Concentration of the DOTP sample: 97.4 %
Storage conditions of test material: room temperature; dark ventilated cabinet
Aqueous Solubility: approximately 0.4 µg/L
Lot/Batch No.: 0022268

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Concentrations of DOTP in water were determined at test start and at test end (48 h). Negative controls and carrier solvent controls were also sampled at test start and end.

The pH, dissolved oxygen concentrations, and temperature were measured in each test vessel at test initiation and weekly thereafter until test termination on day 21. In addition, the dissoloved oxygen concentration, temperature, and pH were measured daily in one vessel of each test concentration and the controls. Temperature was continuously monitored in one replicate vessel of the 0.063 µg a.i./L nominal treatment level (replicate D) throughout the study. Total hardness, alkalinity and specific conductivity were monitored at test initiation and weekly thereafter in one replicate of the highest treatment level and the dilution water control.

Test solutions

Vehicle:
yes
Details on test solutions:
STOCK SOLUTION PREPARATION
A 1.0 mg a.i./mL primary stock solution was prepared as needed by placing 0.0257 g (0.0250 g as active ingredient) of test substance in a 25-mL volumetric flask and bringing it to volume with acetone. The resulting stock solution was observed to be clear and colorless. A 0.010 mg a.i./mL secondary stock solution was prepared by placing 1.0 mL of the 1.0 mg a.i./mL primary stock solution in a 100-mL volumetric flask and bringing it to volume with acetone. In addition, a 0.52 mL/mL solvent stock solution was prepared by placing 52 mL of acetone in a 100-mL volumetric flask and diluting to volume with distilled water.

TEST SOLUTIONS
Prior to test initiation, a 50-mL Glenco® gas-tight syringe on a Harvard Apparatus pump was calibrated to deliver 0.0388 mL of the diluter stock solution (0.010 mg a.i./mL) in to the diluter's chemical mixing chamber during each cycle, which also received 0.388 L of dilution water. The mixing chamber was positioned in an ultrasonic water bath and mixed continuously with a water-driven stir plate and stir bar. The solution contained in the mixing chamber constituted the highest nominal treatment level (1.0 µg a.i./L) and was subsequently diluted (50% dilution factor) to provide the remaining nominal test concentrations: 0.50, 0.25, 0.13, and 0.063 µg a.i./L. During the exposure, a 50-mL Glenco® gas-tight syringe on a Harvard Apparatus pump was calibrated to deliver 0.0388 mL of the 0.25 mL/mL solvent control stock solution to 0.200 L of dilution water each cycle. The resulting solvent control contained the maximum amount of co-solvent (acetone) present in all of the treatment levels tested (0.100 mL/L).

Test organisms

Test organisms (species):
Daphnia magna
Details on test organisms:
Common name: Water flea
Supplier: The test organisms were cultured within the testing facility
Age at study initiation: less than 24-h old
Food: Test organisms were fed a diet consisting of a suspension of green algae (Ankistrodesmus falcatus; 4 x 10E7 cells/mL), introduced at a rate of 3.0 mL algal suspension and 1.0 mL YCT suspension (yeast, cereal leaves, and digested flaked fish food) per test vessel three times daily.
Culture water: well water fortified based on the formula for hard water (U.S. EPA) and filtered through an Amberlite XAD-7 resin column to remove any potential organic contaminants.
Photoperiod: 16 h of light/8 h of darkness.
Illumination: 80 to 100 footcandles, fluorescent bulbs
Temperature: 20 ± 2 °C

Study design

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

Test conditions

Hardness:
170 mg CaCO3/L
Test temperature:
19 to 21 °C
pH:
7.8 to 8.1
Dissolved oxygen:
8.7 to 9.0 mg/L
Nominal and measured concentrations:
Nominal concentrations: 0.063, 0.13, 0.25, 0.50, and 1.0 µg a.i./L
Measured concentrations: 0.039, 0.084, 0.17, 0.35, and 0.76 µg a.i./L

Mean measured concetnrations ranged from 61 to 76% of the nominal levels and maintained the expected concentration gradient. Diluter stock solutions were also measured on day 7 and 21 of the exposure period. Stock concentation were 105 and 114%, respectively of the nominal fortified levels. Analysis of 10 QC samples resulted in measured concentrations which were consistent with the predetermined recovery and ranged from 73.2 to 105% of the nominal fortified levels.
Details on test conditions:
Replicates: 4
Organisms per Replicate: 10
Exposure Vessels: 1.6-L glass battery jars
Exposure Solution Volume: 1.4 L
Photoperiod: 16 hours light / 8 hours dark
Test Parameters: survival, reproduction, and growth
Dilution water: well water fortified based on the formula for hard water (U.S. EPA) and filtered through an Amberlite XAD-7 resin column to remove any potential organic contaminants.

At test initiation, Daphnia magna were impartially selected and distributed to 28 unlabeled 100-mL beakers (intermediate vessels) containing 80 mL of dilution water and several drops of algal food solution. The daphnids were impartially added 2 at a time to each intermediate vessel until each vessel contained 2 organisms. This process was repeated until each intermediate vessel contained 10 organisms. The daphnids were then introduced into the replicate exposure vessels by impartially selecting one of the intermediate vessels containing ten organisms and gently pipetting them one at a time under the surface of the test solution. This process was repeated until 40 Daphnia (10 organisms per replicate vessel) were present at each treatment level and controls.

The number of immobilized adult daphnids and observations of abnormal behavior was recorded daily. Assessments of offspring released were determined on day 7 and three times per week through day 21. Reproduction was determined by first counting and observing adults as they were carefully pipetted from the exposure vessel to a 100-mL beaker containing approximately 50 mL of the respective test solution. After removing the adult daphnids, the remaining exposure solutions in the vessels were then filtered through a fine mesh net into a holding vessel to remove offspring. Offspring were removed from the net and the 100-mL beakers containing the offspring were set aside. The exposure vessels were cleaned and carefully rinsed with water. The original test solutions were returned to the exposure vessels and the adult daphnids were returned by lowering the beaker containing the daphnids into the exposure vessls and then tipping the beaker, allowing the water and daphnia to slowly flow into the test vessel. Following this procedure, the number of offspring for each replicate was counted.

At test termination (day 21), the total body length and dry weight of each surviving adult daphnid was measured. Daphnids were measured from the apex of the head to the base of the shell spine using an Olympus SZ40 dissecting scope. Following the length measurement, daphnids were dried at 100 °C for 24 h, in a Precision oven, prior to being weighed individually (to the nearest 0.01 mg).
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.76 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: survival, reproduction, and growth
Remarks on result:
other: All differences were considered significant at the α=0.05 (95% confidence) level.
Duration:
21 d
Dose descriptor:
LOEC
Effect conc.:
> 0.76 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: survival, reproduction, and growth
Remarks on result:
other: All differences were considered significant at the α=0.05 (95% confidence) level.
Duration:
21 d
Dose descriptor:
EC50
Effect conc.:
> 0.76 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: survival, reproduction, and growth
Remarks on result:
other: All differences were considered significant at the α=0.05 (95% confidence) level.
Details on results:
At termination of the test, survival of daphnids exposed to the treatment levels ranged from 95% to 100% and was not statistically different compared to survival in the pooled controls (98%). Following 21 days of exposure, the cumulative offspring of organisms exposed to the treatment levels was not statistically different compared to the pooled controls. First brood release by daphnids exposed to the treatment levels occurred on test day 8 and was consistent with the performance of the pooled controls. Throughout the 21-d exposure, offspring were observed to be trapped on the surface of the test solution at the hightest treatment level. This observation is indicative of the presence of undissolved test substance and suggests that the functional limit of solubility of the test substance had been exceeded. Mean total body length of daphnids exposed to the treatment levels was not statistically different from the pooled control data. Mean dry weight of daphnids exposed to the 0.17 µg a.i./L treatment level was statistically different from the pooled control value, however it was not considered to be biologically relevent due to the lack of similar response at higher treatment levels.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
At termination, data on survival, reproduction (cumulative offspring produced) and growth (dry weight and total body length) were statistically analyzed to identify treatment-related effects. Analyses were performed using the mean replicate organism response in each treatment group. All statistical analyses were conducted at the 95% level of certainty except in the case of the Shapiro-Wilks Test and the Bartlett's Test, in which the 99% level of certainty was applied.
1. Significant differences in % survival were determined after transformation of the data.
2. Student's t-Test was used to evaluate the endpoints and compare the performance of the dilution water control with that of solvent control organisms. Analyses demonstrated no significant difference between the two control groups for all measured endpoints. Therefore all subsequent statistical analyses were performed using pooled control data.
3. The Shapiro-Wilks Test for normality was used to compare the observed sample distribution with a normal distribution for all endpoints. If the data were not normally distributed, than a non-parametric procedure was used for subsequent analyses.
4. As a check on the assumption of homogeneity of variance, data were analyzed using Bartlett's Test.
5. Survival data were analyzed prior to analysis of the reproduction and growth data; treatment levels at which there were significant adverse effects on survival were excluded from statistical analysis of reproduction.
6. For each endpoint, the performance of organisms exposed to each treatment level was compared with the performace of the pooled control using Williams' Test. Comparisons for survival were performed using Bonferroni t-test. Wilcoxon's Rank Sum Test was used to establish treatment effects on dry weight.

Any other information on results incl. tables

Mean Cumulative Number of Offspring Produced per Daphnid
Mean Measured Concentration (µg a.i./L)
Day Control Solvent Control Pooled Control 0.039 0.084 0.17 0.35 0.76
7 0 0 0 0 0 0 0 0
8 6 4 5 5 6 5 4 4
11 27 23 25 25 29 28 28 26
14 65 56 61 58 66 63 64 61
16 76 66 71 75 77 72 86 76
18 111 105 108 108 112 104 109 105
21 151 146 148 147 152 142 145 144
Mean Total Body Length and Dry Weight of Parental Daphnids at Test End (21-d)
Mean Measured Concentration (µg a.i./L)
  Control Solvent Control Pooled Control 0.039 0.084 0.17 0.35 0.76
Length (mm) 4.93 4.92 4.93 4.90 4.92 4.90 4.95 4.99
Weight (mg) 1.26 1.29 1.27 1.22 1.26 1.15* 1.25 1.23
                 
*Statistically different from pooled control data     
           

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Survival and reproduction in the controls met the validity criteria of >= to 80% survival and > 60 offspring per female.
Conclusions:
Long-term exposure to a saturated solution of DOTP is not expected to adversely affect aquatic organisms.
Executive summary:

In a 21-d toxicity study, Daphnia magna were exposed to DOTP at measured concentrations up to 0.76 µg a.i./L under flow-through conditions. No differences in survival, growth (length and/or dry weight), or reproduction (cumulative young produced) was observed among organisms in this study. The 21-d EC50 value was determined to be >0.76 µg a.i./L, the highest aqueous solubility of DOTP achieved under test conditions. The 21-d LOEC value was determined to be >0.76 µg a.i./L. The 21-d NOEC value was 0.76 µg a.i./L for this study.