Registration Dossier

Administrative data

Endpoint:
fish short-term toxicity test on embryo and sac-fry stages
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985-04-17 to 1985-06-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
other: USEPA. 1972. Proposed Recommended Bioassay Procedure for Egg and Fry Stages of Freshwater Fish. Unpublished manuscript.
Qualifier:
equivalent or similar to
Guideline:
other: ASTM. 1983. Proposed New Standard Practice for Conducting Fish Early Life Stages Toxicity Tests. Draft No. 7.
Deviations:
no
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: 100 %
Storage conditions of test material: in dark at room temperature
Aqueous Solubility: 0.35 mg/L in well water at 25 °C.
Lot/Batch No.: 84-6-20
Physical appearance: clear, colorless liquid

Sampling and analysis

Analytical monitoring:
yes
Details on sampling:
Concentrations of DOTP in test water were determined on days 0, 1, 7, 14, 21, 28, 35, 42, 49, 56, 63, and 71 of the study. Control and DOTP fortified water samples were also determined on each sample day. Concentrations on these days were measured by extraction of the water samples folowed by GLC analysis. Water samples were taken in triplicate on each study day. Each analytical replicate consisted of a 50-mL aliquot from each test system replicate. In addition, reserve samples were collected for all levels on each sample day in pre-washed bottles and stored at room temperature.

Test solutions

Vehicle:
yes
Details on test solutions:
A 2-L Mount and Brungs proportional diluter system with a Hamilton Model 420 syringe dispenser was used for the intermittent introduction of an acetone stock solution of DOTP (13,405 mg/L) to 2 replicate test aquaria per concentration. Flow-splitting chambers divided each of the 5 toxicant concentrations, acetone solvent control, and negative control solutions into the respective replicate test aquaria. A 0.1-mL aliquot of acetone was delivered to 2 L of dilution water in the solvent control mixing chamber, which was equivalent to the maximum amount of solvent used in any of the test solutions. Aerated well water was delivered to the glass aquaria at an average rate of approximately 90 mL/min/replicate, an amount which was sufficient to replace the replicate volume about 6 times in a 24-h period.

Test organisms

Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
Common name: Rainbow trout (obtained as eggs)
Supplier: Trout Lodge, McMillan, WA
Age at study initiation: no data

ACCLIMATION
Photoperiod: 16 hours daylight photoperiod, shielded from UV light exposure

STUDY
From the latter part of the sac-fry stage and until day 29 of growth, all trout were fed live brine shrimp nauplii in combination with a standard commercial fish food 3-4 times per day ad libitum. After growth day 29 and until study termination, the juvenile trout were fed twice daily with commercial fish food ad libitum. All aquaria were siphoned daily to remove fecal material, excess food, and any biological growth on the glass or stainless steel screen.

Study design

Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
60 d
Remarks on exposure duration:
Post-hatch

Test conditions

Hardness:
225-275 mg CaCO3/L
Test temperature:
11 °C
pH:
8.0-8.3
Dissolved oxygen:
7.6-10.4
Nominal and measured concentrations:
Nominal concentrations: 0.02, 0.045, 0.09, 0.18, and 0.35 mg/L

Measured concentrations: 0.014, 0.024, 0.047, 0.15, and 0.28 mg/L
These values ranged from 52 to 83% of the nominal concentrations.
Details on test conditions:
Replicates: 2
Organisms per Replicate: 35
Exposure vessels: 7.0 cm diameter polyethylene cups suspended in treatment and control water within aquaria
Aquaria: glass 51 x 16 cm with a water depth of 25 cm
Temperature: 12 °C by immersion of aquaria in a circulating water bath
Exposure solution volume: 20 L
Photoperiod: 16 h light followed / 8 h dark; fluorescent lighting
Dilution water: Testing facility well water
Test Parameters: Percent hatch, survival, length, and weight
Aeration: No

The biological phase of the study was initiated by distributing 35 randomly-selected eyed rainbow trout eggs into incubator cups in each of the 2 replicate exposure aquaria (70 eggs/concentration). Egg mortality, as discerned by a distinct change in coloration, was recorded daily and dead eggs were removed to prevent fungal growth. When hatching commenced, the number of eggs hatched in each incubation cup was recorded daily until hatching was completed. The time to at least 95% hatch was found to be day 11 of the study. Therefore, the 60-day post-hatch growth period began on day 11, making the study 71 days in duration.

The percent hatch was estimated by the number of fry present on day 11 of the study. Survival data were collected for fry at each growth measurement day (day 5, 30, 45, and 60 post-hatch). Abnormal (sub-lethal) or physical changes and mortality were monitored by visually inspecting each growth chamber daily.

Growth, as determined by standard length of the fry, was determined by a photographic method on study days 41 and 56 (30 and 45 days after complete hatch). At test termination, study day 71 all surviving fish were sacrificed and measured for standard length, then blotted and weighed.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
11 d
Dose descriptor:
other: Maximum Acceptable Toxicant Concentration (MATC)
Effect conc.:
> 0.28 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Remarks on result:
other: All differences were considered significant at the α=0.05 (95% confidence) level.
Duration:
60 d
Dose descriptor:
other: Maximum Acceptable Toxicant Concentration (MATC)
Effect conc.:
> 0.28 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: All differences were considered significant at the α=0.05 (95% confidence) level.
Duration:
60 d
Dose descriptor:
other: Maximum Acceptable Toxicant Concentration (MATC)
Effect conc.:
> 0.28 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
length
Remarks on result:
other: All differences were considered significant at the α=0.05 (95% confidence) level.
Duration:
60 d
Dose descriptor:
other: Maximum Acceptable Toxicant Concentration (MATC)
Effect conc.:
> 0.28 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Remarks on result:
other: All differences were considered significant at the α=0.05 (95% confidence) level.
Duration:
60 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.28 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
weight
Remarks on result:
other: All differences were considered significant at the α=0.05 (95% confidence) level.
Duration:
60 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.28 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
length
Remarks on result:
other: All differences were considered significant at the α=0.05 (95% confidence) level.
Duration:
11 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.28 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
number hatched
Remarks on result:
other: All differences were considered significant at the α=0.05 (95% confidence) level.
Duration:
60 d
Dose descriptor:
NOEC
Effect conc.:
>= 0.28 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: All differences were considered significant at the α=0.05 (95% confidence) level.
Details on results:
HATCH
Hatchability of rainbow trout eggs, when continuously exposed to DOTP for 11 days, was not significantly lower than the percentage hatch of the control eggs.

SURVIVAL
The survival of rainbow trout fry continuously exposed to DOTP was not affected by exposure to any of the test concentrations. There were no abnormal (sub-lethal) effects or behavior observed throughout the study as a result of exposure to DOTP.

LENGTH
Length of trout fry after 60 days of exposure to all test concentrations was not significantly different statistically (P=0.05) when compard to control fish.

WEIGHT
Growth of trout fry, as mesured by wet weight after 60 days of exposure to DOTP, was not significantly reduced statistically (P=0.05) at any test concentration.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Comparison analyses between the water and solvent control were carried out using the measured parameters of hatchability, survival, standard length, and wet weight. Data were compared using one-way analysis of variance (ANOVA) to determine if significant differences (α= 0.05) existed between the control and solvent control. No statistically significant acetone effect was detected. ANOVA and multiple mean comparison for these parameters were made using untreated control animals.

Any other information on results incl. tables

Mean Percentage Hatch, Survival, Length, and Wet Weight of Rainbow Trout Exposed to DOTP
30 Days Post-Hatch 45 Days Post-Hatch 60 Days Post-Hatch
Mean Measured Concentration (mg/L) Mean Hatch (%) Survival (%) Standard Length (mm)   Survival (%) Standard Length (mm)   Survival (%) Standard Length (mm) Mean Wet Weight (g)
Control 100 87 31 (± 2.5) 87 35 (± 3.4) 83 42 (± 3.5) 1.2 (± 0.31)
Solvent Control 97 91 31 (± 2.5) 89 36 (± 3.0) 89 42 (± 3.7) 1.1 (± 0.32)
0.014 100 96 31 (± 2.2) 94 36 (± 2.9) 94 42 (± 4.0) 1.2 (± 0.32)
0.024 100 90 31 (± 2.3) 86 36 (± 3.2) 84 43 (± 3.6) 1.3 (± 0.31)
0.047 100 89 31 (± 3.2) 89 36 (± 3.4) 89 41 (± 4.3) 1.2 (± 0.34)
0.15 100 79 31 (± 2.4) 79 36 (± 3.4) 74 42 (± 4.1) 1.2 (± 0.34)
0.28 100 93 31 (± 2.0)   93 35 (± 3.1)   91 42 (± 3.7) 1.2 (± 0.32)
Notes:
Each value is the mean of data from 2 replicates.        
Solvent control data was not used for statistical analysis because no significant (P=0.05) acetone effect was detected by one-way ANOVA.

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Remarks:
Water quality parameters and performance of control vessels indicate suitable conditions for the performance of this test.
Conclusions:
Long-term exposure to a saturated solution of DOTP is not expected to adversely affect aquatic organisms.
Executive summary:

In a 71-d toxicity study, rainbow trout eggs and fry were exposed to DOTP at measured concentrations up to 0.28 mg/L under flow-through conditions. No differences in hatchability, survival, or growth (standard length and/or wet weight) was observed among organisms in this study. The 60-d post-hatch MATC value was >0.28 mg/L, the highest aqueous solubility of DOTP achieved under test conditions. The 60-d post-hatch NOEC value was >= 0.28 mg/L.