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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study, no deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2011
Report Date:
2011

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:
- Concentrations: 0, 4.1, 8.1, 16, 33, 65 µg/L (nominal conc.)
- Sampling method: Samples from test solutions were removed from the approximate midpoint of each aquarium using a pipette.
- Sample storage conditions before analysis: No data

Test solutions

Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A 2.6 mg/mL diluter stock solution was prepared prior to test initiation and as needed throughout the definitive exposure by placing, for example, approximately 0.1302 g of test substance in a 50-mL volumetric flask and bringing it to volume with dimethylformamide, DMF (CAS No. 68-12-2). The resultant solution was observed to be clear and colorless following preparation.
A 520 μL/mL solvent stock solution was prepared by placing 519 mL of DMF 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 50-mL Glenco® gas-tight syringe was calibrated to deliver 0.0485 mL/cycle of the diluter stock solution (2.6 mg/mL) into the diluter's chemical mixing chamber, which received 1.94 L of dilution water per cycle. The solution contained in the mixing chamber constituted the highest nominal test concentration (65 μg/L) and was subsequently proportionally diluted (50%) to provide the remaining nominal exposure concentrations (33, 16, 8.1 and 4.1 μg/L).
The concentration of DMF in the solution in the mixing chamber and the high test concentration constituted the highest DMF concentration (0.025 mL/L). A Fluid Metering Inc., pump was calibrated to deliver 0.244 mL/cycle of the solvent stock solution (520 μL/mL) to 5.065 L of dilution water per cycle which was subsequently delivered to the solvent control and treatment vessels. The DMF concentration in the solvent control and the treatment levels was 0.025 mL/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 test substance.


- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Dimethylformamide
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): 0.025 mL/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc): The stock solution was clear and colourless

Test organisms

Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnow
- Source: Fathead minnow eggs were obtained from brood stock maintained at Springborn Smithers. The brood stock originated from the U.S. Environmental Research Laboratory, Duluth, Minnesota.

METHOD FOR PREPARATION AND COLLECTION OF FERTILIZED EGGS
- Numbers of parental fish (i.e. of females used to provide required number of eggs): No information
- Method of collection of fertilised eggs: No information
- Subsequent handling of eggs: No information

POST-HATCH FEEDING
- Start date: When hatch was designated as being complete
- Type/source of feed: live brine shrimp nauplii (Artemia salina)
- Amount given: ad libitum
- Frequency of feeding: Three times per day

Study design

Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
32 d
Post exposure observation period:
No post exposure observation period

Test conditions

Hardness:
Total hardness as CaCO3: 68 to 88 mg/L
Test temperature:
25 - 27 °C
pH:
6.4 - 7.2
Dissolved oxygen:
Control: 6.8-8.4 mg/L
Solvent control: 4.9-7.9 mg/L
Test concentrations (4.1, 8.1 33, 65 µg/L): 5.2-8.1 mg/L; 16 µg/L: 2.2.-7.8 mg/L
Salinity:
No information
Nominal and measured concentrations:
Nominal test concentrations: 4.1, 8.1, 16, 33, 65 µg/L
Measured test concentrations: 3.1, 6.2, 15, 31, 59 µg/L
Details on test conditions:
TEST SYSTEM
- Emybro cups: Embryo incubation cups were round glass jars (5 cm O.D., 8 cm high) with Nitex® screen bottoms with 475-μm screen openings
- Test vessel: aquaria
- Type (delete if not applicable): open / closed
- Material, size, fill volume: glass, 30x15x20 cm, 250 ml
- Aeration: no information
- Type of flow-through: intermittent-flow proportional diluter
- Renewal rate of test solution (frequency/flow rate): 90 % replacement of test solution within ca. 7 hours
- No. of fertilized eggs/embryos per vessel: 30
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: max. 0.066 g/L of flowing test solution per day or 0.50 g/L at any time in any replicate exposure aquarium based on control organism wet weight at test termination.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: on site well water, aerated prior to use
- Total organic carbon: no information
- Particulate matter: no information
- Metals, pesticides:Representative samples of the dilution water source were analyzed periodically for the presence of pesticides, PCBs and toxic metals by GeoLabs, Inc., Braintree, Massachusetts. None of these compounds were detected at concentrations considered toxic in any of the water samples analyzed, in agreement with ASTM (2005) standard practices.
- Chlorine: no information
- Alkalinity: 18-24 mg/L as CaCO3
- Conductivity: 450 to 470 μmhos/cm
- Salinity: no information
- Culture medium different from test medium: no
- Intervals of water quality measurement: weekly

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours of light, 8 hours of darkness
- Light intensity: 64-89 footcandles (680-960 lux)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :embryo hatching success, percent of embryos that produced normal larvae (at completion of hatch),
survival, growth and weight of larvae (at test termination(28 days post hatch))

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: 0.063, 0.13, 0.25, 0.5, 1 mg/L
- Results used to determine the conditions for the definitive study: yes

POST-HATCH DETAILS
- Begin of post-hatch period: on day of hatch (test day 4)
- No. of hatched eggs (alevins)/treatment released to the test chamber: 15 per replicate (total of 60 organisms
- Release of alevins from incubation cups to test chamber on day no.: test day 4

FERTILIZATION SUCCESS STUDY
- Number of eggs used: no information
- Removal of eggs to check the embryonic development on day no.: no information
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
32 d
Dose descriptor:
NOEC
Effect conc.:
59 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: hatching success, percent normal larvae at completion of hatch, survival, total length and dry weight of fathead minnow larvae
Duration:
32 d
Dose descriptor:
LOEC
Effect conc.:
> 59 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
other: hatching success, percent normal larvae at completion of hatch, survival, total length and dry weight of fathead minnow larvae
Details on results:
- Larval survival::
Control:93 %
Solvent control: 88 %
Pooled control: 92 %
3.1 µg/L: 87 %
6.2 µg/L: 97 %
15 µg/L: 87 %
31 µg/L: 90 %
59 µg/L: 90 %
- Days to hatch or time to release of young: 4
- Hatching success:
Control: 93 %
Solvent control: 95 %
Pooled control: 94 %
3.1 µg/L: 90 %
6.2 µg/L: 93 %
15 µg/L: 85 %
31 µg/L: 87 %
59 µg/L: 93 %
- Normal larvae at hatch:
Control: 98 %
Solvent control: 99 %
Pooled control: 99 %
3.1 µg/L: 100 %
6.2 µg/L: 100 %
15 µg/L: 87 %
31 µg/L: 100 %
59 µg/L: 98 %
- Length of larvae:
Control: 27.7 mm
Solvent control: 28.2 mm
Pooled control: 28.0 mm
3.1 µg/L: 28.0 mm
6.2 µg/L: 27.7 mm
15 µg/L: 27.8 mm
31 µg/L: 27.6 mm
59 µg/L: 28.5 mm
- Dry weight of larvae:
Control: 0.0497 g
Solvent control: 0.0499 g
Pooled control: 0.0498 g
3.1 µg/L: 0.0509 g
6.2 µg/L: 0.0485 g
15 µg/L: 0.0506 g
31 µg/L: 0.0482 g
59 µg/L: 0.0532 g
Results with reference substance (positive control):
Not applicable

Applicant's summary and conclusion

Validity criteria fulfilled:
not specified
Conclusions:
Exposure of fertilized eggs of pimephales promelas to the test substance at concentrations up to 59 µg/L for 32 days did not affect the embryo hatching success, development of normal larvae, larval survival, length and dry weight of larvae.
Executive summary:

In an early life satage study according to OECD Guideline 210 and OPPTS Draft Guideline 850.1400 fertilized eggs of Fathead Minnow (Pimephales promelas) were exposed to the test substance at nominal concentrations of 4.1, 8.1, 16, 33 and 65 µg/L (measured concentrations: 4.1, 8.1, 16, 33 and 59 µg/L) under flow through conditions. Dimethylformamide was used as a vehicle in a concentration of 0.025 mL/L. After completion of hatch at test day 4 the larvae were exposed for another 28 days to the test substance. The embryo hatching success was assessed and at termination of the test the development of normal larvae, larval survival, length and dry weight of larvae were assessed. No adverse effects were observed. Therefore a NOEC of 59 µg/L and a LOEC of > 59 µg/L were derived.