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Administrative data

short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22nd - 26th June 1992
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Proprietary GLP guideline study

Data source

Reference Type:
study report

Materials and methods

Test guideline
according to
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
yes (incl. certificate)

Test material

Details on test material:
Capa 305 is a colourless liquid. Batch No. 79H. Stored at room temperature. Solubility in water ~400 mg/l. Purity: mixture of polymers.
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable

Sampling and analysis

Analytical monitoring:
Details on sampling:
All samples were taken in duplicate. From each freshly prepared clear saturated solution, a sample of 50 ml was taken for determination of total organic content (TOC). These determinations were done by Centrilab, Soest, The Netherlands. A sample of 1 ml was taken from the old and new test solutions of the highest three concentrations on each day of the study for HPLC analysis.

Test solutions

Details on test solutions:
3.5 g Capa-305 was added to 3.5 l of ISO-water under stirring by an ultra-turrax. Stirring was continued for 10 minutes which resulted in a turbid emulsion. A clear solution was obtained by filtration through a nylon membrane filter (Gelman, Nylaflo, 0.45 µm) under suction. Dilutions were made by making up 1500, 750, 375, 187, 95 and 48 ml of the saturated solution to 3 l with ISO-water. A control with ISO-water was included.

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
Zebra fish (B. rerio) were obtained from a commercial supplier (Ruinemans, Montfoort, The Netherlands). The fish arrived on 10th June 1992. They were held in a plastic container of 40x60x40 cm containing approx 80 l reconstituted ISO-water, which was continuously filtered and aerated. The room temperature was 21±1°C. The fish were fed troutfeed and waterfleas.
The fish used in the final test were 3.0-3.6 cm long and the mean weight was 0.26 g per fish. During the test each aquarium contained 10 fishes. The test aquaria were placed in a climate chamber where the temperature was maintained at 22±1°C, lights on from 6.00am to 10.00pm. The fish were not fed during the test.

Study design

Test type:
Limit test:
Total exposure duration:
96 h
Post exposure observation period:
Not examined.

Test conditions

~250 mg/l
Test temperature:
Dissolved oxygen:
Not determined
Nominal and measured concentrations:
Nominal concentrations: 400, 200, 100, 50, 25, 12.5, 6.2 and 0 mg/l in ISO-water. Based on the TOC determinations the measured exposure levels were calculated as: 319, 160, 80, 40, 20, 10, 5 and 0 mg/l.
Details on test conditions:
In a range finding study with guppies (Poecilia reticulata) it was found that a saturated solution of Capa-305 was toxic to fish, while a ten times dilution was not. Therefore it was decided to carry out the final test with a saturated solution and a 2, 4, 8, 16, 32 and 64 fold dilution with ISO-water. In a study with two other Capa products it was found that the Capa products disappeared from the solution in the presence of fish in 2 days, therefore in this study the test solution was renewed each day.
One test aquarium was used per concentration and each aquarium contained 10 fish. The test solutions were aerated during the test, and renewed after 24, 48 and 72 hours. During the test the pH, dissolved oxygen and temperature were all measured in all test solutions before and after renewal at 0, 24, 48, 72 and 96 hours.
Reference substance (positive control):
potassium bichromate

Results and discussion

Effect concentrationsopen allclose all
96 h
Dose descriptor:
Effect conc.:
150 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: TOC determination of saturated solution
Basis for effect:
Remarks on result:
other: 80 - 320 mg/l
96 h
Dose descriptor:
Effect conc.:
80 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: TOC determination of saturation solution
Basis for effect:
96 h
Dose descriptor:
Effect conc.:
40 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: TOC determination of saturated solution
Basis for effect:
Details on results:
All fish exposed to 319 mg/l died within 24 hours and 70% mortality occurred after 96 hours at 160 mg/l. At lower concentrations no mortality was observed.
In the 80 mg/l group at 72 and 96 h, and in the 160 mg/l at 3 hours, 2 fish (n=10/group) showed hypoactivity. At 319 mg/l all fish (n=6) showed uncontrollable movement after 3 hours. Uncontrolled movement developed in the 160 mg/l group from 24 hours onwards. All fish (n=3) showed hypoactivity after 96 hours exposure to 319 mg/l.
Results with reference substance (positive control):
Once a year an acute toxicity test with zebra fish and potassium bichromate is conducted at the test laboratory. The most recent test was conducted in April 1992, the 96h EC50 was found to be 142 mg/l.
Reported statistics and error estimates:
The 96h LC50 was based on a probit analysis model. The NOEC was assessed as the highest concentration that did not cause a statistically significant different response from controls (Fishers exact test).

Any other information on results incl. tables

The chromatograms of the old and new test solutions showed that no dramatic changes in the relative content of the test medium took place during 24 hours (statistical evaluations were not performed). The results indicate that the fish were exposed to a constant mixture of Capa-305 components.

Applicant's summary and conclusion

Validity criteria fulfilled:
The 96h LC50 of Capa-305 to zebra fish was 150 mg/l, based on measured concentrations.
Executive summary:

The acute toxicity of Capa-305 to zebra fish (B. rerio) in accordance with OECD 203, was based on nominal test concentrations of 400, 200, 100, 50, 25, 12.5, 6.2 and 0 mg/l in ISO-water. The test solutions were renewed each day. Based on the total organic carbon content of the freshly prepared stock solution, the mean measured test concentrations were 319, 160, 80, 40, 20, 10, 5 and 0 mg/l. Based on measured concentrations, the 96h LC50 was 150mg/l. The NOEC for mortality was 80 mg/l and the NOEC for effects on behaviour was 40 mg/l.