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Ecotoxicological information

Short-term toxicity to fish

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Administrative data

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2004-11-15 - 2005-03-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The test had been performed according to relevant guidelines and compliant to GLP. The results are plausible and well documented.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Swiss Ordinance relating to Good Laboratory Practice, adopted February 2nd, 2000 [RS 813.016.5] base on OECD-GLP 1997 (C(97) 186/Final)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
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:
no
Details on sampling:
not applicable

Test solutions

Vehicle:
no
Details on test solutions:
A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test fish up to the highest concentration which could be dissolved in the test water at the loading rate of 100 mg/L.
At the start of the test and prior to each test medium renewal, the test medium was prepared as follows: Due to the low water solubility of the test item, a supersaturated dispersion of the test item with the loading rate of 100 mg/L was prepared by mixing nominal 500 mg of the test item (effective weights between 499.9 and 500.2 mg) into 5000 mL of test water as homogeneously as possible by ultrasonic treatment for 15 minutes and intense stirring. No auxiliary solvent or emulsifier was used. The dispersion was stirred for 96 hours at room temperature in the dark to dissolve a maximum concentration of the test item in the dispersion.
Then, the dispersion of the test item was flltered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 µm) just before the start of the test and prior to each test medium renewal. The undiluted filtrate of the dispersion with the maximum concentration of dissolved test item was used as test medium.
The test medium was prepared just before introduction of the fish (= start of the test) and prior to each test medium renewal.
The limit test was based on the results of a range-finding test and on pre-experiments to determine the solubility of the test item in test water (without GLP).

Test organisms

Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
The study was performed with zebra fish (Brachydanio rerio). The test fish were obtained from Zoohaus Schaub, CH-4410 Liestal, Switzerland. In accordance with the test guidelines, the fish were held in the laboratories of RCC for more than two weeks without any medication. Prior to the test start, they were acclimated for one week to the test water and temperature. During holding and acclimatization until one day before the start of the test, the fish were fed with a commercial fish diet (TETRA MIN Hauptfutter, supplied by TETRA-Werke, D-49304 Melle, Germany). During holding and acclimatization, no fish died in the test fish batch and all fish were healthy.
From the acclimated test fish batch, 10 fish were measured at the start of the test: The mean body Iength of the fish was 2.7 ± 0.1 cm (mean ± standard deviation), the mean body wet weight was 0.19 ± 0.03 g.

Study design

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
not applicable

Test conditions

Hardness:
Total hardness of test water: 196 mg/L as CaCO3
Test temperature:
control: 21-22 °C
treatment: 21-22 °C
pH:
control: 8.2 - 8.7
treatment: 8.2 - 8.6
Dissolved oxygen:
[mg/L]
control: 8.2 - 8.6
treatment: 8.2 - 8.5
Salinity:
not applicable
Nominal and measured concentrations:
Nominal concentration 100 mg/L. A limit test had been performed due to the poor water solubility of the test item.
Details on test conditions:
One glass aquarium with 5 liters test medium was used for each treatment (the single test concentration and the control). The test vessels were labeled with the RCC study number and all necessary additional information to ensure unmistakable identification.
At the start of the test, 7 fish were introduced into each aquarium in random order. The loading rate was 0.26 g fish wet weight per liter of test medium. Thus, the loading rate of the test fish was much lower than the maximum loading rate of 1.0 g fish wet weight per liter test medium indicated by the test guidelines. The test medium and the control were slightly aerated during the test period. The fish were not fed during the test.
A semi-static test with daily test medium renewal was performed to keep the test item concentration in the test medium as constant as possible during the test period of 96 hours. During this semi-static test, the surviving fish were transferred daily into a clean test vessel with freshly prepared test medium.
The test was performed under the following conditions:
Test duration: 96 hours
Water temperature: 21-22 °C during the test period
Light conditions: Photoperiod of 16 hours light and 8 hours darkness (with a 30 minute transition period). Light intensity at light period approximately 80-450 Lux.
Test water: Local tap water (non chlorinated well water of drinking water quality), reduced for total hardness by ion exchange.
Total hardness of test water: 196 mg/L as CaCO3
The test water was aerated prior to the preparation of the test media until oxygen saturation was reached.
Reference substance (positive control):
no

Results and discussion

Effect concentrationsopen allclose all
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Limit test due to poor solubility of the test item
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Limit test due to poor solubility of the test item
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Limit test due to poor solubility of the test item
Details on results:
In the control and in the undiluted filtrate with the loading rate of 100 mg/L, no mortality of test fish or other visible abnormalities were determined during the test period of 96 hours.
Therefore, the 96-hour NOEC (highest concentration tested without toxic effects after the exposure period of 96 hours) and the 96-hour LC0 of the test item to zebra fish were determined to be at the loading rate of 100 mg/L. The 96-hour NOEC and the 96-hour LC0 might even be higher but loading rates of the test item above 100 mg/L were not tested, according to the test guidelines. The 96-hour LOEC (lowest concentration with toxic effects), the 96-hour LC50 and the 96-hour LC100 were clearly higher than the loading rate of 100 mg/L.
In conclusion, the test item had no acute toxic effects on zebra fish up to its solubility limit in test water at the loading rate of 100 mg/L under the present conditions of the test.
No remarkable observations were made conceming the appearance of the test medium. The test medium was clear and non-coloured throughout the test medium renewal periods.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
not applicable

Applicant's summary and conclusion

Validity criteria fulfilled:
yes
Conclusions:
An acute toxicity test on Zebra fish (Brachydanio rerio) had been performed (reliability category 1, compliant to GLP) as a limit test with daily renewal of the test medium (semistatic). The test item is poorly soluble in water. Therfore no analytical determination of the actual test concentration at a nominal concentration of 100 mg/L could be performed.
At these test conditions, the test item proved to be non-toxic to Zebra fish (no deaths or any abnormalities in treatment as well as control group):
NOEC (96h) = 100 mg/L nominal concentration,
LC50 (96h) > 100 mg/L nominal concentration.
Executive summary:

The acute toxicity of the test item to zebra fish (Brachydanio ratio) was determined in a 96-hour semi-static test with daily test medium renewal according to the EU Commission Directive 92/69/EEC, Part C.1 (1992) and the OECD Guideline for Testing of Chemicals No. 203 (1992).

A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test fish up to the solubility limit of the test item in test water at the loading rate of 100 mg/L. Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered. The undiluted filtrate with the maximum concentration of dissolved test item was used as the test medium. Additionally, a control was tested in parallel.

No analytical determination of the test item concentration in the test medium was performed since the analysis of the test item in the filtrates - after comprehensive trials - proved impossible due to the very low solubility of the test item in test water and the matrix of the test water. The biological results are based on the loading rate of the test item of 100 mg/L.

In the control and in the undiluted filtrate with the loading rate of 100 mg/L, no mortality of test fish or other visible abnormalities were determined during the test period of 96 hours.

The 96-hour NOEC (highest concentration tested without toxic effects after the exposure perioct of 96 hours) and the 96-hour LC0 of the test item to zebra fish were determined to be at the loading rate of 100 mg/L. The 96-hour NOEC and the 96-hour LC0 might even be higher but loading rates of the test item above 100 mg/L were not tested, according to the test guidelines. The 96-hour LOEC (lowest concentration with toxic effects), the 96-hour LC50 and the 96-hour LC100 were clearly higher than the loading rate of 100 mg/L.

In conclusion, the test item had no acute toxic effects on zebra fish up to its solubility limit in test water at the loading rate of 100 mg/L under the present conditions of the test.