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Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 FEB 2006 - 28 JUN 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
OECD Guideline for Testing of Chemicals No. 203: "Fish, Acute Toxicity Test", adopted July 17,1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
EEC, Annex to Commission Directive 92/69/EEC of 31 July 1992 adapting to technical progress for the seventeenth time Council Directive 67/548/EEC on the approximation of laws, regulations and administrative provisions relating to the classification, packing and labeling of dangerous substances (Official Journal No. L 383 A, December 29,1992) Part C. 1. "Acute toxicity for fishes"
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 25.0, 35.4, 50.0, 70.7, and 100.0 mg/L
- Sampling method: Directly after preparation in the laboratory the test media were given to analysis. Further samples were analyzed 96 hours after start of the experimental part. These samples were stored under the same conditions as the vessels with zebrafish and afterwards given to analysis
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test media (reconstituted water and test material) were freshly prepared. Therefore, the calibrated flask with the analytical determined test material amount (solvent amount) per nominal concentration and the vehicle, reconstituted water, was treated in an ultrasonic device for 1 hour. The solution was used for the study.
- Controls: Reconstituted water
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No remarkable observations were made concerning the appearance of the solutions of the test material preparations. The test media were clear preparations and stayed unchanged throughout the study.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish (Danio rerio),
- Strain: originated West-Aquarium
- Source: in-house breeding
- Length at study initiation (length definition, mean, range and SD): 2.0 ± 1.0 cm
- Weight at study initiation (mean and range, SD): Body weight per group: 5.7 to 9.3 g

ACCLIMATION
- Acclimation period: > 14 days
- Acclimation conditions (same as test or not): The zebrafish were acclimatized to the conditions of the laboratory for more than 14 days and were kept in reconstituted water.
- Type and amount of food during acclimation: The zebrafish were fed with a commercial flake diet, TetraMin® (Tetra GmbH, Melle). Furthermore the fish were fed with daphnia, or artemia, or frozen mosquito larva.
- Feeding frequency during acclimation: daily
- Health during acclimation (any mortality observed): During the week before testing, the mortality of the test fish batch was lower than 5 %.

FEEDING DURING TEST
The zebra fish were not fed during the study.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
about 267 mg/L CaCO3
Test temperature:
24 - 25 °C
pH:
6.97 - 8.07
Dissolved oxygen:
The dissolved oxygen concentrations were > 60 % oxygen saturation throughout the study.
Nominal and measured concentrations:
nominal: 0, 25.0, 35.4, 50.0, 70.7, and 100.0 mg/L.
mean measured: 0, 1.20, 1.54, 2.35, 3.03 and 4.37 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: all-glass aquaria
- Type (delete if not applicable): The aquaria were closed with Parafilm® laboratory film and a pane of glass.
- Material, size, headspace, fill volume: 9 liters of reconstituted water (control group) or test medium (test material group)
- Aeration: yes
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstututed water
For the reconstituted water the following chemicals (analytical grade) were dissolved in fully demineralized water:
Macro nutrients (mg/L):
CaCl2 * 2H2O: 294.00
MgSO4 * 7H2O: 123.25
NaHCO3: 64.75
KCl: 5.75
Before the start of the experimental part the reconstituted water was prepared. After preparation the water was aerated for 24 hours before using it.
The proportion of Ca to Mg ions was 4:1 and that of Na to K ions 10:1. Hardness: about 15° dH (about 267 mg/L CaC03).
- Ca/mg ratio: 4:1
- Na/K ratio: 10:1

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: Lighting was controlled by a timer to provide a 12 hours light -12 hours dark regime.
- Other: The study was located in an air-conditioned room in the Institute of Toxicology.


EFFECT PARAMETERS MEASURED (with observation intervals if applicable): The behavior and general condition of all zebra fish were checked immediately after the introduction into the test media or reconstituted water, then after three and six hours, and daily thereafter

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.415
- Test concentrations: nominal: 25.0, 35.4, 50.0, 70.7, and 100.0 mg/L.
Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
4.53 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % confidence interval: 3.61 - 5.69 mg/L
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
4.53 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % confidence interval: 3.61 - 5.69 mg/L
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
4.53 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % confidence interval: 3.61 - 5.69 mg/L
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
4.53 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % confidence interval: 3.61 - 5.69 mg/L
Details on results:
- Behavioural abnormalities: Signs of toxicity were seen in the nominal concentrations 50.0 to 100.0 mg/L. They started after 3 hours and lasted up to the end of the experimental part. These consisted of swimming behavior slightly irregular, fish at the water surface, and fish near the bottom of the aquarium.
- Mortality of control: No mortality was seen in the control group.
- Other adverse effects control: No signs of toxicity were seen in the control group.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No remarkable observations were made concerning the appearance of the solutions of the test material preparations. The test media were clear preparations and stayed unchanged throughout the study.
Validity criteria fulfilled:
yes
Conclusions:
The study was conducted under GLP according to OECD Guideline 203 and EU Method C.1 on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies in documentation or deviations from the guidelines, the validity criteria were met. Hence, the results can be considered as reliable to assess the toxicity of the test substance towards daphnids.
Zebrafish (Danio rerio) were exposed to five test material concentrations of nominal 25.0, 35.4, 50.0, 70.7 and 100.0 mg/L beside a control with reconstituted water only for 96 hours in a closed static test system under defined conditions. Signs of toxicity were seen in the nominal concentrations 50.0 to 100.0 mg/L starting after 3 h and lasting up to the end of the experimental part. Deaths occured only in the highest concentration of nominal 100.0 mg/L, after an exposure period of about 24h.
The limit of quantification of the analytical method was 0.06 mg/L. The analytically determined concentrations were between 5.0 - 6.0 % at die start and 3.2 - 3.9 % at the end of the 96 hours exposure period. During the experimental phase of the study, the test material concentrations could not be maintained at ≥ 80 % of the nominal concentrations. Therefore, the LC50 values had to be calculated with the mean analytical concentrations (1.20, 1.54, 2.35, 3.03 and 4.37 mg/L).
Accordingly, the 96h LC50 with 95 % confidence intervals was determined to be 4.53 mg/L (3.61 - 5.69 mg/L) and the 96h NOEC is 1.54 mg/L.
Executive summary:

The objective of this study was to determine the acute fish toxicity of the test material using zebrafish (Danio rerio). The GLP study was performed according to OECD TG 203 and EU Method C.1.

In a stability test, the test material was not stable in an open test system but stable in a closed system. Therefore a closed static test system was chosen. For this purpose, 7 zebrafish per test material group and further 7 zebrafish in a control group were exposed over 96 hours, under defined conditions. The fish were observed for signs of toxicity or death. Zebrafish (Danio rerio) were exposed to aqueous test material solutions of nominal 25.0, 35.4, 50.0, 70.7, and 100.0 mg/L in a closed static system.

 

The limit of quantification of the analytical method was 0.06 mg/L. The analytically determined concentrations were between 5.0 - 6.0 % at die start and 3.2 - 3.9 % at the end of the 96 hours exposure period. During the experimental phase of the study, the test material concentrations could not be maintained at ≥ 80 % of the nominal concentrations. Therefore, the LC50 values had to be calculated with the mean analytical concentrations (1.20, 1.54, 2.35, 3.03 and 4.37 mg/L)

 

No signs of toxicity were seen in the zebrafish exposed to nominal concentrations of 25.0 and 35.4 mg/L. Signs of toxicity were seen in the nominal concentrations 50.0 to 100.0 mg/L. They started after 3 h and lasted up to the end of the experimental part. These consisted of swimming behaviour slightly irregular, fish at the water surface, and fish near the bottom of the aquarium.  Deaths occured only in the highest concentration, after an exposure period of about 24 h.  

For the test material, the following analytical values for zebrafish (Danio rerio) were determined:

24 h  LC50 =  4.53 mg/L  (95 % confidence intervals: 3.61    - 5.69 mg/L)

48 h  LC50 =  4.53 mg/L (95 % confidence intervals: 3.61    - 5.69 mg/L)

72 h  LC50 =  4.53 mg/L  (95 % confidence intervals: 3.61    - 5.69 mg/L)

96 h  LC50 =  4.53 mg/L  (95 % confidence intervals: 3.61    - 5.69 mg/L)

 

The test material, prepared in reconstituted water, was tested in a closed static test system. Under the given conditions of this study the 96h LC50 value for fish was 4.53 mg/L and the 96h NOEC is 1.54 mg/L.

Description of key information

Short-term toxicity to fish: LC50(96h) = 4.53 mg/L for zebrafish (Danio rerio) based on mortality (static, closed, OECD 203, GLP)

Key value for chemical safety assessment

LC50 for freshwater fish:
4.53 mg/L

Additional information

The objective of this study was to determine the acute fish toxicity of the test material using zebrafish (Danio rerio). The GLP study was performed according to OECD TG 203 and EU Method C.1.

In a stability test, the test material was not stable in an open test system but stable in a closed system. Therefore a closed static test system was chosen. For this purpose, 7 zebrafish per test material group and further 7 zebrafish in a control group were exposed over 96 hours, under defined conditions. The fish were observed for signs of toxicity or death. Zebrafish (Danio rerio) were exposed to aqueous test material solutions of nominal 25.0, 35.4, 50.0, 70.7, and 100.0 mg/L in a closed static system.

 

The limit of quantification of the analytical method was 0.06 mg/L. The analytically determined concentrations were between 5.0 - 6.0 % at the start and 3.2 - 3.9 % at the end of the 96 hours exposure period. During the experimental phase of the study, the test material concentrations could not be maintained at ≥ 80 % of the nominal concentrations. Therefore, the LC50values had to be calculated with the mean analytical concentrations (1.20, 1.54, 2.35, 3.03 and 4.37 mg/L)

 

No signs of toxicity were seen in the zebrafish exposed to nominal concentrations of 25.0 and 35.4 mg/L. Signs of toxicity were seen in the nominal concentrations 50.0 to 100.0 mg/L. They started after 3 h and lasted up to the end of the experimental part. These consisted of swimming behaviour slightly irregular, fish at the water surface, and fish near the bottom of the aquarium. Deaths occured only in the highest concentration, after an exposure period of about 24 h.  

For the test material, the following analytical values for zebrafish (Danio rerio) were determined:

24 h  LC50=  4.53 mg/L  (95 % confidence intervals: 3.61 - 5.69 mg/L)

48 h  LC50=  4.53 mg/L (95 % confidence intervals: 3.61 - 5.69 mg/L)

72 h  LC50=  4.53 mg/L  (95 % confidence intervals: 3.61 - 5.69 mg/L)

96 h  LC50=  4.53 mg/L  (95 % confidence intervals: 3.61 - 5.69 mg/L)

 

The test material, prepared in reconstituted water, was tested in a closed static test system. Under the given conditions of this study the 96h LC50value for fish was 4.53 mg/L and the 96h NOEC is 1.54 mg/L.