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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:
2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes
Specific details on test material used for the study:
None.
Analytical monitoring:
yes
Details on sampling:
Samples were taken from the test water (about 1 mL) for each concentration at the start of the experiment, before and after water replacement (48 hour), as well as at the conclusion of the experiment. 300 µL of acetonitrile were added to 300 µL of each of the test samples prior to HPLC analysis, which was conducted immediately after sampling.
Vehicle:
no
Details on test solutions:
80mg of the test substance were weighed, added to 2L of dilution water and stirred for 48 hours and 15 minutes. Furthermore, ad-hoc dissolution work was performed ultrasonically (about 4×5 minutes) during the stirring period. Filtering (GA55,90mm, 0.6µm) was performed after the conclusion of stirring and this filtrate was treated as the stock solution. The tested substance concentration of this stock solution was measured and this was treated as the saturation concentration (28.7 mg/L). Dilution water into which the tested substance had not been added was used for the control group. The preparation of the test medium was performed just prior to the start of the trial. Tested concentrations were all prepared by dilution of the stock solution.
Test organisms (species):
Oryzias latipes
Details on test organisms:
The testing used Oryzias latipes purchased from a vendor. The total length of the test fish was 2.3±1.2cm.

Supplier: Name: Araya Fish Farm
Address: Kyujo-cho 1061, Yama-city, Yamato-Gun, Nara Prefecture, Japan

Domestication and acclimation were performed for 32 days under the same conditions as the test conditions (water quality, temperature, etc.), and using water equivalent to that used in the test. Feed “Oryzias latipes Feed (Kyurin)” was given. No feeding was performed for 24 hours before the start of the experiment. For this test, healthy and visibly normal individuals were selected and used. The mortality rate 7 days before experiment start was 3.4% (i.e. less than 5%). The batch was therefore deemed as usable for testing.

Test fish were selected from the acclimated fish at the start of the experiment. In addition, 7 fish were selected in the same manner, and their weights and lengths measured. The weights were 0.22±0.05 g, and the lengths were 2.9±0.1 cm.

In addition, the test fish (acclimation lot no.: M05C-01) underwent qualification testing with a standard substance (pentachlorophenol sodium salt). The 96-hour LC50 satisfied the range of the standard (0.3~0.7 mg/L). The resistance of this lot was determined to be within the normal range, and so these were provided for testing.
The test values performed in 28 March, 2005 – 1 April, 2005 are shown below: 96-hour LC50 (test value): 0.41 mg/L (Background Data: 0.44±0.09 mg/L)
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
None.
Post exposure observation period:
None.
Hardness:
Not measured
Test temperature:
23.9°C - 24.3°C (min - max)
pH:
7.5 - 7.7 (min - max)
Dissolved oxygen:
6.4 - 8.1 (min - max)
Salinity:
Freshwater
Conductivity:
Not measured.
Nominal and measured concentrations:
Nominal concentrations: 0 ; 2.7 ; 4.9 ; 8.9 ; 15.9 ; 28.7 mg/L (dilutions were made from a unique stock solution at 28.7 mg/L and a spacing factor of 1.8)
Measured concentrations: 0 ; 3.2 ; 5.7 ; 10.0 ; 16.6 ; 34.0 mg/L
Results were based on nominal concentrations because measured concentrations were in the [80-120]% range of the nominal concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 3-L capacity glass vessels
- Material, size, headspace, fill volume: filled with at least 1 L per gram of fish wet weight
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1

TEST MEDIUM
Dechlorinated water (tap water processed with activated charcoal, removing residual chlorine, and fully aerated) was used. When using the dilution water, the presence of residual chlorine was checked by the o-toluidine method. The quality of the dilution water was checked twice a year.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16L:8D
- Light intensity: Not measured

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality

The number of dead Oryzias latipes was checked at experiment start, and after 3, 24, 48, 72, and 96 hours together with recording the observed signs of toxicity or abnormalities. In cases where dead individuals were discovered, these were removed immediately so as not to degrade water quality.
Reference substance (positive control):
yes
Remarks:
Pentachlorophenol sodium salt
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
20.8 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
mortality (fish)
Details on results:
Data on measured concentrations and mortality recorded per day are given in table 1. LC50 was determined by the Probit method. LC50 = 20.8 mg/L
Results with reference substance (positive control):
The test fish (acclimation lot no.: M05C-01) underwent qualification testing with a standard substance (pentachlorophenol sodium salt). The 96-hour LC50 satisfied the range of the standard (0.3~0.7 mg/L). The resistance of this lot was determined to be within the normal range, and so these were provided for testing. The test values performed in 28 March, 2005 – 1 April, 2005 are shown below: 96-hour LC50 (test value): 0.41 mg/L (Background Data: 0.44±0.09 mg/L)
Sublethal observations / clinical signs:

Table 1: fish mortality and mean measured concentrations during the experiment

Nominal concentrations (mg/L)

Mean measured concentrations (mg/L)

Cumulative number of dead fish

24-hour

48-hour

72-hour

96-hour

Control

0

0

0

0

0

2.7

3.15

0

0

0

0

4.9

5.65

0

0

0

0

8.9

9.99

0

0

0

0

15.9

16.62

1

1

1

1

28.7

33.98

6

7

7

7

Validity criteria fulfilled:
yes
Conclusions:
A GLP test following OECD 203 guideline was conducted on Oryzias latipes under semi-static conditions (48-hour renewal). Tested concentrations were 0 ; 2.7 ; 4.9 ; 8.9 ; 15.9 ; 28.7 mg/L. They were in agreement with measured values at 0, 48 hours (both new and old) and 96 hours. The 96h-LC50 was 20.8 mg/L.
Executive summary:

A GLP test following OECD 203 guideline was conducted on Oryzias latipes under semi-static conditions (48-hour renewal). One vessel by concentration was used, in each of which were put seven fish. Tested concentrations were 0 ; 2.7 ; 4.9 ; 8.9 ; 15.9 ; 28.7 mg/L. They were in agreement with measured values at 0 (new), 48 hours (both new and old) and 96 hours (old). Mortality was checked every 24 hour. All fish died in the 28.7 mg/L treatment. One fish died in the 15.9 mg/L treatment. No fish died in any other treatment, including control. The 96h-LC50 was calculated as 20.8 mg/L.

Description of key information

A GLP test following a protocol equivalent to OECD 203 guideline was conducted on Oryzias latipes under semi-static conditions (48-hour renewal). One vessel by concentration was used, in each of which were put seven fish. Tested concentrations were 0 ; 2.7 ; 4.9 ; 8.9 ; 15.9 ; 28.7 mg/L. They were in agreement with measured values at 0 (new), 48 hours (both new and old) and 96 hours (old). Mortality was checked every 24 hour. All fish died in the 28.7 mg/L treatment. One fish died in the 15.9 mg/L treatment. No fish died in any other treatment, including control. The 96h-LC50 was calculated as 20.8 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
20.8 mg/L

Additional information