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Short-term toxicity to fish

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short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
according to guideline
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
July 1992
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
Details on sampling:
Duplicate samples for analysis were taken from the single WAF and the control.
Frequency: at t=0 h and t=96 h
Volume: 2.0 mL
Storage: Not applicable, samples were analysed on the day of sampling
Details on test solutions:
The test item was not completely soluble at the loading rate of 100 mg/L. Thus, preparation of the test solution started with a loading rate of 100 mg/L applying a three-day period of magnetic stirring to reach the maximum dissolution of the test item in medium. Thereafter, the Water Accommodated Fraction (WAF) was collected by means of filtration through a 0.45 µm membrane filter (Supor®, Pall Corporation) and used as test concentration. The test solution was clear and colorless at the end of the preparation procedure. No correction was made for the purity/composition of the test item.
The method of preparation of the test medium was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.
- Controls: Test medium without test item or other additives
Test organisms (species):
Cyprinus carpio
Details on test organisms:
- Common name: carp
- Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.

- Length at study initiation (mean and SD): 2.5 ± 0.1 cm
- Weight at study initiation (mean and SD): 0.52 ± 0.09 g

- Acclimation period: At least 12 days after delivery
- Acclimation conditions (same as test or not): same
- Type and amount of food during acclimation: Pelleted fish food (Essence (300-500 µm), Coppens International bv, Helmond, The Netherlands)
- Feeding frequency during acclimation: Daily
- Health during acclimation (any mortality observed): In the batch of fish used for the test, mortality during the seven days prior to the start of the test was less than 5%.

No feeding from 24 hours prior to the test and during the total test period
Test type:
Water media type:
Limit test:
Total exposure duration:
96 h
1.8 mM (180 mg CaCO3/L)
Test temperature:
20 - 21 °C
7.7 - 8.1
Dissolved oxygen:
≥ 6.6 mg/L (>60% oxygen saturation)
Nominal and measured concentrations:
nominal loading rate: 100 mg/L (WAF)
measured test concentrations: test start: 0.046 mg/L and < LOQ (0.001 mg/L); test end (96 h): < LOQ

The measured concentration of 0.046 mg/L was regarded as not reliable since the solubility limit of the test item has been shown to be < 0.001 mg/L (the LOQ of the analytical method used) during the range-finding test as well as in an acute toxicity study with daphnia and in an algal growth inhibition test (Test Facility Study No. 20148112 and 20148113).
Details on test conditions:
- Test vessel: 10-litres all-glass vessels, containing 8 liters of test medium
- Type (delete if not applicable): open
- Aeration: none
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.46 g fish wet weight/L

- Source/preparation of dilution water: adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis; GEON Waterbehandeling, Berkel-Enschot, The Netherlands)
Composition: CaCl2.2H2O: 211.5 mg/L, MgSO4.7H2O: 88.8 mg/L, NaHCO3: 46.7 mg/L, KCl: 4.2 mg/L.
- Ca/Mg ratio: 4:1
- Culture medium different from test medium: no
- Intervals of water quality measurement:
temperature, dissolved oxygen and pH: daily in all vessels, beginning at the start of the test (day 0)

- Adjustment of pH: no
- Photoperiod: 16 hour photoperiod daily

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
mortality and sub-lethal effects; recorded at 2½, 24, 48, 72 and 96 hours following the start of exposure. In addition, every afternoon from day 0 to observe for any dead or severely distressed fish.

- Spacing factor for test concentrations: not applicable (limit test)
- Range finding study
- Test concentrations: WAFs prepared at loading rates of 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: No mortality or clinical effects were observed at any of the WAFs during the test period. Therefore, the expected LL50 was above the concentration obtained in a WAF prepared at 100 mg/L.
Reference substance (positive control):
Pentachlorophenol at 0.10, 0.22 and 0.46 mg/L
Key result
96 h
Dose descriptor:
Effect conc.:
> 100 mg/L
Nominal / measured:
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality (fish)
Remarks on result:
WAF prepared at a loading rate of 100 mg/L
Key result
96 h
Dose descriptor:
Effect conc.:
>= 100 mg/L
Nominal / measured:
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
other: mortality and sub-lethal effects
Remarks on result:
WAF prepared at a loading rate of 100 mg/L
Details on results:
- Behavioural abnormalities:
- Other biological observations: none; all fish survived and no visible abnormalities were observed.
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none
Results with reference substance (positive control):
- Results with reference substance valid? yes
- LC50: 0.15 mg/L (95 % confidence interval between 0.10 and 0.22 mg/L)
- Other: historical range: 0.10 – 0.46 mg/L
Reported statistics and error estimates:
The NOELR and LL50 were determined directly from the raw data.
Validity criteria fulfilled:
/no mortality of control animals; concentration of dissolved oxygen in all test vessels >60% of the air saturation; test conditions were maintained constant throughout the test

Description of key information

effect value for carp (Cyprinus carpio), (OECD 203): 96-h LL50: > 100 mg/L (WAF)

Key value for chemical safety assessment

Additional information

LL50 for freshwater fish: > 100 mg/L.

The results indicate that the test material is not toxic to fish up to the limits of its solubility (<1 μg/L in test water) and a NOEC and thus a PNEC cannot be determined.