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Ecotoxicological information

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
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:
July 1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
REG (EC) No. 440/2008, May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: nominal 100, 50, 25, 12.5, 6.25 mg/L as WAF and control
- Sampling intervals: start and end of the first and the last renewal period
- Sampling method: samples were taken from test vessels containing fish
- Sample storage conditions before analysis: samples taken at the start of the first and last renewal period were stored for one day after processing (freeze-drying)
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: water accommodated fractions (WAFs)
Supersaturated solutions were prepared by dispersing/dissolving the test item into the test medium (filtered tap water) at nominal concentrations of 100, 50, 25, 12.5 and 6.25 mg/L. These dispersions were shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at the test temperature. The resulting dispersions were opalescent. The non-dissolved test material was removed by filtration through a 0.22-µm filter to provide the WAFs. The filtrates were clear and transparent.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Source: “Black Molly” Fish Farm, Veszprém, Hungary (for the pre-tests) and Ágnes Németh (for definitive test), Veszprém, Hungary
- Length at study initiation (mean and range, SD): 3.0 ± 0.14 cm (2.7-3.3 cm, n= 42)
- Weight at study initiation (mean and range, SD): 0.38 ± 0.03 g (0.34-0.41 g, n= 42)
- Feeding during test: none

ACCLIMATION
- Acclimation period: > 12 days
- Acclimation conditions (same as test or not): same water quality (circulated and filtered tap water)
- Type and amount of food: commercial fish food (TetraMin staple food for ornamental fish, Tetra GmbH, Germany) at 2-10 mL per feeding.
- Feeding frequency: three times per week, until 24 hours before the start of the test
- Health during acclimation (any mortality observed): The fish were healthy. There was no mortality of the population for seven days before the test
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
203 mg/L as CaCO3 (measured)
Test temperature:
21.1-22.0 °C
pH:
7.08-7.52
Dissolved oxygen:
82-98% air saturation value
Nominal and measured concentrations:
nominal loading rates: 100, 50, 25, 12.5, and 6.25 mg/L as WAF
first renewal period (0-24 h):
- freshly prepared test media: 3.99, 3.13, 2.38, 1.57, and 0.11 mg/L
- aged test media (24 h): 1.36, 1.41, 0.47, 0.25 mg/L, and < LOQ (0.08 mg/L).

second renewal period (72-96 h; the two highest loading rates (100 and 50 mg/L) were not analysed since all fish were dead at that time):
- freshly prepared test media: 2.10, 1.13, and 0.30 mg/L
- aged test media (24 h): 0.18, 0.14 mg/L, and < LOQ (0.08 mg/L).

geometric mean measured concentrations: 2.33, 2.10, 0.81, 0.50, mg/L and 0.09 mg/L.

biological results are based on the nominal loading rates.
Details on test conditions:
TEST SYSTEM
- Test vessel: aquaria, containing 5 litres of medium
- Type: not reported (the test material is not volatile)
- Aeration: not reported (oxygen concentration was sufficient during exposure)
- Renewal rate of test solution: daily
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.48 - 0.57 g body weight/L (mean 0.53 ± 0.03 g bw/L)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: circulated and filtered tap water
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 h light, 8 hours dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality and sub-lethal effects; recorded at approx. 3, 6, 24, 48, 72 and 96 hours

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study
- Test concentrations: nominal 100, 10, 1, and 0.1 mg/L
- Results used to determine the conditions for the definitive study: 100% and 25% mortality after 96 hours at 100 and 10 mg/L respectively; no mortality at the lower test concentrations
Reference substance (positive control):
not required
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
28 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% confidence limits: 19-41 mg/L; results are expressed in terms of loading rate
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
6.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: results are expressed in terms of loading rate
Duration:
96 h
Dose descriptor:
LOELR
Effect conc.:
12.5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: one dead fish after 96 h; results are expressed in terms of loading rate
Duration:
96 h
Dose descriptor:
LL0
Effect conc.:
6.25 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: results are expressed in terms of loading rate
Duration:
96 h
Dose descriptor:
LL100
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: results are expressed in terms of loading rate
Details on results:
- Behavioural abnormalities: at the 72-hour observation, one fish showed swimming problems at the nominal loading rate of 25 mg/L; no abnormalities observed at any other concentration and/or time
- Mortality of control: zero
- Other adverse effects control: none

For cumulative mortality data Table 1.
Reported statistics and error estimates:
The 24, 48, 72 and 96 hours LL50 values were calculated by Probit analysis with 95% confidence limits using SPSS PC+ software.
The NOELR, LOELR, LL0 and LL100 were determined directly from the raw data.

Table 1: Cumulative mortality data:


 









































































Nominal loading rate (mg/L)



Cumulative mortality [number dead of fish] *


 

3 h



6 h



24 h



48 h



72 h



96 h



Control



0



0



0



0



0



0



6.25



0



0



0



0



0



0



12.5



0



0



0



0



0



1



25



0



0



0



0



0**



1



50



0



0



0



6



7



-



100



0



0



1



7



-



-



*:  initial population = 7 fish


**: one fish with swimming problems

Validity criteria fulfilled:
yes
Remarks:
no mortality of control animals; concentration of dissolved oxygen in all test vessels >60% saturation
Conclusions:
The test material is toxic to freshwater fish with a 96-hour LL50 between 10 and 100 mg/L (calculated value: 28 mg/L).

Description of key information

96-h LC50 (based on nominal loading rates) 28 mg/L for Brachydanio rerio (new name Danio rerio) (OECD 203, EU C.1)

Key value for chemical safety assessment

LC50 for freshwater fish:
28 mg/L

Additional information