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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:
13 February 2017 - 17 February 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
not applicable
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples were taken in plastic tubes, acidified with pure nitric acid (9 mL sample + 1 mL HNO3) in agreement with the requirements of the Principal
Investigator. To avoid any problems with microparticles, therefore the samples were membrane filtrated before acidification.
Samples were taken at the start and end of the first and last renewal periods from all concentration levels.
Vehicle:
no
Details on test solutions:
Because the test item is a multi constituent substance and poorly soluble in water, test solutions were prepared using a saturated solution method (water accommodated fraction, WAF) according to the Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD No. 23.
Saturated test item solutions with nominal loading rates of 0.625, 1.25, 2.5, 5.0 and 10.0 mg/L were individually prepared by dispersing/dissolving the amount of test item into the test medium (aquarium water) two days before the start of the treatments. These solutions were shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C. The non-dissolved test material were removed by filtration through a fine (0.22 μm) filter to give the 0.625, 1.25, 2.5, 5.0 and 10.0 mg/L nominal lading rate WAFs.
The test solutions were prepared just before introduction of the fish (start of treatments). In absence of stability data the test was performed under semi-static conditions. The frequency of the water renewal periods was 24 hours. Prior to treatment of each renewal period, test item solutions were prepared by the method described above.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
name - zebrafish
Justification - Zebrafish (Brachydanio rerio) is one of the convenient species for acute fish toxicity test.
seven fish per test group
length - 2.7 – 3.3 cm (Body length of animals was in the acceptable range (3.0 ± 0.5 cm))
Acclimatisation: more than 12 days
Fish were bred in a well-known fish farm, under disease- and parasite-controlled conditions. Fish were observed during the acclimatisation and test periods, the fish were healthy. Mortality was less than 5 % of the population for seven days before the test.

The fish were not fed during the test
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Post exposure observation period:
-
Hardness:
152 mg/L (as CaCO3).
Test temperature:
between 20.8 and 22.0°C.
pH:
7.68 – 8.36.
Dissolved oxygen:
In the range of 69 – 99 % of the air saturation value at the temperature used.
Salinity:
-
Conductivity:
-
Nominal and measured concentrations:
Nominal loading rates (WAF) = 0 (control), 0.625, 1.25, 2.5, 5, and 10.0 mg/L.
The measured concentrations for each concentration level at each measurement timepoint was <0.15 mg/L (based on the concentration of copper <0.013 mg Cu/L)
Details on test conditions:
The test duration was 96 hours. One aquarium was used for test groups and for the control group, respectively. Each aquarium comprised 7 fish and 5 L test solution. The animals were not fed during the test. The loading of the test aquaria was less than 1.0 g fish/L test solution at the start of the experiment.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
EL50
Effect conc.:
7.95 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
ca. 5 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Basis for effect:
mortality
Details on results:
No mortality was seen in the control group or in the 0.625, 1.25, 2.5 and 5 mg/L test groups. 1/7 fish was found dead in the highest dose level (nominal 10 mg/L loading rate) at 72 hours, and 6/7 fish in this group were found dead at 96 hours (cumulative mortality).
Behaviour of fish was found as normal in the control group and at the tested concentration levels with the exception of the highest test level of 10.0 mg/L nominal loading rate WAF.
Decreased activity, localization at the bottom of the aquarium, lateral position, fast motility of operculum and immobility was observed at the highest test concentration group of 10.0 mg/L nominal loading rate WAF during the experiment.

The 24h, 48h and 72h LR50 value: > 10.0 mg/L nominal loading rate WAF
The 96h LR50 value: 7.95 mg/L nominal loading rate WAF (95 % conf. limits: 5.62 – 10.44 mg/L nominal loading rate WAF)
The 96h LR100 value: > 10.0 mg/L nominal loading rate WAF
The 96h No-Observed Effect Loading Rate (NOELR): 5.0 mg/L nominal loading rate WAF
The 96h Lowest Observed Effect Loading Rate (LOELR): 10.0 mg/L nominal loading rate WAF
Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of B331 was assessed with acute fish toxicity test on Zebrafish (Brachydanio rerio), over an exposure period of 96 hours in a semi-static system. Under the conditions of this acute fish toxicity study on Zebrafish (Brachydanio rerio) the observed and calculated endpoints for the effect of B331 were the following:
The 24h, 48h and 72h LR50 value: > 10.0 mg/L nominal loading rate WAF
The 96h LR50 value: 7.95 mg/L nominal loading rate WAF (95 % conf. limits: 5.62 – 10.44 mg/L nominal loading rate WAF)
The 96h LR100 value: > 10.0 mg/L nominal loading rate WAF
The 96h No-Observed Effect Loading Rate (NOELR): 5.0 mg/L nominal loading rate WAF
The 96h Lowest Observed Effect Loading Rate (LOELR): 10.0 mg/L nominal loading rate WAF

Description of key information

Zebrafish (danio rerio), 96 hours EL50 = 7.95 mg/L (nominal loading rate). NOEL = 5 mg/L.

Key value for chemical safety assessment

LC50 for freshwater fish:
7.95 mg/L

Additional information

An acute fish toxicity study was conducted (CiToxLAB Hungary, 2017) to assess the short-term effects of B331 on fish. The study was conducted according to OECD test guideline 203 and EC method C1, and in compliance with GLP. The test media was prepared as a water accomodated fraction (WAF) with nominal loading rates of 0.625, 1.25, 2.5, 5 and 10 mg/L; a concurrent vehicle control group (untreated dilution water) was used. Test media were replaced every 24 hours.

Analysis of the test solutions in all dose levels at each sampling timepoint revealed the test material concentration to be below the limit of quantitation of 0.15 mg/L (measured for copper concent by ICP-AES; LoQ = 0.013 mg Cu/L).

Deaths were observed in the 10 mg/L dose level at 72 hours and 96 hours. All other dose levels were considered normal (no mortality and no behavioural abnormalities).

The 96 -hour EL50 was 7.95 mg/L. The 96 -hour NOEL = 5 mg/L.