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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
Study period:
17-4-2000 to 12-7-2000
1 (reliable without restriction)
according to
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
Specific details on test material used for the study:
off white powder, purity 99.15%
Analytical monitoring:
Details on sampling:
The test concentration was verified by chemical analysis. Duplicate medium samples, of 200 mL, were taken from the solvent control at O and 72 hours (fresh media) and 24 and 96 hours (expired media) for analysis. Four samples were taken from the test concentration at the same times
dimethylformamide (DMF)
Details on test solutions:
The test substance was dissolved in dimethylformamide (DMF) to give a primary stock solution with a nominal concentration of 10 mg/mL. This solution was then further diluted with DMF to give a working stock solution containing 1.0 mg/mL. A 2 mL aliquot of the working stock solution was added to diluent water to give a nominal test concentration of 100 µg/L. This provided an excess of test material in diluent water, and was stirred with a magnetic stirrer for approximately30 minutes to give a saturated solution.
This value was obtained using dechlorinated, softened laboratory tap water (diluent water) and may differ from previously determined values obtained in physical/chemical analytical studies, where the test substance is dissolved in ultra pure water.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
Name: Rainbow trout (Oncorhynchus mykiss).
Source: Donnington Fish Farm, Gloucestershire, UK.

Acclimatisation:The stock of fish was obtained from the supplier on 9 May 2000 and acclimatised to test conditions from 15 May 2000. Temperature remained at 15 °C and dissolved oxygen within the range 8.0 to 8.6 mgO,IL in the 14-day period immediately before the study. The fish were fed daily to repletion with commercial trout fry crumb but food was not given during the 24 hour period immediately before exposure or during the exposure period itself. No medication was given during the acclimatisation period and mortalities were recorded as <1 % in the 7 days before the test.

A representative sample of stock fish was measured at the start of the study. The mean standard length was 4.3 cm (SD= 0.1 cm) and the mean weight was 1.33 g (SD= 0.10 g).

Test type:
Limit test:
Total exposure duration:
96 h
Post exposure observation period:
In addition to observations on mortality at 15 minutes and 2, 4, 24, 48, 72 and 96 hours, subjective assessments were made on the type and incidence of sub-lethal effects compared with control fish.
163-178 mg CaCO3 per litre
Test temperature:
Between15 and 16 throughout the test
Between 7.3 and 7.8 throughout the test
Dissolved oxygen:
Between 8.6 mg/L and 9.5 mg O2/L throughout the test
Nominal and measured concentrations:
Nominal concentration:100 ug/L
Measured concentration: 77 ug/L
Reference substance (positive control):
Key result
96 h
Dose descriptor:
Effect conc.:
> 77 µg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks on result:
other: No effects seen up to the limit of water solubiilty

The test substance is described asbeing "very slightly soluble in coldwater" with a determined solubility of lessthan 0.492mg/L using the column elution method.

In line with the recommendations given in the "Revised Draft Guidance Document on AquaticToxicity Testing of Difficult Substances and Mixtures", preliminary  tests were carried out to try to determine the maximum dissolved concentration of the test substance that could be achieved under test conditions, using appropriate media (dechlorinated water,ElendtM4 and algal medium).

The first attempts to create a saturated solution involved direct addition of the test substance to test media, or the use of solvent spikes,followeby stirring for 48hours and then filtering though a 0.45µm membrane. The results of the analysis of the filtrates of the solutions was highly variable, ranging from <LOO (limit of detection: I µg/L) to 69µg/L. It is believed that the variability in the results is because of large excesses of non-dissolved test substance in the primary solutions and losses to the filter membrane.

The second attempt to resolve the solubility of the test substance addressed the problems associated with excess test substance and filter membranes.

 Stock solutions of BS233101-01 were prepared in acetone at 1mg/mLand 5 mg/mL. Aliquots(5mL) of each stock solution were added to two clean,dryglass vessels per medium, whilst the vessels were rotated, to promote the formation of an even coating on the vertical surfaces of the glass interior. The solvent was removed by nitrogen stream until no acetone fumes could be detected.This ensured that the glass represented a reservoir of test substance many times higher than that required to achieve saturation in the volume of water in the vessel.

 The vessels were gently filled with5  litres of the appropriate medium and gently stirred for 48hours with a magnetic stirrer. Duplicate samples were removed for analysis at 24 and 48 hours.

 Based on the variable results obtained in this preliminary work,and following consultation with the Irish HSA, it was agreed that the test material would be added to the water in a solvent carrier in an effort to achieve a more homogenous and stable solution.

Validity criteria fulfilled:
Criteria of effect: The criteria of death employed in this study were (i) absence of respiratory movement and (ii) absence ofresponse to physical stimulation of the caudal peduncle.

The protocol states that a range finding test will be conducted before the definitive test. A range finding test was not done for this study because the solubility of the test compound in the test medium was found to be very low and so was expected to have little, if any, toxicity. Therefore the definitive test was conducted immediately as a limit test.

The protocol states that a limit test will be performed at either: (i) 100-150 mg a.i./1, (ii) a concentration equal to the limit of solubility of the test substance or (iii) the maximum concentration forming a stable dispersion. However, the limit test for this study was conducted at a concentration greater than the limit of solubility of the test substance to ensure saturation of test medium. This method was approved by the HSA.
These deviations from protocol are not believed to have affected the validity of this study

1) No mortality was observed in the control. 2) Test conditions were maintained constant 3) Diss olved oxygen concentration has been at least 60% of the air saturation (> 5mg/L at 22°C). 4) Results were based on the average concentrations
Executive summary:

A study  was performed  to assess  the  acute  toxicity  of BMS  233101-01  to  rainbow  trout (Oncorhynchus mykiss)under semi-static conditions.

 A group of tenjuvenile  fish was exposed to a single concentration,  nominally  100  µg/L, ofBMS233101-01 in water for 96hours.  This concentration was determined tobegreater than thelimitofsolubilityforthetestsubstance,undertheconditionsofthisstudy(44µg/L;seeAppendix 5).Observations weremade onthenumbers ofdeadfishandthe incidence ofsub-lethal effects after

15 minutes and2,4,24,48,72and96hoursexposure.  Asnomortalities orsub-lethal effects werenotedthroughouttheexposureperiod,thefollowingvaluescanbeassumed(meanmeasuredconcentrations ofBMS233IO1-01 areshowninbrackets):


HighesttestconcentrationresultinginO %mortality:Lowesttestconcentrationresultingin I00%mortality:":

LC50 > Limit of solubility (77µg/L)




Mean measuredconcentrationsat0,24, 72 and 96hours remained abovetheestimated limitofsolubilityofBMS233101-01indechlorinatedwater;44µg/L.

Description of key information

In a 96hr acute toxicity study in fish the LD50 value was determined to be >77ug/l. No effects were seen up to the limit of water solubility

Key value for chemical safety assessment

LC50 for freshwater fish:
77 µg/L

Additional information