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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:
1-19 Sep 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
During the limit test samples for analysis were taken from both the limit concentration and the control according to the schedule below.
Frequency:at t=O h and t=96h
Volume: 2ml
Storage:Samples were stored in a freezer until analysis
Additionally, reserve samples of 2 ml were taken from both test solutions for possible analysis. If not already used, these samples were stored in a freezer for a maximum of three months after delivery of the draft report, pending on the decision of the sponsor for additional analysis.
Vehicle:
yes
Remarks:
Adjusted ISO medium with a hardness of 180 mg CaC03 per litre and a pH of 7.7 ± 0.3.
Details on test solutions:
The standard test procedures required generation of test solutions, which should contain
completely dissolved test substance concentrations or stable and homogeneous m.ixtures or
dispersions. The testing of concentrations that disturb the test system should be prevented (e.g.
film of the test substance on the water surface).
The batch of AHU377 C9 tested was a white powder with a purity of 99.82% and completely
soluble in test medium at the concentrations tested.
Preparation of test solutions started with a loading rate of 100 mgll applying 30 minutes of
magnetic stirring followed by a 20-minute treatment period with ultrasonic waves and an
additional 1% hours of stirring to accelerate the dissolving of the test substance in the test
medium. The lower test concentrations for the range-finding test were prepared by subsequent
dilutions of the 100 mgll concentration in test medium. The final test solutions were all clear and
colourless.
Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name:Carp
- Strain:(Cyprinus carpio, Teleostei, Cyprinidae)Linnaeus, 1758
- Source:Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.
- Length at study initiation (length definition, mean, range and SD):3.0 f 0.3 cm
- Weight at study initiation (mean and range, SD):0.84 f 0.21 g
-Feeding:Daily with pelleted fish food (Cyprico Crumble Excellent (300-500 um), Coppens International bv,Helmond, The Netherlands)

ACCLIMATION
- Acclimation period:At least 12 days after delivery
- Acclimation conditions (same as test or not):(tap-water purified by reverse osmosis; GEON
Waterbehandeling, Berkel-Enschot, The Netherlands)
with the following composition:
CaC12.2H20 211.5 mgll
MgS04.7H20 88.8 mg/l
NaHC03 46.7 mgll
KC1 4.2 mgll

Measurements:pH, nitrate and nitrite concentration and ammonia concentration: once a week. Temperature: every day. In addition, pH and temperature were measured before transferring the fish to the test system.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Test temperature:
20-24°C
pH:
6.0-8.5
Dissolved oxygen:
> 60% of air saturation
Details on test conditions:
Test duration: 96 hours
Test type: Static
Test vessels: 20 litres, all-glass, containing 15 litres of test solution
Test medium: Adjusted IS0 medium with a hardness of 180 mg
CaC03 per litre and a pH of 7.7 f 0.3.
Number of fish: 7 per concentration
Loading: 0.39 g fishllitre, i.e. 7 fish per 15 litres of test medium
Illumination: 16 hours photoperiod daily
Aeration:Aeration was introduced after 24 hours of exposure and maintained until the end of the test.
Feeding:No feeding from 24 hours prior to the test and during the total test period
Introduction of fish:Within 10 minutes after preparation of the test media from a holding tank with comparable water quality parameters and pH and temperature differences between test and holding tank media of less than 0.5 unit and 0.5"C.
Euthanasia:At the end of the test the surviving fish were rapidly
killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.




Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
The batch of AHU377 C9 tested was a white powder with a purity of 99.82% and completely
soluble in test medium at the concentrations tested.
Based on the results of a range finding test, a limit test was performed. Preparation of test
solutions started with a loading rate of 100 mgll applying 30 minutes of magnetic stirring
followed by a 20-minute treatment period with ultrasonic waves and an additional 1% hours of
stirring to accelerate the dissolving of the test substance in the test medium. The final test
solution was all clear and colourless.
Seven fish per test group were exposed to the 100 mgll test concentration and a control. The
total test period was 96 hours and samples for analytical confirmation of actual exposure
concentrations were taken at the start and the end of the test.
Analysis of the samples taken from the limit concentration at the start and the end of the test
showed that the measured concentration was in agreement with nominal (102%) and remained
stable during the test (100% of initial). Based on these results, the effect parameters were
.based on nominal concentrations.
The study met the acceptability criteria prescribed by the protocol and was considered valid.
AHU377 C9 induced no visible effects in carp at 100 mgll (NOEC). Hence, the 96h-LC,, was
> 100 mgll.
Results with reference substance (positive control):
This reference test was carried out to check the sensitivity of the test system as used by
NOTOX. The reference substance was pentachlorophenol (PCP, SIGMA, Art. P2604, Batch
06324ED-405).
Concentrations: 0.10, 0.22 and 0.46 mgll in ISO-medium.
Under the conditions of the present test PCP induced no lethal effects in carp at 0.10 mgll. The
96h-LCso for carp exposed to PCP was 0.20 mgll (95 % confidence interval between 0.16 and
0.32 mgll). This effect was already reached within 48 hours of exposure.
The range of the 96h-LCso for carp is generally between 0.10 and 0.46 mgll based on historical
data of reference tests performed approximately every 3 months from April 1988 until the end of
2000, and annually since then. Hence, the sensitivity of carp originating from the present batch
for PCP falls within the range of sensitivities generally observed during the past years.
Validity criteria fulfilled:
yes
Conclusions:
Under the conditions of the present test AHU377 C9 induced no visible effects in carp at 100 mgll (NOEC). Hence, the 96h-LC,, was > 100 mgll.

Description of key information

Under the conditions of the present test AHU377 C9 induced no visible effects in carp at 100 mgl/l (NOEC). Hence, the 96h-LC, was > 100 mg/l.

Key value for chemical safety assessment

LC50 for freshwater fish:
100 mg/L

Additional information