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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:
05 - Oct - 09 Oct 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
GLP compliance:
yes (incl. QA statement)
Remarks:
Staatliches Gewerbeaufsichtsamt Hildesheim, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 2.00-3.56-6.33-11.3-20.0 mg/L, all concentrations were sampled
- Sampling method: samples were taken from freshly prepared media after 0, 24, 48 and 72 h and from the corresponding 24 h aged test media after 24, 48, 72 and 96 h
- Sample storage conditions before analysis: samples were stored at 6 ± 2 °C before preparation, if necessary. All prepared samples were stored at room temperature (in the autosampler) before start of analysis, if necessary.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: for each renewal day: appropriate volumes of the test item were pipetted into measuring flasks filled with 5000 mL of the dilution water. Test media were stirred (130 min, 1100 rpm, room temperature).
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebrafish
- Age at study initiation (mean and range, SD): Zebrafish acclimated at least 12 d were used in the test
- Length at study initiation: 2.67 cm (mean)
- Weight at study initiation: 0.227 g (mean)
- Method of holding: in tap water (filtered on activated charcoal and aerated for at least 24 h to remove chlorine), at 23 ± 2 °C and diffuse light, water was changed at least once per week, dissolved oxygen concentration >= 80 %, hardness: 10 - 250 mg CaCO3/L, pH = 6 - 8.5, food type was Sera Vipan; Sera GmbH, Heinsberg, Germany (4% of fish body weight per feeding day, 3 times per week), fish were not fed 24 h before test start.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
63 mg/L as CaCO3
Test temperature:
22.0 - 23.2 °C
pH:
7.09 - 8.61
Dissolved oxygen:
Oxygen saturation: 77 - 100%
Nominal and measured concentrations:
0 (control), 2.00, 3.56, 6.33, 11.3 and 20.0 mg/L (nominal)
0 (control), 0.914, 1.75, 3.83, 6.52 and 10.9 mg/L (mean measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: glass aquaria of 13 L
- Type: covered with a floating lid (i.e. with aluminium foil) to reduce loss of test item via surface
- Size, fill volume: dimensions: 25.5/18/28 cm, depth of water: 20 cm, fill voluem: 5 L
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): daily
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.318 g fish/L test solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water according to guideline
- Culture medium different from test medium: no
- Intervals of water quality measurement: measured at 0, 24, 48, 72 and 96 h

OTHER TEST CONDITIONS
- Photoperiod: 11 h light/ 13 h dark
- Light intensity: 0.94 to 1.11 µmol photons x m^-2 x s^-1

EFFECT PARAMETERS MEASURED: observations were made after 2, 24, 48, 72 and 96 h for mortality and visual abnormalities

TEST CONCENTRATIONS
- First definitive test: with a nominal test concentration of 22.4 mg/L. 100% mortality occured after 24 h. Therefore a range finding test was performed subsequently.
- Range finding study: yes
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
6.52 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
8.43 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: Geometric mean of LC0 and LC100
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
10.9 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- Mortality of control: no
- Other biological observations: normal behaviour observed at 2, 3.56 and 6.33 mg test item/L; slowed escape reflex or missing escape reflex recorded at 11.3 mg test item/L; at 20 mg test item/L fish were either lying on their side, staggering, hyperventilating or were considered dead
Reported statistics and error estimates:
Due to only partial response after 96 h LC10 and LC20 values were given as a range. For the same reason no calculation of confidence intervals was carried out for the LC-values, which is given as geometric mean between the concentrations resulting in 0 and 100% mortality. The geometric mean measured values after 96 h were used for all corresponding calculations. The concentrations leading to 0 and 100 % mortality (LC0 and LC100) after 96 h were determined directly from the test results. The chosen methods for determination of LCx-values were in accordance with the OECD series on testing and assessment No. 54 (2006).
Sublethal observations / clinical signs:

Table 1: Effect values after 96 h.

Test duration [h] LC10 [mg/L] LC20 [mg/L]
96 6.52 < LC10 < 10.9 6.52 < LC20 < 10.9
LC0 = Highest test item concentration with 0 % mortality after 96 h 6.52
LC100 = Lowest test item concentration with 100 % mortality after 96 h 10.9

Table 2: Cumulative mortality [%]

Nominal test item concentration [mg/L] Cumulative Mortality [%] after different exposure periods [h]
2 24 48 72 96
20 0 0 29 57 100
11.3 0 0 0 0 0
6.33 0 0 0 0 0
3.56 0 0 0 0 0
2 0 0 0 0 0
Control 0 0 0 0 0

Table 3: Measured concentrations and % of nominal concentrations of the test item.

Nominal concentration of the test item Geometric mean measured concentration
mg/L mg/L %
20 10.9 54
11.3 6.52 58
6.33 3.83 61
3.56 1.75 49
2 0.914 46
Validity criteria fulfilled:
yes
Conclusions:
This study resulted in a LC50 (96 h) of 8.43 mg/L (geom. mean measured concentration) based on mortality of D. rerio (OECD 203, GLP).

Description of key information

LC50 (96 h): 8.43 mg/L based on mortality of D. rerio (OECD 203)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
8.43 mg/L

Additional information

One experimental study is available investigating the effects of the substance to freshwater fish. The study was performed according to OECD 203 (GLP) using Danio rerio as test organism under semi-static conditions. Nominal test concentrations of 2.00, 3.56, 6.33, 11.3 and 20.0 mg/L as well as a control were tested. The actual exposure concentrations were verified by GC/FID analysis at the beginning and after 24, 48 and 72 h of the study (fresh and aged media). Since the measured concentrations were < 80% of nominal, the effect concentrations were based on the geometric mean of the measured concentrations. After an exposure time of 96 h a LC50 of 8.43 mg/L resulted based on mortality.