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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:
15 Mar - 19 Apr 2019
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
adopted 17. July 1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
adopted 30. May 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test concentrations at the beginning and end of the test and at medium renewal (every 24 h) in the new and old test solutions. Samples of the control were taken at test start.
- Sample preparation: To a 100 mL of a solution in matrix 6 g NaCl is added, then, the solution was extracted two times with the solvent dichloromethane (2 * 4 mL), the organic phase was collected into a 10 mL flask, after drying with Na2SO4. The flask was filled up to 10 mL with dichloro-methane after addition of 100 µL ISTD stock solution (100 mg/L) and the solution was measured via GC/MSD. Tenfold enrichment was achieved during extraction procedure.
- Storage: All samples were analysed directly after sampling.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: To homogenize the test item, it was melted at 40 °C before use. A WAF was prepared by weighing the nominal load of 6 mg/L, adding the corresponding amount of tap water and thoroughly homogenised by stirring for 24 ±1 h. The resulting solution is filtrated through 0.45 µm cellulose ester filters. During membrane filtration of the water-accommodated fraction (WAF), the filters were renewed after filtration of 500 mL solution. Because the test item is not stable under test conditions, the study was performed under semi-static conditions. For medium renewal after 1, 2- and 3-day(s), fresh test solutions were prepared.
- Controls: Medium without test item
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Source: UBA (Umweltbundesamt, Dessau, Germany) and Dehner Garten Center, 67433 Neustadt, Germany and combined with the offspring of the original animals to one batch.
- Age at study initiation: sexually immature young fish, the offspring of the original animals is used in the test.
- Length at study initiation: 2 ± 1 cm
- Method of breeding: Photo period: 12/12 h, using neon tubes; Temperature: 23 ± 2 °C; Medium: tap water;

ACCLIMATION
- Acclimation period: Before being used for the test, the fish were kept for at least 12 days under test conditions.
- Acclimation conditions (same as test or not): same
- Type and amount of food during acclimation: Warmwater fishfood
- Feeding frequency during acclimation: three times per working day
- Health during acclimation (any mortality observed): During 7 days, no mortality occurred.

FEEDING DURING TEST : no
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
0.88 mmol/L
Test temperature:
23.2 - 23.8 °C (Control)
23.1 - 24.4 °C (Test item concentration)
pH:
8.1 - 8.0 (Control)
7.9 - 8.1 (Test item concentration)
Dissolved oxygen:
7.4 - 9.2 mg/L (Control)
7.7 - 9.1 mg/L (Test item concentration)
Conductivity:
210 µS/cm at 25 °C
Nominal and measured concentrations:
Control and 6 mg/L (nominal)
0.536 (measured concentration)
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type: open
- Material, size, headspace, fill volume: glass aquaria, maximal volume 10 L, 7 L test solution, 3 L headspace
- Aeration: Accomplished with glass tubes
- Renewal rate of test solution (frequency/flow rate): every 24 h
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1 vessel
- No. of vessels per control (replicates): 7
- Biomass loading rate: 0.26 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap water
- Total organic carbon: <0.5 mg/L
- Polycyclic aromatic hydrocarbons: <0.008 µg/L
- Others: Sodium 4.4mg/L, potassium 2.5 mg/L, calcium 32 mg/L, magnesium 2.1 mg/L, aluminium 0.007 mg/L, iron <0.005 mg/L, manganese <0.005 mg/L, ammonium <0.05 mg/L, nitrate 1.4 mg/L, nitrite <0.01 mg/L, chloride 9.2 mg/L, sulphate 10 mg/L, antimony <0.001 mg/L, arsenic <0.001 mg/L, lead <0.001 mg/L, cadmium <0.0001 mg/L, chromium <0.0005 mg/L, copper 0.002 mg/L, nickel <0.001 mg/L, mercury <0.0001 mg/L, selenium <0.001 mg/L, bor <0.01 mg/L, cyanide <0.005 mg/L, fluoride <0.1 mg/L, benzene <0.1 µg/L
- Pesticides and biocides: < LOD mg/L
- Chlorine: chlorinated organic compounds < 1 µg/L
- Culture medium different from test medium: No
- Intervals of quality measurements: Observations were made every 24 h, measuring pH and O2 concentration of the old and new test solution in each vessel and documenting mortalities.

OTHER TEST CONDITIONS
- Photoperiod: 12/12 h
- Light intensity: using neon tubes

EFFECT PARAMETERS MEASURED: Mortality (A fish was considered dead, if no visible movement could be observed, and if touching of the caudal peduncle produced no reaction).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: only one test item concentration
- Range-finder: No Range-finding test was performed
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.536 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 0.536 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- Behavioural abnormalities: No
- Mortality of control: No
Reported statistics and error estimates:
No mortality was observed so no statistical evaluation was performed.
Sublethal observations / clinical signs:

Table 1: Analytical Results: Measured concentrations (purity 26.6 % component 1)

Nominal Concentration Test Item

Measured

Concentration
 t = 0 h

Measured

Concentration t = 24 h old

Measured

Concentration t = 24 h new

Measured

Concentration t = 48 h old

Measured

Concentration t = 48 h new

Measured

Concentration t = 72 h old

Measured

Concentration
t = 72 h new

Measured

Concentration
 t = 96 h old

mg/L

mg/L

mg/L

mg/L

mg/L

mg/L

mg/L

mg/L

mg/L

Blank control

< LOD

--

--

--

--

--

--

--

6

0.478

< LOD

0.591

< LOD

0.556

< LOD

0.518

--

LOD = 0.0584 mg/L (based on the lowest calibration level 0.584 and enrichment factor 10)

Table 2: Biological Results: Mortality

Nominal Conc. in mg/L

Total number of fish used

Number of dead fish

24 h

48 h

72 h

96 h

Blank control

7

0

0

0

0

6

7

0

0

0

0

Neither in the treatment nor in the control any mortality or any sign of abnormal behaviour could be observed.

Table 3: Validity criteria

Criterion from the guideline

Outcome

Validity criterion fulfilled

The mortality in the control(s) should not exceed 10% (or one fish if less than ten are used) at the end of the test

No mortality occured

yes

The dissolved oxygen concentration must have been at least 60 per cent of the air

saturation value throughout the test

87.1 %

yes

 

Validity criteria fulfilled:
yes
Remarks:
For further details please refer to “Any other information on results incl. tables”.

Description of key information

LC50 (96 h): >0.536 mg/L (Danio rerio, OECD 203, Mean measured, Mortality)

Key value for chemical safety assessment

Additional information

One experimental study is available investigating the toxicity of the substance to fish. The study was conducted according to OECD 203 (GLP) using the zebrafish Danio rerio as test species. The test was performed under semi-static conditions at a threshold concentration of 6 mg/L (nominal). The threshhold concentration is based the results of the available experimental data on the acute toxicity to aquatic invertebrates and algae. Due to the low water solubility the test item concentrations were prepared via water-accommodated fractions: 6 mg/L (real load between 6.1 and 6.3 mg/L), adding the corresponding amount of dilution water (tap water) and shaking vigorously for 24 ±1 hours. The resulting solutions were filtrated through 0.45 µm cellulose ester filters. Since the test item was not stable, fresh test solutions were prepared for medium renewal after 1, 2- and 3-day(s). Analytical measurement was done by GC/MSD. The test item consists of two components, ethylene glycol monolaurate (component 1) and ethylene glycol dilaurate (component 2), but only one (component 1) is water soluble. The quantification of the test item was performed using the signal of component 1 only. Due to the test items properties (insufficient stability and adsorbing property), the measured concentration in the aged solution was < LOD for all test item concentrations. Therefore, evaluation was based on the mean measured concentrations in the freshly prepared test solutions. No effects were observed at the mean measured threshold concentration of 0.536 mg/L. Based on these results the fish is not the most sensitive species.