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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:
19 Nov - 27 Nov 2018
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
For further information see section "Any other information on material and methods incl tables".
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
GLP compliance:
yes (incl. certificate)
Remarks:
Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht Rheinland-Pfalz, Mainz, Germany
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Control and test item concentration
- Sampling method: Concentration was measured at the beginning and at the end of the test and on at medium renewal after 24 h in the new and the old solutions.
- Sample storage conditions before analysis: Samples were frozen
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Differential loading: A saturated solution was prepared for the test. This was done by weighing the nominal load of 100 mg/L, adding the corresponding amount of tap water and shaking vigorously for 24 h. The resulting solution was filtrated through 0.45 μm cellulose ester filters. On each day of the experiment, the necessary solutions were freshly prepared.
- Controls: Yes, medium only
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Source: Dehner Garten Center, Neustadt, Germany (date of arrival 05 Jul 2018)
- Length at study initiation: 2 ± 1 cm
- Method of breeding: Vessels: polyethylene aquaria; Medium: tap water; Feeding: two times a day with warmwater fishfood, totalling about 1-2% of body weight per day; Photoperiod 12/12 h; Temperature 23 ± 2 °C

ACCLIMATION
- Acclimation period: at least 12 days
- Acclimation conditions: same as test conditions
- Type and amount of food during acclimation: warmwater fishfood , totalling about 1 - 2% of body weight per day
- Feeding frequency during acclimation: three times a day
- Health during acclimation: mortality was observed after day 7 (3 of 18 animals were found dead -corresponding to 16.7%)

FEEDING DURING TEST: no feeding during the test; 24 h before the start of the test, the test fish were no longer fed.

Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
0.88 (medium) mmol/L
Test temperature:
23.1 – 24.0 °C (deviation within on test 1 °C)
pH:
7.8 - 7.9 (Control)
7.7 - 7.9 (100 mg/L)
Dissolved oxygen:
8.3 - 9.1 mg/L (Control)
8.2 - 9.1 mg/L (100 mg/L)
Conductivity:
210 µS/cm at 25 °C (tap water)
Nominal and measured concentrations:
nominal: Control and 100 mg/L
The measured concentrations are listed in section "Any other information on results incl tables" in table 4-5
Details on test conditions:
TEST SYSTEM
Test vessel:
- Type: Open
- Material, size, headspace, fill volume: glass, 10 L, headspace: 3 L, fill volume: 7 L
- Aeration: Accomplished with glass tubes
- Renewal rate of test solution: every 24 hours
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates):1
- Biomass loading rate: 1 fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap/Drinking water see analysis in table 1 in section "Any other information on material and methods incl tabels"
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Photoperiod: 12/12 hours using neon tubes

EFFECT PARAMETERS MEASURED: Mortality was recorded every 24 h

TEST CONCENTRATIONS
- Range finding study : No pre-test was performed
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
>= 32.23 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 32.23 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
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:
- Mortality of control: No mortality was observed
- Other biological observations: The toxicity against Danio rerio was tested using a semi-static test design. The study was performed as a limit test using the concentration 100 mg/L (nominal). In the treatment and the control, no mortality was observed.

Biological Results:

Table 1: pH-Values

Nominal Concentration in mg/L

pH at t in h (old/new test solution)

0 h

24 h

48 h

72 h

96 h

 

new

old

new

old

new

old

new

old

Blank control

7.8

7.8

7.8

7.8

7.9

7.8

7.8

7.8

100

7.7

7.8

7.8

7.9

7.9

7.8

7.8

7.8

 Table 2: O2 Values

Nominal Concentration in mg/L

O2 in mg/L at t in h (old/new test solution)

0 h

24 h

48 h

72 h

96 h

 

new

old

new

old

new

old

new

old

Blank control

9.1

8.7

8.6

8.5

9.0

8.7

9.0

8.3

100

8.9

8.6

8.6

8.4

9.1

8.6

9.0

8.2

 Tabel 3: Temperature

Nominal Concentration in mg/L

Temperature in °C (old/new test solution)

0 h

24 h

48 h

72 h

96 h

 

new

old

new

old

new

old

new

old

Blank control

24.0

23.3

23.4

23.1

23.3

23.2

23.1

23.2

100

23.3

23.1

23.3

23.2

23.3

23.4

23.2

23.3

 Table 4: Measured Concentrations

Nominal
Conc.

 Test Item

Measured

Conc.
 t = 0 h

Measured

Conc.

 t = 24 h

old solution

Measured

Conc.

 t = 24 h

new solution

Measured

Conc.

 t = 48 h

old solution

Measured

Conc.

 t = 48 h

new solution

mg/L

µg/L

µg/L

µg/L

µg/L

µg/L

Blank control

n. m.

--

--

--

--

100

n. m.

22.19

103.15

80.94

30.04

n. m. = not measured because of a technical defect of the originally planned LC-MS-MS

  Table 5: Measured Concentrations, continuance

Nominal
Conc.

 Test Item

Measured

Conc.

 t = 72 h

old solution

Measured

Conc.

 t = 72 h

new solution

Measured

Conc.

 t = 96 h

old solution

Geometric Mean

Measured

Conc.

additional new

solution

mg/L

µg/L

µg/L

µg/L

µg/L

µg/L

Blank control

--

--

n. d.

--

--

100

12.26

16.44

0.64*

32.23

94.83

*Implausible low measured concentration, an error in sampling was suspected, value excluded from evaluation.

n. d. = not detectable

Analytical Results:

At each measurement the recovery of the QC samples was taken into account because the recovery was not in the range of 97% - 103% of the nominal value. At the beginning the measurement was not yet stable. This can be seen from the area values of the QC sample. Deviating from the study plan, the recovery of QC samples was taken into account although the recovery was not in the range of 97% - 103% of the nominal value. This can be stated as uncritical because it was clear to see that the measurement stabilized later and an additional later measurement was therefore not necessary.

The measured concentration in the freshly prepared solutions was between 16.44 μg/L and 103.15 μg/L and in the old solutions between 0.64 μg/L and 80.94 μg/L. A sampling error is assumed as reason of the implausible low measured concentration of 0.64 μg/L at the end of the test. This value was excluded from evaluation. Reason for the scattering measured concentrations in the test solutions might be the presence of the fish and their excrements.

Table 6: 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 was observed

Yes

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

saturation value throughout the test

92%

Yes

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

Description of key information

LC50 (96h) >32.23 µg/L (Danio rerio, OECD 203, measured geometric mean)

Key value for chemical safety assessment

Additional information

One experimental study is available investigating the toxicity of 4-(octadecylamino)-4-oxoisocrotonic acid (CAS 3077-27-8) to fish. The study was conducted according to OECD 203 (GLP) using the zebrafish Danio rerio as test species (LAUS GmbH, 2018c) under semi-static conditions with daily medium renewal. A nominal test item concentration of 100 mg/L was tested in a limit test. The test solution was prepared by adding appropriate amounts of test item to test medium, followed by intensive stirring and filtration. During the study the actual concentrations in the test medium was measured by HPLC/MS. The measured concentration in the freshly prepared solutions was between 16.44 μg/L and 103.15 μg/L and in the aged test solutions between 0.64 μg/L and 80.94 μg/L, respectively. Therefore the results are given based on the geometric mean of the measured concentrations. Hence the LC50 (96 h) was determined to be > 100 mg/L (nominal) and > 32.23 μg/L (geom. mean measured concentration). The measured water solubility for the test item was determined to be 5.9 mg/L at 25 °C, pH 5. The discrepancy between the low recovery in the test solutions and the water solubility may have been caused by the different test media, pH-values and temperature.