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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
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:
Federal Office for the Environment, Bern, Switzerland
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control and 7 mg/L
- Sampling method: All samples were taken from the approximate center of each test vessel without mixing the test medium. Immediately after sampling, 10 mL methanol per 10 mL sample volume were added to each sample in order to stabilize the latter during the storage period.
- Sample storage conditions before analysis: All samples were stored deep-frozen (at about -20 °C). In pre-experiments for investigation of the storage stability of the samples, the test item proved to be stable under these storage conditions.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test medium of nominal 7.0 mg/L was prepared by weighing the test item into an appropriate volume of test water in the test vessel. The test item was completely dissolved by stirring for 30 min in the closed test vessel. No ultrasonic treatment was applied.
- Eluate: no
- Differential loading: yes
- Controls: yes, test medium control
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): No remarkable observations were made concerning the appearance of the test medium. It was a clear solution throughout the test medium renewal periods of 24 h.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebra fish
- Source: obtained from a breeding culture at the laboratory
- Length at study initiation (length definition, mean, range and SD): 2.3 ± 0.07 cm
- Weight at study initiation (mean and range, SD): 0.08 ± 0.01 g

ACCLIMATION
- Acclimation period: at least one week
- Acclimation conditions (same as test or not): same as test
- Feeding frequency during acclimation: During holding and acclimatization until one day before the start of the test, the fish were fed with a commercial fish diet (Tetra Min® Hauptfutter).
- Health during acclimation (any mortality observed): No mortality was recorded.

FEEDING DURING TEST
- None
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
125 mg/L as CaCO3
Test temperature:
20 - 21 °C
pH:
control: 7.2 - 7.3 (fresh solutions), 6.8 - 7.0 (aged solutions)
7.0 mg/L: 7.2 - 7.3 (fresh solutions), 6.8 - 7.0 (aged solutions)
Dissolved oxygen:
control: 7.3 - 7.5 mg O2/L (fresh solutions), 5.7 - 5.9 mg O2/L (aged solutions)
7.0 mg/L: 7.4 - 7.6 mg O2/L (fresh solutions), 5.8 - 6.0 mg O2/L (aged solutions)
Nominal and measured concentrations:
nominal: control, 7 mg/L
measured: < LOQ, 6.2 mg/L (mean measured concentration; calculated as the arithmetic mean of the four geometric means determined for the four 24 h test medium renewal periods)
Details on test conditions:
TEST SYSTEM
- Test vessel: 5 L aquaria without headspace
- Type (delete if not applicable): closed system
- Aeration: no aeration
- Renewal rate of test solution (frequency/flow rate): every 24 h
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.11 g wet weight/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: according to guideline (reconstituted test water)
- Alkalinity: 0.4 mmol/L
- Ca/mg ratio: 4:1
- Culture medium different from test medium: no, same as test
- Intervals of water quality measurement: The water temperature, pH values and oxygen concentrations were measured for each treatment at the start of the test and once daily during the test in the freshly prepared and aged test medium. At the same dates the appearance of the test medium was recorded. Additionally, the water temperature in the control was continuously recorded using a data logger.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod:16 h light/8 h darkness (30 min transition period)
- Light intensity: 16.5 μE s-1 m-2.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): mortality and visible abnormalities were recorded 3, 24, 48, 72 and 96 h

TEST CONCENTRATIONS
- Justification for using less concentrations than requested by guideline: A threshold approach as developed for chemical substances at the European Commission’s joint Research Centre was applied.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 6.2 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: arithmetic mean of four geometric means for four 24 h renewal periods
Details on results:
- Behavioural abnormalities: not recorded
- Mortality of control: 0%
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no, test solutions were clear and colorless
Sublethal observations / clinical signs:

Table 1: Analytical results of test samples

Sampling day / Sampling age [day/hours]

Nominal Concentration [mg/L]

Determined concentrations [mg/L]

% of nominal

0/0 (fresh)

Control

< LOQ

n.a.

7

6.98

100

1/24 (aged)

Control

< LOQ

n.a.

7

5.91

84

1/0 (fresh)

Control

< LOQ

n.a.

7

6.54

93

2/24 (fresh)

Control

< LOQ

n.a.

7

5.89

84

2/0 (fresh)

Control

< LOQ

n.a.

7

6.46

92

3/24 (aged)

Control

< LOQ

n.a.

7

5.99

86

3/0 (fresh)

Control

< LOQ

n.a.

7

6.38

91

4/24 (aged)

Control

< LOQ

n.a.

7

5.85

84

Table 2: Biological results

Nominal concentration [mg/L]

Mean measured concentration [mg/L]

Number of abnormal and dead fish / number of dead fish

3 h

24 h

48 h

72 h

96 h

Control

-

0/0

0/0

0/0

0/0

0/0

7.0

6.2

0/0

0/0

0/0

0/0

0/0

Validity criteria fulfilled:
yes
Conclusions:
The test item had no acute toxic effects on juvenile zebra fish (Danio rerio) up to the threshold concentration of 6.2 mg/L. According to the threshold approach, it was demonstrated that the test organism fish is not the most sensitive species for the test item and that the 96-hour LC50 for fish is greater than the
threshold concentration of 6.2 mg/L.

Description of key information

LC50 (96 h) > 6.2 mg/L (arithmetic mean measured concentration, Danio rerio, OECD 203)

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
6.2 mg/L

Additional information

One experimental study is available, in which the acute short-term toxicity of the test item to zebra fish (Danio rerio) was determined in a 96 -hour test according to GLP and OECD guideline 203.

A limit test was performed to demonstrate that the test organism fish is not the most sensitive test organism to the test item compared to algae and daphnia. The test method is based on the threshold approach as developed for chemical substances at the European Commission’s joint Research Centre. The selected treshold concentration of 7.0 mg/L corresponded approximately to the lower EC50 value determined in an algae study (OECD 201) and an acute daphnia study (OECD 202).

The lower EC50 value was determined in the algae test to be 1.6 mg/L, based on growth rate.

As the test item is a volatile substance, the test was performed in closed glass vessels (closed system without head space) completely filled with test medium, to avoid potential losses of test item by evaporation. A semi-static test design with daily water renewal was chosen to keep the concentration of the test item as constant as possible during the exposure period and to provide sufficient oxygen saturation in the closed system.

During the test medium renewal period of 24 hours the test item concentration slightly decreased. At the end of the test medium renewal periods, 84 to 86% of the nominal concentration was found.

Due to the decrease of test item concentration, the biological results were based on the mean measured concentration of 6.2 mg/L (calculated as the arithmetic mean of the four geometric means determined for the four 24-hour test medium renewal periods). During the 96-hour exposure period, no death and visible toxic signs were observed in the control group and the exposed group. No behavioral abnormalities were recorded. Based on the arithmetic mean measured concentrations of the test item, the LC50 (96 h) was > 6.2 mg/L.