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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:
27 June 2016 to 26 August 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control and nominal concentration of 12 mg/L
- 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: All samples were stored at 6 ± 2 °C until start of analysis, if necessary. Prepared samples were stored at room temperature until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: An appropriate volume of the test item was pipetted directly into a test vessel filled with an appropriate volume of dilution water. The test media was mixed with an ultraturrax (1 min, 12000 rpm).
- Controls: dilution water without test item
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): none, the test item was clearly dissolved throughout exposure.
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): not reported
- Length at study initiation (length definition, mean, range and SD): 2.76 cm (average)
- Weight at study initiation (mean and range, SD): 0.18 g (average)

ACCLIMATION
- Acclimation period: at least 12 days
- Type and amount of food during acclimation: Sera Vipan; Sera GmbH, 52518 Heinsberg, Germany. The amount of food was 4% of the fish body weight per feeding day.
- Feeding frequency during acclimation: 3 times per week, the test fish were not fed 24 h before test start.
- Health during acclimation (any mortality observed): No mortality was observed. No disease treatments were administered throughout holding and testing.

FEEDING DURING TEST - Fish not fed during the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
10 - 250 mg CaC03/L
measured: 74 mg CaC03/L (measured at Day 0)
Test temperature:
Water temperature in control (continuous monitoring): 22.2 (min) to 22.7 (max) °C, mean: 22.5 °C ± 0.1
Old medium: 22.6 - 22.9°C
New medium: 22.7 - 22.8°C
pH:
old medium: 7.33 - 7.65
new medium: 7.39 - 7.76
Dissolved oxygen:
old medium: 83 - 90 %
new medium: 94 - 100 %
Salinity:
N/A
Conductivity:
174 pS/cm (measured quarterly)
Nominal and measured concentrations:
first definitive study: 7.00 mg/L (nominal); 6.15 mg/L (mean measured)
second definitive study: 12.0 mg/L (nominal); 11.1 mg/L (mean measured)
Details on test conditions:
TEST SYSTEM
- Test vessel: glass aquaria
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: Glass aquaria of 13 L (dimensions: 25.5/18/28 cm), filled with approximately 10 L of dilution water and covered with a floating lid (aluminium foil).
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): daily renewal of the test media
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.13 g fish per litre test solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin filtered on activated charcoal and aerated for at least 24 hours to remove chlorine.
- Alkalinity: 0.60 mmol/L (measured quarterly)
- Culture medium different from test medium: no
- Intervals of water quality measurement: pH-value, temperature and oxygen saturation were measured in all test vessels at the beginning and at the end of the exposure and every 24 hours from fresh and old test media. Total hardness of the water measured at the beginning of the test.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 14 hours
- Light intensity: 7 - 750 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Observations were made after 4, 24, 48, 72 and 96 h for mortality (fish were considered dead if there was no visible movement and if touching of the caudal peduncle produced no reaction) and visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, etc.). For measurements of size and weight the control fish were used at the end of the exposure.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Limit test with only one concentration.
- Test concentrations: Two limit tests with nominal concentrations of 7 mg/L, 12 mg/L, corresponding to 6.15 mg/L and 11.1 mg/L, respectively
- Results used to determine the conditions for the definitive study: Other aquatic toxicity studies (algae and Daphnia) were used to select the test concentrations for the study.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
>= 11.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 11.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
> 11.1 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- Behavioural abnormalities: Yes, slow escape reflex was observed in fish at 6.15 mg/L and lethargy, tumbling, slow escape reflex and missing escape reflex were observed in fish at 11.1 mg/L.
- Mortality of control: no mortality in control observed
- Other adverse effects control: no adverse effects in control
- Abnormal responses: none observed
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The test item was clearly dissolved throughout exposure.
Sublethal observations / clinical signs:

Table 1: Observations in the First Definitive Test (n = 7)

Geometric mean measured test item concentration [mg/L]

Observation

 

Number of fish affected after different exposure periods (hours)

 

 

2

24

48

72

96

6.15

Normal behaviour

7

5

-

-

-

 

Slow escape reflex

-

7

7

7

7

Control

Normal behaviour

7

7

7

7

7

Table 2: Observations in the Second Definitive Test (n = 7)

Geometric mean measured test item concentration [mg/L]

Observation

 

Number of fish affected after different exposure periods (hours)

 

 

4

24

48

72

96

11.1

Lethargic

-

2

2

2

-

 

Tumbling

4

-

-

-

-

 

Slow escape reflex

-

5

5

5

7

 

Missing escape reflex

3

-

-

-

-

Control

Normal behaviour

7

7

7

7

7

Table 3: Cumulative Mortality [%] in the Second Definitive Test (n = 7)

Geometric mean measured test item concentration [mg/L]

Cumulative Mortality [%] after different exposure periods [hours]

                            

4

24

48

72

96

11.1

0

0

0

0

0

Control

0

0

0

0

0

 

Validity criteria fulfilled:
yes
Remarks:
oxygen saturation ≥ 83 %, no mortality in control, the pH value in the control did not deviate by more than one unit, measured concentrations in freshly prepared were in the range of 100 to 106% of the nominal value and 78 to 89% in old media.
Conclusions:
Based on the geometric mean concentration the 96-h LC50 to zebrafish was >11.1 mg/L.
Executive summary:

The acute toxicity of the test item to zebrafish was determined. This study is GLP compliant and follows OECD guideline 203 and EU method C.1. All validity criteria for the study were met. Therefore, it has been assigned a reliability score of 1 (reliable with no restrictions).

An acute toxicity test under semi-static conditions with daily renewal of the test media was conducted with the nominal limit test concentration of 12.0 mg/L corresponding to a geometric mean measured concentration of 11.1 mg/L. The test concentration was chosen based on the ErC50 from an algal study conducted with the substance. The duration of the test was 96 hours. Seven test organisms were exposed to the limit concentration and the control. Water quality parameters (temperature, pH-value and oxygen-saturation) were measured at 0, 24, 48, 72 and 96 hours were determined to be within the acceptable limits. The measured test item concentrations in freshly prepared solutions were in the range of 100 to 106% of the nominal value. In corresponding 24 h aged test media, the measured test item concentrations were between 78 to 89% of nominal. Therefore, all effect values are based on the geometric mean measured concentration of the test item. Based on the geometric mean concentration the 96-h LC50 to zebrafish was >11.1 mg/L.

Description of key information

Based on geometric mean measured concentrations of the test item, the 96h LC50 to zebrafish was > 11.1 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
11.1 mg/L

Additional information

The acute toxicity of the test item to zebrafish was determined (2016).

This study is GLP compliant and follows OECD guideline 203 and EU method C.1. All validity criteria for the study were met. Therefore, it has been assigned a reliability score of 1 (reliable with no restrictions).

An acute toxicity test under semi-static conditions with daily renewal of the test media was conducted with the nominal limit test concentration of 12.0 mg/L corresponding to a geometric mean measured concentration of 11.1 mg/L. The test concentration was chosen based on the ErC50 from an algal study conducted with the substance. The duration of the test was 96 hours. Seven test organisms were exposed to the limit concentration and the control.

The measured test item concentrations in freshly prepared were in the range of 100 to 106% of the nominal value and were between 78 to 89% of the nominal value in the aged media. Therefore, all effect values are based on the geometric mean measured concentration of the test item. An LC50 for fish of >11.1 mg/L was determined.