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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:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
The concentrations of the test item were determined at the start (0 h, and 72 h) and at the end of the exposure intervals (24 h and 96 h) of the tested concentration levels and the control via HPLC-DAD.

PREPARATION OF STANDARDS: A stock solution of 100 mg/L of the test item was prepared in acetonitrile and diluted to 2.0 mg/L with acetonitrile : HPLC water (50 : 50). This solution was further diluted with acetonitrile : HPLC water (50 : 50) and used for calibration (6 concentrations).

PREPARATIONS OF SAMPLES: The test concentrations of 0.625 and 1.25 mg/L were measured undiluted. The test concentrations of 2.50 to 10.0 mg/L and control were diluted wit acetonitrile.

PREPARATION OF FORTIFIED SAMPLES:The fortified samples of 1 x LOQ and blank samples were Samples analysed without dilution. The 50 x LOQ was diluted with acetonitrile (factor 2) and further diluted with acetonitrile : HPLC water (50 : 50; total factor 16).

SAMPLE STORAGE: samples stored in the autosampler at room temperature until analysis. (If necessary, storage at 6 +-2°C).
Vehicle:
no
Details on test solutions:
TEST METHOD: semi-static test with daily renewal of the test media. The test aquaria were covered with a floating lid (i.e. with aluminium foil) to reduce the loss of the test item via the surface.

TEST CONCENTRATIONS: 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L. The concentrations are based on a stability test and a preliminary range finding test

PREPARATION OF TEST MEDIUM: Amounts of test item were weighted and transferred to aquaria filled with appropriate volume of dilution water. The media was mixed via Ultraturrax (1min, 17000 rpm). Thereafte, the test media were covered by floating lid. This procedure was repeatd for each renewal day.

CONTROL: 7 fish in diluation water tested under the same test conditions as the test replicates

TEST VESSELS: Glass aquaria of 13 L filled with 10 L of dilution water covered with floating lid

TEST VOLUME: about 10 L per vessel

AERATION: no aeration was provided in the test vessels
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
- Species: Danio rerio (zebrafish), Gnathostoma, Pisces, Osteichthyes, Teleostei, Cypriniformes, Cyprinidae
- Source: All fish used in the test was gained at test facility from a single brood stock (supplier: Umweltbundesamt, Berlin, Germany)
- Holding: 23 ± 2 °C, diffuse light, water was changed at least 1x per week. DOC > 80 % of the air saturation value.
- Acclimatisation: at least 12 days
- Feeding: 3 times per week, 4% of the fish body weight per feeding day, Food: Sera Vipan; Sera GmbH, 52518 Heinsberg, Germany
- Water: Tap water (local origin), water was filtered on activated charcoal and aerated for at least 24 hours to remove chlorine.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
66 mg CaCO3/L
Test temperature:
21.7 - 22.8 °C
pH:
7.0 - 7.5
Dissolved oxygen:
91 - 100%
Nominal and measured concentrations:
Nominal concentrations : 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L
Measured concentrations: 0.529 - 1.07 - 2.27 - 4.55 - 9.58 mg/L.
Details on test conditions:
TEST METHOD: Semi-static with daily renewal of the test media, the test aquaria were covered with a floating lid (aluminium foil) to avoid the loss of the test item via the surface.

TEST SYSTEM
- Test vessel: Glass aquaria of 13 L filled with 10L of dilution water
- Type: closed: covered with a floating lid (aluminium), to reduce the loss of the test substance.
- Aeration: No aeration in the test vessels.
- No. of fish: 7 zebrafish for each concentration and control. For all study 51 zebrafish used
- Replicates: 1 replicate/test concentration and control
- Introduction of fish: Fish introduced randomly to individual replicates
- Feeding of the fish: fish were not fed 24 h before the test and during the test
- Duration: 96 hours

OTHER TEST CONDITIONS
- Photoperiod: a daily 8 hours photoperiod
- Light intensity: 0.1 - 10 µmol photons . m-2 . s-1 (corresponding to approximetaly 7 - 750 Lux)
- Water temperature: 23 +- 2°C
- Dissolved oxygen concentration: not less than 60% air saturation value

EFFECT PARAMETERS MEASURED:
- Observations intervals: 2, 24, 48, 72 and 96 hours
- Criteria of effects: Fish considered died if there was no visibe movement and if touching of the caudal peduncle produced no reaction. Record were kept of visible abnormalities (e.g. loss of equilibrium, swimming behaviour, respiratory function, pigmentation, ect.)
- Measurement of fish size: for measurement of size and weight the control fish were used at the end of the exposure

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Stability test: yes
- Range finding study: yes
- Nominal test concentrations: 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L
- The concentrations based on a stability test and a preliminary range finding test
- Control: 7 fishes in dilution water (without test item) tested under the same test conditions as the test replicates.

LOADING, LENGHT OF FISH:
- Fish density: in test vessels : 0.128g fish/L test solution.
- Average body length at test end: 2.47 cm
- Average body weight at test end: 0.183g
Reference substance (positive control):
no
Remarks:
No reference item is recommended for this test according to the guidelines
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
4.55 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
3.21 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
2.27 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality

1. Measured Exposure Concentration during the Definitive:

Sampling date 2015-11-30
0h
(Fresh medium)
2015-12-01
24h
(Old medium)
2015-12-03
72h
(Fresh medium)
2015-12-04
96h
(Old medium)
 
Start of analysis 30/11/15 01/12/15 03/12/15 04/12/15  
Nominal concentration of the test item (mg/L) Meas. Conc. (mg/L) % Meas. Conc. (mg/L) % Meas. Conc. (mg/L) % Meas. Conc. (mg/L) % Geometric Mean Measured test item concentration (mg/L)
10.0 10.2 102 9.00 90 - - -   9.58
5.00 5.07 101 4.09 82 - - -   4.55
2.50 2.56 102 1.89 76 2.53 101 2.18 87 2.27
1.25 1.20 96 0.89 71 1.22 98 0.990 79 1.07
0.625 0.595 95 0.454 73 0.595 95 0.486 79 0.529
Control < LOQ < LOQ < LOQ < LOQ < LOQ

Meas. conc.= Measured concentration of the test item, dilution factor taken into account

%= Percent of nominal concentration of the test item

LOQ: Limit of quantification of analytical method (0.4 mg/L test item)

2. Results of Stability test :

Measured concentration and percent of nominal concentrations in the Stability test

Sampling time 0h 24h 48h 7d
Nominal test item concentration (mg/L) Meas. Conc. (mg/L) % Meas. Conc. (mg/L) % Meas. Conc. (mg/L) % Meas. Conc. (mg/L) %
10.0 9.16 92 8.88 97* 8.47 92* 1.03 11*

Meas. conc.= Measured concentration of the test item, dilution factor taken into account

%= Percent of nominal concentration of the test item

*= Based on initial measured concentration

3. Results of Range finding study:

Biological data: in the preliminary test the test concentration decreased more than expected, maybe due to the presence of the fish. Therefore, the definitive study was carried out with daily renewal of the test media.

3.1. Cumulative mortality (%) in the Preliminary test (n=3)

Nominal Test item concentration (mg/L) Cumulative mortality (%)
after different exposure periods (hours)
24h 48h 72h 96h
10.0 100 - - -
1.00 0 0 0 0
Control 0 0 0 0

3.2. Observation in the Test Vessels in the Preliminary Test (n=3)

Nominal test item concentration (mg/L) Observations * Number of fish affected after different exposure periods (hours)
24h 48h 72h 96h
10.0 ( E ) 3 - - -
1.00 ( 1 ) 3 3 3 3
Control ( 1 ) 3 3 3 3

(E)= Exitus lethalis

(1)= Normal behaviour

- = No observations due to 100% mortality

4. Observation in the definitive test: (n=7)

Geometric mean measured concentration
 (mg/L)
Observation Number of fish effected after different exposure periods
 (hours)
2h 24h 48h 72h 96h
9.58 Exitus lethalis 0 7 - - -
hyperventilation 7 - - - -
Fish is lying on its side 7 - - - -
4.55 Exitus lethalis 0 2 4 1 -
hyperventilation 0 0 1 0 -
Missing escape reflex 7 0 0 0 -
Fish is lying on its side 0 0 1 0 -
Lethargy 0 5 0 0 -
2.27 Missing escape reflex 0 7 7 7 7
Normal behaviour 7 0 0 0 0
0.529 Normal behaviour 7 7 7 7 7
Control Normal behaviour 7 7 7 7 7

5. Cumulative Mortality (%) in the definitive test:

Geometric mean measured concentration (mg/L) Cumulative Mortality (%) after different exposure periods (hours)            
2h 24h 48h 72h 96h
9.58 0 100 100 100 100
4.55 0 29 86 100 100
2.27 0 0 0 0 0
1.07 0 0 0 0 0
0.529 0 0 0 0 0
Control 0 0 0 0 0

Validity criteria:

- O2 saturation ≥ 60% (this study ≥ 90%)

- Mortality in the control ≤ 1/7 fish (this study 0/7 fish)

- the pH value in the control group did not deviate by more than one until over the test period.

- The recovery rates at the start of the first exposure interval was between 95 and 102% for the test item of the nominal value. At the end of the first exposure interval, the recovery rates were between 71 and 90 %. The recovery rates at the start of the second exposure interval were between 95 and 101 % of the nominal values. At the end of the second exposure interval, the recovery rates were between 78 and 87 %. Therefore, all effect values were based on the geometric mean measured concentrations of the test item, respectively.

Validity criteria fulfilled:
yes
Conclusions:
The LC50 after 96 hours was 3.21 mg/L (geometric mean measured concentration).
Executive summary:

In the current study the acute toxicity of the test item to zebrafish was assessed according to the OECD guideline 203 and EU Method C.1 (2008) under GLP without significant deviations.

The acute toxicity test was performed under semi-static conditions with daily renewal of the test media with the nominal test item concentration of 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L corresponding to geometric mean measured concentrations of 0.529 - 1.07 - 2.27 - 4.55 - 9.58 mg/L. The duration of the test was 96 hours and 7 test organisms were exposed to each test concentration and the control. The determination of the test concentration was carried out via HPLC-DAD. All validity criteria of the test guideline were met.

The test substance was found to cause lethal effects to zebrafish after 96 hours at a geometric mean measured concentration of 4.55 mg test item/L (LC100) and higher.

The LC50 after 96 hours was 3.21 mg test item/L (2.27 - 4.55 mg/L) (geometric mean measured concentration).

The LC0 after 96 hours was 2.27 mg/L (geometric mean measured concentration).

Description of key information

The test substance has a LC50 for freshwater fish (96 h) of 3.21 mg/L.

Key value for chemical safety assessment

LC50 for freshwater fish:
3.21 mg/L

Additional information

There is one study available assessing the acute toxicity of the test item to zebrafish. In this study the test was performed according to the OECD guideline 203 and EU Method C.1 under GLP without significant deviations.

The acute toxicity test was performed under semi-static conditions and the test media was renewed daily. 7 test organisms were exposed for 96 hours to 0.625 - 1.25 - 2.50 - 5.00 - 10.0 mg/L (nominal concentrations), which correspond to geometric mean measured concentrations of 0.529 - 1.07 - 2.27 - 4.55 - 9.58 mg/L.

All validity criteria of the test guideline were met.

The test substance was found to cause lethal effects to zebrafish after 96 hours at a geometric mean measured concentration of 4.55 mg test item/L (LC100) and higher. The LC50 after 96 hours was 3.21 mg test item/L (2.27 - 4.55 mg/L) (geometric mean measured concentration).