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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:
from 2016-11-23 to 2017-06-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
Each test concentration and the control were analytically verified daily from all freshly prepared and aged media.
Vehicle:
no
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Source: gained at the testing facility from a sigle brood stock
- Length at study initiation (average): 2.77 cm
- Weight at study initiation (average): 0.26 g

ACCLIMATION
- Acclimation period: at least 12 days
- Acclimation conditions: at 23 ± 2 °C and diffuse light (7 - 750 Lux, natural photoperiod). The water was changed at least once per week. The dissolved oxygen concentration was more than 80 % of the air saturation value.
- Type and amount of food during acclimation: Sera Vipan. The amount of food was 4 % of the fish body weight per feeding day.
- Feeding frequency during acclimation: Food was provided 3 times per week. The test fish were not be fed 24 h before test start.
- Health during acclimation: No mortality was observed during this time. No disease treatments were administered throughout holding and testing.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
10 - 250 mg CaC03/L
Test temperature:
22.8 ± 0.6 °C
pH:
6.0 - 8.5
Dissolved oxygen:
>=71%
Salinity:
NA
Conductivity:
181 pS/cm
Nominal and measured concentrations:
Nominal concentrations: 0.500 - 1.00 - 2.00 - 4.00 - 8.00 mg/L
Measured concentrations: 0.266 - 0.498 - 1.10 - 2.41 - 4.79 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: aquaria
- Type: closed, covered with floating lid
- Material, size, headspace, fill volume: glass, no headspace, total volume 13 L, fill volume 10 L
- Aeration: no aeration was provided
- Renewal rate of test solution: daily renewal
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.18 g fish per litre test solution

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Tap water of local origin
- Chlorine: Chlorine was removed with aeration for at least 24 h.
- Alkalinity: 1.00 mmol/L
- Acidity: 0.20 mmol/L
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: not performed
- Photoperiod: A daily 16 hours photoperiod was maintained throughout exposure.
- Light intensity: 7 - 750 lux

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study:
- Results used to determine the conditions for the definitive study: Yes
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
3.59 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
2.41 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.:
> 4.79 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Reported statistics and error estimates:
The LC50-values were calculated by straight line regression. The NOEC and LOEC were provided based on observation of the test organisms, including sub lethal findings, without any statistical calculations. The following software was used for calculations: Microsoft Excel and GraphPad Prism.
Sublethal observations / clinical signs:

Measured Exposure Concentrations

The measured concentrations at the start of all exposure intervals after 0 to 72 hours were between 49 and 119 % of the nominal values. At the end of all exposure intervals after 24 to 96 hours the measured concentrations of the test item were between 31 to 53 %. Since the measured test item concentrations were not within the range of ± 20% of the nominal concentrations, the geometric mean measured concentrations were calculated.

 

All effect levels are given based on the geometric mean measured concentrations of the test item after 96 hours.The LC50 after 96 hours was 3.59 (2.41 - 4.79) mg test item/L.

 

Table 1: Observations in the Definitive Test

(n = 7)

Geometric mean measured test item concentrations [mg/L]

Observation *)

Number of fish effected after different exposure periods [hours]

2

24

48

72

96

4.79

(E)

(4.1)

(2.4)

(2.2)

(2.1)

(1)

0

0

0

0

0

7

1

0

6

0

0

0

3

0

0

0

3

0

2

1

0

0

0

0

0

1

0

1

0

0

2.41

(2.4)

(1)

0

7

0

7

7

0

7

0

7

0

1.10

(1)

7

7

7

7

7

0.498

(1)

7

7

7

7

7

0.266

(1)

7

7

7

7

7

Control

(1)

7

7

7

7

7

 

*) The numbers in brackets correspond to the following observations:

(1) = Normal behaviour

(2.1) = Lethargy

(2.2) = Fish is lying on its side

(2.4) = Slow escape reflex

(4.1) = Hyperventilation

(E) = Exitus lethalis

 

Table 2: Cumulative Mortality [%] in the Definitive Test

(n = 7)

Geometric mean measured test item concentrations [mg/L]

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

2

24

48

72

96

4.79

0

14

57

86

86

2.41

0

0

0

0

0

1.10

0

0

0

0

0

0.498

0

0

0

0

0

0.266

0

0

0

0

0

Control

0

0

0

0

0

 

Validity criteria fulfilled:
yes
Conclusions:
The 96h-LC50 of the test item was determined to be 3.59 mg/L (geometric mean measured test item concentration).
Executive summary:

The acute toxicity of the test item to zebrafish was determined according to the principles of the OECD-Guideline for Testing of Chemicals No. 203 (1992) and EU Council Regulation (EC) No. 440/2008/ Method C.1 (2008). An acute toxicity test under semi-static conditions with daily renewal of the test media was conducted with the nominal test item concentrations of 0.500 - 1.00 - 2.00 - 4.00 - 8.00 mg/L (factor 2) corresponding to geometric mean measured concentrations of 0.266 - 0.498 - 1.10 - 2.41 - 4.79 mg/L. Seven test organisms were exposed to each test concentration and the control. The determination of the test concentrations was carried out via HPLC-DAD on a reversed phase column in isocratic mode using external standard calibration. The measured concentrations at the start of all exposure intervals after 0 to 72 hours were between 49 and 119 % of the nominal values. At the end of all exposure intervals after 24 to 96 hours the measured concentrations of the test item were between 31 to 53 %. Therefore, all effect values are based on the geometric mean measured concentrations of the test item. The 96-h LC50-values was determined to be 3.59 mg/L (geom. mean measured concentration).

Description of key information

In a 96-h semi-static toxicity test with Zebrafish the LC50 value based on mobility of the test substance was found to be 3.59 mg/L (geometric mean measured test item concentration).

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
3.59 mg/L

Additional information

The acute toxicity of the test item to zebrafish was determined according to the principles of the OECD-Guideline for Testing of Chemicals No. 203 (1992) and EU Council Regulation (EC) No. 440/2008/ Method C.1 (2008). An acute toxicity test under semi-static conditions with daily renewal of the test media was conducted with the nominal test item concentrations of 0.500 - 1.00 - 2.00 - 4.00 - 8.00 mg/L (factor 2) corresponding to geometric mean measured concentrations of 0.266 - 0.498 - 1.10 - 2.41 - 4.79 mg/L. Seven test organisms were exposed to each test concentration and the control. The determination of the test concentrations was carried out via HPLC-DAD on a reversed phase column in isocratic mode using external standard calibration. The measured concentrations at the start of all exposure intervals after 0 to 72 hours were between 49 and 119 % of the nominal values. At the end of all exposure intervals after 24 to 96 hours the measured concentrations of the test item were between 31 to 53 %. Therefore, all effect values are based on the geometric mean measured concentrations of the test item. The 96-h LC50-values was determined to be 3.59 mg/L (geom. mean measured concentration).