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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:
2014-05-14 to 2014-05-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This study was performed according to OECD Guideline 203 and EU Method C.1 with GLP compliance. All validity criteria were fulfilled and no deviations were observed.
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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
inspected on September 14, 2010/ signed on February 07, 2011)
Specific details on test material used for the study:
No additional information
Analytical monitoring:
yes
Details on sampling:
- Prior to the proposed test start, the flow through system was equilibrated and samples from all test vessels were taken for chemical analysis. Samples were immediately transferred to the chemical analysis laboratory and measured. A retain sample was stored at ≤-18 °C if an immediate measurement was not possible. If the measured test concentrations did not meet acceptable values of nominal concentrations, the dosing system was re-adjusted. After a further equilibration phase for 24 h with a 5x total exchange of test media, again samples from all test vessels were taken and the procedure was repeated as stated above.
- The experimental phase was started by introducing the test animals after measured test concentrations reached an acceptable range (± 20 % of nominal). During the study, samples of the test media were taken each day.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- For running the flow through system, two stock solutions with nominal concentrations of 2.7 mg/L and 10.1 mg/L were prepared. The density of test item (0.902 g/mL) was accounted for by pipetting appropriate volumes of test item into the dilution water (e.g. 1.11 mL corresponds to 1 g of test item). The stock solution with a nominal concentration of 10.1 mg/L was matching the water solubility of the substance. The stock solutions were prepared by solving appropriate amounts of the test item in dilution water under stirring at room temperature for 24 h. These solutions served as application solutions in the flow through device. To achieve the final concentration in the test vessels, the application solutions were mixed with dilution water in adequate volumes via the dosing pumps.
- Controls: Dilution water only
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebra fish
- Source: Test facility bred; Origin of the used strain of zebrafish: West Aquarium GmbH, 37431 Bad Lauterberg, Germany.
- Size: 2.0 ± 1 cm
- Length at study initiation: 2.4–2.9 cm
- Weight at study initiation: 0.203 ± 0.042

ACCLIMATION
- Acclimation period: 12 days
- Acclimation conditions (same as test or not): The fish were held in water of the same quality as used in the test (purified drinking water) until the start of exposure. Test fish were held for at least 12 days prior to the test under equivalent water quality and illumination conditions to those proposed for use in the test.
- Type and amount of food: Fish were fed ad libitum throughout the holding period with live brine shrimp (Artemia salina) nauplii and ground flake food TetraMin® (Tetra Werke, Melle, Germany).
- Feeding frequency: Once daily
- Health during acclimation (any mortality observed): Only healthy fish without diseases and abnormalities were used in the study. Mortality of the batch was less than 5 % in the week preceding the start of the study.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
Total hardness: 1.1 mmol/L
Test temperature:
23 ± 2 °C; The temperature was in the range of 23.1 and 23.3 °C
pH:
7.85-8.42
Dissolved oxygen:
Oxygen saturation in all test vessels was between 93 and 98 %
Salinity:
No data
Nominal and measured concentrations:
- Nominal test concentrations: 0.313, 0.625, 1.25, 2.50 and 5.00 mg/L
- Mean values of the measured concentrations were calculated to be 0.35, 0.69, 1.74, 2.70 and 5.55 mg/L (111.6, 110.8, 138.8, 108.2 and 111.0 % of nominal concentrations).
Details on test conditions:
TEST SYSTEM
- Test vessel: Test vessels were full glass aquaria of 12 L, containing 10 liters of test solution.
- Aeration: Yes
- Flow-through system: For each vessel, an individual dosage system was used. Dilution water was pumped by a water dosage pump (membrane pump, Prominent, Heidelberg, Germany) into a mixing chamber, placed on a magnetic stirrer. An adequate amount of the stock solution was added into the mixing chamber via a stock solution dosage pump (membrane pump with a stainless steel head, Prominent, Heidelberg, Germany). The prepared test solution with the desired test concentrations flows into the test vessels via flexible tubes. The daily water exchange rate was 5 volumes of the test vessel and flow rate of the dosage pumps was adjusted accordingly. The flow-through system was served by test solutions for eight days before adding the fish. The test animals were introduced in the test vessels when the measured concentrations of the test item achieved an acceptable range.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: Based on the mean weight of 7 fish, a loading of 0.14 g fish/L test medium was calculated.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Purified tap water was used according to the OECD-Guideline 203. The purification included filtration with activated charcoal, passage through a lime-stone column and aeration. The water was aerated to the level of oxygen saturation. The water chemistry data were recorded throughout the study (see table 6.1.1/1 for details)
- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Photoperiod: Test vessels were subjected to a light/dark cycle of 12/12 hours.
- Temperature, pH and oxygen concentration of the water were measured in each vessel directly before adding the fish and afterwards once daily.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Fish size at test start
- Observations on clinical signs and mortality at 3, 24, 48, 72 and 96 h

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Range finding study: Based on existing data for acute fish toxicity for zebrafish (LC50=2.5 mg/L; nominal/measured concentrations), determined within the scope of a pre-test for a BCF study (information provided by the sponsor) five test concentrations of test material were chosen. The highest concentration was limited by the water solubility of the test substance and was 5.0 mg/L. A spacing factor of 2 was applied, resulting in the four additional concentrations of 2.5 mg/L, 1.25 mg/L, 0.625 mg/L, and 0.312 mg/L, plus a water control. This range finding test was already performed under GLP conditions. Since the obtained data were suitable for the determination of a LC50 value, the range finding test served as definite study.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL could not be determined
Duration:
96 h
Dose descriptor:
other: The highest concentration without observed effect
Effect conc.:
0.69 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Clinical signs were observed after 3 h at 2.70, and at 5.55 mg/L (mean measured). After 24 h, treatment with these two concentrations resulted in 100 % mortality.
- Furthermore, clinical signs were observed after 24 h and 48 h, and all fish were found dead after 72 h at a treatment concentration of 1.74 mg/L.
- The LC50 after 96 h was calculated to be 0.89 mg/L for nominal concentrations, and 1.10 mg/L, based on mean measured concentrations.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
Data were statistically analyzed to determine LC10 and LC50 values together with 95 % confidence intervals using Probit-analysis, assuming log-normal distribution of the values. The evaluation of the effects was based on nominal as well as on the mean measured concentrations of the test item. The computer program ToxRat Professional 2.10 was used for statistical evaluations.

Table 6.1.1/2: Clinical signs and mortality during the test period of 96 h

Concentrations

Nominal concentration [mg/L]

Control

0.31

0.63

1.25

2.50

5.00

Mean measured concentration [mg/L]

Control

0.35

0.69

1.74

2.70

5.55

Fish introduced

7

7

7

7

7

7

h of application

Clinical signs [n]

3

nd

nd

nd

nd

7in, 6gr, 5da

7un, 7gr, 7gr, 7da

24

nd

nd

nd

7re, 5su, 7gr, **

-

-

48

nd

nd

nd

3re, 3su, 3gr, **

-

-

72

nd

nd

nd

-

-

-

96

nd

nd

nd

-

-

-

Cumulative mortality [n]

3

0

0

0

0

0

0

24

0

0

0

0

7

7

48

0

0

0

4

7

7

72

0

0

0

7

7

7

96

0

0

0

7

7

7

 

nd.: no symptoms detected; in: inactive; gr: mainly on the ground; da: darker color than the control

un: uncoordinated; su: in a supine position; re: reduced respiratory rate; **: symptoms of anaesthesia

-: 100 % mortality; no further clinical signs detected; For explanation: ‘6gr’ implies 6 fishes (out of 7) were mainly on the ground.

Validity criteria fulfilled:
yes
Conclusions:
Under the test conditions, the 96-hour LC50 for test item was 1.10 mg/L.
Executive summary:

The acute toxicity of the test item to zebra fish (Danio rerio) was conducted according to OECD guideline 203.

Test item was tested at five nominal concentrations of 0.31, 0.63, 1.25, 2.50 and 5.00 mg/L under flow through conditions for 96 h. Untreated dilution water was run in parallel as a control. Seven fish each were used at the test concentrations and in the control.

 

Analysis of test media revealed mean concentrations of test item between 108.2 and 138.8 % of the nominal values. Thus, the effect values were based on mean measured concentrations. The mean measured concentrations were calculated to be 0.35, 0.69, 1.74, 2.70 and 5.55 mg/L.

 

Clinical signs were observed after 3 h at 2.70, and at 5.55 mg/L (mean measured). After 24 h, treatment with these two concentrations resulted in 100 % mortality. Furthermore, clinical signs were observed after 24 h and 48 h, and all fish were found dead after 72 h at a treatment concentration of 1.74 mg/L. The LC50 after 96 h was calculated to be 0.89 mg/L for nominal concentrations, and 1.10 mg/L, based on mean measured concentrations.

 

Under the test conditions, the 96-hour LC50 for test item was 1.10 mg/L, and the highest concentration without observed effect after 96 hours was 0.69 mg/L.

Description of key information

OECD 203, EU Method C.1, GLP, key study, validity 1:

96h-LC50 (Danio rerio) = 1.1 mg/L, based on mean measured concentrations.

Key value for chemical safety assessment

LC50 for freshwater fish:
1.1 mg/L

Additional information

One key study is available to assess the acute toxicity of the test item to zebra fish (Danio rerio) was conducted according to OECD guideline 203.

This study was performed, according to the OECD Guideline 203 and EU Method C.1 with GLP compliance, under flow through conditions for 96 hours. The registered substance was tested at five nominal concentrations of 0.31, 0.63, 1.25, 2.50 and 5.00 mg/L. Untreated dilution water was run in parallel as a control. Seven fish each were used at the test concentrations and in the control. Analysis of test media revealed mean concentrations of test item between 108.2 and 138.8 % of the nominal values. Thus, the effect values were based on mean measured concentrations. The mean measured concentrations were calculated to be 0.35, 0.69, 1.74, 2.70 and 5.55 mg/L. Clinical signs were observed after 3 h at 2.70, and at 5.55 mg/L (mean measured). After 24 h, treatment with these two concentrations resulted in 100 % mortality. Furthermore, clinical signs were observed after 24 h and 48 h, and all fish were found dead after 72 h at a treatment concentration of 1.74 mg/L. Therefore, the LC50 after 96 h was 1.10 mg/L based on mean measured concentrations (95% CL could not be determined), and the highest concentration without observed effect after 96 h was 0.69 mg/L.