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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:
March 19, 2020 - March 23, 2020.
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:
2019
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, OECD Series on Testing and Assessment no. 23
Version / remarks:
2019
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 850.1075 (Freshwater and Saltwater Fish Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Remarks:
Not feasible, see attached background material.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: saturated solution method. Saturated test item solution (nominal loading rate of 100.0 mg/L) was prepared by dispersing/dissolving the amount of test item into the test medium (aquarium water) two days before the start of the experiment. This solution was shaken for about 24 hours at approximately 30°C and then equilibrated for about 24 hours at approximately 20°C. The non-dissolved test material was removed by filtration through a fine (0.22 μm) filter to give the appropriate 100% saturated solution. The maximum amount of test material in the medium was exposed applying this procedure. The test solutions were prepared by appropriate dilution of this stock solution just before introduction of the fish (start of the experiment).
- Eluate: aquarium water
- Controls: The dilution water (circulated and filtered) was used without of addition of the test item.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): no/not specified.
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Strain: Brachydanio rerio
- Source: István Szent University, 2100 Gödöllő, Páter Károly u.1, Hungary.
- Age at study initiation (mean and range, SD): juveniles
- Length at study initiation (length definition, mean, range and SD): 2.5 – 2.7 cm
- Weight at study initiation (mean and range, SD): 0.17 – 0.22 g
- Method of breeding: Fish were bred in a well-known fish farm, under disease- and parasite-controlled conditions. Fish were observed healthy during the acclimatisation and test period.

ACCLIMATION
- Acclimation period: more than 12 days
- Acclimation conditions: same as test
- Type and amount of food during acclimation: not specified; fish were not fed during the test
- Health during acclimation (any mortality observed): Mortality was less than 5 % of the population for seven days before the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
174 mg/L (as CaCO3).
Test temperature:
20.4 – 22.6 °C.
pH:
8.24 – 8.47
Dissolved oxygen:
93 – 100 % of the air saturation value at the temperature used
Nominal and measured concentrations:
Nominal concentrations: 0.0512, 0.128, 0.32, 0.8 and 2.0% saturated solutions (of 100 mg/L loading).
Details on test conditions:
TEST SYSTEM
- Test vessel: 5L glass aquarium vessels
- Material, size, headspace, fill volume: glass, 5L
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): 24h
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): 1
- Biomass loading rate: less than 1.0 g fish/L test solution (see table 3).

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: tap-water purified by reverse osmosis
- Culture medium different from test medium: no
- Intervals of water quality measurement: water quality parameters were kept within the optimum limits for the respective fish species.

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light and 8 hours darkness

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Mortality and other effects at 3, 24, 48, 72 and 96 hours following the start of exposure. In addition, every afternoon from day 0 to observe for any dead or severely distressed fish. Dissolved oxygen content, pH and temperature were measured daily in all vessels from the start of the test (day 0).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study : A preliminary concentration range-finding test was conducted to determine the approximate toxicity of the test item so that appropriate test concentrations could be selected for use in the definitive test. Four fish were exposed to each concentration of the test item plus a control, for 96 hours under semi-static conditions (in absence of stability data). The frequency of the water renewal periods was 24 hours.
- Test concentrations: 0.1, 1, 10, 100 mg/L (nominal).
- Results used to determine the conditions for the definitive study: See table 1. Because significant toxic effect was observed at the highest concentration levels of 1.0, 10.0 and 100.0 % saturated solutions during the preliminary range-finding test and taking into account that the lowest concentration tested should preferably give no effect in the definitive test, therefore a wide concentration range was used, five test concentrations in a geometric series with a separation factor of 2.5 and one untreated control were tested in the main experiment under semi-static conditions.
The nominal concentrations of test item used in the main experiment were: 0.0512, 0.128, 0.32, 0.8 and 2.0% saturated solutions.
Reference substance (positive control):
not required
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
0.17 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
NOELR
Effect conc.:
0.051 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Key result
Duration:
96 h
Dose descriptor:
LOELR
Effect conc.:
0.128 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
- Behavioural abnormalities: Behaviour of fish was found as normal in the control group and at the lowest tested level of 0.0512 % saturated solution. Localization close to the surface of the water, decreased activity, dark colour and fast motility of operculum was observed at the test group of 0.128 % saturated solution during the experiment. At the test item group of 0.32 % saturated solution dark colour, localization at the bottom of aquarium or close to the surface of the water, decreased activity, unbalanced swimming, immobility, fast motility of operculum and gulping was observed during the test. Localization at bottom of aquarium, decreased activity, unbalanced swimming, fast motility of operculum and immobility was observed at the test group of 0.8 % saturated solution during the experiment, then all fish were dead 48 hours after treatment. At the highest concentration of 2.0 % saturated solution localization at the bottom of aquarium or close to the surface of the water, unbalanced and vertical swimming, lateral position and increased activity was recorded during the first 3 hours of the test when all fish were dead 6 hours after treatment.
- Observations on body length and weight:
- Other biological observations:
- Mortality of control: no.
- Other adverse effects control: no.
- Abnormal responses: none observed.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no.






Reported statistics and error estimates:
The 24, 48, 72 and 96 hours LC50 values were calculated by Probit analysis using SPSS PC+ software (based on the measured geometric mean concentrations). The NOEC, LOEC and LC100 were determined directly from the raw data.
Sublethal observations / clinical signs:

Table 4. Summary of endpoints.

Parameter

24h

48h

72h

96h

[% saturated solution of the 100 mg/L nominal loading rate]

LC50
95 % conf. limits

0.60
0.30 – 0.83

0.38
-*

0.27
-*

0.17
0.12 – 0.34

96h LC100

0.32

NOEC

0.0512

LOEC

0.128

Table 5. Cumulative mortality data in the Definitive Test.

Nominal concentration
(% saturated solution)

Cumulative mortality [number of fish]

(initial population = 7 fish)

3h

6h

24h

48h

72h

96h

Control

0

0

0

0

0

0

0.0512

0

0

0

0

0

0

0.128

0

0

0

0

0

1

0.32

0

0

0

2

5

7

0.8

0

0

6

7

-

-

2.0

0

7

-

-

-

-

Table 6. Measured and calculated bodyweight data.

Nominal concentration
(% saturated solution)

Measured weight
of 7 fish

(g)

Calculated mean
weight of 1 fish

(g)

Loading of testing aquarium
(g fish/L testing liquid)

Control

1.37

0.20

0.27

0.0512

1.22

0.17

0.24

0.128

1.20

0.17

0.24

0.32

1.29

0.18

0.26

0.8

1.55

0.22

0.31

2.0

1.24

0.18

0.25

Validity criteria fulfilled:
yes
Remarks:
Constant conditions were maintained in this procedure. The mortality in the controls did not exceed 10 % at the end of the test. The dissolved oxygen concentration throughout the test was above 60% of air saturation value.
Conclusions:
The 96h-LL50 of the test item in zebrafish was found to be 0.17 mg/L and the NOELR 0.0512 mg/L (nominal concentrations).
Executive summary:

The acute toxicity of the test item was assessed with acute fish toxicity test on Zebrafish (Brachydanio rerio), over an exposure period of 96 hours in a semi-static system, according to OECD 203, under GLP conditions. Because significant toxic effect was observed at the highest concentration levels (1.0, 10.0 and 100.0 % saturated solutions) during the preliminary range-finding test (and taking into account that the lowest concentration tested should preferably give no effect in the definitive test, therefore a wide concentration range was used) five test concentrations in a geometric series with a separation factor of 2.5 and one untreated control were tested in the main experiment under semi-static conditions. The nominal concentrations of test item used in the main experiment were: 0.0512, 0.128, 0.32, 0.8 and 2.0 % saturated solutions. According to the given expert statement, the biological results are based on the nominal test item concentrations.

One aquarium was used in each test group and one in the control group. Each aquarium comprised seven fish and five litre test solution. The 24, 48, 72 and 96 hours LC50values of the test item were calculated using Probit analysis, and the NOEC and the LOEC and LC100values were determined from the raw data. All validity criteria were met. Under the conditions of this acute fish toxicity study on Zebrafish (Brachydanio rerio) the observed and calculated endpoints for the effect of DCC were the following:The 96h-LL50 of the test item in zebrafish was found to be 0.17 mg/L (95% CL: 0.12 - 0.34 mg/L) and the NOELR was 0.0512 mg/L (nominal concentrations, based on mortality).

Description of key information

Key study: Method according to OECD 203, GLP study. The 96h-LL50 of the test item in zebrafish (Brachydanio rerio) was found to be 0.17 mg/L (95% CL: 0.12 - 0.34 mg/L) and the NOELR was 0.0512 mg/L (nominal concentrations, based on mortality).

Supporting study: ECOSAR v1.11, accepted calculation method. The substance has a predicted 96h-LC50 = 0.008 mg/L in fish.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
0.17 mg/L

Additional information