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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:
17-09-2014 to 30-10-2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met.
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Definitive test: Nominal test concentrations: 0 (control), 1.02, 1.80, 3.19, 5.65, 10.00 mg/L
Arithmetic Mean Measured concentrations: 0 (ND), 0.1032, 0.2149, 0.3587, 0.5374, 1.070 mg/L
Spacing factor (1.77) determined from range-finding test
Controls: test water without test item but treated in the same way as the test item solutions.
- Sampling method: Triplicate samples for analysis were taken from the mid-layer of each control and all test concentrations at the start of the test and every 24 hours thereafter in old and new solutions until the end of the test (t=96h)
- Sample storage conditions before analysis: Samples were used on day of sampling and analysed as soon as possible after their preparation.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Stock preparation: The required amount of the test substance was weighed and mixed with breeding water and the bottle was filled completely and tightly closed. This preparation was sonicated by sonication for 15 minutes with intense stirring using a magnetic stirrer for 3 hours in the dark and then, it was filtered through a 0.7 μm glass fibre filter. The additional concentrations were prepared by serial dilution of the saturated solution. Five litres of the test solution were prepared for each treatment group and the control group was prepared only with breeding water. The control and test solutions were prepared on the day of exposure.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Not applicable.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): Range Finding Test: Nominal Concentrations: 0 (control), 1 (1:100), 10 (1:10) and 100 mg/L (saturated solution) ; Definitive Test: Nominal test concentrations: 0 (control), 1.02, 1.80, 3.19, 5.65, 10.00 mg/L
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): None. Turbidity testing indicated absence of undissolved test item.
Test organisms (species):
Oryzias latipes
Details on test organisms:
TEST ORGANISM
- Common name: Rice fish (Oryzias latipes)
- Strain: Not reported
- Source: Recognised supplier (listed in full study report)
- Age at study initiation (mean and range, SD): Not reported.
- Length at study initiation (length definition, mean, range and SD): Range Finder: mean: 1.97 ± 0.05 cm (SD) ; range: 1.91 to 2.03 cm and Definitive: mean: 2.03 ± 0.07 cm (SD) ; range: 1.91 to 2.12 cm
- Weight at study initiation (mean and range, SD): Range Finder: mean: 0.18 ± 0.01 g (SD) ; range: 0.173 to 0.190 g and Definitive: mean: 0.19 ± 0.01 g (SD) ; range: 0.181 to 0.201 g
- Method of breeding: Suitable male and female fish, at a ratio of 3 to 2 without any visible abnormalities and of similar size were selected and placed in breeding water of 50 L. Approximately 25 fish (±10%) were bred. Eggs were harvested in breeding chambers and placed in hatching chambers. After hatching, the fry were placed in breeding chambers maintained at 23±1°C and bred.
- Maintenance of the brood fish: 30% of holding water was replaced once a week. The holding room was artificially illuminated; 16-hour light and 8-hour dark. Fish were fed Brine Shrimp in the morning and Top meal in the afternoon. Quantity was ca. 3% bodyweight daily except for weekends when fasted.

ACCLIMATION
- Acclimation period: > 7 days (Actual: 10 days)
- Acclimation conditions (same as test or not): Yes (Temperature: 22.0 to 22.5°C ; dissolved oxygen concentration was maintained at 96.1–98.8% of the saturation value in the range finding study and at 95.7–97.6% of the saturation value in the definitive study).
- Type and amount of food during acclimation: Flaked food or live food (such as brine shrimp nauplii).
- Feeding frequency during acclimation: Daily (with fasting at weekends) ; feeding ceased 24 hours before test initiation.
- Health during acclimation (any mortality observed): Seven days prior to exposure, the mortality of a batch of fish was less than 5% of the population

QUARANTINE (wild caught)
- Duration: Internal quarantine at least 7 days after delivery.
- Health/mortality: None.

FEEDING DURING TEST
- Food type: No.
- Amount: Not applicable.
- Frequency: Not applicable.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
none.
Hardness:
Between 10 and 240 mg/L (expressed as CaCO3) ; actual 70 mg/L
Test temperature:
23 ± 1 ºC (actual 22.0–22.8 ºC)
pH:
7.79-8.00 (fresh media); 7.48-7.72 (expired media)
Dissolved oxygen:
8.38-9.11 mg/L (fresh media); 5.11-8.09 mg/L (or 60.9 to 96.4% ASV) (expired media)
Salinity:
not applicable.
Nominal and measured concentrations:
Range Finding Test:
Nominal Concentrations: 0 (control), 1 (1:100), 10 (1:10) and 100 mg/L (saturated solution)

Definitive Test:
Nominal test concentrations: 0 (control), 1.02, 1.80, 3.19, 5.65, 10.00 mg/L
Arithmetic Mean Measured concentrations: 0 (ND), 0.1032, 0.2149, 0.3587, 0.5374, 1.070 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 5L glass.
- Type (delete if not applicable): closed (aluminium foil)
- Material, size, headspace, fill volume: 600W×300D×400H (mm) dimensions ; ca. 5 L fill volume test media per test vessel (minimum headspace)
- Aeration: no
- Renewal rate of test solution (frequency): 24 h
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): Not applicable
- Biomass loading rate: < 1.0 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Drinking water (certified; filtered and irradiated by UV light)
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported (see certificate of analysis in full study report)
- Pesticides: Not reported (see certificate of analysis in full study report)
- Chlorine: Not reported (see certificate of analysis in full study report)
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: Not reported.
- Culture medium different from test medium: No.
- Intervals of water quality measurement: Water temperature, dissolved oxygen concentration and pH of the test solutions were recorded for the treatment groups and control group prior to initiation of exposure and at 24, 48, 72 (before and after renewal of each test solution) and 96 hours after exposure.

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 16 h light / 8 h darkness
- Light intensity: white fluorescent lamp, intensity not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality and visible abnormality recorded at least the following: 24 h, 48 h, 72 h and 96 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.77 (Definitive)
- Range finding study: yes (semi-static)
- Test concentrations: Range Finder: Nominal Concentrations: 0 (control), 1 (1:100), 10 (1:10) and 100 mg/L (saturated solution)
- Results used to determine the conditions for the definitive study: Yes.
Reference substance (positive control):
yes
Remarks:
copper (II) sulfate (non-concurrent ; documented in full study report)
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
0.718 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: (95% CL: 0.6473-0.7964 mg/L)
Details on results:
- Behavioural abnormalities: See table 1 and 2
- Observations on body length and weight: Not reported
- Other biological observations: Not reported
- Mortality of control: 0%
- Other adverse effects control: No abnormalities observed in the control.
- Abnormal responses: Sub lethal responses are reported in table 1 and 2
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: None. Turbidity measurements indicated the absence of any undissolved test item (transparent)
- Effect concentrations exceeding solubility of substance in test medium: Not applicable. See above.
Results with reference substance (positive control):
- Results with reference substance valid? Yes (non-concurrent ; reliability check documented in the full study report).
- Mortality: Not reported
- LC50: 0.30 mg/L (95% CL 0.25-0.35 mg/L) in 96 h
- Other: In agreement with laboratory historical database
Reported statistics and error estimates:
Statistical analysis was performed using the U.S. EPA authorized program. During the exposure period, a concentration-mortality curve was generated and the LC50 and its 95% confidence limits for mortality at 24, 48, 72 and 96 hours after exposure were analyzed using Probit method and Trimmed Spearman-Karber method.

However, only three decimal points are displayed in the Probit method, therefore, statistical analysis was performed by multiplying the measured values by 10 and the statistical values (LC50) were divided by 10.

The test result is expressed in the same way as significant digits of analytical result. However, only two decimal points are displayed in the Trimmed spearman-Karber method, therefore, statistical analysis was performed by multiplying the measured values by 100 and the statistical values (LC50) were divided by 100.

Table 1.0 – Cumulative mortality

Diluted test item solution [nominal concentration / mg/L]

Arithmetic mean measured concentration

[/ mg/L]

Number of fish

Cumulative Mortality (Mortality %)

 

 

 

1 hr

3 hr

6 hr

24 hr

48 hr

72 hr

96 hr

0 (control)

N.D.

10

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

1.02

0.1032

10

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

1.80

0.2149

10

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

3.19

0.3587

10

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

0 (0)

5.65

0.5374

10

0 (0)

0 (0)

0 (0)

1 (10)

1 (10)

1 (10)

1 (10)

10.00

1.070

10

0 (0)

0 (0)

0 (0)

8 (80)

8 (80)

10 (100)

10 (100)

 

 

 

 

 

 

 

 

 

 

N.D. : Not detected

 

Table 2.0 – Behavioural observations

Diluted test item solution [nominal concentration / mg/L]

Arithmetic mean measured concentration

[/ mg/L]

Number of fish

General symptoms (number of fish)

 

 

 

1 hr

3 hr

6 hr

24 hr

48 hr

72 hr

96 hr

0 (control)

N.D.

10

NOR

(10)

NOR

(10)

NOR

(10)

NOR

(10)

NOR

(10)

NOR

(10)

NOR

(10)

1.02

0.1032

10

NOR

(10)

NOR

(10)

NOR

(10)

NOR

(10)

NOR

(10)

NOR

(10)

NOR

(10)

1.80

0.2149

10

NOR

(10)

NOR

(10)

NOR

(10)

NOR

(10)

NOR

(10)

NOR

(10)

NOR

(10)

3.19

0.3587

10

NOR

(10)

NOR

(10)

NOR

(10)

NOR

(10)

NOR

(10)

NOR

(10)

NOR

(10)

5.65

0.5374

10

NOR

(10)

NOR

(10)

NOR

(10)

NOR (9)

DEA (1)

NOR (9)

NOR (9)

NOR (9)

10.00

1.070

10

NOR

(10)

NOR

(10)

SWI (1)

RES (3)

HYO (6)

HYO (2)

DEA (8)

HYO (2)

DEA (2)

-

 

 

 

 

 

 

 

 

 

 

NOR: Normal

SWI: Swimming on the water surface

RES: Respiration at the water surface

HYO: Hypoactivity

DEA: Death

N.D.: Not detected

Validity criteria fulfilled:
yes
Conclusions:
The 96 hour LC50 for the test item to Oryzias latipes was determined to be 0.7180 (C.I. 0.6473 – 0.7964) mg/L based on arithmetic mean measured concentrations.
Executive summary:

The acute toxicity of the test item to Rice fish (Oryzias latipes) was determined in a 96 hour closed semi-static test according to OECD TG 203 under GLP. Based on the results of preliminary range finding testing, the nominal concentrations of 0 (control), 1.02, 1.80, 3.19, 5.65, 10.00 mg/L with a geometric series factor of 1.77 and minimal headspace were selected for exposure to groups of ten fish. One control containing no test item was also performed. The test total duration was 96 hours and test solutions were renewed on a daily basis. The mortality of the fish was determined by visual observation after 1, 3, 6 24, 48, 72 and 96 hours. Samples taken from the control and all test concentrations were analysed at the start of the test and every 24 hours thereafter in old and new solutions. The test item was renewed at 24 hour intervals within a 96-hour semi-static test system. The actual concentrations were verified by measurement of the test item in the nominal loading rates of 0 (control), 1.02, 1.80, 3.19, 5.65, 10.00 mg/L at 0h, 96h and 24h,48h, 72h of new and old solutions (after and before the renewal). The 24 h aged test media were maintained in the range of ±20% when compared to the initially measured concentrations of the freshly prepared media at 0, 24, 48 and 72 hours. Nevertheless, all test results were calculated based on the arithmetic mean measured concentration. The reason for using the arithmetic mean is the possibility that the concentration in the preparation of the test solutions may vary in this study with replacement of the test solution at 24, 48 and 72-hour intervals. Therefore, the evaluation of the effects was based on the arithmetic mean measured concentrations: 0 (ND), 0.1032, 0.2149, 0.3587, 0.5374, 1.070 mg/L .After the 96-hours exposure, mortality was 0% at 0 to 0.3587 mg/L concentration and 10% and 100% at 0.5374 and 1.070 mg/L concentrations, respectively. Measurements were performed by Gas Chromatography (GC-FID). All relevant validity criteria were fulfilled for the test. The relevant LC50 were: 24h: 0.8181 (C.I. 0.6439 – 1.083) mg/L, 48h: 0.8181 (C.I. 0.6439 – 1.083) mg/L, 72h: 0.7180 (C.I. 0.6473 – 0.7964) mg/L based on arithmetic mean measured concentrations. The 96 hour LC50 was 0.7180 (C.I. 0.6473 – 0.7964) mg/L based on arithmetic mean measured concentrations.

Description of key information

96-h LC50 (fish) = 0.7180 mg/L (C.I. 0.6473 – 0.7964 mg/L) based arithmetic mean measured concentrations, 96-hour, freshwater, OECD TG 203, 2014

Key value for chemical safety assessment

LC50 for freshwater fish:
0.718 mg/L

Additional information

Key data : OECD TG 203, 2014 : The acute toxicity of the test item to Rice fish (Oryzias latipes) was determined in a 96 hour closed semi-static test according to OECD TG 203 under GLP. Based on the results of preliminary range finding testing, the nominal concentrations of 0 (control), 1.02, 1.80, 3.19, 5.65, 10.00 mg/L with a geometric series factor of 1.77 and minimal headspace were selected for exposure to groups of ten fish. One control containing no test item was also performed. The test total duration was 96 hours and test solutions were renewed on a daily basis. The mortality of the fish was determined by visual observation after 1, 3, 6 24, 48, 72 and 96 hours. Samples taken from the control and all test concentrations were analysed at the start of the test and every 24 hours thereafter in old and new solutions. The test item was renewed at 24 hour intervals within a 96-hour semi-static test system. The actual concentrations were verified by measurement of the test item in the nominal loading rates of 0 (control), 1.02, 1.80, 3.19, 5.65, 10.00 mg/L at 0h, 96h and 24h,48h, 72h of new and old solutions (after and before the renewal). The 24 h aged test media were maintained in the range of ±20% when compared to the initially measured concentrations of the freshly prepared media at 0, 24, 48 and 72 hours. Nevertheless, all test results were calculated based on the arithmetic mean measured concentration. The reason for using the arithmetic mean is the possibility that the concentration in the preparation of the test solutions may vary in this study with replacement of the test solution at 24, 48 and 72-hour intervals. Therefore, the evaluation of the effects was based on the arithmetic mean measured concentrations: 0 (ND), 0.1032, 0.2149, 0.3587, 0.5374, 1.070 mg/L .After the 96-hours exposure, mortality was 0% at 0 to 0.3587 mg/L concentration and 10% and 100% at 0.5374 and 1.070 mg/L concentrations, respectively. Measurements were performed by Gas Chromatography (GC-FID). All relevant validity criteria were fulfilled for the test. The relevant LC50 were: 24h: 0.8181 (C.I. 0.6439 – 1.083) mg/L, 48h: 0.8181 (C.I. 0.6439 – 1.083) mg/L, 72h: 0.7180 (C.I. 0.6473 – 0.7964) mg/L based on arithmetic mean measured concentrations. The 96 hour LC50 was 0.7180 (C.I. 0.6473 – 0.7964) mg/L based on arithmetic mean measured concentrations.