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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:
18 February 2013 to 22 February 2013
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
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
At test initiation (0 hour) and test termination (96 hours), duplicate 100 mL samples were removed from each test aquaria and analysed for the test material. Each sample was collected from the approximate midpoint of the vessels. Replicates A and B were frozen and stored until it was delivered to the laboratory for analysis.
A storage stability sample was prepared at 100 mg/L, stored and analysed along with the exposure solutions to identify if there were any effects during the transportation of the exposure solutions.
Vehicle:
no
Details on test solutions:
Concentrations are presented as milligrams of test substance per litre of test solution (mg/L). A nominal 100 mg/L test material solution was prepared by adding 1.100944 g of the test material to a 12 L fish tank and making up to 11 L with carbon filtered water. The solution was left to stir overnight. The stir was stopped approximately four hours prior to transferring the fish. 10 L was transferred to a fresh fish tank using a syphon to avoid transferring any settled test material. The exposure solution was observed to be a clear solution with an oily substance on the surface after being transferred.
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow trout
- Strain: Oncorhynchus mykiss
- Mean wet weight: 1.25 g (range 0.8 to 1.8 g); N = 10
- Mean total length: 49 mm (range 46 to 56 mm); N = 10


ACCLIMATION
- Acclimation period: a minimum of 12 days
- Acclimation conditions (same as test or not): Same as test. Fish were maintained in a 180 L glass stock tank under a photoperiod of 16 hours light and 8 hours of darkness. The stock tanks were filtered using aquarium filters. Stock tank temperatures were maintained at 14 ± 1 °C by aquarium chillers.
- Health during acclimation (any mortality observed): Mortality in the stock fish during the seven days preceding the definitive test was 0%.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Hardness:
222 mg/L
Test temperature:
13.2 - 15.0 °C
pH:
8.02 - 8.63
Dissolved oxygen:
98.5 - 101.5 %
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentration: 100 mg/L
At 0 hour and 96 hours, chemical analysis showed measured concentrations of 1.15 and 0.0532 mg/L, respectively. Geometric mean concentration = 0.25 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: Aquaria
- Material, size, headspace, fill volume: Each aquarium was constructed of glass and silicone adhesive (specific tank dimensions not noted), 10 L capacity.
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.875 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Onsite borehole, carbon filtered to remove potentially harmful organic compounds.
- Total organic carbon: <3 mg/L
- Conductivity: 0.59 mS/cm
- Pesticides and polychlorinated biphenyls: Analysis for pesticides and PCBs in the dilution water revealed all levels to be within the defined acceptable limits.
- Culture medium different from test medium: No
- Intervals of water quality measurement: pH was measured daily using a VWR pH100 meter and combination electrode; the dissolved oxygen concentration was also measured daily using a VWR pHenomenal OXY 11 dissolved oxygen meter. The temperature was continuously monitored with a VWR Minimum Maximum thermometer.

OTHER TEST CONDITIONS
- Adjustment of pH: None reported
- Photoperiod: 16 hours light and 8 hours darkness
- Light intensity: Not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
All aquaria were examined at 0, 3, 24, 48, 72 and 96 hours of exposure: mortalities were recorded and removed; biological observations, including adverse effects (e.g., darkened pigmentation), of the exposed rainbow trout; and observations of the physical characteristics of the test solutions (e.g., presence of precipitate, film on the solution's surface) were made and recorded.
Effects for this study were based on death, defined as the lack of movement by the exposed organisms (i.e., absence of gill movement and reaction to gentle prodding). Fish were defined as moribund with the aid of an observation score chart which allocated a score to commonly seen adverse effects. Moribund fish were removed from the test system and humanely euthanised in order to avoid excessive and prolonged suffering. The decision to euthanise fish was based on an observation score chart, which allocated a score to a range of adverse effects; any fish scoring 8 or higher was considered moribund and was unlikely to survive until the next observation period.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: Not applicable - limit test.
- Range finding study: Yes.
- Test concentrations in the range-finder: 0, 0.1, 1.0, 10 and 100 mg/L (nominal).
- Results used to determine the conditions for the definitive study: Yes. Five test aquaria were established, each containing three fish. Following 96 hours of exposure, all fish survived and no abnormal behaviour was recorded. Based on these results, it was decided to carry out a limit test at a nominal concentration of 100 mg/L.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.25 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
0.25 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Details on results:
BIOLOGICAL RESULTS
Following 96 hours of exposure, no mortality or adverse effects were observed among fish exposed to the control or 0.25 mg/L test material.

EVALUATION OF TEST CONDITIONS
All water quality parameters were unaffected by the concentration of the material tested and remained within acceptable ranges for the survival of rainbow trout. Continuous temperature monitoring established a temperature range of 13.2 to 14.1 °C in test vessels throughout the study. Dissolved oxygen levels were maintained above 60 % as required by the OECD guideline.

ANALYTICAL RESULTS
At 0 hour and 96 hours, chemical analysis showed measured concentrations of 1.15 and 0.0532 mg/L test material, respectively. Given the reduction in measured concentration over the 96-hour exposure period, it was considered justifiable to base the results on the geometric mean measured test concentration. This was calculated to be 0.25 mg/L test material.
Reported statistics and error estimates:
As there was no mortality observed in the limit test there was no need to run any statistics to calculate the median lethal concentrations (LC50).
Sublethal observations / clinical signs:

Table 1: Concentrations of test material measured in the exposure solutions during the 96-hour static acute exposure of rainbow trout (Oncorhynchus mykiss)

Nominal

Concentration

(µg/mL)

Test Material at 0 Hours

(µg/mL)

%

Nominal

Test Material at 96 Hours

(µg/mL) 

%

Nominal 

0

ND

N/A

ND

N/A

100

1.15

1.15

0.0532

0.053

Limit of Detection 0.034 µg/mL

ND = Not detected

N/A = Not applicable

 

Table 2: Geometric mean measured concentrations tested and corresponding cumulative percent and number of mortalities during the 96 hour static acute exposure of rainbow trout (Oncorhynchus mykiss) to the test material

Test Concentration

(mg/L)

Cumulative Mortality (%)*

3 Hours

24 Hours

48 Hours

72 Hours

96 Hours

Control

0

0

0

0

0

(0)

(0)

(0)

(0)

(0)

0.25

0

0

0

0

0

(0)

(0)

(0)

(0)

(0)

*Actual number of mortalities is presented in parentheses.

Validity criteria fulfilled:
yes
Conclusions:
The 96-hour LC50 value of the test material was calculated to be >0.25 mg/L (geometric mean measured concentration). The NOEC was calculated as 0.25 mg/L (geometric mean measured concentration).
Executive summary:

The short term toxicity of the test material to fish (Oncorhynchus mykiss) was investigated in a study conducted in accordance with the standardised guideline OECD 203 under GLP conditions.

Rainbow trout were exposed to a limit value of the test material at a nominal concentration of 100 mg/L for 96 hours under static conditions. A control group was exposed to the dilution water under the same conditions.

This was a limit of solubility test as the material was not soluble in water. The nominal solution was prepared and stirred overnight and allowed to stand for approximately 4 hours. The solution used in the test was transferred using a syphon to avoid the transference of any settled test material.

Following 96 hours of exposure, no mortality or adverse effects were observed among fish exposed to the control or test material. The measured concentrations were very low and therefore the test was reported using the geometric mean measured test concentration.

Under the conditions of the study, the 96-hour LC50 value of the test material was calculated to be >0.25 mg/L (geometric mean measured concentration). The NOEC was calculated as 0.25 mg/L (geometric mean measured concentration).

Description of key information

Study conducted to recognised testing guidelines with GLP certification.

Key value for chemical safety assessment

Additional information

The short term toxicity of the test material to fish (Oncorhynchus mykiss) was investigated in a study conducted in accordance with the standardised guideline OECD 203 under GLP conditions.

Rainbow trout were exposed to a limit value of the test material at a nominal concentration of 100 mg/L for 96 hours under static conditions. A control group was exposed to the dilution water under the same conditions.

This was a limit of solubility test as the material was not soluble in water. The nominal solution was prepared and stirred overnight and allowed to stand for approximately 4 hours. The solution used in the test was transferred using a syphon to avoid the transference of any settled test material.

Following 96 hours of exposure, no mortality or adverse effects were observed among fish exposed to the control or test material. The measured concentrations were very low and therefore the test was reported using the geometric mean measured test concentration.

Under the conditions of the study, the 96-hour LC50 value of the test material was calculated to be >0.25 mg/L (geometric mean measured concentration). The NOEC was calculated as 0.25 mg/L (geometric mean measured concentration).

The study was conducted to GLP and a standardised guideline. It was therefore assigned a reliability score of 1 and considered suitable for assessment as an accurate reflection of the test substance.

The available data are considered to be complete and the result determined, 96 h LC50 >0.25 mg/L, was taken forward for risk assessment.