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Ecotoxicological information

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:
25-01-2002 to 06-04-2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study performed under GLP. All relevant validity criteria were met with acceptable deviations.
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
No positive control conducted as part of the study.
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
yes
Remarks:
No positive control conducted as part of the study.
GLP compliance:
yes (incl. certificate)
Remarks:
inspected: February 2000; signature: April 2000
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 0.28, 0.50, 0.90, 1.6 and 2.8 mg/L (nominal ; based on the results of the pre-study media preparation trial).
Time Weighted Average equivalent concentrations: 0 (control), 0.21, 0.35, 0.69, 1.20, 2.00 mg/L (measured ; based on measured concentrations in the definitive test taken at 24 hour intervals in old/fresh media respectively pre-/post- renewal)
- Sampling method: Water samples were taken from the control and all surviving test groups at 0 (fresh media); 24, 48, 72 (old and fresh media) and 96 (old media) hours for quantitative analysis. Where possible all samples were prepared for analysis on the day of sampling. The prepared test samples may have been stored in a fridge (approximately +4°C) whilst awaiting instrument delays. Duplicate samples were taken and stored frozen (approximately -20°C) for further analysis if necessary.
- Sample storage conditions before analysis: See above.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low aqueous solubility and high purity of the test item, the test concentration used in the definitive test was prepared as a saturated solution from an initial test item dispersion at a concentration of 100 mg/L. An initial dispersed test item concentration of 100 mg/L was employed such that an excess of test item was present to ensure saturation of the test media. 2250 mg test item was dispersed into 22.5 litres of dechlorinated tap water and stirred via propeller stirrer at approximately 2000 rpm at approximately 25°C for approximately 48 hours prior to cooling to approximately 14°C. The aqueous phase (saturated solution) was removed by filtration through a 0.2 µm filter, discarding the initial 1 litre to thoroughly precondition the filter. The saturated solution was prepared in triplicate and pooled to give sufficient volume to conduct the test. Aliquots (2100, 3750, 6750, 12000 and 21000 mL) of this saturated solution (the maximum test concentration employed in the test) were each separately dispersed in dechlorinated tap water prior to adjusting the volume to 21 litres to give the remainder of the test series.
The method employed was determined based on a pre-test media preparation trial. In duplicate with chemical analysis performed for ASTM culture medium. Declorinated and Reconstituted water.
- Evidence of undissolved item (e.g. precipitate, surface film, etc): None reported
Test organisms (species):
Oncorhynchus mykiss (previous name: Salmo gairdneri)
Details on test organisms:
TEST ORGANISM
- Common name: Rainbow Trout (Oncorhynchus mykiss)
- 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): 4.3 cm (SD = 0.3)
- Weight at study initiation (mean and range, SD): 0.71 g (SD = 0.21)
- Method of breeding: Not reported.
- Maintenance of the brood fish: Not reported.

ACCLIMATION
- Acclimation period: 12 days.
- Acclimation conditions (same as test or not): Yes.
- Type and amount of food during acclimation: certified pelleted diet
- Feeding frequency during acclimation: daily (until 24 hours prior to test).
- Health during acclimation (any mortality observed): < 1% mortality in 7 days pre-test

QUARANTINE (wild caught)
- Duration: Not applicable.
- Health/mortality: Not applicable.

FEEDING DURING TEST
- Food type: Not applicable.
- Amount: Not applicable.
- Frequency: Not applicable.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
According to OECD TG 203 guidelines.
Post exposure observation period:
none.
Hardness:
ca. 100 mg/L (expressed as CaCO3)
Test temperature:
14 ºC
pH:
7.4-7.6 (fresh media); 7.6-7.8 (expired media)
Dissolved oxygen:
83-94% ASV (fresh media); 83-96% (expired media)
Salinity:
not applicable.
Nominal and measured concentrations:
Concentrations: 0 (control), 0.28, 0.50, 0.90, 1.6 and 2.8 mg/L (nominal ; based on the results of the pre-study media preparation trial).
Time Weighted Average equivalent concentrations: 0 (control), 0.21, 0.35, 0.69, 1.20, 2.00 mg/L (measured ; based on measured concentrations in the definitive test taken at 24 hour intervals in old/fresh media respectively pre-/post- renewal)
Details on test conditions:
TEST SYSTEM
- Test vessel: 20 L for test and control vessels, glass fibre
- Type (delete if not applicable): open, covered to reduce evaporation
- Material, size, headspace, fill volume: 20L fill volume test media per test vessel
- Aeration: Yes.

- 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: 0.36 g/L (mean)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Drinking water; chlorine removed by activated carbon filtration and partial softening (DI).
- Total organic carbon: Not reported
- Particulate matter: Not reported
- Metals: Not reported
- Pesticides: Not reported
- Chlorine: Not reported
- Alkalinity: Not reported
- Ca/mg ratio: Not reported
- Conductivity: Not reported
- Culture medium different from test medium: No.
- Intervals of water quality measurement: 24 hours.

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 16 h light / 8 h darkness
- Light intensity: Not reported.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : mortality and visible abnormality recorded at 3 h, 24 h, 48 h, 72 h and 96 h.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 1.8
- Range finding study: yes (semi-static) ; 3 organisms per concentration.
- Test concentrations: 0.28 and 2.8 mg/L and blank control
- Results used to determine the conditions for the definitive study: Yes. No mortalities at 0 (control) or 0.28 mg/L. Complete mortality at 24 hours in 2.8 mg/L.
Reference substance (positive control):
no
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.3 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: (95% CL: 1.1 - 1.5 mg/L)
Details on results:
- Behavioural abnormalities: See table 2 and 3
- 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: swimming at surface, swimming at bottom, increased pigmentation, loss of equilibrium, gasping, moribund
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: The pre-study stability analysis indicated that the test item was stable in the test medium in the absence of living organisms. The reason for the decline in test item concentrations over the 24 hour exposure period is therefore unknown but may be due to biological factors.
- Effect concentrations exceeding solubility of substance in test medium: Not applicable

Table 1.0 – nominal and measured concentrations

Nominal Concentration

(mg/L)

Measured concentration of test item (mg/L)

 

Time Weighted Average Concentration (mg/L)

 

0h

24h

 

48h

 

72h

 

96h

 

 

new

old

new

old

new

old

new

old

 

0 Control

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

< LOQ

0.00

0.28

0.264

0.154

0.362

0.152

0.302

0.131

0.287

0.135

0.21

0.50

0.436

0.165

0.560

0.247

0.523

0.224

0.494

0.282

0.35

0.90

0.804

0.485

0.984

0.562

0.985

0.446

0.883

0.529

0.69

1.60

1.31

0.747

1.81

0.925

1.780

0.742

1.57

0.949

1.20

2.80

2.57

1.52

-

-

-

-

-

-

2.00

 

 

 

 

 

 

 

 

 

 

 

Table 2.0 - Cumulative mortality

Time Weighted Average Concentration (mg/L)

Cumulative mortality (n=10)

Total mortality %

 

3h

6h

24h

48h

72h

96h

 

0.00

0

0

0

0

0

0

0

0.21

0

0

0

0

0

0

0

0.35

0

0

0

0

0

0

0

0.69

0

0

0

0

0

0

0

1.20

0

0

1

1

1

4

40

2.00

6

10

10

10

10

10

100

 

 

 

 

 

 

 

 

 

Table 3.0 – Sub-lethal effects/responses

Time Weighted Average Concentration (mg/L)

Sub-lethal effects

Time

 

 

3h

6h

24h

48h

72h

96h

0.00

No abnormalities

 

 

 

 

 

 

0.21

No abnormalities

 

 

 

 

 

 

0.35

No abnormalities

 

 

 

 

 

 

0.69

Swimming at surface

Swimming at bottom

Increased pigmentation and swimming at bottom

 

10/10

9/10

1/10

8/10

2/10

7/10

3/10

 

 

10/10

1.20

Swimming at surface

Swimming at bottom

Increased pigmentation, loss of equilibrium and gasping, moribund

7/10

3/10

10/10

2/9

4/9

3/9

2/9

5/9

2/9

1/9

5/9

 

3/9*

6/6

2.00

loss of equilibrium and gasping, moribund

2/4

2/4**

CM

 

 

 

 

 

 

 

 

 

 

 

 

Where:

* = humanely euthanized and indicated as mortalities

** = humanely euthanized and indicated as mortalities

CM = complete mortality

Validity criteria fulfilled:
yes
Conclusions:
The 96 hour LC50 for the test item to Rainbow Trout (Oncorhynchus mykiss) was determined to be 1.3 (C.I. 1.1 - 1.5) mg/L based on time weighted average concentrations.
Executive summary:

The acute toxicity of the test item to Rainbow Trout (Oncorhynchus mykiss) was determined in a 96 hour semi-static test according to OECD TG 203 and EU Method C.1 guidelines under GLP. Following a preliminary range-finding test (conducted over a range of test concentrations of 0.28 and 2.8 mg/L), fish were exposed, in groups often, to an aqueous solution of the test item at time-weighted mean measured test concentrations of 0.21, 0.35, 0.69, 1.2 and 2.0 mg/L for a period of 96 hours under semi-static test conditions. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in dechlorinated tap water and then removing any undissolved test item by filtration. This saturated solution was then further diluted, as necessary, to provide the remaining test groups. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours. Analysis of the freshly prepared test solutions throughout the definitive test showed measured test concentrations to range from 0.264 to 2.57 mg/L. 24-hour old or expired test media throughout the test showed a marked decline in measured values which ranged from 0.131 to 1.52 mg/L. Pre-study stability analysis indicated that the test item was stable in the test medium in the absence of living organisms. The reason for the decline in test item concentrations over the 24 hour exposure period is therefore unknown but may be due to biological factors. On this basis the test item was not maintained at ±20% of the initial concentrations therefore the effect levels were estimated based on time weighted average concentrations. Under the conditions of this study, the 96h LC50 was 1.3 (C.I. 1.1 – 1.5) mg/L based on TWA concentrations.

Description of key information

LC50 (fish) = 1.3 (C.I. 1.1 – 1.5 ) mg/L based on time weighted average concentrations, 96hour, freshwater, OECD TG 203, 2002

Key value for chemical safety assessment

LC50 for freshwater fish:
1.3 mg/L

Additional information

Key study : OECD TG 201, 2002: The acute toxicity of the test item to Rainbow Trout (Oncorhynchus mykiss) was determined in a 96 hour semi-static test according to OECD TG 203 and EU Method C.1 guidelines under GLP. Following a preliminary range-finding test (conducted over a range of test concentrations of 0.28 and 2.8 mg/L), fish were exposed, in groups often, to an aqueous solution of the test item at time-weighted mean measured test concentrations of 0.21, 0.35, 0.69, 1.2 and 2.0 mg/L for a period of 96 hours under semi-static test conditions. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in dechlorinated tap water and then removing any undissolved test item by filtration. This saturated solution was then further diluted, as necessary, to provide the remaining test groups. The number of mortalities and any sub-lethal effects of exposure in each test and control vessel were determined 3 and 6 hours after the start of exposure and then daily throughout the test until termination after 96 hours. Analysis of the freshly prepared test solutions throughout the definitive test showed measured test concentrations to range from 0.264 to 2.57 mg/L. 24-hour old or expired test media throughout the test showed a marked decline in measured values which ranged from 0.131 to 1.52 mg/L. Pre-study stability analysis indicated that the test item was stable in the test medium in the absence of living organisms. The reason for the decline in test item concentrations over the 24 hour exposure period is therefore unknown but may be due to biological factors. On this basis the test item was not maintained at ±20% of the initial concentrations therefore the effect levels were estimated based on time weighted average concentrations. Under the conditions of this study, the 96h LC50 was 1.3 (C.I. 1.1 – 1.5) mg/L based on TWA concentrations.