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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:
09 April 2017 - 17 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to
Guideline:
other: Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Test material information:
Composition 1
Specific details on test material used for the study:
Solubility in water: 10.8 mg/L at 22°C
Stability in water: Not indicated
Vapour pressure: 3.4 Pa
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all test concentrations (i.e. 10, 18, 32, 56 and 100% of a saturated solution prepared at a loading rate of 100 mg/L) and the control
- Frequency: at the start and the end of the first and the last 24-hour renewal periods
- Sampling method: 3.0 mL from the approximate centre of the test vessels
- Sample storage conditions before analysis: samples were stored in a freezer (≤-15°C) until analysis at the analytical laboratory of the Test Facility
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: direct addition - Preparation of test solutions started with a loading rate of 100 mg/L applying a one-day period of magnetic stirring in a closed vessel to reach the maximum dissolution of the test item in medium. The obtained aqueous mixture was allowed to settle for a period of approximately 1.5 hours. Thereafter, the Saturated Solution (SS) was collected by means of siphoning and used as the highest test concentration. Lower test concentrations were prepared by subsequent dilutions of the SS in test medium.
- Controls: Test medium without test item or other additives
- Evidence of undissolved material: No, all test solutions were clear and colorless at the end of the preparation procedure.
Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: Carp
- Strain: Linnaeus, 1758
- Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands
- Age at study initiation: not reported
- Length at study initiation (mean and SD): 2.2 ± 0.1 cm (final test)
- Weight at study initiation (mean and SD): 0.31 ± 0.06 g (final test)
- Method of breeding: F1 from a single parent-pair bred in UV-treated water

ACCLIMATION
- Acclimation period: At least 12 days after delivery
- Acclimation conditions (same as test or not): yes
- Type and amount of food during acclimation: pelleted fish food (Cyprico Crumble Excellent (300-500 um), Coppens International bv, Helmond, The Netherlands)
- Feeding frequency during acclimation: daily
- Health during acclimation (any mortality observed): mortality during the seven days prior to the start of the test was less than 5%

FEEDING DURING TEST
- No feeding from 24 hours prior to the test and during the total test period.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
180 mg/L (as CaCO3)
Test temperature:
21-22°C
pH:
fresh solutions: 7.6-8.0
used solutions: 7.4-7.9
Dissolved oxygen:
fresh solutions: 8.5-9.2 mg DOC/L
used solutions: 7.3-8.5 mg DOC/L
Nominal and measured concentrations:
Nominal test concentrations (defintive test): 10, 18, 32, 56 and 100% (v/v) of a saturated solution prepared at a loading rate of 100 mg/L
Initial measured concentrations at t=0 were in the range 0.56 to 5.82 mg/L (all concentrations analysed) and decreased to 52-78% of initial after 24 hours.
Initial measured concentrations at t=72 h were in the range 0.55 to 1.83 mg/L (only the 3 lowest test concentrations were analysed) and decreased to 49-54% of initial after 24 hours.

Considering the observed decrease in measured concentrations in used test solutions at 24 and 96 h, geometric mean measured test concentrations were calculated (based on results from the first and the last renewal period) and used to calculate effect parameters.

Geomean measured concentrations: 0.40, 0.76, 1.3, 2,7 and 5.1 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel (material, size, headspace, fill volume): 6.5 litres, all-glass, containing 6.2 litres of test solution. Since the test item was volatile the study was performed in air-tight vessels
- Aeration: not during the test
- Renewal rate of test solution (frequency): every 24 hours
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.35 g fish/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium, formulated using RO-water (tap-water purified by reverse osmosis
- Intervals of water quality measurement: water temperature, pH and dissolved oxygen concentrations were recorded daily in all vessels with surviving fish, beginning at the start of the test (day 0).

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light : 8 hours dark
- Light intensity: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): mortality (3½, 24, 48, 72 and 96 hours following the start of exposure)

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 1.8
- Range finding study
* Test concentrations: 1.0, 10 and 100% of a saturated solution prepared at a loading rate of 100 mg/L (3 fish exposed per concentration)
- Results used to determine the conditions for the definitive study: yes; the expected LC50 was between concentrations obtained in 10 and 100% of the saturated solution prepeared at a loading rate of 100 mg/L
Reference substance (positive control):
yes
Remarks:
pentachlorophenol (PCP) (test performed in February 2017)
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.5 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
mortality
Remarks on result:
other: 95% C.I. 1.1-1.3 mg/L
Details on results:
- Mortality of control: no
- Other adverse effects control: no
- Observed efefcts in test solutions: see table 3 in 'Any other information on results'
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no
- Effect concentrations exceeding solubility of substance in test medium: no
- Other: The LC50 value was already reached after 24 hours of exposure.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Dose levels tested (nominal concentration PCP): 0.10, 0.22 and 0.46 mg/L
- Mortality: no dead fish in 0.10 and 0.22 mg/L test solutions; all fish died within 24 h in 0.46 mg/L test solution
- LC50: 0.32 mg/L (95% C.I. 0.22-0.46 mg/L)
- Other: the sensitivity of carp used in this test was shown to fall within the range of sensitivities generally observed during the past years
Reported statistics and error estimates:
The LC50 values were determined using the maximum likelihood estimation method with the weibits of the percentages of dead fish as function of the logarithms of the corresponding concentrations.

ToxRat Professional v 3.2.1 (ToxRat Solutions® GmbH, Germany) was used to perform the analyses.

Table 1: %SS, Measured and Average Exposure Concentrations

Pinyl iso butyraldehyde alpha, %SS at 100 mg/L

Measured concentration (mg/L)

Average exposure concentration (mg/L)

t=0h (fresh)

t=24h

(old)

t=72h (fresh)

t=96h (old)

10

0.558

0.291

0.551

0.282

0.40

18

1.07

0.620

0.956

0.516

0.76

32

1.85

1.06

1.83

0.887

1.3

56

3.28

2.16

-

-

2.7

100

5.82

4.55

-

-

5.1

- Fish were already dead

Table 2: Incidence of Mortality and Total Mortality During the Final Test

Average conc.
Pinyl iso butyraldehyde alpha (mg/L)

Initial number
of fish

Cumulative mortality

Total
Mortality (%)

3½h

24h

48h

72h

96h

Control

7

0

0

0

0

0

0

0.40

7

0

0

0

0

0

0

0.76

7

0

0

0

0

0

0

1.3

7

0

2

2

2

2

29

2.7

7

7

7

7

7

7

100

5.1

7

7

7

7

7

7

100

 

Table 3: Clinical Effects Observed During the Final Test

Average conc.
Pinyl iso butyraldehyde alpha (mg/L)

Time of recording
(hours)

Specification of effects

Relative number

Control, 0.40

3½-96

No abnormalities

7/7

0.76


24-48

72-96

No abnormalities

Hypoactive, swimming at the bottom

No abnormalities

7/7

7/7

7/7

1.3


24-48

72

 

 

96

 

Immobile

Immobile/loss of equilibrium

Loss of equilibrium

Immobile

No abnormalities

Immobile

No abnormalities

7/7

5/5

3/5

1/5

1/5

1/5

4/5

 

Validity criteria fulfilled:
yes
Remarks:
see 'Overall remarks'
Conclusions:
The 96 h LC50 value in fish (Cyprinus carpio) is 1.5 mg/L, based on mean measured concentrations.
Executive summary:

A study was performed to assess the acute toxicity of the substance to fish (Cyprinus carpio). The study was conducted in accordance with OECD TG 203 and GLP. In a semi-static test set-up in air-tight closed vessels (fill volume: 6.2 L), seven fish per concentration were exposed to 5 concentrations of the substance in test water (10, 18, 32, 56 and 100% of a saturated solution prepared at a loading rate of 100 mg/L) and a control. Throughout the test, test solutions were refreshed in intervals of 24 hours. After 3.5 hours all fish in the two highest treatment groups had died. Hypoactive swimming was observed in the test group at 0.76 mg/L after 24 and 48 hours, whereas no abnormalities were observed after 3.5, 72 and 96 hours. In the test group at 1.3 mg/L, incidences of mortality were observed within 24 hours, as well as loss of equilibrium and/or immobility at all other timepoints. Based on mortality the following effect value was determined: 96 -h LC50 = 1.5 mg/L. All results are expressed in terms of mean measured concentrations. Initial measured concentrations in fresh solutions at test start and at t=72 hours were comparable and decreased in used solutions to 52 -78 % of initial during the first refreshment period of 24 hours, and to 49 -54% of initial during the last refreshment period of 24 hours. Therefore, geometric mean measured concentrations were calculated for the first and last time interval, and these values were used to calculate the 0 -96 h (arithmetic) mean measured concentrations.

Description of key information

A study was performed to assess the acute toxicity of the substance to fish (Cyprinus carpio). The study was conducted in accordance with OECD TG 203 and GLP. In a semi-static test set-up in air-tight closed vessels (fill volume: 6.2 L), seven fish per concentration were exposed to 5 concentrations of the substance in test water (10, 18, 32, 56 and 100% of a saturated solution prepared at a loading rate of 100 mg/L) and a control. Throughout the test, test solutions were refreshed in intervals of 24 hours. After 3.5 hours all fish in the two highest treatment groups had died. Hypoactive swimming was observed in the test group at 0.76 mg/L after 24 and 48 hours, whereas no abnormalities were observed after 3.5, 72 and 96 hours. In the test group at 1.3 mg/L, incidences of mortality were observed within 24 hours, as well as loss of equilibrium and/or immobility at all other timepoints. Based on mortality the following effect value was determined: 96 -h LC50 = 1.5 mg/L. All results are expressed in terms of mean measured concentrations. Initial measured concentrations in fresh solutions at test start and at t=72 hours were comparable and decreased in used solutions to 52 -78 % of initial during the first refreshment period of 24 hours, and to 49 -54% of initial during the last refreshment period of 24 hours. Therefore, geometric mean measured concentrations were calculated for the first and last time interval, and these values were used to calculate the 0 -96 h (arithmetic) mean measured concentrations.

Key value for chemical safety assessment

LC50 for freshwater fish:
1.5 mg/L

Additional information