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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:
24 Aug 2018 - 05 Nov 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: All test concentrations and the control
- Sampling method: 2.0 mL from the approximate centre of the test vessels, taken at the start of the test and after 72 hours from the freshly prepared solutions, and at the first renewal (t=24 h) and the end of the test from the 24-h old solutions.
- 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: Preparation of test solutions started with the highest concentration of 100 mg solids/L (260 mg test item/L) applying a 30-minute period of magnetic stirring to accelerate the dissolution of the test item in test medium. Lower test concentrations were prepared by subsequent dilutions of the highest concentration in test medium. All test solutions were clear and colorless at the end of the preparation procedure
- Controls: Test medium without test item or other additives
- Other relevant information: Test item was completely soluble in test medium at the concentrations tested. A correction was made for the purity/composition of the test substance. A correction factor of 2.6 was used. Concentration/doses are expressed as solids.
Test organisms (species):
Cyprinus carpio
Details on test organisms:
TEST ORGANISM
- Common name: Carp
- Strain: Cyprinus carpio Linnaeus, 1758
- Source: Zodiac, proefacc, "De Haar Vissen", Wageningen University and Research Centre, The Netherlands.
- Length at study initiation: 3.1 ± 0.3 cm
- Weight at study initiation: 0.40 ± 0.15 g
- Method of breeding: F1 from a single parent-pair bred in UV-treated water.
- Total fish used: 51

ACCLIMATION
- Acclimation period: at least 12 days after delivery
- Acclimation conditions: Adjusted ISO medium, formulated using tap-water purified by reverse osmosis.
- Type and amount of food during acclimation: Daily with pelleted fish food (Cyprico Crumble Excellent (300-500 um), Coppens International bv, Helmond, The Netherlands)
- Validity of batch: In the batch of fish used for the test, 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 CaCO3/L
Test temperature:
20 - 21 °C
pH:
7.4 - 7.9
Dissolved oxygen:
New solutions: 9.2 - 9.5 mg/L
Old solutions: 6.7 - 8.1 mg/L
Nominal and measured concentrations:
Nominal: 5.0, 10, 20, 40, 80 mg solids/L
Measured: The concentrations measured at the start and end of the first and last renewal periods were at 116-154% of the nominal test concentrations. Since the recoveries of test item concentrations in the Quality Control (QC) samples ranged between 111 and 157% of the nominal concentrations, it was concluded that the test solutions contained at least the nominal test item concentrations. Based on these results, the effect parameters were expressed based on nominal exposure concentrations. See 'Any other information on results' for details on measured concentrations.
Details on test conditions:
TEST SYSTEM
- Test vessel: 6.5 litres, all-glass, containing 5.0 litres of test solution
- Aeration: no
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 0.56 g fish/Litre
- Introduction of fish: Within 1.5 hours after preparation of the test media from a holding tank with comparable water quality parameters and pH and temperature differences between test and holding tank media of less than 1.0 unit and 1.0°C.
- Euthanasia: At the end of the test the surviving fish were rapidly killed by exposing them to ca. 1.2% ethylene glycol monophenylether in water.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Adjusted ISO medium
- Culture medium different from test medium: no
- Intervals of water quality measurement: Dissolved oxygen content, pH and temperature: 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 h photoperiod daily

EFFECT PARAMETERS MEASURED:
- Mortality and other effects: At 2, 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. Dead fish were removed when observed.
- Dissolved oxygen content, pH and temperature: Daily in all vessels with surviving fish, beginning at the start of the test (day 0).

TEST CONCENTRATIONS
- Range finding study test concentrations: 1.0, 10 and 100 mg solids/L.
- Results used to determine the conditions for the definitive study: yes, LC50 expected to be between 10 and 100 mg solids/L.
Reference substance (positive control):
yes
Remarks:
Pentachlorophenol (performed Mar 2018)
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
28 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Test substance corrected for water content (specified in report as "solids")
Basis for effect:
mortality
Remarks on result:
other: 95% confidence interval: 20-40 mg solids/L
Details on results:
- No mortality or other effects were observed in the control and the three lowest concentrations tested during the exposure period. All fish exposed to the highest concentration were found dead after the first 24 hours of exposure. All fish exposed to 40 mg solids/L were found dead after the 48 hours of exposure. No further clinical effects were observed at any of the concentrations tested.
- The concentrations measured at the start and end of the first and last renewal periods were at 116-154% of the nominal test concentrations. Since the recoveries of test item concentrations in the Quality Control (QC) samples ranged between 111 and 157% of the nominal concentrations, it was concluded that the test solutions contained at least the nominal test item concentrations. Small responses were detected in the samples taken from the control. The source of these responses could not be identified. Considering that the maximum contribution to the lowest test concentration was 0.053% and that no effects were observed in the control throughout the test, this was considered to be negligible.
- All environmental parameters remained within the limits prescribed by the study plan throughout the test.
Results with reference substance (positive control):
The 96h-LC50 for carp exposed to PCP was 0.15 mg/L (95 % confidence interval between 0.10 and 0.22 mg/L).
The range of the 96h-LC50 for carp is generally between 0.10 and 0.46 mg/L based on historical data of reference tests performed approximately every 3 months from April 1988 until the end of 2000, and annually since then. Hence, the sensitivity of carp originating from the present batch for PCP falls within the range of sensitivities generally observed during the past years.
Reported statistics and error estimates:
The LC50 could not be determined using the maximum likelihood estimation method with the probits of the percentages of dead fish as function of the logarithms of the corresponding concentrations. This was because there was no concentration between the highest concentration (A) at which 0% mortality and the lowest concentration (B) at which 100% mortality occurred. Instead, the LC50 was calculated as (AB)½, with A and B being the limits of the 95% confidence interval.

Table 1: Final Test: Test Samples

Time of sampling
[days]

Concentration
[mg solids/L]

Relative to nominal
[%]

Relative to initial
[%]

Nominal

Analyzed

0

0

0.00338

n.a.

 

(fresh)

5

7.39

148

 

 

10

14.9

149

 

 

20

30.8

154

 

 

40

56.9

142

 

 

80

111

139

 

1

0

0.00163

n.a.

n.a.

(old)

5

6.98

140

94

 

10

14.6

146

98

 

20

28.8

144

93

 

40

58.9

147

104

 

80

118

148

106

3

0

0.00156

n.a.

 

(fresh)

5

6.37

127

 

 

10

13.1

131

 

 

20

23.3

116

 

4

0

0.000313

n.a.

n.a.

(old)

5

6.36

127

100

 

10

13.3

133

101

 

20

25.9

129

111

n.a. Not applicable.

 

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

Nominal conc.

(mg solids/L)

Initial
number
of fish

Cumulative mortality

Total
Mortality (%)

2h

24h

48h

72h

96h

Control

7

0

0

0

0

0

0

5.0

7

0

0

0

0

0

0

10

7

0

0

0

0

0

0

20

7

0

0

0

0

0

0

40

7

0

0

7

7

7

100

80

7

0

7

7

7

7

100

Validity criteria fulfilled:
yes
Remarks:
See 'Overall remarks'
Conclusions:
The 96h-LC50 was 28 mg solids/L based on analytically confirmed nominal exposure concentrations (95% confidence interval between 20 and 40 mg solids/L)
Executive summary:

In a study performed in accordance with OECD 203 (1992) and according to GLP principles, the acute toxicity of the test substance to Cyprinus carpio was investigated. The test item consisted of 38.4% solids in water and was completely soluble in test medium at the concentrations tested. A correction factor of 2.6 was used and concentrations/doses are expressed as mg solids/L.

In a semi-static test, carp were exposed for 96 hours to an untreated control and nominal test concentrations of 5.0, 10, 20, 40 and 80 mg solids/L. Measured concentrations at the start and end of the first and last renewal periods were at 116 -154% of the nominal test concentrations. Based on these results, the effect parameters were expressed based on nominal exposure concentrations.

No mortality or other effects were observed in the control and at the three lowest concentrations tested during the exposure period. All fish exposed to the highest concentration were found dead after the first 24 hours of exposure. All fish exposed to 40 mg solids/L were found dead after 48 hours of exposure.

The 96h-LC50 was calculated to be 28 mg solids/L based on analytically confirmed nominal exposure concentrations (95% confidence interval: 20 - 40 mg solids/L). The test met all validity criteria and is considered to be reliable without restriction.

Description of key information

In a study performed in accordance with OECD 203 (1992) and according to GLP principles, the acute toxicity of the test substance to Cyprinus carpio was investigated. The test item consisted of 38.4% solids in water and was completely soluble in test medium at the concentrations tested. A correction factor of 2.6 was used and concentrations/doses are expressed as mg solids/L.

In a semi-static test, carp were exposed for 96 hours to an untreated control and nominal test concentrations of 5.0, 10, 20, 40 and 80 mg solids/L. Measured concentrations at the start and end of the first and last renewal periods were at 116 -154% of the nominal test concentrations. Based on these results, the effect parameters were expressed based on nominal exposure concentrations.

No mortality or other effects were observed in the control and at the three lowest concentrations tested during the exposure period. All fish exposed to the highest concentration were found dead after the first 24 hours of exposure. All fish exposed to 40 mg solids/L were found dead after 48 hours of exposure.

The 96h-LC50 was calculated to be 28 mg solids/L based on analytically confirmed nominal exposure concentrations (95% confidence interval: 20 - 40 mg solids/L). The test met all validity criteria and is considered to be reliable without restriction.

Key value for chemical safety assessment

LC50 for freshwater fish:
28 mg/L

Additional information

96h-LC50: 28 mg/L, based on analytically confirmed test substance corrected for water content (and specified in the report as "solids").