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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
April 7 1986 - August 2 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Test method similar to OECD guideline 203
Principles of method if other than guideline:
- Principle of test: Acute toxicity of the test substance was determined in a 96 h test duration in Fathead Minnows (Pimephales promelas). 2 tests with the same test substance were performed.
- Short description of test conditions: see below (Test conditions)
- Parameters analysed / observed: LC50 and EC50 with corresponding 95% confidence intervals were calculated using the corrected average of the analyzed tank concentrations and the Trimmed Spearman-Karber Method (Hamilton et al.,1977)
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
Test 1 (nominal): 0, 16.2, 25.0, 38.5, 59.3 and 91.2 mg/L
Test 2 (nominal): 0, 15.5, 23.9, 36.8, 56.7 and 87.2 mg/L
- Sampling method: Gas-liquid chromatography (GLC)
- Sample storage conditions before analysis: All samples were analyzed immediately or adequately preserved for later analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A diluted stock of the test substance was obtained in a 18.9 L stainless steel carbonation vessel. Lake Superior water plus the test substance were added to the vessel and blended vigorously.
Test 1: 114 mg/L of diluted stock
Test 2: 110 mg/L of diluted stock
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnows
- Source: EPA Environmental Research Laboratory-Duluth and the University of Wisconsin-Superior campus
- Age at study initiation: Test 1: 32 days old; Test 2: 30 days old
- Length at study initiation (length definition, mean, range and SD): Test 1: 20.3 mm (mean), 1.888 mm (SD); Test 2: 17 mm (mean), 2.874 mm (SD).
- Weight at study initiation (mean and range, SD): Test 1: 0.127 g (mean), 0.037 g (SD); Test 2: 0.077 g (mean), 0.0418 g (SD).
- Method of breeding: Culture chambers were provided with asbestos or plastic pipes (cut in half longitudinally) as spawning substrates, to which the naturally spawned and fertilized embryos became attached to the undersides. The substrates, with intact embryos, were then transferred into another 25ºC bath where hatching ocurred or substrates were removed just prior to hatching at the UW-Superior culture unit, then placed in a rearing bath. Larvae were fed 40 to 48 hr old brine shrimp nauplii (Bio-Marine Research, Inc., Hawthorne, CA or San Francisco Bay Brand, Inc., Newarkk, CA) in excess two times daily (once daily on weekens).


ACCLIMATION
Fish that were 27-44 days old were used in the toxicity tests.
- Health during acclimation (any mortality observed): Only groups of fish having healthy appearance and no history of unusual thermal exposure or abnormally high mortality rate were used for tests.

FEEDING DURING TEST
Test fish were not fed 24 hr before or during a test.
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
Test 1: 45 mg/L (as CaCO3)
Test 2: 44.9 mg/L (as CaCO3)
Test temperature:
Test 1: 25.3 ºC
Test 2: 25.4 ºC
pH:
Test 1: 7.7
Test 2: 7.8
Dissolved oxygen:
Test 1: 6.9 mg/L
Test 2: 6.8 mg/L
Nominal and measured concentrations:
Nominal concentrations: Test 1: 0, 16.2, 25.0, 38.5, 59.3 and 91.2 mg/L; Test 2: 0, 15.5, 23.9, 36.8, 56.7 and 87.2 mg/L
Measured concentrations (aveg.): Test 1: <0.05, 16.1, 25.0, 37.4, 56.2, 85.3 mg/L; Test 2: <0.5, 18.5, 25.5, 32.3, 46.6, 82.1 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Continuous-flow modified mini-diluter (Benoit et al.,1982)
- Fill volume: 1 L
- Type of flow-through: Low-flow
- Renewal rate of test solution (frequency/flow rate): 10 mL/min (14 additions/day)
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: Test 1: 2.54 g/L; Test 2: 1.54 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Lake Superior water filtered through sand and a cotton fiber filter.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Number of dead fish was noted approximately every 24 hr after the beginning of a test, at which time they were also removed. The following observations of fish behaviour and toxic signs were made at 2-8, 24, 48, 72 and 96 hr: Loss of equilibria, general activity, locomotor activities, schooling behaviour, location in tank, startle response, body movements, body pigmentation and body of live fish.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 0.65
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
67.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
Test 1
Basis for effect:
mortality
Remarks on result:
other: LC50 calculated by corrected average of measured concentrations and trimmed spearman-karber method with 95% conf. lim. = 65.2-70.6 mg/L
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
47.8 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
Test 1
Basis for effect:
other: Loss of equilibrium (inability to maintain an upright position when swimming)
Remarks on result:
other: EC50 calculated by corrected average of measured concentrations and trimmed spearman-karber method with 95% conf. lim. = 44.3-51.6 mg/L
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
59 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
Test 2
Basis for effect:
mortality
Remarks on result:
other: LC50 calculated by corrected average of measured concentrations and trimmed spearman-karber method with 95% conf. lim. = 55.4-62.8 mg/L
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
33.5 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Remarks:
Test 2
Basis for effect:
other: Loss of equilibrium (inability to maintain an upright position when swimming)
Remarks on result:
other: EC50 calculated by corrected average of measured concentrations and trimmed spearman-karber method with 95% conf. lim. = 30.9-36.2 mg/L
Details on results:
- Behavioural abnormalities:
Test 1/2: Affected fish lost schooling behaviour were hyperactive and swam in a corkscrew/spiral pattern. They were also overreactive to external stimuli, had increased respiaration, rigid musculature and were darkly colored. Fish lost equilibrium prior to death.
Test 2: samples were not taken at 96 h.

Table1.Mortalities

 

Time (h)

Nominal concentrations (mg/L)

Test 1

Test 2

0

16.2

25

38.5

59.3

91.2

0

15.5

23.9

36.8

56.7

87.2

Initial

20

20

20

20

20

20

20

20

20

20

20

20

24

 

 

 

 

1

20

 

 

 

 

1

20

48

 

 

 

 

1

20

 

 

 

 

1

20

72

 

 

 

 

1

20

 

 

 

 

1

20

96

 

 

 

 

1

20

 

 

 

 

2

20

 

Table 2. Effect

 

Time (h)

Nominal concentrations (mg/L)

Test 1

Test 2

0

16.2

25

38.5

59.3

91.2

0

15.5

23.9

36.8

56.7

87.2

Initial

20

20

20

20

20

20

20

20

20

20

20

20

24

 

 

1

4

10

20

 

 

3

4

15

20

48

 

 

 

1

11

20

 

 

2

4

15

20

72

 

 

 

1

13

20

 

1

3

6

15

20

96

 

 

 

1

17

20

 

 

4

6

20

20

Validity criteria fulfilled:
yes
Remarks:
(mortality in control was 0%; constant conditions were maintained; the dissolved oxygen concentration was within the acceptable range; the concentrations were maintained during the tests).
Conclusions:
The LC50 of test item in Fathead Minnows (Pimephales promelas) at 96 h exposure period was determined to be between 59-67.8 mg/L.
Executive summary:

Two acute toxicity tests in Fathead Minnows (Pimephales promelas) were performed as described in the handbook "Acute Toxicities of Organic Chemicals to Fathead Minnows (Pimephales promelas)".

20 fish per group were exposed for each test to the following nominal concentrations over an exposure period of 96 h in a flow-through system: 0 (control), 16.2, 25.0, 38.5, 59.3 and 91.2 mg/L (test 1) and 0 (control), 15.5, 23.9, 36.8, 56.7 and 87.2 mg/L (test 2). The 96h-EC50 was determined to be between 33.5 -47.8 mg/L and the 96h-LC50 was determined to be between 59 -67.8 mg/L

Endpoint:
short-term toxicity to fish
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance L-Borneol which shares the same functional groups with the substance D-alpha fenchol also has comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose:
read-across source
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
67.8 mg/L
Nominal / measured:
estimated
Conc. based on:
other: Read-across from an analogue (test 1)
Basis for effect:
mortality
Remarks on result:
other: read-across from an analogue for which LC50 = 67.8 mg/L
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
47.8 mg/L
Nominal / measured:
estimated
Conc. based on:
other: Read-across from an analogue (test 1)
Basis for effect:
other: Loss of equilibrium (inability to maintain an upright position when swimming)
Remarks on result:
other: read-across from an analogue for which EC50 = 47.8 mg/L
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
59 mg/L
Nominal / measured:
estimated
Conc. based on:
other: Read-across from an analogue (test 2)
Basis for effect:
mortality
Remarks on result:
other: read-across from an analogue for which LC50 = 59 mg/L
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
33.5 mg/L
Nominal / measured:
estimated
Conc. based on:
other: Read-across from an analogue (test 2)
Basis for effect:
other: Loss of equilibrium (inability to maintain an upright position when swimming)
Remarks on result:
other: read-across from an analogue for which EC50 = 33.5 mg/L
Validity criteria fulfilled:
not applicable
Conclusions:
Based on the read-across approach from the analogue L-Borneol, the LC50 of the test item in Fathead Minnows (Pimephales promelas) at 96 h exposure period was determined to be between 59-67.8 mg/L
Executive summary:

Two acute toxicity tests in Fathead Minnows (Pimephales promelas) were performed on the analogue substance L-Borneol as described in the handbook "Acute Toxicities of Organic Chemicals to Fathead Minnows (Pimephales promelas)". 20 fish per group were exposed for each test to the following nominal concentrations over an exposure period of 96 h in a flow-through system: 0 (control), 16.2, 25.0, 38.5, 59.3 and 91.2 mg/L (test 1) and 0 (control), 15.5, 23.9, 36.8, 56.7 and 87.2 mg/L (test 2). The 96h-EC50 was determined to be between 33.5-47.8 mg/L and the 96h-LC50 was determined to be between 59-67.8 mg/L. Based on these results, the read-across was applied and the 96h-EC50 of the test item was determined to be between 33.5-47.8 mg/L and the 96h-LC50 was determined to be between 59-67.8 mg/L.

Description of key information

Key study: Read-across aproach. Based on the read-across approach from the analogue L-Borneol, the LC50 of the test item in Fathead Minnows (Pimephales promelas) at 96 h exposure period was determined to be between 59-67.8 mg/L.

Key value for chemical safety assessment

LC50 for freshwater fish:
59 mg/L

Additional information

Key study. Read-across approach from experimental data on the analogue substance L-Borneol: Two short-term toxicity tests on fish were performed as described in the handbook "Acute Toxicities of Organic Chemicals to Fathead Minnows (Pimephales promelas)". Based on the read across approach from the experimental data obtained with the analogue substance L-Borneol, the 96h-EC50 in Fathead Minnows (Pimephales promelas) of the test item was determined to be between 33.5-47.8 mg/L and the 96h-LC50 was determined to be between 59 -67.8 mg/L.