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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:
weight of evidence
Study period:
1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study conducted similarly to OECD guideline 203 but without GLP compliance and with minor deviations (no data on acclimation period). The report is sufficiently well documented.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
No data on acclimation period
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 1060, 2120, 3190, 4250 and 5310 μg/L
- Sampling time: 0, 24, 48, 72 and 96 hours
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test solutions was provided as liquid/liquid saturation units by adding excess of test chemical in 14 L of Lake Superior water
- Controls: Dilution water
- Chemical name of vehicle: water
- Concentration of vehicle in test medium: Stock (5310 µg/L liq-liq equil.)
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnows
- Source: ERL-Duluth, USA
- Age at study initiation: 30 days
- Length at study initiation: 16.8 mm (SD=2.7)
- Weight at study initiation: 57 mg (SD=30)
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
44.9 mg CaCO3/L (SD=0.2)
Test temperature:
25.2ºC (SD=0.6)
pH:
7.5 (SD=0.3)
Dissolved oxygen:
6.1 mg O2/L (SD=0.9)
Salinity:
42.7 mg CaCO3/L (SD=0.7)
Nominal and measured concentrations:
Nominal concentrations: 0, 1060, 2120, 3190, 4250 and 5310 μg/L
Average measured concentrations: <195, 1050, 2090, 2720, 3640 and 4820 μg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Six crystallizing dishes were used as test chambers in the electronic diluter with tank volumes of 200 mL; test solution depth: 4-5 cm
- Type (delete if not applicable): Closed
- Type of flow-through: Electronic diluter
- Renewal rate of test solution (frequency/flow rate): Flow rates of 21 mL/min to each tank produced toxicant concentrations of 1050-4820 μg/L
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): One
- No. of vessels per vehicle control (replicates): One
- Biomass loading rate: 2.85 g/L

TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: Lake Superior water
- Preparation of dilution water: Water was passed through sand filters, ultraviolet sterilizers and 55 µm fibre filter. Test waters were then aerated and heated at 25 ± 1 °C
- Intervals of water quality measurement: Tank temperatures (ºC), dissolved oxygen (mg/L) and pH were measured daily in each of the test chambers where test organisms were still alive

OTHER TEST CONDITIONS
- Photoperiod: 16 hour/day light cycle; cool white fluorescent light
- Light intensity: 12-28 lumens

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Behavior and morphological aberrations were observed at 0, 24, 48, 72 and 96 hours
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
3 150 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL: 2920-3400 μg/L
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
1 480 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: Affected fish swam with a corkscrew motion, were hypoactive and underreactive to external stimuli and were darkly colored. They also had convulsions and lost equilibrium prior to death.
- Mortality (96 hour) at <195, 1050, 2090, 2720, 3640 and 4820 μg/L (measured) were 0, 0, 0, 10, 90 and 100%, respectively.
- Effect (96 hour) at <195, 1050, 2090, 2720, 3640 and 4820 μg/L (measured) were observed in 0/10, 0/10, 10/10, 10/10, 10/10 and 10/10 fish respectively.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
- LC50 or EC50 values were calculated using the corrected average of the analyzed tank concentrations and the Trimmed Spearman-Karber Method (Hamilton et al., 1977).
- Mean toxicant concentrations used in the calculations were corrected for analytical spike recoveries

Table 1: Mortalities

 

Hour

Control (0 µg/L)

A (1060 µg/L)

B (2120 µg/L)

C (3190 µg/L)

D (4250 µg/L)

E (5310 µg/L)

Initial

9

10

10

10

10

10

24

0

0

0

1

7

10

48

0

0

0

1

7

10

72

0

0

0

1

9

10

96

0

0

0

1

9

10

Table 2: Effects

Hour

Control (0 µg/L)

A (1060 µg/L)

B (2120 µg/L)

C (3190 µg/L)

D (4250 µg/L)

E (5310 µg/L)

Initial

9

10

10

10

10

10

24

0

0

0

10

10

10

48

0

0

10

10

10

10

72

0

0

10

10

10

10

96

0

0

10

10

10

10

 

Validity criteria fulfilled:
yes
Remarks:
(mortality in control < 10%; flow-through test conditions; dissolved O2 concentration > 60% of air saturation value; results based on measured concentrations)
Conclusions:
The 96h-LC50 of alpha terpinene to fathead minnow (Pimephales promelas) was calculated to be 3150 (2920 -3400) μg/L.

Executive summary:

In an acute aquatic toxicity study performed similarly to OECD guideline 203, groups (10/dose) of fathead minnows (Pimephales promelas) were exposed to alpha terpinene at concentrations of <195, 1050, 2090, 2720, 3640 and 4820 μg/L (measured) for 96 hours under flow-through conditions. Mortality, behaviour and morphological aberrations were observed at 24, 48, 72 and 96 hours. All validity criteria were fulfilled. Mortalities (96 hour) at <195, 1050, 2090, 2720, 3640 and 4820 μg/L (measured) were 0, 0, 0, 10, 90 and 100%, respectively. Treatment-related effects (96 hour) at <195, 1050, 2090, 2720, 3640 and 4820 μg/L (measured) were observed in 0/10, 0/10, 10/10, 10/10, 10/10 and 10/10 fish respectively. The 96 hour LC50, EC50 and NOEC of alpha terpinene to fathead minnow (Pimephales promelas) were calculated to be 3150 (2920 -3400) μg/L, 1480 μg/L and 1050 μg/L, respectively.

 

 

 

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study conducted similarly to OECD guideline 203 but without GLP compliance and with minor deviations (no data on acclimation period). The report is sufficiently well documented.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
No data on acclimation period
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 130, 260, 390, 520 and 650 μg/L
- Sampling time: 0, 24, 48, 72 and 96 hours
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test solutions was provided as liquid/liquid saturation units by adding excess of test chemical in 14 L of Lake Superior water
- Controls: Dilution water
- Chemical name of vehicle: water
- Concentration of vehicle in test medium: Stock (650 µg/L liq-liq equil.)
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnows
- Source: ERL-Duluth, USA
- Age at study initiation: 30 days
- Length at study initiation: 20.9 mm (SD=2.2)
- Weight at study initiation: 128 mg (SD=35)
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
45.2 mg CaCO3/L (SD=0.3)
Test temperature:
25.2ºC (SD=0.2)
pH:
7.7 (SD=0.2)
Dissolved oxygen:
6.7 mg O2/L (SD=0.7)
Salinity:
42.2 mg CaCO3/L (SD=0.9)
Nominal and measured concentrations:
Nominal concentrations: 0, 130, 260, 390, 520 and 650 μg/L
Average measured concentrations: <31, 58, 140, 229, 303 and 424 μg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Six crystallizing dishes were used as test chambers in the electronic diluter with tank volumes of 200 mL; test solution depth: 4-5 cm
- Type (delete if not applicable): Closed
- Type of flow-through: Electronic diluter
- Renewal rate of test solution (frequency/flow rate): Flow rates of 21 mL/min to each tank produced toxicant concentrations of 58-424 μg/L
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): One
- No. of vessels per vehicle control (replicates): One
- Biomass loading rate: 6.4 g/L

TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: Lake Superior water
- Preparation of dilution water: Water was passed through sand filters, ultraviolet sterilizers and 55 µm fibre filter. Test waters were then aerated and heated at 25 ± 1 °C
- Intervals of water quality measurement: Tank temperatures (ºC), dissolved oxygen (mg/L) and pH were measured daily in each of the test chambers where test organisms were still alive

OTHER TEST CONDITIONS
- Photoperiod: 16 hour/day light cycle; cool white fluorescent light
- Light intensity: 12-28 lumens

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Behavior and morphological aberrations were observed at 0, 24, 48, 72 and 96 hours
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
280 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL: 259-303 μg/L
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
179 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: Affected fish swam near the tank bottom, were hypoactive and underreactive to external stimuli, exhibited hemorrhaging and lost equilibrium prior to death.
- Mortality (96 hour) at <31, 58, 140, 229, 303 and 424 μg/L (measured) were 0, 0, 0, 0, 80 and 100%, respectively.
- Effect (96 hour) at <31, 58, 140, 229, 303 and 424 μg/L μg/L (measured) were observed in 0/10, 0/10, 0/10, 10/10, 10/10 and 10/10 fish respectively.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Measured concentrations were lower than nominal concentrations due to volatility.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
- LC50 or EC50 values were calculated using the corrected average of the analyzed tank concentrations and the Trimmed Spearman-Karber Method (Hamilton et al., 1977).
- Mean toxicant concentrations used in the calculations were corrected for analytical spike recoveries

Table 1: Mortalities

 

Hour

Control (0 µg/L)

A (130 µg/L)

B (260 µg/L)

C (390 µg/L)

D (520 µg/L)

E (650 µg/L)

Initial

10

10

9

10

10

10

24

0

0

0

0

7

8

48

0

0

0

0

8

10

72

0

0

0

0

8

10

96

0

0

0

0

8

10

Table 2: Effects

Hour

Control (0 µg/L)

A (130 µg/L)

B (260 µg/L)

C (390 µg/L)

D (520 µg/L)

E (650 µg/L)

Initial

10

10

9

10

10

10

24

0

0

0

10

10

10

48

0

0

0

10

10

10

72

0

0

0

10

10

10

96

0

0

0

10

10

10

 

Validity criteria fulfilled:
yes
Remarks:
(mortality in control < 10%; flow-through test conditions; dissolved O2 concentration > 60% of air saturation value; results based on measured concentrations)
Conclusions:
The 96h-LC50 of d-alpha pinene to fathead minnow (Pimephales promelas) was calculated to be 280 (259 -303) μg/L.

Executive summary:

In an acute aquatic toxicity study performed similarly to OECD guideline 203, groups (10/dose) of fathead minnows (Pimephales promelas) were exposed to d-alpha pinene at concentrations of <31, 58, 140, 229, 303 and 424 μg/L (measured) for 96 hours under flow-through conditions. Mortality, behaviour and morphological aberrations were observed at 24, 48, 72 and 96 hours. All validity criteria were fulfilled.Mortalities (96 hour) at <31, 58, 140, 229, 303 and 424 μg/L (measured) were 0, 0, 0, 0, 80 and 100%, respectively. Treatment-related effects (96 hour) at <31, 58, 140, 229, 303 and 424 μg/L (measured) were observed in 0/10, 0/10, 0/10, 10/10, 10/10 and 10/10 fish respectively. The 96 hour LC50, EC50 and NOEC of d-alpha pinene to fathead minnow (Pimephales promelas) were calculated to be 280 (259 -303) μg/L, 179 μg/L and 140 μg/L, respectively.

 

 

 

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study conducted similarly to OECD guideline 203 but without GLP compliance and with minor deviations (no data on acclimation period). The report is sufficiently well documented.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
No data on acclimation period
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 372, 744, 1120, 1490 and 1860 μg/L
- Sampling time: 0, 24, 48, 72 and 96 hours
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test solutions was provided as liquid/liquid saturation units by adding excess of test chemical in 14 L of Lake Superior water
- Controls: Dilution water
- Chemical name of vehicle: water
- Concentration of vehicle in test medium: Stock (1810 µg/L liq-liq equil.)
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnows
- Source: ERL-Duluth, USA
- Age at study initiation: 32/34 days
- Length at study initiation: 21.8 ± 3.9 mm
- Weight at study initiation: 177 ± 84 mg
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
46.2 mg CaCO3/L (SD=0.3)
Test temperature:
24.4ºC (SD=0.6)
pH:
7.6 (SD=0.10)
Dissolved oxygen:
5.8 mg O2/L (SD=0.5)
Salinity:
39.5 mg CaCO3/L (SD=0.7)
Nominal and measured concentrations:
Nominal concentrations: 0, 372, 744, 1120, 1490 and 1860 μg/L
Average measured concentrations: <150, 178, 386, 595, 928 and 1100 μg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Six crystallizing dishes were used as test chambers in the electronic diluter with tank volumes of 200 mL; test solution depth: 4-5 cm
- Type (delete if not applicable): Closed
- Type of flow-through: Electronic diluter
- Renewal rate of test solution (frequency/flow rate): Flow rates of 21 mL/min to each tank produced toxicant concentrations of 178-1100 μg/L
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): One
- No. of vessels per vehicle control (replicates): One
- Biomass loading rate: 7.08 g/L

TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: Lake Superior water
- Preparation of dilution water: Water was passed through sand filters, ultraviolet sterilizers and 55 µm fibre filter. Test waters were then aerated and heated at 25 ± 1ºC
- Intervals of water quality measurement: Tank temperatures (ºC), dissolved oxygen (mg/L) and pH were measured daily in each of the test chambers where test organisms were still alive

OTHER TEST CONDITIONS
- Photoperiod: 16 hour/day light cycle; cool white fluorescent light
- Light intensity: 12-28 lumens

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Behavior and morphological aberrations were observed at 0, 24, 48, 72 and 96 hours
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
702 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % CL: 619-796 μg/L
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
702 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % CL: 619-796 μg/L
Details on results:
- Behavioural abnormalities: Affected fish were hyperactive and swam in a sporadic manner. They were overreactive to external stimuli and experienced hemorrhaging. Equilibrium loss was observed prior to death.
- Mortality (96 hour) at <150, 178, 386, 595, 928 and 1100 μg/L (measured) were 0, 0, 0, 20, 90 and 100%, respectively.
- Effect (96 hour) at <150, 178, 386, 595, 928 and 1100 μg/L (measured) were observed in 0/10, 0/10, 0/10, 2/10, 9/10 and 10/10 fish respectively.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Measured concentrations were lower than nominal concentrations due to volatility.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
- LC50 or EC50 values were calculated using the corrected average of the analyzed tank concentrations and the Trimmed Spearman-Karber Method (Hamilton et al., 1977).
- Mean toxicant concentrations used in the calculations were corrected for analytical spike recoveries

Table 1: Mortalities

 

Hour

Control (0 µg/L)

A (372 µg/L)

B (744 µg/L)

C (1120 µg/L)

D (1490 µg/L)

E (1860 µg/L)

Initial

10

10

10

10

10

10

24

0

0

0

2

9

6

48

0

0

0

2

9

8

72

0

0

0

2

9

10

96

0

0

0

2

9

10

Table 2: Effects

Hour

Control (0 µg/L)

A (372 µg/L)

B (744 µg/L)

C (1120 µg/L)

D (1490 µg/L)

E (1860 µg/L)

Initial

10

10

10

10

10

10

24

0

0

0

10

10

10

48

0

0

0

2

10

10

72

0

0

0

2

10

10

96

0

0

0

2

9

10

 

Validity criteria fulfilled:
yes
Remarks:
(mortality in control < 10%; flow-through test conditions; dissolved O2 concentration > 60% of air saturation value; results based on measured concentrations)
Conclusions:
The 96h-LC50 of d-limonene to fathead minnow (Pimephales promelas) was calculated to be 702 (619 -796) μg/L.

Executive summary:

In an acute aquatic toxicity study performed similarly to OECD guideline 203, groups (10/dose) of fathead minnows (Pimephales promelas) were exposed to d-limonene (purchased from Aldrich) at concentrations of <150, 178, 386, 595, 928 and 1100 μg/L (measured) for 96 hours under flow-through conditions. Mortality, behaviour and morphological aberrations were observed at 24, 48, 72 and 96 hours. All validity criteria were fulfilled. Mortalities (96 hour) at <150, 178, 386, 595, 928 and 1100 μg/L (measured) were 0, 0, 0, 20, 90 and 100%, respectively. Treatment-related effects (96 hour) at <150, 178, 386, 595, 928 and 1100 μg/L (measured) were observed in 0/10, 0/10, 0/10, 2/10, 9/10 and 10/10 fish respectively. The 96 hour LC50, EC50 and NOEC of d-limonene to fathead minnow (Pimephales promelas) were calculated to be 702 (619 -796) μg/L, 702 (619 -796) μg/L and 386 μg/L, respectively.

 

 

 

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study conducted similarly to OECD guideline 203 but without GLP compliance and with minor deviations (no data on acclimation period). The report is sufficiently well documented.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
No data on acclimation period
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 550, 1090, 1640, 2180 and 2730 μg/L
- Sampling time: 0, 24, 48, 72 and 96 hours
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test solutions was provided as liquid/liquid saturation units by adding excess of test chemical in 14 L of Lake Superior water
- Controls: Dilution water
- Chemical name of vehicle: water
- Concentration of vehicle in test medium: Stock (2730 µg/L liq-liq equil.)
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnows
- Source: ERL-Duluth, USA
- Age at study initiation: 34 days
- Length at study initiation: 19.1 mm (SD=2.1)
- Weight at study initiation: 85 mg (SD=34)
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
no data
Test temperature:
25.4 ºC (SD=0.3)
pH:
7.4 (SD=0.1)
Dissolved oxygen:
6.9 mg O2/L (SD=0.3)
Nominal and measured concentrations:
Nominal concentrations: 0, 550, 1090, 1640, 2180 and 2730 μg/L
Average measured concentrations: <49, 251, 564, 961, 1380 and 1890 μg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Six crystallizing dishes were used as test chambers in the electronic diluter with tank volumes of 200 mL; test solution depth: 4-5 cm
- Type (delete if not applicable): Closed
- Type of flow-through: Electronic diluter
- Renewal rate of test solution (frequency/flow rate): Flow rates of 21 mL/min to each tank produced toxicant concentrations of 251-1890 μg/L
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): One
- No. of vessels per vehicle control (replicates): One
- Biomass loading rate: 4.25 g/L

TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: Lake Superior water
- Preparation of dilution water: Water was passed through sand filters, ultraviolet sterilizers and 55 µm fibre filter. Test waters were then aerated and heated at 25 ± 1ºC
- Intervals of water quality measurement: Tank temperatures (ºC), dissolved oxygen (mg/L) and pH were measured daily in each of the test chambers where test organisms were still alive

OTHER TEST CONDITIONS
- Photoperiod: 16 hour/day light cycle; cool white fluorescent light
- Light intensity: 12-28 lumens

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Behavior and morphological aberrations were observed at 0, 24, 48, 72 and 96 hours
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
720 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95 % CL: 618-839 μg/L
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
688 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % CL: 606-782 μg/L
Details on results:
- Behavioural abnormalities: Affected fish were hypoactive and underreactive to external stimuli. They were also darkly colored and lost equilibrium prior to death.
- Mortality (96 hour) at <49, 251, 564, 961, 1380 and 1890 μg/L (measured) were 0, 0, 10, 90, 100 and 100%, respectively.
- Effect (96 hour) at <49, 251, 564, 961, 1380 and 1890 μg/L (measured) were observed in 0/10, 0/10, 1/10, 10/10, 10/10 and 10/10 fish respectively.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Measured concentrations were lower than nominal concentrations due to volatility.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
- LC50 or EC50 values were calculated using the corrected average of the analyzed tank concentrations and the Trimmed Spearman-Karber Method (Hamilton et al., 1977).
- Mean toxicant concentrations used in the calculations were corrected for analytical spike recoveries

Table 1: Mortalities

 

Hour

Control (0 µg/L)

A (550 µg/L)

B (1090 µg/L)

C (1640 µg/L)

D (2180 µg/L)

E (2730 µg/L)

Initial

10

10

10

10

10

10

24

0

0

1

9

10

10

48

0

0

1

9

10

10

72

0

0

1

9

10

10

96

0

0

1

9

10

10

Table 2: Effects

Hour

Control (0 µg/L)

A (550 µg/L)

B (1090 µg/L)

C (1640 µg/L)

D (2180 µg/L)

E (2730 µg/L)

Initial

10

10

10

10

10

10

24

0

0

1

10

10

10

48

0

0

10

10

10

10

72

0

0

10

10

10

10

96

0

0

1

10

10

10

 

Validity criteria fulfilled:
yes
Remarks:
(mortality in control < 10%; flow-through test conditions; dissolved O2 concentration > 60% of air saturation value; results based on measured concentrations)
Conclusions:
The 96h-LC50 of d-limonene to fathead minnow (Pimephales promelas) was calculated to be 720 (618 -839) μg/L.

Executive summary:

In an acute aquatic toxicity study performed similarly to OECD guideline 203, groups (10/dose) of fathead minnows (Pimephales promelas) were exposed to d-limonene (purchased from Fluorostat) at concentrations of <49, 251, 564, 961, 1380 and 1890 μg/L (measured) for 96 hours under flow-through conditions. Mortality, behaviour and morphological aberrations were observed at 24, 48, 72 and 96 hours. All validity criteria were fulfilled. Mortalities (96 hour) at <49, 251, 564, 961, 1380 and 1890 μg/L (measured) were 0, 0, 10, 90, 100 and 100%, respectively. Treatment-related effects (96 hour) at <49, 251, 564, 961, 1380 and 1890 μg/L (measured) were observed in 0/10, 0/10, 1/10, 10/10, 10/10 and 10/10 fish respectively. The 96 hour LC50, EC50 and NOEC of d-limonene to fathead minnow (Pimephales promelas) were calculated to be 720 (618 -839) μg/L, 688 (606 -782) μg/L and 251 μg/L, respectively.

 

 

 

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1989
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Study conducted similarly to OECD guideline 203 but without GLP compliance and with minor deviations (no data on acclimation period). The report is sufficiently well documented.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
No data on acclimation period
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 450, 900, 1340, 1790 and 2240 μg/L
- Sampling time: 0, 24, 48, 72 and 96 hours
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test solutions was provided as liquid/liquid saturation units by adding excess of test chemical in 14 L of Lake Superior water
- Controls: Dilution water
- Chemical name of vehicle: water
- Concentration of vehicle in test medium: Stock (2240 µg/L liq-liq equil.)
Test organisms (species):
Pimephales promelas
Details on test organisms:
TEST ORGANISM
- Common name: Fathead minnows
- Source: ERL-Duluth, USA
- Age at study initiation: 30 days
- Length at study initiation: 16.3 mm (SD=1.6)
- Weight at study initiation: 59 mg (SD=18)
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
45.1 mg CaCO3/L (SD=0.2)
Test temperature:
25.0ºC (SD=0.2)
pH:
7.6 (SD=0.12)
Dissolved oxygen:
6.3 mg O2/L (SD=0.9)
Salinity:
42.6 mg CaCO3/L (SD=0.7)
Nominal and measured concentrations:
Nominal concentrations: 0, 450, 900, 1340, 1790 and 2240 μg/L
Average measured concentrations: <33, 279, 645, 902, 1360 and 1670 μg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: Six crystallizing dishes were used as test chambers in the electronic diluter with tank volumes of 200 mL; test solution depth: 4-5 cm
- Type (delete if not applicable): Closed
- Type of flow-through: Electronic diluter
- Renewal rate of test solution (frequency/flow rate): Flow rates of 21 mL/min to each tank produced toxicant concentrations of 279-1670 μg/L
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): One
- No. of vessels per vehicle control (replicates): One
- Biomass loading rate: 2.66 g/L

TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: Lake Superior water
- Preparation of dilution water: Water was passed through sand filters, ultraviolet sterilizers and 55 µm fibre filter. Test waters were then aerated and heated at 25 ± 1 °C
- Intervals of water quality measurement: Tank temperatures (ºC), dissolved oxygen (mg/L) and pH were measured daily in each of the test chambers where test organisms were still alive

OTHER TEST CONDITIONS
- Photoperiod: 16 hour/day light cycle; cool white fluorescent light
- Light intensity: 12-28 lumens

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Behavior and morphological aberrations were observed at 0, 24, 48, 72 and 96 hours
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1 210 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL: 1110-1330 μg/L
Key result
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
763 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: Affected fish swam in an almost vertical position, were underreactive to external stimuli and had convulsions. Equilibrium loss was observed prior to death.
- Mortality (96 hour) at <33, 279, 645, 902, 1360 and 1670 μg/L (measured) were 0, 0, 0, 0, 70 and 100%, respectively.
- Effect (96 hour) at <33, 279, 645, 902, 1360 and 1670 μg/L (measured) were observed in 0/10, 0/10, 0/10, 10/10, 10/10 and 10/10 fish respectively.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Measured concentrations were lower than nominal concentrations due to volatility.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
- LC50 or EC50 values were calculated using the corrected average of the analyzed tank concentrations and the Trimmed Spearman-Karber Method (Hamilton et al., 1977).
- Mean toxicant concentrations used in the calculations were corrected for analytical spike recoveries

Table 1: Mortalities

 

Hour

Control (0 µg/L)

A (450 µg/L)

B (900 µg/L)

C (1340 µg/L)

D (1790 µg/L)

E (2240 µg/L)

Initial

9

10

10

10

10

10

24

0

0

0

0

5

9

48

0

0

0

0

6

10

72

0

0

0

0

7

10

96

0

0

0

0

7

10

Table 2: Effects

Hour

Control (0 µg/L)

A (450 µg/L)

B (900 µg/L)

C (1340 µg/L)

D (1790 µg/L)

E (2240 µg/L)

Initial

9

10

10

10

10

10

24

0

0

0

0

7

10

48

0

0

0

10

10

10

72

0

0

0

0

10

10

96

0

0

0

10

10

10

 

Validity criteria fulfilled:
yes
Remarks:
(mortality in control < 10%; flow-through test conditions; dissolved O2 concentration > 60% of air saturation value; results based on measured concentrations)
Conclusions:
The 96h-LC50 of terpinolene to fathead minnow (Pimephales promelas) was calculated to be 1210 (1110 -1330) μg/L.

Executive summary:

In an acute aquatic toxicity study performed similarly to OECD guideline 203, groups (10/dose) of fathead minnows (Pimephales promelas) were exposed to terpinolene at concentrations of <33, 279, 645, 902, 1360 and 1670 μg/L (measured) for 96 hours under flow-through conditions. Mortality, behaviour and morphological aberrations were observed at 24, 48, 72 and 96 hours. All validity criteria were fulfilled.Mortalities (96 hour) at <33, 279, 645, 902, 1360 and 1670 μg/L (measured) were 0, 0, 0, 0, 70 and 100%, respectively. Treatment-related effects (96 hour) at <33, 279, 645, 902, 1360 and 1670 μg/L (measured) were observed in 0/10, 0/10, 0/10, 10/10, 10/10 and 10/10 fish respectively. The 96 hour LC50, EC50 and NOEC of terpinolene to fathead minnow (Pimephales promelas) were calculated to be 1210 (1110 -1330)μg/L, 763 μg/L and 645 μg/L, respectively.

 

 

 

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with national standard methods with acceptable restrictions
Remarks:
Test method according to US EPA (1975): "Methods for acute toxicity tests with fish, macroinvertebrates and amphibiens".
Qualifier:
according to
Guideline:
other: US EPA (1975): "Methods for acute toxicity tests with fish, macroinvertebrates and amphibiens"
Deviations:
not specified
GLP compliance:
no
Analytical monitoring:
not specified
Vehicle:
not specified
Test organisms (species):
Cyprinodon variegatus
Details on test organisms:
TEST ORGANISM
- Common name: Sheepshead Minnows
- Source: Animals were hatched and reared at BMRL or obtained from the EPA's Environmental Research Laboratory, Gulf Breeze, FL.
- Age at study initiation (mean and range, SD): 14-28 days old posthatch
- Length at study initiation (length definition, mean, range and SD): 8 - 15 mm standard length
- Weight at study initiation (mean and range, SD):
- Method of breeding:
- Maintenance of the brood fish:

ACCLIMATION
- Acclimation conditions (same as test or not): Flowing, filtered (10-µm) seawater
- Type and amount of food/Feeding frequency: Fish were fed 24-h Artemia salina nauplii (San Francisco Bay Brand) daily until they were used as test animals.
- Health during acclimation (any mortality observed): Fish mortality during the 48-h period immediately before each test was less than 3% and all fish tested appeared to be in good condition.

FEEDING DURING TEST: No
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
96 h
Test temperature:
25-31 ºC
Salinity:
1.0-3.1 %
Nominal and measured concentrations:
No data on test concentrations
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Material, size, headspace, fill volume: Tests were conducted in either 4-L glass jars that contained 3 L of test solution or 19-L glass jars that contained 15 L
- Aeration: No
- No. of organisms per vessel: 10

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: All dilution water was filtered (5-µm), natural seawater of ambient salinity.
- Intervals of water quality measurement: The dissolved oxygen concentration was measured in each test container at initiation of testing and daily thereafter; pH was measured in the control and the low and high test concentrations at the initiation and after 96 h of testing.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Mortality was recorded at 24, 48, 72, and 96 h.
Reference substance (positive control):
not specified
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
1.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: (95% confidence limits: 1.6-2.2 ppm)
Key result
Duration:
96 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: (95% confidence limits: 1.6-2.4 ppm)
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
2 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: (95% confidence limits: 1.6-2.4 ppm)
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
1.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: (95% confidence limits: 1.0-3.2 ppm)
Reported statistics and error estimates:
Based on the results of the tests, 24-, 48-, 72-, and 96-h LC50's and their 95% confidence limits were calculated when possible. Calculations were performed with a Hewlett Packard Model 9815A calculator programmed (STEPHAN 1977 and 1978 personal communication) to scan the data base and to select the most appropriate of three statistical methods in the following order: moving average angle analysis, probit analysis, or binomial probability. The method selected was determined by the characteristics of the data, that is, the presence or absence of 0% and 100% mortality and the number of concentrations in which >0%<100% mortality occurred. The computer scanned the data, identified the most suited method, and performed the analyses. No correction was made for control mortality.

The LC50 (96 h) was 1.9 ppm (basis for effect: mortality). The NOEC was 1.0 ppm.

Validity criteria fulfilled:
not specified
Remarks:
(No data on control results. There is no evidence that test substance concentration had been maintained throughout the test period).
Conclusions:
The LC50 (96 h) was 1.9 ppm (basis for effect: mortality).
Executive summary:

A marine toxicity static test were conducted at EG&G Bionomics Marine Research Laboratory (BMRL) with sheepshead minnows (Cyprinodon variegatus). Test method was according to US EPA (1975): "Methods for acute toxicity tests with fish, macroinvertebrates and amphibiens". Camphene was tested for 96 hours at 25 -31 ºC. Mortality was recorded at 24, 48, 72, and 96 h. The 96 h-LC50 was 1.9 ppm (basis for effect: mortality). The NOEC was 1.0 ppm.

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
11 January 1993 - 18 February 1993
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Test method was according to OECD guideline 203 and GLP.
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0.4, 0.7, 1.3, 2.3 and 4.2 mg/l
- Sample storage conditions before analysis: samples were analysed immediately.
Vehicle:
no
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Zebrafish
- Source: Aquarium am Aegi, W-3000 Hannover
- Length at study initiation (length definition, mean, range and SD): 2,82 cm
- Weight at study initiation (mean and range, SD): 0.52 g
- Feeding during test: No feeding during test

ACCLIMATION
- Acclimation period: Gem OECD Guideline
- Type and amount of food: The amount of feed was 2% of fish weight. "Tetra Min" of the company, Tetra Werke, Melle.
- Feeding frequency: 6 times a week
- Health during acclimation (any mortality observed): No mortalities observed
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Hardness:
140.8 mg/L CaCO3
Test temperature:
22-23 °C
pH:
7.98 - 8.16
Dissolved oxygen:
91 - 99 %
Nominal and measured concentrations:
Measured concentrations: 2.07, 1.21, 0.43, 0.26 and 0.048 mg/L (mean values)
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 15-L aquariums, filled with 10 L and covered with cellophane foil and cling film on the inert surface.
- Aeration: The dilution water in the feed tank was aerated during the test to achieve an adequate oxygen saturation in the test vessel.
- Renewal rate of test solution (frequency/flow rate): The entire test medium (10 L) was replaced within 2 hours.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): 2.07 mg/L: 3; 1.21 mg/L: 2; Other concentrations: 1.
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Intervals of water quality measurement: At beginning of test, after 24, 48, 72 and 96 h, the water parameters (pH, 02 saturation and temperature) were measured and recorded.

OTHER TEST CONDITIONS
- Photoperiod: 12 vhours light
- Light intensity: Diffuse light (3.0 - 27 µmol/m2s)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): At beginning of test, after 24, 48, 72 and 96 h, mortality and behaviour of fish were observed and recorded (in test and control vessels).
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
0.72 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: (95%CL 0.43-1.21 mg/L)
Duration:
96 h
Dose descriptor:
LC0
Effect conc.:
0.43 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
96 h
Dose descriptor:
LC100
Effect conc.:
1.21 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
0.94 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: (95%CL 0.74-1.19 mg/L)
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
1.21 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: (95%CL 1.04-1.42 mg/L)
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
1.4 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: (95%CL 1.18-1.80 mg/L)
Reported statistics and error estimates:
The confidence interval calculation was based on "Methods of water testing" Volume II (1982), VEB Gustav Fischer Verlag, Jena.
  Cumulative lethalities %  
Concentration (mg/L) 24 (h) 48 (h) 72 (h) 96 (h)
2.07 100 100 100 100
1.21 10 50 80 100
0.43 0 0 0 0
0.26 0 0 0 0
0.048 0 0 0 0
control 0 0 0 0

Table 06.01.01_01. Mortality.

Validity criteria fulfilled:
yes
Remarks:
(Concentration of the dissolved Oxygen: > = 60 %; mortality in the control group < = 10%; analytical monitoring of test substance concentration).
Conclusions:
After 96 hours, the LC50 was 0.72 mg/L for zebra fish.
Executive summary:
An acute toxicity test with Camphene on zebrafish (Brachydanio rerio) in a flow system was performed in accordance with OECD Guideline 203 for 96 hours. It was conducted in the period from 11.01.1993 until 15.01.1993 under GLP conditions. The medium in the test vessel (10 L) was replaced within 2 h. Due to the low water solubility of the substance and its high vapor pressure, the test was conducted in a closed system. The analytical test concentrations were detected during the test: 2.07, 1.21, 0.43, 0.26 and 0.048 mg / L. The mortality and general clinical effects were daily observed and recorded. After 96 hours, the LC50 was 0.72 mg/L.
Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
22 April 1988 - 26 April 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Test method was similar to guideline OECD 203
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
- 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 (665 mg/L) 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 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: 28 days old
- Length at study initiation (length definition, mean, range and SD): 19.1 mm (mean), 0.933 mm (SD)
- Weight at study initiation (mean and range, SD): 0.097 g (mean), 0.0126 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:
43.9 (SD=0.25) mg/L (as CaCO3)
Test temperature:
25.1 (SD=0.62) ºC
pH:
7.9 (SD=0.06)
Dissolved oxygen:
6.9 (SD=0.26) mg/L
Nominal and measured concentrations:
Nominal concentrations: 0, 48.2, 74.1, 114, 176 and 270 mg/L
Measured concentrations (corrected aveg.): <0.99, 40.3, 50.8, 75.9, 124 and 203 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 1 L tank
- Type of flow-through (e.g. peristaltic or proportional diluter): continuous-flow modified mini-diluter (Benoit et al.,1982)
- Renewal rate of test solution (frequency/flow rate): 14.4 volumes per day (10 mL/min)
- No. of organisms per vessel: 20
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- Biomass loading rate: 1.940 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: 1.5
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
102 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: LC50 calculated using corrected average of measured concentrations and trimmed spearman-karber method with 95% conf. lim. = 95.4-109 mg/L
Duration:
96 h
Dose descriptor:
EC50
Effect conc.:
102 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: EC50 calculated using corrected average of measured concentrations and trimmed spearman-karber method with 95% conf. lim. = 95.4-109 mg/L
Details on results:
- Behavioural abnormalities: Affected fish lost schooling behaviour, swam near the tank surface and were hyperactive. They were also overreactive to external stimuli, had increased respiaration and lost equilibrium prior to death.
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: samples were not taken at 96 h. Measured concentrations were much lower than nominal concentrations due to volatility.
Reported statistics and error estimates:
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)

Table1.Mortalities

Time (h)

Nominal concentrations (mg/L)

0

48.2

74.1

114

176

270

Initial

20

20

20

20

20

20

5

-

-

-

-

8

20

24

-

-

-

-

18

20

48

-

-

-

-

18

20

72

-

-

-

-

18

20

96

-

-

-

-

18

20

 

Table 2. Effect

Time (h)

Nominal concentrations (mg/L)

0

48.2

74.1

114

176

270

Initial

20

20

20

20

20

20

5

-

-

-

-

8

20

24

-

-

-

-

18

20

48

-

-

-

-

18

20

72

-

-

-

-

18

20

96

-

-

-

-

18

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 102 mg/L.

Executive summary:

An acute toxicity test in Fathead Minnows (Pimephales promelas) was performed following a method similar to OECD guideline 203. 20 fish per group were exposed over an exposure period of 96 h in a flow-through system to the following nominal concentrations: 0 (control), 48.2, 74.1, 114, 176 and 270 mg/L. Gas-liquid chromatography was used to monitor the concentration throughout the test. LC50 and EC50 with corresponding 95% confidence intervals were calculated using corrected average of measured concentrations and trimmed spearman-karber method. Both 96h-EC50 and 96h-LC50 were determined to be 102 mg/L (95% CL = 95.4-109 mg/L).

Description of key information

Weight of evidence: Data on short-term toxicity to fish of the main constituents are available from experimental data:

Terpinolene: Test method similar to OECD TG 203 (Broderius S, 1990). The 96h-LC50 was determined to be 1.21 mg/L in fathead minnow (Pimephales promelas).

Camphene: Test method according to OECD TG 203 (CABB, 1993). The 96h-LC50 was determined to be 0.720 mg/L in zebrafish (Brachydanio rerio).

D-Limonene (test001): Test method similar to OECD TG 203 (Broderius S, 1990). The 96h-LC50 was determined to be 0.702 mg/L in fathead minnow (Pimephales promelas).

D-Limonene(test002): Test method similar to OECD TG 203 (Broderius S, 1990). The 96h-LC50 was determined to be 0.720 mg/L in fathead minnow (Pimephales promelas).

Alpha terpinene: Test method similar to OECD TG 203 (Broderius S, 1990). The 96h-LC50 was determined to be 3.15 mg/L in fathead minnow (Pimephales promelas).

D-alpha pinene: Test method similar to OECD TG 203 (Broderius S, 1990). The 96h-LC50 was determined to be 0.28 mg/L in fathead minnow (Pimephales promelas).

Cineole: Test method similar to OECD TG 203 (Geiger D L, 1988). The 96h-LC50 was determined to be 102 mg/L in fathead minnow (Pimephales promelas).

Based on the available data on its components, a weight of evidence approach is applied and the 96h-LC50 of the substance in fish is calculated to be 0.89 mg/L.

Key value for chemical safety assessment

LC50 for freshwater fish:
0.89 mg/L

Additional information

Weight of evidence from experimental results with individual components:

Terpinolene: In an acute aquatic toxicity study performed similarly to OECD guideline 203, groups (10/dose) of fathead minnows (Pimephales promelas) were exposed to terpinolene at concentrations of <33, 279, 645, 902, 1360 and 1670 μg/L (measured) for 96 hours under flow-through conditions. Mortality, behaviour and morphological aberrations were observed at 24, 48, 72 and 96 hours. All validity criteria were fulfilled.Mortalities (96 hour) at <33, 279, 645, 902, 1360 and 1670 μg/L (measured) were 0, 0, 0, 0, 70 and 100%, respectively. Treatment-related effects (96 hour) at <33, 279, 645, 902, 1360 and 1670 μg/L (measured) were observed in 0/10, 0/10, 0/10, 10/10, 10/10 and 10/10 fish respectively. The 96 hour LC50, EC50 and NOEC of terpinolene to fathead minnow (Pimephales promelas) were calculated to be 1210 (1110 -1330)μg/L, 763 μg/L and 645 μg/L, respectively.

Camphene: An acute toxicity test with Camphene on zebrafish (Brachydanio rerio)in a flow system was performed in accordance with OECD Guideline 203 for 96 hours. It was conducted in the period from 11.01.1993 until 15.01.1993 under GLP conditions. The medium in the test vessel (10 L) was replaced within 2 h. Due to the low water solubility of the substance and its high vapor pressure, the test was conducted in a closed system. The analytical test concentrations were detected during the test: 2.07, 1.21, 0.43, 0.26 and 0.048 mg / L. The mortality and general clinical effects were daily observed and recorded.After 96 hours, the LC50 was 0.72 mg/L.

D-limonene (Test01): In an acute aquatic toxicity study performed similarly to OECD guideline 203, groups (10/dose) of fathead minnows (Pimephales promelas) were exposed to d-limonene (purchased from Aldrich) at concentrations of <150, 178, 386, 595, 928 and 1100 μg/L (measured) for 96 hours under flow-through conditions. Mortality, behaviour and morphological aberrations were observed at 24, 48, 72 and 96 hours. All validity criteria were fulfilled. Mortalities (96 hour) at <150, 178, 386, 595, 928 and 1100 μg/L (measured) were 0, 0, 0, 20, 90 and 100%, respectively. Treatment-related effects (96 hour) at <150, 178, 386, 595, 928 and 1100 μg/L (measured) were observed in 0/10, 0/10, 0/10, 2/10, 9/10 and 10/10 fish respectively. The 96 hour LC50, EC50 and NOEC of d-limonene to fathead minnow (Pimephales promelas) were calculated to be 702 (619 -796) μg/L, 702 (619 -796) μg/L and 386 μg/L, respectively.

D-limonene (Test02): In an acute aquatic toxicity study performed similarly to OECD guideline 203, groups (10/dose) of fathead minnows (Pimephales promelas) were exposed to d-limonene (purchased from Fluorostat) at concentrations of <49, 251, 564, 961, 1380 and 1890 μg/L (measured) for 96 hours under flow-through conditions. Mortality, behaviour and morphological aberrations were observed at 24, 48, 72 and 96 hours. All validity criteria were fulfilled. Mortalities (96 hour) at <49, 251, 564, 961, 1380 and 1890 μg/L (measured) were 0, 0, 10, 90, 100 and 100%, respectively. Treatment-related effects (96 hour) at <49, 251, 564, 961, 1380 and 1890 μg/L (measured) were observed in 0/10, 0/10, 1/10, 10/10, 10/10 and 10/10 fish respectively. The 96 hour LC50, EC50 and NOEC of d-limonene to fathead minnow (Pimephales promelas) were calculated to be 720 (618 -839) μg/L, 688 (606 -782) μg/L and 251 μg/L, respectively.

Alpha terpinene: In an acute aquatic toxicity study performed similarly to OECD guideline 203, groups (10/dose) of fathead minnows (Pimephales promelas) were exposed to alpha terpinene at concentrations of <195, 1050, 2090, 2720, 3640 and 4820 μg/L (measured) for 96 hours under flow-through conditions. Mortality, behaviour and morphological aberrations were observed at 24, 48, 72 and 96 hours. All validity criteria were fulfilled. Mortalities (96 hour) at <195, 1050, 2090, 2720, 3640 and 4820 μg/L (measured) were 0, 0, 0, 10, 90 and 100%, respectively. Treatment-related effects (96 hour) at <195, 1050, 2090, 2720, 3640 and 4820 μg/L (measured) were observed in 0/10, 0/10, 10/10, 10/10, 10/10 and 10/10 fish respectively. The 96 hour LC50, EC50 and NOEC of alpha terpinene to fathead minnow (Pimephales promelas) were calculated to be 3150 (2920 -3400) μg/L, 1480 μg/L and 1050 μg/L, respectively.

D-alpha pinene: In an acute aquatic toxicity study performed similarly to OECD guideline 203, groups (10/dose) of fathead minnows (Pimephales promelas) were exposed to d-alpha pinene at concentrations of <31, 58, 140, 229, 303 and 424 μg/L (measured) for 96 hours under flow-through conditions. Mortality, behaviour and morphological aberrations were observed at 24, 48, 72 and 96 hours. All validity criteria were fulfilled.Mortalities (96 hour) at <31, 58, 140, 229, 303 and 424 μg/L (measured) were 0, 0, 0, 0, 80 and 100%, respectively. Treatment-related effects (96 hour) at <31, 58, 140, 229, 303 and 424 μg/L (measured) were observed in 0/10, 0/10, 0/10, 10/10, 10/10 and 10/10 fish respectively. The 96 hour LC50, EC50 and NOEC of d-alpha pinene to fathead minnow (Pimephales promelas) were calculated to be 280 (259 -303) μg/L, 179 μg/L and 140 μg/L, respectively.

Cineole: An acute toxicity test in Fathead Minnows (Pimephales promelas) was performed following a method similar to OECD guideline 203. 20 fish per group were exposed over an exposure period of 96 h in a flow-through system to the following nominal concentrations: 0 (control), 48.2, 74.1, 114, 176 and 270 mg/L. Gas-liquid chromatography was used to monitor the concentration throughout the test. LC50 and EC50 with corresponding 95% confidence intervals were calculated using corrected average of measured concentrations and trimmed spearman-karber method. Both 96h-EC50 and 96h-LC50 were determined to be 102 mg/L (95% CL = 95.4-109 mg/L).

Conclusion on test substance: Based on the available data on its components, a weight of evidence approach is applied and the 96h-LC50 of the substance in fish is calculated to be 0.89 mg/L.