Rectified Hydrocarbons by-products Limonene enriched from synthetic process

Short-term toxicity to fish

Link to relevant study record(s)

Reference

Reference 1

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

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 (fish)

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

Sublethal observations / clinical signs:

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.

 

 

 

Reference 2

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

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 (fish)

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

Sublethal observations / clinical signs:

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.

 

 

 

Reference 3

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

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 (fish)

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

Sublethal observations / clinical signs:

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.

 

 

 

Reference 4

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

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 (fish)

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

Sublethal observations / clinical signs:

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.

 

 

 

Reference 5

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

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 (fish)

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 (fish)

Duration:

72 h

Dose descriptor:

LC50

Effect conc.:

2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

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 (fish)

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 (fish)

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.

Sublethal observations / clinical signs:

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.

Reference 6

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

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 (fish)

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 (fish)

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 (fish)

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 (fish)

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 (fish)

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 (fish)

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.

Sublethal observations / clinical signs:

	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.

Description of key information

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

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).

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).

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).

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 0.75 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

0.75 mg/L

Additional information

Weight of evidence from experimental results with individual components:

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.

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.

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.

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 0.75 mg/L.