Fir, Abies alba, ext.

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:

supporting study

Study period:

1989

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Study conducted similarly to OECD guideline 203 with minor deviation: no data on acclimation period. The study was conducted similarly to OECD guideline 203 but there are no details on the solubility data of the substance and the value is not in accordance with the solubility determined in a recent study conducted according to the OECD guideline. In fact, results show that there is no toxicity at the solubilit limit. Mortality at higher nominal rates could be explain by physical effects or by the purity of the acrive substance.

Reason / purpose for cross-reference:

reference to same study

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

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No data

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 420, 840, 1270, 1690 or 2110 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 (2110 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 days
- Length at study initiation: 18.0 ± 2.8 mm
- Weight at study initiation: 84 ± 39 mg

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Post exposure observation period:

None

Hardness:

45.2 ± 0.5 mg/L CaCO3

Test temperature:

24.0 ± 0.9 °C

pH:

7.6 ± 0.1

Dissolved oxygen:

6.6 ± 0.7 mg O2/L

Salinity:

No data

Nominal and measured concentrations:

Nominal concentrations: 0, 420, 840, 1270, 1690 or 2110 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 provided concentrations of 244-1760 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: 4.2 g/L

TEST MEDIUM / WATER PARAMETERS
- Source of dilution water: Lake Superior water
- Alkalinity: 37.4 ± 0.3 mg/L CaCO3
- 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 (°C), 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

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

502 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 402-625 µg/L

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

502 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 402-625 µg/L

Details on results:

- Behavioural abnormalities: Affected fish were hypoactive and underreactive to external stimuli. They also swam with a spiral motion and lost equilibrium prior to death.
- Mortalities (96 hour) at 0, 420, 840, 1270, 1690 or 2110 µg/L (nominal) were 0, 0, 60, 100, 100 or 100% respectively.
- Effects (96 hour) at 0, 420, 840, 1270, 1690 or 2110 µg/L (nominal) were observed in 0/10, 0/10, 6/10, 10/10, 10/10 or 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 2: Mortalities

 

Hour	Control (0 µg/L)	A (420 µg/L)	B (840 µg/L)	C (1270 µg/L)	D (1690 µg/L)	E (2110 µg/L)
Initial	10	10	10	10	10	10
24			3	10	10	10
48			6	10	10	10
72			6	10	10	10
96			6	10	10	10

 

Table 2: Effects

 

Hour	Control (0 µg/L)	A (420 µg/L)	B (840 µg/L)	C (1270 µg/L)	D (1690 µg/L)	E (2110 µg/L)
Initial	10	10	10	10	10	10
24			10	10	10	10
48			6	10	10	10
72			6	10	10	10
96			6	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:

Under the test conditions, 96h-LC50 was reported to be at 0.502mg/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 β-pinene at concentrations of 0, 420, 840, 1270, 1690 or 2110 µg/L (nominal) for 96 hours under flow-through conditions. Mortality, behaviour and morphological aberrations were observed at 24, 48, 72 and 96 hours.

 

Mortalities (96 hour) at 0, 420, 840, 1270, 1690 or 2110 µg/L (nominal) were 0, 0, 60, 100, 100 or 100%, respectively. Treatment-related effects (96 hour) at 0, 420, 840, 1270, 1690 or 2110 µg/L (nominal) were observed in 0/10, 0/10, 6/10, 10/10, 10/10 or 10/10 fish, respectively. Therefore the 96h-LC50 was determined to be at 0.502 mg/L.

This toxicity study is classified as acceptable and satisfies the guideline requirement for acute fish toxicity study.

 

Results Synopsis

 

Test organism: fathead minnows(Pimephales promelas)

Test Type: Flowthrough

 

LC₅₀:  0.502 mg a.i./L                       95% C.I.:  0.402 to 0.625 mg a.i./L}

Endpoint(s) Effected: mortality

Reference 2

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989-09-11 and 1989-07-25

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Study conducted similarly to OECD guideline 203 without GLP statement. Validation applies with restrictions, as some informations are missing and the test substance is not the registered mixture.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

no data on acclimation period

Principles of method if other than guideline:

Two tests were performed in this study.

GLP compliance:

not specified

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No data

Analytical monitoring:

yes

Details on sampling:

First test:
- Concentrations: 0, 372, 744, 1120, 1490 and 1860 µg/L
- Sampling time: 0, 24, 48, 72 and 96 hours

Second test:
- Concentrations: 0, 550, 1090, 1640, 2180 or 2730 µg/L
- Sampling time: 0, 24, 48, 72 and 96 hours

Vehicle:

no

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: no vehicle

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: for the first test = 21.8 +/- 3.9 mm; for the second test = 19.1 ± 2.1 mm
- Weight at study initiation: for the first test = 0.177 +/- 0.084 g; for the second test = 0.085 ± 0.034 g

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

none

Post exposure observation period:

None

Hardness:

First test: 46.2 +/- 0.3 mg CaCO3/L
Second test: no data

Test temperature:

First test: 24.4 +/- 0.6°C
Second test: 25.4 +/- 0.3 °C

pH:

First test: 7.6 +/- 0.10
Second test: 7.4 +/- 0.10

Dissolved oxygen:

First test: 5.8 +/- 0.5 mg O2/L
Second test: 6.9 +/- 0.3 mg O2/L

Salinity:

Not applicable

Nominal and measured concentrations:

First test:
- Nominal concentrations: 0, 372, 744, 1120, 1490 and 1860 µg/L
- Measured concentrations: <150, 178, 386, 595, 928, 1100 µg/L
Second test:
- Nominal concentrations: 0, 550, 1090, 1640, 2180 and 2730 µg/L
- 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 178 to 1350 µg/L for the first test and 215 to 1890 µg/L for the second test.
- No. of organisms per vessel: 10
- No. of vessels per concentration (replicates): One
- No. of vessels per vehicle control (replicates): One
- Biomass loading rate: First test = 7.08 g/L; second test = 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 ± 1 °C
- Intervals of water quality measurement: Tank temperatures (°C), 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

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: First test (95% CL: 619-796 µg/L)

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: Second test (95% CL: 618-839 µg/L)

Details on results:

First test:
- 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 0, 372, 744, 1120, 1490 or 1860 µg/L (nominal) were 0, 0, 0, 20, 90 or 100%, 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.

Second test:
- 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 0, 550, 1090, 1640, 2180 or 2730 µg/L (nominal) were 0, 0, 10, 90, 100 or 100%, 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 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 6.1.1/1: Mortalities in the first test

 

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 6.1.1/2: Mortalities in the second test

 

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

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-LC50was determined to be 0.702 mg/L in the first test and 0.720 mg/L in the second test. The geometric mean between these two results is 0.71 mg/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 two times at differents concentrations for 96 hours under flow-through conditions. Mortality, behaviour and morphological aberrations were observed at 24, 48, 72 and 96 hours.

Two tests were conducted. The 96 hour LC50 of D-limonene to fathead minnow (Pimephales promelas) was calculated to be 702 (619 -796) µg/L and 720 (618 -839) µg/L, respectively. The geometric mean between these results is 0.71 mg/L.

 

Reference 3

Endpoint:

short-term toxicity to fish

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2014-05-23 to 2014-05-23

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: QSAR value. The substance falls into applicability domains of the model QSAR.

Justification for type of information:

QSAR prediction: migrated from IUCLID 5.6

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

QSAR model

Principles of method if other than guideline:

The acute toxicity to fish was determined using a validated QSAR for the Mode of Action in question, based on validated data derived from 96-hour LC50 tests on fish, for which the concentrations of the test item had been determined by chemical analyses over the test period. The QSAR is a simple linear regression of sub-cooled liquid solubility versus 96-h LC50 for fish and is valid within the applicability domain defined in the QMRF.

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Analytical monitoring:

no

Details on sampling:

not applicable

Vehicle:

no

Details on test solutions:

not applicable

Test organisms (species):

other: fish spp.

Details on test organisms:

not applicable

Test type:

other: QSAR model

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

96h-LC50

Post exposure observation period:

not applicable

Hardness:

The QSAR is based on data from studies performed at acceptable hardness to ensure control survival

Test temperature:

The QSAR is based on data from studies performed at between 14 - 25 °C (depending on the species considered)

pH:

The QSAR is based on data from studies performed at acceptable pH between 6.0 - 9.0

Dissolved oxygen:

The QSAR is based on data from studies performed at acceptable oxygen concentrations (generally >60%)

Salinity:

The QSAR is based on data from studies performed on freshwater species

Nominal and measured concentrations:

The QSAR is based on data from studies performed using measured concentrations or with acceptable stability

Details on test conditions:

not applicable

Reference substance (positive control):

no

Remarks:

QSAR model

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

5.38 mg/L

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL [4.18-6.93]

Details on results:

not applicable

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

95% CL [4.18-6.93]
QSAR statistical parameters are given in the QMRF and QPRF

Sublethal observations / clinical signs:

no data

Validity criteria fulfilled:

yes

Remarks:

The substance falls into applicability domains of the model QSAR.

Conclusions:

96h-LC50 for bornyl acetate = 5.38 mg test item/L with 95%-Confidence Limit of 4.18 -6.93 mg test item/L.

Executive summary:

The acute toxicity to fish was determined using a validated QSAR for the Mode of Action in question, based on validated data derived from 96-hour LC50 tests on fish, for which the concentrations of the test item had been determined by chemical analyses over the test period. The QSAR is a simple linear regression of sub-cooled liquid solubility versus 96-h LC50 for fish and is valid within the applicability domain defined in the QMRF.

The acute toxicity to fish of bornyl acetate has been investigated using a QSAR model that predicts fish lethality in an OECD 203 study. Bornyl acetate falls within the applicability domain of the model as demonstrated in the QPRF.

The 96-h LC50 was 5.38 mg test material/L with 95%-Confidence Limit between 4.18 and 6.93 mg test material/L. LC50 were based on mortality. 

Based on the results of this study, bornyl acetate would not be classified as acute 1 to aquatic organisms in accordance with the classification of the CLP.

This toxicity study is acceptable and can be used for that endpoint.

 

Results Synopsis

Test Type: QSAR model

LC50: 5.38 mg test material/L 95%-C.I.: 4.18 to 6.93 mg test material/L

Reference 4

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1989-09-18

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: This study was conducted similarly to OECD Guideline 203 without GLP statement. Validation applies with restrictions, as some informations are missing and the test substance is not the registered mixture.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

no data on acclimation period

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
No data

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 0, 130, 260, 390, 520 or 650 µg/L
- Sampling time: 0, 24, 48, 72 and 96 hours

Vehicle:

no

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

Test organisms (species):

Pimephales promelas

Details on test organisms:

TEST ORGANISM
- Common name: Fathead minnows
- Source: ERL-Duluth, USA
- Age at study initiation: 30-34 days
- Length at study initiation: 20.9 ± 2.2 mm
- Weight at study initiation: 128 ± 35 mg

Test type:

flow-through

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

none

Post exposure observation period:

None

Hardness:

45.2 ± 0.3 mg/L CaCO3

Test temperature:

25.2 ± 0.2 °C

pH:

7.7 ± 0.2

Dissolved oxygen:

6.7 ± 0.7 mg O2/L

Salinity:

Not applicable

Nominal and measured concentrations:

- Nominal concentrations: 0, 130, 260, 390, 520 and 650 µg/L
- Mean measured concentrations: See table 6.1.1/1 in "Any other information on materials and methods incl. tables"

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: Closed
- Type of flow-through: Electronic diluter
- Renewal rate of test solution (frequency/flow rate): Flow rates of 7 mL/min to each tank and 50.4 volume additions per day
- 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
- Alkalinity: 42.2 ± 0.9 mg/L CaCO3
- 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 (°C), 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

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. Relevant endpoint.

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

179 µg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

behaviour

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.
- Mortalities (96 hour) at 0, 130, 260, 390, 520 or 650 µg/L (nominal) were 0, 0, 0, 0, 80 or 100%, respectively. See table 6.1.1/2 in "Any other information on results incl. tables".
- Effects (96 hour) at 0, 130, 260, 390, 520 or 650 µg/L (nominal) were observed in 0/10, 0/10, 0/9, 10/10, 10/10 or 10/10 fish respectively. See table 6.1.1/3 in "Any other information on results incl. tables".
- 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 6.1.1/2: 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 6.1.1/3: 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 was recorded at 280 µg/L (95% CL: 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 1(R)α-pinene at concentrations of 0, 130, 260, 390, 520 or 650 µg/L (nominal) for 96 hours under flow-through conditions. Mortality, behaviour and morphological aberrations were observed at 24, 48, 72 and 96 hours.

 

Mortalities (96 hour) at 0, 130, 260, 390, 520 or 650 µg/L (nominal) were 0, 0, 0, 0, 80 or 100%, respectively. Treatment-related effects (96 hour) at 0, 130, 260, 390, 520 or 650 µg/L (nominal) were observed in 0/10, 0/10, 0/9, 10/10, 10/10 or 10/10 fish respectively.

Based on measured concentrations, the 96h-LC50 was recorded at 280 µg/L (95% CL: 259 - 303 µg/L) for mortality.

Reference 5

Endpoint:

short-term toxicity to fish

Type of information:

(Q)SAR

Adequacy of study:

supporting study

Study period:

2014-05-23 to 2014-05-23

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: QSAR value. The substance falls into applicability domains of the model QSAR.

Justification for type of information:

QSAR prediction: migrated from IUCLID 5.6

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

QSAR model

Principles of method if other than guideline:

The acute toxicity to fish was determined using a validated QSAR for the Mode of Action in question, based on validated data derived from 96-hour LC50 tests on fish, for which the concentrations of the test item had been determined by chemical analyses over the test period. The QSAR is a simple linear regression of sub-cooled liquid solubility versus 96-h LC50 for fish and is valid within the applicability domain defined in the QMRF.

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Analytical monitoring:

no

Details on sampling:

not applicable

Vehicle:

no

Details on test solutions:

not applicable

Test organisms (species):

other: fish spp.

Details on test organisms:

not applicable

Test type:

other: QSAR model

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

96h-LC50

Post exposure observation period:

not applicable

Hardness:

The QSAR is based on data from studies performed at acceptable hardness to ensure control survival

Test temperature:

The QSAR is based on data from studies performed at between 14 - 25 °C (depending on the species considered)

pH:

The QSAR is based on data from studies performed at acceptable pH between 6.0 - 9.0

Dissolved oxygen:

The QSAR is based on data from studies performed at acceptable oxygen concentrations (generally >60%)

Salinity:

The QSAR is based on data from studies performed on freshwater species

Nominal and measured concentrations:

The QSAR is based on data from studies performed using measured concentrations or with acceptable stability

Details on test conditions:

not applicable

Reference substance (positive control):

no

Remarks:

QSAR model

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

0.44 mg/L

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: 95% CL [0.29-0.66]

Details on results:

not applicable

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

95% CL [0.29-0.66]
QSAR statistical parameters are given in the QMRF and QPRF

Sublethal observations / clinical signs:

no data

Validity criteria fulfilled:

yes

Remarks:

The substance falls into applicability domains of the model QSAR.

Conclusions:

96h-LC50 for camphene = 0.44 mg test item/L with 95%-Confidence Limit of 0.29 -0.66 mg test item/L.

Executive summary:

The acute toxicity to fish was determined using a validated QSAR for the Mode of Action in question, based on validated data derived from 96-hour LC50 tests on fish, for which the concentrations of the test item had been determined by chemical analyses over the test period. The QSAR is a simple linear regression of sub-cooled liquid solubility versus 96-h LC50 for fish and is valid within the applicability domain defined in the QMRF.

The acute toxicity to fish of camphene has been investigated using a QSAR model that predicts fish lethality in an OECD 203 study. Camphene falls within the applicability domain of the model as demonstrated in the QPRF.

The 96-h LC50 was 0.44 mg test material/L with 95%-Confidence Limit between 0.29 and 0.66 mg test material/L. LC50 were based on mortality. 

Based on the results of this study, camphene would be classified as acute 1 to aquatic organisms in accordance with the classification of the CLP.

This toxicity study is acceptable and can be used for that endpoint.

 

Results Synopsis

Test Type: QSAR model

LC50: 0.44 mg test material/L 95%-C.I.: 0.29 to 0.66 mg test material/L

Reference 6

Endpoint:

short-term toxicity to fish

Type of information:

calculation (if not (Q)SAR)

Remarks:

Migrated phrase: estimated by calculation

Adequacy of study:

key study

Study period:

2014-05-23 to 2014-05-26

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Calculation method is used ; calculation method applicable for the endpoint.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

Deviations:

yes

Remarks:

Calculation method

Principles of method if other than guideline:

The acute toxicity to fish was determined using a validated QSAR for the Mode of Action in question. The first step of the iSafeRat mixture toxicity calculation employs phase equilibrium thermodynamics in order to determine the concentrations of each constituent within the WAF. This fraction equates to the analyzable fraction of a WAF study.
Within the WAF, the constituents also partition between themselves further reducing the bioavailable fraction and thus the toxicity of the mixture compared to the individual constituents. In the calculation the second step is to remove this non-bioavailable fraction.
The final step is to determine the truly bioavailable fraction of the WAF per constituent. The LC50s of each constituent are already known from literature or calculated using the iSafeRat QSAR model. Each value and calculation has been included as a supporting study in the IUCLID. An additivity approach (based on Chemical Activity of each constituent) is used in order to calculate the Lethal Loading of the WAF.
The method has been validated using data derived from 96-hour LC50 tests on fish, for which the concentrations of the test item had been determined by chemical analyses over the test period. Further to this the lethal loading rate of the WAF is determined by using a series of calculation steps using phase equilibrium thermodynamics and excluding the non-bioavailable fraction.

GLP compliance:

no

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
not applicable

Analytical monitoring:

not required

Details on sampling:

not applicable

Vehicle:

no

Details on test solutions:

not applicable

Test organisms (species):

other: fish spp.

Details on test organisms:

not applicable

Test type:

other: calculation method

Water media type:

freshwater

Limit test:

no

Total exposure duration:

96 h

Remarks on exposure duration:

LL50 (lethal loading rate of WAF)

Post exposure observation period:

not applicable

Hardness:

Hardness is not a necessary component of the WAF calculation

Test temperature:

The Temperature is not a necessary component of the WAF calculation but extremely low or high temperatures could influence the solubility of certain constituents. Therefore, the calculation method is considered acceptable to determine LL50s for fish between 12 and 28°C.

pH:

The pH is not a necessary component of the WAF calculation

Dissolved oxygen:

The oxygen concentration is not a necessary component of the WAF calculation

Salinity:

Salinity is not a necessary component of the WAF calculation. However as the fish QSAR for the constituents calculation was based on data from freshwater studies, the resulting calculation is considered valid for freshwater fish

Nominal and measured concentrations:

The calculation determines measured concentrations

Details on test conditions:

calculation method

Reference substance (positive control):

not required

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

4.7 mg/L

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: typical composition (please refer to confidential section)

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

4.8 mg/L

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: theoritical worst case composition (please refer to confidential section)

Details on results:

not applicable

Results with reference substance (positive control):

not applicable

Reported statistics and error estimates:

not applicable

Sublethal observations / clinical signs:

Table 1: Expected concentrations at this 96h-LL50 (composition 1: typical composition)

constituents	concentration in the WAF (mg.L-1)
limonene	0.93
α-pinene	1.21
β-pinene	0.61
camphene	0.57

Table 2: Expected concentrations at this 96h-LL50 (composition 2: theoretical worst case composition)

constituents	concentration in the WAF (mg.L-1)
limonene	1.06
α-pinene	1.17
β-pinene	0.77
camphene	0.48
bornyl acetate	0.05

Validity criteria fulfilled:

yes

Conclusions:

96h-LL50 for typical composition of Fir Silver oil = 4.7 mg test item/L and 96h-LL50 for theoretical worst case composition of Fir silver oil = 4.8 mg test item/L

Executive summary:

Fir Silver oil is a Natural Complex Substance (UVCB) with a well-defined composition for which the relative percentage and its reported variation of each constituent is known.

Its acute toxicity to fish property has been investigated using an in-house calculation method that replaces an OECD 203 study and guideline for Testing of Chemicals No. 23 (i.e. WAF conditions). Two compositions have been investigated, the “typical” composition proposed by the Lead registrant and a theoretical worst case composition, derived from data supplied in the SIP that maximizes the concentration of the most toxic constituents.

The first step of the iSafeRat mixture toxicity calculation employs phase equilibrium thermodynamics in order to determine the concentrations of each constituent within the WAF. This fraction equates to the analyzable fraction of a WAF study.

Within the WAF, the constituents also partition between themselves further reducing the bioavailable fraction and thus the toxicity of the mixture compared to the individual constituents. In the calculation the second step is to remove this non-bioavailable fraction.

The final step is to determine the truly bioavailable fraction of the WAF per constituent. The LC50s of each constituent are already known from literature or calculated using the iSafeRat QSAR model. Each value and calculation has been included as a supporting study in the IUCLID. An additivity approach (based on Chemical Activity of each constituent) is used in order to calculate the Lethal Loading of the WAF.

The 96-h LL50 was 4.7 mg test material/L for the typical composition of Fir Silver oil, and the 96-h LL50 was 4.8 mg test material/L for the theoretical worst case composition. LL50 were based on mortality. 

Based on the results of this study, Fir Silver oil would not be classified as acute 1 to aquatic organisms in accordance with the classification of the CLP.

This toxicity study is acceptable and can be used for that endpoint.

 

Results Synopsis

Test Type: Calculation method

LL50: 4.7 mg test material/L for typical composition

LL50: 4.8 mg test material/L for theoretical worst case composition

 

Description of key information

Fir Silver oil is a Natural Complex Substance (UVCB) with a well-defined composition for which the relative percentage and its reported variation of each constituent is known. The acute toxicity to fish property has been investigated using an in-house calculation method that mimics an OECD 203 study and guideline for Testing of Chemicals No. 23 (i.e. WAF conditions). The acute toxicity to fish was determined using a calculation method for Mode of Action 1 (non-polar narcotics) for each individual constituent present in the mixture. This algorithm is based on a QSAR model which has been validated to be compliant with the OECD recommendations for QSAR modeling (OECD, 2004). Further to this, the lethal loading rate of the WAF is determined by using a series of calculation steps using phase equilibrium thermodynamics first to determine the analysable fraction (the concentration which should be analysable in a WAF study) and then excluding the non-bioavailable fraction of the remaining constituents. The remaining, bioavailable fraction corresponds to the lethal loading value of the mixture. This approach has been validated using data derived from 96-hour LL50 tests on fish with similar complex substances (OECD 203 study and the OECD guidance document on toxicity testing for difficult substances and mixtures No. 23, i.e. WAF conditions). Two theoretical compositions have been investigated, the “typical” composition proposed by the Lead registrant and a theoretical worst case composition that maximizes the concentration of the most toxic constituents. The 96-h LL50 was 4.7 mg test material/L for the typical composition of Fir Silver oil, and the 96-h LL50 was 4.8 mg test material/L for the theoretical worst case composition. LL50s were based on mortality. 
Conclusion: The 96-h LL50 for fish was calculated at 4.7 mg test material/L for the typical composition and at 4.8 mg test material/L for theoretical worst case composition.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish

Effect concentration:

4.7 mg/L

Additional information

Short-term toxicity to fish is provided in the dataset even if the tonnage band (1 -10 tonnes/year) does not require that endpoint. Indeed that endpoint is fulfilled according to environmental classification rules. Classification should be derived from the most sensible species. Because toxicity data have demonstrated the lowest LC50 values for constituents, it has been decided to calculate fish L(E)C50, in order to ascertain the classification for environment of Fir Silver oil.

 

Fir Silver oil is a Natural Complex Substance (UVCB) with a well-defined composition for which the relative percentage of each constituent is known. Therefore, it has been decided that the ecotoxicity of Fir Silver oil will be derived from knowledge of the constituents, constituent approach.

The mixture ecotoxicity properties may be derived from the ecotoxicity of the individual constituents using the CLP additivity calculation approach. However, CLP additivity approach is calculated on the basis that all the substances are at their maximum solubility and it has been observed that CLP additivity calculations for mode of action 1 compounds are unreasonably conservative when compared to classic WAF studies. This has been proved in a number of cases for natural complex substances. Indeed, natural extract compositions are a mixture of hydrophilic alcohol molecules and hydrophobic terpene molecules. Therefore, when a WAF is performed most of substances fully dissolved in the aqueous phase are the hydrophilic fraction while the hydrophobic fraction (the more toxic elements for MOA 1 substances) may be below their water solubility value.

 

The acute toxicity to fish property for Fir Silver oil has been investigated using an in-house calculation method (iSafeRat WAF module for mixture Toxicity calculation) that mimics an OECD 203 study and guideline for Testing of Chemicals No. 23 (i.e. WAF conditions). Two theoretical compositions have been investigated, the “typical” composition proposed by the Lead registrant and a theoretical worst case composition that maximizes the concentration of the most toxic constituents. The acute toxicity to fish was determined using a calculation method for Mode of Action 1 (non-polar narcotics) for each individual constituent present in the mixture. This algorithm is based on a QSAR model which has been validated to be compliant with the OECD recommendations for QSAR modeling (OECD, 2004).

The first step of the iSafeRat mixture toxicity calculation employs phase equilibrium thermodynamics in order to determine the concentrations of each constituent within the WAF. This fraction equates to the analyzable fraction of a WAF study.

In the calculation the second step is to remove this non-bioavailable fraction. Within the WAF, the constituents also partition between themselves further reducing the bioavailable fraction and thus the toxicity of the mixture compared to the individual constituents. These two reasons explain why ecotoxicity values from WAF studies are always higher for non-polar narcotic mixtures than the calculated values from CLP additivity calculation.

 

The final step is to determine the truly bioavailable fraction of the WAF per constituent. The LC50s of each constituent are already known from literature or predicted using the iSafeRat QSAR model. Each value has been included as a supporting study in the IUCLID. An additivity approach (based on Chemical Activity of each constituent) is used in order to calculate the Lethal Loading of the WAF. Using this approach, the 96-h LL50 for fish was 4.7 mg test material/L for the typical composition of Fir Silver oil, and the 96-h LL50 was 4.8 mg test material/L for the theoretical worst case composition. These LL50 were based on mortality. 

 

The in-house calculation has been designed to mimic the behavior of the mixture in a WAF test where the final toxicity is calculated by additivity of the residual bioavailable fraction of each constituent. This approach has been validated using a Natural Complex Substance similar to Fir Silver oil as presented in the position paper attached to the dossier.

 

Based on the results of this study, Fir Silver oil would not be classified as acutely toxic to aquatic organisms in accordance with the classification of the CLP.

This toxicity prediction has been validated and is considered acceptable to fulfill the fish toxicity endpoint.