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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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 with minor deviation: hardness of the dilution water not determined; no data on acclimation period
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
hardness of the dilution water not determined; no data on acclimation period
Principles of method if other than guideline:
not relevant
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 550, 1090, 1640, 2180 or 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: 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: 19.1 ± 2.1 mm
- Weight at study initiation: 85 ± 34 mg
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
None
Hardness:
Not determined
Test temperature:
25.4 ± 0.3 °C
pH:
7.4 ± 0.10
Dissolved oxygen:
6.9 ± 0.3 mg O2/L
Salinity:
No data
Nominal and measured concentrations:
Nominal concentrations: 0, 550, 1090, 1640, 2180 and 2730 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 215-1890 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 ± 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.:
720 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 618-839 µg/L
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: 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 0, 550, 1090, 1640, 2180 or 2730 µg/L (nominal) were 0, 0, 10, 90, 100 or 100%, respectively.
- Effect (96 hour) at 0, 550, 1090, 1640, 2180 or 2730 µg/L (nominal) were observed in 0/10, 0/10, 1/10, 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

Table 6.1.1/1: 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

1

9

10

10

48

 0

0

1

9

10

10

72

 0

0

1

9

10

10

96

0

1

9

10

10

 

Table 6.1.1/2: 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

10

10

10

10

72

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 96-hour LC50 for d-limonene is less than 1 mg/L in fathead minnows (Pimephales promelas) and therefore it is classified as 'R50 Very toxic to aquatic organisms', according to Directive 67/548/EEC and as ‘Acute Category 1’ according to CLP Regulation (EC) No 1272/2008.
Executive summary:

In an acute aquatic toxicity study performed similarly to OECD Guideline 203, groups (10/concentration) of fathead minnows (Pimephales promelas) were exposed to d-limonene at concentrations of 0, 550, 1090, 1640, 2180 or 2730 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, 550, 1090, 1640, 2180 or 2730 µg/L (nominal) were 0, 0, 10, 90, 100 or 100%, respectively. Treatment-related effects (96 hour) at 0, 550, 1090, 1640, 2180 or 2730 µg/L (nominal) were observed in 0/10, 0/10, 1/10, 10/10, 10/10 or 10/10 fish respectively. The 96 hour LC50 or EC50 of d-limonene to fathead minnow (Pimephales promelas) were calculated to be 720 (618-839) µg/L or 688 (606-782) µg/L, respectively.

 

Therefore, the 96-hour LC50 for d-limonene is less than 1 mg/L in fathead minnows (Pimephales promelas). Thus it is classified as 'R50 Very toxic to aquatic organisms', according to Directive 67/548/EEC and as ‘Acute Category 1’ according to CLP Regulation (EC) No 1272 /2008.

 

 

Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
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 with minor deviation: no data on acclimation period
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
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 ± 0.3 mg/L CaCO3
Test temperature:
24.4 ± 0.6 °C
pH:
7.6 ± 0.10
Dissolved oxygen:
5.8 ± 0.5 mg O2/L
Salinity:
No data
Nominal and measured concentrations:
Nominal concentrations: 0, 372, 744, 1120, 1490 and 1860 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: Closed
- Type of flow-through: Electronic diluter
- Renewal rate of test solution (frequency/flow rate): Saturator flow rate of 21 mL/min to each tank provided toxicant concentrations of 178-1350 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 ± 1 °C
- Alkalinity: 39.5 ± 0.7 mg/L CaCO3
- 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 hours/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
Remarks on result:
other: 619-796 µg/L
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: 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 0, 372, 744, 1120, 1490 and 1860 µg/L (nominal) were 0, 0, 0, 20, 90 or 100%, respectively.
- Effect (96 hour) at 0, 372, 744, 1120, 1490 and 1860 µg/L (nominal) were observed in 0/10, 0/10, 0/10, 2/10, 9/10 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

Table 6.1.1/1: 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

 

 

 

2

9

6

48

 

 

 

2

9

8

72

 

 

 

2

9

10

96

 

 

 

2

9

10

 

Table 6.1.1/2: 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

 

 

 

10

10

10

48

 

 

 

2

10

10

72

 

 

 

2

10

10

96

 

 

 

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 96-hour LC50 for d-limonene is less than 1 mg/L in fathead minnows (Pimephales promelas) and therefore it is classified as 'R50 Very toxic to aquatic organisms', according to Directive 67/548/EEC and as ‘Acute Category 1’ according to CLP Regulation (EC) No 1272/2008.
Executive summary:

In an acute aquatic toxicity study performed similarly to OECD Guideline 203, groups (10/concentration) of fathead minnows (Pimephales promelas) were exposed to d-limonene at concentrations of 0, 372, 744, 1120, 1490 and 1860 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, 372, 744, 1120, 1490 and 1860 µg/L (nominal) were 0, 0, 0, 20, 90 or 100%, respectively. Treatment-related effects (96 hour) at 0, 372, 744, 1120, 1490 and 1860 µg/L (nominal) were observed in 0/10, 0/10, 0/10, 2/10, 9/10 or 10/10 fish, respectively. The 96-hour LC50 (= EC50) of d-limonene to fathead minnow (Pimephales promelas) was calculated to be 702 (619-796) µg/L.

 

Therefore, the 96-hour LC50 for d-limonene is less than 1 mg/L in fathead minnows (Pimephales promelas) and therefore it is classified as 'R50 Very toxic to aquatic organisms', according to Directive 67/548/EEC and as ‘Acute Category 1’ according to CLP Regulation (EC) No 1272/2008.

Endpoint:
short-term toxicity to fish
Type of information:
(Q)SAR
Adequacy of study:
key study
Study period:
04-06 May 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
QSAR prediction
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Qualifier:
equivalent or similar to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Principles of method if other than guideline:
The purpose of this QSAR is to accurately predict the acute toxicity to fish as would be expected in a laboratory experiment following OECD Guideline 203 and EC method C.1 for specific, named modes of action (e.g. nonpolar narcotics) to provide a value that can effectively replace a 96 h LC50 value from an experimental study. The regression based method used to achieve this has been fully validated following the OECD (2004) recommendations.
GLP compliance:
no
Analytical monitoring:
no
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
Not applicable
Test organisms (species):
other: Danio rerio, Oncorhynchus mykiss, Lepomis macrochirus, Pimephales promelas, Oryzias latipes, Leuciscus idus
Details on test organisms:
Not applicable
Test type:
other: QSAR model
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
Not applicable
Hardness:
Not applicable
Test temperature:
14 - 25 °C (depending on the fish species considered)
pH:
6.0 - 8.5
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Nominal and measured concentrations:
Not applicable
Details on test conditions:
Not applicable
Reference substance (positive control):
no
Remarks:
QSAR model
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
0.46 mg/L
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL [0.40-0.52 mg/L]
Details on results:
Not applicable
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
95 % CL [0.40-0.52] QSAR statistical parameters are given in the QMRF and QPRF

None

Validity criteria fulfilled:
yes
Remarks:
The substance falls into applicability domains of the model QSAR
Conclusions:
96-h LC50 for d-limonene = 0.46 mg/L with 95%-Confidence Limit of 0.40-0.52 mg/L

Based on the results of this study, d-limonene is classified:
- dangerous for the environment according to Directive 67/548/EEC "R50 Very toxic to aquatic organisms"
- and hazardous to the aquatic environment according to Regulation (EC) No 1272/2008 (CLP) - Acute Category 1.
Executive summary:

A QSAR prediction was performed to assess the acute toxicity of test item d-limonene to fish. This QSAR has been validated to be compliant with the OECD recommendations for QSAR modeling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following Guideline for Testing of Chemicals No. 203, "Fish Acute Toxicity Test", referenced as Method C.1 of Commission Regulation No. 440/2008. The criterion was the LC50 (Median Effective Concentration), a statistically derived concentration which is expected to cause mortality in 50% of test animals within a period of 96 hours.

 

The QSAR determination of the acute toxicity to fish was based on validated data derived from 96-h LC50 tests on fish, for which the concentrations of the test item had been determined by chemical analyses over the test periods.

 

The 96-h LC50 (based on mortality) is predicted as 0.46 mg/L with 95 %-Confidence Limit between 0.40 and 0.52 mg/L.

 

This toxicity study is considered acceptable and satisfies the guideline requirements for acute fish toxicity study.

Based on the results of this study, d-limonene is classified dangerous for the environment according to Directive 67/548/EEC "R50 Very toxic to aquatic organisms" and hazardous to the aquatic environment according to Regulation (EC) No 1272/2008 (CLP) - Acute Category 1.

Description of key information

The 96-hour LC50 for d-limonene on fish is less than 1 mg/L.

Key value for chemical safety assessment

LC50 for freshwater fish:
720 µg/L

Additional information

Two study reports are 1 QSAR are available for the substance itself and are selected as key studies.

In an acute aquatic toxicity study performed similarly to OECD Guideline 203, groups (10/concentration) of fathead minnows (Pimephales promelas) were exposed to d-limonene at concentrations of 0, 550, 1090, 1640, 2180 or 2730 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, 550, 1090, 1640, 2180 or 2730 µg/L (nominal) were 0, 0, 10, 90, 100 or 100%, respectively. Treatment-related effects (96 hour) at 0, 550, 1090, 1640, 2180 or 2730 µg/L (nominal) were observed in 0/10, 0/10, 1/10, 10/10, 10/10 or 10/10 fish respectively. The 96 hour LC50 or EC50 of d-limonene to fathead minnow (Pimephales promelas) were calculated to be 720 (618-839) µg/L or 688 (606-782) µg/L, respectively.

In an acute aquatic toxicity study performed similarly to OECD Guideline 203, groups (10/concentration) of fathead minnows (Pimephales promelas) were exposed to d-limonene at concentrations of 0, 372, 744, 1120, 1490 and 1860 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, 372, 744, 1120, 1490 and 1860 µg/L (nominal) were 0, 0, 0, 20, 90 or 100%, respectively. Treatment-related effects (96 hour) at 0, 372, 744, 1120, 1490 and 1860 µg/L (nominal) were observed in 0/10, 0/10, 0/10, 2/10, 9/10 or 10/10 fish, respectively. The 96-hour LC50 (= EC50) of d-limonene to fathead minnow(Pimephales promelas) was calculated to be 702 (619-796) µg/L.

A QSAR prediction was performed to assess the acute toxicity of test item d-limonene to fish. This QSAR has been validated to be compliant with the OECD recommendations for QSAR modeling (OECD, 2004) and predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following Guideline for Testing of Chemicals No. 203, "Fish Acute Toxicity Test", referenced as Method C.1 of Commission Regulation No. 440/2008. The criterion was the LC50 (Median Effective Concentration), a statistically derived concentration which is expected to cause mortality in 50% of test animals within a period of 96 hours.

The QSAR determination of the acute toxicity to fish was based on validated data derived from 96-h LC50 tests on fish, for which the concentrations of the test item had been determined by chemical analyses over the test periods.

The 96-h LC50 (based on mortality) is predicted as 0.46 mg/L with 95 %-Confidence Limit between 0.40 and 0.52 mg/L.

All the available data belong to the same range below 1 mg/L.