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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
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 202 with minor deviations (no data on acclimation period). The report is sufficiently well documented.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
(no data on acclimation period)
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 1260, 2520, 3780, 5040 and 6300 μg/L
- Sampling time: 0, 24 and 48 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, stirring in closed vessel
- Controls: Dilution water
- Chemical name of vehicle: water
- Concentration of vehicle in test medium: Stock (6300 µg/L liq-liq equil.)
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: ERL-Duluth, USA
- Age at study initiation: < 24 hours
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
44.6 mg CaCO3/L (SD=0.3)
Test temperature:
19.8ºC (SD=0.3)
pH:
7.8 (SD=0.07)
Dissolved oxygen:
6.5 mg O2/L (SD=0.5)
Salinity:
42.4 mg CaCO3/L (SD=2)
Nominal and measured concentrations:
Nominal concentrations: 0, 1260, 2520, 3780, 5040 and 6300 μg/L
Average measured concentrations: <29, 1360, 2510, 3280, 4570 and 5890 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; stainless steel screen enclosures (forty mesh) were placed in each chamber for the daphnia exposures
- 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 1360-5890 μg/L
- No. of organisms per vessel: 15
- No. of vessels per concentration (replicates): One
- No. of vessels per vehicle control (replicates): One

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 20 ± 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 hours/day light cycle; cool white fluorescent light
- Light intensity: 12-28 lumens

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Final mortality counts were taken at termination. Daphnia were removed from test screens and observed under a dissecting scope to confirm responses.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
1 850 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL: not reliable
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1 850 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: not reliable
Details on results:
- Mortalities (48 hour) at <29, 1360, 2510, 3280, 4570 and 5890 μg/L (measured) were observed in 0/15, 0/15, 15/15, 15/15, 15/15 and 15/15 daphnids respectively.
- Effects (48 hour) at <29, 1360, 2510, 3280, 4570 and 5890 μg/L μg/L (measured) were observed in 0/15, 0/15, 15/15, 15/15, 15/15 and 15/15 daphnids respectively.
Results with reference substance (positive control):
Not applicable
 
Reported statistics and error estimates:
- LC50 or EC50 was calculated using the corrected average of the analyzed tank concentrations and the Trimmed Spearman-Karber Method (Hamilton et al., 1977).
- Mean toxicant concentrations used in the calculations were corrected for analytical spike recoveries

Table 1: Mortalities

 

Hour

Control (0 µg/L)

A (1260 µg/L)

B (2520µg/L)

C (3780 µg/L)

D (5040 µg/L)

E (6300 µg/L)

Initial

15

15

15

15

15

15

24

0

0

0

0

15

15

48

0

0

15

15

15

15

 

Table 2: Effects

Hour

Control (0 µg/L)

A (1260 µg/L)

B (2520µg/L)

C (3780 µg/L)

D (5040 µg/L)

E (6300 µg/L)

Initial

15

15

15

15

15

15

24

0

0

15

15

15

15

48

0

0

15

15

15

15

 

Validity criteria fulfilled:
yes
Remarks:
(immobilisation in control < 10%; dissolved oxygen concentration > 3 mg O2/L)
Conclusions:
The 48h-EC50 of alpha terpinene to daphnia magna was calculated to be 1850 μg/L.
Executive summary:

In an acute aquatic toxicity study performed similarly to OECD guideline 202, groups (15/dose) of daphnia magna were exposed to alpha terpinene at concentrations of <29, 1360, 2510, 3280, 4570 and 5890 μg/L (measured) for 48 hours under flow-through conditions. Mortality and effects were observed at 24 and 48 hours. All validity criteria were fulfilled. Mortalities (48 hour) at <29, 1360, 2510, 3280, 4570 and 5890 μg/L (measured) were observed in 0/15, 0/15, 15/15, 15/15, 15/15 and 15/15 daphnids respectively. Treatment-related effects (48 hour) at <29, 1360, 2510, 3280, 4570 and 5890 μg/L (measured) were observed in 0/15, 0/15, 15/15, 15/15, 15/15 and 15/15 daphnids respectively. The 48 hour LC50, EC50 and NOEC of alpha terpinene to daphnia magna were calculated to be 1850 μg/L, 1850 μg/L and 1360 μg/L respectively.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting 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 202 with minor deviations (no data on hardness of dilution water, acclimation period or reference substance. EC50 value could not be obtained). The report is sufficiently well documented.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
(no data on hardness of dilution water, acclimation period and reference substance. The EC50 value could not be obtained; only the LC50 value was reported).
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 124, 248, 372, 496 and 620 μg/L
- Sampling time: 0, 24 and 48 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, stirring in closed vessel
- Controls: Dilution water
- Chemical name of vehicle: water
- Concentration of vehicle in test medium: Stock (620 µg/L liq-liq equil.)

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: ERL-Duluth, USA
- Age at study initiation: < 24 hours
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
45.4 mg CaCO3/L (SD=0.2)
Test temperature:
20.2ºC (SD=0.2)
pH:
7.8 (SD=0.09)
Dissolved oxygen:
7.8 mg O2/L (SD=0.4)
Salinity:
42.9 mg CaCO3/L (SD=0.1)
Nominal and measured concentrations:
Nominal concentrations: 0, 124, 248, 372, 496 and 620 μg/L
Average measured concentrations: <32, 75.7, 223, 301, 407 and 542 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; stainless steel screen enclosures (forty mesh) were placed in each chamber for the daphnia exposures
- 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 75.7-542 μg/L
- No. of organisms per vessel: 15
- No. of vessels per concentration (replicates): One
- No. of vessels per vehicle control (replicates): One

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 20 ± 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 hours/day light cycle; cool white fluorescent light
- Light intensity: 12-28 lumens

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Final mortality counts were taken at termination. Daphnia were removed from test screens and observed under a dissecting scope to confirm responses.

Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
> 542 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Main test
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 542 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: Main test
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
ca. 1 440 µg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: Based on two additional tests
Details on results:
- Mortalities (48 hour) at <32, 75.7, 223, 301, 407 and 542 μg/L (measured) were observed in 0/15, 0/15, 0/15, 0/15, 0/15 and 2/15 daphnids respectively.
- Effects (48 hour) at <32, 75.7, 223, 301, 407 and 542 μg/L (measured) were observed in 0/15, 0/15, 0/15, 0/15, 0/15 and 2/15 daphnids respectively.
-Additional tests: LC50 and EC50 could not be determined from the results obtained in the main test. Thus, two additional tests were performed. The test concentrations were 920 and 1965 ug/l, respectively. An approximate LC50 value of 1440 ug/l was calculated based on one mortality in the first test and total mortality in the second test.
Results with reference substance (positive control):
Not applicable
 
Reported statistics and error estimates:
- LC50 or EC50 was calculated using the corrected average of the analyzed tank concentrations and the Trimmed Spearman-Karber Method (Hamilton et al., 1977).
- Mean toxicant concentrations used in the calculations were corrected for analytical spike recoveries

Table 1: Mortalities

 

Hour

Control (0 µg/L)

A (124 µg/L)

B (248 µg/L)

C (372 µg/L)

D (496 µg/L)

E (620 µg/L)

Initial

15

15

15

15

15

15

48

0

0

0

0

0

2

Table 2: Effects

Hour

Control (0 µg/L)

A (124 µg/L)

B (248 µg/L)

C (372 µg/L)

D (496 µg/L)

E (620 µg/L)

Initial

15

15

15

15

15

15

48

0

0

0

0

0

2

 

Validity criteria fulfilled:
yes
Remarks:
(immobilisation in control < 10%; dissolved oxygen concentration > 3 mg O2/L)
Conclusions:
The 48h-LC50 of d-alpha pinene to daphnia magna was calculated to be 1440 μg/L.
Executive summary:

In an acute aquatic toxicity study performed similarly to OECD guideline 202, groups (15/dose) of daphnia magna were exposed to d-alpha pinene at concentrations of <32, 75.7, 223, 301, 407 and 542 μg/L (measured) for 48 hours under flow-through conditions. Mortality and effects were observed at 48 hours. All validity criteria were fulfilled. Mortalities (48 hour) at <32, 75.7, 223, 301, 407 and 542 μg/L (measured) were observed in 0/15, 0/15, 0/15, 0/15, 0/15 and 2/15 daphnids respectively. Treatment-related effects (48 hour) at <32, 75.7, 223, 301, 407 and 542 μg/L (measured) were observed in 0/15, 0/15, 0/15, 0/15, 0/15 and 2/15 daphnids respectively. Based on these results, LC50 and EC50 could not be determined. Thus, two additional tests were performed. The test concentrations were 920 and 1965 µg/l, respectively. An approximate LC50 value of 1440 µg/l was calculated based on one mortality in the first test and total mortality in the second test.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
supporting 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 202 with minor deviations (no data on acclimation period). The report is sufficiently well documented.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
(No data on acclimation period)
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 312, 624, 936, 1250 and 1560 μg/L
- Sampling time: 0, 24 and 48 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, stirring in closed vessel
- Controls: Dilution water
- Chemical name of vehicle: water
- Concentration of vehicle in test medium: Stock (1560 µg/L liq-liq equil.)
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: ERL-Duluth, USA
- Age at study initiation: < 24 hours
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
45.5 mg CaCO3/L (SD=0.2)
Test temperature:
19.7ºC (SD=0.5)
pH:
7.9 (SD=0.13)
Dissolved oxygen:
8.0 mg O2/L (SD=0.3)
Salinity:
36.6 mg CaCO3/L (SD=1.5)
Nominal and measured concentrations:
Nominal concentrations: 0, 312, 624, 936, 1250 and 1560 μg/L
Average measured concentrations: <150, 238, 618, 784, 1090 and 1350 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; stainless steel screen enclosures (forty mesh) were placed in each chamber for the daphnia exposures
- 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 238-1350 μg/L
- No. of organisms per vessel: 15
- No. of vessels per concentration (replicates): One
- No. of vessels per vehicle control (replicates): One

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 20 ± 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 hours/day light cycle; cool white fluorescent light
- Light intensity: 12-28 lumens

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Final mortality counts were taken at termination. Daphnia were removed from test screens and observed under a dissecting scope to confirm responses.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
924 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL: not reliable
Details on results:
- Mortalities (48 hour) at <150, 238, 618, 784, 1090 and 1350 μg/L (measured) were observed in 0/15, 0/15, 0/15, 0/15, 15/15 and 15/15 daphnids respectively.
Results with reference substance (positive control):
Not applicable
 
Reported statistics and error estimates:
- LC50 or EC50 was calculated using the corrected average of the analyzed tank concentrations and the Trimmed Spearman-Karber Method (Hamilton et al., 1977).
- Mean toxicant concentrations used in the calculations were corrected for analytical spike recoveries

Table 1: Mortalities

Hour

Control (0 µg/L)

A (312µg/L)

B (624 µg/L)

C (936 µg/L)

D (1250 µg/L)

E (1560 µg/L)

Initial

15

15

15

15

15

15

48

0

0

0

0

15

15

Validity criteria fulfilled:
not specified
Remarks:
(no data on immobilisation was reported)
Conclusions:
The 48h-LC50 of d-limonene to daphnia magna was calculated to be 924 μg/L.
Executive summary:

In an acute aquatic toxicity study performed similarly to OECD guideline 202, groups (15/dose) of daphnia magna were exposed to d-limonene (purchased from Aldrich) at concentrations of <150, 238, 618, 784, 1090 and 1350 μg/L (measured) for 48 hours under flow-through conditions. Only mortality was observed at 48 hours. Mortalities (48 hour) at <150, 238, 618, 784, 1090 and 1350 μg/L (measured) were observed in 0/15, 0/15, 0/15, 0/15, 15/15 and 15/15 daphnids respectively. The 48 hour LC50 of d-limonene to daphnia magna were calculated to be 924 μg/L.

Endpoint:
short-term toxicity to aquatic invertebrates
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 202 with minor deviations (no data on hardness of dilution water and acclimation period). The report is sufficiently well documented.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
(No data on hardness of dilution water and acclimation period)
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 398, 796, 1190, 1590 and 1990 μg/L
- Sampling time: 0, 24 and 48 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, stirring in closed vessel
- Controls: Dilution water
- Chemical name of vehicle: water
- Concentration of vehicle in test medium: Stock (1990 µg/L liq-liq equil.)
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: ERL-Duluth, USA
- Age at study initiation: < 24 hours
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
No data
Test temperature:
20.0ºC (SD=0.4)
pH:
7.9 (SD=0.1)
Dissolved oxygen:
7.8 mg O2/L (SD=0.5)
Nominal and measured concentrations:
Nominal concentrations: 0, 398, 796, 1190, 1590 and 1990 μg/L
Average measured concentrations: <50, 287, 619, 932, 1190 and 1630 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; stainless steel screen enclosures (forty mesh) were placed in each chamber for the daphnia exposures
- 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 287-1630 μg/L
- No. of organisms per vessel: 15
- No. of vessels per concentration (replicates): One
- No. of vessels per vehicle control (replicates): One

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 20 ± 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 hours/day light cycle; cool white fluorescent light
- Light intensity: 12-28 lumens

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Final mortality counts were taken at termination. Daphnia were removed from test screens and observed under a dissecting scope to confirm responses.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
577 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL: 496-672 μg/L
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
421 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: Not reliable
Details on results:
- Mortalities (48 hour) at <50, 287, 619, 932, 1190 and 1630 μg/L (measured) were observed in 0/15, 0/15, 7/15, 15/15, 15/15 and 15/15 daphnids respectively.
- Effect (48 hour) at <50, 287, 619, 932, 1190 and 1630 μg/L (measured) were observed in 0/15, 0/15, 15/15, 15/15, 15/15 and 15/15 daphnids respectively.
Results with reference substance (positive control):
Not applicable
 
Reported statistics and error estimates:
- LC50 or EC50 was calculated using the corrected average of the analyzed tank concentrations and the Trimmed Spearman-Karber Method (Hamilton et al., 1977).
- Mean toxicant concentrations used in the calculations were corrected for analytical spike recoveries

Table 1: Mortalities

Hour

Control (0 µg/L)

A (398µg/L)

B (796 µg/L)

C (1190 µg/L)

D (1590 µg/L)

E (1990 µg/L)

Initial

15

15

15

15

15

15

48

0

0

7

15

15

15

Table 2: Effects

Hour

Control (0 µg/L)

A (398 µg/L)

B (796 µg/L)

C (1190 µg/L)

D (1590 µg/L)

E (1990 µg/L)

Initial

15

15

15

15

15

15

48

0

0

15

15

15

15

Validity criteria fulfilled:
yes
Remarks:
(immobilisation in control < 10%; dissolved oxygen concentration > 3 mg O2/L)
Conclusions:
The 48h-EC50 of d-limonene to daphnia magna was calculated to be 421 μg/L.
Executive summary:

In an acute aquatic toxicity study performed similarly to OECD guideline 202, groups (15/dose) of daphnia magna were exposed to d-limonene (purchased from Fluorostat) at concentrations of <50, 287, 619, 932, 1190 and 1630 μg/L (measured) for 48 hours under flow-through conditions. Mortality and effects were observed at 48 hours. All validity criteria were fulfilled. Mortalities (48 hour) at <50, 287, 619, 932, 1190 and 1630 μg/L (measured) were observed in 0/15, 0/15, 7/15, 15/15, 15/15 and 15/15 daphnids respectively. Treatment-related effects (48 hour) at <50, 287, 619, 932, 1190 and 1630 μg/L (measured) were observed in 0/15, 0/15, 15/15, 15/15, 15/15 and 15/15 daphnids respectively. The 48 hour LC50, EC50 and NOEC of d-limonene to daphnia magna were calculated to be 577 μg/L, 421 μg/L and 287 μg/L respectively.

Endpoint:
short-term toxicity to aquatic invertebrates
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 202 with minor deviations (no data on hardness of dilution water, acclimation period, reference substance and only 3 concentrations tested). The report is sufficiently well documented.
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
(no data on hardness of dilution water, acclimation period and reference substance. Only 3 concentrations tested. However, the highest concentration tested resulted in 100% immobilisation and the lowest concentration tested gave no observable effect).
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 1120, 2230 and 3380 μg/L
- Sampling time: 0, 24 and 48 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, stirring in closed vessel
- Controls: Dilution water
- Chemical name of vehicle: water
- Concentration of vehicle in test medium: Stock (3380 µg/L liq-liq equil.)
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: ERL-Duluth, USA
- Age at study initiation: < 24 hours
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
Not determined
Test temperature:
19.6ºC (SD=0.1)
pH:
7.7 (SD=0.08)
Dissolved oxygen:
6.2 mg O2/L (SD=0.9)
Salinity:
No data
Nominal and measured concentrations:
Nominal concentrations: 0, 1120, 2230 and 3380 μg/L
Average measured concentrations: <31, 940, 2020 and 3020 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; stainless steel screen enclosures (forty mesh) were placed in each chamber for the daphnia exposures
- 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 940-3020 μg/L
- No. of organisms per vessel: 15
- No. of vessels per concentration (replicates): One
- No. of vessels per vehicle control (replicates): One

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 20 ± 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 hours/day light cycle; cool white fluorescent light
- Light intensity: 12-28 lumens

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Final mortality counts were taken at termination. Daphnia were removed from test screens and observed under a dissecting scope to confirm responses.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
2 550 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: 95% CL: not reliable
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
1 380 µg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: not reliable
Details on results:
- Mortalities (48 hour) at <31, 940, 2020 and 3020 μg/L (measured) were observed in 0/15, 0/15 and 13/15 daphnids respectively.
- Effects (48 hour) at <31, 940, 2020 and 3020 μg/L (measured) were observed in 0/15, 15/15 and 15/15 daphnids respectively.
Results with reference substance (positive control):
Not applicable
 
Reported statistics and error estimates:
- LC50 or EC50 was calculated using the corrected average of the analyzed tank concentrations and the Trimmed Spearman-Karber Method (Hamilton et al., 1977).
- Mean toxicant concentrations used in the calculations were corrected for analytical spike recoveries

Table 1: Mortalities

 

Hour

Control (0 µg/L)

A (1120 µg/L)

B (2230 µg/L)

C (3380 µg/L)

Initial

15

15

15

15

48

0

0

0

13

 

Table 2: Effects

Hour

Control (0 µg/L)

A (1120 µg/L)

B (2230 µg/L)

C (3380 µg/L)

Initial

15

15

15

15

48

0

0

15

15

 

Validity criteria fulfilled:
yes
Remarks:
(immobilisation in control < 10%; dissolved oxygen concentration > 3 mg O2/L)
Conclusions:
The 48h-EC50 of terpinolene to daphnia magna was calculated to be 1380 μg/L.
Executive summary:

In an acute aquatic toxicity study performed similarly to OECD guideline 202, groups (15/dose) of daphnia magna were exposed to terpinolene at concentrations of <31, 940, 2020 and 3020 μg/L (measured) for 48 hours under flow-through conditions. Mortality and effects were observed at 48 hours. All validity criteria were fulfilled. Only 3 concentrations were tested based on information from previous aquatic toxicity study. However, the highest concentration tested resulted in 100% immobilisation and the lowest concentration tested gave no observable effect. Mortalities (48 hour) at <31, 940, 2020 and 3020 μg/L (measured) were observed in 0/15, 0/15 and 13/15 daphnids respectively. Treatment-related effects (48 hour) at <31, 940, 2020 and 3020 μg/L (measured) were observed in 0/15, 15/15 and 15/15 daphnids respectively. The 48 hour LC50, EC50 and NOEC of terpinolene to daphnia magna were calculated to be 2550 μg/L, 1380 μg/L and 940 μg/L respectively.

Endpoint:
short-term toxicity to aquatic invertebrates
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, macro invertebrates and amphibiens.
Qualifier:
according to
Guideline:
other: US EPA (1975); Methods for acute toxicity tests with fish, macro invertebrates and amphibiens
Deviations:
not specified
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: the test chemical was added to 500 mL of diluent water in 2-L jars to prepare each test solution.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Solubilizer used (triethylene glycol, ethanol, acetone or dimethylformamide).
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia
- Source: From laboratory stocks cultured at EG&G, Bionomics.
- Age at study initiation (mean and range, SD): Less than 24 hours old.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
173 ± 13 mg/L as CaCO3
Test temperature:
22 ± 1 ºC
pH:
7.4 - 9.4
Dissolved oxygen:
At the beginnig of the test, the dissolved oxygen concentration of diluent water was greater than 60 % of saturation.
The dissolved oxygen concentrations ranged from 6.5-9.1 mg/L for all tests during the 48-hour exposure period.
Nominal and measured concentrations:
Five to eight nominal concentrations were tested.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 500 mL of diluent water in 2-L jars
- No. of organisms per vessel: 15
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water was reconstituted according to U.S. EPA (1975).
- Intervals of water quality measurement: During the test, the dissolved oxygen concentration, pH and temperature of test solution were measured at the initiation and termination of the toxicity tests in the high, middle and low test concentrations and controls.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Observations of test populations were made at 24 and 48 hours of exposure and any mortalities were recorded.
Reference substance (positive control):
not specified
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
41 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: (95 % CL: 27 - 62 mg/L)
Duration:
48 h
Dose descriptor:
LC0
Effect conc.:
8.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
68 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: (95 % CL: 24 - 190 mg/L)
Details on results:
Mortality among water flea control populations never exceeded 10%.

After 48 hours, the LC50 was 41 mg/L with 95% CL: 27-62 mg/L.

Validity criteria fulfilled:
not specified
Remarks:
(There is no evidence that test substance concentration had been maintained throughout the test period).
Conclusions:
After 48 hours, the LC50 was 41 mg/L.
Executive summary:

An acute toxicity static test of alpha pinene was performed on Daphnia magna. The daphnia were exposed to several concentrations of test substance for 48 hours at 22 ± 1 ºC. Test method according to US EPA (1975): Methods for acute toxicity tests with fish, macro invertebrates and amphibiens. After 48 hours, the LC50 was 41 mg/L with 95% CL: 27-62 mg/L.

Endpoint:
short-term toxicity to aquatic invertebrates
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, macro invertebrates and amphibiens.
Qualifier:
according to
Guideline:
other: US EPA (1975); Methods for acute toxicity tests with fish, macro invertebrates and amphibiens
Deviations:
not specified
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Camphene was added to 500 mL of diluent water in 2-L jars to prepare each test solution.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): Solubilizer used (triethylene glycol, ethanol, acetone or dimethylformamide).
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia
- Source: From laboratory stocks cultured at EG&G, Bionomics.
- Age at study initiation (mean and range, SD): Less than 24 hours old.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
173 ± 13 mg/L as CaCO3
Test temperature:
22 ± 1 ºC
pH:
7.4 - 9.4
Dissolved oxygen:
At the beginnig of the test, the dissolved oxygen concentration of diluent water was greater than 60 % of saturation.
The dissolved oxygen concentrations ranged from 6.5-9.1 mg/L for all tests during the 48-hour exposure period.
Nominal and measured concentrations:
Five to eight nominal concentrations were tested.
Details on test conditions:
TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 500 mL of diluent water in 2-L jars
- No. of organisms per vessel: 15
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 1

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dilution water was reconstituted according to U.S. EPA (1975).
- Intervals of water quality measurement: During the test, the dissolved oxygen concentration, pH and temperature of test solution were measured at the initiation and termination of the toxicity tests in the high, middle and low test concentrations and controls.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Observations of test populations were made at 24 and 48 hours of exposure and any mortalities were recorded.
Reference substance (positive control):
not specified
Key result
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: (95 % CL: 13 - 36 mg/L)
Duration:
48 h
Dose descriptor:
LC0
Effect conc.:
< 13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
46 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: (95 % CL: 36 - 60 mg/L)
Details on results:
Mortality among water flea control populations never exceeded 10%.

After 48 hours, the LC50 was 22 mg/L with 95% CL: 13-36 mg/L.

Validity criteria fulfilled:
not specified
Remarks:
(There is no evidence that test substance concentration had been maintained throughout the test period).
Conclusions:
After 48 hours, the LC50 was 22 mg/L.
Executive summary:

An acute toxicity static test of camphene was performed on Daphnia magna. The daphnia were exposed to several concentrations of test substance for 48 hours at 22 ± 1 ºC. Test method according to US EPA (1975): Methods for acute toxicity tests with fish, macro invertebrates and amphibiens. After 48 hours, the LC50 was 22 mg/L with 95% CL: 13-36 mg/L.

Endpoint:
short-term toxicity to aquatic invertebrates
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:
Study conducted similarly to method EPA-821-R-02-012
Qualifier:
equivalent or similar to
Guideline:
other: EPA-821-R-02-012: Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms
Version / remarks:
Fifth Edition. October 2002
Deviations:
not specified
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
no data
Vehicle:
yes
Remarks:
acetone
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: test compound was serially diluted from an initial 0.01% (wt:vol) stock solution prepared in acetone.
- Controls: yes, acetone
- Chemical name of vehicle: acetone
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: colony maintained at the National Academy of Agriculture Science (Suwon, Republic of Korea)
- Age at study initiation: 24 hours
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
not specified
Test temperature:
20 ± 1ºC
pH:
not specified
Dissolved oxygen:
not specified
Salinity:
not specified
Conductivity:
not specified
Nominal and measured concentrations:
Nominal concentrations: 0, 6.25, 12.5, 25, 50, 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 125-ml glass tank
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per vehicle control (replicates): 4

OTHER TEST CONDITIONS
- Photoperiod: 16:8 h light:dark cycle

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Death and immobility were determined at 48 h after treatment.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
8.45 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 7.53-9.51 mg/L
Results with reference substance (positive control):
Not applicable
 
Reported statistics and error estimates:
Percentage mortality of Daphnia was determined and transformed to arcsine square-root values before analysis of variance. Treatment means were compared and separated by Scheffe´ test (SAS Institute 2002). Means (±SE) of untransformed data are reported. The 50% effective concentration values were calculated by probit analysis (SAS Institute 2002).
Validity criteria fulfilled:
not specified
Conclusions:
The 48h-EC50 of alpha terpinene to daphnia magna was reported to be 8.45 mg/L.
Executive summary:

In an acute aquatic toxicity study performed similarly to method EPA-821-R-02-012, groups (5/dose) of daphnia magna were exposed to alpha terpinene at concentrations of 0 (control), 6.25, 12.5, 25, 50 and 100 mg/L for 48 hours. Acetone was used as vehicle and as negative control. Four replicates were conducted. Mortality and effects on mobility were observed at 48 hours. The 48h-EC50 of alpha terpinene to daphnia magna was calculated to be 8.45 mg/L (95% CL: 7.53 - 9.51).

Endpoint:
short-term toxicity to aquatic invertebrates
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:
Study conducted similarly to method EPA-821-R-02-012
Qualifier:
equivalent or similar to
Guideline:
other: EPA-821-R-02-012: Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms
Version / remarks:
Fifth Edition. October 2002
Deviations:
not specified
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
no data
Vehicle:
yes
Remarks:
acetone
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: test compound was serially diluted from an initial 0.01% (wt:vol) stock solution prepared in acetone.
- Controls: yes, acetone
- Chemical name of vehicle: acetone
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: colony maintained at the National Academy of Agriculture Science (Suwon, Republic of Korea)
- Age at study initiation: 24 hours
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
not specified
Test temperature:
20 ± 1ºC
pH:
not specified
Dissolved oxygen:
not specified
Salinity:
not specified
Conductivity:
not specified
Nominal and measured concentrations:
Nominal concentrations: 0, 6.25, 12.5, 25, 50, 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 125-ml glass tank
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per vehicle control (replicates): 4

OTHER TEST CONDITIONS
- Photoperiod: 16:8 h light:dark cycle

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Death and immobility were determined at 48 h after treatment.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
7.85 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 6.86-8.96 mg/L
Results with reference substance (positive control):
Not applicable
 
Reported statistics and error estimates:
Percentage mortality of Daphnia was determined and transformed to arcsine square-root values before analysis of variance. Treatment means were compared and separated by Scheffe´ test (SAS Institute 2002). Means (±SE) of untransformed data are reported. The 50% effective concentration values were calculated by probit analysis (SAS Institute 2002).
Validity criteria fulfilled:
not specified
Conclusions:
The 48h-EC50 of d-limonene to daphnia magna was reported to be 7.85 mg/L.
Executive summary:

In an acute aquatic toxicity study performed similarly to method EPA-821-R-02-012, groups (5/dose) of daphnia magna were exposed to d-limonene at concentrations of 0 (control), 6.25, 12.5, 25, 50 and 100 mg/L for 48 hours. Acetone was used as vehicle and as negative control. Four replicates were conducted. Mortality and effects on mobility were observed at 48 hours. The 48h-EC50 of d-limonene to daphnia magna was calculated to be 7.85 mg/L (95% CL: 6.86 -8.96).

Endpoint:
short-term toxicity to aquatic invertebrates
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:
Study conducted similarly to method EPA-821-R-02-012
Qualifier:
equivalent or similar to
Guideline:
other: EPA-821-R-02-012: Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms
Version / remarks:
Fifth Edition. October 2002
Deviations:
not specified
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
no data
Vehicle:
yes
Remarks:
acetone
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: test compound was serially diluted from an initial 0.01% (wt:vol) stock solution prepared in acetone.
- Controls: yes, acetone
- Chemical name of vehicle: acetone
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Source: colony maintained at the National Academy of Agriculture Science (Suwon, Republic of Korea)
- Age at study initiation: 24 hours
Test type:
not specified
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
not specified
Test temperature:
20 ± 1ºC
pH:
not specified
Dissolved oxygen:
not specified
Salinity:
not specified
Conductivity:
not specified
Nominal and measured concentrations:
Nominal concentrations: 0, 6.25, 12.5, 25, 50, 100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 125-ml glass tank
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per vehicle control (replicates): 4

OTHER TEST CONDITIONS
- Photoperiod: 16:8 h light:dark cycle

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): Death and immobility were determined at 48 h after treatment.
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
7.22 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 6.63-7.86 mg/L
Results with reference substance (positive control):
Not applicable
 
Reported statistics and error estimates:
Percentage mortality of Daphnia was determined and transformed to arcsine square-root values before analysis of variance. Treatment means were compared and separated by Scheffe´ test (SAS Institute 2002). Means (±SE) of untransformed data are reported. The 50% effective concentration values were calculated by probit analysis (SAS Institute 2002).
Validity criteria fulfilled:
not specified
Conclusions:
The 48h-EC50 of l-limonene to daphnia magna was reported to be 7.22 mg/L.
Executive summary:

In an acute aquatic toxicity study performed similarly to method EPA-821-R-02-012, groups (5/dose) of daphnia magna were exposed to l-limonene at concentrations of 0 (control), 6.25, 12.5, 25, 50 and 100 mg/L for 48 hours. Acetone was used as vehicle and as negative control. Four replicates were conducted. Mortality and effects on mobility were observed at 48 hours. The 48h-EC50 of l-limonene to daphnia magna was calculated to be 7.22 mg/L (95% CL: 6.63-7.86).

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
11 September 2012 - 14 September 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: all concentrations and the control were sampled.
- Sampling method: Four replicate samples were taken from each test concentration and one from the control at each analytical occasion. The samples were twofold diluted with acetonitrile before the HPLC analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: A supersaturated solution (nominal loading: 1000 mg/L) was first prepared by mixing an excess of the test item in dilution water (Elendt M4 medium). This mixture was shaken overnight* and then the non-dissolved test material was separated to give the stock solution at the limit of solubility level in the test medium (i.e. 100 % v/v saturated solution). The test solutions of the chosen test concentrations were prepared by appropriate diluting of this stock solution.
The test solutions were prepared just before introduction of test animals (start of the renewal periods) and by a same way at each water renewal period.

* The stock solution used for the pre-conditioning of the test vessels was shaken for about one hour only.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna (Straus)
- Source:Laboratory of Hydrobiology (Central Agricultural Office, Directorate of Plant-, and Soil Protection) 2100 Gödöllő, Kotlán S. u. 3. Hungary. The clone was cultured since 1984 at the Laboratory of Hydrobiology. The clone was originated from the stock of the Hungarian National Health Institute.
- Age at study initiation (mean and range, SD):
- Method of breeding: The Daphnia are bred in the Laboratory of TOXI-COOP ZRT. The Daphnia are fed with concentrated algal suspension of Pseudokirchneriella subcapitata during the holding. Test animals were not fed during the exposure.
- Feeding during test: no

ACCLIMATION
Test animals were bred under similar conditions as that used during the exposure period, therefore additional acclimatisation before the test was not necessary.
Apparently healthy animals were used in this test

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
206.48 mg/L (as CaCO3)
Test temperature:
19.1 – 19.5 °C
pH:
7.74 – 8.02
Dissolved oxygen:
5.75 – 8.59 mg/L
Nominal and measured concentrations:
WAF: control, 13.3 and 18.6, 26.0, 36.4, 51.0, 71.4 and 100 % v/v saturated solution. (nominal loading: 1000 mg/L)
geometric mean of the measured concentrations: Control, 0.48, 0.73, 0.83, 1.12, 1.35, 2.10 and 3.29 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel:glass beaker
- Type (delete if not applicable): In order to minimise the evaporation of the test item the test was performed in a closed system (using sealed test vessels and a maximum volume of test solution to minimise headspace).
- Material, size, headspace, fill volume: fill volume 50 mL of test solution
- Aeration: no
- Renewal rate of test solution (frequency/flow rate): renewal after 24 hours
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4
- No. of vessels per vehicle control (replicates): NA
- Biomass loading rate: 10 mL of test solution / daphnia

TEST MEDIUM / WATER PARAMETERS
- Culture medium different from test medium: no
- Intervals of water quality measurement: 0, 24 hours (aged and new), 48 hours

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16 hours light (artificial illumination) and 8 hours darkness
- Light intensity: not provided

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
NOEC, LOEC, EC50, EC100

TEST CONCENTRATIONS
- Spacing factor for test concentrations: about 1.4
- Justification for using less concentrations than requested by guideline: NA
- Range finding study
- Test concentrations: Control, 1, 3.16, 10, 31.6, and 100 % v/v saturated solution
- Results used to determine the conditions for the definitive study: 0 % immobilization at 31.6% and 100 % immobilization at 100%
Reference substance (positive control):
yes
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
0.96 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.72 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
act. ingr.
Basis for effect:
mobility
Remarks on result:
other: 95% C.I. 0.65 – 0.76 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.48 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
act. ingr.
Basis for effect:
other: immobilisation and sublethal effects
Details on results:
- Behavioural abnormalities: Significant immobility (55 % and 80 % respectively) was detected in the concentrations of 0.73 and 0.83 mg/L. In addition, reduced activity was observed in the case of these concentrations at the animals remained alive. In the control group and in the lowest tested concentration of 0.48 mg/L immobility and any sub lethal effects were not observed. Accordingly the 48-h NOEC was determined to be 0.48 mg/L.
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: no explanation provided, except that the test item is volatile
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- EC50/LC50: 1.03 mg/L

The date of the last study (Study Code: 392.441.3906) with reference item Potassium dichromate was: 04 – 05 September 2012.
Reported statistics and error estimates:
The 24-h and 48-h EC50 values were calculated by Probit analysis using SPSS PC+ software. The NOEC, LOEC and EC100 values were determined directly from the raw data.

Table 1 Calculation of exposure concentrations

Nominal concentration
[% v/v saturated solution]

Measuredconcentration[mg/L]

1. renewal period

2. renewal period

Geometric mean

start

end

start

end

Control
(in Experiment I and II)

not detected

not detected

not detected

not detected

13.3

0.78

0.30

0.82

0.27

0.48

18.6

1.22

0.50

1.19

0.40

0.73

26.0

1.22

0.32

1.80

0.68

0.83

36.4

1.97

0.42

2.43

0.79

1.12

51.0

2.97

0.61

no data*

no data*

1.35

71.4

4.47

0.99

no data*

no data*

2.10

100.0

7.71

1.40

no data*

no data*

3.29

*   Due to the 100 % immobility at 24h these concentrations were not prepared for the second renewal period and thus provided no data for the analytical measurements

 

Table 2: Mobility of Daphnia magna at the Different Concentrations of the Test Item

 

Nominal concentration
[% v/v sat.s.]

Measured concentration
[mg/L]

No. of
Daphnia
tested

No. of immobilised
Daphniaafter

% of immobilised
Daphniaafter

24 h

48 h

24 h

48 h

Exp. I

Control

Not detected

20

0

0

0

0

26.0

0.83

20

6

16

30

80

36.4

1.12

20

14

20

70

100

51.0

1.35

20

20

20

100

100

71.4

2.10

20

20

20

100

100

100.0

3.29

20

20

20

100

100

Exp. II

Control

Not detected

20

0

0

0

0

13.3

0.48

20

0

0

0

0

18.6

0.73

20

1

5

11

55

sat.s.   =   saturated solution

Validity criteria fulfilled:
yes
Remarks:
(immobilisation in control < 10%; dissolved oxygen concentration > 3 mg O2/L)
Conclusions:
After 48 hours, the EC50 was 0.72 mg/L for Daphnia magna.
Executive summary:

In the GLP compliant Klimisch 1 study from Ágh (2012), the acute toxicity of Camphene, technical grade, solid to Daphnia magna was determined according to OECD 202 and EU method C.2 in a semi-static test (24 hour renewal interval) . The test item has a purity of82.6 % by weight and contains 12.4 % Tricyclene as impurity. A supersaturated solution (nominal loading: 1000 mg/L) was first prepared by mixing an excess of the test item in dilution water (Elendt M4 medium). This mixture was shaken overnight and then the non-dissolved test material was separated to give the stock solution at the limit of solubility level in the test medium. This stock solution was further diluted with dilution water to result in following dilutions:26.0, 36.4, 51.0, 71.4 and 100 % v/v saturated solution. Due to immobilization at 24 hours in the lowest test concentration, further dilutions with 13.3 and 18.6% were set up (experiment 2). The concentration of Camphene was experimentally determined. and following geometric mean measured concentrations were obtained: Experiment I: 0.83, 1.12, 1.35, 2.10 and 3.29 mg/L and Experiment II: 0.48 and 0.73 mg/L. After 48 hours, 0,0, 55, 80, 100, 100, 100 and 100% immobilisation was observed in the control and at mean measured 0.48, 0.73, 0.83, 1.12, 1.35, 2.10 and 3.29 mg/L. During the 24 hour aging period of the solutions the concentration of the test item declined to about 1/3 of the initial concentrations. Therefore, the concentrations were based on mean measured concentrations. The NOEC was 0.48 mg/L, the EC50 was calculated to be 0.72 mg/L.

The results of this study are considered relevant and reliable for the risk assessment.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Remarks:
Study conducted according to OECD guideline 202
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Test solutions were prepared by mixing a stock solution of the test substance, which was prepared by mixing and stirring the test substance and water.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Feeding during test: No
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
No data
Test temperature:
20.0ºC
Nominal and measured concentrations:
Nominal concentrations: 350, 230, 160, 104, 69 mg/L (geometric ratio 1.5), control

Details on test conditions:
TEST SYSTEM
- Type (delete if not applicable): closed (with gas phase)
- Material, size, headspace, fill volume: test solution volume 400 mL/concentration (100 mL/vessel)
- Aeration: no
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: dechlorinated tap water

OTHER TEST CONDITIONS
- Photoperiod: room light, 16 hours light / 8 hours dark
Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
200 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 180-230 mg/L
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
> 350 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
104 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Results with reference substance (positive control):
Not applicable
 
Validity criteria fulfilled:
not specified
Conclusions:
The 48h-EC50 of cineole to daphnia magna is determined to be 200 mg/L.


Executive summary:

In an acute aquatic toxicity study performed according to OECD guideline 202 with GLP, groups (20/dose) of daphnia magna were exposed to cineole at concentrations of 0, 69, 104, 160, 230 and 350 mg/L (geometric ratio 1.5) (nominal) for 48 hours under static conditions. Effects based on mobility were observed at 48 hours. An analytical method based on gas chromatography was used to monitor the concentration of cineole in the test solution at the start and at the end of exposure. The 48 hour EC50, EC100 and EC0 of cineole to daphnia magna were calculated to be 200 mg/L, >350 mg/L and 104 mg/L respectively.

Description of key information

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

Terpinolene: Test method similar to OECD TG 202 (Broderius S, 1990). The 48h-EC50 in Daphnia magna was determined to be 1.38 mg/L.

Camphene: Test method according to OECD TG 202 (CABB, 2012). The 48h-EC50 in Daphnia magna was determined to be 0.72 mg/L.

D-Limonene(test002): Test method similar to OECD TG 202 (Broderius S, 1990). The 48h-EC50 in Daphnia magna was determined to be 0.421 mg/L.

Alpha terpinene: Test method similar to OECD TG 202 (Broderius S, 1990). The 48h-EC50 in Daphnia magna was determined to be 1.85 mg/L.

Cineole: Test method according to OECD TG 202 (NITE, 2010). The 48h-EC50 in Daphnia magna was determined to be 200 mg/L.

Based on the available data on its components, a weight of evidence approach is applied and the 48h-EC50 of the substance in Daphanids is calculated to be 0.64 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.64 mg/L

Additional information

Weight of evidence from experimental results with individual components:

Terpinolene: In an acute aquatic toxicity study performed similarly to OECD guideline 202, groups (15/dose) of daphnia magna were exposed to terpinolene at concentrations of <31, 940, 2020 and 3020 μg/L (measured) for 48 hours under flow-through conditions. Mortality and effects were observed at 48 hours. All validity criteria were fulfilled. Only 3 concentrations were tested based on information from previous aquatic toxicity study. However, the highest concentration tested resulted in 100% immobilisation and the lowest concentration tested gave no observable effect. Mortalities (48 hour) at <31, 940, 2020 and 3020 μg/L (measured) were observed in 0/15, 0/15 and 13/15 daphnids respectively. Treatment-related effects (48 hour) at <31, 940, 2020 and 3020 μg/L (measured) were observed in 0/15, 15/15 and 15/15 daphnids respectively. The 48 hour LC50, EC50 and NOEC of terpinolene to daphnia magna were calculated to be 2550 μg/L, 1380 μg/L and 940 μg/L respectively.

Camphene: In the GLP compliant Klimisch 1 study from Ágh (2012), the acute toxicity of Camphene, technical grade, solid to Daphnia magna was determined according to OECD 202 and EU method C.2 in a semi-static test (24 hour renewal interval) . The test item has a purity of82.6 % by weight and contains 12.4 % Tricyclene as impurity. A supersaturated solution (nominal loading: 1000 mg/L) was first prepared by mixing an excess of the test item in dilution water (Elendt M4 medium). This mixture was shaken overnight and then the non-dissolved test material was separated to give the stock solution at the limit of solubility level in the test medium. This stock solution was further diluted with dilution water to result in following dilutions:26.0, 36.4, 51.0, 71.4 and 100 % v/v saturated solution. Due to immobilization at 24 hours in the lowest test concentration, further dilutions with 13.3 and 18.6% were set up (experiment 2). The concentration of Camphene was experimentally determined. and following geometric mean measured concentrations were obtained: Experiment I: 0.83, 1.12, 1.35, 2.10 and 3.29 mg/L and Experiment II: 0.48 and 0.73 mg/L. After 48 hours, 0,0, 55, 80, 100, 100, 100 and 100% immobilisation was observed in the control and at mean measured 0.48, 0.73, 0.83, 1.12, 1.35, 2.10 and 3.29 mg/L. During the 24 hour aging period of the solutions the concentration of the test item declined to about 1/3 of the initial concentrations. Therefore, the concentrations were based on mean measured concentrations. The NOEC was 0.48 mg/L, the EC50 was calculated to be 0.72 mg/L.

D-limonene (Test01): In an acute aquatic toxicity study performed similarly to OECD guideline 202, groups (15/dose) of daphnia magna were exposed to d-limonene (purchased from Aldrich) at concentrations of <150, 238, 618, 784, 1090 and 1350 μg/L (measured) for 48 hours under flow-through conditions. Only mortality was observed at 48 hours. Mortalities (48 hour) at <150, 238, 618, 784, 1090 and 1350 μg/L (measured) were observed in 0/15, 0/15, 0/15, 0/15, 15/15 and 15/15 daphnids respectively. The 48 hour LC50 of d-limonene to daphnia magna were calculated to be 924 μg/L.

D-limonene (Test02): In an acute aquatic toxicity study performed similarly to OECD guideline 202, groups (15/dose) of daphnia magna were exposed to d-limonene (purchased from Fluorostat) at concentrations of <50, 287, 619, 932, 1190 and 1630 μg/L (measured) for 48 hours under flow-through conditions. Mortality and effects were observed at 48 hours. All validity criteria were fulfilled. Mortalities (48 hour) at <50, 287, 619, 932, 1190 and 1630 μg/L (measured) were observed in 0/15, 0/15, 7/15, 15/15, 15/15 and 15/15 daphnids respectively. Treatment-related effects (48 hour) at <50, 287, 619, 932, 1190 and 1630 μg/L (measured) were observed in 0/15, 0/15, 15/15, 15/15, 15/15 and 15/15 daphnids respectively. The 48 hour LC50, EC50 and NOEC of d-limonene to daphnia magna were calculated to be 577 μg/L, 421 μg/L and 287 μg/L respectively.

Alpha terpinene: In an acute aquatic toxicity study performed similarly to OECD guideline 202, groups (15/dose) of daphnia magna were exposed to alpha terpinene at concentrations of <29, 1360, 2510, 3280, 4570 and 5890 μg/L (measured) for 48 hours under flow-through conditions. Mortality and effects were observed at 24 and 48 hours. All validity criteria were fulfilled. Mortalities (48 hour) at <29, 1360, 2510, 3280, 4570 and 5890 μg/L (measured) were observed in 0/15, 0/15, 15/15, 15/15, 15/15 and 15/15 daphnids respectively. Treatment-related effects (48 hour) at <29, 1360, 2510, 3280, 4570 and 5890 μg/L (measured) were observed in 0/15, 0/15, 15/15, 15/15, 15/15 and 15/15 daphnids respectively. The 48 hour LC50, EC50 and NOEC of alpha terpinene to daphnia magna were calculated to be 1850 μg/L, 1850 μg/L and 1360 μg/L respectively.

Cineole: In an acute aquatic toxicity study performed according to OECD guideline 202 with GLP, groups (20/dose) of daphnia magna were exposed to cineole at concentrations of 0, 69, 104, 160, 230 and 350 mg/L (geometric ratio 1.5) (nominal) for 48 hours under static conditions. Effects based on mobility were observed at 48 hours. An analytical method based on gas chromatography was used to monitor the concentration of cineole in the test solution at the start and at the end of exposure. The 48 hour EC50, EC100 and EC0 of cineole to daphnia magna were calculated to be 200 mg/L, >350 mg/L and 104 mg/L respectively.

Conclusion on test substance: Based on the available data on its components, a weight of evidence approach is applied and the 48h-EC50 of the substance in Daphanids is calculated to be 0.64 mg/L.