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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:
supporting study
Study period:
From November 30, 2006 to March 09, 2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
OECD guidelines 202 study with deviations: No graphical representation of percentages of immobilized animals against test concentrations.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
(No graphical representation of percentages of immobilized animals against test concentrations)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
limonene concentration: 98.4%
Batch 703541
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Nominal concentrations (mg limonene/mL): 0.094, 0.21, 0.45, 1.00 and 2.2 corresponding to analytical mean measured values of 0.037, 0.074, 0.16, 0.35 and 1.3, respectively and a control
- Sampling method: Duplicate samples from the freshly prepared test media of all test concentrations and the control were taken at the start of the test; concentrations were analysed at 0 and 48 hours
- Sample storage conditions before analysis: All samples were prepared for analysis directly; remaining part of sample was stored at 0-8 °C
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Test vessels were only opened as often and as long as necessary during the whole period of the study.
- The test medium of the highest test concentration of 2.2 mg/ test item/L was prepared by suspending 12 µL of the test item in 4600 mL of test water (in 2 portions of 2300 mL, each in a 2 L glass flask without headspace and tightly closed with a screw cap) The suspension was stirred for 24 hours to dissolve as much test item as possible. Then, undissolved fractions of test item, possibly existent as a second invisibly thin layer at the surface, were removed by extracting the upper 5 mL of a suspension with pippette. This solution was ussed as a stock solution to prepare the test media of the lower test concentrations. The test media were prepared just before introduction of the daphnia.
- Chemical name of vehicle: no vehicle
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain: Clone 5
- Sex: Female
- Source: Origin: ECT Oekotoxikologie GmbH, Germany ; breeding: laboratories of IBACON, Germany
- Age at study initiation: 7-22.5 hours

ACCLIMATION
- Acclimation period: 7 hours
- Acclimation conditions: Same as test


Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
No
Hardness:
2.5 mmol/L as CaCO3
Test temperature:
20-21 °C
pH:
6.9-7.7
Dissolved oxygen:
8.1-8.8 mg/L
Salinity:
No data
Nominal and measured concentrations:
Sample concentrations (mg limonene/mL) at 0 hour: Control, 0.094, 0.21, 0.45, 1.00 and 2.2
Nominal concentrations (mg limonene/mL) at 0 hour: 0, 0.094, 0.21, 0.45, 1.00 and 2.2
Sample concentrations (mg limonene/mL) at 48 hours: Control, 0.094, 0.21, 0.45, 1.00 and 2.2
Nominal concentrations (mg limonene/mL) at 48 hours: 0, 0.094, 0.21, 0.45, 1.00 and 2.2
Details on test conditions:
TEST SYSTEM
- Test vessel
- Type: Closed
- Material, size, headspace, fill volume: Wide necked Erlenmeyer flask of 300 mL volume
- No. of organisms per vessel: Five
- No. of vessels per concentration (replicates): Four
- No. of vessels per control (replicates): Four

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water: prepared by adding salts in deionised water at nominal concentrations of CaCl2.2H2O- 2 mmol/L; MgSO4.7H2O- 0.5 mmol/L; NaHCO3- 0.75 mmol/L; Kcl- 0.075 mmol/L
- Alkalinity: 0.8mmol/L
- Ca/mg ratio: 4:1
- Conductivity: < 5 µS/cm

OTHER TEST CONDITIONS
- Photoperiod: 16 hours light / 8 hours dark
- Light intensity: 450-500 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility of Daphnia after 24 and 48 hours; pH, dissolved oxygen and water temperature at the start and end of test; light intensity once; behaviour of test item in test water after 24 and 48 hours

Reference substance (positive control):
no
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
0.85 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 0.67-1.07 mg test item/L
Duration:
24 h
Dose descriptor:
EC0
Effect conc.:
0.35 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
EC100
Effect conc.:
> 1.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
NOEC
Effect conc.:
0.35 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
24 h
Dose descriptor:
LOEC
Effect conc.:
1.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.36 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: not determinable
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
0.074 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
1.3 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
0.074 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
0.16 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- After 24 hours: No immobilization of test animals in control and upto 1.0 mg limonene/L; at 2.2 mg/L number of immobilized Daphnia: 17
- After 48 hours: 1 Daphnia immobilized at control at 0.094 mg/L; no immobilization or signs of toxicity at 0.21 mg/L; 5, 6 and 20 daphnia immobilized at 0.45, 1.0 and 2.2 mg/L, respectively
Results with reference substance (positive control):
No data
Reported statistics and error estimates:
None

Table 6.1.3/1: Influence of limonene on the mobility of Daphnia magna

Nominal Concentration (mg test item/L)

No. of Daphnia tested

No. of immobilized Daphnia after

% of immobilized Daphnia after

24 hours

48 hours

24 hours

48 hours

Control

20

0

1

0

5

0.094

20

0

1

0

5

0.21

20

0

0

0

0

0.45

20

0

5

0

25

1

20

0

6

0

30

2.2

20

17

20

85

100

 

Table 6.1.3/2: Dissolved oxygen concentration, pH-values and water temperature in the test media at the start and end of the test

Nominal Concentration (mg test item/L)

Start (0 Hours)

End (48 hours)

pH

O2 (mg/L)

Water temperature (°C)

pH

O2 (mg/L)

Water temperature (°C)

Control

6.9

8.8

20

7.6

8.1

20

0.094

7.2

8.8

20

7.7

8.1

_

0.21

7.3

8.8

20

7.6

8.2

_

0.45

7.3

8.8

20

7.7

8.6

_

1

7.3

8.8

21

7.7

8.5

_

2.2

7.3

8.8

21

7.7

8.7

_

Validity criteria fulfilled:
yes
Remarks:
Control Immobilization Rate: 5.0%; No other Daphnia showed signs of disease or distress; Dissolved Oxygen Concentration => 8.1 mg/L
Conclusions:
The 48-hours EC50 for d-limonene is less than 1 mg/L in Daphnia magna, therefore d-limonene 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 in accordance with OECD Guideline 202 and GLP, groups (5/concentration in 4 replicates) of Daphnia magna were exposed to d-limonene at concentrations of 0 (blank), 0.094, 0.21, 0.45, 1.00, 2.2 mg/L (nominal) for 48 hours under static conditions. Numbers of daphnia no longer capable of swimming were recorded at 24 and 48 hours.

 

Numbers of immobilized daphnia at nominal concentrations of 0 (blank), 0.094, 0.21, 0.45, 1.00, 2.2 mg/L were 0, 0, 0, 0, 0, 0 and 17 at after 24 hours and 1, 1, 0, 5, 6 and 20, respectively after 48 hours.

Therefore, the 48-hour EC50 value was found to be 0.36 mg test item/L.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From December 11 to 14, 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD Guideline 202 without deviation.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
inspected on July 05, 2012 / signed on January 11, 2013
Analytical monitoring:
yes
Details on sampling:
- Single samples for analysis were taken from the control and all test concentrations from replicates without daphnids.
- Frequency of sampling: At the start of the test (t=0 h), at t=24 h (new and old solutions) and the end of the test (t=48 h).
- Sample storage conditions before analysis: Samples were analysed directly after sampling
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Two stock solutions (one for the fresh medium at t=0 h and another for the fresh medium at 24 h) were prepared by slow-stirring. The mixing vessel was a cylindrical glass bottle sealed with screw cap and fitted with a drain port near the bottom for drawing off the saturated solution. The volume of the mixing vessel was approximately 1 L. A magnetic stirring bar was placed in the vessel and 1 L of the test water (2.2.) was added. This was done to use a maximum volume and to minimize head space during preparation. Then an excess of the test item (approximately 3 g) was carefully added directly to the surface of the test water forming a surface layer of about 1-2 mm to maintain optimum surface contact between test item and the water. Mixing was initiated with the vortex in the centre extending maximally around 10 % vessel depth from the top to the bottom of the vessel. The stirring speed was kept as low as possible to maintain mixing of the water phase without dispersing the test substance in the water phase and this was visually observed to be the case. After 24 ± 2 hours of gentle stirring with no standing time (stirring was stopped before drawing off the aqueous phase), the saturated aqueous phase was taken out of the drain port. The first 100 mL were discarded and three samples were taken from the following stock solution and chemically analyzed. Then the stock solution was diluted with test water as necessary into 200-mL volumetric flasks (filled up to the meniscus) to obtain the required test concentrations based on the measured concentration of the stock solution (3.94 mg/L for the fresh medium at t=0 h and 3.15 mg/L for the fresh medium at t=24 h. These concentrations are comparable with slow stir values of maximum aqueous solubility in daphnid medium obtained from previous experiments by different laboratories). Each prepared concentration was inverted several times before filling of test tube (without headspace) to ensure adequate mixing and homogeneity. After filling the vessels were sealed immediately with screwcaps after introduction of daphnids.
The test sample of D-Limonene formed a clear colourless solution and appeared to be completely soluble and stable when mixed with test water at the concentrations prepared.

- Controls: Test water without test substance but treated in the same way as the test substance solutions.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain: Daphnia magna (Straus), clone 5
- Source: LIEBE CNRS UMR 7146 - UFR SciFA Universite de Lorraine Campus Bridoux - Bat.IBISE, 8, rue du General Delestraint - 57070 METZ bred in the Laboratoires des pyrénées.
- Age at study initiation: < 24 h
- Breeding conditions: Daphnids were cultured under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass beakers containing purified drinking water (Composition (mg/L): Calcium 11.5; Magnesium 8.0; Sodium 11.6; Potassium 6.2; Silica 31.7; Bicarbonates 71.0; Sulfates 8.1; Chlorides 13.5; Nitrates 6.3; pH = 7). Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. During the week the stock daphnids were fed daily with a suspension of freshwater algae (mix of 3 algae strains: Chlorella vulgaris - 2.5x10^6 cells/mL/day/daphnid; Desmodesmus subspicatus - 2.5x10^6 cells/mL/day/daphnid and Pseudokirchneriella subcapitata - 5x10^6 cells/mL/day/daphnid. The water was changed once per week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.
- Feeding during test: No feeding

ACCLIMATION
- At least 48 h prior the start of the test daphnids were transferred to OECD test water at the test temperature to be used in the study. Briefly, batches of at least 50 gravid daphnids were separated from the stock population and held at similar temperature and light conditions as used in the test. During this period, daphnids were fed in the same manner as that of the stock population. Only daphnids up to 24 h old were used for the test.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
Total water hardness was approximately 250 mg/L as CaCO3.
Test temperature:
19.4-20.2 °C (average 19.9 °C)
pH:
pH: 6.0-9.0, not varying by more than 1.5 units
Dissolved oxygen:
Oxygen: ≥60 % of the air-saturation value at the end of the test
Salinity:
None
Nominal and measured concentrations:
Nominal concentrations: 0.2, 0.3, 0.4, 0.6, 0.8 and 1.2 mg/L
Measured concentrations: 0.221, 0.263, 0.369, 0.523, 0.659 and 1.014 mg/L (averaged values)
Details on test conditions:
TEST SYSTEM
- Test vessel: All-glass test tubes of approximately 20 mL capacity sealed with screwcaps.
- Type (delete if not applicable): Closed
- Aeration: No aeration of the test solutions occurred throughout the test.
- Renewal rate of test solution: A semi-static test was performed with renewal of test solutions after 24 h, in order to maintain test concentrations within 80-120 % of nominal values since the test substance was volatile.
- No. of organisms per vessel: 5 daphnids per vessel each completely filled with test solution and without headspace
- No. of vessels per concentration (replicates): 4 replicates
- No. of vessels per control (replicates): 4 replicates
- Moreover, 4 abiotic replicates for the control and per test concentration were prepared: two for sampling for analysis of test concentrations from the freshly prepared solutions (t=0 h and t=24 fresh) and two for sampling from the 24 h old solutions (t=24 old and t=48).
- Introduction of daphnids: Daphnids were introduced into the test medium immediately after filling of test tubes with test solutions and the vessels recapped.

TEST MEDIUM / WATER PARAMETERS
- Test water: Reconstituted water, as prescribed by the OECD Guideline 202
- CaCl2.2H20: 11.76 g/L; MgSO4.7H20: 4.93 g/L; NaHCO3: 2.59 g/L; KCl: 0.23 g/L
- Conductivity: < 10 µS/cm

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark
- Light intensity: ca. 400 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- Immobility: Immobility and abnormal behaviour were determined by visual observation after 24 and 48 h. Immobile animals were eliminated from the vessels as soon as they were discovered. The daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of the test vessels.
- pH and dissolved oxygen: At the start of the test (t=0 h), at t=24 h (new and old solutions) and the end of the test (t=48 h) in all vessels.
- Temperature of medium: Measured continuously in a vessel next to the test vessels, over the entire study period, beginning at the start of the test.

TEST CONCENTRATIONS
- Range finding study: 0.3, 0.5, 0.8, 1.2 and 2 mg/L
- Results used to determine the conditions for the definitive study: 0, 0, 0, 90, 100 and 100 % mortality was observed at 0.3, 0.5, 0.8, 1.2 and 2 mg/L.
- Spacing factor for test concentrations: 1.43; Based on the results of a range-finding test, test solutions used in the definitive test were prepared by direct addition of the required amounts of stock solution to the test water to obtain the following nominal concentrations (spaced by a factor of approximately 1.43): 0.2, 0.3, 0.4, 0.6, 0.8 and 1.2 mg/L
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.307 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 0.257-0.354 mg/L
Details on results:
- After 24 h of exposure, immobilizations were 0 % at 0 mg/L, 15 % at 0.3 mg/L, 20 % at 0.2 mg/L, 25 % at 0.4 mg/L, 55 % at 0.6 and 0.8 mg/L, and 100 % at 1.2 mg/L.
- After 48 h of exposure, immobilizations were 0 % at 0 mg/L, 20 % at 0.2 mg/L, 50 % at 0.3 mg/L, 60 % at 0.4 mg/L, 80 % at 0.6 mg/L, 95 % at 0.8 mg/L and 100 % at 1.2 mg/L.
Results with reference substance (positive control):
- In July 2012 (most recent test), the 24 h-EC50 for potassium dichromate was 1.28 mg/L. Hence, the sensitivity of the clone of Daphnia magna was in agreement with the OECD 202 (expected 24 h-EC50: 0.6-2.1 mg/L) at this time.
Reported statistics and error estimates:
Evaluation and statistics:
The evaluation of the effects was based on the nominal and measured test item concentrations. The determination of the EC50 was performed according to the schedule below:
- When for at least two concentrations, immobilization is > 0 % and < 100 %, the data are analyzed by regression to determine the EC50 including the 95 % confidence interval using Probit analysis via the computer program ToxRat.
- When at only one concentration, immobilization is > 0 % and < 100 %, the EC50 is also calculated by Probit analysis via ToxRat but in this case, the highest concentration causing no immobilization and the lowest concentration producing 100 % immobilization are used as confidence limits.
- If at all concentrations immobilization is 0 or 100 %, the EC50 corresponds to the geometric mean of the highest concentration causing no immobilization and the lowest concentration producing 100% immobilization.

At t=24 h and t=48 h immobilization was > 0% and < 100% for at least two concentrations, that is why the data were analyzed by regression to determine the EC50 including the 95 % confidence interval using Probit analysis via the computer program ToxRat.

Table 6.1.3/1: Acute immobilization of daphnids after 24 and 48 hours in the final test

 

Nominal

Concentration mg/L

Replicate

Number of

daphnids

exposed

Response at 24 h

Response at 48 h

Number

Number Total %

Number

Number Total %

Control

1

5

0

0

0

0

2

5

0

0

3

5

0

0

4

5

0

0

0.2

1

5

2

20

2

20

2

5

1

1

3

5

0

0

4

5

1

1

0.3

1

5

0

15

5

50

2

5

1

1

3

5

1

2

4

5

1

2

0.4

1

5

1

25

1

60

2

5

1

5

3

5

3

4

4

5

0

2

0.6

1

5

2

55

3

80

2

5

3

5

3

5

3

5

4

5

3

3

0.8

1

5

4

55

5

95

2

5

1

4

3

5

1

5

4

5

5

5

1.2

1

5

5

100

5

100

2

5

5

5

3

5

5

5

4

5

5

5

 

 

Analytical results: The test item levels were found to be on the whole stable throughout the test. Indeed, concentrations measured in freshly prepared solutions and in 24 h old solutions revealed that the concentration of the test item was satisfactorily maintained within ±20 % of the initial concentration throughout the test, except the first day at 0.8 and 1.2 mg/L and at the end of the test at 0.2 and 0.4 mg/L (nominal). 

Nominal concentrations: 0.2, 0.3, 0.4, 0.6, 0.8 and 1.2 mg/L

Corresponding average exposure concentrations: 0.221, 0.263, 0.369, 0.523, 0.659 and 1.014 mg/L

 

Validity criteria of the study

Controls: In the control, no daphnids became immobilized nor trapped at the surface of the water nor showed signs of stress.

Dissolved oxygen: The dissolved oxygen concentration at the end of the test was 60 % of the air-saturation value in controls and test vessels.

Thus the validity criteria have been respected in the present study.

Validity criteria fulfilled:
yes
Conclusions:
The 48-h EC50 for test item to Daphnia magna is 0.307 mg/L based on measured concentrations.
Executive summary:

A study was performed to assess the acute toxicity of test item d-limonene to Daphnia magna. This study was performed according to OECD Guideline 202 with GLP statement, to assess the 48 h-acute toxicity of the test substance to Daphnia magna, under semi-static conditions.

 

Following a preliminary range finding test and previous tests, twenty daphnids (four replicates, five daphnids per replicate) were exposed to an aqueous solution of the test item at the required nominal test concentrations of 0.2, 0.3, 0.4, 0.6, 0.8 and 1.2 mg/L. The immobility of the daphnids was determined in a semi-static 48 h test by visual observation after 24 and 48 h. The concentrations of the test item were determined by chemical analyses at the start (t=0 h), at t=24 h (new and old solutions) and at the end of the test (t=48 h).

 

The test item levels were found to be on the whole stable throughout the test. Indeed, concentrations measured in freshly prepared solutions and in 24 h old solutions revealed that the concentration of the test item was satisfactorily maintained within ±20 % of the initial concentration throughout the test, except the first day at 0.8 and 1.2 mg/L and at the end of the test at 0.2 and 0.4 mg/L (nominal).  After 24 h of exposure, immobilizations were 0% at 0 mg/L, 15% at 0.3 mg/L, 20% at 0.2 mg/L, 25% at 0.4 mg/L, 55% at 0.6 and 0.8 mg/L, and 100% at 1.2 mg/L. After 48 h of exposure, immobilizations were 0% at 0 mg/L, 20% at 0.2 mg/L, 50% at 0.3 mg/L, 60% at 0.4 mg/L, 80% at 0.6 mg/L, 95% at 0.8 mg/L and 100% at 1.2 mg/L.

24 h EC50: 0.482 mg/L (95% confidence limits: 0.410 -0.575 mg/L)

48 h EC50: 0.307 mg/L (95% confidence limits: 0.257-0.354 mg/L)

 

Therefore, the 48-h EC50 for test item to Daphnia magna is 0.307 mg/L based on measured concentrations.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 July to 01 August 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study performed according to OECD Guideline No. 202. All validity criteria were fulfilled.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Specific details on test material used for the study:
PHYSICO-CHEMICAL PROPERTIES
- Density (20 °C): 0.843 g/mL
- Water solubility: very low
Analytical monitoring:
yes
Details on sampling:
- In order to verify the tested concentrations, samples were collected from the test solutions at the initiation of the test (T = 0 h), at 24 h (old and new test solutions) and at the termination of the test (T = 48 h). In order to freeze the samples for chemical analysis, approx. 6-7 mL were discarded from the test vials and the remaining approx. 35 mL were frozen immediately and stored at -20 ± 2.0 °C until dispatch for chemical analysis. Samples from the test concentrations 0, 19.8, 29.6, 44.4, 66.7 and 100 % were analysed as they covered the concentrations causing 0-100 % effect. Although duplicate samples were collected from each of the test concentrations, only one of these samples was analysed.
- For chemical analysis, a parallel series of test vials without animals was set-up under the same conditions as the test with renewal of test solutions after 24 h.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A stock solution (A) of 1 g/L was prepared by adding 2.65 mL (corresponding to 2.23 g) of the test item to 2.22 L of test medium in a blue cap glass flask without any headspace. The flask was placed on a magnetic stirrer and left in the dark for gentle stirring overnight, approx. 22 h. After stirring, another stock solution (B) was prepared by siphoning the mid fraction from stock solution A. The pH was 7.8 in this stock solution (B). The test solutions were prepared by diluting stock solution B in the test medium.
After 24 h of exposure, this procedure was repeated when preparing fresh test solutions. The pH was 7.7 in stock solution B. As pH in the stock solutions were within 7.8 ± 0.5, no pH adjustment was necessary.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain: Daphnia magna Straus
- Source: A strain of Daphnia magna Straus collected in Langedam, Birkerød, has been cultured at DHI since 1979.
- Age at study initiation: Young animals (less than 24 h)
- Feeding: Culture is fed with Pseudokirchneriella subcapitata three times a day by a peristaltic pump system.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
Hardness was 258 mg/L expressed as CaCO3
Test temperature:
Climate room at 21 ± 0.8 °C in total darkness
pH:
Measured value min.-max. 7.5-7.8
Dissolved oxygen:
Dissolved oxygen saturation at the end of the test was 100 % in all tested concentrations and the control.
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentrations: 0 (control); 19.8, 29.6, 44.4, 66.7 and 100 % of a saturated solution of the test item in test medium
Details on test conditions:
TEST SYSTEM
- Test vessel: 42-mL glass vials sealed with PTFE-coated screw caps
- Renewal rate of test solution: After 24 h of exposure, all test solutions were renewed.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 6
- At the initiation of the test, 41 mL of test solution and 5 animals were added to each vial. A minor head space was present in the test vials and the vials were shaken gently (60 rpm) during the test.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO-medium according to ISO 6341

EFFECT PARAMETERS MEASURED
- Number of immobile animals was recorded after 24 and 48 h.
- Dissolved oxygen and pH were measured at 0 h, at 24 h before and after renewal of test solutions and at 48 h.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
24 h
Dose descriptor:
EC10
Effect conc.:
0.45 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % Cl: 0.32-0.55 mg/L
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
0.84 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % Cl: 0.71-0.99 mg/L
Duration:
48 h
Dose descriptor:
EC10
Effect conc.:
0.42 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % Cl: 0.27-0.46 mg/L
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
0.51 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % Cl: 0.46-0.59 mg/L
Results with reference substance (positive control):
In accordance with the test method, a 24-h acute toxicity test on the reference substance potassium dichromate was performed to check the sensitivity of the test animals. The reference substance was tested at the following concentrations: 0.2, 0.4, 0.7, 1.0, 1.4 and 2.0 mg/L.
- 24 h EC50 value of the reference substance potassium dichromate was 0.6 (0.49- 0.7) mg/L, which is within the range specified in ISO 6341 (0.6-2.1 mg/L).
Reported statistics and error estimates:
The EC values for the test item and the reference substance (potassium dichromate) were calculated by use of the standard procedure Probit Analysis (Version 2.3, 1990.03.20. Statens Naturvårdsverk (National Swedish Environmental Protection Board. The data section).

Table 6.1.3/1: Acute immobilisation of daphnids after 24 and 48 hours exposure

 

Concentration

 

Total number of animals per conc.

 

Number of animals per vessel

 

 

NUMBER OF IMMOBILE

 

24 HOURS

 

48 HOURS

 

Nominal (%)

 

Avg. measured (mg/L)

 

A

C

E

A

C

E

Total

 

A

C

E

Total

 

B

D

F

B

D

F

B

D

F

Control

30

5

5

5

0

0

0

0

0

0

0

0

5

5

5

0

0

0

0

0

0

19.8

0.28

20

5

5

 

0

0

 

0

0

0

 

0

5

5

 

0

0

 

0

0

 

29.6

0.48

20

5

5

 

2

0

 

3

3

0

 

7

5

5

 

1

0

 

3

1

 

44.4

0.72

20

5

5

 

2

2

 

8

5

5

 

20

5

5

 

3

1

 

5

5

 

66.7

1.4

21

6

5

 

4

4

 

16

6

5

 

21

5

5

 

4

4

 

5

5

 

100

2.8

20

5

5

 

5

5

 

20

-

-

 

20

5

5

 

5

5

 

-

-

 

 

 

Analytical results

Table 6.1.3/2: Results from chemical analyses of d-Limonene

Sampling time

 

T = 0 hours (mg/L)

 

T = 24 hours - old (mg/L)

 

T = 24 hours - new (mg/L)

 

T = 48 hours (mg/L)

 

Mean value (mg/L)

 

Control

 

*)

<0.010

<0.010

<0.010

<0.010

19.8 %

0.29

0.27

0.25

0.31

0.28

29.6 %

0.42

*)

0.41

0.61

0.48

44.4 %

0.82

*)

0.63

0.71

0.72

66.7 %

1.5

1.4

1.6

1.2

1.4

100 %

2.7

2.6

2.8

3.1

2.8

*) Vials smashed during freezing or transport

The control (T = 0 h) was not measured as the frozen vial was smashed during transport to Eurofins Environment Sweden AB.

Samples from 29.6 and 44.4 % (T = 24 h before renewal) were measured but as the vials were smashed in the freezer, only very small and insufficient samples were saved from the vials and, therefore, the analyses were not regarded as valid.

The chemical analysis of d-Limonene shows the actual measured test concentrations in the study, which were used in the data analysis of EC10 and EC50 values.

Validity 

Less than 10 % of the control animals were immobilized

The dissolved oxygen saturation at the end of the test was 100% in all tested concentrations and the control.

The 24 h EC50 value of the reference substance potassium dichromate was 0.6 (0.49- 0.7) mg/L, which is within the range specified in ISO 6341 (0.6-2.1 mg/L).

Validity criteria fulfilled:
yes
Conclusions:
The 48 h-EC50 for test item to Daphnia magna is 0.51 mg/L based on the measured test concentrations.
Executive summary:

A GLP study was performed to assess the acute toxicity of test item d-limonene to Daphnia magna according to OECD Guideline 202 under semi-static conditions.

 

Twenty daphnids (four replicates, five daphnids per replicate) were exposed to dilutions of the saturated solution of the test item in test medium at the nominal test concentrations of 19.8, 29.6, 44.4, 66.7 and 100%. All test solutions were renewed after 24 h of exposure. The number of immobile animals was recorded after 24 and 48 h. The concentrations of the test item were determined by chemical analyses at start, 24 and 48 h incubation period.

                       

24 h EC10: 0.45 mg/L (95% Cl 0.32-0.55 mg/L); EC50: 0.84 mg/L (95% Cl 0.71-0.99 mg/L)

48 h EC10: 0.42 mg/L (95% Cl 0.27-0.46 mg/L); EC50: 0.51 mg/L (95% Cl 0.46-0.59 mg/L)

 

Therefore, the 48 h-EC50 for test item to Daphnia magna is 0.51 mg/L based on the measured test concentrations.

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: immobilisation not reported; no data on acclimation period, feeding pattern and reference substance
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
immobilisation not reported; no data on acclimation period, feeding pattern and reference substance
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 conditions
- Controls: Dilution water
- Chemical name of vehicle: No vehicle
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 ± 0.2 mg/L CaCO3
Test temperature:
19.7 ± 0.5 °C
pH:
7.9 ± 0.13
Dissolved oxygen:
8.0 ± 0.3 mg O2/L
Salinity:
No data
Nominal and measured concentrations:
Nominal concentrations: 0, 312, 624, 936, 1250 and 1560 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 250 mL; test solution depth: 4-5 cm
- Type: 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 provided toxicant concentrations of 178-1350 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
- Alkalinity: 36.6 ± 1.5 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):
- Mortalities were observed at 24 and 48 hours
Reference substance (positive control):
no
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
Details on results:
- Mortalities (48 hour) at 0, 312, 624, 936, 1250 or 1560 µg/L (nominal) were observed in 0/15, 0/15, 0/15, 0/15, 15/15 or 15/15 daphnids, respectively.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
- LC50 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 6.1.3/1: Mortalities

 

Hour

Control (0 µg/L)

A (312 µg/L)

B (624 µg/L)

C (936 µg/L)

D (1250 µg/L)

E (1560 µg/L)

Initial

15

15

15

15

15

15

deads 48

 0

 0

15

15

Validity criteria fulfilled:
yes
Remarks:
dissolved oxygen concentration > 3 mg O2/L; mortality in control < 10% but immobilisation not observed
Conclusions:
The 48 hour LC50 of d-limonene to Daphnia magna was calculated to be 924 µg/L. EC50 values were not calculated.
Executive summary:

In an acute aquatic toxicity study performed similarly to OECD Guideline 202, groups (15/concentration) of Daphnia magna was exposed to d-limonene at concentrations of 0, 312, 624, 936, 1250 or 1560 µg/L (nominal) for 48 hours under flow-through conditions. Mortality and effects were observed at 24 and 48 hours.

 

Mortalities (48 hour) at 0, 312, 624, 936, 1250 or 1560 µg/L (nominal) were observed in 0/15, 0/15, 0/15, 0/15, 15/15 or 15/15 daphnids, respectively. The 48 hour LC50 of d-limonene to Daphnia magna was calculated to be 924 µg/L. EC50 values were not calculated.

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, feeding pattern and reference substance
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
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, feeding pattern and reference substance
GLP compliance:
not specified
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 398, 796, 1190, 1590 or 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: no vehicle
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:
20.0 ± 0.4 °C
pH:
7.9 ± 0.1
Dissolved oxygen:
7.8 ± 0.5 mg O2/L
Salinity:
No data
Nominal and measured concentrations:
Nominal concentrations: 0, 398, 796, 1190, 1590 and 1990 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 215-1890 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):
- Mortalities were observed at 24 and 48 hours
Reference substance (positive control):
no
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: 496-672 µg/L
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
Details on results:
- Mortalities (48 hour) at 0, 398, 796, 1190, 1590 or 1990 µg/L (nominal) were observed in 0/15, 0/15, 7/15, 15/15, 15/15 or 15/15 daphnids, respectively.
- Effects (48 hour) at 0-398 or 796-1990 µg/L (nominal) were observed in 0/15 or 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 (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

deads 48

 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

Effects 48

 0

 0

15

15

15

15

Validity criteria fulfilled:
yes
Remarks:
mortality in control < 10% (no immobilisation observed); dissolved oxygen concentration > 3 mg O2/L
Conclusions:
The 48 hour EC50 for d-limonene is less than 1 mg/L in Daphnia magna 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 202, groups (15/dose) of Daphnia magna was exposed to d-limonene at concentrations of 0, 398, 796, 1190, 1590 or 1990 µg/L (nominal) for 48 hours under flow-through conditions. Mortality and effects were observed at 24 and 48 hours.

 

Mortalities (48 hour) at 0, 398, 796, 1190, 1590 or 1990 µg/L (nominal) were observed in 0/15, 0/15, 7/15, 15/15, 15/15 or 15/15 daphnids, respectively. Treatment-related effects (48 hour) at 0-398 or 796-1990 µg/L (nominal) were observed in 0/15 or 15/15 animals, respectively.

Therefore, the 48-hour LC50 or EC50 of d-limonene to Daphnia magna were calculated to be 577 or 421 µg/L.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
(Q)SAR
Adequacy of study:
supporting 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
Remarks:
QSAR value. The substance falls into applicability domains of the model QSAR.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
equivalent or similar to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Principles of method if other than guideline:
The purpose of this QSAR is to accurately predict the acute toxicity to daphnid as would be expected in a laboratory experiment following OECD Guideline 202 and EC method C.2 for specific, named modes of action (e.g. non-polar narcotics) to provide a value that can effectively replace a 48-hour EC50 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):
Daphnia pulex
Details on test organisms:
No data
Test type:
other: QSAR model
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
Not applicable
Hardness:
Not applicable
Test temperature:
Approximately 20-23 °C depending on the species used
pH:
6.0 - 9.0
Dissolved oxygen:
Not applicable
Salinity:
Not applicable
Conductivity:
Not applicable
Nominal and measured concentrations:
Not applicable
Reference substance (positive control):
no
Remarks:
QSAR model
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
ca. 0.62 mg/L
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95 % CL 0.55-0.69
Details on results:
Not applicable
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
95% CL [0.55-069 mg/L] QSAR statistical parameters are given in the QMRF and the QPRF

None

Validity criteria fulfilled:
yes
Remarks:
The substance falls into applicability domains of the QSAR model
Conclusions:
48 h-EC50 for d-limonene = 0.62 mg/L with 95 %-Confidence Limit of 0.55-0.69 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 under 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 daphnids. 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. 202, "Daphnia sp., Acute Immobilisation Test", referenced as Method C.2 of Commission Regulation No. 440/2008. The criterion was the EC50 (Median Effective Concentration), a statistically derived concentration which is expected to cause immobility in 50 % of test animals within a period of 48 hours.

The QSAR determination of the daphnids immobility was based on validated data derived from 48 h tests on daphnids, for which the concentrations of the test item had been determined by chemical analyses over the test periods.

 

The 48 h EC50 is predicted as 0.62 mg/L with 95 %-Confidence Limit between 0.55 and 0.69 mg/L.

 

This toxicity study is considered acceptable and satisfies the guideline requirements for acute daphnid 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 under Regulation (EC) No 1272/2008 (CLP) - Acute Category 1.

 

Results Synopsis

Test Type: QSAR model

EC50: 0.62 mg/L 95%-C.L.: 0.55 to 0.69 mg/L

Description of key information

The substance exhibits an EC50 for freshwater invertebrates of 0.307 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
0.307 mg/L

Additional information

Five experimental data and a valid QSAR prediction are available and are provided as endpoint study records.

Experimental studies investigated the short-term toxicity of substance d-limonene on the freshwater Daphnia magna (less than 24 h old). The studies were performed according to or in a similar way to test methods described by Guideline OECD 202 under semi-static or flow-through conditions. Immobilisation were observed at 24 and 48 hours. The 48h-EC50 are determined in the range 0.307-0.51 mg/L.

Besides, an endpoint value from High Accuracy QSARs is also available. This provides a 48h-EC50 of 0.62 (0.55-0.69) mg/L.

All the available data falls in the aquatic acute toxicity range of 0.1-1.0 mg/L. The lowest acute toxicity value obtained experimentally is given as key value for the chemical safety assessment.