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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:
key study
Study period:
12 December 2018 – 11 January 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0 (control), 1.5, 2.8, 5.7, 11.2, 22.5 and 44.9 mg/L (nominal loading rates)
- Sampling method: During Range finding study and main study, TOC was analyzed on day 0 (fresh) and on day 2 (spent) from samples for each loading rate.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
The test item is a complex mixture (UVCB substance) which was found to be partially miscible in test medium (ISO reconstituted water) according to a lab internal method. Therefore, the Daphnia Test was carried out with aqueous extracts (WAF = Water Accommodated Fraction) at various loading rates of the test item in test medium.
In order to determine the optimum mixing duration to be used for WAF preparation, a water accommodated fraction (WAF) equilibration trail was performed prior to exposure: One individual WAF was prepared at each of three loading levels 1.0, 50.0 and 100 mg/L. At 2, 4, 6 and 24 hours after initiation of mixing, mixing was stopped at respective time points and the solutions were allowed to settle for one hour. A sample of WAF was removed from each loading level and mixing was resumed. The concentration of organic carbon that had solubilized into the WAF from the test item was measured using the TOC analyzer described above.
The test item equilibrium was attained at 4 hour mixing according to the equilibration trail, hence mixing for 4 hours was followed during the test media preparation.
Test media preparation: For every test item concentration, individual WAF was prepared by adding the appropriate amount of test item to the test medium. Each mixing vessel was filled to achieve minimal headspace given the constraints of the vessels and closed with foil-covered stoppers. The mixture was stirred for 4 h (based on equilibration trail) using a magnetic stirrer at minimum of 100 rpm. The mixtures were allowed to settle and equilibrate to test temperature (before removing the test solution). The aqueous portions of WAFs were collected using steri-pipette and transferred to test vessels.
- Controls: ISO reconstituted water only.
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): The test substance was not fully soluble at the tested loading rate. However the method of preparing the WAFs was designed to mimimise exposure to undissolved test material.

Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: Environmental and Molecular Toxicology Division, Department of Zoology, Karnataka University, Karnataka, India.
- Feeding during test: No

ACCLIMATION
- Acclimation period: 25 gravid female daphinds were acclimatized in reconstituted water for 48 hours prior to test (similar for range finding and main study). Offsprings produced on the first day were not used. Progenies produced on day two (less than 24 hours old) were transferred into the test containers.
- Acclimation conditions (same as test or not): same as test.
- Type and amount of food: live algal cells (Pseudokirchneriella subcapitata) at 2 mL per liter.
- Feeding frequency: at the beginning and on Day 2 of acclimatization.
- Health during acclimation (any mortality observed): Not specified.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
212 mg/L as CaCO3
Test temperature:
20.1 - 20.9ºC
pH:
7.39 - 8.51
Dissolved oxygen:
7.39 - 8.14 mg O2/L
Nominal and measured concentrations:
Nominal loading rates: 0 (control), 1.5, 2.8, 5.7, 11.2, 22.5 and 44.9 mg/L WAF

Details on test conditions:
TEST SYSTEM
- Test vessel: rectangular beaker
- Material, size, headspace, fill volume: glass, 100 mL (fill volume).
- Aeration: The dilution water was aerated prior to use for the test so that the dissolved oxygen concentration has reached saturation.
- Renewal rate of test solution (frequency/flow rate): Not applicable (static test)
- 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): Not applicable
- Biomass loading rate: 20 mL of test solution per daphnid.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: ISO reconstituted water prepared as described in the OECD 202 guideline.
- Intervals of water quality measurement: Total hardness was analyzed prior to its use for exposure. Temperature, pH and dissolved oxygen were recorded at test initiation and at the beginning and at the end of test in the treatments and in the control.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours of light and 08 hours dark cycle.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Each test vessel was observed for immobilized daphnids at 24 and 48 hours after the beginning of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker was considered to be immobile. In addition to immobility, any abnormal behavior or appearance was recorded.

VEHICLE CONTROL PERFORMED: Not applicable

RANGE-FINDING STUDY
- Test concentrations: 0.00897, 0.0895, 0.897, 9.1, 44.9 and 89.7 mg/L along with a control group.
- Results used to determine the conditions for the definitive study: No immobilities were recorded in control group and at the tested nominal loading rates of 0.00897, 0.0895 and 0.897 mg/L. Immobilities of 40, 100 and 100% were observed at the nominal loading rates of 9.1, 44.9 and 89.7 mg/L respectively.
Reference substance (positive control):
yes
Remarks:
(Potassium dichromate)
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
16.78 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(water-accommodated fraction loading rate)
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 13.12 - 21.47 mg/L.
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
2.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(water-accommodated fraction loading rate)
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOELR
Effect conc.:
5.7 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
(water-accommodated fraction loading rate)
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities:
- Mortality of control: No mortality was observed over a period of 48 h in the control group.
- Abnormal responses: No clinical signs of toxicity were observed in control and at 1.5 mg/L. Clinical sign of localized on bottom of container and lethargy was observed at 24 hour and 48 hour observation period at 2.8, 5.7,11.2 and 22.5 mg/L. Clinical sign of localized on bottom of container and lethargy was observed at 24 hour observation at 44.9 mg/L.

Results with reference substance (positive control):
- Results with reference substance valid? Yes, the 48 hour EC50 of the reference substance was within the acceptance range confirming the suitability of the test system. EC50 of Potassium dichromate on Daphnia magna is periodically determined. The date of the last study at the date of performing this test was 22-02-2018.
- EC50-48 h: 0.57 mg/L
Reported statistics and error estimates:
Data were analyzed by statistical method (Finney’s probit analysis) to calculate the slopes of the curves and the EL50 with confidence limits. One-way ANOVA was performed for 48 hour immobility for the determination of NOELR and LOELR.

Table 1. Summary of clinical signs and immobilisation during range finding study

Group

Nominal Loading Rate (mg/L)

R

No. of Daphnids per Replicate at Start 0 h

Signs of Toxicity and Immobilisation of Daphnids Observed at

24 h

48 h

Toxic signs

I

Toxic signs

I

G1

0.0

R1

5

N(5)

0

N(5)

0

R2

5

N(5)

0

N(5)

0

G2

0.00897

R1

5

N(5)

0

N(5)

0

R2

5

N(5)

0

N(5)

0

G3

0.0895

R1

5

N(5)

0

N(5)

0

R2

5

N(5)

0

N(5)

0

G4

0.897

R1

5

N(5)

0

N(5)

0

R2

5

N(5)

0

N(5)

0

G5

9.1

R1

5

N(3)

2

B(2)

1

R2

5

N(4)

1

B(2), L(2)

0

G6

44.9

R1

5

B(2)

3

-

2

R2

5

B(1), L(1)

3

-

2

G7

89.7

R1

5

L(1)

4

-

1

R2

5

B(2)

3

-

2

I: Immobilization; N: Normal; B: Localized on Bottom; L: Lethargy; Alphabet outside and values inside the parentheses represent the clinical symptom code and number of Daphnids exhibiting that particular symptom, respectively.

Table 2. Summary of immobilisation at different time points of post exposure during range finding study

Group

Nominal Loading Rate (mg/L)

R

Initial No. of Daphnids

No. of Daphnids immobilized

Cummulative No.

% of Immobilisation

24 h

48 h

24 h

48 h

24 h

48 h

G1

0.0

R1

5

0

0

0

0

0

0

R2

5

0

0

G2

0.00897

R1

5

0

0

0

0

0

0

R2

5

0

0

G3

0.0895

R1

5

0

0

0

0

0

0

R2

5

0

0

G4

0.897

R1

5

0

0

0

0

0

0

R2

5

0

0

G5

9.1

R1

5

2

1

3

4

30

40

R2

5

1

0

G6

44.9

R1

5

3

2

6

10

60

100

R2

5

3

2

G7

89.7

R1

5

4

1

7

10

70

100

R2

5

3

2

Table 3. Summary of clinical signs and immobilisation during main study

Group

Nominal Loading Rate
 (mg/L)

R

No. of Daphnids per replicate at start 0 h

Signs of Toxicity and Immobilisation of Daphnids Observed at

24 h

48 h

Toxic signs

I

Toxic signs

I

G1

0

R1

5

N(5)

0

N(5)

0

R2

5

N(5)

0

N(5)

0

R3

5

N(5)

0

N(5)

0

R4

5

N(5)

0

N(5)

0

G2

1.5

R1

5

N(5)

0

N(5)

0

R2

5

N(5)

0

N(5)

0

R3

5

N(5)

0

N(5)

0

R4

5

N(5)

0

N(5)

0

G3

2.8

R1

5

N(5)

0

N(5)

0

R2

5

N(4), B(1)

0

N(4)

1

R3

5

N(5)

0

N(5)

0

R4

5

N(4)

1

N(4)

0

G4

5.7

R1

5

N(3), L(1)

1

N(2), L(1)

1

R2

5

N(4), B(1)

0

N(4), B(1)

0

R3

5

N(4), L(1)

0

N(3), L(1)

1

R4

5

N(3), L(1)

1

N(3), L(1)

0

G5

11.2

R1

5

N(2), L(2)

1

N(1), L(2)

1

R2

5

N(2), L(1), B(1)

1

N(2),L(1)

1

R3

5

L(1), B(2)

2

L(1), B(2)

0

R4

5

B(1), L(2)

2

L(2)

1

G6

22.5

R1

5

B(2), L(2)

1

L(2)

2

R2

5

L(2), B(1)

2

L(1), B(1)

1

R3

5

L(3), B(1)

1

L(2), B(1)

1

R4

5

L(2)

3

L(2)

0

G7

44.9

R1

5

B(3)

2

-

3

R2

5

L(1), B(1)

3

-

2

R3

5

L(2)

3

-

2

R4

5

L(2)

3

-

2

I: Immobilization; N: Normal; B: Localized on Bottom; L: Lethargy; Alphabet outside and values inside the parentheses represent the clinical symptom code and number of Daphnids exhibiting that particular symptom, respectively.

Table 4. Summary of immobilisation at different time points of post exposure during main study

Group

Nominal Loading Rate

(mg/L)

R

Initial No. of Daphnids

No. of Daphnids Immobilized

Cummulative No.

% of Immobilisation

24 h

48 h

24 h

48 h

24 h

48 h

G1

0

R1

5

0

0

0

0

0

0

R2

5

0

0

R3

5

0

0

R4

5

0

0

G2

1.5

R1

5

0

0

0

0

0

0

R2

5

0

0

R3

5

0

0

R4

5

0

0

G3

2.8

R1

5

0

0

1

2

5

10

R2

5

0

1

R3

5

0

0

R4

5

1

0

G4

5.7

R1

5

1

1

2

4

10

*20

R2

5

0

0

R3

5

0

1

R4

5

1

0

G5

11.2

R1

5

1

1

6

9

30

*45

R2

5

1

1

R3

5

2

0

R4

5

2

1

G6

22.5

R1

5

1

2

7

11

35

*55

R2

5

2

1

R3

5

1

1

R4

5

3

0

G7

44.9

R1

5

2

3

11

20

55

*100

R2

5

3

2

R3

5

3

2

R4

5

3

2

*: Statistical significance of >0.05.

Table 5. TOC analysis during main study

 

Group

 

Nominal Loading rate

 (mg/L)

TOC During Exposure
(mg)

Day 0
(Fresh)

Day 2
(Aged)

G1

0.0

-

-

G2

1.5

0.526

0.700

G3

2.8

1.306

1.196

G4

5.7

1.790

1.916

G5

11.2

6.324

6.276

G6

22.5

8.809

7.007

G7

44.9

35.300

35.23

Table 6. Physico-chemical parameters of test medium during main study

Group

Nominal Loading Rate (mg/L)

R

DO
 (mg/L)

Temperature

(°C)

pH

Day 0

Day 2

Day 0

Day 2

Day 0

Day 2

0 h

48h

0 h

48h

0 h

48h

Fresh

Spent

Fresh

Spent

Fresh

Spent

G1

0

R1

8.08

7.86

20.8

20.6

7.51

7.49

R2

8.11

7.90

20.6

20.6

7.43

7.39

R3

8.06

7.92

20.1

20.8

7.59

8.51

R4

8.14

7.82

20.8

20.4

7.54

7.51

G2

1.5

R1

8.00

7.81

20.1

20.9

7.68

7.58

R2

8.10

7.82

20.9

20.6

7.62

7.66

R3

7.96

7.68

20.1

20.1

7.69

7.54

R4

7.99

7.54

20.7

20.9

7.83

7.63

G3

2.8

R1

7.84

7.62

20.4

20.7

7.81

7.79

R2

7.82

7.68

20.8

20.9

7.84

7.78

R3

7.8

7.71

20.4

20.8

7.89

7.66

R4

7.79

7.61

20.8

20.6

7.80

7.76

G4

5.7

R1

7.79

7.68

20.3

20.8

7.81

7.70

R2

7.82

7.71

20.4

20.8

7.80

7.76

R3

7.83

7.59

20.3

20.4

7.86

7.61

R4

7.76

7.48

20.4

20.8

7.88

7.69

G5

11.2

R1

7.88

7.66

20.8

20.6

7.84

7.76

R2

7.84

7.68

20.3

20.6

7.89

7.81

R3

7.81

7.68

20.8

20.6

7.86

7.63

R4

7.82

7.39

20.6

20.9

7.80

7.68

G6

22.5

R1

7.77

7.54

20.4

20.1

7.89

7.81

R2

7.72

7.58

20.1

20.4

7.9

7.80

R3

7.70

7.54

20.4

20.8

7.91

7.79

R4

7.62

7.42

20.9

20.4

7.87

7.72

G7

44.9

R1

7.89

7.61

20.2

20.4

7.86

7.79

R2

7.91

7.62

20.6

20.2

7.93

7.69

R3

7.93

7.39

20.7

20.1

7.84

7.72

R4

7.68

7.46

20.4

20.5

7.86

7.70

Min

7.39

20.1

7.39

Max

8.14

20.9

8.51

* Min and Max values between 0 and 48 hour

Validity criteria fulfilled:
yes
Remarks:
(Immobilisation in control < 10% (no immobilisation observed); dissolved oxygen concentration > 3 mg O2/L)
Conclusions:
In a short-term toxicity test to Daphnia magna, the 48h-EL50 of the test substance was found to be 16.78 mg/L.


Executive summary:

An acute aquatic toxicity study with Daphnia magna was conducted on test item PINE OIL 50% according to OECD guideline 202, following GLP. The test item is a UVCB substance which was found to be partially miscible in ISO reconstituted water which was used as test medium. Therefore, the test was carried out with aqueous extracts (WAF = Water Accommodated Fraction) at various loading rates of the test item in the test medium. Based on a preliminary WAF equilibration trail, individual WAF solutions for every test item concentration were prepared in closed vessels with minimal headspace and stirred for 4 h. Then, the mixtures were allowed to settle and equilibrate to test temperature and aqueous portions of WAFs were transferred to the test vessels. A range finding study was conducted for 48 h with nominal loading rates of 0.00897, 0.0895, 0.897, 9.1, 44.9 and 89.7 mg/L along with a control group. The immobilization percentages of 40, 100 and 100 % were observed at 9.1, 44.9 and 89.7 mg/L respectively. No immobilization was observed in control and at 0.00897, 0.0895 and 0.897 mg/L. Based on these results, the main test was conducted for 48 h at nominal loading rates of 1.5, 2.8, 5.7, 11.2, 22.5 and 44.9 mg/L along with control group. The test was conducted in static conditions with 4 replicates of 5 daphnids per replicate. Negative control consisted of test medium alone and a solution of potassium dichromate was used as reference substance. No clinical signs of toxicity were observed in control and at 1.5 mg/L. Clinical sign of localized on bottom of container and lethargy was observed at 24 hour and 48 hour observation period at 2.8, 5.7,11.2 and 22.5 mg/L. Clinical sign of localized on bottom of container and lethargy was observed at 24 hour observation at 44.9 mg/L. The immobilization percentages of 10, 20, 45, 55 and 100 % were recorded at the tested nominal loading rates of 2.8, 5.7, 11.2, 22.5 and 44.9 mg/L during the 48 hour observation period. TOC measurements for each nominal loading rate were maintained within ± 20% during the test period. Immobilisation in the control group was 0 % and dissolved oxygen concentration at the end of the test was higher than 7.39 mg/L in the control and treatment groups. Based on these results, the 48 hours No Observed Effect Loading rate (NOELR) and Lowest Observed Effect Loading rate (LOELR) is 2.8 and 5.7 mg/L respectively, and the acute median effective loading rate (EL50) is 16.78 mg/L (95% confidence limits: 13.12 - 21.47 mg/L).

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
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
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
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:
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 and acclimation period). The report is sufficiently well documented.
Qualifier:
equivalent or similar to guideline
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:
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, reference substance and only 3 concentrations tested). The report is sufficiently well documented.
Qualifier:
equivalent or similar to guideline
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
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
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
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
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
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:
supporting study
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
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes (incl. QA statement)
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.

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
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
Remarks:
No information is provided on the report.
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.:
3.45 mg/L
Nominal / measured:
meas. (arithm. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 2.99-4.07 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 gamma terpinene to daphnia magna was reported to be 3.45 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 gamma 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 gamma terpinene to daphnia magna was calculated to be 3.45 mg/L (95% CL: 2.99 -4.07).

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:
guideline study without detailed documentation
Remarks:
Study conducted according to OECD guideline 202
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: analyses of the test substance were performed at beginning and at the end of exposure time.
Vehicle:
no
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Water flea
- Feeding during test: No
Test type:
flow-through
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Test temperature:
20 ± 2ºC
Nominal and measured concentrations:
Nominal concentrations: 0 (control), 1.0, 1.8, 3.2, 5.6 and 10 mg/L


Details on test conditions:
TEST SYSTEM
- Test vessel: 9 L tank
- Type of flow-through (e.g. peristaltic or proportional diluter): running water type (using continuous dilution apparatus using metering pump)
- Renewal rate of test solution (frequency/flow rate): ca. 8 volumes per day (50 mL/min)
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

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.:
1.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 1.6-2.2 mg/L
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
2.9 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 2.5-3.4 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
1 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EC100
Effect conc.:
5.6 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Results with reference substance (positive control):
Not applicable
 
Reported statistics and error estimates:
EC50 and 95% confidence limit were calculated by Probit analysis
Validity criteria fulfilled:
not specified
Conclusions:
The 48h-EC50 of p-cymene to daphnia magna is determined to be 1.9 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 p-cymene at concentrations of 0 (control), 1.0, 1.8, 3.2, 5.6 and 10 mg/L (nominal) for 48 hours under flow-through conditions. Effects based on mobility were observed at 48 hours. An analytical method based on gas chromatography mass spectrometry was used to monitor the concentration of test material in the test solution at the start and at the end of exposure. The 48 hour EC50, NOEC and EC100 of p-cymene to daphnia magna were calculated to be 1.9 mg/L (95% CL: 1.6 -2.2 mg/L), 1 mg/L and 5.6 mg/L respectively.

Description of key information

Key study. Test method according to OECD 202, GLP study. The 48h-EL50 of the test substance to Daphnia magna was found to be 16.78 mg/L (95% confidence limits: 13.12 - 21.47 mg/L).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
16.78 mg/L

Additional information

Supporting studies with individual components: 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.

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

Gamma terpinene: Test method similar to EPA-821-R-02-012 (Park HM, 2011). The 48h-EC50 in daphnia magna was reported to be 3.45 mg/L.

p-cymene: Test method according to OECD TG 202 (MITI, 1998). The 48h-EC50 in Daphnia magna was determined to be 1.9 mg/L.