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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:
17 December 2017 - 31 January 2018
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), 0 (vehicle control) and 50 mg/L
- Sampling method: A known volume of sample (10 mL) was taken in a separating funnel and to this 30 mL of dichloromethane was added.
- Sample storage conditions before analysis: not applicable
Vehicle:
yes
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A quantity of 500.0 mg test item was transferred in 1 mL eppendorf and volume was made up to the mark with acetone to obtain the nominal concentration of 500 mg/mL (stock A). Volume of 10 µL was taken from the stock A, transferred using a micropipette and diluted to 100 mL with reconstituted water (ISO test water) in glass beakers of 600 mL capacity to obtain the nominal test concentration of 50.0 mg test item/L.
- Controls: Reconstituted water and acetone in Reconstituted water (10 µL acetone diluted to 100 mL)
- Chemical name of vehicle: acetone
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): 0.1mL/L
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Source: MicroBio Test Inc, Kleimoer 15, 9030 Mariakerke (Gent), Belgium
- Age of parental stock (mean and range, SD): no data
- Feeding during test: No

ACCLIMATION
- Acclimation period: 48 h
- Acclimation conditions (same as test or not): same as test: mean temperature of 20.0 ± 0.1 °C, mean dissolved oxygen level of 8.62 ± 0.04 mg/L as air saturation value, mean pH of 7.42 ± 0.03 measured daily and kept under a 16 h light and 8 h dark photoperiod during acclimatisation. The total hardness measured was 190.4 mg/L as CaCO3 and light intensity measured was 1330 Lux, on the day of acclimatisation.
- Type and amount of food: live algal cells (Pseudokirchneriella subcapitata)
- Feeding frequency: no specified
- Health during acclimation (any mortality observed): no mortalities reported.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
None
Hardness:
196.0 mg/L as CaCO3
Test temperature:
21.2-21.6ºC
pH:
7.59-7.89
Dissolved oxygen:
8.68-8.94 mg O2/L
Salinity:
No data
Conductivity:
No data
Nominal and measured concentrations:
Nominal concentrations: 0 (control), 0 (vehicle control) and 50 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: glass beaker
- Material, size, headspace, fill volume: glass, 600 mL capacity, 100 mL fill volumen
- Aeration: diluent water was aerated before starting the test so that dissolved oxygen concentration reached saturation.
- Renewal rate of test solution (frequency/flow rate): No 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): 4
- Biomass loading rate: 5 daphnids in 100 mL of test media

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted water (ISO test water)
- Intervals of water quality measurement: Temperature, pH, and dissolved oxygen content (using Multi portable meter) of reconstituted water were recorded at 0 and 48 h. Total hardness of reconstituted water was measured once during acclimatisation and test period using the titrimetric method (IS 3025, 1983).

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h light and 8 h dark cycle
- Light intensity: ranged between 1320 and 1340 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : All test daphnids were observed for immobility and abnormal behaviour or appearance, at 0, 24, and 48 h of exposure. Mobility of the daphnids was assessed by gently swirling the test container for 15 seconds and observing their swimming behaviour. Daphnids unable to swim during the agitation of the test container and remaining settled on the water surface or settled at the bottom of the test vessel were recorded as immobile.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: 0.0 (control), 0.0 [vehicle control (Acetone)], 0.01, 0.1, 1.0, 10.0 and 50.0 mg test item/L.
- Results used to determine the conditions for the definitive study: The percent immobility observed were 0, 0, 0, 0, 0, 0 and 0 at the test concentrations of 0.0 (control), 0.0 [vehicle control (Acetone)], 0.01, 0.1, 1.0, 10.0 and 50.0 mg/L, respectively.
Reference substance (positive control):
yes
Remarks:
(potassium dichromate)
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Behavioural abnormalities: Daphnids exposed to 50.0 mg/L, control and vehicle control (Acetone) did not exhibit any abnormal behavioural symptoms at 24 and 48 h.
- Mortality of control: No mortality was observed over a period of 48 h in the control and vehicle control groups
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Relevant effect levels: The percent immobilisations observed at 48 h were 0, 35, 90, 100 and 100 at the test concentrations of 0.09, 0.20, 0.44, 0.96 and 2.11 mg potassium dichromate/L, respectively.
At 48 h, lethargy was observed in G3 (0.20 mg/L) and G4 (0.44 mg/L). All the daphnids were found immobile at the test concentrations of G5 (0.96 mg/L) and G6 (2.11 mg/L). No immobility and behavioural symptoms were observed in G2 (0.09 mg/L), and G1 (control) group.
- EC50-48 h: 0.24 mg/L

Reported statistics and error estimates:
As the study was conducted as a limit test, probit analysis was not carried out.

Table 1: Immobility Data at 0, 24 and 48 h Exposure Period

Group

Test Concentration

(mg/L)

N° of Replicates

N° of Daphnia/group

Immobility N° and % at

0 h

24 h

%

48 h

%

G1

0.0 (Control)

4

20

0

0

0

0

0

G2

0.0 (Vehicle Control)

4

20

0

0

0

0

0

G3

50.0

4

20

0

0

0

0

0

Table 2: Behavioral Symptoms (Main Study)

Group

Test

 Concentration(mg/L)

Behavioral Symptoms Observed at

0 h

24 h

48 h

R1

R2

R3

R4

R1

R2

R3

R4

R1

R2

R3

R4

G1

0.0 (Control)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

G2

0.0 (Vehicle Control)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

G3

50.0

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

1(5)

 

Behavioral symptom: 1 = Normal

Note: Figures presented outside parentheses refer the clinical symptom and inside parentheses refer the total number of daphnia.

Table 3: Mean Values of Water Parameters of Test Solution

Groups

Test

Concen-tration

 (mg/L)

pH

Temperature (°C)

Dissolved Oxygen (mg/L)

Total Hardness

(as CaCO3)

mg/L water

0 h

48 h

0 h

48 h

0 h

48 h

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

Mean

SD

196.0

G1

0.0 (Control)

7.68

0.065

7.76

0.044

21.4

0.13

21.4

0.05

8.85

0.061

8.83

0.062

G2

0.0

(Vehicle Control)

7.78

0.018

7.82

0.029

21.6

0.06

21.3

0.05

8.74

0.038

8.73

0.039

G3

50.0

7.81

0.000

7.88

0.019

21.5

0.08

21.4

0.06

8.73

0.029

8.71

0.026

Validity criteria fulfilled:
yes
Remarks:
(mortality 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-EC50 of the test substance was found to be greater than 50.0 mg/L.
Executive summary:

An acute aquatic toxicity study with Daphni magna was conducted on test item according to OECD guideline 202, following GLP. A preliminary range finding study was conducted with test concentrations of 0.0 (control), 0.0 (vehicle Control), 0.01, 0.1, 1.0, 10.0 and 50.0 mg/L for a period of 48 h with 70 daphnids, divided into seven groups of 10 daphnids per group. Based on no mortalities found in this preliminary study, the main test was restricted to limit study conducted with single test concentration of 50.0 mg/L. Reconstituted water (ISO test water) was used as test medium. In the previous solubility study the test item was found to be insoluble in reconstituted water and acetone was selected as the vehicle. Thus, the main test was conducted with test concentrations of 0.0 (control), 0.0 (vehicle Control) and 50.0 mg/L for a period of 48 h in static conditions in 4 replicates of 5 daphnids per replicate. The test solutions were not aerated during the exposure. A validated analytical method based on gas chromatography was used to monitor the concentration and stability of the main active ingredients in the test solution at 0 and 48 h of the main study. The active ingredient concentration in the test solution was within acceptable limit (> 80% of nominal concentration). All validity criteria were fulfilled. No immobilisation and no abnormal behavioural symptoms were observed over the period of 48 h at the test concentration of 50.0 mg/L as well as in the control and vehicle control groups. Thus, EC50 (48 h), NOEC and LOEC to Daphnia magna of the test substance were found to be greater than 50.0 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: 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:
EC50
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:
EC0
Effect conc.:
8.8 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
24 h
Dose descriptor:
EC50
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 EC50 was 41 mg/L (basis for effect: mortality) 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 EC50 was 41 mg/L (basis for effect: mortality).
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 EC50 was 41 mg/L (basis for effect: mortality) 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: 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:
EC50
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:
EC0
Effect conc.:
< 13 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
24 h
Dose descriptor:
EC50
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 EC50 was 22 mg/L (basis for effect: mortality) 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 EC50 was 22 mg/L (basis for effect: mortality).
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 EC50 was 22 mg/L (basis for effect: mortality) with 95% CL: 13-36 mg/L.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance alpha-pinene which shares the same functional groups with the main components of the multi-constituent substance reaction mass of fenchene and laevo camphene and dextro camphene and laevo alpha pinene also has comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
41 mg/L
Nominal / measured:
estimated
Conc. based on:
other: Read-across from an analogue
Basis for effect:
mortality
Remarks on result:
other: read-across from an analogue for which EC50 (48 h) = 41 mg/L (95 % CL: 27 - 62 mg/L)
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
8.8 mg/L
Nominal / measured:
estimated
Conc. based on:
other: Read-across from an analogue
Basis for effect:
mortality
Remarks on result:
other: read-across from an analogue for which EC0 (48 h) = 8.8 mg/L
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
68 mg/L
Nominal / measured:
estimated
Conc. based on:
other: Read-across from an analogue
Basis for effect:
mortality
Remarks on result:
other: read-across from an analogue for which EC50 (24 h) = 68 mg/L (95 % CL: 24 - 190 mg/L)
Validity criteria fulfilled:
not applicable
Conclusions:
Based on the read-across approach from the analogue alpha pinene, the EC50(48 h) of reaction mass was 41 mg/L (basis for effect: mortality).
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 EC50 was 41 mg/L (basis for effect: mortality) with 95% CL: 27-62 mg/L.Based on these results, the read-across approach was applied and the 48 h-EC50 of the reaction mass was determined to be 41 mg/L (basis for effect: mortality).

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance camphene which shares the same functional groups with the main components of the multi-constituent substance reaction mass of fenchene and laevo camphene and dextro camphene and laevo alpha pinene also has comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
22 mg/L
Nominal / measured:
estimated
Conc. based on:
other: Read-across from an analogue
Basis for effect:
mortality
Remarks on result:
other: read-across from an analogue for which EC50 (48 h) = 22 mg/L (95 % CL: 13 - 36 mg/L)
Duration:
48 h
Dose descriptor:
EC0
Effect conc.:
< 13 mg/L
Nominal / measured:
estimated
Conc. based on:
other: Read-across from an analogue
Basis for effect:
mortality
Remarks on result:
other: read-across from an analogue for which EC0 (48 h) <13 mg/L
Duration:
24 h
Dose descriptor:
EC50
Effect conc.:
46 mg/L
Nominal / measured:
estimated
Conc. based on:
other: Read-across from an analogue
Basis for effect:
mortality
Remarks on result:
other: read-across from an analogue for which EC50 (24 h) = 46 mg/L (95 % CL: 36 - 60 mg/L)
Validity criteria fulfilled:
not applicable
Conclusions:
Based on the read-across approach from the analogue camphene, the EC50(48 h) of reaction mass was 22 mg/L (basis for effect: mortality).
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 EC50 was 22 mg/L (basis for effect: mortality) with 95% CL: 13-36 mg/L. Based on these results, the read-across approach was applied and the 48 h-EC50 of the reaction mass was determined to be 22 mg/L (basis for effect: mortality).

Description of key information

Key study. Test method according to OECD 202, GLP study. The 48h-EC50 of the test substance to Daphnia magna was found to be greater than 50.0 mg/L.

Supporting studies: Read-across approach. camphene. Test method according to US EPA (1975): "Methods for acute toxicity tests with fish, macroinvertebrates and amphibiens". Based on the read-across approach from the analogue camphene, the EC50 (48 h) of reaction mass to Daphnia magna was determined to be 22 mg/L (basis for effect: mortality).

Supporting studies: Read-across approach. alpha pinene.Test method according to US EPA (1975): "Methods for acute toxicity tests with fish, macroinvertebrates and amphibiens". Based on the read-across approach from the analogue alpha pinene, the EC50 (48 h) of reaction mass to Daphnia magna was determined to be 41 mg/L (basis for effect: mortality).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
50 mg/L

Additional information

Key study. An acute aquatic toxicity study with Daphni magna was conducted on test item according to OECD guideline 202, following GLP. A preliminary range finding study was conducted with test concentrations of 0.0 (control), 0.0 (vehicle Control), 0.01, 0.1, 1.0, 10.0 and 50.0 mg/L for a period of 48 h with 70 daphnids, divided into seven groups of 10 daphnids per group. Based on no mortalities found in this preliminary study, the main test was restricted to limit study conducted with single test concentration of 50.0 mg/L. Reconstituted water (ISO test water) was used as test medium. In the previous solubility study the test item was found to be insoluble in reconstituted water and acetone was selected as the vehicle. Thus, the main test was conducted with test concentrations of 0.0 (control), 0.0 (vehicle Control) and 50.0 mg/L for a period of 48 h in static conditions in 4 replicates of 5 daphnids per replicate. The test solutions were not aerated during the exposure. A validated analytical method based on gas chromatography was used to monitor the concentration and stability of the main active ingredients in the test solution at 0 and 48 h of the main study. The active ingredient concentration in the test solution was within acceptable limit (> 80% of nominal concentration). All validity criteria were fulfilled. No immobilisation and no abnormal behavioural symptoms were observed over the period of 48 h at the test concentration of 50.0 mg/L as well as in the control and vehicle control groups. Thus, EC50 (48 h), NOEC and LOEC to Daphnia magna of the test substance were found to be greater than 50.0 mg/L.

Supporting studies: Read-across approach. camphene. 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 EC50 was 22 mg/L (basis for effect: mortality) with 95% CL: 13-36 mg/L. Based on these results, the read-across approach was applied and the 48 h-EC50 of the reaction mass was determined to be 22 mg/L (basis for effect: mortality).

Supporting studies: Read-across approach. alpha pinene. 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 EC50 was 41 mg/L (basis for effect: mortality) with 95% CL: 27-62 mg/L. Based on these results, the read-across approach was applied and the 48 h-EC50 of the reaction mass was determined to be 41 mg/L (basis for effect: mortality).