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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 September 2013 - 19 September 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Qualifier:
according to
Guideline:
EPA OPPTS 850.1010 (Aquatic Invertebrate Acute Toxicity Test, Freshwater Daphnids)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 0, 6.25, 12.5, 25, 50 and 100 % v/v saturated solution.
- Sampling method: Sampling was performed at the beginning of the experiment and thereafter 24-hour intervals
- Sample storage conditions before analysis: The test item was extracted from the ISO media by Hexane, and then the extracts were immediately subjected to GC analysis
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
Due to the fact, that the test item is poorly soluble in aqueous within the analytical method validation solution preparation method was assess to achieve the saturation concentration (study report 13/037-316AN). Based on these results, the test item was added into ISO media using 100 mg/L test item loading rate. This mixture was stirred vigorously for 4 hours at 20°C degree in tightly closed vessel in order to minimize losses due to evaporation. After 4 hours of equilibration, the mixture was centrifuged at 3000 rpm for 10 minutes, the mixture was poured into a separatory funnel and the undissolved test item was separated from the saturated solution. Test solutions of lower test concentrations were prepared by appropriate dilution of this stock solution. During the analytical method validation the saturation concentration was found to be: 46.2 mg/L (for a nominal loading rate of 100 mg/L).
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Source: National Institute of Public Health. 1097 Budapest Gyáli u. 2-6. Hungary
- Age at study initiation (mean and range, SD): Less than 24 h old
- Feeding during test: No

ACCLIMATION
- Acclimation period: There was no acclimatization because the water used was similar to the culture water
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
249 mg/L (as CaCO3)
Test temperature:
20.3 – 20.7 °C
pH:
7.54 – 7.88
Dissolved oxygen:
7.4 – 7.6 mg/L
Nominal and measured concentrations:
Nominal concentrations: 6.25, 12.5, 25, 50 and 100 % v/v saturated solution.
Measured concentrations (geometric means): 2.15; 4.80; 10.04; 21.01 and 37.26 mg/L.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flasks with stopper.
- Type (delete if not applicable): closed to minimize the loss of the substance from solution.
- Material, size, headspace, fill volume: 100 mL
- Aeration: No.
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water (ISO medium, according to OECD 202)

OTHER TEST CONDITIONS
- Adjustment of pH: No.
- Photoperiod: 16-hour light and 8-hour dark cycle

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
The immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. Those animals not able to swim within 15 seconds after gentle agitation of the test beaker are considered to be immobile.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.0
- Range finding study (1): Open vessels, Ten daphnids (2 replicates); 48 h exposure
- Test concentrations: 0, 0.1, 1, 10, 100 % v/v saturated solution.
- Results used to determine the conditions for the definitive study: Immobilized daphnids: 10/0; 10/0; 10/0; 10/0 and 10/3 respectively.
- Range finding study (2): Closed vessels, Ten daphnids (2 replicates); 48 h exposure
- Test concentrations: 0, 0.1, 1, 10, 100 % v/v saturated solution.
- Results used to determine the conditions for the definitive study: Immobilized daphnids: 10/0; 10/1; 10/0; 10/0 and 10/10 respectively.
Reference substance (positive control):
not required
Remarks:
Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions.
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
14.85 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% CL: 12.34 – 17.87 mg/L
Details on results:
- Other biological observations: Other adverse effects could not be observed during the experiment.
Results with reference substance (positive control):
Potassium dichromate is tested at least twice a year to demonstrate satisfactory test conditions. The date of the last study (Study Code: 13/223-023DA) with reference item Potassium dichromate (batch no.: 0769128) is: 16-17 July 2013. The 24h EC50: 0.82 mg/L, (95 % confidence limits: 0.76 – 0.87 mg/L)
Reported statistics and error estimates:
The 24 and 48 hours EC50 values were determined by Probit analysis with 95 % confidence limits using TOXSTAT software. For the determination of the LOEC and NOEC, the immobilization at the test concentrations was tested on significant differences to the control values by Dunnett’s Test using TOXSTAT software. The 48 hours EC100 values were determined directly from the raw data.

Number and percentage of immobilised animals:

Concentration

Number of treated animals

Immobilised animals

Nominal

% v/v

Measured

mg/L

24 hours

48 hours

Number

Percent

Number

Percent

Control

0.0

20

0

0

0

0

6.25

2.15

20

0

0

0

0

12.5

4.80

20

0

0

0

0

25

10.04

20

2

10

5*

25

50

21.01

20

6*

30

14*

70

100

37.26

20

20^*

100

20*

100

* : statistically significantly different compared to the control values (Dunnett’s Test; α = 0.05)

Concentration of the test solutions:

Nominal

% v/v

Measured test item e, mg/L

Stability

%

0 hours

24 hours

48 hours

Control

Not detected

Not detected

Not detected

-

100

42.2

34.39

35.65

85

50

21.25

22.03

19.81

93

25

10.18

10.48

9.50

93

12.5

4.72

4.94

4.73

100

6.25

2.15

2.27

2.04

95

The difference between the nominal concentrations and the measured concentrations were due to the low solubility of the test item.

The stability of the test item in the biological system varied between 85-100%.

The biological results are based on the measured geometric mean concentrations.

The 24h EC50 value: 21.10 mg/L (95 % confidence limits: 17.75 – 25.09 mg/L)

The 48h EC50 value: 14.85 mg/L (95 % confidence limits: 12.34 – 17.87 mg/L)

The 48h EC100 value: 37.26 mg/L

The 48h No-Observed Effect Concentration (NOEC): 4.80 mg/L

The 48h Lowest Observed Effect Concentration (LOEC): 10.04 mg/L

Validity criteria fulfilled:
yes
Remarks:
(no immobilized animal in control, dissolved oxygen concentration at the end ot the test > 3 mg/L)
Conclusions:
The 48h-EC50 was determined to be 14.85 mg/L in Daphnia magna (based on mobility) in a static closed system.
Executive summary:

An acute immobilisation test on Daphnia magna was performed in accordance with EU Method C2 and OECD 202. Based on preliminary range-finding test, 20 daphnids (5 per replicate) were exposed to nominal concentrations of 6.25, 12.5, 25, 50 and 100 % v/v saturated solution over an exposure period of 48 hours in a closed static system. The corresponding geometric mean test item concentrations were 2.15; 4.80; 10.04; 21.01 and 37.26 mg/L. The difference between the nominal concentrations and the measured concentrations were due to the low solubility of the test item. The immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. The stability of the test item in the biological system varied between 85-100%. All validity criteria were met during this study. Under the conditions of this test, the 48h-EC50 was determined to be 14.85 mg/L in Daphnia magna (based on mobility) in a static closed system.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The analogue substance dextro alpha fenchyl acetate undergoes rapid hydrolysis to acetic acid and dextro alpha fenchyl alcohol which shares the same functional groups with the substance isoborneol and also has comparable values for the relevant molecular properties.
See attached the reporting format.
Reason / purpose:
read-across source
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
11.67 mg/L
Nominal / measured:
estimated
Conc. based on:
other: Read-across from an analogue
Basis for effect:
mobility
Remarks on result:
other: read-across from an analogue for which EC50 (48 h) = 14.85 mg/L
Validity criteria fulfilled:
not applicable
Conclusions:
Based on the read-across approach from the analogue fenchyl acetate, the 48h-EC50 of isoborneol was determined to be 11.67 mg/L in Daphnia magna (based on mobility) in a static closed system.
Executive summary:

An acute immobilisation test on Daphnia magna was performed on fenchyl acetate in accordance with EU Method C2 and OECD 202. Based on preliminary range-finding test, 20 daphnids (5 per replicate) were exposed to nominal concentrations of 6.25, 12.5, 25, 50 and 100 % v/v saturated solution over an exposure period of 48 hours in a closed static system. The corresponding geometric mean analogue substance concentrations were 2.15; 4.80; 10.04; 21.01 and 37.26 mg/L. The difference between the nominal concentrations and the measured concentrations were due to the low solubility of the analogue. The immobility or mortality of the Daphnia was determined by visual observation 24 and 48 hours after the start of the test. The stability of the analogue in the biological system varied between 85-100%. All validity criteria were met during this study. Under the conditions of this test, the 48h-EC50 was determined to be 14.85 mg/L. Based on these results, the read-across approach was applied and the 48h-EC50 of isoborneol in Daphnia magna (based on mobility) was determined to be 11.67 mg/L.

Description of key information

Key study: Read-across approach. Test method EU method C2, OECD 202. GLP study. Based on the read-across approach, the 48h-EC50 of isoborneol was determined to be 11.67 mg/L in Daphnia magna in a static closed system.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
11.67 mg/L

Additional information

Key study: Read-across approach. An acute immobilisation test on Daphnia magna was performed on fenchyl acetate in accordance with EU Method C2 and OECD 202. Based on preliminary range-finding test, 20 daphnids (5 per replicate) were exposed to nominal concentrations of 6.25, 12.5, 25, 50 and 100 % v/v saturated solution (corresponding measured geometric means 2.15; 4.80; 10.04; 21.01 and 37.26 mg/L) over an exposure period of 48 hours in a closed static system. The 48h-EC50 was determined to be 14.85 mg/L. Based on these results, the read-across approach was applied and the 48h-EC50 of isoborneol in Daphnia magna (based on measured concentration and mobility) was determined to be 11.67 mg/L.