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Short-term toxicity to fish

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Endpoint:
short-term toxicity to fish
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 13-Oct-2008 to 10-Dec-2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
This study was performed according to OECD Guideline 203 and EU Method C.1 with GLP certificate. The test method was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000. All validity criteria were fulfilled but the test item was not stable during the test period and no information was provided on the results of the ranged finding test, used to determine the conditions for the definitive study.
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.1 (Acute Toxicity for Fish)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
OECD GLP (inspected from 05th to 09th and 26th to 30th November 2007 / signed on 12th November 2008)
Specific details on test material used for the study:
solubility in water = 1.88 mg/L
Analytical monitoring:
yes
Details on sampling:
- Sampling method: The test item (53.02 mg) was dissolved in ethyl acetate and made up to the mark in a 50 mL volumetric flask to prepare a stock solution with a concentration of 1060 mg/L. Thereof, 1 mL were diluted to 10 mL using ethyl acetate to obtain a working solution with a concentration of
106 mg/L.
- Sample storage conditions before analysis: the samples were stored deep-frozen and protected from light until analysis was performed.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low water solubility of the test item, a dispersion with the loading rate of 100 mg/L was prepared at the start of the test by dispersing 500.1 mg of the test item in 5000 mL of test water. Ultrasonic treatment for 15 minutes and intense stirring were applied. No auxiliary solvent or emulsifier was used. The dispersion was stirred by a magnetic stirrer at room temperature in the dark over 96 hours to dissolve a maximum amount of the test item in the dispersion. After the 96-hour stirring period, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 μm). The undiluted filtrate of the dispersion with the loading rate of 100 mg/L was used as test medium. The test medium was prepared just before the introduction of fish (i.e., start of the test).
- Eluate: test water
- Controls: one control (test water without addition of the test item)
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): not applicable
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): not applicable
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: zebra fish
- Strain: no data
- Source: no data
- Age at study initiation (mean and range, SD): no data
- Length at study initiation (length definition, mean, range and SD): 3.1 +/- 0.17 cm
- Weight at study initiation (mean and range, SD): 0.21 +/- 0.04 g
- Method of breeding: no data
- Feeding during test: no
- Food type: not applicable
- Amount: not applicable
- Frequency: not applicable

ACCLIMATION
- Acclimation period: one week
- Acclimation conditions (same as test or not): same as test
- Type and amount of food: commercial fish diet (Tetra Min Hauptfutter, supplied by TETRA-Werke, 49304 Melle/Germany)
- Feeding frequency: no data
- Health during acclimation (any mortality observed): no fish died in the test fish batch and all fish were healthy

QUARANTINE (wild caught)
not applicable
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
96 h
Remarks on exposure duration:
none
Post exposure observation period:
None
Hardness:
125 mg/L as CaCO3
Test temperature:
The water temperature in the test vessels was maintained at 20-21°C during the test period.
pH:
The pH values in the test medium and the control ranged from 7.3 to 7.4.
Dissolved oxygen:
The oxygen concentration was always 8.2 mg/L or higher (8.2 - 8.6 mg/L), and thus higher than 60% oxygen saturation.
Salinity:
Not applicable
Nominal and measured concentrations:
Nominal concentration: loading rate of 100 mg/L
Measured concentrations: The average concentrations found in the treatment samples were 2.47 mg/L (day 0), 0.228 mg/L (day 2) and below the LOQana (= 0.479 mg test item/L) (day 4).
Details on test conditions:
TEST SYSTEM
- Test vessel: glass test vessel
- Material, size, headspace, fill volume: glass, 5 liters of test medium
- Aeration: the test vessels were slightly aerated during the test period
- Type of flow-through (e.g. peristaltic or proportional diluter): not applicable
- Renewal rate of test solution (frequency/flow rate): not applicable
- No. of organisms per vessel: 7
- No. of vessels per concentration (replicates): 1
- No. of vessels per control (replicates): 1
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: 0.29 g fish wet weight per liter test medium.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reconstituted test water was used in this study. It consisted of analytical grade salts dissolved in purified water to obtain the following nominal concentrations: CaCl2 x 2H2O = 147 mg/L ; MgSO4 x 7H2O = 61.5 mg/L; NaHCO3 = 32.5 mg/L; KCl = 2.9 mg/L; Water Hardness = 125 mg/L as CaCO3; Alkalinity = 0.4 mmol/L. The ratio of Ca:Mg and Na:K was 4:1 and 10:1, respectively, based on molarity. The test water was aerated prior to the preparation of the test medium until oxygen saturation was reached.
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: no data
- Photoperiod: a 16-h light to 8-h dark photoperiod with a 30-min transition period was used
- Light intensity: within the range of 100 to 560 Lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : The test fish were observed for mortality and visible abnormalities after approximately 2.5, 24, 48, 72 and 96 hours test duration.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: not applicable (limit test)
- Justification for using less concentrations than requested by guideline: not applicable
- Range finding study: yes (non-GLP)
- Results used to determine the conditions for the definitive study: the test item was shown to be hydrolytically stable in test water over a period of at least 96 hours and that the solution equilibrium was reached after this time.
Reference substance (positive control):
no
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.51 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
corresponding to a loading rate of 100 mg/L
Remarks on result:
other: No acute effect up to the attainable limit of solubility in test medium.
Duration:
96 h
Dose descriptor:
other: The highest concentration without observed effect
Effect conc.:
>= 0.51 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
corresponding to a loading rate of 100 mg/L
Remarks on result:
other: No acute effect up to the attainable limit of solubility in test medium.
Details on results:
The analytically determined concentration of the test item at the start of the test was 2.5 mg/L. During the test period, a decrease of the test concentration in the test medium was observed. After 48 hours test period 0.23 mg/L were measured and at the end of the test the test concentration was
In the control and in the test medium with the loading rate of 100 mg/L (measured concentration of 0.51 mg/L) all fish survived until the end of the test and no visible abnormalities were observed at the test fish. See table 6.1.1/1 in "Any other information on results incl. tables".

No remarkable observations were made concerning the appearance of the test medium. It was a clear solution throughout the entire test duration.
Results with reference substance (positive control):
Not applicable
Reported statistics and error estimates:
No data

Table 6.1.1/1: Mortality observed at the test fish

Loading rate

Mean measured concentration (mg/L)

Number of abnormal and dead fish / number of dead fish

 

2.5h

24h

48h

72h

95h

Control

-

0/0

0/0

0/0

0/0

0/0

100 mg/L

0.51

0/0

0/0

0/0

0/0

0/0

LC50

> 100 mg/L based on the loading rate

> 0.51 mg/L based on the mean measured concentration

Validity criteria fulfilled:
yes
Conclusions:
The test item had no acute toxic effects on the test fish up to the loading rate of 100 mg/L (mean measured concentration of 0.51 mg/L). The 96-hour LC50 was > 0.51 mg/L and the highest concentration without observed effect after 96 hours was ≥ 0.51 mg/L.
Executive summary:

The acute toxicity of the test item to zebra fish (Danio rerio) was determined in a 96-hour static test according to Commission Regulation (EC) No 440/2008, Part C.1 and the OECD Guideline No. 203 (1992).

A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test fish up to the loading rate of 100 mg/L.

Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered through a 0.45 µm membrane filter. The undiluted filtrate with the maximum concentration of dissolved test item was used as the test medium. Additionally, a control was tested in parallel. The test method was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

The analytically determined concentration of the test item at the start of the test was 2.5 mg/L. During the test period, a decrease of the test concentration in the test medium was observed. After 48 hours test period, 0.23 mg/L were measured and at the end of the test, the test concentration was <LOQ. Since in an analytically pre-experiment the test item was shown to be hydrolytically stable in test water over a period of at least 96 hours and no adsorption onto glass surfaces was observed, the decrease of the test item concentration in the test medium of the definitive test can be attributed to adsorption onto the test fish. The mean measured test item concentration (calculated as the geometric mean over all measurements) was 0.51 mg/L.

Based on the mean measured concentration, the 96-hour LC50 was > 0.51 mg/L and the highest concentration without observed effect after 96 hours was ≥ 0.51 mg/L. In conclusion, the test item had no acute toxic effects on the test fish up to the loading rate of 100 mg/L.

Endpoint:
short-term toxicity to fish
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
[further information is included as attachment to Iuclid section 13]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the structural similarity between the source and the target substances. Their ecotoxicological profiles are expected to be similar because of this structural similarity, on common physico-chemical properties and because they are anticipated to follow the same environmental fate.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Refer to the Test material section of the source and target records.

3. ANALOGUE APPROACH JUSTIFICATION
The results of the available reliable data for the source and target substances are identical for biodegradation in water and short-term toxicity to aquatic invertebrates. No acute effects were observed for both compounds up to the attainable limit of solubility in test medium. The similarity of the structural, physico-chemical properties and ecotoxicity profiles between both substances is pronounced. On this basis, it's considered suitable and scientifically justified to read-across the data between the two substances to fill the short-term toxicity to fish endpoint in the present dossier.

4. DATA MATRIX
See attached document in Iuclid section 13
Reason / purpose:
read-across source
Reason / purpose:
read-across: supporting information
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
> 0.51 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
corresponding to a loading rate of 100 mg/L
Remarks on result:
other: No acute effect up to the attainable limit of solubility in test medium.
Duration:
96 h
Dose descriptor:
other: The highest concentration without observed effect
Effect conc.:
>= 0.51 mg/L
Nominal / measured:
meas. (geom. mean)
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks:
corresponding to a loading rate of 100 mg/L
Remarks on result:
other: No acute effect up to the attainable limit of solubility in test medium.
Validity criteria fulfilled:
not applicable
Conclusions:
Based on data available on the source substance (no observed acute toxic effects on the test fish up to the loading rate of 100 mg/L, corresponding to mean measured concentration of 0.51 mg/L), the 96-hour LC50 was determined to be greater than 0.51 mg/L, the highest attainable test substance concentration in test medium.
Executive summary:

The acute toxicity of the target substance to fish was determined using an experimental study (96-hour static test) performed on a source substance to zebra fish (Danio rerio) according to Commission Regulation (EC) No 440/2008, Part C.1 and the OECD Guideline No. 203 (1992).

A limit test was performed in accordance with the test guidelines to demonstrate that the test item has no toxic effect on the test fish up to the loading rate of 100 mg/L.

Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered through a 0.45 µm membrane filter. The undiluted filtrate with the maximum concentration of dissolved test item was used as the test medium. Additionally, a control was tested in parallel. The test method was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

The analytically determined concentration of the test item at the start of the test was 2.5 mg/L. During the test period, a decrease of the test concentration in the test medium was observed. After 48 hours test period, 0.23 mg/L were measured and at the end of the test, the test concentration was <LOQ. Since in an analytically pre-experiment the test item was shown to be hydrolytically stable in test water over a period of at least 96 hours and no adsorption onto glass surfaces was observed, the decrease of the test item concentration in the test medium of the definitive test can be attributed to adsorption onto the test fish. The mean measured test item concentration (calculated as the geometric mean over all measurements) was 0.51 mg/L.

Based on the mean measured concentration, the 96-hour LC50 was > 0.51 mg/L and the highest concentration without observed effect after 96 hours was ≥ 0.51 mg/L. In conclusion, the source and the target substances are not considered toxic to fish in acute conditions up to the highest attainable test substance concentration in test medium.

Description of key information

Read-Across, EU Method C.1, OECD Guideline 203, GLP, key study, validity 2:

No acute effect up to the attainable limit of solubility in test medium.

96h-LC50 > 0.51 mg/L., corresponding to a loading rate of 100 mg/L.

Key value for chemical safety assessment

Additional information

To assess the short-term toxicity of the registered substance, (-)-(3aR,5aS,9aS,9bR)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan, to fish, two studies are available.

The first study (Harlan, 2009), assessed as the key study, was performed on a source substance (which is the racemic form of the registered/target substance), (±)-(3aR*,5aS*,9aS*,9bR*)-3a,6,6,9a-Tetramethyldodecahydronaphtho[2,1-b]furan, according to OECD Guideline 203 and EU Method C1 with GLP statement, to assess the acute toxicity of the test item to zebra fish (Danio rerio) in a 96-hour static test. A limit test was performed to demonstrate that the test item has no toxic effect on the test fish up to the loading rate of 100 mg/L. Due to the low water solubility of the test item, a dispersion of the test item with the loading rate of 100 mg/L was continuously stirred at room temperature in the dark over 96 hours. Then, the dispersion was filtered through a 0.45 µm membrane filter. The undiluted filtrate with the maximum concentration of dissolved test item was used as the test medium. Additionally, a control was tested in parallel. The test method was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

The analytically determined concentration of the test item at the start of the test was 2.5 mg/L. During the test period, a decrease of the test concentration in the test medium was observed. After 48 hours test period, 0.23 mg/L were measured and at the end of the test, the test concentration was <LOQ. Since in an analytical pre-experiment the test item was shown to be hydrolytically stable in test water over a period of at least 96 hours and no adsorption onto glass surfaces was observed, the decrease of the test item concentration in the test medium of the definitive test can be attributed to adsorption onto the test fish. The mean measured test item concentration (calculated as the geometric mean over all measurements) was 0.51 mg/L. Based on the mean measured concentration, the 96-hour LC50 was > 0.51 mg/L and the highest concentration without observed effect after 96 hours was ≥ 0.51 mg/L. In conclusion, the test item had no acute toxic effects on the test fish up to the loading rate of 100 mg/L.

The second study (Henkel, 1994), assessed as a disregarded study, was performed on the registered substance according to EU directive 92/69/EC with GLP statement. This study was not considered valid because the results were based on nominal concentrations while the measured concentrations were well lower than nominal and the effects observed can be related to physical effects based on the high concentrations tested.

The results of the available reliable data for the source and target substances are identical for biodegradation in water and short-term toxicity to aquatic invertebrates. No acute effects were observed for both compounds up to the attainable limit of solubility in test medium (see ECHA disseminated dossier of the source substance EC#223 -118 -6). The similarity of the structural, physico-chemical properties and ecotoxicity profiles between both substances is pronounced. On this basis, it's considered suitable and scientifically justified to read-across the data between the two substances to fill the short-term toxicity to fish endpoint in the present dossier (see IUCLID section 13 for justification).