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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:
2006-02-27 to 2006-03-06
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study conducted in accordance with the OECD guideline 203. Specificity of Method of analysis is unclear but the exposure medium was renewed every 24h (semi-static). Therefore the expoure of test organisms is expected to be close to the nominal.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
GLP compliance:
yes
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 62 mg/L and 80 mg/L (abiotic test medium solutions)
- Sampling method: Analysis of test element was performed in the test solutions "test medium control" at t= 0 and t=24h (before the first renewal).
- Sample storage conditions before analysis: at 4 +-2°C
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: direct introduction of the test substance in each aquarium and addition of test medium in order to get a 2.5 L test solution.
- Eluate: none
- Differential loading: 28 mg/L, 36 mg/L, 47 mg/L, 62 mg/L, 80 mg/L
- Controls: test medium control
- Evidence of undissolved material (e.g. precipitate, surface film, etc): none
Test organisms (species):
Danio rerio (previous name: Brachydanio rerio)
Details on test organisms:
TEST ORGANISM
- Common name: Danio rerio
- Source: HB developpement élevage, La Fond Garel, 69490 St Forgeux.
- Length at study initiation (length definition, mean, range and SD): 25 to 30 mm
- Feeding during test: none

ACCLIMATION
- Acclimation period:12 days
- Acclimation conditions (same as test or not): same
- Type and amount of food: no data
- Feeding frequency: no data
- Health during acclimation (any mortality observed): no data

Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Post exposure observation period:
none
Hardness:
270 mg/L CaCO3
Test temperature:
23.2 to 25 °C
pH:
7.2 to 7.9
Dissolved oxygen:
78 to 98%
Salinity:
not relevant
Nominal and measured concentrations:
Nominal: 28 mg/L, 36 mg/L, 47 mg/L, 62 mg/L, 80 mg/L
Measured:
- for the 62 mg/l solution: at t= 0, 44.0% which corresponds to 62.5 mg/L when compared to the 100 mg/L solution reference
at t = 24h, 41.3 % which corresponds to 58.66 mg/L
- for the 80 mg/Lsolution: at t= 0, 54.6% which corresponds to 77.55 mg/L when compared to the 100 mg/L solution reference
at t = 24h, 50.4 % which corresponds to 71.59 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: all-glass vessel
- Type (delete if not applicable): open
- Material, size, headspace, fill volume: glass, 15 cm*15cm*20 cm, contains 2.5 liters
- Aeration: no data
- Renewal rate of test solution (frequency/flow rate): 24h
- No. of organisms per vessel: 7
- Biomass loading rate: 0.9 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: theoritical composition 294.0 mg/L CaCl2, 2 H20, 123.3 mg/L MgSO4, 7 H2O, 63.0 mg/L NaHCO3, 5.5 mg/L KCl
- Total organic carbon: 0.02 mg/l

- Intervals of water quality measurement: 24 h

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 14h light /10 h dark
- Light intensity:no data

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
every 24h , the following parameters were checked: pH, dissolved oxygen, and temperature



Reference substance (positive control):
yes
Remarks:
potassium dichromate
Duration:
24 h
Dose descriptor:
LC50
Effect conc.:
>= 62 - <= 80 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
>= 62 - <= 80 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Duration:
72 h
Dose descriptor:
LC50
Effect conc.:
>= 62 - <= 80 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Key result
Duration:
96 h
Dose descriptor:
LC50
Effect conc.:
>= 62 - <= 80 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mortality
Details on results:
- Behavioural abnormalities: fish at concentration 62 mg/L showed abnormal behaviour , they stayed on the side, at the bottom of the aquarium and showed a shortness of breath.
- Observations on body length and weight: no data
- Other biological observations: no data
- Mortality of control: none
- Other adverse effects control: none reported
- Abnormal responses: none reported
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: none
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Mortality:
- LC50: 227 mg/L

Table 6.1.1 -01: Cumulated dead fish as a function of exposure time and concentration

Nominal concentration of test substance

Fish number at the beginning of the test

Cumulated number of dead fish at each observation time

6h

24h

48h

72h

96h

Control

7

0

0

0

0

0

28 mg/L

7

0

0

0

0

0

36 mg/L

7

0

0

0

0

0

47 mg/L

7

0

0

0

0

0

62 mg/L

7

0

0

0

0

0

80 mg/L

7

6

7

7

7

7

Table 6.1.1 -02: TOC analysis in the control medium solutions, 62 mg/L and 80 mg/L at the beginning (T =0) and at the fisrt renewal ( T= 24h)

Test solution 

TOC at T = 0h

TOC at T= 24h

Control fish test medium

0.02

0.09

62 mg/L

44.0

41.3

80 mg/L

54.6

50.4

Validity criteria fulfilled:
yes
Conclusions:
The 96h LC50 of Terpineol multiconstituent on Danio rerio fish was measured to be between 62 and 80 mg/L.
Executive summary:

In a 96-h acute toxicity study, Danio rerio fish were exposed to Terpineol multiconstituent at concentrations of 0 (test medium control), 28, 36, 47, 62 and 80 mg/L} under semi-static conditions.  The 96-h LC50was between 62 and 80 mg/L.  The NOEC value, based on mortality effects, was 62 mg/L.  Sublethal effects were observed at the concentration of 62 mg terpineol multiconstituent /L were observed: abnormal behaviour , the fish stayed on the side, at the bottom of the aquarium and showed a shortness of breath.

 

This toxicity study is classified as acceptable and satisfies the guideline requirements for fish short term toxicity study.

 

Endpoint:
short-term toxicity to fish
Type of information:
calculation (if not (Q)SAR)
Remarks:
Migrated phrase: estimated by calculation
Adequacy of study:
key study
Study period:
2016-03-07 to 2016-03-08
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Remarks:
Calculation method is used ; calculation method applicable for the endpoint.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
Calculation method
Principles of method if other than guideline:
The acute toxicity to fish was determined using a validated QSAR for the Mode of Action in question. The first step of the iSafeRat mixture toxicity calculation employs phase equilibrium thermodynamics in order to determine the concentrations of each constituent within the WAF. This fraction equates to the analyzable fraction of a WAF study.
Within the WAF, the constituents also partition between themselves further reducing the bioavailable fraction and thus the toxicity of the mixture compared to the individual constituents. In the calculation the second step is to remove this non-bioavailable fraction.
The final step is to determine the truly bioavailable fraction of the WAF per constituent. The LC50s of each constituent are already known from literature or calculated using the iSafeRat QSAR model. Each value and calculation has been included as a supporting study in the IUCLID. An additivity approach (based on Chemical Activity of each constituent) is used in order to calculate the Lethal Loading of the WAF.
The method has been validated using data derived from 96-hour LC50 tests on fish, for which the concentrations of the test item had been determined by chemical analyses over the test period. Further to this the lethal loading rate of the WAF is determined by using a series of calculation steps using phase equilibrium thermodynamics and excluding the non-bioavailable fraction.
GLP compliance:
no
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
not applicable
Analytical monitoring:
not required
Details on sampling:
not applicable
Vehicle:
no
Details on test solutions:
not applicable
Test organisms (species):
other: fish spp.
Details on test organisms:
not applicable
Test type:
other: calculation method
Water media type:
freshwater
Limit test:
no
Total exposure duration:
96 h
Remarks on exposure duration:
LL50 (lethal loading rate of WAF)
Post exposure observation period:
not applicable
Hardness:
Hardness is not a necessary component of the WAF calculation
Test temperature:
The temperature is not a necessary component of the WAF calculation but extremely low or high temperatures could influence the solubility of certain constituents. Therefore, the calculation method is considered acceptable to determine LL50s for fish between 12 and 28°C.
pH:
The pH is not a necessary component of the WAF calculation
Dissolved oxygen:
The oxygen concentration is not a necessary component of the WAF calculation
Salinity:
Salinity is not a necessary component of the WAF calculation. However as the fish QSAR for the constituents calculation was based on data from freshwater studies, the resulting calculation is considered valid for freshwater fish
Nominal and measured concentrations:
The calculation determines measured concentrations
Details on test conditions:
calculation method
Reference substance (positive control):
not required
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
ca. 12 mg/L
Nominal / measured:
estimated
Conc. based on:
test mat.
Basis for effect:
mortality
Remarks on result:
other: typical composition (please refer to confidential section)
Details on results:
not applicable
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
not applicable

Table 6.1.1/1: Short-term toxicity to fish values for components.

constituents

96h-LC50 (mg/L)

α-terpineol

69

γ-terpineol

69

β-terpineol

57

terpinolene

0.55

3-terpinen-1-ol

69

borneol

23

fenchol

23

terpinen-1-ol 4

69

limonene

0.46

Table 6.1.1/2: Expected concentrations of components at this 96h-LL50.

constituents

concentration in the WAF (mg/L)

α-terpineol

7.6

γ-terpineol

1.2

β-terpineol

0.63

terpinolene

1.3

3-terpinen-1-ol

0.43

borneol

0.49

fenchol

0.29

terpinen-1-ol 4

0.20

limonene

0.07

Validity criteria fulfilled:
yes
Conclusions:
The 96h-LL50 for Terpineol multiconstituent is estimated at 12 mg test item/L.
Executive summary:

The registered substance is a multiconstituent substance with a well-defined composition for which the relative percentage and the reported variation of each constituent is known.

Its acute toxicity to fish property has been investigated using an in-house calculation method that replaces an OECD 203 study and guideline for Testing of Chemicals No. 203 (i.e. WAF conditions). The worst case composition has been investigated.

The first step of the iSafeRat mixture toxicity calculation employs phase equilibrium thermodynamics in order to determine the concentrations of each constituent within the WAF. This fraction equates to the analyzable fraction of a WAF study.

Within the WAF, the constituents also partition between themselves further reducing the bioavailable fraction and thus the toxicity of the mixture compared to the individual constituents. In the calculation the second step is to remove this non-bioavailable fraction.

The final step is to determine the truly bioavailable fraction of the WAF per constituent. The LC50s of each constituent are already known from literature or calculated using the iSafeRat QSAR model. Each value and calculation has been included as a supporting study in IUCLID. An additivity approach (based on Chemical Activity of each constituent) is used in order to calculate the Lethal Loading of the WAF.

The 96-h LL50 was 12 mg test material/L for the assessed composition. 96h-LL50 were based on mortality. 

This toxicity study is acceptable and can be used for that endpoint.

 

Description of key information

The substance exhibits a LC50 for freshwater fish of 12 mg/L.

Key value for chemical safety assessment

LC50 for freshwater fish:
12 mg/L

Additional information

The registered substance is a multiconstituent substance with a well-defined composition for which the relative percentage of each constituent/impurity is known. The ecotoxicological properties of the registered substance may be derived from the aquatic toxicity values of the individual components using an additivity calculation approach.

It has been observed that CLP additivity calculations for mode of action 1 (MOA 1) compounds are unreasonably conservative when compared to classic experimental studies using Water Accommodated Fractions (WAF), and this has been proved in a number of cases with substances of natural origin. Indeed, substance of natural origin may be composed of hydrophilic alcohol molecules and hydrophobic hydrocarbon terpene molecules. Therefore, when a WAF is performed most of substances fully dissolved in the aqueous phase are the hydrophilic fraction while the hydrophobic fraction (the more toxic elements for MOA 1 substances) may be below their water solubility value. The first step of the iSafeRat mixture toxicity calculation employs phase equilibrium thermodynamics in order to determine the concentrations of each constituent within the WAF. This fraction equates to the analysable fraction of a WAF study. On the other hand, the CLP additivity approach is calculated on the basis that all the substances are at their maximum solubility. Within the WAF, the constituents also partition between themselves further reducing the bioavailable fraction and thus the toxicity of the mixture compared to the individual constituents. In the calculation the second step is to remove this non-bioavailable fraction. These two reasons explain why ecotoxicity values from WAF studies are always higher for non-polar narcotic mixtures than the calculated values from CLP additivity calculation. The final step is to determine the truly bioavailable fraction of the WAF per constituent. The LC50s of each constituent are already known from literature or calculated using the iSafeRat QSAR model. Each value and calculation has been included as a supporting study in the IUCLID. An additivity approach (based on Chemical Activity of each constituent) is used in order to calculate the Lethal Loading of the WAF.

A predicted acute toxicity value to fish is available. The estimated LC50 for freshwater fish is 12 mg/L. This prediction is obtained with QSAR for the Mode of Action 1, based on validated data derived from standard toxicity test, for which the concentrations of the test item had been determined by chemical analyses over the test period. The prediction is sufficiently robust, and is appropriate for the purposes of chemical safety assessment required for REACH.

Besides, one reliable experimental study is available for the registered substance. The toxicity to freshwater fish was studied according to OECD TG No. 203 and GLP. Fish (Danio rerio) were exposed to substance at nominal test concentrations of 0 (test medium control), 28, 36, 47, 62 and 40 mg/L over a test period of 96 hours (semi-static test, daily renewal). Concentrations were verified by TOC determinations at relevant concentrations (62 and 80 mg/L). The endpoint was based on the nominal concentrations. The 96-h LC50 was approximately between 62 and 80 mg/L. Sublethal effects were observed at the concentration of 62 mg of terpineol multiconstituent /L: abnormal behaviour, the fish stayed on the side, at the bottom of the aquarium and showed a shortness of breath.