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Ecotoxicological information

Short-term toxicity to fish

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Reference
Endpoint:
short-term toxicity to fish
Type of information:
calculation (if not (Q)SAR)
Adequacy of study:
key study
Study period:
2020-11-18 to 2020-11-19
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 203 (Fish, Acute Toxicity Test)
Deviations:
yes
Remarks:
Calculation method
Principles of method if other than guideline:
The calculation method used in this study is based on toxic additivity principle. That means the toxic parts of each constituent are added up. Therefore the constituents considered within the mixture should act with a similar MechoA. The MechoA of the consituents are determined using the methodology described by Bauer et al. (2018). Since the constituents of the test item act with the same general MechoA (MechoA 1.1), the calculation method is directly applicable. This calculation method predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following the OECD Test Guideline 203, referenced as EU Method C.1. This approach uses a thermodynamically based module to calculate the water concentrations and the individual toxicity of each of the constituants is first derived from a QSAR model validated to be compliant with the OECD recommandations for QSAR modeling (OECD, 2004, 2007). Then the effective loading rate of the mixture to fish is determined as a Water Accommodated Fraction (WAF) test (OECD, 2002). Method detailed within the position paper written by Bicherel and Thomas (2014).
In brief, the methodology is as follows:
Step 1. Determination of the water solubility of each individual constituent;
Step 2. Determination of the Analytically Measurable Aqueous Phase (AMAP);
Step 3. Determination of the bioavailable toxic fraction of each constituent;
Step 4. Calculation of the global activity of the WAF and determination of the toxic loading rate.
GLP compliance:
no
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
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
3.7 mg/L
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: typical composition (please refer to confidential section)
Key result
Duration:
96 h
Dose descriptor:
LL50
Effect conc.:
2.9 mg/L
Conc. based on:
test mat.
Basis for effect:
mortality (fish)
Remarks on result:
other: theoretical worst case composition (please refer to confidential section)
Details on results:
See tables in "Any other information on results incl. tables".
The graph attached highlights the important of each constituent in order to explain the global toxicity of the WAF of the mixture for the toxic loading rate (E/LL50). The chemical activity (or toxic activity) of the constituents is given on the left vertical axis. The curve indicates the cumulative total (right vertical axis).
This graph indicates limonene, alpha-pinene and sabinene explain ca. 70% of the test item toxicity. Despite its high concentration in the test item, the toxicity part from 1,8-cineol is negligeable.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
not applicable
Sublethal observations / clinical signs:

Table 6.1.1/1: Analytically Measured Aqueous Phase (AMAP) calculated at the predicted LL50




























































ConstituentsConcentration in the WAF (mg/L) for Typical compositionConcentration in the WAF (mg/L) for the "worst case" composition

1,8-cineol


1.4790.640

limonene


0.6141.027

alpha-pinene


0.3490.582

sabinene


0.4340.370

beta-pinene


0.2360.262

para-cymene


0.2240.029

myrcene


0.099-

camphene


0.079-

alpha-thujene


0.071-

gamma-terpinene


0.068-

 


Table 6.1.1/2: Ecotoxicological endpoint values to fish (96h-LC50) used within the calculation method to determine WAF aquatic toxicity to fish


 





























































Constituent



96h-LC50 (mg/L)



Reference



1,8-cineol



110



iSafeRat® prediction



Limonene



0.46



iSafeRat® prediction



alpha-pinene



0.28



iSafeRat® prediction



Sabinene



0.61



iSafeRat® prediction



beta-pinene



0.59



iSafeRat® prediction



para-cymene



0.98



iSafeRat® prediction



myrcene



0.32



iSafeRat® prediction



camphene



0.59



iSafeRat® prediction



alpha-thujene



0.61



iSafeRat® prediction



gamma-terpinene



0.55



iSafeRat® prediction



 

Validity criteria fulfilled:
yes
Conclusions:
96h-LL50 for typical composition of camphor white oil = 3.7 mg test item/L and 96h-LL50 for theoretical worst case composition of camphor white oil = 2.9 mg test item/L
Executive summary:

The ACUTE TOXICITY TO FISH (96-HOUR LL50) of the test item has been determined using the iSafeRat® calculation method for mixtures tested according to the Water Accomodated Fraction (WAF) approach. Each constituents of the test item which have been considered fall within the applicability domain of the model used to determine their individual ACUTE TOXICITY TO FISH (96-HOUR LC50). Moreover, each constituents of the test item act with the same general MechoA. Therefore the calculation method is directly applicable and the final result for the test item can be considered valid for use in risk assessment and classification and labelling.
The ACUTE TOXICITY TO FISH (96-HOUR LL50) of the test item tested according to the WAF method was predicted as a loading rate of 3.7 and 2.9 mg/L based on the typical composition and on the “worst-case” composition, respectively.

Description of key information

iSafeRat® mixture calculation, KREATIS, 2020 :


Typical composition: 96h-LL50 = 3.7 mg/L;


Worst case composition: 96h-LL50 = 2.9 mg/L.

Key value for chemical safety assessment

Fresh water fish

Fresh water fish
Effect concentration:
2.9 mg/L

Additional information

Short-term toxicity to fish is provided in the dataset even if the tonnage band (1-10 tonnes/year) does not require that endpoint. Indeed, that endpoint is fulfilled according to environmental classification rules. Classification should be derived from the most sensible species. Because toxicity data have demonstrated the lowest LC50 values for constituents, it has been decided to calculate fish LL50, in order to ascertain the classification for environment of Camphor white oil.


 


Camphor white oil is a Natural Complex Substance (UVCB) with a well-defined composition for which the relative percentage of each constituent is known. Therefore, it has been decided that the ecotoxicity of Camphor white oil will be derived from knowledge of the constituents, constituent approach.


The mixture ecotoxicity properties may be derived from the ecotoxicity of the individual constituents using the CLP additivity calculation approach. However, CLP additivity approach is calculated on the basis that all the substances are at their maximum solubility and it has been observed that CLP additivity calculations for non-polar narcotic (MechoA 1.1) compounds are unreasonably conservative when compared to classic WAF studies. This has been proved in a number of cases for Natural Complex Substances. Indeed, natural extract compositions are a mixture of hydrophilic alcohol molecules and hydrophobic 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 MechoA 1.1 substances) may be below their water solubility value. In addition, 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. 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.


 


Therefore, the acute toxicity to fish property for Camphor white oil has been investigated using a calculation method (KREATiS, 2020) that predicts the endpoint value which would be expected when testing the substance under experimental conditions in a laboratory following the OECD Test Guideline 203 adapted for testing of a mixture using the WAF method. Two theoretical compositions have been investigated, the typical composition proposed by the Lead registrant and a theoretical worst case composition that maximizes the concentration of the most toxic constituents. The acute toxicity to fish (96h-LL50) was determined using iSafeRat® calculation method adapted for a mixture of compounds with the Mechanism of Action (MechoA) in question (MechoA 1.1, i.e. non-polar narcosis) (Bauer et al., 2018). This method has previously been validated in an internal publication for acute exposure of non-polar narcosis compounds (Bicherel and Thomas, 2014). The algorithm is based on a QSAR model which has been validated to be compliant with the OECD recommandations for QSAR modeling (OECD, 2004, 2007). The lethal loading rate (96h-LL50) of the WAF is determined by using a series of calculation steps using phase equilibrium thermodynamics and excluding the non-bioavailable fraction. This approach is based on validated data derived from 96-hour tests on fish, for which the concentrations of the test item had been determined by chemical analyses over the test period. The LC50s of each constituent were predicted using the iSafeRat QSAR model, and then used to determine the mixture ecotoxicity.


Using this approach, the 96-h LL50 values for fish were 3.7 and 2.9 mg/L for the typical composition and the theoretical worst case composition of Camphor white oil, respectively. These LL50 were based on mortality. 


Based on the results of this study, Camphor white oil would not be classified as acutely toxic to aquatic organisms in accordance with the classification of the CLP.


This toxicity prediction has been validated and is considered acceptable to fulfil the short-term toxicity to fish endpoint.