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Short-term toxicity to aquatic invertebrates

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Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
calculation (if not (Q)SAR)
Remarks:
Estimated by calculation
Adequacy of study:
key study
Study period:
2017-02-10 to 2017-02-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
accepted calculation method
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
calculation method
Principles of method if other than guideline:
The acute toxicity to daphnia 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 EC50s 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 Effective Loading rate of the WAF.
The method has been validated using data derived from 48-hour EC50 tests on aquatic invertebrates, for which the concentrations of the test item had been determined by chemical analyses over the test period. Further to this the effective 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:
not specified
Specific details on test material used for the study:
Not applicable
Analytical monitoring:
not required
Details on sampling:
Not applicable
Vehicle:
no
Details on test solutions:
not applicable
Test organisms (species):
Daphnia magna
Details on test organisms:
not applicable
Test type:
other: calculation method
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
48h-EL50 (effective 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.
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.
Nominal and measured concentrations:
The calculation determines measured concentrations
Details on test conditions:
calculation method
Reference substance (positive control):
not required
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
1.9 mg/L
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: result based on the typical composition given by the supplier of the substance
Details on results:
not applicable
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
not applicable

At this 48-hour EL50 the expected concentrations of each constituent in the mixture (based on thermodynamic calculation) are as follows:

constituents

concentration in the WAF (mg.L-1)

limonene

0.98

elemol

0.22

α-phellandrene

0.25

elemicine

0.069

sabinene

0.069

α-terpineol

0.023

p-cymene

0.040

terpinolene

0.022

methyl eugenol

0.009

α-pinene

0.006

β-phellandrene

0.050

Validity criteria fulfilled:
yes
Conclusions:
48h-EL50 for the typical composition of Elemi Oil = 1.9 mg test item/L.
Executive summary:

Elemi Oil is a Natural Complex Substance (UVCB) with a well-defined composition (>90%). Its acute toxicity to aquatic invertebrates has been investigated using an in-house calculation method that replaces an OECD 202 study and guideline for Testing of Chemicals No. 23 (i.e. WAF conditions). The typical composition provided by the supplier of the substance 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 analysable fraction of a WAF study. In the calculation the second step is to remove this non-bioavailable fraction. 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. The final step is to determine the truly bioavailable fraction of the WAF per constituent. The EC50s of each constituent are already known from literature or predicted using the iSafeRat QSAR model. Each value 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 Effective Loading rate of the WAF.

The 48-h EL50 was 1.9 mg test material/L for the typical composition of Elemi Oil. 

Based on the results of this study, Elemi oil would not be classified as acute 1 to aquatic organisms in accordance with the classification of the CLP.

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

 

Results Synopsis

Test Type: Calculation method

48h-EL50: 1.9 mg test material/L based on the typical composition

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-02-07 to 2017-02-10
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
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Remarks:
2017-01-10
Specific details on test material used for the study:
Not applicable
Analytical monitoring:
yes
Details on sampling:
- Chemical analyses: Single samples for analysis were taken from the control and all test concentrations at the start of the test (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h)
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The study was carried out using WAFs (Water Accommodated Fractions). The WAFs (for fresh media at t=0h and t=24h) were prepared under closed conditions and by slow-stirring to avoid production of a dispersion.
The mixing vessels were cylindrical glass bottles sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. The volume of each mixing vessel was approximately 5 L. A magnetic stirring bar was placed in each mixing vessel and 5.0 to 5.5 L of test water (depending on the brim capacity of the bottles) were added in order to use a maximum volume and to minimise headspace. The loading rates of the test item were weighed on glass slides that afterwards were placed under the surface of the test water contained in the mixing vessels through fishing wire. Then the mixing vessels were closed immediately. The mixing was carried out at a speed that was slow enough not to cause dispersion or emulsification of the undissolved fraction of the test item. Mixing was initiated with the vortex in the centre extending maximally around 10% vessel depth from the top to the bottom of the vessel. After 24 ± 2 hours of gentle stirring in the dark at room temperature, the WAFs were allowed to stand for 1 hour before use. The first 100 mL were removed via the drain port. Then the WAFs were directly added into test vessels that were immediately sealed with screw caps after introduction of daphnids. No small bubble was observed in the test vessels. The test solution in test vessels was observed to be clear and colourless at all loading rates. The test was carried out without adjustment of the pH.

- Controls: Test water without test substance but treated in the same way as the test substance solutions.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain: Daphnia magna (Straus), clone 5
- Source: LIEBE - CNRS UMR 7146 - UFR SciFA - Université de Lorraine Campus Bridoux - Bât. IBISE, 8, rue du Général Delestraint - 57070 METZ, bred in the Laboratoires des Pyrénées et des Landes.
- Reason for selection: Characteristic and common representative of freshwater zooplankton which has been selected as an internationally accepted invertebrate species.
- Daphnids originated from a healthy stock, showing no signs of stress such as mortality, presence of males, ephippia or discoloured animals.
- Age at study initiation: < 24 h
- Breeding Conditions: Daphnids were cultured in the Laboratoires des Pyrénées et des Landes under similar temperature and light conditions as used in the test. The cultivation of the parental daphnids was performed in all-glass vessel containing test water. Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. Daphnids were fed at least three times a week with a suspension of algal cells (Pseudokirchneriella subcapitata) up to 0.1-0.2 mg C.Daphnia.-1day.-1. The water was changed three times a week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.
- Feeding during test: No feeding
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Remarks on exposure duration:
None
Post exposure observation period:
None
Hardness:
Total water hardness was approximately 250 mg/L (as CaCO3)
Test temperature:
between 20.6 and 20.8 °C throughout the test (average value: 20.8°C), and complied with the requirements (20°C ± 2°C, constant within 1°C).
pH:
7.77 - 8.45
pH requirements: 6.0-9.0, not varying by more than 1.5 units
Dissolved oxygen:
7.55 - 8.62 mg O2/L
oxygen: ≥ 60% of the air-saturation value at the end of the test in controls and test vessels.
Salinity:
None
Nominal and measured concentrations:
Nominal : control, 2.0; 2.5; 3.1; 4.0; 5.0
Details on test conditions:
TEST SYSTEM
- Test vessel: All-glass test tubes of approximately 20 mL capacity sealed with screw caps. Each test vessel will be uniquely identified with study code, replicate number, date of experimentation and concentration.
- Aeration: No aeration of the test solutions occurred throughout the test.
- Renewal rate of test solution: A semi-static test was performed with renewal of test solutions after 24 h (because of the properties of the test item)
- No. of daphnids: 20 per treatment group (including controls), divided into 4 groups of 5 animals
- Loading: 5 daphnids per vessel each completely filled with test solution and without headspace.
- Introduction of Daphnids: Daphnids were introduced into the test vessels each completely filled with test solution and without headspace immediately after filling the test vessels with test solutions.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted water as prescribed by the OECD Guideline 202

OTHER TEST CONDITIONS
- Photoperiod: 16 h light : 8 h dark

EFFECT PARAMETERS MEASURED:
- Immobility: Immobility and abnormal behaviour were determined by visual observation after 24 and 48 hours. Immobile animals were eliminated from the vessels as soon as they were discovered. Daphnids were considered to be immobile if they were not able to swim within 15 seconds after gentle agitation of test vessels.
- pH and dissolved O2: At start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h) from all test concentrations and control.
- Temperature of Medium: Measured continuously in a temperature controlled vessel next to the test vessels, over the entire study period, beginning at the start of the test.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: ca. 1.26
- Range finding study: Ten daphnids per concentration (5 per vessel, in duplicate) were exposed to the nominal loading rates 1.0, 2.5, 5.0, 10.0 and 20.0 mg.L-1 and to a control.
- Results used to determine the conditions for the definitive study: The per cent immobility was 0, 0, 0, 100, 100 and 100 (24 h) and 0, 0, 0, 100, 100 and 100 (48 h) at 0, 1.0, 2.5, 5.0, 10.0 and 20.0 mg.L-1, respectively. Based on the results of a range-finding test, the following nominal concentrations: 2.0, 2.5, 3.1, 4.0 and 5.0 mg.L-1. were selected for the definitive test.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
3.895 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
2.972 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Remarks on result:
other: 95% confidence limits: 2.816 - 3.150 mg/L
Details on results:
The evaluation was based on the nominal WAFs concentrations.
After 24 hours of exposure, immobilisation rates were 0% at 2.0, 2.5 and 3.1 mg.L-1 (loading), 70% at 4.0 mg.L-1 (loading) and 100% at 5.0 mg.L-1 (loading). After 48 hours of exposure, immobilisation rates were 0% at 2.0 mg.L-1 (loading), 5% at 2.5 mg.L-1 (loading), 65% at 3.1 mg.L-1 (loading) and 100% at 4.0 and 5.0 mg.L-1 (loading).
The 48-hour EL50 including the 95% confidence interval using Probit-analysis was determined by the computer program ToxRat.
Results with reference substance (positive control):
On January 10, 2017 (most recent test), the 24h-EC50 was 1.22 mg.L-1. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (expected 24h-EC50: 0.6 mg.L-1 to 2.1 mg.L-1) at this time.
Reported statistics and error estimates:
The evaluation of the effects was based on the nominal WAFs concentrations (nominal loading values). The software ToxRat® Professional was used for the determination of the effective concentrations.

Analytical results

The analytical results of this test showed that WAFs were overall stable throughout the test period. Indeed, TOC content in WAFs was satisfactorily maintained within the ± 20% of the initial concentration between the start and the end of each exposure period (t=0h t24hOld and t=24hFresht=48h), except the second day at 2.0 and 2.5 mg.L-1 with gains of 24 and 29%, respectively. It would be difficult to determine the specific cause of this last observation. However, this was considered not to affect the results and the integrity of the study regarding properties of the test item and the nonspecific analytical method.

It should be noted that a WAF is by definition a complex mixture for which the individual concentration of each constituent differs due to its properties (e.g. solubility, adsorption, volatilisation, bioaccumulation…). Due to these differences, interactions between certain constituents of the mixture may occur and affect the behaviour of a given constituent which consequently would not react in the

same way that if it was alone in the mixture.

Therefore, results are provided as Effective Loading Rate 50, according to the OECD No. 23.

 

Table 6.1.3/1: Acute immobilisation of daphnids after 24 and 48 h in the final test

Nominal Concentration*(mg test item.L-1)

Replicate

Number of daphnids exposed

Response at 24h

Response at 48h

Number

Total %

Number

Total %

Control

1

2

3

4

5

5

5

5

0

0

0

0

0

0

0

0

0

0

2.0

1

2

3

4

5

5

5

5

0

0

0

0

0

0

0

0

0

0

2.5

1

2

3

4

5

5

5

5

0

0

0

0

0

0

1

0

0

5

3.1

1

2

3

4

5

5

5

5

0

0

0

0

0

3

3

3

4

65

4.0

1

2

3

4

5

5

5

5

3

3

4

4

70

5

5

5

5

100

5.0

1

2

3

4

5

5

5

5

5

5

5

5

100

5

5

5

5

100

*WAF prepared at the given loading rate.

Water quality parameters and environmental conditions throughout the test:

pH and oxygen concentrations remained within the limits prescribed (pH: 6.0-9.0, not varying by more than 1.5 units; oxygen:≥ 60% of the air-saturation valueat the end of the test), suggesting that pH and oxygen concentrations were not influenced by the test item.

Furthermore, the temperature of the test medium was situated between 20.6 and 20.8°C throughout the test (average value: 20.8°C), and complied with the requirements (20°C ± 2°C, constant within 1°C).

 

Validity criteria of the study:

Controls: In the control, no daphnids became immobilized nor trapped at the surface of the water or showed signs of stress.

Dissolved [O2]: Dissolved oxygen concentration at the end of the test was ≥ 60 % of the air-saturation value in controls and test vessels.

Thus the validity criteria have been fulfilled in the present study.

Validity criteria fulfilled:
yes
Conclusions:
Under the experimental conditions and based on nominal concentrations, the 48-hour EL50 value estimated was 2.972 mg test item.L-1 (loading).
Executive summary:

A study was performed to assess the acute toxicity of test item ELEMI OIL to Daphnia magna and was investigated in a closed semi-static test using Water Accommodated Fractions. The method followed was designed to be compliant with the OECD 202 Guideline and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23). The criterion measured was the EL50 (Median Effective Loading rate), a statistically derived concentration which is expected to cause immobility in 50% of test animals within a period of 48 hours.

 

Following a preliminary range-finding test, twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading values of 2.0, 2.5, 3.1, 4.0 and 5.0 mg.L-1 and to a control. The immobility of the daphnids was determined in a closed semi-static 48-hour test by visual observation after 24 and 48 hours. Concentration of dissolved organic material in the control and the WAFs was checked by TOC analysis at start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h).

 

The analytical results of this test showed that WAFs concentrations were overall stable between the start and the end of each exposure period (t=0h-t24hOld and t=24hFresh-t=48h), within or close to the ± 20% of the initial TOC concentrations values. After 48 hours of exposure, immobilisation rates were 0% at 2.0 mg.L-1 (loading), 5% at 2.5 mg.L-1 (loading), 65% at 3.1 mg.L-1 (loading) and 100% at 4.0 and 5.0 mg.L-1 (loading).

 

Under the experimental conditions and based on nominal concentrations, the 48-hour EL50 value estimated was 2.972 mg test item.L-1 (loading).

Results synopsis:

Test: Toxic effect of ELEMI OIL to Daphnia magna according to OECD guidelines No. 202 and 23.

48h-EL50: 2.972 mg/L (95% Confidence limits: 2.816 - 3.150 mg/L)

Description of key information

Two reliable studies were available to assess the acute toxicity of ELEMI OIL to aquatic invertebrates:

- one test performed according to OECD 202 guideline and under GLP compliance that gave a 48h-EL50 of 2.972 mg/L (95% Confidence limits: 2.816 - 3.150 mg/L)

- a calculation method that replaces an OECD 202 study and guideline for Testing of Chemicals No. 23 (i.e. WAF conditions) that gave a 48h-EL50 of 1.9 mg/L.

Since these two results are reliable, the geometric mean between these two results was used for that endpoint and gave a 48h-EL50 of 2.3 mg/L.

Based on the results of these two studies, Elemi oil would not be classified as acute 1 to aquatic organisms in accordance with the classification of the CLP.

Synopsis of the acute toxicity to aquatic invertebrates:

Test item: ELEMI OIL

48h-EL50 = 2.3 mg/L (geometric mean, key value)

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
2.3 mg/L

Additional information

ELEMI OIL is a Natural Complex Substance (UVCB) with a well-defined composition. The WAF experimental study and the calculation method are based on typical compositions.

It appears that the calculation method gives an accurate prediction (error factor below 2 in comparison to the experimental study) although it is slightly more conservative than the experimental result.

Therefore, this calculation method can be considered as valid to be used alone for other ecotoxicological endpoints.