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Short-term toxicity to aquatic invertebrates

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Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2017-07-17 to 2017-08-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Not relevant.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
no
Principles of method if other than guideline:
Not relevant.
GLP compliance:
yes (incl. certificate)
Remarks:
2017-01-10
Specific details on test material used for the study:
Not applicable.
Analytical monitoring:
yes
Details on sampling:
Samples for analysis were taken from the control, the solvent control and all test solutions at the start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h). Concentration of dissolved organic material was checked by analysis of Total Organic Carbon (TOC). TOC analysis was not performed in compliance with the OECD GLP principles but was adapted to fit the specific parameters of the test item, in accordance with ISO 17025.
Vehicle:
yes
Remarks:
solvent: acetone
Details on test solutions:
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. 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 1 L. A magnetic stirring bar was placed in each mixing vessel completely filled with test water (with a minimum of headspace). The loading rates of the test item were weighed in glass vials and resuspended in acetone. Then the loading rates of the test item/vehicle were vortexed to obtain homogeneous suspensions, and thereafter a specific volume (corresponding to a (limit) solvent concentration of 100 mg.L-1, as recommended in OECD guidance on testing and assessment No. 23) of each preparation was carefully added to the surface of test water contained in the mixing vessels that were closed immediately. The 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. 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 solution. One control series containing acetone (solvent control) at the level used in treatments was also prepared.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Strain: Daphnia magna (Straus), clone 5
- Sex: Female
- 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:
The total water hardness was approximately 250 mg.L-1 (as CaCO3).
Test temperature:
The temperature of the test medium was situated between 20.8 and 20.9°C throughout the test (average value: 20.9°C).
pH:
The pH of this solution was in the range of 6 to 9.
Dissolved oxygen:
Dissolved oxygen concentration at the end of the test was ≥ 60% of the air-saturation value in controls and test vessels
Salinity:
No data.
Conductivity:
No data.
Nominal and measured concentrations:
2, 5, 10, 23 and 50 mg.L-1 (loading rates)
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 (control and loading rate of 100 mg.L-1), divided into 4 groups of 5 animals
- Loading: 5 daphnids per vessel each completely filled with test solution and without headspace.
- Number of replicates: 4 replicates with daphnids per treatment group.
- 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 treatment group.
- 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
- Range finding study: Ten daphnids per concentration (5 per vessel, in duplicate) were exposed to the nominal loading rates 2, 5, 10, 50 mg.L-1 and to a control. Two method of preparation for the WAFs have been tried: heating/sonication and solvent conditions. Highest results were obtained with WAF prepared with solvent.
- Results used to determine the conditions for the definitive study: 100% immobilisation at 48h, at 32 mg/L
Reference substance (positive control):
yes
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
13.916 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Loading rate of test material
Basis for effect:
mobility
Remarks on result:
other: CL95%: 10.893-17.894
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
7.851 mg/L
Nominal / measured:
nominal
Conc. based on:
other: loading rate of test material
Basis for effect:
mobility
Remarks on result:
other: CL95%: 6.445-9.621
Details on results:
After 24 hours of exposure, immobilisation rates were 0% at 2 mg.L-1 (loading), 10% at 5 mg.L-1 (loading), 25% at 10 mg.L-1 (loading), 75% at 23 mg.L-1 (loading) and 100% at 50 mg.L-1 (loading). After 48 hours of exposure, immobilisation rates were 0% at 2 mg.L-1 (loading), 15% at 5 mg.L-1 (loading), 70% at 10 mg.L-1 (loading), and 100% at 23 and 50 mg.L-1 (loading).
The 24 and 48-hour EL50 including the 95% confidence interval using Probit-analysis were determined by the computer program ToxRat.
Results with reference substance (positive control):
On April 4, 2017 (most recent test), the 24h-EC50 was 0.97 mg.L-1. Hence, the sensitivity of the clone of Daphnia magna was in agreement with OECD 202 (1) (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

Concentration of dissolved organic material in the controls 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 revealed that TOC analysis was not specific enough to separate organic carbon related to the solvent from the one of the test item. However, TOC analysis indicated presence of organic compounds that was inversely proportional to the tested loading rates (i.e. TOC content was highest in the solvent control) especially for fresh solutions. TOC analysis also suggested that WAFs concentrations were overall stable.

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. Besides, it was considered that the concentration of the test item in the test solutions did not vary throughout the study duration as each treatment was renewed at t=24h. Therefore, and since the test item was a UVCB substance, the results were based on the nominal test loading rates.

Table : 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

Solvent Control

1

2

3

4

5

5

5

5

0

0

0

0

0

0

0

0

0

0

2

1

2

3

4

5

5

5

5

0

0

0

0

0

0

0

0

0

0

5

1

2

3

4

5

5

5

5

0

0

2

0

10

1

0

2

0

15

10

1

2

3

4

5

5

5

5

2

1

2

0

25

4

3

4

3

70

23

1

2

3

4

5

5

5

5

3

5

5

2

75

5

5

5

5

100

50

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 parameter values throughout the test:

The results of measurement of pH and oxygen concentrations (mg.L-1) are presented in Tables 2 and 3 (see Appendix II). These parameters remained within the limits prescribed by the study plan (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 20.8 and 20.9°C throughout the test (average value: 20.9°C), and complied with the requirements as laid down in the study plan (20°C ± 2°C, constant within 1°C).

Validity criteria of the study:

Validity criteria fulfilled:
yes
Conclusions:
Under the experimental conditions and based on nominal concentrations, the 48-hour EL50 value was estimated to be 7.851 (CL95%: 6.445-9.621) mg test item.L-1 (loading).
Executive summary:

A study was performed to assess the acute toxicity of test item 944452 GALBANUM RDE SUPER INDE to Daphnia magna. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 202, “Daphnia sp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 (amended by Commission Regulation (EU) 2016/266) and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

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, 5, 10, 23 and 50 mg.L-1 and to controls. 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 controls 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 revealed that TOC analysis was not specific enough to separate organic carbon related to the solvent from the one of the test item. However, TOC analysis indicated presence of organic compounds that was inversely proportional to the tested loading rates (i.e. TOC content was highest in the solvent control) especially for fresh solutions. TOC analysis also suggested that WAFs concentrations were overall stable. After 48 hours of exposure, immobilisation rates were 0% at 2 mg.L-1 (loading), 15% at 5 mg.L-1 (loading), 70% at 10 mg.L-1 (loading), and 100% at 23 and 50 mg.L-1 (loading).

All validity criteria were fulfilled. This study is considered as valid for that endpoint.

Results synopsis:

Test: Toxic effect of 944452 GALBANUM RDE SUPER INDE to Daphnia magna according to OECD guidelines No. 202 and 23

48h-EL50: 7.851 mg/L

Description of key information

Under the experimental conditions and based on nominal concentrations, the 48-hour EL50 value was estimated to be 7.851 (CL95%: 6.445-9.621) mg test item.L-1 (loading).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
7.851 mg/L

Additional information

A study was performed to assess the acute toxicity of test item 944452 GALBANUM RDE SUPER INDE to Daphnia magna. The method followed was designed to be compliant with OECD Guideline for Testing of Chemicals No. 202, “Daphnia sp., Acute Immobilisation Test”, referenced as Method C.2 of Commission Regulation No. 440/2008 (amended by Commission Regulation (EU) 2016/266) and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23).

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, 5, 10, 23 and 50 mg.L-1 and to controls. 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 controls 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 revealed that TOC analysis was not specific enough to separate organic carbon related to the solvent from the one of the test item. However, TOC analysis indicated presence of organic compounds that was inversely proportional to the tested loading rates (i.e. TOC content was highest in the solvent control) especially for fresh solutions. TOC analysis also suggested that WAFs concentrations were overall stable. After 48 hours of exposure, immobilisation rates were 0% at 2 mg.L-1 (loading), 15% at 5 mg.L-1 (loading), 70% at 10 mg.L-1 (loading), and 100% at 23 and 50 mg.L-1 (loading).

All validity criteria were fulfilled. This study is considered as valid for that endpoint.