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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 11-09-2017 to 12-12-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
This test was performed according to OECD guideline No. 202 and under GLP conditions.
Qualifier:
according to
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Inspected on 4, 5 and 6 July 2016 / Certificate signed on January 10th, 2017
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Dry area, unopened containers, optimum temp. 11-25°C.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
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 flasks (approximate volume: 100 mL) filled with minimum headspace with test water (from the mixing vessel) and were immediately sealed with screw caps after weighing. After having tried different methods for the preparation of the WAFs in the preliminary study (cf. Appendix V), the following protocol has been selected for the final test: each glass flask was placed in a water bath for 10-15 minutes at approx. 50°C, followed by sonication for approx. 10 minutes. Based on experience on similar substances, the heating/sonication step is a method allowing to remove the paste fragments stuck to the glass of the flasks and to extract the soluble fraction of the test item as much as possible.
Then the mixing vessels were carefully filled with the contents of the glass flasks and thereafter 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.
Analytical monitoring:
yes
Details on sampling:
- Sampling method: Duplicate samples for analysis were taken from the 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) in the control medium and the WAFs. 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:
no
Remarks:
In the final test, heating and sonication were used to remove the paste fragments stuck to the glass of the flasks and to extract the soluble fraction of the test item as much as possible.
Details on test solutions:
Preparation of test solutions (final test) :
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 flasks (approximate volume: 100 mL) filled with minimum headspace with test water (from the mixing vessel) and were immediately sealed with screw caps after weighing. After having tried different methods for the preparation of the WAFs in the preliminary study (cf. Appendix V), the following protocol has been selected for the final test: each glass flask was placed in a water bath for 10-15 minutes at approx. 50°C, followed by sonication for approx. 10 minutes. Based on experience on similar substances, the heating/sonication step is a method allowing to remove the paste fragments stuck to the glass of the flasks and to extract the soluble fraction of the test item as much as possible.
Then the mixing vessels were carefully filled with the contents of the glass flasks and thereafter 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.

Preparation of test solutions (range-finding test):
The range-finding test was carried out using WAFs (Water Accommodated Fractions) of the test item over a range of nominal loading rate of 1, 10, 32 and 100 mg/L and to a control, according two methods of preparation ((a) use of solvent (acetone); (b) heating/sonication). The WAFs (for fresh media at t=0h and t=24h) were prepared in the dark under closed conditions and by slow-stirring.
The mixing vessels were 1 L cylindrical glass bottles (except for the preparation of the lowest loading rate 1 mg.L-1 according (b) method where a 5 L glass bottle was used) sealed with screw caps and fitted with a drain port near the bottom for drawing off the WAFs. A magnetic stirring bar was placed in each mixing vessel completely filled with test water (with a minimum of headspace).
(a) 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 vigorously vortexed for 4 min (despite this, it was not possible to dissolve satisfactory all the beeswax in acetone), and thereafter a specific volume (corresponding to a (limit) solvent concentration of 100 mg/L, as recommended in OECD guidance on testing and assessment No. 23 (4)) of each preparation was carefully added to the surface of test water contained in the mixing vessels that were closed immediately.
(b) The loading rates of the test item were weighed in glass flasks (approximate volume: 100 mL) filled with minimum headspace with test water (from the mixing vessel) and were immediately sealed with screw caps after weighing. Each glass flask was placed in a water bath for 10 minutes at approx. 50°C, followed by sonication for 10 minutes. Then the mixing vessels were carefully filled with the contents of the glass flasks and thereafter were closed immediately.

In both cases (a) and (b), 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 +/- 2.5 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 added into test tubes (without headspace) that were immediately sealed with screw caps after introduction of daphnids. No small bubble was observed in the test tubes. The test was carried out without adjustment of the pH.
The immobility of the daphnids was determined by visual observation after 24 and 48 hours. Concentration of dissolved organic material was checked by analysis of Total Organic Carbon (TOC) in the control medium and the WAFs (only for (b)).

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Controls: Test water without test substance but treated in the same way as the test substance solution.
- Concentration of vehicle in test medium (stock solution and final test solution(s) or suspension(s) including control(s)): In method (a), the maxiumum recommended solvent concentration was carefully added to the surface of test water
- Evidence of undissolved material (e.g. precipitate, surface film, etc.): In the range-finding test, fine particles were observed in the mixing vessels at 10, 32 and 100 mg/L (loading), that’s why the WAFs were filtered.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: 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.
- Feeding before the test
- Food type: suspension of algal cells (Pseudokirchneriella subcapitata)
- Amount: up to 0.1-0.2 mg C.Daphnia.-1day.-1
- Frequency: at least three times a week

Test type:
semi-static
Water media type:
other: Reconstituted water (Elendt M4 medium), as prescribed by OECD Guideline 202
Limit test:
yes
Total exposure duration:
48 h
Hardness:
The total water hardness was approximately 250 mg/L (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:
Between 8.21 (t=0h) and 7.74 (t=48h).
Dissolved oxygen:
Dissolved oxygen concentration at the end of the test was ≥ 60% of the air-saturation value in controls and test vessels.
Nominal and measured concentrations:
100 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: All-glass test tubes of approximately 20 mL capacity sealed with screw caps. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and treatment group.
- Aeration: No aeration of the test solutions occurred throughout the test.

- Renewal rate of test solution (frequency/flow rate): Renewal of test solutions after 24 hours
- No. of organisms per vessel: 5 daphnids per vessel each completely filled with test solution and without headspace
- No. of vessels per concentration (replicates): 4 replicates with daphnids per treatment group
- No. of vessels per control (replicates): 4 replicates with daphnids per treatment group
- No. of vessels per vehicle control (replicates): 2 solvent controls in the range-finding test

OTHER TEST CONDITIONS
- Adjustment of pH: no
- Photoperiod: 16h light / 8h dark

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Immobility and abnormal behaviour were determined by visual observation after 24 and 48 hours. pH and dissolved O2 were measured at start (t=0h), at t=24h (new and old solutions) and at the end of the test (t=48h) from all treatment groups. The temperature of medium was measured continuously in a temperature controlled vessel next to the test vessels, over the study period, beginning at the start of the test.

VEHICLE CONTROL PERFORMED: yes in the range-finding test

RANGE-FINDING STUDY
- Test concentrations: 1, 10, 32 and 100 mg/L
- Results used to determine the conditions for the definitive study:
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Key result
Duration:
48 h
Dose descriptor:
other: Highest nominal loading rate without observed effects
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
- Mortality of control: No mortality observed in the controls
Results with reference substance (positive control):
The 24h-EC50 was 0.92 mg/L.
Reported statistics and error estimates:
No statistical analysis was performed. Effective concentrations were determined directly from the raw data.

Table 1: Concentrations of the test item in test water - Results of the determination of TOC analysis (mg.L-1) - Final test

 

Nominal concentration*

(mg test item.L-1)

Start (t=0h)

 

t=24h

Old

 

t=24h

Fresh

 

End

(t=48h)

 

Mean

Mean

Mean

Mean

Control

1.09

1.09

2.24

2.12

1.16

1.20

0.80

0.80

Control

1.09

2.00

1.23

0.80

100

2.19

2.37

6.80

6.55

5.58

5.55

1.56

1.39

100

2.54

6.29

5.52

1.22

Table 2: Acute immobilisation of daphnids after 24 and 48 hours 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

100

1

2

3

4

5

5

5

5

0

0

0

0

0

0

0

0

0

0

Table 3: pH-values during the final test

 

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

Control

100

Start t=0h

8.48

8.21

t=24h

Old

8.13

7.88

Fresh

8.31

8.10

End t=48h

8.13

7.74

* WAF prepared at the given loading rate.

Table 4: Dissolved oxygen concentrations (mg.L-1) during the final test

 

 

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

Control

100

Start t=0h

8.03

7.52

t=24h

Old

7.83

7.06

Fresh

7.67

7.64

End t=48h

7.58

7.01

* WAF prepared at the given loading rate.

All these dissolved-oxygen concentrations correspond to values > 79% of the air-saturation value.

Validity criteria fulfilled:
yes
Remarks:
In the control, no daphnids became immobilised nor trapped at the surface of the water nor showed signs of stress. Dissolved oxygen concentration at the end of the test was ≥ 60% of the air-saturation value in controls and test vessels.
Conclusions:
Based on nominal concentrations, the 48-hour EC50 and the highest nominal loading rate without observed effects values were estimated to be higher than 100 mg.L-1.
Executive summary:

The study was performed according to OECD guideline 202 with GLP compliance to assess the short-term toxicity of the test substance to aquatic invertebrates.

A limit test was conducted following the results of a range-finding test. Twenty daphnids (four replicates, five daphnids per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item at a nominal loading rate of 100 mg test item.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.

Under the experimental conditions and based on nominal concentrations, the 48-hour EC50 and the highest nominal loading rate without observed effects values were estimated to be higher than 100 mg.L-1.

Description of key information

OECD 202, GLP, key study, validity 1:

48h-EC50 (Daphnia magna) > 100 mg/L based on nominal concentration

Highest nominal loading rate without observed effects > 100 mg/L

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information

One key study is available to assess the short-term toxicity of the registered substance to aquatic invertebrates, Daphnia magna. This study was performed according to OECD guideline 202, with GLP compliance under closed and static conditions for 48 hours.

A limit test was conducted following the results of a range-finding test.Twenty daphnids (four replicates, five daphnids per replicate) were exposed toWater Accommodated Fractions (WAFs) of the test item at a nominal loading rate of 100 mg test item.L-1and 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.

Under the experimental conditions and based on nominal concentrations, the 48-hour EC50 and the highest nominal loading rate without observed effects values were estimated to be higher than 100 mg.L-1.