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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:
2017-10-16 to 2017-12-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted according to the OECD 202 guideline without deviation. All validity criteria were successful
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 applicable
GLP compliance:
yes (incl. certificate)
Remarks:
2017-01-10
Specific details on test material used for the study:
None
Analytical monitoring:
yes
Details on sampling:
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
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. After having tried different methods for the preparation of the WAFs in the preliminary study, the following protocol has been selected for the final test:
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). After heating of the test item sample to approx. 60°C for about 2 hours, 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 10-15 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.

- 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
- 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:
yes
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 20.8 and 21.1 °C throughout the test (average value: 20.9°C), and complied with the requirements (20°C ± 2°C, constant within 1°C).
pH:
7.50 - 8.47
pH requirements: 6.0-9.0, not varying by more than 1.5 units
Dissolved oxygen:
6.70 - 8.46 mg O2/L
All these dissolved-oxygen concentrations correspond to values > 76% of the air-saturation value
Salinity:
None
Nominal and measured concentrations:
100 mg/L (nominal loading rate of the substance)
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: 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 1, 10, 32, 100 mg.L-1 and to a control. Two method of preparation for the WAFs have been tried: heating/sonication and solvent conditions. Both conditions showed no daphnids immobilisation at every tested loading rates.
- Results used to determine the conditions for the definitive study: After the range-finding study, it has been decided to do a limit tests with a loading rate of 100 mg test substance/L for the final test.
Reference substance (positive control):
yes
Remarks:
Potassium dichromate
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
After 24 and 48 hours of exposure, no immobilisation of the test animals was observed in the control and the loading rate of 100 mg.L-1, confirming the observations of the range-finding test.
Based on these results, the 24 and 48-hour EL50 values were therefore > 100 mg/L.
Results with reference substance (positive control):
On August 17, 2017 (most recent test), the 24h-EC50 was 0.92 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:
Not applicable

Analytical results

Due to the complex nature of the substance, it was difficult to well solubilize the substance in the water medium (heating, sonication, mixing without headspace …). Even if the TOC analyses indicated that very few organic compounds were found throughout the test in the WAFs at 100 mg/L, analytical results showed that WAFs concentrations were overall stable between the start and the end of each exposure period (t=0h-t24hOldand t=24hFresh-t=48h). It should be noted that TOC values at 100 mg/L were higher the first day than the ones found the second day of the test. This could be explained by the pre-treatment (heating and sonication), probably more effective the first day on the paste pellet. It can be assumed that a longer time of heating / sonication could be more effective and would conduct to a probable increase of the COD values, but this could lead to considerable heating and potential consequences for stability as described in the OECD GD No. 23. Moreover, test solutions must be treated in the same way, that’s why the same reasonable time of heating / sonication was applied for all treatments during the preparation of the fresh solutions. Moreover, the study was carried out using WAFs of a natural complex substance made of several constituents with different stability and behaviours in aqueous solutions during testing. Therefore, according to the results of the TOC analysis, due to the complex nature of the WAF and since the test item was an UVCB substance, the results were based onthe nominal test loading rates.

Table 6.1.3/1: Results of the determination of TOC analysis (mg/L) - 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

0.55

0.51

1.08

1.05

1.01

1.01

0.97

0.96

Control

0.46

1.02

1.00

0.95

100

8.23

7.75

8.02

8.05

5.30

3.97

4.93

4.95

100

7.27

8.07

2.63

4.96

* WAF prepared at the given loading rate.

Table 6.1.3/2: 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

100

1

2

3

4

5

5

5

5

0

0

0

0

0

0

0

0

0

0

* WAF prepared at the given loading rate.

 

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 loading rates, the 48-hour EL50 was estimated to be higher than 100 mg.L-1. Moreover, the highest nominal loading rate without observed effects was estimated to be at least 100 mg.L-1.
Executive summary:

A study was performed to assess the acute toxicity of test item to Daphnia magna and was investigated 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 study has been performed under GLP compliance.

 

A limit test was performed 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 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).

 

Even if the TOC analyses indicated that very few organic compounds were found throughout the test in the WAFs at 100 mg/L, analytical results showed that WAFs concentrations were overall stable between the start and the end of each exposure period. Therefore, and since the study was carried out using WAFs of an UVCB substance, the results were based on the nominal test loading rates. After 24 and 48 hours of exposure, no immobilisation of the test animals was observed in the control and the loading rate of 100 mg/L.

 

Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 was estimated to be higher than 100 mg.L-1. Moreover, the highest nominal loading rate without observed effects was estimated to be at least 100 mg.L-1.

Description of key information

Under the experimental conditions and based on nominal loading rates, the 48-hour EL50 was estimated to be higher than 100 mg/L. Moreover, the highest nominal loading rate without observed effects was estimated to be at least 100 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
100 mg/L

Additional information

For that endpoint, a study on the registered substance was available. This study was performed to assess the acute toxicity of test item to Daphnia magna and was investigated 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 study has been performed under GLP compliance.

 

A limit test at a nominal loading rate of 100 mg test item/L was performed following the results of a range-finding test. 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). Even if the TOC analyses indicated that very few organic compounds were found throughout the test in the WAFs at 100 mg/L, analytical results showed that WAFs concentrations were overall stable between the start and the end of each exposure period. Therefore, according to the results of the TOC analysis, due to the complex nature of the WAF and since the test item was an UVCB substance, the results were based on the nominal test loading rates.

After 24 and 48 hours of exposure, no immobilisation of the test animals was observed in the control and the loading rate of 100 mg/L.

All validity criteria were fulfilled and the study comply with the requirements of the guideline. This study is therefore considered acceptable for that endpoint.