Registration Dossier

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

Link to relevant study record(s)

Reference
Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2015-04-13 to 2015-08-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Qualifier:
according to
Guideline:
ISO 6341 (Water quality - Determination of the Inhibition of the Mobility of Daphnia magna Straus (Cladocera, Crustacea))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- batch No.: 14822
- Expiration date of the batch: 13 May 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:S tore in cool, dry and ventilated area, away from heat sources, sparks and open flames, protected from light in tightly closed original container.
- Stability under test conditions: 24 months from dispatching date on, under normal storage conditions

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Use of WAFs (Water Accommodated Fractions)

FORM AS APPLIED IN THE TEST (if different from that of starting material)
WAFs (Water Accommodated Fractions)
Analytical monitoring:
yes
Details on sampling:
Concentrations: Nominal loading values 10, 100, 1000 mg of test item /L
Vehicle:
no
Details on test solutions:
The WAFs (for the fresh mediums at t=0h and t=24h) were prepared under closed conditions and by slow-stirring to avoid production of a dispersion.
The mixing vessel was a cylindrical glass bottle sealed with screw cap and fitted with a drain port near the bottom for drawing off the WAFs. The volume of the mixing vessel was approximately 1L. A magnetic stirring bar was placed in each mixing vessel completely filled with test water (without 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 screwcaps after weighing. Each glass flask was placed in a water bath for 5-15 minutes at 70°C (following recommendations of the sponsor), followed by sonication for 5 to 10 minutes. This step was essential to remove the gum pellets stuck to the glass of the flasks (especially at the highest loading rates) and to extract a maximum of soluble fraction of the test item 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 at room temperature, the WAFs were allowed to stand for 1 hour before use and were extracted via the drain port. The first 100 mL were discarded. Then the WAFs were directly added into test tubes (without headspace) that were immediately sealed with screwcaps after introduction of daphnids. No small bubble was observed in the test tubes. The test solution was observed to be clear and colourless at all loading rates. The test was carried out without adjustment of the pH.
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.
- Age of parental stock: < 24 hours old
- Feeding during test :NO
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 beakers containing purified drinking water (Composition (mg.L-1): Calcium 12 – Magnesium 8.0 – Sodium 12 – Potassium 6 – Silica 32 – Bicarbonates 74 – Sulphates 9 – Chlorides 15 – Nitrates 7.3; pH = 7).
Cultures were maintained at a density of 1 adult daphnid per 25 mL of culture medium. During the week the stock daphnids were fed daily with a suspension of freshwater algae (mix of 3 algae strains: Chlorella vulgaris = 2.5x106 cells.mL-1.day-1.daphnid-1, Desmodesmus subspicatus = 2.5x106 cells.mL-1.day-1.daphnid-1 and Pseudokirchneriella subcapitata = 5x106 cells.mL-1.day-1.daphnid-1).
The water was changed once per week. These culture conditions maintained the daphnids in the parthenogenetic reproductive stage.

ACCLIMATION
At least 48 hours prior to the start of the test, gravid daphnids were transferred to OECD test water and held at similar temperature and light conditions as used in the test. During this period, daphnids were fed in the same manner as that of the stock population. Only daphnids up to 24 hours old were used for the test.
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:
approximately 250 mg.L-1 (as CaCO3)
Test temperature:
20 +/- 2°C
pH:
6.0-9.0, not varying by more than 1.5 units
Dissolved oxygen:
≥ 60% of the air-saturation value at the end of the test
Salinity:
No data.
Conductivity:
No data.
Nominal and measured concentrations:
Nominal loading rates: 10, 100 and 1000 mg.L-1.
Details on test conditions:
TEST SYSTEM
- Test vessel: All-glass sealed with screw cap test tubes of approximately 20 mL capacity. Each test vessel was uniquely identified with study code, replicate number, date of experimentation and concentration.
4 replicates with daphnids for the control and per loading rates.
Moreover, abiotic replicates (for analytical monitoring) were prepared for each treatment (in glass flasks filled with minimum headspace with test solutions and sealed with screw caps; approximate volume: 100 mL) in order to avoid disturbing the daphnids at t=24h and to prevent test item losses on opening test tubes.
- Biomass loading rate: 5 daphnids per vessel each completely filled with test solution and
without headspace

TEST MEDIUM / WATER PARAMETERS
Reconstituted water, as prescribed by the OECD Guideline 202

Stock solutions:
a) CaCl2.2H2O 11.76 g.L-1
b) MgSO4.7H2O 4.93 g.L-1
c) NaHCO3 2.59 g.L-1
d) KCl 0.23 g.L-1
An aliquot (25 mL) of each solution (a to d) was added to each litre (final volume) of deionised water (conductivity < 10 μS.cm-1). The pH of this solution was in the range of 6 to 9 and the total water hardness was approximately 250 mg.L-1 (as CaCO3). This
reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value, and then set aside for 2 days without aeration.

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

EFFECT PARAMETERS MEASURED
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.

RANGE-FINDING STUDY
In the range-finding test, ten daphnids per concentration (5 per vessel, in duplicate) were exposed to the loading rates of 0, 10, 50, 100 and 1000 mg.L-1.
- Results used to determine the conditions for the definitive study: 40% of immobilisation at 48h and at a nominal concentration of 1000 mg/L.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (K2Cr2O7)
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Loading rates of test material
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
other: Loading rates of test item
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 nominal test loading rate 10 mg.L-1. Only one daphnid was immobilised at the loading rate 100 mg.L-1. A residual particle of the non-soluble fraction was probably the cause of this immobilisation (physical effect) rather than a potential toxicity due to the test substance (no effects on other daphnids at the same loading rate). On the other hand, 100% immobilisation was observed at 1000 mg.L-1 (loading), suggesting either a toxic effect of the test item (but this data should be taken cautiously because the test item was probably in excess; thus some constituents may have been above their solubility limit) or a physical effect due to residual particles of the non-soluble fraction of the test item (WAFs unfiltered to prevent potential losses of the test item).
Results with reference substance (positive control):
No data.
Reported statistics and error estimates:
None.

Analytical results

Concentration of dissolved organic material in the WAFs was checked by analysis of total organic carbon (TOC) in the control medium and the WAFs at the start, at t=24h (new and old solutions) and at the end of the test. Based on the results of Total Carbon (TC ) and Total Inorganic Carbon (IC) measurements the TOC content of the test samples was determined using the following equation: TOC = TC – IC.

Although every effort was made in a first time to extract and solubilize the soluble fraction of the test item in test water (heating, sonication, mixing without headspace …) and secondly to maintain the concentrations of the WAFs (semi-static and closed conditions without headspace), analytical results of this test indicate that amounts of organic compounds from the test item dissolved in the water phase were not very significant (in fact unquantifiable for the test loading rate of 10 mg.L-1), except at the loading rate of 1000 mg.L-1

Summary of Analytical Results

  Total Organic Carbon
(mg Carbon.L-1)
Nominal
concentration*
(mg test item.L-1)
Start
(t=0h)
t=24h
Old
t=24h
Fresh
End
(t=48h)
Control  N.A. N.A. N.A. N.A.
10 N.A. N.A. N.A. N.A.
100 7.07 4.18 8.65 3.33
1000 33.31 31.84    

N.A.: not applicable

Empty boxes: no further analysis was performed for concentrations in which all daphnids were immobilised at t=24h

* WAF prepared at the given loading rate.

It should be noted that chemical analyses were performed only to provide an indication of the concentration of dissolved organic material in the WAFs and their stability. 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, the ratio of certain constituents may change over time and the composition of the WAFs after 24h will not be exactly that of the fresh solutions. Moreover, 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.

Thus, since the study was carried out using WAFs made of several constituents with different stability and behaviours in aqueous solutions during testing, it was considered acceptable to base

results on nominal loading values.

Acute Immobilisation of Daphnids after 24 and 48 Hours in the Final Test

Nominal
Loading rate
(mg test item.L-1)
Replicate Number of
daphnids
exposed
Response at 24h Response at 48h
Number Total % Number Total %
Control 1 5 0 0 0 0
2 5 0 0
3 5 0 0
4 5 0 0
10 1 5 0 0 0 0
2 5 0 0
3 5 0 0
4 5 0 0
100 1 5 0 5 1 5
2 5 0 0
3 5 0 0
4 5 0 0
1000 1 5 0 100 5 100
2 5 0 5
3 5 0 5
4 5 0 5

Water quality parameter values throughout the test

These test conditions 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 value at the end of the test). It should be noted that a decrease in pH values was observed in particular at the highest WAFs concentrations (in comparison with pH values of the control).

Furthermore, the temperature of the test medium was situated between 20.8 and 21.5 °C throughout the test (average value: 21.3°C), and complied with the requirements as laid down in the study plan (20°C ± 2°C, constant within 1°C).

Validity criteria fulfilled:
yes
Conclusions:
Under the experimental conditions and based on nominal concentrations, the 48-hour EL50 value was estimated to be higher than 100 mg test item.L-1 and the NOELR at least 100 mg test item.L-1.
Executive summary:

A study was performed to assess the acute toxicity of the test item CISTUS CONCRETE to Daphnia magna. The method followed was designed to be compliant with the OECD Guideline for Testing of Chemicals No. 202, "Daphnia sp., Acute Immobilisation Test", referenced as Method C.2 of Commission Regulation No. 440/2008 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 loading rate 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 daphnid per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rate of 0, 10, 100 and 1000 mg test item.L-1. The immobility of the daphnids was determined in a semi-static 48-hour test by visual observation after 24 and 48 hours. Concentration of dissolved organic material in the WAFs was checked by analysis of total organic carbon (TOC) in the control medium and the WAFs at the start, at t=24h (new and old solutions) and at the end of the test.

These chemical analyses were performed only to provide an indication of the concentration of dissolved organic material in the WAFs and their stability.

Analytical results showed that TOC content was not determinable for the test loading rate 10 mg.L-1 and not very significant at 100 mg test item.L-1. However, more significant amounts of organic compounds were founded at the test loading rate 1000 mg.L-1. After 24 and 48 hours of exposure, no immobilisation of the test animals was observed in the control and the nominal test concentration 10 mg.L-1 (loading). Only one daphnid was immobilised at the loading rate 100 mg.L-1 but this has not been a substance related effect. A residual particle of the non-soluble fraction was probably the cause of this immobilisation (physical effect) rather than a potential toxicity due to the test substance (no effects on other daphnids at the same loading rate). On the other hand, 100% immobilisation was observed at 1000 mg.L-1 (loading), suggesting either a toxic effect of the test item (but this data should be taken cautiously because the test item was probably in excess; thus some constituents may have been above their solubility limit) or a physical effect due to residual particles of the non-soluble fraction of the test item (WAFs unfiltered to prevent potential losses of the test item).

Under the experimental conditions and based on nominal concentrations, the 48-hour EL50 value was estimated to be higher than 100 mg test item.L-1 and the NOELR at least 100 mg test item.L-1.

Results synopsis:

Test: Toxic effect of CISTUS CONCRETE to Daphnia magna according to OECD guidelines No. 202.

48h-EL50: > 100 mg/L

Description of key information

Under the experimental conditions and based on nominal loading rates of the test item, the 48-hour EL50 value was determined to be higher than 100 mg test item/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. In this study, the toxic effect of the test item CISTUS CONCRETE to the freshwater invertebrate Daphnia magna was investigated in a closed semi-static test using Water Accommodated Fractions. 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 and with the “Guidance document on aquatic toxicity testing of difficult substances and mixtures” (OECD No. 23). The criterion measured was the EL50, a statistically derived loading rate 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 daphnid per replicate) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rate of 0, 10, 100 and 1000 mg test item.L-1. The immobility of the daphnids was determined in a semi-static 48-hour test by visual observation after 24 and 48 hours. Concentration of dissolved organic material in the WAFs was checked by analysis of total organic carbon (TOC) in the control medium and the WAFs at the start, at t=24h (new and old solutions) and at the end of the test.

These chemical analyses were performed only to provide an indication of the concentration of dissolved organic material in the WAFs and their stability.

Analytical results showed that TOC content was not determinable for the test loading rate 10 mg.L-1 and not very significant at 100 mg test item.L-1. However, more significant amounts of organic compounds were founded at the test loading rate 1000 mg.L-1. After 24 and 48 hours of exposure, no immobilisation of the test animals was observed in the control and the nominal test loading rate 10 mg.L-1 (loading). Only one daphnid was immobilised at the loading rate 100 mg.L-1 but this has not been a substance related effect. A residual particle of the non-soluble fraction was probably the cause of this immobilisation (physical effect) rather than a potential toxicity due to the test substance (no effects on other daphnids at the same loading rate). On the other hand, 100% immobilisation was observed at 1000 mg.L-1 (loading), suggesting either a toxic effect of the test item (but this data should be taken cautiously because the test item was probably in excess; thus some constituents may have been above their solubility limit) or a physical effect due to residual particles of the non-soluble fraction of the test item (WAFs unfiltered to prevent potential losses of the test item).

Under the experimental conditions and based on nominal concentrations, the 48-hour EL50 value was estimated to be higher than 100 mg test item/L and the NOELR at least 100 mg test item/L.

The validity criteria were respected according to the requirements of the guideline. This study was considered as acceptable for that endpoint.