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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not effect the quality of the relevant results.
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
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations:
The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 48 hours.


- Sampling method:
Water samples were taken from the control and all the test groups (replicates R1 – R2 pooled) at 0 and 48 hours for quantitative analysis.

- Sample storage conditions before analysis:
Duplicate samples were taken and stored at approximately -20°C for further analysis if necessary.
Vehicle:
no
Details on test solutions:
TEST WATER:
Reconstituted water was used for all the tests and is defined below:
Reconstituted Water
i)         Stock Solutions
a)  CaCl2.2H2O: 11.76 g/l
b)  MgSO4.7H2O: 4.93 g/l
c)  NaHCO3: 2.59 g/l
d)  KCl : 0.23 g/l
ii)        Preparation
An aliquot (25 ml) of each of solutions a-d was added to each litre (final volume) of deionised water with a conductivity of <5 µS cm-1. The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl and was aerated until the dissolved oxygen concentration was approximately air-saturation value.
The reconstituted water had an approximate theoretical total hardness of 250 mg/l as CaCO3.


RANGE-FINDING TEST:
The test concentration to be used in the definitive test was determined by a preliminary range-finding test.

In the range-finding test Daphnia magna were exposed to a series of nominal test concentrations of 0.10, 1.0, 10 and 100 mg/l. The test material was dissolved directly in water.

An amount of test material (100 mg) was dissolved in reconstituted water and the volume adjusted to 1 litre to give the 100mg/l test concentration from which serial dilutions were made to prepare the 10, 1.0 and 0.10 mg/l test concentrations.

DEFINITIVE TEST:
Based on the results of the range-finding test was conducted at a concentration of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l.

EXPERIMENTAL PREPARATION:
For the purpose of the definitive test the test material was dissolved directly in reconstituted water.

An amount of test material (200 mg) was dissolved in reconstituted water with the aid of ultrasonification for approximately 1 minute and the volume adjusted to 2 litres to give the 100 mg/l test concentration. Aliquots (10, 18, 32, 56, 100, 180, 320 and 560 ml) of this were each further dluted in diluent and the volume adjusted to 1 litre to prepare the test concentrations of 1.0, 1.8, 3.2, 5.6, 10, 18, 32 and 56 mg/l respectively. Each flask was inverted several times to ensure adequate mixing.

















Test organisms (species):
Daphnia magna
Details on test organisms:
The test was carried out using 1st instar Daphnia magna. Daphnia magna were maintained in a laboratory cultures originating from a strain supplied by the Institut National de Recherche Chimique Appliquee (IRCHA), France.

Adult Daphnia were maintained in polypropylene vessels containing approximately 2 litres of reconstituted water in a temperature controlled room at approximately 21°C. The lighting cycle was controlled to give a 16 hours light and 8 hours darkness cycle with 20 minute dawn and dusk transition periods. Each culture was fed daily with a mixture of algal suspension (predominantly Chlorella spp). Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated 24 hours before initiation of the test, the young daphnids produced overnight were then removed for testing.

Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
Not applicable.
Hardness:
The reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaCO3.
Test temperature:
Temperature was maintained at 21ºC ± 1°C throughout the test (water temperature recorded daily throughout the test).
pH:
The reconstituted water had a pH of 7.8 ± 0.2 adjusted (if necessary) with NaOH or HCl.
pH was recorded at the start and termination of the test.
pH in test vessels ranged from 7.8- 7.9
There were no treatment related differences for pH.
Dissolved oxygen:
The reconstituted water was aerated until the dissolved oxygen concentration was approximately air-saturation value.
Dissolved oxygen concentration was recorded at the start and termination of the test.
Concentration dependent differences in oxygen concentration were observed throughout the study
Nominal and measured concentrations:
Pre study recovery and stability conducted showed the test material to be highly unstable under the test conditions employed. This was suspected to be due to possible oxidation of the test material. Therefore, during the definitive study analysis was performed for the parent test material and for the known oxidation product. Analysis of the freshly prepared test solutions at 0 hours was performed immediately due to the unstable nature of the test material under the test conditions.

At 0 hours analysis showed measured concentrations of the parent material to range from 1.11 to 91.5 mg/l (84% to 111% of nominal value), whilst measured concentrations of the oxidation product ranged from 0.0552 to 2.55 mg/l, giving a total concentration of parent material plus oxidation product in the range of 1.17 to 94.1mg/l.
Analysis of the old or expired test solutions sampled at 48 hours showed a decline in the measured concentration of the parent material with values in the range of 0.107 to 11.0 mg/l of nominal whilst an increase in the oxidation product was detected with values ranging from 0.786 to 73.2 mg/l. The concentration of parent test material plus oxidation product was shown to be in the range 0.934 to 84.2 mg/l which was in line with the preliminary stability work conducted. It was considered that as a marked decline was also shown for the total measured concentration, further oxidation products may have formed whcih were not detected during the analysis.

Thus, given that toxicty cannot be attributed to the parent test material or one or more of the oxidation products but to a combination of them all, and certain degradation products may not have been detected during analysis, the EC50 values are based on nominal test concentrations only
Details on test conditions:
EXPOSURE CONDITIONS:
Range finding test:
In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at 21°C with a photoperiod of 16 hours light and 8 hours darkness for a period of 48 hours with 20 minute dawn and dusk transition periods. Information indicated that the test material may decompose in solution when exposed to air. Therefore in order to minimise possible oxidation, 300ml stoppered test vessels completely filled to reduce the headspace were used. After 24 and 48 hours the number of immobilised Daphnia magna were recorded.

The control group was maintained under identical conditions but not exposed to the test material.

Definitive test:
In the definitive study 250ml glass jars containing approximately 200 ml of test solutions were used as opposed to completely filled 300ml stoppered vessels, which were used during the range finding study. The use of different test vessels was considered not to have affected the results of the study as dissolved oxygen concentrations were closely moitored during both the definitive and range finding studies and no significant differences were observed, indicating similiar oxidation rates for both sudies. Further the results from analysis of the test solutions during the definitive study were in line with the pre-study stability analyses which were performed in closed bottles to minimise oxidation of the test material. At the start of the study 10 daphnids were placed in each test and control vessel at random, in the prepared test solutions. Duplicate test vessels were used for each test and control group. The test vessles were then covered to reduce evaporation and maintained in a temperature controlled room at 21°C with a photoperiod of 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods. The daphnids were not individually identiifed, receieved no food during exposure and test vessels were not aerated.
The control group was maintained under identical conditions but not exposed to the test material.

The test preparations were not renewed during the exposure period.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
Any immobilization or adverse reactions to exposure were recorded at 24 and 48 hours after the start of exposure. The criterion of effect used was that Daphnia were considered to be immobilized if they were unable to swim for approximately 15 seconds after gentle agitation.

Reference substance (positive control):
no
Key result
Duration:
48 h
Dose descriptor:
EC50
Effect conc.:
61 mg/L
Nominal / measured:
nominal
Conc. based on:
other: test material and oxidation products
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
other: test material and oxidation products
Basis for effect:
mobility
Details on results:
Range Finding Study
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the range-finding test are given in the attached background information.
No immobilisation was observed at the test concentrations of 0.10, 1.0 and 10 mg/l. However immobilisation was observed at 100 mg/l.


DEFINITIVE TEST:
Immobilisation data:
Cumulative immobilisation data from the exposure of Daphnia magna to the test material during the definitive test are given in the attached background information.

Analysis of the immobilisation data by the trimmed Spearmen - Karber method of Hamiliton (1977) at 0 and 48 hours based on nominal test concentration gave the following results:
24 h EC50: 69 mg/l, 95% confidence liits 63 - 75 mg/l
48 h EC50: 61 mg/l, 95% confidence liits 54 - 69 mg/l

The No Observed Effect Concentration after 24 and 48 hours exposure was 32 mg/l. The No Observed Effect Concentration is based upon zero immobilisation at this concentration.

PHYSICO-CHEMICAL MEASUREMENTS:
Temperature was maintained at approximately 21 ± 1 1°C throughout the test, while there were no treatment related differences in pH, concentration dependent differences in oxygen concentration were observed throughout the study.

VERIFICATION OF TEST CONCENTRATIONS:
Pre study recovery and stability conducted showed the test material to be highly unstable under the test conditions employed. This was suspected to be due to possible oxidation of the test material. Therefore, during the definitive study analysis was performed for the parent test material and for the known oxidation product. Analysis of the freshly prepared test solutions at 0 hours was performed immediately due to the unstable nature of the test material under the test conditions.

At 0 hours analysis showed measured concentrations of the parent material to range from 1.11 to 91.5 mg/l (84% to 111% of nominal value), whilst measured concentrations of the oxidation product ranged from 0.0552 to 2.55 mg/l, giving a total concentration of parent material plus oxidation product in the range of 1.17 to 94.1mg/l.
Analysis of the old or expired test solutions sampled at 48 hours showed a decline in the measured concentration of the parent material with values in the range of 0.107 to 11.0 mg/l of nominal whilst an increase in the oxidation product was detected with values ranging from 0.786 to 73.2 mg/l. The concentration of parent test material plus oxidation product was shown to be in the range 0.934 to 84.2 mg/l which was in line with the preliminary stability work conducted. It was considered that as a marked decline was also shown for the total measured concentration, further oxidation products may have formed whcih were not detected during the analysis.
Conclusions:
The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50 of 61 mg/l.. Correspondingly the No Observed Effect Concentration was 32 mg/l..
Executive summary:

Introduction.

A study was performed to assess the acute toxicity of the test material to Daphnia magna.The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 202,"Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Directive 92/69/EEC.

Methods.

Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed to an aqueous solution of the test material at a concentration of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l for 48 hours under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

Verification of test concentrations

Pre study recovery and stability conducted showed the test material to be highly unstable under the test conditions employed. This was suspected to be due to possible oxidation of the test material. Therefore, during the definitive study analysis was performed for the parent test material and for the known oxidation product. Analysis of the freshly prepared test solutions at 0 hours was performed immediately due to the unstable nature of the test material under the test conditions.

At 0 hours analysis showed measured concentrations of the parent material to range from 1.11 to 91.5 mg/l (84% to 111% of nominal value), whilst measured concentrations of the oxidation product ranged from 0.0552 to 2.55 mg/l, giving a total concentration of parent material plus oxidation product in the range of 1.17 to 94.1mg/l.

Analysis of the old or expired test solutions sampled at 48 hours showed a decline in the measured concentration of the parent material with values in the range of 0.107 to 11.0 mg/l of nominal whilst an increase in the oxidation product was detected with values ranging from 0.786 to 73.2 mg/l. The concentration of parent test material plus oxidation product was shown to be in the range 0.934 to 84.2 mg/l which was in line with the preliminary stability work conducted. It was considered that as a marked decline was also shown for the total measured concentration, further oxidation products may have formed which were not detected during the analysis.

Thus, given the toxicity cannot be attributed to the parent test material or one or more of the oxidation products but to a combination of them all, and certain degradation products may not have been detected during analysis, the EC50 values are based on nominal test concentration only.

The 48-Hour EC50for the test material to Daphnia magna based on nominal test concentrations was 61 mg/l and correspondingly the No Observed Effect Concentration was 32 mg/l.

Description of key information

The acute toxicity of the test material to the freshwater invertebrate Daphnia magna has been investigated and gave a 48-Hour EC50  of 61 mg/l.. Correspondingly the No Observed Effect Concentration was 32 mg/L.

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
61 mg/L

Additional information

Introduction.

A study was performed to assess the acute toxicity of the test material to Daphnia magna.The method followed that described in the OECD Guidelines for Testing of Chemicals (1984) No 202,"Daphnia sp, Acute Immobilisation Test" referenced as Method C.2 of Commission Directive 92/69/EEC.

Methods.

Following a preliminary range-finding test, twenty daphnids (2 replicates of 10 animals) were exposed to an aqueous solution of the test material at a concentration of 1.0, 1.8, 3.2, 5.6, 10, 18, 32, 56 and 100 mg/l for 48 hours under static test conditions. Immobilisation and any adverse reactions to exposure were recorded after 24 and 48 hours.

Results

Verification of test concentrations

Pre study recovery and stability conducted showed the test material to be highly unstable under the test conditions employed. This was suspected to be due to possible oxidation of the test material. Therefore, during the definitive study analysis was performed for the parent test material and for the known oxidation product. Analysis of the freshly prepared test solutions at 0 hours was performed immediately due to the unstable nature of the test material under the test conditions.

At 0 hours analysis showed measured concentrations of the parent material to range from 1.11 to 91.5 mg/l (84% to 111% of nominal value), whilst measured concentrations of the oxidation product ranged from 0.0552 to 2.55 mg/l, giving a total concentration of parent material plus oxidation product in the range of 1.17 to 94.1mg/l.

Analysis of the old or expired test solutions sampled at 48 hours showed a decline in the measured concentration of the parent material with values in the range of 0.107 to 11.0 mg/l of nominal whilst an increase in the oxidation product was detected with values ranging from 0.786 to 73.2 mg/l. The concentration of parent test material plus oxidation product was shown to be in the range 0.934 to 84.2 mg/l which was in line with the preliminary stability work conducted. It was considered that as a marked decline was also shown for the total measured concentration, further oxidation products may have formed which were not detected during the analysis.

Thus, given the toxicity cannot be attributed to the parent test material or one or more of the oxidation products but to a combination of them all, and certain degradation products may not have been detected during analysis, the EC50 values are based on nominal test concentration only.

The 48-Hour EC50for the test material to Daphnia magna based on nominal test concentrations was 61 mg/l and correspondingly the No Observed Effect Concentration was 32 mg/l.