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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:
24 March 2014 to 01 October 2014
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:
yes
Remarks:
environmental measurements taken from only one replicate vessel in line with latest guidelines and with not impact on integrity of the study (see below)
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
environmental measurements taken from only one replicate vessel in line with latest guidelines and with not impact on integrity of the study (see below)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
RANGE-FINDING TEST
- A sample of each test concentration was taken for chemical analysis at 0 and 48 hours.
- All samples were frozen prior to analysis.
- Only concentrations withing the range to be used for the definitive test were analysed.

DEFINITIVE TEST - VERIFICATION OF TEST CONCENTRATIONS
- Water samples were taken from the control and each test group at 0 and 48 hours for quantitative analysis.
- The 0 hours samples were taken from the bulk test preparation and the 48 hour samples were taken from pooled R1-R4 replicates.
- Samples were stored frozen prior to analysis.
- Duplicate samples were taken and stored frozen for further analysis if necessary.
- Only samples at the No Observed Effect Concentration and above were analysed.

DEFINITIVE TEST - TOTAL ORGANIC CARBON ANALYSIS
- Water samples were taken from the control and each test group at 0 and 48 hours.
- Duplicate samples were taken and stored frozen for further analysis if necessary.

Vehicle:
no
Details on test solutions:
- Reconstituted water (ISO medium) was used for the range-finding and the definitive tests and is described in Appendix 3 (attached).
Test organisms (species):
Daphnia magna
Details on test organisms:
- The test was performed using 1st instar Daphnia magna derived from in-house laboratory cultures.
- Adult Daphnia were maintained in 150 mL glass beakers containing Elendt M7 medium (see Appendix 1, attached) in a temperature controlled room at approximately 20 °C.
- The lighting cycle was controlled to give 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.
- Each culture was fed daily with a mixture of algal suspension (Desmodesmus subspicatus) and Tetramin flake food suspension.
- Culture conditions ensured that reproduction was by parthenogenesis.
- Gravid adults were isolated the day before initiation of the test such that the young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
- The diet and diluent water were considered not to contain any contaminant that would affect the integrity or outcome of the study.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Post exposure observation period:
Not applicable
Hardness:
Approximate theoretical total hardness of 250 mg/L as CaCO3 (see Appendix 3, attached)
Test temperature:
20 to 21 °C (see Table 3, attached)
pH:
7.6 to 7.9 (see Table 3, attached)
Dissolved oxygen:
8.8 to 9.1 mg O2/L (see Table 3, attached)
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
RANGE-FINDING TEST
- Nominal concentrations of 1.0, 10 and 100 mg/L.

DEFINITIVE TEST
- Test concentrations of 1.0, 1.8, 3.2, 5.6 and 10 % v/v saturated solution prepared using nominal amount of test item and subsequent dilution.
Details on test conditions:
PRELIMINARY MEDIA PREPARATION TRIAL
- Preliminary solubility work indicated that the test item was practically insoluble in water using traditional methods of preparation such as ultrasonication and high shear mixing.
- Based on this information, the test item was categorised as a ‘difficult substance’ in accordance with the OECD guidance document on aquatic toxicity testing of difficult substances and mixtures (OECD; 2000).
- A media preparation trial was therefore conducted to determine the solubility of the test material under test conditions (see Appendix 4, attached).

RANGE-FINDING TEST
- Conditions to be used in the definitive test were determined by a preliminary range-finding test.
- Daphnia magna were exposed to a series of nominal loading rates of 1.0, 10 and 100 mg/L.
- Nominal amounts of test item (5.0, 50 and 500 mg) were each separately added to the surface of 5 L of test water to give the 1.0, 10 and 100 mg/L loading rates respectively.
- After addition of test item, the test water was stirred by magnetic stirrer for 23 hours using a stirring rate such that a vortex was formed to give a dimple at the water surface.
- After stirring, the test solutions were allowed to stand for one hour prior to removal of any undissolved test item by filtration through a glass wool plug.
- A wide bore glass tube, covered at one end with Nescofilm, was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was then inserted into the glass tube and pushed through the Nescofilm seal.
- A glass wool plug was inserted into the opposite end of the tubing and the aqueous phase removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rates.
- Microscopic observations of the test preparations were performed after filtering and showed there to be no undissolved test item present.
- Ten daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 20 °C.
- The photoperiod during the test was 16 hours light and 8 hours darkness with 20 minute dawn and dusk transition periods.
- Each 250 mL test and control vessel contained 200 mL of test media and was covered to reduce evaporation.
- 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 item.

DEFINITIVE TEST
- Based on the results of the range-finding test, concentrations of 1.0, 1.8, 3.2, 5.6 and 10 % v/v saturated solution were assigned to the definitive test.
- A nominal amount of test item (500 mg) was dispensed onto the surface of 5 L of test water prior to stirring with the aid of a magnetic stirrer for 23 hours at a rate such that a dimple was formed on the surface of the water.
- After stirring, the solution was allowed to stand for one hour prior to removal of any undissolved test item by filtration through a glass wool plug.
- A wide bore glass tube, covered at one end with Nescofilm, was submerged into the vessel, sealed end down, to a depth of approximately 5 cm from the bottom of the vessel. A length of Tygon tubing was then inserted into the glass tube and pushed through the Nescofilm seal.
- A glass wool plug was inserted into the opposite end of the tubing and the aqueous phase removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give a 100 % v/v saturated solution.
- A series of dilutions was made from this saturated solution to give the required test concentrations of 10, 5.6, 3.2, 1.8 and 1.0 % v/v saturated solution.
- Each prepared concentration was inverted several times to ensure adequate mixing and homogeneity.
- The concentration of boron in the test preparations was verified by chemical analysis at 0 and 48 hours (see Appendix 5, attached).
- Total Organic Carbon (TOC) analysis was performed on the test solutions at 0 and 48 hours (see Appendix 6, attached).
- Glass jars (250 mL) containing approximately 200 mL of test preparation were used.
- At the start of the test, five daphnids were placed into test preparations in each test and control vessel at random.
- Four replicate test and control vessels were prepared.
- The test vessels were then covered to reduce evaporation and maintained in a temperature controlled room at approximately 21 °C.
- The photoperiod during the test was 16 hours light (647 to 703 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods.
- The daphnids were not individually identified, received no food during exposure and the test vessels were not aerated.
- The control group was maintained under identical conditions but not exposed to the test item.
- Test preparations were not renewed during the exposure period.

EVALUATIONS
- Any immobilisation or adverse reactions to exposure were recorded at 24 and 48 hours after the start of the test.
- Daphnia were considered to be immobilised if they were unable to swim for approximately 15 seconds after gentle agitation.

WATER QUALITY CRITERIA
- Water temperature was recorded daily throughout the test.
- Dissolved oxygen concentrations and pH were recorded at the start and termination of the test.
- A Hach HQ30d Flexi handheld meter was used to measure pH and dissolved oxygen concentration whilst temperature was measured using a Hanna Instruments HI 93510 digital thermometer.

VORTEX DEPTH MEASUREMENTS
- The vortex depth was recorded at the start and end of the mixing period.

VALIDATION CRITERIA
- No more than 10 % of the control daphnids show immobilisation or other signs of disease or stress (for example, discolouration or unusual behaviour such as trapping at the water surface).
- The dissolved oxygen concentration at the end of the test is ≥ 3 mg/L in the control and test vessels.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (conducted between 05 March 2014 and 07 March 2014; see Appendix 2, attached)
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
2.6 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: carbon content of test item
Basis for effect:
mobility
Remarks on result:
other: 95 % confidence limits 2.3 to 2.8 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
1.9 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: carbon content of test item
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
2.7 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: carbon content of test item
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LC50
Effect conc.:
5.7 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: boron content of test item
Basis for effect:
mobility
Remarks on result:
other: 95 % confidence limits 5.0 to 6.3 mg/L
Duration:
48 h
Dose descriptor:
NOEC
Effect conc.:
3.6 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: boron content of test item
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
LOEC
Effect conc.:
6.3 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: boron content of test item
Basis for effect:
mobility
Details on results:
RANGE-FINDING TEST
- Cumulative immobilisation data from exposure of Daphnia magna during the range-finding test are given in Table 1 (attached).
- No immobilisation was observed at the loading rate of 1.0 mg/L. However, immobilisation was observed at loading rates of 10 and 100 mg/L.
- Based on this information, test concentrations of 1.0, 1.8, 3.2, 5.6 and 10 % v/v saturated solution were selected for the definitive test.
- Chemical analysis of the test preparations at 0 and 48 hours (see Appendix 5, attached) showed measured boron concentrations ranging from 0.022 to 2.3 mg/L.

DEFINITIVE TEST – VERIFICATION OF TEST CONCENTRATIONS
- Chemical analysis of the 3.2, 5.6 and 10 % v/v saturated solution test preparations at 0 and 48 hours (see Appendix 5, attached) showed measured boron concentrations ranging from 0.078 to 0.25 mg/L.
- The measured boron concentrations were equivalent to test item concentration of 3.6 to 11 mg/L based on test item boron content of 2.146 %.

DEFINITIVE TEST – TOTAL ORGANIC CARBON ANALYSIS
- Total Organic Carbon (TOC) analysis of the 3.2, 5.6 and 10 % v/v saturated solution test preparations at 0 and 48 hours (see Appendix 6, attached) showed measured carbon concentration ranging from 1.4 to 3.7 mg/L.
- The measured carbon carbon concentrations were equivalent to test item concentrations of 1.9 to 4.9 mg/L based on test item carbon content of 75.78 %.

DEFINITIVE TEST – IMMOBILISATION DATA
- Cumulative immobilisation data from exposure of Daphnia magna to the test item during the definitive test are given in Table 2 (attached).
- The relationship between percentage immobilisation and nominal concentration at 48 hours is given in Figure 1 (attached).
- Based on carbon content, analysis of immobilisation data by the trimmed Spearman-Karber method at 24 and 48 hours gave EC50 (24 h) 3.8 mg/L (95 % confidence limits 3.4 to 4.2 mg/L) and EC50 (48 h) 2.6 mg/L (95 % confidence limits 2.3 to 2.8 mg/L). The No Observed Effect Concentrations after 24 and 48 hours exposure was 1.9 mg/L. Correspondingly, the Lowest Observed Effect Concentration was considered to be 2.7 mg/L. Due to the nature of the data it was not possible to calculate the slope and error of the response curve at 24 and 48 hours.
- Based on boron content, analysis of immobilisation data by the trimmed Spearman-Karber method at 24 and 48 hours gave EC50 (24 h) 8.7 mg/L (95 % confidence limits 8.0 to 9.6 mg/L) and EC50 (48 h) 5.7 mg/L (95 % confidence limits 5.0 to 6.3 mg/L). The No Observed Effect Concentrations after 24 and 48 hours exposure was 3.6 mg/L. Correspondingly, the Lowest Observed Effect Concentration was considered to be 6.3 mg/L. Due to the nature of the data it was not possible to calculate the slope and error of the response curve at 24 and 48 hours.

VALIDATION CRITERIA
- The test was considered to be valid.
- None of the control daphnids showed immobilisation or other signs of disease/stress.
- Oxygen concentration at the end of the test was ≥ 3 mg/L in the control and test vessels.

WATER QUALITY CRITERIA
- Results of the water quality measurements are given in Table 3 (attached).
- Temperature was maintained at approximately 21 °C throughout the test.
- There were no treatment-related differences for oxygen concentration or pH.
VORTEX DEPTH MEASUREMENTS
- the vortex depth was recorded at the start and end of the mixing period and was observed to be a dimple at the water surface on each occasion.

OBSERVATIONS ON TEST ITEM SOLUBILITY
- Observations on the test media were carried out during mixing and testing of the saturated solution.
- At the start and end of the mixing period, and following a one hour standing period, the 100 % v/v saturated solution was observed to have formed a clear, colourless, water column with test item on the surface.
- Microscopic examination of the aqueous phase showed there to be no micro-particles of test item present and hence the solution was filtered through a glass wool plug (2-4 cm in length).
- Microscopic examination after filtering showed the glass wool plug has removed all of the undissolved test item present.
- For the duration of the test, all control and test cultures were observed to be clear, colourless, solutions.
Results with reference substance (positive control):
- Results from the positive investigation with potassium dichromate were within the normal range for the reference item (see Appendix 2, attached).
Reported statistics and error estimates:
- The EC50 values and associated confidence limits at 24 and 48 hours were calculated by the trimmed Spearman-Karber method (Hamilton et al, 1977) using the ToxCalc computer software package (ToxCalc, 1999).
- When only one partial response is shown the trimmed Spearman-Karber method is appropriate.
Validity criteria fulfilled:
yes
Conclusions:
Exposure of Daphnia magna to the test item gave an LC50 (48 h) value of 2.6 mg/L based on carbon content (95 % confidence limits 2.3 to 2.8 mg/L). The associated No Observed Effect Concentration (NOEC) was 1.9 mg/L and the Lowest Observed Effect Concentration (LOEC) was 2.7 mg/L. Based on boron content the LC50 (48 h) was reported as 5.7 mg/L (95 % confidence limits 5.0 to 6.3 mg/L). The associated NOEC was 3.6 mg/L and the LOEC was 6.3 mg/L.
Executive summary:

GUIDELINE

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202 “Daphnia sp., Acute Immobilisation Test” referenced as Method C.2 of Commission Regulation (EC) No 440/2008.

 

METHODS

Due to the low aqueous solubility and pure nature of the test item, for the purposes of the definitive test, the test medium was prepared as a slow stir saturated solution. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to the test item over a range of nominal concentrations (1.0, 1.8, 3.2, 5.6 and 10 % v/v saturated solution) for 48 hours at a temperature of approximately 21 °C under static conditions. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in culture medium using a magnetic stirrer for 23 hours at a rate such that a dimple was formed at the water surface. After the stirring period, the saturated solution was allowed to stand for one hour prior to the removal of any undissolved test item by filtration through a glass wool plug (first approximate 75-100 mL discarded) to produce a 100 % v/v saturated solution of the test item. This saturated solution was then diluted as necessary to provide the required test groups. The number of immobilised Daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

RESULTS

Chemical analysis of the 3.2, 5.6 and 10 % v/v saturated solution test preparations at 0 and 48 hours showed measured carbon concentrations to range from 1.4 to 3.7 mg/L (equivalent to test item concentrations of 1.9 to 4.9 mg/L based on a carbon content of 75.78 %).

 

CONCLUSION

Exposure of Daphnia magna to the test item gave an LC50 (48 h) value of 2.6 mg/L based on carbon content (95 % confidence limits 2.3 to 2.8 mg/L). The associated No Observed Effect Concentration (NOEC) was 1.9 mg/L and the Lowest Observed Effect Concentration (LOEC) was 2.7 mg/L. Based on boron content the LC50 (48 h) was reported as 5.7 mg/L (95 % confidence limits 5.0 to 6.3 mg/L). The associated NOEC was 3.6 mg/L and the LOEC was 6.3 mg/L.

Description of key information

Exposure of Daphnia magna to the test item gave an LC50 (48 h) value of 2.6 mg/L based on carbon content (95 % confidence limits 2.3 to 2.8 mg/L). The associated No Observed Effect Concentration (NOEC) was 1.9 mg/L and the Lowest Observed Effect Concentration (LOEC) was 2.7 mg/L. Based on boron content the LC50 (48 h) was reported as 5.7 mg/L (95 % confidence limits 5.0 to 6.3 mg/L). The associated NOEC was 3.6 mg/L and the LOEC was 6.3 mg/L (OECD 202).

Key value for chemical safety assessment

EC50/LC50 for freshwater invertebrates:
5.7 mg/L

Additional information

GUIDELINE

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202 “Daphnia sp., Acute Immobilisation Test” referenced as Method C.2 of Commission Regulation (EC) No 440/2008.

 

METHODS

Due to the low aqueous solubility and pure nature of the test item, for the purposes of the definitive test, the test medium was prepared as a slow stir saturated solution. Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to the test item over a range of nominal concentrations (1.0, 1.8, 3.2, 5.6 and 10 % v/v saturated solution) for 48 hours at a temperature of approximately 21 °C under static conditions. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in culture medium using a magnetic stirrer for 23 hours at a rate such that a dimple was formed at the water surface. After the stirring period, the saturated solution was allowed to stand for one hour prior to the removal of any undissolved test item by filtration through a glass wool plug (first approximate 75-100 mL discarded) to produce a 100 % v/v saturated solution of the test item. This saturated solution was then diluted as necessary to provide the required test groups. The number of immobilised Daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

RESULTS

Chemical analysis of the 3.2, 5.6 and 10 % v/v saturated solution test preparations at 0 and 48 hours showed measured carbon concentrations to range from 1.4 to 3.7 mg/L (equivalent to test item concentrations of 1.9 to 4.9 mg/L based on a carbon content of 75.78 %).

 

CONCLUSION

Exposure of Daphnia magna to the test item gave an LC50 (48 h) value of 2.6 mg/L based on carbon content (95 % confidence limits 2.3 to 2.8 mg/L). The associated No Observed Effect Concentration (NOEC) was 1.9 mg/L and the Lowest Observed Effect Concentration (LOEC) was 2.7 mg/L. Based on boron content the LC50 (48 h) was reported as 5.7 mg/L (95 % confidence limits 5.0 to 6.3 mg/L). The associated NOEC was 3.6 mg/L and the LOEC was 6.3 mg/L.