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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:
08 June 2015 - 30 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)
Deviations:
yes
Remarks:
See "Principles of method if other than guideline" for further details on deivations.
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
See "Principles of method if other than guideline" for further details on deivations.
Principles of method if other than guideline:
Deviation 1
- Study plan states daphnia must be less than 24 hours old. Due to an error the daphnia in the range finder test may been upto 24 hours and 10 minuts old. This deviation was considered not to have an adverse effect on the study,

Deviation 2
- Study plan states pH should be within the range of 6 to 9 in the range finding test. At 0 hours in the 1000mg/L loading tate the pH was recorded to be 8.96. As no immobilisation occured in any of the test concentration the high pH was considered not to have an effect on the biological aspect of the study.
- During the initial test the pH was recorded to be 9.21 which 70% immobilisation occured
- A confirmatory test was conducted where 65% immobilisation occurred and the pH was measured at 7.99
- Full definitive test was completed, the pH was measured at 10.01 with no immobilisation.
- As the range finding and definitive test confirms no effect at 100mg/L it is considered that the high pH had no adverse effect on the study.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Samples were taken from the control and each test group from the bulk test preparation at 0 hours and from the pool replicates (replicates R1 - R4) at 48 hours for quantitative analysis.

- Duplicate samples of 6.25, 12.5, 25, 50 & 100mg/L loading rate WAF test groups were taken and stored frozen for further analysis if necessary

- Only samples at the No Observed Effect Loading Rate were analyzed


Vehicle:
no
Details on test solutions:
TEST WATER
- Reconstituted water (ISO medium) used for both the range-finding and definitive tests is defined in Appendix 4 (attached).

PROCEDURE
- Due to the low aqueous solubility and complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF) of the test item.

VALIDATION OF MIXING PERIOD
- Preliminary work (see Appendix 5, attached) was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.

RANGE FINDING TEST
- 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 the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface.
- Stirring was stopped after 23 hours and the mixtures were allowed to stand for one hour.
- 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 inserted into the glass tube and pushed through the Nescofilm seal.
- Microscopic inspections of the WAFs showed no micro-dispersions or undissolved test item present.
-The aqueous phase of WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs.

INITIAL TEST
- Based on the results of the range-finding test an initial experiment was conducted at a nominal loading rate of 100mg/L loading rate WAF to confirm that no immobilisation or adverse reactions to exposure were observed.
- An amount of test item was added to the surface of 1L of test water to give 101mg/L loading rate
- After addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface.
- Stirring was stopped after 23 hours and the mixtures were allowed to stand for one hour.
- 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 inserted into the glass tube and pushed through the Nescofilm seal.
- Microscopic inspections of the WAFs showed no micro-dispersions or undissolved test item present.
-The aqueous phase of WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 101 mg/L loading rate WAFs.

CONFIRMATORY TEST
- Based on the results of the initial test a confimatory test was conducted at a loading rate of 100mg/L loading rate WAF to confirm that no immobilisation or adverse reactions to exposure were observed.
- A nominal amount of test item (250mg) was added to the surface of 2.5L of test water to give 100mg/L loading rate
- After addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface.
- Stirring was stopped after 23 hours and the mixtures were allowed to stand for one hour.
- 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 inserted into the glass tube and pushed through the Nescofilm seal.
- Microscopic inspections of the WAFs showed no micro-dispersions or undissolved test item present.
-The aqueous phase of WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 101 mg/L loading rate WAFs.


DEFINITIVE TEST
- Based on the result of the initial & confirmatory experiment, the following concentration were assigned to the definitve test 6.25, 12.5, 25, 50 & 100mg/L loading rate WAF's.
- A nominal amount of test item (25, 50, 100 & 200 mg) was added to the surface of 4,4,2,2 & 2L respectively of test water to give the 6.25, 12.5, 25, 50, & 100 mg/L loading rate WAF's..
- After addition of the test item, the test water was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface.
- Stirring was stopped after 23 hours and the mixture allowed to stand for one hour.
- 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 inserted into the glass tube and pushed through the Nescofilm seal.
-The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF.
- Microscopic inspection of the WAFs showed there to be no micro-dispersions or undissolved test item present.



Test organisms (species):
Daphnia magna
Details on test organisms:
- The test was carried out 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 2, 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 periods 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 so that young daphnids produced overnight were less than 24 hours old (see deviation).. 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:
None
Test temperature:
22°C
pH:
7.7 to 10 (see deviation)
Dissolved oxygen:
8.6 - 8.9 mg O2/L
Salinity:
Not applicable
Nominal and measured concentrations:
RANGE-FINDING TEST
- Nominal loading rates of 1.0, 10 and 100 mg/L

INITIAL TEST
- Nominal loading rate of 100mg/l


CONFIRMTORY TEST
- Nominal loading rate of 100mg/l

DEFINITIVE TEST
- Nominal loading rate of 6.25, 12.5, 25, 50 & 100 mg/L
Details on test conditions:
RANGE-FINDING TEST
- Daphnids (10) were placed in each test and control vessel and maintained at 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.
- Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation.
- After 24 and 48 hours, the number of immobilised daphnids were recorded.
- The control group was maintained under identical conditions but not exposed to the test item.

INITIAL TEST
- Daphnids (5) were placed in each test and control vessel and maintained at 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.
- Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation.
- After 24 and 48 hours, the number of immobilised daphnids were recorded.
- The control group was maintained under identical conditions but not exposed to the test item.

CONFIRMATORY TEST
- Daphnids (5) were placed in each test and control vessel and maintained at a temperature controlled room at 22°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.
- Each 150 mL test and control vessel contained 100 mL of test media and was covered to reduce evaporation.
- After 24 and 48 hours, the number of immobilised daphnids were recorded.
- The control group was maintained under identical conditions but not exposed to the test item

DEFINITIVE TEST
- 150 mL glass jars containing approximately 100 mL of test preparation were used.
- At the start of the test daphnids (5) were placed in each test and control vessel at random.
- Four replicate test and control vessels were prepared.
- Test vessels were covered to reduce evaporation and maintained in a temperature controlled room at approximately 21 °C with a photoperiod of 16 hours light (200 to 1200 lux) and 8 hours darkness with 20 minute dawn and dusk transition periods.
- 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.
- The test preparations were not renewed during the exposure period
Reference substance (positive control):
yes
Remarks:
potassium dichromate conducted between 23 February 2015 and 25 February 2015 (see Appendix 3, attached)
Key result
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
Key result
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
mobility
Details on results:
RANGE-FINDING TEST
- Cumulative immobilisation data and other observations from the exposure of Daphnia magna to the test item during the range-finding test are given in Table 1 (attached).
- No immobilisation was observed at 1.0, 10 and 100 mg/L loading rate WAF, however sub lethal effecs were observed in the control, 1.0 and 10mg/L Loading Rate WAF's.
- Sub lethal effects were observed at control, 1.0 and 10 mg/L loading rate WAFs.
- Sub lethal effects were trapping at the surface.
- These effects were considered to have had no effect on the study as no sub-lethal effects were observed in the 100mg/L loading rate WAF test concentration
- Based on this information, a nominal test concentration of four replicates of 100mg/L loading rate WAF was selected for the initial experiment. . This experimental design conforms to a 'limit test' to confirm that no immobilisation or adverse reactions to exposure were observed.
- Chemical analysis of the test preparations at 0 hours showed measured aluminium concentration to range from 0.064 to 0.28mg/L. Measured concentration in the range of less than the limit of quatification (LOQ) determined to be 0.031mg/L to 0.17mg/L at 48 hours.
.
INITIAL TEST
- Cumulative immobilisation data and other observations from the exposure of Daphnia magna to the test item during the initial test are given in Table 2 (attached).
- During the test the pH of the 100mg/L loading rate WAF at 0 hours was recorded at 9.21
- At 48 hours 70% immobilisation occured in the 100mg/L loading rate WAF
- Based on the results shown in the initial test it was decided to conduct a confirmatory experiment to ascertain if the high pH was the cause of toxicity.

CONFIRMATORY TEST
- Cumulative immobilisation data and other observations from the exposure of Daphnia magna to the test item during the initial test are given in Table 3 (attached).
- Significant immobilisation was observed at 100mg/L loading rate WAF.
- The initial pH of the 100mg/L loading rate WAF was recorded at 7.99 at 0 hours which was unlike the initial study.
- Test concentration of 6.25, 12.5, 25, 50 & 100mg/L loading rate WAF were selected for the definitive test.

DEFINITIVE TEST - CHEMICAL ANALYSIS OF TEST LOADING RATES
- Chemical analysis of the fresh test preparations at 0 hours showed a measured aluminium concentration of 0.326 mg/L.
- Chemical analysis of the aged test preparation at 48 hours showed a measured aluminium concentration of 0.312 mg/L.
- . Given that toxicity cannot be attributed to a single component or mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

DEFINITIVE TEST IMMOBILISATION DATA
- Cumulative immobilisation data and other observations from exposure of Daphnia magna to the test item during the definitive test are given in Table 4 (attached).
- A single immobilised daphnid was observed at 50mg/L loading rate WAF after 48-hours and was considered to be due to natural causes as less than 10% immobilization occurred.
- There was no immobilisation in 20 daphnids exposed to a 100 mg/L loading rate WAF for a period of 48 hours.
- One daphnid was observed to be immobilised in replicate 1 and was considered to be due to natural causes as less than 10% immobilization occurred.

DEFINITIVE TEST – Sub-Lethal Effects
-Sub-lethal effects of exposure were observed in the 12.5mg/L loading rate replicate 4. These responses were a single daphnid observed to be pale (see Table 4 attached).

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


DEFINITIVE TEST - WATER QUALITY CRITERIA
- The results of the water quality measurements are given in Table 5 (attached).
- Temperature was maintained at approximately 22 °C throughout the test.
- There were no treatment related differences for oxygen concentration
- There were concentration dependent difference in pH throughout the test which were outside of the guideline criteria. This was considered not to have had an adverse effect on the study as no immobilisation occured at 100mg/l loading rate WAF.

DEFINITIVE TEST - VORTEX DEPTH MEASUREMENTS
- The vortex depth recorded at the start and end of the mixing period was observed to be a dimple at the water surface on each occasion.

DEFINITIVE TEST - OBSERVATIONS ON TEST ITEM SOLUBILITY
- Observations were carried out during the mixing and testing of the WAF.
- At the start of the mixing period, the 6.25, 12.5, 25, 50 & 100 mg/L loading rate was observed to be a clear colourless water column with visible test item floating at the surface and through the water column..
- After 23 hours stirring and a 1 hour standing period, the 6.25, 12.5, 25, 50 &100 mg/L loading rate was observed to remain a clear colourless water column with test item on the surface and settled at the bottom.
- Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item present.
- After siphoning and during the test the 6.25, 12.5, 25, 50 & 100 mg/L loading rate was observed to be a clear, colourless solution.
























Validity criteria fulfilled:
yes
Conclusions:
The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated in accordance with OECD Guideline 202 and gave a 48-Hours EL50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.
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 complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test, and inital experiment, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at nominal loading rates of 6.25, 12.5, 25, 50 and 100 mg/L for 48 hours at a temperature of approximately 22°C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

RESULTS

Chemical analysis of the fresh test preparation at 0 hours showed a measured aluminium concentration of 0.326 mg/L. Chemical analysis of the aged test preparation at 48 hours showed a measured test concentration of 0.312 mg/L. The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

CONCLUSION

Exposure of Daphnia magna to the test item gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Description of key information

The acute toxicity of the test item to the freshwater invertebrate Daphnia magna has been investigated in accordance with OECD Guideline 202 and gave a 48-Hours EL50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Effect concentration:
100 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 complex nature of the test item, for the purposes of the test, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following a preliminary range-finding test,and inital experiment,twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (WAF) of the test item at nominal loading rates of 6.25, 12.5, 25, 50 and 100 mg/L for 48 hours at a temperature of approximately 22°C under static test conditions. Immobilization and any adverse reactions to exposure were recorded after 24 and 48 hours.

RESULTS

Chemical analysis of the fresh test preparation at 0 hours showed a measured aluminium concentration of 0.326 mg/L. Chemical analysis of the aged test preparation at 48 hours showed a measured test concentration of 0.312 mg/L.The dissolved test item may have been one or several components of the test item. Given that the toxicity cannot be attributed to a single component or mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.

CONCLUSION

Exposure of Daphnia magna to the test item gave EL50 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.