Registration Dossier

Ecotoxicological information

Short-term toxicity to aquatic invertebrates

Currently viewing:

Administrative data

Link to relevant study record(s)

Referenceopen allclose all

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 June 2015 to 15 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:
Elendt M7 used as dilution water in range-finding test instead of ISO reconstituted water with no impact on results or integrity of the study
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
Elendt M7 used as dilution water in range-finding test instead of ISO reconstituted water with no impact on results or integrity of the study
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Range-finding test: A sample of each loading rate WAF was taken for chemical analysis at 0 and 48 hours in order to determine the stability of the test item under test conditions. All samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analysed.
- Definitive test: Samples were taken from the control and 100 mg/L loading rate W AF test group from the bulk test preparation at 0 hours and from the pooled replicates (replicates R1 to R4) at 48 hours for quantitative analysis. Samples were stored frozen prior to analysis. Duplicate samples were taken and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
TEST WATER
- Elendt M7 medium was used in the range-finding test (see Appendix 2, attached).
- Reconstituted water (ISO medium) was used for the definitive test and is defined in Appendix 4 (attached).
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST SYSTEM
- 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 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 (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 are considered not to contain any contaminant that would affect the integrity or outcome of the study.
- A positive control used potassium dichromate as the reference item. Details of the positive control are given in Appendix 3 (attached). The positive control was conducted between 23 February 2015 and 25 February 2015.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
48 h
Post exposure observation period:
Not applicable
Hardness:
Reconstituted water had an approximate theoretical total hardness of 250 mg/L as CaC03 (see Appendix 4, attached)
Test temperature:
21 to 22 °C (see Table 3, attached)
pH:
7.8 to 8.0 (see Table 3, attached)
Dissolved oxygen:
8.4 to 9.0 mg O2/L (see Table 3, attached)
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
Nominal concentration of 100 mg/L loading rate WAF
Details on test conditions:
PROCEDURE
- Due to the low aqueous solubility and complex nature of the test item, for the purposes of the study 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
- The loading rate 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 loading rates of 1.0, 10 and 100 mg/L.
- Prior to addition of the test item a glass siphon tube was placed in the test media. Nominal amounts of test item (10, 20 and 200 mg) were each separately added to the surface of 10, 2 and 2 L of test water to give the 1.0, 10 and 100 mg/L loading rates respectively. After the 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. The stirring was stopped after 95 hours and the mixtures allowed to stand for 1 hour. A length of Tygon tubing was attached to the top of the glass siphon tube. Microscopic observations made on the WAFs indicated that dispersed test item was present in the 10 and 100 mg/L loading rate water columns and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2 to 4 cm in length, the first approximate 75-100 mL discarded). Microscopic observations of the WAFs were performed after filtering and showed no micro-dispersions to be present, however, visual observation of the 10 mg/L loading rate WAF indicated that the test media was cloudy therefore it was filtered through postlip filter paper.
- In the range-finding test 10 daphnids were placed in each test and control vessel and maintained in a temperature controlled room at approximately 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.

DEFINITIVE TEST
- Based on the results of the range-finding test a "limit test" was conducted at a loading rate of 100 mg/L to confirm that at highest attainable test concentration no immobilization or adverse reactions to exposure were observed.
- Prior to addition of the test item a glass siphon tube was placed in the test media. A nominal amount of test item (200 mg) was added to the surface of 2 L of test water to give the 100 mg/L loading rate. After the 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. The stirring was stopped after 95 hours and the mixture allowed to stand for 1 hour. Microscopic observations made on the W AF indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAF by filtering through a glass wool plug (2 to 4 cm in length). A length of Tygon tubing was attached to the top of the glass siphon tube. A glass wool plug was inserted into the opposite end of the tubing and the WAF removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 100 mg/L loading rate WAF. Microscopic observations of the W AF were performed after filtering through glass wool and showed test item to be present, therefore the WAF was then passed through postlip filter paper in order to ensure removal of undissolved test item.
- As in the range-finding test 150 mL glass beakers containing approximately 100 mL of test preparation were used. At the start of the test 5 daphnids were placed in each test and control vessel at random, in the test preparations. 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 with a photoperiod of 16 hours light (between 200 and 1200 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.
- The test preparations were not renewed during the exposure period.

TEST ORGANISM OBSERVATIONS
- Any immobilisation 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 immobilised if they were unable to swim within 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.
- The pH and dissolved oxygen concentration were measured using a Hach Flexi handheld meter whilst the temperature was measured using a Hanna Instruments HI 93510 digital thermometer.
- The light intensity during the light period was measured using an ATP Instruments Lux meter.

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

VALIDATION CRITERIA
- The results of the test are considered valid if the following performance criteria are met:
(i) No more than 10% of the control daphnids show immobilisation or other signs of disease or stress (e.g. discoloration or unusual behaviour such as trapping at the surface water).
(ii) The dissolved oxygen concentration at the end of the test is equal to or greater than 3 mg/L in the control and test vessels.
Reference substance (positive control):
yes
Remarks:
potassium dichromate (see Appendix 3, attached)
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
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.
Remarks:
loading rate WAF
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
mobility
Details on results:
VALIDATION OF MIXING PERIOD
- Preliminary investigational work (see Appendix 5, attached) indicated that there was a significant increase in the amount of dissolved test item when the preparation period was extended from 23 to 95 hours. Therefore, for the purpose of testing the W AF was prepared using a stirring period of 95 hours followed by a 1-hour settlement period.

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.
- Based on this information, a single loading rate of four replicates, of 100 mg/L, was selected for the definitive test. This experimental design conforms to a "Limit test" to confirm that no immobilization or adverse reactions to exposure were observed.
- Chemical analysis of the 100 mg/L loading rate WAF test preparation at 0 hours (see Appendix 6, attached) showed a measured test concentration of 1.2 mg/L. There was a significant decline in the measured concentration at 48 hours to 0.50 mg/L indicating that the test item was not stable under test conditions.

DEFINITIVE TEST
- Chemical analysis of the fresh test preparation at 0 hours (see Appendix 6) showed the measured test concentration to be 0.19 mg/L. Chemical analysis of the aged test preparation at 48 hours showed a measured test concentration of less than the limit of quantification (LOQ) of the analytical method employed was obtained which was determined to be 0.043 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 a mixture of components, but to the test item as a whole, the results were based on nominal loading rates only.
- Cumulative immobilisation data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in Table 2 (attached).
- There was no immobilisation in 20 daphnids exposed to a 100 mg/L loading rate WAF for a period of 48 hours.
- It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.
- No sub-lethal effects of exposure were observed throughout the 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 equal to or greater than 3 mg/L in the control and test vessels.

WATER QUALITY CRITERIA
- The results of the water quality measurements are given in Table 3 (attached)
- Temperature was maintained at 21 to 22 °C throughout the test.
- There were no treatment related differences for oxygen concentration or pH.
- Throughout the test the light intensity was observed to be in the range 626 to 657 lux.

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 the mixing and testing of the WAF.
- At the start of the mixing period the 100 mg/L loading rate was observed to be clear colourless water column with globules of test item on the surface and dispersed throughout.
- After 95 hours stirring and a 1-hour standing period the 100 mg/L loading rate was observed to be a clear colourless water column with globules of test item on the surface. Microscopic examination of the WAF showed micro-dispersions of test item to be present and therefore it was considered justifiable to remove the W AF by filtering through a glass wool plug (2 to 4 cm in length). Microscopic examination after filtering showed the glass wool plug had not removed all the micro dispersions and the WAF was further filtered through postlip filter paper.
- At the start and throughout the test all control and test solutions were observed to be clear colourless solutions.
Results with reference substance (positive control):
- Results from the positive control with potassium dichromate were within the normal range for this reference item (see Appendix 3, attached)
Reported statistics and error estimates:
STATISTICAL ANALYSIS
- An estimate of the EL50 values was given by inspection of the immobilisation data.
Validity criteria fulfilled:
yes
Conclusions:
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. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.
Executive summary:

GUIDELINE

A study was performed to assess the acute toxicity of the test item toDaphnia magna.The method followed was designed to be compatible with the OECD Guidelines for Testing of Chemicals (April 2004) No 202,"Daphniasp., 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 (W AF). Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (W AF) of the test item at a nominal loading rate of 100 mg/L for 48 hours at a temperature of 21 to 22 °C under static test conditions. Immobilisation 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 the measured test concentration to be 0.19 mg/L. Chemical analysis of the aged test preparation at 48 hours showed a measured test concentration of less than the limit of quantification (LOQ) of the analytical method employed was obtained which was determined to be 0.043 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 a 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. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.

Endpoint:
short-term toxicity to aquatic invertebrates
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Justification for type of information:
See read-across justification attached in Section 13.
Reason / purpose for cross-reference:
read-across source
Duration:
24 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
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.
Remarks:
loading rate WAF
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
mobility

Description of key information

Exposure of Daphnia magna to an analogue 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. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L (OECD 202 and EU Method C.2).

Key value for chemical safety assessment

Additional information

GUIDELINE

A study was performed to assess the acute toxicity of an analogue 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,"Daphniasp., 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 (W AF). Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fraction (W AF) of the test item at a nominal loading rate of 100 mg/L for 48 hours at a temperature of 21 to 22 °C under static test conditions. Immobilisation 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 the measured test concentration to be 0.19 mg/L. Chemical analysis of the aged test preparation at 48 hours showed a measured test concentration of less than the limit of quantification (LOQ) of the analytical method employed was obtained which was determined to be 0.043 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 a 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. It was considered unnecessary and unrealistic to test at concentrations in excess of 100 mg/L.