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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:
09 May 2017 to 02 June 2017
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
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
DEFINITIVE TEST
- Samples were taken from the bulk test preparation of the control and each test group at 0 hours.
- Samples were also taken from the pooled 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.
- Only samples at the No Observed Effect Loading Rate and above were analysed.
Vehicle:
no
Details on test solutions:
TEST WATER
- Reconstituted water (Elendt M7 Medium) used for the range-finding test is defined in Annex 2 (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 Annex 2, attached) in a temperature controlled room maintaining the water temperature at 18 to 22 °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.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
48 h
Hardness:
Not reported
Test temperature:
20 to 22 °C (see Table 2, attached)
pH:
7.8 to 8.0 (see Table 2, attached)
Dissolved oxygen:
8.5 to 8.6 mg O2/L (see Table 2, attached)
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
- Nominal loading rates of 1.0, 10 and 100 mg/L
Details on test conditions:
EXPERIMENTAL DESIGN AND STUDY CONDUCT
- 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 Annex 4, attached) was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.

DEFINITIVE 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 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.
- Stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour.
- Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length).
- 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. 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 1.0, 10 and 100 mg/L loading rate WAFs.
- Microscopic observations of the WAFs were performed after filtering and showed no micro dispersions of test item to be present.

EXPOSURE CONDITIONS
- In the definitive test 250 mL glass jars containing approximately 200 mL of test preparation were used. At the start of the test five 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 maintaining the water temperature at 18 to 22 °C with a maximum deviation of ± 1 °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.
- 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.

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 for approximately 15 seconds after gentle agitation.

WATER QUALITY CRITERIA
- Water temperature was recorded daily throughout the test using a Hanna Instruments H93510 digital thermometer.
- Dissolved oxygen concentrations and pH were recorded at the start and termination of the test using a Hach Flexi handheld meter.
- Light intensity during the light period was measured using an ATP Instruments Lux meter.
- The appearance of the test media was recorded daily.

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

VALIDATION CRITERIA
- Results of the test are considered valid if the following performance criteria are met:
(a) 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).
(b) The dissolved oxygen concentration at the end of the test is ≥ 3 mg/L in the control and test vessels.

MAJOR COMPUTERISED SYSTEMS
- Building management: Delta Control System
Reference substance (positive control):
yes
Remarks:
potassium dichromate (study conducted between 20 October 2016 and 22 October 2016)
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:
CHEMICAL ANALYSIS OF TEST LOADING RATES
- Chemical analysis of the 100 mg/L loading rate WAF test preparations at 0 and 48 hours (see Annex 5, attached) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.10 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.
- 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.

IMMOBILISATION DATA
- Cumulative immobiliSation data and other observations from the exposure of Daphnia magna to the test item during the definitive test are given in Table 1 (attached).
- There was no immobilisation in 20 daphnids exposed to a 100 mg/L loading rate WAF for a period of 48 hours.
- 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 loading rates in excess of 100 mg/L.
SUB-LETHAL EFFECTS
- 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 2 (attached). Temperature was maintained at 20 °C to 22 ºC throughout the test, while there were no treatment related differences for oxygen concentration or pH.
- Throughout the test the light intensity was observed to be in the range 566 to 691 lux.

VORTEX DEPTH MEASURMENTS
- 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 WAFs.
- At the start of the mixing period the 1.0, 10 and 100 mg/L loading rates were observed to be clear colourless water columns with oily globules of test item at the surface.
- After 23 hours stirring and a 1-hour standing period the 1.0 mg/L loading rate was observed to have a small amount of translucent solidified test item suspended in the column and some translucent solidified test item at the water surface.
- The 10 and 100 mg/L loading rates were observed to have translucent solidified test item suspended in the column and translucent solidified test item at the water surface, with the amount of test item increasing with increasing concentration.
- Visual examination of the WAFs showed a significant amount of undissolved test item suspended in the water columns and therefore it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length) with further filtration through 1 sheet of post-lip filter paper. Microscopic examination after filtering showed the glass wool plug and filter paper had removed all the undissolved test item.
- 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):
- The results from the positive control with potassium dichromate were within the normal range for this reference item (see Annex 3, attached).
Reported statistics and error estimates:
STATISTICAL ANALYSIS
- An estimate of the EL50 values was given by inspection of 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 item was prepared as a Water Accommodated Fraction (WAF). Twenty daphnids (4 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 10 and 100 mg/L for 48 hours at a temperature of 20 to 22 °C under static test conditions. The number of immobilised daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

RESULTS

Chemical analysis of the 100 mg/L loading rate WAF test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.10 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ. 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.

Description of key information

Exposure of Daphnia magna to the test item gave EL50 values > 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 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 item was prepared as a Water Accommodated Fraction (WAF). Twenty daphnids (4 replicates of 5 animals) were exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 10 and 100 mg/L for 48 hours at a temperature of 20 to 22 °C under static test conditions. The number of immobilised daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

RESULTS

Chemical analysis of the 100 mg/L loading rate WAF test preparations at 0 and 48 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.10 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ. 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.