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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:
25 April 2016 to 25 May 2016
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:
test temperature deviation with no impact on results or integrity of the study (see below)
Qualifier:
according to guideline
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
test temperature deviation with no impact on results or integrity of the study (see below)
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 from the 100 mg/L loading rate WAF test group at 0 hours.
- Samples were also taken from the pooled replicates (replicates R1-R4) at 24 and 48 hours.
- Samples were stored frozen prior to analysis.
- Duplicate samples were taken and stored frozen for further analysis if necessary.
Vehicle:
no
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.
- 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:
yes
Total exposure duration:
48 h
Post exposure observation period:
Not applicable
Hardness:
Approximate theoretical total hardness of 250 mg/L as CaCO3 (see Annex 4, attached)
Test temperature:
19 to 22 °C (see Table 3, attached)
pH:
7.9 to 8.2 (see Table 3, attached)
Dissolved oxygen:
8.6 to 8.7 mg/L (see Table 3, attached)
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
RANGE FINDING TEST
- Nominal loading rates of 1.0, 10 and 100 mg/L

DEFINITIVE TEST
- Nominal loading rate of 100 mg/L
Details on test conditions:
TEST WATER
- Reconstituted water (ISO medium) used for both the range-finding and definitive tests is defined in Annex 4 (attached).

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 5, attached) was carried out to determine whether stirring for a prolonged period produced significantly higher measured test concentrations in the WAF.

EXPOSURE CONDITIONS
- In the definitive test, 150 mL glass jars 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 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. 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.

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.

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 immobilization 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 07 September 2015 and 23 September 2015; see Annex 3, attached)
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:
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 immobilization was observed at 10 and 100 mg/L loading rate WAF. A single immobilized daphnia was observed in the 1.0 mg/L loading rate, however, as no more than 10% was observed, this was considered to be due to natural causes and considered not to have had an impact on the outcome of the test.
- 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 test preparations at 0 and 48 hours (see Annex 6) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.0088 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.

DEFINITIVE TEST
- Chemical analysis of the test preparations at 0 and 48 hours (see Annex 6, 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 0.0088 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.
- 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.

IMOBILISATION 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 2 (attached).
- No immobilisation was observed in 20 daphnids exposed to a 100 mg/L loading rate WAF for a period of 48 hours.
- Inspection of immobilisation data gave EL50 (24h) > 100 mg/L loading rate WAF and EL50 (48h) > 100 mg/L loading rate WAF. The No Observed Effect Loading rate after 24 and 48 hours exposure 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 ≥ 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 19 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 527 to 552 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 a clear colourless water column with long waxy strings of yellow test item on the surface.
- After 23 hours stirring and a 1-hour standing period the 100 mg/L loading rate was observed to be a clear colourless water column with strings of test item of a yellow waxy nature on the surface.
- Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
- After siphoning and for the duration of the test, the 100 mg/L loading rate was observed to be a clear, colourless solution.
Results with reference substance (positive control):
- Results from the positive control with potassium dichromate were within the normal range for the reference item (see Annex 3, attached)
Reported statistics and error estimates:
- 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 > 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 (WAF). Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fractions (WAF) of the test item at a nominal loading rate of 100 mg/L for 48 hours at a temperature of 19 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 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 0.0088 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. 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 > 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 of > 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

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 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 (WAF). Following a preliminary range-finding test, twenty daphnids (4 replicates of 5 animals) were exposed to a Water Accommodated Fractions (WAF) of the test item at a nominal loading rate of 100 mg/L for 48 hours at a temperature of 19 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 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 0.0088 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. 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 > 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.