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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:
27 March 2017 to 30 March 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:
yes
Remarks:
water quality measurements taken from an additional replicate with no impact on results or integrity of the study (see below)
Qualifier:
according to
Guideline:
EU Method C.2 (Acute Toxicity for Daphnia)
Deviations:
yes
Remarks:
water quality measurements taken from an additional replicate with no impact on results or integrity of the study (see below)
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Remarks:
Total Organic Carbon (TOC) analysis
Details on sampling:
TOTAL ORGANIC CARBON ANALYSIS OF TEST LOADING RATES
- Samples were taken from the bulk test preparation of the control and each loading rate WAF at 0 hours.
- Samples were also taken from the pooled replicates (replicates R1-R4) at 48 hours.
- Samples were analysed immediately after collection.
- Duplicate samples were taken and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
TEST WATER
- Reconstituted water (ISO medium) used for both the range-finding and definitive tests is defined in Annex 2 (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.
- A study to determine the general physico-chemical properties of the test item indicated the water solubility of the test item was less than 1.0 mg C/L. Given this it was considered appropriate to test up to a maximum loading rate of 50 mg/L to prevent overloading of the aqueous phase with undissolved test item.
- A 23-Hour stirring period, followed by a 1-hour standing period was deemed sufficient to ensure equilibration between the test item and aqueous phase.
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
Post exposure observation period:
Not applcable
Hardness:
Not reported
Test temperature:
21 to 22 °C
pH:
7.6 to 7.7 (see Table 2, attached)
Dissolved oxygen:
7.9 to 8.7 mg O2/mL (see Table 2, attached)
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
- Nominal loading rates of 5.0 and 50 mg/L
Details on test conditions:
DEFINITIVE TEST
- Nominal amounts of test item (10 and 100 mg) were each separately added to the surface of 2 L of test water to give the 5.0 and 50 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 23 hours and the mixtures allowed to stand for 1 hour. Microscopic 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 5.0 and 50 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed no undissolved test item remained in the media.
- The carbon concentration in the test preparations were verified by Total Organic Carbon (TOC) analysis at 0 and 48 hours (see Annex 4, attached).

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.

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
Key result
Duration:
48 h
Dose descriptor:
EL50
Effect conc.:
> 50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat. (total fraction)
Remarks:
loading rate WAF
Basis for effect:
mobility
Duration:
48 h
Dose descriptor:
NOELR
Effect conc.:
50 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
mobility
Details on results:
DEFINITIVE TEST
- Total Organic Carbon analysis of the 5.0 and 50 mg/L loading rate WAF fresh test preparations at 0 hours showed measured carbon concentrations of 1.6 to 1.4 mg/L, respectively.
- Analysis of the 5.0 and 50 mg/L aged test preparations at 48 hours showed measured carbon concentrations of less than the limit of quantification (LOQ) of the method employed, which was determined to be 1.0 mg/L.
- Analysis of the control samples at 0 and 48 hours showed carbon concentrations of less than the LOQ (see Annex 4, attached).
- 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 immobilization 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 immobilization in 20 daphnids exposed to a 50 mg/L loading rate WAF for a period of 48 hours.
- Exposure of Daphnia magna to the test item gave an EL50 value of greater than 50 mg/L loading rate WAF. The No Observed Effect Loading Rate was 50 mg/L loading rate WAF.

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 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 598 to 511 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 WAFs.
- At the start of the mixing period the 5.0 and 50 mg/L loading rates were observed to be clear colourless water columns with particles of non-homogenized waxy test item on the surface. After 23 hours stirring and a 1-Hour standing period the 5.0 and 50 mg/L loading rates were observed to be clear colourless water columns with particles of non-homogenized test item on the surface. Microscopic inspection of the WAFs showed undissolved test item to be present and therefore it was considered justifiable to remove the WAFs by filtering through a glass wool plug (2-4 cm in length). Microscopic examination after filtering showed the glass wool plug had removed all of 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 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 greater than 50 mg/L loading rate WAF. The No Observed Effect Loading Rate was 50 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 50 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 complex nature of the test item for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF). A study to determine the general physico-chemical properties of the test item indicated the water solubility of the test item was less than 1.0 mg C/L. Given this it was considered appropriate to test up to a maximum loading rate of 50 mg/L to prevent overloading of the aqueous phase with undissolved test item. 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 5.0 and 50 mg/L for 48 hours at a temperature of 21 to 22 °C under static test conditions. The number of immobilized daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

RESULTS

Total Organic Carbon analysis of the 5.0 and 50 mg/L loading rate WAF fresh test preparations at 0 hours showed measured carbon concentrations of 1.6 to 1.4 mg/L, respectively. Analysis of the 5.0 and 50 mg/L aged test preparations at 48 hours showed measured carbon concentrations of less than the limit of quantification (LOQ) of the method employed, which was determined to be 1.0 mg/L. Analysis of the control samples at 0 and 48 hours showed carbon concentrations 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 greater than 50 mg/L loading rate WAF. The No Observed Effect Loading Rate was 50 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 50 mg/L.

Description of key information

Exposure of Daphnia magna to the test item gave EL50 values of greater than 50 mg/L loading rate WAF. The No Observed Effect Loading Rate was 50 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 50 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,"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 item was prepared as a Water Accommodated Fraction (WAF). A study to determine the general physico-chemical properties of the test item indicated the water solubility of the test item was less than 1.0 mg C/L. Given this it was considered appropriate to test up to a maximum loading rate of 50 mg/L to prevent overloading of the aqueous phase with undissolved test item. 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 5.0 and 50 mg/L for 48 hours at a temperature of 21 to 22 °C under static test conditions. The number of immobilized daphnia and any adverse reactions to exposure were recorded after 24 and 48 hours.

 

RESULTS

Total Organic Carbon analysis of the 5.0 and 50 mg/L loading rate WAF fresh test preparations at 0 hours showed measured carbon concentrations of 1.6 to 1.4 mg/L, respectively. Analysis of the 5.0 and 50 mg/L aged test preparations at 48 hours showed measured carbon concentrations of less than the limit of quantification (LOQ) of the method employed, which was determined to be 1.0 mg/L. Analysis of the control samples at 0 and 48 hours showed carbon concentrations 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 greater than 50 mg/L loading rate WAF. The No Observed Effect Loading Rate was 50 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 50 mg/L.