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Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 January 2017 to 10 February 2017

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:

preliminary investigation treated as limit test (see below)

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

yes

Remarks:

preliminary investigation treated as limit test (see below)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Remarks:

Total Organic Carbon (TOC) analysis

Details on sampling:

CHEMICAL ANALYSIS OF TEST LOADING RATES
- Despite extensive method development work having been conducted, a method of analysis could not be found that could detect measurable concentrations of dissolved test item in a WAF preparation.
- It was therefore considered acceptable to provide samples for Total Organic Carbon (TOC) analysis only.

TOTAL ORGANIC CARBON ANALYSIS
- Water samples were taken from the control and each test group at 0 and 48 hours.
- Samples were stored frozen prior to analysis.
- Duplicate samples were also taken and stored frozen for further analysis, if necessary.
- Only samples at the No Observed Effect Loading Rate 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:

yes

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Hardness:

Not reported

Test temperature:

21 to 22 °C (see Table 2, attached)

pH:

pH 7.9 to 8 (see Table 2, attached)

Dissolved oxygen:

8.4 to 8.8 mg O2/L (see Table 2, attached)

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

- Nominal concentrations of 1.0, 10 and 100 mg/L as loading rate WAFs

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.
- Samples were also provided for Total Organic Carbon (TOC) analysis as a surrogate measure.

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.
- 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. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 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 inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
- The aqueous phase (WAF) was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0, 10 and 100 mg/L loading rate WAFs.

EXPOSURE CONDITIONS
- In the range-finding test, glass beakers (150 mL) containing test preparation (100 mL) were used.
- At the start of the test, five daphnids were placed into test preparation in each of the test and control vessels at random.
- Four replicate test and control vessels were prepared, covered to reduce evaporation, and kept in a temperature controlled room for 48 hours.
- Water temperature was maintained at 18 to 22 °C with a maximum deviation of ±1 °C.
- The photoperiod was 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 to test item and the test vessels were not aerated.
- Test preparations were not renewed during the exposure period.
- The control group was maintained under identical conditions but not exposed to the test item.

Reference substance (positive control):

yes

Remarks:

potassium dichromate used as reference item in study conducted between 20 October 2016 and 22 October 2016 (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:

VALIDATION OF MIXING PERIOD
- Preliminary investigation (see Annex 4, attached) indicated that there was no significant increase in the amount of dissolved test item or total organic carbon when the preparation period was extended for longer than 24 hours.
- The WAF was therefore prepared for testing using a stirring period of 23 hours followed by a 1-hour settlement period.

RANGE FINDING TEST
- Total Organic Carbon (toc) analysis of the test preparations performed at 0 and 48 hours (see Annex 5, attached) showed no significant differences in the amount of carbon present within the 100 mg/L loading rate WAF test vessels when compared to control vessels. Therefore, given the background level of carbon in the control vessels and the low level of carbon in the test vessels, it was considered that all the results were around the limit of quantification of the analytical method.
- The TOC results were consistent with information provided from a study to determine physico-chemical properties of the test item, which indicated the water solubility to be < 1 mg C/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.

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 loading rates of 1.0, 10 and 100 mg/L for a period of 48 hours.
- Exposure of Daphnia magna to the test item gave an EL50 value of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 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 approximately 20 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 404 to 595 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 1.0, 10 and 100 mg/L loading rates were observed to be clear colourless water columns with globules of test item on the surface. After 23 hours stirring and a 1-hour standing period the test groups remained the same. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
- 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 the 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.

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). 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 21 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

Total Organic Carbon analysis of the test preparations performed at 0 and 48 hours showed no significant differences in the amount of carbon present within the 100 mg/L loading rate WAF test vessels when compared to control vessels. Therefore, given the background level of carbon in the control vessels and the low level of carbon in the test vessels, it was considered that all the results were around the limit of quantification of the analytical method.

 

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

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 (OECD 202 and EU Method C.2).

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 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 21 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

Total Organic Carbon analysis of the test preparations performed at 0 and 48 hours showed no significant differences in the amount of carbon present within the 100 mg/L loading rate WAF test vessels when compared to control vessels. Therefore, given the background level of carbon in the control vessels and the low level of carbon in the test vessels, it was considered that all the results were around the limit of quantification of the analytical method.

 

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.