bis(branched-alkyl) 2-{[(1-phenylethyl)amino]methyl}alkanedioate

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:

24 December 2014 to 08 June 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

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 tability 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 1.0 mg/L loading rate WAF test group at 0 hours (taken from the bulk test preparation) and 48 hours (taken from the pooled replicates R1 to R4).
- 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
- Reconstituted water (ISO medium) used for both the range-finding and definitive test is defined in Appendix 4 (attached).

PROCEDURE
- Due to the low 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).

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 nominal loading rates of 0.10 amd 1.0 mg/L.
- A nominal amount of test item (5.0 mg) was added to the surface of 5.0 L of test water to give the 1.0 mg/L loading rate.
- After 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 were allowed to stand for one 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 or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0 mg/L loading rate WAF.
- A dilution was performed to give the 0.10 mg/L loading rate WAF.

DEFINITIVE TEST
- Based on the results of the range-finding test a "limit test" was conducted at a single loading rate of 1.0 mg/L.
- A nominal amount of test item (5.0 mg) were each separately added to the surface of 5.0 L of test water to give the 1.0 mg/L loading rate.
- After 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 were allowed to stand for one 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 or WAF was removed by mid-depth siphoning (the first approximate 75-100 mL discarded) to give the 1.0 mg/L loading rate WAF.

Test organisms (species):

Daphnia magna

Details on test organisms:

- The test was carried out using 1st instar Daphnia magna dervied 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 16 hours light and 8 hours darkness with 20 minute dawn and dusk 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 so that young daphnids produced overnight were less than 24 hours old. These young were removed from the cultures and used for testing.
- The diet and dilutent 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:

None

Hardness:

250 mg/L total theoretical hardness as CaCO3

Test temperature:

21 °C

pH:

7.6 to 7.7

Dissolved oxygen:

8.5 to 9.1 mg O2/L

Salinity:

Not applicable

Nominal and measured concentrations:

RANGE-FINDING TEST
- Nominal loading rates of 0.1 mg/L and 1.0 mg/L

DEFINITIVE TEST
- Nominal loading rate of 1.0 mg/L

Details on test conditions:

RANGE-FINDING TEST
- Daphnids (10) were placed in each test and control vessel and maintained at a temperature controlled room at 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 250 mL test and control vessel contained 200 mL of test media and was covered to reduce evaporation.
- After 24 and 48 hours, the number of immobilised Daphnia magna were recorded.
- The control group was maintained under identical conditions but not exposed to the test item.

DEFINITIVE TEST
- As in the range-finding test, 250 mL glass jars containing approximately 200 mL of test preparation were used.
- At the start of the test daphnids (5) were placed in each test and control vessel at random.
- Four replicate test and control vessels were prepared.
- Test vessels were covered to reduce evaporation and maintained in a temperature controlled room at 21 °C with a photoperiod of 16 hours light (342 to 407 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.

Reference substance (positive control):

yes

Remarks:

potassium dichromate conducted between 23 February 2015 and 25 February 2015 (see Appendix 3, attached)

Key result

Duration:

24 h

Dose descriptor:

EL50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

EL50

Effect conc.:

> 1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

24 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Key result

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

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 immobilisation was observed at 0.10 and 1.0 mg/L loading rate WAF.
- At the 24 hour time point, 6 out of 10 daphnids in the 1.0 mg/L loading rate were observed to be trapped at the surface and, at the 48 hour time point, 1 daphnid in the control group and one in the 1.0 mg/L loading rate were observed to be trapped at the surface.
- Based on this information, a single loading rate of 1.0 mg/L (four replicates) was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no immobilisation or adverse effects to exposure were observed.
- Due to instumentation technical difficulties, no results were available for the range-finding samples. This was considered not to have affected the outcome of the study.

DEFINITIVE TEST - CHEMICAL ANALYSIS OF TEST LOADING RATES
- Chemical analysis of the fresh 1.0 mg/L loading rate test preparation at 0 hours showed that a measured test concentration of 0.244 mg/L was obtained (see Appendix 6, attached).
- Chemical analysis of the aged test preparation at 48 hours showed a measured test concentration of 0.0989 mg/L (see Appendix 6, attached).

DEFINITIVE TEST IMMOBILISATION DATA
- Cumulative immobilisation data and other observations from 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 1.0 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 1.0 mg/L.

DEFINITIVE 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.

DEFINITIVE TEST - WATER QUALITY CRITERIA
- The results of the water quality measurements are given in Table 3 (attached).
- Temperature was maintained at 21 °C throughout the test.
- There were no treatment related differences for oxygen concentration or pH.

DEFINITIVE TEST - VORTEX DEPTH MEASUREMENTS
- The vortex depth recorded at the start and end of the mixing period was observed to be a dimple at the water surface on each occasion.

DEFINITIVE TEST - OBSERVATIONS ON TEST ITEM SOLUBILITY
- At the start of the mixing period, the 1.0 mg/L loading rate was observed to be a clear colourless water column with an oily layer of test item on the surface.
- After 24 hours stirring and a 1 hour standing period, the 1.0 mg/L loading rate was observed to be as at the start of stirring.
- 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 1.0 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 Appendix 3, attached).

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to the test item gave EL50 values of greater than 1.0 mg/L loading rate rate WAF. The No Observed Effect Loading Rate was 1.0 mg/L loading rate WAF. This study showed that the test item was not toxic to Daphnia magna at a concentration in excess of the water solubility.

Executive summary:

GUIDELINE

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method 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 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 Accomodated Fraction (WAF) of the test item at a nominal loading rate of 1.0 mg/L for 48 hours at a temperature of approximately 21°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 fresh 1.0 mg/L loading rate test preparation at 0 hours showed that a measured test concentration of 0.244 mg/L was obtained. Chemical analysis of the aged test preparation at 48 hours showed a measured test concentration of 0.0989 mg/L was obtained.

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 1.0 mg/L loading rate rate WAF. The No Observed Effect Loading Rate was 1.0 mg/L loading rate WAF. This study showed that the test item was not toxic to Daphnia magna at a concentration in excess of the water solubility.

Description of key information

Exposure of Daphnia magna to the test item gave EL50 values of greater than 1.0 mg/L loading rate rate WAF. The No Observed Effect Loading Rate was 1.0 mg/L loading rate WAF. This study showed that the test item was not toxic to Daphnia magna at a concentration in excess of the water solubility (OECD 202 and EU Method C.2).

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1 mg/L

Additional information

GUIDELINE

A study was performed to assess the acute toxicity of the test item to Daphnia magna. The method 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 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 Accomodated Fraction (WAF) of the test item at a nominal loading rate of 1.0 mg/L for 48 hours at a temperature of approximately 21°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 fresh 1.0 mg/L loading rate test preparation at 0 hours showed that a measured test concentration of 0.244 mg/L was obtained. Chemical analysis of the aged test preparation at 48 hours showed a measured test concentration of 0.0989 mg/L was obtained.

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 1.0 mg/L loading rate rate WAF. The No Observed Effect Loading Rate was 1.0 mg/L loading rate WAF. This study showed that the test item was not toxic to Daphnia magna at a concentration in excess of the water solubility.