Tall Oil Fatty Acids, compounds with 2-(2-aminoethoxy) ethanol

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:

21 May 2015 to 11 December 2015

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:

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 TESTS
- 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.
- Due to a technical error during analysis of the range finding samples, no results were obtained.
- In order to determine the stability of the test item under test conditions, concentrations within the range to be used in the definitive test were prepared as in the range finding test and samples provided for analysis.

DEFINITIVE TEST
- Samples were taken from the control and each loading rate WAF test goup at 0 hours (taken from bulk test preparation) and 48 hours (replicates R1 to R4 pooled) 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 (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 TESTS
- Nominal amounts of test item (20 and 200 mg) were each separately added to the surface of 2 L of test water to give the 10 and 100 mg/L loading rates respectively.
- 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.
- Visual observations made on the 100 mg/L loading rate WAF indicated that 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 to 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).
- Undissolved test item was observed to be present in the WAFs after passing through a glass wool plug and hence it was considered appropriate to pass the WAFs through a glass wool plug once again followed by twice through Postlip filter paper to remove as much undissolved test item as possible.
- Observations made on the filtrate showed that undissolved test item still remained and it was considered that further filtration at this point would not have removed any more of the dispersed test item present.
- The aqueous phase or WAF was removed by mid-depth siphoning to give the 10 and 100 mg/L loading rate WAFs.
- Given that 100 % immobilisation was observed in both the 10 and 100 mg/L loading rates, it was considered appropriate to conduct a second range finding test at nominal loading rates of 0.10 and 1.0 mg/L.
- Due to the need to test at relatively low concentrations, the 0.10 mg/L loading rate was prepared as a dilution of the 1.0 mg/L loading rate WAF.
- A nominal amount of test item (5.0 mg) was added to the surface of 5 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.
- Visual observations made on the 100 mg/L loading rate WAF indicated that dispersed test item was present in the water column and hence it was considered justifiable to remove the WAFs by filtration.
- 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 observations of the WAF were performed after filtering and 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.
- The 0.10 mg/L loading rate was produced by dilution of the 1.0 mg/L loading rate WAF.

DEFINITIVE TEST
- Nominal amounts of test item (5.0, 16 and 50 mg) were each separately added to the surface of 5 L of test water to give the 1.0, 3.2 and 10 mg/L loading rates respectively.
- 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.
- Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water columb of the 10 mg/L loading rate and it was considered justifiable to remove this WAF by filtering through a glass wool plug (2 to 4 cm in length). As a precaution, the 3.2 mg/L loading rate was also filtered through a glass wool plug.
- 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 was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 3.2 and 10 mg/L loading rate WAFs.
- Microscopic observations of the WAFs were performed after filtering and showed that no undissolved test item was present in the media.
- The 1.0 mg/L loading rate was removed in the same way but without the addition of the glass wool filtration.
- The 0.10 and 0.32 mg/L loading rates were produced by dilution of the 1.0 mg/L loading rate.

Test organisms (species):

Daphnia magna

Details on test organisms:

- 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 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 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:

None

Hardness:

250 mg/L total theoretical hardness as CaCO3

Test temperature:

21 to 22 °C

pH:

7.8 to 8.1

Dissolved oxygen:

8.6 to 9.0 mg O2/L

Salinity:

Not applicable

Nominal and measured concentrations:

FIRST RANGE FINDING TEST
- Nominal loading rates of 10 and 100 mg/L.

SECOND RANGE FINDING TEST
- Nominal loading rates of 0.10 and 1.0 mg/L.

DEFINITIVE TEST
- Nominal loading rates of 0.10, 0.32, 1.0, 3.2 and 10 mg/L.

Details on test conditions:

RANGE-FINDING TESTS
- Daphnids (10) were placed in each test and control vessel and maintained at a temperature controlled room at 21 to 22 °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
- 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:

48 h

Dose descriptor:

EL50

Effect conc.:

1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: 95% confidence limits 1.4 to 2.0 mg/L loading rate WAF

Duration:

48 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

LOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

RANGE-FINDING TESTS
- Cumulative immobilisation data and other observations from the exposure of Daphnia magna to the test item during the range-finding tests are given in Table 1 (attached).
- No immobilisation was observed at 0.10 mg/L loading rate WAF. However, immobilisation was observed at 1.0, 10 and 100 mg/L loading rate WAF.
- Based on this information loading rates of 0.10, 0.32, 1.0, 3.2 and 10 mg/L were selected for the definitive test.
- In the initial range-finding test, the 10 and 100 mg/L loading rate WAF test preparations were observed to be hazy dispersions after filtration. As a result, microscopic observations were conducted on the immobilised Daphnia, which showed that globules of test item were present under the carapaces.
- Chemical analysis of the stability samples at 0 hours (see Appendix 6, attached) gave measured test concentrations of 0.0718 to 6.18 mg/L.
- Analysis at 48 hours showed measured concentrations of less than the Limit of Quantification (LOQ), which was determined to be 0.0015 mg/L, up to 5.70 mg/L. This showed that the test item remained stable at the higher concentration and proved to be unstable at lower concentrations. Nevertheless, it was considered appropriate to conduct the definitive investigation as a static test.

DEFINITIVE TEST
- Chemical analysis of the fresh test preparations at 0 hours showed that measured test concentrations ranged from 1.16 to 9.08 mg/L.
- Chemical analysis of the aged test preparations at 48 hours showed measured test concentrations ranged from 0.742 to 8.54 mg/L.
- The dissolved test item may have been one or several components of the substance. Given that 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 immobilsation data and other observations from the exposure of Daphnia magna to the test item are given in Table 2 (attached).
- The relationship between percentage immobilisation and concentration at 48 hours is shown in Figure 1 (attached).

Reported statistics and error estimates:

STATISTICAL ANALYSIS
- The EL50 values, associated confidence limits at 24 and 48 hours, the slope of the response curve and its standard error were calculated by Probit analysis using Linear Maximum-Likelihood regression.
- The Lowest Observed Effect Loading Rate and the No Observed Effect Loading Rate at 24 and 48 hours were calculated using the Fisher’s Exact Binomial Test with Bonferroni correction.
- All results were calculated using the ToxRat Professional computer software package (TOXRAT).

SUB-LETHAL EFFECTS

- One daphnia was observed to have reduced movement in the 10 mg/L loading rate WAF and one daphnia was covered in debris in the 0.10 mg/L loading rate WAF group.

VALIDATION CRITERIA

- None of the control daphnids showed immobilisation or other signs of disease or stress.

- Oxygen concentration at the end of the test was ≥ 3 mg/L in the control and test vessels.

- The test was considered to be valid.

WATER QUALITY CRITERIA

- Results of the water quality measurements are given in Table 3 (attached).

- Temperature was maintained at 21 to 22 °C throughout the test.

- There were no treatment releated differences for oxygen concentration or pH.

- Throughout the test the light intensity was observed to be in the range 694 to 730 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, 3.2 and 10 mg/L loading rates were observed to be clear colourless water columns with test item on surface and in strings and globules throughout.

- After 23 hours stirring and a one hour standing period, the 1.0 and 3.2 mg/L loading rates were observed to be clear colourless water columns with test item floating on the surface. The 10 mg/L loading rate was observed to be a slightly hazy water column with test item floating on the surface.

- Visual examination of the WAFs showed that undissolved test item was present in the 10 mg/L loading rate and therefore it was considered justifiable to remove this by filtering through a glass wool plug (2 to 4 cm in length). As a precaution, the 3.2 mg/L loading rate was also filtered through a glass wool plug. Microscopic examination after filtering showed the glass wool plug had removed all of the dispersed test item.

- At the start, and throughout the test, all control and test solutions were observed to be clear colourless solutions.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to Daphnia magna was assessed in accordance with OECD Guideline 202. Exposure of Daphnia magna to the test item gave an EL50 (48 h) value of 1.6 mg/L loading rate WAF (95 % confidence limits 1.4 to 2.0 mg/L loading rate WAF). The No Observed Effect loading rate (NOEL) was determined to be 1.0 mg/L and the Lowest Observed Effect (LOEL) was determined to be 3.2 mg/L.

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 aqueous solubility and complex nature of the test item, the test medium was prepared as a Water Accommodated Fraction (WAF).

Following preliminary range-finding tests, 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 0.10, 0.32, 1.0, 3.2 and 10 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

Chemical analysis of the fresh test preparations at 0 hours showed measured test concentrations to range from 1.16 to 9.08 mg/L. Chemical analysis of the aged test preparations at 48 hours showed measured test concentrations to range from 0.742 to 8.54 mg/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 a mixture of components, but to the test item as a whole, the results were based on loading rates only.

CONCLUSION

Exposure of Daphnia magna to the test item gave an EL50 (48 h) value of 1.6 mg/L loading rate WAF (95 % confidence limits 1.4 to 2.0 mg/L loading rate WAF). The No Observed Effect loading rate (NOEL) was determined to be 1.0 mg/L and the Lowest Observed Effect (LOEL) was determined to be 3.2 mg/L.

Description of key information

The acute toxicity of the test item to Daphnia magna was assessed in accordance with OECD Guideline 202.  Exposure of Daphnia magna to the test item gave an EL50 (48 h) value of 1.6 mg/L loading rate WAF (95 % confidence limits 1.4 to 2.0 mg/L loading rate WAF). The No Observed Effect loading rate (NOEL) was determined to be 1.0 mg/L and the Lowest Observed Effect (LOEL) was determined to be 3.2 mg/L.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

1.6 mg/L

Additional information

No additional data.