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

Toxicity to aquatic algae and cyanobacteria

Link to relevant study record(s)

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 May 2015 to 27 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

SECOND RANGE-FINDING TEST
- A sample of each loading rate WAF was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item over the test duration.
- 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 for quantitative analysis from the control and from the bulk test preparation for each loading rate WAF test group at 0 hours.
- Samples were also taken for quantitative analysis from the pooled replicates at 72 hours.
- All samples were stored frozen prior to analysis.
- Duplicate samples were taken on each occasion and stored frozen for further analysis if necessary.
- Only samples at the No Observed Effect Loading Rate (NOEL) were analysed.

TEST ORGANISM
- Samples were taken at 0, 24, 48 and 72 hours and the cell densities were determined using a Coulter Multisizer Particle Counter.
- To determine the potential effect of the test item on the appearance of algal cells, a sample was removed from each test and control culture (replicates pooled) at the end of the test.
- The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

Details on test solutions:

RANGE-FINDING TESTS
- The loading rates to be used in the definitive test were determined by preliminary range-finding tests.
- The initial range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 10 and 100 mg/L for a period of 72 hours.
- The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.
- Nominal amounts of test item (20 and 200 mg) were each separately added to the surface of 2 L of culture medium to give the 10 mg and 100 mg/L loading rates respectively.
- After addition of test item, the culture medium 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 column and 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 was removed by mid-depth siphoning (the first 75 to 100 mL discarded).
- Undissolved test item was observed to be present in the WAFs after passing through a glass wool plug and it was therefore considered appropriate to pass the WAFs 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.
- An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (5.6 mL) to give the required test concentrations of 10 and 100 mg/L loading rate WAF.
- The control group was maintained under identical conditions but not exposed to the test item.
- At the start of the initial range-finding test, a sample of each test and control culture was removed and the cell density determined using a haemocytometer and light microscope.
- The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under constant illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
- After 72 hours, the cell density of each flask was determined using a haemocytometer and light microscope. It was not possible to monitor algal growth using a Coulter Multisizer Particle Counter due to the presence of dispersed test item.
- The results of the initial range-finding test showed that significant inhibition of growth occurred at both 10 and 100 mg/L loading rate WAF and so a second range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 0.010, 0.10 and 1.0 mg/L for a period of 72 hours.
- Due to the need to test at relatively low loading rates, the 0.010 and 0.10 mg/L loading rate WAF test concentrations were prepared as serial dilutions from the 1.0 mg/L loading rate WAF.
- The test was conducted in 250 mL glass conical flasks each containing 100 mL of test preparation and plugged with polyurethane foam bungs to reduce evaporation. Two replicate flasks were prepared for each control and test concentration.
- Nominal amounts of test item (20 mg) was added to the surface of 20 L of culture medium to give the 1.0 mg loading rate.
- After addition of test item, the culture medium 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 WAF indicated that a significant amount of dispersed test item was present in the water column and 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 was removed by mid-depth siphoning (the first 75 to 100 mL discarded).
- Undissolved test item was observed to be present in the WAFs after passing through a glass wool plug and it was therefore considered appropriate to pass the WAFs through Postlip filter paper to remove as much undissolved test item as possible.
- Observations made on the filtrate showed that all undissolved test item had been removed.
- A series of dilutions was made from the 1.0 mg/L loading rate WAF to give further stock solutions of 0.10 and 0.010 mg/L loading rate WAF.
- An aliquot (450 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (6.2 mL) to give the required test concentrations of 0.010, 0.10 and 1.0 mg/L loading rate WAF.
- The control group was maintained under identical conditions but not exposed to the test item.
- At the start of the second range-finding test, a sample of each test and control culture was removed and the cell density determined using a Coulter Multisizer Particle Counter.
- The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
- After 72 hours, the cell density of each flask was determined using a Coulter Multisizer Particle Counter.

DEFINITIVE TEST
- Based on the results of the range-finding tests, the loading rates assigned to the definitive test were 0.10, 0.32, 1.0, 3.2 and 10 mg/L.
- Nominal amounts of test item (20, 32 and 20 mg) were each separately added to the surface of 20, 10 and 2 L of culture medium to give the 1.0, 3.2 and 10 mg/L loading rates respectively.
- After addition of the test item, the culture medium 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 column and 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 was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 1.0, 3.2 and 10 mg/L loading rate WAFs.
- Microscopic observations of the WAFs were performed after filtering and showed there to be no dispersed test item present.
- Dilutions were made from the 1.0 mg/L loading rate WAF to give further loading rates of 0.32 and 0.10 mg/L.
- An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (2.7 mL) to give the required test concentrations of 0.10, 0.32, 1.0, 3.2 and 10 mg/L loading rate WAF.

Test organisms (species):

Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST SYSTEM
- The test was carried out using Pseudokirchneriella subscapitata strain CCAP 278/4.
- Liquid cultures of Pseudokirchneriella subscapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institue, Oban, Argyll, Scotland.
- Master cultures were maintained in the laboratory by the periodic replenishment of culture medium.
- The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1 °C.
- Prior to the start of the test, sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10E3 cells/mL.
- The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10E4 to 10E5 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

None

Hardness:

Not reported

Test temperature:

24 ± 1 °C

pH:

7.7 to 8.4 during definitive test (see Table 3, attached)

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Nominal and measured concentrations:

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

SECOND RANGE-FINDING TEST
- Nominal loading rates of 0.010 and 0.10 mg/L

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

Details on test conditions:

EXPOSURE CONDITIONS
- As in the range-finding test, 250 mL glass conical flasks were used.
- Six flasks each containing 100 mL of test preparation were used for the control and three flasks each containing 100 mL were used for each treatment group.
- The control group was maintained under identical conditions but not exposed to the test item.
- Pre-culture conditions gave an algal suspension in log phase growth characterised by a cell density of 9.34 * 10E5 cells/mL. Inoculation of 500 mL of test medium with 2.7 mL of this algal suspension gave an initial nominal cell density of 5 * 10E3 cells/mL and had no significant dilution effect on the final test concentration.
- The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

Reference substance (positive control):

yes

Remarks:

potassium dichromate conducted between 12 May 2015 and 15 May 2015 (see Appendix 2, attached)

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

0.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: where ErLx is the loading rate that reduced growth rate by x %

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

1.7 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: where ErLx is the loading rate that reduced growth rate by x %

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

9.5 mg/L

Nominal / measured:

nominal

Conc. based on:

act. ingr.

Basis for effect:

growth rate

Remarks on result:

other: where ErLx is the loading rate that reduced growth rate by x %

Key result

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

2.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: where EyLx is the loading rate that reduced yield by x %

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

2.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: where EyLx is the loading rate that reduced yield by x %

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

2.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: where EyLx is the loading rate that reduced yield by x %

Duration:

72 h

Dose descriptor:

NOELR

Effect conc.:

1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

LOELR

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Details on results:

VALIDATION OF MIXING PERIOD
- Preliminary investigation (see Appendix 4) showed that there was no significant increase in the amount of dissolved test item when the preparation period was extended for longer than 24 hours.
- Therefore, for the purpose of testing, the WAF was prepared using a stirring period of 23 hours followed by a one hour settlement period.

RANGE-FINDING TESTS
- The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item are shown in Tables 1 and 2 (attached).
- The results showed no effect on growth at 0.010 and 0.10 mg/L loading rate WAF. However, growth was observed to be reduced 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.
- Chemical analysis of the 0.10, 1.0 and 10 mg/L loading rate WAF test preparations at 0 hours (see Appendix 5, attached) showed measured test concentrations ranging from 0.033 to 8.8 mg/L.
- A decline in measured test concentration was observed at 72 hours to be less than the LOQ, which was determined to be 0.0024 to 0.042 mg/L. This indicated that the test item was unstable over the test duration.

DEFINITIVE TEST
- Analysis of the test preparations at 0 hours (see Appendix 5, attached) showed measured test concentrations ranging from 0.032 to 7.0 mg/L.
- A decline in measured test concentration was observed at 72 hours to be less than the LOQ, which was determined to be 0.0024 to 0.042 mg/L.
- 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.

GROWTH DATA
- Cell density values determined at each sampling time are given in Table 3 (attached) together with pH values at 0 and 72 hours.
- Daily specific growth rates for the control cultures are given in Table 4 (attached).
- Growth rate and yield values for the control and test cultures after 72 hours are given in Table 5 (attached) together with percentage inhibition values.

OBSERVATIONS ON CULTURES
- After 72 hours there were no abnormalities detected in the control or test cultures at 0.10, 0.32 and 1.0 mg/L loading rate WAFs.
- However, no intact cells were observed to be present in the test cultures at 3.2 and 10 mg/L loading rate WAF.

WATER QUALITY CRITERIA
- The pH values of the control and each test concentration are given in Table 3 (attached).
- Temperature was maintained at 24 ± 1 °C throughout the test.
- The pH value of the control cultures (see Table 3, attached) was observed to increase from 7.8 at 0 hours to 8.5 at 72 hours. The pH deviation in the control cultures was < 1.5 pH units after 72 hours and therefore within the limits given in the test guideline.

VORTEX DEPTH MEASUREMENTS
- The vortex depth was recorded at the start and end of the mixing period and was observed to have formed a dimple at the media surface.

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 mixing all loading rate WAFs were observed to have formed clear colourless media columns with globules of test item dispersed throughout.
- After stirring, and following a one hour standing period, all loading rates were observed to have formed clear colourless media columns with globules of test item settled on the bottom of the mixing vessel and dispersed throughout the media column.
- Given that visual observations showed there to be dispersed test item present it was considered appropriate to remove the aqueous phase by filtration through a glass wool plug.
- Microscopic examination of the WAFs after filtration showed that all the undissolved test item had been removed.
- At the start of the test all control and test cultures were observed to be clear colourless solutions. After the 72 hours test period all control, 0.10, 0.32 and 1.0 mg/L loading rate WAF test cultures were observed to be green dispersions. The 3.2 mg/L loading rate WAF test cultures were observed to be extremely pale green dispersions whilst the 10 mg/L loading rate WAF test cultures 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 ranges for this reference item (see Appendix 2, attached)

Reported statistics and error estimates:

INHIBITION OF GROWTH RATE
- There were no statistically significant differences between the control, 0.10, 0.32 and 1.0 mg/L loading rate WAFs (P ≥ 0.05).
- All other loading rates were significantly different (P < 0.05) and, as a result, the No Observed Effect Loading Rate (NOEL) based on growth rate was 1.0 mg/L loading rate WAF.
- Correspondingly, the Lowest Observed Effect Loading rate (LOEL) based on growth rate was 3.2 mg/L loading rate WAF.

INHIBITION OF YIELD
- There were no statistically significant differences between the control, 0.10, 0.32 and 1.0 mg/L loading rate WAFs (P ≥ 0.05).
- All other loading rates were significantly different (P < 0.05) and, as a result, the No Observed Effect Loading Rate (NOEL) based on growth rate was 1.0 mg/L loading rate WAF.
- Correspondingly, the Lowest Observed Effect Loading rate (LOEL) based on yield was 3.2 mg/L loading rate WAF.

VALIDATION CRITERIA

- Mean cell density of control at 0 hours: 5.15 * 10E3 cells/mL

- Mean cell density of control at 72 hours: 5.27 * 10E6 cells/mL

- The mean coefficient of variation for section by section specific growth rate for the control cultures was 13 % and satisfied the validation criteria given in the OECD guideline, which states the mean must not exceed 35 %.

- The coefficient of variation for average specific growth rate for the control cultures over the test period (0 to 72 hours) was 2 % and satisfied the validation criteria given in the OECD guideline, which states that this must not exceed 7 %.

Validity criteria fulfilled:

yes

Conclusions:

The acute toxicity of the test item to growth of the green alga Pseudokirchneriella subcapitata was assessed in accordance with OECD Guideline 201. Based on growth rate, exposure of Pseudokirchneriella subcapitata to the test item gave an EL50 value of 9.5 mg/L loading rate WAF. The No observed Effect Loading rate (NOEL) was 1.0 mg/L loading rate WAF and the Lowest Observed Effect Loading rate (LOEL) was 3.2 mg/L based on growth rate. Based on yield, exposure of Pseudokirchneriella subcapitatato the test item gave an EL50 value of 2.6 mg/L loading rate WAF. The No Observed Effect Loading rate (NOEL) was 1.0 mg/L loading rate WAF and the Lowest Observed Effect Loading rate (LOEL) was 3.2 mg/L based on yield.

Executive summary:

GUIDELINE

 

A study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" referenced as Method C.3 of Commission Regulation (EC) 761/2009.

 

METHODS

 

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

 

Following preliminary range-finding tests, Pseudokirchneriella subcapitata was 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 (three replicates per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C.

 

Samples of the algal populations were removed daily and cell concentrations determined for each control and treatment group using a Coulter multisizer particle counter.

 

RESULTS

 

Analysis of the test preparations at 0 hours showed measured test concentrations ranged from 0.032 to 7.0 mg/L. A decline in measured test concentrations was observed at 72 hours in the range of less than the Limit of Quantification (LOQ) of the analytical method employed, which was determined to be 0.0024 to 0.012 mg/L.

 

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

 

Based on growth rate, exposure of Pseudokirchneriella subcapitata to the test item gave an EL50 value of 9.5 mg/L loading rate WAF. The No Observed Effect Loading rate (NOEL) was 1.0 mg/L loading rate WAF and the Lowest Observed Effect Loading rate (LOEL) was 3.2 mg/L based on growth rate. Based on yield, exposure ofPseudokirchneriella subcapitatato the test item gave an EL50 value of 2.6 mg/L loading rate WAF. The No Observed Effect Loading rate (NOEL) was 1.0 mg/L loading rate WAF and the Lowest Observed Effect Loading rate (LOEL) was 3.2 mg/L based on yield.

Description of key information

The acute toxicity of the test item to growth of the green alga Pseudokirchneriella subcapitata was assessed in accordance with OECD Guideline 201.  Based on growth rate, exposure of Pseudokirchneriella subcapitata to the test item gave an EL50 value of 9.5 mg/L loading rate WAF. The No observed Effect Loading rate (NOEL) was 1.0 mg/L loading rate WAF and the Lowest Observed Effect Loading rate (LOEL) was 3.2 mg/L based on growth rate. Based on yield, exposure of Pseudokirchneriella subcapitatato the test item gave an EL50 value of 2.6 mg/L loading rate WAF. The No Observed Effect Loading rate (NOEL) was 1.0 mg/L loading rate WAF and the Lowest Observed Effect Loading rate (LOEL) was 3.2 mg/L based on yield.

Key value for chemical safety assessment

EC50 for freshwater algae:

9.5 mg/L

EC10 or NOEC for freshwater algae:

1 mg/L

Additional information

No additional data.