Tall oil, reaction products with ethanolamine

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:

29 December 2014 to 23 January 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:

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.

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.

Vehicle:

no

Details on test solutions:

RANGE-FINDING TEST
- Nominal amounts of test item (20 and 200 mg) were each separately added to the surface of 2 L of culture medium to give 10 and 100 mg/L loading rates.
- 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.
- 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.
- The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 10 and 100 mg/L loading rate WAFs.
- Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
- An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (2.5 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 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
-Nominal amounts of test item (20, 40, 80, 160 and 320 mg) were each separately added to the surface of 2 L of culture medium to give 10, 20, 40, 80 and 160 mg/L loading rates.
- 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.
- 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.
- The aqueous phase or WAF was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 10, 18, 32, 56 and 100 mg/L loading rate WAFs.
- Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
- An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (4.3 mL) to give the required test concentrations of 10, 20, 40, 80 and 160 mg/L loading rate WAF.
- The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours (see Appendix 5, attached).

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 constance 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:

7.6 to 8.6 during definitive test (see Table 2, attached)

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Nominal and measured concentrations:

RANGE-FINDING TEST
- 10 and 100 mg/L (nominal)

DEFINITIVE TEST
- 10, 20, 40, 80 and 160 mg/L (nominal)

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 5.83 * 10E5 cells/mL. Inoculation of 500 mL of test medium with 4.3 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 16 September 2014 and 19 September 2014 (see Appendix 2, attached)

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

> 160 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

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

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

> 160 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

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

Key result

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

> 160 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

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

Details on results:

VALIDATION OF MIXING PERIOD
- Preliminary investigation (see Appendix 4, attached) indicated that there was a significant decline in the amount of dissolved test item when the preparation period was extended from 24 to 96 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 TEST
- The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item are shown in Table 1 (attached).
- The results showed no effect on growth at 10 mg/L loading rate WAF. However, growth was observed to be reduced at 100 mg/L loading rate WAF.
- Based on this information, loading rates of 10, 20, 40, 80 and 160 mg/L were selected for the definitive test.
- Chemical analysis of the 10 and 100 mg/L loading rate WAF test preparations at 0 hours (see Appendix 5, attached) showed measured test concentrations of less than the limit of quantification (LOQ), which were determined to be 0.0054 mg/L and 0.017 mg/L respectively.
- A decline in measured test concentration was observed at 72 hours to be less than the LOQ for both the 10 and 100 mg/L loading rate WAF test preparations.

DEFINITIVE TEST
- Analysis of the 160 mg/L loading rate test preparations at 0 and 72 hours (see Apendix 5, attached) showed measured test concentrations of 0.0074 and 0.0086 mg/L respectively indicating that the test item was stable over the test duration.
- The dissolved test item may have been one or several components of the test item. Given that 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.

GROWTH DATA
- Cell density values determined at each sampling time and pH values at 0 and 72 hours are shown in Table 2 (attached).
- Daily specific growth rates for the control cultures are given in Table 3 (attached).
- Growth rate and yield values for the control and test cultures after 72 hours and percentage inhibition values are given in Table 4 (attached).
- The mean cell densities versus time for the definitive test are presented in Figure 1 (attached).
- Growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were not affected by the presence of test item over the 72-hour exposure period (see Tables 2 and 4, attached).

INHIBITION OF YIELD
- EL10 (0-72 hours) > 160 mg/L loading rate WAF (where EyLx is the loading rate that reduced yield by x %).
- EL20 (0-72 hours) > 160 mg/L loading rate WAF (where EyLx is the loading rate that reduced yield by x %).
- EL50 (0-72 hours) > 160 mg/L loading rate WAF (where EyLx is the loading rate that reduced yield by x %).

OBSERVATIONS ON CULTURES
- All test and control cultures were inspected microscopically at 72 hours.
- No abnormalities were detected in any of the control or test cultures.

WATER QUALITY CRITERIA
- The pH values of the control and each test concentration are given in Table 2 (attached).
- Temperature was maintained at 24 ± 1 °C throughout the test.
- The pH value of the control cultures (see Table 2, attached) increased from 7.8 at 0 hours to 8.6 at 72 hours.
- pH deviation in control cultures was less than 1.5 units after 72 hours and was therefore within the limits given in the test guidelines.

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 both the start and end of the mixing period, and following a 1-hour standing period, all loading rate WAFs were observed to have formed clear colourless water columns with deposits of test item floating at the surface.
- Microscopic examination of the WAFs showed there to be no micro-dispersions of test item present.
- At the start of the test all control and test cultures were observed to be clear, colourless, solutions.
- After the 72-hour test period, all control and test cultures were observed to be green dispersions.

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:

GROWTH RATE
- There were no statistically significant decreases in growth rate between the control and all loading rate WAFs (P ≥ 0.05).
- Therefore, the NOEL based on growth rate was 160 mg/L loading rate WAF.

INHIBITION OF YIELD
- There were no statistically significant decreases in yield between the control and all loading rate WAFs (P ≥ 0.05).
- Therefore, the NOEL based on growth rate was 160 mg/L loading rate WAF.

VALIDATION CRITERIA

- Mean cell density of control at 0 hours: 5.87 * 10E3 cells/mL

- Mean cell density of control at 72 hours: 1.05 * 10E6 cells/mL

- The cell concentration of the control cultures increased by a factor of 178 after 72 hours. This increase was in line with the OECD guideline that states the enhancement must be at least a factor of 16 after 72 hours.

- The mean coeficient of variation for section by section specific growth rate for the control cultures was 25 % 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 3 % and satisfied the validation criteria given in the OECD guideline, which states that this must not exceed 7 %.

Validity criteria fulfilled:

yes

Conclusions:

The effect of the test item on the growth of Psudokirchneriella subcapitata was assessed in accordance with OECD Guideline 201 and gave EL50 values of greater than 160 mg/L loading rate WAF. The No Observed Effect Loading rate was 160 mg/L.

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 a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 10, 20, 40, 80 and 160 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 160 mg/L loading rate WAF test preparations at 0 and 72 hours showed that measured test concentrations of 0.0074 and 0.0086 mg/L respectively were obtained and indicated that the test item was stable over the test duration.

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

The effect of the test item on the growth of Psudokirchneriella subcapitata gave EL50 values of greater than 160 mg/L loading rate WAF. The No Observed Effect Loading rate was 160 mg/L.

Description of key information

The effect of the test item on the growth of Psudokirchneriella subcapitata was investigated using a method compatible with OECD 201 and EU Method C.3and gave EL50 values of greater than 160 mg/L loading rate WAF. The No Observed Effect Loading rate was 160 mg/L.

Key value for chemical safety assessment

EC50 for freshwater algae:

160 mg/L

Additional information

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 a preliminary range-finding test, Pseudokirchneriella subcapitatawas exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 10, 20, 40, 80 and 160 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 160 mg/L loading rate WAF test preparations at 0 and 72 hours showed that measured test concentrations of 0.0074 and 0.0086 mg/L respectively were obtained and indicated that the test item was stable over the test duration.

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

The effect of the test item on the growth of Psudokirchneriella subcapitata gave EL50 values of greater than 160 mg/L loading rate WAF. The No Observed Effect Loading rate was 160 mg/L.