Fatty acids, tall-oil, compds. 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:

25 September 2016 to 23 December 2016

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Freshwater Alga and Cyanobacteria, 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

Remarks:

HPLC-MS using an external standard

Details on sampling:

RANGE FINDING TESTS
- 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 from bulk test preparation at 0 hours for the control and each loading rate WAF test group.
- Samples were also taken from pooled replicates at 72 hours for quantitative analysis.
- All samples were stored frozen prior to analysis. Duplicate samples were taken at each occasion and stored frozen for further analysis if necessary.

Vehicle:

no

Test organisms (species):

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

Details on test organisms:

TEST SYSTEM
- The test was carried out using Pseudokirchneriella subcapitata strain CCAP 278/4. Liquid cultures of Pseudokirchneriella subcapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, 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 ± 2 °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 10 x E03 cells/mL. The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 – 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 104 – 105 cells/mL.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Post exposure observation period:

Not applicable

Hardness:

Not reported

Test temperature:

24 ± 1 °C

pH:

7.7 to 8.8 (see Table 3, attached)

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

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

DEFINITIVE TEST
- Nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L.

Details on test conditions:

CULTURE MEDIUM
- The culture medium used for both the range-finding and definitive tests was the same as that used to maintain the stock culture.
- The culture medium is defined in Annex 3 (attached).

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.

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 weighed onto a glass slide and suspended within 2 liters of culture medium to give the 10 and 100 mg/L loading rates respectively. After the 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. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Visual observations made on the WAFs indicated that a significant amount of 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 (2-4 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). The WAFs were then passed through two filter papers to remove as much undissolved test item as possible. Visual observations of the WAFs after filtering showed dispersed test item remained, however, it was considered that further filtration would not have removed any more.
- An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (10.9 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 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 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 significant inhibition of growth occurred at both 10 and 100 mg/L 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 test concentrations, a single WAF of a nominal loading rate of 1.0 mg/L was prepared from which serial dilutions were made to give the remaining test concentrations.
- A nominal amount of test item (20 mg) was weighed onto a glass slide and suspended within 20 liters of culture medium to give the 1.0 mg/L loading rate. After the 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. 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. The aqueous phase or WAF was removed by mid-depth siphoning (the first 75-100 mL discarded) to give the 1.0 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present. 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 (4.2 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 ºC under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 – 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 loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were assigned to the definitive test.

EXPERIMENTAL PREPARATION
- Nominal amounts of test item (20, 64, 20, 64 and 200 mg) were each separately weighed onto a glass slide and suspended within 20, 20, 2, 2 and 2 liters of culture medium to give the 1.0, 3.2, 10, 32 and 100 mg/L loading rates respectively. After the 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. The stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Visual observations made on the WAFs indicated that a significant amount of 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 (2-4 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). The WAFs were then passed through two filter papers to remove as much undissolved test item as possible. Microscopic examination of the 1.0 and 3.2 mg/L loading rate WAFs showed there to be no micro-dispersions of test item present. Visual observations of the 10, 32 and 100 mg/L loading rate WAFs showed dispersed test item remained, however, it was considered that further filtration would not have removed any more.
- An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (3.4 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L loading rate WAF.

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 7.28 x 10E05 cells per mL. Inoculation of 500 mL of test medium with 3.4 mL of this algal suspension gave an initial nominal cell density of 5 x 10E03 cells per 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.

TEST ORGANISM OBSERVATIONS
- Samples were taken at 24, 46 and 72 hours and the cell densities determined using a haemocytometer and light microscope. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 103 cells/mL) was taken as the starting cell density.
- It was not possible to monitor algal cells using a Coulter Multisizer Particle Counter due to the presence of dispersed test item
- 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.

WATER QUALITY CRITERIA
- The pH of the control and each test concentration was determined at initiation of the test and after 72 hours exposure.
- The pH was measured using a Hach HQ30d Flexi handheld meter.
- Temperature within the incubator was recorded daily.
- The appearance of the test media was recorded daily.

VORTEX DEPTH MEASUREMENTS
- The vortex depth was recorded at the start and end of the mixing period.

COMPARISON OF GROWTH RATES
- The average specific growth rate for a specified period was calculated as the logarithmic increase in biomass using the equation µ = ln Nn – ln N1 / tn – t1 where µ = average specific growth rate from time t1 to tn; N1 = cell concentration at t1; Nn = cell concentration at t0; t1 = time of first measurement; tn = time of nth measurement.
- The average specific growth rate over the test duration was calculated for each replicate control and test item vessel using the nominally inoculated cell concentration as the starting value rather than the measured starting value in order to increase the precision of the calculation.
- In addition the section by section specific growth rate (days 0-1, 1-2 and 2-3) was calculated for the control cultures and the results examined in order to determine whether the growth rate remained constant.
- Percentage inhibition of growth rate for each replicate test item vessel was calculated using the equation Ir = (µc - µt / µc) * 100 where Ir = percentage inhibition of average specific growth rate; µC = mean average specific growth rate for the control cultures; µt = average specific growth rate for the test culture.

COMPARISON OF YIELD
- Yield was calculated as the increase in biomass over the exposure period using the equation Y = Nn – N0 where Y = yield; N0 = cell concentration at the start of the test; Nn = cell concentration at the end of the test.
- For each test concentration and control the mean value for yield along with the standard deviation was calculated.
- The percentage inhibition of yield was calculated using the equation Iy = [(Yc – Yt) / Yc] * 100 where Iy = percentage inhibition of yield; Yc = mean value for yield in the control group; Yt = mean value for yield for the treatment group.

DETERMINATION OF ELx VALUES
- For each individual test vessel (mean values for yield), percentage inhibition (arithmetic axis) was plotted against test concentration (logarithmic axis) and a line fitted by computerized interpolation using the Xlfit software package (IDBS). ELx values were then determined from the equation for the fitted line.
- Where appropriate 95 % confidence limits for the EL50 values were calculated, using the simplified method of evaluating dose-effect experiments of Litchfield and Wilcoxon (1949).

VALIDATION CRITERIA
- The results of the test are considered valid if the following performance criteria are met:
(i) Cell concentration of the control cultures must increase by a factor of at least 16 over the test period.
(ii) The mean of the coefficients of variation of the section by section specific growth rates in the control cultures during the course of the test (days 0-1, 1-2 and 2-3, for 72-Hour tests) must not exceed 35%.
(iii) The coefficient of variation of the average specific growth rate in replicate control cultures must not exceed 7%.

Reference substance (positive control):

yes

Remarks:

potassium dichromate (investigation conducted between 07 December 2015 and 10 December 2015; see Annex 2, attached)

Duration:

72 h

Dose descriptor:

EL10

Effect conc.:

6.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

11 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Duration:

72 h

Dose descriptor:

EL50

Effect conc.:

26 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth rate

Remarks on result:

other: 95 % confidence limits 22-32 mg/L

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.:

1.9 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Duration:

72 h

Dose descriptor:

EL20

Effect conc.:

3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Dose descriptor:

EL50

Effect conc.:

6.8 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

biomass

Remarks on result:

other: 95 % confidence limits 5.6-8.2 mg/L

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 investigational work (see Annex 4, attached) indicated that there was no increase in the amount of dissolved test item when the preparation period was extended for longer than 24 hours.
- For the purpose of testing the WAF was therefore prepared using a stirring period of 23 hours followed by a 1-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 during the range-finding tests are given in Table 1 and Table 2 (attached).
- The results showed no effect on growth at 0.010, 0.10 and 1.0 mg/L loading rate WAF. However, growth was observed to be reduced at 10 and 100 mg/L loading rate WAF.
- Based on this information loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were selected for the definitive test.
- Chemical analysis of the 1.0, 10 and 100 mg/L loading rate WAF test preparations at 0 hours (see Annex 5, attached) showed measured test concentrations of 0.42, less than the LOQ and 0.34 mg/L respectively were obtained. Measured concentrations of 0.40 and less than the LOQ were obtained at 72 hours.
 
CHEMICAL ANALYSIS OF DEFINITIVE TEST LOADING RATES
- Analysis of the test preparations at 0 hours (See Annex 5, attached) showed measured test concentrations to range from 0.49 to 30 mg/L. A significant 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, determined to be 0.079 mg/L, to 0.23 mg/L.
- 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.
 
DEFINITVE TEST GROWTH DATA
- Cell density values determined at each sampling time and pH values at 0 and 72 hours are given in Table 3 (attached).
- 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 and percentage inhibition values are given in Table 5 (attached).
- Mean cell densities versus time for the definitive test are presented in Figure 1 (attached).
- The percentage inhibition values are plotted against loading rate in Figures 2 and 3 (attached)
- From the data given in Tables 2 and 4 (attached), it is clear that the growth rate (r) and yield (y) of Pseudokirchneriella subcapitata (CCAP 278/4) were affected by the presence of the test item over the 72-Hour exposure period.
INHIBITION OF GROWTH RATE
- Statistical analysis of the growth rate data was carried out for the control and all loading rates using one way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955).
- There were no statistically significant differences between the control and 1.0 mg/L loading rate WAF (P≥0.05), however all other loading rates were significantly different (P<0.05) and, therefore 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
- Statistical analysis of the yield data was carried out as described for growth rate data.
- There were no statistically significant differences between the control and 1.0 mg/L loading rate WAF (P≥0.05), however all other loading rates were significantly different (P<0.05) and, therefore the "No Observed Effect Loading Rate" (NOEL) based on yield 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
- The sell concentration of the control cultures increased by a factor of 158 after 72 hours (mean cell density of control at 0 hours was 5.00 x 10E03 cells/mL and mean cell density of control at 72 hours was 7.85 x 10E05 cells/mL).
- This increase was in line with the OECD Guideline that states the enhancement must be at least by a factor of 16 after 72 hours.
- The mean coefficient of variation for section by section specific growth rate for the control cultures was 29 % and hence satisfied the validation criterion 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 – 72 h) was 3 % and hence satisfied the validation criterion given in the OECD Guideline which states that this must not exceed 7 %.
 
OBSERVATIONS ON CULTURES
- All test and control cultures were inspected microscopically at 72 hours.
- After 72 hours there were no abnormalities detected in the control or test cultures at 1.0 and 3.2 mg/L loading rate WAF.
- Some enlarged cells were observed to be present in the 10, 32 and 100 mg/L loading rate WAFs.
- Motile organisms were present in all WAFs.
 
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 ºC throughout the test.
- The pH value of the control cultures (see Table 3, attached) was observed to increase from pH 7.7 at 0 hours to pH 8.4 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and therefore was 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 the start of stirring all loading rate WAFs were observed to be clear colourless media columns with test item adhered to the glass slide suspended within the media column. After stirring, and following a 1-Hour standing period, the 1.0 mg/L loading rate was observed to have formed a clear colourless media column, the 3.2 mg/L loading rate was observed to have formed a very slightly hazy non-homogenous dispersion, the 10 mg/L loading rate was observed to have formed a slightly hazy non-homogenous dispersion, the 32 mg/L loading rate was observed to have formed a hazy non-homogenous dispersion with some test item at the media surface whilst the 100 mg/L loading rate was observed to have formed a very hazy non-homogenous dispersion with test item floating at the media surface.
- As visual observations showed dispersed test item to be present, the aqueous phase was removed by filtration through a glass wool plug and two post lip filter papers. Microscopic examination of the 1.0 and 3.2 mg/L loading rate WAFs showed there to be no micro-dispersions of test item present. Visual observations of the 10, 32 and 100 mg/L loading rate WAFs showed dispersed test item remained, however, it was considered that further filtration would not have removed any more.
- At the start of the test all control, 1.0 and 3.2 mg/L loading rate WAF test cultures were observed to be clear colourless solutions. The 10 mg/L loading rate WAF test cultures were extremely slightly hazy dispersions with some larger particles of precipitated test item throughout, the 32 mg/L loading rate WAF test cultures were slightly hazy dispersions with some larger particles of precipitated test item throughout whilst the 100 mg/L loading rate WAF test cultures were hazy dispersions with some larger particles of precipitated test item throughout. After the 72-Hour test period all control and 1.0 mg/L loading rate WAF test cultures were observed to be pale green dispersions, the 3.2 and 10 mg/L loading rate WAF test cultures were green dispersions. The 32 mg/L loading rate WAF test cultures were slightly hazy dispersions with some larger particles of precipitated test item throughout whilst the 100 mg/L loading rate WAF test cultures were hazy dispersions with some larger particles of precipitated test item throughout.

Results with reference substance (positive control):

- Results from the positive control with potassium dichromate were within the normal ranges for this reference item (see Annex 2, attached).

Reported statistics and error estimates:

STATISTICAL ANALYSIS
- One way analysis of variance incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) and Dunnett's multiple comparison procedure for comparing several treatments with a control (Dunnett, 1955) was carried out on the growth rate and yield data after 72 hours for the control and all test loading rates s to determine any statistically significant differences between the test and control groups.
- All statistical analyses were performed using the SAS computer software package (SAS, 1999 - 2001).

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Pseudokirchneriella subcapitata to the test item gave EL50 (72h) values of 26 mg/L loading rate WAF based on growth rate and 6.8 mg/L loading rate WAF based on yield. No Observed Effect Loading Rates (NOEL) were reported as 1.0 mg/L based on growth rate and 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 followed 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 for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF). Following preliminary range-finding tests, Pseudokirchneriella subcapitatawas exposed to Water Accommodated Fractions (WAFs) of the test item over a range of nominal loading rates of 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks 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 haemocytometer and light microscope.

 

RESULTS

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.49 to 30 mg/L, A significant 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, determined to be 0.079 mg/L, to 0.23 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

Exposure of Pseudokirchneriella subcapitata to the test item gave EL50 (72h) values of 26 mg/L loading rate WAF based on growth rate and 6.8 mg/L loading rate WAF based on yield. No Observed Effect Loading Rates (NOEL) were reported as 1.0 mg/L based on growth rate and yield.

Description of key information

Exposure of Pseudokirchneriella subcapitata to the test item gave EL50 (72h) values of 26 mg/L loading rate WAF based on growth rate and 6.8 mg/L loading rate WAF based on yield. No Observed Effect Loading Rates (NOEL) were reported as 1.0 mg/L based on growth rate and yield (OECD 201 and EU Method C.3).

Key value for chemical safety assessment

EC50 for freshwater algae:

26 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 followed 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 for the purposes of the test the test item was prepared as a Water Accommodated Fraction (WAF). 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 1.0, 3.2, 10, 32 and 100 mg/L (three replicate flasks 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 haemocytometer and light microscope.

 

RESULTS

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 0.49 to 30 mg/L, A significant 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, determined to be 0.079 mg/L, to 0.23 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

Exposure of Pseudokirchneriella subcapitata to the test item gave EL50 (72h) values of 26 mg/L loading rate WAF based on growth rate and 6.8 mg/L loading rate WAF based on yield. No Observed Effect Loading Rates (NOEL) were reported as 1.0 mg/L based on growth rate and yield.