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Toxicity to aquatic algae and cyanobacteria

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Reference
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 May 2017 to 07 June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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.
- 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 the 100 mg/L 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
Details on test solutions:
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).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis 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 10E04 to 10E05 cells/mL.
- A positive control used potassium dichromate as the reference item. Details of the positive control are given in Annex 2 (attached). The positive control was conducted between 28 November 2016 and 01 December 2016.

TEST ORGANISM OBSERVATIONS
- Samples were taken at 22, 46 and 72 hours and the cell densities determined using a Coulter Multisizer Particle Counter. Three determinations were made for each sample. The nominally inoculated cell concentration (5.00 x 10E03 cells/mL) was taken as the starting cell density.
- 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.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
Not applicable
Hardness:
Not reported
Test temperature:
24 ± 1 °C
pH:
7.3 to 7.5 in the definitive test (see Table 2, attached)
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
100 mg/L loading rate WAF
Details on test conditions:
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 TEST
- The loading rate to be used in the definitive test was determined by a preliminary range-finding test.
- The range-finding test was conducted by exposing Pseudokirchneriella subcapitata cells to nominal loading rates of 1.0, 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, 20 and 200 mg) were each separately added to the surface of 20, 2 and 2 L of culture medium to give the 1.0, 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.
- 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, 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 (12.0 mL) to give the required test concentrations of 1.0, 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 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 result of the range-finding test a "limit test" was conducted at a single loading rate of 100 mg/L to confirm that no effect on algal growth was observed.

EXPERIMENTAL PREPARATION
- A nominal amount of test item (200 mg) was added to the surface of 2 L of culture medium to give the 100 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 mixture 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 100 mg/L loading rate WAF. Microscopic inspection of the WAF showed no micro-dispersions or undissolved test item to be present.
- An aliquot (1 L) of the WAF was inoculated with algal suspension (13.9 mL) to give the required test concentration of 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 100 mg/L loading rate WAF 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 characterized by a cell density of 3.61 x 105 cells per mL. Inoculation of 1 L of test medium with 13.9 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.

WATER QUALITY CRITERIA
- The pH of the control and the 100 mg/L loading rate WAF test concentration was determined at initiation of the test and after 72 hours exposure 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.

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 (study conducted between 28 November 2016 and 01 December 2016)
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
loading rate WAF
Basis for effect:
other: yield
Details on results:
VALIDATION OF MIXING PERIOD
- Preliminary investigational work (see Annex 4, attached) indicated that there was no significant 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 TEST
- The cell densities and percentage inhibition of growth values from the exposure of Pseudokirchneriella subcapitata to the test item during the range-finding test are given in Table 1 (attached).
- The results showed no effect on growth at 1.0, 10 and 100 mg/L loading rate WAF.
- Based on this information a single loading rate of six replicates of 100 mg/L was selected for the definitive test. This experimental design conforms to a "limit test" to confirm that no effect on growth was observed.
- Chemical analysis of the 100 mg/L loading rate WAF test preparations at 0 and 72 hours (see Annex 5) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.058 mg/L. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.

CHEMICAL ANALYSIS OF DEFINITIVE TEST LOADING RATES
- Analysis of the 100 mg/L loading rate WAF test preparations at 0 and 72 hours (see Annex 5, attached) showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.058 mg/L were obtained. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ.
- 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 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).
- Mean cell densities versus time for the definitive test are presented in Figure 1 (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 not affected by the presence of the test item over the 72-Hour exposure period.
- It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L.

INHIBITION OF GROWTH RATE
- The ErL10 (0-72h), ErL20 (0-72h) and ErL50 (0-72h) values were all reported as > 100 mg/L loading rate WAF where ErLx is the loading rate that reduced growth rate by x %.
- Statistical analysis of the growth rate data was carried out for the control and 100 mg/L loading rate WAF test group using a Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981). There were no statistically significant differences (P ≥ 0.05), between the control and 100 mg/L loading rate WAF test group and therefore the "No Observed Effect Loading Rate" (NOEL) based on growth rate was 100 mg/L loading rate WAF.

INHIBITION OF YIELD
- The EyL10 (0-72h), EyL20 (0-72h) and EyL50 (0-72h) values were all reported as > 100 mg/L loading rate WAF where EyLx is the loading rate that reduced yield rate by x %.
- Statistical analysis of the yield data was carried out as described for growth rate.
- Statistical analysis of the yield data was carried out as in Section 6.3.3. There were no statistically significant differences between the control and 100 mg/L loading rate WAF (P ≥ 0.05) and therefore the "No Observed Effect Loading Rate" (NOEL) based on yield was 100 mg/L loading rate WAF.

VALIDATION CRITERIA
- The cell concentration of the control cultures increased by a factor of 213 after 72 hours (nominal cell density of control at 0 hours was 5.00 x 10E03 cells/mL and mean cell density of control at 72 hours was 1.06 x 10E06 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 7 % 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 1 % 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.
- There were no abnormalities detected in any of the control or test cultures at 72 hours.

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.

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 WAF.
- At the start of the mixing period the 100 mg/L loading rate WAF was observed to have formed a clear colourless media column with oily globules of test item floating at the media surface. After stirring, and following a 1-Hour standing period, the WAF was observed to have formed a clear colourless media column with an oily layer of test item coating the media surface. Microscopic examination of the WAF 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):
- 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:
DETERMINATION OF ELx VALUES
- ELx values were determined by inspection of the growth rate and yield data after 72 hours.

STATISTICAL ANALYSIS
- A Student’s t-test incorporating Bartlett's test for homogeneity of variance (Sokal and Rohlf, 1981) was carried out on the growth rate and yield data after 72 hours for the control and the 100 mg/L loading rate 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 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF
Executive summary:

GUIDELINE

A study was performed to assess the effect of the test item on the growth of the green algaPseudokirchneriella 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 a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/L (six replicate flasks) 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 100 mg/L loading rate WAF test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.058 mg/L were obtained. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ. 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 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF

Description of key information

Exposure of Pseudokirchneriella subcapitata to the test item gave EL50 values > 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF (OECD 201 and EU Method C.3).

Key value for chemical safety assessment

Additional information

GUIDELINE

A study was performed to assess the effect of the test item on the growth of the green algaPseudokirchneriella 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 a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fraction (WAF) of the test item, at a single nominal loading rate of 100 mg/L (six replicate flasks) 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 100 mg/L loading rate WAF test preparations at 0 and 72 hours showed measured test concentrations of less than the limit of quantification (LOQ) of the analytical method employed, determined to be 0.058 mg/L were obtained. This does not infer that no test item was in solution, just that any dissolved test item was at a concentration of less than the LOQ. 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 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF. It was considered unnecessary and unrealistic to test at loading rates in excess of 100 mg/L loading rate WAF