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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:

22 June 2017 to 21 August 2017

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:

yes

Remarks:

no impact on results or integrity of the study (see below)

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

yes

Remarks:

no impact on results or integrity of the study (see below)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

CHEMICAL ANALYSIS OF TEST LOADING RATES
- Samples were taken from the control, mineral oil control and the 100 mg/L loading rate WAF test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis. Duplicate samples were taken on each occasion and stored frozen for further analysis if necessary.

TOTAL ORGANIC CARBON ANALYSIS
- Analysis of the WAFs was also carried out by Total Organic Carbon (TOC) analysis. Samples were taken from the uninoculated control, mineral oil control and the 100 mg/L loading rate WAF test group at 0 and 72 hours for this analysis (see Annex 7, attached). Duplicate samples were taken 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 4 (attached).

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 10E03 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 – 10E05 cells/mL.

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 ºC

pH:

7.2 to 7.8 (see Table 2, attached)

Dissolved oxygen:

Not reported

Salinity:

Not applicable

Conductivity:

Not reported

Nominal and measured concentrations:

Nominal concentration of 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 5, attached) was carried out to determine whether stirring for a prolonged period produced significantly higher levels of total organic carbon, as an indicator of soluble organic substances, 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 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 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.
- Stirring was stopped after 23 hours and the mixtures allowed to stand for 1 hour. Microscopic 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) to give the 10 and 100 mg/L loading rate WAFs. Microscopic observations of the WAFs were performed after filtering and showed that the filtration had removed all of the dispersed material.
- An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (4.6 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 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 material in the 100 mg/L loading rate.

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 (250 mg) was added to the surface of 2.5 L of culture medium to give the 100 mg/L loading rate. A nominal amount of mineral oil (105.5 mg) was added to the surface of 2.5 L of culture medium to give the mineral oil control at a nominal concentration of 42.2 mg/L. After the addition of the test item and mineral oil, 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 mineral oil control and the 100 mg/L loading rate WAF. Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present. During siphoning of the 100 mg/L loading rate WAF, some large lumps of test item dispersed into the media column, as a result, this preparation was filtered through post lip filter paper as a precautionary measure. Observations conducted on the media at 0 hours showed it to be a clear colourless solution suggesting that the filtration had removed any undissolved test item.
- An aliquot (1 L) of each of the mineral oil control and the 100 mg/L loading rate WAF was separately inoculated with algal suspension (6.7 mL).
- The concentration of boron in the test preparations was verified by chemical analysis at 0 and 72 hours (see Annex 6, attached).
- Total Organic Carbon (TOC) analysis was performed on the test preparations at 0 and 72 hours (see Annex 7, attached).

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, mineral oil 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 7.48 x 105 cells per mL. Inoculation of 1 L of test medium with 6.7 mL of this algal suspension gave an initial nominal cell density of 5.00 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 – 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.

TEST ORGANISM OBSERVATIONS
- Samples were taken at 24, 48 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.

WATER QUALITY CRITERIA
- The pH of the control, mineral oil control and the 100 mg/L loading rate WAF was determined at initiation of the test and after 72 hours exposure.
- The pH was measured using a Hach HQ30d Flexi handheld meter.
- The 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
- ELx values were determined by inspection of the growth rate and yield data after 72 hours.

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; see Annex 3, attached)

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:

EL50

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 5, attached) indicated that there was no significant increase in the amount of total organic carbon 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 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.

DEFINITIVE TEST CHEMICAL ANALYSIS OF TEST LOADING RATES
- Analysis of the test preparation at 0 hours (see Annex 6, attached) showed that a measured concentration of 0.30 mg/L as boron was obtained (equivalent to 16 mg/L as test item), and at 72 hours a measured concentration of 0.33 mg/L as boron was obtained (equivalent to 17 mg/L as test item).
- A measured concentration of boron was found in the control and mineral oil control samples in the 72-hour samples, however, measured concentrations of less than the Limit of Quantification (LOQ) of the analytical method employed were obtained in the corresponding fresh samples at 0 hours. This suggests that the measured concentrations may have been as a result of post sampling contamination and as a result were considered not to have had an impact on the outcome of the test.

DEFINITIVE TEST TOTAL ORGANIC CARBON ANALYSIS
- Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 hours (see Annex 7, attached). Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that the results were all around the Limit of Quantification (LOQ), determined to be 1.0 mg C/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 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).
- From the data given in Tables 2 and 4, 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 following results were determined: ErL10 (0-72h) > 100 mg/L loading rate WAF; ErL20 (0-72h) > 100 mg/L loading rate WAF; ErL50 (0-72h) > 100 mg/L loading rate WAF where ErLx is the loading rate that reduced growth by x%.
- 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 0.625 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 0.625 mg/L loading rate WAF. Correspondingly the "Lowest Observed Effect Loading Rate" (LOEL) based on growth rate was 1.25 mg/L loading rate WAF.

INHIBITION OF YIELD
- The following results were determined: EyL10 (0-72h) > 100 mg/L loading rate WAF; EyL20 (0-72h) > 100 mg/L loading rate WAF; EyL50 (0-72h) > 100 mg/L loading rate WAF where EyLx is the loading rate that reduced yield by x%.
- Statistical analysis of the yield data was carried out as in Section 6.3.3. There were no statistically significant differences between the mineral oil control and 100 mg/L loading rate WAF, 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 108 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 5.39 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 cell concentration of the mineral oil control increased by a factor of 94 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 4.70 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 12% and for the mineral oil control cultures was 17 % 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 for the mineral oil control cultures was 2 % 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.

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 ºC throughout the test.
- The pH value of the control cultures (see Table 2, attached) was observed to increase from pH 7.7 at 0 hours to pH 7.8 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 the mixing period the control was observed to be a clear colourless solution, the mineral oil control was observed to be a clear colourless media column with oily globules of mineral oil test item on the surface and the 100 mg/L loading rate was observed to be a clear colourless media column with an oily slick of test item at the surface.
- After 23 hours stirring and a 1-hour standing period the control was observed to remain as at the start of stirring, the mineral oil control was observed to be a clear colourless water column with oily globules on the surface and the 100 mg/L loading rate was observed to be a clear colourless media column with test item solidified as beige lumps at the surface.
- Microscopic inspection of the WAFs showed no micro-dispersions or undissolved test item to be present.
- During siphoning of the 100 mg/L loading rate WAF, some large lumps of test item dispersed into the media column, as a result, this preparation was filtered through post lip filter paper as a precautionary measure. After siphoning, the control, mineral oil control and the 100 mg/L loading rate test concentration were observed to be clear, colourless solutions, whilst at 72 hours 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.

Reported statistics and error estimates:

- A Student’s t-test was carried out on the growth rate and yield data after 72 hours for the mineral oil 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 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

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.

The test item was a complex mixture containing 42.2 % mineral oil. At the Sponsors request additional vessels were prepared containing mineral oil at the same concentration as in the test item preparation; 42.2 mg/L. 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 at 0 and 72 hours showed that measured concentrations of 0.30 and 0.33 mg/L as boron (equivalent to 16 and 17 mg/L as test item) were obtained respectively.

Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 hours. Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that the results were all around the Limit of Quantification (LOQ), determined to be 1.0 mg C/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 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 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 (OECD 201 and EU Method C.3).

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

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.

The test item was a complex mixture containing 42.2 % mineral oil. At the Sponsors request additional vessels were prepared containing mineral oil at the same concentration as in the test item preparation; 42.2 mg/L. 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 at 0 and 72 hours showed that measured concentrations of 0.30 and 0.33 mg/L as boron (equivalent to 16 and 17 mg/L as test item) were obtained respectively.

Total Organic Carbon (TOC) analysis of the test preparations was performed at 0 and 72 hours. Given the background level of carbon in the control vessels and also the low level of carbon in the test vessels, it was considered that the results were all around the Limit of Quantification (LOQ), determined to be 1.0 mg C/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 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.