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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:
20 August 2015 to 8 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP in accordance with recognised guideline.
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.
- Only concentrations within the range to be used for the definitive test were analysed.

DEFINITIVE TEST - TEST ORGANISM OBSERVATIONS
- Samples were taken at 0, 24, 48 and 72 hours and the cell densities 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 test.
- The shape and size of the algal cells was inspected microscopically and any abnormalities recorded.

DEFINITIVE TEST - CHEMICAL ANALYSIS OF TEST LOADING RATES
- Samples were taken from the control and the 100 mg/L loading rate WAF test group from the bulk test preparation 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.
Vehicle:
no
Details on test solutions:
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 subscapitata 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.
- A nominal amount 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 addition of test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface.
- Stirring was stopped after 23 hours and the mixtures were allowed to stand for one hour.
- Visual observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was 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 was removed by mid-depth siphoning (the first 75 to 100 mL discarded).
- The WAFs were then passed through filter paper to give the 10 and 100 mg/L loading rate WAFS.
- Microscopic inspection of the WAFs showed no micro-dispersions of test item to be present.
- An aliquot (500 mL) of each of the loading rate WAFs was separately inoculated with algal suspension (5.6 mL) to give the required test concentrations of 10 and 100 mg/L loading rate WAF.
- The control group was maintained under identical conditions but not exposed to the test item.
- At the start of the range-finding test a sample of each test and control culture was removed and the cell density determined using a Coulter Multisizer Particle Counter.
- The flasks were then plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
- After 72 hours, the cell density of each flask was determined using a Coulter Multisizer Particle Counter.

DEFINITIVE TEST
- Based on the results of the range-finding tests, a “limit test” was conducted at a single loading rate of 100 mg/L.
- 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 respectively.
- After addition of test item, the culture medium was stirred by magnetic stirrer using a stirring rate such that a vortex was formed to give a dimple at the water surface.
- Stirring was stopped after 23 hours and the mixture was allowed to stand for one hour.
- Observations made on the WAFs indicated that a significant amount of dispersed test item was present in the water column and hence it was 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 was removed by mid-depth siphoning (the first 75 to 100 mL discarded) to give the 100 mg/L loading rate WAF.
- Microscopic inspection of the WAF after filtering showed no micro-dispersions test item to be present.
- An aliquot (1 L) of the WAF was inoculated with algal suspension (9.3 mL) to give the required test concentrations of 100 mg/L loading rate WAF.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
- The test was carried out using Pseudokirchneriella subscapitata strain CCAP 278/4.
- Liquid cultures of Pseudokirchneriella subscapitata were obtained from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institue, Oban, Argyll, Scotland.
- Master cultures were maintained in the laboratory by the periodic replenishment of culture medium.
- The master cultures were maintained in the laboratory under constant aeration and illumination at 21 ± 1 °C.
- Prior to the start of the test, sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10E3 cells/mL.
- The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10E4 to 10E5 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
yes
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
Not reported
Test temperature:
24 ± 1°C
pH:
pH 7.7 to 8.2 during the 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
- 100 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 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 characterised by a cell density of 5.39 x 10E5 cells/mL. Inoculation of 1 L of test medium with 9.3 mL of this algal suspension gave an initial nominal cell density of 5 x 10E3 cells/mL and had no significant dilution effect on the final test concentration.
- The flasks were plugged with polyurethane foam bungs and incubated (INFORS Multitron Version 2 incubator) at 24 ± 1 °C under continuous illumination (intensity approximately 7000 lux) provided by warm white lighting (380 to 730 nm) and constantly shaken at approximately 150 rpm for 72 hours.
Reference substance (positive control):
yes
Remarks:
potassium dichromate conducted between 12 May 2015 and 15 May 2015 (see Appendix 2, attached)
Key result
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EL50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
NOELR
Effect conc.:
100 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 Appendix 4, attached) indicated that there was no significant increase in the amount of dissolved test item obtained when the preparation period was extended for longer than 24 hours.
- The WAF used for testing was therefore prepared using a stirring period of 23 hours followed by a 1 hour settlement period.

RANGE-FINDING TEST
- Cell densities and percentage inhibition of growth values are given in Table 1 (attached).
- The results showed no effect on growth at 10 and 100 mg/L loading rate WAF.
- However, growth was observed to be reduced at 1.0, 10 and 100 mg/L loading rate WAF.
- Based on this information, a single loading rate of sixe replicates of 100 mg/L was selected for the definitive test.
- This test conforms to a “limit test” to confirm that no effect on growth as observed.
- Chemical analysis of the 100 mg/L test preparation at 0 (see Appendix 5, attached) showed a measured test concentration of 0.90 mg/L.
- At 72 hours, a decline to 0.47 mg/L in measured test concentration was observed.
- The decline indicated that the test item was either unstable and/or adsorbing to the algal cells present.

CHEMICAL ANALYSIS OF TEST LOADING RATES FOR DEFINITIVE TEST
- Chemical analysis of the 100 mg/L loading rate WAF at 0 hours (see Appendix 5) showed a measured test concentrations of 1.5 mg/L.
- At 72 hours, a decline to 0.80 mg/L in measured test concentration was observed.
- The dissolved test item may have been one or several components of the test item.
- Given that the toxicity cannot be attributed to a single component or a mixture of components but to the test item as a whole, the results were based on nominal loading rates only.

GROWTH DATA FOR DEFINITIVE TEST
- 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 unnecessary to test at loading rates in excess of 100 mg/L.

INHIBITION OF GROWTH RATE FOR THE DEFINITIVE TEST
- ErL10 (0-72 h) was determined to be >100 mg/L loading rate WAF.
- ErL20 (0-72 h) was determined to be >100 mg/L loading rate WAF.
- ErL50 (0-72 h) was determined to be >100 mg/L loading rate WAF.
- Where ErLx is the loading rate that reduced growth rate by x %.

INHIBITION OF YIELD FOR THE DEFINITIVE TEST
- EyL10 (0-72 h) was determined to be >100 mg/L loading rate WAF.
- EyL20 (0-72 h) was determined to be >100 mg/L loading rate WAF.
- EyL50 (0-72 h) was determined to be >100 mg/L loading rate WAF.
- Where EyLx is the loading rate that reduced yield by x %.
Reported statistics and error estimates:
INHIBITION OF GROWTH RATE IN THE DEFINITIVE TEST
- 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 between the control and 100 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 100 mg/L loading rate WAF.

INHIBIBITION OF YIELD IN THE DEFINITIVE TEST
- Statistical analysis of the yield data was carried out as described for assessment of growth rate inhibition.
- 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

- Data show that the cell concentration of the control cultures increased by a factor of 113 after 72 hours (mean cell density of control at 0 h was 4.98 x 10E3 cells/mL; mean cell density of control at 72 h was 5.63 x 10E5 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 6 % and hence satisfied the validation criterion given in the OECD Guideline which states the mean time 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 DURING THE DEFINITIVE TEST

- All test and control cultures were inspected microscopically at 72 hours.

- After 72 hours there were no abnormalities detected in any of the control or test cultures.

WATER QUALITY CRITERIA DURING THE DEFINITIVE TEST

- 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) was observed to increase from pH 7.7 at 0 hours to pH 8.1 at 72 hours. The pH deviation in the control cultures was less than 1.5 pH units after 72 hours and was therefore within the limits given in the test guideline.

VORTEX DEPTH MEASUREMENTS FOR THE DEFINITIVE TEST

- 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 FOR THE DEFINITIVE TEST

- Observations on the test media were carried out during the mixing and testing of the WAF.

- At both the start of mixing and after stirring, and following a 1-hour standing period, the 100 mg/L loading rate WAF was observed to have formed a clear colourless media column with test item floating at the surface, and test item dispersed throughout the media column after the standing period.

- Given the presence of dispersed test item in the media column, it was considered appropriate to remove aqueous phase by filtration through a glass wool plug.

-Microscopic examination of the WAF after filtration showed there to be no micro-dispersions of the test item present.

- At the start of the test all control and 100 mg/L loading rate WAG 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.

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated and 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.
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 rang-finding tests, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fractions (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 at 0 hours showed a measured test concentration of 1.5 mg/L. A decline in measured test concentration was observed at 72 hours to 0.80 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 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.

Description of key information

Exposure of Pseudokirchneriella subcapitatato 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 (OECD 201 and EU Method C.3).

Key value for chemical safety assessment

EC50 for freshwater algae:
100 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 rang-finding tests, Pseudokirchneriella subcapitata was exposed to a Water Accommodated Fractions (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 at 0 hours showed a measured test concentration of 1.5 mg/L. A decline in measured test concentration was observed at 72 hours to 0.80 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 values of greater than 100 mg/L loading rate WAF. The No Observed Effect Loading Rate was 100 mg/L loading rate WAF.