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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:
1 June 2015 to 25 September 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study conducted to GLP in accordance with recognised guideline
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method C.3 (Algal Inhibition test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Specific details on test material used for the study:
-Physical state: Pale yellow paste
- Analytical purity: 100% product
- Expiry date: 24 April 2017
- Storage condition of test material: Room temperature in the dark
Analytical monitoring:
yes
Details on sampling:
RANGE-FINDING TEST
- A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the stability of the test item under test conditions.
- All samples were stored frozen prior to analysis.

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 – Verification of test concentrations.
- Samples were taken from the control and each 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 analyzed on the day of preparation.
- Duplicate samples were taken at 0 and 72 hours and stored frozen for further analysis if necessary.
Vehicle:
no
Details on test solutions:
RANGE-FINDING TEST
- The test concentrations 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 a series of nominal test concentrations of 0.10, 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.
-The test item was dissolved directly in culture medium.
- A nominal amount of test item (50 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 15 mins and the volume adjusted to 500 mL to give a 100 mg/L stock solution.
-A series of dilution was made from this stock solution to give stock solutions of 10, 1.0 and 0.10 mg/L.
-An aliquot (450 mL) of each stock solutions was separately inoculated with algal suspension (4.3 mL) to give the required test concentrations of 0.10, 1.0, 10 and 100 mg/L.
- The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
- 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, the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg/L.
- A nominal amount of test item (100 mg) was dissolved in culture medium with the aid of ultrasonication for approximately 15 mins and the volume adjusted to 1 L to give a 100 mg/L stock solution.
-A series of dilution was made from this stock solution to give stock solutions of 32, 10, 3.2 and 1.0 mg/L.
-An aliquot (500 mL) of each stock solutions was separately inoculated with algal suspension (6.2 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg/L.
- The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
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 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 ± 1 °C.
- Prior to the start of the test, sufficient master culture was added to approximately 100 mL volumes of culture media contained in conical flasks to give an initial cell density of approximately 10E3 cells/mL.
- The flasks were plugged with polyurethane foam stoppers and kept under constant agitation by orbital shaker (100 to 150 rpm) and constant illumination at 24 ± 1 °C until the algal cell density was approximately 10E4 to 10E5 cells/mL.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
None
Hardness:
Not reported
Test temperature:
24 ± 1°C
pH:
pH 7.6 to 8.6 during the definitive test (see Table 2, attached)
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Nominal and measured concentrations:
RANGE-FINDING TEST
- 0.10, 1.0, 10 and 100 mg/L (nominal)

DEFINITIVE TEST
- 1.0, 3.2, 10, 32 and 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 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 9.48 x 10E5 cells/mL. Inoculation of 500 mL of test medium with 2.6 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)
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
95 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
22 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Remarks on result:
other: (e.g. 95% CL)
Remarks:
18 – 27 mg/L
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
10 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
32 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
biomass
Details on results:
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 0.10 and 1.0 mg/L.
- However, growth was observed to be reduced at 10 and 100 mg/L.
- Based on this information, test concentrations 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 test preparation at 0 hours (see Appendix 4, attached) showed measured test concentrations to range from 72% to 84% of nominal.
- At 72 hours, a decline in the range of 47% to 76% of nominal in measured test concentration was observed.
- Examination of the data could find no cause for the slightly low measured concentrations obtained at 0 hours.
-A 100 mg/L test sample was prepared and analysed immediately to confirm whether it was the frozen storage process which resulted in the low measured concentration.
-This sample gave a measured concentration of 94% of nominal indicating that it may have been the freeze/thaw process which resulted in below nominal concentrations.
-As such all samples were analyzed on the day of preparation for the definitive test.

VERIFICATION OF TEST CONCENTRATIONS FOR DEFINITIVE TEST
- Analysis of the test preparations at 0 hours (see Appendix 4) showed measured test concentrations to range from 80 to 97% of nominal.
- At 72 hours, all measured concentrations were within ± 20% of the nominal concentrations with the exception of the 100 mg/L test sample where a measured concentration of 79% of nominal was obtained.
- Given that the overall concentration to which the algae were exposed to was 88% of nominal, this was considered to be insignificant and as such all results were calculated based on the nominal test concentrations 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).
- Percentage inhibition values are plotted against test concentration in Figure 2 and Figure 3 (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.

INHIBITION OF GROWTH RATE FOR THE DEFINITIVE TEST
- ErC10 (0-72 h) : 17 mg/L
- ErC20 (0-72 h) : 33 mg/L
- ErC50 (0-72 h) : 95 mg/L* (* it was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits).
- Where ErCx is the test concentration that reduced growth rate by x %.

INHIBITION OF YIELD FOR THE DEFINITIVE TEST
- EyC10 (0-72 h) : 7.7 mg/L
- EyC20 (0-72 h) : 9.9 mg/L
- EyC50 (0-72 h) : 22 mg/L; 95% confidence limits 18-27 mg/L
- Where EyCx is the test concentration 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 all test concentrations 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, 1.0, 3.2 and 10 mg/L test concentrations (P ≥ 0.05). However, all other test concentrations were significantly different (P < 0.05) and, therefore, the No Observed Effect Concentration (NOEC) based on growth rate was 10 mg/L.
-The Lowest Observed Effect Concentration (LOEC) based on growth rate was 32 mg/L.

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, 1.0, 3.2 and 10 mg/L test concentrations (P ≥ 0.05) and, therefore, the No Observed Effect Concentration (NOEC) based on yield was 10 mg/L.
- The Lowest Observed Effect Concentration (LOEC) based on growth rate was 32 mg/L.

VALIDATION CRITERIA

- Data show that the cell concentration of the control cultures increased by a factor of 146 after 72 hours (mean cell density of control at 0 h was 4.73 x 10E3 cells/mL; mean cell density of control at 72 h was 6.91 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 30% 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 1% 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 at 1.0, 3.2 and 10 mg/L, however cell clumping was observed in the 32 mg/L test cultures whilst cell clumping and enlarged cells were observed in the 100 mg/L 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 7.6 at 0 hours to pH 8.6 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 guidelines.

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 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, 1.0, 3.2 and 10 mg/L test cultures were observed to be green dispersions. The 32 mg/L test cultures were observed to be pale green dispersions whilst the 100 mg/L test cultures were observed to be extremely pale green dispersions.

Validity criteria fulfilled:
yes
Conclusions:
The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-hour period and gave the following results:
Growth rate: EC50 = 95 mg/L; NOEC = 10 mg/L; LOEC = 32 mg/L
Yield: EC50 = 22 mg/L (95% confidence limits of 18 - 27 mg/L); NOEC = 10 mg/L; LOEC = 32 mg/L
It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.
Executive summary:

INTRODUCTION

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

Following a preliminary rang-finding test,Pseudokirchneriella subcapitatawas exposed to an aqueous solution of the test item at concentrations 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 Coulter Multisizer Particle Counter.

RESULTS

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 80% to 97% of nominal. All measured concentrations at 72 hours were within ± 20% of the nominal concentrations with the exception of the 100 mg/L test sample where a measured concentration of 79% of nominal was obtained. However, given the overall concentrations to which the algae were exposed to was 88% of nominal, this was considered to be insignificant and as such all results were calculate based on the nominal test concentrations only. A decline in measured test concentration was observed at 72 hours to 0.80 mg/L.

Exposure ofPseudokirchneriella subcapitatato the test item gave the following results:

Growth rate: EC50 = 95 mg/L; NOEC = 10 mg/L; LOEC = 32 mg/L

Yield: EC50 = 22 mg/L (95% confidence limits of 18 - 27 mg/L); NOEC = 10 mg/L; LOEC = 32 mg/L

Description of key information

The effect of the test item on the growth of Pseudokirchneriella subcapitata has been investigated over a 72-hour period and gave the following results:

Growth rate: EC50 = 95 mg/L; NOEC = 10 mg/L; LOEC = 32 mg/L

Yield: EC50 = 22 mg/L (95% confidence limits of 18 - 27 mg/L); NOEC = 10 mg/L; LOEC = 32 mg/L

It was not possible to calculate 95% confidence limits for the ErC50 value as the data generated did not fit the models available for the calculation of confidence limits.

Key value for chemical safety assessment

EC50 for freshwater algae:
95 mg/L
EC10 or NOEC for freshwater algae:
10 mg/L

Additional information

INTRODUCTION

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 rang-finding test, Pseudokirchneriella subcapitata was exposed to an aqueous solution of the test item at concentrations 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 Coulter Multisizer Particle Counter.

RESULTS

Analysis of the test preparations at 0 hours showed measured test concentrations to range from 80% to 97% of nominal. All measured concentrations at 72 hours were within ± 20% of the nominal concentrations with the exception of the 100 mg/L test sample where a measured concentration of 79% of nominal was obtained. However, given the overall concentrations to which the algae were exposed to was 88% of nominal, this was considered to be insignificant and as such all results were calculate based on the nominal test concentrations only. A decline in measured test concentration was observed at 72 hours to 0.80 mg/L.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results:

Growth rate:EC50 = 95 mg/L (95% confidence limits not determinable); NOEC = 10 mg/L and LOEC = 32 mg/L.

Yield: EC50 = 22 mg/L (95% confidence limits of 18 – 27 mg/L); NOEC = 10 mg/L and LOEC = 32 mg/L.

EC10 = 17 mg/L