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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:
05 August 2016 to 29 December 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
Analytical monitoring:
yes
Details on sampling:
- Samples were taken from bulk test preparation at 0 hours for the control and each 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 0 and 72 hours 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.
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:
7.6 to 8.1 in the definitive test (see Table 2, attached)
Dissolved oxygen:
Not reported
Salinity:
Not applicable
Conductivity:
Not reported
Nominal and measured concentrations:
PRELIMINARY TEST
- Nominal concentrations of 0.10, 1.0, 10 and 100 mg/L.

DEFINITIVE TEST
- Nominal concentrations of 1.0, 3.2, 10, 32 and 100 mg ai/L.
Details on test conditions:
RANGE FINDING TEST
- The test concentrations to be used in the definitive test were determined by a preliminary range-finding test. The range-finding test was conducted by exposing Pseudokirchneriella subcapitata 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 used 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 and the volume adjusted to 500 mL to give a 100 mg/L stock solution from which a series of dilutions was made to give further stock solutions of 10, 1.0 and 0.10 mg/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (6.9 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 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.
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
- After the conduct of the rage-finding test, information pertaining to the purity of the test item was obtained. At the request of the Sponsor, for the purposes of the definitive test, all test concentrations were corrected for a test item water content of 41%.
- Based on the results of the range-finding test the following test concentrations were assigned to the definitive test: 1.0, 3.2, 10, 32 and 100 mg ai/L.

EXPERIMENTAL PREPARATION
- A nominal amount of test item (169 mg) was dissolved in culture medium and the volume adjusted to 1 L to give a 100 mg ai/L stock solution from which a series of dilutions was made to give further stock solutions of 32, 10, 3.2 and 1.0 mg ai/L. An aliquot (450 mL) of each of the stock solutions was separately inoculated with algal suspension (3.5 mL) to give the required test concentrations of 1.0, 3.2, 10, 32 and 100 mg ai/L.
- The stock solutions and each of the prepared concentrations were inverted several times to ensure adequate mixing and homogeneity.
- The concentration and stability of the test item in the test preparations were verified by chemical analysis at 0 and 72 hours (see Annex 4, 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 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 6.39 x 10E05 cells per mL. Inoculation of 450 mL of test medium with 3.5 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 22, 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 and each test preparation 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.

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). ECx values were then determined from the equation for the fitted line.
- It was not possible to calculate 95 % confidence limits for the EC50 values as the data generated did not fit the models available for the calculation of confidence limits.

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 (test performed between 07 December 2015 and 10 December 2015; see Annex 2, attached)
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
39 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC10
Effect conc.:
8.2 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC20
Effect conc.:
17 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
60 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
other: yield
Details on results:
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 the test concentrations of 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 ai/L were selected for the definitive test.
- Chemical analysis of the 10 and 100 mg/L test preparations at 0 and 72 hours (see Annex 4) showed measured test concentrations to range from 102 % to 111 % of nominal indicating that the test item was stable under test conditions.
- Analysis of the 1.0 mg/L test preparations at 0 and 72 hours showed measured test concentrations of 70 % and 65 % of nominal respectively were obtained. This was considered to be due to the measured concentration of 1.0 mg/L being close to the Limit of Quantification (LOQ) of the analytical method employed.

DEFINITIVE TEST VERIFICATION OF TEST CONCENTRATIONS
- Analysis of the test preparations at 0 and 72 hours (see Annex 4, attached) showed measured test concentrations to range from 81 % to 108 % of nominal and so the results were based on nominal test concentrations 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).
- Percentage inhibition values are plotted against test concentration in Figure 2 and Figure 3 (attached).
- From the data given in Table 2 and Table 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
- It was not possible to calculate ErC20 or ErC50 values as no concentration tested resulted in greater than 20 % inhibition of growth rate.
- 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 (P ≥ 0.05), between the control, 1.0, 3.2 and 10 mg ai/L test concentrations 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 ai/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on growth rate was 32 mg ai/L.

INHIBITION OF YIELD
- Statistical analysis of the yield data was carried out as in Section 6.2.3. There were no statistically significant differences (P ≥ 0.05), between the control, 1.0, 3.2 and 10 mg ai/L test concentrations however all other test concentrations were significantly different (P < 0.05) and, therefore the "No Observed Effect Concentration" (NOEC) based on yield was 10 mg ai/L. Correspondingly the "Lowest Observed Effect Concentration" (LOEC) based on yield was 32 mg ai/L.

VALIDATION CRITERIA
- The cell concentration of the control cultures increased by a factor of 162 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 8.10 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 5 % 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.
- After 72 hours there were no abnormalities detected in the control or test cultures at 1.0, 3.2 and 10 mg ai/L. However cell debris was observed to be present in the test cultures at 32 mg ai/L and very few intact cells were observed to be present in the 100 mg ai/L 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 °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 8.1 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.

OBSERVATIONS ON TEST ITEM SOLUBILITY
- 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 ai/L test cultures were observed to be pale green dispersions whilst the 32 and 100 mg ai/L test cultures were observed to be very pale green dispersions.
Results with reference substance (positive control):
- Results from the positive control with potassium dichromate were within the normal ranges for the reference item.
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 concentrations 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:
Based on growth rate, exposure of Pseudokirchneriella subcapitata to the test item gave an EL50 value of > 100 mg ai/L. The No Observed Effect Concentration was 10 mg ai/L and the Lowest Observed Effect Concentration was 32 mg ai/L. Based on yield, exposure of Pseudokirchneriella subcapitata to the test item gave an EL50 value of 60 mg ai/L. The No Observed Effect Concentration was 10 mg ai/L and the Lowest Observed Effect Concentration was 32 mg ai/L.
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

Following a preliminary range-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 active ingredient (ai)/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 and 72 hours showed measured test concentrations ranged from 81 % to 108 % of nominal and the results were based on nominal test concentrations only.

 

CONCLUSION

Based on growth rate, exposure of Pseudokirchneriella subcapitata to the test item gave an EL50 value of > 100 mg ai/L. The No Observed Effect Concentration was 10 mg ai/L and the Lowest Observed Effect Concentration was 32 mg ai/L. Based on yield, exposure of Pseudokirchneriella subcapitata to the test item gave an EL50 value of 60 mg ai/L. The No Observed Effect Concentration was 10 mg ai/L and the Lowest Observed Effect Concentration was 32 mg ai/L.

Description of key information

Based on growth rate, exposure of Pseudokirchneriella subcapitata to the test item gave an EL50 value of > 100 mg ai/L. The No Observed Effect Concentration was 10 mg ai/L and the Lowest Observed Effect Concentration was 32 mg ai/L. Based on yield, exposure of Pseudokirchneriella subcapitata to the test item gave an EL50 value of 60 mg ai/L. The No Observed Effect Concentration was 10 mg ai/L and the Lowest Observed Effect Concentration was 32 mg ai/L (OECD 201 and EU Method C.3).

Key value for chemical safety assessment

EC10 or NOEC for freshwater algae:
10 mg/L

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

Following a preliminary range-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 active ingredient (ai)/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 and 72 hours showed measured test concentrations ranged from 81 % to 108 % of nominal and the results were based on nominal test concentrations only.

 

CONCLUSION

Based on growth rate, exposure of Pseudokirchneriella subcapitata to the test item gave an EL50 value of > 100 mg ai/L. The No Observed Effect Concentration was 10 mg ai/L and the Lowest Observed Effect Concentration was 32 mg ai/L. Based on yield, exposure of Pseudokirchneriella subcapitata to the test item gave an EL50 value of 60 mg ai/L. The No Observed Effect Concentration was 10 mg ai/L and the Lowest Observed Effect Concentration was 32 mg ai/L.