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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:
22.04.2016-04.01.2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was performed according to the recommended Guidelines (OECD Guidelines for Testing of Chemicals (2006) No 201, "Freshwater Alga and Cyanobacteria, Growth Inhibition Test" and Method C.3 of Commission Regulation (EC) No 761/2009) and GLP. The report fully and accurately reflects the procedures used and data generated. There were no circumstances considered to have affected the integrity of the study or the validity of the data.
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to other study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Freshwater Alga and Cyanobacteria, Growth Inhibition Test)
Version / remarks:
2006
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method C.3 (Algal Inhibition test)
Version / remarks:
(EC) No 761/2009
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Certificate is included in the study report.
Analytical monitoring:
yes
Details on sampling:
- Concentrations: control (0), 6.25, 12.5, 25, 50 and 100% v/v saturated solution.
- Sampling method: Samples were taken from the control and each 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 at 0 and 72 hours.
- Sample storage conditions before analysis: All samples were stored frozen prior to analysis.

Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: In view of the difficulties associated with the evaluation of aquatic toxicity of poorly water soluble test items, a modification of the standard method for the preparation of aqueous media was performed. An approach endorsed by several important regulatory authorities in the EU and elsewhere (ECETOC, 1996 and OECD, 2000), is to expose organisms to a saturated solution of the test item in cases where the test item is of high purity and is poorly soluble in water and in the permitted auxiliary solvents and surfactants. Using this approach, a saturated solution of the test item was prepared.

In accordance with the OECD Guidance Document No 23, in order to avoid the possible complexing of the test item and to prevent complexation-induced nutrient limitations which may impair algal growth, the culture medium used in the modified test was prepared with an additional 198.4 mg CaCl2*2H2O/L in order to increase the hardness to approximately 150 mg as CaCO3/L.

A nominal amount of test item (1100 mg) was dispersed in 11 liters of culture medium with the aid of propeller stirring at approximately 1500 rpm for 48 hours. After 48 hours the stirring was stopped and any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter (first approximate 2 liters discarded in order to pre-condition the filter) to give a 100% v/v saturated solution. A series of dilutions was made from this saturated solution to give further stock solutions of 50, 25, 12.5 and 6.25% v/v saturated solution. An aliquot (500 mL) of each of the stock solutions was separately inoculated with 2.0 mL of algal suspension to give the required test concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution.

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 titanium present in the test preparations were verified by chemical analysis at 0 and 72 hours.

- Controls: The control group was maintained under identical conditions but not exposed to the test item. A positive control used potassium dichromate as the reference item. Exposure conditions and data evaluation for the positive control were similar to those in the definitive test.
- Evidence of undissolved material (e.g. precipitate, surface film, etc): any undissolved test item was removed by filtration through a 0.2 μm Sartorius Sartopore filter prior to use to give a 100% v/v saturated solution.
Test organisms (species):
Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: CCAP 278/4
- Source (laboratory, culture collection): Liquid cultures from the Culture Collection of Algae and Protozoa (CCAP), SAMS Research Services Ltd, Scottish Marine Institute, Oban, Argyll, Scotland
- Method of cultivation: 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 constant illumination at 21 ± 1 °C. For the tests the algae were cultivated in flasks that 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. The culture medium used was prepared with an additional 198.4 mg CaCl2*2H2O/L in order to increase the hardness to approximately 150 mg as CaCO3/L. The culture medium was otherwise the same as that used to maintain the stock culture.

ACCLIMATION
- Acclimation period: 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 10^3 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 10^4 - 10^5 cells/mL.
- Culturing media and conditions (same as test or not): same as test, but without the additional 198.4 mg CaCl2*2H2O/L.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Post exposure observation period:
All test and control cultures were inspected microscopically at 72 hours.
Hardness:
Approximately 150 mg as CaCO3/L, following the recommendations given in Section 3.7 and Annex 4 of the OECD Guidance Document No 23 on the Aquatic Testing of Difficult Substances and Mixtures.
Test temperature:
Temperature was maintained at 24 ± 1 ºC throughout the test.
pH:
The pH value of the test solutions: 7.8-8.7. The pH value of the control cultures was determined to be pH 7.9 at 0 and 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.
Nominal and measured concentrations:
Analysis of the test preparations at 0 and 72 hours showed measured titanium concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.0051 mg/L. This does not infer that no titanium was in solution, just that any titanium present was at a concentration of less than the LOQ. Concentrations of the test item used in the definitive test: 6.25, 12.5, 25, 50 and 100% v/v saturated solution.
Details on test conditions:
TEST SYSTEM
- Test vessel: 250 mL glass conical flasks plugged with polyurethane foam bungs
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: glass, 250 mL, fill volume 100 mL, headspace 150 mL
- Aeration: constant shaking at approximately 150 rpm
- Initial cells density: initial nominal cell density of 5 x 10^3 cells per mL
- Control end cells density: 4.04 x 10^5 cells per mL
- No. of vessels per concentration (replicates): Three flasks for each treatment group
- No. of vessels per control (replicates): Six flasks for the control
- No. of vessels per positive control (replicates): Three flasks for each positive control concentration (potassium dichromate as the reference item at concentrations of 0.25, 0.50, 1.0, 2.0 and 4.0 mg/L)

GROWTH MEDIUM
- Standard medium used: yes, with an additional 198.4 mg CaCl2*2H2O/L in an attempt to overcome any possible chelation effects, following the recommendations given in Section 3.7 and Annex 4 of the OECD Guidance Document No 23 on the Aquatic Testing of Difficult Substances and Mixtures.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: reverse osmosis purified deionized water (Elga Optima 15+ or Elga Purelab Option R-15 BP)
- Culture medium different from test medium: Master cultures maintained in culture medium without the additional 198.4 mg CaCl2*2H2O/L that was used in the range-finding and definitive tests. Otherwise the same.
- Intervals of water quality measurement: The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure. The temperature within the incubator was recorded daily.

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: No. The pH of the control and each test preparation was determined at initiation of the test and after 72 hours exposure.
- Photoperiod: continuous illumination
- Light intensity and quality: intensity approximately 7000 lux, warm white lighting (380 – 730 nm)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: [electronic particle counter] Samples of the algal populations were taken at 0, 24, 48 and 72 hours and cell concentrations determined for each control and treatment group, using a Coulter® Multisizer Particle Counter.
- Other: 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.
Samples were also taken from the control and each test group from the bulk test preparation at 0 hours and from the pooled replicates at 72 hours for quantitative analysis, for the verification of test concentrations.

TEST CONCENTRATIONS
- Range finding study: 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% v/v saturated solution for a period of 72 hours. Two replicate flasks were used for each control and test concentration. The control group was maintained under identical conditions but not exposed to the test item. At the start and end of the range-finding test the cell density of each flask was determined using a particle counter. A sample of each test concentration was taken for chemical analysis at 0 and 72 hours in order to determine the titanium concentration present. All 0-Hour samples were stored frozen prior to analysis. Only concentrations within the range to be used for the definitive test were analyzed.
- Test concentrations in the definitive study: 6.25, 12.5, 25, 50 and 100% v/v saturated solution
- Results used to determine the conditions for the definitive study: The results of the range-finding test showed no effect on growth at the test concentrations of 0.10, 1.0 and 10% v/v saturated solution. However, growth was observed to be reduced at 100% v/v saturated solution. Based on this information test concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution were selected for the definitive test.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
> 100 other: % v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: It was not possible to calculate an ErC50 value as less than 50% inhibition of growth rate occurred at the maximum attainable test concentration of 100% v/v saturated solution. It was not possible to calculate 95% confidence limits for the EC50 values.
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
98 other: % v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
cell number
Remarks:
: Yield
Remarks on result:
other: 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.
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
50 other: % v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
50 other: % v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
cell number
Remarks:
: Yield
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
100 other: % v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
LOEC
Effect conc.:
100 other: % v/v Saturated Solution
Nominal / measured:
nominal
Conc. based on:
test mat. (dissolved fraction)
Basis for effect:
cell number
Remarks:
: Yield
Details on results:
- Exponential growth in the control (for algal test): yes. The cell concentration of the control cultures increased by a factor of 76 after 72 hours. 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.
- Observation of abnormalities (for algal test): After 72 hours there were no abnormalities detected in any of the control or test cultures at 72 hours.
- Colour differences: At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 6.25, 12.5, 25 and 50% v/v saturated solution test preparations were observed to be green dispersions with the exception of the 12.5% v/v saturated solution test replicate R1 which was a pale green dispersion. The 100% v/v saturated solution test preparations were observed to be clear colorless solutions.
- Other: 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 (see Figure 1 as an attached Illustration)
- Any stimulation of growth found in any treatment: Slight increase in growth rate and yield as compared to controls observed in all the replicates of concentrations 25 and 50 % v/v Saturated Solution, slight increase in growth rate observed in a singular replicate of concentration 12.5 % v/v Saturated Solution (see Table 3)
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: Observations on Test Item Solubility: At the start of the test all control and test cultures were observed to be clear colorless solutions. After the 72-Hour test period all control, 6.25, 12.5, 25 and 50% v/v saturated solution test preparations were observed to be green dispersions with the exception of the 12.5% v/v saturated solution test replicate R1 which was a pale green dispersion. The 100% v/v saturated solution test preparations were observed to be clear colorless solutions.
Results with reference substance (positive control):
- Results with reference substance valid? Yes
- Growth rate ErC50 (0 – 72 h): 1.5 mg/L; 95% confidence limits 1.3 – 1.7 mg/L
- Yield EyC50 (0 – 72 h): 0.79 mg/L; 95% confidence limits 0.70 – 0.89 mg/L
- Other: The results from the positive control with potassium dichromate were within the normal ranges for this reference item.
Reported statistics and error estimates:
One way analysis of variance incorporating Bartlett's test for homogeneity of variance and Dunnett's multiple comparison procedure for comparing several treatments with a control 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.

Table 1. Cell Densities and pH Values in the Definitive Test

Nominal Concentration
(% v/v Saturated Solution)
pH Cell Densities* (cells per mL) pH
0 h 0 h 24 h 48 h 72 h 72 h
Control R1 7.9 5.86E+03 1.79E+04 9.67E+04 5.03E+05 7.9
R2 5.15E+03 1.68E+04 6.99E+04 3.80E+05
R3 5.34E+03 1.64E+04 7.12E+04 5.08E+05
R4 5.52E+03 1.66E+04 4.91E+04 3.76E+05
R5 5.30E+03 1.67E+04 5.76E+04 3.70E+05
R6 4.69E+03 1.79E+04 5.58E+04 2.88E+05
Mean 5.31E+03 1.71E+04 6.67E+04 4.04E+05
6.25 R1 7.9 4.85E+03 1.76E+04 8.55E+04 3.56E+05 7.8
R2 5.25E+03 1.99E+04 7.82E+04 4.18E+05
R3 4.82E+03 1.72E+04 8.92E+04 3.86E+05
Mean 4.97E+03 1.83E+04 8.43E+04 3.87E+05
12.5 R1 7.9 4.72E+03 6.50E+03 1.47E+04 1.09E+05 7.8
R2 4.35E+03 1.59E+04 8.00E+04 3.59E+05
R3 4.86E+03 1.84E+04 9.60E+04 4.65E+05
Mean 4.65E+03 1.36E+04 6.36E+04 3.11E+05
25 R1 7.9 3.74E+03 1.68E+04 1.06E+05 4.84E+05 7.8
R2 5.26E+03 1.84E+04 8.85E+04 4.67E+05
R3 4.32E+03 1.64E+04 9.06E+04 4.92E+05
Mean 4.44E+03 1.72E+04 9.49E+04 4.81E+05
50 R1 7.9 4.53E+03 2.04E+04 1.17E+05 5.06E+05 7.8
R2 4.32E+03 2.22E+04 1.30E+05 5.58E+05
R3 4.37E+03 2.15E+04 1.05E+05 5.14E+05
Mean 4.41E+03 2.14E+04 1.17E+05 5.26E+05
100 R1 8.7 3.55E+03 2.25E+04 6.20E+04 1.17E+05 7.8
R2 3.57E+03 1.97E+04 5.28E+04 9.29E+04
R3 3.50E+03 1.92E+04 5.55E+04 9.24E+04
Mean 3.54E+03 2.05E+04 5.67E+04 1.01E+05

* Cell densities represent the mean number of cells per mL calculated from the mean of the cell counts from 3 counts for each of the replicate flasks

R1 - R6 = Replicates 1 to 6

Table 2. Daily Specific Growth Rates for the Control Cultures in the Definitive Test

Daily Specific Growth Rate (cells/mL/hour)
Day 0 - 1 Day 1 - 2 Day 2 - 3
Control R1 0.053 0.070 0.069
R2 0.051 0.059 0.070
R3 0.049 0.061 0.082
R4 0.050 0.045 0.085
R5 0.050 0.051 0.077
R6 0.053 0.047 0.068
Mean 0.051 0.056 0.075

R1 - R6 = Replicates 1 to 6

Table 3. Inhibition of Growth Rate and Yield in the Definitive Test

Nominal Concentration (% v/v Saturated Solution) Growth Rate (cells/mL/hour) Yield (cells/mL)
0 – 72 h % Inhibition 0 – 72 h % Inhibition*
Control R1 0.064 4.97E+05
R2 0.060 3.74E+05
R3 0.064 5.02E+05
R4 0.060 - 3.71E+05 -
R5 0.060 3.64E+05
R6 0.056 2.84E+05
Mean 0.061 3.99E+05
SD 0.003 8.51E+04
6.25 R1 0.059 3 3.51E+05
R2 0.061 0 4.12E+05
R3 0.060 2 3.81E+05
Mean 0.060 2 3.82E+05 4
SD 0.001 3.04E+04
12.5 R1 0.043 30 1.05E+05
R2 0.059 3 3.55E+05
R3 0.063 [3] 4.60E+05
Mean 0.055 0** 3.06E+05 2**
SD 0.011 1.83E+05
25 R1 0.064 [5] 4.80E+05
R2 0.063 [3] 4.62E+05
R3 0.064 [5] 4.88E+05
Mean 0.064 [4] 4.77E+05 [20]
SD 0.001 1.32E+04
50 R1 0.064 [5] 5.02E+05
R2 0.065 [7] 5.54E+05
R3 0.064 [5] 5.09E+05
Mean 0.064 [6] 5.22E+05 [31]
SD 0.001 2.80E+04
100 R1 0.044 28 1.14E+05
R2 0.041 33 8.93E+04
R3 0.041 33 8.88E+04
Mean 0.042 31 9.73E+04 76
SD 0.002 1.42E+04

* In accordance with the OECD test guideline only the mean value for yield for each test concentration is calculated

R1 – R6 = Replicates 1 to 6

SD = Standard Deviation

[Increase in growth as compared to controls]

** Inhibition values for 12.5% v/v saturated solution test concentration calculated based on replicates R2 and R3 only.

Validity criteria fulfilled:
yes
Remarks:
Validation criteria fulfilled: cell concentration increase, mean coefficients of variation of daily growth rates and the coefficient of variation of average specific growth rate in the control cultures
Conclusions:
The effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata has been investigated over a 72-Hour period, using an additional 198.4 mg CaCl2*2H2O/L in the culture medium for the range-finding and definitive tests in an attempt to overcome any possible chelation effects. Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values based on the nominal test concentrations of > 100 % v/v saturated solution for growth rate, as it was not possible to calculate an ErC50 value as less than 50% inhibition of growth rate occurred at the maximum attainable test concentration of 100% v/v saturated solution.

The No Observed Effect Concentration for growth rate was 50 % v/v saturated solution. The Lowest Observed Effect Concentration for growth rate was 100 % v/v saturated solution. The EC50 values for yield were 98 % v/v saturated solution, with a No Observed Effect Concentration of 50 % v/v saturated solution and a Lowest Observed Effect Concentration of 100 % v/v saturated solution for yield. According to the CLP Regulation, classification shall be based on the ErC50 (growth rate) rather than the EyC50 (yield).

Based on the results of this study it was concluded that modifications to the composition of the test media with respect to hardness had an effect on the observed inhibitory effects over the 72-Hour study period. Based on the growth curves of the performed Algal Growth Inhibition studies, the addition of CaCl2*2H2O to the test medium prolongs the period of exponential growth in the concentration 100% v/v Saturated Solution samples from 24 h (original test) to 48 h (modified test).

As there were no cytotoxic effects observed with the test item in any of the tests for genetic toxicity, skin corrosion/irritation or in the aquatic toxicity tests with Daphnia manga, it is thought unlikely that toxicity would be observed towards algae. In the eye irritation tests any adverse effects observed are also thought to be mainly a consequence of the alkaline pH of the test item rather than actual toxicity to the eye.

It is therefore suggested that the inhibitory effects seen in the Algal Growth Inhibition tests might be due to chelation of relevant nutrients by the test item from the culture medium. The test item is used in the selective removal of strontium ions from liquids and therefore has a high affinity for strontium. Strontium has physical and chemical properties similar to those of calcium, which would explain why the test item would have a high affinity for the calcium in the test medium as well.

The nutrient conditions are not static in natural waters, unlike in a strictly controlled laboratory system. Nutrient availability is quickly limited in a closed laboratory set up due to the addition of chelating agents, whereas in natural waters their availability would not be limited as chelated nutrients would be replaced by nutrients from the surrounding water. In addition, water hardness in natural habitats is expected to vary, in contrast to laboratory experiments. It is therefore justified to apply the modified algal test as basis for the classification of the test item.

Based on these results, the substance would not meet aquatic toxicity classification criteria for either Acute Category 1, Chronic Category 1 or Chronic Categories 2 and 3, as the EC50 for growth rate is larger than the maximum test concentration, i.e. the 100% v/v saturated solution. Based on the characteristics of the test item and the results from the ecotoxicity and toxicity testing, the substance will not receive a classification in the Chronic Category 4 either, as it is assumed that titanium in the substance is not going to be highly bioavailable. Therefore, based on the current data there is justification for not classifying the substance as hazardous to the aquatic environment.
Executive summary:

Introduction

The study was performed to assess the effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata. The method followed that described in 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) No 761/2009. Based on the results obtained from a previously conducted Algal Growth Inhibition Test (endpoint: Toxicity to algae and cyanobacteria.001), further tests were conducted in order to determine whether modifications made to the test media reduced the inhibitory effects previously seen.

Methods

Due to the pure, poorly water soluble nature of the test item, the test media was prepared as a saturated solution of the test item.

Following a preliminary range-finding test, Pseudokirchneriella subcapitata was exposed to solutions of the test item at nominal concentrations of 6.25, 12.5, 25, 50 and 100% v/v saturated solution (three replicate flasks per concentration) for 72 hours, under constant illumination and shaking at a temperature of 24 ± 1 °C. The test item solutions were prepared by stirring an excess (100 mg/L) of test item in culture medium using a propeller stirrer at approximately 1500 rpm for 48 hours. After the stirring period any undissolved test item was removed by filtration (0.2 μm Sartorius Sartopore filter, first approximate 2 liters discarded in order to pre-condition the filter) to produce a 100% v/v saturated solution of the test item. This saturated solution was then further diluted as necessary, to provide the remaining test groups.

An additional 198.4 mg CaCl2*2H2O/L was added in the standard culture medium for the range-finding and definitive tests in an attempt to overcome any possible chelation effects. 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 titanium concentrations of less than the limit of quantification (LOQ) of the analytical method employed were obtained which was determined to be 0.00051 mg titanium/L. This does not infer that no titanium was present in the test samples, just that any titanium which was present was at a concentration of less than the LOQ.

Exposure of Pseudokirchneriella subcapitata to the test item gave the following results based on the 0-Hour measured test concentrations:

Growth Rate - EC50 = >100* % v/v Saturated Solution, NOEC = 50 % v/v Saturated Solution, LOEC = 100 % v/v Saturated Solution

Yield - EC50 = 98 % v/v Saturated Solution, NOEC = 50 % v/v Saturated Solution, LOEC = 100 % v/v Saturated Solution

*It was not possible to calculate an ErC50 value as less than 50% inhibition of growth rate occurred at the maximum attainable test concentration of 100% v/v saturated solution.

Based on the results it was concluded that modifications to the composition of the test media with respect to hardness had an effect on the observed inhibitory effects over the 72-Hour study period.

Description of key information

Static 72-hour test for freshwater algae Pseudokirchneriella subcapitata: ErC50 > 100 % v/v Saturated Solution, NOEC = 50 % v/v Saturated Solution (OECD 201, EU Method C.3, GLP)

Key value for chemical safety assessment

Additional information

The effect of the test item on the growth of the green alga Pseudokirchneriella subcapitata has been investigated over a 72-Hour period, using an additional 198.4 mg CaCl2*2H2O/L in the culture medium for the range-finding and definitive tests in an attempt to overcome any possible chelation effects. Exposure of Pseudokirchneriella subcapitata to the test item gave EC50 values based on the nominal test concentrations of > 100 % v/v saturated solution for growth rate, as it was not possible to calculate an ErC50 value as less than 50% inhibition of growth rate occurred at the maximum attainable test concentration of 100% v/v saturated solution.

 

The No Observed Effect Concentration for growth rate was 50 % v/v saturated solution. The Lowest Observed Effect Concentration for growth rate was 100 % v/v saturated solution. The EC50 values for yield were 98 % v/v saturated solution, with a No Observed Effect Concentration of 50 % v/v saturated solution and a Lowest Observed Effect Concentration of 100 % v/v saturated solution for yield. According to the CLP Regulation, classification shall be based on the ErC50 (growth rate) rather than the EyC50 (yield).

Based on the results of this study it was concluded that modifications to the composition of the test media with respect to hardness had an effect on the observed inhibitory effects over the 72-Hour study period.

 

As there were no cytotoxic effects observed with the test item in any of the tests for genetic toxicity, skin corrosion/irritation or in the aquatic toxicity tests with Daphnia manga, it is thought unlikely that toxicity would be observed towards algae. In the eye irritation tests any adverse effects observed are also thought to be mainly a consequence of the alkaline pH of the test item rather than actual toxicity to the eye.

 

It is therefore suggested that the inhibitory effects seen in the Algal Growth Inhibition tests might be due to chelation of relevant nutrients by the test item from the culture medium. The test item is used in the selective removal of strontium ions from liquids and therefore has a high affinity for strontium. Strontium has physical and chemical properties similar to those of calcium, which would explain why the test item would have a high affinity for the calcium in the test medium as well.

 

The nutrient conditions are not static in natural waters, unlike in a strictly controlled laboratory system. Nutrient availability is quickly limited in a closed laboratory set up due to the addition of chelating agents, whereas in natural waters their availability would not be limited as chelated nutrients would be replaced by nutrients from the surrounding water. In addition, water hardness in natural habitats is expected to vary, in contrast to laboratory experiments. It is therefore justified to apply the modified algal test as basis for the classification of the test item.

Based on these results, the substance would not meet aquatic toxicity classification criteria for either Acute Category 1, Chronic Category 1 or Chronic Categories 2 and 3, as the EC50 for growth rate is larger than the maximum test concentration, i.e. the 100% v/v saturated solution. Based on the characteristics of the test item and the results from the ecotoxicity and toxicity testing, the substance will not receive a classification in the Chronic Category 4 either, as it is assumed that titanium in the substance is not going to be highly bioavailable. Therefore, based on the current data there is justification for not classifying the substance as hazardous to the aquatic environment.

However, based on the water solubility test the substance is highly insoluble in water, and can be regarded as a poorly soluble substance. Nevertheless, long-term aquatic studies are not considered necessary due to exposure considerations and aquatic toxicity being unlikely to occur. The substance is only used industrially in highly controlled industrial environments, mainly nuclear facilities and equivalent environments, where all necessary risk management measures are in place. It is not used professionally nor by consumers, nor in any context where its use could be considered as widely dispersive. Due to adopted risk management measures, emissions of the substance to the aquatic compartment during manufacture or during end use are negligible.

As no emissions of the substance to water are expected, the long-term toxicity testing on aquatic invertebrates is waived. The substance is also unreactive. Therefore, there are no data gaps in aquatic toxicity.

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