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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:
6 Dec 2013 - 8 Feb 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD and/or EC guidelines and according to GLP principles.
Remarks:
The observed adverse effect was considered to be due to phosphate deprivation (complexation with cerium and precipitation out of test solution). Phosphate measurements were conducted during the test to investigate this effect of complexation. Because this effect was specifically addressed in the test procedure and can not be relieved (e.g., additional phosphate additions would in their turn result in removal of all cerium from solution, consequently, the algae would not be exposed to the toxicant), there is no reason to reduce the reliability score.
Qualifier:
according to
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: Dissolved Ce concentrations were determined in at least one of the triplicate samples from each treatment per sampling time.
- Sampling method: Triplicate samples were taken from the test media of all test concentrations at the start of the test and at the end of the test (after filtration through a membrane filter, Whatman, Type NC45, pore size 0.45 µm). At the same sampling times, triplicate samples were also taken from the control. For the 72-hour stability samples, additional flasks containing the test medium with algae were incubated for each treatment under the test conditions.
- Sample storage conditions before analysis: room temperature in the dark after sampling until analysis.
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Due to the low solubility of the test item in the test media, a dispersion with the loading rate of 100 mg/L (based on anhydrous cerium ammonium nitrate corrected for purity) was prepared at the start of the test by dispersing 100.6 mg of the test item in 1000 mL of test water.

This preparation was supported by ultrasonic treatment for 15 minutes. The pH of the dispersion was adjusted from 3.0 to 6.5 using 1 M sodium hydroxide solution, followed by intense stirring on a magnetic stirrer over 3 hours in the dark, to dissolve a maximum amount of the test item in the dispersion.

The stirring period of 3 hours was chosen according to the results of a pre-experiment, which showed that the solution equilibrium was reached after this time. In this pre-experiment, similar concentrations of dissolved cerium were analytically measured in filtrates after stirring for 3 and 24 hours; the lowest cerium concentrations were measured after 96 hours of stirring.

At the end of the 3-hour stirring period, the pH of the dispersion was adjusted from 7.0 to 6.5 using 1 M hydrochloric acid solution. The pH adjustment was done to keep as much cerium in solution as possible, while maintaining favorable conditions for algal growth. Afterwards, the dispersion of the test item was filtered through a membrane filter (Schleicher & Schuell, Type NC45, pore size 0.45 µm).

The undiluted filtrate was used as the highest concentrated test medium and as a stock solution for the preparation of the test media of lower test concentrations.
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- Common name: Pseudokirchneriella subcapitata
- Strain: No. 61.81 SAG
- Source (laboratory, culture collection): Collection of Algal Cultures (SAG, Institute for Plant Physiology, University of Göttingen, 37073 Göttingen / Germany).
- Age of inoculum (at test initiation): 4 days
- Method of cultivation: cultivated under test conditions

The inoculum culture was diluted threefold one day before the start of the test to ensure that the algae were in the exponential growth phase when used to inoculate the test solutions.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
15 mg CaCO3/L
Test temperature:
20-22°C
pH:
0 h: 6.5
24 h: 7.5-7.6
48 h: 7.6-8.5
72 h: 8.2-10.4
In the undiluted filtrate, i.e. the only treatment in which a reduction of algal growth rate was observed, pH evolved from 6.5 (day 0) to 7.5 (day 1), 7.6 (day 2), and 8.2 (day 3). The relatively larger pH increase in the other treatments was due to the unaffected algal growth, which unavoidably affects pH of the test medium.
Dissolved oxygen:
not applicable
Salinity:
not applicable
Nominal and measured concentrations:
Test concentrations were: control, dilutions of 1:1000, 1:100, 1:10, and 1:1 of a filtered solution with nominal loading rate of 100 mg anhydrous cerium ammonium nitrate/L.
Dissolved Ce concentrations for all treatments were as follows:
- 0 h (start of testing): < LOQ
- 72 h (end of testing): < LOQ

At the start and end of the preliminary test, no dissolved cerium was detected in any of the treatments. Based on these results and in agreement with the sponsor, no definitive test was performed.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Type (delete if not applicable): covered with glass dish
- Material, size, headspace, fill volume: 50 mL containing 15 mL test solution
- Aeration: continuous stirring by magnetic stirrers
- Initial cells density: 5000 cells/mL

GROWTH MEDIUM
- Standard medium used: yes (reconstituted test water, AAP medium)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Reconstituted test water (AAP Medium) prepared according to the test guidelines. Analytical grade salts were dissolved in sterile purified water
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: The solution with nominal loading rate of 100 mg/L anhydrous cerium ammonium nitate was adjusted to pH 6.5 using 1M NaOH solution after stirring and before filtration. The dilutions of the filtrate were diluted using test medium adjusted to pH 6.5 using 1M HCl solution.
- Photoperiod: continuous light
- Light intensity and quality: 7400 lux

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): inhibition of growth rate and yield (biomass)
- Determination of cell concentrations: electronic particle counter (Coulter Counter, Model Z2).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 10
- Range finding study: yes
- Test concentrations: 1:1000, 1:100, 1:10, and undiluted filtrate
- Results used to determine the conditions for the definitive study: Based on the analytical results (no dissolved cerium detected) and in agreement with the sponsor, only the preliminary test was conducted.
Reference substance (positive control):
yes
Remarks:
potassium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
93 mg/L
Nominal / measured:
nominal
Conc. based on:
other: anhydrous cerium ammonium nitrate
Basis for effect:
growth rate
Remarks on result:
other: 95% CL= 89-98
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
24 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Ce
Basis for effect:
growth rate
Remarks on result:
other: 95% CL= 23-25
Details on results:
At the start and end of the preliminary test, no dissolved cerium was detected in any of the treatments. Based on these results and in agreement with the sponsor, no definitive test was performed. The biological preliminary test results were related to the theoretical concentrations of the test item based on the loading rate.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- EC50: 72-hour EC50 for the growth rate: 0.93 mg/L
Validity criteria fulfilled:
yes
Conclusions:
The effect of cerium ammonium nitrate on the growth of Pseudokirchnerella subcapitata has been investigated over a 72-h period. Only a range finding test was performed as no dissolved cerium could be detected in any of the test solutions at the start and end of testing. Nevertheless, a significant adverse effect on growth rate was observed at the highest (nominal) test concentration, i.e. an indiluted filtrate of a test solution with nominal loading rate of 100 mg/L anhydrous cerium ammonium nitrate (corresponding to 25.56 mg Ce/L). This effect is however concurrent with phosphate depletion observed in the undiluted filtrate (already at the start of the test) and therefore the observed effect is ascribed to phosphate deprivation instead of primary toxicity of cerium (no dissolved cerium observed, i.e. no exposure). The 72-h EC50 values based on growth rate were determined to be 24 mg Ce/L and 93 mg/L anhydrous cerium ammonium nitrate based on nominal concentrations.

Description of key information

The key study (Hefner, 2014c) yielded a 72-h EC50 value (growth rate-based) of 24 mg Ce/L, i.e. 93 mg/L cerium ammonium nitrate, for the unicellular green alga Pseudokirchneriella subcapitata. Because dissolved cerium could not be detected in any of the treatments at the start and end of testing, nominal concentrations were used for calculating the EC50 values. The fact that no dissolved cerium was observed (i.e., no exposure) and the fact that phosphate was depleted already at the start of testing from the test medium in which growth inhibition was observed (i.e., the test medium with the highest nominal loading rate of 100 mg/L cerium ammonium nitrate), resulted in the conclusion that the observed effect on growth is due to phosphate deprivation (in its turn due to heavy complexation of cerium with phosphates) rather than to primary cerium toxicity. Therefore, the effect concentrations will not be taken forward to classification and PNEC derivation. Further testing is not considered useful because the technical issue of phosphate depletion cannot be overcome (phosphate dosing during the test would result in 100% cerium depletion from the test medium and vice versa).

Key value for chemical safety assessment

Additional information

One study on the toxicity of cerium ammonium nitrate to algae was included in this dossier.

The key study from Hefner (2014c) is a 72-h algal growth inhibition test with Pseudokirchneriella subcapitata in which cerium ammonium nitrate was used as test item. The growth rate-based 72-h EC50 was 24 mg Ce/L (corresponding to 93 mg cerium ammonium nitrate/L). However at the start and end of the preliminary (range finding) test, no dissolved cerium was detected in any of the treatments, therefore based on these results and in agreement with the sponsor, no definitive test was performed. The biological preliminary test results were related to the theoretical concentrations of the test item based on the loading rate.

These values will not be taken forward to classification and hazard assessment because the effects on growth were observed to be concurrent with phosphate depletion in the test medium due to complexation with cerium, suggesting that the observed effect on growth inhibition is due to phosphate deprivation rather than direct toxicity of the rare earth. Similar observations were made for trivalent cerium (Ce+III) compounds such as CeCl3 and Ce(NO3)3 (see respective dossiers). For these compounds it was confirmed by modelling calculations using Visual MINTEQ v3.0 that under the conditions of the test all cerium is precipitated as CePO4 whenever phosphate is in excess and vice versa. The good agreement between measured dissolved cerium concentrations at the end of testing and the modelled dissolved cerium concentrations further increase the credibility of these model calculations. Visual MINTEQ calculations can however not be performed for tetravalent cerium (Ce+IV) because Visual MINTEQ focuses on solution chemistry, whereas Ce+IV - according to the Pourbaix diagram (see read across document attached to IUCLID Section 13) - is not stable in solution. However, depending on the conditions of the test medium (pH, redox potential of solution, see Pourbaix diagram), Ce+IV could partly be reduced to Ce+III, which can stay in its turn partly in solution depending on speciation conditions (pH, test medium composition, etc.). The addition of cerium ammonium nitrate to aqueous test media can indeed result in measurable dissolved cerium (Ce+III). This has been demonstrated in the acute toxicity experiments with fish and daphnids (Hefner, 2014a,b). In the fish experiment, a 100 mg/L solution of anhydrous cerium ammonium nitrate at pH 6 (up to maximally 6.4) resulted in a mean measured dissolved Ce concentration of 0.22 mg Ce/L, whereas in the daphnid experiment, a similar solution in daphnid test medium at pH 6.2 (up to maximally 7.2) gave rise to a mean measured dissolved Ce concentration of 3.1 µg Ce/L. This is respectively 0.86 and 0.01% of the nominal Ce loading. However, should there be a comparable reduction of Ce+IV in the algal growth medium, any Ce+III formed would most likely heavily complex with phosphates in the algal test medium and consequently precipitate from the test medium (note that no phosphates are present in the standard fish and daphnid test media). With nominally 1.18E-05 M PO43 - present in the test medium, 1.65 mg Ce/L (Ce+III) could be complexed. Based on the observations in fish and daphnid test medium, it is not expected that so much Ce+III would be formed. Any Ce+III formed can therefore be assumed to precipitate with phosphates in the test medium, explaining why no dissolved cerium could be detected in any of the treatments. The fact that all phosphates were depleted already from the start of the test in the treatment with the nominal loading rate of 100 mg/L anhydrous cerium ammonium nitrate suggests that, on a molar basis, a similar amount of Ce+III and phosphates was present in the test medium, however, part of the phosphate complexation could also be due to Ce+IV (which does not occur in dissolved state). Unfortunately no data are available on the potential contribution of undissolved Ce+IV to phosphate complexation and hence no predictions can be made on this.

Overall, based on abovementioned argumentation, it is considered justified to consider the observed effect on algal growth as a phosphate deprivation effect. This effect occurs due to the limitations of the test system and is not expected to be environmentally relevant. Phosphate deprivation effects may only occur extremely locally at discharge points but are not considered to adversely affect entire algal populations/communities in the receiving (local) ecosystem.