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Toxicity to aquatic algae and cyanobacteria

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Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
As the toxicity of cerium compounds is mainly determined by the extent to which the compound gives rise to bioavailable cerium in the water column, and equally water soluble cerium compounds can be expected to give rise to similar levels of bioavailable cerium under similar conditions, aquatic toxicity data obtained with water soluble cerium compounds are lumped for hazard assessment purposes. In the case of cerium trinitrate, a study on the toxicity of cerium trichloride to algae (Hefner, 2013) was therefore included in this dossier. This study was considered as the key study for endpoint coverage. Another study performed with cerium trichloride (Bringmann and Kühn, 1959) was added as supporting information. Since the latter study is considered of much lower reliability (Klimisch 3), it is not included in the read across results overview below. The full read across justification is attached to IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.63 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: used for recalculation of the results to Ce(NO3)3
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.46 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: used for recalculation of the results to Ce(NO3)3
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.47 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: Ce(NO3)3, anhydrous
Basis for effect:
growth rate
Remarks on result:
other: recalculated
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
1.07 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: Ce(NO3)3, anhydrous
Basis for effect:
growth rate
Remarks on result:
other: recalculated
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 13 Dec 2013 - 11 Mar 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
Deviations:
yes
Remarks:
the phosphate concentration in the test media was analysed, in order to assess potential phosphate depletion.
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
yes
Details on sampling:
- Concentrations: 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
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 153 mg/L (corresponding to 100 mg/L anhydrous cerium trichloride) was sonicated for 15 minutes and continuously stirred at room temperature in the dark over 3 hours. The pH was adjusted to 6.5 and the dispersion was filtered. The dilutions 1:10, 1:22, 1:46, 1:100 and 1:220 of the undiluted filtrate with the loading rate of 100 mg/L anhydrous cerium trichloride were used as test media. Additionally, a control was tested in parallel.

No auxiliary solvent or emulsifier was used.

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 test item concentrations were analytically determined in filtrates after stirring for 3, 24 and 96 hours.

At the end of the 3-hour stirring period, the pH of the dispersion was adjusted from 6.2 to 6.5 using 1 M NaOH solution. 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 a stock solution for the preparation of the test media of lower test item concentrations. For this preparation, the filtrate was diluted with test water (pH adjusted to 6.5 using 1 M HCl). The test media were prepared just before the start of the test (= start of exposure).
Test organisms (species):
Pseudokirchneriella subcapitata (previous names: Raphidocelis subcapitata, Selenastrum capricornutum)
Details on test organisms:
TEST ORGANISM
- 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 (preculture set up 4 days before the start of testing, and diluted 3-fold one day before the start of testing to assure that the algae were in the exponential phase.

ACCLIMATION
- Culturing media and conditions (same as test or not): same as test
- Any deformed or abnormal cells observed: no
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
15 mg/L CaCO3
Test temperature:
23-24°C
pH:
0 h: 6.6-7.1
24 h: 6.6-7.7
48 h: 7.3-8.6
72 h: 7.5-10.2
pH increase was the largest in the control and the treatments where algal growth was not or only minimally affected.
Dissolved oxygen:
not applicable
Salinity:
not applicable
Nominal and measured concentrations:
Test concentrations were: control, dilutions of 1:220, 1:100, 1:46, 1:22, and 1:10 of a filtered solution with nominal loading rate of 100 mg anhydrous CeCl3/L.
Dissolved Ce concentrations were as follows:
- 0 h (start of testing): < LOQ, 0.194, 0.451, 0.925, 1.97 and 4.39 mg Ce/L
- 72 h (end of testing): < LOQ, 0.0017, < LOQ, 0.022, 0.724 and 2.90 mg Ce/L

Over the test duration of 72 h, the concentrations of dissolved Ce decreased (recovery 0.1 to 66% of initially measured concentrations).
Due to the effect of phosphate depletion (see further, when phosphate is in excess of dissolved Ce, all Ce will be precipitated, when dissolved Ce is in excess of phosphate, all phosphate will be precipitated), it was decided to relate the effect concentrations to the initially measured concentrations of dissolved Ce.
Details on test conditions:
TEST SYSTEM
- Test vessel: Erlenmeyer flask
- Type (delete if not applicable): open
- 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 CeCl3 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) :
- Determination of cell concentrations: electronic particle counter (Coulter Counter, Model Z2).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.1
- Range finding study: yes, control + undiluted filtrate (100 mg/L anhydrous CeCl3 as nominal loading rate) and dilutions of 1:10, 1:100 and 1:1000
- Results used to determine the conditions for the definitive study: No significant growth inhibition at 1:100 dilution, and 99.0% growth inhibition at 1:10 dilution.
Reference substance (positive control):
yes
Remarks:
potasssium dichromate
Key result
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
0.63 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth rate
Remarks on result:
other: 95% CL = 0.60-0.66
Key result
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.46 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element (dissolved fraction)
Basis for effect:
growth rate
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
1.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: cerium chloride, anhydrous
Basis for effect:
growth rate
Remarks on result:
other: 95% CL = 1.0-1.2
Duration:
72 h
Dose descriptor:
NOEC
Effect conc.:
0.81 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: cerium chloride, anhydrous
Basis for effect:
growth rate
Details on results:
OTHER EFFECT PARAMETERS
Growth rate-based:
- 72-h LOEC = 0.94 mg Ce/L
- 72-h EC20 = 0.48 mg Ce/L
Yield-based:
- 72-h NOEC = 0.20 mg Ce/L
- 72-h LOEC = 0.46 mg Ce/L
- 72-h EC10 = 0.28 mg Ce/L
- 72-h EC20 = 0.35 mg Ce/L
- 72-h EC50 = 0.52 mg Ce/L

PHOSPHATE DEPLETION
- Phosphate content (mg/L) was measured at the start of testing as well as after 24, 48 and 72 h.
- Limit of quantification was 0.2 mg/L.
- Phosphate levels overall decreased with time due to algal growth in those treatments where no significant growth inhibition was observed.
- In the 1:100 dilution, some phosphate was detectable (but below the LOQ) at the start of testing but no phosphate was detected after 24, 48 and 72 h.
- In the 1:46 dilution and higher test item concentrations, no phosphate was detectable from the start of testing onwards. This suggests that the algal growth inhibition effect is concurrent with phosphate depletion and hence a phosphate deprivation effect rather than an effect of exposure to the dissolved rare earth.

OTHER OBSERVATIONS
- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): no abnormalities observed
- Colour differences: no
- Any stimulation of growth found in any treatment: no
- Effect concentrations exceeding solubility of substance in test medium: no
Results with reference substance (positive control):
ErC50 (0-72h): 0.93 mg/L. The results were within the normal ranges.
Reported statistics and error estimates:
The EC10, EC20 and EC50 values and their 95% confidence intervals were calculated by Probit Analysis using linear maximum likelihood regression and linear weighted regression, respectively.
For determination of NOEC and LOEC average growth rate and yield at the test concentrations were compared to the control values by Williams t-test.

Analysis of phosphate:

At 0 hours, phosphate concentration decreased with increasing test concentration:

  Phosphate concentration (mg/L) at 0h 24h  48h   72h  
control   0.4  0.3  < 0.2  < 0.2
 1:220  0.3  0.2  < 0.2  < 0.2
 1:100  0.1  < 0.2  < 0.2  < 0.2
 1:46  < 0.2  < 0.2  < 0.2  < 0.2
 1:22  < 0.2  < 0.2  < 0.2  < 0.2
  1:10  < 0.2  < 0.2  < 0.2  < 0.2

The phosphate concentration in the test media was measured daily and was shown to decrease with higher concentrations of test item. This is thought to be due to the formation of complexes of the rare earth elements with the dissolved phosphate in the test water (which is a well-known behavior of rare earth elements in the environment). The amount of available phosphate also decreased over time, as it was metabolized by the algae.

Validity criteria fulfilled:
yes
Conclusions:
The effect of cerium trichloride on the growth of Pseudokirchnerella subcapitata has been investigated over a 72h period. The 72-hour NOEC based on growth rate was determined to be 0.46 mg Ce/L (corresponding to 0.81 mg CeCl3/L). The 72-hour EC50 based on growth rate was determined to be 0.63 mg Ce/L (corresponding to 1.1 mg CeCl3/L). Reduced growth rate was most probably due to phosphate depletion rather than due to primary toxicity of the test substance.
Executive summary:

The acute toxicity of cerium trichloride to algae was determined in a 72-hour static test according to the OECD guideline 201 and in compliance with the principles of Good Laboratory Practice.

At the end of the test, 0.1 to 66% of the initially measured concentrations of cerium were found. This is most likely due to the formation of complexes of cerium with the dissolved phosphate in the test water (which is a well-known behavior of rare earth elements in the aquatic environment). At the lowest test concentrations, phosphate was in excess of cerium and, therefore, almost all of the cerium precipitated, whereas phosphate was still measurable. At the higher concentrations, cerium was in excess and, consequently, higher dissolved cerium concentrations were measured, while the entire phosphate was depleted from the start. The 72-hour NOEC based on growth rate was determined to be 0.46 mg Ce/L (corresponding to 0.81 mg CeCl3/L). The 72-hour EC50 based on growth rate was determined to be 0.63 mg Ce/L (corresponding to 1.1 mg CeCl3/L). These reduced growth rates were most probably due to phosphate depletion rather than due to primary toxicity of the test substance.

Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Doubts about extent of exposure to dissolved cerium in the test medium. Test conducted according to method similar to OECD 201. Test results should be used with caution as 1) materials and methods are not clearly described, 2) Ce was not determined in the test media, and 3) it is not clear to what extent the observed effect was an indirect effect such as phosphate deprivation through precipitation of phosphate complexes of Ce.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 201 (Alga, Growth Inhibition Test)
GLP compliance:
no
Analytical monitoring:
no
Details on sampling:
no analytics performed
Vehicle:
no
Details on test solutions:
Test substances were diluted with water from the river Havel at the village Gatow. Test solutions were prepared in closed glass tubes (18 mm diameter).
Test organisms (species):
Scenedesmus sp.
Details on test organisms:
- Algae were cultured in 100 mL medium (570 mg/L KNO3, 200 mg/L CaSO4, 140 mg/L KH2PO4, 90 mg/L MgSO4, 3 mg/L FeCl3) in daylight, at 24°C in 300-mL Erlenmeyer flasks.
- Algae cultures are prepared one week before the test, by adding 10 mL cell suspension (from stock culture) in 100 mL culture medium. These cultures are kept under continuous light (40W Osram light bulbs) at 24°C.
- Before preparing test cultures, the cells are filtered using a membrane filter (0.6-1 µm) and washed with distilled water. The degree of turbidity of the algae in 25 mL distilled water is measured, and expressed as amount of Kieselgur. At the start of the test, turbidity is 20 mg/L Kieselgur.
- During the 96-h exposure period, algae are continuously lighted and stirred at 24°C. After 96 h the degree of turbidity is measured again.
Test type:
static
Water media type:
freshwater
Limit test:
no
Hardness:
12° dH

Test temperature:
23°C
pH:
7.5
Dissolved oxygen:
not applicable
Salinity:
not applicable
Nominal and measured concentrations:
no data
Details on test conditions:
The degree of cell multiplication was measured by photoelectric measurements of the degree of turbidity (biomass).
Duration:
96 h
Dose descriptor:
LOEC
Effect conc.:
0.15 - 0.2 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
biomass
Reported statistics and error estimates:
none
Validity criteria fulfilled:
not applicable
Conclusions:
The lowest concentration at which cerium(3+) trichloride exerts an adverse effect on biomass (growth) of Scenedesmus algae is 0.15-0.20 mg Ce/L. Results cannot be considered reliable because Ce was not analytically determined in the test media, and because it is not clear to what extent the observed effect was an indirect effect such as a phosphate deprivation effect.
Endpoint:
toxicity to aquatic algae and cyanobacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
However, since only biomass-based effect concentrations were reported and Ce concentrations were not analytically determined, the results cannot be considered reliable. Further, it is not clear to what extent the observed effects are due to phosphate deprivation through precipitation of phosphate complexes with Ce.
Qualifier:
according to guideline
Guideline:
ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)
GLP compliance:
not specified
Analytical monitoring:
not specified
Vehicle:
no
Test organisms (species):
Skeletonema costatum
Details on test organisms:
TEST ORGANISM
- Source (laboratory, culture collection): Laboratory of Mariculture, Ocean University of China, Qingdao
- Age of inoculum (at test initiation): 3 days
- Initial cell density 10000 cells/mL (using plate count method)
Test type:
static
Water media type:
saltwater
Limit test:
no
Total exposure duration:
72 h
Hardness:
not reported
Test temperature:
25°C
pH:
initially adjusted to pH 8.0
Dissolved oxygen:
not applicable
Salinity:
not reported (marine)
Nominal and measured concentrations:
Nominal: control + 5, 10, 20, 30, and 40 µmol/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 125-mL glass Erlenmeyer flasks, sterilized (steam, UV irradiation)
- Test vessels randomly arranged in photoautotrophic machine (Eyelatron FL1-160)
- Test flasks were shaken for 5 min each 12 h (110 rpm)
- Initial cells density: 10000 cells/mL
- Control end cells density: not reported
- No. of vessels per concentration (replicates): 3
- No. of vessels per control (replicates): 3

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: artificial seawater
- Test medium: artificial seawater enriched with a modification of f/2 medium, without EDTA
- Composition modified f/2 medium: 74.8 mg/L NaNO3, 4.4 mg/L NaH2PO4, 1 mL trace metals solution and 1 mL vitamin solution (in 1 L of test medium).

OTHER TEST CONDITIONS
- Sterile test conditions: yes
- Adjustment of pH: test solutions were adjusted to pH 8.0 with 1 M NaOH or HCl
- Photoperiod: 14L:10D
- Light intensity and quality: 6000 lux
- Salinity (for marine algae): not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
- Determination of cell concentrations: measured at 0 and 72 h using a hemacytometer

TEST CONCENTRATIONS
- Spacing factor for test concentrations: no fixed spacing factor
- Range finding study: yes, covering several orders of magnitude of test substance concentration
- Results used to determine the conditions for the definitive study: yes
Duration:
72 h
Dose descriptor:
EC50
Effect conc.:
4.16 mg/L
Nominal / measured:
not specified
Conc. based on:
element
Basis for effect:
biomass
Remarks:
area under the growth curve
Reported statistics and error estimates:
EC50 estimated using probit analysis on growth data at the 3rd day.
95% confidence intervals were calculated using the equation given by Neter et al., 1985.
ANOVA and Tukey multiple comparison method were used (p < 0.05).
Validity criteria fulfilled:
not specified
Conclusions:
In this study, the toxicity of Ce (added as Ce(NO3)3) to the marine unicellular alga Skeletonema costatum was investigated in a 72-h growth inhibition study. The 72-h EC50 of Ce was reported to be 4.16 mg Ce/L. However, since no analytical verification was performed, results can not be considered reliable. Further, it is not clear to what extent the observed effects were due to phosphate deprivation.

Description of key information

The key study (Hefner, 2013) yielded a 72-h NOEC and EC50 value (growth rate-based) of 0.46 and 0.63 mg Ce/L for the unicellular green alga Pseudokirchneriella subcapitata (cerium added as CeCl3). However, these values will not be taken forward to PNEC derivation because the effects on growth were observed to be concurrent with phosphate depletion in the test medium due to complexing with cerium, suggesting that the observed effect on growth inhibition is due to phosphate deprivation rather than direct toxicity of the rare earth. This is confirmed by modelling calculations using Visual MINTEQ v3.0. 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). Finally, the results will not be used for classification purposes of Ce(NO3)3 either.

Key value for chemical safety assessment

Additional information

Three studies on the toxicity of cerium to algae were included in this dossier.

The first study of Bringmann and Kühn (1969) investigated the effects of CeCl3 on growth of Scenedesmus algae. The lowest concentration at which cerium exerted an adverse effect on biomass (growth) was 0.15-0.20 mg Ce/L. Results can however not be considered reliable because cerium concentrations in test media were not analytically verified, and because it is not clear to what extent the observed effect was an indirect effect such as a phosphate deprivation effect.

In a second study (Tai et al., 2010), the toxicity of cerium (added as Ce(NO3)3) to the marine unicellular alga Skeletonema costatum was investigated in a 72-h growth inhibition study. The 72-h EC50 of cerium was reported to be 4.16 mg Ce/L. However, since no analytical verification of test substance or element concentrations was performed, results can not be considered reliable. Further, it is not clear to what extent the observed effects were due to phosphate deprivation.

The third study is the study of Hefner (2013). This study was a 72-h algal growth inhibition test with Pseudokirchneriella subcapitata in which CeCl3 was used as test item. The study was assigned a klimisch score of 2 because the study was used as read-across purposes in this dossier. The growth rate-based 72-h EC50 and NOEC were 0.63 and 0.46 mg Ce/L, respectively.

Due to the known issue with phosphate complexing by rare earth elements in algal growth inhibition tests, phosphate concentrations were monitored during this study. It was observed that at the lower test concentrations, all cerium was precipitated shortly after addition to the test medium, because phosphate was in excess. However, at the higher test concentrations, cerium was in excess, complexing all phosphate in the test medium. Algal growth was completely impeded at those test concentrations where all phosphate had disappeared from the test medium from the start of the test already. The ErC50 value (0.63 mg Ce/L) was therefore somewhat lower than the lowest test concentration at which complete immediate phosphate depletion occurred. Therefore, the observed effects are considered to be mostly due to phosphate deprivation instead of direct cerium toxicity. Since all algal growth inhibition tests need to be performed in test media contaning a phosphate source, testing is considered technically not feasible if reliable results on cerium toxicity to algae are to be obtained. Further, the phosphate depletion effect is not considered an environmentally relevant effect, since it would only occur very locally where point source emissions or accidental releases occur, and will never affect an entire ecosystem. Therefore, the effects on algal growth are not taken into account for PNEC derivation and classification. As the study was well conducted and the “phosphate issue” was investigated, it is totally justified to attribute a high Klimisch score to the study.

To further argument the conclusion that the observed toxicity is due to disappearance of phosphate from the test medium as a result of complexing with the rare earth, modelling calculations have been performed using Visual MINTEQ v3.0 using data from the algal growth inhibition study of Hefner (2013). Modelling of cerium speciation was performed as follows:

- All components of the test medium (nominal concentrations) were added to the modelling solution.

- The pH was set to 7.0 (although pH of test solutions was adjusted to 6.5, at the start of testing pH was around 7.0 in most treatments).

- Ionic strength was not set to a fixed value, but the model was allowed to calculate it (default).

- Temperature was set to 23°C (average temperature during the test).

- Five problems were added, using set total cerium levels (i.e., the measured dissolved Ce levels at test initiation: 0.198, 0.46, 0.943, 2.01 and 4.48 mg Ce/L).

- The following aqueous Ce species were modelled by Visual MINTEQ: Ce3+, Ce(CO3)2-, CeCO3+, CeHCO3+2, Ce(SO4)2-, CeSO4+, CeCl+2, CeEDTA-, CeHEDTA, CeOH+2, CePO4, and CeH2PO4+2.

- Two possible solid phases were added for Ce: Ce(OH)3 and CePO4. When solubility products are exceeded in the aqueous solution, the model allows precipitation of these phases. The model does not contain (by default) other solid phases for Ce, although especially at higher pH levels some carbonate precipitation may be expected too. For the modelling exercise presented here, the absence of possible cerium carbonate solid phases does not affect the outcome of the calculations. Note that the nominally added total phosphate (PO4 3-, total) concentration is 1.18E-05 M, hence no more CePO4 than that can be formed.

- Under the abovementioned conditions, the model calculations for dissolved versus precipitated Ce and phosphate can be summarised as follows (measured dissolved Ce at the end of testing was added for comparison):

Ce total (initial measured Ce dissolved) (mg/L) Ce dissolved (model calculation) (mg/L)  % Ce dissolved (model calculation)   Ce dissolved (measured after 72 h) (mg/L) % PO4 3- precipitated (model calculation) 
 0.198 0.00  0.00  0.0017  11.95
 0.46 0.00  0.00  < LOQ  27.80
 0.943 0.00  0.00  0.022  57.03
 2.01 0.35  17.43  0.738  100
 4.48 2.83  63.18  2.96  100

- Ce(OH)3 was not calculated to precipitate under the set conditions of testing, only CePO4 precipitation occurred. Further, when checking phosphate speciation, it became clear that phosphate precipitation was practically entirely due to precipitation with cerium.

- Based on this modelling exercise it is confirmed 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 Ce concentrations at the end of testing and the modelled dissolved Ce concentrations further increase the credibility of the model calculations.