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Toxicity to microorganisms

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Endpoint:
activated sludge respiration inhibition testing
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 activated sludge (Eisner, 2013) was therefore included in this dossier. This study was considered as the key study for endpoint coverage. Another two studies performed with cerium trichloride (Bringmann and Kühn, 1959; Tang et al., 1985) were added as supporting information. Since the latter studies are considered of much lower reliability (Klimisch 3), they are 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
Reason / purpose for cross-reference:
read-across source
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
436 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Ce
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: used for recalculation of the results to Ce(NO3)3
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
91 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Ce
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: used for recalculation of the results to Ce(NO3)3
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
1 016 mg/L
Nominal / measured:
nominal
Conc. based on:
other: anhydrous Ce(NO3)3
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: recalculated
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
212 mg/L
Nominal / measured:
nominal
Conc. based on:
other: anhydrous Ce(NO3)3
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: recalculated
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
8 Jan 2013 - 31 Jan 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
PRE-TEST
- Test item was soluble at a concentration of 8 g test item per liter deionized water before and after pH adjustment from 5.0 to 7.4.
- Test item precipitated and the pH decreased to pH 4.5 after stirring the solution overnight at room temperature.

RANGE FINDER and DEFINITIVE TEST
- To avoid a possible precipitation process, no test item stock solution was used for the test (i.e., direct addition to test water).
- No emulsifiers or solvents were used.
- At the start of the test, appropriate amounts of test item were weighed and added to 234 mL of dilution water. After a short stirring period the pH in each test vessel was adjusted with diluted NaOH solution. Then, 16 mL synthetic sewage and 250 mL activated sludge inoculum was added (sludge concentration 3 g/L).
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: Aerobic activated sludge from a wastewater treatment plant (ARA Ergolz II, Füllinsdorf / Switzerland) treating predominantly domestic wastewater.
- Method of cultivation: Following the recommendations of OECD Guideline No 209 (2010). Sludge was washed three times with chlorine free tap water by centrifugation and the supernatant liquid phase was decanted. Wet to dry weight ratio was determined. Based on this ratio a calculated amount of wet sludge was resuspended in tap water to obtain a concentration equivalent to 3 g dry material per L. During holding period of 2 days prior to use, sludge was fed daily with 50 mL synthetic sewage feed per L and kept at room temperature under continuous aeration.
- Preparation of inoculum for exposure: Again, dry weight was determined and readjusted to 3 g dw/L in the inoculum used for the test (equivalent to 1.5 g/L in the final incubation mixture). pH of inoculum was adjusted from 7.0 to 7.5 using NaOH solution.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
not reported
Test temperature:
20°C
pH:
Start: 7.2-7.6
End: 7.4-7.8
Dissolved oxygen:
Just before measurement of respiration rates, at the end of the incubation period, DO concentrations were at least 7.1 mg/L.
Salinity:
not applicable
Nominal and measured concentrations:
Nominal concentrations tested:
Control + 25.6, 64, 160, 400 and 1000 mg CeCl3 anhydrous per liter
Details on test conditions:
TEST SYSTEM
- Test vessel: 2000 mL glass beakers containing 500 mL of incubation mixture
- No. of vessels per concentration (replicates): 3
- No. of vessels per blank control (replicates): 6
- Dry matter in the test: 3 g suspended solids/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: distilled or deionized water < 1 mg/L DOC

OTHER TEST CONDITIONS
- Adjustment of pH: yes, see above
- Photoperiod: normal laboratory lighting

EFFECT PARAMETERS MEASURED (with observation intervals if applicable):
- After 3 h, a well-mixed sample of each test medium was poured into a BOD flask and not further aerated.
- DO concentration measured using an oxygen electrode (WTW TriOxmatic 300) and an oxygen meter (WTW Oxi 539, Wissenschaftlich-Technische Werkstaetten WTW, Weilheim/Germany).

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2.5
- Range finding study: yes, control + 10, 100 and 1000 mg CeCl3/L
- Results used to determine the conditions for the definitive study: 50% inhibition falls between 100 and 1000 mg CeCl3/L


Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenole (CAS No. 591-35-5
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
436 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Ce
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL = 407-466
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
91 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Ce
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
767 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
anhydrous CeCl3
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Remarks on result:
other: 95% CL = 716-819
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
160 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
anhydrous CeCl3
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Further results based on element:
3-h EC10 = 192 mg Ce/L
3-h EC20 = 254 mg Ce/L
3-h EC80 = 746 mg Ce/L

Results based on test substance (CeCl3, anhydrous):
3-h EC10 = 337 mg/L
3-h EC20 = 447 mg/L
3-h EC80 = 1313 mg/L

- Inhibition of the respiration rates for all tested concentrations was in the range of -1.3 to 66.1% compared to the control, i.e. a marginal stimulatory effect was observed for some concentrations tested.
- No statistically significant inhibitory effect on the respiration rate of activated sludge up to and including the concentration of 160 mg CeCl3/L
- At a concentration of 400 mg CeCl3/L the inhibitory effect increased to 15.6% and reached 66.1% at the highest tested concentration (1000 mg CeCl3/L).
- Concentrations exceeding 1000 mg CeCl3 per liter were not tested.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels:
3-h EC50 = 8 mg/L (95% CI 2-25 mg/L)
Reported statistics and error estimates:
3-h EC10, EC20, EC50 and EC80 values and their 95% confidence limits were derived by Probit Analysis.
The 3-h EC50 for the reference substance was calculated by Weibull Analysis.
NOEC values were derived by per-vessel statistics using the Dunnett t-test.
Validity criteria fulfilled:
yes
Conclusions:
This activated sludge respiration inhibition test with cerium trichloride yielded a NOEC and EC50 of 160 and 767 mg CeCl3/L, respectively, corresponding to a NOEC and EC50 of 91 and 436 mg Ce/L.
Executive summary:

The inhibitory effect of the test item cerium trichloride on the respiration rate of aerobic wastewater microorganisms in activated sludge was investigated in a 3hour respiration inhibition test according to the OECD Guideline for Testing of Chemicals, No. 209, adopted 2010.

The inhibition of the respiration rates for all tested concentrations was in the range -1.3 to 66.1% compared to the control. Up to and including the concentration of 242 mg test item per liter the test item had no statistically significant inhibitory effect on the respiration rate of activated sludge after the incubation period of 3 hours

This activated sludge respiration inhibition test with cerium trichloride yielded a NOEC and EC50 of 160 and 767 mg CeCl3/L, respectively, corresponding to a NOEC and EC50 of 91 and 436 mg Ce/L.

Endpoint:
toxicity to microorganisms, other
Remarks:
Microregma heterostoma (ciliate)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Results of this test are not considered entirely reliable because 1) not all materials and methods are clearly reported, 2) only LOEC is given as an endpoint and 3) no guideline is followed.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The effect of the substance was investigated by monitoring the optical density (i.e., a biomass-related parameter) of a Microregma heterostoma culture (protozoan, ciliate).
GLP compliance:
no
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
- A concentration series was prepared with increasing test substance concentrations.
- Concentrations not reported.
Test organisms (species):
other: Microregma heterostoma (protozoan, ciliate)
Details on inoculum:
- Pre-cultures were started with encysted organisms.
- The organisms were fed with bacteria (24-h cultures of Escherichia coli).
- Inoculum was taken from 48-h pre-cultures.
- Initial density agreed with 200 mg/L kieselgur.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
28 h
Hardness:
not applicable
Test temperature:
27°C
pH:
7.5-7.8
Dissolved oxygen:
not reported
Salinity:
not applicable
Nominal and measured concentrations:
not reported
Details on test conditions:
- 100-mL Erlenmeyer flasks were used (fill-volume 10 mL).
- Turbidity of test solutions was measured electronephelometrically. Adverse effects on bacteria uptake by the ciliates results in a relatively higher turbidity in the test solution compared to the control.
- Before measurement of turbidity, test solutions were acidified to pH 1.
Duration:
28 h
Dose descriptor:
LOEC
Effect conc.:
8 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
other: uptake of bacteria (feeding)
Details on results:
Ce was not measured. Results should be used with caution.
Conclusions:
In this test the effect of Ce (added as CeCl3) on the ciliate Microregma heterostoma was investigated by monitoring the uptake of bacteria by the ciliates (turbidity measurement). The 28-h LOEC was 8 mg Ce/L. Ce was not measured in this test. Results should be used with caution.
Endpoint:
toxicity to microorganisms, other
Remarks:
Tetrahymena pyriformis
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
A Klimisch 3 score was assigned because no statistics were shown. Apart from that highly relevant test organism.
Qualifier:
no guideline followed
Principles of method if other than guideline:
In this study, Tetrahymena pyriformis was exposed to CeCl3 for 60 h and the effect on growth was monitored.
GLP compliance:
not specified
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
Aqueous solutions of RE chlorides were autoclaved before use. Direct spiking in test media.
Test organisms (species):
Tetrahymena pyriformis
Details on inoculum:
Tetrahymena pyriformis, Shanghai strain S1, in the logarithmic period of growth, was used as inoculum.
Culture medium and test medium were a mixture of polypeptone (2%, for bacteriological culture, DAIGO Brand, Japan). and glucose (0.5%).
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
60 h
Hardness:
not reported
Test temperature:
28°C
pH:
7.2
Dissolved oxygen:
not reported
Salinity:
not applicable
Nominal and measured concentrations:
Nominal: Control + 10, 25, 60, 200, 400, 500 mg Ce/L
Details on test conditions:
TEST SYSTEM
- Test vessel: not reported
- Material, size, headspace, fill volume: not reported
- No. of organisms per vessel: not reported
- No. of vessels per concentration (replicates): not reported
- No. of vessels per control (replicates): not reported

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: a mixture of polypeptone (2%, for bacteriological culture, DAIGO Brand, Japan) and glucose (0.5%)
- Culture medium different from test medium: no

OTHER TEST CONDITIONS
- Adjustment of pH: yes, after addition of RE chlorides, to pH 7.2
- Photoperiod: not reported
- Light intensity: not reported

EFFECT PARAMETERS MEASURED (with observation intervals if applicable): population growth as a function of incubation time, binocular microscope
Duration:
60 h
Dose descriptor:
NOEC
Effect conc.:
60 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
growth inhibition
Details on results:
LOEC = 200 mg Ce/L
Conclusions:
In this study, Tetrahymena pyriformis was exposed for up to 60 h to a concentration series of Ce added as CeCl3, yielding a NOEC (visually observed from figure) of 60 mg Ce/L and a LOEC of 200 mg Ce/L, based on population growth. However, because no statistics were performed, NOEC and LOEC values needed to be visually determined from a figure. Therefore the study was not considered reliable.

Description of key information

The key study (Eisner, 2013) was an activated sludge respiration inhibition test performed with the read-across substance CeCl3 and yielding a 3-h EC50 and NOEC value of 436 and 91 mg Ce/L (corresponding to 1016 mg and 212 mg Ce(NO3)3 mg/L), respectively.

Key value for chemical safety assessment

EC50 for microorganisms:
436 mg/L
EC10 or NOEC for microorganisms:
91 mg/L

Additional information

Four studies were identified containing relevant information on the toxicity of cerium to aquatic microorganisms.

The key study (Eisner, 2013) was an activated sludge respiration inhibition study with the read-across substance CeCl3. The study was assigned a klimisch score of 2 because the study was used for read-across purposes in this dossier. The study yielded a 3-h NOEC and EC50 of 91 and 436 mg Ce/L (corresponding to 212 mg and 1016 mg Ce(NO3)3 mg/L), respectively.

Dion (1990) investigated growth inhibition of Pseudomonas putida caused by cerium (added as Ce(NO3)3) and obtained a 16-h EC10 and EC50 value of 1.89 and 3.14 mg Ce/L. This study was considered reliable with restrictions (Klimisch 2). However, as the results of Pseudomonas putida growth inhibition tests should not be used for PNEC derivation unless no data on other relevant organisms or on activated sludge are available, the study was here considered as a supporting study. Indeed, when data are available on both isolated bacteria and whole bacterial populations, the latter is preferred as more representative of the whole functioning of the ecosystem.

In the third study (Tang et al., 1985), Tetrahymena pyriformis was exposed for up to 60 h to a concentration series of cerium added as CeCl3, yielding a NOEC of 60 mg Ce/L and a LOEC of 200 mg Ce/L, based on population growth. However, because no statistics were performed, NOEC and LOEC values needed to be visually determined from a figure. Therefore the study was not considered reliable. Nevertheless, the study supports the key study as the obtained NOEC was quite similar to that of the key study.

Finally, in the study of Bringmann and Kühn (1959), the effect of cerium (added as CeCl3) on the ciliate Microregma heterostoma was investigated by monitoring the uptake of bacteria by the ciliates (turbidity measurement). The 28-h LOEC was 8 mg Ce/L. This study was considered as supporting information and was not considered entirely reliable (Klimisch 3) because not all materials and methods are clearly reported, because only a LOEC is reported, and because no guideline was followed.