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Toxicity to microorganisms

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Reference
Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
7 Jan 2013 - 8 Jan 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
Deviations:
no
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
no
Vehicle:
no
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
RANGE-FINDER TEST
- The attempt to use a stock solution failed since the test item precipitated during pH adjustment of the stock solution.
- Test item amounts of 5.07, 50.29, 500.17, 500.09 and 500.37 mg were weighed and directly transferred to the designated test vessels.
- Test item was mixed into the water (to obtain nominal concentrations of 10, 100 and 1000 mg/L anhydrous cerium ammonium nitrate) by intense stirring for 10 min at room temperature to dissolve a max. amount of test item and/or disperse it as homogeneously as possible.
- No emulsifiers or solvents used.
- After stirring, a clear solution was obtained for all test concentrations.
- After pH adjustment, all loading rates were clearly above the water solubility limit of cerium ammonium nitrate under the test conditions.

DEFINITIVE TEST
- In conformity with the guideline, a definitive test was not necessary since in the range-finding test cerium ammonium nitrate did not show any relevant inhibition of the respiration rate at the highest concentration tested (tested in triplicate).
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- Laboratory culture: aerobic activated sludge from a WWTP (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: 7.8-8.0
Dissolved oxygen:
8.1 mg/L
Salinity:
not applicable
Nominal and measured concentrations:
nominal test item concentrations: control + 10, 100 and 1000 mg/L
Details on test conditions:
TEST SYSTEM
- Test vessel: 2000 mL glass beakers containing 500 mL of incubation mixture
- Volume of microbial inoculum per test mixture: 250 mL
- Volume of synthetic sewage feed per test mixture: 16 mL
- Mixed liquor suspended solids level in the microbial inoculum: 3 g/L
- Mixed liquor suspended solids level in the final test mixtures: 1.5 g/L
- Number of test concentrations: 3 plus control
- Number of replicates per test item concentration: Single test vessel replicates were tested for the two lower concentrations, whereas for the highest test item concentration three replicates were prepared.
- Number of replicates in the control: four blank controls
- Aeration: continuous aeration during the incubation period of 3 h

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: 10
- Range finding study
- Test concentrations: 10 mg/L, 100 mg/L, 1000 mg/L
- Results used to determine the conditions for the definitive study: no definitive study performed
Reference substance (positive control):
yes
Remarks:
3,5-Dichlorophenole (CAS No. 591-35-5)
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 256 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Ce
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 256 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Remarks:
Ce
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
anhydrous
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Remarks:
anhydrous
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Further results based on Ce element:
3-h EC10 = > 256 mg/L
3-h EC20 = > 256 mg/L
3-h EC80 = > 256 mg/L

Results based on test substance (NH4)2Ce(NO3)6:
3-h EC10 = > 1000 mg/L
3-h EC20 = > 1000 mg/L
3-h EC80 = > 1000 mg/L

- At all tested concentrations up to and including 1000 mg/L, inhibition of the respiration rates was in the range of -10.2 to 5.9% compared to the control.
- No statistically significant inhibitory effect on the respiration rate of activated sludge up to and including the concentration of 1000 mg Ce ammonium nitrate/L
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels:
3-h EC50 = 2 mg/L (conf. limits could not be calculated due to mathematical reasons)
Reported statistics and error estimates:
% inhibition of respiration was plotted against logarithm of test substance concentration using suitable software (ToxRat Professional).
NOEC value was derived by per-vessel statistics using the Student t-test (Sachs, 1984). The 3 replicates at 1000 mg/L were compared to the 4 blank control replicates using one-tailed (smaller) hypothesis and alfa = 0.05.
The 3-h EC50 for the reference substance was calculated by Weibull Analysis (Christensen, 1984a,b).
Validity criteria fulfilled:
yes
Conclusions:
This activated sludge respiration inhibition test with cerium ammonium nitrate yielded a NOEC >= and EC50 > 1000 mg (NH4)2Ce(NO3)6/L, corresponding to a NOEC >= and EC50 > 256 mg Ce/L.

Description of key information

The key study (Eisner, 2014) was an activated sludge respiration inhibition study with cerium ammonium nitrate yielding a 3-h NOEC and EC50 of >= 256 mg Ce/L and > 256 mg Ce/L, respectively (corresponding to >= 1000 mg/L and > 1000 mg/L anhydrous cerium ammonium nitrate, respectively).

Key value for chemical safety assessment

Additional information

One study was identified containing relevant information on the toxicity of cerium ammonium nitrate to aquatic microorganisms.

The key study (Eisner, 2014) was an activated sludge respiration inhibition study performed according to OECD guideline 209 with cerium ammonium nitrate yielding a 3-h NOEC and EC50 of >= 256 mg Ce/L and > 256 mg Ce/L, respectively (corresponding to >= 1000 mg/L and > 1000 mg/L anhydrous cerium ammonium nitrate, respectively). The substance is therefore considered not toxic to aquatic microorganisms.