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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Nov - 22 Nov 2012
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:
See test conditions.
Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
- A mixed population of activated sludge micro-organisms was obtained on 21 November 2012 from the aeration stage of the Severn Trent Water Public sewage treatment plant at Loughborough, Leicestershire, UK, which treats predominantly domestic sewage.
- The activated sewage sludge sample was maintained on continuous aeration in the laboratory at a temperature of appoximately 21°C overnight prior to use in the test.
- The SS concentration was equal to 3.0 g/L prior to use.
Test type:
static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
3 h
Hardness:
not stated
Test temperature:
20 ± 2 °C
pH:
Tap water: 8.14
Activated sludge: Pre-test: 6.6, Main test: 7.0
Main test, measured at the end of the test:
- Controls: 7.5 - 7.6
- Positive controls: 7.7 - 8.5
- Test item: 7.6 - 8.5
Dissolved oxygen:
Dissolved oxygen was measured after 30 minutes contact time. In some instances, the initial and final dissolved oxygen concentrations were outside those recommended in the test guidelines (7.0 mg O2/L and 2.0 mg O2/L, respectively). This was considered to have had no adverse effect on the results of the study given that in all cases the oxygen consumption was determined over the linear portion of the oxygen consumption trace.
Nominal and measured concentrations:
10, 100 and 1000 mg/L (nominal, based on anhydrous zirconium acetate)
Details on test conditions:
TEST SYSTEM
- Test vessel: 500 mL (16 mL synthetic sewage, 250 mL tap water or tap water + stock solution, 250 mL inoculum)
- No. of vessels per concentration (replicates): 2
- No. of vessels per control (replicates): 4
- Dry matter in the test: 3 g suspended solids/L

PRE-TEST
- An amount of test item (11590 mg) was dissolved in water and the volume adjusted to 1 liter to give a 2500 mg a.i./L stock solution from which dilutions were made to give 250 and 25 mg a.i./L stock solutions. The 2500 mg a.i./L stock solution was adjusted to pH 7-8. On pH adjustment the test item dropped out of solution and was observed as a cloudy dispersion, however, this was considered acceptable for use in the test. An aliquot (200 mL) of the 25 mg a.i./L stock solution was dispersed with synthetic sewage (16 mL), activated sewage sludge (250 mL) and tap water to a final volume of 500 mL, to give the required test concentration of 10 mg a.i./L. similarly, aliquots (200 mL) of the 250 mg a.i./L and 2500 mg a.i./L stock solutions were used to prepare the test concentrations of 100 and 1000 mg a.i./L. The 1000 mg/L test concentration was prepared in triplicate. The volumetric flasks containing the stock solutions were inverted several times to ensure homogeneity of the stock solutions.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol (CAS No. 591-35-5)
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
zirconium acetate, anhydrous
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Remarks:
zirconium acetate, anhydrous
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 742 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 742 mg/L
Nominal / measured:
nominal
Conc. based on:
element
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
The test item contained 40% anhydrous zirconium acetate (i.e. the active ingredient).
The effect of the test item on the respiration of activated sewage sludge gave a 3-hr EC50 value of greater than 1000 mg a.i./L. The No Observed Effect Concentration (NOEC) after 3 hours exposure was >= 1000 mg a.i./L. It was considered unnecessary and unrealistic to test at concentrations in excess of 1000 mg a.i./L.
At the Sponsors request the test concentrations have also been calculated based on zirconium (%w/w): 10, 100 and 1000 mg a.i./L are equivalent to 8, 74 and 742 mg Zr/L.

The dissolved oxygen concentrations in all vessels were less than 60% of the dissolved oxygen saturation level of 8.9 mg O2/L with the exception of control R1, reference item 10 and 32 mg/L and test item 100 mg a.i./L. This deviation was considerd to have had no adverse effect on the study given that all oxygen consumption values were measured/calculated over the linear portion of the traces.

The coefficient of variation of oxygen update in the control vessel was 4.2% and the specific respiration rate of the controls was 26.89 mg O2 per gram dw of sludge per hour.
Results with reference substance (positive control):
- Results with reference substance valid? yes
- Relevant effect levels:
EC50 = 15 mg/L (95% CI 13-18 mg/L)
Validity criteria fulfilled:
yes
Conclusions:
No statistically significant toxic effects were observed at all test concentrations employed. It was therefore considered justifiable not to perform a definitive test. The relatively large increase in respiration rate observed in the test vessels at 1000 mg a.i./L was considered to be due to metabolism of the acetate component of the test item. The 3-h EC50 and NOEC were determined to be > 1000 mg/L and >= 1000 mg/L anhydrous zirconium acetate, respectively.
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:
Read across from a study performed with zirconium acetate. The read across justification document is attached to IUCLID Section 13.
Reason / purpose for cross-reference:
read-across source
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
inhibition of total respiration
Remarks on result:
other:
Remarks:
Based on the results of the study from Bayliss (2013), performed with the read across substance zirconium acetate, it was concluded that zirconium dinitrate oxide is not expected to present toxicity to aquatic micro-organisms either. The 3-h NOEC of >= 1000 mg/L (anhydrous zirconium acetate) was not recalculated to zirconium dinitrate oxide, but taking into account the fact that the % w/w Zr in zirconium dinitrate oxide is lower than that in zirconium acetate, it can be concluded that the 3-h NOEC for zirconium dinitrate oxide would also be >= 1000 mg/L in a similar study.

Description of key information

The read across substance zirconium acetate did not cause any adverse effects on aquatic microorganisms in the key activated sludge respiration inhibition study performed according to OECD guideline 209 (Bayliss, 2013). The EC50 and NOEC were found to be > 1000 mg/L and >= 1000 mg/L anhydrous zirconium acetate (i.e., the highest test concentration used), respectively. Zirconium acetate and zirconium dinitrate oxide are similar water soluble compounds that are very well soluble in pure water without pH adjustment. However, upon dissolution of zirconium acetate and zirconium dinitrate oxide in environmentally relevant test media and pH being adjusted to environmentally relevant pH levels, zirconium precipitates out of the solution. The similar behaviour of zirconium acetate and zirconium dinitrate oxide is the basis for the read across justification (attached to IUCLID Section 13).

Key value for chemical safety assessment

Additional information