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Endpoint:
activated sludge respiration inhibition testing
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 209 (Activated Sludge, Respiration Inhibition Test
GLP compliance:
yes (incl. certificate)
Analytical monitoring:
yes
Details on sampling:
Samples from stock solution, and from test solutions of selected concentration levels were taken to determine the actual test item concentrations in comparison to the nominally applied concentrations. Samples of all concentrations were taken at the start of the test. Samples of the control, the lowest and the highest test concentration were taken at the end of the exposure period. Test solutions taken at the end of the exposure period were filtered using folded paper filters (pore size: 4 to 7 µm) to remove the microbial inoculum. The filtrates were sent to the Analytical Test Site for determination of the test concentrations.
The sample volume required for analysis was approximately 100 mL per sample.
Details on test solutions:
Reconstituted water according to OECD Guideline No. 203 (Fish, Acute Toxicity Test) was used to wash and to maintain the activated sludge. The reconstituted water contained salts at the following final concentrations:
- CaCl2 * 2 H2O: 294.0 mg/L
- MgSO4 * 7 H2O: 123.25 mg/L
- NaHCO3: 64.75 mg/L
- KCl: 5.75 mg/L

Preparation of stock solution and test solutions:
- A stock solution was prepared by dissolving 4.1298 g of the test item in 2000 mL of deionised water (nominal concentration 2.065 g/L). The stock solution was stirred on a magnetic stirring device until each single test vessel was prepared.
- The test item concentration levels were prepared by combining 16 ml of synthetic sewage feed with corresponding amounts of temperature adapted deionised water and/or stock solution in a way that a volume of 300 ml was obtained. Afterwards 200 ml of the microbial inoculum were added.
- The controls were prepared by combining 16 ml of the synthetic sewage feed with 284 ml of temperature adapted deionised water and adding 200 ml of the microbial inoculum.
- The total mixture volume for each of the test item concentrations and the controls was 500 mL per vessel. Immediately after preparation the test solutions were aerated.
- The test vessels were prepared at intervals of 15 minutes, starting with the first control, followed by the test item concentration levels. Replicates C6B (highest concentration level) and C0B (second control replicate exposed at the end of the testing series) were prepared 17 minutes after the preceding vessel to ensure equal exposure periods (3 hours) and maximum measurement periods (up to 10 minutes) for all vessels when testing the increased number of 14 test vessels.
- At the end of the contact time (test period), the content of the first test vessel (control 1) was poured into the measuring apparatus (consisting of oxygen-meter, flat bottom flask and stirring device) and the respiration rate was determined at two-second intervals over a period of up to 10 minutes. All other test vessels were measured according to this procedure in the same order which was established for the preparation of the test vessels.

Test organisms (species):
activated sludge of a predominantly domestic sewage
Details on inoculum:
Activated sludge from a sewage treatment plant was used as the microbial inoculum for the test. The activated sludge was obtained from the sewage treatment works in Frankfurt/Main Niederrad treating predominantly domestic sewage.
- Date of sampling/ date of arrival at ECT: March 08, 2010
- Washing medium for activated sludge:Reconstituted water (OECD 1992)
- Frequency of washing: three times; between washings the activated sludge was centrifuged for 2 minutes at 1000 rpm
- Storage time before use: 4 days
- Storage temperature: 20 ± 2 °C

During storage 50 mL synthetic sewage feed were added per litre activated sludge and per day. The activated sludge was stirred and aerated during storage. The synthetic sewage feed consisted of the following amounts of substances in 1 litre of deionised water (in g/L):
- Peptone: 16.0
- Meat extract: 11.0
- Urea: 3.0
- NaCl: 0.7
- CaCl2 * 2 H2O: 0.4
- MgSO4 * 7 H2O: 0.2
- K2HPO4: 2.8
The required amount of synthetic sewage feed was prepared within 1 week before use.


Test type:
static
Water media type:
freshwater
Total exposure duration:
3 h
Hardness:
257.2 mg/L CaCO3
Test temperature:
20.6-20.7 degrees Celsius (n=41)
pH:
7.7
Dissolved oxygen:
9.0 mg/L ; 99.0 % saturation
Nominal and measured concentrations:
nominal concentrations (as mg/L BaCL2): 0, 3.2, 10, 32, 100, 320, 1000 mg/L
nominal concentrations (as mg/L test item): 0, 3.8, 11.7, 37.5, 117, 375, 1173 mg/L
measured (nominal of 1000 mg BaCl2/L): t=0: 621.72 mg Ba/L; t=3h: 379.50 mg Ba/L (94% and 58% of nominal, respectively)
Details on test conditions:
- Storage time of the activated sludge before use: 4 days
- pH-value of the activated sludge: 7.9
- Test vessels: 1000 mL glass beakers without cover
- Amount of test mixture per test vessel: 500 mL
- Volume of microbial inoculum per test mixture: 200 mL
- Volume of synthetic sewage feed per test mixture: 16 mL
- Mixed liquor suspended solids level in the microbial inoculum: 4.00 g/L
- Mixed liquor suspended solids level in the final test mixtures: 1.60 g/L
- Number of test concentrations: 6 plus control
- Number of replicates per test item concentration: 2 plus 1 for chemical analysis
- Number of replicates in the control: 2 plus 1 for chemical analysis
- Aeration of test vessels: 50 to 60 litre of air per hour (ambient air, oil-free air-compressor)

An oximeter OXI 530 (WTW GmbH, D-82362 Weilheim) was used to determine the oxygen content of the test solutions. After three hours of incubation (contact time), the contents of the test vessels were poured into flat-bottom flasks. The oxygen electrode was fitted into the flat-bottom flasks which contained a stirrer bar. The flasks were placed onto a magnetic stirrer. The oximeter was connected to an automated data recording device. The oxygen measurements were taken at intervals of two seconds for up to 10 minutes at the end of the contact period for the content of each test vessel.
Reference substance (positive control):
yes
Remarks:
3,5-dichlorophenol
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 000 mg/L
Nominal / measured:
nominal
Conc. based on:
other: BaCl2
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:
other: BaCl2
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
IC50
Effect conc.:
> 943.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: BaCl2
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 943.1 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
other: BaCL2
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
IC50
Effect conc.:
> 622 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Remarks:
total Ba
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 622 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Remarks:
total Ba
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Details on results:
Validity criteria: The control respiration rates did not differ more than 15% compared to each other, i.e., 4.0%.
Results with reference substance (positive control):
The EC50 (3 hours) of 3,5- dichlorophenol was in the accepted range of 5 to 30 mg/L, i.e., 9.1 mg/L
Reported statistics and error estimates:
The respiration rate was calculated from the oxygen consumption of the test solutions as mg O2/L .h (also expressed as mg O2/L. h) between 6.5 and 2.5 mg O2/L. The portion of the respiration curve over which the respiration rate was measured was linear. In order to calculate the inhibitory effect of the test item in the test concentration levels, the respiration rate was expressed as a percentage of the mean of the two control respiration rates RC1 and RC2.
Welch-t test for inhomogeneous variances with Bonferroni-Holm adjustment was used to determine the No Observed Effect Concentration (NOEC). Probit Analysis was used to determine the ECx-values. Since in none of the treatments the respiration rate was inhibited statistically significant compared to the controls, the calculation of an ECx-value was not possible.
The statistical software package ToxRat 2.10 Professional (ToxRat Solutions GmbH, Naheweg 15, D-52477 Alsdorf) is used for these calculations.

 Concentration (mg/L BaCl2) replicate time (h)  Oxygen consumption (mg/L per hour  Inhibition (%)   
80.22  2.81 
3.2  84.86  -2.81 
3.2  83.33  -0.96 
10  3 81.52  1.24   
10  81.52  1.24 
32  79.89  3.21 
32  80.00  3.08 
100  78.65  4.71 
100  80.89  2.00 
320  73.54  10.90 
320  76.86  6.88 
1000  73.56  10.88 
1000  74.21  10.09 
84.86  -2.81 
Validity criteria fulfilled:
yes
Conclusions:
Since the nominal test item concentrations of 10, 32, 100, 320, and 1000 mg BaCl2/L showed no statistically significant inhibitory effect on the respiration rate of the test system, no ECx-values were determined. The concentrations initially measured in the test solutions were in good agreement with the target concentrations; therefore it can be assumed that the activated sludge was exposed to at least 94% of the nominal concentrations throughout the test. Thus, the results of this study demonstrate that the test item did not inhibit respiration of activated sludge at a concentration of up to 1000 mg/L. Toxic effect levels (EC20, EC50 and EC80) are therefore greater than 1000 mg BaCl2/L. The No Observed Effect Concentration (NOEC) is ≥ 1000 mg BaCl2/L, and hence the Lowest Observed Effect Concentration (LOEC) is > 1000 mg BaCl2/L.

The No Observed Effect Concentration (NOEC), when based on measured initial total Ba and BaCl2 concentrations, is ≥ 622 mg Ba/L and ≥ 943.1 mg BaCl2/L, respectively, and hence the Lowest Observed Effect Concentration (LOEC) is > 622 mg Ba/L and > 943.1 mg BaCl2/L, 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:
The justification for read across is attached in IUCLID Section 13.
Reason / purpose:
read-across source
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 622 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Remarks:
Ba
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 622 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
element
Remarks:
Ba
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
EC50
Effect conc.:
> 1 181.8 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
Na(NO3)2
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate
Key result
Duration:
3 h
Dose descriptor:
NOEC
Effect conc.:
>= 1 181.8 mg/L
Nominal / measured:
meas. (initial)
Conc. based on:
test mat.
Remarks:
Na(NO3)2
Basis for effect:
inhibition of total respiration
Remarks:
respiration rate

Description of key information

A read across approach was followed to cover this endpoint, using the activated sludge respiration inhibition study with BaCl2 performed by Egeler et al. (2010). Based on initially measured total Ba-levels in the water column, a  3-h IC50 and 3-h NOEC of > 622 and >= 622 mg Ba/L, respectively, was reported, corresponding to > 943.1 and >= 943.1 mg BaCl2/L, respectively. This result was recalculated to barium dinitrate based on the barium content of both substances (i.e. > 1881,8 and >= 1181,8 mg barium trinitrate/L).

Key value for chemical safety assessment

Additional information

One key study for the read across substance barium chloride was identified and included in this dossier. In the study from Egeler et al. (2010) it was demonstrated that the test item (BaCl2) did not inhibit respiration of activated sludge at a nominal concentration of up to 1000 mg/L. The total fraction of barium in the test medium was measured at the start of the test. At a nominal concentration of 1000 mg BaCl2/L, the measured total barium content was 622 mg Ba/L (equivalent to 943.1 mg BaCl2/L). Therefore, the 3-h IC50 and 3-h NOEC were > 622 and >= 622 mg Ba/L, respectively.