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activated sludge respiration inhibition testing
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
toxicity to microorganisms, other
effects on microbial ecology
Type of information:
experimental study
Adequacy of study:
supporting study
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
no guideline followed
Principles of method if other than guideline:
The study comprises a number of experiments. The first experiment examines the effect of the addition of sodium carbonate on the nitrification performance in a pilot-scale bioreactor consisting of an anoxic tank and two aerobic tanks. A second experiment is carried out in 20L batch bioreactors and assesses the effect of the addition of different buffering chemicals, including sodium carbonate on ammonia oxidizing bacteria (AOB) populations.
GLP compliance:
not specified
Analytical monitoring:
Test organisms (species):
other: ammonia oxidising bacteria population
Details on inoculum:
The ammonia oxidising bacteria (AOB) were obtained from a 300L AOB enrichment bioreactor packed with polyethylene carriers. The influent for the AOB enrichment bioreactor was wastewater collected from a full-scale bioprocess wastewater treatment plant in an industrial park processing diverse petrochemical producst such as cyclohexyl acrylate, acrylonitrile, polyacrylonitrile and vinyl acetate.
The collected wastewater was supplemented with 1000 mg/L of sucrose to enhance the biomass growth onto the carrier material. The seeds for the AOB enrichment reactor was powder activated carbon obtained from the full-scale wastewater treatment plant described above. Every 7 days, 20L of treated supernatant was withdrawn before feeding 20L of influent wastewater. After 30 days, a 57% of ammonia conversion efficiency was attained by the developed AOB populations.

This AOB population was subsequently used in the pilot-scale bioreactor. For the batch experiments, the seeds for the AOB populations were in turn polyethylene carriers taken from the second aerobic tank in the pilot-scale bioreactor.
Test type:
other: flow-through for the pilot-scale bioreactor, static for the batch experiment
Water media type:
Limit test:
Total exposure duration:
150 d
Remarks on exposure duration:
150 days in the pilot-scale experiment, 160h in the batch experiment
Test temperature:
Pilot-scale: 29-34°C
Batch: not specified
Pilot-scale: 7.1 =/- 0.3
Batch: 7 +/- 0.2
Dissolved oxygen:
Pilot-scale: > 2 mg/L in the aerobic tanks
Batch: not specified
Nominal and measured concentrations:
Pilot-scale: buffering in the second aerobic bioreactor with 5% Na2CO3
Batch: buffering with 0.1% Na2CO3
Details on test conditions:
Pilot-scale bioreactor

The pilot-scale moving bed biofilm reactor (MBBR) comprised of an anoxic tank and two following aerobic tanks. The pilot-plant was designed for a treatment capacity of 5m3/d with a nitrate recycle flowrate of 15m3/d, and operated with a total HRT of 20 hours. The anaerobic, anoxic, and aerobic zones comprise 45%, 22%, and 33% of the total reactor volume, respectively. Influent wastewater, return activated sludge (RAS), and recycled flow from the second aerobic tank discharge into the anoxic tank. Wastewater successively flows from the anoxic tank to the first (O1) and second (O2) aerobic tanks and then to the sedimentation tank. The O1 and O2 bioreactors were packed with polyethylene carriers (30% in volume), which were transferred from the AOB enrichment bioreactor (see section on inoculum). Buffering chemicals were added in the O2 bioreactor to maintain a pH of 7.1 +/- 0.3. During this study, 4% NaOH was added as the buffering chemical in Run I and III, while 5% Na2CO3 was used in Run II.

Batch bioreactor

Two 20 L batch reactors were used to evaluate the effects of adding different buffering chemicals on AOB populations. The seeds for the AOB populations were polyethylene carriers obtained from the pilot-scale second aerobic tank (O2) in Run II. The wastewater for the batch experiments was collected from the industrial park described above. During the experiments, pH was controlled at pH 7 +/- 0.2 by adding either 1% NaOH or 0.1% Na2CO3 and samples were taken frequently for ammonia analysis.
Reference substance (positive control):
Details on results:
Nitrification performance:

Pilot-scale bioreactor:
The organic loading of the pilot-scale bioreactor was operated at 1.5 kg COD/m3-day, and the bioreactor was able to achieve a satisfactory COD removal efficiency > 80% during the study. Compared to that observed in the full-scale PACT bioprocess, the nitrification observed in the pilot-scale MBBR bioprocess was with a slightly improvement at an efficiency of 25% in Run I (buffered with NaOH) and with a significant improvement at 41% in Run II (buffered with Na2CO3): 51% during day 110 to 150. These results suggest that nitrification can occur for the petrochemical wastewater treated in this study, leading to an elimination of the possible inhibition effects on nitrification caused by organic compounds and high ammonia concentration presented in the petrochemical wastewater. Furthermore, the significant improvement on nitrification by adding Na2CO3 as a buffering chemical suggests that inorganic carbon limitation can be a potential problem on nitrification in such a low C/N ratio petrochemical wastewater.

Effect on the AOB population:

Full-scale bioreactor:
AOB populations of samples collected from the full-scale bioreactor, AOB enrichment bioreactor, and pilot-scale MMBR bioreactors were evaluated using amoA-based TRFLP analyses. For all the analyses, the expected 48 bp terminal fragment could not be unequivocally detected, due to high background noise for fragments smaller than 100bp. Nevertheless, the electropherograms obtained during this study showed the two bioreactors exhibiting different TRFLP patterns. The TRFLP profiles of the two full-scale aerobic bioreactors (samples A and B) were dominated by reverse terminal fragment (TF) at 135 bp. According to the TF pair signatures, these results suggest that the dominant AOB population in the full-scale bioreactors contains organisms related to the Nm. marina or Nm. communis lineages (48/135 TF signature).

AOB enrichment bioreactor:
For the samples from the AOB enrichment bioreactor, although the reverse TF at 135 bp was observed at day 0, presumably contributed by the seed obtained from the full-scale bioreactor, the forward TF at 219bp and reverse TF at 270bp became dominant after 10 days of acclimation, suggesting that Nm. europaea-like AOB (219/270 TF signature) was able to out-compete other AOB in the enrichment bioreactor.

Pilot-scale bioreactor:
The TRFLP profiles of the pilot-scale first aerobic bioreactor (O1) was dominated by forward TF at 219 bp and reverse TF at 270 bp during this study, suggesting that Nm. europaea-like AOB (219/270 TF signature) was the dominant AOB. For the samples from the pilot-scale second aerobic bioreactor (O2) collected during Run I to III, the dominant AOB populations shifted from Nm. europaea-like AOB (219/270 TF signature) in Run I to Nitrosospira-like AOB (283/206 TF signature) in Run II, and then shifted back to Nm. europaea-like AOB (219/270 TF signature) in Run III, corresponding to the switch of buffering chemical addition from NaOH in Run I to Na2CO3 in Run II, and then back to NaOH in Run III. These results suggest that addition of Na2CO3 in the O2 bioreactor during Run II as a buffering chemical not only improves nitrification efficiency but also influences AOB populations. The importance of alkalinity type and inorganic carbon
limitations on nitrification has been discussed previously, but their effects on AOB populations have never been demonstrated until this study.

Confirmatory experiments in the batch bioreactors:
To further confirm whether the observed AOB population changes in the pilot O2 bioreactor was caused by the switch of buffering chemicals between NaOH and Na2CO3, two batch bioreactors were conducted. In Experiment I (0 to 70h), no observable difference regarding nitrification was found between bioreactors by adding NaOH or Na2CO3 as buffering chemicals, while in Experiment II (70 to 160 h), nitrification of the Na2CO3-buffering bioreactor was considerable faster than that of the NaOH-buffering one. These results confirmed the importance of alkalinity type and inorganic carbon limitations on nitrification.
Additionally, the amoA-based TRFLP profiles of the original seed obtained from the pilot-scale O2 bioreactor during Run II indicated that the dominant AOB was Nitrosospira-like AOB (283/206 TF signature). After 160 h of experiment with NaOH-buffering control, Nm. europaea-like AOB (219/270 TF signature) and 491/491 TF signature were found. In the bioreactor buffered with Na2CO3 for 160 hrs, Nm. europaea-like AOB (219/270 TF signature), however, was not found. The TF signature of 491/491 was also found in the bioreactor buffered with Na2CO3, but with a relatively higher increase in electropherogram intensity compared to that in the NaOH-buffering bioreactor. Although the exact mechanisms are not clear at this time, the results presented in this study demonstrated that addition of different buffering chemicals such as NaOH or Na2CO3 would influence AOB populations.
During biological treatment of a petrochemical wastewater addition of Na2CO3, as a buffering chemical, improve nitrification performance by providing a sufficient inorganic carbon for nitrifying bacteria and influence dominant AOB populations presented in the bioreactors.
Executive summary:

Performance and microbial ecology of nitrifying community during biological treatment of a petrochemical wastewater with a low carbon to nitrogen ratio were investigated in this study. Based on the results obtained from the full-scale, pilot-scale, and batch bioreactors, it was concluded that addition of Na2CO3, compared with addition of NaOH, as a buffering chemical would improve nitrification performance by providing a sufficient inorganic carbon for nitrifying bacteria. In addition to an improvement on nitrification performance, addition of Na2CO3 or NaOH as buffering chemicals would also influence dominant AOB populations presented in the bioreactors. The amoA-based TRFLP results indicate that Nm. europaea-like AOB were found to be dominant during the operation buffered with NaOH, while Nitrosospira-like AOB was responsible for an improved ammonia conversion during the operation buffered with Na2CO3.

Description of key information

Sodium carbonate in the aquatic environment is dissociated into sodium and carbonate ions. Further, both ions originally exist in nature, and their concentrations in surface water are dependent on various factors, such as geological parameters, weathering and human activities. Therefore, there is a continuous source of both ions into the environment, which have been measured extensively in aquatic ecosystems. .

Further, results from the available support study showed that, in a pilot experiment, during biological treatment of a petrochemical wastewater addition of Na2CO3, as a buffering chemical, improve nitrification performance by providing a sufficient inorganic carbon for nitrifying bacteria and influence dominant AOB populations presented in the bioreactors.

Hence the toxicity of sodium carbonate to aquatic microorganisms is expected to be low, as also indicated by the results from the aquatic ecotoxicity studies which showed EC50 greater than the toxicity threshold of 100 mg/L for short-term exposure.

Key value for chemical safety assessment

Additional information