Ozone

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

toxicity to microorganisms, other

Remarks:

Oxygen Uptake Rate (OUR) and ATP measurement

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The main goal was to analyze the effect of ozonation on viability of activated sludge in different systems – activated sludge in distilled water and activated sludge in wastewater. Two viability detection methods, oxygen uptake (OUR) rate and adenosine-5’-triphosphate measurement (ATP), were compared.

GLP compliance:

no

Analytical monitoring:

yes

Details on sampling:

- Concentrations: During ozonation experiments, four ozone doses (~15, ~30, ~40 and ~70 mgO2/L) were transferred to the suspension
(initial volume of 1.5 L) in and ozonation reactor.
- Sampling method: 0.5 L out of 2 L was used as an initial or zero-dose
sample. After transferring a certain dose, a sample was taken.
Ozonation time, depending on the transferred ozone dose,
ranged from ~70 to ~120 seconds.

Vehicle:

no

Details on test solutions:

In the experiments a synthetic phenolic wastewater, corresponding by its composition to the oil-shale semi-coking process effluent after the extraction of phenols with butyl acetate (dephenolated process water) was used (Kamenev et al., 2003). The choice of wastewater was based on fact that the purification of the phenolic effluents from the Estonian oil shale industry is still not adequate. Soluble chemical oxygen demand (COD) of the wastewater was of 3100 mgO2/L and 7-day biochemical oxygen demand (BOD7) of 1300 mgO2/L.

Test organisms (species):

activated sludge

Details on inoculum:

Activated sludge was sampled from a laboratory-scale, continuous stirred tank reactor (CSTR) (volume of 10 L), where it was previously adapted to the phenolic wastewater (approximately 2 years). Hydraulic retention time (HRT) was 2 days and solids retention time (SRT) was between 9 and 10 days. Control and optimization of activated sludge ozonation is based on measurement of mixed liquor volatile suspended solids (MLVSS) and mixed liquor suspended solids (MLSS) by keeping the ratio MLVSS/MLSS constant. The reactor was kept at room temperature (21 ± 1◦C), dissolved oxygen level was maintained between 2–4 mg/L, MLVSS/MLSS was approximately 0.80.

Test type:

other: recirculation system

Water media type:

other: wastewater and distilled water

Limit test:

no

Total exposure duration:

1.5 min

Remarks on exposure duration:

Ozonation time, depending on the transferred ozone dose, ranged from ~70 to ~120 seconds.Ha

Test temperature:

21 ± 1 °C

Dissolved oxygen:

2 and 4 mgO2/L

Nominal and measured concentrations:

Nominal: (~15, ~30, ~40 and ~70 mgO3/L)

Details on test conditions:

TEST SYSTEM
- Test vessel: Batch ozonation experiments were carried out in a 2.6 L cylindrical glass reactor with a conical bottom
- Aeration: After ozonation all samples of AS and AS+WW, were aerated for an hour (dissolved oxygen level was maintained between 2 and 4 mgO2/L) in small batch reactors. Before viability measurement each sample was saturated with oxygen.
- No. of vessels per concentration (replicates): 4 for the activated sludge in distilled water, and 3 replicates for the activated sludge in wastewater.
- No. of vessels per control (replicates):4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: see table 1 below
- Particulate matter: MLSS concentration in the experiment of AS ozonation varied from 1.20 to 1.45 g/L and in AS+WW ozonation from 1.40 to 2.40 g/L.

OTHER TEST CONDITIONS
- Adjustment of pH: no

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : Oxygen uptke rate, ATP

TEST CONCENTRATIONS
- Spacing factor for test concentrations: no constant spacing factor: ca 2

Reference substance (positive control):

no

Duration:

1.5 min

Dose descriptor:

NOEC

Effect conc.:

ca. 42 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: oxygen uptake rate

Remarks on result:

other: AS + WW

Key result

Duration:

1.5 min

Dose descriptor:

NOEC

Effect conc.:

ca. 15 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: oxygen uptake rate

Remarks on result:

other: AS only

Details on results:

see below

Results with reference substance (positive control):

n/a

OUR

The effect of ozone on respiratory activity of activated sludge was determined by measuring the maximum oxygen uptake rate (OUR) expressed in mgO₂L⁻¹h⁻¹of sludge treated with various ozone doses (15–70 mgO₃L⁻¹). For better comparison of the OUR results, the specific oxygen uptake rate (SOUR), expressed in mgO₂gMLVSS⁻¹h⁻¹, was calculated by dividing OUR by MLVSS, as it depends on the concentration of MLVSS. For calculation of exogenous SOUR (SOURex) both endogenous and total SOUR (SOURend and SOURtot, respectively), were measured. SOURex, used for evaluation of sludge viability, was calculated by subtracting SOURend from SOURtot. SOURend is defined as the oxygen consumption in the absence of external substrate (Dawes and Ribbons, 1962). SOURex is oxygen consumption caused by addition of external biodegradable substrate. Initial SOURend of the studied sludge varied between 8 and 12.3 mgO₂gMLVSS⁻¹h⁻¹(Fig. 1). Depending on the ozone dose applied, the measured SOURend, without the addition of external substrate, increased up to 1.8 times compared to initial SOURend (Fig. 1). Ozone is known to be a strong cell lyses agent and thereby reduces the number of viable micro-organisms in activated sludge. If the number of viable micro-organisms is reduced, the SOUR_end should also be reduced, as it depends on the concentration of oxygen consuming cells which use intracellular reserve material for maintenance purposes (Dawes and Ribbons, 1962).

To determine any correlation between the soluble COD generated during ozonation and SOUR_end, ∆COD (COD – COD₀) was plotted against ∆SOUR_end(SOUR_end-SOUR_end0) (Fig. 2).

ATP

Initial specific ATP concentration (SATP) of activated sludge (ATP concentration in zero-dose activated sludge) used for ozonation ranged from 2.30 to 2.80 mgATP gMLVSS⁻¹. SATP concentration, depending on the applied ozone dose, ranged from 1.05 to 1.98 mgATP·gMLVSS⁻¹. Reduction of SATP content in activated sludge due to the ozonation, expressed as relative SATP (SATP/SATP₀), is presented in Figure 3.

Validity criteria fulfilled:

not applicable

Remarks:

no guideline

Conclusions:

The NOEC for O3 was determined with 15 mgO3/L in activated sludge, exposure time 1.5 min.

Executive summary:

This study aimed to analyze the effect of ozonation on viability of activated sludge in different systems – activated sludge in distilled water and activated sludge in wastewater. Ozonation time of the systems, depending on the transferred ozone dose, ranged from ~70 to ~120 seconds. Two viability detection methods, oxygen uptake (OUR) rate and adenosine-5’-triphosphate measurement (ATP), were compared. An immediate viability reduction was detected when only activated sludge was subjected to ozonation, however, ozonation of activated sludge with wastewater, at doses up to 42 mg O3/L did not influence the viability of sludge. A NOEC of 15 mg O3/L was calculated, based on the activated sludge only.

Reference 2

Endpoint:

activated sludge respiration inhibition testing

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because there is no emission to a sewage treatment plant

other:

Justification for type of information:

Ozone is a gas which will evaporate into the air, or react immediately in the water phase with components such as organic material and metal ions. In view of the short half-life (500 to 5000 s, see Gardoni et al., 2012 in section 5.6), ozone generated for the purpose of water ozonation can safely be expected to completely decompose still within the technical environment (i.e. in the sewerage), before reaching the STP or any water bodies. Therefore, the concentration of ozone in surface waters resulting from technical ozone generation can be considered to be negligible. Performing a test on toxicity to microorganisms is not considered to be required.

Description of key information

The study does not need to be conducted, as ozone is unlikely to stay in the water phase, hence, is not expected that the applied ozone will reach a sewage plant. Ozone is unstable, evaporates into the air, and/or reacts with other molecules due to its oxidizing characteristics. Therefore, no PNEC for microorganisms was derived. A supporting study is attached, analyzing the short term toxicity (about 1.5 min exposure) of ozone to activated sludge.