Many human pathogens can be transmitted by waters contaminated by wastewater effluents, which should be disinfected to prevent the spread of pathogenic microorganisms, particularly when the wastewater is used for watering, drinking or bathing purposes [1
Wastewater disinfection is applied to provide protection to humans against exposure to waterborne pathogenic microorganisms. Microbial inactivation is achieved in these processes by induced biochemical changes within the pathogenic microbial population. The nature of these biochemical changes is dependent upon the microbial population and the applied disinfectant. All disinfectants have the ability to involve changes in wastewater composition that many persist after the disinfection process is terminated, though the nature and extent of these changes will be site- and disinfectant-specific. One possible outcome of these chemical changes is an alteration of effluent toxicity [2
Chlorination is the most widely used method for disinfecting the effluents from wastewater treatment plants (WWTPs) and from drinking water treatment plants (DWTPs), but can cause the formation of mutagenic/carcinogenic and toxic by-products that are potentially harmful to human and aquatic organisms [3
Recently, peracetic acid (PAA) has also been proposed as a wastewater disinfectant. Peracetic acid (CH3
H) is a strong oxidant which presents several advantages, including: the treatment being easy to implement (without the need of high investment); the large spectrum of microbial activity even in the presence of heterogeneous organic matter; absence of residual or toxic and/or mutagenic by-products; not requiring dechlorination; presenting low dependency on pH; and short contact time [11
]. Major disadvantages associated with PAA disinfection are the increase of organic content in the effluent due to acetic acid, and thus the potential for microbial regrowth (acetic acid is already present in PAA mixtures and is also formed after PAA decomposition) [12
The disinfection efficiency of PAA in wastewater applications has been demonstrated in the scientific literature [13
]. The formation of disinfection by-products (DBPs) during PAA disinfection was studied by Dell’Erba [17
The European Community’s (EC) environmental regulations aim to reduce the pollution of surface water caused by municipal wastewater (Council Directive 91/271/EEC 1991 as amended by the Commission Directive 98/15/EEC of 27 February 1998) [18
]. This requires the European Union member states to ensure that discharge of urban wastewater and its effects are monitored [20
In order to prevent sanitary hazards related to the uses of recipient water bodies, the current Italian regulations prescribe WWTP effluent emission limits for a wide range of chemical compounds, toxicity, and bacterial discharge, such as Escherichia coli
In Italy the legislation provides the following limits for wastewater: active chlorine concentration below 0.2 mg L−1, lack of acute toxicity and E. coli concentration below 5000 CFU 100 mL−1 (recommended value). In order to respect this value, many WWTPs apply a wastewater disinfection process, because sometimes the E. coli concentration in the effluent is higher than the limit established by the local authorities.
Disinfection techniques should be analyzed case by case, because each effluent shows different chemical and physical characteristics, and, sometimes, the reuse of wastewater (i.e., for agricultural use) is applied [22
In a different way, depending on the treatment adopted, there are different E. coli
removal yields for each different stage of WWTPs [23
It is well known that some disinfectant treatments, despite promoting a reduction in pathogenic organisms and organic contaminants, can also produce toxic and genotoxic compounds, depending on the precursors present in the wastewater and the concentrations of disinfectant used [24
To clarify this issue, a series of toxicity tests, using Daphnia magna, Vibrio fischeri
and Pseudokirchneriella subcapitata
], were performed on wastewater effluent samples taken from two WWTPs.
The toxicity responses were measured in disinfected and undisinfected effluent samples in order to evaluate the changes in toxicity attributable to disinfection processes.
The determination of toxicity in wastewater was carried out using Daphnia magna. The test is based on the observation of the number of immobile organisms after 24 h of exposure to the sample. Immobilization is the effect on the organisms caused by the toxic substances present in the sample.
In addition to the test with Daphnia magna as indicated in D.Lgs 152/2006 and further modifications, acute toxicity tests were carried out using bioluminescent bacteria (Vibrio fischeri) and monocellular green algae (Pseudokirchneriella subcapitata).
The test with Vibrio fischeri was based on the observation of luminescence inhibition after 15 min and 30 min. In the case of Pseudokirchneriella subcapitata the test was based on the observation of inhibition of algal growth after 72 h.
The study of disinfectant agents suitable for microbial population removal without acute toxicity is very important, especially in Italy due to the different enforcement (at a local scale) of E. coli limit values, which, according to national legislation, are recommendations only.
The aim of the present study was to compare two different disinfectants (sodium hypochlorite and peracetic acid) in terms of acute toxicity on the water body and microbial inactivation. Moreover, the aim was to find a dosage range which allows the respect of E. coli limit values, acute toxicity, disinfectant residual and COD (Chemical Oxygen Demand).
The disinfection tests were applied to the effluent from two active sludge WWTPs in northern Italy with small capacities (10,000 to 12,000 PE), and two disinfectant agents (sodium hypochlorite and PAA) were used. These activities provided the assessment of microbiological quality and toxicity of WWTPs effluents in relation to the dosage of sodium hypochlorite and peracetic acid, by means of the use of batch tests.
2. Materials and Methods
2.1. Description of the WWTPs and Characteristics of the Influent
The experimental work were carried out on two urban WWTPs in northern Italy with small capacities (12,000 PE and 10,000 PE respectively).
The urban WWTPs identified by number 1 and 2 are based on a conventional active sludge (CAS) process with a pre-denitrification scheme. They treat domestic, meteoric and industrial sewage collected in a unit drainage system. Table 1
presents the main features of each WWTP.
Both WWTPs are equipped with an emergency disinfection unit with the use of sodium hypochlorite. The contact tanks are built with a longitudinal baffle serpentine flow basin, with a volume of 90 m3 for WWTP 1 and 35 m3 for WWTP 2 (the contact time depends on the flow rate, usually about 30 min for WWTP 1 and 20 min for WWTP 2). There are no flash-mixing tanks.
2.2. Experimental Tests
Laboratory scale disinfection tests were executed (in the WWTPs analyzed) using 40 L of purified effluent taken with an automatic sampler. Each day, three disinfection tests were carried out with different sodium hypochlorite doses, three tests with different peracetic acid doses (PAA) and a reference sample without the dosage of disinfectant agents. pH was measured at the beginning and end of the tests. The solutions were put into a jar test for mixing (Figure 1
) with a rotation speed of 45 rpm.
Disinfection tests were carried out on the effluent of WWTP 1 by testing different disinfectant dosages, while for the WWTP 2 the doses were chosen on the basis of the results obtained for WWTP 1. Table 2
presents the experimental program and the analysis/tests carried out in the work.
2.3. Analytical Methods
The concentrations of COD, BOD5
, Total Nitrogen (TN), Total Phosphorus (TP) and Total Suspended Solids (TSS) were measured according to standard methods for water and wastewater [30
was determined at 20 °C by inoculation of activated sludge from the WWTP. The pH was measured with a probe Sentix 940-3 WTW®
The concentrations of residual chlorine and residual acid peracetic were measured by means of colorimetric tests.
Free residual chlorine was measured with Hach Lange LCK 410 kit.
Total chlorine and peracetic acid residues were measured with the method “Hach 10070 Pillows powder”. In case of PAA the conversion of mg Cl2
to mg PAA L−1
(by multiplying the value of 1.07) is necessary [34
The respirometric tests of oxygen uptake rate (OUR) were carried out according to ISO 8192:2007 [35
The E. coli
concentration was measured using two methods: the number of colonies of E. coli
(CFU) (Standard Method) [30
] and the most probable number of microorganisms per volume (MPN) [36
The CFU method allows the counting of the number of E. coli colonies grown on a membrane on agar soil supplemented with chromogenic substances. The sample is filtered through a cellulose ester membrane of 0.45 μm nominal porosity. After an incubation period of 18–24 h at 44 ± 1 °C, the results are read under ultraviolet light (366 nm). The 4-methylumbelliferyl-β-d-glucuronide (MUG) compound, embedded in the soil, is hydrolyzed by β-glucuronidase of E. coli, releasing 4-methylumbelliferone compound which thus produces ultraviolet light-fluorescent blue-green colonies. The results are expressed as colony forming units per 100 mL sample.
The MPN method expresses results as the most likely number of microorganisms per volume. A dehydrated soil is added to a sample; then the mixture is transferred in boxes that are sealed and then incubated at 36 ± 2 °C for 18–22 h. After the incubation, samples with a yellow color intensity equal to or greater than that of the Quanti-Tray/2000 Comparator are considered positive for coliform bacteria. Yellow samples, which are tested under UV light (365 nm) in a dark room, exhibiting any degree of fluorescence are considered positive for E. coli. The most probable amount of E. coli in 100 mL of the sample can be determined through statistical tables.
The determination of the acute toxicity of the WWTPs effluents was carried out by multispecific ecotoxicological tests: Daphnia magna
at 24 h [37
], Vibrio fischeri
at 15 min and 30 min [38
] and Pseudokirkneriella subcapitata
at 72 h [39
Neonates of Daphnia magna were held in a temperature-controlled room at 20 ± 2 °C illuminated with fluorescent lamps for 16 h d−1. In the acute toxicity tests, daphnids were exposed to samples of different concentrations of wastewater and the tested agent; the immobile daphnids were counted after 24 h of exposure.
The luminescence of reconstituted liquid dried bacteria Vibrio fischeri NRRL-B-11177 was measured on a Microtox mod. 500 luminometer according to the ISO standard. The luminescent bacteria were exposed to wastewater samples for 15 min and 30 min. The percentage inhibition was calculated for each concentration relative to the control.
The green, unicellular algae Pseudokirchneriella subcapitata were cultured in a nutrient solution prepared and kept on an orbital shaker at 100 rpm at a constant room temperature of 23 ± 2 °C and under continuous fluorescent illumination (6000–10,000 lx.). After 72 h the growth of algae was determined by algal density, which was measured by counting algal cells in a Burker counting cell or absorbance measurement with the spectrophotometer. The inhibition of specific growth rates for each concentration was calculated in comparison to the control.
The aim of the present study was to evaluate the ecotoxicological behavior of secondary effluents disinfected with sodium hypochlorite and PAA using three species from different trophic levels of aquatic ecosystems. Moreover, the work is aimed to find a dosage range, which allows the respect of E. coli limit values, acute toxicity, disinfectant residual and COD.
The experimental activities provided assessment of microbiological quality and toxicity of WWTPs effluents in relation to the dosage of sodium hypochlorite and peracetic acid, by means of the use of batch tests.
The results showed that the acute toxicity was increased as the disinfectant residual value increases. Moreover, residual acute toxicity of peracetic acid was higher (2.68 mg L−1) than residual chlorine (0.17 mg L−1). As concerns the chlorination, a good correlation between the content of chloramines and the acute toxicity was observed.
As concerns the WWTP 1, an active chlorine dosage between 30 and 50 mg min L−1 allowed the contemporary respect of the limit of E. coli and a non-toxicity of disinfected effluent and a PAA dosage between 20 and 40 mg min L−1 allowed the contemporary respect of the E. coli limit and a non-toxicity of disinfected effluent. Concerning the WWTP 2, an active chlorine dosage between 20 and 25 mg min L−1 allowed the contemporary respect of the E. coli limit and a non-toxicity of disinfected effluent and a PAA dosage between 20 and 35 mg min L−1 allowed the contemporary respect of the E. coli limit value and a non-toxicity of disinfected effluent.
The results show that with similar disinfectant dosage and comparable initial E. coli concentration, peracetic acid displayed the best performance in terms of microbial removal (with removal yields up to 99.99%).