ARTICLE IN PRESS Ecotoxicology and Environmental Safety 72 (2009) 1076–1081 Contents lists available at ScienceDirect Ecotoxicology and Environmental Safety journal homepage: www.elsevier.com/locate/ecoenv Highlighted Article Physico-chemical, microbiological and ecotoxicological evaluation of a septic tank/Fenton reaction combination for the treatment of hospital wastewaters Josiani Berto, Gisele Canan Rochenbach, Marco Antonio B Barreiros, Albertina X.R Correˆa, Sandra Peluso-Silva, Claudemir Marcos Radetski à ´gicas da Terra e Mar, Itajaı´, SC 88302-202, Brazil Universidade Vale Itajaı´, Centro de Cieˆncias Tecnolo a r t i c l e in f o a b s t r a c t Article history: Received 10 May 2008 Received in revised form 25 November 2008 Accepted December 2008 Available online 23 January 2009 Hospital wastewater is considered a complex mixture populated with pathogenic microorganisms The genetic constitution of these microorganisms can be changed through the direct and indirect effects of hospital wastewater constituents, leading to the appearance of antibiotic multi-resistant bacteria To avoid environmental contamination hospital wastewaters must be treated The objective of this study was to evaluate the efficiency of hospital wastewater treated by a combined process of biological degradation (septic tank) and the Fenton reaction Thus, after septic tank biodegradation, batch Fenton reaction experiments were performed in a laboratory-scale reactor and the effectiveness of this sequential treatment was evaluated by a physico-chemical/microbiological time-course analysis of COD, BOD5, and thermotolerant and total coliforms The results showed that after 120 of Fenton treatment BOD5 and COD values decreased by 90.6% and 91.0%, respectively The BOD5/COD ratio changed from 0.46 to 0.48 after 120 of treatment Bacterial removal efficiency reached 100%, while biotests carried out with Scenedesmus subspicatus and Daphnia magna showed a significant decrease in the ecotoxicity of hospital wastewater after the sequential treatment The use of this combined system would ensure that neither multi-resistant bacteria nor ecotoxic substances are released to the environment through hospital wastewater discharge & 2008 Elsevier Inc All rights reserved Keywords: Hospital wastewater treatment Fenton reaction Hospital wastewater microbiology Pharmaceuticals Ecotoxicity Introduction Hospital establishments make use of large amounts of pharmaceuticals and related products, which originates wastewaters with a complex constitution (Tsakona et al., 2007) After therapeutic use, pharmaceuticals (parent or metabolized compounds) are excreted to the hospital sewage system, and then released to the environment with or without treatment When these wastewaters are treated in the sewage system, most of these compounds are degraded by microbiological processes (Halling-Sorensen et al., 1998), but there are several studies showing that microorganisms are not able to degrade many pharmaceutical compounds (Jorgensen and Halling-Sorensen, 2000; Alexy et al., 2004; Carballa et al., 2004), which can be detected in river and ocean waters (Lee and Arnold, 1983; Calamari et al., 2003), sediments (Lo¨ffler et al., 2005) and soil samples (Christian et al., 2003) It is well established that biologically active compounds like pharmaceuticals pose environmental risks (Ferrari et al., 2004) and in this regard toxic effects have been observed in non-target classes of organisms, such as à Corresponding author Fax: +55 48 33417970 E-mail address: radetski@univali.br (C.M Radetski) 0147-6513/$ - see front matter & 2008 Elsevier Inc All rights reserved doi:10.1016/j.ecoenv.2008.12.002 phytoplankton (Blaise et al., 2006), daphnids (Flaherty and Dodson, 2005), aquatic plants (Pro et al., 2003), insects (RidsdillSmith, 1988), and other species (Halling-Sorensen et al., 1998; Blaise et al., 2006) As some pharmaceuticals are present at low concentrations in environmental samples, a recent review was carried out on the ecotoxicity of pharmaceuticals present in treated urban wastewaters (Blaise et al., 2006) Even at lower concentrations, antibiotics are an example of a notorious public health concern because the genetic constitution of some microorganisms can be changed through the direct and indirect effects of these compounds leading to the appearance of antibiotic multi-resistant bacteria (Davison, 1999; Kola´r et al., 2001; Schwartz et al., 2003) Although a variety of studies addressing methods for the treatment of hospital wastewater constituents have been published (Kajitvichyanukul and Suntronvipart, 2006; Gautam et al., 2007; Jara et al., 2007), few hospitals worldwide treat their wastewaters efficiently, and they are discharged into surface waters or sewage systems (Pru¨ss et al., 1999; Heberer, 2002) Since microbiological degradation has been shown to be only partially effective for this purpose, a combination of microbiological and chemical treatment processes has been proposed to increase the efficiency and decrease the costs of pharmaceutical wastewater treatment (Arslan-Alaton and Balcioglu, 2002; Cokgor et al., 2004; ARTICLE IN PRESS J Berto et al / Ecotoxicology and Environmental Safety 72 (2009) 1076–1081 Gotvajn et al., 2007) Thus, the purpose of this study was to determine the effectiveness of a combined microbiological and chemical (Fenton reaction) process to remove both organic matter and the pathogenic microbiota from hospital wastewaters The efficiency evaluation was carried out by means of time-course measurements of physico-chemical and microbiological parameters of treated hospital wastewater, as well as by comparison of ecotoxicity results obtained with algae and daphnids exposed to raw and treated effluent samples 1077 2.5 Statistical analysis for ecotoxicity tests Statistical analysis was carried out on a microcomputer using the TOXSTAT 3.0 software Responses were presented together with the mean (X) and the coefficient of variation (C.V.) The Williams test (Pp0.05) was used to obtain the lowestobserved-effect concentration (LOEC) after applying Shapiro-Wilk’s test for normality and Hartley’s test for homogeneity of variance In the case of daphnids, Fisher’s exact test was used (Pp0.05) Results Materials and methods 2.1 Hospital wastewater sample collection and analysis Three 2000 mL composite samples were obtained between am and pm at 2-h intervals at three different times Samples were collected in amber glass bottles and stored at 1C for no longer than day Theoretical pharmaceutical concentrations in hospital wastewater were calculated considering the amount of the pharmaceutical used, renal metabolization rate and annual water consumption Periodical physico-chemical and microbiological analyses were carried out (at 0, 30, 60 and 120 min) according to standard methods (APHA et al., 1995) For BOD5 determination of Fenton-treated wastewater samples of a bacterial inoculation of sewage was required 3.1 Antibiotics and bacteria present in the hospital wastewater Quantification of the antibiotics used and the water consumption, together with the renal metabolization rate, allowed us to calculate a theoretical unmetabolized concentration of these compounds in the wastewater, which is shown in Table Besides the residual antibiotics, some pathogenic bacteria from different families were present in the hospital wastewaters (Table 2) 3.2 Microbiological hospital wastewater treatment efficiency 2.2 Hospital wastewater treatment plant description All experiments were carried out at room temperature, to mimic ambient conditions and for economic reasons The hospital wastewater station involves a two-stage secondary biological degradation that occurs in a two-compartment fiber tank (45 m3) that receives non-segregated (but filtered through a grill) hospital wastewater In the first stage (equalization compartment), conventional sedimentation is carried out with a relatively short solids retention time for substrate removal Stage is a separate aerated activated sludge process with a longer solids retention time (30 h) where flocculation and sedimentation take place An on/off cycle of 60 applied to the aerators allows a sludge sedimentation step in this compartment After this two-stage treatment in the hospital wastewater plant, samples were collected to carry out the Fenton reaction in the laboratory In the first step of the hospital wastewater treatment a microbiological process was used to degrade the organic matter in the sewage system The effectiveness of this process is shown in Table The inlet wastewater of the microbiological treatment system showed a characteristic pH of 6.71, and a high value for total solids of 7356 mg/L, whereas the suspended solids concentration was found to be 539 mg/L High values for COD and BOD5 (2480 and 1268 mg/L, respectively), and a high coliform bacteria count of 2.2  108/100 mL were determined in these wastewater samples 3.3 Fenton oxidation efficiency 2.3 Hospital wastewater treatment by Fenton reaction Three wastewater samples (2000 mL) were collected at the outlet of microbiological treatment system From each sample, three replicate sub-samples of 500 mL were taken and these were subject to the Fenton reaction in a batch reactor with pH adjustment to 3.8 by addition of M H2SO4 Ferrous ions (2.88 g) and hydrogen peroxide (30%, dropped at 0.1 mL/min) were added to the batch reactor and the treatment time was 30, 60 and 120 2.4 Ecotoxicity tests 2.4.1 Algal growth inhibition test The algal species used was Scenedesmus subspicatus Chodat (strain 86.81 SAG, Go¨ttingen, Germany) Three algal tests for both, raw and treated effluent samples were conducted according to a standardized protocol (ISO, 1990) with three replicates per concentration (or control) Potassium dichromate was used as a positive control The cell density of the mixture was adjusted to 10,000 cells/mL by dilution with ISO freshwater algal test medium Each test consisted of seven filtered effluent dilutions and a control group The test flasks were incubated on a shaker (100 rpm) with continuous illumination of 70 mE/m2/s (cool-white fluorescent lamps) at 2372 1C After 72 h of incubation, the inhibitory effect based on fluorescent activity was measured at l ¼ 685 nm with a Shimadzu RF-551 (Kyoto, Japan) spectrofluorimeter 2.4.2 Daphnia magna mortality test The 48-h immobilization test with D magna was performed in accordance with a standardized protocol (ISO, 1989) at 2572 1C using 20 individuals per replicate (less than 24 h old) in 50-mL glass beakers with 30 mL of test medium Three different tests (with three replicates per dilution) were performed for both raw and treated effluent samples in order to evaluate the variability of the procedure Each test consisted of seven filtered effluent dilutions and a control group Potassium dichromate was used as a positive control In Table 4, the initial values for some parameters analyzed are shown along with the time-course results of the physico-chemical and microbiological analysis for the hospital wastewater treated by the Fenton reaction 3.4 Ecotoxicity tests Concerning evaluation of hospital wastewater ecotoxicity, two different organisms representing aquatic ecosystems were tested, i.e., algae and daphnids Fig and Table show the results for the algae and daphnids exposed to the raw and treated hospital wastewater Table Theoretical antibiotic concentrations in the hospital wastewater Antibiotics Concentration (mg/m3) Gentamicin Keflin (cephalothin) Keflex (cephalexin) Amoxicillin Ampicillin Benzathine penicillin Crystalline penicillin Procaine benzylpenicillin Kefzol (cefazolin sodium) Rocephin (ceftriaxone) Floxacin 25.52 801.02 300.10 35.12 389.13 434.46 68.20 361.79 85.37 126.91 46.70 ARTICLE IN PRESS 1078 J Berto et al / Ecotoxicology and Environmental Safety 72 (2009) 1076–1081 Table Some bacterial species found in the hospital wastewater sampled in the microbiological treatment step Bacteria Family Classification Acinetobacter sp Aeromonas sp.a Alcaligenes xylosoxidans Citrobacter sp Chryseomonas luteola Enterobacter sp Enterococcus sp Escherichia coli Klebsiella sp.a Kluyvera sp Leuconostoc spp Morganella morgania Pantoea sp Pasteurella sp Pseudomonas sp.a Proteus sp.a Providencia sp Salmonella spp.a Serratia sp.a Staphylococcus sp – Vibrionaceae – Enterobacteriaceae – Enterobacteriaceae Streptococcaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae – Enterobacteriaceae Enterobacteriaceae Pasteurellaceae – Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Enterobacteriaceae Micrococcaceae Coccus and bacillus Gram-negative aerobes/microaerophile Bacillus Gram-negative facultative anaerobes Coccus and bacillus Gram-negative aerobes/microaerophile Bacillus Gram-negatives facultative aerobes Coccus and bacillus Gram-negative aerobes/microaerophile Bacillus Gram-negative facultative aerobes Coccus Gram-positive Bacillus Gram-negative facultative aerobes Bacillus Gram-negative facultative aerobes Bacillus Gram-negative facultative aerobes Coccus Gram-positive Bacillus Gram-negative facultative aerobes Bacillus Gram-negative facultative aerobes Bacillus Gram-negative facultative aerobes Coccus and bacillus Gram-negative aerobes/microaerophile Bacillus Gram-negative facultative aerobes Bacillus Gram-negative facultative aerobes Bacillus Gram-negative facultative aerobes Bacillus Gram-negative facultative aerobes Coccus Gram-positive a High human pathogenicity Table Hospital wastewater quality at the inlet and outlet pipe of the microbiological treatment system and mean removal efficiency (n ¼ 3) Parameters Units Color mg/L Pt-Co mg/L mg/L mg/L mg/L – mg/L mg/L MPN/100 mL MPN/100 mL COD BOD5 Phosphorous Nitrogen pH Total solids (TS) Suspended solids (SS) Total coliforms Thermotolerant coliforms Inlet Outlet 12,08971629 24807413 12687155 28.677.2 85.5714.8 7.2070.5 73837853 546.7764.8 203,000,000715,000 165,000,000713,200 Mean removal efficiency (%) 73087822 39.5 13387267 612751 10.772.0 49.576.7 5.1070.2 23847295 49.6712.4 110,00076000 71,70076800 46.0 51.7 62.6 42.1 – 67.7 90.9 99.9 99.9 Table Hospital wastewater quality before Fenton treatment and after 30, 60, 120 of treatment (n ¼ 3) Parameters (units) Total coliforms (MPN/100 mL) Thermotolerant coliforms (MPN/100 mL) COD (mg/L) BOD5a (mg/L) Values before Fenton treatment 110,00076000 71,70076800 13387267 612.1751.2 Values after Fenton treatment 30 60 120 ND ND 769.0715.9 301.7749.5 ND ND 658.0742.1 232.7722.0 ND ND 120.0723.4 57.7713.3 MPN—most probable number; ND—not detected a pH neutralization and bacterial inoculation were required to carry out this test Discussion Of the different hospital wastewater constituents, antibiotics merit special attention due to their biological activity, which leads to their potential to generate multi-resistant bacteria (Ku¨mmerer, 2004) If we consider the antibiotics present in Table 1, the mean concentration of these compounds in the wastewater is 2.7 mg/L European hospital wastewater discharges have been found to be responsible for an antibiotic concentration of 50 mg/L in municipal wastewater plants (Ku¨mmerer, 2001) Several studies have shown that different classes of antibiotics are found in the environment and many species of organisms can be adversely affected by these compounds (Hirsch et al., 1999; Kolpin et al., 2002; Blaise et al., 2006) But in the case of antibiotic effects, the most dangerous aspect of residual concentrations is the potential generation of multi-resistant bacteria, which will depend on the exposure of the bacteria to antibiotics, even to weak antibiotic concentrations (Guardabassi et al., 1998; Chitnis et al., 2000; Meirelles-Pereira et al., 2002; Schwartz et al., 2003; Ku¨mmerer, 2004) Currently, induction of bacterial resistance that can pose a serious threat to public health has obliged most health care establishments to install an infection control commission to manage this problem, but wastewater treatment generally does ARTICLE IN PRESS J Berto et al / Ecotoxicology and Environmental Safety 72 (2009) 1076–1081 COD and BOD5 values by 42.5% and 50.7%, respectively After 60 and 120 of Fenton treatment, there were additional significant decreases in these parameters (50.8% and 91.0% for COD, and 62% and 90.6% for BOD5, respectively) It is interesting to note that inverting the order of the wastewater treatment sequence leads to the same results in terms of efficiency The use of Fenton oxidation followed by aerobic degradation in sequencing batch reactors to improve the biodegradability of a pharmaceutical wastewater showed an overall COD removal efficiency of 98%, very close to the result shown in our study (Tekin et al., 2006) Result obtained from another study indicated that the photo-Fenton process could be a suitable pretreatment method in reducing the toxicity of pollutants and enhancing the biodegradability of hospital wastewaters treated in a combined photochemical–biological system (Pru¨ss et al., 1999) The Fenton reaction acts as a disinfection system, probably due to the lower pH (3.8) and powerful oxidant hydroxyl radicals generated in this reaction In this sense, low pH is effective in killing the bacteria, but DNA denaturation is assured by hydroxyl radicals, which ensures that plasmid information will not be spread among different species of bacteria by horizontal transmission Furthermore, the BOD5/COD ratio of the wastewater was increased from 0.39 in 30 of treatment to 0.48 in 120 of treatment This is indirect evidence that oxidized intermediate pharmaceuticals are less toxic than parent compounds present in the wastewater before application of Fenton treatment It is generally considered that BODn/COD ratios higher than 0.4 indicate a high biodegradability of the sample (Metcalf and Eddy, 1991) Concerning evaluation of hospital wastewater ecotoxicity, algae are considered to be at the base of the trophic chain in aquatic ecosystems and environmental impacts could lead to the inhibition or stimulation of algal growth The two types of effluents showed opposite effects (Fig 1) While the treated effluent did not show algal toxicity in any of the dilutions tested, the raw effluent showed a biphasic response: increase in algal growth in low effluent dilutions followed by a decrease in algal growth, with an LOEC of 16.0% This finding could be explained by the presence of nutrients and toxicants in the hospital wastewater In the treated wastewater, ecotoxic organic compounds were degraded, while in the raw wastewater ecotoxic organic compounds were present, surpassing stimulation of algal growth by the nutrients A comparison of the toxicity of the two types of wastewaters can be carried out using the LOEC ratios For the algal test this ratio was X4, i.e., treated effluent was times less toxic than the raw effluent In the case of Daphnia magna, the raw hospital wastewater was more ecotoxic (LOEC ¼ 4%) than the treated hospital wastewater (LOEC ratio X100) (Table 5) A comparison of the LOEC ratios showed a value of X25 for the cladoceran organisms Thus, it is clear that daphnids are more sensitive than algae in the assessment of hospital wastewater ecotoxicity Overall, this study revealed that the most employed hospital wastewater treatment, i.e microbiological degradation, is not sufficient to eliminate some pharmaceuticals and bacterial populations, which could be achieved by an additional oxidation not receive special attention In this regard, a horizontal plasmid transmission of genetic contents from resistant bacteria to wild bacteria could trigger an important ecological perturbation Although some studies suggest that the risk of the spread of antibiotic resistance in hospital wastewater is limited (Valles et al., 2004; Tume´o et al., 2008), it is important to avoid the release of hospitalar bacteria to the environment, which could be achieved through efficient antibiotic molecule oxidation, followed by disinfection and DNA denaturation The concentrations of coliform bacteria shown in Table are in the same order of magnitude as the 108/100 mL generally present in municipal sewage systems (Metcalf and Eddy, 1991), but higher than values found by other authors, who have reported values of  105/100 mL (Leprat, 1998) If we consider microbiological hospital wastewater treatment efficiency, this process alone is not sufficient to totally eliminate the organic matter or the bacteria present in the hospital wastewater (Table 3) However, after microbiological treatment, the outlet wastewater samples showed a significant reduction in the values of these parameters, which indicates good removal efficiency Concerning these results, it must be remembered that some pharmaceuticals (e.g., antibiotics and disinfectants) can disturb the wastewater treatment process, which could limit microbiological treatment efficiency (Al-Almad et al., 1999; Ku¨mmerer, 2002) Thus, if we consider that most hospital establishments treat wastewaters using a microbiological process, the presence of multi-resistant bacteria in the treated wastewater and the long-term environmental exposure to low concentrations of antibiotics merit more attention from environmental scientists It is reasonable to assume that some recalcitrant pharmaceuticals and multi-resistant bacteria are present at the outlet of the microbiological wastewater treatment system (Ash et al., 1999; Blaise et al., 2006) Thus, a more powerful oxidant and a simple process (Fenton reaction) were chosen as the second step in the treatment of the hospital wastewater It is clear from Table that the Fenton reaction is very effective in promoting organic matter degradation, since 30 of treatment was sufficient to decrease the mean 400 ∆ Fluorescence 350 300 250 200 150 Raw effluent 100 Treated effluent 50 0% 1% 2% 4% 8% 16% Effluent dilution 32% 1079 64% Fig Fluorescence variation of algae exposed to the raw and treated hospital effluents Table Number of dead Daphnia magna after 48 h of exposure to raw and treated hospital wastewater Dilution (%) Sample Treated wastewater Raw wastewater 0.0 Control 0 Results 1.0 0 2.0 à Statistically significant difference (Pp0.05; Fisher’s exact test) 4.0 4à 8.0 9à 16.0 10à 32.0 10à 100.0 10à LOEC X100.0% 4.0% ARTICLE IN PRESS 1080 J Berto et al / Ecotoxicology and Environmental Safety 72 (2009) 1076–1081 treatment, in this case the Fenton reaction The treatment system used in this study is neither technically complex nor very costly In our opinion, despite some international initiatives from the World Health Organization (Pru¨ss et al., 1999), there is a lack of adequate staff training regarding hospital waste management/treatment issues and the public and environmental risks that might emerge from inappropriate wastewater treatment Thus, in view of the partial efficiency of microbiological treatment, combined systems like that here described should be promoted so that the hospital administration can ensure proper wastewater treatment There are other wastewater treatment process options available for pharmaceutical removal, for example, sorption on powdered activated carbon, reverse osmosis, and oxidation with chlorine and ozone (Heberer, 2002; Ikehata et al., 2006; Kajitvichyanukul and Suntronvipart, 2006; Gagne´ et al., 2008), but generally these processes have some drawback such as high cost, complexity, or hazardous by-products Our results showed that treated hospital wastewater samples were not toxic to the aquatic organisms S subspicatus and D magna within the tested dilutions Since neither microbiological treatment nor the Fenton process is able to remove metals that could be present in the complex mixture of the hospital wastewater, synergistic and antagonistic effects could exist, and prudence must guide the interpretation of correlations between the effluent quality parameters and toxicological profile in the attempt to highlight interactions between these variables Thus, a more complex chemical analysis of the hospital wastewater is necessary in order to establish any correlation Thus, independent of the type of process used to treat hospital wastes (including wastewater sludge), it would be interesting to use chemical analysis and a battery of ecotoxicity tests to evaluate the environmental hazards associated with the residual wastes (Rosa et al., 2001; Mantis et al., 2005) Conclusions Although there was a significant reduction in the wastewater microbiological and organic matter content after the aerobic septic tank treatment step, the remaining microbiota (including multi-resistant bacteria) are sufficient to cause environmental and public health concerns Thus, a low cost chemical oxidation process was carried out to ensure total disinfection of the wastewater and to further reduce the organic content The wastewater treatment by the Fenton reaction for 120 decreased BOD5 by 90.6% and COD by 91.0%, leading to an increase in the wastewater biodegradability (final BOD5/COD ratio of 0.48) No bacterial growth was observed in the treated hospital wastewater samples, while biotests carried out with S subspicatus and D magna showed a significant decrease in the ecotoxicity of the hospital wastewater after the sequential treatment methodology It must be emphasized that the quality of hospital wastewaters discharged to municipal sewerage systems following only a microbiological degradation process, or directly to the environmental compartments, is an issue of critical significance that requires more research from scientists and practical measures from hospital administrations since there will be a continued population growth and an increase in hospital wastewater complexity/generation Acknowledgments This project was financially supported by Universidade Vale Itajaı´ and CNPq (Brazilian National Council for Scientific and Technological 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