Physicochemical,microbiologica land ecotoxicological evaluation of aseptic tankFenton reaction combination for the treament of hospital wastewaters

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Ecotoxicology and Environmental Safety 72 (2009) 1076–1081

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Ecotoxicology and Environmental Safety
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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 Ã
´gicas da Terra e do Mar, Itajaı´, SC 88302-202, Brazil
Universidade do Vale do 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 2 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 min 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 min 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

1. 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: (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;


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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).

3. Results
2. Materials and methods
2.1. Hospital wastewater sample collection and analysis

Three 2000 mL composite samples were obtained between 8 am and 6 pm at
2-h intervals at three different times. Samples were collected in amber glass
bottles and stored at 4 1C for no longer than 1 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 1.
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 2 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 min 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 3.
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 Â 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 6 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 min.

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. 1 and Table 5 show the results for the
algae and daphnids exposed to the raw and treated hospital
wastewater.
Table 1
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


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Table 2
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 3
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 4
Hospital wastewater quality before Fenton treatment and after 30, 60, 120 min 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 min

60 min

120 min

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.

4. 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


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COD and BOD5 values by 42.5% and 50.7%, respectively. After 60
and 120 min 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 min of treatment to 0.48 in 120 min 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 4 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 3 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
3 Â 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 4 that the Fenton
reaction is very effective in promoting organic matter degradation,
since 30 min of treatment was sufficient to decrease the mean

400
∆ Fluorescence

350
300
250
200
150

Raw effluent

100

Treated effluent

50
0
0%

1%

2%

4%
8% 16%
Effluent dilution

32%

1079

64%

Fig. 1. Fluorescence variation of algae exposed to the raw and treated hospital
effluents.

Table 5
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
0

Results
1.0
0
0

2.0

0
2

à Statistically significant difference (Pp0.05; Fisher’s exact test).

4.0
0
4Ã

8.0
0
9Ã

16.0
0
10Ã

32.0
0
10Ã

100.0
0
10Ã

LOEC
X100.0%
4.0%

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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).

5. 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 min
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 do Vale

do Itajaı´ and CNPq (Brazilian National Council for Scientific and
Technological Development-Grant No. 470640/2007-3). C.M.
Radetski acknowledges CNPq fellowship No. 300898/2007-0.

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