ORIGINAL ARTICLE

Cytotoxic and genotoxic evaluation and chemical characterizationof sewage treated using activated sludge and a floating emergent-macrophyte filter in a municipal wastewater treatment plant:a case study in Southern Brazil

Angelica Goldoni • Camila Golfeto • Jane B. Teixeira • Gislaine Blumm •

Camila M. Wilhelm • Franko Teloken • Eloisa Bianchi • Jairo L. Schmitt •

Gunther Gehlen • Marco Antonio S. Rodrigues • Luciano Basso da Silva

Received: 6 June 2013 / Accepted: 8 January 2014

� Springer-Verlag Berlin Heidelberg 2014

Abstract This study was conducted to evaluate the

chemical parameters and the cytotoxic and genotoxic

potential of raw domestic sewage and effluents from

treatment with activated sludge and a floating emergent-

macrophyte filter from a domestic wastewater treatment

plant in the city of Novo Hamburgo, Rio Grande do Sul,

Brazil. The physicochemical analysis revealed that both

treatment systems achieved the legal emission pattern for

biochemical oxygen demand, chemical oxygen demand,

and suspended solids, but ammoniacal nitrogen and E. coli

values were above the limits in the macrophyte treatment

effluent. Phosphorous values were above the maximum

permitted for both treatments. The results obtained from

the Allium cepa test and the micronuclei test in fish did not

demonstrate any significant differences in both cytotoxicity

(mitotic index) and genotoxicity (chromosome aberration

and micronucleus) endpoints between the negative control

group and the exposed groups. However, the comet assay

in fish revealed a DNA damage increase in animals

exposed to the 30 % concentration of the macrophyte

effluent and two concentrations of the activated sludge

treatment effluent (10 and 75 %), which suggests that these

two treatment systems may increase wastewater

genotoxicity.

Keywords Sewage treatment � Activated sludge �Macrophytes � Genotoxicity � DNA damage

Introduction

Population growth and urbanization have led to an

increasing pressure over water resources and a consequent

greater need for appropriate water management practices,

which include low-cost and low-energy solutions for ade-

quate wastewater treatment (Bdour et al. 2009).

According to the National System for Sanitation Infor-

mation (2011), in Brazil, only 37.5 % of municipal waste-

water is treated before being discharged in water bodies. The

most successful and widely used technology for municipal

wastewater treatment is the biological treatment with acti-

vated sludge, in which aerobic microorganisms metabolize

organic matter. This technology requires high electrical

power consumption for pumping and aeration (EPA 2008).

Furthermore, the excess of sludge generated in this system is

a secondary solid waste, and its disposal is a major envi-

ronmental concern (Liu 2003).

Alternatively, many studies have focused on the use of

aquatic macrophytes as candidates to remove pollutants from

industrial and domestic wastewaters (Solano et al. 2004;

Khan et al. 2009; Di Luca et al. 2011; Sani et al. 2013). These

plants are appropriate for wastewater treatment systems,

given their known capacity for nutrient uptake, along with

their ability to create favorable conditions for organic matter

A. Goldoni � E. Bianchi

Programa de Pos-graduacao em Qualidade Ambiental,

Universidade Feevale, Novo Hamburgo, Brazil

C. Golfeto � J. B. Teixeira � G. Blumm

Curso de Ciencias Biologicas, Universidade Feevale,

Novo Hamburgo, Brazil

C. M. Wilhelm

Curso de Biomedicina, Universidade Feevale,

Novo Hamburgo, Brazil

F. Teloken � J. L. Schmitt � G. Gehlen �M. A. S. Rodrigues � L. B. da Silva (&)

Grupo de Pesquisa em Indicadores de Qualidade Ambiental,

Universidade Feevale, RS 239, 2755, Novo Hamburgo,

RS CEP 93352-000, Brazil

e-mail: lucianosilva@feevale.br

123

Environ Earth Sci

DOI 10.1007/s12665-014-3055-6

degradation (Martelo and Borrero 2012). High removal

efficiencies have been obtained for biochemical oxygen

demand (BOD), nitrogen, suspended solids, and coliforms in

wastewater treatment systems with macrophytes (Ansola

et al. 2003; Nahlik and Mitsch 2006). However, the use of

constructed wetlands for wastewater treatment presents

important limitations, mainly the clogging processes that

may limit the system’s lifetime (Zhao et al. 2009; Knowles

et al. 2011) and the fact that sediment-rooted vegetation can

tolerate only relatively shallow water depths (Borne et al.

2013). A novel approach consists in using floating emergent-

macrophyte treatment wetland, in which emergent plants are

grown in a hydroponic matter on floating rafts instead of

being rooted in the sediments. This system offers the

advantage of providing a relatively passive, low-mainte-

nance, and operationally simple wastewater treatment sys-

tem (Headly and Tanner 2012). Furthermore, the digestion of

the excess sludge by plants prevents the problems faced by

the use of wastewater technologies that produce this type of

waste during treatment (Beascoechea and Gonzalez 2005).

Despite the potential advantages of floating emergent-mac-

rophyte systems for the treatment of wastewaters, there has

been little information published to date about their design,

construction, and performance (Headly and Tanner 2012).

Municipal wastewaters are complex mixtures containing

many unknown compounds (Ohe et al. 2004), some of

which are often toxic and will not undergo degradation

during treatment (Claxton et al. 1998). Physicochemical

analyses cannot provide a complete response to the

potential adverse effects of wastewater on wildlife and

humans due to the additive, synergistic, or antagonistic

interactions between the chemicals found in the wastewater

(Dizer et al. 2002; Ahmad et al. 2006; Zegura et al. 2009).

Therefore, the use of bioassays provides a direct and

appropriate measure of toxicity to complement physico-

chemical monitoring, as test organisms respond to all the

compounds in wastewater (Wang et al. 2003). Plant and

fish bioassays using different endpoints (such as cytotox-

icity and genotoxicity biomarkers) are highly suitable

methods to assess the toxicity of complex chemical mix-

tures (Udroiu 2006; Leme and Marin-Morales 2009;

Bolognesi and Hayashi 2011).

Genotoxicity biomarkers, such as the comet assay

(alkaline single-cell gel electrophoresis), the micronucleus

test, and the analysis of chromosome aberrations detect

DNA damage, the initial risk factor in the generation of

carcinogenic, developmental, and reproductive effects

(Ohe et al. 2004; Jha 2008; Rocha et al. 2009; Bolognesi

and Hayashi 2011).

The Sinos River basin, located in the eastern region of

the state of Rio Grande do Sul, includes 32 municipalities

and provides drinking water for 1.4 million inhabitants

(Comitesinos 2009). In this region, one of the main

environmental problems is the discharge of untreated

domestic sewage in tributaries and in the main river, as a

reflex of the lack of appropriate public sewage treatment

systems (Blume et al. 2010). This study combined the use

of bioassays with physicochemical analysis to determine

the effects of raw domestic sewage (RDS) and effluents

treated using activated sludge and a floating emergent-

macrophyte filter (activated process only) in the city of

Novo Hamburgo, using fish and plants as test organisms.

Materials and methods

RDS and treated effluents

The municipal wastewater treatment plant selected for this

study is located in the city of Novo Hamburgo, state of Rio

Grande do Sul, Brazil. This treatment plant serves a pop-

ulation of about 5,000 people and receives an average of

520 m3 of domestic sewage a day. In this area, there are no

factories or hospitals. Originally, the plant had two 570 m3

activated sludge treatment (AST) tanks. In February 2012,

one of these tanks received a floating emergent-macrophyte

filter (FMF system�, developed and patented by the Uni-

versity of Polytechnology in Madrid) with Typha do-

mingensis Pers., commonly known as cattail, placed in

floating rafts. However, because this technology was

applied in a wastewater treatment plant that already exis-

ted, it was not possible to implement all components of this

system, which include two other tanks for clarification and

phosphorous removal. Therefore, the FMF in the study site

is a 17 9 17 9 2.5-m tank covered with T. domingensis

specimens growing in floating rafts, which receives about

90 m3 of domestic sewage a day and has a hydraulic

retention time of 7 days.

Physicochemical analysis

In October 2012, 8 months after the FMF was imple-

mented, samples from RDS and effluents from both sys-

tems used in the treatment plant were collected for the

experiments. For each of the three samples, the following

parameters were assessed using the Standard Methods for

the Examination of Water and Wastewater (APHA 1998):

chemical oxygen demand (COD), biochemical oxygen

demand (BOD), suspended solids, ammoniacal nitrogen,

phosphorous, lead, chromium, copper, and fecal coliforms

(E. coli).

Allium cepa test

Commercial Allium cepa onion bulbs were obtained from

a local market, and the dry parts inside the root

Environ Earth Sci

123

primordia were removed before the test. Onions were

grown in tap water for 48 h and then exposed for 24 h

to the following treatments (five bulbs per treatment):

50 % RDS, 100 % FMF effluent, and 100 % AST

effluent. Previous studies revealed that treatment with

RDS in concentrations higher than 50 % inhibits onion

root growth. A negative control group was kept in tap

water for the same period. After exposure, six roots from

each bulb were removed, fixed in ethanol glacial acetic

acid solution (3:1) for 12 h and kept in 70 % ethanol

until the preparation of slides. In brief, fixed root tips

were hydrolyzed in 1 N HCl (8 min at 60 �C) and

stained with 1 % acetic orcein. Stained root tips were

squashed and analyzed under light microscopy (Nikon

Eclipse E200) for cytogenetic endpoints. The mitotic

index (MI) for each bulb was calculated as the number

of dividing cells/1,000 cells. Similarly, micronucleated

cells were scored from the same number of interphase

cells. For the analysis of chromosome aberrations

(bridges, fragments, and laggard chromosomes), at least

100 cells in anaphase or telophase from each bulb were

evaluated. All slides were coded and examined blind.

Fish bioassay

Fifty-six specimens of Astyanax jacuhiensis (Cope 1894)

(3–4 cm) were obtained from a private breeder and

transported to the laboratory in plastic bags with pres-

surized oxygen. Fish were acclimatized for 48 h and then

equally divided into six treatments: 10 % RDS, 10 and

30 % FMF effluent and 10, 30 and 75 % activated

sludge treatment (AST) effluent. These sublethal con-

centrations were based on a previous test. A negative

control group was kept in dechlorinated tap water for the

same time. After 96 h of exposure, blood samples were

obtained from the caudal vein for the micronucleus test

and the comet assay.

Micronucleus test

Peripheral blood samples were smeared on clean slides,

fixed in absolute methanol for 10 min and stained for

10 min with 5 % Giemsa. For each fish, 2,000 erythro-

cytes were examined under 1,0009 magnification in

coded slides. The criteria for identification of micronu-

clei (MN) were (Schmid 1975): (a) micronuclei should

be smaller than one-third of the main nuclei; (b) micro-

nuclei must not touch the main nuclei and (c) micronu-

clei must not be refractive and should have the same

color and intensity as the main nuclei. Other anomalies,

such as buds, invaginations, and binucleated cells were

recorded as nuclear abnormalities (NA).

Comet assay

The comet assay was performed using a slightly modified

version of the method described by Singh et al. (1988).

From each fish, 1 lL of blood was diluted in 100 lL of

bovine calf serum, and 5 lL of this mixture was added to

95 lL of 0.7 % low melting point agarose and placed on a

slide pre-coated with 1 % normal melting point agarose. A

coverslip was immediately added and the gel was allowed

to solidify in a refrigerator for 10 min. After solidification,

the coverslip was gently slid off, and the slides were placed

in cold, freshly prepared lysis solution [2.5 M NaCl,

100 mM EDTA, 10 mM Tris, pH 10.2, to which 1 %

Triton X-100 and 10 % dimethyl sulfoxide (DMSO) had

been added]. After 10–12 h, slides were placed in an

electrophoresis box filled with fresh electrophoresis buffer

(300 mM NaOH, 1 mM EDTA, pH [ 13) at 4 �C for

25 min to allow for DNA unwinding. Electrophoresis was

performed at 1 V/cm and 300 mA for 25 min. The steps

above were carried out under red light to avoid induction of

DNA damage. After electrophoresis, slides were neutral-

ized with 0.4 M Tris (pH 7.5), air dried for 24 h, fixed

(15 % trichloroacetic acid, 5 % zinc sulfate, and 5 %

glycerol) and stained with silver nitrate (Nadin et al. 2001).

To evaluate DNA damage, 100 cells per individual were

analyzed under light microscopy at 4009 magnification.

Cells were scored visually into five categories according to

the tail length: from undamaged (Type 0) to completely

damaged (Type IV). Based on the arbitrary values assigned

to the different categories (from Type 0 = 0 to Type

IV = 4), a genetic damage index (DI) was calculated as the

sum of nucleoids observed in each damage class multiplied

by the value of the respective class (Pitarque et al. 1999).

Therefore, the total score per individual ranged from 0 (all

cells undamaged) to 400 (all cells maximally damaged).

Damage frequency (DF) was calculated as the mean per-

centage of cells with medium, high and complete damage

(categories II, III and IV) (Palus et al. 1999).

Statistical analysis

ANOVA, followed by Tukey multiple comparison test

when appropriate, was used for statistical analyses. The

Statistical Package for the Social Sciences (SPSS) 15.0 for

Windows was used for analysis, and the level of signifi-

cance was set at p B 0.05.

Results and discussion

The results of the physicochemical analyses of RDS and

effluents of the AST and the FMF treatments are shown in

Table 1. BOD, COD, and suspended solids met legal

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123

criteria for both treatment systems. Ammoniacal nitrogen

and the E. coli values for the FMF treatment were above

the maximum permitted, while the AST met legal criteria

for those parameters. Phosphorous values were above the

maximum permitted for both treatments. Lead and chro-

mium were not detected in any of the samples, while

copper was only detected in the RDS sample. In general,

the activated sludge treatment presented higher removal

efficiency (RE) values than the floating emergent-macro-

phyte system. The AST was able to remove 96.4 % of

BOD, 89 % of COD, 98.5 % of ammoniacal nitrogen,

59.2 % of phosphorous, 87.5 % of suspended solids and

99.9 % of E. coli. Considering the FMF system, the

treatment removed 87 % of BOD, 57.9 % of COD, 49.3 %

of ammoniacal nitrogen, 43.7 % of phosphorous, 79.3 % of

suspended solids and 89.4 % of E. coli.

Table 2 shows the percentage of dividing cells (MI)

and the frequency of micronuclei and chromosome

aberrations in root meristem cells of A. cepa exposed to

RDS and effluents of AST and FMF treatments. Micro-

nucleus frequency was very low (0.00–0.2) in all treat-

ments. MI values ranged from 2.08 % in the 50 % RDS

treatment to 4.02 % in the negative control group, while

the frequency of chromosome aberrations varied from

0.43 % in the 100 % AST to 0.85 % in the 100 % FMF

treatment. However, no significant differences were

found between treatments or the negative control group

in any of the three endpoints under analysis (p [ 0.05).

The results of the fish erythrocyte micronucleus test and

comet assay are shown in Table 3. There were no signifi-

cant differences in the frequency of micronuclei and

nuclear abnormalities between the different treatments or

the negative control group (p [ 0.05; Fig. 1). The comet

assay showed a significant increase in both the damage

index and the damage frequency of fish exposed to 30 %

FMF treatment effluent and 10 and 75 % AST effluent, in

comparison with the negative control group. No significant

differences were found between fish exposed to 10 % RDS,

10 % FMF effluent, 30 % AST effluent and the control

group (Fig. 2).

The analysis of DNA damage is a useful tool to estimate

the genetic risk of integrated exposure to complex mixtures

of chemicals, as it provides information about the integral

effect of all direct and indirect genotoxic substances in

complex liquid samples, such as municipal and industrial

wastes, surface water, and potable water (Lah et al. 2004).

Despite the high genotoxic potential of many industrial

wastewaters, their volumetric emission rates are often low

in comparison with municipal wastewater treatment oper-

ations. The genotoxic hazard of any discharged wastewater

is determined by its loading, which is the product of

genotoxic potential and discharge rate. Therefore, even

though the genotoxic potential of municipal wastewaters is

several fold below that of many industrial wastewaters, the

genotoxic loadings of large municipal wastewater treat-

ment plants may be several orders of magnitude higher

than those obtained from industrial facilities (White and

Rasmussen 1998).

Several studies have investigated the genotoxicity of

substances known to be present in mixed municipal

wastewaters, such as sanitary wastes, pesticides for lawn

and garden care and combustion by-products that enter the

municipal system via surface runoff from roadways. The

genotoxicity detected in municipal wastewater has been

attributed to different substances, such as phenol, cyanide

and heavy metals (Lah et al. 2004), and alkylphenols (Is-

idori et al. 2007). In addition, human feces and urine have

shown mutagenicity due the presence of known mutagens,

such as polycyclic aromatic hydrocarbons (PAHs) as well

Table 1 Physicochemical and microbiological characteristics of raw

domestic sewage (RDS), floating emergent-macrophyte filter (FMF)

and activated sludge treatment (AST) effluents

RDS FMF

effluent

AST

effluent

Emission

patterna

BOD (mg/L) 500.0 65.0 18.0 \80

COD (mg/L) 542.9 228.6 59.5 \260

N–NH3 (mg/L) 82.7 41.9 1.2 \20

Phosphorous

(mg/L)

10.3 5.8 4.2 \3 or 75 %

RE

Suspended solids

(mg/L)

134.0 27.7 16.8 \80

Lead (mg/L) ND ND ND 0.2 mg/L

Chromium

(mg/L)

ND ND ND 0.5 mg/L

Copper (mg/L) 0.011 ND ND 0.5 mg/L

E. coli (mg/L) 32,000,000 3,400,000 16,000 \105 or

95 % RE

ND Not detecteda According to CONSEMA (State Environmental Council) resolution

no. 128 (Rio Grande do Sul 2006)

Table 2 Allium cepa mitotic index and frequency of micronuclei and

chromosome aberrations in bulbs exposed to 50 % raw domestic

sewage (RDS) and 100 % effluents from both treatment systems

Treatment Mitotic

index

MN/1,000

cells

Chromosome

aberrations

Control 4.02 ± 1.69 0.00 ± 0.00 0.51 ± 0.77

50 % RDS 2.08 ± 1.32 0.20 ± 0.45 0.82 ± 0.57

100 % FMF

effluent

2.50 ± 0.78 0.20 ± 0.45 0.85 ± 0.94

100 % AST

effluent

3.22 ± 1.08 0.00 ± 0.00 0.43 ± 0.25

P 0.12 0.55 0.69

Mean ± standard deviation

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123

as heterocyclic aromatic amines and nitro compounds (Ohe

et al. 2004).

In this study, we evaluated the cytotoxic and genotoxic

potential of RDS and effluents from AST and FMF of a

wastewater treatment plant located in Novo Hamburgo,

Brazil, using the A. cepa test, the micronuclei test, and the

comet assay on an indigenous fish species.

In the A. cepa test, the analysis of micronuclei and

chromosome aberrations, as well as the MI, an endpoint for

cytotoxicity, revealed that there were no significant dif-

ferences between the bulbs exposed to effluents and the

negative control group, which suggests that, under the

conditions used in this study, RDS and effluent samples are

neither genotoxic nor cytotoxic according to the A. cepa

test. These results are in agreement with findings reported

by Grisolia et al. (2005), who also did not observe signif-

icant differences between the frequency of aberrant cells in

bulbs exposed to crude sewage and effluents of a municipal

wastewater treatment plant, and by Grover and Kaur

(1999), who found no significant increase in the frequency

of micronuclei in A. cepa roots exposed to sewage extracts

when compared with negative controls. However, in a

study conducted by Nielsen and Rank (1994), wastewater

of a municipal treatment plant induced chromosome

aberrations in A. cepa cells at a significant level. Smaka-

Kincl et al. (1996), investigating the influence of the inlet

and outlet of a sewage treatment plant in A. cepa root cells,

observed a significant decrease in the MI and a significant

increase in the frequency of micronuclei and aberrant cells

in bulbs exposed to both inlet and outlet samples, which

suggests that the results in the literature are conflicting.

Table 3 Comet assay damage index (DI) and damage frequency (DF) and micronuclei (MN) and nuclear abnormalities (NA) frequency in fish

exposed to raw domestic sewage (RDS) and floating emergent-macrophyte filter (FMF) and activated sludge treatment (AST) effluents

Comet assay Micronucleus test

DI DF (%) MN/1,000 cells NA/1,000 cells

Treatment N Mean ± SD Mean ± SD Mean ± SD Mean ± SD

Control 8 126.1 ± 40.8a 34.4 ± 11.9a 0.19 ± 0.26 2.19 ± 2.74

10 % RDS 8 162.8 ± 18.5a,b 45.6 ± 6.9a,b 0.13 ± 0.23 3.81 ± 2.09

10 % FMF effluent 8 152.4 ± 23.8a,b 44.6 ± 10.3a,b 0.06 ± 0.18 1.94 ± 2.37

30 % FMF effluent 8 205.5 ± 30.6b 63.3 ± 16.3b 0.13 ± 0.23 1.19 ± 1.53

10 % AST effluent 8 220.6 ± 65.1b 64.4 ± 19.2b 0.25 ± 0.46 4.31 ± 4.77

30 % AST effluent 8 188.0 ± 61.3a,b 59.9 ± 27.9a,b 0.35 ± 0.38 2.50 ± 1.04

75 % AST effluent 8 204.4 ± 57.5b 64.8 ± 21.7b 0.06 ± 0.18 4.00 ± 3.14

P 0.0013 0.0034 0.84 0.07

a,b Values marked with different letters differ significantly

Fig. 1 Values (mean ± standard deviation) of micronuclei (MN) and

nuclear abnormalities (NA) frequency in fish exposed to raw domestic

sewage (RDS) and floating emergent-macrophyte filter (FMF) and

activated sludge treatment (AST) effluents

Fig. 2 Values (mean ± standard deviation) of comet assay damage

index and damage frequency in fish exposed to raw domestic sewage

(RDS) and floating emergent macrophyte-filter (FMF) and activated

sludge treatment (AST) effluents

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123

The micronucleus test and the comet assay are simple,

sensitive, and relatively inexpensive methods to evaluate

DNA damage in different cell types, and they play an

important role in genetic ecotoxicology (Jha 2008; Rocha

et al. 2009). While the comet assay detects DNA strand-

breaks and alkali-labile sites, DNA damage that can still be

repaired (Jha 2008), the micronucleus test detects chro-

mosome breakage or loss occurring during cell division

(Udroiu 2006).

The results of micronuclei and nuclear abnormalities

suggest that the RDS and both effluents do not induce

aneugenic or clastogenic effects in fish. Very few studies

have addressed the effect of municipal sewage on fish

DNA. Grisolia et al. (2005) evaluated the mutagenic

effect of the final effluent of a municipal wastewater

treatment plant on Tilapia rendalli and Oreochromis

niloticus and did not find any significant increase in

micronuclei frequency of the exposed fish. Grisolia and

Starling (2001) also reported no significant differences in

micronuclei frequencies in fish captured in areas under

influence of sewage treatment plant discharges and in a

reference site.

Comet assay results revealed that the damage index and

the damage frequency found in fish exposed to 30 % FMF

effluent and 10 and 75 % AST effluent were significantly

higher than those found in the negative control group.

These results suggest that, for the activated sludge effluent,

the increase in genotoxicity does not occur in a dose-

dependent manner. Given the fact that the 10 % RDS

sample did not induce significant increases in the damage

index or damage frequency of the exposed fish, both

wastewater treatment systems may actually generate the

genotoxicity of the wastewater. Thus, the present results

are in accordance with other studies, which show that waste

treatment does not always reduce the genotoxicity of an

effluent and, in some instances, may increase genotoxicity

(Claxton et al. 1998; Monarca et al. 2000; Isidori et al.

2007).

Conclusions

Under the experimental conditions of the present study,

RDS and effluent samples submitted to floating emergent

macrophyte and activated sludge treatments did not show

any cytotoxic or genotoxic effects in A. cepa root cells.

Also, mutagenic effects were not detected in erythrocytes

of Astyanax jacuhiensis according to the MN test.

However, the results of the comet assay in fish indicated

that both treatments might increase the genotoxicity of

sewage. In addition, although the floating emergent-

macrophyte system has been in place for only 8 months,

showing a mean removal efficiency lower than the

activated sludge treatment, this study revealed its

potential as an alternative sewage treatment technology.

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