Water Research 103 (2016) 245e255

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Water Research
journal homepage: www.elsevier.com/locate/watres

Review

Quantification of viable helminth eggs in samples of sewage sludge

Maria Carolina Vieira da Rocha*, Monica Eboly Bare

s, Maria Cristina Borba Braga
Parana Federal University, Department of Hydraulics and Sanitation, Water Resources and Environmental Engineering Post-Graduate Program, Curitiba,
Parana, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: For the application of sewage sludge as fertilizer, it is of fundamental importance the absence of path-
Received 23 May 2016 ogenic organisms, such as viable helminth eggs. Thus, the quantification of these organisms has to be
Received in revised form carried out by means of the application of reliable and accurate methodologies. Nevertheless, until the
16 July 2016
present date, there is no consensus with regard to the adoption of a universal methodology for the
Accepted 18 July 2016
Available online 19 July 2016
detection and quantification of viable helminth eggs. It is therefore necessary to instigate a debate on the
different protocols currently in use, as well as to assemble relevant information in order to assist in the
development of a more comprehensive and accurate method to quantify viable helminth eggs in samples
Keywords:
Domestic sludge
of sewage sludge and its derivatives.
Quantification of viable helminth eggs © 2016 Elsevier Ltd. All rights reserved.
Pathogens in sludge
Biossolids
Agricultural use

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
2. Sanitization of sewage sludge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
3. Helminth eggs in sewage sludge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246
4. Purification and quantification of viable eggs from sludge samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
4.1. Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
4.2. Egg separation and concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
4.2.1. Desorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247
4.2.2. Sedimentation/sieving . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
4.2.3. Flotation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248
4.2.4. Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
4.3. Incubation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
4.4. Quantification of viable eggs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
4.4.1. Optical microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
4.4.2. Molecular analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
5. Quality assurance and quality control (QA-QC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254

1. Introduction

The production of sewage sludge is intrinsic to the treatment of

domestic wastewaters, and its use as a fertilizer has been practiced
* Corresponding author. as an alternative to incineration and landfilling. This form of waste
E-mail address: mcarol.dhs@gmail.com (M.C.V. Rocha).

http://dx.doi.org/10.1016/j.watres.2016.07.039
0043-1354/© 2016 Elsevier Ltd. All rights reserved.
246 M.C.V. Rocha et al. / Water Research 103 (2016) 245e255
allocation presents economic and environmental advantages and it
has been adopted in several countries, for example: United States
(USEPA, 1992; Harrison et al., 1999); China (Wang, 1997); Mexico
(Jime
nez et al., 1997; 2004); Spain (Antolín et al., 2005); Brazil
(Andreoli et al., 2008; Ghiniand Bettiol and Ghini, 2011; Bittencourt
et al., 2014), and Australia (Pritchard et al., 2010). However, after the
application of these biosolids on soil, viable pathogenic organisms
that may be present in the sludge can become potentially
infectious.
The sludge from sewage treatment plants is usually stabilized by
either aerobic or anaerobic digestion processes (Hartenstein, 1981).
However, many pathogens remain viable and infectious after sta-
bilization (Reimers et al., 1981; Black et al., 1982; O'Donnell et al.,
1984; Reimers et al., 1986; We
ry et al., 2008), and their presence
in the sludge could often be undetectable. In a study carried out by
We
ry et al. (2008), who studied the inactivation of bacteria in
municipal sludge after composting, it was observed that Campylo-
bacter jejuni, an enteric pathogen present in the sludge, exhibits
higher survival rate than Escherichia coli, which is commonly used
as an indicator of sludge treatment efficiency. Furthermore, enteric
parasite eggs of the genera Ascaris, Trichuris and Toxocara, which are
also known as helminths, are highly resistant to sludge digestion
and their removal, or even inactivation, requires subsequent
disinfection steps. Nevertheless, some of these pathogens maintain
their infective potential even after severe treatment conditions
(Nelson and Darby, 2002). For instance, Maya et al. (2012), evalu-
ated the inactivation rate of eggs of Ascaris lumbricoides, Ascaris
suum, Toxocara canis and Trichuris trichiura, when submitted to
80
C and pH of 12.1, and concluded that less than 25% of these
parasites had been inactivated under these conditions.
Thus, when the aim is to apply the biosolids in soils and prevent
the spread of these pathogens in the environment, regulatory
agencies in several countries have established limits for the con-
centration of helminth eggs in sewage sludge (Swiss Government,
1992; USEPA, 1992 e USA; Journal Officiel, 1998 e France,
European Comission, 2001; NOM-004-SEMARNAT, 2002 e Mexico;
CONAMA, 2006 e Brazil; WHO, 2006). However, there is no general
agreement among researchers, environmental agencies and other
government agencies with regard to the most appropriate method
for the determination of the number of viable eggs in sludge
samples (Nelson and Darby, 2001; National Research Council, 2002;
Szabo and Vargha, 2006; Bare
s, 2010). Hence, in order to produce
more reliable results regarding the identification and quantification
of viable helminth eggs, the methodologies currently in use have to
be reassessed.
Aiming at contributing to the identification of key points that
have mislead researchers to the production of biased results, one of
the goals of this review is to present an evaluation of the meth-
odologies currently in use for the determination of viable helminth
eggs in sewage sludge. This paper also aims at providing a critical
review of different protocols currently in use. In order to fulfill
these aims it was taken into consideration each and every step
necessary for the recovery of viable helminth eggs from sewage
sludge.
2. Sanitization of sewage sludge
The stabilization of organic matter and the assessment of the
biological safety of the biosolids are required for the recycling and
use of sewage sludge in soils (USEPA, 2003; CONAMA, 2006;
Arthurson, 2008; Sidhu and Toze, 2009). Therefore, sludge stabili-
zation processes aim to accelerate the degradation of organic
matter, reduce odors and vector attraction, and ensure that, if
applied to soils, the sludge does not compete for natural resources
in the environment during the degradation of its organic
compounds (Hartenstein, 1981; Singh and Agrawal, 2008).
In order to pose no risk of biological contamination to the
environment, the sludge must be properly sanitized (Reilly, 2001;
Arthurson, 2008). Procedures for the sanitization of sewage
sludge include composting (Pourcher et al., 2005; Cofie et al., 2009),
liming (Czechowski and Marcinkowski, 2006; Tamanini et al.,
2008) and heat treatment (Piterina et al., 2010; Rubio-Loza and
Noyola, 2010). Concerning the correct treatment and final disposal
of sewage sludge, the United States Environmental Protection
Agency, in its guidance Control of Pathogens and Vector Attraction in
Sewage Sludge, defines processes to significantly reduce pathogens
(PSRPs), and processes to further reduce pathogens (PFRPs) in
samples of sewage sludge (USEPA, 2003). In the case of the appli-
cation of these processes, the reduction of pathogens in the sludge
to below detectable levels is expected (Caballero, 1984; Farrel et al.,
1996; USEPA, 2003). Composting, using either in-vessel, static
aerated pile or windrow method, and lime stabilization can be cited
as examples of PSRPs. In the case of composting, the temperature of
the process will reach 70
C and the total period of the process will
vary between 90 and 120 days (Pereira Neto, 1996). In the second
case, sufficient lime will be added to the sewage sludge to raise the
pH to 12, after a 2-h contact time. A sewage sludge treated by these
means is qualified as Class A biosolid with respect to helminth ova,
enteric viruses, and pathogenic bacteria, among other parameters.
Thus, the biosolids produced by both methods may be used in soils
with no site restrictions in the United States, because the resulting
material complies with the pollutant concentration limits estab-
lished in part 503 of the USEPA guidance (USEPA, 2003). Regarding
the PFRPs some techniques can be mentioned, such as: i) heat
drying, in which sludge is dried by direct or indirect contact with
hot gases to reduce the moisture content to 10% or less; ii) heat
treatment, in which liquid sewage sludge is heated to 180
C or
higher, for 30 min; and iii) pasteurization, in which the temperature
of the sewage sludge is maintained at 70
C, or higher, for 30 min or
longer (USEPA, 2003; Lang and Smith, 2008; Romdhana et al.,
2009).
However, to determine whether a disinfection treatment is
effective to remove pathogens, it is necessary to quantify the
number of viable organisms present in the sludge samples before
and after any sanitization treatment. The pathogens that must be
monitored in sewage sludge samples are enteric viruses, primarily
adenoviruses and enteroviruses, thermotolerant coliforms, mainly
Escherichia coli, Salmonella, and helminths in the form of viable
Ascaris eggs, which can become infective under appropriate me-
dium conditions (USEPA, 1992; Straub et al., 1993; Sidhu and Toze,
2009).
Although many pathogens can be found in sludge samples, their
presence does not necessarily indicate that there is a risk of
contamination (Lewis and Gattie, 2002). The risk of contamination
depends on the infectious dose that is required for that organism to
become pathogenic in an individual (Gerba and Smith, 2005). For
instance, the minimum infective dose for helminths is very low and
the contact with or the ingestion of a single viable egg can lead to
the development of a parasite-associated disease (Navarro et al.,
2009). Therefore, the low infectious dose associated with the
resistant structure of helminth eggs makes helminths the primary
target of sewage sludge cleaning techniques. Thus, it is essential to
accurately and rapidly quantify the number of viable helminth eggs
in sludge samples intended for agricultural use.
3. Helminth eggs in sewage sludge
In the field of Sanitary Engineering, helminths are related to the
group of intestinal parasites in which man is the definitive host. The
diseases caused by these organisms are helminthiases and,
 M.C.V. Rocha et al. / Water Research 103 (2016) 245e255
according to World Health Assembly, the parasites are more prev-
alent in less developed countries (WHA, 2001; Jimenez-Cisneros
and Maya-Rendon, 2007).
The development of helminths follows three main stages, that is,
the egg, the larval or juvenile stage and the adult, with the number
of larval stages varying for each species (Parker et al., 2003). After
fertilization, the eggs are released with the feces of the host. If the
eggs find suitable conditions, either by ingestion or contact with an
intermediate host or even find the proper resources to undergo
embryogenesis in the soil, in the case of geohelminths, the cycle
recommences (Pearson, 2002). However, the conditions are hardly
ever ideal and viable eggs remain in the dormant or latent phase, in
which structural resistance is essential to the survival of the or-
ganism (Perry, 1989; Pearson, 2002).
The high resistance of helminth eggs to adverse external con-
ditions can be attributed to their thick shell or wrap, which is
formed by: i) an outer coating. An irregular layer which consists of
lipoproteins covered with mucopolysaccharides; ii) an intermedi-
ate chitinous membrane with variable thickness; and iii) an inner
membrane which consists of lipids (Wharton, 1983). The overlap of
these layers forms a compact structure that is capable of with-
standing stressful situations caused by both physical and chemical
conditions. The first, including heat, and the latter, including oxi-
dants and detergents. These layers can also resist to biological
stresses, such as the action of proteases during the process of sludge
disinfection (Guzm
an et al., 2007).
Among the helminths that are present in sewage sludge, the
nematode Ascaris is the most refractory to treatment processes, and
its inactivation or permanence in treated material can serve as an
indicator of the efficiency of the disinfection process (Maya et al.,
2010; Maya et al., 2012). In fact, Maya et al. (2012) observed that
the species Ascaris lumbricoides and Ascaris suum presented higher
resistance to adverse conditions, such as high temperatures and
alkaline pH, when compared to other helminth eggs of medical
importance, the Toxocara canis and Trichuris trichiura. Thus, nem-
atodes of the Ascaris genus are parasites of great significance for the
control and evaluation of the efficiency of the methods used for the
sanitization of sewage sludge.
4. Purification and quantification of viable eggs from sludge
samples
The first protocols for quantifying viable helminth eggs in
sewage sludge were based on methods of egg isolation from fecal
samples (Faust et al., 1939; Allen and Ridley, 1970). However, the
high dilution of sludge required the adaptation of these methods to
samples containing low concentrations of parasites (Steer et al.,
1974). Therefore, it was necessary the development of new
methods to process large volumes of material and isolate the eggs
in the samples, thus reducing the undesirable components, humic
acids and phenolic compounds, to a minimum (Satchwell, 1986).
Since the early attempts to purify parasite eggs from sludge
samples (Steer et al., 1974; Meyer et al., 1978; Reimers et al., 1981),

s et al., 2010; Rocha, 2015),
until recently (Pecson et al., 2006; Bare
various methods for viable eggs purification and quantification
have been developed or optimized. Despite using different mate-
rials and reagents, these methods basically consist of the following
four steps: (a) sampling of sludge; (b) separation of eggs from the
unwanted material present in the sludge, and maximization of egg
concentration in the sample; (c) incubation, under appropriate
conditions of temperature and oxygen concentration, to induce the
development of larvae; and (d) the analysis and quantification of
viable eggs in the sample, usually by microscopic analysis (Nelson
and Darby, 2001).
Reagents and materials used in each of the quantitative methods
247
vary for each of these steps. These variations are responsible for the
accuracy and precision of the methods, and each method should be
assessed considering the possibility of a higher rate of viable eggs
recovery from the samples.
4.1. Sampling
Sampling procedures should be viewed as an important step
when the development of a protocol is to be taken into consider-
ation. Sludge samples that do not represent the actual diversity of
the environment entail in the disqualification of any further result
obtained by the application of a certain method (USEPA, 2003).
Furthermore, the amount of sludge that is used in the quantifica-
tion assays relates directly to the level of detection achieved
(Bowman et al., 2003).
The USEPA method (2003) that specifies the limit of helminth
eggs in the sludge to be applied in soils or pasture (Class A bio-
solids) is 1 egg/4 g of total solids (TS). Therefore, when an appro-
priate monitoring program is taken into consideration, the size of
the sample should be sufficient to reach the limit of detection of the
method. However, large samples may result in lower rates of egg
recovery. This can be attributed to the interference of the quantity
of the solids with the efficiency of various steps of egg purification.
For instance, Bean and Brabants (2001) applied the USEPA protocol
to inoculated samples of limed and composted biosolids and
compared the recovery of Ascaris lumbricoides. These authors used
two sets of samples, one containing 300 g of TS and another con-
taining 50 g of TS. As a result, it was observed that the recovery of
Ascaris was nearly twice as high when the samples of biosolids
were smaller. The recovery for limed biosolids was 8,0% for the
smaller amount of TS, and 4,4% for the higher content of TS,
whereas for the composted biosolids it was 9,6% in opposition to
4,5%.
Usually, the amount of the test sample should contain an
equivalent mass between 5 g (Reimers et al., 1989) and 50 g (USEPA,
2003) of total solids. However, different concentrations of solids
can be set by the researcher during the development of a new
sampling protocol, and this will be based mainly on the accuracy
and precision of the results to be produced by the assays.
4.2. Egg separation and concentration
In general, methods for the determination of viable eggs utilize
several operations in sequence, such as washing or desorption of
the eggs from the sludge matrix, sedimentation, filtration, flotation
and extraction, among others. These steps are used to guarantee
proper separation of the eggs from other contaminants and to
facilitate the identification and quantification of eggs at a later
stage. Fig. 1 presents a schematic representation of the sequence of
operations required for the purification and quantification of hel-
minth eggs from sludge samples.
4.2.1. Desorption
This is the first step for purification and concentration of hel-
minth eggs from a matrix of compounds in the sludge. The coating
membranes of the helminth eggs are readily adsorbed onto organic
compounds, such as humic and fulvic acids, and polysaccharides
present in the sludge (Gaspard et al., 1994). Trapped within this
matrix, the eggs become refractory to either differential sedimen-
tation or flotation, which are the subsequent steps of purification
and concentration (Meyer et al., 1978; Gaspard et al., 1994).
Moreover, during the process of sludge dewatering, some chemical
coagulants, such as polyacrylamide, may be used, aiming at pro-
ducing more cohesive granules and enhancing the concentration of
total solids in the sludge. Nevertheless, due to its high capacity to
248 M.C.V. Rocha et al. / Water Research 103 (2016) 245e255
Fig. 1. Schematic representation of the main steps for the determination of viable
helminth eggs in samples of sewage sludge.
adsorbing those eggs, the presence of polyacrylamide, or other
coagulant, could interfere with the recovery of helminth eggs from
the matrix of sludge (Zamudio-Pe
rez et al., 2013). Therefore, to
ensure that the eggs are properly isolated, it is necessary to remove
these contaminants.
As recommended by O'Donnell et al. (1984), proteins, such as
lactalbumin, can be effectively used during the desorption process,
and according to Yanko (1987) warm tap water can be used to wash
the samples.
However, as a primary strategy to remove the eggs from the
sludge matrix washing of the samples with solutions of surfactants
is recommended. According to Bowman et al. (2003), non-ionic
detergents, such as Triton®, Tween® 80 and 7X® are commonly
used as surfactants. On one hand, low concentrations of these
substances may effectively desorb the eggs from the sludge
(Reimers et al., 1989; Gaspard et al., 1996; Bowman et al., 2003). On
the other hand, due to probable damage to the integrity of the
membrane of the egg, rinsing the sludge with high concentrations
of a detergent may cause a toxic effect on helminth eggs. As
observed by Jaskoski (1954), the immersion of ascarid eggs in
detergent solutions, such as sodium lauryl sulphate and sodium
dodecylbenzene sulfonate (Nacconol®), at 38
C for 18e36 h, was
destructive for the parasite, since it damaged the integrity of the
eggs. Another example is that presented by Satchwell (1986). This
author evaluated a modified method, that had been originally
presented elsewhere (MAFF, 1977). The assay was carried out using
three detergents; cetylpyridinium chloride, SDS and Nonidet® P40;
at a concentration of 0.8% (w/v) in a washing step prior to filtration.
This was aimed at the recovery of eggs of Taenia saginata which
were inoculated in samples from sewage sludge storage lagoons.
According to the results of the study, the recovery of the eggs from
the samples previously treated with any of those detergents was
lower than those obtained for samples that had not received the
addition of any detergent. The results obtained were 1.7%, 6.4% and
4.4% for the samples treated with cetylpyridinium chloride, SDS
and Nonidet® P40, respectively, versus 10.8% for samples which had
not received detergent during the procedures.
In addition to the concentration, a proper choice of the deter-
gent is a determining factor for the recovery of the parasite eggs.
Bowman et al. (2003) compared the detergents Triton®, Tween® 80
and Linbro 7X® to evaluate their effect on the recovery of Ascaris
eggs. These authors observed a higher rate of parasite eggs recovery
when the 7X® was used in comparison with results produced by
tests carried out with other detergents. It is worth mentioning that
the detergent 7X® does not form a precipitate when in contact with
salt solutions used in the flotation stage. Nevertheless, the authors
did not present comparative values regarded the recovery of the
Ascaris eggs obtained by the use of the detergents previously cited.
4.2.2. Sedimentation/sieving
After desorption, it is important to recover the eggs that are
present in solution and remove coarse residues from the sludge.
This is because these particles affect the subsequent steps of con-
centration and purification of the eggs of the parasites. According to
Meyer et al. (1978), in general, sedimentation followed by sieving is
effective for the removal of coarse residues.
Depending on the characteristics of the material and in order to
allow the solids to sediment after rinsing the samples, the ho-
mogenized solution is put to rest for a period of time ranging from 4
to 12 h or more (Yanko, 1987; Reimers et al., 1989; USEPA, 2003).
However, to increase the efficiency of the desorption, additional
washing steps followed by sedimentation can be carried out after
the removal of the supernatant (USEPA, 2003).
At the end of the sedimentation steps, a new volume of washing
solution is added to the sediment. This sediment contains the eggs
and a large quantity of solid. At this stage, the use of larger sieve
openings, 20e50 mesh, ensures the removal of the coarse fraction
of solids and reduces the interference of other materials in the
flotation step (Black et al., 1982).
It is worth mentioning that, in order to increase the effective-
ness of sedimentation a centrifugation step can be added. However,
taking into consideration the difficulties inherent to the centrifu-
gation of large volumes of samples in a laboratory, this specific step
should only be carried out to process small amounts of samples. In
order to allow the formation of a packed sediment in the bottom of
the centrifuge tubes, after reducing the amount of sludge, a relative
centrifugal force (RCF, or g force) up to 1000g can be applied
(Bowman et al., 2003; USEPA, 2003). However, it is important to
mention that Zdybel et al. (2015), carried out assays to evaluate the
presence and viability of Trichuris, Ascaris and Toxocara eggs in
samples of sludge from different units of wastewater treatment
plants and included a centrifugation step using an RCF of 2500g
after sedimentation and sieving (opening of 200 mesh).
4.2.3. Flotation
After the sieving and sedimentation steps, the eggs are usually
recovered by flotation. This operation is based on the difference of
 M.C.V. Rocha et al. / Water Research 103 (2016) 245e255
the specific gravity (SG) between the eggs and a high density so-
lution (Dryden et al., 2005). The solutions commonly used include
sucrose (Fitzgerald and Fox, 1978), zinc sulphate (Yanko, 1987),
magnesium sulphate (Reimers et al., 1989; Bowman et al., 2003)
and sodium chloride (Gaspard et al., 1996).
To ensure the flotation of the eggs, the density of the flotation
solution must be higher than the density of the eggs. The correct
adjustment of the density of the flotation solutions is a major dif-
ficulty to be surpassed during the development of a method for the
recovery of helminth eggs. For the majority of the parasite eggs, the
specific gravity is between 1.05 and 1.23 (David and Lindquist,
1982; Dryden et al., 2005), and commonly the solutions used pre-
sent specific gravity values of 1.18 for NaCl, 1.20 for MgSO4 and
ZnSO4, and 1.30 for sucrose. Combinations of salts produce solu-
tions of different densities, for instance sodium chloride and zinc
chloride (SG 1.35), and sodium nitrate and sodium thiosulfate (SG
1.45) (Cringoli et al., 2004), which could be useful for the recovery
of heavier eggs, as Trichuris vulpis (1.1453) and Taenia sp. (1.2251).
The selection of the most appropriate flotation solution depends
on both the previous steps of processing the sample and the
parasite species to be recovered. As an example, some saline so-
lutions, such as NaCl, precipitate in the presence of detergents
(Bowman et al., 2003) whereas other solutions, such as sucrose, as a
consequence of its viscosity related to the specific gravity of the
solution (1.30), requires longer periods of centrifugation. However,
it is worth mentioning that sucrose solutions can be used to recover
eggs of higher specific gravity, such as Taenia spp (David and
Lindquist, 1982). Moreover, due to inhibitory characteristics of
some of these salts to the embryonic development of the parasite,
longer periods of contact time between the flotation solution and
the eggs should be avoided (Smith, 1991; Gaspard et al., 1996).
It is importance to mention that the choice of an adequate
flotation solution is central for an effective recovery of helminth
eggs from sludge samples. Bare
s et al. (2010), studied the
improvement of a method used for the quantification of viable
helminths eggs, and evaluated the use of the solution of zinc sulfide
(d ¼ 1.20) for the recovery of viable eggs of Taenia sp. According to
these authors, the methodologies that utilize flotation solutions
with specific gravity of 1.20 are not effective to recover eggs of some
species of helminths, and hence could underestimate the presence
of the parasites in the samples. In this case, and in order to ensure
the recovery of heavier eggs, the authors recommended the use of
flotation solutions with specific gravity of 1.30.
Following the addition of the flotation solution to the extract
containing the eggs, a centrifugation step settles the remaining
solids and allows the eggs to float more rapidly to the surface of the
solution (Dryden et al., 2005). The time for the centrifugation step
is based on the composition of the sludge and influences the
flotation efficiency. Therefore, the approach to determine the
operating conditions of a protocol should consider the optimization
of the recovery of eggs from each sample (Nunes et al., 1994).
4.2.4. Extraction
The goal of this step, besides the quantification of helminth eggs,
is to remove any protein and lipid contaminants from the suspen-
sion of helminth eggs that were obtained after flotation (EPA,
2003). Aiming at the purification of the eggs, lipophilic and hy-
drophilic reagents can be used. The chemical reagents will partition
the sample in two phases, light and heavy, and the parasite eggs
will be concentrated in the bottom of the tubes (Nelson and Darby,
2001). For instance, diethyl ether and the ethyl acetate can be cited
among the lipophilic solutions most widely used (Rude et al., 1987),
whereas the hydrophilic phase commonly consists of acidified
ethanol (EPA, 1992). Although effective in the removal of the con-
taminants present in the suspension of the eggs recovered from the
249
sludge, some reagents can present a detrimental effect on the
viability of the eggs. Originally presented by Allen and Ridley (1970)
as a technique for the purification of parasite eggs from stool
samples, the extraction with formaldehyde-ethanol was used by
Satchwell (1986) during the concentration step of samples of
sewage sludge. This author, used a saline solution of formaldehyde
followed by a solution of diethyl ether, and obtained 40% of removal
of contaminants present in a suspension that contained eggs of
Taenia spp., Ascaris spp. and Trichuris spp. However, in spite of the
reduction of impurities, the authors observed a loss of 95% of the
eggs during the extraction step. This result was mainly related to
the toxic effects of the reagents used.
Thus, aiming at solving the problems caused by chemicals in the
extraction step, an alternative is the substitution of this step by
sieving. This step is recommend by the USEPA (2003) and specifies
the use of a 400 mesh sieve that retains the eggs and, hence, allows
the removal of proteins, lipids and other contaminant molecules.
4.3. Incubation
This step is carried out after the concentration of the helminth
eggs, and allows the development of infective larvae within the
viable eggs (Boisvenue, 1990). The incubation is a critical step
because the larvae can only develop under proper conditions of
temperature and oxygen concentration (Pearson, 2002). Further-
more, the composition of the medium in which the eggs are incu-
bated also influences the larval development of the parasite (Cruz
et al., 2012). Usually, a period of 21e28 days is required for the
full embryonation of the eggs. However, control samples can be
considered and eggs of Ascaris suum are useful in the determination
of the readiness of the samples for quantification, that is, it will be
shown when the majority of the control eggs become embryonated
(USEPA, 2003).
The temperature of incubation influences the time for the
development of the embryo inside the egg (Arene, 1986). In general,
higher temperatures accelerate the development of the eggs,
whereas the incubation time required for larval development is
longer at lower temperatures (Arene, 1986; Cruz et al., 2012). For
instance, the time required to incubate viable eggs at 28
C is, on
average, 12 days to reach the first larval phase (L1) (Cruz et al.,
2012), whereas this time is increased to 17 days when the incu-
bation is carried out at 20
C (Geenen et al., 1999).
For the incubation of helminth eggs, the medium containing
solutions of 0.1 N H2SO4 (Yanko, 1987) and 0.5% or 1% formalin
(Reimers et al., 1989; Oksanen et al., 1990) are widely used. A typical
characteristic of these solutions is the antimicrobial activity, that is,
the property of preventing the growth of fungi and other organisms
that can interfere with the embryonic development of the parasite
(Cruz et al., 2012). Other incubation solutions include distilled
water (Oksanen et al., 1990) and synthetic cell culture media
(Gaspard et al., 1996). However, it was observed that the maturation
of the eggs was delayed when the latter was used. The authors
(Gaspard et al., 1996) concluded that it was due to an inhibitory
effect caused by the organic compounds present in the synthetic
media.
Recently, a novel procedure of incubation of helminth eggs was
presented by Zdybel et al. (2015). This method consists of adding a
step of vacuum filtration using polycarbonate membranes with
4 cm in diameter, and pore size of 12 mm, after those of sedimen-
tation, sieving and flotation. After filtration, the membrane is
covered with glycerol, placed on a glass slide, and the set is incu-
bated at 27
C for 24 h. After this period the eggs identified under
optical microscopy are highlighted and returned to incubation,
which allows the eggs to continue their development. According to
these authors, this procedure has an advantage over the others
250 M.C.V. Rocha et al. / Water Research 103 (2016) 245e255
because the use of this technique provides the means of monitoring
the same eggs over the whole period of development. Nevertheless,
care must be taken regarding the use of glycerol as an incubation
medium, since it can cause partial cut off of the oxygen demanded
for the development of the parasites (Dabrowska et al., 2014).
4.4. Quantification of viable eggs
The quantification of viable helminth eggs is commonly carried
out by optical microscopy (Reimers et al., 1981, 1989; Yanko, 1987;
Gaspard et al., 1996; Bowman et al., 2003); however, attempts to
determine the number of viable eggs using molecular techniques
have also been described (Pecson et al., 2006). Regardless the
method chosen for quantification, this is a critical step because the
number of viable eggs is directly related to the efficiency of the
process of sludge disinfection. Moreover, the number of eggs
recovered is used as a parameter to determine the suitability of the
application of biossolids from sewage sludge in soils (USEPA, 2003).
4.4.1. Optical microscopy
The quantification of the eggs is carried out at magnifications of
40 and 100, and in general, a Sedgwick-Rafter cell, with di-
mensions of 50  20  1 mm and volumetric capacity of 1 mL, is
used for the reading of the samples under optical microscopy. This
cell is easily manipulated and provides reasonable reproducibility
of the tests (Woelkerling et al., 1976).
The quantification of viable eggs using an optical microscope is a
procedure that requires visual acuity due to the presence of inter-
ferents, such as cellulose, yeast, grains of pollen and dirt that can
generate false positive results (Moodley et al., 2008). Thus, a proper
removal of solids and debris from the samples of the sewage sludge,
mainly during the steps of flotation and extraction, is decisive for a
correct visualization and quantification of viable helminth eggs
under optical microscopy. For instance, solid particles, carried with
the supernatant after the flotation step, can be retained during
sieving, and will remain in the suspension of eggs to be incubated.
Therefore, the visualization and quantification of viable eggs may
be hampered as a consequence of the presence of contaminants,
which interferes with the microscopic field under scrutiny (USEPA,
2003). In fact, Bare
s (2010), in a study carried out to evaluate the
inactivation of helminth eggs in sludge samples after a thermal
treatment, described the risk of false positives due to the presence
of contaminants and other microorganisms in sludge samples. The
author highlighted the presence of tardigrades, commonly known
as water bear, that could have been falsely identified as eggs of
hookworms, due to their morphological similarity.
The problems related to the visualization of eggs of Ascaris suum
by optical microscopy was also experienced by Rocha et al. (2014).
These authors studied the accuracy of the USEPA method (2003) for
the quantification of viable eggs in anaerobic sludge, and observed
a low recovery of the eggs of A. suum that were spiked in sludge
samples, i. e., 59,8%. This value is below that of the USEPA protocol
for anaerobically digested sludge, i.e., 75.5% which is regarded to
the quality analysis/control (Bowman et al., 2003). Thus, according
to Rocha et al. (2014), the recovery below 75% could have been
associated with an inadequate removal of solids and debris from
the sludge samples during the steps of desorption, flotation and
sieving, which could have interfered with the visualization of the
eggs of A. suum by optical microscopy. During the quantification,
the analyst must determine both the total number of helminth eggs
of interest and how many of these eggs harbour a larval stage. In the
case of Ascaris, commonly used as an indicator of the presence of
helminth eggs, the quantities are to be reported as eggs that are not
embryonated and eggs that are in the larval stages (USEPA, 2003).
Therefore, the number of viable eggs is presented as the number of
embryonated eggs per gram of total solids.
In the literature six stages of parasite development for Ascaris
are reported: the egg (one cell), four larval stages and the adult. In
order to be considered viable, the egg observed by optical micro-
scopy should house a formed larva (Bowman et al., 2003; USEPA,
2003). Although not being extensively studied, information on
the embryonic stages of Ascaris could be useful for the development
or optimization of a quantitative method. For instance, Cruz et al.
(2012) studied the intermediate stages of development of Ascaris
outside the host, named: one, two, three and four cells, morula, late
morula, blastula, gastrula, pre-larva 1, pre-larva 2, larva 1 and larva
2 stages. According to the results presented by these authors, the
additional stages are potentially indicative of the viability of the egg
and cannot be disregarded during quantification due to the risk of
underestimating the actual number of viable organisms in the
sample. It is worth mentioning that Rocha (2015) observed that the
number of Ascaris eggs incubated is related to the time needed for
the development of these eggs. According to this author, the higher
the quantity of the eggs of the parasite, the larger will be the time
needed for the viable eggs to become a larvae. Hence, depending on
the samples, viable eggs in different stages of development could
be found even after 21 days of incubation. This variation in the time
of embryogenesis could hinder the assessment of the viability of
the parasite. The main stages of the embryogenesis of Ascaris suum,
as observed by Rocha (2015), is presented in Fig. 2.
Nevertheless, it has also to be considered that treatments aim-
ing at the disinfection of sludge can inhibit the development of
helminth eggs. Thus, the observation of distinct stages of devel-
opment of Ascaris eggs, such as morula and blastula, mainly after
the disinfection of sludge samples, do not guarantee the viability of
these eggs. Hence, when optical microscopy is the method of
analysis, for the viability to be accounted for, the infective larvae
must present motility under light stimulus.
There are other methods to quantify and determine the viability
of helminth eggs based on the difference in the capacity of the cells,
alive and dead, of some organisms to adsorb dyes, also known as
vital stain procedures (Victorica and Galva
n, 2003; Dabrowska
et al., 2014; Karkashan et al., 2015). These methods combine
several advantages, such as rapidity and low cost; however, some
difficulties are also presented. For instance, some vital dyes may
lead to the disruption of viable eggs (Karkashan et al., 2015). On the
other hand, the utilization of vital stain procedures has produced
interesting results. As an example, Dabrowska et al. (2014) evalu-
ated a bacterial viability kit, named LIVE/DEAD BacLight Bacterial
Viability Kit (Invitrogen, USA) for the determination of viability of
Ascaris suum, Toxocara canis and Trichuris ova. Considering the
response expected for bacteria, live cells should stain fluorescent
green, whereas dead cells should stain fluorescent red. These au-
thors obtained 93%, 84% and 85% of green-colored eggs of Ascaris
spp., Toxocara spp. and Trichuris, respectively, when the LIVE/DEAD
kit was applied to viable eggs placed in distilled water. They also
obtained results of 95%, 95% and 100% of red-colored eggs of Ascaris
spp., Toxocara spp and Trichuris, considering the assays realized
with non-viable eggs of those parasites. These results indicate good
sensitivity and specificity of response to this solution, however,
further studies are still in need, mainly due to the difficulties to
identify non-viable eggs that do not present any structural damage.
4.4.2. Molecular analysis
The objective of molecular analysis is to determine the presence
of a given organism in a sample by detecting the genetic material of
that organism (Sidhu and Toze, 2009; Girones et al., 2010). Several
molecular markers can be used, however, the most common are
based on ribosomal DNA sequences, which are useful for the
phylogenetic analysis of populations (Olsen et al., 1986; Marande
 M.C.V. Rocha et al. / Water Research 103 (2016) 245e255
Fig. 2. Stages of the embryogenesis of Ascaris suum:a), b) and c) early stages of cell division; d) early morula; e) late morula; f) blastula; g) gastrula; h) larva 1 (L1); i) larva 2 (L2).
Note: eggs without external mammillated protein coat (decorticated).
et al., 2009). To detect the DNA sequence of the organism of in-
terest, the polymerase chain reaction technique (PCR) is generally
used to amplify the target genetic material in a reaction that in-
volves specific primers and a thermostable DNA polymerase (Saiki
et al., 1985).
The use of conventional or endpoint PCR has currently been
used in various laboratories, and sanitary engineering is no
exception. However, for the diagnosis of parasites, this technique
has a major limitation, that is, it can indicate the presence or
absence of a particular species but cannot easily quantify the spe-
cies. However, it is worth mentioning that the contribution of PCR
has been remarkable for the characterisation of different microbial
populations in sludge samples (Choi et al., 2007; Wang and Zhao,
2011), the detection of enterovirus genetic material (Straub et al.,
1995; Shieh et al., 1997) and the diagnosis of Ascaris sp. in host
tissue (Ishiwata et al., 2004).
Conventional PCR, which was used in diagnostic assays, was
enhanced by the advent of quantitative PCR (qPCR). This technique
determines the presence or absence of pathogens and it also
quantifies the organisms in the samples, including sewage sludge
and its derivatives or biossolids. For instance, Pecson et al. (2006)
developed a technique for the quantification of Ascaris eggs using
qPCR amplification of a region of the ribosomal DNA (ITS-1) of the
parasite. According to the authors, only 25% of the samples tested
by qPCR present significant difference in the quantification of eggs
when compared with those obtained by microscopic counting.
In spite of being a very sensitive and efficient technique used for
the quantification of genetic material, there are few studies that
associate quantitative PCR with the detection of viable helminths in
sewage sludge (Pecson et al., 2006; Raynal et al., 2012; Rocha,
2015). The reason regards to the fact that helminth eggs can be
251
quantified by qPCR, however, the use of this technique does not
allow to distinguish between viable and non-viable organisms
present in the samples, as the DNA can remain viable in samples
even after the inactivation of the parasites.
An alternative to provide an effective tool to quantify viable
helminth eggs could be the Reverse Transcriptase-PCR, or RT-PCR,
which could eliminate false-positive results. This is because this
technique uses the RNA, and not the DNA to assess the viability of
one organism.
Nevertheless, until the present date, there is no technique to
successfully relate the quantity of RNA in a sample with the number
of viable eggs of a parasite. Rocha (2015) investigated the use of RT-
qPCR as a tool to determine the presence of viable eggs of Ascaris
suum in samples of domestic sludge. It was observed signal of
amplification in all sludge samples that were evaluated using the
target gene alep1, which is present only in the early stages of the
development of the eggs. These results confirm the RT-qPCR as a
tool for the assessment of the viability of eggs of Ascaris in sewage
sludge. Nevertheless, in spite of the potential for the application of
the method to determine viable eggs of Ascaris, the author was not
able to quantify these eggs using the RT-qPCR technique. This was
mainly due the difficulty related to the construction of a calibration
curve for the method.
Achieving a representative detection limit is also a major diffi-
culty when the qPCR technique is applied to the molecular iden-
tification of helminth eggs. As an example, Pecson et al. (2006)
determined the minimum detection limit (MDL) of qPCR of the
ITS-1 (first internally transcribed spacer) region of ribosomal DNA
of Ascaris, and found out that the value was 3750 ITS-1 rDNA copies.
Considering that the ITS-1 region is present in 2.5  104 copies in a
larvated egg, approximately, the method should be able to
252 M.C.V. Rocha et al. / Water Research 103 (2016) 245e255
detecting and quantifying a single larvated egg in a sample. How-
ever, if other molecular targets are considered, the achievement of
this level of detection is a major challenge. Moreover, other factors
also interfere with the detection limit of the qPCR technique. These
interferences are the quality of the samples (purity, quantity and
integrity of DNA or RNA); the inadequate homogenization of re-
agents; the inaccurate setting of the baseline and threshold of the
calibration curves; and pipetting and in dilution of standards. Thus,
further efforts should be directed to achieving this goal, that is, the
development of a method to quantify helminth eggs that do not
depend on the subjectivity of the microscopic technique.
Methods for the quantification of viable helminth eggs: the
search for a standard method.
Since it was unknown that the chemical compounds used for the
recovery and purification of the parasite eggs in sludge could
interfere with their integrity and viability, the first protocols tested
presented low accuracy (Satchwell, 1986). However, over the years,
to ensure the viability of the eggs that were recovered, several
studies were carried out to determine the optimal processing and
incubating conditions of the samples (Gaspard et al., 1996; Johnson
et al., 1998; Nelson and Darby, 2001; Bare
s et al., 2010; Cruz et al.,
2012). Thus, after the publication of the guidance EPA/625/R-92/
013 (USEPA, 1992), several attempts to develop and standardize a
method for the determination of viable eggs have been carried out
(Gaspard et al., 1996; Huyard et al., 2000; Bean and Brabants, 2001;
Gaspard and Schwartzbrod, 2003; Pecson et al., 2006).
As a consequence of the observations, it has been argued that
there is a need for the standardization of a highly accurate method
to be used for the quantification of helminth eggs in sewage sludge
samples. This method would also have to ensure the reproducibility
of the tests (Bowman et al., 2003) and would have to guarantee the
recovery of the greatest amount of inoculated eggs in a sample.
Moreover, it is essential the detection and quantification of path-
ogens in the biosolids with a known degree of confidence (Sidhu
and Toze, 2009). Additional features include high sensitivity,
rapidity and easy implementation.
In the literature, the methods reported for the determination of
viable helminth eggs can be assessed based on two approaches,
namely: i) split/spike methodologies, in which known concentra-
tions of a given species are added to the sludge samples. The
resulting recovery of viable eggs provides the accuracy of the
method under evaluation; and ii) methodologies that aim to
recover the helminth eggs naturally found in field samples, with no
addition of eggs of any particular species.
Regarding the split/spike methodologies, one of the first
methods to achieve adequate levels of accuracy and precision was
the Tulane method, which was developed by Reimers et al. (1981).
The aim of this study was to assess the presence and density of
resistant forms of parasites in sewage sludge samples from
different treatment plants in the southern states of the USA. The
method developed for the recovery of helminth eggs consisted of a
washing step with the detergent Linbro 7X®, flotation with a so-
lution of MgSO4 (SG 1.2) and incubation in 0.5% formalin solution
(Bowman et al., 2003).
In 1987, Yanko developed a new method for the determination
of viable helminth eggs based on the Tulane method. This new
approach was conceived to use warm tap water for the desorption
of Ascaris eggs from sewage sludge samples. A ZnSO4 solution (SG
1.2) was used for flotation, and after the concentration step, the
eggs were incubated in 0.1 N H2SO4 solution. The limit of detection
of this method was 0.2 Ascaris eggs per gram of solid (Yanko, 1987).
The accuracy of this method was also satisfactory and an average of
88% recovery of eggs from the sludge samples was achieved. In
1992, the USEPA defined the method of Yanko as the standard
method for the quantification of viable eggs in samples of sludge
and wastewater (USEPA, 1992).
In 2003, Bowman and colleagues presented the validation of the
accuracy and precision of the Tulane method. In their tests, the
method presented an egg recovery of 75.5% for anaerobically
digested sludge, 80% for acid-treated sludge and 58% for alkaline-
treated biosolids (Bowman et al., 2003). In the same year, the
USEPA established the new standard method for the quantification
of viable Ascaris eggs in sewage sludge samples based on the Tulane
method. With respect to the original Tulane method, the main
changes included the removal of the extraction step with 10%
household bleach after the incubation period, and incubation of the
recovered eggs in a solution of 0.1 N H2SO4 (USEPA, 2003).
In 2003, Victoria and Galva
n developed a method that does not
require incubation of the recovered eggs. In the approach for the
development of the method, the extraction step was replaced by
filtration through membranes whose pores were 0.8 mm in
diameter. After filtration, the eggs are removed from the mem-
brane and stained with solutions that are indicative of viability,
such as methylene blue and safranin. This method is based on the
observation that non-viable eggs exhibit alterations in cellular
membrane structure that make them permeable to some types of
dyes. The primary advantage of the Victorica and Galv
an method
is the speed with which tests can be performed. The accuracy
achieved by this method was about 77%, which, according to the
authors, was a reasonable result, because it was not necessary to
wait for the development of the parasite larvae and, especially
because a time of only 6 h was necessary to carry out the test.
Nevertheless, it is worth mentioning that when non-invasive
techniques for sludge sanitization are used, such as UV treat-
ment, the eggshell may not be immediately permeable to vital
stained, and care must be taken with regard to the application of
these methods for the determination of the viability of helminth
eggs (Gyawali et al., 2016).
The USEPA protocol, along with the Tulane method, is an
attempt to standardize a procedure for the detection of viable
helminth eggs in samples of sewage sludge. However, when the
accuracy of the method, modified and adopted by the USEPA, was
presented by Bowman et al. (2003), only four matrices of biosolids
were evaluated, namely: i) synox-treated biosolids (acid treatment
process); ii) anaerobically treated biosolids stored in lagoons; iii)
soil-biosolid blends; and iv) biosolids from Chemfix process
(alkaline treatment). Nevertheless, the USEPA method (2003)
generalizes the application of this method for samples of sewage
sludge and compounds (Appendix IEPA/625/R-92/013), with no
specification of the type of sludge that can reach the accuracy
established by the method. The risk of have not defining the types
of sludge for which the method has been validated can result in its
inappropriate application.
In Brazil, for instance, the CONAMA Act n. 375/2006 establishes
that the method specified by USEPA (2003) is the standard for the
quantification of viable Ascaris eggs in sewage sludge samples and
its derivatives. However, there is no mention of whether the
method is valid for anaerobic and/or aerobic sludge neither in the
CONAMA Act nor in the USEPA protocol. Rocha (2015), among other
samples, carried out studies analyzing samples from an activated
sludge system with extended aeration, which is one of the tech-
nologies used for the treatment of sewage sludge in Curitiba, Par-
ana, Brazil. This author obtained a meager accuracy of 16.7% when
sludge samples from that sewage treatment plant were used for the
identification and quantification of Ascaris. It was considered that
this result could have been associated with the characteristics of
the sludge from the aerobic/aerated system in which the settling of
the suspended solids is hindered and can be associated with the
bulking of filamentous microorganisms, a common disturbance of
the activated sludge system.
 M.C.V. Rocha et al. / Water Research 103 (2016) 245e255
Table 1
Features of five analytical methods for the quantification of viable helminth eggs in samples of sewage sludge.
Method Surfactant Flotation Features
Extraction
Tulane Limbro® 7X MgS04 Sieving þ10% NaClOa
Yanko Hot tapwater ZnSO4 Acidic alcohol
Gaspard et al. 0,01% SDS NaCl Ultrasonication þ NaClOa
USEPA Limbro® 7X MgS04 Sieving
de Victoria and Galv

an NaCl 0,85% ZnSO4/MgSO4 Membrane filter
Notes.
a
After the incubation step.
b
For anaerobically digested/lagoon stored sludge.
c
Values of recovery not available, as the method do not perform split/spike assays.
Table 1 presents details of several methods used for the deter-
mination of viable helminth eggs in sewage sludge. The solutions
used in the steps of desorption, flotation, extraction and incubation
are referred to as the main difference among them. Another dif-
ference is that some methods perform the extraction step after the
incubation of the eggs to induce the hatching of the embryonated
eggs, such as the Tulane method (1989) and the method conceived
by Gaspard et al. (1996).
Several other methods, or modifications of stablished methods,
for the determination of the viability of helminth eggs in sewage
sludge samples have been described in the literature (Huyard et al.,
2000; Thomaz-Soccol et al., 2000; Bean and Brabants, 2001).
However, these authors have not presented any data on quality
assurance/quality control (QA/QC) for these methods, either
because the number of replicates performed for the recovery assays
were insufficient, thus the sample was not representative, or
because there is no information with regard to the quality of the
analytical data produced.
5. Quality assurance and quality control (QA-QC)
Over the last decades, several assays aiming at the determina-
tion of the viability of parasite eggs in biosolids have been devel-
oped. However, not even a single method has been universally
accepted, mainly due to a lack of published QA-QC data for these
methods.
The terms quality assurance and quality control are often used
interchangeably to refer to the methods of ensuring the quality of a
service or product. However, according to Konieczka and
Namie
snik (2009), these terms present different meanings.
Whereas QA refers to the planning and implementation of activities
to ensure that the quality requirements of a product or service are
met, QC consists of observation techniques and activities that can
detect and correct possible failures in the process of achieving the
required quality.
Regarding the techniques used for the determination of patho-
gens in sewage sludge samples, quality assurance involves the
following steps: (i) establishment of a representative sampling plan
that covers all the expected variability in the sewage sludge,
including variations due to weather or operational changes; (ii)
description on how the quality of the analytical data can be guar-
anteed during the application of a particular protocol; and (iii)
performance analyses in an independent laboratory that can
guarantee the suitability of the tests (USEPA, 2003).
Quality control ensures that the demands of quality assurance
are satisfied. According to the Standard Methods for the Examina-
tion of Water and Wastewater (APHA, 2013) at least seven essential
elements comprise the QC. These elements are: i) certification of
the competence of the analyst, ii) recovery of organisms in inocu-
lated samples, iii) analysis of positive controls, iv) analysis of
253
Eggs recovery (%) References
Incubation
Formaline 0,5% 75e80b Reimers et al. 1981, Bowman et al. 2003
H2SO4 0,1N 83e92 Yanko 1987
Deionized water NAc Gaspard et al., 1996
H2SO4 0,1N 75e80 USEPA 2003
No incubation 54e77 de Victorica and Galva
n, 2003
negative controls, v) calibration method with standardized sam-
ples, vi) replicate analyses and vii) maintenance of control graphics
and data. However, for the majority of the developed methods, it is
difficult to achieve all these requirements. Hence, these essential
elements should be viewed as aims to be achieved during the
formulation and implementation of a high quality pathogen puri-
fication and quantification method (USEPA, 2003).
In order to certify the quality of the data produced and also to
validate the application of the data, even under different sampling
conditions, the methods available for the quantification of viable
helminth eggs in sludge samples should follow a QA-QC program.
Regarding the QA/QC, the methods for the determination of viable
Ascaris eggs that present quality data and possibility of validation of
the results are Yanko (1987), Reimers et al. (1989) and USEPA
(2003). It is worth mentioning that the USEPA method is the
closest to a standard protocol for the determination of viable Ascaris
eggs in sludge samples and their derivatives. However, the appli-
cation should be considered with caution, thus being fundamental
to perform QA/QC tests for matrices of sludge different from those
validated by the method.
6. Conclusion
This review presents several strategies for the determination of
the viability and quantification of helminth eggs in sewage sludge
samples. However, at present, there is no general agreement on the
direction of the adoption of a universal method for the detection
and quantification of these organisms. The main reason for the lack
of consensus is the absence of information necessary to validate the
quality of the tests. Moreover, the methodologies for the quantifi-
cation and determination of the viability of helminth eggs are
constantly modified by other researchers to increase the egg re-
covery rate from sewage sludge samples. However, these adapta-
tions are rarely validated according to a suitable QA-QC program.
The comments resulting from the analysis of the methods pre-
sented in this review are intended to instigate a debate, whose
results could be used to produce a standard protocol for the
determination and quantification of viable helminth eggs present
in sewage sludge. This is due to a lack of a universal method
entailed in the adoption of procedures that may not be appropriate
and could be related to inaccurate results.
Hence, future researches aimed at the development of a method
that can assure both adequate sampling and analytical control and
that can guarantee high accuracy, speed and precision in the
determination of the viability of helminth eggs in sludge samples
will be of fundamental importance for the development of a stan-
dard protocol of analysis. This would guarantee a more accurate
quantification and could subsidize the development of a universal
method, suitable for sludge samples of diverse composition and
origin.
254 M.C.V. Rocha et al. / Water Research 103 (2016) 245e255
Acknowledgements
The authors acknowledge the funding granted by the Coordi-
nation of Improvement of Higher Education Personnel-CAPES/
Brazil and the Araucaria Foundation for the Scientific Support and
Technological Development of Parana/Brazil for the development of
this research.
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