INACTIVATION OF HELMINTH EGGS WITH SHORT- AND MEDIUM- CHAIN FATTY
ACIDS ALONE AND IN COMBINATIONS AT NATURALLY OCCURRING
CONCENTRATIONS
A Thesis
Presented to the Faculty of the Graduate School
of Cornell University
In Partial Fulfillment of the Requirements for the Degree of
Master of Engineering
by
Dan Zhu
May 2, 2014
INACTIVATION OF HELMINTH EGGS WITH SHORT- AND MEDIUM- CHAIN FATTY
ACIDS ALONE AND IN COMBINATIONS AT NATURALLY OCCURRING
CONCENTRATIONS
by Dan Zhu
This thesis/dissertation document has been electronically approved by the following individuals:
Bowman, Dwight Douglas (Chairperson)
©2014 Dan Zhu
ABSTRACT
Fatty acids are widely occurring in natural fats and dietary oils and they are known to have
antibacterial and antifungal properties. This study assessed the inactivation activity of short- and
medium-chain fatty acids against Ascaris suum eggs, which are routinely used as bio-indicators
to the ovicidal activity of various manure and biosolids disinfection methods due to its inherent
environmental indestructability and prevalence in sludges. Previous research has shown that
the eggs could be easily killed when the pH of the acid solution was below the pKa of the acid,
where most of the acid is in the undissociated form. Expanding on this earlier work, the acetic
acid, butyric acid, valeric acid, and hexanoic acid alone or in combination with naturally
occurring concentration at pH 4, were tested to determine the ability of eggs inactivation at
37°C. The inactivating factor was found to be a mixture of fatty acids. The results suggest
butyric acid (240 mM) and hexanoic acid (16mM) at these low levels which are produced in a
pilot toilet in development are capable of rapid inactivation of helminth eggs.
ACKNOWLEDGEMENTS
I want to express my deep gratitude to Dr. Dwight D. Bowman, who offered me knowledge,
always gave me huge encouragement and support.
Special thanks to Janice L. Liotta, who supported my entire research and brought in so much
fun
Great gratitude goes to all my families, friends, coworkers, and any person who let me realize
the beauty of the world.
It’s a great honor to be a student of Cornell University, really appreciate I got chances to meet
the most wonderful professors, advisors, friends, and coworkers.
CHAPTER 1
INTRODUCTION
1.1. Problem Statement
For the purpose of sustainable development, recycling water is indispensable. Especially for
agricultural production, given the high concentration of organic matter and some necessary
nitrogen and phosphorus for crop growth in the wastewater, reusing water is definitely an
efficient and economic method both for irrigation and fertilization. However, there are many
potential problems due to the imprudent wastewater reuse. Untreated sewage may contain
many animal and human pathogens (e.g. bacteria, helminth eggs, protozoan cysts and viruses),
that could be transmitted through insanitary disposal of wastewater and sludge, and lead to
increasing enteric diseases (Islam N., 2014). This problem is particularly severe in developing
countries, where with poor economic conditions, less ability to establish health defensive
systems, low levels of education, and little in the way of concern for environmental sanitation.
As a result, ascariasis is pandemic in some developing countries, many diseases caused by
fecal pathogens are listed as the main causes of childhood morbidity and mortality, and people
struggling with poor health and poverty, leads to a vicious cycle. According to the World Health
Organization, unsafe water supplies, sanitation and hygiene rank third among the 10 most
significant risk factors for poor health in developing countries. Approximately 3.1% of annual
deaths (1.7 million) and 3.7% of DALYs (Disability Adjusted Life Years) (54.2 million) worldwide
are attributed to unsafe water supply, sanitation and hygiene. Among them, almost all such
associated deaths (99.8%) occurred in developing countries, and 90% of them were children
(SIWI & WHO, 2005; WHO, 2002b). What’s more, the first ranked risk factor of poor health,
malnutrition, is also related to inadequate safe water and sanitation since it’s geographically
associated with poor environmental and hygienic conditions (Islam N., 2014).
The infective pathogens and infectious disease are the main cause of death. Four groups of
pathogens are found in excreta: bacteria, helminths, protozoa and viruses. These pathogens
are related to gastrointestinal diseases giving rise to symptoms such as dysentery, diarrhea,
vomiting, and stomach cramps; they could also affect other organs and lead to severe health
consequences such as malnutrition (Droste, 1997). An adult female A. suum nematode sheds
up to 200,000 eggs daily; these eggs are passed in the feces of the infected individual and are
thus present in wastewater, contaminated soil, and, in some cases, contaminate drinking water
sources. If A. suum egg-contaminated food or water were ingested by humans or other
mammals, the larvae could reach the liver and lung alveoli via bloodstream, resulting in liver
lesion, eosinophilic pneumonia, myelitis, and visceral larva migrans, etc. (Islam N., 2014).
As a result, there are an increasing world-wide concerns about public health and environmental
sanitation risks related to the disposal and reuse of wastewater, searching for the better
disinfection processes is critical for minimizing those risks and improving living conditions.
1.2. Solutions
The swine parasite Ascaris suum is routinely used as a surrogate for the human parasite
Ascaris lumbricoides that is often found in sludge (Paulsrud, B., B. Gjerde, and A. Lundar.
2004.). The nematode Ascaris lumbricoides also releases highly resistant, unembryonated eggs
into the environment, causing about 1.3 billion illnesses worldwide (de Silva, N. R., M. S. Chan,
and D. A. P. Bundy. 1997.). The viability of Ascaris sp. eggs is one of criteria for assessing the
safe disposal of sludge. The Ascaris suum eggs can remain viable anywhere from months to as
many as four or more years in soil, even with repeated freezing and thawing events. The
characters that enable eggs to survive for such a long time on their own are because their
resistance to dehydration, low temperatures, and strong chemicals. The lipid layer of the
eggshell which contains ascarosides is what provides the eggs strong with their ability to resist
these environmental extremes. With this ability of longevity, it is hard to prevent reinfection once
the soil has been contaminated (Larry S. R. & John J, Jr, 2008). Due to its resistance to
biocontrol mechanisms (Capizzi-Banas, S., M. Deloge, M. Remy, and J. Schwartzbrod. 2004),
Ascaris is a model organism for developing environmentally safe disinfection methods (CapizziBanas, S., and J. Schwartzbrod. 2001, Paulsrud, B., B. Gjerde, and A. Lundar. 2004.). Common
methods for inactivating Ascaris eggs in sludge and fecal matter are high temperature, high pH,
or both. The eggs can also be rendered nonviable through natural processes like UV radiation
(Brownell, S. A., and K. L. Nelson. 2006.). Other processes for inactivation include acid
treatment, alkaline stabilization, anaerobic digestion, dehydration, composting, thermal drying,
and disinfection with metal. Many procedures are available for the decontamination of sludge
and manure in agriculture, but are more or less limited by specific energetic problems and costs
(Islam N., 2014). One recommended possible method for controlling Ascaris is the use of shortchain fatty acids (SCFA). The toxicity of SCFA to bacteria, e.g., Escherichia coli (Cherrington, C.
A. M. H., G. R. Pearsonand, and I. Chopra. 1991), fungi (Hatton, P. V., and J. L. Kinderlerer.
1991, Teh, J. S. 1974.), Staphylococcus, and Streptococcus (Nair, M. K. M., J. Joy, P.
Vasudevan.etc. 2005), insects (House, H. L. 1967.), and birds (Donaldson, W. E., and B. L.
Fites. 1970) has been reported. But when at a pH above the pKa, SCFA are far less toxic and
will be degraded by bacteria. Using SCFAs is safe since they are neutralized naturally or by pH
adjustment with the addition of agents such as baking soda. Another advantage of using SCFA
is that fatty acids, which could inactivate the Ascaris eggs, and bioproducts/chemicals with
commercial value could both be generated during the digestion process (Islam N., 2014).
1.3. Overall Objectives
This thesis is based on the fact that short- and medium-chain fatty acids have the potential to
inactivate pathogens in the water, including the Ascaris suum egg. It represents the results of
using certain concentration of different fatty acids alone or in combinations to reduce Ascaris
suum eggs’ viability at certain temperature. The acid pH was determined following the model of
Ascaris suum inhibition (IC50 moles/liter) as a function of pH, and pH~4 was used. The
experiment temperature chose was 37°C which represents the weather condition of tropical
regions and the routine temperatures reached in the anaerobic digestion of manures and
sewage sludges. The main objective of research is to find a safe and effective method for
inactivating parasite eggs in the wastewater, with no or less harmful effects on the environment
(e.g., alteration of the soil pH or potential health effects caused by caustic agents).
1.4. Specific Objectives
1. To describe the morphological changes of Ascaris suum eggs observed during in vitro
incubation for a period of three weeks.
2. To determine the inactivation of Ascaris suum eggs by short- or medium-chain fatty acids
added to water individually in a laboratory environment at low levels mimicking those that have
been produced in a pilot toilet inactivation system.
3. To determine the inactivation of Ascaris suum eggs by short- or medium-chain fatty acids
added to water in various combinations in a laboratory environment.
1.5. Hypotheses
Short- or medium-chain fatty acids combination will increase the Ascaris suum eggs’ inactivation
rate.
CHAPTER 2
LITERATURE REVIEW
2.1.
Sanitation and public health
The World Health Organization states that: “Sanitation generally refers to the provision of
facilities and services for the safe disposal of human urine and feces. Inadequate sanitation is a
major cause of disease world-wide and improving sanitation is known to have a significant
beneficial impact on health both in households and across communities. The word ‘sanitation’
also refers to the maintenance of hygienic conditions, through services such as garbage
collection and wastewater disposal.” Adequate sanitation and hygiene system could not only
ensure public health, but also does a positive impact on economic and poverty reduction.
According to the WHO, every $1 dollar invested in sanitation would yield an economic return
between $3 to $34, depending on the different regions (WHO & UNICEF, 2004.). Many
pathogens exist in the human or animals excreta. Without effective disposal methods to treat
excreta with hygiene practices, pathogens would transmit and contaminate the environment,
furthering the production of more infections. In the areas where sanitation systems are deficient,
fecal pathogens are prevalently distributed in the environment, e.g. surface water, soil,
groundwater, etc. Once the environment is contaminated, individuals could be easily infected
by contacting or ingesting contaminated food or water. Infectious diarrhea, schistosomiasis,
ascariasis, trichuriasis, and hookworms are among the main diseases contributing to the burden
associated with unsafe water, sanitation and hygiene (WHO, 2002b). These diseases affect
close to half the people on the planet at any given time and cause the occupancy of more than
half the hospital beds in the developing world (UN Millennium Project, 2005).
Table 1: Main Fecal Pathogens of Concern for Public Health
Group
Bacteria
Pathogen
Aeromonas spp.
Campylobacter jejuni/coli
Escherichia coli (EIEC,
EPEC, ETEC, EHEC)
Plesiomonas shigelloides
Salmonella typhi/paratyphi
Salmonella spp.
Shigella spp.
Vibrio cholera
Yersinia spp.
Helminths
Disease and Symptoms
Enteritis
Campylobacteriosis-diarrhea, cramps, abdominal
pain, fever nausea, arthritis; Guillain-Barre
syndrome
Enteritis
Enteritis
Typhoid/paratyphoid fever – headache, fever,
malaise, anorexia, bradycardia, splenomegaly,
cough
Salmonellosis – diarrhea, fever, abdominal
cramps
Shigellosis – dysentery, vomiting, cramps, fever;
Reiter’s syndrome
Cholera – watery diarrhea, lethal if severe and
untreated
Yersiniosis – fever, abdominal pain, diarrhea, joint
pains, rash.
Ascaris lumbricoides
Taenia solium/saginata
Trichuris trichiura
Ancylostoma duodenale/
Necator Americanus
Schistosoma spp.
Ascariasis – generally no or few symptoms;
wheezing, coughing, fever, enteritis, pulmonary
eosinophilia
Taeniasis
Trichuriasis – unapparent through vague
digestive tract distress to emaciation with dry skin
and diarrhea
Itch, rash, cough, anemia, protein deficiency
Schistosomiasis, bilharzia
Parasitic
protozoa
Cryptosporidium parvum
Cyclospora cayetanensis
Entamoeba histolytica
Giardia intestinalis
Source: (WHO, 2006b)
Cryptosporidiosis – watery diarrhea, abdominal
cramps and pain
Often asymptomatic; diarrhea, abdominal pain
Amoebiasis – often asymptomatic; dysentery,
abdominal discomfort, fever, chills
Giardiasis – diarrhea, abdominal cramps,
malaise, weight loss
Helminthiasis is infestation with one or more intestinal parasitic worms (roundworms (Ascaris
lumbricoides), whipworms (Trichuris trichiura), or hookworms (Necator americanus and
Ancylostoma duodenale)) (Bethony, J., Brooker, S., et al., 2006). Infected people excrete
helminth eggs in their feces, which then contaminate the water or soil in areas with inadequate
sanitation. Helminths are transmitted to the final host in several ways. The most common
infection is through ingestion of contaminated vegetables, drinking water and raw or
undercooked meat. The infective form can be eggs (for most nematodes) or larvae. Some
larvae of trematodes (specifically the cercaria of schistosomes) can directly penetrate the skin
when an individual is in direct contact with an infested water body (Baron S. 1996). Infection can
cause morbidity, and sometimes death, by compromising nutritional status, affecting cognitive
processes, inducing tissue reactions, such as granulomas, and provoking intestinal obstruction
or rectal prolapse (WHO Website). Soil transmitted helminths produce the most common
infections worldwide, and the causal agents are Ascaris lumbricoides, T. trichiura, and
hookworms (Necator americanus and Ancylostoma duodenale) (Islam N., 2014). Approximately
2 billion people are infected by these helminths, among them 133 million suffer from high levels
of intestinal infections, and 135,000 are estimated to die every year from these infections
(UNICEF, 2006; WHO, 2003). The World Health Organization estimates that A. lumbricoides, T.
trichiura, and hookworms infect respectively 1 billion, 795 million and 740 million individuals
(WHO, 2008c). Most of these infections are attributed to Ascaris, it causes 60,000 deaths per
year, especially among children between 3 and 8 years of age (WHO, 2001b). It has been
estimated that the morbidity caused by Ascaris lumbricoides could be reduced by 29% through
providing safe water, adequate sanitation and hygiene systems (Islam N., 2014).
2.2.
Indicator Organisms
Indicator organisms are often used instead of actual pathogens when monitoring and assessing
behavior of pathogens in the environment. There are two reasons for their use. The first is
some pathogens could be hazardous to laboratory technicians when doing research on. Another
is that pathogens often appear at low concentrations in natural environments, making them
difficult and costly to receive. As a result, indicator organisms are chosen to be used as a
surrogate for these hard-to-get pathogens, making research more reliable, faster and costeffective. There are prerequisites for choosing appropriate and reliable indicator organisms.
Such prerequisites have been widely discussed (Payment and Franco 1993; Mara and Horan
2003; Hach 2000) and mainly include;

be non-pathogenic

having the same origin as the pathogen it is representing

always be present when (and only when) the pathogen is present

exist in high enough numbers to be detected

be easy to measure in the laboratory

be equally persistent or more persistent than the pathogens it is representing (Ingrid,
2012)
Based on these criteria, several organisms have been chosen frequently as indicators of
microbial behavior. Ascaris suum has been used in this study. The swine parasite Ascaris suum
is routinely used as a surrogate for the human parasite (Paulsrud, B., B. Gjerde, and A. Lundar.
2004) and is often found in sludge.
2.3.
Helminth Eggs
Helminths are a polyphyletic group of eukaryotic parasites (Maizels RM, Yazdanbakhsh M,
2003). They are worm-like organisms living in and feeding on or within living hosts, receiving
nourishment and protection while disrupting their hosts’ nutrient absorption, causing weakness
and disease. They are worms measuring from 1mm to several meters in length, which come
from microscopic eggs (US-EPA, 1992). Helminth eggs have highly resistant biological
structures. Their egg shell consists of a variable number of layers each providing mechanical
resistance or protection from toxic compounds. They can remain viable for 1-2 months in crops
and for many months to years in soil, fresh water, and sewage, making them the most resistant
of all pathogen groups (Feachem et al., 1983, Brownell and Nelson 2006), and a good indicator
of pathogen die-off. In addition to being an indicator for other pathogens, helminth eggs
themselves can be highly pathogenic as ingestion of the eggs can lead to severe helminthiases
(WHO 2012a). The eggs are found in wastewater, sludge and excreta in variable amounts,
depending on local health conditions, and have been shown to be most abundant in developing
countries (Schwartzbrod et al. 1989; Jimenez 2007). Studying the abundance and die-off rate of
helminth eggs is therefore very important and much used when assessing health risks
associated with wastewater irrigation in developing and developed countries (Stien and
Schwartzbrod 1990; Hamouri et al. 1999; Amoah et al. 2005).
Ascaris lumbricoides is the giant roundworm of humans, belonging to the phylum Nematoda. An
Ascarid nematode, it is responsible for the disease ascariasis in humans, and it is the largest
and most common parasitic worm in humans. One-sixth of the human population is estimated to
be infected by Ascaris lumbricoides or another roundworm (Harhay MO, Horton J, Olliaro PL,
2010). Ascariasis is prevalent worldwide and more so in tropical and subtropical countries. It
can reach a length of up to 35 cm (Laskey A., 2008). Ascaris eggs have a 3- to 4-µm thick, fourlayer shell that consists of an inner lipoprotein layer (ascaroside layer), a thicker chitin/protein
layer, a lipoprotein vitelline layer, and an outer mucopolysaccharide/protein uterine layer, each
of four layers has a characteristic chemical composition (Islam N., 2014). Two of these, namely
the innermost lipoid membrane and the chitinous shell, have been recognized for many years
(Chitwood, 1937). However, recent evidence supports that the chitinous shell contains both a
protein and a chitin layer, and another protein is to be found either as a separate layer or as a
part of the lipoid membrane (Islam N., 2014). The various layers of the primary envelope will be
considered in the order in which they are formed, beginning with the outermost, and finishing
with the lipoid membrane. The outer layer is usually fully formed by the time the egg has
traversed one-third the length of the uterus. The formation of the fertilization membrane is
followed quickly by the appearance between it and the cytoplasmic surface of a secretion which
rapidly hardens to form the second layer. This optically clear layer is about 3 µ thick, consists
mainly of chitin, although delicate protein fibrils similar in their properties to those of the
fertilization membrane may also be present (Monné and Hönig, 1954b). Ascaris eggs, with their
multilayered structure of chitin and lipid, are among the most resistant of the helminth eggs
(Capizzi-Banas 2004; Brownell and Nelson 2006; Mara et al. 2010), and are therefore often
used as the indicator organism when studying the survival of helminth eggs. The innermost
layer is the ascaroside lipoid layer which provides much of the protection against chemical
attack.
2.4.
Fatty acids and their antimicrobial properties
An organic acid is an organic compound with acidic properties. The most common organic acids
are the carboxylic acids, whose acidity is associated with their carboxyl group – COOH.
Common names used to describe this group of organic compounds include carboxylic, fatty,
volatile fatty, lipophilic, or weak acids. Fatty acid chains differ by length, often categorized as
short to very long. The short-chain and medium- chain fatty acids would be grouped arbitrarily
according to their carbon chain length (Table 2). Short- chains (SCFA) are fatty acids with
aliphatic tails of fewer than six carbons. Medium-chain fatty acids (MCFA) are fatty acids with
aliphatic tails of 6-12 carbons, which can form medium-chain triglycerides (Cifuentes A.,
2013).The individual acids are named systematically from the normal alkane of the same
number of carbon atoms by dropping the final “e” and adding the suffix “oic” (Islam N., 2014).
Table 2: Nomenclature of organic acids (after Streitwieser and Heathcock 1981)
Compound
Common name
Systematic name
Short chain fatty acid
C1 HCOOH
Formic
Methanoic
C2 CH3COOH
Acetic
Ethanoic
Propionic
Propanoic
C4 CH3(CH2)2COOH
Butyric
Butanoic
C5 CH3(CH2)3COOH
Valeric
Pentanoic
C3 CH3CH2COOH
Medium chain Fatty acid
C6 CH3(CH2)4COOH
Caproic
Hexanoic
C7 CH3(CH2)5COOH
Enanthic
Heptanoic
C8 CH3(CH2)6COOH
Caprylic
Octanoic
C9 CH3(CH2)7COOH
Pelargonic
Nonanoic
C10 CH3(CH2)8COOH
Capric
Decanoic
The literature on the effect of surface-active anionic detergents (fatty acids) dates as far back as
the work of Clark (Clark, J.R., 1899) reported in 1899. The antifungal and bactericidal properties
of fatty acids have been extensively investigated (Chattaway, F.W., Thompson, C.C. et al, 1956;
Glassman, H.N., 1948, Prince, H.N., 1959). In general, fatty acids function as anionic surface
agents, and the anionic surfactants are less potent at physiological pH values (Armstrong,
W.McD, 1957, Scherff, T.G., Peck, J.C., 1959, Kabara, J. J.,Swieczkowski, D.M., et al, 1972).
The toxicity of fatty acids to bacteria, e.g., fungi (Hatton, P. V., and J. L. Kinderlerer. 1991, Teh,
J. S. 1974), Streptococcus, and Staphylococcus (Nair, M. K. M., J. Joy, P. Vasudevan, L. etc.
2005.), has been widely reported. Also, there is research showing that fatty acids exhibited
patterns of inhibition against oral bacteria. Formic acid, capric, and lauric acids were broadly
inhibitory for the bacteria. Interestingly, fatty acids that are produced as metabolic end-products
by a number of these bacteria, were specifically inactive against the producing species, while
substantially inhibiting the growth of other oral microorganisms (Huang, C.B., Altimova, Y. et al,
2011). In the food animal industry, organic acids were originally added to animal feeds to serve
as fungistats (Paster, 1979; Dixon and Hamilton, 1981), but in the past 30 years, formic and
propionic acids and various combinations have also been examined for potential bactericidal
activity in feeds and feed ingredients contaminated with foodborne pathogens, particularly
Salmonella spp. (Khan and Katamay, 1969). Although the antibacterial mechanism(s) for fatty
acids are not fully understood, they are capable of exhibiting bacteriostatic and bactericidal
properties depending on the physiological status of the organism and the physicochemical
characteristics of the external environment (Islam N. 2014). Organic acids are more effective
than mineral acids as antimicrobial agents, although they exhibit broad-spectrum antibacterial
activity, the antibacterial efficiency of individual acids varies (Goepfert and Hicks, 1969).
CHAPTER 3
ASCARIS SUUM EGG INACTIVATION USING DIFFERENT
SHORT-CHAIN FATTY ACIDS:
ACID CONCENTRATION, ALONE / IN COMBINATION
3.1. Abstract
Fatty acids are widely occurring in natural fats and dietary oils and they are known to have
antibacterial and antifungal properties. This study assessed the inactivation activity of short- and
medium-chain fatty acids against Ascaris suum eggs, which are routinely used as bio-indicators
to the ovicidal activity of various manure and biosolids disinfection methods due to its inherent
environmental indestructability and prevalent in sludges. Previous research has shown that the
eggs could be easily killed when the pH of the acid solution was below the pKa of the acid,
where most of the acid is in the undissociated form. Expanding on this earlier work, acetic acid,
butyric acid, valeric acid, and hexanoic acid alone or in combination with naturally occurring
concentration at pH 4, were tested to determine the ability of eggs inactivation at 37°C. The
inactivating factor was found to be a mixture of fatty acids. The results suggest Butyric acid (240
mM) and Hexanoic acid (16mM) in combination have potential for the rapid inactivation of
helminth eggs in the water.
3.2. Introduction
Helminth eggs are prevalent in wastewater, sludge and excreta in variable amounts, depending
on local health conditions, and have shown to be most abundant in developing countries,
including Ghana (Schwartzbrod et al. 1989; Jimenez 2007). Diseases caused by ingesting
contaminated food or water are among the main reasons of many morbidity or death. Studying
the abundance and die-off rate of helminth eggs is therefore very important and much used
when assessing health risks associated with wastewater irrigation in developing countries (Stien
and Schwartzbrod 1990; Hamouri et al. 1999; Amoah et al. 2005). Ascaris suum (Goeze, 1782),
a parasite of swine, with the transmission mechanism (fecal/oral), affects millions of pigs and is
responsible for substantial economic losses in many countries (O’Lorcain and Holland, 2000).
Helminth eggs are the most resistant to many types of inactivation and eggs of the genus
Ascaris have the highest resistance and survive under numerous treatment conditions
(Feachem et al., 1983; Gaasenbeek and Borgsteede, 1998; Reimers et al., 1986b). Ascaris
suum eggs are resistant towards most disinfection treatments; in sewage sludge, a treatment
lasting 2 months with an initial pH of 12.5 was required to obtain no viable organisms (Gaspard
et al., 1995). A pH over 10 at temperatures above 10°C was sufficient for inactivation of bacteria
(Allievi et al., 1994) but Ascaris eggs can be inactivated in minutes by temperatures above
60°C. Also, Ascaris eggs can survive for more than 1 year at 40°C (Feachem, 1980). Ascaris
eggs are more resistant to external conditions because of their structural composition of the egg
shell and are permeable only to organic solvents and lipid soluble gases (Fairbairn, 1957).
Exposure of de-shelled or decorticated eggs to a variety of proteolytic, amylolytic and lipolytic
enzymes had no detectable effect on permeability. The resistance of the eggs to many
treatment factors and disinfectants makes Ascaris eggs a conservative indicator organism for
environmental pollution and treatment efficiency (O’Lorcain and Holland, 2000).
Previous research has shown that the eggs could be easily killed when the pH of the acid
solution was below the pKa of the acid, where most of the acid is in the undissociated form
(Butkus, M.A., Hughes, K.T., et al, 2010). There is a clear concentration barrier of SCFAs that
must be reached in order to be toxic, and their concentrations would increase substantially at
pH values slightly above pKa (Figure 1) (Butkus, M.A., Hughes, K.T., et al, 2010). Also, it has
been reported that the effect of fatty acid on the viability of A. suum eggs was dependent on
acid concentration and temperature at which the exposure occurs. As acid concentration and
temperature get higher, there is a marked increase in the killing of eggs by the acids. 37°C is
suggested as the minimum temperature required to achieve total inactivation without other
agents or chemicals being added to the acid in lower concentration under laboratory conditions
(Islam N., 2014). Based on previous research, this study aimed at testing the inactivation ability
of fatty acids at lower concentration with lower pH at 37°C.
Figure 1: Model of Ascaris suum inhibition (IC₅₀ moles/liter) as a function of pH
(Butkus, M.A., Hughes, K.T., et at., 2010).
3.3. Materials and Methods
3.3.1. Collection & cleaning of Ascaris suum eggs:
The unembryonated Ascaris suum eggs used in this study were collected from the intestinal
contents of farm raised pigs at a slaughter house in Pennsylvania. The small intestines
contained adult Ascaris suum and there were eggs in very large numbers in the fecal contents.
Thus, the contents of intestinal tracts was diluted in water and passed through a series of sieves
to remove particulates, and finally the eggs were collected on a 500 mesh sieve. After
collection, the eggs and similar sized particulates were transferred to 4.5 liter buckets, which
were filled with deionized water containing 0.1N H2SO4, to about a depth of 3 cm. Then the eggs
were stored at 4°C with the acidic water being changed regularly.
At the time of use, Ascaris suum eggs in the sediment were further cleaned by centrifugal
flotation with a MgSO4 solution at specific gravity 1.2. The floated eggs were poured over a 500
mesh sieve, and then washed back into a 15 ml conical centrifuge tube. A dilution egg count
method was used for determining the volume of eggs utilized in a given study. According to the
morphological criteria, two kinds of eggs populations in the counting cell were identified: one is
eggs with only a cell wall (composed of an inner lipid layer, an intermediary chitinous layer and
an outer vitellin membrane), another kind of eggs had both a cell wall and the outer uterine
albuminous layer. Both populations are normally present in newly laid eggs. While the outer
layer is made up of secretions deposited as the egg passes through the uterus, it is not evenly
distributed, and even may be absent from some eggs. The outermost layer is believe to assist
the egg in the environment as a protection against UV light due to its dark brown color (Islam N.,
2014).
3.3.2. Acid / acid combinations:
There are four kinds of fatty acids used in the research: acetic acid (C2), butyric acid (C4),
valeric acid (C5) and hexanoic acid (C6). The naturally occurring concentrations of these fatty
acids that were generated in a pilot toilet system under development with the Department of
BEE, Cornell University were respectively: acetic acid (288 mM), butyric acid (240 mM), valeric
acid (16 mM), and hexanoic acid (16 mM) (Lauren Harroff, personal communication). 15
experimental groups were established with different acids or different acid combinations to
represent all combination presented with the toilet system (Table 3).
Table 3: Acid preparation for 15 groups
Group Number
1
Solution
Molarity (mol)
Volume (ml)
Acetic Acid (C2)
0.288
1.8
Butyric Acid (C4)
0.240
2.2
Water
2
Acetic Acid (C2)
0.288
1.8
Valeric Acid (C5)
0.160
0.2
Acetic Acid (C2)
0.288
1.8
Hexanoic Acid (C6)
0.160
0.2
Butyric Acid (C4)
0.240
2.2
Valeric Acid (C5)
0.160
0.2
Butyric Acid (C4)
0.240
2.2
Hexanoic Acid (C6)
0.160
0.2
4.09
97.6
Valeric Acid (C5)
0.160
0.2
Hexanoic Acid (C6)
0.160
0.2
Water
3.99
97.6
Water
6
3.97
98.0
Water
5
3.94
98.0
Water
4
3.97
96.0
Water
3
pH
99.6
4.09
Group Number
7
Solution
Molarity (mol)
Volume (ml)
Acetic Acid (C2)
0.288
1.8
Butyric Acid (C4)
0.240
2.2
Valeric Acid (C5)
0.160
0.2
Water
8
Acetic Acid (C2)
0.288
1.8
Butyric Acid (C4)
0.240
2.2
Hexanoic Acid (C6)
0.160
0.2
Acetic Acid (C2)
0.288
1.8
Valeric Acid (C5)
0.160
0.2
Hexanoic Acid (C6)
0.160
0.2
Butyric Acid (C4)
0.240
2.2
Valeric Acid (C5)
0.160
0.2
Hexanoic Acid (C6)
0.160
0.2
Acetic Acid (C2)
0.288
1.8
Butyric Acid (C4)
0.240
2.2
Valeric Acid (C5)
0.160
0.2
Hexanoic Acid (C6)
0.160
0.2
13
14
15
Acetic Acid (C2)
0.288
0.240
Water
2.2
97.8
0.160
Water
Hexanoic Acid (C6)
1.8
98.2
Water
Valeric Acid (C5)
3.99
95.6
Water
Butyric Acid (C4)
3.89
97.4
Water
12
3.87
97.8
Water
11
3.77
95.8
Water
10
3.91
95.8
Water
9
pH
0.2
99.8
0.160
0.2
99.8
3.95
4.09
4.34
4.08
3.3.3. Exposure of A. suum eggs with fatty acids:
For each experiment groups, about 4200 eggs and 1 ml of the test acids were added into
microfuge tubes, vortexed for 3 seconds and placed in incubator at 37°C in a static condition,
i.e., without shaking or mixing. After two days, the tubes were removed, and centrifuged to pellet
the eggs. Without disturbing the egg pellet, the acid was removed by suction, and the eggs were
washed 6 times with phosphate buffer (10 mM, pH 7.0). The eggs were transferred to 24 well
culture plates with water after washing. Then, the plate wrapped in a wet paper towel and put
into a plastic box that was incubated at 28°C for 25 days. All experiments were carried out in
triplicate.
3.3.4. Assessing the viability percentage of eggs:
After 25 days, 300 ul of 6% sodium hypochlorite (Clorox) was added to each well to remove the
outer albuminous layer from the eggs. After 10 minutes, the eggs were microscopically
examined. Eggs were scored as larvated (viable) or non-larvated (nonviable). The percent of
viability was calculated as the number of viable eggs divided by the total number of eggs
counted. Counting eggs three times for each well, and 100 eggs were scored every time. The
data is expressed as the percentage of viable eggs in the test sample as a percentage of the
viable eggs in the control wells.
3.4. Results
Images of eggs after several of the tests performed show how the eggs were scored based on
their physical appearance. After 25 days incubated at 28°C, 6% sodium hypochlorite (Clorox)
was added into each well to remove the outer coating of eggs for easier visibility. The eggs were
scored as larvated (viable) or non-larvated (not viable). In the control group (Figure 2A), most
eggs were viable, and each viable eggs contains a developing larva. In the case of Group 4 (the
combination of butyric acid and valeric acid) (Figure 2B) there was little effect on the viability of
the eggs, and in the image only 2 eggs in the total of 7 were dead. In the combination of butyric
acid + hexanoic acid in Group 5 (Figure 2C) and in Group 8 (Figure 2D) which represents the
combination of acetic acid + butyric acid + hexanoic acid, the eggs are all dead and contained a
mass of vacuolar cells without a larva, they are all dead eggs. These results suggest eggs
viability would be heavily limited under these two acid combinations treatment (Figure 2).
Figure 2: The appearance of Ascaris suum eggs after several examples of the applied acid
treatments. The eggs in A and B are from groups where the eggs remained viable, and Cand
D show eggs that are all inactivated. (A) Untreated eggs (Group 1) showing 3 viable and one
that is non-viable (on left of image). (B) In treatment Group 4 (the combination of butyric acid
and valeric acid), 5 of the 7 eggs contain developed larvae (the eggs on the left and right are
nonviable). (C) Group 5 (the combination of butyric acid and hexanoic acid) where all eggs
are inactivated. (D) Eggs from Group 8 (the combination of acetic acid, butyric acid and
hexanoic acid) that are all dead and contain a mass of vacuolar cells similar to those shown in
1(C). [Images all presented at 200 magnifications.]
There were 16 groups of eggs that received different treatment regimens at 37°C: 15 treated
groups and one untreated control group (Table 3). The results indicated that single acids had
no significant effect on viability: acetic acid (87.84% viable), butyric acid (93.99% viable), valeric
acid (92.82% viable), and hexanoic acid (94.21% viable) (Figure 3). Out of the 8 pairs of acids
examined, only one pair of acids (butyric and hexanoic) caused a significant decrease in egg
viability (0% viable) (Figure 4). Of the four combinations of triple acids, there were two triplet
groups, acetic, butyric, and hexanoic acids & butyric, valeric, and hexanoic acids, that caused
significant reductions (100% reduction, 0% viable) in egg viability (Figure 5). In addition there
was a significant decrease in egg viability (0% viable) when all four acids (acetic, butyric, valeric
and hexanoic) wer used in combinations at the given concentrations (Figure 5). Overall, the
viabilities were reduced to zero in four groups of the eggs when they were held at 37°C for 48
hours and treated with the acid concentrations as cited in Table 3: butyric & hexanoic; acetic,
butyric, & hexanoic; butyric, valeric, & hexanoic; and acetic, butyric, valeric, and hexanoic
(Table 4).
Viability (%) of A. suum Eggs Treated with Single
Fatty Acids
100.00
93.99
92.82
94.21
13
14
15
Viability (%)
87.84
80.00
60.00
40.00
20.00
0.00
11
12
16
Group Number
Figure 3: Viability (%) A. suum eggs treated with single Fatty acids. Group 12: acetic acid;
Group 13: butyric acid; Group 14: valeric acid; Group15: hexanoic acid. (The data is expressed
as the percentage of viable eggs in the test samples relative to the percentage of viable eggs in
the control wells).
Viability (%) of A. suum Eggs Treated with Pairs of
Fatty Acids
100.00
92.78
87.84
86.73
94.99
89.27
Viability (%)
80.00
60.00
40.00
20.00
0.00
0.00
0
1
2
3
4
5
6
7
Group Number
Figure 4: Viability (%) of A. suum eggs treated with pairs of fatty acids. Group 1:
combination of acetic acid and butyric acid; Group 2: combination of acetic acid and valeric acid;
Group 3: combination of acetic acid and hexanoic acid; Group 4: combination of butyric acid and
valeric acid; Group 5: combination of butyric acid and hexanoic acid; Group 6: combination of
valeric acid and hexanoic acid. (The data is expressed as the percentage of viable eggs in the
test samples relative to the percentage of viable eggs in the control wells).
Viability (%) of A. suum Eggs Treated with
Combinations Containing Three or Four Fatty Acids
97.14
96.49
100.00
Viability (%)
80.00
60.00
40.00
20.00
0.00
0.00
0.00
10
11
0.00
6
7
8
9
12
Group Number
Figure 5: Viability (%) of A. suum eggs when treated with the combinations of three fatty
acids (Groups 7 to 9) or all four fatty acids in combination (Group 10). Group 7:
combination of acetic acid, butyric acid, and valeric acid; Group 8: combination of acetic acid,
butyric acid and hexanoic acid; Group 9: combination of acetic acid, valeric acid and hexanoic
acid; Group 10: combination of butyric acid, valeric acid and hexanoic acid. (The data is
expressed as the percentage of viable eggs in the test samples relative to the percentage of
viable eggs in the control wells).
Table 4: Viability Percentage for 15 groups
Group Number
Acid/Acid Combination
Viability of Control
(%)
1
Acetic Acid (288 mM) + Butyric Acid (240 mM)
87.84
2
Acetic Acid (288 mM) + Valeric Acid (16 mM)
86.73
3
Acetic Acid (288 mM) + Hexanoic Acid (16 mM)
92.78
4
Butyric Acid (240 mM) + Valeric Acid (16 mM)
89.27
5
Butyric Acid (240 mM) + Hexanoic Acid (16 mM)
0.00
6
Valeric Acid (16 mM) + Hexanoic Acid (16 mM)
94.99
Acetic Acid (288 mM) + Butyric Acid (240 mM)
7
96.49
+ Valeric Acid (16 mM)
Acetic Acid (288 mM) + Butyric Acid (240 mM)
8
0.00
+ Hexanoic Acid (16 mM)
Acetic Acid (288 mM) + Valeric Acid (16 mM)
9
97.14
+ Hexanoic Acid (16 mM)
Butyric Acid (240 mM) + Valeric Acid (16 mM)
10
0.00
+ Hexanoic Acid (16 mM)
Acetic Acid (288 mM) + Butyric Acid (240 mM)
11
0.00
+ Valeric Acid (16 mM) + Hexanoic Acid (16 mM)
12
Acetic Acid (288 mM)
87.84
13
Butyric Acid (240 mM)
93.99
14
Valeric Acid (16 mM)
92.82
15
Hexanoic Acid (16 mM)
94.21
Discussion
The concentrations of fatty acids tested in the research reported herein were those naturally
occurring (acetic acid (288 mM), butyric acid (240mM), valeric acid (16 mM), and hexanoic acid
(16 mM)) in a working model of a pilot disinfecting toilet system. The study showed that these
acids when present in water at these given concentrations are capable of 100% inactivation of
the eggs of A. suum when held in a solution of the acids at 37°C for two days (Table 4). The
individual acids were not effective at these lower concentrations, although they are fully active
when examined at higher concentrations at the same pH in similar systems (Butkus et al., 2010;
Islam N, 2014). Also, it appears that not all the acids are required for the 100% inactivation of
the eggs. Based on the comparison of the results of the different acid groups tested (Table 4),
only in those cases where both butyric (butanoic) and caproic (hexanoic) acids were present did
100% inactivation occur. It is important to note that when in combinations, these two acids
killed the eggs at the very low concentrations, concentrations that are generated in the toilet
system of 240 mM butyric and 16 mM caproic.
Previous research has reported that the effects of fatty acids on the viability of A. suum eggs
was dependent on acid concentration and temperature at which the exposure occured. High
acid concentration and high temperature decreased the time required for total inactivation. At
37°C, 100% of eggs treated with 1.5 M pentanoic or hexanoic acid were killed in less than 10
minutes (Islam N., 2014). Previous research mainly tested the inactivation ability of single fatty
acids at high concentrations that are not realistically being produced in the current pilot toilet
system. Thus, this research examined the effects of realistic concentrations of fatty acids at the
levels they are produced in the toilet system, and it appears that the levels generated are fully
sufficient for egg inactivation – at least when the eggs are in water containing the solutions of
acids for 48 hours. As a result, fatty acids and various acid combinations with low concentration
were tested against A. suum eggs in this study. The results of our research showed that fatty
acids in certain combinations have effective inactivation ability even at the lower concentration.
Again, it is worth noting that four combinations of acids that killed the eggs contained both
butyric acid and hexanoic acid. Thus, these two acids somehow when present in combination
seem to provide the conditions necessary for egg inactivation thus, as long as there is a
combination of butyric acid and hexanoic (caproic) acids in the system, the viability of A. suum
eggs would be drastically limited. But under the practical conditions with the current pilot toilet
system, the necessary fatty acid combinations are produced at levels that appear to be
sufficient for the very efficient inactivation of A. suum eggs which serve as the indicator
organism for other helminths eggs that might be present.
For the further research, the system in the pilot toilet with the eggs in the actual waste should be
tested. While the status of fatty acids in the toilet system has been already generated, the
situation of helminth eggs remained uncertain. Also, besides determining whehter the presence
of other organics in the waste may interfere with the inactivation of the eggs, it will also be
important to determine how well this combination of acids works under different temperature
regimens and what the minimum effective doses are for caproic and butyric acids when they are
used in combination. Ultimately, the goal will be to test the inactivation of eggs added to the
waste generated in the toilet system to determine if the effects observed in the laboratory will be
reproduced in the toilet itself. However, the finding that these low concentrations of acid have
major ovicidal effects is very promising in that it clearly shows that the levels of fatty acids
generated in the toilet system seem to have the capability to destroy helminths eggs, and by
extension, many of the other pathogens that are likely to be introduced into these toilets in the
developing world.
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