See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/7211805

Comparison of Techniques for the Detection of Helminth Ova in Drinking

Water and Wastewater

Article in Water Environment Research · March 2006

DOI: 10.2175/106143005X89571 · Source: PubMed

CITATIONS READS

44 415

3 authors, including:

Catalina Maya Blanca Jiménez

Universidad Nacional Autónoma de México Universidad Nacional Autónoma de México
50 PUBLICATIONS 857 CITATIONS 330 PUBLICATIONS 8,492 CITATIONS

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

Removal of Nitrogen View project

Solid wastes View project

All content following this page was uploaded by Blanca Jiménez on 08 December 2020.

The user has requested enhancement of the downloaded file.

 Comparison of Techniques for the
Detection of Helminth Ova
in Drinking Water and Wastewater
C. Maya, B. Jimenez, J. Schwartzbrod
ABSTRACT: Many countries use wastewater for irrigation. The World
Health Organization established, as reuse guidelines, a maximum value of 1
helminth ovum/L for irrigation. Various techniques for enumerating helminth
ova in water have been published. To determine the most adequate method
for Mexico, four techniques were compared: the U.S. Environmental
Protection Agency (U.S. EPA), membrane-filter, Leeds I, and Faust. Two
types of water were used: drinking water and municipal wastewater effluent.
Sensitivity, discrimination coefficients, precision, recovery efficiency, and
cost were determined. In addition, several unseeded wastewater samples were
analyzed. For drinking water, U.S. EPA and the membrane-filter techniques
demonstrated comparable results; however, when wastewater was used, the
membrane technique showed some deficiencies. Because the U.S. EPA
technique may be used for samples with both high and low solids content,
allows for the recovery of helminth ova with different specific gravities, and
has the lowest total cost, it was selected as the best technique. Water Environ.
Res., 78, 118 (2006).
KEYWORDS: analytical techniques, drinking water, helminth ova,
irrigation standards, wastewater reuse.
doi:10.2175/106143005X89571
Introduction
In Mexico, wastewater is an important alternative source for
irrigation in arid or semiarid regions, as in other parts of the world
(Kandiah, 1993). Wastewater is preferred by farmers because it
contains organic matter, nitrogen, and phosphorus, which have fer-
tilizing value and increase crop yields. However, the unrestricted use
of wastewater for irrigation presents a serious health risk through the
dissemination of pathogens, particularly helminth ova. It is estimated
that helminths present the greatest danger in the reuse of wastewater
in agriculture (Cifuentes et al., 1992). To minimize this risk, Mexico
has adopted a national standard for the use of reclaimed wastewater
in irrigation. This standard establishes a fecal coliform limit of less
than 1000 MPN/100 mL in all irrigation water, a maximum of
1 helminth ovum/L for the irrigation of crops eaten uncooked, and
5 ova/L for the irrigation of crops normally cooked before consump-
tion (Official Mexican Legislative, 1997). Unfortunately, there is
still no widely accepted international method for evaluating these
parasites in wastewater. With the aim of selecting the most appro-
priate technique, a comparison study was undertaken, based on in-
ternationally published findings. This comparison was performed
between four techniques: (1) U.S. EPA (Yanko, 1987), (2) mem-
brane filter (Galván et al., 1996), (3) Leeds I (Ayres, 1989), and (4)
Faust techniques (Faust et al., 1939).
All four techniques compared have two general steps. The first
step is to separate, recover, and concentrate the helminth ova from
the sample sediment. In the second step, the helminth ova are
118
identified and counted visually, with the aid of a microscope. The
techniques are categorized in the following two ways:
(1) Based on the means used to separate, recover, and concentrate
the helminth ova; and
(2) Whether the microscopic analysis is of all, or only part, of the
concentrated volume containing the ova.
Another important difference among the techniques is the initial
volume of sample used (Table 1). The U.S. EPA technique uses a 5-L
sample, combining flotation, biphasic, and sedimentation steps to
separate the ova from the water. The microscope readings or ova
counts are performed on the entire mass recovered. The membrane-
filter technique, originally developed for detecting protozoa, uses
flotation and an indirect concentration method of a 1-L sample. The
ova are retained on a cellulose acetate membrane (10-lm pore,
25-mm diameter), which is later dehydrated and treated with ethanol
and is rendered transparent by treatment with glycerol. The ova count
is made on the residual obtained from the whole sample. The Leeds I
technique is based on several successive centrifugation and flotation
steps. The sample size is 1 L for wastewater with high solids content
and 40 L for wastewater with low solids content (filtered using
a micro-wynd device to recover the ova [APHA et al., 1995]). The
final reading is made on only part of the flotation, which is carried out
in a zinc sulphate (ZnSO4) saturated solution (specific gravity 1.18).
The ova are recovered by taking the upper part (positive meniscus) of
the solution from six 15-mL tubes with a slide cover glass (4 per tube),
which is then placed over a slide and quantified under the microscope.
The individual results are added to give the final concentration. The
Faust technique is similar to the Leeds I, and also uses samples sizes
of 1 and 40 L, but involves more flotation, centrifugation, and sedi-
mentation steps. Readings are taken on five aliquots of 50 lL each of
the final sediment, and then the results are averaged and extrapolated
to give the final concentration. Some Mexican laboratories have
modified the Leeds I and Faust techniques by filtering the 40-L
samples through a Micro-wynd system to concentrate such samples,
only when dealing with treated wastewater. For the purpose of this
research, the same modified procedure was used.
Material and Methods
The comparison was performed on synthetic samples. The first was
prepared with drinking water (DW) (well water used for human
consumption without chlorination) and the second with municipal
wastewater treated with a physicochemical process (advanced-
primary-treatment process [APT] [Jiménez et al., 1999]), previously
filtered through a sterilized sieve with a pore size of 20 lm (to
Water Environment Research, Volume 78, Number 2
 Maya et al.

Table 1—Methodological differences among the four techniques.

Means used to separate, recover, Volume of sample used

Technique Sample volume (L) and concentrate helminth ova for microscopic analysis

U.S. EPA 5 Flotation (1.3)a diphasic sedimentation Total

Membrane filter 1 Flotation (1.2)a filtration Total
Leeds I 1 for water with high TSS Centrifugation flotation (1.18)a Only part of the final volume
40 for water with low TSSb of flotation
Faust 1 for water with high TSS Flotation (1.18)a centrifugation 5 aliquots of 50 lL
40 for water with low TSSb sedimentation

a
Zinc sulphate is used for the flotation in all four techniques, varying the specific gravity of the solution.
b
Some Mexican laboratories have modified the Leeds I and Faust techniques by filtering the 40-L samples through a Micro-wynd system
to concentrate such samples, only when dealing with treated wastewater. For the purpose of this research, the same modified procedure
was used.

simulate a wastewater effluent free from helminth ova) (TW). This
water was examined several times to ensure that helminth ova were
absent. A known quantity of Ascaris suum ova was then added to
the water to produce the ‘‘seeded’’ samples. Such samples are called
synthetic samples. Ascaris suum were obtained by dissecting
the uterus of an adult female worm, obtained from a pig in
a slaughterhouse from Mexico city. A short (2 to 3 mm) tract of
uterus, proximal to the vulva (containing fertilized ova), was dis-
sected and the ova suspended in saline solution.
The samples for each technique were prepared using 1, 5, and 40 L
(see Table 1), and, after counting the ova under the microscope in
a Sedgwick Rafter chamber, with a dimension of 50 3 20 mm, a depth
of 1 mm, and a capacity of 1000 lL (Thomas Scientific, Swedesboro,
New Jersey), a known number of Ascaris suum ova was added to each
sample. Because Ascaris ova are larger than most helminths in
wastewater (50 to 80 lm, specific gravity 1.13, Figure 1a), ova
from other genera with different sizes and specific gravities were also
used. A known quantity of Trichuris suis ova (50 to 54 lm in total
length and 26 to 34 lm in width, specific gravity 1.13, Figure 1b)
were obtained also by dissecting the uterus of an adult female worm,
obtained from a pig in a slaughterhouse from Mexico city. In addition,
Taenia solium ova (30 to 40 lm, specific gravity range 1.23 to 1.3,
Figure 1c) were obtained from the mature proglottids from medical
human feces (extracted and suspended in saline solution, in a way
similar to that of Ascaris suum). Ova from these genera were also
added to some samples (type TW) to test the ability and capacity of
each technique to recover and measure these particular ova. Ascaris
ova were selected as the focus of the study because they are the most
common in the world; Figure 2 shows their predominance according
to data from Ellis et al. (1993), Hays (1977), Ogoshi et al. (2000),
Schwartzbrod et al. (1989), and Stott et al. (1997).
Each test was performed using 10 replicate samples. The sen-
sitivity was determined using samples containing 1, 3, and 5 Ascaris
ova/L (AO/L) for each method. Precision was determined by
preparing and analyzing samples containing 5 and 40 AO/L. The
total number of samples analyzed by each technique is shown in
Table 2. Considering the four techniques, the total number of samples
processed was 400 for water used for human consumption (DW) and
400 for water from the effluent of a physicochemical treatment plant,
previously filtered and sterilized (TW). As mentioned earlier, the
recovery of Trichuris suis and Taenia solium ova was used to
evaluate the ability of each technique to measure these smaller and
different specific gravity helminth ova, which are more difficult to
detect (David and Lindquist, 1982).
In addition to the seeded samples and to evaluate the detection
efficiency, several unseeded wastewater samples (WW) were anal-
yzed using each technique: untreated wastewater (10 repetitions) and
effluent from a physicochemical treatment plant, with and without

Figure 1—Typical (a) Ascaris, (b) Trichuris, and (c) Taenia

ova. Images from Treatment and Reuse Group at the
Institute of Engineering, National Autonomous University Figure 2—Average worldwide distribution of helminth ova
of Mexico. in raw wastewater.

February 2006 119

Maya et al.

Table 2—Number of seeded samples analyzed by each Table 3—Sensitivity results obtained for samples with
technique. 1, 3, and 5 Ascaris ova per liter.

Number of Ascaris ova added in each sample Ascaris ova added (AO/L)

1 3 5 25 30 35 40 100 110 120 1 3 5

Number of samples per technique Ascaris ova recovered (AO/L)*

Drinking water 10 10 10 10 10 10 10 10 10 10 Technique Average SD (6) Average SD (6) Average SD (6)
Treated
wastewater 10 10 10 10 10 10 10 10 10 10 U.S. EPA
Total 200 DW 0.6 0.52 2.4 0.52 3.6 0.70
TW 0.7 0.48 2.4 0.52 3.7 0.67
Average 0.7 0.50 2.4 0.52 3.7 0.69
filtration through sand (Jiménez et al., 1999). In this case, for the same
Membrane filter
samples, the detection efficiencies were compared at low suspended-
DW 0.9 0.32 2.6 0.52 4.1 0.88
solids contents (16 and 21 mg/L total suspended solids [TSS]) and
TW 0.3 0.48 1.9 0.57 2.9 0.74
high suspended-solids contents (81 mg/L TSS). The cost and the Average 0.6 0.40 2.2 0.55 3.5 0.81
amount of training required to perform each technique were evalu-
Leeds I
ated to complement the experiment. As a final step, an overall anal-
ysis was carried out, which considered all the results. DW 0 NA 0 NA 0.2 0.42
TW 0 NA 0 NA 0.2 0.42
Average 0 NA 0 NA 0.2 0.42
Results and Discussion
Sensitivity. Sensitivity is defined here as the minimum quantity Faust
of ova per liter that a given technique can detect. The average DW 0 NA 0 NA 0.3 0.48
concentration and the standard deviation measured by each tech- TW 0 NA 0 NA 0.3 0.48
nique in samples containing 1, 3, and 5 Ascaris ova per liter are Average 0 NA 0.0 NA 0.3 0.48
shown in Table 3. In the case of synthetic samples (DW), the greatest
* Average of ten replicates.
sensitivity was obtained with the membrane-filter technique for all 3
AO/L: Ascaris ova per liter.
concentrations, while, for synthetic samples (TW), the U.S. EPA SD: Standard deviation.
technique showed greater recovery. This may be attributed to the DW: Drinking water.
solids concentration in the water, which affects the membrane-filter TW: Treated wastewater.
technique by breaking or exceeding the filtering capacity. By aver- NA: Not applicable.
aging the results for both samples (DW and TW), a slight advantage
may be given to the U.S. EPA technique, with respect to the sen- Where
sitivity. This technique showed an average of 0.7 AO /L 6 0.50 in DC 5 discrimination coefficient;
the samples with an initial concentration of 1 AO /L (average of ten X1 5 mean Ascaris ova with a difference
2.5 (5 AO) or
5
replicates), followed by the membrane-filter technique, which (10 AO);
detected 0.6 AO /L 6 0.40. Xo 5 mean Ascaris ova with a difference , 2.5 (5 AO) or , 5
In fact, the U.S. EPA and membrane filter were the only two (10 AO);
techniques capable of detecting ova in the 1 and 3 AO /L samples, SDx 5 Standard deviation of all the samples;
whereas the other two techniques only detected ova in the 5 AO /L n1 5 Number of samples considered for X1;
samples. The Leeds I and Faust techniques take readings on sub- n0 5 Number of samples considered for X0; and
samples rather than on the whole sample. This could explain their n 5 n1 1 n0.
poor performance in detecting small numbers of Ascaris, even though
both tests use larger volumes of samples (40 L compared with 1 and The discrimination coefficients for differences of 5 and 10 AO are
5 L, for the membrane-filter and U.S. EPA techniques, respectively). summarized in Tables 4 and 5.
Discrimination Coefficient. The discrimination coefficient or In both series, the U.S. EPA and membrane-filter techniques
point-biserial correlation allows to evaluate the ability of each demonstrated the best discrimination coefficients for samples of
technique to distinguish between similar Ascaris ova concentrations drinking water (0.83 and 0.88, respectively, average of the cor-
(differing by 5 or 10 AO). Samples with initial concentrations of 25, responding values, Tables 4 and 5); however, when wastewater was
30 and 35, and 100, 110 and 120 AO were analyzed. First, the values used, the membrane technique showed some deficiencies because of
obtained for 25 and 35 Ascaris ova, as well as those for 100 and 120 the solids content (0.74 compared to 0.84 for the U.S. EPA
ova were subtracted from the mean measured value for 30 and 110 technique). In the case of the Faust technique, sometimes, when five
Ascaris ova, respectively. Then, the resulting values were grouped in 50 lL aliquots from the sediment were analyzed and extrapolated to
two types: those with a difference smaller than 50% (, 2.5 or , 5 give the final concentration, the result was higher than the real value.
AO), and those with a difference equal to or higher than 50% (
2.5 or Thus, the discrimination coefficients are approximately 0.55 to 0.60.

5 AO). Finally, the discrimination coefficient was calculated with To determine the ability of each technique to differentiate intervals of
the following formula (Ebel and Frisbie, 1986; Henrysson, 1971): 5 and 10 AO, the following discrimination classes were set: (1) 30%
rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi bad; (2) .30% , 60% poor; (3)
60% , 80% good; and (4)
80%
X1  X0 n1 n0 optimum. For all the samples and average, the U.S. EPA technique
DC ¼ 3 ð1Þ
SDx nðn  1Þ had the optimum capacity to distinguish between close values.

120 Water Environment Research, Volume 78, Number 2

 Maya et al.

Table 4—Discrimination coefficients in samples containing Table 6—Recovery efficiency and precision from seeded
25, 30, and 35 Ascaris ova. samples with 5 and 40 Ascaris ova.

Discrimination Ascaris ova content in samples

Techniques X1 X0 Sx n1n0 DC class
5 AO/L 40 AO/L
U.S. EPA
Ascaris ova recovered (AO/L)
DW 3.95 1.74 1.23 75 0.80 Optimum
TW 3.97 1.10 1.41 64 0.84 Optimum Technique Average SD (6) Average SD (6)
Membrane filter
U.S. EPA
DW 3.99 1.43 1.48 96 0.87 Optimum
TW 5.45 1.43 3.00 99 0.68 Good DW 3.6 0.70 33.1 2.64
TW 3.7 0.67 32.6 2.79
Leeds I Average 3.7 0.69 32.9 2.72
DW 3.44 1.45 1.18 96 0.55 Poor
Membrane filter
TW 3.33 1.60 1.08 84 0.70 Good
DW 4.1 0.88 36.7 2.06
Faust TW 2.9 0.74 26.1 6.23
DW 5.38 1.18 2.95 99 0.72 Good Average 3.5 0.81 31.4 4.15
TW 4.63 1.23 2.51 96 0.68 Good
Leeds I
DW: Drinking water. DW 0.2 0.42 11.4 2.27
TW: Treated wastewater. TW 0.2 0.42 10.6 1.71
Average 0.2 0.42 11.0 1.99
Faust
Precision. Precision is defined here as the measure of closeness
achieved in results from multiple analyses of a given sample. With DW 0.9 1.45 30.5 8.17
TW 1.2 2.57 26.2 4.34
both the 5 and 40 AO/L samples, the Leeds I method had the lowest
Average 1.1 2.01 28.4 6.26
standard deviations of 6 0.42 and 6 1.99 AO/L, respectively; how-
ever, the recovery was the lowest. Similarly, the highest recovery * Average of ten replicates.
of Ascaris ova in (DW) samples was obtained with the membrane- SD: Standard deviation.
filter technique, but the advantage disappeared when the solids con- AO: Ascaris ova.
centrations increased (TW). The U.S. EPA technique also recovered DW: Drinking water.
a considerable number of ova. For the seeded samples containing TW: Treated wastewater.
5 AO/L, the concentration detected by the U.S. EPA technique was
3.7 6 0.69, followed by the membrane-filter with 3.5 6 0.81, the deviation, as a percentage of the mean values, was generally greater
Leeds I with 0.2 6 0.42, and the Faust with 1.1 6 2.01 ova. For (Table 6).
samples of 40 AO/L, again, the U.S. EPA technique performed best Recovery Efficiency. A summary of the total percent-recovery
with 32.9 6 2.72 AO/L, followed by the membrane-filter with 31.4 6 efficiency of the samples considered in determining sensitivity,
4.15, the Leeds with 11.0 6 1.99, and the Faust with 28.4 6 6.26 ova. discrimination coefficients, and precision, for both types of synthetic
These results also indicate that, at lower concentrations, the standard samples (DW and TW), is shown in Figure 3. The U.S. EPA technique
was the most efficient, with an average recovery of 82.3% 6 6.18,
while the membrane filter recovered 75.1% 6 6.71, the Leeds I only
Table 5—Discrimination coefficients in samples containing
24% 6 16.4, and the Faust 51.2% 6 34.14. On the other hand, the
100, 110, and 120 Ascaris ova.
added Trichuris ova were only recovered with the U.S. EPA and the
Discrimination
membrane-filter techniques, but the added Taenia ova were only
Techniques X1 X0 Sx n1n0 DC class recovered with the U.S. EPA technique, which may be explained by
the use of a denser solution (1.3) that reduces the losses of ova with
U.S. EPA higher specific gravities, such as Taenia.
DW 7.67 3.05 2.73 96 0.85 Optimum
TW 8.71 3.17 3.10 84 0.84 Optimum
Membrane filter
DW 8.80 2.90 2.96 75 0.89 Optimum
TW 5.66 2.55 1.76 75 0.79 Good
Leeds I
DW 5.80 2.84 1.70 75 0.77 Good
TW 5.90 2.63 1.95 91 0.81 Optimum
Faust
DW 16.24 3.1 14.10 64 0.38 Poor
TW 11.84 1.67 9.22 84 0.52 Poor

DW: Drinking water. Figure 3—Percent recovery of Ascaris ova in seeded

TW: Treated wastewater. samples for each technique.

February 2006 121

Maya et al.

Table 7—Comparison of detection efficiencies for activities). The results are presented in Table 8. With the exception
wastewater samples with different TSS concentrations.* of the U.S. EPA technique, costs are greater if the sample has a low
concentration of TSS because of the type of equipment and material
Sample Ova detected TSS Technique required. The number of samples that can be processed simulta-
type (HO/L) (mg/L) used neously is also reduced because additional steps of filtration (micro-
wynd and membranes filters) are required. The technique with the
Wastewater 26 6 2.1 81 U.S. EPA
Wastewater 13 6 3.1 81 Membrane filter
highest costs, for samples with both high and low TSS
Wastewater 11 6 2.5 81 Leeds I modified concentrations, is the Leeds I (U.S. $51.57/ sample), while the
Wastewater 17 6 3.5 81 Faust modified most cost-effective method is the U.S. EPA technique, at U.S.
Physicochemical $39.40/ sample.
treated effluent 1.6 6 0.4 21 U.S. EPA Personnel Expertise. The required period of training to
Physicochemical properly perform each task of the four techniques (from sampling
treated effluent 1 6 0.6 21 Membrane filter to identification) was defined based on the experience of personnel
Physicochemical from the Institute of Engineering, National Autonomous University
treated effluent 0.3 6 0.6 21 Leeds I modified of Mexico. For all four techniques, an analyst with a background
Physicochemical
in biology or parasitology is required. Further training is also ne-
treated effluent 0.3 6 0.6 21 Faust modified
Filtered effluent 0.2 6 0.2 16 U.S. EPA
cessary to identify the most common helminth ova species (Ascaris,
Filtered effluent 0 16 Membrane filter Trichuris, and Taenia) and to distinguish them from algae or detritus.
Filtered effluent 0 16 Leeds I modified The training period, regardless of technique, would be, on average,
Filtered effluent 0 16 Faust modified three to five weeks.
The degree of training, in weeks, required for each task in the four
* A concentration of 81 mg/L of TSS corresponds to a raw techniques is shown in Table 9. The Leeds and Faust techniques
wastewater, while samples with 16 and 21 mg/L of TSS represent require some training in the operation of the micro-wynd system (for
treated wastewater. effluent). The use of the filter for concentrating samples requires skill
and expertise to wash and rinse the filter fibers. Similarly, the mem-
Detection Efficiencies in Wastewater Samples. The average brane-filter technique requires dexterity to avoid tearing the
concentration and standard deviation obtained in unseeded waste- membrane while applying pressure to the filter. In the flotation
water samples (WW) with different TSS concentrations, by each of process of the Leeds I, the analyst must have nimble fingers to avoid
the techniques, is shown in Table 7. Samples were taken from Mexico spilling the concentrated sample.
City raw wastewater, an effluent from an APT plant using aluminum In accordance with the scores given for the level of training in the
sulphate, and a sand-filtered effluent (1.2 mm sand) from the same different processes, it was found that the U.S. EPA technique re-
APT plant (Jiménez et al., 1999). quires the lowest degree of training, in weeks (3.5). The membrane
The U.S. EPA technique detected the greatest number of helminth filter is second in complexity of training required (4) and, finally, the
ova (Ascaris, Trichuris, and Taenia) per liter in the case of raw Leeds I and the Faust Leeds I have the same score (4.5).
wastewater, APT effluent, and filtered effluent. Global Evaluation. The overall rating of the four techniques in
Costs. The costs of the different techniques for determining each of the four areas discussed above is shown in Table 10.
total ova concentration (i.e., without considering the viability Classifying from best to worst, the technique with the lowest rating
analysis for the helminth ova) were determined. The costs were is the most adequate. The final results of the ratings showed that the
calculated based on market prices available in Mexico in 2003 and U.S. EPA technique presented the lowest score (12), followed by the
then transformed to U.S. dollars (a conversion rate of eleven pesos membrane filter (24), Leeds I (31), and Faust (32).
per dollar was used in 2004). The factors evaluated were the
materials (chemicals and reagents used, depreciation of the Conclusions
equipment resulting from use, and energy consumption) and Because very different techniques are currently used to enumerate
human resources (salaries of a specialist, technician, and general helminth ova in water, it is suggested that efforts be increased to
assistant for washing the material, helping in sampling, and similar establish a standardized technique to identify and enumerate hel-

Table 8—Comparison of unit costs (per sample) of four different techniques for the detection of helminth ova*.

Technique

Leeds I Faust Membrane filter U.S. EPA

Type of sample High TSS Low TSS High TSS Low TSS High TSS Low TSS High and low TSS

Unit cost (U.S. $) Per sample Per sample Per sample Per sample Per sample Per sample Per sample
Material 17.75 23.55 14.15 21.28 61.14 16.35 16.76
Human resources 15.35 16.49 10.78 10.78 10.78 10.78 9.0
Person hours 16.36 13.64 16.36 13.64 14.55 12.73 13.64
Total 49.46 53.68 41.29 43.50 41.47 39.86 39.40
Average 51.57 42.40 40.67 39.40

* A conversion rate of eleven pesos per dollar was used in 2004. The cost does not include viability determination.

122 Water Environment Research, Volume 78, Number 2

 Maya et al.

minth ova. This is of great importance, especially for regulating the Table 9—Degree of training, in weeks, required for each
reuse of wastewater in irrigation. Based on the data obtained in this task in the four techniques.
study, it was recommended and selected by the Mexican government
to adopt the U.S. EPA technique as the Mexican standard technique Technique
for the identification and quantification of helminth ova in wastewater
U.S. EPA Membrane filter Leeds I Faust
and reclaimed water for the following reasons:
Tasks IN EF IN EF IN EF IN EF
 Although the U.S. EPA and membrane-filter techniques showed
similar sensitivity and precision when type DW samples were Sampling 0.5 0.5 0.5 0.5 0.5 0.5 0.5 1
treated (with greater values than the Faust and Leeds I), the U.S. Concentration 0.5 0.5 1 1 0.5 1 0.5 1
EPA technique also had the best numbers for type TW samples Flotation 0.5 0.5 0.5 0.5 1 1 1 0.5
(with higher solids contents). Finally, the U.S. EPA technique Identification 2 2 2 2 2 2 2 2
demonstrated a greater recovery over the rest of the procedures. Total 3.5 3.5 4 4 4.0 4.5 4.0 4.5
From a practical point of view, the U.S. EPA technique may be Total (weeks) 3.5 4 4.5 4.5
used for samples with both high and low solids content.
 By using a solution with a specific gravity of 1.3, the U.S. EPA IN: Influent (raw wastewater).
EF: Effluent (from advanced primary treatment)
technique allowed the recovery of helminth ova with specific
* Training time, in weeks.
gravities that range from 1.13 (e.g., Ascaris) to 1.27 (e.g.,
Taenia). For those cases where solutions with lower specific
gravities are used, some ova may be lost during the flotation  In the case of the Faust and Leeds I techniques, it was observed
step. that the advantage of having a 40-L sample is lost when only
 For the U.S. EPA technique, the total costs involved were the one aliquot is used for the reading step. Also, the reading
lowest, and it was estimated to require the shortest time for procedure requires a lot of time. In the case of the membrane-
training personnel. In addition, a larger number of samples can filter technique, handling of the filter cake reduced the final
be analyzed at the same time. recovery rate.
 Even though the four techniques have a similar performance in  In the case of the Faust technique, there were some cases when
the concentration and flotation steps, differences are encoun- the final sediment was averaged and extrapolated to give the
tered in the filtration and managing of the samples and, final concentration, and the result was higher than the real value.
especially, in the reading procedure.
 Although the membrane-filter technique showed a high Acknowledgments
sensitivity, this was obtained only for samples with low solids Authors. C. Maya is a research assistant at the National
contents (almost of drinking quality); thus, it was not adequate Autonomous University of Mexico. B. Jimenez is a senior re-
for raw wastewater or even reclaimed water. searcher at the Institute of Engineering in the National Autonomous

Table 10—Global evaluation of techniques for the detection of helminth ova.

Analysis Unit Parameter U.S. EPA Membrane Leeds I Faust

Sensitivity % Recovery Average 75 68 1.3 2.0

efficiency Rating 1 2 4 3
Discrimination % Interval of 5 AO/L at low 82 78 63 70
between discrimination concentrations
close values % Rating 1 2 3 4
10 AO/L at high 85 84 79 45
concentrations
Rating 1 2 3 4
Precision 6 Standard 5 AO/L 0.69 0.81 0.42 2.01
deviation Rating 2 3 1 4
40 AO/L 2.72 4.15 1.99 6.26
Rating 2 3 1 4
Recovery % Efficiency 82 75 24 51
Rating 1 2 4 3
Costs US$ High TSS 39.40 41.47 49.46 41.29
Rating 1 3 4 2
Low TSS 39.40 39.86 53.68 43.50
Rating 1 2 4 3
Personnel Required level High TSS 1.5 2 2 1.8
of training Rating 1 3 3 2
Low TSS 1.5 2 2.5 2.3
Rating 1 2 4 3
Total score 12 24 31 32

February 2006 123

Maya et al.
University of Mexico, and J. Schwartzbrod is a professor at the
Faculté de Pharmacie in Poincaré University, Nancy, France.
Correspondence should be addressed to Catalina Maya, National
Autonomous University of Mexico, P.O. Box 70-472, 04510.
Mexico, D.F., Mexico; e-mail cmayar@iingen.unam.mx.
Submitted for publication September 17, 2003; revised manuscript
submitted July 26, 2004; accepted for publication September 27,
2004.
The deadline to submit Discussions of this paper is May 15, 2006.
References
American Public Health Association; American Water Works Association;
Water Pollution Control Federation (1995) Standard Methods for the
Examination of Water and Wastewater, 18th ed.; Washington, D.C.
Ayres, R. M. (1989) A Practical Guide for the Enumeration of Intestinal
Helminths in Raw Wastewater and Effluent from Waste Stabilization
Ponds. Leeds University Department of Civil Engineering. Tropical
Public Health Engineering. A Contribution to the International
Drinking Water Supply and Sanitation Decade 1981–1990.
Cifuentes, E.; Blumenthal, U.; Ruiz-Palacios, G.; Bennett, S. (1992) Health
Impact Evaluation of Wastewater Use in Mexico. Public Health Rev.,
19, 243–250.
David, E. D.; Lindquist, W. D. (1982) Determination of the Specific Gravity
of Certain Helminth Eggs Using Sucrose Density Gradient Centrifu-
gation. J. Parasitol. 68 (5), 916–919.
Ebel, R. L.; Frisbie, D. A. (1986) Essentials of Education Measurement.
Prentice Hall: Englewood Cliffs, New Jersey.
Ellis, K. V.; Rodrigues, P. C. C.; Gomez, C. L. (1993) Parasite Ova and
Cysts in Waste Stabilization Ponds. Water Res., 27 (9), 1455–1460.
Faust, E. C.; Sawitz, W.; Tobie, J.; Odem, V.; Peres, C. (1939) Comparative
Efficiency of Various Techniques for the Diagnosis of Protozoa and
Helminths in Faeces. J. Parasitology, 25, 241–262.
124
View publication stats
Galván, M.; Gutierrez, A. L.; De Victorica, J. (1996) Efficiency of Rapid
Quantitative Procedures Adapted for the Analysis of Helminth Eggs in
Irrigation Water. Proceedings of the IAWQ Symposium on Health
Related Water Microbiology. Mallorca, Spain, October 6–10; In-
ternational Association on Water Quality: London, p. 91–96.
Hays, B. D. (1977) Potential for Parasitic Disease Transmission with Land
Application of Sewage Plant Effluents and Sludge. Water Res., 11,
583–595.
Henrysson, S. (1971) Gathering, Analysing, and Using Data on Test Items.
In Educational Measurement, R. L. Thorndike (Ed.); American
Council on Education: Washington, D.C.
Kandiah, A. (1993) Water Resources Development and Management
Service Land and Water Development Division. Food and Agriculture
Organization of the United Nations: Rome, Italy, p. 1–14.
Jiménez, B.; Chávez, A.; Hernández, C. (1999) Alternative Treatment for
Wastewater Destined for Agriculture Use. Water Sci. Technol., 40 (4-5),
355–362.
Official Mexican Legislative (1997) Norms which Establish the Maximum
Levels of Contaminants in Wastewater Discharges, NOM-001-ECOL-
1996. Diario Oficial de la Federación, January 6, p. 67–81.
Ogoshi, M.; Suzuki, Y.; Asano, T. (2000) Non-Potable Urban Water
Reuse—A Case of Japanese Water Recycling. Water, 21, 7–30.
Stott, R.; Jenkins, T.; Shabana, M.; May, E. (1997) A Survey of the
Microbial Quality of Wastewaters in Ismailia, Egypt and the
Implications for Wastewater Reuse. Water Sci. Technol., 35 (11-12),
211–217.
Schwartzbrod, J.; Stien, J. L.; Bouhoum, K.; Baleux, B. (1989) Impact of
Wastewater Treatment on Helminth Eggs. Water Sci. Technol., 21 (3),
295–297.
Yanko, W. A. (1987) Occurrence of Pathogens in Distribution and
Marketing Municipal Sludges, EPA-600/1-87-014. National Technical
Information Service: Springfield, Virginia.
Water Environment Research, Volume 78, Number 2