Evaluation of Occurrence, Concentration, and Removal of Pathogenic Parasites and Fecal Coliforms in Three Waste Stabilization Pond Systems in Tanzania

Abstract

In Tanzania, waste stabilization ponds (WSPs) are employed to treat wastewater, and effluents are used for urban agricultural activities. The use of untreated or partially treated wastewater poses risks of disease transmission, including parasitic and bacterial infections, to exposed communities. Little is known about the occurrence, concentration, and removal of parasites and fecal coliform (FC) bacteria in WSPs in Tanzania. This study evaluates the occurrence and concentration of parasites and FCs in wastewater, the efficiency of WSPs in removing parasites and FCs, and the validity of using FCs as an indicator of parasites. This was a cross-sectional study conducted between February and August 2018. Wastewater samples were collected from three WSPs located in the Morogoro, Mwanza, and Iringa regions. APHA methods were used to test physicochemical parameters. The modified Bailenger method and Ziehl–Neelsen stain were used to analyse parasites. Membrane filtration method was used to analyse FCs. Data were analysed using IBM SPSS version 20. Helminth egg removal ranged from 80.8% to 100%. Protozoan (oo)cyst removal ranged from 98.8% to 99.9%. The Mwanza WSP showed the highest FC reduction (3.8 log units (100 mL)−1). Both the parasites and FCs detected in the effluents of assessed WSPs were of higher concentrations than World Health Organization and Tanzania Bureau of Standards limits, except for helminths in the Morogoro WSP and FCs in the Mwanza WSP. FCs were significantly correlated with protozoa (p

Full text

Research Article
Evaluation of Occurrence, Concentration, and Removal of
Pathogenic Parasites and Fecal Coliforms in Three Waste
Stabilization Pond Systems in Tanzania
Abdallah Zacharia ,
1
Wajihu Ahmada,
2
Anne H. Outwater,
3
Billy Ngasala,
1
and Rob Van Deun
4
1
Department of Parasitology and Medical Entomology, Muhimbili University of Health and Allied Sciences,
Dar es Salaam, Tanzania
2
Department of Chemical and Mining Engineering, University of Dar es Salaam, Dar es Salaam, Tanzania
3
Department of Community Health Nursing, Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania
4
Unit Life Sciences and Chemistry, omas More University of Applied Sciences, Geel, Belgium
Correspondence should be addressed to Abdallah Zacharia; naayz@ymail.com
Received 1 May 2019; Revised 2 September 2019; Accepted 24 September 2019; Published 23 October 2019
Academic Editor: Claudio Cameselle
Copyright ©2019 Abdallah Zacharia et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
In Tanzania, waste stabilization ponds (WSPs) are employed to treat wastewater, and effluents are used for urban agricultural
activities. e use of untreated or partially treated wastewater poses risks of disease transmission, including parasitic and bacterial
infections, to exposed communities. Little is known about the occurrence, concentration, and removal of parasites and fecal
coliform (FC) bacteria in WSPs in Tanzania. is study evaluates the occurrence and concentration of parasites and FCs in
wastewater, the efficiency of WSPs in removing parasites and FCs, and the validity of using FCs as an indicator of parasites. is
was a cross-sectional study conducted between February and August 2018. Wastewater samples were collected from three WSPs
located in the Morogoro, Mwanza, and Iringa regions. APHA methods were used to test physicochemical parameters. e
modified Bailenger method and Ziehl–Neelsen stain were used to analyse parasites. Membrane filtration method was used to
analyse FCs. Data were analysed using IBM SPSS version 20. Helminth egg removal ranged from 80.8% to 100%. Protozoan (oo)
cyst removal ranged from 98.8% to 99.9%. e Mwanza WSP showed the highest FC reduction (3.8 log units (100mL)
−1
). Both the
parasites and FCs detected in the effluents of assessed WSPs were of higher concentrations than World Health Organization and
Tanzania Bureau of Standards limits, except for helminths in the Morogoro WSP and FCs in the Mwanza WSP. FCs were
significantly correlated with protozoa (p<0.01) and predicted protozoa occurrence well (p�0.011). ere were correlations
between physicochemical parameters, parasites, and FC bacteria in the WSP systems. Inadequate performance of these systems
may be due to lack of regular maintenance and/or systems operating beyond their capacity. FC indicators were observed to be a
good alternative for protozoa monitoring, but not for helminths. erefore, during wastewater quality monitoring, helminths
should be surveyed independently.
1. Introduction
In Tanzania, waste stabilization ponds (WSPs) have been
used for municipal wastewater treatment for several de-
cades [1]. Effluents from various WSP systems are used for
urban agricultural activities, such as vegetable gardening in
Iringa [2], and vegetable and rice paddy farming in Moshi
[3] and Morogoro [4]. Using WSP effluent for irrigation
was observed to benefit farmers. For example, in a study
conducted in Morogoro, field plots irrigated with effluents
and without artificial fertilizers were reported to produce
more vegetables and rice than tap water irrigated plots,
with and without artificial fertilizers [5]. Moreover, farmers
using WSP effluents in Moshi were reported to grow paddy
twice a year, as compared to farmers practicing rainwater
agriculture [3].
Hindawi
e Scientific World Journal
Volume 2019, Article ID 3415617, 12 pages
https://doi.org/10.1155/2019/3415617

Despite the abovementioned advantages, the use of
untreated or partially treated wastewater poses a risk of
disease transmission to farmers and the community [6].
Apart from the transmission of pathogenic bacteria, another
public health concern about the use of wastewater is the
transmission of parasitic diseases. Wastewater-associated
pathogenic parasites are considered pathogens of great
public health importance due principally to their environ-
mentally persistent transmissive stages, a low infective dose
(1–10 eggs for helminths, and 10–100 (oo)cysts for protozoa
per person) [7, 8], limited or transient acquired immu-
nity, and morbidity, particularly in immunocompromised
hosts [9].
It has been hypothesized that lack of operation and
maintenance of wastewater treatment systems, such as
WSPs, decreases the removal of pathogens (parasites) and
fecal indicators, thus creating uncertainty about the risks
associated with wastewater use [10]. In Tanzania, many WSP
systems were reported to be dilapidated and receive in-
adequate care [11]. Moreover, there is a paucity of in-
formation about the occurrence of parasites in wastewater
and the efficiency of WSPs in removing parasites from
wastewater. Few studies have evaluated these systems based
on the removal of indicator bacteria. In addition, the Na-
tional Environmental Standards Compendium of the Tan-
zania Bureau of Standards (TBS) specifies coliform bacteria
as an indicator of wastewater pathogens, including waste-
water-transmitted pathogenic parasites [12]. However, re-
cent studies have shown variation in indicator bacteria
correlation with parasites in wastewater, questioning its use
in predicting the occurrence and removal of parasites from
wastewater [13, 14].
e aims of this study were to (1) determine the oc-
currence and concentrations of wastewater-related patho-
genic parasites and fecal coliform (FC) indicator bacteria in
three selected Tanzanian WSPs and (2) evaluate their per-
formance in removing pathogenic parasites and FC in-
dicator bacteria. e correlation between physicochemical
parameters, FC indicator bacteria, and pathogenic parasites
(helminths and protozoa) was also examined.
2. Materials and Methods
2.1. Study Design. is was a cross-sectional study con-
ducted between February and August 2018. ree WSP
treatment systems located in the Morogoro, Mwanza, and
Iringa regions in Tanzania were evaluated.
2.2. Study Sites
2.2.1. Mwanza WSPs. e site in Mwanza is located in
Butuja sub-ward, at 2°28′09.37″S 32°54′35.49″E. e system
is owned by the Mwanza Urban Water Supply and Sani-
tation Authority (MWAUWASA) and serves about 3,500
connected households. e design of this system consists of
2 septage lagoons receiving fecal sludge delivered by vacuum
trucks, 3 anaerobic ponds connected in parallel, 4 facultative
ponds in parallel, and 6 maturation ponds in a series
(Figure 1). is treatment system has the capacity of
receiving 5,000 m
3
·d
−1
of wastewater. Effluent from this
system is discharged into Lake Victoria.
2.2.2. Morogoro WSPs. WSPs in Morogoro are owned and
operated by the Morogoro Urban Water Supply and Sani-
tation Authority (MORUWASA) and are located in Mafisa,
Morogoro (at 6°47′07.28″S 37°40′29.34″E). e treatment
system consists of 2 septage lagoons, 1 anaerobic pond, 1
facultative pond, and 4 maturation ponds connected in a
series (Figure 2). e effluent from the treatment system is
drained into a small river which supports a wetland with
about 8 hectares of rice fields and is used further down-
stream for vegetable irrigation.
2.2.3. Iringa WSPs. e Iringa wastewater treatment plant is
located at 7°45′31.47″S 35°40′12.34″E in Don Bosco. e
treatment system is owned by Iringa Urban Water Supply
and Sanitation Authority (IRUWASA). e system has 2
septage lagoons receiving fecal sludge delivered by vacuum
trucks, 2 anaerobic ponds operating in parallel, 1 facultative
pond, and 2 maturation ponds connected in a series (Fig-
ure 3). e effluent is discharged to free surface wetlands and
then to a stream near Hoho Street and used by local residents
for irrigation and brickmaking.
2.3. Wastewater Sample Collection, Storage, and
Transportation. Wastewater samples were collected for
analysis of physicochemical (1 L), parasitological (10 L,
which was allowed to settle for 2 h and 1 L sediment was
taken), and bacteriological (250 ml) parameters at intervals
of one month for 4 or 5 visits. During each visit to Morogoro,
Mwanza, and Iringa WSPs, a total of 4 samples were col-
lected, one from each sampling point, as indicated in
Figures 1–3.
Wastewater samples for chemical oxygen demand
(COD) and total nitrogen (TN) tests were preserved by using
sulphuric acid at pH of 2; those for parasitological analysis
were preserved by using 10% formaldehyde. All samples
were transported to the respective laboratory in a cool ice
box (at a temperature of 4°C) within 6–36 h after collection.
e samples for physicochemical and bacterial tests were
transported to the University of Dar es Salaam water re-
sources laboratory, and those for parasitological tests were
transported to the Muhimbili University of Health and
Allied Sciences parasitology laboratory. All samples were
analysed immediately upon arrival at the laboratory.
On-site measurement of wastewater flow rates, tem-
perature, and pH was conducted during sample collection.
Wastewater flow rates were measured using a stopwatch and
tracer method to detect the velocity of wastewater within a
channel. e velocities of wastewater were multiplied to the
channel cross-sectional area. e procedure was repeated
thrice and the average was computed. Hydraulic retention
time (HRT) of each system during the study period was
estimated based on the calculated flow rate and designed
volume of the system. Wastewater temperature and pH were
2e Scientific World Journal

determined by using a pH meter (Sartorius, Model: PT-15)
by directly inserting the probe into the wastewater.
2.4. Wastewater Analysis for Physicochemical Parameters.
In order to determine the performance of WSP operations,
physicochemical parameters were measured following
“Standard methods for the examination of water and
wastewater” [15]. ese parameters included COD tested
using the closed reflux colorimetric method (method
5220D), total suspended solids (TSS) using method 2540D,
and TN using method 4500-N C.
2.5. Wastewater Analysis for Microbiological Parameters.
Parasites were analysed by using the modified Bailenger
method (MBM) as described in “Analysis of wastewater for
use in agriculture—A laboratory manual of parasitological
and bacteriological techniques” by Ayres and Mara [16].
Since Cryptosporidium oocysts are very small, they can be
difficult to detect in the MBM, so the smears were stained to
increase visualization, in accordance with the modified
Ziehl–Neelsen (ZN) procedure [17]. Quantification of
parasites was done using the following equation:
n�ax
pv,(1)
where n�number of eggs or (oo)cysts L
−1
of wastewater,
a�number of eggs or (oo)cysts counted, x�volume of the
final product (mL), p�volume examined (0.15 mL for MBM
and 0.05 ml for ZN), and v�original sample volume (L).
Membrane filtration technique was used to detect and
quantify FC bacteria in wastewater samples [16]. Membrane
filters with 0.45 μm pore size and 47 mm diameter and
m-Fecal broth selective media were used. e FC bacteria
results were reported in colony-forming units (cfu)
(100 ml)
−1
of wastewater.
2.6. Data Analysis. Statistical analysis was performed using
statistical software IBM SPSS version 20. Percentage re-
ductions of physicochemical parameters and parasites by the
system or a particular stage of the system were calculated by
using the following equation:
p�(µi−µe) × 100
µi,(2)
where p�percentage reduction, μi�mean concentration of
the influent samples, and μe�mean concentration of ef-
fluent samples.
For statistical analysis and log reduction computation,
FC data were transformed to log units. Log reduction of FCs
was calculated by substituting the average influent log
concentration by the average effluent log concentration.
Shapiro–Wilk tests were used to test for normality of the
data. Data were considered normally distributed at p>0.05.
Kruskal–Wallis test was conducted to compare medians of
parasites (protozoa and helminths) and FC concentrations
between the influent and effluent samples of the three
treatment systems. e nonparametric Spearman rank-or-
der correlation was conducted to determine if there were any
relationships between physicochemical parameters and FC,
protozoan, and helminth concentrations in the WSP
wastewater samples. A simple binary logistic regression
model was performed to assess the ability of the FC indicator
to predict the occurrence of helminths and protozoan
parasites in WSP systems.
From
sewerage
system
From trucks
To the river and
vegetable field
SL
MP3 MP4
MP1 MP2
FP
AP
SL
AP
WW
Figure 3: Schematic presentation of Iringa Municipal waste sta-
bilization ponds. Note: SL �septage lagoon; AP �anaerobic pond;
FP �facultative pond; MP �maturation pond; W �constructed
wetland. MP3 and MP4 are newly constructed maturation ponds,
which were not in operation during the study period. Red dots
indicate sampling points.
From
sewerage
system
SL2
MP3 MP4MP1 MP2FP
AP
SL1
From
trucks
To the river
Figure 2: Schematic presentation of Morogoro waste stabilization
ponds. Note: SL �septage lagoon; AP �anaerobic pond; FP �faculta-
tive pond; MP �maturation pond. Red dots indicate sampling points.
From sewerage
system
Trucks
To Lake Victoria
AP2
FP2
MP6
MP4
FP1
MP5
FP3FP4
MP2
MP1
MP3
AP1
AP3
SLSL
Figure 1: Schematic presentation of Mwanza waste stabilization
ponds. Note: SL �septage lagoon; AP �anaerobic pond; FP �faculta-
tive pond; MP �maturation pond. Red dots indicate sampling points.
e Scientific World Journal 3

3. Results and Discussion
A total of 112 wastewater samples were collected from the
three treatment systems and analysed for the presence and
concentration of pathogenic parasites and FC bacteria.
Twelve wastewater samples, four from each treatment plant,
were analysed for physicochemical parameters.
3.1. Physicochemical Characteristics of Influent and Effluent
Wastewater. e average wastewater flow rates were 6264,
5952, and 4416 m
3
·d
−1
, leading to theoretical HRT of 9, 12,
and 5 d in Morogoro, Mwanza, and Iringa WSPs, re-
spectively. e concentrations of COD, TSS, and TN vary
between the influent and effluent samples of the three WSP
treatment systems. e concentrations were higher in the
influent samples than the effluent samples. e concentra-
tion of COD from the three WSP systems ranged from 420 to
815mg/L in the influents and from 200 to 235 mg/L in the
effluents. e concentration of TSS in the influents from the
three WSP systems ranged from 135 to 494 mg/L and from
57 to 154 mg/L in the effluents. e concentration of TN in
the influents from the three WSP systems ranged from 39 to
65 mg/L and from 32 to 52 mg/L in the effluents. e overall
COD, TSS, and TN removal efficiency ranged from 52.4% in
Morogoro to 71.2% in Iringa WSPs; 58% in Morogoro to
69% in Iringa WSPs; and 19% in Morogoro and Iringa WSPs
to 33% in Mwanza WSPs (Table 1). Despite having a short
theoretical HRT and a higher concentration of TSS in the
influents, Iringa WSPs achieved the highest percentage re-
duction of both COD and TSS compared to the other two
systems. Lower reduction of COD and TSS in the other two
systems could be due to algae growth, decreased HRT due to
sludge accumulation, and systems operating beyond their
designed capacity. e TBS requires that wastewater effluent
quality discharge into the environment has less than 60 mg/L
COD, and 100 mg/L TSS [12]. e three WSP systems pro-
duced effluents with concentrations of COD that exceeded the
set quality standard. Similar results for COD effluent con-
centrations were also reported in other WSPs in Tanzania
[3, 18]. e WSPs in Morogoro achieved the required TSS
effluent quality, while those of Mwanza and Iringa had slightly
higher concentrations.
ere were slight variations in pH and temperature
between the influent and effluent samples of the three WSPs;
the exception was the influent temperatures of Iringa WSPs,
which were much lower than those of the other systems
(Table 1). Within the systems, pH was observed to be higher
in the effluent samples than the influent samples. e pH
ranged from 7.0 to 7.5 for the influents and 8.1 to 8.3 for the
effluents. e increase in pH in WSPs may be attributed to
photosynthesis by pond algae, which consume more CO
2
than can be restored by bacterial respiration, resulting in
carbonate and bicarbonate dissociation. e resulting hy-
droxyl group from carbonate and bicarbonate dissociation
accumulated in the water, leading to raised pH [18, 19]. e
effluent pH qualities were within the TBS limit of 6.5 to 8.5
required for safely discharging wastewater into the envi-
ronment [12]. Except for the Morogoro WSPs, wastewater
temperatures were observed to increase as they passed
through the ponds. Similar results were reported in Lugalo
WSPs by Kaseva et al. [18], and it could be due to the effect of
solar heating. e temperature of the effluents met the TBS
discharge limit of 20–35°C [12].
3.2. Parasite Occurrence in Wastewater of the ree Treatment
Systems. Figure 4 presents photographs of some eggs and
(oo)cysts of identified parasite species found in the waste-
water of the three WSP systems. We identified a total of 11
parasite species. e helminthic parasites were nematodes:
Ascaris lumbricoides, hookworm, Trichuris trichiura and
Trichostrongylus spp, trematodes: Fasciola hepatica, and
cestodes: Taenia spp. e protozoan species included flag-
ellates: Giardia lamblia, amoebae: Entamoeba histolytica and
Entamoeba coli, and coccidia: Cryptosporidium spp and
Isospora spp. All these parasite species except for Isospora
spp have been identified in wastewater in other African
countries, including Tanzania’s neighbouring countries
Kenya and Uganda [20].
Forty out of 56 analysed wastewater samples (71%) were
positive for at least one parasite. e percentage of positive
samples was higher than the results obtained in other studies
conducted in Africa. In Burkina Faso, Kpoda et al. [21]
revealed 36% of samples were positive while assessing hel-
minth and protozoa removal by WSP systems. All 14 (100%)
of our raw/influent wastewater samples were positive for
parasites. Similar findings were obtained by Grimason et al.
[22] in Meze, France, whereby Giardia spp cysts were iso-
lated from all 26 raw wastewater samples; by Stott et al. [23]
in Ismailia, Egypt, whereby helminth eggs were detected in
all raw wastewater samples; and by Konat´
e et al. [24] in
Ouagadougou, Burkina Faso, whereby helminth eggs and/or
protozoan cysts were isolated from all raw wastewater
samples.
e occurrence of parasites in effluent samples of the
three treatment systems was 40%, 80%, and 50% in
Morogoro, Mwanza, and Iringa WSPs, respectively. e high
percentage of parasite occurrence in Mwanza WSP effluent
was contributed by a higher occurrence of Trichostrongylus
spp eggs (60%) than in influent samples (20%). A similar
case was reported by Ellis et al. [25] in Cayman Island WSPs,
whereby hookworm eggs were isolated more frequently in
effluents than influents. e higher occurrence may be due
to the fact that Trichostrongylus spp parasite is multi-host
and can infect a wide range of organisms [26]; hence, there is
a possibility of on-site contamination by other infected
animals in contact with wastewater in the systems.
According to Ellis et al. [25], the reasons for more frequent
detection of parasite eggs in effluents than influents could be
due to the effect of thermal stratification in the final mat-
uration pond, wind mixing of the pond content, and/or
temperatures above 30°C (the case of Mwanza WSPs),
leading to buoyancy of parasites by produced gas bubbles.
Hookworm eggs and Entamoeba coli cysts were detected
in 21% and 61% of all samples, and were the most frequently
recovered helminth and protozoan parasites, respectively.
e results are supported by epidemiological studies
4e Scientific World Journal

conducted in several areas of the country, which show that
hookworm eggs and Entamoeba coli cysts have higher
prevalences than other helminths and protozoan parasites,
respectively [27, 28]. e least frequently identified parasites
were protozoa: Isospora spp, and helminths: Taenia spp and
Fasciola hepatica.
3.3. Parasite Mean Concentration and Removal Efficiencies.
In raw/untreated wastewater, the mean helminth concen-
trations were 12, 67, and 49.5 eggs L
−1
in Morogoro,
Mwanza, and Iringa WSPs, respectively. e helminth
concentrations were lower than those detected in other low-
and middle-income countries (70–3000 eggs L
−1
) and higher
than those recovered in high-income countries (1–9 eggs
L
−1
) [29]. e mean protozoan concentrations were 358, 231,
and 669.5 (oo)cysts L
−1
in Morogoro, Mwanza, and Iringa
WSPs, respectively. Protozoan concentrations were
comparable to those detected in other African countries,
such as reported in Tunisia (250–590 cysts L
−1
) [30], in
Nigeria (190 cysts L
−1
) [31], and in Burkina Faso (147 and
111 cysts/L) [32].
e combined data of raw/untreated wastewater samples
from all treatment systems revealed the overall helminth
mean concentration of 42.9 eggs L
−1
and protozoan mean
concentration of 422.3 (oo)cysts L
−1
. e contribution of
each helminth species in the overall concentrations was
hookworm 32.2%, Ascaris lumbricoides 31.9%, Trichuris
trichiura 15.6%, Trichostrongylus spp 14.0%, and Fasciola
spp 6.3%. Protozoan parasites were Entamoeba coli 76%,
Entamoeba histolytica/dispar 14%, Cryptosporidium spp
7.8%, Giardia spp 2%, and Isospora spp 0.2%.
Entamoeba coli cysts were more frequently identified at
higher concentrations than all other parasite species detected
in all analysed wastewater samples. is protozoan parasite
is a nonpathogenic species of Entamoeba that frequently
(a) (b) (c)
(d) (e) (f )
Figure 4: Images of some identified parasites in wastewater samples collected from either of the three WSP systems: (a) Trichuris trichiura
egg, (b) Ascaris lumbricoides egg, (c) hookworm egg, (d) Fasciola spp egg, (e) Entamoeba coli cyst, and (f ) Cryptosporidium spp oocyst
(photos by first author).
Table 1: Physicochemical characteristics of influents and effluents of wastewater of the three WSP systems.
Parameter Morogoro WSPs Mwanza WSPs Iringa WSPs
Inf Eff % removal Inf Eff % removal Inf Eff % removal
COD (mg/L) 420 200 52.4 575 215 63 815 235 71.2
TSS (mg/L) 135 57 58 311 105 66 494 154 69
TN (mg/L) 39 32 19 57 38 33 65 52 19
pH 7.5 8.3 7.4 8.2 7.0 8.1
Temperature (°C) 29 29 28 30 23 28
Note. COD �chemical oxygen demand; TSS �total suspended solids; TN �total nitrogen; Inf �influents; Eff �effluents.
e Scientific World Journal 5

exists as a commensal parasite in the human gastrointestinal
tract. is parasite was reported because Entamoeba coli can
be confused during microscopic diagnosis with the patho-
genic Entamoeba histolytica. Moreover, when this parasite is
detected in domestic wastewater, it is an indication of un-
hygienic conditions present in the sewered community,
whereby people have consumed fecally contaminated
products with the possibility of pathogenic organisms being
consumed at the same time [33].
Specific concentrations of parasite species in wastewater
may be attributed to their prevalence in the served com-
munities. Parasite species with higher concentrations in
wastewater were observed to have a higher prevalence in
epidemiological studies. For example, Mazigo et al. [27] in
the Mwanza region reported the following prevalences of
parasites among hospitalized patients: hookworm 25.2%,
Ascaris lumbricoides 1.6% and Trichuris trichiura 0.79% for
helminths, and Entamoeba histolytica/dispar 13.6% and
Giardia lamblia 6.9% for protozoa. Another study con-
ducted by Siza et al. [34] to determine the prevalence of soil-
transmitted helminths among school children in the four
regions of Lake Zone showed the following results: hook-
worm 14.6%, Ascaris lumbricoides 3.2%, and Trichuris tri-
chiura 0.3%. Moreover, a study by Venkatajothi [28] in the
Dar es Salaam region to determine the incidence of intestinal
protozoa among school children showed higher prevalence
of Entamoeba coli (56% boys, 66% girls), followed by Ent-
amoeba histolytica (31% boys, 26% girls) and Giardia
lamblia (13% boys and 8% girls).
Parasite removal at each treatment step of the Morogoro,
Mwanza, and Iringa WSPs is presented in Tables 2–4, re-
spectively. In all treatment systems, higher concentrations of
parasites were removed in anaerobic ponds, except for
protozoa in Morogoro WSPs. e anaerobic pond in
Morogoro WSPs was observed to have accumulated sludge
that extended above the surface of the pond as islands
(Figure 5). e sludge accumulation may have contributed
to the inadequate removal of protozoa. e excessive ac-
cumulation of sludge affects pond hydraulics, creating short-
circuiting that may carry parasite eggs, cysts, or oocysts
through to the outlet, or resuspend eggs and (oo)cysts that
have been deposited in the pond sediments [10]. Helminths
were reduced by 93.3%, 96.7%, and 75.8% in the anaerobic
ponds of Morogoro, Mwanza, and Iringa, respectively.
Protozoa decreased from 368 to 303.2, 231 to 12, and 669.5 to
55.3 (oo)cysts L
−1
in the anaerobic ponds of Morogoro,
Mwanza, and Iringa WSPs, respectively. e higher per-
formance of anaerobic ponds for removal of parasites was
also reported by Konat´
e et al. [24] in Burkina Faso. However,
these ponds are prone to sludge accumulation as they receive
raw wastewater, thus reducing their efficiency as observed in
Morogoro WSPs.
Despite the observed decrease in parasite concentrations
in the anaerobic ponds, some parasite species showed
fluctuations in their mean concentrations as they passed
through each WSP treatment stage. In all WSP systems, all
parasite species decreased as they passed through the an-
aerobic ponds, except for Giardia lamblia cysts in Morogoro
WSPs (Table 2). Trichostrongylus spp eggs and
Cryptosporidium spp oocysts in the maturation ponds of
Mwanza and Iringa WSPs, respectively, were found to have
higher concentrations in the effluents than the influents
(Tables 3 and 4). Most hookworm eggs were cleared in the
anaerobic pond in Iringa WSPs, but were observed also in
the effluent of the final maturation pond. In the same system,
Taenia eggs which were not detected in the raw wastewater,
or effluent of anaerobic and facultative ponds, were isolated
in the effluent of the final maturation pond (Table 4). e
main reason for the observed fluctuations in parasite mean
concentrations may be due to the reasons explained in
Section 3.2 paragraph 3.
Effluents of the final maturation ponds in Mwanza and
Iringa WSPs contained both helminth and protozoan par-
asites. e effluent of the final maturation pond in Morogoro
WSPs contained protozoan parasites and no helminth eggs
(100% diminution). e mean helminth concentrations in
effluents was 2.5 and 7.5 eggs L
−1
, which was equivalent to
96.2% and 85% diminution in Mwanza and Iringa WSPs,
respectively. e mean helminth concentrations of effluents
in Mwanza and Iringa WSPs corresponded to an average of
5.8 eggs L
−1
based on data collected by Zacharia et al. [20].
However, studies conducted in Kenya, Tunisia, and Burkina
Faso reported complete removal of helminths in WSP
systems [32, 35–37]. Except for Morogoro WSPs, the other
two systems did not meet the standard set by the World
Health Organization (WHO) of ≤1 egg L
−1
for safe use of
wastewater effluents in agriculture [38].
Protozoan concentrations in effluents were 4.3, 0.3, and 1
(oo)cysts L
−1
in Morogoro, Mwanza, and Iringa WSPs,
respectively. ese concentrations resulted from 98.8% (2
log units), 99.8% (3 log units), and 99.9% (3 log units) re-
ductions of the initial protozoan concentrations of raw/
untreated wastewater in Morogoro, Mwanza, and Iringa
WSPs, respectively. According to the WHO guideline,
protozoa reduction by wastewater treatment systems of 2–3
log units combined with other health protection measures
can achieve the protozoa health-based target for wastewater
reuse in restricted, unrestricted, and localized (drip) irri-
gations [38]. erefore, the target for protozoa pathogen
prevention for people using effluents of the three WSP
systems will be achieved if the attained protozoa removal
efficiencies of the three treatment systems are sustained and
proper health education is given to the exposed
communities.
Hookworm eggs, Trichostrongylus spp eggs, Cryptospo-
ridium spp oocysts, and Entamoeba coli cysts were observed to
persist throughout all treatment stages of at least one WSP
system. Sedimentation is said to be an effective removal
mechanism for helminth eggs and protozoan (oo)cysts in WSP
systems [39]. e process is greatly influenced by the system’s
hydraulic retention time (HRT). However, one of the factors
that may affect HRT includes sludge accumulation [10]. In
addition to that, other design factors may also affect the
parasites’ residence time distribution in the stabilization ponds,
and therefore their sedimentation rate. ese factors include
pond depth, the length-to-width ratio, the configurations of
inlets and outlets, the speed and direction of the wind, strat-
ification caused by diurnal shifts in the temperature at the
6e Scientific World Journal

surface of the pond, and the presence or absence of baffles [39].
us, regular system maintenance (disludging) and monitoring
is very important to enhance WSP parasite removal efficiency.
3.4. Fecal Coliform Bacteria Concentrations and Removal from
Wastewater. To monitor all pathogenic organisms that
could be present in a particular wastewater treatment system
or its effluent is very difficult as it is costly and time-
consuming. Indicator organisms are always employed to
reflect the pathogen level in a wastewater of a particular
treatment system. FCs are bacteria found in the intestines of
warm-blooded animals and are used to indicate the presence
of human pathogens in wastewater [19]. Compared to other
indicators, FC bacteria are the organisms most commonly
used to monitor the removal of pathogens from wastewater
treatment plants [40]. Table 5 presents concentrations of FCs
in influents and effluents of each treatment stage of the three
Table 3: Parasite concentrations and removal in the Mwanza WSP system.
Parasite group Parasite species
Treatment stage
Raw wastewater Anaerobic ponds Facultative
ponds
Maturation
ponds
Helminth species concentration (eggs/L)
Ascaris lumbricoides 19 0 0 0
Hookworm 32 1.8 0.8 0.2
Trichuris trichiura 0 0 0 0
Trichostrongylus spp 8 0.4 12.2 2.3
Fasciola spp 8 0 0 0
Taenia spp 0 0 0 0
Total helminth concentration (eggs/L) 67 2.2 13 2.5
Helminth removal at each stage (%) 96.7 No reduction 80.8
Helminth removal from raw
wastewater (%) 96.7 80.6 96.2
Protozoa species concentrations
((oo)cysts/L)
Giardia lamblia 9 1.6 0.2 0
Entamoeba histolytica/dispar 80 3.2 0 0
Entamoeba coli 127 5.6 1 0
Cryptosporidium spp 12 1.6 0.6 0.3
Isospora spp 3 0 0 0
Total protozoa concentration
((oo)cysts/L) 231 12 1.8 0.3
Protozoa removal at each stage (%) 94.8 85 83.3
Protozoa removal from raw
wastewater (%) 94.8 99.2 99.8
Table 2: Parasite concentrations and removal in the Morogoro WSP system.
Parasite group Parasite species
Treatment stage
Raw
wastewater
Anaerobic
pond
Facultative
pond
Maturation
ponds
Helminth species concentrations (egg/L)
Ascaris lumbricoides 12 0.8 0 0
Hookworm 0 0 0 0
Trichuris trichiura 0 0 0 0
Trichostrongylus spp 0 0 0 0
Fasciola spp 0 0 0 0
Taenia spp 0 0 0 0
Total helminth concentration (egg/L) 12 0.8 0 0
Helminth removal at each stage (%) 93.3 100 —
Helminth removal from raw
wastewater (%) 93.3 100 100
Protozoa species concentrations
((oo)cysts/L)
Giardia lamblia 22 32.4 5.4 0
Entamoeba histolytica/
dispar 26 15.6 0.3 0
Entamoeba coli 248 194.8 47.9 0.8
Cryptosporidium spp 72 60.4 23.7 3.5
Isospora spp 0 0 0 0
Total protozoa concentration ((oo)cysts/L) 368 303.2 77.3 4.3
Protozoa removal at each stage (%) 17.6 74.5 94.4
Protozoa removal from raw wastewater (%) 17.6 79 98.8
e Scientific World Journal 7

systems. e influent of Iringa WSPs had the highest FC
concentration, followed by the Morogoro WSP system.
Observed variations in influent FC concentrations were not
statistically significant, as indicated by the Kruskal–Wallis
test (H(2) �3.01, p�0.22). e same test showed that there
was significant difference in FC concentrations between
effluents of the three WSP systems (H(2) �9.10, p�0.01),
with a mean rank of 9.60 for Morogoro WSPs, 3.00 for
Mwanza WSPs, and 10.50 for Iringa WSPs. e pairwise
comparison test indicated that the difference in FC con-
centrations between effluents of the three WSP systems was
significant between Mwanza WSPs and Morogoro WSPs
(p�0.013), and Mwanza WSPs and Iringa WSPs
(p�0.008). ere was no significant difference between FC
concentrations in effluents of Morogoro WSPs and Iringa
WSPs (p�0.748). e effluent mean FC concentrations
from the three systems were higher than that found by Kihila
et al. [3] in Moshi WSPs (1.00 ×10
3
cfu (100 mL)
−1
), and less
than that obtained by Kaseva et al. [18] in Lugalo WSPs
(8.00 ×10
6
cfu (100 mL)
−1
).
Generally, Mwanza WSPs achieved more FC removal
efficiency (3.81 log units (100 mL)
−1
) than Morogoro WSPs
(2.57 log units (100 mL)
−1
) or Iringa WSPs (1.99 log units
(100 mL)
−1
). e reason for higher FC removal efficiency in
Mwanza WSP systems may be due to the large number of
ponds connected in series and increased wastewater tem-
perature. Moreover, Mwanza WSPs have an estimated HRT
of 12 days, while Morogoro and Iringa WSPs have the
theoretical HRT of 9 days and 5 days, respectively. In WSP
systems, the presence of more ponds connected in a series,
increase in wastewater temperature, and longer HRT in-
creased reduction of bacteria including FCs [39]. Except for
the effluent of Mwanza WSPs, the other systems did not
meet the TBS limit for wastewater discharge of 1.00 ×10
4
cfu
(100 mL)
−1
[12]. According to the WHO guideline, for a
wastewater treatment system with FC removal efficiency
Table 4: Parasite concentrations and removal in the Iringa WSP system.
Parasite group Parasite species
Treatment stage
Raw wastewater Anaerobic
ponds
Facultative
pond
Maturation
ponds
Helminth species concentrations (eggs/L)
Ascaris lumbricoides 10 3 0 0
Hookworm 9.5 0 0 7.5
Trichuris trichiura 20 4.5 0 0
Trichostrongylus spp 10 4.5 0 0
Fasciola spp 0 0 0 0
Taenia spp 0 0 0 0.25
Total helminth concentration (eggs/L) 49.5 12 0 7.75
Helminth removal at each stage (%) 75.8 100 No reduction
Helminth removal from raw wastewater (%) 75.8 100 85
Protozoa species concentrations ((oo)cysts/L)
Giardia lamblia 0 0 0 0
Entamoeba coli 582.5 49.3 7.5 0.75
Entamoeba histolytica/
dispar 72 6 0 0
Cryptosporidium spp 15 0 2 0.25
Isospora spp 0 0 0 0
Total protozoa concentrations ((oo)cysts/L) 669.5 55.3 9.5 1
Protozoa removal at each stage (%) 91.7 82.8 89.5
Protozoa removal from raw wastewater (%) 91.7 98.6 99.9
Figure 5: Anaerobic pond of Morogoro WSPs showing accumulated sludge (photos taken by first author during sample collection).
8e Scientific World Journal

between 2 and 4 log units or effluent FC (E.coli) concen-
tration of 1.00 ×10
3
–1.00 ×10
5
cfu (100 ml)
−1
, its effluent
may be applied in various types of agricultural irrigation, as
illustrated in Table 6 [38]. In this case, the effluent from
Mwanza WSPs can be used for unrestricted or restricted
irrigation. However, other health protection measures, such
as normal household washing of salads and vegetables with
clean and safe water prior to consumption, should be in-
corporated to achieve the set health-based targets.
3.5. Relationship between Physicochemical Parameters and FC
Bacteria and Parasites in WSP Wastewater.Physicochemical
parameters are among the primary factors involved in the
removal and/or destruction of pathogens (parasites and
bacteria) in wastewater [41]. To assess the relationship of
various physicochemical parameters on the concentrations
of FCs, helminths, and protozoa in wastewater as they pass
through the three WSPs, a Spearman rho correlation test was
conducted. COD, TSS, and TN showed positive correlation
with FCs, helminths, and protozoa in the wastewater as they
passed through the treatment systems. e correlation was
statistically significant between COD and the parasitic or-
ganisms (helminths and protozoa). TSS and TN showed
statistically significant relationships with helminths only
(Table 7). is indicates that physicochemical parameters
(COD, TSS, and TN), parasites (helminths and protozoa),
and FC bacteria decrease as wastewater passes through WSP
systems. Similar relationships were observed by other re-
searchers. Wastewater TSS and nitrogen contents were
shown to positively correlate with helminths and protozoa in
WSPs in Spain by Reinoso et al. [41]. Moreover, Jimenez and
Chavez [42] and Tyagi et al. [43] reported positive corre-
lations between TSS and helminth eggs, and TSS and FCs in
WSP wastewater. It has been stated that pathogen particles
can effectively be removed by sedimentation if they are
attached to larger particles, in which case their elimination
from the wastewater correlates with particle removal [44].
at could be the reason for the positive correlation between
TSS and indicator bacteria and parasites in WSP wastewater.
Wastewater pH and temperature correlated negatively
with FC, helminth, and protozoan concentrations in WSP
wastewater. e correlation was statistically significant be-
tween pH and parasitic organisms (helminths and protozoa)
only (Table 7). In wastewater, a negative correlation between
temperature and indicator bacteria was also reported by Liu
et al. [19], while temperature and protozoa were reported by
Molleda et al. [45]. Effects of temperature and pH on survival
and removal of indicator bacteria in WSP have been studied
by Liu et al. [19] and Pearson et al. [46], whereby increased
temperature and pH were associated with decreased fecal
indicator (FC) concentration in WSP systems. e results
indicate that protozoan parasites are more affected by an
increase in pH and temperature than helminths. An ex-
perimental study conducted by Mills [47] in Spain showed
that an increase in pH and temperature resulted in an in-
creased disinfection of Cryptosporidium spp. According to
the author, it is probably as a result of an increase in the
permeability of oocyst walls, allowing highly reactive mol-
ecules such as hydrogen peroxide and ammonia to penetrate
inside the oocysts, increasing their disinfectant effect. e
effect could apply to other protozoan species.
3.6. Relationship between FCs, Protozoa, and Helminths in
WSP Wastewater. e validity of the fecal indicator is de-
termined using the strong correlation of its presence in
wastewater and the presence of human fecal contaminants
(fecal pathogens). To determine the correlation between FCs
and parasites (helminths and protozoa) in wastewater of the
three WSPs, a Spearman rho correlation test was conducted
(Table 8). e test results indicated a strong positive sig-
nificant correlation between FC and protozoa concentra-
tions (r�0.604, p<0.01). Similar results were also obtained
by Levantesi et al. [48] in Europe, Payment and Locas [49] in
Canada, and Reinoso et al. [41] in Spain. Harwood et al. [50]
in the United States did not find a correlation between the
two parameters. e same test indicated that there was no
correlation between FCs and helminths (r�0.051). is
result is in accordance with the result reported by Levantesi
et al. [48] in Europe, whereby no correlation between the
bacteria indicator and helminth eggs was observed. Protozoa
and helminths were observed to have moderate positive
significant correlations (r�0.30 and p�0.04). e positive
correlations between protozoa and the other two types of
organisms (FCs and helminths) indicate that there are
factors affecting the presence of both protozoa and FCs, and
factors affecting the presence of protozoa and helminths in
WSP wastewater. Both FCs and protozoa in WSPs are af-
fected by factors such as temperature, pH, and sunlight
exposure, whereby both FC (bacteria) and protozoa removal
increase at increased temperatures, pH, and sunlight ex-
posure [39]. Sedimentation is the main mechanism for
pathogen removal in WSPs. e rate of pathogen sedi-
mentation in WSPs varies based on their settling velocity.
Protozoan (oo)cysts and helminth eggs have higher settling
Table 5: FC concentration means of raw wastewater and effluents of anaerobic, facultative, and maturation ponds of the three WSP
treatment systems.
Type of wastewater Morogoro WSP Mwanza WSP Iringa WSP
Cfu/100 ml Log units/100 ml Cfu/100 ml Log units/100 ml Cfu/100 ml Log units/100 ml
Raw wastewater 2.07 ×10
8
7.95 1.01 ×10
8
7.43 3.12 ×10
8
8.06
Anaerobic pond(s) 1.14 ×10
8
7.53 4.42 ×10
6
6.52 8.15 ×10
7
7.13
Facultative pond(s) 3.21 ×10
6
6.39 2.73 ×10
5
5.21 2.61 ×10
7
6.56
Maturation pond(s) 5.81 ×10
5
5.38 4.92 ×10
3
3.62 5.73 ×10
6
6.07
Note. WSP �waste stabilization pond; cfu �colony-forming unit.
e Scientific World Journal 9

velocities of 0.026–0.13 m/d and 5–13 m/d, respectively, than
bacteria (0.012 m/d) [39]. e higher settling velocities of
protozoa and helminths lead to higher removal of these
organisms in WSPs than bacteria.
A simple binary logistic regression test was conducted to
assess the ability to use FC indicator bacteria to predict the
presence of helminth eggs and protozoan (oo)cysts in WSP
wastewater. Results indicate that FCs are a good predictor of
protozoa occurrence in WSP wastewater (presence/absence)
[Chi-square �6.516, df�1, and p�0.011]. e predictor
(FC) explains 15.5% (Nagelkerke R
2
) of the variability of
protozoa occurrence, which is significant at the 5% level
[Wald �5.708, p�0.017 (<0.05)]. e odds ratio is 1.733
(95% CI 1.104–2.720). e model correctly predicted 29.4%
of cases where there was an absence of protozoa, and 92.3%
of cases where there was the presence of protozoa, giving an
overall percentage correct prediction rate of 73.2%. ese
results are in accordance with results obtained by Payment
and Locas [49] and Harwood et al. [50, whereby bacteria
indicator/FCs were observed to be a better predictor of
protozoa occurrence in wastewater. e same test showed
that FCs are not a good predictor of helminth presence or
absence in WSP wastewater [Chi-square �0.360, df�1, and
p�0.548]. Table 9 shows the number of correctly and
incorrectly predicted samples for protozoa occurrence in
wastewater of the three WSP systems.
4. Conclusion
e studied WSP reduced pathogenic parasites and FC
concentrations from wastewater. However, they did not
meet either one or both parasitological and microbiological
standards as stated in the WHO guideline and TBS regu-
lations. e Mwanza WSP system achieved the WHO
Table 8: Spearman’s rho correlation test results for FCs, helminths, and protozoa.
Microorganism Number of samples Mean Median FCs Protozoa Helminths
rprprp
FCs (cfu/100 ml) 56 6.8 ×10
7
4.8 ×10
6
— — sc s nc n
Protozoa ((oo)cysts/L) 56 141.6 6.0 0.60 0.000 — — Mc s
Helminths (eggs/L) 56 13.7 0 0.05 0.709 0.30 0.049 — —
Note. cfu �colony-forming unit; r�Spearman’s correlation coefficient; p�is significant level; sc �strong correlation; s �significant; nc �no correlation;
n�not significant; wc �moderate correlation.
Table 6: Bacterial quality requirement for wastewater effluent reuse in agricultural irrigation.
Type of
irrigation
WTS required FC (E.coli)
reduction (log units)
Effluent FC (E.coli)
concentration during
monitoring (cfu/100 ml)
Notes
Unrestricted
4≤10
3
Root crops
3≤10
4
Leaf crops
2≤10
5
Drip irrigation of high-growing crops
Restricted
4≤10
3
Drip irrigation of low-growing crops
3≤10
4
Labour-intensive agriculture
2≤10
5
Highly mechanized agriculture
Note. Table modified from the WHO 2006 guideline. WTS �wastewater treatment system.
Table 7: Spearman’s rho correlation test results for physicochemical parameters and microorganisms (including helminths).
Parameter Helminths (eggs/L) Protozoa ((oo)cysts/L) FCs (cfu/100 ml)
n r prprp
COD (mg/L) 12 0.71 0.009 0.78 0.003 0.53 0.079
TSS (mg/L) 12 0.59 0.041 0.48 0.118 0.48 0.137
TN (mg/L) 12 0.74 0.005 0.26 0.421 0.26 0.420
pH 12 −0.594 0.042 −0.71 0.010 −0.57 0.055
Temperature (°C) 12 -0.367 0.241 −0.57 0.054 −0.50 0.094
Note. COD �chemical oxygen demand; TSS �total suspended solids; TN �total nitrogen; n�number of datasets; r�Spearman’s correlation coefficient. pis
significance level.
Table 9: Protozoa prediction results by FC indicator bacteria in
WSP wastewater.
Observed
Predicted
Protozoa
occurrence
Negative Positive Percentage
correct
Protozoa
occurrence
Negative 5 12 29.4
Positive 3 36 92.3
Overall
percentage 73.2
e cut-off value is 0.500.
10 e Scientific World Journal

standard for FC bacteria effluent concentration for safe reuse
of wastewater in both restricted and unrestricted irrigation.
e effluent FC concentration was also in accordance with
the TBS standard limit for wastewater discharge into the
environment. But the system did not meet the helminth
concentration of ≤1 egg/L, as described in the WHO
guideline. e Morogoro WSP system meets the helminth
standard set by the WHO, but did not meet the WHO and
TBS effluent FC concentration standards. e Iringa Mu-
nicipal WSP system did not achieve either parasitological or
microbiological quality as described by both the WHO
guideline and TBS regulations. In order to reduce the risk of
parasitological and bacterial disease transmission to the
exposed community, measures such as proper operation and
maintenance of WSPs must be taken. ese measures will
enhance WSP efficiency and ensure that the parasitological
and microbial quality of effluents is achieved and sustained.
Some efforts were observed in the Iringa Municipal WSP,
whereby two new maturation ponds were under construc-
tion during the study period. Perhaps the addition of these
two ponds will improve the parasitological and microbio-
logical quality of the effluent of this system to a level that
adheres to the WHO and TBS requirements.
FCs correlated with protozoan (oo)cysts, but not with
helminth eggs. Binary logistic regression results indicated
that FCs are a good predictor of protozoa occurrence in
wastewater but not of helminths. us, FCs are not a reliable
indicator of helminths in wastewater. erefore, during
wastewater monitoring for reuse or discharge into sensitive
receiving water bodies, such as lakes, rivers, and streams,
helminth quality must be surveyed independently.
Data Availability
e data used to support the findings of this study are
available from the corresponding author upon request.
Conflicts of Interest
e authors declare that there are no conflicts of interests.
Acknowledgments
is work was supported by the South Initiatives project
“Pathogen removal from wastewater using sustainable
treatment wetlands,” funded by VLIRUOS, Brussels, Bel-
gium. e authors wish to thank VLIRUOS, Brussels, Bel-
gium, for their financial support. e authors appreciate the
support given by Morogoro, Mwanza, and Iringa Water and
Sanitation Authorities.
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