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Applied and Environmental Microbiology, July 2001, p. 3322-3324, Vol. 67, No. 7
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.7.3322-3324.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Effect of High-Rate Algal Ponds on Viability of
Cryptosporidium parvum Oocysts
S.
Araki,1,*
S.
Martín-Gomez,2
E.
Bécares,1
E.
De
Luis-Calabuig,1 and
F.
Rojo-Vazquez2
Área de Ecología, Instituto del
Medio Ambiente,1 and Departamento de
Patología Animal (Sanidad Animal),2
Universidad de León, 24071 León, Spain
Received 8 December 2000/Accepted 2 May 2001
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ABSTRACT |
The physicochemical conditions of high-rate algal ponds were
responsible for a more than 97% reduction in the infectivity of
Cryptosporidium parvum oocysts in neonatal mice. The use
of semipermeable bags of cellulose showed that pH, ammonia, and/or light seems to be a major factor for the inactivation of oocysts in
wastewater, supporting the importance of alga-based systems for safer
reuse of treated wastewater.
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TEXT |
Present conventional
wastewater treatment is very expensive for application in rural areas
and, moreover, does not guarantee the inactivation of
Cryptosporidium parvum oocysts from sewage (3, 12, 16,
18). The high-rate algal pond (HRAP) is a low-cost wastewater
treatment system designed to achieve two goals: secondary wastewater
treatment and algal biomass production. The HRAP is a combination of
intensified oxidation ponds and an algal reactor. Algae supply the
oxygen demand for bacterial degradation of organic matter, and bacteria
excrete mineral compounds that provide the algae with nutrition. HRAPs
have proved effective in removing organic matter (13) and
in reducing bacterial contamination (8) and the number of
nematode eggs (1), but no data are available on their role
in removing Cryptosporidium oocysts, a subject of special
interest when dealing with rural wastewater. We will focus this study
on the effect of the HRAP physicochemical conditions on the viability
of Cryptosporidium oocysts as measured with a neonatal mouse
infectivity model.
HRAP pilot plants.
Two identical pilot plants fed with urban
wastewater were used for this study (ponds A and B) (Fig.
1). The average physicochemical characteristics of the ponds during the study period are shown in Table
1.
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FIG. 1.
Plant view of the HRAP pilot system: depth, 30 cm;
length, 235 cm; area, 1.54 m2; width, 70 cm; flow
rate, 30 cm/s. HRTs were 3 days for pond A and 10 days for pond B.
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C. parvum oocysts.
Oocysts were obtained from
the feces of an experimentally infected lamb, purified according to the
procedures of Arrowood and Sterling (2), and stored at
4°C in a 2.5% (wt/vol) aqueous potassium dichromate solution until use.
Treatment of oocysts in HRAP.
Regenerated cellulose
semipermeable bags with 14,000-Da porosity were used for the
experiment. These bags allowed oocysts to be in contact with the small
ions present in the water while reducing the effect of other mortality
factors such as bacterial and/or fungal contamination or
predation. The bags were filled with 50 ml of sterile water and
108 oocysts. Two bags were placed in each pond
for 3 (pond A) and 10 (pond B) days. Two other bags without oocysts
were also placed in the ponds to test the osmotic interchange through
the semipermeable membrane, and two bags with oocysts were stored at
4°C in sterile water and used as a control. At the end of the
hydraulic retention time (HRT), the oocysts were washed and centrifuged
with distilled water. The sediment was resuspended and adjusted to
obtain 5 × 105 oocysts/25 µl.
Bioassay for viability.
Two hundred fifty-seven suckling mice
from 21 individual litters (7 to 16 mice/litter) of NMRI mice were
divided in four groups of 3- to 4-day-old mice, day 0 postinfection
(p.i.). Mice in groups A (98 mice) and B (79 mice) were inoculated
intragastrically with 5 × 105 oocysts
recovered from pond A and B bags, respectively. Control mice (group C,
80 mice) also received the same infection dose but with oocysts
preserved under control conditions. The other two litters (group D) (19 mice) were used as uninoculated mice to check that no accidental
infection occurred in the litters during the experiment.
Half of the mice in each group were sacrificed by inhalation of ether 6 days p.i., and the other half were sacrificed 10 days
p.i. to verify
that oocysts were not able to recover their infectivity
after longer
times in the
intestine.
The whole intestinal tract of each mouse was removed, placed in an
individual glass tube with 1 ml of phosphate-buffered saline,
homogenized, and diluted 1/5 in a solution of 0.16% green malachite
and 1% sodium dodecyl sulfate in water. The number of oocysts
per
milliliter was counted at 400× using a Neubauer
slide.
Statistics.
Chi-square tests were used to analyze differences
in percentages of infectivity between mouse groups, and Mann-Whitney U
tests were used to compare physicochemical values and infection
intensities between ponds.
The percentage of infectivity was calculated as the ratio of the number
of infected mice to that of inoculated mice (see Table
2). The number
of oocysts per milliliter of intestinal tract
was used to evaluate the
infection intensity, and the percentage
of infection reduction was
calculated as the difference between
the infection intensity of group C
and that of group A or B, divided
by the infection intensity of group
C.
The infectivity of the oocysts from the HRAP was much lower than that
from the control (Table
2), this
difference being highly
significant (
P < 0.001).
Infectivity in group C (control) was
100% after 6 and 10 days p.i.,
whereas in group A (3 days HRT)
it was 40 and 5% for 6 and 10 days
p.i., respectively, and only
20.5 and 12.5% in group B, respectively
(10 days HRT). The intensity
of infection significantly declined
(
P < 0.001) in mice inoculated
with oocysts exposed to
the HRAP conditions (groups A and B).
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TABLE 2.
Infectivity in neonatal mice inoculated with 5 × 105 C. parvum oocysts after treatment in an
HRAP at an HRT of 3 days (group A) and 10 days (group B)
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Intestines from uninoculated mice (group D) did not contain any
C. parvum oocysts, confirming that no accidental infections
occurred during the experiment. Observed differences between pond
A and
pond B infectivities were not statistically significant
(
P > 0.05). This was in accordance with the lack of
differences
in the measured physicochemical conditions between ponds
(Table
1). It means that the different HRTs assayed did not produce
significant differences in the water characteristics of the ponds,
and
therefore, physicochemical characteristics inside the semipermeable
bags were not different between pond A and pond B. Only conductivity
presented significantly lower values inside the bags (Table
3).
The semipermeable bags used in the experiments excluded predation,
bacterial or fungal infection, and the effect of large
molecules as
potential factors for oocyst inactivation. The lower
conductivity
values for the bags suggest that only small molecules
were able to
diffuse through the membrane, leaving temperature,
pH, small ions
(ammonia, phosphates, etc.), and light as the main
factors potentially
responsible for the significant reduction
in oocyst
infectivity.
With respect to temperature, the oocysts of
C. parvum can
retain their viability and infectivity after freezing (
6)
or
at temperatures above 65°C (
11). In our case, the
water temperature
was very consistent during the experiment (Table
1),
suggesting
that temperature was not an important factor for the
inactivation
of oocysts in the
HRAP.
Using oocysts in semipermeable containers, Robertson et al.
(
15) showed that both high and low pH have a significant
impact
on oocyst viability. The pH in the HRAP was always higher than
8, reaching 9.5 at noon on sunny days as a consequence of the
algal
activity. The pH clearly influences the hydrophobic properties
of the
oocysts, allowing them to autoflocculate or to increase
their adhesion
to other particles (
5).
Light also plays a significant role in the inactivation of
Cryptosporidium oocysts, but its efficiency depends on the
light
type, intensity, and length of exposure. Exposure to UV light
was
responsible for 90 to 99% of oocyst inactivation (
4,
11,
14). White light also seems to have a significant effect on
Cryptosporidium infectivity, achieving 90% reduction in
48 h (
10),
and sunlight clearly inactivates oocysts
in comparison with dark
conditions (
9). In our experiment,
the semipermeable bags were
dangling in the HRAP reactor and
sagging as a consequence of the
water agitation. This means that
bags were erratically moving
from the surface to several centimeters
under the surface during
the study period and that therefore light
could have affected
oocyst
viability.
Another potential factor influencing oocyst inactivation is ammonia
(
7). Due to the high-pH conditions of the HRAP, the
ammonium-ammonia equilibrium tends toward the gas form. This dissolved
ammonia could also have a significant role in inactivating
oocysts.
Results show that inactivation of oocysts by HRAP is higher than that
by conventional wastewater treatment systems. All oocysts
present in
the effluent of activated sludge plants are still active
despite
removal of more than 80% of the influent oocysts (
18).
Using semipermeable bags, Robertson et al. (
17) showed
that,
after 1 week of exposure to different conventional treatments
(activated sludge and trickling filters), none of the environments
tested had a deleterious effect on oocyst viability except the
anoxic
sludge storage
tank.
As with nematode eggs (
1), this work shows that the
physicochemical conditions commonly present in the HRAP seem to be
responsible for the inactivation of more than 97% of the
C. parvum oocysts. The absence of differences between the two
retention
times tested suggests that oocysts lost their infectivity in
a
very short time after contact with the water environment (probably
less than 3 days). Our results strongly support other general
data on
the important role of alga-based systems as a very valuable
treatment
process in the reuse of treated
wastewater.
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FOOTNOTES |
*
Corresponding author. Mailing address: Instituto del
Medio Ambiente, C/ La Serna s/n, León University, 24071 León, Spain. Phone: 34 987 291568. Fax: 34 987 291501. E-mail:
degma{at}unileon.es.
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Applied and Environmental Microbiology, July 2001, p. 3322-3324, Vol. 67, No. 7
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.7.3322-3324.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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