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Removal and fate of Cryptosporidium parvum,Clostridium
perfringens and small-sized centric diatoms (Stephanodiscus
hantzschii) in slow sand filters
Wim A.M. Hijnen
a,
, Yolanda J. Dullemont
b
, Jack F. Schijven
c
, Anke J. Hanzens-Brouwer
a
,
Martine Rosielle
d
, Gertjan Medema
a
a
Kiwa Water Research, P.O. Box 1072, 3430 BB Nieuwegein, The Netherlands
b
Waternet, Provincialeweg 21, 1108 AA Amsterdam, The Netherlands
c
National Institute of Public Health and the Environment, RIVM, P.O. Box 1, 3720 BA Bilthoven, The Netherlands
d
Het Waterlaboratorium, P.O. Box 734, 2003 RS Haarlem, The Netherlands
article info
Article history:
Received 4 October 2006
Received in revised form
19 January 2007
Accepted 24 January 2007
Available online 2 April 2007
Keywords:
Cryptosporidium
Slow sand filtration
Removal
Delayed transport
Surrogates
abstract
The decimal elimination capacity (DEC) of slow sand filtration (SSF) for Cryptosporidium
parvum was assessed to enable quantitative microbial risk analysis of a drinking water
production plant. A mature pilot plant filter of 2.56 m
2
was loaded with C. parvum oocysts
and two other persistent organisms as potential surrogates; spores of Clostridium perfringens
(SCP) and the small-sized (4–7 mm) centric diatom (SSCD) Stephanodiscus hantzschii. Highly
persistent micro-organisms that are retained in slow sand filters are expected to
accumulate and eventually break through the filter bed. To investigate this phenomenon,
a dosing period of 100 days was applied with an extended filtrate monitoring period of 150
days using large-volume sampling. Based on the breakthrough curves the DEC of the filter
bed for oocysts was high and calculated to be 4.7 log. During the extended filtrate
monitoring period the spatial distribution of the retained organisms in the filter bed was
determined. These data showed little risk of accumulation of oocysts in mature filters most
likely due to predation by zooplankton. The DEC for the two surrogates, SCP and SSCD, was
3.6 and 1.8 log, respectively.
On basis of differences in transport behaviour, but mainly because of the high
persistence compared to the persistence of oocysts, it was concluded that both spores of
sulphite-reducing clostridia (incl. SCP) and SSCD are unsuited for use as surrogates for
oocyst removal by slow sand filters. Further research is necessary to elucidate the role of
predation in Cryptosporidium removal and the fate of consumed oocysts.
&2007 Elsevier Ltd. All rights reserved.
1. Introduction
One of the pathogens of major concern for the drinking water
industry is Cryptosporidium, a persistent pathogenic protozoan
and cause of a number of outbreaks of waterborne diarrhoea
documented in the USA and the UK (Richardson et al., 1991;
MacKenzie et al., 1994). Studies have demonstrated the failure
of regular water quality monitoring with Escherichia coli to
indicate the absence of this pathogen in drinking water
(Harwood et al., 2005). This shortcoming of current quality
control is overcome by the use of quantitative microbial risk
assessment (QMRA) to define the microbiological safety of
ARTICLE IN PRESS
0043-1354/$ - see front matter &2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2007.01.056
Corresponding author. Tel .: +31 30 606 9596; fax: +31 30 606 1165.
E-mail address: wim.hijnen@kiwa.nl (W.A.M. Hijnen).
WATER RESEARCH 41 (2007) 2151– 2162
drinking water (Haas, 1983;Medema et al., 2003). In the new
Dutch Drinking Water Decree ‘‘Waterleidingbesluit’’ (Anon-
ymous, 2001) a maximum acceptable annual infection risk of
10
4
has been introduced for pathogens of faecal origin
including Cryptosporidium. The required risk level has to be
demonstrated with QMRA which is based on dose–response
relation in human volunteers and exposure assessment. For
exposure assessment quantitative knowledge about the
presence of pathogenic micro-organisms in the source water
and the capacity of water treatment processes to eliminate
these micro-organisms is required, along with data on
drinking water consumption.
Slow sand filtration (SSF) is one of the oldest water
treatment processes used to produce microbiologically safe
drinking water. Quantitative information of how effective
these filters are in removing pathogens, however, turned out
to be limited (LeChevallier and Au, 2004). It is only since the
last part of the 20th century that some studies were published
on the elimination capacity for protozoan (oo)cysts (Bellamy
et al., 1985;Schuler et al., 1991;Fogel et al., 1993;Timms et al.,
1995). In most of these studies the actual decimal elimination
capacity (DEC; log removal) of slow sand filters for Giardia and
Cryptosporidium (oo)cysts was assessed by dosing peak con-
centrations, resulting in high DEC-values of 4 to more than
6 log in matured and relatively small-sized filter beds. In
contrast with these findings, one study (Fogel et al., 1993)
determined removal of environmental Giardia and Cryptospor-
idium (oo)cysts by full-scale filters by prolonged sampling and
showed breakthrough during periods with low temperature
(o51C) resulting in low average DEC-values of 1.2 and 0.3 log,
respectively. In the Netherlands, slow sand filters are
operated as the last stage of a multiple barrier treatment.
Hence, assessment of the DEC for environmental protozoan
(oo)cysts is not feasible. The removal of spores of sulphite-
reducing clostridia (SSRC; including Clostridium perfringens)as
a surrogate for protozoan (oo)cysts (Payment and Franco,
1993;Hijnen et al., 1997) by full-scale filters was monitored
(Hijnen et al., 2000, 2004). Results showed low DEC for SSRC,
ranging from 0.2 to 1.8 log. Direct comparison of the removal
of dosed spores of C. perfringens and oocysts of C. parvum in a
short sand column (0.5 m) showed 2.3–3.2log removal of the
spores and more than 5 log removal of the oocysts, indicating
that environmental SSRC is a conservative surrogate for
protozoan (oo)cysts (Hijnen et al., 2004). However, the clear
discrepancy between the high removal of dosed spores and
the low removal of SSRC by mature full-scale filters sand casts
doubts on the validity of translating column study results to
the larger full-scale filters. Because spores are very persistent
(Medema et al., 1997), it was hypothesized that spores
accumulate in the filter bed and they might detach which
causes remobilization and delayed breakthrough. This phe-
nomenon was first suggested as cause for the relative low
removal of SSRC by granular activated carbon (GAC) filters
with high SSRC concentrations in the filter bed and the back
wash water (Hijnen et al., 1997). Additional observations
supporting this hypothesis have been described in literature
for protozoan-sized microspheres in soil (Harvey et al., 1995),
1mm fluorescent microspheres in GAC and expanded clay
filters (Persson et al., 2005), aerobic spores in GAC filters
(Mazoua and Chauveheid, 2005) and C. parvum oocyst
transport in sand columns (Harter et al., 2001;Bradford and
Bettahar, 2005;Hijnen et al., 2005) and E. coli and SSRC in the
latter study. Furthermore in a column study, it was demon-
strated that the removal of spores depended on the duration
of the seeding (Schijven et al., 2003). As with SSRC, oocysts of
C. parvum are also persistent, but oocysts are larger by a factor
of 3–4 and have potentially different surface properties. As a
consequence, it remains unknown whether the phenomenon
of reduced elimination by sand filters over time due to
accumulation could apply to these pathogens as well.
To investigate this remaining question a study was
conducted with the objective of describing and quantifying
the removal, accumulation and delayed breakthrough of C.
parvum oocysts in a slow sand filter of some extent (2.56 m
2
)
operated under full-scale conditions mimicking as much as
possible long-term loading conditions. Spores of C. perfringens
as well as a small-sized alga, the centric diatom Stephano-
discus hantzschii, were dosed simultaneously to compare
removal of different kind of persistent micro-organisms and
to investigate the potential use of both organisms as
surrogate for the assessment of protozoan oocysts removal
by SSF (Hijnen et al., 1997; Akiba et al., 2002).
2. Materials and methods
2.1. Slow sand filter and the experimental set up
SSF in the water treatment Leiduin of Water Company
Waternet is the last stage in a treatment train with rapid
sand filtration, ozonation and GAC filtration. The source
water is pre-treated water from the River Rhine after dune
passage and regained by an open collecting system. The filter
used for this study was part of a pilot plant, a dummy system
of the full-scale treatment. The filter has been in operation for
3 years without surface scraping at a flow rate of 1 m
3
h
1
and
a filtration rate of 0.3 m h
1
prior to carrying out the present
study. The influent, GAC filtrate, had a low DOC (1.5 mg C l
1
)
and turbidity (0.1 FTU), a pH of 8.0 and temperature ranging
from 8.2 to 18.8 1C. The filter bed (2.56 m
2
and depth of 1.5 m)
was filled with sand (d
50
¼0.28 mm; porosity of 0.41) on top of
a support of Nicolon cloth (0.7 mm) and 0.4 m gravel pack. The
experimental set up is presented in Fig. 1 and will be
described in detail hereafter.
2.2. Cryptosporidium oocysts
C. parvum oocysts suspensions in watery faeces (Cervine
origin; Moredun scientific Ltd., Midlothian, UK; 2 10
9
) were
used. Each tube of suspension was purified by filtering
through plankton netting with a mesh width of successively
500, 300 and 100 mm, the netting was flushed with sterile
water which was mixed with the filtrate. This suspension was
mixed during 30 min (CAT S50) and subsequently settled
during 30 min. The water was decanted and the sediment was
flushed twice with sterile water. The accumulated water
suspension was centrifuged 10 min (4000 RPM) and the pellet
was concentrated to a volume of approximately 100 ml with
an estimated number of 4.5 10
8
. This pre-treated suspen-
sion was divided into three portions each dosed during 1
ARTICLE IN PRESS
WATER RESEARCH 41 (2007) 2151 – 21622152
week to the influent. For safety reasons the oocyst suspen-
sions were inactivated with UV in a collimated beam
apparatus (calculated UV fluence of X10 mJ cm
2
) prior to
the dosage.
2.3. C. perfringens
A suspension of spores of C. perfringens (SCP strain D10; an
isolate from a patient suffering from diarrhoea caused by food
infection) was prepared with a total number of spores of
2.2 10
9
following a previously described method (Hijnen et
al., 2002). The level of sporulation of the suspension was
almost 100% and tested by enumeration on Perfringens-agar-
base plates (PAB Oxoid CM587) with and without pasteuriza-
tion for 15 min at 60 1C.
2.4. S. hantzschii
S. hantzschii is a small-sized centric diatom (SSCD) with an
average size of 5 mm. SSCD-cells were obtained from a pre-
cultured strain of the Culture Collection of Algae and Protozoa
(CCAP 1079/4; Dunstaffnage Marine Laboratory, Argyll, UK,
Th. Pro
¨schold). The strain was inoculated into a gently stirred
150 ml sterilized diatom medium (DM) for fresh water algae
(Beakes et al., 1988) and incubated for 2–3 weeks at 20721C
with a light–dark cycle of 12:12h (white light intensity of
50 mEm
2
s
1
). The growth and conditions of the algae were
monitored regularly by light microscope. The concentration
of cells in the final suspension of 71.5 10
8
ml
1
was
determined using a haemocytometer.
2.5. Dosage procedure
Appropriate volumes of the pre-treated oocyst, the C. perfrin-
gens and S. hantzschii suspensions were added to 150 l of
drinking water in a 200-l stainless steel (SS) vessel with cover.
This diluted and stirred suspension, which was freshly
prepared every week, was dosed continuously into the mains
of the influent of the filter at a flow rate of 13ml min
1
during
the first two weeks. Because in this vessel biofouling
occurred, from week 3 the oocysts suspension was dosed
(0.7 ml min
1
) separately from a separate 20l suspension in
drinking water prepared fresh weekly in a 30-l SS vessel with
cover and stirrer, cooled in a container with melting ice.
Dosing of the SCP suspension was also from this cooled 20 l
vessel after 7 weeks. The total dosage period was 98 days (14
weeks) in which dosage failed for SCP and SSCD in week 4 and
for oocysts in week 5 (tubing not correctly connected; actual
dosage period was 91 days).
2.6. Large-volume sampling
Large-volume sampling was considered necessary to deter-
mine organism concentration in the filtrate. Water volumes of
1000 l or more were concentrated on-site using a Hemoflow
cross-flow ultrafilter (HF80S; Fresenius, Germany; Simmons
et al., 2001). The water volumes sampled with the Hemoflow
were concentrated to a total volume of 800 ml HF-concen-
trate. Approximately 5% of the HF-concentrate was used to
determine the number of SSRC (including C. perfringens); the
residual HF-concentrate was used to enumerate the number
of oocysts.
ARTICLE IN PRESS
Fig. 1 – Diagram of the experimental set up.
WATER RESEARCH 41 (2007) 2151 – 2162 2153
The concentration of SSCDs in large volumes of effluent
was determined using the Membrane Filtration or MF-
sampler (Hijnen et al., 2000). Algal cells were isolated on
poly-carbonate membrane filters (2 mm; Sartorius) and re-
suspended in tap water by 2min of ultra-sonic treatment (low
energetic; Branson 5200, Danbury, USA). The recovery of this
method was 53–100% (n¼6) determined with chemically
purified centric diatoms in the size range of 6–7 mm (M.
Rosielle, personal communications).
2.7. Sampling program
The filter bed load of C. parvum oocysts and spores of C.
perfringens as SSRC, was determined weekly or more by
sampling the influent of the filter as well as the water volume
above the filter bed. The concentration of S. hantzschii dosed
to the influent was determined as SSCD in the water above
the filter bed. Because of the relative high background
concentration of SSCD in the influent of the pilot plant, the
SSCD concentration was also determined before the dosage
point of S. hantzschii. The effluent of the slow sand filter was
sampled weekly during the dosing period and a period of 22
weeks after the dosage stopped to determine delayed break-
through.
2.8. Analysis
Oocysts of Cryptosporidium in the influent and the HF-
concentrate were enumerated with the EPA method 1623
(USEPA, 2001). Recovery of the HF-concentrate was tested by
addition of 5 ml Colorseed
TM
(BTF Decisive Microbiology,
North Ryde, NSW, Australia) suspension (99 oocysts 71.3).
The HF-concentrate was purified by immuno-magnetic se-
paration (IMS) prior to labelling and microscopic counting.
The adsorbed oocysts were eluted from the beads with HCl
(0.1 N) and this suspension was neutralized with KOH (1N).
The concentrate was collected on a membrane filter (Milli-
pore, 1.2 mm RTTP) and labelled with FITC (2 h at 37 1C). The
membrane was microscopically counted with epi-fluores-
cence microscopy (Leica DM RXA, magnification 312.5).
The concentration of spores of sulphite-reducing clostridia
(SSRC, including C. perfringens) was determined either directly
or after concentration by membrane filtration or HF-filtration.
All samples were pasteurized before enumeration. In the first
weeks SSRC in the effluent was measured by membrane
filtration (1 or 10 l; +47 mm, 0.45 mm pores; Sartorius 13906-
50-ACN) and pasteurization of the membranes (30 min at
70711C; Dutch NEN6567). Given that no SSRC were detected
in these samples, SSRC in the effluent was subsequently
monitored in the HF-concentrate. Fifty millilitres of this
concentrate was pasteurized (15 min at 60711C; ISO-method
6461/2-1986 for C. perfringens spores) prior to cultivation in PAB
medium (Oxoid CM 587) under anoxic conditions during 4874h
at 37711C. A real-time PCR method was used to confirm that
the SSRC colonies observed in the PAB medium were spores of
C. perfringens. Cell material was isolated from the black colonies,
suspended in 1 ml sterile water and subsequently tested with
the real-time PCR for DNA of C. perfringens. The primers which
codes with A-toxine gen of C. perfringens (Yoo et al., 1997)were
used to identify the C. perfringens.
For enumeration of the breakthrough of cells of S. hantzschii
the concentration of SSCD with specific cell morphology and
size (4–7 mm) is determined microscopically in concentrates of
the influent and the effluent, prepared by sedimentation.
Water samples were pretreated with lugol-solution until the
sample was light orange to stop biological activity. These
samples were pored into Hydro-bios sedimentation chambers
(Hydro-bios Apparatenbau, Kiel-Holtenau, GMB) with appro-
priate size (10–50–100 ml) and settled during 24–72 h. Water
was decanted from these tubes and the sediment was
examined microscopically (Olympus IX70, Olympus, Zoeter-
woude, NL; magnification—400 ).
2.9. Enumeration of retained micro-organisms
After the dosage of micro-organisms was stopped, delayed
breakthrough of the dosed micro-organisms was monitored.
After 184 days, when oocyst concentration in the filtrate was
below the detection limit (DL) of the analysis, filtration was
stopped and the water volume above the filter bed was
lowered to the surface. At two locations the saturated filter
bed was scraped with a scoop to sample the Schmutzdecke
(SM; approximately 2–3 mm) and the following 50 cm of the
saturated filter bed was sampled with a soil core sampler
(veenboor; Eijkelkamp, Giesbeek, The Netherlands). This was
repeated after 253 days where sand from the first 5 cm and
from layers deeper than 50 cm was collected. At first the
presence of C. parvum oocysts in the upper part (0–1 cm) was
monitored microscopically by fixing sand grains on a Dynal
slide. The grains were labelled with FITC (2 h at 37 1C) and
completely scanned for oocysts. To determine the concentra-
tion of retained micro-organisms in the sand, 3–4g of the
sampled sand from the two locations in the filter bed was
suspended in 100 ml sterile tap water and treated for 2 min in
a low energetic ultra-sonic cleaner (Bransonic 5510, Branson
ultrasonic, Danbury, USA). The number of micro-organisms
eluted from the sand was enumerated in this sonicated sand
suspension (UTSusp.) with the analytical methods described
above. Oocysts and spores in the SM material and the sand of
the first cm in the filter bed (0–1) were examined in two
separate sub-samples of 3–4 g. A third sub-sample was treated
ultrasonically in 100 ml sterile Laureth-buffer (USEPA, 2001)to
verify the elution efficiency of the ultra-sonic treatment for
oocyst enumeration. Fifty millilitres of the UTSusp. was used
to enumerate oocysts and spores and the other 50ml was
used to enumerate the SSCD with the methods previously
described. Microscopic counting was used to enumerate SSCD
in the UTSusp. and because these suspensions had a high
content of suspended solids, the collected concentrations
must be seen as an indication of the order of magnitude of
centric diatoms in the filter bed. Statistical differences
between concentrations of retained micro-organisms in the
filter bed were analysed with the Student t-test or the
Wilcoxon-test using SPSS (14.0).
2.10. ATP and zooplankton analysis in the sand
The concentration of adenosine tri-phosphate (ATP) in the
sand was measured (Magic-Knezev and van der Kooij, 2004).
The presence of zooplankton in the sand was measured in
ARTICLE IN PRESS
WATER RESEARCH 41 (2007) 2151 – 21622154
separate sand samples of approximately 500 g taken from the
filter bed at the end of the operational time. 30 g of sand was
mixed intensively in tap water. The invertebrates were
separated from the sediment by separation in a MgSO
4
solution in tap water (49 g l
1
). The sand slurry was tested
for the presence of larger invertebrates by filtration over
500 mm sieve and subsequently pored in an Anderson glass
tube (Anderson, 1981) filled with the MgSO
4
solution. After
15–20 min of sedimentation the zooplankton sample
(7800 ml) was taken at the upper sampling port and subse-
quently at the lower sampling port. The suspensions were
sieved subsequently through a 30mm sieve and loaded in a
counting chamber for microscopic examination (Olympus
IX70, Olympus, Zoeterwoude, NL; magnification 100 )to
count the number and identify the zooplankton in the sand.
2.11. Elimination of the micro-organisms and mass
balance
The DEC of the filter bed for the tested micro-organisms was
calculated from the average concentration in the influent ¯
Cin
and the effluent ¯
Cout, the latter calculated by
¯
Cout ¼PNout
PVout
¯
Rout, (1)
where N
out
is the number of oocysts counted, V
out
the
sampled volume (l) and R
out
the recovery in the tested
samples,
DEC ¼log10
¯
Cin
¯
Cout
. (2)
DEC was also calculated from the mass balance by the
following equation:
DECm¼log10
Md
Me
, (3)
where DEC
m
is the elimination capacity on the basis of the
mass balance, Md¼td¯
Cin and Me¼Pn
i¼1tiðCout;iþCout;iþ1Þ=2
the total number of micro-organisms dosed to the influent
and found in the effluent, respectively, t
d
is the time period of
dosing (hours) and dt
i
is the ith time interval of n intervals
between two successive samples with concentrations C
out,i
and C
out,i+1
.
The mass of micro-organisms (numbers) accumulated in
the filter bed M
b
after an operational time of 184 and 253 days
is derived from
Mb¼XMl;n¼X¯
Cl;n1000
dl;nA1000
, (4)
where M
l,n
is the total number of micro-organisms and ¯
Cn;lthe
average concentration of micro-organisms (N ml
1
)innlayers
(l) with a thickness of d
l,n
(m) of the filter bed, respectively,
with a surface area Aof 2.56 m
2
.M
l,n
in the un-sampled layers
was calculated from the average concentration extrapolated
from the concentrations of the next upper and lower layers.
Because of the inaccuracy of the concentrations in the filter
bed, mass balance calculations was not done for SSCD.
2.12. Calculation of collector and sticking efficiencies
Assuming elimination in the filter bed was only due to
attachment and detachment, the sticking efficiency of the
spores and oocysts was calculated from the colloid filtration
model (Yao et al., 1971) described with the equation
LN C
C0
¼3
2
ð1yÞ
dc
aZL, (5)
where d
c
is the diameter of the collector, athe sticking
efficiency, Zthe single collector collision efficiency, and Lthe
length of the column. Zwas calculated with the optimized
equation presented by Tufenkji and Elimelech (2004a). For the
calculations the following parameters values were used: bulk
water density 999.703 kg m
3
; Hamaker constant for bacter-
ium glass water interface 6.2 10
21
J(Rijnaarts et al., 1995);
sizes (m) of spores of C. perfringens 1.5 10
6
and oocysts of C.
parvum 4.9 10
6
(Medema et al., 1998); and r
p
C. perfringens
1270 (Tisa et al., 1982) and oocysts 1045 kg m
3
, respectively
(Medema et al., 1998).
3. Results
3.1. Cryptosporidium removal
Oocysts of C. parvum were dosed to the GAC filtrate, the
influent of the SSF. Based on the low concentration of
environmental oocysts observed in the source water during
the experiment (routine monitoring data, not presented) and
the preceding removal in the rapid sand filter and GAC-
filtration, the background concentration of oocysts in the
influent was negligible. The oocyst concentration measured
in the influent after dosage and in the water above the filter
bed was constant with an average concentration of 314.6
(7161) N l
1
(Table 1; average recovery of 70.6 (713) %; n¼25).
During the dosing period of 98 days and an additional
period of 163 days after dosing had stopped, 26 m
3
of the
filtrate was examined for oocysts. After the first 29 days and
during a period of approximately 50 days, 14 oocysts were
detected in approximately 50% of the samples of the filtrate
(Fig. 2). A second shorter period (25 days) of oocyst break-
through was observed shortly after dosage was stopped. From
the total number of 18 oocysts detected in the volume
sampled during the total monitoring period of 247 days an
average concentration of 0.0016 oocysts l
1
was calculated
(Tab le 1; average recovery of 43.6 (715.8) %; n¼22) resulting
in a DEC-value of 5.3 log.
3.2. Removal of spores of sulphite-reducing clostridia
Also for SSRC the background concentration of environmen-
tal SSRC in the influent was neglected (data derived from
routine sampling and not presented). The average concentra-
tion of spores of C. perfringens (SCP; confirmed with molecular
typing on a few samples) was 1135 (71015) N l
1
(Tab le 1). The
temporary decline in influent concentration after 40 days
(Fig. 2) was solved by switching dosage from the large 150-l
vessel used for S. hantzschii dosage to the smaller and
permanently cooled 20-l vessel used for C. parvum oocysts.
During the first weeks no SCP were observed in effluent
sample volumes of 1 and 10l and breakthrough of these
spores was detected starting from day 36 and further (Fig. 2).
The highest breakthrough of SCP was at day 44, 15 days after
ARTICLE IN PRESS
WATER RESEARCH 41 (2007) 2151 – 2162 2155
the first highest oocyst breakthrough (Fig. 2). Based on the
total number of SCP observed in sampled effluent volume of
2.363 l the average concentration was 0.142 (70.509) SCP l
1
(Tab le 1 ) resulting in a DEC-value of 3.9 log for these
organisms.
3.3. Removal of SSCDs
Compared to the Cryptosporidium and SCP, SSCD (4–7 mm) were
present in higher and more variable concentrations in the
source water of this treatment plant. Concentrations ranged
from o1 up to 5.4 10
5
l
1
. In early spring or late autumn
diatom bloom in the source water reservoir caused peak
concentrations. The average SSCD concentration in the
source water was a factor of approximately 200 higher than
the concentration of SCP and was reduced only slightly
(0.4 log) by the rapid sand filters (data not presented). Because
of these high levels of SSCD in the source water, SSCD were
also determined in the influent before dosage of S. hantzschii
(Fig. 2). The concentrations of SSCD in the filter influent were
highly variable and only slightly below the level of dosed S.
hantzschii. Thus, environmental SSCD concentrations con-
tributed 40–50% on average to the total load of the test filter
with these micro-organisms (Table 1) and could not be
separated from cells of S. hantzschii in the microscopically
enumeration method. The results of SSCD monitoring in the
effluent showed a clear breakthrough (Fig. 2), much larger
than observed for C. parvum oocyst and C. perfringens spores.
After an initial high peak in the effluent, breakthrough
stabilized. Because of the mixed loading of S. hantzschii with
environmental SSCD, and the variability herein, the DEC for
these micro-organisms was not calculated from the average
concentration in the influent and effluent but from the actual
concentrations determined at the same day (n¼12). The
average DEC was 1.870.6 log (range of 0.9–2.6 log).
3.4. Concentration of retained micro-organisms
An important objective of the experiment was to monitor
delayed breakthrough behaviour of oocysts and its potential
surrogates, SCP and SSCD. The filter bed was not disturbed
until breakthrough of oocysts was below the DL (0.002 n l
1
;
Fig. 2). After 184 days sand samples were taken from the filter
bed at two places. This interruption of the filtration process
and locally disruption of the filter bed caused no additional
oocyst breakthrough (Fig. 2). The maximum concentration of
oocysts per ml sand was 160 (7151, n¼6 and range of 57–426)
observed in the top of the filter bed (Fig. 3a) using the method
of elution in sterile water (ultrasonic-treated sand suspen-
sion; UTSusp.) and declined with increasing depths to a DL of
1 oocysts ml
1
after 43 cm. Ultrasonic treatment in 100 ml
laureth buffer (LB) applied to the samples of the SM
and the first centimetres did not increase the oocyst
recovery from the sand (Fig. 3b). When the experiment was
stopped after 253 days of operation the filter bed was sampled
again on two different locations. The concentration of oocysts
in the first 5 cm of the filter bed was lower than the
concentrations determined at day 184 (Fig. 3a). The difference
in oocyst concentration observed after 184 and 253 days
in the layer 1–5 cm was significant (P¼0.03). Due to the
high standard deviations of average concentrations at the top
of both samplings the difference was not significant. All
oocyst concentrations were corrected individually for the
recovery of Colorseed
TM
seeded in the UTSusp. (24.878.1%).
The high performance of the elution method in sterile
water was also demonstrated by direct microscopic counting
of oocysts in the samples. Part of the SM samples and
sand samples of the first centimetre were examined
microscopically for the presence of oocysts. One layer of
sand grains was fixed in the well of a Dynal slide
with a diameter of 9.26 mm and all fields of 0.39mm
2
(average grain number of three) were scanned (total of 518
grains on the top and partly the side). No oocysts were
detected and on the basis of the average grain size of 0.3mm
which results in a grain number of 70,771 N ml
1
of sand and a
scan factor of 0.6 (under and part of the sides of the grains
could not be scanned) a DL of 228 oocysts ml
1
of sand was
calculated, which was slightly higher than the maximum
observed numbers determined by the elution method
(Fig. 3a).
ARTICLE IN PRESS
Table 1 – Concentration of dosed Cryptosporidium oocysts, spores of C. perfringens (SCP) and environmental and small-
sized centric diatoms SSCD (4–7 lm; incl. S. hantzschii)) in the influent and effluent of the slow sand filter
Samples n
a
n+
a
Sampled
vol. (l)
Detected
organisms
Conc. n/lSTD
c
Oocysts in
b
33 33 3.3 733 314.6 161
Oocysts out
b
22 7 26.613 18 0.0016 ND
c
SCP in 20 20 2.0 2270 1135.0 1014.8
SCP out 21 16 2.363 336 0.142 0.509
SSCD in 21 17 1.51 140 92.9 889.4
SSCD in BG
d
17 17 0.98 274 279.6 92.8
SSCD out 29 29 25.94 187 7.22 8.46
a
nand n+, number of samples and number of positive samples.
b
Corrected for recovery.
c
STD, standard deviation; ND, not determined.
d
Background concentration of environmental SSCD (oocysts and SCP below detection limit).
WATER RESEARCH 41 (2007) 2151 – 21622156
Appropriate fractions of the UTSusp. were used to deter-
mine SCP (1–10 ml) and SSCD (50 ml) concentrations. The SCP
concentration in the sand at day 184 was a factor of 100
higher than the oocyst concentration (Fig. 3a and c) and
declined to 1–10 ml
1
in the first 50 cm of the filter bed. The
large difference in accumulation in the top of the filter bed
between oocysts and spores could not be explained by the
difference in influent concentration and observed break-
through (Fig. 2). This indicates that there is a difference in
fate of the retained oocysts and spores. For SCP the
concentrations in the sand sampled at day 253 were in the
same order of magnitude as monitored after 184 days (Fig. 3b).
Hundred percent of a total of 90 SCP colonies which were
molecularly tested, were identified as C. perfringens.
Microscopic counting of SSCD in the UTSusp. was ham-
pered by the high content of suspended solids and thus, the
calculated concentrations show the order of magnitude of the
presence of these organisms in the filter bed. At day 184, low
concentrations of SSCD (o0.3–8.9 N ml
1
) were observed in
the sand. These concentrations were a factor of 10–1000
below the numbers of oocysts and spores (Fig. 3) and indicate
a relative low adsorption capacity of the sand for SSCD, which
agreed well with the high level of breakthrough in this filter
(Fig. 2). At day 253 the estimated concentrations of SSCD in
the SM material (430–2029 N ml
1
) and the sand from the layer
of 1–5 cm (o0.4–492 N ml
1
) were clearly higher than the SSCD
concentrations observed at day 184. A relation with the
autumn bloom of SSCD in the source water is suspected; day
253 was in the beginning of November. Two of the three peak
SSCD concentrations observed in the influent of the filter bed
during this period were monitored in between the filter bed
sampling at days 184 and day 253 (Fig. 2).
3.5. ATP and zooplankton in the sand
The spatial distribution of ATP in the filter bed showed a
higher (micro)biological activity in the SM and the first 5cm of
the bed than deeper in the bed (Fig. 4). ATP concentrations
after 184 and 253 days of operation were similar. The
determination of zooplankton in the sand of the filter bed
revealed high concentrations of Nematoda, Testacea (observed
species Centropyxis,Cyphoderia and Euglypha), Oligochaeta,
Rotatoria (observed species Colurella, Lecane, Lepadella and
other unidentified species), Nauplii and Harpacticoida and
some Cyclopoida in the top 5 cm of the filter bed (Fig. 4). Next
to these organisms Ciliata were detected in lower concentra-
tions at filter depths of 50 and 100 cm.
3.6. Mass balance and CFT parameters
The mass balance for oocysts and spores (in numbers) was
calculated to determine the elimination and accumulation in
the filter bed (Table 2 ). From the calculated number of
organisms dosed to the influent and passed though the filter
bed in the filtrate the DEC
m
was calculated. The DEC
m
for
oocysts and SCP was 4.7 and 3.6 log, respectively, approxi-
mately 0.5 log lower than the DEC calculated from the average
concentrations. The DEC
m
-values are most likely more
accurate than DEC calculated from the average filtrate
concentration where zero counts are included as zero, thus
underestimating the actual average concentration. The accu-
mulated number of C. parvum oocysts M
b
determined in the
filter bed after 184 days was only 1.8% of the total dosed
oocyst number and decreased to 0.2% at day 253. For spores of
C. perfringens, M
b
after 184 and 253 days was 32.5% and 45% of
the dosed spores, respectively. The difference in M
b
between
both sampling dates is most likely caused by the variability in
SCP concentrations determined on four different locations in
the filter bed (Fig. 3d).
The collector efficiency Zfor oocysts in the filter bed of the
present study was a factor of two higher than Zfor spores of C.
perfringens (Table 2 ). Despite the lower Za higher collision
efficiency awas calculated for the removal of the spores,
ARTICLE IN PRESS
Fig. 2 – Concentration of C. parvum oocysts, spores of C.
perfringens and small-sized centric diatoms (SSCD) in the
influent and the effluent of the slow sand filter (DL is
detection limit).
WATER RESEARCH 41 (2007) 2151 – 2162 2157
indicating a higher attachment in the filter bed compared to
the oocysts.
4. Discussion
4.1. Removal of Cryptosporidium and surrogates
The results of the study clearly demonstrate the high capacity
of mature slow sand filters with a well-developed SM for
removal of Cryptosporidium from water. During the prolonged
loading of the pilot filter very few oocysts passed the filter bed
and on the basis of the collected data a decimal elimination
capacity DEC
m
of 4.7 log was calculated. This removal rate
was similar to the removal rates assessed with short term
dosing experiments in slow sand filters operated on labora-
tory and pilot plant scale (Bellamy et al., 1985;Schuler et al.,
1991;Timms et al., 1995;Hijnen et al., 2004). The present
study also showed that slow sand filters have a high capacity
to remove spores of C. perfringens (SCP; DEC
m
of 3.6 log).
Consequently, with the used experimental set up low DEC-
values for both persistent micro-organisms due to delayed
breakthrough could not be demonstrated. The height and
duration of the loading with oocysts and spores in the current
ARTICLE IN PRESS
0
50
100
150
200
250
SM 0.5 3 12.5 27.5 41.5
De
p
th (cm) De
p
th (cm)
ATP ng mL-1
184 days ATP
253 days ATP
0
600
500
400
300
200
100
2.5 50 100
Organisms 30 g-1
Nematoda
Te st a ce a
Polychaeta
Rotatoria
Nauplius
Harpacticoida
Cyclopoida
Ciliophora
Fig. 4 – The ATP concentration (7SD), after 184 and 253 days of operation (at depth of o50 cm, ATPo10 ng ml
1
of sand) and
the zooplankton concentration per 30 g of sand after 260 days at three depths.
Fig. 3 – The concentration (error bars
¼
range of values; DL is detection limit) of C. parvum oocysts and of C. perfringens spores
over the total filter bed (a and c) and the first 3 cm (b and d) of the filter bed after 184 and 253 days of operation (86 and 157
days after finishing with dosing, respectively) with the additional concentrations of oocysts obtained after laureth buffer
elution (+LB; n
¼
2) during the sampling at day 184 (b).
WATER RESEARCH 41 (2007) 2151 – 21622158
experiment was probably not enough to achieve sufficient
accumulation and consequently increase of concentration in
the effluent (Schijven et al., 2003). Moreover, the tested pilot
filter was not pre-loaded with environmental oocysts and
SSRC prior to the experiment, but a pre-loading with
environmental small-sized centric diatoms (SSCD 4–7 mm) is
plausible since the influent contained significant amounts of
environmental SSCD (Fig. 2). This might explain the relatively
low capacity of the filter bed to remove SSCD (DEC of 1.8 log).
4.2. Removal mechanisms and delayed breakthrough
Accumulation and delayed breakthrough of micro-organisms
in filters affecting DEC over time is determined by the ruling
removal mechanisms. It is not likely that in a fixed filter bed
like the slow sand filter, micro-organisms that were removed
by straining are remobilized. Delayed breakthrough is re-
stricted to persistent micro-organisms that are removed by
reversible attachment and show to some degree detachment.
On the basis of the results of this study and observations by
others, each of the mentioned processes, straining, attach-
ment and detachment and survival, will be discussed further
for the removal of the three tested micro-organisms.
Straining as removal mechanism for biocolloids in filters is
expected to depend on the ratio between the colloid and
collector size (d
p
/d
c
) and based on geometric modelling,
straining is expected to be of less importance at ratios of
o0.05 (Sakthivadivel, 1966, 1969) and o0.154 (Herzig et al.,
1970). The ratios for oocysts, SCP and SSCD in the pilot filter of
the current study are 0.018, 0.005 and 0.014–0.0025, respec-
tively, indicating a minor role of straining. In current
literature, however, the significance of straining to colloid
removal of the size of bacteria and protozoa is still in debate.
One study showed the variability of the role of straining and
simple extrapolation of size of grain and microbe is inap-
propriate (Hijnen et al., 2005). The large difference in removal
of SSCD and oocysts, organisms with the same size, in the
current study with a slow sand filter containing a well-
developed SM, supports this conclusion. There are studies
where straining is presented as a significant removal me-
chanism in freshly packed columns (Bradford et al., 2003;
Bradford and Bettahar, 2005;Tufenkji et al., 2004b;Schijven
et al., 2007). The negligible effect of the SM on the oocyst
removal in slow sand filters observed in other studies
(Bellamy et al., 1985;Timms et al., 1995;Hijnen et al., 2004)
question mark the role of straining in SSF. In conclusion,
straining will have contributed to the overall removal of
oocysts, SCP and SSCD in the pilot plant filter with a
prolonged pre-filtering period of 3 years, but is most likely
not the dominating removal mechanism.
Attachment and detachment of biocolloids like micro-
organisms to sand is described by the colloid filtration theory.
The parameter ais the attachment efficiency, calculated from
the removal efficiency and the single-collector removal
efficiency Z, a theoretical parameter based on the physical
processes involved in colloid transport and colloid to collector
interactions. Assuming complete removal by sorption, the
collision efficiency acalculated for the removal of oocysts in
the 1.5 m filter bed was 0.207 (Table 2). This value is slightly
lower than the avalues of 0.26–0.37 observed for C. parvum
oocysts in a column with glass beads of 0.328 mm at
approximately the same hydraulic conditions as the present
study (Tufenkji and Elimelech, 2005). On the basis of a recent
review on Cryptosporidium–sand interactions it was concluded
that physicochemical filtration plays an important role in
oocyst removal in saturated porous media (Tufenkji et al.,
2006). The collision efficiency for the removal of spores of C.
perfringens in the filter bed was 0.310 (Table 2) and SCP
concentrations at the top were higher compared to the oocyst
concentrations (Fig. 3). The difference in a-value calculated
for the removal of spores and oocysts, indicate that attach-
ment of the spores of C. perfringens to the sand was more
efficient than the attachment of the oocysts of C. parvum,an
observation described previously (Hijnen et al., 2005). This
indicates the different surface properties of spores and
oocysts. The zeta-potential of the C. perfringens spores in the
influent was 32.6 (72.5) mV (Schijven et al., 2007), whereas
for oocysts higher zeta-potentials of 6 mV have been
reported by Tufenkji and Elimelech (2005) at pH 8 (similar as
pH of the influent of the present study) and also others (Brush
et al., 1998;Dai and Hozalski, 2003). In the top of the filter bed
a relatively high Fe-hydroxide (0.55 mg/kg) concentration was
determined which will enhance attachment of the mostly
negative charged micro-organisms. The low estimated level of
SSCD in the filter bed indicates unfavourable surface proper-
ties of these micro-organisms for attachment, which is
supported by the observed low removal of SSCD in the pilot
filter and also in the full-scale rapid sand filters (0.4 log), the
first stage of the treatment. Similar low reduction of
fluorescent micro algae (0.4–15 mm) of 0.4–1 log in GAC and
expanded clay filters was reported in literature (Persson et al.,
2005).
Continuation of the breakthrough of oocysts, SCP and SSCD
during approximately 50 days after the dosage was stopped
(Fig. 2) and a secondary peak of oocyst concentration in this
period, an observation also described by others in column
studies (Harter et al., 2001;Hijnen et al., 2005), demonstrate
ARTICLE IN PRESS
Table 2 – Mass balance data (dosed organisms (M
d
) and
organisms in filtrate and filter bed (M
e
;M
b
) and the
colloid filtration parameters (collector efficiency gand
collision efficiency a)
C. parvum C. perfringens
M
d
5.4 10
8
1.9 10
9
M
e
9.8 10
3
5.2 10
5
M
b
184 days
a
9.7 10
6
73.3 10
6
7.0 10
8
74.0 10
8
M
r
184 days (%)
c
1.870.6 32.5717.2
M
b
253 days
b
1.1 10
6
72.6 10
5
8.9 10
8
73.5 10
7
M
r
253 days (%)
c
0.2170.05 4573.3
Z0.0112 0.006
a0.207 0.310
a
Organisms in 0.3 m of the filterbed sampled at two locations.
b
Organisms in 0.05 m of the filter bed sampled at two locations.
c
Recovered mass (% of dosed numbers) in filter bed after 184–253
days of operation.
WATER RESEARCH 41 (2007) 2151 – 2162 2159
that the attachment of these organisms in the filter bed is
reversible. Davies et al. (2005a) demonstrated remobilization
of micro-organisms including C. parvum oocysts retained in
soil in response to changes in ionic strength (from tap water
to rain water). The virtually flat tail of the breakthrough
curves of all three micro-organisms after dosing was stopped
(Fig. 2) indicates a high detachment rate as was described for
SCP (Schijven et al., 2003) and for C. parvum oocysts (Bradford
and Bettahar, 2005) retained in sand column studies.
Finally, for delayed breakthrough the removed micro-
organisms must survive (negligible inactivation rate and no
mass reduction) in the filter bed. The oocysts, spores and
SSCD were primarily retained in the SM and the first 5–10cm
of the filter bed of the present study (Fig. 3), the part with the
increased microbiological activity (Fig. 4) and the highest
accumulation of (in)organic components (data not presented).
Timms et al. (1995) showed similar spatial distribution of
oocysts in a slow sand filter bed monitored immediately after
a dosing experiment and 80% of the oocysts being present in
the top layer of the filter bed. Our study revealed, however, a
low recovery of oocysts in the filter bed determined 86 days
after dosing was stopped (Ta ble 2). In the next 69 days of
filtration a further decline in mass of retained oocysts was
observed. In contrast with these results, the recovery of C.
perfringens spores in the filter bed was significantly higher,
despite the lower mass load and removal rate and no
inactivation of the retained numbers of spores was observed
(Tab le 2 ). The mass accumulation and the log-linear inactiva-
tion or mass reduction rate of spores and oocysts, respec-
tively, in the filter bed (Fig. 5) was estimated from the mass
load, observed removal rate and numbers retained in the filter
bed. The estimated inactivation rate of SCP was 0.005 d
1
(Fig. 5), similar to the inactivation rate assessed for environ-
mental SCP in river water (Medema et al., 1997). The
estimated mass reduction rate of retained oocysts was
0.014–0.02 d
1
(Fig. 5), values lower than estimated from a
study where the mass reduction of retained oocysts in soil
exposed to faecal wastes inoculated with C. parvum oocysts
was determined (Hutchison et al., 2005). From the published
oocyst mass reduction data in the soil of the latter study log-
linear mass reduction rates of 0.136 and 0.153 log d
1
were
estimated. King et al. (2005) showed lower mass reduction
rates of C. parvum oocysts in untreated surface water from a
reservoir; estimated rates were 0.0027, 0.0069 and 0.0076 d
1
at 15, 20 and 25 1C, respectively. The same authors noticed no
reduction occurred in autoclaved samples of the same water
and from their results King et al. (2005) hypothesized an
influential role of predation in oocysts mass reduction.
4.3. Disintegration, predation and inactivation of oocysts
The observed reduction of oocysts in the sand over time can
be caused by disintegration or predation. Microscopic evi-
dence for disintegration of the retained oocysts in this study,
though, was not obtained. The observed oocysts in the filter
bed were intact and only to some degree, differential staining
of the oocysts in the SM with the monoclonal antibody was
noticed. From the results of the zooplankton analysis (Fig. 4)it
was deduced that predation of oocysts was a possible cause of
the large oocyst reduction in the filter bed. Eight different
species were observed in the filter bed in relative high
numbers. Two of these species, Testacae (Testate amoebae)
and Rotatoria, are family of zooplankton species for which
predation of oocysts have been documented (Fayer et al.,
2000;Stott et al., 2001, 2003). The results of the mass recovery
of spores in the filter bed show that spores of sulphite-
reducing clostridia are less susceptible to predation.
The presence of zooplankton like protozoa,Rotifera,Nema-
toda and Oligochaeta in slow sand filters and the significant
role of predation in the removal of bacteria was shown by
several studies (i.e. Lloyd, 1996;Weber-Shirk and Dick, 1997).
Predation as an oocyst reduction mechanism depends on the
presence of suitable predators which is not always the case as
demonstrated by Davies et al. (2005b). In air dried and sieved
surface soil samples from drinking water supply catchment
areas inoculated with C. parvum oocysts they observed hardly
any reduction in total numbers during 180 days of incubation
at 4, 20 and 35 1C. In the absence of predation, inactivation of
the retained oocysts is the only process which will diminish
the risk of delayed breakthrough of reversible attached
infectious oocysts. Inactivation rates of oocysts in the soil
samples of the study of Davies et al. (2005b) estimated by loss
of viability (fluorescence in situ hybridization, FISH) ranged
from 0.015 to 0.022 d
1
. Using cell culture-Taqman PCR assay,
King et al. (2005) demonstrated higher rates in loss of
infectivity of C. parvum oocysts inoculated in surface water
of 0.01, 0.045 and 0.049 d
1
at 15, 20 and 25 1C, respectively. In
addition this inactivation rate adds to the observed mass
reduction rate in reducing the risk of remobilization and
delayed breakthrough of infectious oocysts.
4.4. SSRC and SSCD as surrogates
The results of this study demonstrate clearly differences
between the behaviour of Cryptosporidium oocysts, SSRC and
SSCD in slow sand filters. Based on geometric considerations
removal of oocysts and SSCD by straining is similar and
higher than removal of SSRC by straining, but the importance
of this removal mechanism is uncertain. The present study
ARTICLE IN PRESS
5.0
6.0
7.0
8.0
9.0
10.0
0 50 100 150 200 250 30
0
Operational time (days)
Numbers of organisms (log)
Oocysts no decay
Oocysts decay 0.019 - 0.014
Spores no decay
Spores decay 0.005
Numbers observed in bed
Fig. 5 – Simulated accumulation of mass of spores and
oocysts (numbers) in the filter bed with and without decay
rates estimated from the mass determined after 184 and 253
days of operation.
WATER RESEARCH 41 (2007) 2151 – 21622160
indicates that attachment is of more importance for the
removal of SSRC and oocysts than for the removal of SSCD,
but for all three micro-organisms a high degree of detach-
ment is observed. Finally, oocysts are less persistent than
SSRC and SSCD because of a higher inactivation rate and the
susceptibility to predation. Therefore the risk of accumula-
tion of infectious oocysts in biological active slow sand filters
is expected to be low. This implicates that a lowering of DEC
of slow sand filters for infectious oocysts as observed for SSRC
and SSCD, is less likely to occur. Furthermore, it is assumed
that the environmental oocysts observed by Fogel et al. (1993)
in the filtrate of full-scale slow filters after delayed break-
through and causing low DEC were most likely not infectious.
From these considerations it is concluded that SSRC and to a
larger extent SSCD, are too conservative surrogates for the
assessment of capacity of slow sand filters to eliminate
Cryptosporidium oocysts due to differences in size, surface
properties and persistence. The results of this study suggest
that the higher persistency of both surrogates due to low
inactivation rates and susceptibility to predation is the major
basis for this conclusion.
5. Conclusions
The results of this study demonstrate the high capacity of
mature slow sand filters to remove Cryptosporidium oocysts
and spores of C. perfringens. The observed decimal elimination
capacity DEC
m
was 4.7 and 3.6 log, respectively. The relative
low DEC of 1.8 log observed for the SSCDs with a similar size
as the oocysts was attributed to low attachment and
accumulation followed by delayed breakthrough, a phenom-
enon also observed for SSRC. On the basis of this study, the
risk of delayed breakthrough of infectious oocysts is low
because of a rapid decline in oocyst concentration in the filter
bed most likely caused by predation. The results of this study
indicate that environmental SSCD and SSRC persist longer in
the filter bed. From this finding in combination with the
difference in observed DEC of the filter bed for the three
organisms, it was concluded that SSCD and SSRC are too
conservative parameters to be useful as surrogates for the
assessment of the elimination capacity of slow sand filters for
Cryptosporidium oocysts. Further investigations are necessary
to elucidate the role of predation and the ultimate fate of the
ingested oocysts for Cryptosporidium removal and the effect of
temperature and filter bed scraping on this removal mechan-
ism.
Acknowledgements
This study was funded by Waternet and the joint research
program of the Dutch water companies. The excellent
technical support of Michel Colin (Waternet), Meindert de
Graaf and Carola Blokker (Kiwa Water Reserach), the discus-
sions with Wim Oorthuizen (Dune Water Company South
Holland) and Aleksandra Magic-Knezev (Hetwaterlaborator-
ium) in the project group and the valuable comments of Dr.
Paul van der Wielen (Kiwa Water Research) on the manu-
script, are greatly appreciated.
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