Reduction of Cryptosporidium, Giardia, and Fecal Indicators by

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Reduction of Cryptosporidium, Giardia, and Fecal Indicators by Bardenpho Wastewater Treatment Bradley William Schmitz, Hitoha Moriyama, Eiji Haramoto, Masaaki Kitajima, Samendra Sherchan, Charles P. Gerba, and Ian L Pepper Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b05876 • Publication Date (Web): 30 May 2018 Downloaded from http://pubs.acs.org on May 31, 2018

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Page 1 of 32

Environmental Science & Technology

Reduction of Cryptosporidium, Giardia, and Fecal Indicators by Bardenpho Wastewater Treatment Bradley W. Schmitz!", Hitoha Moriyama#$ Eiji Haramoto%, Masaaki Kitajima§, Samendra, Sherchan!, Charles P. Gerba¥, and Ian L. Pepper¥ ! Department of Civil & Environmental Engineering, National University of Singapore, Block E1A, #07-03, No. 1 Engineering Drive 2, Singapore, 117576 #&Department of Environmental Sciences, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan %&Interdisciplinary Center for River Basin Environment, University of Yamanashi, 4-3-11 Takeda, Kofu, Yamanashi 400-8511, Japan § Division of Environmental Engineering, Faculty of Engineering, Hokkaido University, North 13 West 8, Kita-ku, Sapporo, Hokkaido 060-8628, Japan ! Department of Global Environmental Health Services, Tulane University of Louisiana, 1440 Canal Street Suite 2100, New Orleans, LA, 70112 ¥ Water and Energy Sustainable Technology (WEST) Center, The University of Arizona, 2959 West Calle Agua Nueva, Tucson, Arizona 85745, USA * Corresponding Author: Bradley W. Schmitz Address: JHU/Stantec Alliance, Department of Environmental Health and Engineering, Johns Hopkins University, 615 N. Wolfe Street, Baltimore, MD, USA 21205 Phone: +1 410-955-2452 Fax: +1 410-955-0617 Email: [email protected] KEYWORDS: Cryptosporidium, Giardia, Wastewater Treatment, Bardenpho, Indicator

!

ACS Paragon Plus Environment

"!


Page 2 of 32

ABBREVIATIONS: LRV, log10 reduction value(s); WWTP, wastewater treatment plant(s); IMS, immunomagnetic separation; IFA, immunofluorescent assay; PBS, phosphate buffered saline; qPCR, quantitative polymerase chain reaction; RT, reverse transcription; EPA, Environmental Protection Agency; WAS, waste activated sludge

!


#!

Page 3 of 32




Page 4 of 32

#+!

1.! INTRODUCTION

#"!

Protozoa are an integral aspect of the wastewater microbial community and treatment process

##!

as they prey on bacteria, controlling microbial loads and propagation of pathogens.1 Yet, many

#$!

protozoa commonly found in wastewater and contaminated waters pose serious risks to human

#%!

health when ingested.2-5 The principal protozoa of concern are Cryptosporidium spp., Giardia

#&!

intestinalis, and Cyclospora cayetanensis, all of which can cause severe gastrointestinal illnesses

#'!

and have been associated with outbreaks.5-7 Transmission of waterborne pathogens can have

#(!

severe public health consequences, such as those during the outbreaks of Giardia in Bergen,

#)!

Norway,8 and Cryptosporidium in Milwaukee, Wisconsin and Östersund, Sweden.9,10 Therefore,

#*!

wastewater facilities aim to effectively reduce pathogen numbers to minimize disease

$+!

transmission and prevent outbreaks.

$"!

Cryptosporidium and Giardia (oo)cysts are often robust in wastewaters and difficult to

$#!

remove without stringent treatment. Wastewater facilities employ biological processes during

$$!

secondary stages to promote the removal of nutrients and pathogens. Attached-growth processes,

$%!

such as trickling filters, grow biofilms on medium that decompose organic material as it

$&!

percolates through the system.11,12 Suspended-growth processes, such as activated sludge,

$'!

recycle bacteria-rich slurries that breakdown organic matter.12 During this process, pathogens are

$(!

removed by antagonism, sedimentation, and/or adsorption to flocs that are wasted with solids.12

$)!

Projects analyzing wastewater treatment plants (WWTPs) with these processes often report that

$*!

(oo)cysts are persistent and/or are detected in effluents.13-17 If (oo)cysts remain infectious after

%+!

treatment, transmission can occur in waters leading to public exposure and health risks. Also,

%"!

inadequate water treatment can lead to severe economic consequences, as well as create a lack of

%#!

confidence in municipalities and water industries.6 Still, there are no federal regulations in the

!


%!

Page 5 of 32


%$!

United States for water quality intended for reclamation or reuse, but many states have set

%%!

standards or guidelines.18 Wastewater treatment facilities lacking optimal processes should upgrade technologies to

%&! %'!

produce effluents that are free of pathogens and suitable for water recycling purposes. Our

%(!

previous study examined the reduction of protozoa in WWTPs utilizing activated sludge and

%)!

trickling filter biotowers.19 Recently, these facilities reconstructed their biological secondary

%*!

treatment to five-stage Bardenpho (anaerobic, anoxic, oxic, anoxic, oxic) processes. Bardenpho

&+!

systems are designed similar to activated sludge by recycling mixed liquor, but incorporate two

&"!

aerobic (oxic) stages for nitrification and two anoxic stages for enhanced denitrification.12,20 By

&#!

incorporating an initial anaerobic stage phosphorous removal is enhanced as microbes release the

&$!

nutrient, then take it up for cell functions in subsequent aerobic/oxic conditions, as described for

&%!

the A/O process.12,21 Although Bardenpho systems are designed to improve nutrient reduction,

&&!

the capability to remove pathogens is relatively unknown.

&'!

The present study investigated the incidence and reduction of Cryptosporidium spp., G.

&(!

intestinalis, and C. cayetanensis in wastewater facilities incorporating five-stage Bardenpho

&)!

processes. Data were compared with results from our previous study on the pre-modified

&*!

facilities utilizing activated sludge and trickling filters.19 This is the first study analyzing

'+!

Bardenpho processes with respect to protozoan removal, while comparing large-scale WWTPs

'"!

receiving sewage from the same geographical region within a four-year period. This study

'#!

coincided with our previous report on the reduction of pathogenic viruses in the same samples.22

'$!

Protozoa concentrations in wastewaters were compared with Escherichia coli, somatic coliphage,

'%!

a pepper virus, and human enteric viruses to determine the validity of each target as an

'&!

alternative indicator for the occurrence and/or adequate reduction of protozoa.

!


&!


2. MATERIALS AND METHODS

''! '(!

Page 6 of 32

2.1!Collections of wastewater samples

')!

Wastewater sampling was conducted monthly at four WWTPs located in southern Arizona

'*!

throughout 12-month time periods, as previously described.19,22 In our previous study carried out

(+!

between August 2011 and July 2012, a total of 48 grab samples were collected from Plants A and

("!

B (12 influent and 12 effluent from each plant) that utilized activated sludge and trickling

(#!

filters.19 In the present study, composite samples were collected via 24 h autosamplers (Hach Sigma

($! (%!

900MAX; Loveland, CO) between June 2014 and May 2015 from Plants C and D which utilized

(&!

advanced five-stage Bardenpho processes for secondary treatment (Figure 1). A total of 47

('!

composite samples were collected from Plant C: 12 influent, 11 dissolved-air-flotation (DAF)

((!

effluent, 12 secondary effluent, and 12 final effluent. Plant D consisted of two separate treatment

()!

trains designated East and West. The West train utilized three parallel five-stage Bardenpho

(*!

processes for secondary treatment, whereas the East train utilized a single five-stage Bardenpho

)+!

plus a modified five-stage pseudo-Bardenpho process (Figure 1). A total of 71 composite

)"!

samples were collected from Plant D: 12 influent, 23 primary sedimentation effluent (11 from

)#!

West and 12 from East), 24 secondary treatment effluent (12 from each train), and 12 final

)$!

effluent. All samples (1 L) were collected in sterile plastic bottles, stored on ice, and transported

)%!

to the laboratory, where the samples were processed within 12 h of collection. Characteristics

)&!

and physicochemical parameters of each WWTP are summarized in Table S1.

)'!

2.2!Concentration of protozoa in wastewater samples Protozoa were concentrated from wastewater samples using an electronegative filter method

)(! ))!

capable of also concentrating viruses, but with slight modifications.23 Briefly, the wastewater

!


'!

Page 7 of 32


)*!

samples (100 mL turbid and 1000 mL clear) were passed through the electronegative filter (cat.

*+!

no. HAWP-090-00; Merck Millipore, Billerica, MA) attached to a glass holder (Advantec,

*"!

Tokyo, Japan), followed by an acid rinse and elution of viruses from the filter.24 To recover

*#!

protozoan (oo)cysts, the filter was detached from the glass filter holder, aseptically cut in half,

*$!

placed in a 50-mL conical tube, and stored at -20 oC until further analysis. The filters were vigorously vortexed in the presence of a ball-shaped stirring bar and 10 mL

*%! *&!

of an elution buffer containing 0.2 g/l Na4P2O7 • 10 H2O (Kanto Chemical, Tokyo, Japan), 0.3

*'!

g/l EDTA (C10H13N2O8) • 3 Na • 3 H2O (Wako Pure Chemical Industries, Osaka, Japan), and 0.1

*(!

mL/L Tween 80 (Research Organics, Cleveland, OH) in a 50 mL plastic tube. The water portion

*)!

of the sample was recovered in another 50 mL plastic tube. The same procedure was repeated

**!

twice with 10 and 5 mL of the elution buffer, and 25 mL of the resulting protozoa-concentrated

"++!

sample was obtained.

"+"!

2.3!Immunomagnetic separation (IMS)

"+#!

The 25-mL protozoa-concentrated sample was centrifuged at 2000 ! g for 10 min at 4 oC, the

"+$!

supernatant was removed, and the pellet was suspended with 10 mL of phosphate buffered saline

"+%!

(PBS). The tube was centrifuged again at 2000 ! g for 10 min at 4 oC and the resulting pellet was

"+&!

suspended in 10 mL of PBS. To purify (oo)cysts, the sample was subjected to immunomagnetic

"+'!

separation (IMS) using the Dynabeads GC-Combo (Life Technologies, Carlsbad, CA) following

"+(!

the manufacturer’s protocol. In brief, Dynabeads" anti-Crytposporidium and Dynabeads" anti-

"+)!

Giardia were added to the sample and rotated for an hour at room temperature. Subsequently, the

"+*!

Dynabeads-organism complexes were pelleted using a Dynabeads" MPC"-1 magnet, and the

""+!

supernatant (10 mL) was recovered for the Cyclospora assay. The pellet was resuspended in 10

"""!

mL of PBS, pelleted again using the magnet, and then the supernatant (10 mL) was recovered !


(!


""#!

and combined with the previous supernatant, resulting in 20 mL of sample for the Cyclospora

""$!

assay.

Page 8 of 32

(Oo)cysts were acid-dissociated from the Dynabeads according to the manufacturer’s

""%! ""&!

protocol with slight modification. Briefly, the pellet was resuspended in 50 µL of 0.1 mol/L HCl

""'!

by vortexing. The Dynabeads were pelleted again using the magnet, and the supernatant (50 µl)

""(!

was collected in a tube containing 10 µL of 1.0 mol/L NaOH for neutralization. This procedure

"")!

was repeated, and the supernatant was recovered in the same tube to obtain purified (oo)cysts

""*!

with a final volume of 110 µL. Next, the 20-mL sample for the Cyclospora assay was

"#+!

centrifuged at 2000 ! g for 10 min at 4 oC, and the pellet was resuspended in PBS to obtain a

"#"!

Cyclospora oocyst suspension with a volume of 1.5 mL.

"##!

2.4!Immunofluorescent assay (IFA)

"#$!

Half of the IMS-purified sample was passed through a hydrophobic polytetrafluoroethylene

"#%!

membrane (pore size, 1.0 µm; diameter, 25 mm; Advantec). Then (oo)cysts were stained using

"#&!

EasyStain (BTF, North Ryde, Australia), and counted with a BX60 fluorescence microscope

"#'!

(Olympus, Tokyo, Japan). Cryptosporidium oocysts were round-shaped (4–6 µm diameter),

"#(!

while Giardia cysts were oval-shaped (5–8 µm diameter; 8–12 µm width) with B excitation

"#)!

(wavelength of 450–490 nm). Particles with G excitation (wavelength of 546 nm) were

"#*!

considered algae due to chlorophyll fluorescence and excluded from the count. We previously

"$+!

reported relatively high and stable recovery efficiencies of (oo)cysts from wastewaters using

"$"!

electronegative filtration followed by IMS-IFA ,19 which had comparable efficiency to other

"$#!

reports utilizing the US EPA Method 1622.25-26 It is assumed that recoveries in the current study

"$$!

were similar since the same procedures, equipment, and personnel were used to process

"$%!

wastewater samples.

!


)!

Page 9 of 32

"$&!


2.5!Protozoan nucleic acid extraction

"$'!

The other half of the IMS-purified sample (oo)cysts (55 µL) was used for nucleic acid

"$(!

extraction. For the Cyclospora assay, a 200-µL portion of the 1.5 mL sample was used for

"$)!

nucleic acid extraction. The samples were subjected to ten cycles of freeze–thaw (!80 °C for 10

"$*!

min and 56 °C for 5 min), followed by nucleic acid purification using the QIAamp DNA Mini kit

"%+!

(Qiagen, Hilden, Germany) to obtain 200 µL of DNA extract, and further concentrated using

"%"!

Amicon Ultra-0.5 with a nominal molecular weight limit (NWML) of 30 kDA (Merck Millipore)

"%#!

to obtain a final volume of 30 µL.

"%$!

2.6!PCR detection of protozoa Nested PCR targeting the 18S rRNA gene of Cryptosporidium spp. and semi-nested PCR

"%%! "%&!

targeting the GDH gene of G. intestinalis were carried out as described previously.27-29 For each

"%'!

target, the first PCR was performed in 50 µL of reaction volume containing 5 µL of DNA, 25 µL

"%(!

of Premix Ex Taq Hot Start Version (Takara Bio, Otsu, Japan), and 15 pmol of each primer. The

"%)!

second PCR was performed using the same volumes, except with 2 µL of the first PCR product

"%*!

as the template. Amplification conditions for the first and second PCR were identical (Table S2).

"&+!

SYBR Green-based quantitative PCR (qPCR) was used to target C. cayetanensis-specific

"&"!

single round internal transcribed spacer 2 (ITS-2) gene, as previously described.19,30 Briefly, 5

"&#!

µL of DNA was mixed with 20 µL of PCR mixture containing 12.5 µL of SYBR Premix Ex Taq

"&$!

II (Tli RNaseH Plus) (Takara Bio) and 10 pmol of each primer. At the end of 45 amplification

"&%!

cycles, a melting curve analysis was performed to confirm specific amplification of the target

"&&!

gene. The qPCR reactions for the wastewater samples were performed in duplicate and

"&'!

considered positive only when both tubes fluoresced with sufficient intensity and the average

"&(!

cycle threshold (CT) value was not more than 40, as recommended.31 Primer sequences and

!


*!


"&)!

conditions for all PCR assays are provided in Table S2.

"&*!

2.7!E. coli and somatic coliphages

Page 10 of 32

To determine whether final effluent for each WWTP met microbiological criteria for

"'+! "'"!

recreational waters,32 E. coli was assayed using Colilert (IDEXX Laboratories, Inc., Westbrook,

"'#!

ME) with Quanti-Tray 2000 following manufacturer’s instructions and expressed as most

"'$!

probable number (MPN) per 100 mL. Somatic coliphages were enumerated following the single

"'%!

and double agar layer protocols with E. coli CN-13 (ATCC® no. 700609™) as a host.33 Briefly, 1

"'&!

mL of diluted/raw sample and 500 µL of log-phase host bacteria were mixed with 5 mL of liquid

"''!

2! tryptic soy agar (TSA) and poured onto solidified 2! TSA bottom plates. Plates were left at

"'(!

ambient temperature (22 oC) until the top agar solidified, inverted, incubated at 37 oC for 16 h,

"')!

and counted for plaque-forming units (PFU) per mL.

"'*!

2.8!Statistical analyses Nonparametric Kruskal-Wallis H tests, Wilcoxon Rank Sum post hoc tests (two-tailed), and

"(+! "("!

Student’s t-test were performed with Microsoft Excel for Mac 2015 (Microsoft Corp., Redmond,

"(#!

WA) to determine whether protozoa log10 reduction values (LRV) at WWTPs and treatment

"($!

stages were statistically different (alpha level of 0.05). Spearman’s rank correlation coefficient

"(%!

tests were performed to determine associations between the concentrations of microbial targets at

"(&!

each treatment stage. 3. RESULTS

"('! "((!

3.1!Detection of Protozoa in Wastewater The incidence of Cryptosporidium and Giardia (oo)cysts was detected via IFA microscopy.

"()! "(*!

Cryptosporidium oocysts were detected in 52 out of 118 wastewater samples: 19 of 24 raw

")+!

sewage, 25 of 34 primary effluent, 5 of 36 secondary effluent, and 3 of 24 final effluent (Table

!


"+!

Page 11 of 32


")"!

S3). Giardia cysts were detected in 79 out of 118 wastewater samples: 23 of 24 raw sewage, 34

")#!

of 34 primary effluent, 14 of 36 secondary effluent, and 8 of 24 final effluent (Table S3). The incidence of Cryptosporidium spp. and G. intestinalis was also tested via (semi)nested

")$! ")%!

PCR, whereas C. cayetanensis was tested via SYBR Green-based qPCR. Detection of

")&!

Cryptosporidium spp. by PCR was rare, as only 7 out of 118 samples had positive results (Tables

")'!

S3 and S4). However, Giardia was detected in 51 out of 118 samples with high incidence in raw

")(!

sewage (20 of 24) and primary effluent (21 of 34) wastewaters (Table S3 and S5). C.

"))!

cayetanensis was not detected in any of the samples (Table S3 and Table S6).

")*!

3.2!Reduction of Protozoa Quantifications of Cryptosporidium spp. and G. intestinalis via IMS-IFA detection were used

"*+! "*"!

to determine concentrations in wastewaters. Differentiating viable (oo)cysts with internal

"*#!

structures from those that were empty was difficult using microscopy. Therefore, all (oo)cysts

"*$!

were assumed to be viable and infectious as a conservative approach to estimating the reduction

"*%!

for public health concerns. Reduction values were calculated as the difference in concentrations

"*&!

between influent and effluent sample sets (Tables 1 and 2). However, values were adjusted by

"*'!

substituting detection limits of 40 (influent and primary effluent) or 4 (secondary and final

"*(!

effluents) (oo)cysts/L for negative samples to calculate the lower limit of log10 removals and

"*)!

provide a more-conservative determination of actual removal. Only sample sets with positive

"**!

concentrations above detection limits in inlet wastewater sources were considered when

#++!

calculating LRV (Tables 1 and 2). Previously reported data from Plants A and B were also

#+"!

adjusted by these criteria to maintain consistency for comparisons with WWTPs in the current

#+#!

study. The rates of reduction to concentrations below detection were monitored to evaluate how

#+$!

often treatment resulted in no observable (oo)cysts, suggesting optimal removal of protozoa

!


""!


Page 12 of 32

#+%!

(Table S7). Data from Plants C and D are described below and compared with data from Plants

#+&!

A and B.

#+'!

Cryptosporidium LRV were not significantly different between Plants C and D (Table 1).

#+(!

However, LRV from both Bardenpho facilities were significantly greater than those from Plants

#+)!

A and B (Table 1). Also, Plants C and D more often produced effluent samples with no

#+*!

observable oocysts and concentrations below detectable limits (Table S7). Regarding Giardia, there was also no significant difference in the LRV between Plants C

#"+! #""!

and D (Table 1). Removal by Plant C was significantly greater than both Plants A and B (Table

#"#!

1). However, LRV at Plant D were not significantly different from Plant A that employed

#"$!

activated sludge but were significantly greater than LRV from Plant B that used trickling filters

#"%!

(Table 1). Cyclospora LRV could not be compared as no samples were positive between June 2014 and

#"&! #"'!

May 2015.

#"(!

3.3!Removal of Protozoa Throughout Treatment Processes

#")!

DAF did not result in more efficient removal of protozoa, as LRV for Cryptosporidium

#"*!

oocysts during primary treatment in Plant C were not significantly different than those from

##+!

sedimentation tanks in Plant D (Table 2). In fact, sedimentation tanks had higher LRV of

##"!

Giardia cysts, as mean concentrations remained nearly the same as raw sewage after DAF in

###!

Plant C, but decreased by 0.4 and 0.6 log10 cysts/L following sedimentation in the West and East

##$!

trains in Plant D (Figure 2; Table 2).

##%!

The majority of protozoa removal occurred during secondary treatment. LRV of (oo)cysts

##&!

from Bardenpho processes were significantly greater than those from both sedimentation and

##'!

tertiary treatment in each train (Table 2). In Plant C, Bardenpho treatment resulted in mean LRV

!


"#!

Page 13 of 32


##(!

of 1.86 and 2.49 log10/L of Cryptosporidium and Giardia (oo)cysts, respectively (Figure 2; Table

##)!

2). In Plant D, Bardenpho reduced Cryptosporidium oocysts by 1.63 and 1.49 log10/L and

##*!

Giardia cysts by 2.07 and 1.73 log10/L in the West and East treatment trains, respectively (Figure

#$+!

2; Table 2). Only one Cryptosporidium oocyst was detected in 1 of 12 secondary effluents from

#$"!

each Plant C and Plant D-West, as well as 3 of 12 samples from Plant D-East. However, at least

#$#!

one Giardia cyst was detected in 5 of 12 secondary effluent samples from Plant C, 3 of 12

#$$!

samples from Plant D-West, and 6 of 12 samples from Plant D-East. All other samples did not

#$%!

contain (oo)cysts, as Bardenpho treatment often reduced concentrations to levels below detection

#$&!

limits.

#$'!

Tertiary treatment resulted in minimal physical removal of protozoa, as (oo)cysts were

#$(!

detected in final effluents from all of the sample sets that contained more than one observable

#$)!

cyst (>0.6 log10/L) following secondary treatment. In Plant C, sequential disc filtration did not

#$*!

improve removal as the filter pore sizes (20 "m) were larger than (oo)cysts. Reduction via

#%+!

disinfection could not be assessed since the methods used to detect (oo)cysts did not evaluate

#%"!

infectivity. However, since protozoa are often insensitive to chlorine,4,6 especially

#%#!

Cryptosporidium,34 all (oo)cysts observed via IFA were assumed to be infectious to provide a

#%$!

conservative public health approach. Still, LRV from tertiary treatment could not be calculated,

#%%!

as the both the inlet and outlet water data were censored due to the detection limit being

#%&!

substituted for nearly all samples (Table 2). Secondary effluent, which is the raw feed for tertiary

#%'!

treatment, often did not contain observable (oo)cysts; thus, calculating the mean LRV from

#%(!

sample sets in which the detection limit was substituted for the majority of inlet concentrations

#%)!

would not be appropriate for estimating removal.

#%*!

3.4!Incidence and Reduction of E. coli and Somatic Coliphages

!


"$!


Page 14 of 32

On average, Plants C and D reduced E. coli concentrations by 6.70 and 5.71 log10 MPN/100

#&+! #&"!

mL, respectively (Table 1). Final effluents from each Bardenpho WWTP were below

#&#!

recreational guidelines (126 MPN/100 mL)32 for E. coli in 10 of 11 (91%) samples, with

#&$!

concentrations 0.76 ± 0.22* (n = 12)

> 0.96 ± 0.38* (n = 10)

> 1.67 ± 0.39!! (n = 10)

> 1.52 ± 0.54** (n = 9)

Giardia (cysts/L)

> 2.16 ± 0.51*, (n = 12)

> 1.52 ± 0.62* (n = 12)

> 2.63 ± 0.37** (n = 11)

> 2.44 ± 0.47**, (n = 12)

0.00009

0.048

0.398

1.714

Cyclospora (copies/L)

n/a

n/a

n/a

n/a

E. coli (MPN/100 mL)

n/a

n/a

6.70 ± 0.87 (n = 11)

5.71 ± 1.08 (n = 11)

P valueb

Somatic coliphage n/a n/a 4.75 ± 0.33 4.73 ± 0.28 (PFU/mL) (n = 12) (n = 12) Log10 reduction values (LRV) and standard variations were calculated as the mean difference between influent and effluent sample sets. Only sample sets with positive detection in influent samples were used to calculate values. Detection limit values were substituted for effluent values that were below the detection limit, or not detected at all, in order to calculate the lower limit of log10 removals. Sample sets with substituted values are represented with a ‘greater than’ symbol, as the actual reduction may be greater than calculated. n; number of sample sets used to calculate LRV and standard deviation. Numbers labeled with the same symbol (*, **, or †) are not significantly different - in other words, different symbols denote significant difference. LRVs for Cryptosporidium and Giardia were compared between WWTPs (by row), as calculated by the Kruskal-Wallis H tests and Wilcoxon Rank Sum post-hoc


! "#!

Page 21 of 32


tests (! of 0.05, two tailed). a Data previously reported by Kitajima et al. (2014)19 for Plant A and Plant B were also adjusted by substituting the detection limit for values observed below the limits (see section 3.2). b P value of Student’s t-test (two tailed) comparing the log10 reductions of Cryptosporidium and Giardia at each WWTP. n/a; LRV was not able to be calculated, as Cyclospora was not detected in Plants C and D, no sample sets were positive for Cyclospora in both influent and effluent from Plants A and B, E. coli assays were only performed on effluent samples from Plants A and B, and somatic coliphage were not analyzed for Plants A and B.


! "$!


Table 2: Mean log10 reduction values at treatment stages Cryptosporidium Giardia (oocysts/L) (cysts/L) Plant C > 0.18 ± 0.55 (n = 10) 0.29 ± 0.95 (n = 11) DAF (1o) o * Bardenpho (2 ) > 1.86 ± 0.45 (n = 8) > 2.49 ± 0.52 (n = 11)* Filter + disinfect. (3o) n/a n/a Plant D - West Sedimentation (1o) Bardenpho (2o) Disinfection (3o)

> 0.06 ± 0.26 (n = 9) > 1.63 ± 0.44 (n = 7)* n/a

> 0.48 ± 0.26 (n = 12) > 2.07 ± 0.29 (n = 11)* n/a

Page 22 of 32

E. coli (MPN/100 mL)

Somatic Coliphage (PFU/mL)

0.10 ± 0.57 (n = 10) 5.36 ± 1.55 (n = 10)* 2.57 ± 1.15 (n = 7)

0.45 ± 1.30 (n = 12) 3.37 ± 1.70 (n = 12)* 1.15 ± 0.70 (n = 10)

0.07 ± 0.52 (n = 10) 6.64 ± 0.91 (n = 10)* 0.09 ± 0.65 (n = 3)

0.43 ± 1.26 (n = 12) 4.29 ± 1.40 (n = 12)* 0.23 ± 0.39 (n = 3)

Plant D - East Sedimentation (1o) > 0.02 ± 0.27 (n = 9) 0.60 ± 0.59 (n = 12) 0.05 ± 0.42 (n = 11) 0.06 ± 0.13 (n = 12) Bardenpho (2o) > 1.49 ± 0.39 (n = 10)* > 1.73 ± 0.35 (n = 12)* 3.19 ± 1.46 (n = 11)** 4.09 ± 0.66 (n = 12)* Disinfection (3o) n/a n/a 2.78 ± 1.07 (n = 10)** 0.71 ± 0.53 (n = 10) Log10 reduction values (LRV) and standard variations were calculated as the mean difference between influent and effluent sample sets for each treatment stage. n; number of sample sets used to calculate LRV. Only sample sets with positive detection in inlet waters to the treatment stage were used to calculate values. Detection limit values were substituted for effluent values that were below the detection limit, or not detected at all, in order to calculate the lower limit of log10 removals. Sample sets with substituted values are represented with a ‘greater than’ symbol, as the actual reduction may be greater than calculated. n/a; LRV were not able to be calculated as the both the inlet and outlet water data were censored due to the detection limit being substituted for nearly all samples. Kruskal-Wallis H tests, Wilcoxon Rank Sum post-hoc tests, or Student’s t-tests (! of 0.05, two tailed) were used to compare treatment stages within each train, as well as compare treatment processes across separate trains. * Log10 reduction values from Bardenpho


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treatment are significantly greater than reductions from other processes in the given treatment train. ** Log10 reduction values from Bardenpho and disinfection are not significantly different, but both are significantly greater than sedimentation.


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FIGURES Primary

Influent Sedimentation

Biological Treatment

Plant A

Activated Sludge

Plant B

Trickling Filter

Secondary Sedimentation

Chlorination + Dechlorination Effluent

5-Stage Bardenpho

Plant C

Disc Filtration

DAF

East

Pseudo-Bardenpho

5-Stage Bardenpho

Plant D

West 5-Stage Bardenpho

Figure 1: Schematic of each WWTP. Hollow X ( ) indicates grab sample collection locations. Solid X ( ) indicates 24-hour autosampler collection locations. Adapted from Schmitz, B. W.; Kitajima, M.; Campillo, M. E.; Gerba, C. P.; Pepper, I. L. Virus reduction during advanced Bardenpho and conventional wastewater treatment processes. Environ. Sci. Technol. 2016, 50, 9524-9532; DOI 10.1021/acs.est.6b01384. Copyright 2016 American Chemical Society.

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4.0

Log10 (oo)cysts/liter

3.5 3.0 2.5 2.0 1.5 1.0

Plant C

Plant D

Plant C

Cryptosporidium

Effluent

East 2°

West 2°

East 1°

West 1°

Influent

Effluent

2°

DAF

Influent

Effluent

East 2°

West 2°

East 1°

West 1°

Influent

Effluent

DAF

Influent

0.0 (-)

2°

0.5

Plant D Giardia

Figure 2: Concentrations of Cryptosporidium (red) and Giardia (blue) (oo)cysts in wastewater at different treatment stages in Plants C and D. The boxes represent 50% of the data, the horizontal lines represent the mean, and the lines (“whisker”) extending from the boxes represent maximum and minimum values. Lowest possible concentrations are at the minimum detectable limits: 1.6 log10 (oo)cysts/L (influent, DAF, and primary effluent) or 0.6 log10 (oo)cysts/L (secondary or final effluent). DAF: dissolved-air-flotation effluent; 1o: Primary treatment effluent; 2o: Secondary treatment effluent; (-), not detected.

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Page 26 of 32

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Reduction of Cryptosporidium, Giardia, and Fecal Indicators by

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