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Giardia and Cryptosporidiumremoval from waste-waterby a duckweed (Lemna gibba L.) covered pond

J.A. Falabi, C.P. Gerba and M.M. KarpiscakDepartment of Soil, Water and Environmental Science, Office of Arid Lands Studies, The University of Arizona,

Tucson, AZ, USA

2001/284: received 25 September 2001 and accepted 28 January 2002

J . A . F A L A B I , C . P . G E R B A A N D M . M . K A R P I S C A K . 2 0 0 2 .

Aims: To determine the ability of duckweed ponds used to treat domestic waste-water to

remove Giardia and Cryptosporidium.

Methods and Results: The influent and effluent of a pond covered with duckweed with

a 6 day retention time was tested for Giardia cysts, Cryptosporidium oocysts, faecal coliforms

and coliphage. Giardia cysts and Cryptosporidium oocysts were reduced by 98 and 89%,

respectively, total coliforms by 61%, faecal coliforms by 62% and coliphage by 40%. Therewas a significant correlation between the removal of Giardia cysts and Cryptospordium oocysts

by the pond (P < 0001). Influent turbidity and parasite removal were also significantly

correlated (Cryptosporidium and turbidity, P 005; Giardia and turbidity, P 001).

Conclusions: The larger organisms (parasites) probably settled to the bottom of the pond,

while removal of smaller bacteria and coliphages in the pond was not as effective.

Significance and Impact of the Study: Duckweed ponds may play an important role in

wetland systems for reduction of Giardia and Cryptosporidium.

INTRODUCTION

The ability of wetland and aquatic systems to improve the

quality of waste-water is well documented (Gersberg et al.

1987; Hammer 1989; Kadlec and Knight 1996). Different

studies have shown that aquatic plants, such as water

hyacinth (Eichhornia crassipes L.), water lettuce (Pistia

stratiotes L.) and duckweed (Lemna spp.), are capable of

reducing biochemical oxygen demand (BOD), total suspen-

ded solids (TSS), and nitrogen and phosphorus concentra-

tions in waste-water (Dewedar and Bahgat 1995). However,

little or no information is available on the fate of pathogenic

protozoan parasites from secondarily-treated sewage (acti-

vated sludge) applied to constructed wetlands. The removal

of pathogenic organisms in aquatic or wetland systems couldbe the result of several factors, including natural die-off,

sedimentation, predation and adsorption (Reed et al. 1995).

These factors, in turn, are likely influenced by detention

time and seasonal variability. The objective of this study was

to evaluate the ability of an aquatic system covered with

duckweek to remove protozoan parasites (Giardia and

Cryptosporidium), indicator bacteria and coliphages from

activated sludge effluent. Physical-chemical data such as

temperature, turbidity and pH were measured to determine

whether microbial removal was related to any of these

parameters.

MATERIALS AND METHODS

The constructed wetland facility

The constructed ecosystem research facility (CERF) began

operation in January 1989, and is located adjacent to the

Roger Road Wastewater Treatment plant operated by Pima

County in Tucson, Arizona. The facility is operated by The

University of Arizonas Office of Arid Lands Studies for the

Pima County Wastewater Management Department. The

duckweed pond used in this study is 65 m long, 11 9 m wide

and 26 m deep, with an average influent flow rate of 55

(range 4759) litres min)1. The operational depth of the

pond during this study was 09 m. The average detention

time during the period of this study was estimated at 9 days.

A free-floating aquatic weed called duckweed (Lemna gibba

L.) was the primary plant covering the entire surface of

the pond. The pond receives secondary quality effluent

Correspondence to: C.P. Gerba, Department of Soil, Water and Environmental

Science, Office of Arid Lands Studies, The University of Arizona, Tucson, AZ

85721, USA (e-mail: gerba@ag.arizona.edu).

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(activated sludge) from the Pima County Roger Road

Wastewater Treatment Facility diverted to CERF just prior

to chlorination.

Sample collection and processing

Water samples of influent (waste-water entering the pond)

and effluent (waste-water exiting the pond) were collected

from June 1994 through May 1996 from the duckweed-

covered pond. These samples were analysed for Giardia

cysts and Cryptosporidium oocysts (110 litres), total and

faecal coliforms (50 ml), and coliphages (50 ml). The

samples were usually collected once a month in sterile

plastic bottles and transported on ice to the laboratory for

analysis. Total coliform and faecal coliform bacteria samples

were processed within 6 h, coliphage samples within 72 h

and parasite samples within 48 h.

Detection of Giardia and Cryptosporidium

Volumes of influent and effluent waste-water of 14 litres

were collected directly from the pond in sterile plastic

bottles. Giardia and Cryptosporidium were detected simulta-

neously in the samples using an indirect immunofluores-

cence method (APHA 1992a). The immunofluorescent

method includes three major steps: parasite concentration

into a pellet, pellet floatation to clarify the samples, and

antibody staining for the detection of the parasite using a

microscope with fluorescent light. Giardia cysts and

Cryptosporidium oocysts were concentrated from the water

samples by centrifugation at 1050 g for 15 min in a swingingbucket rotor centrifuge (Jouan, Inc., Winchester, VA, USA).

The pellet provided by initial centrifugation was

resuspended in floated Sheathers solution (APHA 1992a).

The pellet obtained, following floatation, was then distri-

buted on pre-wetted, 25 mm diameter cellulose acetate

filters with a 02 lm pore size (Costar Corp., Pleasanton,

CA, USA). The filters were labelled with fluorescent

antibodies (Hydrofluor Combo Meridian Diagnostics,

Cincinnati, OH, USA). These antibodies are specific for

both Giardia cysts and Cryptosporidium oocysts. During each

assay, positive and negative controls ensured that assay

reagents worked properly. The filters were then examinedmicroscopically. Cysts and oocysts were identified according

to specified criteria: immunofluorescence, size, shape and

internal morphological characteristics (APHA 1992a). The

results were reported as the total number of Giardia and

Cryptosporidium per litre of sample.

Detection of indicator bacteria

Total and faecal coliforms were detected by membrane

filtration on selective media, i.e. mEndo agar (Difco) for

total coliforms and mFC agar (Difco) for faecal coliforms,

according to the Standard Methods (APHA 1992b). If

necessary, samples were diluted using a Tris-buffered saline

solution which was prepared by mixing 1600 ml distilled

water and 632 g Trizma Base (Tris hydroxymethyl amino

methane; Sigma). Various dilutions of the samples werefiltered through 045 lm pore size filters (Gelman Sciences,

Ann Arbor, MI, USA). The filters were then placed on the

selective medium and incubated for 24 h at 37C.

Detection of coliphages

Coliphages were detected by the double layer agar method

described by Adams (1959). The host bacteria used for the

assay was Escherichia coli strain ATTC 15597 (American

Type Culture Collection, Rockville, MD), which detects

both somatic and male specific coliphages. Waste-water

samples were filtered through 0

22 lm pore size filters(Gelman) to remove bacteria that can interfere with the

visualization of the coliphage plaques. The filters were

treated with 3 ml 15% beef extract (Becton Dickinson) to

avoid phage adsorption to the filters. The filtered samples

(1 ml) to be assayed and 1 ml of the host strain culture were

added to the previously melted soft top agar (Tryptic Soy

Broth + 1% agar). All samples were assayed in triplicate.

Plaque enumeration was determined after 18 h of incubation.

Determination of physical/chemical parameters

The influent and effluent water turbidity was determined

using a turbidimeter 2100P (Hach Company, Loveland, CO,USA). The water pH was determined using both pH

indicator strips (EM Science, Gibbstown, NJ, USA) and a

Corning pH meter, model 345 (Corning Inc., Corning, NY,

USA). The water temperature was determined using a

thermometer.

Statistical analysis of the data

The percentage removal of the studied micro-organisms was

calculated using the following formula:

% removal Ninfluent Neffluent 100=Ninfluent

where Ninfluent number of micro-organisms in the influentwaste-water, Neffluent number of micro-organisms in theeffluent waste-water.

Cyst, oocyst, faecal coliform, total coliform and coli-

phage percentage removals were transformed for analysis

using log10(y + 1), where y number of micro-organisms.Arithmetic averages were calculated and correlation coeffi-

cients were developed for turbidity, temperature and each of

the micro-organisms studied using Microsoft Excel Version

50. The average percentage removal of micro-organisms

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was calculated using the above formula. The values of

Ninfluent and Neffluent used were the averaged numbers of

micro-organisms in the influent and effluent.

R E S U L T S A N D D I S C U S S I O N

During the period of this study (JulyMay), 15 sets of

samples were collected for analysis approximately every 35

weeks. The average number of Giardia was 156 cysts l)1 in

the influent and 035 cysts l)1 in the effluent, resulting in an

average percentage reduction of 98%. On average, Cryptos-

poridium oocysts decreased by 89%, with an average number

of 158 oocysts l)1 in the influent and 017 oocyst l)1 in the

effluent (Table 1). Grimalson et al. (1993) studied the

occurrence and removal of Cryptosporidium oocysts in

Kenyan waste stabilization ponds. The oocyst levels they

detected in raw waste-water samples ranged from 212 to

6213 cysts l

)1

. They also observed that the number ofoocysts in the influent from these ponds ranged between 3

and 230 cysts l)1. No Cryptosporidium oocysts were noted in

the final effluent from the 11 ponds studied. The minimum

detention time for the removal of Cryptosporidium oocysts

and Giardia cysts by the stabilization ponds was 37 days.

The average number of total coliforms was 424 106 cfu

(colony-forming units) 100 ml)1 in the influent and

165 106 cfu 100 ml)1 in the effluent. The number of

faecal coliforms averaged 177 106 cfu 100 ml)1 in the

influent and 597 105 cfu 100 ml)1 in the effluent. Total

and faecal coliform bacteria were reduced, on average, by 61

and 62%, respectively, in the duckweed pond. This

compares with the 9099% removal reported for faecalcoliforms in facultative ponds with detention times of

47180 days (EPA 1983).

The average number of coliphages was 1233 pfu (plaque-

forming units) ml)1 in the influent and 742 pfu ml)1 in

the effluent. Coliphage reduction averaged about 40%

(Table 1). Gersberg et al. (1987) found that the total

number of indigenous F-specific bacteriophage (F-specific

RNA and F-specific DNA phages) was reduced by about

99% after passage through a constructed wetland composed

of bulrush planted in gravel with a detention time of

55 days. They also found that poliovirus added to the

wetland was reduced by 999%. During the period of this

study, the temperature of the influent to the duckweed pond

ranged from 21 to 2C, and from 11 to 31C in the effluent.

The turbidity values ranged from 33 to 23

6 NTU

(Nephelometric Turbidity Unit) for the influent and from

104 to 746 for the effluent. The turbidity removal by the

pond ranged from 0 to 49%. The influent pH ranged from

66 to 84 and the effluent pH, from 65 to 82.

Pathogen removal in pond systems is believed to be due to

a variety of factors, among which are temperature, natural

die-off, sedimentation and adsorption (Reed et al. 1995).

Helminths, Ascaris and other parasitic cysts and eggs settle

to the bottom in the quiescent zone of the pond. Duckweed

lacks extensive root systems onto which micro-organisms

can become attached, and they also decrease sunlight below

the duckweed mat; therefore, the removal of micro-organ-isms in duckweed-covered ponds is likely the result of

sedimentation. In this study, the larger the organisms, the

greater the percentage removal (Table 1). The larger

organisms settle more rapidly to the bottom of the pond,

while the removal of viruses was not as effective. Studies by

Reed et al. (1995) on the removal of faecal coliforms and

enteric viruses in multiple-cell pond systems showed a

significant reduction after passage through the pond.

Therewas no correlation between Giardia, Cryptosporidium,

coliform bacteria and coliphage removal, and the water pH

and temperature. There was a weak correlation between the

removal of total coliforms and Cryptosporidium oocysts

(P 010). However, more data are needed to assess the

significance of that correlation. Giardia cyst and Cryptospo-

ridium oocyst removal, and influent turbidity, were signifi-

cantly correlated (P 001 for Giardia and turbidity;P 005 for Cryptosporidium and turbidity). The removalof Cryptosporidium oocysts and Giardia cysts was signifi-

cantly correlated (P < 0001). No correlation existed on

effluent quality and the parameters studied (data not

shown). Rose et al. (1991) compared the occurrence of

Cryptosporidium and Giardia in surface waters and found

Table 1 Average densities and removal of micro-organisms by the duckweed pond

Micro-organisms Influent Effluent

Percentage

removal (%)

Size of organisms

(lm)

Giardia (l)1) 156 (429)* 035 (006) 98 812

Cryptosporidium (l)1) 158 (0325) 017 (005) 89 26

Total coliforms (100 ml)1) 424 106 (822 10

5875 106) 165 10

6 (85 10438 10

6) 61 1115

Faecal coliforms (100 ml)1) 177 106 (13 10553 106) 597 105 (65 103122 106) 62 1115

Coliphages (ml)1) 1233 (6531987) 742 (691454) 40 00450065

*Observed range of values.

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that the concentrations of these two parasites were signifi-

cantly correlated in all the waters analysed (P < 001). The

recovery rate of Giardia and Cryptosporidium may be low

when the waste-water turbidity is high because visualization

of the cysts and oocysts is difficult under the microscope.

There was no correlation between the removal of theparasites and the effluent turbidity.

The duckweed pond was more effective in reducing the

number of protozoan parasites (Giardia 98%; Cryptospori-

dium 89%) than indicator bacteria (total coliform 61%,

faecal coliform 62%) or coliphages. The removal of micro-

organisms in the pond appeared to be related to the size of

the organisms (Table 1).

This study only assessed the physical removal of the cysts

and oocysts, and not their viability. Recently, Araki et al.

(2001) demonstrated that the physicochemical conditions in

high-rate algal ponds were responsible for a 97% reduction

in oocyst viability. Thus, the total reduction of infectiouscysts and oocysts is probably greater than what was

observed.

Aquatic systems for waste-water treatment appear to be

promising as tertiary treatment systems for enteric patho-

gens. Additional detention time could increase the removal

capability of these systems, increasing the detention time,

which would slow the flow rate through the pond, allowing

more time for the micro-organisms to settle before the water

reaches the outlet of the pond.

REFERENCES

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