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. 2007 Feb 2;73(6):2013–2015. doi: 10.1128/AEM.02412-06

Human Enteropathogen Load in Activated Sewage Sludge and Corresponding Sewage Sludge End Products

Thaddeus K Graczyk 1,2,*, Frances E Lucy 3, Leena Tamang 1, Allen Miraflor 4
PMCID: PMC1828822  PMID: 17277215

Abstract

This study demonstrated a significant reduction in the concentrations of Cryptosporidium parvum and Cryptosporidium hominis oocysts, Giardia lamblia cysts, and spores of human-virulent microsporidia in dewatered and biologically stabilized sewage sludge cake end products compared to those of the respective pathogens in the corresponding samples collected during the sludge activation process.


Cryptosporidium parvum, Cryptosporidium hominis, Giardia lamblia, and human-virulent microsporidia (i.e., Encephalitozoon intestinalis, Encephalitozoon hellem, Encephalitozoon cuniculi, and Enterocytozoon bieneusi) are enteric pathogens that inflict considerable morbidity in healthy people and can cause mortality (e.g., Cryptosporidium) in individuals with various immunodeficiencies (6, 18, 21). Their transmissive stages, i.e., oocysts, cysts, and spores, are highly resistant to environmental stressors, and therefore, they are long lasting and ubiquitous in the environment (3, 6, 12). It is believed that spreading sewage sludge on agricultural lands, which has increased during recent years due to economic and environmental reasons (13), facilitates the circulation of Cryptosporidium and Giardia in the environment and contaminates potable waters via surface runoff (15), thus posing a public health threat. However, other studies provided evidences that the sludge activation process significantly reduced (i.e., up to 99.8%) the level of Giardia compared to that of the raw sewage (2, 20). The purpose of the present study was to quantitatively determine and compare the concentrations of viable Cryptosporidium parvum and C. hominis oocysts, G. lamblia cysts, and spores of E. intestinalis, E. hellem, E. cuniculi, and E. bieneusi in sewage sludge during the activation secondary treatment process and in the corresponding sewage sludge end products.

The samples originated from urban wastewater treatment plants: Collooney (54°11′37"N, 08°28′93"W), Strandhill (54°16′89"N, 08°35′95"W), Grange (54°23′53"N, 08°31′79"W), and Keadue (54°03′18"N, 08°09′12"W), Ireland. All plants utilized primary treatment by coarse screening. The Collooney, Strandhill, and Grange plants employed secondary treatment, i.e., sludge activation and sedimentation. The Keadue plant uses a rotating filter and settlement tank. The sewage effluent is then polished using a reedbed, wetland filtration system, with effluent discharging directly to Lake Meelagh. The activated sludge samples (2 liters) were collected directly from the top wastewater layer during the activation process. The corresponding dewatered biologically stabilized sewage sludge cake samples (1 kg) were collected from the top layer of the dewatering beds. At the Keadue wastewater plant, a dewatered biologically stabilized sewage sludge sample was collected as described above in addition to a sample of final wetland filtration effluent (2 liters). Dewatered sludge samples were homogenized with water to a volume of 2 liters. All collected samples were vigorously homogenized, transferred to conical plastic flasks of 2.5-liter capacity, and left overnight at 4oC for gravity sedimentation (1, 5, 16). Fifty milliliters of the top sediment layer was transferred to a plastic 50-ml-capacity tube and centrifuged (3,000 × g, 5 min), the supernatant was decanted, and the resulting pellet was transferred to a 1.5-ml-capacity plastic tube. The solution was mixed with an equal volume of 75% ethanol and stored at 4oC. Alcohol was washed from the pellets by centrifugation (10,000 × g, 10 min) two times using sterile phosphate-buffered saline, and the resulting pellet was subjected to sugar-phenol flotation (1). The pellet was evenly divided into two aliquots. One aliquot was processed for C. parvum, C. hominis, and G. lamblia by multiplexed combined fluorescent in situ hybridization and a direct immunofluorescence assay and the other for human-virulent microsporidia by multiplexed fluorescent in situ hybridization (7-11, 14, 17).

There was a significant reduction of pathogen concentration in dewatered and biologically stabilized sewage sludge cake compared to the concentrations of the respective pathogens in the corresponding sludge samples collected during the activation process (Table 1). Giardia lamblia was found at a significantly higher concentration in activated sewage sludge at Collooney, Strandhill, and Grange (mean, 843 cysts/liter) than Cryptosporidium (mean, 338 oocysts/liter) or microsporidia (mean, 166 spores/liter) (F = 5.2, P < 0.05 [Kruskal-Wallis analysis of variance]). However, the highest concentrations of G. lamblia (i.e., 1,540 cysts/liter) and E. bieneusi (371 spores/liter) were found in the final effluent from the wetland filtration system at Keadue (Table 1). The highest concentration of Cryptosporidium was 650 oocysts/liter in the activated sewage sludge, and the lowest was 43 oocysts/liter (Table 1). Enterocytozoon bieneusi was found in activated sewage sludge samples from all wastewater treatment plants, and the mean concentration (i.e., 224 spores/liter) was significantly higher than the concentration of E. intestinalis (i.e., 51 spores/liter) (F = 13.7, P < 0.02 [Kruskal-Wallis analysis of variance]) (Table 1). The overwhelming majority of oocysts, cysts, and spores identified in sewage sludge-derived samples were viable; the fraction of nonviable pathogens constituted no more than 1%.

TABLE 1.

Effect of the sewage sludge activation process on the removal of viable human enteropathogensa

Wastewater plant Pathogen species Pathogen concn in:
Removal efficiency (%)
AcSS SSC
Collooney Cryptosporidium parvum/ C. hominis 320 110 65.6
Giardia lamblia 1,100 43 96.1
Enterocytozoon bieneusi 245 13 94.7
Encephalitozoon intestinalis 47 5 89.4
Strandhill Cryptosporidium parvum/ C. hominis 43 5 88.4
Giardia lamblia 540 3 99.4
Enterocytozoon bieneusi 31 5 83.9
Grange Cryptosporidium parvum/ C. hominis 650 64 90.2
Giardia lamblia 890 114 87.2
Enterocytozoon bieneusi 247 33 86.7
Encephalitozoon intestinalis 54 3 94.4
Keadue Cryptosporidium parvum/ C. hominis 0* 0
Giardia lamblia 1,540* 5
Enterocytozoon bieneusi 371* 0
a

The concentrations of enteropathogens in activated sewage sludge (in cells/liter) during the activation process and in the corresponding sewage sludge cake samples (in cells/kg) were compared. AcSS, activated sewage sludge; SSC, sewage sludge cake; *, final effluent from wetland filtration treatment.

The present study demonstrated a significant reduction of human enteropathogen load associated with the sewage sludge activation process. The concentration of human pathogens in dewatered and biologically stabilized sewage sludge cake was on average 88% lower than in the same wastewater-derived sludge during its activation process. This pattern was uniform throughout the wastewater treatment plants that utilized sludge activation treatment and for all pathogen groups.

The low level of nonviable enteropathogens indicates that when the pathogen walls become permeable to compounds and the large quantities of microorganisms present in wastewater and sewage sludge, they undergo fast biodegradation. In addition to this, the dewatering and biological stabilization of sewage sludge extend direct exposure of potential human pathogens to environmental stressors such as UV, desiccation, and temperature fluctuations, which, in addition to activation, impact their viability and survival.

Although the present study demonstrated high efficiency of pathogen removal and their relatively low concentration in sewage sludge end products, the detected pathogens were viable. Thus, given the massive amounts of sewage sludge end products utilized by agriculture or deposited in landfills (4, 15, 17, 19), the environmental robustness of these enteropathogens (3, 6, 10, 18), and their high virulence to humans, it should be considered that the environmental contamination derived from such activities presents a serious public health threat.

For the sewage sludge end products to be used without restriction on agricultural lands growing ready-to-eat crops, the pathogen load must be reduced to a level which does not pose an unacceptable public health risk (4, 19). Current risk assessment modeling for cryptosporidiosis and giardiasis acquired through consumption of root crops grown on land where treated sewage sludge end products have legally been applied predicts 50 and 1 disease case per year, respectively (4). Another study predicted up to 25% annual increases in the cryptosporidiosis rate in a community consuming products from agricultural lands where sewage sludge end products were legally applied (19).

Acknowledgments

This study was supported by the Fulbright Senior Specialist Fellowship (grant no. 2225 to T. K. Graczyk), the Johns Hopkins Center in Urban Environmental Health (grant no. P30 ES03819), the Procter & Gamble Foundation, the Johns Hopkins Center for a Livable Future, and the School of Science, Institute of Technology, Sligo, Ireland.

We acknowledge the Sligo County Council and Roscommon County Council for access to and samples from sewage treatment plants.

Footnotes

Published ahead of print on 2 February 2007.

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