ABSTRACT

This study aimed to investigate the occurrence of Cryptosporidium oocysts and Giardia cysts in raw, filtered, and chlorinated waters collected from two drinking water treatment plants (WTP A and WTP B). WTP A uses either direct filtration or flotation-filtration depending on the turbidity of raw water. WTP B has two independent treatment lines, a direct filtration and a conventional treatment line. Cryptosporidium oocysts and Giardia cysts were concentrated by flocculation, identified by direct immunofluorescence microscopy and confirmed by DAPI staining and phase-contrast microscopy. In raw water, the occurrence of cysts was from 75 (WTP A) to 100% (WTP B) of the samples, and of oocysts from 66.6 (WTP A) to 83.3% (WTP B). Both protozoa were detected in water treated by direct filtration (cysts: < 0.27 to 20.0 cysts L^-1; oocysts: < 0.48 to 22.5 oocysts L^-1) and flotation-filtration (cysts: < 0.27 to 5.0 cysts L^-1; oocysts: < 0.48 to 17.5 oocysts L^-1). The absence of cysts and oocysts in chlorinated water does not exclude risks, as the limitations of concentration and identification techniques must be considered, given the low recovery rates, especially in water with low turbidity (15.5 – 72.7% of Giardia; 3.6 – 38.5% of Cryptosporidium). In the raw water samples from WTP A, a moderate correlation was observed between the protozoa, and these with the conventional parameters of water quality. In the raw water samples from WTP B, the correlation was insignificant. These results reinforce the importance of monitoring protozoa in water destined for public supply.

Keywords:
protozoa; direct filtration; flotation-filtration; conventional water treatment

RESUMO

Este estudo teve como objetivo investigar a ocorrência de oocistos de Cryptosporidium e cistos de Giardia em águas brutas, filtradas e cloradas, coletadas de duas estações de tratamento de água potável (ETA A e ETA B). A ETA A utiliza filtração direta ou flotação-filtração dependendo da turbidez da água bruta. A ETA B possui duas linhas de tratamento independentes, uma de filtração direta e outra de tratamento convencional. Os oocistos de Cryptosporidium e cistos de Giardia foram concentrados por floculação, identificados por microscopia de imunofluorescência direta e confirmados por coloração com DAPI e microscopia de contraste de fase. Na água bruta, a ocorrência de cistos variou de 75% (ETA A) a 100% (ETA B) das amostras, e de oocistos de 66,6% (ETA A) a 83,3% (ETA B). Ambos os protozoários foram detectados na água tratada por filtração direta (cistos: ETA A e B < 0,27 a 20,0 cistos L^-1; oocistos: < 0,48 a 22,5 oocistos L^-1) e flotação-filtração (cistos: < 0,27 a 5,0 cistos L^-1; oocistos: < 0,48 a 17,5 oocistos L^-1) na ETA A. A ausência de cistos e oocistos na água clorada não exclui riscos, pois as limitações das técnicas de concentração e identificação devem ser consideradas, dados os baixos índices de recuperação, especialmente em água com baixa turbidez (15,5 – 72,7% de Giardia; 3,6 – 38,5% de Cryptosporidium). Nas amostras de água bruta da ETA A, foi observada uma correlação moderada entre os protozoários e destes com os parâmetros convencionais de qualidade da água. Nas amostras de água bruta da ETA B, a correlação foi insignificante. Esses resultados reforçam a importância de monitorar protozoários em água destinada ao abastecimento público e a otimização dos processos de tratamento de água para produzir água de baixa turbidez.

Palavras-chave:
protozoários; filtração direta; flotação-filtração; tratamento convencional de água

INTRODUCTION

Safe drinking water is a basic human need that contributes to ensuring proper health conditions and quality of life. Inadequate water and wastewater treatment, associated with low-quality public health services and disorderly growth of metropolitan regions, facilitate the transmission of infectious diseases that can have profound social and economic repercussions (^{KARANIS; KOURENTI; SMITH, 2007}36 KARANIS, P.; KOURENTI, C.; SMITH, H. Waterborne transmission of protozoan parasites: a worldwide review of outbreaks and lessons learnt. Journal of Water and Health, v. 5, n. 1, p. 1-38, 2007. https://doi.org/10.2166/wh.2006.002
https://doi.org/10.2166/wh.2006.002... ; ^{SATO et al., 2013}56 SATO, M.I.; GALVANI, A.T.; PADULA, J.A.; NARDOCCI, A.C.; LAURETTO, M.S.; RAZZOLINI, M.T.P.; HACHICH, E.M. Assessing the infection risk of Giardia and Cryptosporidium in public drinking water delivered by surface water systems in Sao Paulo State, Brazil. Science of the Total Environment, p. 442:389-396, 2013. https://doi.org/10.1016/j.scitotenv.2012.09.077
https://doi.org/10.1016/j.scitotenv.2012... ). Water contaminated with pathogenic microorganisms, including bacteria, viruses, and protozoa, can cause diarrhea and vomiting within a few days of ingestion (^{SES/SP, 2013}57 SECRETARIA DA SAÚDE - GOVERNO DO ESTADO DE SÃO PAULO (SES/SP). Doenças transmitidas por alimentos e água. São Paulo: Centro de Vigilância Epidemiológica, 2013.). In immunocompromised individuals, children, and the elderly, such exposure can result in long-term or even fatal infections (^{CHINEN; SHEARER, 2010}16 CHINEN, J.; SHEARER, W.T. Secondary immunodeficiencies, including HIV infection. Journal of Allergy and Clinical Immunology, v. 125, 2 Suppl. 2, p. 195-203, 2010. https://doi.org/10.1016/j.jaci.2009.08.040
https://doi.org/10.1016/j.jaci.2009.08.0... ).

According to the World Health Organization (^{WHO, 2019}70 WORLD HEALTH ORGANIZATION (WHO). Global health observatory data repository 2015. [accessed October 8, 2019]. Available at: https://apps.who.int/gho/data/view.main.inadequatewshv?lang=en/
https://apps.who.int/gho/data/view.main.... ), 89.0% of worldwide deaths from diarrhea are caused by ingestion of contaminated water or inadequate sanitation services. In 2019, 525,000 children aged zero to five years died from diarrhea; in Brazil, the number of deaths totaled 1,318 (^{WHO, 2016}71 WORLD HEALTH ORGANIZATION (WHO). Global health observatory data repository 2016. [accessed October 8, 2019]. Available at: https://apps.who.int/gho/data/view.main.cm1002015world-ch3?lang=en/
https://apps.who.int/gho/data/view.main.... , ²⁰¹⁹70 WORLD HEALTH ORGANIZATION (WHO). Global health observatory data repository 2015. [accessed October 8, 2019]. Available at: https://apps.who.int/gho/data/view.main.inadequatewshv?lang=en/
https://apps.who.int/gho/data/view.main.... ). Brazil also had more than 130 thousand hospitalizations in 2021 due to water-borne diseases (^{DATASUS, 2021}20 DEPARTAMENTO DE INFORMÁTICA DO SISTEMA ÚNICO DE SAÚDE (DATASUS). Tabnet. 2021. [accessed Feb 12, 2023]. Available at: http://tabnet.datasus.gov.br/cgi/tabcgi.exe?logtabnet/log.def
http://tabnet.datasus.gov.br/cgi/tabcgi.... ). Only 51.2% of sewage is treated in Brazil (^{SNIS, 2021}61 SISTEMA NACIONAL DE INFORMAÇÕES SOBRE SANEAMENTO (SNIS). Série Histórica. Brasília: Ministério do Desenvolvimento Regional, 2021.). The state of Espírito Santo, southeastern Brazil, collects 56.9% of domestic wastewater and treats only 45.16% (^{SNIS, 2021}61 SISTEMA NACIONAL DE INFORMAÇÕES SOBRE SANEAMENTO (SNIS). Série Histórica. Brasília: Ministério do Desenvolvimento Regional, 2021.).

Waterborne enteric protozoa, such as Cryptosporidium and Giardia, are among the major etiological agents of diarrhea (^{FLETCHER et al., 2012}24 FLETCHER, S.M.; STARK, D.; HARKNESS, J.; ELLIS, J. Enteric protozoa in the developed world: A public health perspective. Clinical Microbiology Review, v. 25, n. 3, p. 420-449, 2012. https://doi.org/10.1128/CMR.05038-11
https://doi.org/10.1128/CMR.05038-11... ). These parasites are widely distributed in both developed and developing countries (^{BALDURSSON; KARANIS, 2011}4 BALDURSSON, S.; KARANIS, P. Waterborne transmission of protozoan parasites: Review of worldwide outbreaks - An update 2004-2010. Water Research, v. 45, n. 20, p. 6603-6614, 2011. https://doi.org/10.1016/j.watres.2011.10.013
https://doi.org/10.1016/j.watres.2011.10... ; ^{FLETCHER et al., 2012}24 FLETCHER, S.M.; STARK, D.; HARKNESS, J.; ELLIS, J. Enteric protozoa in the developed world: A public health perspective. Clinical Microbiology Review, v. 25, n. 3, p. 420-449, 2012. https://doi.org/10.1128/CMR.05038-11
https://doi.org/10.1128/CMR.05038-11... ). Although the life cycle, sources of contamination, and transmission routes of these pathogens are well known, waterborne disease outbreaks occur every year in several countries (^{KARANIS; KOURENTI; SMITH, 2007}36 KARANIS, P.; KOURENTI, C.; SMITH, H. Waterborne transmission of protozoan parasites: a worldwide review of outbreaks and lessons learnt. Journal of Water and Health, v. 5, n. 1, p. 1-38, 2007. https://doi.org/10.2166/wh.2006.002
https://doi.org/10.2166/wh.2006.002... ). Cryptosporidium spp. were responsible for 60.3% of global diarrhea outbreaks caused by waterborne protozoa in 2004–2010, Giardia spp. were involved in 35.1% of outbreaks, and other protozoa were implicated in 4.5% of cases (^{BALDURSSON; KARANIS, 2011}4 BALDURSSON, S.; KARANIS, P. Waterborne transmission of protozoan parasites: Review of worldwide outbreaks - An update 2004-2010. Water Research, v. 45, n. 20, p. 6603-6614, 2011. https://doi.org/10.1016/j.watres.2011.10.013
https://doi.org/10.1016/j.watres.2011.10... ). In the United States of America (USA), from 1971 to 2006, parasites were responsible for 18.0% of outbreaks associated with drinking water (n = 780), with Giardia intestinalis identified in 86.0% of cases (^{CRAUN et al., 2010}18 CRAUN, G.F.; BRUNKARD, J.M.; YODER, J.S.; ROBERTS, V.A.; CARPENTER, J.; WADE, T.; CALDERON, R.L.; ROBERTS, J.M.; BEACH, M.J.; ROY, S.L. Causes of outbreaks associated with drinking water in the United States from 1971 to 2006. Clinical Microbiology Reviews, v. 23, n. 3, p. 507-528, 2010. https://doi.org/10.1128/CMR.00077-09
https://doi.org/10.1128/CMR.00077-09... ).

Several factors may contribute to the spread of pathogenic protozoa. For instance, high contamination levels in the environment, emergence of highly infective strains, resistance to widely used disinfection processes, small cyst or oocyst size have been shown to facilitate parasite transmission, and weakness of Brazilian regulations regarding the criteria for monitoring protozoa in water for public supply, such as researching protozoa only in raw water and controlling filtration efficiency by analyzing turbidity in filtered water (^{CAREY; LEE; TREVORS, 2004}11 CAREY, C.M.; LEE, H.; TREVORS, J.T. Biology, persistence and detection of Cryptosporidium parvum and Cryptosporidium hominis oocyst. Water Research, v. 38, n. 4, p. 818-862, 2004. https://doi.org/10.1016/j.watres.2003.10.012
https://doi.org/10.1016/j.watres.2003.10... ; ^{RAMIREZ; WARD; SREEVATSAN, 2004}51 RAMIREZ, N.E.; WARD, L.A.; SREEVATSAN, S. A review of the biology and epidemiology of cryptosporidiosis in humans and animals. Microbes and Infection, v. 6, n. 8, p. 773-785, 2004. https://doi.org/10.1016/j.micinf.2004.02.021
https://doi.org/10.1016/j.micinf.2004.02... ; ^{SMITH et al., 2006}60 SMITH, H.V.; CACCIÒ, S.M.; TAIT, A.; MCLAUCHLIN, J.; THOMPSON, R.A. Tools for investigating the environmental transmission of Cryptosporidium and Giardia infections in humans. Trends in Parasitology, v. 22, n. 4, p. 160-167, 2006. https://doi.org/10.1016/j.pt.2006.02.009
https://doi.org/10.1016/j.pt.2006.02.009... ; ^{CARMENA, 2010}12 CARMENA, D. Waterborne transmission of Cryptosporidium and Giardia: detection, surveillance and implications for public health, 3-14. In: MENDEZ-VILAS, A. (Eds.). Current research, technology and education topics in applied microbiology and microbial biotechnology. Spain: Formatex Research Center, 2010. p. 3-14.; ^{RAZZOLINI; SANTOS, BASTOS, 2010}52 RAZZOLINI, M.T.P.; SANTOS, T.F.S.; BASTOS, V.K. Detection of Giardia and Cryptosporidium cysts/oocysts in watersheds and drinking water sources in Brazil urban areas. Journal of Water and Health, v. 8, n. 2, p. 399-404, 2010. https://doi.org/10.2166/wh.2009.172
https://doi.org/10.2166/wh.2009.172... ; ^{BALDURSSON; KARANIS, 2011}4 BALDURSSON, S.; KARANIS, P. Waterborne transmission of protozoan parasites: Review of worldwide outbreaks - An update 2004-2010. Water Research, v. 45, n. 20, p. 6603-6614, 2011. https://doi.org/10.1016/j.watres.2011.10.013
https://doi.org/10.1016/j.watres.2011.10... ; ^{REEVEA et al., 2018}53 REEVEA, N.F.; DIGGLE, P.J.; LAMDEN, K.; KEEGAN, T. A spatial analysis of giardiasis and cryptosporidiosis in relation to public water supply distribution in North West England. Spatial and Spatio-temporal Epidemiology, v. 27, p. 61-70, 2018. https://doi.org/10.1016/j.sste.2018.09.002
https://doi.org/10.1016/j.sste.2018.09.0... ; ^{ZINI et al., 2021}73 ZINI, L.B.; LORENZINI, R.; CAMELO, L.G.G.; GUTTERRES, M. Occurrence of Cryptosporidium and Giardia in surface water supply from 2016 to 2020 in South Brazil. Environmental Monitoring and Assessment, v. 193, p. 496, 2021. https://doi.org/10.1007/s10661-021-09280-y
https://doi.org/10.1007/s10661-021-09280... ; ^{BRASIL, 2021}8 BRASIL. Portaria GM/MS n° 888, de 4 de maio de 2021, altera o Anexo XX da Portaria de Consolidação GM/MS n° 5, de 28 de setembro de 2017, para dispor sobre os procedimentos de controle e de vigilância da qualidade da água para consumo humano e seu padrão de potabilidade. [accessed Jun 20, 2021]. Available at: https://www.in.gov.br/web/dou/-/portaria-gm/ms-n-888-de-4-de-maio-de-2021-318461562
https://www.in.gov.br/web/dou/-/portaria... ). Therefore, periodic monitoring and quantification of pathogenic protozoa in water supply systems are extremely important for the adoption of management measures to reduce health risks and ensure the quality of water distributed to the population (^{ONGERTH, 2013}49 ONGERTH, J.E. The concentration of Cryptosporidium and Giardia in water - the role and importance of recovery efficiency. Water Research, v. 47, n. 7, p. 2479-2488, 2013. https://doi.org/10.1016/j.watres.2013.02.015
https://doi.org/10.1016/j.watres.2013.02... ; ^{SANTOS et al., 2016}55 SANTOS, S.F.O.; SILVA, H.D.; WOSNJUK, L.A.C.; ANUNCIAÇÃO, C.E.; SILVEIRA LACERDA, E.P.; PERALTA, R.H.S.; CUNHA, F.S.; FERREIRA, T.D.S.; GARCIA-ZAPATA, M.T.A. Occurrence and evaluation of methodologies to detect Cryptosporidium spp. in treated water in the central-west region of Brazil. Exposure and Health, v. 8, p. 117-123, 2016. https://doi.org/10.1007/s12403-015-0187-1
https://doi.org/10.1007/s12403-015-0187-... ; ^{LO et al., 2018}41 LO, N.T.; SARKER, M.A.B.; LIM, Y.A.L.; RASHID, M. H.O.; SAKAMOTO, J. Inadequate water treatment quality as assessed by protozoa removal in Sarawak, Malaysia. Nagoya Journal of Medical Science, v. 80, n. 2, p. 165-174, 2018. 10.18999/nagjms.80.2.165
https://doi.org/10.18999/nagjms.80.2.165... ).

In Brazil, the presence of Cryptosporidium and Giardia in clinical samples, food, and animals has been widely reported (^{FRANCO; ROCHA-EBERHARDT; CANTUSIO, 2001}25 FRANCO, R.M.B.; ROCHA-EBERHARDT, R.; CANTUSIO, N.R. Occurrence of Cryptosporidium oocysts and Giardia cysts in raw water from the Atibaia river, Campinas, Brazil. Revista do Instituto de Medicina Tropical de São Paulo, v. 43, n. 2, p. 109-111, 2001. https://doi.org/10.1590/s0036-46652001000200011
https://doi.org/10.1590/s0036-4665200100... ; ^{RAZZOLINI; SANTOS, BASTOS, 2010}52 RAZZOLINI, M.T.P.; SANTOS, T.F.S.; BASTOS, V.K. Detection of Giardia and Cryptosporidium cysts/oocysts in watersheds and drinking water sources in Brazil urban areas. Journal of Water and Health, v. 8, n. 2, p. 399-404, 2010. https://doi.org/10.2166/wh.2009.172
https://doi.org/10.2166/wh.2009.172... ; ^{SATO et al., 2013}56 SATO, M.I.; GALVANI, A.T.; PADULA, J.A.; NARDOCCI, A.C.; LAURETTO, M.S.; RAZZOLINI, M.T.P.; HACHICH, E.M. Assessing the infection risk of Giardia and Cryptosporidium in public drinking water delivered by surface water systems in Sao Paulo State, Brazil. Science of the Total Environment, p. 442:389-396, 2013. https://doi.org/10.1016/j.scitotenv.2012.09.077
https://doi.org/10.1016/j.scitotenv.2012... ; ^{ALMEIDA et al., 2015}2 ALMEIDA, J.C.; MARTINS, F.D.C.; FERREIRA-NETO, J.M.; SANTOS, M.M.; GARCIA, J.L.; NAVARRO, I.T.; KURODA, E.K.; FREIRE, R.L. Occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system, Paraná, Southern Brazil. Revista Brasileira de Parasitologia Veterinária, v. 24, n. 3, p. 303-308, 2015. https://doi.org/10.1590/S1984-29612015051
https://doi.org/10.1590/S1984-2961201505... ; ^{SANTOS et al., 2016}55 SANTOS, S.F.O.; SILVA, H.D.; WOSNJUK, L.A.C.; ANUNCIAÇÃO, C.E.; SILVEIRA LACERDA, E.P.; PERALTA, R.H.S.; CUNHA, F.S.; FERREIRA, T.D.S.; GARCIA-ZAPATA, M.T.A. Occurrence and evaluation of methodologies to detect Cryptosporidium spp. in treated water in the central-west region of Brazil. Exposure and Health, v. 8, p. 117-123, 2016. https://doi.org/10.1007/s12403-015-0187-1
https://doi.org/10.1007/s12403-015-0187-... ). Important studies highlight the occurrence of Giardia and Cryptosporidium in public water sources, such as in the cities of Belo Horizonte (^{LOPES et al., 2017}42 LOPES, A.M.M.B.; GOMES, L.N.L.; MARTINS, F.C.; CERQUEIRA, D.A.; FILHO, C.R.; VON SPERLING, E.; PÁDUA, V.L. Dinâmica de protozoários patogênicos e cianobactérias em um reservatório de abastecimento público de água no sudoeste do Brasil. Engenharia Sanitária e Ambiental, v. 22, n. 1, p. 25-43, 2017. https://doi.org/10.1590/S1413-41522016143529
https://doi.org/10.1590/S1413-4152201614... ), Campinas (^{FRANCO et al., 2016}26 FRANCO, R.M.B.; BRANCO, N.; AMARO, B.C.T.; NETO, R.C.; FIUZA, V.R.S. Cryptosporidium species and Giardia genotypes detected in surface water supply of Campinas, Southeast Brazil, by Molecular Methods. Journal of Veterinary Medicine and Research, v. 3, n. 3, p. 1053, 2016.), Londrina (^{ALMEIDA et al., 2015}2 ALMEIDA, J.C.; MARTINS, F.D.C.; FERREIRA-NETO, J.M.; SANTOS, M.M.; GARCIA, J.L.; NAVARRO, I.T.; KURODA, E.K.; FREIRE, R.L. Occurrence of Cryptosporidium spp. and Giardia spp. in a public water-treatment system, Paraná, Southern Brazil. Revista Brasileira de Parasitologia Veterinária, v. 24, n. 3, p. 303-308, 2015. https://doi.org/10.1590/S1984-29612015051
https://doi.org/10.1590/S1984-2961201505... ), 11 cities in the state of São Paulo (^{BRETERNITZ et al., 2020}9 BRETERNITZ, B.S.; DA VEIGA, D.P.B.; RAZZOLINI, M.T.P.; NARDOCCI, A.C. Land use associated with Cryptosporidium sp. and Giardia sp. in surface water supply in the state of São Paulo, Brazil. Environmental Pollution, v. 266, n. 3, p. 115-143, 2020. https://doi.org/10.1016/j.envpol.2020.115143
https://doi.org/10.1016/j.envpol.2020.11... ), 15 cities in the state of Goiás (^{SILVA; SCALIZE, 2020}58 SILVA, D.P.; SCALIZE, P.S. Detection of Cryptosporidium spp. oocysts and Giardia spp. cysts in surface water destined for public supply in the state of Goiás, Brazil. Engenharia Sanitaria e Ambiental, v. 25, n. 5, p. 777-787, 2020. https://doi.org/10.1590/s1413-4152202020200312
https://doi.org/10.1590/s1413-4152202020... ) and 48 cities in the state of Rio Grande do Sul (^{ZINI et al., 2021}73 ZINI, L.B.; LORENZINI, R.; CAMELO, L.G.G.; GUTTERRES, M. Occurrence of Cryptosporidium and Giardia in surface water supply from 2016 to 2020 in South Brazil. Environmental Monitoring and Assessment, v. 193, p. 496, 2021. https://doi.org/10.1007/s10661-021-09280-y
https://doi.org/10.1007/s10661-021-09280... ).

This study aimed to investigate the occurrence of Cryptosporidium oocysts and Giardia cysts in two public drinking water treatment plants in the metropolitan region of Vitória, Espírito Santo, Brazil. This is the first study on the detection of cysts and oocysts in catchment water and water treatment systems in the State of Espírito Santo. The results provide information for decision making in the management of water resources used for public supply in the region.

MATERIALS AND METHODS

Water collection sites

Water samples were collected from the water treatment plants of Carapina (WTP A) and Vale Esperança (WTP B), located in the Santa Maria da Vitória River and Jucu River basins, respectively (Figure 1). These plants supply water to 1.5 million inhabitants in the metropolitan region of Vitória, Espírito Santo, Brazil. The use and occupation of the soil in the two hydrographic basins are defined by urban, industrial, agricultural and livestock activities. Both rivers receive daily sanitary and industrial effluents, have a high level of siltation, and low vegetation cover.

Description of water treatment plants

WTP A uses either direct filtration (coagulation, filtration, and disinfection) or flotation-filtration (coagulation, flotation-filtration, and disinfection) depending on the turbidity of raw water. Direct filtration is the treatment of choice when turbidity is below 50 NTU. WTP B has two treatment lines that operate independently, a direct filtration line and a conventional treatment line (coagulation, flocculation, decantation, filtration, and disinfection). The water treatment processes and sampling points in WTP A and B are presented in Figure 2. Sampling times were adjusted so that samples could be collected at the beginning of each process.

Detection and enumeration of Cryptosporidium oocysts and Giardia cysts in environmental samples

Samples (10 L) of raw water (n = 24), filtered water (n = 36), and chlorinated water (n = 20) were collected monthly from each sampling point for 12 months (April 2008 to March 2009) and analyzed for the presence of Cryptosporidium oocysts and Giardia cysts. Sample collection, storage, and transportation were performed in accordance with the recommendations of the Guidelines for Collection and Preservation of Water Samples (^{CETESB, 1987}17 COMPANHIA AMBIENTAL DO ESTADO DE SÃO PAULO (CETESB). Guia de coleta e preservação de amostras de água. Brasília: ANA, 1987.) and the Standard Methods for the Examination of Water and Wastewater (^{APHA, 2005}3 AMERICAN PUBLIC HEALTH ASSOCIATION (APHA). Standard Methods for the Examination of Water and Wastewater. Washington: American Public Health Association, 2005.). All analyses were carried out at the Laboratory of Sanitation of the Federal University of Espírito Santo, Vitória, Brazil.

Samples were concentrated in 12L flat-bottomed flasks by the calcium carbonate flocculation method (^{VESEY et al., 1993}68 VESEY, G.; SLADE, J.P.; BYRNE, M.; SHEPERD, K.; FRICKER, C.R. A method for concentration of Cryptosporidium oocysts from water. Journal of Applied Microbiology, v. 75, p. 82-86, 1993. https://doi.org/10.1111/J.1365-2672.1993.TB03412.X
https://doi.org/10.1111/J.1365-2672.1993... ), followed by centrifugation at 3,000 × g for 10 min. This concentration method limits the sample volume to up to 10 L. Pellets were resuspended to 8 mL with elution fluid (1% Tween 80, 1% sodium dodecyl sulfate, 10× PBS, and 0.1% antifoam A). Of the final sample, 10 μL were added to each well slide for identification and quantification of cysts and oocysts. Protozoa were identified by direct immunofluorescence microscopy using the Merifluor C/G kit (Meridian Bioscience, Cincinnati, OH, USA) and confirmed by phase-contrast microscopy with 4,6-diamidino-2-phenylindole (DAPI) (Sigma–Aldrich, St. Louis, MO, USA) staining. Four slides were examined for each sample under an epifluorescence microscope (ZEISS Axioplan HBO 50, excitation wavelength of 450 – 490 nm, 510 nm suppression filter; Oberkochen, Germany) at 200, 400, and 630× magnification. Each slide was viewed in duplicate. Positive and negative controls were also prepared and analyzed.

The detection limit (Equation 1) and concentration (Equation 2) of Cryptosporidium oocysts and Giardia cysts were calculated from the results of the recovery tests according to the formula of Ongerth (2013):

Recovery of Giardia cysts and Cryptosporidium oocysts

Recovery tests were conducted in high-turbidity raw water (65 NTU) and low-turbidity filtered water (0.3 NTU) using the calcium carbonate flocculation method (^{VESEY et al., 1993}68 VESEY, G.; SLADE, J.P.; BYRNE, M.; SHEPERD, K.; FRICKER, C.R. A method for concentration of Cryptosporidium oocysts from water. Journal of Applied Microbiology, v. 75, p. 82-86, 1993. https://doi.org/10.1111/J.1365-2672.1993.TB03412.X
https://doi.org/10.1111/J.1365-2672.1993... ), as described in the previous topic. Cryptosporidium oocysts were purified from feces of newborn calves by sucrose gradient centrifugation, washed with phosphate-buffered saline (PBS), and suspended in PBS containing 10 g L⁻¹ penicillin-streptomycin and 0.01% Tween 20. Isolated oocysts were kindly donated by the Department of Biological Sciences of the Federal University of Triângulo Mineiro, Uberaba, Minas Gerais, Brazil. Giardia cysts were separated from human feces using sucrose gradient solution and suspended in PBS containing 25 μg mL⁻¹ miconazole and 125 μg mL⁻¹ enrofloxacin, according to ^{Roberts-Thompson et al. (1976)}54 ROBERTS-THOMPSON, I.C.; STEVENS, D.P.; MAHMOUD, A.A.; WARREN, K.S. Giardiasis in the mouse: an animal model. Gastroenterology, v. 71, n. 1, p. 57-61, 1976. https://doi.org/10.1016/S0016-5085(76)80097-2
https://doi.org/10.1016/S0016-5085(76)80... . Isolated cysts were kindly provided by the Department of Basic Pathology of the Federal University of Paraná, Curitiba, Brazil. After purification, Cryptosporidium oocysts and Giardia cysts were enumerated by flow cytometry and inoculated into water samples, in triplicate, at two concentrations, 10² and 10³ (oo)cysts L⁻¹. Cysts and oocysts supplied with the kit MeriFluor^® (Meridian Diagnostics, Cincinnati, Ohio, EUA) were used as positive controls, and sterile distilled water was used as negative control. For biosafety reasons, all materials were disinfected with 5% sodium hypochlorite and autoclaved at the end of the experiment. Recovery efficiency (RE) was estimated by the following equation (Equation 3):

Because water samples used to assess recovery efficiency might be naturally contaminated, the samples were also subjected to protozoan quantification prior to inoculation. The number of naturally occurring protozoa was subtracted from the number of (oo)cysts recovered.

Physicochemical and microbial analyses

Water pH, turbidity, temperature, alkalinity, true and apparent color, and free residual chlorine were measured in the field using portable equipment, according to ^{APHA (2005)}3 AMERICAN PUBLIC HEALTH ASSOCIATION (APHA). Standard Methods for the Examination of Water and Wastewater. Washington: American Public Health Association, 2005.. Total coliforms and Escherichia coli were quantified by a chromo-fluorogenic method (Colilert, IDEXX), according to ^{APHA (2005)}3 AMERICAN PUBLIC HEALTH ASSOCIATION (APHA). Standard Methods for the Examination of Water and Wastewater. Washington: American Public Health Association, 2005.. Raw and filtered water were dechlorinated with 1.8% sodium thiosulfate before microbiological analysis. Parameter analyses were performed in triplicate, on 24 samples of raw water, 36 samples of filtered water and 20 samples of chlorinated water.

Statistical analysis

Data were analyzed using descriptive statistics. The Shapiro-Wilk test was applied to assess the normality of the distribution of positional errors. Differences in protozoan concentrations between water sampling points were determined by the nonparametric Mann-Whitney U test (also known as the Wilcoxon rank-sum test). Associations between protozoan concentrations and physicochemical and bacteriological indicators of water quality were assessed by the nonparametric Spearman's correlation test. The level of significance was set at p < 0.05. All statistical analyses were performed using GraphPad Prism version 6.1 (GraphPad Software, La Jolla, CA, USA).

RESULTS

Recovery of Giardia cysts and Cryptosporidium oocysts from turbid water

Recovery efficiencies were determined in high- and low-turbidity water samples. Significant differences (p = 0.0065, high-turbidity; p = 0.0166, low-turbidity) in protozoan recoveries were observed. The highest recoveries were obtained from high-turbidity water (65 NTU): 72.7% (62.5 – 83.3%) for Giardia cysts and 43.0% (20.8 – 65.7%) for Cryptosporidium oocysts. From the low-turbidity sample (0.3 NTU), 36.1% (15.5 – 72.7%) of Giardia and 20.9% (3.6 – 38.5%) of Cryptosporidium were recovered.

Detection of Giardia cysts and Cryptosporidium oocysts in water samples

In raw water supplying WTP A, cysts were detected in 75.0% of samples and oocysts in 66.7%, whereas in water supplying WTP B, cysts and oocysts were found in 100.0 and 83.3% of water samples, respectively. Raw water samples did not differ in Cryptosporidium (p = 0.1190) and Giardia (p = 0.5067) concentrations. Figure 3 shows the boxplot of concentrations of cysts and oocysts in raw, filtered, and chlorinated waters from WTP A and B.

Physicochemical and bacteriological characteristics

Table 1 shows the median physicochemical parameters (turbidity, pH, alkalinity, temperature, and residual chlorine) of raw and treated water from both treatment plants, and Figure 4 shows the concentrations of Cryptosporidium oocysts, Giardia cysts, and E. coli in raw water supplying WTP A and B during the 12-month monitoring period, and the Environmental Protection Agency (EPA) limit of E. coli in raw water for protozoa research. In WTP A, the median concentrations of oocysts, cysts, and E. coli were 25.0 oocysts L⁻¹, 87.5 cysts L⁻¹, and 4.1 × 10² MPN 100 mL⁻¹, respectively. In WTP B, oocysts were detected at 87.5 oocysts L⁻¹, cysts at 100.0 cysts L⁻¹, and E. coli at 3.05 × 10² MPN 100 mL⁻¹, respectively.

In raw water samples from WTP A, a moderate correlation was observed between occurrence of Cryptosporidium and Giardia (r_s = 0.628). Giardia cyst levels were positively correlated with E. coli levels (r_s = 0.637) and true color (r_s = 0.602), where as Cryptosporidium levels showed a positive moderate correlation with total coliforms (r_s = 0.585), E. coli levels (r_s = 0.620), turbidity (r_s = 0.668), true color (r_s = 0.769), and apparent color (r_s = 0.736) (Figure 5A). In samples of raw water supplying WTP B, no correlations were observed between Giardia cyst and Cryptosporidium oocyst levels (r_s = 0.271). Giardia did not correlate with any physicochemical or bacteriological parameter, and Cryptosporidium showed a positive moderate correlation only with total coliforms (r_s = 0.593) (Figure 5B).

DISCUSSION

Recovery of Giardia cysts and Cryptosporidium oocysts

The methodology for detecting Giardia cysts and Cryptosporidium oocysts showed a higher recovery rate in high turbidity water (65 NTU), 72.7% for cysts and 43.0% for oocysts, than in low turbidity water (0.3 NTU), 36.1% for cysts and 20.9% for oocysts. According to ^{LeChevallier e Norton (1995)}38 LECHEVALLIER, M.W.; NORTON, W.D. Giardia and Cryptosporidium in raw water and finished water. Journal AWWA, v. 87, n. 9, p. 54-68, 1995. https://doi.org/10.1002/j.1551-8833.1995.tb06422.x
https://doi.org/10.1002/j.1551-8833.1995... , the presence of suspended particles in raw water helps the precipitation of organisms in the sediment, increasing recovery, especially when flocculation with CaCO₃ is used. On the other hand, excess particles can cover cysts and oocysts, preventing antigen-antibody binding and, consequently, leading to false-negative results (^{VESEY et al., 1993}68 VESEY, G.; SLADE, J.P.; BYRNE, M.; SHEPERD, K.; FRICKER, C.R. A method for concentration of Cryptosporidium oocysts from water. Journal of Applied Microbiology, v. 75, p. 82-86, 1993. https://doi.org/10.1111/J.1365-2672.1993.TB03412.X
https://doi.org/10.1111/J.1365-2672.1993... ; ^{FRANCO et al., 2012}27 FRANCO, R.M.B.; HACHICH, E.M.; SATO, M.I.Z.S.; NAVEIRA, R.M.L.; SILVA, E.D.C.; CAMPOS, M.M.D.C.; NETO, R.C.; CERQUEIRA, D.A.; BRANCO, N.; LEAL, D.A.G. Performance evaluation of different methodologies for detection of Cryptosporidium spp. and Giardia spp. in water for human consumption to meet the demands of the environmental health surveillance in Brazil. Epidemiologia e Serviços de Saúde, v. 21, n. 2, p. 233-242, 2012. https://doi.org/10.5123/S1679-49742012000200006
https://doi.org/10.5123/S1679-4974201200... ; ^{USEPA, 2012}65 UNITED STATES ENVIRONMENTAL PROTECTION AGENCY (USEPA). Method 1623.1 Cryptosporidium and Giardia in Water by Filtration/IMS/FA. Washington: USEPA, 2012. [accessed Dec 02, 2022]. Available at: https://nepis.epa.gov/Exe/ZyPURL.cgi?Dockey=P100J7G4.TXT
https://nepis.epa.gov/Exe/ZyPURL.cgi?Doc... ).

The tests carried out by ^{Vesey et al. (1993)}68 VESEY, G.; SLADE, J.P.; BYRNE, M.; SHEPERD, K.; FRICKER, C.R. A method for concentration of Cryptosporidium oocysts from water. Journal of Applied Microbiology, v. 75, p. 82-86, 1993. https://doi.org/10.1111/J.1365-2672.1993.TB03412.X
https://doi.org/10.1111/J.1365-2672.1993... showed a recovery of Cryptosporidium oocysts of 76% for deionized water, 73.7% for tap water and 75.6% for spring water. ^{Shepherd and Wyn-Jones (1996)}59 SHEPHERD, K.M.; WYN-JONES, A.P. An evaluation of methods for the simultaneous detection of Cryptosporidium oocysts and Giardia cysts from water. Applied and Environmental Microbiology, v. 62, p. 4, p. 1317-1322, 1996. https://doi.org/10.1128/aem.62.4.1317-1322.1996
https://doi.org/10.1128/aem.62.4.1317-13... recovered 71.3% Cryptosporidium oocysts and 72.5% Giardia cysts in river water, and 73.6% oocysts and 77.1% cysts in treated water. In the tests carried out by ^{Cantusio Neto et al. (2010)}47 CANTUSIO NETO, R.; DOS SANTOS, L.U.; SATO, M.I.Z.; FRANCO, R.M B. Cryptosporidium spp. and Giardia spp. in surface water supply of Campinas, Southeast Brazil. Water Science and Technology, v. 62, n. 1, p. 217-222, 2010. https://doi.org/10.2166/wst.2010.312
https://doi.org/10.2166/wst.2010.312... , the recovery rates of Cryptosporidium oocysts were 26.8% and Giardia cysts were 14.3% in the environmental matrix of the study area.

The results found in the recovery tests for Giardia cysts and Cryptosporidium oocysts demonstrate that the efficiency of the concentration and detection techniques depends on the quality of the water used in the tests, the storage time of the water, the method of conservation, the skills of the technical staff, and the different counting techniques adopted.

Detection of oocysts and cysts in raw water

Cryptosporidium oocysts and Giardia cysts were detected with high frequency in water sources that supply the region of Vitória, Espírito Santo, Brazil, throughout the 12-month monitoring period. In raw water supplying WTP A, the occurrence of cysts and oocysts was 75 and 66.6%, respectively. Regarding raw water supplying WTP B, all samples (100.0%) were positive for Giardia cysts and 83.3% of samples were positive for Cryptosporidium oocysts. The high frequencies of detection indicate that current watershed protection measures are ineffective. It is important to highlight that the rivers that supply the Vitória metropolitan region (Santa Maria da Vitória River and Jucu River) cross many agricultural and livestock areas. Therefore, it is probable that water bodies were contaminated with Giardia cysts and Cryptosporidium oocysts excreted by cattle and other animals, which are hosts to these protozoa (^{HANSEN; ONGERTH, 1991}30 HANSEN, J.S.; ONGERTH, J.E. Effects of time and watershed characteristics on the concentration of Cryptosporidium oocysts in river water. Applied and Environmental Microbiology, v. 57, n. 10, p. 2790-2795, 1991. https://doi.org/10.1128/aem.57.10.2790-2795.1991
https://doi.org/10.1128/aem.57.10.2790-2... ; ^{GEURDEN et al., 2004}29 GEURDEN, T.; CLAEREBOUT, E.; VERCRUYSSE, J.; BERKVENS, D. Estimation of diagnostic test characteristics and prevalence of Giardia duodenalis in dairy calves in Belgium using a Bayesian approach. International Journal for Parasitology, v. 34, p. 1121-1127, 2004. https://doi.org/10.1016/j.ijpara.2004.05.007
https://doi.org/10.1016/j.ijpara.2004.05... , ²⁰⁰⁶28 GEURDEN, T.; BERKVENS, D.; GELDHOF, P.; VERCRUYSSE, J.; CLAEREBOUT, E. A Bayesian approach for the evaluation of six diagnostic assays and the estimation of Cryptosporidium prevalence in dairy calves. Veterinary Research, v. 37, p. 671-682, 2006. https://doi.org/10.1051/VETRES:2006029
https://doi.org/10.1051/VETRES:2006029... ; ^{CASTRO-HERMIDA et al., 2009}14 CASTRO-HERMIDA, J.A.; GARCIA-PRESEDO, I.; ALMEIDA, A.; GONZALEZ-WARLETA, M.; COSTA, J.M.; MEZO, M. Detection of Cryptosporidium spp. and Giardia duodenalis in surface water: a health risk for humans and animals. Water Research, v. 43, n. 17, p. 4133-4142, 2009. https://doi.org/10.1016/j.watres.2009.06.020
https://doi.org/10.1016/j.watres.2009.06... ; ^{LIGDA et al., 2020}40 LIGDA, P.; CLAEREBOUT, E.; KOSTOPOULOU, D.; ZDRAGAS, A.; CASAERT, S.; ROBERTSON, L.J.; SOTIRAKI, S. Cryptosporidium and Giardia in surface water and drinking water: Animal sources and towards the use of a machine-learning approach as a tool for predicting contamination. Environmental Pollution, v. 264, p. 1-14, 2020. https://doi.org/10.1016/j.envpol.2020.114766
https://doi.org/10.1016/j.envpol.2020.11... ).

It is essential to define limits for these protozoa in source water to (i) ensure that treatments used by plants are compatible with the microbiological quality of water and (ii) assess the risk of contamination if waters are to be used for recreation. Currently, the Brazilian legislation establishes that Giardia cysts and Cryptosporidium oocysts should be monitored monthly in water catchment areas (for a period of 12 months) when the concentration of E. coli is greater than or equal to 10³ 100 mL⁻¹ and the efficiency of the WTP in removing spores of aerobic bacteria is less than 2.5 log. (BRAZIL 2021). This study preceded the last publication of the Brazilian standard, but it is important to highlight that the concentrations of E. coli in raw water, for the most part, did not exceed the limits established by the EPA and Brazilian regulations for research on protozoa. Protozoan cysts and oocysts were frequently detected in water catchment areas, mainly in the waters of the Jucu River that supplies ETA B.

Cryptosporidium accounts for most waterborne outbreaks of protozoan parasitic diseases even when bacteriological results were in accordance with regulatory standards (^{KARANIS; KOURENTI; SMITH, 2007}36 KARANIS, P.; KOURENTI, C.; SMITH, H. Waterborne transmission of protozoan parasites: a worldwide review of outbreaks and lessons learnt. Journal of Water and Health, v. 5, n. 1, p. 1-38, 2007. https://doi.org/10.2166/wh.2006.002
https://doi.org/10.2166/wh.2006.002... ; ^{BALDURSSON; KARANIS, 2011}4 BALDURSSON, S.; KARANIS, P. Waterborne transmission of protozoan parasites: Review of worldwide outbreaks - An update 2004-2010. Water Research, v. 45, n. 20, p. 6603-6614, 2011. https://doi.org/10.1016/j.watres.2011.10.013
https://doi.org/10.1016/j.watres.2011.10... ; ^{CHECKLEY et al., 2015}15 CHECKLEY, W.; WHITE, A.C.J.; JAGANATH, D.; ARROWOOD, M.J.; CHALMERS, R.M.; CHEN, X.-M.; FAYER, R.; GRIFFITHS, J.K.; GUERRANT, R.L.; HEDSTROM, L.; HUSTON, C.D.; KOTLOFF, K.L.; KANG, G.; MEAD, J.R.; MILLER, M.; PETRI, W.A.JR.; PRIEST, J.W.; ROOS, D.S.; STRIEPEN, B.; THOMPSON, R.C.A.; WARD, H.D.; VAN VOORHIS, W.A.; XIAO, L.; ZHU, G.; HOUPT, E.R. A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for cryptosporidium. The Lancet Infectious Diseases, v. 15, p. 85-94, 2015. https://doi.org/10.1016/S1473-3099(14)70772-8
https://doi.org/10.1016/S1473-3099(14)70... ; ^{EFSTRATIOU; ONGERTH; KARANIS, 2017}22 EFSTRATIOU, A.; ONGERTH, J.E.; KARANIS, P. Waterborne transmission of protozoan parasites: review of worldwide outbreaks—an update 2011–2016. Water Research, v. 114, p. 14-22, 2017. https://doi.org/10.1016/j.watres.2017.01.036
https://doi.org/10.1016/j.watres.2017.01... ). Protozoa and bacteria differ in cell structure, biology, and environmental resistance; thus, the commonly analyzed bacterial groups are not good indicators of the presence of protozoa in water.

According to ^{Benedict et al. (2017)}6 BENEDICT, K.M.; RESES, H.; VIGAR, M.; ROTH, D.M.; ROBERTS, V.A.; MATTIOLI, M.; COOLEY, L.A.; HILBORN, E.D.; WADE, T.J.; FULLERTON, K.E.; YODER, J.S.; HILL, V.R. Surveillance for waterborne disease outbreaks associated with drinking water - United States, 2013-2014. Morbidity and Mortality Weekly Report, v. 66, n. 44, p.1216-1221, 2017. https://doi.org/10.15585/mmwr.mm6644a3
https://doi.org/10.15585/mmwr.mm6644a3... , Cryptosporidium was the second most common cause of both outbreaks and illnesses in USA, demonstrating the continued threat from this chlorine-tolerant pathogen when drinking water supplies are contaminated.

^{De Silva et al. (2016)}21 DESILVA, M.; SCHAFER, S.; KENDALL SCOTT, M.; ROBINSON, B.; HILLS, A.; BUSER, G.L.; SALIS, K.; GARGANO, J.; YODER, J.; HILL, V.; XIAO, L.; ROELLIG, D.; HEDBERG, K. Communitywide cryptosporidiosis outbreak associated with a surface water-supplied municipal water system – Baker City, Oregon. Epidemiology and Infection, v. 144, n. 2, p. 274-284, 2016. https://doi.org/10.1017/S0950268815001831
https://doi.org/10.1017/S095026881500183... claim that, to prevent waterborne outbreaks, it is essential to monitor the quality of both raw water and drinking water and to evaluate the efficiency of current barriers in water treatment plants.

Several factors may affect the quality of source water. Rainfall, for instance, influenced the turbidity of raw water supplying WTP. In the study region, water basins received an average annual rainfall of 1,500 mm, with episodes of heavy and constant rainfall in the summer (^{IEMA, 2020}35 INSTITUTO ESTADUAL DE MEIO AMBIENTE E RECURSOS HÍDRICOS (IEMA). Monitoring Data. 2020. [accessed April 06, 2020]. Available at: https://iema.es.gov.br/qualidadedoar/dadosdemonitoramento/automatica.
https://iema.es.gov.br/qualidadedoar/dad... ). Rainfall was not correlated with the occurrence of protozoa (data not shown), but peaks of cysts, oocysts, turbidity, and coliform bacteria were observed in the rainy season (October to March).

^{Kifleyohannes and Robertson (2020)}37 KIFLEYOHANNES, T.; ROBERTSON, L.J. Preliminary insights regarding water as a transmission vehicle for Cryptosporidium and Giardia in Tigray, Ethiopia. Food and Waterborne Parasitology, v. 19, p. e00073, 2020. https://doi.org/10.1016/j.fawpar.2020.e00073
https://doi.org/10.1016/j.fawpar.2020.e0... comment that it is possible that the concentration of cysts and oocysts is higher in the water source after precipitation. However, other studies that evaluated the presence of Giardia and Cryptosporidium during the seasons of the year reported only a relatively weak correlation, or correlations with only one of the parasites (^{CARMENA et al., 2007}13 CARMENA, D.; AGUINAGALDE, X.; ZIGORRAGA, C.; CRESPO, J.; OCIO, J. Presence of Giardia cysts and Cryptosporidium oocysts in drinking water supplies in northern Spain. Journal of Applied Microbiology, v. 102, n. 3, p. 882-882, 2007. https://doi.org/10.1111/j.1365-2672.2006.03193.x
https://doi.org/10.1111/j.1365-2672.2006... ; ^{MONS et al., 2009}45 MONS, C.; DUMÈTRE, A.; GOSSELIN, S.; GALLIOT, C.; MOULIN, L. Monitoring of Cryptosporidium and Giardia river contamination in Paris area. Water Research, v. 43, n. 1, p. 211-221, 2009. https://doi.org/10.1016/j.watres.2008.10.024
https://doi.org/10.1016/j.watres.2008.10... ; ^{UTAAKER et al., 2019}66 UTAAKER, K.S.; JOSHI, H.; KUMAR, A.; CHAUDHARY, S.; ROBERTSON, L.J. Occurrence of Cryptosporidium and Giardia in potable water sources in Chandigarh, Northern India. Journal of Water Supply: Research and Technology-Aqua, v. 68, n. 6, p. 483-494, 2019. https://doi.org/10.2166/AQUA.2019.157
https://doi.org/10.2166/AQUA.2019.157... ). ^{Davies et al. (2004)}19 DAVIES, C.M.; FERGUSON, C.M.; KAUCNER, C.; KROGH, M.; ALTAVILLA, N.; DEERE, DA.; ASHBOLT, N.J. Dispersion and transport of Cryptosporidium oocysts from fecal pats under simulated rainfall events. Applied and Environmental Microbiology, v. 70, n. 2, p. 1151-1159, 2004. https://doi.org/10.1128/AEM.70.2.1151-1159.2004
https://doi.org/10.1128/AEM.70.2.1151-11... , in a pilot-scale experiment, observed that, after heavy rainfall, floodwater passing through soils without vegetation cover had higher levels of oocysts than floodwater passing through covered soils. In the present study, animal feces containing Giardia cysts and Cryptosporidium oocysts were likely a source of water contamination.

Controversial results have been reported regarding the correlation between occurrence of protozoa and water turbidity. Some authors reported a significant correlation (^{HSU et al., 2000}33 HSU, B.M.; HUANG, C.; HSU, C.L.; HSU, Y.F.; HSU, C.L. Examination of Giardia and Cryptosporidium in water samples and fecal specimens in Taiwan. Water Science & Technology, v. 41, n. 7, p. 87-92, 2000. https://doi.org/10.2166/wst.2000.0119
https://doi.org/10.2166/wst.2000.0119... ; ^{HU, 2002}34 HU, T.L. Giardia cysts and Cryptosporidium oocysts detection in the intake and rapid filter system in a water purification plant. Biotechnology Letters, v. 24, p.1683-1686, 2002. https://doi.org/10.1023/A:1020605501525
https://doi.org/10.1023/A:1020605501525... ; ^{CARMENA et al., 2007}13 CARMENA, D.; AGUINAGALDE, X.; ZIGORRAGA, C.; CRESPO, J.; OCIO, J. Presence of Giardia cysts and Cryptosporidium oocysts in drinking water supplies in northern Spain. Journal of Applied Microbiology, v. 102, n. 3, p. 882-882, 2007. https://doi.org/10.1111/j.1365-2672.2006.03193.x
https://doi.org/10.1111/j.1365-2672.2006... ; ^{BURNET et al., 2014}10 BURNET, J.B.; PENNY, C.; OGORZALY, L.; CAUCHIE, H.M. Spatial and temporal distribution of Cryptosporidium and Giardia in a drinking water resource: implications for monitoring and risk assessment. Science of the Total Environment, v. 472, p. 1023-1035, 2014. https://doi.org/10.1016/j.scitotenv.2013.10.083
https://doi.org/10.1016/j.scitotenv.2013... ; ^{LIGDA et al., 2020}40 LIGDA, P.; CLAEREBOUT, E.; KOSTOPOULOU, D.; ZDRAGAS, A.; CASAERT, S.; ROBERTSON, L.J.; SOTIRAKI, S. Cryptosporidium and Giardia in surface water and drinking water: Animal sources and towards the use of a machine-learning approach as a tool for predicting contamination. Environmental Pollution, v. 264, p. 1-14, 2020. https://doi.org/10.1016/j.envpol.2020.114766
https://doi.org/10.1016/j.envpol.2020.11... ), whereas others reported a lack of correlation (^{MENGE et al., 2001}44 MENGE, J.G.; HAARHOFF, J.; KÖNIG, E.; MERTNES, R.; VAN, D.M.B. Occurrence and removal of Giardia and Cryptosporidium at the Goreangab Reclamation Plant. Water Science & Technology Water Supply, v. 1, n. 1, p. 97-106, 2001. https://doi.org/10.2166/ws.2001.0012
https://doi.org/10.2166/ws.2001.0012... ; ^{BASTOS et al., 2002}5 BASTOS, R.K.X.; VIEIRA, M.B.M.; BRITO, L.A.; BEVILACQUA, P.D.; NASCIMENTO, L.E.; HELLER, L. Giardia sp. cysts and Cryptosporidium spp. oocysts dynamics in Southeast Brazil: occurrence in surface water and removal in water treatment processes. Water Supply, v. 4, n. 2, p. 15-22, 2002. https://doi.org/10.2166/ws.2004.0022
https://doi.org/10.2166/ws.2004.0022... ; ^{HASHIMOTO; KUNIKANE; HIRATA, 2002}32 HASHIMOTO, A.; KUNIKANE, S.; HIRATA, T. Prevalence of Cryptosporidium oocysts and Giardia cysts in the drinking water supply in Japan. Wat Res, v. 36, n. 3, p. 519-526, 2002. https://doi.org/10.1016/s0043-1354(01)00279-2
https://doi.org/10.1016/s0043-1354(01)00... ; ^{RAMO et al., 2017}50 RAMO, A.; CACHO, E.D.; SÁNCHEZ-ACEDO, C.; QUÍLEZ, J. Occurrence of Cryptosporidium and Giardia in raw and finished drinking water in north-eastern Spain. Science of the Total Environment, v. 580, p. 1007-1013, 2017. 10.1016/j.scitotenv.2016.12.055
https://doi.org/10.1016/j.scitotenv.2016... ; ^{NASCIMENTO; GINORIS; BRANDÃO, 2020}46 NASCIMENTO, M.F.; GINORIS, Y.P.; BRANDÃO CCS. Cryptosporidium oocysts removal by upflow direct filtration: pilot scale assessment. Water, v. 12, n. 5, p. 1-14, 2020. https://doi.org/10.3390/w12051328
https://doi.org/10.3390/w12051328... ). Monitoring of protozoan levels in raw and drinking water should not be replaced by turbidity control.

Detection of cysts and oocysts in treated water

Giardia cysts and Cryptosporidium oocysts were detected in filtered water by direct filtration (WTP A and B) and flotation-filtration (WTP A). In WTP A, direct filtration is used to treat low-turbidity water, and flotation-filtration for high-turbidity water. The short time of direct filtration and the lack of clarification prior to filtration may have reduced protozoan removal efficiency. Moreover, an increase in filter washing during periods of high water turbidity reduces treatment efficiency, especially in the first hours after washing (^{LIBÂNIO, 2005}39 LIBÂNIO, M. Fundamento de Qualidade e Tratamento de Água. São Paulo: Átomo, 2005.). The combination of flotation and filtration was not sufficient to improve oocyst removal.

Brazilian drinking water legislation (PRC no. 888/2021, Ministry of Health) states that the turbidity of filtered water should not exceed 0.3 NTU in 95% of samples when the concentration of Cryptosporidium is greater than 1 oocyst L⁻¹ (BRAZIL 2021). The turbidity of water treated in WTP A and WTP B was higher than the limit defined by Brazilian drinking water legislation. In filtered samples containing Cryptosporidium oocysts, protozoan concentration was detected at concentrations above the alert levels of 0.1 oocysts L⁻¹ (^{THE WATER SUPPLY REGULATIONS, 2007}62 THE WATER SUPPLY (WATER QUALITY) REGULATIONS 2000/01. Statutory Instrument 2007 No. 2734. Water, England and Wales. (Amendment) Regulations 2007.) in all samples in which they were identified.

The presence of protozoa in treated water is not uncommon in developed countries. In the United Kingdom, ^{Mason et al. (2010)}43 MASON, B.W.; CHALMERS, R.M.; CARNICER-PONT, D.; CASEMORE, D.P.A. A Cryptosporidium hominis outbreak in North-West Wales associated with low oocyst counts in treated drinking water. Journal of Water and Health, v. 8, n. 2, p. 299-310, 2010. https://doi.org/10.2166/wh.2009.184
https://doi.org/10.2166/wh.2009.184... found an association between the presence of Cryptosporidium in treated drinking water and the 2005 waterborne outbreak. The authors stated that, although low, the oocyst count in treated water (< 0.08 oocysts 10 L⁻¹) was sufficient for infection. ^{Widerström et al. (2014)}69 WIDERSTRÖM, M.; SCHÖNNING, C.; LILJA, M.; LEBBAD, M.; LJUNG, T.; ALLESTAM, G.; FERM, M.; BJÖRKHOLM, B.; HANSEN, A.; HILTULA, J.; LÅNGMARK, J.; LÖFDAHL, M.; OMBERG, M.; REUTERWALL, C.; SAMUELSSON, E.; WIDGREN, K.; WALLENSTEN, A.; LINDH, J. Large outbreak of Cryptosporidium hominis infection transmitted through the public water supply, Sweden. Emerging Infectious Diseases, v. 20, n. 4, p. 581-589, 2014. https://doi.org/10.3201/eid2004.121415
https://doi.org/10.3201/eid2004.121415... detected 0.20 – 0.32 oocysts 10 L⁻¹ of Cryptosporidium in treated drinking water during an outbreak in Östersund, Sweden.

The high costs and methodological limitations of detecting Giardia and Cryptosporidium in water stimulate the search for indirect indicators of these protozoa. However, the scientific community has not yet identified a reliable indicator of protozoan occurrence in water. The United States Environmental Protection Agency (USEPA) established Escherichia coli limits for water sources that, if exceeded, require sampling for Cryptosporidium, but many studies have found no correlation between fecal indicators such as E. coli and Cryptosporidium in water (^{BONADONNA et al., 2002}7 BONADONNA, L.; BRIANCESCO, R.; OTTAVIANI, M.; VESCHETTI, E. Occurrence of Cryptosporidium oocysts in sewage effluents and correlation with microbial, chemical and physical water variables. Environmental Monitoring and Assessment, v. 75, n. 3, p. 241-252, 2002. https://doi.org/10.1023/a:1014852201424
https://doi.org/10.1023/a:1014852201424... ; ^{HARWOOD et al., 2005}31 HARWOOD, V.J.; LEVINE, A.D.; SCOTT, T.M.; CHIVUKULA, V.; LUKASIK, J.; FARRAH, S.R.; ROSE, J.B. Validity of the indicator organism paradigm for pathogen reduction in reclaimed water and public health protection. Applied and Environmental Microbiology, v. 71, n. 6, p. 3163-3170, 2005. https://doi.org/10.1128/AEM.71.6.3163-3170.2005
https://doi.org/10.1128/AEM.71.6.3163-31... ; ^{MONS et al., 2009}45 MONS, C.; DUMÈTRE, A.; GOSSELIN, S.; GALLIOT, C.; MOULIN, L. Monitoring of Cryptosporidium and Giardia river contamination in Paris area. Water Research, v. 43, n. 1, p. 211-221, 2009. https://doi.org/10.1016/j.watres.2008.10.024
https://doi.org/10.1016/j.watres.2008.10... ; ^{NIEMINSKI et al., 2010}48 NIEMINSKI, E.; DURRANT, G.C.; HOYT, M.B.; OWENS, M.E.; PETERSON, L.; PETERSON, S.; TANNER, W.D.; ROSEN, J.; CLANCY, J. Is E. coli an appropriate surrogate for Cryptosporidium occurrence in water? Journal AWWA, v. 102, n. 3, p. 65-78, 2010. https://doi.org/10.1002/j.1551-8833.2010.tb10073.x
https://doi.org/10.1002/j.1551-8833.2010... ). The discrepancy in reports on the correlation between physicochemical and biological parameters can be attributed to differences in water quality, analytical methods, and equipment used for parasite detection (^{VERNILE et al., 2008}67 VERNILE, A.; NABI, A.Q.; BONADONNA, L.; BRIANCESCO, R.; MASSA, S. Occurrence of Giardia and Cryptosporidium in Italian water supplies. Environmental Monitoring and Assessment, v. 152, p. 203-207, 2008. https://doi.org/10.1007/s10661-008-0308-4
https://doi.org/10.1007/s10661-008-0308-... ).

The USEPA suggests that aerobic bacterial spores be utilized as a surrogate for Cryptosporidium, because they are not pathogenic, can be produced and analyzed cheaply and easily in the laboratory, are persistent in the environment, and remain unchanged during transport, sampling, and laboratory analysis (^{USEPA, 2010}64 UNITED STATES ENVIRONMENTAL PROTECTION AGENCY (USEPA). Long Term 2 Enhanced Surface Water Treatment Rule Toolbox Guidance Manual. EPA 815-R-0-16. Washington: USEPA, 2010.). Some aspects of the current study must be considered. The non-detection of cysts and oocysts in chlorinated water samples from WTP A and B does not imply the absence of protozoa (^{ALLEN; CLANCY; RICE, 2000}1 ALLEN, M.J.; CLANCY, J.L.; RICE, E.W. The plain, hard truth about pathogen monitoring. Journal AWWA, v. 92, n. 9, p. 64-76, 2000. https://doi.org/10.1002/j.1551-8833.2000.tb09005.x
https://doi.org/10.1002/j.1551-8833.2000... ; ^{VERNILE et al., 2008}67 VERNILE, A.; NABI, A.Q.; BONADONNA, L.; BRIANCESCO, R.; MASSA, S. Occurrence of Giardia and Cryptosporidium in Italian water supplies. Environmental Monitoring and Assessment, v. 152, p. 203-207, 2008. https://doi.org/10.1007/s10661-008-0308-4
https://doi.org/10.1007/s10661-008-0308-... ). The methods used for Giardia and Cryptosporidium quantification, added to the small sample volume, resulted in low recovery efficiencies from low-turbidity waters. Factors related to water quality and chemical compounds used in water treatment processes, such as iron and aluminum coagulants, polymers, and chlorine, may interfere with parasite separation and detection with antibodies (^{USEPA 2001}63 UNITED STATES ENVIRONMENTAL PROTECTION AGENCY (USEPA). Method 1623: Cryptosporidium and Giardia in water by filtration/IMS/FA. EPA 821-R-01-025. Washington: USEPA, 2001.). The determination of protozoa viability is important, because low numbers of viable cysts and oocysts in water can present risks (^{EHSAN, 2015}23 EHSAN, A. Water-borne transmission of Cryptosporidium and Giardia in Belgium and Bangladesh. 2015. 193 f. Thesis (Doctor in Veterinary Science) — Faculty of Veterinary Medicine, Ghent University, Merelbeke, 2015.). However, commonly used methodologies do not assess infectivity.

The results showed that direct filtration and flotation-filtration alone are not effective in removing protozoa from waters supplying WTP A and B; and post-treatment with chlorine does not guarantee a reduction of infection risks. The already proven resistance of Cryptosporidium oocysts and Giardia cysts to chlorination combined with the methodological limitations in detecting protozoa in chlorinated water reinforces the importance of continuous monitoring and determination of the viability of cysts and oocysts in drinking source water and the need for preventive and corrective measures to minimize watershed contamination.

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