Inactivation of Cryptosporidium across the wastewater treatment train: Recycled water fit for purpose (Phase II) Brendon King, Stella Fanok, Renae Phillips, Melody Lau, Ben van den Akker and Paul Monis Project 8OS – 8012 Final Report – March 2015 1 Table of Contents 1 INTRODUCTION ..................................................................................................................................................................... 19 1.1 OVERVIEW ............................................................................................................................................................................................... 19 1.2 CRYPTOSPORIDIUM AND CRYPTOSPORIDIOSIS .................................................................................................................................... 20 1.3 CRYPTOSPORIDIUM SPECIES PRESENT IN THE AUSTRALIAN COMMUNITY ..................................................................................... 21 1.4 CURRENT METHODS FOR ISOLATION AND DETECTION FOR CRYPTOSPORIDIUM .......................................................................... 22 1.5 ASSESSMENT OF CRYPTOSPORIDIUM INFECTIVITY ............................................................................................................................ 23 1.5.1 In vitro assays for assessing viability of Cryptosporidium ........................................................................................... 23 1.5.2 Human infectivity assays ........................................................................................................................................................... 25 1.5.3 Animal models ............................................................................................................................................................................... 25 1.5.4 Infection of Cell Culture ............................................................................................................................................................. 27 1.5.5 Focus detection method ............................................................................................................................................................. 29 1.6 CRITICAL PROCESSES AFFECTING CRYPTOSPORIDIUM INFECTIVITY ............................................................................................... 31 1.6.1 Heat (temperatures >37°C) ...................................................................................................................................................... 31 1.6.2 Freezing and desiccation .......................................................................................................................................................... 31 1.6.3 Ammonia ......................................................................................................................................................................................... 32 1.6.4 Hydrological parameters .......................................................................................................................................................... 32 1.6.5 Predation and microbial activity ........................................................................................................................................... 34 1.6.6 Inactivation by temperature (temperatures 2-37°C) .................................................................................................... 34 1.6.7 Inactivation by sunlight ............................................................................................................................................................. 35 1.6.8 Media filtration ............................................................................................................................................................................. 38 1.6.9 Dissolved air flotation ................................................................................................................................................................ 39 1.6.10 Membrane filtration .............................................................................................................................................................. 40 1.6.11 Activated sludge and other secondary wastewater treatment processes ........................................................ 40 1.6.12 Lagoons / waste stabilization ponds .............................................................................................................................. 41 1.6.13 Oxidant-based disinfection .................................................................................................................................................. 42 1.6.14 Ultraviolet light ....................................................................................................................................................................... 43 1.6.15 Combined processes ............................................................................................................................................................... 44 1.6.16 Heat .............................................................................................................................................................................................. 44 1.6.17 Other ............................................................................................................................................................................................ 45 1.7 DESCRIPTION OF THE WASTE WATER TREATMENT PLANTS CHOSEN FOR INVESTIGATION ....................................................... 45 1.7.1 Western Treatment Plant ......................................................................................................................................................... 45 1.7.2 Altona WWTP ................................................................................................................................................................................ 45 1.7.3 Mt Martha WWTP ........................................................................................................................................................................ 46 1.7.4 Aldinga WWTP .............................................................................................................................................................................. 46 2 1.7.5 Glenelg WWTP ............................................................................................................................................................................... 46 1.8 OBJECTIVES OF THE STUDY ................................................................................................................................................................... 47 2 METHODOLOGY ..................................................................................................................................................................... 48 2.1 WASTEWATER TREATMENT PLANT (WWTP) SELECTION AND SAMPLING STRATEGY.............................................................. 48 2.2 SAMPLING SCHEDULES AND SAMPLE COLLECTION ............................................................................................................................ 49 2.3 ROUTINE DATA COLLECTION ................................................................................................................................................................ 59 2.4 CONCENTRATION AND ISOLATION OF CRYPTOSPORIDIUM FROM WWTP SAMPLES ................................................................... 59 2.5 ENUMERATION OF CRYPTOSPORIDIUM OOCYSTS AND DETERMINATION OF OOCYST INFECTIVITY BY THE MODIFIED FOCUS DETECTION METHOD .............................................................................................................................................................................................. 60 2.5.1 Sampling and sample pre-treatment ................................................................................................................................... 60 2.5.2 Establishment of an HCT-8 monolayer for infection ...................................................................................................... 60 2.5.3 Dissociation of Cryptosporidium sp. oocysts from IMS Dynabeads .......................................................................... 61 2.5.4 Excystation pre-treatment of Cryptosporidium sp oocysts and their application to the HCT-8 cell monolayers...................................................................................................................................................................................................... 62 2.5.5 Staining the HCT-8 monolayer for foci detection ............................................................................................................ 63 2.6 OUTDOOR SOLAR INACTIVATION STUDIES ........................................................................................................................................ 65 2.7 STATISTICAL ANALYSES ........................................................................................................................................................................ 67 2.7.1 Calculation of oocyst inactivation ......................................................................................................................................... 68 2.7.2 Calculation of log10 reduction values (LRVs) for total