Development of a standarised protocol for analyses somatic coliphages in sludge, soil and treated biowastes [Final Report]

Abstract

An initial desk study indicated that many biosolids contain human virus, but also that even in the more contaminated ones, they are not very abundant and that they are difficult to recover and quantify. Moreover, feasible methods for detecting infectious viruses are only available for enteroviruses, and it is well known that infectious viruses are needed for risk assessment. The need of indicators seemed obvious, as it was clear that the traditional bacterial indicators are not a good option for predicting presence and behaviour of human viruses in biosolids, biowastes and soils. Bacteriophages appeared as more suitable indicators, and among the proposed groups of phages, somatic coliphages aroused as those potentially more useful attending to their numbers in biosolids, feasibility of the detection methods and for sharing behaviour with viruses in biosolid and biowastes processing. Consequently, at present, somatic coliphages appear as a very useful indicator. Moreover, feasible standardised methods (ISO (adopted by CEN) and USEPA) for detection and quantification of somatic coliphages suspended in water, and consequently in aqueous solutions, are available. As well, literature available indicated that as human viruses, bacteriophages present in biosolids tend to be either included in or adsorbed to particles. Consequently, an extraction steep is necessary. There is a relatively abundant literature regarding extraction of human viruses and bacteriophages from solids. The methods for extracting bacteriophages (and also human viruses) from solids require the following steeps: homogenization, elution, clarification and decontamination. Described methods mostly vary in the elution steep. In a pair of papers comparing several elution methods showed elution with beef extract as the more efficient one. However the described methods required optimization in the various steeps. The optimization of the various steeps was the main aim of this work. Unfortunately, inoculation of biosolids with known concentrations of viruses (as it is feasible with water samples) are not mimicking what happens in the real world because of inclusion and adsorption of phages in/to solid particles. Therefore, it was decided to perform the experiments of optimization of the extraction method with matrixes containing concentrations as high as possible of naturally occurring somatic coliphages. The studied matrixes were raw sludge, digested and dewatered sludge, selected (for its content of somatic coliphages) compost and soil contaminated with raw sewage. Some of these matrixes contained very homogeneous and steady numbers of phages and consequently it was possible to compare modifications of each one of the steeps of the extraction method and how they affected the efficiency of recovery. The method was optimised, published in scientific literature (Guzman et al. 2007. J. Virol. Methods 144: 41-48) and a draft of a “standard method” for the extraction of somatic coliphages from biosolids, biowastes and soils has been presented for approval to the CEN/TC308/WG1/TG5. The non-convenience of inoculating these sort of matrices, since as said previously, the inoculated material does not mimic what happens in nature, complicated the performance of validation studies. Reference materials are needed for validation studies. To get round this problem, a few natural biosolids were tested as potential reference materials. For it, the matrices were distributed in a great number of containers, and placed at 4ºC. Then, somatic coliphages were enumerated after different days of storage from two containers and also from two subsamples of each one of the containers. This allowed testing the intra and inter-container homogeneity and the time elapsed without significant descent in the number of phages detected. Digested-dewatered sludge probed to be an excellent reference material lasting in perfect condition for at least 2 months. This will allow to make validation multilaboratory studies and have a reference material for “in lab” quality control. In fact, a small validation study of the extraction method with three laboratories was performed with satisfactory results. A few experiments done with other phages, as for example F-specific RNA bacteriophages, indicate that the method will also be applicable to other phages. As well, it may be useful for extracting human and animal viruses, though this should be further verified. In conclusion, a feasible, fast and low cost method for determining somatic coliphages from biosolids, biowastes and soils is available. Besides the feasibility of the methods for extraction, detection and enumeration, somatic coliphages have, in our opinion, several advantages to follow the higienization processes of sludges as well as to have an indication of the viral contamination of these solid matrixes.