Title: Frequency Of Reovirus Detection In Biosolids: Comparison Of The EPA CFR 503 Technique To Integrated Cell Culture - Real Time PCR
Project Type: Research
Start Date: 03/01/2006
End Date: 02/28/2007
Congressional District: NH01
Focus Categories: Methods, Water Quantity, Groundwater
Keywords: Biosolids, viruses
Principal Investigator: Margolin, Aaron B.
Federal Funds: $ 25,000
Non-Federal Matching Funds: $ 51,356
Abstract: Biosolids are defined by the EPA as the residue generated during treatment of domestic sewage that meets federal and state standards for beneficial use (EPA, 1992). Biosolid disposal by land application is a controversial issue because of the potential public health threat from pathogens, such as Salmonella, Cryptosporidium and enteric viruses. The EPA 503 regulations distinguish between two classes of biosolids. Class B biosolids are processed by methods such as digestion, drying, composting, and lime stabilization. Class B biosolids are restricted in the sites that they can be land-applied, but can be applied within the restrictions without further testing. Some places that class B biosolids are allowed to be applied are agricultural land not used for food crops and disturbed mining areas. They can not be used in areas that are open to the public.
Class A biosolids are not restricted and can be applied to any application such as fertilizer for food crops or sold in bags as fertilizer for the public. In order for class A biosolids to be approved for land application they need to be tested for either fecal coliforms or Salmonella, which are both bacteria. Class A biosolids must be treated according to one of the 9 approved processes. If they are not treated by one of the approved processes, alternatively they can be evaluated for viable helminth ova and enteric viruses to assess the sanitary quality. Enteric virus testing is performed by a method known as a plaque assay, which in a non-molecular method that was developed more then 30 years ago.
Sludge that is directly land applied may lead to viral contamination of crops or groundwater, especially since viruses tend to be associated with solids (Metcalf, 1995). Viruses that are particle associated are subject to desorption and migration following heavy rain and similar weather events (Metcalf, 1995).
It is very difficult to detect viruses in biosolids using a plaque assay because many viruses do not form plaques or will only form plaques after several passages on a cell line (Fong, 2005). Additionally, plaques can not conclusively identified as virus by visual inspection because they could be caused by either a virus or varied toxic substances found in sludge. These toxins can cause a clearing of the cell layer resulting in a false positive (Schmidt 1978). Hence, evaluating the public health risk associated with the land application of biosolids using the plaque assay method originally mandated in the CFR 503 may yield results which underestimate the true concentration of virus in the material. Modern advances in molecular diagnostic techniques have lead to an alternative method that combines the use of cell culture with that of the polymerase chain reaction assay (PCR), collectively known as integrated cell culture-PCR. This method permits detection of viruses that do not form plaques and would otherwise go undetected. Utilization of this methodology, along with real-time PCR, permits rapid and reliable detection of viruses that were not evaluated in biosolids when the original CFR 503 regulations were developed; yielding a more accurate evaluation of the potential public health threat that may exist from the land application of Class A biosolids.
Progress/Completion Report, PDF