Data presented demonstrate the relatively high multiplicity of solids-associated virus in field samples, i.e., wastewater, sludge, and soils. Influent, effluent, and chlorinated effluent samples showed 16.1 to 100% of the total virus demonstrated in samples to be solids associated. Three techniques for freeing solidsassociated virus are described and compared. Using sonication of solids and polyethylene glycol concentration, virus was demonstrated in fully digested sludge (60 days at 34 C), sand at the site of a sewer leak, and dried sludge cake and mud 900 m downstream from a sewage disposal site. These data emphasize the inadequacy of virus concentration techniques that do not include the processing of solids. In situ elution failed to free solids-associated virus.

No abstract

The distribution of solids-associated viruses in wastewater was studied to determine the effect of treatment processes on viruses associated with solids. Solids less than 0.3 pim in diameter were separated from the liquid phase of each sample by using a continuous-flow centrifuge. The percentage of virus associated with solids larger than 0.3 ,um decreased from 28% in the influent to 3.4% in unchlorinated effluent, and this was accompanied by a 92% decrease in the total concentration of virus. These results indicate that the original solids-associated virus as well as virus that is secondarily adsorbed to mixed liquor-suspended solids is lost during clarification. The total concentration of virus was reduced by 82% by chlorination, and the percentage of virus associated with solids increased to 7.7% upon chlorination, indicating some protection due to association with particles larger than 0.3 ,um. When a suspension of fecal particles and a 0.22-,um filtrate of a fecal homogenate were sonicated, a threefold increase in virus titer was observed in each. This demonstrated that viruses may be attached to particles smaller than 0.22 ,um. Thus, small viral aggregates or viruses attached to submicron particles represented the major portion of solids-associated virus in treated sewage. Viruses associated with solids in water may be protected from inactivation by chlorine and by natural inactivating factors (3, 6, 11, 13). Viruses adsorb to various organic and inorganic solids, and this adsorption is highly dependent on such factors as pH, solids concentration, and metal ion composition (2,9, 10). These findings suggest that virions associated with solids in water may be of primary importance since they would resist inactivation and be transported into receiving waters and possibly back to humans.

Five cases of primary amoebic meningoencephalitis associated with swimming in freshwater lakes have been recorded in Florida over the past 14 years. The present study demonstrated that pathogenic Naegleria, the causative agent, is relatively widespread. Twelve of 26 lakes sampled only once yielded the amoeba. Populations in three of five lakes sampled routinely reached levels of one amoeba per 25 ml of water tested during the hot summer months. Overwintering in freshwater lake bottom sediments was demonstrated, showing that thermaldischarge pollution of waters plays a miniscule, if any, role in the maintenance of pathogenic Naegleria in nature in this semitropical area.

Poliovirus in fecal homogenates was used to determine the protection against inactivation by chlorination afforded virus that was occluded within particulates. Virus that was closely associated with or occluded within small fecal particulates was protected. A fourfold increase in combined residual chlorine was required to achieve the same degree of inactivation for occluded virus as for free or secondarily adsorbed virus. A combined chlorine residual of 6.6 mg/liter was necessary to achieve 50% inactivation in 15 min at pH 8.0 and 220C in a particulate suspension containing occluded virus compared to 1.4 mg/liter for free virus. These differences were found to be relatively small compared to differences due to the presence of dissolved organics or between free and combined chlorine residuals. The results suggest different mechanisms of protection due to adsorption and occlusion. The presence of viruses in treated wastewater and receiving waters has led to concern about long-range transport of human viruses in the environment. The fact that viruses can survive wastewater treatment and chlorination has led to speculation on how they escape inactivation. One means of protection may be the occlusion of virions within particulate matter. Viruses adsorb to organic and inorganic solids under many conditions (2, 9, 13, 14, 18) and retain their infectivity (10, 12). There is evidence that some viruses adsorbed to clays and organic solids are afforded protection from inactivation by chlorine (J. C. Hoff, paper presented at the Conference on the Evaluation of Microbiology Standards for Drinking Water, 13-14 April 1977, EPA Office of Water Supply, Washington, D.C.) and seawater (6, 15). However, data are not available on the additional protection that may result when viruses remain occluded within fecal particulates, the form in which they are introduced into the sewerage system. The objective of this study was to quantify the protection against chlorine inactivation afforded viruses that are occluded within or tightly bound to the original fecal material.