Persistence of poliovirus 1 in soil and on vegetables grown in soil previously flooded with inoculated sewage sludge or effluent.

Land disposal of sewage sludge and effluent is becoming a common practice in the United States. The fertilizer content and humus value of such wastes are useful for agricultural purposes, and the recycling of sewage onto the land eliminates many of our stream pollution problems. The potential exists for crops grown in such irrigated soil to be contaminated by viruses that may be present in the sewage. Studies were initiated to determine viral persistence in soil and on crops grown under natural conditions in field plots that had been flooded to a depth of 1 inch (2.54 cm) with poliovirus 1-inoculated sewage wastes. Lettuce and radishes were planted in sludge- or effluent-flooded soil. In one study, the vegetables were planted 1 day before flooding, and in another they were planted 3 days after the plots were flooded. Survival of poliovirus 1 in soil irrigated with inoculated sewage sludge and effluent was determined during two summer growing seasons and one winter period. The longest period of survival was during the winter, when virus was detected after 96 days. During the summer, the longest survival period was 11 days. Poliovirus 1 was recovered from the mature vegetables 23 days after flooding of the plots had ceased. Lettuce and radishes are usually harvested 3 to 4 weeks after planting.     

Poliovirus removal from primary and secondary sewage effluent by soil filtration.

Adsorption of poliovirus from primary sewage effluent was similar to that from secondary sewage effluent in both batch soil studies and experiments with soil columns 240 cm long. Virus desorption by distilled water was also similar in a soil column that had been flooded with either primary or secondary effluent seeded with virus. These results indicated that absorption of poliovirus from primary effluent and virus movement through the soil were not affected by the higher organic content of primary sewage effluent.     

Poliovirus removal from primary and secondary sewage effluent by soil filtration.

Adsorption of poliovirus from primary sewage effluent was similar to that from secondary sewage effluent in both batch soil studies and experiments with soil columns 240 cm long. Virus desorption by distilled water was also similar in a soil column that had been flooded with either primary or secondary effluent seeded with virus. These results indicated that absorption of poliovirus from primary effluent and virus movement through the soil were not affected by the higher organic content of primary sewage effluent.     

Survival of enteroviruses in rapid-infiltration basins during the land application of wastewater.

The downward migration through soil of seeded poliovirus type 1 and echovirus type 1 and of naturally occurring enteroviruses during infiltration of sewage effluent through rapid-infiltration basins was investigated. After 5 days of flooding, the amount of seeded poliovirus type 1 that had migrated 5 to 10 cm downward through the soil profile was found to be 11% of that remaining at the initial burial depth. The amount of echovirus type 1 determined to have moved an equal distance was at least 100-fold less. Migration of naturally occurring enteroviruses during infiltration of sewage effluent through soil could not be measured with accuracy because of the possibility of virus survival from previous applications of effluent. The rate of inactivation for seeded poliovirus 1 and echovirus 1 buried in the infiltration basins ranged between 0.04 and 0.15 log10 units per day during the time when the basins were flooded. Inactivation of these same seeded virus types and of indigenous enterovirus populations in the infiltration basins during the drying portion of the sewage application cycle ranged between 0.11 and 0.52 log10 units per day. The rate of virus inactivation was dependent upon the rate of soil moisture loss. These results indicate that drying cycles during the land application of wastewater enhance virus inactivation in the soil.     

Survival of enteroviruses in rapid-infiltration basins during the land application of wastewater

The downward migration through soil of seeded poliovirus type 1 and echovirus type 1 and of naturally occurring enteroviruses during infiltration of sewage effluent through rapid-infiltration basins was investigated. After 5 days of flooding, the amount of seeded poliovirus type 1 that had migrated 5 to 10 cm downward through the soil profile was found to be 11% of that remaining at the initial burial depth. The amount of echovirus type 1 determined to have moved an equal distance was at least 100-fold less. Migration of naturally occurring enteroviruses during infiltration of sewage effluent through soil could not be measured with accuracy because of the possibility of virus survival from previous applications of effluent. The rate of inactivation for seeded poliovirus 1 and echovirus 1 buried in the infiltration basins ranged between 0.04 and 0.15 log10 units per day during the time when the basins were flooded. Inactivation of these same seeded virus types and of indigenous enterovirus populations in the infiltration basins during the drying portion of the sewage application cycle ranged between 0.11 and 0.52 log10 units per day. The rate of virus inactivation was dependent upon the rate of soil moisture loss. These results indicate that drying cycles during the land application of wastewater enhance virus inactivation in the soil.     

Virus movement in soil during saturated and unsaturated flow

Virus movement in soil during saturated and unsaturated flow was compared by adding poliovirus to sewage water and applying the water at different rates to a 250-cm-long soil column equipped with ceramic samplers at different depths. Movement of viruses during unsaturated flow of sewage through soil columns was much less than during saturated flow. Viruses did not move below the 40-cm level when sewage water was applied at less than the maximum infiltration rate; virus penetration in columns flooded with sewage was at least 160 cm. Therefore, virus movement in soils irrigated with sewage should be less than in flooded groundwater recharge basins or in saturated soil columns. Management of land treatment systems to provide unsaturated flow through the soil should minimize the depth of virus penetration. Differences in virus movement during saturated and unsaturated flow must be considered in the development of any model used to simulate virus movement in soils.     

Poliovirus survival and movement in a sandy forest soil.

Movement of poliovirus I (Chat) through nonsterile core samples of a sandy forest soil was monitored, using several regimens of loading with either dechlorinated final effluent from an operating activated sludge treatment plant or distilled water. Stimulated cycles of rainfall and effluent applications, resulting in ionic gradients, were shown to affect virus movement. Such studies indicate that poliovirus applied in effluents may move considerable distances through this soil after rainfall. Survival of poliovirus in the soil at 4 and 20 C has been monitored for 84 days. During this period, the capacity of the virus to migrate is unchanged.     

Poliovirus survival and movement in a sandy forest soil.

Movement of poliovirus I (Chat) through nonsterile core samples of a sandy forest soil was monitored, using several regimens of loading with either dechlorinated final effluent from an operating activated sludge treatment plant or distilled water. Stimulated cycles of rainfall and effluent applications, resulting in ionic gradients, were shown to affect virus movement. Such studies indicate that poliovirus applied in effluents may move considerable distances through this soil after rainfall. Survival of poliovirus in the soil at 4 and 20 C has been monitored for 84 days. During this period, the capacity of the virus to migrate is unchanged.     

Virus movement in soil during saturated and unsaturated flow.

Virus movement in soil during saturated and unsaturated flow was compared by adding poliovirus to sewage water and applying the water at different rates to a 250-cm-long soil column equipped with ceramic samplers at different depths. Movement of viruses during unsaturated flow of sewage through soil columns was much less than during saturated flow. Viruses did not move below the 40-cm level when sewage water was applied at less than the maximum infiltration rate; virus penetration in columns flooded with sewage was at least 160 cm. Therefore, virus movement in soils irrigated with sewage should be less than in flooded groundwater recharge basins or in saturated soil columns. Management of land treatment systems to provide unsaturated flow through the soil should minimize the depth of virus penetration. Differences in virus movement during saturated and unsaturated flow must be considered in the development of any model used to simulate virus movement in soils.     

Impact of Fertilizing with Raw or Anaerobically Digested Sewage Sludge on the Abundance of Antibiotic-Resistant Coliforms,Antibiotic Resistance Genes,and Pathogenic Bacteria in Soil and on Vegetables at Harvest

The consumption of crops fertilized with human waste represents a potential route of exposure to antibiotic-resistant fecal bacteria. The present study evaluated the abundance of bacteria and antibiotic resistance genes by using both culture-dependent and molecular methods. Various vegetables (lettuce, carrots, radish, and tomatoes) were sown into field plots fertilized inorganically or with class B biosolids or untreated municipal sewage sludge and harvested when of marketable quality. Analysis of viable pathogenic bacteria or antibiotic-resistant coliform bacteria by plate counts did not reveal significant treatment effects of fertilization with class B biosolids or untreated sewage sludge on the vegetables. Numerous targeted genes associated with antibiotic resistance and mobile genetic elements were detected by PCR in soil and on vegetables at harvest from plots that received no organic amendment. However, in the season of application, vegetables harvested from plots treated with either material carried gene targets not detected in the absence of amendment. Several gene targets evaluated by using quantitative PCR (qPCR) were considerably more abundant on vegetables harvested from sewage sludge-treated plots than on vegetables from control plots in the season of application, whereas vegetables harvested the following year revealed no treatment effect. Overall, the results of the present study suggest that producing vegetable crops in ground fertilized with human waste without appropriate delay or pretreatment will result in an additional burden of antibiotic resistance genes on harvested crops. Managing human exposure to antibiotic resistance genes carried in human waste must be undertaken through judicious agricultural practice.     

Adsorption of enteroviruses to soil cores and their subsequent elution by artificial rainwater.

The adsorption and elution of a variety of human enteroviruses in a highly permeable, sandy soil was studied by using cores (43 by 125 mm) collected from an operating recharge basin on Long Island. Viruses studied included field and reference strains of polioviruses types 1 and 3 and reference strains of coxsackie virus B3 and echovirus types 1 and 6. Viruses suspended in treated sewage effluent were allowed to percolate through soil cores, and the filtrate was assayed for unadsorbed viruses. To determine the likelihood of desorption and mobilization, soil-bound viruses were subjected to a rinse with either treated sewage effluent or simulated rainwater which reflected the anion, cation, and pH characteristics of a typical northeastern United States rainfall. The results demonstrated that all polioviruses tested, including both reference and field strains, adsorbed extremely well to cores. Adsorption was somewhat reduced when clean, unconditioned soils were used. Soil-bound poliovirus strain LSc was not significantly mobilized by flooding columns with either a sewage effluent or rainwater rinse. One virus was mobilized by both types of rinses. The amount of viruses mobilized by rainwater rinses ranged from 24 to 66%. Variable adsorption-elution results were observed with other enteroviruses. Two guanidine-resistant mutants of poliovirus LSc demonstrated a soil adsorption-elution profile different from that of the parent strain. The data support the conclusion that soil adsorption-elution behavior is strain dependent and that poliovirus, particularly strain LSc, represents an inappropriate model.     

High levels of microbial contamination of vegetables irrigated with wastewater by the drip method.

The public health aspects of the use of wastewater in agriculture and the effects of the drip irrigation method on the contamination of vegetables were studied. The method used was to simulate enteric microorganisms' dissemination by contaminated irrigation water in the field. The vegetables were irrigated with an effluent inoculated with a high titer of traceable microorganisms: poliovirus vaccine and a drug-resistant Escherichia coli. The dissemination of the marker organisms in the field was followed, and the effects of certain manipulations of the drip irrigation method on the contamination of the crops by the effluent were examined. It was shown that drip irrigation under plastic sheet cover with the drip lines placed either on the soil surface or buried at a depth of 10 cm significantly reduced crop contamination from inoculated irrigation water even when massive doses of bacteria and viruses were used. The microbial contamination was found to persist in the irrigation pipes and in the soil for at least 8 and 18 days, respectively. The data indicate that the recovery of the marker organisms was affected by soil texture and environmental conditions.     

High levels of microbial contamination of vegetables irrigated with wastewater by the drip method.

The public health aspects of the use of wastewater in agriculture and the effects of the drip irrigation method on the contamination of vegetables were studied. The method used was to simulate enteric microorganisms' dissemination by contaminated irrigation water in the field. The vegetables were irrigated with an effluent inoculated with a high titer of traceable microorganisms: poliovirus vaccine and a drug-resistant Escherichia coli. The dissemination of the marker organisms in the field was followed, and the effects of certain manipulations of the drip irrigation method on the contamination of the crops by the effluent were examined. It was shown that drip irrigation under plastic sheet cover with the drip lines placed either on the soil surface or buried at a depth of 10 cm significantly reduced crop contamination from inoculated irrigation water even when massive doses of bacteria and viruses were used. The microbial contamination was found to persist in the irrigation pipes and in the soil for at least 8 and 18 days, respectively. The data indicate that the recovery of the marker organisms was affected by soil texture and environmental conditions.     

Virus movement in soil columns flooded with secondary sewage effluent.

Secondary sewage effluent containing about 3 X 10(4) plaque-forming units of polio virus type 1 (LSc) per ml was passed through columns 250 cm in length packed with calcareous sand from an area in the Salt River bed used for ground-water recharge of secondary sewage effluent. Viruses were not detected in 1-ml samples extracted from the columns below the 160-cm level. However, viruses were detected in 5 of 43 100-ml samples of the column drainage water. Most of the viruses were adsorbed in the top 5 cm of soil. Virus removal was not affected by the infiltration rate, which varied between 15 and 55 cm/day. Flooding a column continuosly for 27 days with the sewage water virus mixture did not saturate the top few centimeters of soil with viruses and did not seem to affect virus movement. Flooding with deionized water caused virus desorption from the soil and increased their movement through the columns. Adding CaCl2 to the deionized water prevented most of the virus desorption. Adding a pulse of deionized water followed by sewage water started a virus front moving through the columns, but the viruses were readsorbed and none was detected in outflow samples. Drying the soil for 1 day between applying the virus and flooding with deionized water greatly reduced desorption, and drying for 5 days prevented desorption. Large reductions (99.99% or more) of virus would be expected after passage of secondary sewage effluent through 250 cm of the calcareous sand similar to that used in our laboratory columns unless heavy rains fell within 1 day after the application of sewage stopped. Such virus movement could be minimized by the proper management of flooding and drying cycles.     

Virus movement in soil columns flooded with secondary sewage effluent.

Secondary sewage effluent containing about 3 X 10(4) plaque-forming units of polio virus type 1 (LSc) per ml was passed through columns 250 cm in length packed with calcareous sand from an area in the Salt River bed used for ground-water recharge of secondary sewage effluent. Viruses were not detected in 1-ml samples extracted from the columns below the 160-cm level. However, viruses were detected in 5 of 43 100-ml samples of the column drainage water. Most of the viruses were adsorbed in the top 5 cm of soil. Virus removal was not affected by the infiltration rate, which varied between 15 and 55 cm/day. Flooding a column continuosly for 27 days with the sewage water virus mixture did not saturate the top few centimeters of soil with viruses and did not seem to affect virus movement. Flooding with deionized water caused virus desorption from the soil and increased their movement through the columns. Adding CaCl2 to the deionized water prevented most of the virus desorption. Adding a pulse of deionized water followed by sewage water started a virus front moving through the columns, but the viruses were readsorbed and none was detected in outflow samples. Drying the soil for 1 day between applying the virus and flooding with deionized water greatly reduced desorption, and drying for 5 days prevented desorption. Large reductions (99.99% or more) of virus would be expected after passage of secondary sewage effluent through 250 cm of the calcareous sand similar to that used in our laboratory columns unless heavy rains fell within 1 day after the application of sewage stopped. Such virus movement could be minimized by the proper management of flooding and drying cycles.     

Bacteriological quality of crops irrigated with wastewater in the Xochimilco plots, Mexico City, Mexico

Xochimilco county plots (Mexico City), one of the most fertile agricultural areas in the Valley of Mexico, produce a large portion of the fresh vegetables consumed in the city. These plots are generally irrigated with domestic wastewater, and for this reason, it was deemed important to examine and evaluate the bacteriological quality of the water, soil, and vegetables from these plots that are harvested and marketed. The soils were also examined for the classical parameters such as nitrates, ammonia, etc., and organic matter and texture. The crops selected for this study were radishes, spinach, lettuce, parsley, and celery because they are usually consumed raw. The highest bacterial counts were encountered in leafy vegetables, i.e., spinach (8,700 for total coliform and 2,400 for fecal coliform) and lettuce (37,000 for total coliform and 3,600 for fecal coliform). Statistically significant differences in bacterial counts between rinsed and unrinsed edible portions of the crops were observed even in rinsed vegetables, and high densities of fecal coliform were detected, indicating that their consumption represents a potential health hazard. The total coliform values found in irrigation water ranged from 4 X 10(4) to 29 X 10(4), and for fecal coliform the values ranged from 5 X 10(2) to 30 X 10(2).     

Effect of soil permeability on virus removal through soil columns

Laboratory experiments were performed on four different soils, using 100 cm long columns, to determine the extent of virus movement when wastewater percolated through the soils at various hydraulic flow rates. Unchlorinated secondary sewage effluent seeded with either poliovirus type 1 (strain LSc) or echovirus type 1 (isolate V239) was continuously applied to soil columns for 3 to 4 days at constant flow rates. Water samples were extracted daily from ceramic samplers at various depths of the column for the virus assay. The effectiveness of virus removal from wastewater varied greatly among the different soil types but appeared to be largely related to hydraulic flow rates. At a flow rate of 33 cm/day, Anthony sandy loam removed 99% of seeded poliovirus within the first 7 cm of the column. At flow rates of 300 cm/day and above, Rubicon sand gave the poorest removal of viruses; less than 90% of the seeded viruses were removed by passage of effluent through the entire length of the soil column. By linear regression analyses, the rate of virus removal in soil columns was found to be negatively correlated with the flow of the percolating sewage effluent. There was no significant difference in rate of removal between poliovirus and echovirus in soil columns 87 cm long. The rate of virus removal in the upper 17 cm of the soil column was found to be significantly greater than in the lower depths of the soil column. This study suggests that the flow rate of water through the soil may be the most important factor in predicting the potential of virus movement into the groundwater. Furthermore, the length of the soil column is critical in obtaining useful data to predict virus movement into groundwater.     

Effect of soil permeability on virus removal through soil columns.

Laboratory experiments were performed on four different soils, using 100 cm long columns, to determine the extent of virus movement when wastewater percolated through the soils at various hydraulic flow rates. Unchlorinated secondary sewage effluent seeded with either poliovirus type 1 (strain LSc) or echovirus type 1 (isolate V239) was continuously applied to soil columns for 3 to 4 days at constant flow rates. Water samples were extracted daily from ceramic samplers at various depths of the column for the virus assay. The effectiveness of virus removal from wastewater varied greatly among the different soil types but appeared to be largely related to hydraulic flow rates. At a flow rate of 33 cm/day, Anthony sandy loam removed 99% of seeded poliovirus within the first 7 cm of the column. At flow rates of 300 cm/day and above, Rubicon sand gave the poorest removal of viruses; less than 90% of the seeded viruses were removed by passage of effluent through the entire length of the soil column. By linear regression analyses, the rate of virus removal in soil columns was found to be negatively correlated with the flow of the percolating sewage effluent. There was no significant difference in rate of removal between poliovirus and echovirus in soil columns 87 cm long. The rate of virus removal in the upper 17 cm of the soil column was found to be significantly greater than in the lower depths of the soil column. This study suggests that the flow rate of water through the soil may be the most important factor in predicting the potential of virus movement into the groundwater. Furthermore, the length of the soil column is critical in obtaining useful data to predict virus movement into groundwater.     

Elimination of human enteric viruses during conventional waste water treatment by activated sludge

The present study was undertaken to determine if viruses were selectively eliminated during waste water treatment. Human enteric viruses were detected at all steps of treatment in a conventional activated sludge waste water treatment plant. Liquid overlays and large volume sampling with multiple passages on BGM cells permitted the detection of poliovirus (serotypes 1, 2, and 3), coxsackievirus B (serotypes 1, 2, 3, 4, and 5), and echovirus (serotypes 3, 14, and 22), as well as reoviruses. The mean virus concentration was 95.1 most probable number of infectious units per litre (mpniu/L) in raw sewage, 23.3 in settled water, 1.4 in effluent after activated sludge treatment, and 40.3 mpniu/L in sludge samples. All samples of raw sewage and settled water, 79% of effluent water, and 94% of sludge samples contained viruses. The mean reduction was 75% after settling and 98% after activated sludge treatment. Poliovirus type 3 was rarely isolated after the activated sludge treatment, but was still detected in about one-third of the sludge samples. Reoviruses and coxsackieviruses were detected at similar rates from all samples and appear to be more resistant to the activated sludge treatment than poliovirus type 3. Poliovirus types 1 and 2 were present in almost every sample of raw sewage and settled water and still found in about half of the effluent and sludge samples, indicating a level of resistance similar to that of reoviruses and coxsackieviruses.     

Poliovirus retention in soil columns after application of chemical- and polyelectrolyte-conditioned dewatered sludges

The transport of poliovirus type 1 (strain LSc) was studied in Red Bay sandy loam columns that were treated with chemical- or polyelectrolyte-conditioned dewatered sludges and then leached with natural rainwater under saturated flow conditions. Poliovirus was concentrated in the alum and ferric chloride sludges that were produced following the flocculation of virus-seeded raw sewage. Virtually complete inactivation of the virus was observed following the flocculation of raw sewage or the stabilization of alum and ferric chloride sludges with lime at pH 11.5. Poliovirus was also concentrated in polyelectrolyte-conditioned dewatered sludge that was produced from virus-seeded, anaerobically digested sludge. Despite the saturated flow conditions for a sustained period, no viruses were detected in the leachates of the soil columns that were treated with these chemical and chemically treated sludges. Since the viruses were mostly associated with the solids in these sludge samples, it is believed that they were immobilized along with the sludge solids in the top portion of the soil columns.