Occurrence of rotaviruses and enteroviruses in recreational waters of Oak Creek, Arizona

Recent epidemiological studies have shown a relationship between swimming in recreational waters meeting bacteriological standards and gastroenteritis with a suggested viral etiology. No previous studies have been conducted in the United States on the occurrence of human pathogenic enteric viruses in freshwater recreational areas. The presence of enteroviruses and rotaviruses was investigated in Oak Creek, Arizona, a heavily used recreational area. Water samples were filtered through positively charged filters (168–1555 I.), eluted with beef extract, and assayed for human enteroviruses and rotaviruses. Eighteen of the 41 recreational water samples were positive for enterovirus or rotavirus. Of these, nine samples exceeded the Arizona State recommended limit of 1 PFU 40 l⁻¹ for full body contact in effluent dominated recreational waters. Several virus positive samples met the recommended fecal coliform standards (200 CFU 100 ml⁻¹) for recreational waters indicating the inadequacy of bacterial standards for monitoring viral water quality. The isolation of the pathogenic enteric viruses (i.e., poliovirus 1, echovirus 1, coxsackievirus B1 and B6 and rotavirus) from this popular recreational water demonstrates the potential for transmission of viral disease.     

An improved microdesulphonation/gas liquid chromatography procedure for the determination of linear alkylbenzene sulphonates in U.K. rivers

An improved microdesulphonation/gas liquid chromatography (GLC) procedure is described for the specific determination of μg l⁻¹ levels of linear alkylbenzene sulphonates (LAS) in aqueous environmental samples including sewage, sewage effluent and surface waters. The LAS is concentrated from samples as its methylene blue complex by a large-scale solvent extraction and is then freed from potential interferences by a series of clean-up steps, i.e. ion-exchange chromatography, hydrolysis and solvent extraction, prior to its desulphonation with a concentrated phosphoric acid reagent. The resulting alkylbenzene hydrocarbons are recovered and quantitatively determined by a capillary GLC technique with the aid of internal standards (primary and secondary alkylbenzene sulphonate isomers) added at the initial concentration stage.The introduction of the clean-up stages, particularly a selective extraction of the LAS as the l-methylheptyl amine salt into hexane, has resulted in GLC traces that are free from major interferences and in which it is possible to readily identify LAS isomers on the basis of the relative retention times. The procedure has a limit of detection of less than 10 μg l⁻¹ LAS in a sample and allows the quantification of sub-μg l⁻¹ levels of individual isomers (C₉–C₁₅ homologues). The mean recovery of a C₁₂ LAS internal standard through the complete procedure is 91% for the environmental samples analysed.This procedure, together with a non-specific methylene blue colorimetric method (for determining anion surface active material), has been used in a monitoring exercise to establish the levels of LAS and methylene blue active substances (MBAS) in U.K. rivers and the River Rhine. A mean MBAS level of 0.15 mg l⁻¹ was found at the U.K. river sites selected (35 samples), of which only 26% on average was attributable to LAS by microdesulphonation/GLC analysis. However, the levels of LAS and its contribution to the total MBAS in rivers was found to vary with the nature of the sampling location, i.e. depending whether it was above, below or in the vicinity of a sewage effluent discharge. The distribution of the LAS isomers at these sites also showed differences that could be explained in terms of their relative biodegradabilities.     

Persistence of nucleic acid markers of health-relevant organisms in seawater microcosms: Implications for their use in assessing risk in recreational waters

In the last decade, the use of culture-independent methods for detecting indicator organisms and pathogens in recreational waters has increased and has led to heightened interest in their use for routine water quality monitoring. However, a thorough understanding of the persistence of genetic markers in environmental waters is lacking. In the present study, we evaluate the persistence of enterococci, enterovirus, and human-specific Bacteroidales in seawater microcosms. Two microcosms consisted of seawater seeded with human sewage. Two additional seawater microcosms were seeded with naked Enterococcus faecium DNA and poliovirus RNA. One of each replicate microcosm was exposed to natural sunlight; the other was kept in complete darkness. In the sewage microcosms, concentrations of enterococci and enterovirus were measured using standard culture-dependent methods as well as QPCR and RT-QPCR respectively. Concentrations of human-specific Bacteroidales were determined with QPCR. In the naked-genome microcosms, enterococci and enterovirus markers were enumerated using QPCR and RT-QPCR, respectively. In the sewage microcosm exposed to sunlight, concentrations of culturable enterococci fell below the detection limit within 5 days, but the QPCR signal persisted until the end of the experiment (day 28). Culturable enterococci did not persist as long as infectious enteroviruses. The ability to culture enteroviruses and enterococci was lost before detection of the genetic markers was lost, but the human-specific Bacteroidales QPCR signal persisted for a similar duration as infectious enteroviruses in the sewage microcosm exposed to sunlight. In the naked-genome microcosms, DNA and RNA from enterococci and enterovirus, respectively, persisted for over 10 d and did not vary between the light and dark treatments. These results indicate differential persistence of genetic markers and culturable organisms of public health relevance in an environmental matrix and have important management implications.     

Medically-derived (131)I in municipal sewage effluent

This work presents ¹³¹I (t_½ = 8.04 d) concentrations in sewage effluent from the Stony Brook Water Pollution Control Plant (WPCP), a small plant serving a regional thyroid cancer treatment facility in Stony Brook, NY, USA. The concentrations detected in sewage effluent ranged from 1.8 ± 0.3 to 227 ± 2 Bq L⁻¹. The primary source of ¹³¹I is excreta from thyroid cancer inpatients treated at the Stony Brook University Medical Center. Based on several time series measurements following known inpatient treatments, the mean sewage half-life (T_s) of iodine is 3 d in this plant. The T_s, analogous to a radioactive half-life, describes the time it takes for half of a wastewater component to be removed from a WPCP. Flow recycling, or activated sludge, used to maintain bacterial populations necessary for sewage treatment causes iodine to remain in this plant far longer than its hydraulic retention time. The experimental results suggest that most ¹³¹I entering the Stony Brook WPCP leaves in sewage effluent, not in sewage sludge. Patient treatments can result in continuous discharges of ¹³¹I to surface waters where it can be used as a tracer of sewage-derived material and to understand the behavior of ¹³¹I in aquatic environments.     

Concentrations of nitrilotriacetate and certain metals in Canadian wastewaters and streams: 1971–1975

At 13 Ontario cities, representing a variety of populations and sewage treatment processes, grab samples were taken from the sewage treatment plant influent and effluent and from the receiving stream above and below the sewage outfall. The samples, taken once each month from November 1971 to March 1975 were analyzed for nitrilotriacetate (NTA), 9 metals, and phosphorus. From April 1971 to January 1973 household detergents in Canada contained an average of 6% NTA; after March 1973 they contained 15%. This increased usage of NTA was reflected in the NTA content of sewage influents, which rose from a median level of 1.3 mg l⁻¹ before the change to 3.2 mg l⁻¹ after the change. NTA levels in sewage effluent also increased somewhat, but much of the NTA disappeared in the sewage treatment processes. Even with the increased usage of NTA, the receiving streams below the sewage outfall contained only low levels of NTA: 97% of all samples during this period contained less than 0.5 mg l⁻¹, and the median concentration was 0.05 mg l⁻¹. Phosphorus concentrations in the sewages decreased at the time detergent compositions changed. Comparison of metal concentrations before and after the change, as well as metal-NTA correlation coefficients, failed to show clear evidence of an association between NTA concentration and metal concentration.     

Recovery of polluted lakes. a swedish research program on the effects of advanced waste water treatment and sewage diversion

In order to elucidate the effects of the effluents from advanced wastewater treatment plants for phosphorus removal, the National Swedish Environment Protection Board during 1972 started a program for analysing the conditions in a number of different recipient lakes. This study of recovery of polluted lakes also includes some recipient lakes from which sewage effluent will be completely diverted. This paper presents the program, some new methods used and the treatment plants and recipient lakes involved. The project. including 18 lakes and 15 sewage treatment plants, is running in cooperation with 13 Provincial Governments and 16 Municipalities. The efficiency of the sewage treatment plants, is continuously monitored by a new method called Minitest, and the nutrient loadings from these plants as well as from other sources are calculated.As the water quality of many recipient lakes showed great variations during the vegetation period (see Fig. 2), a need for a frequent sampling exists in these lakes.In order to simplify the sampling procedure sampling on the shore (after pumping water from the sampling point) has been introduced. Data obtained after analysing samples taken from a boat and samples from the same area taken on the shore after pumping through a 600 m plastic tube showed good agreement (see Table 2).The lake water samples are preserved by freezing, Analyses after rapid thawing showed that this method can be used also when determining from cell material easily leaking ions such as PO₄, NO₃ etc. (see Tables 3 and 4).The sewage treatment plants, the recipient lakes and some background data are presented in Tables 5 and 6. The water quality of the lakes is illustrated with the correlation between chlorophyll A and suspended solids (Fig. 3). The correlation coefficient = + 0–88. The first sign of lake recovery has been observed after an 85 per cent reduction of the waste phosphorus load. Post-precipitation (A1-sulphate) decreased the yearly load from 2000 kg P to 300 kg, which gave a 50 per cent reduction of the P content of the recipient water. Also the chlorophyll values decreased (Table 7).The role of nitrogen and phosphorus as algal growth limiting nutrients has been studied by using an algal assay procedure. N limited the growth of Selenastrum capricornutum in of the samples. In filtered lake water P was the growth limiting nutrient at total-P values below 0–05 mg 1⁻¹. Above 0·1 mg P 1⁻¹ N played the principal role. Between these values the growth was primarily limited by P or N or chelating agents (Fig. 4).     

Removal of indigenous rotaviruses during primary settling and activated-sludge treatment of raw sewage

An eight month study of indigenous rotavirus removal during primary settling and activated sludge treatment of raw sewage was made in a plant in Houston, Texas treating 1.5 million gal day⁻¹. An average reduction of 44–55% was obtained by primary settling and a 93–99% reduction was achieved in final chlorinated effluents. Composite sampling at 1 h intervals over a 24 h period indicated average removals of 85% compared to a misleading 6% indicated by one set of grab samples of raw sewage and effluent collected simultaneously. Quantification of rotaviruses was made by immunofluorescent foci counts 24 h after addition of sample concentrates to coverslip cultures of fetal rhesus kidney cells. Rotaviruses varied from 40–5101⁻¹ of raw sewage and from 0 to 25 in the final chlorinated effluent.     

Enterovirus types in Israel sewage

Two methods were used for the isolation and identification of enteroviruses in sewage from various communities in Israel. Out of 489 isolates from sewage at different places by the picked plaque method, 74% were polioviruses, 10% were Coxsackie Type B, Coxsackie A9 or Echo 9 and 16% were Echo or other enterovirus strains. Samples taken from sewage (26 grab samples and 26 gauze pads) were tested for polio and non-polioviruses by a neutralization test. An average of 37% of the daily grab and gauze pad samples were identified as polioviruses.     

Detection of somatic phages, infectious enteroviruses and enterovirus genomes as indicators of human enteric viral pollution in surface water

In the present study, we aimed to determine whether the concentrations of somatic coliphages, infectious enteroviruses or the detection of enterovirus genomes were associated with the detection of human pathogenic viruses in surface water. Four French rivers were sampled monthly or semimonthly for the quantitative detection of somatic coliphages, infectious enteroviruses and the qualitative RT-PCR detection of enterovirus, hepatitis A virus, Norwalk I viruses, Norwalk II viruses, astrovirus and rotavirus genomes over 12 months. All the 68 water samples tested were positive for the quantitative detection of somatic coliphages (range of concentrations: 4 x 10(2) to 1.6 x10(5) FUl(-1)). Infectious enteroviruses were isolated by a cell culture system in only two (3%) of the 68 concentrated water samples tested, whereas enterovirus genomes were detectable in 60 (88%) of the same samples. A positive RT-PCR detection of the genome of hepatitis A virus, Norwalk-like virus genogroup II, astrovirus, rotavirus and Norwalk-like virus genogroup I was demonstrated, respectively, in 1.5% (1/68), 1.5% (1/68), 3% (2/68), 0% and 0% of the 68 concentrated water samples tested. All of these four water samples were positive for the detection of enterovirus genomes, whereas only one of them was positive for the isolation of enteroviruses on cell culture. Moreover, the genomic detection of human pathogenic viruses appeared not to be statistically associated with the concentration levels of somatic coliphages in the 68 concentrated water samples tested (Wilcoxon rank test; P=0.14). Taken together, our findings indicate that the quantitative detection of somatic coliphages and the isolation of enteroviruses on cell culture are not suitable parameters for the control of the viral contamination in surface water, whereas the detection of enterovirus genomes may be useful for predicting the presence of waterborne viruses.     

Distribution of enteroviruses in sediments contiguous with a deep marine sewage outfall

The distribution of enteroviruses, coliforms, fecal coliforms and fecal streptococcus discharged from a raw sewage outfall pipe 3.6 km from shore at a depth of 44 m was studied. Enteroviruses and indicator bacteria were only isolated from water in an area within 200 m of the outfall. However, viruses were isolated from sediments as far as 3.6 km from the outfall at recreational bathing beaches. The concentration of enteroviruses in the sediment at the outfall boil ranged from 112 to 78 PFU l⁻¹ and 0 to 30 PFU l⁻¹ at the bathing beach. Indicator bacteria were not detected in the water or sediment at the bathing beach. Analysis of sediments for viruses may give a better idea of the long term distribution of sewage discharged from the outfall pipe.     

Oxygen utilization and biochemical stabilization rate constants for a brewery effluent

Composite samples of brewery effluents were obtained from a brewery in Benin City, Nigeria. They were analysed for their biochemical oxygen demand (BOD) values at 20°C for definite periods in days. From the values obtained, oxygen utilization and biochemical stabilization rate constants, k and K, respectively, were determined. The mean values obtained for them were 0.37 day^‐1 and 0.16 day^‐1 respectively. The ultimate BOD (L_o) value for the brewery effluent was 757.1 mg/l, and the ratio of the 5‐day BOD (BOD₅) to the Ultimate BOD (L_o) (i.e., BOD₅/L_o) was found to be 0.85.     

Behaviour in a river and dam of coliform bacteria with transferable or non-transferable drug resistance

The water in a river and the dam into which it flows was assayed for total coliforms and coliforms with transferable (R⁺) or non-transferable (R^-) resistance to five antimicrobial drugs. The effluent of a sewage works is discharged into the river 10 km upstream of the dam. The ratio of resistant to total coliforms decreased between the sewage works and the dam, but increased between the influent and effluent of the dam. This ratio was higher in the dam effluent than in the river upstream of the sewage works. No difference was detected in the survival of drug-sensitive and known R⁺ and R^- strains in distilled water or saline, or in dialysis bags immersed in the river or dam. Selection of those R⁺ strains resistant to a wide spectrum of drugs occurred in the river and dam. Immersed dialysis bags containing donor and recipient strains were used to demonstrate R factor transfer in the waters concerned. Three out of ten R⁺ strains isolated from the effluent of the dam yielded positive results. These donors were resistant to a wide spectrum of drugs but all determinants were transmitted at a relatively low frequency in the water. One donor had a higher frequency of transfer at 20 than at 37°C. The water pollution significance of these findings is discussed.     

Use of bituminous coal for concentration of enteroviruses from sewage and effluent

A method has been developed for concentration of enteroviruses from untreated and treated domestic wastewater using bituminous coal bed as a virus adsorbent. A bed made from 1.5 g of 120 mesh coal powder was used for concentrating enteroviruses from 100 ml of clarified sewage at different pH values with and without addition of AlCl3. To enhance the adsorption of viruses, requisite quantities of aluminium chloride were added so that a final concentration of 0.0005 M could be achieved. At pH 3.0 maximum adsorption (82.8%) of poliovirus type 1 from artificially contaminated clarified sewage was observed without addition of AlCl3. However, at pH 5.0 maximum virus adsorption of 98.7% was achieved after addition of aluminium chloride. An average recovery of 86.9% of adsorbed viruses at pH 5.0 was achieved from coal bed with 3% flocculating beef extract at pH 9.5. This method for concentration of enteroviruses incorporating use of coal was compared with that of Millipore membrane filter method applied to raw sewage and clarified sewage. The results obtained from the methodology using coal as adsorbent was subjected to Student's "t" test and it was observed that its efficiency is confirmed for recovery of enteroviruses from raw and nonclarified sewage. These results are also comparable with that obtained with MF method. The results presented in this paper are indicative of the potential of this method for both treated and raw sewage.     

Viruses and bacteriophages

Many of the enteric viruses which are transmitted from person to person by thefecal-oral route are found in raw and treated wastewater, and because of their persistence under adverse conditions may also be found in slightly polluted waters.There is no routine examination procedure of water and wastewater for entero-viruses,mainly because of the cumbersome isolation techniques, high cost and the need for highly skilled laboratory personnel.Phages are specific to single species of bacteria, are known for many entericbacteria, and are very often used for final identification of enteric pathogenic bacteria.Coliphages are provalent in raw and treated sewage as well as in polluted water, where enteric viruses may also be found.Coliphages were often mentioned as possible viral indicators in polluted water.To be a perfect indicator, they should comply with minimum criteria as follows: (a) they should be present wherever human enteric viruses are present: (b) the coliphage numbers recovered should be equal to or larger than those of enteric viruses recovered: (c) the coliphages should be at least as resistant as enteric viruses to adverse environmental conditions: (d) isolation and quantification of the coliphage should be faster and less expensive than isolation of the enteroviruses.Comparative studies show that the coliphage to enterovirus ratio in wastewater is about 10³:1. Levels of poliovirus 1 (attenuated) to coliphage f2 remained stable for a few months in oxidation pond effluents.f2 coliphage exhibited higher resistance to chlorination than poliovirus 1 (attenuated). When the two strains were kept in water of different quality, f2 survived longer. In addition, all coliphage counts were completed within 24 h. while those of enteroviruses required about a week. Results indicate very strongly that coliphages can be used as viral indicators and this is already the practice in a few European and other countries.     

An in situ method to compare the potential for periphyton productivity of lotic habitats

A simple field method for measuring relative net productivity in flowing waters is described. To assess its sensitivity and practicality, the eutrophication potential of a tertiary sewage treatment plant effluent was assayed on the Fort River of Amherst, Massachusetts. Two sets of three 10 m lexan tubes (1.25 cm i.d.) were anchored to the river bed 100 m upstream and downstream from the sewage plant outfall. Data collection began following a 2 week colonization period for the periphyton community. Substrate primary productivity (mg O₂ min⁻¹) was determined by subtracting inflow dissolved oxygen concentrations from these levels at the discharge ends of each tube and multiplying the difference by the flow rate. The downstream tubes demonstrated an enhanced response at a 0.01 level of significance to an increment of 0.005 and 0.1 ppm of phosphorous and nitrogen respectively. Data analysis revealed a highly significant regression of the net productivity on stream flow which was adjusted for by an analysis of covariance. The productivity of the downstream station was distinctly higher (0.4522 mg O₂ min⁻¹) then the value of the upstream station (0.2584 mg O₂ min⁻¹). The protocol thus affords a biological summation of the sewage effluent through comparison with the control station replicates.     

Distribution of antidepressant residues in wastewater and biosolids following different treatment processes by municipal wastewater treatment plants in Canada

The fate of 14 antidepressants along with their respective N-desmethyl metabolites and the anticonvulsive drug carbamazepine (CBZ) was studied in 5 different sewage treatment plants (STPs) across Canada. Using two validated LC-MS/MS analytical methods, the concentrations of the different compounds were determined in raw influent, final effluent and treated biosolids samples. Out of the 15 compounds investigated, 13 were positively detected in most 24-h composite raw influent samples. Analysis showed that venlafaxine (VEN), its metabolite O-desmethylvenlafaxine (DVEN), citalopram (CIT), and CBZ were detected at the highest concentrations in raw influent (up to 4.3 μg L⁻¹ for DVEN). Cumulated results showed strong evidence that primary treatment and trickling filter/solids contact has limited capacity to remove antidepressants from sewage, while activated sludge, biological aerated filter, and biological nutrient removal processes yielded moderate results (mean removal rates: 30%). The more recalcitrant compounds to be eliminated from secondary STPs were VEN, DVEN and CBZ with mean removal rates close to 12%. Parent compounds were removed to a greater degree than their metabolites. The highest mean concentrations in treated biosolids samples were found for CIT (1033 ng g⁻¹), amitriptyline (768 ng g⁻¹), and VEN (833 ng g⁻¹). Experimental sorption coefficients (K_d) were also determined. The lowest K_d values were obtained with VEN, DVEN, and CBZ (67-490 L kg⁻¹). Sorption of these compounds on solids was assumed negligible (log K_d ≤ 2). However, important sorption on solids was observed for sertraline, desmethylsertraline, paroxetine and fluoxetine (log K_d > 4).     

Evaluation of a sampler for liquid effluents

A design of automatic sampler for liquid effluent streams is described, which is based on the use of a rapidly circulating pumped stream of the liquid effluent. This stream passes through an interceptor vessel from which sub-samples may be taken iso-kinetically to provide a variety of sample types (e.g. snap, composite, flow-related). This interceptor may be conveniently located and may also be used to hold monitoring probes (pH, ion selective electrodes) which will provide continuous information on the composition of the effluent stream.This sampler system has been evaluated using simulated liquid effluents, chosen to represent cases in which it is difficult to acquire a truly representative sample. The cases chosen were:

1. (a) water containing up to 10,000 mg 1⁻¹ of finely divided inorganic solid;

2. (b) water containing 2000 mg l⁻¹ of sewage solids

3. (c) water containing dissolved, volatile organic solutes;

4. (d) water containing dispersed oil.

In all cases it proved practical to obtain samples which were representative in composition of the liquid being tested; comparison in all cases was with snap samples taken simultaneously from appropriate points in the loop system.The sampler appears to be generally suitable for use with most types of liquid effluents. The several components of the sampler should be constructed of materials which are inert to the effluent.     

A method for monitoring mineralization of 14C-labeled compounds in aqueous samples

Previously reported laboratory methods for measuring the rate of ¹⁴CO₂ release from an aqueous sample require a series of replicate growth flasks. At chosen times, some of these are sacrificed in order to quantitate the ¹⁴CO₂ released. The method presented here requires only one culture flask containing 200 ml of sample with labeled substrate, and one control-replacement flask containing 200 ml of sterile sample with labeled substrate. At each sampling time, $\text{1}\text{40}\text{th}$ of the liquid and headspace gas is removed from the modified 500 ml Erlenmeyer culture flask for ¹⁴CO₂ recovery. This method was used to study the mineralization of labeled amino acids, starch, n-hexadecane and anthracene by microorganisms in river water, and of n-hexadecane and n-hexadecanoic acid by microorganisms in sewage effluent.     

A method for monitoring mineralization of C-labeled compounds in aqueous samples

Previously reported laboratory methods for measuring the rate of ¹⁴CO₂ release from an aqueous sample require a series of replicate growth flasks. At chosen times, some of these are sacrificed in order to quantitate the ¹⁴CO₂ released. The method presented here requires only one culture flask containing 200 ml of sample with labeled substrate, and one control-replacement flask containing 200 ml of sterile sample with labeled substrate. At each sampling time, of the liquid and headspace gas is removed from the modified 500 ml Erlenmeyer culture flask for ¹⁴CO₂ recovery. This method was used to study the mineralization of labeled amino acids, starch, n-hexadecane and anthracene by microorganisms in river water, and of n-hexadecane and n-hexadecanoic acid by microorganisms in sewage effluent.     

Concentration of adenovirus from seawater

Factors influencing adenovirus 5 recovery from seawater by virus concentrator methods were determined. A 19,000-fold concentration of 25 gal samples with a theoretical recovery efficiency of 90% was possible with input multiplicities of 1000 TCID₅₀ units ml⁻¹. Pre-treatment of orlon and cellulose acetate filters with beef extract or tween 80 solutions promoted adenovirus passage during sample clarification. Adenovirus adsorbed to textile and epoxy fiberglass filters at acid pH. Adsorption to textile filters was enhanced by 0–05 m MgCl₂. No salt enhancement was necessary for adsorption to epoxy fiberglass filters. Adenovirus was recovered from adsorbent filters following elution with 3% beef extract solution adjusted to pH 9.0. Adenovirus was reconcentrated from beef extract eluates by aqueous polymer phase separation. Actual recovery of 106 PFU of adenovirus from 50 gal of a waste treatment plant effluent was made with the modified virus concentrator procedure developed in the study.