Detection of enteroviruses near deep marine sewage outfalls

Vol. 9, pp. 246--2-19 Pergamon Pros Lid 197~. Printed in Great Britain

~}25X 78/rOL'~]-0246 $02.(X) 0

Idartne Pollution Bullettn.

Detection of Enteroviruses Near Deep Marine Sewage Outfalls T. D. EDMOND, G. E. SCHAIBERGER and C. P. GERBA*

Department of Microbiology, University of Miami School of Medicine, Miami, FL 33152, U.S.A. and *Department of Virology and Epidemiology, Baylor College of Medicine, Houston, TX 77030, U.S.A.

At the present time there are approximately 160 million gallons per day of municipal sewage being discharged into the waters off the southeastern coast of Florida. Present in these sewage effluents are human pathogenic viruses whose fate in marine waters is not completely understood. Virus surveillance in waters receiving domestic wastewater discharge has concentrated on estuarine areas where water quality and depth are significantly different from the deep marine ouffalls. Using the membrane filter adsorption technique, viruses were detected in the vicinity of deep marine outfalls discharging both raw and chlorinated, secondarily treated sewage.

This investigation is a primary report on the virological studies of the Southeast Florida Ocean Outfall Study (SEFOOS) being conducted under the sponsorship of the United States Environmental Protection Agency. The project as a whole is designed to evaluate the current impact of deep, ocean sewage outfalls on the receiving system and their relationship to human health. The results of the study hopefully will be used by local and state authorities to design the most appropriate treatment procedures for wastewater before being discharged into marine waters. The southeast coast of Florida is a rapidly urbanizing, 16 km wide strip, extending from Palm Beach to the Florida Keys and bordered on the west by the Everglades (Berg, 1974). Regional sewage treatment plants are slowly approaching their design capabilities as there is approximately 160 mgd of effluent from l0 ocean outfalls between Palm Beach and Viriginia Key. These constitute raw, primary, secondary, and chlorinated secondary outfalls that approach and invade the currents of the Gulf Stream. Shoreward eddies off the Gulf Stream, with the combination of wind and wave action, occasionally allow surface effluent plumes to intersect the bathing waters along adjacent shorelines, creating bacterial concentrations in excess of state standards (Lee & McGuire, 1973). Field studies designed to assess the occurrence of pathogenic human enteric viruses are being conducted at selected sewage outfalls (Fig. 1) discharging raw (Miami Beach) and secondary treated (activated sludge plus chlorination, Miami and Hollywood) sewage. These data will be used to evaluate virus concentration methodology for the open marine environment and the occurrence of viruses in the vicinity of outfalls discharging both treated and untreated domestic wastewater. 246

Materials and Methods

Figure 1 illustrates the location of the study sites; mgd discharge rates, and depth of the three outfalls. The Miami outfall discharges approximately 55 mgd (1.77 × l0 s 1. day -l) of secondarily treated (activated sludge), chlorinated effluent at a depth of 5 m. This outfall is currently being extended to a depth of 37 m, 1.6 km from shore. The Miami Beach outfall discharges 47 mgd (1.78 x 108 1. day -I) of untreated sewage at a depth of 44 m. The amount of wastewater being discharged is highly seasonal and dependent on tourist influx during the winter months. The Hollywood outfall lies in 28 m of water, discharging approximately 27 mgd (9.8x107 1. day -1) of secondary treated and chlorinated wastewater.

Bacterial assays Water samples for bacterial assays were collected in sterile glass bottles 1 m below the surface at the outfall boil. All samples were kept cool in an ice chest until returned to the laboratory. Total coliform, faecal coliform and faecal streptococci determinations (Table 2) were performed by the membrane filtration technique, according to Standard Methods for the Examination of Water and Wastewater (American Public Health Association, 1976). Filters with rated pore size diameters of 0.45 Hm were used for total coliform analyses. Location of ocean

I Hollywood

outfalls

27M G 0.v,.28 m

Atlantic Ocean Miami Beach Miami

47 MGD x-44m iSMGD~5 m

¢

Florida

I

/t

z~

of Fig. 1 Map of study area.

. t

Volume 9/Number 9/September 1978

Membrane filters (type H C W G , Millipore Corp., Bedford, Mass.) with a retention pore size of 0.7 /~m and a surface opening diameter of 2.4/am were used for faecal coliform assays. This filter has been demonstrated to provide the optimum pore structure for faecal coliform recovery (Green et al., 1975). M-Endo MF broth, M-FC broth and KF streptococcus agar were used for the enumeration of total coliforms, faecal coliforms and faecal streptococci, respectively. The incubation temperature for total coliform analysis was 37°C, while for faecal coliforms the incubation time was 5 h at 35°C followed by 18 h at 44.5°C in a water bath. Faecal streptococci samples were incubated at 35°C for 48 h. A 30-40°7o increase in recovery efficiency was noted when the faecal coliform group was incubated in the aforementioned manner as opposed to an incubation period of 24 h at 44.5°C. These findings are in agreement with Green et al. (1977) whose determinations were performed with chlorinated sewage effluents. Viral assays

Virus in 1 to 19 1. samples of seawater or sewage was concentrated by adsorption to and elution from 142-mm diameter epoxy fiberglass disc filters (Duo-Fine filters, Filterite Corp., Timonium, Md.) in a 3.0- and 0.45-/~m series. The water samples were adjusted to pH 3.5 with 0.2 N HC1 and 0.00015 M A1C13 prior to passage through the adsorbent filters (Payment et al., 1976). After pressurized passage of the samples through the filters, virus was eluted with 50 ml of pH 11.5, 0.005 M glycine buffer which was then neutralized and frozen until assay. When sample sizes between 190 and 800 !. were processed, a modification of the virus concentration method described by Payment et al. (1976) was used. The sample was pretreated in the same manner, but was pressurized through 10-inch, 3.0- and 0.45-/am filter cartridges with pleated membranes. Utilizing a flow rate between 22 and 37 1. min -I, as much as 800 1. of seawater at an outfall site could be processed. The final field eluate was 1 to 2 1., neutralized to a pH of 7.2 and transported to the laboratory on ice for reconcentration. Reconcentration of the initial eluate was accomplished as described by Farrah et al. (1977). This involved adsorption of the virus to aluminum hydroxide flocs followed by elution using buffered foetal calf serum. The eluate was then further reduced in volume by hydroextraction, yielding final assayable volumes of 2 0 - 3 0 ml. Samples were collected within 10 m of the outfall boil at a depth of 1 m. The overall recovery efficiency of this method using seeded poliovirus type 1 (strain LSc) in 190 1. of seawater was 56°7o. This is within the efficiency range reported by Payment et al. (1976). Viral assays were performed by the plaque-forming unit (PFU) method (Wallis & Melnick, 1967) using the BGM, CV-1 and Vero cell lines which were passaged, grown and maintained as previously described (Melnick & Wenner, 1969). The sampling procedures and sampling methodologies are optimized and largely selective for the detection of enteroviruses (Melnick & Wenner, 1969; Payment et al., 1976; Farrah et al., 1977). Thus, the enteric viruses isolated are referred to as enteroviruses, even though this was not confirmed by neutralization

with specific antisera. Samples were assayed after being made isotonic and after addition of foetal calf serum to a final concentration of 2°70. The concentrated samples were placed on cell layers (1.5 ml per 75 cm" cell surface area) for 90 min to allow for viral adsorption. The cell layers were then overlaid with a nutrient agar and incubated at 37°C. The overlaid cell cultures were examined daily for 10-14 days for the presence of plaques. The results are reported as PFU per 400 1. of seawater. Salinity was measured with an AO refractometer (.AO Instruments Corp., Buffalo, N.Y.). Salinity ranged from 38.82 to 36.05 g/kg at the Miami Beach outfall, 24.20 to 34.00 g/kg at the Miami outfall, and 34.8 to 36.00 g/kg at the Hollywood outfall.

Results and Discussion The public health significance of human enteric viruses in marine waters has not been fully evaluated due to the lack of sensitive and quantitative methods for their concentration. However, the presence of enteric viruses in sewage-polluted waters, the continued occurrence of outbreaks of infectious hepatitis, and the possibility of other waterborne viral diseases (Cliver, 1971; Craun & McCabe, 1973) demonstrate the need for definitive information on these outfalls. Previous studies on the occurrence of enteric viruses in marine waters were limited to shallow coastal areas that were often grossly polluted (Metcalf & Stiles, 1965; Metcalf et al., 1974; De Flora et al., 1975). In addition, quantitative techniques were usually not available and virus recovery efficiencies were not reported. Recently, quantitative methods have become available for concentrating large volumes of estuarine waters harbouring enteric viruses (Payment et al., 1976; Farrah et al., 1977). To date, only one extensive field study has been performed using these methods to monitor naturally occurring enteroviruses (Goyal et al., 1977). This study was performed in shallow estuarine water along the Texas coast, where the water quality is significantly different than the oceanic waters along the southeast coast of Florida. As evidenced by Table 1, appreciable quantities of enteroviruses have been isolated from the untreated wastewater outfall at Miami Beach. Also, several viruses were isolated from the secondary, chlorinated outfalls at Miami and Hollywood. High concentrations of faecal bacteria were also present in the vicinity of the Miami Beach outfall (Table 2). Chlorination of the secondarily treated effluent at the Miami and Hollywood outfalls reduced the levels of faecal bacteria to almost TABLE 1 Number of enterovirus isolates from selected marine outfalls (PFU/400 1. sample). Sampling date (1977) May June August September October November

Hollywood

Miami Beach

Miami

0 3 I 0

42 24 27 37 28 21

0 2 0 0 3 I

247

Marine Pollution Bulletin TABLE

2

Results of bacteriological surveillance of selected ocean outfalls (colonies/100 ml). Total coliforms Sampling dates (1977) May June August September October November

M* 8 2

14 6 9 0

MB*

l a x 10a 1.6x 10a 1.3×10 4 1.2x10 4 1.2×10 a 1.5 × 104

Faecal coliforms

H*

M

-

0

0 0 11 4

0 3 0 l 0

MB -

l a x 104 1.0×10 4 0.9×10 a l . l × 1 0 "~ 1.2 x 104

Faecal Streptococci H

M

MB

H

-

0 0 6 2

0

~

12 13 29 33

4.4× 103 0.5×103 3.9× 103 4.9x 103

6 11 18 3

*M = Miami; MB = Miami Beach; H = Hollywood.

below detectability. Unlike shallow coastal waters, ah appreciable amount of dilution occurs before discharged sewage reaches the surface. However, viruses could still be detected at the surface boil using newly developed concentration methodology. Recently, standards have been proposed for viral quality of recreational water. Melnick (1976) recommended consideration of a limit of one infectious unit of virus per 10 gallons (ca. 37.8 1.) of recreational water, whereas Shuval (1976) proposed a standard of no detectable virus in 10-gallon (ca. 37.8 1.) samples. These standards were exceeded in all of the samples taken in the vicinity of the Miami Beach outfall, which discharges untreated sewage. The standards were not exceeded near those outfalls discharging secondarily treated, chlorinated effluent. Because of the lack of quantitative epidemiologic data, all such standards are arbitrary and reflect limitations of current detection methodology for enteric viruses in water rather than disease risk. Still, they may be conservative when considering such factors as: (1) the efficiency of our concentration method was approximately 50%, (2) very low concentrations of enteroviruses may cause infection in susceptible hosts (Westwood & Sattar, 1976), and (3) current concentration and detection methods are optimized for enteroviruses and are not capable of recovering adenoviruses, infectious hepatitis virus and rotaviruses, which may also be present in sewage discharges. The results of this study, while preliminary in nature, indicate that pathogenic human enteric viruses are present in significant numbers in and around non-treated sewage outfalls as well as in those secondarily treated, chlorinated sewage effluents. There was almost a 4 log10 reduction in faecal coliforms in the vicinity of the outfalls discharging treated chlorinated effluent as compared to the one discharging raw sewage. In contrast, the average viral concentration was only 1-2 log10 less. Outfalls discharging chlorinated, secondarily treated sewage resulted in a significant reduction in the amount of virus being discharged, but virus was not completely removed. Such reductions obviously decrease the chance of viruses reaching recreational and shellfishharvesting areas. Further studies are underway to determine if viruses can survive long enough to reach these areas and the role of suspended solids in the hydrotransportation of viruses being discharged from deep marine outfalls. We are greatly obligated to Ms. Carmen I. Gomez for her technical assistance and to Captain Clifford M. Shoemaker of the Orea IIL

248

This work was sponsored as part of the Southeast Florida Ocean Outfall Study, supported by grant no. R-804, 749-015B01 from the United States Environmental Protection Agency. American Public Health Association (1976). Standard Methods .for the Examination of Water and Wastewater, 14th edn. New York: American Public Health Association. Berg, G. (1974). Regional problems with sea outfall disposal of sewage on the coast of the U.S. In DSSO International Symposium, A. L. H. Gaeson Gameson (ed.), pp. 19-75. New York: Pergamon Press. Cliver, D. O. (1971). Transmission of viruses through foods. Critical Rev. environ. Control, 1,551-579. Craun, C. F. & McCabe, L. J. (1973). Review of the cases of water borne disease outbreaks. J. Am. War. Wks. Ass., 65, 74-83. De Flora, S., De Renzi, G. P. & Badolati, G. (1975). Detection of animal viruses in coastal seawater and sediments. AppL MicrobioL, 30,472-475. Farrah, S. R., Goyal, S. M. Gerba, C. P., Wallis, C. & Melnick, J. L. (1977). Concentration of enteroviruses from estuarine water. Appl. environ. MicrobioL, 33, 1192-1196. Gerba, C. P., Smith, E. M. & Melnick, J. L. (1977). Development of a quantitative method for detecting enteroviruses in estuarine sediments. AppL environ. Microbiol., 34, 158-163. Gerba, C. P., Smith, E. M., Schaiberger, G. E. & Edmond, T. D. (1977). Field evaluation of methods for the detection of enteric viruses in marine sediments. In Determination of Microbial Biomass and Activities in Sediments (Proc. Symp. Am. Soc. Testing and Materials), in press. Goyal, S. M., Gerba, C. P. & Melnick, J. L. (1977). Prevalence of human enteric viruses in coastal canal communities. J. Wat. Pollut. Control Fed., in press. Green, B. L., CIausen, E. & Litsky, W. (I975). Comparison of the new Millipore HC with conventional membrane filters for the enumeration of fecal coliform bacteria. AppL Microbiol., 30, 697-699. Green, B. L., Clausen, E. & Litsky, W. (1977). Two-temperature membrane filter method for enumerating fecal coliform bacteria from chlorinated effluents. Appl. environ. MicrobioL, 33, 1259-1264. Lee, T. N. & McGuire, J. B. (1973). The use of ocean outfatls for marine waste disposal in southeast Florida's coastal waters. Coastal Zone Management Series, bull. 2. Melnick, J. L. (1976). Viruses in water: an introduction. In Viruses in Water, Berg, G., Bodily, H. L., Lennette, E. H., Melnick, J. L. & Metcalf, T. G. (eds.), pp. 3-11. Washington: American Public Health Association. Melnick, J. L. & Wenner, H. A. (1969). Enteroviruses. In Diagnostic Procedures for Viral and Rickettsial Infections, 4th edn., Lennette, E. H. & Schmidt, N. J. (eds.), pp. 529-602. New York: American Public Health Association. Metcalf, T. G. & Stiles, W. (1965). The accumulation of enteric viruses by the oyster, Crassostrea virginica. J. infect. Dis., 115, 68-76. Metcalf, T. G., Wallis, C. & Melnick, J. L. (1974). Virus enumeration and public health assessments in polluted surface water contributing to transmission of virus in nature. In Virus Survival in Water and Wastewater Systems, Malina, J. F., Jr. & Sagik, B. P. (eds.), pp. 57-70. Austin: Center for Research in Water Resources. Payment, P., Gerba, C. P., Wallis, C. & Melnick, J. L. (1976). Methods for concentrating viruses from large volumes of estuarine water on pleated membranes. Wat. Res., 10,893-896. Schaub, S. A. & Sagik, B. P. (1975). Association of enteroviruses with natural and artificially introduced colloidal solids in water and infectivity of solids associated virions. AppL MicrobioL, 30, 212-222. Shuval. H. I. (1976). Water needs and usage: the increasing burden of enteroviruses on water quality. In Viruses in Water, Berg, G.,

Volume9/Number 9/September 1978 Bodily, H. L., Lennette, E. H., Melnick, J. L. & Metcalf, T. G. (eds.), pp. 12-26. Washington:AmericanPublic Health Association. Wallis, C. & Melnick, J. L. (1967). Concentration of enteroviruses on membrane filters. J. ViroL, 1,472-477.

Westwood, J. C. N. & Sattar, S. A. (1976). The minimal infective dose. In Virusesin Water. Berg, G., Bodily, H. L., Lennette, E. H., Melnick, J. L. & Metcalf, T. G. (eds.), pp. 61-69. Washington: American Public Health Association.

Marine Pollution Bulletin, Vol. 9, pp. 249-251 I~ Pergamon Press Ltd. 1978. Printed in Great Britain

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Phthalate Ester Plasticizers, DDT, DDE and P olychlorinated Biphenyls in Biota from the Gulf of Mexico C. S. GIAM, H. S. C H A N and G. S. NEFF Department o f Chemistry, Texas A & M University, College Station, T X 77843, U.S.A.

The levels of phthalate ester plasticizers, DDT, DDE and polychlorinated biphenyls (PCBs) were determined in the tissues of 18 species of marine organisms from the northwestern Gulf of Mexico. Low levels of the most widely used phthalate, di-(2-ethylhexyl) phthalate, were found in the majority of the samples; no other phthalates were detected. DDT, D D E and PCBs were found in all samples, but at somewhat lower levels than those found in our 1971 survey. A decrease in p,p'-DDT/p,p'-DDE ratios relative to 1971 was also noted. In earlier studies, concentrations of DDT and DDE (DDTs) and of polychlorinated biphenyls (PCBs) in biota from the Gulf of Mexico were determined; these chlorinated hydrocarbons were found in all samples analysed (Giam et al., 1972,1973,1974). As the phthalate ester plasticizers have been in wide use (Autian, 1973; Mathur, 1974) and have production volumes exceeding those of the chlorinated hydrocarbons, their presence in Gulf biota was suspected. In view of this possibility and of the absence of systematic studies to quantitate the phthalates in marine biota, a programme to determine phthalate levels in Gulf biota was initiated. The necessary low background, high sensitivity procedures were developed (Giam et aL, 1975) as an extension of those used for the chlorinated hydrocarbons (Giam & Wong, 1972) and allow the simultaneous analysis of the DDTs, PCBs and phthalates. Thus, the levels of DDTs and PCBs were determined along with those of the phthalates for comparison with the levels of chlorinated hydrocarbons found in previous studies as well as with current phthalate levels.

Procedure Samples were collected with a net or hook and line and were wrapped in pre-cleaned foil or placed in cleaned Mason jars. They were then frozen and kept at or below 0°C until analysis. If possible, the skin or shells of biota samples were dissected off and only inner or subdermal tissues were used. Cleaning procedures and analytical methods have

been reported in detail elsewhere (Giam et al., 1975). Samples were weighed into a tared blender or Mason jar and homogenized with 100 ml of acetonitrile. The extract was filtered and the extraction repeated. The combined acetonitrile extracts were diluted with 650 ml of purified salt water (5070) and extracted with 100 ml of methylene chloride-petroleum ether (1:5) in a 1 1. separatory funnel. The organic layer was separated, washed with 50 ml of salt water and dried with sodium sulphate. The dried extract was transferred to a Kuderna-Danish evaporative concentrator with the aid of 10 ml of isooctane. After evaporation to less than 10 ml, the residue was subjected to Florisil chromatography. For samples of greater than 0.5 g lipid, 2.2 cm i.d. columns containing 35 g of Florisil were used. These columns were eluted sequentially with 200-ml portions of 6, 15 and 2007/0 diethyl ether in petroleum ether. The fractions were concentrated to approximately 5 ml and were analysed by gas chromatography (GC) with an electron capture detector for the chlorinated hydrocarbons, D E H P and DBP respectively. For samples of less than 0.5 g lipid, 1.0 cm i.d. columns containing 10 g of Florisil were used. These columns were eluted with 40 ml of petroleum ether for chlorinated hydrocarbons and with 40 ml of 20°70 ether in petroleum ether for D E H P and DBP. The compounds were identified by GC retention times and confirmed by alkaline hydrolysis and chemical derivatization (Giam et al., 1976a).

Results and Discussion Samples were obtained from the locations mapped in Fig. I. Near-shore areas were sampled most extensively as their generally higher levels of contamination relative to open-ocean sites made them more likely areas for the detection of the phthalates. As shown in Table 1, di-(2-ethylhexyl) phthalate (DEHP) was detected in the majority of the samples. However, dibutyl phthalate (DBP) which was found in water and sediment samples from the Gulf (Glare et al., 1976b) was not present above the detection limit of 0.1 ng g-L DDTs and PCBs were found in all samples. 249