61
Journal of’ Virological Methods, 40 (1992) 67 16 ,c> 1992 Elsevier Science Publishers B.V. ! All rights reserved / 0166-0934192jSO5.00
VIRMET 01397
Processing procedures for recovering enteric viruses from wastewater sludges Ronald Virology
Branch,
E. Stetler,
Mary Ellen Morris
Microbiology
Research Division. Environmental Cincinnati, OH (USA)
(Accepted
and Robert
S. Safferman
Monitoring
S~~stems Laboratory,
12 May 1992)
_ Summary A powdered beef extract specially formulated for recovering viruses from environmental samples and designated as beef extract V was evaluated using indigenous and viral seeded wastewater sludge samples. When beef extract V was used to process activated and aerobically digested sludge solids, virus recoveries were shown to be similar to other methods that used commercially available supplemented beef extract. When used to process other sludge solids (primary and activated without primary clarification), cytotoxicity resulted in the BGM cell line used for virus assay. When these sludge solids were processed with the supplemented commercially marketed beef extract cell toxicity did not occur. Metal concentrations in the processed sewage sludge eluates were analyzed, but based on the levels observed they could not be shown as the source of the cytotoxicity. This did not exclude possible synergistic cytotoxic effects or organometal complexes. The commercially marketed beef extract was supplemented with either a floccing aid (FeClj), a filter aid (Celite) or a floe prepared from paste beef extract. The paste beef floe supplement proved to be the most useful and reliable method for processing for viruses from wastewater sludge solids. Viruses;
Wastewater
sludge;
Elution-concentration
process;
Correspondence to: R.E. Stetler, Virology Branch, Microbiology Monitoring Systems Laboratory, Cincinnati, OH, USA.
Detection
Research Division,
Environmental
68
introduction Commercially marketed powdered beef extract (CBE) has been used to elute viruses adsorbed to solid matrices. Eluted viruses have then been effectively concentrated from CBE by the organic flocculation procedure of Katzenelson et al. (1976), in which the viruses are adsorbed to the floe formed upon acidification of the CBE solution. Reducing sample volume has been an essential and practical step in this process of recovering waterborne viruses. Subsequent changes in the manufacturing process have improved the clarity of CBE in solution, but the altered product precluded processing of the eluate by the organic flocculation procedure. Upon acidification of the refined CBE. floe concentration ievels necessary for virus adsorption failed to form (Hurst et al.. 1984). This has led to the development of other mechanisms to reduce sample volumes. The method was modified for virus recovery from tap and wastewaters by the addition of a floccing agent (Payment et al., 19X4) and an inorganic filter aid (Dahling and Wright, 1986b, 1988). The method was also modified for use with sludge solids by the addition of a preformed flnc prepared from acidification and centrifugation of a paste beef extract solution (Safferman et ai., 1988). A new powdered beef extract specially formulated for virus studies, beef extract V (BEV), recently became available which, upon acidi~cat~on, produces adequate anlounts of floe for virus adsorption (Dahl~ng and Wright, 1988; Hurst, 1990). We evaluated BEV, CBE supplemented with paste beef ftoc. CBE supplemented with the floccing aid, FeCl3, and CBE supplemented with the filter aid, Celite, as to their efficiency for recovery and concentration of viruses from municipal wastewater sludge solids.
Materials and Methods
Virus assays were carried out using the continuous African green monkey kidney cell line designated BGM (Dahling et al., 1974). Propagation and assay procedures have been described previously (Dahling and Wright, 19X&+). Poliovirus type 1 (Mahoney LP) was used to seed the activated sludge samples,
A nonfloccing commercially marketed beef extract powder (lot no. 05401. Gibco Diagnostics, Madison, WI) and a floccing beef extract powder specially formulated for virus studies, beef extract V (lot no. CF)DVLB, BBL, Cockeysville, MD), were used for elution of sludge solids. The beef extracts were prepared as 10% (w/v) solutions in distilled water buffered with 1.34 g Na2HP04. 7Hz0 and 0.12 g citric acid per 100 ml. The supplemental floe was
69
prepared from a 3% (w/v) solution MI) in distilled water as previously
of paste beef extract (Difco, Inc., Detroit, described (Safferman et al., 1988).
Test procedure
Activated and primary sludge samples were collected from sewage treatment plants (STP) in southwestern Ohio. Samples of activated sludge without prior primary clarification and aerobically digested sludge were collected from the Trumansburg STP in New York. Sludge samples were shaken and lOO-ml aliquots were removed to process for indigenous virus recovery. Additional aliquots were removed from the activated sludge samples collected at the southwestern Ohio STPs. These samples were seeded with poliovirus type 1 and processed concomitantly with the nonseeded samples. The sludge extraction procedure, described previously (Berg et al., 1982, 1984; Berman et al., 1981; Safferman et al., 1988) is outlined briefly. To each lOO-ml sample, AlCls was added to a final concentration of 0.0005 M and the sample acidified to pH 3.5. After mixing for 30 min, the sample was centrifuged at 2500 x g for 15 min and the supernatant discarded. Sludge pellets were suspended in 100 ml of either buffered 10% CBE or buffered 10% BEV. After 30 min mixing, all samples were centrifuged at 10000 x g for 30 min. Supernatants were pressure filtered through a sterile stack of Filterite membrane filters (3.0 pm, 0.45 ;lrn, and 0.25 pm) placed in order of increasing pore size. To each volume of filtered eluate, a sufficient volume of sterile, distilled water was added to reduce the beef extract concentration to 3%. The CBE eluates were supplemented as follows: one group received floe prepared from 100 ml of 3% paste beef extract, the second group received 0.05 M FeC13 at 0.5 ml/l00 ml, and the third group received Celite at 0.1 g/l00 ml. BEV filtered eluates were not supplemented. All filtered eluates, except those supplemented with Celite, were aciditied to pH 3.5 and stirred for 30 min. After centrifuging at 2500 x g for 15 min, the pellets were solubilized in 0.15 M Na2HP04. 7H20 at l/20 the volume of the diluted liltered eluate and kept at 4°C until assayed. Celite supplemented filtered eluates were acidified to pH 4.0, stirred 10 min and centrifuged as above. The pellet was suspended in 0.15 M Na2HP04. 7Hz0, mixed for 25 min and recentrifuged. The supernatant was decanted and kept at 4°C until assayed. Lowry protein analysis
BGM cell growth was determined by protein analysis. BGM cell monolayers, cultured in glass bottles for 24 h, were rinsed twice with phosphate buffered saline (PBS). Two bottles were drained, air-dried, recapped, and stored for Otime protein determination. Two other bottles received fresh medium to serve as controls. The remaining bottles received medium plus 1.0 ml of a test solution in duplicate. All bottles were incubated for 72 h at 37°C after which the bottles were drained, rinsed twice with PBS, and air-dried. The cell
IO
concentration in each bottle was then analyzed (Oyama and Eagle, 1956).
by the Lowry protein
method
Inorganic chemical analyses for metats were carried out by inductively coupled plasma (ICP) tests (U.S. environmental Protection Agency, 1983). Metal concentrations were determined on IO% solutions of BEV and CBE and on 3% BEV concentrated eluates of activated and primary sludges.
Results Evaluation of the four concentration procedures with activated sludge samples seeded with poliovirus indicated that EEV yielded the highest mean virus recovery (Tables 1 and 2). Virus recovery from these seeded sludges averaged 83% with BEV. Average recoveries with CBE supplemented with floe, FeC13, and Celite ranged from I6 to 60% lower than that with BEV (Table 2). The Celite method, developed for virus recovery from water and wastewater, was the least effective. TPlBLE 1 Comparison of virus recoveries from activated and aerobically digested sludges using beef extract V and floe-supplemented nonlloccing beef extract in combination with the organic flocculation method Sewage
treatment plant
Sludge type
Poliovirus seeded
Indigenous virus (PFU/lOO ml)
Sample Poliovirus Beef extract Beef extract V Beef extract no. inoculum plus flOCi’ (%, recovery) (PFU~~~~ ml) (% recovery) piUS
Beet extract v
floe”
Sycamore
activated
1 2 3
Harrison
activated
I 2 3
Little Miami activated
1 2 3
Trumansburg
646 640 800
64 61 55
72 80 99
22 21 12
I6 9 8
I960 1400 700
48 63 9s
IX 83 96
20 20 12
63 43 I3
I200 I400 1200
63 67 83
81 68 X8
15 29 23
0 0 3
94 122 I90 I38 99
57 91 I80 I IO 54
aerobically 1 digested 2 3 4 5
~‘~onfloccing beef extract supplemente~i with paste beef floe.
71 TABLE 2 Virus recovery from IOO-ml samples of seeded and nonseeded activated sludge using nonfloccing beef extract with the floe supplement. Celite or F&l3 procedures as floccing aids ---. Indigenous virus Sewage Sample Poliovirus seeded (PFU: 100 ml) treatment no. plant ___- -Beef Beef Poliovirus Beef Beef Beef Beef extract extract extract extract extract inoculum extract plus FeC& plus floe plus Celite PlUS plus plus (PFU,: 100 ml) (% recovery) (O/Orecovery) (X recovery) floe Celite FeCli --_-...-._~__ --.-._---..__--___~~~~~-.~~.-.. 12 38 22 12 6 Sycamore I 646 64 23
640 800
67 55
13 116
48 34
21 I2
IO 4
t
I 2 3
1960 1400 700
48 63 95
I8 IX 3s
40 45 x2
20 20 12
2 6
22 19
1
7
Little Miami 1 2 3
I200 1400 1200
63 61 83
21 28 40
61 68 79
15 29 23
4 8 7
I5 21 I5
Harrison
__--
Indigenous virus recovery from the above activated sludge samples presented a different virus recovery pattern (Table I). Only activated sludge samples from Harrison STP showed higher virus recoveries with BEV. Although seeded activated sludge samples from the Little Miami STP showed little difference in poliovirus recoveries with either BEV or floe-supplemented CBE, processing sludge samples for indigenous virus with BEV resulted in virus detection in only one of three samples. FeC13 was at least twice as efficient as Celite in all nine of the poliovirus seeded samples and in seven of the nine samples tested for indigenous viruses (Table 2). In comparison with floe-supplemented CBE, the FeCl, method usually displayed lower recovery efficiencies with some instances of highly significant differences in recoveries. Processing the BEV eluates from primary sludge by the Katzenelson organic flocculation procedure yielded concentrates that were cytotoxic to the BGM cell line. The organic flocculation procedure, applied to BEV eluates from activated sludge processed without prior primary clarification, also resulted in concentrates that were partially or totally cytotoxic. Processing the same sludges with the floe-supplemented CBE caused no cytotoxicity (Table 3). When aerobically digested sludges were processed with BEV and flocsupplemented CBE, no cytotoxicity was observed. In these live samples, higher numbers of indigenous viruses were obtained with floe-supplemented CBE (Table 1). During virus assay, when cytotoxicity was observed with certain BEV sludge concentrates, metals were considered as the source, because they are known to be concentrated in sewage sludges (Lake et al., 1989). In Table 4, comparisons are shown between metal concentrations found in buffered 10% solutions of
TABLE 3 Comparison of virus recoveries from primary sludge and activated sludge processed without primary clarification using beef extract V and tloc-supplemented nontloccing beef extract in combination with the organic flocculation method -__-__-._ -_-_ __ Slildge Simple Nonfloccing beef extract Beef extract v type” no. supplcmente~~ with paste beef bloc (PHJ 100 ml)” (Pi-Y!,‘lOO ml) f%mary
Activated without primary clarificaliori
I ?
39’ X5
I
701 44X 490 325 34.: b35
7 3 4 5 6 7
8
1.oxic 175 IX3 73 53 6X 32 h
Iox 70
“Primary sludge was collected fl-ant Sycamore STP and activated without primary cl~fri~ic~~ti~~li from Trumansburg STP. “Activated sludge samples 2 through X formed partial toxicity in the cell monolayer.
TABI,E 4 Metals analysts of beef extracts and sludge eluates in mg/l” Elcmcnt
10% CBEh
10% BEV’
Al As (‘a Cd Co Cr Cu K ME MI1 Na Ni
0.3 0.2 5.1 C 0.2 0.01 0.09 1.5 1230 II 0.02 7140 0.1
P
2t00
0.8 0.3 I%.(1 0.09 0.7 o.06 0. I 4.5 706 I4 0.02 5350 0.5 2040 O..? I.6 0.2 0.2 0.3
Fe
Pb Se sn V Zn
0.3 0.3 0.06 1.0
Activated sludge cluatc” 7.0 0.2 5.0 0.01 0. I3 0.06
Primary sludge eluate” ICI.7 0.17 5.0 0.03 0.38 0.15
0.7
2.5
11.2
26.0
35.5 1.4 0.07 4315 0.15 3225 0.3 0.5 0.09 0.18 0.9
40.4 2.0 0.05 4170
0.3 .:I’5 0.9 0.S 0.25 0.28 4.2
“(_oncetltration of metals determined bq inductively coupled plasma analysis. hCBE -= commercially marketed beef extract. ‘BEV -= beef extract V. “Sludges eluted with 10% BEV. cluatc diluted to 3% BEV. concentrated by organic flocculation and suspended in phosphate buffer. “‘Not detected.
73
‘TABLE
5
Comparison ___Element
Cd Cr Se
of cytotoxic
metal
concentrations
Concentration causing in RGM cells (mgil)” IO IO 13
with natural concentrations in primary sludge __~~__ ____ Concentration in primary sludge cytotoxicity &ate (mgjl)’ 0.03 0.15 0.5
“Concentration of metals causing cytotoxicity determined by Lowry protein method. hConcentration of metals determined by inductively coupled plasma analysis of the 10% beef extract V eluate diluted to 3%. concentrated by organic flocculation and suspended in phosphate buffer.
CBE and BEV, as well as in concentrated eluates of activated and primary sludge solids processed with BEV. It was noted that three metals, cadmium, chromium and selenium, normally considered toxic, were at higher concentrations in BEV than in CBE. No cytotoxic effect was observed, however, when 10% BEV itself was tested on BGM cells. Additionally, when 10% BEV was reduced to 3% and concentrated by organic flocculation, the resulting solution was noncytotoxic. Of the three metals, only chromium was higher in primary sludge eluate. It was determined that 10 mg/l of chromium was required to cause cytotoxicity in BGM cells, which was 67 times greater than the concentration of chromium in the primary sludge eluate (Table 5). Both sludge concentrated eluates show a large increase in aluminum that is due to the AlC13 added initially to aid binding of the free virus to the sludge solids. Table 4 also shows that copper and zinc are highest in the primary sludge eluate. Neither metal was cytotoxic when BGM cells were exposed to 3 mg/l of copper or 5 mg/ I of zinc, individually or combined.
Discussion The change in the manufacturing process used to produce powdered beef extract did not affect adversely the elution capability of the beef extract, but it markedly decreased the effectiveness of the organic flocculation procedure (Payment et al., 1984; Hurst et al., 1984). Modifications to the concentration technique by supplementing CBE resulted in a more laborious procedure. Development of BEV for virus studies generated a product of which the floe formation was similar to that obtained in beef extract solutions prior to this manufacturing change (Dahling and Wright, 1988; Hurst, 1990). The cytotoxicity observed with the concentrated eluates obtained from processing solids from primary sludge treatment systems and an activated sludge system processed without prior primary clarification precluded the use of BEV. Hurst and Goyke (1983) reported that there was no cytotoxicity with filtered eluates of primary and activated sludges; however, the concentrates of
these eluates could be cytotoxic and nonassayable. BEV gave exceptionally good recoveries from the Harrison STP activated sludges (Table 1). The poor recoveries seen in the Little Miami STP activated sludges may be related to cytotoxicity, although it was not evident on examining the BGM test cells. The difference in recoveries also may be related to the type of virus population present in the sludge solids. Morris and Waite (1980) reported organic flocculation concentration efficiency could be affected by virus serotype. Gerba et al. (1980), studying adsorptive properties of sludges, concluded that they could vary greatly for different virus serotypes and strains. Our results show the necessity of evaluating concentration procedures with different sludges to determine the more effective processing method. The FeC13 and Celite alternative methods to organic flocculation were developed primarily for virus recovery from water and wastewater rather than from sewage sludge eluates. Dahling and Wright (1988) reported no differences in virus recovery from wastewater between the FeC13 and Celite procedures. However, our tests showed that FeC& and Celite were not as effective as the floe-supplemented CBE (Safferman et al., 1988) for concentrating viruses from sludge eluates. A comparison of the FeC13 and Celite procedures revealed a marked difference in the level of virus recovery (Table 2). The increased level of organics in the sludge eluates may interfere with virus adsorptionl’elution with the Celite. Virus-FeC13 binding, similar to AI/Mg binding (Berman et al.. 1981). and pH conditions below the isoelectric point of many viruses may have resulted in more favorable virus recovery from sewage sludge eluates with FeC13. The BGM cell line, used extensively for environmental virus monitoring. has been used to test cytotoxicity of water and wastewater samples (Kfir and Prozesky, 1981). In our study, using the Lowry protein procedure to determine cell growth, BGM cell monolayers were exposed to varying concentrations ot metals to determine their cytotoxicity, and this cytotoxic concentration was compared to levels of the metals found in cytotoxic BEV concentrated eluates of primary sludge. The levels found in these eluates were too low to cause the observed cytotoxicity. Hurst and Goyke (1983) suggested that metals may exert a synergistic cytotoxic effect; however, no synergistic effects for copper and zinc were observed in our study. This does not exclude the possibility of a synergistic effect by different metal combinations or of more than two metals. Another possibility, which was not investigated, is the formation o( organometal complexes that transforms metals into more toxic and bioavailable forms (Karapanagiotis et al.. 1990).
References Berg, G.. Berman, D. and Safferman. K.S. (1982) A method for concentrating viruses recovcrctl from sewage sludges. Can. J. Microbial. 2X. SY- 556. Berg. G.. Safferman. R.S.. Dahling. D.R.. Berman. D. and Hurst. C‘.J. (1984) I;.S. EPA Manual of
75 Methods for Viroiogy. Environmental Monitoring and Support Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH. EPA-600/4-84-013. Berman, D., Berg, 6. and Safferman, R.S. ( 1981) A method for recovering viruses from sludges. J. Viral. Methods 3, 2833291. Dahling, D.R. and Wright, B.A. (1986a) Optimization of the BGM cell line culture and viral assay procedures for monitoring viruses in the environment. Appl. Environ. Microbial. 51, 790 812. Dahling. D.R. and Wright, B.A. (1986b) Recovery of viruses from water by a modified flocculation procedure for second-step concentration. Appt. Environ. Microbioi. 51, 13266133 I. Dahling. D.R. and Wright, B.A. (1988) A comparison of recovery of virus from wastewaters by beef extract-Celite, ferric chloride and filter concentration procedures. J. Viral. Methods 22, 337.-346. Dahling. D.R., Berg, G. and Berman, D. (1974) BGM, a continuous cell line more sensitive than primary rhesus and African green kidney cells for the recovery of viruses from water. Health Lab. Sci. II, 275-282. Gerba, C.P.. Goyal, SM., Hurst, C.J. and LaBeile, R.L. (1980) Type and strain dependence of enterovirus adsorption to activated sludge, soils and estuarine sediments. Water Res. 14, I l971198. Hurst, C.J. (1990) Field methods for concent~ting viruses from water samples. In: G. Craun (Ed.), Methods for the Investigation and Prevention of Waterborne Disease Outbreaks. U.S. Environmental Protection Agency, Washington, D.C. SPA-400~1-90-005a. Hurst, C.J. and Goyke, T. (1983) Reduction of interfering cytotoxicity associated with wastewater sludge concentrates assayed for indigenous enteric viruses. Appl. Environ. Microbial. 4h, l33139. Hurst, C.J., Dahling, D.R., Safferman, R.S. and Coyke, T. (1984) Comparison of commercial beef extracts and similar materials for recovering viruses from environmental samples. Can. J. Microbial. 30, 1253-1263. Kara~~nagiotis, N.K., Sterritt. R.M. and Lester, J.N. (1990) Heavy metal binding by the polymeric organic fractions of sewage sludges. Environ. Poll. 67, 259-278. Katzenelson, E., Fattal, B. and Hostovesky, T. (1976) Organic flocculation: an efficient second-step concentration method for the detection of viruses in tap water. Appl. Environ. Microbial. 32, 638-639. Kfir, R. and Prozesky, O.W. (1981) Detection of toxic substances in water by means of a mamlnaljan cell culture technique. Water Res. 1.5,553-559, Lake, D.L., Kirk, P.W.W. and Lester, J.N. (1989) Heavy metal solids association in sewage sludges. Water Res. 23, 285-29 I. Morris, R. and Waite, W.M. (1980) Evaluation of procedures for recovery of viruses from water - I. Concentration systems, Water Res. 14, 791 793. Oyama, V.I. and Eagle, H. (1956) Measurement of cell growth in tissue culture with a phenol reagent (Folin-Ciocalteau). Proc. Sot. Exptl. Biol. Med. 91, 305-307. Payment, P., Fortin, S. and Trudel, M. (1984) Ferric chloride ~occulation for non~occulatin~ beef extract preparations. Appl. Environ. Microbial. 47, 591-592. Safferman, R.S., Rohr, M.E. and Goyke, T. (1988) Assessment of recovery efficiency of beef extract reagents for concentrating viruses from municipal wastewater sludge solids by the organic flocculation procedure. Appl. Environ. Microbial. 54. 309.-316. U.S. ~nvironmentai Protection Agency (1983) Method 200.7 Inductively coupled plasma-~~tornic emission spectro~hotometric method for trace element analysis of water and wastes. In: Methods for Chemical Analysis of Water and Wastes. EPA-600/4-79-020, revised March, 1983.
Journal of’ Virological Methods, 40 (1992) 67 16 ,c> 1992 Elsevier Science Publishers B.V. ! All rights reserved / 0166-0934192jSO5.00
VIRMET 01397
Processing procedures for recovering enteric viruses from wastewater sludges Ronald Virology
Branch,
E. Stetler,
Mary Ellen Morris
Microbiology
Research Division. Environmental Cincinnati, OH (USA)
(Accepted
and Robert
S. Safferman
Monitoring
S~~stems Laboratory,
12 May 1992)
_ Summary A powdered beef extract specially formulated for recovering viruses from environmental samples and designated as beef extract V was evaluated using indigenous and viral seeded wastewater sludge samples. When beef extract V was used to process activated and aerobically digested sludge solids, virus recoveries were shown to be similar to other methods that used commercially available supplemented beef extract. When used to process other sludge solids (primary and activated without primary clarification), cytotoxicity resulted in the BGM cell line used for virus assay. When these sludge solids were processed with the supplemented commercially marketed beef extract cell toxicity did not occur. Metal concentrations in the processed sewage sludge eluates were analyzed, but based on the levels observed they could not be shown as the source of the cytotoxicity. This did not exclude possible synergistic cytotoxic effects or organometal complexes. The commercially marketed beef extract was supplemented with either a floccing aid (FeClj), a filter aid (Celite) or a floe prepared from paste beef extract. The paste beef floe supplement proved to be the most useful and reliable method for processing for viruses from wastewater sludge solids. Viruses;
Wastewater
sludge;
Elution-concentration
process;
Correspondence to: R.E. Stetler, Virology Branch, Microbiology Monitoring Systems Laboratory, Cincinnati, OH, USA.
Detection
Research Division,
Environmental
68
introduction Commercially marketed powdered beef extract (CBE) has been used to elute viruses adsorbed to solid matrices. Eluted viruses have then been effectively concentrated from CBE by the organic flocculation procedure of Katzenelson et al. (1976), in which the viruses are adsorbed to the floe formed upon acidification of the CBE solution. Reducing sample volume has been an essential and practical step in this process of recovering waterborne viruses. Subsequent changes in the manufacturing process have improved the clarity of CBE in solution, but the altered product precluded processing of the eluate by the organic flocculation procedure. Upon acidification of the refined CBE. floe concentration ievels necessary for virus adsorption failed to form (Hurst et al.. 1984). This has led to the development of other mechanisms to reduce sample volumes. The method was modified for virus recovery from tap and wastewaters by the addition of a floccing agent (Payment et al., 19X4) and an inorganic filter aid (Dahling and Wright, 1986b, 1988). The method was also modified for use with sludge solids by the addition of a preformed flnc prepared from acidification and centrifugation of a paste beef extract solution (Safferman et ai., 1988). A new powdered beef extract specially formulated for virus studies, beef extract V (BEV), recently became available which, upon acidi~cat~on, produces adequate anlounts of floe for virus adsorption (Dahl~ng and Wright, 1988; Hurst, 1990). We evaluated BEV, CBE supplemented with paste beef ftoc. CBE supplemented with the floccing aid, FeCl3, and CBE supplemented with the filter aid, Celite, as to their efficiency for recovery and concentration of viruses from municipal wastewater sludge solids.
Materials and Methods
Virus assays were carried out using the continuous African green monkey kidney cell line designated BGM (Dahling et al., 1974). Propagation and assay procedures have been described previously (Dahling and Wright, 19X&+). Poliovirus type 1 (Mahoney LP) was used to seed the activated sludge samples,
A nonfloccing commercially marketed beef extract powder (lot no. 05401. Gibco Diagnostics, Madison, WI) and a floccing beef extract powder specially formulated for virus studies, beef extract V (lot no. CF)DVLB, BBL, Cockeysville, MD), were used for elution of sludge solids. The beef extracts were prepared as 10% (w/v) solutions in distilled water buffered with 1.34 g Na2HP04. 7Hz0 and 0.12 g citric acid per 100 ml. The supplemental floe was
69
prepared from a 3% (w/v) solution MI) in distilled water as previously
of paste beef extract (Difco, Inc., Detroit, described (Safferman et al., 1988).
Test procedure
Activated and primary sludge samples were collected from sewage treatment plants (STP) in southwestern Ohio. Samples of activated sludge without prior primary clarification and aerobically digested sludge were collected from the Trumansburg STP in New York. Sludge samples were shaken and lOO-ml aliquots were removed to process for indigenous virus recovery. Additional aliquots were removed from the activated sludge samples collected at the southwestern Ohio STPs. These samples were seeded with poliovirus type 1 and processed concomitantly with the nonseeded samples. The sludge extraction procedure, described previously (Berg et al., 1982, 1984; Berman et al., 1981; Safferman et al., 1988) is outlined briefly. To each lOO-ml sample, AlCls was added to a final concentration of 0.0005 M and the sample acidified to pH 3.5. After mixing for 30 min, the sample was centrifuged at 2500 x g for 15 min and the supernatant discarded. Sludge pellets were suspended in 100 ml of either buffered 10% CBE or buffered 10% BEV. After 30 min mixing, all samples were centrifuged at 10000 x g for 30 min. Supernatants were pressure filtered through a sterile stack of Filterite membrane filters (3.0 pm, 0.45 ;lrn, and 0.25 pm) placed in order of increasing pore size. To each volume of filtered eluate, a sufficient volume of sterile, distilled water was added to reduce the beef extract concentration to 3%. The CBE eluates were supplemented as follows: one group received floe prepared from 100 ml of 3% paste beef extract, the second group received 0.05 M FeC13 at 0.5 ml/l00 ml, and the third group received Celite at 0.1 g/l00 ml. BEV filtered eluates were not supplemented. All filtered eluates, except those supplemented with Celite, were aciditied to pH 3.5 and stirred for 30 min. After centrifuging at 2500 x g for 15 min, the pellets were solubilized in 0.15 M Na2HP04. 7H20 at l/20 the volume of the diluted liltered eluate and kept at 4°C until assayed. Celite supplemented filtered eluates were acidified to pH 4.0, stirred 10 min and centrifuged as above. The pellet was suspended in 0.15 M Na2HP04. 7Hz0, mixed for 25 min and recentrifuged. The supernatant was decanted and kept at 4°C until assayed. Lowry protein analysis
BGM cell growth was determined by protein analysis. BGM cell monolayers, cultured in glass bottles for 24 h, were rinsed twice with phosphate buffered saline (PBS). Two bottles were drained, air-dried, recapped, and stored for Otime protein determination. Two other bottles received fresh medium to serve as controls. The remaining bottles received medium plus 1.0 ml of a test solution in duplicate. All bottles were incubated for 72 h at 37°C after which the bottles were drained, rinsed twice with PBS, and air-dried. The cell
IO
concentration in each bottle was then analyzed (Oyama and Eagle, 1956).
by the Lowry protein
method
Inorganic chemical analyses for metats were carried out by inductively coupled plasma (ICP) tests (U.S. environmental Protection Agency, 1983). Metal concentrations were determined on IO% solutions of BEV and CBE and on 3% BEV concentrated eluates of activated and primary sludges.
Results Evaluation of the four concentration procedures with activated sludge samples seeded with poliovirus indicated that EEV yielded the highest mean virus recovery (Tables 1 and 2). Virus recovery from these seeded sludges averaged 83% with BEV. Average recoveries with CBE supplemented with floe, FeC13, and Celite ranged from I6 to 60% lower than that with BEV (Table 2). The Celite method, developed for virus recovery from water and wastewater, was the least effective. TPlBLE 1 Comparison of virus recoveries from activated and aerobically digested sludges using beef extract V and floe-supplemented nonlloccing beef extract in combination with the organic flocculation method Sewage
treatment plant
Sludge type
Poliovirus seeded
Indigenous virus (PFU/lOO ml)
Sample Poliovirus Beef extract Beef extract V Beef extract no. inoculum plus flOCi’ (%, recovery) (PFU~~~~ ml) (% recovery) piUS
Beet extract v
floe”
Sycamore
activated
1 2 3
Harrison
activated
I 2 3
Little Miami activated
1 2 3
Trumansburg
646 640 800
64 61 55
72 80 99
22 21 12
I6 9 8
I960 1400 700
48 63 9s
IX 83 96
20 20 12
63 43 I3
I200 I400 1200
63 67 83
81 68 X8
15 29 23
0 0 3
94 122 I90 I38 99
57 91 I80 I IO 54
aerobically 1 digested 2 3 4 5
~‘~onfloccing beef extract supplemente~i with paste beef floe.
71 TABLE 2 Virus recovery from IOO-ml samples of seeded and nonseeded activated sludge using nonfloccing beef extract with the floe supplement. Celite or F&l3 procedures as floccing aids ---. Indigenous virus Sewage Sample Poliovirus seeded (PFU: 100 ml) treatment no. plant ___- -Beef Beef Poliovirus Beef Beef Beef Beef extract extract extract extract extract inoculum extract plus FeC& plus floe plus Celite PlUS plus plus (PFU,: 100 ml) (% recovery) (O/Orecovery) (X recovery) floe Celite FeCli --_-...-._~__ --.-._---..__--___~~~~~-.~~.-.. 12 38 22 12 6 Sycamore I 646 64 23
640 800
67 55
13 116
48 34
21 I2
IO 4
t
I 2 3
1960 1400 700
48 63 95
I8 IX 3s
40 45 x2
20 20 12
2 6
22 19
1
7
Little Miami 1 2 3
I200 1400 1200
63 61 83
21 28 40
61 68 79
15 29 23
4 8 7
I5 21 I5
Harrison
__--
Indigenous virus recovery from the above activated sludge samples presented a different virus recovery pattern (Table I). Only activated sludge samples from Harrison STP showed higher virus recoveries with BEV. Although seeded activated sludge samples from the Little Miami STP showed little difference in poliovirus recoveries with either BEV or floe-supplemented CBE, processing sludge samples for indigenous virus with BEV resulted in virus detection in only one of three samples. FeC13 was at least twice as efficient as Celite in all nine of the poliovirus seeded samples and in seven of the nine samples tested for indigenous viruses (Table 2). In comparison with floe-supplemented CBE, the FeCl, method usually displayed lower recovery efficiencies with some instances of highly significant differences in recoveries. Processing the BEV eluates from primary sludge by the Katzenelson organic flocculation procedure yielded concentrates that were cytotoxic to the BGM cell line. The organic flocculation procedure, applied to BEV eluates from activated sludge processed without prior primary clarification, also resulted in concentrates that were partially or totally cytotoxic. Processing the same sludges with the floe-supplemented CBE caused no cytotoxicity (Table 3). When aerobically digested sludges were processed with BEV and flocsupplemented CBE, no cytotoxicity was observed. In these live samples, higher numbers of indigenous viruses were obtained with floe-supplemented CBE (Table 1). During virus assay, when cytotoxicity was observed with certain BEV sludge concentrates, metals were considered as the source, because they are known to be concentrated in sewage sludges (Lake et al., 1989). In Table 4, comparisons are shown between metal concentrations found in buffered 10% solutions of
TABLE 3 Comparison of virus recoveries from primary sludge and activated sludge processed without primary clarification using beef extract V and tloc-supplemented nontloccing beef extract in combination with the organic flocculation method -__-__-._ -_-_ __ Slildge Simple Nonfloccing beef extract Beef extract v type” no. supplcmente~~ with paste beef bloc (PHJ 100 ml)” (Pi-Y!,‘lOO ml) f%mary
Activated without primary clarificaliori
I ?
39’ X5
I
701 44X 490 325 34.: b35
7 3 4 5 6 7
8
1.oxic 175 IX3 73 53 6X 32 h
Iox 70
“Primary sludge was collected fl-ant Sycamore STP and activated without primary cl~fri~ic~~ti~~li from Trumansburg STP. “Activated sludge samples 2 through X formed partial toxicity in the cell monolayer.
TABI,E 4 Metals analysts of beef extracts and sludge eluates in mg/l” Elcmcnt
10% CBEh
10% BEV’
Al As (‘a Cd Co Cr Cu K ME MI1 Na Ni
0.3 0.2 5.1 C 0.2 0.01 0.09 1.5 1230 II 0.02 7140 0.1
P
2t00
0.8 0.3 I%.(1 0.09 0.7 o.06 0. I 4.5 706 I4 0.02 5350 0.5 2040 O..? I.6 0.2 0.2 0.3
Fe
Pb Se sn V Zn
0.3 0.3 0.06 1.0
Activated sludge cluatc” 7.0 0.2 5.0 0.01 0. I3 0.06
Primary sludge eluate” ICI.7 0.17 5.0 0.03 0.38 0.15
0.7
2.5
11.2
26.0
35.5 1.4 0.07 4315 0.15 3225 0.3 0.5 0.09 0.18 0.9
40.4 2.0 0.05 4170
0.3 .:I’5 0.9 0.S 0.25 0.28 4.2
“(_oncetltration of metals determined bq inductively coupled plasma analysis. hCBE -= commercially marketed beef extract. ‘BEV -= beef extract V. “Sludges eluted with 10% BEV. cluatc diluted to 3% BEV. concentrated by organic flocculation and suspended in phosphate buffer. “‘Not detected.
73
‘TABLE
5
Comparison ___Element
Cd Cr Se
of cytotoxic
metal
concentrations
Concentration causing in RGM cells (mgil)” IO IO 13
with natural concentrations in primary sludge __~~__ ____ Concentration in primary sludge cytotoxicity &ate (mgjl)’ 0.03 0.15 0.5
“Concentration of metals causing cytotoxicity determined by Lowry protein method. hConcentration of metals determined by inductively coupled plasma analysis of the 10% beef extract V eluate diluted to 3%. concentrated by organic flocculation and suspended in phosphate buffer.
CBE and BEV, as well as in concentrated eluates of activated and primary sludge solids processed with BEV. It was noted that three metals, cadmium, chromium and selenium, normally considered toxic, were at higher concentrations in BEV than in CBE. No cytotoxic effect was observed, however, when 10% BEV itself was tested on BGM cells. Additionally, when 10% BEV was reduced to 3% and concentrated by organic flocculation, the resulting solution was noncytotoxic. Of the three metals, only chromium was higher in primary sludge eluate. It was determined that 10 mg/l of chromium was required to cause cytotoxicity in BGM cells, which was 67 times greater than the concentration of chromium in the primary sludge eluate (Table 5). Both sludge concentrated eluates show a large increase in aluminum that is due to the AlC13 added initially to aid binding of the free virus to the sludge solids. Table 4 also shows that copper and zinc are highest in the primary sludge eluate. Neither metal was cytotoxic when BGM cells were exposed to 3 mg/l of copper or 5 mg/ I of zinc, individually or combined.
Discussion The change in the manufacturing process used to produce powdered beef extract did not affect adversely the elution capability of the beef extract, but it markedly decreased the effectiveness of the organic flocculation procedure (Payment et al., 1984; Hurst et al., 1984). Modifications to the concentration technique by supplementing CBE resulted in a more laborious procedure. Development of BEV for virus studies generated a product of which the floe formation was similar to that obtained in beef extract solutions prior to this manufacturing change (Dahling and Wright, 1988; Hurst, 1990). The cytotoxicity observed with the concentrated eluates obtained from processing solids from primary sludge treatment systems and an activated sludge system processed without prior primary clarification precluded the use of BEV. Hurst and Goyke (1983) reported that there was no cytotoxicity with filtered eluates of primary and activated sludges; however, the concentrates of
these eluates could be cytotoxic and nonassayable. BEV gave exceptionally good recoveries from the Harrison STP activated sludges (Table 1). The poor recoveries seen in the Little Miami STP activated sludges may be related to cytotoxicity, although it was not evident on examining the BGM test cells. The difference in recoveries also may be related to the type of virus population present in the sludge solids. Morris and Waite (1980) reported organic flocculation concentration efficiency could be affected by virus serotype. Gerba et al. (1980), studying adsorptive properties of sludges, concluded that they could vary greatly for different virus serotypes and strains. Our results show the necessity of evaluating concentration procedures with different sludges to determine the more effective processing method. The FeC13 and Celite alternative methods to organic flocculation were developed primarily for virus recovery from water and wastewater rather than from sewage sludge eluates. Dahling and Wright (1988) reported no differences in virus recovery from wastewater between the FeC13 and Celite procedures. However, our tests showed that FeC& and Celite were not as effective as the floe-supplemented CBE (Safferman et al., 1988) for concentrating viruses from sludge eluates. A comparison of the FeC13 and Celite procedures revealed a marked difference in the level of virus recovery (Table 2). The increased level of organics in the sludge eluates may interfere with virus adsorptionl’elution with the Celite. Virus-FeC13 binding, similar to AI/Mg binding (Berman et al.. 1981). and pH conditions below the isoelectric point of many viruses may have resulted in more favorable virus recovery from sewage sludge eluates with FeC13. The BGM cell line, used extensively for environmental virus monitoring. has been used to test cytotoxicity of water and wastewater samples (Kfir and Prozesky, 1981). In our study, using the Lowry protein procedure to determine cell growth, BGM cell monolayers were exposed to varying concentrations ot metals to determine their cytotoxicity, and this cytotoxic concentration was compared to levels of the metals found in cytotoxic BEV concentrated eluates of primary sludge. The levels found in these eluates were too low to cause the observed cytotoxicity. Hurst and Goyke (1983) suggested that metals may exert a synergistic cytotoxic effect; however, no synergistic effects for copper and zinc were observed in our study. This does not exclude the possibility of a synergistic effect by different metal combinations or of more than two metals. Another possibility, which was not investigated, is the formation o( organometal complexes that transforms metals into more toxic and bioavailable forms (Karapanagiotis et al.. 1990).
References Berg, G.. Berman, D. and Safferman. K.S. (1982) A method for concentrating viruses recovcrctl from sewage sludges. Can. J. Microbial. 2X. SY- 556. Berg. G.. Safferman. R.S.. Dahling. D.R.. Berman. D. and Hurst. C‘.J. (1984) I;.S. EPA Manual of
75 Methods for Viroiogy. Environmental Monitoring and Support Laboratory, U.S. Environmental Protection Agency, Cincinnati, OH. EPA-600/4-84-013. Berman, D., Berg, 6. and Safferman, R.S. ( 1981) A method for recovering viruses from sludges. J. Viral. Methods 3, 2833291. Dahling, D.R. and Wright, B.A. (1986a) Optimization of the BGM cell line culture and viral assay procedures for monitoring viruses in the environment. Appl. Environ. Microbial. 51, 790 812. Dahling. D.R. and Wright, B.A. (1986b) Recovery of viruses from water by a modified flocculation procedure for second-step concentration. Appt. Environ. Microbioi. 51, 13266133 I. Dahling. D.R. and Wright, B.A. (1988) A comparison of recovery of virus from wastewaters by beef extract-Celite, ferric chloride and filter concentration procedures. J. Viral. Methods 22, 337.-346. Dahling. D.R., Berg, G. and Berman, D. (1974) BGM, a continuous cell line more sensitive than primary rhesus and African green kidney cells for the recovery of viruses from water. Health Lab. Sci. II, 275-282. Gerba, C.P.. Goyal, SM., Hurst, C.J. and LaBeile, R.L. (1980) Type and strain dependence of enterovirus adsorption to activated sludge, soils and estuarine sediments. Water Res. 14, I l971198. Hurst, C.J. (1990) Field methods for concent~ting viruses from water samples. In: G. Craun (Ed.), Methods for the Investigation and Prevention of Waterborne Disease Outbreaks. U.S. Environmental Protection Agency, Washington, D.C. SPA-400~1-90-005a. Hurst, C.J. and Goyke, T. (1983) Reduction of interfering cytotoxicity associated with wastewater sludge concentrates assayed for indigenous enteric viruses. Appl. Environ. Microbial. 4h, l33139. Hurst, C.J., Dahling, D.R., Safferman, R.S. and Coyke, T. (1984) Comparison of commercial beef extracts and similar materials for recovering viruses from environmental samples. Can. J. Microbial. 30, 1253-1263. Kara~~nagiotis, N.K., Sterritt. R.M. and Lester, J.N. (1990) Heavy metal binding by the polymeric organic fractions of sewage sludges. Environ. Poll. 67, 259-278. Katzenelson, E., Fattal, B. and Hostovesky, T. (1976) Organic flocculation: an efficient second-step concentration method for the detection of viruses in tap water. Appl. Environ. Microbial. 32, 638-639. Kfir, R. and Prozesky, O.W. (1981) Detection of toxic substances in water by means of a mamlnaljan cell culture technique. Water Res. 1.5,553-559, Lake, D.L., Kirk, P.W.W. and Lester, J.N. (1989) Heavy metal solids association in sewage sludges. Water Res. 23, 285-29 I. Morris, R. and Waite, W.M. (1980) Evaluation of procedures for recovery of viruses from water - I. Concentration systems, Water Res. 14, 791 793. Oyama, V.I. and Eagle, H. (1956) Measurement of cell growth in tissue culture with a phenol reagent (Folin-Ciocalteau). Proc. Sot. Exptl. Biol. Med. 91, 305-307. Payment, P., Fortin, S. and Trudel, M. (1984) Ferric chloride ~occulation for non~occulatin~ beef extract preparations. Appl. Environ. Microbial. 47, 591-592. Safferman, R.S., Rohr, M.E. and Goyke, T. (1988) Assessment of recovery efficiency of beef extract reagents for concentrating viruses from municipal wastewater sludge solids by the organic flocculation procedure. Appl. Environ. Microbial. 54. 309.-316. U.S. ~nvironmentai Protection Agency (1983) Method 200.7 Inductively coupled plasma-~~tornic emission spectro~hotometric method for trace element analysis of water and wastes. In: Methods for Chemical Analysis of Water and Wastes. EPA-600/4-79-020, revised March, 1983.
