Bacteriophages as particle migration indicators in subsurface environments

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1 Tracers and Mdelling in Hydrgelgy (Prceedings f the TraM'2000 Cnference held at Liège, Belgium, May 2000). IAHS Publ. n. 262, Bacteriphages as particle migratin indicatrs in subsurface envirnments KEITH G. KENNEDY CHYN-Hydrgelgy Centre, University f Neuchatel, Emile-Argand 11, CH-2007 Neuchatel, Switzerland Abstract Bacteriphages can assist characterizatin f hydrgelgical settings and dcument cntaminant behaviur. They emulate viral and facilitated/assisted transprt. Field research was dne in Switzerland in periglacial aquifers under natural gradients t 65 m and in a grandirite shear zne t 1.7 m. Phages had distinct and quantifiable breakthrugh. Particle first arrivals were earlier r similar t slutes. Phage C/C 0. ra was ntably earlier than slute Tr/Tr 0. lmx. Typical retardatin factrs (RF) were frm 0.2 t 0.9. In the prus media, transprt rates were frm 40 t 440 m day" 1, phage relative breakthrughs (RBs) were frm t 72% and C/C 0. max frm 3e" 10 t 4e" 4. Three shear zne diple tests illustrated an pen path, as well as fault guge dminated, respnses. RBs were frm 0.15 t 41% and C/C 0. max frm 7e" 4 t 2e" 1. Lw particle attenuatin was in channels discvered in thin (<2 m) aquifer layers and in ne directin in the shear zne. INTRODUCTION Predicting migratin behaviur f cllidal particles and slutes in hetergeneus subsurface envirmnents is the key t prtecting aquifers and develping a sund grundwater management strategy. Tday it is cmmnly based n scaled-dwn reginal numerical mdels r simplistic Darcy's law calculatins limited t hmgeneity assumptins. Pathgenic health issues, such as thse related t virus r bacterial behaviur, are based frequently n scenaris frm dramatically scaled-up clumn test results. Actual field verificatin fr grundwater prtectin is rare fr biparticles except in karst (Ddrfliger, 1996). Applying recently imprved field methds and instrumentatin (e.g. Schnegg & Kennedy, 1998; Kennedy et al, 1999) and bacteriphage analysis (e.g. Rssi & Kâss, 1998) can result in cst-effective and timely data dcumenting particle and slute migratin. These data are pertinent input t grundwater prtectin and validatin f mdel cdes applied t water supply aquifers. There are few well-dcumented and published case histries that prvide bicllid transprt rates in prus media and fractured subsurface envirnments. With the exceptin f the extensive wrk at the Cape Cd site lead by the US Gelgical Survey (e.g. Harvey et al., 1989), there are few aquifer-scale field experiments related t drinking water in prus media ver significant distances. Use f bacteriphages dcumenting migratin in tighter fractured rck (e.g. Mackay et al, 1993, 2000) is als limited.

2 152 Keith G. Kennedy The bjective f the wrk reprted here was t determine bacteriphage efficacy (a) in Swiss prductive prus media aquifers, (b) in lwer permeability fractured envirnments and (c) cmpared t mre cnventinal "cnservative" slute tracers. C O O r / /, 1 \l 20 M 40 Kappelen (kap) 7 tests- 10 t 90 days 7 wells - 15 m deep 7 wells - 9 m deep Gradient Particle Arrival: 20 m-11 h 60 m - 36 h Wilerwald (WW) 16 day test 4 wells f-25 used Gradient D7-64mD High phage recvery (In channel) \ Particle Arrival: 14 m-1.5 hurs 64 m - 7 hurs I C6-14m I (In channel) '. CD-34m \ (Off channel) 50 M B4 Injectin well Dublet wells Single well A 11 j Flw Directins O I 11C 11A Pl.-' 11D - - ' " a 11B Ô ÏÔM 20 11B - inj'ntr Tests 1,2,3 11A- ini'n fr tests 4,5,6 6 tests-1 t 20 days " '- 5 wells - 10 m t 30 m deep Active flw -1 t 6 m tnick Variable seasnal gradient Particle Arrival: 10 m-0.6 h (high wt) 22 m - 2 h (lw wt) SWITZERLAND *A Bis de Finges (BdF) 5 t 28 day tests Wall t shear: 5 t 10 m Lw permeability fracture Flw rate: 1 t 2 ml/mm Particle Arrival: 5 t 10 h Observatin brehles Grimsel (gam) Fig. 1 Research site lcatins and well/brehle instrumentatin layut. DATA SOURCES The data in this paper are frm field research experiments dne in Switzerland (Fig. 1) frm 1997 thrugh 1999 at three prus media research areas and in ne grandirite shear zne (GAM) used fr research at NAGRA's (Swiss Natinal Nuclear Waste Dispsal Cperative) Grimsel Test Site (GTS) in the Alps. In prus media, relatively prlific peri-glacial aquifers were investigated under natural gradient cnditins ver distances frm 10 t 64 m at depths frm abut 5 t 15 m at sites BdF, KAP and WW (Fig. 1). Tracer "ccktails" with the flurescent dye uranine and up t three different bacteriphages were injected and thereafter dissipated ppsite the aquifer active interval using clsed-circuit flw circulatin. Uranine analysis was cmmnly dne inline, nsite and field results guided sampling actins. Individual samples were taken fr cmparative bacteriphage and uranine cncentratins frm dedicated lines in each well cmmnly circulating at frm 2 t 4 1 min" 1. In the grandirite shear zne, three flw fields were evaluated in 1999 using diple cnfiguratins with similar injectin/ex tractin flw rates (~1 t 2 ml m" 1 ). Separatin between the brehle shear znes tested was 1.2 and 1.7 m. Each test had t have different flurescent slute and bacteriphage tracers because f residual backgrunds. Tracers were input as single step cncentratins circulating ppsite the injectin interval fr frm 16 t 30 h. Recvery frm diple extractin was restricted t match inflw rates and was measured fr up t 16 days frm the brehle utflw.

3 Bacteriphages as particle migratin indicatrs in subsurface envirnments 153 METHODS Field flurimeters (Schnegg & Kennedy, 1998) riginally develped fr karst were mdified t prvide adequately sensitive and rapid results frm (a) dwnhle, (b) inline flw during well and brehle pumping/circulatin, and (c) discrete aliquts/samples. The reslutin was 0.1 t 0.5 ppb with either rapid (each 10 s fr 15 h) r lng-term acquisitin capacity (each 4 min fr 15 days) fr multi-flurescent Table 1 Prus media aquifer respnse characteristics using bacteriphages. K Test Hydraulic Depth Wells: Uranine respnse: Bacteriphage respnse: (m s"') n. gradient Injec- Mni- Apart t t, m -r r Phage t a r max RF ç RB Att'n (fn-1) (m) tin tr (m) (h) (h) (h) (hj (%) (%) Kappelen (fn-kap) e e e le" t 10 t e- 05 nt adequately sampled present, nt quantified H40 nne detected 5e- w 6 60 < e- M < e t 7 63 present, nt quantified present, nt quantified Se" nne detected na 3e-' na na t le' e t 0 J 1 H le" e 0 S e- 06 nne detected 1 Bis de Finges (fn-bdf) t 0.003, f 6,, D l l B t l 2 3e- 03 t t 12 1 IB >le" C 1 ID t,.,, D ne t 12 1 IB 1 ID e- 06 3e- 04 H40 11C e' 05 H40 MS2 1 ID le" 03 H40 MS e- 04 4e- 03 H e- 07 3e" Se' 07 le" e- 05 4e e* 1.4 4c t 6 11A ne e- 04 H4 < e Wilerwald (fn-ww) H e" C6 14 < le- 04 Psf e~ MS e e- H Se* t t 7 B4 CD e- 05 Psf Se" >le" 2 MS e~ H le' D e' 0S Psf Se" MS c fn-1 Values apprximate based n field characterizatin, testing dne by thers, and this authr (Kappelen), ver the past tw t 10 years. fn-kap Tests 3, 7 used ne injectin, six mnitring wells. If n well number, n tracers detected. Separate paper (Kennedy et ai, submitted). fn-bdf Tests 1, 2, 3: used injectin (1 IB), sampling fur wells (11, 11A, 11C, 1 ID). N tracers detected in wells 11 r 11 A. Test 4: injectin (HA), sampling at three wells (11, 11C, 1 ID). N tracers detected in wells 11 r 1 ID. Separate paper (Kennedy & Scheurch, submitted). fn-ww Test with new micrsphere/phage techniques and cmparative behaviur (three wells). Separate paper (Kennedy & Niehren, submitted).

4 154 Keith G. Kennedy tracer detectin (uranine, sulphrhdamine, naphthinate). Access irregularity required data t be retrievable in real time, remtely (telecm-retrievable) and als pst-testing (PC-card). Bacteriphages (B) are nn-pathgenic, nn-txic bilgical tracers. They are viruses f a specific hst bacteria (HB) ranging in size frm 20 t 350 nm. They are cmmnly injected with high cncentratins (>le 10 plaque frming units (pfu) ml" 1 ), have a large percentage remval in the subsurface, yet are useful due t their lw detectin level (0.5 pfu mf 1 ) and uniqueness. Field samples are analysed in a micrbilgy labratry fr each B/HB system using an adaptatin f the classical duble-layer technique n Petri dishes. Cncentratins are visible dish pint cunts. Bacteriphage behaviur was cmpared t slute tracer respnses in a manner similar t methds described riginally by Harvey et al. (1989). This included cmparing relative accumulatin (C/C max ), maximum dimensinless cncentratins ([C/CJmax r [Tr/Tr] max ), retardatin factr (RF) the rati f particle vs slute maximum times, relative breakthrugh (RB) particle mass recvered cmpared t slute recvery (nrmalized t 100% using nn-reactive slute assumptins), equivalent t mass recvery under diple cnditins, and attenuatin (1 minus RB). RESULTS Results frm the prus media sites (Fig. 1) are given in Table 1. Example respnses are in Figs 2-5. Channelled, preferential pathway respnses were able t be distinguished frm the tracer data (Fig. 2). Transprt rates were high ( m day" 1 based n t an ). Directin was nt cnsistent with gradient at BdF. Particle and slute arrivals (? ait ) were similar ptentially due t lw slute detectin limits. RF values, with ne exceptin, ranged frm 0.2 t 0.9 as shwn by relative abundance (C/C m ax) plts (Figs 2, 3 and 5). The exceptin was in BdF 4 (Fig. 3) with a transprt rate f % O, d"x O C-UR D - UR - - D - - c - H40 ^r D-H40 i Well C URCmax h ; > WeilD~ÛFÏ Cmax "rf I Lg-Lg Plt Bis de Finqes Test 1: Depth: 6 t 12 m Injectin time: 0.2 h Separatin: C ~ 11 m D -22 m Slute: Uranine (UR) Bacteriphage: H40 1 Time since injectin (hr) Fig. 2 Bis de Finges: Test 1 relative abundance Wells 11C, 1 ID.

5 Bacteriphages as particle migratin indicatrs in subsurface envirnments 155 abut 440 m day" 1 and a RF f 1.2. Where different phages were used, their RF values were similar (BdF 2, WW). Hwever, RB differed with phage type by a factr f at BdF fr H40 vs MS2 and by a factr f frm 2.5 t 15 at WW fr H40 vs MS2 and Psf2. Where phages were detected, C/C-max ranged frm 3e" 10 t 4e" 4 accrding t subsurface setting and phage type. 7>/7V-max fr uranine detected ranged frm 5e" 6 t 4e" 3. RB values at WW and BdF were significantly higher than at KAP likely attributable t the reduced xygen envirnment at KAP rather than different 0.1 Time since injectin (hr) Fig. 3 Bis de Finges: Test 4 relative abundance Well 11C. Phage x <x *3 1X3 Well K3.2 ~ 20 m x ; Uranine J cp Xb x x x Time since Injectin (h) Kappelen Test 7; Slute: Uranine (UR) Bacteriphage: H40 1.E-06, 1.E-08 1.E-10 1.E-02 1.E-04 pan CD g 'c 1.E-06 Well K4.2 ~ 21 m "W Time since injectin (h) Uranine 288 Fig. 4 Kappelen: Test 7 dimensinless cncentratin Wells K3.2, K4.2.

6 156 Keith G. Kennedy 100% -SÛT- TJX--''..«) % ' cp, Lg-Lg Plt I K3.2: UR -90 h 10% A K3.2 Phage -27 h "K4.2 Riâgë~?7 h".i K4.2: UR -83 h x CO E 1% / / /O' 0 A \ I? 9 10 Time since injectin (h) 100 Kappelen Test 7: Depth: 4 t 9 m Injectin time: 1.5 h Separatin: - 20 m Slute: Uranine (UR) Bacteriphage: H40 Fig. 5 Kappelen: Test 7 relative abundance Wells K3.2, K4.2. K3.2 UR K4.2 UR --BK3.2 - H40 A-K4.2 H hydrgelgical characteristics. At WW, RB drpped with distance by a factr f frm 12 t 85 ver 50 m in the channel and by the same ver nly 20 m ff the channel. Hydraulic cnductivity and setting equally affected particle attenuatin. At KAP, RB drpped by a factr f 4 ver 40 m. Phage attenuatin ranged frm 28 t 99.99%. Atypical attenuatin ccurred in thin layers (~1 t 2 m) fund during this wrk at bth BdF (<30% at 10 m) and WW (-60 t 85% at 14 m). GAM shear zne (Fig. 1) test results are shwn in Table 2. Example respnses are in Figs 6 and 7. Transprt rates, nrmalized fr induced diple flw rate, were frm 3.6 t 5.4 m day" 1 (based n f arr ). Particle arrival was earlier but is likely similar t the slutes based n extraplated flurmeter sensitivity. RF values were 0.4 and 0.7 in PT2 and 3 and 0.9 in PT1. Phage C ma x always preceded slute 7V max. PT1 (Fig. 6) had an atypical RB f 41 and a C7Cn-max f 20% suggesting an pen path ver the 1.2 m distance. Tests (PT2, PT3 Fig 7) had typically high RBs (99.7, 99.9%) and lw C/C-max (0.04, 0.07%) as expected in a fault guge filled shear zne. Phage C/C-max ranged frm 4e" 3 t 2e" 1. Tr/Tr 0 fr the three slutes used was frm 2e" 1 t 4e" 1. Lwer permeability parts f the shear (PT2, PT3) had reduced diple particle and slute Table 2 GAM shear zne respnse characteristics using bacteriphages. Diple brehles: Flw rates: Slute respnse Bacteriphage respnse: Injectitin used tin tin (m) "(h) (h) (h) Extrac Test Slute injec- Extrac Apart Tr Phage ^arr (h) 'max RF (ml h") (ml h") GAM GAM TP F 95- GAM TP F 95- GAM Uranine e +ul H e naphthina 91 te 3 Sulph- 94 rhdamine C C -, RB (%) Att'n (%) e- 01 MS2 < e e- 0 ' H e

7 Bacteriphages as particle migratin indicatrs in subsurface envirnments % Shear diple test PT1 : Injectin time: 20 h Shear separatin: 1.2 m Flw rate: ml/min Slute: Uranine (UR) Bacteriphage: H40 j Phage H40 recvery 43% 0 Time since injectin (hr) Fig. 6 GAM shear zne: Test PT1 dimensinless cncentratin and mass recvery. f\ Phage \H6 " 58% SRB recvery 0.07% SRB(C/C) SRB (M/M) + H6(C/C) - H6(M/M) Shear diple test PT3: Injectin time: 17.5 h Shear separatin: 1.2 m Flw rate: ml/min Slute: Sulphrhdamine (SRB) Bacteriphage: H6 Phage H6 recvery 09% Q Time since injectin (h) Fig. 7 GAM shear zne: Test PT3 dimensinless cncentratin and mass recvery. capture with ttal recvery varying accrding t diple efficiency and tracers. There was n evident crrelatin between distance r flw rate and either RB r RF. Acknwledgements The authr thanks the Swiss Natinal Research Fundatin (FNS) and Swiss Natinal Nuclear Waste Dispsal Cperative (NAGRA) fr funding

8 158 Keith G. Kennedy and supprt. Critical input and guidance came frm the University f Neuchatel Micrbilgy Labratry (M. Grb, P. Rssi) and Hydrgelgy Centre (I. Miiller, F. Zwahlen, P.-A. Schnegg, M. Schiirch), NAGRA (P. Marschall, T. Baer), Slexperts AG (T. Fierz, T. Trick), and ETH's Water Resurces Management Institute (W. Kinzelbach, S. Niehren). The wrk wuld nt have been pssible t cmplete withut their assistance. REFERENCES Dôrfliger, N. (1996) Advances in karst grundwater prtectin strategy using artificial tracer tests and multi-attribute vulnerability mapping (EPIK methd). PhD, CHYN-Hydrgelgy Centre, University f Neuchatel, Switzerland. Harvey, R., Gerge, L., Smith, R. & LeBlanc, D. (1989) Transprt f micrspheres and indigenus bacteria thrugh a sandy aquifer: results f natural- and frced-gradient tracer experiments. Wat. Resur. Res. 23, Kennedy, K., Niehren, S., Schnegg, P.-A., Rssi, R., Kinzelbach, W. & Miiller, I. (1999) Mdern cllid and slute detectin methds in prus and fractured media labratry and field scale examples. Pster abstract in: Prc. EGS Annual Meeting (The Hague, The Netherlands). Mackay, L., Gillham, R. & Cherry, J. (1993) Field experiments in fractured clay till 2. Slute and cllidal transprt. Wat. Resur. Res. 29, Mackay, L., Sanfrd, W. & Strng, J. (2000) Field-scale migratin f cllidal tracers in a fractured shale saprlite. Grundwater, accepted pending minr revisins. Rssi, P., Dôrfliger, N., Kennedy, K., Miiller, I. & Aragn, M. (1998) Bacteriphages as surface and grund water tracers. J. Hydrl. Earth Sys. Sci. 2(1), Rssi, P. & Kâss, W. (1998) Phages. In: Tracing Technique in Hydrgelgy (ed. by W. Kâss), , Balkema, Rtterdam. Schnegg, P.-A. & Kennedy, K. (1998) A new brehle flurmeter fr duble tracer tests. In: Prceedings Mass Transprt in Fractures, Aquifers and Aquitards (Cpenhagen). University f Cpenhagen, Denmark.

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