SURVIVAL OF VIRUSES IN CARSTIC FRACTURED ROCK ENVIRONMENT

Mihael Bricelj

HUMAN HEALTH HAZARDOUS VIRUSES

virus type family disease

Norwalk virus (NV)*** Calciviridae acute epidemic gastroenteritis

Norwalk-like viruses*** Calciviridae acute epidemic gastroenteritis

Sapporo virus (SV) Calciviridae gastrointestinal disturbances

Hepatitis A (HAV)** Picornaviride hepatitis

Hepatitis E (HEV)* »HEV-similar viruses« hepatitis

Rotaviruses (RV)** Reoviridae gastrointestinal disturbances

Echovirus (EV) Picornaviridae gastrointestinal disturbances

Enteroviruses (HEV-Nr.)** Picornaviridae gastrointestinal disturbances

Poliovirus (PV) Picornaviridae infantile paralysis

*in undeveloped countries 1-2% mortality in population and 30% mortality of pregnant women

** various virus strains resistant to chlorinated water*** these viruses cause yearly only in USA disease symptoms among 23 to 40 millions

inhabitants. about 50.000 hospitalizations and death of 310 pacients

Survival of some enteric viruses in tap water

0 50 100 150 200 250 300 350

days (2 log10 reduction)

4 oC20 - 23 oC

HepatitisA virus

Poliovirus

Adenovirus 41

Adenovirus 40

Astrovirus

Survival of some enteric viruses on surfaces (aluminium)

0 10 20 30 40 50 60 70days (2 log10 reduction)

20 - 23 oC4 oC

Adenovirus 41

HumanRotavirus

Poliovirus

Hepatitis Avirus

400 – 800nm

• Because of very special propagation of bacterial viruses, within the host bacteria, bacteriophages represent a valuable tracer in underground tracing experiments. In the environment they act as an inert complex macromolecule, build-up mostly of DNA and protein envelope, with special morphology and size between 400 and 800 nm. They are relatively stable in environment and survive for a long time.

BACTERIOPHAGE TRACER

• P22H5 virulent mutant of • Salmonella typhimurium strain LT2 • (NIB 22)

• ... because the phages of Salmonella typhimurium

• had been rarely encountered in surface waters (N.D SEELEY & S.B. PRIMROSE, 1982).

injection point sampl. point distancem

Tgh

vtgm/day

Recovery%

Kapsia Kiveri 39,000 233.0 4,017 -

Smokovskavala

Rižana 3,880 348.0 268 0.006

Hotična Rižana 12,450 482.0 620 4.5

Lurbach Hammerbach 3,000 53.4 1,348 2.5

Kačji potok Radeščica 19,200 511.4 901 3.0

Kačji potok Obrh 20,000 704.6 681 -

Bajer Krupa 6,000 99.1 1,453 -

Vrčice Krupa 6,000 156.8 818 -

Movražkavala

Ara 800 80.8 237 -

Movražhavala

Mlini 1,000 94.6 165 3.4

Movražkavala

Sopot 1,043 145.1 173 -

Subsurface injection of bacteriophageto usaturated zone

Tracer experiment a avp av. Qinj tg vtg recovery

OtlicaTrnovski gozd

mm m3 sec-1 h m/day %

October 1993 1.3 x 102 15.4 2.81 94.7 253.9 0.780

April 1994 8.3 x 103 11.8 6.33 99.6 241.4 0.012

August 1995 1.0 x 105 2.8 0.51 678.2 35.5 0.007

400 – 800nm

Decimal diluting test tubes (FS – Na Cl 0,85%)

Sample10-2 10-4 10-6 10-8 10-9 10-10

Test tubewith soft

agar contains

3 ml of soft agar0.2 ml host bacterium0.1 ml sample (directlyor from dilution)

Mixing

Petri dish

plaques

KAPSIA - KIVERI

0

1

2

3

4

5

6

0 50 100 150 200 250 300 350 400 450

hours

pfu

ml-1

231232233hoursConcent.sampleMPNAgar overlaytg

Kiveri39 kmKapsia1. April 19843.7 x 10149.38 L

SpringDistanceSinkholeDate ofinjection

Number ofparticles

Injectedquantity

tg

0

5

10

15

20

25

30

35

0 100 200 300 400 500 600

time from injection (h)

phag

e (p

fu/m

l)

0

5

10

15

20

25

30

35

Q (l

/s)

phage tg waterflow

Tracing curve of phage P22H5 appearance in spring of Hubelj in octobre 1993

EXPERIMENTAL FIELDEXPERIMENTAL FIELD SITESITE SINJI VRHSINJI VRH

EXPERIMENTAL FIELD SITEEXPERIMENTAL FIELD SITE SINJI VRHSINJI VRH

EXPERIMENTAL FIELD SITEEXPERIMENTAL FIELD SITE SINJI VRHSINJI VRH

Tracer Amount Solutionlithium chloride 300 g

zinc sulphate 250 g

sulfonic acid 10 mg

pyranine 10 g

naphthionate 20 g

uranine 1.5 g

sulforodamine B 5 g

microspheres 10 mL

potassium chloride 300 g 1.0 L

deuterium (90 %) ~ 0.2 L

bacteriophage P22H5 1.2 x 1015 pfu 4.0 L

rinsing water for the several tracers 2.2 L

Σ 10.4 L

3.0 L

EXPERIMENTAL FIELD SITEEXPERIMENTAL FIELD SITE SINJI VRHSINJI VRH

• 10 segments• l=1.5 m • A=2.2 m2

system for water samplingsystem for water samplingplastic tilt

2.2m2

1.5m

EXPERIMENTAL FIELD SITEEXPERIMENTAL FIELD SITE SINJI VRHSINJI VRH

SP3 - P22H5 bacteriophage

1,0E+00

1,0E+01

1,0E+02

1,0E+03

1,0E+04

1,0E+0526.9.03 4.1.04 13.4.04 22.7.04 30.10.04 7.2.05 18.5.05 26.8.05

date

pfu

ml-1

P22H5

SP6 - P22H5 bacteriophage

1,0E-01

1,0E+00

1,0E+01

1,0E+02

1,0E+03

1,0E+04

1,0E+0526.9.03 4.1.04 13.4.04 22.7.04 30.10.04 7.2.05 18.5.05 26.8.05

days

pfu

ml-1

P22H5

Bacteriophage P22H5 - Sampling point 4

1,0E-01

1,0E+00

1,0E+01

1,0E+02

1,0E+03

1,0E+04

1,0E+05

1,0E+06

1,0E+07

1,0E+08

1,0E+0928.6.03 14.1.04 1.8.04 17.2.05 5.9.05 24.3.06 10.10.06 28.4.07 14.11.07

time (days)

p.f.u

./ml

Bacteriophage P22H5 - Sampling point 5

1,0E+00

1,0E+01

1,0E+02

1,0E+03

1,0E+04

1,0E+05

1,0E+06

1,0E+07

1,0E+08

1,0E+0928.6.03 14.1.04 1.8.04 17.2.05 5.9.05 24.3.06 10.10.06 28.4.07 14.11.07

time/days

p.f.u

./ml

Bacteriophage P22H5 on SP 4 and 5

1,0E-01

1,0E+00

1,0E+01

1,0E+02

1,0E+03

1,0E+04

1,0E+05

1,0E+06

1,0E+07

1,0E+08

1,0E+09

1,0E+10

26.9.03 13.4.04 30.10.04 18.5.05 4.12.05 22.6.06 8.1.07 27.7.07 12.2.08

time (days)

p.f.u

./ml

Sampling point 4 Sampling point 5

Sampling point Appearance of tracer (days)

Max. value inpfu/ml

Appear. of max. value (days)

Last + result

1 7.0 2.1E+03 11.1 64

2 7.0 2.9E+02 7.0 648

3 22.1 5.7E+02 50.0 1261

4 5.0 1.1E+08 5.0 1482

5 4.1 3.1E+09 4.1 1482

6 8.0 4.3E+04 11.1 676

7 13.4 1.4E+04 30.9 550

8 7.0 6.5E+04 7.0 550

9 22.1 1.7E+03 24.9 21410 8.0 4.9E+03 40.0 178

at 07.11.2007 (1482 days after injection) ( SP 4 ) ( SP5 )pfu ml-1 8.0E-01 3.3E+00

0 200 400 600 800 1000 1200 1400 1600

days

1

2

3

4

5

6

7

8

9

10

11

12

sam

plin

g po

ints

The appearance of phage P22H5

WRL - 14WLL - 9

SP10 - 178

SP 9 - 214

SP 8 - 550

SP 7 - 550

SP 6 - 676

SP 5 - 1482

SP 4 - 1482

SP 2 - 648

SP 3 - 1261

SP 1 - 64

CONCLUSIONS

BACTERIOPHAGES ARE APPROPRIATE PARTICULATE TRACERS FOR RESEARCH OF POLLUTANT TRANSPORT IN FRACTURED AND KARSTIFIED ROCKS

DATA OF COLUMN EXPERIMENTS WITH VARIOUS SUBSTRATES IN LABORATORY CONDITIONS CONFIRM THAT SOME HUMAN HEALTH HAZARDOUS VIRUSES, APPEARING IN POLLUTED WATER, ARE EQUALLY SENSITIVE TO ENVIRONMENTAL FACTORS AS BACTERIOPHAGES

COMPARATIVELY LONG-CONTINUED TIME OF BACTERIOPHAGE SURVIVAL IN FRACTURED AND KARSTIFIED ROCKS (> 4 YEARS) PROVE THAT THE KARST ENVIRONMENT COULD BE VERY VULNERABLE FOR POLLUTION WITH HUMAN HEALTH HAZARDOUS VIRUSES

THANK YOU!