# Presentation: 530811

## Environmental factors and chemical and microbiological constituents related to the presence of viruses in ground water from small public water supplies in southeastern Michigan

- In cooperation with USEPA, Office of Research and Development
- Donna Francy, Rebecca Bushon, Emma Luzano — USGS Columbus
- Julie Stopar — USGS Michigan
- G. Shay Fout — USEPA

## OBJECTIVES

- Determine (in small public GW supplies)
- occurrence of viral pathogens and indicators
- adequacy of indicators as predictors for presence of viruses
- factors that affect the presence of viruses and indicators

**Notes:**
Viruses may be more of a risk to GW than other microorganisms because of their 
small size and persistence in the environment.
Small supplies are not regulated like larger supplies. (serve >25 and <3,300 people) 
Sampling is done less frequently.
Occurrence—same frequency as that found in other studies??
Factors--geology of overlying deposits, land use, well depth, 
population density, distance to septic or animal source, 
water chemistry

## APPROACH

-    38 drinking-water wells in glacial aquifer in Michigan
-    Each well was sampled 1 to 5 times over a  2-year period

**Notes:**
Community, non-community wells

## APPROACH

- Water-quality samples were collected once per well
**majors, nutrients, boron, DO**

- For viruses, we  filtered 2,000 liters of GW

## Viruses by

**Viruses by**
**cell culture**

**Viruses by RT-PCR **
** ****Enterovirus**
** ****Reovirus**
** ****Rotavirus**
** ****Hepatitis A**
** ****Norwalk **

**Notes:**
Buffalo Green Monkey Kidney cells
At first we used a 2 multiplex reactions for 5 enteric viruses.  Because of
 QC failure, switched to single reactions and only enterovirus and hepatitis A.

## BACTERIAL INDICATOR METHODS

- Ultraviolet wavelength

- Visible spectrum

**Total coliforms,**
***E. coli, *****and**
**enterococci**

## COLIPHAGE BY TWO METHODS

- Presence/absence method

**Notes:**
IPTG induces the enzyme in the host E. coli, -galactosidase, while X-gal acts 
as a substrate for the enzyme. Once the phage infect the host, the host cell is 
lysed and a stable indolyl product of X-gal that is dark blue is visible within 
each plaque. 

## RT-PCR QUALITY CONTROL RESULTS

- 169 regular and 32 replicate spikes
- Of 32 replicate pairs for HAV, four were not in agreement
- 3 were negative in one replicate and inconclusive in the other
- 1 was positive in one replicate and negative in the other
- Eight types of QC samples

**Notes:**
Shows that results are not always reproducible, even samples collected at the 
same well on the same date.  
Low concentrations of viruses in ground water or intermittent occurrence of 
viruses in GW

## RT-PCR RESULTS (by sample)

## BACTERIAL INDICATORS AND CULTURABLE VIRUSES

## COLIPHAGE

## VIRUSES AND INDICATORS (sample results)

- Only 2 out of 11 virus-positive samples were also positive for an indicator
- 1 had culturable viruses _and_ enterococci
- 1 had enterovirus by RT-PCR _and_ total coliforms

## VIRUSES AND INDICATORS(well results)

- 9 wells were positive for viruses
- 2 wells had enterovirus and HAV on different sampling days
- Viruses positive wells with indicators
- 2 – total coliforms
- 1 – *E. coli*
- 1 – enterococci
- 1 – F-specific coliphage

## CATEGORICAL VARIABLES

- Sampling condition– GW or GW and tank
- Sewage-system type
- Distance to Septic
- Type of nearest surface water
- Nitrate plus nitrite
- Ammonia plus organic nitrogen
- Chloride
- Chloride to bromide ratios

**Notes:**
How well are related to the presence of total coliforms, other indicators, or enteric viruses

## Sewerage-system type

- p = 0.088

**Notes:**
Not significant for total coliforms and other indicators
One sided Fisher’s exact test.

## Presence of nitrate plus nitrite

- p = 0.086

**Notes:**
Also significant for other indicators, but not total coliforms
Chloride related to total colifiorms
Chloride/bromide ratios related to TC and viruses

## CONTINUOUS VARIABLES

**Notes:**
ORANGE are significantly related to TC at alpha = 0.1

## Iron concentrations

- p = 0.062

- p = 0.121

**Notes:**
For viruses, the p value was 0.722

## CONCLUSIONS

- Results on viruses in GW are not reproducible
- Viruses were found in 9 of the 38 wells sampled (23.7 percent)
- Repeat sampling at the well is needed
- Indicators were fair predictors of the presence of viruses in wells (5/9), but not in samples (2/11)
- Related variables—sewerage system type, nitrate plus nitrite, chloride, DOC, iron

**Notes:**
Even samples collected at the same well on the same day

## Donna FrancyU.S. Geological SurveyColumbus, Ohio

- dsfrancy@usgs.gov
- 614/430-7769