What's in the Water?

What’s in the water? Links between irrigation water quality and food safety. Presented by Dr Floris van Ogtrop Faculty of Science Sydney Institute of Agriculture Problem

• We know - Irrigation water is potentially a source of food-borne pathogens on fresh produce

• We don’t (always) know – when and where food-borne pathogens are present in Australia’s agricultural water resources

• We need to identify: • pathogen presence in irrigation source waters • environmental conditions associated with increased chance of finding pathogens in source waters Disease in Pathogens humans & released in animals environment

The Irrigation Contamination Pathogens Pathogens in on crops Problem water

Irrigation Outbreaks potentially linked to irrigation water

Two US examples where irrigation water implicated

• US: 2006, E. coli in fresh bagged spinach (Gelting et al., 2011) • Outbreak strain identified in river water • Wet conditions lead to increased recharge to groundwater and potentially facilitated transfer of contamination into ground water • US: 2018, E. coli in fresh Cos Lettuce Gelting, R.J., Baloch, M.A., Zarate- Bermudez, M.A. and Selman, C., 2011. Irrigation water issues potentially • Outbreak strain identified in river water related to the 2006 multistate E. coli O157: H7 outbreak associated with • Speculation that source was from stirred up sediment spinach. Agricultural Water but more likely from feedlots Management, 98(9), pp.1395-1402. Source of contamination is still highly speculative Outbreaks potentially linked to irrigation water

• Australian outbreaks suspected to be linked to water . 2016, salad greens? . 2006, 2016, rockmelons? •  No published direct link of outbreak to irrigation water though pathogenic Salmonella

spp. were identified (Munnoch et al., 2009) Munnoch, S.A., Ward, K., Sheridan, S., Fitzsimmons, G.J., Shadbolt, C.T., Piispanen, J.P., Wang, Q., Ward, T.J., Worgan, T.L.M., Oxenford, C. and Musto, J.A., 2009. A multi-state outbreak of Salmonella Saintpaul in Australia associated with cantaloupe consumption. Epidemiology & Infection, 137(3), pp.367-374. Need more data to help focus industry efforts Challenges

• Water is transient/flowing/dispersive • Water is a vehicle for transfer rather than reservoir • Very hard to trace back pathogens to a water source What are we doing?

1. Characterising the diversity of microbial populations in irrigation water and sediment sources 2. Identifying associated probabilities of finding pathogens/toxins, to provide guidance for the fresh produce industry 3. Lab experiments to understand processes occurring in the field Bacteria Algae Fungi Which microbes are found in irrigation

Cyanobacteriawater sources? Protozoa Monitor Microbial Populations

• Spatial sampling of water and sediment . Watersheds Upper Hawkesbury River Werribee Others? . Water types – source water Rivers, tributaries, on-farm dams, others • Upstream/downstream of agricultural activities, up to on- farm level Monitor Microbial Populations

• Monitoring over time Seasonal changes Rainfall events • Environmental drivers of microbial contamination vary through time • Irrigation applications not consistent through year Hawkesbury Water Sampling

1. Sites are a mix of water sources: Dams, main channel, river,… 2. Number of sites increasing as local growers and other water users get involved Sample Processing - Lab

5L of sample +

Al(SO4)2

Colilert DNA Extraction Kit Sample Processing - Field

• Water quality variables measured with YSI EXO2 WQ sonde at point of sampling and confirmed in lab

• Data also acquired from councils, government, and non-government organisations Summary - Environmental variables

Microbial Water quality Environmental Site characteristics populations parameters data Bacteria Temperature Streamflow Coordinates Cyanobacteria pH Rainfall Catchment Protozoa Turbidity Air temperature Water source Algae Salinity Relative humidity Site survey Fungi Dissolved oxygen Solar radiation fDOM Chlorophyll a Phycocyanin Summary - Quantification Methods

• Colilert • Semi-quantitative, coliforms (yellow) and E. coli (fluorescent) • Fluorescing sample processed through DNA extraction kit to determine if pathogenic • PCR/qPCR • Targeting specific pathogens, multiplex • E. coli genes (from Colilert) • Protozoa: Cryptosporidium, Giardia, Cyclospora • NGS • 16S sequencing at WSU facility • Overall ecology, diversity of system Identify Influences & Determine Correlations

• Integrate data: . Water and sediment sampling . Industry data . Government monitoring programs • Statistical correlations • Development of model based on data Results – March, June 2018 Colilert Results –2018 – Water E. coli CFU/100ml Presumptive Salmonella spp.

March June September September September Site Type Water Sediment Bardenerang Tributary 46.5 - Brewers Storage 193.5 435.2 55.6 3.1 + Grose River 21.1 1.0 + Hanna River 131.7 517.2 648.8 4.0 + Inalls Storage 48.8 248.9 54.8 <1 - Longneck Wetland 435.2 <1 + Nepean River 115.3 16.1 30.1 2.0 - Pitt Wetland 95.9 770.1 + Punt River 22.3 65.7 36.4 15.8 + Screek River 14.6 34.1 29.2 3.1 + WSU Storage 107.6 137.6 9.7 5.2 + YarraL Storage 56.5 12.8 + Next Steps

• Continue sampling Hawkesbury region to cover entire growing year • More intense sampling over summer period • Identify additional on-farm storages to sample • Other irrigation regions? • Begin collecting data from external organisations • Sydney Water, SunWater, local councils, growers • Report on preliminary results • 16S sequencing of samples collected so far Sediments as reservoirs for pathogens – Lab study

• Prolong survival in sediment compared to water • Viruses: <1 day to 1 week for viruses7 • Bacteria: Days to multiple weeks4 • Dependent on water/sediment quality parameters Sediments as reservoirs for pathogens

• Evaluate the effect of varying flow rates on re-suspension of surrogate organisms from sediments into overlying irrigation water • Clay and sand sediments • MS2 as a surrogate for enteric viruses • E. coli surrogate for enteric bacteria • Varying flow rates • Determine if re-suspension of organisms is a potential public health problem Methods

• Characterize the re-suspension of viral and bacterial surrogates from sediments into overlying water • Simulated irrigation canal using a flume (1.6 m x 0.15 m) • Clay and sand sediments • Varying flow rates: 0.41, 0.73 and 1.46 liters per second (L/s) Flume Results – PFU/CFU per ml resuspension

• E. coli in sand sediments and clay sediments • E. coli in clay sediment was a significantly greater concentration per mL than E. coli in sand sediment at the highest flow rate • Varying flow rates • MS2 at 1.46 L/s had significantly greater PFU/mL than 0.41 L/s • E. coli at 1.46 L/s in clay sediments had significantly greater CFU/mL than 0.73 L/s Conclusions – what does this mean for you?

• Relating this to sediments in irrigation canals • Large particle sizes (gravel and sand) characterized in grab samples from canals in US versus finer sediments found in Australia • More force needed to re-suspend particles and particle-associated pathogens in course versus fine sediments Sediments as reservoirs for pathogens – Field studies

Positive coliform QuantiTray Salmonella spp.? Results –2018 – Sediment

E. coli CFU/100ml Presumptive Salmonella spp.

• Initial testing indicates E. coli and presumptive Salmonella spp.

• Further testing to confirm “bad” E. coli and Salmonella spp. present

• Monthly testing to determine whether seasonal variation Recommendations - sediment

• Increased water sampling after: • Canal dredging • Heavy rainfall/high wind • Events that would disturb bed sediments

• Field-scale evaluation of re-suspension from sediments • Larger scale study will provide a better understanding of the dynamics in the field

• Flume experiments using high speed video/capture of fluorescent E. coli Overall recommendations - “know your catchment” • Some questions to ask when thinking about water quality • Where’s you water coming from? • Where are potential sources of contamination? • What time of year is the likelihood of contamination highest? • How often should you sample, Hipsey, M.R. and Brookes, J.D., 2013. Pathogen management in surface waters: where and when? practical considerations for reducing public health risk. In Current Topics in Public Health. InTech. Bringing it all together

• Irrigation water quality drivers: Field studies . Rainfall . Temperature . Salinity Lab studies • Compliance with regulations: . FSMA: 126 E. coli cfu / 100mL Local knowledge . Irrigation recommendation • Australian potential: . Freshcare: 100 E. coli cfu / 100mL Thanks

• Project team • Emily White • Dr. Hannah Sassi • Dr. Katarzyna Safianowicz • Prof. Robyn McConchie