Pathogen Pathways − Contamination of water bodies via artificial drainage
3. Methods
3.1 Site description
A research site, to investigate the impacts of intensive dairy farming on the qualities of drainage and surface runoff waters, has been established on a naturally poorly drained Pallic Soil, the Tokomaru silt loam, at Massey University’s No. 4 Dairy Farm, near Palmerston North. The site has a total of 8 plots (each 40 m x 40 m). Each plot has an isolated mole-pipe drainage network, which drains into a central dam. Four of these plots (Plots A, B, C and D) where used for the current study. (Figure 1).
Figure 1: The trial site on No.4 Dairy Farm, Massey University. An example of one of the sampling pits is shown with the dam in the background
At the site of this current study, a sustainable land-based treatment system for farm dairy effluent, called “deferred irrigation”, is also being developed and evaluated. Four plots on the left side of the dam (including Plots A and B; refer to the diagram in Figure 1) are fertilised and grazed according to the farm’s normal management programme, whilst the another four plots (including Plots C and D) on the right side of the dam receive effluent from the aerobic pond in accordance with the “deferred irrigation” scheduling criteria. The plots that receive effluent are also grazed, and have a crop of forage removed annually.
3.2 Rainfall, drainage and runoff volume measurement
At the corner of each plot (Figure 1), a sampling pit has been excavated and a V-notch weir placed at the exit of the pipeline to monitor drainage flow rates and facilitate the sampling of drainage events for subsequent measurements of water quality.
Figure 2: On the left is a view a V-notch weir and Starlog water level recorder for monitoring subsurface drainage flow and on the right is a tipping bucket meter with an Odyssey data recorder for monitoring surface runoff flow
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All pits have been instrumented with data loggers to provide continuous measurements of flow rate. Drainage flow rates are measured using V-notch (22.5o angle) weirs, and either Starlog or Odyssey instruments are used to measure and record water level (Figure 2). Surface runoff flow rates from sub-plots (each 5 m x 10 m) are monitored using a tipping bucket meter (1 litre/tip) and an Odyssey data recorder (Figure 2; image on the right).
3.3 Temperature, rainfall and the soil water relations for experimental period
Soil water balance
The soil water balance was calculated using the method described by Moir et al. (2000). Daily rainfall was measured at the site, and the method of Priestly and Taylor (1972) was used to calculate the daily reference crop evaporation.
Figure 3 shows the calculated soil water balance for the 2003 drainage season at the experimental site. When the soil water deficit is zero the soil is at field capacity and any further rainfall results in drainage or surface runoff. In order to generate drainage and runoff samples for analyses, this study was conducted during a period when soil water deficits were at, or close to, zero. The experimental period for this study (25 September-5 October, 2003) is indicated in Figure 3. It should also be noted that minimum temperatures fell below zero and were close to zero on the mornings following 6 October 2003 when the experiments involving the simulated rainfall events were conducted.
Figure 3: Daily maximum/minimum air temperature, natural rainfall and soil water deficit for the trial site during the 2003 winter drainage season
Rainfall simulation
A rainfall simulator was used to enable drainage samples to be collected at specific time intervals following a grazing event by dairy cows. Data collected from the 2003 drainage season from natural rainfall events provided information on the quantity and intensity of rainfall required to generate surface runoff. This information was used to assist with the design of the rainfall simulator. Figure 4 shows the layout of the rainfall simulator, which consists of 18 Seninger Miniwobbler (No.10) nozzles. A view of the simulator operating is provided in Figure 5.
At each simulation, water was applied to an area up to approximately 200 m2 at an average application depth of approximately 12 mm. The layout of nozzles provided more rainfall in the centre 75 m2, where the application depth was approximately 18 mm. As the rainfall simulator was centred over the 50 m2 surface runoff sub-plots, these sub-plots received applications of 18 mm of water. Observations of earlier natural storm events indicated that “instantaneous” rainfall intensities of approximately 0.5 mm/min, or greater, are required to generate runoff, as long as the soil water deficit is zero. In the current study, each 18 mm simulated rain event was achieved using two applications of 9 mm at an approximate rate of 1.3 mm/min applied about 15 minutes apart.
Figure 4: Schematic diagram (bird’s-eye view) showing the layout of the rainfall simulator
Figure 5: View of the rainfall simulator operating over a surface runoff plot
3.4 Microbiological Methods
Thermotolerant Campylobacter
Enumeration of thermotolerant Campylobacter (hereafter referred to as Campylobacter) was by a two-step most probable number (MPN) technique (le Roux et al., 2001) in which a primary selective enrichment in Exeter broth was followed by a secondary selective enrichment on blood-free modified charcoal cefoperazone deoxycholate agar (mCCDA). Faecal and water (surface runoff and subsurface drainage) samples (1 L) were collected using aseptic technique and were processed for MPN analysis within six hours of collection.
A three-tube (that is, 3 tubes per dilution), ten-fold dilution series was inoculated into Exeter broth. Water aliquots of 1 ml were inoculated directly into the triplicate tubes, whereas 10 ml and 100 ml aliquots were concentrated by filtration through 0.45μm membrane filters that were then transferred to triplicate tubes. Ten gram subsamples of freshly voided faeces were mixed in 90 ml of peptone saline, and 1 ml of each 10-fold dilution was inoculated into triplicate Exeter broths. Inoculated Exeter broths with less than 1 cm headspace were incubated aerobically at 37°C for 4 hours as pre-enrichment, followed by 42°C for a further 44 hours enrichment. A loopful of growth from each enrichment tube was streaked to a parallel mCCDA plate and incubated in a microaerophilic atmosphere for 48 hours of secondary selective enrichment.
The presence of thermotolerant Campylobacter was confirmed by growth at 42°C, colonial morphology, microscopic examination of a wet mount, Gram stain and oxidase test. A positive control C. jejuni was included. Campylobacter isolates obtained were stored at -70°C. Three-tube MPN tables were used to determine the concentration of Campylobacter in the original sample.
Faecal Coliforms
Within 12h of collection, chilled sub-samples of drainage and runoff waters, and faeces were analysed for E. Coli using the Colilert and QuantitrayTM (IDEXX, USA) methods. Trays were incubated at 35°C for 24 hours and E. Coli identified under UV light (366nm). The concentration of E. Coli was determined using MPN tables supplied by the manufacturer.
Giardia and Cryptosporidia
Screening of faecal specimens
Faecal specimens were treated with a formal saline/ether cocktail to remove fats and faecal debris. Oocysts were concentrated along with heavy faecal debris by centrifugation. Immunomagnetic Separation (IMS) of oocysts from the heavy faecal debris was performed using the Dynabead® GC-Combo (Dynal®) kit set as per the manufacturer’s instructions. Paramagnetic polystyrene beads coated with monoclonal antibody specific for G. intestinalis or C. parvum captured the oocysts from the faecal debris. Separation of the bead/oocyst complex from the faecal debris was accomplished by using a magnet.
Fifty microlitre aliquots of sample were spotted onto painted slides and allowed to air dry. Positive and negative controls were included. The slides were fixed in acetone for ten minutes before staining with fluorescein conjugated monoclonal antibody (CellLabs) to Giardia and Cryptosporidium. One drop of counterstain (Eriochrome Black solution) was also added to each well and the slide incubated at room temperature in the dark for 30 minutes. The slides were briefly washed in PBS pH 7.4 before a drop of 0.4 mg/mL solution of DAPI stain was added to each well and incubated for 1 minute at room temperature. PBS was used to wash the slides before mounting and examination with an epifluorescent microscope under UV-light. The number of Giardia cysts and Cryptosporidium oocysts were recorded and the results expressed as number per 100 L.
Faecal specimens were screened for Giardia and Cryptosporidium oocysts using Fluoroscein isothiocyanate (FITC)-labelled monoclonal antibody and examined with epifluorescent microscopy. Each specimen was graded on a scale from one to three, with a zero score representing no oocysts detected and the value three representing greater than 10 oocysts per field of view at 250 x magnification.
Processing of water samples
Water sample collection and filter processing
Between 24 and 150 litres of drainage water was collected from V-notched weirs and filtered through a yarn-wound polypropylene cartridge Micro-Wynd II DPPPY-1 filter (Cunno Filter System). The filter was cut lengthways, the plastic core removed, the filter fibres shredded and placed into a stomacher bag. Eluting solution was added to the stomacher bag and the fibres homogenised for two five-minute intervals. The filter fibres were rung by hand to express as much of the liquid as possible before discarding. The filter eluent was centrifuged at 4,150 rpm for 10 minutes using a Sorvall RC C3 Plus centrifuge fitted with a 6000A swing out rotor. The supernatant was discarded, the pellet resuspended with eluting solution, transferred to a 50 mL conical centrifuge tube and made to a final volume of 50 mL. The sample was further centrifuged for 10 minutes at 1,050G using a Sorvall RT7 centrifuge. The supernatant was discarded and the pellet was suspended to give a total volume of 1 mL.
Sample purification
One hundred microlitres of 10X SL-buffer-A and 100 mL of 10X SL-buffer-B was added to the 1 mL filter concentrate and a 20 mL suspension of immunomagnetic beads (DynabeadsÒ GC-Combo) was added. The sample tube was rotated for one hour (approximately eight revolutions per minute) at room temperature using a mixer (Barnstead-Thermolyne LabQuake). A MPC-1 magnetic separator was used to isolate the bead-oocysts complex by placing the tube in the separator. The separator was gently rocked by hand end-to-end through approximately 90o for two minutes with approximately one tilt per second. The supernatant was discarded and the pellet +resuspend in 1 mL of 1X SL-buffer-A. This wash was repeated twice using the magnetic separator and 1X SL-buffer-A. The supernatant was discarded, the pellet resuspended in 50 mL of 0.1M HCl, vortexed for 10 seconds and allowed to dissociate from the beads for ten minutes. The MPC-1 separator was used to separate the beads from the oocysts which were collected. Five microlitres of a 1.0M NaOH was added to the oocysts suspension to neutralise the acid.
Sample staining
Fifty microlitre aliquots of sample were spotted onto painted slides and allowed to air dry. Positive and negative controls were included. The slides were fixed in acetone for ten minutes before staining with fluorescein conjugated monoclonal antibody (CellLabs) to Giardia and Cryptosporidium. One drop of counterstain (Eriochrome Black solution) was also added to each well and the slide incubated at room temperature in the dark for 30 minutes. The slides were briefly washed in PBS pH 7.4 before a drop of 0.4 mg/mL solution of DAPI stain was added to each well and incubated for 1 minute at room temperature. PBS was used to wash the slides before mounting and examination with an epifluorescent microscope under UV-light. The numbers of Giardia cysts and Cryptosporidium oocyst were recorded and the results expressed as number of per 100 L.
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