Sampling methods

2.1 Soils in-situ

2.1.1 Collection of soil samples

The goal in collecting soil samples is to provide a small volume of soil for analysis that is representative of the whole volume of soil within the area of interest. There are a number of different ways to accomplish this goal. This section describes the minimum sampling requirements for information purposes and outlines some of the more detailed techniques.

To be representative of the area of interest, the sample is comprised of a number of sub-samples. The sub-samples must include the depth of soil that would normally be tilled. For most nutrients and other regulated parameters this depth is approximately 15 centimeters (6 inches). This depth is appropriate even where no tillage is used, as it represents the part of the soil where most roots are present and most nutrient absorption occurs. The exception to this are the soil nitrate samples, which are collected at a depth of 30 cm (12 inches) to include nitrate which may have been leached from the surface soil to the lower part of the rooting zone. To collect sub-samples at the proper depth, the easiest equipment to use is a sampling tube or auger, which is simply inserted to the proper depth and then removed, bringing the sample with it. A shovel or spade can be used instead, but it is more labour intensive than a sampling tube or auger, and it is much more difficult to keep each sub-sample of uniform size.

The pattern of sub-sample collection must provide a representative sample of the entire area. It means: 1) sufficient sub-samples (e.g. cores) must be collected, 2) the sub-samples must be collected so as not to introduce bias into the representative sample, 3) the area being sampled must be reasonably uniform, and 4) the area being sampled must not be too large.

2.1.2 Number of sub-samples

There is significant small scale variation in the nutrient content of soil, so a relatively large number of sub-samples must be collected to provide an accurate average value. In small fields (less than 5 hectares, 12.5 acres) collect a minimum of twenty sub-samples (about 50 - 75 cm³ each), and mix them together thoroughly to produce the composite sample that is to be submitted for analysis. In larger fields (more than 5 hectares, 12.5 acres), at least two additional cores must be taken for each additional hectare (2.5 acres).

2.1.3 Avoiding bias

In areas that have been farmed, the largest source of variation in soil nutrient content is the past management of the field, so any sampling design must take this into account. Any areas that have received different nutrient applications in the past, if this is known, should be sampled separately. This includes areas where manure, fertilizer or lime have been stockpiled, laneways, and old exercise yards. These areas may have much higher nutrient contents than the field as a whole. Also avoid sampling dead furrows, as the sample would include a significant portion of subsoil which is normally much lower in nutrient content. Other factors that could affect the nutrient content include topography, soil texture, erosion, or large variations in crop yield.

2.1.4 Maximum field size

Even in fields that appear uniform, there can be significant variation in nutrient content. Normally, the largest area that can be represented by a single sample is ten hectares (25 acres). Larger fields should be divided, where possible, according to previous field boundaries. Failing this, divisions should be made according to distance from existing or previous barns or manure storages, or according to topography or soil type.

A single representative composite sample for fields larger than 10 hectares, up to a maximum of 20 hectares (50 acres), may be permitted where there is evidence to show that the nutrient content of the field is uniform (within 15% of the mean value), and that the management of the field is uniform.

Where samples for metal analysis must be collected (for example, where application of sewage biosolids is planned), a single representative composite sample for a field size of up to 40 hectares (100 acres) is permitted.

2.1.5 Sample collection

The best way to ensure a representative sample of the field is to traverse the field in a zig-zag pattern, collecting grab or sub-samples (e.g., cores) randomly from the entire field area. Take separate samples for analysis from any parts of the field that differ markedly in appearance of the soil or the crop. Clean equipment between collecting different samples to avoid cross-contamination (see Section 2.5).

In some situations, particularly for high value crops, more intensive sampling techniques are used. If this practice is used, each sampling area should be treated as a small field for the purposes of defining any application limitations. Further information on sampling techniques can be found in OMAFRA Publications 611 (Soil Fertility Handbook) and 811 (Agronomy Guide for Field Crops).

Collect the soil sub-samples in a clean pail or other suitable clean container, break up the lumps and mix the soil well. Place a sub-sample of the soil in an appropriate, properly labeled container for shipment to the accredited analytical laboratory.

Where non-agricultural source materials are to be applied to soils, it is recommended that the sampling locations be geo-referenced. At a minimum, this means providing the GPS co-ordinates of one corner of the field, and the distance and direction of the area encompassed by the samples. While co-ordinates for each sub-sample are not required, this can be included with the sample information.

2.1.6 Sample handling

Once a representative soil sample has been taken, it may be necessary to fill a variety of analysis - specific containers for shipping to a laboratory. For example, samples to be analyzed for trace contaminants will require special handling and containers that are specific to each contaminant. The laboratory will provide information on the handling requirements for trace contaminants. The receiving laboratory may have a preference for the type of container, for example, plastic bags or plastic lined paper are generally favoured because they keep each sample separate from the other, and prevent moisture from soaking any information sheets included with the samples.

No special handling is required for samples to be analyzed for pH, metals, or most nutrients. It is desirable to air dry the samples if they are to be held for a long period of time. The samples should be held in a cool, dry location. The exception to this is samples to be analyzed for nitrate nitrogen, which must be cooled to below 10°C (preferably to below 4°C), and kept cool until analysis. If the samples for nitrate nitrogen are stored at room temperature, nitrification can occur within the sample and distort the analytical results.

2.2 Sampling liquid prescribed materials in storage tanks, storage

Lagoons and stabilization ponds

This section applies to sampling liquid prescribed materials for nutrients and material quality. In the case of NASM, these sections also apply to sampling for pathogens where the sample location recommended is immediately after the treatment process. For pathogen testing, special instructions regarding containers, additional sample handling and shipping requirements are described in section 2.5.2.

2.2.1 Requirements for agitated and non-agitated facilities

Liquid materials pose special challenges in collecting representative samples. Most of the liquid materials considered for application to land are suspensions rather than true liquids, and tend to settle into layers of varying density and nutrient content. Where distinct layers are to be applied to land in separate applications (for example, the supernatant and the settled solids from a lagoon), each layer must be sampled separately. Alternatively, if all layers are to be applied at the same time, the material must be agitated prior to application to produce a relatively uniform mixture. The following provides a summary of the different approaches required when sampling from a storage tank or lagoon that is agitated and from a facility that is not agitated.

2.2.1.1 Agitated storage tanks and lagoons

Agitation can be carried out using a mechanical mixing device, aeration device or using a hauling truck’s pumping system. It must provide a well mixed material for sampling where the solids concentration is homogeneous throughout the material to be sampled.

Agitation of a storage tank or lagoon can be carried out on a continuous basis, or only done prior to sampling. When used prior to sampling, agitation must be carried out for a period of time long enough to result in homogeneous mixture of material for sampling.

For large storage lagoons, agitation can be provided at the outlet section of the lagoon only, provided the volume agitated is at least equal to the volume to be removed between sampling events.

2.2.1.2 Non-agitated storage tanks and lagoons

Non-agitated storage tanks or lagoons will likely have at least two distinct layers (settled solids and supernatant). Special care is required to obtain representative samples from materials that have stratified.

When sampling from a tank or lagoon with at least two layers, and where each layer is to be land applied separately, separate samples must be taken and analyzed for each layer to be land applied. The depth of each layer must be determined before sampling to identify the sampling depth. The depth can be determined using a weighted tape measure, probe line or other suitable measuring device.

When sampling from a tank or lagoon with at least two layers where the layers will be land applied simultaneously, the sample taken must have a representative volume from each layer to be land applied.

2.2.1.3 Stabilization ponds

For stabilization ponds where the settled solids are to be land applied, samples of the settled solids must be taken before land application. The samples taken should be representative of the material to be removed for land application.

2.2.2 Sample number and size

A minimum of five sub-samples must be collected and composited for each storage facility for each sampling event. The number of sub-samples required is based on the volume removed for land application per sampling period. Table 2-1 provides a summary of the minimum number of sub-samples required per composite sample.

Table 2-1. Minimum Number of Sub-Samples per Composite for Different - Land Application Volumes

Normally one 500 mL sample bottle will suffice for nitrogen, phosphorus and total solids analyses. An additional 500 mL sample bottle, which can be taken from the mixture already collected, is required for metals analyses when these are required. Separate bottles are required for mercury and pathogen analyses. Sample bottles must not be filled more than 1/2 to 2/3 full, so that there is enough headspace in the bottle to allow for the build-up of pressure and prevent bursting. For additional guidance on sampling for pathogens see Section 2.5.2.

2.2.3 Sampling locations

There are five possible types of sampling locations for liquid prescribed materials:

• as the material is being loaded onto the hauling or application equipment;
• from an open storage tank;
• from an enclosed storage tank;
• from a storage lagoon; and,
• from a stabilization pond.

The following provides information on the requirements for sampling from the different locations. Section 2.2.4 provides information on the sampling equipment and methods that can be used at each sampling location.

2.2.3.1 Sampling at the hauling or other application equipment

Samples of liquids can readily be taken from taps or valves situated on pipe-work from the storage facility to a truck or other application equipment or from a tap on the truck or application equipment. Where this option is available and enables a representative sample to be taken, the pipe-work should be flushed clear of static or stagnant material prior to taking a sample. The period of time required to flush the pipe-work will depend on the length and diameter of the pipe, the available “head” (i.e. the pressure exerted by the bulk of material stored above the sample line) and, if a pump is fitted, the pump rate.

The minimum number of sub-samples required should be taken evenly spaced out over the time that the material is being transferred to the hauling or other application equipment. Only one sub-sample per load should be taken.

2.2.3.2 Sampling from enclosed storage tanks

If the tank is agitated and has a sampling line, samples can be taken from the sampling line. The line should be flushed prior to taking a sample to remove any static or stagnant material.

Sampling material in a non-agitated enclosed tank should be carried out from at least one sampling port (or hatch) at the top of the tank. If more than one hatch is available, samples should also be taken from these locations. Sampling locations should not be located close to inflow pipes or other inlets.

Sampling material from a sampling port at the top of the tank may be hazardous, depending on the material stored in the tank and the potential for toxic or explosive vapours in the headspace of the tank. Before proceeding to sample from the top of an enclosed tank, sampling personnel should follow all appropriate health and safety procedures, including, but not limited to, the following:

• review all information concerning the tank, such as the type and capacity of tank, condition of tank, and known/suspected contents;
• inspect the ladder, stairs, catwalk or other structure to be used to access the sampling port to ensure that they will support the person(s) doing the sampling;
• review all safety procedures and emergency contingency plans with regard to potential toxic or explosive vapours in the tank headspace;
• if the tank is metal, ensure that the tank is properly grounded; and
• remove all sources of ignition from the immediate area.

Where toxic or explosive vapours are likely to be present in the tank headspace, air quality measurements should be taken and sampling from the top of the tank should only proceed if the readings meet acceptable air quality standards. In addition, before sampling commences, the tank headspace should be cleared of any toxic or explosive vapours using a high volume explosion proof blower.

If there is the potential that sampling from these types of facilities requires a confined space entry, appropriate health and safety measures must be followed and training may be required. For further information on health and safety information related to sampling from enclosed tanks, refer to the Ontario Ministry of Labour’s Confined Spaces Guideline.

2.2.3.3 Sampling from open storage tanks

If the tank is agitated and has a sampling line, samples can be taken from the sampling line. The line should be flushed prior to taking a sample to remove any static or stagnant material.

Sampling material in a non-agitated open tank should be carried out from the top of the tank. Where there is a walkway over an open tank, the locations may consist of randomly selected sites below the walkway. If there is no walkway, the sites should be randomly selected around the perimeter of the tank. Reasonable effort should be made to take samples from the whole area of the tank. Sampling locations should not be located close to inflow pipes or other inlets.

While the potential for toxic or explosive vapours is not as great as for enclosed tanks, they may still occur above an open tank. In addition, there are potential health and safety issue with structures used to access the tank for sampling. Sampling personnel should, therefore, follow the same safety procedures and take the same safety precautions as outlined for sampling from the top of an enclosed tank in the previous section

2.2.3.4 Sampling from storage lagoons and stabilization ponds

Sampling materials from storage lagoons and stabilization ponds should be conducted using either a transect or grid sampling approach. The following describes these two methods:

• Transect Method: When using the transect method, two transects should be carried out: one (1) transect across the length of the lagoon; and (1) transect perpendicular to the other across the width. The point where both transects intersect should be near the centre of the lagoon. Sampling along each transect should be carried out at a minimum of five (5) pre-determined sampling locations (e.g. take samples every 15 m along the transect).
• Grid Method: When using a grid approach, the lagoon should be divided into blocks and sampling should be carried out at a minimum of five (5) predetermined locations equally spaced within the blocks. Additional sampling at a greater number of equally spaced locations within a greater number of blocks will improve the accuracy of the results.

For stabilization ponds, sewage biosolids can also be sampled from dewatered piles prior to land application as an alternative to sampling from the stabilization pond itself.

2.2.4 Sampling equipment and methods

The type of sampler used is dependent on where the sample is to be taken and if it is from an agitated or non-agitated storage facility. The following provides a brief description of the type of samplers commonly used, where they can be used and the methodology for each. Table 2-2 presents a summary of the type of equipment appropriate for different sampling requirements.

For all sampling, sub-samples must be collected using clean equipment and placed in a non-metallic container, which is covered except when the next sub-sample is being added. Mix the resulting composite sample thoroughly to ensure homogeneity. The composite sample can then be transferred to clean sample bottles for analysis.

Table 2-2. Summary of Sampling Equipment

Additional information can be found in Sections 2.4 and 2.5 of the Protocol with regards to field quality control and sample bottle requirements and equipment cleaning.

2.2.4.1 Bucket-toss method

This method is appropriate when sampling:

• an agitated storage tank or lagoon; or
• supernatant from a non-agitated storage tank or lagoon.

Attach a rope to a small plastic bucket and then throw the bucket out into the tank or lagoon, let it sink and then carefully pull the bucket back. When sampling supernatant, make sure that it does not contain surface scum or solids and that the sample is taken from approximately the midpoint of the supernatant depth.

Swirl the bucket and then pour about 1 litre of the contents into a second clean plastic bucket. Repeat this from different locations around the perimeter of the tank or lagoon until the required number of sub-samples has been taken.

2.2.4.2 Dipper method

This method is appropriate when sampling:

• an agitated storage tank or lagoon; or
• supernatant from a non-agitated storage tank or lagoon.

Tape a clean plastic bottle securely to a pole that is long enough to reach over any scum collected at the edge of the tank or lagoon. Remove the required number of sub-samples and pour them into a clean plastic bucket. When sampling supernatant, make sure that the sub-samples are taken from approximately the midpoint of the supernatant depth. Once all of the sub-samples have been taken, mix the bucket contents and transfer to sample bottles for analysis.

2.2.4.3 Kemmerer sampler

This type of sampler is appropriate when sampling:

• settled solids from a non-agitated storage tank or lagoon; or,
• settled solids from a stabilization pond.

This type of sampler consists of a cylindrical body with seals on the side of the cylinder that are closed using a weight. The sample procedure for this equipment is as follows:

• Lower the sampler carefully to the desired depth, allowing the line for the trigger to remain slack at all times.
• When the desired depth is reached, trigger the sampler to collect the sample by sending the weighted messenger down.
• Transfer the sample to a non-metallic container.
• When all sub-samples are taken, mix the contents of the non-metallic container and transfer to the sample bottle (s) for analysis.

2.2.4.4 Sludge judge

This method is appropriate when sampling:

• settled solids from a non-agitated storage tank or lagoon;
• supernatant and settled solids simultaneously for non-agitated tanks and lagoons when the layers are to be land applied at the same time; or,
• settled solids from a stabilization pond.

The sampler consists of plastic pipe in sections (typically 1.5 m long each) with screw type fittings. Various diameters of sampler are available, and typically the 1.3 cm (1/2 inch) diameter sampler is appropriate. The procedure for this sampler is as follows:

• Lower the Sludge Judge to the bottom of the tank or lagoon.
• When the bottom has been reached, the pipe will fill to the surface level. This will seat the check valve, trapping a column of the material. The sampler may need a quick ‘jolt’ to fully seat the valve.
• The Sludge Judge is removed clear of the material and the material released by touching a pin extending from the bottom section against a hard surface.
• Transfer the sample to a non-metallic container.
• When all sub-samples are taken, mix the contents of the non-metallic container and transfer to the sample bottle (s) for analysis.

2.2.4.5 COLIWASA method

A Composite Liquid Waste Sampler (COLIWASA) is appropriate for:

• taking settled solids samples at depth from non-agitated tanks or lagoons; or,
• taking a sample of the supernatant and settled solids simultaneously for non-agitated tanks and lagoons when the layers are to be land applied at the same time.

This method is only suitable for use in tanks and lagoons that are less than 4.5 m deep.

A COLIWASA consists of a long tube with a stopper at one end, attached by a rod running the length of the tube to a locking mechanism at the other end. Manipulation of the locking mechanism opens and closes the sampler by raising and lowering the stopper.

The procedure for using a COLIWASA is as follows:

• Put the sampler in the open position and slowly lower it into the material to be sampled to the bottom of the tank or lagoon.
• Push the sampler tube downward against the stopper to close the sampler and lock the sampler in the closed position.
• Slowly withdraw the sampler from the tank or lagoon and discharge the contents of the sampler into a plastic bucket.
• Repeat the procedure until the required number of sub-samples has been taken.
• Mix the contents of the bucket and transfer a composite sample to a sample bottle for analysis.

2.3 Sampling solid prescribed materials

2.3.1 Sampling solid prescribed materials in piles or large containers

Sampling from large piles of materials, such as solid manure, food manufacturing residuals or paper mill biosolids, can pose problems with respect to obtaining samples that are representative of the piles. It is difficult to collect any samples other than surface samples. Since some materials have a tendency for fine and coarse fractions to separate when piled, surface samples are not likely to be representative. Because many piled materials have a large amount of inherent variability, collecting a representative sample will be difficult at any time.

The preferred method of sampling piles is for samples to be obtained from different depths and mixed together such that the resulting composite sample is representative of the pile. This is most easily accomplished when the storage is being emptied, as sub-samples can be collected as the material is being loaded. If the material is being loaded by means of a conveyor belt, the sample should be taken in a single motion using a scoop across the complete cross section of the stream to obtain a representative sample.

If the piles must be sampled in situ, then some form of equipment to extract cores from the entire depth of the pile will be necessary, including those shown in the table below. Dewatered materials, which contain polymer, may vary in consistency and may be too condensed to permit sampling with augers or thief samplers and a shovel may be the only option. Loosely packed granular materials will more easily permit use of the sample devices available:

2.3.1.1 Sampling equipment options for solid NASM

• Shovels, trowels or hand scoops - for collecting samples from a stockpile or a flowing stream of solid material from conveyors. A clean shovel is used to access deeper layers of a pile and sterile scoops are available at laboratory supply companies for collecting sub-samples for pathogen testing, and are particularly useful for CP1 NASM materials.
• Sample Augers – rotating core devices with spiral blades for sampling compacted materials in cross section at various depths. Augers are available at hardware and laboratory supply stores.
• Triers – a tube cut in half lengthwise with sharpened tip to allow the sampler to cut into sticky materials such as dewatered and lime stabilized sewage biosolids. Triers are available at laboratory supply companies.
• Thief Samplers – two slotted concentric tubes made of stainless steel or brass. Rotation of the inner tube will open and close the tube to collect sub-samples at various depths.

Solid materials can be highly variable in their chemical analyses or pathogen levels. It is therefore necessary that for such materials at least 10 sub-samples be collected and composited to form the final sample that is submitted for analysis. To accurately characterize the material, subsamples must include material from all depths of the pile. If the subsample cannot be collected by coring or otherwise collected in such a way as to include material from the entire depth of the pile, additional subsamples must be collected so that material from all depths of the pile is proportionally represented in the composite sample. Each composite sample should not represent more than 500 tonnes of dry matter. Place the sub-samples in a clean container that can be covered or sealed between sample additions to prevent moisture loss. Once all the sub-samples have been collected, empty them onto a large surface (of appropriate composition) for mixing.”

The most efficient way of obtaining a representative sample from the composite sample is to mix and chop the material with a clean shovel, then divide the pile into quarters. Discard two opposite quarters, combine the remaining two, and repeat the process until a composite sample of the desired size is obtained. The composite sample should total approximately 1 kg. The same result can be obtained by taking small sub-samples from all sections of the sample until a sample of approximately 1 kg has been obtained. Alternate methods approved by a recognized standards organization, such as American Society for Testing and Materials (ASTM), International Organization for Standards (ISO) and Bureau de Normalisation du Québec (BNQ) may also be followed. Place the composite sample in a container or bag (see Table 2-3, then place in an appropriate container for shipping to the laboratory for analysis.

For pathogen analyses there are additional steps must be followed as described in section 2.5.2 to maintain sample sterility and avoid contamination while obtaining a composite sample. Additionally, the samples must be stored on ice and shipped to and received by the testing lab and analyses initiated within 30 hours.

2.3.2 Sampling solid prescribed materials from continuous processes and unloaders

In some situations it may be necessary or desirable to sample a material that is resulting from a continuous process or from an unloader. It is likely that proper sampling from the material stream will produce more accurate and representative samples at less cost than sampling of the final large pile or hopper. The main principle in sampling material streams is that the sample must be representative of the entire stream.

Discharges from a belt should be sampled with a scoop or shovel which has been chosen or fabricated to match the width and general contour of the belt as closely as possible. Sub-samples can be taken at any convenient point along the belt as long as the entire width of the belt is being sampled or the sample should be taken in a single motion across the complete cross section of the flowing stream. Any fines or liquid present on the belt must be included in the sample.

For any sampling strategy for material streams, the sampling frequency and the number of sub-samples combined into composite samples depends on the variability of the material over time. Possibilities for taking representative sub-samples include taking samples every hour over eight to twenty four hours (depending on the process schedule) and combining the sub-samples to form daily composites, as well as taking daily sub-samples for a week and combining them into a weekly composite. Since the sampling period and number of samples will vary for each process, it is important that sampling personnel be familiar with the variability of the material stream both over time and at different locations in the process. The sampling program must result in characterization of this variability as well as the ability to provide a representative sample of the material.

Often, solid discharges fall into a hopper or storage area directly from a process. In these situations, long-term composites may be obtained by sampling the material after it has accumulated. Random sub-samples can be taken from the hopper or storage area provided that the sampling strategy results in samples that are representative of the material. It may be necessary to mix materials prior to sampling if separation of materials has occurred in the container.

2.4 Field quality control (QC)

Table 2-3 provides a summary of the field quality control procedures that must be used for sampling for nutrient management activities

Table 2-3. Field Quality Control Procedures

2.5 Cleaning and prevention of cross contamination

For all forms of sampling, equipment and containers must be cleaned and rinsed between collection of separate samples for analysis (that is, between sites, locations or sampling times), such that cross contamination of samples is minimized. Thorough washing of equipment with soap or detergent followed by a thorough rinse with clean water (preferably distilled or deionized) should be adequate for the standard parameters to be analyzed.

2.5.1 Special cleaning procedures for sampling for trace organic analysis

Analysis for trace organic compounds is not normally required; however there may be situations where materials proposed for land application are suspected of containing specific trace organic compounds due to the particular process used to produce them. Special considerations regarding prevention of cross contamination apply when sampling for trace organic constituents. The basic methodology for sampling for trace organics is the same as that for inorganics described in the preceding sections. However, samplers must adhere to the following additional procedures:

a) Control of Cross-contamination

Sampling for trace organic contaminants requires special techniques to avoid contamination both from other samples and from sampling equipment and containers. Where potentially dangerous levels of contaminants are suspected, the sampler should wear protective gloves made of solvent-resistant material (e.g., latex). Neither gloves nor bare hands should contact the sample directly.

b) Equipment Cleaning Procedures

The sampler must carefully clean all sampling equipment which contacts material directly (i.e. samplers, corers, knives) between sites. The recommended cleaning procedure is as follows:

1. Remove adhering particles of the material by scrubbing with dilute laboratory soap solution.
2. Rinse thoroughly with distilled water.
3. Rinse with acetone.^{footnote 2[2]}
4. Rinse with hexane.^{footnote 2[2]}
5. Allow equipment to air-dry before sampling. Do not use a paper towel or cloth.

The sampler should place all sub-samples in a stainless steel bowl and mix the material prior to placing it in the sample jars. The bowl and mixing spoon/rod must be cleaned as per the wash/rinse procedure described above. When analysing for volatile organic compounds (VOCs), sub-samples must not be mixed or composited, as these processes cause losses of the compounds of interest. Rather, the sub-samples should be placed immediately into the appropriate containers.

c) Sample Containers and Sample Preservation

Solvent (hexane and /or acetone) rinsed, wide-mouthed amber-coloured glass jars with foil or Teflon lined lids are suitable for all classes of organic compounds (including PAH's, PCB's, pesticides, and VOC's).

The samples, with lids screwed on tightly, must be kept cool (preferably refrigerated, otherwise in coolers out of the direct sunlight) until delivery to the analytical laboratory.

2.5.2 Procedure for sampling for pathogens

When sampling prescribed materials for microbial pathogens the sampler should wear appropriate personal protective equipment, including gloves. Gloves protect the sampler and the sample from contamination. In the case of sampling for pathogens, a special focus on sterile techniques to avoid contamination needs to be maintained. Neither gloves nor bare hands nor non-sterile sampling equipment should contact the sample directly. Care must be taken to control microbial contamination of the sample, both from other samples and from sampling equipment.

To collect samples from tanks, lagoons, containers, stockpiles and conveyors, sampling devices will be required, as described in sections 2.2 and 2.4 and Table 2-2. Sample devices that come in direct contact with the sub-samples to be analyzed must be clean and sterile.

a) Equipment Cleaning and Sterilization Procedures

Thorough cleaning and rinsing of sampling apparatus is required, as stated in section 2.7. The sampler must carefully clean all sampling equipment which contacts material directly (i.e. samplers, corers, knives) between sample sites. The recommended cleaning procedure is as follows:

1. Remove adhering particles of the material by scrubbing with brush using dilute soap or detergent solution;
2. Rinse thoroughly with potable water;
3. Rinse again with distilled or deionized water, if possible.

For pathogen analyses, and particularly in the case of sampling for CP1 non-agricultural source materials testing, the equipment must be sterile. Stainless steel equipment is recommended as surfaces can be effectively cleaned and easily sterilized. Wood apparatus cannot be sterilized adequately. Equipment can be sterilized by following the manufacturer’s instructions on autoclaves or steam cleaners, however in the absence of autoclaves or steam cleaners, sampling devices such as scoops and shovels can be sterilized by the following procedure:

1. dip clean scoop or other device in a 1:10 dilute solution of household bleach (~5% sodium hypochlorite) for a contact time of 1-minute to sterilize the device between samples;
2. rinse the chlorine off using deionized or distilled water before sampling; or purge the device through the desired sample a number of times prior to collecting the sample. Do not dry the surface with paper towel or cloth.

Alternatively, pre-sterilized plastic scoops of various sizes and other devices are available at laboratory supply companies, thus eliminating the need to sterilize sampling equipment on site.

b) Preparing Composite Samples and Sample Containers

Analytical laboratories and laboratory supply companies typically supply sterile, sealed, plastic sample bottles or bags for collecting samples for microbiological testing. Container capacity should be no larger than 500 mL to ensure that material cools quickly. Typically no more than 200 mL sample volume is required for the microbiological analyses.

Composite samples may be required in many cases such as with solid materials, in which a minimum of 5 to 10 sub-samples are required (section 2.2.2. To adequately mix a composite sample for microbiological analysis, the mixing must be done in a sealed sterile plastic bag or other sterile vessel. The sterile bags of approx. 1–L capacity are available from laboratory supply companies and are convenient for hand-mixing and hand-quartering the samples in a sterile environment. After `kneading` the composite sample for three of four times (basically consistent with the `quartering` method described in section 2.3.1.1) a number of small sub-samples can be removed using a sterile scoop and placed in the sterile container to make up the 200 mL sample for analysis.

c) Sample Shipping and Handling-Storage

Samples must be shipped via overnight service on the day they are collected, and shipped and stored at 4±3°C, without freezing. Sample processing for bacteria (E. coli and Salmonella) analyses must be initiated within 30 hours of sample collection. For protozoan parasites (Cryptosporidium and Giardia) analyses must be initiated within 48 hr. For Helminth ova testing samples can remain refrigerated (4 ±3°C) for up to one month prior to analysis. Samples for enteric virus analyses may be stored at -18°C for up to 2 weeks before analysis is initiated.