Sampling and Analysis Protocol for Ontario Regulation 267/03 Made under the Nutrient Management Act, 2002 - Data Quality Requirements

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| Introduction | Sampling Methods | Laboratory Analysis |
| Data Quality Requirements - Part One | Data Quality Requirements - Part Two |
| Acronyms | Glossary |

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4.3 Analysis - Land Applied Materials

4.3.1 Hydrogen Ion (pH)

Matrix: Land Applied Materials (Non-agricultural source materials)

Analysis

Non-agricultural source materials with pH less than 6.0 or pH greater than 8.5, should not be applied to crops when they are being grown.

Method Principle

pH is determined with a standard glass electrode pH meter.

Sample Preparation

Solid: Prepare aqueous slurry in the ratio of 1g sample: 9 mL water. Stir or shake for approximately 20 minutes, allow suspension to settle and then determine pH of liquid fraction.

Liquid/Slurry: Decant, filter or centrifuge a portion of sample, then determine the pH of the liquid fraction.

Instrumentation

pH electrode and pH meter compensated to 25°C. Accuracy and reproducibility to 0.2 pH unit with a range of 0 - 14 and equipped with temperature compensation.

Laboratory QC Samples per Run

3 Buffer Checks, Sample Replicate and In-House Matrix Check.

Method Performance Criteria

Bias: ± 0.2 pH units

Precision: ± 0.2 pH units

Reference Method

MOE/LaSB - E3137 (solid)

MOE/LaSB - E 3218 (liquid/slurry)

Notes

1. Sample preservation - store samples in refrigerator (4 - 10°C).
2. Maximum sample storage time: 14 days.

4.3.2 Total Dry Matter/Total Solids

Matrix: Land Applied Materials

Analysis

Accurate determination of the dry matter content of land-applied materials is necessary to calculate application rates on a moist basis.

Method Principle

A portion of sample is weighed as received, dried for 16 hours at 105 ± 5°C, cooled and reweighed. The percent total solid is determined.

Sample Preparation

Disaggregate the sample and pass through a 2 mm sieve. Take subsamples (10 - 25 g) of this mixture, and dry in an oven at 105 ± 5°C for 16 hours to a constant weight. Cool and reweigh sample to determine total solids as a percent of the fresh weight.

Instrumentation

Balance, capable of weighing ±0.01 g.

Laboratory QC Samples per Run

Calibration Check and Sample Replicate. 

Method - Performance Criteria

Bias: 100 ± 10%

Precision: ± 10%

Reference Method

MOE/LaSB-3139

4.3.3 Total Organic Matter (Volatile Solids)

Matrix: Solid or Liquid Land Applied Materials

Analysis

Method Principle

A portion of ground sample is dried for 16 hours at 105°C ± 5°C, then muffled at 475°C ± 25°C for 4 hours. The weight loss, and per cent ash are determined.

Sample Preparation

Disaggregate the sample and pass through a 2 mm sieve, then take and grind a sub-sample to pass through a 0.355 mm sieve.

Heat the sub-sample in a muffler oven at 475°C ± 25°C for 4 hours.

Determine the weight loss, and per cent ash.

Instrumentation

Muffle furnace. Balance, capable of weighing ± 0.01 g.

Laboratory QC Samples per Run

Calibration Check and Sample Replicate.

Method - Performance Criteria

Accuracy: 100 ± 10%

Precision: ± 10%

Reference Method

MOE/LSB-3139

4.3.4 Total Kjeldahl Nitrogen (TKN)

Matrix: Land Applied Materials

Analysis

Total Kjeldahl nitrogen in land applied materials is determined to provide a basis for calculating the organic nitrogen portion of the material (by subtracting the ammonium N from the total). Note: The TKN determination does not include the oxidized nitrogen parameters NO2 and NO3

Method Principle

Amino nitrogen in organic materials is converted to ammonium by digestion in the presence of strong acid, salt and a catalyst. Ammonium content, which will also include ammonia and ammonium in the sample before digestion, is determined by colourimetry, ammonia selective electrode or titration.

Sample Preparation

Test the samples as received. Determine the dry matter content of the material separately.

Sample is mixed with H₂SO₄, K₂SO₄, and cupric sulphate (catalyst), and heated to 400^°C to convert organically bound nitrogen to NH₄. Note: Cupric sulphate may be replaced with phenate/sodium hypochlorite

Instrumentation

Autoanalyzer, colourimeter

Laboratory QC Samples per Run

Method Blank, Matrix Matched In-House Control or CRM, Calibration Check and Sample Replicate.

Method Performance Criteria

Inter- and intra-lab precision must be within ± 10% of the mean of samples from all accredited labs.

Reference Method

AOAC 978.02

MOE/LaSB – E3116

Notes

1. Sample Preservation - Store samples in refrigerator (4 - 10°C). If extended storage is required (>10 days), freeze the samples.

2. Method does not completely account for oxidized forms of nitrogen such as nitrate, nitrite, or nitrogen in heterocyclic ring compounds. Nitrogen determination by combustion (Dumas method) may give better results in materials with significant contents of these forms of nitrogen.

4.3.5 Ammonia and Ammonium - Nitrogen

Matrix: Land Applied Materials

Analysis

Ammonium nitrogen in land applied materials (which includes both ammonium and ammonia nitrogen) is determined to provide an estimate of plant available nitrogen, as well as a basis for calculating the organic nitrogen portion of the material (by subtracting the ammonium N from the TKN).

Method Principle

Ammonium plus ammonia nitrogen is extracted from the sample in a KCl solution.  Ammonium content in the extract is determined by colourimetry, using a modified Berthelot reaction.

Sample Preparation

Test the samples as received. Determine the dry matter content of the material separately.

Sample is mixed with 2M KCl solution, shaken, and then centrifuged or filtered.

Instrumentation

Autoanalyzer, colourimeter

Laboratory QC Samples per Run

Method Blank, Matrix Matched In-House Control or CRM, Calibration Check and Sample Replicate.

Method Performance Criteria

Inter- and intra-lab precision must be within ± 10% of the mean of samples from all accredited labs.

Reference Method

USEPA 352.2

Notes

Ammonia specific electrode may be used as an alternate method, and will be less subject to interference from discolouration of the extract.

4.3.6 Nitrate and Nitrite - Nitrogen

Matrix: Land Applied Materials

Analysis

Samples are analyzed for nitrate nitrogen and nitrite nitrogen to assess the nitrogen immediately available to plants. Some non-agricultural source materials may contain significant amounts of nitrate and nitrite nitrogen.

Method Principle

Samples are analyzed using an automated colourimetric procedure which entails converting nitrate to nitrite, and then analyzing the sample for nitrite.

Nitrate is reduced to nitrite by heating an aliquot of sample with hydrazine in alkaline media; this reaction is catalyzed by the addition of cupric ion. Subsequently, an azo dye is formed in acid media by diazotizing sulphanilamide with nitrite and coupling the product with N (1-naphthyl) ethylenediamine dihydrochloride. The absorbance of the light red azo dye is measured at 520 nm and the concentration of nitrate nitrogen plus nitrite nitrogen is determined by comparison with a similarly treated series of mixed Standards.

Sample Preparation

Liquid/Slurry: A supernatant of the settled sample is used for this analysis. Samples (if frozen) are thawed to room temperature prior to analysis. Highly turbid samples should be filtered to prevent clogging of the analyser fittings. Sewage sludge samples should be centrifuged prior to analysis.

Solid: Under Development

Instrumentation

Colourimeter

Laboratory QC Samples per Run

Method Blank, In-House Standard, Calibration Check and Sample Replicate.

Method Performance Criteria

Bias: 100 ± 10%

Precision: 10%

Reference Method

MOE/LaSB - E3366

Notes

1. Store samples in a refrigerator (4 – 10°C).
2. Maximum storage time, 7 days.

4.3.7 Organic Nitrogen

Matrix: Land Applied Materials

Analysis

The organic nitrogen portion of the material is calculated by subtracting the ammonium N from the TKN of the sample.

Method Principle

Ammonium plus ammonia nitrogen, as determined in a KCl extract of the sample, is subtracted from the Total Kjeldahl Nitrogen in the sample. The difference is the organic N.

Sample Preparation

Extraction as defined for ammonium and total N.

Instrumentation

As defined for ammonium and total N.

Laboratory QC Samples per Run

As defined for ammonium and total N.

Method Performance Criteria

As defined for ammonium and total N.

Method Reference

N/A

Notes

If the total N content of the material has been determined by combustion (dumas method) rather than wet digestion, the nitrate content of the material should also be determined and the nitrate as well as the ammonium content should be deducted from the total N to determine the organic N.

4.3.8 Metals – Cd, Cr, Co, Cu, Pb, Mo, Ni, and Zn

Matrix: Land Applied Materials (Non-agricultural source materials)

Analysis

This analysis is required as specified in the regulation.

Method Principle

A portion of sample is extracted with a heated, strong mixed acid solution, brought to volume with pure deionized water and analyzed using a spectrometric technique.

Sample Preparation

Solid (i.e. dewatered biosolids, filter cake - Method E3071)

Digest a portion of previously air dried, ground and sieved sample with concentrated Nitric acid/Hydrochloric acid mixture (1:3) by heating at 50°C for 1 hour and then at 95°C for another 3 hours. Adjust volume with pure deionized water, decant/filter and then analyze.

Liquid/slurry (i.e. liquid biosolids, likely 1% to 10% solids - Method E3071)

Weigh an aliquot of homogenized sample. Digest with concentrated Nitric acid/Hydrochloric acid mixture (1:3) by heating at 50°C for 1 hour and then at 95°C for another 3 hours.  Adjust volume with pure deionized water, decant/filter and then analyze. Report results on dry weight basis.

Clear Liquid (i.e. supernatant, less than 1% solids - Method E3094)

Digest an aliquot of sample with concentrated Nitric acid/Hydrochloric acid mixture (1:3) by heating at 50°C for 1 hour and then at 95°C for another 3 hours. Adjust volume with pure deionized water, decant/filter and then analyze.

Instrumentation

ICP/AES

DCP, ICP/MS, flame AAS and graphite furnace AAS with suitable matrix modifiers may be used.

Laboratory QC Samples per Run

Method Blank, Matrix Matched In-House Control or CRM, Calibration Check and Sample Replicate.

Method Performance Criteria

Analyte	RDL (μg/g)	Acceptable Bias (%)	Precision (Reproducibility) (% RDS)
Cadmium	2.0	± 20	± 20
Chromium	106	± 20	± 20
Cobalt	15	N/A	N/A
Copper	76	± 20	± 20
Lead	50	± 20	± 20
Molybdenum	2.5	N/A	N/A
Nickel	18	± 20	± 20
Zinc	185	± 20	± 20

* Bias is based upon the certified reference material, such as, WWS-26 from Environmental Resource Associate or EPA Quality Control Sample, municipal digested sludge (SPL # 2900).

Reference Method

MOE/LaSB - E3071 for liquid/slurry and solid; MOE/LSB - 3094 for Clear Liquid.

Notes

1. Sample Preservation
   • Clear Liquid Samples - Preserved with nitric acid to less than pH 2.
   • Solid/Slurry/Solid Sample - Store samples in refrigerator (4 - 10°C).
2. Maximum Sample Storage Time: 60 days

4.3.9   Mercury

Matrix: Land Applied Materials (Non-agricultural source materials)

Analysis

This analysis is required for non-agricultural source material only.

Method Principle

Mercury in the sample is converted to the inorganic form by acid digestion process. The inorganic mercury in aqueous solution is then reduced with stannous chloride, and analyzed by CV AAS.

Sample Preparation

Solid (e.g. dewatered sludge, filter cake - Method E3058)

Digest a portion of previously air dried ground and sieved sample with 50% v/v Aqua Regia (hydrochloric acid/Nitric acid - v/v 3:1) in the presence of potassium permanganate by heating within a temperature range of 90°C to 110°C for 1 hour and 15 minutes. Treat excess permanganate with hydroxylamine sulphate. Reduce inorganic mercury with stannous chloride prior to analysis. Adjust volume with pure deionized water, decant/filter and then analyze. Report results on dry weight basis.

Liquid/Slurry (e.g. liquid sludge, likely 1% to 10% solids - Method E3058)

Digest weighed aliquot of homogenized (well mixed) sample with 50% v/v Aqua Regia (hydrochloric acid/Nitric acid - v/v 3:1) in the presence of potassium permanganate by heating within a temperature range of 90°C to 110°C for 1 hour and 15 minutes. Treat excess permanganate with hydroxylamine sulphate. Reduce inorganic mercury with stannous chloride prior to analysis. Adjust volume with pure deionized water, decant/filter and then analyze. Report results on dry weight basis.

Clear Liquid (e.g. supernatant, less than 1% solids - Method E 3060)

Digest an aliquot of homogenized (well mixed) sample with concentrated sulphuric acid/nitric acid (1.2:0.5) in the presence of potassium persulphate and potassium dichromate for 2 hours at 87°C ± 3°C. Adjust volume with pure deionized water, decant/filter and then analyze.

Instrumentation

Cold Vapour Flameless Atomic Absorption (CV-FAAS)

Laboratory QC Samples per Run

Method Blank, Matrix Matched In-House Control or CRM, Calibration Check and Sample Replicate.

Method Performance Criteria

Analyte	RDL (μg/g)	Acceptable Bias (%)	Precision (Reproducibility) (% RDS)
Mercury	0.5	± 20	±20

 * Bias is based upon the certified reference material, such as, CRM 145R (sewage sludge mixed origin) or BE - 1 (sewage sludge).

Reference Method

MOE/LaSB - E3060 (clear liquid), MOE/LaSB E3058 (slurry and solid)

Notes

Sample Preservation

1. Clear Liquid Sample - Preserve 250 mL sample with 0.5 - 1.0 mL concentrated nitric acid and 5 - 10 drops of 5% potassium dichromate solution. This should lower pH to below 2.0 and give the sample a permanent yellow colour. Liquid Slurry/Solid Sample – Store samples in refrigerator (4 - 10°C).
2. Maximum Sample Storage Time: 15 days.

4.3.10 Arsenic and Selenium

Matrix: Land Applied Materials (Non-agricultural source materials)

Analysis

This analysis is required for non-agricultural source material only.

Method Principle

A portion of sample is digested in oxidizing acid mixture to convert all forms of arsenic and selenium to arsenate (AsO₄)^3- and selenate (SeO₄)^2- respectively. The arsenate and selenate are then reduced with sodium borohydride to arsine and hydrogen selenide which are then analyzed by flameless AAS.

Sample Preparation

Solid - (e.g. dewatered sludge, filter cake - Method E3091)

Digest a portion of previously air dried ground and sieved sample with concentrated Nitric acid/Hydrochloric acid mixture (1:3) at 50 °C for 1 hour and then at 95 °C for an additional 3 hours. Adjust volume with pure deionized water, decant/filter and then analyze.

Liquid/Slurry (e.g. liquid sludge, likely 1% to 10% solids - Method E3091)

Digest a weighed aliquot of sample with concentrated Nitric acid/Hydrochloric acid mixture (1:3)  at 50 °C for 1 hour and then at 95 °C for an additional 3 hours. Adjust volume with pure deionized water, decant/filter and then analyze. Report results on dry weight basis.

Clear Liquid (e.g. supernatant, less than 1% solids) (Method E 3302)

Digest an aliquot of liquid with concentrated Nitric acid/Hydrochloric acid mixture (1:3) at 50 °C for 1 hour and then at 95 °C for an additional 3 hours. Adjust volume with pure deionized water, decant/filter and then analyze.

Instrumentation

Hydride - Flameless Atomic Absorption Spectrophotometry (HYD-FAAS)

Laboratory QC Samples per Run

Method Blank, Matrix Matched In-House Control or CRM, Calibration Check and Sample Replicate.

Method Performance Criteria

Analyte	RDL (μg/g)	Acceptable Bias (%)	Precision (Reproducibility) (% RDS)
Arsenic	7.5	± 20	± 20
Selenium	1.4	± 20	± 20

 * Bias is based upon the certified reference material, such as, CRM 145R (sewage sludge mixed origin) or BE - 1 (sewage sludge).

Reference Method

MOE/LaSB - E3302 (clear liquid), MOE/LaSB E3091 (liquid/slurry and solid)

Notes

1. Sample Preservation
   Clear Liquid Sample - Preserve sample with nitric acid
   Liquid/Slurry/Solid Sample - Store samples in refrigerator (4 - 10 °C)
2. Maximum Sample Storage Time: 30 days.

4.3.11 Total Phosphorus, Potassium, Sodium and Boron

Matrix: Land Applied Materials

Analysis

Total phosphorus and potassium content of land applied materials are determined to estimate the amount of plant available nutrients being applied to land. Sodium or boron content may need to be determined for land applied materials suspected of containing high concentrations of these elements, on a case by case basis.

Method Principle

A portion of sample is extracted with a heated, strong mixed acid solution, brought to volume with pure deionized water and analyzed using a spectrometric technique.

Sample Preparation

Solid: Digest a portion of previously dried and homogenized sample with concentrated Nitric acid/Hydrochloric acid mixture (1:3) by heating at 50°C for 1 hour and then at 95°C for another 3 hours. Adjust volume with pure deionized water, decant/filter and then analyze.

Liquid/slurry: Weigh an aliquot of homogenized (well mixed) sample and then digest with concentrated Nitric acid/Hydrochloric acid mixture (1:3) by heating at 50°C for 1 hour and then at 95°C for another 3 hours.  Adjust volume with pure deionized water, decant/filter and then analyze.

Instrumentation

ICP/AES

Laboratory QC Samples per Run

Method Blank, Matrix Matched In-House Control or CRM, Calibration Check and Sample Replicate.

Method Performance Criteria

Inter- and intra- lab precision must be within ±10% of the mean of samples from all accredited labs.

Reference Method

Under development

4.3.12 E. coli

Matrix: Sewage Biosolids and other Non-Agricultural Source Materials (NASM)

Analysis

Municipal sewage biosolids and any other material, other than untreated septage, that contains human body waste are required to be sampled and analyzed for E.coli. prior to land application

Method Principle

A volume of buffered dilution water is added to a weighed amount of biosolids and processed with a Stomacher™ or equivalent. Serial dilutions are then prepared using the supernatant. Serial dilutions are then plated out on mFC-BCIG agar (or other selective agar) and incubated for required time and the required temperature. Alternatively an equivalent estimate most probable number (MPN) statistical method can be used to quantify culturable organisms.

Sample Preparation

Add an amount of buffered dilution water to a weighed mass of biosolids material. Process in Stomacher™ or equivalent technology for 2 minutes. Decant off supernatant.

Sample Shipping and Storage

1. Samples must be collected in sterile bottles of no larger 500 mL capacity with sample volumes of approx. 200 mL to permit rapid cooling of sample.
2. Samples must be shipped via overnight service on the day they are collected, and shipped and stored on ice and at 4±3°C, without freezing.
3. Sample processing for bacteriological analysis must be initiated within 30 hours of sample collection.

Instrumentation

Classical microbiological techniques - selective agar, biochemicals for confirmation.

Laboratory QC Samples per Run

Positive and negative controls run with each set of samples, and addition of spiked samples for recovery.

Method Performance Criteria

Between analyst precision (method duplicate) and within analyst precision (analyst duplicate) and inter-technician comparative reading data points must be within 95% confidence limits (Standard Methods, 2005).

Reference Method

MOE/LaSB - E3433

Notes

1. Sample storage - ice bath/temperature
2. Analysis must be performed within 30 hours.

4.3.13 Culturable Salmonella spp.

Matrix: Sewage biosolids and any other material, other than untreated septage, that contains human body waste

Analysis

NASM to be considered of CP1 pathogen quality are required to be sampled and analysed as per the regulation.  Such material may not be utilized as a CP1 material where densities of viable salmonellae are at or above 3 most probable number (MPN) per 4-g sample (dry weight) or per 100-mL if aqueous NASM sample of < 1% total solids on a wet weight basis. A minimum of 100 mL or 4-g dry wt sample must be analyzed to report a non-detect.

Method Principle

The sample is diluted, homogenized and inoculated into selective growth media and all Salmonella spp. are enumerated statistically using the most probable number method (MPN), which involves making the required number of serial decimal dilutions of the original sample in three or five replicates to obtain a statistical estimate of salmonellae density. The specific method protocol must meet the performance criteria described below.

Sample Preparation

Liquid:  A minimum of 100-mL or 4-gram sample volume must be accommodated by a modification of standard MPN procedures by increasing sample volumes or by concentration by filtration or other suitable mechanism. 

Solid: A volume of buffered dilution water is added to a weighed amount of NASM and homogenized. Sterile. high-volume plastic bags can be used for homogenization, unless the sample contains hard debris that may pierce the plastic bag. Salmonella spp. are enumerated from the sample using the 3-tube MPN method of 3- replicates carried in 3 or more serial decimal dilutions. A minimum of 100 mL or 4-g sample must be analysed to report a non-detect.  

Instrumentation

Filter apparatus, incubators, sterile tubes, sterile, sealed, high capacity (e.g., 1-L) plastic bags (e.g., Stomacher™ bags or equivalent).

Sample Shipping and Storage

1. Samples must be collected in sterile bottles of no larger volume than 500 mL to permit rapid cooling of sample, with approx. 200 mL of sample required.
2. Samples must be shipped via overnight service on the day they are collected, and shipped and stored and at 4±3°C, without freezing.
3. Sample processing must be initiated within 30 hours of sample collection.

Laboratory QC Samples per Run

One method blank and one spiked positive control for every 20 samples, or each week during which samples are analyzed if 20 or fewer samples are analyzed.

Performance Criteria

Detection limits of Salmonella spp. in all matrices (biosolids and any other material, other than untreated septage, that contains human body waste.) are expected to be 3 MPN Salmonella MPN per gram dry weight of material based on validation work (Laboratory Methods Validation Study for Pathogens in Pulp and Paper Mill Biosolids and other NASM, Univ. of Guelph, Final Report for MOE, February 28, 2007).

Reference Methods

1. APHA 1992.  Standard methods for the examination of water and wastewater. Method 9260 D. American Public Health Association.

And sample preparation for sludges:

2. US EPA Report No: EPA/625/R-092/013. (Revised July 2003) Environmental Regulations and Technology: Control of pathogens and vector attraction in Sewage Sludge. Appendix F: Sample preparation for fecal coliform tests and Salmonella sp. analysis. Cincinnati, OH: Office of Research and Development.

4.3.14 Total Culturable Enteroviruses (a subset of total enteric viruses)

Matrix: Sewage biosolids and any other material, other than untreated septage and Category B compost, that contains human body waste

Analysis

Sewage biosolids and any other material, other than untreated septage, that contains human body waste may be tested to be considered of CP1 pathogen quality. This material cannot be deemed CP1 material if the level of total culturable enteroviruses exceeds 1 plaque forming units (pfu) per 4-g dry weight (wt). A minimum of 4-g sample on a dry wt basis must be analysed to report a non-detect. Category B compost is not required to be tested for total culturable enteroviruses to be considered CP1 pathogen quality.

Principle method

Solids: Culturable enteroviruses are first adsorbed to solids under select solution conditions (such as pH) and concentrated by centrifugation followed by removal/desorption and solids removed by centrifugation by change in solution conditions.

Solids and liquids: Culturable enteroviruses in the centrate liquid after extraction from solids or viruses directly in the liquid NASM samples are concentrated by organic-flocculation and assayed by counting plaque forming units (pfu) on Buffalo Green Monkey (BGM) kidney cell cultures or equivalent cell-culture technology. This procedure may take approximately 16 days.

Sample Preparation

Solids: Samples are first conditioned using AlCl₃ at pH 3.5 (or equivalent) to adsorb viruses to solids fraction and concentrated by centrifugation. Then viruses are desorbed from solids by physicochemical means and the aqueous fraction (containing the viruses) is clarified by centrifugation followed by serial or stacked membrane filtrations down to 0.2 µm. Total culturable viruses in the liquid fraction may be further concentrated by organic-flocculation and centrifugation if necessary.

Instrumentation

pH meter, refrigerated centrifuge, stirrer/stir bars, membrane filtration apparatus, BGM (or equivalent) tissue cultures, tissue culture rocker apparatus and incubators with appropriate gas cylinders.

Storage and Handling of samples

Sample bottles should not exceed 500 mL in size to permit rapid cooling of sample after collection. Samples should be shipped and stored frozen (-18°C) for up to 2 weeks prior to analysis. After processing, the virus eluate must be stored at 4±3°C and the viral assay must be completed within 24 hrs, or freeze at -70°C. Once thawed (rapidly at 37°C) keep at 4°C less than 24 hr until viral assay is complete.

Laboratory QC Samples per Run

One method blank and one spiked positive control for every 20 samples, or each week during which samples are analyzed if 20 or fewer samples are analyzed. Enteroviruses are required for positive controls, for example attenuated Poliovirus or Echovirus. At time of viral assay, at least 10% of the isolates should be confirmed by second passage on tissue cultures.

Method Performance Criteria

Under development. Detection limit expected is 1 virus particle 4-g (dry wt) sample based on work by U.S. EPA (Yanko 1987 Report No. EPA/600/1-87/014). Inter-laboratory precision testing resulted in a total standard deviation for all sludges and all labs for this method of 0.41 (as a log10 value).

Reference methods

1. ASTM (2004) Method D 4994-89. Standard practice for recovery of viruses from wastewater sludges. Procedure A. In:  Annual Book of ASTM Standards- Section 11. Water and environmental technology. West Conshohocken, PA: American Soc. for Testing and Materials.

2. USEPA Report No. EPA/625/R-092/013. (Revised July 2003). Environmental Regulations and Technology: Control of pathogens and vector attraction in Sewage Sludge. Appendix H: Method for the recovery and assay of total culturble viruses from sludge. Cincinnati, OH:.

For liquid samples organic-flocculation procedure:

3. USEPA Publication No. EPA/600/R-95/178.1996. Virus monitoring protocol for the ICR (Information Collection Requirements Rule); In "ICR microbial laboratory manual".Washington, DC.

4.3.15 Viable Helminth Ova

Matrix: Sewage biosolids and any other material, other than untreated septage and Category B compost, that contains human body waste

Analysis

Sewage biosolids and any other material, other than untreated septage, that contains human body waste to be considered of CP1 pathogen reduction quality must be tested. Such material cannot be deemed a CP1 category material if the level of viable parasitic helminth ova exceeds 1 ovum per 4-g dry weight (wt). A minimum 4-g sample must be analysed in its entirety to report a non-detect. Category B compost is not required to be tested for viable helminth ova to be considered CP1 pathogen quality.

Principle Method

Ascaris ova are removed from material by washing, blending, screening and concentrated by settling followed by density gradient centrifugation. The fraction containing Helminth ova are collected, washed and concentrated by centrifugation, and then incubated 3-4 weeks in dilute H₂SO₄ (or equivalent) until control samples of Ascaris ova become embryonated (larval stages visible). Total and viable counts are determined by microscopic counting of total ova and those embryonated (viable) ova.

Sample Preparation

Samples are diluted in buffered solution followed by further processed by blending in buffered surfactant solution and coarse screening. After screened ova are settled out, they are concentrated by density gradient centrifugation using MgSO₄ (or equivalent). The fraction containing ova is further screened and concentrated by centrifugation. The final samples are incubated 3-4 weeks at 26°C in dilute H₂SO₄ (or equivalent) until ova become embryonated.

Sample Storage and Handling

Sample bottles should not exceed 500 mL in volume to permit rapid cooling of sample after collection. Samples should be immediately refrigerated and can be stored refrigerated for up to 1 month. Do not freeze as this may interfere with recovery efficiency due to changes in buoyant density.

QA/QC

One method blank and one spiked positive control for every 20 samples, or each week during which samples are analyzed if 20 or fewer samples are analyzed.

Performance Criteria

Detection limits are expected to be 1 viable ovum per 4- or 10-g dry wt sample mass based on work by U.S. EPA (Yanko 1987 Report No. EPA/600/1-87/014) and Gantzer et al. (2001 Wat Res 35(16):3763), respectively.

Reference method

1. USEPA Report No: EPA/625/R-092/013. (Revised July 2003) Environmental Regulations and Technology: Control of pathogens and vector attraction in Sewage Sludge. Appendix I: Test method for detecting, enumerating and determining the viable of Ascaris ova in sludge. U.S. EPA Office of Research and Development, Cincinnati, OH P.p. 166-172.

4.3.16 Cryptosporidium Oocysts

Matrix: Non-Agricultural Source Materials that are not sewage biosolids and do not contain human body waste

Analysis

NASM that are not sewage biosolids and does not contain human body waste that wish to demonstrate CP1 pathogen reduction quality' are required to be sampled and analysed. Such material may not be applied to land where levels of Cryptosporidium are detected in a 4-g sample (dry wt) or a 100-mL aqueous sample. Detection limits are expected to be at or better than 25 oocysts per 4-g or 100mL sample. (Rose et al., 2004. Water Environment Research Foundation (WERF), Report No. 00-PUM-2T). A minimum of 100 mL for liquid NASM or 4-g dry wt sample solid sample must be analyzed to report a non-detect.

Method Principle

Cryptosporidium oocysts are concentrated from the sample, purified using immunomagnetic separation or equivalent technology and enumerated either by immunofluorescent and differential interference contrast (DIC) microscopy, quantitative PCR, or technology shown to produce equivalent results. The specific method protocol must meet the performance criteria described below. A method may be chosen to simultaneously detect Cryptosporidium and Giardia, provided all performance criteria are met.

Sample Preparation

Liquid:  A volume of liquid is concentrated by centrifugation or other suitable mechanism.

Solid: A volume of buffered dilution water is added to a weighed amount of NASM.

Cryptosporidium oocysts are purified using immunomagnetic separation or equivalent technology. Cryptosporidium oocysts are enumerated either by immunofluorescent and differential interference contrast (DIC) microscopy, quantitative PCR, or technology shown to produce equivalent results.

Instrumentation

Centrifuges, immunomagnetic separation apparatus, real-time (quantitative) PCR thermocycler and software, or appropriate microscope

Laboratory QC Samples per Run

One method blank and one spiked positive control for every 20 samples, or each week during which samples are analyzed if 20 or fewer samples are analyzed.

Sample Shipping and Storage

1. Samples must be shipped via overnight service on the day they are collected, and shipped and stored and at 4±3 °C without freezing.
2. Sample processing must be initiated within 48 hours of sample collection.
3. The laboratory must complete purification and detection within one work day to minimize the time that any target organisms present in the sample sit in eluate or concentrated matrix. Detection must occur within 72 hours of purification.

Method Performance Criteria

As per US EPA Methods 1622 and 1623

Method acceptance criteria are shown in Table 1. The initial and ongoing precision and recovery criteria are based on the results of spiked samples.  Analysis of a minimum of 4 samples spiked with 100 to 500 oocysts is required to calculate the initial precision and recovery.

The acceptance criteria for mean recovery is 13 to 143% based on the following equation:

R= 100 x [(N_sp - N_{s) ÷} T]

Where:

R is the percent recovery

N_sp is the number of oocysts detected in the spiked sample

N_s is the number of oocysts detected in the unspiked sample

T is the true value of the oocysts spiked

The maximum relative standard deviation of at least 5 samples with recovery between 24 and 100 % must be no greater than 55.

The maximum relative percent difference (precision) is 67. This is calculated according to the following equation:

RPD = 100 x [(|N_MS – N_MSD|) ÷ X_mean]

Where:

RPD is the relative percent difference

N_MS is the number of oocysts detected in the matrix spike

N_MSD is the number of oocysts detected in the matrix spike duplicate

X_mean is the mean number of oocysts detected in the matrix spike and duplicate

Table 4.3.16. Quality Control Acceptance Criteria for Cryptosporidium (US EPA, 2001)

Performance Test	Acceptance Criteria
Initial Precision and Recovery
Mean recovery (percent)	24-100
Precision (as maximum relative standard deviation)	55
Ongoing precision and recovery (percent)	11-100
Matrix spike/matrix spike duplicate (for method modifications)
Mean recovery1 (as percent)	13-143
Precision (as maximum relative percent difference)	67

1. The acceptance criteria for mean MS/MSD recovery serves as the acceptance criteria for MS recovery during routine use of the method

2. Some sample matrices may prevent the acceptance criteria from being met

Reference Method

US EPA Method 1622 or 1623 for water may be modified for use in NASM matrices. MOE Method E3463 for abattoir waste water may be used. In any case, the method performance criteria must be met for initial precision and recovery.

4.3.17 Giardia cysts

Matrix: Non-Agricultural Source Materials that are not sewage biosolids and do not contain human body waste

Analysis

NASM that are not sewage biosolids and do not contain human body waste, that wish to demonstrate CP1 pathogen reduction quality, are required to be sampled and analysed. Such material may not be applied to land where levels of Giardia are detected in a 4-g sample (dry wt) or a 100-mL aqueous sample. Detection limits are expected to be at or better than 25 cysts per 4-g or 100mL sample. (Rose et al. 2004. WERF Report No. 00-PUM-2T). A minimum of 100 mL for liquid NASM or 4-g dry wt solid sample must be analyzed to report a non-detect.

Method Principle

Giardia cysts are concentrated from the sample, purified using immunomagnetic separation or equivalent technology and enumerated either by immunofluorescent and differential interference contrast (DIC) microscopy, quantitative PCR, or technology shown to produce equivalent results. The specific method protocol must meet the performance criteria described below.

Sample Preparation

Liquid: A volume of liquid is concentrated by centrifugation or other suitable mechanism.

Solid: A volume of buffered dilution water is added to a weighed amount of NASM. Giardia cysts are purified using immunomagnetic separation or equivalent technology. Giardia cysts are enumerated either by immunofluorescent and differential interference contrast (DIC) microscopy, quantitative PCR, or technology shown to produce equivalent results.

Instrumentation

Centrifuges, immunomagnetic separation apparatus, real-time (quantitative) PCR thermocycler and software, or appropriate microscope

Sample Shipping and Storage

1. Samples must be shipped via overnight service on the day they are collected, and shipped and stored and at 4±3 °C, without freezing.
2. Sample processing must be initiated within 48 hours of sample collection.
3. The laboratory must complete purification and detection within one work day to minimize the time that any target organisms present in the sample sit in eluate or concentrated matrix. Detection must occur within 72 hours of purification.

Laboratory QC Samples per Run

One method blank and one spiked positive control for every 20 samples, or each week during which samples are analyzed if 20 or fewer samples are analyzed.

Method Performance Criteria

As per US EPA Methods 1622 and 1623

Method acceptance criteria are shown in Table 1. The initial and ongoing precision and recovery criteria are based on the results of spiked samples. Analysis of a minimum of 4 samples spiked with 100 to 500 cysts are required to calculate the initial precision and recovery.

The acceptance criteria for mean recovery is 13 to 143% based on the following equation:

R= 100 x [(N_sp - N_{s) ÷} T]

Where:

R is the percent recovery

N_sp is the number of cysts detected in the spiked sample

N_s is the number of cysts detected in the unspiked sample

T is the true value of the cysts spiked

The maximum relative standard deviation of at least 5 samples with recovery between 24 and 100 % must be no greater than 55.

The maximum relative percent difference (precision) is 67. This is calculated according to the following equation:

RPD = 100 x [(|N_MS – N_MSD|) ÷ X_mean]

Where:

RPD is the relative percent difference

N_MS is the number of cysts detected in the matrix spike

N_MSD is the number of cysts detected in the matrix spike duplicate

X_mean is the mean number of cysts detected in the matrix spike and duplicate

Table 4.3.17. Quality Control Acceptance Criteria for Giardia (US EPA, 2001)

Performance Test	Acceptance Criteria
Initial Precision and Recovery
Mean recovery (percent)	24-100
Precision (as maximum relative standard deviation)	49
Ongoing precision and recovery (percent)	14-100
Matrix spike/matrix spike duplicate (for method modifications)
Mean recovery1 (as percent)	15-118
Precision (as maximum relative percent difference)	30

3. The acceptance criteria for mean MS/MSD recovery serves as the acceptance criteria for MS recovery during routine use of the method

4. Some sample matrices may prevent the acceptance criteria from being met

Reference Method

US EPA Method 1622 or 1623 for water may be modified for use in NASM matrices. MOE Method E3463 for abattoir waste water may be used. In any case, the method performance criteria must be met for initial precision and recovery.

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| Introduction | Sampling Methods | Laboratory Analysis |
| Data Quality Requirements - Part One | Data Quality Requirements - Part Two |
| Acronyms | Glossary |

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