Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites

1Department of Chemistry and Chemical Engineering, Royal Military College of Canada, 2Analytical Services Unit, School of Environmental Studies, Queen's University
Published 11/28/2014

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Biochar is a carbon-rich material used as a soil amendment with the ability to sustainably sequester carbon, improve substrate quality and sorb contaminants. This protocol describes the 17 analytical methods used for the characterization of biochar, which is required prior to large scale implementation of these amendments in the environment.

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Denyes, M. J., Parisien, M. A., Rutter, A., Zeeb, B. A. Physical, Chemical and Biological Characterization of Six Biochars Produced for the Remediation of Contaminated Sites. J. Vis. Exp. (93), e52183, doi:10.3791/52183 (2014).


The physical and chemical properties of biochar vary based on feedstock sources and production conditions, making it possible to engineer biochars with specific functions (e.g. carbon sequestration, soil quality improvements, or contaminant sorption). In 2013, the International Biochar Initiative (IBI) made publically available their Standardized Product Definition and Product Testing Guidelines (Version 1.1) which set standards for physical and chemical characteristics for biochar. Six biochars made from three different feedstocks and at two temperatures were analyzed for characteristics related to their use as a soil amendment. The protocol describes analyses of the feedstocks and biochars and includes: cation exchange capacity (CEC), specific surface area (SSA), organic carbon (OC) and moisture percentage, pH, particle size distribution, and proximate and ultimate analysis. Also described in the protocol are the analyses of the feedstocks and biochars for contaminants including polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), metals and mercury as well as nutrients (phosphorous, nitrite and nitrate and ammonium as nitrogen). The protocol also includes the biological testing procedures, earthworm avoidance and germination assays. Based on the quality assurance / quality control (QA/QC) results of blanks, duplicates, standards and reference materials, all methods were determined adequate for use with biochar and feedstock materials. All biochars and feedstocks were well within the criterion set by the IBI and there were little differences among biochars, except in the case of the biochar produced from construction waste materials. This biochar (referred to as Old biochar) was determined to have elevated levels of arsenic, chromium, copper, and lead, and failed the earthworm avoidance and germination assays. Based on these results, Old biochar would not be appropriate for use as a soil amendment for carbon sequestration, substrate quality improvements or remediation.


Biochar is a carbon-rich by-product produced during the pyrolysis of organic matter 1. Interest, both publicly and academically, in adding biochar to soils, stems from its ability to improve soil quality and plant growth 2, 3, sustainably sequester carbon 4, and sorb harmful contaminants 2, 3, 5-7 whilst simultaneously offering alternatives for waste management and energy production by pyrolysis.

Biochars are being produced by numerous companies and organizations worldwide via different pyrolysis systems. Materials used for biochar production include (but are not limited to) woodchips, animal manure and construction wastes1. These differences are expected to alter the biochars’ physical and chemical properties and thus their ability to improve substrates, promote long-term stability and increase sorption capabilities. Additionally, during the pyrolysis process the biochar may become unintentionally contaminated with metals, PAHs and PCBs as a result of contaminated feedstocks or inappropriate pyrolysis conditions. Therefore, before biochar can be applied on a large scale to the environment as a soil amendment, careful characterization of the biochar for contaminants, specific surface area, cation exchange capacity, earthworm avoidance and germination and others suggested by the International Biochar Initiative (IBI) must be conducted. In 2013, the first Standardized Product Definition and Product Testing Guidelines for Biochar, which sets standards for biochar physical and chemical characteristics, was published and made publically available.

Research has shown that biochar produced at a commercial greenhouse in Odessa, ON, Canada has the ability to significantly improve plant growth in intensely degraded soils and sorb persistent organic pollutants (POPs) such as PCBs 2, 3. This biochar has been produced from three different feedstocks (i.e. organic matter sources) via a boiler system where the heat generated is used to warm their greenhouse operation during winter months.

This study provides characterization data pertinent to the production of biochar in a biomass boiler, and the use of biochar as a soil amendment. The objective of this study is to thoroughly characterize the physical, chemical and biological characteristics of six biochars according to standards set by the IBI in their Standardized Product Definition and Product Testing Guidelines (Version 1.1) (2013). These characteristics will be linked, where possible, to the performance of each biochar as agricultural amendments and their ability to sorb contaminants.

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NOTE: Chemical analyses were conducted at the Analytical Services Unit (ASU) in the School of Environmental Studies at Queen’s University (Kingston, ON). The ASU is accredited by the Canadian Association for Laboratory Accreditation (CALA) for specific tests listed in the scope of accreditation. Other analyses, including greenhouse trials, were conducted at The Royal Military College of Canada (Kingston, ON) in the Department of Chemistry and Chemical Engineering.

1. General Considerations

  1. To ensure quality assurance and quality control, analyze an analytical blank and an analytical duplicate, a sample duplicate and a standard reference material with each batch of samples (maximum batch size 10) for the methods in the protocol.
  2. Establish duplicate samples when sub-sampling from the original sample and go through the same preparation as the unknown samples. Ensure that duplicate values are within 20% of each other or repeat the analysis. Ensure that analysis outcomes of the blanks are below detection limits for the corresponding method. Standard reference material limits depended on the individual method but ensure that they are generally within 15–30% of the expected value.
    NOTE: In many of the methods described in the protocol, details are included on the suggested order of sample analysis including calibrants, blanks, high and low standards, and unknown samples. This is to ensure no cross contamination between samples and ensure a high standard to QA/QC.
    NOTE: Six biochars were produced at a commercial greenhouse and analyzed for chemical, physical and biological parameters. The names of each biochar reflect their production parameters or feedstock source (Table 1).

2. Test Category A: Basic Biochar Utility Properties

  1. Moisture and Organic Matter Content
    1. Use the loss on ignition procedure outlined by Nelson and Sommers (1996).
      1. Include a sample duplicate and standard reference material (Ottawa Sand) for every 10 unknown samples.
      2. Label 50-ml beakers with heat resistant marker, oven dry them at 105 °C, allow them to cool then record weight.
      3. Weigh 2 g of air-dried sample into the oven-dried beaker Dry sample at 105 °C for 24 hr, then remove from the oven and allow to cool.
      4. Once cool, weigh the beaker and the sample (X = weight of dried sample - weight of beaker).
      5. Place the sample in the muffle furnace and heat for 16 hr covering at 420 °C. Remove the sample from the furnace and allow to cool. Weigh the beaker with sample again and record the weight (Y = weight of ashed sample - weight of beaker).
      6. Perform the following calculations:
        i) Loss on Ignition = X-Y
        ii) % Moisture = ((Sample Weight – X)/Sample Weight) x 100%
        iii) % Organic Matter = (Loss on Ignition/X) x 100%
  2. Proximate and Ultimate Analysis
    NOTE: For proximate/ultimate analysis, four samples were analyzed: Low, High, Standard Fuel and High 2. PAH analysis was carried out on Low, High, and Standard Fuel. These were chosen as representative of the biochars produced since 2012.
    1. Conduct Proximate and Ultimate analyses at a commercial facility based on methods: ASTM D3172-13 8 and D3176-09, Standard Practice for Proximate and Ultimate9 Analysis of Coal and Coke, respectively.
  3. pH
    1. Calibrate the pH probe daily before use with calibration standards.
    2. Add 0.25 g biochar to 25 ml distilled, deionized water.
    3. Shake manually for 2 min, then centrifuge for 3,000 x g for 5 min.
    4. Collect supernatant into glass test tube and measure pH.
  4. Particle Size Distribution
    1. Analyze all samples in triplicate via progressive dry sieving adapted from ASTM D5158-98 10 using seven U.S. Standard sieves and pan (4.7, 2.0, 1.0, 0.50, 0.25, 0.15, and 0.0075 mm)
      1. Record the weight of each empty sieve and stack the sieves in order from pan to 4.7 mm with the 4.7 mm sieve being at the top.
      2. Place 60 g of biochar in the 4.7 mm sieve, place the lid on top and secure the stack of sieves on the shaker.
      3. Shake for 10 min and record the weight of each sieve. Report the data in an excel file as percent remaining in each sieve.

3. Test Category B: Toxicant Reporting

  1. Germination Tests
    1. Use the seed germination testing method outlined by Solaiman et al. (2012) 11.
      1. Use filter paper and potting soil as positive controls.
      2. Ensure that the respective weights of each treatment is 3 g of biochar, 10 g of potting soil, and 1 piece of filter paper.
        NOTE: These values are based on volume in the Petri dish so that each dish is ~50% full (by volume).
      3. Into the Petri dishes (8.5 cm in diameter), place five Cucurbita pepo spp. pepo (pumpkin) seeds and 50 Medicago sativa (alfalfa) seeds into each treatment.
      4. Using a graduated cylinder add 15 ml of water to all Petri dishes, then cover them with their respective lids.
      5. Place the Petri dishes for germination under a 14:10 hr (day:night) fluorescent photoperiod and maintain temperature at 27 ºC (±6 ºC).
      6. After seven days record the number of seeds germinated. Report results as % germinated per Petri dish. Measure the root length of germinated seeds using a ruler. Report root lengths as a sum for each Petri dish (cm/Petri dish).
  2. Earthworm Avoidance
    1. Store Eisenia fetida in a healthy soil matrix comprised of peat moss and potting soil and maintain soil moisture at ~30%.
    2. Use earthworm avoidance method described by Li et al. (2011). Choose worms ranging from 0.3–0.6 g in size.
      1. For this assay, use six avoidance wheels (Figure 1) or similar structure to those outlined in Environment Canada’s Acute Avoidance Test (Environment Canada, 2004).
      2. Mix biochars separately using a spade and bucket with potting soil at a rate of 2.8% (by weight).
      3. Fill each of the six compartments with 120 g of soil or soil/biochar mixture, with every other compartment serving as an unamended control (Figure 1) i.e. soil without biochar. Add 10 worms to the round middle compartment.
      4. Expose the worms for 48 hr keeping the avoidance wheel covered with aluminum foil to prevent worm escape. Maintain temperature conditions for the avoidance wheels between 20–25 °C. Monitor the soil moisture and maintain at ~30%.
      5. After 48 hr remove the worms and record their location in the avoidance wheel, i.e. if they are in the i) amended or ii) unamended compartments. Do not reuse worms for future testing.
  3. Polycyclic Aromatic Hydrocarbons (PAHs)
    1. Analyze PAHs by solvent extraction and GC-MS based on EPA 8270 12.
  4. Polychlorinated Biphenyls (PCB) Concentration
    1. Dry samples (10 g) overnight at 25 °C for 18–24 hr then grind them to a fine powder (particle size < 0.15 mm) with 10 g sodium sulphate and 10 g Ottawa sand.
    2. Include one analytical blank (Ottawa sand), one control (a known amount of PCB standard) and one analytical duplicate sample for every 10 unknown samples.
    3. Place 2 g sample into Soxhlet thimble and add 100 µl decachlorobiphenyl (DCBP) as an internal surrogate standard.
    4. Extract samples in a Soxhlet apparatus for 4 hr at 4–6 cycles per hour in 250 ml of dichloromethane.
    5. Using a gas chromatograph equipped with a micro-63Ni electron capture detector (GC/μECD), a fused silica capillary column (30 m, 0.25 mm ID × 0.25 μm film thickness) and appropriate software analyze biochar extracts for total Aroclors. Use helium as the carrier gas at a flow rate of 1.6 ml/min. Use Nitrogen as the makeup gas for the electron capture detector (ECD). Report values as μg/g dry weight.
  5. Metal Analysis
    1. Air-dry samples for 18–24 hr and grind into a fine powder (particle size < 0.15 mm) with a mortar and pestle.
    2. Using reagent grade concentrated acids, heat 0.5 g of the sample in 2 ml 70% (w/w) nitric acid and 6 ml 38% (w/w) hydrochloric acid, until the volume is reduced to 1–2 ml. Then make-up the solution to 25 ml in a volumetric flask using distilled, deionized water, filtered through a Whatman No. 40 filter paper.
    3. Analyze samples using a simultaneous inductively coupled plasma atomic emission spectrometer (ICP-AES) with the following standards/controls (see step Analyze multi-element ICP standards and check % error and correlation coefficients of the calibration curves. Standards are purchased in custom blends with many elements in each standard. Each element has a 3 point calibration curve (for example cadmium is run at 0, 0.1, 1.0 and 5 ppm). Verify curves with calibration check standards. Recalibrate approximately every 18 samples.
      1. Add internal standards (indium and scandium) ‘on line’ with samples to verify instrument stability. Analyze samples with additional quality control standards including certified reference materials (Bush, Branches and Leaves; White Cabbage and Spinach), method blanks (add acids to an empty digestion tube and treat them as described in 3.5.2 above), analytical duplicates, and field duplicates.
  6. Mercury
    1. Ensure the instrumentation meets the criteria outlined in US EPA Method 7473 and allows for direct mercury measurement
    2. Weigh 100 mg of ground air-dried biochar (particle size < 0.15 mm) into quartz or nickel weigh boats.
    3. Use an ICP-AES stock solution of 1,000 µg/ml Hg and 5% hydrochloric acid in double deionized water (DDI) to make working stocks (5 µg/ml, 1 µg/ml, 0.1 µg/ml, 0.01 µg/ml) and calibration standards.
    4. Use a cleaned empty boat as a method blank. Analyze samples starting with a Method blank, Low QC (20 ng Hg – 20 µl of 1 µg/ml Hg), Blank, High QC (200 ng Hg – 40 µl of 1 µg/ml Hg), Blank, Blank, Standard Reference Material (MESS-3), Blank, MESS-3, Blank, Sample 1, Blank, Sample 2, Blank, Sample 2 dup, Blank, Sample 3, Blank, etc.
    5. Place the boats in the instrument chamber where the sample will thermally decompose in a continuous flow of oxygen.
      NOTE: The combustion products will then be carried off in the oxygen flow and then further decomposed in a hot catalyst bed. Mercury vapors will be trapped on a gold amalgamator tube and subsequently desorbed for spectrophotometric quantitation at 254 nm.

4. Test Category C: Biochar Advanced Analysis and Soil Enhancement Properties

  1. Ammonium as Nitrogen
    NOTE: The method makes use of the Berthelot reaction wherein ammonium salts in the solution react with phenoxide. Addition of sodium hypochlorite causes the formation of a green-colored compound. Sodium nitroprusside is added to intensify the color.
    1. Weigh 5 g of ground air-dried sample (particle size < 0.15 mm) into a 125-ml Erlenmeyer flask. Add 50 ml of 2 M (0.01% (V/V) KCl. Put the flasks on a rotating shaker for 1 hr at 200 rpm. After shaking is complete, filter the samples through Whatman No. 42 filter paper into 100-ml plastic vials.
    2. Prepare Reagent Solutions:
      1. Alkaline Phenol — measure 87 ml of liquefied phenol into 1-L volumetric filled 2/3 with DDI water. Add 34 g NaOH, make up to volume with DDI water.
      2. Hypochlorite Solution — using 100-ml graduated cylinder measure 31.5 ml of commercial bleach (5–10%) and fill to 100 ml with DDI water. Transfer to bottle and add 1.0 g of NaOH pellets and allow them to dissolve.
      3. EDTA solution — dissolve 32 g of di-sodium EDTA and 0.4 g NaOH in a 1-L volumetric filled 2/3 with DDI water. Add 0.18 g nitroprusside and dissolve by shaking. Make up to volume with DDI water and add 3 ml Triton (10%).
    3. Make calibration standards (0.1, 0.2, 0.3, 0.5, 1.0, and 2.0 µg/ml N Concentration) using reagent grade NH4Cl and DDI water. Prepare QC reference standard from a reagent grade source of ammonium chloride different from the source used to make the standards. Use double deionized water as the blanks.
    4. Begin running the autoanalyzer. Design each run to start with the High Standard (2.0 µg/ml N) x 2, Calibration Standards (high to low), Method Blank, High Standard, Low Standard (0.1 µg/ml N) x 2, Wash Water, QC Reference Sample x 2, Samples, Sample duplicate, and High Standard., and Wash Water.
      NOTE: The autoanalyzer software will automatically calculate concentrations in the extract.
    5. Calculate the Biochar Concentration = (Extract Concentration x 50 ml (KCl)) / 5 g Biochar Sample.
  2. KCl Extractable Nitrite and Nitrate by Autoanalyzer
    NOTE: The Griess Ilosvay colorimetric method utilizes the reaction of nitrite ions with sulfanilamide under acidic conditions to form a diazo compound. The compound further reacts with N-1-naphthylethylenediamine dihydrochloride to form a magenta azo dye. Nitrate in the sample is converted to nitrite through exposure to a reducing agent (in this case a copper-cadmium reducing column). This gives a measure of the nitrate + nitrite concentration in the sample.
    1. Weigh 5 g of ground air-dried sample (particle size < 0.15 mm) into 125-ml Erlenmeyer flask. Add 50 ml of 2 M (0.01% (V/V)) KCl. Put the flasks on a rotating shaker for 1 hr at 200 rpm. After shaking is complete, filter the samples through Whatman No. 42 filter paper into 100-ml plastic vials.
    2. Allow reagents (Ammonium chloride and Color Reagent) to warm to room temperature.
    3. Turn on colorimeter to let the lamp warm up. Stored within the auto analyzer are reagent lines labeled Ammonium chloride, Color Reagent and Water; start the pump and allow water to run through the system, check all pump-tubing lines for proper function.
    4. Once the system has equilibrated, place lines in the respective reagents and allow to run for 5–10 min. Turn on the chart recorder. Wait for baseline to stabilize, and set to the 10th chart unit.
    5. Prepare 100 µg/ml nitrate and nitrite QC Stock Standards from KNO3 and NaNO2 and DDI water, respectively. To make a 10 µg/ml Intermediate Standard, add 5 ml of 100 µg/ml stock solution to 50-ml volumetric flask and make up to volume with 0.01% KCl. To make Calibration Standards combine 0.01% KCl and the 10 µg/ml intermediate standard prepared in 25-ml volumetric flasks to make calibration standards (0.05, 0.2, 0.5, 1.0, 1.5, 2 µg/ml NO3 or NO2). Use KCl for method blanks.
    6. Prepare spikes using 5 g of Ottawa sand (inert material) and add 0.05 ml of the appropriate 1,000 µg/ml QC standard for an end result of 10 mg N/kg sample. Make a combined NO3 + NO2 spike by spiking a single sample with 0.025 ml of each 1,000 µg/ml QC standard stock. Prepare one sample spike per run by spiking 5.0 g of the unknown biochar sample with 0.025 ml of the appropriate 1,000 µg/ml QC standard stock.
    7. Begin running analysis. Include a full set of calibration standards, two QC Reference Samples, at least two KCl blanks, and at least two Nitrite Standards, a set of Ottawa Sand Spikes and blanks and a Sample Spike in each run.
      NOTE: Standards may be rerun as markers between every 5 unknown samples and to verify the values for preparation of the standard curve.
    8. Repeat the 2.0 µg/ml standard at the end of each run. Run duplicate samples at a minimum rate of 10%. Run Nitrite + Nitrate analysis first, followed by the Nitrite analysis.
    9. Record on the Nitrite Nitrate Worksheet peak heights of all standards, QC checks and samples. Use the number of chart units as the measurement of height. To calibrate the instrumentation, use the relative heights of the standards. Ensure that the R2 value lies above 0.99, if not re-run the standards.
    10. Calculate the concentration of the samples using the formula:
      Extract Concentration = (Peak Height - Intercept of the Calibration Curve/Calibration Curve Slope) x Dilution
      Biochar Concentration = (Extract Concentration x 50 ml (KCl)) / 5 g Biochar Sample
    11. Subtract the estimated nitrite concentration from the nitrate plus nitrite concentration to calculate nitrate.
  3. Extractable Phosphorous (2% Formic Acid Extraction)
    NOTE: The auto analyzer software automatically calculates concentrations. The software reports calibration information, goodness of fit of the calibration curve, concentrations for all samples, calibrants, blanks and QC samples that have been run.
    1. Prior to analysis store samples in a clean glass container or sterile plastic bag. Keep samples refrigerated and analyze within two weeks or keep frozen for up to one year.
    2. Make all standards and QC standard with the same extraction fluid that is used for the samples. Use Estuarine Sediment as a standard reference material and in every bath of samples include two blanks to be extracted.
    3. Using a 1-L volumetric filled to 750 ml with DDI water, add 20 ml (98–99%) formic acid and fill to volume with DDI water.
    4. Add 1.0 g of ground air-dried sample (particle size < 0.15 mm) into a 125-ml Erlenmeyer flask. Add 50 ml of 2% formic acid solution. Put the flasks on sonicator for 10 min, then transfer onto rotating shaker for 1 hr at 200 rpm. After shaking, filter samples using Whatman No. 42 filter paper into another set of 125-ml Erlenmeyer flasks.
    5. Prepare Standards and Spikes:
      1. Prepare a 1,000 µg/ml QC Stock Standard from potassium dihydrogen orthophosphate and DDI water. Use the QC Stock Standard to make the Calibration Standards (5 µg/ml, 1 µg/ml, 0.5 µg/ml, 0.2 µg/ml, 0.1 µg/ml). Use 0.100 ml of the QC Standard to make the QC Spike. To make a QC Standard Check, add 0.100 ml of the QC Stock Standard to a 50-ml volumetric flask and make it up to volume with KCl.
        NOTE: This is a 0.2 µg/ml dilution concentration.
      2. Use Estuarine sediment as a QC Reference Sample. Use 0.01% KCl as the method blank.
    6. Analyze on the autoanalyzer system. Set samples up as Primer (High Standard (0.5 µg/ml), Calibrants (5 µg/ml, 1 µg/ml, 0.5 µg/ml, 0.2 µg/ml, 0.1 µg/ml), Blank, Null, High Standard (0.5 µg/ml), Low Standard (0.1 µg/ml), Low Standard (0.1 µg/ml), Null, QC (Reference Sample/ Estuarine Sediment), QC (Reference Sample/Estuarine Sediment), Method Blank, Sample 1, Sample 2, Sample 2 Dup, Sample 3 etc., High Standard, Null.
    7. In every batch of samples also extract two blanks: one is a calibration blank and it is to be placed in the standard rack of the autosampler, the other is a method blank and it is to be placed in the sample tray.
  4. Specific Surface Area
    NOTE: Analysis for Brunauer-Emmett-Teller (BET) surface area was conducted in the Chemical Biological Radio Nuclear (CBRN) Protection Lab at RMC. The method utilizes N2 gas sorption analysis at 77 K in a relative pressure range from 0.01 to 0.10 after degassing at 120 °C for a minimum of 2 hr. A duplicate sample was analyzed for every 6 unknown samples. Samples are not ground into powdered form prior to analysis.
    NOTE: Degassing times and pressures are specific to instrument manufacturer and the method provided has been validated previously with high temperature activated carbons.
  5. Cation Exchange Capacity (CEC)
    1. Follow the sodium acetate method for CEC described by Laird and Fleming (2008) to calculate CEC.
      1. Include one analytical blank (DDI water), standard reference material (Ottawa Sand) and duplicate for every 10 samples.
      2. Prepare saturating solution (1 M NaOAc pH 8.2) by dissolving 136.08 g of NaOAC.3H2O in 750 ml distilled, deionized water. Adjust the pH to 8.2 by adding acetic acid or sodium hydroxide. Dilute to 1 L with DDI water.
      3. Prepare first rinsing solution (80% isopropanol (IPA)) by combining 800 ml IPA with 200 ml distilled, deionized water. Then prepare the second rinsing solution (100% IPA).
      4. Prepare the replacing solution (0.1 M NH4Cl) by dissolving 5.35 g NH4Cl into 1 L distilled, deionized water.
      5. Weigh 0.2 g of sample (air dried, not ground) into a 30-ml centrifuge tube. At the same time, weigh 0.5 g of the same air dried sample into a pre-weighed aluminum drying pan. Place the sample in the aluminum drying pan in the oven at 200 °C for 2 hr, cool it in a desiccator and then weigh again to determine the water content of the air-dried sample. Use this sample to calculate the water content correction factor, F (step
      6. Add 15 ml of the saturating solution, vortex, then centrifuge at 3,000 x g for 5 min. Decant and carefully discard the supernatant to ensure no sample is lost. Repeat this step two more times.
      7. Add 15 ml of the first rinsing solution. Vortex and centrifuge at 3,000 x g for 5 min. Decant and carefully discard the supernatant. Repeat this step several times, each time measuring the electrical conductivity of the supernatant solution. When the conductivity of the supernatant drops below the conductivity of NaOAc saturated with IPA (~6 µS/cm), switch to the second rinsing solution. Continue to rinse the sample until the conductivity of the supernatant drops below 1 µS/cm.
      8. Allow the sample to air dry in a fume hood, then add 15 ml of the replacing solution. Vortex and centrifuge at 3,000 x g for 5 min. Decant and save the supernatant into a 100-ml volumetric flask. Repeat this step three more times, each time saving the supernatant into the same volumetric flask. Then bring the volumetric to 100 ml with distilled, deionized water.
      9. Analyze the sodium content via inductively coupled plasma-atomic emission spectrometry (ICP-AES) as previously described.
      10. Perform the following calculations:
        F = (weight of oven dried, air dried sample - weight of air dried sample)
        C = Na concentration (mg/L) in the 100-ml volumetric flask
        W = weight (g) of air-dry sample added to centrifuge tube
        CEC = (C x 0.435)/(W x F)(cmol/kg)

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Representative Results

A summary of all results including a comparison to the criteria set by the IBI 13 can be found in Tables 1 (summary), 2 (New, High, Low, Third Feedstock and High-2 biochars) and 3 (Old biochar). All biochars and feedstocks used in 2012 and 2013 (Table 2) were well within the criterion set by the IBI and there were little differences among biochars. Old biochar (Table 3), the first biochar submitted for testing, was made from used shipping pallets and construction wastes and was determined to have elevated levels of the metals arsenic, chromium, copper, and lead. Old biochar also had the lowest levels of organic carbon (63.2%) as determined by loss on ignition. This biochar had the highest levels of extractable phosphorus (850 mg/kg) and CEC (34.8 cmol/kg), as well as the highest percentage of fine particles (<0.5 mm, 48%). Old biochar was also the only biochar to fail the germination test (Figure 3) and it was determined that Eisenia fetida (soil invertebrate) significantly avoided the 2.8% Old biochar amendment, whereas they preferred the 2.8% amendment of the New biochar (Figure 2).

Test Category A: Basic Biochar Utility Properties

Biochar production via pyrolysis is essentially the carbonization of biomass. The carbonization process allows for the transformation of structured organic molecules of wood and cellulose materials into carbon, or carbon-containing residues, which are often aromatic in nature 14-18. Carbonization is obtained through the elimination of water and volatile substances from the biomass feedstock, due to the action of heat during the pyrolysis process 19. All of the biochars produced at the commercial greenhouse contained a relatively low moisture percentage (<5%) with the exception of Old biochar. All biochars are categorized by the IBI as Class A (>60%) in terms of their composition of organic carbon as a result of complete carbonization of the feedstock material via pyrolysis. Thus due to the high percentage of organic carbon, all biochars produced have a low percentage of ash (<2.5%), which is the inorganic or mineral component of the biochar 13. Although these low ash biochars do not provide substantial amounts of nutrients directly to the soil as do their high-ash biochar (often made from manures and bones) counterparts; the carbon content of these biochars is much higher and therefore they have higher long-term nutrient retention abilities 20-22.

The hydrogen to carbon ratio (H:C) is a term often used to measure the degree of aromaticity and maturation of the biochar, which has been linked to their long-term stability in the environment 18. For biomass feedstock containing cellulose and lignin, the H:C ratios are approximately 1.5. However, pyrolysis of these materials at temperatures greater than 400 °C is expected to produce biochars with H:C ratios <0.5. It has been reported that an H:C ratio < 0.1 indicates a graphite-like structure in the biochar 23. All biochars in this report have H:C ratios less than 0.02, indicating that these biochars are highly aromatic in nature and will have long-term stability in the environment.

Soil pH is a measure of soil acidity, and unfortunately many agricultural soils in Canada and worldwide are acidic (pH < 7), meaning that they are not ideal for crop growth. Biochars with an alkaline pH (> 7), such as those being produced at the greenhouse, can be added to acidic soils to increase the soil pH to levels that are more appropriate for plant growth.

Another important soil characteristic for plant growth is particle size distribution (PSD). Biochars that have a higher percentage of coarse particles may favorably increase soil aeration and prevent biochar movement into the subsoil over time, thereby increasing the length of time biochar offers benefits to plant growth 24. However, smaller particle sizes are favored for biochars that are being produced for remediation purposes with the intent to sorb contaminants and minimize their bioavailability, as contaminants are more easily able to access pore space for binding 3,25,26. Also smaller particles sizes increases the number of biochar particles per unit volume of soil which is favorable for contaminant sorption27. As in a previous study3, fine particles are defined as those < 0.25 mm and coarse particles as > 0.5 mm. The biochars named New-, High- and Third Feedstock have a high proportion of coarse particles (~98%), and a low proportion of fine particles (~2%). The biochar produced at a slightly lower temperature, had 89% coarse and 11% fine particles sizes. All of these biochars may offer substantial improvements to soil texture and aeration especially in degraded or clay type soils. The Old biochar had a PSD that differed substantially from the others, having 52% coarse and 48% fine particles. A biochar with this PSD may be preferable for use at contaminated sites, where contaminant sorption is the primary focus.

Test Category B: Toxicant Reporting

Biological testing of biochar is important to assess the toxicity (if any) of these materials to soil invertebrates and plants. To date, there is little existing literature on the potential impact of biochar on terrestrial organisms and their associated response, and often the literature that does exist presents conflicting results. Exposure to contaminants may inhibit earthworms ability to perform essential soil functions such as decomposition, nutrient mineralization, and soil structure improvements 28. New biochar showed no detrimental effects on the earthworm Eisenia fetida as assessed by earthworm avoidance, however worms significantly avoided Old biochar (Figure 2). Germination assays are a technique used to evaluate the toxicity of a particular material to plants. Potting soil served as a better control than filter paper as the filter paper often encouraged mold formation. Pumpkin and alfalfa seeds germinated well with 67% ± 12% and 81% ± 6% germination, respectively. Roots also proliferated well with average lengths after seven days being 14 cm ± 0.6 cm and 55 cm ± 8 cm for pumpkins and alfalfa, respectively. As with the earthworm avoidance studies Old biochar showed toxicity to plants and all other biochars evaluated showed no detrimental effects to seed germination as measured by percent germination and root length after seven days (Figure 3).

Although some types of biochar have the potential to sorb organic contaminants and reduce their toxicity in the environment, careful characterization of the biochar is required to ensure that it does not contain harmful contaminants such as PAHs, PCBs, and metals as a result of contaminated feedstocks or pyrolysis conditions. None of the biochars produced at the greenhouse had PAH concentrations exceeding IBI guidelines. Old biochar was determined to have elevated levels of PCBs and the metals arsenic, chromium, copper, and lead, however none of the biochars produced from the other two biomass materials contained metals above IBI guidelines. Old biochar was produced from used shipping pallets and construction wastes which is likely the source of the metal contamination. Although Old biochar would not be suitable for use in agricultural soils or home gardens, all other biochars could be used for these purposes.

Test Category C: Biochar Advanced Analysis and Soil Enhancement Properties

Biochars containing a high concentration of ammonium and nitrate may be applied to agricultural soils to offset the requirements for synthetic fertilizers. However, if biochar contains an excess of these nitrogen compounds then application on a large scale could increase the atmospheric N2O concentration and contaminate drinking water sources with nitrates. None of the biochars studied contained elevated amounts of ammonium or nitrate.

Phosphorus is an essential component for many physiological processes related to proper energy utilization in both plants and animals. Biochars with moderate amounts of available phosphorus will act as important plant fertilizers. In Ontario, soils containing 15–30 mg/kg phosphorus are considered low, 31–60 mg/kg moderate, and 61–100 mg/kg high. Old biochar was highest in available phosphorus at 850 mg/kg and may not be suitable for adding to soils already classified as high in phosphorus. However, all other biochars tested had a much lower amount of available phosphorus and would not be expected to cause problems when added at rates up to 10% (w/w).

The components of biochar (except moisture) that are released during pyrolysis are referred to as volatile matter. These components are typically a mix of short and long chain hydrocarbons, aromatic hydrocarbons with minor amounts of sulfur. Volatile matter was determined via proximate analysis which also determines the moisture and ash content of biochars (section 2.2). The volatile content affects the stability of the material 29, N availability and plant growth 30. In theory, biochars high in volatile matter are less stable and have a higher proportion of labile carbon that provides energy for microbial growth and limits the availability of nitrogen necessary for plant growth. A study by Deenik et al., (2010) considered 35% volatile matter to be high (inducing nitrogen deficiency), and 10% volatile matter to be low. All biochar in this report contained less than 20% volatile matter, and hence would not be expected to limit plant growth. Proximate analysis determination of volatile matter is most important for biochars with low ash concentrations such as those produced at the commercial greenhouse.

Specific surface area (SSA) is a measure of the porosity of a biochar. It includes not only the external biochar surface area, but also the surface area within the pore spaces and is an important characteristic used to predict the ability of a biochar to sorb organic contaminants. Contaminant sorption has been attributed to π-π interactions (attractive, non-covalent binding) between the aromatic ring(s) of the contaminant and those of the biochar 31. Activated carbon (AC) is a charcoal-like material that is treated during its production to maximize its porosity and therefore has higher SSAs than most biochars. Although all the of biochars presented in this report have SSAs in the 300 m2/g range (i.e. much less than that of AC; ~800 m2/g), as reported in Denyes et al., 2012 and 2013, the biochars, Old and New, have both shown significant potential to serve as a soil amendment for the remediation of PCBs.

Cation exchange capacity (CEC) is a measure of the number of cations (positively charged ions) that a soil particle is capable of holding at a given pH. The ability of the soil to hold cations is due to electrostatic interactions with negatively charged sites on the surface of a particle, such as hydroxyl (OH-) and carboxyl (COO-) groups 32, 33. The CEC of the soil can be linked to the ability of the soil to hold nutrients and retain cations from fertilizers which are essential for plant growth. Also, many environmental contaminants such as lead, cadmium and zinc have positive charges; therefore soils with a high CEC may function to prevent the leaching of these contaminants into drinking water sources. Biochars have been reported to increase the CEC of soils, due to the slow oxidation of the biochar surface which increases the number of negatively charged sites, and therefore may reduce fertilizer requirements and immobilize positively charged contaminants in soils 32. Typically, sandy soils have a CEC between 1–5 cmol/kg, loam soils 5–15 cmol/kg, clay type soils >30 cmol/kg and organic matter 200–400 cmol/kg. The methods for determining the CEC of biochar are still in their infancy and therefore should be considered in relative terms. The CEC of the biochars produced at the greenhouse are higher than the CEC of PCB-contaminated soils (Denyes et al., 2012), but lower than compost amended soils.

Figure 1
Figure 1. Earthworm avoidance wheel. The wheels are produced from steel and the worms are allowed to move throughout the compartments via multiple holes which are approximately 5 cm in diameter.

Figure 2
Figure 2. Earthworm avoidance assay of Old and New type biochars. The biochar titled “Old” was produced via construction wastes, whereas the biochar titles “New” was produced from sawdust materials. * indicates a significant difference between unamended potting soil and potting soil amended with 2.8% of either biochar (p < 0.05).

Figure 3
Figure 3. Percent germination of two different plant species. Pumpkin (Cucurbita pepo spp. pepo) and alfalfa (Medicago sativa) were grown in triplicate in various biochars produced at a commercial greenhouse for seven days. Old and New refer to biochars made from different feedstocks, whereas Low and High refer to different temperatures of pyrolysis. * indicates significantly difference from the controls (potting soil and filter paper).

Sample  Feedstock  Pyrolysis  Temperature Organic Matter  (LOI) pH  CEC PSD PSD SSA
Coarse Fine
°C  cmol/kg  % % m2/g 
Old  1 >700  63.2 9.3 34.8 51.7 48.3 373.6
New  2 700 97.8 9 16 98.7 1.3 324.6
Low Temp  2 500 96.7 8.7 15.9 86.2 13.8 336.9
High Temp  2 >700  97.9 8.4 11.1 98.1 1.9 419.5
Third Feedstock  3 700 96.2 9.6 13.2 97.6 2.4 244.4
High Temp-2  3 >700  97.1 9.1 17.1  97.9 1.9  428
LOI: Loss on Ignition, CEC: Cation Exchange Capacity, PSD: Particle Size Distribution, SSA: Specific Surface Area

Table 1. Feedstock type, pyrolysis temperature and physical characteristics of the six biochars.

Requirement IBI  Biochar Feedstock Range Unit
Criteria  Range
Test Category A: Basic Biochar Utility Properties - Required for All Biochars
Moisture Declaration <0.1–4.3 %
Organic Carbon Class 1 > 60% 96.2–97.8 (LOI) %
Class 2 > 30% 92.44–97.93(Pro/Ult)
Class 3 > 10 < 30%
H:Corg  0.7 max 0.01–0.02 Ratio
Total Ash Declaration 1.38–2.26 %
Total N Declaration 0.28–1.06 %
pH Declaration 8.4–9.6 pH
Particle Size Distribution Declaration 86–98 % Coarse 
Test Category B: Toxicant Reporting- Required for All Feedstocks
Germination Pass/Fail Pass
Earthworm Avoidance Declaration No Avoidance
Polyaromatic Hydrocarbons (PAHs) 6–20 <2.0 mg/kg
Polychlorinated Biphenyls (PCBs) 0.2–0.5 <0.1 mg/kg
Arsenic 12–100 <1.0 <1.0 mg/kg
Cadmium 1.4–39 <1.0 <1.0 mg/kg
Chromium 64–1,200 <2.0 <2.0–2.6 mg/kg
Cobalt 40–150 <1.0 <1.0 mg/kg
Copper 63–1,500 3.6-6.5 <2.0–5.9 mg/kg
Lead 70–500 <2.0–2.7 <2.0–8.1 mg/kg
Mercury 1,000–17,000 <5.0–294 ng/g
Molybdenum 5–20 <2.0 <2.0 mg/kg
Selenium 1–36 <10 <10 mg/kg
Zinc 200–7,000 5.6–56.2 7.8–30.5 mg/kg
Chlorine Declaration mg/kg
Sodium Declaration 137-878 <75-770 mg/kg
Test Category C: Biochar Advanced Analysis and Soil Enhancement Properties- Optional for All Biochars
Mineral N (Ammonium and Nitrate) Declaration <0.2–6.1 mg/kg
Total Phosphorus Declaration 69.5–276 52.5–74 mg/kg
Available Phosphorus Declaration 9–80 mg/kg
Volatile Matter Declaration 12.47–19.09 %
Specific Surface Area Declaration 244–428 m2/g
Cation Exchange Capacity Declaration 11.1–17.1 cmol/kg

Table 2. Summary Criteria and Characteristics for New, High, Low, Third and High-2 Biochars and Feedstocks. All biochars listed in this table are produced from similar feedstocks at the same pyrolysis facility.

Requirement IBI  Biochar Range Feedstock Range Unit
Test Category A- Basic Biochar Utility Properties - Required for All Biochars
Moisture Declaration 20 %
Organic Carbon Class 1 > 60% 63.2 (LOI) %
Class 2 > 30%
Class 3 > 10 < 30%
H:Corg  0.7 max Ratio
Total Ash Declaration %
Total N Declaration %
pH Declaration 9.3 pH
Particle Size Distribution Declaration 52 % Coarse 
48 % Fine
Test Category B: Toxicant Reporting- Required for All Feedstocks
Germination Pass/Fail Fail
Earthworm Avoidance Declaration Avoided
Polyaromatic Hydrocarbons (PAHs) 6–20 mg/kg
Polychlorinated Biphenyls (PCBs) 0.2–0.5 1.2 mg/kg
Arsenic 12–100 167 <1.0 mg/kg
Cadmium 1.4–39 <1.0 <1.0 mg/kg
Chromium 64–1,200 206 <20 mg/kg
Cobalt 40–150 5.3 <5.0 mg/kg
Copper 63–1,500 558 <5.0 mg/kg
Lead 70–500 314 <10 mg/kg
Mercury 1,000–17,000 <5.0 ng/g
Molybdenum 5–20 <2.0 <2.0 mg/kg
Selenium 1–36 <10 <10 mg/kg
Zinc 200–7,000 498 <15 mg/kg
Chlorine Declaration mg/kg
Sodium Declaration 6460 <75 mg/kg
Test Category C: Biochar Advanced Analysis and Soil Enhancement Properties- Optional for All Biochars
Mineral N (Ammonium and Nitrate) Declaration 2.6 mg/kg
Total Phosphorus Declaration mg/kg
Available Phosphorus Declaration 850 mg/kg
Volatile Matter Declaration %
Specific Surface Area Declaration 373.6 m2/g
Cation Exchange Capacity Declaration 34.8 cmol/kg

Table 3. Summary Criteria and Characteristics for Old Biochar and Feedstock. The biochar listed in this table was produced from construction wastes at the same pyrolysis facility as the biochars listed in Table 2.

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All of the methods listed in the protocol have been carefully validated and extensively used for soils. As biochar characterization is still in its infancy, the effectiveness of these methods for the carbon-rich substrate was largely unknown. Hence, although these methods themselves are not novel, their application to routinely characterize biochar is. In terms of quality assurance/ quality control, there were no issues among any of the methods with respect to the blanks being below detection limits or the recoveries being correct for the standard reference materials. This indicates that these methods are suitable to be used for the characterization of biochar and other charcoal-like materials. Many different methods have been used to characterize biochars in the literature20, 34-41 however, as biochar becomes increasingly accepted as a soil additive, routine methods are required.

Cation exchange capacity was the only method in which difficulty arose. The method for calculating the CEC of a sample is dependent on the weight of sample and the concentration of sodium in that given weight. Biochar has a very low density and therefore does not pelletize at the bottom of the tube after centrifugation, as soil does. Therefore, when decanting and discarding the supernatant in steps 6 and 7 of the method (4.4), it is important to not lose any of the biochar sample. Pipetting the solution from the centrifuge was required to avoid any sample loss.

Other analytical methods were easily adapted from soil methods. Ultimate and proximate analysis is specific to biochar and similar products such as coal, and hence is not normally available in laboratories which routinely analyze soils. Another method (ASTM D1762) is available, for the determination of moisture, volatile matter, and ash in charcoal made specifically from wood. This method would also have also been suitable for proximate analysis. When determining loss on ignition for percent organic matter and percent moisture some may choose to perform these analyses at temperatures greater than 420 °C, especially if the biochars in question are produced via very high temperatures of pyrolysis. In the case this particular study 420 °C was sufficient to completely ash all biochars, and although not discussed this temperature was sufficiently high to ash even activated carbon.

Working with biological organisms such as plants and worms can often be challenging. Selecting the appropriate study organisms is of particular importance. The soil invertebrate Eisenia fetida is used frequently as a terrestrial organism model in contamination experiments because this species is capable of surviving at high concentrations of organic contaminants, is very well researched, and is ecologically relevant in many areas of the globe 2, 28, 42-46. Soil invertebrates play an important role in the soil matrix, as they degrade organic matter, cycle nutrients, and transfer water. The plant species’ alfalfa (M. sativa) and pumpkin (C. pepo) were chosen for the germination assays as they are commonly grown in Canada and have been used in our complimentary work on contaminant remediation 2, 3, 47. Greenhouse conditions for germinating seeds need to be carefully monitored to ensure proper functioning of lighting and to avoid extreme temperature fluctuations.

The characterization of biochar is essential to its successful application as measured parameters will indicate the effectiveness of different biochars for different applications (i.e. whether a biochar is appropriate for contaminant sequestration, soil quality improvement, contaminant remediation etc.). Because the methods detailed here are widely available for soil analysis, they are a cost-effective means for characterization of biochars, and should be widely employed prior to large-scale application of biochar in the field.

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The authors declare that they have no competing financial interests.


This work was funded by the Government of Canada’s Federal Economic Development Agency (FedDev) Applied Research and Commercialization Extension to Queen’s University (Dr. Allison Rutter and Dr. Darko Matovic). Sincerest thank you to Burt’s Greenhouses (Odessa, ON) for providing the biochars. Special thanks to Yuxing Cui of the CBRN Protection Group at RMC and staff of the ASU and Zeeb Lab for their ongoing support.


Name Company Catalog Number Comments
Biochar Burt's Greenhouses All six biochars were produced at Burt's Greenhouses via BlueFlame Boiler system
NaOAc Fisher Scientific E124-4 Dissolving 136.08 g of NaOAC.3H2O in 750 ml distilled, deionized water (DDI water)
Acetic Acid Fisher Scientific A38-212
Sodium Hydroxide Fisher Scientific SS284-1
Isopropanol Fisher Scientific A416P4 80% IPA: 800 ml IPA with 200 ml DDI water.
NH4Cl Fisher Scientific A649500 Dissolving 5.35 g NH4Cl into 1 L DDI water. 
Alumminum Drying Pan Fisher Scientific 08-732-110
Drying Oven Fisher Scientific 508N0024 200 °C for 2 hr.
Desiccator Fisher Scientific 08-595A
Balance Mettler 1113032410
Saturating Solution Fisher Scientific 06-664-25
Vortex Barnstead/Thermolyne 871000536389   
Centrifuge International Equipment Company 24372808 3,000 x g for 5 min.
Rinsing Solution Fisher Scientific (Ricca Chemistry Company) 06-664-24
Conductivity Meter WESCAN 88298
Replacing Solution Fisher Scientific 06-664-24
ICP-AES Varian EL00053841
ASAP 2000 Surface Area Analyser  Cavlon 885 Degassing at 120 °C for a minimum of 2 hr.
Muffle Furnace Fisher Scientific 806N0024 Heat for 16 hr covering at 420 °C.
pH Meter Fisher Scientific 1230185263
Sieve Fisher Scientific 2288926 4.7 mm sieve being at the top.
Sieve Skaker Meinzer II 0414-02 Shake for 10 min.
Sodium Sulphate VWR EM-SX0761-5
Ottawa Sand Fisher Scientific S23-3
Soxhlet Apparatus Fisher Scientific (Pyrex) 09-557A 4 hr at 4–6 cycles/hr.
DCBP Suprlco Analytical 48318   
Dichloromethane Sigma Aldrich 40042-40855-U
6890 Plus Gas Chromatograph Micro 63 Ni ECD Agilent US00034778
Helium AlphaGaz SPG-NIT1AL50SMART
Nitrogen AlphaGaz SPG-HEL1AL50SMART
Mortor and Pestle Fisher Scientific (CoorsTeh) 12-948G
Nitric Acid Fisher Scientific 351288212
No. 40 Filter Paper Fisher Scientific (Whatman) 09-845A
Quartz/Nickel weigh boats Fisher Scientific 11-474-210
DMA-80 ATS Scientific 5090264
98–99% Formic Acid Sigma Aldrich 33015-1L 1 L volumetric filled to 750 ml with DDI water add 20 ml formic acid and fill to volume with DDI water.
Sonicator Fisher Sientific 15338284
Rotating Shaker New Brunswick Scientific (Innova 2100) 14-278-108 1 hr at 200 rpm.
No. 42 Filter Paper Fisher Scientific (Whatman) 09-855A
WhirlPacks Fisher Scientific R55048
Potassium Dihydrogen Orthophospahte Fisher Scientific 181525
2 M KCl Fisher Scientific P282100
Plastic Vials Fisher Scientific 03-337-20
Ammonium Chloride Fisher Scientific PX05115 Allow to warm up to room temperature
Colour Reagent Fisher Scientific 361028260 Allow to warm up to room temperature
Colorimeter Fisher Scientific 13-642-400 Turn on to let the lamp warm up and run for 5 min.
ASEAL Auto Analyzer 2 SEAL 4723A12068
Liquified Phenol Fisher Scientific MPX05115 Alkaline Phenol: Measure 87 ml of liquefied phenol into 1-L volumetric filled 2/3 with DDI water. Add 34 g NaOH, make up to volume with DDI water.
NaOH Fisher Scientific S318-3
Commercial Bleach Retail Store Hypochlorite Solution: Using 100-ml graduated cylinder measure 31.5 ml of commercial bleach and fill to 100 ml with DDI water.
NaOH Pellets Fisher Scientific S320-1
Disodium EDTA Sigma Aldrich E5124
Sodium Hyprchlorite Fisher Scientific SS290-1
Triton (10%) Fisher Scientific BP151-100
Sodium Nitroprusside Fisher Scientific S350-100
Ammonium Salts Fisher Scientific A637-10
Phenoxide Fisher Scientific AC388611000
Eisenia Fetida The Worm Factory
Spade Retail Store
Bucket Retail Store
Potting Soil Retail Store
Avoidance Wheel Environment Canada Constructed by a modified design from Environment Canada’s Acute Avoidance Test.
Alumminum Foil Fisher Scientific 01-213-100
Petri Dishes Fisher Scientific 08-757-11 8.5 cm in diameter.
Pumpkin Seeds Ontario Seed Company (OSC) 2055
Alfalpha Seeds Ontario Seed Company (OSC) 6675
Centrifuge Tubes (30 ml) Fisher Scientific  22-038-906
Beakers (50 ml) Fisher Scientific (Pyrex) 02-540G Oven dry at 105 °C.
Beakers (30 ml) Fisher Scientific (Pyrex) 20-540C
Erlenmeyer Flasks (125 ml) Fisher Scientific (Pyrex) S76106C
Volumetric Flask (100 ml) Fisher Scientific (Pyrex) 10-211C
Estuarine Sediment National Insititute of Standards 1546A Standard Reference Material
Bleach Clorox Ultra (5–10% sodium hypochlorite)



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