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

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 3.5.3.1). 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 10^th 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 4.4.1.10).
      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)