Effects of Unoxidized Biochars, Oxidized Biochars, Zinc and Compost on Cadmium Uptake in Spinach

Effects of Unoxidized Biochars, Oxidized Biochars, Zinc and Compost on Cadmium Uptake in Spinach | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Effects of Unoxidized Biochars, Oxidized Biochars, Zinc and Compost on Cadmium Uptake in Spinach Bárbara Samartini Queiroz Alves, Luiz Arnaldo Fernandes, Randal J Southard This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3857978/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Cadmium (Cd) is a toxic element that can be consumed by humans through food, posing a public health problem. Application to soil of zinc chloride (ZnCl 2 ), and amendments, such as compost and biochar, have been proposed as solutions to prevent Cd from entering the food chain. Biochar is a stable carbon-rich charcoal that is produced by an eco-friendly method. The objective of this research was to compare the effectiveness of the treatments as remediation materials for an agricultural soil enriched with Cd. Pot experiments were conducted with spinach and soil material collected in the Salinas Valley of California, for three consecutive life-cycles of spinach. We utilized five biochars (pre-selected in laboratory) in their unoxidized and oxidized (with hydrogen peroxide - H 2 O 2 ) versions, at application rates of 1% and 5% by volume. Cd uptake by spinach was compared to uptake in soil alone (control), soil + 25 mg/kg Zn, and soil + 5% poultry litter compost by volume. Compared to the control, some biochars significantly reduced Cd uptake and increased yield. Compost was also a successful amendment and may be the most cost-beneficial for farmers, although biochar produces carbon credits. The Zn treatment generally reduced Cd uptake, but also reduced yield. To achieve uptake values close to the CODEX limit (4 mg/kg dry wt.) it is necessary to lime all treatments to approximately pH 7. The oxidation of biochars with H 2 O 2 did not decrease Cd uptake. Biochar Cd removal Oxidizing with H2O2 Spinach Waste management Soil chemistry Highlights Biochar shows promise in reducing Cd uptake. Higher application rates of biochar did not consistently reduce Cd uptake, and the effect varied depending on the type of biochar used. The Zn treatment reduced Cd uptake but had variable effects on yield. Compost was found to be the most cost-beneficial solution for farmers, but biochar has potential benefits in terms of carbon credits. Oxidation of biochars with H 2 O 2 did not significantly decrease Cd uptake. Introduction Cadmium (Cd) is a nutritionally non-essential persistent metal element that can accumulate in the food chain (i.e., half-life of 10–30 years in the kidneys) and is often toxic to plants, animals, and microorganisms at very low concentrations (Åkerblom et al. 2007 ; UNEP 2010 ). In humans, 90% of Cd exposure in the non-smoking population is through food, where cereals, vegetables, and potatoes represent more than 80% of exposure (Järup and Åkesson 2009 ; Clemens et al. 2013 ). Global average weekly intake from food is estimated to be within 1.9 and 3.0 µg/kg body weight (Sigel et al. 2013 ). Although only 3–5% of the ingested Cd is absorbed, it is estimated that a large portion of the global population has already exceeded the tolerable intake dose of 2.5 µg/kg body weight weekly (EFSA 2012 ). The reported main health effects brought on by excessive Cd uptake are renal dysfunction, osteoporosis, and cancer (WHO 2010 ). The development of effective and agronomically feasible solutions to prevent Cd from entering the food chain is imperative (Hamid et al. 2019 ). The Salinas Valley in California is one of the most productive agricultural areas in the USA (production value of about US $ 13,000 per acre (CDFA, 2015 ). Many soils in the valley are derived from the Monterey Shale, an organic, and phosphate-rich sedimentary rock from the Miocene (Filippelli 2011 ), and have naturally high Cd concentrations (up to 35 mg/kg) (Lund et al. 1981 ). The main crops in the Salinas Valley are leafy vegetables, strawberries, cauliflower, and wine grapes. Among these, spinach ( Spinacia oleracea ) is of concern, because it is known to be a Cd accumulator crop (Wolnik et al. 1985 ), capable of translocating Cd from the roots to the shoots at concentrations higher than in non-accumulating species. The mechanism of uptake (not specific for spinach) is related to a protein that also transports Zn and Fe through the root membrane, followed by translocation to the shoot (Grant et al. 1998 ; Rascio and Navari-Izzo 2011 ; Sterckeman and Thomine 2020 ). Smith and Hartz ( 2016a ) showed that Cd uptake by spinach is roughly proportional to the soil Cd content. Alexander et al. ( 2006 ) showed that the uptake can vary by up to 40%, depending on the spinach variety. In a 2013 field survey, the Salinas Valley agricultural soils contained concentrations up to 9 mg/kg total Cd (the world average is 0.1–0.5 mg/kg) (UNEP 2010 ). Spinach planted in soils with high Cd-concentration had leaf mean concentrations above levels considered safe (up to 1.90 mg/kg, fresh wt.), which is more than 10 times the average concentration found in spinach grown in other areas (Giclas 2017 ) and almost 10 times more than the CODEX limit. The CODEX limit is established by the joint initiative of the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) for food standards, which is set at 0.2 mg/kg fresh wt. (~ 4 mg/kg dry wt.) (FAO/WHO 2011 ). These high Cd levels led to a recall of baby spinach by the California Department of Public Health in 2015 (News Desk 2015 ). Solutions have been proposed, such as high rates of Zn application (25–50 mg/kg of elemental Zn on a soil dry wt.) (Paul and Chaney 2017 ) to compete for Cd uptake (McKenna et al. 1993 ), liming the soil to increase pH (Cd precipitates from soil solution in alkaline pH) (Chaney et al. 2009 ), and remediation with organic amendments (Rehman et al. 2018; Simmler et al. 2013 ), such as compost (Bashir et al. 2020 ) or biochar (Chen et al. 2020 ; Qiu et al. 2020 ). Biochar, is a versatile, low-cost, carbon-rich, porous material produced from the thermal carbonization of the biomass under oxygen limited conditions (Igalavithana et al. 2017 ). As a versatile material, biochar can be modified to maximize Cd retention (Sizmur et al. 2017 ). It is expected that oxidation of biochar surface to maximize oxygenated functional groups will increase Cd retention (Frišták et al. 2015 ). An oxidizing method that has been becoming popular is washing with hydrogen peroxide (H 2 O 2 ) (Huff and Lee 2016 ). The intent of this study is to investigate Cd uptake by spinach from soil amended with biochar, ZnCl 2 , and poultry litter compost additions. Further, we investigated appropriate application rates, and how long remediation methods affect Cd uptake. Our focus is on the effectiveness of biochar as a remediation material for agricultural soils enriched with Cd. The specific objectives were: (i) evaluate Cd uptake by spinach (shoots) comparing pre-selected biochars with ZnCl 2 or compost applications; (ii) evaluate effects of two application rates (1% and 5%) of biochar; (iii) evaluate how treatments change over three consecutive life-cycles of the spinach; and (iv) compare unoxidized biochars with their oxidized (with H 2 O 2 ) versions. We hypothesize: (i) biochar will be more effective to reduce the accumulation of Cd in spinach than ZnCl 2 application or compost; (ii) higher biochar application rates will more strongly reduce Cd uptake; and, (iii) since Cd inputs can be maintained from weathering of parent material, it is likely to be necessary to reapply biochar over time. Materials and Methods Soil sampling and characterization The soil material collection site was selected based on previous research. Smith and Hartz ( 2016b ) studied a variety of soils in the Salinas Valley with Cd concentrations of 0.5-8 mg kg − 1 . Their research suggested that growers not grow spinach in soils with concentrations above 2.5 mg kg − 1 (data not published). We selected a soil with a Cd concentration closest to this threshold. Soil material was collected from the 0–25 cm depth in a field mapped as Elder loam that grew spinach in May 2017 near Greenfield, California (36°17'33.92"N, 121°15'55.80"W). The USDA classification of the Elder series is coarse-loamy, mixed, superactive, thermic Cumulic Haploxerolls. Soil material was mixed, air-dried to a constant weight, manually ground with mortar and pestle, and sieved to pass 2 mm for subsequent laboratory analysis. For soil characterization, pH and electric conductivity (EC) were measured in distilled water (1:1 w/w) after 90 min equilibration. Total C and N were determined by dry combustion-elemental analyzer Costech ECS 4010 instrument. Total elemental concentration and plant available (extractable) concentration of Ca, Cd, Cr, Cu, Fe, Mg, Mn, Mo, Ni, P, and Zn were determined after microwave acid digestion using US EPA method 3050B (US EPA 1996 ) and by diethyl-enetriaminepentaacetic acid (DTPA) with pH 7.3 extraction method (Embrapa 1997 ) and measured using inductively coupled plasma mass spectrometry – ICPMS - Agilent Technologies. Extractable P was obtained using Olsen extractant (pH 8.5), and Ca, Mg, K, Na were extracted using 1 M ammonium acetate (pH 7), and measured by atomic absorbance spectrometry (AAS) using a graphite furnace (Perkin Elmer Analyst 800). The sum of cations extracted by ammonium acetate was used to calculate soil cation exchange capacity (CEC sum ) (Ross and Ketterings 2011 ). Water holding capacity (WHC) and soil density were determined with a fraction of soil used to fill the pots (sieved to pass 4 mm to preserve some of the soil structure). WHC was determined by placing approximately 10g of soil into a funnel lined with a water-saturated filter paper. Water was added slowly to the sample until the sample was saturated and water began to drip from the funnel. When the drip stopped, the sample was weighed to determine the mass of water retained, and WHC was calculated based on the mass difference between the initial and final soil water content. Soil density was estimated using a graduated cylinder and tapping 10 times until particles settle in and density determined by the weight divided by the volume times 100. Particle size distribution was measured by laser diffraction method according to Eshel et al. ( 2004 ) using Coulter LS-230 Particle Size Analyzer. Biochar acquisition, selection and characterization Biochar acquisition, selection, and characterization were described elsewhere (Alves et al. 2021a ). Five biochars pre-tested in our laboratory for maximum Cd retention were selected for the greenhouse trials. We had a special interest in SSB 700 because it was rich in Zn (sludge elemental content is presented in Table 1 of supplemental information - SI). Previous research demonstrated: 1) the reduction of Cd uptake by spinach using composted Zn-rich sewage sludge (Chaney et al. 2006 ); 2) the efficiency of Zn-rich biochar in reducing Cd (Peng et al. 2018 ); and 3) the combination of Zn and biochar being better than biochar or Zn alone (Farooq et al. 2020 ). Biochars were: almond shell 500 o C (ASB 500–1), walnut shell biochar 900 o C (WSB 900) sewage sludge biochar 700 o C (SWB 700) and two engineered coconut shell biochars at 600 o C (CSB 600 – m2 and CSB 600 – m3) (Table 1 ) and their corresponding oxidized versions ASB 500–1 H 2 O 2 , WSB 900 H 2 O 2, SSB 700 H 2 O 2 , CSB 600 - m2 H 2 O 2, and CSB 600 – m3 H 2 O 2 . Companies were identified by letters to prevent their identification. Table 1 – Biochars experimental matrix and description of production methods. # Sample ID Feedstock Temp. ( o C) Production method Production Company Residence time Post production modification 1 ASB 500–1 Almond Shell 500 Fast pyrolysis A 45min None 2 WSB 900 Walnut Shell 900 Gasification B 2 min None 3 SSB 700 Sewage Sludge 700 Slow pyrolysis UC Davis Lab 5 hours None 4 CSB 600 - m2 Coconut Shell 600 Slow pyrolysis C 3 Days Yes, proprietary information 5 CSB 600 - m3 Coconut Shell 600 Slow pyrolysis C 3 Days Yes, proprietary information Biochar oxidation 1.3 L of each biochar was placed in an 18.93 L bucket and 3 L of hydrogen peroxide (H 2 O 2 ) 30% certified ACS grade was added. The mixture was left overnight to react and manually mixed for the next 36h. Samples were filtered and washed with 3L of Barnstead Nanopure (BNP) water and then air-dried. Sample characterization is presented in Alves et al. ( 2021a ). Compost and Zinc amendments Poultry manure compost was obtained from the Russell Ranch Sustainable Agriculture Facility, a division of the Agricultural Sustainability Institute at the University of California, Davis ( http://asi.ucdavis.edu/programs/rr ). Its chemical characteristics are presented in Table 2 - SI. Zn (ZnCl 2 ) in powder form were purchased from Fisher Scientific, certified ACS grade. Greenhouse experimental design for the three life-cycles Soil material < 4 mm was transferred to the greenhouse, mixed with treatments in large plastic bags and added to single 4.9 L tree pots (12.7 x 30.5 cm) containing plastic bags to prevent leaching. Each pot was filled by volume (3.8 L of soil + treatment) using a graduated cylinder with the subsequent addition of 100 mg/dm 3 of P and 126.23 mg/dm 3 of K as KH 2 PO 4 as a starter fertilizer. Two non-simultaneous batches were conducted: 1) with the five best biochars pre-tested by adsorption isotherms for Cd retention, and 2) with their corresponding oxidized versions also pre-tested in the lab (Alves et al. 2021a ). Each batch consisted of 13 treatments: the five biochars added to the soil at application rates of 1 and 5% v/v (10 biochar treatments), soil + 25 mg/kg of Zn (ZnCl 2 ), soil + compost (5% v/v), and one control of non-amended soil. The rationality for using 1% and 5% biochar application rate (v/v) was based on extensive literature review which showed that 0–10% is a viable application (Alves et al. 2021b ). Lime treatments were initially not included (see below) because the original pH of soil was already above 7 (soils in the Salinas Valley are frequently limed) (Smith and Hartz 2016a ). Further, Chaney et al. ( 2009 ) proposed that pH between 6.5 and 7 is optimal because at lower pH Cd become highly phytoavailable and at higher pH Zn become less available, which could affect crop growth. All treatments were conducted in quadruplicate (T = 26, n = 104 for the two batches), and pots were randomly moved weekly in a block design to minimize greenhouse climate differences. Pots were watered with reverse osmosis (RO) water with the aim of maintaining 60% water holding capacity and fertigated with ammonium nitrate (NH 4 NO 3 − 300 mg/dm 3 of N per growth cycle). During the experiment, the temperature was maintained at 27 o C ± 2 o C for a 16h period of light. The Silver Whale variety of spinach was selected due to commercial availability and common use by farmers in the region. Seedlings were grown in inert media (vermiculite) for 14 days. After this period, the most vigorous seedlings were manually transplanted to the center of the pots and grew for four more weeks (one plant per pot). The rationality to use one plant per pot was to be able to maintain a mass balance control and to avoid competition between plants. This process was repeated for each life-cycle. Harvest cycle longevity of 42 days was determined by a pre-experimental trial (data not shown). After 42 days, plants were starting to show reproductive parts, and the most common spinach grown in the Salinas Valley is baby spinach (spinach harvested in a young phase). Shoots were harvested by cutting the stem at the soil surface and subsequently washed with RO water. This was repeated for three consecutive life-cycles using the same pot for each treatment. Post-growth analysis After harvesting (after each life-cycle), soil samples were analyzed for total content of C and N, pH, EC, and DTPA extraction for Cd and Zn. Plant wet and dry yields (after drying for 72h at 60 o C) were recorded, and dry plants were acid digested using aqua regia (3 HCl: 1 HNO 3 ), following (Uddin et al. 2016 ), and analyzed for Cd and Zn using ICPMS. Statistical analysis To identify statistically significant differences among treatments we used a non-parametric statistical test, given the high variability of the data. The nonparametric Kruskal-Wallis test was applied to identify differences among treatments, while the nonparametric Mann-Whitney was applied to identify differences between the biochar application rates (1 and 5%), both at the 5% significance level. Statistical analysis was conducted using software R Studio version 4.1.1. Results and Discussion Soil characterization Soil texture was classified as a loam (USDA soil texture classification), with the particle size distribution of sand, silt, and clay of 48%, 42% and 10%, respectively. Other results from the soil analysis include: 1) pH of 7.35; 2) EC of 1.01 mS/cm; 3) bulk density of 1.42 g/cm 3 ; 4) WHC of 34.13 mass %; 5) Total C% and N% of 0.69% and 0.09% respectively; 6) P extractable concentration by Olsen of 14.92 mg/kg; 7) Ca, Mg, K, Na extractable concentration by 1 M ammonium acetate of 3012.66 mg/kg, 200.12 mg/kg, 202.78 mg/kg and 97.44 mg/kg respectively; 8) CEC sum of 17.52 cmolc/kg; 9) Total concentration of P, Ca, Cd, Fe, Mg, Ni and Zn by acid digestion of 9.3 g/kg, 0.47 g/kg, 2.30 mg/kg, 21.95 g/kg, 0.55 g/kg, 26.05 mg/kg and 74.50 mg/kg. 10) Plant available concentration of K, Ca, Cd, Fe, Mg, Ni and Zn by DTPA (pH 7.3) extraction of 1.2 mg/kg, 26.72 mg/kg, 0.74 mg/kg, 41.91 mg/kg, 2.24 mg/kg, 1.47 mg/kg and 3.10 mg/kg. According to these results, the total concentration of Cd is well above global concentration of 0.1–0.5 mg/kg (UNEP 2010 ) and well above quality levels of < 0.5 mg/kg and prevention levels of < 1.3 mg/kg established by the US EPA SW 848 (Kabata-Pendias 2011 ). Unoxidized biochars Results from the three life-cycles for the unoxidized biochars are shown in Table 2 . Table 2 – Unoxidized biochars batch--mean values for three life cycles. Treatments Soil Plant pH EC Cd Zn Yield Cd Zn (g dry wt.) mS/cm mg/kg mg/kg Soil (control) 5.68 a 3.47 b 0.71 a 2.88 a 0.09 a 28.14 ab 141.36 ab Compost 5.97 b 3.03 a 0.62 a 4.59 a 0.12 b 25.07 ab 186.49 ab Zinc Chloride 5.79 ab 2.97 a 0.71 a 7.77 b 0.05 a 21.74 a 328.77 b ASB 500 - 1 1% 5.70 a 3.74 b 0.66 a 3.21 a 0.08 a 31.34 ab 181.23 ab ASB 500 - 1 5% 5.92 b 3.27 ab 0.63 a 3.12 a 0.10 ab 32.46 ab 142.67 ab CSB 600 - m2 1% 5.79 ab 3.54 b 0.66 a 3.24 a 0.08 a 30.88 ab 119.11 a CSB 600 - m2 5% 5.90 b 3.67 b 0.66 a 3.42 a 0.05 a 46.74 b 167.52 ab CSB 600 - m3 1% 5.42 a 3.47 b 0.72 a 3.32 a 0.08 a 45.13 b 159.55 ab CSB 600 - m3 5% 5.76 ab 3.27 ab 0.66 a 2.93 a 0.06 a 32.85 ab 146.92 ab SSB 700 1% 5.84 b 3.49 b 0.71 a 4.16 a 0.07 a 37.70 b 151.10 ab SSB 700 5% 6.04 b 4.39 b 0.65 a 10.40 b 0.12 b 23.65 a 163.20 ab WSB 900 1% 5.76 ab 3.69 b 0.72 a 3.71 a 0.10 ab 19.84 a 67.34 a WSB 900 5% 6.46 b 3.68 b 0.51 a 2.94 a 0.07 a 17.25 a 84.37 a Mean values followed by the same letter in the column are not statistically different by the Kruskal-Wallis test (p<5%). Soil properties According to Table 2 , there were statistical differences between treatments for pH and EC. For pH, soil (control), ASB 500–1 1% and CSB 600 – m3 1% treatments showed the lowest values, that significantly differed from Zinc, CSB 600 - m2 1%, CSB 600 – m3 5% and WSB 900 1%, that subsequently significantly differed from the rest of the treatments. For EC, the soil that had one of the lowest pH, had one of the highest EC. As expected, the most saline biochar (SSB 700 = 6.86 mS/cm) had the highest EC in soil. Even though EC values exceed the threshold of spinach salt tolerance reported by FAO ( 1985 ) of 2 mS/cm, we could still grow healthy plants, but the increased salinity may have had some impact on yield. The DTPA extractable Cd concentration ranged from 0.51 mg/kg in WSB 900 5% to 0.72 mg/kg in WSB 900 1% and there was no significant difference in Cd in soil, showing that although some treatments were able to retain Cd, the available extractable Cd is consistent. The DTPA extractable Zn concentration ranged from 2.88 mg/kg in soil to 10.40 mg/kg in the SSB 700 5% treatment. The high concentration of Zn in soil in this last treatment was expected because the sewage sludge used to make this biochar has a high concentration of Zn (1000 mg/kg) as shown in Table 2 in SI. As expected, the Zinc treatment produced the next highest Zn enrichment (7.77 mg/kg), followed by compost (4.59 mg/kg). Plant yield and uptake In terms of yield (dry wt.), a few treatments significantly differed from the control (0.09 g), where the highest yield was found in Compost treatment and SSB 700 5% (both with 0.12 g). The only treatments that produced mean (three life-cycles) yields above the control were Zn and SSB 700 5%, ASB 500–1 5% and WSB 900 1%. It is important to mention that all plants in SSB 700 5% treatment died in the first life cycle, which we attribute to phytotoxicity present in this biochar. This toxicity was transient because three plants grew in the next life cycle and four plants in the last two life cycles (new plants were transplanted after each harvesting for all treatments). We believe washing this biochar before its use may prevent this toxicity. For Cd concentration in spinach (dry wt.), few treatments reduced Cd uptake when compared to control (mean of all life-cycles was 28.14 mg/kg), that is Compost, Zinc, SSB 700 5%, WSB 900 1% and WSB 900 5% (25.07; 21.74; 23.65; 19.84; and 17.25 mg/kg, respectively). Seven treatments: ASB 500–1 1%, ASB 500–1 5%, CSB 600 – m2 1%, CSB 600 – m2 5%, CSB 600 – m3 1%, CSB 600 – m3 5% and SSB 700 1% resulted in Cd uptake above the control. In treatments with large Cd uptake we observed some leaf Cd toxicity in the form of rolled leaves (Benavides et al. 2005 ; Qin et al. 2020 ). And none of the treatments resulted in Cd concentrations below CODEX limits (4 mg/kg dry wt.) in any life-cycle (varied from 8.12 mg/kg to 64.08 mg/kg) (full data presented in Table 3 - SI). Paul and Chaney ( 2017 ) showed that compost and zinc treatments significantly reduced Cd uptake but also significantly reduced yield, when compared to the control. In our case, Compost significantly increased yield and reduced Cd uptake (but not significantly), while Zinc treatment reduced yield (not significantly) and reduced Cd uptake (significantly). All treatments increased Cd uptake by the 3th life cycle including the control, likely because of soil acidification due to fertilization with NH 4 NO 3 and roots exudates. pH started before planting on an average of 6.80 (all treatments) and finished in the third life-cycle on an average of 5.12 (all treatments). This shows that Salinas Valley soil needs to be constantly limed to approximately pH 7. Thus, our hypothesis that the treatments will need to be reapplied over time could not be verified, once the pH dropped. For Zn uptake, the largest uptake was found, as expected, in the zinc and SSB 700 treatments. Similar to Paul and Chaney ( 2017 ), Zn uptake in zinc treatment was greater than reported potentially phytotoxic concentrations (> 200 mg/kg) in leaves. For the other treatments, we observed no evidence of deficiency or phytotoxicity in any treatment (e.g., necrosis or yellowing). We did not observe a clear competitive correlation among Cd and Zn uptake. For example, zinc treatment had the highest Zn uptake but not the lowest Cd uptake, and the WSB 900 1% treatment had the lowest Zn uptake and one of the lowest Cd uptake, whereas, the CSB 600 – m2 5% treatment had the highest Cd uptake, and a high uptake of Zn. Overall, SSB 700 5% treatment was the “best” because it produced higher yield and lower Cd uptake than the control. Interestingly, similar to Yang et al. ( 2017 ), for SSB 700 treatment the biochar application rate of 5% produced higher yields and lower Cd uptake than the application rate of 1%. On the other hand, similar to Simmler et al. ( 2013 ), where lower application rates produced higher yields, the CSB 600 – m2 1% treatment produced higher yield and less Cd uptake than CSB 600 – m2 5% treatment. Lastly, similar to Bian et al. ( 2013 ), the CSB 600 – m3 1% and WSB 900 1% treatments produced higher yield and more Cd uptake than CSB 600 – m3 5% and WSB 900 5% treatments. These contrasting results show that the relationships among biochar application rates, Cd uptake, and crop yield are not straight forward. Higher application rates of some biochars increase adsorption of Cd, preventing plant uptake, but also increase the adsorption of essential plant nutrients, and reduce yields. Our results are not completely surprising because the biochars we used a wide array of chemical and physical properties (Alves et al 2021a ). We suggest the use of artificial intelligence to try to predict biochar behavior in soil. In terms of financial, Zinc application can be more expensive than biochar and compost methods (i.e., a ton of ZnSO 4 , biochar and compost may cost roughly $ 500, $ 200, and $ 70 respectively; prices calculated in 2018). Compost use to be more affordable than biochar, but since the development of long-term removal carbon credits market, biochar prices have been significantly reduced The downside of using Zn is that it may increase Cd availability input, because it is a byproduct of Zn production (Hetherington et al. 2008 ). Globally, Cd contaminated sites are usually related to geogenic Zn mining or smelter sources (Chaney et al. 2005 ). Finally, greenhouse plant uptake results did not follow lab isotherm results (Alves et al. 2021a ). In the lab, the Cd-retention order was SSB 700 > CSB 600 – m2 > ASB 500–1 > CSB 600 – m3 > WSB 900 treatments. While here there was no clear trend between maximum Cd retention and lower Cd uptake, which varied depending on the harvest. These results can be explained by biochar application rates and interactions between soil and biochar. Finally, we recommend future soil-plant based research to predict the most efficient biochar and to test lower application rates, since the rates we apply may not be economically viable. Oxidized biochars Results from the three life-cycles for the oxidized biochars are shown in Table 3 . Table 3 – Oxidized biochars batch--mean values for three life cycles. Treatments Soil Plant pH EC Cd Zn Yield Cd Zn (g dry wt.) mS/cm mg/kg mg/kg Soil (control) 6.46 ab 2.84 a 0.60 a 2.19 a 0.38 a 20.10 ab 76.65 ab Compost 6.14 a 3.26 a 0.64 a 4.91 ab 0.28 a 13.77 a 91.13 ab Zinc Chloride 6.13 a 3.06 a 0.63 a 4.05 ab 0.69 b 17.44 a 106.23 b ASB 500 -1 H 2 O 2 1% 6.40 a 2.87 a 0.61 a 2.45 a 0.34 a 18.84 a 74.57 ab ASB 500 -1 H 2 O 2 5% 6.32 a 2.71 a 0.59 a 1.95 a 0.32 a 17.14 a 98.54 ab CSB 600 - m2 H 2 O 2 1% 6.68 ab 2.45 a 0.63 a 2.15 a 0.34 a 18.92 ab 94.46 ab CSB 600 - m2 H 2 O 2 5% 6.07 a 2.35 a 0.63 a 2.47 a 0.31 a 22.71 b 100.13 b CSB 600 - m3 H 2 O 2 1% 6.27 a 2.83 a 0.60 a 2.18 a 0.40 ab 15.88 a 75.02 ab CSB 600 - m3 H 2 O 2 5% 5.72 a 2.94 a 0.71 a 2.92 a 0.29 a 23.18 b 118.25 b SSB 700 H 2 O 2 1% 6.04 a 3.28 a 0.67 a 3.44 ab 0.37 a 20.58 ab 94.65 ab SSB 700 H 2 O 2 5% 6.46 ab 3.64 a 0.66 a 7.11 b 0.50 ab 12.19 a 109.15 b WSB 900 H 2 O 2 1% 6.39 a 2.91 a 0.62 a 2.51 a 0.45 ab 18.48 a 80.63 ab WSB 900 H 2 O 2 5% 7.02 b 2.44 a 0.51 a 2.32 a 0.51 ab 14.42 a 57.28 a Mean values followed by the same letter in the column are not statistically different by the Kruskal-Wallis test (p<5%). Soil properties According to Table 3 , generally, pH was slightly higher (average of 6.31 for all treatments) when comparing to unoxidized batch (average of 5.85 for all treatments), which was not expected since we used the same soil that was sampled at the same time and was stored in five gallons plastic buckets, it may be caused by changes in soil properties while stored. With the oxidation with H 2 O 2 the majority of the biochars had their pH reduced, except for the WSB 900 H 2 O 2 treatment that despite having its pH reduced, continued to be alkaline. For example, the CSB 600 - m2 H 2 O 2 biochar had its pH reduced from 7.05 to 4.02 (Alves et al. 2021a ). Thus, apart from WSB 900 H 2 O 2 5% treatment that had pH significantly higher after all harvests, in comparison to the control, most of the other treatments had pH bellow the control (full data in Table 3 – SI). The oxidation also significantly reduced the biochar EC (salts were removed by the washing process). The salinity reduction was more pronounced in some (e.g., WSB 900 original EC 6.02 mS cm − 1 and H 2 O 2 EC 0.93 mS cm − 1 ) than in others (e.g., SSB 700 original EC 6.86 mS cm − 1 and H 2 O 2 EC 2.32 mS cm − 1 ) (Alves et al. 2021a ). Surprisingly, there were no statistical differences among treatments for EC, although SSB 700 H 2 O 2 5% treatment the most saline (3.64 mS/cm) and CSB 600 – m2 H 2 O 2 5% the least saline (2.35 mS/cm), but not very much different from the soil control (2.84 mS/cm). Salinity also slightly increased over the three life-cycles, probably due to the fertigation effect. Even though EC reached 4.81 ± 0.62 mS/cm in the 3rd life-cycle for SSB 700 H 2 O 2 1%, we could still grow plants, although there may have been an unknown yield effect, as in the case with the unoxidized biochar. For DTPA extractable elements in soil, no treatment statistically differed from the control for Cd concentration, again, showing some consistency. Overall, the lowest mean concentration of Cd was obtained with WSB 900 H 2 O 2 5% treatment (0.51 mg/kg) in all three life-cycles while mean concentrations of 0.71 mg/kg were found in all three life-cycles for CSB 600 H 2 O 2 5%. For extractable Zn in soil, statistical differences were found among treatments. SSB 700 H 2 O 2 5% (7.11 mg/kg) statistically differed from control (2.19 mg/kg). Results show that the modification with H 2 O 2 must have removed the phytotoxicity initially found in its unoxidized version. Plant yield and uptake In terms of yield (dry wt.), there were statistical differences among treatments. Zinc treatment was overall the best treatment in terms of yield for the three harvests (mean weight for all life-cycles, 0.69 g dry wt.). The WSB 900 H 2 O 2 5% treatment was the second-best treatment followed by SSB 700 H 2 O 2 5% (0.51 g and 0.50 g, respectively). Note that the spinach was not killed in the first harvest of the SSB 700 H 2 O 2 5% treatment, as was the case with the unoxidized biochar. The lowest yields were obtained with compost, followed by CSB 600 – m3 H 2 O 2 5% (0.28 and 0.29 g, respectively). Interestingly, in the unoxidized batch compost produced a higher (but not significantly) yield than the zinc treatment, while the oxidized batch produced lower yield. For Cd concentrations in spinach (dry wt.), when comparing to the unoxidized batch, more treatments could reduce Cd uptake when comparing to control (mean from all treatments: 20.10 mg/kg). That is, SSB 700 H 2 O 2 5% (12.19 mg/kg) < compost (13.77 mg/kg) < WSB 900 H 2 O 2 5% (14.42 mg/kg) < CSB 600 - m3 H 2 O 2 1% (15.88 mg/kg) < ASB 500 -1 H 2 O 2 5% (17.14 mg/kg) < zinc (17.44 mg/kg) < WSB 900 H 2 O 2 1% (18.48 mg/kg) < ASB 500–1 H 2 O 2 1% (18.84 mg/kg) < CSB 600 – m2 H 2 O 2 1% (18.92 mg/kg) could reduce Cd uptake. In general, concentrations were lower in the oxidized batch probably due to the higher initial pH values when compared to the unoxidized batch. The closest Cd uptake in spinach we obtained to the CODEX limit was 5.18 mg/kg with compost and 7.22 mg/kg with SSB 700 H 2 O 2 5% in the 3rd life-cycle. For both SSB 700 H 2 O 2 5% and compost treatments, increased application rates might achieve CODEX limits. The SSB 700 H 2 O 2 5% treatment had the advantage of also increasing the yield by 30.89% when compared to the control. The SSB 700 biochar, if proven to remove phytotoxicity after washing with water may also be a very economically feasible solution, because currently the Monterey Wastewater treatment facility spends money to place the sludge in a landfill, and if they could invest in a biochar pyrolizer, maybe could produce a biochar cost effective and a feasible solution to address spinach grower issues with Cd. Important to say that we observed a wrinkled leaf appearance associated with Cd toxicity in treatments with large Cd uptake, i.e., CSB 600 - m2 H 2 O 2 5% and CSB 600 - m3 H 2 O 2 5%. Surprisingly, Cd uptake increased from the 1st to the 2nd life-cycle (mean all treatments) and reduced from the 2nd to the 3rd life cycle (mean all treatments) coming back to the 1st life-cycle levels, even though the pH reduced over time, showing that maybe the fertilization had some effect in Cd uptake in spinach. In the unoxidized batch the highest Zn uptake was verified in the zinc treatment that was significantly higher when looking mean values for all life-cycles. In the oxidized batch, CSB 600 - m3 H 2 O 2 5%, SSB 700 H 2 O 2 5%, zinc and CSB 600 - m2 H 2 O 2 5% had higher mean concentration than the control. No evidence of Zn deficiency was observed in the unoxidized batch. Again, we did not verify a clear competition between Cd and Zn in this batch, since CSB 600 – m3 H 2 O 2 5% treatment had the highest Zn uptake and the highest Cd mean uptake. Overall, SSB 700 H 2 O 2 5% was the “best” biochar treatment with the highest yield among the biochar treatments, and the lowest Cd uptake. Other treatments that might also be effective for Cd remediation purposes are 1) WSB 900 H 2 O 2 5% that also had a higher yield than control, lower Cd uptake; and 2) zinc that reduced Cd uptake and increased yield compared to the control. Interestingly, the SSB 700 H 2 O 2 and WSB 900 H 2 O 2 biochar treatments with the application rate of 5% produced higher yield and lower Cd uptake than the application rate of 1%. Nevertheless, CSB 600 – m3 H 2 O 2 1% treatment had higher yield and lower Cd uptake than their 5% treatment counterparts, showing that adding more biochar may not decrease Cd uptake and increase yield. Our results indicate that the greater biochar application rate did not always reduce Cd uptake, possibly because some biochars are sources of trace elements (data not shown). Thus, increasing the biochar application rate may add more Cd to the soil than its retention capacity of metals already available in the soil. Finally, greenhouse results again did not follow lab results (Alves et al. 2021a ). In the lab, the Cd retention order by treatment was WSB 900 H 2 O 2 > ASB 500–1 H 2 O 2 > CSB 600 – m3 H 2 O 2 > CSB 600 – m3 H 2 O 2 > SSB 700 H 2 O 2 . Here the greatest average Cd retention in biochar was obtained with SSB 700 H 2 O 2 5% (12.19 mg/kg) > WSB 900 H 2 O 2 5% (14.42 mg/kg) > CSB 600 – m3 H 2 O 2 1% (15.88 mg/kg) > ASB 500 -1 H2O2 5% (17.14 mg/kg). The higher Cd retention by the poultry manure compost and SSB 700 H 2 O 2 treatment can be attributed to the high concentrations of polar functional groups in these materials (Alves et al 2021a ). Poultry manure compost is rich in N (Table 2 - SI), while SSB 700 is rich is N, S (Alves et al. 2021a ) and Zn (Table 1 - SI). N and S form very stable bonds with Cd. The formation of these bonds can be explained by the concept of “hardness” or “softness” (Pearson 1963 ). Cd is a soft acid, i.e., an electron acceptor atom of low positive charge and large size and has several easily excited outer electrons. A soft acid is polarizable and covalently binds to soft bases such as N- and S-containing surface functional groups (amine, amide, and thione) (Sparks 2003 ). Cd also binds strongly to oxygenated functional groups (e.g. -OH, C = O, C-O), so by oxidizing biochar, we could enrich it with O as well. We speculate that a modification with amination would also be a good option to maximize biochar retention, but that is a modification that would increase biochar’s cost significantly and we thought that wouldn’t be a viable solution compared to compost and zinc application. Conclusions Even though no treatment produced Cd uptake levels below the CODEX limit (4 mg/kg dry wt.) after three life cycles, the best treatments when considering yield and Cd uptake were WSB 900 5% and poultry manure compost 5% in the unoxidized batch and SSB 700 H 2 O 2 5% and zinc in the oxidized batch. WSB 900 is C rich and highly aromatic, with high metal adsorption capacity including Cd + 2 (Alves et al 2021a ) while, poultry manure compost and SSB 700 H 2 O 2 are rich in N or S, which form very stable bonds with Cd. Our hypothesis that biochars are more effective than other proposed solutions (ZnCl 2 and compost) was not verified since the lowest mean Cd uptake (5.18 mg/kg) was obtained by compost in the oxidized batch. Our hypothesis that higher application rates would reduce Cd uptake was not verified, and the effect was biochar-dependent. Some of the biochars may be sources of Cd. The zinc treatment had mixed results, in the unoxidized batch it reduced Cd uptake but also reduced yield, while in the oxidized batch it increased yield and reduced Cd uptake in spinach, showing that further studies must be conducted to understand this solution. Compost use to be the most cost-beneficial for farmers, but since the establishment of the carbon credits marked, biochar has becoming cost-beneficial as well. The hypothesis that the material would have to be reapplied over time also was not able to be verified, because pH reduced over time. Although by planting in consecutive growing-harvest cycles we observed that treatment effects seem to be durable, lasting through the third cycle. Our research also showed that Salinas Valley soil needs to be limed to approximately pH 7 to maximize treatments effects. Further studies need to be conducted to achieve safe plants to be consumed. Finally, our hypothesis that oxidation of biochars would increase Cd retention was also not verified. Oxidation effects were very biochar dependent and increase Cd retention only in SSB 700. However, it is not clear if the benefits achieved by the oxidation came from the EC and phytotoxicity reduction or from the increase in O content itself, since SSB 700 1% and SSB 700 H 2 O 2 1% were not so different. We recommend the utilization of this biochar washed with water in another greenhouse study to verify if the phytotoxicity is removed. In general, the oxidation with H 2 O 2 was not as beneficial as we initially expected -- neither in the laboratory (Alves et al. 2021a ) nor at the greenhouse. Finally, we recommend the use of artificial intelligence to predict biochar effects in soil, once they are very biochar-dependent. Declarations Ethics approval and consent to participate This article does not contain any studies with human participants or animals performed by any of the authors. Availability of data and materials All data is provided in the supplemental material. Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This work was financially supported by Coordenação de aperfeiçoamento de Pessoal de Nível Superior – CAPES Foundation – Brazil Author´s contributions Barbara Samartini Queiroz Alves: writing the original draft and data analysis; Luiz Arnaldo Fernandes: Data analysis; Randal J Southard: Visualization and revision of the paper. All the authors contributed to the final review of the manuscript. Acknowledgments We thank Snow Seeds for donating the seeds and acknowledge Dr. Bryan Jenkins and his team from the UCD Biomass Lab for allowing us to perfect the production of the sewage sludge biochars. Finally, we thank Dr. Richard F. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3857978","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":275280551,"identity":"9f39acf9-1620-4cab-9b7a-6b0963cf7aa9","order_by":0,"name":"Bárbara Samartini Queiroz Alves","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYBACAwYGxgNAWg7CNgCJJRDUwgDSYky6lsQGqHrCWsz5Dz84zFNRl77h+OGNn3kK6hj42XMM8GqxnJFmcJjnzOHcDWfSiqV5DA4zSPa8wa/F4AaDwcGZbQdyN9zgMZCcYXAAKELAFoPzxz8cnPmvLt3gBo/xzxkGdQz2BLUcyDE48LGBOQGoxUzigwEzg4EEQb/kFBz4cOyw4cwzaWUWH4D+kjjzrACvFnP+4xsfJNTUyfMdP7z5RsKfOjn+9uQNeLVgAB7SlI+CUTAKRsEowAoA351LuP9PX+4AAAAASUVORK5CYII=","orcid":"","institution":"University of California-Davis","correspondingAuthor":true,"prefix":"","firstName":"Bárbara","middleName":"Samartini Queiroz","lastName":"Alves","suffix":""},{"id":275280552,"identity":"907b5751-1695-49d9-b854-8456076c783f","order_by":1,"name":"Luiz Arnaldo Fernandes","email":"","orcid":"","institution":"Federal University of Minas Gerais","correspondingAuthor":false,"prefix":"","firstName":"Luiz","middleName":"Arnaldo","lastName":"Fernandes","suffix":""},{"id":275280553,"identity":"0c4a2640-14a3-42ea-b972-025523fd796e","order_by":2,"name":"Randal J Southard","email":"","orcid":"","institution":"University of California-Davis","correspondingAuthor":false,"prefix":"","firstName":"Randal","middleName":"J","lastName":"Southard","suffix":""}],"badges":[],"createdAt":"2024-01-12 19:29:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3857978/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3857978/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":56667723,"identity":"104c4e66-0700-4b78-8422-5f840c6bb9c7","added_by":"auto","created_at":"2024-05-17 13:45:07","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":747772,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3857978/v1/89f814c4-5436-4956-802d-46fff6949348.pdf"},{"id":51802346,"identity":"c60a3c75-ec80-42e3-888f-3a04c4863307","added_by":"auto","created_at":"2024-02-29 09:29:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":273358,"visible":true,"origin":"","legend":"","description":"","filename":"Paper2SIV3.docx","url":"https://assets-eu.researchsquare.com/files/rs-3857978/v1/898191035601d9ad204320a9.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of Unoxidized Biochars, Oxidized Biochars, Zinc and Compost on Cadmium Uptake in Spinach","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eBiochar shows promise in reducing Cd uptake.\u003c/li\u003e\n \u003cli\u003e\u0026nbsp;Higher application rates of biochar did not consistently reduce Cd uptake, and the effect varied depending on the type of biochar used.\u003c/li\u003e\n \u003cli\u003eThe Zn treatment reduced Cd uptake but had variable effects on yield.\u003c/li\u003e\n \u003cli\u003eCompost was found to be the most cost-beneficial solution for farmers, but biochar has potential benefits in terms of carbon credits.\u003c/li\u003e\n \u003cli\u003eOxidation of biochars with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e did not significantly decrease Cd uptake.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eCadmium (Cd) is a nutritionally non-essential persistent metal element that can accumulate in the food chain (i.e., half-life of 10\u0026ndash;30 years in the kidneys) and is often toxic to plants, animals, and microorganisms at very low concentrations (\u0026Aring;kerblom et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; UNEP \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). In humans, 90% of Cd exposure in the non-smoking population is through food, where cereals, vegetables, and potatoes represent more than 80% of exposure (J\u0026auml;rup and \u0026Aring;kesson \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Clemens et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Global average weekly intake from food is estimated to be within 1.9 and 3.0 \u0026micro;g/kg body weight (Sigel et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Although only 3\u0026ndash;5% of the ingested Cd is absorbed, it is estimated that a large portion of the global population has already exceeded the tolerable intake dose of 2.5 \u0026micro;g/kg body weight weekly (EFSA \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The reported main health effects brought on by excessive Cd uptake are renal dysfunction, osteoporosis, and cancer (WHO \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2010\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e The development of effective and agronomically feasible solutions to prevent Cd from entering the food chain is imperative (Hamid et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The Salinas Valley in California is one of the most productive agricultural areas in the USA (production value of about US\u003cspan\u003e$\u003c/span\u003e13,000 per acre (CDFA, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Many soils in the valley are derived from the Monterey Shale, an organic, and phosphate-rich sedimentary rock from the Miocene (Filippelli \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2011\u003c/span\u003e), and have naturally high Cd concentrations (up to 35 mg/kg) (Lund et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e1981\u003c/span\u003e). The main crops in the Salinas Valley are leafy vegetables, strawberries, cauliflower, and wine grapes. Among these, spinach (\u003cem\u003eSpinacia oleracea\u003c/em\u003e) is of concern, because it is known to be a Cd accumulator crop (Wolnik et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e1985\u003c/span\u003e), capable of translocating Cd from the roots to the shoots at concentrations higher than in non-accumulating species. The mechanism of uptake (not specific for spinach) is related to a protein that also transports Zn and Fe through the root membrane, followed by translocation to the shoot (Grant et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Rascio and Navari-Izzo \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Sterckeman and Thomine \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Smith and Hartz (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e) showed that Cd uptake by spinach is roughly proportional to the soil Cd content. Alexander et al. (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) showed that the uptake can vary by up to 40%, depending on the spinach variety.\u003c/p\u003e \u003cp\u003eIn a 2013 field survey, the Salinas Valley agricultural soils contained concentrations up to 9 mg/kg total Cd (the world average is 0.1\u0026ndash;0.5 mg/kg) (UNEP \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Spinach planted in soils with high Cd-concentration had leaf mean concentrations above levels considered safe (up to 1.90 mg/kg, fresh wt.), which is more than 10 times the average concentration found in spinach grown in other areas (Giclas \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) and almost 10 times more than the CODEX limit. The CODEX limit is established by the joint initiative of the Food and Agriculture Organization (FAO) and the World Health Organization (WHO) for food standards, which is set at 0.2 mg/kg fresh wt. (~\u0026thinsp;4 mg/kg dry wt.) (FAO/WHO \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). These high Cd levels led to a recall of baby spinach by the California Department of Public Health in 2015 (News Desk \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSolutions have been proposed, such as high rates of Zn application (25\u0026ndash;50 mg/kg of elemental Zn on a soil dry wt.) (Paul and Chaney \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) to compete for Cd uptake (McKenna et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1993\u003c/span\u003e), liming the soil to increase pH (Cd precipitates from soil solution in alkaline pH) (Chaney et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e), and remediation with organic amendments (Rehman et al. 2018; Simmler et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), such as compost (Bashir et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) or biochar (Chen et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Qiu et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBiochar, is a versatile, low-cost, carbon-rich, porous material produced from the thermal carbonization of the biomass under oxygen limited conditions (Igalavithana et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). As a versatile material, biochar can be modified to maximize Cd retention (Sizmur et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). It is expected that oxidation of biochar surface to maximize oxygenated functional groups will increase Cd retention (Frišt\u0026aacute;k et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). An oxidizing method that has been becoming popular is washing with hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) (Huff and Lee \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe intent of this study is to investigate Cd uptake by spinach from soil amended with biochar, ZnCl\u003csub\u003e2\u003c/sub\u003e, and poultry litter compost additions. Further, we investigated appropriate application rates, and how long remediation methods affect Cd uptake. Our focus is on the effectiveness of biochar as a remediation material for agricultural soils enriched with Cd. The specific objectives were: (i) evaluate Cd uptake by spinach (shoots) comparing pre-selected biochars with ZnCl\u003csub\u003e2\u003c/sub\u003e or compost applications; (ii) evaluate effects of two application rates (1% and 5%) of biochar; (iii) evaluate how treatments change over three consecutive life-cycles of the spinach; and (iv) compare unoxidized biochars with their oxidized (with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) versions. We hypothesize: (i) biochar will be more effective to reduce the accumulation of Cd in spinach than ZnCl\u003csub\u003e2\u003c/sub\u003e application or compost; (ii) higher biochar application rates will more strongly reduce Cd uptake; and, (iii) since Cd inputs can be maintained from weathering of parent material, it is likely to be necessary to reapply biochar over time.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSoil sampling and characterization\u003c/h2\u003e \u003cp\u003eThe soil material collection site was selected based on previous research. Smith and Hartz (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016b\u003c/span\u003e) studied a variety of soils in the Salinas Valley with Cd concentrations of 0.5-8 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. Their research suggested that growers not grow spinach in soils with concentrations above 2.5 mg kg\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e (data not published). We selected a soil with a Cd concentration closest to this threshold. Soil material was collected from the 0\u0026ndash;25 cm depth in a field mapped as Elder loam that grew spinach in May 2017 near Greenfield, California (36\u0026deg;17'33.92\"N, 121\u0026deg;15'55.80\"W). The USDA classification of the Elder series is coarse-loamy, mixed, superactive, thermic Cumulic Haploxerolls.\u003c/p\u003e \u003cp\u003eSoil material was mixed, air-dried to a constant weight, manually ground with mortar and pestle, and sieved to pass 2 mm for subsequent laboratory analysis. For soil characterization, pH and electric conductivity (EC) were measured in distilled water (1:1 w/w) after 90 min equilibration. Total C and N were determined by dry combustion-elemental analyzer Costech ECS 4010 instrument. Total elemental concentration and plant available (extractable) concentration of Ca, Cd, Cr, Cu, Fe, Mg, Mn, Mo, Ni, P, and Zn were determined after microwave acid digestion using US EPA method 3050B (US EPA \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) and by diethyl-enetriaminepentaacetic acid (DTPA) with pH 7.3 extraction method (Embrapa \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e1997\u003c/span\u003e) and measured using inductively coupled plasma mass spectrometry \u0026ndash; ICPMS - Agilent Technologies. Extractable P was obtained using Olsen extractant (pH 8.5), and Ca, Mg, K, Na were extracted using 1 M ammonium acetate (pH 7), and measured by atomic absorbance spectrometry (AAS) using a graphite furnace (Perkin Elmer Analyst 800). The sum of cations extracted by ammonium acetate was used to calculate soil cation exchange capacity (CEC\u003csub\u003esum\u003c/sub\u003e) (Ross and Ketterings \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWater holding capacity (WHC) and soil density were determined with a fraction of soil used to fill the pots (sieved to pass 4 mm to preserve some of the soil structure). WHC was determined by placing approximately 10g of soil into a funnel lined with a water-saturated filter paper. Water was added slowly to the sample until the sample was saturated and water began to drip from the funnel. When the drip stopped, the sample was weighed to determine the mass of water retained, and WHC was calculated based on the mass difference between the initial and final soil water content. Soil density was estimated using a graduated cylinder and tapping 10 times until particles settle in and density determined by the weight divided by the volume times 100. Particle size distribution was measured by laser diffraction method according to Eshel et al. (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2004\u003c/span\u003e) using Coulter LS-230 Particle Size Analyzer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eBiochar acquisition, selection and characterization\u003c/h2\u003e \u003cp\u003eBiochar acquisition, selection, and characterization were described elsewhere (Alves et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). Five biochars pre-tested in our laboratory for maximum Cd retention were selected for the greenhouse trials. We had a special interest in SSB 700 because it was rich in Zn (sludge elemental content is presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e of supplemental information - SI). Previous research demonstrated: 1) the reduction of Cd uptake by spinach using composted Zn-rich sewage sludge (Chaney et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2006\u003c/span\u003e); 2) the efficiency of Zn-rich biochar in reducing Cd (Peng et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e); and 3) the combination of Zn and biochar being better than biochar or Zn alone (Farooq et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Biochars were: almond shell 500\u003csup\u003eo\u003c/sup\u003eC (ASB 500\u0026ndash;1), walnut shell biochar 900\u003csup\u003eo\u003c/sup\u003eC (WSB 900) sewage sludge biochar 700\u003csup\u003eo\u003c/sup\u003eC (SWB 700) and two engineered coconut shell biochars at 600\u003csup\u003eo\u003c/sup\u003eC (CSB 600 \u0026ndash; m2 and CSB 600 \u0026ndash; m3) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) and their corresponding oxidized versions ASB 500\u0026ndash;1 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, WSB 900 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2,\u003c/sub\u003e SSB 700 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e, CSB 600 - m2 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2,\u003c/sub\u003e and CSB 600 \u0026ndash; m3 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Companies were identified by letters to prevent their identification.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u0026ndash; Biochars experimental matrix and description of production methods.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e#\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSample ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFeedstock\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTemp. (\u003csup\u003eo\u003c/sup\u003eC)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eProduction method\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eProduction Company\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eResidence time\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003ePost production modification\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eASB 500\u0026ndash;1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAlmond Shell\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eFast pyrolysis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eA\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e45min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWSB 900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eWalnut Shell\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e900\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGasification\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eB\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e2 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSSB 700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSewage Sludge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e700\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSlow pyrolysis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eUC Davis Lab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e5 hours\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eNone\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSB 600 - m2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCoconut Shell\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSlow pyrolysis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3 Days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eYes, proprietary information\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCSB 600 - m3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eCoconut Shell\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e600\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eSlow pyrolysis\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003eC\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e3 Days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eYes, proprietary information\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eBiochar oxidation\u003c/h2\u003e \u003cp\u003e1.3 L of each biochar was placed in an 18.93 L bucket and 3 L of hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) 30% certified ACS grade was added. The mixture was left overnight to react and manually mixed for the next 36h. Samples were filtered and washed with 3L of Barnstead Nanopure (BNP) water and then air-dried. Sample characterization is presented in Alves et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCompost and Zinc amendments\u003c/h2\u003e \u003cp\u003ePoultry manure compost was obtained from the Russell Ranch Sustainable Agriculture Facility, a division of the Agricultural Sustainability Institute at the University of California, Davis (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://asi.ucdavis.edu/programs/rr\u003c/span\u003e\u003cspan address=\"http://asi.ucdavis.edu/programs/rr\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Its chemical characteristics are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e - SI. Zn (ZnCl\u003csub\u003e2\u003c/sub\u003e) in powder form were purchased from Fisher Scientific, certified ACS grade.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGreenhouse experimental design for the three life-cycles\u003c/h3\u003e\n\u003cp\u003eSoil material\u0026thinsp;\u0026lt;\u0026thinsp;4 mm was transferred to the greenhouse, mixed with treatments in large plastic bags and added to single 4.9 L tree pots (12.7 x 30.5 cm) containing plastic bags to prevent leaching. Each pot was filled by volume (3.8 L of soil\u0026thinsp;+\u0026thinsp;treatment) using a graduated cylinder with the subsequent addition of 100 mg/dm\u003csup\u003e3\u003c/sup\u003e of P and 126.23 mg/dm\u003csup\u003e3\u003c/sup\u003e of K as KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e as a starter fertilizer. Two non-simultaneous batches were conducted: 1) with the five best biochars pre-tested by adsorption isotherms for Cd retention, and 2) with their corresponding oxidized versions also pre-tested in the lab (Alves et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e). Each batch consisted of 13 treatments: the five biochars added to the soil at application rates of 1 and 5% v/v (10 biochar treatments), soil\u0026thinsp;+\u0026thinsp;25 mg/kg of Zn (ZnCl\u003csub\u003e2\u003c/sub\u003e), soil\u0026thinsp;+\u0026thinsp;compost (5% v/v), and one control of non-amended soil. The rationality for using 1% and 5% biochar application rate (v/v) was based on extensive literature review which showed that 0\u0026ndash;10% is a viable application (Alves et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021b\u003c/span\u003e). Lime treatments were initially not included (see below) because the original pH of soil was already above 7 (soils in the Salinas Valley are frequently limed) (Smith and Hartz \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2016a\u003c/span\u003e). Further, Chaney et al. (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2009\u003c/span\u003e) proposed that pH between 6.5 and 7 is optimal because at lower pH Cd become highly phytoavailable and at higher pH Zn become less available, which could affect crop growth. All treatments were conducted in quadruplicate (T\u0026thinsp;=\u0026thinsp;26, n\u0026thinsp;=\u0026thinsp;104 for the two batches), and pots were randomly moved weekly in a block design to minimize greenhouse climate differences.\u003c/p\u003e \u003cp\u003ePots were watered with reverse osmosis (RO) water with the aim of maintaining 60% water holding capacity and fertigated with ammonium nitrate (NH\u003csub\u003e4\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e \u0026minus;\u0026thinsp;300 mg/dm\u003csup\u003e3\u003c/sup\u003eof N per growth cycle). During the experiment, the temperature was maintained at 27\u003csup\u003eo\u003c/sup\u003eC\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u003csup\u003eo\u003c/sup\u003eC for a 16h period of light. The Silver Whale variety of spinach was selected due to commercial availability and common use by farmers in the region. Seedlings were grown in inert media (vermiculite) for 14 days. After this period, the most vigorous seedlings were manually transplanted to the center of the pots and grew for four more weeks (one plant per pot). The rationality to use one plant per pot was to be able to maintain a mass balance control and to avoid competition between plants. This process was repeated for each life-cycle. Harvest cycle longevity of 42 days was determined by a pre-experimental trial (data not shown). After 42 days, plants were starting to show reproductive parts, and the most common spinach grown in the Salinas Valley is baby spinach (spinach harvested in a young phase). Shoots were harvested by cutting the stem at the soil surface and subsequently washed with RO water. This was repeated for three consecutive life-cycles using the same pot for each treatment.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePost-growth analysis\u003c/h2\u003e \u003cp\u003eAfter harvesting (after each life-cycle), soil samples were analyzed for total content of C and N, pH, EC, and DTPA extraction for Cd and Zn. Plant wet and dry yields (after drying for 72h at 60\u003csup\u003eo\u003c/sup\u003eC) were recorded, and dry plants were acid digested using aqua regia (3 HCl: 1 HNO\u003csub\u003e3\u003c/sub\u003e), following (Uddin et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and analyzed for Cd and Zn using ICPMS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eTo identify statistically significant differences among treatments we used a non-parametric statistical test, given the high variability of the data. The nonparametric Kruskal-Wallis test was applied to identify differences among treatments, while the nonparametric Mann-Whitney was applied to identify differences between the biochar application rates (1 and 5%), both at the 5% significance level. Statistical analysis was conducted using software R Studio version 4.1.1.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cdiv id=\"Sec11\"\u003e\n \u003ch2\u003eSoil characterization\u003c/h2\u003e\n \u003cp\u003eSoil texture was classified as a loam (USDA soil texture classification), with the particle size distribution of sand, silt, and clay of 48%, 42% and 10%, respectively. Other results from the soil analysis include: 1) pH of 7.35; 2) EC of 1.01 mS/cm; 3) bulk density of 1.42 g/cm\u003csup\u003e3\u003c/sup\u003e; 4) WHC of 34.13 mass %; 5) Total C% and N% of 0.69% and 0.09% respectively; 6) P extractable concentration by Olsen of 14.92 mg/kg; 7) Ca, Mg, K, Na extractable concentration by 1 M ammonium acetate of 3012.66 mg/kg, 200.12 mg/kg, 202.78 mg/kg and 97.44 mg/kg respectively; 8) CEC\u003csub\u003esum\u003c/sub\u003e of 17.52 cmolc/kg; 9) Total concentration of P, Ca, Cd, Fe, Mg, Ni and Zn by acid digestion of 9.3 g/kg, 0.47 g/kg, 2.30 mg/kg, 21.95 g/kg, 0.55 g/kg, 26.05 mg/kg and 74.50 mg/kg. 10) Plant available concentration of K, Ca, Cd, Fe, Mg, Ni and Zn by DTPA (pH 7.3) extraction of 1.2 mg/kg, 26.72 mg/kg, 0.74 mg/kg, 41.91 mg/kg, 2.24 mg/kg, 1.47 mg/kg and 3.10 mg/kg.\u003c/p\u003e\n \u003cp\u003eAccording to these results, the total concentration of Cd is well above global concentration of 0.1\u0026ndash;0.5 mg/kg (UNEP \u003cspan\u003e2010\u003c/span\u003e) and well above quality levels of \u0026lt;\u0026thinsp;0.5 mg/kg and prevention levels of \u0026lt;\u0026thinsp;1.3 mg/kg established by the US EPA SW 848 (Kabata-Pendias \u003cspan\u003e2011\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec12\"\u003e\n \u003ch2\u003eUnoxidized biochars\u003c/h2\u003e\n \u003cp\u003eResults from the three life-cycles for the unoxidized biochars are shown in Table \u003cspan\u003e2\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv\u003e\n \u003cp\u003eTable 2 \u0026ndash; Unoxidized biochars batch--mean values for three life cycles.\u0026nbsp;\u003c/p\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.242424242424242%\" rowspan=\"4\"\u003e\n \u003cp\u003eTreatments\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"37.37373737373738%\" colspan=\"4\"\u003e\n \u003cp\u003eSoil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.38383838383838%\" colspan=\"3\"\u003e\n \u003cp\u003ePlant\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"12.32876712328767%\" rowspan=\"2\"\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.32876712328767%\" rowspan=\"2\"\u003e\n \u003cp\u003eEC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.95890410958904%\" rowspan=\"2\"\u003e\n \u003cp\u003eCd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.32876712328767%\" rowspan=\"2\"\u003e\n \u003cp\u003eZn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.17808219178082%\"\u003e\n \u003cp\u003eYield\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.068493150684931%\" rowspan=\"2\"\u003e\n \u003cp\u003eCd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.80821917808219%\" rowspan=\"2\"\u003e\n \u003cp\u003eZn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" rowspan=\"2\"\u003e\n \u003cp\u003e(g dry wt.)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.254237288135593%\"\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.254237288135593%\"\u003e\n \u003cp\u003emS/cm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"28.8135593220339%\" colspan=\"2\"\u003e\n \u003cp\u003emg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.67796610169491%\" colspan=\"2\"\u003e\n \u003cp\u003emg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.742268041237114%\"\u003e\n \u003cp\u003eSoil (control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e5.68 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e3.47 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.24742268041237%\"\u003e\n \u003cp\u003e0.71 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e2.88 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e0.09 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.34020618556701%\"\u003e\n \u003cp\u003e28.14 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.402061855670103%\"\u003e\n \u003cp\u003e141.36 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.742268041237114%\"\u003e\n \u003cp\u003eCompost\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e5.97 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e3.03 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.24742268041237%\"\u003e\n \u003cp\u003e0.62 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e4.59 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e0.12 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.34020618556701%\"\u003e\n \u003cp\u003e25.07 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.402061855670103%\"\u003e\n \u003cp\u003e186.49 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.742268041237114%\"\u003e\n \u003cp\u003eZinc Chloride\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e5.79 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e2.97 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.24742268041237%\"\u003e\n \u003cp\u003e0.71 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e7.77 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e0.05 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.34020618556701%\"\u003e\n \u003cp\u003e21.74 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.402061855670103%\"\u003e\n \u003cp\u003e328.77 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.742268041237114%\"\u003e\n \u003cp\u003eASB 500 - 1 1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e5.70 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e3.74 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.24742268041237%\"\u003e\n \u003cp\u003e0.66 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e3.21 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e0.08 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.34020618556701%\"\u003e\n \u003cp\u003e31.34 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.402061855670103%\"\u003e\n \u003cp\u003e181.23 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.742268041237114%\"\u003e\n \u003cp\u003eASB 500 - 1 5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e5.92 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e3.27 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.24742268041237%\"\u003e\n \u003cp\u003e0.63 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e3.12 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e0.10 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.34020618556701%\"\u003e\n \u003cp\u003e32.46 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.402061855670103%\"\u003e\n \u003cp\u003e142.67 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.742268041237114%\"\u003e\n \u003cp\u003eCSB 600 - m2 1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e5.79 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e3.54 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.24742268041237%\"\u003e\n \u003cp\u003e0.66 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e3.24 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e0.08 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.34020618556701%\"\u003e\n \u003cp\u003e30.88 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.402061855670103%\"\u003e\n \u003cp\u003e119.11 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.742268041237114%\"\u003e\n \u003cp\u003eCSB 600 - m2 5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e5.90 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e3.67 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.24742268041237%\"\u003e\n \u003cp\u003e0.66 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e3.42 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e0.05 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.34020618556701%\"\u003e\n \u003cp\u003e46.74 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.402061855670103%\"\u003e\n \u003cp\u003e167.52 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.742268041237114%\"\u003e\n \u003cp\u003eCSB 600 - m3 1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e5.42 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e3.47 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.24742268041237%\"\u003e\n \u003cp\u003e0.72 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e3.32 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e0.08 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.34020618556701%\"\u003e\n \u003cp\u003e45.13 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.402061855670103%\"\u003e\n \u003cp\u003e159.55 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.742268041237114%\"\u003e\n \u003cp\u003eCSB 600 - m3 5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e5.76 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e3.27 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.24742268041237%\"\u003e\n \u003cp\u003e0.66 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e2.93 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e0.06 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.34020618556701%\"\u003e\n \u003cp\u003e32.85 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.402061855670103%\"\u003e\n \u003cp\u003e146.92 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.742268041237114%\"\u003e\n \u003cp\u003eSSB 700 1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e5.84 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e3.49 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.24742268041237%\"\u003e\n \u003cp\u003e0.71 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e4.16 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e0.07 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.34020618556701%\"\u003e\n \u003cp\u003e37.70 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.402061855670103%\"\u003e\n \u003cp\u003e151.10 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.742268041237114%\"\u003e\n \u003cp\u003eSSB 700 5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e6.04 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e4.39 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.24742268041237%\"\u003e\n \u003cp\u003e0.65 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e10.40 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e0.12 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.34020618556701%\"\u003e\n \u003cp\u003e23.65 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.402061855670103%\"\u003e\n \u003cp\u003e163.20 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.742268041237114%\"\u003e\n \u003cp\u003eWSB 900 1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e5.76 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e3.69 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.24742268041237%\"\u003e\n \u003cp\u003e0.72 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e3.71 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e0.10 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.34020618556701%\"\u003e\n \u003cp\u003e19.84 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.402061855670103%\"\u003e\n \u003cp\u003e67.34 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"24.742268041237114%\"\u003e\n \u003cp\u003eWSB 900 5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e6.46 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e3.68 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.24742268041237%\"\u003e\n \u003cp\u003e0.51 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"9.278350515463918%\"\u003e\n \u003cp\u003e2.94 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43298969072165%\"\u003e\n \u003cp\u003e0.07 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.34020618556701%\"\u003e\n \u003cp\u003e17.25 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.402061855670103%\"\u003e\n \u003cp\u003e84.37 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u0026nbsp;Mean values followed by the same letter in the column are not statistically different by the Kruskal-Wallis test (p\u0026lt;5%).\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eSoil properties\u003c/p\u003e\n \u003cp\u003eAccording to Table \u003cspan\u003e2\u003c/span\u003e, there were statistical differences between treatments for pH and EC. For pH, soil (control), ASB 500\u0026ndash;1 1% and CSB 600 \u0026ndash; m3 1% treatments showed the lowest values, that significantly differed from Zinc, CSB 600 - m2 1%, CSB 600 \u0026ndash; m3 5% and WSB 900 1%, that subsequently significantly differed from the rest of the treatments. For EC, the soil that had one of the lowest pH, had one of the highest EC. As expected, the most saline biochar (SSB 700\u0026thinsp;=\u0026thinsp;6.86 mS/cm) had the highest EC in soil. Even though EC values exceed the threshold of spinach salt tolerance reported by FAO (\u003cspan\u003e1985\u003c/span\u003e) of 2 mS/cm, we could still grow healthy plants, but the increased salinity may have had some impact on yield.\u003c/p\u003e\n \u003cp\u003eThe DTPA extractable Cd concentration ranged from 0.51 mg/kg in WSB 900 5% to 0.72 mg/kg in WSB 900 1% and there was no significant difference in Cd in soil, showing that although some treatments were able to retain Cd, the available extractable Cd is consistent.\u003c/p\u003e\n \u003cp\u003eThe DTPA extractable Zn concentration ranged from 2.88 mg/kg in soil to 10.40 mg/kg in the SSB 700 5% treatment. The high concentration of Zn in soil in this last treatment was expected because the sewage sludge used to make this biochar has a high concentration of Zn (1000 mg/kg) as shown in Table \u003cspan\u003e2\u003c/span\u003e in SI. As expected, the Zinc treatment produced the next highest Zn enrichment (7.77 mg/kg), followed by compost (4.59 mg/kg).\u003c/p\u003e\n \u003cp\u003ePlant yield and uptake\u003c/p\u003e\n \u003cp\u003eIn terms of yield (dry wt.), a few treatments significantly differed from the control (0.09 g), where the highest yield was found in Compost treatment and SSB 700 5% (both with 0.12 g). The only treatments that produced mean (three life-cycles) yields above the control were Zn and SSB 700 5%, ASB 500\u0026ndash;1 5% and WSB 900 1%. It is important to mention that all plants in SSB 700 5% treatment died in the first life cycle, which we attribute to phytotoxicity present in this biochar. This toxicity was transient because three plants grew in the next life cycle and four plants in the last two life cycles (new plants were transplanted after each harvesting for all treatments). We believe washing this biochar before its use may prevent this toxicity.\u003c/p\u003e\n \u003cp\u003eFor Cd concentration in spinach (dry wt.), few treatments reduced Cd uptake when compared to control (mean of all life-cycles was 28.14 mg/kg), that is Compost, Zinc, SSB 700 5%, WSB 900 1% and WSB 900 5% (25.07; 21.74; 23.65; 19.84; and 17.25 mg/kg, respectively). Seven treatments: ASB 500\u0026ndash;1 1%, ASB 500\u0026ndash;1 5%, CSB 600 \u0026ndash; m2 1%, CSB 600 \u0026ndash; m2 5%, CSB 600 \u0026ndash; m3 1%, CSB 600 \u0026ndash; m3 5% and SSB 700 1% resulted in Cd uptake above the control. In treatments with large Cd uptake we observed some leaf Cd toxicity in the form of rolled leaves (Benavides et al. \u003cspan\u003e2005\u003c/span\u003e; Qin et al. \u003cspan\u003e2020\u003c/span\u003e). And none of the treatments resulted in Cd concentrations below CODEX limits (4 mg/kg dry wt.) in any life-cycle (varied from 8.12 mg/kg to 64.08 mg/kg) (full data presented in Table \u003cspan\u003e3\u003c/span\u003e - SI).\u003c/p\u003e\n \u003cp\u003ePaul and Chaney (\u003cspan\u003e2017\u003c/span\u003e) showed that compost and zinc treatments significantly reduced Cd uptake but also significantly reduced yield, when compared to the control. In our case, Compost significantly increased yield and reduced Cd uptake (but not significantly), while Zinc treatment reduced yield (not significantly) and reduced Cd uptake (significantly). All treatments increased Cd uptake by the 3th life cycle including the control, likely because of soil acidification due to fertilization with NH\u003csub\u003e4\u003c/sub\u003eNO\u003csub\u003e3\u003c/sub\u003e and roots exudates. pH started before planting on an average of 6.80 (all treatments) and finished in the third life-cycle on an average of 5.12 (all treatments). This shows that Salinas Valley soil needs to be constantly limed to approximately pH 7. Thus, our hypothesis that the treatments will need to be reapplied over time could not be verified, once the pH dropped.\u003c/p\u003e\n \u003cp\u003eFor Zn uptake, the largest uptake was found, as expected, in the zinc and SSB 700 treatments. Similar to Paul and Chaney (\u003cspan\u003e2017\u003c/span\u003e), Zn uptake in zinc treatment was greater than reported potentially phytotoxic concentrations (\u0026gt;\u0026thinsp;200 mg/kg) in leaves. For the other treatments, we observed no evidence of deficiency or phytotoxicity in any treatment (e.g., necrosis or yellowing). We did not observe a clear competitive correlation among Cd and Zn uptake. For example, zinc treatment had the highest Zn uptake but not the lowest Cd uptake, and the WSB 900 1% treatment had the lowest Zn uptake and one of the lowest Cd uptake, whereas, the CSB 600 \u0026ndash; m2 5% treatment had the highest Cd uptake, and a high uptake of Zn.\u003c/p\u003e\n \u003cp\u003eOverall, SSB 700 5% treatment was the \u0026ldquo;best\u0026rdquo; because it produced higher yield and lower Cd uptake than the control. Interestingly, similar to Yang et al. (\u003cspan\u003e2017\u003c/span\u003e), for SSB 700 treatment the biochar application rate of 5% produced higher yields and lower Cd uptake than the application rate of 1%. On the other hand, similar to Simmler et al. (\u003cspan\u003e2013\u003c/span\u003e), where lower application rates produced higher yields, the CSB 600 \u0026ndash; m2 1% treatment produced higher yield and less Cd uptake than CSB 600 \u0026ndash; m2 5% treatment. Lastly, similar to Bian et al. (\u003cspan\u003e2013\u003c/span\u003e), the CSB 600 \u0026ndash; m3 1% and WSB 900 1% treatments produced higher yield and more Cd uptake than CSB 600 \u0026ndash; m3 5% and WSB 900 5% treatments. These contrasting results show that the relationships among biochar application rates, Cd uptake, and crop yield are not straight forward. Higher application rates of some biochars increase adsorption of Cd, preventing plant uptake, but also increase the adsorption of essential plant nutrients, and reduce yields. Our results are not completely surprising because the biochars we used a wide array of chemical and physical properties (Alves et al \u003cspan\u003e2021a\u003c/span\u003e). We suggest the use of artificial intelligence to try to predict biochar behavior in soil.\u003c/p\u003e\n \u003cp\u003eIn terms of financial, Zinc application can be more expensive than biochar and compost methods (i.e., a ton of ZnSO\u003csub\u003e4\u003c/sub\u003e, biochar and compost may cost roughly \u003cspan\u003e$\u003c/span\u003e500, \u003cspan\u003e$\u003c/span\u003e200, and \u003cspan\u003e$\u003c/span\u003e70 respectively; prices calculated in 2018). Compost use to be more affordable than biochar, but since the development of long-term removal carbon credits market, biochar prices have been significantly reduced The downside of using Zn is that it may increase Cd availability input, because it is a byproduct of Zn production (Hetherington et al. \u003cspan\u003e2008\u003c/span\u003e). Globally, Cd contaminated sites are usually related to geogenic Zn mining or smelter sources (Chaney et al. \u003cspan\u003e2005\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eFinally, greenhouse plant uptake results did not follow lab isotherm results (Alves et al. \u003cspan\u003e2021a\u003c/span\u003e). In the lab, the Cd-retention order was SSB 700\u0026thinsp;\u0026gt;\u0026thinsp;CSB 600 \u0026ndash; m2\u0026thinsp;\u0026gt;\u0026thinsp;ASB 500\u0026ndash;1\u0026thinsp;\u0026gt;\u0026thinsp;CSB 600 \u0026ndash; m3\u0026thinsp;\u0026gt;\u0026thinsp;WSB 900 treatments. While here there was no clear trend between maximum Cd retention and lower Cd uptake, which varied depending on the harvest. These results can be explained by biochar application rates and interactions between soil and biochar.\u003c/p\u003e\n \u003cp\u003eFinally, we recommend future soil-plant based research to predict the most efficient biochar and to test lower application rates, since the rates we apply may not be economically viable.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\"\u003e\n \u003ch2\u003eOxidized biochars\u003c/h2\u003e\n \u003cp\u003eResults from the three life-cycles for the oxidized biochars are shown in Table \u003cspan\u003e3\u003c/span\u003e.\u003c/p\u003e\n \u003cdiv\u003e\n \u003cp\u003eTable 3 \u0026ndash; Oxidized biochars batch--mean values for three life cycles.\u0026nbsp;\u003c/p\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"100%\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.181818181818183%\" rowspan=\"4\"\u003e\n \u003cp\u003eTreatments\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.42424242424242%\" colspan=\"4\"\u003e\n \u003cp\u003eSoil\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"39.39393939393939%\" colspan=\"3\"\u003e\n \u003cp\u003ePlant\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15%\" rowspan=\"2\"\u003e\n \u003cp\u003epH\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"8.75%\" rowspan=\"2\"\u003e\n \u003cp\u003eEC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.75%\" rowspan=\"2\"\u003e\n \u003cp\u003eCd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15%\" rowspan=\"2\"\u003e\n \u003cp\u003eZn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15%\"\u003e\n \u003cp\u003eYield\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15%\" rowspan=\"2\"\u003e\n \u003cp\u003eCd\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.5%\" rowspan=\"2\"\u003e\n \u003cp\u003eZn\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" rowspan=\"2\"\u003e\n \u003cp\u003e(g dry wt.)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.647058823529413%\"\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.294117647058824%\"\u003e\n \u003cp\u003emS/cm\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.8235294117647%\" colspan=\"2\"\u003e\n \u003cp\u003emg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"38.23529411764706%\" colspan=\"2\"\u003e\n \u003cp\u003emg/kg\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003eSoil (control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e6.46 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.142857142857143%\"\u003e\n \u003cp\u003e2.84 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\"\u003e\n \u003cp\u003e0.60 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e2.19 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e0.38 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e20.10 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e76.65 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003eCompost\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e6.14 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.142857142857143%\"\u003e\n \u003cp\u003e3.26 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\"\u003e\n \u003cp\u003e0.64 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e4.91 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e0.28 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e13.77 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e91.13 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003eZinc Chloride\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e6.13 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.142857142857143%\"\u003e\n \u003cp\u003e3.06 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\"\u003e\n \u003cp\u003e0.63 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e4.05 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e0.69 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e17.44 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e106.23 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003eASB 500 -1 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e6.40 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.142857142857143%\"\u003e\n \u003cp\u003e2.87 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\"\u003e\n \u003cp\u003e0.61 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e2.45 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e0.34 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e18.84 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e74.57 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003eASB 500 -1 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e6.32 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.142857142857143%\"\u003e\n \u003cp\u003e2.71 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\"\u003e\n \u003cp\u003e0.59 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e1.95 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e0.32 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e17.14 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e98.54 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003eCSB 600 - m2 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e6.68 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.142857142857143%\"\u003e\n \u003cp\u003e2.45 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\"\u003e\n \u003cp\u003e0.63 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e2.15 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e0.34 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e18.92 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e94.46 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003eCSB 600 - m2 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e6.07 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.142857142857143%\"\u003e\n \u003cp\u003e2.35 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\"\u003e\n \u003cp\u003e0.63 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e2.47 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e0.31 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e22.71 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e100.13 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003eCSB 600 - m3 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e6.27 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.142857142857143%\"\u003e\n \u003cp\u003e2.83 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\"\u003e\n \u003cp\u003e0.60 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e2.18 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e0.40 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e15.88 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e75.02 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003eCSB 600 - m3 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e5.72 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.142857142857143%\"\u003e\n \u003cp\u003e2.94 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\"\u003e\n \u003cp\u003e0.71 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e2.92 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e0.29 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e23.18 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e118.25 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003eSSB 700 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e6.04 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.142857142857143%\"\u003e\n \u003cp\u003e3.28 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\"\u003e\n \u003cp\u003e0.67 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e3.44 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e0.37 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e20.58 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e94.65 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003eSSB 700 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e6.46 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.142857142857143%\"\u003e\n \u003cp\u003e3.64 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\"\u003e\n \u003cp\u003e0.66 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e7.11 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e0.50 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e12.19 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e109.15 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003eWSB 900 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e1%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e6.39 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.142857142857143%\"\u003e\n \u003cp\u003e2.91 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\"\u003e\n \u003cp\u003e0.62 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e2.51 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e0.45 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e18.48 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e80.63 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.367346938775512%\"\u003e\n \u003cp\u003eWSB 900 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u0026nbsp;\u003c/sub\u003e5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e7.02 b\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"7.142857142857143%\"\u003e\n \u003cp\u003e2.44 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"11.224489795918368%\"\u003e\n \u003cp\u003e0.51 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e2.32 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e0.51 ab\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.244897959183673%\"\u003e\n \u003cp\u003e14.42 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.285714285714286%\"\u003e\n \u003cp\u003e57.28 a\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"0%\" height=\"19\"\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003cp\u003e\u0026nbsp;Mean values followed by the same letter in the column are not statistically different by the Kruskal-Wallis test (p\u0026lt;5%).\u003c/p\u003e\n \u003c/div\u003e\n \u003cp\u003eSoil properties\u003c/p\u003e\n \u003cp\u003eAccording to Table \u003cspan\u003e3\u003c/span\u003e, generally, pH was slightly higher (average of 6.31 for all treatments) when comparing to unoxidized batch (average of 5.85 for all treatments), which was not expected since we used the same soil that was sampled at the same time and was stored in five gallons plastic buckets, it may be caused by changes in soil properties while stored.\u003c/p\u003e\n \u003cp\u003eWith the oxidation with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e the majority of the biochars had their pH reduced, except for the WSB 900 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment that despite having its pH reduced, continued to be alkaline. For example, the CSB 600 - m2 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e biochar had its pH reduced from 7.05 to 4.02 (Alves et al. \u003cspan\u003e2021a\u003c/span\u003e). Thus, apart from WSB 900 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% treatment that had pH significantly higher after all harvests, in comparison to the control, most of the other treatments had pH bellow the control (full data in Table \u003cspan\u003e3\u003c/span\u003e \u0026ndash; SI).\u003c/p\u003e\n \u003cp\u003eThe oxidation also significantly reduced the biochar EC (salts were removed by the washing process). The salinity reduction was more pronounced in some (e.g., WSB 900 original EC 6.02 mS cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e EC 0.93 mS cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) than in others (e.g., SSB 700 original EC 6.86 mS cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e EC 2.32 mS cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) (Alves et al. \u003cspan\u003e2021a\u003c/span\u003e). Surprisingly, there were no statistical differences among treatments for EC, although SSB 700 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% treatment the most saline (3.64 mS/cm) and CSB 600 \u0026ndash; m2 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% the least saline (2.35 mS/cm), but not very much different from the soil control (2.84 mS/cm). Salinity also slightly increased over the three life-cycles, probably due to the fertigation effect. Even though EC reached 4.81\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62 mS/cm in the 3rd life-cycle for SSB 700 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 1%, we could still grow plants, although there may have been an unknown yield effect, as in the case with the unoxidized biochar.\u003c/p\u003e\n \u003cp\u003eFor DTPA extractable elements in soil, no treatment statistically differed from the control for Cd concentration, again, showing some consistency. Overall, the lowest mean concentration of Cd was obtained with WSB 900 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% treatment (0.51 mg/kg) in all three life-cycles while mean concentrations of 0.71 mg/kg were found in all three life-cycles for CSB 600 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5%.\u003c/p\u003e\n \u003cp\u003eFor extractable Zn in soil, statistical differences were found among treatments. SSB 700 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% (7.11 mg/kg) statistically differed from control (2.19 mg/kg). Results show that the modification with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e must have removed the phytotoxicity initially found in its unoxidized version.\u003c/p\u003e\n \u003cp\u003ePlant yield and uptake\u003c/p\u003e\n \u003cp\u003eIn terms of yield (dry wt.), there were statistical differences among treatments. Zinc treatment was overall the best treatment in terms of yield for the three harvests (mean weight for all life-cycles, 0.69 g dry wt.). The WSB 900 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% treatment was the second-best treatment followed by SSB 700 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% (0.51 g and 0.50 g, respectively). Note that the spinach was not killed in the first harvest of the SSB 700 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% treatment, as was the case with the unoxidized biochar. The lowest yields were obtained with compost, followed by CSB 600 \u0026ndash; m3 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% (0.28 and 0.29 g, respectively). Interestingly, in the unoxidized batch compost produced a higher (but not significantly) yield than the zinc treatment, while the oxidized batch produced lower yield.\u003c/p\u003e\n \u003cp\u003eFor Cd concentrations in spinach (dry wt.), when comparing to the unoxidized batch, more treatments could reduce Cd uptake when comparing to control (mean from all treatments: 20.10 mg/kg). That is, SSB 700 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% (12.19 mg/kg)\u0026thinsp;\u0026lt;\u0026thinsp;compost (13.77 mg/kg)\u0026thinsp;\u0026lt;\u0026thinsp;WSB 900 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% (14.42 mg/kg)\u0026thinsp;\u0026lt;\u0026thinsp;CSB 600 - m3 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 1% (15.88 mg/kg)\u0026thinsp;\u0026lt;\u0026thinsp;ASB 500 -1 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% (17.14 mg/kg)\u0026thinsp;\u0026lt;\u0026thinsp;zinc (17.44 mg/kg)\u0026thinsp;\u0026lt;\u0026thinsp;WSB 900 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 1% (18.48 mg/kg)\u0026thinsp;\u0026lt;\u0026thinsp;ASB 500\u0026ndash;1 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 1% (18.84 mg/kg)\u0026thinsp;\u0026lt;\u0026thinsp;CSB 600 \u0026ndash; m2 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 1% (18.92 mg/kg) could reduce Cd uptake. In general, concentrations were lower in the oxidized batch probably due to the higher initial pH values when compared to the unoxidized batch.\u003c/p\u003e\n \u003cp\u003eThe closest Cd uptake in spinach we obtained to the CODEX limit was 5.18 mg/kg with compost and 7.22 mg/kg with SSB 700 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% in the 3rd life-cycle. For both SSB 700 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% and compost treatments, increased application rates might achieve CODEX limits. The SSB 700 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% treatment had the advantage of also increasing the yield by 30.89% when compared to the control. The SSB 700 biochar, if proven to remove phytotoxicity after washing with water may also be a very economically feasible solution, because currently the Monterey Wastewater treatment facility spends money to place the sludge in a landfill, and if they could invest in a biochar pyrolizer, maybe could produce a biochar cost effective and a feasible solution to address spinach grower issues with Cd.\u003c/p\u003e\n \u003cp\u003eImportant to say that we observed a wrinkled leaf appearance associated with Cd toxicity in treatments with large Cd uptake, i.e., CSB 600 - m2 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% and CSB 600 - m3 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5%. Surprisingly, Cd uptake increased from the 1st to the 2nd life-cycle (mean all treatments) and reduced from the 2nd to the 3rd life cycle (mean all treatments) coming back to the 1st life-cycle levels, even though the pH reduced over time, showing that maybe the fertilization had some effect in Cd uptake in spinach.\u003c/p\u003e\n \u003cp\u003eIn the unoxidized batch the highest Zn uptake was verified in the zinc treatment that was significantly higher when looking mean values for all life-cycles. In the oxidized batch, CSB 600 - m3 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5%, SSB 700 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5%, zinc and CSB 600 - m2 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% had higher mean concentration than the control. No evidence of Zn deficiency was observed in the unoxidized batch. Again, we did not verify a clear competition between Cd and Zn in this batch, since CSB 600 \u0026ndash; m3 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% treatment had the highest Zn uptake and the highest Cd mean uptake.\u003c/p\u003e\n \u003cp\u003eOverall, SSB 700 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% was the \u0026ldquo;best\u0026rdquo; biochar treatment with the highest yield among the biochar treatments, and the lowest Cd uptake. Other treatments that might also be effective for Cd remediation purposes are 1) WSB 900 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% that also had a higher yield than control, lower Cd uptake; and 2) zinc that reduced Cd uptake and increased yield compared to the control.\u003c/p\u003e\n \u003cp\u003eInterestingly, the SSB 700 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and WSB 900 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e biochar treatments with the application rate of 5% produced higher yield and lower Cd uptake than the application rate of 1%. Nevertheless, CSB 600 \u0026ndash; m3 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 1% treatment had higher yield and lower Cd uptake than their 5% treatment counterparts, showing that adding more biochar may not decrease Cd uptake and increase yield. Our results indicate that the greater biochar application rate did not always reduce Cd uptake, possibly because some biochars are sources of trace elements (data not shown). Thus, increasing the biochar application rate may add more Cd to the soil than its retention capacity of metals already available in the soil.\u003c/p\u003e\n \u003cp\u003eFinally, greenhouse results again did not follow lab results (Alves et al. \u003cspan\u003e2021a\u003c/span\u003e). In the lab, the Cd retention order by treatment was WSB 900 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;ASB 500\u0026ndash;1 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;CSB 600 \u0026ndash; m3 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;CSB 600 \u0026ndash; m3 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;SSB 700 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e. Here the greatest average Cd retention in biochar was obtained with SSB 700 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% (12.19 mg/kg)\u0026thinsp;\u0026gt;\u0026thinsp;WSB 900 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% (14.42 mg/kg)\u0026thinsp;\u0026gt;\u0026thinsp;CSB 600 \u0026ndash; m3 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 1% (15.88 mg/kg)\u0026thinsp;\u0026gt;\u0026thinsp;ASB 500 -1 H2O2 5% (17.14 mg/kg).\u003c/p\u003e\n \u003cp\u003eThe higher Cd retention by the poultry manure compost and SSB 700 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e treatment can be attributed to the high concentrations of polar functional groups in these materials (Alves et al \u003cspan\u003e2021a\u003c/span\u003e). Poultry manure compost is rich in N (Table \u003cspan\u003e2\u003c/span\u003e - SI), while SSB 700 is rich is N, S (Alves et al. \u003cspan\u003e2021a\u003c/span\u003e) and Zn (Table \u003cspan\u003e1\u003c/span\u003e - SI). N and S form very stable bonds with Cd. The formation of these bonds can be explained by the concept of \u0026ldquo;hardness\u0026rdquo; or \u0026ldquo;softness\u0026rdquo; (Pearson \u003cspan\u003e1963\u003c/span\u003e). Cd is a soft acid, i.e., an electron acceptor atom of low positive charge and large size and has several easily excited outer electrons. A soft acid is polarizable and covalently binds to soft bases such as N- and S-containing surface functional groups (amine, amide, and thione) (Sparks \u003cspan\u003e2003\u003c/span\u003e). Cd also binds strongly to oxygenated functional groups (e.g. -OH, C\u0026thinsp;=\u0026thinsp;O, C-O), so by oxidizing biochar, we could enrich it with O as well. We speculate that a modification with amination would also be a good option to maximize biochar retention, but that is a modification that would increase biochar\u0026rsquo;s cost significantly and we thought that wouldn\u0026rsquo;t be a viable solution compared to compost and zinc application.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eEven though no treatment produced Cd uptake levels below the CODEX limit (4 mg/kg dry wt.) after three life cycles, the best treatments when considering yield and Cd uptake were WSB 900 5% and poultry manure compost 5% in the unoxidized batch and SSB 700 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 5% and zinc in the oxidized batch. WSB 900 is C rich and highly aromatic, with high metal adsorption capacity including Cd\u003csup\u003e+\u0026thinsp;2\u003c/sup\u003e (Alves et al \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e) while, poultry manure compost and SSB 700 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e are rich in N or S, which form very stable bonds with Cd. Our hypothesis that biochars are more effective than other proposed solutions (ZnCl\u003csub\u003e2\u003c/sub\u003e and compost) was not verified since the lowest mean Cd uptake (5.18 mg/kg) was obtained by compost in the oxidized batch.\u003c/p\u003e \u003cp\u003eOur hypothesis that higher application rates would reduce Cd uptake was not verified, and the effect was biochar-dependent. Some of the biochars may be sources of Cd. The zinc treatment had mixed results, in the unoxidized batch it reduced Cd uptake but also reduced yield, while in the oxidized batch it increased yield and reduced Cd uptake in spinach, showing that further studies must be conducted to understand this solution. Compost use to be the most cost-beneficial for farmers, but since the establishment of the carbon credits marked, biochar has becoming cost-beneficial as well.\u003c/p\u003e \u003cp\u003eThe hypothesis that the material would have to be reapplied over time also was not able to be verified, because pH reduced over time. Although by planting in consecutive growing-harvest cycles we observed that treatment effects seem to be durable, lasting through the third cycle. Our research also showed that Salinas Valley soil needs to be limed to approximately pH 7 to maximize treatments effects. Further studies need to be conducted to achieve safe plants to be consumed.\u003c/p\u003e \u003cp\u003eFinally, our hypothesis that oxidation of biochars would increase Cd retention was also not verified. Oxidation effects were very biochar dependent and increase Cd retention only in SSB 700. However, it is not clear if the benefits achieved by the oxidation came from the EC and phytotoxicity reduction or from the increase in O content itself, since SSB 700 1% and SSB 700 H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e 1% were not so different. We recommend the utilization of this biochar washed with water in another greenhouse study to verify if the phytotoxicity is removed. In general, the oxidation with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e was not as beneficial as we initially expected -- neither in the laboratory (Alves et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2021a\u003c/span\u003e) nor at the greenhouse. Finally, we recommend the use of artificial intelligence to predict biochar effects in soil, once they are very biochar-dependent.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data is provided in the supplemental material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was financially supported by Coordena\u0026ccedil;\u0026atilde;o de aperfei\u0026ccedil;oamento de Pessoal de N\u0026iacute;vel Superior \u0026ndash; CAPES Foundation \u0026ndash; Brazil\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eAuthor\u0026acute;s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBarbara Samartini Queiroz Alves: writing the original draft and data analysis; Luiz Arnaldo Fernandes: Data analysis; Randal J Southard: Visualization and revision of the paper. All the authors contributed to the final review of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Acknowledgments\u003c/p\u003e\n\u003cp\u003eWe thank Snow Seeds for donating the seeds and acknowledge Dr. Bryan Jenkins and his team from the UCD Biomass Lab\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003efor allowing us to perfect the production of the sewage sludge biochars. Finally, we thank Dr. Richard F. Smith, UC Cooperative Extension, Monterey County, CA, and Dr. Tim Hartz from UC Davis for helping us to select sampling site and for providing research guidance.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e\u0026Aring;kerblom, S., E. B\u0026aring;\u0026aring;th, L. Bringmark, and E. 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Elliott. 1981. \u0026ldquo;Occurrence of Naturally High Cadmium Levels in Soils and Its Accumulation by Vegetation.\u0026rdquo; \u003cem\u003eJournal of Environmental Quality\u003c/em\u003e 10 (4): 551\u0026ndash;56.\u003c/li\u003e\n\u003cli\u003eMcKenna, I. M., R. L. Chaney, and F. M. Williams. 1993. \u0026ldquo;The Effects of Cadmium and Zinc Interactions on the Accumulation and Tissue Distribution of Zinc and Cadmium in Lettuce and Spinach.\u0026rdquo; \u003cem\u003eEnvironmental Pollution\u003c/em\u003e 79 (2): 113\u0026ndash;20.\u003c/li\u003e\n\u003cli\u003eNews Desk. 2015. \u0026ldquo;CA Baby Spinach Recalled for Elevated Levels of Cadmium.\u0026rdquo; \u003cem\u003eFood Safety News\u003c/em\u003e, September 2015.\u003c/li\u003e\n\u003cli\u003ePaul, A. L. D., and R. L. 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Cooperative Bulletin No. 493.\u0026rdquo; In: \u003cem\u003eRecommended Soil Testing Procedures for the Northeastern United States\u003c/em\u003e, 75\u0026ndash;85.\u003c/li\u003e\n\u003cli\u003eSigel, A., H. Sigel, and R. K. O. Sigel. 2013. \u003cem\u003eCadmium: From Toxicity to Essentiality\u003c/em\u003e. \u003cem\u003eCadmium: From Toxicity to Essentiality\u003c/em\u003e. Vol. 11.\u003c/li\u003e\n\u003cli\u003eSimmler, M., L. Ciadamidaro, R. Schulin, P. Madej\u0026oacute;n, R. Reiser, L. Clucas, P. Weber, and B. Robinson. 2013. \u0026ldquo;Lignite Reduces the Solubility and Plant Uptake of Cadmium in Pasturelands.\u0026rdquo; \u003cem\u003eEnvironmental Science \u0026amp; Technology\u003c/em\u003e 47 (9): 4497\u0026ndash;4504.\u003c/li\u003e\n\u003cli\u003eSizmur, T., T. Fresno, G. Akg\u0026uuml;l, H. Frost, and E. 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Washington, DC.\u003c/li\u003e\n\u003cli\u003eUSDA. 1925. \u0026ldquo;Soil Survey of the Salinas Area, California.\u0026rdquo;\u003c/li\u003e\n\u003cli\u003eWHO. 2010. \u0026ldquo;Exposure to Cadmium: A Major Public Health Concern.\u0026rdquo; \u003cem\u003ePreventing Disease Through Healthy Environments\u003c/em\u003e, 3\u0026ndash;6.\u003c/li\u003e\n\u003cli\u003eWolnik, K. A., F. L. Fricke, S. G. Capar, M. W. Meyer, R. D. Satzger, E. Bonnin, and C. M. Gaston. 1985. \u0026ldquo;Elements in Major Raw Agricultural Crops in the United States. 3. Cadmium, Lead, and Eleven Other Elements in Carrots, Field Corn, Onions, Rice, Spinach, and Tomatoes.\u0026rdquo; \u003cem\u003eJournal of Agricultural and Food Chemistry\u003c/em\u003e 33 (5): 807\u0026ndash;11.\u003c/li\u003e\n\u003cli\u003eYang, X., K. Lu, K. McGrouther, L. Che, G. Hu, Q. Wang, X. Liu, et al. 2017. \u0026ldquo;Bioavailability of Cd and Zn in Soils Treated with Biochars Derived from Tobacco Stalk and Dead Pigs.\u0026rdquo; \u003cem\u003eJournal of Soils and Sediments\u003c/em\u003e 17 (3): 751\u0026ndash;62.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Biochar, Cd removal, Oxidizing with H2O2, Spinach, Waste management, Soil chemistry","lastPublishedDoi":"10.21203/rs.3.rs-3857978/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3857978/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCadmium (Cd) is a toxic element that can be consumed by humans through food, posing a public health problem. Application to soil of zinc chloride (ZnCl\u003csub\u003e2\u003c/sub\u003e), and amendments, such as compost and biochar, have been proposed as solutions to prevent Cd from entering the food chain. Biochar is a stable carbon-rich charcoal that is produced by an eco-friendly method. The objective of this research was to compare the effectiveness of the treatments as remediation materials for an agricultural soil enriched with Cd. Pot experiments were conducted with spinach and soil material collected in the Salinas Valley of California, for three consecutive life-cycles of spinach. We utilized five biochars (pre-selected in laboratory) in their unoxidized and oxidized (with hydrogen peroxide - H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) versions, at application rates of 1% and 5% by volume. Cd uptake by spinach was compared to uptake in soil alone (control), soil + 25 mg/kg Zn, and soil + 5% poultry litter compost by volume. Compared to the control, some biochars significantly reduced Cd uptake and increased yield. Compost was also a successful amendment and may be the most cost-beneficial for farmers, although biochar produces carbon credits. The Zn treatment generally reduced Cd uptake, but also reduced yield. To achieve uptake values close to the CODEX limit (4 mg/kg dry wt.) it is necessary to lime all treatments to approximately pH 7. The oxidation of biochars with H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e did not decrease Cd uptake.\u003c/p\u003e","manuscriptTitle":"Effects of Unoxidized Biochars, Oxidized Biochars, Zinc and Compost on Cadmium Uptake in Spinach","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-29 09:28:58","doi":"10.21203/rs.3.rs-3857978/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"834d52a7-94f7-42da-9255-805878ef12c6","owner":[],"postedDate":"February 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-17T13:37:00+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-29 09:28:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3857978","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3857978","identity":"rs-3857978","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}