Induced changes of pyrolysis temperature on the physicochemical traits of sewage sludge and on the potential ecological risks

Biochar from sewage sludge is a low-cost sorbent that may be used for several environmental functions. This study evaluates the induced effects of pyrolysis temperature on the physicochemical characteristics of sewage sludge (SS) biochar produced at 350 (SSB350), 450 (SSB450) and 600 (SSB600), based on the metal enrichment index, metal mobility index (MMI), and potential ecological risk index (PERI) of Cd, Cu, Pb, and Zn. Increased pyrolysis temperature reduced the biochar concentration of elements that are lost as volatile compounds (C, N, H, O, and S), while the concentration of stable aromatic carbon, ash, alkalinity, some macro (Ca, Mg, P2O5, and K2O) and micronutrients (Cu and Zn), and toxic elements such as Pb and Cd increased. Increasing the pyrolysis temperature is also important in the transformation of metals from toxic and available forms into more stable potentially available and non-available forms. Based on the individual potential ecological risk index, Cd in the SS and SSB450 were in the moderate and considerable contamination ranges, respectively. For all pyrolysis temperature biochar Cd was the highest metal contributor to the PERI. Despite this, the potential ecological risk index of the SS and SSBs was graded as low.

The proximate analysis [moisture, ash, volatiles, and fixed carbon (FC)] of the sewage sludge (SS) and sewage sludge biochar (SSB) were determined using different procedures. The moisture content was determined by the weight loss of the sample as it was heated to 150 °C. The volatile content was determined as the sample was heated from 150 to 750 °C in a muffle (Linn-Elektro Therm model N 480 D). The ash content of each sample was measured by dry combustion in a muffle furnace at 750 °C for 6 h 29 . Fixed carbon was calculated according to the formula 2: Characteristics of the mineral fractions. The total metal concentration in the biochar was determined in 5 cm of macerated and homogenized samples deposited in 20 cm acrylic capsules. sealed with a 0.2 mm thick polypropylene film. The samples were analyzed by the method 6200 30 using energy dispersive portable X-ray fluorescence (PXRF) spectrometry (Brucker, model Titan 600).
The metal concentration was also determined in a 3050B extract 31 by atomic absorption spectroscopy (Varian, model FS 240F). Samples of 0.5 g of SS and SSB were digested in 5 mL of 2 M HNO 3 solution together with 2 mL H 2 O 2 (30%) and the volume was made up to 50 mL with Milli-Q water.  www.nature.com/scientificreports/ Physicochemical characteristics. The feedstock and SSB samples were suspended in deionized water and 1 M KCl (1:10 m/v ratio), stirred for 30 min and allowed to stand for 5 min, then assessed for pH (H2O) and pH (KCl) , respectively, using a pH meter (Hanna, model HI 3221). The electroconductivity (EC) was analyzed using a conductivity meter (Tecnal, model 4 MP). Cation exchange capacity (CEC) was measured as described by Ref. 32 . In summary, 25 g of each biochar sample and 125 mL of 1 M NH 4 OAc were transferred into 200 mL vessels and shaken on a reciprocal shaker for 15 h. The vessel contents were poured through a filter paper-fitted Buchner funnel. Each flask containing biochar was rinsed four times with 25 mL NH 4 OAc to remove biochar stuck onto container sides and the leachate was discarded. The biochar on the filter paper was rinsed eight times by adding 25 mL of 95% CH 3 CH 2 OH to remove the excess NH 4 adsorbed . The NH 4 + adsorbed in the biochar was displaced with 1 M KCl. The leachate was transferred to a 250 mL volumetric flask and the volume was made up to 250 mL with 1 M KCl. The concentration of NH 4 + in the KCl extract was determined by Spectro colorimetry analysis (PerkinElmer Model Lambda 25 UV/Vis) at ƛ = 400 nm. The concentration of NH 4 + was determined in both the sample and the blank KCl extraction solution. The concentration of NH 4 + was calculated using the Nessler method 33 according to Eq. (3): where CEC is the cation exchange capacity (cmol c kg-−1 ); NH 4 + Extractant is the concentration of NH 4 + adsorbed by the biochar (mg L −1 ) and NH 4 + Blank is the concentration of NH 4 + in the blank extractant solution (mg L −1 ). Potential ecological risk index (PERI). The potential ecological risk index (PERI) proposed by Hakanson 27 was used to evaluate the potential ecological risk of PTEs in biochar produced at different temperatures. The method takes into consideration the toxic level, total concentration, and ecological sensitivity to PTEs 36 . The PERI was calculated according to the steps described by Eqs. (6, 7 and 8):

Speciation of potential toxic metals (PTEs
where Cf is the contamination factor, a measure of the degree of pollution on PTE; Cm and Cn are the concentrations of each PTE in the mobile (F1 + F2 + F3) and stable fractions (F4 + F5 + F6) respectively; T r is the biological toxic factor for individual metals: Zn (1), Cu (5), Pb (5), and Cd (30) 27 ; E R is the potential ecological risk index of a single element; PERI is the potential ecological risk index of the overall contamination. The values of Cf, E R , and PERI were used to assess the risk of metal in the SS and the different pyrolysis temperature SSB s .

Results and discussion
Ultimate and proximate analysis. When compared to biochar produced from other feedstocks, sew- The reduction of C and O in the biochar occurs mostly by the volatilization of the elements as CO, CO 2 , H 2 O, and hydrocarbon during pyrolysis 40 . Additional loss of O alone or associated with H occurs during pyrolysis due to the reduction of the hydroxyl (-OH) functional groups, dehydration, and condensation processes 41 . Decreases in the H content with increasing pyrolysis temperature have also been reported for other feedstocks 5,12 . Nitrogen is lost mainly by the volatilization of different nitrogen groups such as NH 4 -N or NO 3 -N at low temperatures 42 , and pyridine at temperatures > 600 °C 43 . The decrease in S with temperature has been reported in other studies 44,45 . The loss of S from the biochar is due to sulfur containing volatile organic compounds. Organic sulfur losses to the vapor phase during pyrolysis have been primarily identified as carbonyl sulfide 46 .
The reflex of the pyrolysis temperature on the reduction of C, H, N, O, and S is the change in key biochar treatments such as the H/C molar ratio, an index of the biochar aromaticity and stability; the O/C molar ratio, an index of polarity or the abundance of polar oxygen-containing surface functional groups 47 ; and the C/N molar ratio, an index of inorganic N release from organic matter when biochar is incorporated into soils 48 .
The H/C and O/C molar ratios reduced with pyrolysis temperature and ranged from 2.11 ± 0.04 (SS) to 0.61 ± 0.02 (SSB 600 ), and from 0.24 ± 0.02 (SS) to 0.03 ± 0.01 (SSB 600 ). Plotting the H/C and O/C molar ratios in a van Krevelen diagram (Fig. 2) shows the reduction of the molar ratio with the increase of pyrolysis temperature. This result is in agreement with the results reported by Zhang et al. 49 , where evaluation of biochar from cow manure produced at 300, 400, 500, 600, and 700 °C also reported H/C and O/C molar reduction with increase of temperature and attributed the results to the formation of stable aromatic structures. According to Ahmad et al. 9 , at pyrolysis temperatures up to 480 °C, the decrease in H/C and O/C molar ratio with increasing temperature is due to the loss of carboxylic and phenolic functional groups that are responsible for the CEC; above 480 °C, the reduction occurs due to the processes of dehydration and deoxygenation, which reduce H-and O-containing functional groups.
The C/N molar ratio of the SS (7.63 ± 0.37) did not differ from those of SSB 350 (7.50 ± 0.37) and SSB 450 (7.17 ± 0.24), but it was reduced in the SSB 600 (7.04 ± 0.20) as a result of the formation of compounds rich in C and poor in volatile N 50,51 . According to Jindo et al. 52 , the biochar from lignocellulosic material ranged from 40 to 256 ;thus, the low C/N ratio of the biochar in this study revealed the SSB potential as a source of N for plants.
Proximate analysis typically involves the determination of volatile matter, moisture, fixed carbon (FC), and ash 53 . Pyrolysis reduced the volatile content by up to 83% (from SS 36.6 ± 0.7 to SSB 600 6.3 ± 0.3) due to the transformation of compounds containing O-C=O into gas 53 , leading to an increase in the concentration of Si, Al, and Fe oxides with increasing temperature. Working with sewage sludge pyrolyzed at temperatures from 300 www.nature.com/scientificreports/ to 900 °C 54 also yielded a reduction in volatile compounds with increasing temperature. The moisture content of the SS (7.3 ± 0.0%) reduced by approximately five times as much as the SSB 600 (1.5 ± 0.2%; Table 1). The moisture reduction was attributed to water evaporation and loss of pyrolytic volatiles 55 relative to SS. The content of FC, carbon remaining after loss of moisture and free volatile materials of the SSB 600 (13.2 ± 1.0%) was about seven times higher than that obtained for SS (2.0 ± 0.4%). Working with biochar produced at different pyrolysis temperatures 5,47 also found similar FC trends. The plot of H/C and FC content (Fig. 3) indicated that the SS material has more H relative to FC, while the biochar produced at high pyrolysis temperatures has less H relative to FC. Working with biochar derived from macaúba endocarp pyrolyzed at temperatures from 200 to 700 °C 56 , reported a similar relationship between H/C and FC. The reduction in the H/C ratio at higher temperatures also results from the breakup of oxygen-containing functional groups, such as carboxyl, carbonyl, and methoxyl, and the formation of aromatic compounds 57 .
Ash accounted for between 72.7% and 81.5% of the proximate analysis components ( Table 1). The ash content in the biochar (SSB 350 72.5 ± 2.6%, SSB 450 74.5 ± 0.6% and SSB 600 78.9 ± 0.7%) was higher than in the SS (55.4 ± 1.9%; Table 1). These results are similar to the one reported by Regkouzas and Diamadopoulos 60 , that studying SSB produced at 300 (63.97%), 500 (77.44%) and 700 (81.15%) also found increased ash concentration with pyrolysis temperature and values closed to the ones found in this study. The pyrolysis process impacts not only the biochar ash concentration but also the quality of the material produced, which will be discussed in the following sections. Physicochemical characteristics. Pyrolysis at different temperatures promoted significant physicochemical changes in the feedstock (Table 2). When compared to feedstock pH (pH H2O 4.5 and pH KCl 4.2), the biochar acidity was reduced to 1.3 pH H2O units (pH 4.8 SSB 350 , pH 5.7 SSB 450 , and pH 5.8 SSB 650 ) and 1.2 pH KCl units (pH 4.4 SSB 350 , pH 5.3 SSB 450 , and pH 5.4 SSB 650 ) with increasing pyrolysis temperature. The increase in biochar pH with thermal treatment has been attributed to the loss of acidic functional groups (carboxyl, hydroxyl, or formyl) on the biochar surface 1 . The increase in biochar alkalinity is due to the separation of alkali elements (Ca, Mg, and K) from organic constituents during pyrolysis 45 , which contributes to the potential liming effect. When studying biomass from SS pyrolyzed at 300, 500, and 700 °C 60 , also reported an increase in pH with increasing pyrolysis temperature.
Biochar EC, an estimator of the amount of total dissolved salts in the sample, is one of several biochar properties influenced by the feedstock source and pyrolysis conditions, such as temperature, residence time, and activation treatment 61,62 . The EC of the SS (4.0 ± 0.0 dS m −1 ) was higher than that observed for SSB 350 (2.2 ± 0.0 dS m −1 ),    Table 2). During SS pyrolysis, the ash content increases, whereas the solubility of salts and metals decreases 63,64 . This occurs because the water-soluble concentrations of K + , Ca 2+ , Mg 2+ , and P increase in biochar produced up to 200 °C, but above that temperature it is likely that they will form crystals like whitlockite [(Ca, Mg) 3 (PO 4 ) 2 ]. At pyrolysis temperatures over 500 °C they will be incorporated into the silicon structure, forming less soluble salts 58,65 . The cation exchange capacity (CEC) is one of the most important biochar characteristics because it indicates the potential of the material to attract positively charged ions per unit of mass 66 . In this study, the CEC of the SSB 350 (6.3 ± 0.1 cmol c kg −1 ) and SSB 450 (6.4 ± 0.0 cmol c kg −1 ) were higher than those obtained for both SS and SSB 600 (6.0 ± 0.1 cmol c kg −1 ; Table 2). Biochar produced at temperatures up to 480 °C tends to have higher CEC because some acidic oxygenated functional groups, such as phenolic acid and carboxyl groups, are retained 67 . In contrast, biochar produced at temperatures above 480 °C has lower CEC 68 .
Mineral composition. The total concentrations of some mineral elements are shown in Table 3. The reduction of C, H, S, O, moisture, and volatile content with increasing temperature shows a positive correlation with the increase in concentrations of nonvolatile elements normalized for oxides, such as SiO 2 , Al 2 O 3 , Fe, CaO, and P 2 O 5 , which are the main mineral components of SS and SSBs. The SiO 2 concentrations in SSB 450 (43.49 ± 0.11%) and SSB 600 (40.85 ± 0.11%) were higher than the values observed for SS (33.37 ± 0.09%) and SSB 350 (33.30 ± 0.29%). In contrast, the increase in Al 2 O 3 concentration in the biochar with increasing pyrolysis temperature can be described by linear regression (Al 2 O 3 % = 0.0097 (Temperature) + 7.3453, R 2 = 0.86).
The concentration of Al 2 O 3 found in this study was lower than the 17.2% and 29.6% reported by Fan et al. 69 in biochar produced from a cyclic activated sludge system (CSS) process and an applied membrane bioreactor (KSS). The higher Al content was attributed to the presence of inorganic solids from the WWTP. The presence of SiO 2 , Al 2 O 3 , and Fe in biochar is generally associated with the presence of soil material or chemicals used in the coagulation step of SS treatment 70 .
The main macronutrients present in the feedstock were CaO, P 2 O 5 , and MgO, and Fe, Zn, Mn, and Cu were the main micronutrients. The P 2 O 5 concentration (from 2.06 ± 0.01% SS to 2.72 ± 0.05% SSB 650 ), and K 2 O (from 0.37 ± 0.0% SS to 0.46 ± 0.01% SSB 600 ) increased with pyrolysis temperature ( Table 3). The effect of pyrolysis on the MgO content was negligible, and there is no clear explanation for CaO reduction (from 2.80 ± 0.00% SS to 2.24 ± 0.01% SSB 450 ) with increasing temperature. The MEI of the macronutrients followed the sequence P 2 O 5 > K 2 O > MgO > CaO ( Table 4). The increase in metal enrichment with temperature is due to the  www.nature.com/scientificreports/ decomposition of organic matter, which results in the release of the metals associated with organic compounds, and loss of volatile content 18 . Biochar produced from organic residues such as SS has the potential to present high concentrations of PTE, and their content increases with pyrolysis temperature as they form inorganic salts, hydroxides, oxides, and/or sulfides 18,71 . Similar findings were obtained in this study (Tables 4 and 5), in which the concentration and MEI values of Cu, Zn, Pb, Mn, and Fe increased in line with pyrolysis temperature due to the loss of volatile materials and moisture 3 , and the high boiling points of PTEs 72 .
The concentration of PTEs in the biochar followed the sequence Fe (from 5.05 ± 0.02% SS to 6.18 ± 0.05% SSB 600 ) > Zn (from 650 ± 30 mg kg −1 SS to 1120 ± 38 mg kg −1 SSB 600 ) > Mn (450 ± 30 mg kg −1 SS and 570 ± 30 mg kg −1 SSB 600 ) > Cu (from 290 ± 0 mg kg −1 SS to 520 ± 10 mg kg −1 SSB 600 ; Table 4). The higher Fe concentration in the SS (5.05%) compared to the other PTEs is related to the addition of ferric chloride during sludge aerobic digestion 73 . The MEI separated the micronutrients into two groups; metals with higher atomic mass (Cu and Zn) were enriched in higher proportions than those of lower atomic mass (Mn and Fe). The PTE concentrations in the SS and biochar used in this study are in agreement with the pollutant control standard of the International Biochar Initiative Guidelines 74 (Cu from 143 to 6000 mg kg −1 and Zn from 416 to 7400 mg kg −1 ).
The Pb concentration ranged from 50 mg kg −1 (SS) to 80 mg kg −1 (SSB 600 ; Table 3), and the MEI ranged from 1.13 (SSB 350 ) to 1.65 (SSB 450 ). Based on frequency, toxicity, and potential exposure, Pb is the second most dangerous element behind arsenic (As) 75 . However, the Pb concentration was below the lower limit (121-300 mg kg −1 ) reported by IBI 74 . Moreover, despite the relevance of the data for content and enrichment of PTE, it must be considered that the estimation of the total metal content is insufficient to assess metal bioavailability, environmental risk, and toxicity, which are controlled by their chemical species rather than their absolute quantities in the samples 76,77 .  Table 5. Effect of the pyrolysis temperature on contamination factor (Cf), potential ecological risk coefficient (Er) and potential ecological risk Index (PERI) of the sewage sludge (SS) and sewage sludge biochar produced at 350 (SSB 350 ), 450 (SSB 450 ) and 600 (SSB 600 ). www.nature.com/scientificreports/ Metal fractionation and metal mobility index. The geochemical forms of PTEs in the environment determine their bioavailability, ecotoxicity, diffusion in mobile forms, and consequently their fate in the environment 71 . Metal fractionation is the term used to identify and quantify the different operationally defined species forms or phases in which an element occurs 78 . The fractionation scheme used in this study for quantification of PTEs was based on operationally defined fractions for the following pools: water-soluble (F1), exchangeable (F2), carbonate, (F3), Fe-Mn oxides (F4), organic (F5), and residual (F6) fractions. The process used in this study for determination of metal availability in the SS and SSBs was adapted from the classifications of Ref. 18 . The metals present in the F1, F2, and F3 fractions were classified as bioavailable because they are readily released to the environment. The metals in the F4 and F5 fractions were classified as potentially bioavailable because they are leachable only under very rigorous conditions. The metals in F6 were classified as non-bioavailable because they are unlikely to leach and degrade under natural conditions.

Sample
The metal concentrations resulting from USEPA 3050 are given by the sum of all fractions. The extractable fractions of the metals from the SS and SSBs, as well as their mobility index, are shown in Figs. 4, 5, 6 and 7. The total Cd concentration of the SSB 600 (1.58 ± 0.02 mg kg −1 ) was ~ 2.5 fold higher than that of the SS (0.65 ± 0.02 mg kg −1 ; Fig. 4a). In contrast to Ref. 49 , where up to 73.3% of the Cd in cow manure biochar produced at 300, 400, 500, 600, and 700 °C was present in the directly toxic and bioavailable fraction, in this study, bioavailable and toxic forms represented 58.11 ± 1.19% (SS), to 54.86 ± 2.18% (SS 350 ), and 75.41 ± 6.67% (SSB 450 ). All Cd present in SSB 600 was on the non-bioavailable form (Fig. 4a). Based on the MEI, the Cd present in SSB 600 poses no risk to humans or microorganisms, whereas SS, SSB 350, and SSB 450 have the potential to cause environmental toxicity (Fig. 4b).  www.nature.com/scientificreports/ The Cu concentration of SSB 600 (470.14 ± 2.14 mg kg −1 ) was ~ 1.5-fold greater than that in the SS (329.53 ± 4.21 mg kg −1 ; Fig. 5a). Copper binds readily to organic constituents, forming a highly stable complex 79 . This was observed in this study; in the SS, 2.94 ± 0.22% of Cu was of the toxic bioavailable form, and 90.15 ± 1.37% was of the potentially bioavailable form (Fig. 5a). As the pyrolysis temperature increased, most of the Cu was distributed in potentially bioavailable (SSB 350 77.77 ± 2.12%, SSB 450 73.07 ± 2.47%, and SSB 600 70.69 ± 0.29%) and non-bioavailable (SSB 350 21.81 ± 2.19%, SSB 450 24.96 ± 2.64%, and SSB 600 27.01 ± 0.34%) forms. These results are similar to those reported by Lu et al. 80 where study of SSBs produced at 300, 400, 500, 600, and 700 °C showed that most of the Cu was concentrated in the potentially available and non-available fractions. In this study, 96.3-100% of the potentially available Cu was in the organic fraction. The Cu enrichment in the biochar increased in line with the pyrolysis temperature (SSB 350 0.42%, SSB 450 1.92%, and SSB 600 2.31%); however, since most of the metal is of potentially bioavailable and non-bioavailable forms, this enrichment is not associated with its increase in ecotoxicity (Fig. 5b). Moreover, the Cu MEI in the SSBs was lower than that observed in the SS.
Zinc content in the SSB 600 (665.82 mg kg −1 ) was 1.4 times higher than the SS (487.40 mg kg −1 ), and its distribution in the biochar fraction was greatly influenced by the pyrolysis temperature (Fig. 6a) (Fig. 6b). According to Li et al. 81 , the low mobility of Cr, Ni, Cu, Zn, Cd, Pb, and Hg in sewage sludge biochar is due to their alkaline properties.    Fig. 7a). Similar to this study, evaluation by Wang et al. 59 of biochar produced at temperatures of 300, 500, and 700 °C from hydrothermal pretreatment with pyrolysis (HTP) suggested that up to 99.97% of the Pb was in the residual fraction. Although Pb had the lowest MMI of all the metals studied, the index increased in line with temperature (SS 0.00 ± 0.00, SSB 350 0.00 ± 0.00, SSB 450 0.00 ± 0.00 and SSB 600 5.50 ± 0.78; Fig. 7b). The predominance of Pb in the residual fraction may be associated with the combination of the metal with primary minerals in the SS 59 .
Risk analysis of potentially toxic elements. The risk to the environment and organisms posed by PTEs present in the SS and SSBs produced at different pyrolysis temperature was assessed by calculating the PERI (Table 5). Among the PTEs, Cd and Zn were the only metals that presented Cf values in the range of contamination. Cadmium Cf values increased in line with pyrolysis temperature, and the risk of contamination ranked from moderate for SS (1.39 ± 0.07) and SSB 350 (1.22 ± 0.11), to considerable for SSB 450 (3.64 ± 0.53), while Zn Cf values reduced in line with pyrolysis temperature, and only SS (1.01 ± 0.10) had a Cf value in the range for moderate contamination (Tables 5 and 6). The Pb and Cu Cfs were below the contamination values. According to Ref. 18 , the Cf of individual PTEs measures the degree of pollution by individual heavy metals, and its value is inversely proportional to its leaching potential.
The index of potential ecological risk individuals, given by Er, is a function of the biological toxicity factor of individual PTEs 27 . Cadmium, present in the SS (41.65 ± 2.01) and SSB 450 (109.28 ± 15.89), was the only metal with the individual potential ecological risk index value in the moderate (SS 41.65 ± 2.01) to considerable (SSB 450 109.28 ± 15.89) contamination range. The pyrolysis temperature had a significant effect on reduction of the Er values for the other metals studied ( Table 5).
The potential ecological risk index (PERI) measured the degree of superposition of various harmful PTEs on organisms and the environment 81 . As reported in other studies 59,82 , the increase in pyrolysis temperature had a positive benefit on reducing the PERI value (SSB 350 36.80 ± 3.16%, and SSB 600 0.51 ± 0.04%) as compared with SS (42.81 ± 2.08%). The SSB 450 (109.50% ± 15.89) presented a PERI value of 155.76%, higher than that for SS, because of the increase in Cd availability. Despite the SSB 450 PERI value, the four PTEs have values that suggest a low potential ecological risk for utilization of biochar.

Conclusions
The effects of pyrolysis temperature on sewage sludge biochar physicochemical properties were evaluated. The pyrolysis temperature affects the ultimate and proximate composition, the stability, aromaticity, and polarity of the biochar produced at different temperature. Moreover, the pyrolysis temperature also influenced the concentration of inorganic macro (Ca, Mg, P 2 O 5 , and K 2 O), micronutrients (Cu and Zn), and some toxic elements such as Pb and Cd. The pyrolysis temperature also has an important contribution in the transformation of metals from more toxic and available forms into more stable nontoxic and non-available forms. Based on the individual potential ecological risk index, Cd in the SS and SSB 450 was in the moderate and considerable contamination ranges, respectively, and was the metal with the highest contribution to the PERI. Despite this contribution, the potential ecological risk index of the SS and SSBs was graded as low-risk. Table 6. Grading of contamination factor (Cf), the potential ecological risk coefficient (Er) and potential ecological risk index (PERI). www.nature.com/scientificreports/