Synthesis, Characterization and Ecotoxicity Evaluation of Biochar-Derived Carbon Dots from Spruce Tree, Purple Moor-Grass and African Oil Palm

: Biochar-derived C-Dots from Picea, Molinia caerulea and Elaeis guineensis were synthesized through a hydrothermal process, and their physicochemical and optical characteristics and environmental effects were compared. These C-Dots were characterized by techniques such as Attenuated Total Reﬂection–Fourier Transform Infrared (ATR-FTIR), UV-Vis spectrophotometry, ﬂuorescence spectroscopy, dynamic light scattering (DLS), Z potential, and High-Resolution Transmission Electronical Microscopy (HR-TEM). The ecotoxicity tests were performed using the Microtox™ test, making this study one of the few that use this method. The C-Dots from Molinia caerulea showed the best quantum yield (QY) of 8.39% and moderate ecotoxicity, while Elaeis guineensis has the lowest QY (2.31%) but with zero toxicity. Furthermore, the C-Dots from Picea presents good optical properties but showed high toxicity and limits its use. Finally, all C-Dots showed functional groups that could be biofunctionalized with biomolecules, especially C-Dots from Molinia caerulea and Elaeis guineensis show potential for use in the development of optical biosensors. Transform Infrared (ATR-FTIR) in a Perkin Elmer instrument between 4000 cm − 1 and 650 cm − 1 frequency range and 32 running scans at a resolution of 4 cm − 1 . The stability of the synthesized C-Dots at a concentration of 1000 ppm was examined through Dynamic light scattering (DLS) and Z potential techniques on a Nanoplus-3. The measurement was performed using 0.2 µ m ﬁltered solutions in a B0631009 cell, with water as a dispersant (Refractive Index: 1.3328). The shape and size of the C-Dots were taken using High-Resolution Transmission Electronical Microscopy (HR-TEM) on a FEI-Tecnai F20 Super Twin TMP. great


Introduction
Non-pollutant energy access is one of the sustainable development goals defined by the United Nations (UN) to achieve sustainability by 2030 [1], so many research efforts have been focused mainly on the use of plant biomass for energy and biofuels production from high temperature deconstructive methods such as pyrolysis, gasification, and thermal liquefaction [2][3][4][5]. Pyrolysis is a method with complete absence of oxygen and process temperatures around 300-700 • C. The three main products from pyrolysis are synthesis gas (syngas), bio-oils, and biochar [6]. Syngas and bio-oils are widely used for electricity, heat, and biofuel production while biochar is used in bioremediation and amendments processes in soils [6][7][8]. However, the use of biochar is limited due to the variability of the physicochemical properties that depend on the original biomass and the thermal process conditions. Therefore, alternatives for the use of biochar are necessary for the integral sustainability of the pyrolysis process and to close biological cycles following circular economy principles [9,10].
Biochar from the pyrolysis process usually exhibits a porous and semi-crystalline structure with a large surface area. This kind of structure maintains the surface chemical

Synthesis of Biochar-Derived C-Dots
Biochar-derived C-Dots were synthesized using previously reported methods by Placido et al. [13,21] employing a hydrothermal method in presence of a strong oxidation reagent (KMnO 4 ). First, the biochar was shattered into small pieces using a mortar and then passed through a 425 µm sieve (Woven Wire, Endecotts), the sieved biochar was used for the C-dots production. The depolymerization reaction was performed with 10 g of biochar mixed with a solution of KMnO 4 10% (w/v) in a 500 mL Erlenmeyer flask. The depolymerization reaction was carried on an autoclave (SA-300H, Sturdy, Wilmington, DE, USA) at 121 • C and 15 psi for 60 min. After reaction, the biochar solutions were centrifuged (SL 8, Thermo Scientific) for 5 min at 1000 rpm to separate the liquid (supernatant) and solid phases (remaining biochar). The supernatant was filtered with a 0.22 µm syringe filter (Merck Millipore, Burlington, VT, USA). The filtered liquid was mixed with Acetone and centrifugated (SL 8, Thermo Scientific) again (1000 rpm, 5 min) until the production of two liquid phases. The upper liquid phase was withdrawn, and later roto evaporated (BM500, Yamato, Santa Clara, CA, USA) at 85 • C and 750 HPa. The Carbon-dots were purified by using overnight dialysis (3.5 KDa MWCO, Standard RC Dry Dialysis, Labs Spectra/PorTM 3) and freeze-dried (18LC-16WW-TS, Thermo Scientific) for 32 h. The newly synthesized C-Dots were kept in solid state at 4 • C until use.

Characterization of Biochar-Derived C-Dots
C-Dots were characterized by various spectroscopic and morphological techniques. The fluorescence emission and excitation spectra and the UV-Vis spectra were performed at C-Dots concentration of 500 ppm in a Varioskan Lux (Thermo scientific, SkanIt Software 4.1) on a 200 µL working volume microplates (Falcon™ non-treated black 96-well) and 20 • C with 1 nm optical step. The identification of C-Dots functional groups was performed through Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) in a Perkin Elmer instrument between 4000 cm −1 and 650 cm −1 frequency range and 32 running scans at a resolution of 4 cm −1 . The stability of the synthesized C-Dots at a concentration of 1000 ppm was examined through Dynamic light scattering (DLS) and Z potential techniques on a Nanoplus-3. The measurement was performed using 0.2 µm filtered solutions in a B0631009 cell, with water as a dispersant (Refractive Index: 1.3328). The shape and size of the C-Dots were taken using High-Resolution Transmission Electronical Microscopy (HR-TEM) on a FEI-Tecnai F20 Super Twin TMP.

Quantum Yield Measurement
The quantum yield (QY) of the synthesized C-Dots was calculated through comparative method, using quinine sulfate in 0.5 M H 2 SO 4 and based on previous reports [15,16]. The QY was calculated according to the following equation: where suffix "ref" and "sam" refers to the standard reference and sample, respectively. "I" is the integrated fluorescent emission intensity, "n" is the refractive index of the solvent and "A" is the intensity of absorbance. All the measurements were done at maximum intensity.

Ecotoxicity Assay
The C-Dots aquatic toxicity was evaluated via an ecotoxicity assay performed on a Microtox™ Model 500 Analyzer. The Microtox™ test is based on the measurement of Vibrio fischeri bioluminescence changes which correspond to cell viability, specifically the toxicity is related to fluorescence inhibition of the bacteria [52]. An 81.9% Basic Test with 9 double dilutions was applied, starting from the concentration of 1000 ppm for all the C-Dots samples. The results were calculated, taking into account the Gamma function after 5 and 15 min, and were reported as EC50, that is, the concentration that causes the 50% inhibition of the fluorescence of the microorganism [53]. Gamma [54] as: where I c is the average light reading of filtrates of the control solutions and I t is the light reading of a filtrate of a particular concentration of the test material.

Biochar Characterization
The characteristics of the three types of biochar are shown in Table 1. All materials have alkaline pH, a characteristic attributed to the thermal decomposition of hydroxyl bonds and other weak bonds within the biochar structure produced under medium to high temperatures of synthesis [55]. The biochars exhibited variable nitrogen (N) and sulfur (S) content, which is related to the initial content of the source and the temperature of the synthesis process. It has been reported that with increasing temperatures, more N and S were lost in the material [56,57]. The loss of N in the biochars was attributed to the removal of N via volatilization at relatively low temperatures (~200 • C) [57]. Some works suggest that the pyrolysis temperature is an important factor that influences the speciation of sulfur in the biochar, since there is a much higher fraction of sulfur in organosulfur form for biochar produced at high temperatures, while the biochars at lower temperatures have sulfate, organosulfur, and sulfide [56,58]. those reported for biochar obtained from dairy manure [60], hardwood [61], eucalypt [62], willow [63][64][65], and bagasse from 14 agriculture waste [66]. The three materials reported in this study stand out for having a higher amount of sulfur than some of those reported in the literature [67].
The N and S content is important because these heteroatoms are associated with aromatic rings forming various functional groups on the biochar surfaces [55,68], which influences the properties of the material and its different applications [67,69]. Specifically, in the production of C-Dots from biochar, the N and S heteroatoms can be manipulated to act as electron donors or acceptors, and this process affects the characteristics and optical properties of the C-Dots that are obtained [70].
For the determination of the presence of functional groups on the biochar surface and the C-Dots, the Fourier transform infrared spectroscopy (FTIR) was used. The biochars FTIR spectrum of biochar derived from Picea, Molinia caerulea and Elaeis guineensis (Figure 1) doesn't show peaks with high intensities, which is supported by several authors [71][72][73][74], and it's due to the pyrolysis process, which is performed in biomass to produce biochar, because it promotes the rupture of several functional groups and the progressive carbonization of the material. However, all the spectra showed similar peaks at 744, 813, 877, 1574, and 1700 cm −1 . The peaks at 744, 813, and 877 cm −1 are associated with the carbon linkages, more specifically C-H bending, and the peaks at 1574 and 1700 cm −1 are associated with carboxyl groups. Besides, the Molinia caerulea and Elaeis guineensis biochar shows a C-O group (1111 cm −1 ) that corresponds to a secondary alcohol, while the Picea biochar shows the C-O group (1196 cm −1 ) associated with a tertiary alcohol. These groups are mostly found in biochar that are obtained from sources that tend to be polar, hydrophilic, and relatively reactive, such as lignocellulosic feedstocks. Furthermore, the oxygen-containing functional groups have variable charge, at a higher pH, the carboxylic acids give up protons and become negatively charged (-COO − ) [71,75,76].
The spectra of Molinia cerulea and Elaeis guineensis show peaks at 2000 and 2100 cm −1 , corresponding to triple bond of alkynes that are part of the lignocellulose [77][78][79]. Finally, only the biochar of Elaeis guineensis show the presence of asymmetric (2910 cm −1 ) and symmetric (2850 cm −1 ) C-H stretching bands associated with aliphatic functional groups, which decrease in intensity due to structural changes occurred during pyrolysis that produce a decrease in aliphatic compounds [80,81]. The spectra obtained from the biochar of Picea, Molinia caerule and Elaeis guineensis, don't show high intensities due to the carbonization processes to which the biomass is subjected. In addition, the functional groups are related to lignocellulosic feedstocks such as those used in this work. Finally, it is suggested that the differences found in the spectra may be due to the conditions of the thermal conversion process of biochar, since many authors [21,82] report that variables Processes 2021, 9, 1095 6 of 16 such as reactor type, heating rate, temperature, residence time, oxygen concentration, etc. are significant variables that can affect the characteristics of biochar produced.

Chemical Characterization
The FTIR spectra of the three biochar-derived C-Dots are depicted in Figure 2. These spectra, unlike those obtained for biochar, show an increase in the intensity of the bands and a change in their position, which suggests an increase in the amount (per unit volume) of some functional groups, and a change in the state of hybridization or electronic distribution of molecular bonds. Furthermore, the absence of the bands between 1700 and 2100 cm −1 suggests the formation of other functional groups due to the hydrothermal process with KMnO 4 . The bands between 700 and 900 cm −1 shown in the spectra are associated with con C -H bending, as are the spectra of biochar. The existence of C-O-C bonds leads to the absorption peak at 990 cm −1 for Elaeis guineensis C-Dots and 1045 cm −1 for Picea and Molinia caerulea C-Dots [83,84], and the peak at 1236 cm −1 is associated with C-O stretching [85,86]. One of the most intense peaks of all the spectra occurs at 1370 cm −1 which corresponds to C-N [87][88][89]; the bands located around 1550-1750 cm −1 are assigned, for some authors to C=N [89,90], while other authors associated these bands with the stretching vibration of C=C and carbonyl bonds (C=O) [83][84][85]91]. Moreover, the band around 1700 cm −1 could indicate the vibration of the carbonyl bond of a carboxylic acid, however, because it's weak, it could represent the deprotonation of the group and the apparition of the carboxylate group (COO − ) [20]. The presence of carbonyl groups could allow, in future works, the functionalization of these carbonaceous materials with molecules that have amine groups available through the carbodiimide technique.
Finally, the peaks displayed between 2850 cm −1 and 2950 cm −1 are from C-H stretching vibration [91][92][93], and the broad absorption band within 3100-3600 cm −1 derives from the stretching vibration of hydroxyl bonds (O-H) and N-H [83,84]. This data suggest that the C-Dots obtained from Picea, Molinia caerulea and Elaeis guineensis present large hydrophilic groups indicating the formation of water-soluble materials, and the presence of carbonyl moieties suggest that the C-Dots could be slightly negatively charged.

Morphology and Stability
The HR-TEM and the DLS were utilized to investigate the morphology and average size of the biochar-derived C-Dots. The size distribution obtained by measuring the hydrodynamic diameter of C-Dots (1000 ppm) derived from Picea, Molinia caerulea and Elaeis guineensis; by DLS indicates that these nanoparticles have a total hydrodynamic diameter of 113 ± 13.4, 122.7 ± 2, and 105.4 ± 7.1 nm and the polydispersity index are 0.19 ± 0.01, 0.18 ± 0.04, and 0.2 ± 0.03, respectively. Besides, the DLS graphs show that the three types of C-Dots are bimodal and have a second population of particles with hydrodynamic diameter less than 5 nm. However, HR-TEM images (Figure 3) reveal that the three types of the extracted biochar nanoparticles (Picea, Molinia caerulea and Elaeis guineensis) are mono-dispersed, and they have a nearly spherical shape with an average size of 4.2 ± 1.2, 6.2 ± 1.9 and 2.5 ± 0.7 nm, respectively (Figure 3) (the size distribution was determined with 100 particles, which are founded in various TEM images related to different zones in the sample). Besides, the HR-TEM images show some aggregation between the particles, which explains the total hydrodynamic diameter measured with DLS. It is comparable to other reported works [94][95][96]. C-Dots derived from Elaeis guineensis biochar are smaller than the other particles and the C-Dots derived from Molinia caerulea biochar have the greatest variability in size. However, since all the particles are in the range less than 10 nm, they are considered C-Dots. The Z potential is an indicator of nanoparticles stability since it is a measure of the charge repulsion or attraction between the particles from its surface to the boundary of the diffuse layer. The measurements affording a value of -58.85 ± 7.7, −48.7 ± 1.2, and −56.2 ± 6.2 for C-Dots synthetized from Picea, Molinia caerulea and Elaeis guineensis, respectively. These values indicate that the nanoparticles have great stability.

Optical Properties
The fluorescence spectra (Figure 4) show the maximum emission wavelength (λ em ) at similar value: 430, 420, and 420 nm for Picea, Molinia caerulea and Elaeis guineensis, respectively. The maximum excitation wavelength is also similar, ranging from 300 nm to 320 nm. The optimal excitation and emission wavelength combination are shown as an insert of Figure 4. All the obtained C-Dots show a brownish yellow color under daylight, and blue emission under UV light.
The Z potential is an indicator of nanoparticles stability since it is a measure of the charge repulsion or attraction between the particles from its surface to the boundary of the diffuse layer. The measurements affording a value of -58.85 ± 7.7, −48.7 ± 1.2, and −56.2 ± 6.2 for C-Dots synthetized from Picea, Molinia caerulea and Elaeis guineensis, respectively. These values indicate that the nanoparticles have great stability.

Optical Properties
The fluorescence spectra (Figure 4) show the maximum emission wavelength (λem) at similar value: 430, 420, and 420 nm for Picea, Molinia caerulea and Elaeis guineensis, respectively. The maximum excitation wavelength is also similar, ranging from 300 nm to 320 nm. The optimal excitation and emission wavelength combination are shown as an insert of  Usually, C-Dots exhibit an excitation-dependent photoluminescence that are related to state defects [17]. This behavior is particular for Elaeis guineensis whose intensity decrease between 360 nm and 400 nm. In this range is also a red shift photoluminescence that could be explained by the surface oxidation degree as a theory for the fluorescence origin [23]. Some authors found these defects can trap excitons, and the radiation from the recombination of trapped excitons causes the red-shift emission [97][98][99]. The band gap between the lowest unoccupied molecular orbital and the highest occupied molecular orbital reduces with the increase of oxygen content on the C-dots surface, that is, red-shifted emission is derived from increasing surface oxidation degree [23,100]. For Picea and Molinia caerulea, there is no visible red shift phenomenon, and the emission peak is preserved for the different excitation wavelengths, just changing its intensity. This behavior could be explained by the surface functional groups as an alternative theory for the fluorescence origin [23]. According to that, surface states are correlated to surface functional groups, such as C = O and C = N. Those groups are present in all the C-dots ( Figure 2) and Usually, C-Dots exhibit an excitation-dependent photoluminescence that are related to state defects [17]. This behavior is particular for Elaeis guineensis whose intensity decrease between 360 nm and 400 nm. In this range is also a red shift photoluminescence that could be explained by the surface oxidation degree as a theory for the fluorescence origin [23]. Some authors found these defects can trap excitons, and the radiation from the recombination of trapped excitons causes the red-shift emission [97][98][99]. The band gap between the lowest unoccupied molecular orbital and the highest occupied molecular orbital reduces with the increase of oxygen content on the C-dots surface, that is, red-shifted emission is derived from increasing surface oxidation degree [23,100]. For Picea and Molinia caerulea, there is no visible red shift phenomenon, and the emission peak is preserved for the different excitation wavelengths, just changing its intensity. This behavior could be explained by the surface functional groups as an alternative theory for the fluorescence origin [23]. According to that, surface states are correlated to surface functional groups, such as C=O and C=N. Those groups are present in all the C-dots ( Figure 2) and were proved to be closely related to the fluorescence [101,102]. The functional groups can introduce new energy levels and further produce new electron transitions [23].
The UV-Vis spectra ( Figure 5) were investigated at a concentration of 500 ppm. Picea and Molinia caerulea show the same behavior, a characteristic slight absorption signal at 244 nm, while Elaeis guineensis shows a slight absorption signal at 244 nm and a more visible signal at 280 nm. Absorption signals between 240 nm and 290 nm are attributed to the π-π* or n-π* transitions and suggest the presence of C=O, C=C, and C=N surface functional groups [103]. On the other hand, the signal at 244 nm could be attributed precisely to n-π* Processes 2021, 9, 1095 9 of 16 transitions and could explain the slightly blue shift in the fluorescence spectra of Picea and Molinia caerulea when they are exciting with longer wavelengths [16,104].  [105]) as the reference. Table 2 shows the comparison of several biomass-derived C-Dots QYs and excitation wavelengths with the results obtained in this work. Moreover, for C-Dots derived from biomass using the hydrothermal method, the QYs can range from 2.3% to 21.7% depending mostly on its size, carbon precursor, and processing parameters [106][107][108]. It is suggested that the high quantum yield obtained for the C-Dots synthesized from Molinia caerulea, is because the biochar from this source is one with the highest nitrogen and sulfur content (Table 1), compared to Picea and Elaeis guineensis, which influence the production of C-Dots with heteroatoms and are related to the increase in fluorescence intensity and its quantum yield [109][110][111]. For instance, Zhu et al. [99], obtained C-Dots doped with nitrogen and synthesized from citric acid and ethylenediamine with a QY of 80%. Likewise, several authors have shown an increase in the QY of C-Dots through doping with nitrogen or sulfur heteroatoms [112][113][114][115][116][117].

Ecotoxicity
In the present work, the aquatic toxicity analysis was carried out because environmental applications of C-Dots are projected (detection of heavy metals, pesticides, and endocrine disruptors) but also to determine the toxic potential that this nanomaterial may have at the end of its life cycle, by encountering water sources and aquatic biota. The Microtox™ test results for the different C-Dots samples are shown in Figure 6. The gamma value equal to unity represents the EC50 in percentage value, which can be seen to be met and exceeded for very low values of Picea (2.35% with slightly erratic values) and higher values of Molinia caerulea (41.92%). In the case of Elaeis guineensis C-Dots, the unit value is not reached, and the projected slope implies that extremely high values would be necessary to cause any toxic effect (858.6%). The different samples present EC50 values in concentration units, taken at 15 min of the test of 23.5 mg/L, 419.2 mg/L and 8586 mg/L for Picea, Molinia caerulea and Elaeis guineensis C-Dots respectively. According to these results, we can classify them as highly toxic (Picea C-Dots), moderately toxic (Molinia caerulea C-Dots) and non-toxic (Elaeis guineensis C-Dots) [123].
The toxicity of nanomaterials, in general, is a growing field of study, which includes initiatives like US NanoEPA [124] and EU NanoSafety cluster [125]. Toxicity depends on starting material, dose, size, surface chemistry, and durability; additionally, various routes of exposure are considered with different effects on organisms [126]. Picea C-Dots present a marked toxicity, which can be explained by the characteristic mentioned previously and their smaller size (Figure 3). Most studies focused on the safety of nanomaterials agree that size is the main factor of toxicity of nanomaterials with respect to the material on a macroscopic scale, mainly due to their effects through biological barriers, in the surface area and the density of absorption; this triggers endocytosis/exocytosis, oxidative stress, reactive oxygen species (ROS), cell damage and apoptosis [127].
C-Dots have a lower toxicity than quantum dots, traditionally synthesized from heavy metals [127]. However, ecotoxicological evaluations of C-Dots are scarce, despite being a priority test in terms of safety. Few studies have been carried out in bioindicator organisms such as microcrustaceans (Daphnia magna) and zebra fish (Danio rerio), showing that concentrations up to 200 mg/L can be cleared without acute toxic effects [128]. However, in the present work, it was found that Picea C-Dots are toxic for Vibrio fischeri at lower values. This shows that the Microtox™ assays are more sensitive [123] and that more data is needed at the ecotoxicological level to follow the in vivo testing process. The damage routes of C-Dots in these organisms are related to oxidative stress [128]. However, several authors agree that carbonaceous nanomaterials have little or no bioaccumulation capacity, so there would be no chronic toxicity [127]. Future work is required to further clarify these findings.

Conclusions
This work shows that Picea C-Dots have excellent optical properties. However, they exhibit high ecotoxicity, which limits their applications. Molinia caerulea and Elaeis guineensis C-Dots have low or no toxicity, but Elaeis guineensis C-Dots has the lowest fluorescence intensity with respect to the others. All the C-Dots have high values of negative Z potential, which indicate colloidal stability. Besides, they have excellent chemical groups on the surface that would allow for easy functionalization to other molecules, so they have the potential, especially Molinia caerulea and Elaeis guineensis C-Dots, for use in the development of optical biosensors with applications in the detection of environmental contaminants and pathogens. However, the optical properties (quantum yield) could be optimized to improve their performance. These results allow for the encouragement of the valorization of agro-industrial waste for its transformation into biochar and its use beyond soil amendment. Finally, future works could include more studies focused on the optimization of the optical properties of C-Dots, as well as the study of their ecotoxicology that allows a safe application of these nanomaterials in different fields of knowledge.