Biochar Extracts Can Modulate the Toxicity of Persistent Free Radicals in the Nematode Caenorhabditis elegans

: As an effective soil amendment, biochars require a comprehensive ecological evaluation before they can be widely used in agriculture because endogenous contaminants, such as environmentally persistent free radicals (EPFRs), certainly pose an ecological risk to soil invertebrates. In this study, Caenorhabditis elegans ( C. elegans ) was used as a model organism to investigate the neurotoxicity of two rice straw biochars pyrolyzed at 500 and 700 ◦ C. After 24 h exposure to unwashed biochar, washed biochar, and leaching ﬂuids (supernatants), the neurobehavioral parameters of C. elegans were determined in a liquid toxicity test. The results showed that the washed 700 ◦ C biochar particles signiﬁcantly impaired locomotion and prolonged the defecation interval at a biochar concentration of 4 g · well − 1 , while the unwashed biochar and supernatants caused no apparent impairment. Supporting this, electron paramagnetic resonance (EPR) results showed that the intensity of EPFRs in unwashed 700 ◦ C biochar was stronger than that of the corresponding washed particles. This indicates that, in the liquid test, the EPR signal alone is not indicative of particle toxicity. The accessibility and activity of the EPFRs should be considered. Dissolved organic matter (DOM) was observed in the leaching ﬂuids. The neurotoxic activity of the washed biochar was alleviated after the re-addition of leaching ﬂuids to the washed biochar, suggesting that the dissolved organic materials modulate the reactivity of the EPFRs in the liquid phase. This study suggests that the leaching process may increase the risk of biochar when used in the ﬁeld environment.


Introduction
Biochar is an environmentally friendly carbonaceous material to improve soil quality [1,2]. Biochar is expected to mitigate global warming through carbon sequestration [3]; enhance crop production by improving soil quality, including fertility and water balance [4,5]; and sequester contaminants due to its absorbability [6]. However, biochar can also be critical to organisms due to endogenous contaminants [7]. Although conventional endogenous contaminants in biomass-derived biochars, such as polycyclic aromatic hydrocarbons (PAHs) and metals, have not posed significant toxicity risks to soil organisms due to their relatively low concentrations [8][9][10]. An emerging contaminant, environmentally particles were prepared by adding 40 mg of unwashed biochar to 30,000 µL of sterilized double-distilled water (sddH 2 O), then dried and labeled as P500 and P700. To obtain the supernatant, 40 mg of unwashed biochar was added to 15,000 µL of sddH 2 O and shaken for 24 h at 20 • C without light; the leachate was passed through a 0.45 µm filter and labeled as S500 and S700. All supernatants were stored at 4 • C.
Hydroxyl radicals (•OH) were scavenged with 5,5-dimethyl-1-pyrroline N-oxide (DMPO, Aladdin). Briefly, for unwashed and washed biochar, 4 mg of solid powder was mixed with 300 µL of sddH 2 O, vortexed for 1 min, and the solid powder was allowed to settle. Then, 100 µL of 0.3 M fresh DMPO was mixed with 100 µL supernatant of the solid samples; for liquid samples, 100 µL of the sample was mixed with 100 µL of fresh DMPO. Finally, 50 µL of each sample was used to determine the free radical content by absorption in a micropipette (1.5 mm in outer diameter, 0.5 mm in wall thickness, and 125 mm in length). Electron paramagnetic resonance (EPR) signals were recorded using an EPR spectrometer (X-Band A300-6/1, Bruker, Billerica, MA, USA) [8]. All EPR detection experiments were performed at room temperature.
The DOM concentration in the supernatants was determined with a fluorescence excitation emission matrix (EEM). Spectra were recorded with an excitation wavelength range of 200-400 nm and an emission wavelength range of 250-550 nm using an F-7000 FL spectrophotometer (HITACHI, Hitachi, Ibaraki, Japan) at a scan speed of 12,000 nm·min −1 and a voltage of 550 V. All supernatants were freshly prepared before testing, as described above.
For the supernatant tests, 150 µL was added to 150 µL of exposure medium (to obtain the same solid-liquid ratio of 40 mg: 30,000 µL was used to obtain the washed biochar). C. elegans worms were synchronized and cultured to L4 stage at 20 • C on NGM plates before being transferred to the 24-well exposure plates. All test plates were cultured at 20 • C for 24 h, and C. elegans were transferred to new NGM plates before neurotoxic assays.

Neurotoxic Assays
Neurobehavioral experiments, including autonomic and sensory behaviors, were performed as previously described [15,27]. For the autonomic behavioral tests, a single worm was transferred to a new NGM plate covered with OP50, and the crawling path was observed for 20 s under a microscope (Nikon SMZ 1500, Tokyo, Japan). Body bends were counted as changes in the direction of movement in the sinusoidal motion of the worm, and the relative length of movement was measured using the NIS-Elements D software (Nikon Eclipse E100, Tokyo, Japan) and normalized to the average body size of each group. The defecation intervals were calculated as the time between two contractions of the posterior body wall under the microscope. For the mechanical sensory behavior, the anterior tip of the worm was touched 10 times with a fine hair, and each stimulus response was recorded as a reversal movement. The chemotaxis assay is based on the worm's attraction to sodium chloride (NaCl), as its memory is associated with feeding. Ninety-six mm chemotaxis assay plates were prepared without NaCl and stored at 4 • C (Text S2). Twenty-four hours before an assay, an agar plug (approximately 5 mm × 5 mm × 3 mm) containing 100 mM NaCl was placed on the NaCl spot, with the NaCl spot and the control spot equidistant from the starting spot (3 cm), and their distance was 4 cm. The agar plug was removed just before the assay, then 1 µL 0.5 M sodium azide was added to the NaCl spot and the control spot. Thirty worms were transferred to the starting spot and incubated at 20 • C for 1 h. Then, the number of nematodes in the circular area (r = 1.5 cm) of the NaCl spot and the control spot was counted as N n and N c , respectively. The chemotaxis index was calculated as the difference between the number of N n and N c divided by the total number. The test order for each day was body bending, relative movement length, touch response, and defecation; the chemotaxis test was constantly tested on a different day and was not performed with the four endpoints mentioned above. For the body bending, defecation, and mechanical sensory assays, at least 10 worms were randomly selected from each exposure concentration. All experiments were repeated at least three times.

The Autonomic Behavior Assay of Selected Supernatant
Supernatants S500 and S700 were selected to test their potential effects after a short exposure time (12 h, test 1 in Figure 1). Autonomous behavior was performed as described in Section 2.4. Next, S500 or S700 was added to the M9 buffer containing OP50 (see Section 2.3), and body curvature and relative movement length were determined after 12 h of exposure. In test 2 ( Figure 1), C. elegans were first treated with S500 or S700 for 12 h, then P500 and P700 were added to the appropriate portion of the supernatant (S+P500 and S+P700). Additional controls were exposed to P500 and P700 only (C+P500 and C+P700). The concentration of P500 and P700 was 4 mg·well −1 , and the corresponding supernatants were used. mm chemotaxis assay plates were prepared without NaCl and stored at 4 °C (Text S2). Twenty-four hours before an assay, an agar plug (approximately 5 mm × 5 mm × 3 mm) containing 100 mM NaCl was placed on the NaCl spot, with the NaCl spot and the control spot equidistant from the starting spot (3 cm), and their distance was 4 cm. The agar plug was removed just before the assay, then 1 μL 0.5 M sodium azide was added to the NaCl spot and the control spot. Thirty worms were transferred to the starting spot and incubated at 20 °C for 1 h. Then, the number of nematodes in the circular area (r = 1.5 cm) of the NaCl spot and the control spot was counted as Nn and Nc, respectively. The chemotaxis index was calculated as the difference between the number of Nn and Nc divided by the total number. The test order for each day was body bending, relative movement length, touch response, and defecation; the chemotaxis test was constantly tested on a different day and was not performed with the four endpoints mentioned above. For the body bending, defecation, and mechanical sensory assays, at least 10 worms were randomly selected from each exposure concentration. All experiments were repeated at least three times.

The Autonomic Behavior Assay of Selected Supernatant
Supernatants S500 and S700 were selected to test their potential effects after a short exposure time (12 h, test 1 in Figure 1). Autonomous behavior was performed as described in Section 2.4. Next, S500 or S700 was added to the M9 buffer containing OP50 (see Section 2.3), and body curvature and relative movement length were determined after 12 h of exposure. In test 2 ( Figure 1), C. elegans were first treated with S500 or S700 for 12 h, then P500 and P700 were added to the appropriate portion of the supernatant (S+P500 and S+P700). Additional controls were exposed to P500 and P700 only (C+P500 and C+P700). The concentration of P500 and P700 was 4 mg·well −1 , and the corresponding supernatants were used.

Statistical Analysis
The statistical significance of the changes was determined using GraphPad Prism 7 (GraphPad Software, San Diego, CA, USA) and the one-way ANOVA test (Holm-Sidak's multiple comparison test). Probability levels of 0.05 (*) and 0.01 (**) were considered statistically significant. All neurobehavioral data are expressed as mean ± SEM (standard error of the mean). Neurobehavioral figures were generated using Graphpad Prism 7. For the EPR signal and fluorescence of DOM, the figures were generated using Origin 2018 (OriginLab Corporation, Northampton, MA, USA).

Only Washed Particles from 700 • C Pyrolysis Caused Neurobehavioral Changes
To investigate the neurotoxic potential of different biochars on C. elegans, two different pyrolysis temperatures (500 and 700 • C) were chosen for biochar preparation. C. elegans were exposed to unwashed biochars, washed biochar particles, and supernatants. After 24 h exposure to unwashed biochars and supernatants, none of the neurological parameters tested was significantly altered by any biochar concentration ( Figure S1 in Supplementary). For washed biochar particles, body curvatures decreased by 15.1% (p < 0.01) and 21.0% (p < 0.01) after treatment with 1 mg·well −1 and 4 mg·well −1 of P700, respectively ( Figure 2B). After exposure to 2 and 4 mg·well −1 of P700, the defecation interval increased by 13.3% (p < 0.01) and 20.9% (p < 0.01), respectively ( Figure 2C). Relative movement length, touch response, and chemotaxis did not change for any particle exposures tested ( In the literature, the avoidance behavior of earthworms to biochar could be alleviated in the liquid phase [28], and the phytotoxicity of corncob biochar could also be reduced by washing out the water-soluble toxic compounds [29]. However, the question arises: In the literature, the avoidance behavior of earthworms to biochar could be alleviated in the liquid phase [28], and the phytotoxicity of corncob biochar could also be reduced by washing out the water-soluble toxic compounds [29]. However, the question arises: how does biochar change during the washing process? Thus, to find the underlying cause of the phenotypical observations, we compare the different characteristics before and after washing the biochar (details are shown in Figure S2, Tables S1 and S2).
The consequences of the reduced rate of body bends could be an impaired ability to reach food and avoid stressful or risky places. The prolonged defecation interval was considered a possibility of neuronal damage [30]. In this study, unwashed biochar had no adverse effects on nematodes, while the washed particle fraction at 700 • C caused significant inhibition of the autonomic behavior. These results contradict our initial assumption that EPFRs could trigger the production of free oxygen radicals (•OH) in solution and, therefore, exhibit higher toxicity in solution than on the NGM plate. Compared to our previous study using solid agar surface plates for exposure, in which 500 • C rice biochar with highintensity EPFRs decreased locomotion behavior and prolonged the defecation interval of C. elegans [15], the present study found that the two unwashed biochars did not significantly affect the behavioral parameters tested. According to the literature, earthworm avoidance behavior towards biochar was mitigated in the liquid phase [28], and the phytotoxicity of biochar was also reduced by washing [29]. However, the question remains on how biochar changes during the washing process. To find the reason for the phenotypic observations, we compare the different traits before and after washing the biochar (details are shown in Figure S2, Tables S1 and S2).

EPFRs Reactivity Might Play a Crucial Role in Neurotoxicity
Many contaminants, such as PAHs and metals, remain or are formed during the pyrolysis of biochar [11]. Our previous work with plate cultures showed that PAHs and metals were not the main cause of the observed neurotoxicity due to their low content in biochar [15]. EPFRs have been found in biochars and have been shown to have the potential to inhibit grain seedling growth and modulate C. elegans behavior [8,14,15]. In this word, EPFRs were also determined in all samples before and after washing. The g-factor and EPR signal intensity parameters can be used to identify the type of free radicals and the concentration of EPR-active species [8,31]. As shown in Figure 3, both the unwashed biochar and the washed biochar particles exhibited significantly higher intensities than the blank control. The supernatants did not show any EPFR signals. With increasing pyrolysis temperature, the EPR intensity of the unwashed biochar decreased from 4.8 × 10 5 spins·mg −1 (500 • C) to 5.9 × 10 4 spins·mg −1 (700 • C), and the same phenomenon was observed for the washed particles ( Figure 3A,C). The toxicity experiments showed that only P700 decreased body bends and prolonged defecation intervals ( Figure 2B,C). At the same time, P500 and B500, with the strongest EPR intensities, did not affect the neurobehavior of C. elegans. Moreover, Yang, et al. [32] showed that the degradation of 700 • C biochar with low EPR intensity was even better than that of 500 • C biochar with relatively high intensity of EPR in the first 100 h, indicating that the 700 • C biochar is likely to be more reactive. Thus, a stronger EPR signal does not necessarily indicate higher toxicity; rather, the reactivity of EPFRs should be considered.
The EPFRs could stimulate the formation of •OH in an aqueous solution, causing a deleterious effect [8,33,34]. In this work, •OH was trapped using DMPO, and the typical spectrum of the DMPO-OH adduct is a quartet shape with a relative intensity ratio of 1:2:2:1 [8]. As shown in Figure 3B,D, both unwashed (B500 and B700) and washed biochar (P500 and P700) exhibit strong DMPO-OH signals. This indicates that both 500 • C and 700 • C biochar can induce the production of •OH. Researchers shows that •OH is the main cause of organic chemical degradation and biochar toxicity [8,35], indicating that •OH might be the reason for the toxicity. However, only P700 showed a significant inhibitory effect (Figure 2), suggesting that •OH may not be responsible for this observation. The EPFRs could stimulate the formation of •OH in an aqueous solution, causing a deleterious effect [8,33,34]. In this work, •OH was trapped using DMPO, and the typical spectrum of the DMPO-OH adduct is a quartet shape with a relative intensity ratio of 1:2:2:1 [8]. As shown in Figure 3B,D, both unwashed (B500 and B700) and washed biochar (P500 and P700) exhibit strong DMPO-OH signals. This indicates that both 500 °C and 700 °C biochar can induce the production of •OH. Researchers shows that •OH is the main cause of organic chemical degradation and biochar toxicity [8,35], indicating that •OH might be the reason for the toxicity. However, only P700 showed a significant inhibitory effect (Figure 2), suggesting that •OH may not be responsible for this observation.

DOM Can Modulate the Toxicity of EPFRs
Dissolved organic material plays an essential role in the formation and stability of EPFRs and can affect EPFR activity [13,36,37]. In this work, DOM was observed in all supernatants leached from biochar using the EEM spectrometer, and the peaks at the excitation/emission wavelength pair (Ex/Em) of S500 and S700 were 270 nm/395 nm and To test whether DOM can modulate the toxicity of EPFRs, supernatants and particles of 500 °C and 700 °C biochar were selected to determine the effect of DOM. In test 1, the nematodes were directly exposed to the supernatant for 12 h. The aim is to check whether the nematode adapts to the DOM environment after 24 h exposure since no impairments were observed (unshown data). The relative movement length and body bends of nematodes exposed to S700 increased significantly compared to the control ( Figure 5A,C). In addition, there was a significant difference between S500 and S700, and exposure to S700 improved locomotion. In test 2, nematodes were first exposed to supernatants for 12 h, then corresponding particles were added for another 12 h. After exposure to C+P500 and C+P700, the rate of body bends decreased compared to the control, but there was no significant difference between P500 and P700 ( Figure 5B). The results demonstrate that the washed particles are harmful to the nematodes. After 12 h exposure to the supernatant and 12 h exposure to the particles, the body bends of the nematodes exposed to S+P500 were significantly different from C+P500 but not significantly different from the control. The supernatant S500 significantly reduced the damage caused by EPFRs in the particles. A comparison of DOM levels and EPFR intensities in biochar, particles, and supernatants indicates that DOM can modulate the toxicity of EPFRs. Applying solid-state 13 C NMR, Liu, et al. [40] showed that biochar DOM contains abundant small aromatics with fused rings, aliphatic C, and carboxyl-C, and that waterextractable DOM can reach up to 0.5-40 mg C g −1 biochar. This coarse characterization is similar to that of humic substances. Pyrolyzing manure, protein-like and humic acid-like substances were the major components of DOM. The proteinaceous matter first decreased and then increased with an increase in the pyrolysis temperature, while an opposite trend was observed for the humic acid-like matter [41]. By sequential extraction, Liu, et al. [42] confirmed the humic-like character of DOM extractable from biochars. They showed that water-extractable DOM components are mostly composed of low aromatic and highly hydrophilic fulvic acid-like substances, which are the most labile and diversified components. This statement was confirmed by recent studies [43][44][45], which also detected fulvic acid-like compounds. In contrast, Tomczyk, et al. [46] found that extractable DOM from biochars produced from potato and raspberry stems was predominantly constituted of substances of large molecular weight and high aromaticity. This illustrates that there is (still) no unifying model that describes under which conditions, and of what quality, leachable organic carbon is produced by the pyrolysis of biowaste. However, some effects of humic and humic-like substances on soil organisms can be predicted, which are relevant for the present study since mainly fulvic acids interact with organisms [45,47].
Humic substances primarily reduce the bioavailability of organic chemicals, metals, and nanomaterials to C. elegans, and the extent of the reduction depends on the quantity and quality of the humic substances [21,46,48,49]. In addition, Lieke, et al. [50] found that fulvic acid protects fish gills from oxidative stress, probably by directly scavenging reactive oxygen species (ROS). Based on the above studies, DOM in unwashed biochar and supernatant can be expected to modulate the fate and reactivity of EPFRs. However, humic substances in higher internalized concentrations can block exporter proteins [51] and disproportionately increase bioconcentrations of persistent organic pollutants [52]. This could be detrimental if these persistent organic pollutants can also be toxic via the release of EPFRs.
To test whether DOM can modulate the toxicity of EPFRs, supernatants and particles of 500 • C and 700 • C biochar were selected to determine the effect of DOM. In test 1, the nematodes were directly exposed to the supernatant for 12 h. The aim is to check whether the nematode adapts to the DOM environment after 24 h exposure since no impairments were observed (unshown data). The relative movement length and body bends of nematodes exposed to S700 increased significantly compared to the control ( Figure 5A,C). In addition, there was a significant difference between S500 and S700, and exposure to S700 improved locomotion. In test 2, nematodes were first exposed to supernatants for 12 h, then corresponding particles were added for another 12 h. After exposure to C+P500 and C+P700, the rate of body bends decreased compared to the control, but there was no significant difference between P500 and P700 ( Figure 5B). The results demonstrate that the washed particles are harmful to the nematodes. After 12 h exposure to the supernatant and 12 h exposure to the particles, the body bends of the nematodes exposed to S+P500 were significantly different from C+P500 but not significantly different from the control. The supernatant S500 significantly reduced the damage caused by EPFRs in the particles. A comparison of DOM levels and EPFR intensities in biochar, particles, and supernatants indicates that DOM can modulate the toxicity of EPFRs. It is evident that DOM has modulatory effects on environmental substances and their effects on organisms; however, the direction of modulation is not uniform. In a model analysis, Steinberg and Brüggemann [53] showed that humic substances can react in seemingly opposite ways, depending on their structure and chemical characterization. In fact, the effect of organic matter is a trade-off between promotion and inhibition due to its diverse nature, and this contrasting effect appeared to be intrinsic. Moreover, dissolved humic substances could facilitate fish life in rather acidic environments and prolong the lifespan of C. elegans [17]. Therefore, it is not unlikely that DOM, which is leachable from biochar, can protect nematodes from damage by EPFRs. However, a promoting effect of It is evident that DOM has modulatory effects on environmental substances and their effects on organisms; however, the direction of modulation is not uniform. In a model analysis, Steinberg and Brüggemann [53] showed that humic substances can react in seemingly opposite ways, depending on their structure and chemical characterization. In fact, the effect of organic matter is a trade-off between promotion and inhibition due to its diverse nature, and this contrasting effect appeared to be intrinsic. Moreover, dissolved humic substances could facilitate fish life in rather acidic environments and prolong the lifespan of C. elegans [17]. Therefore, it is not unlikely that DOM, which is leachable from biochar, can protect nematodes from damage by EPFRs. However, a promoting effect of S700 was observed in test 1, while no significant reduction in toxicity was observed in test 2. The reason may be the different nature and reactivity of EPFRs in the solid particles and the leached DOM between 500 • C and 700 • C, which should be clarified in future studies

Conclusions
The present study aimed to determine the reactivity of EPFRs in different biochars with or without the presence of different DOM. Increased neurotoxicity was only present in EPFRs with relatively high reactivity (P700). Although the potential risks of EPFRs in biochar have been a growing concern, our findings suggest that the presence of organic matter in the environment can significantly influence the toxicity of EPFRs, potentially overestimating the danger they pose. The DOM released from biochar can alleviate the harm from biochar particles, such as through (1) decreasing the probability of direct contact between biochar and C. elegans, and (2) partially quenching EPFRs by some active components of DOM. This may be the reason why EPFRs have been overlooked and understudied. To better understand how environmental factors affect the reactivity of EPFRs, future research should focus on using different types of biochar and developing methods to quantify reactivity. In addition, this study was only considering short-term exposure; long-term exposure may lead to chronic toxicity or affect the next generations, which also should be of more concern in further studies. This could contribute to developing effective strategies for reducing the risks associated with EPFRs and promoting the safe use of biochar in various applications.

Conflicts of Interest:
The authors declare no competing interests.