3.1 Only washed particles from 700°C pyrolysis caused neurobehavioral changes
To explore the neurotoxic potential of the different biochars on C. elegans, three different pyrolysis temperatures including 500, 700, and 1000°C were selected to prepare biochars, C. elegans was exposed directly to unwashed biochars, washed biochars particles, and supernatant (the water-soluble content). After the exposure to unwashed biochar, none of the tested neurological parameters were significantly changed by any biochar concentration (Fig. 2A, 2D, 2G, 2J, and 2M). For washed biochar particles, body bends decreased by 15.1% (p < 0.01) and 21.0% (p < 0.01) after treatment at 1 mg·well− 1 and 4 mg·well− 1 of P700, respectively (Fig. 2E). After exposed 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) (Table S3, Fig. 2H), respectively. P1000 even significantly improved the relative move length (p < 0.01). The touch response and chemotaxis index did not change with any tested particles exposures. None of the supernatant exposure scenarios resulted in any significant impairment of neurobehavioral changes (Fig. 2C, 2F, 2I, 2L, and 2O).
Consequences of a decreased rate of body bends could be an impaired ability to reach food and to avoid stressful or dangerous locations, and the prolonged defecation interval time was considered a damaged possibility to neurons (Lieke et al., 2015; Sun et al., 2015). In this work, unwashed biochars had no adverse effects on the nematodes, while the washed particle fraction of 700°C caused significantly autonomic behaviors inhibition. These results contradict our initial hypotheses, which stated that EPFRs could induce free-radical (•OH) production in solution, thus having higher toxicity in solution than on the NGM plate. Compared to our previous study that using solid agar surface plates for exposure, 500°C rice biochar with the high-intensity EPFRs decreased locomotion behavior and extended the defecation interval of C. elegans (Lieke et al., 2018), the present work identified that all three unwashed biochars did not significantly influence the tested behavior parameters. In literature, the avoidance behavior of earthworms to biochar could be alleviated in the liquid phase (Li et al., 2011; Brtnicky et al., 2021) and the phytotoxicity of biochar could also be reduced by washing (Intani et al., 2019). However, the question arises, in which way 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 biochar (details are shown in Fig. S1, Table S1, and S2).
3.2 EPFRs reactivity might play a crucial role in neurotoxicity
Many pollutants remain or form in the pyrolysis process of biochars, such as PAHs and metals (Lian and Xing, 2017). Our previous work with plate cultures showed that PAHs and metals were not the main reason for the observed neurotoxicity, due to their low contents in biochars (Lieke et al., 2018). EPFRs have been found in biochars and proved to have the potential to inhibit the growth of cereal seedlings and modulate the behavior of C. elegans (Liao et al., 2014; Lieke et al., 2018; Liu et al., 2018). In this paper, EPFRs were also determined in all samples before and after washing. The parameter of g-Factor and EPR signal intensity could be used to identify the type of free radicals and the concentration of the EPR active species (Dellinger et al., 2007; Liao et al., 2014). As can be seen from Fig. 3, both unwashed biochars and particles had a significantly higher intensity than the blank control. The supernatants featured no EPFR signals. With increasing pyrolysis temperature, the EPR intensity of the unwashed biochars decreased from 4.8×105 spins·mg− 1 (500°C) to 5.9×104 spins·mg− 1 (700°C) and continuously decrease to the approximate intensity of the blank (1000°C), and the same phenomenon was observed for the particles (Fig. 3A and 3C). The toxicity experiments showed that only P700 decreased the body bends and prolonged the defecation period (Fig. 2E and 2H), however, P500 and B500 with the strongest EPR intensities did not lead to any effect on the neurobehaviors of C. elegans. Moreover, Yang et al. (2016) showed that the degradability of the 700°C biochar with low EPR intensity was even better than the 500°C biochar with relatively high EPR intensity in the first 100 hours, which implies that the 700°C biochar is likely to be more reactive. Therefore, the stronger EPR signal does not necessarily indicate higher toxicity, the reactivity of EPFRs should be better concerned.
EPFRs could stimulate the generation of •OH in an aqueous solution and thus cause a negative effect (Khachatryan et al., 2011a and 2011b; Liao et al., 2014). In this paper, •OH was captured by using DMPO and the typical spectrum of DMPO-OH adduct is a quartet shape with a relative intensity ratio of 1:2:2:1 (Liao et al., 2014). As can be seen from Fig. 3 (B, D, and F), both unwashed (B500 and B700) and washed biochars (P500 and P700) have strong DMPO-OH signals. Researchers demonstrated that •OH was the main reason for the degradation of organic chemicals and the toxicity of biochar (Liao et al., 2014; Wen et al., 2014). It indicated that both 500°C and 700°C biochars could induce •OH production, thus causing toxicity to C. elegans. However, only P700 shows significant inhibition effects, which implies •OH might not be responsible for this observation.
3.3 DOM is able to modulate the toxicity of EPFRs
DOM plays an essential role in the formation and stability of EPFRs and may influence the EPFR activity (Paul et al., 2004; Huang et al., 2019; Pan et al., 2019). In this paper, DOM in all supernatants was observed by EEM spectrometer, and peaks at excitation/emission (Ex/Em) wavelength pair of S500 and S1000 were 270 nm/395 nm and 250 nm/380 nm, respectively (Fig. 4). Peaks at these two Ex/Em pairs are commonly related to the existence of humic acid-like substances (Chen et al., 2003; Helms et al., 2013). The relative fluorescence intensity of S500 was stronger than S700, which presented the amount of DOM leached from B500 was higher than B700. It is mentioned in the literatures that humic substances mainly decrease the bioavailability of organic chemicals, metals, and nanomaterials on C. elegans, and the decreasing extent depends on the quantity and quality of humic substances (Haitzer et al., 1999; Menzel et al., 2005; Collin et al., 2016). Moreover, Lieke et al (2021) found that humic substance protects the fish's gills from oxidative stress, probably by directly scavenging the reactive oxygen species (ROS). Based on the above researches, it is able to assume that DOM in unwashed biochar and supernatant may modulate the fate and reactivity of EPFRs.
To verify that DOM could modulate the toxicity of EPFRs, the supernatants and particles of 500°C and 700°C biochar were chosen to determine the effect of DOM. In test 1, nematodes were exposed to supernatant directly for 12 h. The relative move length and body bends of nematodes exposed to S700 were significantly increased compared to the control (Fig. 5A and 5C). Furthermore, there was a significant difference between S500 and S700, and the exposure to S700 led to enhanced locomotion abilities. For test 2, nematodes were exposed to supernatants firstly for 12 h, and corresponding particles were added to expose for another 12 h. After being exposed to C + P500 and C + P700, the rate of body bends decreased compared with the control, but there was no significant difference between P500 and P700 (Fig. 5B). The results verified that washed particles could cause injury to nematodes. After 12 h-exposure to the supernatant and 12 h-exposure to the particles, the body bends of nematodes exposed to S + P500 were significantly different from C + P500, but not significantly different from the control. It identified that S500 can significantly reduce the damage from EPFRs in particles. Comparing the DOM contents and EPFRs intensities in biochars, particles, and supernatants, it is concluded that DOM can modulate the toxicity of EPFRs.
DOM appears to have a modulating effect on the influence of the environmental substance. Steinberg and Brüggemann (2001) demonstrated that the effect of organic matter is a trade-off between promotion and inhibition due to its diverse nature, and this contrasting effect seemed to be intrinsic. Moreover, dissolved humic substances could facilitate fish's life and prolong the lifespan of C. elegans (Steinberg et al., 2007). Therefore, the DOM of biochars might protect nematodes from the harm of EPFRs. However, S700 was found to have a promoting effect in test 1, but test 2 did not show a significant ability to reduce toxicity. One reason could be the different type and reactivity of EPFRs and DOM between 500°C and 700°C, which needs to be elucidated in future studies.