Contrasting environmental impacts of nano-biochar and conventional biochar on various organisms

1 Contrasting environmental impacts of nano-biochar and 1 conventional biochar on various organisms 2 Monika Raczkiewicz1, Aleksandra Bogusz2, Bo Pan3, Baoshan Xing4, Patryk Oleszczuk1* 3 4 1Department of Radiochemistry and Environmental Chemistry, Faculty of Chemistry, 3 Maria 5 Curie-Skłodowska Square, 20-031 Lublin, Poland 6 2Department of Ecotoxicology, Institute of Environmental Protection - National Research 7 Institute, Ks. Troszynskiego St. 9, Warsaw 01-693, Poland 8 3Faculty of Environmental Science and Engineering, Kunming University of Science and 9 Technology, Kunming, 650500, People's Republic of China 10 4 Stockbridge School of Agriculture, University of Massachusetts, Amherst, MA 01003, United 11 States 12 13 Corresponding author: Patryk Oleszczuk, [email protected] 14 2 Abstract 15 16 The environmental hazards of nanobiochar (n-BC) require attention due to limited 17 knowledge. This study is the first to explore the effects of biochar size reduction across various 18 organisms, including bacteria (Allivibrio fischeri), plants (Lemna minor, Lepidium sativum), 19 and invertebrates (Daphnia magna, Folsomia candida). Bulk biochar (b-BC) and n-BC were 20 applied in both liquid and solid-phase tests to assess its ecotoxicity. The resulting leachates 21 were tested at concentrations of 2, 10, and 100 mg/L for organisms like Daphnia magna and 22 Lemna minor. In the solid-phase tests, b-BC and n-BC were added to standard soil at 23 concentrations of 1% and 5% to evaluate toxicity in Folsomia candida and at concentrations of 24 1% to evaluate toxicity in Lepidium sativum.We found n-BC to be significantly more toxic (by 25 18% to 2886%) to A. fischeri than bulk biochar (b-BC), with toxicity increasing over time. Low 26 doses (1%) of both b-BC and n-BC did not cause mortality or inhibit reproduction in F. candida, 27 though b-BC enhanced reproduction (by 30% to 56%) compared to n-BC. At a 5% dose, both 28 b-BC and n-BC inhibited reproduction F. candida, with n-BC being 0.5 to 1.8 times more toxic. 29 Neither b-BC nor n-BC immobilized D. magna, but both inhibited reproduction (by 28% to 30 35%). The nanoscale dimensions of n-BC facilitated bioaccumulation in D. magna, leading to 31 adhesion on the organism’s body. The n-BC had a greater impact on plants, both b-BC and n32 BC were non-toxic to L. minor, but all n-BC inhibited root growth in L. sativum. These findings 33 highlight the importance of considering biochar size, feedstock, and pyrolysis conditions when 34 evaluating environmental risks, ensuring safe use in sustainable agriculture. Future directions 35 for this study should focus on exploring the long-term environmental impacts of n-BC, 36 including its persistence and transformation in various ecosystems. Further research is needed 37 to elucidate the mechanisms driving its increased toxicity and bioaccumulation across different 38 organisms. Additionally, studies should evaluate the effects of varying feedstocks, pyrolysis 39 3 conditions, and functionalization strategies to optimize n-BC's environmental safety and 40 efficacy in sustainable agriculture. 41 Keywords: toxicity; leachates; sewage sludge; agriculture residues; biomass; particle size; 42 nanoscale 43 4 1. INTRODUCTION 44 As global environmental challenges continue to grow, nanotechnology emerges as a 45 pivotal tool for developing innovative solutions to pollution in water and soil. Among these 46 advancements, nanobiochar stands out as a promising material due to its remarkable ability to 47 adsorb both organic and inorganic contaminants. Nanobiochar (n-BC) represents an innovative 48 form of well-known carbonaceous material called biochar (BC) obtained through pyrolysis. The 49 n-BC, defined as BC with particle sizes ranging from 1 to 100 nm (Rashid et al., 2023) is 50 currently at the forefront of research. The current approaches to n-BC production and utilization 51 include environmentally friendly synthesis methods and highlighting energy-efficient 52 techniques according to the directions of green chemistry in nanotechnology (Lyu et al., 2018). 53 Among various synthesis n-BCs methods, such as sonication (Oleszczuk et al., 2016) or 54 conventional grinding followed by sieving the aqueous suspension of n-BC (Li et al., 2017), 55 recently ball milling stands out as the most commonly employed technique (Naghdi et al., 56 2017). 57 The increased interest in n-BC can be attributed to n-BC’s unique properties determined 58 by particle size like higher specific surface area (Huang et al., 2020), reduced hydrodynamic 59 radius, more negative zeta potential (Song et al., 2019), increased presence of oxygen (O)- 60 containing functional groups, and the occurrence of carbon defects compared to pristine 61 (regular) BC(Weber and Quicker, 2018). These distinctive properties potentially affect n-BC 62 adsorption capacity to trace metals (TMs) and organic contaminants (e.g., polycyclic aromatic 63 hydrocarbons - PAHs) (Ramanayaka et al., 2020), which can be used for wastewater treatment 64 and immobilization of contaminants. However, by changing the mentioned properties during 65 ball milling, the strength of interaction between hydrophobic organic contaminants and BC may 66 decrease, making previously inaccessible PAHs, more available and mobile (Wang et al., 2017). 67 Releasing these toxic substances into the environment and inducing harmful effects on the 68 5 ecosystem. At the same time, more functional groups created during ball milling may increase 69 the affinity (Liu et al., 2022) of n-BC to TMs and simultaneously decrease the TM’s mobility. 70 Moreover, n-BC exhibits a higher critical coagulation concentration (CCC) than other 71 carbonaceous nanomaterials (e.g. carbon nanotubes or graphene oxides) (Song et al., 2019), 72 which induces it greater aggregation or accumulation and colloidal stability (Sun et al., 2018). 73 This better aggregation and stability of n-BC than other carbon materials may influence the 74 toxicological character of nanoparticles. Aggregated forms of nanoparticles can have distinct 75 biological effects compared to individual nanoparticles (Sani et al., 2023). For example, the 76 reduced bioavailability of aggregated nanoparticles may influence their interactions with 77 biological systems (Zhang, 2014). 78 Considering that bulk biochar (b-BC) and n-BC are primarily used in agriculture and 79 soil remediation (Pathak et al., 2024; Pradhan et al., 2024; Sarma et al., 2024; Vadakkan et al., 80 2024), it is crucial to assess their impact on plants and different groups of organisms. The 81 physicochemical characterization and the contaminant content are insufficient for evaluating 82 the environmental risks associated with n-BC (Ndoung et al., 2021; Osman et al., 2024). This 83 is due to the complex interactions between n-BC and various environmental matrices, as well 84 as the dynamic nature of contaminant release. Furthermore, the potential for bioaccumulation 85 in ecosystems underscores the need for comprehensive ecotoxicological studies and long-term 86 monitoring to fully understand their impacts. While chemical analyses still remain essential and 87 give an overall look at possible behavior and indirect and potential risk of n-BC, 88 ecotoxicological tests can complement information about the direct risk and offer valuable 89 insights into the direct effects on living organisms, such as their growth, behavior, reproduction, 90 and overall physiological health over the long term (Domene et al., 2015; Oleszczuk et al., 91 2013). In this context, the biological assays should not be seen as a replacement for chemical 92 analyses but rather as a supplementary tool, which extends our knowledge about potential 93 6 threats and additionally allowing the explanation of interactions between various contaminants, 94 providing crucial evidence in the assessment process. 95 Numerous studies have examined the effects of b-BC on organisms and plants 96 (Godlewska et al., 2021; Kim et al., 2020; Marcińczyk et al., 2024; Wei et al., 2024). These 97 studies have revealed that b-BC can have varying impacts on microorganisms, plants, and soil 98 invertebrates (Kim et al., 2020; Marcińczyk et al., 2024; Wei et al., 2024) and the effect very 99 often depends on the soil types. Currently, studies specifically focused on the ecotoxicity of n100 BCs are extremely limited, highlighting the novelty and significance of our research. There is 101 a lack of information regarding the influence of n-BC depending on feedstocks and pyrolysis 102 conditions on different ecosystems. It is very crucial to explain the effect of the initial 103 parameters of b-BC in the context of the toxicological properties of n-BC. A critical knowledge 104 gap exists regarding whether size-related changes in n-BC properties influence potential 105 toxicity. Addressing this gap is necessary for predicting n-BC's environmental impacts based 106 on pristine BC properties and for the eventual widespread use of n-BC. 107 The aim of this study was to assess the impact of n-BC on various groups of organisms 108 like bacteria (Alivibrio fischerii), plants (Lemna minor, Lepidium sativum), and invertebrates 109 (Daphnia magna, Folsomia candida), specifically examining their effects on viability, 110 reproduction, and growth. Evaluating organisms from various trophic groups can provide 111 comprehensive information on the potential risks of introducing n-BC into the environment. 112 Understanding interactions among organisms at different trophic levels is crucial for predicting 113 n-BC's environmental impacts. Analyzing these interactions may reveal changes in ecosystem 114 dynamics and potential cascading effects. 115 7 2. MATERIALS AND METHODS 116 2.1. Bulk and nano-biochars 117 Detailed information about production conditions and physicochemical properties of bulk118 (b-BC) and nano-biochar (n-BC) was presented in our previous work (Raczkiewicz et al., 119 2024b). The b-BCs used in this experiment were obtained from willow (WL), rice husk (RH), 120 oilseed rape (OSR), and various sewage sludges 1-3 (SSL 1-3) collected from three wastewater 121 treatment plants. The b-BCs were produced at 550°C under a nitrogen atmosphere. The WL122 derived b-BC were produced at additional temperatures of 450°C, 650°C, and 750°C to 123 examine the temperature-dependent effects on b-BC properties (Tomczyk et al., 2020), which 124 may influence n-BC properties and ecotoxicity. The SSL-derived b-BC was also produced in a 125 carbon dioxide atmosphere, which may alter b-BC properties and toxicity (Kończak et al., 2020) 126 Afterward, the resulting b-BCs were frozen (-80°C ) and ground using a ball milling machine 127 (PM100, Retsch Corporation, German) to achieve BC particle sizes smaller than 100 nm 128 according to the method described previously (Raczkiewicz et al., 2024b). The contaminants 129 content (PAHs and TMs,) and their bioavailability are presented in our previous work 130 (Raczkiewicz et al., 2024a). 131 2.2. Ecotoxicological tests 132 The effect of the b-BC and n-BC on tested organisms was evaluated in liquidand solid133 phase tests. The “liquid-phase tests” investigate the impact of leachates obtained from BC on 134 organisms (mainly water organisms) and can be called indirect test because the part of chemical 135 (leached from solid) is evaluated, whereas solid-phase tests examine the direct effect of the 136 whole material on the tested organism. Five ecotoxicological tests were employed to assess and 137 compare the ecotoxicity of b-BC and n-BC. The liquid phase (leachate) was prepared according 138 to the EN 12457-2 protocol (2003). The b-BC and n-BC were mixed with deionized water at a 139 8 proportion of 1:10 (solid:liqiud, w/v) and were subsequently shaken on a horizontal shaker for 140 24 hours (Model 358A, ELPINSC+, Poland). The resulting extract was filtered through a 0.45 141 μm syringe filter. 142 2.3. Liquid phase test 143 The evaluation of the leachate toxicity to Alivibrio fischerii was performed based on 144 Microtox® tests using a Microtox M500 analyzer (Microtox M500, Modern Water, UK) 145 according to the test protocol (SDI, 1992). Before the test, the pH was adjusted to the range of 146 6-8 using 0.1M HCl/NaOH to ensure test accuracy. Luminescence inhibition was measured 147 after 5 and 15 minutes of exposure of A. fischeri to the leachates. The Microtox Omni software 148 was employed for the analysis of the results. 149 The toxicity assessment of b-BC and n-BC to Daphnia magna was conducted following the 150 standard procedures according to OECD 202 (OECD, 2004) and OECD 211 guidelines (OECD, 151 1998). The immobilization and reproduction of D. magna are the endpoints of this test. To 152 assess the impact of b-BC and n-BC on immobilization, three concentrations (2, 10, and 100 153 mg/L) of each b-BC or n-BC were prepared by adding specific amounts in deionized water. In 154 exposure containers, five young daphnids were placed in 100 mL of the test solution, with three 155 replicates for each concentration of b-BC or n-BC. The containers without BCs were used as a 156 control. Test conditions, including light and temperature, were consistent with the culture 157 conditions, and the exposure medium was not aerated. Following the immobilization test, the 158 impact of b-BC and n-BC on reproduction was further assessed for selected b-BC/n-BC (where 159 the effect of the adhesion induced by n-BC was observed under a microscope). Two 160 concentrations of b-BC or n-BC (2 and 10 mg/L) were selected. A young female D. magna was 161 exposed to each concentration for 21 days, and at the end of the test, the total number of living 162 offspring produced by the exposed D. magna was assessed. 163 9 In order to assess the toxicity of b-BC and n-BC solutions to aquatic plants, the duckweed 164 Lemna minor was used according to the procedure proposed by OECD 221 guidelines (OECD, 165 2006). The primary objective of the test was to quantify substance-related effects on vegetative 166 growth over a test period, based on assessments of selected measurement variables, including 167 frond number and total frond area. Three concentrations of b-BC or n-BC (2, 10, and 100 mg/L) 168 were applied. For each concentration, 3 colonies with 3-4 visible fronds each were placed into 169 measurement vessels with a total volume of 100 mL. The organisms were exposed in growth 170 medium for 3 weeks prior to evaluating the particular endpoints. The cultures were incubated 171 in a temperature-controlled environment (at 24 ± 2°C) under continuous illumination with 172 fluorescent lamps). The frond counting was manually performed at the beginning of the test (0 173 days) and after 2, 5, and 7 days using the ImageJ 1.54 software (ImageJ 1.54, Java, USA). The 174 average specific growth rate (μi) was calculated for each replicate after 7 days, to assess the 175 health status of plants following equation: 176 μ𝑖=ln(𝑁𝑗) − ln⁡(𝑁𝑖) t 177 Where µi-average specific growth rate from time i to j, Nimeasurement variable in the test 178 or control vessel at the time I, Nj-measurement variable in the test or control vessel at the time 179 j, t-time period from i to j. 180 2.4. Solid phase test 181 An ecotoxicological test with Collembola (Folsomia candida) was performed following the 182 standard procedure based on OECD 232 guidelines (OECD, 2016). The test examines the 183 mortality and reproduction of F. candida. A detailed description of the test is presented in our 184 previous publications (Kołtowski et al., 2017; Oleszczuk et al., 2019; Tomczyk et al., 2021). 185 Briefly, the adult individuals (aged 10-12 days) were placed in a petri dish with standard soil 186 (OECD) mixed with 1% and 5% of b-BC or n-BC. The standard soil (OECD) was used as a 187 16 nutrients such as potassium, phosphorus, and calcium (Hossain et al., 2020), which are 336 beneficial for plant growth. Consequently, a higher ash content in n-BC may result in a greater 337 availability of nutrients for L. minor (Bisane et al., 2023). 338 3.3. Toxicity to Daphnia magna 339 The Daphnia magna immobilization test revealed that none of the tested concentrations 340 caused statistically significant immobilization of these organisms for b-BCs and n-BCs (Table 341 S3). However, detailed microscopic observations of the organisms revealed a significant 342 adherence of BC’s particles to the bodies of D. magna (Fig. 4). This adherence may pose long343 term risks, potentially leading to chronic toxicity. The adherence effect was not observed in 344 the system with b-BC. Following this observation, n-BC and b-BC representing different 345 feedstocks were selected for a reproduction test to assess the long-term effects of the particles. 346 Both n-BC and b-BC affected the reproduction of D. magna (Table 1), but a higher reduction 347 compared to control was observed for n-BC than b-BC, especially in the system with a higher 348 concentration of particles. The harmful effect was observed for n-BC for two applied 349 concentrations, while for b-BC the effect was only noted for the highest concentration of 350 particles. Reproduction inhibition clearly depended on the feedstock used for BC production. 351 Except for b-BC-WL-750 and n-BC-WL-750, higher reproduction inhibition was observed for 352 n-BC than b-BC (Table 1). Regarding the age of first reproduction, it was generally noted that 353 the values were very close to those of the control group. However, for n-BC-OSR-550and b354 BC-Wl-750, the age of first reproduction was significantly longer (>11 days) compared to the 355 control (9 days). This delay may be explained by the potential presence of inhibitory 356 compounds in the specific feedstocks or by the larger impact of these particular BC on the 357 organisms' overall energy budget, possibly due to the leaching of specific TMs or organic 358 compounds that interfere with reproductive timing. 359 17 3.4. Toxicity to Lepidium sativum 360 No notable impact or trend was detected regarding the inhibition of Lepidium sativum seed 361 germination (Table S6). Other studies report that under stress conditions, the addition of n-BC 362 can increase the germination rate of plants. Xin et al. (2024) observed maximum increases in 363 germination rates of 24.8% and 20.8% for BC’s nanoparticles and BC, respectively, compared 364 to the control group. In contrast, Zhang et al.(2020) did not observe any effect of BC’s 365 nanoparticles at different concentrations (0.5, 5, and 50 mg/L) on the seed germination rates of 366 rice and tomato. 367 Nevertheless, a statistically significant effect (p≤ 0.05) of BCs was observed on root growth 368 inhibition (Fig. 5). When comparing different feedstocks, only the SSL-derived b-BC (b-BC369 SSL1-550, b-BC-SSL2-500, and b-BC-SSL3-500) among the tested b-BC exhibited toxicity 370 towards L. sativum, causing root growth inhibition ranging from 18% to 23%. In contrast, the 371 agricultural residues-derived b-BC (b-BC-RH-550 and b-BC-OSR-550) and WL-derived b-BC 372 (b-BC-WL-550) produced at the same temperature were found to be non-toxic and even 373 stimulated the root growth by 6% to 15%. SSL-derived b-BC produced at lower temperatures 374 (500-550°C) tends to have higher concentrations of volatile organic compounds (VOCs), which 375 can disrupt root development (Goldan et al., 2022). Additionally, SSL-derived b-BC has lower 376 carbon content and higher levels of ash (Raczkiewicz et al., 2024b). Higher ash content is 377 associated with an increased presence of inorganic contaminants, such as heavy metals, which 378 can be toxic to plants (Goyal et al., 2020) and in BC can also cause fluctuations in soil pH and 379 electrical conductivity (EC), creating an unfavorable environment for plant growth (Marra et 380 al., 2018). Increasing the pyrolysis temperature also enhanced the positive effect of b-BC on L. 381 sativum, which correlated with a reduction in the content of the contaminants in these BC (Fig. 382 7b) (Table S4). Statistical analysis revealed a strong negative correlation between the Ctotal 383 PAHs and the root growth of L. sativum. It was previously suggested that PAHs contained in 384 18 BCs could be responsible for their negative impact on plants (Godlewska et al., 2022; Jajoo, 385 2017; Stefaniuk and Oleszczuk, 2016). Additionally, a positive correlation was observed 386 between the carbon content (C) and root growth, while a negative correlation was found 387 between the nitrogen content (N) in b-BC and root growth. These correlations could be 388 attributed to the differing physicochemical properties of the BC associated with its chemical 389 composition. Higher C content may enhance soil structure, water retention, and nutrient 390 availability, thereby promoting plant growth (Murtaza et al., 2024). On the other hand, the N 391 content in BC may influence microbial activity in the soil, which could also affect plant growth 392 (Liu et al., 2018). Insufficient N levels may lead to adverse effects such as inhibited plant 393 growth, explaining the observed negative correlation. Furthermore, replacing the carrier gas 394 from N2 to CO2 showed no significant impact (p ≤ 0.05) on the inhibition of L. sativum root 395 growth. These findings align with similar trends observed in previous studies regarding the 396 influence of pyrolysis conditions on BC’s toxicity (Kończak et al., 2020; Sun et al., 2023). 397 The reduction of BC’s size to the nanoscale resulted in a shift from a stimulatory effect to 398 toxicity (BC-WL, BC-RH, BC-OSR) or a significant increase in the inhibition of L. sativum 399 root growth (Fig. 5). The Tukey test results in the Figure 5 indicate that n-BC generally causes 400 higher root growth inhibition in L. sativum compared to bulk biochar b-BC, as indicated by the 401 higher percentage inhibition values for n-BC bars. Statistically significant differences in root 402 growth effects are denoted by different letters. Zhang et al. (2020) indicated that BC’s 403 nanoparticles could pose a potential threat to biota, primarily due to phenolic compounds 404 deposited on the larger BC’sspecificsurface area, which have been shown to negatively impact 405 plants. Therefore, the risk posed by n-BC to biota likely originates from these phenolic 406 compounds on its increased surface. All n-BCs displayed a consistent trend of changes similar 407 to those observed with b-BC concerning the feedstock used and pyrolysis conditions. Statistical 408 analysis indicated that the physicochemical factors determining toxicity are the same in both b409 19 BC and n-BC (Table S4). However, due to the reduction in size, harmful substances may be 410 released more easily. These trends between b-BC and n-BC were also observed for F. candida 411 (Fig. 6). Furthermore, n-BCs demonstrated significantly higher EC values compared to b-BCs 412 (Table S2) (Raczkiewicz et al., 2024b). These elevated EC values may exceed the tolerance 413 levels of plants to salt presence, especially as BC concentration increases. Numerous studies 414 have reported that high EC value resulting from soil amendments with BC is a key factor 415 contributing to the detrimental effects of BC on L. sativum (Gell et al., 2011; Muhammad and 416 Hussain, 2010; Nhan et al., 2019). 417 3.5. Toxicity to Folsomia candida 418 Both b-BC and n-BC, when added to soil at two different doses did not affect the mortality 419 of F. candida (Table S7), nevertheless, they did influence their reproduction (Fig. 6). At 1% 420 dose, b-BC stimulated the reproduction of F. candida in the range from 9% to 90%. Concerning 421 the feedstock used, the most visible effect was observed for SSL-derived b-BCs, while the 422 lowest values were noted for b-BC-OSR-550. An increase in pyrolysis temperature resulted in 423 a gradual increase in reproduction with the highest temperature yielding a reproduction rate 424 25% higher than that observed at the lowest temperature (Fig. 6). Changing the carrier gas from 425 N2 (b-BC-SSL3-500) to CO2 (b-BC-SSL3CO2-500) did not affect reproduction of F. candida 426 (Fig. 6). 427 The addition of 1% of n-BC had a less favorable impact on the stimulation of F. candida 428 reproduction to their respective bulk counterparts (Fig. 6a). The reproduction stimulation of n429 BC was from 38% to 45% lower for WL-derived n-BCs, from 30% to 44% for SSL-derived n430 BCs, and from 46% to 56% for agricultural residues-derived n-BC (n-BC-RH-550 and n-BC431 OSR-550) compared to corresponding b-BC. The observed trend in response to the applied 432 feedstock and pyrolysis parameters for n-BC exhibited a strong correlation with the trend noted 433 for b-BC. 434 20 An increase of the b-BC dose to 5% significantly diminished the stimulatory effect observed 435 at lower doses (Fig. 6), resulting in the inhibition of F. candida reproduction for WL- (from 4% 436 to 16%) and for agricultural residues-derived b-BC (from 35% to 60%) (Fig. 6b). Only SSL437 derived b-BC did not negatively affect the reproduction of F. candida, showing no significant 438 difference from the control. An increase pyrolysis temperature, which reduced toxicity and 439 replacing N2 with CO2, which showed no significant effect, exhibited a consistent trend as 440 observed at lower b-BC dose. 441 At higher dose of n-BC, a similar trend was observed as for b-BC regarding to the applied 442 feedstock and pyrolysis conditions (Fig. 6b). The n-BCs exhibited a greater toxic effect on F. 443 candida than corresponding b-BCs. In general, the reproduction inhibition was from 62% to 444 81% higher for WL-derived n-BC and from 15% to 51% for agricultural waste-derived n-BCs 445 than b-BCs. 446 The observed toxic or stimulatory effects were significantly correlated (p ≤ 0.05) with the 447 levels of contaminants in the BC, including PAHs and TMs (Fig. 7). This observation aligns 448 with findings from previous researchers (Eom et al., 2007; Sverdrup et al., 2002), who 449 demonstrated the sensitivity of springtails (Folsomia candida and Folsomia fimetaria) to PAHs, 450 particularly regarding reproduction juvenile. In the case of the 5% dose of b-BC, another 451 potential explanation for the detrimental impact on reproduction could be the pH levels. Prior 452 studies (Luo et al., 2022; Styrishave et al., 2010) have shown that the alkaline pH may 453 negatively affect F. candida, especially in terms of reproduction. The optimal pH for the 454 growth and reproduction of F. candida falls within the range of 5 to 6. The pH values for the 455 tested b-BC ranged from 8.2 to 11. With the addition of 1% b-BC, the pH impact was minimal 456 and did not cause a toxic effect, but rather a stimulating one. This could be due to the relatively 457 low concentration of b-BC, which might not significantly alter the overall pH or create 458 conditions that are harmful to the organisms. Instead, the small amount of b-BC might provide 459 21 essential nutrients or create a mildly favorable environment that enhances reproduction. In the 460 case of n-BC, the pH after size reduction decreased to the range of 7.4 to 10. For n-BC, besides 461 the ability to adsorb nutrients, it may be easier for the particles to penetrate organisms or 462 accumulate, increasing the chance of introducing contaminants that could have a direct toxic 463 effect. Additionally, nano-sized particles may more easily deposit in tissues/cells than macro464 sized particles, which pass through the organism and are excreted or not even absorbed. 465 Nanoparticles, due to their small size, are more prone to accumulation, potentially causing 466 long-term adverse effects on organisms (Rajput et al., 2020). Due to its larger specific surface 467 area and size effect, n-BC exhibits greater adsorption properties than b-BC. When added to soil, 468 n-BC can adsorb essential native nutrients from the soil solution, leading to their depletion. This 469 process can induce the translocation of nutrients into deeper soil layers due to the enhanced 470 migratory capabilities of n-BC, which are associated with their greater surface charge in soil 471 profiles, thereby diminishing nutrient retention within the surface horizons (Li et al., 2024). 472 Consequently, the reduced physical availability of these elements in the surface layers for F. 473 candida may exacerbate toxicological effects. Our study showed that low doses of n-BC (1% 474 dose) do not lead to significant soil nutrient depletion sufficient to inhibit reproduction or cause 475 mortality in adult individuals, but it is evident that reproduction is reduced compared to b-BC 476 (Fig. 6a). The observed detrimental effect at elevated doses of n-BC (5% dose) may be 477 attributable to the adsorption capacity of n-BC surpassing the natural replenishment rate of soil 478 nutrients at this concentration. Furthermore, a higher dose of n-BC is associated with increased 479 nutrient adsorption, which then migrates deeper into the soil profile, thereby reducing their 480 availability in the surface horizons. The pronounced opposing effect of BC, attributable to the 481 applied dose (stimulation vs. detrimental effect) may suggest the existence of a threshold 482 concentration of n-BC/b-BC beyond which its presence in the soil becomes toxic to springtails. 483 While lower doses of n-BC may still confer benefits without reaching this threshold, the 484 22 cumulative effect of higher doses could prove detrimental. Bielská et al. (2018) observed that 485 with increasing doses of BC, the toxicity towards F. candida also increased. In their study, 486 reproduction rates in soil with the addition of 1% wood and rice husk BC were comparable to 487 those of control soil. However, when the BC dose was increased to 10%, reproduction rates 488 decreased by 38% and 27% for wood BC and rice husk BC, respectively, compared to control 489 soil. Moreover, the elevated content of dissolved organic carbon (DOC) in n-BC may influence 490 its ecotoxicity by enhancing the bioavailability and mobility of toxic substances (like PAHs or 491 TMs) associated with DOC (Ma et al., 2021). This is confirmed by the statistical analysis of 492 correlations between DOC and reproduction of F. candida (Fig. 8c). Despite the reduced 493 organic-solvent extractable (Ctotal) and freely dissolved (Cfree) PAHs content in n-BC than in b494 BC, the increased specific surface area and DOC content in n-BC facilitate enhanced 495 interactions between PAHs in the soil-n-BC system. 496 4. CONCLUSION 497 This study provides a systematic assessment of the ecotoxicological effects of nanobiochar 498 (n-BC) across multiple biological systems, elucidating the environmental implications of its 499 application. The findings highlight the pivotal role of feedstock properties, pyrolysis conditions, 500 and particle size in influencing BC’s interactions with organisms. The key outcomes are as 501 follows: 502 ⎯ Bacteria: n-BC exhibited significantly higher toxicity to A. fischeri compared to bulk 503 biochar (b-BC), likely due to enhanced surface activity, leaching of toxic substances, 504 and lower pH. 505 ⎯ Aquatic and terrestrial plants: While n-BC was non-toxic to L.minor, promoting 506 frond growth potentially due to the release of bioavailable nutrients, it inhibited root 507 growth in L. sativum, possibly as a result of elevated electrical conductivity (EC) 508 surpassing the plant’s salt tolerance. 509 23 ⎯ Soil invertebrates: Higher concentrations of n-BC induced reproductive toxicity to 510 F.candida, which may be attributed to increased dissolved organic carbon (DOC) and 511 deeper soil migration of n-BC particles. 512 ⎯ Aquatic invertebrates: Exposure to elevated doses of n-BC inhibited D. magna’s 513 reproduction and resulted in notable particle adhesion to the organisms’ surfaces, 514 indicating potential bioaccumulation and long-term toxic effects. 515 Our research reveals the pivotal role of feedstock and particle size in determining the 516 environmental risks of biochar. By methodically analyzing both bulkand nano-sized 517 biochar across various trophic levels, we unlock new perspectives on the ecological impacts 518 of biochar usage. These insights can drive future innovations in the development and 519 responsible application of biochar technologies within environmental management. 520 Ultimately, our study underscores the imperative of integrating multiple factors, such as 521 feedstock, pyrolysis conditions, and particle size, when evaluating the toxicological profiles 522 of biochar. Additionally, it highlights the urgency for continued research to fully grasp the 523 environmental ramifications and to devise strategies for the safe, sustainable deployment of 524 biochar materials. Further investigations should focus on long-term ecological monitoring, 525 mechanistic studies of biochar-organism interactions, and the development of guidelines for 526 the safe and sustainable deployment of biochar in environmental management. 527 528 529 Acknowledgments 530 The project was funded by the National Science Centre granted based on the decision number 531 DEC2021/42/A/ST10/00161. 532 Competing interests 533 The authors have no relevant financial or non-financial interests to disclose. 534 24 Author Contributions 535 Conceptualization was performed by M.R and P.O.; Material preparation, data collection and 536 analysis were performed by M.R. and A.B.; The first draft of the manuscript was written by 537 M.R. and all authors commented on previous versions of the manuscript. All authors read and 538 approved the final manuscript. 539 5. REFERENCES 540 Bala, S., Garg, D., Thirumalesh, B.V., Sharma, M., Sridhar, K., Inbaraj, B.S., Tripathi, M., 2022. 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