The dose-dependent effects of dissolved biochar on C. elegans: insights into the physiological and transcriptomic responses

Abstract

As the benefits of biochar amendment for soil remediation have been widely recognized, the potential risk of downward nematode migration has received increasing attention. Dissolved biochar (DBC) is an essential component of biochar that is easily absorbed and utilized by organisms. However, the effect of DBC on nematodes remains unclear. This study aimed to assess the effect of DBC on Caenorhabditis elegans. The response of C. elegans to different DBC concentrations (0, 250, 500, and 1000 mg L⁻¹) was investigated using culture assays and RNA-seq analysis. The results revealed a hormetic effect of DBC, with low concentrations (250–500 mg L⁻¹) promoting growth and high concentrations (≥ 1000 mg L⁻¹) inhibiting growth. Meanwhile, DBC affected nematode movement and neuromuscular function. Transcriptome analysis revealed a dose-dependent increase in the number of differentially expressed genes (DEGs), with key changes related to metabolism, the stress response, and cellular processes. Weighted gene coexpression network analysis (WGCNA) revealed that gene modules, such as dyf-11, ins-16, and hsp-12.6, were strongly correlated with traits such as body size and reproduction. Additionally, genes involved in ciliary function, insulin signaling, and neurotransmitter biosynthesis were affected, highlighting the impact of DBC on growth and movement regulation. These findings suggest the need to carefully manage biochar application in agriculture to balance its benefits and potential risks to soil organisms like nematodes.

Keywords

Dissolved biochar (DBC) / C. elegans / Hormetic effect / Transcriptome analysis

References

[1] AkellaJS, WlogaD, KimJ, StarostinaNG, Lyons-AbbottS, MorrissetteNS, DouganST, KipreosET, GaertigJ. MEC-17 is an α-tubulin acetyltransferase. Nature, 2010, 467: 218-U111.

[2] AkhmanovaA, SteinmetzMO. Control of microtubule organization and dynamics: two ends in the limelight. Nat Rev Mol Cell Biol, 2015, 16: 711-726.

[3] AlharbiHA, AlotaibiKD, El-SaeidMH, GiesyJP. Polycyclic Aromatic Hydrocarbons (PAHs) and metals in diverse biochar products: effect of feedstock type and pyrolysis temperature. Toxics, 2023, 1114.

[4] Aquino-Nunez W, Mielko ZE, Dunn T, Santorella EM, Hosea C, Leitner L, McCalla D, Simms C, Verola WM, Vijaykumar S, et al. (2020) cnd-1/NeuroD1 Functions with the Homeobox Gene ceh-5/Vax2 and Hox Gene ceh-13/labial To Specify Aspects of RME and DD Neuron Fate in Caenorhabditis elegans. G3 Genes Genomes Genet 10: 3071–3085. https://doi.org/10.1534/g3.120.401515

[5] BakerRH, BrittonC, RobertsB, LoerCM, MatthewsJB, NisbetAJ. Melanisation of Teladorsagia circumcincta larvae exposed to sunlight: a role for GTP-cyclohydrolase in nematode survival. Int J Parasit, 2012, 42: 887-891.

[6] BurghesAHM, VaessinHEF, de la ChapelleA. Genetics—the land between Mendelian and multifactorial inheritance. Science, 2001, 293: 2213-2214.

[7] ChatzispyrouIA, AldersM, Guerrero-CastilloS, PerezRZ, HaagmansMA, MouchiroudL, KosterJ, OfmanR, BaasF, WaterhamHR, et al.. A homozygous missense mutation in ERAL1, encoding a mitochondrial rRNA chaperone, causes Perrault syndrome. Hum Mol Genet, 2017, 26: 2541-2550.

[8] ChuDS, LiuHB, NixP, WuTF, RalstonEJ, YatesJR, MeyerBJ. Sperm chromatin proteomics identifies evolutionarily conserved fertility factors. Nature, 2006, 443: 101-105.

[9] Da CostaVB, OguraAP, AlexandreDS, SoaresMB, AlleoniLRF, EspíndolaELG, PintoTJD. How much biochar is safe? Exploring potential ecotoxicological consequences for soil invertebrates and plants. Appl Soil Ecol, 2024, 2029.

[10] DongMY, JiangMY, HeLZ, ZhangZR, GustaveW, VithanageM, NiaziNK, ChenB, ZhangXK, WangHL, et al.. Challenges in safe environmental applications of biochar: identifying risks and unintended consequence. Biochar, 2025, 720.

[11] Ellegaard-JensenL, JensenKA, JohansenA. Nano-silver induces dose-response effects on the nematode Caenorhabditis elegans. Ecotox Environ Safe, 2012, 80: 216-223.

[12] FujiwaraM, TeramotoT, IshiharaT, OhshimaY, McIntireSL. A novel zf-MYND protein, CHB-3, mediates guanylyl cyclase localization to sensory cilia and controls body size of Caenorhabditis elegans. PLoS Genet, 2010, 6. e1001211

[13] GuY, JiangY, ChenX, LiL, ChenH, ChenJ, WangC, YuJ, ChenC, LiH. Generation of environmentally persistent free radicals on photoaged tire wear particles and their neurotoxic effects on neurotransmission in Caenorhabditis elegans. Environ Int, 2024, 186. 108640

[14] GuggenbergerG, RodionovA, ShibistovaO, GrabeM, KasanskyOA, FuchsH, MikheyevaN, ZrazhevskayaG, FlessaH. Storage and mobility of black carbon in permafrost soils of the forest tundra ecotone in Northern Siberia. Glob Change Biol, 2008, 14: 1367-1381.

[15] HemmatzadehM, MohammadiazarmH, GhasemiSA, RezaieA. Influence of humic acids on growth performance, bravery, stress resistance, antioxidant activity, and immune gene expression in male Siamese fighting fish (Betta splendens). Aquacult Rep, 2024.

[16] HouS, WangJ, DaiJ, BoussafirM, ZhangC. Combined effects of earthworms and biochar on PAHs-contaminated soil remediation: a review. Soil Ecol Lett, 2022.

[17] HuangKW, HsuKC, ChuLY, YangJM, YuanHS, HsiaoYY. Identification of Inhibitors for the DEDDh Family of Exonucleases and a Unique Inhibition Mechanism by Crystal Structure Analysis of CRN-4 Bound with 2-Morpholin-4-ylethanesulfonate (MES). J Med Chem, 2016, 59: 8019-8029.

[18] Inglis PN, Blacque OE, and Leroux MR (2009). Functional Genomics of Intraflagellar Transport-Associated Proteins in C. elegans. In: King SM, Pazour GJ (eds.) Cilia: Model Organisms and Intraflagellar Transport, (San Diego: Elsevier Academic Press Inc), pp 267–304

[19] JefferyS, van de VoordeTFJ, HarrisWE, MommerL, Van GroenigenJW, De DeynGB, EkelundF, BrionesMJI, BezemerTM. Biochar application differentially affects soil micro-, meso-macro-fauna and plant productivity within a nature restoration grassland. Soil Biol Biochem, 2022.

[20] JiaX, ZhangB, HanY, GuanJ, GaoH, GuoP. Role of reactive oxygen species (ROS) on biochar enhanced chromium phytoremediation in the soil-plant system: exploration on detoxification mechanism. Environ Int, 2025, 199109471

[21] JiangSF, HaoHC, ChenS, ChenYL, JiangH. Mechanistic elucidation and tuning pathway of environmentally persistent free radicals during sewage sludge pyrolysis. J Anal Appl Pyrolysis, 2024, 17910.

[22] JohnstoneIL. Cuticle collagen genes-: expression in Caenorhabditis elegans. Trends Genet, 2000, 16: 21-27.

[23] KimD, LandmeadB, SalzbergSL. HISAT: A fast spliced aligner with low memory requirements. Nat Methods, 2015, 12: 357-U121.

[24] KimuraK, MamaneA, SasakiT, SatoK, TakagiJ, NiwayamaR, HufnagelL, ShimamotoY, JoannyJF, UchidaS, et al.. Endoplasmic-reticulum-mediated microtubule alignment governs cytoplasmic streaming. Nat Cell Biol, 2017, 19: 399-406.

[25] KumariT, PhogatD, PhogatJ, ShuklaV. Biochar & fly ash amendments lower mortality and increase antioxidant activity in chlorpyrifos-exposed earthworms. Appl Biol Chem, 2024, 6765.

[26] LecroiseyC, MariolMC, SchwabY, LabouesseM, SégalatL, GieselerK. DYC-1 and ZYX-1, new actors of muscle degeneration in Caenorhabditis elegans. Neuromusc Disord, 2009, 19: 639-639.

[27] LeeI, KnickerbockerAC, DepewCR, MartinEL, DicentJ, MillerGW, BucherML. Effect of altered production and storage of dopamine on development and behavior in C. elegans. Front Toxicol, 2024, 617.

[28] LehmannJ. Bio-energy in the black. Front Ecol Environ, 2007, 5: 381-387.

[29] LehmannJ, RilligMC, ThiesJ, MasielloCA, HockadayWC, CrowleyD. Biochar effects on soil biota—a review. Soil Biol Biochem, 2011, 43: 1812-1836.

[30] LettreG, HengartnerMO. Developmental apoptosis in C. elegans: a complex CEDnario. Nat Rev Mol Cell Biol, 2006, 7: 97-108.

[31] LiJ, ChenYX, ZhangGL, RuanWB, ShanSJ, LaiX, YangDL, YuZG. Integration of behavioural tests and transcriptome sequencing of C. elegans reveals how the nematode responds to peanut shell biochar amendment. Sci Total Environ, 2020, 70711.

[32] LiB, DeweyCN. RSEM: Accurate Transcript Quantification from RNA-Seq Data with or without a Reference Genome. BMC Bioinform, 2011, 12323.

[33] LiHJ, LiH, ZuoN, LangD, DuW, ZhangP, PanB. Can the concentration of environmentally persistent free radicals describe its toxicity to Caenorhabditis elegans? Evidence provided by neurotoxicity and oxidative stress. J Hazard Mater, 2024, 4699.

[34] LiH, LiuY, TaoYuanLY, LiT, YanJ, YangC. Insights into the characteristics and toxicity of microalgal biochar-derived dissolved organic matter by spectroscopy and machine learning. Sci Total Environ, 2024, 957. 177648

[35] LiL, MaR, YuanY, YaoQ, HanY, CaoH, QiJ. Neurotoxicity induced by aged microplastics from plastic bowls: abnormal neurotransmission in Caenorhabditis elegans. Sci Total Environ, 2024, 952. 175939

[36] LianF, XingBS. Black Carbon (Biochar) in water/soil environments: molecular structure, sorption, stability, and potential risk. Environ Sci Technol, 2017, 51: 13517-13532.

[37] LianF, XingBS. From Bulk to Nano: formation, Features, and Functions of Nano-Black Carbon in Biogeochemical Processes. Environ Sci Technol, 2024, 58: 15910-15925.

[38] LianF, YuWC, WangZY, XingBS. New insights into black carbon nanoparticle-induced dispersibility of goethite colloids and configuration-dependent sorption for phenanthrene. Environ Sci Technol, 2019, 53: 661-670.

[39] LiekeT, ZhangXC, SteinbergCEW, PanB. Overlooked Risks of Biochars: persistent Free Radicals trigger Neurotoxicity in Caenorhabditis elegans. Environ Sci Technol, 2018, 52: 7981-7987.

[40] LiuH, DuX, LiY, HanX, LiB, ZhangX, LiQ, LiangW. Organic substitutions improve soil quality and maize yield through increasing soil microbial diversity. J Clean Prod, 2022.

[41] LiuX, GeP, LuZ, YangR, LiuZ, ZhaoF, ChenM. Reproductive toxicity and underlying mechanisms of fine particulate matter (PM(2.5)) on Caenorhabditis elegans in different seasons. Ecotoxicol Environ Saf, 2022, 248114281.

[42] LiuH, WangY, WangSX, WuJ, WangYL. Release characteristics of biochar-derived dissolved organic matter and its impact on Cr adsorption and reduction. RSC Adv, 2024, 14: 38171-38182.

[43] LiuMY, LiuXC, HuYL, ZhangQ, FarooqU, QiZC, LuLT. Mobility of biochar-derived dissolved organic matter and its effects on sulfamerazine transport through saturated soil porous media. Environ Sci Process Impacts, 2024, 26: 2264-2278.

[44] LongXX, YuZN, LiuSW, GaoT, QiuRL. A systematic review of biochar aging and the potential eco-environmental risk in heavy metal contaminated soil. J Hazard Mater, 2024, 472. 134345

[45] LoveMI, HuberW, AndersS. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol, 2014, 15550.

[46] MaSQ, JingFQ, SohiSP, ChenJW. New insights into contrasting mechanisms for PAE adsorption on millimeter, micron- and nano-scale biochar. Environ Sci Pollut Res, 2019, 26: 18636-18650.

[47] ManiR, EzhumalaiD, MuthusamyG, NamasivayamE. Neuroprotective effect of biogenically synthesized ZnO nanoparticles against oxidative stress and β-amyloid toxicity in transgenic Caenorhabditis elegans. Biotechnol Appl Biochem, 2024, 71: 132-146.

[48] MatsunagaY, HondaY, HondaS, IwasakiT, QadotaH, BenianGM, KawanoT. Diapause is associated with a change in the polarity of secretion of insulin-like peptides. Nat Commun, 2016, 78.

[49] MitchumMG, HusseyRS, BaumTJ, WangXH, EllingAA, WubbenM, DavisEL. Nematode effector proteins: an emerging paradigm of parasitism. New Phytol, 2013, 199: 879-894.

[50] MurphyCT, McCarrollSA, BargmannCI, FraserA, KamathRS, AhringerJ, LiH, KenyonC. Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature, 2003, 424: 277-284.

[51] NawaM, MatsuokaM. The method of the body bending assay using caenorhabditis elegans. Bio Protocol, 2012, 2e253.

[52] NepalJ, AhmadW, MunsifF, KhanA, ZouZY. Advances and prospects of biochar in improving soil fertility, biochemical quality, and environmental applications. Front Environ Sci, 2023, 1117.

[53] NgLYF, AriffinH, Yasim-AnuarTAT, FaridMAA, HassanMA. High-Energy Ball Milling for High Productivity of Nanobiochar from Oil Palm Biomass. Nanomaterials, 2022, 1211.

[54] PakSC, KumarV, TsuC, LukeCJ, AskewYS, AskewDJ, MillsDR, BrommeD, SilvermanGA. SRP-2 is a cross-class inhibitor that participates in postembryonic development of the nematode Caenorhabditis elegans: initial characterization of the clade L serpins. J Biol Chem, 2004, 279: 15448-15459.

[55] PerniM, van der GootA, LimbockerR, van HamTJ, AprileFA, XuCK, FlagmeierP, ThijssenK, SormanniP, FuscoG, et al.. Comparative Studies in the A30P and A53T α-Synuclein C. elegans Strains to Investigate the Molecular Origins of Parkinson’s Disease. Front Cell Dev Biol, 2021, 910.

[56] PhilippTM, GongW, KohnleinK, OhseVA, MullerFI, PriebsJ, SteinbrennerH, KlotzLO. SEMO-1, a novel methanethiol oxidase in Caenorhabditis elegans, is a pro-aging factor conferring selective stress resistance. BioFactors, 2022, 48: 699-706.

[57] PierceSB, CostaM, WisotzkeyR, DevadharS, HomburgerSA, BuchmanAR, FergusonKC, HellerJ, PlattDM, PasquinelliAA, et al.. Regulation of DAF-2 receptor signaling by human insulin and ins-1, a member of the unusually large and diverse C. elegans insulin gene family. Genes Dev, 2001, 15: 672-686.

[58] PrevoB, MangeolP, OswaldF, ScholeyJM, PetermanEJG. Functional differentiation of cooperating kinesin-2 motors orchestrates cargo import and transport in C. elegans cilia. Nat Cell Biol, 2015, 17: 1536-1545.

[59] ProdanaM, SilvaC, GravatoC, VerheijenFGA, KeizerJJ, SoaresA, LoureiroS, BastosAC. Influence of biochar particle size on biota responses. Ecotox Environ Safe, 2019, 174: 120-128.

[60] QinYX, LiGY, AnTC, YangZF. Advances in ecological and health risks of biochar during environmental applications. Chin Sci Bull-Chin, 2021, 66: 5-20.

[61] RamakrishnanK, OkkemaPG. Regulation of C. elegans Neuronal Differentiation by the ZEB-Family Factor ZAG-1 and the NK-2 Homeodomain Factor CEH-28. PLoS ONE, 2014, 917.

[62] RombolàAG, GreggioN, FabbriD, FacchinA, TorriC, PulcherR, CarliniC, BaluganiE, MarazzaD, ZannoniD, et al.. Changes of labile, stable and water-soluble fractions of biochar after two years in a vineyard soil. Environ Sci-Adv, 2023, 2: 1587-1599.

[63] Sanchez-HernandezJC, CaresXA, PérezMA, del PinoJN. Biochar increases pesticide-detoxifying carboxylesterases along earthworm burrows. Sci Total Environ, 2019, 667: 761-768.

[64] SandhuA, BadalD, SheokandR, TyagiS, SinghV. Specific collagens maintain the cuticle permeability barrier in Caenorhabditis elegans. Genetics, 2021.

[65] SanyalS, WintleRF, KindtKS, NuttleyWM, ArvanR, FitzmauriceP, BigrasE, MerzDC, HébertTE, van der KooyD, et al.. Dopamine modulates the plasticity of mechanosensory responses in Caenorhabditis elegans. Embo J, 2004, 23: 473-482.

[66] SinghRP, AhsanM, MishraD, PandeyV, AnupamaYA, KhareP. Ameliorative effects of biochar on persistency, dissipation, and toxicity of atrazine in three contrasting soils. J Environ Manage, 2022, 30311.

[67] TianYX, GuoX, MaJ, LiuQY, LiSJ, WuYH, ZhaoWH, MaSY, ChenHY, GuoF. Characterization of biochar-derived organic matter extracted with solvents of differing polarity via ultrahigh-resolution mass spectrometry. Chemosphere, 2022, 307. 135785

[68] TomisawaS, HojoE, UmetsuY, OhkiS, KatoY, MiyazawaM, MizuguchiM, KamiyaM, KumakiY, KikukawaT. Overexpression of an antimicrobial peptide derived from C. elegans using an aggregation-prone protein coexpression system. AMB Express, 2013, 38.

[69] WangZW, LiJ, ZhangGL, ZhiYC, YangDL, LaiX, RenTZ. Characterization of Acid-Aged Biochar and Its Ammonium Adsorption in an Aqueous Solution. Materials, 2020, 1317.

[70] WoodruffGC, WillisJH, PhillipsPC. Dramatic evolution of body length due to postembryonic changes in cell size in a newly discovered close relative of Caenorhabditis elegans. Evol Lett, 2018, 2: 427-441.

[71] XuYL, LuXF, SuGJ, ChenX, MengJ, LiQQ, WangCX, ShiB. Scientific and regulatory challenges of environmentally persistent free radicals: from formation theory to risk prevention strategies. J Hazard Mater, 2023.

[72] YangJN, ZhaoYL, WangYW, WangHF, WangDY. Toxicity evaluation and translocation of carboxyl functionalized graphene in Caenorhabditis elegans. Toxicol Res, 2015, 4: 1498-1510.

[73] YangF, WangCP, SunHW. A comprehensive review of biochar-derived dissolved matters in biochar application: production, characteristics, and potential environmental effects and mechanisms. J Environ Chem Eng, 2021, 910.

[74] YuS, AveryL, BaudeE, GarbersDL. Guanylyl cyclase expression in specific sensory neurons: a new family of chemosensory receptors. Proc Natl Acad Sci USA, 1997, 94: 3384-3387.

[75] YuYJ, ChenHB, HuaX, DangY, HanYJ, YuZL, ChenXC, DingP, LiH. Polystyrene microplastics (PS-MPs) toxicity induced oxidative stress and intestinal injury in nematode Caenorhabditis elegans. Sci Total Environ, 2020, 7268.

[76] YuY, ChenH, HuaX, WangC, DongC, XieD, TanS, XiangM, LiH. A review of the reproductive toxicity of environmental contaminants in Caenorhabditis elegans. Hyg Environ Health Adv, 2022.

[77] ZhangG, ZhangQ, SunK, LiuX, ZhengW, ZhaoY. Sorption of simazine to corn straw biochars prepared at different pyrolytic temperatures. Environ Pollut, 2011, 159: 2594-2601.

[78] ZhangFS, LiYX, ZhangGX, LiW, YangLS. The importance of nano-porosity in the stalk-derived biochar to the sorption of 17β-estradiol and retention of it in the greenhouse soil. Environ Sci Pollut Res, 2017, 24: 9575-9584.

[79] ZhangA, GuanZ, OckermanK, DongP, GuoJ, WangZ, YanD. Regulation of glial size by eicosapentaenoic acid through a novel Golgi apparatus mechanism. PLoS Biol, 2020, 18. e3001051

[80] ZhangY, ZhaoC, ZhangH, LuQ, ZhouJJ, LiuR, WangSZ, PuYP, YinLH. Trans-generational effects of copper on nerve damage in Caenorhabditis elegans. Chemosphere, 2021, 28413.

[81] ZhangR, ZhangR, ZimmermanAR, WangH, GaoB. Applications, impacts, and management of biochar persistent free radicals: a review. Environ Pollut, 2023, 327. 121543

[82] ZhaoL, RuiQ, WangDY. Molecular basis for oxidative stress induced by simulated microgravity in nematode Caenorhabditis elegans. Sci Total Environ, 2017, 607: 1381-1390.

[83] ZhuR, Chin-SangID. C.elegans insulin-like peptides. Mol Cell Endocrinol, 2024, 58511.

Funding

National Natural Science Foundation of China(42207048)

The Agricultural Science and Technology Innovation Program(CAAS-202408-04)

RIGHTS & PERMISSIONS

The Author(s)