Caenorhabditis elegans presents functioning, biologically relevant phenotypes and is frequently used as a bioindicator of toxicity. However, most C. elegans in vivo effect-assessment methods are laborious and time consuming. Therefore, we developed a novel method to measure the oxygen consumption rate of C. elegans as a sublethal endpoint of toxicity. This protocol was tested by exposing 50 larval stage one C. elegans individuals for 48 h (at 20 °C) to different concentrations of two toxicants i.e. benzylcetyldimethylammonium chloride (BAC-C16) and cadmium (Cd). Following exposures, the oxygen consumption rate of the C. elegans individuals were measured using the high-throughput functionality of the Seahorse XFe96 Extracellular Flux Analyzer. Dose-response curves for BAC-C16 (R2 = 0.93; P = 0.001) and Cd (R2 = 0.98; P = 0.001) were created. Furthermore, a strong, positive correlation was evidenced between C. elegans oxygen consumption rate and a commonly used, ecologically relevant endpoint of toxicity (growth inhibition) for BAC-C16 (R2 = 0.93; P = 0.0001) and Cd (R2 = 0.91; P = 0.0001). The data presented in this study show that C. elegans oxygen consumption rate can be used as a promising functional measurement of toxicity.

Agricultural Research Council Institute for Tropical and Subtropical Crops Private Bag X11208 Nelspruit 1200 South Africa

Centre of Excellence for Nutrition North West University Potchefstroom South Africa

Czech University of Life Sciences Faculty of Agrobiology Food and Natural Resources Department of Plant Protection Kamycka 129 165 21 Prague Czech Republic

Ecossa Giselastrasse 6 82319 Starnberg Germany

Human Metabolomics Faculty of Natural Sciences North West University Potchefstroom 2520 South Africa

Pediatric Epidemiology Department of Pediatrics University Medicine Leipzig Leipzig Germany

Unit for Environmental Sciences and Management North West University Private Bag X6001 Potchefstroom 2520 South Africa

University of Bielefeld Department of Animal Ecology Konsequenz 45 33615 Bielefeld Germany

See more in PubMed

Hägerbäumer A, Höss S, Heininger P, Traunspurger W. Experimental studies with nematodes in ecotoxicology: an overview. J. Nematol. 2015;47:11–27. PubMed PMC

Hunt PR. The C. elegans model in toxicity testing. J. Appl. Toxicol. 2017;37:50–59. doi: 10.1002/jat.3357. PubMed DOI PMC

Boyd WA, McBride SJ, Rice JR, Snyder DW, Freedman JH. A high-throughput method for assessing chemical toxicity using a Caenorhabditis elegans reproduction assay. Toxicol. Appl. Pharmacol. 2010;245:153–159. doi: 10.1016/j.taap.2010.02.014. PubMed DOI PMC

Scanlan LD, et al. Counting Caenorhabditis elegans: protocol optimization and applications for population growth and toxicity studies in liquid medium. Sci. Rep. 2018;8:904. doi: 10.1038/s41598-018-19187-3. PubMed DOI PMC

Harlow PH, et al. The nematode Caenorhabditis elegans as a tool to predict chemical activity on mammalian development and identify mechanisms influencing toxicological outcome. Sci. Rep. 2016;6:22965. doi: 10.1038/srep22965. PubMed DOI PMC

O’Reilly LP, Luke CJ, Perlmutter DH, Silverman GA, Pak SC. C. elegans in high-throughput drug discovery. Adv. Drug Del. Rev. 2014;0:247–253. doi: 10.1016/j.addr.2013.12.001. PubMed DOI PMC

Jung S-K, et al. Multi-endpoint, high-throughput study of nanomaterial toxicity in Caenorhabditis elegans. Environ. Sci. Technol. 2015;49:2477–2485. doi: 10.1021/es5056462. PubMed DOI PMC

Höss, S. & Williams, P. Ecotoxicity testing with nematodes in Nematodes as environmental indicators (eds Michael J. Wilson & Thomais Kakouli-Duarte) 208–224 (CABI Publishing, 2009).

Kohra S, et al. A rapid respiratory toxicity test using Caenorhabditis elegans with an oxygen electrode system. J. Health Sci. 2002;48:269–272. doi: 10.1248/jhs.48.269. DOI

Zuo Y-T, et al. Toxicity of 2,6-dichloro-1,4-benzoquinone and five regulated drinking water disinfection by-products for the Caenorhabditis elegans nematode. J. Hazard. Mater. 2017;321:456–463. doi: 10.1016/j.jhazmat.2016.09.038. PubMed DOI

Schouest K, Zitova A, Spillane C, Papkovsky DB. Toxicological assessment of chemicals using Caenorhabditis elegans and optical oxygen respirometry. Environ. Toxicol. Chem. 2009;28:791–799. doi: 10.1897/08-083.1. PubMed DOI

Bérard A, et al. Soil microbial respiration and PICT responses to an industrial and historic lead pollution: a field study. Environ. Sci. Pollut. R. 2016;23:4271–4281. doi: 10.1007/s11356-015-5089-z. PubMed DOI

Bownik A. Physiological endpoints in daphnid acute toxicity tests. Sci. Total Environ. 2020;700:134400. doi: 10.1016/j.scitotenv.2019.134400. PubMed DOI

Tang H, et al. Earthworm (Eisenia fetida) behavioral and respiration responses to sublethal mercury concentrations in an artificial soil substrate. Appl. Soil Ecol. 2016;104:48–53. doi: 10.1016/j.apsoil.2015.12.008. DOI

Luz, A. L., Smith, L. L., Rooney, J. P. & Meyer, J. N. Seahorse Xfe24 extracellular flux analyzer‐based analysis of cellular respiration in Caenorhabditis elegans. Curr. Protoc. Toxicol., 25.27.21–25.27.15 (2015). PubMed PMC

Koopman M, et al. A screening-based platform for the assessment of cellular respiration in Caenorhabditis elegans. Nat. Protoc. 2016;11:1798–1816. doi: 10.1038/nprot.2016.106. PubMed DOI PMC

Luz AL, et al. Mitochondrial morphology and fundamental parameters of the mitochondrial respiratory chain are altered in Caenorhabditis elegans strains deficient in mitochondrial dynamics and homeostasis processes. PloS ONE. 2015;10:e0130940. doi: 10.1371/journal.pone.0130940. PubMed DOI PMC

Porta-de-la-Riva M, Fontrodona L, Villanueva A, Cerón J. Basic Caenorhabditis elegans methods: synchronization and observation. JoVE. 2012;64:e4019. PubMed PMC

Höss S, Schlottmann K, Traunspurger W. Toxicity of ingested cadmium to the nematode Caenorhabditis elegans. Environ. Sci. Technol. 2011;45:10219–10225. doi: 10.1021/es2027136. PubMed DOI

Uppaluri S, Brangwynne CP. A size threshold governs Caenorhabditis elegans developmental progression. P. Roy. Soc. B - Biol. Sci. 2015;282:20151283. doi: 10.1098/rspb.2015.1283. PubMed DOI PMC

Hansen M, et al. A role for autophagy in the extension of lifespan by dietary restriction in C. elegans. PLoS Genet. 2008;4:e24. doi: 10.1371/journal.pgen.0040024. PubMed DOI PMC

De Cuyper C, Vanfleteren JR. Oxygen consumption during development and aging of the nematode Caenorhabditis elegans. Comp. Biochem. Physiol. 1982;73:283–289. doi: 10.1016/0300-9629(82)90071-8. DOI

Braeckman BP, Houthoofd K, Vanfleteren JR. Assessing metabolic activity in aging Caenorhabditis elegans: concepts and controversies. Aging Cell. 2002;1:82–88. doi: 10.1046/j.1474-9728.2002.00021.x. PubMed DOI

Braeckman BP, Houthoofd K, De Vreese A, Vanfleteren JR. Assaying metabolic activity in ageing Caenorhabditis elegans. Mech. Ageing Dev. 2002;123:105–119. doi: 10.1016/S0047-6374(01)00331-1. PubMed DOI

Braeckman BP, et al. No reduction of energy metabolism in Clk mutants. Mech. Ageing Dev. 2002;123:1447–1456. doi: 10.1016/S0047-6374(02)00085-4. PubMed DOI

Suda H, Shouyama T, Yasuda K, Ishii N. Direct measurement of oxygen consumption rate on the nematode Caenorhabditis elegans by using an optical technique. Biochem. Biophys. Res. Commun. 2005;330:839–843. doi: 10.1016/j.bbrc.2005.03.050. PubMed DOI

Höss S, et al. Interlaboratory comparison of a standardized toxicity test using the nematode Caenorhabditis elegans (ISO 10872) Environ. Toxicol. Chem. 2012;31:1525–1535. doi: 10.1002/etc.1843. PubMed DOI

Hanna S, et al. Feasibility of using a standardized Caenorhabditis elegans toxicity test to assess nanomaterial toxicity. Environ. Sci. Nano. 2016;3:1080–1089. doi: 10.1039/C6EN00105J. DOI

ISO10872. Water quality — determination of the toxic effect of sediment and soil samples on growth, fertility and reproduction of Caenorhabditis elegans (Nematoda) (International Organization for Standardization, 2010).

Connell DW, Yu QJ, Verma V. Influence of exposure time on toxicity—An overview. Toxicology. 2016;355–356:49–53. doi: 10.1016/j.tox.2016.05.015. PubMed DOI

Chen P, Martinez-Finley E, Bornhorst J, Chakraborty S, Aschner M. Metal-induced neurodegeneration in C. elegans. Frontiers in Aging. Neuroscience. 2013;5:1–11. PubMed PMC

Schertzinger G, Zimmermann S, Grabner D, Sures B. Assessment of sublethal endpoints after chronic exposure of the nematode Caenorhabditis elegans to palladium, platinum and rhodium. Environ. Pollut. 2017;230:31–39. doi: 10.1016/j.envpol.2017.06.040. PubMed DOI

Traunspurger W, et al. Ecotoxicological assessment of aquatic sediments with Caenorhabditis elegans (nematoda)—a method for testing liquid medium and whole‐sediment samples. Environ. Toxicol. Chem. 1997;16:245–250.

Van Kessel W, Zaalberg RB, Seinen W. Testing environmental pollutants on soil organisms: a simple assay to investigate the toxicity of environmental pollutants on soil organisms, using CdCl2 and nematodes. Ecotoxicol. Environ. Saf. 1989;18:181–190. doi: 10.1016/0147-6513(89)90079-1. PubMed DOI

Teixeira-Castro A, et al. Serotonergic signalling suppresses ataxin 3 aggregation and neurotoxicity in animal models of Machado-Joseph disease. Brain. 2015;138:3221–3237. doi: 10.1093/brain/awv262. PubMed DOI PMC

Gruber J, Ng LF, Poovathingal SK, Halliwell B. Deceptively simple but simply deceptive – Caenorhabditis elegans lifespan studies: considerations for aging and antioxidant effects. FEBS Lett. 2009;583:3377–3387. doi: 10.1016/j.febslet.2009.09.051. PubMed DOI

Järup L, Åkesson A. Current status of cadmium as an environmental health problem. Toxicol. Appl. Pharmacol. 2009;238:201–208. doi: 10.1016/j.taap.2009.04.020. PubMed DOI

Vellinger C, et al. Comparison of arsenate and cadmium toxicity in a freshwater amphipod (Gammarus pulex) Environ. Pollut. 2012;160:66–73. doi: 10.1016/j.envpol.2011.09.002. PubMed DOI

Van Aardt WJ, Fourie H, Pretorius M, Louw R, Van Dyk H. The effects of stirring, population levels and a potential anti-nematodal product on the respiration of second-stage juveniles of Meloidogyne incognita measured with different technology. Nematology. 2016;18:1053–1061. doi: 10.1163/15685411-00003014. DOI

Kawasaki I, Jeong M-H, Yun Y-J, Shin Y-K, Shim Y-H. Cholesterol-responsive metabolic proteins are required for larval development in Caenorhabditis elegans. Mol Cells. 2013;36:410–416. doi: 10.1007/s10059-013-0170-2. PubMed DOI PMC

Lev I, Bril R, Liu Y, Ceré LI, Rechavi O. Inter-generational consequences for growing Caenorhabditis elegans in liquid. Philosophical Transactions of the Royal Society B: Biological Sciences. 2019;374:20180125. doi: 10.1098/rstb.2018.0125. PubMed DOI PMC

Stiernagle, T. Maintenance of Caenorhabditis elegans in WormBook [Fay, D (ed)]. WormBook. (WormBook, 2006).

Agilent. Agilent Seahorse XF instruments overview and selection guide (2018).

SAS. SAS/STAT User’s Guide (SAS Institute, 2013).

Ghasemi A, Zahediasl S. Normality tests for statistical analysis: a guide for non-statisticians. Int. J. Endocrinol. Metab. 2012;10:486–489. doi: 10.5812/ijem.3505. PubMed DOI PMC