Environmental Science and Pollution Research

, Volume 21, Issue 18, pp 10803–10814 | Cite as

Comparative study of non-invasive methods for assessing Daphnia magna embryo toxicity

  • Matthew C. Stensberg
  • Michael Anthony Zeitchek
  • Kul Inn
  • Eric S. McLamore
  • D. Marshall Porterfield
  • Maria S. SepúlvedaEmail author
Research Article


Embryos, unlike adults, are typically sessile, which allows for an increase in the available metrics that can be used to assess chemical toxicity. We investigate Daphnia magna development rate and oxygen consumption as toxicity metrics and compare them to arrested embryo development using four different techniques with potassium cyanide (KCN) as a common toxicant. The EC50 (95 % CI) for arrested development was 2,535 (1,747–3,677) μg/L KCN. Using pixel intensity changes, recorded with difference imaging, we semi-quantitatively assessed a decrease in development rate at 200 μg/L KCN, threefold lower than the arrested development lowest observed effect concentration (LOEC). Respirometry and self-referencing (SR) microsensors were two unique techniques used to assess oxygen consumption. Using respirometry, an increase in oxygen consumption was found in the 5 μg/L KCN treatment and a decrease for 148 μg/L, but no change was found for the 78 μg/L KCN treatment. Whereas, with SR microsensors, we were able to detect significant changes in oxygen consumption for all three treatments: 5, 78, and 148 μg/L KCN. While SR offered the highest sensitivity, the respirometry platform developed for this study was much easier to use to measure the same endpoint. Oxygen consumption may be subject to change during the development process, meaning consumption assessment techniques may only be useful only for short-term experiments. Development rate was a more sensitive endpoint though was only reliable four of the six embryonic developmental stages examined. Despite being the least sensitive endpoint, arrested embryo development was the only technique capable of assessing the embryos throughout all developmental stages. In conclusion, each metric has advantages and limitations, but because all are non-invasive, it is possible to use any combination of the three.


Daphnia magna Arrested embryo development Difference imaging Respirometry Self-referencing sensing 



The authors would like to acknowledge Eric Karplus from Sciencewares, Inc. for assistance with the DVIT software. This work was supported by the National Science Foundation (CBET- 0854036).


  1. Abe T, Saito H, Niikura V, Shigeoka T, Nakano Y (2000) Embryonic development assay with Daphnia magna: application to toxicity of chlorophenols. Water Sci Technol 42(7–8):297–304Google Scholar
  2. Abe T, Saito H, Niikura Y, Shigeoka T, Nakano Y (2001) Embryonic development assay with Daphnia magna: application to toxicity of aniline derivatives. Chemosphere 45(4–5):487–495. doi: 10.1016/s0045-6535(01)00049-2 CrossRefGoogle Scholar
  3. Anderson BG (1946) The toxicity thresholds of various sodium salts determined by the use of Daphnia magna. Sew Works J 18(1):82–87Google Scholar
  4. Archibald F, Methot M, Young F, Paice MG (2001) A simple system to rapidly monitor activated sludge health and performance. Water Res 35(10):2543–2553. doi: 10.1016/s0043-1354(00)00542-x CrossRefGoogle Scholar
  5. Berg JM, Tymoczko JL, Stryer L (2007) Biochemistry, 6th edn. W. H. Freeman and Company, New YorkGoogle Scholar
  6. Cairns J, Heath AG, Parker BC (1975) The effects of temperature upon the toxicity of chemicals to aquatic organisms. Hydrobiologia 47(1):135–171. doi: 10.1007/bf00036747 CrossRefGoogle Scholar
  7. Chang DJ, Zubal IG, Gottschalk C, Necochea A, Stokking R, Studholme C, Corsi M, Slawski J, Spencer SS, Blumenfeld H (2002) Comparison of statistical parametric mapping and SPECT difference imaging in patients with temporal lobe epilepsy. Epilepsia 43(1):68–74. doi: 10.1046/j.1528-1157.2002.21601.x CrossRefGoogle Scholar
  8. Chatni MR, Porterfield DM (2009) Self-referencing optrode technology for non-invasive real-time measurement of biophysical flux and physiological sensing. Analyst 134(11):2224–2232. doi: 10.1039/b903092a CrossRefGoogle Scholar
  9. Chatni MR, Maier DE, Porterfield DM (2009) Evaluation of microparticle materials for enhancing the performance of fluorescence lifetime based optrodes. Sensors Actuators B Chem 141(2):471–477. doi: 10.1016/j.snb.2009.06.052 CrossRefGoogle Scholar
  10. Cheng J, Flahaut E, Cheng SH (2007) Effect of carbon nanotubes on developing zebrafish (Danio rerio) embryos. Environ Toxicol Chem 26(4):708–716. doi: 10.1897/06-272r.1 CrossRefGoogle Scholar
  11. Dodson SI, Caceres CE, Rogers DC (2010) Cladocera and other Branchiopoda. In: Thorp JH, Covich AP (eds) Ecology and classification of North American freshwater invertebrates. Aquatic ecology, 3rd edn. Academic Press, OxfordGoogle Scholar
  12. Echtay KS, Murphy MP, Smith RA, Talbot DA, Brand MD (2002) Superoxide activates mitochondrial uncoupling protein 2 from the matrix side. Studies using targeted antioxidants. J Biol Chem 277(49):47129–47135. doi: 10.1074/jbc.M208262200 CrossRefGoogle Scholar
  13. Embry MR, Belanger SE, Braunbeck TA, Galay-Burgos M, Halder M, Hinton DE, Leonard MA, Lillicrap A, Norberg-King T, Whale G (2010) The fish embryo toxicity test as an animal alternative method in hazard and risk assessment and scientific research. Aquat Toxicol 97(2):79–87. doi: 10.1016/j.aquatox.2009.12.008 CrossRefGoogle Scholar
  14. EPA US (1980) Ambient water quality criteria for cyanides. EPA US, WashingtonGoogle Scholar
  15. EPA US (2002) Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms, 5th edn. EPA US, WashingtonGoogle Scholar
  16. EPA US (2006) National recommended water quality criteria. EPA US, WashingtonGoogle Scholar
  17. Ewell WS, Gorsuch JW, Kringle RO, Robillard KA, Spiegel RC (1986) Simultaneous evaluation of the acute effects of chemicals on seven aquatic species. Environ Toxicol Chem 5(9):831. doi: 10.1897/1552-8618(1986)5[831:seotae];2 CrossRefGoogle Scholar
  18. Ferrell RT, Himmelblau DM (1967) Diffusion coefficients of nitrogen and oxygen in water. J Chem Eng Data 12(1):111–115. doi: 10.1021/je60032a036 CrossRefGoogle Scholar
  19. Folt CL, Chen CY, Moore MV, Burnaford J (1999) Synergism and antagonism among multiple stressors. Limnol Oceanogr 44(3):864–877CrossRefGoogle Scholar
  20. Forget-Leray J, Landriau I, Minier C, Leboulenger F (2005) Impact of endocrine toxicants on survival, development, and reproduction of the estuarine copepod Eurytemora affinis (Poppe). Ecotoxicol Environ Saf 60(3):288–294. doi: 10.1016/j.ecoenv.2004.06.008 CrossRefGoogle Scholar
  21. Good CD, Johnsrude IS, Ashburner J, Henson RN, Friston KJ, Frackowiak RS (2001) A voxel-based morphometric study of ageing in 465 normal adult human brains. NeuroImage 14(1 Pt 1):21–36. doi: 10.1006/nimg.2001.0786 CrossRefGoogle Scholar
  22. Gopalakrishnan S, Thilagam H, Raja PV (2007) Toxicity of heavy metals on embryogenesis and larvae of the marine sedentary polychaete Hydroides elegans. Arch Environ Contam Toxicol 52(2):171–178. doi: 10.1007/s00244-006-0038-y CrossRefGoogle Scholar
  23. Gray MA, Teather KL, Metcalfe CD (1999) Reproductive success and behavior of Japanese medaka (Oryzias latipes) exposed to 4-tert-octylphenol. Environ Toxicol Chem 18(11):2587. doi: 10.1897/1551-5028(1999)018<2587:rsaboj>;2 Google Scholar
  24. Handy RD, Depledge MH (1999) Physiological responses: their measurement and use as environmental biomarkers in ecotoxicology. Ecotoxicology 8(5):329–349. doi: 10.1023/a:1008930404461 CrossRefGoogle Scholar
  25. Heugens EH, Tokkie LT, Kraak MH, Hendriks AJ, Van Straalen NM, Admiraal W (2006) Population growth of Daphnia magna under multiple stress conditions: joint effects of temperature, food, and cadmium. Environ Toxicol Chem 25(5):1399–1407. doi: 10.1897/05-294r.1 CrossRefGoogle Scholar
  26. Huang RS, Lin CJ, Isaacs HS (2006) A difference-imaging technique used to study streaking corrosion of aluminum alloys AA7075 and AA8006 in chloride solution. Electrochem Solid St 9(2):B11–B14. doi: 10.1149/1.2140503 CrossRefGoogle Scholar
  27. Jaafarzadeh N, Hashempour Y, Ahmadi Angali K (2013) Acute toxicity test using cyanide on Daphnia magna by flow-through system. J Water Chem Technol 35(6):281–286. doi: 10.3103/s1063455x13060076 CrossRefGoogle Scholar
  28. Kast-Hutcheson K, Rider CV, LeBlanc GA (2001) The fungicide propiconazole interferes with embryonic development of the crustacean Daphnia magna. Environ Toxicol Chem 20(3):502–509CrossRefGoogle Scholar
  29. Khangarot BS, Das S (2009) Toxicity of mercury on in vitro development of parthenogenetic eggs of a freshwater cladoceran Daphnia carinata. J Hazard Mater 161(1):68–73. doi: 10.1016/j.jhazmat.2008.03.068 CrossRefGoogle Scholar
  30. Kuhtreiber WM, Jaffe LF (1990) Detection of extracellular calcium gradients with a calcium-specific vibrating electrode. J Cell Biol 110(5):1565–1573. doi: 10.1083/jcb.110.5.1565 CrossRefGoogle Scholar
  31. Kungolos A, Hadjispirou S, Petala M, Tsiridis V, Samaras P, Sakellaropoulos GP (2003) Toxic properties of cyanide, chromium and organotin compounds and their interactions on Daphnia magna. In: Lekkas TD (ed) Proc Int Conf Env Sc. Proceedings of the International Conference on Environmental Science and Technology. Univ Aegean, Athens, pp 515–522Google Scholar
  32. Laban G, Nies LF, Turco RF, Bickham JW, Sepulveda MS (2010) The effects of silver nanoparticles on fathead minnow (Pimephales promelas) embryos. Ecotoxicology 19(1):185–195. doi: 10.1007/s10646-009-0404-4 CrossRefGoogle Scholar
  33. LeBlanc GA, Mu X, Rider CV (2000) Embryotoxicity of the alkylphenol degradation product 4-nonylphenol to the crustacean Daphnia magna. Environ Health Perspect 108(12):1133–1138CrossRefGoogle Scholar
  34. Lussier SM, Gentile JH, Walker J (1985) Acute and chronic effects of heavy-metals and cyanide on Mysidopsis bahia (Crustacea, Mysidacea). Aquat Toxicol 7(1–2):25–35. doi: 10.1016/0166-445x(85)90034-7 CrossRefGoogle Scholar
  35. Martins JC, Saker ML, Teles LFO, Vasconcelos VM (2007) Oxygen consumption by Daphnia magna Straus as a marker of chemical stress in the aquatic environment. Environ Toxicol Chem 26(9):1987–1991. doi: 10.1897/07-051r.1 CrossRefGoogle Scholar
  36. McLamore ES, Porterfield DM, Banks MK (2009) Non-invasive self-referencing electrochemical sensors for quantifying real-time biofilm analyte flux. Biotechnol Bioeng 102(3):791–799. doi: 10.1002/bit.22128 CrossRefGoogle Scholar
  37. McLamore ES, Diggs A, Calvo Marzal P, Shi J, Blakeslee JJ, Peer WA, Murphy AS, Porterfield DM (2010a) Non-invasive quantification of endogenous root auxin transport using an integrated flux microsensor technique. Plant J 63(6):1004–1016. doi: 10.1111/j.1365-313X.2010.04300.x CrossRefGoogle Scholar
  38. McLamore ES, Jaroch D, Chatni MR, Porterfield DM (2010b) Self-referencing optrodes for measuring spatially resolved, real-time metabolic oxygen flux in plant systems. Planta 232(5):1087–1099. doi: 10.1007/s00425-010-1234-6 CrossRefGoogle Scholar
  39. McLamore ES, Stensberg M, Yale G, Ochoa-Acuna H, Sepulveda M, Sun X, Akkus O, Porterfield DM (2010c) A difference imaging technique for monitoring real time changes in morphology within the cell, tissue, and organism spatial domain. In: Cullum BM, Porterfield DM, Booksh KS (eds) Smart biomedical and physiological sensor technologies Vii, vol 7674. Proceedings of SPIE. doi: 10.1117/12.851694
  40. McLamore ES, Garland JL, Mackowiak C, Desaunay A, Garland N, Chaturvedi P, Taguchi M, Dreaden K, Catechis J, Ullman JL (2014) Development and validation of an open source O2-sensitive gel for physiological profiling of microbial communities. J Micro Meth 96:62–67CrossRefGoogle Scholar
  41. Mu X, LeBlanc GA (2002a) Environmental antiecdysteroids alter embryo development in the crustacean Daphnia magna. J Exp Zool 292(3):287–292. doi: 10.1002/jez.10020 CrossRefGoogle Scholar
  42. Mu XY, LeBlanc GA (2002b) Developmental toxicity of testosterone in the crustacean Daphnia magna involves anti-ecdysteroidal activity. Gen Comp Endocrinol 129(2):127–133. doi: 10.1016/s0016-6480(02)00518-x CrossRefGoogle Scholar
  43. Ohta T, Tokishita S, Shiga Y, Hanazato T, Yamagata H (1998) An assay system for detecting environmental toxicants with cultured cladoceran eggs in vitro: malformations induced by ethylenethiourea. Environ Res 77(1):43–48. doi: 10.1006/enrs.1997.3783 CrossRefGoogle Scholar
  44. O’Mahony FC, O’Donovan C, Hynes J, Moore T, Davenport J, Papkovsky DB (2005) Optical oxygen microrespirometry as a platform for environmental toxicology and animal model studies. Environ Sci Technol 39(13):5010–5014. doi: 10.1021/es048279 CrossRefGoogle Scholar
  45. Palma P, Palma VL, Fernandes RM, Bohn A, Soares A, Barbosa IR (2009a) Embryo-toxic effects of environmental concentrations of chlorpyrifos on the crustacean Daphnia magna. Ecotox Environ Safe 72(6):1714–1718. doi: 10.1016/j.ecoenv.2009.04.026 CrossRefGoogle Scholar
  46. Palma P, Palma VL, Matos C, Fernandes RM, Bohn A, Soares AM, Barbosa IR (2009b) Effects of atrazine and endosulfan sulphate on the ecdysteroid system of Daphnia magna. Chemosphere 74(5):676–681. doi: 10.1016/j.chemosphere.2008.10.021 CrossRefGoogle Scholar
  47. Porterfield DM (2007) Measuring metabolism and biophysical flux in the tissue, cellular and sub-cellular domains: recent developments in self-referencing amperometry for physiological sensing. Biosens Bioelectron 22(7):1186–1196. doi: 10.1016/j.bios.2006.06.006 CrossRefGoogle Scholar
  48. Prabhakaran K, Li L, Borowitz JL, Isom GE (2002) Cyanide induces different modes of death in cortical and mesencephalon cells. J Pharmacol Exp Ther 303(2):510–519. doi: 10.1124/jpet.102.039453 CrossRefGoogle Scholar
  49. Roex EWM, Van Gestel CAM, Van Wezel AP, Van Straalen NM (2000) Ratios between acute aquatic toxicity and effects on population growth rates in relation to toxicant mode of action. Environ Toxicol Chem 19(3):685. doi: 10.1897/1551-5028(2000)019<0685:rbaata>;2 CrossRefGoogle Scholar
  50. Sanchez BC, Ochoa-Acuna H, Porterfield DM, Sepulveda MS (2008) Oxygen flux as an indicator of physiological stress in fathead minnow (Pimephales promelas) embryos: a real-time biomonitoring system of water quality. Environ Sci Technol 42(18):7010–7017. doi: 10.1021/Es702879t CrossRefGoogle Scholar
  51. Sawyer PL, Heath AG (1988) Cardiac, ventilatory and metabolic responses of two ecologically dissimilar species of fish to waterborne cyanide. Fish Physiol Biochem 4(4):203–219. doi: 10.1007/BF01871746 CrossRefGoogle Scholar
  52. Scherer C, Seeland A, Oehlmann J, Muller R (2013) Interactive effects of xenobiotic, abiotic and biotic stressors on Daphnia pulex—results from a multiple stressor experiment with a fractional multifactorial design. Aquat Toxicol 138–139:105–115. doi: 10.1016/j.aquatox.2013.04.014 CrossRefGoogle Scholar
  53. Sobral O, Chastinet C, Nogueira A, Soares A, Goncalves F, Ribeiro R (2001) In vitro development of parthenogenetic eggs: a fast ecotoxicity test with Daphnia magna? Ecotox Environ Safe 50(3):174–179. doi: 10.1006/eesa.2001.2088 CrossRefGoogle Scholar
  54. Soetaert A, Moens LN, Van der Ven K, Van Leemput K, Naudts B, Blust R, De Coen WM (2006) Molecular impact of propiconazole on Daphnia magna using a reproduction-related cDNA array. Comp Biochem Physiol C Toxicol Pharmacol 142(1–2):66–76. doi: 10.1016/j.cbpc.2005.10.009 CrossRefGoogle Scholar
  55. Soetaert A, Vandenbrouck T, van der Ven K, Maras M, van Remortel P, Blust R, de Coen WM (2007) Molecular responses during cadmium-induced stress in Daphnia magna: integration of differential gene expression with higher-level effects. Aquat Toxicol 83(3):212–222. doi: 10.1016/j.aquatox.2007.04.010 CrossRefGoogle Scholar
  56. Stensberg MC, Wei Q, McLamore ES, Porterfield DM, Wei A, Sepulveda MS (2011) Toxicological studies on silver nanoparticles: challenges and opportunities in assessment, monitoring and imaging. Nanomedicine (Lond) 6(5):879–898. doi: 10.2217/nnm.11.78 CrossRefGoogle Scholar
  57. Stensberg MC, Madangopal R, Yale G, Wei Q, Ochoa-Acuna H, Wei A, McLamore ES, Rickus J, Porterfield DM, Sepulveda MS (2014) Silver nanoparticle-specific mitotoxicity in Daphnia magna. Nanotoxicology 8(8):833–842. doi: 10.3109/17435390.2013.832430 CrossRefGoogle Scholar
  58. Szela TL, Marsh AG (2005) Microtiter plate, optode respirometry, and inter-individual variance in metabolic rates among nauplii of Artemia sp. Mar Ecol Prog Ser 296:281–289. doi: 10.3354/meps296281 CrossRefGoogle Scholar
  59. Ton SS, Chang SH, Hsu LY, Wang MH, Wang KS (2012) Evaluation of acute toxicity and teratogenic effects of disinfectants by Daphnia magna embryo assay. Environ Pollut 168:54–61. doi: 10.1016/j.envpol.2012.04.008 CrossRefGoogle Scholar
  60. Toumi H, Boumaiza M, Millet M, Radetski CM, Felten V, Fouque C, Ferard JF (2013) Effects of deltamethrin (pyrethroid insecticide) on growth, reproduction, embryonic development and sex differentiation in two strains of Daphnia magna (Crustacea, Cladocera). Sci Total Environ 458–460:47–53. doi: 10.1016/j.scitotenv.2013.03.085 CrossRefGoogle Scholar
  61. Warnau M, Iaccarino M, DeBiase A, Temara A, Jangoux M, Dubois P, Pagano G (1996) Spermiotoxicity and embryotoxicity of heavy metals in the echinoid Paracentrotus lividus. Environ Toxicol Chem 15(11):1931–1936. doi: 10.1897/1551-5028(1996)015<1931:Saeohm>2.3.Co;2 CrossRefGoogle Scholar
  62. Wiench K, Wohlleben W, Hisgen V, Radke K, Salinas E, Zok S, Landsiedel R (2009) Acute and chronic effects of nano- and non-nano-scale TiO2 and ZnO particles on mobility and reproduction of the freshwater invertebrate Daphnia magna. Chemosphere 76(10):1356–1365. doi: 10.1016/j.chemosphere.2009.06.025 CrossRefGoogle Scholar
  63. Wozniak PR, Alard C, Udalski A, Szymanski M, Kubiak M, Pietrzynski G, Zebrun K (2000) The optical gravitational lensing experiment monitoring of QSO 22371+0305. Astrophys J 529(1):88–92. doi: 10.1086/308258 CrossRefGoogle Scholar
  64. Zhang L, Gibble R, Baer KN (2003) The effects of 4-nonylphenol and ethanol on acute toxicity, embryo development, and reproduction in Daphnia magna. Ecotox Environ Safe 55(3):330–337. doi: 10.1016/s0147-6513(02)00081-7 CrossRefGoogle Scholar
  65. Zitova A, O’Mahony FC, Cross M, Davenport J, Papkovsky DB (2009) Toxicological profiling of chemical and environmental samples using panels of test organisms and optical oxygen respirometry. Environ Toxicol 24(2):116–127. doi: 10.1002/tox.20387 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Matthew C. Stensberg
    • 1
    • 2
  • Michael Anthony Zeitchek
    • 1
    • 2
  • Kul Inn
    • 1
    • 2
  • Eric S. McLamore
    • 3
  • D. Marshall Porterfield
    • 1
    • 2
  • Maria S. Sepúlveda
    • 2
    • 4
    Email author
  1. 1.Department of Agriculture and Biological EngineeringPurdue UniversityWest LafayetteUSA
  2. 2.Birck-Bindley Physiological Sensing FacilityPurdue UniversityWest LafayetteUSA
  3. 3.Agricultural and Biological Engineering DepartmentUniversity of FloridaGainesvilleUSA
  4. 4.Department of Forestry and Natural ResourcesPurdue UniversityWest LafayetteUSA

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