Skip to main content

Screening the Toxicity and Biodegradability of Petroleum Hydrocarbons by a Rapid Colorimetric Method

Archives of Environmental Contamination and Toxicology volume 68, pages 342–353 (2015)Cite this article

Abstract

Crude oil and petroleum products have a wide variety of hazardous components with high toxicity and low biodegradability. Certain dyes change their colors by intercepting electron transfer reactions during the transformation processes. This study applied resazurin and 2,6-dichlorophenol-indophenol indicators for a rapid screening biodegradation capability and toxicity response to various petroleum products such as motor oil, diesel, gasoline, and phenol. Colorimetry tests were performed in test tubes, and the absorbance values were measured over time. We observed different discoloration profiles after degradation tests using Bacillus subtilis inoculum. Phytotoxicity assays were also performed to compare colorimetric screening assays with a conventional toxicity testing with plants (seed germination). The results indicated that biotransformation of oils can increase its overall toxicity. Intermediate byproducts can be formed through biodegradation and thereby increase the toxicity of oils. The assessment of acute toxicity has shown that phenol is extremely toxic to petroleum-biodegrading microbial communities. Low molecular-weight gasoline was considered biodegradable, but it also exhibited a high acute toxic effect, mainly due to its high solubility and the presence of more volatile compounds that can penetrate cells and potentially damage cellular structures.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

USD 39.95

Price includes VAT (USA)
Tax calculation will be finalised during checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  • Aas E, Baussant T, Balk L, Liewenborg B, Andersen OK (2000) PAH metabolites in bile, cytochrome P4501A and DNA adducts as environmental risk parameters for chronic oil exposure: a laboratory experiment with Atlantic cod. Aquat Toxicol 51:241–258

    CAS  Article  Google Scholar 

  • Aksmann A, Tukaj Z (2008) Intact anthracene inhibits photosynthesis in algal cells: a fluorescence induction study on Chlamydomonas reinhardtii cw92 strain. Chemosphere 74:26–32

    CAS  Article  Google Scholar 

  • Attiogbe FK, Glover-Amengor M, Nyadziehe KT (2007) Correlating biochemical and chemical oxygen demand of effluents—a case study of selected industries in Kumasi, Ghana. West Afr J Appl Ecol 11:110–118

    Google Scholar 

  • Ayers RS, Westcot DWA (1999) Qualidade da água na agricultura. UFPB, Campina Grande

    Google Scholar 

  • Barron MG, Podrabsky T, Ogle S, Ricker RW (1999) Are aromatic hydrocarbons the primary determinant of petroleum toxicity to aquatic organisms? Aquat Toxicol 46:253–268

    CAS  Article  Google Scholar 

  • Belpoggi F, Soffritti M, Maltoni C (1995) Methyl-tertiary-butyl ether (MTBE)—a gasoline additive—causes testicular and lymphohematopoietic cancers in rats. Toxicol Ind Health 11:119–149

    CAS  Article  Google Scholar 

  • Bentley A, Atkinson A, Jezek J, Rawson DM (2001) Whole cell biosensors—electrochemical and optical approaches to ecotoxicity testing. Toxicol In Vitro 15:469–475

    CAS  Article  Google Scholar 

  • Bishop E (1972) Indicators. Pergamon, Oxford

    Google Scholar 

  • Bopp SK, Lettieri T (2007) Gene regulation in the marine diatom Thalassiosira pseudonana upon exposure to polycyclic aromatic hydrocarbons (PAHs). Gene 396:293–302

    CAS  Article  Google Scholar 

  • Bossert I, Bartha R (1984) The fate of petroleum in soil ecosystems. In: Atlas RM (ed) Petroleum microbiology. Macmillan, New York, pp 435–474

  • Brock TD, Madigan MT, Martinko JM, Parker J (1994) Biology of microorganisms. Pearson Education, San Francisco

    Google Scholar 

  • Byth HA, McHunu BI, Dubery IA, Bornman L (2001) Assessment of a simple, non-toxic Alamar blue cell survival assay to monitor tomato cell viability. Phytochem Anal 12:340–346

    CAS  Article  Google Scholar 

  • Cai Z, Zhou Q, Peng S, Li (2010) Promoted biodegradation and microbiological effects of petroleum hydrocarbon by Impatiens balsamina with strong endurance. J Hazard Mater 183:731–737

    CAS  Article  Google Scholar 

  • Carls MG, Holland L, Larsen M, Collier TK, Scholz NL, Incardona JP (2008) Fish embryos are damaged by dissolved PAHs, not oil particles. Aquat Toxicol 88:121–127

    CAS  Article  Google Scholar 

  • Chapelle FH (1984) Groundwater microbiology and geochemistry. Wiley, New York

    Google Scholar 

  • Charles J, Sancey B, Morin-Crini N, Badot P, Degiorgi F, Trunfio G et al (2011) Evaluation of the phytotoxicity of polycontaminated industrial effluents using the lettuce plant (Lactuca sativa) as a bioindicator. Ecotoxicol Environ Saf 74:2057–2064

    CAS  Article  Google Scholar 

  • Chen H, Juang R (2008) Recovery and separation of surfactin from pretreated fermentation broths by physical and chemical extraction. Biochem Eng J 28:39–46

    Article  Google Scholar 

  • Choi H, Rauh V, Garfinkel R, Tu Y, Perera FP (2008) Prenatal exposure to airborne polycyclic aromatic hydrocarbons and risk of intrauterine growth restriction. Environ Health Perspect 116:658–665

    CAS  Article  Google Scholar 

  • Culbertson JB, Valiela I, Pickart M, Peacock EE, Reddy CM (2008) Long-term consequences of residual petroleum on salt marsh grass. J Appl Ecol 45:1284–1292

    Article  Google Scholar 

  • Dawson RMC, Elliott DC, Elliott WH, Jones KM (1986) Data for biochemical research. Oxford Science, Oxford

    Google Scholar 

  • Dehghan-Noudeh G, Housaindokht M, Bazzaz BSF (2005) Isolation, characterization, and investigation of surface and hemolytic activities of a lipopeptide biosurfactant produced by Bacillus subtilis ATCC 6633. J Microbiol 43:272–276

    Google Scholar 

  • Difco (1984) Difco manual. Difco, Detroit

    Google Scholar 

  • Du J, Mehler WT, Lydy MJ, You J (2012) Toxicity of sediment-associated unresolved complex mixture and its impact on bioavailability of polycyclic aromatic hydrocarbons. J Hazard Mater 203:169–175

    Article  Google Scholar 

  • Dutka B (1989) Short-term root elongation toxicity bioassay—methods for toxicological analysis of waters, wastewaters and sediments. NWRI, Ontario

    Google Scholar 

  • Ertürk MD, Saçan MT (2013) Assessment and modeling of the novel toxicity data set of phenols to Chlorella vulgaris. Ecotoxicol Environ Saf 90:61–68

    Article  Google Scholar 

  • Euliss K, Ho CH, Schwab AP, Rock S, Banks AK (2008) Greenhouse and field assessment of phytoremediation for petroleum contaminants in a riparian zone. Bioresour Technol 99:1961–1971

    CAS  Article  Google Scholar 

  • Faksness LG, Brandvik PJ, Sydnes LK (2008) Composition of the water accommodated fractions as a function of exposure times and temperatures. Mar Pollut Bull 56:1746–1754

    CAS  Article  Google Scholar 

  • Feuston MH, Low LK, Hamilton CE, Mackerer CR (1994) Correlation of systemic and developmental toxicities with chemical component classes of refinery streams. Fund Appl Toxicol 22:622–630

    CAS  Article  Google Scholar 

  • Foght JM, Westlake DWS (1987) Biodegradation of hydrocarbons in freshwater. In: Vandermeulen JH, Hrudey SR (eds) Oil in freshwater: chemistry, biology, countermeasure technology. Pergamon Press, New York, pp 217–230

  • Gamo M, Oka T, Nakanishi J (2003) Ranking the risk of 12 major environmental pollutants that occur in Japan. Chemosphere 53:277–284

    CAS  Article  Google Scholar 

  • Gong G, Zheng Z, Chen H, Yuan C, Wang P, Yao L et al (2009) Enhanced production of surfactin by Bacillus subtilis E8 mutant obtained by ion beam implantation. Food Technol Biotechnol 47:23–31

    Google Scholar 

  • Guerin TF, Mondido M, McClenn B, Peasley B (2001) Application of resazurin for estimating abundance of contaminant-degrading micro-organisms. Lett Appl Microbiol 32:340–345

    CAS  Article  Google Scholar 

  • Gustavson KE, Sonsthagen SA, Crunkilton RA, Harkin JM (2000) Groundwater toxicity assessment using bioassay, chemical, and toxicity identification evaluation analysis. Environ Toxicol 15:421–430

    CAS  Article  Google Scholar 

  • Haigh DS (1995) Fate and effects of synthetic lubricants in soil: biodegradation and effect on crops in field studies. Sci Total Environ 168:71–83

    CAS  Article  Google Scholar 

  • Hanson KG, Desai JD, Desai AJ (1993) A rapid and simple screening technique for potential crude oil degrading microorganisms. Biotechnol Technol 7:745–748

    CAS  Article  Google Scholar 

  • Harwood AD, Landrum PF, Lydy MJ (2013) Bioavailability-based toxicity endpoints of bifenthrin for Hyalella azteca and Chironomus dilutus. Chemosphere 90:1117–1122

    CAS  Article  Google Scholar 

  • Heiskanen A, Yakovleva J, Spegel C, Taboryski R, Koudelka-Hep M, Emneus J et al (2004) Amperometric monitoring of redox activity in living cells: comparison of menadione and menadione sodium bisulfite as electron transfer mediators. Electrochem Commun 6:219–224

    CAS  Article  Google Scholar 

  • Hesterberg T, Bunn WB (2006) A critical assessment of studies on the carcinogenic potential of diesel exhaust. Crit Rev Toxicol 36:727–776

    CAS  Article  Google Scholar 

  • Hu G, Li J, Zeng G (2013) Recent development in the treatment of oily sludge from petroleum industry: a review. J Hazard Mater 261:470–490

    CAS  Article  Google Scholar 

  • Huesemann MH (1995) Predictive model for estimating the extent of petroleum hydrocarbon biodegradation in contaminated soils. Environ Sci Technol 29:7–18

    CAS  Article  Google Scholar 

  • Hutchins SR, Sewell GW, Kovacs DA, Smith GA (1991) Biodegradation of aromatic hydrocarbons by aquifer microorganisms under denitrifying conditions. Environ Sci Technol 25:68–76

    CAS  Article  Google Scholar 

  • Incardona JP, Collier TK, Scholz NL (2004) Defects in cardiac function precede morphological abnormalities in fish embryos exposed to polycyclic aromatic hydrocarbons. Toxicol Appl Pharmacol 196:191–205

    CAS  Article  Google Scholar 

  • Jedrychowski W, Perera FP, Tang D, Stigter L, Mroz E, Flak E et al (2012) Impact of barbequed meat consumed in pregnancy on birth outcomes accounting for personal prenatal exposure to airborne polycyclic aromatic hydrocarbons: birth cohort study in Poland. Nutrition 28:372–377

    CAS  Article  Google Scholar 

  • Kavitha V, Palanivelu K (2004) The role of ferrous ion in Fenton and photo-Fenton processes for the degradation of phenol. Chemosphere 55:1235–1243

    CAS  Article  Google Scholar 

  • Khan RA, Ryan P (1991) Long term effects of crude oil on common murres (Uria aalge) following rehabilitation. Bull Environ Contam Toxicol 46:216–222

    CAS  Article  Google Scholar 

  • Konidari CN, Tzouware SM, Bowman LE, Karayannis MI (1992) Kinetic and mechanistic study of the reaction of 2,6-dichlorophenol-indophenol and cysteine. Talanta 39:863–868

    CAS  Article  Google Scholar 

  • Labouriau LG, Agudo M (1987) On the physiology of seed germination in Salvia hispanica L. I. temperature effects. An Acad Bras Ciênc 59:37–56

    Google Scholar 

  • Liste HH, Felgentreu D (2006) Crop growth, culturable bacteria, and degradation of petrol hydrocarbons (PHCs) in a long-term contaminated field soil. Appl Soil Ecol 31:43–52

    Article  Google Scholar 

  • Luo W, Verweij RA, Gestel CAM (2014) Determining the bioavailability and toxicity of lead contamination to earthworms requires using a combination of physicochemical and biological methods. Environ Pollut 185:1–9

    CAS  Article  Google Scholar 

  • Mariscal A, Lopez-Gigosos RM, Carnero-Varo M, Fernandez-Crehuet J (2009) Fluorescent assay based on resazurin for detection of activity of disinfectants against bacterial biofilm. Appl Microbiol Biotechnol 82:773–783

    CAS  Article  Google Scholar 

  • Martienssen M, Fabritius H, Kukla S, Balcke GU, Hasselwander E, Schirmer M (2006) Determination of naturally occurring MTBE biodegradation by analysing metabolites and biodegradation by-products. J Contam Hydrol 87:37–53

    CAS  Article  Google Scholar 

  • McClellan RO (1986) Health-effects of diesel exhaust—a case-study in risk assessment. J Occup Environ Hyg 47:1–13

    CAS  Google Scholar 

  • Moore SF, Dwyer RF (1974) Effects of oil on marine organisms a critical assessment of published data. Water Res 8:819–827

    CAS  Article  Google Scholar 

  • Morales CG (2004) Ensayos toxicológicos y métodos de evaluación de calidad de agua: Estandarización, intercalibración, resultados y aplicaciones. IMTA, Ciudad de Mexico

    Google Scholar 

  • Myers MS, Landahl JT, Krahn MM, McCain BB (1991) Relationships between hepatic neoplasms and related lesions and exposure to toxic chemicals in marine fish from the United States west coast. Environ Health Perspect 90:7–15

    CAS  Article  Google Scholar 

  • Neff JM (2002) Bioaccumulation in marine organisms. Effect of contaminants from oil well produced water. London, Elsevier

    Google Scholar 

  • Nicolich MJ, Simpson BJ, Murray FJ, Roth RN, Gray TM (2013) The development of statistical models to determine the relationship between aromatic-ring class profile and repeat-dose and developmental toxicities of high-boiling petroleum substances. Regul Toxicol Pharm 67:10–29

    Article  Google Scholar 

  • Nitschke M, Ferraz C, Pastore GM (2004) Selection of micro-organisms for biosurfactant production using agroindustrial wastes. Braz J Microbiol 35:81–85

    Article  Google Scholar 

  • O’Brien J, Wilson I, Orton T, Pognan F (2000) Investigation of the Alamar blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 267:5421–5426

    Article  Google Scholar 

  • Okoh AI, Trejo-Hernandez MR (2006) Remediation of petroleum hydrocarbon polluted systems: exploiting the bioremediation strategies. Afr J Biotechnol 5:2520–2525

    CAS  Google Scholar 

  • Oluwole OS, Makinde SCO, Philips DA (2005) The impact of spent engine oil pollution on the growth of Celosia argentea. Lagos State University Press, Ojo

    Google Scholar 

  • Paixão JF, Nascimento IA, Pereira SA, Leite MBL, Carvalho GC, Silveira JSC et al (2007) Estimating the gasoline components and formulations toxicity to microalgae (Tetraselmis chuii) and oyster (Crassostrea rhizophorae) embryos: an approach to minimize environmental pollution risk. Environ Res 103:365–374

    Article  Google Scholar 

  • Pardeshi SK, Patil AB (2008) A simple route for photocatalytic degradation of phenol in aqueous zinc oxide suspension using solar energy. Sol Energy 82:700–705

    CAS  Article  Google Scholar 

  • Pasco N, Baronian KH, Jeffries C, Hay J (2000) Biochemical mediator demand—a novel rapid alternative for measuring biochemical oxygen demand. J Microbiol Biotechnol 53:613–618

    CAS  Article  Google Scholar 

  • Poulton SW, Krom MD, Rijn JV, Raiswell R (2002) The use of hydrous iron(III) oxides for the removal of hydrogen sulphide in aqueous systems. Water Res 36:825–834

    CAS  Article  Google Scholar 

  • Prince RC, Walters CC (2007) Biodegradation of oil hydrocarbons and its implications for source identification. Academic, Toronto

    Google Scholar 

  • Roy S, Hens D, Biswas D, Kumar R (2002) Survey of petroleum-degrading bacteria in coastal waters of Sunderban Biosphere Reserve. World J Microbiol Biotechnol 18:575–581

    CAS  Article  Google Scholar 

  • Salanitro JP, Dorn PB, Huesemann MH, Moore KO, Rhodes LM, Jackson R et al (1997) Crude oil hydrocarbon bioremediation and soil ecotoxicity assessment. Environ Sci Technol 31:1769–1776

    CAS  Article  Google Scholar 

  • Sancey B, Trunfio G, Charles J, Badot PM, Crini G (2011) Sorption onto crosslinked cyclodextrin polymers for industrial pollutants removal: an interesting environmental approach. J Inclusion Phenom Macrocycl Chem 69:377–382

    Article  Google Scholar 

  • Schmidt TC, Morgenroth E, Schirmer M, Effenberger M, Haderlein SB (2002) Oxygenates in gasoline: environmental aspects. ACS, Washington

    Google Scholar 

  • Shiloh MU, Ruan J, Nathan C (1997) Evaluation of bacterial survival and phagocyte function with a fluorescence-based microplate assay. Infect Immunol 65:3193–3198

    CAS  Google Scholar 

  • Sikkema J, Debont JAM, Poolman B (1995) Mechanisms of membrane toxicity of hydrocarbons. Microbiol Rev 59:201–222

    CAS  Google Scholar 

  • Skládal P, Morozova NO, Reshetilov AN (2002) Amperometric biosensors for detection of phenol using chemically modified electrodes containing immobilized bacteria. Biosens Bioelectron 17:867–873

    Article  Google Scholar 

  • Skotti E, Anastasaki E, Kanellou G, Polissiou M, Tarantilis PA (2014) Total phenolic content, antioxidant activity and toxicity of aqueous extracts from selected Greek medicinal and aromatic plants. Ind Crop Prod 53:46–54

    CAS  Article  Google Scholar 

  • Smith CF, Townsend DE (1999) A new medium for determining the total plate count in food. J Food Prot 62:1404–1410

    CAS  Google Scholar 

  • Sreenivasan PK, Tambs G, Gittins E, Nabi N, Gaffar A (2003) A rapid procedure to ascertain the antimicrobial efficacy of oral care formulations. Oral Microbiol Immunol 18:371–378

    CAS  Article  Google Scholar 

  • Sturve J, Hasselberg L, Falth H, Celander M, Forlin L (2006) Effects of North Sea oil and alkylphenols on biomarker responses in juvenile Atlantic cod (Gadus morhua). Aquat Toxicol 78:73–78

    Article  Google Scholar 

  • Sun PY, Gao ZH, Cui WL (2007) Development and application of the oil finger identification. Ocean Press, Beijing

    Google Scholar 

  • Tang J, Lu X, Sun Q, Zhu W (2012) Aging effect of petroleum hydrocarbons in soil under different attenuation conditions. Agric Ecosyst Environ 149:109–117

    CAS  Article  Google Scholar 

  • Timur S, Pazarlio N, Pilloton R, Telefoncu A (2003) Detection of phenolic compounds by thick film sensors based on Pseudomonas putida. Talanta 61:87–93

    CAS  Article  Google Scholar 

  • United States Environmental Protection Agency (USEPA) (2003) Low emission nonroad diesel engines and fuel—proposed rule. Air Quality, Health, and Welfare Effects, USAEPA

  • Valberg PA, Watson AY (1996) Analysis of diesel-exhaust unit-risk estimates derived from animal bioassays. Regul Toxicol Pharm 24:30–44

    CAS  Article  Google Scholar 

  • Valerio ME, García JF, Peinado FM (2007) Determination of phytotoxicity of soluble elements in soils based on a bioassay with lettuce (Lactuca sativa). Sci Total Environ 378:63–66

    CAS  Article  Google Scholar 

  • Wang L, Zheng B, Meng W (2008) Photo-induced toxicity of four polycyclic aromatic hydrocarbons, singly and in combination, to the marine diatom Phaeodactylum tricornutum. Ecotoxicol Environ Saf 71:465–472

    CAS  Article  Google Scholar 

  • Wilkinson S, Klar J, Applegarth SP (2006) Optimizing biofuel cell performance using a targeted mixed mediator combination. Electroanalysis 18:2001–2007

    CAS  Article  Google Scholar 

  • Xu X, Zhu X (2004) Treatment of refectory oily wastewater by electro-coagulation process. Chemosphere 56:889–894

    CAS  Article  Google Scholar 

  • Xu S, Song J, Yuan H, Li X, Li N, Duan L et al (2011) Petroleum hydrocarbons and their effects on fishery species in the Bohai Sea, North China. J Environ Sci China 23:553–559

    CAS  Article  Google Scholar 

  • Yang S, Zhu W, Wang J, Chen Z (2008) Catalytic wet air oxidation of phenol over CeO2–TiO2 catalyst in the batch reactor and the packed-bed reactor. J Hazard Mater 153:1248–1253

    CAS  Article  Google Scholar 

  • Yari A, Kargosha K (2006) Simple photometric determination of free cyanide ion in aqueous solution with 2,6-dichlorophenolindophenol. Eur J Chem 4:329–337

    CAS  Google Scholar 

  • Yoshida N, Hoashi J, Morita T, McNiven SJ, Nakamura H, Karube I (2001) Improvement of a mediator-type biochemical oxygen demand sensor for on-site measurement. J Biotechnol 88:269–275

    CAS  Article  Google Scholar 

  • Zhao J, Wang M, Yang Z, Wang Z, Wang H, Yang Z (2007) The different behaviors of three oxidative mediators in probing the redox activities of the yeast Saccharomyces cerevisiae. Anal Chim Acta 597:67–74

    CAS  Article  Google Scholar 

Download references

Acknowledgments

Our research group acknowledges Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Fundação para o Desenvolvimento da UNESP, Programa de Formação de Recursos Humanos em Geologia do Petróleo e Ciências Ambientais Aplicadas ao Setor de Petróleo & Gás e de Biocombustíveis, and Sao Paulo State University (UNESP) for financial support.

Author information

Authors and Affiliations

  1. Departamento de Bioquímica e Microbiologia, Instituto de Biociências, UNESP – University Estadual Paulista, Avenida 24 A, 1515 – Bela Vista, Rio Claro, SP, CEP 13506-900, Brazil

    Renato Nallin Montagnolli, Paulo Renato Matos Lopes & Ederio Dino Bidoia

Authors
  1. Renato Nallin Montagnolli
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paulo Renato Matos Lopes
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ederio Dino Bidoia
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ederio Dino Bidoia.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Montagnolli, R.N., Lopes, P.R.M. & Bidoia, E.D. Screening the Toxicity and Biodegradability of Petroleum Hydrocarbons by a Rapid Colorimetric Method. Arch Environ Contam Toxicol 68, 342–353 (2015). https://doi.org/10.1007/s00244-014-0112-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00244-014-0112-9

Keywords

  • PAHs
  • Diesel
  • Biodegradation
  • Gasoline
  • Petroleum Hydrocarbon