RESEARCH PROPOSAL

Researcher: Abraham Ogheneruemu Ekperusi M.Sc. Environmental Quality Management (Pollution Studies/Ecotoxicology) De artment o! "nimal and Environmental #iology$ %aculty o! &i!e Sciences$ 'niversity o! #enin$ #enin (ity. )el: *+*,,+-.++/ E0mail: e1 erusia23gmail.com

Su ervisor:

Prof. Felix I. Aigbodion De artment o! "nimal and Environmental #iology$ %aculty o! &i!e Sciences$ 'niversity o! #enin$ #enin (ity.

RESEARCH I LE )he 2iodegradation otentials o! three s ecies o! earth4orms and 2acteria on crude oil$ diesel and 1erosene in the 5iger Delta.

I! RO"#C IO! %inding a universally acce ta2le method or techni6ues !or cleaning the environment has 2ecome a serious concern to etrochemical industries and scientist in the 7. st century$ since gro4ing research has sho4n that chemical dis ersant 4hich is hitherto used !or cleaning the environment during s ills oses a threat to 2enthic organisms and 2ene!icial micro0organisms 4hich lay a great role in ecotoxicology assessment and in the cleaning rocess o! etrochemicals. 8hile micro0organisms and lants have sho4n a strong ros ect in cleaning o! the environment over the years$ the delicate nature o! micro2es and the long duration o! trees challenge in creating micro2ial seeding and real time monitoring o! ose a hyto0

assessment es ecially in 5igeria. )hese challenges have resulted in slo4 ace o! degradation o! ollutants in the environment. 8ith these challenges$ scientists are turning to macro0organisms in the soil and their otentials in cleaning the environment. 9! all the macro0organisms inha2iting soil$ earth4orms have 2ecome the leading s ecies 4ith ros ect !or removing contaminants !rom the environment. RESEARCH $#ES IO!S )he research see1s to ossi2ly ans4er the !ollo4ing 6uestions. .. (an earth4orms hel environment: 7. 8hich s ecies o! earth4orms are most suita2le !or 2ioremediation o! etrochemicals: ,. 8ill 2ioremediation rate increase i! earth4orms and micro2es 4or1 together synergistically: /. 8hat is the !ate o! the toxic su2stances ta1en 2y earth4orms: "re they trans!ormed or are accumulated in the tissues o! the earth4orms: RESEARCH O%&EC I'ES )he research see1s to determine the rate and e!!ectiveness o! hydrocar2on degradation otentials o! selected s ecies o! earth4orms alone and earth4orms 4ith micro2es on crude oil$ diesel and 1erosene in the 5iger Delta. to remove ollutants or contaminants !rom the

LI ERA #RE RE'IE( Earth4orms are no4 highly regarded as a rich 2iological resource 4ith a multitude o! uses having commercial and environmental a among researchers in various otentials o! earth4orms. "meh et al.$ (7*.7)$ conducted an investigation on the e!!ect o! earth4orm ( Eudrilus eugeniae) on the 2ioremediation o! used engine oil contaminated soil$ amended 4ith oultry manure. %or initial used engine oil concentration o! -$ .*$ .- and 7* g/1g soil$ the dro in total etroleum hydrocar2on 4as !ound to 2e 0.;.<.$ 7*.+7$ ,/.;+ and ,;.7+= res ectively a!ter /7 days o! treatment. >o4ever$ the rate o! 2ioremediation 4as !ound to increase 4ith increase in earth4orm concentration. ?orna et al.$ (7**-)$ conducted a study to determine the im act o! earth4orms (Allolobophora chlorotica and Aporrectodea caliginosa) 2iotur2ation on the distri2ution and availa2ility o! @inc in the soil. )hey re orted that the e!!ects o! earth4orm 2iotur2ation 4ere most ronounced a!ter .A- days. %or A. chlorotica$ total and (a(l70exchangea2le @inc concentrations in the olluted layers 4ere lo4er than 4ithout earth4orms. )hara1an et al.$ (7**-)$ in this study 4e investigated the otential o! using olychlorinated lications. )here is a gro4ing interest arts o! the 4orld regarding the 2ioremediation

earth4orms (Eisenia foetida) to e!!ect the 2iotrans!ormation o!

2i henyls (P(#Bs) in sludge and sediment !rom the Ralston Street &agoon (RS&) S'PER%'5D site in Cary$ Dndiana. Cas chromatogra hy results demonstrated an +*= reduction in P(# level 4ithin .-*07**days. "nalysis o! earth4orms sho4ed elevated P(# levels in the 4orm. )hese results suggest vermicom osting may 2e a via2le o tion !or on0site in situ 2ioremediation and site clean0u . Schae!er and %ilser (7**;)$ investigated earth4orms ( Eisenia foetida$ Allolobophora chlorotica$ and Lumbricus terrestris) on the degradation o! a crude oil contaminated soil and/or additives (organic material) enhance the micro2ial degradation o! etroleum hydrocar2ons in soil. )hey re orted a signi!icant decrease in the )P> concentration in treatments 4ith earth4orms$ com ared 4ith the sam les 4ithout 4orms. 8hereas 7<...= reduction o! the )P> concentration 4as recorded in the

4orm treatments 4ithout additives$ only A.;-= 4as reduced in the treatments 4ithout 4orms. &ue romchai et al., (7**7)$ studied the interactions o! earth4orms ( Pheretima hawayana) 4ith indigenous and 2ioaugmented P(#0degrading 2acteria. Results sho4ed that a earth4orms a roximately -*= o! the P(#s 4ere removed in the to <cm using a lied alone 4ere e!!ective only !or removing P(#s !rom the to ,cm o! earance o! com2ination o! earth4orms and 2io0augmentation$ 4hereas 2io0augmentation or the soil columns. "!ter < 4ee1s$ the treatment em loying 2io0augmentation 4ith P(#0degrading 2acteria and earth4orms resulted in signi!icant disa P(#s at all de ths res ectively. (outiEo0Con@Fle@ et al.$ (7*.*)$ 4or1s 4ith Eisenia foetida on the removal o! anthracene and <$ .*0anthra6uinone. /.= removal o! anthracene 4as !ound !or treatments 4ithout earth4orms and <,= !or those 4ith earth4orms. Grishna et al., (7*..)$ investigated the 2ioremediation o! Phenol 2y earth4orms (Eisenia foetida$ Eudrillus eugenia and Anantapur). )heir results indicated that Eisenia foetida and Eudrillus eugenia 4ere the 2est tolerant s ecies$ 4hile the Anantapur s ecies 4as !ound to 2e least tolerant. Eudrillus euginea sho4ed an increased u ta1e o! initial o! 7*$ /* H ;* o! +* H .** m and a decrease to hal! the m$ 4hen o2served a!ter 7/ hours. henol concentration ercent at higher concentration

RESEARCH )E HO"OLO*+ S,ud- Area )he study 4ill 2e conducted at the ostgraduate la2s o! the De artment o! "nimal and Environmental #iology and De artment o! Micro2iology$ %aculty o! &i!e Sciences$ 'niversity o! #enin$ #enin (ity$ 5igeria. Colle.,ion of Samples Colle.,ion and Iden,ifi.a,ion of Ear,h/orms )hree s ecies o! earth4orms 4ill 2e collected and used !or the ex eriment. )he earth4orms 4ill 2e collected or urchased. )he earth4orms 4ill 2e identi!ied using taxonomic 1eys i! they are collected. Plant and !ood 4aste 4ill 2e collected !or !eeding. Soils Soil sam les com rising sand$ clay and loam com onents 4ill 2e collected !or the ex eriment. H-dro.arbons 7*litres each o! crude oil$ diesel and 1erosene 4ill also 2e collected !or the ex eriment as 4ell. Isola,ion0 Cul,i1a,ion and Iden,ifi.a,ion of )i.robes Micro2e I " s eci!ic 2acterial s ecies 4ill 2e collected !rom etrochemical sites at the Se lat Petrochemical (om any$ Sa ele$ Delta State. )he hydrocar2on utili@ing 2acteria (>'#) !rom the etrochemical sites 4ill 2e isolated$ cultivated and identi!ied !rom the im acted soil 4ith nutrients agar in an incu2ator. )he >'# !or the ex eriment 4ill 2e isolated and identi!ied. Su2culture 4ill 2e carried out to get ure isolates or strains o! s ecies o! desired s ecies !or the ex eriment. Ddenti!ication 4ill 2e carried out 2ased on mor hological characteristics$ motility test$ gram staining and 2iochemical reactions$ 4ith the hel Determinative #acteriology. Experimen,al Se,up Pers ex columns 4ill 2e used !or the ex erimental setu . )he columns 4ill 2e cut underneath hori@ontally 4ith a ra@or 2lade to allo4 some levels o! 4ater loss !rom the columns. Soil Arrangemen, Measured levels o! soil 4ill 2e sieve in a -mm !ilter and dried. Standard measures o! the soil 4ill 2e laced inside each o! the Pers ex columns. o! #ergeyJs Manual o!

)he soil is then ma1e moist 4ith the addition o! distil 4ater to the 4ater holding ca acity o! the soil. )he clay$ sand$ loamy contents etc 4ill 2e measured rior to the ex eriment. Soil Con,amina,ion Pers ex column soil microcosm (seven each) 4ill 2e contaminated or mixed thoroughly 4ith measured volumes o! crude oil$ diesel and 1erosene at levels that 4ill not have lethal e!!ects on the earth4orms. ((rude oil K A$ Diesel K A and Gerosene K A). (ontaminated soil 4ill 2e ex osed !or one 4ee1 2e!ore a and micro2e. Ino.ula,ion Ear,h/orms )hree di!!erent s ecies o! earth4orms 4ill 2e used !or the ex eriment. Purchased or collected earth4orms 4ill 2e identi!ied using taxonomic 1eys and they 4ill 2e cleaned$ laced in a !ilter a er !or 7/hours to clear their gut rior to the ex eriment. )he earth4orms 4ill 2e 4eighed 2e!ore inoculated into the contaminated soil. "2out - each o! the three di!!erent s ecies o! earth4orms 4ill 2e inoculated into three soil microcosm 4ith crude oil$ diesel and 1erosene contaminated soils 4hile the !ourth Pers ex !or each o! the crude oil$ diesel and 1erosene 4ithout earth4orms 4ill serve as control !or the ex eriment$ ma1ing a total .7 Pers ex columns 4ith ex erimental setu . )he earth4orm and the contaminated soil 4ill have some com ost on to as !ood materials !or the earth4orms. Ear,h/orms and )i.robe "nother set o! Pers ex columns soil microcosm 4ill 2e ut in lace$ 4ith each having - earth4orms and a s eci!ic micro2e to 4or1 synergistically 4ith the earth4orms in the contaminated soil containing crude oil$ diesel and 1erosene ma1ing it a total o! < setu . )he entire setu 4ill 2e monitored daily and 4ater 4ould 2e added to maintain the moisture level needed !or the survival o! earth4orms. Ph-si.o2Chemi.al Parame,ers Physical and chemical arameters 4ill 2e measured. Some o! the arameters to 2e measured 4ill include 2ut not limited to oxygen$ moisture$ nitrate$ tem erature$ etc 4ill 2e ta1en daily. hos hate$ >$ lication o! earth4orms

Samples Anal-ses Soils sam les 4ill 2e collected !rom the soil microcosm in three dimensions. .). Earth4orms and hydrocar2on grou s. 7). Earth4orms$ micro2es and hydrocar2on grou s. ,). (ontrol containing only hydrocar2on grou s !or analysis on a monthly 2asis !or the three months eriod o! the ex eriment. )otal etroleum hydrocar2on ()P>)$ 4ill 2e determined !rom measured volumes o! the soil microcosm using Cas0&i6uid (hromatogra hy. "!ter the entire three months eriod$ the earth4orms 4ill 2e sorted out$ counted$ clean and 4eigh. )hen the earth4orms 4ill 2e crushed and grinded and also ta1en !or analysis !or )P> using Cas 0 &i6uid (hromatogra hy. S,a,is,i.al Anal-ses Data 4ill 2e summari@ed using "nalysis o! variance$ ("59L") or the Golmogorov 0 Smirnov one sam le test and ratio !or deviation. )he data 4ill 2e analyse statistically using the SPSS .; so!t4are ac1age. "#RA IO! OF RESEARCH )he research is going to ta1e three months or <* days. Pre0research and ost0 research lanning 4ill amount in total to a2out !ive months. "ll things 2eing e6ual$ the research ractical 4ill start in %e2ruary 7*., and ends in " ril$ 4hile statistical analysis and dra!t re ort 4ill 2e availa2le in Muly. A! ICIPA E" FI!"I!*S )he antici ated !indings !or this research includes D! earth4orms alone can clean etrochemical su2stances. >o4 !ast and e!!icient earth4orms can clean etrochemical su2stances. )he rate o! 2ioremediation 4hen earth4orms and micro2es 4or1s synergistically. 8hat is the !ate o! the chemical su2stances I are they accumulated in the tissues o! the earth4orms or they are degraded or trans!orm into other use!ul materials in the tissues o! the earth4orms. )he research intends to sho4 the hydrocar2on degrading a2ility o! the s ecies and their otentials as 2ioremediation agents !or cleaning etroleum im acted environments. S ecies 4ith high degrading a2ility can 2e enhanced into micro2ial seeding !or 2ioremediation 4or1s in the 5iger Delta.

"ISSE)I!A IO! OF FI!"I!*S Dn!ormation !rom the ecological$ 2iological and ollution studies !rom this research ho ely 4ill 2e disseminated in scienti!ic con!erences organi@ed 2y 2odies such as the 5igerian Environmental Society$ 5igerian Society !or Ex erimental #iology$ SE)"($ and Society !or Ex erimental #iology$ etc. )he !indings 4ill 2e u2lished in nota2le Nournals 2oth local and international li1e 5igerian Mournal o! Environmental Science$ Dnternational Mournal o! Environmental Sciences$ Environmental Pollution$ etc. AC3!O(LE"*E)E! 8herever the research is re orted 2y the author$ re!erence and ac1no4ledgement 4ill 2e given to the relevant 2odies or organi@ations that contri2uted to the success o! the research. "c1no4ledgement relating to the ac6uisition o! the chemicals used$ la2oratory !or the analysis and the source o! !unding !or the 4or1. E$#IP)E! 4)A ERIALS !EE"E" FOR HE S #"+ &i6uid0Cas (hromatogra hy Digital Soil > Meter I %erry0Morse$ 'S". Dissecting microsco e Digital 4eighing 2alance Digital thermometer Moisture (ontent "naly@er I Ceneral )ools$ 'S". (ar2on "naly@er 0 Ceneral )ools$ 'S". Dncu2ator #ea1ers Reagents "gar medium (om ost Earth4orms 7*&itres o! (rude oil$ diesel and Gerosene Sieve !ilter I -mm. 5etting material Ra@or 2lade Distil 4ater

%#"*E )he estimated amount needed !or the roNect is stated 2elo4. S/5 . 7 , / ; A + < .* Dtems (rude oil Diesel Gerosene Pers ex columns Sieve !ilter -mm 5etting materials Distil 4ater Earth4orms (hromatogra hic analyses Statistical analyses 'nit 7* litres 7* litres 7* litres 7/ 7 -yards 7* litres .** ieces ;* sam les "59L"$ Signi!icant Di!!erence$ Golmogorov0 .. .7 ., Smirnov )rans ortation &ogistics Digital Soil > Meter 7 (%erry0Morse$ 'S") Miosture (ontent 7 "naly@er ./ ..; .A (Ceneral 5.7$7** 5-*** 57/$/** 57-$*** 5.;$+** 5,*$*** o,al !5650788 )ools$ 'S") (ar2on "naly@er 7 (Ceneral )ools$ 'S") Reagents !or micro2e - ty es 5.-$*** 57*$7** 5.;$*** 'nit Price 5.A5.A5.-* 5.-** 57$-** 5-** 5.757-* 5.*$*** )otal 5,$-** 5,$-** 5,$*** 5,;$*** 5-*** 57-** 57$-** 57-$*** 5;**$*** 5,*$***

5.*$.** 5+***

culture Printing o! Re ort I / co ies each K + 57.** Dra!t H %inal Miscellaneous &ogistics co ies and

EXPERIMENTAL SETUP
Analysis using Chromatography Soil Treatments Earthworms Sample for Analysi s !" #ays$ Sample for Analysi s %" #ays$ Sample for Analysi s &" #ays$ Total Sample s Amount N

N'""""(Sampl e

Earthworms ) Cru*e oil only 1 2 3 Earthworm 1 + Crude oil Earthworm 2 + crude oil Earthworm 3 + Crude oil Earthworms ) #iesel +nly 4 5 6 Earthworm 1 + Diesel Earthworm 2 + Diesel Earthworm 3 + Diesel Earthworms ) ,erosene +nly 7 " # Earthworm 1 + erose!e Earthworm 2 + erose!e Earthworm 3 + erose!e Earthworms ) Cru*e oil ) Mi-ro.es 10 Earthworm 1 + Crude oil + $icro%e Earthworm 2 + Crude oil + $icro%e Earthworm 3 + Crude oil + 5 earthworms 5 earthworms 5 Sample Sample Sample 3 N30,000 5 earthworms 5 earthworms 5 earthworms Sample Sample Sample Sample Sample Sample Sample Sample Sample 3 3 3 N30,000 N30,000 N30,000 5 earthworms 5 earthworms 5 earthworms Sample Sample Sample Sample Sample Sample Sample Sample Sample 3 3 3 N30,000 N30,000 N30,000 5 earthworms 5 earthworms 5 earthworms Sample Sample Sample Sample Sample Sample Sample Sample Sample 3 3 3 N30,000 N30,000 N30,000

11

Sample

Sample

Sample

N30,000

12

Sample

Sample

Sample

N30,000

$icro%e

earthworms

Earthworms ) #iesel ) Mi-ro.es 13 14 15 Earthworm 1 + Diesel + $icro%e Earthworm 2 + Diesel + $icro%e Earthworm 3 + Diesel + $icro%e Earthworms ) ,erosene ) Mi-ro.es 16 Earthworm 1 + erose!e + $icro%e Earthworm 2 + erose!e + $icro%e Earthworm 3 + erose!e + $icro%e Control Contaminate* Soil +nly$ 1# Crude oil No earthworms No earthworms No earthworms Sample 1 N10,000 5 earthworms 5 earthworms 5 earthworms Sample Sample Sample 3 N30,000 5 earthworms 5 earthworms 5 earthworms Sample Sample Sample Sample Sample Sample Sample Sample Sample 3 3 3 N30,000 N30,000 N30,000

17

Sample

Sample

Sample

N30,000

1"

Sample

Sample

Sample

N30,000

20 21

Diesel erose!e /rin*e* Earthworms for Analysis

Sample Sample

1 1

N10,000 N10,000

22

&ri!ded earthworm + crude oil

1 earthworm 'rom crude oil 1 earthworm 'rom diesel 1 earthworm 'rom (erose!e

Sample

N10,000

23

&ri!ded earthworm + Diesel &ri!ded earthworm + erose!e

Sample

N10,000

24

Sample

N10,000

)otal Sample s

60

N%""0"""