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1 w y wz 9«( 3y) 191~198, 006 J. of the Korean Society for Environmental Analysis Microtox w w m sƒ Á½»Áû * w w» vgp l, * w» y k Microtox Biological Toxic Assessment of Soils Treated with Sewage Sludge Sang-Jun Park, Seungki Kim, and Jae-jak Nam* Doping Control Center, Korea Institute of Science & Technology, Seoul , Korea *National Institute of Agriculture Science & Technology, Suwon , Korea The toxicity of soils treated with sewage sludge was evaluated using Microtox instrumental analysis. In soils treated with highly polluted sewage sludge from industrial zone, the bioassay revealed a high potential toxicity in proportion to the amount of sewage sludge. In soils treated with highly polluted sewage sludge from municipal zone contained the low level of heavy metals, PAHs. The effect of Microtox concentration was well correlated with the concentration of heavy metals and PAHs on soils. This bioassay technique can be a sensitive method of soil quality evaluation and useful for discriminating the quality of soil following potential risk assessment. Key words : bioassay, heavy metals, polycyclic aromatic hydrocarbons(pahs), effect of concentration( ) 1. w 75% swwš š Á w n ƒ w y w, n» ù wš ù, x ³ w w n»» w»ƒ š w y w š. w d w ky 1~3) w ƒ. d» wš, ¼ Soxhlet ù» q w w z w w j m v(high performance liquid chromatography)» j m v y (gas chromatography-flame ionization detector, GC-FID) w w w,» j m v (gas chromatography-mass spectrometry, GC-MS) w ¾ ƒ w. 4)» ü w» w» w w w w j q w e w š. w y 5), y» yw. w y» wwš,» ü {» w. w w», y w v Ÿ»(Inductively Coupled Plasma Atomic Emission Spectrometer, ICP-AES) w w. ù Hg, As, Se, B, Sb { y, ³ ³ x. y 6) w w w yw xk y g k e w To whom correspondence should be addressed. xodid4@hanmail.net

2 19 Á½»Áû w w ƒ š. 7) Microtox w (bioassay) w Ÿ Vibrio fisheri Ÿ w d w w sƒ» w ü w š w ùkü r ƒ š. Microtox Ÿ ü w ATP z Luciferase w y j, w w ü ATP ƒ yw. Microtox w Ÿ w w sƒw» p m ù n w PCBs, PAHs, metals, sulfur ƒ w w w. 8,9) w m (5, 50, 100 ton/ ha) microtox basic test, solid phase test, basic solid phase test, basic test for aqueous extract w w, ICP-AES GC-MS»» w PAHs w microtox w w..1. x. Microtox Ÿ l (Vibrio fischeri) AZUR (Carlsbad, CA, USA) l w. l y y (reconstitution solution) AZUR l w, Ÿ l 1 y y 1mL Ÿ l y y j w. (diluent) Azur l w š, v w w. r (Phenol) Aldrich (Milwaukee, WI, USA) l w r 50 mg 500 ml v j w g 100 ug/ml w t w. Millipore (Bedford, MA, USA) Mill-Q water system w 183 MO w ƒ k w.» sodium sulfate, methylene chloride, n-hexane Fiser Scientific (Fair lawn, NJ, USA) w, dimethylsulfoxide(dmso) Junsei (Chemical Co., Ltd, Japan) w w. ICP-AES w m w (ternary solution) nitric acid(hno 3 ) Fisher Scientific(Fair lawn, NJ, USA), sulfuric acid(h SO 4 ) Duksan (Ansan, Kyunggi, Korea), perchloric acid(hclo 4 ) Samchn (Columbus, OH, USA) l w HNO 3 : H SO 4 : HClO 4 = 10: 1: 4 w w. GC-MS dichloromethane, cyclohexane ƒƒ Fisher Scientific Junsei l w w...»» x w microtox»» Microtox Analzyer(AZUR Environmental, USA) model 500 w. -0 C Ÿ o y y k z 4C x o w microtox mw d w. š w w m d w» w ICP-AES(GBC-Integra XMP, Australia) w w, w PAHs d w» w GC-MS(GCQ, Finnigan, USA) w w..3. x Basic test m 15 g (diluent) 30 ml 4 w w w, solid phase test m 15 g (diluent) 30 ml 10 z filter column w microtox manual w. basic solid phase test m 15 g (diluent) 30 ml 10 w microtox w, basic test for aqueous extract test m ƒ (diluent, solvent) w. m 15 g 30 ml k(0 ± C) 48 k g o w,» w solvent m 50 g sodium sulfate 50 g, methylene chloride 150 ml metallic copper 5 mg 00 rpm 4 k w z (Watman No. ) w»»(turbo vap, Zymark) methylene chloride k z dimethylsulfoxide(dmso) ww %ƒ w microtox manual

3 Microtox w w m sƒ 193 d w. w m yw, ph ƒ e y w w. ph m w m 1 g distilled water 5 ml w w. ICP-AES w m m 5 g 4 M HNO 3 e 5 ml š 80 o C 16 e w d w m g w ternary solution(hno 3 :H SO 4 :HClO 4 = 10:1:4) 0~30 ml š q w e (kieldatherm, Gerhardt, Germany)» 1 50 o C, z 375 o C ƒ w z w d w w. w m PAHs ƒƒ mw p» w w. m soxhlet w y z f m w 1 ml w z w. GC-MS w PAHs w» w US EPA SW ) w w. 0 g 0-50 g y ùp yww z m š soxhlet w 16 w. dichloromethane 00 ml w. ü t ƒ z y ùp f m g z, z» w w. ml cyclohexane y z e f j m v w w. f m w 1 ml w z GC-MS w w. 3. š 3.1. w m yw w m ph w Table 1 ùkü. y w m (MSS) ph w 5.4, 4.8, 4.5, œ w m (ISS) 4.9, 4.9, 4.6. ƒ ISS Cu ƒƒ 138.3, 199.9, 39.9 mg/kg, Ni 4., 4.6, 8. mg/ kg ùkû. ph MSS ISSƒ ùkü. MSS ISS w ƒ š, ISS MSS. Ibekwe ph w š w w m ƒ ƒw š w š, 11) Stuman and McBride Blasiak and Harter phƒ m y w w e š w ph 5.5 ƒ š w. w 1,13) m w e w yw» w ph w v ƒ, Microtox l w yw» w ph 6~8 w w Microtox Manual mw. 3.. w m PAHs GC-MS w 16ƒ PAHs w Table ùkü. MSS ISS w 16ƒ PAHs MSS ISS r naphthalene MSS w 38.3, 86.6, 4.5 ug/kg, ISS 440.4, 801.1, ug/kg, acenaphthene MSS 18.8, 8.4, 10.5 ug/kg, ISS 18.1, 33.7, 59.4 ug/kg, fluorene MSS 3.4, 18.4, 31.0 ug/kg, ISS 46.8, Table 1. ph and Concentration of heavy metals on soils treated with the sewage sludges(mg/kg). Sewage Sludges Levels (ton/ha) ph Cr Ni Cu Pb Cd Zn Fe Mn MSS ISS Municipal Sewage Sludge, Industrial Sewage Sludge

4 194 Á½»Áû Table. Concentrations of PAHs on soils treated with the sewage sludges(ug/kg). Sewage Sludges Levels (ton/ha) NaP A Acy B Ace C Fle D Phe E Ant F Fla G Pyr H Chr I BaA J BbF K BkF L BaP M IcP N DaA O BgP P MSS ISS Municipal Sewage Sludge, Industrial Sewage Sludge. A B C D E F G H I NaPhthalene, Acenaphthylene, Acenaphthene, Fluorene, Phenanthrene, Anthracene, Fluoroanthene, Pyrene, Chrysene, J K L M N O Benzo(a)anthracene, Benzo(b)fluoroanthene, Benzo(k)fluoroanthene, Benzo(a)pyrene, Indeno(1,,3-cd)pyrene, Dibenzo(a,h) anthracene, P Benzo(ghi)perylene. 75.8, 167. ug/kg, phenanthrene MSS 7.5, 1.3, 7.8 ug/kg, ISS 313.8, 435.8, ug/ kg, anthracene MSS 75.3, 1.4, 9.0 ug/kg, ISS 48.4, 7.5, 17.1 ug/kg, pyrene MSS 593.5, 15.7, ug/kg, ISS 590.9, 568.6, ug/kg ùkû. û naphthalene 50~430 ug/kg w š w, acenaphthylene (,, w ) 0 ug/kgü š š w. x 5) naphthalene ISS j w, MSS w w š, acenaphthylene MSS 19.9, 13.6, 11.7 ug/kg, ISS 1.9, 16.4, 34.0 ug/kg e w. š PAHsƒ y w š, y ³ ù p, PAHs w j ƒ Microtox w m w PAHs w Microtox mw w» w Basic Test, Solid Phase Test, Basic Solid Phase Test, Basic Test for Aqueous Extract(Diluent Extract, Solvent Extract) mw w. Table 3 w m ƒ w ùkü. Basic test w MSS 448.1, 430., g/l, ISS 130.6, 135., g/l š Solid Phase Test MSS 190.9, 00., 187.5g/L, ISS 135.1, 137.6, g/l, Basic Solid Phase Test MSS 98., 100.5, 99.5 g/l, ISS 7.1, 73.4, 80.5 g/ L. w ƒ ùkü basic test for aqueous extract MSS 45.6, 40.4, 36.1 g/l, ISS 170.5, 104.7, 73. g/l w m sƒ w» w basic test for aqueous extract ƒ ww. w»yw Table 3. Comparison of Microtox media Q with different method. Sewage Sludges Levels (ton/ha) Basic Test Solid Phase Test Basic Solid Phase Test Basic Test for Aqueous Extract Diluent Extract Solvent Extract MSS ISS Q concentration : (g/l), 1 MSS : Municipal Sewage Sludge, ISS : Industrial Sewage Sludge

5 Microtox w w m sƒ 195 sƒw» w å basic test for aqueous extract d w MSS 49.4, 43.3, 15.7 g/l, ISS 198.7, 14., 70.9 g/l» w Ÿ (Vibrio fischeri) w ƒ., w basic test for aqueous extract ƒ dw š, w basic test for aqueous extract PAHs ƒ dw. Fig. 1. The relationship between heavy metals and the Microtox on soil treated with the sewage sludges(diluent Extract).

6 196 Á½»Áû 3.4. Diluent extract Diluent extract Fig. 1 ùkü. (r ) Cu , Cr , Ni 0.791, Zn , Cd 0.560, Fe 0.637, Mn 0.69 ƒ š, Pb ƒ. Cu, Cr, Ni, Zn, Cd, Fe w diluent extract û w ùkü š, Mn w y ùkü., ICP-AES diluent extract ƒ š,» sƒ (bioassay) microtox sƒ w PAHs solvent extract GC-MS mw PAHs» w m w w. w MSS, ISS w š w PAHs w 3z w š, z w. GC-MS w PAHs solvent extract z w Fig. ùkü. Naphthalene, phenathrene, fluoroanthene, pyrene, benzo(h)fluoroanthene, benzo(ghi)perylene PAHs ƒ, phenathrene 0.594, benzo(ghi)perylene (r ) ùkü š, PAHs ƒ acenaphthene 0.544, fluorene 0.611, benzo(a)anthracene (r )ƒ Fig.. The relationship between PAHs and the Microtox test on soil treated with the sewage sludges(solvent Extract).

7 Microtox w w m sƒ 197. MSS ISS PAHs solvent extract z mw Fig. ùkü, (r ) ù w w ü y y w mw ¼w PAHs w solvent extract ƒ û m w z w» w ICP-AES GC-MS w w m PAHs w ƒƒ Table 1 Table ùkü š, ƒ microtox basic test for aqueous extract (diluent extract, solvent extract) mw Table 3 ùkü. PAHs (p)ƒ 0.01 û. Cu > Ni > Cr > Mn > Fe > Zn > Cd. ICP-AES mw Fe > Mn > Zn > Pb > Cu > Ni > Cr w ƒ w microtox dw, m w w x ù. m yww w» w mw ¼w w microtox Ÿ ( ) e w w» sƒ»» w sƒw». PAHs benzo(a)anthracene (r )ƒ ƒ š, fluorene > phenathrene > acenaphthene > benzo(ghi)perylenetns ƒ. PAHs m ü ƒ w., ƒ PAHs 5, PAHs solvent extract (r ) 0.58 š, Table 4 ùkü. yw PAHs, ¼w microtox e w w. Table 4. Significance of difference between heavy metals, PAHs and Microtox. Heavy Metals PAHs 4. w s» ù w n»ƒ y w w ƒ š. p w PAHs w ƒ š w yw w» w w microtox w. x w w yw w m microtox»» (ICP-AES, GC-MS) mw w w. microtox w» w w m w, wš w m PAHs w» w» w. w ƒ microtox w, basic test for aqueous extract(diluente extract, solvent extract) PAHs w. w w (r ) Cu:0.871, Ni:0.794, Cr:0.784, Zn:0.576, Cd:0.56, Fe: 0.637, ùkü, Mn 0.69 ùkü. PAHs (r ) acenaphthene:0.544, fluorene: 0.611, phenanthrene:0.594, benzo(a)anthracene:0.655, benzo(ghi)perylene:0.500 ùkü r p a Cu ** p< Ni ** p< Cr ** p< Zn ** p= Cd 0.56 ** p= Fe ** p< Mn 0.69 ** p< Ace C ** p= Fle D ** p= Phe E ** p=0.000 BaA J ** p< BgP P ** p=0.001 Total PAHs 0.58 ** p=0.000 C Acenaphthene, D Fluorene, E Phenanthrene, J Benzo(a) anthracene, P Benzo(ghi)perylene, a Prob., **: P<0.01.

8 198 Á½»Áû š, 16ƒ ƒ yw PAHs (r ) š x 1. y, 00, y.. y, 001, s» xy. 3. y, 001, s» xy. 4. Bernard S. Crimmins and Joel E. Baker, Improved GC/ MS methods for measuring hourly PAH and nitro- PAH concentrations in urban particulate matter, 006, Atmospheric Environment 40, û, û,, w, w m wz, 1998, 31(3), US EPA, 1990, Microwave assited acid digestion of sediments, sludge, soils, and oils, SW-846, Method 3051, Washington, DC, USA. 7. û, ³, w, w y wz, 00, 11(4), Salizzato, M., B. Pavoni, A.V. Ghirardini, P.F. Ghetti, Sediment toxicity measured using Vibrio fischeri as related to the concentrations of organic(pcbs, PAHs) and inorganic (Metals, sulphur) pollutants, 1998, Chemosphere 36, Ringwood, A.H., M.E. DeLorenzo, P.E. Ross, A.F. Holland, Interpretation of Microtox solid-phase toxicity tests: The effects of sediment composition, Environ. Toxicol. Chem., 1997, 16, US EPA, 1996, Test Methods for Evaluating Solid waste, Physical/Chemical Methods: Semivolatile Organic compounds by Gas Chromatography/Mass spectrometry(gc/ms), SW-846, Method 870C, Washington, DC, USA. 11. Ibekwe, A. M., J. S. Angle, R. L. Chaney, P. Van Berkum, Sewage sludge and heavy metal effects on nodulation and nitrogen fixation of Legumes J. Environ. Qual,, 1995, 4, McBride, M. B, Growing food crops on sludgeamended soils: Problems with the U.S. Environmental Protection Agency method of estimating toxic metal transfer, Environment Toxicity and Chemistry, 1998, 17(11), McBride, M. B., B. K. Richards, T. Steenhuis, and G. Spiers, Molybdenum uptake by forage crops grown on sewage sludge-amended soils in the field and greenhouse, J. Environ. Qual., 000, 9,