Science, Faculty of
Chemistry, Department of
DSpace
UBCV
Kuipers, Bianca
Doctor of Philosophy - PhD
University of British Columbia
Polychlorinated biphenyls (PCBs) and polycylic aromatic hydrocarbons (PAHs) are widespread environmental contaminants, and their presence in the environment is a cause for concern because they are linked to a variety of toxic effects. In this thesis, some aspects of the microbial degradation of PCBs and alkyl PAHs are investigated. Analytical methods for the extraction and analysis (by using gas chromatography coupled to mass spectrometry (GC/MS)) of PCBs were developed and tested. The PCB content of three sediment cores taken from lakes in Cambridge Bay (Nunavut) was analyzed, but no evidence for in-situ microbial dechlorination of PCBs was found. A variety of approaches to the development of microbial enrichment cultures were tested. The most successful were developed by using 4-bromobenzoate, as an amendment to stimulate dehalogenation activity, and sediment collected from Esquimalt Harbour (British Columbia) as an inoculum source. The dechlorination of Aroclor 1260 in soils from Resolution Island (Nunavut) and Saglek (Labrador) by using a variety of enrichment cultures was investigated. Extensive dechlorination of highly-chlorinated PCB congeners was achieved, and the most abundant products from Aroclor 1260 dechlorination were tetrachlorobiphenyls. A method to determine the identity of one major dechlorination product by using MS/MS was developed, and the dechlorination product was found to be 24-24- tetrachlorobipheny 1. Some factors that could affect the extent and rate of PCB dechlorination were investigated. It was found that autoclaving pre-incubated cultures prior to inoculation with PCB-dechlorinating enrichment cultures increased the rate and extent of PCB dechlorination. It was also found that dechlorination of PCBs that had been present in Resolution Island soil for a number of years was not as extensive as dechlorination of PCBs that were freshly added to Resolution Island soil. A variety of incubation temperatures were tested, and the most rapid and extensive PCB dechlorination occurred at 30 °C. The dechlorination of three nona- and four octa-chlorobiphenyl congeners, both singly, and in various combinations, was also investigated. It was established that the dominant dechlorination pathway involved the removal of meta chlorines, although some para and some ortho dechlorination was also observed. The rates and products of PCB dechlorination varied widely, depending on the mixture of congeners present. This is the first time the dechlorination of single octa- and nona-chlorobiphenyl congeners has been reported, and also the first time that the dechlorination of various combinations of these congeners has been investigated. Extensive degradation of penta- and tetra-CBs to biphenyl was also found to occur. A method to detect alkylated polycylic aromatic hydrocarbons (alkyl PAHs) in complex hydrocarbon mixtures was developed, and by using this method, the alkyl PAH content of several hydrocarbon mixtures was determined. Degradation of the alkyl PAH portion of hydrocarbon-contaminated Hall Beach (Nunavut) soil was demonstrated.
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MICROBIAL DEGRADATION OF POLYCHLORINATED BIPHENYLS AND ALKYLATED POLYCYCLIC AROMATIC HYDROCARBONS by BIANCA KUIPERS B . S c , Un ive r s i t y o f A u c k l a n d , N e w Zealand, 1991 M . Sc. (Hons.) , Univers i ty o f A u c k l a n d , N e w Zealand, 1993 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F D O C T O R O F P H I L O S O P H Y i n T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Department o f Chemis t ry) W e accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F B R I T I S H C O L U M B I A N o v e m b e r 1999 © B i a n c a Ku ipe r s , 1999 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of C-Kem\'st:r The University of British Columbia Vancouver, Canada Date I S&c^hxS l^S^ DE-6 (2/88) Abstract Polychlor ina ted biphenyls ( P C B s ) and p o l y c y l i c aromatic hydrocarbons ( P A H s ) are widespread environmental contaminants, and their presence i n the environment is a cause for concern because they are l i nked to a variety o f tox ic effects. In this thesis, some aspects o f the mic rob i a l degradation o f P C B s and a l k y l P A H s are investigated. A n a l y t i c a l methods for the extraction and analysis (by us ing gas chromatography coupled to mass spectrometry ( G C / M S ) ) o f P C B s were developed and tested. The P C B content o f three sediment cores taken f rom lakes i n Cambr idge B a y (Nunavut) was analyzed, but no evidence for in-si tu mic rob i a l dechlor inat ion o f P C B s was found. A variety o f approaches to the development o f mic rob i a l enrichment cultures were tested. The most successful were developed by us ing 4-bromobenzoate, as an amendment to stimulate dehalogenation act ivi ty, and sediment col lected from Esqu ima l t Harbour (Br i t i sh C o l u m b i a ) as an i nocu lum source. The dechlor inat ion o f A r o c l o r 1260 i n soils f rom Reso lu t ion Island (Nunavut) and Saglek (Labrador) by us ing a variety o f enrichment cultures was investigated. Ex tens ive dechlor inat ion o f h ighly-chlor ina ted P C B congeners was achieved, and the most abundant products f rom A r o c l o r 1260 dechlor inat ion were tetrachlorobiphenyls. A method to determine the identity o f one major dechlor inat ion product by us ing M S / M S was developed, and the dechlor inat ion product was found to be 24-24-tetrachlorobipheny 1. Some factors that cou ld affect the extent and rate o f P C B dechlor inat ion were investigated. It was found that autoclaving pre-incubated cultures pr ior to inocula t ion w i t h P C B - d e c h l o r i n a t i n g enrichment cultures increased the rate and extent o f P C B ii dechlor inat ion. It was also found that dechlor inat ion o f P C B s that had been present i n Reso lu t ion Island so i l for a number o f years was not as extensive as dechlor inat ion o f P C B s that were freshly added to Reso lu t ion Island so i l . A variety o f incubat ion temperatures were tested, and the most rapid and extensive P C B dechlor inat ion occurred at 30 ° C . The dechlor inat ion o f three nona- and four octa-chlorobiphenyl congeners, both s ingly , and i n various combinat ions, was also investigated. It was established that the dominant dechlor inat ion pathway invo lved the removal o f meta chlorines, a l though some para and some ortho dechlor inat ion was also observed. The rates and products o f P C B dechlor inat ion var ied w ide ly , depending on the mixture o f congeners present. T h i s is the first t ime the dechlor inat ion o f single octa- and nona-chlorobiphenyl congeners has been reported, and also the first t ime that the dechlor inat ion o f various combinat ions o f these congeners has been investigated. Extens ive degradation o f penta- and te t ra -CBs to b iphenyl was also found to occur. A method to detect a lkylated p o l y c y c l i c aromatic hydrocarbons ( a l k y l P A H s ) i n complex hydrocarbon mixtures was developed, and by us ing this method, the a l k y l P A H content o f several hydrocarbon mixtures was determined. Degradat ion o f the a l k y l P A H por t ion o f hydrocarbon-contaminated H a l l B e a c h (Nunavut) so i l was demonstrated. iii Table of Contents Abstract ii Table of Contents iv List of Tables viii List of Figures xi List of Abbreviations xiv Acknowledgements xvi Introduction - General 1 1. Introduction: PCBs 2 1.1 Structure and nomenclature 2 1.2 P h y s i c a l and chemica l properties 4 1.3 Format ion 5 1.4 Uses 6 1.5 P C B s as environmental contaminants 6 1.6 T o x i c i t y 7 1.7 Canad ian regulations 9 1.8 P C B s i n the Canad ian A r c t i c 10 1.9 M e t a b o l i s m o f P C B s 13 1.9.1 A e r o b i c degradation 13 1.9.2 A n a e r o b i c degradation 14 1.9.3 Sequential anaerobic/aerobic P C B degradation 16 1.10 Other destruction technologies 17 1.11 A n a l y s i s 18 1.11.1 Genera l 18 1.11.2 Ex t rac t ion 19 1.11.3 Separation 19 1.11.4 Detec t ion 21 1.12 Object ives 23 2 Materials and Methods for Investigation of PCB Dechlorination 24 2.1 Mate r ia l s 24 2.2 Sample co l lec t ion and storage 24 2.3 M e d i u m preparation 25 2.4 Pre- incubat ion and conf i rmat ion o f methanogenic condit ions 28 2.5 Ex t rac t ion o f P C B s from soi l and m e d i u m 29 2.5.1 Ex t rac t ion o f P C B s from s o i l 29 2.5.2 Ex t rac t ion o f P C B s f rom m e d i u m 31 2.5.3 Soxhle t extract ion o f P C B s f rom so i l 32 iv 2.6 W o r k - u p o f so i l extracts. 33 2.7 G a s chromatography 34 2.8 M a s s spectrometry and data interpretation 35 2.9 Qua l i ty control 42 3 Development of Anaerobic PCB Enrichment Cultures and Analysis of Sediment Cores 44 3.1 Deve lopment o f enrichment cultures 44 3.1.1 Different A r o c l o r mixtures as putative substrates w i th a variety o f inocu la 44 3.1.1.1 Introduction 44 3.1.1.2 Exper imenta l 45 3.1.2 U s i n g amendments to stimulate dechlor inat ion act ivi ty 46 3.1.2.1 Introduction 46 3.1.2.2 Exper imen ta l 47 3.1.3 Inocula t ion w i t h an A r o c l o r 1242 dechlorinator 48 3.1.4 A n a e r o b i c A r c t i c i n o c u l u m - Cambr idge B a y 49 3.1.5 A r c t i c i n o c u l u m - Saglek 49 3.2 Tes t ing for dechlor inat ing act ivi ty 50 3.3 Results and d iscuss ion 52 3.4 Cul tures selected for further w o r k 55 3.5 A n a l y s i s o f sediment cores f rom Cambr idge B a y 56 3.5.1 A i m 56 3.5.2 Exper imen ta l 56 3.5.3 Resul ts and discuss ion 57 4 The Effects of Different Enrichment Cultures, Autoclaving and Weathering on Aroclor 1260 Dechlorination 64 4.1 Introduct ion 64 4.2 Exper imenta l 65 4.2.1 Dech lo r ina t ion o f weathered and unweathered A r o c l o r 1260 i n Reso lu t ion Island so i l by us ing four different enrichment cultures 65 4.2.2 The effect o f autoclaving pr ior to inocula t ion on A r o c l o r 1260 degradation 67 4.2.3 Further experiments o n weathered and unweathered A r o c l o r 1260 i n Reso lu t i on Island so i l 68 4.2.4 Dech lo r ina t ion o f weathered A r o c l o r 1260 i n soils f rom Reso lu t ion Island and Saglek 70 4.3 Resul ts and discuss ion 70 4.3.1 Re la t ive rates o f weathered and unweathered A r o c l o r 1260 dechlor ina t ion 70 4.3.2 The effects o f autoclaving pr ior to inocula t ion on A r o c l o r 1260 degradation . . . 80 4.3.3 Further experiments on weathered and unweathered A r o c l o r 1260 i n Reso lu t ion Island so i l 85 4.3.4 Dech lo r ina t ion o f weathered A r o c l o r 1260 i n soils f rom Reso lu t ion Island and Saglek 89 4.3.5 Impl icat ions 92 5 Temperature Effects 93 5.1 Introduction 93 5.2 Me thods 93 5.3 Resul ts and discuss ion 94 6 Reductive Dechlorination of Nonachlorobiphenyls and Selected Octachloro-biphenyls 102 6.1 Introduction 102 6.2 Exper imenta l 103 6.3 Resul ts 103 6.4 D i s c u s s i o n I l l 7 Dechlorination of Selected PCB Congeners: Singly and in Combination 116 7.1 Introduction 116 7.2 Me thods 117 7.3 Resul ts and D i s c u s s i o n 119 7.3.1 Cul tures w i t h single octachlorobiphenyl congeners and various combinat ions o f octachlorobiphenyl congeners 119 7.3.2 D i scus s ion : octachlorobiphenyl congeners 125 7.3.3 Incubations w i t h single nonachlorobiphenyl congeners and various combinat ions o f nonachlorobiphenyl congeners 129 7.3.4 D i scuss ion : nonachlorobiphenyl congeners 130 7.3.5 Incubations w i t h a combina t ion o f four octa- and three nona-chlorobiphenyl congeners 132 7.3.6 D i scus s ion : a l l seven congeners i n combina t ion 135 7.3.7 Incubation o f decachlorobiphenyl (23456-23456-decaCB) at 30 ° C 136 7.3.8 Incubations w i t h pentachlorobiphenyl and tetrachlorobiphenyl congeners . . . . 136 7.3.9 D i scus s ion : penta- and tetra-chlorobiphenyl congeners 140 7.4 Summary 142 8 Identification of the Most Abundant Dechlorination Product Found in Aroclor 1260 Incubations, by Using MS/MS 143 8.1 Introduction 143 8.2 Exper imenta l 146 8.2.1 Op t imiza t i on o f C I D condit ions 146 8.2.1.1 Op t imiza t i on o f C I D exci ta t ion voltage 146 8.2.1.2 O p t i m i z a t i o n o f C I D exci tat ion t ime 147 8.2.2 The effect o f concentration on the fragmentation o f 24-24- and 245-2-tetrachlorobiphenyl 147 8.2.3 Da ta analysis 147 8.3 Results and discuss ion 148 8.3.1 The effect o f C I D exci tat ion amplitude on the fragmentation o f 24-24- and 245-2-tetrachlorobiphenyl 148 8.3.2 The effect o f C I D exci tat ion t ime on the fragmentation o f 24-24- and 245-2-tetrachlorobiphenyl 151 vi 8.3.3 The effect o f concentration o n the fragmentation on 24-24- and 245-2-tetrachlorobiphenyl 154 8.3.4 Identifying the major dechlor inat ion product present i n A r o c l o r 1260 incubations . . 156 9 Detection and Biodegradation of Alkyl PAHs in Arctic Soils 158 9.1 Introduction 158 9.1.1 Structure 158 9.1.2 Phys i ca l and chemica l properties 160 9.1.3 T o x i c i t y 161 9.1.4 Dis t r ibu t ion i n the environment 161 9.1.5 P A H s i n fuel/hydrocarbon contaminat ion 162 9.1.6 M e t a b o l i s m o f P A H s 163 9.1.7 M e t a b o l i s m o f a l k y l P A H s 165 9.1.8 A n a l y s i s 165 9.1.8.1 Separat ion 165 9.1.8.2 Retent ion index system 166 9.1.8.3 Detec t ion 167 9.1.9 D i e s e l fuel contaminat ion i n the A r c t i c 168 9.1.10 Object ives 168 9.2 Exper imenta l 168 9.2.1 Mater ia l s 168 9.2.2 M e t h o d development 169 9.2.3 S o i l sample co l lec t ion 170 9.2.4 S o i l extract ion 170 9.2.5 Sample analysis 171 9.2.6 P re l imina ry degradation experiments 172 9.2.7 M o d i f i e d degradation experiments for Saglek so i l 173 9.3 Results and discuss ion 174 9.3.1 O p t i m i z a t i o n o f exci tat ion voltages for M S / M S 174 9.3.2 B i o p i l e s 178 9.3.3 P re l iminary degradation experiments 180 9.3.4 M o d i f i e d degradation experiments for H a l l B e a c h so i l 184 9.4 Summary 184 10 Conclusions and Further Work 188 References 193 vii List of Tables Table 1-1: P h y s i c a l properties o f P C B homologs 4 Table 2-1: Components o f stock solutions used to make R A M M 26 Table 2-2: S o i l extraction results 30 Table 2-3: M e d i u m extraction results 32 Tab le 2-4: A r o c l o r 1260 concentrations i n soi ls 33 Table 2-5: P C B s eluted i n different eluent fractions 34 Table 2-6: P C B congeners identif ied i n this w o r k 36 Table 3-1: Ini t ial enrichment cultures 46 Table 3-2: Enr ichment cultures w i t h amendments 48 Tab le 3-3: Enr i chmen t culture results 53 Table 3-4: P C B s found i n Core 2 (Sewage outfall lake) 58 Table 3-5: P C B s found i n Core 3 (Sewage outfall lake) 60 Table 4-1 : Summary o f dechlor inat ion experiments described i n Sect ion 4.2.1 66 Tab le 4-2: Exper imen ta l pro tocol for determining the effect o f au toc laving o n A r o c l o r 1260 dechlor inat ion 68 Table 4-3: Dech lo r ina t ion o f weathered and unweathered A r o c l o r 1260 i n Reso lu t ion Island so i l 69 Table 4-4: Changes i n h o m o l o g dis tr ibut ion ( M o i %) i n B K 2 4 cultures 72 Tab le 4-5 : Changes i n h o m o l o g dis t r ibut ion ( M o i %) i n B K 3 0 cultures 73 Table 4-6: Changes i n h o m o l o g dis tr ibut ion ( M o i %) in B K 7 2 cultures 74 Table 4-7: Changes i n h o m o l o g dis tr ibut ion ( M o i %) i n B K 8 1 cultures 75 Table 4-8: Changes i n h o m o l o g dis tr ibut ion ( M o i %) i n controls 76 Table 4-9: Changes i n homolog dis tr ibut ion ( M o i %) in B K 8 1 cultures that were not autoclaved pr ior to inocula t ion 81 Table 4-10: Changes i n homolog dis tr ibut ion ( M o i %) i n controls that were not autoclaved pr ior to inocula t ion 82 Table 4-11: Changes i n h o m o l o g dis tr ibut ion ( M o i %) i n unweathered and weathered A r o c l o r 1260 i n Reso lu t i on Island so i l 86 viii Table 4-12: Changes i n homolog distr ibution ( M o i %) i n weathered A r o c l o r 1260 i n Saglek so i l 90 Table 4-13: Changes i n homolog dis tr ibut ion ( M o i %) i n weathered A r o c l o r 1260 i n Reso lu t ion Island so i l 91 Table 5-1: M o i percent distr ibution o f P C B homologs i n 21 ° C incubations 95 Table 5-2: M o i percent distr ibution o f P C B homologs i n 30 ° C incubations 95 Table 5-3: M o i percent distr ibution o f P C B homologs i n 37 °C incubations 96 Table 5-4: M o i percent dis tr ibut ion o f P C B homologs i n 42 ° C incubations 96 Table 6-1: M a j o r products formed i n B K 2 4 and B K 7 2 cultures after 16 weeks 105 Table 7-1: A m o u n t s and combinat ions o f congeners added to enrichment cultures i n different experiments 118 Table 7-2: M o i percent distr ibution over t ime o f single oc tachlorobiphenyl congener substrates and their dechlor inat ion products 119 Table 7-3: A m o u n t (jag) o f octachlorobiphenyl substrate dechlorinated after 8 weeks o f incubat ion 120 Tab le 7-4: M o i percent dis tr ibut ion over t ime o f octachlorobiphenyl substrate mixtures and their products 121 Table 7-5: M o i percent dis tr ibut ion o f single nonachlorobiphenyl congener substrates and their dechlor inat ion products 129 Table 7-6: M o i percent distr ibution o f nonachlorobiphenyl congener substrate mixtures and their dechlor inat ion products. 130 Table 7-7: M o i percent dis tr ibut ion o f four o c t a C B and three n o n a C B substrates and their dechlor inat ion products 133 Table 7-8: M o i percent dis tr ibut ion over t ime o f decachlorobiphenyl substrate 136 Table 7-9: M o i percent dis tr ibut ion over t ime o f single penta- and te t ra -CB congener substrates and their dechlor inat ion products 137 Table 7-10: M o i percent distr ibution over t ime o f 246-24-pentaCB, 246-2- te t raCB and 24-24- te t raCB substrate mixtures and their dechlor inat ion products 138 Table 7-11: M o i percent dis tr ibut ion o f 246-24-pentaCB and 2345-2346-oc tasCB and their dechlor inat ion products 140 Table 9-1: S u m m a r y o f some properties o f P A H s 160 IX Table 9-2: Concentrat ions o f some P A H s found in crude and refined oi ls 163 Table 9-3: M / z values used to select for a l k y l P A H s in the ion trap 170 Table 9-4: E x c i t a t i o n voltages selected for compound identif icat ion 177 Table 9-5: A l k y l P A H s found i n so i l f rom Cambr idge B a y biopi les 179 Table 9-6: A l k y l P A H s found i n jet fuel 181 Table 9-7: A l k y l P A H s found i n H a l l B e a c h so i l 185 x List of Figures Figure 1-1: B i p h e n y l molecule showing number ing o f a l l ten possible ch lor ina t ion sites .2 F igure 1-2: 24-26-tetrachlorobiphenyl ( I U P A C no. 51) 3 F igure 1-3: Structures o f 3,4-3,4-tetraCB and 2,3,7,8- tetrachlorodibenzo-p-dioxin 8 F igure 1-4: M a p o f Canada 12 Figure 1-5: A e r o b i c P C B degradation 14 Figure 1-6: A n a e r o b i c P C B dechlor inat ion 15 Figure 1-7: M a s s spectra o f P C B homologs 22 Figure 2-1: A r o c l o r 1260 chromatogram .41 Figure 3-1: Changes i n P C B congener dis tr ibut ion after treatment w i t h i n o c u l u m B K 8 1 . . 51 Figure 4-1: Disappearance over t ime o f penta-, hexa- and hepta-chlorobiphenyl congeners o f both weathered and unweathered A r o c l o r 1260 i n so i l treated w i t h two different enrichment cultures, B K 2 4 and B K 8 1 77 Figure 4-2: Disappearance o f h e x a C B s i n autoclaved and unautoclaved soi ls over a per iod o f 8 weeks. (Results are presented for both weathered and unweathered A r o c l o r 1260) 83 Figure 4-3: Disappearance o f hep taCBs i n autoclaved and unautoclaved soi ls over a per iod o f 8 weeks. (Results are presented for both weathered and unweathered A r o c l o r 1260) 84 Figure 4-4: Appearance o f t e t raCB congeners over an incubat ion per iod o f 8 weeks i n Reso lu t ion Island soi ls containing weathered and unweathered A r o c l o r 1260 87 Figure 4-5: Disappearance o f (a) h e x a C B s and (b) hep taCBs i n weathered and unweathered A r o c l o r 1260 over an incubat ion per iod o f 8 weeks 88 Figure 5-1: Disappearance o f A r o c l o r 1260 components over t ime at different temperatures 98 Figure 5-2: Appearance o f dechlor inat ion products over t ime at different temperatures . . . 99 Figure 5-3: Degradat ion rates o f A r o c l o r 1260 at different temperatures 100 Figure 6-1: Dech lo r ina t ion o f 23456-234-oc taCB after 16 weeks 106 XI Figure 6-2: Dech lo r ina t ion products, o f 2345-2346-oc taCB i n a B K 2 4 culture over t ime. . 107 F igure 6-3: Dech lo r ina t ion o f 2345-2346-oc taCB after 16 weeks 108 Figure 6-4: Dech lo r ina t ion products o f 234456-245-oc taCB i n a B K 2 4 culture over t ime 109 Figure 6-5: Dech lo r ina t ion o f 23456-245-oc taCB after 16 weeks 110 Figure 6-6: Proposed order o f removal o f the first three chlorines f rom 23456-234-o c t a C B 112 Figure 7-1: Disappearances o f 23456-234-oc taCB and 2345-2356-oc t aCB when incubated s ing ly and i n combina t ion w i t h other congeners 122 Figure 7-2: Disappearances o f 2345-2346-oc taCB and 23456-245-oc t aCB when incubated s ing ly and i n combinat ion w i t h other congeners 123 F igure 7-3: Disappearance over t ime o f a l l four o c t a C B congeners together i n the same incubat ion 124 Figure 7-4: 23456-234-oc taCB and 2345-2356-oc taCB 126 Figure 7-5: 2345-2346-oc taCB and 23456-245-oc taCB 127 Figure 7-6: Disappearances o f n o n a C B s when incubated s ingly and i n combina t ion . . 131 F igure 7-7: Disappearances o f o c t a C B and n o n a C B congeners when incubated s ing ly and i n combina t ion 134 Figure 7-8: Dech lo r ina t ion o f 246-24-pentaCB, 24-24- te t raCB and 24-26- te t raCB, showing format ion o f b iphenyl as the major dechlor inat ion product 139 Figure 8-1: Structures o f 24-24- te t raCB and 245-2- te t raCB 143 F igure 8-2: M a s s spectra o f (a) 24-24- te t raCB and (b) 245-2- te t raCB under E I operat ing condi t ions for the V a r i a n Saturn ion trap G C / M S 145 Figure 8-3: Fragmentat ion o f (a) 24-24- te t raCB and (b) 245-2- te t raCB w i t h different resonant exci ta t ion energies 149 Figure 8-4: Fragmentat ion o f (a) 24-24- te t raCB and (b) 245-2- te t raCB w i t h different non-resonant exci ta t ion energies 150 Figure 8-5: Fragmentat ion o f (a) 24-24- te t raCB and (b) 245-2- te t raCB, w i t h 0.6 V resonant exci ta t ion energy, as a function o f exci tat ion t ime 152 xii Figure 8-6: Fragmentat ion patterns for (a) 24-24- te t raCB and (b) 245-2- te t raCB, w i t h 100 V non-resonant exci ta t ion energy, as a function o f exci tat ion t ime 153 Figure 8-7: Fragmentat ion o f (a) 24-24- te t raCB and (b) 245-2- te t raCB w i t h different concentrations 155 Figure 8-8: Fragmentat ion o f 24-24- te t raCB, the major product f rom A r o c l o r 1260 incubations, and 245-2- te t raCB, w i t h a non-resonant exci tat ion energy o f 100 V and an exci ta t ion t ime o f 30 ms 157 Figure 9-1: C h e m i c a l structures o f some unsubstituted P A H 159 Figure 9-2: C h e m i c a l structures o f some alkyl-substi tuted P A H s 159 Figure 9-3: T y p i c a l ox ida t ion pathways for P A H s 164 F igure 9-4: M a s s spectra obtained for 1-methylnaphthalene by us ing different resonant exci ta t ion voltages 175 Figure 9-5: M a s s spectra obtained for 3,6-dimethylphenanthrene by us ing different resonant exci ta t ion voltages 176 Figure 9-7: T o t a l i o n chromatogram o f jet fuel before and after incubat ion w i t h a jet fuel enrichment culture 182 Figure 9-8: Selected ion moni tor ing o f d imethy l - and monoethyl-naphthalenes present i n jet fuel before and after incubat ion for four weeks w i t h an enrichment culture at 4 ° C .183 Figure 9-9: Percent o f or ig ina l C2-naphthalenes remaining i n H a l l B e a c h so i l after 8 weeks o f incubat ion w i t h a H a l l B e a c h enrichment culture at 4 ° C 186 Figure 9-10: Percent o f or iginal C3-naphthalenes remaining in H a l l B e a c h so i l after 8 weeks o f incubat ion w i t h a H a l l B e a c h enrichment culture at 4 ° C 186 Figure 9-11: Percent o f or ig ina l monomethylphenanthrenes remain ing i n H a l l B e a c h so i l after 8 weeks o f incubat ion w i t h a H a l l B e a c h enrichment culture at 4 ° C 187 Figure 9-12: Percent o f or ig inal C2-phenanthrenes remaining i n H a l l B e a c h so i l after 8 weeks o f incubat ion w i t h a H a l l Beach enrichment culture at 4 ° C 187 xiii List of Abbreviations A G C automatic gain control A h aryl hydrocarbon B . C . B r i t i s h C o l u m b i a b.p. b o i l i n g point C B ch lorobiphenyl C E P A Canad ian Envi ronmenta l Protect ion A c t C I D co l l i s ion- induced dissociat ion C I - M S chemica l ion iza t ion - mass spectrometry D E W Distant E a r l y W a r n i n g E C D electron capture detector E P A Envi ronmenta l Protect ion A g e n c y F I D flame ion iza t ion detector G C gas chromatograph(y) G C / M S gas chromatography / mass spectrometry H P L C h igh performance l i q u i d chromatography i .d . internal diameter I U P A C International U n i o n o f Pure and A p p l i e d Chemis t ry m.p. mel t ing point M S mass spectrometry M S / M S mass spectrometry / mass spectrometry M W molecular weight n.a. not available n.d. not detected N R C N a t i o n a l Research C o u n c i l (Canada) P A H ( s ) p o l y c y c l i c aromatic hydrocarbon(s) P C B ( s ) polychlor inated biphenyl(s) P D A photodiode array p p m parts per m i l l i o n R A M M reduced anaerobic minera l m e d i u m xiv r.f. radio frequency S aqueous solubi l i ty S D standard deviat ion S I M selected i o n moni tor ing T I C total i o n current U B C Un ive r s i t y o f B r i t i s h C o l u m b i a uv ultra v io le t uv-v i s ultra v io le t -v i s ib le Acknowledgements I would like to express my sincere thanks to my research supervisors, Dr. Bi l l Cullen and Dr. Bi l l Mohn, for their guidance, encouragement and support during the course of my PhD. I would also like to thank the members of my supervisory committee, Dr. David Chen and Dr. Giinter Eigendorf for their interest and support. Dr. Ken Reimer gave me the opportunity to go on several exciting trips to the Arctic, and I am extremely grateful to him for this. I would also like to thank Dr. Barbara Zeeb for assistance with collecting the sediment cores from Cambridge Bay, and Mr. Gordon Stewart for supplying microbial enrichment cultures for the alkyl P A H work. I am also grateful to Dr. Elena Polishchuk in Biological Services for always being interested in helping me, and to Mr. Rob Christian for assistance in solving problems with the Saturn ion trap. Many present and past members of my research group have helped me both professionally and by their friendship, these include Vivian Lai, Iris Koch, Andrew Mosi, and Chris Simpson. Other friends in the chemistry department that deserve a special mention for their efforts at enticing me away from my work include Elizabeth Cheu, Gay Yuyitung and Krystyna Skupinska. I would also like to thank the many other friends I have made during my stay in Canada for making my time here so enjoyable, and Peter Awram and my family for their constant encouragement and support. xvi Introduction General Envi ronmen ta l contaminants are substances that cause a net detrimental effect on the environment. M a n y different contaminants are present i n the environment i n great quantities, and m u c h effort and research is usual ly required i n order to f ind effective ways o f r emov ing or remediat ing these contaminants. Th is thesis is concerned w i t h two different classes o f environmental contaminants, polychlor inated biphenyls ( P C B s ) and alkylated p o l y c y l i c aromatic hydrocarbons (a lky l P A H s ) . These classes o f chemicals are both considered tox ic , and both are present as pollutants i n the Canad ian A r c t i c . Th i s thesis investigates some aspects o f the mic rob ia l degradation o f these chemicals , w i t h a v i e w to the eventual remediat ion o f contaminated sites. 1 1. Introduction: Polychlorinated Biphenyls (PCBs) 1.1 Structure and nomenclature Polychlor ina ted biphenyls ( P C B s ) are a class o f chlorinated hydrocarbons that have two to ten chlor ine atoms attached to a b iphenyl nucleus (Figure 1-1). St r ic t ly speaking, monochlor ina ted biphenyls are not polychlor inated biphenyls ; however , they are members o f the same chemica l class, and U . S . court rul ings have declared monochlorob iphenyls to be P C B s for E P A enforcement purposes (1 ,2 ) . Figure 1-1: Biphenyl molecule showing numbering of all ten possible chlorination sites. There are 209 different mono- and poly-chlor inated b iphenyl compounds possible , and these are referred to as congeners. W h e n P C B s are grouped according to the degree o f chlor inat ion, these groups are ca l led homologs . (e.g. the tetrachlorobiphenyl homologs , the pentachlorobiphenyl homologs) . P C B s o f a g iven h o m o l o g w i t h different chlorine substitution posi t ions are isomers. Ind iv idua l P C B congeners are c o m m o n l y g iven a systematic name that identifies the posi t ions o f chlor ine substitution and the degree o f chlor inat ion. Several different convent ions can be used i n assigning systematic names. F o r example , 2 ,2 ' ,4 ,6 ' -2 te trachlorobiphenyl , 2 ,4 ,2 ' ,6 ' - te t rachlorobiphenyl and 24-26-tetrachlorobiphenyl a l l refer to the same congener. The systematic names used i n this thesis list the chlor ine substitution posi t ions on the first r ing , separated by a hyphen f rom the chlor ine substitution posi t ions on the second r ing. One example, 24-26-tetrachlorobiphenyl (24-26- te t raCB) is shown i n Figure 1-2. There are also two different number ing systems i n use for P C B congener identif icat ion, both o f w h i c h arrange a l l the congeners i n order and assign them a number from 1 to 209. The first system was proposed by Ba l l s chmi te r and Z e l l i n 1980 (3). The second number ing system (the I U P A C one) differs f rom the Ba l l schmi te r and Z e l l system for 11 o f the 209 congeners. Th i s thesis uses systematic names and the I U P A C numbering system to identify P C B congeners (Figure 1-2). CI CI CI Figure 1-2: 24-26-tetrachlorobiphenyl (IUPAC no. 51). 3 1.2 Physical and chemical properties P C B s have l o w water solubi l i t ies and l o w vapour pressures, and are non-polar and l i poph i l i c . A summary o f key phys ica l properties o f P C B s , monochlorob iphenyls and b iphenyl is presented i n Table 1-1. Table 1-1: Physical properties of PCB homologs. Adapted from Erickson (4). Homolog Melting Boiling Vapour Water Log octanol-group point (°C) point (°C) pressure solubility at water (Pa) at 25°C 25°C (g/m3) partition coefficient B i p h e n y l 71 256 4.9 9.3 4.3 M o n o C B s 25-77.9 285 1.1 4.0 4.7 D i C B s 24.4-149 312 0.24 1.6 5.1 T r i C B s 28-87 337 0.054 0.65 5.5 T e t r a C B s 47-180 360 0.012 0.26 5.9 P e n t a C B s 76.5-124 381 2.6 x 10"3 0.099 6.3 H e x a C B s 77-150 400 5.8 x 10" 4 0.038 6.7 H e p t a C B s 122.4-149 417 1.3 x 10" 4 0.014 7.1 O c t a C B s 159-162 432 2.8 x 10"5 5.5 x 10" 3 7.5 N o n a C B s 182.8-206 445 6.3 x 10"6 2.0 x l O " 3 • 7.9 D e c a C B 305.9 456 1.4 x 10"6 7.6 x 10"4 8.3 4 1.3 Formation There are no k n o w n natural sources o f P C B s . P C B s were first synthesized at the end o f the nineteenth century, commerc ia l product ion began i n 1929, and large-scale product ion started i n 1945. Product ion i n N o r t h A m e r i c a and Europe was stopped i n the late 1970s after P C B s were banned i n those places, but product ion i n Eastern Europe and Russ i a cont inued unt i l the early 1990s (5). C o m p l e x mixtures conta ining many different degrees o f ch lor ina t ion were synthesized by bu lk chlor inat ion o f b iphenyl w i t h chlor ine gas. The average degree o f chlor inat ion i n the product mixture depended either on the durat ion o f the chlor inat ion process or on fractional d is t i l la t ion o f the product mixture (6). Reac t ion condi t ions tended to favour specific substitution reactions, so mixtures have typica l congener composi t ions . A variety o f trade names were used for these commerc ia l mixtures , depending on w h i c h company produced them. Some o f the most c o m m o n trade names used were A r o c l o r , Kanech lo r , C l o p h e n , Phenoclor and Fenchlor . M o s t o f the P C B s used i n Canada were produced by Monsan to Industrial Chemica l s C o m p a n y i n the U S A , under the trade name A r o c l o r . The names o f particular A r o c l o r mixtures reflect the average degree o f ch lor ina t ion o f the congeners i n that mixture . F o r example, A r o c l o r 1260 contains 6 0 % chlor ine by mass, w h i l e A r o c l o r 1221 contains on ly 2 1 % chlor ine by mass. The exact compos i t ion o f A r o c l o r mixtures var ied sl ight ly from batch to batch, but it has been found that batch to batch var ia t ion is not very significant (7). A r o c l o r 1260 has been found to contain over 75 different congeners, and consists o f approximate ly 12% pentachlorobiphenyls , 4 2 % hexachlorobiphenyls , 3 8 % heptachlorobiphenyls , 7% octachlorobiphenyls and 1% nonachlorobiphenyls (8, 9). 5 PCBs can also be formed inadvertently as by-products in a wide variety of chemical processes that contain chlorine and hydrocarbon sources (10). 1.4 Uses When PCBs were first produced, they were hailed as "the new wonder chemical". Among other things, they are very stable chemically and thermally, and have high electrical resistance. As a consequence of these properties, PCBs had a wide variety of industrial applications. They were commonly used as dielectric fluids in capacitors and transformers, and also as heat transfer fluids, lubricating oils, hydraulic fluids, fire retardants, paint additives, and in pesticides, carbonless copy paper, adhesives and plastics (11). 1.5 PCBs as environmental contaminants Because of their extensive use and stability, there is widespread PCB contamination in the environment. It has been estimated that approximately 1 billion kilograms of PCBs have been released into the environment (12). There is a fairly uniform global distribution of PCBs, with localized hot spots of contamination, usually due to local environmental inputs. Widespread, low-level releases on a global scale have occurred through non-contained PCB uses such as in inks. There are also many examples of localized PCB spills from, for example, transformer oil fluid, which have lead to areas with high localized levels of contamination. Because of their low volatility and low water solubility, over 99% of environmental PCB mass is found in soil (12). PCBs in the environment can be transported by air, water and animals, including fish and birds. The 6 primary mode of global PCB distribution has been atmospheric transport (13, 14). One theory, referred to as the cold condensation theory, suggests that the earth acts as a giant condenser, wherein PCBs from warmer, more tropical areas are volatilized and then condense and accumulate in colder, polar regions (15, 16). PCBs can truly be called ubiquitous pollutants as they have been found almost everywhere, in air, water and soil, as well as in almost all the animal species that have been investigated, especially in birds (17) and mammals, including humans (18). PCBs do not degrade readily in the environment and they are lipophilic, so they tend to bioaccumulate and magnify in the food chain (19). Uptake and retention of individual PCB congeners in animals depends on the animal species involved, and differences in susceptibility to degradation and bioaccumulation depend on the structural arrangements of particular congeners (20). Human exposure to PCBs is mainly via low-level food contamination (21). Arctic inhabitants have among the highest levels of PCBs recorded, due to their exposure to PCBs through the Arctic food chain (22, 23). There has been a significant decline in environmental and human PCB levels since their production was banned in the late 1970s (24, 25). 1.6 Toxicity PCB toxicity is a subject of much controversy. It has been shown that toxicity varies widely with both the homolog class and the individual congener concerned (26-28). It also depends on the level of PCB exposure, and interactive effects with other PCBs and other chemicals (29). Since PCBs are broad-acting toxins (i.e. they can have 7 a wide range o f tox ic effects), accurate r isk assessment has been elusive (30). P C B tox ic i ty is often d iv ided into two m a i n classes: D i o x i n - l i k e effects, and n o n d i o x i n - l i k e effects. Dioxin-like effects: Cop lanar P C B s (i.e. non- or mono-ortho substituted congeners) have d i o x i n - l i k e tox ic effects. The most tox ic P C B s i n this class contain 2 para, 2 meta, and no ortho chlor ines (31), and are very s imi la r i n structure to 2,3,7,8-tetrachlorodibenzo-/?-d i o x i n (Figure 1-3). The toxic i ty is the result o f the P C B b ind ing to, and activating, a cytosol ic A h receptor protein. Th i s affinity for the A h receptor may be a consequence o f the relat ively h i g h electron affinities o f these planar P C B s (32). T y p i c a l d i o x i n - l i k e effects i n rodents include chloracne, dermal lesions, body weight loss, thymic atrophy, immunosuppress ion , hepatotoxicity, reproductive and developmental tox ic i ty , endocrine disrupt ion, and induct ion o f cytochrome P450 enzymes. CI CI o Non dioxin-like effects: The non-d iox in - l ike toxic effects o f P C B s observed i n laboratory animals inc lude neurotoxici ty, carcinogenici ty, endocrine disrupt ion (5, 34), and disrupt ion o f thyro id hormone homeostasis (30). 8 Some reproductive and developmental problems in particular wildlife populations appear to be related to PCB contamination (33). In humans, increased levels of some serum enzymes, chloracne and dermal lesions, possible hepatic damage, and respiratory problems have been linked to PCB exposure (29). Studies on occupational PCB exposure and cancer have not provided conclusive evidence that this exposure caused increased incidences of cancer (35), however, these studies have been subject to a lot of criticism (36). Recent studies suggest that PCB mixtures can induce liver tumours in rats, and that different Aroclor mixtures have different potencies (37). 1.7 Canadian regulations PCB levels in Canada are stringently regulated because of concerns about potential toxicity. Soils containing PCB levels above 50 ppm are regulated by the Canadian Environmental Protection Act and have to be treated as hazardous waste. The Arctic Environmental Strategy's Action on Waste component led to a two-tiered DEW Line Cleanup Criterion for PCBs which requires that anything containing PCB levels above 5 ppm (Tier II) must be removed from contact with the Arctic ecosystem. Arctic PCB levels between 1 and 5 ppm (Tier I) may be landfilled. The Canadian water quality guideline recommended for the protection and maintenance of coastal and estuarine aquatic organisms is a maximum concentration of 0.01 ug/L, and the Canadian freshwater PCB guideline is 0.001 ug/L (38). 9 1.8 PCBs in the Canadian Arctic There is evidence o f widespread P C B contaminat ion i n the Canad ian A r c t i c . P C B s have been found i n A r c t i c plants (39-41), invertebrates and fish (42, 43), mammals (44, 45), and humans (22). Breast-fed Inuit infants have been found to have P C B b lood levels that are be l ieved to be causing h igh immune response impairment and increased infect ion rates (46). Some o f this P C B contaminat ion cou ld be due to long-range atmospheric transport o f P C B s into the A r c t i c region (15, 16, 41), but there are also numerous abandoned mi l i t a ry sites that contain h igh levels o f P C B contaminat ion (47). P C B s were c o m m o n i n m u c h o f the electr ical equipment used at the radar stations w h i c h formed the Distant Ea r ly W a r n i n g ( D E W ) sites and the Polevaul t L i n e . There is evidence to suggest that these mi l i t a ry sites are acting as a loca l source for the aerial transport o f P C B s to the nearby tundra (48). Reso lu t ion Island (Nunavut , Canada, 6 2 ° N , 6 5 ° W ) and Saglek (Labrador, Canada, 5 8 ° N , 6 4 ° W ) , both former mi l i t a ry radar facil i t ies, are two areas that have considerable quantities o f so i l contaminated w i t h A r o c l o r 1260. The locat ion o f these sites is shown i n F igure 1-4. Reso lu t ion Island was abandoned i n 1974, and studies conducted i n 1993 show that, almost 20 years later, Reso lu t ion Island contains an estimated 2801 m o f so i l containing P C B s over the 50 p p m l i m i t regulated by the Canad ian Env i ronmen ta l Protect ion A c t ( C E P A ) , i nc lud ing some areas w i t h P C B levels above 500ppm. A further estimated 4215 m 3 o f so i l is over the T ie r II D E W L i n e Cleanup Cr i t e r ion o f 5 p p m (wh ich means it must be removed f rom contact w i t h the 10 A r c t i c ecosystem), and an addit ional 4011 m o f soi l is over the T ie r I D E W L i n e Cleanup Cr i t e r ion o f l p p m . S imi l a r ly , it has been estimated that Saglek has over 1000 m 3 o f so i l conta ining P C B s above the 50 p p m C E P A leve l , and over 4 4 0 0 m 3 o f so i l between 1 and 50 ppm. A s w e l l as this, analysis o f sculpins col lected o f f shore f rom Saglek indicate that P C B s from the beach have made their way into the marine food cha in (47). A l t h o u g h Saglek lies South o f 6 0 ° N , its cl imate and ecology are typ ica l o f A r c t i c regions. Because the remote locations o f these sites make so i l transportation costs p roh ib i t ive ly expensive, the feasibil i ty o f on-site b ioremediat ion o f P C B contaminat ion is being investigated. A large por t ion o f the w o r k presented i n this thesis was performed as part o f this b ioremedia t ion project. 11 12 1.9 Metabolism of PCBs A t one t ime it was thought that P C B s d id not undergo biodegradation. H o w e v e r , i n the past 15 to 20 years, it has been shown that mic rob ia l degradation o f P C B s is possible. There are two very different classes o f P C B mic rob ia l degradation: aerobic and anaerobic. 1.9.1 Aerobic degradation A e r o b i c P C B degradation has been studied i n detail , and is covered i n many reviews (49-51). The pathway o f aerobic P C B degradation is out l ined i n F igure 1-5. Aerobes oxygenate the carbon r ing , and sometimes minera l ize the compounds complete ly . A e r o b i c P C B degraders are generally only able to degrade P C B congeners w i t h up to four chlorines on the b iphenyl r ing (52, 53), however, some aerobic bacterial strains have been found to be able to degrade selected penta-, hexa- and even a few hepta- chlorobiphenyls as w e l l (54, 55). The poss ib i l i ty o f aerobic P C B degradation is determined and l imi t ed by the posi t ions o f chlor inat ion. M o s t aerobic P C B degraders u t i l i ze 2,3-dioxygenase attack, so this site on the b iphenyl r ing needs to be avai lable , i.e., not chlorinated. Some aerobic P C B degraders are also capable o f 3,4-dioxygenase attack on the b iphenyl r ing (56, 57). A e r o b i c bacteria are not capable o f degrading the vast majority o f congeners present i n A r o c l o r 1260. 13 1: Dioxygenase , 0 2 , N A D P H 2: Dehydrogenase 3: Dioxygenase , O2, N A D P H 4: Hydratase, H 2 0 Figure 1-5: Aerobic PCB degradation. F u n g i have also been reported to aerobical ly degrade P C B s (58, 59). H o w e v e r , this act ivi ty is on ly observed at relat ively l o w concentrations (up to 10 ppm) (60). 1.9.2 Anaerobic degradation M o s t o f the more-highly chlorinated P C B congeners ( in general, those w i t h more than 4 chlor ine substituents) require dechlor inat ion before aerobic degradation can take place (61). Dech lo r ina t ion (shown i n Figure 1-6) requires anaerobic condi t ions , and has been observed both in the f ie ld and i n the laboratory (62-65). Anae rob ic degradation does not destroy P C B s , but turns them into less-highly chlorinated congeners. The anaerobic m i c r o b i a l dechlor inat ion o f P C B s i n the environment was first d iscovered over ten years ago (66), but studies that show the anaerobic degradation o f A r o c l o r 1260 under control led condi t ions , and reveal the effectiveness o f P C B enrichment cultures on 14 A r o c l o r 1260-contaminated anaerobic sediments, have been publ i shed on ly recently (65, 67-69). R H R" Figure 1-6: Anaerobic PCB dechlorination. The predominant sites o f dechlor inat ion are at the meta and para posi t ions o f the b ipheny l r ing . M a n y different dechlor inat ion patterns have been discovered, and these have been classif ied according to the type o f dechlor inat ion act ivi ty observed (70). F o r example , "process N " dechlor inat ion is defined as removal o f doubly and s ing ly f lanked meta chlorines only , wh i l e "process M " dechlor inat ion refers to r emova l o f a l l meta chlorines (71). These different activit ies that are observed are thought to be due to the presence o f different bacterial populat ions w i t h different P C B dechlor inat ion capabil i t ies. In general, rates o f P C B dechlor inat ion decrease as the degree o f ch lor ina t ion increases. The most l i m i t i n g resource to microorganisms i n anaerobic environments is often the presence o f electron acceptors that w i l l a l l o w these organisms to o x i d i z e the abundant reduced substrates that usual ly accumulate i n anaerobic environments. P C B -dechlor inat ing microorganisms are thought to use P C B s as terminal electron acceptors, a l l o w i n g re-oxidat ion o f metabolic intermediates. Dechlor ina t ion consumes electrons, 15 and thus P C B s are a resource for microorganisms that have the enzymatic capacity to transfer electrons to them. It is very diff icul t to isolate active dechlorinators (61) and attempts to isolate P C B - d e c h l o r i n a t i n g microorganisms have not yet been successful. A r o c l o r 1248 spiked into sediment has been successfully dechlorinated i n an anaerobic bioreactor system (72). Several studies have investigated factors inf luencing the rate o f P C B dechlor inat ion, and these have been rev iewed i n detai l (70). Recent ly , studies have investigated the addi t ion o f various amendments such as FeSC>4 (73), various P C B congeners (74), brominated biphenyls (75, 76) and halogenated aromatics (77) i n order to stimulate anaerobic P C B dechlorinat ion. A n a e r o b i c dechlor inat ion does not destroy P C B s completely , but it has been s h o w n to s ignif icant ly reduce the tox ic i ty o f P C B s (78, 79), and it makes P C B s avai lable for further degradation by aerobic P C B degraders. 1.9.3 Sequential anaerobic/aerobic P C B degradation In order to be minera l ized completely, h igh ly chlorinated P C B s need to undergo two sequential m i c r o b i a l degradation processes. The first process, w h i c h is anaerobic, transforms the h igh ly chlorinated P C B s into less-highly chlorinated P C B s . In the second (subsequent) process, these less-highly chlorinated P C B s can be degraded further, by aerobic P C B degraders. The successful anaerobic/aerobic sequential degradation o f A r o c l o r 1242 has been reported (80). Successful sequential degradation o f A r o c l o r 1260 has not yet been reported. 16 1.10 Other destruction technologies M a n y non-b io log ica l destruction technologies for P C B s have been investigated, and on ly a very b r i e f ove rv i ew is g iven here. The most c o m m o n l y used disposal technology is incinerat ion. Th i s requires very carefully control led temperatures i n order to a v o i d the formation o f other tox ic compounds such as chlorinated d iox ins and chlorinated furans. A variety o f chemica l ways to degrade P C B s have also been investigated. F o r example , the complete and rapid dechlor inat ion o f A r o c l o r s 1254 and 1260 by us ing a pa l l ad ium- i ron b imeta l l i c system has been reported (81, 82). Patents exist for treating P C B - c o n t a m i n a t e d groundwater by us ing i ron f i l ings (83), and for treating P C B s by adding hydrogen transfer agents such as potassium formate i n the presence o f a catalyst such as p a l l a d i u m (84), but there is no publ ished information on the successful treatment' o f P C B - c o n t a m i n a t e d so i l w i t h any o f these systems. S o m e alternative disposal technologies combine chemica l and b i o l o g i c a l methods. These technologies generally use a chemica l method to dechlorinate P C B s ( in order to make them more susceptible to aerobic mic rob ia l P C B degradation), f o l l owed by b io log i ca l treatment w i t h aerobic P C B - d e g r a d i n g cultures (85). In one method, A r o c l o r 1260 i n transformer o i l has been dechlorinated by uv irradiation, and the dechlorinated products are then degraded by aerobic bacteria (86). There are no literature reports on the treatment o f contaminated so i l by us ing this method. 17 1.11 Analysis 1.11.1 General There is a w ide variat ion i n the complex i ty o f P C B analyses performed. The simplest P C B analyses give a total P C B concentration, and this total concentration is usual ly based on the quantif ication o f a few selected compounds i n a part icular P C B mixture . T h i s method depends on the correct identif icat ion o f the P C B mixture concerned (e.g. A r o c l o r 1260 or A r o c l o r 1254), and assumes the overa l l congener d is t r ibut ion is very s imi la r to the standard P C B mixture identif ied. T h i s assumption is often not v a l i d i n environmental samples, and this method o f P C B quantif icat ion is real ly on ly semi-quantitative. M o r e detailed P C B analyses can group P C B s accord ing to the number o f chlor ines on the b iphenyl r ing , and give the total concentrations for each h o m o l o g class. The most detailed P C B analysis is congener-specific analysis, i n w h i c h every ind iv idua l P C B congener is identif ied and quantified separately. Congener-specif ic analysis o f A r o c l o r mixtures poses a challenge, as these mixtures consist o f many compounds w i t h very s imi la r structures, so there is often not m u c h difference i n the e lu t ion characteristics or mass spectra o f the compounds that need to be dis t inguished. Congener-specif ic analysis is l imi ted by the degree o f separation o f congeners, and almost a l l methods used for separating P C B congeners f rom each other s t i l l result i n several co-elut ing congeners for the complex A r o c l o r mixtures. In these cases, congener-specific analysis reports and identifies each P C B peak ind iv idua l ly , but some peaks represent several congeners. Env i ronmenta l samples are often signif icantly changed f rom commerc i a l formulat ions because o f weathering effects such as vo la t i l i za t ion and redis tr ibut ion o f 18 less h igh ly chlorinated congeners, and i n some cases because o f mic rob ia l degradation. Because o f these differences, and the fact that the tox ic i ty o f P C B s varies w i d e l y between different congeners, there is a need to think o f P C B s i n terms o f ind iv idua l congeners rather than complex mixtures , and so congener specific analysis should be the analysis technique o f choice wherever possible. The analyt ical chemistry o f P C B s has been covered extensively i n the literature (4), so only a b r i e f ove rv iew w i t h emphasis o n topics relevant to this thesis is g iven here. 1.11.2 Extraction P C B s i n environmental samples are extracted by several different techniques, depending largely o n the m e d i u m to be extracted. S o i l and sediment samples are often extracted w i t h s imple solvent extraction techniques (i.e. shaking or sonication), but many other techniques such as Soxhlet extraction, supercri t ical f lu id extraction, thermal desorption and microwave-assis ted extraction have also been used (87-90). Water samples are c o m m o n l y extracted us ing l i q u i d - l i q u i d extraction techniques (91), but other techniques such as solid-phase extraction have also been shown to be effective (92). A n i m a l and plant tissue extraction techniques include acetonitrile back part i t ion (93), solvent extraction (94) and base digest ion (95). 1.11.3 Separation Since many P C B isomers have very s imi lar structural characteristics, it is a challenge to m a x i m i z e the separation o f P C B isomers so as to obtain as many w e l l -defined peaks as possible. Temperature-programmed gas chromatography is usual ly the 19 method o f choice. Several different co lumns can be used for P C B separation. The most w i d e l y used co lumns are those w i t h bonded non-polar methyl s i l icone (e.g. D B - 1 ) or 5 % phenyl me thy l s i l icone (e.g. D B - 5 , SE-54 ) phases. Different types o f co lumns can cause different P C B combinat ions to co-elute, so some separation techniques use mul t ip le co lumns (96-98). Peaks have also been k n o w n to change retention order, depending o n the type o f c o l u m n used (99). Other techniques that are concerned on ly w i t h specific congeners can use specific columns i n order to enhance separation o f the congeners o f interest (for example , the planar, d iox in - l i ke P C B s are often separated from a larger mixture) (100, 101). It is possible to purchase a l l 209 P C B congeners i nd iv idua l ly , but this is extremely expensive (depending on the congener, 5 m g o f a single congener can cost up to U S $ 150) and is not w i t h i n the budget o f most laboratories. Instead, compound ident if icat ion is usual ly performed by compar ison o f relative retention t imes w i t h A r o c l o r standards. Re la t ive retention times for a l l 209 congeners on several different co lumns have been publ i shed (102-104). Chromatograms o f A r o c l o r standards have also been publ ished, and the major peaks can be easily identified, as each different A r o c l o r mixture has a very characteristic pattern o f peaks (8). High-performance l i q u i d chromatography ( H P L C ) can also be used for P C B separation (105, 106). 20 1.11.4 Detection P C B s separated by G C are c o m m o n l y detected by electron capture detection ( E C D ) . E C D has a very h igh sensit ivity for P C B s , and the detector response is propor t ional to the number and locat ion o f chlorines on the b iphenyl r ing (107). Howeve r , peaks are identif ied solely o n relative retention t imes and interfering or co -elut ing compounds cannot be detected or identif ied. Quantitative results are based on relating peak areas to corresponding peak areas produced by k n o w n quantities o f commerc ia l P C B mixtures (8, 9). Th i s has led to many problems, especial ly i n the analysis o f c o m p l e x environmental mixtures (108). M a s s spectrometry is being used as a P C B detector more and more frequently (109-111). It is less sensitive than E C D for more h igh ly chlorinated congeners (112) but it has several advantages that outweigh this decrease i n sensit ivity. The degree o f ch lor ina t ion o f a particular peak can be assigned, and interfering compounds can be easi ly ident if ied, since mass spectra o f P C B s have very characteristic chlor ine isotope patterns ( shown i n Figure 1-7). Selected i o n moni tor ing ( S I M ) can enhance the select ivi ty and sensit ivity o f P C B determination, but care must be taken w i t h this technique as it is on ly acceptable for quantitative analysis when the i o n o f interest does not over lap w i t h any interfering ions. M S / M S has also been used i n order to separate P C B s f rom complex environmental samples (113). Indiv idual congener assignments s t i l l have to be made by comparisons w i t h relative retention times o f standards, however , since it is s t i l l d i f f icul t to dis t inguish between isomers o f a specific h o m o l o g group by mass spectrometry. 21 Isomer differentiation by mass spectrometry has been attempted recently, and researchers have found some differences i n characteristic fragmentation patterns o f some isomer groups. In particular, i n a study i n v o l v i n g 5 different tetrachlorinated P C B isomers and modulated resonant excitat ion, Lausev ic et al. (114) discovered that non-ortho and mono-ortho substituted P C B s tend to lose 2 chlorine atoms w h e n they fragment, w h i l e di-ortho substituted P C B s tend to lose only 1 chlor ine atom. They also found that it was possible to dis t inguish between two co-elut ing congeners (one te t raCB, one pen t aCB) by us ing chemica l ion iza t ion (115). The general order o f stabili ty o f ions depends on the degree and dis tr ibut ion o f ortho substitution (111). 1.12 Objectives The objectives o f this work were: (i) T o develop mic rob i a l enrichment cultures capable o f dechlor inat ing P C B s i n A r c t i c soi ls , especial ly soi ls f rom Reso lu t ion Island and Saglek that contained weathered A r o c l o r 1260. (i i) T o investigate some o f the factors that can affect P C B degradation, i n part icular the use o f different enrichment cultures, weathering o f P C B s i n so i l , and incubat ion temperature. ( i i i ) T o determine the extent o f dechlor inat ion achieved by treatment o f the weathered P C B s present i n Reso lu t ion Island and Saglek soils w i t h the enrichment cultures developed i n (i), and to identify the major dechlor inat ion products formed. (iv) T o determine i f dechlor inat ion o f single, h ighly-chlor inated P C B congeners is possible, and i f so, to determine the dechlor inat ion pathways taking place. 23 2. Materials and Methods for Investigation of PCB Dechlorination 2.1 Materials Standard A r o c l o r mixtures, analyt ical test mixtures and single P C B congeners were purchased from Accustandard . A d d i t i o n a l P C B reference standard mixtures were obtained f rom the Na t iona l Research C o u n c i l o f Canada. Techn ica l grade A r o c l o r 1260 and A r o c l o r 1242 were donated by B C H y d r o . H P L C - g r a d e hexane, acetone and dichloromethane were purchased from Fisher Scient i f ic . S o d i u m sulfate (assurance grade) was obtained f rom B D H chemicals l imi ted . F l o r i s i l (60-100 mesh) was obtained f rom Fisher Scient i f ic . Gas was purchased f rom Praxair , and the gas m i x used inside the anaerobic chamber consisted o f COa:N2:H2 i n a ratio o f 5:85:10. 1 L amber glass jars fitted w i t h Tef lon- l ined l ids used for so i l and sediment co l lec t ion were obtained commerc i a l l y ( I - C H E M Ltd) and are certified free o f organic materials. Stainless steel scoops used for sample co l lec t ion were pre-washed i n Kapt -c lean solvent, baked at 110 ° C for 12 hours, and stored i n baked a l u m i n u m fo i l to preclude organic contaminat ion. The stainless steel scoops were suppl ied by Norwes t Labs . A l l compounds used i n the reduced anaerobic minera l m e d i u m (Sect ion 2.3) were purchased from A l d r i c h or Fisher Scient i f ic . 2.2 Sample collection and storage P C B - c o n t a m i n a t e d soils f rom Reso lu t ion Island (Nunavut , 62 ° N , 65 ° W ) , Saglek (Labrador, 58 ° N , 64 ° W ) , and Cambr idge B a y (Nunavut , 69 ° N , 105 ° W ) , uncontaminated so i l f rom Reso lu t ion Island and pond sediments f rom Jericho 24 (Vancouver , B r i t i s h C o l u m b i a ) were col lected by us ing stainless steel scoops, and placed i n 1 L ( I - C H E M ) amber glass jars. S o i l sampl ing was restricted to the upper 10 c m o f so i l and care was taken to obtain representative samples. So i l s were kept at ambient temperatures i n the A r c t i c (for up to 4 days), transported to U B C i n coolers w i t h ice-packs, and stored at U B C i n a co ld r o o m kept at 4 ° C . S o i l was s ieved through a 2 m m -mesh sieve i n order to remove stones, and m i x e d thoroughly i n order to ensure homogeneity. Contaminated soi l from Saglek and sediment from Esqu imal t Harbour (Br i t i sh C o l u m b i a ) were p rov ided by the Env i ronmenta l Sciences Group o f the R o y a l M i l i t a r y Co l l ege i n K i n g s t o n (Ontario). Saglek so i l was sieved through a 2 m m - m e s h sieve i n the lab and m i x e d thoroughly. Vancouve r soils and sediments containing P C B s were p rov ided by B C H y d r o . A l l soi ls and sediments were stored i n a co ld r o o m kept at 4 ° C . The so i l f rom Saglek was a sandy loam, and that f rom Reso lu t ion Island was a sand (Canadian System o f S o i l Class i f icat ion) . Deta i led phys ica l properties o f these soi ls have been reported elsewhere (116). The locations o f the A r c t i c sites and Esqu ima l t are shown i n F igure 1-4. 2.3 Medium preparation Reduced anaerobic minera l m e d i u m ( R A M M ) was prepared as described by Shel ton and Tiedje (117), and modi f i ed according to M o r r i s et al. (64). The components o f the stock solutions used are l isted i n Table 2-1. The trace elements stock solut ion was prepared by d i s so lv ing ferrous chlor ide i n 10 m L cone. H C 1 , d i lu t ing to 1 L , and adding remaining trace elements. The v i tamins 25 stock solu t ion was prepared by adding the vi tamins l isted to 1 L de ionized water. The p H was adjusted to 7.00 w i t h H C 1 , and then naphthoquinone i n N a O H was added to a concentrat ion o f 0.2 g /L . The vi tamins stock solut ion was stored i n the freezer and sma l l quantities were filter-sterilized before use. Table 2-1: Components of stock solutions used to make RAMM. M a j o r Salts S tock So lu t ion C o m p o u n d Concentrat ion (g /L) N H 4 C 1 53.4 M g C l 2 . 6 H 2 0 2.0 C a C l 2 . 2 H 2 0 1.5 Trace Elements Stock Solu t ion C o m p o u n d Concentrat ion (g /L) F e C l 2 . 4 H 2 0 0.4 M n C l 2 . 4 H 2 0 0.5 H3BO3 0.05 Z n C l 2 0.05 C u C l 2 . 2 H 2 0 0.03 C o C l 2 . 6 H 2 0 0.5 N a 2 S e 0 3 0.05 N i C l 2 . 6 H 2 0 0.05 N a 2 M o 0 4 . 2 H 2 0 0.01 26 Table 2-1 cont. V i t a m i n s Stock So lu t ion C o m p o u n d Concentrat ion (g /L) B i o t i n 0.02 F o l i c ac id 0.02 L i p o i c ac id 0.06 Th iamine 0.05 R i b o f l a v i n 0.05 N i c o t i n i c ac id 0.05 P y r i d o x a l - H C l 0.10 Pantothinic ac id 0.05 Cyanocobo lamine 0.05 p - A m i n o b e n z o i c ac id 0.05 R A M M was prepared as fo l lows : M a j o r salts stock solut ion (10 m L ) , 3.36 m L o f 1000 m M K 2 H P 0 4 , 0.64 m L o f 1000 m M K H 2 P 0 4 , 1 m L trace elements and 1 m L resazurin were added to 990 m L dis t i l led water. The mixture was sparged w i t h N 2 for 20 minutes, after w h i c h t ime per iod on ly the headspace was flushed w i t h N 2 . N a 2 S was added to give a concentrat ion o f I m M , and the m e d i u m was dispensed into 100 m L serum bottles. These bottles had a N 2 headspace, and were cr imp-sealed w i t h butyl rubber stoppers. The sealed bottles were autoclaved at 120 ° C for 20 minutes. Af te r the solutions had cooled back d o w n to r o o m temperature, pyruvate (0.24 m L o f a 2 M anaerobic stock solution), stock v i tamins (0.1 m L ) and t i tanium citrate (final 27 concentrat ion 0.1 m M ) were added (by sterile syringe) to every l O O m L o f m e d i u m . In experiments where cultures were first pre-incubated and then autoclaved pr ior to inocula t ion , the pyruvate, v i tamins and t i tanium citrate were added after this f inal autoclaving. 2.4 Pre-incubation and confirmation of methanogenic conditions Pre l iminary experiments indicated that the A r c t i c soils that were to be treated had s t i l l not become methanogenic after 6 months o f incubat ion w i t h R A M M , so an alternative way o f ach iev ing methanogenic condit ions was required. A n a e r o b i c sediment f rom a duck pond i n Jericho Park (Vancouver , B . C . ) was col lected and added to A r c t i c so i l for pre-incubation. A e r o b i c A r c t i c so i l and anaerobic Jericho sediment was weighed into serum bottles or B a l c h tubes under aerobic condit ions. These bottles or tubes were then placed in an anaerobic chamber, R A M M was added, and they were cr imp-sealed w i t h butyl rubber stoppers. Bott les or tubes containing so i l , sediment and R A M M were pre-incubated at 21 ° C i n the dark unt i l methanogenic condi t ions were established. Methanogenesis was conf i rmed by injecting headspace gas into a gas chromatograph equipped w i t h a Haysep D B , 100-120 mesh, 10' x 1/8' packed c o l u m n and a thermal conduct iv i ty detector. The injector and detector temperatures were set to 140 ° C , the oven temperature was held at 30 ° C and the c o l u m n pressure was 2 k g / m . The f l o w rate o f the carrier gas h e l i u m was set to 30 m L / m i n and the thermal conduct iv i ty detector was set to a current o f 140 m A . Methane was identif ied by compar ison w i t h the retention t ime o f standard methane gas. 2.5 Extraction of PCBs from soil and medium A n efficient extraction technique that w o u l d a l l o w for rapid extract ion o f P C B s from large numbers o f so i l cultures was required. Standard E P A methods for extract ion o f P C B s f rom sediments invo lve extraction w i t h dichloromethane (Methods 3510 and 3520), acetone/hexane (Methods 3540 and 3541), and acetone/dichloromethane ( M e t h o d 3550). 2.5.1 Extraction of PCBs from soil Three different extraction procedures were tested i n triplicate. A l l tests were done on Reso lu t ion Island so i l that contained 170 parts per m i l l i o n (ppm) weathered A r o c l o r 1260. S o i l portions (1 g) were weighed out accurately and used i n the f o l l o w i n g procedures. In Procedure 1, the so i l was sequentially extracted wi th three 2 - m L portions o f hexane. In Procedure 2, the s o i l was sequentially extracted w i t h three 2 - m L port ions o f acetone. In Procedure 3, the so i l was sequentially extracted w i t h two 2 - m L port ions o f acetone fo l l owed by two 2 - m L portions o f hexane. In each o f the three procedures, the appropriate solvent was added by Pasteur pipette. The so i l and solvent were m i x e d by us ing a vortex mixe r for 30 seconds, and then centrifuged for 2 minutes at 3000 rpm. The solvent was removed by Pasteur pipette and placed i n a clean test tube. E a c h solvent por t ion was w o r k e d up separately and analyzed for P C B content (see sections 2.6 to 2.8 for details o n work -up and analysis). The results for triplicate trials are g iven i n Tab le 2-2. 29 Table 2-2: Soil extraction results. Procedure Ext rac t ion P C B s Extracted (ppm) M e a n ( S D ) 1 Hexane , Extract 1 68.0 (4.5) 1 Hexane, Extract 2 14.6(0.8) 1 Hexane, Extract 3 2.7 (0.6) 1 Total Extracted 85.5 (4.1) 2 Acetone , Extract 1 135.6(2.5) 2 Acetone , Extract 2 30 .0(2 .3) 2 Ace tone , Extract 3 2.5 (0.2) 2 Total Extracted 168.2 (0.4) 3 Ace tone , Extract 1 136.8(2.2) 3 Acetone , Extract 2 29.0 (2.0) 3 Hexane , Extract 1 3.8 (1.5) 3 Hexane, Extract 2 0.18 (0.02) 3 Total Extracted 169.2 (0.4) It can be seen from the results shown i n Table 2-2 that the extractions us ing hexane alone d i d not give very h igh extraction efficiencies. The extractions us ing acetone alone and acetone fo l lowed by hexane both resulted i n very h igh extraction efficiencies, w i t h good precis ion. Since the acetone i n the P C B samples had to be removed and replaced by hexane pr ior to injection into the G C , it was decided to use procedure 3. 30 2.5.2 Extraction of PCBs from medium Procedure 3 (Sect ion 2.5.1) is an effective extraction technique for so i l samples, but the majority o f samples i n this w o r k were incubated w i t h m e d i u m as w e l l , and so it was necessary to determine i f the m e d i u m needed to be extracted as w e l l as the so i l . T w o different extract ion procedures were tested. In procedure 1, the l i q u i d portions o f three incubat ion cultures were removed from the so i l by Pasteur pipette. The l i q u i d was p laced i n separating funnel and extracted w i t h two 5 - m L portions o f hexane. Ext rac t ion was performed by shaking v igorous ly for two minutes, a l l o w i n g the aqueous and hexane layers to separate, and r emov ing the hexane layer. The so i l was extracted w i t h 2 X 2 m L o f hexane, as described i n the previous procedure (2.5.1). Hexane portions were worked up separately and analyzed for P C B content. Procedure 2 was s imi la r to procedure 1, but the l i q u i d portions were extracted w i t h dichloromethane instead o f hexane, and the so i l portions were extracted w i t h acetone instead o f hexane. Resul ts for these extractions are g iven i n Table 2-3. The results show that the P C B fraction present i n the aqueous por t ion is probably not significant enough to warrant the extra t ime-consuming extractions i nvo lved . However , because less h igh ly chlorinated, more water-soluble P C B dechlor inat ion products might be lost preferentially i n the aqueous fractions, the above tests were repeated at a later date w i t h so i l and m e d i u m that contained P C B dechlor inat ion products. The results were very s imi la r to the ones shown i n Table 2-3, however, so it was concluded that extraction o f the aqueous por t ion was not necessary. Table 2-3: Medium extraction results. Sample extracted Procedure Extract number Solvent Concentra t ion Extracted M e a n ( S D ) S o i l 1 1 Hexane 66.9 (3.9) S o i l 1 2 Hexane 14.7 (0.9) M e d i u m 1 1 Hexane 0.68 (0.04) M e d i u m 1 2 Hexane n.d. S o i l 2 1 Ace tone 135.5 (2.4) S o i l 2 2 Ace tone 28.1 (2.1) M e d i u m 2 1 Dichloromethane 0.70 (0.04) M e d i u m 2 2 Dichloromethane n.d. n.d.: not detected 2.5.3 Soxhlet extraction of PCBs from soil Soi l s f rom Reso lu t ion Island and Saglek were extracted by us ing Soxhle t extraction i n order to determine the total P C B concentrations. Port ions (approximately 10 g each) o f undried so i l were weighed accurately. S o d i u m sulphate (3 g) and 10 |ug decachlorobiphenyl were added to each port ion. The decachlorobiphenyl was added as a solut ion i n acetone, and was used as an internal standard. Soxhlet extraction i n v o l v e d re f luxing dichloromethane (135 m L ) for 16 hours. The dichloromethane was then removed by rotary evaporation and the round-bottomed flasks were r insed thoroughly w i t h hexane. The hexane was evaporated d o w n to approximately 0.2 m L by b l o w i n g air onto the sample, and the 0.2 m L sample was passed through a F l o r i s i l c o l u m n (6.0 c m 32 long x 0.55 m m wide) by us ing hexane as eluent. 2 m L samples were col lec ted for further analysis. D r y weights o f so i l were obtained w i t h so i l samples that were weighed before and after oven-drying at 1 2 0 ° C for 48 hours. A r o c l o r 1260 concentrations found i n the soi ls are presented i n Table 2-4. Table 2-4: Aroclor 1260 concentrations in soils. S o i l Source A r o c l o r 1260 Concentra t ion Reso lu t ion Island 185 p p m (dry weight) Saglek 127 p p m (dry weight) 2.6 Work-up of soil extracts S o i l extracts were evaporated d o w n to approximately 0.2 m L and then p laced o n a F l o r i s i l c o l u m n ( i .d . 5.5 m m ) containing 6 c m F l o r i s i l and 2 c m sod ium sulphate. The amount o f eluent that needed to be col lected i n order to ensure quantitative recovery o f the P C B s was determined experimental ly. Hexane was added to the top o f the c o l u m n , and three 1.5-mL fractions o f eluent were col lected. The different eluent fractions were col lected and analyzed separately. Th i s procedure was performed i n tr iplicate, and results are presented i n Table 2-5. 33 Table 2-5: PCBs in different eluent fractions. Eluent fraction % o f total P C B s eluted 1 99 .6(0 .1) 2 0.41 (0.01) 3 trace These results show that the majority o f P C B s are eluted i n the first 1.5 m L fraction o f hexane. A smal l proport ion o f P C B s are eluted i n the second 1.5 m L fraction. Howeve r , i f 3 m L o f hexane is col lected rather than 1.5 m L , the addi t ional increase i n sample v o l u m e w i l l decrease the concentration o f P C B s to be analyzed, so it was decided to col lect 1.5 m L o f eluent. N e w batches o f F l o r i s i l were checked before use to ensure that 1.5 m L o f eluent was s t i l l a suitable vo lume to use. 2.7 Gas chromatography P C B samples were analyzed by us ing a gas chromatograph ( V a r i a n Star 3400) fitted w i t h a D B 5 - m s c o l u m n (30 m x 0.25 m m x 0.25 um) and coupled to a mass spectrometer (Va r i an Saturn 4 D ion trap). The sample vo lume injected was 2.5 u,L, the temperature o f the injector was held at 260 ° C , and the temperature o f the transfer l ine was 280 ° C . Several temperature programs were tested in i t i a l ly i n order to determine an appropriate one. There over 80 different congeners present i n A r o c l o r 1260, and it was desirable to separate as many as possible o f these. M a n y different temperature programs were tested, the cri teria used to choose the best temperature program were (a) good separation and (b) a m i n i m a l total t ime per run. The opt imal c o l u m n temperature program 34 chosen for A r o c l o r 1260 was as fo l lows : 104 ° C for 3 minutes, increased at 20 ° C / minute to 180 ° C , increased at 2.5 °C / minute to 278 ° C , held for 3 minutes, increased at 22 ° C / minute to 300 °C and held at 300 ° C for 1 minute. Th i s separated A r o c l o r 1260 into 63 well-separated peaks, and co-elut ing peaks were not separated by us ing a longer, more gradual temperature program. Th i s temperature program was considered op t imal for the samples to be run. A r o c l o r 1242 contains a m u c h less h igh ly chlorinated mixture o f P C B s than A r o c l o r 1260, so a different temperature program was chosen as being more appropriate for A r o c l o r 1242. In this program the c o l u m n was he ld at 104 ° C for 3 minutes, increased at 20 ° C / minute to 160 ° C , increased at 2.5 ° C / minute to 221 ° C , increased at 50 ° C / minute to 290 ° C and then held at 290 ° C for 3 minutes. 2.8 Mass spectrometry and data interpretation P C B s were detected by us ing a mass spectrometer (Va r i an Saturn mode l 4 D ion trap). P C B s have very characteristic mass spectra because o f the cluster o f peaks generated as a result o f the combina t ion o f chlor ine isotopes. In this w o r k the mass spectrum o f each G C peak was investigated i n order to determine whether the peak corresponded to a P C B , and i f so, to determine the number o f chlor ine substituents. Ind iv idua l congener assignments were made by compar ison w i t h A r o c l o r standards (Aroc lo r s 1221, 1242, 1254 and 1260), and by compar ison w i t h publ i shed relative retention t imes on a D B 5 - m s c o l u m n (103). The ident if icat ion o f major product congeners that were not present i n any o f the A r o c l o r mixtures used was conf i rmed by purchasing i nd iv idua l standards and determining their relative retention t imes, and also by sp ik ing these standards into the samples. The peak number, relative retention times, 35 structures and I U P A C identities o f P C B s identif ied i n this w o r k are g iven i n Tab le 2-6, and a typ ica l A r o c l o r 1260 chromatogram showing the peak numbers o f the most abundant peaks is shown i n Figure 2-1. The relative retention times are calculated as (the retention t ime o f the congeners) / (the retention t ime o f decachlorobiphenyl) . In some instances where two peaks are v i s ib le i n chromatograms, but are not w e l l separated, the t w o peaks are reported separately under the same peak number. Table 2-6: PCB congeners identified in this work. Peak N u m b e r Rela t ive Retent ion I U P A C identity Structure (posi t ion T i m e o f chlorines) 1 0.1991 1 2 2 0.2184 2 3 3 0.2207 3 4 4 0.2304 4,10 2-2, 26 5 0.2461 7,9 24, 25 6 0.2528 5,6 23, 2-3 7 0.2571 8 2-4 8 0.2662 14 35 9 0.2691 19 26-2 10 0.2811 12 34 11 0.2867 13,18 3-4, 25-2 12 0.2880 17 24-2 13 0.2910 15 4-4 14 0.2936 27,24 26-3, 236 15 0.3011 16,32 23-2, 26-4 16 0.3089 34,23 35-2, 235 17 0.3126 54,29 26-26, 245 18 0.3187 26 25-3 36 Table 2-6 cont. 19 0.3201 25 24-3 20 0.3233 50 246-2 21 0.3270 31,28 25-4, 24-4 22 0.3305 21 234 23 0.3342, 0.3356 53,20,33 25-26, 23-3 , 34-2 24 0.3388 51 24-26 25 0.3420 22 23-4 26 0.3457 45 236-2 27 0.3529 46 23-26 28 0.3572 69,73 246-3, 26-35 29 0.3585 39 35-4 30 0.3604 43.52 235-2, 25-25 31 0.3639 49 24-25 32 0.3665 47,48,75 24-24, 245-2, 246-4 33 0.3745 104 246-26 34 0.3801 44 23-25 35 0.3836 59 236-3 36 0.3876 37 34-4 37 0.3914 71,41 26-34, 234-2 38 0.3946 64 236-4 39 0.3964 68 24-35 40 0.3988 96 2326-26 41 0.4015 40,103 23-23, 246-25 42 0.4055 57 235-3 43 0.4092 100 246-24 44 0.4111 67 245-4 45 0.4164 94 235-26 46 0.4175 63 235-4 47 0.4228 74 245-4 37 Table 2-6 cont. 48 0.4255 102 245-26 49 0.4290, 0.4303 70,95,66 25-34, 236-25, 24-34 50 0.4381 91 236-24 51 0.4413 55 234-3 52 0.4469 155 246-246 53 0.4506 60,56 234-4, 23-34 54 0.4536 92 235-25 55 0.4562 84 236-23 56 0.4592 89 234-26 57 0.4618 101,90 245-25, 235-24 58 0.4666 99 245-24 59 0.4738 119 246-34 60 0.4744 119,150 246-34, 236-246 61 0.4794 83 235-23 62 0.4834 152 2356-26 63 0.4869 86,97 2345-2, 245-23 64 0.4885 125 345-26 65 0.4920 117 2356-4 66 0.4960 87,115 234-25, 2346-4 67 0.4981 148,81 235-246, 345-5 68 0.5005 85 234-24 69 0.5024 120 245-35 70 0.5043 136 236-236 71 0.5083 110,154 236-34, 245-246 72 0.5123 77 34-34 73 0.5224 82 234-23 74 0.5248 151 2356-25 75 0.5307, 0.5318 135,144 235-236, 2346-25 38 Table 2-6 cont. 76 0.5350 147,124 2356-24, 345-25 77 0.5368 109 234-34 78 0.5408 139 2346-24 79 0.5424 149,123 236-245, 345-24 80 0.5459 140 234-246 81 0.5489 118,143 245-34, 2345-26 82 0.5547 134 2356-23 83 0.5553 133 235-235 84 0 .5614,0 .5635 131,114 2346-23, 2345-4 85 0.5643 122 345-23 86 0.5662 188 2356-246 87 0.5713 146 235-245 88 0.5780 184 2346-246 89 0.5814 153 245-245 90 0 .5828 ,0 .5852 132,105 234-236, 234-34 91 0.5990 179,141 2356-236, 2345-25 92 0.6004 N o t identif ied 93 0 .6076 ,0 .6105 , 0.6124 137,176,130 2345-24, 2346-236, 234-23 94 0.6193, 0.6220 160,138,158 23456-3 , 234-245, 2346-34 95 0.6247 186 23456-26 96 0.6316 129,178 2345-34, 2356-235 97 0.6407 175,126 2346-235, 345-34 98 0.6458 182,187 2345-246, 2356-245 99 0.6543 183 2346-245 100 0.6623 128 234-234 101 0.6690, 0.6717 167,185 245-345, 23456-25 102 0.6837 174 2345-236 39 Table 2-6 cont. 103 0.6909 177 2356-234 104 0.6954 202 2356-2356 105 0.6989 171 2346-234 106 0 .7045,0 .7061 156,173 2345-34, 234-345, 23456-3 107 0.7085 201 2346-2356 108 0.7117 157 234-345 109 0.7202 172 2345-235 110 0.7224 197 2346-2346 111 0.7325 180,193 2345-245,2356-345 112 0.7389 191 2346-345 113 0.7480 200 23456-236 114 0.7672 169 345-345 115 0.7779 170,190 2345-234, 23456-34 116 0.7843 198 23456-235 117 0.7907 199 2345-2356 118 0.8009 203,196 23456-245, 2345-2346 119 0.8230 N o t identif ied 120 0.8297 189 2345-345 121 0.8462, 0.8489 208,195 23456-2356,23456-234 122 0.8620 207 23456-2346 123 0.8884 194 2345-2345 124 0.8940 205 23456-345 125 0.9485 206 23456-2345 126 1.000 209 23456-23456 40 Three-point calibration curves were constructed for all congeners either by using pure congeners, where available, or by using the concentrations of congeners present in Aroclor standards. Data for the concentrations of congeners present in Aroclor standards was supplied by Williams (118). Mass spectra were usually quantified by summing the total ion current (TIC) for each peak. This method was used as the degree of fragmentation varied between congeners, and the TIC summed all the fragments as well as the molecular ions. In a few examples where PCB concentrations were very low, selected ion monitoring (SIM) of the molecular ion peaks was used instead of the TIC to determine and quantify PCBs, as this improved the detection limits. The relevant experimental procedures state when selected ion monitoring was used for PCB determination. Detection limits were determined for a range of di- to nona-CB congeners, and they ranged from 0.05 pg to 0.5 pg for TIC, and 0.01 pg to 0.15 pg for SIM. Detection limits varied considerably between different PCB homologs, and also between isomers of a given homolog class. The detection limit was defined to be the concentration at which the signal to noise ratio was greater than 3 to 1. 2.9 Quality control Many checks were made on the validity and reproducibility of the experimental data. Blanks were run at regular intervals to ensure that there was no PCB contamination in the glassware, in any of the materials being used, or in the GC/MS. Most experiments included replicate samples, and several experiments were performed in triplicate. When the GC/MS was in use it was re-tuned at least once every 24 hours, and standard solutions were run before and after samples to be calibrated. The GC column resolution 42 was moni tored against the peak separation required and when it was not acceptable the c o l u m n was either cut or replaced. In order to check the va l id i ty o f results several standard reference materials were tested. A P C B Ca l ib ra t ion C h e c k solut ion f rom Accus tandard contained 21 different P C B congeners o f k n o w n concentration. A P C B W i n d o w D e f i n i n g M i x t u r e f rom Accus tandard contained 20 different P C B congeners o f k n o w n concentration. Four different standards f rom the Na t iona l Research C o u n c i l o f Canada ( N R C ) ( C L B - 1 , C L B -2, C L B - 3 and C L B - 4 ) each contained 14 or 15 different P C B congeners. These mixtures were used to conf i rm the identities o f P C B congeners, and P C B congeners that were also present i n m y experimental samples were quantified i n order to check quantitation accuracy. Quanti ta t ion results were always w i t h i n an error range o f 7 % or less o f the g iven concentrations, and P C B congeners had been correctly identif ied i n a l l cases. 43 3. Development of Anaerobic PCB Enrichment Cultures and Analysis of Sediment Cores Because we d i d not have access to any P C B - d e c h l o r i n a t i n g cultures, the first step i n this w o r k was to try to develop enrichment cultures capable o f dechlor inat ing A r o c l o r 1260. Several different approaches were used i n attempts to enr ich anaerobic P C B degraders. 3.1 Development of enrichment cultures 3.1.1 Different Aroclor mixtures as putative substrates with a variety of inocula 3.1.1.1 Introduction A series o f enrichment cultures that contained either A r o c l o r 1260 or A r o c l o r 1242 as putative substrates for dechlorinat ing microorganisms were set up. A r o c l o r 1260 was used i n h a l f the enrichment cultures as it is the P C B mixture that needs to be remediated i n Reso lu t ion Island and Saglek soils . A r o c l o r 1242 was used i n the other h a l f o f the enrichment cultures as it should be more readily dechlorinated by bacteria than A r o c l o r 1260 (61). A r o c l o r 1242 consists ma in ly o f t r i - and tetra-chlorobiphenyls, whereas A r o c l o r 1260 consists ma in ly o f hexa- and hepta-chlorobiphenyls. It was reasoned that since A r o c l o r 1242 should be more readily dechlorinated than A r o c l o r 1260, it might be easier to f ind dechlorinat ing act ivi ty w i t h A r o c l o r 1242, and then transfer this act ivi ty to A r o c l o r 1260 at a later date. Sites w i t h his tor ical P C B contaminat ion were considered to be the most l i ke ly sources o f P C B degraders, so a l l the different PCB-con tamina t ed sediments or soils 44 avai lable at the t ime were used as i n o c u l u m sources. PCB-con t amina t ed sediment was col lected f rom Esqu imal t Harbour (Br i t i sh C o l u m b i a ) and P C B - c o n t a m i n a t e d so i l was col lected f rom Reso lu t ion Island (Nunavut) . V a r i o u s PCB-con t amina t ed soils and sediments f rom sites i n V a n c o u v e r (Br i t i sh C o l u m b i a ) were p rov ided by B C H y d r o , and these were combined to form a single " B C H y d r o " i nocu lum. Enr ichment cultures were g rown on a mixture o f so i l f rom Reso lu t ion Island and anaerobic sediment f rom Jericho. Reso lu t ion Island so i l was used because this is one o f the soi ls that needs to be remediated. Jericho sediment was added to this so i l since pre l iminary experiments showed that Reso lu t ion Island so i l on its o w n d id not develop methanogenic condi t ions , even after 6 months o f incubat ion under anaerobic condit ions. 3.1.1.2 Experimental Reso lu t ion Island so i l (12.5 g) and anaerobic sediment f rom Jericho Park (12.5 g), were m i x e d i n serum bottles w i t h 40 m L reduced anaerobic minera l m e d i u m ( R A M M ) prepared as described i n Chapter 2 (Sect ion 2-3). 125 u L o f a 200 m g / m L A r o c l o r solut ion (either A r o c l o r 1260 or A r o c l o r 1242) i n acetone was added to the serum bottles, to give a f inal P C B so i l concentration o f 1000 ppm. Soi l s or sediments (30 g) to be used as inocu la were placed i n Er lenmeyer flasks, and 100 m L o f R A M M was added to each flask inside an anaerobic chamber. The flasks were shaken w e l l , and 10 m L portions o f R A M M were transferred to the serum bottles as inocula . Cont ro l s had an extra 10 m L o f R A M M added instead o f inocu lum. Table 3-1 shows the various combinat ions o f A r o c l o r mixtures and inocu la used. Cultures were incubated stationary at 30 ° C i n the dark. 45 Table 3-1: Initial enrichment cultures. Culture Inocu lum Source A r o c l o r used B K 2 1 Esqu imal t 1242 B K 2 2 B C H y d r o 1242 B K 2 3 Reso lu t ion Island 1242 B K 2 4 N o n e (control) 1242 B K 2 5 N o n e (control) 1242 B K 2 6 Esqu imal t 1260 B K 2 7 B C H y d r o 1260 B K 2 8 Reso lu t ion Island 1260 B K 2 9 N o n e (control) 1260 B K 3 0 N o n e (control) 1260 3.1.2 Using amendments to stimulate dechlorination activity 3.1.2.1 Introduction After 5 months there was no evidence o f any dechlor inat ion act ivi ty i n cultures B K 2 1 to B K 3 0 , so a second set o f enrichment cultures was set up. Some changes were made to the approach out l ined i n section 3.1.1.2. V a r i o u s amendments were added i n an attempt to stimulate dechlor inat ion act ivi ty (74, 77), and cultures were pre-incubated before the i n o c u l u m was added. The pre- incubat ion step was added i n order to establish methanogenic condi t ions before inocula t ion, i n case the P C B dechlorinators were strict anaerobes that cou ld not survive the in i t ia l pre-methanogenic condit ions i n the serum bottles. S ince the amendments were expensive and only very l imi t ed quantities were avai lable, on ly one i n o c u l u m source and one A r o c l o r mixture was tested. The i n o c u l u m used came f rom Esqu imal t Harbour sediment, as this was the on ly i n o c u l u m source that 46 had been col lected and stored under anaerobic condit ions. The P C B mixture used was A r o c l o r 1260. A higher ratio o f Reso lu t ion Island so i l to Jericho sediment than i n the previous set o f enrichment cultures was used since it was found that this higher ratio w o u l d s t i l l establish methanogenic condit ions i n a short t ime per iod. The f inal so i l concentrat ion o f A r o c l o r 1260 used was also lower than the one used previous ly , i n case the h igh concentrat ion o f A r o c l o r 1260 used previous ly inhibi ted the organisms o f interest. One treatment was autoclaved before inocula t ion, i n order to e l iminate anaerobes that had established themselves i n the pre-incubation step before inocula t ion occurred. 3.1.2.2 Experimental Reso lu t ion Island so i l (12.5 g) and anaerobic sediment f rom Jericho (7.5 g) were m i x e d i n serum bottles w i t h 40 m L reduced anaerobic minera l m e d i u m ( R A M M ) . The serum bottles were cr imp-sealed w i t h butyl rubber stoppers, and soil /sediment mixtures were pre-incubated for two weeks, at w h i c h t ime methanogenesis was detected. A r o c l o r 1260 (to give a f inal soil /sediment concentration o f 50 ppm) and 10 m L o f microorganisms anaerobically eluted ( in an anaerobic chamber, us ing R A M M ) f rom sediment col lected f rom Esquimal t Harbour (Br i t i sh C o l u m b i a ) were added to a l l enrichment cultures. Cont ro ls had an addit ional 10 m L o f R A M M added instead o f i nocu lum. Cul tures were either left unamended, or amended w i t h 5 m g o f 2 - 3 4 - t r i C B , 26-d i C B , 25-34- te t raCB or 4-bromobenzoate. Table 3-2 gives a summary o f the var ious treatments, a l l o f w h i c h were carried out i n duplicate. Cultures were incubated stationary at 22 ° C i n the dark. 47 Table 3-2: Enrichment cultures with amendments. Cultures A m e n d m e n t Au toc l aved? Inocu lum B K 7 0 , B K 7 1 N o n e Y e s Esqu ima l t B K 7 2 , B K 7 3 N o n e N o Esqu ima l t B K 7 4 , B K 7 5 2 , 6 - d i C B N o Esqu ima l t B K 7 6 , B K 7 7 2 ' , 3 , 4 - t r i C B N o Esqu imal t B K 7 8 , B K 7 9 2 ,3 ' ,4 ' ,5 - te t raCB N o Esqu imal t B K 8 0 , B K 8 1 4-bromobenzoate N o Esqu imal t 3.1.3 Inoculation with an Aroclor 1242 dechlorinator T w o inocu la that had previous ly been k n o w n to dechlorinate A r o c l o r 1242 were p rov ided by John Quensen o f M i c h i g a n State Univers i ty . They were o ld , however , and the current levels o f dechlor inat ion act ivi ty were not k n o w n . Experimental Serum bottles that contained anaerobic sediment from Jericho (20 g) and R A M M (45 m L ) were pre-incubated for two weeks, after w h i c h t ime A r o c l o r 1260 (to give a f inal sediment concentrat ion o f 120 ppm) and 5 m L o f microorganisms anaerobical ly eluted f rom the enrichment cultures p rov ided were added. T w o different enrichment cultures ( B K 1 1 0 and B K 1 1 2 ) were set up from the two different inocu la provided . T w o controls ( B K 111 and B K 1 1 3 ) were also set up. Cultures were autoclaved three t imes after pre-incubat ion, and controls had 5 m L addit ional R A M M added instead o f i n o c u l u m . A l l cultures were incubated stationary at 22 ° C i n the dark. 48 3.1.4 Anaerobic Arctic inoculum - Cambridge Bay A n a e r o b i c PCB-con t amina t ed sediment f rom a sewage outfal l lake i n Cambr idge B a y (Nunavut) was col lected. Th i s sediment was used as an i n o c u l u m to search for P C B dechlorinators. Incubations were done at both 4 ° C and at 22 ° C , since it was postulated that potential P C B degraders f rom the A r c t i c cou ld be psychrophi l ic . Experimental: Cambr idge B a y sediment (20 g) was placed i n serum bottles inside an anaerobic chamber. R A M M (50 m L ) and A r o c l o r 1260 (to give a final sediment concentrat ion o f 120 ppm) were added, and the bottles were sealed w i t h butyl rubber stoppers. Cont ro l s were autoclaved three times. B K 1 1 4 and its control ( B K 1 1 5 ) were incubated at 4 ° C , B K 1 1 6 and its control ( B K 1 1 7 ) were incubated at 22 ° C . 3.1.5 Arctic Inoculum - Saglek Enr ichment cultures us ing PCB-con tamina t ed so i l and anaerobic sediment col lected f rom two different sites at Saglek (Labrador) were also set up i n an attempt to get an A r c t i c source o f P C B dechlorinators. Experimental: Serum bottles containing Reso lu t ion Island so i l (10 g), Jericho sediment (5 g) and R A M M (40 m L ) were pre-incubated and then autoclaved. N o A r o c l o r 1260 was added as the Reso lu t ion Island so i l used already contained 170 p p m A r o c l o r 1260. Inocu lum (10 m L ) was anaerobical ly eluted f rom Saglek so i l and added to the bottles. B V 2 0 0 contained i n o c u l u m from marine sediment and B V 2 0 1 contained i n o c u l u m f rom lake sediment. Cul tures were incubated stationary at 22 ° C i n the dark. 49 3.2 Testing for dechlorinating activity S o i l f rom a l l the enrichment cultures that were set up was sampled at regular intervals. Se rum bottles were shaken w e l l , opened inside the anaerobic chamber, and portions o f so i l (approximately 2 g) were removed for analysis. S o i l extract ion and P C B analysis was performed as described previously (Chapter 2, Sections 2-5 to 2-8). Chromatograms were inspected for any evidence o f dechlorinat ion. T h i s i n v o l v e d searching for the appearance or increase o f less-highly chlorinated P C B s , accompanied by a significant decrease i n more-highly chlorinated P C B congeners relative to decachlorobiphenyl . F igure 3-1 shows an example o f typica l changes that occur i n P C B congener dis t r ibut ion as a result o f incubat ing A r o c l o r 1260 w i t h an anaerobic P C B -dechlor inat ing enrichment culture. The top chromatogram (a) shows a so i l extract before dechlor inat ion has occurred, a typica l A r o c l o r 1260 profi le . The bot tom chromatogram (b) shows an extract f rom the same batch o f so i l after incubat ion w i t h enrichment culture B K 8 1 . The A r o c l o r 1260 profi le has disappeared and the h igh ly chlorinated P C B congeners have been replaced w i t h less-highly chlorinated congeners that were not prev ious ly present. 50 (a) Before treatment 2000000 1800000 1600000 ® 1400000 c ° 1200000 a> a: 1000000 o> :§ 800000 m r2 600000 400000 -200000 601 901 1201 1501 1801 2101 2401 2701 3001 3301 3601 Scan N u m b e r (b) Af ter treatment 4000000 3500000 3000000 a> in O 2500000 in a> * 2000000 a> > « 1500000 a> a. 1000000 500000 0 901 1201 1501 1801 2101 2401 2701 3001 3301 3601 Scan N u m b e r Figure 3-1: Changes in PCB congener distribution after treatment with inoculum BK81. 51 3.3 Results and discussion F o r many months there was no evidence o f any dechlor inat ion act ivi ty i n the enrichment cultures. Af ter incubat ion periods ranging from 10 to 16 months, however , a w ide range o f dechlorinat ing act ivi ty was found between the different enrichment cultures. T h i s range o f dechlor inat ion act ivi ty is summar ized i n Table 3-3. The most extensive dechlor inat ion in i t i a l ly occurred i n cultures B K 8 0 and B K 8 1 . These cultures had both been amended w i t h 4-bromobenzoate, w h i c h suggests that, o f a l l the treatments tested here, addi t ion o f 4-bromobenzoate is the most effective way to stimulate P C B dechlorinators. The cultures containing unamended Esqu imal t inocu la had the second-highest rate o f P C B dechlor inat ion (cultures B K 7 2 and B K 7 3 ) , suggesting that none o f the other amendments (used i n cultures B K 7 4 to B K 7 9 ) were useful i n s t imulat ing P C B dechlorinators, and autoclaving ( B K 7 0 and B K 7 1 ) was not useful either. The other cultures that showed h igh P C B dechlorinat ing act ivi ty were one o f the A r o c l o r 1242 controls ( B K 2 4 ) and one o f the A r o c l o r 1260 controls ( B K 3 0 ) . S ince the controls were uninoculated, this act ivi ty is probably due to contaminat ion o f the controls by another culture inside the anaerobic chamber (wh ich is not sterile). It is also possible that the P C B - d e g r a d i n g culture present i n the control came f rom the Jer icho or Reso lu t ion Island sediment, but this is un l ike ly , since the other controls and the other incubations that contained these sediments together w i t h A r o c l o r 1260 d i d not show any dechlor inat ing act ivi ty. Such contaminat ion d i d not occur i n later experiments when shorter incubat ion periods were used for enrichment cultures. A l l cultures that were incubated w i t h A r o c l o r 1242 as the dechlor inat ion substrate ( B K 2 1 to B K 2 5 ) showed some evidence o f dechlorinat ion, but none o f the cultures f rom 52 the same set of 16-month incubations that were incubated with Aroclor 1260 as a dechlorination substrate showed any dechlorination activity (apart from the control BK30, which was discussed on the previous page). This corroborates the hypothesis (stated in section 3-1-1-1) that it is easier to find dechlorinating activity when the dechlorination substrate is Aroclor 1242 than when it is Aroclor 1260. A small amount of dechlorination activity was found in enrichment cultures BK74 to BK79. Because it was concluded that the amendments were not useful in stimulating dechlorination activity, and because other enrichment cultures with Aroclor 1260 and the same inoculum (BK26, BK70 and BK71) did not show any evidence for dechlorination, it is possible that the pre-incubation step along with the lack of autoclaving prior to inoculation was more favourable for the development of dechlorinating organisms. Table 3-3: Enrichment culture results. Culture Incubation period Results (amount of dechlorination) Notes BK21 16 months Small BK22 16 months Small BK23 16 months Small BK24 16 months Large Transferred BK25 16 months Small BK26 16 months None BK27 16 months None BK28 16 months None BK29 16 months None BK30 16 months Large Transferred 53 Table 3-3 cont. BK70 10 months None BK71 10 months None BK72 10 months Large Transferred BK73 10 months Large Combined with BK72 BK74 10 months Small BK75 10 months Small BK76 10 months Small BK77 10 months Small BK78 10 months Small BK79 10 months Small BK80 10 months Very large Lost due to explosion BK81 10 months Very large Transferred BK110 6 months None BK111 6 months None BK112 6 months Small BK113 6 months None BK114 14 months None BK115 14 months None BK116 14 months None B K 117 14 months None BV200 12 months None BV201 12 months None The cultures obtained from Michigan State University (BK110 and B K 112) did not show much evidence of Aroclor 1260 dechlorination after 6 months of incubation. This suggests that either the cultures were old and no longer viable, or that the Aroclor 1242 dechlorinating activity was not transferred successfully to the congeners present in 54 A r o c l o r 1260. N o evidence o f dechlor inat ion was found i n any o f the cultures conta ining A r o c l o r 1260 and A r c t i c (Resolu t ion Island, Cambr idge B a y or Saglek) inocula . 3.4 Cultures selected for further work Unfortunately the B K 8 0 culture was lost, as the serum bottle exp loded due to bui l t -up pressure. A l l other enrichment cultures that produced a large amount o f P C B dechlor inat ion (i.e. cultures B K 8 1 , B K 7 2 / 7 3 , B K 3 0 and B K 2 4 ) were transferred for further use. Cul tures were transferred as fo l lows : Serum bottles containing Reso lu t ion Island so i l (12.5 g), Jericho sediment (7.5 g) and R A M M (80 m L ) were sealed w i t h buty l rubber stoppers inside an anaerobic chamber, and pre-incubated at 22 ° C unt i l methanogenic condi t ions were established. The serum bottles were then autoclaved, and after c o o l i n g to r o o m temperature, A r o c l o r 1260 i n acetone (to give a f inal A r o c l o r 1260 concentrat ion o f 100 p p m i n soi l) and 4-bromobenzoate i n methanol (to give a f inal 4-bromobenzoate concentration o f 5 ppm) were added. The enrichment cultures to be transferred were shaken w e l l , and after the so i l had settled, 10 m L o f R A M M was removed f rom the enrichment cultures and used to inoculate the freshly-prepared serum bottles (inside an anaerobic chamber). A l l transferred cultures were incubated stationary at 22 ° C i n the dark. Cul ture B K 2 4 , w h i c h had in i t i a l ly been g rown on A r o c l o r 1242, was transferred to A r o c l o r 1260. Results f rom further experiments (reported i n Chapter 4) show that B K 2 4 dechlorinates the congeners present i n A r o c l o r 1260. The B K 2 4 and B K 7 2 / 7 3 transfers were incubated at 22 ° C rather than the previous 30 ° C . Cul tures w h i c h showed 55 no dechlor inat ion or on ly a smal l amount o f dechlor inat ion were stored for possible future invest igat ion, but have not been transferred or sampled again. 3.5 Analysis of sediment cores from Cambridge Bay 3.5.1 Aim The a i m o f this w o r k was to investigate the P C B depth profi les o f core samples taken f rom lakes i n Cambr idge B a y (Nunavut) . It was o f particular interest to determine i f there was evidence for in situ m ic rob i a l degradation. 3.5.2 Experimental Three core samples were taken from two different lakes i n the C A M - M radar station i n Cambr idge B a y . T w o o f these cores were col lected f rom the sewage outfal l lake, and the th i rd f rom the d r ink ing water lake. Cores were taken w i t h a G l e w m i n i -corer, and C o r e 1 (from the freshwater lake) and Core 2 (from the sewage outfal l lake) were s l i ced at 2 c m intervals. Core 3 (from the sewage outfal l lake) was s l i ced at 1 c m intervals. Sediment was transferred direct ly into plastic wh i r lpak bags, and stored and transported to the lab i n coolers w i t h ice packs. Once i n the lab, sediment was removed f rom the plast ic bags into glass petri dishes, and freeze-dried. Freeze-dried samples were weighed accurately, and 10 p g decachlorobiphenyl was added to each sample as an internal standard. A blank sample was run w i t h each set o f core samples to ensure there was no P C B contaminat ion. P C B s were extracted f rom the so i l by us ing Soxhle t extract ion (16 hours, w i t h 135 m L dichloromethane). The dichloromethane was then removed by rotary evaporation and the round-bottomed flasks were r insed thoroughly 56 w i t h hexane. The hexane was evaporated d o w n to approximately 0.2 m L , and passed through a F l o r i s i l co lumn. Eluent was col lected and made up to 1.5 m L by elut ing addi t ional hexane through the F l o r i s i l co lumn . Samples were placed i n autosampler v ia l s and analyzed by us ing G C / M S w i t h selected i o n moni tor ing , as described i n Chapter 2 (Sect ion 2-8). 3.5.3 Results and discussion N o P C B s were found i n the core sample taken from the d r ink ing water lake, or i n any o f the blank samples. Core 2 (from the sewage outfall lake) contained some P C B s , but on ly at very l o w concentrations. The results for the different sections o f Core 2 are presented i n Table 3-4. Due to the very l o w levels o f P C B s , only the presence or absence o f a congener was noted. The P C B congeners present i n Core 2 correspond to major components o f A r o c l o r 1242. W h e n the depth profi le is examined, it can be seen that not a l l congeners were detected at a l l depths o f the core. T w o peaks, corresponding to 24-25-te t raCB and either or both o f 26-3- and 2 3 6 - t r i C B , were only found i n the deeper sections. Ano the r congener, 236-34-pentaCB, was only present i n sections closer to the surface. The var ia t ion observed between different sections o f the core does not correspond to the k i n d o f var ia t ion that w o u l d be expected i f m i c r o b i a l dechlor inat ion o f A r o c l o r 1242 were occurr ing (i.e., the disappearance o f more-h ighly chlorinated congeners w i t h a corresponding increase i n less-highly chlorinated product congeners was not observed). Thus it is concluded that this core does not provide any evidence for in situ m i c r o b i a l degradation o f A r o c l o r 1242 i n the lake. 57 CN CN OO oo SO tzi CM Q CN CN C/3 1) 1 5 CN CN ix CN in CN 5H m CN CN CN i i C O CN CN IT) Tj" CN CN ON So i i CN CN CN m CN CN CN CN • CN i m CN Ti-r o i to CN o as <u o 58 The concentrations o f P C B congeners found i n Core 3 (also f rom the sewage outfal l lake) are presented i n Table 3-5. It can be seen that the P C B compos i t ion o f Core 3 is very different f rom Core 2, even though the two cores were taken from the same lake. The most abundant congeners present i n Core 3 are 23456-2356-nonaCB and/or 23456-234-o c t a C B (peak no. 121), and 23456-2345-nonaCB (peak no. 125). A r o c l o r 1268 is the on ly A r o c l o r that contains substantial amounts o f these congeners (17) (119). H o w e v e r , the other major congeners that should be present i n A r o c l o r 1268 (for example , the congeners that correspond to peaks n o . l 17 and 118) are either not detected i n the core, or are on ly present i n very smal l amounts. It is un l ike ly that the congeners corresponding to peaks 117 and 118 were dechlorinated i n substantial amounts w h i l e the congeners corresponding to peaks 121 and 125 were not. Because o f this, it was conc luded that there was no A r o c l o r 1268 present i n the P C B mixture . M o s t o f the other relat ively abundant P C B congeners present i n Core 3 correspond to congeners found i n A r o c l o r 1254, and a few correspond to congeners found i n A r o c l o r 1260. It was conc luded that the P C B mixture present i n Core 3 originated ma in ly from A r o c l o r 1254, w i t h a smal l propor t ion o f A r o c l o r 1260. It is possible that the h igh relative concentrations o f the h ighly-chlor ina ted P C B s (corresponding to peaks 121 and 125) are the result o f weathering. Less -h igh ly chlorinated P C B congeners have a higher water so lub i l i ty and are more vola t i le than more-highly chlorinated P C B congeners, and these differences can result i n an increase i n the proport ion o f more-highly chlorinated P C B congeners relative to less-highly chlorinated P C B congeners i n environmental samples. The relative enrichment o f more-h ighly chlorinated h o m o l o g groups i n lake sediments has been reported prev ious ly (120). 59 NO r -n.d. n.d. n.d. © cn © CN © © <0.1 in © <0.1 © © in NO n.d. n.d. o n.d. cn CN © <0.1 ON © © © <0.1 c n CN © NO © •4- m n.d. n.d. ON o n.d. oo o cn © <0.1 cn © CN O CN © <0.1 't <0.1 © rA ^ n.d. <0.1 in © n.d. oo © © <0.1 cn O CN © CN O <0.1 © o r--© CN c n n.d. n.d. NO o n.d. NO o o <0.1 cn © © CN o <0.1 © © ON © r-^ ( N < 1—< n.d. n.d. i n © n.d. © CN © <0.1 CN © <0.1 CN © <0.1 NO © c n © 1 O n.d. n.d. in © n.d. NO o <0.1 n.d. cn © <0.1 © <0.1 © CN © CN o 9-10 n.d. c n o NO © n.d. © <0.1 n.d. cn © <0.1 <0.1 ro> ON o <0.1 © ON l 00 <0.1 n.d. m o n.d. © cn © n.d. © CN © CN © <0.1 CN O c n o oo i <0.1 <0.1 © n.d. © © © oo © cn © m © <0.1 NO CN © oo © i NO n.d. n.d. NO © o CN © o oo o © o <0.1 o CN CN c n O NO t in n.d. n.d. 00 © in © cn CN NO © © 00 © © in © <0.1 ON CN O NO © m • o n.d. CN © CN in NO NO © CN NO © OO OO NO •<*• i c n n.d. n.d. c n CN n.d. ON in NO © ON 00 © CN in 00 c n ON © (cm) c n i CN n.d. n.d. c n n.d. cn NO cn © CN ON o oo oo in ON © ; depth CN i n.d. n.d. 9.51 n.d. o NO oo in © NO CN * ON 10.1 NO ,—i CN Sampl* t O n.d. n.d. 47.0 n.d. 52.4 14.8 cn 21.6 ON 20.4 19.1 85.4 12.3 16.2 Congener identities 3-4, 25-2 23-2, 26-4 25-4, 24-4 25-26, 23-3, 34-2 235-2, 25-25 24-25 24-24, 245- 2, 246- 4 23-25 236-4 245-4 245-26 25-34, 236-25, 24-34 236-24 234-4, 23-34 Peak No. in CN c n CN o cn cn CN cn cn 00 cn oo ON o i n c n i n 60 o <0.1 in in © r o O VO © C N O Tj" © ON TT o Tf o o C N CO o n.d. C N © VO © VO r o ON © c--© r o C N © r-o ON r o v o © C N VO C N C O o r o Tf o o V <0.1 o 00 © C N © C N © C O O C N O C N O C N © o ON o o V ON o o o V ©' o © TI-o r o O TT; © © V r o © r o C N O ON o o o V C N o o V o o v q C N © C N © TT © © © Tf" ON o o o C N o in o r o TT o r o O <0.1 o V VD O C N © C N © r o © o V C N O 00 © C N o OO o v o o o V O C N o -6 c <0.1 C N © © C N © r o © C O © o V o ON o C N O ON o 00 o v q o o o o V d <0.1 r o o O C N C O O Tf O VO O © V ON o ON o r o C O Tt Tf O in o Ti-e d <0.1 c o © r o TT o Tf © in © © r o © VO r o O r o Tt ON C N C N O C N o C N © C N O C N C N r -o in © OO o C N © m ©' in C N VO o C N r o C N CO o r o r o in o C N o r o © TT © C N in TT C N in TT o VO © © in o CO r o o o Tf m TT VO Tf VO o o TT o V O © r o TT v q 00 © wo © v o © ON in in o in C N Tf; in Tl" C N TT C O o o V C N r O r o ON r o C N r o t--Tt-' TT m © in C N i—t v d C O C N Tt C N ON o o o o V in v q r o r o in TT in in in o o TT o CN in CN in o d O in CN C N i—i Tt C N c d m v q T|-TT v d TT TT v o v d o C N CN C N r o r o C N ON C N in in v q C N c d ON o C N v q 00 r o C N i n in i> r o C N Tj" C N in C N o o ON C O Tf C N C N C N v d C O r o c> 15.4 in Tf >-i ON o o r o in r o C N v d TT © C N C N r o CN o d C N O ON v d o o 00 ON ON Tt C N TT - - i TT ON ON r o 235-25 r o C N i VO r o C N in" T|-C N C N in in -3- c o C N C N T|-C N • in TT C N C N i in Ti-r o in" TT C N 1 l VO Tt" Ti-r o r o C N C N TT C N i Tf r o C N VO r o C N VO r o C N ., VO TT TT r o C N i i v o m r o T)-CN C N m C N 1 v o in r o CN in" TT Tf C N C N 1 1 v o m r o TJ-C N r o ^ v o TT CN i m in T|-TT CO C N C N in Tf C N i in CO C N in v o TT C O T|-C N C N r o m 4 4 Tt" CO C O C N C N C N VO r o in C N C N v o i n in Ti-r o r o C N C N ^ VO Tf r o C N C N r o i i r \ l i n v o i Tf Tf TT r o r o r o C N C N C N TI-in i n in r--i n o o in Tf VO VO VO OO VO O Tf ON t-- o o 00 ON O 00 ON ON r o ON 61 K O o I ro o v q o C N c d C N c d C N c d n.d. n.d. o <0.1 n.d. 13.3 O 12.0 ro C N o ro c d C N c d C N c d C N c d c d V o o c d C N c d - d d ON C N c d v q o Tt o C N c d c d V c d V c d V c d V in c d c d V c d V in ON in c d o d 00 o ro o C N c d c d c d V c d V c d V in c d c d V c d V o d ro c d in ro C N r - ; r— c d C N c d v o c d in c d c d VO C N o Tt c d ro o d in c d VO r-~: v o o ro O C N c d c d V c d V <d V c d V ro c d C N c d - d d vo o d C N c d t--ro c d O - d d • d d c d V • d d - d d ro c d c d - d d v q o d C N c d o o r~~ C N ON c d C N c d v o c d ON c d - d d in C N ro m c d c d C N c d o d o v o o cn c d c d V Tt c d C N c d • d d ON c d ro c d C N c d o c d V o o C N O in c d C N c d Tt c d ro c d c d V v q c d V C N c d v q o d C N c d o d Tt; ro o in c d ro c d C N c d ro c d • d d v q • d d o ON - d d C N o d Tt ro' v o O cn c d ro c d C N c d C N c d c d V ON c d Tt c d - d d Tt • d d C N ON ro 00 C N in c d o Tt o ro c d Tt o Tt in c d • d d v q o d • d d v q o d 12.0 O v o c d ro c d Tt c d r--o in r-c d - d d o d - d d in ON 16.1 Tt 00 c d Tt c d ON c d C N ro C N ~ • d d o o c d • d d ro 14.4 in o o o in c d c d VO c d o V r--c d • d d o o ON • d d c d 94.3 o o v d ro c d ON ON C N o d in ON ro Tt c d 00 v d • d d ON i ( r—1 • d d 00 23456-3, 234-245, 2346-34 v o m Tt Tt C N C N in v o Tt m ro cn C N C N in Tt C N 1 VO Tt ro C N Tt ro C N i Tt ro C N VO ro C N in Tt ro C N Tt ro C N i VO m ro C N m Tt ro • Tt ro C N in in Tt Tt C N ro i i in v o Tt in ro ro C N C N Tt Tt ro ro C N 1 i VO in in Tt Tt ro ro C N C N in v o Tt in ro ro C N C N VO „ VO in v o in Tt m Tt Tt ro ro ro ro C N C N C N C N • VO m v o Tt Tt ro ro C N C N i VO in in Tt Tt ro ro C N C N Tt ON OO ON ON ON O O C N O ro O OO o in C N C N C N in C N o +-» CD O C • d d 62 The t r i - and te t ra-CB congeners present i n Core 3 do not correspond to expected dechlor inat ion products o f A r o c l o r 1254. Observed dechlor inat ion pathways usual ly exhibi t either meta or para dechlorinat ion, or a combina t ion o f the two (67), and most o f the t r i - and te t ra -CB congeners present i n Core 3 do not correspond to possible meta or para dechlor inat ion products o f A r o c l o r 1254 congeners. Because o f this, it was conc luded that there is no evidence for in situ P C B dechlor inat ion i n Core 3. 63 Chapter 4 The Effects of Different Enrichment Cultures, Autoclaving and Weathering on Aroclor 1260 Dechlorination 4.1 Introduction Several o f the enrichment cultures described i n Chapter 3 are capable o f dechlor inat ing P C B s present i n A r o c l o r 1260. It is o f interest to investigate this dechlor ina t ion i n more detai l , and to determine w h i c h o f the enrichment cultures promote the most extensive and rapid dechlor inat ion o f A r o c l o r 1260. In this chapter, the effects o f four different enrichment cultures on A r o c l o r 1260 dechlor inat ion are compared. The effect o f autoclaving soi ls and sediments after pre-incubation but pr ior to inocula t ion w i t h an enrichment culture is also investigated. Pub l i shed studies o n A r o c l o r 1260 dechlor inat ion report either dechlor ina t ion i n sediments that have been contaminated w i t h A r o c l o r 1260 for a number o f years (121) (69), or the dechlor inat ion o f recently added A r o c l o r 1260 i n sediments (67) (65). It is possible that weathered P C B s (i.e., P C B s that have been present i n sediments or soi ls for a number o f years) behave differently towards mic rob ia l dechlor inat ion than P C B s that have only recently been added to soils , because o f the reduced b ioava i lab i l i ty o f weathered P C B s (122). Consequent ly it is o f interest to determine the effects that weathering o f P C B s i n so i l may have on their dechlor inat ion by direct ly compar ing dechlor inat ion o f weathered A r o c l o r 1260 w i t h that o f recently added A r o c l o r 1260 i n s imi la r soi ls . In this chapter, several comparisons are made between the degradation o f unweathered and weathered A r o c l o r 1260 i n Reso lu t ion Island so i l . T h i s means that comparisons are made between so i l that has on ly recently had A r o c l o r 1260 added to it i n 64 the laboratory, and so i l that has a (long-term) history o f A r o c l o r 1260 contaminat ion i n the f ie ld . It is especial ly important to determine i f the enrichment cultures that have been developed can promote extensive dechlor inat ion o f the weathered P C B s found i n soils f rom Reso lu t i on Island and Saglek. In this chapter, the dechlor inat ion o f weathered P C B s i n both Reso lu t ion Island so i l and Saglek so i l is reported and discussed. 4.2 Experimental 4.2.1 Dechlorination of weathered and unweathered Aroclor 1260 in Resolution Island soil by four different enrichment cultures S o i l slurries were prepared by m i x i n g Reso lu t ion Island so i l (1.25 g) w i t h anaerobic Jericho Park sediment (0.75 g) and 4 m L R A M M . B a l c h tubes were c r imp-sealed w i t h butyl rubber stoppers inside an anaerobic chamber i n an atmosphere o f C 0 2 : N 2 : H 2 o f 5:85:10. Af ter two weeks, when methanogenic condi t ions had been established, the tubes were autoclaved. T w o different sets o f experiments were set up i n paral le l for both the weathered and unweathered (i.e., freshly spiked) treatments, i n order to test four different anaerobic enrichment cultures, B K 2 4 , B K 3 0 , B K 7 2 and B K 8 1 . The development o f these enrichment cultures was described i n Chapter 3. Inocu lum (1 m L ) and decachlorobiphenyl (1.0 |o.g) were added to the so i l containing weathered A r o c l o r 1260 [these cultures had a f inal so i l concentration o f 106 p p m A r o c l o r 1260 (wet weight)] . Inocu lum (1 m L ) , decachlorobiphenyl (1.0 u,g) and A r o c l o r 1260 [0.20 m g , to give a f inal so i l concentration o f 100 p p m (wet weight)] were added to the uncontaminated so i l . A r o c l o r 1260 and decachlorobiphenyl were added f rom 65 concentrated stock solutions i n acetone. Cont ro ls had 1 m L o f extra m e d i u m added instead o f i n o c u l u m . A summary o f the different experimental treatments is g iven i n Table 4-1 . Table 4.1: Summary of dechlorination experiments described in Section 4.2.1. Sample I D Reso lu t ion Island so i l used Inocu lum A r o c l o r 1260 added F i n a l A r o c l o r 1260 Concentra t ion Decach lo ro -b iphenyl added B K 2 4 Uncontaminated B K 2 4 100 p p m 100 p p m a 1 p p m B K 3 0 Uncontaminated B K 3 0 100 p p m l O O p p r n 3 1 p p m B K 7 2 Uncontamina ted B K 7 2 100 p p m 100 p p m a 1 p p m B K 8 1 Uncontaminated B K 8 1 100 p p m 100 p p m a 1 p p m B K C 1 Uncontamina ted N o n e 100 p p m 100 p p m a 1 p p m B K C 2 Uncontamina ted N o n e 100 p p m 100 p p m a 1 p p m RI24 Contaminated B K 2 4 N o n e 106 p p m b 1 p p m RI30 Contaminated B K 3 0 N o n e 106 p p m " 1 p p m RI72 Contaminated B K 7 2 N o n e 106 ppm" 1 p p m RI81 Contaminated B K 8 1 N o n e 106 p p m b 1 p p m R I C 1 Contaminated N o n e N o n e 106 p p m " 1 p p m R I C 2 Contaminated N o n e N o n e 106 ppm" 1 p p m a: unweathered A r o c l o r 1260 b: weathered A r o c l o r 1260 66 Soi l s were m i x e d by us ing a vortex mixer , and entire cultures conta ining inocu la were frozen at 0, 2, 4, 8, 12, 16, and 24 weeks; controls were frozen at 0, 8, 16, and 24 weeks. Samples were extracted, w o r k e d up and analyzed as described i n Chapter 2. 4.2.2 The effect of autoclaving prior to inoculation on Aroclor 1260 degradation. S o i l slurries were prepared by m i x i n g Reso lu t ion Island so i l (1.25 g) w i t h anaerobic Jer icho Park sediment (0.75 g) and 4 m L R A M M . B a l c h tubes were c r imp-sealed w i t h butyl rubber stoppers inside an anaerobic chamber i n an atmosphere o f C 0 2 : N 2 : H 2 o f 5:85:10. Af ter two weeks, when methanogenic condi t ions had been established, one set o f B a l c h tubes was autoclaved at 120 °C for 20 minutes, w h i l e the other set was left unautoclaved. W h e n the autoclaved tubes had coo led to r o o m temperature, i n o c u l u m (1 m L o f B K 8 1 ) was added to both treatments. Cont ro ls had l m L m e d i u m added instead o f i nocu lum. One set o f autoclaved and one set o f unautoclaved experiments used Reso lu t ion Island so i l that was contaminated w i t h weathered A r o c l o r 1260. The A r o c l o r 1260 concentration i n these so i l mixtures was 106 p p m (wet weight) , and decachlorobiphenyl was added to give a so i l concentration o f 1 p p m . A second set o f autoclaved and unautoclaved experiments used uncontaminated Reso lu t ion Island so i l . A r o c l o r 1260 w i t h decachlorobiphenyl was added to these experiments, to give a f inal so i l concentrat ion (wet weight) o f 100 p p m A r o c l o r 1260 and 1 p p m decachlorobiphenyl . A r o c l o r 1260 w i t h decachlorobiphenyl was added from a concentrated stock solu t ion i n acetone. A l l B a l c h tubes were m i x e d w i t h a vortex mixe r and incubated stationary at 21 ° C . Ent i re cultures were frozen at 0, 2, 4, 6, 8, 10, 12, 16 and 24 weeks. Samples were 67 extracted, w o r k e d up and analyzed as described i n Chapter 2. The different treatments used i n this set o f experiments are summar ized i n Table 4-2. Table 4.2 Experimental protocol for determining the effect of autoclaving on Aroclor 1260 dechlorination. Sample I D Reso lu t ion Island so i l used Inoculum added A u t o -claved? A r o c l o r 1260 added F i n a l A r o c l o r 1260 concentration Decach lo ro -b iphenyl added B K 8 1 a Uncontamina ted B K 8 1 Y e s 100 p p m 100 p p m 1 p p m B K C l a Uncontamina ted N o n e Y e s 100 p p m 100 p p m 1 p p m B K 8 1 b Uncontamina ted B K 8 1 N o 100 p p m 100 p p m 1 p p m B K C l b Uncontamina ted N o n e N o 100 p p m 100 p p m 1 p p m R I 8 1 a Contaminated B K 8 1 Y e s N o n e 106 p p m 1 p p m R I C 8 1 a Contaminated N o n e Y e s N o n e 106 p p m 1 p p m RI81b Contaminated B K 8 1 N o N o n e 106 p p m 1 p p m R I C 8 1 b Contaminated N o n e N o N o n e 106 p p m 1 p p m 4.2.3 Further experiments on weathered and unweathered Aroclor 1260 in Resolution Island soil S o i l slurries were prepared by m i x i n g Reso lu t ion Island so i l (12.5 g) w i t h anaerobic Jericho Park sediment (7.5 g) and 40 m L R A M M i n serum bottles. These serum bottles were cr imp-sealed w i t h Tef lon- l ined butyl rubber stoppers inside an anaerobic chamber w i t h an atmosphere o f C02:N2:H-2 o f 5:85:10. Af ter two weeks, when 68 methanogenic condit ions had been established, the bottles were autoclaved. T w o different experiments were set up i n paral le l . One experiment used weathered A r o c l o r 1260 i n Reso lu t ion Island so i l , and had a f inal so i l concentration o f 106 p p m A r o c l o r 1260 (wet weight) . The other experiment used unweathered (i.e., freshly spiked) A r o c l o r 1260 i n Reso lu t i on Island so i l . A r o c l o r 1260 was added to this experiment to g ive a final so i l concentrat ion o f 100 p p m (wet weight) A r o c l o r 1260. A r o c l o r 1260 was added f rom concentrated stock solutions i n acetone. 5 m L o f i n o c u l u m ( B K 7 2 ) was added to the cultures. Cont ro ls had 5 m L o f extra m e d i u m added instead o f i nocu lum. So i l s were m i x e d w i t h a vortex mixer , and incubated at 30 ° C . A l l incubations were performed i n tr iplicate, and serum bottles were subsampled at t ime zero, and again after 8 weeks o f incubat ion. Samples were extracted, worked up and analyzed as described prev ious ly (Chapter 2, Sections 2-5 to 2-8). A summary o f the different treatments used is g iven i n Table 4-3. Table 4-3: Dechlorination of weathered and unweathered Aroclor 1260 in Resolution Island soil. Sample I D Reso lu t ion Island so i l used Inoculum added A r o c l o r 1260 added F i n a l A r o c l o r 1260 concentrat ion 201a,b,c" Uncontaminated B K 7 2 100 p p m 100 p p m 202a,b,c* Uncontaminated N o n e 100 p p m 100 p p m 203a,b,c* Contaminated B K 7 2 N o n e 106 p p m 204a,b,c* Contaminated N o n e N o n e 106 p p m * a,b,c refer to triplicate cultures 69 4.2.4 Dechlorination of Aroclor 1260 in weathered soils from Resolution Island and Saglek S o i l slurries were prepared for incubat ion as described i n section 4.2.1, us ing either Reso lu t ion Island so i l or Saglek so i l , both o f w h i c h contained weathered A r o c l o r 1260 (final so i l concentrations (wet weight) o f A r o c l o r 1260 were 106 p p m and 68 p p m respectively). Mod i f i ca t i ons to the experimental procedure were made as fo l lows : butyl rubber stoppers were replaced w i t h Tef lon- l ined butyl rubber stoppers i n an attempt to decrease the loss o f P C B s experienced previously; decachlorobiphenyl was omit ted and samples were made up to 1.00 m L i n a vo lumet r ic flask after extraction and clean-up. Incubations were inoculated w i t h enrichment culture B K 8 1 , and controls had 1 m L extra R A M M added instead o f i nocu lum. Incubations were stopped after 0 weeks and 8 weeks. A l l treatments o f Saglek so i l were done i n tr iplicate. Sample extraction, work-up , and analysis was performed as described prev ious ly (Chapter 2, Sections 2-5 to 2-8). 4.3 Results and Discussion 4.3.1 Relative rates of weathered and unweathered Aroclor 1260 dechlorination Four different enrichment cultures were used to investigate relative rates o f A r o c l o r 1260 dechlorinat ion. A l l cultures, except for controls, showed evidence o f dechlor inat ion. Incubations w i t h cultures B K 2 4 , B K 7 2 and B K 8 1 showed extensive dechlor inat ion, w h i l e incubations w i t h culture B K 3 0 on ly showed a sma l l amount o f dechlor inat ion act ivi ty. Dech lor ina t ion act ivi ty was indicated by an increase or appearance o f less-highly chlorinated P C B s (mainly te t raCBs) (Tables 4-4 to 4-7). One 70 control also showed evidence o f slight dechlor inat ion after 24 weeks, but the other three controls d i d not (Table 4-8). A l l treatments showed the disappearance o f P C B congeners, but this was not taken as conclus ive evidence for dechlor inat ion act ivi ty since the mass balances showed that not a l l o f the P C B s were accounted for. 71 > o a NO cn oo ,—1 C O cn cn cn cn cn 4> ON NO o NO ON 00 O 00 o in © cn in © o in CN od o cn o 00 r--S '—» s K SI 3 o cs >• o s PS o o NO cn in ON cn © cn in o © cn in NO cn © '—i in *—i o oo m ON in o NO 00 NO NO cn cn NO ON ON •2 ST s I «C3 U J 3 es c CN O B © P3 U OH in oo 5 © W) o in oo O - H o NO u r" Q o r--NO CN in CQ U S-H H 1/3 m u y i s oo CN CN NO o o NO l N l " i—i ,—i o ON Vi u a x u C/3 U o O CN in U a o 00 ON NO cn O cn NO CN ON >—; ON cn 00 ON "St cn cn NO o © CN cn cn o o 05 OS > © s p< 0 S Tt ON in ON v d o d v d CN CN co co '—i '—( 5C C N ON CN V O Tt CO o o in m co Tt in © CN ON c d v d c d c d c d Tt *—' OO s '—a s o c cu <u SI es Tt co 93 >• O s Tt C O ON C O >—; Tt © in v d CO c d '—i in '—i Tt C N o l > o o v d c d CO Tt C N C N <—i o o 03 I T5 s-« s SB cu T t C N J2 o a o pa u PH CN CO v o o o in CN 00 Tt in o o Tt o d in ON o <—< '—i CN Tt c d r - -C O C N ON C N © V O CO v o 00 >—i O o C O o o ON c d Tt CO C N CO Tt C O v d c d c d o C/3 CQ O 'EH H CQ o in U H on C/3 CQ PH in CQ O +-* CO C/5 CQ U cd -t—< o O C/3 PQ U cd O o u Pi et © S cu Pi v q in c n in v d C N Tt in m Tt o o in v o OO CO c n r o in c n v o r o o o in ON c n c n 00 Tt ON O o c n Tt C N v d c d »3 a cq T3 cu s-cu 0) 5 CU o O S Pi c n o o r-- m CN ON c n v q Tt CN r-; o c d c n in v d c n in c d r-- ON CN oo C N 00 ON r--C N VO O OO ON VO VO in Tt c d o S 1^ ^>, s v d T3 cu s-cu cu S3 cu cu CC cu cu ° c o E o -= P2 U CC CQ U 5 VO * c-» C N ON ,—1 r - , 1 v o v o T t in O c d o C N o Tt c n C N C N c d C N Tt Tt in C N c d VO CC PQ O H c n ON o o C N o c n o o ON in 00 00 Tt c n © c n v d c d c d C N c n c n o CC CQ U Cd a CD H CC CQ U cd cu PH CC CQ U cd X CD cc CQ U cd -t—> A , CD CC CQ U cd CC CQ O cd C o cu > o cu cu ffi ffi o £ pi S3 > o r-- CN o ON NO in CN o ON in cn cn C D cn in o cn ,—c cn cn ON >—1 >—; •—1 < in o o NO cn 00 cn CN NO o ON to S S i - A o es 4> J2 m cn NO oo o ON CN in CN 00 in NO cn o * in r-- r--CD C D O ?° es > © £ cn [--; C D NO NO cn in cn 00 oo oo NO NO •2 to 1^ bo o 1^ to ^ • tool R a u ii es a a a CN * ° o etf> o o « in cn cn in ON NO in CN CN oo NO ON CN cn CN cn NO CN C D oo u 00 m u Q H 00 PQ U 03 s-c +J <U H oo m u oi oo U 03 X X oo u 03 +^ OH <u 00 m u 03 +-» O O on CP u 03 S3 O ON OO NO NO ON NO CN OO NO NO <—J NO o in 00 NO CD cn CN C D cn cn NO C D C D O ii > o u ii Pi et > o E cu Pi r-- ro CN r--vd CN O CN o cn CN r—1 .2 cu cu T t ( N CN ON in m Tt _ o 00 CN r--o CN ON CN ON Tt cn © oo T3 cu cu et cu o 4: o I t o 05 1.3 Co cu 5 I cu u cu -a cs cu c J2 cu cu o o E cu Pi CC cu cu T t CN 3 cu cu two o "o E o J3 CQ U PH CC PQ U CC PQ u cd l-l CU H CN 0 O _ H m Tt in Tt Tt vd in ro 1—1 in 1—1 CQ U cd CD PH PQ U cd cu X CC PQ U cd CU CC CQ O cd -»-» o o ON vo © o 0 O 0 Tt CN 0 O vd Tt r-H cn in cn CN CN cn cn O r-- 00 ON Tt Tt in 1—1 0 O Tt Tt in cd' cn CN CN cn in ON 00 O cn CN CN in in '— 1 O Tt ON ON O CN cn CN in vd cd cd CN cn cn 0 CQ O cd G o cu > o cu cu Pi 100 80 -60 -o 40 -20 -P e n t a C B s g T ime (weeks) 1 g 24 Unweathered control Weathered control BK(24) unweathered BK(24) weathered BK(81) weathered BK(81) weathered Figure 4-1: Disappearance over time of penta-, hexa- and hepta-chlorobiphenyl congeners in both weathered and unweathered soil treated with two different enrichment cultures, BK24 and BK81. 11 0 8 16 24 0 8 Time (weeks) 1 6 2 4 -#— Unweathered control — • - Weathered control - A - BK(24) unweathered - • - BK(24) weathered • BK(81) unweathered - • - BK(81) weathered Figure 4-1: Disappearance over time of penta-, hexa- and hepta-chlorobiphenyl congeners in both weathered and unweathered soil treated with two different enrichment cultures, BK24 and BK81. 11 Since the best mass balances obtained for incubations showing a large degree o f dechlor inat ion act ivi ty were for the incubations that used cultures B K 2 4 and B K 8 1 , the data f rom these two experiments was investigated i n more detail. In order to compare the effects o f the different treatments and controls w i t h each other, the disappearance over the s ix month t ime per iod o f penta-, hexa- and hepta-chlorobiphenyls, the most abundant P C B homologs i n the or ig ina l samples, is shown i n Figure 4-1. These results clearly show that the most extensive dechlor inat ion occurred dur ing the first 8 weeks, and al though dechlor inat ion was s t i l l be ing observed after s ix months, the rate had decreased substantially. S imi l a r observations were made by S o k o l et a l . (123) for the dechlor inat ion o f weathered A r o c l o r 1248 i n sediments, but i n their work , the most extensive dechlor inat ion was reported to occur over a longer per iod o f t ime. There is a marked difference between the extents o f dechlor inat ion observed for treatments w i t h i n o c u l u m B K 8 1 i n unweathered and i n weathered so i l (Table 4-7, Figure 4-1). Th i s difference may be due to the decreased b ioava i lab i l i ty o f weathered P C B s . The mixture o f weathered P C B s present i n Reso lu t ion Island so i l had a higher propor t ion o f more-highly chlorinated (hepta-, octa- and nona-chloro) congeners and a lower propor t ion o f the less-highly chlorinated (hexa- and penta-chloro) congeners than is expected i n A r o c l o r 1260 (Tables 4-4 to 4-8), w h i c h cou ld poss ib ly contribute to the lower rate o f dechlor inat ion i n weathered so i l as w e l l . (The A r o c l o r 1260 used for the unweathered experiments had a lower proport ion o f the more h igh ly chlorinated P C B s than was found i n the weathered A r o c l o r 1260). A smal l difference between the extent o f dechlor inat ion observed i n unweathered and weathered P C B s i n so i l was also observed for incubations inoculated w i t h enrichment culture B K 2 4 , but this difference is probably 78 not significant, g iven that the so i l containing unweathered P C B s gave m u c h poorer P C B recoveries than the so i l containing weathered P C B s . The major product formed i n a l l cultures had a retention t ime that corresponded to either 24-24- te t raCB or 245-2-te t raCB (these two congeners co-elute o n the c o l u m n used). Exper iments described i n Chapter 7 conf i rm that this major product is 24-24-te t raCB. The other major products formed were ( in order o f abundance) 24-26- te t raCB, 246-24-pen taCB, 24-25- te t raCB and 25-26- te t raCB. Tr i ch lo rob ipheny l peaks that corresponded to 26-4 or 2 3 - 2 - t r i C B (co-eluting), 24-4 or 2 5 - 4 - t r i C B (co-eluting) and 25-2 - t r i C B were detected, al though i n m u c h smaller quantities than the tetrachlorobiphenyl products. 2-4, 4-4, 2-2 and 2 - 6 - d i C B were also detected, as were 2 - C B and 4 - C B , al though these monochlorobiphenyls were only present i n very smal l quantities. The slight dechlor inat ion found i n one o f the controls is most l i ke ly the result o f contaminat ion o f the control , because even though controls were autoclaved pr ior to incubat ion, they were sealed i n an anaerobic chamber w h i c h was not sterile. Unfortunately the P C B mass balances determined for the 6-month incubat ion series were not very good, ranging f rom 6 3 % to 73% recovery i n the experiments w i t h unweathered P C B s i n so i l , and f rom 80%) to 88%) i n the experiments conta ining weathered P C B s i n so i l (Tables 4-4 to 4-8). The better mass balances obtained for the weathered experiments are probably a consequence o f the weathered P C B s be ing more t ightly bound to the so i l and therefore less l i ke ly to sorb to the butyl rubber stoppers, or to the greater propor t ion o f more-highly chlorinated, less volat i le P C B congeners present i n the weathered treatments. 79 4.3.2 The effect of autoclaving prior to inoculation on Aroclor 1260 degradation. The results obtained for P C B dechlor inat ion i n so i l that was autoclaved p r io r to addi t ion o f the i n o c u l u m (or, for controls, prior to addi t ion o f an extra 1 m L R A M M ) are presented i n Tables 4-7 and 4-8. The results obtained for so i l that was not autoclaved pr ior to addi t ion o f the i n o c u l u m (or addi t ion o f an extra 1 m L R A M M for controls) are presented i n Tables 4-9 and 4-10. Figures 4-2 and 4-3 show the disappearances o f hexa-and hep t a -CB congeners i n the autoclaved and unautoclaved inoculated incubations and controls for both weathered A r o c l o r 1260 and freshly added A r o c l o r 1260. It can be seen from these figures and from Tables 4-7 and 4-9 that i n a l l instances the inoculated incubations that were not autoclaved showed at least 20 to 30 % less disappearance o f hepta- to n o n a - C B congeners than the inoculated incubations that were autoclaved. The differences between the autoclaved and unautoclaved uninoculated control incubations (Figures 4-2 and 4-3 and Tables 4-8 and 4-10) were very smal l i n compar ison . T h i s means that the observed differences are not due to increased vola t i l i ty o f P C B s dur ing the autoclaving process. Rather, it suggests that autoclaving cou ld el iminate m i c r o b i a l populations that might otherwise compete w i t h the P C B dechlorinators, or it c o u l d help make nutrients bound i n the so i l or i n the biomass more accessible to P C B degraders. In response to these results, sediments were autoclaved pr ior to inocula t ion w i t h enrichment cultures i n a l l subsequent experiments. 80 •2 SI I"? a s o c es > o B Pi 0 s co o C N oo co r~1 CN CD co 00 00 in CN "3- CN CN CN — in 00 oo ON o o CN in l< O NO CD • *—1 00 CO 00 o 00 Ik s a S-I -a J2 © oo m o 00 ON o o CO 1/0 CO CD t-- oo CO CD co in NO CO CD CD CD 1 — 1 ' 1 in O ?° •S 1^ es > © a Pi CN CO O CO ON i n ON NO NO CN CN •2 -kt 3 1^ I? tO s? ON s o c -a u •** es a oo CN 6 0 J * o CU) o o n u a. CO CO in r—« r- o CN 00 o ^ H CO © 1/0 CN 00 NO I/O >—1 CO 00 «—i CD ND CN in OO CO in NO CN ON o in OO CD CD CO CN CD CO CO NO CD O 00 CP u 00 00 CQ CQ 03 S-H -t—» o H 03 CO m u 03 X 0) on CQ U 03 OH K 00 CQ O 03 -t-» O O oo CQ U 03 a o > o u Pi •2 ft, I"? cu > a s o K <u <U 4: R O 1ST It c •2 s Co I "3 bo o to cx> 5 •2 s •a I-cu E es cu T3 cu s-cu 03 CU a P 73 O s cu Pi J2 cu cu C N cu cu 03 o s cu Pi J2 cu cu T t J2 cu cu o wo _© "3 S © pa u C N in r o C N v o O C N r o C N in v d O Tt r o VO OO r o 00 in r o in C N C N r--o o ON m c d o o C N C N ON o r o in in OO O0 o © ' o Tt v d in C N c d c d i n T—1 o o o 00 C N o C N o o C N C N c d c d C N CO C N C N ON 00 C N O o o O ON o ON Tt in in OO in o o C N CN in c d r-ON o C N r-- ON v o r-Y-H in v q ^ H 00 Tt 00 r--o C N r o C N Tt v d c d c d C N r o r o o PQ U H m PQ U cd VH CO H PQ u cd -t—» cu PH on PQ U cd X cu X m PQ U cd -*-> PH CU m PQ U cd -4-* o O m PQ U cd a o cu > o cu cu Pi I o ro o O, < c o O I _ If 03 O $> 1 § I o | ° I 8 3 E < o CD O 00 o o CD O LO o o co o CM 1 ^ 8 0> o S CSJ JS is £ T ~ o c (i o o gj o | ° I 8 111! o I ro c 3 73 CD 1 E J K 8 8 JE £ 5 "5? 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O CJ O ^ 3 cu a cu i c e a ^ 3 cu k, cu •s a cu 84 4.3.3 Further experiments on weathered and unweathered Aroclor 1260 in Resolution Island soil A further experiment compar ing the dechlor inat ion o f weathered A r o c l o r 1260 and unweathered A r o c l o r 1260 i n Reso lu t ion Island so i l was carr ied out i n order to conf i rm results discussed i n Sect ion 4.3.1 w h i c h indicated that A r o c l o r 1260 dechlor inat ion was possible for both unweathered and weathered A r o c l o r 1260 i n Reso lu t ion Island so i l , and suggested that unweathered A r o c l o r 1260 dechlor inat ion was more extensive than weathered A r o c l o r 1260 dechlorinat ion. The results discussed i n Sect ion 4.3.1 were obtained w i t h serum bottles that had been sealed w i t h buty l rubber stoppers, and accurate mass balances were not produced. In an attempt to improve the mass balances, the butyl rubber stoppers were replaced w i t h Tef lon- l ined buty l rubber seals i n these experiments, i n the be l i e f that the P C B s , especial ly the less h igh ly chlorinated, more vola t i le ones, cou ld be sorbed by the butyl rubber stoppers. Af t e r the stoppers were changed there was a marked improvement i n the mass balances achieved -this t ime they were excellent, ranging f rom 9 1 % to 104%. The results for this set o f experiments are g iven i n Table 4-11. Cont ro l s d i d not show any signs o f dechlorinat ion. The results obtained for the incubations after 8 weeks are comparable w i t h the results obtained previous ly for the same t ime per iod , except that m u c h more tetrachlorobiphenyl dechlor inat ion products were detected. T h i s corroborates the hypothesis that te t raCBs were being produced but not detected quanti tat ively i n the previous set o f experiments. Figure 4-4 shows the appearance o f t e t raCB congeners after 8 weeks o f incubat ion, and Figure 4-5 shows the disappearance o f hexa- and hep ta -CB 85 ha O > © S Pi CO oo 1/0 CO Tt CO © ON vd VO VO vo Tt Tt © cu , ^ 1-5 a cu cu cu T t CN VO o Tt oo CO ON CN CO Tt I/O 1/0 co^ CN o o —' cd cd cd cd o . V —' ON •' S —' CO o VO r- CN 00 ON Tt , —; vd CO CO cd Tt Tt vd o in in vd CN vd cd ON h a c C3 1 CU cu s; a K •2 a 4: co ©° 1^ to cu K 5 I c—I Co CU u cu i JS « CU 13 cu u cu i-n es cu a 1 o © eS > © s cu Pi CC cu cu Tt CN CO Tt Tt Tt CN 5 ^ © o "© E o pa u PH CC PQ O ' JH H oo CN o oo CN in ON OO VO o CO cd vo 00 CN cc PQ U cd -t—» CO H CO CO in OO ON TT CN cd cd ©_ vo p 00 Tt ON m oo I/O CN in CN CN O ON in CN o Tt CN cc PQ U cd +-* 0 co PH CN - H oo oo o ON cd CO CC PQ O cd X co HI CO CO ro cc PQ U cd —^» OH CU X r-, O VO PQ U cd o O CN ^ OO CN o in © 00 Tt OO cd 00 cd 00 in r--Tt VO oo 00 Tt in m oo oo VO oo cd © , cd cd ON vo CN CN ro vo Tt vd od CN cd 2 CN . O i - H 00 in oo CC PQ O cd o CN CN ON Tt o , ON 00 ON CU > © cu cu Pi A p p e a r a n c e of T e t r a C B C o n g e n e r s <a o c OJ re 0 QL a (TJ I 30 c a; o 0 70 50 10 -10 J Unweathered Weathered Unweathered Weathered Resolution Resolution Soil Control Soil Control Island Soil Island Soil Figure 4-4: Appearance of tetraCB congeners in weathered and unweathered Resolution Island soil over an incubation period of 8 weeks (mean ±SD, n=3). congeners after 8 weeks o f incubation. F r o m these results it can be seen that there is a significant difference between the dechlorinat ion o f unweathered A r o c l o r 1260 and weathered A r o c l o r 1260 i n Reso lu t ion Island so i l . Different h o m o l o g classes show a range o f 15 to 3 5 % less dechlor inat ion i n the weathered A r o c l o r 1260 than i n the unweathered A r o c l o r 1260. These results show that laboratory experiments need to use weathered contaminated soi ls i n order to predict dechlor inat ion act iv i ty i n sites that have his tor ical A r o c l o r 1260 contaminat ion. 87 (a) H e x a C B s 8 1 0 0 j Unweathered Weathered Unweathered Weathered Soil Resolution Island Resolution Island Soil Control Control Soil Soil (b) H e p t a C B s Unweathered Weathered Unweathered Resolution Island Resolution Island Soil Control Soil Soil Weathered Soil Control Figure 4-5: Disappearance of (a) hexaCBs and (b) heptaCBs in weathered and unweathered Aroclor 1260 over an incubation period of 8 weeks (mean +SD, n= 4.3.4 Dechlorination of weathered Aroclor 1260 in soils from Resolution Island and Saglek Resul ts f rom experiments on the dechlor inat ion o f weathered A r o c l o r 1260 i n soils f rom Reso lu t ion Island and Saglek are summarized i n Tables 4-12 and 4-13. The results obtained for incubations w i t h Reso lu t ion Island so i l show a substantial decrease i n the concentrations o f penta-, hexa-, hepta- and oc t a -CBs , accompanied by the accumula t ion o f less-highly chlorinated P C B congeners, ma in ly te t raCBs. These results are consistent w i t h other results (presented i n Sections 4.3.1 and 4.3.3) obtained for incubations w i t h this so i l . The total extent o f dechlor inat ion is s l ight ly higher than for the experiments reported i n Sect ion 4.3.1 even though the same incubat ion temperature is used, and this is consistent w i t h observations that the dechlorinat ing cultures become increasingly more efficient over successive transfers. Weathered A r o c l o r 1260 i n Saglek so i l showed a far higher extent o f dechlor inat ion than it d i d i n the Reso lu t ion Island so i l , w i t h on ly 2 7 % o f the P C B congeners found at the start o f the incubations s t i l l being present after 8 weeks ( in compar i son to 40%o for Reso lu t ion Island so i l , and 9 7 % for the controls) (Tables 4-12 and 4-13). Howeve r , when the differences i n P C B concentrations o f the two different soi ls are taken into account, it can be see that the total amount o f dechlor inat ion is very s imi lar , i.e. 99 p p m for the Saglek so i l and 101 p p m for Reso lu t ion Island so i l . T h i s w o u l d br ing the concentration o f A r o c l o r 1260 i n Saglek so i l to 38 p p m , w h i c h is w e l l be low the l i m i t o f 50 p p m set out by regulations ar is ing f rom the Canad ian Env i ronmen ta l Protect ion A c t , but Reso lu t ion Island so i l is s t i l l above the l imi t , at 69 p p m . It should be noted that the A r o c l o r 1260 congeners removed have been replaced by 89 O S Pi in cn NO ON NO ON e NO - H O r-H r- ON Zl 11 r- o r-H O CN + O a o s 4: bo o 1-3 =0 CNJ o a o U 00 P3 .2 00 &o o es > © s Pi u C u .3 o 00 .3 es 3 £ O two o "o s o pq U 0* m cn cn OO IT) '—1 ON cn ON 00 O O O ,_J y ^—^ CN ON rl- ON r-~- NO ON *-H NO 0 NO cn 0 cn <—1 CN ^ H CN CN O OO o £ o 00 o o CO CQ U H o o o 00 + + cn cn ON ON o 00 ON CN ON H 0 00 cn in NO ,—< cn OO '— 1 CN CN ON cn o © m NO o 00 in ON 00 to CQ CO 03 5 (3 OH in NO CO CQ U 03 x CD CN cn ON NO CN 2 CN in NO ON ON r-in O O CN, CN ^-CN NO cn NO CN •* CO CQ O -*-» PH <D CO CQ U 03 -t-» O o CN © 00 r- cn in —^1 cn >—; cn CN O ,—1 O , ^ O ^—j cn NO cn NO cn r - CN in cn in CN CN CN NO 00 ON CN CN ON ON 06 O in in OO NO NO CN NO 00 O cn cn in o CO CQ O 03 S3 o NO d CN 00 ON o o NO in ON m ON o r< © CL) > O o 03 > © s CU OS cu cu S3 cu u .cu v q o o o Tt r o r o C N o r o o v o v o V O o o v o C N o 1 o 1 C N • O • V O o + o to C J 3 a #© '•** 03 X ! S eu C o c o U o co , ^ cu , ^ a cu £ 1 c o a is CO O 5 00 CQ xi to CJ cu CC _<! cu cu 00 J2 cu cu o 03 > o s cu s o 0 s cu cu a cu 2 cu cu 00 1/1 a V* 03 X ! s cu 5 o CC cu cu 6* <© E © XI CQ C J PH o V O C N CO O N OO r o o OO o o O N O N o O Tt © C N o v o ON V O 00 cn VO V O Tt o o C N Tt o d C N cn '—1 '—< v o <—i v q r o <-< o d Tt V O ON V O I/O r o 00 C N + Tt C N m + cc CQ U J-i H CC CQ CC CQ cd eg CU H cu PH CC CQ U cd X cu X CQ U cd OH CU CC CQ u cd -*-» o O 00 . Tt r o 00 V O V O r o r o °) C N v o l> O 1/0 C N v d Tt c d 1 ^ H r o 1 Tt ON O o o o v o o o O r o o r - ; 00 c--C N CN 00 v o v d o d c d Tt I/O ON 00 c-» V O r o o o o r o ON v o o o VO © c d Tt C N r o c d ON r - H ^ H v o ON CC CQ U cd CJ o Z >H CU > o CJ CU h igh concentrations o f less h igh ly chlorinated tetrachlorobiphenyl congeners. H o w e v e r , tox ic i ty studies have shown that the tox ic i ty o f P C B mixtures can decrease s ignif icant ly after dechlor inat ion (78, 79). The treatments w i t h Saglek so i l were performed i n tr iplicate and the reproducibi l i ty o f the results was excellent. 4.3.5 Implications The results presented i n this chapter clearly show that weathered A r o c l o r 1260 i n soils that were in i t i a l ly aerobic can be degraded by anaerobic enrichment cultures. T h i s is an encouraging development for those consider ing bioremediat ion strategies for the treatment o f PCB-con t amina t ed so i l . The most extensive dechlor inat ion takes place i n a relat ively short per iod o f t ime (8 weeks), after w h i c h the rate o f dechlor inat ion s lows d o w n substantially. Th i s is important information to consider when p lann ing the t ime scales o f b ioremedia t ion procedures. The results presented i n sections 4.3.1 and 4.3.3 also show that the effect o f weathering needs to be taken into account, and i n some instances weathering may l imi t P C B dechlorinat ion; al though this effect can vary considerably between different locations and P C B concentrations. In particular, the results presented i n this chapter have shown that the enrichment cultures described i n Chapter 3 are capable o f extensive dechlor inat ion o f the weathered P C B congeners found i n Reso lu t ion Island and Saglek soils . 92 Chapter 5: Temperature Effects 5.1 Introduction The w o r k described i n this chapter was carried out i n order to investigate the effect o f temperature on the rate and extent o f A r o c l o r 1260 degradation. Because anaerobic dechlor inat ion o f A r o c l o r 1260 is a relat ively s low process, it is important to f ind the op t imal temperature at w h i c h incubations should be run i n order to get the highest possible rates o f degradation. In general, the rate o f a chemica l react ion increases w i t h increasing temperature as described by the Arrhenius equation. The m i c r o b i a l dechlor inat ion o f P C B s is expected to be an enzyme-catalyzed reaction; however , and wh i l e enzyme reactions f o l l o w this increase i n rate wi th increasing temperature up to a certain point , their act ivi ty decreases s ignif icant ly above some cr i t ica l temperature, because o f protein denaturation. There are very few literature reports on the effect o f temperature on m i c r o b i a l reductive dechlor inat ion o f P C B s (61, 124, 125) and there are no literature reports on the effect o f incubat ion temperature on the reductive dechlor inat ion o f A r o c l o r 1260. 5.2 Methods Sediment slurries were prepared i n 2 5 - m L B a l c h tubes by m i x i n g 1.25 g o f Saglek so i l contaminated w i t h 109 p p m o f A r o c l o r 1260 (wet weight) , and 0.75 g o f anaerobic pond sediment, w i t h 4 . 5 m L R A M M . Tubes were cr imp-sealed w i t h Te f lon - l i ned bu ty l rubber stoppers inside an anaerobic chamber, and were autoclaved after 14 days o f pre-incubat ion. T h e tubes were then inoculated (0.5 m L ) w i t h an enrichment culture ( B K 8 1 ) . 93 Cont ro l cultures that contained extra m e d i u m instead o f i n o c u l u m were also prepared, and a l l incubat ions were carried out stationary and i n the dark. T h e incubat ion temperatures used were 21 ° C , 30 ° C , 37 ° C , 42 ° C and 55 ° C . Ent i re incubations were stopped at 0, 2, 4, 8 and 16 weeks by p lac ing the B a l c h tubes i n a freezer. The 8-week t ime point was sampled i n triplicate, i.e. w i t h three separate incubations. Ex t rac t ion , work-up and data analysis was performed as described i n Chapter 2 (Sections 2-5 to 2-8). 5.3 Results and Discussion The changes i n the P C B homolog profiles over t ime for the incubations at 21 ° C , 30 ° C , 37 ° C and 42 ° C are summarized i n Tables 5-1 to 5-4. The results for the 55 ° C incubations are not presented as no dechlor inat ion was detected. The most rapid and extensive dechlor inat ion occurred at 30 ° C . Incubations at 21 ° C also showed extensive dechlor inat ion, but the incubations kept at 37 ° C showed on ly a sma l l degree o f dechlor inat ion, and the incubations at 42 ° C showed almost no dechlor inat ion. There was no evidence for any dechlor inat ion i n any o f the incubations kept at 55 ° C . N o n e o f the controls showed any signs o f dechlor inat ion after 8 weeks o f incubat ion, but both the 21 ° C controls and the 30 ° C controls showed a smal l amount o f dechlor inat ion after 16 weeks o f incubat ion. N o monochlorobiphenyls were detected i n any o f the incubations, but trace levels o f b iphenyl were detected i n both the 30 °C and the 21 ° C incubations after 16 weeks. One o f the tr iplicate 21 °C incubations showed the appearance o f a large propor t ion o f d ich lorobiphenyls after 8 weeks o f incubat ion, but no d ichlorobiphenyls were detected i n any o f the other incubations. Th is result suggests that the i n o c u l u m transferred into this 94 to K •2 s •S bo o o 8 •2 s K O no CD I 00 co M CD CD NO ^ o CD t-i > o oo O CO , i M o CD i-i % ^ > o oo O CO RT M O CD t-i !> O oo O CO CD CD 2 * 0 0 CN CO M CD co CD CD CO CD CD CO ^! CD CD 00 £ CO CD <D CO M CD CO M CD CD bfl O o a ON CN CN co u n CD to K •2 s Co •S bo o o 8 . •2 3 -0 ^3 J*. i "O CN NO CO M CD CD TJ" NO CN O co _^ M O CD VH > O oo O TT | C N m TT O ON TT in NO O T f © co , i M O CD i-i % * > o : CN NO T f TT T f NO co _ M O CD t-i ? O oo U CN © T f TT CN o d CO , i ^ O CD >H !> O O U ro ON I co CN CN NO CO CD CN TT © CN O ON © 00 M CD CD ON 0O CN m NO © co © co CO M CD CD 0O 5 OO ONl CN ON oo in CN OO CO M CD CD 00 j£ ON ON | hi-O ON CO ON in co CO M CD <D NO CO OO NO I V£>| CO CN 00 | CN CO M CD CD 00 NO in ON CD CD T f O O O | CN O T f 00 | CN Tt 00 o o i3 CO ffl o S i CO o CO Iffl cc! CD H CO ICQ u CD OH co |PQ U cd PH CD in O R--O in O" CN TT O T f COl in O T f O T t NO O CN NO oo in ON O co |PQ U KS o 95 cd I GO ° 3 c 3 ^ I ^ o CD SH !> O oo O on ^! CD CD on CD CD c o o CD 5 * on CD oo > on CD CD OO 5 on CD CD on CD CD T t £ CN on M CD CD on CD CD O £ 00 o on on CN ro ON VO 00 V O on CQ O cd ai CD r--•2 s o •S CN bo o ^» o S o >« 8 K •2 s c CJ ft i vo CD on , i M O CD (-, > o oo U CD CD OO on M CD CD c/n CD CD OX) a o X on on cd U i n r o vd ON o i n I m o Tt p © vo r o I oo | ON r o r o Tt ON © r o I oo ON r o oo I Tt Tt |ro on PQ U on PQ O cd i-i +-> CD H on PQ U cd CD PH cc PQ O cd CD 00 cd on PQ U cd CI o 96 tube had a different mic rob ia l compos i t ion from the other inocula , even though the same bottle o f i n o c u l u m was used for a l l incubations. Th i s highlights a p rob lem that can sometimes arise when an undefined m i x e d culture i n sediment is used as an i n o c u l u m source. The incubations i n this section were a l l separate incubations i n separate tubes (rather than one larger incubat ion for each temperature that was subsampled over t ime). The experiments were planned this w ay partly i n order to have many separate repetitions o f the same incubat ions, thus avo id ing skewing a who le set o f exper imental data because o f differences i n the inocula . The data for these experiments can be d iv ided into two categories: P C B s that are or ig ina l ly present i n A r o c l o r 1260, and P C B s that are dechlor inat ion products o f A r o c l o r 1260. It is then possible to plot the total decrease over t ime o f A r o c l o r 1260 components i n the mixture (Figure 5-1), and the total increase over t ime o f A r o c l o r 1260 dechlor inat ion products (Figure 5-2). It should be noted that this way o f d i v i d i n g the data cou ld s l ight ly underestimate the total dechlor inat ion activi ty, as some components present i n A r o c l o r 1260 can also be dechlor inat ion products o f other components o f A r o c l o r 1260 (e.g. the first dechlor inat ion step o f 2345-2356-oc taCB is expected to fo rm 2356-245-hep taCB, w h i c h is also a component o f A r o c l o r 1260). H o w e v e r , other experiments reported i n this thesis (Chapters 4, 6 and 7) have shown that most dechlor inat ion products found arise f rom complete meta- dechlor inat ion o f congeners (e.g. 2345-2356-oc t aCB is l i ke ly to end up as 24-26- te t raCB, not the hep t aCB) . T h i s accumula t ion o f meta- dechlor inat ion products is conf i rmed i n the above experimental results, w h i c h do not show large increases i n possible intermediate dechlor inat ion products, but instead show the appearance o f large quantities o f a l l the possible products 97 o f complete meta dechlorinat ion. These results mean that almost no overlap between dechlor inat ion products o f A r o c l o r 1260 and congeners or ig ina l ly present i n A r o c l o r 1260 is expected. 20 H 1 r > , 0 4 8 12 16 Time (weeks) Figure 5-1: Disappearance of Aroclor 1260 components over time at different temperatures. (Mean ± SD (n=3) at r= 8 weeks). F r o m F igure 5-1 it can be seen that the biggest decrease i n A r o c l o r 1260 components occurs at 30 ° C , and that this decrease is s ignif icant ly bigger than the decrease observed for incubations at 21 ° C . There is also a significant difference between the amount o f dechlor inat ion observed i n the incubat ion kept at 37 ° C and its control . Af te r 16 weeks, the rate o f disappearance o f A r o c l o r 1260 components i n the 21 ° C and 30 ° C incubations has decreased substantially, but it has not yet stopped 98 completely . I f the curves i n Figure 5-1 are extrapolated, it appears that when degradation stops, a m u c h lower percentage o f A r o c l o r 1260 w i l l remain i n the 3 0 ° C incubat ion than i n the 21 ° C incubat ion. The appearance and increase i n P C B dechlor inat ion products over t ime at the various temperatures tested is shown i n Figure 5-2. Once again, the extent o f dechlor inat ion at 30 ° C is signif icantly higher than the extent o f dechlor inat ion at 21 ° C . There is also a significant difference between the inoculated incubations and the controls that were kept at 21 ° C , 30 ° C and 37 ° C . 80 PCBs 60 -total 40 ~ o 65 20 " "21 C •30 C "37 C "42 C •21 C Control •30 C Control "37 C Control •42 C Control 0 4 8 12 16 T i m e (weeks ) Figure 5-2: Appearance of dechlorination products over time at different temperatures. (Mean ± SD (n=3) at t = 8 weeks). The m a x i m u m rate o f degradation A r o c l o r 1260 congeners was calculated and plotted against the incubat ion temperature (Figure 5-3). Th i s data supports the 99 conc lus ion that the observed degradation is enzymatic , since it in i t i a l ly increases w i t h temperature, and then drops o f f sharply after a certain cr i t ica l temperature. The degradation that was observed i n the 21 ° C and 30 °C controls after 16 weeks, w h e n taken i n combina t ion w i t h the 42 °C control for 16 weeks, shows a s imi la r pattern i n degradation rates. T h i s supports the hypothesis that the smal l degree o f dechlor inat ion observed i n the controls is also enzymatic and so probably comes f rom contaminat ion o f the controls dur ing their preparation inside the anaerobic chamber, w h i c h is not sterile. Cont ro ls had been autoclaved once pr ior to incubat ion, but these results show that addi t ional autoclaving is required for complete steri l izat ion. Q 15 20 25 30 35 40 45 Temperature (degrees Celcius) Figure 5-3: Degradation rates of Aroclor 1260 at different temperatures. 100 One interesting discovery from other w o r k on the effect o f temperature on P C B dechlor inat ion has been that changes i n temperatures can change the pathway o f dechlor inat ion (125). Related w o r k i n our lab (126) has also shown that decreasing the incubat ion temperature to 15 ° C can lead to the detection o f dechlor inat ion products not found at higher temperatures. There was no significant var ia t ion i n the dechlor inat ion products detected at the temperatures studied i n the present work . 101 Chapter 6 Reductive Dechlorination of Nonachlorobiphenyls and Selected Octachlorobiphenyls 6.1 Introduction It has been well established that microbial dechlorination of Aroclor 1260 can take place both in the environment and under laboratory condition (69, 127-128). Some studies have also quantified the extent of dechlorination of octa- and nona-chlorobiphenyls in Aroclor 1260 mixtures (65, 67, 130). However, because of the complexity of both the starting Aroclor 1260 mixture and the product mixture formed, it is impossible to identify specific products of, and degradation pathways for, specific congeners in the general mixture. Several studies have investigated the dechlorination of single PCB congeners under anaerobic conditions (131-135), but these have all focused on PCBs with six or fewer chlorine substituents. The dehalogenation of decachlorobiphenyl over time has also been reported (136); however, the products were only tentatively identified, and not quantified. There are no published reports on the microbial degradation of single PCB congeners with 8 or 9 chlorines showing the determination of dechlorination products. In this work the anaerobic dechlorination of all possible nonachlorobiphenyls [23456-2345-nonaCB (IUPAC# 206), 23456-2346-nonaCB (IUPAC# 207) and 23456-2356-nonaCB (IUPAC# 208)) and four of the most common octachlorobiphenyls found in Aroclor 1260 [(23456-234-octaCB (IUPAC# 195), 2345-2346-octaCB (IUPAC# 196), 2345-2356-octaCB (IUPAC# 199) and 23456-245-octaCB (IUPAC# 203)] was investigated. 102 6.2 Experimental Sediment slurries were prepared in 25 mL Balch tubes by mixing 1.25 g of aerobic sand and 0.75 g of anaerobic pond sediment with 4 mL R A M M . Tubes were crimp-sealed inside an anaerobic chamber, and were autoclaved after incubation for 14 days, during which time methanogenesis was detected. Single PCB congeners (23456-2345-nonaCB, 23456-2346-nonaCB, 23456-2356-nonaCB, 23456-234-octaCB, 2345-2346-octaCB, 2345-2356-octaCB or 23456-245-octaCB) (100 pg) were added to the tubes from concentrated stock solutions in acetone, and the tubes were inoculated (0.5 mL) with an enrichment culture. Control cultures that contained an extra 0.5 mL medium instead of inoculum were also prepared, and all incubations were carried out stationary at 21 °C in the dark. Entire cultures (1 control culture and 1 inoculated culture) were frozen at 0, 4, 8, 12 and 16 weeks. After thawing, the cultures were extracted and analyzed as described previously (Chapter 2, Sections 2-5 to 2-8). The detection limit for biphenyl was 8 pg/L, and the quantitation limit for biphenyl was 25 pg/L. 6.3 Results 23456-2345-nonaCB and all four octachlorobiphenyl congeners tested yielded dechlorination products after only 4 weeks of incubation. 23456-2346-nonaCB and 23456-23 56-nonaCB showed evidence of dechlorination after 12 weeks of incubation. In each case, the major product that accumulated over time resulted from removal of all meta chlorines. The extent of dechlorination after sixteen weeks varied widely from congener to congener, although the results obtained for the two different cultures used 103 were reasonably consistent for each congener (Table 6-1). M a s s balances were calculated for the total P C B s detected i n each sample and it was found that the total recovery o f P C B s ranged from 9 2 % to 9 7 % after 16 weeks. Since an entire culture was used for each sample, there were four replicate cultures for each treatment. The results indicate consistent dechlor inat ion rates and specificit ies i n each set o f replicate cultures. N o degradation products were detected i n any o f the control cultures after 16 weeks o f incubat ion. 23456-234-octaCB: Dechlor ina t ion products o f 23456-234-oc taCB were detected after on ly four weeks o f incubat ion w i t h both B K 2 4 and B K 7 2 . The products found after 16 weeks and the dechlor inat ion pathway suggested by their format ion are shown i n Figure 6-1. The numbers i n parentheses show the proportions o f the different products (as m o i % o f total P C B s present) after incubat ion w i t h enrichment culture B K 2 4 . Resul ts obtained for incubat ion w i t h B K 7 2 suggested an ident ical dechlor inat ion pathway, w i t h the except ion that no 246-23-pentachlorobiphenyl was detected. The relative proport ions o f products were also s l ight ly different. Trace amounts (8-25 p g / L ) o f b iphenyl were detected after 12 weeks i n B K 2 4 cultures and after 16 weeks i n B K 7 2 cultures, but were not detected i n control cultures. 104 43 NO a o o NO » o H O 0 S O T J O C3 -*-» 00 00 0 s -60 C5 • i-H C '3 a Pi in 00 JO 00 CQ u • NO CN I T j -CQ U I i TT CN CQ U • N O CN i NO TJ-CN CQ U l CN i NO Tt" CN CN r-CQ TJ-CN CQ CN CQ TT CN CQ CN CQ TJ-CN CQ CN CQ TT CN CQ CN CQ T f CN CQ-NO Tt-© NO 00 ON T f ON T f CO CN i NO i n Tt-co CN CN TJ-' ON © i n © i n oo CN T t CO NO NO CN ON NO CN 00 NO T t CO CN i i n Tt-co CN i n ON NO NO ON NO i n CO CN i i n T f CO CN © Tj " © i n © 00 CN CO NO NO i n TJ-CN I NO i n T i -co CN 00 © CN ON © o NO o6 O N 00 ON i n TJ-co CN i NO i n TJ-co CN CN N O O N 00 ON NO T j -CO CN i NO m TJ" co CN ON © NO ON i n ON NO i n CO CN i NO i n TJ-CO CN 105 CI CI CI CI °-0--Q--c i c i 23456-234-CB (95%) I CI CI CI CI a 2346-234-CB (0.04%) Biphenyl (trace) Figure 6-1: Dechlorination of23456-234-octaCB after 16 weeks. Relative amounts of congeners in a BK24 culture are given in parentheses. 106 2345-2346-octaCB: The amounts o f the most abundant metabolites detected for 2345-2346-oc taCB over the t ime per iod investigated are shown i n Figure 6-2. Results are g iven for incubat ion w i t h enrichment culture B K 2 4 , but the relative amounts o f products detected i n culture B K 7 2 were very s imi lar . A proposed pathway for the formation o f a l l detected products is shown i n Figure 6-3. Trace amounts o f b iphenyl were detected after 12 weeks o f incubat ion. Time (weeks) Figure 6-2: Dechlorination products of congener 2345-2346-octaCB in a BK24 culture over time. 107 CI. CI CI CI CI CI C l -C l 245-24-CB (trace) C l -Cl CI' 2345-2346-CB (82%) CI CI 2346-245-CB (trace) C! CI 245-246-CB (0.54%) CI CI. CI CI, -CI CI 246-24-CB (17%) CI CI 246-25-CB (trace) CI CI. C l -24-24-CB (0.54%) CI 24-26-CB (0.17%) A. Biphenyl (trace) Figure 6-3: Dechlorination of2345-2346-octaCB after 16 weeks. Relative amounts of congeners in a BK24 culture are given in parentheses. 108 2345-2356-octaCB: N o heptachlorobiphenyl products were detected i n cultures conta ining 2345-2356-oc taCB. Trace amounts o f 2356-24-hexachlorobiphenyl were detected, and no pentachlorobiphenyl compounds were detected. The major dechlor inat ion product found after sixteen weeks was 24-26-tetrachlorobiphenyl (3 .39% and 4 .24% o f total P C B s for cultures B K 2 4 and B K 7 2 respectively). Trace amounts o f b iphenyl were detected after 12 weeks o f incubat ion. 23456-245-octaCB: The t ime sequence o f product format ion f rom 23456-245-o c t a C B over the 16-week sampl ing per iod is shown i n Figure 6-4. A proposed pathway for dechlor inat ion o f 23456-245-oc taCB, w i t h the proportions o f the various products at 16 weeks, is shown i n Figure 6-5. Trace amounts o f b iphenyl were detected after 12 weeks o f incubat ion. Figure 6-4: Dechlorination products of23456-245-octaCB in a BK24 culture over time. 109 CI, CI CI ci ci ci 23456-245 (62%) CI CI 23456-24 (trace) CI Ci 2346-245 (0.18%) CI CI CI CI 245-246 (0.35%) ci ci. ci ci 245-26 (trace) CI CI CI CI CI CI CI CI CI 246-24 (37%) 245-24 (trace) ci a CI CI ci CL ci ci 25-26 (0.02%) CI 24-26 (0.19%) 24-24 (0.29%) 24-25 (0.1 1%) Biphenyl (trace) Figure 6-5: Dechlorination of congener 23456-245-octaCB after 16 weeks. Relative amounts of products in a BK24 culture are given in parentheses. 110 23456-2345-nonaCB: N o octa-, hepta- or hexachlorobiphenyls were detected i n the cultures containing 23456-2345-nonaCB. The major metabolite detected was 246-24-pentachlorobiphenyl (1 .01% and 0 .81% for cultures 24 and 72 respectively after 16 weeks). B i p h e n y l was also detected at trace levels after 16 weeks o f incubat ion. 23456-2346-nonaCB: N o octachlorobiphenyls or hepta-chlorobiphenyls were detected i n the cultures w i t h 23456-2346-nonaCB. 246-246-hexa-chlorobiphenyl was detected after 4 weeks, and at 16 weeks this comprised 0 .17% and 0 .15% o f the total m o l % for enrichment cultures B K 2 4 and B K 7 2 respectively. 246-24 pentachlorobiphenyl (0 .01% and 0.02%), 24-26-tetrachlorobiphenyl (1.16%, 3.15%), 24-24-tetrachlorobiphenyl (0.22, 0.39%) and b iphenyl (trace, trace) were also found. 23456-2356-nonaCB: Cultures containing 23456-2356-nonaCB also d i d not fo rm detectable amounts o f octa- or heptachlorobiphenyls. 246-236-hexachlorobiphenyl was found i n trace amounts, and 246-26-pentachlorobiphenyl (0 .95%, 0.70%) was the on ly pentachlorobiphenyl detected. 24-24-tetrachlorobiphenyl (1 .71%, 1.69%) and 24-26-tetrachlorobiphenyl (1.39%, 0.88%) were also found. B i p h e n y l was detected after 12 weeks o f incubat ion. 6.4 Discussion T h i s study clear ly shows, for the first t ime, the reductive dechlor inat ion by anaerobic microorganisms o f single octa- and nonachlorobiphenyls , and the specific dechlor inat ion products formed. W h e n a l l o f the proposed pathways for dechlor inat ion are examined , dechlor inat ion o f doubly f lanked meta chlorines is the predominant in i t i a l step o f dechlor inat ion, fo l lowed by dechlor inat ion o f s ingly f lanked meta chlorines. Th i s i l l is consistent w i t h Process N dechlor inat ion as described by Bedard and Quensen (70), w h i c h has a dechlor inat ion specifici ty for f lanked and doubly f lanked meta chlor ines . It should also be noted that w i t h i n the different molecules containing doubly f lanked chlorines, a definite sequence o f dechlor inat ion is observed, depending on the pattern o f chlor ine substitution. F o r example, i f one o f the rings contains f ive chlorines, the first chlor ine r emoved is always a meta chlor ine on this r ing. Figure 6-6 shows the order o f removal o f the three different doubly f lanked meta chlorines i n congener 195. The present results conf i rm suggestions that substituents on one r ing affect dechlor inat ion on the other r ing (128). Fo r example, when the results obtained for congeners 196 and 199 are compared, it can be seen that the first chlor ine removed i n both these congeners is f rom the same pos i t ion o f ident ical ly substituted rings, yet the rates o f r emova l (as evidenced by the total removal o f the congeners over t ime) are very different. T h i s difference i n dechlor inat ion rates is attributed to the different chlor ine configurat ion on the r ing where dechlor inat ion is not taking place. 3rd CI CI CI CI 2nd 1st C I C I 23456-234-oc tach lo rob ipheny l (Congener 195) Figure 6-6: Proposed order of removal of the first three chlorines from 23456-234-octaCB. 112 Ortho and para dechlor inat ion was also observed, but usual ly on ly after a l l meta chlorines were removed. F o r most cultures and congeners, there was more ortho dechlor inat ion than para dechlor inat ion observed. In a few instances, however , para dechlor inat ion was more prevalent than ortho dechlorinat ion. F o r example , for congeners 203 and 195, the tetrachlorobiphenyl products formed differ, but both are expected to come f rom the c o m m o n 246-24-pentaCB precursor. It is possible that these differences i n act ivi ty are due to differences i n the anaerobic mic rob i a l populat ions present. The per iod o f incubat ion required before ortho and para dechlor inat ion occurred also var ied between cultures. In some examples ortho and para dechlorinat ing act ivi ty was observed after on ly eight weeks, w h i l e i n the other cultures us ing the same single congener, ortho and para dechlor inat ing act ivi ty appeared on ly after 12 or 16 weeks. T h i s once again suggests dechlor inat ion activities o f different mic rob ia l populations present i n the in i t i a l inocula . E v e n though some products accumulated i n substantial quantities, there were also smal l amounts o f many others present. The presence o f i someric products o f one congener indicate that even though dominant pathways are clear ly observed, the dechlor inat ion pathways are branched (i.e., the order o f dechlor inat ion sequence is varied). In the present w o r k no trace o f any t r i - , d i - or monochlorobiphenyls was found. S imi l a r results were reported for the dechlor inat ion o f 234-234 and 2 3 6 - 2 3 6 - h e x a C B (133). H o w e v e r , several other studies that started w i t h less h igh ly chlor inated P C B s that were substituted w i t h chlorines on on ly one o f the two b iphenyl rings have indicated the format ion o f t r i - , d i - and monochloro-b iphenyl products (131, 132). In the present study, 113 traces of biphenyl were found in most cultures after 12 weeks, and in all cultures after 16 weeks. This suggests that even though intermediates between tetrachlorobiphenyls and biphenyl were not detected, they were probably formed in quantities below the detection limit, and were further dechlorinated to biphenyl. This is the first evidence for complete anaerobic dechlorination of nona- and octaCBs. It is interesting to note that in the incubations with the nonachlorobiphenyls, octa-and hepta-chlorobiphenyl products were not detected. Also, the major penta- and hexa-CB products that would be expected to accumulate, by analogy with results obtained for octaCB dechlorination, (246-24-pentaCB for congener 206, 246-246-hexaCB for congener 207 and 246-26-pentaCB for congener 208), did not always accumulate. Rather, a much higher proportion of tetrachlorobiphenyl products was formed. It is possible that the initial dechlorination step of nonaCBs (e.g. 23456-2356-nonaCB) is a slower process than the subsequent dechlorination of the octa-, hepta-, hexa- and pentaCB products, explaining the accumulation of tetraCB products rather than the penta-or hexaCB products. In contrast, for three of the octachlorobiphenyls studied, the accumulation of 246-24-pentaCB occurred, so it is possible that in this case the ortho or para dechlorination of this pentachlorobiphenyl is slower than the initial meta dechlorination of the octachlorobiphenyl. With the fourth octachlorobiphenyl studied, 2345-2356-octaCB, 246-24-pentaCB did not accumulate as it was not a possible dechlorination product. In this case the product of meta dechlorination was 24-26-tetraCB. It should be noted that PCB concentrations used in these experiments greatly exceeded concentrations of octa- and nonaCBs found in most contaminated environments. 114 The results o f other experiments investigating the dechlor inat ion o f A r o c l o r 1260 w i t h the same enrichment cultures exhibi t the same dechlor inat ion patterns (e.g. predominant ly Process N ) as those found w i t h the single congener experiments reported here (Chapters 4 and 5). Th i s suggests that A r o c l o r mixtures f o l l o w s imi la r dechlor inat ion pathways to the single congeners, and confirms the relevance o f s tudying single congeners as a means o f e lucidat ing dechlor inat ion pathways i n more complex mixtures. These results give a greater understanding o f what may be happening to ind iv idua l octa- and n o n a - C B congeners i n anaerobic b io log ica l systems conta ining mixtures o f different congeners (e.g. as i n A r o c l o r 1260). The products detected suggest dechlor inat ion pathways specific to particular congeners. F o r a l l congeners examined dechlor inat ion was possible, and for some congeners a large fraction o f the in i t ia l compound was transformed. 115 Chapter 7 Dechlorination of Selected PCB Congeners: Singly and in Combination 7.1 Introduction The results presented i n Chapter 6 established that the m i c r o b i a l enrichment cultures are capable o f dechlorinat ing single octa- and nonachlorobiphenyl congeners. In the present chapter, the dechlor inat ion o f various combinat ions o f these congeners was investigated, i n order to see i f the extent or pathways o f dechlor inat ion differ when congeners, are incubated i n combina t ion w i t h others rather than s ingly. The dechlor inat ion o f one pentachlorobiphenyl congener and two tetrachlorobiphenyl congeners, both s ingly and i n combina t ion w i t h other congeners, was also investigated. Th i s was done because the incubations o f single congeners described i n the previous chapter had shown accumulat ion o f one major product (either a tetra- or pentachlorobiphenyl , depending on the single congener under investigation) and a trace amount o f b iphenyl , but no t r i - , d i - or monoch lorob ipheny l products. Other experiments (reported i n Chapter 5) show that dechlor inat ion rates increase s ignif icant ly w h e n cultures are incubated at 30 ° C rather than at 21 ° C , so these experiments were a l l carried out at 30 °C rather than the 21 ° C used i n the previous single congener incubations (Chapter 6). The dechlor inat ion o f decachlorobiphenyl at 30 ° C was also investigated, since previous incubations at 21 ° C (results not shown) had not shown the format ion o f any dechlor inat ion products. 7.2 Methods Sediment slurries were prepared i n 2 5 - m L B a l c h tubes by m i x i n g 0.65 g o f Reso lu t ion Island sand and 0.35 g o f anaerobic pond sediment w i t h 2 m L R A M M . Tubes were sealed w i t h Tef lon- l ined butyl rubber stoppers, and cr imp-sealed inside an anaerobic chamber. The tubes were autoclaved after incubat ion for 14 days, dur ing w h i c h t ime methanogenesis was detected. S ingle P C B congeners were added to the tubes i n var ious combinat ions (Table 7-1) f rom concentrated stock solutions i n acetone. Af ter addi t ion o f the P C B congeners, the tubes were inoculated (0.1 m L ) w i t h an enrichment culture ( B K 8 1 ) . Con t ro l cultures that contained an extra 0.1 m L m e d i u m instead o f i n o c u l u m were also prepared, and a l l incubations were carried out stationary at 30 ° C i n the dark. Unfortunately, examinat ion o f one set o f experiments revealed that there was no dechlor inat ion after 8 weeks o f incubat ion, so it was decided that the i n o c u l u m used had not been v iable . The two remaining inoculated incubations for each experiment were re-inoculated at this t ime per iod w i t h an addi t ional 0.2 m L o f a fresh enrichment culture ( B K 8 1 ) , and an addi t ional 0.2 m L R A M M was added to the one remain ing control . These re-inoculated cultures were sampled after an addi t ional 4 and 8 weeks o f incubat ion, and the control was sampled after 8 weeks. In the results section, t ime = 4 weeks and t ime = 8 weeks refer to the incubat ion times after re- inoculat ion. T i m e = 0 weeks refers to the congener dis t r ibut ion at the end o f the in i t i a l experiment i n w h i c h no dechlor inat ion was observed. 117 Table 7-1: Amounts and combinations of congeners added to enrichment cultures in different experiments. Exper iment Congener A m o u n t added (u,g) N u m b e r 1 23456-234-oc taCB 7.5 2 2345-2346-oc taCB 9.1 3 2345-2356-oc taCB 12.0 4 23456-245-oc taCB 5.9 5 23456-2345-nonaCB 9.1 6 23456-2346-nonaCB 14.0 7 23456-23 5 6 - n o n a C B 3.2 8 23456-23456-decaCB 20.0 9 23456-234-oc taCB 7.5 2345-2346-oc taCB 9.1 2345-2356-oc taCB 12.0 23456-245-oc taCB 5.9 10 23456-2345-nonaCB 9.1 23456-2346-nonaCB 14.0 23456-2356-nonaCB 3.2 11 23456-234-oc taCB 7.5 2345-2346-oc taCB 9.1 2345-2356-oc taCB 12.0 23456-245-oc taCB 5.9 23456-2345-nonaCB 9.1 23456-2346-nonaCB 14.0 23456-2356-nonaCB 3.2 12 246-24-pentaCB 112 13 2345-2346-oc taCB 9.1 246-24-pentaCB 6.8 14 2345-2346-oc taCB 9.1 23456-245-oc taCB 5.9 15 23456-234-oc taCB 7.5 2345-2356-oc taCB 12.0 16 246-2-te t raCB 100 17 24-24-te t raCB 100 18 246-24-pentaCB 37 246-2- te t raCB 33 24-24-te t raCB 33 19 246-24-pentaCB 112 246-2- te t raCB 100 24-24-tetraCB 100 7.3 Results and Discussion 7.3.1 Cultures with single octachlorobiphenyl congeners and various combinations of octachlorobiphenyl congeners A l l cultures w i t h single octachlorobiphenyl congeners (Exper iments 1 -4, Table 7-1) showed extensive dechlor inat ion after only 4 weeks o f incubat ion, and the extent o f this dechlor inat ion had increased after 8 weeks o f incubat ion. In a l l cases, the accumula t ion o f a single major dechlor inat ion product was observed. N o n e o f the controls showed any evidence o f dechlorinat ion. Da ta are shown i n Table 7-2, w h i c h shows the extent o f dechlor inat ion and a l l dechlor inat ion products detected for the single octachlorobiphenyl cultures. Table 7-2: Moi percent distribution over time of single octachlorobiphenyl congener substrates and their dechlorination products. Exper iment N u m b e r Congener Identity M o i % dis t r ibut ion o f congeners 0 weeks 4 weeks 8 weeks 0 weeks C o n t r o l 8 weeks C o n t r o l 1 2 3 4 5 6 - 2 3 4 - C B 100 74.9 66.0 100 100 2 4 6 - 2 4 - C B 0 25.1 34.0 0 0 2 2 3 4 5 - 2 3 4 6 - C B 100 59.9 49.6 100 100 2 4 5 - 2 4 6 - C B 0 1.9 6.2 0 0 2 4 6 - 2 4 - C B 0 38.2 44.2 0 0 3 2 3 4 5 - 2 3 5 6 - C B 100 77.8 61.9 100 100 2 3 5 6 - 2 4 5 - C B 0 0 4.6 0 0 2 4 - 2 6 - C B 0 22.2 33.5 0 0 4 2 3 4 5 6 - 2 4 5 - C B 100 73.3 60.4 100 100 2 3 4 6 - 2 4 5 - C B 0 0 3.2 0 0 2 4 5 - 2 4 6 - C B 0 0 2.0 0 0 2 4 6 - 2 4 - C B 0 26.7 34.4 0 0 119 The percent dechlor inat ion o f the single octachlorobiphenyl congeners ranges f rom 34.0 % for 23456-234-oc taCB to 50.4 % for 2345-2346-oc taCB. S l igh t ly different in i t i a l concentrations o f congeners were used. The total amounts o f o c t a C B substrates dechlorinated over the 8 week incubat ion per iod are g iven i n Table 7-3. A l l incubations w h i c h inc luded a combina t ion o f two different o c t a C B congeners (Exper iments 14 and 15, Table 7-1) or a combina t ion o f a l l the above four o c t a C B congeners (Exper iment 9, Table 7-1) also showed extensive dechlor inat ion after on ly 4 weeks, and for most o f the congeners the degree o f dechlor inat ion after 8 weeks was even greater. Resul ts are g iven i n Table 7-4. M o i percent values for two o f the o c t a C B s used are reported as a combined number, as these congeners co-elute. Table 7-3: Amount (/ug) of octachlorobiphenyl substrate dechlorinated after 8 weeks of incubation. Exper iment Congener Identity Ini t ial A m o u n t N u m b e r Concentra t ion (u.g) Dechlor ina ted (ug) 1 2 3 4 5 6 - 2 3 4 - C B 7.5 5.0 2 2 3 4 5 - 2 3 4 6 - C B 9.1 4.5 3 2 3 4 5 - 2 3 5 6 - C B 12.0 7.4 4 2 3 4 5 6 - 2 4 5 - C B 5.9 5.9 120 Table 7-4: Moi percent distribution over time of octachlorobiphenyl substrate mixtures and their products. Exper iment N u m b e r Congener Identity M o i % dis t r ibut ion o f congeners 0 weeks 4 weeks 8 weeks 0 weeks C o n t r o l 8 weeks C o n t r o l 14 2 3 4 5 - 2 3 4 6 - C B + 2 3 4 5 6 - 2 4 5 - C B 100 78.9 68.5 100 100 2 3 4 6 - 2 4 5 - C B 0 0 0.1 0 0 2 3 5 6 - 2 4 5 - C B 0 0 0.4 0 0 2 3 4 6 - 2 4 - C B 0 0.4 0.9 0 0 2 4 5 - 2 4 6 - C B 0 1.8 3.9 0 0 2 4 6 - 2 4 - C B 0 18.8 22.9 0 0 B i p h e n y l 0 0 3.3 0 0 15 2 3 4 5 6 - 2 3 4 - C B 38.9 24.9 22.4 39.0 38.3 2 3 4 5 - 2 3 5 6 - C B 61.1 51.4 42.4 61.0 61.7 2 3 4 5 - 2 3 6 - C B 0 1.8 2.0 0 0 2 3 5 6 - 2 4 5 - C B 0 4.0 4.2 0 0 2 3 4 - 2 3 6 - C B 0 0.7 0.8 0 0 2 3 6 - 2 4 5 - C B 0 1.2 1.2 0 0 2 3 5 6 - 2 4 - C B 0 0.7 0.7 0 0 2 4 6 - 2 4 - C B 0 11.7 15.7 0 0 2 4 - 2 6 - C B 0 3.6 1.8 0 0 2 4 - 4 - C B 0 0 0.3 0 0 2 6 - 2 - C B 0 0 0.4 0 0 4 - 4 - C B 0 0 0.9 0 0 B i p h e n y l 0 0 4.7 0 0 9 2 3 4 5 6 - 2 3 4 - C B 21.1 15.2 9.0 21.0 21.3 2 3 4 5 - 2 3 4 6 - C B + 2 3 4 5 6 - 2 4 5 - C B 43.7 32.1 33.9 43.3 43.5 2 3 4 5 - 2 3 5 6 - C B 35.2 28.2 24.4 35.6 35.2 2 3 4 5 - 2 3 6 - C B 0 0 0.8 0 0 2 3 5 6 - 2 4 5 - C B 0 0 2.2 0 0 2 3 6 - 2 4 5 - C B 0 0 0.5 0 0 2 3 5 6 - 2 4 - C B 0 0 0.8 0 0 2 4 5 - 2 4 6 - C B 0 0 1.2 0 0 2 4 6 - 2 4 - C B 0 14.7 18.1 0 0 2 4 - 2 6 - C B 0 9.8 9.1 0 0 121 The disappearances over t ime o f 23456-234- and 2345-2356-oc taCB when incubated s ingly, and the corresponding disappearances over t ime o f the same congeners when incubated i n combina t ion w i t h each other, and i n combina t ion w i t h a l l the o c t a C B congeners tested, are shown i n Figure 7-1. Figure 7-2 shows the t ime course o f results obtained w i t h 2345-2346- and 23456-245-oc taCB incubated s ingly and i n combina t ion . 50 4 40 -I , , 0 4 8 Time (weeks) — • — 23456-234-octaCB (Incubated singly) - + -23456-234-octaCB (Incubated with 2345-2356-octaCB) ------ - 23456-234-octaCB (All four octaCBs incubated together) X— 2345-2356-octaCB (Incubated singly) X - - 2345-2356-octaCB (Incubated with 23456-234-octaCB) - - - X- - • 2345-2356-octaCB (All four octaCBs incubated together) Figure 7-1: Disappearances of23456-234-octaCB and 2345-2356-octaCB when incubated singly and in combination with other congeners. 122 0 4 8 Time (weeks) —•—2345-2346-octaCB (Incubated singly) —*—23456-245-octaCB (Incubated singly) —e—2345-2346-octaCB + 23456-245-octaCB (Average of results for single incubations) _ _o_ - 2345-2346-octaCB + 23456-245-octaCB (Incubated together) - - -o- - - 2345-2346-octaCB + 23456-245-octaCB (All four octaCBs incubated together) Figure 7-2: Disappearances of2345-2346-octaCB and 23456-245-octaCB when incubated singly and in combination with other congeners. The percent disappearance o f the o c t a C B congeners over t ime when they are a l l incubated together is shown i n Figure 7-3. D) C 'E "<5 E CD or 100 90 80 70 60 50 40 - - - o Time (weeks) 23456-234-octaCB .o--- 2345-2346-octaCB + 23456-245-octaCB 2345-2356-octaCB Figure 7-3: Disappearance over time of all four octaCB congeners together in the same incubation. 124 7.3.2 Discussion: Octachlorobiphenyl congeners The percent dechlorinations reported for the incubations w i t h single o c t a C B congeners at 30 ° C (Table 7-2) are m u c h higher than the ones reported i n Chapter 6 (Table 6-1) for the same congeners after 16 weeks o f incubat ion at 21 ° C . Th i s is not surprising, however , as the incubat ion temperature is 9 ° C higher i n the present experiments, and the in i t ia l concentrations o f congeners used i n the experiments reported i n Chapter 6 are m u c h higher (50 ppm) than the ones used here ( 6 - 1 2 ppm) . The absolute amounts o f congener dechlorinated i n these 30 ° C incubations (Table 7-3) are very different f rom what w o u l d have been expected by direct compar i son w i t h the previous experiments incubated at 21 ° C (Table 6-1, Chapter 6). In the experiments performed at 21 ° C , there was a very wide range i n the extent o f dechlor inat ion for the different congeners, w i t h 23456-245-oc taCB and 2345-2346-o c t a C B showing m u c h more extensive dechlor inat ion than 23456-234-oc taCB and 2345-2 3 5 6 - o c t a C B . In the 30 ° C incubations reported here (Table 7-3), the amount o f dechlor inat ion detected for the various congeners does not vary nearly as much . The total amount ( in ug) o f 23456-234-oc taCB dechlorinated over 8 weeks at 30 ° C is very s imi la r to the total amount dechlorinated over the same t ime per iod at 21 ° C , but the total amount o f 2345-2346-oc taCB dechlorinated at 30 ° C is more than double the amount dechlorinated at 21 ° C , and the total amounts o f both 2345-2346-oc taCB and 23456-245-o c t a C B dechlorinated are m u c h lower at 30 ° C than at 21 ° C . T h i s var ia t ion i n dechlor inat ion act ivi ty between different congeners at different temperatures suggests that the enzymes i n v o l v e d i n dechlor inat ion are less selective at 30 ° C than at 21 ° C . 125 W h e n the effects o f incubat ing congeners together rather than s ingly are investigated (Figure 7-1), it can be seen that there is only a smal l difference between the extent o f dechlor inat ion o f 23456-234-oc taCB and 2345-2356-oc taCB w h e n they are incubated separately. W h e n they are incubated together w i t h each other, however , the extent o f dechlor inat ion o f 23456-234-oc taCB increases, w h i l e the extent o f dechlor inat ion o f 2345-2356-oc taCB decreases. T h i s same difference is observed when the two congeners are incubated i n a mixture containing four different o c t a C B s (Figure 7-1). T h i s suggests that, i n a mixture o f congeners, 23456-234-oc taCB is preferentially dechlorinated, and the dechlor inat ion o f 2345-2356-oc taCB is inhibi ted. 23456-234-o c t a C B contains 3 doubly-f lanked meta chlorines, wh i l e 2345-2356 on ly contains one doubly-f lanked meta chlor ine (the other two meta chlorines are on ly s ingly flanked) (Figure 7-4). It is possible that doubly-f lanked meta dechlor inat ion occurs more readily than s ingly-f lanked meta dechlor inat ion w i t h the enrichment cultures used i n the present w o r k (Chapter 6, Sect ion 6-4), and this may expla in the observed difference i n dechlor inat ion selectivity. C I CI C I C I C I C I 2 3 4 5 6 - 2 3 4 - o c t a C B 2 3 4 5 - 2 3 5 6 - o c t a C B Figure 7-4: 23456-234-octaCB and 2345-2356-octaCB. 126 T h i s suggested explanat ion for the observed differences i n dechlor inat ion act ivi ty is tested again i n the experiments where a l l four o c t a C B congeners are incubated i n combina t ion (Figure 7-3). In this example, 2345-2346-oc taCB and 23456-245-oc taCB both contain two doubly f lanked meta chlorines (Figure 7-5), so us ing the reasoning suggested above, the 2345-2346-oc taCB/23456-245-oc taCB combina t ion w o u l d be expected to have a dechlor inat ion rate in-between those o f 2345-2346-oc taCB and 23456-2 3 4 - o c t a C B , when i n combina t ion w i t h these congeners. Th i s expectation holds at the 4-week t ime interval , but at the 8-week t ime interval , the 2345-2346/23456-245-oc taCB combina t ion does not fit the pattern. Since the results for the 8-week t ime interval do not fit the trend o f increasing dechlor inat ion over t ime (i.e. it w o u l d appear that less, rather than more, dechlor inat ion occurred after 8 weeks as compared to 4 weeks) , it cannot be determined at this stage i f the observed differences are significant. Further experiments w i l l be necessary i n order to conf i rm these results. C I C I CI C I C I 2345 -2346 -oc t aCB 2 3 4 5 6 - 2 4 5 - o c t a C B Figure 7-5: 2345-2346-octaCB and 23456-245-octaCB. Ill F r o m the results o f the two co-elut ing congeners (Figure 7-2), it appears that less dechlor inat ion takes place for the two congeners i n combina t ion than w h e n they are separate. It should be noted that the overa l l dechlor inat ion has not decreased, but the dechlor inat ion o f each ind iv idua l congener i n the mixture has decreased. T h i s decrease i n dechlor inat ion o f the ind iv idua l congeners cou ld be due to the increased concentration o f P C B s i n the combined mixture , an effect that is discussed i n more detail for other congeners i n section 7.3.9 o f this chapter. It is also interesting to compare the differences i n the dechlor inat ion products f rom the different experiments (Tables 7-2 and 7-4). A l l the incubations i n v o l v i n g single congeners showed the formation o f one major dechlor inat ion product (the product formed from the remova l o f a l l the meta chlorines) , and no product ion o f b iphenyl . W h e n congeners were incubated together, the format ion o f the major meta dechlor inat ion product s t i l l occurred, but a few extra dechlor inat ion products, w h i c h were not detected when congeners were incubated separately, were also found. In experiment 9 (Table 7-4), where a l l four o c t a C B congeners were incubated together, 2345-236-hep taCB and 2 3 5 6 - 2 4 - h e x a C B were found, and they were not detected i n any o f the single congener incubations. In addi t ion, the amount o f 24-26- te t raCB, the meta dechlor inat ion product o f 2345-2356-oc t aCB, was not found i n sufficient quantities to account for the total reduct ion o f 2345-2356-oc taCB i n the mixture . Th i s suggests that the combina t ion o f congeners is inh ib i t ing complete or rapid meta dechlor inat ion o f this congener. Ano the r difference found was i n experiment 14 (Table 7-4), the incubat ion that contained both 2345-2346-oc taCB and 23456-245-oc taCB. W h e n these congeners were incubated 128 together, b iphenyl was detected after 8 weeks o f incubat ion. W h e n they were incubated separately, no b iphenyl was detected. 7.3.3 Incubations with single nonachlorobiphenyl congeners and various combinations of nonachlorobiphenyl congeners Cultures w i t h 23456-2345-nonaCB and 23456-2356-nonaCB showed evidence o f dechlor inat ion after only 4 weeks o f incubat ion, and the extent o f this dechlor inat ion had increased after 8 weeks. 23456-2346-nonaCB d id not show evidence o f any dechlor inat ion after 4 weeks o f incubat ion, but showed a very smal l amount o f dechlor ina t ion at 8 weeks. The results obtained for cultures w i t h n o n a C B s incubated s ingly are presented i n Table 7-5. N o n e o f the controls showed any evidence o f dechlor inat ion. Table 7-5: Moi percent distribution of single nonachlorobiphenyl congener substrates and their dechlorination products. Exper iment N u m b e r Congener Identity M o i % distr ibut ion o f congeners 0 weeks 4 weeks 8 weeks 0 weeks C o n t r o l 8 weeks C o n t r o l 5 2 3 4 5 6 - 2 3 4 5 - C B 100 92.4 83.4 100 100 2 3 4 5 - 2 3 4 6 - C B 0 1.5 1.5 0 0 2 4 6 - 2 4 - C B 0 6.1 15.1 0 0 6 2 3 4 5 6 - 2 3 4 6 - C B 100 100 99.9 100 100 2 4 6 - 2 4 6 - C B 0 0 0.1 0 0 7 2 3 4 5 6 - 2 3 5 6 - C B 100 94.5 90.9 100 100 2 4 6 - 2 6 - C B 0 5.5 9.1 0 0 129 Cultures w h i c h contained a l l three n o n a C B congeners also showed dechlor inat ion o f 23456-2345-nonaCB and 23456-2356-nonaCB after 4 weeks o f incubat ion, and dechlor inat ion o f a l l three n o n a C B congeners after 8 weeks. The results obtained for the incubations that inc luded a l l three n o n a C B s are presented i n Table 7-6. Table 7-6: Moi percent distribution of nonachlorobiphenyl congener substrate mixtures and their dechlorination products. M o i % distr ibut ion o f congeners Exper iment Congener 0 weeks 4 weeks 8 weeks 0 weeks 8 weeks N u m b e r Identity C o n t r o l C o n t r o l 10 2 3 4 5 6 - 2 3 4 5 - C B 34.9 26.6 25.9 34.9 34.7 2 3 4 5 6 - 2 3 4 6 - C B 53.5 53.4 53.1 53.9 54.1 2 3 4 5 6 - 2 3 5 6 - C B 11.7 9.8 9.4 11.3 11.2 2 4 6 - 2 4 6 - C B 0 0 0.2 0 0 2 4 6 - 2 6 - C B 0 2.1 2.4 0 0 2 4 6 - 2 4 - C B 0 8.1 9.0 0 0 The percent disappearances over t ime o f the three n o n a C B congeners w h e n incubated separately and when incubated together are shown i n Figure 7-6. 7.3.4 Discussion: Nonachlorobiphenyl congeners The results obtained for single congener incubations o f n o n a C B s i n the present experiments at 30 ° C (Table 7-5) differ f rom those obtained at 21 ° C (Chapter 6, Table 6-1), i n that 23456-2346-nonaCB only showed a very smal l degree o f dechlor inat ion i n the present work , but it showed a m u c h higher degree o f dechlor inat ion i n previous experiments incubated at 21 ° C . The overa l l percent dechlor inat ion o f 23456-2345- and 130 23456-2356-nonaCB was higher i n the present set o f experiments, but once again (c.f. 7.3.2), the total amount o f congener dechlorinated was lower due to the lower amount o f congener in i t i a l ly present i n the present experiments. —*—23456-2345-nonaCB (Incubated singly) - -x- - 23456-2345-nonaCB (Incubated together with all three nonaCBs) —e—23456-2346-nonaCB (Incubated singly) _ _ 23456-2346-nonaCB (Incubated together with all three nonaCBs) —&—23456-2356-nonaCB (Incubated singly) _ - 23456-2356-nonaCB (Incubated together with all three nonaCBs) Figure 7-6: Disappearances of nonaCBs when incubated singly and in combination. It is interesting to note that a l l three n o n a - C B s show a greater extent o f dechlor inat ion when they are incubated together than when they are incubated separately. Th i s result is found for a l l n o n a C B s i n both the 4-week and 8-week incubations, 131 indicat ing that this result is real, and not just an artifact caused by, for example , i n o c u l u m variations. 7.3.5 Incubations with a combination of four octa- and three nona-CB congeners One set o f incubations that was set up contained a combina t ion o f a l l seven octa-and n o n a - C B congeners used i n the previous ly described experiments. The results for these incubations are presented i n Table 7-7. The percent disappearance over t ime o f these congeners for both the incubations o f the congeners on their o w n , and for the incubations o f the congeners combined i n a mixture , are plotted i n Figure 7-7. It should be noted that 2345-2346-oc taCB and 23456-2 4 5 - o c t a C B co-elute, and 23456-234-oc taCB and 23456-2356-nonaCB co-elute. T h i s means that the results for the degradation o f these congeners i n the mixture have to be presented together. In order to make comparisons between experiments where congeners were incubated separately and experiments where they were incubated together, a l ine w h i c h averages the results for co-elut ing congeners i n the single congener experiments has been plotted. 132 Table 7- 7: Moi percent distribution of four octaCB and three nonaCB substrates and their products. Exper imen t N u m b e r Congener Identity M o i % dis t r ibut ion o f congeners 0 weeks 4 weeks 8 weeks 0 weeks C o n t r o l 8 weeks C o n t r o l 11 2 3 4 5 6 - 2 3 4 - C B + 2 3 4 5 6 - 2 3 5 6 - C B 14.3 13.3 8.9 14.4 14.2 2 3 4 5 - 2 3 4 6 - C B + 2 3 4 5 6 - 2 4 5 - C B 26.7 20.0 13.6 26.9 26.1 2 3 4 5 6 - 2 3 4 5 - C B 12.7 11.7 7.8 12.5 12.5 2 3 4 5 6 - 2 3 4 6 - C B 27.1 26.1 23.5 26.9 27.4 2 3 4 5 - 2 3 5 6 - C B 19.2 16.3 14.0 19.3 19.8 2 4 6 - 2 4 6 - C B 0 0 1.0 0 0 2 4 6 - 2 6 - C B 0 0 2.9 0 0 2 4 6 - 2 4 - C B 0 7.1 8.2 0 0 2 4 - 2 4 - C B 0 4.2 7.9 0 0 2 4 - 2 6 - C B 0 1.3 12.1 0 0 133 50 -I-0 4 Time (weeks) 8 —&—23456-234-octaCB + 23456-2356-nonaCB (Incubated singly) _ ^y. _ 23456-234-octaCB + 23456-2356-nonaCB (Incubated in combination with all 7 congeners) —e—2345-2346-octaCB + 23456-245-octaCB (Incubated singly) _ _c- _ 2345-2346-octaCB + 23456-245-octaCB (Incubated in combination with all 7 congeners) —*—23456-2345-nonaCB (Incubated singly) - -x- - 23456-2345-nonaCB (Incubated in combination with all 7 congeners) —•—23456-2346-nonaCB (Incubated singly) --•--23456-2346-nonaCB (Incubated in combination with all 7 congeners) —x—2345-2356-octaCB (Incubated singly) - -x- - 2345-2356-octaCB (Incubated in combination with all 7 congeners) Figure 7-7: Disappearances of octaCB and nonaCB congeners when incubated singly and in combination with each other. 7.3.6 Discussion: A l l seven congeners in combination 23456-2345-nonaCB and 23456-2346-nonaCB both show a greater degree o f dechlor inat ion when they are incubated i n the mixtures containing a l l seven congeners than when these congeners are incubated s ingly (Figure 7-7). T h i s difference is seen after 4 weeks o f incubat ion, and has increased by 8 weeks. W h e n these results are compared w i t h the results presented i n Figure 7-6, it can be seen that the increase i n dechlor inat ion o f these n o n a C B congeners i n the mixture containing 4 o c t a C B s as w e l l as 3 n o n a C B s is greater than the increase i n dechlor inat ion o f these n o n a C B s i n the mixture conta in ing on ly 3 n o n a C B s . T h i s suggests that despite the fact that o c t a C B s are generally considered to be easier to degrade (61), their presence does not have any inhib i tory effect on n o n a C B dechlor inat ion, i n fact, the presence o f o c t a C B s appears to stimulate n o n a C B dechlor inat ion. The results obtained for the degradation o f 2345-2356-oc taCB show a decreased rate o f degradation for the congener i n the mixture as opposed to the congener on its o w n (Figure 7-7). Th i s is consistent w i t h previous results for this congener shown i n F igure 7-1. F o r the co-elut ing congeners 2345-2346-oc taCB and 23456-245-oc taCB, and 23456-234-oc t aCB and 23456-2356-nonaCB, less degradation is seen after 4 weeks o f incubat ion i n the mixture than is seen when the congeners are incubated s ingly (Figure 7-7). In contrast, more degradation is seen i n the mixture after 8 weeks o f incubat ion than when the congeners are incubated singly. W h e n the dechlor inat ion products produced i n experiment no. 11 (Table 7-7) are investigated, it can be seen that the amount o f 246-24-pentaCB found at 8 weeks is m u c h 135 lower than w o u l d be expected i f on ly meta dechlor inat ion were taking place. In contrast, the amount o f 24-26- te t raCB found is m u c h higher than w o u l d be expected. T h i s was not found i n any o f the other incubations, and it suggests that reasonably extensive para dechlor inat ion o f 246-24-pentaCB is taking place"in this mixture o f congeners. 7.3.7 Incubation of decachlorobiphenyl (23456-23456-decaCB) at 30°C Decach lo rob ipheny l was incubated at 30 ° C for a per iod o f 8 weeks. Table 7-8 shows the results obtained i n these incubations. N o dechlor inat ion products were detected after 4 weeks o f incubation, after 8 weeks a very smal l amount (0.4%) o f 246-2 4 6 - h e x a C B was found. T h i s contrasts w i t h s imi la r experiments incubated at 21 ° C (results not shown), i n w h i c h no dechlor inat ion products o f decachlorobiphenyl were detected after 24 weeks o f incubat ion. Table 7-8: Moi percent distribution over time of incubations containing decachloro-biphenyl (23456-23456-decaCB). Exper iment N u m b e r Congener Identity M o i % distr ibut ion o f congeners 0 weeks 4 weeks 8 weeks 0 weeks C o n t r o l 8 weeks C o n t r o l 8 2 3 4 5 6 - 2 3 4 5 6 - C B 100 100 99.6 100 100 2 4 6 - 2 4 6 - C B 0 0 0.4 0 0 7.3.8 Incubations with pentachlorobiphenyl and tetrachlorobiphenyl congeners A l m o s t a l l dechlor inat ion experiments reported i n this thesis show the accumulat ion o f a few specific te t raCB and pen taCB products. The experiments reported i n this section were designed to determine i f further dechlor inat ion o f these tetra- and 136 pen ta -CBs cou ld take place by us ing the enrichment cultures, and i f so, to investigate the effects o f congener combina t ion and concentration on this dechlor inat ion. The extents o f dechlor inat ion and a l l dechlor inat ion products found for the single congener incubations are g iven i n Table 7-9. N o n e o f the single congener incubations showed the presence o f any dechlor inat ion products after 4 weeks o f incubat ion. B o t h 246-24-pen taCB and 24-24-te t raCB showed some dechlor inat ion after 8 weeks o f incubat ion, but 246-2- te t raCB d i d not show any signs o f dechlor inat ion after this t ime per iod. 24-24- te t raCB underwent a smal l amount o f ortho dechlor inat ion, to give 24-4-t r i C B . 246-24-pen taCB showed m u c h more extensive dechlorinat ion, g i v i n g a large amount o f b iphenyl as the predominant dechlor inat ion product. Th i s means that both ortho and para dechlor inat ion took place i n this incubat ion. Table 7-9: Moi percent distribution over time of single penta- and tetra-CB congeners substrates and their dechlorination products. Exper iment N u m b e r Congener Identity M o i % distr ibut ion o f congeners 0 weeks 4 weeks 8 weeks 0 weeks C o n t r o l 8 weeks C o n t r o l 12 2 4 6 - 2 4 - C B 100 100 66.6 100 100 2 6 - 4 - C B 0 0 13.1 0 0 2 4 - 4 - C B 0 0 3.5 0 0 B i p h e n y l 0 0 16.8 0 0 16 2 4 6 - 2 - C B 100 100 100 100 100 17 2 4 - 2 4 - C B 100 100 99.3 100 100 2 4 - 4 - C B 0 0 0.7 0 0 137 The m o i percent distributions o f a l l P C B s found i n experiments where the penta-C B and te t ra -CB congeners were incubated together i n a mixture are g iven i n Table 7-10. Figure 7-8 shows chromatograms obtained for these incubations at t ime = 0 weeks and at t ime = 8 weeks. These chromatograms clearly show the relative decreases i n the congeners, and the formation o f b iphenyl as the major dechlor inat ion product. Table 7-10: Moi percent distribution of incubations 246-24-pentaCB, 246-2-tetraCB and 24-24-tetraCB mixtures and their dechlorination products. M o i % distr ibut ion o f congeners Exper iment Congener 0 weeks 4 weeks 8 weeks 0 weeks 8 weeks N u m b e r Identity C o n t r o l C o n t r o l 18 2 4 6 - 2 4 - C B 32.9 32.8 19.8 32.7 32.6 2 4 6 - 2 - C B 33.7 33.8 33.0 33.6 34.0 2 4 - 2 4 - C B 33.4 33.4 26.4 33.7 33.4 2 6 - 4 - C B 0 0 2.4 0 0 2 4 - 2 - C B 0 0 0.9 0 0 2 6 - 2 - C B 0 0 0.5 0 0 4 - 4 - C B 0 0 1.8 0 0 B i p h e n y l 0 0 15.2 0 0 19 2 4 6 - 2 4 - C B 33.0 33.1 30.2 32.9 33.0 2 4 6 - 2 - C B 33.7 33.3 33.0 33.6 33.7 2 4 - 2 4 - C B 33.3 33.6 25.5 33.5 33.3 2 6 - 4 - C B 0 0 0.9 0 0 2 4 - 2 - C B 0 0 0.4 0 0 2 6 - 2 - C B 0 0 0 0 0 4 - 4 - C B 0 0 0.7 0 0 B i p h e n y l 0 0 9.2 0 0 138 t = 0 weeks 180000 160000 + 140000 120000 + I 100000 O) (0 80000 60000 40000 20000 0 246-2-tetraCB 24-24-tetraCB 246-24-pentaCB 101 201 301 401 501 601 701 Retention time 801 901 1001 J2 re E Ut CO t = 8 weeks 1600 1400 1200 1000 800 600 400 200 0 246-2-tetraCB biphenyl 101 201 301 24-24-tetraCB 246-24-pentaCB 401 501 601 701 Retention time 801 901 1001 1101 Figure 7-8: Dechlorination of246-24-pentaCB, 24-24-tetraCB and 246-2-tetraCB, showing formation of biphenyl as the major dechlorination product 139 The final experiment i n this set investigated the dechlor inat ion o f 246-24-pen t aCB i n the presence o f 2345-2346-oc taCB. 246-24-pentaCB is expected to be the major meta dechlor inat ion product o f 2345-2346-oc taCB. The results obtained for this experiment are presented i n Table 7-11. Table 7-11: Moi percent distribution of246-24-pentaCB and 2345-2346-octaCB and their dechlorination products. M o i % dis t r ibut ion o f congeners Exper iment Congener 0 weeks 4 weeks 8 weeks 0 weeks 8 weeks N u m b e r Identity C o n t r o l C o n t r o l 12 2 3 4 5 - 2 3 4 6 - C B 50.0 27.3 25.7 49.6 49.0 2 4 6 - 2 4 - C B 50.0 72.7 49.1 50.4 51.0 2 6 - 4 - C B 0 0 4.7 0 0 4 - 4 - C B 0 0 1.6 0 0 B i p h e n y l 0 0 18.9 0 0 7.3.9 Discussion: PentaCB and tetraCB congeners The m a i n difference that is found when the penta- and te t raCB congeners are incubated s ingly or i n a mixture is i n the dechlor inat ion o f 24-24- te t raCB. W h e n this congener is incubated s ingly, only a very l imi ted degree o f dechlor inat ion is detected. In the incubations o f congener mixtures , however , 24-24- te t raCB shows m u c h more extensive dechlor inat ion. These results are the same for both concentrations o f combined congener incubations tested. 24-24- te t raCB d i d not produce any b iphenyl w h e n incubated alone, but when incubated i n combina t ion w i t h the other two congeners, a significant amount o f b iphenyl was produced f rom this congener. T h i s shows that i n these experiments the presence o f the other two P C B congeners had a marked effect on the dechlor inat ion o f this particular congener. The on ly difference between experiments 18 and 19 was the total concentrat ion o f P C B s present i n the incubations. A s expected, the percent dechlor inat ion i n experiment 18, the experiment w i t h the lower concentration o f P C B s , was higher, but the total amount o f P C B dechlorinated was higher for the higher concentration o f P C B s i n experiment 19. B o t h experiments showed a s imi lar dis t r ibut ion o f dechlor inat ion products. In the final experiment that i n v o l v e d both 246-24-pentaCB and 2345-2346-o c t a C B , it can be seen that after 4 weeks o f incubat ion, over 4 5 % o f the o r ig ina l 2345-2 3 4 6 - o c t a C B had dechlorinated to 246-24-pentaCB, but no dechlor inat ion o f p e n t a C B had taken place (Table 7-11). Af ter 8 weeks o f incubation, a further smal l percentage o f 2345-2346-oc taCB was dechlorinated, and a large proport ion o f the 246-24-pen taCB had complete ly dechlorinated, g iv ing 18.9% biphenyl i n the final mixture. T h i s shows that the enrichment cultures used are capable o f further dechlor inat ion o f 246-24-pen taCB to b iphenyl i n a mixture that s t i l l contains a significant proport ion o f more-h ighly chlorinated congeners that require meta dechlorinat ion. N o n e o f the experiments reported here i n v o l v i n g incubat ion o f penta- or te t ra-CBs showed any dechlor inat ion o f these congeners after 4 weeks o f incubat ion, but many o f them showed extensive dechlor inat ion to b iphenyl after 8 weeks o f incubat ion. T h i s means that the in i t ia l lag phase for ortho and para dechlor inat ion o f these congeners is longer than the one for meta dechlor inat ion o f octa- and n o n a - C B s . In many instances, the rate o f meta dechlor inat ion o f octa- and n o n a - C B s decreased substantially between 4 141 and 8 weeks. Since the further ortho and para dechlor inat ion only appears for the first t ime after 8 weeks o f incubation, it is not possible f rom these experiments to determine i f this dechlor inat ion has yet reached its m a x i m u m rate. One possible explanat ion for the lag i n ortho and para dechlor inat ion act ivi ty as opposed to meta dechlor inat ion act ivi ty c o u l d be that different mic rob i a l populat ions are i n v o l v e d i n these different types o f dechlor inat ion, and that the in i t ia l meta dechlorinat ing popula t ion is replaced by other ortho and para dechlor inat ing populations over the t ime course o f the experiments. 7.4 Summary T h e results presented i n this chapter show that the rates and products o f P C B dechlor inat ion can vary wide ly , depending on the mixture o f congeners present. Some results suggest that the presence o f congeners w i t h doubly-f lanked meta chlorines inhibi t dechlor inat ion o f congeners w i t h s ingly- or unflanked meta chlorines. Exper iments performed w i t h n o n a C B s and o c t a C B s mixtures suggest that the presence o f o c t a C B s stimulates the dechlor inat ion o f n o n a C B s . It has also been shown that extensive degradation o f penta- and te t raCB congeners to b iphenyl can occur, even when other, more h igh ly chlorinated congeners are s t i l l present. T h i s is the first t ime that experiments w i t h combinat ions o f a few selected h igh ly chlorinated P C B congeners have been reported. 142 Chapter 8 Identification of the Most Abundant Dechlorination Product Found in Aroclor 1260 Incubations, by Using MS/MS 8.1 Introduction The majority o f dechlor inat ion products o f A r o c l o r 1260 found i n the present w o r k (Chapters 4 and 5) were tetrachlorobiphenyls that cou ld be identif ied f rom their mass spectra. Speci f ic isomers were identif ied by compar ison o f their relative retention t imes w i t h those o f tetrachlorobiphenyl congeners found i n A r o c l o r mixtures . T h e most abundant dechlor inat ion product found after the incubat ion o f A r o c l o r 1260 w i t h the enrichment cultures had a retention t ime that cou ld correspond to either 24-24- te t raCB or 245-2- te t raCB, as these two congeners co-elute. The structures o f 24-24- te t raCB and 245-2- te t raCB are shown i n Figure 8-1. C I C I C I C I C I 24-24-Te t rach lorob iphenyl 245-2-Tet rach lorob iphenyl Figure 8-1: Structures of24-24-tetraCB and 245-2-tetraCB. 143 The mass spectra o f both isomers show a range o f ions from m / z 290 to 298 (due to the molecula r i o n cluster), accompanied by a range o f masses corresponding to the loss o f 1, 2 or more chlorines (Figure 8-2). A l t h o u g h a l l tetrachlorobiphenyl mass spectra are expected to be s imi lar , Lausev ic et al. (114) have shown that te t raCBs w i t h no or one ortho chlor ine can be dis t inguished f rom te t raCBs w i t h two or three ortho chlorines on the basis o f different fragmentation patterns (under certain resonant exci ta t ion condit ions i n a quadrupole i o n trap, t e t raCBs w i t h no or one ortho chlor ine tend to lose 1 C I , whereas te t raCBs w i t h two or three ortho chlorines tend to lose 2 CI) . 24-24- te t raCB and 245-2- te t raCB, however , are both di-ortho substituted P C B s , and so the techniques developed by Lausev i c et al. do not a l l o w for differentiation between these two congeners on the basis o f their mass spectra. It is possible that the different isomers cou ld have different fragmentation characteristics depending on the co l l i s ion- induced dissociat ion ( C I D ) exci ta t ion condi t ions. The w o r k presented i n this chapter was performed to determine i f it is possible to d is t inguish between these two congeners by using M S / M S , and i f so, to determine w h i c h congener corresponds to the dechlor inat ion product found i n A r o c l o r 1260 incubations. A r ev iew o f the basic pr inciples o f i o n trap mass spectrometry is g iven i n a paper by M a r c h (137). In the present work , two different types o f C I D exci ta t ion i n an i o n trap mass spectrometer were tested. The first type, non-resonant exci ta t ion, uses a l o w frequency r.f. potential appl ied to the end caps o f the trap i n order to increase the potential energy o f a l l the ions i n the trap. Th i s increase i n potential energy is then 144 (a) 2 4 - 2 4 - t e t r a C B 1000000 \-800000 600000 400000 200000 -2C1 -1C1 140 160 180 200 220 240 m/z 260 280 300 320 (b) 2 4 5 - 2 - t e t r a C B 250000 200000 150000 •% 100000 50000 0 --2C1 --3C1 -1C1 1 _ 1 Ii •! ll , Ii. , , In , i Ii, , 140 160 180 200 220 240 260 280 300 320 m/z Figure 8-2: Mass spectra of (a) 24-24-tetraCB and (b) 245-2-tetraCB under EI operating conditions for the Varian Saturn ion trap GC/MS. transformed into translational kinet ic energy, w h i c h is i n turn converted into internal v ibra t ional energy upon subsequent co l l i s ions o f the ions w i t h the h e l i u m gas i n the i o n trap. The second type o f i o n excitat ion, resonant excitat ion, applies a h igh frequency r.f. potential to the end caps o f the ion trap. The frequency o f the resonant exci ta t ion r.f. potential generated matches the osc i l la t ion frequency o f the selected ion . In both k inds o f i o n exci ta t ion, chemica l bonds i n the ions may be broken i f the ions acquire enough vibrat ional energy dur ing the exci ta t ion experiment. T h i s fragmentation leads to the appearance o f lower mass fragments i n the mass spectra. 8.2 Experimental 8.2.1 Optimization of CID conditions Sequential M S / M S experiments were performed by us ing " T o o l k i t V e r s i o n 1.0" software. " T o o l k i t " is a program suppl ied by V a r i a n that generates waveforms for the isola t ion and fragmentation o f ions i n the i o n trap. A l l experiments were performed w i t h automatic gain control ( A G C ) w i t h a target value o f 10 000. The mass range scanned was f rom m / z 150 to m / z 310, w i t h a scan t ime o f 0.6 seconds. 8.2.1.1 Optimization of CID excitation voltage In order to determine the effect o f C I D exci ta t ion voltage on the fragmentation o f 24-24- and 245-2-tetrachlorobiphenyl, a range o f different non-resonant and resonant exci ta t ion voltages were appl ied to single congener standards o f 24-24- te t raCB and 245-2- te t raCB. The C I D t ime was set 20 ms, and 2.5 u L o f 10 p p m P C B standards were 146 injected, separately, into the G C . The G C was temperature-programmed as described i n Chapter 2 (Sect ion 2-7). Non-resonant exci ta t ion voltages tested ranged f rom 0 - 100 V i n 10 V intervals. Resonant exci tat ion voltages in i t i a l ly tested ranged f rom 0 - 3.6 V i n 0.4 V intervals. The exci tat ion w i n d o w for the resonant exci ta t ion was then narrowed, and exci ta t ion voltages f rom 0.2 V to 1.8 V were tested at intervals o f 0.2 V . 8.2.1.2 Optimization of CID excitation time The C I D exci ta t ion times were var ied from 10 - 40 ms i n 10 ms intervals for selected exci ta t ion voltages. The selected non-resonant exci ta t ion voltages were 90 V and 100 V , and the selected resonant exci ta t ion voltages were 0.6 V , 0.8 V and 1.0 V . In order to test the reproducibi l i ty o f the results, experiments for the op t imized C I D voltages and exci ta t ion t imes were performed i n triplicate. 8.2.2 The effect of concentration on the fragmentation of 24-24-tetraCB and 245-2-tetraCB A range o f concentrations o f 24-24- te t raCB and 245-2- te t raCB (from 0.1 p p m to 100 ppm) were subjected to the selected exci ta t ion voltage (100 V non-resonant) and exci ta t ion t ime (30 ms). 8.2.3 Data analysis M a s s spectra were analyzed by separating the five different mass clusters corresponding to (i) the molecular ions (m/z 290 - 298), (i i) loss o f 1 chlor ine (m/z 255-262), ( i i i ) loss o f 2 chlorines (m/z 220-226), ( iv) loss o f 3 chlorines (m/z 185-190), or (v) 147 loss o f a l l 4 chlorines (m/z 150-154). These different mass clusters were integrated and the relative propor t ion (as a percent contribution) o f each mass range to the total i o n current ( T I C ) was calculated. Vol tage ranges that gave the greatest difference i n these relative proport ions between the two te t raCB isomers were selected for further w o r k (sections 8.2.1.2 and 8.2.2). 8.3 Results and Discussion 8.3.1 The effect of CID excitation energy on the fragmentation of 24-24-tetraCB and 245-2-tetraCB The resonant exci ta t ion fragmentation curves for 24-24- te t raCB and 245-2-te t raCB are s h o w n i n Figure 8-3. N o fragmentation occurs at a C I D o f 0.2 V , an increase i n fragmentation occurs at a C I D o f 0.6 V , and quite extensive fragmentation occurs at a C I D o f 1.4 V . The results for exci tat ion energy variations on the fragmentation process for 24-24- te t raCB and 245-2- te t raCB are shown i n Figure 8-4. Nei ther t e t raCB congener fragments at a non-resonant exci tat ion energy o f 70 V , but at a non-resonant exci ta t ion energies o f 90 and 100 V , 245-2- te t raCB fragments more extensively than 24-24- te t raCB. Resonant C I D exci ta t ion amplitudes o f 0.6 V , 0.8 V and 1.0 V and non-resonant C I D exci ta t ion ampli tudes o f 90 V and 100 V were chosen for further w o r k as these corresponded to the largest differences i n fragmentation between 24-24- te t raCB and 245-2- te t raCB. The mass clusters corresponding to the molecular ions (m/z 290-298), the loss o f 1 chlor ine (m/z 255-262), and the loss o f 2 chlorines (m/z 220-226) showed a large degree o f var ia t ion between the two congeners at the various exci ta t ion energies tested. (b) 2 4 5 - 2 - T e t r a C B Resonant Excitation Energy (V) Figure 8-3: Fragmentation of (a) 24-24-tetraCB and (b) 245-2-tetraCB with different resonant excitation energies. 149 (a) 24-24-TetraCB 1 • 0 9 -o 0 8 -1- 0 7 -*o 0 6 -c o 0 5 -u 0 4 -CO >_ 0 3 -Li_ 0 2 -0 1 -0 • 70 80 90 100 Non-Resonant Excitation Energy (V) m/z mass clusters - • - 2 9 0 - 2 9 8 " • - 2 5 5 - 2 6 2 220-226 185-190 150-154 (b) 245-2-TetraCB 70 80 90 100 Non-Resonant Excitation Energy (V) Figure 8-4: Fragmentation of (a) 24-24-tetraCB and (b) 245-2-tetraCB with different non-resonant excitation energies. 150 The mass clusters corresponding to the loss of 3 chlorines (m/z 185-190) and the loss of 4 chlorines (m/z 150-154) did not show much variation between the isomers. 8.3.2 The effect of CID excitation time on the fragmentation of 24-24-tetraCB and 245-2-tetraCB The effect of excitation time on the fragmentation of 24-24- and 245-2-tetraCB with a resonant excitation energy of 0.6 V is shown in Figure 8-5. At a 10 ms excitation time, 81% of 24-24-tetraCB and 70% of 245-2-tetraCB are detected as molecular ions (m/z 290-298). When this time is increased to 40 ms, only 69% of 24-24-tetraCB and 55% of 245-2-tetraCB are detected as molecular ions. The difference in the amount of fragmentation of the two congeners has increased with increasing CID excitation time. Fragmentation of 24-24-tetraCB and 245-2-tetraCB with increasing excitation times at 0.8 V and 1.0 V resonant excitation energies followed the same trend. Fragmentation observed at different CID excitation times and a non-resonant excitation energy of 100 V is shown in Figure 8-6. As with the resonant excitation experiments, the degree of fragmentation increases with increasing excitation time. At 100 V excitation energy and 10 ms excitation time, 71% of the TIC is due to molecular ions (m/z 290 - 298) for 24-24-tetraCB, in contrast to only 53% for molecular ions of 245-2-tetrCB. When the excitation time is increased to 40 ms, these values drop to 22% for 24-24-tetraCB and 8% for 245-2-tetraCB, respectively. Experiments performed with a 90 V non-resonant excitation energy gave similar results. Excitation times that corresponded to the largest differences in the observed fragmentation between the two tetraCB isomers were selected. 151 (a) 24-24-TetraCB o c o Q_ O c 0 O L-100% 80% 60% 40% 20% 0% U 100% 80% 60% 40% 20% J 0% 10 20 30 40 Resonant Excitation Time (ms) (b) 245-2-TetraCB 10 20 30 40 Resonant Excitation Time (ms) m/z mass clusters • 220-226 • 255-262 13290-298 H U L J ^ m/z mass clusters • 220-226 • 255-262 • 290-298 Figure 8-5: Fragmentation of (a) 24-24-tetraCB and (b) 245-2-tetraCB, with 0.6 V resonant excitation energy, as a function of excitation time. 152 (a) 24-24-TetraCB 100% 80% 60% 40% 20% 0% 10 20 30 40 Non-Resonant Excitation Time (ms) m/z mass clusters • 220-226 1255-262 1290-298 (b) 245-2-TetraCB 100% 80% o o 60% *-< c CD O 40% L. O CL 20% 0% • • ^^^^^ m/z mass clusters • 220-226 • 255-262 • 290-298 10 20 30 40 Non-Resonant Excitation Time (ms) Figure 8-6: Fragmentation ratios for (a) 24-24-tetraCB and (b) 245-2-tetraCB, with 100 V non-resonant excitation energy, as a function of excitation time. 153 The greatest difference between the fragmentation for the two different isomers is found for a 100 V non-resonant exci tat ion w i t h a 30 ms exci tat ion t ime, where 3 3 % o f 24-24- te t raCB and 16% o f 245-2- te t raCB are detected as molecular ions. Thus , 100 V non-resonant exci ta t ion w i t h 30 ms exci ta t ion t ime was chosen for appl ica t ion to samples that had undergone m i c r o b i a l dechlor inat ion. 8.3.3 The effect of concentration on the fragmentation of 24-24-tetraCB and 245-2-tetraCB It was important to determine i f the results reported i n 8.3.1 and 8.3.2 were appl icable over a range o f concentrations, because the concentration o f dechlor inat ion products i n incubations can vary wide ly . The concentration o f congener present was found to have no effect on the fragmentation o f 24-24- and 245-2- te t raCB. Thus the op t imized method for dis t inguishing between the two congeners is appl icable over the range o f concentrations tested (0.1 p p m to 100 ppm). The concentrations o f the most abundant dechlor inat ion product found i n A r o c l o r 1260 dechlor inat ion experiments reported i n this thesis usual ly range f rom 10 p p m to 60 ppm, ly ing w e l l w i t h i n the concentration range p rov id ing reliable differentiation between 24-24- and 245-2- te t raCB. 154 (a) 24-24-tetraCB O i -O c o 0.6 0.5 0.4 0.3 o re 0.2 0.1 0 x — * — * -20 40 60 80 100 Concentration (ppm) 120 m/z mass clusters — 290-298 -m- 255-262 - ± - 220-226 -*- 185-190 — « — 150-154 o c o o re (b) 245-2-tetraCB 20 40 60 80 100 Concentration (ppm) 120 m/z mass clusters -•-290-298 -•-255-262 -A-220-226 -H-185-190 150-154 Figure 8-7: Fragmentation of (a) 24-24-tetraCB and (b) 245-2-tetraCB with different concentrations 155 8.3.4 Identification of the major dechlorination product present in Aroclor 1260 incubations Once condit ions for isomer differentiation had been op t imized , samples f rom f ive different A r o c l o r 1260 incubat ion experiments were analyzed. These samples were introduced into the mass spectrometer by G C , and subjected to 100 V (non-resonant) for an exci ta t ion per iod o f 30ms. The result ing mass spectra were analyzed as described i n section 8.2.3, and the results obtained were compared w i t h those obtained for the single congener standards (24-24-tetraCB and 245-2- te t raCB). A l l f ive samples tested were clearly shown to contain entirely or ma in ly 24-24-te t raCB as the dechlor inat ion product. The fragmentation o f 24-24- te t raCB standards, the fragmentation o f the experimental dechlor inat ion products, and the fragmentation o f 245-2- te t raCB standards is shown i n Figure 8-8. The proportions o f ions found i n the major dechlor inat ion product l ie w i t h i n the experimental range found for 24-24- te t raCB, and are s ignif icant ly different f rom the ion dis tr ibut ion found for 245-2- te t raCB. 156 CD O CD 0-100% 80% 60% 40% 20% m/z mass clusters • 220-226 • 255-262 • 290-298 24-24-tetraCB Major product 245-2-tetraCB standard from incubations standard Figure 8-8: Fragmentation of24-24-tetraCB, the major product from Aroclor 1260 incubations, and 245-2-tetraCB with a non-resonant excitation energy of 100 V and an excitation time of 30 ms. Because other experiments reported i n this thesis (Chapters 4 - 7 ) have shown that the predominant dechlor inat ion i n the cultures used i n this w o r k occurs at the meta posi t ion, 24-24- te t raCB was to be the expected dechlor inat ion product. The five major dechlor inat ion products tested originated from three different enrichment cultures ( B K 2 4 , B K 7 2 and B K 8 1 ) , and i n a l l cases 24-24-tetraCB was shown to be present. T h i s w o r k shows that it is possible to dis t inguish between P C B isomers by us ing M S / M S , and it is l i k e l y that this w o r k can be expanded to dis t inguish between other co-elut ing or c lose ly elut ing P C B congeners as w e l l . 157 Chapter 9 Detection and Biodegradation of Alkyl PAHs in Arctic Soils 9.1 Introduction T h i s section is concerned w i t h the analysis and biodegradation o f another class o f environmental pollutants, a lkylated p o l y c y c l i c aromatic hydrocarbons (a lky l P A H s ) . 9.1.1 Structure P o l y c y c l i c aromatic hydrocarbons ( P A H s ) are a class o f organic compounds that possess two or more fused aromatic rings. The compounds that have on ly hydrogen atoms attached to the aromatic rings are generally referred to as parent, or unsubstituted, P A H s . T y p i c a l examples o f lower molecular weight P A H s are shown i n Figure 9-1. The hydrogen atoms can be replaced w i t h a number o f different chemica l functional groups (e.g. a l k y l , a lkoxy , C I , B r , O H , SO3, NH2), g i v ing a w ide variety o f substituted P A H s . The number ing shown i n Figure 9-1 shows the numbers used for naming substituted P A H S . P A H s that have one or more a l k y l groups attached to the aromatic r ing are ca l led a l k y l P A H s . E x a m p l e s o f some a l k y l P A H s referred to i n this work are g iven i n Figure 9-2. 158 naphthalene acenaphthene acenaphthylene 8 9 l 8 10 1 5 4 fluorene 5 9 4 anthracene 8 9 phenanthrene Figure 9-1: Chemical structures of some unsubstituted PAHs. 3-Methylphenanthrene 2 ,7-Dimethylphenanthrene Figure 9-2: Chemical structures of some alkyl-substituted PAHs. 9.1.2 Physical and chemical properties: A summary o f some important phys ica l and chemica l properties o f selected parent and a l k y l P A H s is presented i n Table 9-1. It should be noted that the P A H s have l o w water so lubi l i ty , and increasing a l k y l substitution decreases water so lubi l i ty . Increasing a l k y l substitution has vary ing effects on mel t ing and b o i l i n g points. Table 9-1: Summary of some properties of PAHs. C o m p o u n d M W (g/mol) m.p. (°C) b.p. (°C) S (25 ° C g /m 3 ) Naphthalene 128 80.5 178 31 1 -methyl 142 -22 245 28 2-methyl 142 34.6 242 25 1,4-dimethyl 156 7.7 262 11.4 Phenanthrene 178 101 339 1.1 Anthracene 178 216 340 0.045 9-methyl 192 82 355 0.261 9 ,10-dimethyl 206 182 n.a. 0.056 n.a. not available Abbreviations: MW, molecular weight; m.p., melting point; bp., boiling point; S, aqueous solubility. Data from Mackay et al (138) 160 9.1.3 Toxicity Some P A H s are k n o w n carcinogens and mutagens (139, 140), and the Canad ian Env i ronmenta l Protect ion A c t ( C E P A ) lists P A H s as a class on its Pr io r i ty Substance L i s t (141). L o w levels o f P A H s have been shown to be acutely tox ic to aquatic organisms (141, 142), and P A H s have been shown to induce tumours and cel lular abnormali t ies i n fish (142, 143). Several P A H s have been shown to be carcinogens i n mammal s (143-146), and several P A H s are potent inducers o f cytochrome P 4 5 0 (142). Potent ia l ly hazardous consequences o f cytochrome P450 induct ion include mutagenesis, carcinogenesis and attenuation o f endogenous cel lular processes. The locat ion o f a l k y l groups around the r ing structure may strongly influence carcinogenici ty and other tox ic effects. F o r example , l ,12-dimethylbenz[a]anthracene is not carcinogenic w h i l e 7,12-dimethylbenz[a]anthracene is one o f the most potent P A H carcinogens k n o w n (147). In a rev iew on P A H toxic i ty , Lee et al (148) report that some alkylated P A H s are less tox ic than the unsubstituted analogues, wh i l e others are more tox ic . 9.1.4 Distribution in the environment P A H s are relat ively stable and i n soils and are less easy to degrade than many other organic compounds . The two m a i n processes that generate P A H s are h igh temperature combus t ion and l o w to moderate temperature diagenesis o f organic matter (142, 149). The most significant g lobal source o f P A H contaminat ion i n the environment is incomplete combus t ion o f organic material (141, 142, 150, 151). A t h igh temperatures, unsubstituted P A H s are formed almost exc lus ive ly , so P A H s from a pyrogenic source are usual ly characterized by the dominance o f unsubstituted (parent) P A H s (149, 152). 161 PAHs are also present in substantial amounts in fossil fuels (141, 142, 153), and in contrast to pyrogenic PAHs, petrogenic PAHs are generally characterized by higher concentrations of alkylated PAHs than parent, or unsubstituted PAHs (149, 154, 155). Diesel fuel in particular contains high concentrations of methylated naphthalenes relative to unsubstituted naphthalenes (156). Oil spills and effluent from fuel-processing industries thus represent important local sources of alkyl P A H contamination (157). 9.1.5 PAHs in fuel/hydrocarbon contamination Crude oil and most refined petroleum products are extremely complex mixtures of many thousands of organic compounds. Typical crude petroleums may contain from 0.2 % to more than 7 % PAHs. The chemical composition of the P A H fraction of oils and fuels has been studied in great detail (158-159). Table 9-2 shows the concentrations of PAHs with four or fewer rings found in two crude oils and two refined oils. Ratios of several parent PAHs to alkylated PAHs have been shown to vary widely depending on the age and source of the oil, and so these ratios have been used as geochemical markers (160) to identify, among other things, the sources oil spills in the environment. 162 Table 9.2 Concentrations of some PAHs found in crude and refined oils. Concentra t ion (mg/kg, ppm) C o m p o u n d South Lou i s i ana crude K u w a i t crude N o . 2 fuel o i l B u n k e r C residual o i l Naphthalene 400 400 4000 1000 1 -Methylnaphthalene 800 500 8200 2800 2-Methylnaphthalene 900 700 18900 4700 Dimethylnaphthalenes 3600 2000 31100 12300 Trimethylnaphthalenes 2400 1900 18400 8800 Fluorenes 200 <100 3600 2400 Phenanthrene 70 26 429 482 1 -Methylphenanthrene 111 - 173 43 2-Methylphenanthrene 144 89 7677 828 Fluoranthene 5.0 2.9 37 240 Pyrene 3.5 4.5 41 23 Adapted from Neff (142) 9.1.6 Metabolism of PAHs The abi l i ty o f bacteria and fungi to degrade unsubstituted P A H s conta ining three or fewer arene rings has been w e l l documented (161-165), and these lower molecula r weight P A H s can be degraded rapidly i n sediments. General ly , the rate o f degradation o f P A H s is inversely proport ional to the number o f rings i n the P A H molecu le , and so the 163 higher molecula r weight P A H s are m u c h more recalcitrant to biodegradation (163, 164). A e r o b i c bacterial metabol i sm o f unsubstituted P A H s can i nvo lve incorporat ion o f molecular oxygen by dioxygenase enzymes to fo rm cw-dihydrodio ls , f o l l owed by dehydrogenation to fo rm dihydroxyla ted intermediates. R i n g f i ss ion o f these d ihydroxyla ted intermediates is catalyzed by dioxygenases, and minera l iza t ion to CO2 and H2O is then sometimes possible. F u n g i are also capable o f P A H metabol i sm (166, 167). Different pathways discovered for the aerobic mic rob ia l ca tabol ism o f P A H s are shown i n F igure 9-3. O-GHucoside . O H Non-enzymatic rearrangement R -Phenol R - 4 O H Dehydrogenase R H -O H r^K NAD+ NADH H as-Dihydrodiol . O H ^ O H Catechol O-Qucuronide O-Sulfate R -C O O H k ^ C O O H cis, cis-Muconic acid 'ortho-fission meta-fission R -X H O C O O H O H 2-Hydroxymuconic semialdehyde Figure 9-3: Typical oxidation pathways for PAHs (adapted from Cerniglia (161)). 164 9.1.7 Metabolism of alkyl PAHs In contrast to the wel l -s tudied mic rob i a l degradation o f parent P A H s , there is very li t t le literature o n the degradation o f alkylated P A H s . Studies have reported that the number o f a l k y l groups present can reduce the rate o f degradation (163), and one study has also shown that the pos i t ion o f the a lky l groups affects degradation rates (168). These studies are supported by data w h i c h shows that f ie ld samples w i t h h is tor ica l hydrocarbon inputs generally have a higher proport ion o f higher a lkylated P A H s over unsubstituted or lesser substituted P A H s than more recent hydrocarbon inputs do (169). The in i t i a l site o f oxygen incorporat ion i n a l k y l P A H s can be either on the aryl r ing , as discussed i n Sec t ion 9.1.6 for non-a lky l P A H s , or o n the a l k y l s ide-chain (170-173) . 9.1.8 Analysis 9.1.8.1 Separation G a s chromatography ( G C ) and h igh performance l i q u i d chromatography ( H P L C ) are the most c o m m o n l y used methods o f P A H separation (148, 174, 175). Ana lyses o f P A H s i n fuel mixtures pose an analyt ical challenge due to the complex i ty o f the mixtures and the relat ively smal l proportions o f P A H s present. Preparative chromatography or fractionation is usual ly performed pr ior to analysis. These methods resolve target compounds w e l l , but sample preparation is t ime-consuming and can be prone to losses. 165 9.1.8.2 Retention Index System Lee et a l (176, 177) have developed a retention index system for the analysis o f P A H s by temperature-programmed gas chromatography. The system is based on a series o f P A H internal standards (naphthalene, phenanthrene, chrysene and picene), and from the retention t imes o f these internal standards, relative retention indices for other P A H s are calculated us ing the formula: I = 100 [ ( t R x - t R z ) / ( t R z + I - t R z ) ] + lOOz Where I is the relative retention index, z and z + 1 are the numbers o f aromatic r ings i n the bracket ing standards, tRX is the retention t ime o f the compound o f interest, and tRZ and tRZ+i are the retention times o f the standards that bracket the e lut ion t ime o f the compound o f interest. Re la t ive retention indices have been assigned to a wide variety o f P A H s (178-180) and are often used to identify P A H s i n mixtures i n the absence o f pure analyt ical standards. S ince there are on ly a l imi ted number o f commerc ia l ly avai lable a l k y l P A H standards, many o f the a lky l P A H s that have been assigned relative retention indices i n this system have so far only been identif ied as C2-naphthalenes, C3-naphtalenes, etc., rather than by their particular posi t ional isomer. 166 9.1.8.3 Detection A variety of detectors have been used for P A H analysis. Flame ionization (FID) and mass spectrometry (MS) detectors are commonly used in conjunction with GC separation (148, 181). Ultra-violet/visible (UV-VIS), photodiode array (PDA), fluorescence and (less frequently) MS detectors have been linked with H P L C sample separation (148). The GC detector of choice is MS, as it not only provides increased sensitivity compared to FID, but it also provides structural information to confirm identification of the analyte. Pure standards for most alkyl PAHs are not commercially available, so MS detection is necessary for the identification of alkyl PAHs in environmental samples of unknown composition. It should be noted that although M S provides information on molecular ions and fragmentation products, which allows determination of the molecular weight and degree of alkylation of PAHs, MS often allows positive identification of an isomer class of PAHs, but does not distinguish between the individual isomers. Some work has been done to distinguish between different P A H isomers by using chemical ionization-mass spectrometry (CI-MS) and tandem-MS techniques that exploit differences in chemical reactivity between parent P A H isomers (182-185). Ion-trap MS detection has the additional analytical advantage that it can be run in selective ion monitoring mode (SIM), which allows the isolation of PAHs of interest from complex environmental samples without the need for the fractionation or preparative chromatography mentioned previously. The principles behind the MS/MS techniques used in this chapter are explained in Chapter 8, Section 8.1. 9.1.9 Diesel fuel contamination in the Arctic Several sites i n the Canadian A r c t i c are contaminated w i t h h igh levels o f diesel fuel. These sites include Cambr idge B a y and Saglek. Pi lot-scale b iopi les were bui l t i n Cambr idge B a y i n Ju ly , 1996 i n an attempt to treat hydrocarbon-contaminated fuel (186), and a land-farming experiment was conducted at Saglek i n order to assess the m i c r o b i a l r emova l o f hydrocarbon contaminat ion (187). B io remedia t ion o f so i l contaminated w i t h P A H s has been discussed (188), but most studies only look at the disappearance o f parent P A H s . A few studies also report the loss o f a l k y l P A H s over t ime (189-192). 9.1.10 Objectives The objectives o f this w o r k were: 1. T o develop M S / M S techniques capable o f investigating the dis t r ibut ion o f a l k y l P A H s i n some hydrocarbon-contaminated A r c t i c soils . 2. T o moni tor the fate o f alkylated P A H s i n jet fuel and A r c t i c soils dur ing hydrocarbon bioremediat ion. 9.2 Experimental 9.2.1 Materials A l k y l P A H standards (1-methylnaphthalene, 2-methyl-naphthalene, 1,4,-dimethylnaphthalene, 1,8-dimethyl-naphthalene, 2,6-dimethyl-naphthalene, 2,3,5-tri-methylnaphthalene, 2-methyl-phenanthrene, 3,6-dimethyl-phenanthrene and 1-methyl-anthracene; 9 5 - 9 9 % purity) were obtained from A l d r i c h and Accus tandard . S o d i u m sulfate (Assurance grade) was obtained from B D H Chemica l s L t d . H P L C grade hexane 168 purchased f rom Fisher Scient i f ic . A m b e r bottles used for so i l storage were obtained f rom I -Chem and were certified free o f organic contaminants. Enr ichment cultures g rown on jet fuel and H a l l B e a c h so i l were developed and suppl ied by G o r d o n Stewart (Department o f M i c r o b i o l o g y and Immunology , U B C ) . 9.2.2 Method development G C w i t h i o n trap M S detection was used for the analysis o f a l k y l P A H s i n jet fuel and so i l extracts. Chromatographic condit ions were established for G C to ensure adequate separation o f the P A H s being analyzed. A n M S / M S procedure capable o f ident ifying a lky l P A H s i n the environmental samples studied was developed. Th i s was done by investigating the fragmentation o f a l k y l P A H s i n the i o n trap by subjecting them to a variety o f both resonant and non-resonant exci ta t ion voltages. The m / z values used to select for a l k y l P A H s i n the i o n trap are give i n Table 9-3. Fragment masses scanned ranged f rom m / z 20 to m / z 220, and the scan t ime was 0.6 seconds. Non-resonant exci tat ion voltages in i t i a l ly tested ranged f rom 0 V to 90 V i n 10 V intervals. Resonant exci ta t ion voltages in i t i a l ly tested ranged f rom 0 V to 3.6 V i n 0.4 V intervals. These exci ta t ion w i n d o w s were subsequently narrowed, and exci ta t ion voltages w i t h i n the revised ranges were tested at intervals o f 1.0 V (non-resonant) and 0.04 V (resonant). Resu l t ing mass spectra were investigated and voltage ranges that gave both a strong molecular i o n signal and distinct fragmentation signals were selected. 169 Table 9-3: M/z values used to select for alkyl PAHs in the ion trap. A l k y l P A H G r o u p m / z value Monomethylnaphthalenes 142 C2-naphthalenes 156 C3-naphthalenes 170 Monomethylphenanthrenes , Monomethylanthracenes 192 C2-phenanthrenes, C2-anthracenes 206 9.2.3 Soil sample collection S o i l samples from the biopi les i n Cambr idge B a y , Nunavu t (69 ° 0 7 ' N , 105 ° 0 7 ' W ) were col lected w i t h a metal corer and placed i n amber glass jars. Surface so i l samples f rom Saglek, Labrador (58 ° 2 3 ' N , 62 ° 35 ' W ) were col lected w i t h a l u m i n u m spatulas and placed i n amber glass bottles. Care was taken to obtain representative samples i n a l l cases. A l l so i l f rom Saglek stored i n a co ld r o o m kept at 4 ° C . S o i l f rom Cambr idge B a y was stored at r oom temperature. 9.2.4 Soil extraction S o d i u m sulfate was added to the so i l (2.0 g sod ium sulfate per 1.0 g soi l ) before extraction i n order to dry it. The sod ium sulfate was m i x e d thoroughly into the so i l by us ing a spatula. Hexane (8 m L ) was added, and the tubes were m i x e d on a vortex m i x e r for 1 minute. Tubes were then sonicated for 30 minutes, and left to stand at r oom temperature for 30 hours. Af ter this t ime per iod, test tubes were centrifuged, and 1 m L hexane was removed and placed i n an autosampler v i a l . 170 9.2.5 Sample analysis The sample (2.5 p L ) was injected by autosampler into a V a r i a n Star 3400 G C , w h i c h was equipped w i t h a 30 m D B 5 - m s c o l u m n (internal diameter 0.15 m m , f i l m thickness 0.25 um). The oven was programmed as fo l lows : the in i t ia l temperature was 50 ° C . F o l l o w i n g injection, the oven was he ld at this temperature for 2 minutes, and then ramped to 280 ° C at a rate o f 5 ° C per minute. The oven was then he ld at 280 ° C for 10 minutes. The injector was he ld at 260 ° C , and the transfer l ine was he ld at 280 ° C . H e l i u m was used as the carrier gas. A V a r i a n Saturn i o n trap M S detector was used. A l l searches for a lkyla ted P A H s were carried out by us ing both M S and M S / M S . The target value used for M S experiments was 30 000, and the target value used for M S / M S experiments was 10 000. The i o n trap temperature was held at 290 ° C . A standard sample conta ining naphthalene, phenanthrene and chrysene was a lways run by M S pr ior to running M S / M S samples i n order to determine at what retention t imes the different a lkylated P A H s w o u l d elute. The mass spectrometer acquis i t ion program was then set up i n order to acquire alkylated naphthalenes i n one w i n d o w , and alkylated phenanthrenes or anthracenes i n the next w i n d o w . Exc i t a t ion voltages used for M S / M S are described i n the results section (8.3.1), and the C I D t ime was 20 ms. Peak integration was always performed o n selected ions rather than on the T I C due to the complex mixture o f hydrocarbons present i n a l l experimental samples. A l k y l P A H s present i n soils or jet fuel were firstly identif ied as substituted naphthalenes or as substituted phenanthrenes or anthracenes by inspect ion o f their mass spectra. M a s s spectra also determined the degree o f substitution. Ind iv idua l isomers were then identif ied wherever possible by compar ison o f relative retention t imes by us ing 171 the Lee retention index. Samples were run with and without naphthalene / phenanthrene / chrysene spikes in order to confirm isomer identities. 9.2.6 Preliminary degradation experiments Experiments were set up in order to determine if alkyl PAHs were degraded during bioremediation treatments of hydrocarbon contamination. Jet fuel was chosen as it has a composition similar to that of many Arctic fuel spills. Hydrocarbon-contaminated soil from Saglek was chosen because preliminary experiments showed a relatively high proportion of alkylated PAHs in the hydrocarbon mixture (when compared with hydrocarbon-contaminated soils collected from other Arctic sites). Phosphate buffer was prepared by adding 22.71 g K 2 H P 0 4 , 8.78 g of K H 2 P 0 4 and 11.04 g of NH4CI to 400mL of deionized water (56). Mineral salts were prepared by adding 3.90g M g S 0 4 , 1.00 g MnSO 4 .H 2 0, 0.20 g FeS0 4 .7H 2 0 and 0.06 g CaCl 2 .2H 2 0 and 1 drop of concentrated H 2 S 0 4 to 200 mL of deionized water (56). Phosphate-buffered mineral salts medium (56) was then prepared by adding 31 mL of the phosphate buffer to 364 mL of deionized water. This solution was autoclaved, after which 1 mL filter-sterilized mineral salts and 0.1 mL vitamins (prepared as described in Chapter 2, Section 2-3) were added. Inoculum that had been developed from jet fuel enrichment cultures was used. Cultures were set up in Balch tubes sealed with Teflon-lined butyl rubber stoppers. Two sets of experiments were set up and run in parallel. The first set contained 4 mg jet fuel and 4 mL phosphate-buffered mineral salts medium, the second set contained 4 g (wet weight) soil collected from Saglek and 0.5 mL phosphate-buffered 172 minera l salts m e d i u m . H a l f the test tubes f rom each set o f experiments were autoclaved, the other h a l f were left unautoclaved. Th i s was done so that results f rom autoclaved and unautoclaved controls and cultures c o u l d be compared, to assess whether the microbes already present i n the so i l were responsible for a significant degree o f degradation. Inocu lum (0.1 m L ) was added to three autoclaved tubes and three unautoclaved tubes i n each set o f experiments. Three autoclaved controls and three unautoclaved controls for each set o f experiments were left uninoculated. Tubes were incubated at 4 ° C , and cultures were frozen at 0 weeks and 4 weeks. Phenanthrene was added as a recovery standard to a l l tubes pr ior to extraction. 9.2.7 Modified degradation experiments for Saglek soil A set o f 18 B a l c h tubes, each containing 4.0 g o f so i l col lected f rom Saglek, was prepared. A minera l salts solut ion was prepared as fo l lows : 1109 m g N H 4 C I , 116 m g N a H 2 P 0 4 and 187 m g N a 2 H P 0 4 were added to l O m L water. 2 0 u L o f 1 0 M N a O H was added to the salts so lu t ion i n order the adjust the final p H o f the H a l l B e a c h so i l to 7.0. Salts solut ion (44 .6uL) was added to each tube. Decach lorob iphenyl was added to a l l tubes as an internal standard. H a l f the tubes were then autoclaved, the other h a l f were left unautoclaved. Inoculum ( 5 0 u L ) was added to 3 autoclaved treatments and 3 unautoclaved treatments. The i n o c u l u m used came f rom enrichment cultures g r o w n o n H a l l B e a c h so i l , and was prepared by spinning 7 0 m L o f culture for 20 m i n at 6000 rpm. The 1.5 m L cel ls col lected were reconstituted i n 3 0 m L o f water. Three autoclaved controls and 3 unautoclaved controls were left uninoculated. The remain ing sets o f tr ipl icate tubes for the autoclaved treatment and the unautoclaved treatment were frozen 173 at t ime zero. Tubes were incubated i n a co ld r o o m kept at 4 ° C , and incubations were stopped after 8 weeks by freezing the samples. 9.3 Results and discussion 9.3.1 Optimization of excitation voltages for MS/MS A series o f mass spectra col lected for 1-methylnaphthalene wi th different exci ta t ion voltages are shown i n F igure 9.4. F igure 9.4 (a) shows a mass spectrum col lected by using a non-resonant excitat ion voltage o f 10V, w h i c h gives the molecular i o n on ly . A mass spectrum col lected by using a resonant excitat ion voltage o f 7 0 V is shown i n F igure 9.4 (b). T h i s spectrum shows fragmentation only , and no molecular i on . T h e mass spectrum col lec ted by using a resonant exci tat ion o f 4 0 V is shown i n F igure 9.4 (c). The mass spectrum shown i n F igure 9.4 (c) gives a good idea o f the molecular i o n as w e l l as dist inct ive a l k y l P A H fragmentation, so a resonant excitat ion voltage o f 4 0 V was chosen for further work . A series o f mass spectra col lected for 3,6-dimethylphenanthrene w i t h different exci ta t ion voltages are shown i n F igure 9.5. A mass spectrum obtained by us ing a 10 V non-resonant excitat ion voltage is shown in F igure 9.5 (a). T h i s mass spectrum shows the molecular ion , w i t h almost no fragmentation. In figure 9.5 (b), a mass spectrum obtained by us ing a 90 V non-resonant excitat ion voltage, a lot o f fragmentation and no molecu la r i o n is seen. The f inal non-resonant exci ta t ion voltage chosen for further w o r k w i t h this compound was 72 V , and F igure 9.5 (c) shows the mass spectrum obtained by us ing a 72 V excitat ion voltage wi th 3,6-dimethylphenanthrene. Exc i t a t ion voltages for a l l compounds were op t imized so that mass spectra showing both a molecular i on and some characteristic fragmentation ( l ike the examples g iven i n Figures 174 a ) 1 0 • V N o n - R e s o n a n t E x c i t a t i o n E n e r g y > Pi 5 0 0 0 0 <u 4 0 0 0 0 | -a 3 0 0 0 0 h 2 0 0 0 0 10 0 0 0 0 1 0 0 1 2 0 1 4 0 m / z b ) 7 0 V N o n - R e s o n a n t E x c i t a t i o n E n e r g y > Pi c > ea Pi 1 8 0 0 0 0 h 1 6 0 0 0 0 1 4 0 0 0 0 | -1 2 0 0 0 0 -1 0 0 0 0 0 h 8 0 0 0 0 6 0 0 0 0 4 0 0 0 0 2 0 0 0 0 0 8 0 1 0 0 1 2 0 1 4 0 m / z c ) 4 0 V N o n - R e s o n a n t E x c i t a t i o n E n e r g y o o o o h 6 0 0 0 0 h 4 0 0 0 0 h 2 0 0 0 0 h 1 2 0 1 4 0 m / z Figure 9-4: Mass spectra obtained for 1-methylnaphthalene by using different non-resonant excitation voltages. 175 a ) 1 0 V N o n - R e s o n a n t E x c i t a t i o n E n e r g y >> 8 0 0 0 0 0 7 0 0 0 0 0 C CD 6 0 0 0 0 0 +-* C 5 0 0 0 0 0 M 4 0 0 0 0 0 CD > 3 0 0 0 0 0 « 2 0 0 0 0 0 CD 1 0 0 0 0 0 0 c CD CD > -4-t CO CD 2 0 0 2 2 0 b ) 9 0 V . N o n - R e s o n a n t E x c i t a t i o n E n e r g y Cfl C CD CD > CD 2 0 0 2 2 0 c ) 7 2 V N o n - R e s o n a n t E x c i t a t i o n E n e r g y 16 0 0 0 0 14 0 0 0 0 12 0 0 0 0 10 0 0 0 0 8 0 0 0 0 6 0 0 0 0 -4 0 0 0 0 -2 0 0 0 0 -0 1 0 0 1 2 0 1 4 0 1 6 0 m / z 1 8 0 2 0 0 2 2 0 Figure 9-5: Mass spectra obtained for 3,6-dimethylphenanthrene by using different non-resonant excitation voltages. 176 9.4 (c) and 9.5 (c)) were produced. Table 9.4 summarizes these results, showing the in i t i a l voltage range selected for further testing for a l l compounds , and then the f inal voltages selected as g i v i n g op t imal fragmentation patterns for each compound . Table 9.4 Excitation voltages selected for compound identification. Non-Resonant Exc i t a t ion Resonant E x c i t a t i o n Init ial Range F i n a l Vol tage Ini t ial Range F i n a l Vo l t age 1 -methylnaphthalene 36-45 40 0.4-0.76 0.44 2-methylnaphthalene 36-45 40 none* 1,4-dimethylnaphthalene 48-57 50 0.04-0.4 0.32 1,8-dimethylnaphthalene 48-57 50 0.2-0.56 0.40 2,6-dimethylnaphthalene 48-57 50 0.04-0.4 0.12 2,3,5 -trimethylnaphthalene 50-59 54 0.04-0.4 0.20 2 -methylphenanthrene 26-35 30 1.2-1.56 1.32 3,6-dimethylphenanthrene 70-79 72 0.2-0.56 0.32 1 -methylanthracene 6-15 10 none* * none of the voltages tested showed suitable fragmentation patterns. Non-resonant voltages were chosen for further experiments as the op t ima l voltage for i somer identif icat ion was consistent between P A H isomers substituted w i t h the same number o f a l k y l groups (e.g. for a l l dimethylnaphthalenes tested, the op t imal non-resonant exci ta t ion voltage was found to be 5 0 V ) . The resonant exci ta t ion voltages were 177 found to be m u c h more variable between P A H isomers wi th the same number o f a l k y l substituents. Because o f these results, resonant excitat ion was not used i n further experiments, as it was decided that resonant excitat ion voltages are too isomer-specif ic to a l l ow detection o f unknown a l k y l P A H isomer peaks i n the soils . 2-Methylphenanthrene and 1-methylanthracene d i d not fragment as expected by us ing non-resonant excitat ion. The loss o f the methyl group was not detected. Instead, on ly m / z 191 and 190 fragments were detected at h igh voltages. A non-resonant voltage that gave a dist inct molecular i on peak w i t h no fragmentation was chosen for further w o r k w i t h these isomers. Resonant exci ta t ion o f 2-methylphenanthrene d i d produce the expected loss o f a methyl group, but for reasons mentioned above, resonant exci ta t ion was not used i n further experiments. 9.3.2 Biopiles The identities, relative retention t imes and reference relative retention t imes for alkylated P A H s found i n the b iop i le so i l are shown i n Table 9-5. These results conf i rm that a lkylated P A H s are present i n this hydrocarbon contamination, and so it is important to k n o w the fate o f these alkylated P A H s during the course o f the b ioremedia t ion treatment. Unfortunately, samples obtained f rom the Cambr idge B a y biopi les dur ing the t ime course o f the b ioremedia t ion experiment had been stored at r oom temperature i n K i n g s t o n pr ior to being sent to U B C , so these soils w o u l d probably not g ive an accurate reflection o f the mic rob i a l degradation that had taken place at l o w temperatures. T o avo id this p rob lem, mic rob i a l degradation experiments us ing jet fuel and hydrocarbon-178 contaminated A r c t i c soils were conducted i n the laboratory instead, so that sample storage and handl ing condit ions cou ld be control led more carefully. Table 9.5 Alkylated PAHs found in soil from Cambridge Bay biopiles. P A H i d Rela t ive Retent ion T i m e Literature Re la t ive Retent ion T i m e 2-Methylnaphthalene 220.77 220 .22 3 1 -Methylnaphthalene 223.58 2 2 3 . 0 1 3 2,6/2,7-Dimethylnaphthalene 240.43 240 .28 3 1,7-Dimethylnaphthalene 242.78 242 .77 a 1,3/1,6-Dimethylnaphthalene 243.33 243 .30 3 2,3/1,4-Dimethylnaphthalene 246.11 246 .03 3 1,2-Dimethylnaphthalene 248.60 248 .50 3 C3-Naphthalene 260.55 N o n e Trimethylnaphthalene 261.66 261 .50 a C3 -Naphthalene 264.73 N o n e C4 -Naphthalene 283.41 N o n e 3 -Methylphenanthrene 318.84 319 .19 3 2-Methylphenanthrene 319.85 319 .93 3 9/4-Methylphenanthrene 322.70 322.81" 1 -Methylphenanthrene 323.55 323 .64 a 2,7-Dimethylphenanthrene 340.31 339.23" a: Vass i la ros et al (177); b: Lee et al (176). 179 9.3.3 Preliminary degradation experiments The identities of the alkylated PAHs found in the jet fuel are given in Table 9-6. Jet fuel that was incubated at 4 °C with a jet fuel enrichment culture showed that none of the alkylated PAHs that had originally been present were detectable after 4 weeks. In contrast, there was no significant decrease in the ratio of alkylated PAHs to internal standard in any of the controls. A chromatogram obtained for jet fuel at time 0, and a chromatogram obtained after 4 weeks of incubation with an enrichment culture are shown in Figure 9-7. The chromatograms obtained for the controls before and after 4 weeks of incubation were very similar to the chromatogram shown for jet fuel at time 0. The distribution of dimethylated naphthalenes in the samples at time 0 and after 4 weeks of incubation with an enrichment culture is shown in Figure 9-8. The results were very similar for all replicates. Hydrocarbon-contaminated Hall Beach soil that was incubated at 4 °C with a jet fuel enrichment culture did not show any evidence for alkyl P A H degradation (or for any hydrocarbon degradation) after 4 weeks of incubation. These cultures had been very wet and consequently not well aerated, so it was postulated that the conditions were not favourable for aerobic microbial degradation. 180 Table 9-6 Alkyl PAHs found in jet fuel. P A H Rela t ive Retent ion T i m e Reference Rela t ive Retent ion T i m e 2-Methylnaphthalene 221.18 220 .22 3 1 -Methylnaphthalene 223.80 2 2 3 . 0 1 a 2-Ethylnaphthalene 238.62 2 3 8 . 5 5 a 2,6 or 2,7-Dimethylnaphthalene 240.66 240 .28 3 1,3 or 1,6-Dimethylnaphthalene 243.08 243 .30 a 2,3 or 1,4-Dimethylnaphthalene 246.43 2 4 6 . 0 3 3 1,2-Dimethylnaphthalene 248.85 248 .50 a C3-Naphthalene 260.72 N o n e 2,3,6-Trimethylnaphthalene 264.92 265 .09 3 3 -Methylphenanthrene 318.93 319 .19 s 2-Methylphenanthrene 319.78 319 .93 3 9 or 4-Methylphenanthrene 322.64 3 2 2 . 8 1 3 1-Methylphenanthrene 323.57 323 .64 a C2-Phenanthrene 336.55 N o n e 2,7-Dimethylphenanthrene 340.19 3 3 9 . 0 1 b 1,9- or 4,9-Dimethylphenanthrene 343.20 344.34" a: Vass i la ros et al (177) b: W i l l i a m s et al (159) 160000 140000 ^ 120000 § 100000 > to CD OH a) 0 w e e k s 250 750 1250 1750 Scan Number 2250 2750 250000 £> 200000 '35 c £ 150000 c .1 100000 n Qi 50000 b) 4 w e e k s 250 750 1250 1750 Scan Number 2250 2750 Figure 9-7: Total ion chromatogram ofjet fuel before and after incubation with a jet fuel enrichment culture. 182 a) 0 weeks 16000 -i 14000 -£ 12000 § 10000 +-» £ > — 8000 -<o v 6000 re / / 4000 -2000 - A 0 - / y \l 1850 1900 1950 2000 2050 2100 2150 2200 Scan Number b) 4 weeks 14000 12000 4 '55 10000 c • £ 8000 > 6000 '•5 © 4000 OH 2000 0 1850 1900 1950 2000 2050 Scan Number 2100 2150 2200 Figure 9-8: Selected ion monitoring of dimethylnaphthalenes present in jet fuel before and after incubation for four weeks with an enrichment culture at 4 °C. 183 9.3.4 Modified degradation experiments for Hal l Beach soil Extens ive degradation o f a lky l P A H s i n H a l l B e a c h so i l was found i n both the autoclaved and the unautoclaved inoculated cultures. N o significant differences i n the ratio o f a l k y l P A H s to decachlorobiphenyl were found for either set o f uninoculated cultures. Table 9-7 lists the a l k y l P A H s found i n the H a l l B e a c h so i l , and the percentages o f a l k y l P A H s remain ing i n the so i l for the different treatments after 8 weeks o f incubat ion are shown i n Figures 9-9 to 9-12. These results clearly show that, under the experimental condi t ions used, a l k y l P A H s are degraded a long w i t h the rest o f the hydrocarbon mixture when an i n o c u l u m enriched on a hydrocarbon source is used. 9.4 Summary A technique i n v o l v i n g non-resonant exci ta t ion o f the parent i o n dur ing M S / M S was developed for the identif icat ion o f a l k y l P A H s i n complex environmental samples. The non-resonant exci tat ion energy used was selected according the parent P A H and the degree o f a lkyla t ion . B y us ing this technique, alkylated P A H s were identif ied i n jet fuel , hydrocarbon-contaminated so i l f rom biopi les i n Cambr idge B a y , and hydrocarbon-contaminated so i l f rom H a l l Beach . Extens ive degradation o f the alkylated P A H s present i n both the jet fuel and the so i l f rom H a l l B e a c h was shown. 184 Table 9-7: Alkyl PAHs found in Hall Beach soil. P A H Rela t ive Retent ion T i m e Literature Rela t ive Retent ion T i m e 2,6/2,7-Dimethylnaphthalene 240.50 240 .28 3 1,3/1,6-Dimethylnaphthalene 243.41 243 .30 3 1,4/2,3-Dimethylnaphthalene 246.17 246 .03 3 C3-Naphthalene 258.43 258 .8 b C3-Naphthalene 261.38 261 .50 3 C3-Naphthalene 262.09 262 .13 3 C3-Naphthalene 263.95 N o n e 2,3,5 -Trimethylnaphthalene 265.89 265 .90 c C3-Naphthalene 270.52 N o n e C4-Naphthalene 275.72 N o n e C4-Naphthalene 276.71 N o n e C4-Naphthalene 280.90 281 .05 b C4-Naphthalene 283.40 283 .40 b C4-Naphthalene 285.96 N o n e 1 -Methylacenaphthene 266.82 266 .57 3 3 -Methylphenanthrene 318.89 319 .19 3 2-Methylphenanthrene 319.87 319 .93 3 9/4-Methylphenanthrene 322.73 322 .81 3 C2-Phenanthrene 330.77 N o n e Dimethylphenanthrene 334.96 336.00" a: Vass i l a ros et al (177) b: W i l l i a m s e^e7 / (159) c: L e e et al (176) 140 -oo c 120 ' c ro 100 -E o 80 cc c 60 cu o J - 40 -<D Q_ 20 -o -J Unautoclaved Control Autoclaved Control Unautoclaved Inoculated Experiment Autoclaved Inoculated Experiment Figure 9-9 Percent of original dimethylnaphthalenes remaining in Hall Beach soil after 8 weeks of incubation with a Hall Beach enrichment culture at 4 °C. (Mean ± SD, n=3). 120 OJO c 100 'c 'ro 80 E o 60 cc c 40 O) u CL 20 1 Unautoclaved Autoclaved Unautoclaved Autoclaved Control Control Inoculated Inoculated Experiment Experiment Figure 9-10 Percent of original Cynaphthalenes remaining in Hall Beach soil after 8 weeks of incubation with a Hall Beach enrichment culture at 4 °C. (Mean ± SD, n=3). 186 120 100 c c 80 'ra E 60 a> CC H—• 40 -c CU u 20 -i-a> 0 --20 - Unautoclaved Autoclaved Unautoclaved Autoclaved Control Control Inoculated Inoculated Experiment Experiment Figure 9-11 Percent of original monomethylphenanthrenes remaining in Hall Beach soil after 8 weeks of incubation with a Hall Beach enrichment culture. (Mean ± SD, n=3). 120 c "E 'ra E cu al *J c CD O i_ 0) CL 100 80 60 40 20 0 _ 2 0 J Unautoclaved Autoclaved Unautoclaved Autoclaved Control Control Inoculated Inoculated Experiment Experiment Figure 9-12 Percent of original C2-phenanthrenes remaining in Hall Beach soil after 8 weeks of incubation with a Hall Beach enrichment culture. (Mean ± SD, n=3). 187 Chapter 10 Conclusions and Future Work The m a i n objective o f the work reported i n this thesis was to investigate the anaerobic m i c r o b i a l dechlor inat ion o f P C B s . The dechlor inat ion o f h igh ly chlor inated P C B s i n soi ls f rom the Canadian A r c t i c was investigated, and dechlor inat ion pathways o f h ighly-chlor ina ted P C B congeners were determined. The aerobic m i c r o b i a l degradation o f a l k y l P A H s i n A r c t i c soils that contain hydrocarbon contaminat ion was also investigated. * In order to investigate P C B dechlorinat ion, effective extraction and analysis techniques were required. In this thesis, a few different solvent extract ion procedures were tested, and a procedure that extracts s o i l by us ing acetone and hexane was s h o w n to be effective. A method for the efficient separation o f P C B congeners o f interest by us ing temperature-programmed G C was developed, and appropriate M S procedures for compound detection were chosen. The P C B content o f three sediment cores taken from lakes i n Cambr idge B a y (Nunavut) was analyzed, and P C B s were found i n two o f the three sediment cores. A l t h o u g h the P C B concentration and congener distr ibution var ied w i t h sediment depth, no evidence for in situ m ic rob i a l dechlor inat ion o f P C B s was found. A variety o f approaches to the development o f mic rob ia l enrichment cultures were tested, and several enrichment cultures capable o f dechlorinat ing P C B s were developed. The enrichment cultures that in i t i a l ly showed the most dechlor inat ion act ivi ty used 4-bromobenzoate as an amendment to stimulate dehalogenation act ivi ty, and sediment col lected from Esqu ima l t Harbour (Br i t i sh C o l u m b i a ) as an i n o c u l u m source. 188 It was important to establish i f the enrichment cultures developed i n this w o r k are capable o f dechlor inat ing the P C B s present in A r c t i c soi ls , as a substantial por t ion o f this w o r k is part o f a larger project a imed at the bioremediat ion o f P C B - c o n t a m i n a t e d Saglek and Reso lu t ion Island soils . Four o f the mic rob i a l enrichment cultures were chosen to investigate dechlor inat ion o f A r o c l o r 1260 in A r c t i c soils . Extens ive dechlor inat ion o f h ighly-chlor ina ted P C B congeners was achieved i n both Reso lu t ion Island so i l and Saglek so i l by us ing the enrichment cultures. It was found that dechlor inat ion o f P C B s that had been present i n Reso lu t ion Island so i l for a number o f years (i.e. weathered P C B s ) was not quite as extensive as dechlor inat ion o f P C B s that were freshly added to Reso lu t ion Island so i l (i.e. unweathered P C B s ) . P C B dechlor inat ion was accompanied by the accumula t ion o f less-highly chlorinated P C B congeners, and the most abundant products f rom A r o c l o r 1260 dechlor inat ion were tetrachlorobiphenyls. A pre- incubat ion step (to achieve methanogenic condit ions) was inc luded i n the treatments. T h i s is the first report showing dechlor inat ion o f P C B s i n soils that had been aerobic pr ior to treatment. In order to make the dechlor inat ion process as efficient as possible , some factors that cou ld affect the extent and rate o f P C B dechlor inat ion were investigated. It was found that autoclaving pre-incubated cultures pr ior to inocula t ion w i t h P C B enrichment cultures increased the rate and extent o f P C B dechlorinat ion. A variety o f incubat ion temperatures were also tested, and it was found that the most rapid and extensive P C B dechlor inat ion by us ing the enrichment cultures developed i n the present w o r k occurred at 30 ° C . 189 One o f the most abundant A r o c l o r 1260 dechlor inat ion products gave a G C peak that c o u l d correspond either to 24-24- te t raCB, to 245-2- te t raCB, or to both these te t raCBs. A n M S / M S method that can dis t inguish between these two isomers was developed, and the dechlor inat ion product was found to be 24-24-tetrachlorobiphenyl . The dechlor inat ion o f three nona- and four octa-chlorobiphenyl congeners, both s ingly , and i n var ious combinat ions, was also investigated. It was established that the dominant dechlor inat ion pathway involves the removal o f a l l meta chlorines, a l though some para and some ortho dechlor inat ion was also observed. The experiments that investigated the dechlor inat ion o f various mixtures o f octa- and n o n a C B s showed that the rates and products o f P C B dechlor inat ion can vary, depending on the mixture o f congeners present. Th i s is the first t ime that dechlor inat ion o f single octa- and nona-chorobiphenyl congeners has been reported, and also the first t ime that the dechlor inat ion o f var ious combina t ions o f these congeners has been investigated. Further experiments i n v o l v i n g combinat ions o f tetra- and pen t a -CB congeners i n combina t ion w i t h each other and also i n combina t ion w i t h more-h ighly chlorinated congeners showed that extensive degradation o f penta- and te t ra-CBs to b iphenyl can occur, even when other, more-h ighly chlorinated congeners are present. In summary, the w o r k on P C B dechlor inat ion presented i n this thesis has shown that h ighly-chlor ina ted P C B s i n soils f rom Reso lu t ion Island and Saglek can be successfully dechlorinated to a mixture o f P C B s w i t h a m u c h lower degree o f ch lor ina t ion (predominantly te t raCBs) . It was also established that the dominant dechlor inat ion pathways invo lve the removal o f a l l meta chlorines, but para and ortho 190 dechlor inat ion was also found, and complete dechlor inat ion o f P C B congeners to b iphenyl was also shown. Further Research: The w o r k described i n this thesis has led to several different projects w h i c h are already underway. The enrichment cultures that were developed i n this w o r k are n o w being used for the treatment o f PCB-con t amina t ed so i l i n pi lot-scale bioreactors and field experiments at Saglek. Th i s w o r k is being carr ied out by researchers associated w i t h the R o y a l M i l i t a r y Co l l ege i n K i n g s t o n . Sequential anaerobic/aerobic treatment experiments o f PCB-con tamina t ed so i l are be ing carr ied out i n col labora t ion w i t h E m m a Master (Department o f M i c r o b i o l o g y and Immunology , U B C ) . The essential requirements for successful P C B dechlor inat ion (e.g. m e d i u m compos i t ion , addi t ion o f anaerobic so i l f rom Jericho) are being investigated i n more detail by V i v i a n L a i (Department o f Chemis t ry , U B C ) , i n order to s impl i fy eventual bioreactor requirements as m u c h as possible. The effect o f a range o f P C B concentrations o n P C B dechlorinat ion, and the use o f lower incubat ion temperatures (down to 7 ° C ) is also being investigated. The results f rom experiments that used different combinat ions o f P C B congeners showed that dechlor inat ion is affected by congener structures and combinat ions. Further w o r k investigating the dechlor inat ion o f more combinat ions o f congeners is suggested, i n order to conf i rm the interactions suggested by the present work . In the Chapter o n a l k y l P A H s , M S / M S techniques that identify a l k y l P A H s i n complex hydrocarbon mixtures were developed. These techniques were then used to 191 identify a l k y l P A H s present i n a jet fuel mixture , i n b iopi les from Cambr idge B a y , and i n hydrocarbon-contaminated soi l f rom H a l l Beach . The mic rob i a l degradation o f a l k y l P A H s i n jet fuel and i n H a l l B e a c h so i l was investigated, and it was shown that a l k y l P A H s were degraded a long w i t h the rest o f the hydrocarbon mixture . It was important to establish that the a l k y l P A H s are degraded, as a l k y l P A H s are generally more resistant to degradation than most other hydrocarbons present i n fuel spi l ls . It w o u l d be interesting for further w o r k to moni tor this degradation more c losely , for example to look at the concentrations o f a l k y l P A H s i nvo lved , and to sample more frequent t ime points i n order to determine i f the different a l k y l P A H s degrade at different rates. Ideally, samples f rom actual f i e ld experiments should be investigated. 192 References (1) Pe r lman , H . L . "Accelerated Decision", Before the Administrator U.S. E.P.A., Washington, D.C., Re: Dow Chemical Company, Respondent. Docket No. TSCA (16(a))-l September 22, 1980. 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Chemistry
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Microbial degradation of polychlorinated biphenyls and alkylated polycyclic aromatic hydrocarbons
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