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Polycyclic aromatic hydrocarbons in still creek sediments : distributions, concentrations and possible… Morton, Teresa Anne 1983

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POLYCYCLIC AROMATIC HYDROCARBONS IN STILL CREEK SEDIMENTS: DISTRIBUTIONS, CONCENTRATIONS AND POSSIBLE SOURCES By TERESA ANNE MORTON B.Sc, The University of B r i t i s h Columbia, 197 7 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF CHEMISTRY We accept t h i s thesis as conforming to i. the required standards THE UNIVERSITY OF BRITISH COLUMBIA October 1983 © Teresa Anne Morton, 1983 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the 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 , I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l no t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department o f Chemistry The U n i v e r s i t y o f B r i t i s h Co lumbia 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3 Date September 1983 DE-6 (3/81) ABSTRACT The extent of p o l y c y c l i c aromatic hydrocarbon (PAH) contamination was determined i n the stream sediments of an urban watershed, and some possible sources were investigated. A n a l y t i c a l methodology was adapted from the l i t e r a t u r e f o r deter-mination of p o l y c y c l i c aromatic hydrocarbons i n sediments. A l k a l i n e digestion of samples was followed by solvent-solvent p a r t i t i o n and F l o r i s i l chromatography. An alumina column clean-up effected p r e l i m i -nary separation of compounds before determination using high pressure l i q u i d chromatography (HPLC). Some extracts were also analyzed on a c a p i l l a r y gas chromatography system and by c a p i l l a r y gas chromatography -mass spectrometry (GCMS) to provide addi t i o n a l q u a l i t a t i v e informa-t i o n as a r e s u l t of the much improved re s o l u t i o n . Samples of stream sediments were obtained from S t i l l Creek i n the Brunette River watershed. Analysis f o r PAH i n sediments from f i v e sampling s i t e s (sampled two consecutive years) revealed that a l l sed i -ments contained a wide range of PAH. While the p r o f i l e of the extracts (by HPLC) was remarkably s i m i l a r from s i t e to s i t e , and from the f i r s t to second year, the amounts of PAH present varied by two orders of magnitude among s i t e s . Levels of i n d i v i d u a l compounds quantitated ranged from mid part per b i l l i o n (55 ppb) to mid part per m i l l i o n (38.5 ppm) . PAH i d e n t i f i e d by HPLC analysis (eleven compounds) were mainly non-alkylated parent compounds, but GCMS data indicated that many alk y l - s u b s t i t u t e d or heteroatom p o l y c y c l i c s were also present. Preliminary investigations of PAH l e v e l s found i n oligochaetes - i i i -indicated bioconcentrations of some compounds, and suggested a possible competition for PAH among high organic content sediments and the l i v e oligochaetes present. Investigation of possible sources of p o l y c y c l i c s aromatic hydro-carbons to stream sediments included street surface contaminants and crankcase o i l . Street sediments exhibited HPLC e l u t i o n p r o f i l e s s t r i k -ingly s i m i l a r to those of stream sediments. Levels of PAH found were s l i g h t l y lower than, but comparable to, concentrations i n stream sediments, and the same i n d i v i d u a l compounds were quantitated i n each type of sample. Thus the str e e t sediments were determined to be an important source of PAH to stream sediments, with transport occurring through urban street runoff. Crankcase o i l demonstrated a l i n e a r buildup of PAH with mileage over approximately the f i r s t 2000 km, then a l e v e l i n g o f f occurred. Based on HPLC p r o f i l e s , used crankcase o i l contributes to str e e t surface contamination. Regression analysis of t o t a l PAH l e v e l s i n various samples f a i l e d to provide s i g n i f i c a n t c o r r e l a t i o n s with l o c a t i o n of sampling point (on stream), t r a f f i c volume at sampling point, land use ( f o r st r e e t sediment samples) or sediment c h a r a c t e r i s t i c s . However, sediment c h a r a c t e r i s t i c s ( p a r t i c l e s i z e , organic content) were i d e n t i f i e d as being the most important of the factors considered. Implications o f the presence of a PAH burden i n the stream sedi-ments (comparable to other older, larger urban watersheds) were discussed with r e l a t i o n to natural systems. Recommendations were made for study to further elucidate PAH sources and transport to stream sediments. Research Supervisor - i v -ACKNOWLEDGEMENTS The author would l i k e to express gratitude to Dr. K.J. Ha l l f o r h i s guidance and support i n the completion of t h i s t h e s i s . Gratitude i s due to many people who contributed time and e f f o r t , and advice towards t h i s t h e s i s work. Thanks are expressed to Dr. J.P. Kutney and to Dr. G.S. Bates for the generous use of t h e i r lab f a c i l i -t i e s . Dr. B. Dunn (Cancer Research) i s thanked for donating a n a l y t i c a l standards and providing advice. C o l l e c t i o n of crankcase samples was kindly c a r r i e d out by G. Morton, R. Morton, V. Williams and D. Davies. Thanks are expressed to the following for assistance i n analysis; Dr. B.R. Worth and R. Carlson (HPLC assistance), T. Ma (GCMS a n a l y s i s ) , and V. Gujral ( c a p i l l a r y GC a n a l y s i s ) . Special acknowledgement i s due to D.V. Williams f o r h i s patience and understanding, which contributed greatly to the success of t h i s t h e s i s . F i n a n c i a l support was provided by an NSERC grant to Dr. H a l l to study pollutants from non-point sources. - v -TABLE OF CONTENTS Page Abstract i i Acknowledgements i v Table of Contents v L i s t of Figures v i i L i s t of Tables x i Chapter I - Introduction, Purpose and Scope of Thesis 1 A. Introduction 1 B. Purpose and Scope of Thesis 2 Chapter II - Li t e r a t u r e Review 6 A. Physical and Chemical Properties of PAH 6 B. PAH i n the Environment 7 1. Sources to the Environment 7 2. PAH i n Air and Water 9 3. PAH i n Food 17 C. PAH i n Sediments 21 1. Determination of PAH Sources 21 2. Natural PAH Levels 23 3. Point Sources 28 4. Non-Point Sources 29 5. Summary 36 D. Assessment of PAH Exposure 37 E. Analysis of PAH 43 1. Introduction 43 2. Ex t r a c t i o n Procedures 43 3. P a r t i t i o n and Clean-up .45 4. High Pressure Liquid Chromatography (HPLC) . . . . 47 5. Gas Chromatography (GC) 56 6. Gas Chromatography Mass Spectrometry (GCMS). . . . 62 Chapter III - Experimental Techniques 66 A. Outline of A n a l y t i c a l System 66 1. Scope 66 2. A n a l y t i c a l Scheme 66 3. Instrumental Analysis 67 4. Data Analysis 68 B. Sampling 68 1. Sediments 68 2. Street Surface Contaminants 68 3. Oligochaetes 68 4. Crankcase O i l 71 - v i -Page C. General Procedures 71 1. Glassware 71 2. Solvents 71 3. Other Chemicals and Supplies 72 4. Determination of P a r t i c l e Size and Organic Content 72 5. Standards 73 D. Extraction and Clean-up Procedures 73 1. Sediments and Street Surface Contaminants 73 2. Oligochaetes 77 3. Crankcase O i l 77 E. Recovery Studies 77 F. Instrumental Analysis 79 1. Liquid S c i n t i l l a t i o n Counting 79 2. HPLC 79 3. UV Spectrophotometry 79 4. GC 81 5. GCMSDS 81 G. S t a t i s t i c s Treatment 82 Chapter IV - Results and Discussion 84 A. Development of A n a l y t i c a l Scheme 84 1. Introduction 84 2. Choice of Target Standards 84 3. Choice of A n a l y t i c a l Method 85 4. Development of LC System 86 5. Recoveries 90 6. Sampling Rationale 93 7. Comments on A n a l y t i c a l Methodology 96 B. Analysis of Stream Sediments 98 1. Sediment Ch a r a c t e r i s t i c s 98 2. HPLC Analysis 99 3. C a p i l l a r y GC Analysis 113 4. GCMS Analysis 127 5. S t a t i s t i c s Treatment 142 6. Summary 145 C. Street Sediments 152 1. Sediment C h a r a c t e r i s t i c s 152 2. HPLC Analysis 155 3. S t a t i s t i c s Treatment 155 4. Discussion 165 D. Oligochaetes 167 E. Crankcase O i l 169 F. Summary of Results: Sources and Fates of PAH 175 Chapter V - Summation, Implications and Recommendations 178 A. Summation 178 B. Implications 180 C. Recommendations 180 References 182 - v i i -LIST OF FIGURES Page Figure 1-1 Possible Pathways for Movement of PAH i n an 3 Urban Watershed 1-2 Relationships Investigated i n this Thesis 5 3-1 Sampling Locations 69 3-2 Analysis Scheme 3-3 Alumina Separation of P o l y c y c l i c Extract 74 3- 4 Aliquots Taken for Liquid S c i n t i l l a t i o n Counting 76 4- 1 L i n e a r i t y of UV Detector, 280 nm 78 4-2 Naphthalene Recoveries, 3 T r i a l s 89 4-3 B(a)P Recoveries - No Alumina Preparation 92 Procedures, 3 T r i a l s 4-4 B(a)P Recoveries - Alumina Deactivation Problems, 92 3 T r i a l s 4-5 P a r t i c l e Size D i s t r i b u t i o n and Organic Matter, 94 D(S)('78) and D(N)('79) 4-6 P a r t i c l e Size D i s t r i b u t i o n and Organic Matter, 101 D(N)('78) and D(N)('79) 4-7 P a r t i c l e Size D i s t r i b u t i o n and Organic Matter, 101 L('78) and L('79) 4-8 P a r t i c l e Size D i s t r i b u t i o n and Organic Matter, 102 W('78) and L('79) 4-9 P a r t i c l e Size D i s t r i b u t i o n and Organic Matter, 102 G('78) and G('79) 4-10 % Organic Matter (0M) vs. % S i l t and Clay, (d) 103 4-11 Chromatogram of Douglas (N) Extract 106 (a) ('78) I I I (b) ('79) I I I 4-12 Chromatogram of Willingdon Extract 107 (a) ('78) I I I (b) ('79) III - V l l l -Page 4-13 Chromatogram of Douglas (N) Extract 108 (a) ('78) IV (b) ('79) IV 4-14 Chromatogram of Lougheed Extract 109 (a) ('78) IV (b) ('79) IV 4-15 Chromatogram of Douglas (N) Extract 110 (a) ('78) V (b) ('79) V 4-16 Chromatogram of Lougheed Extract 111 (a) ('78) V (b) ('79) V 4-17 Lougheed Extract at Two Mobile Phase Concentra- 112 tions (a) 60%, L('79) I I I (b) 70%, L('79) III 4-18 C a p i l l a r y GC Chromatogram, D(S)('79) 121 (a) III 2 y l (b) I I I 0.2yl (c) IV (d) V 4-19 C a p i l l a r y GC Chromatogram, D(S)(*78) 123 (a) I I I (b) IV (c) V 4-20 C a p i l l a r y GC Chromatogram, W('78) 125 4-21 GCMS of D(S)('78) III 133 (a) III (b) IV (c) V f II (a) TI chromatogram (b) TI chromatogram, scan #1080 to #1440 (c) spectrum, scan #1406 (d) TI chromatogram, scan #1800 to #2060 (e) spectrum, scan #1818 GCMS of D(S)('79) III (a) TI chromatogram (b) screen f o r masses 17 8, 202, 228, 166, 216 and 279 4-22 f II 136 - i x -Page 4-23 GCMS of D(S)('79) IV 138 (a) TI chromatogram (b) TI chromatogram, scan #920 to #1160 (c) spectrum, scan #955 (d) TI chromatogram, scan #1200 to #1440 (e) spectrum, scan #1308 4-24 GCMS of D(S)('79) V 141 (a) TI chromatogram 4-25 D i s t r i b u t i o n of Levels of Individual PAH In 146 Sediments 4-26 D i s t r i b u t i o n of Levels of Equivalent B(a)P i n 146 Sediments 4-27 P a r t i c l e Size D i s t r i b u t i o n and Organic Matter, 153 C _ and C 2 4-28 P a r t i c l e Size D i s t r i b u t i o n and Organic Matter, 153 Gr_ and Gr 2 4-29 P a r t i c l e Size D i s t r i b u t i o n and Organic Matter, 154 1^ and I 5 4-30 P a r t i c l e Size D i s t r i b u t i o n and Organic Matter, 154 R _ and R 2 4-31 HPLC Analysis of Street Sediment R 2 157 (a) I I I (b) IV (c) V 4-32 HPLC Analysis of C 2 159 (a) I I I (b) IV (c) V 4-33 HPLC Analysis of 1^ 161 (a) I I I (b) IV (c) V 4-34 HPLC Analysis of Gr 2 163 (a) H I (b) IV (c) V - x -4-35 Crankcase O i l Sample DD (a) accumulated distance 874 km (b) accumulated distance 1665 km (c) accumulated distance 2623 km 4-36 Crankcase O i l Sample RM (a) accumulated distance 895 km (b) accumulated distance 1943 km (c) accumulated distance 2545 km 4-37 Eq B(a)P vs accumulated distance - DD 4-38 Eq B(a)P vs accumulated distance - RM - x i -LIST OF TABLES Page Table 2-1 PAH i n Petroleum and Petroleum Products 8 2-2 PAH Levels i n Air 10 2-3 PAH i n Water 11 2-4 PAH i n Aquatic Organisms 13 2-5 PAH i n Mussels 15 2-6 PAH i n Various Plant Products (yg/kg) 18 2-7 PAH i n F i e l d Crops Grown i n Controlled Climate, 18 Greenhouse or Open F i e l d s (yg/kg) 2-8 PAH i n Smoked Foods (yg/kg) 20 2-9 PAH i n Sediments (yg/kg) 24 2-10 PAH i n Sediments, Boston as a Point Source 33 2-11 Estimation of B(a)P Intake (per person/year) 38 i n Hungary 2-12 Estimates of B(a)P Intake, From an Occupational 40 Viewpoint 2-13 PAH Levels i n Foods Used to Calculate Human 41 Exposure Due to Food Consumption 2-14 Estimates of Allowable Daily Intake 42 2-15 Column Chromatography Systems 46 2-16 HPLC Systems Used for PAH Analysis 49 2-17 Column Packings Used i n GC Analysis of PAH 57 2-18 C a p i l l a r y Columns Used for PAH Analysis 60 2- 19 Examples of GCMS Systems Used for PAH Analysis 63 3- 1 Sampling Locations 70 3-2 HPLC Conditions 80 3- 3 C a p i l l a r y GC Conditions 80 4- 1 V a r i a t i o n i n Retention Time and Peak Area 87 - x i i -Page 4-2 Limit of Detection for PAH Standards with 91 UV Detector 4-3 El u t i o n Pattern for B(a)P From Alumina 95 4-4 P a r t i c l e Size Ranges 100 4-5 PAH Quantitated i n Stream Sediments 105 4-6 Comparison of Conditions for Determination of 114 PAH Retention Indices 4-7 Retention Indices for Target PAH 116 4-8 Tentative I d e n t i f i c a t i o n of Peaks by Retention 117 Index ( I ) , D(S)('79) 4-9 Tentative I d e n t i f i c a t i o n of Peaks by Retention 118 Index ( I ) , D(S)('78) 4-10 Tentative I d e n t i f i c a t i o n of Peaks by Retention 119 Index ( I ) , D(S)('78) 4-11 Scope of GCMS Analysis 128 4-12 Results of GCMS Analysis - D(S)(*78) III 130 4-13 Results of GCMS Analysis - D(S)('79) 131 4-14 Values of Variables Examined i n Regression 144 Analysis; "eq B(a)P", "km", and " t r a v o l " 4-15 PAH Levels and Equivalent B(a)P for Street 156 Sediments 1. CHAPTER I INTRODUCTION, PURPOSE AND SCOPE OF THESIS A. INTRODUCTION Throughout t h i s t h e s i s " p o l y c y c l i c aromatic hydrocarbons" r e f e r s to compounds of two or more si x membered aromatic rings, and i s abbre-viated PAH, or shortened as p o l y c y c l i c s . Abbreviations f o r the many pol y c y c l i c s are contained, along with t h e i r structures, i n Appendix 1. P o l y c y c l i c s are of concern due to t h e i r usefulness as i n d i c a t o r s of wide ranging contamination of environments. Many of the PAH are carcinogens, varying widely i n t h e i r carcinogenic strength (Appendix 1), and may act i n combination. Alkylated or hetero-substituted poly-c y c l i c s are found i n conjunction with non-substituted PAH and have potential to be even stronger carcinogens. Sources of p o l y c y c l i c s to the environment include natural inputs ( o i l seeps, microbial transformation and perhaps forest f i r e s ) and the far greater anthropogenic sources ( o i l s p i l l s , combustion of f u e l s , production of coke). Emissions of benzo(a)pyrene (B(a)P), a potent carcinogen, were estimated worldwide at over 5000 tons per year (Suess, 1976). P o l y c y c l i c s can be deposited i n aquatic sediments through sedimen-tation of atmospheric p a r t i c u l a t e s , runoff from catchment areas, and ( i n urban watersheds) runoff from streets and curbs. Sediments near urbanized areas are p o t e n t i a l l y the "sink" for PAH r e s u l t i n g from f u e l combustion (heating, power generation, vehicular exhaust), used l u b r i -cating o i l s , asphalt wear p a r t i c u l a t e s , or t i r e wear products, e i t h e r • .2. d i r e c t l y or r e s u l t i n g from street run-off (see Figure 1-1). Due to the potential of stream sediments for acting as the contam-inant "sink" for a watershed, and for providing an averaging of poly-c y c l i c input, l e v e l s and d i s t r i b u t i o n s of PAH in sediments can provide useful information on source and importance of inputs. The build-up of carcinogenic compounds in aquatic systems ( i n c l u d -ing sediments) i s being studied due to the possible detrimental e f f e c t s on aquatic ecosystems (bacteria, benthic invertebrates, f i s h , and species dependent on f i s h ) . These e f f e c t s d i r e c t l y impact on human systems (exposure to carcinogens through ingestion or skin contact) and i n d i r e c t l y by threatening the d i v e r s i t y and strength of natural systems providing food. In a study by H a l l _et al. (1976) the water q u a l i t y of an urban t r i b u t a r y (Brunette River System) was investigated i n order to assess i t s c o n t r i b u t i o n to water q u a l i t y i n the lower Fraser River. Concen-tratio n s and d i s t r i b u t i o n s of trace metals and of selected organo-ch l o r i n e compounds were determined throughout the Brunette watershed. The researchers u t i l i z e d analysis of sediments (lake, r i v e r and stream), s t r e e t surface deposits, and storm waters to elucidate inputs to the Brunette system. Impact of various land uses on l e v e l s of contaminants was examined for s t r e e t deposits and stream sediments. B. PURPOSE AND SCOPE OF THESIS The objectives of t h i s t hesis s h a l l be to: 1) adapt or develop a n a l y t i c a l methodology appropriate for the type of sample encountered; 2) determine the extent and nature of the PAH contamination i n , Figure 1-1. Possible pathways for movement of PAH i n an urban watershed 4. p r i m a r i l y , selected S t i l l Creek sediments, (and associated benthic organisms); 3) examine PAH contamination i n str e e t sediments and crankcase o i l to investigate potential sources of p o l y c y c l i c s to S t i l l Creek stream sediments, and (by in v e s t i g a t i n g physical c h a r a c t e r i s t i c s of sed i -ments) attempt to i d e n t i f y environmental factors which regulate PAH d i s t r i b u t i o n . The study w i l l be designed to complement the work by Hall et a l . i n the Brunette watershed but s h a l l be r e s t r i c t e d to the S t i l l Creek section. Relationships investigated are depicted i n Figure 1-2. 5. Figure 1-2. Relationships investigated i n t h i s t h e s i s . 6. CHAPTER II LITERATURE REVIEW A. PHYSICAL AND CHEMICAL PROPERTIES OF PAH P o l y c y c l i c aromatic hydrocarbons (PAH) are organic compounds con-taining two or more fused benzene rings with two or more carbon atoms being common to two or more r i n g s . Non-aromatic rings may be included i n the structure, for example fluorene, and there may be substituted groups ( a l k y l , n i t r o , hydroxyl) on one or more r i n g s . Appendix 1 l i s t s some of the major p o l y c y c l i c s , including t h e i r structures and carcino-genic p o t e n t i a l s . Rules f o r naming these compounds have been adopted by IUPAC and are contained i n The Ring Index (Patterson et_ al., 1960). A summary of these naming and numbering rules are given i n Appendix 1. Abbreviations for PAH names which are used i n t h i s thesis are also included i n Appendix 1. In many references to PAH i n the l i t e r a t u r e , benzo(a)pyrene (B(a)P) i s given as representative of the PAH c l a s s . B(a)P's h i s t o r y as the f i r s t i s o l a t e d p o l y c y c l i c carcinogen and i t s strength as a carcinogen have resulted i n many researchers concentrat-ing t h e i r analysis on t h i s one compound. In quantitative terms, how-ever, B(a)P i s often a very minor component of the p o l y c y c l i c f r a c t i o n i n environmental samples. Physical properties of various PAH are tabulated i n Appendix 2 (melting and b o i l i n g points and molecular weights). The aromatic character of these fused rings r e s u l t s i n r e l a t i v e l y stable compounds (and corresponding extended l i f e t i m e s i n some environmental systems). PAH tend to undergo s u b s t i t u t i o n rather than addition. It i s suggested that addition i n the 'K' region (9, 10 bond of phenanthrene) may be 7. important i n carcinogenesis. For a complete review of p o l y c y c l i c hydrocarbon chemistry, see Clar (1964). For l a t e r work see Bjifrseth and Dennis (1980). PAH undergo photo-oxidation i n c e r t a i n atmospheric conditions, r e s u l t i n g i n potential interactions with urban a i r po l l u t a n t s (reviewed i n N.A.S., 1972). B. PAH IN THE ENVIRONMENT 1. Sources to the Environment The load of PAH i n the environment includes material from both natural and anthropogenic sources. Petroleum products and coal contain p o l y c y c l i c s , sometimes to substantial l e v e l s (see Table 2-1). The le v e l s of B(a)P and of t o t a l PAH vary considerably from crude o i l s to highly refi n e d o i l s (Hermann et a l . , 1980; Tomkins et al., 1980). Even automobile fuels contain widely d i f f e r e n t amounts of B(a)P and aromatics (Newhall et a l . , 1973; Zaghini et a l . , 1973; Pederson et a l . , 1980). In general, products having higher average molecular weights contain higher PAH l e v e l s . Additional natural sources of PAH include forest f i r e s (Youngblood and Blumer, 1975), and perhaps volcanic action. The most s i g n i f i c a n t mechanisms for introducing PAH into the environment are the combustion of f o s s i l f u e l s , production of coke, and the c a t a l y t i c cracking of petroleum products. Combustion products from heat and power generation are then deposited i n aquatic or atmospheric systems. Suess (1976) estimated the emissions of B(a)P to the atmo-sphere, based on 1966-1969 data, as a staggering t o t a l emission of 5044 tons per year. On a world-wide basis, heating and power generation accounted for 52% of the B(a)P load while i n d u s t r i a l processes account Table 2-1. PAH In petroleum and petroleum products. Material Aromatic Content B(a)P(ug/kg) PAH(pg/g) Reference crude o i l , from coal tar d i s t i l l a t i o n 2x10 5 -700 Hermann et a l . , 1980 petroleum d i s t i l l a t e A B 840 <10 78 54 crankcase o i l new old < 5 6x10 ** nd 15 white o i l , medicinal < 5 0.64 crude o i l , from shale 2.5X101* Tomkins et a l . , 1980 crude o i l 2.6xl0 3 d i e s e l f u e l , from shale 38 unleaded f u e l A B 25 38 % nd 800 Newhall et a l . , 1973 unleaded f u e l A B C 48 48 48 (c9-c10) (benzene) 70 900 nd Zaghini et a l . , 1973 leaded f u e l A B 40 40 40 (mixture) (benzene) (0-xylene) 120 4 1 Pedersen et a l . , 1980 nd = not detected 9 . for 21%. Vehicular emissions, although important i n urban areas, only contribute about 1% to the estimated t o t a l . 2. PAH i n A i r and Water Numerous studies have been c a r r i e d out to determine the extent of PAH contamination i n the environment. Results of a few of the more recent studies are summarized i n Tables 2-2 and 2-3. In t h e i r study of aerosols over Lake Michigan, Strand and Andren (1980) sampled on 12 cruises over 26 months. By examining the si z e of the aerosol p a r t i c u l a t e s i t was determined that the major PAH source i s anthropogenic combustion. These p a r t i c l e s are deposited i n the water micro-layer (top 300 Um), awaiting eventual adsorption and sedimenta-t i o n i n the Lake. Greenburg (1980) sampled a i r over urban New Jersey and the PAH found i n a sample from Newark are l i s t e d i n Table 2-2. The PAH l e v e l s were found to vary throughout the year, being lower i n summer. This may be due to greater v o l a t i l i z a t i o n , more photochemical decomposition of PAH and smaller amounts of f u e l used i n r e s i d e n t i a l heating. The presence of cyclopenta(c,d)pyrene i s of note due to i t s mutagenic properties and r e l a t i v e l y high concentrations i n carbon black (Neal and T r i e f f , 1972; Wallcave et _al., 1975). The concentrations of PAH i n drinking water are generally low (ng/1) but are s t i l l monitored c l o s e l y due to possible r i s k from continuous exposure to carcinogens. Olufsen (1980) i d e n t i f i e d about 30 PAH in Norwegian drinking water ranging from < 0.05 ng/1 to 3.1 ng/1 Ph (see Table 2-3). A f t e r having investigated PAH l e v e l s i n Ottawa tap water, Benoit 10. Table 2-2. PAH l e v e l s i n a i r . PAH l e v e l (ng/m 3) a range (times found) l e v e l (ng/m ) F l 0.8 0-2.2 (9) Ph 0.4 0-1.0 (8) A 0.4 0-1.0 (8) F 0.9 0-1.7 (10) 2.3BF 1.0 0-4.1 (8) P 0.9 0-4.2 (10) B(a)A 0.5 0-2.5 (8) 0.7 Per 0.1 0-1.7 (3) Tr i 0.1 0-0.5 (6) B(a)P 0.2 0-1.8 (2) 1.5 Ind(l,23-cd)P 0.1 0-0.9 (1) B( ghi)Per 0.3 0-2.0 (2) 3.3 B(k)F 1.3 Cor 1.2 Cyclopenta(cd)-pyrene 0.5 a - Strand and Andren, 1980. b - Greenburg et a l . , 1980. 11. Table 2-3. PAH i n water PAH l e v e l ( n g / l ) a l e v e l A ( n g / l ) b l e v e l B (ng/1) N 2.9 5.8 0.9 2-meN 1.4 1-meN 1.1 i 5.8 J 0.6 acenaphthene 0.82 F l 0.72 0.4 0.5 Ph 3.1 A 0.35 lr 1.2 } 0.3 2-meA 0.06 4,5 dimePh 0.30 1-mePh 0.37 3.0 0.6 F 2.6 0.8 0.3 P 1.1 0.4 0.07 B(a)Fl <0.05 B(b)Fl <0.05 4-meP <0.05 1-meP <0.05 B( a) A 0.49 ] Chr/Tri 0.79 J 0.2 > n.d. B(b)F 0.21 B( j)F/B( k)F 0.07 B(e)P 0.20 B(a)P <0.05 a - Norweigan tap water; Olufsen, 1980. b - average of two treated drinking water samples, Eastern Ontario A - heavy population and industry B - low population and industry - Benoit et a l . , 1979. 12. ejt al. (1979) sampled drinking water at s i x municipal water treatment plants i n Eastern Ontario. The PAH l e v e l s varied considerably i n the raw water but l e s s so i n treated waters. The land use and population density i n the water basins seem to a f f e c t the PAH concentrations (see Table 2-3). A basin with a large population and s i g n i f i c a n t i n d u s t r i a l a c t i v i t y (basin A) shows higher PAH l e v e l s than basin B, with small population and l i t t l e i n d u s t r i a l development. Levels of p o l y c y c l i c s i n aquatic organisms have been studied for s h e l l f i s h and f i n f i s h around the world. Table 2-4 l i s t s a few examples of concentrations i n marine tissue samples found off Eastern United States and i n a freshwater system i n Ontario. Kalas et_ _al. (1980) investigated the incidence of PAH i n the marsh s n a i l Cipangopaludina chinesis and found p o l y c y c l i c s present up to the low ppm l e v e l (Table 2-4, c o l . I ) . The marsh environment for t h i s s n a i l was i n a heavily populated area near major i n t e r c i t y highways, and the s n a i l i s considered to be p o l l u t i o n tolerant. In contrast, Brown and Pancirov (1979; Table 2-4, c o l . III-VI) were unable to detect many of t h e i r target p o l y c y c l i c s i n bottom-feeding f i s h from Baltimore Canyon. Those PAH quantitated occurred i n the low ppb range. Similar l e v e l s were determined i n a previous study of s h e l l f i s h and f i n f i s h o f f the Eastern US coast (Pancirov and Brown, 1977). In a d d i t i o n to the p o l y c y c l i c l e v e l s found i n clams as l i s t e d i n Table 2-4, c o l . VII-IX, the researchers also analyzed a lake trout from northern Canada, i n which they detected no PAH. The majority of studies of p o l y c y c l i c s i n s h e l l f i s h deal with the l e v e l s of B(a)P i n mussels. Dunn and Stich (1975) proposed that mussels should be used to monitor contamination of marine environments, Table 2-4. PAH In Aquatic Organisms PAH r e f . a Col. I II r e f . b III IV V VI r e f . VII c VIII IX P 37.00 2.50 2 2 4.1 nd 1.0 12 nd meP nd 1.00 1 nd 2.7 nd nd 2.5 nd B(a)P 25.10 nd nd nd nd nd 0.3 nd nd B(a)A nd nd 1.1 0.3 0.3 1 nd N nd 2.00 meN nd 0.50 A,Ph 134.80 3.00 meA, mePh 60.80 0.70 F 97.50 3.00 B(a)Fl 216.00 nd B(b)Fl 217.50 nd Chr 302.80 3.00 binaphthyl 181.90 nd Per 25.90 nd Cor 110.90 nd r e f . a Kalas jat al. (1980), yg/kg, dry wt. c o l . I - l e v e l s i n marsh s n a i l II - marsh sediment r e f . b Brown and Pancirov (1979), ug/kg, wet wt, c o l . I l l - summer flounder IV - scup V - sea scallops VI - red hake r e f . c Pancirov and Brown (1977) ug/kg wet wt. c o l . VII - clam VIII - clam IX - clam 14. using B(a)P as an in d i c a t o r . In t h e i r sampling of mussels from various s i t e s , a c o r r e l a t i o n was found between i n d u s t r i a l a c t i v i t y , urban development, or presence of creosote, and B(a)P l e v e l s . In some areas creosoted p i l i n g s seemed to contribute s i g n i f i c a n t l y to B(a)P l e v e l s , while untreated sewage/urban runoff were possible sources i n other areas (Dunn and Stich, 1976a). The B(a)P l e v e l s tabulated i n Table 2-5 demonstrate the v a r i -a b i l i t y encountered. It appears that the background concentration of B(a)P i s 0.1 to 1.0 ppb, with mussels from highly contaminated areas containing over 2 ppm (Bj<t>rseth et_ al., 1979). Mackie et_ al. (1980) examined PAH and a l i p h a t i c hydrocarbon l e v e l s i n mussels from 27 sampling s i t e s around Scotland. They concluded that, due to the slow depuration of larger molecular weight PAH from mussels, the mussel watch was of li m i t e d value i n monitoring current marine contamination. Dunn and S t i c h (1976b) found that i n n a t u r a l l y contaminated mussels, B(a)P had a h a l f - l i f e of approximately two weeks i n clean water. This i s much longer than the one to three days generally allowed for elimination of b a c t e r i a l contamination before marketing. Pathobiology of aquatic organisms ( b r i e f l y reviewed by Sonstegard and Leatherland, 1980) serves as an i n d i c a t i o n of potential p o l l u t i o n problems i n the aquatic environment, and can provide a monitoring system for the presence of a carcinogen. Examination of thousands of f i s h (those caught currently and museum specimens from the 1950's) have provided information on several types of tumours and related disorders. Gonadal tumours i n g o l d f i s h and carp suggest induction of tumours by a p o l l u t i o n source, as do increased incidences of l i p tumours i n bottom-1 5 . Table 2-5. B(a)P i n mussels Location B(a)P yg/kg (wet wt.) Comments/Reference Norway 74 15 km from f e r r o - a l l o y smelter/ Bjorseth et a l . , (1979) 2707 next to smelter Vancouver, Canada 0.1 open P a c i f i c / Dunn and Stich (1975) 2 outer harbour, Vancouver, B.C. 18 wharf and dock areas 42 poorly flushed i n l e t , False Creek, Vancouver, B.C. C a l i f o r n i a , U.S. 0.1-8.2 sit e s from i s o l a t e d areas to heavily populated harbours/ Dunn and Young (1976) Mass., U.S. <0.5 unpolluted area / Pancirov and Brown (1977) 0.5 polluted harbour Scotland 50 average of 29 s i t e s , range 1-329/Mackie et a l . , (1980) 16. feeding white suckers found around an i n d u s t r i a l complex i n Lake Ontario. F i s h tumour pathology and i n d u s t r i a l p o l l u t i o n were investigated i n the outflow area of the Buffalo River into Lake Erie (Black et a l . , 1980). Applying the Ames tes t f o r mutagens i n the sediment pinpointed a source of mutagens at the outflow of the l o c a l dye industry. Sedi-ments c o l l e c t e d at t h i s point contained twenty PAH, i n c l u d i n g B(a)P and B(a)A. HPLC traces of extracts from sediments, invertebrates and f i s h demonstrate strong s i m i l a r i t i e s , e s p e c i a l l y once extracts were acid washed (interferences perhaps due to basic a n i l i n e dyes). Examination of some of the f i s h able to survive i n the highly contaminated outflow area revealed gonadal abnormalities, l i p tumours and some highly malig-nant dermal tumours. Black _a_t _al. (1980) determined that PAH were concentrated by s i x times i n tubifex, and twenty times i n carp, with respect to the sed i -ment l e v e l s . This bioaccumulation of p o l y c y c l i c s was also noted i n mussels by Dunn and Stich (1976a), and by Mackie ja_t al. (1980) as discussed above. Kalas et_ al. (1980) quantitated s p e c i f i c PAH i n both sediments and s n a i l s (see Table 2-4, c o l . I - I I ) . Calculated on dry weight of mollusc soft parts, concentrations of PAH were up to 200 times higher than i n sediments. Exceptions to this were found with naphthalene (also meN and dimeN), biphenyl and meP, where none were detected i n the s n a i l . Bioaccumulation of p o l y c y c l i c s can be predicted by t h e i r l i p o p h i -l i c i t y , expressed as t h e i r octanol:water p a r t i t i o n i n g f a c t o r . Southworth et a l . (1980) investigated the predicted and actual bio-accumulation of diB(a,h)ac, B(a)ac, acridine and quinoline i n fathead 17. minnows. Actual bioaccumulation of the higher molecular weight compounds never reached the predicted l e v e l s but evened out within 24 hours due to formation of metabolites. Exposure of Daphnia pulex to the same four compounds resulted i n concentrations very c l o s e l y match-ing those predicted. Obviously each organism reacts i n a d i f f e r e n t way to various compounds. While Southworth et al. (1980) dealt with bioaccumulation of compounds from water the same concentration e f f e c t has been i n v e s t i -gated between sediments and organisms. Bindra and H a l l (1979) analyzed the d i s t r i b u t i o n of several trace metals i n waters, organisms and sedi-ments. Metal l e v e l s i n benthic organisms were generally found to be related to sediment concentrations, however, organism contamination was lower than expected i n some heavily contaminated sediments, and higher i n "clean" sediments. Bindra and Hall postulated that t o t a l lead, for example, experienced competitive adsorption between l i v e organisms and fine organic matter. Sediments with lower amounts of f i n e s , and lower organic content would have higher lead concentrations i n associated organisms (which o f f e r organic adsorption s i t e s ) . 3. PAH i n Food There has been considerable question as to whether bio-synthesis i s a source of the global PAH background. Levels of p o l y c y c l i c s i n plants seem to depend on the extent of urban/industrial development i n the immediate area, but even very r u r a l regions have a PAH background. When studying the l e v e l s of eight PAH i n Hungarian grain Soos, (1974) found that concentrations were higher i n grain from i n d u s t r i a l areas than from r u r a l areas (Table 2-6). 18. Table 2-6. PAH In Various Plant Products (yg/kg) Food/Reference F P Chr B(a)P B(e)P diB(a,h)A l,2BPer B( a) A F r u i t s Vegetables /So6s, 1980 1.5 3.0 Wheat i n d u s t r i a l r u r a l 1.1 0.6 2.1 0.2 0.23 0.08 0.29 0.15 0.18 0.15 0.03 nd 0.03 nd 0.40 0.22 Barley i n d u s t r i a l r u r a l 1.3 0.3 Rye r u r a l /So6s, 1974 0.15 — Table 2-7. PAH i n F i e l d Crops Grown i n Controlled Climate, Greenhouse or Open F i e l d s , yg/kg. Food 3 B(a)P B( e)P Per Anthan-threne B(ghi)Per DiB(a,h)A Cor Lettuce seeds nd nd nd nd nd nd nd controlled -climate " greenhouse 4.2 3.7 0.4 0.2 2.5 0.4 0.6 f i e l d 4.3 4.2 0.4 0.2 2.2 0.6 0.7 Rye controlled - nd nd nd nd nd nd nd climate greenhouse 3.4 1.6 0.9 0.1 0.9 0.1 0.1 f i e l d 1.2 0.64 0.10 0.06 0.34 0.1 0.1 Soybeans cont r o l l e d -climate nd nd nd nd nd nd nd greenhouse 4.3 3.1 0.3 1.5 0.1 0.4 a - Grimmer and Duvel, 1972. 19 . Some investigators have grown plants under co n t r o l l e d conditions to determine whether PAH bio-synthesis takes place. As Schamp and van Wassenhove (1972) point out, great care has to be taken to avoid f a l s e p o s i t i v e s . Grimmer and Duvel (1972) grew various crops (tobacco, l e t t u c e , rye, and soybeans) i n open f i e l d s , greenhouse, and controlled climate chambers. The controlled climate chamber had an elaborate f i l t e r system for i t s a i r supply and was used with an a i r l o c k and sp e c i a l clothes for any personnel entering the room. As indicated i n Table 2-7, crops grown i n open f i e l d s or greenhouse showed s i m i l a r , low l e v e l s of seven p o l y c y c l i c s while those grown i n the s p e c i a l chambers contained none of those PAH. Smoked meats and f i s h have been extensively investigated with respect to B(a)P le v e l s and, more recently, l e v e l s of other poly-c y c l i c s . Table 2-8 l i s t s the r e s u l t s obtained from some of the l a t e r surveys of smoked foods. Levels of PAH i n smoked foods can vary considerably. Joe et a l . (1979) found no samples with B(a)P and the highest t o t a l PAH was f i v e ppb i n a sample of f r a n k f u r t e r s . In contrast to those r e s u l t s , Larsson (1982) analyzed samples of "home-smoked" herring (Table 2-8 l i s t s commercially smoked herring) for B(a)P and found 128 ppb i n the skin and 11.3 ppb i n the f l e s h of one sample. The t o t a l of a l l quantitated PAH f o r that sample was 1,100 ppb. Commercial smoking procedures r e s u l t i n much lower but s t i l l s i g n i f i c a n t p o l y c y c l i c l e v e l s . D i f f e r -ent types of f i s h or meat seem to develop varying concentrations of p o l y c y c l i c s on smoking. In a survey of Canadian foods, Panalaks (1976) determined B(a)P l e v e l s ranging from 0 to 15 ppb. The other most frequently found PAH labia 2-6. PAH In Saokcd Foodi (Mg/kg) Food Ph A F F B(.)A Chr/Trl « b ) F B(J)F « k ) F « « ) F B(a)F Far Ind(1.2,3cd)P B(ghl)Per dlB(a,h)F dlB(a,j)A Cor 7,12dlHeB(a)A Barring* Mackerel Eel Salaon 138 112 108 23 29 22 20 S.5 30 25 19 3.3 26 2.8 16 12 3.7 2.8 2.0 1.0 4.7 3.9 2.0 1.0 0.7 0.7 0.4 0.2 1 > 1.3 0.5 0.2 -> 0.5 0.6 . 0.3 0.1 1.2 1.0 0.6 0.4 0.2 0.2 0.1 0.2 0.3 0.4 0.9 nd 0.1 0.5 0.3 nd . , b nd ad 50 2.0 nd nd nd 0.5 nd nd nd Bologna nd 2.0 nd - 2.0 -Frankfurter* nd 0.2 • 0.2 2.0 - 0.5 - 1.0 - nd • Baa 0.5 - nd 0.5 nd 1.0 0.6 4.0 • Bacon • nd - 0.5 nd nd nd -Beef, Saokad Fort, " tKrr log, " Ft ah, " Gouda Cheaaa 8.0 nd 20 1.0 20 1.5 0.2-0.3 15 0.5 nd 0.2-90 nd -5.0 8.0 10.0 m nd 30 nd 1.0 0.5 nd - 2.0 nd . nd 25.0 Oyetara Ojretere, Saokad 15-30 25 1.6 20 20.0 5.0 4.0 1.0 38.0 Bacon0 Kipper* Chaaaa 7.8 2.4 4.2 0.30 0.35 0.30 0.05 0.1P 0.15 0.05 0.10 0.20 2.5 2.7 nd nd 3 nd * - Larson, 1982, Sweden b - Panalaka, 1976, Canada c - Crosby et a l . 1981, B r i t a i n . 21. were B(a)A (0-30 ppb) and B(e)P (0-16 ppb). Smoked oysters had high l e v e l s of B(b)F, B(a)A, B(e)P, 7,12-dimeB(a)A and Per. In studies of PAH l e v e l s r e s u l t i n g from c h a r c o a l - b r o i l i n g , inves-ti g a t o r s examined e f f e c t s of percent fat i n the food, length of time food i s b r o i l e d and distance between heat source and the food. L i j i n s k y and Ross (1967) determined that the p o l y c y c l i c s produced increased as meat percent f a t increased. Analysis of hamburger b r o i l e d i n a gas or e l e c t r i c oven resulted i n no PAH formation while charcoal b r o i l i n g i n a no-drip pan resulted i n only traces of a few PAH and no B(a)P. Bories (1979) reports that the lowest B(a)P (0.5 ppb) i n charcoal b r o i l e d steak i s obtained by l i g h t l y b r a i s i n g the steak at 10 cm from the charcoal. In general, the more heavily smoked a food i s , the higher the PAH l e v e l . External smoke generation (smoke produced i n a separate chamber from food) r e s u l t s i n lower p o l y c y c l i c concentrations. The control over smoking conditions i n commercial smoking establishments r e s u l t s i n much lower PAH formation when compared to most home-smoked foods. Charcoal b r o i l i n g of high-fat foods at close range r e s u l t s in elevated PAH concentrations. C. PAH IN SEDIMENTS 1. Determination of PAH Sources Concentrations and d i s t r i b u t i o n s of p o l y c y c l i c s i n marine or freshwater sediments are of considerable i n t e r e s t as in d i c a t o r s of natural PAH l e v e l s i n non-contaminated areas, or as records of poly-c y c l i c input to aquatic systems i n more developed areas. Several approaches have been used to determine the important 22. sources of PAH i n a given sediment sample. The techniques include geological surveys, comparisons of a l k y l homologue abundances, and an analysis of sedimentary cores. It i s often possible to determine input of PAH to sediments from a point or semi-point source by c o r r e l a t i n g p o l y c y c l i c l e v e l s with d i s t -ance of sampling points from given source(s). Also, samples taken near or far from urban centres should indicate the PAH loads. S i m i l a r l y , samples taken i n south or north hemispheres should r e f l e c t the greater population and i n d u s t r i a l development i n the northern hemisphere. The PAH composition of sediments i s often very s i m i l a r from one s i t e to another. However, the r e l a t i v e abundances of non-alkylated p o l y c y c l i c s and the various a l k y l homologues are d i s t i n c t i v e for p a r t i -cular PAH sources. It has been found that the p o l y c y c l i c s from petro-genic sources (c o a l , petroleum o i l s , shales) commonly have three- or four- carbon side chains with l i t t l e or no non-alkylated PAH. (See LaFlamme and Hites (1978) for a review of methods to determine the sources of PAH i n recent sediments.) P o l y c y c l i c s derived from combus-t i o n processes have very s i m i l a r q u a l i t a t i v e d i s t r i b u t i o n regardless of the f u e l . However, differences i n r e l a t i v e abundances of alkylated and non-alkylated PAH are seen from f u e l to f u e l and with differences i n combustion temperature. Low combustion temperatures (eg. cigarettes) r e s u l t i n an abundance of a l k y l p o l y c y c l i c s . (As mentioned above, petrogenic p o l y c y c l i c s derived through low temperatures over very long periods of time are almost e n t i r e l y alkyl-substituted.) Combustion carried out at very high temperatures, as i n carbon black furnaces, r e s u l t s i n non-alkylated PAH. The degree of a l k y l a t i o n encountered i n the PAH f r a c t i o n of a 23. sample i s often indicated through a l k y l homologue p l o t s , where the r e l a t i v e abundance of each homologue i s arranged by increasing t o t a l carbon number of the s u b s t i t u t i o n . Various isomers of a s i n g l e molecular weight parent may be included i n a given pl o t , and d i s t i n c -t i o n between s i n g l e or multiple s u b s t i t u t i o n of the parent i s n ' t generally made (Blumer and Youngblood, 1975; or Giger and Blumer, 1974). Also, Blumer et_ al. (1977) examined aza-arene homologue serie s i n Massachusetts sediments. As an a d d i t i o n a l t o o l to help develop understanding of PAH deposition i n recent sediments, sediment cores are analyzed and the layers dated to c o r r e l a t e PAH l e v e l s with estimated inputs. A great deal of information can be gained from sediment cores, e s p e c i a l l y i f there has been minimal mixing. 2. Natural PAH Levels Considerable e f f o r t has been made to determine the "natural" l e v e l s of PAH i n sediments. Investigations have been c a r r i e d out on numerous surface sediments and on several sets of sediment cores. Analysis of water and sediments of a northern Ontario wilderness lake was c a r r i e d out by Brown and Starnes (1978). Low l e v e l s (ppb) of nineteen PAH were determined i n the sediment (see Table 2-9, column I ) . From the low r e l a t i v e abundance of methyl-substituted p o l y c y c l i c s i n comparison to the parent compound, petroleum contamination was ruled out. Several other possible sources of PAH to t h i s remote, undeveloped lake were considered; atmospheric f a l l - o u t of contamination from distant urban-industrial centres, biogenic generation i n - s i t u , and pol y c y c l i c s from forest f i r e s . Of these, the biogenic formation and Table 2-9. PAH In Sediments ( Pg/kg) ref .a ref .b ref .c ref d ref .e ref .1 ref .£ ref .t I Polycyclic Col.I II III IV V VI VII VIII IX X XI XII XIII XIV XV XVI XVII XVIII XIX XX XXI XXII XXIII XIV Ph 5000 43 7 nd 2 6 300 10 2175 150 230 140 1880 260 53 42 8 340 210 3500 F 38 15000 120 1 nd 0.6 3 950 2 2135 15 870 1260 3000 650 420 172 23.5 130 130 11 420 390 10000 B(e)P 13 90 nd 6974 14 610 530 1730 280 420 149 33.2 210 150 3900 B(a)P 28 250 nd 6313 16 340 350 1610 270 443 99 30.2 160 190 270 B(b)F 25 1 5587 22 1 340 380 26 B(k)F 25 33000 200 1 nd 0.6 500 U50 nd v 2540 7 , ft270 1140 3520 580 |693 260 38.3 B( j)F 8 J J 1 Per 20 35 750 1658 nd 130 150 450 150 151 45 4.0 40 30 660 B(a)Fl 60 nd 30 nd 190 210 920 150 112 38 26.1 B(b/c)Fl 80 nd 120 150 610 90 88 22 32.2 B(ghl)F 14 70 100 230 50 153 35 15.3 Tr l 10 i 300 nd > 416' 29 | Chr 23 I J V 510 1140 2200 330 356 143 24.4 |l60 200 12 B(a)A 7 21000 80 2 nd 1 13 90 nd 2160 21 I i B(c)Ph 39 nd P 23 13000 100 1 nd 0.6 3 950 2 1517 6 650 610 2860 430 383 138 20.3 120 120 7 380 330 7200 IndF 230 240 550 120 80 50 54.5 B(ghl)Per 28 8703 nd 640 730 1660 360 335 154 54.5 Anthanthrene 25 nd 40 100 280 70 nd nd Cor 6 50 nd 190 260 400 110 323 76 A 40 nd 728 nd 30 30 340 70 30 20 450 N me-N Acenaphthene 32 nd Fl 150 nd raePh/MeA 490* 25* 769 16 150* 90* 820* 160* meP <2 157 nd 650 650 1690 360 Ind(l,2,3-cd)P 700 nd dlB(a,c/a,h)A 65 nd 1970 nd *raePh Brown and Starnes, (1978); Wilderness Lake. LaFlamme and Hites, (1978); dry wt. basis c o l . I I Charles River (Boston, MA) II I Gulf of Maine IV Yosemite s o i l (wooded) V Nevada s o i l (desert) VI Alaska sediment ( i n t e r t i d a l ) VII Rio lea, Amazon River system Wakeham et a l . , (1980a); Greifensee Lake c o l . VIII 0-3 cm, approximate values IX 63-68 cm, approximate values Bj<j>rseth et al., (1979); Saudafjord sediments, dry wt. c o l . X 0-2 cm, next to smelter XI 0-2 cm, 15 km from smelter Grimmer and Bohnke, (1975b); Grosser Ploner Sea, dry wt. c o l . XII 5-7 cm, undeveloped shore XIII 54-56 cm, undeveloped shore XIV 4-6 cm, developed shore XV 60-63 cm, developed shore Muller et a l . , (1977); Lake Constance c o l . XVT 0-1 cm XVII 14-15 cm XVIII Enrichment f a c t o r , see text. Hites __t_ _ _ i l . , (1977); Buzzards Bay, dry wt. c o l . XIX 0-4 cm, 1970 XX 20-24 cm, 1900 XXI 38-42 cm, 1850 Giger and Schaffner, (1978); dry wt. c o l . XXII Lake sediment XXIII River sediment XXIV Street dust 26. forest f i r e s were indicated as the most probable sources. S i g n i f i c a n t forest f i r e haze was observed i n the region of the lake. LaFlamme and Hites (1978) ca r r i e d out a survey of PAH i n recent sediments on a global basis to determine the baseline l e v e l s of poly-c y c l i c s . Sediments sampled ranged from the highly contaminated Charles River (mid ppm range PAH, see Table 2-9, column II) to near p r i s t i n e sediments i n Alaska (low ppb range PAH, column VI). In a l l samples the qu a l i t a t i v e pattern of PAH d i s t r i b u t i o n was found to be very s i m i l a r . The composition of the p o l y c y c l i c f r a c t i o n was usu a l l y Ph (12%); F(16%); P(15%); molecular weight 228 including B(a)A, Chr, T r i and B(c)Ph, (23%); and molecular weight 252 including BF, BP and Per (35%). This d i s t r i b u t i o n of compounds i n the p o l y c y c l i c f r a c t i o n (and the abundances of a l k y l homologues) indicate combustion as an important PAH source. Increasing p o l y c y c l i c l e v e l s with increasing proximity to i n d u s t r i a l i z e d areas i n d i c a t e mainly anthropogenic sources. Two sets of compounds form d i s t i n c t exceptions to the general pattern; a l k y l phenanthrenes and Per. The a l k y l phenanthrenes are l a r g e l y comprised of l-methyl-7-isopropyl-phenanthrene (retene) and also 1,7-dimethyl phenanthrene (pimanthrene). These compounds are perhaps derived from a b i e t i c or pimaric acid, both components of pine r o s i n . Samples from several s i t e s with nearby pine forest a l l contained these alkylphenanthrenes and also other r o s i n components. Work by Wakeham et a l . (1980b) on sediments of four lakes i n d i c a t e the presence of retene and pimanthrene, as mentioned above by LaFlamme and Hites (1978), as well as other phenanthrene homologues. Wakeham et a l . propose that the phenanthrene series may be formed by diagenesis i n recent sediments by microbial action (perhaps dehydrogenation of 27. steroids) on n a t u r a l l y occurring compounds. In the cores studied, Ph was always present at much higher l e v e l s than any homologues, but the importance of the a l k y l compounds increased with increasing depth. The presence of retene and pimanthrene s p e c i f i c a l l y was related to the extent of coniferous cover i n the surrounding area. Lake Washington sediments (Seattle area) demonstrated much higher l e v e l s of these two p o l y c y c l i c s than the sediments of three Swiss lakes, where con i f e r s have been r e l a t i v e l y scarce. Wakeham et a l . (1980b) also t e n t a t i v e l y i d e n t i f i e d a s e r i e s of PAH (three to f i v e aromatic rings) i n Lake Washington sediments which they suggest could be derived from pentacyclic t r i t e r p e n e s . Successive dehydrogenation (again possibly microbial mediation) of these compounds has been demonstrated to occur and would account for the v a r i e t y of p o l y c y c l i c s present i n these very recent sediments. The presence of perylene i n the vast majority of sediments studied by LaFlamme and Hites (1978) was i n accordance with work done by Aizenshtat (1973) on sediments, shale and peat. Aizenshtat found that, i n order for Per to be formed and preserved i n sediment, a source of material i s required (perhaps terrigenous extended quinone pigments), then rapid conversion under reducing conditions, and preservation of Per, probably by ir-bonding with t r a n s i t i o n metals. In t h e i r survey of baseline PAH concentrations, LaFlamme and Hites (1978) found that the group of C 2 Q H 1 2 isomers was generally composed of mainly benzofluoranthenes and benzopyrenes. However i n several samples they found very high l e v e l s of perylene, up to 97% of the t o t a l PAH f r a c t i o n . Samples containing such high l e v e l s of Per were often found in the Amazon River system (Table 2-9, column VII) although Per has 28. been found i n most other sediments around the world. LaFlamme and Hites sampled addi t i o n a l non-reducing sediments i n the Amazon River region ( f l o o d plains) and found no s i g n i f i c a n t amounts of perylene. This gives support to Aizenshtat's proposals of diagenetic transformation of terrigenous pigments under reducing conditions. 3. Point Sources In some cases i t has been possible to determine a point source f o r the input of p o l y c y c l i c s to an aquatic system by quantitating PAH l e v e l s i n the sediments at various sampling s i t e s . Dunn and Stich (1976a) sampled sediments near the Iona sewage treatment plant near Vancouver, B.C. As the sample s i t e s were moved further from the sewage o u t f a l l , the l e v e l of B(a)P i n the sediments f e l l sharply; 121 yg/kg dry weight next to the o u t f a l l compared to 0.7 yg/kg 5 km from the o u t f a l l . Bj4>rseth jajt _al. (1979) determined l e v e l s of over twenty PAH i n sediments at various points along Saudafjord i n Norway (Table 2-9, column X, XI). A f e r r o a l l o y smelter has been i n operation at the head of the Inlet since 1923, discharging waste from scrubbers into the f j o r d without treatment. It was found that, f o r most of the compounds, concentrations f e l l o f f steeply as sampling s i t e s were moved from the smelter, so that "uncontaminated" sediments had only 1% of the PAH l e v e l s determined for sediments next to the smelter. Relative abundances of Ph, however, increased i n uncontaminated sediments as was observed by LaFlamme and Hites (1978) and Wakeham _at a l . (1980b). As another example of studies of suspected point sources of poly-c y l i c contamination, Gump _2t &L. (1977), sampled water and sediment 29. near a r e f i n e r y tanker j e t t y . Water samples indicated a very s l i g h t increase i n hydrocarbon load compared to that of uncontaminated s i t e s . The sediments, however, demonstrated a s i g n i f i c a n t increase i n hydro-carbons, including approximately 800 ppb B(a)P. The contribution of recent coal mining to p o l y c y c l i c input to aquatic systems was investigated by John __t ____. (1979). The system chosen was the Severn Drainage i n Wales which includes several r i v e r s running through c o a l f i e l d s and exposed o i l shales, as well as receiving e f f l u e n t from heavy industry (chemicals and s t e e l ) . By determining the p o l y c y c l i c l e v e l s (9 compounds) i n sediments from various s i t e s i n the system, i t was found that l e v e l s were highest i n areas which had had recent coal mining a c t i v i t y . This i s of some concern since there may be a s i g n i f i c a n t swing to the use of coal as f u e l rather than o i l , possibly r e s u l t i n g i n higher inputs of PAH to the environment. 4. Non-Point Sources Sediment cores were studied by Grimmer and Bohnke (1975b) to compare PAH l e v e l s encountered near an undeveloped forested lakeshore, and near a well built-up lakeshore ( i n c l u d i n g highway and railway) on Grosser Pioner Sea. The cores taken extended back to approximately 1910, and were quantitated for PAH, elemental analysis and metal concentrations. It was found that the p o l y c y c l i c l e v e l s have remained constant over the s i x t y - f i v e year span covered by the core from the undeveloped southern shore. PAH l e v e l s increased f i v e times over the same time i n the core from the more highly developed area (Table 2-9, column XII-XV). The authors dismiss v e h i c l e emissions as the main source for these increased l e v e l s since B(ghi)Per and Cor were present 30. at very low concentrations, and r a t i o s of these two PAH to B(a)P were very d i f f e r e n t from that found i n car exhaust. S i m i l a r l y , petroleum wasn't considered an important p o l y c y c l i c source due to lower than expected l e v e l s of S-containing PAH. Grimmer and Bohnke concluded that l o c a l coal combustion was mainly responsible f o r higher l e v e l s . This conclusion was supported by a decrease i n p o l y c y c l i c concentrations i n surface layers of cores from the developed shore, c o i n c i d i n g with the recent replacement of coal as f u e l for the l o c a l railway engines. As a type of follow-up study, Muller et a l . (1977) examined con-centrations of heavy metals and PAH i n sediment cores from Lake Constance. Cores of lake sediment (mainly derived from the Rhine River) were taken to a depth of 40 cm, with 25 cm corresponding to approximately 1900. In order to compare the increases i n concentration among the p o l y c y l i c s and the heavy metals, an enrichment factor was defined. The enrichment f a c t o r i s the r a t i o of the component's highest l e v e l found since 1900 to i t s concentration i n the 1900 l a y e r . Some of the metals (K, L i , Fe, Cu, Cr, Co, Ni) showed no change i n t h e i r con-centrations over the span of the cores, having enrichment factors close to 1. Four other metals (Zn, Cd, Hg, Pb) however, showed s i g n i f i c a n t l y increased concentrations having enrichment factors ranging from 2.96 f o r Zn to 4.35 f o r Pb. On examination of PAH l e v e l s over the same time i t became evident that t h e i r enrichment factors were much higher (Table 2-9, column XVI-XVIII). The lowest increase was noted for Per (enrich-ment factor 4.0) which as discussed previously, i s often found i n s i g n i f i c a n t l e v e l s i n uncontaminated lake sediments. B(ghi)F was another PAH with somewhat lower enrichment factor (15.3). A l l other eleven p o l y c y c l i c s quantitated showed enrichment f a c t o r s between twenty 31. (P, 20.3) and nearly s i x t y (IndP, 56.9). Since there i s such a strong c o r r e l a t i o n between metal l e v e l s (Cd, Hg, Pb, Zn) and PAH, the authors propose a common source; the combustion of c o a l . Coal ash has been shown to contain high l e v e l s of Pb, Cd, Zn, Cu, and Hg, as well as a wide range of p o l y c y c l i c s . Muller _ej_ ______ set f o r t h two possible explanations for the much higher enrichment factors found for PAH: 1) P o l y c y c l i c s and metals are released i n s i m i l a r proportions and deposited i n s o i l s , but PAH are more e a s i l y "leached" than metals, thus r e s u l t i n g i n much greater sediment l e v e l s . 2) P o l y c y c l i c s are also produced as veh i c l e emissions. The second proposal was discounted on the basis of the presence of benzonaphthothiophene (enrichment factor 25.6) i n sediments and i t s absence i n v e h i c l e exhaust. Also, the r a t i o B(a)P: B(e)P i s inappro-priate for a s i g n i f i c a n t petroleum hydrocarbon input. Thus the increase i n l e v e l s of both metals and PAH i s a t t r i b u t e d l a r g e l y to the combustion of c o a l . Hites jat &L. (1977) examined sediments i n Buzzards Bay, Massachusetts at d i f f e r e n t depths (Table 2-9, column XIX-XXI). The t o t a l PAH found i n the sediment layer r e f l e c t s the production of PAH by f u e l combustion. As can be seen, the PAH concentrations rose sharply from 18 50 to 1900 but stayed r e l a t i v e l y constant t i l l 1970. This l e v e l l i n g o f f during the l a s t seventy years p a r a l l e l s a change from burning wood and coal (producing greater amounts of PAH) to o i l and gas (which r e s u l t i n lower PAH emissions). The s o i l s of Nova Scotia and sediments of the Gulf of Maine were sampled by Hites et_ j i l . (1980). With the Boston area i n mind as a semi-point source, samples were taken downwind as far as northern Nova 32. Scotia, including transects across the Gulf of Maine and Massachusetts Bay. Some deep ocean sediments were also taken. The re s u l t s are summarized i n Table 2-10. As can be seen, t o t a l PAH l e v e l s i n Nova Scotia s o i l s and the deep ocean sediments are low i n contrast to the very high l e v e l s i n Charles River sediments. PAH concentrations increased with increasing proximity to the Boston area i n the transect across Massachusetts Bay and i n deep ocean samples. Hites and co-workers suggest that the major source of p o l y c y c l i c s to the study are i n the p a r t i c u l a t e matter from various f u e l s burned i n the Boston area. Larger particulates w i l l be deposited near the combustion source, being washed o f f buildings and streets into l o c a l "sinks", such as the Charles River and Massachusetts Bay. These heavily contaminated sediments may be slowly transported to nearby basins (possibly explaining the variable PAH le v e l s i n the Gulf of Maine). PAH associated with small p a r t i c l e s may be transported much further, accounting for the f a l l - o f f i n PAH concentrations i n deep ocean samples and low values i n Nova Scotia s o i l s . In a study of a p p l i c a t i o n of c a p i l l a r y GC to PAH analysis, Giger and Schaffner (1978) included analysis of lake and r i v e r sediments, a i r and r i v e r p a r t i c u l a t e s and street dust (Table 2-9, column XXII-XXIV). The d i s t r i b u t i o n of p o l y c y c l i c s i n each of these samples was generally s i m i l a r . The a i r particulates lacked many of the lower molecular weight PAH, however t h i s may be a r e s u l t of the sampling technique. In considering the quantitative data, the authors consider PAH from street dust to be the Important factor i n sediments which receive urban run-o f f . Wakeham et a l . (1980a) continued t h i s i n v e s t i g a t i o n of the 3 3 . Table 2-10. PAH i n Sediments, Boston as a Point Source Sample Site Total PAH (ug/kg, dry wt.) ! Comment s Nova Scotia Gulf of Maine Massachusetts Bay Charles R., Boston Deep Ocean 50 540 500 870 200 160 830 3400 8500 120000 18 97 160 median of 10 s o i l samples increasing proximity to Boston increasing proximity to Boston a - Hites et a l . , (1980). 34, possible sources of p o l y c y c l i c s to sediments, looking at cores from three Swiss Lakes and Lake Washington. They found, as expected, higher PAH concentrations i n sediments near urban areas than i n sediments with input from r u r a l areas. Alkylated PAH were very common and the abund-ance of the homologues decreased r a p i d l y with an increase i n carbon number i n the side chain. Also, l i n e a r p o l y c y c l i c s such as anthracene were very much l e s s common i n comparison to non-linear, or angular, isomers such as Ph. The enrichment of PAH found by Wakeham et a l . (1980a) over appro-ximately the l a s t hundred years ranged from f i v e times (Lake Lucerne) to f o r t y times (Lake Zurich), i n d i c a t i n g that, although atmospheric transport of p o l y c y c l i c s adsorbed to a i r p a r t i c u l a t e s could account for q u a l i t a t i v e s i m i l a r i t i e s , other factors must contribute to quantitative d i f f e r e n c e s . (See Table 2-9, column VIII-IX, for PAH l e v e l s i n Lake Greifensee core.) Street dust may contain PAH derived from asphalt p a r t i c l e s , t i r e wear p a r t i c l e s , atmospheric f a l l - o u t , vehicular exhaust p a r t i c u l a t e s , motor o i l or l u b r i c a t i n g grease. Wakeham et a l . (1980a) found strong q u a l i t a t i v e c o r r e l a t i o n between the p o l y c y c l i c f r a c t i o n i n street dust and r i v e r or lake sediments, s i m i l a r molecular weight range and a l k y l s u b s t i t u t i o n pattern, and preponderance of unsubstituted PAH. In comparisons between street dust samples taken from asphalt roads and those from cement roads the q u a l i t a t i v e d i s t r i b u t i o n of p o l y c y c l i c s was the same, but asphalt roads c o n s i s t e n t l y c a r r i e d several times the PAH loading of cement roads. Street dust was shown to comprise p a r t i c l e s up to 500 pm i n diameter while v e h i c l e exhaust p a r t i c u l a t e s were gener-a l l y smaller than 50 urn i n diameter. The authors concluded from the 35. above study that the PAH input to r i v e r or lake sediments from street dusts ( e s p e c i a l l y from asphalt surface) was of major importance i n urban areas. High l e v e l s of contamination i n street surface dusts were reported i n an extensive study of the subject by Sartor _et _al. (1974). Sedi-ments from 12 American c i t i e s were analyzed for oxygen demand (chemical and biochemical), a l g a l nutrients, heavy metals, pesticides and PCB's and coliform b a c t e r i a . PCB's were found i n a l l sediments at l e v e l s approximately twenty times those of the organochlorine p e s t i c i d e s . Most of the contaminants were associated with the small f r a c t i o n of organic fines ( l e s s than 10% of the t o t a l sediment weight). In a com-parison of pollutant loadings on asphalt and concrete surfaces, asphalt surfaces were consistently about 80% more heavily loaded than concrete s t r e e t surfaces. This may o f f e r a d i f f e r e n t perspective on the high PAH content of street dust from asphalt streets as mentioned i n Wakeham et a l . (1980a) . The incidence of p o l y c y c l i c s i n used crankcase o i l s has been of concern due to the carcinogenicity of some isomers, and the huge volumes of used crankcase o i l which are released to the environment each year. Payne et al. (1980) tested used o i l for mutagenicity with the Ames t e s t . Contrary to t h e i r expectations, the major f r a c t i o n responsible for mutagenicity was not that containing B(a)P or B(a)A, the c l a s s i c carcinogens which had been found i n used o i l s . The factors a f f e c t i n g the buildup of PAH i n crankcase o i l were investigated by Handa _____ al. (1979), concentrating on car mileage and o i l mileage. It was found that each car demonstrated a d i f f e r e n t rate of B(a)P accumulation i n o i l , but that the accumulation i s d i r e c t l y 36. proportional to o i l mileage. Handa and co-workers were able to r e l a t e the B(a)P accumulation to engine age; an equation was eventually developed incorporating a l i n e a r function of car mileage and a quadratic function of o i l mileage. MacKenzie and Hunter (1979) used s u l f u r - s p e c i f i c detection for GC analysis of urban stormwater runoff, crankcase o i l s (used and unused), f u e l o i l s , and r i v e r sediments. P r o f i l e s were determined for each of these, and the used crankcase o i l p r o f i l e correlated most c l o s e l y with that of r i v e r sediment. Their i n v e s t i g a t i o n s indicated that the l i g h t e r diaromatics (eg. benzthiophene) were l o s t due to natural weathering but that the larger molecular-weight s u l f u r compounds (associated with particulates) could r e a d i l y be transported i n urban runoff. 5. Summary In summary, the l e v e l s of p o l y c y c l i c aromatic hydrocarbons i n sediments predating the I n d u s t r i a l Revolution are extremely low (low ng/g range). In these e a r l y sediments there are several groups of PAH (Per, Ph and i t s a l k y l homologues, and pentacyclics from triterpenes) which appear to be of diagenetic o r i g i n . It i s possible that forest f i r e s also contributed to these low l e v e l s . Recent sediments, however, generally r e f l e c t the increase i n i n d u s t r i a l a c t i v i t y since approximately 1800. Whether these elevated l e v e l s of p o l y c y c l i c s are due to increased petroleum input, urban and i n d u s t r i a l combustion f a l l o u t or increased vehicular t r a f f i c , i s a mat-ter of some question. The methods for determining the source(s) for a PAH residue have been reviewed, and examples given. In sampling areas 37. where a point or semi-point source (such as creosoted p i l i n g s , urban sewage e f f l u e n t , smelter scruber wastewater, coal mining or exposed o i l shales) i s n ' t i d e n t i f i e d , atmospheric transport of PAH adsorbed to p a r t i c u l a t e s appears to be the main p o l y c y c l i c source. Systems receiv-ing urban runoff may get the largest PAH load from street dust ( e s p e c i a l l y from asphalt roads). D. ASSESSMENT OF PAH EXPOSURE The complexity of the PAH component i n environmental samples i s well appreciated and becoming more evident with each improvement i n a n a l y t i c a l technique. In addition to compositional complexity, the e f f e c t s of the PAH ( i n d i v i d u a l l y , i n combination, or with other compon-ents) are extremely d i f f i c u l t to determine. E f f o r t s have been made, however, to assess (to the best of our knowledge) the r i s k s to human health posed by p o l y c y c l i c s . Following extensive surveys of PAH l e v e l s i n Hungarian food-s t u f f s , Soos (1980) estimated the dietary B(a)P load i n Hungary. The c a l c u l a t i o n s are summarized i n Table 2-11. It can be seen that, i f the p o l y c y c l i c l e v e l i n f r u i t s or vegetables are representative, the vast majority of the B(a)P i n d i e t s i s a t t r i b u t a b l e to f r u i t s and vege-tables. Smoked meats and meat products contribute only one percent to the estimated B(a)P load. Considering mainly B(a)P exposure through a i r p o l l u t i o n , Bridbord _at al. (1976) assess the potential exposure for several occupations and l i f e s t y l e s . A person smoking a pack of c i g a r e t t e s each day would be receiving a B(a)P load under half of that encountered by a worker in a restaurant or an airplane (where r e c i r c u l a t e d a i r becomes "enriched" 38. Table 2-11. Estimation of B(a)P Intake (per person/year) i n Hungary 3 Food Average B(a)P l e v e l Average B(a)P intake % of Estimated (ug/kg)/range (ug/person/year) Total Intake F r u i t s 1.5 (0.92-1.73) 101.5 (62.3-117.1) 21 Vegetables 3.0 (1.30-4.40) 258 (118.8-378.4) 54 Edible o i l s 4.8 17.3 4 Margarines 4.7 9.4 2 Bread 0.66 62.2 13 Baker's ware 0.70 8.2 2 Floury products 0.87 3.0 1 of sweets industry Smoked Meats 0.67 (0.60-0.74) 6.3 (5.6-6.9) 1 and meat products Smoked cheese 0.85 -0.1 -G r i l l e d meat <0.05 - -'Pure' coffee 0.4 9.6 2 extract Total < 475.6 (290.2-612.2) 100 a - Soos, 1980. 39. with c i g a r e t t e PAH). Workers i n coke f a c t o r i e s , or using coal tar pitch , experience by far the greatest r i s k (see Table 2-12). A more comprehensive attempt at quantitating PAH exposure i s made by Sandodonato _____ al. (1980). Urban PAH concentrations i n ambient a i r are used to cal c u l a t e a figure f or d a i l y intake of B(a)P and t o t a l PAH from a i r . Likewise, l e v e l s of PAH contamination of drinking water sources are used to ca l c u l a t e the water-related d a i l y load. Assessment of dietary B(a)P (or PAH) intake i s complicated by the dif f e r e n c e s i n food o r i g i n , method of preparation and r e l a t i v e amounts of food consumed. See Table 2-13 for a summary of the p o l y c y c l i c con-centrations used by the authors to ca l c u l a t e intake. The estimated p o l y c y c l i c load for a i r , water and food are combined into a t o t a l d a i l y intake. C a l c u l a t i o n of an allowable d a i l y intake (ADI), r e l i e s on findings from animal carcinogenieity tests (see Table 2-14). Assumptions are made with respect to dose response r e l a t i o n s h i p s f or PAH, a c t i v a t i o n of carcinogenic p o l y c y c l i c s and a p p l i c a b i l i t y of animal tests i n order to assess the p o t e n t i a l f or cancer i n humans. In Table 2-14, the calculated allowable d a i l y intake has been l i s t e d f o r B(a)P, diB(a,h)A and two PAH mixtures. In comparison to estimated d a i l y B(a)P load, i t can be seen that the food contribution may exceed the ADI by three to 30 times. Smoking (one pack per day) exceeds the ADI by approximately eight times. These assessments i n d i -cate that d i e t a r y intake may be the most important source of PAH f o r a non-smoker. The d a i l y dietary exposure calculated by So&s (1980) i s i n agreement with the upper range of the d a i l y d i e t a r y exposure estimated by the Santodonato assessment. 40. Table 2-12. Estimates of B(a)P Intake, From an Occupational Viewpoint 3 B(a)P intake Pg/day Cigarette equivalents packs/day Smoking, 1 pack/day 0.4 1 Coke oven workers Topside exposures 180 450 Side & bench exposures 70 175 Coal tar p i t c h worker 750 1875 Airplane p i l o t s Transatlantic f l i g h t s 0.93 2.3 Domestic cross country 1.38 3.5 Employee i n restaurant 0.8 2 Person l i v i n g near expressway 24 hr/day adverse meteorology 0.02 0.05 Commuter on expressway 2 hr/day adverse meteorology 0.04 0.10 Exposure to ambient B(a)P l e v e l s 8 hrs/day 0.02 0.05 a - Bridbord et a l . , 1976. Table 2-13. PAH Levels i n Foods Used to Calculate Human Exposure Due to Food Consumption 3 Food B(a)P Total PAH vegetable o i l s and margarine 0.2 — 6.8 2.1 - 136 f i s h and other aquatic foods smoked trace - 6.6 5.2 - 162 non-smoked 0 1.8 - 3.2 smoked meats and meat products trace - 3.6 1.5 - 150 cooked meat charcoal b r o i l e d hamburger 0 - 2.6 0.3 - 43.9 steak 4.4 - 50.4 70 - 183.7 barbequed 3.5 - 10.5 37.5 - 186.1 f r u i t s nd - 29.7 grains and cereal products 0.1 - 60 sugar and adjuncts 0.2 - 72.0 vegetables nd - 24.3 beverage nd — 21.3 a - Santodonato et a l . , 1980. Table 2-14. Estimate of Allowable Daily Intake 3 Estimated B(a)P, Total PAH exposures (yg/day) Source B(a)P Total PAH a i r water food (smoking)(1 pack/day) 0.0095 - 0.0435 0.0011 0.16 - 1.6 0.4 0.207 0.029 1.6 - 16 Allowable Daily Intake (ng/day) Compound Route ADI (ng/day) B(a)P Dietary 47 B(a)P Intratracheal 48 B(a)P Dermal 4.7 PAH mixture Oral 43 PAH mixture Dermal 4.2 DiB(ah)A Oral 108 Exposure i n Relation to ADI B(a)P (ng) Proportion ADI Water A i r Food Smoking 48 1.1 9.3 - 43.5 160 - 1600 400 1.0 0.02 0.02 - 0.91 3.3 - 33 8.3 Santodonata et a l . , 1980. 43. E. ANALYSIS OF PAH 1. Introduction Due to the considerable complexity of the p o l y c y c l i c f r a c t i o n s i n sediments, analysis methods have generally been long and complex procedures. Since e a r l y instrumental methods involved very low s e l e c t -i v i t y ( t h i n layer chromatography or paper chromatrography), the clean-up procedures were required to remove most of the i n t e r f e r i n g compounds. With the greater resolving power of HPLC and GC, analysis schemes have been s i m p l i f i e d to a large extent. The improved resolu-tion, however, often reveals a much greater complexity i n environmental samples than was indicated by low r e s o l u t i o n methods. Thus, work on analysis schemes aims to 1) stream-line sample workup and 2) achieve adequate r e s o l u t i o n of important p o l y c y c l i c s once interferences have been eliminated. The usual approach to analysis of PAH i n sediments involves e x t r a c t i o n of the analytes from the sample; separation of the analytes from any aqueous phase; clean-up by column chromatography; and perhaps gross separation by a d d i t i o n a l column chromatography before f i n a l separation by instrumental methods. 2. E x t r a c t i o n Procedures Several of the older methods are adapted from methods used for the analysis of PAH i n foods. The work of Grimmer and Bohnke (1979) described a method based on a IUPAC recommended method for p o l y c y c l i c s In meat. Dried sewage sludge or s o i l samples are extracted by r e f l u x -ing with acetone. Another example of the adaptation of food-related methods i s the procedure used by Dunn (1976). Samples (tis s u e or s e d i -ments) are refluxed with a l k a l i n e ethanol i n order to extract the 44. organics and to saponify l i p i d s . A l k a l i n e methanol has also been used to saponify extracts ( B i e r i _=_t 1978). Soxhlet e x t r a c t i o n of sediments i s widely used; the e x t r a c t i o n solvent and the r e f l u x time vary widely. Giger and Blumer (1974) developed a very thorough procedure to prepare sediment samples f o r analysis by UV-Vis spectrophotometry and MS. The sediment i s Soxhlet extracted f or 24 hours with benzene then methanol i s added and the mixture Soxhlet refluxed for a further 24 hours. La Flamme and Hites (1978) also used t h i s procedure. Keizer _a_t al. (1978) combined the benzene and methanol, and reduced the time to a t o t a l of 18 hours. Brown and Starnes (1978) used a 6 hour Soxhlet using benzene only. In e f f o r t s to simplify the Giger and Blumer (1974) procedure to be used with high r e s o l u t i o n c a p i l l a r y GC, Giger and Schaffner (1978) used dichloromethane to Soxhlet extract the dry sediments and reduced the time to 6 hours. MacLeod et a l . (1982) reported on i n t e r l a b o r a t o r y comparisons of determinations of trace hydrocarbons i n sediments, conducted by the Northwest and Alaska F i s h e r i e s Centre for the National Oceanic and Atmospheric Adminstration. Two sets of samples were sent to p a r t i c i -pating labs; f o r the f i r s t set of determinations the extraction methods included Soxhlet, r e f l u x , sonication, and tumbler-shaker extraction. In an e f f o r t to improve the comparability of r e s u l t s from d i f f e r e n t labs, Soxhlet or tumbler-shaker extraction were s p e c i f i e d for the second set of determinations. P a r t l y due to these requirements, r e s u l t s from the second round of analyses showed improved reproduci-b i l i t y . It was shown that Soxhlet e x t r a c t i o n of dri e d sediments with dichloromethane i s equivalent to dewatering the sediment using 45. methanol, then extracting with methanol-dichloromethane i n a tumbler shaker. 3. P a r t i t i o n and Clean-Up Aft e r the p o l y c y c l i c s have been extracted from the sediment the extract i s usually cleaned up with some type of solvent-solvent p a r t i -t i o n . If the extract s t i l l contains water, the PAH are extracted into iso-octane (Dunn, 1976; Brown and Starnes, 1978), pentane (Giger and Blumer, 1974; Keizer et al., 1978) or hexane (LaFlamme and Hites, 1978; B i e r i et _al. , 1978). Interfering compounds are l e f t behind i n the hydrocarbon solvent when i t i s p a r t i t i o n e d with dimethylsulfoxide (DMSO)(Natush and Tomkins, 1978; Dunn, 1976), nitromethane (LaFlamme and Hites, 1978) or aqueous N,N-dimethyl-formamide (DMF) as recommended by Wallcave et_ al. (1971). Usually water i s added to these f r a c t i o n s to allow the PAH to be back-extracted into hydrocarbon solvent. Some of the column clean-up methods used are l i s t e d i n Table 2-15. F l o r i s i l i s generally used to separate a l i p h a t i c s f o r aromatics (Dunn, 1976) while s i l i c a or alumina are used as clean-up and/or separation columns. S i l i c a columns were used for clean-up by LaFlamme and Hites (1978) with hexane as eluent. B i e r i et_ al. (1978) also used s i l i c a but eluted the p o l y c y c l i c s with benzene. Brown and Starnes (1978) cleaned up the PAH f r a c t i o n on alumina using a cyclohexane-benzene-methanol system. An alumina system used by S o r r e l l and Reding (1979) r e s u l t s i n three f r a c t i o n s , separating B(a)P/B(e)P, Chr/Tri, and B(b)F, B(k)F from B(a)P. Several procedures for i s o l a t i n g PAH e x p l o i t the d i f f e r i n g proper-t i e s of the adsorbants i n order to obtain clean p o l y c y c l i c residues. 46. Table 2-15. Column Chromatography Systems Column Type E l u t i o n System Function Reference Alumina hexane-benzene-chloroform separation of PASH, PAH, and PANH V a s s i l i a r o s e t a l . , 1982a Bio-beads d ichloromethane clean-up as above S i l i c a pet.ether-pet.ether a c e t o n i t r i l e clean-up Guerrero et a l . , 1976 Alumina cyclohexane-benzene -methanol clean-up Pancirov & Brown, 1977 Alumina pentane through to dichloromethane separation of some PAH (three fractions) and clean-up S o r r e l l & Reding, 1979 S i l i c a Sephadex cyclohexane iso-propanol clean-up 11 tt Grimmer & Bohnke, 1975 F l o r i s i l iso-octane-benzene separation of a l i p h a t i c s , aromatics Dunn, 19 7 6, and Howard, 1979 47. LaFlamme and Hites (1978) use Sephadex LH-20 gel permeation, then silica/alumina i n a single column, charge transfer complex formation followed by further cleanup on a column of s i l i c a by i t s e l f , and concluding with f r a c t i o n a t i o n of the PAH on alumina. With the use of high r e s o l u t i o n chromatographic methods (HPLC or c a p i l l a r y GC) several of these steps can be dropped. Giger and Schaffner (1978) and Grimmer and Bohnke (1979) both use s i l i c a and Sephadex for clean-up, although i n d i f f e r e n t orders. Giger and Schaffner (1978) t e n t a t i v e l y i d e n t i f i e d the interferences (those that pass through s i l i c a but are removed with Sephadex LH-20) as long chain wax esters. MacLeod et al. (1982) recommend following s i l i c a with Sephadex i n order to eliminate alipha-t i c s and also highly alkylated PAH. As long as analysis of only parent p o l y c y c l i c s or mono- to t r i - a l k y l PAH are required, needless i n t e r f e r -ences i n the c a p i l l a r y separations are avoided. 4. High Pressure L i q u i d Chromatography (HPLC) Several techniques have been applied to the i d e n t i f i c a t i o n and quantitation of PAH i n environmental samples. The various techniques have to be able to deal with the large numbers of d i f f e r e n t PAH present i n most samples, the p a r t - p e r - b i l l i o n l e v e l s which must be detected, and the separation of carcinogenic and non-carcinogenic isomers. HPLC has been applied to the analysis of PAH in samples i n the l a s t few years. A persistent problem has been the separation of isomers such as Ph/A, B(a)A/Chr/Tri, B(e)P/B(b)F/B(k)F/B(a)P and B(ghi)Per/lnd(l,2,3,c,d)P. Wise et al. (1980) examined the properties of nine HPLC columns (eight C 1 8 , one amino column). Seventeen PAH (fourteen of the EPA p r i o r i t y pollutants) were run on these columns. 48. The amino column generally separates by number of rings while C l g columns are more s e l e c t i v e . The C 1 8 columns tested were Lichrosorb RP-18, Micropak CH-10, Micropak MCH-10, Nucleosil 10 C l g , P a r t i s i l 5 ODS, Radial PAK A, Vydac 201 TP reverse phase and Zorbax ODS. The Vydac column demonstrated quite d i f f e r e n t s e l e c t i v i t y to the PAH than any of the other columns and w i l l separate 18 PAH very well, including the more d i f f i c u l t PAH. The authors recommend using the amino column for separations by ring size and then taking fractions for analysis on a reverse phase system (preferably Vydac or Zorbax). The use of gradient e l u t i o n can r e s u l t i n improved peak shape and better r e s o l u t i o n . Ogan _et _al. (1979) used an a c e t o n i t r i l e - w a t e r system to optimize separation of a group of 16 PAH. A chemically bonded stationary phase for HPLC, phthalimido-propylsilane (PPS), was prepared for PAH separations (Hunt et a l . , 1977). It i s able to separate B(k)F and Per, and B( e)P/B( cd)F, but not B(e)P/3-me Col. The vast majority of PAH HPLC analyses are done on C l g columns. Some representative studies are summarized i n Table 2-16. A c e t o n i t r i l e water and methanol-water are the most widely used solvent systems (methanol-acetonitrile-water i s also used). The number of PAH determined v a r i e s from two (Guerrero _at al., 1976) to 17 (Crosby et a l . , 1981). It i s i n t e r e s t i n g to note that only one group i s using the Vydac columns recommended by Wise (1980). In many cases only a few PAH are analyzed, avoiding the problem of quantitating isomers. If LC retention time i s the only basis used f o r peak i d e n t i f i c a t i o n , r e s u l t s may not be accurate due to co-eluting p o l y c y c l i c s . The study by Wise et a l . (1980) didn't consider C 8 reversed phase Table 2-16. HPLC Systems Used for PAH A n a l y s i s . Sample Type Column Solvent System Detect ion System Comments Reference O i l s & Fats L iChrosorb SRP18 CH,0H:H 90 95:5 f luorescence : ex3 65,em410. •detect 5 PAH, some i n t e r f e r e n c e from sample Van Heddeghem et a l . , (1980) Smoke concentrate P a r t i s i l 10 ODS 11 CH,0H:CH,CN:H,0 35 : 35 : 30 f luorescence : ex280,em390. •cons iderab le i n t e r -fe rences from samples with e a r -l i e r PAH S i l v e s t e r (1980) Beer RP-18, 5nm CH,OH:CH,CN:H 90 47 .5 : 47 .5 : 5 UV at 287, f luorescence : ex287,em370. •4 PAH s t u d i e d , f l u o r -escence has much l ess i n t e r f e r e n c e Joe et a l . , (198T) S h e l l f i s h pBondapak-Cjg CH,0H:H,0 75 : 25 UV at 383. •no chromatograms inc luded •B(a)P, B(ghi)P only Guerrero et a l . , (1976) Smoked, charcoa l b r o i l e d meats Vydac ODS CH,0H:H 90 87 : 13 UV at 254, f luorescence : ex320-400, em400-700. • s e l e c t i v i t y improved by use of two de t e c -to r s •9 PAH detected but not a l l we l l - reso l ved Panalaks (1976) Meats, smoked L iChrosorb ODS P a r t i s i l PPS CH,0H:THF:H,0 80 : 8 : 12 f luorescence : ex290,em430, ex282,em457. •17 PAH detec ted •marked decrease i n i n t e r f e r e n c e s wi th f luorescence detector •meB( )A i n t e r f e r e , a l so B(b)F/Per • i n t e r f e r ence o f B (a )P/diB(a ,h)A, a l so B(b)F/B(ghi)F/7 -meB(a)A Crosby et a l . , (1981) Sediments, oys te r s L iChrosorb RP-18, 5\im CH,:H,0 70: 30 f luorescence , var ious e x c i -t a t i o n , emis-s i on wavelengths •analyzed for P,A, B(a)P, B(a)A, B(k)F , B(b)F, B (ghi )Per Obana et a l . , (1981^ 50. columns, or smaller p a r t i c l e s i z e C 1 8 columns. The C 8 columns can have better hydrocarbon coverage on the s i l i c a backbone, giving a more hydrophobic surface. This may r e s u l t i n d i f f e r e n t s e l e c t i v i t y f o r PAH. Smaller p a r t i c l e sizes can r e s u l t i n better chromatography, again g i v i n g p o t e n t i a l f o r improved analysis of p o l y c y c l i c s . In e f f o r t s to improve r e s o l u t i o n on HPLC columns various research-ers have investigated very small diameter columns, following the same move as i n c a p i l l a r y columns for GC. Since the resistance to mass transfer i s about 101* times greater i n l i q u i d phase than i n gas phase, the columns should be even smaller i n t e r i o r diameter than GC columns. Several approaches to t h i s problem have been taken, including open columns with chromatographic phase coated on the i n t e r i o r walls, and very small p a r t i c l e s of packing materials i n micro-bore columns. In either case the c a p i l l a r y HPLC columns requires low dead volume sample introduction, connections, and detector c e l l s . Scott and Kucera (1979) compared peak shapes for various detector c e l l volumes and the reduc-t i o n of band width with lower c e l l volumes can be e a s i l y demonstrated. One benefit of such low volume system i s the reduction i n mobile phase requirements; flow rates are t y p i c a l l y ul/min as compared to ml/min i n a more conventional HPLC system. Although sample capacity i s very low, much higher r e s o l u t i o n can be obtained, u s u a l l y at the expense of analysis times. Tsuda et_ al. (1978) coated the walls of a c a p i l l a r y tube (0.06 mm) with the reverse phase of octadecylsilane. A mixture of 7 PAH stand-ards was separated on a column with about 600 plates/metre. In i s o c r a t i c e l u t i o n , i t required 90 minutes to elute pyrene at 1 u&/min. This could be reduced to 20 minutes by the use of gradient e l u t i o n . 51. The authors suggest that a monolayer of stationary phase may improve mass transfer. Also they note that k' i s generally under 2 while i t should be 10 or greater f or adequate separation of complex mixtures. Hirata and Novotny (1979) used a packed m i c r o - c a p i l l a r y column to separate PAH i n a coal tar extract. The column was 55 m long and 0.070 mm i n t e r i o r diameter, packed with 0.03 mm basic alumina-octade-c y l s i l a n e . Using a stepwise gradient from 80% methanol i n water, through methanol to 3% dichloromethane i n methanol, separation of the coa l tar was achieved i n about 17 hours. Sixteen d i f f e r e n t p o l y c y c l i c s were i d e n t i f i e d out of the f i f t y to hundred peaks present. Scott and Kucera (1979) also applied microbore packed columns f o r the separation of p o l y c y c l i c s i n coal tar. The column was packed with P a r t i s i l 0DS-2 and was used with an i s o c r a t i c acetonitrile-water system at 5 ul/min. The coal tar sample was run for over twenty hours, r e s u l t i n g i n over 120 peaks i n the chromatogram. The l a s t peak to be eluted had a k' of 60 and was s t i l l well separated from preceding peaks. Continuing research i n c a p i l l a r y LC columns may r e s u l t i n important separation c a p a b i l i t i e s for complex p o l y c y c l i c mixtures. The column and the solvent system are used to manipulate the el u t i o n of analytes but temperature control i s often ignored. Chmielowiec and Sawatzky (1979) studied the e f f e c t s of temperature control on PAH separations. The control of the system temperature can r e s u l t i n changing e l u t i o n orders f o r some p o l y c y c l i c p a i r s . Thus, peaks which co-elute at one temperature (3,4-BF and 9,10-dimeB(a)A at 25°C) w i l l be resolved at a d i f f e r e n t temperature (45°C or p a r t i a l r e s o l u t i o n at 15°). Temperature gradients during a run w i l l r e s u l t i n 52. improved peak shape f o r l a t e r - e l u t i n g peaks, and may change e l u t i o n orders. HPLC i s used f o r PAH an a l y s i s because e l u t i o n times f o r some larger p o l y c y c l i c s can be much shorter than for packed-column GC, some isomer pa i r s are better resolved than by GC analysis and the detection systems o f f e r extra s e n s i t i v i t y and s e l e c t i v i t y . A comparison was made by Christiansen and May (1978) of u l t r a -v i o l e t and fluorescence detectors for HPLC analysis of PAH. They noted that both systems have good s e n s i t i v i t y f o r PAH, and fluorescence detectors can be used to increase s e n s i t i v i t y . At 254 nm a UV detector's s e n s i t i v i t y to PAH would range from 0.16 ng i n j e c t e d f o r N to 0.025 ng injected for Ph. Fixed wavelength detectors can provide an order of magnitude greater s e n s i t i v i t y at a given wavelength than do variable wavelength detectors. Since p o l y c y c l i c s absorb strongly at 2 54 nm, a fixed wavelength detector at 254 nm can provide comparable s e n s i t i v i t y to the fluorescence detector, although less s e l e c t i v i t y . It has been found that UV detectors i n series can be used to aid in i d e n t i f i c a t i o n of peaks and i n the se l e c t i v e detection of various p o l y c y c l i c s . S o r r e l l and Reding (1979) used three UV detectors i n series enabling them to s e l e c t i v e l y monitor a given p o l y c y c l i c i n a co-eluting PAH peak, or i n a poorly resolved peak. Even f o r peaks that weren't subject to interferences ( p o l y c y c l i c or other) the choice of wavelength allowed quantitation at the optimum wavelength, lowering the detection l i m i t s i n some cases. The detection l i m i t for the i n d i v i d u a l p o l y c y c l i c s varied from 0.2 5 to 1 ng/1. Additional information to aid i n the i d e n t i f i c a t i o n of s p e c i f i c p o l y c y c l i c s i s the UV spectrum of the compound obtained from a selected 53. f r a c t i o n f o r spectrophotometry. The spectra f o r PAH can, however, be quite s i m i l a r for isomers and alkylated PAH. UV spectra of peaks i n a sample can be obtained by c o l l e c t i n g f r a c t i o n s for spectrophotometry; stopping the mobile phase at the top of a peak and using a scanning UV detector to determine the spectrum while the peak i s s t i l l i n the flow c e l l ; or performing rapid scanning of a wide wavelength range during the e n t i r e run. The l a s t approach i s the easiest operationally and can y i e l d the greatest amount of informa-t i o n . Rapid scanning UV detectors generally use e i t h e r a d i f f r a c t i o n grating to produce scans or a photodiode array to monitor many d i f f e r -ent wavelengths at once. With appropriate data processing, these systems can provide UV spectra of the LC eluent at any given time or, i n pseudo three-dimensional p l o t s , spectra over several hundred wave-lengths for the entire LC run (absorbance vs wavelength vs time). This allows the separated peaks to be i d e n t i f i e d by t h e i r spectra. Resolu-t i o n of co-eluting peaks can be improved by choosing to monitor absorb-ance at the X of the analytes of i n t e r e s t . S e l e c t i v i t y can also be max J J improved by monitoring the f i r s t d e r i v a t i v e of the absorbance and setting dA/dt at zero for interferences. Being able to monitor at optimized wavelengths can also r e s u l t i n improved s e l e c t i v i t y . A l l the possible manipulations of the enormous amounts of the data allow much more information to be obtained, e s p e c i a l l y i n the separa-ti o n of complex mixtures. Denton et_ al. (1976) used an o s c i l l a t i n g m i r r o r - d i f f r a c t i o n grating to s p l i t the incident beam into the various wavelengths to be scanned before going to flow c e l l . They were able to obtain uniform r e s o l u t i o n over the whole range (200-930 nm) with a f u l l 54. spectrum of the chosen range each second. An array of 256 s o l i d state diodes was used by Milano et a l . (1976) as an HPLC detector. Half of these (128) were used to determine spectra to store data over longer LC runs. With the data processing used (on tape) the spectra were c o l l e c t e d at an e f f e c t i v e rate of 1 per 2 seconds; t h i s could be changed with other data processing systems. Milano et a l . (1976) found that the use of the f i r s t d e r i v a t i v e of the LC trace was very useful for deconvoluting peaks. Dessy _st _al. (1976) developed an LC detector based on a photodiode array similar to that used by Milano et_ _al. (1976) but with a xenon source rather than deuterium. Fibre o p t i c s were also employed, to reduce transmission and r e f l e c t i o n los ses• The system i s capable of 20 s p e c t r a / s e c , and was used f o r detection of low l e v e l s of carcinogen metabolites i n mouse urine. The UV spectra of metabolites can aid i n t h e i r i d e n t i f i c a t i o n . S e l e c t i v i t y i s obtained with the fluorescence detector by choosing optimum ex c i t a t i o n and emission wavelengths for selected compounds. Thus, by using an e x c i t a t i o n at 271 nm Ph can be detected when i t i s only one-tenth the concentration of anthracene present (Christiansen and May, 1978). In order to quantitate anthracene i n the presence of ten times as much Ph, e x c i t a t i o n at 325 nm i s used. Under these conditions the detector can "tune out" the more concentrated compound and detect the trace component. When dealing with environmental samples, many compounds w i l l absorb UV r a d i a t i o n , but fewer w i l l fluoresce. This r e s u l t s i n cleaner chromatograms, allowing the quantitation of PAH i n the presence of other UV absorbers. An example of the s e l e c t i v i t y of fluorescence detection was 55. provided by wheals _____ a l . (1975) i n t h e i r analysis of f i v e PAH at various excitation/emission wavelength combinations. In a mixture of A,N,F,P and Per, conditions can be set, f o r example, to detect only N (ex 275, em 320), or A, F and P (ex 335 em 385), or Per only (ex 410, em 470). This type of s e l e c t i v i t y was applied to analysis of engine o i l s for p o l y c y c l i c s . Time-resolved fluorescence detection was investigated f o r s e l e c t i -v i t y and s e n s i t i v i t y i n HPLC separation of PAH mixtures by Richardson __t _____. (1980). Laser-induced fluorescence was monitored at 0 to 45 nanosecond (ns) delays, r e s u l t i n g i n s e l e c t i v i t y towards those poly-c y c l i c s with long fluorescence l i f e t i m e s (eg. F, P, or B(l l , 1 2 ) P e r ) . Detection l i m i t s of approximately 20 pg were obtained for F and P. This highly s e n s i t i v e and highly s e l e c t i v e method was used f o r analysis of F i n a coal g a s i f i c a t i o n f r a c t i o n . At 0 ns delay F i s l o s t i n surrounding fluorescence peaks, but at 45 ns delay, the F peak can be c l e a r l y seen. Fluorescence spectra of PAH can be of considerable value i n iden-t i f y i n g the i n d i v i d u a l p o l y c y c l i c s . Fox and Staley (1976) c o l l e c t e d peaks f o r determination of fluorescence spectra. F i r s t and second derivatives of the fluorescence spectra can aid i n detecting trace components and p o t e n t i a l interferences. Certain precautions are necessary for the quantitative fluorescent detection of p o l y c y c l i c s (Fox and Staley, 1976). A n a l y t i c a l standards are required i n order to construct c a l i b r a t i o n curves since quantita-t i o n may be affected by solvent composition, the system of f i l t e r s used, fluorescence e f f i c i e n c y and l i f e t i m e , and e x t i n c t i o n c o e f f i c i e n t of the sample. Quenching by oxygen may also be a problem. 5 6 . Combining the advantages of both UV and fluorescence detectors, Christiansen and May (1978) recommend a UV fixed wavelength detector at 254 nm followed by a fluoresence detector to provide s e l e c t i v i t y . HPLC analysis of PAH o f f e r s the advantages of separation of some troublesome isomer p a i r s , s e n s i t i v e detection, possible i d e n t i f i c a t i o n by recording UV or fluorescence spectra and e l u t i o n of higher molecular weight p o l y c y c l i c s . Disadvantages include some per s i s t e n t isomer separation problems, a b i l i t y to separate a r e l a t i v e l y low number of compounds, and some operational problems with detectors. Research i s continuing on greater s e l e c t i v i t y and separation power for HPLC columns. It i s anticipated that improved detector systems (greater s e n s i t i -v i t y and s e l e c t i v i t y , more spectral information from a single run) w i l l strengthen the whole HPLC system f o r PAH a n a l y s i s . 5. Gas Chromatography (GC) GC i s used extensively f o r the separation and detection of PAH. The separation of isomers on GC columns (packed, and to a much smaller extent, c a p i l l a r y ) i s a s i g n i f i c a n t problem. See Table 2-17 f o r some examples. In t h e i r method f o r analysis of p o l y c y c l i c s , Grimmer and Bohnke (1975a) use a 5% 0V101 column with flame i o n i z a t i o n detector. This system has been used by many i n v e s t i g a t o r s . The OV101 systems w i l l not separate B(a)A/Chr/Tri or the benzofluoranthenes. The authors state that 0V17 packing w i l l provide the B(a)A/Chr separation and c a p i l l a r y column systems are necessary for the benzofluoranthenes. Several of the Dexil s e r i e s of packings have been evaluated for Table 2-17. Column Packings Used i n GC Analysis of PAH TYPE OF SAMPLE COLUMN TYPE CONDITIONS INTERFERENCES REFER-ENCES High-protein foods, o i l s , fats 57% OV101 2 & 3 rings; 120° 240° @ l°/min 4-7 rings, 250° iso-thermal B( a) A/Chr/Tri, B(b)F/B(k)F/ B(ghi)F Grimmer & Bo hnke, (1975) Chlorinated water samples 1.95% SP2401, 1.5% SP2250 182° iso-thermal photo-ionization detection not mentioned Oyler et a l . , 1978 Smoked and charbroiled food 5% 0V101 260° iso-thermal B(a)P/Per, p a r t l y resolved Panalaks, 1976 Atmospheric samples Dexil 300, 400, 410. 0V101 for GC-MS 165° - 295° at 4°/min De x i l 300 Ph/A, B(a)A/ Chr/Tri, B(a)P/ B(e)P, B(ghi)Per/ anthanthrene Dexil 400,410 Ph/A, Chr/ T r i , B(k)F/B(b)F, B(ghi) Per/anth-anthrene Lao et a l . , 1975 Standards only Liquid c r y s t a l BPhBT (see text) 270° to 290° iso-thermal J a n i n i et a l . , 1976 Sediments 2% Dexil 300 100° to 300° Chr/B(a)A Cretney et a l . , 1980 58. p o l y c y c l i c analysis by Lao et_ al. (1975). The greater thermal s t a b i -l i t y of these phases makes them l o g i c a l candidates for chromatographing the higher molecular weight PAH. As can be seen i n Table 2-17, there are several groups of p o l y c y c l i c s which are unresolved on both Dexil 300 and Dexil 400. Dexil 400 can separate, f o r example, B(a)P from Chr or T r i and B(a)P from B(e)P. Since this packing can achieve the same separations as Dexil 410, but with shorter analysis times, Dexil 400 was the packing of choice for Lao and co-workers. Since so many conventional packing materials have trouble separat-ing important p o l y c y c l i c s , considerable in t e r e s t has been shown i n l i q u i d c r y s t a l s as stationary phases. J a n i n i _e_t al. (1976) reported using the l i q u i d c r y s t a l N,N'-bis(p-phenylbenzylidine)a,a'-bi-p-tolui-dine (BPhBT) for PAH a n a l y s i s . Separation of isomers depends on t h e i r length-to-breadth r a t i o ; molecules having a larger r a t i o ( r o d - l i k e molecules) are retained longer. For example, diB(a,c)A, B(ghi)Per, diB(a,h)A, picene and pentacene were a l l baseline resolved at 270°C. There are some serious l i m i t a t i o n s to the use of l i q u i d c r y s t a l s , including a narrow dynamic range (225° to 270° with optimum at 270°C), r e l a t i v e l y low column e f f i c i e n c i e s , some problems with column bleed, short l i f e t i m e s and no data on alkylated PAH. These problems vary among l i q u i d c r y s t a l s and new compounds may be developed to minimize these d i f f i c u l t i e s . Grob and Grob (1975) compared a packed column and a c a p i l l a r y column, both OVl, for PAH analysis. The greater e f f i c i e n c y of the c a p i l l a r y column i s immediately evident; the packed column indicated that a water sample contained 118 PAH compounds, while the c a p i l l a r y column separated 490 components. 59. The great increase i n r e s o l u t i o n i n going from packed to c a p i l l a r y columns reduces the separation problems but i t can be seen from Table 2-18 that there are s t i l l i nterferences. It i s sometimes d i f f i c u l t to assess separation due to lack of information provided by various authors. Also, no s i n g l e system w i l l be able to separate a l l the PAH present in a complex sample, but should separate the important poly-c y c l i c s (e.g. Chr/Tri/B(a)A, or B(e)P/B(a)P) . It i s often the a l k y l a -ted PAH that are extremely d i f f i c u l t to separate (Vassilaros et a l . , 1982a; Lee___tj_l., 1979a; Bjcj>rseth et al., 1979). In studying the e f f e c t of py r o l y s i s temperature on the production of methylated A and methylated Ph , Adams _3_t a l . (1982) used both the non-polar 0V101 and the polar Carbowax 20M to separate the methylated PAH. They weren't able to use the 0V101 f o r separation of l-meA/9-mePh, or 4-mePh/4,5dimePh. One of the problems with using c a p i l l a r y columns f o r PAH i s that, with so many peaks resolved, they then have to be i d e n t i f i e d . The i d e n t i f i e d peak also requires confirmation, preferably by c a p i l l a r y GC-MS. Lee (1979a) developed a system of retention indices for PAH on a c a p i l l a r y SE52 column. A retention index system based on four p o l y c y c l i c s (N, Ph, Chr, picene) shows better r e p r o d u c i b i l i t y (and l e s s s e n s i t i v i t y to changes i n conditions) than the system based on n-alkanes. The indices were i n s e n s i t i v e to column bore s i z e , f i l m thick-ness or the temperature program used. Over two hundred PAH were included i n the retention index, including some alkylated or nitrogen substituted p o l y c y c l i c s . The retention index system was used i n the c a p i l l a r y G.C. analysis of PAH i n f i s h tissue (Vassilaros _3_t _a_L., 1982a). Up to 17 compounds 60. Table 2-18. Ca p i l l a r y Columns Used for PAH Analysis TYPE OF SAMPLE COLUMN TYPE CONDITIONS SOME POSSIBLE INTERFERENCES REFERENCES Mussels 50 m SE 54 120° - 250° mePh/meA, Chr/Tri, B( j)F/B(k)F, diB(a)A/diB(ah)A Bj<j>rseth et a l . , 1979 Smoked meats 55 m SE 54 165° - 225° Chr/Tri, B(j)F/ B(k)F Larsson, 1982 Sediments 20 m SE 52 60° - 250° Chr/Tri Giger & Shaffner 1978 Fish tissue 13-20m SE 52 fused s i l i c a 50° - 250° Ind-P/diBA, alk y l a t e d PAH Vassilaros et a l . , 1982a Atmospheric samples 40 m SE 30 190° - 290° meChr/B(a)A/Tri, mePh/meA, rneBQF/ B()P Choudhury and Bush, 1981 Maize 50 m SE 54 180° - 250° None mentioned for quantitated PAH Winkler et a l . , 1977 Ai r samples 19 m SE 52 70° - 250° m ePh/meA, meBP/ meBF Lee et a l . , 1977 Standards only 12 m SE 52 50° - 250° dimeN, B(b)Fl/meP, B(a)A/B(a)acridine Chr/Tri Lee et al., 1979 Water samples 35 i + 60 i 0V1 25° - 175° 25° - 250° None mentioned Grob and Grob, 1975 P y r o l y s i s products OVIOI, 50 m and 25 m Carbowax 20 M, fused s i l i c a 150° - 230° for OVIOI 100° - 200° for carbo-wax mePh, meA Adams et a l . , 1982 Air samples OV 17 100° - 260° None mentioned Gr immer et a l . , 1980 61. were i d e n t i f i e d by cap GC and confirmation. A BASIC program (written for Hewlett-Packard 5880) was employed to provide tentative i d e n t i f i c a -t i o n through the r e t e n t i o n index system. F i s h extracts were chromato-graphed on fused s i l i c a columns and the effluent s p l i t (to FPD and FID) to enable analysis of s u l f u r heterocycles among the PAH. A nitrogen-phosphorus detector was also used to i d e n t i f y nitrogen heterocycles. Vassilaros and co-workers (1982a) report a detection l i m i t of < 0.2 ppb (wet wt) for PAH i n f i s h . Winkler et al. (1977) reported a s e n s i t i v i t y of 0.25 ng B(a)P by c a p i l l a r y GC while Christiansen and May (1978) report detection l i m i t s of 0.007 to 0.21 ng for HPLC fluorescence or UV detectors. C a p i l l a r y GC's much greater r e s o l u t i o n allows small peaks (possibly shoulders of much larger peaks by HPLC) to be i d e n t i f i e d and quantitated. In general, c a p i l l a r y GC systems allow detection i n the low part per b i l l i o n range (Giger and Schaffner, 1978 and Bj<t>rseth et al., 1979). Futoma et al., (1981) concluded that 0.1 to 30 ng of i n d i v i d u a l PAH can be detected by FID, the most common detector f o r p o l y c y c l i c s . The use of fused s i l i c a columns should r e s u l t i n better chromato-graphy of p o l y c y c l i c s due to fewer active s i t e s . Columns with bonded phases should provide further chromatographic improvements, as well as extended l i f e t i m e s due to resistance to stripping l i q u i d phase by i n j e c t i o n solvents. The FID and mass spectrometer are by f a r the most widely used GC detectors for PAH. The FID i s sens i t i v e to organics but gives l i t t l e information f o r peak i d e n t i f i c a t i o n . As mentioned above, NPD and FPD are also employed for s e l e c t i v e detection of heterocycles (Vassilaros et a l . , 1982a). The photoionization detector (PID) has been applied to 62. p o l y c y c l i c analysis (Oyler et al., 1978) with ten to f o r t y times the s e n s i t i v i t y to PAH. Since t h i s detector has l i t t l e s e n s i t i v i t y to solvents such as a c e t o n i t r i l e or water, f r a c t i o n s from LC ana l y s i s were injected d i r e c t l y . The authors found v a r i a t i o n s i n detector l i n e a r i t y and r e p r o d u c i b i l i t y with prolonged use. These drawbacks may be reduced with improved detector design. 6. Gas Chromatography Mass Spectrometry (GCMS) The GC MS combination has been widely used to provide i d e n t i f i c a -t i o n of p o l y c y c l i c s . Table 2-19 l i s t s some of the applications of GCMS to various sample types. Under conditions of e l e c t r o n impact i o n i z a -tion , PAH have very simple spectra, with l i t t l e fragmentation (Seversen jat a l . , 1976; Hase _et _ a l . , 1976). The M"1" ion i s very stable and quite intense, with a much less intense ( M - l ) + . A double charged ion at (M-2)/2e - i s often large and i n substituted PAH there may be peaks at (M-C 2 H 2 ) 2 + ' The lack of fragmentation under electron impact i o n i z a t i o n makes i t very d i f f i c u l t to d i s t i n g u i s h isomers. Thus f o r isomers that d i f f e r i n t h e i r carcinogenic p o t e n t i a l , the best possible separation by c a p i l l a r y G.C. i s recommended. Lane __t_ a l . (1980) experimented with a quadrupole system with chemical i o n i z a t i o n carried out at atmospheric pressure. When using charge transfer conditions (benzene as chemical i o n i z a t i o n reagent) some isomer pairs could be distinguished on the basis of d i f f e r e n t (M+l)/M r a t i o s . N,Fl, 9-fluorenone, Ph, A, F, and P were studied. Shushan et a l . (1979) investigated linked-scan monitoring of metastable ions to d i s t i n g u i s h isomers of molecular weight 228 (Chr, T r i , B(a)A, B(b)A). In th i s study B and E are linked (B/E i s constant) to transmit 63. Table 2-19. Example of GCMS Systems Used f o r PAH Ana l y s i s . Type of Sample Type of Column MS System Comments Reference water samples 11m SE54 Finnigan 4023, EI used as evaluation of EPA protocol Nowicki et a l . , 1980 standards only -' TAGA 2000 APCI-M, CI mobile, r e a l time sampling Lane et a l . , 1980 f l y ash 30m SE54 HP 5985, SIM complements HPLC with fluorescence Zelensky etal., 1980 combustion effluents Dexil 300 Finnigan 3200, CI, methane confirmation of PAH Strup et a l . , 1976 a i r samples 30m SE52 HP 5982A mixed charge ex-change - chemical i o n i z a t i o n to determine i s o -meric PAH Lee et a l . , 1979b sediments 13-20m SE52 HP 5985 confirmation of PAH Vassilaros et a l . , 1982a. 64. ions with m/e = 228. An integrating ion monitor was set to detect m/e = 228 peak as well as any r e s u l t i n g ions (daughter ion s ) . After examining the r e l a t i v e i n t e n s i t i e s of the daughter ions, only M, (M-l) and (M-2) were eventually monitored. It was found that the r a t i o s of r e l a t i v e i n t e n s i t i e s (M-2)/(M-l) and (M-l)/M are d i s t i n c t i v e f o r each isomer and these r a t i o s can be rela t e d to the number of hydrogen atoms i n each molecule which are capable of benzo isomers (B(b)A,0; B(a)A,l; Chr,2; T r i , 3 ) . Thus the four interactions could be i d e n t i f i e d on the basis of the two r a t i o s (M-2)/(M-l) and (M-l)/M. Using mixed charge exchange-chemical i o n i z a t i o n MS of peaks from c a p i l l a r y GC, Lee j=_t al. (1979b) were able to d i s t i n g u i s h between isomers of unsubstituted PAH i n meA/mePh and meF regions. Using a chemical i o n i z a t i o n reagent con s i s t i n g of 5-10% methane i n argon, quite d i f f e r e n t (M+l) +/M r a t i o s are obtained for A and Ph, as examples. In methylated p o l y c y c l i c s , the PAH isomer can be determined but the posi-tion of the methyl group on the backbone structure cannot be i d e n t i -f i e d . Ten nanograms per PAH i s required f or a spectrum under these conditions (compare to less than one nanogram required for most f l u o r e -scence HPLC dete c t o r s ) . Single Ion Monitoring (SIM) as i n Lane et a l . (1978) can detect as low as one nanogram of compound. Zelensky et a l . (1980) compared r e s u l t s obtained by HPLC with fluorescence to those obtained with SIM c a p i l l a r y GCMS. It was found that the fluorescence system was more s e n s i t i v e but resulted i n some m i s - i d e n t i f i c a t i o n s . They recommend HPLC-fluorescence for screening and GCMS f o r confirmation. A great number of studies on PAH use GC with GCMS as confirmation. The GCMS i s usually c a r r i e d out in the electron impact mode. Despite 65. somewhat lower s e n s i t i v i t y to PAH, the vast amount of s t r u c t u r a l i n f o r -mation provided by c a p i l l a r y GCMS makes i t the method of choice at present (Futoma et a l . , 1981; Lee and Wright, 1980). Even the most e f f i c i e n t c a p i l l a r y GC systems w i l l not be able to separate a l l the PAH present i n environmental samples but the important isomer pairs should be separable. 66. CHAPTER III EXPERIMENTAL TECHNIQUES A. OUTLINE OF ANALYTICAL SCHEME 1. Scope In order to quantitate the extent of PAH residues i n the sediments of the S t i l l Creek system, samples of stream sediments were taken at d i f f e r e n t points along the creek, once i n 1978 and again i n 1979. Preliminary investigations into p o l y c y c l i c l e v e l s incorporated into the sediment organisms was also c a r r i e d out. Since st r e e t run-off was suspected as a source of contamination to the S t i l l Creek system, str e e t surface contaminants were sampled i n the second year of samp-l i n g . And f i n a l l y the contribution of crankcase o i l to street contami-nations was investigated through sampling crankcase o i l at various mileages. As a d d i t i o n a l data, the percent organic content and the p a r t i c l e s i z e d i s t r i b u t i o n of both stream sediments and s t r e e t deposits were determined. A set of twenty PAH were chosen as target compounds i n the analy-t i c a l scheme, these p o l y c y c l i c s were reported i n environmental studies by other researchers. 2. A n a l y t i c a l Scheme Stream sediments, s t r e e t surface deposits and sediment organisms (mainly oligochaetes) were a l l extracted according to the scheme developed f o r sediments and tissues by Dunn (1976). This procedure involves a basic hydrolysis ( i n ethanol) of the sediment, p a r t i t i o n of p o l y c y c l i c s into iso-octane, and clean-up of the extract with F l o r i s i l 67. chromatography and a DMSO iso-octane p a r t i t i o n . A preliminary separa-tion of the PAH was carried out on an alumina column ( S o r r e l l et a l . , 1977) before analysis by HPLC. Crankcase o i l samples were analyzed using an abbreviated form of the extraction procedure for sediments. 3. Instrumental Analysis Sediment extracts were analyzed by reversed-phase HPLC with UV detection. Retention times of the extract peaks r e l a t i v e to retention times of standards ( i n j e c t e d either before or a f t e r the sample) were determined at two mobile phase compositions. These retention times, combined with comparisons of absorbance r a t i o s (absorbance2 5 i + : absorbance28o) a n <* separation into three fract i o n s by alumina chroma-trography, allowed tentative i d e n t i f i c a t i o n of sample peaks to be made. The peaks were c o l l e c t e d (with a larger i n j e c t i o n size) during HPLC separations f o r determination of t h e i r UV spectra. Thus peak i d e n t i f i -cation was based on retention times, UV absorbance r a t i o s and UV spectra. Several sediments were analyzed by c a p i l l a r y GC with a flame i o n i -zation detector. Standard solutions were injected to act as markers and some of the peaks were t e n t a t i v e l y i d e n t i f i e d using the retention index developed by Lee ____ a l . (1979a). GCMSDS (DS = data system) analysis (with c a p i l l a r y column) was ca r r i e d out on a few samples to screen for the twenty target compounds or, i n one case, to examine a l l major peaks for the presence of PAH. 68. 4. Data Analysis Correlations were examined between l e v e l s of s p e c i f i c p o l y c y c l i c s (or also t o t a l quantitated PAH), watershed c h a r a c t e r i s t i c s and various sediment parameters. The t o t a l area of the LC chromatograms (considered as equivalent B(a)P) was also examined with respect to cor r e l a t i o n s with watershed and sediment c h a r a c t e r i s t i c s . The l e v e l s of PAH i n street deposits and i n organisms were treated s i m i l a r l y . Crankcase o i l p r o f i l e s were compared to those of sediments f o r s i m i l a r i t i e s and differences. B. SAMPLING 1. Sediments Stream sediments were sampled at f i v e d i f f e r e n t s i t e s , using some of the locations sampled by researchers H a l l , Yesaki and Chan (1976). The sampling locati o n s are l i s t e d i n Table 3-1 and indicated i n Figure 3-1. Samples were obtained with a metal scoop and stored i n double p l a s t i c bags. Sediments were frozen u n t i l required f o r analysis and after thawing and mixing, a weighed subsample taken for e x t r a c t i o n . 2. Street Surface Contaminants Sampling locati o n s f o r street surface contaminants are also l i s t e d i n Table 3-1 and indicated i n Figure 3-1. Samples were obtained i n September 1979 using a metal spoon and were frozen i n p l a s t i c bags. After thawing and mixing, a weighed subsample was taken for an a l y s i s . 3. Oligochaetes Samples of stream sediments f o r oligochaetes were taken at the »R2 wry. D ( S ) , ( N ) \ ?4 PGr. LAKE, scale in kilometers Figure 3-1 Sampling Location 70. Table 3-1. Sampling Locations Equivalent 3 Location/ Abbre-Land Use v i a t i o n Description Metals Organics Sediments Lougheed L(»78) 49°15'57"N 123°01»05"W 32 21 L('79) Gilmore G('78) 49°15'40"N 123°00'48"W 31 18 G(*79) Willingdon W('78) 49015'33"N 123o00'13"W 30 17 W('79) Douglas D(N)('78) 49°15'38"N 122°58'59"W 35 12 D(N)('79) D(S)('78) D(S)('79) Street Surface I n d u s t r i a l Willingdon north of 401 If interchange Douglas Road at S t i l l Creek I 5 I 5 Commercial C l Canada Way at Boundary Road C l c l c 2 Willingdon at Lougheed c 2 c 2 (Brentwood Shopping Centre) Residential * i E.14 Ave.(Blk.E. of Renfrew) R l R l R 2 E.16 Ave. (between Renfrew and Rupert Street) Green Space G r l Forest Lawn Cemetary G r G l Gr 2 Robert Burnaby Park G 2 G 2 a H a l l et al. 1976. Equivalent sampling s i t e s f o r metals and f o r chlorinated hydrocarbons. 71. same time as the 1979 stream sediments. Organisms and coarse organic de t r i t u s were removed from the sediment with a 0.61 u sieve. The oligochaetes were preserved with small amounts of formaldehyde, and r e f r i g e r a t e d . 4. Crankcase O i l Crankcase o i l was sampled unused and then at i n t e r v a l s of appro-ximately 500 miles (800 km) for up to 5000 miles (8000 km). Samples were c o l l e c t e d through Teflon tubing under a s l i g h t vacuum, and stored (frozen) i n glass v i a l s u n t i l required for a n a l y s i s . C. GENERAL PROCEDURES 1. Glassware Glassware was solvent rinsed a f t e r use and washed with detergent and d i s t i l l e d water. Each piece was rinsed i n a chromic acid bath, and a f t e r thorough r i n s i n g with tap water and d i s t i l l e d water, heated over-night at 150°C. Normal lab glassware was used with the exception of the column for alumina chromatography, which was constructed with a water-cooling jacket. The column was 1 cm i . d . by 25 cm length for packing, with a reservoir and a Teflon stopcock. F l o r i s i l chromatography was c a r r i e d out using a 4 cm i . d . glass column of 40 cm length, also with a Teflon stopcock. 2. Solvents Benzene and ethanol were a n a l y t i c a l reagent grade while dimethyl-sulphoxide (DMSO), dichloromethane and pentane were spectral q u a l i t y . Iso-octane was d i s t i l l e d i n glass over sodium. The methanol or a c e t o n i t r i l e for HPLC were Fisher HPLC grade and were f i l t e r e d (0.5 u f i l t e r s ) before use. Water for HPLC mobile phase was d i s t i l l e d and f i l t e r e d (0.5u f i l t e r ) . 3. Chemicals and Supplies Potassium hydroxide and granular anhydrous sodium s u l f a t e were a n a l y t i c a l reagent grade. Alumina was supplied by BDH (Merck, neutral alumina for column chromatography) and was prepared by heating each portion (20 g) at 150°C f o r 16 hours. Af t e r being removed from the oven, alumina was stored in an evacuated dessicator u n t i l use. F l o r i s i l (100-200 mesh) was prepared according to Dunn (1976), by separating out the fines and washing with water to remove any sodium s u l f a t e . After removing water by r i n s i n g with methanol and drying overnight, the F l o r i s i l was activated at 250°C for 18 hours and cooled before being deactivated to 2% water. F l o r i s i l was prepared i n batches of about 250 g. The f l u o r used f o r l i q u i d s c i n t i l l a t i o n counting i n recovery studies was Bray's solu t i o n , chosen for i t s compatibility with both organic and aqueous phases. Bray's s o l u t i o n i s comprised mainly of dioxane with PP0 (2,5-diphenyl-oxazole, 0.4%) as the primary solute. Secondary solute i s M2P0P0P (2 ,2,1( 1,4-phenylene)bis[ 4-^nethyl-15-phenyloxazole], 20%); ethylene g l y c o l (20%), methanol (10%) and naphathalene (6%) are also added. 4. Determination of P a r t i c l e Size and Organic Content Wet sediments and st r e e t surface contaminants were heated at 110°C 73. to dry thoroughly, and t h i s weight was used i n dry weight c a l c u l a t i o n s . The dried sample was disaggregated i n a mortar and seived, r e s u l t i n g i n four f r a c t i o n s ; coarse sand and larger (>0.589 mm), medium sand (0.589-0. 250 mm), fine or very fine sand (<0.250-0.074 mm) and s i l t or clay (<0.074 mm). Weight of sediment i n each f r a c t i o n was determined. Percent organic matter was calculated by determining the portion of the dry sediment sample which was l o s t on ashing at high temperature (600°C). 5. Standards A n a l y t i c a l standards were obtained f or approximately f i f t y p o l y c y c l i c s . The majority of these were donated through the kind generosity of Bruce Dunn, of Cancer Research. The remaining PAH were ordered from K and K Fine and Rare Chemicals. Solutions of the p o l y c y c l i c s ( s i n g l y or as mixture) for HPLC analysis were dissolved i n a c e t o n i t r i l e (HPLC grade). D. EXTRACTION AND CLEAN-UP PROCEDURES 1. Sediments and Street Surface Contaminants The procedure for extraction of p o l y c y c l i c s from sediment was taken from Dunn (1976), with some minor modifications (Figure 3-2). A weighed sample was refluxed with K0H (5 g), ethanol (100 ml) and b o i l -ing chips for 1.5 hours. Liquid extract was decanted o f f the s o l i d material and f i l t e r e d through a glass wool plug into a 2 l i t r e separa-tory funnel. Sediment was washed with two further portions of ethanol (2 x 50 ml), which were added to funnel. After addition of water (150 ml), the extract was partitioned with iso-octane (3 x 200 ml). These 74. basic hydrolysis of weighed sediment iso-octane p a r t i t i o n F l o r i s i l chromatography DMSO / iso-octane p a r t i t i o n alumina chromatography HPLC (UV) cap. GC cap. GC MS DS Figure 3-2 Analysis Scheme 75. combined organic portions were washed with another four volumes of water (4 x 200 ml). A F l o r i s i l column (30 g F l o r i s i l topped with 60 g Na 2S0i t) was prepared and the iso-octane f r a c t i o n passed through the column, then the a l i p h a t i c s were washed o f f the column with further iso-octane (2 x 100 ml). P o l y c y c l i c s were eluted from F l o r i s i l with benzene (3 x 100 ml) and the benzene was removed from the extract by evaporating on a rotary evaporator to near dryness (5 ml), a d d i t i o n of iso-octane (50 ml) and reducing the volume once again to 5 ml. The iso-octane PAH extract was parti t i o n e d with DMS0 ( 3 x 5 ml) i n order to eliminate some fluorescent interferences. PAH were back-extracted into iso-octane by ad d i t i o n of water (30 ml) to the combined DMSO portions and p a r t i t i o n i n g with fresh iso-octane (2 x 10 ml). Any res i d u a l DMSO was removed with water washes (3 x 20 ml) and extract dried through a bed of sodium sulphate (10 g). Extract volume was reduced to approximately 0.5 ml f o r alumina chromatography. At this point an alumina column was included as a preliminary separation of the p o l y c y c l i c s (see Sorrel _____ al., 1977). The alumina column was prepared by wetting very l i g h t l y packed alumina (16 ml) with pentane (15 ml), and topping with Na 2S0 L f (2 cm). After r i n s i n g with dichloromethane (20 ml), the column was reconditioned with pentane (20 ml). The p o l y c y c l i c extract was added to top of column followed by pentane from two small rinses of the fl a s k . Pentane from the f i r s t e l u t i o n ( I , 30 ml - see Figure 3-3) was used to rin s e the f l a s k . The f i r s t and second f r a c t i o n s (II, 25% CH 2C1 2 i n pentane, 20 ml) were discarded, while the t h i r d , fourth and f i f t h f r a c t i o n s ( I I I 50% Ch^C^ in pentane, 25 ml; IV 50% CH 2C1 2, 5 ml then 75% CH 2C1 2 i n pentane, 30 ml; V CHj-Clj) , 60 ml) were c o l l e c t e d and the solvent evaporated to near 76. alumina column (16 ml) with extract (0.5 ml) elute I pentane (30 ml) - discarded I I 25% CH 2C1 2 i n pentane (20 ml) -discarded I I I IV 50% CH 2C1 2 i n 50% CH 2C1 2 i n pentane (25 ml) pentane (5 ml) then -Ph 75% CH 2C1 2 i n -A pentane (30 ml) -P -F -B(e)P -B(a)P - F l -Chr -2-mePh -B(a)A -3,6-dimePh -Per -9,10-dimeA -B(ghi)Per - T r i -B(a)Fl - B ( a , j ) a c r i d i n e -9-meA V CH2 CX2 (60 ml) - B(b)F - B(k)F Figure 3-3 Alumina Separation of P o l y c y c l i c Extract 77. dryness on a rotary evaporator. Extracts were made up i n 1.0 ml CHgCN for HPLC analysis. 2. Oligochaetes Once the oligochaetes were removed from the sediment with tweezers, they were washed with ethanol to remove water and then dried at 110°C overnight. Dried oligochaetes were ground c a r e f u l l y with mortar and pestle to a fine powder. The extraction of ground organisms continued as f o r sediments. 3. Crankcase O i l A subsample (1 ml) of the crankcase o i l was p a r t i t i o n e d with DMSO ( 3 x 1 ml) then water (5 ml) was added to the combined DMSO f r a c t i o n s . PAH were back-extracted i n t o iso-octane ( 2 x 5 ml) which was dried through Na 2S0 l f before being replaced with a c e t o n i t r i l e (1 ml) for HPLC. E. RECOVERY STUDIES A sediment sample was s piked with 0.5 yCi (0.5 ml) as [7,10- 1 1 +C] B(a)P or as [ 1(4, 5,8)- 1 1 +C]N and extracted as per regular extraction procedure sediments. Sub-samples of extracts from d i f f e r e n t steps i n the procedure were dissolved i n Bray's solution and counted for radio-a c t i v i t y on a LS8000 L i q u i d S c i n t i l l a t i o n Counter (Figure 3-4). To determine recoveries, i t was necessary to make corrections for the r a d i o a c t i v i t y removed at each step i n the procedure. Recovery studies indicated problems with preparation of both F l o r -i s i l and alumina. Standardized procedures for preparing the adsorbents (see General Procedures C-3) resulted i n reproducible chromatography. 78. PROCEDURE ALIQUOT TAKEN SOLVENT Basic hydrolysis of spiked sediment iso-octane p a r t i t i o n F l o r i s i l chromatography DMSO / iso-octane p a r t i t i o n alumina chromatography 'A" equivalent volume of spik-ing solution (toluene) "B" a f t e r d i g e s t i o n (ethanol) "C" before F l o r i s i l (iso-octane) "D" a f t e r F l o r i s i l (iso-octane, benzene) "E" before alumina (iso-octane) *F", I+V a f t e r alumina (pentane, CH_C1_) Figure 3-4 Aliquots Taken for Liquid S c i n t i l l a t i o n Counting 79. F. INSTRUMENTAL ANALYSIS 1. L i q u i d S c i n t i l l a t i o n Counting Liquid s c i n t i l l a t i o n counting was c a r r i e d out using a Beckman LS8000 series counter with a Si l e n t 700 data p r i n t e r . A series of seven standards and one background sample were incorporated into the program i n order to construct a quench curve. Counting e f f i c i e n c y was determined f o r each sample and was indicated by the appropriate "H-number". This allowed automatic quench compensation to be applied to the samples. The preset counting time was 10.00 min. and data reported as normalized disintegrations per minute. 2. HPLC HPLC analyses were c a r r i e d out on a Waters LC system. Model 6000A pumps provided mobile phase flow and were con t r o l l e d by a model 720 System C o n t r o l l e r . Injections were made on the U6K i n j e c t o r . Peaks were detected at 280 mm and 254 mm, with the 440 UV detector. The s i g n a l from 280 mm detector c e l l was integrated by the p r i n t e r - p l o t t e r , model 730 Data Module. Detector parameters were selected manually and wavelengths changed by s e l e c t i n g appropriate f i l t e r s . An a n a l y t i c a l u-Bondapak-C 1 8 column (lOu, 30 cm x 5 ram i.d.) was preceded by a Corasil-Cjg guard column (40p, 4 cm x 7 mm i . d . ) . E l u t i o n was i s o c r a t i c . Conditions used are l i s t e d i n Table 3-2. 3. UV Spectrophotometer UV spectra were obtained for a l l the PAH standards a v a i l a b l e Table 3-2. HPLC Conditions Mobile phase: 60% CH3CN 40% H_0 or 70% CH3CN 30% H_0 Flow: 1.0 ml/min Detector: 280 nm 254 nm AUFS: 0.02 or as required Chartspeed: 0.5 cm/min Area r e j e c t i o n : 100 Noise r e j e c t i o n : 50.000 Table 3-3 C a p i l l a r y GC Conditions Inlet temperature: 225°C Detector temperature: 325°C Oven temperature: - program 40°C f o r 2.00 min 2.00°C/min to 250°C hold 20.00 min post run at 300°C f o r 5.00 min Chart speed: - program 0 to 4.00 min: 0.5 cm/min 4.00 to 60.00 min: 0.25 cm/min 60.00 to 88.00 min: 2.00 cm/min 88.00 to 130.00 min: 0.5 cm/min Attentuation: 2+0 Threshold: 0 Peak width: 0.02 81. (approximately f i f t y compounds). Spectra were also used to aid i n id e n t i f y i n g peaks co l l e c t e d from HPLC f r a c t i o n s . The samples were run on a Cary 15 instrument using a 10 mm path-length. Fractions c o l l e c t e d from HPLC runs were placed i n small volume (approximately 0.7 ml) 10 mm c e l l s . Generally standards or c o l l e c t e d f r a c t i o n s were scanned 400 nm to 220 nm at 5 mm/sec. S e n s i t i v i t y was set at "4" and spectra run at ei t h e r 1 or 0.1 AUFS, as required. 4. C a p i l l a r y GC Several of the samples run on HPLC were also run on a c a p i l l a r y GC system. A Hewlett-Packard 5880A GC with a c a p i l l a r y s p l i t l e s s i n j e c -t i o n system was used with a flame i o n i z a t i o n detector. The fused s i l i c a c a p i l l a r y column was a Hewlett Packard methyl s i l i c o n e (SE54) , 0.31 mm i . d . x 20 m. Samples were injec t e d i n CHC1 3. Conditions used are l i s t e d i n Table 3-3. A post-run heat up was included to prevent interference by l a t e - e l u t i n g peaks i n subsequent runs. 5. GCMSDS GCMSDS analysis was ca r r i e d out on a few of the sediment extracts. An HP 5840 GC with a c a p i l l a r y column was used i n an HP 5985 GCMSDS. The s p l i t l e s s i n j e c t i o n technique was used on an HP SE54 column 30 m x 0.31 mm i . d . The system was cal i b r a t e d for mass with an i n j e c t i o n of perfluorotributylamine. Decafluorotriphenylphosphine (200 ng) was used as i n t e r n a l standard. The GC was programmed from 60°C (held for one minute) to 290°C at 8° per minute. 8 2 . G. STATISTICS TREATMENT Data from HPLC a n a l y s i s of samples was c o l l e c t e d and analyzed f o r possible c o r r e l a t i o n s , using the Triangular Regression Package computer program (Le & T e n i s c i , 1977). This package (revised Oct., 1978) permits a p p l i c a t i o n of regression analysis to various sets of data. Three routines were used to trea t the data: 1) INMSDC - treatment of raw data to produce means, standard devia-tions and c o r r e l a t i o n s , 2) SIMREG - provides simple l i n e a r regression equations, 3) STPREG - step wise regression - performs simples and mul t i p l e regression, also step wise backwards and forwards regression an a l y s i s . The f i n a l regression equation i s i n the form y = b + b,y, + + b y J o l ^ l nrm where b = constant and m = number of v a r i a b l e s . Output from the STPREG routine includes several parameters which allow assessment of the accuracy of regression equation: 1) R 2 - squared m u l t i p l e regression c o e f f i c i e n t - R 2 takes values between 0 and 1, f i t of equation to data improves as R 2 approaches 1, 2) F - p r o b a b i l i t y - p r o b a b i l i t y of obtaining an R 2 value greater than or equal to the value calculated, i f the F - p r o b a b i l i t y i s les s than 0.05, in d i c a t e s that R 2 i s s i g n i f i c a n t l y d i f f e r e n t from zero. Missing data was flagged by use of the code "-99" i n the raw data. In most cases, names were assigned to va r i a b l e s used i n regression 83. a n a l y s i s . Data sets requiring STPREG were generally run using backwards stepwise regression, i . e . forcing a l l variables into the equation at the f i r s t step and allowing the i n s i g n i f i c a n t v a r i a b l e s to be eliminated one at a time. F i l e s were created for required data under the names STREAM, STREET, BUGS, and OILS to apply to the four types of samples analyzed. 84. CHAPTER IV RESULTS AND DISCUSSION A. DEVELOPMENT OF ANALYTICAL SCHEME 1. Introduction In t h i s chapter the r e s u l t s of the experiments outlined i n Chapter III are presented and discussed. Chromatograms (HPLC or c a p i l l a r y GC) are included as required to i l l u s t r a t e r e s u l t s or as t y p i c a l examples. Conditions for each figure may be found i n Appendix 3. 2. Choice of Target Standards Twenty target PAH were chosen such that they s a t i s f i e d the follow-ing requirements: 1) standard material was available; 2) compounds chromatograph on HPLC system used i n experiments; 3) peaks were separ-able on LC column; 4) peaks detectable at both.280 nm and 254 nm; 5) compound eluted from alumina; 6) compound not s i g n i f i c a n t l y s p l i t between alumina fra c t i o n s ; 7) values for absorbance r a t i o s a v a i l a b l e ; 8) PAH had been found i n environmental samples previously; 9) some of the important isomers included; 10) EPA p r i o r i t y pollutants included. Of the compounds targeted 11 are on the EPA p r i o r i t y p o llutant l i s t . The twenty compounds eventually selected (see Figure 3-3 for d i s t r i b u t i o n of the target compounds among the three alumina fractions) were separable at a mobile phase of 60% CHgCN (with three pairs of interferences at higher ambient temperatures) but some co-eluted at 70% CH3CN. See Appendix 3. As well, meA and mePh absorb very weakly at 280 nm, making the r a t i o of absorbance d i f f i c u l t to determine. 8 5 . 3. Choice of A n a l y t i c a l Method P r i o r to s e l e c t i o n of basic h y d r o l y s i s / d i g e s t i o n of sediments as the a n a l y t i c a l approach, other methods were investigated. Samples were o r i g i n a l l y prepared using the Soxhlet extractions described by Giger and Blumer (1974), involving 24-hour extraction with methanol and then the same with benzene. This method was time-consum-ing and required large volumes of solvent. Also the HPLC chromatograms of crude extracts were very complex, so various column clean-up methods were investigated. F l o r i s i l was used with iso-octane to apply crude sediment extract to column while PAH were eluted with benzene. P o l y c y c l i c s were su c c e s s f u l l y eluted, however very l i t t l e f r a c t i o n a t i o n was obtained. Alumina systems were also explored, s t a r t i n g with the system described by Giger and Blumer (1974). PAH were eluted from an alumina column (packed i n pentane) with increasing strength of dichloromethane i n pentane. The majority of the p o l y c y c l i c s encountered i n sediment extracts eluted very quickly, r e s u l t i n g i n l i t t l e improvement in HPLC chromatograms. Various alumina systems were devised using petroleum ether to pack the alumina and then increasing percentages of benzene to elute poly-c y c l i c s . It was found that the e l u t i o n patterns were very s e n s i t i v e to the a c t i v i t y of the alumina. Taking into account the d i f f i c u l t i e s encountered with the extrac-tion system and the alumina clean-up, a combination of the extraction and preliminary clean-up steps from Dunn (1976) and the alumina column chromatography from S o r r e l l _ 3 t _al. (1977) was adopted. This allowed ex t r a c t i o n of the sediments i n 1.5 hours rather than 48 hours, and with 86. smaller volumes of solvent. The alumina system developed by S o r r e l l et a l . allowed separation into three f r a c t i o n s several of the p o l y c y c l i c s which would otherwise i n t e r f e r e during HPLC an a l y s i s ( i . e . B(a)P/B(e)P/B(k)F/B(b)F). 4. Development of LC System O r i g i n a l LC instrumentation comprised a s i n g l e M-6000 pump, syringe i n j e c t o r , a 254 nm detector and a s t r i p - c h a r t recorder. As updated modules were made a v a i l a b l e a system was f i n a l l y assembled with two pumps and a c o n t r o l l e r , loop i n j e c t o r , multi-wavelength detector and a data processor (see Section III F - l , Table 3-2). During the time the LC system was limited to one pump, gradients were developed by adding the stronger eluent through a dropping funnel to the s t i r r e d mobile phase. This approach lacked r e p r o d u c i b i l i t y since flow from the dropping funnel was d i f f i c u l t to adjust accurately. A more reproducible r e s u l t was obtained by drawing the stronger eluent i n t o the mobile phase ( i n a sealed flask) from an unsealed f l a s k . As mixed mobile phase was pumped out, the resultant vacuum drew the stronger eluent i n t o the s t i r r e d mobile phase. It was found that t h i s gave reproducible gradients, with a s l i g h t l y convex p r o f i l e . When two pumps and a system c o n t r o l l e r were i n s t a l l e d , gradients of various shapes were e a s i l y obtained, with each pump supplying a d i f f e r e n t eluent. Eluents investigated include CHgOH or CHjCN i n combination with H£0. A small percentage of iso-propanol was used with CHgOH gradients i n order to sharpen up l a t e r - e l u t i n g peaks. Eventually i s o c r a t i c e lution was chosen i n order to e x p l o i t greater retention time r e p r o d u c i b i l i t y and to eliminate turn-around time between samples. 87. Table 4-1. V a r i a b i l i t y i n Retention Time and Peak Area Retention Standard Standard Sample Time Deviation/(%) Peak Area Deviation/(%) Ph 8.20 8.20 8.71 0.6 (7%) 8.20 9.85 8.20 8.92 F l 8.06 0.04 (0.5%) 4.69 0.73 (13%) 7.93 6.10 7.93 5.71 D(N)('79)III 8.20 0.04 (0.5%) 8.23 8.16 8.43 0.04 (0.5%) 8.43 8.36 10.53 0.07 (0.6%) 10.46 10.40 18.91 0.10 (0.5%) 18.80 18.71 run #12 7 7.95 1.02 -9%* #130 7.95 1.13 #127 8.65 5.71 -8%* #130 8.65 6.19 #127 9.42 5.48 +6%* #130 9.42 5.17 *percent difference wrt #130 88. Other parameters investigated include column temperature, stop-scan chromatography, recycle, flow rate and i n j e c t i o n s i z e . Increased column temperature (achieved with a heating tape i n a glass tube) resulted i n shorter retention times and sharper l a t e r peaks but, since a proper column heater wasn't a v a i l a b l e , temperature control was not continued due to r e p e a t a b i l i t y problems. Stop-scan chromatography was applied to a sediment extract (Perkin Elmer HPLC) but no fin e d e t a i l i n UV spectra was obtained. Recycle chromatography (where two i n t e r f e r i n g peaks are eluted through the LC column several times) was investigated but was not very useful f or sediment extracts since only e a r l y - e l u t i n g peaks can be treated i n this way. The LC conditions f i n a l l y adopted incorporated i s o c r a t i c e l u t i o n at two d i f f e r e n t mobile phase concentrations (CHgCN-^O), monitored at two wavelengths. Flow rate (1.0 ml/min) and i n j e c t i o n s i z e (1.0 to 10 pi) were optimized for the sample extracts. Detector l i n e a r i t y was determined for both wavelengths and a log-log plot of response versus amount injected i s presented i n Figure 4-1. Response on 280 nm detector i s expressed as peak height i n Figure 4-1; response at 254 nm demonstrates s i m i l a r behaviour. Repeatability of retention time was good f o r a seri e s of i n j e c -tions, with standard deviation i n the range 0.5%. (See Table 4-1 for examples.) Variations i n retention times were experienced with changes i n ambient temperature, hence the use of a mixed standard injected before or a f t e r each i n j e c t i o n of an extract. Response (peak area quantitated at 280 nm) demonstrated greater v a r i a b i l i t y (standard devi-a t i o n about 10%) as shown i n Table 4-1. 4000 2000 peak height (mm) 1000 600 400 200 h _L 0.2 0.4 0.6 0.8 1.0 2 Naphthalene (yg) J L_ 8 10 Figure 4-1. L i n e a r i t y of UV Detector, 280 nm. 90. The l i m i t of detection (LOD) for the twenty target PAH vary according to t h e i r UV c h a r a c t e r i s t i c s . In general however, 1 to 2 ng PAH i s required to obtain a 1% f u l l scale d e f l e c t i o n at 0.02 AUFS. (At this attenuation noise i s l e s s than 0.5% f u l l scale.) See Table 4-2 for LOD at 280 nm or 254 nm. These LOD r e f l e c t a detectable p o l y c y c l i c l e v e l i n sediments from the low to middle part per b i l l i o n range (wet weight) . 5. Recoveries Sediment samples were spiked with 1 1 +C B(a)P and ll*C N as described i n section III-E. The spiking l e v e l was 0.29 Pg/g to 0.58 Pg/g (wet weight). The amounts of labeled p o l y c y c l i c were counted at s i x points through the procedure (see Figure 3-4). It was found that counting e f f i c i e n c y varied from point to point, l a r g e l y as a r e s u l t of high c o l o r a t i o n or p a r t i c u l a t e matter i n , most seriously, subsample B ( a f t e r d i g e s t i o n ) . Although both p o l y c y c l i c s o r i g i n a l l y demonstrated v a r i a b l e recovery through F l o r i s i l , N recovery consistently dropped to less than 10% at point D ( a f t e r F l o r i s i l ) . See Figure 4-2 f o r N recoveries over three t r i a l s . Investigations of the problem indicated that heavy loss of N was occurring during rotary evaporation of F l o r i s i l column eluent. No changes were made to the procedure but l i m i t a t i o n s to the scope of the procedure were taken into account. B(a)P recoveries through F l o r i s i l were highly v a r i a b l e (ranging from 4% to 90%) u n t i l a standardized procedure was i n s t i t u t e d f o r pre-paration of the adsorbent ( a c t i v a t i o n overnight at 250°C, deactivation 2%, see Section I II C-3) . Once chromatography on F l o r i s i l was made consistent, B(A)P demonstrated o v e r a l l recoveries of 62% (standard Table 4-2. Limit of Detection f o r PAH Standards With UV Detector PAH LOD (280) a LOD (254) b Ph 1.8 A 1.1 a-ng PAH required f o r a 1% response (AUFS 0.020) at 280 nm. P 2.3 Chr 1.8 F 0.67 b-ng PAH required f o r a 1% response (AUFS 0.020) at 254 nm. B(a) A 0.59 B(a)P 1.1 B(e)P 0.98 B(b)F 1.4 B(k)F 1.5 Per 2.8 B( ghi)Per 2.4 F l 2.4 2-mePh 27 0.027 3,6-dimePh 2.4 9,10-dimeA 50 0.29 9-meA 7.4 0.40 T r i 2.2 B(a)Fl 1.8 diB(a,j)Ac 0.43 92. Figure 4-2. Naphthalene Recoveries, 3 T r i a l s . Figure 4-3. B(a)P Recoveries - No Alumina Preparation Procedure, 3 T r i a l s A before d i g e s t i o n B a f t e r d i g e s t i o n C before f l o r i s i l D a f t e r f l o r i s i l E before alumina F a f t e r alumina deviation 13%) (see Figure 4-3). However problems subsequently devel-oped with control over the a c t i v i t y of the alumina, r e s u l t i n g i n the recovery p r o f i l e s i n Figure 4-4. B(a)P was being eluted i n f r a c t i o n I I , and therefore not c o l l e c t e d for counting (nor analysis i n non-spiked sediments). Proper a c t i v a t i o n procedures for alumina ( s e c t i o n III C-3) f i n a l l y produced reproducible chromatography and high recoveries (96% average, see Table 4-3). With systems i n place f o r preparation of both adsorbents, B(a)P recovery through the en t i r e procedure averaged 84% over three t r i a l s . * This o v e r a l l recovery of 84% for B(a)P i s i n good agreement with B(a)P recoveries reported by Obana et al. (1981) at 81-91%, or Giger and Blumer (1974) at 88%. Dunn and Stich (1976a), using b a s i c a l l y the same procedure as i n t h i s t h e s i s work, had 60-80% recoveries. Giger and Blumer (1974) with t h e i r very thorough f r a c t i o n a t i o n procedure (see Section II E-2, II E-3) reported recoveries of Ph as only 39%, and various other PAH exhibiting recoveries between that and the 88% for B(a)P. In general, data on r e p e a t a b i l i t y i s not often provided i n the l i t e r a t u r e . 6. Sampling Rationale Stream sediments were sampled to provide a p r o f i l e of p o l y c y c l i c concentrations i n the "sink" of the watershed. The sampling l o c a t i o n s were chosen to correspond to those used by H a l l _at_ al., 1976 (Section III A - l ) . H a l l and co-workers studied trace metals and chlorinated hydrocarbons i n the Brunette River watershed (of which S t i l l Creek i s the largest tributary) since i t drains a t y p i c a l urban area, with some *A reagent blank was carried through the procedure, Including the alumina chromatography. HPLC analysis of t h i s blank resulted i n a few small early peaks, elu'ting before any peaks of i n t e r e s t . Table 4-3. E l u t i o n Pattern of B(a)P From Alumina T r i a l 1 2 3 4 Standard Deviation II % B(a)P III IV 10 100 85 100 100 7.5 strong i n d u s t r i a l input. S t i l l Creek drains areas of a l l four major land-use types; commercial, r e s i d e n t i a l , i n d u s t r i a l and greenspace. Five sampling points were chosen f o r stream sediments from those used by Hall and co-workers. The stream was sampled at three points along i t s main branch and at one point on a secondary branch. The sampling point furthest downstream was sampled on both shores of the creek. Each stream sampling point was sampled i n two consecutive years i n order to investigate annual v a r i a t i o n . Sample s i t e s f o r street sediments (also selected from those estab-li s h e d by H a l l _____ a l . , 1976) were chosen to be representative of the land use types encountered i n the Brunette River (and to a smaller extent i n S t i l l Creek) drainage (Table 3-1). Oligochaetes present i n samples were analyzed to investigate possible "competition" relationships between PAH l e v e l s i n sediments and l e v e l s i n organisms. Samples of crankcase o i l were analyzed to examine the p o s s i b i l i t y of used engine o i l acting as a source of p o l y c y c l i c s to the S t i l l Creek system. O i l samples were taken from the engine crackcase a f t e r various mileages to provide p r o f i l e s of PAH build-up over time. 7. Comments on A n a l y t i c a l Methodology Choice of HPLC as the main instrumental method determined the sample preparation procedures and the s p e c i f i c PAH studied (section IV A-2). The dec i s i o n to use HPLC followed consideration of 1) instrumen-tation a v a i l a b i l i t y , 2) instrumental s e n s i t i v i t y to PAH, 3) chromato-graphic resolving power. Instrumentation a v a i l a b l e consisted of packed column GC and HPLC with UV detection. S e n s i t i v i t y of UV detectors to p o l y c y c l i c s i s generally 0.1 to 1 ng injected (see Table 4-2) while FID response for PAH would range from 1 to 30 ng required per compound (s e c t i o n II E-5). HPLC - UV i s thus the better choice with respect to s e n s i t i v i t y . The t h i r d consideration, r e s o l u t i o n , i s important i n p o l y c y c l i c analysis since so many isomers e x i s t f o r the higher molecu-l a r weight compounds. Separation a b i l i t i e s of HPLC and GC are discussed i n section II E-4 to II E-6, but HPLC has the important a b i l i t y to resolve some isomers that would co-elute i n GC an a l y s i s . (Alumina chromatography r e l i e v e s some of HPLC's remaining interference problems.) C a p i l l a r y columns (GC) allow so much improvement i n r e s o l u t i o n of PAH i n comparison to HPLC or packed GC that, had the instrumentation been a v a i l a b l e at the s t a r t of t h i s study, i t probably would have been used rout i n e l y . Retention indices and confirmation of i d e n t i f i c a t i o n by GCMS r e s u l t i n a powerful method. Considerations of safety would r e s u l t i n changes i n several aspects of the sample preparation when further work with p o l y c y c l i c s i s planned. The use of benzene (a human carcinogen) would be reduced i f possible, s u b s t i t u t i n g toluene or dichloromethane f o r e l u t i o n of PAH from f l o r i s i l (section II E-3). Also DMSO w i l l r e a d i l y transport PAH through the skin, providing a possible route f o r ingestion of the carcinogenic p o l y c y c l i c s . Special precautions should be taken to prevent skin contact with DMSO solutions of PAH. In comparison with l i t e r a t u r e methods, the a n a l y t i c a l methodology described f o r t h i s thesis u t i l i z e s a v a i l a b l e f a c i l i t i e s to provide a reproducible, e f f i c i e n t sample workup. The extraction procedure (re-flux with basic ethanol) as adopted from Dunn (1976) allows preparation 98. of a wet sediment i n only 1.5 hours, using small solvent volumes. Pro-cedures used by many researchers (Giger and Blumer, 1974; LaFlamme and Hi t e s , 1978; Keizer _at al., 1978) involved highly time-consuming Soxhlet extractions, which may be required for older compacted s e d i -ments but don't appear necessary f o r l o o s e l y packed stream or street sediments. Solvent-solvent p a r t i t i o n and then clean-up of extract by running through f l o r i s i l eliminates the need to reduce the iso-octane volume, saving time. The combination of f l o r i s i l and then alumina column chromatography re s u l t s i n an extract suitable for HPLC analysis. The alumina system (adopted from S o r r e l l and Reding, 1979) i s designed for use with HPLC detection on reverse-phase column, separating B(a)P from B(e)P, B(k)F, B(b)F and separating Chr from T r i . A s i z e - e x c l u s i o n clean-up was not required for the system used i n t h i s thesis work but was used, f o r example, by LaFlamme and Hites (1978), Vassilaros _3j_ a l . (1982a), or Giger and Schaffner (1978), a l l of whom used GC analysis systems with l e s s s e l e c t i v e flame Ionization detection. The HPLC column used was not Zorbax or Vydac, as recommended by Wise _3_t j a l . . (1980), but the lower p-Bondapak C l g r e s o l u t i o n was p a r t i a l l y overcome by use of the alumina separation. PAH extracts were eluted i s o c r a t i c a l l y , as with the majority of studies using HPLC instrumentation (see Table 2-16). Recoveries of B(a)P through the a n a l y t i c a l procedure (as discussed above) are reproducible and quite acceptable for environmental samples. B. ANALYSIS OF STREAM SEDIMENTS 1. Sediment C h a r a c t e r i s t i c s Subsamples of each stream sediment were taken for determination of 99. organic content and the p a r t i c l e size d i s t r i b u t i o n . The p a r t i c l e size ranges used are presented i n Table 4-4 (along with abbreviations for p a r t i c l e s i z e fractions) while p r o f i l e s of s i z e d i s t r i b u t i o n (and percent organic matter) are graphed i n Figures 4-5 to 4-8. Samples from the same s i t e s f o r 1978 and 1979 are compared i n each fi g u r e . With the exception of samples from Lougheed, sediment c h a r a c t e r i s t i c s were remarkably s i m i l a r i n the two years' samples. Sediments from S t i l l Creek at Douglas Avenue were taken at the north and south ends of a f l o a t i n g weir. Figures 4-5 and 4-6 show the greater percentage of fine sand and clay i n the south end sediments where the water current i s somewhat slower. Willingdon samples (Figure 4-8) were composed mainly of s i l t s , while Gilmore sediments were generally coarse sand or small gravel. As expected, sediment organic matter co r r e l a t e s with percent s i l t and clay (% d i n Figure 4-10). 2. HPLC Analysis Sediment extracts were analyzed on reversed phase columns with acetonitrile-water mobile phase as described in III A-3. Chromatograms of sediments are very s i m i l a r when comparing sediments from the same sampling s i t e . Figure 4-11 shows chromatograms of f r a c t i o n I II of Douglas Avenue samples taken two consecutive years. S i m i l a r i t i e s are also found i n the l a s t two alumina f r a c t i o n s for Douglas Avenue sedi -ments (Figures 4-13 and 4-15). The differences found are i n r e l a t i v e i n t e n s i t i e s of the peaks and i n the amounts of p o l y c y c l i c matter pre-sent. On examination of UV spectra of peaks that matched retention times of standards, differences are found from 1978 to 1979. For a peak with the same r e l a t i v e r e t ention time (RRT) maxima of UV spectra 100. Table 4-4. P a r t i c l e Size Ranges range (mm) > 0.589 0.589-0.250 0.250-0.074 < 0.074 s o i l type coarse sand medium sand fine & very fin e s i l t and clay sand designation a b c d 40 30 20 10 D(S)('79) • D(S)('79) O D(S)('78) OM Figure 4-5. P a r t i c l e Size D i s t r i b u t i o n and Organic Matter. D(S)('78) and D(S)('79) Figure 4-6. P a r t i c l e Size D i s t r i b u t i o n and Organic Matter. D(N)(*78) and D(N)('79) 102. Figure 4-8. P a r t i c l e Size D i s t r i b u t i o n and Organic Matter. W('78) and W('79) 103. Figure 4-10. % Organic Matter (OM) vs. % S i l t and Clay (d) 104. were often s l i g h t l y s h i f t e d , perhaps due to dif f e r e n c e s i n degree of a l k y l a t i o n . No consistent pattern i n UV spectra changes from 1978 to 1979 was observed. Extracts from d i f f e r e n t sampling s i t e s showed some s i m i l a r i t i e s i HPLC traces. This can be demonstrated by comparing alumina f r a c t i o n III from several s i t e s . See traces i n Figures 4-11, 4-12 and the 70% trace i n Figure 4-17. By f a r the largest differences among the sediments was i n the amount of PAH matter present. If the response to UV detection for eac sediment i s normalized per gram of dry weight, t h i s range of response can be e a s i l y demonstrated. Comparing the Douglas 1979 extract to Gilmore extract f or the same year, Douglas gives a response over two orders of magnitude greater than Gilmore. Following procedure outlined i n III A-3 (Instrumental Analysis) the sediment extracts were screened for the twenty target PAH. It was found that In a s i g n i f i c a n t number of cases, the peaks showed appro-priate chromatography for i d e n t i f i c a t i o n as a target p o l y c y c l i c , but the UV spectra indicated the presence of other UV absorbers. This problem was encountered with the more highly contaminated sediments such as Douglas (S) and Willingdon, while "cleaner" extracts (eg. Gilmore) exhibited few such interferences. PAH quantitated i n the ten sediment extracts are l i s t e d i n Table 4-5. Some of the p o l y c y c l i c s quantitated were not included i n the o r i g i n a l set of 20 compounds but have been i d e n t i f i e d through r e l a t i v e retention times, UV absorbance r a t i o s and UV spectras of standard and sample. Table 4-5. PAH Quantltated i n Stream Sediments Sediment PAH ppm (dry wt) D(S)('78) Chr 0.29 B(a)P 1.79 B( b)F 0.47 G('78) B(ghi)F 0.080 P 1.06 B(ghi)Per 0.21 B(k)F 0.099 diB(ai)Ph 0.017 W(*78) Chr 3.99 B(e)P 0.85 B(ghi)Per 1.08 diB(a,i)Ph 0.055 L('78) P 8.22 B(e)P 3.40 D(N)(»79) B(ghi)Per 0.26 D(S)('79) T r i 37.6 B(ghi)Per 38.5 L('79) 2-mePh 3.18 B(b)F 0.057 W( '79) B(a)P 3.14 B(ghi)Per 1.49 106. i " Figure 4-11. Chromatogram of Douglas (N) Extract a) ('78)111 b) ('79)111 107. Figure 4-12. Chromatogram of Willingdon Extract a) ('78)111 b) ('79)111 r Figure 4-13. Chromatogram of Douglas (N) Extract a) ('78)IV b) ('79)IV Figure 4-14. Chromatogram of Lougheed Extract a) ('78)IV b) ('79)IV «' 1; :: 110. Figure 4-15. Chromatogram of Douglas (N) Extract a) ('78)V b) ('79)V 111. t Figure 4-16. Chromatogram of Lougheed Extract a) ('78)V b) ('79)V 112. 113. 3. C a p i l l a r y GC Analysis Extracts of several sediments were analyzed on a c a p i l l a r y GC with FID i n order to exploit the confirmatory advantages of a d i f f e r e n t detector, and most Importantly, to e x p l o i t the considerable resolving powers of the fused s i l i c a c a p i l l a r y column. The extracts Douglas (S) ('78), Douglas (S)('79) and Willingdon ('78) were run on the c a p i l l a r y system described i n Section III F-4. Standards were also run In order to provide r e t e n t i o n time information. Peaks in the mixed standards were i d e n t i f i e d by the use of a retention index system designed for use with p o l y c y c l i c s . Lee _at_ al. (1979a) investigated various retention index systems f o r the c a p i l l a r y a nalysis of PAH but found r e p r o d u c i b i l i t y problems with the system In use for other compounds. Lee and co-workers devel-oped a set of standard p o l y c y c l i c s to define t h e i r retention index (see II E-5) and Vassilaros (1982b) evaluated performance under a wide v a r i e t y of conditions. The p o l y c y c l i c s N, Ph, Chr, and picene are used as markers for the retention index system under s p e c i f i e d temperature-programmed e l u t i o n conditions. The time between these standards i s divided into 100 units and e l u t i o n times of other PAH calculated to 0.01 u n i t . Vassilaros et_ al. (1982b) examined the e f f e c t s of column i n t e r n a l diameter, temperature program, and i n i t i a l temperature, on the i n d i c e s . The r e l a t i v e r e t e n t i o n times maintained good r e p r o d u c i b i l i t y despite v a r i a t i o n s . See Table 4-6 for a compari-son of the standard conditions used by V a s s i l a r o s and co-workers and those used i n t h i s study. It was found that the d i f f e r e n t programming rate and, more importantly, the d i f f e r e n t column coating, r e s u l t i n g i n small differences between the retention indices published and those 114. Table 4-6. Comparison of Conditions f o r Determination of PAH Retention Indices. Parameter Vas s i l a r o s et a l . (1982b) This Study i n i t i a l temp (°C) 40 40 i n i t i a l time (min) 2 2 program rate (°C/min) 4 2 f i n a l temp (°C) 265 250 f i n a l time (min) — 20 column type SE-52 SE-54 column dimensions 20m x 0.31mm 20m x 0.31mm standards used N I = 200.00 Ph I = 300.00 Ph 300.00 Chr 400.00 Chr 400.00 T r i 400.00 picene 500.00 B(ghi)Per 501.32 c a r r i e r gas H 2 He 115. calculated on the SE-54 column. Table 4-7 l i s t s the r e t e n t i o n indices for the standards eluted i n alumina f r a c t i o n III and f r a c t i o n IV. (See Appendix 3 for chromato-grams.) As can be seen from the peak i d e n t i f i c a t i o n i n I I I , r e t e n t i o n data i s n ' t available for a l l p o l y c y c l i c s ( e s p e c i a l l y isomers of alky-lated PAH). The d i f f e r e n c e s i n I ^ ^ v a l u e s (a,b) l i s t e d are due to a 2 minute iso-thermal period incorporated at the beginning of the program i n ref b (Vassilaros et a l . , 1982b). Correlation between I ,. and r e f retention Indices for standard mixtures for t h i s study i s good (0.01% to 1.2% for ST III) although matching i s not perfect. Terminating the oven temperature program at 250°C (rather than continuing to 265°C) makes the r e t e n t i o n index system inapplicable to c a l c u l a t i n g I values for peaks beyond that point (eg. B(ghi)Per i n ST IV), although e l u t i o n orders w i l l be the same. As pointed out by Vassilaros ejt a l . (1982b) absolute agreement i n I values from lab to lab cannot be expected, however the system i s extremely useful i n i d e n t i f y i n g p o l y c y c l i c s by e l u t i o n order, e s p e c i a l l y important for the various isomers. In Table 4-8 through 4-10, retention indices (I) are l i s t e d f o r the samples analyzed by c a p i l l a r y GCFID. Since peaks with tentative I-value match are l i s t e d , often only a small f r a c t i o n of the peaks i n a sample i s represented. (For example, i n sample W('78)IV, I-values were calculated for 41 peaks while only 16 peaks appear i n Table 4-10). For compounds included i n the set of 20 target PAH, i t was known i n which alumina f r a c t i o n to expect the peak, however that information wasn't available for the other p o l y c y c l i c s with retention i n d i c e s , hence a given PAH may be i d e n t i f i e d i n two f r a c t i o n s of a sample. GCMS analysis of sediment extracts w i l l be discussed more f u l l y i n Table 4 -7. Retention Indices f o r Target PAH PAH Standard Retention Time (min) I(calc'd) T a  I r e f I b r e f Peak I d e n t i f i c a t i o n I I I 52.11 265.66 268.17 269.955 F l 62.91 300.00 300.00 300.000 Ph 63.79 302.79 301.69 301.364 A 72.79 331.41 329.13 9-meA 73.91 334.98 a Lee et a l . , 1979a 74.70 337.49 76.98 344.74 344.01 344.471 F b V a s s i l a r o s et a l . , 1982b 78.47 349.48 P 79.32 352.18 351.22 351.484 B( a) F l c trace amount 83.91 366.77 366.74 84.72 c 369.35 94.36 400.00 400.00 T r i 99.06 414.94 108.71 445.63 450.73 B(e)P 490.66 diB(a,j)Ac 2-mePh 3,6-dimeA 9,10-dimePh IV 92.58 398.13 398.50 B(a)A 93.21 400.00 400.00 Chr 107.99 447.57 453.44 B(a)P 108.77 450.08 456.22 Per 501.32 B(ghi)Per Table 4-8. Tentative I d e n t i f i c a t i o n of Peaks by Retention Index ( I ) , D(S)('79) Frac t i o n Retention Time I(calc'd) T 8 * r e f 57.68 287.91 287.69 61.52 300.16 300.00 64.11 308.42 308.79 66.20 315.09 315.19 66.53 316.14 316.37 67.57 319.46 319.46 67.89 320.48 320.17 69.05 324.18 323.90 70.60 329.12 329.13 71.67 332.54 332.59 73.85 339.49 339.38 74.82 342.59 342.45 78.80 355.28 355.49 80.19 359.72 359.91 80.70 361.34 361.38 83.30 369.64 369.39 86.91 381.15 381.56 87.06 381.63 381.85 88.14 385.08 385.35 90.39 392.25 392.50 92.85 400.10 400.00 94.39 405.01 405.35 98.88 419.34 419.39 53.14 269.31 269.67 54.26 272.89 272.57 68.04 316.84 216.37 70.14 323.53 323.33 72.96 332.54 332.59 84.27 368.62 368.62 84.56 369.54 369.39 84.98 370.88 370.86 89.72 386.00 386.36 94.08 399.91 400.00 96.13 406.45 406.54 96.81 408.62 408.30 98.45 413.85 413.78 99.55 417.36 417.16 100.70 421.03 421-.12 101.28 422.88 422.87 105.67 436.88 436.82 47.44 255.24 255.48 52.46 271.26 271.39 62.12 302.07 302.22 66.37 315.63 315.19 69.00 324.02 324.46 77.21 350.21 350.30 80.37 360.29 360.73 82.14 365.94 366.10 94.79 406.29 406.54 102.99 432.45 432.32 Tentative I d e n t i f i c a t i o n MS Correlation I I I IV 1,2,3,4,5,6,7,8-octa-hydroanthracene Ph phenanthr id ine 1- phenylnaphthalene l,2,3,10b-tetrahydro F 3-mePh 2- mePh 1-mePh 9-meA phenylnaphthalene 1,2,3,6,7,8-hexahyd ropyrene 6- phenylquinoline 9,10di-meA 9-me-10-ethyl Ph benzo[ kl] xanthene B(b)Fl 5,12-dihydronaphthacene 9,10-dimethyl-3-e thylphenanthrene 1-ethylP B[c] a c r i d i n e T r i l,2'-binaphthyl 12-meB(a)A trans-l,2,3,4,4a,9a-hexahydrodibenzothiophene 2.3.5- trimethylindole l,2,3,10b-tetrahydro-F 1-meA phenylnaphthalene 1- methyl-7-isopropyl-Ph B(b)Fl 4.5.6- trihydroB(de)A B( a) A Chr 7- B( de) anthrene naphthacene 2-meB( a) A 9-me-10-phenyl Ph 2,2'-biquinoline 1-me Chr l,12dimeB(a)A 2,3-dimeindole cls-l,2,3,4 t4a,9a-hexa-hyd rod ibenzo thiophene benz(h)qulnoline 1- phenylnaphthalene 2- methylacridine 9-n-propylPh m-terphenyl p-ter phenyl 7-benz(de)A 1,3-dimeTri Ph 3-mePh 1-mePh MS at 354.75,Mass 202,P (C 1 3H 1 0N_) Chr Mass243,10-meB( a) a c r i d i n ( CmH 1 Q0 5) 1-me Chr a - Lee et a l . 1979a. 118. Table 4-9. Tentative I d e n t i f i c a t i o n of Peaks by Retention Index ( I ) , D(S)('78) Fraction Retention Time I(calc'd) T a * r e f Tentative I d e n t i f i c a t i o n MS Correlation I I I 62.86 299.84 300.00 Ph Ph 65.00 306.67 306.76 1,2,3,4-tetrahydrocarbazole 68.95 319.21 319.46 3-mePh me-Ph 70.38 323.75 323.90 1-mePh me-Ph 71.93 328.68 328.99 9-n-butylfluorene 73.02 332.15 332.59 2-phenylnaphthalene 76.13 342.04 342.45 6-phenylquinoline phenylquinoline 76.86 344.36 344.01 F F 79.17 351.71 351.22 P P 81.22 358.23 358.53 B[lmn] phenanthridine 82.04 360.83 360.73 m-terphenyl 82.28 361.60 361.38 B[kl]xanthene 85.00 370.25 370.15 2-mePh 99.08 415.02 414.87 1-n-butylP IV 53.34 269.95 269.67 trans-l,2,3,4,4a,9a- -no MS run 56.47 hexahydrodibenzothiophene 280.80 280.48 xanthene 62.78 300.06 300.00 Ph 66.54 312.06 312.13 carbazole 68.04 316.84 316.37 1,2,3,10b-tetrahydro-carbazole 72.14 329.92 329.69 4,5,9,10-tetrahydroP 78.69 350.82 350.30 9-n-propylPh 83.32 365.59 366.10 p-terphenyl 84.95 370.79 370.86 4,5,6-trihydroB(de)A 88.52 382.17 382.09 9-phenylcarbazole 89.35 384.82 385.35 1-ethylP 89.72 386.00 386.34 2,7-dimeP 90.37 388.08 388.38 l,l*-b i n a p h t h y l • 93.90 399.34 400.00 Chr 95.68 405.01 405.35 l,2'-binaphthyl 96.72 408.33 408.30 naphthacene 98.47 413.91 413.78 2-meB(a)A 98.84 415.10 414.87 1-n-butylpyrene 99.50 417.20 417.16 9-me-10-phenyl Ph 99.84 418.29 418.10 3-me Chr 100.68 420.96 420.83 4-me Chr 104.22 432.26 432.32 1,3-dime T r i V 52.60 271.55 271.39 cis-l,2,3,4,4a,9a- -no MS run hexahydrod ibenzo thiophene 53.52 274.48 274.59 4,4'-dimethylbiphenyl 65.86 313.84 313.97 9-ethylcarbazole 69.20 324.50 324.46 2-methylacridine 75.82 345.62 345.78 9-isopropylPh 76.87 348.81 348.54 9-n-hexylFl 83.71 370.79 370.86 4,5,6-trihydroB(de)A 87.34 382.37 382.09 9-phenylcarbazole 93.39 401.67 401.81 benz( a) carbazole 104.51 437.14 436.82 l,12-dlmeB(a)A a - Lee et a l . , 1979a. 119. Table 4-10. Tentative I d e n t i f i c a t i o n of Peaks by Retention Index ( I ) , W('78). Fraction Retention Time I(calc'd) T 8  I r e f Tentative I d e n t i f i c a t i o n I I I 57.64 287.78 278.69 1,2,3,4,5,6,7,8-octahydroA 61.46 299.97 300.00 Ph 64.33 309.12 309.25 benzo(f)quinoline 67.84 320.32 320.17 2-mePh 68.76 323.26 323.33 1-meA 69.00 324.02 323.90 1-mePh 70.57 329.03 328.99 9-n-butylFl 71.63 332.41 332.59 2-phenylnaphthalene 73.82 339.40 339.38 1,2,3,6,7,8-hexahydroP 74.77 342.43 342.45 6-phenylquinoline 80.18 359.68 359.91 9-methyl-10-ethylFh 80.70 361.34 361.38 benzo[ kl] xanthene 81.50 363.90 364.22 4H-benzo[de f]carba zole 82.15 365.97 366.10 p-terphenyl 82.49 367.05 367.04 ll-tneB(a)Fl IV 68.02 316.78 316.37 1,2,3, lObtetrahydroF 77.54 347.15 347.47 2-phenylindole 78.67 350.75 350.30 9-n-propylPh 84.09 368.04 367.97 9,10-diethylPh 84.34 368.84 368.67 l-me-7-isopropylPh 84.95 370.79 370.86 4,5,6-trihydroB[de]A 88.49 382.08 382.09 9-phenylcarbazole 94.03 399.75 400.00 Chr 95.70 405.08 405.35 l,2'-binaphthyl 96.77 408.49 408.30 naphthacene 98.54 414.14 414.37 l-meB(a)A 99.57 417.42 417.56 8-meB(a)A 99.92 418.52 418.72 5-meB(a)A 100.71 421.06 421.12 2,2'-biquinoline 104.28 432.45 432.32 1,3-dimeTri 105.65 436.82 436.82 l,12-dimeB(a)A V 54.26 272.89 272.57 2,3,5-trimethylindole 64.73 306.28 306.78 1,2,3,4-tetrahydrocarbazole 66.53 312.03 312.12 carbazole 67.44 314.93 315.19 1-phenylnaphthalene 68.53 318.41 318.01 9-n-propylFl 77.84 348.11 348.54 9-n-hexylFl 84.05 367.92 367.97 9,10 diethylPh 84.35 368.87 368.67 l-me-7-isopropylPh 84.59 369.64 369.64 4-meP 93.11 396.82 396.54 cyclopenta[cd]pyrene 98.52 414.07 414.37 l-meB(a)A 101.28 422.88 422.87 1-me Chr a - Lee et a l . , 1979a. 120. Section IV B-4, but i t s u t i l i t y f o r confirming GCFID i d e n t i f i c a t i o n s i s demonstrated i n Tables 4-8 and 4-9. The D(S)('79) extract was screened at masses of the 13 target compounds eluted i n alumina f r a c t i o n III, confirming the assignment of a peak at I = 355.28 (9,10-dimeA versus P). F r a c t i o n IV was screened for p o l y c y c l i c s through the whole range of masses. Many PAH compounds i d e n t i f i e d through GCMS weren't included i n the table of retention indices so c o r r e l a t i o n s don't appear i n Table 4-7. GCMS screening of the alumina f r a c t i o n V was again r e s t r i c t e d to the two target compounds, B(k)F and B(b)F. The only other sediment f r a c t i o n analyzed by GCMS i s D(S)('78)111, allowing confirmation of Ph, (and me-Ph) F,P, and phenyl quinoline (Table 4-9). Chromatograms ( c a p i l l a r y GC) are presented i n Figures 4-18, 4-19 and 4-20. The number of peaks resolved on the c a p i l l a r y column i s many times greater than that resolved by HPLC. For example, for W('78)111 54 peaks are integrated on the c a p i l l a r y system while 14 peaks are detected by HPLC. The possible co-elution of p o l y c y c l i c s on HPLC was indicated by the number of peaks which had appropriate retention times but t h e i r UV spectra didn't match those of the standard PAH. The traces from each of the three sediments were compared, f r a c -tion by f r a c t i o n , for s i m i l a r i t i e s i n GC el u t i o n patterns. Alumina f r a c t i o n I I I demonstrates strong s i m i l a r i t i e s between D(S)('79) and W('78) (see Figures 4-18 and 4-20) with many peaks having the same rete n t i o n i n d i c e s , while the f r a c t i o n I I I f o r D(S)('78) shares only a few si m i l a r I values with the other two. The fourth f r a c t i o n i s highly consistent across a l l the sediment extracts, with at l e a s t ten large peaks being common to a l l chromatograms. Fraction V, however, i s v a r i -able, with no observable c o r r e l a t i o n among the ex t r a c t s . The fra c t i o n s Figure 4-18. C a p i l l a r y GC Chromatogram, D(S)('79) a) III , 2ul b) I I I , 0.2 ul Figure 4-19. Ca p i l l a r y GC Chromatogram, D(S)(*78) a) III b) IV ~ 4 4-19 C a p i l l a r y GC Chromatogram, D(S)('78) c) V Figure 4-20. Capillary GC Chromatogram, W('78) a) III b) IV I r ho c) C a p i l l a r y GC Chromatogram, W('78) Continued c) V ho ON 127. showing s i m i l a r p r o f i l e s Indicate a common source f o r the p o l y c y c l i c s , as expected from HPLC traces, and as indicated by workers examining PAH p r o f i l e s i n an urban environment (Wakeham et a l . , 1980a). The d i f f e r -ences i n p r o f i l e s demonstrated by the V f r a c t i o n extracts, or by D(S)III from one year to the next, are more d i f f i c u l t to explain. Perhaps the p o l y c y c l i c s eluted i n f r a c t i o n V are i n d i c a t i v e of. the presence or absence of a p a r t i c u l a r small p o l y c y c l i c source. Build-up time may also be a determining factor In the development of PAH p r o f i l e s , r e s u l t i n g i n differences i n the various extracts. Stream flow c h a r a c t e r i s t i c s could also have an e f f e c t on PAH fractio n s since f l o o d conditions may scour sediments and r e s u l t i n r e d i s p o s i t i o n of po l y c y c l i c s along the stream. It should be pointed out that the c a p i l l a r y GC chromatograms can-not be compared q u a n t i t a t i v e l y to HPLC r e s u l t s due to changes i n t o t a l extract volume. 4. GCMS Analysis Four sediment f r a c t i o n s were analyzed by GCMS (see section III F- " 5). D(S)('79) was screened for target PAH i n fractions I II and V while f r a c t i o n IV was examined i n d e t a i l f o r a wide range of p o l y c y c l i c s . Also, D(S)('78)111 was screened for the masses of the target PAH. De t a i l s are given i n Table 4-11. Injections were made i n chloroform and masses scanned from 30 to 500. In some cases hydroxy- or al k y l - s u b s t i t u t e d PAH were picked up i n the screen for target PAH since the parent ion mass occurred i n t h e i r spectra. An example of t h i s i s seen i n Table 4-12, where isomers of hydroxy Ph or hydroxy A were i d e n t i f i e d i n D(S)('78)111. 128. Table 4-11. Scope of GCMS Analysis. Sample Fraction Mass PAH D(S)('78) III 166 178 192 202 206 216 228 252 279 F l Ph,A 9-meA, 2-mePh P,F 3,6-dimePh, 9,10-dimeA B(a)Fl T r i B(e)P d i B ( a , j ) a c r i d i n e D(S)('79) III -as f o r D(S)('78) -as f or D(S)('78) IV 142-302 -screened f o r p o l y c y c l i c s i n EPA/NIH mass sp e c t r a l data base V 252 B(k)F, B(b)F 129. Retention indices were calculated for the MS peaks by i d e n t i f y i n g Ph at mass 178 and Chr or T r i at mass 228, then comparing scan numbers with those of the peaks i n chromatograms of standards III and IV. Many PAH were found (including p o l y c y c l i c s containing oxygen, s u l f u r or nitrogen) for which no I-value was a v a i l a b l e . For those peaks which were i d e n t i f i e d by GCMS and a retention index, assignment of isomers was possible. It i s t h i s confirmation of basic structure as well as the i d e n t i f i c a t i o n of d i s t i n c t PAH isomers which makes the retention index system so use f u l . Although generally fewer peaks were i d e n t i f i e d when GCMS was used to screen for target PAH, as compared to peaks i d e n t i f i e d using c a p i l l a r y GC r e s u l t s and the retention system, the de t a i l e d screen for p o l y c y c l i c s (D(S)('79)IV) resulted i n i d e n t i f y i n g nearly three times as many peaks as the GCFID system. Many of the peaks i d e n t i f i e d weren't included i n the retention index table. Some of the i d e n t i f i c a t i o n s made by c a p i l l a r y GC-FID (and I value) were not confirmed by GCMS ana-l y s i s . The possible reasons for t h i s include; 1) several peaks having the same or s i m i l a r I values; 2) the compounds indicated by the reten-tion index are not necessarily found i n the EPA/NIH mass spectral data base; 3) the I values are not exactly transferable due to dif f e r e n c e s i n c a p i l l a r y columns, and oven temperature programming. Almost h a l f (40%) of the peaks found i n the d e t a i l e d search of f r a c t i o n IV are alkyl-substituted p o l y c y c l i c s while 20% contain a hetero atom, most often oxygen ( i n hydroxy groups). The a l k y l substi-tutions are normally methyl, occasionally e t h y l . HPLC a n a l y s i s of D(S)('78) i d e n t i f i e d only three PAH, a l l i n fra c -tions IV or V, allowing no comparison between GCMS and HPLC r e s u l t s . Table 4-12. Results of GCMS Analysis. D(S)('78)111. Mass GCMS I d e n t i f i c a t i o n •••MS T a  i r e f Compound^ 178 Ph,A hydroxy-Ph, hydroxy-A 300 300 Ph 194 303.55 194 hyd roxy-Ph, hyd roxy-A 303.95 210 dihydroxy-A 307.13 192 mePh, meA 315.60 319.46 3-mePh 192 mePh, meA phenanthr id ine 319.88 320.17 2-mePh 179 320.55 208 mixture of 320.63 1) phenanthridine 2) 3 * - ( t r i m e t h y l s i l o x y l ) -acetophenone 3) methoxyA 4) methoxyPh 206 dimePh, dimeA 335.40 206 dimePh, dimeA 336.83 206 dimePh, dimeA 337.13 3.6- dimePh 2.7- dimePh 206 dimePh, dimeA 337.43 206 dimePh, dimeA 340.13 206 dimePh, dimeA 341.25 206 dimePh, dimeA 342.23 206 dimePh, dimeA 342.53 202 P,F B(gh i ) F , l - ( l , l - d i m e t h y l e t h y l ) - 4 -phenoxybenzene, cyclopenta[cd] pyrene 343.95 344.01 F 226 347.33 202 P,F 353.55 351.22 P 216 me-P, B(a)Fl, B(b)Fl 383.25 216 as above 389.55 a - Lee et a l . , 1979a b - as confirmed by I value from r e f . a 131. Table 4-13. Results of GCMS Analysis. D(S)('79). Fraction! Mass GCMS I d e n t i f i c a t i o n *MS L r e f Compound III IV 166 178 178 202 192 192 202 206 202 202 228 228 228 228 228 142 142 168 168 180 178 178 194 194 192 211 208 208 194 206 206 206 206 202 202 230 216 244 216 234 228 243 258 Fl Iph.A ,P,F mePh ]mePh meA meA dimePh, dlmeA Chr, Chr, , Chr, T r i , Chr, T r i , Chr, T r i , T r i , B(c)Ph B(c)Ph B(c)Ph B(c)Ph B(c)Ph meN meN 3- mebiphenyl 4- mebiphenyl 9-meFl Ph,A Ph,A dimeFl dimeFl mePh 4-(l-methylethyl)-N- phenylbenzamine methoxyPh methoxyPh 2-phenyl-lH-pyrolo-[2,3-6]pyridine dimePh, dlmeA dimePh, dimeA dimePh, dlmeA dimePh, dimeA P.F P.F p-ter phenyl B(b)Fl 1,3,8-trihydroxy-9H-xanthen-9-one meP B( b) naptho[ 2,1-d] -thiophene B(a)A, Chr, T r i meB(a)acridine l,3,6-trihydroxy-8-methyl-9H-xanthen-9-one 273.80 289.97 300.00 317.46 318.88 324.43 340.71 340.83 347.20 354.75 389.21 400.00 454.23 454.93 462.96 237.66 238.94 257.12 258.25 286.94 300.00 301.42 305.68 312.78 314.34 318.03 319.74 320.31 323.57 337.63 339.62 340.33 341.32 343.03 350.13 363.05 365.88 379.24 370.43 395.14 400.00 406.50 408.91 268.17 300.00 319.46 323.90 339.23 344.01 351.22 391.39 400.00 254.81 288.21 300.00 301.62 337.83 339.23 343.01 351.22 366.01 366.74 370.15 389.37 400.00 Fl Ph 3-mePh 1-mePh 2,7-dimePh B(c)Ph T r i 3-mebiphenyl 2-meFl Ph A 3.6- d imePh 2.7- dimeA F P p-terphenyl B(a)Fl 2-meP B(b)naphtho[2,3-d] thiophene Chr Continued. 132. Table 4-13. Continued. Fraction Mass GCMS Identification lVLS T a  x r e f Compound IV cont'd 248 242 258 242 254 256 256 252 252 252 252 266 266 266 276 meB( b)naphtho[ 2,3-d] -thiophene meChr, meTri, meB(a)A 9,10-dimethyldiB[b,h]-[l,6] naphthyridlne meChr, meTri, meB(a)A 2,2'-binaphthyl dimeB(c)Ph, dimeB(a)A, ethylChr as above B( )F, B( )P, Per B( )F, B( )P, Per B( )F, B( )P, Per B( )F, B( )P, Per meB( j) aceanthrylene meB( j)aceanthrylene meB(j)aceanthrylene B(ghi)Per, Ind[ 1,2,3-cd]P,diB[def, mnoJChr isomer of above isomer of above B( )Chr, diB( )A, B(a)naphthacene, pentacene, pentaphene, diB( )Ph B(ghi)Per, Ind[l,2,3-cd]P, diB[def,mno]Chr as above as above diB( )Chr, B( )Per, naphtho[ l,2-e]P Cor 413.03 416.01 419.71 421.69 423.11 431.49 432.63 439.30 443.99 451.09 455.06 456.63 459.47 462.73 480.34 412.08 416.32 422.87 423.91 432.32 440.92 443.99 450.73 453.44 481.87 7-meB( b) naphtho [2,l-d]-thiophene 1-meTri 1-meChr 2,2*-binaphthyl 1,3-dimeTri B(j)F B(k)F B(e)P B(a)P Ind(l,2,3-cd)P 276 276 278 276 276 276 302 300 475.51 483.04 483.89 489.00 490.92 491.56 515.70 525.92 486.81 Pentacene V 252 B( )F, B( )P, Per 3 peaks a - Lee et a l . , 1979a. 133. a) Figure 4-21. GCMS of D(S)('78)111 a) TI chromatogram b) TI chromatogram, scan #1080 to #1440 tee -i 80 60 H 40 "] e J i 1T9 e ) •can 11406 4 0 6? 1 94 S I 6 3 1 01 U S j , e 139 1E2 i t s 100 i to -40 -20 -4 0 £.0 J... ' i ' ' i—r"<—i 1 i 1 i 1 i — 1 ' 1 > r ,:. eo I O O i£C M O i * o i so i o o LS2 ie-4 354 CJr——l 1 1 ' • -.•-,(, 240 2-tO -'bo ?2C 340 181! 1 ' I j n 1 ; 'I J v '-- is ' 20 ie4 0 i s t o le'sc 1 ?' i C' 1''•,c, l ? t c'- i- O » O 01 L1 w O - O L.'** 4-21 GCMS of D(S)('78) I I I Continued (c) spectrum, scan #1406 (d) TI chromatogram, scan #1800 to #2060 135. lie© ee H 6© 4© -2© -© lie© i 2© t e) scan #1818 191 4© 102 51 fc':;; 115 126 13? 152 I 1 1 1" 'I 224 T*-1—r"» 2 2 0 ,. 4© i" i ' ' " I " ' ' i ' 6© 80 10© 12© - i — i — r 14© 160 I S © 2©© 80 H 6© 4 0 2© © J 2 6 0 4 2J r — • — l — I — ^ - T 2 4 © 2 6 © i — 1 — i — r 3 © © ' — i — ' — i — ' — i — 1 — i — — 1 — r 3 2 © 2 4 © 3 6 © - r — • — r — — r — 1 — r 4 © © 4 2 © 4-21 GCMS of D(S)('78) I I I Continued e) spectrum, scan #1818 136. 1 16666 a) ] ill .•i . v j i , t 1.1 I 1 — i 1 : 1 1 — — r 1 — — i 1 1 1 i 2 8 3 566 850 1133 1416 1699 1983 2 2 6 6 2 5 4 9 2832 3115 3 3 9 9 Figure 4-22. GCMS of D(S)('79)III a) TI chromatogram 137. ? e o g e 177 .7 17B.6 6©7 227.7 228.6 i3?e: 165-7 166.6 216.6 116666 I 1 aT •'I A 1 1 1 1 T 1 r- r-£66 35* 1133 14 16 1699 1933 £366 2549 I . 1 1 7 1 r 1 1 r~ i l l 31 15 33 9'! 4?4 1 4 6 0 2 116666 4-22 GCMS of D(S)('79) III Continued b) screen for masses 178, 202, 228, 166, 216, and 279 1 1 r 1 1 ! 1 1 I Figure 4-23. GCMS of D(S)('79)IV a) TI chromatogram b) TI chromatogram, scan #920 to #1160 5 HITS: REFERENCE FRN 1136© SCAN 9ES 139. » 1 LFRN 3008 SPECT 3932 MW= 216 C17H12 .8?06 1 lH-BenzoEbDfluorene (8Cl'?.:n 1 00 -80 -60 -40 -20 -0 - J .. .1 ' 1 1 1 4 0 ' l 1 80 , i 1-20 i 1 i 1 1 60 i •• • 2 0 0 , , , ,. 1 ..._,„_,„T 24 0 230 - -7 " '1 ' | ' | d) '. I 1 • 1 I V v _ i T 1 1 1 1 1 1 1 1 1 1 1 i r -1200 12 2 0 124 0 1 2 6 G 1 2 8 G 1 3 0 G 1 3 2 0 1 3 4 0 1 3 6 G 1 3 8 0 1 4 G 0 1 4 2 f 14 4 o 4-23 GCMS of D(S)(*79) IV Continued c) spectrum, scan #955 d) TI chromatogram, scan #1200 to 1440 10 HITS: fiVERGGEB SPECTRUM (FRN 11300 +1368 • 1 L F R H 3011 S P E C 7 633 MM* £42 ri'-HM .9313 Chrysen*, 5-ir.ethyl- (8CI9C I :• 4-23 GCMS of D(S ) ( '79) IV Continued e) spectrum, scan #1308 141. — i 1 r 2 1 5 421 64'. 1502 17 IS 1' —1 1 1 :14S 2264 2 5 7 ? Figure 24. GCMS of D(S)('79)V a) TI chromatogram 142. The two p o l y c y c l i c s quantitated by HPLC i n D(S)('79) are also found i n GCMS analysis although I values don't match as expected. T o t a l ion (TI) currents traces f or the four extracts are presented in Figures 4-21 through 4-24. For D(S)('78)111 the TI i s shown i n Figure 4-21 a) a section of the chromatogram i s selected i n b), then the spectrum ( i n c) of scan #1406 (I = 303.95) i d e n t i f i e s the peak as hydroxy A or hydroxy Ph. From a d i f f e r e n t section of the TI chromato-gram i n d) the spectrum of peak at scan #1818 (I = 336.83) indicates dimeA or dimePh. Selected ions were monitored as indicated i n Figure 4-22-b) to pick up the target PAH i n D(S)('79)111. The d e t a i l e d exami-nation of peaks i n D(S)('79)IV i s i l l u s t r a t e d by the examples i n Figure 4-23. TI chromatograms and spectra are presented for B(a)Fl and 1 me-T r i . 5. S t a t i s t i c s Treatment Data on stream sediments was analyzed using the Triangular Regression Package program (see section III-G) to examine possible c o r r e l a t i o n s between various sample c h a r a c t e r i s t i c s and l e v e l s of poly-c y c l i c s found. The f i l e STREAM contained data for the variables set out below f o r the ten sediment samples. 1) "eqB(a)P" - equivalent B(a)P ppm - integrated area under HPLC chromatogram expressed as B(a)P. Normalized to a 1 u l i n j e c t i o n of a 1 ml extract, 1 ppm B(a)P dry wt corresponds to 85.5 area u n i t s . Hence the t o t a l integrated area (normalized to area/g dry wt) over 85.5 equals eqB(a)P. It i s acknowledged that t h i s expression of "area under the curve" doesn't take Into account d i f f e r e n t response factors (area response/ng injected) for 143. d i f f e r e n t p o l y c y c l i c s ; contributions to the t o t a l area by non-PAH components; or any a f f e c t s of chromatography on response f a c t o r s . Nonetheless i t provides a useful technique for quantitating an approximate " t o t a l PAH" i n a sample. (See Table 4-14.) 2) "sum PAH" - the sum of PAH ( i n ppm) quantitated f o r that sample. 3) to 13) - concentrations ( i n ppm) of the i n d i v i d u a l p o l y c y c l i c s ; B(ghi)Per, P, T r i , B(e)P, B(k)F, B(a)P, B(b)F, B(ai)Ph, Chr, 2-mePh, B(ghi)F. 14) "% OM" - percent organic matter. 15) to 18) - sediment size d i s t r i b u t i o n ; % a, % b, % c, and % d. 19) "year" - 1978 or 1979, assigned a value of 1 or 2 r e s p e c t i v e l y . 20) "km" - distance i n kilometres of the sampling point upstream from the entrance to Burnaby Lake (see Table 4-14). 21) " t r a v o l " - t r a f f i c volume - volume (vehicles per 24 hour period) at the nearest junction to sampling point. Volumes were assumed to have been increasing at a rate of 5% per year (as i s documented fo r bridge t r a f f i c 1977 to 1978). See Table 4-14. Values for the three variables which are discussed for the f i r s t time i n t h i s s ection ("eqB(a)P", "km", and " t r a v o l " ) are l i s t e d i n Table 4-14. The f i r s t a p p l i c a t i o n of the TRP set as the independent v a r i a b l e s those numbered 14 to 21 above; the sediment c h a r a c t e r i s t i c s (organic matter, s i z e d i s t r i b u t i o n ) , the year the sediment was c o l l e c t e d , the p o s i t i o n of the sampling point on the stream, and the d a i l y t r a f f i c volume. The dependent va r i a b l e s were set as "eqB(a)P", "sum PAH", as well as the seven quantitated p o l y c y c l i c s and each was examined for c o r r e l a t i o n separately. For these runs both forward and backward 144. Table 4-14. Values of Variables Examined i n Regression Analysis; "eqB(a)P", "km", and " t r a v o l " . Sample "eqB(a)P" i n ppm "km" " t r a v o l " Source Sample Point Volume L ('78) 19.72 5.83 BC a Lougheed @ Boundary 25,983 G ('78) 1.48 3.98 Burnaby Gilmore @ Lougheed 7,330 W ('78) 4.50 3.28 Willingdon S. of Juneau 19,830 D(S)('78) 23.46 1.61 ti Douglas @ S t i l l Creek 11,647 D(N)('78) 2.68 1.61 Douglas @ S t i l l Creek 11,647 L ('79) 12.59 5.83 27,350 G ('79) 0.77 3.98 7,716 W ('79) 26.23 3.28 20,874 D(S)('79) 88.12 1.61 12,260 D(N)('79) 3.07 1.61 12,260 a - BC Ministry of Transport, Communications and Highways b - M u n i c i p a l i t y of Burnaby, Highways Department. 145. regression was used. In a l l cases, regression analysis equations collapsed to a constant. In other words no s i g n i f i c a n t c o r r e l a t i o n among the v a r i a b l e s e x i s t s . By examining the steps of the regression a n a l y s i s , i t was found that the l a s t variables to be forced out of the equations included "% d" and "km". A subset of independent v a r i a b l e s was constructed; "% OM", "% d", "km", and " t r a v o l " . Analysis was run against these with f i r s t "eqB(a)P" and then "sum PAH" as the dependent v a r i a b l e s . Again, only constants were produced as the regression equations. "% d" (percent f i n e s i l t or clay) was c o n s i s t e n t l y one of l a s t v a r i a b l e s forced out. Since the amount of fine p a r t i c u l a t e s i s generally r e l a t e d to a sediments organic content ("% OM"), i t i s n ' t s u r p r i s i n g that i t should have some importance to the PAH load i n the sample. The lack of s i g n i f i c a n t c o r r e l a t i o n s may be due to inadequate numbers of sampling s i t e s , or to considering too few v a r i a b l e s , or not i n c l u d i n g important v a r i a b l e s . Perhaps quantitation of s p e c i f i c compounds over a l l the samples (as compared to at most five) would y i e l d more meaningful r e s u l t s . 6. Summary Quantitative r e s u l t s of HPLC analysis have been set out i n Table 4-5 for s p e c i f i c PAH, and are l i s t e d i n Table 4-14 expressed as equiva-lent B(a)P. The values f o r s p e c i f i c compounds range from 55 ppb to a high of 38.5 ppm (dry wt b a s i s ) . The average of a l l 21 values i s 5.04 ppm but two-thirds of the quantitations f a l l between 55 ppb and 1.79 ppm. See frequency chart, Figure 4-25. When considering values of eqB(a)P, much the same pattern emerges, with 50% of the sediments 146. 16 14 12 10 h 8 r 6 L _ l L 0- 2-2 4 X X 4- 6- 8- 10-6 8 10 12 i n d i v i d u a l PAH, ppm I I 32- 34-34 36 • _ J 36-38 • _X_ 38-40 Figure 4-2 5. D i s t r i b u t i o n of Levels of Individual PAH i n Sediments 3 h 2 h 1 L I 0-5 L 5-10 X X X X X 10- 15- 20- 25- 30-15 20 25 30 35 •i fcfc 80-85 _1 L 85- 90-90 95 eq B(a)P, ppm Figure 4-26. D i s t r i b u t i o n of Levels of Equivalent B(a)P i n Sediments. 147. eqB(a)P values f a l l i n g i n the range 0.77 ppm to 4.50 ppm, with a single high value of 88.12 for D(S)('79) (as expected from high l e v e l s of T r i and B(ghi)Per). Figure 4-26 graphs t h i s d i s t r i b u t i o n . In comparing r e s u l t s obtained i n t h i s study to those obtained by other researchers, sampling areas resembling the urban S t i l l Creek watershed were chosen i f possible. As discussed i n d e t a i l i n section II C, Table 2-9, sediment PAH l e v e l s were determined i n the Charles River of Boston (LaFlamme and Hites, 1978), the developed shore of a European lake (Grimmer and Bohnke, 1975b), Buzzards Bay, Mass. (Hites et a l . , 1977), and r i v e r sediment (Giger and Schaffner, 1978). The values f o r B(ghi)Per and T r i i n D(S)('79) sediment (each i s approxi-mately 38 ppm, dry wt) are higher than any other i n d i v i d u a l reported concentrations, although l e v e l s of 15 ppm (F) and 13 ppm (P) are found in the Charles River (LaFlamme and Hites, 1978). Reported concentra-tions f o r the other p o l y c y c l i c s quantitated i n S t i l l Creek sediments are i n the same range as t h i s study's r e s u l t s , but are generally s l i g h t l y lower. The 38 ppm concentrations of PAH i n D(S)('79) are possibly due to the combination of a f l o a t i n g weir across the creek immediately downstream to the sampling point and the slower current on the south shore, allowing s i g n i f i c a n t amounts of contamination to s e t t l e out at t h i s point. C o l l e c t i o n of sediments samples at the Douglas weir caused small " s l i c k s " of o i l to r i s e to the creek surface at every move of the sample shovel. LaFlamme and Hites (1978) examined the d i s t r i b u t i o n of p o l y c y c l i c s i n sediments from around the world as discussed i n Section II C-2. The majority of the sediments contained p o l y c y c l i c s i n the r e l a t i v e abund-ance (by molecular weight, MW; Ph (MW178) 12%, F (MW202) 16%, P (MW202) 148. 15%, while MW228 isomers comprised 23%, and the remaining 35% was made up of MW252 isomers. Similar i n t e r p r e t a t i o n of data for S t i l l Creek i s d i f f i c u l t since complete molecular weight d i s t r i b u t i o n s aren't a v a i l -able, but several estimations are possible. Quantitative stream sediment data (as a r e s u l t of HPLC analysis) indicates that the most frequently quantitated MW fractio n s are MW252 (3 0% of a l l quantitated PAH) while MW276 accounts for 24% of the deter-minations, and a further 10% are of MW228 isomers. Summing the concen-t r a t i o n s of these f r a c t i o n s across a l l stream sediments, MW228 accounts for the greatest amount, MW276 isomers contribute nearly as much, while MW252 compounds are quantitated at t o t a l concentrations approximately one-quarter those of MW228 or MW276 PAH. It should be noted that there are many peaks present i n HPLC chromatograms which could not be quanti-tated, often due to co-elution of peaks, or no standard available f or matching. Molecular weight d i s t r i b u t i o n data i s most extensive for the sample D(S)('79) where a l l three fractions underwent some form of GCMS an a l y s i s . The frequency of occurrence of each molecular weight was calculated for D(S)('79). The MW252 isomers accounted for 10% of a l l peaks while MW228, MW276, and MW206 compounds were each responsible for 9% of the peaks detected. The f r a c t i o n MW202 was 7% of a l l peaks. The remaining peaks spanned molecular weights from MW142 to MW302. Since alumina fractions III and V were only screened for s p e c i f i c masses (Table 4-11) the molecular weight p r o f i l e s w i l l not be completely accurate. In comparison with data presented by LaFlamme and Hites (1978) the S t i l l Creek analyses confirm the importance of fractio n s with molecular 149. weight 252 (eg. BP, BF, Per) and molecular weight 228 (eg. T r i , Chr, B(a)A), and to lesser extent MW202(F,P). In addition, isomers of MW276 (eg. B(ghi)Per, Ind(l,2,3-cd)P) and MW206 (eg. dimeA, DimePh) made s i g n i f i c a n t contributions to S t i l l Creek sediments. Incidence of Ph (MW178) was not nearly as high f o r S t i l l Creek as for LaFlamme and Hites data. S i m i l a r i t i e s between the two studies support the basic conclusion of LaFlamme and Hites, that the major PAH source to sedi-ments i s combustion. The lack of quantitative GCMS data may explain the decreased importance of MW178 or MW202 f r a c t i o n s to the S t i l l Creek system. The high l e v e l s of po l y c y c l i c contamination i n S t i l l Creek, e s p e c i a l l y the D(S)('79) sample, may provide a greater range of PAH (hence the importance of the MW276 f r a c t i o n ) . F i n a l l y , the d i f f e r e n t MW p r o f i l e s may indica t e a s l i g h t l y d i f f e r e n t combination of combustion sources than found i n areas sampled by LaFlamme and Hites (1978). Giger and Schaffner (1978) reported that P and F are the most abundant compounds i n the r i v e r or lake sediment, or street dust which they sampled. As well, B(a)P and B(e)P were generally present i n approximately a one-to-one r a t i o . S t i l l Creek sediment quantitations (HPLC data) do not confirm P,F as most abundant, perhaps due to peak coeluting problems preventing a l l compounds being i d e n t i f i e d . The only quantitative data a v a i l a b l e on B(a)P, B(e)P l e v e l s indicates that each of these two isomers are present i n si m i l a r concentration ranges (0.5 to 4.0 Pg/g). This concentration ranges compares to the upper end of B( )P data presented by Giger and Schaffner (see Table 2-9, c o l . XXII-XXIV). Several q u a l i t a t i v e aspects of the PAH d i s t r i b u t i o n s found i n t h i s work can be compared to the reports discussed i n section IIC. The 150. degree of a l k y l a t l o n has been used to i n d i c a t e the source of p o l y c y c l i c contamination, since petrogenic sources r e s u l t i n highly alkylated compounds with three to four carbons i n side-chains, while high temper-ature combustion (e.g. carbon black furnaces) produces v i r t u a l l y no a l k y l p o l y c y c l i c s ) . Examination of GC and GCMS data f o r t h i s study indicates a wide v a r i e t y of a l k y l PAH present, but mainly methyl- or dimethyl- compounds. This d i s t r i b u t i o n r u l e s out e i t h e r petrogenics or high-temperature combustion as the main sources to the S t i l l Creek sediments. Discussion by Grimmer and Bohnke (1975b) of sediment p o l y c y c l i c concentrations encompasses several factors that can be addressed with reference to S t i l l Creek r e s u l t s . Grimmer and Bohnke compared sediment l e v e l s of B(ghi)Per and Cor to those found i n vehicular exhaust, which contains s i g n i f i c a n t amounts of these two compounds. As well, the r a t i o of B(a)P to the l e v e l s of B(ghi)Per and Cor i s very high in auto-mobile emissions. The t h i r d d i s t i n g u i s h i n g feature was the absence of benzo [b]naphtho w( 2,1-d)thiophene, generally derived from l u b r i c a t i o n o i l s rather than car exhausts. Applying the se c r i t e r i a , the high l e v e l of B(ghi)Per supports vehicular t r a f f i c as a source although the lack of quantitative data f o r Cor make assessment of r a t i o s among i t , B(a)P and B(ghi)Per impossible. The i d e n t i f i c a t i o n of benznaphthothiophene isomers (GCMS data), however, indicates l u b r i c a t i n g o i l contamination of sediments. It i s reasonable to assume that both factors (vehicular emission and l u b r i c a t i o n o i l s ) contribute to PAH l e v e l s . Investigations by Dunn and Stich (1976) into B(a)P l e v e l s around creosote p i l i n g s , and i n the outflow area of Iona sewage treatment 151. plant, were able i d e n t i f y point sources of B(a)P contamination. No such is o l a t e d source was found i n S t i l l Creek sediments. Equivalent B(a)P l e v e l s didn't co r r e l a t e with the sampling point on the stream ("km"), and land use data for points upstream i s not extensive enough to allow determinations of point sources. The highly s i m i l a r p r o f i l e s among sediment extracts i s i n d i c a t i v e of a general p o l y c y c l i c s source. The study of Brunette River watershed ( i n c l u d i n g S t i l l Creek) by H a l l et a l . (1976) analyzed sediments for trace metals and organo-c h l o r i n e compounds. The r e s u l t s f o r lead and PCB's were compared to eqB(a)P l e v e l s for equivalent sample s i t e s . Sediment lead l e v e l s tended to c l u s t e r around 400 ppm except f o r the Douglas s i t e , where lead was present at 840 ppm. Thus lead l e v e l s showed no d i r e c t corre-l a t i o n with eqB(a)P concentrations, however, annual v a r i a t i o n i n contaminant l e v e l s i s great enough that simultaneous sampling would be valuable. Values for PCB concentration demonstrated considerable scatter. Hall and co-workers report that the middle reach of S t i l l Creek (where sampling points for t h i s t h e s i s work are situated) i s the most heavily contaminated section of the Brunette system. Certainly the range of p o l y c y c l i c quantitations i s comparable to values reported i n other highly urbanized areas (e.g. Charles River, LaFlamme and Hites, 1978) . The p o l y c y c l i c s i d e n t i f i e d include a number of carcinogens, including B(a)P, B(k)F, B(b)F, B(ghi)Per, and cyclopenta(cd)pyrene (see l i s t i n g of carcinogenic potentials i n Appendix 1). Quantitation of carcinogen load to environment i s n ' t possible with the data available here, but p o t e n t i a l f o r a d d i t i o n a l human exposure to these compounds i s demonstrated by t h e i r presence i n the sediments. Estimations of 152. carcinogenic impact are further complicated by the possible i n t e r a c t i o n of the various PAH and also t h e i r action combined with other cancer causes. The a p p l i c a t i o n of s t a t i s t i c s programs to the quantitative data resulted i n no s p e c i f i c c o rrelations among sediment sample character-i s t i c s and p o l y c y c l i c l e v e l s . Other f a c t o r s which could have been investigated include: a) s i t e c h a r a c t e r i s t i c s - flow/current parameters, and the extent of "competition" for xenobiotics by sediment organisms; b) stream c h a r a c t e r i s t i c s - extent of most recent r a i n f a l l and b u i l d -up time since that r a i n f a l l , e f f e c t s of the changes i n season, and length of curb (or street side) drained by that section of stream; c) watershed c h a r a c t e r i s t i c s - patterns of f u e l use i n the watershed, auto exhaust input to watershed, and a quantitation of i n d u s t r i a l or commercial landuse, e s p e c i a l l y upstream of s p e c i f i c sampling s i t e s . C. STREET SEDIMENTS 1. Sediment Ch a r a c t e r i s t i c s P a r t i c l e s i z e d i s t r i b u t i o n and organic matter content were deter-mined for street sediment samples. Size ranges used are the same as described i n Section IV B - l (Table 4-4). D i s t r i b u t i o n s are plotted i n Figures 4-27 to Figure 4-30 and i t can be seen that the sediments have very s i m i l a r p r o f i l e s , with roughly equivalent amounts of the three larger fractions and approximately ten percent fine s i l t and c l a y . Percent organic matter c l o s e l y c o r r e l a t e s with % d ( f i n e s i l t and 153. Figure 4-28. P a r t i c l e Size D i s t r i b u t i o n and Organic Matter, Gr, and 154. Figure 4-30. P a r t i c l e Size D i s t r i b u t i o n and Organic Matter, R_ and R 2. 155. c l a y ) . Street sediments demonstrate much l e s s v a r i a t i o n sample to sample than i s found i n S t i l l Creek sediments, and organic matter i s generally much lower. 2. HPLC Analysis Extracts f o r the eight street sediments (three f r a c t i o n s each) were analyzed by HPLC as described i n Section I I I F-2. Extract i n j e c -t ions were eit h e r preceeded or followed by i n j e c t i o n s of mixed stand-ards . The l e v e l s of quantitated PAH are set out i n Table 4-15, as well as the calculated "equivalent B(a)P" for each sediment. In comparison to l e v e l s i n stream sediments, st r e e t samples have somewhat lower but comparable PAH l e v e l s , and fewer PAH were quantitated. Wakeham et a l . (1980a) reported that material on asphalt-paved st r e e t s c o n s i s t e n t l y contained several times the PAH load of material c o l l e c t e d on concrete s t r e e t surfaces. Unfortunately, s t r e e t samples i n t h i s study weren't chosen to allow consideration of th i s f a c t o r . Examples of HPLC traces of s t r e e t samples can be found i n Figures 4-31 through 4-34. A q u a l i t a t i v e comparison of these p r o f i l e s to those of stream sediments (Figures 4-11 through 4-16) indicate that the p r o f i l e s have many s i m i l a r i t i e s . The same peaks are present i n the majority of the extracts, and most differences are due to varying peak heights. Street and stream extracts, f r a c t i o n IV, have highly s i m i l a r p r o f i l e s . 3. S t a t i s t i c s Treatment Regression analysis (as described i n Section II-G) was applied to 156. Table 4-15. PAH Levels and equivalent B(a)P f o r Street Sediments. Sediment PAH Level (ppm) eqB(a)P (ppm) R l B(k)F 0.056 5.81 R2 P 9,10-dimeA B(ghi)Per 0.70 6.50 0.32 2.31 C l B(ghi)Per 0.63 10.11 c 2 P 0.45 4.11 B(e)P B(ghi)Per 0.65 0.78 \ T r i B(ghi)Per P 0.25 0.20 5.98 3.64 15.61 G r l Gr 2 0.62 P T r i B(k)F 3.52 1.16 0.16 6.92 157. 158. 4-31 HPLC Analysis of Street Sediment R 2 Continued c) v a) I I I b) IV 160. 4-32 HPLC Analysis of C 2 Continued a) I I I b) IV 162. 4-33 HPLC Analysis of I u Continued c) V Figure 4-34. HPLC Analysis of Gr_. a) I I I b) IV 164. 4-34 HPLC Analysis of Gr 2 Continued. c) V 165. the street sediment r e s u l t s to investigate possible c o r r e l a t i o n s . Parameters investigated are l i s t e d below. 1) "eqB(a)P", 2) "sum PAH" - t o t a l concentration of quantitated PAH, 3) to 8) - concentrations of the i n d i v i d u a l p o l y c y c l i c s , 9) "Land use" - four land uses (Gr, R, C, I) assigned values 1 to 4 respectively, 10) "% OM" - percent organic matter, 11) to 14) - sediment size d i s t r i b u t i o n , % a to % d, 15) " t r a v o l " - t r a f f i c volume - vehicles per 24 hour period at nearest monitored junction to sampling points. T r a f f i c volumes may be s l i g h t l y low due to differences i n l o c a t i o n of sampling point and the t r a f f i c monitor, but r e l a t i v e volumes from one sampling s i t e to another should be accurate. Both forward and backward stepwise regression were run on these parameters with each of the following being set as the dependent v a r i -ables i n turn; eqB(a)P, sum PAH, and the i n d i v i d u a l p o l y c y c l i c values. Most t r i a l s collapsed to a constant ( i . e . no s i g n i f i c a n t c o r r e l a t i o n ) but sum PAH was expressed i n terms of % b with an R 2 value of 0.89. Percent organic matter was eliminated i n the penultimate step, i n d i c a t -ing i t s strong contribution. Since both % b and % OM are physical sediment c h a r a c t e r i s t i c s , indications are that the p o l y c y c l i c load i s at l e a s t to some extent determined by the composition of the sediment. 4. Discussion Comparison of s t r e e t extract p r o f i l e s to those of sediment 166. extracts indicated a s i g n i f i c a n t q u a l i t a t i v e s i m i l a r i t y . Quantitations of p o l y c y c l i c s were i n the same range as for sediments. Contribution of s t r e e t sediments PAH to stream deposits appears l i k e l y i n view of q u a l i t a t i v e s i m i l a r i t y and loading l e v e l . Wakeham et a l . (1980a) reported that street dust had the same molecular weight range and simi-l a r a l k y l homologue plots to r i v e r sediments, thus leading them to conclude that street run-off forms an important input to r i v e r sedi-ments. The street sediments sampled by Wakeham et a l . (1980a) demon-strated a preponderance of non-alkylated PAH. The quantitated poly-c y c l i c s i n S t i l l Creek watershed street sediments (see Table 4-15) also tend to be the parent compound, confirming t h i s observation. Alkylated p o l y c y c l i c s were i d e n t i f i e d i n GCMS analysis of stream sediments but no comparable data i s a v a i l a b l e for street sediments. In a study by Zurcher __t j i l . (1980), which investigated the t o t a l hydrocarbon l e v e l s i n highway run-off and i n secondary e f f l u e n t from a sewage treatment plant, an important contribution of highway run-off material to the e f f l u e n t was established. Correlations of hydrocarbon content with lead l e v e l s also implicated vehicular exhaust. Asphalt road surfaces (studied by Wakeham et a l . , 1980a) consist-ently c a r r i e d greater p o l y c y c l i c loads than cement roads. Other contaminants ( p e s t i c i d e s and PCB's) were also i n higher concentrations on asphalt surfaces than on cement surfaces i n an extensive survey of road deposits by Sartor jajt al. (1974). Street surface samples i n the S t i l l Creek study were c o l l e c t e d without regard to whether the surface was asphalt or concrete, or the age of the asphalt. However, one sample of asphalt was extracted (Soxhlet extraction) to investigate t h i s possible PAH source. HPLC analysis resulted i n only a s i n g l e 167. peak, i n c o n t r a d i c t i o n to complexity of asphalt sample analyzed by Wakeham et a l . (1980a). Other factors which could have been assessed i n analysis of street deposits include lead (or metal) content of sediments, asphalt c o n t r i -bution, build-up time p r i o r to sampling, t r a f f i c volumes for s p e c i f i c s i t e s sampled, and a more precise expression of land use. Levels of eqB(a)P for street deposits c o l l e c t e d i n t h i s study, and concentrations of lead and PCB's from the previous study by Hall and co-workers (1976) were compared to determine any c o r r e l a t i o n s . For either contaminant (Pb or PCB), l e v e l s i n greenspace samples were lower than for any other land use, but otherwise the r e s u l t s showed no s i g n i -f i c a n t c o r r e l a t i o n to p o l y c y c l i c l e v e l s determined in t h i s study. D. OLIGOCHAETES Samples for oligochaetes (benthic invertebrates) were obtained at the same time (and the same si t e s ) as the sediment samples i n 1979. The oligochaetes were separated from the r e s t of the sediment and analyzed for p o l y c y c l i c s . Results of these analyses are only p r e l i m i -nary due to deactivation problems with alumina chromatography. B(a)P (used to monitor alumina e l u t i o n behaviour) was eluted too quickly, so some p o l y c y c l i c s i n oligochaete extracts (and i n corresponding sediment extracts) were inadvertently discarded i n alumina fractions I and I I . Comparisons between obligochaetes and the sediments are therefore l i m i -ted to those peaks which were c o l l e c t e d . No attempt was made to account f o r the oligochaete c o n t r i b u t i o n to sediment p o l y c y c l i c l e v e l s for this comparison. Qu a l i t a t i v e comparisons of HPLC traces indicates that only some of 168. the peaks present i n the sediments are found i n the oligochaetes at that sampling point. This observation points to the p o s s i b i l i t y of s e l e c t i v e uptake of PAH by oligochaetes. Sediments with higher PAH l e v e l s tend to share e a r l i e r peaks with oligochaetes while i n "cleaner" samples the l a t e r - e l u t i n g peaks were i n common i n both sediments and oligochaetes. Equivalent B(a)P i s much higher f o r the oligochaetes than for the sediments, perhaps a r e f l e c t i o n of ass o c i a t i o n of p o l y c y c l i c s with organic matter. Also, the eqB(a)P f o r oligochaetes from Gilmore and Douglas (N) are higher than for the more heavily contaminated sed i -ments . As discussed i n Section II B-2, l e v e l s of p o l y c y c l i c s i n aquatic organisms are dependent on the PAH contamination i n the environment, and on the organism i t s e l f . F i s h caught i n the ocean depths generally have PAH l e v e l s below detection l e v e l s , while p o l l u t i o n tolerant s n a i l s contained p o l y c y c l i c s at mid ppm concentrations (see Table 2-4). Bio-concentration of PAH from the surrounding environment varies from an average concentration factor of 6 f o r tubifex, through 20 f o r carp (Black et a l . , 1980). Each compound i s accumulated to a d i f f e r e n t l e v e l . Assessment of bioconcentration by oligochaetes i n S t i l l Creek sediments can only be preliminary, yet yi e l d s some in t e r e s t i n g r e s u l t s . Area under the HPLC trace (normalized to gram dry material) (eqB(a)P) was calculated for each oligochaete extract and the corresponding se d i -ment ext r a c t . The r a t i o of area (oligochaete) to area (sediment) varied tremendously from 1.8 to 127. The highest r a t i o s were obtained from Gilmore (very low organic content) and Douglas (N) (also low organic 169. content). This observation could be explained by the competition f o r PAH occurring between sediment organic matter and l i v e organisms, as postulated by Bindra and H a l l (1979) for explanation of lead d i s t r i b u -tions i n sediments. E. CRANKCASE OILS Study of p o l y c y c l i c s i n crankcase o i l s involved taking samples (at approximately 800 km i n t e r v a l s ) from two d i f f e r e n t v e h i c l e s . O i l from a four c y l i n d e r Toyota car was sampled over 8000 km (RM), while a V-8 Mustang (newly reconditioned engine) was driven 6200 km (DD) during the sampling period. As can be seen from Figures 4-35 and 4-36, the same peaks are present i n each subsample, and the o i l s from both vehicles have the same p r o f i l e . Equivalent B(a)P f o r sample DD was c o n s i s t e n t l y greater than that for RM extracts by a factor of approximately f i v e . Research i n the automotive industry indicates that f u e l composition, engine condition, and combustion temperatures (and to a lesser extent, o i l composition) e f f e c t the quantities of PAH produced. The build-up of p o l y c y c l i c s i n o i l (DD) i s clear i n Figure 4-37 over the f i r s t section of highway d r i v i n g . Once freeway d r i v i n g began, however, the accumulation dropped off and some scatter of r e s u l t s i s evident. The lower PAH l e v e l s may be due to higher engine temperatures during constant highway d r i v i n g . Inadvertent sampling of sludge may explain the one high value i n the freeway d r i v i n g section. In the RM sample series (Figure 4-38) i t i s also clear that the build-up i s occurring. It can be seen that a change i n o i l but not the f i l t e r w i l l reduce o i l p o l y c y c l i c l e v e l s considerably but not eliminate Figure 4-35. Crankcase O i l Sample DD. a) accumulated distance 874 km b) accumulated distance 1665 km 171. vi A i ., m r/-. I Crankcase Oil Sample DD Continued c) accumulated distance 2623 km a) a) accumulated distance 895 km b) accumulated distance 1943 km 4-36 Crankcase O i l Sample RM Continued c) accumulated distance 2545 km 174. Eq B(a)P, ppm 1000 2000 3000 4000 5000 6000 7000 accumulated distance, km 4-37 Eq B(a)P vs accumulated distance - DD Eq B(a)P 1000 2000 3000 4000 5000 6000 7000 4-38 Eq B(a)P vs accumulated distance - RM 175. them. (Unused o i l contained no UV-detectable peaks.) Some peaks concentrated i n o i l s are also present i n sediment samples i n d i c a t i n g possible c o n t r i b u t i o n to sediment p o l y c y c l i c l e v e l s . Peaks i n o i l s tend to be e a r l i e r eluting than the bulk of the sediment extract peaks, suggesting that other sources are also s i g n i f i c a n t f o r sediments. Comparison of r e s u l t s discussed here and those reported by Handa et^ a l . (1979) i n section II C-4 gives agreement with the increase i n PAH due to increased o i l mileage. Also, the d i f f e r e n t accumulation rates found between the two tests run for t h i s thesis Is expected from work done by Handa and co-workers. However, i f car (engine) mileage i s important, RM samples would be expected to contain the higher poly-c y c l i c l e v e l s , rather than DD samples. EqB(a)P f o r o i l samples was found to increase as a l i n e a r function of o i l mileage as compared to a quadratic function as determined by Handa et al.. Disagreement not-withstanding, the dependence of p o l y c y c l i c l e v e l s on o i l mileage was confirmed. One aspect of o i l sample p r o f i l e s which wasn't explored i n t h i s thesis work was the analysis of extracts with the s u l f u r - s p e c i f i c flame photometric detector. This approach could provide more e a s i l y charac-terized p r o f i l e s , aiding i n determining fate of crankcase o i l i n the urban watershed, as described by MacKenzie and Hunter (1979). F. SUMMARY OF RESULTS, SOURCES AND FATES OF PAH Data and i n t e r p r e t a t i o n from previous sections w i l l be summarized here, and the thesis work as a whole w i l l be discussed b r i e f l y . Investigations i n t o l e v e l s and d i s t r i b u t i o n of p o l y c y c l i c s i n 176. S t i l l Creek stream sediments demonstrated that the PAH f r a c t i o n i s very sim i l a r from one sediment to another. The most s t r i k i n g difference among stream sediments i s the range of concentration of p o l y c y c l i c s to be found (eqB(a)P varies by two orders of magnitude). PAH concentra-tions are comparable to those found i n other urban watersheds, although the highest quantitations are greater than any others reported. Possible sources of t h i s p o l y c y c l i c f r a c t i o n include s t r e e t sedi-ments, atmospheric pa r t i c u l a t e f a l l - o u t , v e h i c l e exhaust, l u b r i c a t i n g o i l s , and crankcase o i l s . Examination of stream sediment and street sediment HPLC elu t i o n p r o f i l e s indicates a very strong s i m i l a r i t y between the two types of samples. Quantitated PAH are s i m i l a r i n i d e n t i t y and i n l e v e l for the two sediment types, although the maximum l e v e l s i n stream sediments are not matched by stre e t sediments. Street surface deposits thus appear to be one of the main p o l y c y c l i c sources to stream sediments. The higher concentrations determined f o r the stream may be due to the stream acting as a "sink" for the watershed, or the stream may also receive d i r e c t PAH input from atmospheric p a r t i -culate f a l l - o u t or dumping of l u b r i c a t i o n (crankcase) o i l s . Crankcase o i l extracts demonstrated p r o f i l e s s i m i l a r to street surface samples, but were less well related to stream sediments. This f a c t implicates crankcase o i l as a source of PAH to street sediments, but indicates that both street and stream sediments receive a d d i t i o n a l inputs or are modified by microbial and chemical processes during transport to streams. The indicated transport of p o l y c y c l i c s from streets to stream systems i s supported by Giger and Schaffner (1978), and Wakeham et a l . (1980a), since both groups have determined strong s i m i l a r i t i e s between 177. s t r e e t surface material and watershed sediments. These researchers contend that urban watersheds receive a major portion of their PAH input from street surfaces. Assessment of sources to street sediments i n the Brunette River watershed i s more d i f f i c u l t , since emphasis was placed on analysis of S t i l l Creek sediments. The presence of B(ghi)Per (often quantitated i n S t i l l Creek) i s i n d i c a t i v e of vehicle exhaust while benzonaphthothio-phene isomers are re l a t e d to l u b r i c a t i n g o i l s residues (Grimmer and Bohnke, 1975b). Degree of a l k y l a t i o n (and abundance of alkylated polycyclics) i n stream sediments implicates low to medium temperature combustion as a major source to street materials, and hence to stream sediments. Preliminary analysis of oligochaetes was of in t e r e s t due to the evidence of bioconcentration of c e r t a i n compounds. Degree of concen-t r a t i o n varied by over 100 times, and may be controlled by a competi-t i o n f o r p o l y c y c l i c s between sediment organic matter and l i v e organisms. 178. CHAPTER V SUMMATION, IMPLICATIONS AND RECOMMENDATIONS A. SUMMATION This thesis work investigated the extent of PAH contamination i n selected stream sediments of an urban watershed, S t i l l Creek, and examined some of the possible sources of p o l y c y c l i c s to sediments. The f i r s t section of the thesis concerned the a n a l y t i c a l methodo-logy necessary f o r determining PAH i n sediments. An extraction system was adapted from Dunn (1976) and the re s u l t i n g extract was cleaned-up using alumina chromatography from S o r r e l l __t al. (1977). Samples were analyzed by HPLC, and several of the extracts were also analyzed by GC and GCMS f o r a d d i t i o n a l q u a l i t a t i v e information. In the second part, samples were obtained of stream sediments from S t i l l Creek, (including one set of oligochaete samples), st r e e t sedi-ments from the Brunette River watershed, and crankcase o i l samples. In stream sediments, PAH residues were found i n each sample with l e v e l s of ind i v i d u a l compounds varying from mid part per b i l l i o n (55 ppb) to mid part per m i l l i o n (38.5 ppm). Although the majority of the eleven com-pounds i d e n t i f i e d by HPLC were not al k y l - s u b s t i t u t e d , GCMS analysis indicated that many a l k y l - and hetero-subtituted p o l y c y c l i c s were present. A l k y l s u b s t i t u t i o n was generally methyl or et h y l . The compo-s i t i o n of the PAH f r a c t i o n was very s i m i l a r from s i t e to s i t e , with the greatest v a r i a t i o n occurring i n the concentrations found. Application of regression analysis to the data (eqB(a)P, i n d i v i d u a l PAH, sediment c h a r a c t e r i s t i c s , t r a f f i c volumes, point on stream) did not res u l t i n any s t a t i s t i c a l l y s i g n i f i c a n t c o r r e l a t i o n s but indicated that sediment 179. c h a r a c t e r i s t i c s were important. Analysis of s t r e e t sediments revealed PAH present at s l i g h t l y lower l e v e l s than i n stream sediments. P r o f i l e s of street sediments were sim i l a r to those of the stream sediments, i n d i c a t i n g that street surface runoff i s one of the main source of p o l y c y c l i c s to stream sedi-ments. Average l e v e l s of B(a)P i n each land use increased as generally expected (lowest l e v e l s i n greenspace, highest in i n d u s t r i a l area) although regression analysis pointed to sediment c h a r a c t e r i s t i c s as being important. Investigations of p o l y c y c l i c buildup i n crankcase o i l s revealed a l i n e a r increase of eqB(a)P with mileage at f i r s t and then a l e v e l i n g o f f . P r o f i l e s of o i l extracts corresponded more c l o s e l y to those of street surface materials than to stream sediments. Preliminary r e s u l t s of analysis of oligochaetes indicated that bioconcentration was occurring (accumulations of up to two hundred times i n comparison to sediments) and that concentration of PAH i n organisms may be controlled by competition between organisms and sed i -ment organic matter. In summary, PAH were found to be present i n a l l samples taken i n Brunette River watershed, at l e v e l s comparable with other highly urban-ized areas. Street surface materials were determined to be an important source of p o l y c y c l i c s to the S t i l l Creek sediments. Used crankcase o i l probably contributes s i g n i f i c a n t l y to st r e e t contamina-ti o n , while other po t e n t i a l sources include v e h i c l e exhaust, wear pa r t i c u l a t e s from asphalt or t i r e s and atmospheric p a r t i c u l a t e s from combustion of organic materials. 180. B. IMPLICATIONS Levels of PAH i n sediments are comparable to those found i n other urban watersheds, often those with a much longer h i s t o r y of develop-ment. The po t e n t i a l load of contaminants deposited i n sediments from a large metropolitan area can provide a s i g n i f i c a n t input to aquatic systems. Since such pollutants include carcinogens (eg. PAH) and heavy metals, there i s considerable concern for the health of such productive systems as streams and d e l t a s . P o t e n t i a l e f f e c t s include increased tumour incidence, lowered feeding or reproductive success, or reduced m o b i l i t y . In order to maintain p r o d u c t i v i t y and d i v e r s i t y of these "sink" areas, expensive management strategies may be required to reduce contaminant loads. With respect to input of p o l y c y c l i c s , i t may be necessary to reduce PAH i n vehic l e exhaust, maintain close control on waste crankcase o i l s , or reduce emissions of p o l y c y c l i c s (generally associated with p a r t i c u l a t e s ) r e s u l t i n g from i n c i n e r a t i o n , heating or power generation. C. RECOMMENDATIONS Results from t h i s thesis elucidate some aspects of PAH contamina-tio n of S t i l l Creek, and provide a data base for further inv e s t i g a -t i o n s . a) A n a l y t i c a l methodology could be s i m p l i f i e d for determination of po l y c y c l i c s i n sediments. Extraction of dried sediments with dichloromethane ( V a s s i l a r o s et a l . . 1982a) should be examined. Analysis of extracts by c a p i l l a r y GC, using GCMS as confirmation) would provide improved r e s o l u t i o n and, with GCMS, a d d i t i o n a l 181. s t r u c t u r a l confirmation. Further refinement to the understanding of PAH inputs to stream sediments could be obtained through quantitating the street sur-face contribution to streams. This could be done by monitoring PAH loads i n urban stormwater, c o r r e l a t i n g r e s u l t s to size of catchment areas and curb lengths. Quantitation of land-use would aid i n these investigations as well as for street surface materials. Bioconcentration of PAH i n benthic organisms should be researched, including e l u c i d a t i o n of the apparent s e l e c t i v i t y of oligochaetes i n concentrating s p e c i f i c compounds. The indicated competition between organisms and sediments requires further research. 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"Poly-nuclear Aromatic Hydrocarbons i n Vehicle Exhaust Gas", i n National Combined Farm, Construction and I n d u s t r i a l Machinery and Fuels and Lubricants Meetings, Society of Automotive Engineers, Paper # 730836, 12 pages. Zelenski, S.G., Hunt, G.T. and Pangaro, N., (1980). "Comparison of SIM GC/MS and HPLC f o r the Detection of Polynuclear Aromatic Hydro-carbons i n Fly Ash Collected From Stationary Combustion Sources", i n A. Bj<t>rseth and A.J. Dennis (eds.), Polynuclear Aromatic  Hydrocarbons: Chemistry and B i o l o g i c a l E f f e c t s , B a t t e l l e Press, Columbus, Ohio, p. 589-597. Zurcher, F., Thuer, M. and Davis, J.A., (1980). "Importance of P a r t i -culate Matter on the Load of Hydrocarbons of Motorway Runoff and Secondary E f f l u e n t s " , i n Hydrocarbons and Halogenated Hydrocarbons  i n the Aquatic Environment, Environmental Science Research, Volume 16, Plenum Press, New York, p. 373-385. 194. APPENDIX 1 - Abbreviations, naming and numbering r u l e s , structures and carcinogenic a c t i v i t y . This appendix contains a tabulation of major p o l y c y c l i c s discussed i n the thesi s , including abbreviations used i n the text and i n tables; names and structures; as well as an i n d i c a t i o n of carcinogenic a c t i v i t y . A summary of naming and numbering rules i s provided, condensed from The Ring Index (Patterson et a l . , 1960). 1. The names of p o l y c y c l i c hydrocarbons with maximum number of non-cumulative double bonds end i n "ene". Examples include anthracene, chrysene, fluoranthene, fluorene, naphthalene, phenanthrene, pyrene and triphenylene. 2. The names of hydrocarbons containing f i v e or more fused benzene rings i n a s t r a i g h t l i n e a r arrangement are formed i n a st r a i g h t l i n e a r arrangement are formed from a numerical pref i x followed by "acene". Examples: pentacene, hexacene. 3. For purposes of numbering, the structures are oriented so that the greatest number of rings are i n a horizontal row, and the maximum number of rings i s i n the upper righthand quadrant. Numbering sta r t s with the carbon atom (not common to 2 or more rings) i n the most counter-clockwise position i n the upper right quadrant. Numbers are assigned i n a clockwise d i r e c t i o n and carbon atoms common to two or more rings are omitted. As examples see the numbering of pyrene or triphenylene. Anthracene and phenanthrene have non-standard numbering systems. 195. 4. Compounds which are comprised of a d d i t i o n a l rings (with f i v e or more members) are named by a f f i x i n g "benzo" (one six-membered ring) or "naphtho" (two fused rings) to "ene" compounds l i s t e d i n rule 1. The largest "ene" compound possible should be chosen, with the simplest p r e f i x possible. For example, dibenzoanthracene (diB(a,c)A) rather than naphthophenanthrene. 5. Isomers of "ene" compounds with a d d i t i o n a l rings are distinguished by assigning l e t t e r s to the sides of the o r i g i n a l compound. Lett e r s begin with the "1-2" side and continue around the periphery of the structure, assigning a l e t t e r to each side a v a i l a b l e f o r fusion. When several naming options e x i s t , the combination fo the lowest number and the e a r l i e s t alphabetic l e t t e r s h a l l be used. Data on carcinogenic a c t i v i t y of various p o l y c y c l i c s i s taken from a compilation by Lee e_t _al. (1981). Although studies are not completely comparable due to differences i n animal species, age, d i e t , dose range and method of administration, the information provides a useful guideline to PAH carcinogenicity. Major parent p o l y c y c l i c s have been included i n t h i s appendix as well as some a l k y l - s u b s t i t u t e d PAH. Carcinogenicity i s indicated by "o" i n studies where none of the test animals developed tumours; "+" i n cases where up to 33% tumour incidence was found (weakly carcinogenic); "++" for studies i n which over 33% of the animals developed tunours (strongly carcinogenic). 196. ABBREVIATION NAME CARCINOGENIC ACTIVITY STRUCTURE acenaphthene o A anthracene o 5 io 4 meA 9-meA dimeA 9,10dimeA methylanthracene 9-me t h ylanthrac ene d imethylanthracene 9,10-dimethylanthracene anthranthene 0 o/+ 659 B(a)A benzo(a)anthracene + C O ? 4-meB(a)A 6- meB( a) A 7- meB(a)A 5,12-dimeB(a)A B(b)F 4-methylbenzo(a)anthra-cene 6- methylbenzo(a)anthra-cene 7- methylbenzo(a)anthra-cene 5,12-dimethylbenzo(a)-anthracene benzo(b)fluoranthene o/+ ++ -H-o ++ B(ghi)F benzo(ghi)fluoranthene o 197. ABBREVIATION NAME CARCINOGENIC ACTIVITY STRUCTURE B( j ) F benzo(j)fluoranthene ++ B(k)F benzo(k)fluoranthene -H-B(a)Fl benzo(a)fluorene o B(b)Fl benzo(b)fluorene -B(ghi)Per benzo(ghi)perylene + 6<g> B(a)Ph benzo( &) phenanthrene + B(a)P benzo(a)pyrene ++ 4- meB(a)P 5- meB(a)P 8-meB(a)P l,3-dlmeB(a)P 4- methylbenzo(a)pyrene 5- methylbenzo(a)pyrene 8-methylbenzo(a)pyrene l,3-dimethylbenzo(a)-pyrene ++ + o •H-198. ABBREVIATION NAME CARCINOGENIC ACTIVITY STRUCTURE B(e)P benzo(e)pyrene o/+ Chr chrysene + 1-meChr 5- meChr 6- meChr Col Cor 1-methylchrysene 5- rae th yl c hr ysene 6- methylchrysene cholanthrene coronene o/+ ++ + ++ o/+ - cyclopenta[ c,d]pyrene + diB(a,j)Ac dibenzo(a,j)acridine + diB(a,c)A d ibenzo(a,c)anthracene + diB(a,h)A d ibenzo(a,h)anthracene + 7-mediB(a,h)A 7-methyldibenzo(a,h)-anthracene ++ 199. ABBREVIATION NAME CARCINOGENIC ACTIVITY STRUCTURE diB(a , j )A d ibenzo(a , j )anthracene + diB(a, i )Ph dibenzo(a,i)phen-anthr ene (picene) o F 2-meF IndF Fl fluoranthene 2-methylfluoranthene indenofluoranthene fluorene 0 + o 7 8 *»c l 1 Ind[l,2,3-cd]P indenof1,2,3-cd]pyrene + 5 ^ N naphthalene o Per perylene o CCS 5 A> 1of^^^^^' Ph phenanthrene o 200. ABBREVIATION NAME CARCINOGENIC ACTIVITY STRUCTURE 2-mePh 2-methylphenanthrene -3,6-dimePh 3,6-dimethyl-phenanthrene P pyrene 0 b 5 1-meP 1-methylpyrene 0 T r i triphenylene 0 2 1-meTri 1-methyl triphenylene o 7 201. APPENDIX 2 - Physical Properties of PAH ABBREVIATION MOLECULAR WEIGHT MELTING3 POINT BOILING 3 POINT A 178.24 216.2 340 B(b)F 252.32 168 -B( j ) F 252.32 166 -B(k)F 252.32 217 480 B(a)Fl 216.29 189 413 B(a)P 252.32 255 448 B(c)Ph 228.30 68 -diB(a,c)A 278.36 205 -diB(a,h)A 278.36 269 -diB(a,i)A 278.36 263 -diB(a,j)A 267.36 197 -diB(b,h)Ph 267.36 257 -F l 166.23 116 294 F 202.26 111 375 N 128.19 80.55 218 Per 252.32 278 -Ph 178.24 101 340 P 202.26 156 393 Picene 278.36 367 518 T r i 228.30 119 425 a - CRC Handbook of Chemistry and Physics, 1975. 202. APPENDIX 3 - Chromatography Conditions, Chromatograms of Mixed Standards 1. HPLC traces - Chromatography conditions are set out i n section I I I F-2 and i n Table 3-2. Additional d e t a i l s are provided ( l i s t e d by figure number) i n Tables A and C below. Absorbance at 254 nm i s the upper trace (dotted or dashed lin e ) i n each figure. 254 nm absorbance increased towards the bottom of the page, and i s o f f s e t s l i g h t l y h o r i z o n t a l l y from the 280 nm trace. (AUFS = 0.020). 280 nm absorbance ( s o l i d l i n e ) i s integrated, and i n t e g r a t i o n marks and retention times ( i n minutes) are included i n f i g u r e s . (AUFS = 0.020). 2. C a p i l l a r y GC traces - Chromatography conditions are found i n I I I F-3 and i n Table 3-3. Additional d e t a i l s are l i s t e d by f i g u r e number i n Table B below. Table A - HPLC Conditions Figure 4-11 to 4-17 FIGURE SAMPLE INJECTION %CH,CN NUMBER SIZE ( p i ) 4-11 a) D(N)('78)III D(N)('79)III W('78)111 W( '79)111 D(N)('78)IV D(N)('79)IV L('78)IV L('79)IV D(N)('78)V 5 70 b) 3 4-12 a) 5 b) 3 4-13 a) 10 b) I *• 4-14 a) 5 ** b) 1 4-15 a) 10 b) D(N)('79)V 5 4-16 a) L('78)V L('79)V L('78)III L('79)III 5 b) 5 4-17 a) 3 60 b) 3 70 203. Table B - C a p i l l a r y GC Conditions Figure 4-14 to 4-20. FIGURE SAMPLE INJECTION COMMENTS NUMBER SIZE (ul) 4-18 a) D(S)('79)III 2 b) III 0.2 c) IV 2 d) V 2 4-19 a) D(S)('78)III 2 chart speed 0.5 cm/min b) IV 2 c) V 2 4-20 a) W( '78)111 2 b) IV 2 c) V 2 Table C - HPLC Conditions Figure 4-31 to 4-3 6. FIGURE NUMBER SAMPLE INJECTION SIZE (ul) % CH3CN AUFS 254 nm AUFS 280 nm 4-31 a) R 2 I I I 5 70 0.02 0.02 b) IV 5 " c) V 5 4-32 a) C2 III 5 *' b) IV 5 M c) V 5 4-33 a) lit I I I 5 " b) IV 5 c) V 5 tf 4-34 a) Gr 2 III 10 M b) IV 5 tt c) V 5 " 4-35 a) DD 874km 1 0.200 0.200 b) 1665km 1 •• c) 2623km 1 it •• 4-36 a) RM 895km 1 0.050 0.050 b) 1943km 1 tt c) 2545km 1 3. Mixed Standards, HPLC-Mixed standards were run for each of the three alumina f r a c t i o n s . Chromatograms are included i n t h i s appendix for each standard at 60% CH.CN and at 70% CHoCN. 4. Mixed Standards, c a p i l l a r y GC - Chromatograms of mixed standards corresponding to each alumina f r a c t i o n are included. Conditions set out i n section I I I F-3 and Table 3-3. •JB •7. •M n <-.j • n r ^z. CO Standards Eluted i n Alumina F r a c t i o n I I I , 60%, HPLC 2 0 5 . Standards Eluted i n Alumina Frac t i o n IV, 60% HPLC 206. Standards Eluted i n Alumina Frac t i o n V, 60%, HPLC CD i n 2-m«Ph 9. 10-dimeA ' r - T i Standards Eluted i n Alumina F r a c t i o n I I I , 70%, HPLC 208. Standards Eluted i n Alumina Frac t i o n IV, 70%, HPLC 209. i I Standards Eluted i n Alumina F r a c t i o n V, 70%, HPLC Standards Eluted in Fraction I I I , c a p i l l a r y GC B(a)P Chr B(«)A Per Standards Eluted in Fraction IV, c a p i l l a r y GC 1 BO>)F > i r i Standards Eluted i n Fraction V, c a p i l l a r y GC 

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