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Toxicity of anionic surfactants in a primary effluent : identification, characterization and removal Bradley, James Craig 2004

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Toxicity of Anionic Surfactants in a Primary Effluent: Identification, Characterization and Removal by  JAMES CRAIG B R A D L E Y  B . A . S c . Mining and Mineral Process Engineering, University of British Columbia, 2000  A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F THE REQUIREMENTS FOR THE D E G R E E OF  M A S T E R OF APPLIED  SCIENCE  in T H E F A C U L T Y OF G R A D U A T E STUDIES (Department of Civil Engineering)  We accept this thesis as conforming to the required standard  T H E UNIVERSITY O F BRITISH C O L U M B I A August 13, 2004  © James Craig Bradley, 2004  THE UNIVERSITY OF BRITISH C O L U M B I A  F A C U L T Y OF G R A D U A T E STUDIES  Library Authorization  In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  /?.(>%. Name of Author (ple/OUe print)  Title of Thesis:  J /fx/os/c SufA,A^-k  p^/A,  Degree:  Department of  £g<f  The University of British Columbia » 1  Vancouver, BC  A.  Year:  [ Canada  e>¥  Date (dd/mm/yyyy)  j/\£^ri.  fnnAasy  MX? / 1  Abstract  ii  Abstract This research project was undertaken to identify, characterize and remove anionic surfactant induced toxicity from a primary effluent.  The anionic surfactants, present in the  primary effluent, were first separated into low, medium and high molecular weight fractions using solid phase extraction columns and gradient methanol elution. B y separating the anionic surfactants, on the basis of molecular weight, the relative toxicities of each fraction could be determined. A colourimetric method was developed that was used to measure the concentration of anionic surfactants as methylene blue active substances ( M B A S ) .  This method required less  time and less reagents than the conventional method outlined in Standard Methods ( A P H A et al, 1992). Finally, the M i c r o t o x ™ system was used to measure the toxicity o f the whole sample and of the different molecular weight fractions.  Using the methods developed for the present study, the anionic surfactant concentration and associated toxicity in a primary effluent were determined.  These determinations were  performed on two sampling events at the Lions Gate Primary Wastewater Treatment Plant. In each sampling event primary effluent was collected in the morning, afternoon and night.  MBAS  concentrations in the primary effluent increased throughout the day and ranged from 1.20 m g / L M B A S to 9.34 m g / L M B A S .  The anionic surfactant concentrations were highest in the medium  molecular weight fraction and lowest in the high molecular weight fraction. The toxicity of the primary effluent was shown to increase as the concentration of anionic surfactants increased. The toxicity associated with anionic surfactants was highest in the high molecular weight fraction. While the high molecular weight fraction was the most toxic fraction, it contained the lowest anionic surfactant concentration.  A screening study was conducted to provide a preliminary indication of the feasibility of using either partitioning to abiotic bio-solids, biological treatment, alum coagulation/flocculation with gravity settling, ozonation, or air flotation to remove anionic surfactants and the associated toxicity from a primary effluent. produced  anionic  surfactant  Partitioning to abiotic bio-solids and biological treatment removals  of  64%  and  96%,  respectively.  Alum  coagulation/flocculation with gravity settling removed 46% of the anionic surfactants, while  Abstract  ozone removed 95% o f the anionic surfactants. surfactants.  iii  A i r flotation removed 77% o f the anionic  Toxicity studies were conducted using the samples treated with ozone and air  flotation only. The toxicity studies revealed that ozonation slightly increased the toxicity o f the treated whole sample, possibly due to the formation o f by-products from the oxidation process. O n the other hand, air flotation reduced the toxicity o f the whole sample significantly.  In  addition, air flotation removed all o f the measurable toxicity, using the methods described in the present study, from the elution fraction corresponding to the high molecular weight fraction. These preliminary results indicate that air flotation may be an effective interim means o f reducing both anionic surfactants and anionic surfactant induced toxicity from the Lions Gate Wastewater Treatment Plant primary effluent.  Table of Contents  iv  Table of Contents Abstract  ii  Table of Contents  '..iv  List of Tables  :  List of Figures  viii ix  Acknowledgements  x  Chapter 1 Introduction  1  Chapter 2 Literature Review  4  2.1 Summary of Literature Review  4  2.2 Anionic Surfactants  4  2.3 Toxicity of Anionic Surfactants  5  2.3.1  Toxicity o f Biodegradation By-Products  7  2.4 Methods for the Removal of Anionic Surfactants Contained in Wastewater 2.4.1 Bio-treatment 2.4.2 Physical and Chemical Treatment 2.5 Measurement of Anionic Surfactants 2.5.1 Recovery o f M B A S 2.6 Fractionation of Anionic Surfactants 2.6.1 Preservation of M B A S on S P E Columns 2.7 Measurement of Toxicity 2.7.1 Limitations in the Measurement o f Toxicity Using M i c r o t o x ™ . 2.8 Previous Characterization and Treatment Studies at Lions Gate Chapter 3 Analytical Procedures and Experimental Setups 3.1 Analytical Procedures 3.1.1 Sample Collection... A ) Collection from W W T P B) Collection Following Treatment  ,  7 7 8 9 9 —9 10 11 12 13 16 ....16 18 18 18  3.1.2 Filtration  19  3.1.3 Sample Storage  19  A ) Whole Sample Storage  19  B) S P E Sample Storage  19  C) Waste Activated Sludge Storage  20  3.1.4 Solid Phase Extraction  20  A ) Solid Phase Extraction Apparatus  20  B) Reagent Preparation  21  C) Activation  22  D) Analyte Loading  23  E) C o l u m n Washing  23  Table of Contents  v  F) Elution  ...24  G) Reconstitution  25  Methanol Evaporation from the S P E Column Extracts  25  Standard Volume of the S P E Extracts  ,...25.  H) Additional Notes on Solid Phase Extraction  28  3.1.5 M B A S Analysis.....  28  A ) Organic Extraction  29  B) Aqueous Back-wash  31  C) Measurement  32  3.1.6 M i c r o t o x ™ Analysis  34  3.1.7 Methanol Concentration  36  3.1.8 Total Suspended Solids Analysis  37  3.2 Experimental Set-up and Analytical Procedures for Treatment Studies  37  I Bio-Treatment  39  A ) Biological Degradation  39  B) Partitioning to Abiotic Bio-Solids  39  II A l u m Coagulation/Flocculation with Gravity Settling  40  III Ozonation  40  IV A i r Flotation  41  3.2.7  42  Analytical Procedures  Chapter 4 Development of Analytical Methods  44  4.1 Methylene Blue Active Substances Method Development  44  4.1.1 Short-Term Sample Storage  44  4.1.2 Working M B A S Concentration Range  45  4.1.3  Precision of the M B A S Analytical Method  46  A ) Precision of the M B A S Analysis for the Standards  47  B) Precision Following Sample Preparation Procedures  47  4.1.4 Recovery of Analyte During The M B A S Analytical Procedure  48  4.1.5  50  Impacts of Sample Preparation Procedural Steps on M B A S Recovery  4.1.5.1  Positive Interferences of  Sample  Preparation Procedural Steps on  MBAS  Recovery A ) Filtration  50 :  50  B) S P E  •.  51  4.1.5.2 Negative M B A S Interferences  52  A ) Glass Wool  52  B) Sodium Chloride  53  4.1.5.3 Potential Loss of M B A S  55  A ) S P E Underflow and Wash Underflow  55  B) S P E Extract Reconstitution  56  4.1.5.4 Recovery of M B A S to Elution Fractions  ...57  4.2 M i c r o t o x ™ Method Development  58  4.2.1  M i c r o t o x ™ Acute Reagent Reconstitution  58  4.2.2  Standard Zinc Toxicant  59  4.2.3  S P E Reconstitution Water  4.2.4  S P E Correction Factors  , '.  59 60  Table of Contents  4.2.5  vi  Residual Methanol in the Reconstituted S P E Extracts  A ) Methanol Evaporation  „.  ;  61 62  B) Toxicity of Methanol  63  Chapter 5 Sampling Program & Characterization of Primary Effluent  64  5.1 Sampling at The Lions Gate W W T P  64  5.2 Characterization of the Lions Gate W W T P primary effluent  65  5.2.1  MBAS  65  5.2.2  Toxicity  68  5.2.3  Toxicological Impact  73  Chapter 6 Treatability Studies 6.1  Sampling  A ) Lions Gate W W T P B) U B C Pilot Plant 6.2 Removal of M B A S I Bio-Treatment A ) Biological Degradation  75 :  76 76 76 76 77 77  B) Partitioning to Abiotic Bio-Solids II A l u m Coagulation/Flocculation with Gravity Settling  77 78  III Ozonation IV A i r Flotation  78 78  6.3 Removal of Toxicity  79  6.3.1  Ozonation  80  6.3.2  A i r Flotation.....  80  Chapter 7 Conclusions  82  Chapter 8 Recommendations....  84  List of References  85  Table of Contents  vii  A P P E N D I X A : Analytical Method Development o f Microtox'™  ..90  A ) Standard Zinc Toxicant  90  B) Methanol Toxicity to M i c r o t o x ™  90  C) Impact of Laboratory water on the Bioluminescence of Vibrio fischeri A P P E N D I X B : S P E Bioluminescence Correction Factors  ....92 93  A P P E N D I X C : Lions Gate Characterization Study  94  A ) M B A S - December 16, 2003  94  B) M B A S - February 3,  95  ,h  2004  rd  C) M B A S - February 26, 2004  96  D) Methanol - December 16, 2003  97  E) Methanol - February 3,  98  th  th  rd  2004  F) Toxicity - December 16, 2003  99  ,h  G) Toxicity - February 3,  rd  2004  105  H) Toxicity - February 26, 2004  111  th  A P P E N D I X D : U B C Pilot Plant A) M B A S  112 ...  ..112  B) Toxicity  113  C) Raw data  114  A P P E N D I X E : Treatability Study  117  A) M B A S  117  B) Toxicity  121  List of Tables  viii  List of Tables Table 1: Homologue distributions o f some L A S formulations  11  Table 2: Toxicity o f some L A S formulations measured using the M i c r o t o x ™ system  12  Table 3: Summary o f the procedures used during the S P E method  27  Table 4: Summary of the M B A S method procedures  33  Table 5: Precision o f the M B A S analytical method found using M B A S standards  47  Table 6: Precision o f M B A S method following sample preparation procedures.  48  Table 7: Recovery o f M B A S following 2 or 3 extractions  49  Table 8: M B A S concentrations in blank reconstituted S P E column extracts.  51  Table 9: The effects o f glass wool on M B A S recoveries  52  Table 10: The effect o f sodium chloride on M B A S recovery following filtration  53  Table 11: The effect o f sodium chloride on M B A S recovery following S P E  54  Table 12: Recoveries o f M B A S to the S P E underflow streams  55  Table 13: M B A S recovery following methanol evaporation  57  Table 14: Laboratory water and percent light reduction  60  Table 15: Correction factors - blank S P E column  61  Table 16: Methanol toxicity measured using the M i c r o t o x ™ system  63  Table 17: Sample storage and analysis times - characterization  65  Table 18: M B A S concentration measured in the Lions Gate W W T P primary effluent  ...66  Table 19: Toxicity contained in the Lions Gate W W T P primary effluent  69  Table 20: Rationale for selecting treatment methods  75  Table 21: Date on which sample analyses were conducted during the treatment study  76  Table 22: Treatability study: M B A S removal efficiencies  77  Table 23: Treatability study: toxicity before and after treatment  ,  80  List of Figures  ix  List of Figures Figure 1: Structure o f linear alkylbenzenesulfonate Figure 2: Flowchart o f analytical procedures  5 .  17  Figure 3: Solid phase extraction apparatus  21  Figure 4: M i c r o t o x ™ apparatus  34  Figure 5: Cuvette and reagent vial at an angle to vertical  36  Figure 6: Flowchart o f treatment and analytical procedures  38  Figure 7: Ozonation apparatus  41  Figure 8: A i r flotation experimental set-up  42  Figure 9: M B A S standard curve.  46  Figure 10: Cumulative M B A S recovery versus elution fraction  58  Figure 11: Residual methanol concentrations versus heating time  62  Figure 12: M B A S versus time - whole filtered primary effluent  66  Figure 13: Average M B A S content in each o f the elution fractions  67  Figure 14: Toxicity versus time - whole filtered primary effluent  69  Figure 15: Toxicity o f whole filtered primary effluent versus M B A S concentration.  70  Figures 16 & 17: IC20' versus elution fractions - December 16, 2003  71  Figures 18 & 19: IC2o's versus elution fractions - February 3 and 26, 2004  72  S  Figures 20 & 21: Average toxicity and average M B A S measured during all sampling events. ..73 Figure 22: Toxicological impact of elution fractions.  74  Acknowledgements  x  Acknowledgements The author wishes to acknowledge the following individuals and agencies for their contributions to this project:  • •  Dr. Pierre Berube for bringing objective realism and wisdom to the pursuit o f science. Susan Harper for expertly handling all matters related to the safe and efficient operation of the laboratory.  •  Paula Parkinson for her infinite patience, her keen and varied interests, and her astute interpretations.  •  M r . Robert N g and M r . Paul vanPoppelen ( G V R D ) for contributing to the successful outcome of the present study.  •  The G V R D and the Department of Policy and Planning for funding this project.  Chapter 1. Introduction  1  Chapter 1 Introduction The Lions Gate Wastewater Treatment Plant ( W W T P ) provides primary treatment to wastewater generated by approximately 160 000 people (Bailey and Elphick, 2001). Located in North Vancouver, the plant discharges effluent to the outer harbour o f Burrard Inlet at First Narrows.  Separate storm water and wastewater collection systems are in place.  In 2001, the  average daily flow to the Lions Gate W W T P was 92 M L D , and the dry-weather flow to the plant was 86 M L D ( G V R D , 2001).  Monthly trout bioassays are conducted to monitor the acute toxicity o f the primary effluent from the Lions Gate W W T P .  In 2001, four of the bioassays were indicative o f toxicity  events ( G V R D , 2001). Similar results were obtained in 1995, 1996 and 1999 ( G V R D , 2001). A toxicity event was considered to have occurred when the  LC50  LC50  was below 100% ( V / V ) .  The  is that concentration o f the effluent (percent by volume) resulting in 50% mortality o f the  species tested. It follows that the lower the percent concentration required for 50% mortality, the more toxic is the sample. The results o f the bioassays prompted studies to be undertaken in 2000 and 2002 to further investigate the causes of toxicity.  The 2000 study revealed that anionic  surfactants, contained in the primary effluent, were the predominant cause o f the toxicity to rainbow trout that were used in the bioassays  (Bailey and Elphick, 2001). In a follow up study  conducted in 2002, toxicity tests and toxicity identification and evaluation (TIE) studies were performed using Lions Gate primary effluent.  Again, anionic surfactants were identified as the  predominant cause o f toxicity to rainbow trout that were used in the bioassays ( E V S , 2003). In both the 2000 and 2002 studies, ammonia was found to contribute to the toxicity contained in the primary effluent from the Lions Gate W W T P . However, ammonia contributed significantly less to the toxicity than did the anionic surfactants.  Long-range plans indicate that secondary treatment will be implemented at the Lions Gate W W T P , and it is known that secondary treatment effectively removes the toxicity induced by anionic surfactants. However, an interim treatment method is sought that can be configured  2  Chapter 1. Introduction  within the existing primary plant until such time when secondary treatment can be provided. T o address this need, the present study was undertaken.  The objectives o f the present study were three-fold. The first objective was to develop analytical methods that could be used to quantify the concentration o f anionic surfactants in the primary effluent, to fractionate the anionic surfactants on the basis o f molecular weight, and to measure the toxicity o f the whole and discrete molecular weight fractions. The second objective was to characterize the primary effluent  from the Lions Gate W W T P .  The parameters  characterized included both the concentration o f anionic surfactant and the toxicity contained in the whole samples as well as in the discreet molecular weight fractions. The third objective was to screen potential treatment methods to determine the effectiveness o f the method at removing both anionic surfactants and toxicity. The treatability study was designed to screen the treatment methods, and it was a small portion of the overall work completed as part o f the present study.  T o achieve the stated objectives four main tasks were performed.  1.  The literature pertaining to anionic surfactants was reviewed to first identify analytical methods that could be used to fractionate anionic surfactants on the basis o f molecular weight and to measure the concentration o f anionic surfactants. Secondly, the toxicity o f anionic surfactants to fish was determined. Finally, potential treatment methods were identified.  2.  Analytical procedures were developed and validated.  The first analytical procedure  was used to fractionate anionic surfactants on the basis o f molecular weight.  The  second procedure was used to determine the concentration o f anionic surfactants in each molecular weight fraction and in the whole sample.  Finally the third analytical  procedure was used to measure the toxicity of each molecular weight fraction and that of the whole sample. 3.  Lions Gate primary effluent was characterized in terms o f its anionic surfactant concentration and its associated toxicity.  4.  A preliminary treatability study was conducted using four treatment methods for the removal o f anionic surfactants and induced toxicity.  The methods included bio-  treatment, alum coagulation/flocculation with gravity settling, ozonation, and air flotation.  Chapter I. Introduction This thesis is organized into eight sections. materials relevant to the present study.  3  Chapter 2 consists o f a literature review o f  Chapter 3 describes the experimental methods and  procedures used in the present study. Chapter 4 contains a discussion o f the development o f the new analytical methods used in the present study. characterization of the primary effluent.  Chapter 5 consists o f the results from the  Chapter 6 includes the results o f the treatability study.  Finally, chapter 7 offers conclusions arising form this research project, and chapter 8 provides recommendations for future research.  Chapter 2. Literature Review  4  Chapter 2 Literature Review 2.1 Summary of Literature Review  Detergents products.  are commonly used in residential, commercial and industrial cleaning  Anionic  formulations.  surfactants are the main active  Linear alkylbenzenesulfonate  surfactant used in detergent formulations.  components  in household  detergent  ( L A S ) is the most common type o f anionic  L A S is highly biodegradable and is hydrophobic.  L A S has previously been shown to be toxic to fish, and M i c r o t o x ™ has previously been used as a surrogate for the standardized rainbow trout toxicity bioassay.  Anionic surfactants can be  measured as methylene blue active substances ( M B A S ) and can be partitioned into discrete molecular weight fractions following passage through a solid phase extraction column. Previous studies have identified anionic surfactants as a cause o f toxicity in the effluent from the Lions Gate W W T P , and treatment studies were undertaken to evaluate methods o f removing anionic surfactants from the Lions Gate effluent.  2.2 Anionic Surfactants  Linear alkylbenzenesulfonates ( L A S ) are synthetic anionic surfactants. A surface active agent, or surfactant for short, functions to alter the surface tension between two or more substances. L A S has a strongly hydrophobic linear carbon chain to which a hydrophilic, anionic group is attached.  In the presence o f anionic surfactants, the surface tension between two  immiscible substances is lowered, and foams, bubbles, dispersions or emulsions are formed. For example, when anionic surfactants are used to wash clothes, the hydrophobic chains attach to dirt or grease while the hydrophilic groups remain in water.  In this way, dirt and grease can be  removed from the surface being cleaned, as the dirt and grease is suspended in and removed with the water. Figure 1 shows the structure o f a L A S molecule.  Chapter 2. Literature Review  5  CH3-(CH2)n-CH-(CH2)m-CH3  V S03Na  where n + m = 7 to 11  Figure 1: Structure of linear alkylbenzenesulfonate. From [Matthijs, E . , and De Henau, FL, 1987] Approximately 2.5 million tonnes of L A S were consumed worldwide in 1995 (Ferrer et al.,  1996).  L A S was introduced in the early 1960's to replace the recalcitrant branched  alkylbenzenesulfonates  (ABS).  This change  brought about a reduction in foaming and  persistence in the receiving environment caused by A B S .  L A S solutions are typically discharged  to wastewater collection systems and eventually to wastewater treatment facilities where influent concentrations of L A S typically range from 1-7 mg L A S / L  (Rapaport et al., 1987 appearing in  W H O , 1996).  The  hydrophobic chain of L A S can be of varied length and structure.  commercial preparations of L A S have linear chains of 10 to 14 carbons in length  Typical  (Painter and  Zabel, 1989, appearing in W H O , 1996). The hydrophobicity of a L A S molecule increases as the length of the carbon chain increases.  The structure of a L A S molecule differs by the placement  of the phenyl group on the carbon chain. L A S molecules that have the same chain length, but have different placements of the phenyl group, are referred to as isomers.  L A S molecules that  have different chain lengths are referred to as homologues.  2.3 Toxicity of Anionic Surfactants  The following discussion on aquatic toxicity, induced by anionic surfactants, deals only with L A S , since L A S is the most prevalent surfactant in detergents and cleaning products for both industrial and residential use ( W H O ,  1996).  Chapter 2. Literature Review  The  6  aquatic toxicity of L A S depends on many factors, but generalities have been  established which qualitatively describe trends in aquatic toxicity. In general, aquatic species are sensitive to surfactants and will avoid areas where surfactants are present. depends on its carbon chain length (homologues).  The toxicity of L A S  L A S molecules with longer chain lengths are  typically more toxic than those with shorter chain lengths.  When freshwater fish were exposed  to L A S having carbon chain lengths ranging from 8 to 15 carbons, the L C o ranged from 125 to 5  0.1 m g / L L A S , respectively ( W H O , 1996). The acute L C  5 0  (dose of L A S that kills 50% of the  test organisms) is used to describe the toxicity of a substance.  In another study, a two-fold  decrease in fish and Daphnia toxicity resulted when the carbon chain length of L A S was decreased from 12 to 11 carbons. acute 96-hour (Brown et al,  LC50  In the case of Rainbow Trout (Onchorynchus mykiss), the  ranged from 0.36 m g / L L A S , for L A S with chain lengths of 12.6 carbons  1978 appearing in W H O , 1996) to 2.1, 3.4, and 4.7 m g / L L A S , for L A S with  chain lengths of 11.6 carbons (Wakabayshi et al,  1986; Wakabayshi et al,  1984 all appearing in  W H O , 1996). O n the other hand, saltwater organisms are generally more sensitive to L A S than are freshwater organisms.  Fortunately, L A S concentrations are generally lower in marine  environments, due to dilution ( W H O ,  1996). When marine fish were exposed to L A S that had  carbon chain lengths of 11.7 carbons, the acute  LC50 ranged  from 0.05 to 7 m g / L L A S ( W H O ,  1996). In addition, the toxicities of other substances can be increased when combined with L A S . Enhanced toxicities to aquatic organisms were observed in studies where L A S was combined with fuel oil, cadmium, copper or zinc (Hokanson and Smith, 1971; Part et al, 1985; Swedmark and Granmo, 1981; Tsai and M c K e e , 1978; all reported in W H O , 1996).  L A S was shown to  increase the transfer of cadmium or fuel oil across the gill membranes of fish resulting in increased toxicities.  The toxicity of L A S to aquatic organisms varies with species and is dependent upon the carbon chain length of L A S .  Toxicity has been shown to increase as the attachment of the  phenyl group is moved from the center of the carbon chain to the end, but the position of the phenyl group is less significant to toxicity than is increased carbon chain lengths (Swisher, 1987).  Long-term and acute toxicity screening can be applied to test the effects of L A S on  aquatic organisms. In the case of long-term tests, it is important to note that solids and/or food particles can adsorb L A S thereby lowering the amount of L A S in solution.  Chapter 2. Literature Review  7  Significant physiological responses and impairment of normal gill physiology can lead to mortality when fish are exposed to various concentrations of L A S ( W H O , 1996; Randall et al, 1996; Swisher, 1987). Decreased viability of the gills, separation o f the gill structures, decreased oxygen transfers, and increased ventilation rates are all reported to occur when fish are exposed to L A S with doses ranging from 0.39 m g / L L A S to 36 m g / L L A S (Maki, 1979 as reported by W H O , 1996; Part et al,  2.3.1  1985 as reported by W H O , 1996; Zaccone et al, 1985).  Toxicity of Biodegradation By-Products  Biodegradation is an effective means of removing anionic surfactants from wastewaters as discussed in Section 2.4.  Biodegradation of L A S using mixed bacterial cultures changes the  three distinct structures making up a molecule of L A S : the chain, the ring and the sulfonate linkage (Swisher, 1987). A s a result, many intermediate degradation by products are formed, as the parent L A S molecule undergoes biodegradation.  Intermediate biodegradation by-products  have been found to contain a carboxyl group at the end of the altered alkyl chain.  This  carboxylated by-product was found to be three to four times less toxic than the parent L A S compound (Kolbener et al, 1995; Swisher et al., 1978 as reported by W H O , 1996).  2.4 Methods for the Removal of Anionic Surfactants Contained in Wastewater  2.4.1 Bio-treatment  Wastewater treatment plants provide varying degrees of removal of L A S during both primary and secondary treatments.  Interestingly, it is reported that with favorable aerobic  conditions up to 50% of the L A S present in wastewater can be biodegraded in wastewater collection networks before entering W W T P s (Moreno et al., 1990 appearing in W H O , 1996). A significant fraction (15-30%) of the L A S entering wastewater treatment plants can be removed via adsorption to primary sewage solids (Fauser et al., 2003; Giger et al., 1989 and Prats et al, 1993 both appearing in W H O , 1996; Swisher, 1987).  During secondary treatment, the main  mechanism for L A S removal is biodegradation where removals of 80-85% are typically reported (Fauser et al, 2003; Kolbener et al,  1995).  A s a result, complete removal of the parent L A S  Chapter 2. Literature Review  8  molecule is possible in an aerobic biological treatment system (Kolbener et al., 1995). However, under anaerobic conditions L A S does not undergo biodegradation (Holt and Bernstein, 1992).  A s discussed in Section 2.3, the toxicity of L A S tends to increase as the carbon chain length of the L A S molecule increases.  Fortunately, as the carbon chain length increases, so too  does the rate at which the L A S molecule is biodegraded (Divo, 1976 as reported by Swisher, 1987). Therefore, during biodegradation the more toxic, higher molecular weight fractions are removed from solution before the less toxic, lower molecular weight fractions. In addition, the more toxic, higher molecular weight fractions are more readily adsorbed to solids due to their greater hydrophobic characteristics.  2.4.2 P h y s i c a l a n d C h e m i c a l T r e a t m e n t  A l u m coagulation/flocculation with gravity settling was considered as a treatment method for the removal of anionic surfactants from the effluent of the Lions Gate W W T P . The findings of a previous study in which alum coagulation/flocculation with gravity settling was used to treat the primary effluent from the Lions Gate W W T P are presented in Section 2.8.  The effects of  L A S on alum coagulation/flocculation of a synthetic wastewater has also been studied.  Fettig  and Ratnaweera (1993) reported that L A S had no significant affect on the coagulant dose needed to achieve maximum particle or turbidity removals (Fettig and Ratnaweera, 1993).  Beltran et al,  (2000) investigated the kinetics of L A S decomposition by ozonation.  It  was found that the p H and the amount of organic material initially present in the L A S solution influenced the removal of L A S (Beltran et al,  2000).  Beltran et al,  (2000) observed higher  oxidation rates at higher pH's, and suggests that hydroxyl radicals were more abundant at higher pH's.  L A S tends to react faster with hydroxyl radicals than it does with molecular ozone.  high organic concentrations, the oxidation rate of L A S tends to decrease.  At  The oxidation of  inorganic and organic compounds, other than L A S , present in wastewaters likely consumes the radicals thus reducing the amount of hydroxyl radicals that are available to oxidize the L A S molecules.  9  Chapter 2. Literature Review 2.5 Measurement of Anionic Surfactants  Anionic surfactants can be measured as methylene blue active substances ( M B A S ) .  In  the M B A S test, aqueous anionic surfactants combine with aqueous methylene blue to form an ion pair which is then extracted into an immiscible chloroform liquid.  The ion pair remains in  the chloroform. Subsequently, an aqueous back-wash is used to remove excess methylene blue and positive interferences from the chloroform. Positive interferences, with poor extractability, are effectively  removed by the aqueous backwash.  Positive interferences include organic  sulfonates, sulfates, carboxylates, phenols, inorganic cyanates, nitrates and chlorides.  Negative  interferences include cationic surfactants, cationic materials and particulate matter. Following back-wash, the volume of chloroform is made up to a standardized volume.  Finally, the  concentration of M B A S is determined spectrophotometrically at a wavelength of 652 nm. The conventional approach used to measure M B A S is presented in Standard Methods 5540 C ( A P H A et al., 1992). T o overcome some o f the limitations o f Standard Method 5540 C , Chitikela, et al, (1995) developed another method (Chitikela, et al, 1995) (see Section 3.1.5).  2.5.1 Recovery of M B A S  Many researchers have presented methods to improve the recovery o f M B A S during sample collection and analysis.  Marcomini et al,  (1987) added sodium chloride to samples  before filtration to improve the recovery o f L A S following filtration. Subsequently, the filtercake was rinsed with methanol which was added to the filtrate (Marcomini et al,  1987).  To  preserve samples containing L A S , Matthijs and De Henau (1987) added formaldehyde to sample bottles which were subsequently filled with wastewater and then stored at 4 ° C .  Both the  formaldehyde and low temperature acted to slow or stop the biodegradation o f L A S (Matthijs and De Henau, 1987).  2.6 Fractionation of Anionic Surfactants  Solid phase extraction (SPE) can be used to fractionate anionic surfactants on the basis o f hydrophobicity. Hydrophobic surfactant molecules adsorb to the S P E column packing material.  Chapter 2. Literature Review  10  Highly hydrophobic molecules, which have long carbon chains and higher molecular weights, are strongly adsorbed while the less hydrophobic molecules are more weakly adsorbed to the S P E column packing material.  Solvents o f decreasing polarity can then be used to elute  surfactant molecules from the packing material. A s the polarity o f the solvents are decreased, the dissolving power increases, and the more hydrophobic surfactant molecules can be eluted. In this way, surfactants can be separated on the basis of hydrophobicity and therefore molecular weight.  A  study was performed by E V S Environmental Consultants (2003) in which S P E  columns were used to separate the anionic surfactants, contained in the Lions Gate primary effluent, into discrete fractions on the basis o f hydrophobicity. Solutions containing methanol in water were used as solvents to elute the adsorbed anionic surfactants from the S P E packing material ( E V S , 2003).  A 65% solution o f methanol in water was used to elute the less  hydrophobic material.  A 90% solution o f methanol in water was used to elute the more  hydrophobic material having longer carbon chains.  According to the directions supplied by the manufacturer o f the S P E columns used in the present study, a wash step helps to eliminate interfering substances that are either weakly adsorbed to the packing material or are entrained in the packing material (Supelco, 1997).  2.6.1 Preservation of MBAS on SPE Columns  Molecules o f L A S are highly biodegradable, particularly those having longer carbon chains. Therefore, proper sample storage is o f the utmost importance to preserve the form and concentration o f L A S .  Petrovic and Barcelo (2000) observed a significant decrease in M B A S  concentrations for wastewater samples when stored for 30 days at 4 ° C ; these losses were greatest for the L A S molecules having longer carbon chains (Petrovic and Barcelo, 2000). O n the other hand, minimal losses of L A S molecules were observed after S P E columns, containing L A S extracted from wastewater, were stored for 7 days at room temperature. observed when similar columns were stored at - 2 0 ° C for 30 days.  N o losses were  Chapter 2. Literature Review  11  2.7 Measurement of Toxicity  Monthly rainbow trout toxicity studies are used to monitor the toxicity o f the Lions Gate effluent ( G V R D , 2001).  However, a number o f other organisms can be used to evaluate the  toxicity o f anionic surfactants. toxicity o f liquid samples.  The M i c r o t o x ™ system uses Vibrio fischeri to evaluate the  The bioluminescence of Vibrio fischeri is indicative o f their  metabolic activity and provides an indication o f the relative toxicity o f a test sample.  By  comparing the bioluminescence of the test samples to that o f a control, the IC20 or IC50 can be found. The IC20 and IC50 represent the concentrations o f solution (% V / V ) or o f analyte (mg/L) that causes a 20% and 50% reduction in bioluminescence, respectively.  Two  acute toxicity test protocols can be used with the M i c r o t o x ™ system (Microbics  Corp, 1992). The first type is the detailed basic test protocol in which the highest test sample concentration is 45% by volume. The second type is the 100% test protocol in which the highest test sample concentration is 90% by volume.  The detailed basic test protocol provides more  reliable results than does the 100% test protocol.  The M i c r o t o x ™ system has been previously used to determine the toxicity o f L A S . The  IC50 al,  of L A S , having a chain length of 12 carbons, was found to be 14.29 mg/L L A S (Gutierrez et  2002).  Distributions o f L A S carbon chain lengths and molecular weights and associated  toxicities are presented in Tables 1 and 2.  Table 1: Homologue distributions o f some L A S formulations Molecular Percentage of homologue distribution Product Weight C14 C C10 C„ C C, 8.0 LAS-255 255 n.d. 0.2 8.3 24.7 55.8 LAS-242 242 n.d. 0.6 28.8 32.9 23.2 4.5 240 0.4 0.8 LAS-240 10.1 37.0 31.5 20.2 LAS-236 236 LAS-232 232 9  I 2  n.d. not detected [adapted from Vives-Rego et al,  1991]  3  Chapter 2. Literature Review  12  Table 2: Toxicity of some L A S formulations measured using the Microtox IC o in mg/L Product 5 min 15 min 30 min 24.2 10.4 LAS-255 8.1 LAS-242 19.3 12.6 11.5 LAS-240 13.4 10.5 10.2 20.4 LAS-236 25.1 20.4 LAS-232 25.6 20.2 20.2  system.  5  [adapted from Vives-Rego et al,  The range of 30-minute  IC50 values  1991]  obtained above is 8.1 to 20.4 m g / L L A S , depending  on the carbon chain length (Vives-Rego et al., 1991). A s the carbon chain length and molecular weight increase, so too does the toxicity (see section 2.3).  2.7.1 Limitations in the Measurement of Toxicity Using M i c r o t o x ™  The presence of methanol and specific ions, ions originally present in the sample or added during sample preparation, can both impact the measurement o f toxicity using the M i c r o t o x ™ system.  Ions, such as sodium, that are either intentionally added during sample preparation (see Section 2.5.1) or that are originally present in the sample, may affect the bioluminescence of Vibrio fischeri (Carlson-Ekwall and Morrison, 1995 as reported in Dizer et al,  2002).  example, the bioluminescence of Vibrio fischeri, measured using the M i c r o t o x ™ increased in the presence of N a ,  +  For  system,  K or M g ions, and decreased in the presence of heavy metals +  +  (Bitton, 1983; Carlson-Ekwall and Morrison, 1995; Watanabe et al,  1991; all reported in Dizer  et al, 2002). In addition, chloride ions may react with dissolved metal ions reducing both the inhibitory effect and the perceived toxicity of the metal ions.  Substances present in the matrix may induce or inhibit bioluminescence of the test organism (Dizer et ah, 2002); therefore, appropriate sample preparation is of the utmost importance. Methanol added during sample preparation (see Section 3.1.4 F and G) may affect the bioluminescence o f Vibrio fischeri.  Chapter 2. Literature Review  13  The results of short-term, acute toxicity tests are to be interpreted with care. Firstly, the M i c r o t o x ™ test uses a single species o f bacteria. Secondly, short-term, acute tests are unable to measure the effect of the toxicant over a longer period o f time, such as the period o f time required to degrade the toxicant in the aquatic environment (Vives-Rego et al., 1991).  Despite  these drawbacks, the M i c r o t o x ™ test offers a number o f advantages when compared to other test organisms.  The M i c r o t o x ™ test system is relatively inexpensive when compared to other  toxicity tests. In addition, it rapidly produces results, and it is relatively easy to use.  2.8 Previous Characterization and Treatment Studies at Lions Gate  In 2000, Bailey and Elphick, (2001) undertook a study of four o f the W W T P s in the G V R D , including the Lions Gate W W T P , to establish i f the effluent from these W W T P s was toxic to the aquatic life in the receiving water bodies. Effluent samples were collected from the Lions Gate W W T P from August to November, 2000 during dry-weather conditions. Samples o f the effluent were screened for acute toxicity using juvenile rainbow trout. Samples that caused 50% or greater mortality in the juvenile rainbow trout were subjected to further testing.  Samples  that were subjected to further testing underwent a Toxicity Identification and Evaluation (TIE) program.  In the T I E program, the samples underwent physical and chemical manipulations to  first determine the general'properties o f the toxicant and second to specifically identify the toxicant.  The T I E study suggested that anionic surfactants in the effluent o f the Lions Gate  W W T P were the predominant cause o f toxicity to rainbow trout. contribute to toxicity.  Ammonia was found to  However, ammonia contributed less to the toxicity than did anionic  surfactants (Bailey and Elphick, 2001).  In 2002, a follow-up study was initiated to further investigate the sources o f toxicity present in the effluent o f the Lions Gate W W T P .  This follow-up study was undertaken to  investigate the impact o f chemical substances of interest on the toxicity to three marine species. Samples o f Lions Gate primary effluent were collected between April and November 2002 during dry-weather conditions.  Half o f the samples collected were chlorinated, as is normal  plant practice during the summer season.  The marine species that were considered in the study  were the topsmelt (Atherinops affinis), blue mussel (Mytilus galloprovincialis) and giant kelp  Chapter 2. Literature Review  14  (Macrocystis pyrifera). Although the study by Bailey and Elphick (2001) indicated that anionic surfactants were responsible for the toxicity of the effluent from the Lions Gate W W T P , the contribution to toxicity from other possible contaminants/parameters was considered during the follow-up study. The contaminants/parameters that were investigated included metals, mercury, chlorine,  weak  acid dissociable  ( W A D ) cyanide,  sulphides,  ammonia, phthalate  esters,  polychlorinated biphenyls (PCB), 4-nonylphenol, dichlorodiphenyltrichloroethane (pp'-DDT), p H , dissolved oxygen, suspended solids, chemical oxygen demand ( C O D ) and temperature. The follow-up study confirmed that anionic surfactants significantly contributed to the toxicity while the contributions from ammonia and the other parameters were of a much lesser extent ( E V S , 2003).  In the follow-up study conducted in 2002 by E V S Environmental Consultants, the anionic organic toxicant was retained on hydrophobic S P E columns.  Gradient methanol elution  was used to separate the anionic surfactants into three separate molecular weight fractions. The anionic organic toxicant was predominantly recovered in the high molecular weight fractions. Intermediate  molecular weight fractions were determined to be non-toxic,  although they  contained a high concentration of anionic surfactants measured as M B A S . It was shown that the high molecular weight fractions had a lower M B A S concentration but a higher toxicity.  It was  suggested that the anionic surfactants in the high molecular weight fractions are less polar and more hydrophobic, and, therefore, more likely to be toxic ( E V S , 2003).  A bench-scale treatability study of the primary effluent from the Lions Gate W W T P was undertaken from late August until early November 2002. The goal of the treatability study was to determine the feasibility of removing M B A S  from the primary effluent using chemical  precipitation. Lime arid calcium chloride were initially evaluated to determine M B A S removal by precipitation from both the influent and effluent at the Lions Gate W W T P . lime and calcium chloride were necessary to remove M B A S .  High dosages of  In addition, adsorption and or  coagulation of surfactant molecules with suspended solids, and not chemical precipitation, was found to be the mechanism responsible for the M B A S removals. For these two reasons, lime and calcium chloride were abandoned in favor of examining treatment using alum and ferric chloride coagulation/flocculation with gravity settling. Jar testing revealed that the optimal dose of alum  Chapter 2. Literature Review  15  (as A l ) , or ferric chloride (as Fe) for the removal of M B A S using coagulation/flocculation with gravity settling was 40 m g / L .  Using alum, a 50% reduction in M B A S concentrations was  achieved in both the influent and effluent. 96-hour L C  5 0  96-hour trout toxicities were reduced from an average  of 45% ( V / V ) to 91% ( V / V ) and 77% ( V / V ) following treatments using alum and  ferric chloride, respectively ( C H 2 M H I L L , 2002).  Chapter 3. Analytical Procedures and Experimental Setups  16  Chapter 3 Analytical Procedures and Experimental Setups Analytical procedures were developed and four treatment methods were used to fulfill the objectives of the present study.  First, analytical procedures were developed to preserve and  fractionate the anionic surfactants present in a primary effluent, to measure the concentration o f the anionic surfactants and to measure the anionic surfactant induced toxicity. These analytical procedures are presented in Sections 3.1.1 to 3.1.8. In the second part o f the present study, four treatment methods were used to remove anionic surfactant induced toxicity from a primary effluent.  The  treatment  methods  that  were  used  included  bio-treatment,  alum  coagulation/flocculation with gravity settling, ozonation, and air flotation. The experimental setups used during the treatability study are presented in Sections 3.2.1 to 3.2.IV.  3.1 Analytical Procedures The analytical procedures used in the present study are detailed in the following sections: (Section 3.1.1 - sample collection), (Section 3.1.2 - filtration), ( Section 3.1.3 - sample storage), (Section 3.1.4 - solid phase extraction), (Section 3.1.5 - M B A S ) , (Section 3.1.6 - M i c r o t o x ™ ) . Figure 2 illustrates the order in which the analytical procedures were performed. It also provides the names o f samples and procedures to which reference is made in this thesis.  Chapter 3. Analytical Procedures and Experimental Setups Figure 2: Flowchart of analytical procedures.  Sample to be Analyzed  Whole Sample | Storage  65% S P E Column Extract  75% S P E Column Extract  65% S P E Reconstituted Extract  75% S P E Reconstituted Extract  90% S P E Column Extract  Samples Kept Separate for Subsequent Analyses  Microtox™  MBAS  17  Chapter 3. Analytical Procedures and Experimental Setups  3.1.1  18  Sample Collection  A) Collection from W W T P  Primary effluent samples were collected from the Lions Gate W W T P and from the U B C Pilot Plant.  Waste activated sludge was also collected from the U B C Pilot Plant.  Primary  effluent collected from the U B C Pilot Plant was used for method development while the waste activated sludge collected from the U B C Pilot Plant was used in the treatability study.  Samples of Lions  Gate primary effluent  were  collected downstream of the de-  chlorination mixers. A steel bucket was used to draw samples from the final effluent weir. The bucket was rinsed three times with primary effluent before samples were collected.  Samples of U B C Pilot Plant primary effluent were collected from a sampling port located on the primary clarifier.  The sampling port and associated pipes were flushed with primary  effluent prior to sampling. A steel bucket was used to collect samples. The bucket was rinsed three times with primary effluent before samples were collected.  A s discussed in Section 3.2.1, U B C Pilot Plant waste activated sludge was used in the treatability study (see Chapter 6).  The sludge samples were collected from a sampling port  located on the membrane activated sludge tank. The sampling port and associated pipes were first flushed with waste activated sludge. samples.  A graduated plastic container was used to collect  The container was rinsed three times with waste activated sludge before the sample  was collected.  B) Collection Following Treatment  Following treatment as outlined in Chapter 6, samples were immediately collected and preserved in accordance with the procedural steps illustrated in  Figure 2.  Further details  regarding sample collection following treatment are provided in Sections 3.2.1 through to 3.2.IV and in Section 3.2.7.  Chapter 3. Analytical Procedures and Experimental Setups  19  3.1.2 Filtration  A l l samples were filtered prior to storage or S P E column loading as illustrated in Figure 2. Samples were filtered using V W R #413 ( V W R International, West Chester, P A . ) qualitative filter papers. The filtrates were used in M B A S and toxicity tests. The filter cake was discarded, as only soluble anionic surfactants were o f interest in the present study. The filter papers were prewashed to remove any adventitious anionic surfactants (see Section 4.1.5.1.A). The washing procedure consisted o f soaking the filter papers for approximately 10 minutes in a beaker containing de-ionized water, obtained from a M i l l i - Q water system (Molsheim, France). The deionized water was then drained and replaced with fresh de-ionized water. The beaker and filter papers were then sonicated for 10 minutes.  The de-ionized water was once again drained and  replaced with fresh de-ionized water a total o f three times.  The wet filter papers were then  wrapped in aluminum foil until used.  3.1.3 Sample Storage  A) Whole Sample Storage  Filtered whole samples were stored in brown glass jars with Teflon lined lids.  In all  cases, the brown glass jars were rinsed twice with sample. When transported, the glass jars were stored along with ice packs in coolers. A t the lab, glass jars containing sample were stored at 4 °C until needed. In every instance, attempts were made to minimize the storage time particularly for unfiltered whole samples.  B) S P E Sample Storage  S P E columns have been previously shown to preserve L A S molecules (see 2.6.1).  Section  For this reason, S P E columns were used to store anionic surfactants during the  characterization and treatability studies presented in Chapters 5 and 6.  In the case o f the  characterization study, S P E columns were loaded on-site at the Lions Gate W W T P in an attempt to minimize the biodegradation of analyte. The S P E columns were activated, loaded and washed  Chapter 3. Analytical Procedures and Experimental Setups  according to the procedures detailed in section 3.1.4.  20  The loaded columns were wrapped in  aluminum foil, transported in a cooler containing ice packs and stored at 4 ° C until needed.  C) Waste Activated Sludge Storage  Waste activated sludge samples collected from the U B C Pilot Plant were stored as described in Section A above, for whole sample storage.  3.1.4 Solid Phase Extraction  Solid phase extraction consists o f five steps: activation, analyte loading, column washing, analyte elution, and reconstitution.  A) Solid Phase Extraction Apparatus  A standard 10-port vacuum box (J.T. Baker company, Phillipsburg, N.J.) solid phase extraction system was used. Figure 3 illustrates the 10-port vacuum box solid phase extraction system with all o f the supporting apparatus used in the present study.  Stopcocks were used to  control the flow through each column. 75 m L reservoirs were used to increase the volumetric capacity o f each S P E column.  Standard laboratory 4 m L test tubes were used to collect the  eluants from the S P E column. The 4 m L test tubes are subsequently referred to as elution vials. A gas flow meter was used to monitor and control the flow induced by the vacuum that was applied to the 10-port vacuum box (see Section C below).  S u p e l c l e a n ® L C - 1 8 S P E columns  (Supelco, Bellefonte Pennsylvania) were used in the present study.  Each column had 0.5 g o f  reversed phase packing material and a volume o f 6 m L . The reversed phase packing material retains non-polar to moderately polar analytes from aqueous solutions. was obtained from Fisher Scientific (Hampton, N H ) .  H P L C grade methanol  Chapter 3. Analytical Procedures and Experimental Setups  21  Figure 3: Solid phase extraction apparatus.  B) Reagent Preparation  Four methanol solutions were prepared for use in the S P E procedures. The following solutions o f methanol in de-ionized water, obtained from a M i l l i - Q water system (Molsheim, France), were prepared: 40%, 65%, 75% and 90% ( V / V ) .  A solution containing 40% methanol  in de-ionized water ( V / V ) was used to wash the loaded column. The three other solutions (65%, 75% and 90% methanol in water) were used to sequentially extract the analyte from the column. The purpose and rationale for selecting these solutions is discussed in Sections 2.6 and 4.1.5.4.  Chapter 3. Analytical Procedures and Experimental Setups  22  C) Activation  The S P E column packing material was activated before a sample was loaded. Activation leaves a thin film of water-miscible solvent on the packing material which promotes better contact between the aqueous sample matrix and the hydrophobic solid phase packing material thereby increasing the recovery of analyte (Supelco, 1997). A S P E column was positioned in the 10-port vacuum box, as shown in Figure 3. A vacuum was applied to the 10-port vacuum box and the resulting gas flow rate was adjusted to provide a liquid flow rate through the S P E column of 5 m L per minute or less. A gas flow meter was used to monitor and control the liquid flow rate.  (The gas flow meter settings were determined in previous trials, and the settings are  dependent upon the number of S P E columns being used in the 10-port vacuum box at one time.) T o this S P E column, 1 m L of 100% methanol was added. Once half of the methanol added was drawn through the packing material, the vacuum was stopped for approximately 1 minute by closing the stopcock. This enabled the methanol to contact all of the packing material for a given amount of time. The vacuum was then resumed and the remaining methanol was drawn through the packing material. Another 1 m L aliquot of 100% methanol was then added to the column and drawn through the packing material without stopping the vacuum.  Following methanol  addition, 1 m L of de-ionized water, obtained from a M i l l i - Q water system (Molsheim, France), was added to the column.  Once half of the de-ionized water was drawn through the packing  material, the vacuum was once again stopped for approximately stopcock.  1 minute by closing the  This enabled the de-ionized water to contact all of the packing material for a given  amount of time. packing material.  The vacuum was resumed and the remaining water was drawn through the Another 1 m L aliquot of de-ionized water was then added to the column.  When 1 to 2 mm of de-ionized water remained on top of the packing material, the vacuum was stopped by closing the stopcock.  This thin layer of de-ionized water was left on top of the S P E  packing material to prevent the S P E packing material from drying. If the packing material dried before the sample was added, it was necessary to repeat the activation procedure, as the analyte would not be effectively retained by the hydrophobic packing material.  Chapter 3. Analytical Procedures and Experimental Setups  23  D) Analyte Loading  The S P E columns were loaded by passing filtered samples (see Section 3.1.2) through an activated S P E column. Since only the material that can be retained on the S P E column packing material was of interest, the S P E column underflow was discarded.  A total of 50 m L of filtered sample was loaded onto a S P E column.  This volume was  selected since M B A S losses in the S P E column underflow were shown to be negligible at M B A S concentrations encountered in the primary effluent from the Lions Gate W W T P (see  Section  4.1.5.3 A ) . Initially, 3 m L of filtered sample was added directly to the S P E column. A 75 m L reservoir was then fitted to the S P E column and the remaining 47 m L of sample was added to the reservoir. The vacuum was controlled to achieve a liquid flow rate of 5 m L per minute or less through the S P E column. Had all of the filtered sample been added to the 75 m L reservoir, the S P E column packing material may have gone to dryness.  When the vacuum was first applied,  the S P E column packing material could have gone to dryness as the 6 m L of air, contained above the S P E column packing material, was first drawn through the S P E column packing material before the filtered sample was drawn from the 75 m L reservoir into the S P E column.  After the sample was drawn through the packing material, the packing material was dried. The packing material was dried by continuing the vacuum for approximately 30 seconds after all of the sample had been drawn through the packing material.  E) Column Washing  Washing was done to remove weakly retained and entrained materials from the packing material. Six-1 m L aliquots of a solution having 40% methanol in water ( V / V ) was used to wash the S P E columns. A 1 m L aliquot of the wash solution was washed over the sides of the 75 m L reservoir and drained into the S P E column. The S P E packing material was dried by continuing the vacuum for approximately 30 seconds before the next 1 m L aliquot of solution was added.  Chapter 3. Analytical Procedures and Experimental Setups  24  The S P E column wash underflow was discarded since weakly retained and entrained materials, present in the S P E column packing material, were not of interest in the present study.  F) Elution  The anionic surfactant components adsorbed to the S P E column packing material can be eluted into discrete fractions based on the hydrophobic nature of the components (see Section 2.6).  Using gradient methanol elution, the anionic surfactants were separated into low, medium  and high molecular weight fractions.  The loaded S P E column was positioned above an elution vial in the sample rack (see Figure 3).  The vacuum induced liquid flow rate through the S P E column was adjusted using a  flow meter to achieve a liquid flow rate not exceeding 5 m L per minute. T o the S P E column, a total of 2-1 m L aliquots of solution containing 65% methanol in water ( V / V ) were added. Initially, 1 m L of eluting solution (65% methanol in water) was added to the S P E column, and as soon as half of the eluting solution was drawn through the packing material, the vacuum was stopped for approximately 1 minute by closing the stopcock. This enabled the eluting solution to contact all of the packing material for a given amount of time. The vacuum was resumed and the remaining eluting solution was drawn through the packing material. The packing material was dried by continuing the vacuum for approximately 30 seconds before the next 1 m L aliquot of eluting solution was added.  The second aliquot of eluting solution was passed through the  packing material without stopping the vacuum. Following the removal of all traces of eluting solution from the packing material, the packing material was then dried by continuing the vacuum for approximately 30 seconds. Next, the vacuum was stopped by closing the stopcock. The S P E column was then moved above an unused elution vial in the sample rack.  The above procedure was repeated with a solution having 75% methanol in water. When applied after the solution having 65% methanol in water, a 75% methanol in water eluting solution can be used to elute the anionic surfactant components that exhibit intermediate hydrophobic characteristics (i.e., intermediate molecular weights).  The above procedure was  once again repeated with a solution having 90% methanol in water.  When applied after the  Chapter 3. Analytical Procedures and Experimental Setups  25  eluting solution having 75% methanol in water, the 90% eluting solution can be used to elute the anionic surfactants that exhibit high hydrophobic characteristics (i.e., high molecular weights).  G) Reconstitution  Methanol Evaporation from the SPE Column Extracts  . The methanol used in the elution process needed to be evaporated from the S P E column extracts to eliminate interferences with the M i c r o t o x ™ toxicity test results (see Section 2.7.1). T o do this, the S P E column extracts were transferred from the elution vials into 50 m L beakers. Two-1 m L aliquots of 100%) methanol were used to rinse each elution vial ensuring complete transfer o f the analyte from the elution vials to the 50 m L beakers.  The addition of 100%)  methanol also acted to lower the boiling point of the methanol contained in the S P E column extract (Stecher, 1968).  The 50 m L beakers were placed in a water bath at 88-90 ° C for 4  minutes at which time 5 m L of de-ionized water, obtained from a M i l l i - Q water system (Molsheim, France), was added. This solution remained in the water bath for an additional 20 minutes. The purpose of this evaporation procedure was to remove as much of the methanol as possible without allowing the contents of the beakers to go to dryness. The S P E column extracts must not go to dryness during the evaporation step; otherwise, the analyte may be baked onto the 50 m L beakers resulting in poor analyte recovery.  Therefore, the S P E column extracts were  watched closely during the evaporation procedure to ensure that the beakers did not go to dryness.  Standard Volume of the SPE Extracts  Reconstitution of the S P E column extract to a known standard volume is required so that subsequent toxicity and M B A S analyses can be conducted. The evaporated S P E column extracts were transferred from the beakers into 25 m L volumetric flasks.  Each beaker was rinsed three  times with de-ionized water, obtained from a M i l l i - Q water system (Molsheim, France), to ensure complete transfer of M B A S from each beaker to each volumetric flask.  The volume in  the volumetric flask was made up to 25 m L by adding de-ionized water. The solution contained  Chapter 3. Analytical Procedures and Experimental Setups  26  in the volumetric flask was subsequently referred to as the reconstituted S P E extract. It should be noted that a concentration factor o f 2x was achieved through the extraction procedure. Recall that 50 m L o f sample was passed through a S P E column.  A 10 m L screw top test tube was  rinsed three times with the reconstituted S P E extract before it was filled and the contents stored for subsequent toxicity analysis (see Section 3.1.6).  The remaining reconstituted S P E extract  was stored in a 25 m L volumetric flask for M B A S analysis (see Section 3.1.5).  A summary o f the procedures used during the S P E method is presented in Table 3.  Chapter 3. Analytical Procedures and Experimental Setups  27  Table 3: Summary o f the procedures used during the S P E method.  Step 1 — Activation Add 1 mL of 100% methanol to the SPE column and apply the vacuum to produce a flow rate of 5 mL per minute through the column (subsequently, use this flow rate). Stop the vacuum for 1 minute when half of the methanol has been drawn through the SPE column. Continue the vacuum and allow the remaining methanol to be drawn through the packing material. Add 1 mL of 100% methanol to the column and apply the vacuum until the methanol is drawn through the packing material. Repeat the above procedure using de-ionized water, obtained from a Milli-Q water system (Molsheim, France), in place of 100% methanol, and, for the second-1 mL aliquot of water, stop the vacuum when 1 to 2 mm of water remain on the top^of the packing material.  Step 2 ~ Analyte Loading Add approximately 3 mL of filtered sample to the SPE column. Place the 75 mL reservoir above the column and add the remaining filtered sample (47 mL) to the reservoir. Apply the vacuum at a rate of 5 mL per minute or less until the packing material goes to dryness.  Step 3 -- Column Washing Apply vacuum, and in succession, wash 6-1 mL aliquots of 40% methanol in water over the sides of the reservoir allowing the solution to drain into the SPE column. Allow the packing material to go to dryness before adding the next aliquot.  Step 4 - Elution Position the SPE column above an elution vial. Apply the vacuum, and add 1 mL of 65% methanol in water. Stop the vacuum for 1 minute when half of the 65% methanol in water has been drawn through the packing material. Allow SPE packing material to go to dryness before adding another 1 mL aliquot of 65% methanol in water. Let column go to dryness. Stop vacuum and move SPE column above a new elution vial. Repeat above procedure using solutions of 75% and 90% methanol in water.  Step 5 ~ Reconstitution Add contents of elution vial to a beaker. Rinse elution vial with 2-1 mL aliquots of 100% methanol adding therinseto the beaker. Heat the contents of the beaker in a 88-90°C water bath. After 4 minutes, add 5 mL of de-ionized water, obtained from a Milli-Q water system (Molsheim, France), to the beakers and continue to heat for 20 minutes. Do not allow the contents of the beakers to go to dryness Transfer the contents of the beaker into a 25 mL volumetric flask. Rinse the beaker 3 times with de-ionized water adding therinseto the volumetric flask. Rinse a 10 mL screw top test tube 3 times with reconstituted SPE extract. Fill therinsedtest tube with reconstituted SPE extract and store the test tube for toxicity analysis. Store the remaining SPE extract in the 25 mL volumetric flask for MBAS analysis.  Chapter 3. Analytical Procedures and Experimental Setups  28  H) Additional Notes on Solid Phase Extraction  Often an air bubble in the frit o f the S P E column prevented the passage o f liquid through the packing material. B y tapping the column, the bubble could often be freed. Failing this, it was sometimes necessary to close the stopcock to allow the vacuum td build-up within the vacuum box. When the stopcock was opened, the air bubble was often freed due to the greater downward force exerted by the increased vacuum.  To ensure an airtight seal between the stainless steel body o f the 10-port vacuum box and the white diaphragm attached to the lid o f the 10-port vacuum box, the white diaphragm was wetted with water.  Downward force on the lid, held for approximately 20 seconds, helped to  establish an airtight seal as the vacuum in the 10-port vacuum box was built-up.  3.1.5 M B A S Analysis  The procedure developed by Chitikela et al,  (1995) to measure the concentration o f  M B A S was used as the basis for developing a suitable method for use in the present study. The method that was developed as part o f the present study requires less sample volume, less chloroform, less glassware and less labour than does the Standard Method 5540 C ( A P H A et al, 1992).  In addition, fewer organic extractions are required than are required by the method  developed by Chitikela et al, (1995).  In the present study, the working M B A S concentration for the analytical procedure described below ranged from 0 m g / L L A S to 4 m g / L L A S . Within this range, the relationship between absorbance at a wavelength o f 652 r)m and M B A S (measured as m g / L L A S ) was linear.  The M B A S procedure consists o f three steps: organic extraction, aqueous back-wash, and measurement.  The theoretical basis o f the method is discussed in Section 2.5.  discussion, sample refers to any aqueous phase intended for M B A S analysis.  In the following In the context o f  the present study, the term, sample, corresponds to filtered whole sample, stored whole sample or any one o f 65%, 75%, or 90% reconstituted S P E extracts (see Figure 2).  29  Chapter 3. Analytical Procedures and Experimental Setups  Fresh chloroform refers to uncontaminated, reagent chloroform.  American Chemical  Society ( A C S ) chloroform was obtained from Fisher Scientific (Hampton, N H ) . It should be pointed out that High Performance Liquid Chromatography ( H P L C ) grade chloroform should not be used in the M B A S test as preservatives, used to preserve the chloroform, may interfere with the colourimetric development of the methylene blue. Chloroform, on the other hand, refers to reagent grade chloroform containing extracted methylene blue-anionic surfactant ion pairs.  A) Organic Extraction  The organic extraction step was performed in 50 m L Pyrex round bottomed vials. The vials were capped with Teflon coated lids. The vials were first rinsed 6 times with tap water and twice with de-ionized water obtained from a M i l l i - Q water system (Molsheim, France). The vials were then baked in a muffle oven for one hour at 450 - 500 ° C . Fresh chloroform was used to rinse the vials before samples were added.  T o a vial, 5 m L of sample was added.  Dilutions were necessary when the expected  M B A S concentration exceeded 4 m g / L . T o the vial, 1 drop of the alcoholic phenolphthalein indicator (Standard Method 5540 C ( A P H A et al., 1992)) and one drop o f 1 N N a O H were added.  The vial was swirled until the solution turned a uniform pink colour.  1 N H2SO4 was  then added drop wise until the pink colour of the solution disappeared and the solution became colourless.  The vial was swirled after each drop of 1 N H2SO4 was added. T w o m L of fresh  chloroform and 2 m L of methylene blue solution were then added to the vial.  Methylene blue  solution was prepared as outlined in Standard Methods 5540 C ( A P H A et al., 1992) using methylene blue (basic blue 9) obtained from Matheson, Coleman and Bell (Norwood, O H ) . The vial was capped and mixed using a vortex mixer for 30 seconds. Mixing caused the organic and aqueous phases to emulsify.  The vial was allowed to sit for 2 to 3 minutes.  During this time, the two phases would  usually separate except for small droplets adhered to the vial wall. A t this stage, complete phase separation would be ideal but not necessary.  If complete phase separation occurred, a sharp  separation between organic and aqueous phase could be made.  However, i f complete phase  Chapter 3. Analytical Procedures and Experimental Setups  30  separation did not occur, an emulsion was evident. The vial was then gently swirled to ensure a more complete separation o f the phases.  This was necessary as droplets o f aqueous phase and  organic phase, adhered to the walls o f the vial, would be present in the opposite phase.  The  chloroform, containing the surfactant-methylene blue ion pairs, was extracted from the bottom o f the vial using a Pasteur pipette.  The extracted chloroform was transferred to a second vial.  A  small residual amount o f chloroform was left in the bottom o f the first vial to prevent the transfer of any o f the aqueous phase to the second vial.  During the first extraction step, the aqueous phase may contain anionic surfactant molecules that have not yet formed ion pairs with methylene blue. Transferring these unpaired surfactant molecules would result in a reduction o f the analyte recovery. When complete phase separation did not occur, less chloroform was present in a separate phase. chloroform was transferred to the second vial.  Therefore, less  Invariably, when complete phase separation did  not occur, some aqueous phase, emulsified with the chloroform, was likely transferred to the second vial. However, every attempt was made to minimize the transfer o f aqueous phase during the first step o f the extraction.  The organic extraction step was repeated by adding 2 m L of fresh chloroform to the first vial. The vial was capped and mixed for 30 seconds, and then allowed to sit for 2 to 3 minutes. The vial was then gently swirled to produce a sharper separation of organic and aqueous phases. Emulsions were, generally, less problematic, as greater phase separation usually occurred following the second extraction.  In the event that good phase separation occurred, all of the  chloroform was extracted and transferred to the second centrifuge tube.  A s this is the final  organic extraction, a small amount of aqueous phase may be extracted to ensure complete recovery of all the chloroform. The potential transfer of some o f the aqueous phase, during the second extraction, is not problematic since complete recovery o f chloroform is more likely. Also, the aqueous back-wash step, described below, removes the residual unpaired aqueous methylene blue that may be present. In the event that good phase separation did not occur, all o f the separated chloroform and all o f the emulsion was extracted to the second vial. This ensured that all o f the chloroform containing anionic surfactant-methylene blue ion pairs was recovered.  Chapter 3. Analytical Procedures and Experimental Setups  31  B) A q u e o u s Back-wash  T o the 4 m L o f chloroform contained in the second centrifuge tube, 10 m L o f aqueous wash solution was added.  The aqueous wash solution was prepared in accordance with  Standard Method 5540 C ( A P H A et al., 1992) using sodium phosphate, monobasic monohydrate' obtained from E M Science (Gibbstown NJ). The second vial was then capped and mixed using the vortex mixer for 30 seconds.  Ideally, the two phases would separate after the vial was  allowed to sit for 2 to 3 minutes. The vial was then gently swirled. If a sharp phase separation occurred, a new Pasteur pipette was used to extract the chloroform to a third vial.  It is o f the  utmost importance that none o f the aqueous phase be transferred with the chloroform to the third vial.  The presence o f any aqueous phase in the third vial would interfere with the absorbance  reading obtained during the measurement step described below.  If persistent emulsions were present after the vial was allowed to sit for 2 to 3 minutes, glass wool plugs and sodium sulfate anhydrous were used together to remove aqueous phase from the chloroform. Sodium sulfate anhydrous is often used to remove aqueous phase from organic extracts. Ohio).  Glass wool was obtained from Ohio Valley Specialty Chemical (Marieatta,  Small glass wool plugs were inserted into the stem of a glass funnel.  The glass wool  plugs were rinsed with fresh chloroform to remove adventitious surfactants.  Sodium sulfate  anhydrous was obtained from E M Science (Gibbstown, NJ.) and enough reagent was placed on top o f the glass wool plug to just cover the top surface.  Together, the glass wool plugs and  sodium sulfate anhydrous functioned to remove aqueous phase from the chloroform as it was being transferred from the second vial to the third vial.  Following aqueous backwash, one organic back-extraction was performed to ensure complete recovery o f all methylene blue anionic surfactant ions pairs to the organic phase.  To  the second vial, 2 m L o f fresh chloroform was added, the solution was mixed for 30 seconds, then allowed to sit for 2 to 3 minutes and then gently swirled. The chloroform was transferred to the third vial as described above.  The third vial contained a total o f 6 m L o f chloroform  following completion o f the aqueous backwash procedural steps.  Chapter 3. Analytical Procedures and Experimental Setups  32  C) Measurement  Fresh chloroform was added to the third vial to increase the volume to 20 m L . If a glass wool plug was used in the aqueous backwash procedural steps, the fresh chloroform was filtered through the plug to ensure that all the anionic surfactant-methylene blue ion pairs were transferred to the third vial. The 20 m L o f chloroform was then mixed to ensure both uniform colour distribution and reliable absorbance measurements.  A new Pasteur pipette was used to rinse and partially fill a 1 cm square cuvette with the chloroform contained in the third vial.  A square cuvette minimizes interferences due to  differences in alignment o f round and possibly scratched cuvettes.  The outer surface o f the  cuvette was wiped and dried with a methanol soaked lab glassware wipe.  High performance  liquid chromatography grade methanol was obtained from Fisher Scientific (Hampton, N H ) . A Turner 690 spectrophotometer (Dubuque, I A . ) was zeroed on a process blank that had gone through the above  MBAS  analytical procedure.  Using a Tuner 690  spectrophotometer  (Dubuque, IA.) the absorbance o f each sample was measured at a wavelength o f 652 n m and compared to that o f a standard curve to determine the M B A S concentration. M B A S Standards were prepared using Dodecylbenzenesulfonic acid sodium salt ( M W = 348.48) obtained from Fluka (Buchs S G Switzerland).  It should be noted that it is best i f the third vial is graduated, as the volume o f chloroform can be accurately increased to 20 m L . If graduated vials are unavailable, a strip o f lab marking tape placed at the level equivalent to a volume o f 20 m L is sufficient. A mark placed on the tape where the readings should be sighted will help increase reproducibility. The tape can be baked onto the vial by placing the marked centrifuge tube in a 40 ° C drying oven for 20 minutes.  Table 4 summarizes the procedures used in the M B A S method.  Chapter 3. Analytical Procedures and Experimental  Setups  33  Table 4: Summary of the M B A S method procedures.  Step 1 ~ Organic Extraction Place 5 m L of sample in the first 50 m L vial. A d d 1 drop phenolphthalein to the sample and 1 N N a O H drop wise until solution turns pink. A d d 1 N H2SO4 drop wise until solution turns colourless. A d d 2 m L of fresh chloroform. A d d 2 m L of methylene blue. M i x for 30 seconds. Let sit 2 to 3 minutes to separate phases and gently swirl to ensure complete separation. Extract chloroform and transfer into a second 50 m L vial using a Pasteur pipette. Repeat the extraction in the first centrifuge tube once using 2 m L of fresh chloroform.  Step 2 — Aqueous Backwash To second vial, containing approximately 4 m L of extracted chloroform from step 1, add 10 m L of wash solution to the second vial. M i x for 30 seconds, allow phases to separate and swirl. Extract chloroform and transfer it to a third 50 m L vial using a new Pasteur pipette. T o second vial, add 2 m L of fresh chloroform, mix, allow the phases to separate and extract to third vial.  If persistent emulsions are present, use a plug of glass wool to filter the chloroform extract as it is being transferred into the third vial.  Step 3 — Measurement To the third vial, containing approximately 6 m L of extracted chloroform from step 2, make up the volume in the third vial to 20 m L by adding fresh chloroform. Measure the absorbance at 652 n m using a standard curve.  Chapter 3. Analytical Procedures and Experimental Setups 3.1.6  Microtox  34  Analysis  A detailed description of the procedures and the materials used in the Microtox™ test system are outlined in the owners manual (Microbics Corporation, 1992). The detailed basic test protocol, recommended by the equipment manufacturer, was used (see Section 2.7). Figure 4 illustrates the Microtox™ apparatus used in the present study.  The bioluminescence of the bacteria contained in the reconstituted acute reagent decreases with time (Microbics Corporation, 1992). For this reason, once a vial of acute reagent is reconstituted, it has a shelf life of 2 hours. During the present study, six detailed basic test protocols could be performed within approximately 1.5 hours. Any acute reagent remaining after this time was discarded to ensure that the shelf life was not exceeded during a test run.  When following the detailed basic test protocol, outlined in the Microtox  users manual,  many test configurations are possible. In the present study, the detailed basic test protocol was  Chapter 3. Analytical Procedures and Experimental Setups  35  followed with three samples being run in parallel. Each of the three samples were analyzed with one control and four serial dilutions of the sample. concentrations that were  A s a result, the whole filtered sample  examined included 5.625%,  11.250%, 22.500%, and 45.000%.  Similarly, in the reconstituted S P E column extracts, where a concentration factor o f 2x was used, the sample concentrations examined were 11.250%, 22.500%, 45.000% and 90.000%.  The data reduction formulae, used to calculate the toxicity of a sample, are presented in the M i c r o t o x ™ users manual (Microbics Corporation, 1992). The IC20 represents the sample concentration or analyte concentration which inhibits the bioluminescence of the Vibrio  fischeri  by 20%. The IC2o's were calculated and presented in the present study. When reconstituted S P E extracts were tested, correction factors were determined as part of the present study and applied to the bioluminescent values measured using the M i c r o t o x ™ instrument (see Section 4.2.4). These correction factors accounted for the influence of foreign substances introduced from the S P E column packing material.  Although the M i c r o t o x ™  users manual provides a detailed description of the test  protocols, it does not adequately emphasize the importance of proper reagent reconstitution. T o achieve reproducible results, it is of the utmost importance that the acute reagent  be  reconstituted properly. Specifically, the reconstitution solution must be poured - as quickly as is possible - from the cuvette containing the reconstitution solution into the reagent vial.  In the  present study, this was accomplished by holding both the cuvette and reagent vial at an angle to vertical, and inserting the cuvette into the reagent vial - so that the open end of the cuvette was lined up over the open end of the reagent vial (see Figure 5).  The reconstitution solution was  rapidly transferred to the reagent vial by inverting the cuvette over the reagent vial. The reagent vial was then swirled 3 to 4 times. Next, the activated reagent was transferred from the reagent vial into the cuvette that previously contained the reconstitution solution.  Finally, the cuvette  was immediately placed in the reagent well of the M i c r o t o x ™ instrument (see Figure 4).  Chapter 3. Analytical Procedures and Experimental Setups  36  Figure 5: Cuvette and reagent vial at an angle to vertical.  The  Microtox  users manual (Microbics Corporation, 1992) does not adequately  emphasize the sensitivity of Vibrio fischeri to temperature. A n increase in the temperature will increase the bioluminescence. Therefore, the cuvettes should be transferred to the read well of the instrument as quickly as possible without excessive handling. During the present study, physical contact with the bottom of the cuvette (e.g. wiping the cuvettes with laboratory wipes or handling the cuvettes for more than just a few seconds) was shown to increase the bioluminescence.  3.1.7 Methanol Concentration An HP 6890 series gas chromatogram (GC) with a flame ionization detector (Richmond, BC) was used to determine the methanol concentration present in the reconstituted SPE extracts. The GC was operated with a split ratio of 10:1, an injection volume of 1.0 uL, an inlet heater temperature of 150 °C, and a flame ionization detector operated at 250 °C. A n Agilent J & W  Chapter 3. Analytical Procedures  and Experimental  Setups  37  DB624 capillary column (Palo Alto, C A ) with dimensions of 28.0 m x 530 urn x 3.0 jam was used.  The column was operated in the constant flow mode having a flow o f 35 cm/sec.  The  oven set point was 35 ° C for 4 minutes after which a 20 ° C / m i n u t e ramp occurred until the oven temperature reached 100 ° C . The total run time was 7.25 minutes.  3.1.8 Total S u s p e n d e d S o l i d s A n a l y s i s  The total suspended solids concentrations were measured for the bio-treatment methods used in the treatability study presented in Chapter 6.  The procedure outlined in Standard  Methods, 2540 D ( A P H A et al., 1992) was followed to determine the total suspended solids.  3.2  Experimental Set-up and Analytical Procedures for Treatment Studies  Bench-scale treatment methods were considered as part of the present study.  The  methods were selected based on their documented or expected abilities to remove anionic surfactants from wastewater (see Sections 2.4 and Chapter 6). The flowchart presented in Figure 6 illustrates the treatment and analytical procedures followed.  Chapter 3. Analytical Procedures and Experimental  Setups  Figure 6: Flowchart of treatment and analytical procedures. Raw Primary Effluent  *Microtox™ only applied to ozone and air flotation treatments  38  Chapter 3. Analytical Procedures and Experimental  Setups  39  I Bio-Treatment  A) Biological Degradation  Five hundred m L o f waste activated sludge from the U B C Pilot Plant was aerated for 10 minutes using a coarse bubble diffuser. The aerated waste activated sludge was added to a 2 L beaker. Next, 500 m L of Lions Gate effluent was added to the beaker containing the activated sludge. Aeration was applied for 30 minutes. After 30 minutes o f aeration, the total suspended solids concentration was measured (see Section 3.1.8).  Approximately 400 m L o f the mixed  liquor was transferred from the beaker into two-200 m L centrifuge tubes. The mixed liquor was centrifuged for 5 minutes at 3000 R P M .  The supernatant present at the top o f the centrifuge  tubes was defined as the treated effluent from the biological treatment test. The treated effluent was subject to further procedural steps and analyses as discussed in section 3.2.7.  B) Partitioning to Abiotic Bio-Solids  Five hundred m L o f waste activated sludge was aerated for 10 minutes using a coarse bubble diffuser. The aerated waste activated sludge was added to a 2 L beaker. Five grams (1% by weight) o f sodium azide was added to the 500 m L o f waste activated sludge to inactivate the biological solids (Berube, 2000). This mixture was then mixed for five or ten minutes to ensure that inactivation o f the biological solids was complete.  Five hundred m L o f primary effluent from the Lions Gate W W T P was added to the beaker containing the inactivated sludge. The mixture was aerated for 30 minutes. After 30 minutes o f aeration, total suspended solids concentration was measured (see Section 3.1.8). Approximately 400 m L o f the mixed liquor was transferred from the beaker into two-200 m L centrifuge tubes. The mixed liquor was centrifuged for 5 minutes at 3000 R P M . The supernatant present at the top of the centrifuge tubes was defined as the treated effluent from the partitioning to abiotic biosolids test. The treated effluent was subject to further procedural steps and analyses as discussed in Section 3.2.7.  Chapter 3. Analytical Procedures and Experimental Setups  40  II A l u m Coagulation/Flocculation with Gravity Settling  A l u m coagulation/flocculation with gravity settling tests were performed using a jar testing apparatus (Phipps and Bird, Richmond Virginia). Eight hundred m L o f primary effluent from the Lions Gate W W T P was added to a 1 L beaker, to which alum was added. A stock 100 m g / m L alum solution was prepared in advance, and 4 m L o f the stock solution was added. This resulted in an alum concentration o f 40 m g / L (as A l ) present in the beaker.  A previous study  showed that this dose was the most effective at removing M B A S (see Section 2.8).  Immediately following the addition o f alum, the p H o f the solution was adjusted to target a p H o f 7.0 by adding six drops o f 6 N N a O H .  The solution was rapidly stirred at 300 R P M for  60 seconds followed by a slow mix at 50 R P M for five minutes. Following mixing, the solution was settled for 30 minutes.  Following settling, the supernatant present at the top o f the 1 L beaker was defined as the treated effluent from the alum settling test. The treated effluent was subject to further procedural steps and analyses as discussed in Section 3.2.7.  III Ozonation  2 L of primary effluent from the Lions Gate W W T P was added to a 2 L Erlenmeyer flask. The flask was fitted with a bung having an inlet and an exit port. Ozone was added to the system through the inlet port. The off gas exited the system through the exit port. Residual ozone in the off gas was captured using potassium iodide (KI) traps.  The sample was ozonated for ten  minutes at the highest capacity o f the ozone generator (Azco Industries, Surrey, B C . ) .  Figure 7  depicts the experimental set-up used.  The whole ozonated sample was defined as the treated effluent.  The treated effluent was  subject to further procedural steps and analyses as discussed in section 3.2.7.  Chapter 3. Analytical Procedures and Experimental Setups  41  Figure 7: Ozonation apparatus.  IV Air Flotation  400 m L o f primary effluent from the Lions Gate W W T P was added to a 500 m L glass graduated cylinder. A fine bubble diffuser was placed at the bottom o f the cylinder. Figure 8 depicts the experimental set-up used. A i r was introduced at such a rate that a stable froth layer formed on the liquid surface.  Sufficient air flow was required to generate a stable froth.  Insufficient or excessive air flow rates would result in a froth that collapsed making froth removal impossible. The froth layer was removed by applying vacuum to a fine tipped Pasteur pipette. The Pasteur pipette was positioned in the froth layer just above the liquid/froth interface. A i r flotation and froth removal were applied for five minutes. Both the initial and final volumes o f the liquid were noted. The froth product removed with the vacuum was discarded.  Chapter 3. Analytical Procedures and Experimental Setups  42  After air flotation, the sample remaining in the graduated cylinder was defined as the treated effluent.  The treated effluent was subject to further procedural steps and analyses as  discussed in Section 3.2.7. Figure 8: A i r flotation experimental set-up.  3.2.7 Analytical Procedures  The M B A S concentrations in the whole filtered raw Lions Gate primary effluent and in the 65%, 75% and 90% reconstituted S P E extracts were determined in triplicate (see Figure 6). The toxicities contained in the whole filtered raw Lions Gate primary effluent and in the 90% reconstituted S P E extract were determined in triplicate using the M i c r o t o x ™ system.  The initial M B A S concentration contained in the bio-treatment systems was determined using a mass balance approach. The contributions o f M B A S in the whole and 65%, 75% and  Chapter 3. Analytical Procedures and Experimental Setups  43  90% elution fractions from both the waste activated sludge and the Lions Gate primary effluent were considered.  The treated effluents from the partitioning to abiotic bio-solids, biological treatment, alum coagulation/flocculation with gravity settling, ozonation and air flotation treated effluent samples were analyzed in triplicate for whole filtered M B A S and M B A S in the reconstituted S P E extracts (65%, 75% and 90% elution fractions) as illustrated in Figure 6.  The toxicity contained in the treated effluents  from the ozonation and air flotation  treatment methods was determined using the M i c r o t o x ™ system.  The whole filtered samples  and the 90% reconstituted S P E extracts were analyzed in triplicate. The toxicity contained in the treated effluents  from the partitioning to abiotic bio-solids, the biological, and the alum  coagulation/flocculation with gravity settling tests were not measured. When conducting these treatment tests, additional material (i.e., abiotic sludge containing sodium azide (see Section 3.2.I.B), waste activated sludge (section 3.2.LA) or alum (section 3.2.II)) was introduced into the primary effluent matrix.  A s a result, the characteristics o f the primary effluent matrix was  changed making it impossible to establish the impact o f the treatment on the original primary effluent matrix. matrices.  Therefore, toxicity measurements were not performed on these modified  Chapter 4. Development of Analytical  AA  Methods  Chapter 4 Development of Analytical Methods T o fulfill the objectives o f the present study, analytical methods were developed to quantify the concentration o f anionic surfactants in a primary effluent, to fractionate the anionic surfactants on the basis o f molecular weight and to measure the toxicity o f the whole and discrete molecular weight fractions. The analytical methods needed to be reliable and compatible with each other. Determining the best set o f sample preparation procedures and operating conditions, for both the M B A S and the M i c r o t o x ™ tests, required a significant amount o f work.  4.1 Methylene Blue Active Substances Method Development  A new analytical method was developed to measure M B A S concentrations in the present study.  A number o f tasks were performed as part of the method development.  First, sample  storage procedures, working M B A S concentration ranges and the precision o f the method were investigated, to establish the conditions within which meaningful and reproducible M B A S data could be obtained.  Second, a series o f quality control tests were performed to ensure that the  new method was reproducible while producing high analyte recoveries. sample preparation procedures on M B A S  recovery was investigated.  Finally, the effect o f Sample preparation  procedures consisted o f the preliminary steps required to prepare a sample for analysis.  4.1.1 Short-Term Sample Storage  M B A S concentrations in the whole filtered samples were measured as soon as was possible following sample collection. The time interval between sample collection and sample analysis was due to the time spent preparing the sample for analysis and to the time spent transporting the sample from the collection site to the Environmental Engineering Laboratories at UBC.  However, as discussed in Section 2.6.1, L A S has been shown to degrade rapidly i f not  stored properly.  T o investigate the impact o f short-term sample storage, the concentration o f  M B A S in a preserved sample was compared to that o f an unpreserved sample, after a one-hour period had elapsed.  Short-term preservation procedures were considered to minimize the  Chapter 4. Development of Analytical  Methods  45  degradation of M B A S over a one-hour period. The unpreserved samples were simply stored at 5 ° C . Formaldehyde was added to the preserved samples, and the mixture was stored at 5 ° C (see Section 2.5.1).  Following collection, one primary effluent  sample from the U B C Pilot Plant was  immediately preserved on site with 1% (W/W) formalin (37% formaldehyde in water)  (Fisher  Scientific, Fair Lawn, NJ). A second sample, collected at the same time, was not preserved with formaldehyde.  Both samples were filtered on-site and stored for 1 hour at 5 ° C before whole  filtered M B A S tests were conducted in the Environmental Engineering Laboratory at U B C . The sample preserved with formaldehyde was found to have an M B A S concentration of 5.9 m g / L , while the control sample had an M B A S concentration of 6.3 mg/L. The raw data is presented in Table D.3 in Appendix D . The results of this investigation indicate that M B A S biodegradation did not occur during the time interval between sample collection and analysis. Therefore, when the sample was immediately filtered and stored at 5 ° C , formaldehyde addition was not required to preserve M B A S .  Greater M B A S stabilities can be achieved using S P E columns and refrigeration as a preservation method. A s presented in Section 2.6.1, M B A S biodegradation does not occur when a sample is loaded onto a S P E column and stored at 4 ° C (Petrovic and Barcelo, 2000). Therefore, samples loaded onto a S P E column can be stored for a few days prior to analysis.  4.1.2 Working M B A S Concentration Range  A s presented in Section 2.5, the concentration of M B A S in a sample is determined spectrophotometrically by measuring the absorbance at a wavelength of 652 um.  MBAS  standards were prepared using a 1 000 m g / L L A S (Fluka, Buchs S G Switzerland) stock solution prepared with de-ionized water, obtained from a M i l l i - Q water system (Molsheim, France).  A  linear relationship was observed between the M B A S concentration and the absorbance over an M B A S concentration range from 0 to 4 mg/L.  Figure 9 shows this linear relationship.  Chapter 4. Development of Analytical  Methods  46  Figure 9: M B A S standard curve.  M B A S Standard Curve  MBAS (mg/L)  Throughout the present study, samples were diluted with de-ionized water, obtained from a M i l l i - Q water system (Molsheim, France), prior to analysis when the M B A S concentration of samples were expected to exceed 4 m g / L .  4.1.3 Precision of the M B A S Analytical Method  The precision of the M B A S analytical method was determined.  Precision provides an  indication of the quality of the data by considering the variability of the results obtained from multiple analyses of a given sample.  The standard deviation associated with multiple analyses  was used as a measurement of the precision. A precise analytical procedure is characterized by a small standard deviation. The type of sample (i.e., whole sample versus L A S standard) and the nature of the sample preparation procedures (i.e., SPE) used may affect the precision of the analytical method. The precision associated with the M B A S analytical method was determined by considering the standard deviation of the results obtained when developing the standard calibration curve, when analyzing the reconstituted S P E extracts and when analyzing the whole sample. This enabled the impact of sample preparation on the precision of the M B A S analytical method to be quantified.  Chapter 4. Development of Analytical  Al  Methods  A) Precision of the M B A S Analysis for the Standards  The precision o f the M B A S analytical method was determined using standards containing 0.5, 1, 2 and 4 m g / L L A S . These standards were prepared on December 16, February 3  rd  and 26,  th  th  2003, and  2004. The raw data is presented in Tables C l , C.2 and C.3 (Appendix C ) .  Outliers were not included in the determination o f precision. values determined on March 1  Outliers included all the M B A S  presented in Table C.3 (Appendix C ) . A standard curve was  st  prepared from the data presented in Tables C l , C.2 and C.3 (except the outliers o f March 1 ). st  Table 5 below contains the number o f samples considered, the average M B A S concentration measured, and the standard deviation.  Table 5: Precision o f the M B A S analytical method found using M B A S standards. Concentration of Standard (mg/L L A S ) 2.0 0.5 1.0 4.0 Number of Replicates 4 5 2 3 Mean Concentration (mg/L M B A S ) 0.48 0.92 1.85 4.13 0.02 Standard Deviation (mg/L M B A S ) 0.04 0.19 0.16  The highest standard deviation is approximately 0.2 m g / L M B A S as found in the 1 m g / L LAS  standard.  This means  that, for the analysis o f standards prepared using various  concentrations o f L A S , 68.3% of the measured values will be within 0.2 m g / L M B A S o f each other assuming the measured values are normally distributed. For the purposes o f the present study, which focuses on relative trends rather than absolute values, the variability is considered to be acceptable.  B) Precision Following Sample Preparation Procedures  A s part o f the Lions Gate W W T P effluent characterization study presented in Chapter 5, the M B A S concentrations were measured in triplicate in the whole sample and in the 65%, 75% and 90% reconstituted S P E extracts.  Standard Method 1030 C was followed to determine the  precision o f the M B A S measurements that had been performed in triplicate ( A P H A et al, 1992). (Note, the whole sample analysed on February 3, 2004 was not done in triplicate, and it was, therefore,  not  procedures).  included in the  determination o f precision following  sample preparation  Table 6 below contains the number o f replicates, the average range o f measured  Chapter 4. Development of Analytical  Methods  48  M B A S concentrations and the calculated standard deviations for both the whole samples and the reconstituted S P E column extracts.  Table 6: Precision of M B A S method following sample preparation procedures.  Number of Replicates Average Range (mg/L M B A S ) Standard Deviation (mg/L M B A S )  M B A S Analysis Following SPE 21 0.44 0.26  Whole Sample M B A S Analysis 4 0.65 0.39  Sample preparation procedures, such as filtration and S P E and reconstitution induces some variation in the measured M B A S values i.e., increases the standard deviation.  The  standard deviations of the results obtained following sample preparation procedures are greater than those found following the analysis of standards as presented above.  The decrease in the  precision is likely due to the greater number of procedural steps involved in these sample preparation procedures.  The whole sample analysis exhibited a substantially higher variation. This was likely due to the persistent emulsions which occurred during the analysis of whole samples. plugs were used to break these emulsions.  Glass wool  A s discussed in Section 4.1.5.2.A, glass wool plugs  impact the results from the M B A S analysis.  The  highest  standard  approximately 0.4 m g / L M B A S .  deviation  following  sample  preparation  procedures  was  This means that approximately 68.3% of the measured M B A S  values will be within 0.4 m g / L M B A S assuming the measured values are normally distributed. For the purposes of the present study, which focuses on relative trends rather than absolute values, this variability is considered to be acceptable.  4.1.4  Recovery of Analyte During The M B A S Analytical Procedure  Both Standard Methods 5540 C ( A P H A et al, Chitikela et al,  1992) and the method developed by  (1995) recommend the use of three organic extraction steps, followed by one  aqueous backwash and two additional organic back-extraction steps.  Organic extraction and  Chapter 4. Development of Analytical  Methods  49  organic back-extraction steps are used to recover analyte to the organic phase while aqueous backwash steps are used to remove excess methylene blue and some interferences from the organic phase (see Section 2.5).  It is well known that greater analyte recoveries and more  reproducible results are achieved as the number of organic extraction steps increases.  However,  more time is spent completing the analysis as the number of extractions increases.  The  feasibility of using only two organic extractions and two aqueous backwashes was considered.  A  test was designed to determine the M B A S  recovery following just two organic  extractions, one aqueous backwash and one organic back-extraction.  A 1 m g / L standard L A S  (Fluka, Buchs S G Switzerland) solution was prepared. T w o analyses were performed on this standard L A S solution.  The first analysis used 2 organic extractions followed by one aqueous  backwash and 1 organic back-extraction.  The second analysis used 3 organic extractions  followed by one aqueous backwash and 2 organic back-extractions.  Each analysis  was  conducted four times. M B A S recoveries obtained using the two test conditions are presented in Table 7.  Table 7: Recovery of M B A S following 2 or 3 extractions. Trial 1 . 2 3 4 Average  Two extractions.followed by one back-extraction Recovery (%) 91 100 102 96 97 ± 8  Three extractions followed by two back-extractions Recovery (%) 102 96 92 111 100 ± 13  ± corresponds to the 95% confidence interval  The recoveries were 97 ± 8% and 100 ± 13 % following 2 or 3 organic extractions, respectively. In addition, the 95% confidence interval for two organic extractions was 8%. This narrow confidence interval indicates that the use of two extractions and one organic backextraction generates reproducible results. Given the high recovery of analyte (97%) and the narrow confidence interval ( ± 8%) obtained, the procedure which consists of using two organic extractions, one aqueous backwash and one organic back-extraction was deemed suitable for use in the present study.  Chapter 4. Development ofAnalytical Methods  50  4.1.5 Impacts of Sample Preparation Procedural Steps on M B A S Recovery  Sample preparation procedural steps, shown in Figure 2, were used to ready samples for analysis using the M B A S method.  A s discussed below, some sample preparation procedural  steps have been identified that increase or decrease the recovery of M B A S in the sample being prepared for analysis using the M B A S method.  4.1.5.1 Positive Interferences of Sample Preparation Procedural Steps on M B A S Recovery  Standard Methods ( A P H A et al,  1992) cautions the reader of potential contamination  from adventitious surfactants adsorbed to filter media. Adventitious surfactants are those that are adsorbed to unwashed filter media, for example. interference.  Adventitious surfactants act as a positive  Positive interferences were found in the filter papers and in the S P E column  packing material used in the present study.  A) Filtration  Preliminary investigations, undertaken as part of the present study, indicated that the filter papers used to filter raw effluent could act to increase the M B A S concentration of a filtered sample. Initially, 60 m L of de-ionized water, obtained from a M i l l i - Q water system (Molsheim, France), was used to rinse a V W R #413 ( V W R International, West Chester, P A . ) filter paper. Next, approximately 60 m L of de-ionized water, obtained from a M i l l i - Q  water system  (Molsheim, France), was filtered through the pre-rinsed filter paper. The filtrate was analyzed for M B A S . This analysis was performed in duplicate. The concentration of M B A S in the filtrate was 0.06 and 0.07 m g / L M B A S .  Although the M B A S concentrations are very low, they are  consistently greater than 0 m g / L M B A S .  These results suggest that the filtration process can  impact the results.  These preliminary results prompted an additional test to be conducted which was designed to investigate the effectiveness o f a more thorough filter paper wash procedure used to  Chapter 4. Development of Analytical  Methods  51  remove adventitious surfactants from filter papers. The procedure consisted o f pre-washing and sonicating the filter papers as outlined in section 3.1.2. B y following this procedure, no M B A S was detected in the filtrate.  B) SPE  S P E columns and gradient methanol elution were used in the present study to separate anionic surfactants on the basis o f hydrophobicity, which for anionic surfactants is an indication of the molecular weight.  Since filtered sample is passed through the S P E column packing  material, any adventitious surfactants adsorbed to the S P E column packing material could influence M B A S recoveries in the reconstituted S P E extracts.  The potential M B A S contamination from a new S P E column was investigated. This was done by passing 50 m L o f de-ionized water, obtained from a M i l l i - Q water system (Molsheim, France), through an activated S P E column according to the procedure outlined in Section 3.1.4. Following the S P E wash procedure, elution was carried out using solutions containing 65%, 75%, and 90% methanol in de-ionized water ( V / V ) as described in Section 3.I.4.F. The elution extracts were evaporated and reconstituted using de-ionized water to give a final volume o f 25 m L as described in Section 3.I.4.G. Trace concentrations o f M B A S were detected in the blank reconstituted S P E column extracts as presented in Table 8.  Table 8: M B A S concentrations in blank reconstituted S P E column extracts.  Fraction 65% Reconstituted SPE Extract 75% Reconstituted SPE Extract 90% Reconstituted SPE Extract  MBAS Measured in Reconstituted SPE Column Extracts (mg/L) 0.09* 0.07* 0.08*  value obtained by interpolation  A more thorough S P E column wash/activation procedure to eliminate the presence o f adventitious surfactants was not developed.  The M B A S measured in the reconstituted S P E  column extracts was very low, and no correction factors were applied. For the purposes o f the present study, which focuses on relative trends rather than absolute values, this  MBAS  Chapter 4. Development of Analytical  concentration is considered to be  acceptable.  Methods  In addition, effects  52  of a more rigorous  wash/activation procedure on the surface properties of the S P E packing material were unknown. The standard procedure outlined by the S P E column manufacturer was used (Supelco, 1997) to activate the reverse phase packing material.  4.1.5.2 N e g a t i v e M B A S I n t e r f e r e n c e s  M B A S recoveries decreased when either glass wool or sodium chloride was used during sample preparation procedures.  A) Glass Wool  Glass wool filter plugs were used to break persistent emulsions by removing aqueous phase from organic phase as discussed in Section 3.I.5.B.  Persistent emulsions are commonly  encountered when biologically treated wastewaters are analysed using the M B A S method. The effect of glass wool plugs on M B A S recovery was investigated by filtering a 1 m g / L L A S Fluka (Buchs  S G Switzerland) solution through a glass wool plug and measuring the  concentration in the filtrate. Method 5540 C ( A P H A et al,  MBAS  1992) was followed to measure the  concentration of anionic surfactants, as this series of investigations preceded the implementation of the modified M B A S method used in the present study (Section 3.1.5). The concentration of M B A S in the filtrate was then compared to that of a 1 m g / L L A S Fluka (Buchs S G Switzerland) control solution that had not been filtered. The results are presented in Table 9.  Table 9: The effects of glass wool on M B A S recoveries. Trial 1 2 3 4 Average  Set 1 Glass Wool Recovery (%) 94 83 84 82 86 ± 1 5  Set 2 No Glass Wool Recovery (%) 100 98 100 102 100 ± 3  ( ± 95% confidence interval)  Chapter 4. Development ofAnalytical Methods  53  Based on a 95% confidence interval, the use of glass wool plugs did not significantly affect the recovery of M B A S .  However, the recovery of M B A S  was consistently  lower  (approximately 14% lower) in those samples that had been passed through glass wool plugs. In addition, a narrower confidence interval was achieved for the samples that had not been filtered through glass wool plugs.  Therefore, glass wool plugs should only be used when necessary to  break-up persistent emulsions.  Persistent emulsions would otherwise interfere with the accurate  determination of absorbance measured using a Turner 690 spectrophotometer (Dubuque, IA.) with a wavelength of 652 nm.  B) Sodium  Chloride  Marcomini et al,, (1987) reported that the recovery of M B A S during filtration and S P E could be increased by adding sodium chloride to the sample prior to filtration or extraction. After filtering the sample, Marcomini et al,  (1987) rinsed the filter paper and the S P E column  packing material with a solvent capable of eluting adsorbed M B A S . Therefore, Marcomini et al. (1987) measured both the dissolved and adsorbed fractions of M B A S simultaneously.  The effects of sodium chloride addition on M B A S recovery following filtration and S P E were investigated.  In the present study, 8% N a C l (W/W) was added to a 1 m g / L L A S Fluka  (Buchs S G Switzerland) solution. This solution was then filtered through a washed filter paper (see Section 3.1.2). M B A S recoveries were compared to those of a control solution consisting of a 1 m g / L L A S Fluka (Buchs S G Switzerland) solution to which sodium chloride had not been added. The results are presented in Table 10.  Table 10: The effect of sodium chloride on M B A S recovery following filtration 8% NaCl (w/w) + 1 mg/L L A S 1 mg/L L A S Control Trial Recovery (%) Recovery (%) 92 1 67.5 2 95.5 71 69 ± 2 2 94 ± 2 2 Average  Based on a 95% confidence interval, the addition of sodium chloride did not significantly affect the recovery of M B A S after the sample had been filtered.  However, the recovery of  M B A S was consistently lower (approximately 25% lower) in those samples to which sodium  Chapter 4. Development of Analytical  Methods  54  chloride had been added prior to filtration. These results are somewhat contradictory to those reported by Marcomini et al, (1987). However, in the present study, only soluble M B A S was considered. MBAS.  Marcomini et al,  (1987) considered both soluble and particulate (i.e., adsorbed)  These adsorbed M B A S species were subsequently eluted from the filter paper and  added to the soluble M B A S contained in the filtrate.  The effect of sodium chloride addition on the recovery of M B A S following S P E elution was also investigated.  In the present study, 8% N a C l (WAV) was added to a 1 m g / L L A S Fluka  (Buchs S G Switzerland) solution. This solution was then loaded onto an activated S P E column as described in Section 3.1.4. Elution was carried out using solutions containing 80%, 85%, and 90%) methanol in water ( V A / ) .  The cumulative M B A S recoveries from all three fractions were  compared to that o f a control solution consisting of a 1 m g / L L A S (Fluka, Buchs S G Switzerland) solution to which sodium chloride had not been added. The results are presented in Table 11.  Table 11: The effect of sodium chloride on M B A S recovery following S P E Trial 1 2 Average  NaCl (8% W/W) + 1 mg/L L A S Cum. Recovery (%) 61 43 52 ± 114  1 mg/L L A S Cum. Recovery (%) 71.5  While not based on a 95% confidence interval, a trend was identified in which lower recoveries of M B A S were obtained for the samples to which sodium chloride had been added. The use of a preliminary M B A S analytical method accounts for the low M B A S  recoveries  presented in Table 11.  Consistently, the addition of sodium chloride to sample solutions before filtration or S P E decreased the recovery o f M B A S .  In addition, sodium ions are a known interference with the  bioluminescence of Vibrio fischeri an organism that is used in the M i c r o t o x ™ analytical method. For these two reasons, sodium chloride was not used in the present study.  Chapter 4. Development of Analytical  Methods  55  4.1.5.3 Potential L o s s of M B A S  The potential loss of analyte in the S P E underflow and wash underflow streams was investigated.  The potential loss of analyte during the S P E extract reconstitution procedure was  also investigated.  A) SPE Underflow and Wash Underflow  During S P E column loading, sample is drawn through the S P E column packing material, and anionic surfactants are retained on the hydrophobic packing material. The remainder of the sample leaves the column through the bottom of the column. underflow (see Section 3.1.4.D).  This stream is named the S P E  A wash step is then performed to remove weakly retained and  entrained matter from the S P E column packing material.  The wash solution, containing 40%  methanol in water ( V / V ) , passes through the packing material and leaves the column through the bottom of the column. This stream is named the S P E wash underflow (see Section 3.1.4.E). The S P E underflow and the S P E wash underflow streams both have the potential to contain M B A S that should otherwise be retained by the S P E column packing material and recovered following gradient methanol elution. T o establish i f M B A S is lost to these streams, primary effluent from the U B C Pilot Plant was loaded onto a S P E column, and the M B A S concentrations of the S P E underflow and wash underflow streams were measured.  The results are presented in Table 12.  Raw data is presented in Tables D.4 and D.5 (Appendix D).  Table 12: Recoveries of M B A S to the S P E underflow streams. Sample Description U B C Pilot Plant - Nov 26, 2003 U B C Pilot Plant - Dec 11, 2003  Recovery (%) of M B A S to SPE Underflow Streams SPE Wash Underflow SPE Underflow 4 3.4 ' 7.6  The M B A S contained in the S P E underflow streams represented a potential loss in analyte recovery of approximately 3.7%. However, the potential loss of M B A S in the S P E wash underflow stream was much larger. It is likely that a portion o f the M B A S lost to the underflow streams consisted of either weak hydrophobic anionic surfactant molecules interferences (see Section 2.5).  or of positive  However, further testing would be necessary to address this  Chapter  hypothesis.  4. Development  of Analytical  Methods  56  Weak hydrophobic anionic surfactant molecules were not o f interest in the present  study, as they have not been identified as being highly toxic (see Section 2.3).  B) SPE Extract  Reconstitution  The potential loss of analyte during the procedure used to reconstitute the S P E column extracts was investigated (see  Section 3.1.4.G).  The reconstitution process consisted  evaporating the methanol contained in the S P E extracts.  of  While the removal o f methanol is  important for accurate determination o f M B A S toxicity using the M i c r o t o x ™ system, heating the extracts may bake the M B A S onto the glassware. It is of the utmost importance that the extracts do not go to dryness during the evaporation step.  Otherwise, low recoveries o f M B A S will  occur.  The effects o f the methanol evaporation procedure (see Section 3.1.4.G), used in the present study, on the recovery o f M B A S  in the reconstituted S P E column extracts  was  investigated. Fifty m L o f a solution containing 1 m g / L L A S Fluka (Buchs S G Switzerland) was loaded onto an activated S P E column. Gradient methanol elution was carried out using 2 m L o f 70% and 90% solutions o f methanol in water ( V / V ) .  These elution solutions were arbitrarily  chosen to limit the number o f samples requiring analysis.  Next, 2 m L o f 100% methanol was  used to rinse any residual analyte from the elution vial into a beaker containing the S P E column extract.  The 100%) methanol was used to rinse the S P E elution vial ensuring complete M B A S  transfer, and to lower the boiling point o f the methanol contained in the S P E column extract (see Section 3.1.4.G).  The S P E column extract was then evaporated for 4 minutes at which time 5  m L of de-ionized water, obtained from a M i l l i - Q water system (Molsheim, France), was added to the beaker. The de-ionized water was added to ensure that the S P E column extract did not go to dryness while being evaporated for an additional 20 minutes. These tests were done in duplicate. M B A S recoveries and the concentration o f methanol present in each o f the reconstituted S P E column extracts are presented in Table 13.  Chapter 4. Development of Analytical  Methods  57  Table 13: M B A S recovery following methanol evaporation. Elution Fraction 70% 90%  Cum. Recovery (%) 44 100  Trial 1 Methanol Cone. (mg/L) 79 N.D.  Cum. Recovery (%) 62 111  Trial 2 Methanol Cone. (mg/L) 158 N.D.  N . D . = Non-detect  Using the evaporation method outlined in Section 3.1.4.G, high M B A S recoveries were achieved.  The concentration of methanol in the reconstituted samples was relatively low.  At  these concentrations, methanol does not produce a toxic response in the bioluminescence of Vibrio fischeri measured with the M i c r o t o x ™ system (see Sections 2.7.1 and 4.2.5.A).  4.1.5.4 Recovery of M B A S to Elution Fractions  A s discussed in Section 3.1.4.F, gradient methanol elution solutions containing 65%, 75% and 90% methanol in water ( V / V ) were used in the present study. These elution fractions were chosen based on the recovery of M B A S to each fraction following S P E column gradient methanol elution.  The 65%, 75% and 90% methanol elution solutions represent cumulative  analyte recoveries of approximately 35%, 75% and 95%, respectively as illustrated in Figure 10. Primary effluent from the U B C Pilot Plant was used in this investigation.  Similar recoveries of  M B A S to each elution fraction were expected to be found when the Lions Gate primary effluent was analyzed.  Chapter 4. Development of Analytical  Methods  58  Figure 10: Cumulative M B A S recovery versus elution fraction. 140  Elution Fraction % Methanol in Water (VA/) 95% Confidence Intervals  4.2 M i c r o t o x ™ Method Development  The results obtained from the M i c r o t o x ™ system can be impacted by the effects o f the sample preparation procedures. T o identify and eliminate (or minimize) the effect o f the sample preparation procedures, a number of investigations were undertaken.  4.2.1  M i c r o t o x ™ Acute Reagent Reconstitution  Proper acute reagent reconstitution is o f the utmost importance in achieving reproducible results. A method for acute reagent reconstitution is outlined in Section 3.1.6. During the course of the present study, it was observed that the bioluminescence o f the controls would decrease between 30 to 40% when improper acute reagent reconstitution procedures were used.  A  significant decrease in the bioluminescence o f the controls can mask the effect of a toxicant contained in a sample.  Chapter 4. Development of Analytical  Methods  59  When the acute reagent is properly reconstituted, the light levels o f the controls should not decrease substantially during a 15-minute acute toxicity test. A substantial decrease in the bioluminescence o f the controls during a 15-minute acute toxicity test may indicate that the acute reagent was not reconstituted as described in Section 3.1.6.  4.2.2  Standard Zinc Toxicant  A s recommended by the M i c r o t o x ™ system manufacturer, a zinc sulphate solution can be used as a standard toxicant to verify that the M i c r o t o x ™ system is performing as expected. The toxicity o f a solution containing 50.8 m g / L of zinc sulphate was assessed. The resulting IC50 was 7.0 ± 4 . 1 m g / L ZnSC>4 ( ± corresponds to the 95% confidence interval). The normal IC50 range is between 5 and 12 m g / L ZnSC>4 (Microbics Corporation, 1992).  The results indicated  that the M i c r o t o x ™ system was performing as expected. Figure A . l ( A P P E N D I X A ) contains a plot of the data obtained from the standard zinc toxicant investigation.  4.2.3  S P E Reconstitution Water  Preliminary tests suggested that the type o f water used to reconstitute the S P E column extracts could decrease the bioluminescence o f the Vibrio fischeri toxicity test system.  used in the M i c r o t o x ™  The effects o f de-ionized water, obtained from a M i l l i - Q water system  (Molsheim, France), and distilled de-ionized water on the bioluminescence of Vibrio fischeri  was  investigated further.  The impact o f de-ionized water, obtained from a M i l l i - Q water system (Molsheim, France), and distilled de-ionized water, on the bioluminescence o f Vibrio fischeri, were tested using the detailed basic test protocol (Microbics Corporation, 1992). The results are presented in Table 14, and the raw data is found in Table A . 1 ( A P P E N D I X A ) .  Chapter 4. Development of Analytical  Methods  60  Table 14: Laboratory water and percent light reduction. Sample De-ionized water* Distilled De-ionized  +  Percent Light Reduction (%) 5.625% 11.25% 22.5% 45% 0.7 1.8 0.6 6.0 5.8 9.6 16.5 2.9  * Analysis done in duplicate because o f pipetting error. +  Analysis done in triplicate.  A s presented in Table 14, de-ionized water, obtained from a M i l l i - Q water system (Molsheim, France), had the least affect on the bioluminescence o f Vibrio fischeri measured using the M i c r o t o x ™  system.  Therefore, de-ionized water, from a M i l l i - Q water system  (Molsheim, France), was used in all subsequent sample preparation and analytical procedures used in the present study.  4.2.4 SPE Correction Factors  S P E columns were used to retain and fractionate anionic surfactants on the basis of hydrophobicity and molecular weight (see Section 2.6).  The S P E columns used in the present  study contained 0.5 g o f reverse-phase packing material.  Preliminary trials indicated that  substances eluted from blank packing materials induced a toxic response measured using the M i c r o t o x ™ system.  Attempts were made to remove the substances inducing toxicity by thoroughly rinsing the S P E column during the activation procedure. However, the blank S P E column extract continued to induce toxicity in the M i c r o t o x ™ test, despite thorough rinsing.  The technical support  personnel at Microbics Corporation recommended that a series o f correction factors could be used to adjust the results from the M i c r o t o x ™ test to account for the toxic responses induced by the blank S P E columns.  The first step in determining the magnitude o f the correction factors was to establish the percent reduction in the bioluminescence o f Vibrio fischeri, measured using the M i c r o t o x ™ system, when blank reconstituted S P E column extracts were analyzed. A n activated S P E column was loaded with 50 m L o f de-ionized water, obtained from a M i l l i - Q water system (Molsheim, France).  Extraction was carried out using solutions having 65%, 75% and 90% methanol in  Chapter 4. Development of Analytical  water ( V / V ) .  Methods  61  The extracts were evaporated and made up to a volume o f 25 m L .  These  procedures followed those outlined in Section 3:1.4.  Table 15 lists the magnitude o f the reduction in the bioluminescence of Vibrio  fischeri,  measured using the M i c r o t o x ™ system for different dilutions. Values are reported as the percent decrease  in bioluminescence resulting from the blank S P E column extract.  (APPENDIX  Table B . l  B) contains the percent reductions in bioluminescence, measured using the  M i c r o t o x ™ system, for all three trials.  Table 15: Correction factors - blank S P E column. Sample 65% Fraction 75% Fraction 90% Fraction  5.625(%) 0.77 0.36 0  Light decrease (%) 11.25(%) 22.5(%) 1.14 2.71 1.54 . 1.94 2.28 5.30  45(%) 7.49 6.26 13.23  The 45% dilution exhibited the greatest decrease in bioluminescence for all three elution fractions.  O f the three elution fractions, the 90% elution fraction had the greatest induced  toxicity.  In subsequent tests using samples o f reconstituted S P E column extracts, correction factors, corresponding to the respective dilution and elution fraction, were used to correct the measured 15-minute bioluminescence values.  4.2.5  Residual Methanol in the Reconstituted S P E Extracts  Methanol was used in the present study to separate anionic surfactants into discrete fractions, and to rinse analyte from the elution vials into beakers containing the S P E extracts (see Section 3.1.4.F and G). However, methanol is known to inhibit the bioluminescent bacteria used in the M i c r o t o x ™ system (see Section 2.7.1). Therefore, the methanol in the S P E extracts must be removed before toxicity analyses can be performed.  Chapter 4. Development of Analytical  Methods  62  A) Methanol Evaporation  A methanol evaporation procedure was developed to reduce the methanol concentration in the reconstituted S P E column extracts without decreasing the recovery o f analyte. A series o f tests were performed using solutions containing methanol in water at concentrations equivalent to those used in the S P E elution procedures (see Section 3.1.4.F).  A s the concentration o f  methanol in water increases, the boiling point of methanol decreases (Stecher, 1968).  For this  reason, a second series of tests were performed in which the concentration of methanol in water was increased by adding 100% methanol (see Section 3.1.4.G).  Solutions respectively.  1 and 2 contained 4 m L o f 70% and 90% methanol in water  (V/V),  Solutions 3 and 4 contained 2 m L o f 100% methanol plus 2 m L o f 70% and 90%  methanol in water ( V / V ) , respectively.  These solutions were heated in a water bath having a  temperature o f approximately 90 ° C . After 4 minutes of heating, 5 m L o f de-ionized water, obtained from a M i l l i - Q water system (Molsheim, France), was added to the solutions. solutions were heated for an additional 15 to 30 minutes.  The  The methanol concentrations in the  solutions were determined at the end o f the different heating periods. The results are presented in Figure 11.  Figure 11: Residual methanol concentrations versus heating time 2500 i  :  0  15  20  25  Heating Time (minutes)  30  Chapter 4. Development of Analytical Methods  63  Following 20 minutes of heating, solutions 3 and 4 had the lowest residual concentrations of methanol. The residual methanol concentrations after 20 minutes o f heating were 316 m g / L , 79 mg/L, not detected, and 8 mg/L, respectively for solutions 1, 2, 3, and 4, respectively.  Following 20 minutes of heating, the estimated decrease in the bioluminescence o f Vibrio fischeri,  corresponding to a methanol concentration o f 8 m g / L found in solution 4, is 0.2% (see  Section 4.2.5.A). This decrease is very low and considered acceptable given the purpose o f the present study which is to identify trends rather than obtain absolute values.  Therefore, the  addition o f 2 m L o f 100% methanol to the S P E extracts followed by a heating time o f 20 minutes was used in the present study as outlined in Sections 3.1.4.F and G .  B) Toxicity of Methanol  The toxicity o f methanol to the bioluminescent bacteria used in the M i c r o t o x ™ system was investigated.  Three methanol in water solutions were prepared. Following the 100% test  procedure (see Section 2.7), the toxicity o f methanol to Vibrio fischeri  was determined using the  M i c r o t o x ™ toxicity test system. Table 16 contains the results o f the investigations, and the raw data is presented in Figures A . 2 , A . 3 , A . 4 (for methanol concentrations o f 791, 1582 and 3955 mg/L, respectively) ( A P P E N D I X A ) .  Table 16:, Methanol toxicity measured using the Microtox Residual amount of methanol in solution 791 mg/L methanol 1582 mg/1 methanol 3955 mg/L methanol  system.  15 min IC o (mg/L methanol)  15 min IC o (mg/L methanol)  2 300* 1 200 1 700  11 700* 12 000* 3 800*  2  * Values obtained by extrapolation.  5  Chapter 5. Sampling Program & Characterization  of Primary Effluent  64  Chapter 5 Sampling Program & Characterization of Primary Effluent Lions Gate primary effluent was characterized on three occasions.  Grab samples were  collected in the morning, evening and night on two sampling events, while a morning grab sample was collected on the third occasion.  This third sample was used in treatment studies  presented in Chapter Six.  The primary effluent  was  characterized in terms o f the concentration o f anionic  surfactants (measured as M B A S ) and the anionic surfactant induced toxicity (measured using the M i c r o t o x ™ system).  These parameters were measured in the whole filtered samples, and in the  65%, 75% and 90% reconstituted S P E extracts.  U B C Pilot Plant primary effluent was also characterized in terms of the concentration o f anionic surfactants and the anionic surfactant induced toxicity. These parameters were measured in whole filtered samples, and in the 65%, 75% and 90%) reconstituted S P E extracts. The results of the U B C Pilot Plant primary effluent characterization study are presented in Appendix D .  5.1 Sampling at The Lions Gate W W T P  Characterization studies were undertaken using the primary effluent from the Lions Gate WWTP.  The primary effluent was sampled on December 16,  11:30 P M and on February 3,  rd  ,h  2003 at 9:30 A M , 6:30 P M , and  2004 at 9:45 A M , 6:45 P M and 11:30 P M - Both sampling events  were performed during periods o f rain. 26,  th  A grab sample was taken on the morning o f February  2004 for use in the characterization study (current Chapter) and in the treatability study that  is presented in Chapter Six.  For the grab samples collected on December  16,  th  2003 and February 3,  rd  2004,  preliminary sample preparation was done on-site at the Lions Gate W W T P to minimize the biodegradation o f M B A S . 3.1.2).  Approximately 250 m L o f primary effluent was filtered (see Section  O f the filtered sample, 150 m L was withdrawn and loaded onto three S P E columns as  Chapter 5. Sampling Program & Characterization  of Primary Effluent  65  outlined in Section 3.I.4.D. The S P E columns were wrapped in aluminum foil and stored along with the remaining whole filtered sample (see Section 3.1.3). A l l subsequent sample preparation procedures and analyses were performed at U B C in the Environmental Engineering Laboratory.  th Sample handling procedures, for the grab sample collected on February 26,  2004, are outlined  in Section 6.1 . A . Table 17 contains an accounting o f sample storage and analysis times. Table 17: Sample storage and analysis times - characterization. Sample Collection 9:30 A M Dec 16 6:30 P M Dec 16 11:30 P M Dec 16 9:45 A M Feb 3 6:45 P M Feb 3 11:45 P M Feb 3 8:00 A M Feb 26  M B A S Analysis afternoon Dec 16 evening/night Dec 16 night Dec 17 afternoon Feb 3 night/morning Feb 3/4 night Feb 4 Feb 27  Microtox Analysis afternoon Dec 16 morning Dec 18 afternoon Dec 18 afternoon Feb 4 morning Feb 5 afternoon Feb 5 Feb 27 1M  Methanol Analysis night Dec 18 night Dec 18 night Dec 18 night Feb 5 night Feb 5 night Feb 5 N.A.  5.2 Characterization of the Lions Gate W W T P primary effluent  The results from the characterization study o f the Lions Gate W W T P primary effluent are presented in the sections below. The results are presented in terms of: M B A S concentration and the induced toxicity.  5.2.1 M B A S  M B A S was measured in the whole filtered samples and in the 65%, 75% and 90% reconstituted S P E elution fractions. Cumulative recoveries to each o f the three elution fractions were calculated based on the M B A S concentration measured in the whole filtered sample.  In general, cumulative recoveries were high with the exception o f the morning sample taken on February 3,  rd  2004.  The poor recoveries observed for the reconstituted S P E column  extracts prepared from the sample collected on the morning o f February 3,  rd  2004 are likely due  to an analytical error. These results are considered to be outliers and have not been included in subsequent discussions or in plots o f the data presented in this Chapter.  The results are  Chapter 5. Sampling Program & Characterization  summarized in Table 18 and illustrated in Figure 12.  of Primary  Effluent  66  (Detailed results can be found,in Tables  C l , C.2 and C.3 ( A P P E N D I X C).  Table 18: M B A S concentration measured in the Lions Gate W W T P primary effluent. Date  Morning Cum. MBAS (mg/L) Rec. (%)  Sample  Evening Cum. MBAS (mg/L) Rec. (%)  Nie ht MBAS (mg/L)  Cum. Rec. (%)  Dec 6.40 ± 1.32 Whole 1.20 ±0.35 430 ± 0.60 16 Dec 1.10 ± 0 . 2 4 1.10 ± 0.33 0.42 ± 0.22 35 26 65% 16 Dec 3.90 ±0.91 0.76 ± 0 . 1 0 98 2.80 ± 1.30 91 75% 16 Dec 106 0.38 ±0.21 100 1.10 ± 0.14 90% 0.10±0.10 16 9.34 8.63 Feb 3 Whole 4.39 outlier outlier 26.4 2.86 ± 0 . 8 9 65% 2.28± 1.85 Feb 3 outlier 94.7 5.75± 0.89 outlier 5.89± 1.94 Feb 3 75% outlier 1.60 ± 0 . 3 2 1.35± 0.45 outlier 113 Feb 3 90% Whole 3.71 ± 1.05 Feb 26 65% 1.70 ±0.35 45.8 Feb 26 2.39 ±0.82 110.2 Feb 26 75% 0.39 ±0.21 Feb 26 90% 120.8 ± corresponds to the 95% confidence interval of the measurements made, n=3  Figure 12: M B A S versus time - whole filtered primary effluent. 10  0 -I 07  1  .  1  1  11  .  1  .  1  .  1  15  .  1  —  .  —  .  19  Time (hours) n=3 for Dec 16 & Feb 26, 95% confidence interval n=1 for Feb 3.  .  1 —  23  17 79 96 30.6 92.2 107  Chapter 5. Sampling Program & Characterization  of Primary Effluent  67  The M B A S concentration ranges in the morning between 1.20 m g / L and 4.39 mg/L, and increases throughout the day ranging between 6.4 m g / L and 9.34 m g / L late at night. sampling events, which occurred on December 16, place during periods o f rain.  th  2003 and February 3,  rd  The  2004, both took  Storm water and seawater infiltration is known to significantly  increase the wet-weather flows to the Lions Gate W W T P .  A s a result, the characteristics of the  Lions Gate W W T P effluent is impacted by rain events.  It can be expected that the dilution  effect, caused by rain events, can reduce the M B A S concentration in the wastewater. difference  in the M B A S  concentrations  observed between the two  The  sampling events can  potentially be explained by the difference in the magnitude o f the rain intensity on both o f these days which causes storm water and seawater infiltration. Despite the difference in the M B A S concentrations between the two days, there is a trend exhibited in which M B A S concentration increases throughout the day and into the night.  The M B A S content o f the 65%, 75% and 90% elution fractions, reported as a percentage of the whole filtered mass of M B A S , was determined for the morning, evening and night sampling events. The percent of the whole filtered mass of M B A S , in each elution fraction, was averaged for the two sampling events and results are presented in Figure 13. Since the morning sample collected on February 3,  rd  2004 contained outliers in the reconstituted S P E extracts, the  data obtained from the morning sample collected on February 26,  th  2004 was used to replace the  outliers.  Figure 13: Average M B A S content in each o f the elution fractions. 100  ,  65  75  90  Elution fraction (% methanol in water V/V) 95% confidence interval shown  Chapter 5. Sampling Program & Characterization  of Primary  Effluent  68  As presented in Figure 13, the percentage o f the whole filtered mass o f M B A S in each o f the elution fractions was similar for each sampling time (i.e., morning, evening, and night) (based on a 95% confidence interval). The distribution o f M B A S observed in the three elution fractions likely reflects collection system.  the original surfactant formulations discharged to the wastewater  Recall from Table  1 that many anionic surfactant formulations have  somewhat normal distributions, centered around a particular molecular weight as is also the case in Figure 13. The observed M B A S distribution confirms the work done by E V S (2003) in which the concentration o f M B A S , found in the high molecular weight fraction, was lower than the concentration found in either the low or medium molecular weight fractions (see Section 2.8).  The results o f the M B A S analysis are further discussed in Section 5.2.3.  5.2.2 Toxicity  Methanol concentrations were measured in the samples prepared for toxicity tests, and the concentrations are presented in Tables C.4 and C.5 ( A P P E N D I X C). The highest methanol concentrations measured in the reconstituted S P E extracts was 478 m g / L .  A s presented in  section 4.2.5.B, at this methanol concentration, the bioluminescence o f Vibrio fischeri estimated to decrease by 6.2%.  is  This value is not expected to significantly impact the toxicity  measurements, as it is just one o f three replicates.  15-minute acute toxicities were determined for the whole filtered sample and for the 65%, 75% and 90% reconstituted S P E extracts on both December 16, 2004 for the morning, evening and night sampling events.  th  2003 and February 3,  O n February 26,  th  rd  2004 only the  whole filtered sample and the 90% reconstituted S P E extract toxicities were determined. The results are summarized in Table 19. Raw data is presented in Figures C . 3 to C.28 (Appendix C ) .  The toxicities are presented in terms of the percent o f sample volume and in terms o f the concentration of M B A S required to produce a 20% reduction in the bioluminescence o f Vibrio fischeri  measured using the M i c r o t o x ™ system. With this nomenclature, a lower percentage, or  a lower concentration, is indicative o f a sample exhibiting greater toxicity.  Chapter 5. Sampling Program & Characterization  of Primary Effluent  69  Table 19: Toxicity contained in the Lions Gate W W T P primary effluent. Date  Sample  Morning lC mg/L I C % (Vol) MBAS 18.93±5.62 0.23±0.10 100*±212 0.42*±0.92 36.26±37.94 0.28±0.30 0.02±0.04 18.02±27.18 26.61±7.37 1.17±0.32 outlier outlier outlier outlier ' outlier outlier 16.94±5.48 0.63±0.27 68.15*±35.39 0.27*±0.20 20  20  Dec 16 Dec 16 Dec 16 Dec 16 Feb 3 Feb 3 Feb 3 Feb 3 Feb 26 Feb 26  Whole 65% 75% 90% Whole 65% 75% 90% Whole 90%  Evening I C mg/L IC %(Vol) MBAS 13.59±5.58 0.58±0.25 109*±61 1.2*±0.72 33.36±9.05 0.93±0.50 34.44±17.06 0.13±0.10 1.14±0.22 13.17±2.53 43.08±16.04 0.98±0.87 1.30±1.14 21:99±17.83 10.27±6.22 0.16±0.10 20  2 0  Night IC  20  % (Vol)  9.08±0.71 52*±36.87 17.64±23.19 17.82±7.97 11.81±1.60 44.56±25.51 22.80±26.90 8.42±7.42  1C mg/L MBAS 0.58±0.13 0.58*±0.45 0.69±0.92 0.20±0.09 1.10±0.15 1.27±0.83 1.31±1.56 0.11±0.10 20  measurements, n= * value obtained by extrapolation The toxicity contained in the whole filtered primary effluent from the Lions Gate W W T P versus time is presented in Figure 14.  Figure 14: Toxicity versus time - whole filtered primary effluent. 40 8 CD CO CD O  — • — December 16, 2003 •• O • February 3, 2004 - - T — February 26, 2004  35  30  o  T3 CD  25  •o  CD CD C  \l  Z.  20  z  15  (-1 c CD O CD  10  0. 07:00:00  11:00:00  15:00:00  19:00:00  23:00:00  Time (hours) 95% Confidence intervals shown  The percent o f sample volume needed to cause an  IC20, measured using  the Microtox  TM  system, ranges from 17% to 27% ( V / V ) in the morning, and decreases throughout the day ranging from 9% to 12% late at night.  A s presented in Figure 14, the whole filtered primary  effluent became more toxic throughout the day and into the night.  Storm water and seawater  Chapter 5. Sampling Program & Characterization  of Primary Effluent  70  infiltration is known to significantly increase the wet-weather flows to the Lions Gate W W T P . A s a result, the characteristics o f the Lions Gate W W T P effluent is impacted by rain events. It can be expected that the dilution effect, concentration in the wastewater.  caused by rain events, can reduce the  MBAS  Therefore, the difference in the anionic surfactant induced  toxicities contained in the whole filtered samples on the two sampling events can potentially be explained by the difference in the magnitude o f the rain intensity on both o f these days which causes storm water and seawater infiltration. Despite the difference in the toxicities between the two days, there is a trend exhibited in which toxicity increases throughout the day and into the night.  A s presented in Figure 15, a linear relationship was observed between the  IC20  values  (expressed as percent o f sample) and M B A S concentrations in the whole filtered sample. The slopes o f the lines, for the two days, are relatively similar. While wet-weather conditions may influence the magnitudes of the M B A S  concentrations and  IC20  values, the toxicological  response (slope o f the lines) to an increasing M B A S concentration is similar for both sampling events.  Figure 15: Toxicity of whole filtered primary effluent versus M B A S concentration.  1  40 "i  o S D  December 16, 2003 February 3, 2004 February 26, 2004 30  "CD O  TD CD  "S 20 CD C  V  "c  CD  i t CD  IO  A  C CD  5£  CD CL-  10  MBAS (mg/L as LAS MW = 348.48) 95% Confidence intervals shown  Chapter 5. Sampling Program  & Characterization  of Primary  Effluent  71  Figures 16 and 17 are plots o f IC2o's, expressed as percent volume and as m g / L M B A S versus elution fractions for December 16,  th  2003.  Similarly, Figures 18 and 19 are plots o f  IC2o's, expressed as percent volume and as m g / L M B A S versus elution fractions for February 3, and February 26,  th  2004.  The results from February 3,  rd  rd  2004 are outliers and have not been  included in the plots (see Section 5.2.1).  Figures 16 & 17: IC20' versus elution fractions - December 16, 2003. S  Figure 16.  Figure 17.  120 •  g-100  ••o'—  > cu  Morning Evening Night  E 80 O >  c 60 cu  o  CD  ^ 40 CN  O  20  65  75  90  Elution fraction (% methanol in water VA/)  65  75  90  Elution fraction (% methanol in water V/V)  The IC o's expressed in terms o f mg/L M B A S (Figure 17) were calculated based on the 2  IC20, as percent volume, for a given sample and the corresponding M B A S concentration for that sample. However, the relationship between the magnitude of the values for the IC20 expressed as percent volume (Figure 16) and as m g / L M B A S (Figure 17) is not consistently similar.  This  discrepancy is likely due to the error associated with the measured M B A S concentrations and  IC20 values determined using the M i c r o t o x ™ system. Tables 18 and 19 contain the experimental uncertainty, at the 95% confidence interval, associated with the measured M B A S concentrations and toxicities.  Despite the discrepancies that are likely a result o f experimental uncertainties,  overall trends can be observed.  Chapter 5. Sampling Program  & Characterization  of Primary  Effluent  72  Figures 18 & 19: IC^o's versus elution fractions - February 3 and 26, 2004. Figure: 18. 5 0  T~  :  Figure: 19. = n  Elution fraction (% methanol in water VA/)  2.0  n  ,  i  =  Elution fraction (% methanol in water VA/)  The trends exhibited in Figures 16 and 18 show that the toxicity o f the reconstituted S P E extracts, measured using the M i c r o t o x ™ increases.  system, increases as the eluting solvent strength  Similarly, the trends exhibited in Figures 17 and 19 show that the toxicity o f the  reconstituted S P E extracts is greatest for the 90% elution fraction. These results indicate that the 90% elution fraction is consistently the most toxic component o f the whole filtered primary effluent.  These findings are consistent with the discussion presented in Section 2.3 which  indicates that hydrophobic anionic surfactants are more toxic to aquatic organisms. In addition, these findings support the results obtained from a study conducted by E V S (2003) (see Section 2.8).  In the study conducted by E V S (2003), toxicity tests were conducted using three marine  species as the test organisms.  The more hydrophobic (i.e., higher molecular weight) fractions  were found to be more toxic to the test organisms.  The results from the present study and those from the E V S (2003) study suggests that the Vibrio fischeri  used in the M i c r o t o x ™ test system respond in a similar manner to other aquatic  organisms (i.e., A. affinis,  M. galloprovincialis  and M. pyrifera)  when exposed to anionic  surfactants. The toxicity analysis results are further discussed in Section 5.2.3.  Chapter 5. Sampling Program & Characterization  of Primary Effluent  73  5.2.3 Toxicological Impact  The average toxicities and the average M B A S concentrations, for the sampling events o f December 16,  th  2003 February 3,  rd  and 26, 2004, are presented in Figures 20 and 21. th  Figures 20 & 21: Average toxicity and average M B A S measured during all sampling events.  Figure 20: Average Toxicity  Figure 21: Average M B A S  00 CO CO  o X  o  c •(/) O)  CD CD  CO  <  _l  co CD  CO  < E  o  65  75  90  Elution fraction (% methanol in water VA/) 95% Confidence intervals shown  65  75  90  Elution fraction (% methanol in water VA/) 95% Confidence intervals shown  The 90% elution fraction was identified as being the most toxic o f the three elution fractions studied (see Section 5.2.2). In addition, it was found that the 90% elution fraction had the lowest amount o f M B A S (see Section 5.2.1).  These results indicate that a relatively large  proportion of the toxicity that is associated with the primary effluent is due to a relatively small proportion o f the total M B A S concentration. These finding are consistent with the discussion presented in Section 2.3 that indicates that the high molecular weight anionic surfactants are more toxic to aquatic organisms. These findings also confirm the previous work o f E V S (2003) (see Section 2.8).  Chapter 5. Sampling Program & Characterization  of Primary Effluent  74  The toxicological impact of the different elution fractions is defined as the percent of M B A S in excess of that amount of M B A S which corresponds with the  IC20  The calculated  values for the toxicological impact of the different elution fractions are presented in Figure 22.  Figure 22: Toxicological impact of elution fractions. 800  ljl  6 0 t H  co ±=  CuS.  400 S  CD  M-  <=  ° -s C £ <D ^  200  HoOX v co CD Q. co CD c  Elution fraction (% methanol in water VA/) 95% Confidence intervals shown  While not based on a 95% confidence interval, the 90% elution fraction appears to have the greatest toxicological impact of the three elution fractions.  Even though the relative  abundance of M B A S contained in the 90% elution fraction is low (see Figure 21), the toxicity o f the 90% elution fraction is the highest of the three elution fractions (see Figure 20). Therefore, the 90% elution fraction has the greatest toxicological impact of the elution fractions studied. The molecular structure of the anionic surfactant molecules contained in the 90% elution fraction likely accounts for this observation. In the 90% elution fraction, high molecular weight anionic surfactant molecules are likely present (see Section 2.3).  For this reason, the 90% elution  fraction was the subject o f further investigations as reported in Chapter Six.  Chapter 6. Treatability Studies  75  Chapter 6 Treatability Studies A treatability study was conducted in which various physical/chemical and bio-treatment methods were considered to remove both anionic surfactants and anionic surfactant induced toxicity from a primary effluent.  The treatability study was designed to screen the treatment  methods, and it was a minor aspect in the overall work completed as part of the present study. The four treatment methods considered were bio-treatment, alum coagulation/flocculation with gravity settling, ozonation and air flotation.  The experimental procedures used as part o f the  treatability study are presented in Section 3.2. discussed below and in Section 2.4.  The basis for selecting these four methods is  Table 20 contains a summary o f the rationale for selecting  each of the treatment methods considered.  Table 20: Rationale for selecting treatment methods Treatment Method  Rationale  I Bio-Treatment A) Partitioning to Abiotic Bio-Solids B) Biological Degradation  II Alum Coagulation/Flocculation with Gravity Settling III Ozonation IV Air Flotation  Documented ability of solids to adsorb and remove L A S from solution (see Section 2.4.1). Documented ability of bio-solids to degrade up to 85% of L A S while producing less toxic by-products (see Sections 2.3.1 and 2.4.1). Findings from a previous study indicated that a 50% reduction in M B A S concentration was possible (see Section 2.8). Documented ability to oxidize L A S (see Section 2.4.2). Favourable surface chemistry. Pre-concentrates anionic surfactants.  A literature search did not find any studies that investigated the removal o f anionic surfactants  from  wastewater  using  air  flotation.  However,  because  of  their  surface  characteristics, anionic surfactants are expected to adsorb to the surface o f air bubbles (Rubio et al,  2002).  Subsequently, the air bubbles and adsorbed surfactants are expected to rise to the  surface o f the liquid where they form a froth layer that can be removed.  Chapter 6. Treatability Studies  76  6.1 Sampling  Table 21 contains an accounting o f sample storage times. Table 21: Date on which sample analyses were conducted during the treatment study. February 27  February 26 Filtration and SPE  Microtox™  1,2,3,4,5,6,7  1,2,3  Whole Sample MBAS 1,2,3,4,5,6,7  Fractionated MBAS 1 (n=l&2)  February 28  March 1  Fractionated M B A S  Fractionated M B A S  1 (n=3) 2,3  4,5,6,7  1 = raw filtered effluent, 2 = air flotation effluent, 3 = ozonation effluent, 4 = U B C Pilot Plant waste activated sludge, 5 = biological effluent, 6 = partitioning to abiotic bio-solids effluent, 7 = alum coagulation/flocculation with gravity settling effluent, n = number o f replicates.  A)  Lions Gate W W T P  Primary effluent was obtained from Lions Gate W W T P on the morning o f February 26,  th  2004 for use in the treatability study. 8 L o f primary effluent was collected from the Lions Gate W W T P (see Section 3.1.1). The primary effluent was stored in 2-4 L brown glass bottles at 4 ° C until needed.  B) U B C Pilot Plant  A waste activated sludge sample was collected from the U B C Pilot Plant on the morning of February 26, B).  th  2004. This sample was used in the bio-treatment test (see Sections 3.2.I.A and  1.5 L of waste activated sludge was collected from the U B C Pilot Plant, and was stored in a  2 L glass bottle at 4 ° C until needed.  6.2 Removal of M B A S  The M B A S removal efficiency was determined by comparing the M B A S concentrations in the treated effluent to that present in the primary effluent before treatment. For bio-treatment where the primary effluent was diluted with waste activated sludge, the starting M B A S concentrations were calculated using a mass balance approach (see Section 3.2.7). The results from the treatability study are presented in Table 22.  Tables E . l through to E.8 (Appendix E)  11  Chapter 6. Treatability Studies  include the raw M B A S data for the different treatments considered.  Each treatment  was  conducted once, but M B A S measurements were done in triplicate.  Table 22: Treatability study: M B A S removal efficiencies. Sample Description Whole filtered sample 65% 75% 90%  Partitioning to solids 64 ± 9 7 56 ± 42 100 ± 9 5 67 ± 100  Removal Efficiency (%) Biodegradation Alum Ozone 96 ± 8 3 46 ± 3 2 95 ± 3 9 85 ± 4 9 63 ± 3 8 94 ± 2 9 98 ± 9 5 49 ± 4 3 97 ± 4 8 95 ± 100 0±73 82 ± 7 8  Air Flotation 77 ± 3 6 66 ± 2 5 77 ± 4 4 74 ± 7 7  ± Corresponds to 95% confidence interval  I Bio-Treatment  A) Biological Degradation  Biological treatment resulted in the highest overall M B A S removal efficiency approximately 96% of the M B A S being removed during treatment.  with  The suspended solids  concentration was 1850 mg/L. The M B A S removal efficiency is slightly higher than removal efficiencies reported in the literature in which L A S removals o f 80-85% are reported (Fauser et al, 2003; Kolbener et al,  1995).  O f the 96% of the M B A S removed, a large fraction was likely removed simply by the M B A S molecules partitioning to bio-solids.  N o attempt was made to quantify the relative  contribution of both biological degradation and partitioning to bio-solids to the overall removal of M B A S .  B) Partitioning to Abiotic Bio-Solids  Overall, a 64% reduction in the M B A S partitioning to abiotic bio-solids alone.  concentration could be achieved through  Chapter 6. Treatability Studies  78  Bio-treatment was performed as a control to verify that the high M B A S removal efficiencies reported in the literature can be achieved (Fauser et al, 2003; Kolbener et al, 1995). However, bio-treatment methods are not a viable interim solution at the Lions Gate W W T P , as bio-treatment methods do not fulfill the objective of being an interim treatment method.  II Alum Coagulation/Flocculation with Gravity Settling  Overall, alum coagulation/flocculation followed by gravity settling resulted in poor M B A S removals.  The overall M B A S removal in the whole filtered sample was 46%.  This  relatively low removal efficiency is consistent with those reported in a previous study (see Section 2.8). O f particular interest is the complete absence of any M B A S removal from the 90% elution fraction. Therefore, the presence of high molecular weight anionic surfactant molecules, following treatment with alum coagulation/flocculation with gravity settling, may contribute to the residual toxicity contained in the treated effluent measured in a previous study using primary effluent  from the Lions Gate W W T P  ( C H 2 M H I L L , 2002).  Although this previous study  confirmed that alum coagulation/flocculation with gravity settling can remove some of the MBAS  from a primary effluent,  results obtained in the present study suggest that alum  coagulation/flocculation followed by gravity settling is not capable of removing the more toxic, high molecular weight fractions of the M B A S .  III  Ozonation  Ozone effectively removed M B A S from the primary effluent with removal efficiencies comparable to those that could be achieved with biological treatment.  However, the M B A S  contained in the 90% reconstituted S P E extract was not as effectively removed in comparison to that which could be removed using biological treatment.  IV Air Flotation  A i r flotation was effective at removing M B A S from the Lions Gate W W T P primary effluent.  Approximately 66 to 77% of the M B A S present in the different elution fractions was  Chapter 6. Treatability Studies  removed via air flotation.  79  M B A S removals in the 75% and 90% elution fractions were higher  than observed for the 65% elution fractions. This is likely due to the greater affinity of the more hydrophobic M B A S ,  contained in the 75% and 90% elution fractions, for the surface o f rising  air bubbles. The froth formed by the rising air bubbles and the associated partitioned M B A S was removed from the surface of the liquid.  The volume of froth removed from the air flotation  system during treatment was equivalent to approximately 16% of the total volume treated. This waste stream, containing the recovered anionic surfactants, will require further treatment. Biological treatment of the pre-concentrated waste stream may be a viable treatment method, since the volume of wastewater requiring treatment is significantly reduced following air flotation. However, further studies, beyond the scope of the present study, are needed.  6.3 Removal of Toxicity  The M i c r o t o x ™ system was used to measure the toxicity contained in samples of the primary effluent from the Lions Gate W W T P before and after treatment.  Gradient methanol  elution using 65% and 75% methanol in water preceded elution using 90% methanol in water. However, the toxicities were only determined in the whole filtered sample and in the 90%> elution fraction.  The  90% elution fraction was the only fraction tested because previous results  indicated that the 90% elution fraction was more toxic than the other elution fractions (see Section 5.2.3).  Toxicities were measured following treatment by ozonation and air flotation.  Toxicities  were not determined following bio-treatment and alum coagulation/flocculation with gravity settling.  A s part of these treatments, foreign materials (i.e., alum, bio-solids or sodium azide)  were added to the primary effluent.  A s a result, it was not possible to precisely determine the  impact of treatment on the original primary effluent matrix.  The results of the treatability study are presented in Table 23.  The raw data, used to  calculate the toxicity values, is presented in Figures E . l through to E.6 (Appendix E). A nontoxic response is one in which greater than 100% sample by volume would be required to cause a 20% reduction in bioluminescence.  Chapter 6. Treatability Studies  80  Table 23: Treatability study: toxicity before and after treatment. 15minIC (%V/V) 2 0  Sample  Whole filtered sample 90%  Untreated Effluent  Treated Ozone  Treated Air Flotation  17 ± 5  12± 1  62 ± 2 3  Non-Toxic Non-Toxic 68* ± 3 5 ± corresponds to 95% con!idence intervals  Percent Decrease in Toxicity (%) Treated Treated Air Ozone Flotation (5 ± 5 )  45 ± 2 4  100  100  * value obtained by extrapolation ( ) denotes an increase in toxicity  6.3.1 Ozonation  Ozone treatment rendered the 90% elution fraction non-toxic (i.e., greater than 100% sample by volume would be required to cause a 20% reduction in bioluminescence).  However,  the toxicity o f the whole filtered sample increased slightly, although not significantly, based on a 95% confidence interval. It is possible that by-products o f the oxidation process were formed and were responsible for the increase in toxicity. These results are consistent with those found by Monarco, et al., (2000) in which the toxicity of a secondary effluent increased following ozonation (Monarco, et al., 2000).  However, further investigation in this area was beyond the  scope of the present study.  6.3.2 A i r Flotation  A i r flotation significantly reduced the toxicity of the treated whole filtered sample by 45 ± 24%, based on a 95% confidence interval. Following treatment, the 90% reconstituted S P E extract was rendered non-toxic (i.e., greater than 100% sample by volume would be required to cause a 20% reduction in bioluminescence).  The more hydrophobic anionic surfactant  molecules, contained in the 90% reconstituted S P E extract, may be preferentially removed from solution by partitioning to the rising air bubbles forming a froth that is removed from the surface. It should be noted that the removed froth product likely contains a high concentration of anionic surfactants.  Therefore, this waste stream will likely require further treatment.  Biological  treatment o f the pre-concentrated waste stream may be a viable treatment method, as the volume  Chapter 6. Treatability Studies  81  of wastewater requiring treatment is significantly reduced following air flotation and since biological treatment resulted in the highest M B A S removals as found in the present study.  Chapter 7.  Conclusions  82  Chapter 7 Conclusions A series of analytical methods were developed to quantify the concentration o f anionic surfactants in the primary effluent, to fractionate the anionic surfactants on the basis o f molecular weight, and to measure the toxicity o f the whole and discrete molecular weight fractions.  A  modified methylene blue active substances ( M B A S ) test was used to quantify the concentration of anionic surfactants contained in the primary effluent o f the Lions Gate W W T P .  Solid phase  extraction columns and gradient methanol elution were used to fractionate the anionic surfactants on the basis of hydrophobicity and, therefore, molecular weight.  Following sample preparation  procedures, the modified M B A S method produced results with good precision and with high recoveries. The toxicity of the whole and discrete molecular weight fractions was determined by measuring the bioluminescence o f Vibrio fischeri response o f the Vibrio fischeri,  using the M i c r o t o x ™ toxicity test system. The  used in the M i c r o t o x ™ system, to anionic surfactants was  consistent with the responses o f three marine aquatic organisms to anionic surfactants ( E V S , 2003).  The primary effluent from the Lions Gate W W T P was characterized in terms of the concentration o f anionic surfactants and the anionic surfactant induced toxicity contained in whole samples and in discrete molecular weight fractions.  The concentration o f M B A S ,  contained in the primary effluent o f the Lions Gate W W T P , was found to increase throughout the day.  A s the concentration o f M B A S increased, so too did the toxicity.  The concentration o f  M B A S contained in the three reconstituted S P E elution fractions was greatest in the 75% fraction, followed by the 65% fraction and lowest in the 90%> fraction. Despite having the lowest amount o f M B A S , the toxicity o f the material contained in the 90%> reconstituted S P E elution fraction was the highest. in excess of the  A  IC20, was  In addition, the toxicological impact, defined as the percent o f M B A S greatest for the 90% elution fraction.  treatability study was conducted to investigate  the removal o f M B A S  associated toxicity from the Lions Gate W W T P primary effluent.  and the  The treatability study was  Chapter 7.  Conclusions  83  designed to screen the treatment methods, and it was a minor aspect in the overall work completed as part o f the present study. Four treatment methods were considered: bio-treatment, alum coagulation/flocculation with gravity settling, ozonation, and air flotation.  Biological  treatment resulted in the highest M B A S removals followed by ozonation, air flotation and finally alum coagulation/flocculation with gravity settling.  A i r flotation effectively  decreased the  toxicity o f the whole filtered sample by 45%, and the toxicity o f the most toxic components o f the M B A S (i.e., the 90% elution fraction) by 100%. further treatment, had a significantly reduced volume.  The recovered froth product, requiring  Chapter 8. Recommendations  84  Chapter 8 Recommendations  When Vibrio fischeri are exposed to fractionated anionic surfactants (on the basis of hydrophobicity and therefore molecular weights), the toxicity trends determined using the M i c r o t o x ™ test system were similar to those found in a previous study in which three marine aquatic organisms were used ( E V S , 2003). The M i c r o t o x ™ system is fast and relatively simple to use. For these reasons, the continued use of the M i c r o t o x ™ system is recommended.  Full treatment using activated sludge is not a viable interim treatment method at Lions Gate W W T P .  A i r flotation, however, appears to be a useful means of pre-concentrating the  toxicant and reducing anionic surfactant induced toxicity from a primary effluent.  Further  studies investigating the efficiency o f toxicant removal, using air flotation and subsequent treatment of the recovered foam, are warranted.  List of References  85  List of References A P H A , A W W A , and W E F . 1992. Standard Methods for the Examination  of Water and  Wastewater, 18th ed. American Public Health Association, American Water Works Association and the Water Environment Federation. Bailey, H . C . , J.R. Elphick. 200i. Acute Toxicity Identification Evaluation of G V S & D D Wastewater Treatment Plant Effluents. Prepared for the Greater Vancouver Regional District, Burnaby, B C , by E V S Environment Consultants, North Vancouver, B C . March, 2001. Beltran, F . J . , J.F. Barcia-Araya, P . M . Alvarez. 2000. Sodium Dodecylbenzenesulfonate Removal from Water and Wastewater. 1. Kinetics of Decomposition by Ozonation.  Industrial Engineering  Chemistry Research vol 39: 2214-2220.  Berube, P. 2000. High Temperature Biological Treatment of Foul Evaporator Condensate for Reuse. Ph.D. diss., The University of British Columbia. Bitton, G . 1983. Bacterial and Biochemical Tests for Assessing Chemical Toxicity in the Aquatic Environment: A Review. CRC Crit. Rev. Envion.  Control vol 13: 51-67.  Appearing in Dizer, H . , E . Wittekindt, B . Fischer, P.D. Hansen. 2002. The Cytotoxic and Genotoxic Potential of Surface Water and Wastewater Effluents as Determined by Bioluminescence, umu-assays and selected Biomarkers. Chemosphere vol 46 no. 2: 225233. Brown V . M . , F . S . H . Abram, L . J . Collins. 1978. The Acute Lethal Toxicity to Rainbow Trout of an L A S Surfactant and of its Residues and Degradation Products. Tenside Surfactants Detergents vol 15 no. 2: 57-59. Appearing in World Health Organization Geneva. 1996. Environmental Health Criteria 169 Linear Alklylbenzene Sulfonates and Related  Compounds. International Programme On Chemical Safety Carlson-Ekwall, C . E . A . , G . M . Morrison. 1995.  INCHEM.  Contact Toxicity of Metals in Sewage Sludge:  Evaluation of Alternatives to Sodium Chloride in the Microtox Assay.  Environmental  Toxicology and Chemistry, vol 14: 17-22. Appearing in Dizer, H . , E . Wittekindt, B . Fischer, P.D. Hansen. 2002. The Cytotoxic and Genotoxic Potential of Surface Water and Wastewater Effluents as Determined by Bioluminescence, umu-assays and selected Biomarkers. Chemosphere vol 46 no. 2: 225-233. C H 2 M H I L L . 2002. Bench-Scale M B A S Treatability Study at Lions Gate W W T P ,  December  2002. Chitikela, S., S.K. Dentel, H . E . Allen. 1995. Modified Method for the Analysis of Anionic Surfactants as Methylene Blue Active Substances. Analyst vol 120: 2001-2004.  List of References  86  Divo, C . 1976. Survey on Fish Toxicity and Biodegradability of L A S . Riv. Ital. Sost. Grasse vol 53: 33-93, 439, 440, 445, 454, 619, 752, 753, 757, 759. Appearing in Swisher, R. D . 1987. Surfactant Biodegradation,  Second Edition.  New York: Marcel Dekker, Inc. pg.  445. Dizer, H . , E . Wittekindt, B . Fischer, P.D. Hansen. 2002. The Cytotoxic and Genotoxic Potential of Surface Water and Wastewater Effluents as Determined by Bioluminescence, umuassays and selected Biomarkers. Chemosphere vol 46 no. 2: 225-233. E V S . 2003. Toxicity Tests Using Marine Species and Identification of Causes of Toxicity in Effluent from the Lions Gate Wastewater Treatment Plant. Prepared for the Greater Vancouver Regional Districe ( G V R D ) , Burnaby, B C by E V S Environment Consultants, North Vancouver, B C . Fauser, P., J. Vikelsoe, P.B. Sorensen, L . Carlsen. 2003. Phthalates, nonylphenols and L A S in an alternately operated wastewater treatment plant—fate modelling based on measured concentrations in wastewater and sludge. Water Research vol 37 no. 6: 1288-1295. Ferrer, J., A . Moreno, M . T . Vaquero, L . Cornelias. 1996. Monitoring of " L A S " (Linear Alkylbenzene Sulfonate) In Direct Discharge Situations: Untreated Sewage and on Sludge Amended Soils. In 4 World Surfactant Congress vol 3: 99-100. th  Fettig, J., H . Ratnaweera. 1993. Influence of Dissolved Organic Matter on Coagulation/Flocculation of Wastewater by A l u m . Water Science Technology vol 27 no. 11: 103-112. Giger, W . , A . C . Alder, P . H . Brunner, A . Marcomini, H . Siegrist. 1989. Behaviour of L A S in Sewage and Sludge Treatment and in Sludge-Treated Soil. Tenside Surfactants Detergents vol 26: 95-100. Appearing in World Health Organization Geneva. 1996. Environmental Health Criteria 169 Linear Alklylbenzene Sulfonates and Related  Compounds. International Programme On Chemical Safety  LNCHEM.  Greater Vancouver Regional District. 2001. Quality Control Annual Report For Greater Vancouver Sewerage and Drainage District Volume 1. Gutierrez, M . , J. Etxebarria, L . de las Fuentes. 2002. Evaluation of Wastewater Toxicity: Comparative Study Between Microtox and Activated Sludge Oxygen Uptake Inhibition. Water Research vol 36:919-924. Hokanson, K . E . F . , L . L . Smith. 1971. Some Factors Influencing Toxicity o f Linear Alkylate Sulfonate ( L A S ) to the Bluegill. Trans Am Fish Soc vol 100: 1-12. Appearing in World Health Organization Geneva. 1996. Environmental Health Criteria 169 Linear Alklylbenzene Sulfonates and Related Compounds. International Programme  Chemical Safety  INCHEM.  On  List of References  87  Holt, M . S . , S . L . Bernstein. 1992. Linear Alkylbenzenes in Sewage Sludges and Sludge Amended Soils. Water Resources vol 26 no. 5: 613-624. Kolbener, P., U . Baumann, T. Leisinger, A . M . Cook. 1995. Nondegraded Metabolites Arising From The Biodegradation o f Commercial Linear Alkylbenzenesulfona'te ( L A S ) Surfactants in a Laboratory Trickling Filter. Environmental  Toxicology and Chemistry  vol 14 no. 4: 561-569. Maki, A . W . 1979. Respiratory Activity o f Fish as a Predictor o f Chronic Fish Toxicity Values for Surfactants. In: L . L . Marking, R . A . Kimerle eds. Aquatic Toxicology Philadelphia, Pennsylvania, American Society for Testing and Materials, pgs. 77-95 ( A S T M S T P 667). Appearing in World Health Organization Geneva. 1996. Environmental Health Criteria 169 Linear Alklylbenzene Sulfonates and Related Compounds. International .  On Chemical Safety  Programme  INCHEM.  Marcomini, A . , S. Capri, W . Giger. 1987. Determination o f Linear Alkylbenzenesulphonates, Alkylphenol Polyethoxylates and Nonylphenol in Wastewater by High-Performance Liquid Chromatography After Enrichment on Octadecylsilica. Journal of Chromatography  vol 403: 243-252.  Matthijs, E . , H . De Henau. 1987. Determination o f Linear Alkylbenzensulfonates in Aqueous Samples, Sediments, Sludges and Soils Using H P L C . Tenside Surfactants Detergents vol 24 no. 4: 193-199. Microbics Corporation. 1992. Microtox  Manual  Volume 2 Detailed Protocols.  Carlsbad,  California: Microbics Corporation. Monarca, S., D . Feretti, C . Collivignarelli, L . Guzzella, I. Zerbini, G . Bertanza, R. Pedrazzani. 2000. The Influence of Different Disinfectants on Mutagenicity and Toxicity o f Urban Wastewater. Water Research vol 34 no. 17: 4261-4269. Moreno, A . , J. Ferrer, J . L . Berna. 1990. Biodegradability o f L A S in a Sewer System. Tenside Surfactants Detergents vol 27 no. 5: 312-315. Appearing in World Health Organization Geneva. 1996. Environmental Health Criteria 169 Linear Alklylbenzene Sulfonates and Related Compounds. International Programme  On Chemical Safety  INCHEM.  Painter, H . A . , T . Zabel. 1989. The Behaviour o f L A S in Sewage Treatment. Tenside Surfactants Detergents vol 26 no. 2: 108-115. Appearing in World Health Organization Geneva. 1996. Environmental Health Criteria 169 Linear Alklylbenzene Sulfonates and Related Compounds. International Programme  On Chemical Safety TNCHEM.  Part, P., O . Svanberg, E . Bergstrom. 1985. The Influence o f Surfactants on G i l l Physiology and Cadmium Uptake in Perfused Rainbow Trout Gills. Ecotoxicol  Environ  Saf vol 9: 135-  144. Appearing in World Health Organization Geneva. 1996. Environmental Health Criteria 169 Linear Alklylbenzene Sulfonates and Related Compounds. Programme  On Chemical Safety  INCHEM.  International  88  List of References  Petrovic, M . , D . Barcelo. 2000. The Stability of Non-Ionic surfactants and Linear Alkylbenzene Sulfonates in a Water Matrix and on Solid-Phase Extraction Cartridges. Fresenius  Jounal  and Chemistry vol 368: 676-683. Prats, D . , F . Ruiz, B . Vazquez, D . Zarzo, J . L . Berna, A . Moreno. 1993. L A S Homolog Distribution Shift During Wastewater Treatment and Composting: Ecological Implications. Environmental  Toxicology and Chemistry vol 12: 1599-1608. Appearing in  World Health Organization Geneva. 1996. Environmental Health Criteria 169 Linear Alklylbenzene Sulfonates and Related Compounds. International Programme On Chemical Safety  INCHEM.  Randall, D.J., C . J . Brauner, R . V . Thurston, J.F. Neuman. 1996. Water Chemistry at the G i l l Surfaces o f Fish and the Uptake o f Xenobiotics. Toxicology of Aquatic Physiological,  Cellular, and Molecular  Approaches.  Pollution  Edited by E . W . Taylor. Cambridge:  Cambridge University Press, pgs. 1-16. Rapaport, R . A . , W . D . Hopping, W . S . Eckloff. 1987. Monitoring L A S in the Environment: Abstracts and Programme. Eighth Annual Meeting of the Society of Toxicology and Chemistry (SETAC),  Pensacola,  Florida.  Environmental  New York: Society o f  Environmental Toxicology and Chemistry. Appearing in World Health Organization Geneva. 1996. Environmental Health Criteria 169 Linear Alklylbenzene Sulfonates and Related Compounds. International Programme  On Chemical Safety  INCHEM.  Rubio J . , M . L . Souza, R . W . Smith. 2002. Overview o f Flotation as a Wastewater Treatment Technique. Minerals  Engineering  vol 15:139-155.  Stecher, Paul G , editor. 1968. The Merck Index Eigth Edition.  Rattway, N.J.: Merck & C o . , Inc.  pg.671. Supelco. 1997. Instructions for Using S u p e l c l e a n ™ Solid Phase Extraction Tubes. Supelclean LC-18 6cc 0.5g Packing  Slip. Bellefonte, P A : Sigma-Aldrich Company.  Swedmark, M . , A . Granmo. 1981. Effects o f Mixtures o f Heavy Metals and a Surfactant on the Development o f C o d ( Gadus morhua L.). Rapp P-VReun  Cons Int Explor Mer vol 178:  95-103. Appearing in World Health Organization Geneva. 1996. Environmental Health Criteria 169 Linear Alklylbenzene Sulfonates and Related Compounds. Programme  On Chemical Safety  International  INCHEM.  Swisher, R . D . 1987. Surfactant Biodegradation,  Second Edition.  New York: Marcel Dekker, Inc.  pgs. 17-39,444-445,618-621. Swisher, R . D . , W . E . Gledhill, R . A . Kimerle, T . A . Taulli. 1978. Carboxylated Intermediates in the Biodegradation of Linear Alkylbenzene Sulfonates ( L A S ) . In Proceedings o f the 7th International Congress on Surface Active Substances, Moscow, June 1976. Moscow, Teknol Vody vol 4: 218-230. Appearing in World Health Organization Geneva. 1996.  89  List of References  Environmental Health Criteria 169 Linear Alklylbenzene Sulfonates and Related Compounds. International Programme  On Chemical Safety  INCHEM.  Tsai, C . F . , R . A . M c K e e . 1978. The Toxicity to Goldfish o f Mixtures o f Chloramines, and L A S (linear alkylate sulfonate). In Technical Report No. 44; PB-280-554.  College Park,  Maryland: University o f Maryland, Water Resources Research Center. Appearing in World Health Organization Geneva. 1996. Environmental Health Criteria 169 Linear Alklylbenzene Sulfonates and Related Compounds. International Programme On Chemical Safety  INCHEM.  Vives-Rego, J . , J. Martinez, A . Calleja. 1991. Aquatic Toxicity o f Linear and Branched A l k y l Benzene Sulphonates Assessed by Mortality of Natually Occcuring Bacteria, Daphnia and Microtox Test. Tenside Surfactants Detergents vol 28: 31-34. Wakabayashi, M . , M . Kikuchi, Y . Naganuma, H . Kawakara. 1984. Acute Toxicity o f Some Detergents in Fish. Ann Rep Tokyo Metrop Res Inst Environ Prot [Tokyo Kogai Kenkyujo Nempo]: 114-118 (in Japanese). Appearing in World Health Organization Geneva. 1996. Environmental Health Criteria 169 Linear Alklylbenzene Sulfonates and Related Compounds. International Programme On Chemical Safety  INCHEM.  Wakabayashi, M . , S. Onizuka. 1986. Some Factors Affecting Toxicity o f Chemicals to Fish. Ann Rep Tokyo Metrop Res Inst Environ Prot [Tokyo Kogai Kenkyujo Nempo]: 102-104 (in Japanese). Appearing in World Health Organization Geneva. 1996. Environmental Health Criteria 169 Linear Alklylbenzene Sulfonates and Related Compounds. Programme  On Chemical Safety  International  INCHEM.  Watanabe, H . , H . Inabe, J.W. Hasting. 1991. Osmoregulation o f Bioluminescence Expression o f P. phosphoreum is Related to Gyrase Activity. In P . E . Stenley, L . J . Kricka, (editors). Biouminescene  and Chemiluminescence  Current Status. Wiley, Chichester, pgs. 43-46.  Appearing in H . Dizer, E . Wittekindt, B . Fischer, P.D. Hansen. 2002. The Cytotoxic and Genotoxic Potential o f Surface Water and Wastewater Effluents as Determined by Bioluminescence, umu-Assays and Selected Biomarkers. Chemosphere vol 46: 225-233. World Health Organization Geneva. 1996. Environmental Health Criteria 169 Linear Alklylbenzene Sulfonates and Related Compounds. International Programme On Chemical Safety  INCHEM.  Zaccone, G . , S. Fasulo, L o , P. Cascio, A . Licata. 1985. Patterns of Enzyme Activities in the Gills of the Catfish Heteropneustes fossilis Na-Alkyl-Benzenesulphonate  (Bloch) Exposed to the Anion-Active Detergent  ( L A S ) . Histochemistry  vol 82: 341-343. Appearing in  World Health Organization Geneva. 1996. Environmental Health Criteria 169 Linear Alklylbenzene Chemical Safety  Sulfonates INCHEM.  and Related  Compounds.  International  Programme  On  APPENDIX  A: Analytical Method Development of  MicrotoxTM  APPENDIX A: Analytical Method Development of Microtox A) Standard Zinc Toxicant  Figure A . 1: Standard Zinc Toxicant IC  50  -15 Minute Acute Toxicity (n=3) 50.8 mg/L Z n S 0  0.8  0.6  1.0  1.2  4  1.4  Log Cone.  •o  Trial 1, y = 0.6803X - 0.7954, I = 0.9328  T  Trial 3. y = 0.7690x - 0.7722, I = 0.9764  2  Trial 2, y = 0.6268X - 0.7593, I = 0.9950 2  2  B) Methanol Toxicity to M i c r o t o x ™  Figure A . 2 : Methanol Toxicity to M i c r o t o x ™ (791 mg/L Methanol)  791 mg/L methanol _ o «r ) E -0.5 E ro  1 0.5  1 1  1.5  -  r—  2  2  _i  j? -1-5  y = 0.8517x- 2.6993  Log (cone)  •r = 0.9843  90  TM  APPENDIX  A: Analytical Method Development  ofMicrotoxTM  Figure A . 3 : Methanol Toxicity to M i c r o t o x ™ (1582 m g / L Methanol)  1582 mg/L methanol 0 -0.2 d)  1.5  0.5  £ -0.4  a -0.6  y = 0.5973x- 1.7193 f = 0.909  r? -0.8 _J  -1 -1.2 Log (cone)  Figure A . 4 : Methanol Toxicity to M i c r o t o x ™ (3955 m g / L Methanol)  3955 mg/L methanol 0  . i  )  0.5  1  1.5  /  2  2  7a "0.5 E E O)  -1  O)  o  •1.5  V =  *  -2 Log (cone)  1.6486X-3.2777 I = 0.9889 2  91  APPENDIX  C)  A:  A nalytical Method Development  of  MicrotoxTM  Impact of Laboratory water o n the B i o l u m i n e s c e n c e of Vibrio fischeri  Table A . l : Effect o f Laboratory Water Source on Bioluminescence - Results  Laboratory water s o u r c e a n d b i o l u m i n e s c e n c e - results  Sample  Concentration (%) 5.625 11.25 22.5 45 Reduction in Bioluminescence (%)  Milli-Q Trial 1  11.2  0  1.2  0  Milli-Q Trial 2  0.8  1.5  2.4  .1.2  Distilled De-ionized water Trial 1  4.6  9.5  12.3  19.3  Distilled De-ionized water Trial 2  4  6.2  10.6  17.3  Distilled De-ionized water Trial 3  0  1.7  5.9  12.8  92  APPENDIX  B: SPE Bioluminescence  Correction  Factors  APPENDIX B: SPE Bioluminescence Correction Factors Table B . l : Solid Phase Extraction Blank Correction Factors - Trials 1, 2 and 3  January 22, 2003 Blank SPE CoUmn Extracts: Trials 1,  Sample 65-1 65-2 65-3 75-1 75-2 75-3 90-1 90-2 90-3  Concentration (%) (2x cone. Factor) 22.5 45 90 11.25 Reduction in Bioluminescence (%) 2.2 0 0 5.95 3.41 3.57 9.68 0 6.84 2.31 0 2.36 6.74 0 1.16 5.81 2.22 2.44 -1.14 4.76 -2.42 7.27 2.23 1.23 0 3.33 10.11 -3.23 -1.04 5.75 8.89 18.56 11.02 1.17 1.09 3.68  93  APPENDIX  C: Lions Gate Characterization  Study  94  APPENDIX C: Lions Gate Characterization Study A) M B A S - December 16, 2003 th  Tables C.I: M B A S in Lions Gate effluent December 16,  1  .MBA'SIStandardS-.  MBAS Cone. (mg/L) 0 0.5 1 4 0 0.25 0.5 1  Date Dec Dec Dec Dec Dec Dec Dec Dec  16th 16th 16th 16th 17th 17th 17th 17th  th  2003  December 16th 9:30 AM  Absorbance  Sample  Multiplication Factor  0 0.019 0.023 0.152 0 0.009 0.016 0.031  65-1 75-1 90-1 65-2 75-2 90-2 65-3 75-3 90-3 whole-1 whole-2 whole-3  0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 1 1 1  MBAS Absorbance Concentration (mg/L) 0.034 0.051 0.008 0.021 0.056 0.004 0.03 0.056 0.002 0.037 0.042 0.048  0.49 0.71 0.15 0.32 0.78 0.09 0.44 0.78 0.07 1.06 1.19 1.34  . . . . DecemberliBth 6:30 PM  Sample  Multiplication Factor  65-1 75-1 90-1 65-2 75-2 90-2 65-3 75-3 90-3 whole-1 whole-2 whole-3  0.50 0.83 0.50 0.83 1.25 0.50 1.25 1.25 0.50 1.67 1.67 1.67  MBAS Absorbance Concentration (mg/L) 0.068 0.103 0.023 0.048 0.098 0.033 0.03 0.078 0.022 0.101 0.092 0.091  0.94 2.33 0.34 1.12 3.33 0.48 1.09 2.67 0.33 4.57 4.17 4.13  December 16th 11:30 PM  Sample  Multiplication Factor  65-1 75-1 90-1 65-2 75-2 90-2 65-3 75-3 90-3 whole-1 whole-2 whole-3  1.25 1.25 0.50 0.83 1.25 0.50 0.83 1.25 0.50 2.50 2.50 2.50  MBAS Absorbance Concentration (mg/L) 0.03 0.121 0.081 0.038 0.125 0.077 0.05 0.104 0.085 0.103 0.093 0.087  1.09 4.08 1.11 0.90 4.21 1.05 1.16 3.52 1.16 6.98 6.32 5.93  APPENDIX  B) M B A S - F e b r u a r y 3 ,  r d  2004  Tables C.2: M B A S in  Gate effluent February 3,  MBAS Standards  MBAS Cone. (mg/L) 0 0.5 1 2 4 1  Date Feb Feb Feb Feb Feb Feb  3rd 3rd 3rd 3rd 3rd 4th  95  C: Lions Gate Characterization Study  rd  2004  F e b r u a r y ^ 9:45 AM  Absorbance  Sample  Multiplication Factor  Absorbance  MBAS Concentration (mg/L)  0 0.018 0.036 0.067 0.144 0.041  65-1 75-1 90-1 65-2 75-2 90-2 65-3 75-3 90-3 Whole  0.83 0.63 0.50 0.83 0.63 0.50 0.83 0.83 0.50 2.50  0.049 0.079 0.013 0.051 0.097 0.011 0.045 0.059 0.013 0.063  1.13 1.38 0.17 1.18 1.70 0.15 1.04 1.37 0.17 4.39  February 3rd 6:45 PM  Sample  Multiplication Factor  Absorbance  MBAS Concentration (mg/L)  65-1 75-1 90-1 65-2 75-2 90-2 65-3 75-3 90-3 Whole  2.50 2.50 0.63 2.50 2.50 0.63 2.50 2.50 0.63 5.00  0.037 0.074 0.083 0.041 0.083 0.097 0.021 0.096 0.094 0.062  2.56 5.16 1.45 2.84 5.80 1.70 1.43 6.71 1.64 8.63  February 3rd 11:30 PM  Sample  Multiplication Factor  Absorbance  MBAS Concentration (mg/L)  65-1 75-1 90-1 65-2 75-2 90-2 65-3 75-3 90-3 Whole  2.50 2.50 0.83 2.50 2.50 0.83 2.50 2.50 0.83 5.00  0.037 0.083 0.067 0.04 0.077 0.055 0.047 0.087 0.053 0.067  2.56 5.80 1.56 2.77 5.37 1.27 3.26 6.08 1.23 9.34  APPENDIX  C: Lions Gate Characterization  Study  96  C) MBAS - February 26, 2004 th  Tables C.3: M B A S in Lions Gate effluent February 26,  th  2004  February 26th Lions G a t e Effluent  M B A S Cone. (mg/L)  Date  Absorbance  Sample  0 0.017  whole-1  0  F e b 27th  0.5 1 2  F e b 27th F e b 27th F e b 27th  4  F e b 27th  0.036 0.068 0.155  M B A S S t a n d a r d s M a r c h 1st M B A S Cone. (mg/L)  whole-2 whole-3 65-1  Multiplication Factor 1.67 1.67  Absorbance  0.085  MBAS Concentration (mg/L)  1.67 1.25  0.074 0.093 0.05  3.76 3.27 4.11 1.66  75-1  1.25  0.082  2.72  90-1  0.50  0.029  0.38  65-2  1.25  0.056  1.86  75-2  1.25  0.072  2.39  Date  Absorbance  90-2  0.50  0.023  0.31  0 0.5 1  M a r c h 1st M a r c h 1st  65-3 75-3  1.25 1.25  0.048 0.062  1.59 2.06  M a r c h 1st  0 0.03 0.047  90-3  0.50  0.036  0.48  2  M a r c h 1st  0.104  4  M a r c h 1st  0.186  APPENDIX D) Methanol - December 16,  C: Lions Gate Characterization  th  Study  2003  Figure C . l and Tables C.4: Methanol concentrations December 16,  Methanol Standard Curve  y = 1.9075X FT = 0.8749  0  20  40  60  80  100  Methanol Cone. (mg/L)  Reconstituted SPE Extracts 930 AM. Dec 16,2003  Sample  97  methanol cone. (mg/L)  65-1 226.32* 75-1 19.66 90-1 N.D. 65-2 N.D. 75-2 5.56 90-2 N.D. N.D. 65-3 75-3 11.43 N.D. 90-3 •concentration obtained by extrapolation N.D. Non-detect  Reconstituted SPE Extracts 6 30 PM, Dec. 16. 2003  Sample  methanol cone. (mg/L)  65-1 154.81* 75-1 22.18 90-1 N.D. N.D. 65-2 75-2 N.D. 90-2 N.D. 65-3 32.92 75-3 N.D. 90-3 477.85* 'concentration obtained by extrapolation N.D. Non-detect Reconstituted SPE Extracts 11:30 PM, Dec. 16, 2003  Sample 65-1 75-1 90-1 65-2 75-2 90-2 65-3 75-3 90-3 N.D. Non-detect  methanol cone. (mg/L) 25.43 79.21 N.D. N.D. N.D. N.D. N.D. N.D. N.D.  th  2003  APPENDIX  C: Lions Gate Characterization  Study  98  E) Methanol - February 3, 2004 rd  Figure C.2 and Tables C.5: Methanol concentrations February 3,  Methanol Standard Curve 1500 re 1000  9>  V = 2.4255X FT = 0.9915 0  100  200  300  400  500  Methanol Cone. (mg/L)  Reconstituted SPE Extracts 9:45 AM. Feb. 3. 2004 Sample 65-1 75-1 90-1 65-2 75-2 90-2 65-3 75-3 90-3 N.D. Non-detect  methanol cone. (mg/L) 9.80 12.70 N.D. N.D. N.D. N.D. N.D. N.D. N.D.  Reconstituted SPE Extracts 6 45 PM. Feb 3, 2004 Sample 65-1 75-1 90-1 65-2 75-2 90-2 65-3 75-3 90-3 N.D. Non-detect  methanol cone. (mg/L) N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 83.00  Reconstituted SPE Extracts 11:30 PM. Feb. 3. 2004  Sample 65-1 75-1 90-1 65-2 75-2 90-2 65-3 75-3 90-3 N.D. Non-detect  methanol cone. (mg/L) N.D. N.D. N.D. N.D. N.D. N.D. N.D. 14.30 54.30  rd  2004  APPENDIX F) T o x i c i t y - D e c e m b e r 16,  C: Lions Gate Characterization  th  Study  2003  Figure C.3: 9:30 A M , 65% elution fraction, Dec. 16, 2003. 0.2 •  Log Cone.  • o •  Trial 1, y = 0.7182x -1.9565, I = 0.6105 2  Trial 2, y = 0.9084X - 1.9396, I = 0.6929 Trial 3, y = 0.2914X - 1.2697, I = 0.4284 2  2  99  APPENDIX  C: Lions Gate Characterization  100  Study  Figure C.6: 9:30 A M , whole filtered sample, Dec. 16, 2003.  -1.6 -I 0.6  1  1  1  1  1  1  0.8  1.0  1.2  1.4  1.6  1.8  Log Cone.  • o •  Trial 1, y = 1.2679x - 2.2673,1 = 0.9942 Trial 2, y = 1.1657x -2.1148, l = 0.9889 Trial 3, y = 1.1602x - 2.0099,1 = 0.9905 2  2  2  Figure C.7: 6:30 P M , 65% elution fraction, Dec 16, 2003.  APPENDIX  1.0  C: Lions Gate Characterization  Study  1.2  1.8  1.4  1.6  101  2.0  Log Cone.  • o •  Trial 1, y = 0.9942x - 2.5789, I = 0.8657 Trial 2, y = 0.6990x - 2.0954, I = 0.8770 Trial 3, y = 0.8295x - 2.2458, I = 0.9853 2  2  2  Figure C.8: 6:30 PM, 75% elution fraction, Dec. 16, 2003.  1.0  1.2  • o T  1.4  1.6 Log Cone.  Trial 1, y = 1.5382x -3.0177, I = 0.9627 Trial 2, y = 1.3785X - 2.6944, I = 0.9680 Trial 3, y = 1.2653x - 2.4699, I = 0.9795 2  2  2  1.8  2.0  APPENDIX  C: Lions Gate Characterization  102  Study  Figure C I O : 6:30 P M , whole filtered sample, Dec 16, 2003.  0.6  0.8  • o  T  1.0  1.2 Log Cone.  Trial 1, y = 1.7061X- 2.6643, I = 0.9999 Trial 2, y = 1.2380x- 1.9416, I = 0.9060 Trial 3, y = 1.4331X- 2.1750, I = 0.9945 2  2  2  1.4  1.6  1.8  APPENDIX  C: Lions Gate Characterization  Study  Figure C . l l : 11:30 PM, 65% elution fraction, Dec. 16, 2003. o.o  •o •  Trial 1, y = 1.3302x - 2.7873, I = 0.9944 Trial 2, y = 1.5186X - 3.0932, I = 0.9567 Trial 3, y = 1.2977x - 2.9925, I = 0.9281 2  2  2  Figure C.12: 11:30 PM, 75% elution fraction, Dec. 16, 2003. 0.6  • o •  Trial 1, y = 1.0927x- 1.6941,1 = 0.9974 Trial 2, y = 0.9224X - 1.6843, I = 0.9967 Trial 3, y = 1.6277x- 2.9587, I = 0.9961 2  2  2  103  APPENDIX  C: Lions Gate Characterization  104  Study  Figure C . 13: 11:30 P M , 90% elution fraction, Dec. 16, 2003.  -1.0 + 1.0  1  1  1  r  1.2  1.4  1.6  1.8  2.0  Log Cone.  •  Trial 1, y = 1.1017x - 1.8762, I = 0.9966  o  Trial 2, y = 0.8906X - 1.7302, I = 0.9882  •  Trial 3, y = 1.1911X -2.1683, I = 0.9878  2  2  2  Figure C.14: 11:30 P M , whole filtered sample, Dec. 16, 2003. 0.4  0.6  0.8  1.0  1.2  Log Cone.  •  Trial 1, y = 1.1022X- 1.6457, I = 0.9973  0  Trial 2, y = 1.1013X- 1.6736, I = 0.9983  •  Trial 3, y = 1.0778X- 1.6305, I = 0.9856  2  2  2  1.4  1.6  1.8  APPENDIX  G) Toxicity - February 3,  rd  C: Lions Gate Characterization  Study  2004  Figure C.15: 9:45 A M , 65% elution fraction, Feb 3, 2004. -0.4  •o •  Trial 1, y = 0.4332x - 1.6366,1 = 0.6091 Trial 2, y = 0.8607x - 2.1955, I = 0.8893 Trial 3, y = 0.3578x - 1.4814, I = 0.4848 2  2  2  Figure C.16: 9:45 A M , 75% elution fraction, Feb 3, 2004.  • o •  Trial 1, y = 0.9247X - 1.9170, I = 0.9247 Trial 2, y = 0.9794X - 1.9329, I = 0.9989 Trial 3, y = 1.1832X- 2.3480, I = 0.9939 2  2  2  105  APPENDIX C: Lions Gate Characterization Study Figure C.17: 9:45 AM, 90% elution fraction, Feb 3, 2004.  • o •  Log Cone. Trial 1, y = 0.9581x- 1.9795, I = 0.9167 Trial 2, y = 0.7734X - 1.5826, I = 0.9496 Trial 3, y = 0.7039X - 1.5723, I = 0.9705 2  2  2  Figure C.18: 9:45 AM, whole filtered sample, Feb 3, 2004. -0.2  • o •  Trial 1, y = 0.6662X - 1.5236, I = 0.8626 Trial 2, y = 0.7279x - 1.6291. I = 0.9554 Trial 3, y = 0.5484x - 1.4114, I = 0.9414 2  2  2  106  APPENDIX  C: Lions Gate Characterization  107  Study  Figure C.19: 6:45 P M , 65% elution fraction, Feb 3, 2004.  2.0  • o T  Trial 1, y = 1.7280X- 3.4942, I = 0.9634 Trial 2, y = 1.0759x- 2.2718, I = 0.9336 Trial 3, y = 1.1044X- 2.4429, 12= 0.5460 2  2  Figure C.20: 6:45 P M , 75% elution fraction, Feb 3, 2004. 0.8  •  0 •  Log Cone. Trial 1, y = 1.3291x -2.1246, I = 0.9862 Trial 2, y = 1.4769x - 2.7356, I = 0.9677 Trial 3, y = 1.4253X -2.5731, I = 0.9963 2  2  2  APPENDIX  C: Lions Gate Characterization  108  Study  Figure C.21: 6:45 PM, 90% elution fraction, Feb 3, 2004. 0.4 -,  -0.8 -I 1.0  1  !  1  1.2  1.4  1.6  ;  ! 1.8  :  ;  1 2.0  Log Cone. Trial 1, y = 0.7615x- 1.2635, I = 0.9697 Trial 2, y = 0.7620x - 1.4120, I = 0.9989 Trial 3, y = 0.9436X - 1.6160, I = 0.9811 2  o T  2  2  Figure C.22: 6:45 PM, whole filtered sample, Feb 3, 2004.  0.6  0.8  • o T  1.0  1.2 Log C o n e .  Trial 1, y = 0.9488X Trial 2, y = 0.9373X Trial 3, y = 0.9090X  -  1.6300, I = 0.9826 1.6807, I = 0.9990 1.6215, I = 0.9814 2  2  2  1.4  1.6  1.8  109  APPENDIX C: Lions Gate Characterization Study Figure C.23: 11:30 PM, 65% elution fraction, Feb 3, 2004.  -i  1.0  1  1  1  1  1  1.2  1.4  1.6  1.8  2.0  Log Cone.  •  Trial 1, y = 1.2935x- 2.6126,1 = 0.9742  o  Trial 2, y = 1.2400x- 2.7687,1 = 0.9864  •  Trial 3, y = 1,5778x - 3.1627,1 = 0.9954  2  2  2  Figure C.24: 11:30 PM, 75% elution fraction, Feb 3, 2004.  CO  E E  CO  O  o> o  •  Trial 1, y = 1.1996x- 2.1086, l = 0.9950  0  Trial 2, y = 1.2089X- 2.4715, l = 0.9928  T  Trial 3, y = 0.9915x- 1.7732, l = 0.9958  2  2  2  APPENDIX  C: Lions Gate Characterization  Study  110  Figure C.25: 11:30 P M , 90% elution fraction, Feb 3, 2004. 0.4  -0.8 -I 1.0  • o T  1  1  1  1.2  1.4  1.6  •  r1.8  •  :  1 2.0  Log Cone.. Trial 1, y = 0.8501X- 1.3732, I = 0.9976 Trial 2, y = 0.7232X - 1.3709, I = 0.9958 Trial 3, y = 0.7499x - 1.1645, I = 0.9994 2  2  2  Figure C.26: 11:30 P M , whole filtered sample, Feb 3, 2004.  -1.2 0.6  0.8  1.0  1.2 Log Cone.  • o •  Trial 1, y = 0.9815x- 1.6321,1 = 0.9866 Trial 2, y = 1.0904x- 1.7680,1 = 0.9932 Trial 3, y = 1.1058x- 1.8146,1 = 0.9946 2  2  2  1.4  1.6  1.8  APPENDIX  C: Lions Gate Characterization  Study  111  H) Toxicity - February 26, 2004 tn  Figure C.27: Morning, 90% elution fraction, Feb 26, 2004. -0.4 n  :  -1.2 1.0  1.2  1.4  1.6  1.8  2.0  Log Cone. Trial 1, y = 0.6953x - 1.8756, I = 0.9690 2  o  Trial 2, y = 0.3508x - 1.2744, I = 0.9980  T  Trial 3, y = 0.3329x - 1.1789, I = 0.4401  2  2  Figure C.28: Morning, whole filtered sample, Feb 26, 2004.  o.o-:  •—i  0.6  0.8  1.0  1.2  Log Cone.  •o  Trial 1, y = 0.9850x - 1.8585, I = 0.9537  T  Trial 3, y = 0.6637x - 1.3734, I = 0.9346  2  Trial 2, y = 0.7251X- 1.5021, I = 0.9039 2  2  1.4  1.6  1.8  APPENDIXD:  UBC Pilot Plant  112  APPENDIX D: UBC Pilot Plant The U B C Environmental Engineering Pilot Plant is located on the south campus of U B C where it draws wastewater from the south sewer trunk.  The south sewer collects domestic  wastewater mainly from Acadia Park family housing and Hampton Place developments.  A) M B A S  The U B C Environmental Engineering Pilot Plant offered a convenient location from which primary effluent and waste activated sludge could be obtained. The primary effluent was used in method development and quality control investigations  (Sections 4.1.1,  4.1.5.3.A,  4.1.5.4). The waste activated sludge was used in treatment studies that are discussed in Chapter Six. M B A S concentrations contained in the U B C Pilot Plant primary effluent are presented in Table D . l . Tables D.3, D.4, and D.5 contain the raw data for the M B A S measured on November 21,  st  26  th  and December 11 * 2003 respectively.  Table D.6 contains the raw data for the M B A S  concentration of the waste activated sludge.  Table D . l : U B C Pilot Plant primary effluent: M B A S concentration. Sample  Date  m  Primary Effluent  Nov 26, '03  Primary Effluent  Dec 11,'03  Primary Effluent Waste Activated Sludge  Nov 21,  Feb 26, '04  60%  65%  0.8  M B A S Concentration (mg/L) 90% 70% 75% 2.2  1.0 2.6 ± 0 . 6 n=2 1.4 0.17 ± 0 . 0 4 n=3  ± corresponds to the 95% confidence interval  3.1 ± 0 . 6 n=3 3.4 0.02 ± 0.07 n=3  0.7 ± 3 . 2 n=2 0.64 0.04 ± 0.05 n=3  Whole Sample 6.1 ± 2 . 5 n=2 7.2 ± 0 n=2 6.4 0.5 ± 0.06 n=3  APPENDIX  D:  UBC Pilot Plant  113  The distribution of M B A S in each elution fraction is presented in Figure D . l . Figure D . l :  M B A S distribution in elution fractions - U B C Pilot Plant  60  65  75  90  Elution fraction (% methanol in water V/V) 95% Confidence interval shown  The distribution of M B A S between the three elution fractions (65%, 75%, and 90%) is similar to the distributions observed during the morning, evening and night sampling events that took place at the Lions Gate W W T P (see Section 5.2.1).  B) Toxicity  The toxicity contained in three elution fractions using the U B C pilot plant effluent was measured using the M i c r o t o x ™ system and results are presented in Table D.2. The raw data is presented in Figure D.2.  Table D.2: U B C Pilot Plant primary effluent toxicity Date  Sample  Nov 26, TO Nov 26, X)3 Nov 26, m  65% 75% 90%  IC (% V / V ) 11 6 8 20  ic  20  (mg/L M B A S ) 0.3 0.2 0.04  The U B C Pilot Plant reconstituted S P E extracts appear to contain greater toxicity than the toxicity contained in the Lions Gate W W T P (see Section 5.2.2).  APPENDIXD:  UBC Pilot Plant  114  C) Raw data Table D.3: U B C Pilot Plant - N o v 21, 2003 Primary Effluent U B C Pilot Plant primary effluont - Nov. 2 1 . 20U3  Sample 0.5 mg/L std. 1.0 mg/L std. 2.0 mg/L std. 60-1 70-1 90-1 65-1 75-1 90-1 Whole Sample Whole Sample + formaldehyde  MBAS Multiplication Absorbance Concentration Factor (mg/L) 0.016 0.04 0.07 0.5 0.063 0.8 0.037 1 1.25 0.07 1.25 2.2 0.07 2.2 1.25 1.25 0.111 3.6 0.7 1.25 0.028 0.112 1 6.3 1  0.106  5.9  Table D.4: U B C Pilot Plant - N o v 26, 2003 Primary Effluent U B C Pilot Plant primary effluent - N o v . 2 6 . 2 0 0 3 I  Sample 2 mq/L std. 4 mq/L std. 75-1 90-1 65-2 75-2 65-3 75-3 90-3 Whole Sample Whole Sample SPE underflow  MBAS Multiplication Absorbance Concentration Factor (mq/L) 0.071 0.164 0.021 3 5 1 2.5 0.013 0.039 2.6 2.5 0.052 3.4 2.5 2.5 0.038 2.5 2.5 0.046 3 0.5 2.5 0.005 7.2 5 0.056 7.2 5 0.056 1 0.009 0.3  APPENDIXD:  UBC Pilot Plant  115  Table D.5: U B C Pilot Plant - Dec 11, 2003 Primary Effluent  U B C Pilot Plant primary effluent - D e c 11, 2 0 0 3  Sample 1 mg/L std. 65% 75% 90% Whole Sample SPE Underflow 40% Wash Underflow  MBAS Multiplication Absorbance Concentration Factor (mq/L) 0.03 25/14 0.031 1.43 1.25 0.11 3.45 0.5 0.049 0.64 0.155 5/3 6.43 1 0.006 0.22 0.5  0.017  0.49  Table D.6: U B C Pilot Plant - Feb 26, 2004 filtered waste activated sludge February 26th Pilot Plant Sludge  Sample whole-1 whole-2 whole-3 65-1 75-1 90-1 65-2 75-2 90-2 65-3 75-3 90-3 Non-detect  Multiplication Factor 1.00 1.00 1.00 0.50 0.50 0.50, 0.50 0.50 0.50 0.50 0.50 0.50  MBAS Absorbance Concentration (mg/L) 0.02 0.53 0.022 0.58 0.021 0.56 0.017 0.18 0 0.00 0.006 0.06 0.014 0.15 0.00 N.D. 0.002 0.02 0.17 0.016 0.005 0.05 0.005 0.05  APPENDIX  D: UBC Pilot Plant  116  APPENDIXE:  Treatability Study  117  APPENDIX E: Treatability Study A)  MBAS Table E . l : M B A S standards. MBAS Standards Feb 27th  MBAS Cone. (mg/L) 0 0.5 1 2 4  Date Feb Feb Feb Feb Feb  -  »  Absorbance  27th 27th 27th 27th 27th  0 0.017 0.036 0.068 0.155  MBAS Standards March 1st MBAS Cone. (mg/L). 0 0.5 1 2 4  Date March March March March March  Absorbance  1st 1st 1st 1st 1st  0 0.03 0.047 0.104 0.186  Table E.2: Lions Gate primary effluent (untreated) 8:00 A M Feb 26, 2004 February 26, 2004 Lions Gate primary effluent  Sample  Multiplication Factor  Absorbance  whole-1 whole-2 whole-3 65-1 75-1 90-1 65-2 75-2 90-2 65-3 75-3 90-3  1.67 1.67 1.67 1.25 1.25 0.50 1.25 1.25 0.50 1.25 1.25 0.50  0.085 0.074 0.093 0.05 0.082 0.029 0.056 0.072 0.023 0.048 0.062 0.036  MBAS Concentration (mg/L) 3.76 3.27 4.11 1.66 2.72 0.38 1.86 2.39 0.31 1.59 2.06 0.48  APPENDIXE:  Treatability Study  118  Table E.3: U B C pilot plant waste activated sludge (untreated) Feb 26, 2004 I  February 26 2004 UBC Pilot Plant waste activated sludge (untreated)  Sample whole-1 whole-2 whole-3 65-1 75-1 90-1 65-2 75-2 90-2 65-3 75-3 90-3 Non-detect  Multiplication Factor  Absorbance  1.00 1.00 1.00 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50  0.02 0.022 0.021 0.017 0 0.006 0.014 N.D. 0.002 0.016 0.005 0.005  I  MBAS Concentration (mq/L) 0.53 0.58 0.56 0.18 0.00 0.06 -0.15  0.00 0.02 0.17 0.05 0.05  Table E.4: Partitioning to abiotic bio-solids (treated) February 26, 2004 February 26, 2004 Abiotic solids contact (treated)  Sample whole-1 whole-2 whole-3 65-1 75-1 90-1 65-2 75-2 90-2 65-3 75-3 90-3 Nondetect  Multiplication Factor  Absorbance  1.67 1.67 1.67 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50  0.003 0.033 0.016 0.039 0.001 0.002 0.038 N.D. 0.012 0.039 N.D. 0.007  MBAS Concentration (mq/L) 0.13 1.46 0.71 0.41 0.01 0.02 0.40 0.00 0.13 0.41 0.00 0.07  APPENDIXE:  Treatability Study  Table E.5: Biological treatment (treated with activated sludge) February 26, 2004 February 26, 2004 Biological treatment with activated sludge  Sample  Multiplication Factor  whole-1 1.00 whole-2 1.00 whole-3 1.00 65-1 0.50 75-1 0.50 0.50 90-1 65-2 0.50 75-2 0.50 90-2 0.50 65-3 0.50 75-3 0.50 90-3 0.50 Non-Detect N.A. Not Available  Absorbance 0.006 N.D. N.A. 0.011 0.001 0.001 0.012 0.004 0.002 0.016 0.001 0  MBAS Concentration (mq/L) 0.16 0.00 N.A. 0.12 0.01 0.01 0.13 0.04 0.02 0.17 0.01 , 0.00  Table E.6: A l u m coagulation/flocculation with gravity settling (treated) February 26, 2004 February 26, 2004Alum.cpagulation/floculation with gravity settling (treated)  Sample  Multiplication Factor  Absorbance  whole-1 whole-2 whole-3 65-1 75-1 90-1 65-2 75-2 90-2 65-3 75-3 90-3  1.00 1.00 1.00 0.83 0.83 0.50 0.83 0.83 0.50 0.83 0.83 0.50  0.075 0.072 0.081 0.035 0.065 0.05 0.033 0.083 0.037 0.04 0.062 0.036  MBAS Concentration (mq/L) 1.99 1.91 2.15 0.61 1.14 0.52 0.58 1.45 0.39 0.70 1.08 0.38  119  APPENDIX  E: Treatability Study  Table E.7: Ozonation (treated) February 26, 2004 February 26, 2004 Ozonation (treated)  Sample  Multiplication Factor  Absorbance  whole-1 whole-2 whole-3 65-1 75-1 90-1 65-2 75-2 90-2 65-3 75-3 90-3  1.00 .1.00 1.00 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50  0.008 0.007 0.005 0.006 0.007 0.003 0.005 0.007 0.01 0.012 0.002 0.002  MBAS Concentration (mq/L) 0.21 0.19 0.13 0.08 0.09 0.04 0.07 0.09 0.13 0.16 0.03 0.03  Table E . 8: A i r flotation (treated) February 26, 2004 Februffg2gf2004 Air flotation (treated)  Sample  Multiplication Factor  Absorbance  whole-1 whole-2 whole-3 65-1 75-1 90-1 65-2 75-2 90-2 65-3 75-3 90-3  1.00 1.00 1.00 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50 0.50  0.031 0.036 0.03 0.042 0.037 0.012 0.04 0.043 0.003 0.046 0.045 0.008  MBAS Concentration (mq/L) 0.82 0.95 0.80 0.56 0.49 0.16 0.53 0.57 0.04 0.61 0.60 0.11  120  APPENDIXE:  121  Treatability Study  B) Toxicity  Figure E . 1: 90% reconstituted S P E extract Lions Gate primary effluent (untreated) February 26, 2004  Figure E.2:  Whole filtered sample Lions Gate primary effluent (untreated) February 26, 2004  o.o  • O •  Trial 1, y = 0.9850x - 1.8585,1 = 0.9537 Trial 2, y = 0.7251x -1.5021,1 = 0.9039 Trial 3, y = 0.6637x - 1.3734, I = 0.9346 2  2  2  1.2 Log Cone.  -1— 1.4  APPENDIXE:  122  Treatability Study  Figure E.3: 90% reconstituted S P E extract ozonation (treated) February 26, 2004  • o •  Trial 1, y = 0.2470X - 1.6872,1 = 0.7227 Trial 2 Trial 3, y = 0.3423x - 2.5183, I = 0.2405 2  2  Figure E.4: Whole filtered sample ozonation (treated) February 26, 2004 0.2  •  o •  Trial 1, y =1.1804x- 1.8917, I = 0.9997 Trial 2, y = 1.1687x- 1.8724, I = 0.9998 Trial 3, y = 1.1090x- 1.7725, I = 0.9838 2  2  2  -1.2 0.6  0.8  1.0  1.2  Log Cone.  1.4  —i— 1.6  1.8  APPENDIXE:  Treatability Study  123  Figure E.5: 90% reconstituted S P E extract air flotation (treated) February 26, 2004 -0.8  •  o T  Trial 1, y = 0.5985x -2.1385, l = 0.7849 Trial 2, y = 0.5809x -2.0903, l = 0.8436 Trial 3, y = 0.7970X -2.5994, l = 0.8376 2  2  2  -2.0 1.0  1.2  1.4  1.6  Log Cone.  Figure E.6: Whole filtered sample air flotation (treated) February 26, 2004 -0.5  -r  1.8  2.0  

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