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Biodegradation and Environmental

Fate of Nonylphenol

A thesis submitted to the

Division of Graduate Studies and Research

of the University of Cincinnati

in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

in Chemical Engineering

from the Department of Chemical and Materials Engineering

of the College of Engineering

August 2004

Marcus A. Bertin

B.S., (ChemE), University of Cincinnati

Cincinnati, Ohio, 2001

Under the Advisement of

Dr. Panagiotis G. Smirniotis Abstract

Concern for the fate of nonylphenol (NP) has increased in recent years due to reports that it is an endocrine disrupting compound and that it is persistent in the environment. The biodegradation of NP was examined through the use of microcosms and respirometers. NP biodegradation was examined under aerobic, nitrate reducing, sulfate reducing, and methanogenic conditions. Through the use of gas chromatography- mass spectroscopy, the technical mixture of NP was differentiated into 23 isomers. Since no standards are available, a novel technique was used to quantify the isomers of NP.

Under aerobic conditions, biodegradation rates for some isomers differed significantly, indicating that some isomers are more resistant to biodegradation. Comparisons between known isomer structures and biodegradation rates show that correlations exist between the branching of the alkyl chain and biodegradability. Under anoxic conditions, NP degradation using cultures obtained from the anaerobic digester of a local wastewater treatment plant did not occur. This result explains the high NP concentration typical of anaerobic digesters.

Acknowledgements

I thank the US EPA Traineeship Grant Program, which provided the funding for

my tuition and stipend to complete by studies. I thank the US EPA, National Risk

Management Research Laboratories, Land Remediation and Pollution Control Division

which provided the laboratory supplies and equipment necessary to conduct my work. I

thank my research committee of Dr. Peter Smirniotis, Dr. Neville Pinto, Dr. Greg Sayles,

and Dr. Marc Mills. Dr. Smirniotis, my research advisor, I thank for his advice and guidance. I thank Dr. Pinto for taking the time to review and critique my work. I thank

Dr. Sayles for taking the time to discuss experimental designs and help with manuscript development, but most importantly for giving me the opportunity to work with the EPA.

I especially thank Dr. Mills for his countless hours of help and discussions about nearly every aspect of my research.

I thank Tracy Dahling for frequently providing an extra set of hands in the lab and her help with the total biomass analysis. I thank Ron Herrmann for his many discussions about microbiology and total biomass. I thank John Haines for his tutelage concerning basic microbiological techniques and the use of respirometers.

On a personal note, I thank Tracy McCullough, my fiancée, for her support, patience, and understanding over the last few years. Without her support, I would have never made it.

Table of Contents

1. Background...... 7

1.1. Introduction...... 7

1.2. Uses, Production, and Structure...... 7

1.3. Source of NP to the Environment ...... 10

1.4. Endocrine Disruption and Reasons for Concern...... 12

1.5. Biodegradation Studies ...... 14

1.6. Summary...... 16

2. Method Development for Analysis of Nonylphenol...... 17

2.1. Objective...... 17

2.2. Review of Previous Methods...... 17

2.3. Method Development...... 18

3. Enrichment by the Use of Aerobic Respirometers ...... 20

3.1. Materials and Methods...... 20

3.2. Results and Discussion ...... 21

4. Aerobic Biodegradation of Nonylphenol in Microcosms: Estimation of Rate by

Individual Nonylphenol Isomers...... 27

4.1. Abstract...... 27

4.2. Introduction...... 27

4.3. Materials and Methods...... 30

4.4. Results and Discussion ...... 33

5. Biodegradation of NP Using Anaerobic Respirometers ...... 44

5.1. Abstract...... 44

5.2. Introduction...... 44

5.3. Materials and Methods...... 47

5.4. Results and Discussion ...... 50

6. Summary, Conclusions, and Future Work...... 56

6.1. Summary...... 56

6.2. Conclusions...... 59

6.3. Future Work...... 60

7. Bibliography ...... 63

8. Appendix 1: Analysis of Nonylphenol and Octylphenol in Bioslurry and Sediment by Full Scan Gas Chromatography/Mass Spectrometry (GC/MS)...... 71

8.1. Scope and Application ...... 71

8.2. Summary of Method ...... 71

8.3. Interferences and Potential Problems...... 72

8.4. Safety and Waste Management...... 72

8.5. Equipment, Reagents and Supplies...... 74

8.6. Sample Collection, Preservation and Handling ...... 76

8.7. Quality Control ...... 76

8.8. On going QC...... 77

8.9. Calibration...... 77

8.10. Procedure ...... 81

8.11. Routine Analysis...... 85

8.12. Troubleshooting Guide ...... 87

8.13. Tables and Validation ...... 90

8.14. References...... 94

9. Appendix 2: Total Biomass Analysis ...... 96

9.1. Summary of Method ...... 96

9.2. References...... 97

10. Appendix 3: Preparation of Microbiological Media...... 98

10.1. Introduction...... 98

10.2. Preparation of RST Basal Microbiological Media...... 98

10.3. Preparation of the Reduced Anaerobic Microbiological Media (RAMM)... 99

10.4. References...... 102

List of Tables

Table 4-1. Initial total biomass and NP concentrations that were present in the

inoculum for each condition before the start of the experiment...... 34

Table 5-4-2. Comparison of NP isomer for this study to that of Wheeler et al. and Kim

et al...... 37

Table 9-7-1. Method Parameters...... 90

Table 9-7-2. Retention Times (RT) and Electron Impact Ions ...... 91

Table 9-7-3. Preparation os Stock Standards...... 92

Table 9-7-4. Concentrations of Calibration Standards (Made using 10mL volumetric

flasks)...... 92

Table 9-7-5. Amounts of Calibration Standards on Column Based on Table 4

Concentrations...... 92

Table 9-7-6. DFTPP Abundance Criteria ...... 93

List of Figures

Figure 1-1. The structure of NPE where n is the number of ethoxylates in the

ethoxylate chain...... 8

Figure 1-2. Several possible isomers found in the technical mixture of NP...... 9

Figure 1-3. The biodegradation pathway of APEs to AP begins with progressive

removal of ethoxylate units...... 11

Figure 1-4. Comparison of one possible isomer of NP to 17β-estrodial...... 12

Figure 3-1. The oxygen consumption curve for a culture degrading NP as the sole

carbon and energy source...... 22

Figure 3-2. Carbon dioxide uptake curve and illustration of calculation of maximum

theoretical cell yield...... 23

Figure 3-3. Comparison of the chromatogram of NP isomers in the technical mixture

and after 25 days of degradation...... 25

Figure 4-1. TIC comparison of NP isomers at T=0 days and T=98 days...... 34

Figure 4-2. Least squares regression of first order biodegradation for each condition.

...... 35

Figure 4-3. Peak notation used for NP isomers...... 36

Figure 4-4. Estimated first order rate constants for the biodegradation of individual NP

isomers...... 38

Figure 4-5. Isomer group designation adapted from Wheeler et al. (1997)...... 39

Figure 4-6. Biodegradation rates for each isomer normalized to the initial total

biomass for that condition...... 40

Figure 4-7. Comparison between the average of normalized biodegradation rate and the estrogenic activity of the isomers relative to 17 β-estradiol (E2)...... 42

Figure 5-1. Average metabolite gas production of the quadruplicates for the first 40 days of the experiment...... 51

Figure 5-2. Total biomass numbers are shown here for every sampling event...... 52

Figure 5-3. Isomer profile of NP based on chromatographic response...... 53

1. Background

1.1. Introduction

Nonylphenol (NP) is the most persistent and estrogenically active metabolite of nonylphenol polyethoxylate (NPE). NPE belongs to a class of nonionic surfactants called alkylphenol ethoxylates (APEs) which have been in use since the 1940s. APEs, the majority being NPEs, are one of the high production volume surfactants used in the

United States. APEs have an estimated yearly production of over 450 million lbs

(Naylor, Mieure et al. 1992). Since approximately 80% of APEs produced are nonylphenol polyethoxylates (NPEs), up to 360 million lbs yr-1 of NPEs can be sent to wastewater treatment plants (WWTPs) in the US. A recent USGS survey of water from

139 streams across the US reported that nonionic detergent metabolites were present in

70% of the streams (Kolpin, Furlong et al. 2002). APEs were the fourth highest class out of fifteen compounds in regards to detection frequency, but were present at the highest total concentration. Of the APEs analyzed, NP was found at the highest maximum level,

40 µg/L. NP is also classified as an endocrine disrupting compound (EDC). NP has been shown to bind to the estrogen receptor resulting in estrogenic responses in a wide range of wildlife (Servos 1999). Due to their ubiquitous nature, their persistence in the environment, and their estrogenic activity, the fate of these compounds has recently become a growing concern.

1.2. Uses, Production, and Structure

Alkylphenolic compounds are mainly produced for three categories of uses. The major use is as alkylphenol polyethoxylates (APEs) for industrial non-ionic surfactants

7 and institutional detergents. This includes the use of APEs in the paper and pulp industry for paper de-inking and in other industries as a metal degreasing agent. A second use is in phosphite antioxidants in the rubber and plastic industries. A third category includes the use of alkylphenolic compounds as an additive to fuel oils and other miscellaneous uses (ChemExpo 1998). There are several other non-ionic surfactants that could be used as alternatives to APEs, but they are more difficult to manufacture in a pure state. In order to replace APEs a more expensive mixture of surfactants is often necessary to achieve similar properties.

APEs are produces by reacting branched-chain alkylphenols with ethylene oxide to produce a hydrophilic ethoxylate side chain. Depending on the application, the number of ethoxylates in the chain can vary between 1 and 50. The majority of APEs are produces in the form of NPEs. Figure 1-1 shows the general structure of NPEs.

Figure 1-1. The structure of NPE where n is the number of ethoxylates in the ethoxylate chain.

The NP used to produce the ethoxylated surfactant is a complex mixture of isomers due to variations in the branching of the akyl side chain. Figure 1-2 shows some

8 of the possible isomers of NP. There are 47 theoretically possible isomers of NP (Thiele,

Heinke et al. 2004). This complex branching is a result of the production method. The nonyl group is first produced by polymerizing propylene into a trimer. Rearrangement can occur during polymerization resulting in additional isomers. Alkylation of phenol with the propylene trimer results in a mixture of highly branched olefins with the alkyl chain substituted at the para position.

Figure 1-2. Several possible isomers found in the technical mixture of NP.

Wheeler et al. (1997) separated and characterized 22 isomers from a technical mixture of NP based on the branching of the alkyl chain using high resolution gas chromatography and mass spectroscopy (Wheeler, Heim et al. 1997). Thiele et al. (2004) synthesized 10 NP isomers and determined their structure using NMR. These isomers were then compared to the isomer found in the technical mixture used by Wheeler et al.

(1997), and six isomer groups were characterized by the branching of the alpha and beta carbons on the nonyl chain.

1.3. Source of NP to the Environment

The majority of the uses for APEs results in their discharge to wastewater treatment plants (WWTPs). Once in a WWTP, the APEs are biotransformed in the secondary (aerobic) treatment. The biotransformation pathway begins with attack and successive removal of units from the ethoxylate chain (Ball, Reinhard et al. 1989), (Ahel,

Giger et al. 1994). Figure 1-3 illustrates the biodegradation pathway of APE to AP.

Studies of octylphenol ethoxylate biotransformation by a bacterial culture developed from sewage have shown that progressive removal of the ethoxylate group occurs either by oxidation of the terminal alcohol and cleavage of the newly formed carboxylic acid or by ether cleavage (Ball, Reinhard et al. 1989). The biotransformation results in byproducts with a shortened ethoxylate chain, alkylphenoxy carboxylic acids, and APs.

The AP, specifically NP, has been shown to be the most persistent of the biotransformation products and orders of magnitude more toxic than the parent compound (Scott and Jones 2000).

Figure 1-3. The biodegradation pathway of APEs to AP begins with progressive removal of ethoxylate units.

WWTPs in the US achieve average removal rates of 97% for NPEs (Naylor

1995). However, while WWTPs are able to achieve a high percentage of primary biodegradation of nonylphenolic compounds, they typically do not achieve complete mineralization (Maguire 1999). The solubility of NPEs decline and the octanol/water partitioning coefficient increases as the ethoxylate chain is shortened with NP having the lowest solubility in water, 5.4 mg/L, and the highest partioning coefficient, 4.5 (Ahel and

Giger 1993; Ahel and Giger 1993). Therefore, the majority of the partially degraded compound partitions to the organic solids and the sludge. However, due to the large throughput of WWTPs, significant quantities of NP are discharged with the aqueous effluent. After discharge to the receiving body of water, again due to their relatively high hydrophobicity, the NP can partition to the organic sediment (Ying, Williams et al.

2002). Over time, the NP can accumulate in the sediment, and concentrations in sediments near WWTP outfalls have been observed at levels between 10 mg/kg (Bennie

1999) to 40 mg/kg (Ferguson, Bopp et al. 2003).

1.4. Endocrine Disruption and Reasons for Concern

NP was recognized as an endocrine disruptor on accident when the NP used as an additive in plastic centrifuge tubes interfered with studies being conducted with estrogen- sensitive MCF7 breast cancer cells (Soto, CJusticia et al. 1991). Concern for the fate of

APEs has increased significantly due to the evidence from subsequent studies that their metabolites are persistent in the environment (Ying, Williams et al. 2002), that they can bind to the estrogen receptors of humans and wildlife (Jobling and Sumpter 1993), and have estrogenic effects on wildlife. White et al. (1994) has shown that at high enough concentrations APs can illicit a biological response similar to 17β-estradiol (White,

Jobling et al. 1994). Figure 1-4 shows a comparison of a possible NP isomer to 17β- estradiol. A survey of surface waters in northeast Spain reported that NP was ubiquitous,

NP reached levels of 600 µg/L, and vitellogenin induction occurred in male carp exposed at all sampling points (Sole, Lopez de Alda et al. 2000). Vitellogenin induction was reported to increase relative to the proximity of WWTPs.

Figure 1-4. Comparison of one possible isomer of NP to 17β-estrodial.

The estrogenicity of NPE is inversely proportional to the length of the ethoxylate chain with NP having an estrogenicity of orders of magnitude greater than NPE with an ethoxylate chain length of nine (Jobling and Sumpter 1993), (Routledge, Sheahan et al.

1998). However, the estrogenic effect of alkylphenolic compounds appears to be additive

(Soto, Sonnenschein et al. 1995), (Jobling, Sheahan et al. 1996), so alkylphenolic compounds in the environment must be viewed as a complex mixture (Ying, Williams et al. 2002).

Recently evidence has been brought forward that NP is commonly found in human foods. NP was examined in edible mollusks, in the Adriatic Sea and levels were reported up to 696 ng/g fresh weight (Ferrara, Fabietti et al. 2001). The study reported a general contamination of the Adriatic Sea even a significant distance from the coast. A

German study reported that NP is ubiquitous in foods and found levels of NP in all 24 different food types surveyed, ranging from 0.1 to 19.4 µg/kg (Guenther, Heinke et al.

2002). The study concluded that the average person ingests 7.5 µg/day of NP.

One study concluded that a alkyl group with a tertiary alpha carbon on an otherwise unhindered phenol ring results in the greatest estrogenic activity (Routledge and Sumpter 1997). Little work has been conducted to better understand the estrogenic activity of the individual isomer. Kim et al. (2004) fractionated seven isomers from a commercial mixture of NP and synthesized one isomer and compared their estrogenicity using a recombinant yeast screen assay (Kim, Katase et al. 2004). All the isomers had tertiary alpha carbons and distinct differences in estrogenic activity were reported.

Therefore, the isomer configurations surveyed by Kim et al. (2004) are most likely to have the highest estrogenicity.

1.5. Biodegradation Studies

Studies on the biodegradation of APEs have reported a wide range of removal

efficiencies and biodegradation rates. Primary, aerobic degradation of NPEs have been

shown to have a halflife of 4 days in water and 10 days in sediments (Yoshimura 1986).

Field studies and laboratory work have showed that under aerobic conditions

microorganisms are able to degrade APE metabolites (Ekelund, Granmo et al. 1993),

(Staples, Williams et al. 1999). Under anaerobic, methanogenic conditions, NP1E and

NP2E were degraded to NP, but the NP was not shown to be further degraded (Ejlertsson,

Nilsson et al. 1999).

Although many studies have investigated the aerobic and anaerobic degradation

of APEs, few have examined the aerobic and anaerobic fate of the metabolic byproducts, especially the APs. Under aerobic laboratory conditions, activated sludge cultures were able to readily degrade NP at a temperature of 28ºC, but lower temperatures were found to considerably decrease the elimination capacity of the culture (Tanghe, Devriese et al.

1998). The activated treatment of WWTPs and the waterways that contain NP, rarely reach the higher temperatures that were shown to be optimal for degrading NP.

Degradation studies of NP in sewage sludge aggregates showed that complete mineralization of NP was possible, but that degradation could be closely correlated to oxygen penetration into the aggregates (Hesselsoe, Jensen et al. 2001). Hesselsoe et al.

(2001) suggested from their findings that the majority of the NP degradation took place in the aerobic region of the aggregate. Several studies have shown that alkylphenolic compounds are more persistent in sediments (Ying, Williams et al. 2002). Schroger et al.

(2001) showed that NPE metabolites are persistent in the environment than the parent

compound (Schroder 2001).

Through in situ studies, APs were shown to be produced in sediments as

metabolites from the degradation of APEs and that the APs are well preserved in dated

sediment cores from Japan (Isobe, Nishiyama et al. 2001). Isobe et al. (2001) also

reported that the highest level of AP in sediments correlates to the 1970s and that the

recent decrease can be attributed to increased legal regulation on industrial wastewaters.

Dated sediment cores from Venice were analyzed and concentrations of 110 µg/kg NP for 1972 were found, again illustrating the persistence of these compounds (Marcomini,

Pojana et al. 2000). The half life of NP in sediments has been estimated to be in the range of 20 to 60 yrs by analyzing dated sediment cores (Shang, MacDonald et al. 1999),

(Ferguson, Bopp et al. 2003).

A single isomer of NP found in technical mixtures was synthesized and found to be resistant to biodegradation in lake water and sediment samples (Lalah, Schramm et al.

2003). In addition to studies showing the persistence on NP in the environment, evidence

of a recalcitrant NP isomer has been shown (Hawrelak, Bennett et al. 1999). Hawrelak et

al. (1999) examined the presence of NP in recycled paper plant sludge after various

holding times and periods after land application. Through visual interpretation of the NP

chromatograms an increase in proportion of an isomer peak to the others was reported

indicating a more recalcitrant NP isomer. Although their work did not examine the

individual isomers in detail, they did show the possibility of recalcitrant isomers.

A recent study conducted on anaerobic microcosms showed that biodegradation

of NP is possible. After one year of enrichment and acclimation and a temperature of

30°C, halflives for NP of 46.2 to 69.3 days were reported (Chang, Yu et al. 2004).

However, studies have shown that NP is persistent in anaerobic sediments (Ying,

Williams et al. 2002).

1.6. Summary

Due to the popularity of APEs for variety of uses, their metabolites are found in surface waters and sediments throughout the industrialized world. A large body of work exists showing the estrogenic and toxic effects the metabolites of these compounds can have on the wildlife (Servos 1999). While few studies have shown that the most persistent of these compounds, NP, can be degraded in laboratory conditions using enriched and acclimated cultures, many more studies give evidence of their persistence in the environment. Additional work is needed to determine and understand the fate of NP in the environment. Of particular interest is the difference in biodegradation rates of the

NP isomers.

2. Method Development for Analysis of Nonylphenol

2.1. Objective The first step for this research was to develop a method for the extraction and

analysis of NP in both aqueous bioslurries and in sediments. The procedure was required

to yield good extraction efficiencies for both matrixes. A simple extraction procedure

was needed that does not require expensive equipment. A gas chromatogram with a mass

spectrometer (GC/MS) was available for use and GC/MS is able to achieve good

separation of NP isomers (Wheeler, Heim et al. 1997), (Thiele, Gunther et al. 1997).

Therefore the extraction technique needed to produce sample extracts suitable for GC injection. Since microcosms where used for a portion of this work, the extraction technique and analysis had to be developed for use with small volume samples, less than

10 mL. A detailed description of the method developed for the analysis of NP can be found in Chapter 9, Appendix 1.

2.2. Review of Previous Methods A review of the previous methods in the literature showed that many techniques

have been developed for the analysis of NP and NPEs in both aqueous samples and in

sediments. Early techniques used steam distillation with iso-octane followed by analysis

with high performance liquid chromatography (Kubeck and Naylor 1990). The use of

steam distillation requires large samples, up to 1 L, and expensive distillation equipment.

Liquid chromatographic techniques are popular where analysis for alkylphenol

ethoxylates (APEs) is desired (Ahel and Giger 1985), (Marcomini and Giger 1987),

(Scullion, Clench et al. 1996), (Kibbey, Yavaraski et al. 1996), (Shang, MacDonald et al.

1999), (Takina, Daishima et al. 2000), (Schroder 2001), (Jeannot, Sabik et al. 2002).

Since this research will not examine APEs and an HPLC is not available for use, another

method of analysis was needed.

GC/MS has been used for analysis of NP in various matrixes (de Voogt, de Beer

et al. 1997), (Ding and Tzing 1998), (Ferguson, Iden et al. 2000), (Kuch and Ballschmiter

2001), (Kojima, Tsunoi et al. 2003). Some of these use an extraction technique based on a solid phase extraction which is relatively simple and yields good extraction efficiencies

(de Voogt, de Beer et al. 1997), (Kuch and Ballschmiter 2001). However, the solid phase extraction technique can only be used with relatively clean or filtered samples and cannot be used effectively for thick bioslurries or sediments. High recoveries of NP from aqueous samples have been shown by the use of liquid-liquid extractions (Bennie,

Sullivan et al. 1997). However, when liquid-liquid extraction was conducted on sediment samples, low extraction efficiencies of NP were observed (Ferguson, Iden et al. 2000).

This is most likely due to analyte becoming trapped within the sediment pores.

2.3. Method Development A novel method was developed to be used for the extraction of NP from both

bioslurries and sediments. Due to the limitation of liquid-liquid extraction to remove

analyte from with the pore structure, a single phase solvent extraction technique was

developed. This technique was adapted from a method used to extract phospholipids

from cell cultures (Dobbs and Findlay 1993). Before the extraction, a surrogate standard

is added for later determination of extraction efficiency. To perform the extraction, a 7

mL aqueous sample or a 7 g sediment sample with 7 mL of distilled, deionized water is

mixed with 10.5 mL of methanol and 10.5 mL of methylene chloride to form a single

phase in which the analyte is soluble. Since the NP is soluble in this phase, the NP will

18 not be trapped in the sediment pores. The samples are shaken for 12 hours in a reciprocating shaker at 100 rpm. To separate the single phase into a solvent and aqueous phase, 10.5 mL water and 10.5 mL of methylene chloride are added. The solvent phase, totaling 21 mL, is removed by pipetting and dried by passing it through an anhydrous sodium sulfate column. After the extract is dried, it is ready for analysis by GC/MS. A 1 mL aliquot is placed in a GC vial and internal standards are added.

The GC method used to quantitate NP was developed to yield good chromatographic separation of the isomers of NP. With the use of the mass spectrometer’s selected ions monitoring mode, 23 isomers of NP were differentiated. A novel technique for the quantification of the isomers was developed because individual isomer standards are not commercially available. A calibration curve was created by assigning each peak the value of the total NP in the calibration standard. For example, the 10 mg/L standard was analyzed and the response for each peak was correlated to the value of 10 mg/L. A calibration curve was developed each isomer peak using this method. Total NP was determined by averaging the value of each of the isomers.

3. Enrichment by the Use of Aerobic Respirometers

3.1. Materials and Methods

Aerobic Respirometer Experiments. Aerobic respirometers were used to enrich a culture capable of degrading NP as a sole carbon and energy source. Samples taken from the activated sludge (secondary treatment) and anaerobic digester of a local WWTP combined in equal proportions. The combined sludge was then diluted 1:1 with a basic minimal media as described by Selton et al. (1984) (Shelton and Tiedje 1984). The respirometers were constructed with 250 mL of the combined media/sludge and NP at a concentration of 1000 mg/L. The respirometers were connected to an N-Con Comput-Ox

Computerized Respirometer, Model# WB512. The respirometers operate with a vial of

KOH suspended in the headspace which removes carbon dioxide as it is produced by the culture. The removal of carbon dioxide by the KOH trap and oxygen by the culture from the headspace results in a drop in pressure within the respirometer bottle. As the pressure drops, the computer signals a metering valve to dose a known volume of oxygen into the respirometer bottle. The computer maintains a continuous log of oxygen consumption data. The KOH traps are frequently changed and by measuring the pH of the trap, the amount of carbon dioxide removed can be calculated. As oxygen consumption leveled off, indicating a reduction in biological activity, the cultures were diluted again 1:1 with

fresh media and additional NP was added to the respirometers.

Most Probable Number of NP Degraders. A technique known as MPN or

Most Probable Number was used to enumerate the number of NP degraders in a culture

(Haines, Wrenn et al. 1996), (Wrenn and Venosa 1996). This technique uses 96 well plates with each well containing a small volume of sterile media and substrate. A robot is

20 used to repetitively dilute a small sample of culture across the plate. By determining the number of equal proportion dilutions needed to remove degraders from a sample, a statistical calculation can be employed to enumerate the number of active NP specific degraders in the culture.

3.2. Results and Discussion After four generations of enrichment, a culture was developed that was able to degrade 1000 mg/L of NP in 40 days. The number of degraders in the culture was low at

106 active degrader cells/mL as enumerated by the MPN procedure (Haines, Wrenn et al.

1996), (Wrenn and Venosa 1996). As shown in Figure 3-1, oxygen consumption followed a curve typical for a culture with a limited substrate. The oxygen consumption decreased as available carbon sources were removed. Through biological activity and physical dilution a culture was enriched to use NP as a sole carbon and energy source.

Lag time became shorter with each enrichment as the as the culture became more acclimated to consuming the NP.

2 12 O l 10

mmo 8 3rd Enrich (Best Fit) 6 3rd Enrich Rep A 4 3rd Enrich Rep B

0 0 100 200 300 400 500 600 700 800 time (hrs)

Figure 3-1. The oxygen consumption curve for a culture degrading NP as the sole carbon and energy source.

Figure 3-2 shows an illustration comparing the carbon dioxide uptake for the third and fourth enrichment. The difference in the curves for enrichment 3 and 4 can be attributed to remaining carbon sources from the original inoculation. Also illustrated in

Figure 3-2 is a method used for calculating the maximum theoretical cell yield assuming

NP is the only remaining carbon source. Cell yield is defined as the ratio between the mass of biomass produced to the mass of substrate consumed (Tchobanoglous, Burton et al. 2003). By calculating the stoichiometric maximum quantity of carbon dioxide production and comparing that to the actual production, the maximum theoretical cell yield was determined. The maximum theoretical cell yield was calculated to be 0.25, which is very low when compared to most biological systems in WWTPs and the environment. Typical cell yield for a WWTP is between 0.4 and 0.5. The low yield of

NP degraders may account for the low removal efficiency of NP in WWTPs. The

22 secondary treatment of most WWTPs have a retention time of only a few hours, therefore any organisms that do not reproduce fast enough will be washed out.

Figure 3-2. Carbon dioxide uptake curve and illustration of calculation of maximum theoretical cell yield.

Although WWTPs in the U.S.A. have an average removal efficiency of 97% for

NPEs in the aqueous stream (Naylor 1995), high concentrations of NP have been seen in the anaerobic digester sludge (Bennie, Sullivan et al. 1986), (Bennie 1999), (Pryor, Hay et al. 2002). Due to the high hydrophobicity and low aqueous solubility of NP (Ahel and

Giger 1993; Ahel and Giger 1993) the majority of NP remaining after biodegradation in secondary treatment may partition to the organic solids in the sludge. This partioning of

NP to the sludge may give artificially high removal efficiencies when only the aqueous stream is considered. The removal of NP from the aqueous stream cannot be considered a biological removal unless a mass balance of all nonylphenolic compounds in a WWTP

is constructed for all streams. Since anaerobic digesters operate as continuous stirred

tank reactors, the high concentrations of NP seen in the sludge may indicate a low

biodegradation rate of NP. Even with the high aqueous removal efficiencies, the high

throughput of WWTPs may result in a significant mass of NP still being discharged to the receiving bodies of water in the aqueous effluent.

In addition to the persistence of NP, recent work has shown that some isomers of

NP may be more resistant to biodegradation than others. Evidence of recalcitrant NP isomers has been reported by Hawrelak et al. (1999) (Hawrelak, Bennett et al. 1999).

Their work examined the presence of NP in recycled paper plant sludge after various

holding times and periods after land application. Although their work did not examine

the individual isomers in detail, they did show the possibility of recalcitrant isomers by

visual interpretation of the NP peaks in their chromatograms. Figure 4-3 contains two

chromatograms comparing NP in the technical mixture used in this study and the NP

remaining in the respirometers after 40 days of biodegradation. From this figure, it is

clearly evident that certain NP isomers are more resistant to biodegradation than others.

Unfortunately, the structure of all the NP isomers has not been determined, nor

have all of the isomers been separated or individually synthesized. In addition, standards

for the individual isomers are not yet available. Thiele et al. (2004) has proposed a

structure for each isomer in a technical mixture, after following up on the work or

Wheeler et al. (1997), through the use of GC-MS and NMR (Wheeler, Heim et al. 1997),

(Thiele, Heinke et al. 2004). Thiele et al. (1997) also verified the structure of some of

these isomers through synthesis of those isomers. With additional research it may

24 become possible to manufacture a mixture of isomers that is easier for microorganisms to biodegrade.

Cal Solution 7 21008 Cal Solution 18-Oct-200222:01:58 210917 cal_7 2: SIR of 9 Channels EI+ 163.00 100 1.50e7

0 Time 41.54 42.04 42.54 43.04 43.54 44.04 44.54 45.04 45.54 46.04 46.54

N3HB1 from NP rate exp 20718 21023 N3HB1 24-Oct-200202:07:15 21023 n3hb1 2: SIR of 9 Channels EI+ TIC 100 9.20e6

0 Time 41.58 42.08 42.58 43.08 43.58 44.08 44.58 45.08 45.58 46.08 46.58

Figure 3-3. Comparison of the chromatogram of NP isomers in the technical mixture and after 25 days of degradation.

The low cell yield of NP biodegrading organisms may be the reason for poor NP biodegradation rates in WWTPs. By using a batch system, this study showed that cell yields for NP degraders are significantly lower than for typical microorganisms in

WWTPs. Since the secondary treatments of WWTPs have a normal retention time of

25 only a few hours, it is likely that NP degraders are out competed by faster growing organisms and their number are kept low by washout.

Additional work is needed to determine verify these finding in WWTPs and to perform studies that help to close the mass balance on NP in WWTPs. Currently there is very little work done on the fate of NP and its precursors in the anaerobic digesters of

WWTPs. Study should be conducted to examine the biodegradation of NP under the anaerobic conditions seen in WWTPs and in sediments exposed to NP.

4. Aerobic Biodegradation of Nonylphenol in Microcosms: Estimation of Rate by Individual Nonylphenol Isomers

4.1. Abstract

Microcosms were used to investigate the biodegradation of a technical mixture of nonylphenol (NP) in an aerobic environment. The microcosms operated at three active conditions, which differed only by inoculum source, and one killed control. Through the use of gas chromatography-mass spectroscopy, the technical mixture of NP was differentiated into 23 isomers and rate constants were estimated for total NP and for each

NP isomer. The halflife for the total mixture of NP was less than 17 days for two of the three active conditions. For the active conditions, biodegradation rates for some isomers differed significantly, indicating that some isomers are more resistant to biodegradation.

Halflives for the NP isomers ranged from 10 days to more than 100 days. Comparisons between known isomer structures and biodegradation rates show that correlations exist between the branching of the alkyl chain and biodegradability. This suggests that the development of a technical mixture with increase degradability may be possible.

4.2. Introduction Alkylphenol polyethoxylates (APEs), a class of nonionic surfactants, have been in use since the 1940s and are one of the high production volume surfactants used in the

United States. APEs have an estimated yearly production of over 450 million lbs

APEs are biotransformed in the secondary (aerobic) treatment and in the anaerobic

digester. The average removal rate of NPEs in US WWTPs was 97% (Naylor 1995).

The biotransformation pathway begins with removal of the ethoxylate chain (Ball,

Reinhard et al. 1989), (Ahel, Giger et al. 1994). The biotransformation of APEs results in byproducts with shortened ethoxylate chain, alkylphenoxy carboxylic acids, and alkylphenols. AP, specifically nonylphenol (NP), has frequently been shown to be the

most persistent of the biotransformation products. NP is also more toxic and orders of

magnitude more estrogenically active than the parent compound (Scott and Jones 2000).

Concern for the fate of APEs has increased significantly due to the evidence that

their metabolites are persistent in the environment (Ying, Williams et al. 2002), that they

can bind to the estrogen receptor (Jobling and Sumpter 1993), and have estrogenic effects

in the wildlife. White et al. (1994) has shown that at high enough concentrations APs can

illicit a biological response similar to 17β-estradiol (White, Jobling et al. 1994).

WWTPs are typically able to achieve primary biodegradation of nonylphenolic

compounds, but often do not achieve complete mineralization (Maguire 1999). Since

nonylphenol (NP) has a low solubility in water, 5.4 mg/L and a relatively high octanol- water partition coefficient (log Kow) of 4.48, (Ahel and Giger 1993; Ahel and Giger

1993) the majority of the undestroyed NP partitions to the organic solids and sludge.

However, due to the large throughput of WWTPs, over time a significant mass of NP is discharged with the aqueous effluent. After discharge to the receiving body of water, the relatively high hydrophobicity of NP will drive NP into the organic sediment (Ying,

Williams et al. 2002). Over time, the NP can accumulate in the sediment, and concentrations in sediments near WWTP outfalls have been observed at levels from 10

mg/kg (Bennie 1999) to 40 mg/kg (Ferguson, Bopp et al. 2003). Several studies have

shown that alkylphenolic compounds are persistent in sediments (Ying, Williams et al.

2002). The half life of these compounds has been estimated through the use of dated

sediment cores to be in the range of 20 to 60 years (Ferguson, Bopp et al. 2003), (Shang,

MacDonald et al. 1999).

Due to the method of production, NP exists as a complex mixture of isomers. NP is produced from an alkylation reaction of a propylene trimer with phenol. A technical mixture was differentiated into 22 isomers by high-resolution gas chromatography and mass spectoscopy (Wheeler, Heim et al. 1997), (Thiele, Heinke et al. 2004). By examining the mass spectra for each individual isomer, Wheeler et al. (1997) speculated the branching of the alpha and beta carbon of the nonyl chain, and the isomers were characterized into five groups based on their branching. Theile et al. (2004) used NMR to determine the structure of ten synthesized isomers and determine the corresponding isomers in the technical mixture. Eight isomers of NP were fractionated by high- performance liquid chromatography, and recombanent yeast assays were run on individual isomers to determine their estrogenic response relative to estrodiol (Kim,

Katase et al. 2004). NMR was used to determine the structure of the isomers.

Little is known about the persistence and biodegradation rates of the individual isomers of NP. The structure of the NP isomers greatly affects their estrogenicity.

Isomers with a tertiary substituted alpha carbon on an otherwise unhindered chain are more estrogenic than isomers without a tertiary substituted alpha carbon (Routledge and

Sumpter 1997). The results from this study suggest that under aerobic conditions the

29 most estrogenic isomers, as determined by Kim et al. (2004), experience faster biodegradation rates than other isomers of NP.

4.3. Materials and Methods Aerobic Microcosm Experiments. Sacrificial aerobic microcosms were set up using three active (viable) conditions and one killed control. All conditions were setup identically, except for inoculum source. Samples for inocula were taken from (1) the anaerobic digester of a WWTP receiving a mixture of industrial and domestic effluent

(Condition 1), (2) the anaerobic digester of a domestic WWTP (Condition 2), and (3) the activated treatment of a domestic WWTP (Condition 3). A slurry was prepared for each condition using 25% vol/vol inoculum source in a basic minimal media as described by

Selton et al. (1984) (Shelton and Tiedje 1984). NP is a viscous liquid and was dissolved in pentane to ensure consistent dosing to the microcosm vials. NP in pentane was first added to sterile microcosm vials to yield 1000 mg/L of NP in the final volume of the microcosm. The sacrificial microcosms were loosely capped and the pentane was allowed to evaporate. An inoculum (7 mL) was added to each vial. A sterile control

(Condition 4) was prepared by combining equal parts of the other three inocula. The sterile control slurry was autoclaved twice at 121°C for one hour and 10 mg/L of sodium azide was added before aseptically dispensing to sterile microcosm vials. The microcosms were prepared for eleven timepoints in triplicate. The microcosms were capped with a U-shaped needle inserted though the septa to allow the headspace to exchange freely with the atmosphere without contamination. The microcosms were placed in a gastight box and maintained at a temperature of 20°C and continuously agitated on an oscillating shaker at 200 rpm. A 10 ml/min stream of humidified oxygen

30 was continuously purged though the box to limit evaporation and to ensure aerobic conditions. The microcosms were sacrificially sampled in triplicate at times 0, 7, 14, 21,

28, 35, 42, 49, 56, 63, and 98 days.

Chemicals. The nonylphenol was a technical grade mixture of isomers (Sigma

Aldrich, 29,085-8, St. Louis, MO). The surrogate standard was 2, 4, 6 tribromophenol

(Supelco, 47960-U, Bellefonte, PA). The internal standard was acenapthene-d10

(Accustandard, Z-014J, New Haven, CT). All solvents were pesticide grade (Fisher

Scientific, Hanover Park, IL). Anhydrous sodium sulfate columns were constructed using large volume, disposable pipettes, conditioned glass wool and approximately 2 g of anhydrous sodium sulfate (Fisher Scientific, Hanover Park, IL). Columns were pre-dried in a 100°C oven for 12 hours before use.

Chemical Analysis. All liquid samples were extracted by a single-phase extraction technique (Dobbs and Findlay 1993), (EPA 2004). A single-phase extraction technique allowed the extract to be used for both NP analysis and for determination of the

Total Biomass in the sample. To perform the extraction, the 7 mL aqueous samples were first mixed with 10.5 mL of methanol and 10.5 mL of methylene chloride to form a single phase in which the analyte is soluble. The samples were shaken for 12 hours in a reciprocating shaker at 100 rpm. To separate the single phase into a solvent and aqueous phase, 10.5 mL water and 10.5 mL of methylene chloride were added. The solvent phase, totaling 21 mL, was removed by pipetting and dried by passing it through an anhydrous sodium sulfate column. Extracts were then split for the two analyses with 10

31 mL for total biomass analysis and the remainder for NP analysis. Method blanks consisted of distilled, de-ionized water that was processed through the entire sampling and extraction procedure prior to analysis.

NP analysis was performed on an Agilent 6890 GC (Palo Alto, CA) equipped with a 60 meter, MDN-5S capillary column (Supelco, 2-4392, Bellefonte, PA), an autosampler, and an Agilent 5971 Mass Selective Detector. The carrier gas was helium at a constant flow rate of 1 mL/min. Samples were injected in splitless mode. The oven temperature was initially set at 50°C for 1 min, increased to 100°C at a rate of 20°C/min, increased to 175°C at a rate of 1.5°C/min, increased to 315°C at a rate of 20°C/min and held for 6 minutes. Internal standard (acenapthene-d10) was added to samples at a concentration of 25 mg/L prior to injection. Surrogate recoveries of 2, 4, 6 tribromophenol were 75 - 90.0%. NP calibration range was 1 mg/L to 50 mg/L of NP with a minimum detection limit of 0.5 mg/L (EPA 1998).

NP was differentiated into 23 separate isomer peaks (see Figure 5-1 and Table 5-

1) by examining the chromatograms for the ions characteristic of NP and using the isomer group notation developed by Wheeler et al. (Wheeler, Heim et al. 1997). Wheeler et al. characterized 22 para-NP isomers into five groups based on the branching of the alpha and beta carbons on the nonyl chain. Due to differences in chromatographic methods and the source of NP, a unique isomer peak notation was developed for this study. The peak notation used in this research is shown in Figure 5-1. A novel technique was employed to quantitate the separate isomers, since standards do not exist for individual NP isomers. A calibration curve was created by assigning each peak the value of the total NP in the calibration standard. For example, the 10 mg/L standard was

32 analyzed and the response for each peak was correlated to the value of 10 mg/L. A calibration curve was developed each isomer peak using this method. Total NP was determined by averaging the value of each of the isomers.

Biological Analysis. Total phospholipids biomass was determined according to

Dobbs (1993) as a measure of viable biomass concentration initially present in the microcosms (nmol phospholipids/mL) (Dobbs and Findlay 1993).

Data Analysis. Total NP and NP isomer concentration were fit to a first order biodegradation model to yield rate constants with 95% confidence intervals. Beta-Hat analysis was used to determine if a statistical difference exists between the rate constants of the individual isomers within each condition (Khorasani and Milliken 1982), (Johnson and Milliken 1983), (Hinds and Milliken 1987).

4.4. Results and Discussion The initial total biomass and the NP background level in the inoculum are shown in Table 1. The background level of NP was insignificant (< 0.4%) compared to the initial concentration of NP added to the microcosms. Figure 4-1 compares a total ion chromatogram (TIC) of NP at zero and 98 days. From this figure, the occurrence of biodegradation of NP is clearly evident. Total NP in the microcosms was reduced by

95% in less than 80 days in two of the three active treatments.

Table 4-1. Initial total biomass and NP concentrations that were present in the inoculum for each condition before the start of the experiment. Initial Total Biomass NP Conc (mg/L) of

Source (nmol/mL phospholipids) Inoculum Condition Anaerobic digester receiving 23.2 3.8 1 industrial and domestic effluents Condition Anaerobic digester receiving 20.2 0.7 2 domestic effluents Condition Activated Sludge receiving 6.0 0.7 3 domestic effluents Condition Mixture of condition 1, 2, and 3 6.6 1.8 4

Figure 4-1. TIC comparison of NP isomers at T=0 days and T=98 days.

NP was analyzed at mg/L concentrations as both total NP and differentiated into

23 isomer groups. Biodegradation rates were observed to follow first order reaction kinetics. Studies of NP degraded by aerobic microbes from river sediments also showed that biodegradation of NP could be modeled by first order kinetics (Yuan, Yu et al.

2004). The curves in Figure 4-2 are shown with 95% confidence intervals. The difference in rates between Condition 3 and the other two active treatments, listed in

Figure 4-2, was attributed to variation of the initial biomass in the inoculums.

Normalizing the rates of the active conditions to the initial biomass yields 0.0018,

0.0024, and 0.0021 mL/nmol *day for condition 1, 2, and 3, respectively.

Figure 4-2. Least squares regression of first order biodegradation for each condition. Open dots are the mean of the replicate set, and error bars are 95% confidence intervals for the replicate set, the solid line is the fitted curve, dashed lines are 95% confidence intervals for the replicate set, the solid line is the fitted curve, dashed lines are 95% confidence intervals for the curve.

By comparison of the chromatograms in Figure 4-1, it is evident that some of the isomers are being degraded at different rates and that isomer 2 is relatively undegraded.

Evidence of recalcitrant NP isomers has been shown by Hawrelak et al. (1999)

(Hawrelak, Bennett et al. 1999). Their work examined the presence of NP in recycled paper plant sludge after various holding times and periods after land application.

Although their work did not examine the individual isomers in detail, they did show the

35 possibility of recalcitrant isomers by visual interpretation of the NP peaks in their chromatograms.

The NP used in the commercial production of surfactants consists of multiple isomers due to the method of production. NP is produced by the alkylation of phenol with nonene. The nonene, also called a propylene trimer, is a complex mixture of isomers. Currently analysis of individual NP isomers is difficult, because analytical standards do not exist for each of the individual isomers, only for technical mixtures of isomers. Through the use of a high-resolution gas chromatograph and mass spectrometer,

Wheeler et al. (1997) achieved resolution of 22 NP isomers (Wheeler, Heim et al. 1997).

Six distinct isomer groups were characterized by the branching of the alpha and beta carbons on the nonyl chain (Thiele, Heinke et al. 2004).

Figure 4-3. Peak notation used for NP isomers. Each line represents an ion (m/z) characteristic to NP.

Table 5-4-2. Comparison of NP isomer for this study to that of Wheeler et al. and Kim et al. Primary Qualifier Wheeler et al. Wheeler et al. Peak # Ion (m/z) Ion (m/z) Group # Peak # 1 121 107, 163 5 1 2 135 107 1* _∏ 3 135 107, 121 1 2 4 135 107, 121 1 3 5 135 - 1 5+4a 6 149 107, 121 2 4b 7 135 - 1 7 8 149 107, 121 2 6 9 135 - 1* _∏ 10 149 107, 121 2 8 _† 11 107 135 _∏ 12 135 107 1 10 13 121 107, 163 5 9 14 163 107, 121 4 11 15 149 - 3 12c 16 121 163 5 12b 17 135 - 1* 12a 18 163 107, 121 4 13 19 135 - 1* _∏ 20 135 - 1 14 21 107 121 3 15b 22 149 107, 121 2+3 16+18 23 135 107 1 17 * - Peak not designated to this group by Wheeler, but m/z spectra fits. † - Peak does not fit into a group defined by Wheeler et al. ∏ - Peak not labeled by Wheeler et al.

This work uses the group notation developed Wheeler et al. (1997), but uses a

novel peak notation, because four peaks that were not present in Wheeler's work are

found in the technical mixture used for this study. Figure 4-3 contains a selected ion

chromatogram illustrating the peak notation. Table 4-2 is provided as a reference

between the peak notation used in this study and the notation used by Wheeler et al.

(1997) and lists the primary ions used to quantify each isomer.

Figure 4-4. Estimated first order rate constants for the biodegradation of individual NP isomers. Heavy shaded bars are condition 1, hatched bars are condition 2, lightly shaded bars are condition 3, open bars are condition 4, and error bars are 95% confidence intervals of estimated rates.

For each isomer in the four conditions, first-order rate constants were estimated

(Figure 4-4). Beta-Hat analysis of isomer rates described whether a difference in the behavior of the isomers exists. The killed control (Condition 4) showed only slight loss of NP and no statistical difference in the rate constants for each isomer. This implies that there was no loss of NP due to biological activity. The active treatments all showed

- 38 – strong statistical differences in the rate constants of the isomers within a condition,

implying that the removal of NP was due to biological activity.

Figure 4-5. Isomer group designation adapted from Wheeler et al. (1997).

The isomers for NP are organized into five groups based on the configuration of

their alpha and beta carbons on the nonyl chain, as illustrated in Figure 4-5 (Wheeler,

Heim et al. 1997). Comparing the isomer biodegradation rates determined in this study

by isomer group, it is evident that the isomers of certain groups are degraded slower than

the others. Group IV and Group V isomers, isomer 12, 13, 16, and 18, and three of the

eleven isomers in Group I have consistently slower biodegradation rates than isomers

belonging to the other groups, as shown in Figure 4-4. Isomer 2, a Group I isomer,

showed little biodegradation over the course of the experiment. The isomers in Group II,

Group III, and the majority of isomers in Group I have the fastest rates of biodegradation.

The branching pattern of isomers of Group IV and V may be more difficult to degrade.

Alkyl-branched alkanes have been shown to be less susceptible to biodegradation and

- 39 – certain branching patterns of alkanes may confer resistance to biological attack

(Alexander 1973), (Pirnik 1977). Steric inhibition of oxidizing enzymes caused by alkyl branching located near the terminal end of an alkyl chain could result in decreased biodegradability (Schaeffer, Cantwell et al. 1979). In the case of NP, some isomers may be more sterically hindered than others, which may explain the varying rates of isomer biodegradation.

Figure 4-6. Biodegradation rates for each isomer normalized to the initial total biomass for that condition. Solid bars are condition 1, hatched bars are condition 2, and open bars are condition 3.

The profile of the biodegradation rates is comparable across conditions as shown in Figure 4-6 where the isomer degradation rates are normalized to initial biomass. This normalization assumes that the percentage of microorganisms in the microcosms responsible for NP degradation is the same in all conditions. Comparing changes in isomer profile may be useful as a tool to measure the extent of degradation in environmental samples, especially in sediments where NP concentrations may be

- 40 – heterogeneous. A similar method for determining biodegradation in the field has been

used for monitoring remediation of oil spills (Venosa, Suidan et al. 1996), (Mills,

McDonald et al. 1999). The method used for oil spills is to analyze the change in hydrocarbon profile relative to a recalcitrant compound within the oil mixture and relate the change to the extent of degradation irrespective of total mass of contaminant in the sample.

Other studies have also shown that NP can be degraded aerobically (Ekelund,

Granmo et al. 1993), (Tanghe, Devriese et al. 1998), (Staples, Williams et al. 1999).

However, WWTPs are not achieving complete removal of nonylphenolic compounds

(Naylor 1995) and significant quantities of NP are still being found in the environment

(Kolpin, Furlong et al. 2002). Comparing the work presented in this study to the work of others implies that even partial degradation of NP has the potential to significantly reduce

the overall estrogenic activity of the remaining NP. Kim et al. (2004) fractionated NP

isomers by high performance liquid chromatography and tested the estrogenic activities

of some NP isomers relative to 17β-estradiol (Kim, Katase et al. 2004). In Figure 4-7,

the estrogenic activity of nine isomers as determined by Kim et al. (2004) is compared

with the average, biomass normalized, biodegradation rates of the isomers determined in

this study. The four most estrogenic isomers from the work of Kim et al. (2004) are

Group I and Group II isomers (corresponding to isomer 9, 23, 20, and 22, in order of

decreasing estrogenic activity). Isomer 9 is one of the most rapidly degraded isomers,

and isomers 23, 20, and 22 are also degraded relatively quickly.

According to the work of Routledge et al. (1997), a single tertiary branched alkyl

group on an otherwise unhindered phenol ring is optimal for estrogenic activity. Even

- 41 – though Kim et al. (2004) did not examine any isomers from Group III or IV, it is most

likely that they possess less estrogenic activity than isomers from the other groups. The

research presented in this study compared with the work of Kim et al. (2004) and

Routledge et al. (1997) supports that the NP isomers with the highest estrogenic activity

are the isomers that are biodegraded the fastest under the conditions of this study.

Therefore, the estrogenic activity of the NP remaining after partial degradation may be

lower than that of the non-degraded isomer mixture.

Figure 4-7. Comparison between the average of normalized biodegradation rate (open bars) and the estrogenic activity (black bars) of the isomers relative to 17 β-estradiol (E2). Error bars are 95% confidence interval of the averaged rate. * - Values for the estrogenic activity of the NP isomers were taken from the work of Kim et al. (2004).

- 42 –

This work provides a possible tool for monitoring the occurrence and extent of biodegradation of NP in environmental samples. The isomer rate profile described here may not be indicative of every biological population degrading NP in the environment.

However, the three sources used for this study did show similar patterns, and some shift in the isomer profile should be evident irregardless of the microbial population. More research is needed to determine if this trend occurs in the more complex conditions of the environment such as various redox conditions, microbial population, nutrient loadings, and temperatures.

Future work is needed to explain the relationship between biodegradability, estrogenic activity, and isomer structure. With greater understanding of the isomers it may be possible to develop a technical mixture of NP that is more readily biodegraded.

There also appears to be potential for partial biodegradation to result in a significant decrease in estrogenic activity and this will be examined further. One study has recently shown that NP can be degraded under various anaerobic conditions by microorganisms enriched from river sediment (Chang, Yu et al. 2004). Addition studies will be conducted to examine the relative biodegradability of NP isomers under anaerobic conditions.

43 5. Biodegradation of NP Using Anaerobic Respirometers

5.1. Abstract The fate of nonylphenol (NP) was investigated under three anaerobic conditions using respirometers. Quadruplicate respirometers were maintained under nitrate reducing, sulfate reducing, and methanogenic conditions. Samples taken from local wastewater treatment plants (WWTPs) were used as inoculums. The volume and rate of metabolite gas production by each respirometer was recorded for the duration of the experiment. Samples were taken for NP and total biomass analysis on days 0, 7, 14, 21,

28, 35, 42, 49, 56, 63, 98, and 207. Metabolite gas production and total biomass data indicated that the cultures were active for the duration of the experiment. NP levels were constant and there was no shift in the isomer profile of NP indicating that no degradation occurred under these anaerobic conditions.

5.2. Introduction Alkylphenol ethoxylates, 80% of which are nonylphenol ethoxylates (NPEs), have seen wide spread use as a non-ionic surfactant. By the nature of their use, NPEs are commonly discharges to wastewater treatment plants (WWTPs) where they are biotransformed. The biotransformation of NPEs results in byproducts with shortened ethoxylate chain, nonylphenoxy carboxylic acid, and nonylphenol (NP). NP has frequently been shown to be the most persistent of the biotransformation products

(Maguire 1999), (Ying, Williams et al. 2002).

The fate of NP has been given increased attention in recent years, since NP has been shown to bind to estrogen receptors (Jobling and Sumpter 1993), due to strong evidence that their metabolites are persistent in the environment (Ying, Williams et al.

44 2002), and that NP, at high enough concentrations, can illicit a biological response

similar to 17β-estradiol (White, Jobling et al. 1994). NP has also been reported to be

more toxic and orders of magnitude more estrogenically active than the surfactant

product (Scott and Jones 2000).

WWTPs typically do not achieve complete mineralization of nonylphenolic

compounds, but only achieve primary biodegradation (Maguire 1999). NP has a

relatively high octanol-water partition coefficient (log Kow) of 4.48 and a low solubility in water, 5.4 mg/L (Ahel and Giger 1993; Ahel and Giger 1993), therefore the majority of the remaining NP partitions to the organic solids in the sludge. Concentrations of NP in the digester sludge are typically high. NP levels in anaerobic sludge from New York were reported to be as high as 1840 mg/kg on a dry weight basis (Pryor, Hay et al. 2002).

However, due to the large volume of water treated by WWTPs, a significant mass of NP may be discharged over time in the aqueous effluent. After entering the receiving water body, the relatively high hydrophobicity of NP will cause NP to partition to the organic sediment (Ying, Williams et al. 2002). The discharged NP can accumulate in the sediment, and concentrations in sediments near WWTP outfalls have been observed at levels from 10 mg/kg (Bennie 1999) to 40 mg/kg (Ferguson, Bopp et al. 2003). The half life of NP in sediments has been estimated through the use of dated cores to be in the range of 20 to 60 yrs (Ferguson, Bopp et al. 2003), (Shang, MacDonald et al. 1999).

Few studies have examined the fate of NP under anaerobic conditions. Under methanogenic conditions, NP1E and NP2E were degraded to NP, but the NP was not shown to be further degraded (Ejlertsson, Nilsson et al. 1999). Degradation studies of NP in initially anaerobic, sewage sludge aggregates exposed to air showed that complete

45 mineralization of NP was possible, but that degradation was closely correlated to oxygen penetration into the aggregates (Hesselsoe, Jensen et al. 2001). Hesselsoe et al. (2001) suggested from their findings that the majority of the NP degradation took place in the aerobic region of the aggregate.

A recent study conducted with microcosms and using cultures derived from river sediment showed that anaerobic biodegradation of NP is possible. After one year of enrichment and acclimation and at a temperature of 30°C, halflives for NP of 46.2 to 69.3 days were reported (Chang, Yu et al. 2004). However, multiple studies have shown that

NP is persistent in anaerobic sediments found in the environment (Ferguson, Iden et al.

2001), (Shang, MacDonald et al. 1999), (Marcomini, Pojana et al. 2000), (Isobe,

Nishiyama et al. 2001). Most sediment is anoxic and the majority of NP is expected to partition to the sediment, therefore to understand the fate of NP in the environment, more knowledge is needed concerning the anaerobic biodegradation of NP.

Though biodegradation of NP has been shown to be possible with an acclimated culture under anoxic conditions, the objective of this study was to examine the fate of NP under conditions found in an anaerobic digester and in sediments. Non-acclimated sludge from an anaerobic digester was used as the inocula. Anaerobic digesters are typically operated using methanogenic conditions and sediments can be found with nearly any redox conditions. This study was conducted using nitrate reducing, sulfate reducing, and methanogenic conditions. More knowledge is needed about the fate of NP under these conditions.

46 5.3. Materials and Methods Anaerobic Respirometer Experiments. Anaerobic respirometers were operated

under three redox conditions and one sterile control. The respirometers were placed

under nitrate reducing, sulfate reducing, or methanogenic conditions with the addition of

40 mmol of sodium nitrate for nitrate reducing, 40 mmol of sodium sulfate for sulfate

reducing, and sodium bicarbonate for methanogenisis. All conditions were constructed

identically, except for the addition of electron acceptors. Inocula were collected from the

anaerobic digester of a WWTP receiving a mixture of industrial and domestic effluent. A

bioslurry was prepared for each condition using 25% vol/vol inoculum source in a basic

minimal media as described by Selton et al. (1984). The redox indicating dye, resazurin

was added at a concentration of 5 mM to indicate whether anoxic conditions were

maintained. A quadruplicate set of sterile controls with no added electron acceptor were

prepared by autoclaving twice at 121°C for one hour and 5*10-4 M 2-bromoethane- sulfonic acid was added to inhibit methanogenisis (Oremland and Capone 1988).

NP is a viscous liquid and was dissolved in pentane to ensure consistent dosing to the respirometer bottles. NP in pentane was first added to sterile microcosm vials to yield

10 mg/L of NP in the final volume of the respirometer. The respirometers were loosely capped and the pentane was allowed to evaporate. The respirometers were inoculated and assembled in an anaerobic chamber and were connected to a respirometer unit (AER-

200 Respirometer, CES Inc., Fayetteville, AR) to continuously record gas production volumes. The respirometer unit counted metabolite gas bubbles of a known volume passing through a u-shaped tube. The respirometers were magnetically stirred at 200 rpm and maintain at a temperature of 22°C +/- 2°C. The respirometers were operated in quadruplicate and were sampled and analyzed at times 0, 7, 14, 21, 28, 35, 42, 49, 56, 63,

47 98, and 207 days. As gas production leveled off in the nitrate and sulfate reducing

conditions, additional electron acceptor of either 20 mmol of sodium nitrate (Fisher

Scientific, BP360, Hanover Park, IL) or anhydrous sodium sulfate (Fisher Scientific,

S429, Hanover Park, IL) was added as appropriate to the nitrate and sulfate reducing

conditions.

Chemicals. The nonylphenol was a technical grade mixture of isomers (Sigma

Aldrich, 29,085-8, St. Louis, MO). The surrogate standard was 2, 4, 6 tribromophenol

(Supelco, 47960-U, Bellefonte, PA). The internal standard was acenapthene-d10

(Accustandard, Z-014J, New Haven, CT). All solvents were pesticide grade (Fisher

Scientific, Hanover Park, IL). Anhydrous sodium sulfate columns were constructed

using large volume, disposable pipettes, conditioned glass wool, and approximately 2 g

of anhydrous sodium sulfate (Fisher Scientific, S429, Hanover Park, IL). Columns were pre-dried in a 105°C oven for 12 hrs before use.

Chemical Analysis. All samples were extracted and analyzed using the methods developed by Bertin et al. (Submitted), but are summarized as follows. The samples were extracted by a single-phase extraction technique (Dobbs and Findlay 1993), (EPA

2004). A single-phase extraction technique allowed the extract to be used for both NP analysis and for determination of the Total Biomass in the sample. The solvent phase was dried by passing it through an anhydrous sodium sulfate column before analysis.

Extracts were then split for total biomass analysis and for NP analysis. Method blanks consisted of distilled, de-ionized water that were processed through the entire sampling and analytical procedure.

48 NP analysis was performed on an Agilent 6890 GC (Palo Alto, CA) equipped with a 60 meter, MDN-5S capillary column (Supelco, 2-4392, Bellefonte, PA), an autosampler, and an Agilent 5971 Mass Selective Detector. The carrier gas was UHP helium at a constant flow rate of 1 mL/min. Samples were injected in splitless mode.

The oven temperature was initially set at 50°C for 1 min, increased to 100°C at a rate of

20°C/min, increased to 175°C at a rate of 1.5°C/min, increased to 315°C at a rate of

20°C/min and held for 6 minutes. Internal standard (acenapthene-d10) was added to samples at a concentration of 25 mg/L prior to injection. NP calibration range was 1 mg/L to 50 mg/L of NP with a minimum detection limit of 0.5 mg/L (EPA 1998).

Surrogate recoveries of 2, 4, 6 tribromophenol were 75 - 90.0%.

NP was differentiated into 23 separate isomer peaks, as described by Bertin et al.

(submitted) by examining the chromatograms for the ions characteristic of NP. A novel technique was employed to quantitate the separate isomers, since standards do not exist for individual NP isomers. A calibration curve was created by assigning each peak the value of the total NP in the calibration standard. For example, a standard made with 10 mg of technical grade NP in one liter of dichloromethane was analyzed and the response for each peak was correlated to the value of 10 mg/L. A calibration curve was developed for each isomer peak using this method. Total NP was determined by averaging the value of each of the isomers.

Biological Analysis. Total phospholipids biomass was determined according to

Dobbs (1993) as a measure of viable biomass concentration initially present in the microcosms (nmol phospholipids/mL). Through extraction of the phospholipid fatty acid

49 content of an environmental sample, the total biomass can be determined, and an

approximation of the cell concentration can be made.

5.4. Results and Discussion The background level of NP in the inoculum was significant (approximately 33%)

compared to the initial concentration of NP added to the respirometers. Therefore, the

background level of NP was taken into account when calculating NP degradation. Since

the inocula were obtained from a WWTP receiving industrial effluents containing NPEs,

a high concentration of NP in the anaerobic digester sludge was expected. Gas

production data (Figure 6-1) showed that all active cultures were viable. Figure 5-1 shows the first 40 days of the experiment to illustrate the increase of gas production after addition of electron acceptor and the reduction of gas production as electron acceptor is depleted. Under nitrate and sulfate reducing conditions, gas production began to level off within four days of starting the experiment and quickly resumed after additional electron acceptor was added to the respirometer on day 10. Additional electron acceptor was added to a concentration of 20 mM to the nitrate and sulfate reducing conditions weekly for the first four weeks and every two weeks thereafter until the tenth week, resulting in continued metabolite gas production.

Figure 5-1. Average metabolite gas production of the quadruplicates for the first 40 days of the experiment. The sharp increases in production at 11, 18, and 25 days correspond to the addition of electron accepter.

To confirm that that anoxic conditions were maintained, the redox indicating dye,

resazurin, was added to the respirometers. Resazurin will change from colorless under

anoxic conditions to pink when exposed to oxygen. By visual examination, all of the

respirometers maintained anoxic conditions for the duration of the experiment. This

observation combined with the continuous production of metabolite gases implies that the

microorganisms in the respirometers were active under anaerobic conditions over the course of the experiment.

Total biomass was analyzed at every sampling event. Figure 5-2 shows the concentration of total phospholipids in the respirometers for the length of the experiment.

The biomass in the respirometers diminished over time as expected, because the cultures were not feed after the start of the experiment. Therefore it can be implied that the microorganisms had removed all of the easily degraded carbon sources and the surviving organisms were those able to degrade the remaining and more difficult carbon sources.

Figure 5-2. Total biomass numbers are shown here for every sampling event. Error bars depict 95% confidence intervals for the four replicate respirometers at each timepoint.

Even though the microorganisms present in the respirometers were active over the course of the experiment, there was no removal of NP based on statistical interpretation of total NP data. Figure 5-3 shows comparisons of isomer response ratios taken from the chromatogram peaks at 7, 68, and 207 days. The response for each isomer peak in Figure

6-3 was normalized to the response for isomer 1. It has been reported that a significant shift in the isomer profile will occur after biodegradation of NP under aerobic conditions and that a change in the isomer profile can indicate biodegradation (Hawrelak, Bennett et al. 1999), (Bertin submitted). It is possible that NPEs in the inocula were being metabolized to NP which obscured any biodegradation of NP. However, neither the active conditions nor the killed control showed loss of NP or a significant difference in the isomer profile indicating that no biodegradation of NP occurred under these conditions. This result supports the work of Ejlertsson et al. (1999), who reported that

NPE2 and NPE1 were degraded under anaerobic (methanogenic) conditions forming NP, but determined that NP was not further degraded under this condition.

Figure 5-3. Isomer profile of NP based on chromatographic response. All isomers are normalized to the response of isomer 1. Time of 7 days is represented by the black bars, 68 days by the open bars, and 207 days by the hatched bars.

No studies have been found to report NP degradation rates or removal efficiencies for anaerobic digesters. However, NP concentrations in WWTP sludge have been reported by various studies ranging from 8.4 to 4000 mg/kg on a dry weight basis

(Waldock and Thain 1986), (Bennie 1999) suggesting that removal efficiencies may be low. Since the study presented used anaerobic digester sludge as an inoculum source, the results suggest that the removal efficiency in digesters may be low.

The respirometers were also operated under nitrate and sulfate reducing conditions to examine the effect of these conditions on NP biodegradation. It is possible for microorganisms in sediments to also be exposed to these redox conditions in addition to methanogenic. Although, Chang et al. (2004) recently reported that highly enriched cultures derived from river sediments were able to achieve degradation of NP under anaerobic conditions after one year of acclimation to NP. In their study, NP degradation was studied under nitrate reducing, sulfate reducing, and methanogenic conditions with the former showing an inhibited rate and the second showing the fastest biodegradation rate (Chang, Yu et al. 2004). Even though the work of Chang et al. (2004) showed that anaerobic degradation of NP is possible, it has been reported that NP is persistent in the environment, especially in anoxic conditions (Maguire 1999), (Ying, Williams et al.

2002). One reason Chang et al. (2004) achieved biodegradation of NP and this study did not, could be that biodegradation of NP is not favored until the more easily degraded compounds are removed. In sediments, and especially in WWTPs, there is always an excess of easily degraded carbon sources available for microorganisms which may explain the long observed halflives.

54 Biodegradation of NP has been reported under laboratory conditions, but NP has

also been reported to be persistent in the environment. Therefore, additional work needs to be conducted both in laboratories and in the field to determine if anaerobic degradation of NP occurs in WWTPs and in sediments. If the biodegradation of NP does occur in

WWTPs and in sediments, more research is needed to examine the individual isomers of

NP and determine if some isomers are more susceptible to biodegradation than others.

55 6. Summary, Conclusions, and Future Work

6.1. Summary Studies were conducted using microcosms and respirometers to investigate the biodegradation behavior of NP and its fate in the environment. Previous studies have shown that NP can be readily biodegraded under laboratory conditions (Staples, Williams et al. 1999), (Chang, Yu et al. 2004). However, it has often been shown that NP is persistent in the environment (Shang, MacDonald et al. 1999), (Marcomini, Pojana et al.

2000), (Isobe, Nishiyama et al. 2001), (Schroder 2001), (Ferguson, Bopp et al. 2003). In addition, one study has shown that when biodegradation does occur there may be more recalcitrant isomers (Hawrelak, Bennett et al. 1999).

Aerobic respirometers were used to enrich a culture on a sole carbon source of

NP. Samples taken from a WWTP’s secondary (aerobic) treatment and anaerobic digester sludge were used as the inocula for this study. After four generations of enrichment, a culture free of extraneous carbon sources was developed. The enriched culture was able to achieve 95% biodegradation of NP at a concentration of 1000 mg/L in

40 days. This study showed that NP can be biodegraded and used by microorganisms as a sole carbon and energy source. However, under the conditions of this study, it was determined that the maximum theoretical cell yield for an NP degrading culture is relatively low. The low yield of NP degraders means that few cells will be produced by a culture degrading NP, and the likelihood of these microorganisms being out competed in other matrixes is high. Since the secondary treatment of most WWTPs have a retention time of only a few hours, any microorganisms that do not reproduce fast enough may be washed out of the system.

56 The enrichment study also showed qualitative evidence of recalcitrant isomers, supporting the findings of Hawrelak et al (1999). Comparisons of the chromatograms of the technical mixture of NP isomers to the remaining NP in the enrichment culture after

25 days showed that six isomers peaks were being biodegraded at a slower rate. This evidence prompted a further investigation into the degradation rate of the individual NP isomers.

A study using aerobic microcosms was conducted to determine the degradation rates of NP and the individual NP isomers by non-acclimated cultures taken from

WWTPs. The technical mixture of NP was differentiated into 23 isomers by GC/MS, and the biodegradation rates for each isomer were estimated. Through the use of the statistical method, Beta-Hat, the biodegradation rates of a number of the individual NP isomers were found to be statistically different. Knowledge of the varying rates of isomer biodegradation can be used as a tool to measure biodegradation in the environment. If the isomer profile of the source of NP to the environment is known, then the amount of biodegradation can be measured by monitoring shifts in the isomer profile.

A similar technique has been used successfully for monitoring the remediation of oil spills (Venosa, Suidan et al. 1996), (Mills, McDonald et al. 1999).

Comparing the NP isomer structures proposed by Wheeler et al. (1997) and

Thiele et al. (2004) (Wheeler, Heim et al. 1997), (Thiele, Heinke et al. 2004) to the estimated biodegradation rates of the isomers showed that the basic structure effects the biodegradability of the isomers. Making a second comparison between the estrogenic activity of the isomers determined by Kim et al. (2004) (Kim, Katase et al. 2004) and the isomer biodegradation rates demonstrates that the isomers with the highest estrogenic

57 activity are the most susceptible to biodegradation. Therefore the residual material may be less estrogenic than the source material. Alternatively, with increased understanding of the biodegradation rates of the isomers under other conditions, it may be possible to develop a technical mixture of isomers that is more readily biodegraded.

The final study was conducted with anaerobic respirometers. Samples taken from the anaerobic digesters of a local WWTP were diluted with microbiological media and

NP was added to a concentration of 10 mg/L. The respirometers were operated under one of three redox conditions (nitrate reducing, sulfate reducing, or methanogenic). Gas production was monitored and all the samples were analyzed for total biomass concentration throughout the experiment. Based on the gas production numbers, the cultures were viable. However, no biodegradation or change in the isomer profile was observed during the 207 days of the experiment.

Although Chang et al. (2004) was able to achieve biodegradation of NP using a culture that was enriched and acclimated for one year (Chang, Yu et al. 2004), the rest of the literature suggests that if biodegradation does occur in anaerobic conditions found in

WWTPs and sediments that it is very slow. The halflife of NP in sediments has been calculated by examining dated sediment cores to range from 20 years (Ferguson, Bopp et al. 2003) to 60 years (Shang, MacDonald et al. 1999). The respirometers were operated at various redox conditions to mimic those found in the environment. The inoculum used in this study was not acclimated, therefore the methanogenic respirometers should closely resemble the anaerobic digester of a WWTP. Sediments in the environment can be found with several possible redox conditions. Although the cultures were active under these conditions, again, no biodegradation was observed.

58 Due to the high Kow of NP (Ahel and Giger 1993; Ahel and Giger 1993), the

majority of non-degraded NP in a WWTP is expected to partition to the sludge and be

sent to the anaerobic digester. Based on the results of this study it is likely that the

removal efficiencies of the anaerobic digesters in WWTPs are low.

6.2. Conclusions The result of these studies shows that biodegradation of NP occurs aerobically

and was not shown to occur anaerobically with non-acclimated cultures. However, when

biodegradation does occur, some NP isomers are more recalcitrant. The structural

features that lead to higher biodegradability appear to be the same features that have a

high potential for estrogenic activity.

Due to its high octanol-water partitioning coefficient, the majority of NP in the

environment is expected to be found in sediments. Aquatic sediments are typically

anaerobic and NP has been shown to be more persistent under anoxic conditions.

Therefore biodegradation of NP in the environment is expected to be very slow. This

conclusion is backed by the high concentrations and the ubiquitous nature of NP found in

streams and rivers.

Since some NP isomers have been shown to biodegrade faster than others, it may

be possible to develop a mixture of NP isomers that is more easily biodegraded.

Currently, ethoxylated NPs are commonly used as a surfactant because they are

significantly less expensive to manufacture and have more desirable properties than other

choices. There are 46 theoretically possible NP isomers (Thiele, Heinke et al. 2004) and the technical mixture only contained 23 differentiated isomers. It may be economically feasible to produce an isomer mixture that is more susceptible to rapid biodegradation.

59 6.3. Future Work This research adds to the limited knowledge about the biodegradation and the

environmental fate of NP. However, the knowledge gained here also raises more

questions about nonylphenol. Knowledge about the fate of NP in WWTPs and sediments

would improve greatly by research into several additional topics.

The major difficultly in the research of NP and its isomers is the lack of analytical

standards for the individual isomers. The development of individual NP isomer standards

would significantly aid the research effort into the biodegradation, environmental fate,

and estrogenic activity of the NP isomers. Standards for each isomers of NP would allow

for more accurate quantification of NP. They would allow for more detailed

understanding of the various rates of biodegradation for the different isomers. A more

detailed understanding of the estrogenic activity of NP could be developed. With the

development of individual standards, studies could be conducted to gain more knowledge

about the specific structures that are associated with the highest estrogenic activity, and

those structures that are most susceptible to biodegradation.

Although aerobic biodegradation of NP is possible, complete mineralization is

unlikely to occur during the residence time of the secondary treatment in a WWTP. Even

though the majority of NP partitions to the biosolids and is sent to the anaerobic digester,

a significant concentration of NP is detected in the sediments downstream of the WWTP

effluent. Using cultures derived from the anaerobic digester of WWTPs, no

biodegradation of NP occurred under anaerobic conditions after 207 days. Since a

different consortium of microorganisms is expected in sediments, additional work on

anaerobic biodegradation should be conducted using cultures derived from anoxic sediments. The long half-lives for NP observed through the use of dated sediment cores

60 suggests that biodegradation of NP in anoxic sediments will be slow (Shang, MacDonald

et al. 1999), (Ferguson, Bopp et al. 2003). The development of anaerobic enrichment

cultures would be valuable. Chang et al. (2004) showed that it is possible to enrich a

culture capable of biodegrading NP. However, no examination of the individual NP

isomers was made. Future work should conducted to develop an enriched culture of NP

degrading microorganisms and determine if the isomers of NP are biodegraded at various

rates as was seen under aerobic conditions.

This research has shown that some NP isomers are more recalcitrant than others.

Kim et al. (2004) began examining the estrogenic activity of nonylphenol isomers by

isolating nine isolating and assaying them for estrogenic activity. Comparison of these

studies implies that the same structural features that lead to faster rates of biodegradation

are also responsible for increased estrogenic activity. Additional work is needed to

further examine the relationships between isomer structure, estrogenic activity, and

biodegradation rates.

Since the major concern with NP in the environment is its estrogenic activity,

determining if the net estrogenic activity of the mixture of NP isomers after partial

biodegradation would be valuable. A study could be conducted where NP is extracted

after various degrees of biodegradation. The extracted NP could then be assayed for estrogenic activity and any net change in estrogenic activity could be determined.

The major concern with NP in the environment is due to its recalcitrant nature and its high estrogenic activity. Since WWTPs are designed to treat extremely large volumes of water containing an enormous number compounds and contaminates from industrial and domestic wastes, it is not feasible to redesign there operation to treat a single class of

61 compounds. Therefore, it would be best to conduct research to help increase the understanding of the fate of NP in WWTPs and the environment and better understand its effects on humans and wildlife.

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70 8. Appendix 1: Analysis of Nonylphenol and Octylphenol in Bioslurry and Sediment by Full Scan Gas Chromatography/Mass Spectrometry (GC/MS)

8.1. Scope and Application 8.1.1. This procedure describes an extraction method for use with aqueous and sediment samples to allow for analysis of nonylphenol and octylphenol. The extraction procedure is relatively simple and does not require costly equipment. The use of gas chromatography equipped with a mass spectrometer (GC/MS) makes this a very sensitive measurement procedure. These compounds or isomers are qualitatively and quantitatively determined by this method.

8.1.2. The method detection limit (MDL) and the reporting limit (RL) for these compounds are listed in Table 1. This standard operating procedure (SOP) has been tested on bioslurry and sediment samples.

8.1.3. This method utilizes an 8.0 ml aqueous bioslurry or sediment sample with a 12 h extraction period. The alkylphenol molecule can be detected on the GC/MS and the concentration measured.

8.2. Summary of Method 8.2.1. For NP and OP single phase solvent extraction technique (SPSE) is used for bioslurry and soil samples.

8.2.2. Single Phase Solvent Extraction (SPSE) Technique - A measured amount of bioslurry or sediment is mixed with methanol and methylene chloride to form a single phase, in which NP and OP are miscible. The extraction samples are shaken for 12 hours. Methylene chloride and water are added and the mixture is broken into two phases. The bottom, organic phase, is removed as the extract. This extract is dried using sodium sulfate and then analyzed by GC/MS.

8.2.3. The target compounds are identified by comparing the sample mass spectra to a known standard. The target compounds are quantitated using the quantitation ions of the target compounds utilizing the internal standards that are associated with the normal ABN analysis. The internal standards used are acenaphthene-d10 and phenanthrene-d10. The final report issued for each sample lists total concentration of NP and OP if detected, or RLs if not detected, in µg/ml for bioslurry and µg/Kg for sediment samples.

8.2.4. Any sample that produces analytical numbers greater than the linear dynamic range of the standard curve for each congener will require dilution. No concentration of sample extracts will be done and the analysis of these analytes will be flagged and labeled below the linear dynamic range.

71 8.2.5. Nonylphenol and octylphenol concentrations will be determined according to the following equation:

CgMS(/µ ml)× Vsolvent(ml) Cgi(/µ ml)= Vmsample()l

where, Ci = concentration of NP or OP in sample (µg/ml) CMS (µg/ml) = concentration of NP or OP measured by a calibrated GC/MS

8.2.6. Preservation of the extracts will be done by sealing in screw-capped vials, PTFE-lined caps and refrigerated at 4°C. Vial weights will be recorded before storage and checked before analysis. Weight loss of the extraction solvent will be made up by adding solvent. Holding time will be less than 40 days, but analysis will be done as soon as possible.

8.3. Interferences and Potential Problems 8.3.1. Method interferences may be caused by contaminants in solvents, reagents, glassware, and other apparatus that lead to discrete artifacts or elevated baseline in the selected ion current profiles. All of these materials are routinely demonstrated to be free from interferences by analyzing laboratory reagent blanks under the same conditions as the samples.

8.3.2. All glassware is scrupulously cleaned. All glassware is detergent washed in hot water and rinsed with distilled water. The glassware is then dried, and heated in an oven at 250°C for 15 to 30 minutes. All glassware is subsequently cleaned with methylene chloride. Detergents containing alkylphenolic compounds must not be used.

8.3.3. All reagents and solvents should be of high purity to minimize interference problems.

8.3.4. Matrix interferences may be caused by contaminants that are co-extracted from the sample. The extent of matrix interferences will vary considerably from sample source to sample source, depending upon variations of the sample matrix.

8.3.5. High concentrations (low biodegradation) of added alkylphenol will require running samples initially undiluted to determine method/extraction efficiency (recovery of surrogate), followed by dilution to obtain measurements of the alkylphenol in the linear range.

8.4. Safety and Waste Management 8.4.1. The toxicity and carcinogenicity of each reagent used in this method has not been precisely defined; however, each chemical compound is treated as a

72 health hazard. From this viewpoint, exposure to these chemicals is reduced to the lowest possible level.

8.4.2. All flammable solvents will be stored in AWBERC Rm. 709 combustible cabinet until needed for use. Any spill of alkylphenol from sample extracts will be of relatively low concentration but proper gloves should be worn during cleanup. Standard solutions contain a much higher concentration of alkylphenol and extra caution should be executed during any spill. The spill should be cleaned up using either methylene chloride or methanol and paper towels. Hands should be protected with a pair of nitrile gloves under a pair of “Silver Shield” solvent resistant gloves. All waste produced during cleanup should be placed in the hood to allow solvent to evaporate, and then waste placed in appropriate containers.

8.4.3. Methylene Chloride (CAS #75-09-2) is a liquid with a sharp penetrating and non-residual odor. Users should avoid heat, sparks and open flames. Routes of entry include inhalation and ingestion. If acetone enters eyes, they should be flushed with water for at least 15 min. If swallowed, vomiting should be induced if possible. Inhalation should be avoided by use in a hood, but if inhaled, move to fresh air.

8.4.4. Methanol (CAS #67-56-1) is a colorless flammable liquid with a mild gasoline like odor. Users should avoid high temperatures, sparks, open flames and static charge. Routes of entry are by inhalation, skin and ingestion. Overexposure symptoms include nausea, headache and dizziness. If swallowed, do not induce vomitting and keep head below hips. Contact with eyes should be followed by flushing with water for at least 15 min.

8.4.5. Pentane (CAS #109-66-0) is a colorless flammable liquid with a mild gasoline like odor. Users should avoid high temperatures, sparks, open flames and static charge. Routes of entry are by inhalation, skin and ingestion. Overexposure symptoms include nausea, headache and dizziness. If swallowed, do not induce vomiting and keep head below hips. Contact with eyes should be followed by flushing with water for at least 15 min.

8.4.6. Nonylphenol (CAS #25154-52-3) is a clear, odorless, and highly viscous fluid. Routes of entry include ingestion and skin contact.

8.4.7. Octylphenol (CAS #27193-28-8) is an odorless, white, and fluffy crystalline solid. Routes of entry include ingestion and skin contact.

8.4.8. Nonylphenol and octylphenol are solubilized in methylene chloride or methanol depending on how they are to be used. Nonylphenol and octylphenol standard solutions (see Section 8.1.1) are made in methylene chloride.

8.4.9. The GC/MS operates at high temperatures. Extreme caution should be observed when operating the instruments or handling instrument

73 components.

8.4.10. Safety equipment that should be worn at all times includes lab coats, safety glasses and gloves for hand protection. For further information, see Health and Safety Plan in Rm. 424/426.

8.4.11. All waste (both liquids and solids) will be handled in the proper format as described in Section 12.

8.5. Equipment, Reagents and Supplies 8.5.1. Equipment and Supplies

8.5.1.1. Grab sample bottle - amber glass, fitted with teflon lined screw cap. If amber bottles are not available, the samples should be protected from light. Soil samples are collected in amber jars.

8.5.2. Glassware and Miscellaneous Supplies

8.5.2.1. Vials - 60-mL amber glass vials, 20 ml amber glass vials, 12 ml amber glass vials, and 2-mL amber glass, autosampler vials with teflon- lined caps.

8.5.2.2. Syringes - 0.2 mL, 0.5 mL, 1.0 mL and 5.0 mL (±5%).

8.5.2.3. Micro-syringes - 100 µL, 50 µL, 25 µL and 10 µL (±5%).

8.5.2.4. Pipetters: - 50.0-250.0 µl SMI Gravimetric capillary pipetter and glass capillaries and5.0-30.0 µl SMI Gravimetric capillary pipetter (VWR, #53499-458) and capillaries (VWR, #53499-410)

8.5.2.5. Analytical balance accurate to 0.1 mg; reference weights traceable to Class S or S-1 weights.

8.5.2.6. pH meter - Calibrate according to manufacturer's instructions.

8.5.2.7. NIST Thermometer or one meeting the requirements of NIST to calibrate laboratory thermometers.

8.5.2.8. Class A volumetric glassware.

8.5.2.9. Na2SO4 columns - pack small amount of glass wool into a clean, large volume Pasteur pipette (Fisher #XXX). Add approximately 1.5g Na2SO4 that has been heated to 105°C for 24 hours and subsequently stored in a desiccator. Na2SO4 columns are prepared before the day that the extraction procedure is conducted and stored in a desiccator

8.5.3. Reagents

74 8.5.3.1. When compound purity is assayed to be 99.9% or greater, the weight may be used without correction to calculate the concentration of the stock standard. Calibrate an analytical balance using reference weights (Class S or S-1) daily. All weights and concentrations listed in this SOP are corrected to at least 99.9% purity. Example: A weight of 0.511 g of compound X that is assayed to be 98% pure is recorded as 0.501 g of compound X after correction.

8.5.3.2. Solvents - methanol, and methylene chloride - Pesticide quality or equivalent.

8.5.3.3. ABN Custom Mix (2,4,6 - tribromophenol, 2- fluorobiphenyl, 2- fluorophenol, phenol- d6, nitrobenzene- d5 and p- terphenyl- d14 - surrogates; Source: Supelco Inc.; Custom Mix Lot #: LA 72649).

8.5.3.4. Nonylphenol (NP-Target isomer mix; Source: Aldrich Chemical Company; Purity: 90%, Cat. #: 29,085-8).

8.5.3.5. 4-(tert-Octyl)phenol (OP- Target compound; Source: Aldrich Chemical Company; Purity: 97%, Cat. #: 29,082-3).

8.5.3.6. ABN Internal Standard Mix (acenaphthene-d10, chrysene-d12, 1,4- dichlorobenzene- d4, naphthalene- d8, perylene- d12, phenanthrene- d10- internal standards; Source: Accustandard, Inc.; Cat. # Z-014J, Lot # B0040156).

8.5.3.7. Reagent water - Unless otherwise noted, references to water are understood to mean reagent grade water as described in NRMRL- LRPCD-05.SOP.011.00, Reagent Water Quality and Monitoring.

8.5.3.8. Silica sand combusted at 450ºC to remove organic interferents.

8.5.3.9. ACS grade, anhydrous sodium sulfate with no interferents above the RL for all compounds.

8.5.4. GC/MS System

8.5.4.1. Gas Chromatograph (GC) System - An analytical system complete with a temperature programmable gas chromatograph (Hewlett-Packard - Model 6890) and all required accessories including syringes, analytical columns, autosamplers and gases. The injection port must be designed for split/splitless when using the capillary columns.

8.5.4.2. Analytical column - An analytical column (Supelco SPB-5, 30 m length x 0.25 mm ID, and 0.10 µm film thickness or equivalent.) suitable for the analysis of target alkylphenols.

8.5.4.3. Mass Spectrometer (MS) System - A MS system capable of scanning

75 45-500 amu every two seconds or less, using 70 electron volts in the electron impact mode, and producing a mass spectrum which meets all the criteria in Table 7 when 50 ng of decafluorotriphenylphosphine (DFTPP) is injected through the GC inlet.

8.5.4.4. Data Backup Device - A data archival unit to archive data using zip disk/CD media. The laboratory has capabilities to store and retrieve data using other devices such as ZIP100 PlusTM and CD writers.

8.5.4.5. Data System - HP ChemstationTM must be interfaced to the mass spectrometer that allows the continuous acquisition and storage on machine-readable media of all mass spectra obtained throughout the duration of the chromatographic program. The Chemstation has software that allows searching any GC/MS data files for specific mass (m/z) and plotting such m/z abundances versus time or scan number. This type of plot is defined as an Extracted Ion Current Profile (EICP). HP Chemstation software allows integrating the abundances in any EICP between specified time and scan number limits. The software must be configured to flag any result that has been manually integrated and allow the audit trail to be “on”.

8.5.4.6. DFTPP/NP/OP/Surrogates/Internal Standards Ions for Full Scan Data (see Table 2 for primary and secondary ions associated with the target compounds).

8.6. Sample Collection, Preservation and Handling 8.6.1. All sample extracts are stored in a refrigerator at 4°C (± 2°C) or less until requested for analysis. The sample extracts must be analyzed within 40 days of extraction.

8.7. Quality Control 8.7.1. A-Initial demonstration, which include precision and accuracy (P&A) study and MDL:

8.7.1.1.An initial demonstration of the laboratory capability to generate data of acceptable quality is made. A precision and accuracy (P&A) study must be performed whenever a major modification is made to this method.

8.7.1.2.For a precision and accuracy (P&A) study, a check standard containing the NP and OP at the concentration as specified in section 8.2 is prepared. 200 µL of the check standard is added to each of six 30 g clean soil samples. The check samples are then analyzed according to the procedures in Sections 9 and 10.

8.7.1.3.The average percent recovery (X), and the standard deviation (σ) of the recoveries is calculated for each analyte. Establish the QC limits

76 with a confidence interval of 99.7% (3 σ). Equations for standard deviation and average percent recovery are shown in Attachment 1.

8.7.1.4.Method Detection Limit Test: The method detection limit test is performed in accordance with the definition and procedure outlined in 40 CFR Part 136, Appendix B.

8.8. On going QC 8.8.1. Matrix Spike: 8.8.1.1.Spike accuracy for each matrix is monitored and updated regularly. Records are maintained of spiked matrix analyses and the average percent recovery (X)¯ and the standard deviation of the percent recovery (σ) is calculated. This procedure maintains a 99% confidence interval from X±3¯ σ control limits for spike compounds.

8.8.2. Surrogates: 8.8.2.1.All samples are spiked with surrogate standard spiking solution as described in Section 8. An average percent recovery of the surrogate compound and the standard deviation of the percent recovery are calculated. This procedure maintains a 99% confidence interval from X±3¯ σ control limits for surrogate compounds. Each sample will be spiked with surrogates (2,4,6-tribromophenol - section 8.1) to evaluate the method performance. See Table 2.

8.8.3. Internal Standards: 8.8.3.1.Each extract will be spiked with a series of internal standards (acenaphthene-d10 and phenanthrene-d10) to evaluate instrument performance. The calculation expressing the change of the area response of the internal standard and the retention time shift change is shown in Attachment 1 under Internal Standard Calculations.

8.8.4. Reagent Blank: 8.8.4.1.A reagent blank is extracted each day that extractions are performed. Water and silica sand are used for bioslurry and sediment extractions, respectively, every day of extraction to investigate for system/laboratory contamination.

8.9. Calibration 8.9.1.1.All calibration standard preparations should be noted in an appropriate laboratory notebook as maintained by xx.SOP.xxx.xx. All stock solutions are prepared with methylene chloride unless otherwise stated.

8.9.2. Alkylphenols Surrogate Spiking Solution

8.9.2.1.Surrogate standard solution (Solution C - Table 3) consisting of 5000

77 µg/mL of 2,4,6-tribromophenyl, 2-fluorobiphenyl, 2-fluorophenol, phenol-d6, nitrobenzene-d5 and p-terphenyl-d 14 is added to all samples (Tables 3 - 5, Section 8.3). Surrogate compound is added at a concentration of 25 µg/ml of final extract (i. e., 100 µL of a 5000 µg/mL solution is added to 20 ml of final extract).

8.9.2.2.Preparation of Alkylphenols Surrogate Solution - To a 10 mL volumetric flask add exactly 5.00 mL of commercially available custom mix ABN surrogate solution Lot # LA72649 from Supelco that contains 10,000 µg/mL of each of the following in methylene chloride: 2,4,6-tribromophenol, 2-fluorobiphenyl, 2-fluorophenol, phenol-d6, nitrobenzene-d5, p-terphenyl-d14. Note that only 2,4,6-tribromophenol is used to calculate extraction efficiency. The 10 mL volumetric flask is then filled to 10 mL marking with methylene chloride and shaken vigorously. Spike all samples with 100 µL of the 5000 µg/mL alkylphenols surrogate solution. This will result in 25 ng of each surrogate on column per 1 µL injection.

8.9.3. Calibration Standards

8.9.3.1.Calibration stock standard solution is prepared from standard materials or purchased as certified solutions. Stock standard Solution A (10,000 µg/ml NP), Solution B (1000 µg/ml OP), and Solution C (5000 µg/ml ABN Surrogate Standards) are prepared and aliquots of those solutions are diluted to prepare Levels 1 through 7 as shown in Table 3.

8.9.3.2.Prepare stock standard Solution A by adding to a 50 ml volumetric flask, 0.556 g of NP and bringing to a volume of 50 ml with methylene chloride to yield 10,000 µg/ml NP.

8.9.3.3.Prepare stock standard Solution B by adding to a 50 ml volumetric flask, 0.052 g of OP and bringing to a volume of 50 ml with methylene chloride to yield 10,000 µg/ml OP.

8.9.3.4.Preparation of Calibration Standards - Each of the working alkylphenols standard solutions is prepared from the above stock standard solutions A, B, and C (see section 8.1 for preparation of solution C) as shown in Table 3.

8.9.4. DFTPP Standard

8.9.4.1.A 10 µg/ml solution of DFTPP is prepared in a 25 ml volumetric flask by adding 1.0 ml of DFTPP at 250 µg/ml, using a 1 ml syringe, and then bringing the volume to 25.0 ml with methylene chloride. The DFTPP standard is then stored in 12 ml Teflon sealed vial.

8.9.5. Internal Standards

78 8.9.5.1.The internal standards (IS) to be used are acenaphthene-d10 and phenanthrene-d10, although the stock standard also contains 1,4- dichlorobenzene-d4, naphthalene-d8, chrysene-d12 and perylene-d12at the same concentration. 2.5 ml of internal standard stock solution at 4000 µg/ml is diluted to 5 ml in methylene chloride to obtain a working internal standard solution at a concentration of 2000 µg/mL. Spiking 12.5 µl of this solution into a sample extract of 1.0 mL results in an internal standard concentration of 25 µg/ml. The internal standards used for quantitation and compounds they are quantifying are shown in Table 6.

8.9.6. PFTBA System Tuning

8.9.6.1.Before any samples or calibration standards are analyzed, the GC/MS instrument is tuned for the mass spectrum of perfluorotributylamine (PFTBA). Significant changes in any of the analyzer parameters (especially entrance lens, and electron multiplier) must be recorded before analyzing any samples.

8.9.6.2.Copies of the spectrum scan and the profile scan (m/z 69, 219 and 502) are made and filed in the QC binder for that instrument.

8.9.6.3.The acceptance criteria for (PFTBA) is:

Mass 69: 100% Mass 50: 0.3-5% Mass 131: 20-120% Mass 219: 20-120% Mass 414: 0.3-10% Mass 502: 0.3-10%

8.9.7. DFTPP Performance Test

8.9.7.1.Before analyzing any samples, 1 µL of DFTPP standard solution (10.0 µg/ml) is injected and analyzed. A background corrected mass spectra of DFTPP is obtained to confirm that the m/z criteria in Table 7 (Section 13) are achieved. If the criteria are not achieved, the mass spectrometer is re-tuned with PFTBA and the DFTPP test repeated until all criteria have been met. The performance criteria must be achieved before samples, blanks, or standards are analyzed.

8.9.7.2.Copies of the DFTPP system tuning results generated are to be included in the QC data package accompanying the samples. Also, copies of the GC/MS autotune and manual tune reports are generated, when needed, to be included in the Instrument Performance binder.

8.9.8. Quantitation of Alkylphenols

79 8.9.8.1.The quantitation of alkylphenols is accomplished by the comparison of the sample chromatogram to those of alkylphenol standards and a matching of the mass spectrum. The responses of the same isomer groups of the sample chromatogram as the alkylphenol standard must be chosen. Once the pattern has been identified, an equal weighting of each characteristic isomer group peak is assigned. The amount of alkylphenol is calculated using the individual calibration factor for each of the 8 to 12 characteristic peaks chosen and the concentration is determined using each of the characteristic peaks, finally those 8 to 12 concentrations are averaged to determine the concentration of the alkylphenol.

8.9.9. Initial Calibration

8.9.9.1.1. Prepare calibration standards at a minimum of five concentration levels for each parameter of interest. Using injections of 1 µL, calibration standards at the concentrations specified in Table 5 are analyzed and a five to seven point calibration is generated. A report is prepared tabulating the area of the primary characteristic ions (m/z), the concentration for each compound, and the average response factor (RRFs). Relative response factors are calculated for each compound and sample calculations are shown in Attachment 1 under Relative Response Factors

8.9.9.1.2. The % Relative Standard Deviation (RSD) criteria applies to all the compounds listed in Table 1. An acceptable initial calibration should have the method specific parameters RSDs no greater than 35%. Since NP is a mixture of several isomers and since the laboratory reports concentration of total NP based on the sum of 12 isomers, a minimum of 10 isomers should meet this criteria. If the RRF value over the working range is constant (less than 35% RSD), the RRF is assumed invariant and the analysis may begin. If not, re-inject the point(s) that appears to be the cause of the large variation. If the calibration standard does not pass, proceed with column and/or mass spectrometer maintenance, prepare new solutions, and/or replace insert, column, and septum. Then proceed with initial calibration again. If the chromatography continues to be poor change the injection volume to 2 µl and re-evaluate performance of the calibration standards. If performance is acceptable, collect the data being consistent to use 2 µL injections throughout. Table 5 will have to be adjusted taking account the 2 µL injection (all the numbers in Table 5 will be multiplied by 2). The calibration curves must also be adjusted to account for the 2 µL injection. Due to the nature of the isomeric mixtures involved, the chromatography results will be best with new columns, injectors, and a clean source. A sample

80 equation for determination of the RSD is in Attachment 1 under Percent Relative Standard Deviation.

8.9.9.2.The initial calibration package must consist of the following: (i) quantitation reports for the calibration standards; (ii) the calibration report; and (iii) the time-stamped RRF update based on the average response factors. A sample equation for determination of the average response factors is in Attachment 1 under Average Response Factors.

8.9.9.3.If time remains on the day of analysis (that is, less than 24 hours from the time of DFTPP injection) after meeting the initial calibration acceptance criteria, samples may be analyzed. It is necessary to analyze a continuing calibration standard. Always quantify sample results against the average response factor obtained from the initial calibration.

8.9.10. Continuing Calibration Check (CCC)

8.9.10.1.1. Each RRF in the initial calibration curve is verified on each working day and after each set of samples before the expiration of the 24 hour clock by the measurement of a mid-level calibration standard (Level 3 or 4, Table 5, section 8). The CCC QC criterion (%D: < 25%) applies to all the compounds listed in Table 1 except for NP. A minimum of 10 of the 12 NP isomers must meet this criterion. If the response for any of the site specific target parameters varies from the predicted response by less than or equal to 25%, the initial calibration is acceptable. If these criterion fails another CCC must be ran, if this fails a new Initial Calibration curve must be made and the samples re-analyzed. If the CCC criterion is met additional samples can be analyzed for an additional 24 hours, at the end of analysis another CCC must be analyzed and QC criterion must be met for that data to be valid. If the CCC continues to pass, additional samples can be analysed with a CCC processed every 24 hours and at the end of analysis. Finally, quantify sample results against the average response factor obtained from the initial calibration. A sample equation for determination of %D is in Attachment 1 under Percent Difference.

8.9.10.2. The continuing calibration check standard data package must consist of the following: (i) quantitation report for the continuing calibration check standard; (ii) the continuing calibration check report; and (iii) the time-stamped RRF update. Copies of the CCC reports should be included in the instrument performance binder.

8.10. Procedure 8.10.1. Extraction of bioslurry and sediment samples by Single Phase Solvent Extraction (SPSE)

81 8.10.1.1. Begin with 8 ml of bioslurry or sediment in a 60 ml amber glass vial. Add 100.0 µl of prepared surrogate solution (Solution C, section. 8.1).

8.10.1.2. Add 20 ml of methanol and 10 ml of methylene chloride and seal with Teflon lined cap. A single phase should form.

8.10.1.3. Shake at 150 oscillations per min and at room temperature (20- 25ºC) for 12 hours.

8.10.1.4. After shaking, add 10 ml of water and 10 ml of methylene chloride. The vials are then vigorously hand shaken and are centrifuged at 1200 rpm for 5 min at 20ºC. Two phases should be present.

8.10.1.5. After the centrifugation, the bottom organic layer is pulled using a Pasteur pipette and the solvent passed through a Na2SO4 column (as described in Section 5.2.9) into a 20.0 ml vial. The vial is sealed with a Teflon-lined cap and labeled.

8.10.1.6. The extract is then weighed and preserved at 4ºC if analysis cannot begin immediately. If possible, samples should be analyzed immediately after all extracts have been completed.

8.10.1.7. At least one method blank and laboratory control sample will be prepared per set of samples corresponding to a single time point experimental run. Eight ml of reagent water will serve as a method blank. The laboratory control sample (blank spike) will be prepared by spiking a known concentration of the target analytes to the clean culture medium.

8.10.1.8. Spike all method blanks, laboratory control samples, check standards, and extracted samples with internal standard and immediately before processing on the GC/MS. The internal standard (Solution D) is added at 12.5 µl per 1.0 ml sample in each GC vial (acenapthalene-d10 and phenanthrene-d10 stock at 2000 µg/ml in methylene chloride).

8.10.1.9. After determining extraction efficiency, dilutions of extracts may be necessary to make sure that NP and OP are measured in their linear dynamic range on the GC/MS. No concentration of extracts will be done.

8.10.2. Dry Weight Determination of Sediments - Results are always reported on a dry weight basis, unless instructed otherwise. A portion of the homogenized sample must be weighed out for this determination at the same time as the portion used for analytical determination.

82 8.10.2.1. Immediately after weighing the sample for extraction, weigh 5 - 10 g of the sample into a tared crucible or an evaporating dish. Weigh to the nearest 0.01 g.

8.10.2.2. Calibrate oven temperature with a reference thermometer. Place the sample containers in the oven at a temperature of 105 °C for a minimum of 12 hours.

8.10.2.3. Remove the samples from the oven and allow cooling in a desiccator.

8.10.2.4. Re-weigh the samples and record the weights.

8.10.2.5. Calculate the % Moisture as shown in Attachment 1.

8.10.3. Gas Chromatography/Mass Spectrometry

8.10.3.1. The following are the gas chromatographic conditions:

8.10.3.1.1. Gas Flows

Helium Carrier: 0.8 ml/min in constant flow mode.

8.10.3.2. Temperature

8.10.3.2.1. Column Oven: Initial Temperature: 30ºC Ramp at Level 1 at rate of 10 C/min to 160ºC Hold for 12 min Ramp at Level 2 at rate of 25 C/min to 300ºC Hold for 6 min Final Temperature: 300ºC Total Run Time: 38.6 min. 8.10.3.2.2. Injector: 290ºC 8.10.3.2.3. Detector: 250ºC

8.10.3.3. Injection Volume

8.10.3.3.1. Injection Volume: 1.0 µl, splitless

8.10.3.4. After the GC/MS performance tests in Sections 8.6 and 8.7 are conducted, the system is calibrated daily as described in Sections 8.9 and 8.10.

8.10.3.5. Internal standard calibration is used. The internal standard mixture is added (see section 9.2.3) to the extract immediately prior to injection into the instrument.

83 8.10.3.6. One microliter of the sample extract or standard is injected into the GC/MS system.

8.10.3.7. If the absolute amount of a target compound exceeds the working range of the GC/MS system (see Level 5 in Table 5, section 8.3), the extraction efficiency is calculated and the sample extract is diluted and re-analyzed.

8.10.4. If there are two or more analyses for a particular fraction due to sample dilution, the analyst must determine which are the best data to report on the sample summary results sheet.

8.10.4.1. All qualitative and quantitative measurements are performed as described in Section 10. When not being analyzed, extracts are stored in the freezer at -10°C or less and protected from light in crimp top vials equipped with un-pierced Teflon-lined septa.

8.10.5. Qualitative and Quantitative Analysis

8.10.5.1. Mass spectra are obtained for the primary m/z and one or two other masses for the compounds listed in Table 2. The following criteria should be met to make a qualitative identification:

8.10.5.1.1. The characteristic masses of each parameter of interest must maximize in the same scan or within one scan of each other.

8.10.5.1.2. The retention time must fall within ± 20 seconds of the retention time of the reference compound.

8.10.5.1.3. The relative peak intensities of the characteristic masses must fall within 70-130% (60-140% when several isomers co-elute within a few scans) of the relative intensities of these masses in a reference mass spectrum. The reference mass spectrum must be obtained from the daily CCC.

8.10.5.1.4. Structural isomers that have the same mass spectra and less than 20 seconds difference in retention time can be explicitly identified only if the baseline to valley height between the isomers is less than 25% of the sum of two peak heights. NP results are reported based on the sum of all isomers. If there are less than the optimum number of isomers for NP due to poor resolution, low concentration or some not present just add up the isomers that are present making sure the same isomers are chosen for each compound in the calibration curve and samples analyzed. No correction is made for QC recovery data.

84 8.11. Routine Analysis 8.11.1. The concentration in the bioslurry or sediment sample is calculated using the relative response factor (RRF) determined by the following equation:

CgMS(/µ ml)× Vsolvent(ml) Cgi(/µ ml)= Vmsample()l

where, Ci = concentration of NP or OP in sample (µg/ml) CMS (µg/ml) = concentration of NP or OP measured by a calibrated GC/MS

8.11.2. The bioslurry results are reported in micrograms per milliliter (µg/ml) and sediment sample results are reported in micrograms per kilogram (µg/Kg). These results are reported without any corrections for recovery data.

8.11.3. Analytical Sequence

8.11.3.1. Prepare a sequence that includes all QC samples and field samples. The first sample to be analyzed is a 1 µL injection of 10 ng/µL DFTPPThe passing DFTPP mass spectrum data is filed as previously described.

8.11.3.2. The calibration standards (see section 8.3) or a continuing calibration standard (at Level 3 or 4, Table 3, section 8.3) are analyzed next. Verify that all analytes have been properly identified and quantified. Using software programs, manually integrate as necessary. NP is a complex mixture. Therefore check how each isomer is integrated and perform manual integrations, if necessary. Print quantitation reports for the calibration standards.

8.11.3.3. Update the calibration file and print a calibration report. Review the report for calibration outliers and make area (RRF) corrections if the numbers are due to faulty automated integration. If corrections have been made, update the calibration file and regenerate a calibration report. An acceptable initial calibration should meet the criteria specified in section 8.8. Alternatively, re-analyze "nonconforming" calibration level(s) and repeat the above procedures.

8.11.3.4. If the initial calibration data are acceptable, generate the calibration report. If time does not remain during the 24-hour period beginning with injection of the instrument performance check solution (DFTPP), a new injection of the instrument performance check solution must be made followed by the analysis of a continuing calibration standard (Level 3, Table 3, section 8.3.) The instrument performance check solution must meet the criteria in Table 7, Section 13 and the

85 continuing calibration standard must meet the percent difference (%D) criteria specified in section 8.9. Update and generate the continuing calibration file report.

8.11.3.5. Cool the oven to the initial temperature of 30°C. The next samples to be analyzed should be in the following recommended sequence: Lab Blank, samples, and diluted samples.

8.11.3.6. After every 10 samples analyzed and at the initiation and end of a sequence, calibration will be confirmed using calibration midpoints (standard Level 3 or 4, Table 3, section 8.3)) as calibration checks.

8.11.3.7. Archive all the raw data promptly to the appropriate backup media.

8.11.3.8. Generate quantitation reports for all samples following data system and Chromatographic Peak Interpretation SOP (CRL SOP HK014). Generate the final data. Copies of the original and final sequence listings should be included in the data packages as well as in the Instrument Sample Log binders.

8.11.4. Data Reduction

8.11.4.1. Review the quantitation reports for all samples making sure all surrogate compounds and internal standards have been properly quantitated. Check for integration errors.

8.11.4.2. Review the quantitation reports for all samples. Delete any false positive method specific parameters results and results that are less than the method detection limits listed in Table 1.

8.11.4.3. Create sample header and miscellaneous information files for all samples in the analytical sequence.

8.11.4.4. Update the reference spectra with the current daily calibration standard (Level 3, Table 5, section 8.3).

8.11.4.5. Be sure the blank sample data have been properly reviewed. Then generate a QC form listing field samples extracted with the associated lab blank sample.

8.11.4.6. Generate analysis data sheets for blank, and field samples. Review the final results.

8.11.4.7. On a daily basis archive all the processed data files to their original directories in the appropriate backup media.

8.11.5. Data Package Assembly

86 8.11.5.1. Create a tuning and calibration deliverables package with the following: copies of sequence files, DFTPP report, initial and continuing calibration reports, and quantitation reports for calibration standards.

8.12. Troubleshooting Guide 8.12.1. Symptom: Inadequate Abundance at High Masses

8.12.1.1. Probable Causes:

Dirty or contaminated ion source, electron multiplier, or quadrupole rod surfaces.

Potentials of ion source elements at wrong values due to open or short circuits.

Faulty ion source electronics, detector electronics or power supply.

Abnormally high pressure (greater than 1.0 X 10-4 torr) causing ionization difficulties. Check for presence of air at m/z 28 and 32 (CO2 at m/z 44) indicating vacuum system leak.

8.12.2. Symptom: Improper Isotope Ratios

8.12.2.1. Probable Causes:

High "background" levels of undesired substances (Contamination from earlier sample injections) which contribute additional abundance at the isotope mass. Bake out the ion source assembly and condition the column.

8.12.2.2. Resolution of adjacent masses set improperly: higher than normal ratios due to poor resolution (peaks too wide) or lower ratios due to over resolution (narrow peaks).

8.12.3. Symptom: Low Overall Sensitivity

8.12.3.1. Probable Causes:

Dirty or contaminated ion source, electron multiplier or quadrupole rod surfaces.

Electron multiplier with low gain.

Abnormally high pressure (greater than 1.0 X 10-4 torr) causing ionization difficulties. Check for pressure of air at m/z 28 and 32 (CO2 at m/z 44) indicating vacuum system leak.

87 Low emission current from filament. Check that the direct probe is properly adjusted.

8.12.4. Symptom: Poor Reproducibility

8.12.4.1. Probable Causes:

Loose or intermittent connection either to a printed circuit or to one or more ion source or quadrupole elements inside the analyzer assembly.

Pressure or flow fluctuations - Check using ion gauge. For autotune, check the PFTBA vial. Refill the vial, if necessary. Pressure with PFTBA valve open should be between 1x10-6 and 4x10-6 torr.

8.12.5. Symptom: High Background

8.12.5.1. Probable Causes:

Dirty or contaminated ion source, electron multiplier or quadrupole rod surfaces. Bake out the source.

Column or septum bleed, as evidenced by an intense peak at m/z 73, 147, 207 or 221, should not have an abundance of greater than 5000 counts under AUTOTUNE conditions. Replace the faulty septum and condition the column.

Pump Oils - In any oil diffusion pumped vacuum system there is the potential for diffusion pump oil and/or mechanical pump oil to be present in the system. This is highly unlikely under normal conditions.

"Yesterday's" Samples - There is the possibility that some previously injected sample can still be present in the vacuum system long after it was thought to be evacuated. This phenomenon depends on sample volatility, temperature, etc.

Contamination in a recently cleaned vacuum system - After any venting of a vacuum system for maintenance, there is the potential for introducing new substances into the vacuum system. Some substances are normal and can be pumped out, while others require more cleaning or baking.

Solvents used in the cleaning process: These will be present for a while but should be pumped out as heat is applied to the vacuum system.

88 Water absorbed on the metal surfaces while vented. This will pump out with heat.

"Fingerprints" - Heavy organic substances from inadequate clean room procedures may not be pumped out and may require source cleaning.

8.12.6. Symptom: Mass Spectrometer Does Not Respond

8.12.6.1. Probable Causes:

The mass spectrometer electronics are not on - Check the switch inside of the front panel.

Secondary fuse blown - Check the secondary fuses on the rear of the mass spectrometer and replace the faulty fuse or fuses.

Board Failure

89 8.13. Tables and Validation

Table 9-8-1. Method Parameters

PARAMETER CAS# MDL Reporting Limit* (µg/ml) (µg/ml)

Nonylphenol (Target Compound) 11066-49-2 1 3

Octylphenol (Target Compound) 140-66-9 .01 .05

2,4,6-Tribromophenol (NP Surrogate)

Acenaphthene-d10 (Internal Std.)

Phenanthrene-d10 (Internal Std.) Calibration Criteria: IC (%RSD < 35%) and CCC (%D < 25%). Currently, LCS spike recovery limits are used to assess site sample data quality. * - Reporting Limit amounts are calculated from Table 5 Level 1 concentrations assuming a 1 µL injection of a 1 mL extract solution obtained from extracting 8 ml of bioslurry or 8 grams of dry sediment with SPSE technique.

Table 9-8-2. Retention Times (RT) and Electron Impact Ions

Peak RT (minutes) Primary Ion Secondary Ions Octylphenol 135 107, 91 NP Isomer 1 48.35 121 107, 163 NP Isomer 2 48.58 135 107 NP Isomer 3 49.00 135 107, 121 NP Isomer 4 49.20 135 107, 121 NP Isomer 5 49.58 135 - NP Isomer 6 49.92 149 107, 121 NP Isomer 7 49.80 135 - NP Isomer 8 49.87 149 107, 121 NP Isomer 9 50.23 135 - NP Isomer 10 50.28 149 107, 121 NP Isomer 11 50.79 107 135 NP Isomer 12 50.90 135 107 NP Isomer 13 51.08 121 107, 163 NP Isomer 14 51.19 163 107, 121 NP Isomer 15 51.55 149 - NP Isomer 16 51.55 121 163 NP Isomer 17 51.55 135 - NP Isomer 18 51.76 163 107, 121 NP Isomer 19 51.76 135 - NP Isomer 20 52.31 135 - NP Isomer 21 52.48 107 121 NP Isomer 22 52.80 149 107, 121 NP Isomer 23 53.11 135 107 2,4,6-Tribromophenol (NP 64 Surrogate) Acenaphthene-d10 (Internal Std.) 164 140 Phenanthrene-d10 (Internal Std.) 188 94, 160

Table 9-8-3. Preparation os Stock Standards Surrogate Internal Solution Nonylphenol Octylphenol Standards Standard A 1000 µg/ml ------B --- 1000 µg/ml ------C ------5,000 µg/ml --- D ------2,000 µg/ml Stock solutions as prepared in Section 8.3

Table 9-8-4. Concentrations of Calibration Standards (Made using 10mL volumetric flasks). Calibra- Vial Label Desired Spike Vol. (µl) Desired Spike Vol Desired Spike Vol. tion NP of 1000 OP Conc. (µl) of 1000 SS* (µl) of Standard Conc. (µg/ml) NP (µg/ml) (µg/ml) OP Conc. 1000 Level (µg/ml) (µg/ml) µg/ml SS* 1 1NPOPSS 1 10 1 10 1 10 2 5NPOPSS 5 50 5 50 5 50 3 10NPOPSS 10 100 10 100 10 100 4 20NPOPSS 20 200 20 200 20 200 5 30NPOPSS 30 300 30 300 30 300 6 40NPOPSS 40 400 40 400 40 400 7 50NPOPSS 50 500 50 500 50 500 This table assumes dilution of spike volume in 10 ml of methylene chloride. * - ABN Surrogates include: 2,4,6-tribromophenol, 2-fluorobiphenyl, 2-fluorophenol, phenol-d6, nitrobenzene-d5, p-terphenyl-d14.

Table 9-8-5. Amounts of Calibration Standards on Column Based on Table 4 Concentrations. MSP/ LV 1 LV 2 LV 3 LV 4 LV 5 LV 6 LV 7 Surrogate (ng) (ng) (ng) (ng) (ng) (ng) (ng) NP 1 5 10 20 30 40 50 Octylphenol 1 5 10 20 30 40 50 ABN Surrogates* 1 5 10 20 30 40 50 ABN Internal 25 25 25 25 25 25 25 Standards** *ABN Surrogate Solution includes: 2,4,6-tribromophenol, 2-fluorobiphenyl, 2-fluorophenol, phenol-d6, nitrobenzene-d5, p-terphenyl-d14. **ABN Internal Standard Solution includes: acenaphthene-d10, chrysene-d12, 1,4-dichlorobenzene-d4, naphthalene-d8, perylene-d12, phenanthrene-d10.

Table 9-8-6. DFTPP Abundance Criteria Mass Relative Abundance Criteria Purpose of Checkpoint1 (m/z) 51 30 - 80% of the base peak low mass sensitivity 68 <2% of mass 69 low mass resolution 69 Mass 69 Relative abundance Low mass resolution 70 <2% of mass 69 Low mass resolution 127 25 - 75% of the base peak low-mid mass sensitivity 197 <1% of mass 198 mid-mass resolution base peak, 100% relative 198 mid-mass resolution and sensitivity abundance 199 5 - 9% of mass 198 mid-mass resolution and isotope ratio 275 10 - 30% of the base peak mid-mass high sensitivity 365 >0.75% of the base peak baseline threshold 441 Present and < mass 443 high mass resolution 442 40.0- 110.0% of mass 198 high mass resolution and sensitivity 443 15 - 24% of mass 442 high mass resolution and isotope ratio All ions are used primarily to check the mass measuring accuracy of the mass spectrometer and data system, and this is the most important part of the performance test. The three resolution checks, which include natural abundance isotope ratios, constitute the next most important part of the performance test. The correct setting of the baseline threshold, as indicated by the presence of low intensity ions, is the next most important part of the performance test. Finally, the ion abundance ranges are designed to encourage some standardization to fragmentation patterns.

93 8.14. References

Barber, L., and others, USGS, Analysis of Endocrine Disrupting Compounds, Draft, May 5, 1999.

Burke, J.A. "Gas Chromatography for Pesticide Residue Analysis; Some Practical Aspects", J. Assoc. Off. Anal. Chem. 1965, 48, 1037.

Carcinogens - Working with Carcinogens; U.S. Department of Health, Education and Welfare. Center for Disease Control. National Institute for Occupational Safety and Health, Publication No. 77-206, August 1977.

Code of Federal Regulations, 40 CFR Part 136, Appendix B.

Eichelberger, J.W.; Harris, L.E.; Budde, W.L. "Reference Compound to Calibrate Ion Abundance Measurement in Gas Chromatography/Mass Spectrometry" Analytical Chemistry 1975, 47, 995.

Extraction of Soil Samples Using Dionex ASE 200, USEPA Region 9 Draft SOP, January 11, 1999.

Interlaboratory Method Study for EPA Method 625- Base/Neutrals, Acids and Pesticides, Final Report for EPA Contract 68-03-3102.

McNair, N.M.; Bonelli, E.J. Basic Chromatography;, Consolidated Printing: Berkeley, CA, 1969; p. 52.

Methods 330.4 (Titrimetric, DPD-FAS) and 330.5 (Spectrophotometric, DPR) for Chlorine, Total Residual, Methods for Chemical Analysis of Water and Wastes, U.S. Environmental Protection Agency, Environmental Monitoring and Support Laboratory, Cincinnati, Ohio 45268, March 1979. EPA-600/4-79-020

Olynyk, P.; Budde, W.L.; Eichelberger, J.W. "Method Detection Limit for Methods 624 and 625", Unpublished report, October 1980.

OSHA Safety and Health Standards, General Industry, (29 CFR 1910), Occupational Safety and Health Administration, OSHA 2206 (Revised, January 1976).

94 Provist, L.P.; "Interpretation of Percent Recovery Data" American Laboratory 1983, 15, 58-63.

Sampling and Analysis Procedures for Screening of Industrial Effluents for Priority Pollutants: May 1977, Revised April 1977; U.S. Environmental Protection Agency. Environmental Monitoring Support Laboratory, Cincinnati, Ohio 45268. Available from Effluent Guidelines Division, Washington, DC 20160.

Standard Practices for Sampling Water, American Society for Testing and Materials, Philadelphia. ASTM Annual Book Standards, Part 31, D3370-76.

95 9. Appendix 2: Total Biomass Analysis

9.1. Summary of Method Total biomass analysis was used as a simple and reliable method for determining

the phospholipid concentration of a sample. The phospholipid concentration is directly

proportional to the density of living cells in the sample. By performing the total biomass

analysis throughout an experiment, the growth and decline of the cell population within a

culture can be monitored. The total biomass analysis was particularly helpful for this

study because the same extraction procedure for NP analysis could be used for total

biomass analysis. Therefore, a sample could be extracted and simply split into portions

for each analysis. The following is a brief summary of the method. For a more detailed

explanation of the method refer Chapter 40 of the Handbook of Methods in Aquatic

Microbial Ecology by Dobbs and Findlay (1993) (Dobbs and Findlay 1993).

After an extracted sample is obtained, the total extractable lipid is dissolved in

chloroform. The dissolved extractable lipid sample is then split into triplicate sub-

samples such that each sub sample contains between 3 and 15 nmol of phosphate. The

sub-samples are placed in clean 2 ml ampules and dried with nitrogen. 450 µl of

saturated persulfate is added to each ampule. The ampules are seal using a Bunsen

burner and placed in a 100°C oven overnight. The samples are cooled, opened, and 100

µl of ammonium molybdate solution is added. After a 10 minute rest, 450 µl of malachite green solution is added and the samples are allowed to rest for 30 minutes.

Each sub-sample is transferred to a semi-microcuvette and read using a spectrophotometer at an absorbance at 610 nm.

96 9.2. References

97 10. Appendix 3: Preparation of Microbiological Media

10.1. Introduction Microbiological media was used to provide essential minerals and trace metals.

Experiments for this study were conducted using both aerobic and anaerobic conditions.

A separate media was used for each condition. The following recipes explain the

procedure for making each media.

10.2. Preparation of RST Basal Microbiological Media This is the basic media used for all aerobic experiments. This media recipe was

provided by Tamara Marsh.

RST Basal Microbiological Media

Solution per Liter Minerals 20 ml Trace Metals 5 ml Vitamins 10 ml NaHCO3 10g

To 1 liter deionized distilled water, add the Minerals, Trace Metals, and Vitamins Solutions with continuous mixing on a stir plate. Heat the media to boiling while mixing. Once boiling has started, continue boiling for 10 min while continuously stirring with a magnetic stir plate. Remove from heat, place in cool water bath, and bring temperature o down to 30 C. Once cooled, add the NaHCO3, and mix well. The final medium should have a clear, gray color when complete. If a large amount of precipitates form overnight, filter the medium using Whatman No. 1 paper. Media can be sterilized by autoclaving at 15 psia and 121°C for 20 minutes.

RST Minerals Solution (store at 4ºC) Component g/L NaCl 40 NH4CL 50 KCl 5 KH2PO4 5 . MgSO4 7H20 10 . CaCl2 2H2O 2

98 RST Trace Metals Solution (store at 4ºC) Component g/L Nitrilotriacetic Acid 2 Adjust to pH 6 with KOH . MnSO4 H2O 1 Fe(NH4)2(SO4)2 0.8 . CoCl2 6H20 0.2 . ZnSO4 7H2O 0.2 . CuCl2 6H2O 0.02 . NiCl2 6H2O 0.02 . Na2MoO4 2H2O 0.02 Na2SeO4 0.02 Na2WO4 0.02

RST Vitamins Solution (store at 4ºC) Component mg/L Pyrixodine .HCl 10 Thiamine .HCl 5 Riboflavin 5 Calcium Pantothenate 5 Thioctic Acid 5 p-Aminobenzic Acid 5 Nicotinic Acid 5 Vitamin B12 5 Biotin 2 Folic Acid 2 Mercaptoethanesulfonic Acid (MESA) 10

10.3. Preparation of the Reduced Anaerobic Microbiological Media (RAMM) The following recipe was used for all anaerobic experiments conducted in this

study. This recipe is adapted from Shelton and Tiedje (1984) (Shelton and Tiedje 1984).

Reduced Anaerobic Microbiological Media (RAMM)

Solution per Liter Solution 1 10 ml Solution 2 10 ml Solution 3 1 ml Resazurin Solution 0.5 ml

After Boiling: NaHCO3 2.4 g Na2S 0.24 g

To 1 liter deionized, distilled water, add the above solutions in the amount listed

while continuously mixing on a stir plate. Heat the media to boiling while mixing. Once

boiling has started, continue boiling for 5min while continuously blanketing with

nitrogen. Remove from heat, place in cool water bath, and bring temperature down to

30ºC, while continuing to blanket with nitrogen gas. Once cooled, add the NaHCO3 and

Sulfide Solution, mix, and place in anaerobic chamber. Due to the nature of this media some precipitation will form overnight, so stir or shake media before dispensing.

Precipitation will not effect biological growth.

The final medium should have a gray color when complete. If any pinkish tint is present,

the medium is no longer reduced and this indicates the presence of oxygen.

RAMM Solution 1 Component g/L KH2PO4 27.2 K2HPO4 34.8 (Adjusted to pH 7.0 with HCl or NaOH.)

RAMM Solution 2 Component g/L NH4CL 53.5 . CaCl2 2H2O 7.85 . MgCl2 2H2O 10.15 . FeCl2 4H2O 2.0

100 RAMM Solution 3 Component g/L . MnCl2 H2O 0.5 H3BO3 (Boric Acid) 0.05 ZnCl2 0.05 . CuCl2 6H2O 0.03 . Na2MoO4 2H2O 0.01 . CoCl2 6H20 0.5 . NiCl2 6H2O 0.05 Na2SeO4 0.05

Resazurin Solution Component g/50 ml Resazurin 0.1

Redox Potential and the use of Resazurin

Resazurin is the most common redox indicator used in anaerobic cultivation, because it is

generally nontoxic to bacteria and is effective at final concentrations of 1 to 2 ug/ml.

When incorporated into media, this indicator first undergoes an irreversible reduction

step to resorufin, which is pink at pH values near neutrality. At higher pH, it remains

blue. This first reduction step can occur when media are heated under an oxygen-free

atmosphere. Typically, resorufin (pink color) does not form from resazurin (purple-blue

color) when the medium is boiled (unless a medium component acts as a mild reductant).

The second reduction step to dihydroresorufin (which is colorless) has a midpoint

potential (Eo’) of –51 mV, so the resorufin/hydroresorufin redox couple becomes

completely colorless at an Eh below –110 mV. This usually requires the addition of a reducing agent to the medium.

101 Preparation of Cysteine-Sulfide Reducing Agent

1. Boil 110 ml of water under N2 or N2/CO2 stream. Seal and bring inside anaerobic

chamber.

2. Weigh out more than 2.5 g sodium sulfide crystals and place in chamber. Rinse

Na2S·9H2O crystals with water to remove oxidation products that collect on the crystals’

surface. Weigh out 2.5 g of rinsed sodium sulfide crystals.

3. Weight out 2.5 g cysteine hydrochloride and bring into chamber (This powder must be

covered when going through air-lock as it is very light and will be blown around during

the vacuum process)

4. Inside chamber:

a. Mix the sodium sulfide crystals into 100 ml of water

b. Add the cysteine hydrochloride

c. Once thoroughly dissolved dispense into 5 or 10 ml aliquots (In small serum

bottles or Balch test tubes) and seal with a rubber stopper and aluminum crimp

seal.

5. Autoclave for 20 minutes at 121° C.

6. Add 0.1 ml to every 10 ml of media.

10.4. References

Shelton, D. R. and J. M. Tiedje (1984). "General method for determining anaerobic biodegradation potential." Applied and Environmental Microbiology 47: 850-857.

102