## January 2009 

# Targeted National Sewage Sludge Survey

Sampling and Analysis Technical Report

# U.S. Environmental Protection Agency Office of Water (4301T) 1200 Pennsylvania Avenue, NW Washington, DC 20460

EPA-822-R-08-016TNSSS Sampling and Analysis Technical Report i January 2009 

### Acknowledgments and Disclaimer

The Targeted National Sewage Sludge Survey was made possible by the assistance and cooperation of numerous staff wo rking at each of the sewage tr eatment facilities involved. The staffs of the facilities contact ed during the course of this survey were, without exception, knowledgeable, friendly, helpful, and deserved ly proud of their effo rts to protect the environment and serve their local constituencies.

This technical report has been reviewed and approved for publication by the Office of Science and Technology. This report was prep ared with the support of Battelle Memorial Institute, Computer Sciences Corporation, and their subcontractors, und er the direction and review of the Office of Science and Technology. EPA woul d like to thank the Alexandria Sanitation Authority, Alexandria, VA, who opened their doors and assisted EPA and contractor staff during hands-on training in sampling techniques in July 2006.

Neither the United States government, nor any of its employees, contractors, subcontractors, or other employees makes any warranty, expressed or im plied, or assumes any legal liability or responsibility for any third part y’s use of, or the result s of such use of, any information, apparatus, product, or process discusse d in this report, or re presents that its use by such a third party would not infr inge on privately owned rights.TNSSS Sampling and Analysis Technical Report ii January 2009TNSSS Sampling and Analysis Technical Report iii January 2009 

### Table of Contents

Page

Acknowledgments and Disclaimer ................................................................................................. . i Executive Summary ....................................................................................................................v Section 1 Background and Organization .......................................................................................1 1.1 Regulatory and Surveys History............................................................................1 1.2 Targeted National Sewage Sludge Survey ............................................................1 1.3 Content of this Report ...........................................................................................3 Section 2 Survey Objective and Design.........................................................................................4 2.1 Survey Objective ...................................................................................................4 2.2 Target Population ..................................................................................................4 2.3 Stratification ..........................................................................................................4 2.4 Facillity Selections ................................................................................................5 Section 3 Sample Collection..........................................................................................................7 3.1 Training .................................................................................................................7 3.2 Sample Collection .................................................................................................7 3.3 Site-specific Deviations.......................................................................................88 3.4 Representative Samples.........................................................................................9 3.5 Field Duplicates.....................................................................................................9 3.6 Sample Labeling and Tracking..........................................................................100 3.7 Packing and Shipping Samples to the Repository...............................................10 3.8 Storage and Shipments to Laboratories...............................................................11 3.9 Shipping Issues....................................................................................................12 3.10 Sampling Summary .............................................................................................13 3.11 Equipment Blanks ...............................................................................................13 Section 4 Sample Analyses..........................................................................................................15 4.1 Analytes of Interest..............................................................................................15 4.2 Analytical Techniques.........................................................................................18 4.3 Laboratories.........................................................................................................18 4.4 Method Modifications .........................................................................................19 4.4.1 Ensuring Consistent Method Sensitivity ..................................................19 4.4.2 Anions in Sewage Sludge .......................................................................201 4.4.3 Modified GC/MS Procedures for Semivolatile Organics.........................21 4.4.4 PBDE Analyses ........................................................................................23 4.4.5 Pharmaceutical Analyses..........................................................................24 4.4.6 Steroid and Hormone Analyses ................................................................26 4.4.7 Focusing Quality Control on the Survey-specific Analytes of Interest....28 Section 5 Data Review Procedures..............................................................................................2 9 5.1 General Review Procedures ................................................................................29 5.2 QC Acceptance Criteria.......................................................................................30 5.3 Data Qualifiers and Database ..............................................................................30 5.4 Data Review Findings .........................................................................................31 5.4.1 Reporting Limits.......................................................................................31 5.4.2 Calculation Errors.....................................................................................32 5.4.3 Blank Contamination................................................................................32 5.4.4 Extract Dilution ........................................................................................33TNSSS Sampling and Analysis Technical Report iv January 2009 5.4.5 Matrix Spikes............................................................................................34 5.4.6 Interferences .............................................................................................34 5.4.7 Recovery Issues ........................................................................................37 5.5 Revised Results for BDE-209 .............................................................................38 Section 6 Survey Results .............................................................................................................39 6.1 Summary Results.................................................................................................39 6.2 Investigation of Results for Metals......................................................................42 6.3 Comparison of Metals Results to Current Standards ..........................................43 6.4 Analytical Completeness.....................................................................................43 6.5 Analytical Sensitivity ..........................................................................................44 6.6 Equipment Blank Evaluation...............................................................................47 6.6.1 Semivolatile Organics and PAHs .............................................................47 6.6.2 Metals .......................................................................................................48 6.6.3 PBDEs.......................................................................................................49 6.6.4 Anions.......................................................................................................49 6.7 Field Duplicate Results........................................................................................51 6.7.1 Anions.......................................................................................................52 6.7.2 Metals .......................................................................................................52 6.7.3 Semivolatile Organics and PAHs .............................................................53 6.7.4 PBDEs.......................................................................................................54 6.7.5 Pharmaceuticals ........................................................................................54 6.7.6 Steroids and Hormones.............................................................................56 6.7.7 Results in Liquid Samples ........................................................................58 6.8 Matrix Spike and Duplicate Results....................................................................59 6.8.1 Matrix Spike Results for Anions ..............................................................60 6.8.2 Duplicate Results for Anions....................................................................62 6.8.3 Matrix Spike Results for Metals...............................................................62 6.8.4 Duplicate Results for Metals ....................................................................65 6.8.5 MS/MSD Results for Organics.................................................................67 6.8.6 MS/MSD Results for PBDEs....................................................................69 6.8.7 Qualification of Sample Re sults based on MS/MSD Results...................70 6.8.8 Labeled Compound Recoveries for Isotope Dilution Methods ................71 Section 7 References ..................................................................................................................72

Appendix A Solids Leaching Procedure for Anions....................................................................73 Appendix B Method Modifications for the PBDE Analyses.......................................................74 Appendix C QC Acceptance Criteria...........................................................................................75TNSSS Sampling and Analysis Technical Report v January 2009 

### Executive Summary

This Sampling and Analysis Technical Report (“Technical Re port”) describes the sampling and analysis activities conducted by EPA in support of the Targeted National Sewage Sludge Survey (TNSSS). The TNSSS was de signed to: 1) obtain updated occurrence information on nine analytes of potential conc ern, and 2) obtain occurrence information on a number of contaminants of emerging interest identified by EPA and the National Research Council (NRC). The objective of the survey was to obtain nationa l estimates of the concentrations of these pollutants in sewage sl udge for use in assessing if exposures may be occurring and whether those levels may be of concern.

Final sewage sludge is define d as the liquid, solid, or semi-s olid residue generated during the treatment of domestic sewage , receiving secondary treatment or better, in a treatment works, which may include sewage sludge processed to m eet land application standards. The publicly owned treatment works (POTWs) included in the su rvey were selected without consideration of their sewage sludge use or disposal practices.

For this survey, EPA focused its efforts on POTWs that treat more than one million gallons of wastewater per day (MGD). This group of facilities collectively generates approximately 94 percent of the wa stewater flow in the nation. To be eligible for the survey, EPA also required that a POTW be located in the contiguous United States and employ secondary treatment or better. From the 3,337 POTWs that met the criteria, EPA statistically selected 74 facilities in 35 states for the survey and collected biosolids samples from those facilities. Whether the facility recycles the sewage sludge to land or disposes of it via incineration or surface disposal was not a consider ation for selecting a facility for inclusion in the survey. By using statistical methods, the con centration measurements can be extrapolated to the entire population of 3,337 POTWs.

EPA collected samples between A ugust 2006 and March 2007. EPA collected 84 samples of sewage sludge from 74 facilities, one from each of 64 POTWs, as well as two samples at the remaining ten facilities (either be cause the facility had more than one treatment system and produced two types of final sewage sludge, or for quality assurance purposes. EPA conducted analysis of sewage sludge sample s for 145 analytes, including four anions (nitrite/nitrate, fluoride, water-extractable pho sphorus), 28 metals, four polycyclic aromatic hydrocarbons, two semi-volatiles, 11 flame retardants, 72 pharmaceuticals, and 25 steroids and hormones.

The survey used both well-established multi- laboratory validated EPA procedures as well as three analytical methods that were deve loped or updated for the survey. The two new methods are single-lab validated methods for pharmaceuticals (EPA Method 1694), and steroids and hormones (EPA Method 1698). The updated multi-lab validated method is for flame retardants (EPA Method 1614).

EPA took steps to ensure that the results we re comparable across al l of the facilities sampled. The percent solids in the various se wage sludge samples range from 0.14 to 94.9. To ensure comparability of results, all sample results are reported on a dry-weight basis.TNSSS Sampling and Analysis Technical Report vi January 2009 EPA subjected all of the anal ytical results generated by the laboratories to data review procedures. These procedures used review pr otocols to ensure that the results met EPA’s objectives for data quality.

This Technical Report includes the numb er of samples in which each analyte was reported, along with minimum and maximum measurements. Reported concentrations and frequency of detects are limited by the sensitivity of the analytical methods used. Some analytes were found in all 84 samples, while others were found in none or only a few of the sewage sludge samples. The minimum concentration is the lowest value report ed as present in any sample. EPA did not report a minimum or maxi mum value for those analytes that were not detected (i.e., a situation that occurred for some of the pharmaceuticals, steroids and hormones). For these situations, EPA used “ND” to indicat e that the minimum and maximum values were “not detected.” The maximum concentration is the highest value reported as present in any sample.

Briefly, the survey found:

 The four anions were found in every sample.

 27 metals were found in virtually every samp le, with one metal (antimony) found in no less than 72 samples.

 Of the six semivolatile organics and polyc yclic aromatic hydrocarbons, four were found in at least 72 samples, one was found in 63 samples, and one was found in 39 samples.

 Of the 72 pharmaceuticals, three (i.e., cyprof loxacin, diphenhydramine, and triclocarban) were found in all 84 samples and nine were found in at least 80 of the samples. However, 15 pharmaceuticals were not found in any sample and 29 were found in fewer than three samples.

 Of the 25 steroids and hormones, three st eroids (i.e., campester ol, cholestanol, and coprostanol) were found in all 84 samples and six steroids were found in at least 80 of the samples. One hormone (i.e., 17 α -ethynyl estradiol) was not found in any sample and five hormones were found in fewer than six samples.

 All of the flame retardants except one (BDE- 138) were found every sample or all but one sample.

It is not appropriate to speculate on th e significance of the results until a proper evaluation has been completed and reviewed. EP A plans to evaluate the pollutants identified by the survey as being present in sewage sludge. As its first priority, using the survey information, EPA has begun assessing the nine pollutants identif ied from the 2003 biennial review as needing updated concentration informati on and molybdenum to determine whether additional action may be necessary. Later this year, EPA expects to initiate evaluations of other pollutants in the survey that may warrant further consideration. The evaluations will depend on the availability of data needed to c onduct the evaluations.TNSSS Sampling and Analysis Technical Report 1 January 2009 

### Section 1 Background and Organization

1.1 Regulatory and Surveys History

Sewage sludge is the solid, semisolid, or liq uid organic material that results from the treatment of domestic wastewat er by municipal wastewater tr eatment plants, also known as publicly owned treatment works (POTWs). The U.S. Environmental Protection Agency (EPA) uses the terms sewage sludge and biosolids intercha ngeably, but others often refer to biosolids as sewage sludge that has had additi onal processing for land application.

Section 405(d) of the Clean Water Act (CWA) requires that the EPA establish requirements for the use or disposal of sewage sl udge. The Standards for the Use or Disposal of Sewage Sludge are found at Part 503 of Section 40 of the Code of Fede ral Regulations (40 CFR 503, hereafter simply “Part 503”).

These regulations establish numeric limits , management practices, and operational standards to protect public health and the environment. Sewage sludge is typically used by land applying to fertilize crops or reclaim mined lands , or disposed either by landfilling or surface disposing, or by incinerating. States may adopt a dditional or more stringent regulations for the use or disposal of biosolids.

Additionally, Section 405(d) of the CWA requires EPA to revi ew existing sewage sludge regulations at least ever y two years (i.e., biennial review). The purpose of such reviews is to identify additional toxic pollutants, and promulga te regulations, if need ed, for those pollutants consistent with the requirements set forth in the CWA.

1.2 Targeted National Sewage Sludge Survey

The Agency periodically conduc ts surveys to determine what may be present in sewage sludge. EPA has conducted three previous sewage sludge surveys: 1) a 40-city survey in 1982 to develop information on the fate and effects of prio rity pollutants in wastewater treatment plants and estimates of pollutant concentrations in se wage sludge; 2) a National Sewage Sludge Survey in 1988-1989 to gather information on sewage sl udge use or disposal pr actices and to obtain updated information on the concentration of ove r 400 pollutants in the Nation’s sewage sludge; and 3) a National Sewage Sludge Survey in 2001 to obtain updated national estimates of dioxins and dioxin-like compounds in sewage sludge managed by land application.

In conducting the 2003 biennial review (68 FR 75531), EPA identified 15 analytes that needed further evaluation. EPA subsequently re duced the list of analyt es to nine based on a biosolids exposure and hazard assessment. EPA also determined it needed updated concentration data for more refined risk asse ssment and risk characterization of these nine pollutants (see Table 1).TNSSS Sampling and Analysis Technical Report 2 January 2009 Table 1. 2003 Biennial Revi ew Analytes Requiring Additional Information

Analyte Type

Nitrate Anion Nitrite Anion Barium Metal Beryllium Metal Manganese Metal Silver Metal Fluoranthene PAH Pyrene PAH 4-Chloroaniline Semivolatile organic

Inclusion of pollutants in th e TNSSS does not reflect a determ ination that their presence in sewage sludge adversely affects human health or the environment. Rather, EPA decided that updated or new concentration data were needed to assess exposure and help in evaluating whether levels of these pollutants in sewage sludge present environmental or human health

concerns.

Given the national scope of the survey, EPA e xpanded the list of analytes to reflect the Agency’s interest in collecting concentration data for other pollutants. The expanded list included 24 additional metals that could be analyzed at little extr a cost at the same time as the four metals (barium, beryllium, manganese, and silver) included in the list of nine pollutants above; molybdenum because of the Agency’s inte rest in determining the need for a revised numeric standard for it in land-applied biosolids; and other analytes because of their widespread incidence and use, as well as emerging concern. The latter category included:

 benzo( a )pyrene (found in coal tar, automobile exhaust fumes, tobacco and wood smoke, charbroiled food, and burnt toast);

 2-methylnaphthalene (found in nonstructural ca ulking compounds and sealants, synthetic resins, rubber adhesives, and wall coverings);

 bis (2-ethylhexyl) phthalate (wid ely used as a plasticizer in manufacturing of items such as cosmetics, toys, tools, and laboratory equipment);

 fluoride (used in topical and systemic therapy for preventing tooth decay, as well as many other uses);

 water-extractable phospho rus (correlated with phosphorus concentration in runoff from soils amended with manure a nd biosolids and an effective indicator of loss that may contribute to algae buildup in surface waters);

 11 polybrominated diphenyl ethers (PBDEs). Four of the PBDEs were of most interest because of available human health information that may be useful for future risk evaluation efforts. PBDEs are used as flam e retardants in a wide array of products, including building materials, electronics, furnishings, motor vehicles, plastics, polyurethane foams, and textiles; and

 97 pharmaceuticals, steroids, and hormones because of broader emerging interest in these analytes.TNSSS Sampling and Analysis Technical Report 3 January 2009 EPA began sampling in August 2006, using the procedures described in Section 3, and completed sampling in March 2007. Analyses of survey samples were conducted as described in Section 4.

1.3 Content of this Report

This report describes the design, sampling, a nd analysis activities for the TNSSS. The report addresses the following topics:

 Survey Objective and Design

 Sample Collection

 Sample Analyses

 Data Review Procedures

 Survey Results

 References

 AppendicesTNSSS Sampling and Analysis Technical Report 4 January 2009 

### Section 2 Survey Objective and Design

2.1 Survey Objective

The TNSSS was designed to: 1) obtain update d occurrence information on nine analytes of potential concern, and 2) obt ain occurrence information on a number of contaminants of emerging interest identified by EPA and the Na tional Research Council (NRC) that may be present in sewage sludge generated by POTWs.

2.2 Target Population

For this survey, EPA focused its efforts on POTWs that treat more than one million gallons of wastewater per day (MGD). This group of facilities collectively generates approximately 94 percent of the wa stewater flow in the nation. To be eligible for the survey, EPA also required that a POTW be located in the contiguous United States and employ secondary treatment or better. EPA selected PO TWs meeting the criteria from information in the 2004 Clean Water Needs Survey and the 2002 versi on of the Permit Compliance System. From the 3,337 POTWs that met the criteria in either data source, EPA used a stratified random sampling design to statistically select 74 facili ties in 35 states for the survey and collected biosolids samples from those facilities. Whethe r the facility land applies the sewage sludge or disposes of it via incineration or surface disposal was not a consideration for selecting a facility for inclusion in the survey. By using statistica l methods, the concentration measurements can be extrapolated to the enti re population of 3,337 POTWs.

2.3 Stratification

EPA selected POTWs for inclusion in th e survey using a random sampling design stratified for flow. EPA divi ded the 3,337 facilities in the sample popul ation into three categories, based on their design flow:

 Flow rate of 1 to 10 MGD;

 Flow rate of 10 to 100 MGD; and

 Flow rate of greater than 100 MGD

EPA then selected a proportionate number of POTWs from each of the above stratum at random.

POTWs with flow rates less than 1 MGD were not included in the survey. However, the combined flows of all such fac ilities represent less than 6% of the total flow of all POTWs nationwide.TNSSS Sampling and Analysis Technical Report 5 January 2009 2.4 Facility Selections

EPA invited 80 POTWs to voluntarily particip ate in the survey. The initial written invitation was followed by a telephone call. Th ese communications outlined the nature of the survey, the analytes of interest, and the timef rame for completion. EPA also assured each facility that samples sent to the laboratories fo r analysis would be submitted as “blind” samples, such that the results from any given sample could not be associated with a particular facility.

These communications identified facilities that did not meet the criteria for POTW selection in the peer-reviewed survey design and outlined above. Some POTWs provided only partial treatment of their wast ewater, while others employed wastewater lagoons which do not typically produce sewage sludge, as defined, on a routine basis. Ultimately, EPA eliminated eleven POTWs and found five replacement POTWs. As a result, EPA selected 74 POTWs that met the stated criteria. The rationale for each replacement POTW is included in Table 2.

Table 2. POTWs Selected for Sampling Facility Name and Flow Group Flow Stratum City ST Status

Sugar Creek WWTP 1 <MGD <10 Alexander City AL Replacement for Coley Creek; Sugar Creek is on the same system and completes the processing of Coley Creek’s partially-treated sewage sludge Aldridge Creek WWTP 1 <MGD <10 Huntsville AL Original selection Phoenix WWTP 10 <MGD <100 Phoenix AZ Original selection Valley Sanitary District STP 1 <MGD <10 Indio CA Original selection San Francisco > 100 MGD San Francisco CA Original selection El Estero WWTP 1 <MGD <10 Santa Barbara CA Original selection Santa Rosa 1 <MGD <10 Santa Rosa CA Original selection Stockton Water Quality Plant > 100 MGD Stockton CA Original selection Los Angeles County Sanitation District 10 <MGD <100 Whittier CA Original selection Boulder WWTP 1 <MGD <10 Boulder CO Original selection South Windsor 1 <MGD <10 South Windsor CT Original selection Three Oaks WWTF 1 <MGD <10 Estero FL Original selection Orange County Northwest WRF 1 <MGD <10 Orlando FL Original selection Tampa 1 <MGD <10 Tampa FL Original selection Albany 10 <MGD <100 Albany GA Original selection Americus-Mill Creek 1 <MGD <10 Americus GA Original selection Boone STP 1 <MGD <10 Boone IA Original selection

Calumet Water Reclamation Plant > 100 MGD Chicago IL Replacement for MWRDGC North Side WWTP; Calumet is on the same system and completes the processing of North Side’s partially-treated sewage sludge Plainfield WWTP 1 <MGD <10 Plainfield IL Original selection Lake County DPW, New Century STP 1 <MGD <10 Vernon Hills IL Original selection Dupage County-Knollwood STP 1 <MGD <10 Wheaton IL Original selection Blucher Poole WWTP 1 <MGD <10 Bloomington IN Original selection William Ross Edwin WWTP 10 <MGD <100 Richmond IN Original selection Parsons 1 <MGD <10 Parsons KS Original selection Topeka 10 <MGD <100 Topeka KS Original selection Mayfield WWTP 1 <MGD <10 Mayfield KY Original selection Eunice 1 <MGD <10 Eunice LA Original selection Jefferson Parish East Bank WWTP 1 <MGD <10 Marrero LA Original selection Nantucket 1 <MGD <10 Nantucket MA Original selection Salisbury 1 <MGD <10 Salisbury MD Original selection Mechanic Falls Treatment Plant 1 <MGD <10 Mechanic Falls ME Original selection Benton Harbor-St. Joseph WWTP 1 <MGD <10 St. Joseph MI Original selection Wixom WTP 1 <MGD <10 Wixom MI Original selection Festus Crystal City STP 1 <MGD <10 Crystal City MO Original selection Elizabeth City WWTP 1 <MGD <10 Elizabeth City NC Original selection Hillsborough WWTP 1 <MGD <10 Hillsborough NC Original selectionTNSSS Sampling and Analysis Technical Report 6 January 2009 Table 2. POTWs Selected for Sampling Facility Name and Flow Group Flow Stratum City ST Status Beatrice 1 <MGD <10 Beatrice NE Original selection

Wildwood Lower WTF 10 <MGD <100 Cape May Court House NJ Original selection

Middlesex County Utility Authority WRC > 100 MGD Sayreville NJ Original selection Verona TWP DPW 1 <MGD <10 Verona NJ Original selection Buffalo > 100 MGD Buffalo NY Original selection Canajoharie WWTP 1 <MGD <10 Canajoharie NY Original selection Geneva A-C Marsh Creek STP 1 <MGD <10 Geneva NY Original selection NYC DEP - Jamaica WPCP 10 <MGD <100 New York City NY Original selection

North Tonawanda STP 1 <MGD <10 North Tonawanda NY Original selection

Clermont County Commissioners 1 <MGD <10 Batavia OH Original selection Bedford 1 <MGD <10 Bedford OH Original selection Metropolitan Sewer District Little Miami WWTP 10 <MGD <100 Cincinnati OH Original selection

Northeast Ohio Regional Sewerage District Southerly WWTP > 100 MGD Cleveland OH Replacement for Easterly; Southerly is on the same system and completesthe processing of Easterly’s partiallytreated sewage sludge Delaware County Alum Creek WWTP 1 <MGD <10 Delaware OH Original selection Mingo Junction STP 1 <MGD <10 Mingo Junction OH Original selection Duncan Public Utilities Authority 1 <MGD <10 Duncan OK Original selection City of Klamath Falls WWTF 1 <MGD <10 Klamath Falls OR Replacement for South Suburban Western Westmoreland Municipal Authority 1 <MGD <10 Irwin PA Original selection

Allegheny County Sanitary Authority 1 <MGD <10 Pittsburgh PA Original selection Greater Pottsville Ar ea Sewer Authority 1 <MGD <10 Pottsville PA Original selection Punxsutawney 1 <MGD <10 Punxsutawney PA Original selection South Kingstown WWTF 1 <MGD <10 Narragansett RI Original selection Plum Island WWTP 10 <MGD <100 Charleston SC Original selection Lawson Fork WTP 1 <MGD <10 Spartanburg SC Original selection Elizabethton 1 <MGD <10 Elizabethton TN Original selection Amarillo 10 <MGD <100 Amarillo TX Original selection

Dallas Southside WWTP > 100 MGD Dallas TX Replacement for Dallas Central; Southside is on the same system and completes the processing of Dallas Central partially-treated sewage sludge Trinity River Authority of Texas 1 <MGD <10 Ellis County TX Original selection Fredericksburg 1 <MGD <10 Fredericksburg TX Original selection Odo J. Riedel Regional WWTP 1 <MGD <10 Schertz TX Original selection Wagner Creek WWTP 1 <MGD <10 Texarkana TX Original selection Tyler Southside WTP 1 <MGD <10 Tyler TX Original selection Spanish Fork City Corporation 1 <MGD <10 Spanish Fork UT Original selection Buena Vista 1 <MGD <10 Buena Vista VA Original selection

Everett City SVC Center MVD 10 <MGD <100 Everett WA Original selection; lagoon was sampled during dredging operations Beaver Dam 1 <MGD <10 Beaver Dam WI Original selection Elkins WWTP 1 <MGD <10 Elkins WV Original selection Huntington 10 <MGD <100 Huntington WV Original selection Facilities originally selected but not sampled

Coley Creek WWTP Alexander City AL Partial treatment – replaced by Sugar Creek City of Peoria, Beardsley WWTF Peoria AZ Partial treatment – dropped Osceola Osceola AR Lagoon – dropped Red Bluff WWTP Red Bluff CA Lagoon – dropped

MWRDGC North Side WRP Chicago IL Partial treatment – replaced by Calumet Water Valley Water Valley MS Lagoon – dropped Northeast Ohio Regional S D Easterly WWTP Cleveland OH Partial treatment – replaced by Southerly

South Suburban Sanitary District Klamath Falls OR Lagoon – replaced by City of Klamath Falls Saluda Hampton SC Lagoon - dropped

Dallas Central WWTP Dallas TX Partial treatment – replaced by Southside Moses Lake WWTP Moses Lake WA Lagoon - droppedTNSSS Sampling and Analysis Technical Report 7 January 2009 

### Section 3 Sample Collection

EPA collected samples of the final treated sewage sludge at each of the 74 POTWs that ultimately participated in the TNSSS. EPA deve loped a sampling and analysis plan that was peer-reviewed and describes the sample collection procedures in detail. EPA revised the plan periodically during the survey to address the cha nging list of facilities and to add updated contact information for the laboratories that performe d the analyses. As noted in Section 1.2, EPA sampled between August 2006 and March 2007.

3.1 Training

Prior to the start of sampli ng, the biosolids samplers were trained by the contractor. The contractor, with assistance from the Alexandria Sanitation Authority, Al exandria, VA, provided the samplers with instructions on sampling techniques, sample point selection, required paperwork, sample packing, and shipping techniques. The samplers also toured the Alexandria Sanitation Authority to become familiar with t ypical sewage treatment processes. The tour included demonstrations and hands-on traini ng in the collection of sewage sludge. Demonstrations included how to collect a range of samples, from liquid to dewatered sewage sludge.

3.2 Sample Collection

EPA began the sample collection process by identifying the number and nature of the types of sewage sludge produced at each faci lity. This effort took place during telephone conversations with the plant staff well in adva nce of sampling. Detail s were confirmed with plant staff upon gaining access to the final treated sewage sludge. Access to the treated sewage sludge was generally not difficult. However, in several instances, the samplers worked with plant staff to obtain samples from difficult locations where there might be safety concerns. Two facilities required that their pe rsonnel collect the actual samples. These instances are described in Section 3.3.

Grab samples were collected using sampling e quipment appropriate to the type of sewage sludge (liquid or solid) and the analytes of inte rest. To avoid or minimize contamination from sampling equipment, plastic equipment was used to collect samples for analyses of metals and anions, and stainless steel equipment was used to collect samples for analys es of all the organics.

Liquid samples were collected as free-flowi ng materials from storage tanks, transfer lines, taps, and hoses. After purging any lines us ed to collect samples, liquid samples were collected directly into the final sample containe rs shown in Table 3. Where possible, plant staff turned on mixing equipment in any storage tanks pr ior to sampling so that the collected liquids were representative of the bulk sewage sludge.

Solid samples included dewatered sewage sludg e. These samples were collected from a belt press, filter press, drying bed, centrifuge, co mpost pile, or other sour ce on site. The sampler collected small grab samples from multiple areas of any large piles, or multiple grabs from any continuous processes (e.g., belt press). Small grabs were composited in a large pre-cleanedTNSSS Sampling and Analysis Technical Report 8 January 2009 container of appropriate construc tion, mixed well, and the mixed sa mple was transferred into the final sample containers (see Table 3). Several ki lograms of material were collected for each type of treated sewage sludge and mi xed. Any mixed material that remained after all the sample containers were filled was returned to the sewage sludge process for disposal.

Grabs of solid samples for anions and metals analyses were collected with a large precleaned plastic serving spoon and mixed in a pr e-cleaned plastic wastebasket. Grab solid samples for organics analyses we re collected using a pre-cleane d stainless steel scoop and mixed in a pre-cleaned stainless steel bowl (8 to 12 qua rts). Separate sampling equipment was used for each facility and all equipment was cleaned prior to shipment to the facility.

Sample containers were purchased from co mmercial suppliers w ho provided certificates of analysis for common contaminants of interest (e.g., metals, semivolatile organics, pesticides, PCBs). The cleaning procedures applied by the vendors were pres umed to be sufficient for the other analytes in the survey fo r which routine testing by the vendor was not performed. At least two containers of each type were used to prepare equipment blanks as an overall check on possible contamination (see the discussion of equipm ent blanks later in Section 3.11). Both the high density polyethylene (HDPE) and glass containers were wi de-mouth designs, sealed with screw caps containing a polytetrafl uorothylene (PTFE) lid liner.

Table 3. Sample Containers for Solid and Liquid Sewage Sludge, by Analysis Fraction Analysis Fraction Solid Sample Container Liquid Sample Container Metals 500-mL wide-mouth HDPE 500-mL wide-mouth HDPE Polycyclic Aromatic Hydrocarbons (PAHs) and Semivolatiles (as one analytical fraction) 500-mL wide-mouth glass 1000-mL wide-mouth glass

Inorganic Anions 500-mL wide-mouth HDPE 500-mL wide-mouth HDPE Polybrominated Diphenyl Ether Congeners 500-mL wide-mouth glass 1000-mL wide-mouth glass

Antibiotics and Drugs 500-mL wide-mouth glass 1000-mL wide-mouth glass Steroids and Hormones 500-mL wide-mouth glass 1000-mL wide-mouth glass Archive Samples - for use in the event of breakage, lab accident, or for future EPA studies 2 500-mL wide-mouth HDPE and 4 500-mL wide-mouth glass 2 500-mL wide-mouth HDPE and 4 1000-mL wide-mouth glass

Total Containers per Sampling Point 12 12

3.3 Site-specific Deviations

Site-specific conditions at two facilities required modifications to the equipment protocols. At one facility, th e sewage sludge was discharged fr om a two-story tower with a belt press directly into a dump truck parked below. Access to the bed of the truck was not practical, even if the discharge was stopped temporarily. Therefore, the sta ff at this facility routinely collected samples in a polyethylene container m ounted on the end of a length of PVC pipe. In the presence of the EPA-contract or sampler, the facility sta ff inserted the device into the discharge from an opening in the tower, collected a small grab sample, and pulled the device back through the opening. Successive grab sa mples were collected through that opening and quickly placed into either the stainless steel or plastic compositing cont ainers held by the EPAcontactor sampler. The sampler carried the cont ainers down from the tower, mixed the samples in the compositing containers as described a bove, and then placed the samples into the appropriate final containers. The total contact time of each grab sample with the polyethylene sampling device was on the order of 30 seconds.TNSSS Sampling and Analysis Technical Report 9 January 2009 At a second facility, sewage sludge was di scharged into a roll-off dumpster body in a narrow building which allowed limited access. In the presence of the EPA sampler, one of the facility staff collected the sample in a 5-gallon pl astic bucket lined with a trash bag. A stainless steel scoop was used to remove aliquots of se wage sludge from the center of the bucket and transfer the material to a st ainless steel bowl. Once that portion was removed, a plastic spoon was used to transfer the sewa ge sludge to a plastic bowl.

3.4 Representative Samples

The TNSSS was designed to collect sewage sl udge samples that were representative of various types of sewage sludge. For bulk se wage sludge, collecting representative samples presented a challenge at some facilities. For ex ample, at one facility that composted its final sewage sludge, samples were collected from one of the long piles of sewage sludge mixed with woods chips. The piles were upwards of 50 feet long and over 6 feet high, with sides sloping up at roughly a 45 degree angle. Samples were collecte d from the oldest sections of the rows at the facility to represent the length of the typical co mposting period at the fac ility, which ranges from one to six months, depending on the season.

Samples of biosolids materials were taken by di gging into the side of the compost pile at roughly six points along its length, on both sides of the pile, a foot or more off the ground to avoid materials in contact with the concrete substrate. Materi als removed from the pile often contained large chunks of wood or small branches. Because these materials would not fit into the sample containers, they were removed from the compositing containers before mixing the bulk sample. Once the bulk sample was well mixed, the samples were transferred to the final sample containers. This procedure was repe ated twice: 1) for samples for the organic parameters, using stainless stee l equipment and glass containers, and 2) for the metals and anions, using plastic equipment and containers.

At another facility which produced liquid se wage sludge, samples were collected from a catwalk atop a 1-million gallon storage tank. Sewage sludge was introduced into the tank by water cannon with a 4-inch diam eter discharge nozzle. Plan t personnel turned on the water cannon and throttled back the flow to a relativ e trickle and the sampler held each sample container in the edge of the stream until it was fu ll. The containers were capped once they were full and wiped down before packing. Neither of th ese situations means that the samples were not representative or that the Agency can not rely on the results obtained. It simply points out the complexities and challenges with sampling sewage sludge generated by the variety of treatment processes and management op tions available nationally.

3.5 Field Duplicates

The sampling plan called for collection of field duplicate samples at 10% of the facilities. A field duplicate sample is a second sample collected at the facility using similar procedures and equipment as the original sample for quality c ontrol purposes. The result s of the field duplicate sample can be compared to the results of the or iginal sample as a means of assessing the overall precision of the sampling and analysis processes.TNSSS Sampling and Analysis Technical Report 10 January 2009 Note: The 10% frequency for field duplicates is a common, but arbitrary, choice designed to balance the cost of the additional samples agai nst the desire to assess the precision of the sampling and analysis processes.

Eight facilities were originally selected fo r collection of field duplicates. This number was ultimately reduced to six because two of the facilities at which field duplicates were to be collected were dropped from the survey and not replaced (for reasons de scribed in Section 2).

Separate EPA sample numbers were assigne d to the field duplicates so they were not identifiable by the laboratories as duplicates. The results of the field duplicate analyses are discussed in Section 6.

3.6 Sample Labeling and Tracking

The EPA contract samplers labeled each container with a preprinted EPA Sample Number. An example label is shown at the right. The EPA sample number was specific to each sewage sludge sample at the facility.

In addition to labeling each container, the samplers prepared an EPA Traffic Report that documente d the origin of the samples. The sampler recorded the name of the facility, date of sampling, sampler’s name, and shipping airbill number on the traffic report. The numbers of sample contai ners of each type (e.g. , four plastic and eight glass) that were collected were recorded. The tr affic report prepared at the site allowed EPA to track shipments of samples to the EPA Samp le Repository at Microbac Laboratories in Baltimore, MD.

3.7 Packing and Shipping Samples to the Repository

The sample containers were packed for shi pping using procedures described in the peerreviewed sampling and analysis plan. Each samp le container was either encased in bubblewrap bag or layers of bubblewrap sheeting to preven t its movement during shipping. Samples were packed into sturdy plastic ice ches ts. All of the samples from a si ngle site could be packed, with ice and bubblewrap, in one 48-quart ice chest, or two 28-quart ice chests, depending on availability.

The samplers purchased ice near each facili ty, or the POTW provided ice, and packaged it in one-gallon self-sealing pl astic bags. Approximately one pound of ice was used for each sample container (e.g., four bags containing tw o pounds of ice each were used to cool eight samples in a 28-quart ice chest). To prevent leakage during shipping, each ice chest was lined with bubble wrap and two trash bags. Samples and ice were packed into the inner bag, and then tied or sealed shut with tape. Additional pack ing materials were placed around the inner bag, if needed, and the outer bag sealed shut. The comp leted traffic report was placed in a plastic bag and affixed to the underside of the lid of the ice chest with either tape or a plastic airbill pouch.

E ach cooler was shut with a layer of duct tape placed horizontally across the seam between the ice chest and its lid. Packaging tape or filament tape was applied to the cooler vertically, one band near each end of the ice chest, to secure the lid. EPA Sample No. 68408

POTW Sewage sludge

Date collected ___________

Sampler Initials ___________TNSSS Sampling and Analysis Technical Report 11 January 2009 Ice chests were shipped overnight from fu ll-service FedEx offices to the EPA Sample Repository. Samples collected from multiple facilit ies in a given day were shipped at the end of the day, or depending on the logistics, sent sepa rately from different FedEx locations. Each sample shipment was tracked through the carrier ’s web site and EPA confirmed receipt at the repository.

Over the course of the survey, samples fr om two facilities were hand carried to the repository in Baltimore, because the facilities we re located nearby. These samples were packed in a similar fashion as those sent by FedEx, except that no airbills were prepared.

3.8 Storage and Shipments to Laboratories

When samples arrived at the sample reposit ory, the staff inspected the ice chests for external damage or leakage (none occurred) an d placed them in one of two walk-in freezers dedicated to EPA samples and maintained at - 11°C. Freezing at - 11°C reduces microbiological activity and the rates of any chemical reactions that might lead to changes in the sample.

To streamline the shipping l ogistics and manage both shipping and analytical costs, EPA shipped batches of 15 to 20 samples from the repository to the contract laboratories for analyses. EPA prepared new traffic reports that listed the samples in each shipment. Additional shipments were sent to the laboratories as more facilities were sampled. In all, six shipments were sent to the laboratory performing the an alyses of metals, anions, and or ganics, with the last shipment being the two samples collected at the last facility. For the PBDEs, pharmaceuticals, and steroids and hormones analyses, more samples had been collected and stored at the repository by the time those analyses began. Ultimately, th ree shipments were made to the laboratory performing the PBDE analyses and three shipment s were made to the laboratory performing the pharmaceuticals, steroids, and hormones analyses.

During the packing process prior to shipme nt, EPA examined the samples for signs of breakage, including cracked gla ss jars and cracked lids. Al though some cracking and breakage did occur in jars or lids, as described below, it was observed th at neither cross-contamination occurred due to frozen conditi ons nor were any samples lost or not available for analysis.

Only three incidents of breakage were observe d and these containers were not shipped to laboratories, but were packaged in separate plasti c bags and returned to the freezer. The jars may have cracked during freezing, as the cracked jars were all of the 1000-mL size used for liquid samples. While the samplers took care not to f ill any of the jars to more than 90% of their capacity, the high water content of the liquid se wage sludge samples could lead to greater expansion during freezing. This may have result ed in a greater risk of breaking the 1000-mL glass jars than their 500-mL count erparts used for solid samples.

All samples sent from the repository were shipped frozen, with la rge quantities of ice added to all coolers. The labor atories inspected the samples on r eceipt and reported that all the coolers still contained ice. The laboratories also reported that a small number of samples (<15) were received with cracked lids or jars, which may have cracked during sh ipping. In each case, the laboratory transferred the sample to a suit able clean container or otherwise protected the contents, such that no samples were lost as a result of shipping. The laboratories stored theTNSSS Sampling and Analysis Technical Report 12 January 2009 samples frozen until analysis. Each laboratory retu rned a copy of the traffi c report, with the date and time of sample receipt documented on the form.

Methods for the analysis of pharmaceuticals, steroids, and hormones in sewage sludge were not available when sample collection be gan in August 2006. Therefore, samples were stored frozen (-11°C) until methods for these an alytes were available. Except for the two samples collected from one plant in Marc h 2007, all of the samples analyzed for pharmaceuticals, steroids, and hormones were stored frozen for 11 to 15 months.

For the purpose of this survey, EPA assumed th at all the analytes of interest were stable in sewage sludge samples stored at -11°C. Becau se the analyses for metals involved the “total recoverable” concentrations (e.g., all of the metal that can be recovered during digestion with a strong acid), holding frozen samples for extended periods is not a concern because the metals will be recovered even if there were any residu al microbiological or chemical activity in the frozen samples. For the anions and semivola tile organics, EPA also did not anticipate any substantive changes in the concentrations of th ese analytes during storage due to the relatively low storage temperature.

The samples analyzed for pharmaceuticals, steroids, and hormones were held in storage longer than any of the other samples. The stabil ities of these analytes have not been studied by EPA. However, EPA began a holding time st udy of pharmaceuticals in aqueous samples and sewage sludge in late 2008 that may provide some data to assess the freezer storage stability of these analytes in the near future.

3.9 Shipping Issues

EPA tracked each sample shipment through the carrier’s web site, or confirmed receipt by contacting the laboratory directly. Only two shipping problems occurred, a surprisingly small number, given the number of shipments involved. In one instance, the laboratory performing the metals and anions analyses was sent the inco rrect number of sample containers, which was resolved. In a second instance, the airbills applied to two sim ilar ice chests containing samples for different laboratories were switched and the wrong types of containers were sent to each laboratory. The error was discovered when the sa mples were received at each laboratory the next morning. Both laboratories kept the samples frozen until they were able to ship them to the intended recipient.TNSSS Sampling and Analysis Technical Report 13 January 2009 3.10 Sampling Summary

The overall scope of the sample collecti on effort is summarized in Table 4 below.

Table 4. Summary Sampling Statistics People Travel Shipping A total of 12 samplers visited 74 plants and collected 84 samples. This included 6 field duplicates and additional samples at 4 plants that produced 2 types of final sewage sludge. 46 one-way airline flights More than 150 containers of sampling supplies and equipment were shipped to field locations during the survey. Over 3,100 pounds samples and ice packed in 108 coolers were shipped to the EPA Sample Repository in Baltimore, MD, via FedEx. An additional 4 coolers were hand delivered to the repository.

Each sampler visited between 2 and 14 plants and collected 12 jars of sewage sludge per plant, for a total of 1,002 jars. 12 samplers spent 107 days on the road and drove over 19,000 miles EPA shipped 427 jars to commercial labs for the analyses of metals, anions, organics, PBDEs, pharmaceuticals, steroids, and hormones. Samples from each site remain in an archive at the repository for possible future analyses.

3.11 Equipment Blanks

All of the equipment that came into contact with samples during the collection process was made of stainless steel (for organics) or plastic (for metals and anions) and was used for only one facility to avoid potential cr oss-contamination between sites. Prior to sending equipment to the field, all of the stainless steel and plasti c scoops, spoons, and bowls were washed thoroughly with a non-phosphate detergent, rinsed three times with tap wa ter, rinsed once with reagent water, inverted and air dried. Once dry, stainle ss steel equipment was wrapped in aluminum foil and plastic equipment was sealed in plastic bags . No field cleaning of equipment was performed during the survey. Liquid samples were placed di rectly into appropriate containers, while solids samples were placed in appropriate c ontainers using scoops and spoons.

There is no relevant clean solid “reference ma trix” for sewage sludge that could be easily used to prepare equipment blanks for the variet y of analytes in this survey. Therefore, equipment blanks were created as follows. Two se ts of the relevant equipment were sent to each laboratory performing analyses of anions, meta ls, semivolatiles organics, and PBDEs (e.g., two stainless steel compositing bowls, two stainless stee l scoops, and two glass jars for the organics). For the semivolatile organics and PBDEs, there we re two styles of bowls, based on availability, and two sizes of glass jars (500-mL and 1000-mL wide-mouths). Because the glass jars were purchased with certificates of analysis for co mmon organics, EPA did not anticipate any effect due to the jar size. Therefore, EPA assembled one blank using each style of stainless steel bowl and included one jar size with one style bowl and the other jar size with the other bowl. For the plastic equipment, EPA used only one style of plastic compositing bowl and one size high density polyethylene resin (HDPE) jar, so four identical sets of equipment were sent to the laboratory performing the metals and anions analyses (two sets for each class of analytes).

Each laboratory used the equipment to prep are the equipment blank by rinsing each piece with the solvents or solutions us ed to prepare field samples. Fo r organics, including PBDEs, the laboratories poured the same volumes of extracti on solvents used for samples over the scoop into the bowl. The analyst carefully swirled the solvent in the bowl to contact the majority of the inner surface, and then poured it into the glass jar. For metals, the laboratory used the acidic sample digestion solution to contact all of the plastic materials. For the anions, the laboratory used reagent water to “extract” the samples in a similar fashion. The laboratory treated theTNSSS Sampling and Analysis Technical Report 14 January 2009 solvent or solution as if it came from a nominal size field sample and the reported the results accordingly, on a dry-weight basis.

The results for these equipment blanks, or ri nsates, represent worst-case estimates of the potential contributions of the equi pment to the final sample results. The worst-case nature of the estimate reflects the fact that the samples were solids, and even with vigorous mixing during the compositing steps, not all of the surface area of the sample contacted the entire surface of the bowl. The samples themselves did not contain solvents, or acidic solutions, so the potential transfer of contaminants would be expected to be much less than using the sample preparation solutions. Finally, any contaminants that we re transferred from the equipment would be associated with the bulk composite and the final sample sent to the laboratory was only a portion of the total material in the bow l. In practice, 6 to 8 liter s of solid sewage sludge were composited to provide material to fill eight 500 -mL glass jars, or partially fill eight 1000-mL jars, for the organics analyses. For the metals and anions, only four plastic 500-mL jars were filled. The remainder of the composited material was returned to the sewage sludge disposal

process.

The results of the equipment blank analyses for the anions, metals, and semivolatile organics are discussed in detail in Section 6.6. Based on EPA’s e xperience with the semivolatile organic analytes of interest in the survey, and given the fact that EPA had exhausted the supply of one of the types of glass containers used to collect samples, EPA opted not to submit equipment blanks to the laboratory analyzing the pharmaceuticals, steroids, and hormones. There was no issue with organic compounds in general regarding contam ination of equipment (e.g., through ambient air, equipment supplier, or laboratory contamination). Thus, the Agency does not believe that contamination with pharm aceuticals, steroids and hormones would have been an issue.TNSSS Sampling and Analysis Technical Report 15 January 2009 

### Section 4 Sample Analyses

4.1 Analytes of Interest

The TNSSS was designed to do two things: 1) obtain updated occurrence information on nine pollutants of potential concern, and 2) obtain occurren ce information on a number of contaminants of emerging interest identified by EPA and the National Re search Council (NRC).

As discussed in Section 1, EPA identified nine pollutants (shown in bold in Table 5) for further evaluation of occurrence in sewage sludge. This evaluation was based on an assessment of chemical pollutants for which EPA had adequa te data (e.g., human health benchmark values, and information on fate and transport in the environment).

Given the national scope of the survey, EPA e xpanded the list of analytes to reflect the Agency’s interest in collecting concentration data for other chemicals (see Tables 5 and 6). The expanded list included 24 additional metals that could be analyzed at little extra cost at the same time as the four metals (barium, beryllium, mang anese, and silver) included in the list of nine pollutants above; molybdenum because of the Agency ’s interest in determining the need for a revised numeric standard for it in land-applied bi osolids; and other analytes because of their widespread incidence and use and emergi ng concern. The latter category included:

 benzo( a )pyrene (found in coal tar, automobile exhaust fumes, tobacco and wood smoke, charbroiled food, and burnt toast);

 2-methylnaphthalene (found in nonstructu ral caulking compounds and sealants, synthetic resins, rubber adhesi ves, and wall coverings);

 bis (2-ethylhexyl) phthalate (w idely used as a plasticizer in manufacturing of items such as cosmetics, toys, tool s, and laboratory equipment);

 fluoride (used in topical and systemic ther apy for preventing tooth decay, as well as many other uses);

 water-extractable phospho rus (correlated with phosphorus concentration in runoff from soils amended with manure and bioso lids and an indicator of loss that may contribute to algae buildup in surface waters);

 11 polybrominated diphenyl ethers (PBDEs). Four of the PBDEs were of most interest because of available human health information that may be useful for future risk evaluation efforts. PBDEs are used as flame retardants in a wide array of products, including building ma terials, electronics, furnishings, motor vehicles, plastics, polyurethane foams, and textiles; and

 97 pharmaceuticals, steroids, and hormones b ecause of broader emerging interest in these analytes.TNSSS Sampling and Analysis Technical Report 16 January 2009 Table 5. Primary Target Analytes for the TNSSS, by Analyte Class Analyte Class Analyte Aluminum Manganese Antimony Mercury* Arsenic* Molybdenum* Barium Nickel* Beryllium Phosphorus Boron Selenium* Cadmium* Silver Calcium Sodium Chromium* Thallium Cobalt Tin Copper* Titanium
Iron Vanadium Lead* Yttrium Metals

Magnesium Zinc* Benzo(a)pyrene 2-Methylnaphthalene Polycyclic aromatic hydrocarbons (PAHs) Fluoranthene Pyrene Other semivolatile organics bis (2-Ethylhexyl) phthalate 4-Chloroaniline
Fluoride Water-extractable phosphorus Inorganic anions Nitrate Nitrite 2,4,4'-TrBDE (BDE-28) 2,2',3,4,4',5'-HxBDE (BDE-138) 2,2',4,4'-TeBDE (BDE-47) 2,2',4,4',5,5'-HxBDE (BDE-153) 2,3',4,4'-TeBDE (BDE-66) 2,2',4,4',5',6-HxBDE (BDE-154) 2,2',3,4,4'-PeBDE (BDE-85) 2,2',3,4,4',5',6-HpBDE (BDE-183) 2,2',4,4',5-PeBDE (BDE-99) 2,2',3,3',4,4',5,5',6,6'-DeBDE (BDE-209) Polybrominated diphenyl ethers (PBDEs), including the Tetra, Hexa, Penta, and Deca congeners

2,2',4,4',6-PeBDE (BDE-100) The 9 pollutants in bold are those selected in the December 2003 Biennial Review * Metals currently regulated at 40 CFR 503

Among the other “new and emerging contamin ants” of concern in the NRC report were various pharmaceuticals, steroids, and hormones for which several EPA organizations were developing methods at the time that the TN SSS was being planned. EPA included certain pharmaceuticals, steroids and hormones in th e TNSSS for which analytical methods were developed. Given the time required to develop and test new methods, EPA proceeded with the sample collection effort for the TNSSS as descri bed in Section 3, and stored samples for the analyses of these analytes of interest until su ch time as the new methods for these classes of compounds were more fully developed. The drugs, antibiotics, steroids, and hormones added to the TNSSS are shown in Table 6.TNSSS Sampling and Analysis Technical Report 17 January 2009 Table 6. Pharmaceuticals, Steroids, and Hormones Included in the TNSSS Analyte Class Analyte Anhydrochlortetracycline Ofloxacin Anhydrotetracycline Ormetoprim Azithromycin Oxacillin Carbadox Oxolinic acid Cefotaxime Oxytetracycline Chlortetracycline Penicillin G Ciprofloxacin Penicillin V Clarithromycin Roxithromycin Clinafloxacin Sarafloxacin Cloxacillin Sulfachloropyridazine Demeclocycline Sulfadiazine Doxycycline Sulfadimethoxine Enrofloxacin Sulfamerazine 4-Epianhydrochlortetracycline Sulfamethazine 4-Epianhydrotetracycline Sulfamethizole 4-Epichlortetracycline Sulfamethoxazole 4-Epioxytetracycline Sulfanilamide 4-Epitetracycline Sulfathiazole Erythromycin Tetracycline Flumequine Triclocarban Isochlortetracycline Triclosan Lincomycin Trimethoprim Lomefloxacin Tylosin Minocycline Virginiamycin Antibiotics and their degradation products, disinfectants, and other antimicrobials

Norfloxacin 1,7-Dimethylxanthine Diphenhydramine Acetaminophen Fluoxetine Albuterol Gemfibrozil Caffeine Ibuprofen Carbamazepine Metformin Cimetidine Miconazole Codeine Naproxen Cotinine Norgestimate Dehydronifedipine Ranitidine Digoxigenin Thiabendazole Digoxin Warfarin Other drugs

Diltiazem Campesterol Epi-coprostanol Cholestanol Ergosterol Cholesterol β -Sitosterol Coprostanol β -Stigmastanol Steroids

Desmosterol Stigmasterol

Androstenedione Estriol

Androsterone Estrone

17 α -Dihydroequilin 17 α -Ethynyl estradiol Equilenin Norethindrone Equilin Norgestrel

17 α -Estradiol Progesterone

17 β -Estradiol Testosterone Hormones

β -Estradiol-3-benzoateTNSSS Sampling and Analysis Technical Report 18 January 2009 4.2 Analytical Techniques

Table 7 presents the analytic al techniques applied to samples in the TNSSS. The target reporting limits in the table are based on a consen sus of what might be achievable in a sewage sludge sample. The actual reporting limits achieved ar e discussed in Section 6.5.

Table 7. Analytical Methods or Techniques

Analyte Class Method or Technique Target Reporting Limit (dry weight)

28 Metals, including mercury ICP/AES, ICP/MS, and CVAA (EPA Methods 200.7, 2 00.8, and 245.1) 3 to 4 mg/kg

4 Polycyclic aromatic hydrocarbons (PAHs) and 2 semivolatiles (as one analytical fraction) GC/MS, with selected io n monitoring (SIM), after solvent extraction and gel permeation chromatography (GPC) cleanup (EPA SW-846 Method 8270C) 100 to 300 μg/kg

4 Inorganic anions, including waterextractable phosphorus (WEP) EPA Methods 340.2, 353.2, and 365.3, after leaching of the solid sample with reagent water with a study-specific protocol 2 to 8 mg/kg

11 PBDE Congeners* High resolution GC/MS, draft EPA Method 1614 5 to 200 ng/kg

72 Pharmaceuticals High performance liquid chromatography (HPLC) with tandem MS/MS detection, using an early draft of EPA Method 1694† Not specified

25 Steroids and hormones High resolution GC/MS, using an early draft of EPA Method 1698† Not specified

* The list of target PBDE analytes was limited to the following 11 PBDE congeners: 28, 47, 66, 85, 99, 100, 138, 153, 154, 183, and 209, which include those identified in the method as being of potential environmental or public health significance. There are some differences between the specifics of the method and the proc edures used for the TNSSS (see Section 4.4.4 of this report).

† The laboratory solicitation and contract were issued prior to the December 2007 formal release of EPA Methods 1694 and 1698 and as a result, there are some differences between the specifics of those methods and the procedures used for the TNSSS (see Sections 4.4.5 and 4.4.6 of this report).

mg/kg = milligrams per kilogram μg/kg = microgr ams per kilogram ng/kg = nanograms per kilogram

As indicated, the survey used both well -established multi-laboratory validated EPA procedures as well as three anal ytical methods that were devel oped or updated for the survey. The two new methods are single-lab validated methods for pharmaceuticals (EPA Method 1694), and steroids and hormones (EPA Method 1698). The updated multi-lab validated method is for flame retardants (EPA Method 1614). These thr ee methods have not yet been promulgated at 40 CFR Part 136 for compliance monitoring in CWA programs, including the analysis of sewage sludge.

4.3 Laboratories

EPA awarded competitive-bid analytical contracts to the following commercial laboratories:

 Columbia Analytical Services for the anal yses of the metals, anions, and PAHs and semivolatile organics

 Severn Trent Laboratories (now part of Test America) for the analyses of the PBDEs

 Axys Analytical Services for the analyses of pharmaceuticals, steroids, and hormones.TNSSS Sampling and Analysis Technical Report 19 January 2009 4.4 Method Modifications

From an analytical standpoint, sewage sludge is a challenging matrix. The concentrations of pollutants present in samples vary depending on th e nature of the inputs to the treatment plant. In addition to the pollutants of interest, sewage sludge contai ns a number of other components that are potential interferences in the analyses of the pollutants of interest. These components include lipids and other na turally occurring materials, as well as materials that may be added to the sewage during processing (e.g., surfactants, ferric chloride, pol ymeric colloids, or lime). These components can manifest themselves as in terferences at all stages of the analytical process, from sample preparation through the determinative analysis.

Another analytical challenge with a national survey of sewa ge sludge is that the various treatment process and disposal practices used na tionwide lead to differences in the moisture content of the final sewage sludge sent for use or disposal. Some of the facilities from which samples were obtained in the TNSSS produce liquid final sewage sludge, while others produce solid sewage sludge. Among the sewage sludge th at were pourable liquids, the percent solids (hereafter percent solids) content ranged from less than 1% to a bout 4%, across treatment plants. For the solids, the percent solids content ranged fr om 5% to 99%. These differences in the form (liquid vs. solid) of the sewage sludge and the range of moisture or solid contents have direct effects on the analyses of the samples. The diffe rences also affect how the data for the survey can be interpreted.

Recognizing these challenges, EPA structured the laboratory subcont racts for the various analyses to achieve the most uniform results acro ss facilities as practical . These modifications are described below.

4.4.1 Ensuring Consistent Method Sensitivity

Many analytical methods applicable to bios olids instruct the laboratory to prepare a specific known weight of a solid material for analysis (e.g., some methods for organics specify using 30 g of sample). However, that samp le aliquot may contain significant amounts of moisture. Those same methods may treat samples that are pourable liquids as if they contain little or no solids, and specify using a known volume, such as 1 L, for the analysis, although that volume may contain measurable solids as well. These differences in how liquid and solid samples are prepared and analyzed, as well as the differences in the amount of solids or moisture in the two types of samples, mean that any m easure of method sensitiv ity (e.g., a reporting limit or a detection limit) will depend on the initial mass or volume chosen for analysis and its moisture content.

EPA considered these effects on sensitivity and comparability when it planned the TNSSS. EPA minimized the potenti al sensitivity differences by in structing the la boratories to determine the percentage of solids (percent soli ds) of each sample first, and then use that information to select a portion of the sample fo r the analysis that contains the method-specified sample weight or volume on a dry-weight basi s. In addition, even when the laboratories prepared liquid samples using procedures de signed for aqueous samples (e.g., liquid-liquid extraction with an organic solvent), they were in structed to report the results in weight/weight units (e.g., ng/kg,  g/kg, or mg/kg) appropriate for the class of analyte, adjusted for the moisture content of the sample (e.g., 100% dry sample).TNSSS Sampling and Analysis Technical Report 20 January 2009 The laboratory that performed the analys es of the metals, anions, and PAH and semivolatile organics was instructed to determin e the percent solids separately for each class of analytes. EPA examined the percent solids data from each class for the first 35 samples and determined that there was no statistically signifi cant difference between the three measurements on each EPA sample (see Section 6.7.7). Nevertheless, in order to ensure the most consistent sensitivity across the survey, EPA instructed th e laboratory to continue to determine percent solids on each sample for each class fo r the remainder of the project.

4.4.2 Anions in Sewage Sludge

Methods for determining anions (e.g., nutrien ts) in sewage sludge samples require that the anions be dissolved in water and separated from the solid material. In planning the TNSSS, EPA considered several approaches to preparing the sewage sludge samples for the analysis of anions, including:

 EPA Method 1685, Nitrate/Nitrite-N in Water and Biosolids by Automated Photometry, Draft January 2001;

 EPA Method 1688, Total Kjeldahl Nitrogen in Water and Biosolids by Automated Colorimetry with Preliminary Semi-aut omatic Digestion, Draft January 2001;

 A water extraction (or leaching) procedur e developed at the Pennsylvania State University (Vadas, P. A. and Kleinman, P. J. A., 2006).

The two draft EPA methods cited above have onl y been validated in a single lab. Neither of these methods has been promulgated at 40 CFR Part 136 for compliance monitoring in CWA programs, nor for the analysis of sewage sludge samples. As a result, few, if any, laboratories routinely run samples using those draft methods.

The method developed at Penns ylvania State University was published in th e literature and has been used by several mid-western states that regulate the appl ication of manure and biosolids to agricultural lands. Ho wever, the authors only used that procedure for the analysis of phosphorus, and not the other anions.

After reviewing the Vadas and Kleinman l eaching procedure, EPA concluded that the leachate was amenable to analyses of the anions of interest in the TNSSS by existing commonly used EPA methods that are a pproved at 40 CFR 136. Therefor e, EPA decided to use the leaching procedure to prepare all of the sewage sludge samples and have the anion analyses performed using the following EPA methods:

 Method 340.2, Fluoride, Potentiometric, Ion Selective Electrode, March 1983

 Method 353.2, Nitrate-Nitrite, Colorimetr ic, Manual Cadmium Reduction, March 1983

 Method 365.3, Phosphorus, All Forms, Colori metric, Ascorbic Acid, Two Reagent, March 1983TNSSS Sampling and Analysis Technical Report 21 January 2009 Appendix A of this report provides th e TNSSS-specific directi ons for the leaching procedure. Briefly, th e procedure involves:

 Determining the percent solids of the orig inal sample using standard procedures

 Weighing a sample aliquot equal to 0.5 g (dry weight) into a plastic bottle

 Adding 100 mL of reagent water

 Shaking the bottle on a shaker table for 60 minutes at 70 RPM

 Centrifuging the mixture for 10 minutes at 2000 RPM

 Filtering the sample by gravity through a Whatman #2 filter

 Preserving the aqueous leachate sample by adding H 2 SO 4 to pH <2

 Analyzing the leachate sample within 48 hour s (the holding time for nitrate/nitrite)

 Reporting all results in mg/kg, based on the or iginal 0.5-g sewage sludge sample weight.

The leaching procedure includes steps that will preserve the relative proportions of nitrate and nitrite after the sewage sludge sample is leached (e.g., acid preservation and analysis within 48 hours). However, it is not pos sible to determine whether or not the leaching process itself resulted in oxidation of nitrite to nitrate, or vi ce versa. Therefore, EPA decided that nitrate and nitrite would be analyzed as the combined parameter nitrate/nitrite, usi ng Method 353.2. This is approach is acceptable for determining better ex posure scenarios for nitrate/nitrite during land applicable of biosolids.

In addition, the analytical approach for the survey included determining the element phosphorus (P) as part of the suite of meta ls. The water-extractable phosphorus (WEP) determined using the leaching procedure above is a useful predictor of the concentrations of phosphorus that might be available for runoff from land to which sewage sludge has been applied. The ratio of the two forms of phosphorus (WEP/P) is an indication of the proportion of the total phosphorus applied that ma y contribute to runoff. That ratio may be of interest to those states that regulate land app lication of sewage sludge.

Note: The reader is cautioned about making compar isons between the anion results from this survey and data from other sources that may have used different procedures to leach the anions from the sample. EPA’s use of dry- weight reporting units may facilitate such comparisons, but other differences among leaching procedures may still influence the results.

4.4.3 Modified GC/MS Procedures for Semivolatile Organics

Gas chromatography, coupled with mass sp ectrometric detecti on (GC/MS), is the backbone of the analytical methods for many organic pollutants, including the PAHs and semivolatile organics of interest in the TNSSS. The most common form of GC/MS analysis is known as “full scan” GC/MS and involves examini ng all of the mass fragments in a wide mass range that exit the GC column and reach the MS detector. A typical GC/MS method will scan a mass range from 35 to 450 atomic mass units (amu) once every second. The masses of any materials that exit the GC column in that ra nge will be recorded and used to identify the pollutants of interest.TNSSS Sampling and Analysis Technical Report 22 January 2009 Some EPA GC/MS methods incl ude several hundred target an alytes, and while full-scan GC/MS is a powerful technique, it involves tradeoffs in sensitivity and selectivity in order to be applied to large number of analytes simultaneous ly. Full-scan GC/MS can also be subject to interferences from other materials in the sample that are not of interest.

Some of these “co-extracted” interferences include biolipid s that can be removed from the sample extracts using established cleanup pro cedures. One such procedure is gel permeation chromatography (GPC), which segregates the re latively small pollutant molecules from the larger lipids and other interfer ences on the basis of molecular size. Based on our experience in two previous sewage sludge surveys, EPA require d every sample extract analyzed for the PAHs and semivolatile organics be subjec ted to GPC cleanup before analysis.

When a GC/MS instrument is operated in full-scan mode, it scans the entire mass range very quickly and there is relatively little time to observe the results at a ny given mass within that range. This places some practical limits on the sensitivity of the procedure. However, those limitations can be overcome by using the technique known as selected ion monitoring (SIM). In SIM, the MS instrument only looks for a small su bset of masses (ions) in the overall mass range. These masses are ones associated with the list of target analytes, and any other masses that exit the GC column are simply ignored. The ions th at are ignored may be those associated with interferences, including biolipids, or those from analytes in a full-scan method that are not of interest for a given project. Because the MS can spend more time looking for fewer masses, the sensitivity of the instrument for those pollutant s with those masses can increase 10-fold or more over that of a full-scan procedure.

EPA examined the results for the first 50 samples that were analyzed by full-scan GC/MS and found that for many of those samples, the re porting limits achieved by the laboratory were much higher than anticipated. In other words, analytes were reported as “not detected” at concentrations greater than the reporting limits desired for the survey. Many of the increased reporting limits were due to large amounts of interferences that remained in the sample extract even after GPC cleanup, which required dilution of the extract. The labor atory also diluted a smaller number of samples in order to get one or two target analytes (usually bis [2-Ethylhexyl] phthalate) within the instrument calibration range , resulting in a loss of sensitivity for other analytes present at much lower concentrations.

In response to these early findings, EPA re quired that the labora tory reanalyze the extracts for about 35 samples using a SIM proc edure. That procedure included masses that represented the four PAHs and two semivolatile organi cs that are target analytes in the survey, as well as the surrogate compounds and internal st andards used in the full-scan method. The masses used in the SIM procedure are shown in Table 8. Based on successful analyses of these extracts, the SIM procedure was the only GC/MS procedure employed for the analysis of the remaining samples in the survey.

Table 8. Selected Ion Monitoring Parameters for Organic Analytes Type Analyte Quantitation Mass Approximate Retention Time (min)* 4-Chloroaniline 127 9.71 2-Methylnaphthalene 142 10.49
Fluoranthene 202 15.04 Pyrene 202 15.32 Target Analytes

bis (2-Ethylhexyl) phthalate 149 16.61TNSSS Sampling and Analysis Technical Report 23 January 2009 Table 8. Selected Ion Monitoring Parameters for Organic Analytes Type Analyte Quantitation Mass Approximate Retention Time (min)* Benzo(a)pyrene 252 18.12

Nitrobenzene-d 5 82 8.67 2-Fluorobiphenyl 172 10.94 Surrogates p-Terphenyl-d 14 244 15.49 1,4-Dichlorobenzene-d 4 152 7.90 Naphthalene-d 8 136 9.59 Acenaphthene-d 10 164 11.77
Phenanthrene-d 10 188 13.57 Chyrsene-d 12 240 16.63 Internal Standards

Perylene-d 12 264 18.18 *Retention times are specific to the GC column and operating conditions used for these samples. Retentions times on other columns or instruments will differ. These data are presented so lely to illustrate the relationships among the target analytes, surrogates, and internal standards.

4.4.4 PBDE Analyses

The original plan for the survey was to an alyze for all 209 of the PBDE congeners using the latest draft of EPA Method 1614, a high reso lution GC/MS isotope dilution procedure. Method 1614 is a highly sensitiv e procedure that can determine PBDEs at the part per trillion (ng/kg) levels in solid samples. It employs at least five clea nup techniques, including GPC, to remove interferences from sample extracts. The method employs isotope dilution quantitation, in which PBDE congeners synthesized using only carbon 13 ( 13 C), a stable (nonradioactive) isotope of carbon, are added to th e sample prior to extract. Th ese isotopically labeled congeners do not occur in nature and are used as intern al standards to quantif y the unlabeled PBDE congeners. Because the labeled congeners are ca rried through the entire sample preparation, cleanup, and analysis process, they can be used to correct for any loss of analytes during the overall analysis, providing a more accurate result for each unlabeled target analyte. For this reason, EPA uses isotope dilution in many of the 1600-series methods, including those for dioxins, furans, PCBs, and PBDEs.

Shortly after work began on the first batch of survey samples, the laboratory and EPA realized that additional efforts would be requi red to adequately determine PBDEs in sewage sludge. Ultimately, EPA agreed to permit the use of a range of modifications to the original plan to overcome as many analytical difficulties as pr actical in the time permitted for the TNSSS. Appendix B contains a detailed li st of those modifications. The most significant modifications

were:

 Due to the high levels of some congeners and some interferences, the initial sample size was reduced from 10 g to 2 g for extracti on, and ultimately to 0.2 g (dry weight).

 Samples were not spiked with labe led congeners prior to extraction.

 The sample extracts were concentrated to a final volume of 10 mL after extraction.

 A 1-mL aliquot of the extract wa s removed for spiking and cleanup.

 Labeled congeners were spiked into the 1-mL extract, which was then carried through the following cleanup steps described in the method: silica gel, GPC, and alumina. The remaining 9 mL of extract were retained in case dilutions or a dditional cleanups were required.

 After cleanup, extracts were analyzed by HRGC/HRMS.TNSSS Sampling and Analysis Technical Report 24 January 2009  The list of target an alytes was limited to the follo wing 11 PBDE congeners: 28, 47, 66, 85, 99, 100, 138, 153, 154, 183, and 209, which includes those identified in the method as being of potential envi ronmental or public health significance.

 Data were examined to determine if a larger sample size (greater than 0.2 g) was required to achieve the desired sensitivity, or if the extract required dilution to keep analytes within the calibration range. Th e latter was much more common.

 If congeners were present a bove the upper limit of the calib ration range, the laboratory evaluated whether or not the peaks had saturate d the detector. If sa turation did not occur, the results were reported, but flagged in the database as exceeding the calibration range. This approach reduced the dilutions that had to be analyzed for each sample to a practical number.

 Because the labeled congeners are not spiked into the samples before extraction, matrix spike and matrix spike duplicate samples we re prepared and analyzed periodically to provide an estimate of extraction efficiency.

 Results for the 11 congeners were corrected fo r any losses of analyt es that occur during the many cleanup steps, but this is not equivale nt to the true isotope dilution quantitation procedure in the original method because it do es not correct for the efficiency of the sample extraction procedures.

These steps were successful in overcomi ng challenges inherent in sewage sludge analyses, and produced useful data for the pur poses of the TNSSS. However, these steps involved some trade-offs, most notably the loss of true recovery correction of isotope dilution quantitation and the degree to which the very sm all sample size represents the bulk material.

4.4.5 Pharmaceutical Analyses

The analysis of the pharmaceuticals was perf ormed using an early draft of EPA Method 1694. EPA Method 1694 employs high performance liquid chromatography (HPLC) to separate the analytes of interest and tandem mass spect rometry (MS/MS) to detect them. HPLC (or simply LC) is a technique that allows the analys is of polar compounds in polar solvents. It has advantages over gas chromatographic (GC) methods for the pharmaceuticals because GC methods involve introducing the anal ytes into the instrumentation in a gaseous form and many of the pharmaceuticals are not easily volatilized. Some have boiling points that are above the operating temperatures of a GC system and others will break down when heated (i.e., they are “labile”).

Tandem mass spectrometry involves the use of two quadrapole mass spectrometers in series, with a collision cell betw een them, such that selected ions produced in the first MS unit are directed into the collision cell and further frag mented before being sent to the second MS for detection. The only ions passed through the collisi on cell are those selected by the instrument as representing the analytes of interest. These frag ments, or “product” ions, are characteristic of the “precursor” compound and are used to positively id entify the analyte in the presence of other analytes and potential interferen ces. The MS/MS detector can be operated in an ionization mode that produces positive ions from the analytes of interest, or in a mode that produces negative ions. The method for pharmaceuticals involves four analytical fractions, three of which operate in the positive ionization mode and one of wh ich uses the negative ionization mode. As employed in this method, the tandem MS unit is operated at unit mass resolution (e.g., it can distinguish between masses that differ by one atomic mass unit).TNSSS Sampling and Analysis Technical Report 25 January 2009 The pharmaceuticals method employs solven t extraction procedures to isolate the analytes of interest from the sewage sludge samp les. Many of the analytes are weak acids or weak bases that ionize in aqueous solutions, losi ng or gaining a proton fr om a water molecule. The extraction procedures in the pharmaceutical me thod involve adjusting the pH of the sample to provide more favorable c onditions for isolating the anal yte from the sample matrix.

An aliquot of each sewage sludge sample containing a consistent dry weight of solids is mixed with a phosphate buffer solution with a pH of 2. This pH adjustment causes the ionized acid forms of the analytes of interest to gain protons and become neutrally charged molecules that are less soluble in water than their ionized fo rms and more soluble in a polar organic solvent.

A suite of stable isotopically labeled standa rds (forms of the anal ytes that do not occur naturally) is spiked into the sample and the sample is further mixed. The analyst adds acetonitrile to the buffered sample, ultrasonic ally agitates the mixture for 30 minutes, and centrifuges the solution to separate the solvent extract from the solids. After decanting and collecting the acetonitrile, the an alyst performs a second extrac tion of the solids with fresh aqueous buffered acetonitrile, and a third extracti on using acetonitrile alone. All three acid extracts are combined for cleanup.

The base extraction is conducted in a sim ilar fashion, using a second aliquot of the original sample, but the pH of the sample is adjusted to 10 with an ammonium hydroxide solution. Three extractio ns are performed, two with aqueous buffered acetonitrile and a third extraction with acetonitrile alone. All th ree base extracts are combined for cleanup.

The combined acid extract is concentrated to remove the acetonitr ile and prepared for cleanup by adding disodium EDTA and diluting the solution to 200 mL with reagent water. The aqueous solution is processed through a solid-pha se extraction (SPE) cartridge, which traps the analytes of interest. Potential interferences are removed by eluting the cartridge with reagent water and discarding that eluant. The analytes of interest are eluted from the cartridge with methanol, followed by 1:1 acetone:methanol. The eluant is evaporated to near dryness, reconstituted in methanol, spiked with the method-specified intern al standards, and brought to a final volume of 4 mL with a 0.1% formic acid buffer solution.

The base extract is subjected to a simila r SPE cleanup procedure, but the cartridge is eluted with methanol, followed by 2% formic acid in methanol. The combined eluant is evaporated to near dryness, reconstituted in me thanol, spiked with the method-specified internal standards, and brought to a final volume of 4 mL with a 0.1% formic acid buffer solution.

Four separate LC/MS/MS analyses are performed for the pharmaceuticals: three on the acid extract and one on the base extract. Separate chromatogra phic conditions are associated with each of the four fractions. The LC/MS/MS instrument is operated in the multiple reaction monitoring (MRM) mode, which monitors a series of precursor/product ion transitions that are characteristic of each target analyte.

The labeled analytes spiked into the samp le prior to extraction are used to perform isotope dilution quantitation for all of the target analytes that ha ve labeled analogs. For those target analytes for which labeled analogs are not readily available, quantitation is performed using the labeled analog of a similar compound in th at fraction. As a resu lt, all of the targetTNSSS Sampling and Analysis Technical Report 26 January 2009 analyte concentrations ar e corrected for the recovery of the labeled analogs, thus accounting for extraction efficiencies and losses during cleanup.

The approach to ensuring more consistent method sensitivity desc ribed in Section 4.4.1 of this report was applied to the pharmaceutical analyses as well.

4.4.6 Steroid and Hormone Analyses

The analysis of the steroids and hormones was performed using an early draft of EPA Method 1698. EPA Method 1698 employs GC to sepa rate the analytes of interest and high resolution mass spectrometry (HRMS) to detect them. As employed in this method, the mass spectrometer achieves a resolution of at least 5,000. The target analytes in the method have molecular weights that range fr om about 100 to 500, such that th e MS can disti nguish between analytes with molecular weight s that differ by 0.02 to 0.1 atomic mass units, and identify them in the presence of poten tial interferences.

The steroids and hormones method also employs solvent extraction pr ocedures to isolate the analytes from the sewage sludge samples. An aliquot of each solid sample containing a consistent dry weight of solids is spiked with a su ite of stable isotopically labeled standards. The sewage sludge samples that are firm solids ar e extracted in a Soxhlet extractor with 60:40 acetone:hexane. The extract is spli t into two portions, with 1/25th of the extract used for steroids analysis and the other 24/25th used for the horm ones analysis. This split ratio is used to compensate for the fact that the steroids and thei r interferences are present at higher levels in the samples than the hormones.

The sewage sludge samples that are pourable liquids are extracted using a liquid-liquid solvent extraction with methylene chloride. The extracts of these samples also are split into two portions, with a split ratio of 1:99, for the steroids and hormones respectively.

For both forms of sewage sludge (liquid and solid), the separate extract portions are subjected to cleanup using a la yered alumina-Florisil (LAF) column. Following cleanup, each sample extract is concentrated to approximately 0.1 mL and the extract solvent is exchanged to pyridine.

In order to be amenable to GC analysis , the steroids and hormones are converted to compounds that are more volatile than their native forms. Both the steroids and hormones are derivatized to their trimethyl-silyl ethers using N,O-bis(trimethylsilyl) trifluoroacetamide with trimethylchlorosilane (BSTFA:TMCS). The deri vatized extracts are concentrated and spiked with the method-specified internal standards a nd the final extract volum e is adjusted to 500 μ L.

Separate GC/HRMS analyses are performed fo r the steroids and for the hormones, using the same chromatographic conditions for each anal ytical fraction, but runn ing the two fractions at different dilutions. The GC/HRMS instrument is operated at a mass resolution of at least 5,000, and at least two exact masses are monitored for each target analyte.

As with the pharmaceuticals method, the labeled analytes spiked into the sample prior to extraction are used to perform isotope dilution quantitation for all of the target analytes that have labeled analogs. For those target analytes for which labeled analogs are not readily available,TNSSS Sampling and Analysis Technical Report 27 January 2009 quantitation is performed using th e labeled analog of a similar compound in that fraction. As a result, all of the target analyt e concentrations are corrected fo r the recovery of the labeled analogs, thus accounting for extraction e fficiencies and losses during cleanup.

EPA worked with the laboratory to adjust th e procedures for the steroids and hormones to address problems encountered early in the survey. In particular, the laboratory modified one of the cleanup procedures. As written, the ster oids and hormones method involves solvent extraction of the sample and processes that ex tract through a layered al umina-Florisil (LAF) cleanup column before splitting the extract into two unequal portions (1/25 and 24/25) for analyses of steroids and hormones, respectively. However, some of the steroids and hormones are present in both extract portions and during the early part of the survey, the high levels of steroids were creating analyt ical challenges during the anal ysis of the hormones fraction.

The laboratory proposed a solution to the pr oblem that involved modifying the order in which the extract was subjected to cleanup and sp lit into two portions. In stead of running the entire extract through the LAF column and then splitting it into two unequal portions, the raw extracts were split first and then run through se parate LAF cleanups, one for steroids and one for hormones. The LAF cleanup for the steroids was run as described in th e original procedure.

The laboratory modified the LAF procedure for the hormones to remove most of the steroids from the hormone frac tion. The steroids were eluted from the LAF column with methylene chloride and that eluant was discarded. The hormones then were eluted from the column with methanol, as described in the original procedure.

The laboratory tested the met hod modifications prior to star ting analyses of the survey samples by extracting large samples of sewage sludge from another so urce (e.g., not a TNSSS sample), spiking the extracts with the hormones of in terest to ensure that they were present, and splitting each extract into two por tions. One of the portions was run through the LAF column as described in the method. The other portion was s ubjected to the modified LAF procedure. Both extract portions were analyzed for hormones by GC/HRMS and the results were compared. The change to the LAF procedure dramatically reduced the levels of steroids that remained in the hormone fraction, and improved th e results for the hormones.

As noted in Section 4.2, the target analyte list for the TNSSS was based on a variety of factors, but was not driven by lists of anal ytes in any individual methods. EPA had included Mestranol as a target analyte in early versions of the survey plan for the TNSSS. However, Desogestrel was never listed as a target analyte for the survey. Therefore, this modification to the LAF cleanup procedure sacrificed one hormone in favor of improvements for all the other target hormones. Given the difficulties inherent in sewage sludge analyses, EPA judged this to be a reasonable compromise.TNSSS Sampling and Analysis Technical Report 28 January 2009 4.4.7 Focusing Quality Control on the Survey-specific Analytes of Interest

Because the analytes of interest for the TN SSS often are a very limited subset of the total number of analytes listed in some of the rele vant methods, EPA did not require the laboratories to consider those other analytes in either prep aring or evaluating the qua lity control operations associated with these samples. For example, EPA only required the la boratory to spike the analytes of interest for this survey into such QC samples as matrix spike/matrix spike duplicates and ongoing precision and recovery (OPR) samples, not all the analytes that may be listed in a given method. Alternatively, if the laboratory ch ose to use spiking solu tions that contained additional analytes beyond those in Tables 5 and 6, EPA did not require them to assess the results for those non-survey target analytes or take corrective actions if those non-survey analytes failed to meet the acceptance criteria.

This approach allowed the laboratories to focus their efforts on the survey-specific analytes of interest in the face of the analytical challenges presented by sewage sludge matrices. It also allowed EPA to control the costs fo r the analyses to some degree by eliminating reanalyses related to potential QC failures associat ed with non-target anal ytes. It also reduced the costs for EPA’s data review efforts described in Section 5.TNSSS Sampling and Analysis Technical Report 29 January 2009 

### Section 5 Data Review Procedures

5.1 General Review Procedures

EPA assessed the results for all of the samp les analyzed during the survey using wellestablished procedures described in this se ction. The analysis involved in the TNSSS was complex and a number of analytical challenges were faced. Biosolids is one of the most challenging environmental matrices known due to the high solids content and matrix interferences present. When c onducting analyses in sewage sludge matrices it is expected that some results will have to be qualified to accura tely reflect the uncertainty of the values.

EPA subjected all laboratory results to a comprehensive review for completeness and compliance with project and method specifications to ensure that the data met the objectives of the survey. A multi-stage review process was used and designed to identify and correct data deficiencies as early as po ssible and maximize the amount of usable data generated.

Trained staff reviewed the data using estab lished review process de signed to identify and correct data deficiencies as ear ly as possible and maximize the am ount of usable data generated during the TNSSS. EPA encoded the data quality information gathered during the review in the final results database using a se ries of qualifiers and reasons. EPA did not exclude data unless a result was flawed such that no reasonable use could be made of it. The four stages of the review process are described below.

In the first stage of the data review pro cess, EPA performed a data completeness check. Specifically, EPA evaluated elements in the labor atory submission to verify that results for specified samples were provided, th at data were reported in the co rrect format, and that relevant information, such as preparation and analysis lo gs, was included in the data package. EPA initiated corrective action procedures to resolve any deficiencies identified.

The second stage of the data review pro cess focused on an instrument performance check. EPA verified that calibra tions, calibration verifi cations, standards, a nd calibration blanks were analyzed at the appropriate freque ncy and met method or survey performance specifications. Corrective action procedures were in itiated to resolve any deficiencies identified.

The third stage of the data review proce ss focused on a laboratory performance check. EPA verified that the laboratory correctly performed the required analytical procedures and was able to demonstrate a high level of precision and accuracy. During this stage, EPA evaluated quality control (QC) elements such as the ongo ing precision and recovery (OPR) tests, method blanks, and other QC operations. Again, corrective action procedur es were initiated to resolve any deficiencies identified.TNSSS Sampling and Analysis Technical Report 30 January 2009 In the fourth stage of the data review process, EPA examined method/matrix performance data to discern whether any QC failures resulted from laboratory performance or difficulties with the method or sample matrix. EPA evaluated labeled compound and surrogate spike results and other performance data. The reviewers also verifi ed that proper sample dilutions were performed and that necessary sample cleanup steps were take n. As with previous steps, corrective action procedures were initiated with the laborat ory to resolve any deficiencies identified.

The objective of the data review process was to document the qua lity of all of the data in the TNSSS and identify any limitations that might a ffect their end use. The EPA database on the mainframe contains data qualifiers applied to results from the TNSSS, individually, and by analyte class.

5.2 QC Acceptance Criteria

As noted in Section 4, the anal ytes of interest for this su rvey were subject to quality control. Appendix C presents a summary of th e QC acceptance criteria fo r all of the analyte classes.

The laboratory performing the analyses for the anions, metals, PAHs and semivolatiles used its own in-house limits routinely applied to soil samples (as opposed to aqueous samples), except as noted in Appendix C. The laboratory prepared liquid sewage sludge samples using procedures applicable to aque ous samples (e.g., a liquid-liquid solvent extraction procedure for the organics) and the laboratory ran an aqueous laboratory control sample (LCS) and applied acceptance limits appropriate to that set of proc edures. The laboratory derived their acceptance limits for solid samples from historical data for sewage sludge samples. Given the difficulties evident in the analyses of the se wage sludge samples, one might e xpect that statistically derived acceptance criteria for sewage sludge samples would differ from those for soil samples. Therefore, recoveries falling sli ghtly outside of the limits for soil are not necessarily fatal flaws in these analyses. However, for the sake of tr ansparency, EPA noted all recovery problems in the database.

The laboratory performing the analysis of the PBDEs using draft EPA Method 1614 employed the default acceptance limits in the draft method.

The methods used for the pharmaceutical, steroid, and hormone analyses include QC acceptance criteria. The acceptance criteria includ e statistically derived limits that are based on data from a single laboratory. As a result, the acceptance limits for some QC operations are fairly wide (e.g., 5 – 200% for labeled compound r ecoveries). The laboratory that performed the pharmaceutical, steroid, and hormone analyses for the TNSSS was also the laboratory that helped EPA develop these methods. Therefor e, EPA believes that the use of the singlelaboratory specifications is a reasonable approach.

5.3 Data Qualifiers and Database

After reviewing each data package, the re viewers and the database development staff created an analytical database in MS Access th at contained all field sa mple results from the survey. Appendix D of this report contains a tabl e with the data applied to the results during the review process for TNSSS pollutants and entered into the database.TNSSS Sampling and Analysis Technical Report 31 January 2009 At intervals during development and upon co mpletion of the database, EPA performed various checks to verify the accuracy of the da tabase, including checks for consistent analyte names, CAS numbers, and data qualifier flags. After completing the review of the results for each analytical fraction, EPA uploaded the data fr om an Access database to the EPA mainframe using standardized procedures. The final database for the survey includes all of the qualifiers that were applied to the individual sample results.

Note: Except for the “exclude” qualifier in the database, the presence of data qualifiers is not intended to suggest that data are not useable . Rather, the qualifiers are designed to caution the user about an aspect of the da ta that does not meetthe acceptance criteria originally established for the project.

5.4 Data Review Findings

The data review process was crucial in identi fying the sensitivity issues associated with the full-scan GC/MS analyses discussed in Sectio n 4. By examining the first sets of sample results shortly after they were delivered by the laboratory, EPA was able to take corrective action and instituted the use of selected ion monitoring for all PAH analys es in the survey to achieve the needed analytical sensitivity.

The data review process uncovered only a fe w other issues with data quality. Typical issues included:

 Reporting unit issues

 Calculation errors for specific samples

 Blank contamination

 Extract dilution issues

 Matrix spike issues

 Interference issues

 Recovery issues

Specific examples of these issues are presented in Section 5.4.1 to 5.4.7.

5.4.1 Reporting Limits

As expected, some samples with low per cent solids were prepared and analyzed as aqueous samples. However, when reporting th e results for the earliest such samples, the laboratory performing the metals analyses provided results in the weight /volume units typically applied to aqueous samples. When contacted by the data reviewers regarding this error, the laboratory revised these results a nd reported them on the basis of th e dry weight of solids in the samples, as originally requested.TNSSS Sampling and Analysis Technical Report 32 January 2009 The laboratory analyzing the pharmaceuticals, steroids, and hormones reported all the sample results on a dry-weight basis, as require d, including those samples that were pourable liquids. However, the laboratory used reporting units of nanogram s per gram (ng/g), instead of the method-specified units of nanograms per kilogr am (ng/kg). This error was discovered during the early stages of the review of these data. However, because the units of ng/g are equivalent to μg/kg, after consulting the laboratory and consideri ng the concentrations of the analytes in the samples, EPA decided to accept the numerical results in ng/g (e.g., EPA kept the numbers reported by the lab), but changed the units in th e EPA database to μg/kg (i.e., 100 ng/g is equal to 100 μg/kg).

5.4.2 Calculation Errors

EPA noted that the fluoride results for two sewage sludge samples appeared to be much higher than any other results for this analyte. EPA contacted th e laboratory and asked them to check these two results. The la boratory reported that the raw data for these two results were off by two orders of magnitude, due to a transcription error. The laboratory corrected the error and resubmitted the results for those two samples. Th e corrected results were included in the survey database.

5.4.3 Blank Contamination

EPA examined the results for each analyte in every sample and compared the results to the concentrations of analytes found in each of th e method blanks associated with each batch of samples. For all of the analyt e classes, the concentrations of the analytes found in the method blanks were generally well below the c oncentrations in the field samples.

In some cases, there were low levels of anal ytes in some method blanks. For all of the analytes in the TNSSS, EPA used a common appr oach to evaluating blank contamination known simply as “the 5x and 10x rules.” Under routin e circumstances, EPA qualified a field sample result if the concentration of an analyte was not at least 10 time s the amount found in the blank. The rationale for the 10x rule is that under the worst of circumstances, in which the material found in the blank is coming from a source within the laboratory, the am ount in the blank would only represent 10% of the amount in the field sample , and that small contribut ion is likely within the overall measurement error. If the amount in the sample is between 5x and 10x the amount in the blank, EPA normally will qualify the sample re sult as a maximum value because the potential contribution from the laboratory could be as high as 20%. Below 5x the amount in the blank, EPA considered the field sample result to be a non-detect at the nominal reporting limit.

The method blanks for the steroids and horm ones presented greater challenges. Three of the steroid and hormone analytes, Cholesterol, Stigmasterol, and β -Sitosterol, were frequently found in the method blanks, and a fourth analyte, Progesterone, was occasionally found in those blanks. During the course of the survey, the la boratory determined that the steroids were adhering to the glassware used to prepare sample s, perhaps originating from an earlier sewage sludge sample prepared in the same glassware. Although the laboratory took steps to improve its glassware cleaning procedures to prevent such “ carryover,” they were not able to completely eliminate the occurrence of some ta rget analytes in the method blanks.TNSSS Sampling and Analysis Technical Report 33 January 2009 Therefore, EPA flagged the steroids and hor mones results, where applicable. Normally, EPA evaluates results for samples that have been diluted for analysis by multiplying the blank result (which is usually not d iluted) by the dilution factor for the sample. For example, if a sample extract is diluted by a factor of 50 to bring one or more target analytes within the calibration range of the instrument, EPA will mul tiply the concentration of an analyte found in the blank by that dilution factor of 50 before comparing the blank and the field sample results. That approach represents a worst-case assumpti on that the contaminant may be present in the solvent used to extract the sample s, thus using more of that solv ent to dilute the extract would add more of the contaminant.

In the case of the steroids and hormones, the laboratory checked its reagent water and extraction solvents and found no such contamina tion. As noted above, the laboratory surmised that the material in the blanks was being transf erred from the surface of the glassware used to prepare the blank, but it originated from an earlier sample proce ssed with the same glassware. Thus, the amount, or concentration, found in the blank may represent the material that the sample extract might pick up from similar glassware, but there would be no likely contribution from the solvents themselves, and thus no need to consid er the dilution factors of the samples. The dilution factors for samples in t hus survey often were quite high.

Even after instituting this minor change to the data review procedures, there were still 24 instances in which the levels in the blank led EPA to consider the results for one or more of the steroids to be a non-detect. These 24 instances re presented 14 field samples, with 1 to 3 analytes affected in each of those samples. In many cases , the original result for the field sample was not only less than 5x the blank results it was actually 2x to 3x lower than the amount found in the blank. Of the 14 affected samples, 5 were in th e first batch of 12 sample s analyzed and reported. The 6 subsequent batches of samples had lower le vels of these analytes in the blanks, with 2 batches having no samples set to non-detects and 2 more batches with only 1 difficult analyte. These improved blank results are evidence of the effectiveness of the revised glassware cleaning procedures instituted by the laboratory.

5.4.4 Extract Dilution

Surrogate compounds were added to each samp le for the semivolatile and PAH analysis as a measure of sample extraction efficiency. Ho wever, if the sample contained large amounts of the target analytes, the laboratory often had to dilute the sample extract to bring the results for that analyte within the instrument’s calibra tion range. If the dilution factor was high enough, the surrogates may have been present in the diluted extract at such low levels that they could not be detected. Without measurable results for the surr ogate, EPA could not evaluate the efficiency of the extraction procedures for that sample.

An important part of the data review process was to ensure that results for a given sample included those where the surrogates could be meas ured as well as results where all of the analytes were within the calibration range. This often requires that the la boratory provide results from multiple analyses of the sample at diffe rent dilution factors. EPA worked with the laboratory analyzing the semivola tiles and PAHs to obtain data from as many analyses as practical and minimize situations in which only the most dilute analysis (without observable surrogates) was provided. However, the excep tionally high levels of bis (2-Ethylhexyl)TNSSS Sampling and Analysis Technical Report 34 January 2009 phthalate in some samples made the situation mo re difficult. EPA flagged in the database any samples in which the surrogates were diluted out.

5.4.5 Matrix Spikes

Due to the often high and variable levels of many analytes in the samples, some of the MS/MSD samples were spiked at levels too low to provide useful recovery data. This issue occurred frequently for the anions, metals, semivol atile organics and PAHs. It also was an issue for the PBDE analyses when EPA added matrix sp ike analyses to those analyses to make up for the method modifications that eliminated the us e of true isotope dilution quantitation (see Section 4.4.4). Matrix spike samples were not required for the pharmaceutical, steroid, and hormone analyses because those methods use isot ope dilution quantitation and therefore provide a sample-specific recovery correction of every analyte.

The difficulty in generating useful matrix spike results was exacerbated by the different approaches to calculating MS/MSD recovery pr ovided in different methods. EPA initially qualified the results for samples associated with “under spiked” MS/MSD samples as estimates. However, EPA recalculated the recoveries of the spiked analytes using an alternative equation designed to address this issue (s ee Section 6.7) and the bias and precision of th e recalculated results were further evaluated.

5.4.6 Interferences

All of the methods used in the TNSSS include d some form of identification criteria for the analytes of interest. Thes e criteria included absorption or emission wavelengths, retention times, and mass spectrometric criteria. The GC/HRMS method for the steroids and hormones involves monitoring two characteristic ions fo r each analyte. As with other many EPA GC/HRMS methods (e.g., Method 1613B, 1614, and 1668A), the responses for both of those ions are used to quantify the concentration of the analyte in the sample, using an approach called dual-ion quantitation.

The method for the steroids and hormones stip ulates that the ratio of the abundances of the two ions must be within a ce rtain percentage of the theoretical ratio for the analyte. The theoretical ratio is determined from natural abundances of the exact masses of all of the component elements that make up both of the io ns monitored for the analyte. The acceptance criterion in the method for the steroids and hormones is a ± 30% window around the theoretical ion abundance ratio for the analyte.

The purpose of this criterion is to ensure that the analytes of interest are adequately separated from one another and from any potential interfering compounds. As described in the method, if the ion abundance ratio is not met, th e analyte cannot be positively identified. The method instructs the analyst to not e any instances where both ions from the analyte are present, but the ion abundance ratio accepta nce criterion is not met. This approach to reporting results allow the data users to decide how best to use the data.TNSSS Sampling and Analysis Technical Report 35 January 2009 The most likely reason that the ion ab undance ratio does not meet the acceptance criterion is that there is a positive interference for one of the two ions monitored. That positive interference increases the response for that ion, a ffecting the ion abundance ratio (IAR). Because the areas (abundances) for the peak s representing the two ions are added together to calculate the concentration of the analyte, the positive interferen ce translates into a higher reported concentration for the analyte of interest. T hus, the reported result is an estimated maximum possible concentration (EMPC) for the analyte.

There were 129 instances of EMPCs in the ster oid and hormone results out of the total of 2100 results, or about 6% of all steroid and hormone results. In all, 76 of the 84 survey samples had one or more analytes qualifi ed as an EMPC, and 17 of the 25 analytes were affected. The frequencies at which EMPCs were reported are shown in Table 9. In some instances, the observed ion abundance ratio was only marginally outside of the method acceptance limits, and in other instances, the differences were much greater.

Table 9. Frequency of Estimated Maximum Possible Concentrations (EMPCs) Analyte # of EMPCs Reported Analyte # of EMPCs Reported Campesterol 50 Androstenedione 3

Estrone 21 β -Stigmastanol 3 Testosterone 11 Equilin 3 Stigmasterol 10 17 α -Dihydroequilin 1 Ergosterol 6 Androsterone 1
Desmosterol 5 β -Estradiol 3-benzoate 1 β -Sitosterol 4 Equilenin 1 Norethindrone 4 Progesterone 1 Norgestrel 4

The affected analytes include both steroi ds and hormones, and therefore represent separate analytical runs in the method.

The degree to which the reported results fo r an EMPC exceed the actual concentration cannot be determined exactly because a number of factors are involved, in cluding the effect of the interferences and the fact that the results are quantified by isot ope dilution. In order to place some bounds on the likely effects, EPA examined th e survey results for Testosterone in detail. The 11 EMPCs for Testosterone have reported concentrations that range from 34.8 to 2040 μg/kg. The reported ion abundance ratios for thes e 11 Testosterone results ranged from 0.01 to 2.34, with the laboratory’s QC acceptance limits fo r the ratio being 2.38 to 4.42. The lowest ion abundance ratio (0.01) was associated with the highest reported re sult (2040 μg/kg), but there is no apparent relationship between the other ratios and results.

Although the actual area s of the two peaks monitored for each analyte are presented in the hard copy raw data provided by the laboratory, these peak areas are not readily accessible in the electronic data. Rather th an retrieving several hundred peak areas (2 peaks for each of 129 EMPCs) from the raw data, entering them into a spreadsheet, and checking for data entry errors, EPA took an alternative approach.

Although the actual peak areas were not in an accessible electronic format, EPA had the ion abundance ratios themselves in the database. In order to investigate the likely effects of the interferences, EPA arbitrarily assigned the area of the first peak a value of 10,000 area counts.TNSSS Sampling and Analysis Technical Report 36 January 2009 Using the reported ion abundance ratios, we ca lculated the correspond ing area of the second peak. As described in the method, the concentrati on of an analyte is calc ulated from the sum of the areas of both peaks, so we summed the two “dummy” areas for each sample result. EPA also calculated the area of the second peak using the theoretical ion abundance ratio for the analyte and summed those two areas. Since the sample concentration is proportio nal to the sum of the areas, we compared the two calculated sums to dete rmine a factor that could be used to convert the reported result to the result that might have been calculated without the positiv e interferences that affected the ion abundance ratio. The resu lts for the 11 EMPCs for Testosterone are shown in Table 10.

Table 10. Potential Effects of Ion Abundance Ratio on Reported Concentrations of Testosterone

Sample Dummy Abundance Mass 1 Reported IAR Dummy Abundance Mass 2 Sum of Dummy Masses Ratio of Sum from Reported IAR to that from Theoretical IAR Reported Conc. Adjusted Conc. 1 10,000 0.01 1,000,000 1,010,000 0.012813046 2040 26.1 2 10,000 0.27 37,037 47,037 0.275127374 97.9 26.9 3 10,000 0.54 18,519 28,519 0.453781513 238 108 4 10,000 0.55 18,182 28,182 0.459203036 701 322 5 10,000 0.68 14,706 24,706 0.523809524 46.9 24.6 6 10,000 1.2 8,333 18,333 0.705882353 65.7 46.4 7 10,000 1.22 8,197 18,197 0.711181770 42.3 30.1 8 10,000 1.4 7,143 17,143 0.754901961 122 92.1 9 10,000 1.99 5,025 15,025 0.861302380 238 205 10 10,000 2.04 4,902 14,902 0.868421053 34.8 30.2 11 10,000 2.34 4,274 14,274 0.906657274 67.3 61.0

The results in Table 10 are sorted by the reported ion abundance ratios. For ease of discussion, the samples addressed in this table were numbered 1 to 11, in order of reported ion abundance ratio. The sample numbers have no other significance. As can be seen for Sample 1, the potential implications of the ion abundance ra tio could be dramatic. However, for many of the other samples, the effects ar e far less apparent. The reported concentrations already include the recovery correction inherent in isotope dilution, so the adjust ed concentrations include the same degree of correction.

EPA performed similar calculations for the 50 reported EMPCs for Campesterol. Because none of the observed ion abundance ratios we re as far from the theoretical ratio for this analyte, the overall effects on the reported resu lts were smaller. The applicable conversion factors for Campesterol range from 0.173 to 0.907, m eaning that the results for this analyte might be reduced by factors from 1.1 to 6. Give n the time required, EPA did not perform these calculations for all the other analytes in which EMPC results were reported by the laboratory.

Note: The adjusted concentrations shown in Table 10 illustrate the potential change to the reported results. However, the calculations shown assume that th e ion abundance ratio problem is the result of a simple positive interference in one of the two ions for the analyte. The actual cause may be more complicated. Therefore, EPA included the original results reported by the laboratory in the survey database, not the adjusted concentrations described above. EPA flagged each result in the database that did not meet the method-specified ion a bundance ratio as an “EMPC.”TNSSS Sampling and Analysis Technical Report 37 January 2009 5.4.7 Recovery Issues

Three other analytical issues were noted that had significant pot ential effects on the survey data for the pharmaceuticals, steroids, and hormones. EPA identified each such instance in the EPA database with the qualifier EXCLUDE to prevent those results from being used to determine the national estimates of the con centrations of polluta nts in biosolids.

Two of those issues involved recoveries of the isotopically labeled analytes used to perform isotope dilution quantitati on. The third issue involved the “internal standards” added to the sample extracts immediately prior to inst rumental analyses and used to measure the recoveries of the labeled analytes.

There were 27 instances where EPA excluded pharmaceutical results from the database, involving 15 analytes. For 13 of those analytes , EPA excluded the results for 3 samples because the labeled compounds for those analytes were not recovered from the samples. These were not simply instances of lower than expected recoveries of the labeled compounds, but rather, no recovery at all (zero). The in ability to recover the labeled compound spiked into the sample suggests a significant analytical problem beyond the routine anal ytical challenges presented by sewage sludge samples. All three samples we re extracted on the same date and analyzed together several days later.

There were two instances where the resu lts for the analyte Fluoxetine were excluded because the labeled compound for this analyt e was not recovered from those samples.

The remaining 12 results were excluded fo r the analyte Minocycline. All 12 instances came from the same extraction batch and were excluded because the laboratory did not recover the native (unlabeled) analyte in the laboratory control sample (LCS, also known as an ongoing precision and recovery sample, or OPR) that was prepared at the same time as this batch of 12 samples. The laboratory did not report numeri cal results for Minocycline in these 12 samples and flagged all of the Minocycline results with an “NQ” flag in the database, indicating that the laboratory was not able to quantify the analyte.

There were 65 instances of steroid a nd hormone results being excluded from the database, involving 14 analytes. In 42 of those instances, involvi ng 7 analytes, the data were excluded because the laboratory fo und only trace levels (e.g., extremely low) of the internal standard, Pyrene-d 10 , added to the sample extract immedi ately before analysis and used to measure the recovery of the labeled compounds a dded to the sample before extraction. Trace levels of the internal standard occurred in 6 samples analyzed in the same batch, suggesting a possible problem with the addition of the internal standard (e.g., the auto mated injection of the internal standard may have failed or been incomplete).

The remaining 23 excluded results involved 7 other analytes where there was no recovery of the labeled compound in the same 6 samples. In these cases, the issue was not simply that the recovery could not be measured because of the ve ry low level of the internal standard present, but rather that there was no measurable signal for the labeled compound itself.TNSSS Sampling and Analysis Technical Report 38 January 2009 5.5 Revised Results for BDE-209

In March 2008, the laboratory that performed the PBDE an alyses in 2007 prepared a new set of calibration standards for the PBDEs, compar ed the responses of the new standards to data from the standards used for the TNSSS analyses, a nd found that the results for one of the target congeners (BDE-209) were markedly different. The laboratory also compared the results to a standard from a second source and found similar differences, ultimately tracing the difference to a vial of the original BDE-209 standard that was mislabeled by the manufacturer.

After discussions with the laboratory, EPA decided that the BDE-209 results could be recalculated based on the responses in the single-point calibration veri fication standard analyzed with each batch of samples and that this recalcul ation would mathematically adjust the results to a more accurate value. This verification standard was prepared from a separate source from the initial calibration standards and was not affected by labeling erro r. The laboratory recalculated the BDE-209 results for all 84 field samples and the 2 equipment blanks and resubmitted the corrected data in late May 2008. The resubmitted BDE-209 results were reviewed and uploaded to the survey database on the EPA mainframe.TNSSS Sampling and Analysis Technical Report 39 January 2009 

### Section 6 Survey Results

6.1 Summary Results

Table 11 provides a summary of the results for all 84 samples in the first phase (i.e., all analytes excluding pharmaceuticals, steroids, and hormones) of the survey, listing the number of samples in which each analyte was reporte d, along with the minimum and maximum concentrations. Tables 12 and 13 provide the results for the pharmaceuticals and steroids and hormones, respectively. All sample results ar e reported on a dry-weight basis, based on the percent solids in the original sample. The perc ent solids in the various sewage sludge samples range from 0.14 to 94.9. The units for pollutants vary with the class of anal yte, as shown. This summary includes the results for the six fiel d duplicate samples and the four POTWs that generate more than one type of sewage sludge. The minimum concentration is the lowest value reported as present in any sample. EPA did not report a minimum or maximum value for those analytes that were not detected. That situati on only occurred for some of the pharmaceuticals, steroids and hormones, and EPA used “NA” to indicate that the minimum and maximum values were “not applicable.”

Table 11. Summary of Results for Metals, Anions, Organics, and PBDEs Observed Dry-weight Concentration Class Analyte Units # Detects Minimum Maximum Solids Percent Solids % 84 0.43 93.5 Fluoride 84 7.6 234 Nitrate/Nitrite 84 1.6 6,120 Water-extractable phosphorus mg/kg 84 11.0 9,550 Anions

WEP ratio unitless 84 0.00065 0.33920 Aluminum 84 1400 57,300 Antimony 72 0.45 26.6 Arsenic* 84 1.18 49.2 Barium 84 75.1 3,460 Beryllium 83 0.04 2.3 Boron 80 5.70 204.0 Cadmium* 84 0.21 11.8 Calcium 84 9,480 311,000 Chromium* 84 6.74 1160 Cobalt 84 0.87 290 Copper* 84 115 2,580 Iron 84 1,575 299,000 Lead* 84 5.81 450 Magnesium 84 696 18,400 Manganese 84 34.8 14,900 Mercury* 84 0.17 8.3 Molybdenum* 84 2.51 132 Nickel 84 7.44 526 Phosphorus 84 2,620 118,000 Selenium* 84 1.10 24.7 Silver 84 1.94 856 Sodium 84 154 26,600 Thallium 80 0.02 1.7 Tin 78 7.50 522 Titanium 83 18.50 7,020 Vanadium 84 2.04 617 Yttrium 84 0.70 26.3 Metals

Zinc* mg/kg

84 216 8,550TNSSS Sampling and Analysis Technical Report 40 January 2009 Table 11. Summary of Results for Metals, Anions, Organics, and PBDEs Observed Dry-weight Concentration Class Analyte Units # Detects Minimum Maximum 4-Chloroaniline 63 51 5,900 2-Methylnaphthalene 39 10 4,600 Fluoranthene 77 45 12,000 Pyrene 72 44 14,000 bis (2-Ethylhexyl) phthalate 84 657 310,000 Organics (PAHs andSemivolatiles) Benzo( a )pyrene  g/kg

64 63 4,500 BDE-28 84 2,200 160,000 BDE-47 84 73,000 5,000,000 BDE-66 84 1,800 110,000 BDE-85 84 3,200 150,000 BDE-99 84 64,000 4,000,000 BDE-100 84 13,000 1,100,000 BDE-138 56 1,900 40,000 BDE-153 84 9,100 410,000 BDE-154 84 7,700 440,000 BDE-183 84 2,100 120,000 PBDEs

BDE-209 ng/kg

83 150,000 17,000,000 * Metals currently regulated at 40 CFR 503

Table 12. Summary of Results for Pharmaceuticals Observed Dry-weight Concentration Analyte Units # Detects Minimum Maximum Percent Solids % 84 0.14 94.9 Acetaminophen 2 1,120 1,300 Albuterol 1 23.2 23.2 Anhydrochlortetracycline 1 125 125 Anhydrotetracycline 52 94.3 1,960 Azithromycin 80 10.2 6,530 Caffeine 39 65.1 1,110 Carbadox 0 NA NA Carbamazepine 80 8.74 6,030 Cefotaxime 0 NA NA Chlortetracycline 1 1,010 1,010 Cimetidine 74 7.59 9,780 Ciprofloxacin 84 74.5 47,500 Clarithromycin 45 8.68 617 Clinafloxacin 0 NA NA Cloxacillin 0 NA NA Codeine 20 9.59 328 Cotinine 39 11.4 690 Dehydronifedipine 19 3.48 24.6 Demeclocycline 3 96 200 Digoxigenin 0 NA NA Digoxin 0 NA NA 1,7-Dimethylxanthine 4 1,130 9,580 Diltiazem 69 1.39 225 Diphenhydramine 84 36.7 5,730 Doxycycline 76 50.8 5,090 Enrofloxacin 14 12.1 66 4-Epianhydrochlortetracycline 0 NA NA 4-Epianhydrotetracycline 31 126 2,160 4-Epichlortetracycline 1 974 974 4-Epioxytetracycline 8 35.7 54.9 4-Epitetracycline 80 47.2 4,380 Erythromycin-total 77 3.1 180 Flumequine 0 NA NA Fluoxetine μg/kg

79 12.4 3,130 Gemfibrozil μg/kg 76 12.1 2,650TNSSS Sampling and Analysis Technical Report 41 January 2009 Table 12. Summary of Results for Pharmaceuticals Observed Dry-weight Concentration Analyte Units # Detects Minimum Maximum Ibuprofen 54 99.5 11,900 Isochlortetracycline 1 3,140 3,140 Lincomycin 3 13.9 33.4 Lomefloxacin 2 33.3 39.8 Metformin 6 550 1,160 Miconazole 80 14.2 9,210 Minocycline 32 351 8,650 Naproxen 44 20.9 1,020 Norfloxacin 29 99.3 1,290 Norgestimate 0 NA NA Ofloxacin 83 73.9 58,100 Ormetoprim 1 5.91 5.91 Oxacillin 0 NA NA Oxolinic Acid 1 39.4 39.4 Oxytetracycline 29 18.6 467 Penicillin G 0 NA NA Penicillin V 0 NA NA Ranitidine 46 3.83 2,250 Roxithromycin 3 14.3 22.8 Sarafloxacin 2 179 1,980 Sulfachloropyridazine 2 35.9 58.7 Sulfadiazine 3 22.9 140 Sulfadimethoxine 5 3.58 62.2 Sulfamerazine 1 5.61 5.61 Sulfamethazine 2 21.5 23.2 Sulfamethizole 0 NA NA Sulfamethoxazole 30 3.91 651 Sulfanilamide 8 191 15,600 Sulfathiazole 1 21 21 Tetracycline 81 38.3 5,270 Thiabendazole 58 8.42 239 Triclocarban 84 187 441,000 Triclosan 79 430 133,000 Trimethoprim 24 12.4 204 Tylosin 0 NA NA Virginiamycin 15 43.5 469 Warfarin 0 NA NA NA = Not applicable, because the analyte was not reported in any sample

Table 13. Summary of Results for Steroids and Hormones Observed Dry-weight Concentration Analyte Units # Detects Minimum Maximum Percent Solids % 84 0.14 94.9
Androstenedione 32 108 1,520
Androsterone 50 21.3 1,030 Campesterol 84 2,840 524,000 Cholestanol 84 3,860 4,590,000 Cholesterol 81 18,700 5,390,000 Coprostanol 84 7,720 43,700,000
Desmosterol 58 2,730 94,400

17 α -Dihydroequilin 1 98.4 98.4 Epicoprostanol 83 868 6,030,000 Equilenin 1 60.6 60.6 Equilin μg/kg

15 22.3 107 Ergosterol 53 4,530 91,900

17 α -Estradiol μg/kg

5 16.1 48.8TNSSS Sampling and Analysis Technical Report 42 January 2009 Table 13. Summary of Results for Steroids and Hormones Observed Dry-weight Concentration Analyte Units # Detects Minimum Maximum

17 β -Estradiol 11 22 355 β -Estradiol 3-benzoate 18 30.2 1850

17 α -Ethinyl-estradiol 0 NA NA
Estriol 18 7.56 232
Estrone 60 26.7 965
Norethindrone 5 21 1,360 Norgestrel 4 43.8 1,300 Progesterone 19 143 1,290 β -Sitosterol 73 24,400 1,640,000 β -Stigmastanol 83 3,440 1,330,000 Stigmasterol 76 11,000 806,000
Testosterone 17 30.8 2,040 NA = Not applicable, because the analyte was not reported in any sample

6.2 Investigation of Results for Metals

After compiling the results from the first phase of the survey, EPA investigated the potential causes for the maximum results for calcium, iron, phosphorus, and silver. By reviewing the sampler’s field notes and ultim ately contacting the POTWs by telephone, EPA found that:

 The maximum result for calcium (311,000 mg/kg) was for a sample of Class A sewage sludge produced by a process known as advanced alkaline stabilizat ion with subsequent drying. The alkaline stabilization process i nvolves addition of la rge amounts of lime (calcium carbonate) to the material. The final sewage sludge is sold as a soil amendment.

 The maximum concentrations of iron (299,000 mg/kg) and elemental phosphorus (118,000 mg/kg) occurred in the same sample. The facility from which this sample was collected adds ferric chloride during its wast ewater treatment process to reduce the level of phosphorus in its effluent discharge. This tr eatment step results in high levels of iron and phosphorus in the sewage sludge from this plant.

 The maximum result for silver (856 mg/kg) o ccurred in a sewage sl udge sample from a POTW that employs a “complete mix activated sewage sludge proces s” and disposes of its sewage sludge by incineration. There are two major industrial dischargers to this POTW, but neither employs silver. The plant is not aware of any ot her instances of high silver results in their sewage sludge. There were no apparent calcula tion or transcription errors. The laboratory noted that this resu lt was determined by ICP/MS, and when the laboratory re-examined its result for the ICP/AES analysis of the same sample, silver was present in that analysis at about 900 mg/kg, thus seem ingly confirming the ICP/MS results.TNSSS Sampling and Analysis Technical Report 43 January 2009 6.3 Comparison of Metals Results to Current Standards

As noted in Section 1 of this report, the sewage sludge regulations at Part 503 include standards for land application of nine metals. These standard s are based on the dry-weight concentrations. Table 14 illustrates the maximum results from this survey for the nine metals.

Table 14. Comparison of Survey Maximums to Existing Regulatory Limits Dry-Weight Concentration in mg/kg Pollutant Land application ceiling Survey Maximum Number of TNSSS Results Over Ceiling
Arsenic 75 49.2 0 Cadmium 85 11.8 0 Copper 4,300 2,580 0 Lead 840 450 0 Mercury 57 8.3 0 Molybdenum 75 132 2 Nickel 420 526 3 Selenium 100 24.7 0 Zinc 7,500 8,550 1 Maximum results that exceed the land application ceiling are shown in bold.

Note: It is critical to note that the selection of facilities to be sampled in this survey was not based on whether they managed their sewage sludge by land application, nor was a goal of this survey to assess compliance. In fact , a number of the facili ties disposed of their sewage sludge by incineration or placement in a landfill, and those facilities need not meet the ceiling concentrations shown in Table 14.

As shown in bold in Table 14, three metals had observe d concentrations in this survey that exceeded the land application ceiling concen trations (molybdenum, nick el, and zinc). The maximum observed concentrations for all other re gulated metals were below the land application ceiling concentrations.

Five samples in this survey, collected from f our facilities, contained metals that exceeded the land application limits (two of those samples were a pair of field dup licates collected from one facility). One sample exceeded the limits for both molybdenum and nickel. Of the four facilities involved, one incinerates its sewage slu dge on site and the other three send their sewage sludge to landfills. None of th e facilities that actually dispose of their sewage sludge by land application exceeded the limits.

6.4 Analytical Completeness

“Completeness” is a quality assurance measure of the number of samples collected compared to the number of useable results produc ed. Although the laboratories experienced a number of difficulties with the samples from this survey and not all of the results met all of the acceptance criteria in the applicable analytical methods, laboratories made acceptable efforts to overcome these challenges and ad equately document any QC issu es encountered. In all cases laboratories provided acceptable documenta tion for every sample in the survey.TNSSS Sampling and Analysis Technical Report 44 January 2009 6.5 Analytical Sensitivity

As noted earlier in this report, the two previous na tional sewage sludge surveys experienced analytical sensitivity challenges for so me analytes. This was largely due to the coextracted interferences present in the challenging matrix and the wide variation in the solids content of treated sewage sludge disposed of nationwide. EPA designed the TNSSS for practical analytical sensitivity, taking the steps outlined in Sec tion 4 to ensure that the results were directly comparable across facilities in the survey.

Table 7 lists the target reporting limits for a ll classes of analytes in the survey. Those targets were based on EPA’s decisi on of what was practical in se wage sludge. The anions were found in every sample and all but six metals were found in the 100 percent of the survey samples (see Table 11). Thus, sensitivity is not a concern. Therefore, the focus of analytical sensitivity is on the actual reporting limits for the other an alyte classes and on the metals that were not found in every sample (i.e., antimony, beryllium, boron, th allium, tin, and titanium). Table 15 provides a comparison of those target limits with the actual reporting limits.

Table 15. Analytical Sensitivity Analyte Class Target Reporting Limit (dry weight) Actual Reporting Limit for Non Detects Antimony 0.05 mg/kg Beryllium 0.02 mg/kg Boron 5 mg/kg Thallium 0.02 mg/kg Tin 5 mg/kg Metals 3 to 4 mg/kg

Titanium 2 mg/kg 4-Chloroaniline 2-Methylnaphthalene Fluoranthene Pyrene Benzo(a)pyrene 10 μg/kg

(by SIM) PAHs and semivolatiles 100 to 300 μg/kg

bis (2-Ethylhexyl) phthalate NA PBDEs 5 to 200 ng/kg BDE 138 5000 ng/kg Anions 2 to 8 mg/kg All anions detected in all samples NA NA = Not applicable, analyte reported in all samples in the survey

As can be seen in Table 15, the laboratories not only met, but far exceeded, the majority of the sensitivity targets. The reporting limits for boron and tin were only slightly higher than the target range. Four of the six samples in wh ich tin was not reported in clude situations where the results for tin in the sample s and their associated method bla nk differed by less than a factor of five. As part of the data review process de scribed in Section 5, the ti n results were reset in those four samples to non-detects at the nominal reporting level.

Bis (2-Ethylhexyl) phthalate is a common la boratory contaminant and EPA reviewed the results for all of the method blanks to ensure that the laboratory was not the source of the bis (2- Ethylhexyl) phthalate in the samples. Alt hough the laboratory reported bis (2-Ethylhexyl) phthalate in many of the method blanks for organi cs, the levels in the samples are two to four orders of magnitude higher than the levels in the blanks, indicating that sensitivity was not an issue.

The reporting limits for the other five organics were well below the target range using the selected ion monitoring modifica tions described in Section 4. The full-scan GC/MS resultsTNSSS Sampling and Analysis Technical Report 45 January 2009 included in the database are for samples in which the majority of the analytes were present above the original target reporting limits in Table 11, thus sensitivity was not an issue for those samples.

All of the PBDE congeners except BDE- 138 and BDE-209 were detected in all of the survey samples, often at high levels relative to the calibration range of the method. There was only one non-detect for BDE-209. BDE-138 was not detected in 30 of the survey samples with the reporting limit of 5,000 ng/kg. Th at reporting limit is significan tly higher than the original target, but reflects the need to adjust the sample size and extract dilution to accommodate the very high levels of the other c ongeners that the laboratory reporte d in the survey samples, which were often 10 to 20 times higher than the BDE- 138 concentrations. Therefore, sensitivity was not a significant issue for the PBDE analyses.

Because the methods for the pharmaceuticals, steroids, and hormones were under development at the time the TNSSS began, EPA di d not set any goals for sensitivity for these analytes. The reporting limits are based on the “Minimum Levels” in the methods, which were not optimized for the analysis of sewage sludge samples, but apply to all solid matrices. The Minimum Level is the concentration in the sample that is equivalent to the concentration of the lowest calibration standard analyzed by the labo ratory. When used as a reporting limit, that value is adjusted for the nominal sample size and the moisture content of the sample (i.e., it is a dry-weight concentration). The advantage of the approach used for the TNSSS to ensure consistent sensitivity is that the reporting limit for each analyte was the essentially the same for all the samples, regardless of the moisture content of the original sample.

The Minimum Levels for the 72 pharmaceuticals vary by analyte, and range from 2 μg/kg for analytes such as Albuterol, Digoxin, a nd Erythromycin, to 1,000 μg/kg for 1,7-Dimethylxanthine. The Minimum Levels for the st eroids and hormones ra nged from 21 μg/kg for Testosterone, to 2,500 μg/kg for Cholesterol. Table 16 presents the Minimum Levels for the pharmaceuticals and Table 17 presents the Minimum Levels for the steroids and hormones.TNSSS Sampling and Analysis Technical Report 46 January 2009 Table 16. Minimum Levels for the Pharmaceuticals Minimum Level μg/kg (dry-weight) 2 4 10 20 Albuterol Digoxin Diltiazem Erythromycin-total Roxithromycin Sulfadimethoxine Cimetidine Dehydronifedipine Diphenhydramine Ormetoprim Oxolinic Acid Ranitidine Sulfamerazine Sulfamethazine Sulfamethizole Sulfamethoxazole Azithromycin Carbadox Carbamazepine Clarithromycin Cotinine Flumequine Fluoxetine Gemfibrozil Miconazole Ofloxacin Sulfachloropyridazine Sulfadiazine Sulfathiazole Thiabendazole Trimethoprim Warfarin Cloxacillin Codeine Enrofloxacin Lincomycin Lomefloxacin Naproxen Norgestimate Oxacillin Penicillin G Triclocarban

Minimum Level μg/kg (dry-weight) 40 100 400 1,000 Ciprofloxacin* 4-Epioxytetracycline 4-Epitetracycline Cefotaxime Chlortetracycline Clinafloxacin Doxycycline Isochlortetracycline Oxytetracycline Penicillin V Tetracycline Tylosin Sarafloxacin† 4-Epianhydrotetracycline 4-Epichlortetracycline Anhydrochlortetracycline Anhydrotetracycline Caffeine Demeclocycline Digoxigenin Ibuprofen Norfloxacin Sulfanilamide Metformin ▲ 4-Epianhydrochlortetracycline Acetaminophen Minocycline Triclosan 1,7-Dimethylxanthine

There were 3 analytes with unique MLs. They were grouped with other analytes in this table for simplicity. The actual MLs are shown below. * Actual ML = 35 μg/kg † Actual ML = 91 μg/kg ▲ Actual ML = 200 μg/kg

Table 17. Minimum Levels for the Steroids and Hormones Minimum Level μg/kg (dry-weight) 21 42 104 17 α -Dihydroequilin 17 α -Estradiol 17 α -Ethinyl-estradiol 17 β -Estradiol Androsterone Equilenin Equilin Estriol Estrone Norethindrone Testosterone β -Estradiol 3-benzoate Norgestrel Androstenedione Progesterone

Minimum Level μg/kg (dry-weight) 500 1,500 2,500 Campesterol Cholestanol Coprostanol Epicoprostanol Stigmasterol β -Sitosterol β -Stigmastanol Cholesterol Desmosterol ErgosterolTNSSS Sampling and Analysis Technical Report 47 January 2009 There were 15 pharmaceuticals and 1 hormone th at were not detected in any sample. EPA examined the supporting QC data, such as the OPR samples and labeled compound recoveries, looking for indicati ons of methodological issues.

There were several pharmaceutical analytes th at exhibited occasional low recoveries in an OPR aliquot, no recoveries in an OPR aliquot, or low labe led compound recoveries in a sample. For example, 1,7-Dimethylxanthine wa s only found in 4 samples, and there were 12 samples (prepared in one batch) th at were associated with an OP R aliquot with low recovery of this analyte. The other 72 samples were analyzed in 6 other batches asso ciated with acceptable OPR recoveries for 1,7-Dimethylxanthine.

There were 13 results for Warfarin where the sample exhibited low labeled compound recovery and EPA qualified the non- detect results for the analyte. There were no issues with labeled compound recoveries or OPR recoveries for Warfarin in the other 71 samples in the survey. Therefore, EPA does not believe that th e fact that Warfarin wa s not detected in any sample is an indication of a methodological chal lenge (i.e., that the method did not work). Rather, it may be related to method sensitivit y or breakdown of this analyte during sewage

treatment.

For the steroids and hormones, 17 α -Ethinyl-Estradiol was the one analyte that was not reported in any sample. EPA noted four instan ces where the labeled compound associated with this analyte was not recovered at all, leading EPA to exclude t hose four non-detect results from the survey database. In three other instances , EPA noted that the recovery of the labeled compound was low, but there was some recovery. However, EPA did not note OPR issues for this analyte, which suggests that in those specific samples there may be issues associated with the specific biosolids samples being analyzed rather than pervasive analytical problems.

6.6 Equipment Blank Evaluation

Equipment blanks were prepared as describe d in Section 3.11 in each of the laboratories that analyzed sewage sludge samples in the fi rst phase of the TNSSS. Equipment blanks were prepared for anions, metals, semivolatile organi cs and PAHs, and PBDEs. Equipment blanks were not prepared for the pharmaceuticals, ster oids, and hormones in the second phase of the TNSSS.

EPA evaluated the results for the equipment blanks by comparing them to their associated method blanks and to the sample results . This was done to determine if any analytes of concern were present at levels that might affect EPA’s use of the data.

6.6.1 Semivolatile Organics and PAHs

For semivolatile organics and PAHs, the labo ratory reported only one target analyte in either equipment blank, BEHP, at a concentration of 10  g/kg, based on a nominal 10-g sample weight. However, BEHP is a common laborator y contaminant and the method blank associated with both equipment blanks was reported to contain 11  g/kg. In addition, all of the field sample results for BEHP were orders of magnitude hi gher than either the method blank or equipment blank results, ranging from 657 to 310,000  g/kg. Therefore, there is no evidence that the compositing equipment contributed BEHP to any of the field samples.TNSSS Sampling and Analysis Technical Report 48 January 2009 6.6.2 Metals

The laboratory reported low concentrations of eight elements in the two equipment blanks. Table 18 presents the resu lts for the two equipment blanks . The reported concentrations are based on the nominal 1-g dry-weight aliquot used for the field samples. The laboratory reported all other metals as non-dete cts in the two equipment blanks.

The concentrations of some of the meta ls in the equipment blanks exceeded the concentrations in the associated method blank, although in some instances only marginally. For example, the laboratory reported nickel in the method blank at 0.05 mg/kg, and at 0.1 mg/kg in one equipment blank, but nickel was not detected in the other equipment blank. The laboratory reported lead in one equipment blank at 0.03 mg/kg, which is only marginally above the laboratory’s reported detection limit of 0.02 mg/kg for lead, and lead was not detected in the other equipment blank. Therefore, the equipm ent blank results are not unexpected, or of significant concern.

Table 18. Equipment Blank Metal Results Concentration (mg/kg) Analyte Equipment Blank 1 Equipment Blank 2 Barium 27.2 27.9 Calcium 26.9 22.3 Copper 1.33 1.56 Lead 0.03 ND Magnesium 2.8 2.1 Manganese 0.07 ND Nickel 0.1 ND Zinc 3.3 6.7 ND = not detected

The concentrations in the field samples ge nerally were several orders of magnitude higher than those in the equipm ent blanks (see Table 11). However, EPA took a conservative approach to evaluating the potential impact of the equipment blanks. EPA compared the results for each analyte in each of the solid samples ag ainst the results for both equipment blanks and flagged in the database any solid sample result that was not at least five times higher than the result in either of the two equipment blanks.

Barium was the only metal analyte affected, w ith the results in four solid sewage sludge samples less than five times the results in the equipment blanks (27.2 and 27.9 mg/kg, respectively). Those results are shown in Table 19. The value of 5 is a multiplier that is often used for the evaluation of blanks . It indicates that the amount of contaminant in the equipment blank might account for 20% or more of a given field sample result.

Table 19. Comparison of Sample Results and Equipment Blank Results for Barium Ratio of Sample Result to Equipment Blank Result Sample Result (mg/kg Sample/Equipment Blank 1 Sample/Equipment Blank 2

68320 128 4.7 4.6 68343 75.1 2.8 2.7 68344 78.6 2.9 2.8 68393 134 4.9 4.8TNSSS Sampling and Analysis Technical Report 49 January 2009 The fact that the results for these four sa mples are less than five times the equipment blank results is not clear evid ence that any portion of the barium in the samples was actually derived from the sampling equipment. As noted in Section 3, equipment blanks for solids are conceptually different than t hose for aqueous samples and sampling equipment. In the 2001 NSSS, equipment blanks were prepared by placing wet sand in the compositing bowls used for collecting samples. Aliquots of the sand had been analyzed for dioxins, furans, and PCBs and the sand was found to be free of these analytes, so it could be mixed with reagent water to simulate wet sewage sludge. Equipment blanks prepared in that manner in the 2001 survey demonstrated that the stainless steel equipment used to collect samples did not contribute any dioxins, furans, or PCBs.

In this survey, sand could not be used for equipment blanks because it contains metals. No readily available solid reference material is free of metals and resembles sewage sludge, so it was not practical to prepare equipment blanks for solids that mimic the way that the sample comes in contact with the sampling equipment.

6.6.3 PBDEs

The laboratory did not detect any of the PBDE congeners in the two equipment blanks above their nominal reporting limits. The repor ting limits for these blanks ranged from about 100 ng/kg to 20,000 ng/kg, for the various congeners, based on a nominal sample size of 10 g. However, because the sample size for the field samples was reduced to 0.2 g, EPA examined the equipment blank in greater detail. In addition to using reporting limits that were based on the low-point of the instrument calibration, the laboratory estimated the signal-to-noise based detection limits for each analyte in each sample. Those estimated detection limits were markedly lower than the nominal reporting limits (e.g., 1 ng/kg versus 100 ng/kg). The low levels of PBDEs detected in the equipment blanks were similar to those reported in the method blanks, indicating that the sampling equi pment did not contribute addi tional PBDEs to the samples.

6.6.4 Anions

The equipment blanks for the anions anal ysis contained low levels of fluoride and nitrate/nitrite. The results for both analytes in both equipment blanks were below the laboratory’s reporting limits, but a bove their detection limits, and the laboratory reported the results as “estimated.” Neither analyte was detected in the met hod blanks associated with the equipment blanks or the field samples. Table 20 presents the results for the anions in the two equipment blanks.

Table 20. Equipment Blank Anion Results Concentration (mg/kg) Analyte Equipment Blank 1 Equipment Blank 2 Nitrate/Nitrite 5.2 1.9 Fluoride 11 18TNSSS Sampling and Analysis Technical Report 50 January 2009 The equipment blank results for water-extract able nitrate/nitrite ar e two to five times lower than the laboratory’s nominal reporting lim it. The concentration of water-extractable nitrate/nitrite in the field samples ranges from 1.6 mg/kg to more than 6,100 mg/kg. Of 41 sewage sludge samples that contained less than 10 mg/kg of nitrate/nitrite , 29 of those are solid samples that came in contact with the plastic sampling equipment. However, the other 12 samples with less than 10 mg/kg nitrate/nitrite are liquid sewage sludge that was never in contact with the equipment. Because of this, EPA conclude d that nitrate/nitrite levels observed in blanks were acceptable for the purposes of this report.

Fluoride is added to drinking water by many municipal water systems for its dental decay prevention benefits. It also is present in toot hpastes and mouthwashes th at are rinsed down the drain after use. Fluoride is soluble in water, whic h is, in fact, the basis of the analytical results for this survey, as the samples were leached with reagent water as described in Appendix A, and the leachate was analyzed for all of the anions. Figure 2 is a plot of th e fluoride concentrations versus the percent solids in each survey sample.

Water is removed from sewage sludge by a va riety of means, includ ing several types of presses, centrifugation, and air dryi ng. Given the solubi lity of fluoride, one would expect that water removed by mechanical means such as pr esses and centrifugation w ould take some portion of fluoride with it, leaving the solids with lower concentrations then at the start of the water removal process.

Figure 2. Plot of Fluoride Concentration versus Percent SolidsTNSSS Sampling and Analysis Technical Report 51 January 2009 As can be seen in Figure 2, ther e is an inverse relationship betw een solids and fluoride in these samples, with most of the highest fluoride concen trations in the samples with the lowest solids

contents.

Note: All of the fluoride results are reported on a dry- weight basis in this survey, so the results are directly comparable across samp les with different percent solids.

The presence of fluoride in the two equipmen t blanks may indicate that the reagent water rinse did not remove some of the fluoride in the ta p water used to wash the equipment. As with the nitrate/nitrite results, the amounts of fluoride reported in the two equipment blanks are 2 to 4 times lower than the laboratory’s reporting limits. EPA treated the fluoride results in the field samples in the same fashion described above fo r nitrate/nitrite, flagging any results in solid samples less than five times the higher equipment blank result as an estimate, but retained the data in the database. A total of 62 fluoride results for solid samples were flagged in the database. However, there also were 12 liquid samples with fl uoride concentrations in that same range of up to five times the higher equipment blank result . These samples never contacted the sampling equipment, so the fluoride in them ca nnot be attributed to the equipment.

As noted in Section 3, the sampler mixed a large quantity of sewage sludge in each plastic bowl, and only placed a portion of that materi al in jars for analysis. The moisture in the original sewage sludge samples was not free flowing for the solid samples, thus it would not contact the surfaces of the equipment in the same way that the reagent water used to prepare the anions equipment blank did. EPA does not believe that the qualifiers applied to these results significantly compromise data usability.

6.7 Field Duplicate Results

As part of the quality assurance effort, EP A collected field duplicate samples to assess the overall precision of the sampling and analysis approach for the survey. Of the 80 facilities originally selected for sampling, 8 were chosen at random for collection of a field duplicate. As described in Section 3, two of those facilities were not sample d and not replaced, and one other field duplicate was collecte d at a different plant th an originally planned.

The tables in this section present the rela tive percent differences (RPDs) between the two results in each of the six pairs of field duplicate samples, for each analytical class. The RPD is used as the measure of precision because both resu lts from the pair are measured concentrations and there is no “true” concentration to be us ed in the comparison. The formula for RPD is shown below where Result 1 and Result 2 repres ent the concentrations reported in the two samples in each pair. The vertical bars in the numerator indicate it is the absolute value of the difference, and the factor of 100 c onverts the value to a percentage.

In cases where both of the results in a fi eld duplicate pair were non-detects, there was little point in comparing the sample-specific repo rting limits. EPA indicated these instances with ND, for “not detected.” When only one of the re sults in a field duplicate pair was a non-detect, EPA did not calculate the RPD and indicated these instances with NC, for “not calculated.” 100

2 2)Result1(Result 2Result1Result RPD   

TNSSS Sampling and Analysis Technical Report 52 January 2009 EPA did not establish a formal acceptance lim it for the RPD of field duplicates in this project. However, 50% is often used as a defa ult limit that reflects the sum of the anticipated analytical variability and the variabil ity in the sample collection process.

6.7.1 Anions

Table 21 summarizes the field duplicate results for the anions. The majority of the RPD values for the anions are less than 20%, and are well within the expected variability of laboratory duplicate analyses. Using the default limit of 50% for field duplicates, the 16 of 24 observed RPD values in Table 21 th at are below 20% indicate better than anticipated results for these QC samples.

Table 21. Comparison of Field Duplicate Results for Anions Relative Percent Difference (%) Analyte Pair 1 Pair 2 Pair 3 Pair 4 Pair 5 Pair 6 Fluoride 11.9 18.3 8.0 35.1 8.1 3.6 Nitrate/Nitrite 19.0 17.1 16.1 15.7 2.2 36.2 Water-Extractable Phosphorus 84.3 26.0 33.4 39.5 9.0 20.5 Total Solids 8.1 0. 2 0.8 58.7 0.9 1.0

The RPD values above 20% occur primarily for the water-extractable phosphorus (WEP) and most of the analytes in Pair 4. The WEP resu lts are more variable than those for fluoride or nitrate/nitrite. All three analytes are extracted from the sewage sludge at the same time using the same leaching procedure. The WEP differences between some of the duplicate pairs may simply be due to variability.

Field duplicate Pair 4 exhibited large RPD va lues for the anions and all other analytes (see Tables 21 – 26). EPA examined the sample collection information and found that this was a liquid sewage sludge collected from a large stor age tank. Because of safety concerns at the facility, the sampler observed as on e of the facility staff opened a series of valves, flushed liquid sewage sludge through the piping, and collected each aliquot of the first sample, then collected the second sample. The percent solids results fo r these two samples are 1.85% and 1.01% in the aliquots use for the anions analyses. However, the percent solids in the aliquots used for the metals analyses are 4.27% and 1.01%, while the aliquots for the orga nics are 0.61% and 3.2% respectively. These data for to tal solids suggest that liquid se wage sludge was not particularly homogeneous and the sampling procedures used for this facility did not result in true duplicate samples.

6.7.2 Metals

Table 22 presents the field duplicate results for the metals. The majority of the RPD values for the metals (125 of 174) are less than 20%, and 135 RPD values are less than 30%. As with the anions, results for field duplicate Pair 4 are markedly higher than the other five pairs.TNSSS Sampling and Analysis Technical Report 53 January 2009 Table 22. Comparison of Field Duplicate Results for Metals Relative Percent Difference (%) Analyte Pair 1 Pair 2 Pair 3 Pair 4 Pair 5 Pair 6 Aluminum 0.0 1.3 4.2 100.5 7.7 0.7 Antimony ND 3.6 3.0 97.5 19.3 2.3 Arsenic 6.2 0.0 4.5 109.2 30.8 7.3 Barium 4.1 59.8 4.6 100.7 10.8 1.5 Beryllium 0.0 3.6 0.0 83.3 25.0 7.4 Boron 13.7 2.9 1. 6 115.1 12.6 0.1 Cadmium 7.1 1.7 3.7 106.1 31.5 7.3 Calcium 4.3 1.9 2.5 112.2 11.2 1.5 Chromium 7.5 6.8 27.9 120.3 29.4 0.9 Cobalt 2.7 0.2 4.6 110.4 22.4 9.1 Copper 3.4 1.7 5.4 100.7 10.3 2.9 Iron 4.7 3.6 2. 5 106.5 9.6 0.6 Lead 7.1 0.6 3. 9 105.4 30.5 8.5 Magnesium 10.5 0.7 4.9 112.5 8.5 1.2 Manganese 4.8 5.4 3.1 105.8 12.1 1.0 Mercury 25.6 95.3 2.7 89.5 11.8 7.1 Molybdenum 9.1 5. 0 0.5 112.6 31.2 8.4 Nickel 5.1 3.3 4. 3 107.9 27.7 11.4 Phosphorus 4.7 0. 6 4.3 106.1 11.0 0.6 Selenium 2.7 1.1 12.5 118.7 31.3 9.8 Silver 5.0 1.8 10.2 95.4 8.8 16.9 Sodium 1.6 41.8 4.2 121.6 5.2 3.2 Thallium 6.1 3.1 0.0 112.5 41.4 0.0 Tin 6.6 0.5 ND 94.1 9.3 23.5 Titanium 9.9 3.9 27.2 96.8 4.6 16.2 Vanadium 8.3 4.1 3.6 100.7 28.6 8.7 Yttrium 8.1 1.6 4.3 107.5 28.0 8.2 Zinc 4.6 1.6 5. 4 106.5 10.7 1.5 Total Solids 0.4 1. 2 0.4 123.5 2.7 1.0 ND = not detected in either sample in the duplicate pair

6.7.3 Semivolatile Organics and PAHs

Table 23 presents the field duplicate results for the semivolatile organics and PAHs. A total of 29 of 42 RPDs are less than 20% and 31 of 42 RPDs are less than 50%. The issues with Pair 4 are apparent in the organics data as well (see Section 6.7.7).

Table 23. Comparison of Field Duplicate Re sults for Semivolatile Organics and PAHs Relative Percent Difference (%) Analyte Pair 1 Pair 2 Pair 3 Pair 4 Pair 5 Pair 6 2-Methylnaphthalene 12.7 36.6 ND ND ND 17.1 4-Chloroaniline NC 7. 4 8.0 41.8 12.7 5.0 Benzo(a)pyrene 2.4 3.1 ND 143.9 8.7 17.0 bis (2-Ethylhexyl) phthalat e 16.7 3.6 0.0 157.8 15.9 10.8 Fluoranthene 16.7 0. 0 0.0 144.3 2.1 14.2 Pyrene 0.0 6.9 ND 128.9 5.7 8.9 Total Solids 1.2 4. 3 1.2 136.0 10.6 1.0 ND = not detected in either sample in the duplicate pair NC = not detected in one sample in the duplic ate pair and therefore RPD was not calculatedTNSSS Sampling and Analysis Technical Report 54 January 2009 6.7.4 PBDEs

Table 24 presents the field duplicate results for the PBDEs. A total of 36 of 72 RPD values are less than 20% and 58 of 72 are less th an 40%. Field duplicate Pair 4 exhibits higher RPD values than then other five pairs, but the differences are not as marked as for the other analyte classes. It is possible that the difference may be a reflec tion of the smaller sample size used for the PBDE analyses compared to the othe r classes. Due to the concentrations of PBDEs in biosolids, which can interfere with analysis, the laboratory extracted just 0.2 g of the sewage sludge, compared to samples of up to 10 g for ot her samples and analytes. Extracting a smaller sample eliminated the need for repeated dilution of the sample, resulted in fewer burdens to (i.e., didn’t overwhelm) the laboratory equipm ent, and made quantitation possible.

Table 24. Comparison of Field Duplicate Results for PBDEs Relative Percent Difference (%) Analyte Pair 1 Pair 2 Pair 3 Pair 4 Pair 5 Pair 6 BDE-28 19.6 16.7 11. 2 42.4 21.9 19.4 BDE-47 16.7 22.2 9. 1 35.3 26.3 9.5 BDE-66 28.0 10.0 6. 5 25.6 2.7 14.6 BDE-85 45.3 25.6 2. 3 40.0 30.8 20.2 BDE-99 17.4 15.1 19. 0 48.6 27.8 1.0 BDE-100 14.7 22.2 9.5 48.6 34.2 14.6 BDE-138 NC 34.3 ND 44.6 ND 8.0 BDE-153 18.2 15.4 11.1 45.0 27.9 8.7 BDE-154 21.1 15.0 13.3 45.1 28.6 10.5 BDE-183 16.9 18.2 10.3 46.2 28.6 19.4 BDE-209 33.0 32.7 4.9 44.7 41.9 25.0 Total Solids 4.6 7. 1 2.5 38.9 28.2 0.0 ND = not detected in either sample in the duplicate pair NC = not detected in one sample in the duplic ate pair and therefore RPD was not calculated

6.7.5 Pharmaceuticals

Table 25 presents the field duplicate results fo r the pharmaceuticals. As noted earlier, in cases where both of the results in a field dupli cate pair were non-detect s, EPA indicated these instances with ND, for “not detected.” There are 270 instances of NDs for the pharmaceuticals.

When only one of the results in a field duplicate pair was a non-detect, EPA did not calculate the RPD and indicated these instances w ith NC, for “not calculated.” There were 18 instances of NCs for the pharmaceuticals. In Table 25, bold is used to indicate all of the RPD values that exceeded 50%.

Table 25. Comparison of Field Duplicate Results for Pharmaceuticals Relative Percent Difference (%) Analyte Pair 1 Pair 2 Pair 3 Pair 4 Pair 5 Pair 6 Acetaminophen ND ND ND ND ND ND Albuterol ND ND ND NC ND ND Anhydrochlortetracycline ND ND ND ND ND ND Anhydrotetracycline 13. 9 NC 11.2 31.8 22.6 15.3 Azithromycin 14.8 9.4 9.38 50.9 25.6 18.9 Caffeine ND ND 3.71 72.4 21.4 ND Carbadox ND ND ND ND ND ND Carbamazepine 18.4 6. 0 9.32 38.2 11.1 19.7 Cefotaxime ND ND ND ND ND NDTNSSS Sampling and Analysis Technical Report 55 January 2009 Table 25. Comparison of Field Duplicate Results for Pharmaceuticals Relative Percent Difference (%) Analyte Pair 1 Pair 2 Pair 3 Pair 4 Pair 5 Pair 6 Chlortetracycline ND ND ND ND ND ND Cimetidine 3.6 34.4 73.1 116 24.5 17.0 Ciprofloxacin 38.4 5.5 0.54 50.7 2.0 5.5 Clarithromycin ND ND 20.6 19.0 ND 6.4 Clinafloxacin ND ND ND ND ND ND Cloxacillin ND ND ND ND ND ND Codeine ND ND ND 39.4 NC ND Cotinine ND 10 10.5 NC 27.3 19 Dehydronifedipine ND ND 27.8 27.3 ND ND Demeclocycline ND ND ND ND ND ND Digoxigenin ND ND ND ND ND ND Digoxin ND ND ND ND ND ND Diltiazem NC 34.1 50.5 32.9 46.4 2.96 1,7-Dimethylxanthine ND ND ND ND ND ND Diphenhydramine 22.0 0. 8 11.9 15.8 29.5 6.23 Doxycycline 48.1 27. 4 15.7 17.4 15.8 4.55 Enrofloxacin ND ND ND 18.9 7.2 ND 4-Epianhydrochlortetracycline ND ND ND ND ND ND 4-Epianhydrotetracycline ND NC 2.5 36.3 35.0 5.30 4-Epichlortetracycline ND ND ND ND ND ND 4-Epioxytetracycline ND ND ND ND ND NC 4-Epitetracycli ne 37.6 27.8 24.4 55.2 3.24 18.2 Erythromycin-total 51.7 44.1 2.25 32.5 138 22.2 Flumequine ND ND ND ND ND ND Fluoxetine 25.5 10.1 40.3 47.9 7.5 18.5 Gemfibrozil 3.4 13.1 7.8 71.1 4.7 2.6 Ibuprofen ND 2.2 14.2 119.1 3.0 0 Isochlortetracycline ND ND ND ND ND ND Lincomycin ND ND ND ND NC ND Lomefloxacin ND ND ND ND ND ND Metformin ND ND ND ND ND ND Miconazole 5.7 1.80 ND 2.3 187 33.8 Minocycline ND ND 9.2 ND 5.1 6.2 Naproxen ND 12.0 11.8 68.5 15.5 NC Norfloxacin ND ND 18.2 59.2 1.8 ND Norgestimate ND ND ND ND ND ND Ofloxacin 15.9 16.5 3.9 29.0 11.2 12.3 Ormetoprim ND ND ND ND ND ND Oxacillin ND ND ND ND ND ND Oxolinic acid ND ND ND ND ND ND Oxytetracycline ND NC ND ND NC 9.04 Penicillin G ND ND ND ND ND ND Penicillin V ND ND ND ND ND ND Ranitidine ND ND NC 121.5 2.1 ND Roxithromycin ND ND 4.03 ND ND ND Sarafloxacin ND ND ND ND ND ND Sulfachloropyridazine ND ND ND NC ND ND Sulfadiazine ND ND ND ND ND ND Sulfadimethoxine ND ND ND NC ND ND Sulfamerazine ND ND ND ND ND ND Sulfamethazine ND ND ND ND ND ND Sulfamethizole ND ND ND ND ND ND Sulfamethoxazole ND ND ND 56.3 ND ND Sulfanilamide ND ND ND ND ND NDTNSSS Sampling and Analysis Technical Report 56 January 2009 Table 25. Comparison of Field Duplicate Results for Pharmaceuticals Relative Percent Difference (%) Analyte Pair 1 Pair 2 Pair 3 Pair 4 Pair 5 Pair 6 Sulfathiazole ND ND ND ND ND ND Tetracycline 15.2 1. 6 5.09 2.3 0.52 13.1 Thiabendazole ND ND 20 0.8 0.30 0.49 Triclocarban 6.1 10. 2 0 13.2 7.31 5.90 Triclosan 5.7 12.4 37.3 38.3 26.9 41.0 Trimethoprim ND ND 30.8 ND ND NC Tylosin ND ND ND ND ND ND Virginiamycin ND 22.1 NC ND NC ND Warfarin NC ND ND ND ND ND Total Solids 1.7 4.0 2.84 88.0 6.21 3.54 ND = Not detected in both samples in the pair NC = Not calculated because one of the results was a non-detect RPD values greater than 50% are shown in bold

.
EPA calculated 143 RPD values for the pharm aceuticals, not including those for the total solids in each sample. Of the 143 RPDs, 16 ex ceeded 50%, although some only marginally (e.g., 50.5, 50.7, and 50.9%). Of the 16 values greater than 50%, 11 occurred in Field Duplicate Pair 4. As discussed above, EPA believe s that the RPD values in Pair 4 reflect differences in the two samples of liquid sewage sludge collected at that facility, with the RPD fo r the total solids at 88%.

Many of the 270 instances where an analyte was not detected in either sample in the field duplicate pair are a function of the low freque ncy at which some of the pharmaceuticals were detected. For example, as shown in Table 12, Acetaminophen was only detected in 2 of the 84 survey samples. Therefore, its frequency of occurrence was only 2.38%. The chance that a field duplicate sample would be collected at any of th e 74 POTWs in the survey was 8.1% (6 out of 74 plants). The likelihood of detecting Acetaminophe n in both samples in a field duplicate pair is on the order of 0.2% (e.g., 2.38% x 8.1%). Therefore, the fact that Acetam inophen is listed as ND in Table 25 for all 6 field dupl icate pairs is not surprising.

In contrast, Ciprofloxacin was reported in al l 84 samples from the survey, including the 6 field duplicate samples. Therefore, EPA was ab le to calculate an RPD value for each field duplicate pair in Table 25. Except for field dupli cate Pair 4, the RPDs for Ciprofloxacin indicate good precision (four of the RPD values are less than 6%).

6.7.6 Steroids and Hormones

Table 26 presents the field duplicate re sults for the steroids and hormones.TNSSS Sampling and Analysis Technical Report 57 January 2009 Table 26. Comparison of Field Duplicate Results for Steroids and Hormones Relative Percent Difference (%) Analyte Pair 1 Pair 2 Pair 3 Pair 4 Pair 5 Pair 6

Androstenedione ND 9.7 ND ND 83.3
Androsterone ND ND 3.4 ND NC 28.8 Campesterol 0 10.4 5.1 88.9 96.6 17.1 Cholestanol 11.1 9.3 6.2 97.0 32.8 7.2 Cholesterol 0.0 3.4 2.5 99.4 48.0 3.7 Coprostanol 13.4 14.5 3.8 110.4 39.8 13.6
Desmosterol 20.0 3.2 14.8 96.7 9.0 23.1

17 α -Dihydroequilin ND ND ND NC ND ND Epicoprostanol 17.3 38.4 13.4 109.7 32.8 5.6 Equilenin ND ND ND ND ND ND Equilin ND ND 13.4 ND ND 5.7 Ergosterol 38.4 ND 33.6 93.8 24.4 43.0

17 α -Estradiol ND ND ND ND NC ND

17 β -Estradiol ND ND ND 119.5 27.5 ND β -Estradiol 3-benzoate ND ND ND 88.3 ND ND

17 α -Ethinyl-estradiol ND ND ND ND ND ND
Estriol ND ND 90.0 NC 21.0 NC

Estrone ND 8.3 3.1 0.6 4.5
Norethindrone ND ND ND ND ND ND Norgestrel ND ND ND ND ND ND Progesterone ND 20.5 ND ND ND 65.9 β -Sitosterol 38.7 8.8 16.2 87.4 44.4 3.2 β -Stigmastanol 39.0 3.8 14.3 87.0 33.1 30.6 Stigmasterol 25.5 5.1 34.5 83.6 61.0 2.5
Testosterone ND ND ND ND ND NC Total Solids 1.7 4.0 2.8 88 6.2 3.5 ND = Not detected in both samples in the pair NC = Not calculated because one of the results was a non-detect RPD values greater than 50% are shown in bold

.

There were 6 instances of NCs for the ster oids and hormones. For the steroids and hormones, EPA calculated 74 RPD va lues, not including those for the total solids in each sample. Of those 74 RPDs, 17 exceeded 50%. There were 70 instances where EPA did not calculate an RPD value because both results for the analyte we re non-detects (listed as ND in Table 26). As discussed for the pharmaceuticals, the prevalence of the ND entries in Table 26 is largely a function of the frequency of o ccurrence of the analytes across all samples. For example, Norgestrel was only reported in 4 of 84 sample s from the survey (4.76%), yielding a very low likelihood it would be found in both samples from a field duplicate pair. The occurrence of Cholesterol in 81 of 84 samples is not surprising, given that it is excreted by humans. That high frequency of occurrence enabled us to calculate an RPD value for all 6 field duplicate pairs.

Of the 17 RPD values for steroids and horm ones greater than 50%, 12 occurred in Field Duplicate Pair 4. EPA believes that the RPD valu es reflect differences in the two samples of liquid sewage sludge collected at that facility. The differences are evident in the total solids contents of each sample, where the RPD is 88%.TNSSS Sampling and Analysis Technical Report 58 January 2009 6.7.7 Results in Liquid Samples

The results for all classes of analytes support EPA’s conclusion that the sampling procedures were appropriate a nd effective in collecting sewa ge sludge for the TNSSS. The exception was for the facility from which Field Duplicate Pair 4 was collected. EPA believes that the variability of the field duplicate results for Pair 4 is not typical of the variability observed for the other liquid samples in the surve y, but may reflect site -specific conditions.

Given the variability of the results for Field Duplicate Pair 4 shown in the tables above, EPA examined the results for those two samples in greater detail, comparing the two samples from that one site to the liquid sewage sludge samples from other sites. EPA examined the percent solids data from the anions, metals, and semivolatile organics analyses for all 19 liquid sewage sludge samples collected during the survey. Because the results for the PBDEs, pharmaceuticals, steroids, and hormones were deliv ered later in the TNSSS effort, EPA did not include them in this more detailed analysis of liquid sample results.

EPA examined the percent solids results gene rated during analyses of the anions, metals, and semivolatile organics in all 19 liquid sewage sludge samples (19 samples x 3 classes = 57 measurements in all). Based on the observed distribution of resu lts, the data were transformed by taking the natural log of the results and subjec ting them to an F-test. The null hypothesis was that the three aliquots from each of the two sample s in Pair 4 did not have significantly different variances from the variances of the three aliquots in each of the other liquid samples, indicating that they came from the same population. Table 27 presents the log-transformed percent solids data for all 19 liquid sewage sludge samples.

Table 27. Log-transformed Percent Solids Data for Liquid Sewage Sludge Samples Natural Log of % Solids Liquid Sample Anions Metals Organics Variance of Logs 1 1.284 1.747 0.425 0.450 2 3.795 3.773 3.795 0.000 3 0.560 0.560 1.621 0.376 4 4.126 4.126 4.126 0.000 5 1.543 0.270 1.085 0.416 6 0.482 0.482 1.456 0.316 7 - FD 0.615 1.452 -0.494 0.953 8 - FD 0.010 0.010 1.163 0.443 9 2.434 2.407 2.477 0.001 10 2.425 2.434 2.370 0.001 11 1.677 1.128 0.920 0.153 12 -0.844 -0.693 -0.942 0.016 13 0.293 0.647 0.604 0.037 14 0.815 0.658 0.888 0.014 15 0.182 0.182 0.191 0.000 16 -0.673 -0.844 -0.892 0.013 17 0.270 0.207 0.182 0.002 18 2.072 1.991 1.652 0.049 19 0.788 -0.041 0.775 0.226 FD = Field duplicate pairTNSSS Sampling and Analysis Technical Report 59 January 2009 Table 28 presents the results of the F-test . The pooled within -sample variance (the standard deviation squared) is markedly greater for the two field duplicate samples, compared to the remaining liquid sewage sludge samples in th e survey. The F-ratio in Table 28 is greater than the critical value of F, leading to the conclusion that th e variances in the percent solids results for the field duplicate samples are greater than would be expected by chance from samples in a single population.

Table 28. F-test Results for Fiel d Duplicates and Other Liquid Samples Pooled Variance (field duplicates) 0.698 Pooled Variance (non-field duplicates) 0.122 F-ratio 5.731 F-critical 2.650

p-value 0.001229

Therefore, the variability exhibited by the field duplicate results for Pair 4 is not typical of the variability that is apparent for the other liquid samples in the survey. The results for Pair 4 are not an indication that the sampling procedures used for the survey are inappropriate for liquid samples. Rather, the results for Pair 4 may refl ect site-specific conditions. However, both sets of results for all of the field dupl icate pairs, including Pair 4, are included in the survey database.

6.8 Matrix Spike and Duplicate Results

Matrix spike (MS) samples and matrix spike duplicate (MSD) samples, or matrix spike samples and unspiked duplicate (DUP) samples, we re prepared with batches of field samples analyzed for the anions, metals, semivolatile or ganics and PAHs, and the PBDEs analyzed as described in Section 4 without isotope diluti on quantitation. These QC samples served to demonstrate the applicability of the methods to the matrices in question, e.g., liquid and solid sewage sludge. The use of an MSD versus a DUP is generally called out in the method, with MS/MSD pairs being the norm in methods for organics, and MS and DUP samples being the norm in methods for metals and ot her inorganic such as anions. The results for those analyses are discussed below by analyte class. Appendix C presents the QC acceptance limits used by the laboratories.

Because the methods used for the pharmaceuticals, steroids, and hormones use isotope dilution quantitation, those methods do not require that separate MS/MSD samples be analyzed. Rather, the recoveries of the isotopically labeled analytes spiked into every sample are monitored and used to correct the results for the target analytes.

The MS/MSD results for the TNSSS are discussed in the subsections that follow, by analyte class. The labeled compound recoveries for the isotope dilution me thods are discussed in Section 6.8.8.TNSSS Sampling and Analysis Technical Report 60 January 2009 6.8.1 Matrix Spike Results for Anions

The laboratory prepared eight sets of matrix spike samples for the anion analyses. Table 29 presents the results in terms of the percent r ecovery of each analyte spiked into the sample.

Table 29. Matrix Spike Recoveries for Anions in Sewage Sludge Matrix Spike Recovery (%) Analyte MS1 MS2 MS3 MS4 MS5 MS6 MS7 MS8 Fluoride 93 74 66 82 93 60 68 77 Nitrate/Nitrite 98 101 103 95 100 90 101 96 Water-Extractable Phosphorus -24 88 75 102 90 95 80 95

The laboratory’s acceptance limits for matrix spike recoveries were 75 - 125%, and 18 of 24 recoveries in Table 29 met those limits. The laboratory reported the recovery of WEP in MS3 as 74.8%, before rounding. That value is only ma rginally out of the sp ecification, and rounds to 75%, the lower limit of the laboratory’s acceptance range.

The laboratory reported the r ecovery of WEP in one matrix spike (MS1) as -24%. Negative recoveries are not physic ally possible and the reported recovery is a function of the manner in which it is calculated. Although most recent published EPA methods explicitly include example calculations for QC parameters such as recovery, some older analytical methods do not. EPA Methods 340.2, 353.2, and 365.3, used fo r the anion analyses, are among those methods without example calculations. In the absence of project-speci fic requirements, the laboratory performing the anions analyses reli ed on formulae from the SW-846 methods manual from EPA’s Office of Solid Waste. Chapter One of the manual includes definitions of a number of commonly used terms and provides the equation for the calculation of recovery shown below:

where:

%R = percent recovery

x s = measured value for spiked sample

x u = measured value for unspiked sample K = known value for the spike in the sample

This same basic formula appears in many individual EPA methods. The remainder of this report refers to this equation as the “tra ditional approach” to cal culating recovery. As written, K is often interpreted as the amount of material spik ed into the sample. That interpretation ignores any “background” concentrat ion of the analyte in the unspiked sample. In practice, the equation produces recoveries that appear reas onable and generally meet expectations in those samples where the backgro und concentration of the analyte is either very low, or where the amount spiked into the samp le is much greater than the background amount. However, the laboratory calculates negative recoveries any time the result in the spiked sample is less than that in the unspiked sample, even if that is a function of inhomogeneity in the original sample. Given the Law of Conservation Mass, whereby matter cannot be created or destroyed, negative recoveries are physically impossible. K ) u x s (x100 %R 

TNSSS Sampling and Analysis Technical Report 61 January 2009 Laboratories commonly prepare MS samp les without extensive knowledge of the background levels of any target analytes in sp ecific samples. Therefore, a laboratory may blindly spike an amount that is similar to that already in the sample and the equation above will perform poorly.

Given the frequency at which negative and unr ealistic recoveries are reported using the equation above, EPA developed an alternative calc ulation that considers the result found in the matrix spike sample in comparison to what wa s found in the unspiked sample plus the amount spiked. The alternative equation is as follows:

where:

%R = percent recovery C s = measured value for spiked sample C u = measured value for unspiked sample C n = nominal spike added to the sample

Eliminating the subtraction operation in th e numerator of the equations prevents the occurrence of any negative values. Moving the concentration of the unspiked sample to the denominator more effectively addresses th e issue of the “back ground” concentration.

In the case of the first MS sample in Table 29, the water-extractab le phosphorus result in the MS sample (271 mg/kg) was less than the re sult in the unspiked sample (317 mg/kg), despite adding 189 mg/kg of phosphorus, leading to a ne gative recovery (-24.3%) using the traditional approach, as follows:

(100 x (271 - 317))/189 = -4600/189 = -24.3%

Using the alternative equation above and the same laboratory results, the r ecovery in the matrix spike is calculated as: 100 x (271/(317 + 189) = 27100/506 = 53.6%

While the recovery of 53.6% is still below the acceptance limits used by the laboratory, it is a more rational expression of the results in this sample.

The alternative equation increased the recovery of WEP dramatically in this example. However, it did not cause the recoveries that already appear reasonabl e to exceed the acceptance limits when they did not otherwise do so. For example, Table 29 lis ts the recovery of nitrate/nitrite in MS3 as 103%. This value is rounded down from 103.3652% determined via the traditional calculation. Using the alternativ e equation, the recovery is 103.3466%, a trivial difference that is removed when the results are rounded to the nearest whole percentage. Given this trivial difference, EPA did not recalculate every recovery reporte d by the laboratory, but focused this discussion on th e negative recoveries alone. 100x

n C u C s C %R  TNSSS Sampling and Analysis Technical Report 62 January 2009 6.8.2 Duplicate Results for Anions

The laboratory prepared eight sets of duplicate sample an alyses for anions. Table 30 presents the results shown in terms of the rela tive percent difference (RPD) for each analyte in the sample. Although the laboratory used accepta nce limits expressed to the nearest whole percentage, Table 30 presents the RPDs to one decimal place to illustrate the small differences between many of the duplicate pair results.

Table 30. Duplicate Precision for Anions in Sewage Sludge Relative Percent Difference (%) Analyte Dup 1 Dup 2 Dup 3 Dup 4 Dup 5 Dup 6 Dup 7 Dup 8 Fluoride 0.9 2.7 4.5 14.6 5.4 12.2 0.4 14.6 Nitrate/Nitrite 3.6 0.0 23.0 17.1 2.2 8.3 6.9 10.7 Water-extractable Phosphorus 6. 1 5.2 18.8 4.6 9. 4 6.8 4.1 11.1

The laboratory’s acceptance limit for precisi on (RPD) is 20% and al l but one result in Table 30 met that limit. The highest RPD value reported by the laboratory was 23.0% for nitrate/nitrite in Duplicate 3. This RPD was only slightly outside of the 20% limit.

Except as discussed above, the recovery data in Table 29 and the precision data in Table 30 demonstrate that the methods were generally accurate and precise when applied to sewage sludge samples.

6.8.3 Matrix Spike Results for Metals

The laboratory prepared 15 sets of matrix sp ike samples for the metals analyses. Table 31 presents the matrix spike recoveries in th ree parts (Tables 31A, 31B, and 31C), rounded to one-tenth of a percent, to illustrate some of the smaller differences.

Because calcium and magnesium are common components of soils and occur at levels that vary widely, the laboratory did not spike these two metals into solid samples, and they do not appear in Table 31. However, calcium and magnesium also are not major metals of concern in this survey.

The laboratory analyzed mercur y separately from any other metals. Therefore, the results for mercury appear at the bottom of the table beca use the laboratory prepared only 9 matrix spike samples for mercury and did not necessarily use the same field samples for the mercury matrix spike analyses as for the other metals.

During the survey, as the laboratory gained experience with the sewa ge sludge samples, they adjusted the amounts of some metals spiked into each matrix spike sample in an effort to account for the background concentrations. For example, for aluminum, the spiking concentrations ranged from about 400 mg/kg to about 3200 mg/kg, while copper spiking levels ranged from about 40 mg/kg to 4000 mg/kg. These adjustments to the spiking levels were not always successful in ad dressing recovery issues.TNSSS Sampling and Analysis Technical Report 63 January 2009 Aluminum and iron presented problems with reported rec overies. The laboratory also reported negative recoveries for phosphorus, ag ain a function of the assumption that the background concentrations are much lower than the spiking levels. Therefore, Table 31 includes the recoveries reported by the la boratory using the tr aditional calculation fr om the methods (in the MS# columns) along with the alternative calc ulation described above (i n the ALT# columns). Using the alternative calculation, all of the negative recoveries reported by the laboratory become positive values. In a ddition, many of the very high re coveries (e.g., over 250%) are greatly reduced in magnitude.

Table 31A. Matrix Spike Recoveries for Metals in Sewage Sludge, Calculated in the Traditional Fashion and with an Alternative Equation Recovery (%) Analyte MS1 ALT1 MS2 ALT2 MS3 ALT3 MS4 ALT4 MS5 ALT5 Aluminum 177.7 102.4 334.5 104. 2 68.8 98.5 400 114.9 443.9 107.9 Antimony 44.3 45.7 41.1 43.7 75.7 75.7 41.5 42.0 46.4 47.4 Arsenic 102.0 101.8 102.0 101.7 101.5 101.4 111.1 110.6 101.6 101.5 Barium 99.2 99.7 98.8 99.5 101.9 101.0 96.3 98.2 117.5 105.7 Beryllium 108.7 108.3 99.7 99.7 102.3 102.3 112.4 112.0 101.2 101.2 Boron 105.1 103.5 106.5 105.9 110. 6 108.4 110.6 108. 9 103.7 103.0 Cadmium 110.7 105.9 117.4 108.6 112.9 111.5 121.8 119.6 110.7 107.9 Chromium 93.9 98.8 113.7 105.0 110.5 106.9 118.8 110.5 118.1 102.4 Cobalt 101.1 101.0 102.9 102.7 107.6 107.3 116.3 115.7 115.7 104.0 Copper 83.2 98.5 59.7 96.7 79.5 97.9 40.0 95.2 173.3 104.7 Iron -203.0 98.0 1543.8 111.3 - 71.4 96.5 265.0 103.4 156.2 100.8 Lead 111.3 104.4 108.3 103.0 111.1 108.6 118.9 113. 6 109.5 105.7 Manganese 78.2 97.9 135.9 108.1 91.7 99.7 81.0 95.1 101.3 100.4 Molybdenum 103.0 102.6 108.7 105.7 111.5 110.0 110.3 110.0 107.5 106.9 Nickel 101.7 101.3 105.5 103.8 105. 6 105.1 118.3 114. 7 102.5 102.2 Phosphorus -101.5 96.9 -123.5 91.2 -203.9 90.1 -46.0 91.7 -41.8 96.0 Selenium 102.3 102.0 101.4 101.3 102.8 102.6 112.9 112.2 103.5 103.2 Silver 68.0 90.7 93.8 98.2 103.3 101.4 83.1 92.3 101.8 101.1 Thallium 104.8 104.7 106.2 106.2 107.4 107.4 108.9 108.8 108.4 108.4 Tin 96.6 96.8 95.4 95.9 95. 6 95.9 91.3 91. 6 81.1 82.6 Titanium 47.3 57.7 29.4 43.2 16.3 21.9 55.5 61.1 10.8 14.8 Vanadium 101.4 101.0 105.9 104.3 107.3 106.4 117.7 115.1 101.4 101.3 Yttrium 102.2 101.8 102.4 101.9 106.0 105.6 119.6 115.5 103.7 102.7 Zinc 50.8 97.1 41.2 96.6 99. 9 100.0 72.0 94. 1 125.3 101.9 Mercury 106.0 101.3 76.0 97.0 110.8 104.1 106.6 103.2 62.5 94.4

Table 31B. Matrix Spike Recoveries for Metals in Sewage Sludge, Calculated in the Traditional Fashion and with an Alternative Equation Recovery (%) Analyte MS6 ALT6 MS7 ALT7 MS8 ALT8 MS9 ALT9 MS10 ALT10 Aluminum 236.2 104.3 194.9 104.3 653.0 353.7 202.0 103.2 485.0 325.1 Antimony 72.4 72.9 74.8 75.8 69.1 69.4 47.9 49.1 74.3 74.5 Arsenic 103.0 102.8 102.8 102.7 140.3 137.8 100.3 100.2 119.1 118.5 Barium 103.7 101.8 107.8 104.2 119.5 118.8 92.6 96.9 103.0 102.9 Beryllium 93.0 93.1 95.6 95.8 98.7 98.7 117.5 116.7 100.9 100.9 Boron 105.7 104.8 102.6 101.9 119. 5 119.1 103.4 102. 3 127.0 126.3 Cadmium 110.3 108.8 107.2 106.3 119.9 119.3 111.8 109.0 107.7 107.6 Chromium 93.5 96.7 100.0 100.0 116.9 116.3 88.6 96.6 184.4 171.2 Cobalt 102.4 102.3 102.3 102.2 118.8 118.2 99.6 99.6 110.3 110.2 Copper 111.3 101.1 182.6 103.6 216.4 191.8 38.4 93.6 304.4 247.3 Iron 94.7 99.8 1167.5 103.9 2126 465.7 -1312.7 92.5 250.4 214.3 Lead 107.2 105.7 110.1 108.8 329.8 263.9 131.2 113. 6 166.5 157.6 Manganese 98.8 99.5 99.3 99.7 206.4 186.1 10.1 93.7 102.7 102.6 Molybdenum 109.6 108.4 108.2 107.3 101.9 101.8 100.6 100.5 107.1 106.7 Nickel 100.3 100.3 99.8 99.8 150. 0 145.0 103.2 102. 4 184.9 171.3TNSSS Sampling and Analysis Technical Report 64 January 2009 Table 31B. Matrix Spike Recoveries for Metals in Sewage Sludge, Calculated in the Traditional Fashion and with an Alternative Equation Recovery (%) Analyte MS6 ALT6 MS7 ALT7 MS8 ALT8 MS9 ALT9 MS10 ALT10 Phosphorus -63.0 96.5 243.4 102. 0 360.6 263.5 -282. 7 91.1 320.6 238.4 Selenium 103.4 103.1 101.7 101.6 111.7 111.3 101.8 101.7 114.4 113.6 Silver 56.8 85.1 33.4 84.0 95.8 96.0 67.7 87.3 114.2 112.1 Thallium 105.9 105.9 105.7 105.7 108.9 108.9 104.7 104.7 103.9 103.9 Tin 92.5 93.1 85.4 86.1 94. 0 94.0 99.7 99. 7 83.6 83.6 Titanium 13.7 20.7 22.5 29.5 91.0 91.0 85.0 90.8 79.0 79.0 Vanadium 98.9 99.0 115.8 106.7 169.7 162.1 109.6 105.8 139.8 136.8 Yttrium 101.9 101.7 99.7 99.8 140.0 137.4 107.0 105.6 100.1 100.1 Zinc 95.7 99.5 109.4 101.1 230. 8 201.3 92.0 92. 2 160.4 153.4 Mercury 236.7 145.5 116.2 115. 6 124.9 109.0 91.4 94.4 -- --

Table 31C. Matrix Spike Recoveries for Metals in Sewage Sludge, Calculated in the Traditional Fashion and with an Alternative Equation Recovery (%) Analyte MS11 ALT11 MS12 ALT12 MS13 ALT13 MS14 ALT14 MS15 ALT15 Aluminum 229.9 184.4 500.0 108.6 -395.9 75.8 301.9 107.3 147.5 101.9 Antimony 77.7 77.7 55.9 56.7 75.2 75.6 54.7 56.1 85.1 85.4 Arsenic 101.2 101.2 95.9 96.1 69. 7 70.5 -8.2 56.4 105.8 105.6 Barium 107.7 107.6 95.2 98.0 47.7 82.3 115.1 103.5 120.7 108.8 Beryllium 101.4 101.4 100.1 100.1 94.5 94.5 91.9 92.6 109.4 109.2 Boron 119.5 118.4 100.1 100.1 88. 7 90.7 99.8 99. 8 111.6 106.8 Cadmium 104.4 104.4 108.7 107.4 101.9 101.7 55.5 76.9 114.1 111.9 Chromium 105.3 105.3 102.8 101.6 77.4 87.6 118.9 108.7 130.0 104.7 Cobalt 103.5 103.5 100.9 100.9 95.8 96.0 102.5 102.3 108.2 107.8 Copper 155.8 153.7 62.1 97.8 -204.1 74.7 181. 8 104.0 178 105.9 Iron 505.6 224.6 250 102.6 -1573. 6 77.4 1209.8 109.4 599.9 103.2 Lead 100.0 100.0 108.9 106.0 62.2 82.6 120.5 107. 6 114.9 109.8 Manganese 100.3 100.3 92.1 99.1 0.0 89.0 178.0 104.1 124.0 105.9 Molybdenum 105.3 105.3 108.8 107.2 99.3 99.3 -40.9 50.9 112.9 110.5 Nickel 105.0 105.0 102.8 102.3 91. 0 92.3 104.7 103. 8 116.2 109.5 Phosphorus 384.0 169.0 -80.2 96. 2 -142.0 76.9 105. 9 100.6 260.0 102.3 Selenium 100.3 100.3 93.1 93.5 91.8 92.2 -98.2 38.9 107.8 107.3 Silver 90.9 91.8 94.5 97.2 41.0 79.4 79.2 93.5 91.0 97.4 Thallium 99.4 99.4 104.5 104.5 101.0 101.0 95.4 95.6 111.6 111.6 Tin 90.2 90.3 88.9 90.0 91. 8 92.2 84.0 84. 3 99.4 99.4 Titanium 90.5 90.8 35.7 51.4 23.9 26.8 27.0 28.3 40.8 47.7 Vanadium 100.9 100.9 109.1 108.1 92.6 93.7 102.5 101.9 106.9 105.0 Yttrium 100.0 100.0 107.8 106.9 108.2 106.2 89.1 91.2 104.8 104.4 Zinc 461.3 237.9 86.1 98.5 - 106.1 77.7 181.8 104.9 138.0 103.8

Using either calculation, many of the metals exhibited a small positive bias. A total of 12 the 25 spiked metals have mean traditional recoveries between 101% and 115%, while 14 of 25 metals have mean alternative recoveries in th e same range. Five metals exhibited a slight negative bias using the traditiona l calculation, with mean recove ries ranging from 80% to 97%.

Antimony, phosphorus, and titanium exhibited m ean traditional recoveries below 70%. The alternative calculation dramatically altered the mean recovery of phosphorus, raising it from 39% to 120%, by virtue of eliminating the large number of negative values in the traditional calculation.TNSSS Sampling and Analysis Technical Report 65 January 2009 The mean recovery of titanium was only 43% in these 15 matrix spike samples, with only four values in the acceptable range. Similarly, the mean recovery of antimony was 62% in these 15 matrix spike samples, with only five values in the acceptable range. Neither titanium nor antimony is among the nine pollutants initially selected for this survey (see Section 1), nor one of the metals that currently has a regu latory standard in sewage sludge.

6.8.4 Duplicate Results for Metals

Over the course of the survey, the laborator y analyzed 15 samples for metals in duplicate, to assess analytical precision. Table 32 presents the results for those duplicate analyses. The laboratory prepared and analyzed only seven dupli cates for mercury, and as with the matrix spike results, not necessarily using the same field samples as for the other metals. The exception is for Duplicate 8, where the results for mercury are from the same sample as for all the other metals. Table 32 is divided into several parts. The vast majority of the RPD values are less than the acceptance limit of 30%. The exceptions are almost exclusively in Duplicates 8, 10, and 11. All three of these samples were liquid sewage sludge.

Table 32A. Duplicate Precision for Metals in Sewage Sludge Relative Percent Difference (%) Analyte Dup1 Dup2 Dup3 Dup4 Dup5 Dup6 Dup7 Dup8 Aluminum 14.4 6.2 0.7 13.6 3.1 8.1 1.4 142.1 Antimony 8.3 4.5 ND 21.9 18.8 10.0 6.8 146.3 Arsenic 2.5 0.0 8.6 6.8 3.2 10.0 4.4 145.0 Barium 2.3 0.8 3.1 1.4 1.7 5.8 1.4 140.3 Beryllium 6.3 0.0 8.0 18.2 3.8 4.4 5.4 141.9 Boron 13.8 2.8 6.5 13. 9 5.0 6.1 4.2 141.4 Cadmium 1.3 4.8 3.4 5.2 0.6 3.1 6.4 151.0 Calcium 1.2 3.9 0.7 17.1 1.8 6.1 1.1 139.0 Chromium 0.7 2.2 4.7 13.6 3.8 8.0 5.0 140.0 Cobalt 0.4 2.4 4.3 10.1 4.7 5.3 9.3 144.4 Copper 3.2 3.0 2.6 2.1 0.7 5.5 0.9 138.1 Iron 0.7 5.4 1.5 7. 3 1.4 7.5 0.9 140.9 Lead 3.9 4.0 3.7 0. 5 0.5 1.6 7.2 150.7 Magnesium 4.3 8.5 0.7 27.3 4.3 6.6 1.4 140.6 Manganese 2.3 6.2 1.8 3.2 1.2 5.6 1.9 141.0 Molybdenum 1.2 4.2 3. 3 14.1 0.9 16.2 5.0 134.1 Nickel 1.2 2.3 4.3 2.9 3.7 9.3 5.5 143.6 Phosphorus 1.6 0.0 2.8 2.4 0.6 5.9 1.2 141.1 Selenium 3.6 3.4 2.6 3.3 1.8 9.1 2.8 141.0 Silver 12.4 7.6 2.1 1.7 1.5 8.0 15.9 139.5 Sodium 1.0 10.4 3.3 2.0 2.0 4.4 1.0 134.1 Thallium 0.0 0.0 0.0 22.2 4.0 2.9 4.3 167.0 Tin 1.0 2.3 3.3 9. 1 0.7 2.1 5.8 ND Titanium 51.1 22.5 1.4 18.2 4.3 1.2 1.0 142.5 Vanadium 1.1 3.2 4.1 2.9 6.6 3.1 11.9 148.9 Yttrium 0.0 3.8 3.5 6.1 2.6 3.7 4.3 149.4 Zinc 3.2 0.6 0.8 0.5 0.8 5.9 1.3 141.1 Mercury 17.5 2.5 28.7 14.1 5.8 29.9 -- 136.1TNSSS Sampling and Analysis Technical Report 66 January 2009 Table 32B. Duplicate Precision for Metals in Sewage Sludge Relative Percent Difference (%) Analyte Dup9 Dup10 Dup11 Dup12 Dup13 Dup14 Dup15 Aluminum 5.0 148.3 94. 0 7.4 20.4 2.5 0.1 Antimony 20.5 97.6 30. 9 6.3 21.9 11.2 10.7 Arsenic 1.8 89.8 41. 7 0.4 7.7 18.8 7.3 Barium 3.2 147.9 43. 6 0.2 21.8 3.5 2.3 Beryllium 6.2 81.1 ND 0.0 15.2 30.9 0.0 Boron 8.7 135.6 71.2 8.3 11.5 3.5 0.7 Cadmium 2.5 105.9 38. 9 1.2 17.5 33.1 11.6 Calcium 3.1 140.0 82. 1 0.4 24.6 2.7 2.4 Chromium 2.9 83.9 43. 1 4.3 23.9 1.6 1.9 Cobalt 2.2 98.0 17. 8 0.0 27.0 1.8 9.5 Copper 1.9 148.2 44. 9 0.3 23.1 1.2 2.1 Iron 3.0 144.6 91.8 4.4 19.8 0.6 1.3 Lead 2.3 89.9 43.6 1.9 28.9 5.0 8.8 Magnesium 3.4 129.8 51. 1 3.7 19.1 0.8 1.6 Manganese 2.3 144.9 87. 6 0.7 13.5 2.3 2.3 Molybdenum 1.9 91.2 48.0 4.2 6.1 46.0 6.0 Nickel 0.6 89.6 38.2 0.4 32.5 7.9 11.2 Phosphorus 2.4 137.7 63.4 0.9 11.3 1.5 2.0 Selenium 0.0 63.9 50. 0 1.3 2.2 45.2 8.6 Silver 2.7 118.2 36. 5 0.0 26.8 2.5 4.6 Sodium 1.9 122.4 5. 5 1.3 21.3 2.4 1.3 Thallium 2.2 66.7 ND 8.0 6.5 43.7 0.0 Tin 6.2 ND 48.4 1. 3 21.0 7.6 9.1 Titanium 12.6 ND 68. 5 55.3 11.1 1.0 4.4 Vanadium 7.3 89.8 53. 1 2.6 20.4 1.1 10.5 Yttrium 2.4 84.3 46. 9 5.7 2.4 32.7 8.2 Zinc 1.7 148.8 94.5 0.1 19.9 2.2 1.6 Mercury -- -- -- 8.6 18.9 -- --

The differences apparent in the three liquid sewage sludge samples in Table 32 (duplicates 8, 10, and 11) cannot be attributed to differences betwee n the percent solids results in different containers, as was suggested for the field duplicate samples earlier. Each of these duplicates was prepared at th e laboratory from the single 500 -mL HDPE container of sewage sludge from the particular POTW.

The laboratory homogenized each sample before removing the two aliquots (the original sample and the duplicate), but thos e procedures may not have been entirely adequate for samples with very low solids contents, or the samples may have settled between collection of the two aliquots. In addition, the laborat ory only measured the percent so lids of each sample once, for the original aliquot. Therefore, if the duplicate aliquot used for the metals analysis had slightly different solids content than that of the origin al sample aliquot, this would not be known or reflected in the dry-weight results in this survey.

EPA does not include laboratory duplicate analys es in a survey database, since that would provide two results for each sample chosen for th is laboratory QC test. Therefore, while the RPD values in Table 32 may be useful in diagno sing laboratory issues, they do not influence the survey data EPA may use.TNSSS Sampling and Analysis Technical Report 67 January 2009 6.8.5 MS/MSD Results for Organics

Organic contaminants such as the PAHs and semivolatiles of interest in this survey are generally less common in environmental samples. Therefore, most methods for organics specify using MS and MSD samples as the means of asse ssing the applicability of the method to the matrix of interest, rather than a single MS sample and a duplicate sample analysis. The advantage of spiking the analytes into both QC samples is that it avoids the difficulty of comparing non-detect results to a ssess precision. If th e laboratory only analyzes an unspiked duplicate sample and a compound is not found, there is no numerical result th at can be compared to the original sample result, which may also be a non-detect. While one can compare reporting limits for non-detects, those limits may differ for le gitimate reasons that do not reflect analytical precision.

The laboratory prepared and an alyzed seven sets of MS/MSD samples for the organics in this survey. They prepared some of the MS /MSD samples in conjunction with the full-scan GC/MS analyses and prepared others, later in th e survey, with the select ed ion monitoring (SIM) analyses. Five of the seven MS/MSD pairs were solid samples and two were liquid samples. The laboratory employed separate acceptance limits for samples analyzed as liquids versus those analyzed as solids. The results of al l seven sets are summarized in Table 33.

Table 33. MS and MSD Recovery and Precision for Organics MS/MSD 1 (solid) MS/MSD 2 (solid) MS/MSD 3 (solid)

Analyte MS Rec MSD Rec RPD MS Rec MSD Rec RPD MS Rec MSD Rec RPD 2-Methylnaphthalene 55 60 9 19 11 56 120 143 18 4-Chloroaniline 89 98 10 -480 -503 5 671 685 2 Benzo(a)pyrene 104 112 7 -641 -589 9 131 115 14 bis (2-Ethylhexyl) phthalate 149 226 41 -8024 -9476 17 -7160 -4296 50 Fluoranthene 62 63 3 -2101 -1980 6 93 105 12 Pyrene 51 61 17 -613 -754 21 105 146 32 MS/MSD 4 (liquid) MS/MSD 5 (liquid) MS/MSD 6 (solid) MS Rec MSD Rec RPD MS Rec MSD Rec RPD MS Rec MSD Rec RPD 2-Methylnaphthalene 62 67 9 99 108 8 109 110 1 4-Chloroaniline 66 80 19 97 95 2 75 85 12 Benzo(a)pyrene 67 80 18 169 94 58 117 120 3 bis (2-Ethylhexyl) phthalate -72 16 314 -1362 -275 133 267 416 44 Fluoranthene 43 44 2 290 119 84 124 135 8 Pyrene 57 66 15 278 100 94 123 141 14 MS/MSD 7 (solid) MS Rec MSD Rec RPD 2-Methylnaphthalene 92 97 5 4-Chloroaniline 53 57 7 Benzo(a)pyrene 83 95 14 bis (2-Ethylhexyl) phthalate 4622 4785 4 Fluoranthene 78 97 22 Pyrene 77 91 17TNSSS Sampling and Analysis Technical Report 68 January 2009 The recoveries in Table 33 exhi bit some of the same issues as for the anions and metals, including large negative recoveries for some anal ytes, particularly bis(2- Ethylhexyl) phthalate. The laboratory adjusted the amounts of the analytes spiked into samples over the course of the survey. However, the within-sample variabilit y affected many of the recoveries, resulting in some negative recovery values. Therefore, EPA recalculated the MS/MSD recoveries and RPDs using the alternative recovery equation describe d earlier. Table 34 presents the recalculated results, rounded to one-tenth of a percent, to illustrate some of the smaller differences.

Table 34. Alternative MS and MSD Recovery and Precision for Organics MS/MSD 1 (solid) MS/MSD 2 (solid) MS/MSD 3 (solid)

Analyte MS Rec MSD Rec RPD MS Rec MSD Rec RPD MS Rec MSD Rec RPD 2-Methylnaphthalene 66.6 70.4 5. 5 67.6 64.2 5.2 109.7 120.8 9.6 4-Chloroaniline 94.1 99.1 5.2 41.3 38.9 6.0 670.6 685.0 2.1 Benzo(a)pyrene 104.0 111.7 7. 1 61.3 64.0 4.3 113.0 106.0 6.4 bis (2-Ethylhexyl) phthalate 112.3 131.6 15.8 71. 7 66.7 7.2 82.2 89.2 8.2 Fluoranthene 76.1 77.1 1.3 40.3 43.6 7.9 98.6 101.0 2.4 Pyrene 71.1 76.8 7.7 70.8 65.0 8.5 101.1 110.0 8.4 MS/MSD 4 (liquid) MS/MSD 5 (liquid) MS/MSD 6 (solid) MS Rec MSD Rec RPD MS Rec MSD Rec RPD MS Rec MSD Rec RPD 2-Methylnaphthalene 63.1 68.5 8. 2 99.3 107.6 8.0 108.9 109.9 0.9 4-Chloroaniline 78.5 87.2 10.5 98.3 97.4 0.9 89.6 93.8 4.6 Benzo(a)pyrene 74.8 84.5 12. 2 142.1 96.0 38.7 114.8 117.4 2.2 bis (2-Ethylhexyl) phthalate 73.9 87.3 16.6 68.1 91. 8 29.6 115.3 129.0 11.2 Fluoranthene 69.1 69.6 0.7 183.3 108.3 51.4 114.1 120.6 5.5 Pyrene 70.3 76.7 8.7 171 99.9 52.5 114.6 126.1 9.6 MS/MSD 7 (solid) MS Rec MSD Rec RPD 2-Methylnaphthalene 92.5 97.3 5.1 4-Chloroaniline 91.2 91.9 0.8 Benzo(a)pyrene 90.0 97.2 7.7 bis (2-Ethylhexyl) phthalate 526.1 541.5 2.9 Fluoranthene 88.3 98.6 11.0 Pyrene 88.0 95.3 8.0

As Table 34 illustrates, all of the negativ e recovery values are eliminated and the exceptionally large negative and positive recove ries for bis (2-Ethyl hexyl) phthalate were reduced as well. With the notable exceptions of 4-chloroaniline in MS/MSD 3 and bis (2- Ethylhexyl) phthalate in MS/MSD 7, the recoveries range from 39% to 183%.

A total of 74 out of 84 rec overies are less than 125% and 70 of 84 recoveries are in the range of 70% to 130%. Only 4 recoveries are below 50%. A total of 38 of 42 RPD values are less than 20%, with the other 4 RPDs between 30% and 53%. Th ese alternative recovery data demonstrate that the analytical methods employe d for this survey exhibit precision and bias within expected norms for th e analysis of organics.TNSSS Sampling and Analysis Technical Report 69 January 2009 6.8.6 MS/MSD Results for PBDEs

The laboratory performed the PBDE anal yses using EPA Method 1614, which normally employs isotope dilution quantitation of the analytes of interest. Because every sample is spiked with the labeled compounds and their recoveries are measured in every sample, isotope dilution methods do not require the analysis of MS/MSD aliquots to assess bias. However, as noted in Section 4, the survey involved a number of modifications to th e published method in order to overcome analytical challenges presented by the sewage sludge samples. One of these modifications was that the laborat ory did not spike the labeled co mpounds into the sample before extraction, but rather spiked the raw sample extrac ts. Using this modification, the laboratory was able to successfully analyze the survey samples, but their data on labeled compound recovery does not include an assessment of extraction effi ciency. Therefore, in conjunction with that modification, the laboratory agreed to prep are MS/MSD aliquots with each batch of field samples analyzed by the modified procedure. Ul timately, four sets of MS/MSD analyses were performed. Table 35 summarizes the MS/MSD results for the PBDEs.

Table 35. MS/MSD Recovery and Precision for PBDEs MS/MSD 1 MS/MSD 2 Analyte MS Rec MSD Rec RPD MS Rec MSD Rec RPD BDE-28 91 128 19 61 54 4.5 BDE-47 0 3430 0 0 0 0 BDE-66 0 197 0 26 0 0 BDE-85 102 202 23 44 18 7.1 BDE-99 0 1750 0 0 0 0 BDE-100 146 547 23 0 0 0 BDE-138 144 180 15 104 91 7.4 BDE-153 91 256 22 0 0 0 BDE-154 95 224 20 0 0 0 BDE-183 104 123 8.7 54 35 9.8 BDE-209 171 2910 18 0 0 0 MS/MSD 3 MS/MSD 4 MS Rec MSD Rec RPD MS Rec MSD Rec RPD BDE-28 99 100 1.8 91 60 35 BDE-47 215 121 5.4 298 0 0 BDE-66 98 98 1 107 69 35 BDE-85 102 105 3.2 113 61 41 BDE-99 172 116 3.6 315 0 0 BDE-100 118 98 5.1 128 2.9 51 BDE-138 141 94 20 123 84 34 BDE-153 94 102 4.4 106 36 45 BDE-154 96 101 3.5 105 40 44 BDE-183 106 98 3.8 88 58 34 BDE-209 261 143 8.7 291 179 28 The laboratory reported recoveries as zero (0) any time the calculated recovery was negative, and reported the RPD as 0 when ei ther of the recovery values was reported as 0.

As noted in Table 35, the laboratory’s repor ting practices included substituting zero (0) for any calculated negative recoveries, as well as for the RPD when either recovery value in the MS/MSD pair is reported as 0. There are 16 negative recoveries repor ted as 0 for first 2 MS/MSD pairs and only 2 negative recoveries for the last 2 MS/MSD pairs. This is because the laboratory increased their spiking levels for th e later analyses. There are also a number of reported recoveries well over 100%, ra nging as high as 3430% in one case.TNSSS Sampling and Analysis Technical Report 70 January 2009 Given these recoveries and RPDs, EPA perf ormed the alternative calculations described in Sections 6.7.1 to 6.7.5 for the PBDE data. Thos e alternative recoveries and RPDs are shown in Table 36, rounded to one-tenth of a percent, to illustrate some of the smaller differences.

Table 36. Alternative MS/MSD Recovery and Precision for PBDEs MS/MSD 1 MS/MSD 2 Analyte MS Rec MSD Rec RPD MS Rec MSD Rec RPD BDE-28 95.7 116.1 19. 3 81.5 51.7 44.8 BDE-47 96.2 126.0 26. 8 89.0 77.3 14.1 BDE-66 72.3 124.7 53. 2 82.9 76.5 8.1 BDE-85 100.3 126.4 23. 0 88.3 82.1 7.2 BDE-99 94.3 121.2 24. 9 84.1 78.0 7.6 BDE-100 103.1 129.9 23.0 83.8 71.7 15.5 BDE-138 127.0 148.4 15.5 101.9 94.6 7.5 BDE-153 98.7 123.8 22.6 84.5 76.8 9.6 BDE-154 98.6 120.5 20.0 85.7 77.6 9.9 BDE-183 101.8 111.5 9.1 78.4 71.4 9.4 BDE-209 99.9 120.4 18.6 94.0 79.5 16.7 MS/MSD 3 MS/MSD 4 MS Rec MSD Rec RPD MS Rec MSD Rec RPD BDE-28 99.5 99.5 0. 0 91.8 64.5 35.0 BDE-47 107.2 101.6 5. 4 120.8 68.7 55.0 BDE-66 98.5 98.1 0. 5 106.2 74.1 35.5 BDE-85 101.3 103.3 2. 0 109.0 72.1 40.8 BDE-99 104.1 100.4 3. 6 121.3 66.5 58.4 BDE-100 104.2 98.6 5.6 110.0 65.3 51.1 BDE-138 136.7 164.4 18.4 121.1 86.1 33.8 BDE-153 96.8 100.3 3.5 103.0 65.5 44.5 BDE-154 99.3 101.7 2.4 102.2 65.2 44.2 BDE-183 103.8 98.8 5.0 89.3 63.3 34.1 BDE-209 111.9 102.6 8.7 172.4 130.1 27.9

Using the alternative calculat ions, all 18 of the negative valu es originally reported by the laboratory as “0” were eliminated. The recalcul ated recoveries range from 52% to 172%. The laboratory employed acceptance limits of 50-150% for MS/MSD recoveries, and all but the one recalculated recovery of 172.4% fa ll within that range. The calculated RPD values range from 0% to 58.4%. Only three recalculated RPD va lues were above the laboratory’s acceptance limit of 50%.

As with the other analytical classes, these alternative recovery data demonstrate that the analytical methods employed for this survey exhi bit precision and bias within expected norms for the analysis of PBDEs.

6.8.7 Qualification of Sample Results based on MS/MSD Results

During review of the sample results, EP A qualified any MS/MSD results reported by the laboratory that fell outside of the relevant accepta nce limits. Those data qualifiers were carried over into the results database. However, the re sults of the alternative calculations shown in Section 6.7 for all classes of analytes demonstrat e that the shortcomings in MS/MSD recoveries and precision are largely a function of the calculations in many EPA methods.TNSSS Sampling and Analysis Technical Report 71 January 2009 Therefore, while retaining the data qualifiers that indicate that the recoveries and/or RPD reported by the laboratories fell outside of the acceptance limits, EPA also added a qualifier to affected samples to indicate that the alternative calcul ations suggest that method performance in the sewage sludge matrix is not an immediat e concern. This new qualifier is “ACAP,” for “Alternative Calculation indicates Acceptable Performance,” as stated in the database.

6.8.8 Labeled Compound Recoveries for Isotope Dilution Methods

As noted elsewhere in this report, EPA’s isotope dilution methods spike labeled analogs of the target analytes into each sample and mon itor the recoveries of t hose labeled compounds as a measure of method performance. Therefore, EPA’s isotope dilution methods do not require that the laboratory prepare separate matrix sp ike samples. Rather than assessing extraction efficiency and other aspects of method performan ce on 5% of the samples (i.e., 1 out of every 20 samples is used to create an MS/MSD pair), the isotope dilution met hods generate extraction efficiency data on 100% of the samples, and use those data to correct the final results for each analyte for the recovery of its labeled compound.

During the data review process, EPA checked the recovery of every labeled compound in every field sample and QC samples against the method acceptance criteria for that analyte. When the labeled recovery fell outside of the acceptance criteria, EPA flagged in the database the sample results for the associated unlabeled analytes.TNSSS Sampling and Analysis Technical Report 72 January 2009 

### Section 7 References

NRC, 2002. Biosolids Applied To Land: Advancing Standards And Practices , National Research Council of the National Academies. July 2002.

USEPA, 2003. United States Federal Register. 68 FR 75531. Wednesday December 31, 2003, pp. 75531-75552.

Vadas, P. A. and Kleinman, P. J. A., 2006. Effect of Methodology in Estimating and Interpreting Water-Extractable Phosphorus in Animal Manures , J. Environ. Qual., 35: 1151-159.TNSSS Sampling and Analysis Technical Report 73 January 2009 

### Appendix A Solids Leaching Procedure for Anions

1. Using the percent solid s determined by drying an aliquot of the sample of known weight overnight at 103 - 105°C and reweighing, weigh out a sample aliquot equal to 0.5 g dry weight in a 200- or 250-mL wide-m outh (screw-top) plastic bottle.

2. Add reagent water until the total mixture mass is 100.5 g. The resultant solids:solution ratio is 0.5 g solids:100-mL solution, or 1:200.

3. Seal bottles with screw top caps and place in a standard laboratory shaker set at 70 revolutions per minute for 60 minutes.

4. Upon completion of agitation, centrifuge s ealed bottles at 2000 rpm for 10 minutes.

5. Gravity filter the concentrate from step 4 us ing Whitman #2 filter paper. (Suggest 150-mm circular filters folded and placed in simple plastic lab funnels.)

6. Adjust the pH of the filtrate to pH<2 with H 2 SO 4 to preserve the nitrate/ nitrite in the filtrate and store at 4°C until analysis.

7. The holding time for nitrate/nitrite is 48 hours, so samples must be analyzed for all three analytes within 48 hours of preservation.

8. All sample results will be repor ted in mg/kg on the basis of th e original 0.5-g sludge sample.

Adapted from Vadas, P. A. and Kleinman, P. J. A., 2006.TNSSS Sampling and Analysis Technical Report 74 January 2009 

### Appendix B Method Modifications for the PBDE Analyses

The following table summarizes the modifica tions made to the sample preparation and analysis procedures described in EPA Method 1614 for the analysis of the PBDEs. EPA and the laboratory instituted these modifications to addre ss the interferences present in extracts of the samples, and the relatively high levels of some PBDEs in the samples.

The table provides a reference to the releva nt section of the draf t method, the original method specification, and the revised a pproach employed for this survey.

Method Section Topic Original Met hod Specification Revised Approach

11.5.1 Sample size 10 g dry weight As little as 0.2 g dry weight 11.5.2 Spiking labeled standards Spike into sample befo re extraction Spike sample extract before cleanup 12.6 Macroconcentration of extract 3 – 4 mL 10 mL

13 Cleanups May use GPC, silica gel, alumina, or Florisil, if needed. Must use silica gel, GPC, and alumina, in that order 13.2.3 GPC cleanup Process 5 mL of extrac t Process 1 mL of the 10 mL extract 1.1.1 and 17 Target analytes All 209 possi ble BDE congeners Only 11 congeners: BDE-28 BDE-47 BDE-66 BDE-85 BDE-99 BDE-100 BDE-138 BDE-153 BDE-154 BDE-183 BDE-209 17.1 Isotope dilution quantitation 11 congeners determined by true isotope dilution, and the remaining congeners by internal standard. The internal standards are the labeled congeners for other PBDEs that are added to the samples before extraction. 11 congeners quantified using labeled standards for 8 of those 11 congeners, all spiked into the extract before cleanup. All results are corrected for losses during the cleanup steps, but not for initial extraction efficiency. 17.1 Matrix spike samples Not used, due to isotope dilution Added periodic MS/MSD to provide data on extraction efficiency 17.5 Calibration range All results must be within the calibration range, or must be diluted to bring them within range Results for some congeners like BDE-209 flagged “E” in the database if above the calibration range but not high enough to saturate the detector system.TNSSS Sampling and Analysis Technical Report 75 January 2009 

### Appendix C QC Acceptance Criteria

QC Acceptance Criteria for the Targeted National Sewage Sludge Survey Analytical Fraction QC Parameter Analyte Acceptance Limits (%) Fluoride 85-115 Phosphorus 85-115 LCS Nitrate/Nitrite 90-110 MS Recovery All analytes 75-125 Anions

Duplicate Precision (RPD) All analytes 20 Aluminum 58-142 Antimony 12-223 Arsenic 77-123 Barium 82-118 Beryllium 77-122 Boron 56-144 Cadmium 80-121 Calcium 79-121 Chromium 78-121 Cobalt 80-120 Copper 82-118 Iron 50-150 Lead 79-121 Magnesium 77-123 Manganese 80-120 Mercury 60-123 Molybdenum 72-128 Nickel 81-119 Phosphorus NA Selenium 76-124 Silver 61-139 Sodium 56-145 Thallium 76-124 Tin NA Titanium 40-160 Vanadium 76-124 Yttrium NA LCS for solid samples

Zinc 79-120 LCS for liquid samples All metals 85 -115 All metals, except as noted below 70-130

Mercury 60-128 Tin 50-150 MS/MSD Recovery

Titanium 50-150 Metals

MS/MSD Precision (RPD) All metals 30 4-Chloroaniline 39-95 2-Methylnaphthalene 12-159 Fluoranthene 47-139 Pyrene 52-129 bis (2-Ethylhexyl) phthalate 39-174 LCS

Benzo(a)pyrene 49-144 4-Chloroaniline 10-104 2-Methylnaphthalene 39-116 Fluoranthene 49-130 Pyrene 58-110 bis (2-Ethylhexyl) phthalate 53-172 Organics

MS/MSD Recovery for solid samples

Benzo(a)pyrene 50-126 NA = Not applicable. The commercial reference material used for the LCS does not have certified values for these analytes.TNSSS Sampling and Analysis Technical Report 76 January 2009 QC Acceptance Criteria for the 2006–2007 Targeted National Sewage Sludge Survey Analytical Fraction QC Parameter Analyte Acceptance Limits (%) 4-Chloroaniline 10-62 2-Methylnaphthalene 10-109 Fluoranthene 10-150 Pyrene 10-136 bis (2-Ethylhexyl) phthalate 10-150 MS/MSD Recovery for liquid samples

Benzo(a)pyrene 10-152 MS/MSD Precision (RPD) All analytes, all matrix types 40 Nitrobenzene-d 5 35-128 2-Fluorobiphenyl 43-133 Organics

Surrogate Recovery p-Terphenyl-d 14 49-137 LCS All analytes 25-150 All, except as shown below 25-150 Labeled Compound Recovery 13 C-BDE-209 20-200 MS/MSD Recovery All analytes 50-150 PBDEs

MS/MSD Precision (RPD) All analytes 50

QC Acceptance Criteria for Pharmaceuticals for the Targeted National Sewage Sludge Survey Analyte VER (%) OPR (%)
Acid-Extractable Fraction - Po sitive Electrospray Ionization Acetaminophen 70 -130 50 -120 Azithromycin 70 -130 33 -120 Caffeine 70 -130 50 -124 Carbadox 70 -130 33 - 144 Carbamazepine 70 -130 21 - 137 Cefotaxime 70 -130 8 - 186 Ciprofloxacin 70 -130 50 - 120 Clarithromycin 70 -130 8 - 154 Clinafloxacin 70 -130 5 - 200 Cloxacillin 70 -130 5 - 200 Codeine 70 -130 34 - 129 Cotinine 70 -130 50 - 124 Dehydronifedipine 70 -130 42 - 120 Digoxigenin 70 -130 8 - 183 Digoxin 70 -130 5 - 148
Diltiazem 70 -130 11 - 120 1,7-Dimethylxanthine 70 -130 50 - 138 Diphenhydramine 70 -130 48 - 120 Enrofloxacin 70 -130 50 - 125 Erythromycin 70 -130 50 - 158 Flumequine 70 -130 36 - 200
Fluoxetine 70 -130 49 - 125 Lincomycin 70 -130 5 - 120 Lomefloxacin 70 -130 17 - 120
Miconazole 70 -130 27 - 120 Norfloxacin 70 -130 50 - 135 Norgestimate 70 -130 36 - 120 Ofloxacin 70 -130 50 - 200 Ormetoprim 70 -130 50 - 120 Oxacillin 70 -130 5 - 200TNSSS Sampling and Analysis Technical Report 77 January 2009 QC Acceptance Criteria for Pharmaceuticals for the Targeted National Sewage Sludge Survey Analyte VER (%) OPR (%) Oxolinic Acid 70 -130 42 - 124 Penicillin G 70 -130 5 - 200
Penicillin V 70 -130 5 - 200 Roxithromycin 70 -130 38 - 120 Sarafloxacin 70 -130 17 - 200 Sulfachloropyridazine 70 -130 50 - 200 Sulfadiazine 70 -130 5 - 200 Sulfadimethoxine 70 -130 50 - 120 Sulfamerazine 70 -130 50 - 148 Sulfamethazine 70 -130 50 - 142 Sulfamethizole 70 -130 50 - 120 Sulfamethoxazole 70 -130 50 - 120 Sulfanilamide 70 -130 5 - 189 Sulfathiazole 70 -130 41 - 120
Thiabendazole 70 -130 50 - 120 Trimethoprim 70 -130 50 - 126 Tylosin 70 -130 16 - 149 Virginiamycin 70 -130 5 - 189 Tetracyclines Anhydrochlortetracycline 70 -130 50 - 135 Anhydrotetracycline 70 -130 7 - 141 Chlortetracycline 70 -130 45 - 172 Demeclocycline 70 -130 5 - 200 Doxycycline 70 -130 22 - 166 4-Epianhydrochlortetracycline 70 -130 18 - 120 4-Epianhydrotetracycline 70 -130 5 - 200 4-Epichlortetracycline 70 -130 40 - 150 4-Epioxytetracycline 70 -130 50 - 142 4-Epitetracycline 70 -130 50 - 173 Isochlortetracycline 70 -130 5 - 200 Minocycline 70 -130 5 - 176 Oxytetracyclin 70 -130 50 - 183 Tetracycline 70 -130 50 - 155 Acid-Extractable Fraction - Negative Electrospray Ionization Gemfibrozil 70 -130 50 - 120 Ibuprofen 70 -130 50 - 120 Naproxen 70 -130 50 - 120
Triclocarban 70 -130 50 - 120
Triclosan 70 -130 50 - 120 Warfarin 70 -130 50 - 120 Base-Extractable Fraction - Po sitive Electrospray Ionization

Albuterol 70 -130 50 - 133 Cimetidine 70 -130 5 - 120 Metformin 70 -130 50 - 149
Ranitidine 70 -130 24 - 160TNSSS Sampling and Analysis Technical Report 78 January 2009 QC Acceptance Criteria for Pharmaceutical Labeled Compounds for the Targeted National Sewage Sludge Survey Analyte VER (%) OPR (%) Recovery in Samples (%)

Acid-Extractable Fraction - Po sitive Electrospray Ionization

13 C 2 - 15 N-Acetaminophen 70 -130 5 - 200 19 - 200

13 C 3 -Caffeine 70 -130 5 - 200 31 - 200

13 C 3 - 15 N-Ciprofloxacin 70 -130 5 - 200 37 - 181 Cotinine-d 3 70 -130 5 - 120 5 - 145

13 C 2 -Erythromycin 70 -130 50 - 120 23 - 120
Fluoxetine-d 5 70 -130 50 - 126 40 - 148

13 C 6 -Sulfamethazine 70 -130 5 - 157 12 - 120

13 C 6 -Sulfamethoxazole 70 -130 50 - 146 40 - 129
Thiabendazole-d 6 A 70 -130 50 - 146 32 - 140

13 C 3 -Trimethoprim 70 -130 50 - 177 50 - 172 Tetracyclines

Thiabendazole-d 6 A 70 -130 50 - 120 30 - 132 Acid-Extractable Fraction - Negative Electrospray Ionization Gemfibrozil-d 6 70 -130 38 - 122 21 - 123

13 C 3 -Ibuprofen 70 -130 28 - 122 29 - 127

13 C-Naproxen-d 3 70 -130 34 - 131 14 - 132

13 C 6 -Triclocarban 70 -130 5 - 172 5 - 147

13 C 12 -Triclosan 70 -130 5 - 168 5 - 153 Warfarin-d 5 70 -130 50 - 177 50 - 200 Base-Extractable Fraction - Po sitive Electrospray Ionization

Albuterol-d 3 70 -130 35 - 121 39 - 141 Metformin-d 6 70 -130 5 - 141 5 - 200

A Thiabendazole-d 6 is used as a labeled analog in both the tetracyclines and the acid

extractable-positive electrospray fractions, with separate acceptance criteria.TNSSS Sampling and Analysis Technical Report 79 January 2009 QC Acceptance Criteria for Steroids and Hormones for the Targeted National Sewage Sludge Survey Analyte VER (%) OPR (%)

Androstenedione 70 - 130 5 - 200

Androsterone 70 - 130 50 - 121 Campesterol 70 - 130 40 - 200
Cholestanol 70 - 130 50 - 164
Cholesterol 70 - 130 5 - 200 Coprostanol 70 - 130 34 - 200

Desmosterol 70 - 130 5 - 200

17 α -Dihydroequilin 70 - 130 45 - 151 Epicoprostanol 70 - 130 50 - 197 Equilenin 70 - 130 5 - 200 Equilin 65 - 135 5 - 200 Ergosterol 50 - 150 5 - 200

17 α -Estradiol 70 - 130 50 - 120

17 α -Ethinyl estradiol 70 - 130 50 - 123

17 β -Estradiol 70 - 130 50 - 176 β -Estradiol-3-benzoate 70 - 130 5 - 189

Estriol 70 - 130 5 - 193

Estrone 70 - 130 50 - 173

Norethindrone 70 - 130 45 - 200 Norgestrel 70 - 130 46 - 200 Progesterone 70 - 130 5 - 200 β -Sitosterol 70 - 130 5 - 200 β -Stigmastanol 70 - 130 29 - 200 Stigmasterol 70 - 130 50 - 200

Testosterone 70 - 130 50 - 136

QC Acceptance Criteria for Steroid and Hormone Labeled Compounds for the Targeted National Sewage Sludge Survey Analyte VER (%) OPR (%) Recovery in Samples (%)
Cholesterol-d 7 70 - 130 50 - 120 50 - 120

17 α -Ethinyl estradiol-d 4 70 - 130 50 - 120 50 - 120

17 β -Estradiol-d 4 70 - 130 50 - 120 29 - 132
Norethindrone-d 6 70 - 130 37 - 120 12 - 120 Norgestrel-d 6 70 - 130 36 - 120 7 - 120 Progesterone-d 9 70 - 130 5 - 200 5 - 200