In cooperation with the U.S. Department of Agriculture, Natural Resources 

Conservation Service and the Lenawee Conservation District 

Chemical and Microbiological Water Quality of 

Subsurface Agricultural Drains during a Field Trial of 

Liquid Dairy Manure Effluent Application Rate and 

Varying Tillage Practices, Upper Tiffin Watershed, 

Southeastern Michigan 

By Sheridan Kidd Haack and Joseph W. Duris 

Open-File Report 2008–1189 

U.S. Department of the Interior 

U.S. Geological Survey 
U.S. Department of the Interior 

DIRK KEMPTHORNE, Secretary 

U.S. Geological Survey 

Mark D. Myers, Director 

U.S. Geological Survey, Reston, Virginia 2008 

For product and ordering information: 

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its natural and living resources, natural hazards, and the environment: 

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Suggested citation: 

Haack, S.K., and Duris, Joseph W., 2008, Chemical and microbiological water quality of subsurface 

agricultural drains during a field trial of liquid dairy manure effluent application rate and varying tillage 

practices, Upper Tiffin Watershed, southeastern Michigan: U.S. Geological Survey Open-File Report 2008–

1189, 38 p. 

Any use of trade, product, or firm names is for descriptive purposes only and does not imply 

endorsement by the U.S. Government. 

Although this report is in the public domain, permission must be secured from the individual 

copyright owners to reproduce any copyrighted material contained within this report. 

ii
Contents 

Abstract ................................................................................................................................................................................ 1

Introduction ......................................................................................................................................................................... 2

Purpose and Scope ........................................................................................................................................................ 4

Management Practices ................................................................................................................................................. 4

Chemical and Microbiological Indicators of LDME Transport to Subsurface Drains ....................................... 6

Conditions at the Study Area ........................................................................................................................................ 7

Sampling and Analytical Methods .................................................................................................................................. 8

Sampling Procedures ..................................................................................................................................................... 9

Chemical Analyses ......................................................................................................................................................... 9

Microbiological Analyses ........................................................................................................................................... 10

Bacteria Enumeration and Preservation .............................................................................................................. 10

Microbiological Indicators of LDME Transport to Subsurface Drains ........................................................... 10

Immunological Test for O157 Antigen ............................................................................................................... 10

DNA Extraction ...................................................................................................................................................... 13

Polymerase Chain Reaction (PCR) ..................................................................................................................... 13

Multiplex PCR for Shiga-Toxin-Producing E. coli ............................................................................................ 13

PCR for Bovine Bacteroidetes Gene ................................................................................................................. 14

Quality Assurance/Quality Control ..................................................................................................................... 14

Results of Chemical and Microbiological Analyses ................................................................................................... 14

Specific Conductance and pH .................................................................................................................................... 15

Bacteria Concentrations ............................................................................................................................................. 15

Nutrient Concentrations .............................................................................................................................................. 16

Chemical Indicators of LDME Transport to Subsurface Drains ........................................................................... 22

Microbiological Indicators of LDME Transport to Subsurface Drains ............................................................... 26

Study Limitations .............................................................................................................................................................. 28

Summary and Conclusions ............................................................................................................................................. 29

References Cited .............................................................................................................................................................. 31 

Appendix ............................................................................................................................................................................ 36 

Figures 

 1. Map showing location of Upper Tiffin Watershed Conservation Effects Assessment Project area ....... 3

Tables 

 1. Treatment definitions and abbreviations ............................................................................................................. 8 

 2. Rainfall during the course of the field trial .......................................................................................................... 8 

 3. Methods of nutrient analysis and reporting limits ............................................................................................. 9 

 4. Water-quality field data and bacteria concentrations for LDME on date of application and 

for all tile-water samples ...................................................................................................................................... 11 

 5. Nutrient concentrations in LDME, field blank, and tile-water samples ....................................................... 17 

 6. U.S. Environmental Protection Agency Ambient Water-Quality Criteria and 25th percentiles for 

nutrients in Ecoregion VI (Corn Belt) and Level III Ecoregion 57 (Huron/Erie Lake Plain) ........................ 21 

iii
 7. Results of antibiotic and wastewater chemical analyses for LDME, pre-application sample, 

and NT8000 treatment (Tile 6) over time ............................................................................................................ 23 

 8. Results of wastewater-chemical analyses for field blank and for all treatments after the first 

significant rainfall event ........................................................................................................................................ 25 

 9. Results of analyses for microbiological indicators in subsurface drain water for NT8000 

treatment before and after addition of liquid dairy manure effluent ............................................................ 27 

 10. Results of analyses for microbiological indicators in LDME and all treatments before LDME 

application and at 6 days post-application ........................................................................................................ 28 

 1–1. Wastewater-method compound names, U.S. Geological Survey National Water Quality 

Laboratory reporting limits, and possible compound uses or sources ........................................................ 36

 1–2. Antibiotic analytes by liquid chromatography/mass spectrometry .............................................................. 38

Conversion Factors and Abbreviated Water-Quality Units 

 Multiply By To obtain

Length 

inch (in.) 2.54 centimeter (cm) 

ft (ft) 0.3048 meter (m) 

mile (mi) 1.609 kilometer (km) 

Area 

acre 0.4047 hectare (ha) 

Volume 

gallon (gal) 3.785 liter (L) 

milliliter (mL) 0.03381 ounce, fluid (fl. oz) 

microliter (μL) 0.0001 milliliter (mL) 

Flow rate 

gallon per acre (gal/acre) 9.354 liter per hectare (L/ha) 

gallon per acre per year 

[(gal/acre/)yr] 

9.354 liter per hectare per year 

[(L/ha/)yr] 

Mass 

pound (lb) 0.4536 kilogram (kg) 

pound per gallon (lb/gal) 119.826 gram per liter (g/L) 

iv
Temperature in degrees Celsius (°C) may be converted to degrees Fahrenheit (°F) as follows: 

°F=(1.8×°C)+32 

Specific conductance is given in microsiemens per centimeter at 25 degrees Celsius (μS/cm at 25°C). 

Small volumes are reported in milliliters (mL) and microliters (μL). Very small masses are given in nanograms (ng; a nanogram is 

one billionth of a gram). Pore sizes of filters are given in micrometers (μm). 

Concentrations of chemical constituents in water are given in milligrams per liter (mg/L), micrograms per liter (μg/L), millimolar 

(mM) or micromolar (μM). 

Bacteria concentrations are given in colony-forming units per 100 milliliters (CFU/100 mL). 

v

Chemical and Microbiological Water Quality of 

Subsurface Agricultural Drains during a Field Trial of 

Liquid Dairy Manure Effluent Application Rate and 

Varying Tillage Practices, Upper Tiffin Watershed, 

Southeastern Michigan 

By Sheridan Kidd Haack and Joseph W. Duris 

Abstract 

A field trial was done in the Upper Tiffin River Watershed, in southeastern Michigan, to 

determine the influence of liquid dairy manure effluent (LDME) management practices on the 

quality of agricultural subsurface-drain water. Samples from subsurface drains were analyzed for 

nutrients, fecal-coliform and Escherichia coli (E. coli) bacteria, antibiotics, chemicals typically 

detected in wastewater, and the occurrence of genes indicating the presence of shiga-toxinproducing E. coli, or of bovine-specific Bacteroidetes bacteria. Samples were collected from 

November 2, 2006, to March 20, 2007, from eight subsurface drains under field plots that received 

no LDME and no tillage (controls) or received 4,000 or 8,000 gallons per acre (gal/acre) of LDME 

and either no tillage or two different types of tillage. The two types of tillage tested were (1) 

ground-driven, rotary, subsurface cultivation and (2) rolling-tine aeration. Samples were collected 

before LDME application and at 4 hours, and 1, 2, 6, 7, and 14 days post-application. 

Nutrient concentrations were high in subsurface-drain water throughout the field-trial 

period and could not be attributed to the field-trial LDME application. Of the 59 drain-water 

samples, including those collected before LDME application and control samples for each date, 56 

had concentrations greater than the U.S. Environmental Protection Agency (USEPA), Ecoregion VI 

recommended surface-water criterion for total phosphorus, and all samples had concentrations 

greater than the recommended total nitrogen criterion. Nitrate + nitrite nitrogen concentration 

exceeded 20 milligrams per liter for every sample and contributed most to the total nitrogen 

concentrations. Substantial increases in drain-water concentrations of organic and ammonia 

nitrogen and total phosphorus were found for all treatments, including controls, at 14 days postapplication after 0.84 inch of rainfall over 2 days. 

E. coli concentrations exceeded the USEPA recreational-water-quality single-sample 

criterion of 235 colony forming units per 100 milliliters in only 3 of 56 samples. Of these three 

samples, two were collected within 1 day post-LDME application from the treatment receiving 

8,000 gal/acre LDME with no tillage (NT8000). The third sample was from the rolling-tine aerator 

treatment with 4,000 gal/acre LDME application rate after the first significant rainfall. 

1
Two wastewater chemicals and two bacterial genes (eaeA and stx1) detected in the LDME, 

but absent in field blank or pre-application samples, were detected in the 4-hour or 1-day postapplication NT8000 samples. No LDME-associated chemicals were detected in later samples from 

the NT8000 treatment, and none were detected in samples from other treatments after the first 

significant rainfall. 

Results of this field trial were somewhat equivocal with respect to the influence of LDME 

concentration and tillage practices on subsurface-drain water quality, both immediately after 

LDME application and in the longer term, after significant rainfall. Interpretation of study findings 

is limited by the fact that treatments were not replicated, and flow rate or discharge from the 

subsurface drains was not measured. Nevertheless, study results provide useful information about 

nutrient and bacteria concentrations in subsurface drains during the non-growing season. In 

addition, study results demonstrate some potential for the use of chemical and microbiological 

indicators of LDME transport to subsurface drains. 

Introduction 

The lower parts of the Bean Creek Watershed (Hydrologic Unit Code 04100006106) and 

the Lime Creek Watershed (Hydrologic Unit Code 04100006107), in southern Hillsdale and 

Lenawee Counties, Michigan, are part of the headwaters of the Tiffin River (fig. 1). Land use in 

these watersheds is predominantly agricultural: approximately 75 percent in the Bean Creek 

Watershed and approximately 91 percent in the Lime Creek Watershed. Soils are fine textured and 

require subsurface drainage for agricultural use. Historically, subsurface drainage systems were 

formed with clay tiles installed at depth in a field. Currently, various types of plastic piping are 

used, but the term “tile drains” or “tiles” is still in use. Manure from livestock operations is landapplied to cropland. In soils of the region, liquid manure has the potential to move through the soil 

profile and into subsurface tile drains through soil cracks and wormholes, a process referred to as 

“preferential flow” (Shipitalo and Gibbs, 2000; Hoorman and Shipitalo, 2006). Thus, manure and 

manure-associated chemicals and bacteria have the potential of moving offsite to surface waters 

through subsurface drainage tiles. 

In the past 5 years, the watersheds in the Tiffin River headwaters have been subject to 

establishment and expansion of livestock operations, particularly large dairies. Parts of Bean Creek 

and Lime Creek fail to meet Michigan Water-Quality Standards for Escherichia coli (E. coli) 

bacteria. The Michigan Department of Environmental Quality (MDEQ) has established Total 

Maximum Daily Loads (TMDLs) for E. coli for parts of these watersheds (Michigan Department of 

Environmental Quality 2003a,b) and has identified agricultural tile drains as one potential 

contributor of E. coli to surface water in these watersheds. MDEQ identified stormwater runoff, 

sewage outfalls, and possible connections of sewage or septic-system waste to storm or agricultural 

drains (referred to in the TMDL documents as “illicit connections”), as other potential sources of 

E. coli. A volunteer monitoring group called the Environmentally Concerned Citizens of South 

Central Michigan has also documented instances of discharge of manure and nutrients through tile 

lines into surface waters of Bean and Lime Creeks. The Medina Drain, which flows into Bean 

Creek, has been placed on Michigan’s 303(d) list of impaired water bodies; nuisance odors, 

phosphorous, nuisance plant growth, and organic enrichment are identified as specific impairments. 

2
In 2004, the Natural Resources Conservation Service (NRCS) began a 3-year project in the 

Lime and Bean Creek Watersheds, collectively called the Upper Tiffin Watershed. The Upper 

Tiffin Watershed was designated a Special Emphasis watershed, funded under the National 

U.S. Department of Agriculture Conservation Effects Assessment Project (CEAP; 

http://www.nrcs.usda.gov/Technical/nri/ceap/). The NRCS established a partnership agreement 

with the Lenawee Conservation District (LCD) to assist with implementation of the watershed 

project. The Upper Tiffin Watershed Project seeks to determine which combination of manure 

management practices will mitigate the risk of offsite movement of nutrients and bacteria from 

subsurface discharge. Manure management practices that were evaluated included three types of 

tillage and three rates of manure application. A field trial was established in 2006. 

To help identify the potential for offsite movement of nutrients and bacteria through 

subsurface drainage under various manure management practices, the U.S. Geological Survey 

(USGS), in cooperation with the NRCS and the LCD, evaluated concentrations of nutrients, fecalcoliform bacteria, and E. coli bacteria in liquid dairy manure effluent (LDME) and subsurfacedrain-water samples for the Upper Tiffin Watershed Project field trial. In addition, to help identify 

potential indicators of dairy manure contamination of water, the USGS analyzed LDME and 

selected subsurface-drain-water samples for chemicals found in many wastewaters, for antibiotics 

used by humans and in animal agriculture, and for genes of bacteria often associated with cattle. 

Figure 1. Location of Upper Tiffin Watershed Conservation Effects Assessment 

Project area. 

0 510 MILES

0 5 10 KILOMETERS

Tiffin River watershed,

Michigan and Ohio

Bean Creek

watershed Lime Creek

watershed

Hillsdale County Lenawee County

County linework data from Michigan Resource Information System, 1998.

Hydrologic data from Michigan Department of Environmental Quality 1:24,000 scale data, 1998.

Hydrography linework from National Hydrography Dataset Plus 1:100,000 scale data, 2005

Explanation

84°30' W 84°00' W

42°00' N

41°45' N MICHIGAN

MICHIGAN OHIO

OHIO

Upper Tiffin watershed

Streams

3
Drain-water samples analyzed for these constituents were collected both immediately after LDME 

application and on subsequent dates, especially after a significant rainfall event (for the purposes of 

this study, a storm total greater than 0.5 in.). 

Purpose and Scope 

The primary purpose of this report is to describe the concentration of nutrients and fecal 

indicator bacteria in subsurface agricultural drainage water over a period of 2 weeks after various 

combinations of LDME application rate and tillage practices. A secondary purpose is to describe, 

in a subset of these samples, the occurrence of chemicals and microbiological constituents that 

might indicate LDME transport to subsurface drains. 

The field trial investigated three different management practices within a field subdivided 

into eight plots. The first was application to soil with no tillage (NT). The second was tillage with a 

ground-driven, rotary, subsurface cultivator that disturbed the soil profile to a depth of 2 to 3 in. 

The third was tillage with a rolling-tine aerator that disrupts the top 6 to 8 in. of soil. In each case, 

the plot was tilled immediately before the LDME application. In this field trial, LDME either was 

not applied, was applied at the rate of 8,000 gal/acre, or was applied at half that rate. Samples of 

LDME were collected from September 27, 2006, to November 2, 2006. Samples of tile water were 

collected after the growing season, from November 2, 2006, to March 20, 2007. 

This report includes (1) concentrations of organic, ammonia, and nitrate + nitrite nitrogen 

and total and orthophosphorus, (2) concentrations of antibiotics and of other chemicals typically 

detected in wastewater, (3) concentrations of Escherichia coli (E. coli) bacteria, (4) data on the 

occurrence of genes indicating the presence of shiga-toxin-producing E. coli, and (5) data on the 

occurrence of a genetic marker indicating the presence of bovine-specific Bacteroidetes bacteria. 

Management Practices 

Factors that might influence manure movement to subsurface drains include the amount or 

rate of manure applied and tillage practices that influence the degree to which preferential 

flowpaths are established or disrupted. The amount of manure applied under agricultural best 

management practices is governed by factors such as the water and nutrient content of the manure, 

the water-holding capacity of the soil, and existing levels of nutrients in the soil (Michigan 

Department of Agriculture, 2007). 

Manure contains substantial amounts of nitrogen, mostly in organic forms. Some of this 

organic nitrogen is converted to ammonia nitrogen during storage. When applied to soil, some of 

this ammonia nitrogen volatilizes to air immediately, but some moves into the soil; the latter is 

bound by soil particles and taken up by plants for growth. The organic form of nitrogen also tends 

to remain bound to the soil; however, bacteria may degrade the organic forms of nitrogen and may 

convert ammonia nitrogen to nitrate and nitrite, which are very soluble in water and which may be 

lost to subsurface drains. How much nitrogen may be lost by these various processes depends on 

the methods of manure application, weather conditions, the type of soil, the type of crop, and the 

season (Bakhsh and others, 2005). During the growing season, plants may take up ammonia 

nitrogen, but during fallow seasons, nitrate may be lost to subsurface drainage. 

Manure also typically contains substantial amounts of phosphorus, again much of it in 

organic forms. Phosphorus transformations and interactions with soils are complex (Sharpley and 

others, 2003), and the exact forms of phosphorus in soils are difficult to determine. Historically, 

phosphorus leaching from soil to the subsurface was considered negligible, owing to the affinity of 

phosphorus for the soil matrix. In the past, therefore, concern for water contamination with 

4
phosphorus focused primarily on overland runoff of phosphorus attached to soil particles or of 

soluble phosphorus in surface-runoff water (Sharpley and others, 2003). Agricultural management 

practices (conservation tillage; see below) designed to reduce soil losses by runoff appear to have 

been effective in reducing phosphorus concentrations in the Maumee River (to which the Tiffin 

River is a tributary) during 1976–95. However, recent research indicates that newer agricultural 

practices, including the growth of animal feeding operations, have contributed to phosphorus 

surplus at the farm and watershed scale (Sharpley and others, 2003). Calhoun and others (2002) 

found that soluble phosphorus (Bray P1 test) measured from 1996 to 1998 in soils of the Maumee 

and Sandusky River Watersheds was significantly greater than concentrations determined from 

1953 to 1982. Nationally, soil phosphorus has increased in many parts of the country (Sharpley and 

others, 2003). The relation between soil phosphorus measurements and the potential for loss of 

phosphorus by surface runoff, or to subsurface flow, is complex, and depends on soil properties, 

the forms of phosphorus applied, weather conditions, plant growth, and management practices 

(Sharpley and others, 2003). Michigan Generally Accepted Agricultural Management Practices 

(GAAMPs; Michigan Department of Agriculture, 2007) for manure management and use give 

specific recommendations for the levels of plant-available nitrogen and phosphorus that should be 

applied to soils in any form (fertilizer or manure or both) and recommend manure and soil analyses 

regularly to avoid exceeding these levels. 

Conservation tillage consists of a variety of no-till or reduced-tillage approaches that were 

designed to reduce soil and nutrient runoff to surface water and retain water and nutrients for plant 

growth (Lemunyon and Gross, [n.d.]). However, earthworm burrows and cracks (macropores) are 

more prevalent in land that is subject to conservation tillage (Shipitalo and others, 2000; Shipitalo 

and Gibbs, 2000). Earthworm burrows or cracks in soil may serve as direct transport routes for 

rapid movement of land-applied pesticides, nutrients, bacteria, or manure to subsurface drainage 

tiles (Shipitalo and others, 2000; Shipitalo and Gibbs, 2000). Rapid transport may take place within 

minutes or hours of application, and it is particularly an issue when liquid manure is applied to 

already wet soils or when significant rainfall occurs soon after application. Several studies have 

shown that chemicals that might normally bind tightly to the soil matrix (for example, ammonia 

nitrogen, phosphorus, or pesticides) move rapidly through preferential flow pathways without 

interacting with the soil (Sims and others, 1998; Shipitalo and others, 2000; Simard and others, 

2000; Geohring and others, 2001; Hodgkinson and others, 2002; Stamm and others, 2002; de Jonge 

and others, 2004; Toor and others, 2004; Schelde and others, 2006; Ball Coehlo and others, 2007). 

Likewise, research also indicates rapid bacteria movement to tile lines by macropores after liquid 

manure application (Joy and others, 1998; Cook and Baker, 2001; Jamieson and others, 2002; Ball 

Coelho and others, 2007). Chemicals such as nitrate, which are readily soluble, are less affected by 

preferential flow pathways. Very recently, several studies have documented particle-associated 

phosphorus movement through macropores, especially during significant rainfall after extended dry 

periods (Simard and others, 2000; de Jonge and others, 2004; Schelde and others, 2006; Gentry and 

others, 2007). Even if transport of chemicals to subsurface drainage lines does not occur 

immediately by preferential flow pathways, this transport mechanism still permits rainfall or 

irrigation to move chemicals below the root zone—where they are not available for plant uptake 

during the growing season—and remain available for movement to tile drains when the growing 

season ends and soils become more saturated (Shipitalo and others, 2000). Manure application with 

equipment that disrupts earthworm burrows and cracks may prevent direct transport to the 

subsurface and provide more time for plant uptake or for diffusion of manure and 

5
manure-associated chemicals and bacteria into the soil matrix (Goehring and others, 2001; 

Jamieson and others, 2002; Shipitalo and Gibbs, 2000; Thiagarajan and others, 2007). 

Chemical and Microbiological Indicators of LDME Transport to Subsurface Drains 

Distinguishing manure sources of nutrients or bacteria from other sources, such as sewage 

or septic systems, would be helpful in applying the appropriate practices to those source areas and 

more efficiently achieving water-quality standards and reducing water-quality impairments in the 

Upper Tiffin Watershed. Several water constituents and microbiological indicators could 

potentially be used to distinguish manure pollution from pollution by other sources. As reviewed in 

Cimenti and others (2007), these include physical or genetic characterization of fecal-coliform, 

E. coli, or enterococci bacteria; detection of nonpathogenic but host-specific microbes or their 

genes; direct testing for host-specific pathogens or their genes; and testing for chemicals associated 

with specific types of fecal pollution or with animal-specific waste streams. The best approach to 

source determination involves several methods and a multiple-lines-of-evidence approach (Cimenti 

and others, 2007). 

One means of distinguishing pollution sources is to evaluate chemicals unique to those 

sources. Chemicals used every day in homes, industry, and agriculture can enter the environment in 

wastewater. A 1999 study by the USGS (http://toxics.usgs.gov/regional/emc/index.html) showed 

that a broad range of chemicals found in residential, industrial, and agricultural wastewaters 

commonly occurs in mixtures at low concentrations (typically less than 1 μg/L) in surface waters of 

the United States. The chemicals include human and veterinary drugs (including antibiotics), 

detergent metabolites, cleaning-product fragrances, plasticizers, agricultural and urban-use 

insecticides or herbicides, fecal sterols, and fire retardants. In Huron County, Mich., a USGS study 

detected both human-use and veterinary-use antibiotics in stream water but not in ground water 

(Duris and Haack, 2004). Other chemicals indicating domestic, industrial, and agricultural 

influences on water quality also were detected in the Huron County study. Some of these chemicals 

may be good indicators of sources of pollution (Cimenti and others, 2007), but little is known about 

their general occurrence in manure or their transport through soil. However, because antibiotics are 

used widely in animal agriculture, and because they have been detected in manure, several studies 

of antibiotic fate and transport in manured soils have been done (Burkhardt and others, 2005; Kay 

and others, 2004, 2005a, b; Hamscher and others, 2005). In addition, the sorption of commonly 

used veterinary antibiotics to soils under various conditions has been investigated (Rabolle and 

Spliid, 2000; Boxall and others, 2002; Schlüsener and Bester, 2006; Kahle and Stamm, 2007). In 

general, tetracycline antibiotics bind strongly to soil (Rabolle and Spliid, 2000; Hamscher and 

others, 2005) but sulfonamide antibiotics exhibit more complex transport, which is influenced by 

the pH, ionic strength, and nature of soils (Boxall and others, 2002; Kay and others, 2004, 

2005a, b; Thiele-Bruhn and others, 2004; Hamscher and others, 2005; Burkhardt and others, 2005; 

Kahle and Stamm, 2007). Field studies indicate that the addition of antibiotics with manure may 

change the antibiotics’ transport properties (Burkhardt and others, 2005), that sulfonamide 

antibiotics may accumulate in, and subsequently leach from, manured soils (Hamscher and others, 

2005), and that tillage may decrease leaching of sulfonamide antibiotics by preferential flow 

pathways (Kay and others, 2004, 2005a, b). At least one study has indicated that soil-bound 

tetracycline antibiotics may retain their antibacterial activity (Chandar and others, 2005). 

Another means of distinguishing pollution sources is to evaluate microorganisms or 

microbial genes unique to those sources. For example, shiga-toxin producing EC (STEC) may 

potentially indicate nonhuman sources. STEC are a major cause of gastrointestinal disease in 

6
humans, and some strains may lead to severe disease such as hemolytic uremic syndrome (HUS) or 

hemorrhagic colitis (Nataro and Kaper, 1998). STEC include E. coli O157:H7, for which the 

dominant reservoir is cattle (Boerlin and others, 1999; Bach and others, 2002). However, other 

STEC may come from other animals. Transmission between wild and domestic animals and 

humans may occur through contaminated food or water. The health effects of STEC are owing to 

several genes, and variations in shiga-toxin genes may indicate the potential animal source of 

contamination as well as the potential severity of disease (Beutin and others, 1993). In addition, 

Bacteroidetes bacteria may indicate different animal sources. The Bacteroidetes are more 

numerous than E. coli in intestinal samples, and a gene-based test that is specific for the 

Bacteroidetes associated with cattle was recently developed (Bernhard and Field, 2000). 

 Conditions at the Study Area 

The study area is within the Upper Tiffin Watershed in Lenawee County, Mich. (fig. 1). 

Soils of the field site are primarily Blount loam soils (fine, illitic, mesic, Aeric Epiaqualfs) on a 

3–7 percent slope. Eight field plots were established overlying 8, 200-ft-long, 4-in. diameter PVC 

tiles, installed 32 in. below land surface at 50-ft horizontal intervals. Tiles were installed in 1993 

and were connected to a larger drainage system for the entire field. Each PVC tile was modified in 

2006 by connecting a 4-in. PVC riser at the terminal end, approximately 2 months before field trial 

initiation, to allow for water-sample collection. 

The field site is on an active agricultural field that for the last 8 years had received liquid 

dairy manure effluent (LDME) at a rate of 8,000 gal/acre/yr. The field had been planted with 

typical rotations of corn and soybeans during that time and had received no tillage. The field had 

been in crop production for approximately 50 years and had received inorganic fertilizers before 

receiving LDME. Silage corn had been grown on the field before the field trial, and corn stubble 

(less than 15 percent cover) was present on the field plots during the field trial. In addition, manure 

application had taken place approximately 3 months before the field trial, in accordance with the 

management plan for the farm. Tile-drain flow for these plots was not measured. Tile-drain flow is 

a function of antecedent moisture, crop status, and soil characteristics (Baker and others, 2007). 

Occasional field observations indicated no water in these tile drains from September 27 to 

October 13, during which time a cumulative total of 1.6 in. of rain fell. From October 14 until the 

beginning of the field trial on November 2, water was generally present in the tiles. In this latter 

interval, removal of the summer’s corn crop, as well as two rainfall events with greater than 0.5 in. 

of rain in a 24-hour period, likely contributed to the presence of water in the tiles. There was no 

rainfall in the 5 days preceding LDME application. Calculations that assume the LDME is 

100 percent liquid indicate that application of LDME at 4,000 gal/acre is roughly equivalent to 

0.15 in. of rain per acre. 

The LDME applied to this field is analyzed on a routine basis in accordance with Michigan 

Generally Accepted Agricultural and Management Practices for Manure Management and 

Utilization (Michigan Department of Agriculture, 2007). The LDME typically has around 

1.0 percent solids, 10 lb/gal (1.2 g/L) of total nitrogen, 8.8 lb/gal (1.1 g/L) of ammonia nitrogen, 

and 0.2 lb/gal (24 mg/L) of orthophosphorus. 

This field trial investigated three management practices. The first was applying to soil 

without tillage (NT). The second was tillage with a ground-driven, rotary, subsurface cultivator that 

disturbed the soil profile to a depth of 2 to 3 in. (Dyna-Drive; DD). The third was tillage with a 

rolling-tine aerator that disrupted the top 6 to 8 in. of soil (AerWay; AW). Tillage immediately 

preceded LDME application. In this field trial LDME either was not applied at all, or was applied 

7
at the rate of 8,000 gal/acre or at half that rate. The eight plots with individual drainage tiles were 

assigned these treatments as indicated in table 1. 

Table 1. Treatment definitions and abbreviations. 

[gal/acre, gallons per acre] 

Plot/Tile number Tillage method Application rate

(gal/acre) Abbreviation 

1 Dyna Drive 8,000 DD8000 

2 Dyna Drive 4,000 DD4000 

3 Control None C1 

4 Aerway 8,000 AW8000 

5 Aerway 4,000 AW4000 

6 Not tilled 8,000 NT8000 

7 Not tilled 4,000 NT4000 

8 Control None C2 

Sampling and Analytical Methods 

Samples of the applied LDME, as well as tile-water samples, were collected on November 

2, 2006 before the application of LDME (pre-application), then after the application on the same 

date (4 hours). Similarly, samples were collected from all tiles on November 3 (1 day), November 

4 (2 days), November 8 (6 days), November 9 (7 days), and November 16 (14 days). Some sample 

dates were chosen specifically to evaluate the effects of rainfall on subsurface drain water quality. 

Rainfall amounts during the field trial are listed in table 2. The NT8000 treatment tile (tile 6) was 

additionally sampled on December 21, 2006, and both the NT8000 treatment tile (tile 6) and the 

Control 1 treatment tile (tile 3) were sampled on March 20, 2007, to gain further temporal 

perspective on nutrient concentrations. 

Table 2. Rainfall during the course of the field trial. 

 [All results reported in inches; days with no rainfall not reported] 

Date Rainfall amount 

10/26/2006 0.07 

10/27/2006 .58 

10/28/2006 .06 

11/7/2006 .55 

11/8/2006 .01 

11/10/2006 .17 

11/11/2006 .24 

11/15/2006 .24 

11/16/2006 .60 

11/24/2006 .01 

11/30/2006 .88 

12/1/2006 .90 

12/12/2006 .63 

12/14/2006 .01 

12/17/2006 .01 

12/21/2006 .33 

8
Sampling Procedures 

Tile-water samples were collected in accordance with procedures in the USGS National 

Field Manual (NFM) (U.S. Geological Survey, variously dated). For nutrients and bacteria 

analyses, a peristaltic pump and polyethylene tubing were used to draw water from the sampling 

port on each subsurface tile. For other chemical analyses, silicon tubing was used. Tubing was 

cleaned between samples with successive rinses of distilled water, 1 percent soap solution, 

0.005 percent bleach solution, distilled water, and at least 500 mL of water from the next tile. 

LDME samples were collected as grabs from the effluent access on the slurry spreading tank. 

Samples for chemical analysis were filtered and/or preserved in the field as per procedures 

described in the USGS NFM, and, for nutrients, in the references listed in table 3. 

Table 3. Methods of nutrient analysis and reporting limits. 

[mg/L, milligrams per liter; ASF, automated segmented flow] 

 Nutrient Method Reporting limit 

(mg/L) Reference 

Ammonia + organic N Colorimetry, ASF, 

microkjeldahl 0.10 Patton and Truitt, 2000 

Ammonia, as N Colorimetry, salicylatehypochlorite .02 Fishman, 1993 

Nitrite + nitrate, as N Colorimetry, ASF, cadmium 

reduction – diazotization .06 Fishman, 1993 

Phosphorus as P Colorimetry, ASF, 

microkjeldahl .04 Patton and Truitt, 1992 

Orthophosphate as P Colorimetry, 

phosphomolybdate .006 Fishman, 1993 

Chemical Analyses 

All samples were analyzed at the USGS National Water Quality Laboratory (NWQL), 

Denver, Colo., for the nutrients listed in table 3. Organic + ammonia nitrogen and total phosphorus 

were analyzed on whole (unfiltered) water samples. Ammonia nitrogen, nitrate + nitrite nitrogen, 

and orthophosphorus were analyzed on filtered water samples. Field blank samples for nutrient 

analyses were collected on November 16, 2006. Field blanks were processed by passing inorganic 

blank water through the entire sampling apparatus, in the field, after a between-sample cleaning per 

established protocols in the USGS National Field Manual. Selected samples were sent to the USGS 

NWQL for Schedule 1433 (filtered)/8033 (unfiltered, for LDME samples only) “Wastewater 

Analysis” (table 1–1) and the USGS Kansas Organic Research lab for Schedule LCAN, antibiotics 

analysis (table 1–2). The following samples were tested: LDME (November 2, 2006); field blank 

(November 16, 2006); pre-application (Control 2, November 2, 2006); all treatments except 

Control 1 on November 8, 2006; and NT8000 additionally on November 2, 3, 4, and 9, and 

December 21, 2006. 

9
Microbiological Analyses 

Tile-water samples were analyzed by use of standard membrane-filtration methods (APHA 

and others, 1998) for detection of fecal-coliform (FC) bacteria (mFC medium) and E. coli

(NA-MUG medium). In addition, two gene-based assays were done to evaluate the presence of 

shiga-toxin-producing E. coli or the presence of bovine-specific Bacteroidetes. 

Bacteria Enumeration and Preservation 

All media was prepared according to manufacturer's instructions. For each water sample 

50-, 5-, and 0.5-mL volumes were filtered though a 0.45-μm nylon membrane filter that was 

transferred to mFC medium and incubated at 44.5oC for 24 hours. If growth was uncountable at 

these dilutions, further ten-fold serial dilutions were made to obtain countable growth. Because the 

second set of ten-fold dilutions of necessity took place on the second day, after evaluating 

overnight growth on the previous day’s filters, such samples exceed typical holding times and 

concentrations are thus indicated as estimated (E) in table 4. Bacteria enumeration was based on 

preparations with 20 to 80 colonies or was calculated from multiple dilutions in the case of nonideal counts. After enumeration of FC colonies, the filter with the appropriate range of colonies 

was transferred to NA-MUG medium and incubated at 37oC for 4 hours. Fluorescent colonies were 

counted as Escherichia coli. Growth from the 50-mL mFC filter was transferred to 1 mL phosphate 

buffered saline (PBS) with a final concentration of 20 percent glycerol, and was frozen at -70oC for 

further analysis; this set of processed samples is hereafter referred to as the “FC stock.” In addition, 

samples from all treatments on November 2 and 8, 2006, and from the NT8000 treatment 

additionally on November 3, 4, 9, and 16 were directly preserved by centrifuging 10 mL of the 

sample for 15 minutes at 4,500 revolutions per minute. The supernatant was decanted and the pellet 

was resuspended in 1 mL of phosphate buffered saline with 20 percent glycerol. Stocks of the 

LDME were made by adding 20 mL of LDME to sterile 50-mL polyethylene tubes and mixing 1:1 

with 40 percent glycerol to create a final concentration of 20 percent glycerol. All stocks were 

frozen and stored at -70oC until analysis. 

Microbiological Indicators of LDME Transport to Subsurface Drains

These microbiological assays were done to help identify whether fecal bacteria present in 

the tile drains could be from a cattle source. One set of tests was done to evaluate whether shigatoxin-producing E. coli (STEC) were present. Tests targeted the E. coli O157 serotype by two 

different assays. Tests also targeted a suite of toxin genes carried by typical STEC. Positive results 

might indicate a human-health concern and a bovine source. A second test analyzed for bovinesource Bacteroidetes. Positive results would indicate a bovine source. 

Immunological Test for O157 Antigen 

For every sample, 100 μL of FC stock was inoculated into Reveal for E. coli O157:H7 

medium and the 8-hour test was performed according to manufacturer’s instructions (Neogen, 

Lansing, Mich.). This test uses anti-O157 antibodies and detects all H serotypes of E. coli O157; 

therefore, all positive results are classified as detection of E. coli O157. 

10
Table 4. Water-quality field data and bacteria concentrations for LDME on date of 

application and for all tile-water samples. 

[LDME, liquid dairy manure effluent; μS/cm, microsiemens per centimeter at 25 degrees Celsius; CFU, 

colony forming units; mL, milliliters; ND, not detected; E, estimated because of non-ideal holding times] 

Sample 

identification 

Sample 

date 

Specific 

conductance 

(μS/cm) 

 pH 

Fecal-coliform 

bacteria 

(CFU/100 mL) 

Escherichia 

coli 

(CFU/100 mL) 

LDME 11/2/2006 4,000 6.8 E1,030,000 E600,000 

Pre-application samples 

Tile 1 - DD8000 11/2/2006 1,230 7.7 1 1 

Tile 2 - DD4000 11/2/2006 1,250 7.6 130 150 

Tile 3 - C1 11/2/2006 1,180 7.6 100 10 

Tile 4 - AW8000 11/2/2006 1,040 8.3 ND ND 

Tile 5 - AW4000 11/2/2006 1,040 7.5 6,900 100 

Tile 6 - NT8000 11/2/2006 1,280 7.5 6 1 

Tile 7 - NT4000 11/2/2006 1,220 8.4 19 19 

Tile 8 - C2 11/2/2006 1,360 7.8 260 180 

4 hours post-application 

Tile 1 - DD8000 11/2/2006 1,250 7.6 ND ND 

Tile 2 - DD4000 11/2/2006 1,290 7.6 ND ND 

Tile 3 - C1 11/2/2006 1,210 7.5 2 2 

Tile 4 - AW8000 11/2/2006 1,090 7.5 ND ND 

Tile 5 - AW4000 11/2/2006 1,060 7.5 3 ND 

Tile 6 - NT8000 11/2/2006 1,370 7.4 E35,000 7,000 

Tile 7 - NT4000 11/2/2006 1,250 7.5 ND ND 

Tile 8 - C2 11/2/2006 1,370 7.5 ND ND 

1 day post-application 

Tile 1 - DD8000 11/3/2006 1,190 7.5 ND ND 

Tile 2 - DD4000 11/3/2006 1,230 7.8 ND ND 

Tile 3 - C1 11/3/2006 1,190 7.8 ND ND 

Tile 4 - AW8000 11/3/2006 1,070 7.7 ND ND 

Tile 5 - AW4000 11/3/2006 999 7.8 ND 6 

Tile 6 - NT8000 11/3/2006 1,270 7.5 1,290 400 

Tile 7 - NT4000 11/3/2006 1,200 7.6 ND ND 

Tile 8 - C2 11/3/2006 1,310 7.5 ND ND 

11
Table 4. Water-quality field data and bacteria concentrations for LDME on date of 

application and for all tile-water samples.—Continued 

[LDME, liquid dairy manure effluent; μS/cm, microsiemens per centimeter at 25 degrees Celsius; CFU, 

colony forming units; mL, milliliters; ND, not detected; E, estimated because of non-ideal holding times] 

Sample 

identification 

Sample 

date 

Specific 

conductance 

(μS/cm) 

 pH 

Fecal-coliform 

bacteria 

(CFU/100 mL) 

Escherichia 

coli 

(CFU/100 mL) 

2 days post-application 

Tile 1 - DD8000 11/4/2006 1,190 7.6 7 5 

Tile 2 - DD4000 11/4/2006 1,230 7.7 2 2 

Tile 3 – C1 11/4/2006 1,160 7.6 2 2 

Tile 4 - AW8000 11/4/2006 1,080 7.5 ND ND 

Tile 5 - AW4000 11/4/2006 988 7.5 2 2 

Tile 6 - NT8000 11/4/2006 1,280 7.5 127 80 

Tile 7 - NT4000 11/4/2006 1,220 7.6 ND ND 

Tile 8 - C2 11/4/2006 1,380 7.5 8 8 

6 days post-application – Rainfall event 

Tile 1 - DD8000 11/8/2006 1,220 7.6 26 13 

Tile 2 - DD4000 11/8/2006 1,280 7.3 29 14 

Tile 3 - C1 11/8/2006 1,140 7.5 15 6 

Tile 4 - AW8000 11/8/2006 982 7.4 838 520 

Tile 5 - AW4000 11/8/2006 945 7.4 35 21 

Tile 6 - NT8000 11/8/2006 1,230 7.3 145 120 

Tile 7 - NT4000 11/8/2006 1,200 7.5 18 17 

Tile 8 - C2 11/8/2006 1,310 7.5 3 2 

7 days post-application 

Tile 1 - DD8000 11/9/2006 1,250 7.4 ND ND 

Tile 2 - DD4000 11/9/2006 1,280 7.5 ND ND 

Tile 3 - C1 11/9/2006 1,150 7.6 ND ND 

Tile 4 - AW8000 11/9/2006 1,020 7.7 700 30 

Tile 5 - AW4000 11/9/2006 992 7.7 118 40 

Tile 6 - NT8000 11/9/2006 1,250 7.5 100 ND 

Tile 7 - NT4000 11/9/2006 1,200 7.6 ND ND 

Tile 8 - C2 11/9/2006 1,280 7.6 12 ND 

12
Table 4. Water-quality field data and bacteria concentrations for LDME on date of 

application and for all tile-water samples.—Continued 

[LDME, liquid dairy manure effluent; μS/cm, microsiemens per centimeter at 25 degrees Celsius; CFU, 

colony forming units; mL, milliliters; ND, not detected; E, estimated because of non-ideal holding times] 

Sample 

identification 

Sample 

date 

Specific 

conductance 

(μS/cm) 

 pH 

Fecal-coliform 

bacteria 

(CFU/100 mL) 

Escherichia 

coli 

(CFU/100 mL) 

14 days post-application – Rainfall event 

Tile 1 - DD8000 11/16/2006 599 8.1 50 25 

Tile 2 - DD4000 11/16/2006 640 8.0 74 44 

Tile 3 - C1 11/16/2006 654 7.9 20 16 

Tile 4 - AW8000 11/16/2006 680 7.9 82 70 

Tile 5 - AW4000 11/16/2006 697 7.9 90 70 

Tile 6 - NT8000 11/16/2006 709 7.8 90 50 

Tile 7 - NT4000 11/16/2006 680 7.8 62 34 

Tile 8 - C2 11/16/2006 624 7.8 82 50 

DNA Extraction 

DNA was extracted from 200 μL of frozen source stock (representing 2 mL of original 

water from the tile, 10 mL of the 50-mL mFC culture, or 100 μL of LDME stock). DNA was 

extracted by use of the QIAamp DNA stool mini kit (Qiagen, Valencia, Calif.) according to 

manufacturer’s instructions. 

Polymerase Chain Reaction (PCR) 

PCR was done by use of a PerkinElmer GeneAmp PCR System 2400 Thermal Cycler 

(Perkin Elmer, Boston, Mass.). All reaction mixtures were 25 μL final volume. PCR fragments 

were separated on 2 percent agarose gels in Tris Acetate EDTA (TAE) buffer and stained for 

15 minutes in 0.5 μg/mL ethidium bromide solution. Fragments were visualized by use of a 

Foto/prep transilluminator (Fotodyne, Hartland, Wis.), imaged with a Kodak EDAS 290 Zoom 

digital camera and analyzed with the Kodak 1D-gel image analysis software. 

Multiplex PCR for Shiga-Toxin-Producing E. coli 

This analysis was designed to detect four genes: eaeA, stx1, stx2 (Gannon and others, 1992; 

Fagan and others, 1999) and the 16S rDNA of E. coli (Sabat and others, 2000). The latter gene was 

used as an internal positive control. All reagents were from Applied Biosystems (Foster City, 

Calif.). Each 15-μL PCR reaction mixture contained (final concentration) 1× Buffer II PCR 

reaction buffer, 3 mM MgCl2, 0.2 mM dNTPs, 0.1 μg/μL bovine serum albumin (BSA), eaeA, stx2 

and stx1 primers (0.05 μM), E. coli 16S rDNA primers (0.025 μM), AmpliTaq Gold Polymerase 

(0.1 unit/μL), and from 1 to 100 ng of DNA. A separate PCR assay was used to detect the rfbO157 

gene for the E. coli O157 serotype (Maurer and others, 1999). This is a confirmation assay for the 

Reveal test. All reagents were from Applied Biosystems (Foster City, Calif.). Each 15-μL reaction 

mixture contained (final concentration) 1 × Gold Buffer, 2 mM MgCl2, 0.2 mM dNTPs, 0.1 μg/μL 

13
BSA, 0.1 μM of forward and reverse primers, 0.1 unit/μL of AmpliTaq Gold polymerase, and from 

1 to 100 ng of template DNA. All reagents were from Promega (Madison, Wis.). 

PCR for Bovine Bacteroidetes Gene 

Extracted DNA was amplified with primers described in Bernhard and Field (2000). PCR 

was performed on 1–10 ng of DNA. Each 15-μL reaction contained the after (final concentrations): 

1× colored GoTaq buffer, 2.5 mM MgCl2, 0.2 mM dNTPs, 0.1 μg/μL bovine serum albumin, 

0.3 μM of CF128F primer, 0.3 μM of BAC708R primer, and 0.1 unit/μL of GoTaq Polymerase 

(Promega, Madison, Wis.). A touchdown DNA amplification was used with the after temperature 

cycles as per Fogarty and Voytek (2005): 94oC for 3 minutes; 10 cycles of 94oC for 30 seconds, 

63oC for 30 seconds (decreasing by 1oC each cycle), and 72oC for 30 seconds; and 20 cycles of 

94oC for 30 seconds, 53oC for 30 seconds, and 72oC for 1 minute and 30 seconds. 

Quality Assurance/Quality Control 

Several steps were taken to assure the quality of each PCR reaction, after recommendations 

of the USEPA (2004). For approximately every 20 samples of any given PCR reaction, PCR 

positive controls (extra reaction with 10 ng of DNA from the positive control strain), and PCR 

negative controls (blank water replaced template DNA) were included. Environmental matrix 

issues were addressed by including a matrix spike (addition of positive DNA to a previously 

analyzed negative sample) approximately every 40 reactions for the STEC multiplex and bovine 

Bacteroidetes gene assays. PCR inhibition controls (internal controls) were run with every reaction 

of the STEC multiplex PCR. 

Results of Chemical and Microbiological Analyses 

As described previously, LDME might move to tile drains by rapid transport through 

preferential flow pathways or by slower leaching through the soil matrix. In studies by others, if 

preferential flow pathways existed, transport typically occurred within a few hours to a day after 

LDME application. Subsequent leaching could occur at any time that the water-holding capacity of 

the soil was exceeded, usually after a rainfall event. Therefore, sample collection was designed to 

evaluate tile-drain water quality before LDME application and in both the short term (4 hours to 

1 day) and in the longer term, especially after rainfall events. On each sampling date, tile-drain 

water was analyzed in the field for specific conductance (SC) and pH. Specific conductance is a 

measure of the concentration of ions in a solution, and pH indicates the degree of acidity or 

alkalinity of the sample. Both measures were chosen to possibly indicate the movement of LDME 

from the surface application to the tile drains. In addition, fecal-coliform and E. coli bacteria were 

enumerated in subsurface drain water, and concentrations of these bacteria were compared to 

various water-quality standards. Various forms of nitrogen and phosphorus were determined, 

patterns in nutrient concentrations with respect to LDME application and rainfall were evaluated, 

and nutrient concentrations were compared to USEPA-recommended water-quality criteria. The 

presence of chemical and microbiological indicators of LDME transport to subsurface drains was 

evaluated in one tile drain over the duration of the field trail and in all tile drains after the first 

significant rainfall event. 

14
Specific Conductance and pH 

Neither specific conductance nor pH gave a clear indication of short-term or longer term 

LDME movement to subsurface tiles under any treatment (table 4). The LDME on the day of 

application had an SC of 4,000 μS/cm. The SC of subsurface drain water before LDME application 

ranged from 1,034 to 1,361 μS/cm. For most of the sampling dates, changes in SC were within the 

range of accuracy for the SC probes (± 3–5 percent). On November 16, 2006, however, subsurface 

drains were full to overflowing, and SC values ranged from 599 to 709 μS/cm for all treatments. 

These SC values are approximately half of pre-application SC and most likely indicate the dilution 

of subsurface water with rainwater. 

The LDME on the day of application had a pH of 6.8. The pre-application pH for 

subsurface drain water for all treatments ranged from 7.5 to 8.4. After application of LDME, there 

was no obvious trend in pH with respect to treatment. After heavy rainfall on November 16, 2006, 

pH increased by about 0.5 unit for all treatments. 

Bacteria Concentrations 

Both fecal-coliform and E. coli bacteria were enumerated. E. coli are members of the fecalcoliform group. In the LDME, E. coli made up 59 percent of fecal coliforms. Among the tile 

samples, E. coli ranged from less than 1 percent of fecal coliforms to (more typically) 

60–100 percent of fecal coliforms. The LDME contained around 1,000,000 fecal coliforms, and 

600,000 E. coli, per 100 mL. 

One pre-application drain (tile 5, table 4) exhibited a relatively high concentration of fecalcoliform, but not E. coli, bacteria. Some fecal-coliform bacteria may occur naturally in the 

environment, and fecal-coliform bacteria and E. coli may persist from prior manure applications 

(Jamieson and others, 2002). This information may explain the presence of these bacteria in preapplication tile samples. 

The only subsurface drain for which fecal-coliform and E. coli bacteria concentrations 

increased after LDME application was tile 6 (NT8000 treatment, table 4). The results for the 

NT8000 treatment appear to indicate rapid movement of bacteria to tile-drain water after LDME 

application. At 4 hours post-application, both fecal-coliform and E. coli concentrations were very 

high for the NT8000 treatment in comparison to pre-application concentrations and to 

concentrations in other treatment tile drains. 

There was only one indication of the potential for rainfall to promote the movement of 

LDME bacteria to subsurface drains. Bacteria concentrations in tile 4 (AW4000 treatment, table 4) 

increased above prior levels 6 days after LDME application, after a rainfall event. For this 

treatment, fecal-coliform concentrations, but not E. coli concentrations, remained elevated on the 

seventh day. 

In Michigan, surface waters are required to meet recreational water-quality criteria during 

the recreational season (May through September). For recreational waters, the concentration of 

E. coli permitted for full-body contact during the recreational season is 235 CFU/100 mL in a 

single water sample (USEPA) or, for the State of Michigan, 300 CFU/100 mL as a geometric mean 

of three samples. In this field trial, sampling was not done in such a way that the geometric mean 

could be calculated as required by the State of Michigan. In addition, sampling was done during the 

nonrecreational season. Therefore, recreational water-quality standards serve only as a reference 

against which bacteria concentrations can be compared. Two treatments had samples that exceeded 

the USEPA single-sample standard: NT8000 at 4 hours and 1 day post-application, and AW8000 at 

15
6 days post-application, after a rainfall event (table 4). No sample from a control tile exceeded 

these standards. 

Under the National Pollutant Discharge Elimination System (NPDES) the State of 

Michigan has issued a general permit for large, concentrated animal-feeding operations and for 

other animal-feeding operations that request coverage (http://www.deq.state.mi.us/documents/deqwater-npdes-generalpermit-MIG019000.pdf). Discharges that violate or that contribute to violation 

of Michigan Water Quality Standards are prohibited. The data from this field trial indicate that 

LDME application, combined with some management practices, may influence bacteria 

concentrations in subsurface drains. Bacteria leaching may occur immediately after application or 

subsequently in association with rainfall events, as others have observed (Joy and others, 1998; 

Geohring and others, 2001; Jamieson and others, 2002; Ball Coelho and others, 2007). However, in 

this field trial, subsurface-drain samples were not collected at a point of outflow to surface water. 

Concentrations of bacteria or other measured constituents might be different at the point where tile 

drains discharge to surface water. 

Nutrient Concentrations 

The LDME was analyzed on three dates to evaluate variability in nutrient concentrations 

(table 5). Concentrations of nutrients in LDME or in any type of manure may vary with holding 

time, frequency of application, number of animals contributing, relative solids percentage, and a 

wide array of environmental factors. As noted previously, the LDME is analyzed routinely and 

typically has around 1.0 percent solids, 10 lb/gal (1.2 g/L) of total nitrogen, 8.8 lb/gal (1.1 g/L) of 

ammonia nitrogen, and 0.2 lb/gal (24 mg/L) of orthophosphorus. For the three samples reported in 

table 5, concentrations of organic + ammonia nitrogen (roughly equivalent to total nitrogen), 

ammonia nitrogen, and orthophosphorus were consistent with those previous analyses by other 

laboratories, indicating that sampling the LDME from the applicator did not result in obvious bias 

in nutrient concentrations. Most nutrients in manure are in organic forms, and nitrate is not usually 

present in any abundance. The data in table 5 are consistent with this distribution of the various 

forms of nutrients. 

A field blank sample was analyzed for all nutrients (table 5). No nitrate + nitrite nitrogen, 

total phosphorus, or orthophosphorus were detected in the field blank, but organic + ammonia 

nitrogen and ammonia nitrogen were detected. These detections might indicate that a contaminant 

such as a soil particle may have been introduced into the sample bottle by handling. These 

detections could also indicate that cleaning of the sampling apparatus did not remove all 

contamination from the previous sample. However, ammonia nitrogen was rarely detected in 

subsequent samples; and because all samples were collected by means of the same between-sample 

cleaning procedure as for the field blank, it would appear that there was no routine contamination 

with ammonia nitrogen due to the sampling procedure. The tile 3 Control 1 treatment exhibited 

anomalously high pre-application concentrations of organic + ammonia nitrogen and of total 

phosphorous, indicative of possible sample contamination. However, because both these analyses 

were done on unfiltered water samples, the relatively high concentrations of organic + ammonia 

nitrogen and total phosphorous may simply reflect the presence of naturally occurring particulate 

material in the sample. Nevertheless, the tile 3 C1 pre-application sample is marked in table 5 as 

possibly subject to contamination. In the tables and charts that follow, the concentrations of 

organic + ammonia nitrogen and ammonia nitrogen detected in the field blank have not been 

subtracted from the reported values. 

16
Table 5.  Nutrient concentrations in LDME, field blank, and tile-water samples. 

[LDME, liquid dairy manure effluent; ND, not detected; C, sample possibly contaminated; E, estimated concentration; 

all concentrations in milligrams per liter] 

Site 

identification 

Sample 

date 

Organic + 

ammonia 

nitrogen 

Ammonia 

nitrogen 

Nitrate + 

nitrite nitrogen 

Orthophosphorus Total 

phosphorus 

LDME 9/27/2006 1,300 1,320 0.07 0.562 30 

LDME 10/11/2006 930 860 .07 .058 25.8 

LDME 11/2/2006 800 519 ND 195 195 

Field Blank 11/16/2006 .5 .04 ND ND ND 

Pre-application samples 

Tile 1 - DD8000 11/2/2006 .61 ND 44.0 .038 .06 

Tile 2 - DD4000 11/2/2006 9.8 ND 39.3 .102 3.53 

Tile 3 - C1 11/2/2006 C120 ND 52.6 .084 C49.3 

Tile 4 - AW8000 11/2/2006 .93 ND 47.3 .093 .22 

Tile 5 - AW4000 11/2/2006 1.3 ND 46.2 .092 .29 

Tile 6 - NT8000 11/2/2006 1.3 ND 53.8 .061 .58 

Tile 7 - NT4000 11/2/2006 .67 ND 41.0 .104 .12 

Tile 8 - C2 11/2/2006 1.1 ND 48.8 .232 .36 

4 hours post-application 

Tile 1 - DD8000 11/2/2006 .53 ND 44.1 .035 ND 

Tile 2 - DD4000 11/2/2006 .78 ND 38 .084 .09 

Tile 3 - C1 11/2/2006 4.2 ND 52.5 .068 2.15 

Tile 4 - AW8000 11/2/2006 .98 ND 46.3 .089 .27 

Tile 5 - AW4000 11/2/2006 1.6 ND 45 .094 .34 

Tile 6 - NT8000 11/2/2006 3.2 .05 52.5 .008 .53 

Tile 7 - NT4000 11/2/2006 .63 ND 41.1 .119 .12 

Tile 8 - C2 11/2/2006 .63 ND 44.6 .126 .15 

1 day post-application 

Tile 1 - DD8000 11/3/2006 .53 ND 43.6 .036 .06 

Tile 2 - DD4000 11/3/2006 .6 ND 38.1 .091 .09 

Tile 3 - C1 11/3/2006 .53 ND 43.7 .035 .06 

Tile 4 - AW8000 11/3/2006 .84 ND 48.3 .099 .16 

Tile 5 - AW4000 11/3/2006 .73 ND 43.7 .103 .13 

Tile 6 - NT8000 11/3/2006 .66 .16 55.1 .072 .09 

Tile 7 - NT4000 11/3/2006 .61 ND 40.3 .12 .1 

Tile 8 - C2 11/3/2006 .86 ND 46.2 .138 .2 

17
Table 5.  Nutrient concentrations in LDME, field blank, and tile-water samples.—Continued 

[LDME, liquid dairy manure effluent; ND, not detected; C, sample possibly contaminated; E, estimated concentration; 

all concentrations in milligrams per liter] 

Site 

identification 

Sample 

date 

Organic + 

ammonia 

nitrogen 

Ammonia 

nitrogen 

Nitrate + 

nitrite nitrogen 

Orthophosphorus Total 

phosphorus 

2 days post-application 

Tile 1 - DD8000 11/4/2006 0.68 ND 53.8 0.05 0.16 

Tile 2 - DD4000 11/4/2006 .69 ND 38.6 .095 .34 

Tile 3 - C1 11/4/2006 .82 ND 54.8 .05 .19 

Tile 4 - AW8000 11/4/2006 .64 ND 47.5 .102 .12 

Tile 5 - AW4000 11/4/2006 .68 ND 42.8 .106 .12 

Tile 6 - NT8000 11/4/2006 .6 .09 55.3 .057 .17 

Tile 7 - NT4000 11/4/2006 .86 ND 40.7 .131 .2 

Tile 8 - C2 11/4/2006 .97 ND 49.9 .22 .28 

6 days post-application – Rainfall Event 

Tile 1 - DD8000 11/8/2006 .93 ND 43.5 .024 .09 

Tile 2 - DD4000 11/8/2006 1.5 .03 40 .059 .19 

Tile 3 - C1 11/8/2006 .77 ND 49.8 .042 .15 

Tile 4 - AW8000 11/8/2006 1.5 ND 41.1 .081 .25 

Tile 5 - AW4000 11/8/2006 1.9 ND 42.5 .095 .41 

Tile 6 - NT8000 11/8/2006 1 ND 54.4 .057 .2 

Tile 7 - NT4000 11/8/2006 .71 ND 41.7 .053 .08 

Tile 8 - C2 11/8/2006 .78 ND 44.3 .052 .08 

7 days post-application 

Tile 1 - DD8000 11/9/2006 .51 ND 43.2 .024 ND 

Tile 2 - DD4000 11/9/2006 1.4 ND 38.5 .056 .19 

Tile 3 - C1 11/9/2006 3.8 ND 50.6 .035 2.31 

Tile 4 - AW8000 11/9/2006 .79 ND 42.8 .081 .08 

Tile 5 - AW4000 11/9/2006 .92 ND 43.7 .093 .11 

Tile 6 - NT8000 11/9/2006 .58 ND 53.2 .049 .11 

Tile 7 - NT4000 11/9/2006 .73 .62 39.9 .149 .12 

Tile 8 - C2 11/9/2006 1.5 ND 40.8 .067 .39 

18
Table 5.  Nutrient concentrations in LDME, field blank, and tile-water samples.—Continued 

[LDME, liquid dairy manure effluent; ND, not detected; C, sample possibly contaminated; E, estimated concentration; 

all concentrations in milligrams per liter] 

Site 

identification 

Sample 

date 

Organic + 

ammonia 

nitrogen 

Ammonia 

nitrogen 

Nitrate + 

nitrite nitrogen 

Orthophosphorus Total 

phosphorus 

14 days post-application – Rainfall Event 

Tile 1 - DD8000 11/16/2006 3.5 0.12 25.5 1.54 2.48 

Tile 2 - DD4000 11/16/2006 4.9 .12 29.4 1.4 2.9 

Tile 3 - C1 11/16/2006 3.7 ND 33.9 .67 1.63 

Tile 4 - AW8000 11/16/2006 4.2 E.12 27.7 .582 1.93 

Tile 5 - AW4000 11/16/2006 3.9 .04 21 .738 1.8 

Tile 6 - NT8000 11/16/2006 3.6 .08 28 .735 1.46 

Tile 7 - NT4000 11/16/2006 4.1 .05 29.5 .936 1.83 

Tile 8 - C2 11/16/2006 3.6 .05 36.8 1.23 1.98 

51 days post-application 

Tile 6 - NT8000 12/21/2006 1.6 ND 52.3 .037 .47 

171 days post-application 

Tile 3 - C1 3/20/2007 5.4 .41 43.9 .079 1.63 

Tile 6 - NT8000 3/20/2007 1.9 .03 43.5 .033 .57 

Nutrient concentrations in tile water did not conclusively indicate short-term preferential 

flow of LDME to tile drains. Concentrations of organic + ammonia nitrogen, nitrite + nitrate 

nitrogen, and ortho- and total phosphorus measured within 4 hours to 1 day after LDME 

application were within the range of pre-application values for all treatments (table 5), indicating 

no immediate influence of LDME application on tile-drain water quality for these constituents. 

Ammonia nitrogen was detected in the NT8000 samples immediately after, and for 2 days after, 

LDME application. This was the only treatment to exhibit this result. With only this exception, 

concentrations of all forms of nutrients measured within 4 hours to 1 day after LDME application 

were generally similar among all treatments, including control treatment plots, to which no LDME 

was applied. Therefore, antecedent conditions appeared to have more influence on short-term 

nutrient concentrations than did the LDME application. 

Nutrient concentrations also did not indicate an effect of LDME application or management 

practice on tile water quality after rainfall events. After the rainfall event at 6 days post-application 

(approximately 0.56 in. of rain within 24 hours), there was little change in tile-water nutrient 

concentrations with respect to previous samples nor any obvious pattern in nutrient concentrations 

among treatments receiving LDME in comparison to those receiving no LDME. However, after the 

rainfall event at 14 days post-application (approximately 0.84 in. of rain within 24 hours), organic 

+ ammonia nitrogen, orthophosphorus, and total phosphorus concentrations increased above 

average concentrations for all prior sampling dates in every tile. In addition, ammonia nitrogen was 

present in every tile, including Controls, whereas it was not typically present in every tile on prior 

sampling dates. Because increases in the concentrations of these constituents also occurred in tiles 

draining Control plots to which no LDME was applied, this effect cannot be attributed to LDME 

19
application or management practice. Finally, nitrate + nitrite nitrogen concentrations decreased in 

every tile, including Controls. This result may reflect dilution by rainwater. 

Very recently, several studies have documented particle-associated phosphorus movement 

to subsurface drains, especially during significant rainfall events after extended dry periods 

(Simard and others, 2000; de Jonge and others, 2004; Schelde and others, 2006; Gentry and others, 

2007). This recent research indicates that particles may be released from soil when the ionic 

strength of the soil water decreases or when pH increases (de Jonge and others, 2004; Schelde and 

others, 2006). Both water-quality changes were observed for the 14-day samples for all treatments 

(table 4), and are likely due to the influx of rainwater. It is common to estimate particulate 

phosphorus as the difference between phosphorous measured in unfiltered samples (in this study, 

total phosphorous) and phosphorous measured in filtered samples (in this study, orthophosphorus) 

(Simard and others, 2000; Toor and others, 2004). If this calculation is done for the data reported in 

table 5, the median particulate phosphorous for all samples preceding the 14 days post-application 

rainfall event is 0.062 mg/L. In contrast, for the samples collected at 14 days after application, the 

median particulate phosphorus concentration estimated by this method is 0.95 mg/L. 

Additional samples were collected from the NT8000 treatment at 51 days and 171 days 

post-LDME application to provide some longer term perspective on nutrient concentrations 

(table 5). A sample was also collected from the Control 1 treatment at 171 days post-application. 

No further LDME was applied during this time. Nevertheless, for both the NT8000 and Control 1 

treatments, concentrations for all constituents in March 2007 were similar to those noted during the 

field trial. 

The USEPA has established recommended total phosphorus and total nitrogen water-quality 

criteria for streams and rivers (United States Environmental Protection Agency, 2000). These 

criteria contain USEPA’s recommendations to States and authorized Tribes for use in establishing 

their water-quality standards consistent with section 303(c) of Clean Water Act. These values are 

not at this time incorporated into water-quality standards by the State of Michigan. At this time, 

these values serve only as a reference against which stream-water quality may be compared. 

Table 6 lists these values for Aggregate Ecoregion VI (Corn Belt and Northern Great Plains), into 

which the Maumee River Watershed falls; however, this ecoregion covers several states, including 

parts of South Dakota, Nebraska, and Minnesota. The 25th-percentile values for the entire Region 

VI were taken as the recommended criteria for total nitrogen and total phosphorus. There are 

currently no recommended criteria for organic + ammonia nitrogen or nitrate + nitrite nitrogen. If 

criteria were to be recommended, it is possible the 25th-percentile values would be used, so these 

are reported in table 6. In addition, the USEPA lists 25th-percentile values for total phosphorous, 

total nitrogen, organic + ammonia nitrogen, and nitrate + nitrite nitrogen for the subregion that 

includes specifically the Maumee River Watershed and a region surrounding Saginaw Bay 

(Level III Ecoregion # 57, Huron/Erie Lake Plain). Although the USEPA has not recommended 

criteria for nutrients at the subregional level, the 25th-percentile values for the Level III Ecoregion 

#57 more likely reflect specific water-quality conditions in the Maumee River Watershed. 

20
Table 6. U.S. Environmental Protection Agency Ambient Water-Quality Criteria and 

25th percentiles for nutrients in Ecoregion VI (Corn Belt) and Level III Ecoregion 57 

(Huron/Erie Lake Plain). 

[All concentrations in milligrams per liter; --, not applicable] 

 Region 

Water-quality criteria 25th percentiles 

Total 

phosphorus 

Total 

nitrogen 

Organic + 

ammonia 

nitrogen

Nitrate + 

nitrite 

nitrogen

Total 

nitrogen 

Total 

phosphorus 

Ecoregion VI 0.0763 2.18 0.591 0.633 2.18 0.0763 

Level III 

Ecoregion 57 -- -- .65 .897 1.91 .070 

Field-trial results indicate potential for water-quality degradation, based on USEPA nutrient 

water-quality criteria, if concentrations of nutrients in subsurface drain outfalls to surface water are 

similar to those measured. Nutrient concentrations in tile-drain water were greater than the USEPA 

criteria for most samples (tables 5 and 6). Total nitrogen (organic+ ammonia nitrogen + nitrate + 

nitrite nitrogen) was greater than the recommended water-quality criterion for Ecoregion VI and the 

25th percentiles for all nitrogen forms for Level III Ecoregion #57 in all samples. Nitrate + nitrite 

nitrogen concentrations were the major contributor to the relatively high nitrogen concentrations. 

The median nitrate + nitrite nitrogen concentration for all samples was 43.55 mg/L (range, 21– 

55.3 mg/L). The median organic + ammonia nitrogen concentration for all samples was 0.89 mg/L 

(range, 0.51–9.8 mg/L). Among all samples and dates, only four did not have concentrations 

greater than the USEPA Ecoregion VI criterion, or the Level III Ecoregion # 57 25th percentiles for 

total phosphorus. The median total phosphorus concentration for all samples and all dates was 

0.195 mg/L (range, 0.03–3.53 mg/L). The median orthophosphorus concentration for all samples 

and dates was 0.091 mg/L (range, 0.024–1.54 mg/L). 

Although the field-trial results were inconclusive about the influence of LDME on tile-drain 

nutrient water quality in either the short or long term, field-trial results indicated significant 

concentrations of nutrients in tile water during the non-growing season, similar to previous studies 

(Gentry and others, 2007; Owens and Shipitalo, 2006). The field trial demonstrated that antecedent 

nutrient concentrations were relatively high in tile water and that a rainfall event exceeding 0.5 in. 

may result in increases in organic + ammonia nitrogen, ammonia nitrogen, orthophosphorus, and 

total phosphorous concentrations and decreases in nitrate + nitrite nitrogen concentrations in 

subsurface drains. Nitrate + nitrite nitrogen accounted for most of the nitrogen in tile water during 

the field trial. Nitrate concentrations in tile water are influenced by flow conditions and cropping 

strategies (Bakhsh and others, 2005). It is possible that nitrate + nitrite nitrogen concentrations do 

not remain as high throughout the year as those measured during the field trial, especially during 

the growing season, as noted by Bakhsh and others (2005). Flow rate or discharge of water from 

the subsurface drains was not measured, and nutrient concentrations were not determined at an 

outfall to surface water. It is therefore not possible to estimate the potential load of nutrients 

delivered to receiving waters. Loading rate for various nutrients may vary with tillage, season, and 

cropping strategy (Bakhsh and others, 2005; Schelde and others, 2006), and this type of treatment 

effect could not be determined by the field-trial design. Nevertheless, the field trial yielded 

important information about nutrient concentrations in tile drains during the non-growing season. 

21
Future studies of nutrient concentrations in tile drains would benefit from treatment replication, 

measurement of tile-drain discharge, and analysis of seasonal or crop effects. 

Chemical Indicators of LDME Transport to Subsurface Drains 

Various pharmaceutical, antibiotic, and wastewater chemical compounds (tables 1–1 and 

1–2) were analyzed for in a blank sample, in the LDME, in a pre-application tile-water sample, and 

in two categories of treatment tile-water samples. First, the occurrence of these chemicals was 

evaluated over time after LDME application for the NT8000 treatment. Second, the occurrence of 

the chemicals in tile water from all treatment plots and one control plot was evaluated at 6 days 

post-LDME application, after the first significant rainfall event. 

In all, 1 pharmaceutical, 4 antibiotics, and 12 wastewater chemicals (WWCs) were detected 

and quantified in the LDME (table 7). The potential sources of the antibiotics and WWCs detected 

in the LDME are listed in tables 1–1 and 1–2. The four antibiotics detected are commonly used in 

animal agriculture, so their detection is not unexpected. Analgesics such as ibuprofen may be used 

for animal comfort and to reduce inflammation. The WWCs detected in the LDME (table 7) could 

readily come from typical on-farm practices and sources. Specifically, the cleaning products might 

come from any type of cleaning procedure for which wash water is added to the LDME. The 

detergent degradates may also be included directly in a variety of cleaning fluids. The fecal sterols 

are products of animal digestion. 

None of the antibiotics detected in the LDME was detected in a treatment subsurface drain 

sample. Among the antibiotics detected in the LDME, previous research by others would indicate 

that oxytetracycline would bind tightly to the soil matrix, and that the sulfonamide antibiotics 

(sulfadimethoxine and sulfamethazine) would bind less strongly. The concentrations of antibiotics 

detected in the LDME are lower than concentrations observed, or artificially prepared, in other 

studies (Kay and others, 2004, 2005a, b; Burkhardt and others, 2005). Therefore, it is not surprising 

that the applied antibiotics were not detected in the subsurface drain samples. Tylosin, another 

antibiotic widely used in animal agriculture, binds tightly to soil (Boxall and others, 2002) and 

degrades rapidly (Schlüsener and Bester, 2006). Nevertheless, it was detected in the pre-application 

sample. The detection of tylosin in the pre-application sample may be the result of previous LDME 

applications to the field. 

No WWC detected in the LDME was quantified in NT8000 tile water at a concentration 

exceeding the reporting level. However, the analysis method can confirm the identity of a given 

compound at concentrations less than the reporting level, even if the quantification is uncertain 

(Zaugg and others, 2001). Several WWCs were detected in the NT8000 tile water at concentrations 

less than the reporting level. These are indicated by a “+” in table 7. 

One chemical detected in the LDME (phenol) was also detected in the field blank (table 8). 

Three chemicals (phenol, diethoxynonylphenol, and diethoxyoctylphenol) detected in the LDME 

also were detected in the pre-application sample, possibly reflecting previous LDME applications. 

Of the 12 WWCs detected in the LDME 2 chemicals (the fecal sterols cholesterol and indole) were 

detected in the NT8000 sample collected 1 day after LDME application, but not in a blank or preapplication sample. These chemicals might be inferred to indicate LDME transport from the current 

application to tile water. Subsequent NT8000 samples did not indicate clear evidence of any 

additional LDME-chemical transport. 

22
Table 7.  Results of antibiotic and wastewater chemical analyses for LDME, pre-application 

sample, and NT8000 treatment (Tile 6) over time. 

[LDME, liquid dairy manure effluent; PA, pre-application sample; ND, not detected; +, detected but not quantified 

at a concentration greater than the reporting level; concentrations in micrograms per liter] 

Compound name LDME PA 

Numbers of days post-LDME application for 

NT8000 treatment – Tile 6 

1 2 6 7 51 

Pharmaceuticals 

Ibuprofen 4.5 ND ND ND ND ND ND 

Antibiotics 

Sulfadimethoxine 1.9 ND ND ND ND ND ND 

Sulfamethazine 1.2 ND ND ND ND ND ND 

Oxytetracycline 1.6 ND ND ND ND ND ND 

Lincomycin 1.2 ND ND ND ND ND ND 

Tylosin ND 0.2 ND ND ND ND ND 

Wastewater chemicals 

Cleaning products 

Phenol 649 + + ND ND ND + 

Detergent degradates 

Monoethoxynonylphenol 

329 ND ND ND ND ND ND 

Diethoxy-nonylphenol 507 + + ND ND ND ND 

Monoethoxyoctylphenol 

18 ND ND ND ND ND ND 

Diethoxy-octylphenol 26 + ND ND ND ND ND 

4-nonylphenol ND + + ND ND ND + 

Fecal sterols 

Cholesterol 381 ND + ND ND ND ND 

Coprostanol 456 ND ND ND ND ND ND 

Indole 5.2 ND + ND ND ND ND 

Methyl indole 230 ND ND ND ND ND ND 

β-sitosterol 692 ND ND ND ND ND ND 

β-stigmastanol 1,070 ND ND ND ND ND ND 

Polycyclic aromatic hydrocarbons 

Anthracene ND ND ND ND ND ND + 

Benzo[a]pyrene ND ND ND ND ND ND + 

Fluoranthene ND ND ND ND ND ND + 

Phenanthrene ND ND ND ND ND ND + 

Pyrene ND ND ND ND ND ND + 

23
Table 7.  Results of antibiotic and wastewater chemical analyses for LDME, pre-application 

sample, and NT8000 treatment (Tile 6) over time.—Continued 

[LDME, liquid dairy manure effluent; PA, pre-application sample; ND, not detected; +, detected but not quantified 

at a concentration greater than the reporting level; concentrations in micrograms per liter] 

Compound name LDME PA 

Numbers of days post-LDME application for 

NT8000 treatment – Tile 6 

1 2 6 7 51 

Wastewater chemicals—Continued 

Other 

1,4 dichlorobenzene ND + ND + + + + 

Benzophenone ND ND ND + ND ND + 

DEET ND ND ND ND ND ND + 

Carbazole ND ND ND ND ND ND + 

HHCB ND ND ND ND ND ND + 

Isophorone ND ND ND ND ND ND + 

Menthol ND ND ND ND ND ND + 

Methyl salicylate 1.8 ND ND ND ND ND + 

Metolachlor ND ND + + + + ND 

Tributyl phosphate ND ND ND ND ND ND + 

Triphenyl phosphate ND ND ND ND ND + ND 

24
Table 8. Results of wastewater-chemical analyses for field blank and for all treatments 

after the first significant rainfall event. 

[Concentrations in micrograms per liter; NT, not tested; ND, not detected; +, detected but not quantified at a 

concentration greater than the reporting level] 

Compound name Field blank DD8000 

Tile 1 

DD4000 

Tile 2 

AW8000 

Tile 4 

AW4000 

Tile 5 

NT8000 

Tile 6 

NT4000 

Tile 7 

C2 

Tile 8 

Pharmaceuticals 

Ibuprofen NT ND ND ND ND ND ND ND 

Antibiotics 

Sulfadimethoxine NT ND ND ND ND ND ND ND 

Sulfamethazine NT ND ND ND ND ND ND ND 

Oxytetracycline NT ND ND ND ND ND ND ND 

Lincomycin NT ND ND ND ND ND ND ND 

Tylosin NT ND ND ND ND ND ND ND 

Wastewater chemicals 

Cleaning products 

Phenol + + ND ND ND ND ND ND 

Detergent degradates 

Mononethoxynonylphenol ND ND ND ND ND ND ND ND 

Diethoxy-nonylphenol ND ND ND ND ND ND ND ND 

Monoethoxyoctylphenol ND ND ND ND ND ND ND ND 

Diethoxy-octylphenol ND ND ND ND ND ND ND ND 

4-Nonylphenol ND ND ND ND ND ND ND ND 

Fecal sterols 

Cholesterol ND ND ND ND ND ND ND ND 

Coprostanol ND ND ND ND ND ND ND ND 

Indole ND ND ND ND ND ND ND ND 

Methyl indole ND ND ND ND ND ND ND ND 

β-Sitosterol ND ND ND ND ND ND ND ND 

β-Stigmastanol ND ND ND ND ND ND ND ND 

Other Compounds 

1,4-dichlorobenzene + + + + + + + + 

Acetophenone ND + ND ND ND ND ND ND 

Benzophenone ND + + ND + ND ND ND 

Methyl salicylate + ND ND ND ND ND ND ND 

Metolachlor ND ND ND + + + ND ND 

Triphenyl phosphate ND ND + ND ND ND ND ND 

25
A variety of other chemicals not detected in the applied LDME were detected in some 

subsequent NT8000 samples. Two of these additional chemicals (1,4-dichlorobenzene and DEET) 

also were detected in the field blank (table 8), again indicating potential contamination for these 

constituents. The other chemicals detected could feasibly come from some typical on-farm practice. 

In particular, the herbicide metolachlor is used on corn and soybeans, and its detection in 

subsurface drains would not be unexpected. Most of the other chemicals could be associated with 

cleaning products or possibly fuels. 

To evaluate whether rainfall might influence chemical transport from LDME to subsurface 

drains, the occurrence of antibiotics and WWCs was tested for all treatments at 6 days postapplication on November 8, 2006 (table 8). This sample date followed the first significant rainfall 

event. No LDME chemical except phenol (also detected in the field blank sample) was detected in 

any of the November 8 samples from any treatment. Additional WWCs not detected in the LDME 

were detected in some treatment samples. Of these, two also were detected in the field blank 

sample. 

The antibiotic and WWC results do not indicate a substantive contribution of the LDME to 

subsurface drain water over the course of the field trial, either through short-term preferential flow 

pathways or after the first significant rainfall event. This result is consistent with the nutrient results 

presented previously. It should be noted that the second rainfall event did influence nutrient 

concentrations (table 5); however, no antibiotics or WWCs were analyzed for that sample date. In 

addition, it is possible that the 1-day post-application sampling (for treatment NT8000, table 7) was 

not soon enough to detect immediate transport by preferential flow. Finally, many of these 

chemicals may bind tightly to soil particles, but tile-water analyses were done on filtered water 

samples. Nevertheless, field-trial results demonstrated that a variety of chemicals associated with 

LDME, or with other farming practices, may be present in subsurface drains and might serve as 

indicators of transport from the surface to tile drains. Further study of the factors affecting transport 

behavior of these chemicals including their persistence in soil and the conditions under which they 

are transported to subsurface drains is needed. 

Microbiological Indicators of LDME Transport to Subsurface Drains 

The LDME on the date of application was analyzed for microbiological indicators of 

LDME transport to subsurface drains. All indicators except the stx2 gene were detected in the 

LDME (table 9). The E. coli O157 serotype was detected in bacteria from the LDME by both an 

immunological analysis and a DNA-based analysis. The stx1 and eaeA genes also were detected in 

bacteria from the LDME. The stx1 gene has been associated with bovine sources but has rarely 

been associated with E. coli isolated from humans with gastrointestinal illness (Beutin and others, 

1993; Boerlin and others, 1999). The stx2 gene is frequently associated E. coli causing human 

illness. The stx2 gene was not detected in bacteria from the LDME. The eaeA gene is often found 

in E. coli bacteria in association with several other genes responsible for the symptoms of human 

gastrointestinal illness (Nataro and Kaper, 1998). The eaeA gene was detected in bacteria from the 

LDME. The Bacteroidetes bovine-specific genetic marker was also detected in DNA from the 

LDME. These results indicate that bacteria and genes often detected in cattle were present in the 

LDME. The presence of the most pathogenic form of E. coli, E. coli O157:H7 carrying the 

stx2 gene, is not indicated by these data. 

26
Table 9. Results of analyses for microbiological indicators in subsurface drain water for NT8000 

treatment before and after addition of liquid dairy manure effluent. 

[LDME, liquid dairy manure effluent; PA, pre-application sample; ND, not detected; +, detected; IT, immunological 

test; DT, DNA-based test; NT, not tested – no E. coli were present] 

LDME PA Time elapsed since LDME application for NT8000 Treatment–Tile 6 

4 hours 1 day 2 days 6 days 7 days 14 days 

E. coli O157 - IT + ND NT NT NT NT NT NT 

E. coli O157 - DT + ND ND ND ND ND ND ND 

eaeA gene + ND + + + + NT + 

stx1 gene + ND + ND ND ND ND ND 

stx2 gene ND ND ND ND ND ND ND ND 

Bacteroidetes marker + ND ND ND ND ND ND ND 

Microbiological analyses indicate a shift in the types of E. coli present in the NT8000 tile 

after LDME application (table 9). Subsurface drain water for the NT8000 treatment was analyzed 

before LDME application and at 4 hours, and 1, 2, 6, 7 and 14 days post-application (table 9). The 

eaeA gene was detected in every NT8000 post-application sample in which E. coli were detected. 

The eaeA gene was not detected in pre-application tile water for the NT8000 treatment, even 

though E. coli were present in pre-application tile water. In addition, the stx1 gene, often found in 

STEC E. coli from cattle and present in the LDME, was detected at 4 hours post-application in 

NT8000 tile water but not in NT8000 tile water before application. The NT8000 tile also contained 

greater concentrations of E. coli after LDME application, than before application (table 4). 

However, the bovine-Bacteroidetes marker was not detected in any NT8000 sample. The test for 

the Bacteroidetes marker is done on DNA isolated directly from the water sample and is less 

sensitive than the test for E. coli genes. The test for the E. coli genes is done on bacteria grown 

from the water sample. The growth process creates greater numbers of the bacteria carrying the 

genes, so the potential for detecting the genes is enhanced over the direct-DNA-extraction method. 

In addition, Bacteroidetes are believed to die off very quickly in the environment. These field-trial 

results indicate that large numbers of Bacteroidetes bacteria from LDME application were not 

transported to subsurface drain water for the NT8000 treatment, either within 4 hours postapplication or over longer time periods. 

To evaluate whether rainfall might influence LDME transport to subsurface drains, tile 

water from all treatments was analyzed for all indicators before LDME application, and at 6 days 

post-application, after the first rainfall event (table 10). Among the pre-application drain-water 

samples, only the eaeA gene was detected, and only for one treatment (DD4000, table 10). 

However, at 6 days after application and the rainfall event, the eaeA gene was detected in bacteria 

from subsurface drain water from 4 of the 8 treatment plots: DD8000, DD4000, AW8000, and 

NT8000. The eaeA gene was absent in bacteria from subsurface drain water for each of the Control 

plots. These results might indicate transport of bacteria associated with the applied LDME to the 

subsurface drains after the rainfall event at 6 days post-application; however, no other indicators of 

LDME bacteria were detected. The bovine-Bacteroidetes marker was not detected in any sample. 

However, even though the stx2 gene was not detected in the LDME, it was detected in tile water 

from the AW8000 treatment at 6 days post-application, after the rainfall event. Taken together, 

these results do not clearly indicate whether the microbiological indicators of LDME contamination 

detected in drain water were from the current LDME application. As noted previously, E. coli may 

27
persist from prior manure applications (Jamieson and others, 2002). The detection of the eaeA gene 

in a pre-application tile-water sample and the detection of the stx2 gene in tile water when it was 

not detected in the LDME could indicate a persistent population of E. coli in soils of the treatment 

plots. Nevertheless, tile water did contain some microbiological indicators of bacteria from 

probable bovine sources. These indicators may prove useful in future studies, but much more 

information needs to be obtained regarding bacteria and gene persistence and transport through 

these soils. 

Table 10. Results of analyses for microbiological indicators in LDME and all treatments before 

LDME application and at 6 days post-application. 

[LDME, liquid dairy manure effluent; ND, not detected; +, detected; DT, DNA-based test] 

Treatment 

DD8000 DD4000 AW8000 AW4000 NT8000 NT4000 C1 C2 

Before application 

E. coli O157 - DT ND ND ND ND ND ND ND ND 

eaeA gene ND + ND ND ND ND ND ND 

stx1 gene ND ND ND ND ND ND ND ND 

stx2 gene ND ND ND ND ND ND ND ND 

Bacteroidetes marker ND ND ND ND ND ND ND ND 

6 days post-application 

E. coli O157 - DT ND ND ND ND ND ND ND ND 

eaeA gene + + + ND + ND ND ND 

stx1 gene ND ND ND ND ND ND ND ND 

stx2 gene ND ND + ND ND ND ND ND 

Bacteroidetes marker ND ND ND ND ND ND ND ND 

Study Limitations 

This field trial was done only during the non-growing season; nutrient concentrations might 

be different during the agricultural growing season, which also would be the time when algal 

growth in receiving streams would be most readily influenced by soluble nutrients. In addition, 

flow rate or discharge of water from the subsurface drains was not measured. It is therefore 

impossible to estimate the potential load of nutrients or bacteria delivered to receiving waters. 

Loading rate for various nutrients may vary with tillage, season, and cropping strategy (Bakhsh and 

others, 2005; Schelde and others, 2006), and this type of treatment effect could not be determined 

by the field-trial design. This field trial did not involve replication of treatments, so caution in 

interpreting results is warranted. In addition, the first post-application samples were collected at 

about 4 hours, and this may have been too long to observe very rapid losses to tile drains, if these 

did indeed occur. Chemical and microbiological indicators of potential LDME transport to 

subsurface drains were analyzed only on selected samples. A more complete analysis of all 

samples, which was not economically practical for this field trial, might have clarified subtle 

patterns of occurrence in the selected sample set. 

Future studies would benefit from treatment replication so that statistical significance of 

differences in concentrations of nutrients or bacteria could be evaluated. In addition, measurement 

of tile-drain discharge and enhanced sampling in the initial time period after LDME application 

would be beneficial. Future studies might benefit from the addition of dyes or inorganic tracers, 

28
such as bromide, to the LDME before application to help separate the influence of residual 

chemicals or bacteria surviving from previous applications from the influence of the newly applied 

LDME. Finally, continuations of similar studies over multiple seasons and crop rotations would 

help to determine the importance of these variables. 

Summary and Conclusions 

A field trial was done in the Upper Tiffin River Watershed, in southeastern Michigan, to 

determine the influence of liquid dairy manure effluent (LDME) management practices on the 

quality of agricultural subsurface-drain water. Samples from subsurface drains were analyzed for 

nutrients, fecal-coliform and Escherichia coli (E. coli) bacteria, antibiotics, chemicals typically 

detected in wastewater, and the occurrence of genes indicating the presence of shiga-toxinproducing E. coli, or of bovine-specific Bacteroidetes bacteria. Samples were collected from 

November 2, 2006, to March 20, 2007, from subsurface drains under eight field plots that received 

no LDME and no tillage (two Control plots), or received 4,000 or 8,000 gallons per acre (gal/acre) 

of LDME after no tillage, or after either of two different types of tillage (six Treatment plots). The 

two types of tillage tested were (1) ground-driven, rotary, subsurface cultivation, and (2) rollingtine aeration. 

The purpose of the field trial was to obtain initial information on whether manure 

management practices can affect the transport of nutrients and bacteria from LDME to subsurface 

drains. Water samples were collected from the eight subsurface drains before LDME application 

and at 4 hours, and 1, 2, 6, 7, and 14 days post-application. Samples collected at 4 hours and 1 day 

post-application were evaluated for evidence of immediate transport of nutrients and bacteria by 

potential preferential flow pathways. Samples at 6 and 14 days post-application were timed to 

follow rainfall. In addition, the potential of wastewater chemicals and bacterial genes as indicators 

of LDME transport to subsurface drains was evaluated. The occurrence of antibiotics, wastewater 

chemicals, and bacterial genes was evaluated at 4 hours, and 1, 2, 6, 7, and 14 days post-application 

in subsurface-drain water from the plot receiving 8,000 gal/acre of LDME with no tillage 

(NT8000). In addition, all drains were analyzed for these constituents both before LDME 

application and at 6 days post-application, after the first significant rainfall event. 

Nutrient concentrations were high in tile-drain water throughout the field-trial period. In all, 

56 drain-water samples were analyzed. Of these, 53 samples exceeded the U.S. Environmental 

Protection Agency (USEPA) water-quality criteria for total phosphorus, and all samples exceeded 

the total nitrogen criterion for Ecoregion VI (Corn Belt). Nitrate + nitrite nitrogen concentrations 

exceeded 20 mg/L for every sample and contributed most to the total nitrogen concentrations. 

These results include eight samples collected before LDME application as well as two control 

samples for each date, representing drain water from plots that received no LDME and no tillage. 

Therefore, the high nutrient concentrations measured were not a result of the LDME application 

during the field trial and reflect antecedent conditions and prior management practices. The 

management practices tested during the field trial did not affect the nutrient concentrations in 

subsurface drain water during the field-trial period. The field trial was done during the nongrowing season only. As shown by others (Bakhsh and others, 2005; Schelde and others, 2006) 

nutrient concentrations in subsurface drains may vary with season and crop. 

Nutrient concentrations did not indicate an effect of LDME application on tile-water quality 

either immediately after application or after subsequent rainfall events. Significant rainfall did, 

however, influence nutrient concentrations in tile water. After approximately 0.84 in. of rainfall in 

24 hours, organic + ammonia nitrogen, orthophosphorus, and total phosphorus concentrations 

29
increased above average levels for all prior sampling dates in every treatment tile. In addition, after 

this rainfall event, ammonia nitrogen was present in every tile, including Controls, whereas it was 

not typically present in every tile on prior sampling dates. Finally, nitrate + nitrite nitrogen 

concentrations decreased in every tile, including Controls. Because similar changes occurred in 

tiles draining plots to which LDME was, or was not applied, nutrient patterns after rainfall cannot 

be attributed to the LDME application. 

E. coli concentrations exceeded the USEPA recreational-water-quality single-sample 

criterion of 235 CFU/100 mL in only 3 of 56 samples. Of these three samples, two were from the 

NT8000 treatment at 4 hours and 1 day post-LDME application. The NT8000 treatment was the 

only treatment for which post-application E. coli concentrations increased to levels exceeding 

water-quality standards immediately after LDME application. The NT8000 treatment also was the 

only treatment for which ammonia was detected at 4 hours, 1 day, and 2 days post-application. The 

changes in subsurface-drain-water microbiology and nutrient chemistry immediately after the 

LDME application may indicate transport of LDME to the tile drain for this treatment. No other 

treatment indicated any immediate change in nutrient or bacteria concentrations after LDME 

application. The third sample for which E. coli concentrations exceeded the single-sample standard 

was the AW8000 treatment after the first significant rainfall event. There was no additional 

evidence of rainfall-mediated movement of E. coli bacteria to any tile drain. 

Both chemical and microbiological indicators of possible LDME transport to subsurface 

drains were analyzed in the LDME, in a pre-application tile-water sample and in two categories of 

treatment tile-water samples. First, the occurrence of these indicators was evaluated over time after 

LDME application for the NT8000 treatment. Two bacterial genes (eaeA and stx1) and two fecal 

sterol chemicals, found in the LDME, but not detected in the pre-application NT8000 tile-water 

sample or in a field blank, were detected in water samples from the 4-hour or 1-day postapplication NT8000 treatment. These findings may indicate rapid transport of the LDME to tile 

water for this treatment, and they are consistent with a large increase in bacteria concentrations 

after LDME application for this treatment. Only the NT8000 treatment was analyzed for chemical 

or microbiological indicators immediately after LDME application; therefore, general patterns of 

chemical or microbiological indicator occurrence in the short term after LDME application cannot 

be evaluated. Nevertheless, results for the NT8000 treatment indicate some potential for the use of 

chemical or microbiological indicators to track preferential flow of LDME to subsurface drains. In 

addition, other chemicals not found in the LDME but possibly associated with farming practices 

(such as the herbicide metolachlor) were detected in some samples. These chemicals also indicate 

transport from surface application to subsurface drains, even though they may not specifically 

indicate LDME. 

The influence of rainfall on the occurrence of these chemical and microbiological indicators 

in tile water also was evaluated. Tile water from all treatment plots and one control plot was 

evaluated pre-LDME application and at 6 days post-LDME application, after the first significant 

rainfall event. No chemical present in the LDME but not in the blank or pre-application samples 

was detected. However, after the rainfall event, the eaeA gene, present in the LDME, was detected 

in tiles draining three plots to which LDME was applied but was not detected in these tiles before 

application. The eaeA gene was not detected in tiles draining control plots. 

Two chemicals and one microbiological indicator of LDME transport also were present in 

tile-water samples collected before the LDME was applied. For one treatment (DD4000), the eaeA 

gene was present both pre-application and after the rainfall event. In addition, the stx2 gene, not 

present in the LDME, was detected in drain water from the AW8000 treatment after the rainfall 

30
event. Chemicals may bind tightly to, or leach slowly from, soil. E. coli may persist from prior 

manure applications (Jamieson and others, 2002). This information may explain the presence of 

LDME chemicals or of E. coli genes in pre-application tile samples or the presence of the genes or 

chemicals in tile-water samples when they were not detected in current-application LDME. 

Although chemical and microbiological indicators of LDME transport to subsurface drains were 

somewhat equivocal with respect to the influence of LDME in tile-drain water quality, the 

detection of indicators in tile water confirms that they may be useful in future studies. However, 

much more needs to be known about the factors that influence their persistence and fate in soil and 

subsurface drainage. 

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34
35

Appendix 1 

_____________________________________ 
Table 1–1.  Wastewater-method compound names, U.S. Geological Survey National Water 

Quality Laboratory reporting limits, and possible compound uses or sources. 

[RL, Laboratory reporting limit; reporting limits in micrograms per liter; PAH, polycyclic aromatic hydrocarbon; 

possible use or sources from Zaugg and others, 2001] 

 Analyte Synonym RL Possible use or sources 

1,4-Dichlorobenzene 0.5 Moth Repellant, fumigant, deodorant 

1-Methylnaphthalene .5 2-5 percent of gasoline, diesel fuel or crude oil, 

pesticide adjuvant 

2,6-Dimethylnaphthalene .5 Present in diesel/kerosene (trace in gasoline) 

2-Methylnaphthalene .5 2-5 percent of gasoline, diesel fuel or crude oil, 

pesticide adjuvant 

3-beta-Coprostanol 2 Carnivore fecal indicator 

3-Methyl-1(H)-indole Skatole 1 Fragrance, stench in feces and coal tar 

3-tert-Butyl-4-hydroxy anisole BHA 5 Antioxidant, general preservative 

4-Cumylphenol 1 Nonionic detergent metabolite 

4-n-Octylphenol 1 Nonionic detergent metabolite 

4-tert-Octylphenol 1 Nonionic detergent metabolite 

5-Methyl-1H-benzotriazole 2 Antioxidant in antifreeze and deicers 

Acetophenone .5 Fragrance in detergent and tobacco, flavor in 

beverages 

Acetyl hexamethyl 

tetrahydronaphthalene 

AHTN .5 Musk fragrance (widespread) 

Anthracene .5 Wood preservative, component of tar, diesel or 

crude oil 

9,10-Anthraquinone .5 Manufacturing of dye/textiles, seed treatment, bird 

repellant 

Benzo[a]pyrene Benz[a]pyrene .5 Regulated PAH, used in cancer research 

Benzophenone .5 Fixative for perfumes and soaps 

beta-Sitosterol 2 Plant sterol 

beta-Stigmastanol Stigmastanol 2 Plant sterol 

Bisphenol A 1 Manufacturing of polycarbonate resins, antioxidant, 

flame retardant 

Bromacil .5 Herbicide, noncrop usage 

Tribromomethane Bromoform .5 Wastewater ozonation byproduct, 

military/explosives 

Caffeine .5 Beverages, diuretic 

Camphor .5 Flavor, odorant, ointments 

Carbaryl 1 Insecticide, crop and garden use 

Carbazole .5 Manufacturing of dyes, explosives and lubricants 

Chlorpyrifos .5 Insecticide, termite and pest control 

Cholesterol 2 Fecal indicator, plant sterol 

Cotinine 1 Primary nicotine metabolite 

36
Table 1–1.  Wastewater-method compound names, U.S. Geological Survey National Water 

Quality Laboratory reporting limits, and possible compound uses or source.—Continued 

[RL, Laboratory reporting limit; reporting limits in micrograms per liter; PAH, polycyclic aromatic hydrocarbon; 

possible use or sources from Zaugg and others, 2001] 

 Analyte Synonym RL Possible Use or Sources 

Diazinon 0.5 Insecticide, ants and flies 

Dichlorvos 1 Insecticide, pet collars 

d-Limonene .5 Fungicide, antimicrobial, antiviral, fragrance 

Fluoranthene .5 Component of coal tar and asphalt 

Hexahydrohexamethylcyclopentabenzopyran HHCB .5 Musk fragrance 

Indole .5 Pesticide inert ingredient, fragrance in coffee 

Isoborneol .5 Fragrance in perfume 

Isophorone .5 Solvent for lacquer, plastic, oil, silicone and resin 

and some pesticides 

Isopropylbenzene Cumene .5 Manufacturing of phenol/acetone, fuels and paint 

thinner 

Isoquinoline .5 Flavors and fragrances 

Menthol .5 Cigarettes, cough drops, liniment, mouthwash 

Metalaxyl .5 Herbicide, fungicide, general use pesticide 

Methyl salicylate .5 Liniment, food, beverage, sun block 

4-Nonylphenol, total para-Nonylphenol 5 Nonionic detergent metabolite, pesticide adjuvant 

Metolachlor .5 Herbicide 

N,N-Diethyl-meta-toluamide DEET .5 Insect repellant, urban use on mosquitoes 

Naphthalene .5 Fumigant, moth repellent, component of gasoline 

Nonylphenol, diethoxy- (total) NPEO2 5 Nonionic detergent, pesticide adjuvant 

Nonylphenol, monoethoxy- (total) NPEO1 5 Nonionic detergent, pesticide adjuvant 

Octylphenol, diethoxy- OPEO2 1 Nonionic detergent 

Octylphenol, monoethoxy- OPEO1 1 Nonionic detergent metabolite 

para-Cresol 1 Wood preservative 

Pentachlorophenol 2 Herbicide, fungicide, wood preservative, termite 

control 

Phenanthrene .5 Manufacturing of explosives, component of tar, 

diesel and crude oil 

Phenol .5 Disinfectant, used in the manufacturing of many 

products 

Prometon .5 Herbicide, noncrop, before blacktop 

Pyrene .5 Component of coal tar and asphalt 

Tetrachloroethylene PCE .5 Solvent, degreaser, veterinary anthelmintic 

Tri(2-butoxyethyl)phosphate .5 Flame retardant 

Tri(2-chloroethyl)phosphate .5 Plasticizer, flame retardant 

Tri(dichlorisopropyl)phosphate .5 Flame retardant 

Tributyl phosphate .5 Antifoaming agent, flame retardant 

Triclosan 1 Disinfectant, antimicrobial 

Triethyl citrate Ethyl citrate .5 Cosmetics and pharmaceuticals 

Triphenyl phosphate .5 Plasticizer, resin wax, roofing paper 

37
38

Table 1–2.  Antibiotic analytes by liquid 

chromatography/mass spectrometry. 

[CAS, Chemical Abstract Service; RL, laboratory reporting 

level in micrograms per liter] 

 Compound name CAS number RL 

Pharmaceuticals 

Carbamazepine 61336-70-7 0.005 

Ibuprofen 69-53-4 .005 

Macrolides 

Anhydro-erythromycin -- .008 

Erythromycin 114-07-8 .008 

Roxithromycin 80214-83-1 .005 

Tylosin 1401-69-0 .005 

Virginiamycin 11006-76-1 .005 

Quinolones 

Ciprofloxacin 85721-33-1 .005 

Enrofloxacin -- .005 

Lomefloxacin 98079-51-7 .005 

Norfloxacin 70458-96-7 .005 

Ofloxacin 83380-47-6 .005 

Sarafloxacin 98105-99-8 .005 

Sulfonamides 

Sulfachlorpyridazine 80-32-0 .005 

Sulfadiazine 68-35-9 .050 

Sulfadimethoxine 122-11-2 .005 

Sulfamethazine 57-68-1 .005 

Sulfamethoxazole 723-46-6 .005 

Sulfathiazole 72-14-0 .020 

Tetracyclines 

Chlorotetracycline 57-62-5 .010 

Anhydrochlorotetracycline 4497-08-9 .010 

Doxycycline 564-25-0 .010 

Oxytetracycline 79-57-2 .010 

Tetracycline 64-75-5 .010 

Anhydro-tetracycline 13803-65-1 .010 

Other 

Lincomycin 154-21-2 .005 

Ormetoprim 6981-18-6 .005 

Trimethoprim 738-70-5 .005