Environ Monit Assess DOI 10.1007/s10661-014-3683-z

A new sampler for stratified lagoon chemical and microbiological assessments M. R. McLaughlin & J. P. Brooks & A. Adeli

Received: 7 June 2013 / Accepted: 3 February 2014 # Springer International Publishing Switzerland (outside the USA) 2014

Abstract A sampler was needed for a spatial and temporal study of microbial and chemical stratification in a large swine manure lagoon that was known to contain zoonotic bacteria. Conventional samplers were limited to collections of surface water samples near the bank or required a manned boat. A new sampler was developed to allow simultaneous collection of multiple samples at different depths, up to 2.3 m, without a manned boat. The sampler was tethered for stability, used remote control (RC) for sample collection, and accommodated rapid replacement of sterile tubing modules and sample containers. The sampler comprised a PVC pontoon with acrylic deck and watertight enclosures, for a 12 VDC gearmotor, to operate the collection module, and vacuum system, to draw samples into reusable autoclavable tubing and 250-mL bottles. Although designed primarily for water samples, the sampler was easily modified to collect sludge. The sampler held a stable position during deployment, created minimal disturbance in the water Journal article number J-12192 of the Mississippi Agricultural and Forestry Experiment Station. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable. This work was prepared by employees of the US Government as part of their official duties and is in the public domain and may be used without further permission. M. R. McLaughlin (*) : J. P. Brooks : A. Adeli United States Department of Agriculture, Agricultural Research Service, Genetics and Precision Agriculture Research Unit, P.O. Box 5367, Mississippi State, MS 39762-5367, USA e-mail: [email protected]

column, and was readily cleaned and sanitized for transport. The sampler was field tested initially in a shallow fresh water lake and subsequently in a swine manure treatment lagoon. Analyses of water samples from the lagoon tests showed that chemical and bacterial levels, pH, and EC did not differ between 0.04, 0.47, and 1.0 m depths, but some chemical and bacterial levels differed between winter and spring collections. These results demonstrated the utility of the sampler and suggested that future manure lagoon studies employ fewer or different depths and more sampling dates. Keywords Bacteria . Lagoon . Microbes . Nutrients . Remote control sampler . Water Introduction Lagoons are used worldwide for treatment of domestic sewage and animal wastes (Keffala et al. 2013). In the Mid-South USA, anaerobic lagoons (ASAE Standards 2011) are the predominant manure treatment technology used by confined swine feeding operations (McLaughlin et al. 2009). Chemical components, nutrients, and bacterial loads may be stratified in the water column of anaerobic swine manure lagoons (Lovanh, et al. 2009; Cook et al. 2010). Potential stratification should be considered when characterizing nutrient and bacterial loads for development of appropriate lagoon management options. Chemical, nutrient, and bacterial levels in swine manure treatment lagoons in Mississippi in the Mid-South USA have been routinely characterized from shallow lagoon surface water samples

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collected near the edges of lagoons (McLaughlin et al. 2009), and stratified water sample data were not available from these lagoons. The need to obtain data on spatial and temporal changes in chemical, nutrient, and bacterial levels in lagoon water in the Mid-South required that a different method of water sample collection be developed. A method enabling collection and comparison of samples from different depths in the water column necessarily requires contending with deeper water further from the bank. Conventional means of manual sample collection from a boat carry inherent risk and may disturb water column stratification. Water sample collections from manure treatment lagoons also carry added risk, from potential exposure to zoonotic pathogens. Bacterial species including Clostridium perfringens, Escherichia coli, Campylobacter spp., Listeria spp., and Salmonella spp. are known to occur in Mid-South swine lagoons (McLaughlin and Brooks 2009; McLaughlin et al. 2009). Various samplers have been developed and used for assessing either water quality or sludge buildup in manure treatment lagoons (Mitchell and Dickey 1973; Kroes, et al. 1987; Miller and McGhee 2011). In addition to samplers, several remotely controlled selfpropelled unmanned boats have been developed for mapping sludge and water quality profiles (Raman et al. 2004; National Hog Farmer 2005; Robert 2006; Singh et al. 2008; Kaizu et al. 2011). These unmanned boats, some incorporating global positioning system receivers, and sonar technologies, provided a safer alternative to conventional methods which used gridbased manual measurements and required two people in a flat-bottomed boat (Westerman et al. 2008; Classen et al. 2011). These remotely controlled unmanned boats utilized either propeller-driven or airboat designs; the former disturbs stratification and is subject to fouling (Singh et al. 2008), and the latter is difficult to hold in a stationary position (Kaizu et al. 2011). Other studies have utilized moored stationary or cable and pulleyoperated floating platforms for sampling water quality and gas emissions and for monitoring meteorological conditions (Mitchell and Dickey 1973; Aneja et al. 2000; Harper et al. 2000, 2004; DeSutter and Ham 2005; Lovanh et al. 2009). Water sample collectors incorporated into these and other protocols often utilized commercially available water bottle-type remotely actuated closed drop samplers (Johnson 1995) that were lowered and raised

manually or by remotely controlled winches and subsequently required manual emptying or replacement between samples and locations (Mitchell and Dickey 1973; Chen et al. 2003; Harper et al. 2004; Raman et al. 2004: Miller and McGhee 2011). Some collectors utilized peristaltic pumping systems to draw lagoon water samples (Loughrin et al. 2010), whereas others utilized vacuum systems ranging from single pipette bulbs (up to 7 mL capacity) to hand-pumped systems capable of collecting larger (up to 250 mL) volumes (McLaughlin et al. 2006, 2009). The pumping and vacuum systems also required emptying or replacement of sample containers between samples, but because the respective containers were relatively inexpensive compared with commercial drop samplers, replacement was practical, indeed critical in avoiding contamination of microbiological samples. The goal of the present study was to assess stratification of bacteria and nutrients in a 1-m water column of a relatively shallow lagoon, where previous studies had used only surface water samples to partially characterize the bacterial, chemical, and nutrient quality of the water. The objective of the present study was to develop a new water sampler capable of simultaneous collection of shallow lagoon water samples at multiple depths, from near the surface to near the sludge layer, at the same location in the lagoon, without redeployment between samples, and without requiring a manned boat. Requirements for the sampler also included: a capacity for water sample collections without using multiple, expensive, commercial water samplers; capacity to hold a stable position during sample collection with minimal disturbance to the water column during deployment and operation; and capacity to be readily transported, deployed, and sanitized.

Methods Sampler design Earlier studies employed hand-operated samplers for collecting single samples of surface water (Fig. 1). The “dipper” (Fig. 1a, c) had a capacity of up to 7 mL (McLaughlin et al. 2006), and the “floater” (Fig. 1b, d) up to 250 mL (McLaughlin et al. 2009). The dipper, made by replacing the blade on a tree pruning tool with opposing aluminum T (┤├) jaws and attaching a springloaded burette clamp to hold a pipette, was suspended

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over the lagoon surface and operated using the rope to close and open the T jaws. The floater comprised a PVC float and paint roller fitted with a stainless steel laboratory shaker clamp to hold the sample bottle. A sterile glass collection tube clamped to the float drew water into sterile tubing and a Luer fitting on the sample bottle, using vacuum pressure from a microbiologically filtered vacuum line, and a hand vacuum pump. Concepts developed in these hand-operated samplers were incorporated in the design of the new sampler, including: positional stability through extendable tethered control, rather than free floating and self-propelled; a vacuum system for drawing samples; and capacity for rapid and easy replacement of (inexpensive) sample containers and tubing. Additional concepts that were incorporated in the new sampler were adopted from others as described earlier, including: wireless electronic remote control of sample collection modules and sonar capability for assessing water depth and possibly sludge depth. Translucent acrylic sheet material (1.27 cm thick, 96.5 cm long, and 61 cm wide) was used for the deck

because it offered rigidity, stability, visibility, a smooth surface for easier cleaning, and suitable machining characteristics for the cuts and drilling that were required in securing modules and sample bottles. All modules were assembled, and the respective enclosures and other components positioned on the deck to optimize and coordinate their individual and combined functions and to balance the total weight (front to back and side to side) before attachment to the deck. Balance was achieved on a solid flat surface by balancing the deck on a 21-mm o.d. pipe alternately positioned longitudinally or horizontally, at the center lines under the deck and placing the modules and components on top of the deck to attain equilibrium front to back and side to side, respectively. The power and vacuum/remote control module enclosures were set tightly into openings cut in the deck to secure them and to lower the center of gravity of the completed platform. The weight of the deck was determined and flotation required for a pontoon to support the deck was estimated using the equation for displacement of a cylinder:

M d ¼ Lπr2 fR where M d ¼ the mass ðkgÞ of displacement; L ¼ the length ðcmÞ of the ðpipeÞ cylinder ðignoring differences in curved and straight sectionsÞ; r ¼ the radius ðcmÞ of the pipe;  f ¼ a conversion factor 1 m3 106 cm−3 ; and  R ¼ the density of fresh water 998:2 kg m−3 at 20¨C :

Electronic and pneumatic components for the new sampler (Table 1) were assembled into modules comprising the power source (battery, main power switch, and indicator lamp), the vacuum system (pump, gauge, vacuum switch, relay, solenoid valves, and manifold), the sample depth system (gearmotor and relay), and the remote control system (receiver, antenna, and indicator light panel). Modules were enclosed separately or together, in the case of the vacuum and remote control systems, in watertight plastic enclosures with clear plastic lids (Fig. 2). Enclosure bulkhead penetrations for vacuum ports, strain relief connectors for electrical wiring, and the gearmotor shaft were sealed to be watertight. It was determined that a pontoon made of 15.24 cm i.d. ASTM D3034 PVC sewer pipe (four cylinders,

ð1Þ

44.5 cm each on two sides and 30.5 cm each on two ends, connected by four ¼ bend ASTM D3034 PVC elbows at the corners) was sufficient to float the completed sampler. The deck was secured to the pontoon by four (two on each side) UV-resistant heavyduty plastic cable ties (Fig. 2). Support arms for sample collection tubing were measured and marked, and the tubing attached so that each support arm assembly could be rapidly and accurately clamped to the gearmotor shaft to provide a precise depth control for each sample at full vertical deployment. A visual marker was added to each sample collection support arm, in the form of a flag made of colored tape affixed to the top end of each arm (Fig. 2d). A sonar transducer (Table 1) was clamped just below the water line at the inside front of the pontoon (Fig. 2). The

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Fig. 1 Hand-operated samplers on extendable poles: a, c “Dipper” and b, d “Floater”

transducer signal was communicated via a sealed extension cable taped to the tether and connected to a receiving unit (Fishfinder) housed in a handheld plastic toolbox (Fig. 2a) on the bank. The sonar unit displayed water depth and temperature data on a grayscale liquid crystal screen. The remote control system comprised an 8-channel 9-VDC transmitter and 12-VDC receiver programmed to “flip-flop” so that pressing once on a control switch of the transmitter signaled the respective single-pole double-throw (SPDT) relay in the receiver to close, thus supplying power to its respective component module. A subsequent press of the respective transmitter control switch reversed the previous action, cutting power to the respective component. Six remote control channels were used in the present study (Table 2). The gearmotor module also utilized a double-pole double-throw (DPDT) relay to reverse DC direction and gearmotor shaft rotation (Fig. 3a) in lowering and raising the support arm and tubing assembly (Fig. 3b). The reversible gearmotor was controlled using SPDT and DPDT relays with common (C), normally closed (NC), and normally open (NO) contacts. Nonenergized

positions are shown as solid arrows and energized positions as dashed arrows in the figures. Transmitter channels were set to flip-flop, throwing respective receiver relay circuits with each press of the button. Separately controlled vacuum lines attached to different lengths of sample collection tubing on the sampling arm (Fig. 3b) allowed independent and simultaneous collection of different depth samples. To accomplish multiple sample collections at defined depths with minimal disturbance to the water column, support arms for attachment to the shaft of the reversible gearmotor were made initially from 1.27 cm diameter polypropylene rod and later from 13 mm i.d.×1.5 m long crosslinked polyethylene (PEX) pipe. The support arm assembly was held by a burette clamp attached to the gearmotor shaft. This enabled rapid replacement of used support arm/collection tubing with pre-assembled sterile modules in the field. The gearmotor was operated by remote control to rotate (0.59 rpm) the support arm counterclockwise or clockwise to lower or raise the arm, respectively (Fig. 3). When the arm was rotated into the water, collection tubing (4.8 mm i.d. silicone in appropriate lengths), with open ends fixed at different

Environ Monit Assess Table 1 Electronic and pneumatic components used in building the remotely controlled sampler

Component

Part number

Source

555110

R&D Batteries, Inc., Burnsville, MN

Battery 12 VDC, 12 A h−1, sealed, rechargeable Battery charger N CON Systems 15-515

Cat-153948

Fisher Scientific Co., Pittsburgh, PA

Enclosures 10X8X6 clear hinge PVC

NSN-TL1G2461475

GSA Global Supply, Arlington, VA

8X6X4 clear hinge PVC

G2461475

6X6X4 clear hinge PVC

G2461467

Gearmotor Dayton 12 VDC, 0.45 rpm

1LNG3

W.W. Grainger, Lake Forest, IL

01230 01231

Remote Control Technology, Kirkland, WA

Part-91F5541 Part-13M2991

Newark, Chicago, IL

25197 99-91

Argon Office Supplies, Inc., San Bruno, CA

Part-MM-210 Part-WG-151

Reid Supply Company, Muskegon, MI

BR420-1903

VWR International, LLC., Suwanee, GA

MSC-5649918 MSC-07420987

MSC Industrial Supply Co, Inc., Melville, NY

RC components 9 VDC 8-ch 27 MHz transmitter 12 VDC 8-ch 27 MHz receiver antenna, cable, mount, power supply Relays, DPDT 12 VDC, IDEC RR2P-ULDC12V 8-pin relay socket, IDEC SR2P-06 Sonar Eagle Cuda 242 Fishfinder Lowrance extension cable Vacuum manifold and gauge Splitter, 1/4 in. NPT ports, 3/8 in. NPT inlet 30 in. HG/0 ga, 50 mm (2 in.) d¼in. NPT Vacuum pump 12 VDC Various parts Switch NV-2C-8 J max 83kPa (12 psi) Solenoid valves, NC, 12 VDC, 3-way Norgren 10–32 pneumatic fittings ½" MPT strain relief connectors

positions on the arm, provided depth precision (Fig. 3b). Collection tubing was fixed to the support arm with masking tape to enable rapid assembly and disassembly. Sanitized support arm and autoclave-sterilized tubing assemblies were prepared in the laboratory. In the field, used assemblies (support arm and collection tubing) were replaced with fresh assemblies between collection sites at the lagoon. Collection tube inlets were open, whereas outlets were terminated with female Luer fittings complementary to male Luer fittings in the lids of sample bottles. Wide-mouth sample bottles (250 mL sterile polypropylene) were secured using laboratory shaker platform clamps bolted to the deck (Fig. 2). The vacuum source line and pump were protected from possible microbial aerosol contamination by a 0.22-μm

inline filter (Fig. 4b) and by liquid traps (a second 250-mL polypropylene bottle) in each collection line (Fig. 2f). Samples were drawn into the collection tubing by a remotely controlled vacuum pump operating at approximately −50 kPa (Fig. 4). The vacuum pump was attached to a manifold (made from schedule 40 PVC pipe and end caps) that was fitted with a pressure gauge and pressure switch (Fig. 4b). Individual remote control of three vacuum collection tubes was provided using a four-way manifold/splitter and three-way solenoid valves, which could be operated simultaneously or independently in any sequence (Fig. 4b). The vacuum system was housed inside a sealed enclosure (Fig. 2), which further dampened operating noise of the

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Fig. 2 New remotely controlled (RC) sampler: a transport, b deployment, c, d operation, and e, f components

relatively quiet pump, therefore, indicator lamps of different colors were added to electrical circuits (for the

Table 2 Remote control (RC) channel assignments RC channel

Function assignment

1

Counterclockwise rotation of the gearmotor shaft

2

Clockwise rotation of the gearmotor shaft

3

Vacuum pump on/off

4

Unused

5

Unused

6

Solenoid valve, vacuum line, sample collector 1

7

Solenoid valve, vacuum line, sample collector 2

8

Solenoid valve, vacuum line, sample collector 3

pump and for each solenoid valve) to provide visual confirmation when the respective circuits were activated (Fig. 4a). Circuitry for the vacuum system used SPDT and DPDT relays, 3-way solenoid valves, and a vacuum pressure relay. All relays used C, NC, and NO contacts (nonenergized=solid arrows, energized=dashed arrows in figures). For vacuum pump control (Fig. 4a), transmitter channel-3, was set to “flip-flop,” alternately closing or opening the respective receiver relay (three) circuit. An indicator light signaled power to the pump. A pressure switch de-energized the circuit below −50 kPa. Transmitter channel-6, set to flip-flop, controlled receiver relay (six) to operate a three-way solenoid valve. An indicator light signaled power to the solenoid and vacuum pressure in the line. Solenoid valves (S1, S2, and S3)

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Fig. 3 Sample tubing support arm control diagrams (not to scale): a circuitry and b operation

were closed (solid lines) to the collector when nonenergized and open (dashed arrows) to the collector when energized (Fig. 4b). Channels 6, 7, and 8 were programmed for individual control of vacuum lines (V1, V2, and V3) for simultaneous or sequential collections. An articulated floating tether and a transport trailer were also constructed from scheduled 40 PVC pipe and fittings (Fig. 2). Both were made using common pipe sizes and fittings. Each pipe section of the articulated tether was in the shape of an “L” at one end and had a “T” fitting at the other end. The short leg of each “L” comprised a smaller diameter male threaded pipe that was attached through the larger diameter “T” fitting at the end of the respective adjacent section in the tether. This “L” in “T” arrangement served as a flexible joint. Each threaded “L” section was simultaneously sealed from water and affixed in its complementary “T” by a removable threaded cap, the outside diameter of which was slightly greater than the i.d. of the complementary “T” fitting. Water sealing at the “T” end of each pipe section was accomplished by gluing a PVC plug inside the end of the pipe before gluing on the “T” fitting. The

tether was attached to the front of the pontoon by four heavy-duty plastic cable ties and could be folded up or disassembled into separated sections for transport. The length of the tether could be adjusted in 1.5 m increments by adding or removing sections. The trailer was constructed on a frame of 34 mm o.d. pipe with 48 mm o.d. pipe (as rollers) slipped over bottom cross members and rear side guide posts (Fig. 2b). The rollers aided in launching and reloading the pontoon. The trailer wheels were 6.4 cm wide and 23 cm diameter pneumatic rubber tires on plastic rims and were fitted on 1.27 cm diameter steel rod axles, which extended the full width of the trailer to provide additional strength to the PVC frame. Stratification simulation Tank tests were conducted to assess stratification disturbance during sample arm deployment and sample collection (Fig. 5a). Stratified layers of 0, 2.5, and 5.0 % NaCl solutions in tap water containing Bright Dyes 101101 (Kingscote Chemicals, Miamisburg, OH) yellow/green fluorescent dye (one 1.32 g tablet/227 L) at relative rates of 1.0, 0.75, and 0.5, respectively, were

Fig. 4 Vacuum system control diagrams (not to scale): a circuitry and b air handler

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b AVERAGE INTEGRATED DENSITY

a

45000 40000

35000 30000

___ Before Sampler Deployed y = 35665x + 3882.4 R² = 0.9851

- - - After Sampler Deployed y = 33831x + 4954.8 R² = 0.9607

25000

…… Standard Curve

20000

y = 34349x + 5473.2 R² = 0.9508

15000 10000

5000 0 0.00

0.25

0.50

0.75

1.00

DYE TRACER RATE

Fig. 5 Stratification simulation test: a tank before sampler arm deployed (bottom inset showing positions of sampler intake and tank ports after sampler arm deployed) and b results (error bars=±one SD; n=3)

formed by sequential addition from the bottom of a (14.5 cm wide × 114 cm across × 99 cm high) tank through a vertical PVC inlet pipe. Pre-deployment samples (20 mL) were drawn by syringe at three depths through serum bottle stopper-sealed tank ports on the right front of the tank, followed immediately by sampler arm deployment and sampler (50 mL) collections from the same three depths at locations immediately adjacent to the tank ports (Fig. 5a inset). All samples were transferred to individual wells (3.0 mL ea) of 24-well plastic tissue culture plates and sample fluorescence quantified using an AlphaImager (Alpha Innotech, San Leandro, CA) and 595/55 nm filter, with image capture and spot density analysis software. Means from image analysis data of water samples collected before and after sample arm deployment were compared with a standard curve of the different density NaCl dye tracer solutions collected before their addition to the tank (Fig. 5b).

retained a permanent red-purplish color (Fig. 2c), indicative of mature phototrophic (purple sulfur bacteria) lagoons (Chen et al. 2003). Nutrient-rich lagoon water was land applied on the farm for irrigating and fertilizing summer grass hay crops. The annual lagoon cycle involved reducing the water volume from April through September to utilize this resource and subsequently accommodate increased rainfall water volume from October through March, when nutrient management plans preclude land application of lagoon water. Earlier studies, using water samples collected during irrigation or drawn from the lagoon surface, had documented chemical, nutrient, and bacterial levels in the lagoon (Adeli and Varco 2001; McLaughlin and Brooks 2009; McLaughlin et al. 2009), but temporal changes and chemical, nutrient, and bacterial levels at different depths had not been studied. Lagoon water samples collection

Field test sites Before deploying the remotely controlled sampler for swine lagoon water sample collection, the sampler and transport trailer (Fig. 2a) were field tested at a local fresh water lake (Fig. 2b). Subsequently, the sampler was field tested in collection of lagoon water samples from a single-stage 3.2 ha anaerobic swine manure lagoon on a commercial sow farm in east central Mississippi. Fresh manure slurry flowed into the lagoon from 13 confinement barns. Barns housed 220 to 1,000 gilts for breeding and gestation or 384 sows and 3,500 piglets for farrowing. The lagoon had been used for 17 years and

Lagoon water samples were collected on 26 Jan and 9 Mar 2009 at three depths (0.04, 0.47, and 1.0 m), by deploying the sampler such that the 1.0 m samples were collected just above the sludge layer. Six samples were collected at each depth on both dates. Samples were collected on each date at the same six locations, three each equidistantly spaced along two opposing sides of the lagoon. Three locations were proximal to the barns and influent flow and three were distal to the barns and influent flow. For each location, a separate set of collection tubing (pre-assembled module, tubing cut to length for each respective sampling depth and taped to a PEX

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pipe support arm), was lowered from the collection platform by remote control (Fig. 3). A different set of bottles, tubing, and PEX pipe was used at each sampling location. The sampler was moved between sample locations using the four-wheel PVC trailer (Fig. 2a), from which the sampler was launched and retrieved by backing the trailer into the water and floating the sampler off and on, respectively, (Fig. 2b) with the aid of the floating articulated PVC tether (Fig. 2c). Filled sample bottles were removed from the sampler, capped with sterile lids and placed in a cooler on ice immediately after collection. Water samples were returned to the laboratory, where they were held at 5 °C until processed, within 3 to 4 h after collection. Safety considerations Although no direct comparisons with other sampling methods were made in the present study, development of the remotely controlled sampler was intended to offer a safer alternative to manned boats for water column sample collections. This safety consideration is especially significant in wastewater lagoons where, in addition to the obvious drowning hazard, workers must also be aware of potential biohazards, such as zoonotic bacteria and other pathogens. With regard to pathogens, operation of the remotely controlled sampler was not without some risk, as deployment of the trailer, floating tether, and floating sampler necessarily resulted in all three being in contact with lagoon wastewater. Therefore, operators utilized appropriate personal protective equipment (PPE), which at a minimum included waterproof boots and gloves. Although the sampler did not generate aerosols during its operation, operators may be protected from occasional splashing during sampler launch and recovery by wearing disposable coveralls and face and eye protection, such as provided by a respirator and goggles. In the present study, operators routinely practiced double gloving, which not only provided added protection against tearing, but more importantly, enabled faster replacement of contaminated (outer) gloves with clean gloves when changing tubing modules between sample collections. All contaminated disposable PPE and other disposables, such as paper towels and sanitary wipes, were collected in biohazard bags, which were double bagged, returned to the laboratory, sterilized by autoclaving (121 °C, 1.06 kg cm−2, 45 min), and appropriately

disposed as solid waste. Reusable sample collection tubing and ported caps from sample collection bottles were rinsed with water at the lagoon site, placed in a biohazard bag, double bagged, and returned to the laboratory for sterilization by autoclaving (121 °C, 1.06 kg cm−2, 15 min). Rubber boots and the tether, sampler, and trailer were pressure rinsed with clean water and sanitized at the lagoon site after each day’s use. Clean water was transported to the site in a 95-L polypropylene tank and pumped using a 110-VAC water pump powered through an AC/DC inverter connected to the 12-VDC system of the pickup used to transport the trailer and sampler. Pressure-rinsed equipment was sanitized using alkyldimethylbenzyl ammonium chloride (Oasis 144, ECOLAB, St. Paul, MN) following the manufacturer’s recommendations. Sanitizer was applied using a dedicated, 3.8-L, manual pump, garden-type sprayer. Chemical and microbial analyses Water samples from each date, location, and depth were processed individually. Aliquots of each were tested separately for nutrient and bacterial content. Chemical properties (pH and EC) and nutrient concentrations (milligrams per liter inorganic and organic C, total N, P, K, Ca, Mg, Na, Cu, Fe, Mn, and Zn) were determined as previously published (McLaughlin et al. 2009). Bacterial levels, either cfu (colony forming units) 100 mL−1 for enterococci, staphylococci, C. perfringens, and E. coli, or MPN (most probable number) 100 mL−1 for Campylobacter spp., Listeria spp., and Salmonella spp., were determined using cultural and molecular methods as previously published (McLaughlin et al. 2009, 2010). Statistical analyses Bacterial counts (cfu or MPN 100 mL−1) were Log10 transformed to stabilize the variance. Transformed bacterial counts and raw chemical and nutrient data were analyzed using SAS (v9.2, SAS Institute, Inc., Cary, NC) MIXED model of sampling dates (two) using a repeated measures design with location (six) and depth (three) as repeated variables and a heterogeneous autoregressive covariance structure. Main effects were compared by F tests, and differences between means were compared at the P≤0.05 level (SAS 2010).

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Results and discussion Sampler operation and performance Total cost of the new sampler, including six sample collection modules, sample bottles and transport trailer, was less than US$2,000. The most expensive components, the remote control transmitter and receiver, totaled less than US$400. The new lagoon water sample collector was readily transported, deployed, retrieved, and sanitized. Test results from deployment of the sampler arm in an artificially stratified water column indicated only slight disturbance in the water column (Fig. 5b). Deployment, retrieval, and movement between locations at the lagoon could be handled by one operator, although two operators made the processes more manageable, especially following the first deployment of the day, after which the tether was contaminated with lagoon effluent and occasional flotsam. The new sampler was easier to operate than the first generation dipper sampler (Fig. 1a), because the new sampler floated and did not need to be held suspended over the lagoon. Furthermore, using the new sampler it was possible to collect much larger sample volumes (200 to 250 versus 5 to 7 mL) and at greater depths (up to 1 m in the present study) versus 5 to 10 cm with the dipper. The new sampler was also an improvement over the second generation floater sampler (Fig. 1b), because it could collect a larger number of samples from greater and varying depths and did not require being hoisted into and out of the lagoon. Although the new sampler used the same sample bottles as the floater it could easily accommodate larger volumes either by using larger sample containers, as described later for sludge sample collections, or by allowing the primary sample bottle to fill and spill over into the secondary bottle, although the latter is not recommended, as the secondary bottle was designed to function primarily as a trap to prevent water flow into the vacuum system. The present study reports results using three sampling depths, but given that two channels on the remote control system were not used in the tested design, the sampler could be easily modified to accommodate up to five samples. Constraints to the maximum sample depth attainable by the present design were: (1) the support arms (composition and length); (2) the collection tubing (i.d. and length); and (3) and the capacity of the vacuum pump. The PEX pipe used in the support arms in the present study may be too flexible for longer lengths and

deeper samples. Preliminary tests with 1.27 cm diameter polypropylene rod showed this material to be unsuited for repeated use. Although it offered the advantage of being autoclavable, it was relatively more flexible and expensive than the PEX pipe and was warped by repeated autoclaving. The limits of weight and torque imposed by the support arm on the gearmotor shaft were not tested in the present study and although the design functioned without problems, longer, heavier arms for deeper samples may require a larger capacity gearmotor. Added weight from longer support arms, a larger gearmotor, or greater sample volumes would likely require adjustments in module alignments for stable flotation in a next-generation larger capacity sampler. Potential options to 90° vertical rotation of a longer horizontal arm for sampling deeper water include: using a weighted line with attached sample collection tubing deployed from a winch in place of a sample arm; using opposing pressure rollers to move a vertical sample arm; and a cable, winch and pulley system to move a vertical sample arm; with the latter being more amenable to rapid in-field replacement with sterile sample arm and tubing modules, if required for microbiological samples. Similarly, the collection tubing and vacuum pump were capable of lifting water a maximum vertical distance of 2.33 m (92 in. and would require modification for greater depths. Such modification could include smaller i.d. tubing for deeper water-only samples, or larger diameter tubing and a higher capacity vacuum pump for watery sludge samples. Although constructed using sealed watertight enclosures and bulkhead penetrations, the balanced deck and pontoon design were stable and floated with the deck at an even height above the water line, such that water never lapped the deck or any of the seals on electronic modules (Fig. 2). The deck and modules were, however, routinely pressure rinsed with clean water and sanitizer after each day’s use in the lagoon, so the watertight seals were necessary. The sonar system was utilized only in the first lagoon deployment. Utilizing the sonar required that the sonar communication cable attached to the tether be connected to a display unit on land, which added steps to the sample collection process. During operation, however, the sampler was readily deployed by sensing the bottom of the lagoon with the flexible support arm and positioning the sampler to draw 1.0 m deep samples at or just above the sludge interface, which made it unnecessary to use the sonar depth finder. A weather and

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environmental monitoring and logging station at the lagoon was available to supply lagoon water temperature data, so that aspect of the sonar probe was also unnecessary in the present study. Although not necessary to the present study and not used in subsequent deployments, the sonar transducer was retained for possible future uses, such as in assessing sludge profiles or collecting deeper samples (National Hog Farmer 2005; Robert 2006; Singh et al. 2008; Kaizu et al. 2011). Collection of sludge Collection of 1.0 m deep lagoon water samples at or just above the sludge interface proceeded smoothly, but if the collection inlet was deployed below the sludge interface, collections were complicated by sediment or debris small enough to pass through the collection tubing, but too large to pass through the Luer fittings on the sample bottles. On such occasions, it was necessary to retrieve the sampler, clear or replace the clogged lines and sample bottle lid, and redeploy the sampler. The Luer fittings functioned well for water samples, were quickly and easily connected and disconnected in the field, and could be autoclaved between uses, but when collection of sludge was the objective it was necessary to replace these fittings with larger i.d. fittings. Modification of the sampling module to include larger (13 mm i.d.) fittings, larger (1 L) wide-mouth sample bottles (with larger lids for larger fittings), and larger (13 mm i.d.) collection tubing (collecting directly through the PEX pipe support arm) enabled collection of watery sludge samples at the sludge interface. The water sampler, however, was not designed for or capable of collecting solid sludge samples from deeper in the sludge layer and sludge samples were not included in nutrient and microbial analyses reported here. Nutrient and bacteria levels in sludge from this lagoon have been discussed (McLaughlin et al. 2012). Chemical and microbiological data No date and depth interactions were observed in the statistical analysis (Table 3). Analysis of lagoon water sample depth data across dates showed no significant differences in pH and EC among depths, but both chemical parameters showed differences between collection dates. Lagoon water pH and EC were higher in samples collected 9 Mar than those collected 26 Jan. Both

parameters were within the ranges reported from shallow surface samples of sow farm lagoons in the region (McLaughlin et al. 2009). Results of nutrient data analysis followed this same trend in that no differences were found among sample means from the three sample depths for any of the nutrients measured, but levels of several nutrients including C (inorganic, organic, and total), N, K, Mg, Na, and Zn differed between collection dates (Table 3). In every instance where nutrient levels differed between collection dates, levels were higher in the 9 Mar spring samples than in the 26 Jan winter samples. Among the nutrients with levels that did not differ statistically between collection dates (P, Ca, Cu Fe, and Mn), all had numerically higher levels on 9 Mar (Table 3). Taken together, this suggests a pattern of reloading and concentrating of nutrients from winter to spring. During this time, fresh manure is flushed into the lagoon in recirculated lagoon water, but no lagoon water or nutrients are removed because land application is not permitted. Increasing temperatures during this period may also be a factor. Average daily lagoon water temperatures were 8.8 and 19.0ºC on 26 Jan and 9 Mar, respectively. Nutrient solubility and microbial activity increase with increasing temperature, which has been recognized as a significant factor influencing lagoon nutrients (Harper et al. 2000; DeSutter and Ham 2005). Analysis of bacterial count data showed results similar to those observed with nutrient levels, in that no differences were found between sample depths (Table 3). Analyses of bacterial count data between sampling dates, however, showed two bacterial groups (C. perfringens and E. coli) with higher levels on 9 Mar, two groups (enterococci and Salmonella spp.) with lower levels on 9 Mar, and three groups (staphylococci, Campylobacter spp., and Listeria spp. ) with no significant differences between counts on 26 Jan and 9 Mar. Swine manure lagoons are complex dynamic environments for bacteria and no simple explanation for change or lack of change in bacterial populations was apparent in comparisons of two sampling dates. As observed in analysis of nutrient levels, a lagoon water temperature increase from winter to spring could have been a factor, but one not so easily described with only two dates to compare, especially as the bacterial populations did not all respond in the same way. These results suggest the need for a longer term study of bacterial levels with a larger number of sampling dates covering all the seasonal changes typical of lagoons in the region.

Environ Monit Assess Table 3 Chemical and microbiological analyses of stratified swine manure lagoon water samples Variable (units)

Analysis by depth (n=12) 0.04 m Mean

Analysis by date (n=18)

0.47 m ±SE

Mean

1.0 m

P>F

±SE

Mean

±SE

26 Jan 2009

09 Mar 2009

Mean

±SE

Mean

±SE

P>F

Chemical properties pH

8.04

0.03

8.02

0.04

8.00

0.04

ns

7.91

0.01

8.14

0.01

***

EC (mS cm−1)

4.83

0.14

4.90

0.09

4.89

0.11

ns

4.53

0.04

5.22

0.03

***

Inorganic C

488

9

488

8

493

9

ns

462

3

516

2

***

Organic C

415

19

415

17

430

20

ns

366

7

473

8

***

Total C

903

27

902

24

922

28

ns

829

10

990

7

***

16

451

15

456

15

ns

402

2

504

1

***

Carbon fractions (mg L−1)

Total fertilizer nutrients (mg L−1) N

453

P

46

1

47

1

57

7

ns

49

5

51

1

ns

K

412

6

416

7

423

6

ns

402

4

432

3

***

Total macronutrients (mg L−1) Ca

53

1

52

1

67

10

ns

56

7

59

2

ns

Mg

21

1

21

1

23

1

ns

20

1

24

0

***

Na

215

3

219

3

223

3

ns

212

2

226

2

***

Cu

99

8

119

27

137

26

ns

114

25

122

8

ns

Fe

1,683

172

1,628

126

2,545

605

ns

1,686

387

2,218

200

ns

Mn

183

17

177

12

326

111

ns

220

75

237

21

ns

Zn

782

121

926

191

1,040

224

ns

677

137

1,154

140

* ***

Total micronutrients (μg L−1)

Bacterial indicators (Log10 cfu 100 mL−1) Clostridium perfringens

6.05

0.05

6.10

0.05

6.14

0.07

ns

6.01

0.04

6.18

0.05

Enterococcus spp.

5.12

0.03

5.17

0.04

5.09

0.07

ns

5.20

0.03

5.05

0.04

**

Escherichia coli

5.68

0.18

5.62

0.17

5.84

0.15

ns

5.40

0.15

6.03

0.04

***

Staphylococcus spp.

6.70

0.10

6.63

0.05

6.77

0.08

ns

6.66

0.08

6.74

0.04

ns

Bacterial pathogens (Log10 MPN 100 mL−1) Campylobacter spp.

5.01

0.03

4.92

0.09

4.95

0.07

ns

4.96

0.05

4.96

0.06

ns

Listeria spp.

3.01

0.14

3.05

0.11

3.18

0.12

ns

2.98

0.09

3.17

0.10

ns

Salmonella spp.

2.00

0.40

2.25

0.18

1.84

0.29

ns

2.81

0.11

1.25

0.19

***

Least squares means, standard errors (SE) and probability of greater F in analysis of variance (P=0.05); samples at three depths and six locations per date ns not significant *P≤0.05; **P≤0.01; ***P≤0.001

Summary A new remotely controlled water column sampler was designed, constructed, and tested for use in a study of chemical, nutrient, and bacterial stratification in a relatively shallow swine manure lagoon in

the Mid-South USA. The sampler was designed to collect multiple samples simultaneously from different depths in the water column. The new sampler was deployed and operated with minimal disturbance to the water column. The sampler displayed positional stability during sample collection and was

Environ Monit Assess

easily recovered and reloaded with new sample bottles between sampling positions in the lagoon. Designed to collect water samples, the sampler was easily modified to collect watery sludge at the sludge interface; however, the sampler was not designed for or capable of collecting samples from deeper in the sludge layer. The new sampler could be readily sanitized and transported between deployments. Water quality tests showed that pH, EC, nutrient concentrations, and bacterial levels did not differ significantly between samples collected at 0.04, 0.47, and 1.0 m depths, but differences between samples collected in winter and those collected in early spring were significant for five of ten nutrients and four of seven bacterial groups. Results demonstrated the utility of the new sampler in collection of samples from multiple depths, showed no stratification of nutrients or bacteria within the depths sampled, and suggested that a more extensive study of nutrients and bacteria could omit collections from 0.47 m but should include a greater range of dates and seasons. Acknowledgments The authors are grateful for swine farm lagoon access provided by a private cooperator and for the technical assistance of Cindy Smith and Renotta K. Smith for microbiological laboratory work; Tim Fairbrother, Mary Hardy, and Walter Woolfolk for chemical analyses of lagoon water nutrients; Stan Malone for advice on electrical relay circuitry; and Debbie Boykin for statistical consultation.

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A new sampler for stratified lagoon chemical and microbiological assessments.

A sampler was needed for a spatial and temporal study of microbial and chemical stratification in a large swine manure lagoon that was known to contai...
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