Environ. Sci. Technol. 2010, 44, 2365–2371
Estrogenicity and Nutrient Concentration of Surface Waters Surrounding a Large Confinement Dairy Operation Using Best Management Practices for Land Application of Animal Wastes N A N C Y W . S H A P P E L L , †,* KEVIN H. ELDER, AND MARK WEST‡ Animal Metabolism-Agricultural Chemicals Research Unit 1605 Albrecht Blvd. Biosciences Research Laboratory Red River Valley Agricultural Research Center, USDA Fargo, North Dakota, 58105
Received December 3, 2009. Revised manuscript received February 2, 2010. Accepted February 9, 2010.
The impact of a confinement dairy operation (>2000 head) using best management practices for land application of animal wastes, on estrogenic activity (E-Screen), estrogens, and nutrients of associated surface waters and tile drain runoff were evaluated. Farm tile drain and creek samples were collected from the drainage region: above and below a municipal wastewater treatment plant located upstream from the dairy; and downstream from animal housing, parlor, and fields receiving applied wastes. Fifty-four thousand tons of waste (from ∼1000 milking head) were applied to ∼809 ha from April to July. Maximum estradiol equivalents (E2Eqs) present in tile drain samples (e0.257 ng/L) were 2-fold maximum creek E2Eqs, but only 25% of the proposed no observable effect concentration for E2 (1 ng/L). Relative manure slurry estrogen concentrations were estrone >17R-E2 > 17β-E2. Creek nutrient concentrations were similar above and below the dairy, with higher concentrations found in tile drain samples: tile ammonia ranged from 50% chance of rainfall exceeding 1/2 in./1.3 cm); monitoring of soils to assess potential for preferential flow, pooling or runoff of liquid manure; no more than 1/2 in./1.3 cm of liquid manure applied per acre for fields that are tile drained; 100 ft/30.5 m buffer (or 30 foot/ 9.1 m vegetated buffer) between manure application and state waters; and complete record keeping of analyses and date, rate and method of application (USDA-NRCS, Code 633 and 590, refs 7, 8). The objectives of this study were to determine (1) estimates of nutrient and estrogen loading from dairy waste application, (2) the magnitude and timing of area precipitation events, and (3) nutrient and estrogenicity contents of surface waters from surrounding creek and tile drain water samples from the drainage of a large commercial dairy that employed best management practices for land application of dairy waste. This project was possible through a partnership established with a large confinement dairy operator, a federal agency (U.S. Department of Agriculture, Agricultural Research Service), and a state department of agriculture.
Experimental Section System Description. Surface waters surrounding a large confinement dairy operation (Figure 1; >2000 milking head) located in the midwestern United States were sampled in April, June, and July in a period when dairy wastewater was land applied. Parlor wash waste, press effluents, and solids were applied to fields by center pivot irrigation, drag-hose application and incorporation, and manure spreader in April and May of 2008. Center pivot irrigation using parlor wash wastes continued through July. Rates of application were in compliance with best management practices for nutrient loading (7, 8) and each field received only a single application by entrenchment. Fields were characterized by degree slopes (i.e., a 2% slope is a 2 ft/0.61 m change in elevation over 100 ft/30.5 m) and soil type. Fields consisted predominantly of Blount loams on 0-2% and 2-6% slopes, followed by fields of Pewamo silty clay loams on 0-2% slopes, and small areas of Glynwood loams on 2-6% slopes. A creek running parallel to the dairy farm was sampled upstream 10.9 km from the dairy farm both above (C1; Figure 1) and below (C2) a municipal wastewater treatment plant (0.105 million gallons/ da or 0.397 million liters/da), and downstream from the dairy facility and fields receiving dairy wastewater (C3 and C4, Figure 1). There are no allowed agricultural wastewater discharges or agricultural return flows from irrigation in this watershed. Field tile drain water was sampled at two locations (TD1 and TD2). Tile drain water enters the creek between C3 and C4, with a contribution of less than 0.1% of creek volume at that location. Waste was applied no closer than 100 ft/30.5 m from the tile drain. A 12 foot (3.6 m) vegetative buffer zone ran parallel between tile drain and planted crops. The Tile Drain 1 site (TD1) was 150 ft/45 m from the tile system’s origin, with receiving water coming from one 60 acre/ 0.243 km2 field and possible drainage from a domestic septic system. Fields associated with TD1 received application of VOL. 44, NO. 7, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
2365
FIGURE 1. Schematic of water sampling sites. Four creek sites, and two tile drain sites.
FIGURE 2. Schedule of field application of dairy waste and precipitation events. Metric tons of waste (dry matter, approximated on analyses of source samples) were applied on indicated dates to fields draining to respective sites. waste on 5/17/08. The Tile Drain 2 site (TD2) was a continuation of the tile system on which TD1 was located, but was approximately 1 mile/1.6 km downstream of TD1 and 0.55 mile/0.9 km above the creek entrance. Fields associated with TD2 (358 acres/1.45 km2) received dairy waste applications from 5/1 to 5/10. The field emptying into TD1 received ∼50% of dry matter per acre that the TD2 acreage received (Figure 2). Applied wastes in Figure 2 represent waste from ∼1000 of the cows in residence, field-applied to 1996 acres/8.1 km2. Fields did not receive manure from grazing cattle because all cows were maintained in a free-stall barn and did not have access to fields. Manure stored in three concrete lined storage units, press effluent stored in two claylined storage units and animal housing were ∼0.4 km from the creek. Sampling sites were marked by rebar placed in the banks of the creek or tile drain. Water temperature, width, column height (depth measured across the width at g3 places 2366
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 7, 2010
(with the exception of TD 1 and 2 on 7/15 because of insufficient water), and estimated flow rate based on measuring water flow and cross-sectional path (9) were recorded. Sampling, Extraction, and Analyses. The farmer, accompanied by an agent of the Federal Fish and Wildlife Services, collected triplicate water samples from each site on 4/20/08, 4/30/08, 6/4/08, and 7/15/08. Samples were collected by hand, in 2 L HDPE bottles (precleaned to Environmental Protection Agency (EPA) standards, EP, VWR International, West Chester, PA) filled six inches below the water surface (exception, tile drains when water was too shallow) while standing in the creek or beside the tile drainage ditch, with container submerged upstream from the collector. After agitation, samples were subdivided into two 500 mL HDPE sample bottles (EP, A. Daigger, Vernon Hills, IL) which were then frozen for estrogen analyses; the remaining oneliter portion was preserved with acid for nitrate/ite, ammonia, and total phosphorus determinations (stored at 4 °C prior to shipment, U.S. EPA methods 353.1, 350.1, and 365.4, respectively (10)). Waste materials were collected from the drag hose applicator during field application on 5/16/08 (at three separate times). The material being applied to the fields represented a collection of wastes accumulated during the previous 6 months. Processing, to remove bedding sand, was via a McLanahan sand manure separator. Briefly, wastes were screw pressed through a 500 µm screen with the resulting liquid fraction held in a long-term (primary) storage lagoon. Lagoon waste (primary and secondary lagoons) and pressed solids were also analyzed by a commercial lab for dry matter, total Kjeldahl nitrogen, phosphorus and potassium using the methods described by Peters (11). Water Samples. Water samples were extracted and extracts were evaluated for estrogenicity by E-Screen as described by Shappell (12). Briefly, samples were thawed, shaken, and 250 mL filtered through packed glass wool, followed by solid phase extraction on a cartridge containing a mixture of hydrophobic and hydrophilic packing (OASIS HLB, Waters, Milford, MA). Extracts were eluted with a series
of organic solvents, evaporated to dryness under N2, and the residue resuspended in water for analysis using the E-Screen assay. The MCF-7 BOS, estrogen-dependent cell line (derived from a human mammary epithelial carcinoma) was used to evaluate sample extract estrogenicity relative to 17-β estradiol. Extracts were tested over a wide range of concentrations (15-fold concentrated to 128-fold diluted from original sample/environmental concentration). Specificity of estrogenicity in positive samples was confirmed by E2-receptor antagonist ICI 182,780 (Tocris, Ellisville, MO) (13, 14). Surface water samples with the highest 17β-estradiol equivalents (E2Eqs) and nanopure extraction blanks were analyzed by LC MSMS as previously described (ref 15 text and Supporting Information). The same sample extracts prepared for E-Screen were prepared for injection (20 µL of 781 fold original concentration) to contain internal standards (deuterated 17β-E2, estrone, and estriol at final concentrations of 10 pg/µL; and ethinyl estradiol at 20 pg/µL). Parent and three fragment ions were monitored in the ESI mode using a quadrupole-time-of-flight mass spectrometer (15) (Waters, Beverly, MA). Dairy Waste Samples. Dairy waste collected at the time of application (5/16/08) was extracted according Hanselman et al. (16) (source of SPE columns: Carbograph 150 mg, Grace, Deerfield, IL; Bakerbond C18, 1000 mg, JT Baker, Phillipsburg, NJ; ODS Sep Pak, 350 mg, Waters, Milford, MA), and assessed for estrogenic activity (E-Screen using 143-500 fold dilutions of the original concentrations), and for chemical composition (LC MSMS using sample extracts concentrated 1.7-50 times those of the original concentrations). The instrument limits of quantitation (LOQ) were 2 pg/µL on column for all but estriol, which was 5 pg/µL. Limits of detection were 50% of respective LOQs. Limit of detection in original samples depend on the matrix and sample concentration factors. Statistical Methods. Data analyses for this observational study was conducted using SAS/STAT software (17). A general linear model was fitted to observed concentrations of phosphorus, nitrate, ammonia, and E2Eqs using the PROC MIXED procedure. The REPEATED statement was used with the GROUP option to account for heterogeneity of variance among dates and between creek and tile sites. The fitted model included Date, Site and the interaction of Date and Site. The CONTRAST statement was used to compare specific groups of sites (e.g., Tile vs Creek) across dates and for each sampling date. Tukey’s Multiple Comparison procedure was used to provide post hoc comparisons of all possible pairs of site means for each date using a macro written and described by Saxton (18).
Results and Discussion Estrogens in Surface Water. Farms using best management practices apply animal wastes to fields to meet N and P needs, and must monitor tile drains to ensure that runoff is not occurring during application. The timing of field application of waste material is dependent on a number of variables including snowmelt, field moisture content, weather forecast, proposed planting date, and availability of personnel. As a consequence, wastes are typically applied over an extended period of time, rather than in a brief discrete time frame. Figure 2 shows the rainfall and schedule of field application of waste (tons of dry matter, grouped by the upstream-most creek site or tile drain into which the fields would drain). In an experimental world where all parameters could be optimized, and timing, duration, and intensity of precipitation events could be predicted, an additional sampling point in mid-May would have been included. Estrogenic activities of surface waters measured by E-Screen (E2Eqs) and rates of water discharge are plotted in Figure 3. All estrogenic activity was confirmed as E2 receptor dependent, because coincu-
FIGURE 3. Estradiol equivalents of surface waters and estimated discharge volumes. E-Screen estradiol equivalents of extracted water samples over four sampling dates, means ( standard deviations from three separate grab samples (per site per date). Site values with different letters within a sampling date, are different (analyses of least-squares means, P < 0.05) A. Estimated discharge rates for sample sites. Tile Drain 1 had no flow on 7/15/08. pNOEC (proposed no observable effect concentration) for estradiol in fish. bation of estrogenic water samples with ICI 182,780 prevented cellular proliferation. Estrogenic activities of all sites were below the proposed no observable effect concentration of 1000 pg/L for estradiol (pNOEC) in fish (19). Of note is the fact that mean E2Eq were not different along the creek (P > 0.20, within and across sampling dates), whether upstream (C1) or downstream (C2) from the municipal wastewater treatment plant (MWTP), or the dairy facility (C3, C4). Creek mean E2Eqs ranged from 44-120 pg/L. In July, estrogenicity of water decreased to 0.05) explain variation of E2Eqs over and above sampling site, date and their interaction, and therefore were not used to adjust means for comparisons. Comparison of tile drain and creek samples indicated that tile drain samples contained greater E2Eqs than creek samples (P < 0.01) within, and across, sampling dates. Comparing specific sites, estrogenic activity of TD1 was significantly (P < 0.05) greater than all creek samples, except the June sampling, 104-257 pg/L E2Eqs, while TD2 samples were similar (66-140 pg/L; P > 0.05) to creek values for all but the July sampling (180 pg/L). Perhaps the elevation of E2Eqs at the TD1 site was due to the proximity of the sampling site to the origin of the tile drain (∼150 ft or 45.7 m from the origin of the tile drain). Nichols et al. (20) indicated that grass filter strips have the capacity to prevent estradiol release in runoff, and a 12 foot/3.6 m vegetative buffer was in place VOL. 44, NO. 7, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
2367
TABLE 1. Estimated Estradiol Equivalents of Applied Dairy Waste dairy wastea
E2Eq ng/Lb
SD (c
fold pLOECd
dry matter % ( SDe
E2Eq DM µg/kgf
grab 1 grab 2 grab 3
1476 1742 1032
84 253 89
148 174 103
2.85% ( 0.014 3.03% ( 0.042 1.76% ( 0.015
51.8 57.5 58.6
a Waste samples were collected on 5/16/08, at three times over the application period. b Sum of E-screen found in both the Carbograph wash fraction and the ethyl acetate elution from the final column (ODS). c Standard deviations are based on the highest COV from either extract tested. d Based on the 10 ng/L pLOEC (19). e Dry matters were based on quadruplicate measurements. f Assumption that E2Eqs are constant per unit of dry matter (DM).
in our study. Another explanation of higher E2Eqs values at TD1 may be contamination from human waste treated in a neighboring residential septic system. Input of E2Eqs from such a source would be expected to remain relatively constant. To evaluate this theory, the values obtained on the April 20th sampling were adjusted for the decrease in discharge rates found at the April 30th and June 4th sampling. The resultant predicted E2Eqs for TD1 would be 198 and 413 pg/L E2Eqs, while the observed values were 173 and 257 pg/L (April 30th, June 4th), with predicted being greater than actual. By July there was essentially no discharge from either tile drain sampling site, and TD1 E2Eq values were lower than any previous time, whereas TD2 samples were higher (P < 0.05), peaking at this time. In making statistical comparisons of the TD1 and TD2 samples, the variability of E-screen values should be considered. Overall, the variability for the triplicate samples taken from each site was fairly low, with a mean of all sample sites’ COVs of 28%. In contrast, variability was higher in tile drain samples for the June and July sampling (44%) when the depth of water in the ditches draining the tile drain was limited. The discharge rates for tile drain samples were essentially zero for the July sampling. The low volume resulted in inclusion to varying amounts of sediment and particulate material in samples. To allow for this heterogeneity of variance, the GROUP option in the REPEATED statement of PROC MIXED was used. Statistical analyses comparing E2Eqs from TD1 to TD2 found TD1 > TD2 for both April samples, but the reverse for the July sampling, TD1 < TD2 (P < 0.001). Again, while E2Eqs were detected in all but one sampling site in July, all values from all dates and sample sites were below the pNOEC of 1000 pg/L for estradiol. Analyses by LC MSMS were used to identify the chemicals responsible for the estrogenicity detected by the E-Screen assay. Prior to LC MSMS analyses, possible concentrations of estrogens were calculated based on E2Eqs measured by E-Screen and the relative estrogenicity of each estrogen, in order to determine if concentrations would fall within our instrument detection limits. While the E-Screen reflects a sum of biological activity of all estrogens present, calculations were performed assuming that 100% of the estrogenic activity of a given samples was caused by each estrogen separately. Thus, the greatest observed E2eqs (0.257 ng/L) expressed as 17 β-E2 equivalents would represent only ∼ 0.4 pg/µL in an extract concentrated approximately 1500 times; this concentration of 17β-E2 is below the limit of detection by LC MSMS. If estrone or 17R-E2 alone were responsible for the observed E2Eqs, then we would expect to find 100 times the amount of 17β-E2 because estrone and 17R-E2 have only 1% of the estrogenic potency of 17β-E2 (in the E-Screen application). The resulting value, ∼40 pg/µL, was well within the limit of quantitation by LC MSMS. Samples from nanopure water control extractions, and sites C2, TD1, and TD2 were analyzed by LC-MSMS. Matrix interference caused ion suppression and samples were diluted to 750 fold original concentrations, at which point deuterated internal standards were then quantifiable. Twenty microliter injection volumes failed to yield any detectable estrogens, suggesting that 17 2368
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 7, 2010
β-E2 may have been the predominant source of E2Eqs present. This data does not rule out the presence of other estrogenic compounds. Estrogens in Applied Waste. In order to ensure that, in fact, dairy waste contained estrogenic activity at the time of application, waste samples collected at application were extracted by the method of Hanselman et al. (16). The mean E2Eq of the three dairy waste samples was 1417 ng/L, or ∼60 µg/kg dry matter (DM, Table 1). Using the method reported by Hanselman et al. (16), estrogens were analyzed only in the final eluent collected after three sequential SPE steps using three different SPE chemistries. However, we found that estrogenic activity was present (50-70% of the final eluent) in the first SPE wash phase (Carbograph). The estrogenic activity in the Carbograph wash could have been the result of unretained estrogen conjugates present in the matrix or due to overloading the cartridges. Subsequent work by H. Hakk, using radiolabeled 17 β-E2 in the absence of matrix, has indicated that 50% of the applied radioactivity was also lost in the first Carbograph column washes (personal communication), indicating overloading was not a factor in the incomplete recoveries of estradiol. Literature reports of estrogen concentrations in dairy waste were converted to E2Eqs for comparison to study results. Hanselman et al (16) evaluated flushed dairy manure wastewater sampled on their university research farm on five consecutive days (above method). Converting the mean estrogen concentrations they determined to E2Eqs, their wastewater contained 625 ng/L E2Eqs. Assuming that the concentrations reported by Hanselman et al (16) underestimate the actual E2Eqs (∼40% due to loss on Carbograph washes), then their adjusted mean concentration should have been approximately 1062 ng/L E2Eqs. This value is similar to the 1417 ng/L we reported. Converting chemical data of Raman et al. (21) to estrogenic equivalents, the sum of estrogens in an analysis of dairy waste holding pond material would be 2150 ng/L E2Eqs. Other reports have yielded chemical concentrations of estrogens that result in lower E2Eqs, ranging from 30 to 319 ng/L for dairy lagoon waste (22). However, the report by Sarmah et al. (22), is from dairies in New Zealand, where management practices are very different from those in the United States, including a seasonal milking schedule. Regardless of differences in management practices, it is obvious that the amount of wash water used will seriously impact the reported concentrations. Just as enzyme activity is reported on a mg of protein and unit of time basis, reporting E2Eqs concentrations in animal wastes on a dry matter basis will help compensate for differences due to water content. On a dry matter basis, the mean E2Eqs of the dairy wastewater from Hanselman’s (16) study was 77 µg/kg DM, a value highly consistent with the 60 µg/kg DM reported here. In a review paper of steroids in animal waste (23), 17 β-E2 ranged from 113 to 239 µg /kg DM for waste from dairy holding ponds, solid and semisolid dry stacked wastes, with coefficients of variations of 12 and 92%, respectively (n > 20 per mean). These data indicate some degree of consistency of estrogen concentrations in waste on a DM basis, with means varying no more than 2-fold.
TABLE 2. Estimated Concentrations of Estrogens Detected by LC MSMSa dairy waste
17r-E2 ng/L
estrone ng/L
17r-E2 µg/kg DM
estrone µg/kg DM
grab 1 grab 2 grab 3
700 1100 700
3200 3900 1000
25 36 39
112 129 58
a 17 β-estradiol, estriol, and ethinyl estradiol were not detected. Carbograph extracts were not analyzed. Values are provided to give relative concentrations, rather than absolute concentrations, as matrix effects and detection limits hindered LC MSMS performance.
Using these data as support for an assumption of relative constancy for E2Eqs on a dry matter basis, the application rate of E2Eqs in our study was 32 mg E2Eqs/acre (7.91 g/km2), or 63 g E2Eqs applied to 1996 acres (8.08 km2). In comparison, values from dairy manure (21, 24) when converted to E2Eqs results in approximately 381 mg E2Eqs per acre (DM basis). Johnson’s value is ten times greater than the value reported here, and reflects the difference in estrogenic activity of fresh manure versus processed or aged waste (25). Similar findings were mirrored in the concentration change of 17 β-E2 in fresh manure (153 µg/kg DM) versus dairy waste held for 3 months (18 µg/kg DM) (25). The relative abundances of specific estrogens present in applied dairy waste were measured by LC MSMS (Table 2). These values do not include estrogens that eluted in the Carbograph wash fraction. Ion suppression due to matrix effects required dilution of samples to ∼2× pre-extract concentrations. Only estrone and 17R-E2 were detected in these samples, with estrone present at greater concentrations. No 17β-E2 was detected. Others (25, 26), have reported similar findings in treated dairy waste, indicating the relative lability of 17β-E2. Colucci et al. (27) reported conversion of 17β-E2 to estrone, even in the presence of autoclaved soil. When dairy waste is fresh, higher concentrations of 17R-E2 have been reported than in aged or processed wastes; in addition, fresh dairy waste will contain measurable 17β-E2, but no estriol has been reported (16, 21, 22). Hutchins et al. (28) found lower concentrations of estrone and 17R-E2 in dairy lagoons than those reported in Table 2, but also found Rand β-E2 isomers to be present at similar concentrations. It is unclear if extraction method differences, maturity of waste lagoons, and or dry matter content account for most of these differences. No attempts were made to analyze for estrogen conjugates, due to their relatively limited estrogenicity (from 500 to 80 000 lower activity than 17β-E2 as assessed by E-Screen) (29). Water Nutrient Concentrations. Nutrient content of wastes effects potential rate of application. The nutrient content of applied wastes are presented in Supporting Information (SI) Table S1. Application rates of N, P, and K were all at, or below commonly recommended rates. Nutrient data, water temperature, and cross sectional area of sampling sites are presented in Figure 4. Water temperatures at creek sites were consistently warmer than those measured in tile drain samples. This is expected, as tile drains are fed by subsurface water flow. Discharge rates were similar to those reported by Dodds and Oakes (30) for creeks and prairie streams, ranging from 1 to 100 ft3/sec and 0 to 0.3 ft3/sec (0.0283 to 2.83 m3 and 0 to 0.0085 m3), respectively (Figure 3). Nitrate/ite concentrations were higher in samples from tile drains than creek samples across all dates (P < 0.0001, maxima of 14.8 versus 0.8 mg/L for tile drain versus creek, respectively). Maximal nitrate/ite concentrations were well below concentrations which would raise environmental alarm. For example, the EPA maximum contaminant level goal for nitrate/ite in drinking water is 10 mg/L (EPA CFR 40) (31), and the highest tile drain samples were 14.8 mg/L, and 10.4 mg/L in TD1 and TD2, respectively, collected in
FIGURE 4. Water chemistry, water temperature, and cross sectional area of sampling sites. Nitrate/ite, ammonia, and total phosphorus concentrations (means ( standard deviations from three separate samples) and cross-sectional areas of creek or tile drain at time of sampling. Temperature in degrees C. Limits of quantitation were 0.1, 0.05, and 0.01 mg/L for nitrate/ite, ammonia, and phosphorus, respectively. Within a sampling date, bars with different letters are different based on analyses of least-squares means (P < 0.05). June. Kolodziej et al. (5) reported a range of 9.5-350 mg/L for nitrate in tile drain samples found on dairy farms. While nitrate/ite were consistently higher in tile drain samples than creek samples, both within and across dates (P < 0.05); ammonia results varied by sampling date and site (P < 0.05). Ammonia was below assay detection limits (0.05 mg/L) at all sites sampled in April except TD1. The June sampling yielded detectable ammonia concentrations at all sites, and these were higher (P < 0.05) in ammonia concenVOL. 44, NO. 7, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
2369
trations than other dates across all sites except July TD1. Mean creek ammonia concentrations fell back to below assay limits in July, while tile drain samples were still elevated (0.70 TD1 > 0.15 TD2, P < 0.05). Peak ammonia concentration from tile drain was 0.7 mg/L versus 0.2 mg/L in creek samples. Limits for ammonia concentrations in aquatic warm water habitat streams are dependent on duration of exposure, pH, and water temperature, and are cited as 4.16 mg/L at 20 °C, and pH 7.5 (32). While July tile drain values rose to 0.7 mg/L ammonia, the permitting limits for summer release of ammonia by some US MWTPs is 19 mg/L. Sample pH values are reported in SI Table S2. Phosphorus concentrations ranged from 0.025 to 0.340 mg/L, and were similar to those previously reported in creeks (0.013 to 0.234 mg/L) (30). For both April sampling dates, all creek phosphorus concentrations were lower than TD1 (P < 0.05), and TD2 phosphorus was intermediate to both creek and TD1. In June, tile drain values were higher than creek (P < 0.05). The greatest values were measured in July, at times of lowest discharge rates. While July phosphorus values for TD1 and TD2 were not different, the mean for C3 was equivalent to TD2 and higher than all other creek concentrations (P < 0.05). Phosphorus concentrations mirrored those of nitrate/ite, in that TD1 were higher than TD2, with the exception of the June phosphorus sample. Limits for phosphorus in surface water and discharge concentration limits for MWTPs have yet to be established in the U.S. Typically regional permits designate total maximum daily loads based on total phosphorus loading and monitored phosphorus concentrations. Warm water goals of 1 mg/L are typically met, and even the highest P values from tile drains (0.34 mg/L) were ∼30% of the mean for 25 Canadian MWTP (1.3 mg/L) (33) and 10% of typical discharge concentrations from a local municipal wastewater treatment plant (3 mg/L, MWTP, Fargo, ND). Creek P values were in line with reported 25% values for the decade in Ecoregion VI of 0.076 mg/L (EPA, Table 2, Median stream values, 2000; ref 34). In conclusion, no differences in estrogenic activity or nitrate/ite, ammonia, and phosphorus concentrations were detected in surfaces waters upstream and downstream of a large confinement dairy facility. The highest E2Eqs detected were measured in tile drain samples, but concentrations of E2Eqs in those samples were approximately 1/3 lower than the proposed no effect concentration for estradiol. Water quality (nitrate/ite, ammonia, and total phosphorus) were similar regardless of sampling site along the creek, and were within regional norms. It appears that use of best management practices for land application of dairy waste results in negligible nutrient and estrogenic contributions to surface waters associated with large dairy operations.
Acknowledgments Appreciation is extended to Dr. Ana Soto and Dr. Carlos Sonnenschein, who graciously provided cells and access to their laboratories to learn E-screen methodology. We would like to acknowledge the dedicated efforts of Mr. Lloyd Billey in sample extraction and E-screen analyses; excellent technical skills of Grant Harrington in operation of the LC/MSMS; and Dr. Heldur Hakk’s pioneering efforts in method development for quantification of steroids by LC/MS-MS. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.
Supporting Information Available Methodology for estimation of flow rates for creek and tile drains; N, P, and K concentrations of applied dairy waste, and pH of water samples are provided in Supporting 2370
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 44, NO. 7, 2010
Information. This material is available free of charge via the Internet at http://pubs.acs.org.
Literature Cited (1) Erb, R.; Chew, B.; Keller, H. Relative concentrations of estrogen and progesterone in milk and blood, and excretion of estrogen in urine. J. Anim. Sci. 1977, 45 (3), 617–26. (2) Mellin, T. N.; Erb, R. E.; Estergreen, V. L. Quantitative estimation and identification of estrogens in bovine urine. J. Dairy Sci. 1965, 48 (7), 895–902. (3) Guilette, L. J.; Gross, T. S.; Masson, G. R.; Matter, J. M.; Percival, H. F.; Woodward, A. R. Developmental abnormalities of the gonad and abnormal sex hormone concentrations in juvenile alligators from contaminated and control lakes in Florida. Environ. Health Perspect. 1994, 102 (8), 680–88. (4) Nichols, D. J.; Daniel, T. C.; Moore, P. A., Jr.; Edwards, D. R.; Pote, D. H. Runoff of estrogen 17 β-estradiol from poultry litter applied to pasture. J. Environ. Qual. 1997, 26 (4), 1002–06. (5) Kolodziej, E. P.; Harter, T.; Sedlak, D. L. Dairy Wastewater, aquaculture, and spawning fish as sources of steroid hormones in aquatic environment. Environ. Sci. Technol. 2004, 38 (23), 6377–84. (6) Soto, A. M.; Calabro, J. M.; Prechtl, N. V.; Yau, A. Y.; Orlando, E. F.; Daxenberger, A.; Kolok, A. S.; Guillette, L. J., Jr.; le Bizec, B.; Lange, I. G.; Sonnenschein, C. Androgenic and estrogenic activity in cattle feedlot effluent receiving water bodies of eastern Nebraska, USA. Environ. Health Perspect 2004, 112 (3), 46–52. (7) USDA-Natural Resource Conservation Service, Conservation practice standard: Waste utilization code 633, 2005. http:// efotg.nrcs.usda.gov/references/public/AL/tg633.pdf. (8) USDA-Natural Resource Conservation Service, Conservation practice standard: Nutrient management code 590, 2008. http:// efotg.nrcs.usda.gov/references/public/IA/IA590Dec08.pdf (Accessed February 25, 2010). (9) Michaud, J. P. A Citizens’ Guide to Understanding and Monitoring Lakes and Streams; Department of Ecology Publication Office: Washington State Department of Ecology, 1991. (10) US-EPA. Methods for chemical analysis of water and wastes, EPA/ 600/4-79/020. nitrate/ite 353.1, ammonia 350.1, and phosphorus 365.4, 1983. http://nepis.epa.gov/Exe/ZyPURL.cgi?Dockey) 30000Q10.txt (Accessed February 25, 2010). (11) Recommended Methods of Manure Analysis. Bulletin A3769; Peters, J., Ed.; University of Wisconsin Extension: Madison, WI, 2003; http://learningstore.uwex.edu/pdf/A3769.pdf. (12) Shappell, N. W. Estrogenic activity in the environment: Municipal wastewater effluent, river, ponds, and wetlands. J. Environ. Qual. 2006, 35 (1), 122–32. (13) Wakeling, A. E.; Dukes, M.; Bowler, J. A potent specific pure antiestrogen with clinical potential. Cancer Res. 1991, 51, 3867–73. (14) Rasmussen, T. H.; Nielsen, J. B. Critical parameters in the MCF-7 cell proliferation bioassay (E-Screen). Biomarker. 2002, 7, 322–36. (15) Shappell, N. W.; Billey, L. O.; Forbes, D.; Matheny, T. A.; Poach, M. E.; Reddy, G. B.; Hunt, P. G. Estrogenic activity and steroid hormones in swine wastewater through a lagoon constructedwetland system. Environ. Sci. Technol. 2007, 41, 444–50. (16) Hanselman, T. A.; Graetz, D. A.; Wilkie, A. C.; Szabo, N. J.; Diaz, C. S. Determination of steroidal estrogens in flushed dairy manure wastewater by gas chromatography-mass spectrometry. J. Environ. Qual. 2006, 35, 695–700. (17) SAS/STAT Software, Version 9.2 of the SAS System for Windows; SAS Institute Inc.: Cary, NC, Copyright 2002-2008. (18) Saxton, A. M. A macro for converting mean separation output to letter groupings in Proc Mixed. In Proceedings of the 23rd SAS Users Group International; SAS Institute: Cary, NC, 1998, 1243-46. (19) Young, W. F.; Whitehouse, P.; Johnson, I.; Sorokin, N. Proposed Predicted No Effect Concentrations (PNECs) for Natural and Synthetic Steroid Oestrogens in Surface Waters, Environment Agency R & D Technical Report P2-T04/1; England and Wales Environment Agency: Bristol, 2002; pp 93-95. (20) Nichols, D. J.; Daniel, T. C.; Edwards, D. R.; Moore, P. A., Jr.; Pote, D. H. Use of grass filter strips to reduce 17β-estradiol in runoff from fescue-applied poultry litter. J. Soil Water Conserv. 1998, 53, 74–77. (21) Raman, D. R.; Williams, E. L.; Layton, A. C.; Burns, R. T.; Easter, J. P.; Daugherty, A. S.; Mullen, M. D.; Sayler, G. S. Estrogen content of dairy and swine wastes. Environ. Sci. Technol. 2004, 38, 3567–73. (22) Sarmah, A. K.; Northcott, G. L.; Leusch, F. D. L.; Tremblay, L. A. A survey of endocrine disrupting chemicals (EDCs) in municipal sewage and animal waste effluents in the Waikato region of New Zealand. Sci. Total Environ. 2006, 355, 135–44.
(23) Hanselman, T. A.; Graetz, D. A.; Wilkie, A. C. Manure-borne estrogens as potential environmental contaminants: A review. Environ. Sci. Technol. 2003, 37, 5471–78. (24) Johnson, A. C.; Williams, R. J.; Matthiessen, P. The potential steroid hormone contribution of farm animals to freshwaters, the United Kingdom as a case study. Sci. Total Environ. 2006, 362, 166–78. (25) Zheng, W.; Yates, S. R.; Bradford, S. A. Analysis of steroid hormones in a typical dairy waste disposal system. Environ. Sci. Technol. 2008, 42, 530–35. (26) Raman, D. R.; Layton, A. C.; Moody, L. B.; Easter, J. P.; Sayler, G. S.; Burns, R. T.; Mullen, M. D. Degradation of estrogens in dairy waste solids: Effects of acidification and temperature. Trans. ASAE 2001, 44 (6), 1881–88. (27) Colucci, M. S.; Bork, H.; Topp, E. Persistence of estrogenic hormones in agricultural soils: I. 17β-estradiol and estrone. J. Environ. Qual. 2001, 30, 2070–76. (28) Hutchins, S. R.; White, M. V.; Hudson, F. M.; Fine, D. D. Analysis of lagoon samples from different concentrated animal feeding operations for estrogens and estrogen conjugates. Environ. Sci. Technol. 2007, 41, 738–44. (29) Gadd, J. B.; Tremblay, L. A.; Northcott, G. L. Steroid estrogens, conjugated estrogens and estrogenic activity in farm dairy effluents. Environ. Pollut. 2010, 158, 730–36.
(30) Dodds, W. K.; Oakes, R. M. Controls on nutrients across a prairie stream watershed: Land use and riparian cover effects. Environ. Manage. 2006, 37, 634–46. (31) US-EPA. 40 CFR Ch. I (7-1-02 Edition), 141.62 Maximum contaminant levels for inorganic chemicals, Nitrate nitrite maximum contaminant level In Part 141 National Primary Drinking Water Regulations. 2002. http://edocket.access.gpo. gov/cfr_2002/julqtr/pdf/40cfr141.51.pdf. (32) US-EPA, Office of Water. Draft 2009 update aquatic life ambient water quality criteria for ammonia - freshwater. Document No. EPA-822-D-09-001, 2009. http://www.epa.gov/waterscience/ criteria/ammonia/2009update.pdf (Accessed February 25, 2010). (33) Servos, M. R.; Bennie, D. T.; Burnison, B. K.; Jurkovic, A.; McInnis, R.; Neheli, T.; Schnell, A.; Seto, P.; Smyth, S. A.; Ternes, T. A. Distribution of estrogens, 17β-estradiol and estrone, in Canadian municipal wastewater treatment plants. Sci. Total Environ. 2005, 336, 155–70. (34) US-EPA, Office of Water. Ambient water quality criteria recommendations. Information supporting the development of state and tribal nutrient criteria for rivers and streams in nutrient ecoregion VI. Document No. EPA-822-B-00-017, 2000. http:// www.epa.gov / waterscience / criteria / nutrient / ecoregions / rivers/rivers_6.pdf (Accessed February 25, 2010).
ES903669M
VOL. 44, NO. 7, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
2371
