Grantee Research Project Results

Final Report: Transport/Fate/Ecological Effects of Steroids from Poultry Litter & Evaluations of Existing/Novel Management Strategies

EPA Grant Number: R833418
Title: Transport/Fate/Ecological Effects of Steroids from Poultry Litter & Evaluations of Existing/Novel Management Strategies
Investigators: Fisher, Daniel J. , Kane, Andrew S. , Staver, Kenneth , Yonkos, Lance T. , VanVeld, Peter , Klauda, Ronald J.
Institution: Wye Research and Education Center , Maryland Department of Natural Resources , Virginia Institute of Marine Science , School of Medicine at the University of Maryland
EPA Project Officer: Aja, Hayley
Project Period: August 1, 2007 through June 30, 2010 (Extended to July 31, 2012)
Project Amount: $694,736
RFA: Fate and Effects of Hormones in Waste from Concentrated Animal Feeding Operations (CAFOS) (2006) RFA Text | Recipients Lists
Research Category: Endocrine Disruptors , Human Health , Safer Chemicals

Objective:

The objectives of the proposed study were to determine the environmental transport, fate and ecological effects of fecal sex steroids following agricultural field application of poultry manure. Previous studies at our Wye Research and Education Center (WREC) laboratory have demonstrated that: (1) fecal steroids in poultry manure reach receiving waters via rain-induced runoff; (2) this runoff is sufficiently estrogenic to feminize male fish; and (3) differences in agricultural management strategies can affect steroid concentrations in runoff and receiving waters.

Our work under the 2007 EPA CAFO research grants addressed remaining questions concerning environmental persistence and bioactivity of steroids upon reaching surface waters and further investigated effects of agronomic practices on mitigating resultant environmental steroid loads. This was accomplished using laboratory, controlled research field, and in situ watershed investigations. Steroids and estrogenicity in aqueous manure mixtures (lab-generated and field-collected) were monitored over time to determine degradation rates. In all analyses, particular attention was paid to determining proportions of fecal steroids, most importantly 17β-estradiol (E2) and estrone (E1). Fish were exposed to aqueous manure mixtures in laboratory assays to determine the effects of steroid degradation on bioactivity as measured by two vitellogenin (VTG) assays and two novel in-vitro estrogen-inducible reporter-gene assays.

A final multi-litter fish exposure was conducted to compare various analytical techniques for measuring estrogens and estrogenicity (GC/MS/MS, LC/MS/MS, UPLC/MS/MS, VTG blood plasma ELISA, VTG mRNA, K_BLUC, and E-Screen). Effects of agricultural management practices on rates of transport of fecal steroids and nutrients to surface waters via rain-induced runoff were investigated using adjacent 33-acre research fields cropped variously under standard No-till practices of direct surface manure application, or by a novel Sub-surface Manure Injection (SS) technique and a currently used Turbo-Till^® (TT) (Great Plains, Salina, KA) manure application practice. Turbo-Till is the name of the particular tillage equipment used at the WREC for this study. The genralized terminology for this type of tillage is “verticle tillage.” Finally, Maryland Biological Stream Survey (MBSS) protocols were applied to agriculturally influenced watersheds to assess community and population level disturbances resulting from fecal steroid exposure. Based on results from the early MBSS sampling effort, largemouth bass sampling for intersex (testicular oocytes) was conducted in lakes on the Delmarva Peninsula in Maryland and Delaware.

Summary/Accomplishments (Outputs/Outcomes):

Maryland Biological Stream Survey (MBSS)

Results from the MBSS sampling conducted in 2007 and 2009 showed fair to good stream health in all four streams with Fish IBI (FIBI) and Benthic IBI (BIBI) scores ranging from 3 to 5 (Table 1). The IBI scores are based on a condition scale that ranges from 1 (very poor) to 5 (good), with scores below 3.0 indicating poor/very poor biological condition [Southerland, et al., 2005]. Stream segments with IBIs above 3 are comparable to sentinel/reference site conditions said to meet MDDNR expectations (fair to good). From a fish and benthic community standpoint, all of the sampled streams were rated fair to good. The stream biological community health metrics were equal to or greater than those calculated for a number of yearly samplings of Skeleton Creek, a DNR sentinel reference site [Becker, et al., 2010]. Many components of agriculture runoff may not drastically impact lotic (flowing systems) because any contaminants are transient there. In contrast, such runoff could have greater impacts to lentic (lake) environments where the biota are exposed for a longer time period (contaminants do not flow away quickly). This is why MDDNR and WREC decided to begin sampling lakes and ponds in Maryland and Delaware for largemouth bass to determine if intersex (testicular oocytes) could be identified as in rivers in the mid and western parts of Maryland and nationally by Alvarez, et al. [2009], Blazer, et al. [2007], and Hinck, et al. [2009].

Lake and River Sampling of Largemouth Bass (LMB) on the Delmarva Peninsula

Vitellogenin (VTG) in blood plasma was not detected in any male largemouth bass (LMB). Intersex (testicular oocytes [TO] or the presence of eggs in male bass gonads) was observed in fish from all six lakes in 2008 (Table 2, Figure 1). These lakes drain watersheds dominated by agriculture. Collectively, 38 individuals (63%) were found to possess TO with lake-specific TO prevalences ranging from 40% to 88% and average severity indices ranging from 0.11 to 0.37. In 2009, a total of 34 mature male largemouth bass were collected from Tuckahoe Lake and from the Pocomoke River in spring (pre-spawn) and again in summer (post-spawn). Occurrence of intersex in Tuckahoe Lake fish ranged from 33% (summer) to 50% (spring). In the Pocomoke River, occurrence of intersex ranged from 40% (spring) to 80% (summer). Site average severity indices for the two systems ranged from 0.16 to 0.53. Collectively, of 94 male Delmarva LMB assessed over the 2 years, 54 (57%) were found to possess TO with site specific prevalences ranging from 33% to 88%. Prevalence of TO in Delmarva largemouth bass was similar to reported levels of intersex in largemouth bass and smallmouth bass from the Western Shore of Maryland and nationally [Blazer, et al., 2007; 2011; Iwanowicz, et al., 2009; Hinck, et al., 2009]. Severity indices, however, were generally lower than those reported in smallmouth bass collected from the Potomac River system, but higher than those reported for smallmouth bass from minimally impacted reference sites outside of the Potomac Drainage [Blazer, et al., 2011]. Seasonal differences in TO prevalence were inconsistent with Tuckahoe LMB having greater TO prevalence in spring vs. summer and Pocomoke LMB having greater TO prevalence in summer vs. spring. Severity was greater in both systems in summer compared to spring; however, low numbers of organisms and high variability preclude assessing statistical significance. These are the first reported findings of intersex in waters of the Delmarva Peninsula.

Bioassays and Estrogen/Estrogenicity Degradation Studies

Measurement of estrogens and estrogenicity in exposure aquaria over time using poultry litter from various sources in 2010 allowed us to investigate changes in estrogenic activity over time. Results were consistent with degradation experiments using runoff from litter amended fields in 2008 and 2009. Consistently, measured E2 and E1 levels (GC/MS/MS) in poultry litter solutions were low at exposure initiation (T0) and increased during the 9-day fish exposure period (T9) before decreasing by day 28 (T28) (Figure 2). Estrogenicity (E-Screen) followed an identical pattern with activity increasing from T0 to T9 before decreasing at T28. In each instance, estrogenicity was explained entirely by measured E2 levels. This is not surprising given that E2 is the most potent vertebrate estrogen. Although differences in amplitude of measured concentration/activity varied between poultry litter sources, the pattern of response was consistent across all integrators. The explanation for increases in measured estrogen concentrations and increases in estrogenicity relate to the abundance of conjugated estrogens present in poultry litter. These water soluble conjugated steroids go readily into solution, but have minimal estrogenic activity. Microbial degradation, however, converts conjugates to their more estrogenic parent compounds. In this way, detectible estrogens and estrogenicity increase as a result of microbial activity.

Adult male Fathead minnows were exposed to the same poultry litter aqueous solutions from various sources described above (2010). Again, results of vitellogenesis from this experiment were consistent with vitellogenesis experiments using runoff from litter amended fields in 2008 and 2009. The process of vitellogenesis involves up-regulation of vitellogenin (VTG) mRNA within liver hepatocytes which leads to manufacture of VTG protein within cytoplasm and eventually transport in plasma. VTG was induced in all three litters tested (Figure 3). As one would expect, there is a strong relationship between measured VTG mRNA in liver and VTG protein in plasma sampled from any given fish (Figure 4). Mean fathead minnow plasma VTG concentrations measured at T9 correlated very strongly with measured E2 concentrations (GC/MS/MS) in exposure aquaria averaged over the 9- day exposure interval (Figure 5). Interestingly, significant induction of plasma VTG was measured in treatments where analytically measured E2 was below 2.0 ng/L, a very low environmental concentration. This may mean that “biological” measurement of estrogenicity may be equally or perhaps more sensitive than chemical analytical methods.

Of the estrogen analytical methods investigated in the 2010 multi-source poultry litter aqueous exposures, Gas Chromatography Dual Mass Spectrometry and Liquid Chromatography Dual Mass Spectrometry proved to be equally sensitive for measuring environmental samples containing poultry litter. Results from these methods correlated very well and both had low detection limits (Figure 6). In contrast, Radioimmunoassay and Ultra Performance Liquid Chromatography methods had difficulty with measuring poultry litter samples due to matrix interferences and did not correlate well with the other chemical analytical methods (Figures 7 and 8). In addition, both VTG analytical methods measuring fish responses discussed above were sensitive in detecting estrogenicity at the very low levels detected by the more traditional chemistry methods and correlated well with each other (Figure 4). Likewise, In-vitro Estrogenicity Analysis (K_BLUC Reporter-Gene Assay and E-screen Proliferation Assay) using estrogen-inducible reporter-gene cell lines also proved equally sensitive to the chemical methods and correlated well with each other (Figure 9) and with the best chemical methods (Figure 10). In vitro assays are sensitive indicators of estrogenicity with detection limits often below those of analytical methods. Likewise, induction of VTG mRNA and resulting VTG protein are sensitive measures of estrogenicity and can yield indications of exposure to estrogens at or near analytical limits of quantitation.

Agricultural Management (Tillage) Practices and Their Effects on Nutrient and Steroid Runoff

State of Maryland New Nutrient Management Regulations

On October 15, 2012 Maryland’s New Nutrient Management Regulations went into effect (http://www.mda.maryland.gov/pdf/finalnmregs.pdf) (COMAR 15.20.07). The new management regulations are part of Maryland’s Watershed Implementation Plan required by EPA to meet EPA’s goals to avoid future consequences of nutrient runoff. The regulations state that ...”organic nutrient sources shall be injected or incorporated as soon as possible, but no later than 48 hours after application.” The data generated by Dr. Ken Staver (WREC) and the other Co-PIs at WREC for this EPA CAFO study on the efficacy of alternative tillage practices were used to help provide the scientific basis for this decision. Three separate litter applications were examined during this project. No-till (NT) management is a soil conservation tillage technique whereby the field is not tilled following litter application on the surface while Turbo-till (TT) is vertical tillage practice where the litter is lightly tilled and mixed into the top few inches of soil after litter application. In Subsurface Injection (SS) using a Poultry Litter Subsurfer, circular blades cut a furrow in the soil in which the litter is dropped. The cut piece then falls back into the furrow and is rolled smooth. With this method, there is much less surface disruption and the litter is buried a few inches below the surface. As discussed below, both vertical tillage (TT) and Subsurface Litter Injection (SS) provided reductions in steroid concentrations and nutrient concentrations and loads in runoff from fields compared to No-till (NT). This is a major finding of the EPA CAFO study. The new nutrient Maryland Nutrient Management Regulations requiring litter incorporation or injection should result in a significant reduction in nutrients and other water soluble contaminants (e.g., steroids) to the Chesapeake Bay.

Steroids - One of the advantages of incorporating litter into the soil is that the soils have a greater capacity for water retention and less initial runoff. For example, during the the first two major precipitation events of 2008 and 2009 there was no runoff from the TT field and significant runoff from the NT field. The more important finding of this study was that runoff concentrations of estrogens and estrogenicity were reduced from the TT and SS incorporation practices compared to NT (Table 3). The average percentage reduction of estrogens and estrogenicity for TT over the 3 years of testing (2008 – 2010) compared to NT was 41% and 62%, respectively. For the SS tillage practice studied in 2011 the average reduction in estrogens compared to NT was 77%. These values represent large reductions in estrogens and estrogenicity from poultry litter amended fields simply by incorporating the litter into the soil.

Nutrients - The high density of poultry production on the Maryland Eastern Shore means that any successful strategy to reduce agricultural impacts on Chesapeake Bay includes effective practices for reducing nutrient losses associated with application of poultry litter to crop land. The challenge is to reduce nutrient losses without exacerbating sediment losses, which have been dramatically reduced in Maryland through widespread adoption of no-till methods. Vertical tillage (Turbo-till (TT) in the current study) is a new tillage practice that leaves plant residues on the soil surface while providing some incorporation of applied material. The effect of vertical tillage versus no-till on nutrient losses from corn production systems was compared during the 2008-2010 growing seasons in adjacent natural field-scale watersheds located at the Wye Research and Education Center (WREC) in the Wye River drainage basin, which is a sub-estuary of Chesapeake Bay. In addition, a new technique using a prototype device (Subsurfer) being developed by USDA-ARS that places poultry litter approximately 20 cm below the soil surface was compared to no-till during the 2011 growing season. Surface runoff volume varied widely seasonally and annually depending on precipitation patterns, but was relatively similar under the different tillage treatments except for immediately after litter application when tillage tended to reduce runoff volume. Area-normalized runoff volume was approximately 17-18 % of total precipitation; averaging approximately 22 cm/yr. Vertical tillage did increase sediment losses by approximately 50 percent relative to no-till with most of the increase occurring early in the growing season. But overall sediment losses were very low for crop land, averaging 156 kg/ha/yr from the vertical tillage watershed versus 102 kg/ha/yr from the no-till watershed during the 3-year comparison. Despite the higher sediment losses from the tilled watershed, vertical tillage sharply reduced dissolved nutrient losses when runoff occurred soon after poultry litter application. Average surface N losses during May were approximately fivefold higher from the no-till versus the tilled watershed but relatively similar the rest of the year. Runoff P losses also were much higher from the no-till watershed early in the growing season, with the effect lasting longer than for N. P losses were dominated by dissolved forms, which comprised approximately 90 % of total P losses from both watersheds. Vertical tillage reduced P losses during May-June by approximately 50 percent. Surface runoff P losses from the tilled watershed averaged 1.8 kg/ha from May through September as compared to 2.9 kg/ha from the no-till watershed with losses the rest of the year being very similar from the two watersheds. During the 1 year of evaluation of the Subsurfer (Subsurface injection) technique of application, the observed patterns of nutrient loss were similar to those measured during the vertical tillage/no-till comparison with the injection of poultry litter resulting in lower runoff nutrient concentrations early in the growing season. An extreme runoff event in August resulting from tropical storm Irene made 2011 a somewhat atypical year, but even during this large event, the Subsurfer application technique reduced dissolved nutrient losses in comparison to no-till. Overall, vertical tillage using a Turbo-till and subsurface application of poultry litter using the Subsurfer were found to be practices capable of reducing growing season losses of N and P in comparison to no-till associated with applications of poultry litter. These reductions in nutrient losses were achieved with only minimal increase in sediment or sediment-related nutrient losses. The reduction in dissolved nutrient losses early in the growing season may be especially important because the reductions were primarily of algal-available forms of N and P at a time when algal growth potential in receiving waters is high.

Table 1. Fish and Benthic IBI scores for streams sampled in 2007 and 2009.

Creek/Run	Year	MD DNR MBSS Site Designation	Fish IBI	Condition	Benthic IBI	Condition
Herring Run	2007	UPCK-211-X-2007	5.00	Good	4.43	Good
Watts Creek	2007	UPCK-212-X-2007	4.67	Good	5.00	Good
Tuckahoe Creek	2009	TUCK-450-H-2009	3.33	Fair	5.00	Good
Skeleton Creek	2000	UPCK-113-S-2000	2.00	Poor	3.00	Fair
Skeleton Creek	2001	UPCK-113-S-2001	2.67	Poor	4.43	Good
Skeleton Creek	2002	UPCK-113-S-2002	3.33	Fair	3.86	Fair
Skeleton Creek	2003	UPCK-113-S-2003	2.33	Poor	4.14	Good
Skeleton Creek	2004	UPCK-113-S-2004	4.00	Good	4.14	Good
Skeleton Creek	2007	UPCK-113-S-2007	3.00	Fair	3.86	Fair
Skeleton Creek	2009	UPCK-113-S-2009	3.00	Fair	3.29	Fair

Table 2. Prevalence and severity of testicular oocytes (TO) in male largemouth bass (Micropterus salmoides) collected from lakes on the Delmarva Peninsula during 2008 and 2009. Site average severity indices presented as mean ± standard deviation.

*Site*	*Sample Date*	n	*Testicular Oocyte*
*Site*	*Sample Date*	n	*Prevalence*	*Severity Index*
*2008*
Hearns Pond, DE	04/30/08	8	88%	0.22 ± 0.16
Moore’s Lake, DE	04/30/08	10	80%	0.37 ± 0.38
McColley Pond, DE	05/01/08	9	67%	0.25 ± 0.26
Williston Lake, MD	05/15/08	11	73%	0.24 ± 0.31
Smithville Lake, MD	05/22/08	10	40%	0.11 ± 0.23
Tuckahoe Lake, MD	05/29/08	12	42%	0.22 ± 0.48
*2009*
Tuckahoe Lake, MD	05/12/09	10	50%	0.16 ± 0.21
Tuckahoe Lake, MD	08/19/09	9	33%	0.25 ± 0.42
Pocomoke River, MD	05/14/09	10	40%	0.33 ± 0.56
Pocomoke River, MD	08/20/09	5	80%	0.53 ± 0.40

Table 3. Relative reduction of fecal estrogen concentrations as estrone (GC/MS/MS) and estrogenicity (K_BLUCin-vitro estrogen-inducible reporter-gene assay) in runoff following poultry litter application using different litter application practices. Reductions are in comparison to No-Till application.

Year	Tillage Practice		% Reduction in fecal estrogens from No-till	% Reduction in estrogenicity from No-till
2002	No Till	Conventional Till	40%	Not measured
2008	No Till	Turbo Till	58%	66%
2009	No Till	Turbo Till	28%	71%
2009	No Till	Turbo Till	Sample lost	66%
2010	No Till	Turbo Till	38%	43%
2011	No Till	Sub-Surface Injection (Grab)	70%	Not measured
2011	No Till	Sub-Surface Injection (Composite)	84%	Not measured

Figure 1. Testicular oocytes (TO) in largemouth bass (Micropterus salmoides) from the Delmarva Peninsula, USA. Clustered (A) and diffuse (B) previtellogenic oocytes (arrows) within epithelium of testicular tubules (Bar = 50 mm; H&E stain)

Figure 2. Changes in estrogens (GC/MS/MS) and estrogenicity (E-Screen) in fish exposure aquaria as indicated by samples measured at various time intervals. Exposure solutions were generated using poultry litter from three different integrators. Estrogenicity (solid black line) is reported as E2 equivalents (EEQ) with values indicated in graph as ng/L.

Figure 3. Fathead minnow VTG mRNA (A) and plasma protein (B) induction following 9-d 2010 exposures to aqueous solutions of three different poultry litters.

Figure 4. Comparison of fathead minnow VTG mRNA in liver hepatocytes and plasma VTG of fathead minnows after nine-day aqueous estrogenic exposures in 2010.

Figure 5. Comparison of fathead minnow plasma VTG and mean E2 (GC/MS/MS) concentration over the nine-day exposure interval in 2010.

Figure 6. Comparison between GC/MS/MS (ng/L) and LC/MS/MS (ng/L) methods for measurement of estrone (E1) in samples from fish exposures.

Figure 7. Comparison of E2 as measured by GC/MS/MS (ng/L) and RIA (ng/L) as measured in fish exposures.

Figure 8. Comparison of E1 as measured by GC/MS/MS (ng/L) and UPLC/MS/MS (ng/L) as measured in fish exposures to samples made with poultry litter.

Figure 9. Comparison of total estrogenicity (EEQ) between K_BLUC and E-Screen in vitro assays in samples from fish exposures.

Figure 10. Comparison of total estrogenicity (EEQ) as measured by the E-Screen in vitro assay and E2 as measured by GC/MS/MS in samples from fish exposures.

Conclusions:

Runoff from poultry litter amended fields and from aqueous litter exposures generated in the laboratory were found to be estrogenic to both adult and larval fish. Vitellogenin (VTG), a potent protein biomarker for endocrine disruption due to estrogens, was induced in male fathead minnows in both exposure scenarios. Male fish do not produce VTG unless exposed to some external source of estrogens. VTG can be measured directly in blood plasma or by measuring the induction of VTG-mRNA in fish livers. Both techniques proved to be equally sensitive in detecting VTG. In addition, larval fathead minnows exposed to poultry litter from runoff produced adult fish whose sex was harder to determine than fish from control treatments. Consistently, measured 17β-estradiol (E2) and estrone (E1) levels in poultry litter solutions were low at exposure initiation and increased during fish exposure periods before decreasing by day 28. Estrogenicity followed an identical pattern with activity increasing before decreasing at day 28. Estrogenicity was still measureable at day 28. In each instance, estrogenicity was explained entirely by measured E2 levels. This is not surprising given that E2 is the most potent vertebrate estrogen.

Of the estrogen and estrogenicity analytical methods compared in this study using split samples, Gas Chromatography Dual Mass Spectrometry and Liquid Chromatography Dual Mass Spectrometry proved to be equally sensitive for measuring environmental samples containing poultry litter. In contrast, Radioimmunoassay and Ultra Performance Liquid Chromatography methods had difficulty with measuring poultry litter samples due to matrix interferences. VTG analytical methods measuring fish responses were sensitive in detecting estrogenicity at the very low levels detected by the more traditional chemistry methods. Likewise, In-vitro Estrogenicity Analysis (K_BLUC Reporter-Gene Assay and E-screen Proliferation Assay) using estrogen-inducible reporter-gene cell lines also proved equally sensitive to the chemical methods. Both VTG and In-vitro Estrogenicity Analysis can yield indications of exposure to estrogens at or near chemical analytical limits of quantitation and the In-vitro method can do this without requiring fish exposures.

A large part of this project involved field sampling to detect possible endocrine disruptive effects in poultry litter influenced streams and lakes on the Delmarva Peninsula. Rigorous sampling of four small streams in agriculturally dominated watersheds did not show degraded systems when compared to reference streams. Many components of agriculture runoff may not drastically impact lotic (flowing systems) because any contaminants are transient there. In contrast, such runoff could have greater impacts to lentic (lake) environments where the biota are exposed for a longer time period (contaminants do not flow away quickly). Largemouth bass sampled from six lakes and ponds in Maryland and Delaware and a large river in Maryland showed male fish with gonads containing eggs (intersex). Intersex prevalences ranged from 40% to 88% similar to prevalence values from other parts of the country. These are the first reported findings of intersex in waters of the Delmarva Peninsula.

Finally, our investigations into the benefits of using alternative poultry litter tillage practices to reduce nutrient and steroid runoff from litter amended fields found significant reductions in these contaminants from practices that incorporated the litter into the soil compared to No-till litter applications. Both vertical tillage (Turbo-till) and subsurface injection (Poultry Litter Subsurfer) dramatically reduced runoff or nutrients, especially dissolved phosphorous, and steroids. On October 15, 2012 Maryland’s New Nutrient Management Regulations went into effect. The new management regulations are part of Maryland’s Watershed Implementation Plan required by EPA to meet EPA’s goals to avoid future consequences of nutrient runoff. The regulations state that ...”organic nutrient sources shall be injected or incorporated as soon as possible, but no later than 48 hours after application.” Organic nutrient sources include animal manure. The data generated by Dr. Ken Staver and the other Co-PIs at WREC for this EPA CAFO study on the efficacy of alternative tillage practices were used to help provide the scientific basis for this decision.

References:

Alvarez DA, Cranor WL, Perkins SD, Schroeder VL, Iwanowicz LR, Clark CC, Guy CP, Pinkney AE, Blazer VS and Mullican JE. 2009. Reproductive health of bass in the Potomac, USA drainage: Part 2. Seasonal occurrence of persistent and emerging organic contaminants. Environ Toxicol Chem 28:1084-1095.

Becker AJ, Stranko SA, Klauda RJ, Prochaska AP, Schuster JD, Kashiwagi MT and Graves PH. 2010. Maryland Biological Stream Survey’s Sentinel Site Network: A Multi-purpose Monitoring Program. Maryland Department of Natural Resources, Monitoring and Non-tidal Assessment Division, Annapolis, MD. 39 pp. http://www.dnr.maryland.gov/streams/pdfs/2010SentinelSiteReport.pdf

Blazer VS, Iwanowicz LR, Iwanowicz DD, Smith DR, Young JA, Hedrick JD, Foster SW and Reeser SJ. 2007. Intersex (testicular oocytes) in smallmouth bass Micropterus dolomieu from the Potomac River and selected nearby drainages. J Aquat Anim Health 19:242–253.

Blazer VS, Iwanowicz LR, Henderson H, Mazik PM, Jenkins JA, Alvarez DA and Young J. 2012. Reproductive endocrine disruption in smallmouth bass (Micropterus dolomieu) in the Potomac River basin: Spatial and temporal comparisons of biological effects. Environ Monit Assess 18:4309-34.

Fisher DJ, Staver KW, Yonkos LT, Ottinger MA and Pollock S. 2005. Poultry Litter-Associated Contaminants: Environmental Fate and Effects on Fish. Final Report. Maryland Center for Agro-Ecology, Inc. Queenstown, MD. 63 pp.

Hinck JE, Blazer VS, Schmitt CJ, Papoulias DM and Tillitt DE. 2009. Widespread occurrence of intersex in black basses (Micropterus spp.) from U.S. Rivers, 1995-2004. Aquat Toxicol 95:60-70.

Iwanowicz LR, Blazer VS, Guy CP, Pinkney AE, Mullican JE, Alvarez DA. 2009. Reproductive health of bass in the Potomac, USA drainage: Part 1. Exploring the effects of proximity to wastewater treatment plant discharge. Environ Toxicol Chem 28:1072-1083.

Southerland MT, Rogers GM, Kline MJ, Morgan RP, Boward DM, Kazyak PF, Klauda RJ and Stranko SA. 2005. New Biological Indicators to Better Assess the Condition of Maryland Streams. Publication # DNR-12-0305-0100. Maryland Department of Natural Resources, Monitoring and Non-Tidal Assessment Division, Annapolis, MD. 69 pp. http://www.dnr.maryland.gov/streams/pdfs/ea-05-13_new_ibi.pdf

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Publications
Type	Citation	Project	Document Sources
Journal Article	Yonkos LT, Friedel EA, Fisher DJ. Intersex (testicular oocytes) in largemouth bass (Micropterus salmoides) on the Delmarva Peninsula, USA. Environmental Toxicology and Chemistry 2014;33(5):1163-1169.	R833418 (2011) R833418 (Final)	Abstract from PubMed Abstract: Wiley-Abstract Exit

Progress and Final Reports:

Original Abstract

The perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.