Intersex and other Indicators of Endocrine Disruption throughout the

Northeast

Luke R. Iwanowicz, Vicki S. Blazer, Kelly L. Smalling

USGS, Leetown Science Center, National Fish Health Research Laboratory USGS, New Jersey Water Science Center

April 26, 2017

What is Intersex?

An abnormal condition characterized by the presence of male and female reproductive components in a gonad that should be exclusively male OR female.

So what is Normal?

FEMALE MALE

Mature sperm

Immature oocytes

Intersex Male

(testicular oocytes)

Emerging Contaminants (Chemicals of Emerging Concern; CECs)

• Synthetic or natural compounds that are not commonly monitored or regulated, but are present in the environment and lead to adverse effects

• Chemically engineered to have a biological effect at low concentrations and may inadvertently effect non-target organisms sub ppb concentrations

• Contains the group of chemicals called “endocrine disruptors”

Sewage treatment facilities

Confined (intensive) animal production

Run-off… Pesticides and other emerging contaminants

Phytoestrogens

Many others…………………………..

Estrogens in the Environment

2003 Potomac Watershed: Cause for Concern?

• Multiple bacterial pathogens

• Parasite/fungal infections

• Skin lesions • Largemouth Bass

virus • High prevalence of

intersex

• Immunosuppression • Causes?

Land-use Characteristics Intersex Prevalence Intersex Severity

r2 p r2 p

Human population density 0.39 0.10 0.42 0.08 Number of WWTP 0.22 0.24 0.34 0.13 WWTP Flow 0.32 0.15 0.63 0.02 Percent agriculture 0.63 0.02 0.50 0.05 Tot. Animal Feeding Operations 0.28 0.17 0.56 0.03 Poultry houses 0.27 0.18 0.50 0.05 Total animals 0.27 0.18 0.48 0.06 Animal density 0.49 0.05 0.58 0.03

Site Human Density WWTP WWTP

Flow % Ag AFO Animal

Numbers Intersex

Gauley River

0.06 0 0 0.5 0 464 11.3% 0.02 (0.07)

South Branch Petersburg

0.07 3 0.95 16.4 296 (296)

1,450,120 74.3% 0.97 (0.95)

South Branch Moorefield

0.07 4 1.43 15.2 497 (496)

7,384,685 54.5% 0.50 (0.50)

South Branch Springfield

0.08 5 1.93 15.2 565 (562)

8,719,093 82.2% 1.02 (0.76)

Shenandoah North Fork

0.28 50 1.59 32.7 1,174 (960)

11,757,596 90.0% 1.16 (0.78)

Shenandoah Mainstem

0.43 101 25.66 32.6 3,655 (2,539)

33,928,442 93.0% 1.64 (0.93)

Shenandoah South Fork

0.56 19 20.84 35.9 2,029 (1,176)

14,788,173 100.0% 1.83 (0.65)

Conococheague Creek (lower)

0.69 13 8.31 50.3 10 (1)

1,819,225 87.5% 1.03 (0.78)

Associations of Land-use with Intersex Spawning Study (Chesapeake Bay Drainage)

Blazer et al (2012) Reproductive endocrine disruption in smallmouth bass (Micropterus dolomieu) in the Potomac River basin: spatial and temporal comparisons of biological effects. Environmental Monitoring and Assessment, 184: 4309-4334

To understand the population level effects of endocrine disruption resulting from exposure to contaminants in the Chesapeake Bay Watershed

• Identify and quantify the sources, fate, transport, distribution, and exposure of EDCs to fish and wildlife.

• Evaluate the effects EDCs on fish and wildlife in the environment.

• Determine the mechanism(s) and thresholds for adverse effects on fish and wildlife species.

• Develop appropriate assessment tools and models for risk evaluation of EDCs on natural resources.

Northeast Region FWS NWRs

• Site selection –19 Refuges

Assabet River, Back Bay, Blackwater, Cherry Valley, Erie, Great Bay, Great Meadows, Great Swamp, John Heinz, Mason Neck, Missisquoi, Montezuma, Moosehorn, Ohio River Islands, Patuxent, Rappahannock, Sunkhaze, Umbagog, and Wallkill

Iwanowicz et al. 2015. Ecotox Environ Safety. 124: 50-59.

Target species

• Smallmouth bass (10 per sex)

• Largemouth bass (10 per sex)

Sample dates

• Sampling occurred during Late August thru late October

– 2008 – Rivers – 2009 – Rivers – 2010 – Impoundments

Fish collection

• Pulsed DC electroshocking • Angling when necessary

Field Sampling

• Morphometric data collected

• External health assessment

• Blood collected for plasma

– Vitellogenin

• Internal organs removed and preserved in Z-Fix

– Histopathology Intersex

• Liver and anterior kidney collected for possible gene transcription work

• Water samples for in vitro bioassays

Field Sample Processing

Sample collection

Sample analysis

• Histopathology - Intersex (testicular oocytes in males)

• ELISA - Plasma vitellogenin

• In vitro bioassays – nuclear receptor activation

– Estrogenicity, androgenicity & glucocorticoid receptor translocation

Smallmouth bass intersex prevalence

• Intersex ranged from 60- 100% (composite = 85%)

• 118 males evaluated in total

• No differences between up and downstream locations

• Previous work has identified intersex as low as 10-14%

OH/WV ME NJ VT ME PA/NJ NH/ME

Estrogenicity Results

• Estrogenicity measured at a number of locations

• Highest concentrations measured in WWTP effluent (over 5ng/L)

• PNOEL = 0.73ng/L

• 36% of 38 non-WWTP samples were estrogenic

• 79% of those were above the PNOEL

General Comments

• Evidence of estrogenic endocrine disruption in waters of FWS Northeast Region NWRs

• Apparent differences between SMB and LMB – SMB intersex 85% (Hinck et al 2009: 33%) (Blazer et al. 2014 > 90%) – LMB intersex 27% (Hinck et al 2009: 18%)

• The current study did not comprehensively evaluated water quality or site specific emerging contaminants

• Further research may be warranted to identify the sources and causes (chemical?) biological effects

The Full Cast (Co-authors NWR Study)

L.R. Iwanowicz, V.S. Blazer, A.E. Pinkney, C.P. Guy, A.M. Major, K. Munney, S. Mierzykowski, S. Lingenfelser, A. Secord, K. Patnode, T.J. Kubiak, C. Stern, C.M. Hahn, D.D. Iwanowicz, H.L. Walsh,

and A. Sperry

ED in New Jersey

• Sites selected based on previous monitoring data (WQ and/or bio) and land-use

• 101 sites sampled in fall

under base-flow – 16 reservoirs – 85 river sites – Estrogenicity only in

water

THANK YOU! FOR MORE INFORMATION

ksmall@usgs.gov

Funding provided by US Fish and Wildlife Service Division of Environmental Quality, USGS Priority Ecosystems, Contaminant Biology, Ecosystems and Cooperative Water Programs, NJ Department of Environmental Protection.