Intersex in Bass “Emerging” Contaminant

Issues

Vicki S. BlazerU.S Geological Survey

National Fish Health Research LaboratoryLeetown Science Center, Kearneysville, WV

Sampling With WV DNRu Spring –Summer 2002 – Fish kills and fish

lesionsu Randomly examined a variety of fish species

focusing on the external lesions– Variety of causes – bacterial, parasitic, noninfectious– Suggested some environmental stressors that may

cause immunosuppression and increased disease susceptibility

Sampling 2003

uMore comprehensive fish health assessments of bass from 7 sites

uNecropsy-based, plasma collection, histologic (microscopic) analysis of all organ

uFindings included:– High parasite loads at some sites– Oxidative damage in a number of

organs– Intersex

Intersex in Fish

ôIncreasingly there are reports of wild and laboratory-exposed fishes with intersexôIntersex (ovotestis; testisova) is a general

term for gonadal abnormalities most often noted microscopically, occasionally macroscopicallyôMost reports involve observation of female

germ cells or immature oocytes within a predominantly male gonadôIncludes malformed/intersex reproductive

ducts; large areas of ovarian tissue; testicular tissue within ovaries

Intersexin Normally Gonochorist Fishes

u Suggested as a marker of endocrine disruption

uMost often associated with exposure to estrogenic compounds

u Has been reported in a number of fish species

33% (6)72% (11)80% (10)South Branch857% (7)70% (10)14% (7)Cacapon733% (6)ND0% (3)North River6

67% (12)90% (10)60% (5)South Branch536% (11)69% (13)NDSouth Branch440% (10)85% (13)0% (4)South Branch325% (8)75% (12)NDNorth Fork250% (4)100% (7)33% (6)South Branch1

Summer 2004% Intersex

Spring 2004% Intersex

Summer 2003% IntersexRiver SystemSite

#

Frequency of Intersex in Smallmouth Bass

Questions

u How widespread is it within the Potomac and ultimately Chesapeake drainage?

u Is it somehow “normal” for smallmouth bass?– Or are there other indications of exposure?

u How does the incidence in the Potomac compare to other, out of basin drainages?

u Does it affect reproductive capacity and hence eventually population levels

Sampling in the Potomac Watershed

Ovotestis Severity Index

Score 1Single oocyte

Score 2More than 1 oocyte

Not associated - multifocal

Ovotestes Severity Index

Score 3 Cluster>1 but <5 associated oocytes

Score 4 Zonalmultiple clusters or

>5 associated oocytes

Fall 2005Prevalence of Intersex

Potomac Basin

Out of Basin

Fall 2005Severity of Intersex

Potomac Basin

Out of Basin

Other Biomarkers of ExposureVitellogenin

ôVitellogenin is the serum/plasma phospholipoglycoprotein precursor to egg yolkôNormally found in measurable amounts

only in the blood of sexually mature female (egg-laying) vertebratesôEstrogen stimulates the liver to produce

vitellogenin which travels to the ovaries via bloodstream and is sequestered by developing oocytes

Vitellogenin in Male Fish

ôMales have the gene to produce vitellogenin - usually not turned onôExposure to natural or synthetic

estrogens from human and agricultural sources, phytoestrogensand a variety of estrogen mimics can turn the genes on

Vitellogenin in Male Bass in the Potomac

69%23%Blue Plains

0.810.3013%100%LowerMonocacy

0.850.3250%80%UpperMonocacy

0.620.1350%90%LowerConcocheague

1.240.3950%100%UpperConcocheague

FemaleGSI

Male GSI

IncidenceVitellogenin

IncidenceIntersex

Site

A Problem??

u IntersexuCirculating vitellogenin in male fishuOther histologic observationsuApparent immunosuppression

– Opportunistic infections– Lesions– Fish kills

Cause(s) in the Potomac

ôEstrogenic chemicals are most likely culpritôUnlikely that there is one causeôMixture of contaminants from various sourcesôCause(s) may not be the same in all areasôHuman wastewater, agricultural runoff, industrial,

atmospheric deposition

v Sewage treatment facilities

v Confined (intensive) animal production

Chemicals“Emerging Contaminants”

ôAgricultural Sources - AnimalôNatural and synthetic hormones

ôPesticides and herbicidesôAgricultural and household use

ôPersonal care productsôPharmaceuticalsôSynthetic Hormones – birth control, hormone

replacement therapyôAntimicrobials – soap, detergent, toothpasteôFragrances, organic UV filters, DEET

“Emerging Contaminants”Pharmaceutical, Hormones, Organic

Wastewater Contaminants

ôThese are chemicals that are produced to have biological effects at low levels

ôFor many that end up in human sewage and ultimately the aquatic environment, aquatic organisms may be constantly exposed

ôNational Reconnaissance (Kolpin et al. 2002)ôMeasured 95 in water from 139 streams in 30 statesôMost downstream of urban areas or livestock

productionôMedian of 7, as many as 38, in a given sample

Are there EDCs in the South Branch?

Brominated flame retardantBDE100Brominated flame retardantBDE 99Brominated flame retardantBDE 47

Pesticidecis-NonachlorPesticidetrans-NonachlorPesticidecis-ChlordanePesticidetrans-Chlordane

Pesticide/ FungicideHexachlorobenzeneInsecticideLindane (γ-HCH)

Chlorinated anti-bacterialTriclosanOrganophosphate insecticideChlorpyrifos

Pesticide/ HerbicideTrifluralinPersonal Care/ContraceptiveEthynyl estradiol (EE2)

CategoryContaminant

Questions That Need to be Addressed

ôInteractive effects of the complex mixturesIn vitro studies and experimental exposures suggest

many of these compounds are additive in estrogenicity

ôWhat ends up in the sediment?Levels of estrogenic activity have been found to be

much higher (532 to 748-fold) in sediments than in the overlying water, suggesting these chemicals accumulate in the bed sediment phase (Peck et al 2004).

Effects of IntersexIndividual and Population Levels

u Studies with wild roach (Rutilus rutilus)u Intersex males

– Produced up to 50% less milt (per gram)– Reduced percent of motile sperm– Lowered ability of sperm to successfully

fertilize eggs and produce viable offspring

Research Needs

u Need to identify the contaminants and sourcesu Need a risk assessment of various landuse

influencesu Need to understand exposure times and routes

– Does exposure at a young age predispose the fish for the rest of its life to lower reproductive capacity and increased disease susceptibility?

ôNeed to understand that the intersex problem is just one part of a complex issue

Acknowledgementsv USGS NFHRL

Alison Griffin Kathy SpringDeb Cartwright Luke IwanowiczBlake Martin

v West Virginia DNR

v Maryland DNR

v Virginia DGIF

v US Fish Wildlife Service

v US Army Corps of Engineers

v Tennessee TechMartha Wells

v Texas Tech UniversityReynaldo Patino

v USGS Water Resource Division

v US EPA

v Virginia Institute of Marine Science

v West Virginia University