SUMMARY OF ECOLOGICAL STUDIES WITHIN THE TITTABAWASSEE RIVER FLOODPLAIN

Presentation to the Community Advisory Committee Professor Matthew Zwiernik 20 June 2011 Studies in Support of Ecological Risk

 Rita Seston  Timothy B. Fredricks  Sarah J. Coefield  Dustin L. Tazelaar  Jeremy N. Moore  David W. Hamman  Mike M. Fales  Megan Barker  Michael N. Nadeau  Melissa S. Shotwell  Steph Plautz  William Folland  Patrick W. Bradley  Steve Bursian Ph.D  John P. Giesy, Ph.D. and many more… Research Contributors

 Cooperating landowners (~60)  Chippewa Nature Center  Shiawassee National Wildlife Refuge  Mike Bishop (Alma College)  U.S.Fish and Wildlife Service (East Lansing)  Sean Kennedy, Environment Canada  The Dow Chemical Company Technical Support Group (analytical laboratory) Overview of Presentation

Mink TCDF feeding study

Mammalian Mink TCDF and lab studies 4PeCDF toxicokinetics

Mink TCDD, TCDF and 4PeCDF reproduction study

MSU ecological AhR ligand binding field studies domain sequencing

Avian species sensitivity lab Injected egg studies hatchability

Injected egg exposure biomarker

Exposure biomarker response in field collected birds

Field Studies: Furan Exposure and Effects

 Initiated Spring 2004  Completed Fall 2008 (> 20,000 hours in the field)  Designed to provide decision makers with the most accurate information possible pertaining to the exposure and possible impacts of dioxin-like contaminants on resident wildlife of the Tittabawassee river basin  An expansion of the MDEQ screening level ERA  Multi-year  Multi-species  Site-specific  Multi-lines of evidence

Tittabawassee River Ecological Studies

 Song birds  Fish eating birds  Raptors  Migratory waterfowl  Mink Field Based Furan Exposure and Effects Studies

 Dietary exposure assessment  Tissue based exposure assessment  Individual and population health measurements

Field studies

 Dietary exposure assessment  Tissue based exposure assessment  Individual and population health measurements

Field studies

 Dietary exposure assessment  Tissue based exposure assessment  Individual and population health measurements

MSU ecological studies

 Aquatic & terrestrial exposure pathways evaluated  2004 – 2008 Tittabawassee River (and reference areas)  2006 expanded to Saginaw River Mink Dietary exposure

 Stomach content analysis (presented)  Scat analysis Mink Dietary exposure

• Fish • Tittabawassee River • Alexander, 1977 • Crayfish • site specific • 10% • Muskrat • 9% • 2% • Small Mammals • 3% • Amphibians • 4% • 4% • Vegetation • 8%

• 8% • 52%

• 19%

• 73% • 8% Mink dietary exposure • Mink Dietary Items

100

80 Reference Area T-River 60

40 TEQ (ppt)

20

0

Muskrat Sediment Crayfish egetation Forage fish V Amphibian Small mammal Example of concentrations of Dioxin-like contaminants in food web items

100 90 Crayfish tissue Error bars represent one 80 (composite samples) standard deviation 70

60

50

40

30

20 N=3 2 4 5 3 10

0 Sanford Chippewa Nature Smiths Crossing Tittabawassee Freeland Festival Imerman Park Shiawassee Veterans Dow Light House 250 SAN CenterCNC SC TownshipTTP Park ParkFFP IP WildlifeSNWR Refuge MemorialVMP Park DLH Sum Mamm TEQ

CRAYFISH Terrestrial beetle CRAYFISH 200 tissue 150 (composite samples) mammalian TEQ / kg / kg TEQ mammalian

ng 100

50

Mean N=4 5 3 2 4

0 Sum Mamm TEQ Sum Mamm TEQ Sum Mamm TEQ Sum Mamm TEQ Sum Mamm TEQ Sum Mamm TEQ Sum Mamm TEQ Sum Mamm TEQ Sum Mamm TEQ

INSECTA - INSECTA - INSECTA - INSECTA - INSECTA - INSECTA - INSECTA - INSECTA - INSECTA - COLEOPTERAReferenceCOLEOPTERA COLEOPTERA COLEOPTERATittabawasseeCOLEOPTERA COLEOPTERA COLEOPTERA COLEOPTERASaginawCOLEOPTERA Sanford Chippewa Nature Smiths Crossing Tittabawassee Freeland Festival Imerman Park Shiawassee Veterans Dow Light House ReferenceCenter TittabawasseeTownship Park Park Wildlife Refuge SaginawMemorial Park Upstream   TERRESTRIAL  INVERTEBRATE   Downstream Dioxin Equivalents in Wild Mink Liver

• 450 (ng/kg) • 400 • 350 • 300 • 250

WHO-mammalian • 200 • 150 • 100 50 0 Reference areas Tittabawassee River Mink liver TEQ Mink liver • Mink Exposure assessment

• Liver and Dietary HQs (TEQ)

8.0 Dietary Based HQs 7.0 Liver Based HQs 6.0

5.0

HQ 4.0 3.0 2.0 1.0 0.0 Mink Individual and Population health

 Track surveys  Visual observations  Trapping success  Mink scat Mink Individual and Population health

• Habitat Suitability and Abundance Chippewa Tittabawassee Pine River River River Habitat Suitability 100%=Excellent 70% 59% 51% 0% = Poor # Mink / km 1.46 0.44 0.96

Study areas have appropriate habitat and support mink year round Mink Collection Locations

SEX - FEMALE MALE

Department of Animal Science Mink Individual and Population health  Body weight Mink Necropsy  Body length  Sex  Age  Liver weight  Brain weight  Baculum length  Placental scaring  Nutritional status  Histology • Liver • Brain • Kidney • Jaw Mink Individual and Population Health

Difference between Individual Health Measurement Reference and Tittabawassee River animals Body Weight No Body Length No Age No Organ weights No Nutritional Status No Tissue histology No

Population Health

Abundance No Age Structure No Male to Female ratio No Placental Scaring No Results summary: Mink field studies

 Exposure  Identified dibenzofurans are entering the food web  Two furans make up > 80 of the dietary exposure  Exposures are great enough to be of concern  Data pertaining to the toxicity of site-specific contaminants is limited  Individual and population health  No difference between sites  Mink appear healthy (size, age, nutritional status)  No adverse measures for any endpoints  Mink abundance is not different between sites  Male to Female ratio not different between sites  Abundance and demographics are indicative of stable lightly harvested population Questions Resulting from Mink Field Studies

 Site-specific Individual and population health parameters suggest a healthy, lightly harvested mink population, however, the predicted daily dietary exposure and measured tissue concentrations were greater than those observed in some laboratory studies were adverse effects were seen.

 Mink: PCB-based TRVs • Zwiernik et al. 2008. ET&C, 27(10) Mink laboratory exposures: TCDF feeding study

Objective  Compare the effect levels for reproductive success and Jaw lesions for mink exposed to 2378-TCDF or PCB126  2378-TCDF is a major component of Tittabawassee River exposure profile Mink laboratory exposures: TCDF feeding study

Results  No reproductive effect for TCDF TEQ concentrations 10x greater than PCB126 TEQ concentrations that produced complete reproductive failure  Zwiernik et al. 2009, IEAM  Beckett et al. 2008, Arch Env Contam & Tox  2378-TCDF appears to be significantly less potent than previously thought Mink laboratory exposures: kinetic study

Objectives  Quantify accumulation and kinetics of 2378-TCDF and 23478-PCDF individually and as a mixture (zwiernik et al. 2008)  Assess EROD/MROD assays as metrics of exposure (Moore et al. 2009)  Assess mRNA as a metric of exposure (Zhang et al. 2009)  Examine relationship of mink scat to body burden (Zwiernik et al. 2008) Mink laboratory exposures: kinetic study

Results  2378-TCDF does not bioaccumulate (8hr half-life) Zwiernik et al. 2008, Tox Sci  Laboratory Bioaccumulation Factors were in agreement with field measures  Confirmation of predicted dietary exposure based on liver data Mink laboratory exposures: relative potency study

Objectives  Determine if TCDD, PeCDF and TCDF affect reproductive performance of female mink and the survival and growth of their offspring  Determine the relative potency of TCDF and 4PeCDF compared to TCDD based on various endpoints Mink laboratory exposures: Relative Potency

Endpoints measured  Adult survival  Kit birth weight  Kit survival  Litter birth weight  Histopathology  Kit body weight  Brain, liver, heart, kidneys, heart, spleen,  3 week, 6 week, 12 adrenal glands, thyroid week, and 27 week gland, thymus, lymph  Adult organ weight nodes, mandible  Kit organ weight  Number of females whelping  Adult body weight  Number of kits per litter  Number of kits per female Mink laboratory exposures: Relative Potency

Results  Mink exposed to TCDD, PeCDF, and TCDF at concentrations 4 times greater than high end field exposure were unaffected in terms of reproductive success and survivability of offspring Overall Conclusions of Mink laboratory Studies

 TCDF was much less potent than thought  TCDF was quickly degraded in mink  Laboratory BAFs matched the field diet to tissue ratio  PeCDF, and TCDF did not effect the reproduction or the kit survivability at concentrations 4 times greater than the maximum possible field exposure  All lines of evidence are in agreement Kingfisher Kingfisher

Department of Animal Science Kingfisher

Department of Animal Science Great blue heron

Challenges •Kingfisher Burrow Sites

Dietary exposure assessment •2005 Burrows •2006 Burrows •2007 Burrows

•Interim Action Sites Results Summary: Belted Kingfisher

 Kingfisher furan exposure is significantly greater downstream of Midland  Kingfisher dioxin-like exposure is 70% furans  Kingfisher population health measurements are not different between study and reference sites Great Horned Owl

Dietary exposure assessment Prey remains (2005-08) Small mammals and passerines (2003-08) Tissue-based exposure assessment Addled eggs (2005-08) Nestling and adult plasma (2005-08) Individual and population health (2005-08) Clutch size Hatching success Fledging success Banding and telemetry Great horned owl

Department of Animal Science Great horned owl

Department of Animal Science Results Summary: Great Horned Owl

 Great horned owl dioxin-like exposure is significantly greater downstream of Midland (13pg TEQ/ml in adult plasma)  Great horned owl dioxin-like exposure is >90% furans in the diet and >80% furans in the plasma  Great horned owl population health within the basin is good and not different between study and reference sites Avian Field Study Results

 Dibenzofurans are entering the food web  Diet and tissues measurements agree  Abnormalities including those associated with dioxin-like exposure were not observed in >6000 birds handled  Species that should be present are present  Despite more than 20,000 person-hours in the field and the conscription of more than 6,350 animals we were unable to identify with any certainty the presence of any adverse effects associated with exposure to the contaminants of concern Questions Resulting from Avian Field Studies

 If wild birds are as sensitive as a chicken, egg concentrations are approaching levels of concern  Passerines: TCDD/chicken TRVs – Fredericks et al. 2010, Arch Env Contam & Tox  Laboratory studies suggest 1000 fold difference in s sensitivity between species for TCDD  Avian toxicity data lacking for site-relevant congeners (2378-TCDF, 23478-PeCDF) Study Sequence

Mink TCDF feeding study

Mammalian Mink TCDF and lab studies 4PeCDF toxicokinetics

Mink TCDD, TCDF and 4PeCDF reproduction study

MSU ecological AhR ligand binding field studies domain sequencing

Avian species sensitivity lab Injected egg studies hatchability

Injected egg exposure biomarker

Exposure biomarker response in field collected birds

Avian species sensitivity

A molecular basis for differences in sensitivity to TCDD- like compounds among avian species has been suggested

Avian species sensitivity based on genetic sequence of AhR ligand binding domain:  Most sensitive (Chicken)  Moderately sensitive (Ringneck pheasant)  Least sensitive (Japanese quail)

 Do these sensitivity classes hold for TCDF & 4-PeCDF? Avian species sensitivity studies

 Ah-receptor sequencing  64 species present in TR basin  Egg injection with 3 species sensitivity classes  PeCDF  TCDF  TCDD  Field to lab comparisons of molecular and biochemical responses Ah-receptor sequencing study

 The AhR LBD was sequenced in 64 avian species  3 individuals of each species collected from Tittabawassee River  2 species were chicken like, 32 were pheasant like and 30 were Japanese quail like Biomarker response in field collected birds

 Moderate sensitivity  bluebird, tree swallow, house wren  Least sensitivity  great blue heron, kingfisher  Validation of species sensitivity study results with species-specific EROD assay results for field collected birds Avian egg injection study

 Egg injection studies were conducted to confirm proposed avian sensitivity classification using TCDD, 2378-TCDF and 23478-PeCDF Chicken > ring-necked pheasant > quail

Avian egg injection results

 Relative species sensitivity confirmed  Chicken = most sensitive; quail = least sensitive (range ~ 100-fold)  Rank order of potency differed among species  Chicken : TCDF > TCDD > PeCDF  Pheasant : PeCDF > TCDF > TCDD  Quail : PeCDF > TCDF > TCDD  TCDF metabolized by the developing embryo of Japanese quail Effect new avian research may have on ERA

 Predicting sensitivity based on AhR LBD allows for a more accurate, species specific effect concentration  Allow for the selection of representative sensitivity classes in the field  We can now potentially develop congener specific and sensitivity category specific effect concentrations long-term passerine monitoring (Tittabawassee River)

 Five year study banding more than 5,000 resident birds and then follow their survival (bluebird, tree swallow, house wren)  Adult survival rates as great as 85%  No differences in survival rates between study and reference areas  Survival rates were similar to or greater than rates reported in the literature Overall Conclusions for Tittabawassee River Studies

 Contaminants of concern are entering the food web  We were unable to identify with any certainty any furan associated adverse effects despite a herculean effort by many, many dedicated individuals  TCDF appears to be much less toxic than would be predicted by its TEF in mammals and PeCDF appears to be more toxic than predicted for birds  Preliminary data suggests that avian species sensitivity may be predictable based on the AhR LBD Matthew Zwiernik 3270 Anthony Hall Michigan State Univ. [email protected] 517-749-5243 Riverwildlife.msu.edu Questions?