Ecological Studies at the Kingston Ash Spill Site Marshall Adams, Mark Greeley, & John Smith - Oak Ridge Natl Lab Tyler Baker and Hill Henry - TVA Bill Hopkins - Virginia Tech Univ. Dave McKinney & Bobby Brown - TWRA Ryan Otter- Middle TN. State Univ. Marcy Sousa - Univ. of Tennessee Shea Tuberty - Applachian State Univ. Fish Birds HerpsSmall mammals

The major components of the Ecological studies include: - Fish Health and metal bioaccumulation - Marshall Adams, Oak Ridge Natl. Lab - Fish reproduction and early life history development - Mark Greeley, ORNL - Terrestrial animal health and metal bioaccumulation - Hill Henry TVA and Bill Hopkins, Virginia Tech. Turtles –TWRA. - Metal bioaccumulation in benthic marcroinvertebrates - John Smith, ORNL and Tyler Baker, TVA - Small mammal health and metal bioaccumulation - Marcy Sousa, Univ. Tennessee - Metal bioaccumulation in fish – Shea Tuberty, Applachian State Univ. - Overall health and bioaccumulation of metals in mussels - Dave McKinney and Bobby Brown, TWRA & Ryan Otter, Middle TN State Univ. - Reservoir spring sport fish survey- Tyler Baker - TVA

Integration of Biological Exposure and Effects over Landscape of fly ash spill Birds Fish Small mammals Fly Ash Spill Exposure to fly ash - Feeding type - Home range - Habitat type - Trophic level Sentinel Wildlife Species Herps Adult aquatic insects Aquatic insect nymphs Mollusks Benthic macroinvertebrates

Objectives of Fish Studies Overall objective is to determine if fly ash exposure is causing short-term, intermediate-, and/or long term health effects on representative (sentinel) fish populations in the vicinity of the spill and at downstream sites Evaluate relationships between levels of metals in fish tissue and various indicators of fish health to access cause and effect relationships

Bioaccumulation 25 metals + Hg Fly Ash Spill Exposure of Fish to Metals Whole fish MuscleLiverOvary Focus on sentinel species - sunfish - bass - catfish - crappie Analysis of Fish Health Assess Effects and Causality (different trophic levels and home ranges ) Physio- logical Repro- ductive Histo- path Bio- energetic

Study Design Sample Sites 7 sampling sites including 3 reference and 3 sites below the spill area Study species - 4 species represented by different trophic levels and home ranges largemouth bass (piscivorous), sunfish (omnivore), catfish (benthic scanvenger), crappie (omnivore, piscivorous) - bioaccumulation in gizzard shad to access role in food chain transfer of metals to predator species including fish and birds Sampling frequency - in spring for reproductive fitness (sunfish, largemouth bass, crappie), bioaccumulation of metals in ovary and liver - in fall for fish health and bioaccumulation (LMB, sunfish, channel catfish) Parameters measured - for reproduction= fecundity, egg size and condition, vitellogenic oocytes - for bioaccumulation= 25 metals in fillets, whole body, liver, ovaries - for fish health= indicators of condition, physiological, hematological, histopathological, and bioenergetic responses and disease/parasites

CRM 1.5 CRM 7 ERM 7 (Ref) CRM 7 ERM 0.9 ERM 3 Sample locations for baseline bioaccululation and fish health studies Little Emory (Ref) CRM 25.0 (ref)

Ecological Relevance Early warning, sensitivity, specificity Physiological Histopathological Reproductive (spring study) - Clutch size - Steroid hormones - Gonad condition - Egg quality - Oocyte atresia Indicators of: - organ dysfunction - electrolyte homeostasis - carbohydrate metabolism - protein metabolism - hematology - liver - gill - spleen - ovary Bioenergetic - condition factor - liver-somatic index - visceral-somatic index - lipid index - feeding & nutrition Measures of Fish Health

EPA Tier 1 to Tier 2 trigger level for toxicity monitoring State water Quality criterion (1.5 mg./kg DW)

Total lesion score of gills Gill Histopathology (short-intermediate term response)

Additional Fish Studies Metal bioaccumulation Shea Tuberty - Applachian State Univ. - 3 fish species (LMB, channel catfish, redear sunfish) sampled at 3 Emory River sites, 3 Clinch River sites, and TRM individuals of each species sampled from each site every 3 months - Metal levels analyzed in muscle, ovary, spleen, liver, and stomach

Objectives of Terrestrial Animal Studies - determine levels of metals in tissues of representative birds, amphibians, and reptiles near the ash spill site and at reference areas - evaluate potential relationship in selected species between levels of metals in body tissues, eggs, and nestlings and indicators of reproductive success Osprey on nest Collecting heron eggs from nest - Overall objective is to determine if fly ash exposure is causing effects on terrestrial wildlife species

Study Design Sample Locations Great blue herons - 2 impacted sites, 1 reference site Osprey - multiple nests on Emory, Clinch, and Tennessee Rivers Tree swallows - 3 impacted sites, 2 reference sites using established colonies Amphibians (frogs and toads) - 3 impact sites, 2 reference sites Reptiles (turtles) - multiple sites on Emory, Clinch & TN. Rivers, 1 reference Sample Number and Analysis Osprey and GBH - metal levels in 5-8 eggs/site Tree swallows - metal levels in 8-16 eggs per site; 1 nestling per nest box; clutch size, nestling condition, & mortality recorded Amphibians – 5-10 per site/species - whole-body concentrations Turtles- sampling throughout summer – metal levels in muscle (TWRA) and blood (TVA)

Selenium in tree swallows

Selenium in fish-eating birds

Objectives of Terrestrial Mammal Studies - Determine differences in condition of racoons captured at spill site compared to reference areas for a suite of health responses - Evaluate relationships between levels of metals in various organs and tissues and the health status of individuals and various organs - Determine if antemortem samples (hair, blood) can be used for long-term biological monitoring of metal exposure Performing necropsy

Study Design Parameters measured - Gross Necropsy - tissue examinations for evidence of necrosis, fibrosis, parasites, disease, etc. - Histopathology – liver, kidney, lungs, adrenal, gonads, brain, eye - Blood chemistry and hematology - Complete CBCs and biochemistry panel - Metal bioaccumulation – analysis of 25 metals on hair, subcut fat, muscle, liver, kidney, liver, gonad, brain, and blood Sample collection & processing - 15 adult racoons captured in vicinity of spill area and 5 at reference - individuals anesthetized in field for blood collection and then euthanized in lab for complete necropsies and histopathological analysis Histopathology Hematology-CBC

Objective of Aquatic Invertebrate Studies Evaluate the magnitude and spatial extent of metal exposures to aquatic macroinvertebrates Snail- Pleurocera canaliculatum Mayfly nymph - Hexagenia bilineata

Study design Two widespread and abundant species of aquatic invertebrates sampled: the snail Pleurocera canaliculatum and the mayfly Hexagenia bilineata Five main sampling sites: Emory River – Two sites adjacent to or downstream of the spill site, one reference site upstream of Little Emory River Clinch River – One site downstream of the Emory River and reference site upstream of the Emory River

Collection and Processing Procedures Snails Replicate snail samples collected by hand 76-hr gut depuration in lab Tissue extracted and analyzed for metals ________________________________________ Adult mayflies collected with sweep net Adults sorted by developmental stage and sex to evaluate metal loading among stage & sex Adults Mayflies Composite samples of nymphs collected with Peterson grab Nymphs Gut content depuration Additional composite samples collected for comparing metal loading with/without gut contents

Selenium and Mercury in Mayfly Nymphs

Objective of Freshwater mussel studies Investigate the short - and long-term effects of the coal ash spill on native Tennessee mussels Individually tagged mussels Mussels in cages Processing of mussels on board

Study Design Field Studies - mussels from non-impacted reference areas individually tagged and transplanted in cages at various sites in Emory river - mussels deployed for various periods of time before harvested and analyzed for effects of fly ash exposure - endpoints measured are mortality, health index, body burdens of metals, glycogen content, and metallothionein Laboratory studies - ash-amended (dredged) sediments placed in static renewal laboratory tanks - mussels from non-impacted reference sites placed in tanks with ash and sampled periodically over 4 months exposure - endpoints measured are mortality, health index, body burdens of metals, glycogen content, and metallothionein

Integration of Ecological Studies

BIOACCUMULATIONHEALTH INDICATORSREPRODUCTION Whole BodyMuscleLiverGonads/eggsHistopath Blood Chemistry Health Assessment Fecundity/ clutch size Egg conditionOther FISH Largemouth Bass Bluegill Channel Catfish Crappie Gizzard Shad SMALL MAMMALS Raccoon BIRDS Osprey Great Blue Heron Tree Swallow AMPHIBIANS Chorus Frog Spring Peeper American Toad REPTILES Snapping Turtle Musk Turtle Softshell Turtle MACROINVERTS Mayfly nymphs Mayfly adults Snails (Pleur. sp.) MUSSELS (nestlings) (hatching success) Responses Measured by Ecological Groups

Ash Periphyton Adult mayflies emergence Piscivorous birds Metals, detritus, periphyton Tree swallow GB heronOsprey Mayfly nymph Sunfish LM bass Shad Racoon Mussel Summary of Current Food Chain Studies Frogs Se +4 Se -2 To periphyton

Summary/Synthesis Ecological studies are integrated across spatial and temporal scales with sample sites above (reference), at, and below the ash spill area Food chain studies emphasize the transfer of fly ash- associated metals among critical components of the food chain leading to top predators and to humans Some ecological studies (fish, racoons, tree swallows, mussels) also focus on the causal relationships between metal exposure and animal health which is important in ecological risk assessment and in establishing the basis of sound environmental regulatory decisions Multiple lines of evidence including representative species from several ecological groups and response indicators at several levels of biological organization should be used when assessing effects of environmental stressors on ecological systems. Such information is critical to the ecological risk assessment process