Ecological Studies at the Kingston Ash Spill SiteMarshall Adams, Mark Greeley, & John Smith - Oak Ridge Natl LabTyler Baker and Hill Henry - TVABill Hopkins - Virginia Tech Univ.Dave McKinney & Bobby Brown - TWRARyan Otter- Middle TN. State Univ.Marcy Sousa - Univ. of TennesseeShea Tuberty - Applachian State Univ.

Fish

Birds

Herps Small 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 aquaticinsectsAquatic insect

nymphsMollusks

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

Wholefish

Muscle Liver Ovary

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 Sites7 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

CR

M 7

ERM 7 (Ref)

CRM 7ERM 0.9

ERM 3

Sample locations for baselinebioaccululation and fish health studies

Little Emory (Ref)

CRM 25.0 (ref)

Ecological Relevance

Ear

ly w

arn

ing

, sen

siti

vity

, sp

ecif

icit

y 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

Selenium in Muscle Tissue

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

Bluegill Channel Catfish Largemouth Bass

Se

(mg

/kg

dry

wt)

Downstream

Mercury in Muscle Tissue

0.00

0.20

0.40

0.60

0.80

1.00

1.20

Bluegill Channel Catfish Largemouth Bass

Hg

(m

g/k

g d

ry w

t)

ERM8.0 ERM2.0 ERM0.05 CRM1.5

Downstream

EPA Tier 1 to Tier 2trigger level for toxicity monitoring

State waterQuality criterion(1.5 mg./kg DW)

0

2

4

6

8

10

Bluegill LMB Catfish

ERM 8.0 ERM 2.0 ERM 0.05 CRM 1.5

Downstream

To

tal l

esio

n s

core

of

gill

sGill 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 564

- - 3- 6 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 DesignSample LocationsGreat blue herons - 2 impacted sites, 1 reference siteOsprey - multiple nests on Emory, Clinch, and Tennessee RiversTree swallows - 3 impacted sites, 2 reference sites using established colonies Amphibians (frogs and toads) - 3 impact sites, 2 reference sitesReptiles (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 recordedAmphibians – 5-10 per site/species - whole-body concentrationsTurtles- 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 thespill site, one reference site upstream of Little Emory RiverClinch River – One site downstream of the Emory River and reference site upstream of the Emory River

Collection and Processing Procedures

Snails

Replicate snail samplescollected by hand

76-hr gut depurationin lab

Tissue extracted andanalyzed for metals

________________________________________

Adult mayfliescollected with sweep net

Adults sorted by developmentalstage and sex to evaluate metal loading among stage & sex

Adults

Mayflies

Composite samples ofnymphs collected withPeterson grab

Nymphs

Gut content depuration

Additional composite samplescollected for comparing metalloading with/without gut contents

Mercury

Site

Hg

(g

/g d

ry w

gt)

0.0

0.2

0.4

0.6

0.8

Non-purgedPurged

*

**

Fish diet - Reproductive failure in sensitive species (U.S. DOI 1998)*

Selenium

Site

Se

(g

/g d

ry w

gt)

0

2

4

6

8

10

Non-purgedPurged

** Fish diet - accumulation in eggs (Ohlendorf et al. 2008)

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 DesignField 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

BIOACCUMULATION HEALTH INDICATORS REPRODUCTION

Whole Body Muscle Liver Gonads/eggs HistopathBlood

ChemistryHealth

AssessmentFecundity/clutch size

Egg condition Other

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 adultsSnails (Pleur. sp.)

MUSSELS

(nestlings) (hatching success)

Responses Measured by Ecological Groups

Ash

Periphyton

Adult mayflies

emergence

Piscivorous birds

Metals, detritus,periphyton

Treeswallow

GB heron Osprey

Mayflynymph

Sunfish

LM bass Shad

Racoon

Mussel

Summary of Current Food Chain Studies

Frogs

Se+4Se-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 foodchain 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 animalhealth which is important in ecological risk assessment and in establishing the basis of sound environmental regulatory decisions

Multiple lines of evidence including representative species fromseveral ecological groups and response indicators at several levels of biological organization should be used when assessingeffects of environmental stressors on ecological systems. Suchinformation is critical to the ecological risk assessment process

Populationstructure

Community

Time scale

YearsMonths

Days/weeksHours/days

Minutes

INDIVIDUAL POPULATION--COMMUNITY

SUB-ORGANISMAL

Early warning

Biomolecular

BiochemicalImmunological

Histopath

Sensitive

EXPOSURE

Population fitness

Physiological

Reproductive

Ecologically

Fly Ash

Rapid response

EFFECTS

Response

significant

Assessment of Fish Health

Exposure to

Natural Resource Damage Assess.

Ecological Risk Assessment