NORWEGIAN UNIVERSITY OF LIFE SCIENCES

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2111 2005

Epigenetics and Ecotoxicology Challenges and Applications in

Radioecology

Deborah OUGHTON

Norwegian University of Life Sciences, Aas, Norway

[email protected]

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NORWEGIAN UNIVERSITY OF LIFE SCIENCES

Overview

Epigenetics and Ecotoxicology (vs human applications)

Implications for Radioecology:

Protection of the Environment from ionising radiation

Areas of Application – COMET EU Field studies - Komi case study

Deborah Oughton: DoReMiMunich, 2013

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Over 1500 review papers on epigenetics and

biomedicine

Only 2-3 in ecology and ecotoxicology

Increasing focus in ecotoxicology due to

applications in laboratory and field studies

Epigenetics and Ecotoxicology

Nature 441 2006

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Characteristics of genetic, epigenetic, and metabolic pathways by which environmental stressors can

influence gene expression

Susceptible to

environment ally induced change

Reproducible Reversible Persistent after

stressor is removed

Heritable

Genetic Limited Partially No Yes Yes

Epigenetic Yes Yes Yes Yes Yes

Metabolic Yes Yes Yes No No

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Head et al., ET&C, 31, 2012

Laboratory tests need to go F2/F3

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Applicable Species, Patterns and Mechanisms

Vertebrates: high and mechanisms appear to be

conserved across species (mice, rodent models – plus zebrafish, polar bears, …)

Plants: Yes, CZG or CZZ sites, widespread Invertebrates: wide variataion

– C elegans - no CpG methylation – Bee and wasps – yes

– Fruit fly - CpT and CpA – Daphnia – yes

– Earthworms (lumbricus rubellus) - yes

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Vandegehuchte and Janssen, Ecotoxicology, 2011

Different methylation

distributions suggest that DNA methylation may have

different functions in different

organisms.

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Implications for Ecotoxicogy

Temporal disconnection between cause and effect

Evidence and mechanisms for field observed effects

Biomonitoring and biomarker assay

(broad indicator of accumulated stress, Deb-tox models)

Risk Assessment: impacts can be both positive and negative

(acclimation/adaptation)

an additional parameter in the existing suite of endpoints/assays

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Implications for Ecosystem Change, Inter and

Intraspecies biodiversity, resistance and adaptation

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Majority of ecotox studies on non radiological stressors

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The International Commission on Radiological Protection (ICRP)

“ If man is adequately protected then other living things are also likely to be sufficiently protected” [ICRP, 1977],

Deborah Oughton: MINA410 EnvironmentalRadiobiology, 2013

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The International Commission on Radiological Protection (ICRP)

“ The Commission believes that the standard of environmental control needed to protect man to the

degree currently though desirable will ensure that other species are not put at risk. Occasionally,

individual members of non-human species might be harmed, but not to the extent of endangering whole species or creating imbalance

between species. At the present time, the Commission concerns itself with mankind’s environment only…." [ICRP, 1991],

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Lethal dose to different species from acute radiation doses

Figure 3.1 Comparative radiosensitivity of different organisms demonstrated as the acute lethal dose ranges (reproduced from UNSCEAR 1996).

Reproduction 20-100x more sensitive

10⁰ 10¹ 10² 10³ 10⁴

Mammals Birds

Higher plants Fishes

Amphibians Reptiles

Crustaceans

Insects

Mosses, lichens, algae Bacteria Protozoa Molluscs

Viruses

Acute lethal dose (Gy)

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Papers from Pentreath and Woodhead (1998- )

Report from International Union of Radioecologists (IUR) 2000 IAEA Report on ethical

considerations (2003) Issues:

Situations where humans are absent (e.g., disposal)

Not compatible with management of other environmental stressors Needs to be demonstrated

Background: Towards a Framework for

Radiological Protection of Non-Human Species

EU 6th-7th Framework Project s: FASSET, ERICA, PROTECT, STAR, COMET www.erica-project.org ; www.star-radioecology.org

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NORWEGIAN UNIVERSITY OF LIFE SCIENCES Deborah Oughton: MINA410 EnvironmentalRadiobiology, 2013

Emerging consensus that radiation protection needs to address the effects of ionising radiation on non-human species (IUR, 2001)

EU 6th-7th Framework Project s: FASSET, ERICA, PROTECT, STAR, COMET www.erica-project.org ; www.star-radioecology.org

ICRP 208 (2007) Environmental Protection - the Concept and Use of Reference Animals and Plants www.icrp.org

IAEA Safety Standards www.iaea.org

Requirement for enormous amount of information on transfer, uptake and effect of ionising radiation (especially for wild animals)

Background: Towards a Framework for

Radiological Protection of Non-Human Species

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Isotope Laboratory Departmen

t of Plant Environme

ntal Sciences

e

Release of radionuclides

Single effect endpoint:

Cancer induction (Sv)

Several effect endpoints:

Reproduction, sexual maturation Human risk assessment Ecological risk assessment

Protection of individuals Protection of populations/communities

Human vs Ecological Assessment

One species Multiple species

Reference Man Reference Animals and Plants

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O u g h to n e t a l. E R IC A D 7 a , 2 0 0 8

Deborah Oughton: DoReMi Munich, 2012

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Wildlife defies Chernobyl radiation

By Stephen Mulvey BBC News

« It contains some of the most contaminated land in the world, yet it has

become a haven for wildlife - a nature reserve in all but name. »

20 April 2006

Chernobyl 'not a wildlife haven'

By Mark Kinver

Science and nature reporter BBC News

«

The idea that the exclusion zone around the Chernobyl nuclear power plant has

created a wildlife haven is not scientifically justified, a study says.

»

14 August 2007

What is Harm? (Slide courtesy of Tom Hinton)

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Epigenetics and Radioecology

Attracting attention in radiobiology – but NOT at present a main research area in EU projects

The few published laboratory and field studies mechanism occurs in non-human species

Links to «Non-Targeted Effects»

(e.g., bystander and genomic instability)

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Fukushima Butterflies

Hiyama et al, Nature, 2012

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Epigenetics in EU COMET

Laboratory and Field Studies

Initial focus on DNA methylation (links to reproduction effects)

Field Sites: Chernobyl and Fukushima

Test organisms: Plants, Zebrafish (lab only), earthworms (frogs)

Laboratory Studies: need to be multigeneration Field considerations:

– Abundance of test organsim – Sampling season

– Field sampling and transportation protocols – …

– DNA methylation one of many other parameters….

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Field Study Uncertainties

Influence of confounding factors such as other contaminants, weather, food or nutrient availability?

Reliability of dose rate measurements in laboratory and field condition

Spatial variability (e.g. in dose and dose rate due to contamination) and species mobility in the field

Extrapolation from laboratory tests to field situations

Relationship of biomarkers/endpoints to long‐term population effects?

The shape of dose‐response curves, particularly at low dose and dose rates?

Hormesis, non‐targeted mechanisms such as bystander effects and genomic stability?

Field Ecology Uncertainties Workshop, Lancaster, 2013 https://wiki.ceh.ac.uk/x/wACwCw

Deborah Oughton: MINA410 Field studies intro, 2013

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Fukushima Evacuation Area about 30km From F1

Photo by Tetsuo Yasutaka

1.5μSv/h

Slide from: Wataru Naito

Research Institute of Science for

Safety and Sustainability (RISS)

www.eu.neris.net

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Photo by Tetsuo Yasutaka

Dose level was reduced, but what extent?

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Decontamination is going on..

Photo by Tetsuo Yasutaka

In-Situ or Temporary Storage → Interim Storage → Final

Disposal Site

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Field Study Uncertainties

Influence of confounding factors such as other contaminants, weather, food or nutrient availability?

Reliability of dose rate measurements in laboratory and field condition

Spatial variability (e.g. in dose and dose rate due to contamination) and species mobility in the field

Extrapolation from laboratory tests to field situations

Relationship of biomarkers/endpoints to long‐term population effects?

The shape of dose‐response curves, particularly at low dose and dose rates?

Hormesis, non‐targeted mechanisms such as bystander effects and genomic stability?

Field Ecology Uncertainties Workshop, Lancaster, 2013 https://wiki.ceh.ac.uk/x/wACwCw

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Komi Project, Vodny Republic

Between 1931 and 1956 the Vodny area in the Komi Republic, Russia, was the main site of Soviet radium production.

Wastes from the industry caused contamination of the

environment, leading to high levels of radionuclides, heavy metals and rare-earth elements in the surroundings.

Project objective to study chronic effects on terrestrial

ecosystem (i.e., impacts on plants and soil invertebrates at a population and community level).

Transportation of radium concentrate in wooden barrels, 1940 (Evseeva et al 2000)

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Site Assessment and Biomarkers (contaminated site and reference site)

Species diversity: macrofauna taxonomic identification

Metabarcoding of soil DNA - community diversity

Microorganism – Adaptation to metal (and radiation)

Soil invertebrate diversity - earthworm barcoding

Earthworm biomarkers - epigenetics (DNA methylation)

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Site Assessment and Biomarkers (contaminated site and reference site)

Soil characteristics (chemistry, geological, etc..)

Radionuclide and metal analysis of soil, plants and organisms

Dosimetry

Other earthworm biomarkers: DNA damage (COMET), Apoptosis

(TUNEL, Apopstain); bystander, qPCR, …

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Summary: Challenges within Ecological Risk Assessment

Fundamental research is needed to address:

1. the occurrence of chemical-induced epigenetic modifications at environmentally realistic exposure concentrations in ecotoxicologically relevant species, 2. phenotypic and population-level effects of these modifications

3. the transmission of these changes to subsequent non- exposed generations.

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Vandegehuchte and Janssen, Ecotoxicology, 2011

Need for collaboration on method development

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C o rr e la ti o n s b e tw e e n c a u se a n d e ff e ct s ch a lle n g in g i n e n v ir o n m e n ta l sc ie n ce

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Deer Rat Bee

Earthworm Pine tree Grass

Duck Frog Trout Flat fish Crab

Macroalga

Reference organisms:

ICRP « Reference Animals and Plants »

Typical, accessible, documented, various sizes and life cycles, measurable dose-effect

Generic virtual entities to serve as points of comparison to assess exposure and effects

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NORWEGIAN UNIVERSITY OF LIFE SCIENCES ²

9

V o d n y

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Chernobyl : A Wildlife Reservation?

The accident had large detrimetal effects on the environment: Forest death; change in genetic changes in mammals (1-3 yrs)

Medium term: recovery of

ecosystem, but changes pine to birch forest.

Long term ecosystem effects overshadowed by the positive benefits from human evacuation

Wormwood Forest

A Natural History of Chernobyl Mary Mycio

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W h it e S to rks P rze w a lski ’s h o rse s A lb in o sw a llo w s (p h o to : T .A M o u sse a u )

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NORWEGIAN UNIVERSITY OF LIFE SCIENCES Fauna

Flora

ECOSYSTEMMan Life support Services

Linear Transfers Cyclic Transfers + Effects

Low doses Chronic exposures Multiple stressors

PAST NOW

Man Sources

Environment Environment

Biogeochemical

Radiation Protection: From Anthropocentric to Ecocentric

Brechignac, 2004; Brechignac et al. 2012

Context

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Deinococcus radiodurans

… and RADIATION QUALITY, DOSE RATE, ETC

(Sparrow, 1961; Harrison and Andersen, 1996)

Factors influencing radiosensitivity in non-human biota

Initial infliction of DNA damage

– eg. DNA content, chromosome volume, oxygen, FR scavangers, DNA packaging

Checkpoint control mechanisms and DNA repair

– e.g., Homologous recombination repair (HR) and non homologous endjoining (NHEJ), [Mn]/[Fe] ratios

Induction of cell death

– e.g. necrosis, apoptosis and mitotic death Tissue regeneration

Cell cycle sensitivity Life Stage

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ntal Sciences

e

Release of radionuclides

Single effect endpoint:

Cancer induction (Sv)

Several effect endpoints:

Reproduction, sexual maturation Human risk assessment Ecological risk assessment

Protection of individuals Protection of populations/communities

Human vs Ecological Assessment

One species Multiple species

Reference Man Reference Animals and Plants

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E c o to x ic o lo g y

Important questions in exotoxicology

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< 200 µG/h

Coniferous Deciduals

After Smith & Beresford, 2005

500 up to 2000 µGy/h 120 km²

No more reprod.

capacity Dried needles Morphological modifications

Morphological modifications

Growth & reproduction perturbations

Morphological perturbations 2000 up to 5000 µGy/h

38 km²

Death of growth zones Terminal vanishment

Morphological modifications

> 5000 µGy/h

4 km² Deat within a few days Partial dammage

Effects on forests

Chernobyl effects on wildlife

Effects of radiation

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The “Red Forest”: Shift in ecosystem structure

Pine stands were replaced by grasses, with a slow invasion of hardwoods

Chernobyl: Effects in Plants

• Morphological mutations (e.g. leaf gigantism)

UNSCEAR, 2001

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•

60 to 90% of initial contamination captured by plant canopies

•

Majority washed off to soil and litter within several

weeks

•

Populations of soil

invertebrates reduced 30-fold, reproduction strongly impacted

Chernobyl: Effects in Soil Invertebrates

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Natural background

10^-2

Dose rate (µGy/h)

10^-1 1 10 100 1000 10000

Nuclear installation in normal operation

TERRESTRIAL PLANTS MOSSES, LICHENS, MUSHROOMS

AQUATIC PLANTS BACTERIA

PROTOZOA

BIRDS

CRUSTACEANS MOLLUSCS

FISHES

INSECTS

MAMMALS

AMPHIBIANS REPTILES

Enough data to derive « no effect dose rates » No data

Experimental investigations of dose-effects relationships (external)

Radiation effects on wildlife: Knowledge Gaps

^D^e^b^o

rah Oughton: ERR Stockhom2010

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A few species studied

Data mostly derived from short- term studies

Data mostly derived from acute doses (and dose rates)

Data essentially derived from external exposure situations (γ irradiation)

Data essentially derived from

observations up to individual level

State of the art on radiation effects on animals and plants

More species (biodiversity) Long-term (trans-

generational)

Low doses and dose rates Internal contamination

Observations at

population, community and ecosystem level

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O u g h to n e t a l. E R IC A D 7 a , 2 0 0 8

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Population effects

Data related to individual organisms : what link to population ?

• Individual mortality => mortality rate => population density

• Fertility => reproduction rate => population density

Toxicology Ecology

Ecotoxicology

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UMB and IoB Pilot Fieldwork

A small joint field-expedition with Russian and Norwegian scientists

was carried out in July 2012 (prior to the official start of the project)

Activities:

– mapping radionuclide and metal concentrations in the area.

– Analysis of the type and diversity of soil invertebrates.

– To design the extensive fieldwork sampling in 2013.

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Olesya Vakhrusheva (IoB), Anna Kaneva (IoB) and Yevgeniya Tomkiv (UMB) taking background gamma radiation measurements at one of the soil and

invertebrate sampling sites.

Photo: Elena Belykh (IoB)

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Pilot study: invertebrate density (Lapied, unpublished)

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Mean µGy/hr: Clayey 17, Sandy soil 14; control 0.05-0.1

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Deer Rat Bee

Macroalga

Ecological Relevance:Reference Animals and Plants

Need ecosystem relevant set of reference organisms (decomposers, primary producers, predators, etc..)

Need better understanding of DIFFERENCES in radiosensitivity between species – more variation in lower trophic levels

See Brechignac et al ICRER Alonzo et al, 2009

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Case Study : The Earthworm

The earthworm (Esenia fetida) as a test species (Exposure and dosimetry)

Effect studies

Ecological relevance

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NORWEGIAN UNIVERSITY OF LIFE SCIENCES Deer

Rat Bee

Macroalga

Reference organisms:

ICRP « Reference Animals and Plants »

• Recognized test species in chemical toxicity studies; standardised tests

(OECD)

• Potentially high exposure to radionuclides

• Important role in soil ecosystems

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ntal Sciences

Why me?

Important role in the food web Important for the soil fertility

Eat dead organic material

•Increases the bioavailability of nutrients for other organisms

They make burrows in the soil

•Increase the aeration and water drainage

•Mixing organic and inorganic components of the soil

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“An earthworm is not just an earthworm”

The total number of species is estimated to exceed 2000 Three major ecological groups of earthworm have been

identified based on the feeding and burrowing behaviours of the different species.

E. fetida

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Earthworm anatomy and physiology

Earthworms are segmented The principal excretory organs are the nephridia They have no specialized respiratory organs: They respire over the whole body surface

– Thin cuticle covered with mucus

Coelomocytes; immune cells in the coelom

The circulatory system is closed with a blood vessel running along the dorsal and ventral surface of the

digestive tract

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Reproduction in earthworms

Esenisa fetida are hermaphrodites with separate testes and ovaries that function simultaneously

During mating they crossfertilize

Spermatozoa are transferred to spermatechae

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Life cycle: Eisenia fetida

The life-cycle of E.fetida is relatively short

– approximately 11 to 16 weeks from cocoon to sexual maturity (20°C)

The life span is rather long – E.fetida has been kept in

the laboratory for about 4

½ years

– Can be reproductively active for more than 500 days

2-5 cocoons per worm per week

1-6 hatchlings per cocoon Growth and sexual

maturation: 8-12 weeks

Adult

Juvenile

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Earthworm dose-response studies

Life stage, generation Exposure time

Cocoons 3 weeks

Adult (F0) reproduction

13 weeks Juvenile (F1)

growth /maturation

11 weeks Adult (F1)

reproduction

13 weeks

38 14

91 21 35 56

24 F1 Growth and maturation Adult F1 Reproduction Adult F0 reproduction

Hatching of F1 juveniles

9 0

13

28 56

4 8 3 5 8 11 16 20

63 77 112 140 168

Dose rates: 0.01 – 40 mGy/hr

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Earthworm Reproduction Study

Irradiation (0.1 – 43 mGy/hr), 13 weeks There was no radiation induced effect on

– Viability, cocoon production rate, Sexual maturation rate in the F1 generation

Significant effects on cocoon hatchability

0% 0%

0 20 40 60 80 100

1-4 5-8 9-13

Weeks of exposure

% Hatchability

Control 0.18 mGy/h 1.7 mGy/h 4.2 mGy/h 11 mGy/h 43 mGy/h

Hatchability of F0 cocoons

Hertel-Aas et al., Radiation Research, 2007

0 10 20 30 40 50 60 70

Control 0.19 mGy/h 1.7 mGy/h 4 mGy/h 11 mGy/h 43 mGy/h

# F1 hatchlings per adult F0

**

*

**

• Reduction in the total number of offspring produced by each F0

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Life-cycle trait variations in earthworm species

Dendrobaena octaedra

– Epigeic

– Reproduction – pathenogenesis (clonal, rapid adaptation)

– Frost tolerant

Lumbricus terrestris

– Anecic

– Sperm storage up to 12 months after mating

– Cocoon incubation median 1.5 yrs (up to 5 yrs)

– 1 hatchling per cocoon

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