Application of Biomarkers and Application of Biomarkers and BioindicatorsBioindicatorsfor Marine Environmental Monitoringfor Marine Environmental Monitoring

EAS Congress 2009

Doris W.T. AURudolf S.S.Wu, Paul K.S. Shin

(bhdwtau@cityu.edu.hk)

City University of Hong Kong

Manila

Outline• What is biological monitoring? and

Why bother?• Application of biomarkers and

bioindicators for different management purposes

• Successful examples in coastal ocean monitoring

Molecules

OrganellesCells

TissuesOrgans

Organisms

Levels of

Biological O

rganizatio

n

CommunitiesEcosystems

Organ systems

Biochemical

Populations

Minutes / Hours Days Months Years

Time until manifestation

Environmental stresses

Bio-monitoring : Use biological responses at different biological organization levels (biomarkers and bioindicators) to indicate significant environmental changes

Application of integrative biomarkers for ERA

Adam 2003

• Biomarkers are biological responses at lower levels of biological organization (molecular, biochemical and physiological responses) that can provide direct evidence of exposure to stressors

relatively sensitive (short-term response) to stressors, serves as early warning indicators of impaired organism health.

• Bioindicators are biological responses at higher levels of organization (individuals, populations, communities)

relatively insensitive (long-term response) to stressors but have higher ecological relevance

• Chemicals may exist in different forms, while bioavailability and toxicity varies considerably with different chemical forms

• Presence ≠ available ≠ effect ≠ adverse effects• Interaction of chemicals (additive, synergistic or

antagonistic) may be important yet difficult to determine

• Major problems exist in assessing or predicting biological effects from chemical / physical data

Bio-monitoring : Why bother ?

Chemical vs Biological monitoring Strength & Weakness

Chemical Monitoring

• Bioavailability not known• No indication of biological effects• Large variations with time• Indirect interpretation of results• Costly and laborious in sampling• Lower Conc., making analysis

difficult

• Comparable between locations/regions

• Accurate• Continuous measurement is

normally easy• More established techniques

Biological Monitoring

• Indicates bioavailability• Provides clues for biological effects• Time integration built in• Direct interpretation of results• Less expensive and easier• Higher conc., making analysis easier

• Different biota may not allow direct comparison of results

• Less accurate• Continuous measurement difficult

• Less established

1. Identify exposure to certain chemicals2. Monitor spatial and temporal changes in pollution 3. Provide early warning to environmental

deterioration4. Indicate occurrence of adverse ecological

consequences.

Biological indicators developed for different management purposes

In the last two decades, a number of biological indicators have been successfully developed and adopted by various national and international marine pollution monitoring programs in North America, Europe, Australia and New Zealand (e.g. NOAA, USEPA, ICES, WFD, OECD, OSPAR, Mussel Watch)

1. Identify exposure to certain chemicals

• Brain acetycholinesterase(AChE) activity for neurotoxic pesticides

• Liver EROD / CYP1A enzyme activity for PAHs/PCBs

Cl

Cl Cl

Cl

P

OO

SNH2

CH3

CH3

Methamidophos

B(a)P

Enzyme assay

organophosphate pesticides, carbamates

1. Identify exposure to certain chemicals

• Vitellogenin for endocrine disruptive compounds (EDCs)

• Metallothionein for metals

Cu-metallothionein Zn-metallothionein

DNA Adducts, DNA Strand Breakage• Indicates levels of genotoxicants in water• Can be measured by alkaline unwinding assay or single cell gel

electrophoresis (Comet assay)

1. Identify exposure to certain chemicals

Body Burden of Metals & Trace Organics• Indicates time-integrated values of bio-available fractions of metals

and trace organics in the environment

The “Mussel Watch” program

2. Monitor spatial and temporal changes in pollution

0

200

400

86 87 88 89 90 91 92 93

DDTPCB

PAH

DDTPCBPAH

Data from O’Connor & Beliaeff (1995)

Annual means of trace organic in mussels at 154 sites in coastal waters of USA

Fish Pathology• Indicate general quality of the environment, and related to

contamination levels of heavy metals, PAHs, PCBs and DDT

Fin erosion Skeletal deformities Skeletal deformities

Liver tumorEpidermal hyperplasia / papilloma

3. Provide early warning to environmental deterioration

Lysosomal Integrity of Mussels• Indicate pollution-induced stress on mussels• Measured by neutral red assay• Used in biomonitoring programs in Europe and USA (e.g. NOAA,

ICES, MED POL, North Seas, WHO-FAO, Hong Kong)

3. Provide early warning to environmental deterioration

Imposex in Marine Snails• Change of sex from females to males (imposex) indicate levels of

tributyl tin (TBT, an antifouling chemical) • Highly sensitive (threshold: 0.5 ng/L in water or body burden of 10

ng/g)• Easy to determine, quantified by e.g. % imposex, relative penis

size, etc

CH2CH2CH2CH3

SnCH3CH2CH2CH2 CH2CH2CH2CH3

Cl

3. Provide early warning to environmental deterioration

e.g. Changes of species diversity

Gymnodinium

eutrophic

4. Indicate occurrence of adverse ecological consequences

Mesodinium

Community Indicators• Indicate general “health”

of a community– Species richness – Species diversity (H’)– Evenness ( J)– Dominance– Similarity – SAB (Species, Abundance,

Biomass) curves– ABC (Abundance Biomass

Comparison) plot– Multivariate statistics

Skeletonema

A Case study in Hong Kong

Review of 53 bioindicators

13 selection criteria

13 Shortlisted bioindicators

Identify informationgap

Field & Lab validations

13 Selected bioindicators

A US$1.1 M study commissioned by

Environmental Protection

Department, Hong Kong

2001-03

I. Identify exposure to certain chemicals1) EROD, (Ethoxyresourufin-O-deethylase) in fish liver

II. Monitor spatial and temporal changes in pollution2) Body burden of metals and trace organics in barnacles and mussels

III. Provide early warning to environmental deterioration1) Fin erosion of fish2) Epidermal hyperplasia/papilloma of fish3) Condition Factor (CF) of fish4) Hepatosomatic Index (HSI) of fish5) Gonadosomatic Index (GSI) of fish6) Lysosomal integrity of mussels7) Imposex of gastropods

IV. Indicate occurrence of adverse ecological consequences1) Diversity indices2) Log normal distribution3) ABC (Abundance Biomass Comparison) plots4) Multivariate statistics of species composition

The 13 selected biomarkers/bioindicators

Development of a cumulative Index

• Integrate different measures at different biological scales to provide an overall message (good for laymen)

• Rapid evaluation by users of the state of the ecosystem

• Provide early warning of changes• Can be used to make quantitative spatial and

temporal comparisons

Integration of measures into a single index

d j = Σ S iji= 1

n

d j = cumulative index for zone jS ij =  weighted score for zone ji = measure ( 1….n) 

Conclusion• Many biological indicators have been successfully used

in a cost-effective way in N. America, Europe, Australia and New Zealand, including Hong Kong

• However, applications of bio-monitoring in Asia have been extremely limited

• High national priority should be accorded to adopt and validate the use of these bio-indicators in the local seas, so as to improve the cost-effectiveness and environmental relevance of the current monitoring programs in the region

Selection Criteria

1. Ecological relevance2. Ecological importance3. Sensitivity4. Specificity5. Dose-response

relationship6. Reproducibility7. Confounding factors

8. Representativeness9. Scale of applicability10. Easy to understand11. Overseas experience12. Technical difficulty13. Cost-effectiveness