Biomarkers provide a concise way to monitor aquatic environments. Current monitoring methods have limitations, but biomarkers can indicate exposure to pollutants and their effects. Biomarkers are measured at different biological levels from cells to whole organisms. They are used globally to assess contaminant impacts and identify stressors. In India, biomarkers have been applied to coastal waters to study effects of metals, oil, and pesticides on species. Biomarkers are a valuable tool for environmental monitoring when chosen carefully based on sensitivity, specificity, and other factors.

1. Biomarkers in Aquatic Environment
Monitoring
Presented by
Kantharajan G
Ph.D Scholar
ICAR - Central Institute of Fisheries Education
Versova, Mumbai - 61
12-Jan-18

2. Limitations of current methods
Physical/Chemical monitoring
 contaminant monitoring
 may not be bioavailable
 the effect of mixture of pollutants
 no interaction with the organism & ecosystem
Biological monitoring
 abundance/disappearance
(concept of Bio-indicator)
 very low specificity
Toxicological Studies
Logical constraints
 size and no. of organism in the lab
 long-term study – determining the ultimate effect of chemical exposure
that occur at larval phase on the reproductive output of fish
 field – animal avoids the contaminant hotspot
 ability to mimic natural stressors (hypoxia, predator/prey
relationship food limitation) & complex natural exposure (oil spill)
(Hook et al., 2014

3. ‘Concepts from the field of medicine to predict the health & function of ecosystem’
Physician
Bio-metric measurements
Blood test
Imaging
Pathological analysis
Biological measurements
Sub-organism level changes
Analytical chemistry
Envt. Manager
Non-chemical stressors
Predict change
Organ function Ecosystem function
Avoid loss
Holistic, Integrated or Both?

4. ‘‘ Almost any measurement reflecting an interaction between a biological
system and a potential hazard, which may be chemical, physical, or
biological. The measured response may be functional and physiological,
biochemical at the cellular level, or a molecular interaction’’ (WHO, 1993).
‘‘ A xenobiotically-induced variation in cellular or biochemical
components or processes, structures, or functions that is measurable in a
biological system or sample’’ (McCarty et al., 2002).
‘‘ The changes can be interpreted as an early response to
environmental pollution, before longer-term or higher level
changes become apparent’’ (Lam and Gray, 2003).
Bio-marker

5. Bio-Monitoring
Sediment
Water
Contaminant
Bioaccumulation (Neutral fraction )
Biotransformation
Specific Interaction
(active fraction)
T
o
x
i
c
e
f
f
e
c
t
s
Population
Whole organism
Organ
Cell
Protein
Ecosystem
Community
Immediate time response
High specificity
(-)
(+)
(Modified from Conti et al. 2008; Narbonne & Michel, 1993)
BM

6. Reliable, repeatable, relatively cheap &
easy to perform
Sensitive to pollutant exposure and/or effects
Well defined baseline data to distinguish between
natural variability & contaminant-induced stress
Correlation with the health or fitness of the organism / Ecologically relevant
(Van der Oost et al., 2003; Svendsen et al., 2004; Huggett et al., 1992; Ho
What to
choose?

7. Data Monitoring
Programme
Further analysisData signt. Different from Ref.
site
Physico-chemical
properties
Lower Oxygen & higher Chl. in the
surface water
Surveys to ascertain
statistical relevant spatio-
temporal difference
Easily measured chemicals
in water & sediments
If levels are higher than reference
sites test for effects using BM of
effect
Examine changes at
population or higher level
Benthic assemblages
Multi-variate analyses suggest
causative agent: test with BM of
effect
Examine relationship
between spatio-temporal
changes & specific agents
Bioassays (subcellular, cell
based, organism based) to
measure total toxicity for
specific toxicants or
complex mixture of
chemicals
Advanced analysis for specific
toxicants (eg: Dioxins)
If elevated levels use of
specific biomarkers
(eg: genotoxicity/ED)
Biomarkers of general
stress (growth)
BM indicates stress response in
organism (Lam & Gray, 2003)
Where to use ?

8. External exposure
& dose
Internal dose
Uptake in critical
organ
Target dose in less
susceptible group
Target dose in
susceptible group
Occurrence of critical
effect
Occurrence of disease
Target dose for
critical organ
Biomarker of pollutant exposure Biomarker of pollutant effectContaminant
Monitoring
Modified from Nordberg (2010)
Types of Bio-Marker

9. ‘’Markers which indicate that exposure of an individual or organism to a
xenobiotic has occurred & to what extent’’ (Editorial-Biomarkers. 1996)
Metabolites or adducts indicating interaction with the biological system / If
Parent compound, remain unchanged – no interaction
Used to assess the amount of a chemical that is present within the body
Early response to contaminants & are highly specific
Bio-Marker of internal
dose
Bio-Marker of biological
effective dose
Types
 Most reliable and precision
 Combined with external exposure assessment
 (Eg: Cd in blood and urine)
Eg: DNA adducts in the case of Cr
adducts are formed when an activated chemical
species binds covalently to DNA
Not enough details regarding its reacting
(Mussali-Galante et al., 2013)
(i) Bio-Markers of Exposure of Pollutant

10. Bio-marker Measurement and Indication References
Bile fluorescent
aromatic
compounds (FACs)
Metabolites of PAHs measured in the bile of fish, can
reflect exposure to oil
Myers et al. 1991;
Myers et al. 1994
Cytochrome
P4501A mRNA
(CYP1A) or protein
An inducible isoform of the cytochrome p450 family
that is measured in various tissues of fish, bivalves
and seabirds exposed to oil, and other chemicals that
are Ah receptor agonists
Collier et al. 1996;
Roberts et al. 2004
Ethoxyresorufin-o-
deethylase (EROD),
Aryl hydrocarbon
hydroxylase
Catalytic activities of Cytochrome P4501A enzyme Huggett et al. 2003;
MartinezGomez et
al. 2009
Vitellogenin (vtg) Egg yolk precursor protein induced on exposure to a
broad range of estrogenic compounds
Folmar et al. 1996;
Denslow et al. 2004
Metallothioneins Metal-binding proteins that are induced on exposure
to certain metals (Cd, Hg)
Roberts et al. 2005;
Sakuragui et al.
2013; Williams &
Gallagher 2013
Common biomarkers in Aquatic Envt. Monitoring
(Exposure category)
(Hook et al., 2014)

11. Background:
Measuring hepatic
EROD activity
 Exxon Valdez oil spill, 1989 in Alaska
 CYP1A genes - induced by a larger PAHs, PCBs, dioxins & difurans which is
measured by EthoxyResorufin-O-Deethylase (EROD) activity
 Study was undertaken by Esler et al. 2017 from 2011 to 2014 to determine the
evidence of oil exposure persistence in Halequin ducks.
Methods:
0.5 g liver sample
2006,
2007,
2009,
2011,
2013
2014
Case study: Cessation of oil exposure in Harlequin ducks after the
Exxon valdez oil spill: cytochrome P4501A biomarker evidence

12. Fig: Average (±standard error) EROD activity of Harlequin duck
captured in Alaska in 2006, 2007, 2009, 2011, 2013 and 2014.
Result: Conclusion:
 In 2013, CYP1A induction was
similar between oiled and
unoiled areas – indicates the
lack of measurable exposure
to residual Exxon Valdez oil.
 Period of exposure of this
duck to lingering oil was
between 22 yr and 24 yr.
 lack of CYP1A induction in
2013 and 2014, it is assumed
that oil exposure was no
longer occurring at that time
and effects of oil exposure
can be considered to have
ceased.
Note: Sampling period with asterisk indicate statistical difference in
EROD activity.
Continued…

13.  Indicators of physiological or biochemical changes as a consequence of
exposure
 Can be effectively incorporated with other diagnostic markers of fish &
analytical chemistry approaches to provide evidence
 Helps in monitoring of disease progression and prognosis
Direct measures
(e.g. DNA damage)
Indirect measures
(e.g. the impact on sub-
cellular lysosomes)
Types
(ii) Bio-Markers of Effect of pollutant
(Gil & Pla, 2008)

14. Common biomarkers in Aquatic Envt. Monitoring
(Effect category)
Bio-marker Measurement and Indication References
Heat-shock proteins
(i.e. HSP 90)
Proteins that are induced in tissues of aquatic organisms as a
generalized response to stress, including exposure to
chemicals, hypoxia, and temperature
Downs et al. 2006
Markers of oxidative stress
(heme oxygenase,
superoxide dismutase,
glutathione, catalase, lipid
peroxidation)
Enzymes, cofactors in metabolic products that can be
quantitatively altered on exposure to pollutants. Oxidative
stress is an adverse cellular and tissue level response to a
variety of contaminants and non-chemical stressors
Ahmad et al. 2006;
Nogueira et al.
2013; Patil and
David 2013; Pereira
et al. 2013;
& Gallagher 2013
Markers of lysosomal
membrane stability
Sub-cellular organelles containing hydrolytic enzymes that are
sensitive to toxicity leading to membrane rupture and leakage
(metals)
Ringwood et al.
2004; Edge et al.
2012
Condition indices
(hepatosomatic index,
gonadal indices)
Decreases in organ weight relative to whole body weight can
reflect organ toxicity or disease
Johnson et al.
2008a; Blazer et
al. 2012
Circulating hormone levels Levels of circulating hormones can be used to measure normal
sexual and reproductive behaviour
Blazer et al. 2012
DNA damage measures
(induction of DNA repair enzymes,
presence of PAH-DNA adducts)
DNA damage is a reflection of exposure and genotoxic effects Balk et al. 2011
Triglyceride levels, IGF1, The levels of lipids (such as triglycerides) and of growth
hormones (such as IGF1) can be used as a metric of the animals
energy reserves
Balk et al. 2011
(Modified from Hook et al., 20

15. Note: the arrow presents likely up - or downregulation following metal exposure;
suitable tissues are given in between brackets: b = blood, g = gill, l = liver, m = muscle, r = gonads.
Chemical, Biochemical, Physiological and other alterations in
response to metal exposure in fish
(Kroon et al., 2017

16. Widely accepted but this approach has to be validated with clearly defined and
measurable effects at organ, tissue level.
Yield info about both exposure and pathway of injury.
Much stronger in the recognition of exposure than in the identification of effects (van
Straalen & Feder, 2011).
Microarray approach – to characterize response of a contaminant in lab and field
Used to differentiate exposed and unexposed animals at very low concentration of
complex mixture (Garcia-Rayero et al., 2008)
(iii) Super Biomarker (Genome enabled biomarker)
Disadvantage:
1. Difficulty in interpreting the data – for the contaminant concentration of No
Observable Transcriptional Effect Level
2. Linking the measured gene expressions to ecologically relevant effects

17. Discovery &
Screening
Confirm. assay
devment.
Validation
(analytical &
Clinical)
Regulatory
approval &
adoption
Degree of evidence required
1. Discovery and screening
for novel biomarkers
(Proteomics, Metagenomics)
2. The development of specific
assays for the analysis of the
identified biomarkers
3. Acceptable in terms of its sensitivity,
specificity, accuracy, precision using a
specified technical protocol
4. Acceptably identifies, measures,
or predicts the concept of interest
(https://biomarkerbay.com/; FDA, 2016)
Development of Bio-Marker

18. (i) Sensitivity and Specificity
(ii) Ecological relevance
(Lam, 2009; Amiard et al., 2006; Diana et al.,
 EROD activity in fish - in vivo biomarker of exposure to PAHs
 Excellent correlative results have been obtained between EROD activity &
exposure to certain contaminants
 A change in EROD activity actually constitutes a significant (harmful) biological
effect ?
 MT in oysters also showing response to other non-metal stressors including
handling stress
(iii) Intrinsic Biotic factor
(iv) Baseline data for comparison – case of stress markers
(v) Seasonal variation of Biomarkers
 Muscle & gill – appropriate for all seasons
Challenges in Biomarker approach

19. (vi) Acclimation/recovery/repair
‘Organisms have the ability to evade
environmental stress and repair damage’
 Mussels were exposed to environmentally
realistic concentrations of B[a]P under
laboratory conditions
 Increasing B[a]P concentrations resulted in
elevated DNA adduct levels after 3–6 days of
exposure, but, importantly, this pattern of
dose-related increase disappeared after 12
days
Continued…
Field-based study (Xu et al.
1999)
 Perna viridis - transplanted from a ‘‘clean’’ site
to a number of ‘‘polluted’’ sites
 DNA adducts in the gill tissues quantified using
a 32P-postlabeling technique.
 Adduct levels - related to tissue concentrations
of Benzo [a] Pyrene as well as total PAHs.
Lab study (China et al. 2001)
* Indicates significant difference from the control

20. Use of multiple Biomarker, across multiple levels and multi species (Hook et al., 2014; )
– Oil pollution : EROD activity, Oxidative stress markers, Changes in lipid ratios
Biomarker validation:
Lab test – to determine cause-effect relationship between
stressors and biomarkers
Field studies – to test the strength of the relationship under
variable degree of complexity (Colin et al., 2015)
Biomarker tools can be combined with other traditional techniques to
make ecological relevance and area specific pollution management
(Downs et al., 2012)
Choosing biomarkers & organs that show weak
seasonal variability
Overcoming the challenges

21. Spatial – temporal extent of contaminants
- Induction of CYP1A or EROD activity – distribution of oil spill in a particular area,
extent of its effect and existence in the system (Exxon valdez oil spill/Micronesia fuel oil
spill/Prestige oil spill)
Predicting the health of organism
- Quantitative changes in Biomarker level – to examine the health of organism
Identifying stressors within complex scenarios contributing to ecosystem
decline – to avoid mis-management
Tools for determining the efficacy of management efforts in real time
(Downs et al., 2012; Lam & Gray,
Responses are rapid
- Transcriptomic changes can be measured after an hour of contaminant exposure)
Applications of Biomarker

22. ChinaUSA
Europe Union
India
S.Korea
Japan
Indonesia
Malaysia
Thailand
(Tracy et al., 2013; Dalzochio et al., 2016;
Scarlet, 2015; Katelyn et al., 2014;
Brazil
Mozambique
Australia
Antarctic
- Application
- R & D
CCAMLR
(Cetaceans)
OSPAR
15 Countries
NOAA,
USEPA, USGS
EPD
(Hong Kong SAR)
Independent
researcher group
Biomarkers in Envt. Assessment - Global overview

23.  Heavy metals, petroleum residues & pesticides reported in coastal waters, sediments and biota
Studies BM/Technique Status Reference
BM response in
coastal waters
Cronia contracta: AchE – Neurotoxin BM, Goa
R & D
Gaidonde et al., 2006
Cronia contracta: Impairment of DNA integrity:
Genotoxin BM, Goa
Sarkar et al., 2008
Perna viridis: Oxidative stress BM, Goa Jena et al., 2009
Morula granulata: Impairment of DNA integrit:
Genotoxin BM, Goa
Sarkar et al., 2014
Saccostrea cucullata: genotoxic effects by Comet assay
and oxidative stress - Goa
Sarkar et al., 2017
Inland waters
hsp27 – Coliform contamination / hsp47 – Organic
pollution (under validation)
R & D ICAR-CIFRI, 2017
 COMAPS (Coastal Ocean Monitoring and Prediction System) and ICMAM (Integrated Coastal
Mapping and Management): only to document the concentration of chemical constituents
(25 parameters) from 1991-92 onwards
 The application of bivalve BM can be incorporated in regular monitoring programme
(Verlecar et al., 2006)
 No consideration of BM usage in the regular coastal monitoring programmes & other
devastating events including the recent Ennore oil spill, 2017.
Status in India

24. Conclusion
 Developmental activities without any environmental concern
 Monitoring the environment for management
 Biomarker – an excellent tool for the pollutant exposure assessment &
effect prediction
 Benefits were recognized in developed countries – yet to be realized in
developing countries
 Integrated/Holistic approach – showing promising results

25. 1. Amiard, J. C., Amiard-Triquet, C., Barka, S., Pellerin, J., & Rainbow, P. S. (2006). Metallothioneins in aquatic invertebrates:
their role in metal detoxification and their use as biomarkers. Aquatic Toxicology, 76(2), 160-202.
2. Ching, E. W., Siu, W. H., Lam, P. K., Xu, L., Zhang, Y., Richardson, B. J., & Wu, R. S. (2001). DNA adduct formation and
DNA strand breaks in green-lipped mussels (Perna viridis) exposed to Benzo [A] Pyrene: dose-and time-dependent
relationships. Marine Pollution Bulletin, 42(7), 603-610.
3. COMAPS. http://www.incois.gov.in/portal/comaps/home.jsp
4. Conti, M. E. (2008). Biomarkers for environmental monitoring. In: Biological Monitoring (p.30). Southampton, UK: WIT
Press.
5. Dalzochio, T., Rodrigues, G. Z. P., Petry, I. E., Gehlen, G., & Da Silva, L. B. (2016). The use of biomarkers to assess the
health of aquatic ecosystems in Brazil: A review. International Aquatic Research, 1-16.
6. Diana, M., Catarina, V., Vanessa, M., & Mário, S. D. (2017). Seasonal changes in stress biomarkers of an exotic coastal
species - Chaetopleura angulata (Polyplacophora) - Implications for biomonitoring. Marine Pollution Bulletin, 120(1–2),
401-408.
7. Downs, C. A., Ostrander, G. K., Rougee, L., Rongo, T., Knutson, S., Williams, D. E., ... & Richmond, R. H. (2012). The use
of cellular diagnostics for identifying sub-lethal stress in reef corals. Ecotoxicology, 21(3), 768-782.
8. Editor. (1996). Editorial-Biomarkers, 1: 1-2. DOI: 10.3109/13547509609079340.
9. Esler, D., Ballachey, B. E., Bowen, L., Miles, A. K., Dickson, R. D., & Henderson, J. D. (2017). Cessation of oil exposure in
Harlequin ducks after the Exxon Valdez oil spill: Cytochrome P4501A biomarker evidence. Environmental Toxicology and
Chemistry, 36(5), 1294-1300.
10. FDA. (2016). Biomarker Qualification-Developing and Obtaining Regulatory Acceptance of a New Biomarker. In: FDA case
study - A collaborative effort to qualify a novel biomarker. Retrieved from
https://www.fda.gov/downloads/Drugs/DevelopmentApprovalProcess/DrugDevelopmentToolsQualificationProgram/UCM531
522.pdf.
11. Gaitonde, D., Sarkar, A., Kaisary, S., Silva, C. D., Dias, C., Rao, D. P., ... & Patill, D. (2006). Acetylcholinesterase activities
in marine snail (Cronia contracta) as a biomarker of neurotoxic contaminants along the Goa coast, West coast of
India. Ecotoxicology, 15(4), 353-358.
12. Garcia-Reyero, N., Adelman, I., Liu, L., & Denslow, N. (2008). Gene expression profiles of Fathead Minnows exposed to
surface waters above and below a sewage treatment plant in Minnesota. Marine Environmental Research, 66, 134–136.
13. Gil, F., & Pla, A. (2001). Biomarkers as biological indicators of xenobiotic exposure. Journal of applied toxicology, 21(4),
245-255.
References

26. 14. Hook, S. E., Gallagher, E. P., & Batley, G. E. (2014). The role of biomarkers in the assessment of aquatic ecosystem
health. Integrated Environmental Assessment and Management, 10(3), 327-341.
15. Huggett, R. J., Kimerle, R. A., Mehrl, P. M., Bergman, H. L., Dickson, K. L., Fava, J. A., McCarthy, J. F., Parrish, R., Dorn, P.
B., McFarland, V., Lahvis, G. (1992). Introduction. In: R. J. Huggett, R. A. Kimerle, P. M. Mehrle, H. L. Bergman
(Eds.), Biomarkers: Biochemical, Physiological, and Histological Markers of Anthropogenic Stress. Boca Raton, FL, USA:
Lewis Publishers.
16. ICAR-CIFRI. (2017). 2016-17 annual report of the ICAR-CIFRI. Retrieved from
http://www.cifri.ernet.in/AR/CIFRI_Annual%20Report%202016-17.pdf
17. ICMAM. http://www.icmam.gov.in/mandate.htm
18. Katelyn, J. E., Katherine, A. D., Stuart, L. S., Anthony, C. R., & Emma, L. J. (2014). A biomarker of contaminant exposure
is effective in large scale assessment of ten estuaries. Chemosphere, 100, 16-26.
19. Kroon, F., Streten, C., & Harries, S. (2017) A protocol for identifying suitable biomarkers to assess fish health: A
systematic review. PLoS ONE 12(4): e0174762.
20. Lam, P. K. (2009). Use of biomarkers in environmental monitoring. Ocean & Coastal Management, 52(7), 348-354.
21. Lam, P. K., & Gray, J. S. (2003). The use of biomarkers in environmental monitoring programmes. Marine Pollution
Bulletin, 46, 182–186.
22. McCarty, L.S,. Power, M., & Munkittrick, K. R. (2002). Bioindicators versus biomarkers in ecological risk assessment.
Human and Ecological Risk Assessment, 8, 159-64.
23. Mussali-Galante, P., Tovar-Sánchez, E., Valverde, M., & Castillo, E. R. D. (2013). Biomarkers of exposure for assessing
environmental metal pollution: from molecules to ecosystems. International Journal of Environmental Pollution, 29, 117-
140.
24. Nordberg, G. F. (2010). Biomarkers of exposure, effects and susceptibility in humans and their application in studies of
interactions among metals in China. Toxicology Letters, 192(1), 45-49. DOI: 10.1016/j.toxlet.2009.06.859.
25. Norwegian Environment Agency. (2013). Biomarkers for Environmental Monitoring. Retrieved from
http://www.miljodirektoratet.no/Documents/publikasjoner/M88/M88.pdf.
26. Sarkar, A., Bhagat, J., & Sarker, S. (2014). Evaluation of impairment of DNA in marine gastropod, Morula granulata as a
biomarker of marine pollution. Ecotoxicology and Environmental Safety, 106, 253-261.
Continued…

27. 27.Sarkar, A., Bhagat, J., Sarker, M. S., Gaitonde, D. C., & Sarker, S. (2017). Evaluation of the impact of bioaccumulation
of PAH from the marine environment on DNA integrity and oxidative stress in marine rock oyster (Saccostrea cucullata)
along the Arabian Sea coast. Ecotoxicology, 26(8), 1105-1116.
28.Scarlet, M. P. J. (2015). Biomarkers for assessing benthic pollution impacts in a subtropical estuary, Mozambique.
Thesis submitted to University of Gothenburg, Mozambique. p. 46.
29.Svendsen, C., Spurgeon, D. J., Hankard, P. K., & Weeks, J. M. (2004). A review of lysosomal membrane stability
measured by neutral red retention: is it a workable earthworm biomarker?. Ecotoxicology and Environmental
Safety, 57(1), 20-29.
30.The hazard assessment of cetaceans by the use of a non-destructive biomarker in skin biopsy. 30, September, 2011.
In Commission for the Conservation of Antarctic Marine Living Resources. Retrieved from
https://www.ccamlr.org/en/wg-cemp-92/47.
31.Tracy, K. C., Michael, W. L. C., Doris, W. T. A., & Philip, S. R. (2013). Biomarkers currently used in environmental
monitoring. In: C. Amiard-Triquet, P.S. Rainbow, J.C. Amiard (Eds.), Ecological Biomarkers: Indicators of
Ecotoxicological Effects (pp. 385-409). Florida, USA: CRC Press.
32.Van der Oost, R., Beyer, J., & Vermeulen, N. P. (2003). Fish bioaccumulation and biomarkers in environmental risk
assessment: A review. Environmental Toxicology and Pharmacology, 13(2), 57-149.
33.Van Straalen, N. M., & Feder, M. E. (2011). Ecological and evolutionary functional genomics-How can it contribute to
the risk assessment of chemicals?. Environmental Science and Technology, 46(1), 3–9.
34.Verlecar, X. N., Pereira, N., Desai, S. R., & Jena, K. B. (2006). Marine pollution detection through biomarkers in marine
bivalves. Current Science, 1153-1157.
35.WHO. (1993). International Programme on Chemical Safety - Biomarkers and Risk Assessment: Concepts and
Principles. Retrieved from http://www.inchem.org/documents/ehc/ehc/ehc155.htm
36.Xu, L., Zheng, G. J, Lam, P. K., & Richardson, B. (1999). Relationship between tissue concentrations of polycyclic
aromatic hydrocarbons and DNA adducts in green-lipped mussels (Perna viridis). Ecotoxicology, 8(2),73–82.
Continued…

28. • Thank You
Thank You