Marine Pollution Bulletin 46 (2003) 182186 www.elsevier.

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The use of biomarkers in environmental monitoring programmes

Paul K.S. Lam a, John S. Gray
a

a,b,*

Department of Biology and Chemistry, City University of Hong Kong, 81 Tat Chee Avenue, Kowloon Tong, Hong Kong SAR, PR China b Department of Biology, University of Oslo, PB 1064 Blindern, 0316 Oslo, Norway

Abstract The monitoring of biological eects has recently become an integral component of environmental monitoring programmes as a supplement to the commonly used contaminant monitoring. Over the years, many biomarkers have been developed that are claimed to be ecient at providing an early warning of deleterious eects on biological systems and for estimating biological eects due to contaminants. Although biomarkers are potentially useful, they have a number of important limitations. In this paper, we examine some of the key assumptions behind the theory and practice of use of biomarkers, and propose a scheme, which may facilitate decisions by environmental managers as to how and when to use biomarkers in their monitoring programmes. 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Environmental monitoring; Eect biomarker; Exposure biomarker; Bioassays

In the early phase of environmental monitoring of coastal areas most programmes consisted of the measurement of physical and chemical variables and only occasionally were biological variables incorporated. In the water column routine measurements included temperature, salinity, and oxygen concentrations, usually nutrients and some chemical contaminants such as heavy metals, which were perceived to be relatively easy to measure. In some programmes Secchi disc measurements of water transparency gave useful information in relation to algal blooms and primary production using the C14 method were often done. Sediment monitoring involved sampling the benthos with quantitative grab samplers and measurements included sediment grain size distributions, organic matter content and some contaminants, again usually heavy metals. Such programmes gave useful information on levels of contamination but with the exception of the benthic fauna of sediments, did not give information on eects of the contaminants on biota. In the 1960s concerns arose as to the eects of orga-

* Corresponding author. Address: Department of Biology and Chemistry, City University of Hong Kong, 81 Tat Chee Avenue, Kowloon Tong, Hong Kong SAR, PR China. Tel.: +852-2784-4325; fax: +852-2788-7406. E-mail address: bhjsgray@cityu.edu.hk (J.S. Gray).

nochlorine chemicals on the marine environment, especially DDT and PCBs. Yet routine measurements in the water column could not be made as concentrations were nearly always below the detection limits and such analyses, even if possible, required expensive apparatus and skilled analysts. With the realization that herbicides, insecticides and antifouling agents were also likely to cause eects in the marine environment attention turned to eects monitoring, rather than contaminant monitoring. Researchers concentrated on development of methods that could provide early warning of eects on biota caused by the wide variety of contaminants present in the marine environment. Such indicators were called biomarkers. There are many denitions of a biomarker. Typically, biomarkers are dened as quantitative measures of changes in the biological system that respond to either (or both) exposure to, and/or doses of, xeonbiotic substances that lead to biological eects. Although not explicitly contained in most denitions, the use of the term biomarker or biomarker response is often restricted to cellular, biochemical, molecular, or physiological changes that are measured in cells, body uids, tissues, or organs within an organism and are indicative of xenobiotic exposure and/or eect. Changes that occur at the organismic, population and assemblage levels more usually referred to as bioindicators (hence sometimes the term bioindicator organisms is used),

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although there is still no clear consensus on this issue. One possible reason for limiting the term biomarkers to sub-organismic changes is that one of the functions of biomarkers is supposedly to provide early warning signals of biological eects, and that it is generally believed that sub-organismic (molecular, biochemical and physiological) responses tend to precede those that occur at organismic, or higher levels. The use of biomarkers in providing early warning signals for environmental deterioration necessitates the inclusion of biological changes that are indicative of exposure to a specic agent (referred to as exposure biomarkers), even if these changes may not be directly linked to harmful (toxic) eects in the target organism. Biological changes occurring in organisms and caused by contaminants are called eect biomarkers. By the mid 1980s a wide range of biomarkers had been developed and suggested for use in monitoring programmes. Yet, there was little agreement between researchers on which were the best or most appropriate techniques. As a result a workshop was held, the Oslo Workshop, under the auspices of UNESCO-IOC where a wide range of methods were tested blind and a team of statisticians analyzed the results independently. Not surprisingly some methods were found to be highly variable and were unable to detect the known gradients of contamination, whereas others were reliable (Bayne et al., 1988). A further workshop was held at Bremerhaven with essentially similar ndings, but where a wider range of biomarkers was tested (Stebbing and Dethlefsen, 1992). ICES also established a working group to study the application of biomarkers (ICES, 1997) and in 1995 OSPARCOM/ICES agreed on a biological monitoring programme that was put into eect in the N. Sea (JAMP, 1998a,b). To our knowledge this is the only programme that so far, has integrated a suite of biomarkers into routine monitoring of coastal waters. The national and international monitoring programmes take many years to arrive at an agreed programme, yet biomarker research does not stop. On the contrary continuously new methods are being developed that are claimed to be more sensitive than previous methods, or more reliable or can detect new eects that had not been observed before. Thus a manager is faced with the dilemma of deciding which biomarker methods should be used and under what circumstances; and has to answer the question of what useful information can be derived from biomarker responses observed in monitoring programmes. We believe that this topic is important and has not been discussed in detail. Here, we (1) examine the major assumptions/issues associated with the theory behind the biomarker approach in environmental monitoring and the assumptions that need to be made in putting the approach into practice; and (2) provide some suggestions of how and when to use which biomarkers.

1. What are the assumptions/issues? 1.1. Biomarkers are eective early warning signals of adverse biological eects For biomarkers to be useful in providing early warning of future eects on biological systems, it is conceivable that some false positives will occur, that is an eect is indicated by statistical analysis when in fact one is not present. Thus in order for the biomarker approach to be cost-eective, the number of false positives (i.e. making a Type I error) needs to be small, while false negatives, indicating no eect when there is in fact one present (a Type II error), must be kept to an absolute minimum. In environmental terms making Type II errors is more serious. The eectiveness of a biomarker, as an early warning signal provider or a screening tool for important environmental samples or media, decreases with the number of false negatives as well as false positives. A sensitive biomarker would be expected to produce more false positives. Thus, an ideal biomarker for monitoring purposes should have optimal rather than maximum sensitivity. 1.2. Biomarkers can indicate biological eects, while chemistry-based surveillance system cannot The question here depends crucially on what is meant by an eect. If it means an observable change in the biological system, then the biomarker is, by denition, capable of providing this information. However, if an eect, in the context of biomarker research, means an observable change in the target system that has a signicant biological consequence (see also the denitions of exposure biomarker and eect biomarker earlier), and then the conventional biomarkers are not always eective. For an eect to have a signicant biological consequence, it should result in a decrease in the Darwinian tness of individual organisms or a reduction in the useful functions (i.e. goods and services) provided in the case of an ecosystem. There is evidence that many biomarker responses are not directly associated with real harmful effects in the target organism(s) and there are at least two reasons for this. Firstly, some biomarkers are designed merely to indicate exposure. Secondly, some observed biological changes (biomarker responses) are eectively compensated or repaired by the biological systems concerned (e.g. Ching et al., 2001). In general, an observed biomarker response probably indicates exposure, but an apparent lack of response could either be due to an absence of inducers (e.g. the levels of toxic agents are not high enough to elicit a response) or an ecient repair system occurs within the organism. In addition, whether a particular biomarker response is observable or not depends not only on the level of exposure, but the timing of the measurement taken after the exposure.

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Although it is true that chemically-based monitoring programmes cannot provide direct information on biological eects, the chemistry data are still useful in predicting potential biological eects if prior investigations (including those associated with biomarker research) have been successful in relating contaminant levels in specic environmental media (water, sediment or biota) with responses in biological systems i.e. derivation of threshold eects levels (such as predicted no eect concentrations, PNECs) from ecotoxicological observations (Lam and Gray, 2001). Under this circumstance, the use of chemical data to make predictions of eects in biological systems may conceivably be cost-eective, provided that the analytical procedures can be performed routinely at a suciently low cost. Recent advances in the automation and miniaturization of analytical equipment, due primarily to advances in computer technology and power electronics, will also prove invaluable in this endeavour. 1.3. Biomarkers are more eective in revealing overall toxicities of complex mixtures Environmental managers are faced with many questions. Two common ones are: (1) Is a particular system under stress? (2) Is a particular system under stress due to a specic agent or operation? We believe that a biomarker or biomarkers that is/are designed to indicate general stress would be useful for the former situation where there is often no information on which stressors are important. Here, useful biomarkers are likely to be of a higher level of biological organization, e.g. physiological biomarkers that relate to feeding, growth, or reproduction of individual organisms or the presence/ absence of certain species in a biological assemblage (if a broader denition of biomarker is adopted). In contrast, a specic biomarker would be more useful in dealing with the second question as the biomarker(s) will be employed to ascertain the eects due to a number of dened agents of interest. These specic biomarkers utilise the fact that some chemicals can only exert toxic eect when bound to a specic site of toxic action (receptor). These receptors may be parts of cells conned to certain tissues, or can be nucleic acids or specic regions of proteins within nerve synapses or membranes distributed among the cells of an organism. Biomarker responses of this type are often developed into standardised or semi-standardised tests to measure toxicity and are known as bioassays. Examples of these bioassays (or biomarker assays) include assays on acetylcholinesterase activity for pesticide exposure; receptor binding assays for dioxin-like compounds (Giesy et al., 2002); and radioimmunoassay or enzyme-linked immunoabsorbent assay for aquatic toxins, etc. In the case where an indicator for the general state of the environment is needed, the biomarker approach is, in most

cases, instructive. However, in the case where the agents in question are known or dened, the choice between a chemistry-based or a biomarker-based monitoring strategy becomes less clear. The nal decision will depend on the precise question(s) asked and ultimately will have to rely on an informed cost-benet analysis. 1.4. Biomarkers are economical Some key chemical contaminants (e.g. dioxins) are extremely expensive to analyse (up to US$500 per sample from certain commercial laboratories). Thus routine monitoring of such chemicals is prohibitively expensive. One suggested solution is to employ bioassays that indicate that organisms have been exposed to such chemicals. Only when the bioassay indicates there is a problem are the contaminants in question sampled and analysed.

2. The suggested approach Fig. 1 shows a suggested approach to eective use of biomarkers in routine monitoring programmes. To our knowledge such a programme has not been put into eect anywhere. Yet we believe there are strong arguments for introducing such monitoring. The basic monitoring programme includes commonly measured variables of a physico-chemical nature, and easily measured contaminants in water, sediments and organisms. Sediment-living benthic assemblages are sampled since they have been shown to exhibit sensitive responses to contaminant and organic enrichment gradients. Nearly all coastal monitoring programmes that have the objective of measuring the quality of the environment record such variables. We suggest that it is cost-eective to employ bioassays at the sub-cellular, cellular, or organismic level to measure total toxicity for specic toxicants or complex mixture of chemicals not amenable to common analytical procedures. In addition a suite of biomarkers of general stress should be employed. Biomarkers of this type are used in the N. Sea monitoring programme. The monitoring programme must have a suciently large number of sites so that statistical analyses can be done to compare responses at impacted sites with controls. Often sampling design is not considered carefully enough with the result that it is not possible to compare sites, (or times) with sucient statistical power, (see Nicholson et al., 1992 for a good example). If signicant dierences are found between control and impacted sites in the measured variables then the next phase of the analysis starts. Decreased oxygen concentrations in bottom water and raised chlorophyll a concentrations in surface waters suggest that eutrophication is the cause. Gray et al.

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Fig. 1. Diagram showing a suggested approach to the eective use of biomarkers in routine monitoring programmes. The biomarkers used are shown in italics.

(2002) suggest ways for the manager to follow up on assessing the extent and eects of eutrophication. Should the level of contaminants in water, sediments or organisms be found to be higher in impacted than control sites, biomarkers should be employed to measure the eects on organisms. Suggested biomarkers are those that indicate changes associated with a decrease in the Darwinian tness of individual organisms and/or a reduction in eciency of ecosystem processes, and thus are likely to cause real harm to target organism(s) or ecological system(s). There are not many unequivocal examples of eect biomarkers. Biomarkers that can indicate signicant impairment on feeding, growth, reproduction and survivorship are good examples. For the benthic assemblages routine monitoring will involve multivariate statistical analyses to compare control with impacted sites. The methods available today enable scientists to relate contaminant data to species distribution data, e.g. PRIMER (Clarke and Warwick, 1994). Yet the results are merely correlative and do not necessarily indicate cause and eect. Further studies are needed and again biomarkers of eect should be employed. If the bioassays suggest that there are problems with unmeasured contaminants then more detailed analyses are needed. Finally, if biomarkers of general stress in-

dicate a problem one needs to examine the contaminant concentration within the organisms and conduct bioassays in case the eects are caused by contaminants that have not been measured. The nal phase of the monitoring is that should biomarkers of eect show responses to specic contaminants in a given organism then one sets in motion population level monitoring for eects on that organism. This implies that there is a sound understanding of the biology of the organism used in the study of the eect biomarker and that the organism is suitable for monitoring of the eld population. Typical species used in biomarkers of eect monitoring programmes are mussels and demersal sh such as ounders. For the benthic assemblages the next phase is to examine the links between spatial and temporal eects in the assemblage and the agents thought to be responsible for the changes. This phase is rarely done if ever, and multivariate analyses have unfortunately been the endpoint for monitoring. For example in monitoring of eects of oil on benthic assemblages the eects found are correlated with increased concentrations of heavy metals, barium (used in the drilling process) and components of oil. The causative agent is not known and on the Norwegian continental shelf regulation has been to reduce discharges of oil and barium by prohibiting

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P.K.S. Lam, J.S. Gray / Marine Pollution Bulletin 46 (2003) 182186 Ching, E.W.K., Siu, W.H.L., Lam, P.K.S., Xu, L., Zhang, Y., Richardson, B.J., Wu, R.S.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, 603610. Clarke, K.R., Warwick, R.M., 1994. Changes in marine communities: an approach to statistical analysis and interpretation. Plymouth Marine Laboratory, Plymouth, UK. Giesy, J.P., Hilscherova, K., Jones, P.D., Kannan, K., 2002. Cell bioassays for detection of aryl hydrocarbon (AhR) and estrogen receptor (ER) mediated activity in environmental samples. Marine Pollution Bulletin 45, 316. Gray, J.S., Bakke, T., Beck, H-J., Nilssen, I., 1999. Managing the environmental eects of the Norwegian oil and gas industry: from conict to consensus. Marine Pollution Bulletin 38, 525530. Gray, J.S., Wu, R.S.S., Or, Y.Y., 2002. Eects of hypoxia and organic enrichment on the marine coastal environment. Marine Ecology Progress Series 238, 249279. ICES, 1997. ICES review of the status of biological eects techniques relative to their potential application programmes. ICES Cooperative Research Report, No. 222, 1220. JAMP (Joint Assessment and Monitoring Programme), 1998a. JAMP guidelines for general biological eects monitoring. Oslo and Paris Commissions, p. 15. JAMP (Joint Assessment and Monitoring Programme), 1998b. JAMP guidelines for contaminant-specic biological eects monitoring. Oslo and Paris Commissions, p. 38. Lam, P.K.S., Gray, J.S., 2001. Predicting the eects of toxic chemicals in the marine environment. Marine Pollution Bulletin 42, 169 173. Lyndon, A., 2002. Intersex sh found in UK estuaries. Marine Pollution Bulletin 44, 722. Nicholson, M.D., Fryer, R.J., Ross, C.A., 1992. Designing monitoring programmes for detecting temporal trends in contaminants in sh and shellsh. Marine Pollution Bulletin 34, 821826. Stebbing A.R., Dethlefsen, V., 1992. Introduction to the Bremerhaven workshop on the biological eects of contaminants. Marine Ecology Progress Series 91, 18.

discharges of drilling cuttings, (Gray et al., 1999). Whilst this has been eective in this case there is a danger that the perceived agent causing the problem is wrongly attributed and the solution to the problem may be ineffective and or nor cost-eective. The nal phase of further monitoring is probably that requiring most attention today. Should raised levels of hard to measure organic contaminants be found then a suite of biomarkers for genotoxicity, mutagenicity and endocrine disrupters need to be applied. It was thought that apart from eects of TBT endocrine disrupters were not a problem in open waters with high dilution factors for contaminants. Yet recently it has been found that endocrine disrupters are aecting sh populations in 12 estuaries in UK (Lyndon, 2002). We believe that biomarkers for genotoxicity, mutagenicity and endocrine disruptors should be key aspects of a biomonitoring programme, but in order to be cost-eective we see their employment in a sequential context.

Acknowledgements PKSL acknowledges support of a strategic research grant of the City University of Hong Kong (7001345).

References
Bayne, B.L., Clarke, K.R., Gray, J.S., 1988. Background and rationale to a practical workshop on biological eects of pollution. Marine Ecology Progress Series 46, 15.