Persistent pollutants from the environment cause impairments in animals and plants and in severe cases produce irreversible damage to individuals, population community and ecosystem. These irreparable changes begin in individuals as very mild changes (if the dosage of the pollutant is not very acute) and then advance to a stage where there is no retrieval from it. In the beginning stages all the affected individuals show some signals and symptoms which can be read and assessed properly and treated in right time so that all succeeding impairments could be prevented. These early warning signals shown by individual entities are called biological markers or biological indicators (biomarkers). Proper diagnosing and understanding of these biomarkers are important to save individuals and so population, community and ecosystem from pollution related multitude of problems.
A biomarker is a quantifiable time dependent or dose dependent biological response or toxic effect elicited by a xenobiotic substance at the individual level or below (Mayer et al., 1992; Walker, 1998). It is a measure of exposure to a toxic substance (Timbrell, 1998), or of toxic effect at the histological, biochemical, physiological, morphological or behavioral level (Jamil, 2001). Another definition is "a change induced by a contaminant in the biochemical or cellular component of a process, structure or function that can be measured in a biological system" (NRC, 1989). Because biomarkers are sought as diagnostic tools that are more easily measured than the underlying phenomenon, they should be quantifiable, sensitive, non-invasive, specific, and easy to measure; and they should relate to the biochemical mechanisms, and work at realistic doses (Walker, 1998, Timbrell, 1998). The most relevant biomarkers are those that are essential to normal function of cells, tissues, or the organism (Handy et al., 2003). It is advisable to have multiple biomarkers to characterize concentrations of different contaminants, adverse effects due to contaminants and to quantify toxic damage than it is to consider a single response (Handy et al., 2003). Biomarkers can be used to track each phase of the dose-response continuum, from exposure through irreversible effect or to resultant disease (Fox, 1993; Schmidt, 2006).
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There are many analytical methods available to detect and quantify the amount of toxins or their breakdown products in different animal species. But the accurate measurement of these chemicals depends on a thorough understanding of their chemistry and their actions. According to Walker (1998), the four most desirable characteristics for biomarker assays are sensitivity, specificity, simplicity and stability.
Timbrell (1998) divided biomarkers into following three groups
biomarker of exposure;
biomarker of response or toxic effect;
biomarker of susceptibility. (fig. 1)
Biomarkers of exposure are important in toxicology, because they are indicators of
internal dose, or the amount of chemicals absorbed by the different systems of an organism or the presence of toxic substances or its metabolites in the body.
For example instead of continuously measuring trace metal concentrations in sediment, water and biota, the concentration of metal binding protein metallothionein can be measured in tissues of organisms or instead of measuring tissue concentration of organophosphorous pesticides, it can be assessed as cholinesterase in blood samples (Handy et. al., 2003).
Biomarkers of exposure can be divided into two groups
Biomarkers of internal dose
Biomarkers of effective dose
Biomarkers of internal dose give a measure of the occurrence of a toxin in an organism. This is measured as the amount of toxin or its metabolite in the system or in body fluids. Metabolites derived from glutathione conjugation are one of the most important groups belonging to this category. Glutathione (GSH) detoxifies reactive chemicals to which biological systems are exposed and are excreted as sulphur containing metabolites (Timberell, 1998). Biomarkers of effective dose measure the interactions of toxicants and some specific molecular or cellular targets in the body such as protein receptors (Schmidt, 2006).
Biomarkers of toxic response or toxic effect are measured as health impairment or as early disease precursors or as a disease. It can be described as different cellular responses following exposure to toxic chemicals.
Biomarkers of susceptibility measure the variability expressed by different individuals when exposed to the same chemical or same environmental stress due to their differences in genetic make up. Biological or genetic differences make some individuals more susceptible to environmentally induced diseases than others.
Always on Time
Marked to Standard
A number of enzymes or other molecules exhibit polymorphisms and this gives a measure to the magnitude and type of this response (Gulumian et al., 2007).
Some advantages of biomarkers
they indicate biologically available contaminants rather than the inert form of contaminants
if a biomarker measures the induction of enzymes (it shows that a defence mechanism has been activated), and it involves energy expenditure that consequently affects an individual's reproduction and growth,this can translate into a population-level effect seen in the next generation (Walker, 1998)
a suite of biomarkers can indicate even unsuspected contaminants from the environment
they can detect persistent contaminants in biological systems after they have been degraded in the environment
biomarker analyses are inexpensive in many cases and may be easier to perform than other chemical analyses (Handy et. al., 2003)
When an organism is exposed to increasing levels of a toxin, it passes through three distinct physiological stages:
the stressed state and physiologically compensable state;
a phase in which a toxic effect exceeds the organism's ability to compensate, but in which the effects are reversible;, and
a stage where the damage is irreversible and death may occur.
The different physiological stages of xenobiotic chemicals effects in the body of an organism can be measured by looking at different biomarkers. The best biomarker is one that can detect and convey the presence of toxins at the first physiological phase. But it is desirable to have biomarkers that can detect the presence of toxin at least at the second phase.
Many types of biomarkers have been developed for different groups of animals including birds both in the field and in the lab, but most are destructive. Biomarkers that depend on collecting eggs during brooding or sacrificing animals to obtain organs and tissues are known as destructive methods. Biomarkers can be assessed non-destructively too because chemical contaminants can affect both morphological and behavioural traits of an organism (McCarthy and Secord, 2000; Bortolotti et al., 2003). So the presence and effects of contaminants can be effectively assessed non-destructively by evaluating their morphology and behaviour. Other non-destructive biomarkers can reflect the concentrations of contaminants evaluated in organs or tissues of animals found dead, or in feces, feathers, fur, skin and blood of live animals, and in abandoned eggs (Fossi, et al., 1999; Marsili et al., 1996), by assessing the quality of some of their ornamental traits, or by observing and quantifying some of their daily activities and behaviours. Nondestructive biomarkers are preferable in many ways.
They are necessary when rare, protected, or threatened species are to be studied and it is an important measure in wildlife conservation (Walker, 1998)
They permit simple and quick sampling, with minimal stress to individuals and population
They are useful when repeated sampling is required over long periods (Hatch et al., 2002; Casini et al., 2003).
Large number of individuals can be studied without population reduction (Walker, 1998)
They are ethical and humane
Materials that can be obtained non-invasivelyfor biomarker analysis include excreta, abandoned eggs, shed feathers, breath (exhalation). Blood, live feathers, skin and secretions of preen glands can also be used for biomarker estimation but here the procedure is invasive but non destructive. Morphological measurements and recording of reproductive success are other non-destructive indicators of condition. This chapter reviews some of the materials that can be collected non-destructively in the laboratory and in the field for birds that live in organochlorine-polluted areas. The two most important organochlorine groups included in this review are DDT and its metabolites, and PCB congeners.
Non invasive procedures
These are biomarkers that can be estimated in materials and tissues such excreta, shed feathers, abandoned eggs, morphological measurements, expiratory gases etc. Biomarkers can also be estimated by determining reproductive rates, growth of the individual or examination of external features like pigmentation of feathers, beak and feet and general appearance of external features and also their behaviours e.g., nest building patterns, nest attentiveness, nest abandonment etc). Additionally, ectoparasite load on individuals or in their nests can be measured. The biomarkers described below, some are already used in the field and in the laboratory and some are in the very beginning stage of study and use. Some of the described ones are biomarkers of exposure whereas others are indicators of effects. Most these need more research to refine them and to demonstrate their effectiveness and acceptability.
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Feathers can be used to directly estimate the presence and concentrations of organochlorine contaminants (Jaspers et al., 2006; 2007[refs?]. Feather pigmentation pattern can be used as a qualitative tool [ref], and concentration of pigments can be used as a quantitative tool for estimating the presence of contaminants in many birds [refs]. Feather colours can represent either the expression of pigments, or the refraction of light caused by the structure of the feather or by a combination of both (Cornell lab., 2007). There are three different kinds of pigments found in birds - melanins, carotenoids and pophyrines. Melanins give rise to colours like darkest black to reddish browns to grey and tan. Melanocytes are formed in the neural crest and migrate to the dermis and supply melanosomes to the growing feathers (Rutz, et al. 2004). Melanine is formed by metabolism of the amino acid tyrosine (Fox, 1935). Birds must acquire carotinoids through their diet either by directly eating plants or indirectly by eating animals that have consumed plants. The colour of carotinoids range from bright yellow to yellow and from light orange to bright orange (Cornell lab., 2007). So quantity and quality of carotinoids in the diet, the proper digestion, absorption and metabolism of this pigment is important in maintaining the feather colour of that particular individual's feathers. Porphyrins are intermediate metabolites of heme synthesis produced by modifying amino acids. Porphyrins are responsible for a range of colors, including pink, browns, reds, and greens. Consequently, any contaminant-induced interference in the formation of melanocytes during development, carotinoid acquisition or metabolism, or heme metabolism can affect the colour pattern of feathers.
McCarthy and Secord (2000) confirmed that examination of variations in ornamental traits was an efficient method to evaluate effects of chemical contaminants in the environment. They studied patterns of plumage colour in subadult female tree swallows in their first breeding season. They found that subadults in colonies in contaminated areas had significantly more adult-type blue-green color than did subadults from other sites. Bortolotti et al. (2003) exposed captive American Kestrels to dietary PCBs and observed them during breeding and winter seasons. At both times they observed sex specific differences in both colour and carotenoids between controls and PCB-exposed birds.
Dauwe et al. (2005) and Jaspers et al. (2007) estimated the presence of PCBs and organochlorine pesticides in bird feathers. Dauwe et al. (2005) could detect most of the PCB congeners and DDT and their metabolites that occurred in fat samples of 27 adult great tits in their feathers, also. Furthermore, the concentration of organochlorines detected in the feathers were significantly positively correlated to what they could detect in the fat samples collected during breeding season. Jaspers et al. (2007) could detect PCBs and DDTs in single tail feathers of Common Moorhen (Gallinula chloropus). After analyzing eight different terrestrial and aquatic species, they recommended feathers as a non destructive biomonitoring tool, although they couldn't predict the exact body burden from the observed contaminant concentrations in the feather samples. Behrooz et al. (2009) analysed museum preserved tail feathers of 37 birds belonging to 18 species from Persian Gulf region. They could identify many organochlorine pesticides such as DDT and its metabolites, hexachlorobenzene (HCB), Î±, Î² and Î³-hexachlorocyclohexane (HCH) isomers, as well as PCB congeners.
Although the use of feathers in biomarker studies for the accumulation of organochlorines has received limited attention, feathers are more widely studied to document metal contamination [refs? 'e.g. 'reviewed by **** (20**) ].
The induction of cytochrome P4501A (CYP1A) can be used as a biochemical endpoint to measure the presence of polyhalogenated aromatic compounds in birds. The induction of CYP1A is measured in liver microsomes, as isthe rate of catalysis of CYP1A substrates such as ethoxyresorufin or activities of ethoxyresorufin -O-dealkylase (EROD), benzyloxyresorufin-O-dealkylase (BROD) or pentoxy benzyloxyresorufin-O-dealkylase (PROD) (Elliott et al., 1996; Smits et al., 2000; Custer et al., 2001; Papp et al., 2005) also in the liver. However, birds must be sacrificed to obtain liver samples. But CYP1A activity can also be measured by caffeine breath test (CBT) through use of radiolabelled substrate (14C-caffine) (Feyk and Giesy, 1996; Feyk et al., 2000). Feyk et al. (2000) used radiolabelled substrate (14C-caffine) to measure in vivo CYP1A activity twice during the development of common tern chicks fed for 21 days with fish spiked with different amounts of 3,3',4,4',5-pentachlorobiphenyl (PCB 126) and 2,2',4,4',5,5'-hexachlorobiphenyl (PCB153) such that the diet contained an average of 23, 99 or 561 pg of 2,3,7,8-tetrachlorodibenzo-p-dioxin equivalents per gram fish. The CBT successfully detected PCB treatment differences by the presence of caffeine N-demethylation especially during week one. But there was greater induction of EROD activity than demethylation of caffeine. Numata et al. (2007) studied caffeine metabolism as a potent measure of CYP1A induction in two bird species, paradise shelducks and southern black backed gulls, They concluded that caffeine metabolism was a potentially useful non-destructive biomarker for CYP1A induction in wild birds.
Eggshell and membranes
One of the most serious effects of DDTs on avian wildlife is thinning of their eggshells, which is one of the most serious impediments to reproductive success (Lundholm, 1997; Gill et al., 2003). Almost 40 years after banning the chemical from North America the effect of those chemicals are still evident in many parts of this continent. Shell thickness is an important transgenerational biomarker that can be evaluated to assess avian exposure to endocrine disrupting chemicals (EDCs) (Fossi et al., 1999). This biomarker is known as "Index of Ratcliff", which relates the DDT levels in maternal tissue to eggshell thickness (Peakall, 1992; Fossi et al., 1999).
Chorioallantoic membranes (CAM) have been successfully used by many researchers to estimate maternal contaminant exposure and predict chemical concentrations of offspring in multiple species of birds and reptiles (Cobb et al., 1995; 2003; Pastor et al., 1996; Barger et al., 2003; Pepper et al., 2004). This highly vascularised extraembryonic membrane is used by developing embryo for respiration, nutrient transport, and waste storage. The membrane is discarded with shell at the time of hatching (Pepper et. al., 2004). This can be collected and can be used for contaminant analysis. Cobb et al. (1995) detected DDTs and PCBs in the CAMs from great blue heron eggs. Pastor et al. (1996) studied pipping eggs of Audouin's gulls for the distribution of organochlorine pollutants among yolks, embryos, and CAMs and to assess the usefulness of CAMs as surrogates for predicting whole egg contaminant burdens. Their results showed that CAMs could provide good predictions only for hexachlorocyclohexanes, PCB52 and DDD, as those pollutants were almost completely excreted to the CAM. Barger et al. (2003) studied the effect of PCBs and endosulfan in chicken eggs, membranes, and adults and suggested that CAM can be used to estimate avian exposure to PCBs and resultant biological response.
The excreta of birds is the cloacal mixture of feces and urine (Goymann, 2002). It consists of water, food residue, bacteria, nitrogenous waste products and secretions of the intestines and liver. Since it contains the liver secretions, it will accumulate all the associated metabolic waste products, pollutants and pollutant detoxification products. Therefore, feces are an ideal material to use for biomarker assays.
Many hormones and their metabolic byproducts can also be found in the excretory wastes. One such hormone is the stress hormone, corticosterone (CORT).
Physical and chemical changes in the environment or any kind of stress to a bird can induce the production of corticosterone by affecting the hypothalamic-pituitary-adrenal (HPA) axis. Love et al. (2003) found that PCBs acquired through diet can induce fluctuations in corticosterone. Franceschini et al. (2008) studied adult female and nestling tree swallows exposed to different levels of contamination with polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in the environment. They found that fluctuations in corticosterone levels reflected environmental contaminant concentrationst. Goymann et al. (2002) concluded that birds' excreta can be used to analyze cortisone hormones instead of using blood plasma. So corticostrone measurements can be done noninvasively in excreta and semi-invasively in blood.
Another metabolite that can be measured in both blood and excreta is porphyrin, whose profile can be estimated as a biomarker of exposure to organochlorines.. Porphyrins are tetrapyrrolic pigments with a characteristic absorption spectrum that includes an intense brand of absorbance at about 400 nm and a diagnostic spectrum that makes their detection and quantification both sensitive and specific (Smith, 1975; Casini et al., 2003). Porphyrins are intermediate metabolites of heme synthesis. The heme part of hemoglobin consists of iron atoms that are attached to porphyrins. Porphyrins consist of five- numbered ring structures called pyrrols with interconnections (methane bridges) and side chains attached to pyrrols. The particular porphyrin found in hemoglobin is protoporphyrin IX, which can be distinguished from other porphyrins based on side chains - four methyls (-CH3), two vinyls (-C=CH2) and two propionic acids (-CH2 - CH2 -COOH). In the synthesis of heme, a glycine is activated by association with pyridoxal phosphate and combines with succinate, which has been in turn activated by association with coenzyme A. The activated glycine and succinate combine under the influence of an enzyme called Î´-amnolevulinic acid synthetase, to form Î´-amnolevulinic acid (ALA). Two ALA molecules are combined to form the monopyrrole porphobilinogen (PBG) and four PBG molecules are assembled together to form the large, ring-shaped uroporphyrinogen. Following some modification of this ring molecule involving mainly the clipping of its projecting side chains, we arrive at the structure known as protoporphyrinogen IX and then protophorphyrin IX.
Succinyl CoA + glycine
ALA Dehydrase (ALAD)
URO Decarboxylase (UROD)
+Fe Heme synthetase
(Maclean, 1978; Peakall, 1992)
Some classes of environmental contaminants including PCBs can interfere with the synthesis of heme by directly interfering with enzymes (UROD or ALAD) of biosynthesis or by increasing the rate of oxidation of intermediary porphyrinogens. Other contaminants interfere with the enzymes uroporphyrinogen decarboxylase and coproporphyrinogen oxidase, resulting in the accumulation of Highly Carboxylated Porphyrins (HCPs). So porphyrins or their oxidative byproducts such as coproporphyrins and uroporphyrins or HCPs can be accumulated in blood, erythropoietic tissues (the liver and kidneys) and are excreted via urine (Casini, et al., 2001). Fox et al., (1988) measured the concentrations HCPs in liver tissues of adult herring gulls collected from the Great Lakes region during the early 1980s and found that they were positively correlated with the amount of halogenated aromatic hydrocarbons including PCBs in the environment. Japanese quails (Coturnix coturnix japonica) treated with Phenochlor DP6 and Aroclor 1260 showed porphyrin accumulation in the liver and other organs (Casini et al., 2003). In tree swallow studies, Bishop et al. (1999) found that concentrations of uroporphyrin in the liver were correlated with amounts of PCB congener 118 in nestlings. Pulp and paper mill effluents have also been found to elevate HCPs in tree swallows (Wayland et al., 1998). Kuzyk et al., (2003) detected uroporphyrin, copropophyrin and protoporphyrin in the livers of black guillemots exposed to PCBs in marine sediments, but they didn't find any variation with respect to Î£PCB exposure. Porphyrins can be detected in minute amounts, so they can be used as a very sensitive biomarker and possibly as an early warning signal of exposure in the laboratory and field (Fox et al., 1988, Fossi, et al., 1996). Porphyrins can be detected by a rapid fluorimetric assay or by High Performance Liquid Chromatography (HPLC).
Preen gland secretion (preen oil)
Preen glands or uropygial glands are located at the base of tail feathers in birds and secrete preen oil that is used for grooming and cleaning of feathers. They are found in almost all groups of birds. In passerines, the preen oil's most important components are mixtures of homologous monoesters made up of long chain acids and alcohols (Haribal et al., 2005). Yamashita et al. (2007) estimated PCB concentrations and profiles in the preen glands and corresponding abdominal adipose tissue collected from 30 seabirds (2 orders, 3 families, 10 genera, 13 species) and found a weak but significant correlation between the PCB concentrations in the oil and abdominal adipose tissue. The authors collected preen gland oil (50 mg) by wiping the gland with KimwipeÂ® paper tissues.
Even moderate doses of PCBs and other persistent organochlorine contaminants, can disrupt the endocrine system and impair vertebrate reproduction and development (Colborn and Smolen, 1996). These compounds are neurotoxic, so they can induce changes in behaviour (Walker, 2003), physiological changes, and functional differences (Tanabe, 2002). Many chemicals appear to mimic vertebrate steroids, and interact with steroid receptors (Ottinger et al., 2001). These chemicals can lead to reduced egg production, change in egg size, eggshell thinning, decreased fertility (Lundholm 1997; Verboven, 2009) and are particularly harmful during the embryonic, foetal and early postnatal periods (Fossi, et al., 1999). Sublethal doses in adult birds can reduce parental attentiveness and cause abnormal reproductive behaviour (Barron et al., 1995), ultimately resulting in reduced reproductive success. So the variables of reproductive success are important ultimate measures of the effects of chlorinated hydrocarbon contaminants in birds (Smits, et al., 2000; 2005). This can be measured from nest building behaviour to all different stages of reproduction. Reproductive parameters that can be measured are effects on fertility (Barron et al., 1995), date of clutch initiation (McCarthy and Secord 1999b; Fernie et al., 2001), clutch size, sequence length (the length of time to complete the egg laying), brood size (Gill et al., 2003), incubation period, hatching success, sex ratio, nest abandonment, burial of eggs in the nest lining (McCarthy and Secord, 1999b), feeding rates and amount, nestling period, nestling development and fledging success.
Nest building behaviour of birds is initiated and enhanced by hormones. Nests are ephemeral but are made with greatest ingenuity and care to ensure that their eggs and young are getting ultimate comfort, adequate warm environment and protection during development. Tree swallows line their nests with feathers. The number of feathers in the lining will determine the level of insulation of the nest cups and thus ensure better growth of nestlings and protection from ectoparasites (Lombardo et al., 1995). So another behavioural aspect that can be assessed is the number of feathers in the nest lining in different nest boxes in different sites.. McCarthy and Secord (1999a) found that the number of nest-lining feathers at first egg, the number of nest-lining feathers at the last egg and the number of nest-lining feathers at hatching were significantly higher at uncontaminated sites than in PCB-contaminated sites. Even though qualitatively, nest construction was similar at all sites, with a tightly woven grass mat filling the bottom of the nest liner and a nest cup lined with feathers, the nests at control sites had greater mass and more feathers than two contaminated sites, and also varied with PCB contamination of sites. Their observations confirmed that the behaviors of parents were affected by PCB contamination.
Many studies have reported the toxicity of dietary PCBs expressed as reproductive and developmental impairment including effects on fertility, egg production, hatching success, and chick growth. Verboven et al. (2009) found that there is variation in the allocation of resources to eggs by mothers exposed to pollutants. They found that females of Larus hyperboreus who had high levels of different organochlorines in their plasma laid smaller eggs, and egg size correlated negatively with the amount of pollutants. Rutkowska and Cihon (2002) reported that brood sex ratio of zebra finches was male-biased when females received low quality food and also when food quantity was poorer. The quality of food will be lowered when females are exposed to pollutants so that it could lead to skewed sex ratio.
Male Japanese quails treated with PCB hydroxylated congener 30 had a longer latency to mount the control females and were significantly less successful in copulation than reference males (Ottinger et al., 2001). Studies on tree swallows by McCarthy and Secord (1999b) in several PCB contaminated sites along Hudson River found that reproductive success at PCB contaminated sites was significantly impaired due to high levels of nest abandonment during incubation and reduced hatchability of eggs. Halbrook and Arenal (2003) monitored the accumulation PCBs in European starlings (Sturnus vulgaris) at nest boxes constructed at two PCB exposed and two reference sites during the breeding season and observed their productivity (number of chicks produced per nest) and adult nest attentiveness behaviour (provisioning behaviour). Reduced adult nest attentiveness behaviour and decreased chick survival were observed at PCB sites. Nest success was substantially reduced in a tree swallow population breeding in chlorinated hydrocarbon-contaminated habitats due to nest abandonment by parents (Harris and Elliot, 2000). Reproduction of captive American kestrels was suppressed when adult birds were exposed to PCBs (Fernie et al., 2001). Hernandez et al. (2008) studied temporal and regional trends of OC pesticide and PCB levels in eggs of the Spanish Imperial Eagle (Aquila adalberti) collected in Spain between 1972 and 2003 in three populations central, western, and DonËœana (DonËœana being the most polluted of three) and found that significantly lower values for egg volume and breadth as well as Ratcliffe Index after DDT use than during the pre-DDT period; and eggs were significantly smaller when DDE levels increased. Clutch size in DonËœana varied according to DDE concentrations with the highest DDE concentrations found in clutches consisting of one egg. The fertility rate was lowered in clutches with DDE levels greater than 4.0 ?g/g. This was a consequence of hatching failure and fewer fledglings.
Many studies have found that reproductive rates of tree swallows are reduced by organochlorine contaminants (Custer et al., 1998; 2003; 2005; McCarthy and Secord 1999a, Bishop et al., 1999). Studies by Fernie et al. (2001) on American kestrels suggest that there was reproductive loss through the mortality of nestlings whose parents had been exposed in ovo to PCBs. The mechanism was suggested to be altered parental behaviour or reduced nestling viability. Neurotoxic compounds can reduce the ability of prey species to avoid predation. Such chemicals also lead to aberrant behaviour that can attract the attention of predators (Walker, 2003). Thus, overall survival and success of the affected population will be impaired by these kinds of contaminants. Consequently, it is necessary to assess the status of the population and to develop definite nondistructive biomarkers from the list of parameters available that can be used to detect the exposure of a population or populations to these contaminants.
Morphology and growth
Exposure to PCB and other organochlorines can result in decreased growth because both hypothalamo-pituitary and thyrotropin-thyroid axes required for normal growth can be impacted by organochlorine pollutants (Gould et al. 1997). Many studies have found that the thyroid axis has been affected by PCBs (Gould et al. 1997). An alteration in growth can affect all morphological features. Effects of PCB 126 on chickens, American kestrels and common terns were studied by Hoffman et al. (1998) from eggs to hatching after air cell injection on day 4, found beak defects in all three species.Defectsin kestrels were observed at a concentration of 0.3 ppb in kestrels. They occurred at higher concentrations in other two species.
Bustnes, et al. (2002) noticed a correlation between organochlorine pollutants and wing feather asymmetry in Arctic breeding Glaucous Gulls (Larus hyperboreus, Gunnerus). They found asymmetry in the lengths of the third primary in the left and right wing. The authors suggested that fluctuating asymmetry could serve as a potential measurement in predicting the early effects of organochlorine pollution on bird populations.
Another condition that can be assessed in relation to this is the number of common ectoparasites such as fleas (Siphonaptera) and blow fly larvae (Calliphoridae, Diptera) (Thomas and Shutler, 2001). These ectoparasites are commonly found in bird nests and on nestlings and adult birds.
Bird populations are sensitive to PCBs in the environment as they can cause neurological damage, and this can affect their overall behaviour (Walker, 2003). One such behaviour that can be affected is bird song. DeLeon and Dhondt (2008) found that song quality of black-capped Chickadees (Poecile atricapillus) was impaired by the presence of PCB. They looked at differences in the interval ratio (a measure of song quality) among locations (those with PCBs present vs. reference sites), and the interval ratio variation between and within individuals. They found that the average interval ratio was lower in the low PCB site (P<0.001) than in the higher PCB site. On this basis, they suggested that song learning but not song production appears to be affected by PCBs. Their research provided a possible non-invasive bioindicator of effects for sublethal PCB levels in passerines.
Residues of persistent chemicals and CYP450 can be measured in skin samples taken from animals and birds (Walker, 1998). A less invasive technique is the phytohaemagglutinin skin-testing technique of avian immunocompetence (Smits et al., 1999; 2002). This test is evaluates the proliferation, differentiation and response potential of circulating T-lymphocytes to an injected mitogen (Smits et al., 1999; Grasman, 2002). When PHA is injected into skin, a swelling will develop in the injected area within 12- 24 h. due to proliferation and differentiation of T-cells, cytokine production and accumulation of other white cell corpuscles and fluid. In the past, this was compared with the effect of a placebo treatment, i.e., an injection of phosphate buffered saline (PBS) into an alternate site. However, recently researchers are omitting this step to avoid extra stress to the organism undergoing the tests (Smits 1999; Grasman, 2002). In birds the injections can be made into wing web, wattle, dewlap or interdigitary skin (Grasman, 2002). Smits et al. (2002), found that PCB-exposed American kestrels had a greater response to PHA than controls, when analysis was done with sex as a significant factor and plasma triidothyonine (T3) as a significant co-variate.
Biomarkers in blood
Blood can be obtained easily from birds (invasive but non-destructive) and many biomarker assays can be performed on blood. Clinical haematology and biochemistry are valuable tools in assessing the health of birds (Sergent et al., 2004). Hematological variables that can be used as indicators of immune structure and function and the health status of birds include total red blood cell count, total leucocytes (WBC), % heterophils, % lymphocytes, % monocytes, % eosinophils and their ratio to one another.
DNA alterations including strand breakage, adducts and sister chromatid exchanges (SCE) can be detected in blood cell nuclei. Blood can also serve as a biomarker for poly aromatic hydrocarbon (PAH) induction. Protein responses including mixed function oxidases (MFO), protein inductions and stress proteins, haemoglobin adducts, various enzyme inductions and presence of aminolevulinic acid dehydratase (ALAD) are another suite of biomarkers that can be detected in blood as a consequence of PAHs exposure in birds. Presence of metabolic products like porphyrin and other by-products of heme synthesis and immunological changes can also be detected in blood following exposure to PAHs (Fossi, et al., 1999)
Immunological changes (immunomodulation):
Different studies have proved that immune system is very sensitive to environmental contaminants (Grasman et al., 2000, Grasman and Fox, 2001; Smits and Bortolotti, 2001; Grasman, 2002; Smits et al., 2002; Finkelstein et al., 2003; Holloway et al., 2003). Exposure to PCBs and other organochlorines from the environment makes all animals including birds more susceptible to many disease-causing epizootics. This is because the toxic chemicals modulate the immunology of organisms and this will lead to reduced resource availability for many other physiological functions including resistance to disease and infections (Wong et al., 1992; Grasman, 2002).
An organism's immunological status can be assessed by the mass, volume and structure of immunological organs like thymus, spleen and bursa of fabricius and blood cells. Tissues other than blood cells require sacrificing of the animals but blood cells and blood chemistry can give faultless information on the status of immune state of an animal. A very simple and reliable method to assess immune state of an organism in the blood is by cell counts (Grasman, 2000a). Different white blood cells (WBC) are important in cell-mediated, antibody-mediated and non-specific immunity. Total WBC and differential counts are reliable indicators of immune status and general health of organisms. A variation in the number of a type of WBC shows that there is some variation in the health of an organism and in most cases it indicates that the organism is fighting an infection. Grasman et al., (1996, 2000b) observed elevated ratios of heterophils to lymphocytes in Caspian tern chicks exposed to dioxin-like chemicals including PCBs. Heterophils and lymphocytes are the most numerically abundant WBCs in avian circulatory system. Grasman and Fox (2001) in a study with young Caspian terns found that plasma PCB and DDE concentrations and percentages of monocytes were negatively correlated and percentages of basophils and the concentrations of chemicals in plasma were positively correlated. Bustnes et al., (2004) counted heterophils and lymphocytes and their correlation with blood concentrations of organochlorines (p'p' DDE and 7 different PCB congeners (PCB 101, 99, 118, 153, 138, 180 and 170)) in Glaucous gulls (Larus hyperboreus) in 1997 and 2001. They found significant or near significant (0.1 > p < 0.001) positive relationships between the OCs and levels of heterophils in the blood for both sexes in 1997 and for male gulls in 2001. The levels of lymphocytes and OC concentrations were positively correlated in both sexes in 1997.
Serum chemistry values that can be assessed to evaluate the immunological status include ionic content (calcium, phosphorus, sodium, potassium, magnesium, iron, chloride etc.) glucose content, uric acid, cholesterol, enzymes like amylase, alanine amino transferase (ALT), aspartic amino transferase (AST), creatine kinase (CK), alkaline phosphatase, sorbitol dehyrogenase etc., total bilirubin, total CO2 , and total serum proteins (albumin and globulin and their ratios). Relative and total amounts of plasma Î±, Î², and Î³ globulins are affected by different types of infections, inflammations and by physiological conditions of an organism. Grasman et al., (2000a) found that in pre-fledging herring gulls from Great Lakes region concentrations of Î²2 globulin increased as a function of PCB and DDT levels in the tissues. In terns, PCB concentrations were negatively correlated with Î± globulins and positively associated with Î²1 globulins.
There are in vitro and in vivo tests available to test birds' immunological functional levels . The most commonly used in vivo immune tests in birds are the phytohemagglutinin (PHA) for T -cell mediated immunity (Grasman and Fox, 2001; Grasman, 2002; Smits et al., 2002) and sheep red blood cell (SRBC) hemagglutination assay (Grasman and Fox, 2001; Grasman, 2002) or dinitrophenol-keyhole limpet (DNP-KLH) test (Smits et. al., 2001) for antibody-mediated immune responses. Grasman and Fox (2001) in a study (mentioned before) with young Caspian terns found that there was a strong negative correlation between PHA response and plasma PCB and a lesser degree of negative correlation with DDE in the plasma. But the total antibody titres following immunization with SRBC showed positive association with plasma PCB and DDE.
Phagocytic properties of white blood cells such as macrophages can be used as another potential immunological indicator (Fournier et al., 2000). Lymphocyte proliferation and phagocyctosis can be performed ex vivo in cryopreserved avian peripheral blood cells and can be performed in a single blood sample (Finkelstein et al., 2003).
Blood collection is not favoured by many bird lovers as the technique is invasive and can cause distress to birds. Becker et al., (2006) suggested a non-invasive method to collect blood from breeding birds. A larval instar of a blood-sucking bug Dipetalogater maximus (Heteroptera) was introduced into common tern (Sterna hirudo) nest within a hollowed artificial egg. The hole was covered with gauze so that the bugs could collect blood from the brood patch of breeding adults. This can be done without trapping or handling the breeding birds. The authors claimed to have collected sizable amounts of blood by the above method.
The presence of plasma esterase activities in blood is a sure indicator and signal of potential problems of organophosphate (OP) and carbamate pesticides (Strum et al., 2008; Peakell, 1992). These pesticides act directly on the nervous system by inhibiting acetylcholine esterase (AChE). AChE is the enzyme that helps in the Acetylcholine (ACh) mediated synaptic transmission. ACh is a neurotransmitter in both the peripheral nervous system (PNS) and central nervous system (CNS) in many organisms including humans and also one of many neurotransmitters in the autonomic nervous system (ANS) and the only neurotransmitter used in the somatic nervous system.
Summary, Implications, and Recommendations
Biomarkers provide a quantitative measure of the effect of pollutants on biota that cannot be obtained by just measuring the amount of pollutants in the environment. The long term goal of biomarker research is to find a suitable end point that gives the presence of one pollutant or another at a very preliminary stage of exposure in the individuals of a population. It should be possible to extrapolate measured biomarker to other populations so that any deterioration of the community should be prevented at a very early stage or at a stage when easy recovery is possible. Rapidly responding biochemical markers can work as indicators of any change, which if not monitored can lead to irreparable changes to the populations, community and ecosystem at large.
Biomarkers are important in the field of surveillance, biological conservation, environmental hazard assessment and regulations and ecological remediation (McCarthy and Shugart, 1990). Effective environment management requires knowledge of the movement and fate of contaminants in the different compartments the natural system (Shugart, 1990) including the physical, chemical and biotic compartments. The biomarkers are an integrated measure of the contaminants' effects in the biotic compartment. So biomarkers are very important in wildlife toxicology. Consequently, testing should involve minimal sacrificing of individuals of any population. In this review, I have identified many alternative biomarkers that can be used in the field. The task ahead is to refine those different methods and find a few which are best suitable ethically and economically and work realistically. An ideal biomarker should provied an early warning signal and best be able to predict the true situation of pollutants in the field. Another important aspect in ecotoxicology to find suitable biomonitor or sentinel species - species in which we can test the biomarkers. The biomonitor species should be neither extremely sensitive to pollutants nor highly tolerant. Birds are excellent models for biomonitoring. They are easy to observe and are found in almost all habitats.They are sensitive to toxicants, occupy various trophic positions in the food web, and have well defined life histories and behavioural patterns. So an important task in the field of ecotoxicology is to find a suite of biomarkers that are ethically acceptable, technically sound, environmentally correct and economically feasible.