Research and Development s11

MINISTRY OF AGRICULTURE, FISHERIES AND FOOD CSG 15 Research and Development Final Project Report (Not to be used for LINK projects)

Two hard copies of this form should be returned to: Research Policy and International Division, Final Reports Unit MAFF, Area 6/01 1A Page Street, London SW1P 4PQ An electronic version should be e-mailed to [email protected]

Project title Links between biomarkers of PAH-exposure in flounder, and susceptibility to disease.

MAFF project code A1125

Contractor organisation CEFAS FISHERIES LABORATORY and location

Total MAFF project costs £ 225,000

Project start date 01/04/98 Project end date 03/06/01

Executive summary (maximum 2 sides A4)

1. Executive Summary The Joint assessment and monitoring programme (JAMP) under the auspices of the Oslo and Paris commissions (OSPAR) and International Council for the Explotarion of the Seas (ICES) recommends the integrated use of biological indicators for the assessment of pollution and quality status of areas. Biomarkers are measures of response and effect that represent a potential ‘early warning system’ that upon detection may allow remediating action to be taken before population or ecosystem level effects become apparent. From the regulatory perspective it is essential that a wide range of biomarkers are evaluated to determine sensitivity, reproducibility and ease of application. In this way UK monitoring programmes can be planned and managed more cost effectively. If biomarkers are viewed as an ‘early warning system’ on a continuum of increasing effects it is important to gain a better understanding of their relationship to the development of more severe and potentially irreversible pathologies. Since the polycyclic aromatic hydrocarbons (PAHs) are a widely studied group of organic compounds that are commonly detected in the UK's estuaries and coastal areas, this study used molecular, chemical and histopathological biomarkers to identify the effects of dose specific PAH concentrations. Furthermore since an increasing amount of environmental data suggest that such potentially genotoxic contaminants are the causative agents of disease in fish (Vethaak and Wester 1996) the use of a bacterial challenge model in tandem with biomarker techniques was developed as an indicator of the effects of PAH exposure upon the susceptibility of fish to pathogenic micro-organisms. PAHs are extensively metabolised in fish and therefore measurement of parent PAHs in tissues by standard analytical methods alone does not provide a complete assessment of PAH exposure. More firmly establishing the relationship between biomarkers of exposure and effect following exposure to PAHs is therefore an essential prerequisite to their use in monitoring programmes. By assessing a range of biomarkers

CSG 15 (Rev. 12/99) 1 Project Links between biomarkers of PAH-exposure in flounder, MAFF A1125 title and susceptibility to disease. project code

relative to each other, at measured levels of PAH exposure this project makes an important contribution to the understanding of biomarker data and its interpretation in an environmental context.

The key developments and outputs from this study are:

(I) Development of a methodology to dose contaminants via an oral route of exposure. This will be extremely valuable for use in future studies. (II) The ICES methodology for measuring Ethoxyresorufin-o-deethylase (EROD) activity was established and compared with the original CEFAS methodology (III) Two of the main biomarkers used in previous monitoring programmes were compared, EROD activity, estimates the induction of an enzyme system that catalyses a variety of chemical pollutants, and fluorescence analysis can be used to measure PAH metabolites in the bile. (IV) Genetic damage caused by chemical pollution may have serious consequences for fish populations therefore three separate biomarkers of genetic damage were also compared. (V) A methodology was developed for conducting disease challenge trials to investigate the potential impact of chemicals upon the disease resistance of fish populations: Two separate methodologies one with direct injection of pathogenic organisms in saline and one using co-habitation exposure (more technically difficult) were evaluated. (VI) The study results have been presented as a poster at SETAC Brighton 2000, two further posters were presented at PRIMO 2001. A paper is being finalised for Aquatic Toxicology.

Key findings  EROD is a sensitive but variable biomarker and may be inhibited at exposure concentrations > 100mg kg-1 based on oral exposure. Data must be considered in association with other biomarkers such as bile metabolites in order to avoid misinterpretation.  Bile metabolite analysis showed a clear dose response relationship with PAH exposure. The indications are that with one months continuous exposure the bile metabolite level reached a plateau that remains unchanged over longer exposure periods.  DNA adduct formation was only detected at exposure to concentrations greater than 50 mg kg-1 over 6 months but measurable effects are likely to persist for longer periods.  The comet and micronuclei assays (both are measures of genetic damage), showed dose related effects following exposure of fish to PAHs. The comet analysis of flounder sperm provided a sensitive indicator of genotoxic effects of PAHs (a 5mg kg-1 oral dose produced significant DNA damage after 6 months exposure). Both techniques have the advantage of being able to be applied non-destructively.  The histopathology data indicated dose related effects but these occurred at relatively high doses. It is concluded that the lower levels of PAH contaminantion that are more representative of the UK marine environment would require exposure periods in excess of one year in order for significant changes in histopathology to occur.  Selective use of the Electron microscope coupled with histochemical analysis is recommended to identify and characterise the early cellular changes following contaminant exposure.  The importance of the parasite fauna of flounder as a potential biomarker of contaminant exposure was highlighted by the significant dose related decrease in the number of the microsporean species glugea after a 1 month exposure of the host fish to PAH concentrations > 0.1 mg kg-1.  In the disease challenge studies fish exposed to PAHs and subsequently injected with the bacterium Vibrio anguillarum showed better survival than did those from control groups. This unexpected ‘protective effect’ did not prove to result from the toxicity of the PAH to Vibrio anguillarum but may be due to immunostimulation.  A dose related decrease in the natural commensal bacterial assemblage associated with the fish was measured. The potential for using the bacterial flora as an indicator of contaminant exposure warrants further consideration as it may represent a cost effective, sensitive and rapid assessment technique.

CSG 15 (1/00) 2 Project Links between biomarkers of PAH-exposure in flounder, MAFF A1125 title and susceptibility to disease. project code

Scientific report (maximum 20 sides A4)

Introduction Polycyclic aromatic hydrocarbons (PAHs) are a common and widespread class of environmental contaminants found in marine sediments and waters associated with urbanised estuarine and coastal areas (Meador et. al., 1995; Woodhead et. al., 1999). An increasing amount of environmental data suggest that such potentially genotoxic contaminants are the causative agents of disease in fish (Vethaak and Jol 1996; Vethaak and Wester 1996). Such studies have highlighted the need to develop and calibrate techniques associated with pollution monitoring in living organisms. PAHs are extensively metabolised in fish (Meador et. al., 1995) and therefore measurement of parent PAHs in tissues by standard analytical methods alone does not provide an adequate assessment of PAH exposure. The use of biomarkers indicative of PAH exposure has become widespread since they may provide early warning of potential ecosystem degradation, an indication of bioavailability of contaminants in the environment and the defence responses of exposed organisms (Goksøyr and Förlin, 1992; Goksøyr, et. al., 1996). Biomarkers may be whole animal, cellular and molecular indicators of exposure to, and effects of, contaminants upon organisms (Livingstone, 1993; Livingstone, et. al., 1997).

The mode of action of contaminants upon organisms and hence the biomarker result may be significantly affected by the route of contaminant exposure into the organism (Lee et. al., 1972; Meador et. al., 1995). The route of PAH exposure to benthic fish that has received most attention is via the sediment (French et. al., 1996). Previous laboratory and field experiments that have correlated pathological changes and biomarker responses with known concentrations of PAHs have therefore focused on sediment exposure (Stein et. al., 1990; Vethaak and Jol, 1996; Vethaak and Wester, 1996). However uptake in the food is another pathway for the entry of contaminants into an organism that may have an associated toxicological risk (McElroy and Sissons, 1989). Ingestion of prey organisms that have been contaminated, along with any sediment itself (in benthic fish species), may lead to the accumulation of toxic compounds if they and/or the sediments are contaminated (Maccubbin et. al., 1985; McElroy and Sissons, 1989; Pizza and O’Connor, 1983). Ingestion of contaminants via food consumed was therefore considered a representative and significant route of uptake which should be given further consideration, and this view is supported by a number of studies. Uptake of contaminants from the sediment also represents a significant route of contaminant uptake however earlier mesocosm studies conducted at CEFAS demonstrated that laboratory mesocosms incorporating contaminated sediments have problems associated with rapid contaminant loss and the development of anoxic conditions when the size of the system is constrained. For these reasons the PAHs were dosed via the food.

The aim of this study was to investigate the response of biomarkers and pathological conditions in the European flounder (Platichthys flesus L.) associated with known levels of ingested PAH contamination and consider the implications of the results for environmental monitoring studies. A further aim of the project was to assess the threshold concentrations of contaminants that are likely to cause significant histopathology and reduce resistance to pathogenic microorganisms. Two short term studies of 1 and 4 months duration were conducted in order to calibrate the biomarker measurements. A main dosing study of 11 months duration with an intermediate sampling period after 6 months was also conducted. A further 6 month experiment using the same experimental set-up was also initiated for assessing biomarker response to PAHs during the main reproductive period. A series of range finding studies using intraperitoneal injection of PAH were also conducted in order to optimise a pathogen challenge protocol for use in the assessment of contaminant effects upon immunocompetence.

All feeding studies used a proprietary pelleted feed, dosed with PAH at a range of concentrations. Tissues were subsequently analysed for biomarker response and histopathology

Biomarkers

Three commonly applied biomarkers for detecting PAH exposure in fish are:

(i) Ethoxyresorufin-o-deethylase (EROD) activity, estimates the induction of cytochrome P450-dependent mono-oxygenases, a group of inducible enzymes that catalyse both xenobiotic and endogenous substrates (including PAHs) in fish (Di-Giulio, et. al., 1995). The in vitro catalytic activity of EROD has been used as a means to measure CYP1A induction under both laboratory and field conditions (Kirby et. al., 1999).

(ii) Fluorescence analysis of PAH metabolites in the bile. Generally the biotransformation rate of PAHs in fish is rapid, this means that PAHs do not accumulate to a high degree in the tissues. Analysis of the tissue concentration of PAHs in fish from contaminated sites may therefore provide a misleading impression of exposure. However additional information on the uptake of PAHs can be obtained by analysis of the bile of exposed fish since this will contain a number of water soluble, non-toxic and readily excreted PAH derivatives which can be analysed by fluorescence spectrometry. Analysis of bile metabolites is a simple tool for assessing hydrocarbon metabolism and is widely used to estimate PAH exposure (Britvic et. al., 1993; Ariese et. al., 1993).

(iii) The formation of DNA adducts in the liver (Goksøyr, et. al., 1996; Reichart et. al., 1998). The biotransformation of PAH via the MFO system can lead to the formation of highly reactive electrophilic metabolites which can covalently bind to cellular macromolecules including DNA (Varanasi et. al., 1989). This modification of DNA by reactive PAH metabolites is considered to be one of the initiating steps in chemical carcinogenesis. The detection of DNA adducts has been found to be a valuable biomarker of genotoxic exposure in the aquatic environment (Reichart et. al., 1998; Ericson et. al., 1998; Lyons et. al., 1999). The quantitative analysis of DNA adducts provides a measure of the biologically effective dose reaching a critical target site and thus integrates the multiple toxicokinetic factors (i.e. bioavailability, metabolism and detoxification) involved in genotoxic exposure. Currently, the 32P-postlabelling technique is the most sensitive technique available for the detection of DNA adducts (Randerath et. al., 1981) and was therefore applied in this study.

PAH absorption

EROD Cytochrome metabolism P450 in ER. Induction measured as EROD

DNA adducts Measurement of the binding of Bile reactive intermediates Measurement of BaP to DNA metabolites in the bile

reactive intermediates

Figure 1 Schematic of the potential sequence of metabolic steps that lead to cellular changes that can be measured by each of the three main biomarkers used in this study.

All these techniques are regularly used in the detection of PAH contamination and are recommended Joint Assessment Monitoring Programme (JAMP) biomarker techniques (Stagg, 1998). Further techniques to investigate DNA damage include the micronucleus and the comet assay. In this study the use of pathological changes in selected tissues was integrated with the biomarker analysis to allow a more complete interpretation of the response of flounder to PAH exposure.

(iv) The micronucleus assay is employed to test for the genotoxic activity of chemicals. Unlike human red blood cells fish erythrocytes are nucleated. Micronuclei are small “satellite” nuclei of the main nucleus that are formed following anaphase by the condensation of chromosomal fragments or whole chromosomes not included in the main nucleus (Al-Sabati and Metcalfe, 1995). The high rate of formation of erythrocytes enables a relatively rapid assessment of exposure to genotoxic stimuli when the cells are appropriately stained.

(v) The Comet analysis technique is employed for the visualiation of DNA damage in individual cells (Fairbairn et. al., 1995). PAHs can reduce the size of DNA by causing strand breakages. After the DNA is uncoiled by alkaline denaturation and subjected to electrophoresis the resulting damaged strands can be stained and visualised. Different tissues can be analysed in this way, in this study both blood and sperm were tested, which allows comparison of uptake, distribution and effects of a contaminant and both tissues can be sampled non-destructively. This latter point means that the

technique can be effectively applied to gain a better understanding of the time-course of contaminant effects without the need to use large numbers of experimental organisms.

(vi) Histopathology The use of histological biomarkers of toxic injury, dysfunction and carcinogenesis is now well established. These provide powerful tools to detect and characterise the biological end points of toxicant and carcinogen exposure. In particular, several categories of hepatocellular pathology are now regarded as reliable biomarkers of toxic injury and are considered to be representative biological endpoints of contaminant exposure (Myers et al., 1987). Consequently the liver has attracted the most attention as a target organ for biological effects monitoring programmes in both Europe and the North America (Kranz & Dethlefsen, 1990; Köhler, 1989; Köhler et al., 1990; Bucke & Feist, 1993; Myers et al., 1991, 1994). Other tissues and organs also exhibit lesions following exposure to xenobiotics. These responses have been recognised following controlled exposure under experimental conditions (Hendricks et al., 1985) as well as in fish exposed to environmental contamination (Bucke et al., 1989).

Feeding studies Experimental set-up

Fish A stock of healthy 0-group flounder (Platicthys flesus) were obtained from Port Erin Marine Laboratory. The fish were grown on into their second year under standardised laboratory conditions before the start of the experiments.

Feeding regime All fish were fed a diet of nutra marine 4mm pellets (Trouw aquaculture) during acclimation to the tanks and during the experiment. Fish were fed once a day at a ration of 0.9% body weight/day. This was calculated by weighing all of the fish at the beginning of the study. During the 6 and 11 month exposures the fish were weighed every month and the ration adjusted accordingly. At this initial ration level all of the pellets were consumed within 10 minutes of feeding therefore there was little if any wasted feed.

Tanks The 1 month exposure was carried out with 20 fish per exposure held in glass fibre tanks with a through-flow temperature controlled water system. The 6 and 11 month, experiments were conducted in glass fibre tanks held inside the laboratory in constant tempertaure rooms with both through flow and a pumped recirculated water system. The water was supplied directly from the tidal River Crouch, following settlement and filtration at ambient temperatures. The set up involved a tiered system of 3 tanks. 15 fish were maintained in each of the three tanks giving a total of 45 fish per exposure (4 exposure groups in all). Fish were introduced into the tanks two weeks before the initiation of the experiment to allow for acclimation to test conditions. In each exposure group 15 fish were sacrificed at 6 months and the remainder at 11 months.

Physical parameters - In all experiments, 33.5 ±1‰ salinity sea-water was supplied to the tanks at 2.5L per min. In the one month studies the incoming water temperature was held at a constant temperature of 10±1°C. This led to a tank temperature of 12.5°C ±2°C in all experiments. The artificial light regime was the maintained as the current, daily periodicity. The tanks were thoroughly cleaned at the mid-point of the study. All waste was removed daily.

Dosing of contaminated food

A mix of four PAHs were selected to be used, on the basis of their common occurrence in water, sediment and flounder prey items in the marine environment: benzo(a)pyrene (BaP), benzanthracene (BaA), phenanthrene and pyrene (Baumard, et. al., 1998; Law et. al., 1997; Woodhead, et. al., 1999). The PAHs were applied to pelleted food (Trouw aquaculture, nutra marine 04) using hexane as a carrier solvent. The dosing apparatus set- up was a modification of that used by Fernandez et. al., (1998) for loading chemicals onto pelleted food. Each PAH in the mix was equally represented, by weight.

To account for losses of PAH during the food mixing process 20% extra of the total nominal PAH concentration was added to each concentration. To reflect the wide range of PAH concentrations measured in the marine environment from background levels to those measured at heavily contaminated industrial sites (Woodhead, et al., 1999) (and additionally to include high treatment levels as a positive control) pelleted food was nominally dosed at; 0.1, 1, 5, 10, 50, 100, 300, 500 mg/kg and a control dose, (hexane dosed food only) in the 1 month exposure. In the 6 and 11 month exposures fish were supplied with feed that had been dosed with PAHs at 0.1, 10 and 50mg/kg, or was left undosed for the control fish. Table 1 shows the analysis of the food by HPLC to determine the actual amounts of PAH in the dosed food. At all concentrations other than 300mg/kg the actual concentrations were higher than the nominal concentration. The PAH concentrations in the dosed food were within 15% of the target concentration, with the exception of the 10 and 300mg/kg dose.

The blanked concentration was calculated from Pyrene and Phenanthrene which were found in detectable concentrations in the undosed food at 2.5µg/kg and 9.65µg/kg respectively.

An initial siting study confirmed that there was no appreciable loss of PAH from the food pellets upon immersion in the water over a two hour period. This indicated that due to the normally rapid consumption rate of the pellets the loss of PAH upon addition to the tanks would be negligible

Table 1. Concentrations of PAH in P. flesus feed and projected uptake after dosing with four compounds; BaP, BaA, Phenanthrene and Pyrene. Results expressed in ppm (mg kg-1). Expected Measured PAH % change from Expected dose of 4 PAHs per Recovery Blanked expected fish (nominal) 1 month 6 months - 0.00 - 0 0.10 0.11 10.00 0.001 0.03 1.0 1.1 13.00 0.009 1.57 5.0 5.4 7.80 0.043 - 10 12 19.90 0.086 - 50 55 9.98 0.43 15.67 100 111 11.19 0.86 - 300 389 29.80 2.57 - 500 558 11.68 4.29 -

Experiment termination and analysis Upon the termination of each of the exposure studies the fish were removed from their tanks and killed by a blow to the head followed by destruction of the brain. Samples of liver tissue were stored in liquid nitrogen, for EROD and DNA adduct analysis. The gall bladder was removed and the bile was drained into a vial for cryogenic storage until analysis could be conducted. A fillet of muscle from the dorsal side of each fish was stored frozen until used for chemical analysis. Samples for histopathology were removed by dissection, these included; Liver, spleen, kidney, gill, gonad, intestine and muscle.

EROD activity Analysis was performed according to Stagg and Mcintosh (1998). The rate of catalytic breakdown of a substrate was reported relative to the protein content of the liver tissue sample. The protein normalised enzyme activity was compared for each exposure group and control in each of the experiments conducted.

1-hydroxy pyrene bile metabolites The method employed for bile metabolite analysis was synchronous fluorescence spectrometry (SFS) for the glucuronide conjugate (Ariese et. al., 1993). In the present study the gall bladder was removed from the flounder and directly frozen in liquid nitrogen before storage at –80°C. All samples were analysed within 3 months. Other studies have shown that little change in the metabolite concentration occurs even after 12 months storage under similar conditions, (Ariese et. al., 1993). The concentration of metabolites present in each of the exposure groups were compared with control levels to determine whether increases occurred following various exposure regimes.

32P-postlabelling assay to DNA adduct formation DNA was isolated from hepatic tissue of individual fish. DNA adducts were determined using an assay technique described previously (Jones et al., 1991). Adducted and normal nucleotides were labelled separately, using labelling buffer containing radioactive -32P. The adducted nucleotides were separated from their normal undamaged counterparts using thin layer chromatography (TLC). The levels of DNA adduct radioactivity were then determined using a radioanalytical scanning system. Upon the quantification of both the adducted nucleotides and the normal nucleotides the relative adduct labelling values were calculated and converted to the number of adducted nucleotides per 108 undamaged nucleotides. Appropriate negative and positive DNA controls were analysed throughout the studies as described by Harvey and Parry, (1998). Positive control experiments for the formation of DNA adducts were conducted. This ensured that when compared with the mixed compound fed fish the DNA adduct profiles could be accurately interpreted, i.e the profiles of individual compounds could be accurately identified. The number of adducted nucleotides per 108 undamaged nucleotides was compared for each exposure group and control fish to determine whether differences were statistically different.

Micronucleus assay Blood was removed from the fish using a heparinised syringe/needle via the caudal vein. Blood samples were then mixed with Phosphate buffered saline in a 50:50 solution. A 20µl aliquot was pipetted onto a glass slide and a smear made by drawing a second glass slide down the first. After drying and fixing the slides were stained in giemsa stain and following mounting, 2,000 erythrocytes were counted per field of view of a high power microscope and micronuclei and abnormal (irregular shaped) nuclei were noted. Micronuclei formation was only assessed in the higher doses of the 1 month exposure and the 6 and 11 month exposures. Micronuclei numbers were compared between exposure groups to determine if exposure increasd the incidence of occurrence.

Comet assay DNA damage in cells can be visualised by staining the material and subjecting it to electrophoresis. Shorter damaged sections of DNA in the nucleus migrate further relative to the (longer) undamaged strands. The stained DNA appears ‘comet-like’ with a dense area of undamaged nuclear DNA and a ‘tail’ of shorter damaged material . The amount of DNA in the ‘tail’ and within the more dense nuclear material can be measured after appropriate staining using a fluorescence microscope or other methods and the ratio compared for samples from different treatment groups. Two different measures of DNA damage were used in this study: In preliminary experiments conducted using a four month exposure, the degree of DNA migration from 50 nuclei per sample was scored on an arbitary scale and the mean rank for each treatment group compared. In the reproduction study the mean tail moment (tail length x fluorescence intensity of the tail) was compared for each treatment group. In the reproduction study blood and sperm samples were analysed and compared using this technique.

Analysis of parent compounds in fish muscle by HPLC Both biota and fish food samples were extracted in n-pentane. After thoroughly shaking the samples they were allowed to separate, and the extract was then collected in a 100 ml conical flask containing solvent-rinsed anhydrous sodium sulphate to remove water any residual. The process was repeated twice. The two extracts were combined, and evaporated down to <1 ml before transfer into a glass sample vial sealed with a crimped top vial cap prior to HPLC analysis. Each sample batch contained a procedural blank, and a reference material.

The PAH were separated by HPLC and detected by a fluorescence detector. The PAH tissue results are reported as mg/kg for each of the four PAHs dosed.

Histopathology Fixed tissue samples were dehydrated and processed to paraffin wax. The resulting tissue blocks were cut into thin sections and then stained by standard protocols with haematoxylin and eosin. Sections were examined on a high-power microscope.

Specimens for electron microscopy (6 month exposure only) were fixed, stained and infiltrated with a medium hardness epoxy resin. One micron semi-thin sections were evaluated by light microscopy. Subsequently, representative ultrathin sections (90nm) were cut, mounted on copper grids and stained with uranyl acetate and Reynolds lead citrate. Sections were examined using a (JEOL 1210) transmission electron microscope.

Statistical Analysis The biomarker results were analysed either by one way analysis of variance following logarithmic transformation to normalise the data or by the nonparametric (or distribution free) equivalent Kruskal Wallis test. Following the initial analysis to determine if there was an exposure related effect an appropriate multiple range test was used to indicate which groups differed significantly from each other. All data was analysed using the Statgraphics package (Manugistics Inc.)

Results General All exposure groups remained in good health throughout the study with the exception that a number of fish in the 50 mg/kg exposure group died during the 10th and 11th months of exposure leading to the early termination of this experiment. However the condition of all groups including those in which mortality was observed remained good.

EROD analysis Levels of EROD activity in fish from the 1, 6 and 11 month exposures are displayed in Figures 2 to 5. After 1 month exposure to PAH contaminated food mean hepatic EROD activity in males and females was significantly increased by exposure to PAHs at all exposure concentrations from 0.1 to 50 mg/kg (p<0.05). Following a 1 month exposure to PAH concentrations of 100, 300 and 500 mg/kg EROD levels were not significantly elevated with respect to control groups in either males or females. The one month high and low dose experiments were run separately therefore figures 2 and 3 show two control groups, one for each experiment. After 6 months exposure, EROD activity was not significantly different between control and treatment groups for both male and females (p>0.05). The mean hepatic EROD activity of flounder in the 11 month exposure is displayed in Figure 5. The EROD activity was not significantly elevated in either the male or female groups at each treatment level with respect to the control.

Figure 2. EROD activity in male flounder following 1 month exposure to PAH contaminated food (second control value relates to 100-500 mg/kg exposure levels)

80 n i m

/ 60 o r p

g 40 m / M p

D 20 O R E 0 Control Control 0.1 1 5 10 50 100 300 500 log PAH dose m g/Kg food

Figure 3. EROD activity in female flounder following 1 month exposure to PAH contaminated food

80 n i m

/ 60 o r p

m 40 / M p

D 20 O R E 0 Control Control 0.1 1 5 10 50 100 300 500 log PAH dose m g/Kg food

(second control value relates to 100-500 mg/kg exposure levels)

Figure 4. Mean EROD activity (with 95% confidence limits) in male and female flounder following 6 month exposure to PAH contaminated food

30 % 5 9 (

m 20 / o

) males r L p

C g females m

/ 10 M p

O 0 R E Control 0.1 5 50 Exposure concentration of PAH (m g/kg)

Figure 5. Mean EROD activity (with 95% confidence limits) in male and female flounder following 11 month exposure to PAH contaminated food

) L C 50 % 5 9 40 ± Male (

n i 30 Female m / o r p

20 g m / 10 M p

R control 0.1 5 E PAH dose (mg/kg)

Bile metabolite analysis Levels of bile metabolites for fish from the 1 month feeding exposure are displayed in Figure 6. The results show a significant dose response relationship (p<0.05) between oral PAH exposure and subsequent metabolite excretion. Comparison of the bile metabolite concentration between the same treatment groups following one and 6 months indicated that there was a similar dose response relationship.

Figure 6. Mean (and 95% confidence limits) levels of 1-OH pyrene equivalent bile metabolites following 1 month exposure to PAH contaminated food. - )

1 80000 L

60000 5 e 9

t y = 107.5x + 1955.6 i ± l (

e 40000 R = 0.9825 b n a t e r e y

m 20000

e l i H b O 0 0.01 0.1 1 10 100 1000 log PAH dose (mg/kg)

DNA adducts No DNA adducts were detected in pooled samples of fish from the 1 month exposure groups. The mean levels of hepatic DNA adducts detected in fish from the 6 month exposure study are displayed in Table 2. After 6 months no DNA adducts were detected in fish fed either the control diet or the 0.1 mg kg-1 PAH spiked food. A single fish had adducted DNA nucleotides in the 5 mg kg-1 exposure group and 10 of the 15 fish in the 50 mg kg-1had adducts present. The results from positive controls (fish exposed in vivo to 500 mg kg-1 PAH mix for 4 months and fish that received DNA treatment in vitro with a diol-expoxide of BaP) suggest that the DNA adduct measurements were detecting the biotransformation of BaP to a DNA reactive metabolite (Figure 7).

Table 2. Levels of hepatic DNA adducts, expressed as adducted nucleotides per 108 normal nucleotides detected ±SE, in flounder following 6 months exposure to PAH contaminated food. Feeding regime Control 0.1 mg kg-1 5 mg kg-1 50 mg kg-1 ND ND 1.4 ±1.4 5.9 ± 2.7 *ND: not detectable under current assay conditions Figure 7. (A-D): Typical DNA adduct profiles produced following the 32P-postlabelling of hepatic DNA from; (A) In vivo positive control, flounder dosed with PAH mix in food at 500 mg kg –1 for 4 months. (B) 6 month feeding exposure, flounder dosed with control food only. (C) 6 month feeding exposure, flounder dosed with PAH mix in food at 50 mg kg –1. (D) In vitro positive control consisting of flounder hepatic DNA treated with 1.5 mM B[a]P diol epoxide.

A B C D

Micronuclei - The results of the micronuclei counts for fish in the control and exposures, 100, 300 and 500 mg/kg are displayed in Figure 8. Micronuclei were only present in the cells of fish that were in PAH exposed groups (100 – 500mg/kg). However although there was a significant treatment related difference compared to the control (p<0.05) there was no clear dose related difference between treatment groups.

Figure 8. Mean (and standard deviation) numbers of abnormal nuclei and micronuclei in flounder erythrocytes following 1 month exposure to PAH contaminated feed.

S abnormal nuclei ± ( 4 s

l micronuclei l e c

3 0 0 0 2

2 r e p

t 1 n u o

c 0 control 100 300 500 dose (mg/kg)

Following a dosing period of 4 months to treatment levels of 100 and 500 mg/kg there was a significant difference between each treatment group as well as to the control (p<0.01). Following a 6 month exposure period there was no significant increase in the number of micronuclei detected in fish exposed to either 0.1 or 50 mg/kg of PAH.

Comet assay In the initial 4 month sighting study blood cell nuclei were assigned to one of five categories dependent upon the spread of nuclear material around the nucleus. The greater the spread of material the higher the category and hence the higher the associated score. A total of fifty nuclei were scored per sample. When the level of DNA damage in the haemocytes collected from fish in the exposure groups (100 and 500 mg/kg) is compared with the controls (Figure 9), there is a statistically significant difference between those exposed to PAH and the controls (p< 0.01).

Figure 9 Mean (and SD) score for spread of nuclear material (arbitary units) for fish exposed to different treatment levels of PAHs for 4 months.

24 s t i n u

y 16 r a t i b r a 8

0 Control 100 mg/kg 500 mg/kg

Parent compound muscle analysis Results of the analysis of flounder muscle by HPLC are displayed in Table 3 for exposure periods of 1, 6 and 11 months respectively. Parent PAHs were only found in the muscle at exposures of 1mg/kg and above. Compounds accumulated in order of their decreasing solubility in the water phase and increasing solubilty in lipid (i.e Phenanthrene – Pyrene – BaA – BaP) and all four PAHs were found to accumulate at exposures of 300mg/kg and above. A dose-related elevation of the phenanthrene and pyrene concentration was measured even in fish muscle from the low dose groups. The concentration of phenanthrene in the tissue of the highest treatment group (50 mg kg-1) was equivalent to the concentration measured in the liver of flounder sampled in the Mersey during 1997. This fact indicates that the PAH dose range used in this study is representative of the levels of PAH exposure in some UK estuaries and coastal waters.

Table 3. Mean muscle concentration of PAH ppm (mg kg-1) after 1 months exposure to PAH contaminated food. Dose (mg/kg) Phenanthrene Pyrene BaA BaP Control ND* ND ND ND 0.1 ND ND ND ND 1 0.002 ND ND ND 5 0.006 ND ND ND 10 0.008 0.003 ND ND 50 0.052 0.009 ND ND 100 0.037 0.004 ND ND 300 0.079 0.010 0.004 0.005 500 0.150 0.020 0.020 0.007 6 MONTHS Control ND* ND ND ND 0.1 0.002 ND ND ND 5 0.009 ND ND ND 50 0.044 0.008 ND ND 11 MONTHS Control ND* ND ND ND 0.1 0.002 ND ND ND 5 0.005 ND ND ND

*ND: below limit of detection ±SD only shown where they were above the limit of detection Histology The prevalence of each category of vacuolation in the liver of fish from the 1, 6 and 11 month studies did not appear to be dose related. However, the absence of class 1 vacuoles in the liver of fish from both the control and treatment groups in the 6 month exposure suggests that a common factor such as seasonal condition or the composition of the artificial diet may have been responsible. Other liver pathologies were observed in the 1

month study. These consisted of one case of lipoidosis in the control group and in the 50 mg kg-1 exposure group two cases of coagulative necrosis, one of lymphocytic infiltration and one of basophillic adenoma.

In the fish exposed to PAH for 6 months three cases of vacuolated foci of cellular alteration (FCA) were noted in the liver of those fish from the 5 mg kg-1 treatment. No other liver pathology was noted.

In other tissues a variety of pathologies were noted, however only a few of the observed differences between treatment groups appeared to be related to exposure to PAHs. The pathologies observed are recorded in Table 4, those of particular interest are highlighted in bold.

In the one month studies samples of kidney and spleen showed abnormality in the melanomacrophage centres (MMCs). This consisted of grossly enlarged macrophages and was often associated with dilated capillaries in the renal haematopoietic tissues. The prevalence of MMC pathology in the kidney and spleen was highest for the control group (7 of the 20 fish, 35%), with the prevalence in the exposed groups ranging from 15% to 25%. Aneurysms were observed in the secondary lamellae of the gills of fish from the 100, 300 and 500 mg kg-1 (5, 15, and 10% respectively) treatment groups. The gastric gland cells of the stomach showed some vacuolation in 5% of the fish exposed to 100 and 300 mg kg-1 respectively however none was apparent in the 500 mg kg-1 treatment.

The most prominent gonadal pathology observed was that of atresia with an associated inflammatory response. The intersex condition was detected in one female fish from the 5 mg kg-1 dose group. In addition, it was noted that pre-vitellogenic oocytes were found in control females (15%) but not in the exposed groups.

Following 6 months exposure to PAHs there was a high prevalence of MMC pathology of the spleen (40%) observed in fish in the 50 mg kg-1 exposure group with reduced prevalence (13%) in the 0.1 and 5 mg kg-1 exposures. However at this time MMC pathology was also elevated in the control group (mean 30%). Vacuolation of gastric gland cells was seen in 40% of the 50 mg kg-1 exposure group with 13% and 7% prevalence recorded in the 0.1 and 5 mg kg-1 exposure groups respectively, the controls appeared normal. In the gonad tissue 13% atresia was observed in the 0.1 mg kg-1 treatments. Aneurysms were again observed in the secondary lamellae of the gills with 53, 20 and 27% occurrence in fish from PAH treatments of 0.1, 5 and 50 mg kg-1 respectively, whilst control levels were 13%. No other abnormalities were observed in the control or other treatment groups.

After 11 months exposure some gonad atresia was present in all treatment groups and the control although predominently in the control and lowest treatment group (0.1 mg kg-1). Gill aneurysms were also present at higher levels (37%) in the 0.1 and 5 mg kg-1 groups compared to the control (20%).

Parasite prevalence The only parasite noted was the microsporidian parasite Glugea sp. In fish exposed to 0.1, 1, 5, 10 and 50 mg kg-1 PAH for 1 month the prevalence was 10, 5, 15, 0, 25 and 15% respectively. At exposures of 100-500 mg kg-1 , occurrence of Glugea sp. was lower (mean 8 %) than in control fish (30 %). In samples taken after 6 month exposure, Glugea sp. was only recorded in fish from the 0.1 mg kg-1 and control at 13 % and 20% occurrence respectively. After 11 months exposure there was no obvious relationship between Glugea numbers and the PAH treatment level.

Table 4. Percentage prevalence of histopathologies observed in various tissues of flounder exposed to PAHs for exposure periods of 1, 6 and 11 months

Necrosis Kidney Spleen Focal Dilated Glugea Haem. Gonad Gill Gut Skin Exposure period MMC MMC inflamm. capillaries Spleen path path. path path and dose * 1 month

Control NR 35 35 NR NR NR NR 0 NR NR NR 0.1 mg/kg NR 20 20 NR NR NR NR 5 NR NR NR 1 mg/kg NR 20 15 NR NR NR NR 0 NR NR NR 5 mg/kg NR 25 25 NR NR NR NR 5 NR NR NR 10 mg/kg NR 25 25 NR NR NR NR 0 NR NR NR 50 mg/kg NR 25 25 NR NR NR NR 0 NR NR NR

1 month Control 0 15 25 5 25 30 0 0 0 0 0 100 mg kg-1 0 15 15 10 20 5 0 0 5 5 0 300 mg kg-1 0 35 35 0 50 10 0 15 15 5 5 500 mg kg-1 20 10 10 0 35 10 25 15 10 0 0

6 month Control 0 33 27 0 0 20 0 0 13 0 20 0.1 mg kg-1 0 13 13 0 13 13 0 13 53 13 _ 5 mg kg-1 0 13 13 0 7 0 7 0 20 7 33 50 mg kg-1 0 40 40 7 0 0 0 0 27 40 7

11 month Control NR 17 17 3 17 23 3 13 20 3 3 0.1 mg kg-1 NR 17 30 0 17 17 7 20 37 0 0 5 mg kg-1 NR 23 23 7 20 20 0 7 37 0 0 *Abnormally dilated capillaries in the haematopoietic tissues of the spleen

EM Targeted EM of the liver from the 6 month exposure groups demonstrated the presence of numerous foci of lipid vacuoles and glycogen rossettes in the hepatic cytoplasm of control fish. Rough Endoplasmic Reticulum (RER) and mitochondria were distributed around the edges of these cells and nuclei contained marginalised heterochromatin. In contrast, in the 50mg kg-1 exposed fish, there was less glycogen present, the lipid vacuoles were larger and there was a marked increase in the amount of rough endoplasmic reticulum.

Figure 10. Transmission electron microscope image showing the vacuoles (*) in the hepatocytes of control fish (a) and those exposed to 50mg kg-1 PAH for a 6 month period (b). The arrow in (b) indicates the well developed endoplasmic reticulum as compared to that in (a). (a) CSG 15 (1/00) 16 Project Links between biomarkers of PAH-exposure in flounder, MAFF A1125 title and susceptibility to disease. project code

Reproduction experiment Introduction Endocrine disruption by chemicals in vertebrates including fish has been well documented (Stahlschmidt-Allner et al., 1997). The metabolism of various contaminants including PAHs, by fish and other vertebrates involves the cytochrome P450-dependent monooxygenase enzyme system. The same enzyme system is also involved in steroid metabolism. Therefore PAHs are likely to interfere with the normal functioning of this metabolic pathway. In addition because many contaminants including PAHs are lipophilic they are likely to accumulate in lipid rich organs such as the liver and the gonads. Accumulation of PAH in the maturing gonad could have serious consequences for the development of gametes and the potential for transfer of effects to subsequent generations. This part of the study therefore focuses on the potential for genetic damage in fish exposed to PAH via the diet. Methodology Eighteen month old fish were maintained for six months (November to April) in outdoor tanks under natural seasonal conditions. This was done to ensure that the fish received the appropriate environmental cues that are necessary to induce spawning. Filtered seawater was supplied at ambient temperatures at 2.5L per min. There were three treatment groups of fish fed PAH contaminated pellets at doses of 0.1, 5 and 50 mg kg-1 of feed respectively and a control fed undosed pellets. At the end of the six month exposure period fish were sacrificed and tissues were sampled for histology and biomarker analysis as previously described.

Results The comet bioassay was conducted on samples of sperm and blood and the tail moment endpoint was compared for samples from each of the exposure groups. There was a significant increase (p<0.05) in

The tail moment value for exposure groups of 5 and 50 mg kg-1 relative to the control and 0.1 mg kg-1 treatment for the sperm and blood (Figures 11 and 12). This indicates exposures of 5 and 50 mg kg-1 were producing genotoxic effects in both these tissues.

Figure 11. Comparison of the genotoxicity of PAH exposure via the diet, as visualised by the mean tail moments in the Comet assay of sperm samples isolated from flounder following a 6 month exposure period.

25 e c n e

c 20 ) l s i e t a r t n

o f e u

o 15 l

y o t x i

i 10 g e a t t n n e i l

i 5 a t ( 0 C 0.1 5 50 -1 PAH treatm ent group m g kg Figure 12. Comparison of the Genotoxicity of PAH exposure via the diet, as visualised by the mean (95 % CL) tail moments in the Comet assay of blood samples isolated from flounder following a 6 month exposure period.

c 8 n e c ) s l i e t

a 6 r t n

o f e u o l

y o t x

m s h l t n i g e a t t n

n 2 e i l

l i a t ( 0 C 0.1 5 50 PAH treatment group mg kg-1

The gonad tissue was weighed for both males and females and was expressed as a percentage of the total body weight. The resulting gonadosomatic indices (GSIs) were compared between exposure groups. Only the female GSIs were significantly different between groups (p<0.05). The only difference however was that the mean GSI of the 0.1 mg kg-1 exposure group was larger than that of the 50 mg kg-1 group. In all other exposures the GSIs were comparable (Figure 13).

Figure 13. Comparison of the effect of PAH exposure via the diet, upon the mean (95 % CL) gonadosomatic index of female flounder following a 6 month exposure period.

e 0.4 d n i

S 0.2 G 0 C 0.1 5 50 PAH dose mg kg-1 of food

Sections of the female gonad were scored for atresia of eggs. Although there was an increase in the number of cases of atresia in the two highest exposure groups this was not statistically significant.

Discussion The comet assay detected genotoxic effects at lower concentrations than either the micronucleus assay or the assessment of DNA adducts. However these two latter measures were conducted in an earlier dosing study when the fish were at an earlier stage of maturation and this may have contributed to this difference. Nevertheless the comet assay appears to be a sensitive technique.

Significant changes in the GSI or atresia were not detected. Both these measures however reflect more acute exposure to contaminants whereas the comet assay is a better measure of more subtle chronic effects that are of no less importance.

Bacterial Challenge Experiment Introduction In order to evaluate links between disease, environmental pollution and biomarker response, appropriate models for the experimental exposure of fish to contaminants and a subsequent challenge infection of fish, as an indicator of contaminant effects, need to be developed. Studies into the immunocompetence of fish have indicated the likely impairment of immune function with increasing contaminant exposure (Zelikoff,1994). Arkoosh et. al. (1998) found that mortality in feral fish exposed to Vibrio anguillarum in the laboratory (caught during their outmigration) was greater in groups from an urban estuary compared to those from a non-urban estuary. Juvenile salmon from the urban estuary had been exposed to higher levels of PAHs and PCBs. The study concluded that contaminant associated immunodysfunction in juvenile chinnok salmon may lead to increased infection by a virulent marine bacterium.

Three preliminary experiments were conducted to establish baseline data and to allow refinement of the pathogen challenge. The final experiment used the challenge on fish that have been dosed with contaminants over an 11 month period.

Aim The aim of the current study was therefore to provide a working methodology for a disease challenge in fish in association with the use of a battery of biomarkers to provide more detailed information of the effects of PAHs upon immune impairment / dysfunction.

Methods A preliminary contaminant exposure study was set up to identify a suitable challenge organism, route and dose and demonstrate an increase in susceptibility to this organism in fish exposed to the compounds of interest. Vibrio anguillarum (ATCC-43306) was found to effectively induce mortalities in flounder when injected intramuscularly. Cohabitant exposure, the most realistic and least stressful route of bacterial exposure was found to be ineffective for transmission of V. anguillarum. Bath challenge was subsequently ruled out, as it’s

mechanism of infection, adhesion and infiltration, is the same. In contaminant trials intramuscular delivery of PAHs via acetone was found to be the most effective delivery route, rather than gavage or intraperitoneal (I/P) injection of corn oil. The bacterial challenge methodology was validated to elucidate whether the dose, of 10 x106 colony forming unit (cfu) per fish, was appropriate for the fish size and stocking density of the final test system.

The final bacterial challenge experiment was run in parallel to the previous feeding studies used to obtain biomarker data, fish were fed the same feed as those in these studies with two tanks per treatment. Furthermore, commensal bacteria were assessed during the six month interim sampling period by taking kidney swabs onto seawater agar and incubating for two to three days at 20C. Total bacteria present were assessed and percent plate coverage estimated qualitatively. Kidney swab samples were taken from dead fish from each treatment group and slide agglutination and API 20E biochemical tests were used to confirm the presence of V. anguillarum. No V. anguillarum were found in fish that survived during the final challenge.

The main findings in the studies were: Commensal bacteria were present in lower numbers in contaminant dosed fish (six month interim sampling). This could be due to the contaminants having an inhibitory or toxic effect on the bacteria or result from the stimulation of the immune system of the fish, causing reduction in the numbers of normally tolerated organisms.

The final bacterial challenge showed a negative relationship between PAH exposure and mortality (Figure 14). It is not clear whether this is due to the toxicity of the PAHs, an immunostimulatory response or possibly a change in the commensal strucure preventing establishment and growth of V. anguillarum.

Small scale plate studies were undertaken to try to clarify the interactions between PAH, commensals and V. anguillarum. They revealed no apparent direct toxicity from PAH to V. anguillarum. Furthermore, gut fauna from PAH exposed fish was not found to inhibit the growth of V. anguillarum. However, an element of caution is required in the interpreation of the toxicity tests since it is difficult to evaluate true exposure of V. anguillarum to PAH in the tissue and no account was made of the toxicity of various PAH metabolites that may be present.

Figure 14 Percentage mortality of flounder following intramuscular injection with V. anguillarum after an 11 months exposure to PAH.

0 C C 0 0 5 5 0 . . 0 o o 1 1 m m n n m m t t g g r r g g o o / / K / / K l l K K g g g g

General Discussion The methodology for five biomarker techniques was developed and refined during the course of this study. EROD activity was one the methods used in this study subject to the greatest variability due to seasonal and sex related factors. Male flounder were consistently found to have higher EROD activity than females, this agrees with previous studies (Goksøyr, et al., 1996). The lack of significant differences between exposure regimes and EROD activity in the 6 month study is in contrast to the results obtained in the 1 month exposure. It is possible that activity of the MFO system was lower at 6 months since it coincided with the main reproductive period. Similar inhibition has been noted by Goksøyr, et al., (1996) who found that no CYP1A induction was observed in sexually mature female flounder fed gelatin capsules containing BaP. A significant negative correlation has also been observed between MFO specific activity and percent fertilisation for female starry flounder (Spies et al., 1985). The highest EROD activity measured in this study (one month exposure to 0.1-5 mg kg-1) was significantly lower than values for wild fish sampled from polluted estuarine sites sampled during UK environmental monitoring programmes (Kirby et. al, 1999). In this study, exposure of fish to PAH concentrations >100 mg kg-1 over a one month period inhibited EROD activity, suggesting that the field observations may be due to longer exposures to lower concentrations or to combined exposure to a range of different contaminants. Since EROD levels in this study following 11 months exposure to 5 mg kg-1 PAH were lower than those measured in the field, this suggests that (if PAHs alone were responsible for the field observations) the PAH exposure concentrations in the field are likely to fall between 5 – 100 mg kg-1. The lack of significant differences between exposure regimes and EROD activity in the 6 month study is in contrast to the results obtained in the 1 month exposure. It is possible that activity of the MFO system was lower at this time since it coincided with the main reproductive period. Similar inhibition has been noted by Goksøyr, et al., (1996) who found that no CYP1A induction was observed in sexually mature female flounder fed gelatin capsules containing BaP. A significant negative correlation has also been observed between MFO specific activity and percent fertilisation for female starry flounder (Spies et al. 1984).

PAH metabolites in the bile were produced at levels that reflect the exposure regime. This was also the case for exposure concentrations above 100 mg kg-1 in which EROD activity was inhibited. This suggests that metabolic pathways other than those measured by the EROD assay were still functioning. This latter point also makes a strong case for not relying entirely on a single biomarker result but rather to consider a range of responses when interpreting field data.

In this study genetic damage was measured using three different techniques all of which consider the integrity of the DNA structure. The amount of DNA adducts resulting from a given exposure can be influenced by a number of factors, these include the level and duration of exposure, the degree of absorbtion and transport of contaminants, the balance between the production of carcinogenic metabolites and their detoxification and the ability of cells to repair the adducts. Adducted DNA nucleotides were present in a single fish exposed to 5mg kg –1 for 6 months, however, 10 of the 15 fish in the 50 mg kg-1exposure had adducts present. Therefore although PAHs were being metabolised the threshold level for adduct formation lies between 5 and 50 mg kg-1

in a 6 month exposure to PAHs. In samples of Dab (Limanda limanda) caught off the rivers Humber and Tyne the level of DNA adducts was comparable to that measured in the 50 mg kg-1exposure in this study (Kirby et al., 2000). However it must be noted that species differences may give rise to different levels of adduct formation following the same level of contaminant exposure. The histopathology results also confirmed that there were no obvious cellular abnormalities, associated with genotoxicity of the PAHs with the exposure regimes employed in this study. The micronuclei assay did not show any significant differences between different exposure groups in the 6 month study and the threshold effect concentration was 500 mg kg –1for a 4 month exposure. The comet assay however proved a more sensitive technique. A significant increase in the amount of damaged DNA was measured at an exposure concentration of 5 mg kg –1after a 6 month exposure to PAH. This technique also identified differences between sperm samples taken from fish exposed to 5 mg kg –1 PAH after the same exposure period. This latter point is of particular relevance since it may have implications for heritable changes and viability of the next generation.

Whilst a range of tissues were considered in the histopathology assessment the dosage levels and route of exposure employed in this study was below the threshold for a clear dose response to occur. There was some evidence of dose related effects at the higher PAH (100-500 mg kg –1)doses in some of the shorter term experiments and some of these conditions were also observed following 6 months exposure to 50 mg kg-1 PAH in the diet (e.g vacuolation in gastric gland cells and anuerysms in the gills). The results of other studies suggest that the more severe pathologies in flounder populations (e.g. neoplastic nodules in the liver) increase with age of the fish, this suggests that if these pathologies are related to contaminant exposure, an exposure period of several years may be required (Vethaak and Jol, 1996).

Targeted EM of control and 50mg kg-1 dosed fish after 6 months exposure showed a significant proliferation of Rough endoplasmic reticulum suggesting heightened protein production. If this response is due to protein proliferation in the mono oxygenase enzyme system responsible for detoxification of contaminants this was not reflected in the EROD levels measured. Further EM studies of samples from other exposure groups, coupled with comprehensive biochemical analysis are required to clarify this phenomenon.

Summary The results of this study indicate that the five biomarkers used can provide valuable and complimentary information with respect to the interpretation of the previous contaminant exposure history of fish populations around the UK coast. The exposure route employed in this study is not usually considered in most contaminant studies but at realistic environmental concentrations of PAH in the diet, significant responses were measured. Dietary exposure to contaminants may also be the most significant route of exposure to predominantly pelagic species.

Uptake of PAHs in the diet also produced inhibition in oocyte development and an increase in the genetic damage present in sperm. These changes may have important consequences for successive generations and therefore warrant further study that specifically focuses on early developmental stages.

Clear responses were seen in all of the biomarkers used and these correlated with early observations of dose related histopathogy. These findings indicate that biomarkers such as those employed here are vital tools for use in monitoring programmes, providing an early warning of the onset of more serious disease conditions. The disease challenge protocol developed as part of this study provides an extremely valuable tool for the assessment of contaminant related reduction in disease resistance of fish populations that may have detrimental implications for commercial fish stocks. Further work using this methodology is recommended to develop our knowledge of the factors influencing the onset and development of infectious disease within fish populations.

References Al-Sabti, K., Metcalfe, C. D., 1995. Fish micronuclei for assessing genotoxicity in water. Mutat. Res. 343, 121- 135.

Ariese, F., Kok, S. J., Verkaik, M., Gooijer, C., Velthorst, N. H., Hofstraat, J. W., 1993. Synchronous fluorescence spectrometry of fish bile: A rapid screening metod for the biomonitoring of PAH exposure. Aquat. Toxicol. 26, 273-286.

Baumard, P., Budzinski, H., Garrigues, P., Sorbe, J. C., Burgeot, T., Bellocq, J., 1998. Concentrations of PAHs in various marine organisms in relation to those in sediments and to trophic level. Mar. Pollut. Bull. 36, 951- 960.

Britvic, S., Lucic, D., Kurelec, B., 1993. Bile fluorescence and some early biological effects in fish as indicators of pollution by xenobiotics. Environ Toxicol Chem. 12, 765-773.

Bucke, D; Feist, SW. Histopathological changes in the livers of dab, Limanda limanda (L.). Journal of Fish Diseases 16 (4) 281-296 1993

Di-Giulio, R. T., Behar, J. V., Carlson, D. B., Hasspieler, B. M., Watson, D. E., Forlin, L., Andersson, T., 1995. Determinants of species susceptibility to oxidative stress: A comparison of channel catfish and brown bullhead. Mar. Environ. Res. 39, 175-179.

Ericson, G., Lindesjoo, E., Balk, L., 1998. DNA adducts and histopathological lesions in perch (Perca fluviatilis) and northern pike (Esox lucius) along a polyclclic aromatic hydrocarbon gradient on the swedish coastline of the Baltic Sea. Can. J. Fish. Aquat. Sci. 55, 815-824.

Fernandez, J. D., Denny, J. S., Tietge, J. E., 1998. A simple apparatus for administering 2,3,7,8- tetrachlorodibenzo-p-dioxinto commercially available pelletized fish food. Environ. Toxicol. Chem. 17, 2058- 2062.

Fairbairn, D. W., Olive, P. L., O’Neill, K. L., 1995. The comet assay: a comprehensive review. Mut. Res. 339, 37-59.

French, B. L., Reichert, W. L., Hom, T., Nishimoto, M., Sanborn, H. R., Stein, J. E., 1996. Accumulation and dose-response of hepatic DNA adducts in English sole (Pleuronectes vetulus) exposed to a gradient of contaminated sediments. Aquat. Toxicol. 36, 1-16.

Goksøyr, A., Förlin, L., 1992. The cytochrome P-450 system in fish, aquatic toxicology and environmental monitoring. Aquat. Toxicol. 22, 287-312.

Goksøyr, A., Beyer, J., Egaas, E., Grøsvik, B. E., Hylland, K., Sandvik, M., Skaare, J. U., 1996. Biomarker responses in Flounder (Platicthys flesus) and their use in pollution monitoring. Mar. Pollut. Bull. 33, 36-45.

Harvey, J. S., Parry, J. M., 1998. Application of the 32P-postlabelling assay for the detection of DNA adducts: False positives and artefacts and their implications for environmental biomonitoring. Aquat. Toxicol. 40, 293- 308. Hendricks, JD; Meyers, TR; Shelton, DW; Casteel, JL; Bailey, GS: Hepatocarcinogenicity of benzoapyrene to rainbow trout by dietary exposure and intraperitoneal injection. Journal of the National Cancer Institute 74 (4) 839-851 1985

Jones, N. J., Kadhim, M. A., Hoskins, P. L., Parry, J. M., Waters, R., 1991. 32P-postlabelling analysis and micronuclei induction in primary lung cells exposed to tobacco particulate matter. Carcinogenesis. 12, 1507- 1514

Kohler A. Regeneration of contaminant induced liver lesions in flounder-experimental studies towards the identification of cause-effect relationships. Aquat. Toxicol 14:203-232 (1989)

Koehler, A. 1990. Identification of contaminant-induced cellular and subcellular lesions in the liver of flounder (Platicthys flesus) caught at differently polluted estuaries. Aquat Toxicol., 16: 271-294.

Krantz, H. and Dethlefsen, V. (1990). Liver anomalies in dab Limanda limanda from the southern North Sea with special consideration given to neoplastic lesions. Dis. Aquat. Org. 9, 171-185

Kirby, M. F., Matthiessen, P., Neall, P., Tylor, T., Allchin, C. R., Kelly, C. A., Maxwell, D. L., Thain, J. E., 1999. Hepatic EROD activity in flounder (Platichthys flesus) as an indicator of contaminant exposure in English estuaries. Mar Pollut. Bull. 38, 676-686.

Kirby, M. F., B.P. Lyons, MJ Waldock, RJ. Woodhead, F. Goodsir, RJ Law, P. Matthiessen, P., Neall, C., Stewart, JE. Thain, T. Tylor and SW Feist. 2000. Biomarkers of polycyclic aromatic hydrocarbon (PAH) exposure in fish and their application in marine monitoring. Science Series Technical Report No. 110.

Kohler, Gunther, A ; Moller, H; Anders, K; Pluta, H.J.; Cameron, P; Harms, U; Soffker, K: (1990) Report on the interdisciplinary research project "Fish diseases in the Wadden Sea". Source: ICES CM/E:29. 10 p., figs.

Law, R. J., Dawes, V. J., Woodhead, R. J., Matthiessen, P., 1997. Polycyclic aromatic hydrocarbons (PAH) in seawater around England and Wales. Mar. Pollut. Bull. 34, 306-322.

Lee, R. F., Sauerheber, R., Hobbs, G. H., 1972. Uptake, metabolism and discharge of polycyclic aromatic hydrocarbons by marine fish. Marine. Biol. 17, 201-208.

Livingstone, D. R. 1993. Biotechnology and pollution monitoring: Use of molecular biomarkers in the aquatic environment. J. Chem. Technol. Biotechnol. 57, 195-211.

Livingstone, D. R., Förlin, L., George, S. G., 1997. Molecular biomarkers and toxic consequences of impact by organic pollution in aquatic organisms. In: Water quality and stress indicators in marine and freshwater ecosystems. Linking levels of organisation (individuals, populations, communities). D. W. Sutcliffe (Ed), pp 154-171.

Lyons, B. P., Stewart, C., Kirby, M. F., 1999. The detection of biomarkers of genotoxin exposure in the European flounder (Platichthys flesus) collected from the River Tyne Estuary. Mutat. Res. 446, 111-119

Maccubbin, A. E., Black, P., Trezeciak, L., Black, J. J., 1985. Evidence for polynuclear aromatic hydrocarbons in the diet of bottom feeding fish. Bull. Environ. Contam. Toxicol. 34, 876-882.

McElroy, A. E., Sissons, J. D., 1989. Trophic transfer of Benzo[a]pyrene metabolites between benthic marine organisms. Mar. Environ. Res. 28, 265-269.

Meador, J. P., Stein, J. E., Reichert, W. L., Varanasi, U., 1995. Bioaccumulation of polycyclic aromatic hydrocarbons by marine organisms. Rev. Environ. Contam. Toxicol. 143, 79-165.

Myers M.S.L.D.Rhodes, and B.B.McCain. 1987. Pathologic anatomy and patterns of occurrence of hepatic neoplasms, putative preneoplastic lesions, and other idiopathic hepatic conditions in English sole (Parophrys vetulus) from Puget Sound, washington. J. Natl. Cancer Inst. 78:333-363.

Myers M.S, Landhal JT, Krahn MM, McCain BB. Relationships between hepatic neoplasms and related lesions and exposure to toxic chemicals in marine fish from the U.S. West Coast. Environ Health Perspect 90:7-15 (1991).

Myers M.S, Stehr CS, Olson O, Johnston LL, McCain BB, Chan S-L, Varanasi U. Relationships between toxicopathic hepatic lesions and exposure to chemical contaminants in English sole (Pleuronectes vetulus), starry flounder (Platicthys stellatus) and white croaker (Genyonenus lineatus) from selected marine sites on the Pacific Coast, U.S.A Environ Health Perspect 102:200-215 (1994)

Pizza, J. C., O’Connor, J. M., 1983. PCB dynamics in Hudson river striped bass. II. Accumulation from dietary sources. Aquat. Toxicol. 3, 313-327.

Randerath, K., Reddy, M. V., Gupta., R. C., 1981. 32P-postlabelling test for DNA damage. Pro. Natl. Acad. Sci. USA. 78, 6126-6129.

Reichert, W. L., French, B. L., Stein, J. E., 1999. Biological effects of contaminants; Measurement of DNA adducts in fish by 32P-postlabelling. ICES Techniques in Marine Environmental Sciences. 25, 1-45.

Spies RB, Rice DW, Montagna PA, and Ireland RR. (1985) Reproductive Success, Xenobiotic Contaminants and Hepatic Mixed-Function Oxidase (MFO) Activity in Platicthys stellatus Populations fronm San Francisco Bay. Marine Environmental Research 17 (1985) 117-121.

Stahlschmidt-Allner, P., Allner, B., Rombke, J., Knacker, T., 1997. Endocrine disruptors in the aquatic environment. Environ. Sci & Pollut. Res. 4, 155-162.

Stagg, R. M., McIntosh, A., 1998. Biological effects of contaminants: Determination of CYP1A-dependent mono-oxygenase activity in dab by fluorimetric measurement of EROD activiy. ICES. 23 pp1-

Vethaak, A. D., Jol, J. G., 1996. Diseases of flounder Platicthys flesus in Dutch coastal and estuarine waters, with particular reference to environmental stress factors. I. Epizootiology of gross lesions. Dis. Aquat. Org. 26, 81-97.

Vethaak, A. D., Wester, P. W., 1996. Diseases of flounder Platicthys flesus in Dutch coastal and estuarine waters, with particular reference to environmental stress factors. II Liver Histopathology. Dis. Aquat. Org. 26, 99-116.

Woodhead, R. J., Law, R. J., Matthiessen, P., 1999. Polycyclic aromatic hydrocarbons in surface sediments around England and Wales, and their possible biological significance. Mar. Poll. Bull. 38, 773-790.

Zelikoff, J.T., 1994. Fish Immunotoxicology. In: Dean, J.H., Luster, M.I., Munson, A.E. and Kimber, I. (eds). Immunotoxicology and Immunopharmacology, 2nd edition. Raven Press, New York, pp. 71-94.

Please press enter

CSG 15 (1/00) 25