a preliminary investigation into the use of biomarkers for the monitoring of an ocean outfall

A Preliminary Investigation into the Use of Biomarkers for the Monitoring of an Ocean Outfall

Peter R. L. Mosse*

Gippsland Water, P.O. Box 348, Traralgon, Victoria, 3844 Australia

Carolyn M. Brumley, Jorma T. Ahokas, and Douglas A. Holdway

Key Centre for Applied and Nutritional Toxicology, RMIT-City Campus, G. P. 0 Box 2476V, Melbourne, Victoria, 3001, Australia

The use of several biomarkers of exposure for monitoring a treated mixed effluent from the Latrobe Valley Ocean Outfall was examined. Sand flatheads (Platycephalus bassensis) were exposed in the laboratory to two dilutions of effluent (1.3 and 2.5%, v/v) for three days. Cytochrome P450 content, 7-ethoxyresorufin-0-deethylase (EROD) activity, and biliary metabolites of polycyclic aromatic hydrocarbons (PAH), 2-chlorosyringaldehyde and 2,6-dichlorosyringaldehyde, were measured. There were no statistically significant differences in cytochrome P450 content or EROD activity between control and exposed fish. Analysis of the bile by gas chromatography/mass spectrometry of fish exposed to 2.5% effluent found no increases above background in the levels of PAHs, or specific chlorinated phenolics that are found in bleached hardwood pulp effluents. 0 7996 by John Wiley & Sons, Inc.

INTRODUCTION

Traditionally two approaches have been used to monitor effluent discharges. The first involves the determination of the levels and types of specific compounds in the effluent, and is generally associated with licences that specify limits for particular chemicals. The second approach involves ecological surveys that compare population and community structures, either before and after commencement of discharge, or at discharge and reference sites. While the first approach to monitoring de- scribes the chemical composition of effluents, it pro- vides no information on the synergistic or antagonistic effects that chemicals in a mixture may have on biological systems. It is also becoming increasingly difficult, and expensive, to measure all of the chemicals that com- prise a complex effluent. The second approach, al-

* To whom correspondence should be addressed

though the ultimate arbiter of environmental impact, is slow to yield results and any change is often masked by the highly variable nature of the ecosystem in both space and time. This slow response time makes short-term management of treatment and disposal processes, based on ecological surveys, almost impossible.

An alternative approach to monitoring effluent discharges is now emerging. This approach is based on the premise that, for there to be a detectable impact at a population or community level, there must first have been an impact on individual organisms. It may be possible for this impact to be measured using biochemical and physiological markers, or biomarkers, which may thus provide early warning of change, before the onset of serious pathological damage. The use of biomarkers also offers the prospect of measuring sensitive biological responses to chemical exposure at the organism level without the delay of ecological studies, thereby allowing appropriate process management steps to be rapidly initiated.

Environmental Toxicology and Water Quality: An International Journal, Vol. 11 (1996) 113-1 19 0 1996 by John Wiley & Sons, Inc. CCC 1053-4725/96/02113-07

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11 4 MOSSE ET AL.

Most persistent organic contaminants are fat soluble and require metabolic conversion to increase their water solubility before they can be excreted by living organisms. As such the vertebrates, and to a lesser extent, the invertebrates, have developed a two-phase detoxication system that facilitates the excretion of lipophilic chemicals. In the first phase of detoxication, a cytochrome P450 mixed function oxidase (MFO) enzyme system inserts an atom of oxygen into the organic molecule, thereby producing a more water-soluble product. Increases in the activities of MFOs, as measured by ethoxyresorufin-O-deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH) activities, have been used in fish to monitor exposure to chemical contaminants (Andersson et al., 1988; Lindstrom-Seppa and Oikari, 1989; Porter et al., 1989; Holdway et al., 1994; Martel et al., 1994).

In the second phase of detoxication, one of several biotransformation enzymes may conjugate an endoge- nous compound, such as sulphate or uridinedi phosphate (UDP)-glucuronic acid, to the lipophilic chemical to complete its biotransformation to a water-soluble metabolite. Many of these transformed molecules are then released into the bile, where they may be concen- trated before excretion. The quantification of these biliary metabolites can provide a sensitive biomarker of exposure to the parent chemical (Lech et al., 1973). Metabolites of pollutants extracted from fish bile have been used previously as specific indicators of exposure to pulp mill effluent (Oikari and Anas, 1985; Lindstrom- Seppa and Oikari, 1989) and polycyclic aromatic hydrocarbon (PAH) contaminated sediments (Krahn et al., 1987; 1993; Deshpande, 1989).

In 1989 and 1990 Ahokas et al. (1994) sampled Euro- pean carp (Cyprinus carpio) from Lake Coleman, Aus- tralia, to determine whether organochlorine contaminants from treated pulp mill effluents were affecting carp detoxication systems. The study found elevated levels of hepatic EROD, compared with fish from a reference location, which correlated well with site ad- sorbable organic halogen (AOX) and muscle dioxin levels. Lake Coleman is a shallow lake in South Eastern Victoria that, prior to commissioning of the Latrobe Valley Ocean Outfall in July 1992, received treated effluent from the Dutson Downs treatment facility. The treatment facility consists of an interconnected series of anaerobic and aerobic lagoons. The main treatment lagoon underwent extensive remodeling in 19891 1990 and now operates with a total detention time of around 60-90 days. At the time of the original study the facility treated a mixture of domestic waste and pulp and paper mill effluent. In July 1992 waste water containing low levels of benzene, toluene, xylene, and low molecular weight PAHs from the ESSO-BHP oil and gas operations in Bass Strait were also introduced to the system.

Thus at present the system treats waste waters derived from three sources: domestic waste from the towns of the Latrobe Region (- 15 ML/day), pulp and paper mill effluent from the Australian Paper Mill at Maryvale (-15 ML/day), and formation water from the Esso- BHP offshore oil drilling operations in Bass Strait (-9 ML/day). The final treated effluent is now discharged to Bass Strait via a shallow water outfall diffuser ap- proximately 1.2 km offshore at a depth of around 15 m (Mosse, 1993; Donlon and Mosse, 1994).

In addition to the changes described above, the quality of the effluent has changed since the earlier study as judged by the aggregate parameters AOX and color. The mean AOX levels have fallen from 2.38 mg/L in 1992 to 1.56 mg/L in 1994. Similarly, the average color has fallen from 1260 Pt/Co units in 1992 to 610 Pt/ Co units in 1994 (Gippsland Water, unpublished data). Thus the present study was designed to investigate possible effects of the treated effluent on the detoxication systems of a marine species in a situation where exposure could be fully controlled.

This paper presents the results of determinations of cytochrome P450 content, EROD activity, and an analysis of biliary metabolites of PAHs and specific chlorinated phenolics from sand flathead (Platycepha- lus bassensis) exposed in the laboratory to two dilutions (1.3 and 2.5% v/v) of treated effluent from the Latrobe Valley Ocean Outfall. The 1 : 75 (1.3%) dilution was chosen since this was the minimum design dilution of the outfall diffuser, and the 1 : 40 (2.5%) dilution was chosen as an extreme case, being roughly one half the design dilution of the outfall diffuser.

MATERIALS AND METHODS

Chemicals

Phenanthroline, 7-ethoxyresorufin, bovine serum albu- min (BSA), and dithiothreitol (DTT) were obtained from Sigma, USA; glucose-6-phosphate (G-6-P), glucose-6-phosphate dehydrogenase (G-6-P dehydrogenase), and nicotinamide adenine dinucleotide phosphate (NADP) were obtained from Boehringer Mannheim, Germany. Resorufin was purchased from Kodak, USA. Tetrachloronaphthalene (TCN) was purchased from ICN Biomedicals, USA; 2,4,6-tribromophenol came from Alderich, USA. All solvents used were of analytical grade.

Fish

Sand flathead were caught by hook and line from Port Phillip Bay, Victoria, in April 1994. The mean (-+SD) length and weight of the fish was 258 * 19 mm and

BIOMARKERS FOR MONITORING OCEAN OUTFALL 115

94 k 23 g, respectively. Fish were kept on board the research vessel in 500 L polyethylene bins supplied with flowthrough seawater and, upon arrival at the aquarium facilities, were transferred to a 5000 L con- crete holding tank supplied with flowthrough seawater at a rate of 120 L/min. After a 20 h acclimatization period, fish were transferred into 80 L treatment tanks that were supplied with flowthrough seawater at 375 mL/min. Five fish were allocated to each tank and were allowed to acclimatise for a further 50 h.

Effluent

Treated effluent was collected at the discharge point of the Latrobe Valley Ocean Outfall in cleaned 44 gallon drums that had been thoroughly rinsed with the effluent. The sealed drums were road freighted to the aquarium facility and stored at 18-19.5"C.

Effluent Exposure

The effluent dosing system was a modified version of the multichannel gravity operated system described by Connor and Wilson (1972). Seawater and effluent were mixed to the required dilution and stored in a 1000 L polyethylene reservoir from where it was pumped to a constant level header tank. Each treatment tank drew from a separate flow line through a glass flow meter connected to an adjustable height flow control chamber.

Fish were exposed to diluted effluent at a concentration of I : 40 or 1 : 75 (vlv) for 3 days. Control fish received seawater alone. At the commencement of the exposure period there was a change in color in the tanks receiving the effluent; however, this stabilized over the first few hours of exposure. There were no noticeable changes in fish behavior during this time or during the rest of the exposure period. One fish died in one of the 1 : 40 exposure tanks. All tanks had similar ranges for water temperature (16- 17"C), dissolved oxygen (84-90% saturation), and pH (8.0-8.2).

Sampling

At the end of the exposure period, each fish was killed by a sharp blow to the head. The abdomen was quickly opened and the liver was removed to liquid nitrogen. Bile was aspirated from the gallbladder with a syringe and stored in liquid nitrogen until required for analysis. Of the 30 fish used in this study, 4 were males and 26 were females. The skewed male to female ratio (2 : 13) of this study is in keeping with previous research using this fish and may be due to size selection at the time of fishing.

Microsome Preparation

Livers were processed to a microsomal suspension and assayed for the activity of EROD and cytochrome P540 content using previously published standard methods (Holdway et al., 1993). To describe these methods briefly; livers were homogenized in a 20% (wlv) solution of 0.1M phosphate buffer (pH 7.4), containing 1 mM DTT, 0.1 mM phenanthroline, 0.1 mM KCI, and 1 mM EDTA. The homogenate was centrifuged at 12,000 x gfor 20 min and the supernatant further centrifuged at 100,000 x g for 1 h. The microsomal pellet was resuspended in 0.1M phosphate buffer (pH 7.41, containing 20% glycerol (w/v), and then stored at -80°C until analysis.

Cytochrome P450 assay

P450 content was analyzed by the dithionite reduced difference spectrum of carbon monoxide (CO) bubbled samples, according to the method of Matsubara et al. (1976), with modifications from Rutten et al. (1987) and described in Brumley et al. (1995). Samples were read on a Shimadzu UV-3000 dual wavelengthldouble beam spectrophotometer. An extinction coefficient of 104 mM-l cm-' was used (Matsubara et al., 1976).

EROD Assay

EROD assays were performed in triplicate at 30°C and pH 7.6. Ethoxyresorufin was dissolved in I : 1 metha- nol : DMSO and diluted to working concentration in 0.1 M Tris buffer. The incubation mixture (total volume 1 mL) consisted of 0.25 mL of an NADPH re- generating system (2.5 mM MgCI,, 200 mM KCI, 6 mM glucose-6-phosphate, 1.25 mM NADP, and 10 U glucose-6-phosphate dehydrogenase), Tris buffer, 1.2 mg of BSA, and 0.1 mL of the microsomal suspension. The reaction was started with the addition of 7-ethoxyresorufin (final concentration: 1 pM), incubated for 10 min, and terminated by the addition of ethanol. Sam- ples were centrifuged and the supernatants analysed on a Hitachi F-4500 fluorescence spectrophotometer at excitation/emission wavelengths of 530/585 nm against a resorufin standard curve.

Bile Fluorescence

Fluorescence was used in this work as a screening method for the presence of PAHs and their metabolites in flathead bile. Bile analysis was carried out in five fish exposed to 2.5% effluent and the level of biliary metabolites compared with two unexposed fish. Bile quantity was measured, internal standard 2,4,6 tribromophenol(O.5 pg) was added, and then the sample was

116 MOSSE ET AL.

diluted to 1 mL with Milli-Q water. A 25 p L aliquot was removed and diluted to 1 mL prior to measuring fluorescence on a Hitachi F-4500 fluorescence spectrophotometer. The excitation/emission wavelength pairs 290/335, 280/385, 256/380, and 380/430 nm were selected (Hellou and Payne, 1987; Krahn et al., 1993; Upshall et al., 1993), and correspond to the analysis of naphthyl glucuronides, conjugate cleaved naphthyls, phenanthrenes and their metabolites, and benzo(a) pyrene and its metabolites, respectively.

Cleavage of the glucuronide and sulphate conjugates was achieved by incubating the diluted bile samples with P-glucuronidase (4000 U) and aryl sulphatase (32,000 U) in 0.3M sodium acetate buffer, pH 5 , at room temperature for 24 h. The fluorescence of the bile samples was again recorded at the above wavelength pairs.

Biiiary Metabolite Extraction and Analysis

Bile samples were extracted with ethyl acetate (two times 2 mL), and the organic fractions were combined and evaporated under nitrogen. Acetate derivatives of the conjugate-cleaved metabolites were prepared by the addition of acetic anhydride (100 pL) and pyridine (50 pL), and heating at 70°C for 20 min. Samples were then washed with 0.5M HC1 (0.5 mL) and the organic layer was analysed using a Shimadzu QP-2000 gas chromatograph/mass spectrometer (GC/ MS) after the addition of the internal standard tetrachloronaphthalene (0.5 pg). The conditions used were as follows: 2 p L injections were made onto a 30 m X 0.25 mm DB-1 coated fused silica column (J&W, USA) with injection port, mass spectrometer source, and interface at 250°C.

Selected ion monitoring was used for the detection of acetate derivatives of chlorinated hardwood effluent chemicals and standards. The chemicals monitored were 2-chlorosyringaldehyde (216/218), 2,6 dichlorosyringaldehyde (250/252/254), and their respective biliary metabolites, 2-chloro- (260/201) and 2,6-dichloro- 3,5-dimethoxy-4-hydroxybenzyl alcohol (294/296/ 298); standards 2,4,6-tribromopheno1(330/332) and tetrachloronaphthalene (266) as previously used in studies of sand flathead exposure to bleached hardwood effluent (Haritos et al., 1995; Brumley et al., unpublished data). In addition, derivatized bile extracts were analyzed by gas chromatography/mass spectral acquisition where eluting chemicals were scanned between 50 and 650 amu each second. The mass spectra resulting from the chromatogram peaks of the bile samples of both exposed and control fish were compared with the library of mass spectra installed on the QP-2000 GC/ MS and resulted in a similarity index to known chemicals where the spectra matched.

Data Analysis

Data were analyzed using a two-way analysis of vari- ance (ANOVA) from the SuperANOVA software package.

RESULTS AND DISCUSSION

The results for EROD activity and cytochrome P450 content are shown in Table I. There were no differences in EROD activity between fish exposed to the different dilutions of effluent and control fish ( p = 0.48). Hepatic cytochrome P450 content was also not significantly different between exposed and control fish ( p = 0.99). In neither case were there any differences in the response of the sexes to effluent exposure ( p = 0.64 for EROD and p = 0.42 for cytochrome P450). However, the small number of males sampled would have made any differences, if present, hard to detect.

There were no significant differences between fish exposed to effluent and control fish in the levels of bile fluorescence due to the presence of PAH metabolites, with or without cleavage of conjugates (Table 11). This could be due either to low PAH content in the diluted effluent or to an insufficient exposure period for PAHs to achieve steady-state concentration in the fish. Bile fluorescence has been previously shown to be a very sensitive indicator of PAH exposure in fish, and has been correlated with specific toxic PAHs such as benzo(a)pyrene (Hellou and Payne, 1987; Britvic et al., 1993; Krahn et al., 1993; Upshall et al., 1993). However, most of these studies were carried out using fish that had experienced long-term exposure to polluted water or sediment.

Selected ion monitoring of exposed and control samples did not detect any of the specific chemicals and metabolites of bleached hardwood pulp effluent such as 2-chlorosyringaldehyde (2-CSA) or 2,6-dichlorosyringaldehyde. As the detection limits for these chemicals were less than 5 ng/mL (ppb) by the GUMS technique and the recovery of internal standard 2,4,6-tribromophenol in this study was 85%, there was adequate sensitivity to detect low levels of these effluent chemicals. In a study of the biliary metabolites of flathead exposed to 0.5% simulated bleached eucalyptus pulp effluent, metabolites of 2-CSA were easily detectable after 4 days exposure (Brumley et al., unpublished data).

Gas chromatography/mass spectral acquisition of the extracted bile revealed no obvious difference between chromatograms of exposed and control fish. There were no peaks of any significance in the spectra of the treated fish that were not present in those of the control fish (Fig. I ) . A mass spectral library


TABLE 1. Mean (&E) of hepatic EROD activity and cytochrorne P450 content in flathead exposed to dilutions of treated effluent compared with clean water-exposed control fish

EROD Cytochrome P450 Exposure N (nmol/rnin/rng protein) (rnrnol/rng protein)

Control 1:75(1.3%) 1 : 40 (2.5%)

10 10 10

0.015 (0.002) 0.013 (0.003) 0.023 (0.003)

0.151 (0.013) 0.126 (0.018) 0.162 (0.01 1)

database comparison indicated the presence of cholesterol-like compounds (e.g., cholest-5-en-3-01 derivatives) and hydrocarbon derivatives (e.g., 5-octadecene, 3-eicosene) in the bile of both treated and control fish.

The results of the biomarker determinations on flathead exposed to the treated effluent show no clear evidence of induction of detoxication enzymes or the accumulation of biliary metabolites. There were no significant differences in EROD activities between fish exposed to effluent and control fish. This is in contrast to a previous field study (Ahokas et al., 1994), which found significant elevations of EROD in European carp endemic to Lake Coleman, the previous receiving water for the effluent, prior to commissioning of the present ocean outfall. This difference in response can probably be attributed, in part, to the progressive im- provement in the quality of the effluent, which in turn reflects changes in production processes at the Austra-

TABLE II. Results of total fluorescence in the bile of sand flathead exposed to 2.5% dilution of effluent and controls

Prior To Enzymatic Cleavage ~~

PAH Class

Average Fluorescence (Units)

Treated (2.5%) Controls

Naphthyl conjugates 5.8 Naphthyls 7.6 Phenanthrenes 7.6 Benzo(a)pyrenes 0.9

5.5 8.3 7.6 0.8

After Enzymatic Cleavage ~~

Average Fluorescence (Units)

PAH Class Treated (2.5%) Controls

Naphthyl conjugates 29.1 29.4 Naphth yls 14.9 15.8 Phenanthrenes 7.7 7.7 Benzo(a)pyrenes I .o 1 .o

lian Paper, Maryvale, mill. Discharge of the poorer quality effluent to Lake Coleman over a period of 25-30 years has caused persistent organochlorines to accu- mulate in the sediments (Ahokas et al., 1994). Different exposure times and routes, as well as species differences in sensitivity may also have contributed to the different responses. Curtis et al. (1993) studied biomarker responses in different species of fish along the length of the Willamette River in Oregon and found both EROD and cytochrome P450 levels reflected site contamination. However, there was considerable spe-

A

2

I c

I I 0 12.3 Mins 19.0 22!0

Fig. 1. Total ion chromatograms of solvent-extracted, acetate derivatized bile samples from sand flathead (A) exposed to 2.5% effluent for 3 days and (B) unexposed control. Analytes were detected by the mass spectrometer scanning between 650 and 50 amu. Numbered peaks were tentatively identified by mass spectral matching (>60% similarity) between the library and sample spectra. Peak 1 : 5-octadecene; peaks 2 and 3: cholesterol derivatives.

11 8 MOSSE ET AL.

cies variability and unspecified environmental effects that masked the identification of point source discharges (including a pulp and paper mill). The study highlighted some of the difficulties in field application of this methodology, in particular the inability to control exposure. In another study of a modern bleached kraft pulp mill site, differential denaturation of hepatic P450 and P420 occurred in some fish that resulted in differential inactivation of EROD and poor correlation with organochlorine body burden (Kloepper-Sams and Swanson,1992). No such degradation of P450 was found in this study. Laboratory studies, such as the one presented here, are able to control exposure and may therefore offer a better approach to assessing environmental impact at an early stage. An elevated response can only be taken as an indicator at this time since the link between elevated EROD and cytochrome P450 levels and frank pathology is still not clearly estab- lished (e.g., Curtis et al. 1993).

Exposure periods in experimental studies vary. While Martel et al. (1994) and Porter et al. (1989) chose 96 and 90 h exposures, respectively, induction has been shown clearly to occur in as little as 24 h (Bowman and Rand, 1980; Kleinsmith and Kish, 1988). The exposure period of 72 h used in this study was selected to be within this range and to reflect what might reason- ably be considered to be about that for which fish might be continuously exposed in the field. The concentra- tions of effluent selected for exposure of the fish were the design dilution of the outfall (75 : 1) and one half of that dilution. The effluent discharged from the Latrobe Valley Ocean Outfall actually achieves a minimum ini- tial dilution of 100 : 1, and dilutions of 1000 : 1 within 100 m of the diffuser (Mosse, 1993).

Other fields studies investigating the impact of the treated effluent from the Latrobe Valley Ocean Outfall on the receiving environment have found no statistically significant evidence for the bioaccumulation of extractable organic halogens (EOX); Haynes et al. 1995a) or dioxins (Haynes et al. 1995b) in transplanted cultured mussels (Mytilus edulis). Similarly, in a laboratory study, there was no evidence that fillets obtained from sand flathead exposed to two dilutions of effluent (2.3 and 2.5% effluent) could be distinguished on the basis of odor from fillets obtained from control fish exposed to seawater (Mosse and Kowarsky, 1995).

In conclusion, the results reported here provide no evidence of induction of liver detoxication enzymes for a species of marine fish exposed under laboratory conditions to a treated effluent that originated from pulp and paper mill and petroleum industry waste waters.

The authors would like to thank Ms. V. S. Haritos and Ms. J. Butty for technical assistance.

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a preliminary investigation into the use of biomarkers for the monitoring of an ocean outfall

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