A Protocol for Identifying Suitable Biomarkers to Assess Fish Health: a Systematic Review

RESEARCH ARTICLE A protocol for identifying suitable biomarkers to assess fish health: A systematic review

Frederieke Kroon1☯*, Claire Streten2☯, Simon Harries2☯

1 Australian Institute of Marine Science, Townsville, Queensland, Australia, 2 Australian Institute of Marine Science, Arafura Timor Research Facility, Brinkin, Northern Territory, Australia

☯ These authors contributed equally to this work. * [email protected] a1111111111 Abstract a1111111111 a1111111111 a1111111111 a1111111111 Background Biomarkers have been used extensively to provide the connection between external levels of contaminant exposure, internal levels of tissue contamination, and early adverse effects in organisms. OPEN ACCESS

Citation: Kroon F, Streten C, Harries S (2017) A Objectives protocol for identifying suitable biomarkers to To present a three-step protocol for identifying suitable biomarkers to assess fish health in assess fish health: A systematic review. PLoS ONE 12(4): e0174762. https://doi.org/10.1371/journal. coastal and marine ecosystems, using Gladstone Harbour (Australia) as a case study. pone.0174762 Editor: James P. Meador, Northwest Fisheries Methods Science Center, UNITED STATES Prior to applying our protocol, clear working definitions for biomarkers were developed to Received: October 23, 2016 ensure consistency with the global literature on fish health assessment. First, contaminants Accepted: March 15, 2017 of concern were identified based on the presence of point and diffuse sources of pollution

Published: April 12, 2017 and available monitoring data for the ecosystem of interest. Second, suitable fish species were identified using fisheries dependent and independent data, and prioritised based on Copyright: © 2017 Kroon et al. This is an open access article distributed under the terms of the potential pathways of exposure to the contaminants of concern. Finally, a systematic and Creative Commons Attribution License, which critical literature review was conducted on the use of biomarkers to assess the health of fish permits unrestricted use, distribution, and exposed to the contaminants of concern. reproduction in any medium, provided the original author and source are credited.

Data Availability Statement: All relevant data are Results/Discussion within the paper and its Supporting Information We present clear working definitions for bioaccumulation markers, biomarkers of exposure, files. biomarkers of effect and biomarkers of susceptibility. Based on emission and concentration Funding: This study was supported by the information, seven metals were identified as contaminants of concern for Gladstone Har- Gladstone Healthy Harbour Partnership. The bour. Twenty out of 232 fish species were abundant enough to be potentially suitable for bio- funders had no role in study design, data collection and analysis, decision to publish, or preparation of marker studies; five of these were prioritised based on potential pathways of exposure and the manuscript. susceptibility to metals. The literature search on biomarkers yielded 5,035 articles, of which

Competing interests: The authors have declared 151met the inclusion criteria. Based on our review, the most suitable biomarkers include that no competing interests exist. bioaccumulation markers, biomarkers of exposure (CYP1A, EROD, SOD, LPOX, HSP, MT,

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DNA strand breaks, micronuclei, apoptosis), and biomarkers of effect (histopathology, TAG: ST).

Conclusion Our protocol outlines a clear pathway to identify suitable biomarkers to assess fish health in coastal and marine ecosystems, which can be applied to biomarker studies in aquatic ecosystems around the world.

Introduction Globally, the coastal environment is the ultimate sink for many contaminants and their associated breakdown products [1]. Contaminants can enter the coastal environment via aquatic and atmospheric transport from point sources such as shipping ports, industrial waste, sewage outfalls, and stormwater drains, and from diffuse sources such as agricultural and urban runoff [1]. The presence of contaminants has the potential to affect the quality and uses of the coastal environment including direct uses such as fisheries, tourism, and recreation [2]. Assessing the impacts of contaminants on the health of aquatic organisms and ecosystems is challenging due to the presence of multiple stressors and the complexity of ecosystems [3, 4]. Biomarkers have been used extensively to provide the connection between external levels of contaminant exposure, internal levels of tissue contamination, and early adverse effects in organisms [5–7]. As such, they are considered ‘early warning’ signals that have the potential to detect an effect in target biota prior to one being observed at the population, community or ecosystem level [3, 5, 7]. Hence, the use of biomarkers can be a critical line of evidence to understand relationships between stressors and effects on coastal resources, and to prevent detrimental impacts of contamination on ecosystem structure and function [3–5]. Fish species are generally considered to be one of the key elements for the assessment of the quality of aquatic ecosystems [5]. First, fish are ubiquitous in almost all aquatic environments with resident fish comprising a critical component of the community exposed to contaminants [5]. Second, fish have high ecological relevance in the aquatic environment due to their influence on food web structure, nutrient cycling and energy transfer. Fish are also an important protein source for humans, and the exposure and effects of contaminants on this food source is of general interest to consumers. Third, the taxonomy, basic life history, and physiology of fish are generally well understood, allowing for targeted studies on internal levels of tissue contamination and early adverse effects. Importantly, however, considerable variation exists among fish species with regards to their contaminant exposure patterns, their basic physiological features, and ultimately their response to environmental contaminants [6]. Hence, fish species used for aquatic health assessment need to be selected based on the potential pathways of exposure to the contaminant of concern, and the biological response that is measured or proposed as a biomarker [6]. In this study, we present a protocol for identifying suitable biomarkers to assess fish health in coastal and marine ecosystems (Fig 1). We outline three steps that are required as part of this protocol, using Gladstone Harbour (Australia) as a case study. First, we identify potential contaminants of concern based on the presence of point and diffuse sources of pollution and available monitoring data for the ecosystem of interest. Second, we identify the most appropriate fish species suitable for biomarker studies based on abundance records from fisheries dependent and independent studies, combined with species-specific biological information

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Fig 1. Conceptual diagram outlining a three-step protocol for identifying suitable biomarkers to assess fish health. Rectangles represent literature reviews, ovals represent data lists generated. The protocol is applied using Gladstone Harbour (Australia) as a case study. https://doi.org/10.1371/journal.pone.0174762.g001

that may affect exposure pathways to the contaminants of concern. Next, we conduct a systematic and critical literature review [8] of the biomarkers available to assess the health of fish exposed to the contaminants of concern. Combined, our protocol outlines a clear pathway suitable for biomarker studies that can be used to assess health of aquatic organisms in ecosystems around the world.

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Materials and methods Definition of biomarkers Various definitions of biomarkers are used in the scientific literature resulting in confusion about their meanings [5, 6, 9]. To ensure that our use of the term ‘biomarker’ is consistent with that used in fish health assessments worldwide [5, 6], we reviewed the literature in Web of ScienceTM for definitions of biomarkers used in assessments of fish health specifically, and aquatic ecosystem health more broadly. Based on our findings, we developed clear working definitions of biomarkers for this study.

Study area Gladstone Harbour (Fig. 3.1 in [10]) is located near the City of Gladstone in central Queens- land, Australia. The harbour is exposed to various point and diffuse sources of pollution, including shipping, industrial waste, sewage outfalls, stormwater drains, and agricultural and urban runoff. The Harbour is the location of the Port of Gladstone, a major bulk commodity port with 1,648 ships visiting and 100 MT total throughput in the financial year 2014/15 (http://www.gpcl.com.au/). The Port of Gladstone is one of the three major hubs for the export of coal in Queensland [11], and services major industries in the Gladstone region, including mining, engineering, construction, and manufacturing. The City of Gladstone had a population of 66,097 in 2014; the potential impact of stormwater, and urban runoff more broadly, on Gladstone Harbour is currently unknown [10]. The Gladstone area is serviced by seven sewage treatment plants (STP), of which two discharge into watercourses connected to Gladstone Harbour [12]. The two river basins discharging into Gladstone Harbour, the Calliope and the Boyne, contribute additional suspended sediment, nutrients, and pesticides, derived from upstream agricultural land uses [13, 14].

Contaminants of concern in water and sediment To identify potential contaminants of concern in water and sediment of Gladstone Harbour, we considered point and diffuse sources of pollution and assessed available monitoring data. First, we examined the National Pollutant Inventory (NPI, [15]) which records annual emissions and transfers of 93 pollutants from facilities around Australia under the National Envi- ronment Protection Measures legislation. We extracted facilities, as well as pollution emission and transfer records, for the Gladstone region for 2014/2015. Potential pollutants of concern for Gladstone Harbour were identified based on the NPI inventory and volumes released into water each year. The NPI data do not provide information on the level of exposure, toxicity or the fate of these pollutants in the environment. To evaluate if these NPI pollutants are of environmental concern we reviewed publicly available monitoring and environmental assessment reports from 2005 onwards [10, 16–31]. Where possible, contaminant concentrations in water and sediment were compared against Australian guidelines for water and sediment quality [32–34]. Concentrations of contaminants detected in water and/or sediment in Gladstone Harbour, other than the NPI ones, were also reviewed.

Selection of suitable fish species To identify fish species suitable for biomarker studies that could be used to assess fish health, we considered both fisheries dependent [35–39] and independent [40–44] data published from 2005 onwards to generate a list of fish species present for Gladstone Harbour. Commercial fishing data [37] were extracted for two fishing areas in and around Gladstone Harbour, namely S30 and S31 [39]. In addition, we examined catch data from the Queensland Government shark

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control program [44] from locations nearby Gladstone Harbour (Tannum Sand). Using this list, we identified 20 fish species that are abundant in Gladstone Harbour year-round. To ensure consistency with the latest nomenclature, the names of these 20 fish species were checked against the most recent accepted synonym for scientific name in FishBase (ver 01.2016) (http://www. fishbase.org/), and the accepted common name in Australian Fish Names Standard AS 5300– 2015 [45]. Relevant information for identifying the most suitable fish species for future biomarker studies was subsequently collated for each of the 20 fish species from established references [46–48] and internet resources (FishBase ver 01.2016, http://www.fishbase.org/; Austral- ian Museum, http://australianmuseum.net.au/), including (i) potential pathways of exposure to contaminants of concern (e.g. habitat preferences, temporal movement and/or migration patterns, feeding mode, and trophic level in the food web), and (ii) life history characteristics, such as age, size, weight and sex, that may affect such exposure pathways as well as the responses to the contaminants of concern.

Selection of suitable fish biomarkers To conduct a systematic review and meta-analyses of the global literature of the use of biomarkers in fish health assessment, we followed an established protocol (PRISMA [8], Fig 2). Specifically, we conducted a thorough literature search to develop a database of fish biomarkers in order to assess their potential use in Gladstone Harbour. The search was performed in Web of Science™ in August 2016 and covered the years 1980–2016. The search included the following terms: fish, biomarkerÃ, pollutÃ, health, conditÃ, bioaccumulÃ, metalÃ, aluminÃ, cadmium, copper, gallium, lead, selenium, and zinc. Additional records were identified through other sources (e.g. reports in the grey literature, etc.). Following the removal of duplicate records, the

Fig 2. PRISMA flowchart providing the steps of data collection for the systematic review of fish biomarkers to assess fish health. The review focussed on the contaminants of concern identified for Gladstone Harbour (Australia). https://doi.org/10.1371/journal.pone.0174762.g002

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remaining publications were screened based on study organisms and contaminants of concern. Records that did not examine the effects of contaminants of concern on coastal and/or marine fish were subsequently removed. Studies on freshwater fish species were excluded given that the speciation and behaviour of metals, and consequently the uptake pathways and sites of bioaccumulation, differ markedly in fresh and marine waters [49–51]. Full text articles were obtained for the remaining records where possible, and assessed for eligibility for inclusion in the qualitative synthesis of the potential use of fish biomarkers for assessing fish health in Gladstone Harbour. Criteria for exclusion included non-fish, non-priority contaminants, in vitro studies, pathways of metal application not environmentally relevant (i.e. injections), metal concentrations not moni- tored (field), measured (lab), or significantly different between treatments, scientific name of fish species not given, full text unavailable, conference abstract only, and text not in English. To identify potential suitable biomarkers for fish health assessment in Gladstone Harbour, the findings of eligible papers were tabulated against individual fish species for the contaminants of concern.

Results and discussion Definition of biomarkers Based on our review of the international literature [7, 52–59], we agree with previous authors that definitions of biomarkers are rather diffuse (S1 Table) [5, 6, 9]. For example, many definitions of biomarkers of exposure include both body burden as well as early response to contaminants. Simi- larly, definitions for biomarkers of effects generally refer to biochemical and physiological alterations resulting from exposure to a contaminant, but sometimes also include changes ranging from molecular, behavioural, and up to ecosystem level. To ensure consistency with the global literature on fish health assessment, we developed the following clear working definitions of biomarkers for this study (see also Figure 3 in [5]): Bioaccumulation markers = Analytical/chemical indicators inside an organism or its products (also referred to as body burden) [5], Biomarkers of exposure = Markers which indicate an early biochemical response has occurred following exposure of an individual or organism to a contaminant [3, 59], Biomarker of effect = Measurable biochemical, physiological or other alterations within tissues or body fluids of an organism that can be recognized as associated with an established or possible health impairment or disease [3, 5], Biomarker of susceptibility = Inherent or acquired ability of an organism to respond to the challenge of exposure to a specific xenobiotic substance, including genetic factors and changes in receptors which alter the susceptibility of an organism to that exposure [5]. In our systematic review, we specifically focus on bioaccumulation markers, biomarkers of exposure, and biomarkers of effect.

Contaminants of concern in water and sediment Twenty-seven facilities listed their emissions in the NPI database in the Gladstone region in 2014/2015 (S2 Table); the water treatment plant in Agnes Water is the only one not located around Gladstone Harbour and is not further considered. Of the 93 substances that are required to be reported in the NPI, 46 are emitted into the air (44), water (22), and land (14), and 25 are transferred by these 26 facilities (S3 Table). Based on the NPI inventory and volumes released into water each year, potential contaminants of concern for Gladstone Harbour include nutrients (ammonia; total nitrogen, TN; total phosphorus, TP), heavy/trace metals (arsenic, As; cadmium, Cd; chromium, Cr; copper, Cu; lead, Pb; manganese, Mn; nickel, Ni; and zinc, Zn), chlorine, cyanide, and fluoride (Table 1).

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Table 1. Potential contaminants of concern in Gladstone Harbour based on emission and monitoring reports. Contaminants in bold are those prioritised as contaminants of concern for Gladstone Harbour, and are further considered in identifying suitable fish biomarkers for assessing fish health. Potential contaminant of concern Information References Emission Monitoring Ammonia X X [10, 15, 21, 22, 24] Total nitrogen (TN) X X [10, 15, 21, 22, 24] Total phosphorus (TP) X X [10, 15, 21, 22, 24] Chlorophyll a X [24] Turbidity X [10, 21, 22, 24] Chlorine X [15] Cyanide X X [15, 18, 19, 26] Fluoride X X [15, 18, 19, 26, 27] Aluminium (Al) X [10, 17±24, 26, 27] Arsenic (As) X X [10, 15, 17±24, 26, 27] Cadmium (Cd) X X [10, 15±24, 26, 27] Chromium (Cr) X X [15, 17±24, 26, 27] Cobalt (Co) X [17, 21, 22, 24, 27] Copper (Cu) X X [10, 15±24, 26, 27] Gallium (Ga) X [17, 21, 22, 24] Iron (Fe) X [17±19, 21±24, 26, 27] Lead (Pb) X X [10, 15, 17±19, 21±24, 26, 27] Manganese (Mn) X X [10, 15±18, 21, 22, 24, 25, 27] Mercury (Hg) X [17±19, 21±24, 26, 27] Nickel (Ni) X X [10, 15±24, 26, 27] Selenium (Se) X X [15, 17±22, 24, 26, 27] Zinc (Zn) X X [10, 15±20, 22±24, 26, 27] Tributyltin (TBT) X [18, 19, 23, 26, 27, 31] Polycyclic aromatic hydrocarbon compounds (PAHs) X [10, 18, 19, 23, 24, 26, 27] Polychlorinated biphenyls (PCBs) X [18, 23, 27, 31] Chlorinated hydrocarbons X [18, 23, 27, 31] Semi volatile organic compounds (SVOCs) X [18, 23, 27, 31] Total petroleum hydrocarbons (TPHs) X [18, 27, 31] Benzene, toluene, ethylbenzene and xylene (BTEX) X [18, 27, 31] Organochlorine and organophosphorus pesticides X [18, 27, 31] Herbicides, carbamate pesticides, and insecticides X [18, 27, 31] https://doi.org/10.1371/journal.pone.0174762.t001

Monitoring information for Gladstone Harbour water and/or sediment was available for the following contaminants: nutrients; heavy/trace metals; tributyltin (TBT); chlorine, cyanide and fluoride; polycyclic aromatic hydrocarbons (PAHs); polychlorinated biphenyls (PCBs); chlorinated hydrocarbons; semi-volatile organic compounds (SVOC); total petroleum hydrocarbons (TPHs); benzene, toluene, ethylbenzene and xylene (BTEX); organochlorine and organophosphorus pesticides; and herbicides, carbamate pesticides and insecticides (Table 1) [10, 16–31]. In contrast, we were unable to find monitoring information for pharmaceuticals and personal care products. These contaminants were not further assessed to identify contaminants of concern for this review, but if present in Gladstone Harbour may contribute to fish biomarker responses [60, 61]. In Gladstone Harbour, aqueous concentrations of nutrients (e.g. TN, TP) and associated indicators (e.g. turbidity and Chl a) have been reported above national guideline values [10, 21, 24, 62]. While potential contaminants of concern for the Gladstone Harbour environment

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(Table 1), we did not further consider nutrients given that potential impacts likely manifest themselves through changes in aquatic food web structure rather than eco-toxicological pathways. Furthermore, monitoring information on specific N and P constituents rather than TN and TP is required to assess potential eco-toxicological effects (e.g. nitrite [63], nitrate [64]). Based on emission and concentration information in the environment (S3–S5 Tables), the metals aluminium (Al), Cd, Cu, gallium (Ga), Pb, selenium (Se), and Zn were prioritised as contaminants of concern for Gladstone Harbour (Table 1). Concentrations of these metals in water and/or sediment were generally elevated in inner harbour sites compared to reference sites, with those of Al, Zn, and Cu on occasion exceeding their respective national guideline values (S4 and S5 Tables) [16, 17, 19–24, 27–31]. In contrast, elevated concentrations of As, cobalt (Co), Cr, iron (Fe), mercury (Hg), Mn, and Ni are thought to be derived from natural rather than anthropogenic sources [10, 16–27], and are not considered contaminants of concern. In addition, national guideline values for Hg were exceeded in only two out of 1,295 sediment samples over seven years of monitoring [17, 19, 23, 27, 29–31]. Tributyltin (TBT) concentrations in Gladstone Harbour sediment have exhibited a generally decline between 2001 and 2012 to levels well below the national guideline values (S4 and S5 Tables) [19, 27, 31]. Since 2005, TBT has been detected in only five out of 870 sediment samples with concentrations well below the national guideline values [27, 31]. Concentrations of TBT in the water column have not been measured since 2002 [19], because the environmental risk of TBT was expected to decline with the chemical being phased out in the 2000s [18]. Hence, for this study TBT was not considered a contaminant of concern (Table 1).Fluoride, chlorine and cyanide are emitted by industry around Gladstone Harbour (S3 Table), but monitoring information is only available for fluoride and cyanide (S4 and S5 Tables). These inorganic elements are not considered a contaminant of concern for this study (Table 1), as aqueous concentrations of cyanide were all below reporting limits, while those of fluoride were considered to have a low likelihood of adverse ecological effects [19, 27]. Sediment PAHs concentrations in Gladstone Harbour were considerably lower than their low national guideline values, and showed no exceedances of these values in four studies from 2005 to 2012 (S6 Table) [19, 23, 27, 31]. The most recent study detected 11 individual PAHs in 12 to 75% of sediment samples, respectively, with the maximum sediment concentration of total PAHs 59 times lower than the low sediment guideline value [27]. The highest total PAHs concentration reported for Gladstone Harbour sediment was still a magnitude lower than the low national sediment guideline value [31]. Hence, PAHs were not considered a contaminant of concern for this study (Table 1). Polychlorinated biphenyls (PCBs), chlorinated hydrocarbons and SVOCs were not detected in Gladstone Harbour sediment in surveys conducted in 2009 (S7 Table) [31]. Long chain TPHs were more frequently detected than short chain TPHs, but total TPHs concentrations were almost four times below the low sediment guideline value (S8 Table) [27, 31]. Very few detections of BTEX have been reported (S8 Table) [27, 31]. Combined, the low prevalence and concentrations of these compounds excluded them as a contaminant of concern for fish health assessments (Table 1). Finally, organochlorine and organophosphorus pesticides, herbicides, carbamate pesticides and insecticides have not been detected in the sediments of Gladstone Harbour (S9–S11 Table) [27, 31]. These two studies analysed more than a 1,000 sediment samples for over 60 different pesticides. Hence, for this study these contaminants were not considered contaminants of concern (Table 1).

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Selection of suitable fish species A total of 232 fish species were identified for locations in and around Gladstone Harbour from both fisheries dependent [35–39] and independent [40–44] studies, as well as from catch data from the Queensland Government shark control program [44] (S12 Table). Scientific names were not available for some species, potentially resulting in a higher number of species listed than actually present. Based on catch and abundance records, 20 fish species were identified as potentially suitable for biomarker studies (Table 2). This list includes Sand Whiting (Sillago ciliata), as earlier surveys report this as the most prevalent fish caught by recreational fishers between 1990 and 2004 [65]. The potential pathways of exposure to contaminants of concern are likely to differ for at least some of these 20 fish species, based on habitat use, migration and movement patterns, and feeding modes and trophic levels (Table 2) [46–48] (http://www.fishbase.org/; http:// australianmuseum.net.au/). Only one of these 20 species, namely Scomberomorus queenslandicus, appears to be restricted to one habitat (coastal), with all other species occurring in two or more habitats. This multi-habitat use is reflected in the migration patterns of the 15 species for which this information is known, with amphidromous, catadromous, diadromous, and oceanodromous patterns all being represented. Only Lethrinus laticaudis and S. ciliata are considered non-migratory. Movements and feeding modes of all but five species, namely Carchar- hinus melanopterus, Eleutheronema tetradactylum, Herklotsichthys castelnaui, Mugil cephalus, and S. queenslandicus, are demersal. All 18 species for which the trophic level is known are carnivores, except for Liza argentea and M. cephalus. Information on life history characteristics was not as readily available for these 20 fish species (Table 2) [46–48] (http://www.fishbase.org/; http://australianmuseum.net.au/). Maximum sizes for individual species ranged from 20 cm Standard Length (SL) (Ambassis marianus) to 200 cm Total length (TL) (C. melanopterus, E. tetradactylum and Lates calcarifer). Many of the 14 species for which weight and/or age information is available have maximum ages of 10 years or more. Labile patterns of sexual development such as protandry and protogony occur in at least five of the 20 species. This information, combined with our systematic review on fish biomarkers (see next section), was used to identify fish species suitable for biomarker studies that could be used to assess fish health in Gladstone Harbour.

Selection of suitable fish biomarkers Our systematic review of the literature identified a total of 5,862 publications, including 832 duplicate records (Fig 2; S1 Text). Following screening of the 5,030 records on study organisms and contaminants of concern, 981 records remained for further assessment of eligibility. Based on availability of full text articles and other eligibility criteria, a total of 151 publications were included in the qualitative synthesis of fish biomarkers. In general, the response of fish biomarkers are assessed using three different types of exposure studies, namely (i) in fish har- vested from contaminated compared to reference field sites, (ii) in caged fish exposed to contaminated versus reference field conditions, and (iii) in fish exposed to controlled laboratory conditions (including toxicity testing) of contaminated water, sediment or food items. Bioaccumulation markers. Our literature review identified a total of 118 publications on bioaccumulation markers for the identified contaminants of concern (Al, Cd, Cu, Ga, Pb, Se, and Zn) in coastal and marine fish (S13 Table). This includes bioaccumulation studies for seven out of the 20 fish species suitable for biomarker studies in Gladstone Harbour (Table 2), namely A. australis, A. berda, E. coioides, L. equulus, M. cephalus, P. fuscus and P. indicus [66– 75]. Of these 118 studies, 72 were field studies with wild or caged fish collected from contaminated and reference sites, and 46 were controlled laboratory exposure studies.

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Table 2. Exposure and life history characteristics of twenty fish species abundant in and around Gladstone Harbour. Abbreviations: TL = total length, SL = standard length, FL = fork length, ID = insufficient data. Fish species Exposure characteristics Life history characteristics Scienti®c name Common Habitat Migration Movement Feeding Mode / Size Weight Sex Name Trophic level and Age Acanthopagrus Yellow®n Coastal, Diadromous, pre- Schooling, Carnivore (worms, max 65cm max 3.7± Protandrous. australis Bream estuarine spawning from demersal molluscs, TL 4.5kg; 14 Sexually mature river to coast crustaceans, yrs at 3±4 yrs and echinoderms, 22cm TL ascidians, and small ®sh) Acanthopagrus Pikey Bream Marine, Oceanodromous Demersal Carnivore (worms, max 90cm max 3.2kg; Protandrous. Sex berda freshwater, molluscs, TL; 14 yrs change at 19.1 TL brackish crustaceans, common and 1.95 yrs echinoderms, and 35cm TL small ®sh) Ambassis Maclaey's Freshwater, ID Schooling, ID max 10cm ID ID marianus Glass®sh brackish demersal SL; common 6cm SL Carcharhinus Blacktip Marine, Amphidromous Reef Carnivore (Prefers max ID (mis- Viviparous, melanopterus Reef Whaler brackish associated ®shes but also 200cm TL reported as placental, mature pelagic crustaceans, 13.5 kg in at 90-120cm TL cephalopods and FishBase) other molluscs) Eleutheronema Blue Freshwater, Amphidromous Loose schools, Carnivore (mainly max max 145kg Protandrous, tetradactylum Thread®n inshore, pelagic-neritic on pony®sh, other 200cm males at 24±47 estuarine, ®sh, crustaceans, TL; cm FL, intersex at marine molluscs) common 25±46 cm FL and 50cm TL females at 28±72 cm FL Epinephelus Goldspotted Brackish, ID Solitary, Carnivore (small max max 15kg; Protogynous. coioides Rockcod marine, rocky demersal ®shes, shrimps, 120cm TL 22 yrs Sexually mature sea beds, cephalopods and (25±30 cm) coral reefs crabs) Epinephelus Long®n Marine, reef- ID Solitary, Carnivore (shrimp, max 40cm ID ID quoyanus Rockcod associated demersal small ®shes, TL worms and crabs) Herklotsichthys Southern Estuarine, Coastal waters to Schools, ID max 20cm ID Oviparous castelnaui Herring marine upper estuaries pelagic-neritic SL, common 14cm SL Lates calcarifer Barramundi Freshwater, Diadromous, Demersal Carnivore (®shes, max max 60kg; Protandrous. estuarine, freshwater to shrimps, cray®sh, 200cm 20 yrs Sexually mature coastal estuaries (males) crabs and aquatic TL, at 55 cm TL and insects) common 3±5 yrs (males), 5 150cm TL yrs (females) Leiognathus Common Freshwater, Amphidromous Schooling, Carnivore Max 28cm ID ID equulus Pony®sh brackish, demersal (polychaetes, small TL, marine crustaceans, small common ®shes and worms) 20cm TL Lethrinus Grass Marine, Non-migratory Schooling, Carnivore (®sh and max 56cm ID ID laticaudis Emperor brackish, demersal crustaceans). TL, reef- common associated 35cm TL (Continued)

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Table 2. (Continued)

Fish species Exposure characteristics Life history characteristics Scienti®c name Common Habitat Migration Movement Feeding Mode / Size Weight Sex Name Trophic level and Age Liza argentea Goldspot Freshwater, Catadromous Schooling, Omnivorous ®lter max 45cm ID Oviparous Mullet brackish, demersal feeder (detritus, TL, marine micro-algae, common ®lamentous algae, 18.5cm and benthic TL organisms) Lutjanus Mangrove Freshwater, Oceanodromous Demersal Carnivore (®shes, max 150 max 14.5 ID argentimaculatus Jack estuarine, crustaceans) cm TL, kg, 39 yrs marine, reef- common associated 80 cm TL Lutjanus Stripey Coastal, ID Schooling, Carnivore max 40cm max 20 Multiple spawner carponotatus Snapper marine, reef- demersal (zoobenthos, TL, yrs associated benthic common crustaceans, ®sh) 30cm TL Mugil cephalus Sea Mullet Freshwater, Coastal spawning Schooling, Omnivorous ®lter max max 12kg, Sexually mature estuarine, migrations benthopelagic feeder 100cm 16 yrs at 3 to 4 yrs marine (phytoplankton, SL, macroalgae, common detritus, and 50cm SL benthic organisms) Platycephalus Dusky Estuarine, ID Demersal, Active foragers, max at least Gonochoristic or fuscus Flathead marine regular contact ambush predators, 120cm TL 15kg protandrous. with bottom (small ®sh, small Sexual mature at crustaceans, 1.2 yrs and 47cm cephalopods, and (males), and at polychaete worms) 2±5 yrs and 56.8cm (females) Platycephalus Bartail Brackish, Oceanodromous Demersal, Carnivore (®sh, max max 3.5kg Mature at 40cm indicus Flathead marine, reef regular contact benthic 100cm TL associated with bottom crustaceans) TL, common 60cm TL Pomadasys Barred Estuarine, Spawners form Demersal Carnivorous (®sh, max 80.0 max 6kg Oviparous, length kaakan Javelin inshore, shoals near river crustaceans) cm TL, at maturity 35cm marine, reef- mouths during the common TL associated winter 50.0 cm TL Scomberomorus School Coastal Oceanodromous Schooling, Carnivore max max 12.2 ID queenslandicus Mackerel seasonal inshore pelagic (zooplankton, ®sh, 100cm kg migration benthic FL, crustaceans, common cephalopods) 50.0 to 80.0 m FL Sillago ciliata Sand Estuarine, Non-migratory Schooling, Carnivores max 51cm max 1.4 Sexually mature Whiting coastal, demersal (benthic TL kg, 22 yrs at 24 cm FL marine polychaetes, (males) and 26 crustaceans, and cm FL (females) molluscs) https://doi.org/10.1371/journal.pone.0174762.t002

Bioaccumulation was determined primarily in gill, liver and muscle tissues, with whole fish bodies and other tissues such as brains, intestines, and gonads used less often. The majority of field studies (45 studies) examined bioaccumulation in muscle tissue, mostly to determine potential risks of fish consumption to humans. Surprisingly, a large number of field studies (47 studies) did not provide information on the specific life history stages examined. In contrast,

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in laboratory studies liver tissue (29 studies) and juveniles (33 studies) were most commonly analysed, to examine detoxification, metabolism and secretion of metals in life history stages considered most vulnerable to contaminant exposure. The exposure pathways most commonly examined were sediment contamination in field studies (59 studies) and aqueous contamination in laboratory studies (33 studies). The most common analytical techniques to measure metal bioaccumulation in fish tissues were inductively coupled plasma mass spectrometry (ICP-MS), inductive coupled plasma atomic emission spectrometry (ICP-AES) (also referred to as inductively coupled plasma optical emission spectrometry, ICP-OES), and atomic absorption spectroscopy (AAS). Bioaccumulation factors (BAFs) for individual fish species, calculated as the metal concentration in the organism (or tissue) divided by that in the sediment or water, were reported in only six out of the 118 studies [76–81]; none of these were on fish species identified as suitable

for biomarker studies in Gladstone Harbour. Bioaccumulation factors for sediment (BAFsed) and water (BAFwater) were mostly determined for Cd, Cu and Zn, with BAFsed values for Cd ranging from 0 in Cathorops spixii [76] to 521 in Liza microlepis [79], for Cu from 0.13 for Stro- mateoides argenteus [81] to 304 for Liza saliens [80], and for Zn from 0.21 in Johnius belengeri [78] to 447 in L. saliens [80]. The values for BAFwater for Cd ranged from 1,048 in Cynoglossus sinicus to 21,333 in Trypauchen vagina, for Cu from 987 for Argyrosomus argentatus to 2,242 in Setipinna taty, and for Zn from 371 in Johnius belengeri to 1,651 in Leiognathus rivulatus [78]. The high values for BAFsed and BAFwater for Cd, Cu and Zn suggest a high bioaccumulation potential for these metals, in fish muscle and liver tissue in particular. Of the seven contaminants of concern, bioaccumulation of Cd, Cu, Pb and Zn in fish tissues was most often examined, in both field and laboratory studies. In contrast, bioaccumulation of Al and Se was examined much less frequently, or in the case of Ga not at all. Metal bioaccumulation, however, does not always reflect environmental metal concentrations [66, 67, 83, 84], and can vary with fish species [67, 74, 82, 85–87], tissue [66, 67, 73, 86], and life history stage [70, 82], with exposure pathways [82, 88, 89], with season [90, 91], and can be influenced by metal speciation and bioavailability [73, 85, 92]. Hence, when using biomarkers to assess fish health, metal bioaccumulation should be used in conjunction with other biomarkers to indicate whether biological responses are related to bioaccumulation. Biomarkers of exposure. Biotransformation enzymes: Phase I. Biotransformation is the process by which an organism alters a xenobiotic compound to enable its excretion. Phase I reactions are the major pathway for the biotransformation of lipophilic compounds and include oxidative, reductive, and hydrolytic reactions where a polar group is either introduced or unmasked (via the addition of an oxygen atom), rendering the xenobiotic molecule less active and more water-soluble and allowing it to be more readily excreted. Most Phase I reactions are catalysed within the microsomal mono-oxygenase (MO) enzyme system and are dependent on the heme protein cytochrome P450 (cyt P450). These heme proteins are located predominantly in the liver, but also present in other fish organelles and tissues [5]. Cyt P450 isozyme induction is triggered when specific xenobiotic compounds bind to the aromatic hydrocarbon (Ah) receptor protein complex and the heat-shock protein 90 (HSP 90), causing HSP 90 to be released. Total cytochrome P450 (cyt P450). Exposure to certain xenobiotic compounds, such as PAHs and PCBs, can show a strong and highly specific induction in the cyt P450 isozymes and a significant corresponding elevation in total cyt P450 levels (S14 Table). In L. calcarifer, exposure to different metal concentrations in sediment did not result in differences in cyt P450 levels [93]. The use of total cyt P450 as a biomarker, however, has been somewhat superseded by techniques that target specific isozymes such as cytochrome P450 1A (CYP1A) which allow

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more specific discrimination of causal agents. Hence, total cyt P450 was not considered a suitable biomarker for fish health assessments in Gladstone Harbour. Cytochrome P450 1A (CYP1A). Cytochrome P450 1A is a class of cyt P450 isozymes responsible for the biotransformation of a number of compounds including PAHs, halogenated aromatic hydrocarbons (HAHs), PCBs, and dioxins. CYP1A protein levels respond to specific xenobiotic compounds and are generally more responsive to contaminants than other CYP isozymes. CYP1A protein levels can be determined cheaply via immunological tests such as enzyme-linked immunosorbent assays (ELISA) or via histochemical techniques. Xenobiotic- related elevations in CYP1A proteins are usually preceded by an increase in CYP1A mRNA which may also be used as a biomarker. Four different methods have been used to assess cytochrome P450 1A (CYP1A) activity as a biomarker of fish health when exposed to metal contamination; all of these studies have used sediment or water as the toxicant pathway (S14 Table). CYP1A activity has been measured in liver, gonads, gills and skin. CYP1A activity in gonads and skin did not change in response to metal contamination [86, 94]. A single study measuring CYP1A activity (using immunoreac- tivity) in gills found that enzyme activity varied with cell type and not site [95]. Assessment of CYP1A activity in the liver using the same method also revealed no significant differences in CYP1A activity between sites contaminated with metals, even though an upregulation of the cyt P450 system was detected in the same tissue using the EROD bioassay [95]. The authors [95] proposed this difference was due to immunochemical assays being less sensitivity than catalytic assays. Ten studies measured CYP1A in the liver using catalytic activity (8 studies) and mRNA concentrations (2 studies) (S14 Table). Seven studies identified a significant positive or negative change in CYP1A activity when fish were exposed to contaminated sediment. CYP1A activity in fish studied in situ was higher when exposed to sediments with elevated metal concentrations; the differential activity of this enzyme could be used to separate effects of organic contamination from metal contamination [96, 97]. The changes in CYP1A activity, however, did not necessarily correlate with metals levels in the water column or metals accumulated in fish tissue [86], as shown by the mixed response observed in some studies [97, 98]. The absence of a relationship between CYP1A activity and metal concentrations is possibly due to the sediments also being contaminated with organic contaminants which are known to induce the CYP1A enzyme due to its role in detoxification of PAHs [96, 97, 99]. Alternatively, the absence of a correlation between cytochrome c system and total concentrations of all metals in the sediment may be due to this enzyme having metal specific responses. For example, it is suppressed by Hg and Cd [100, 101], while Cu induces the system [95]. This complex relationship between CYP1A activity and metals means the suitability of this enzyme as a biomarker is dependent on the ability to resolve responses to individual metals from the suite of metals present in the ecosystem, as is the case in Gladstone Harbour. In addition, CYP1A activity is influenced by season, tissue, life stage and sex and importantly, salinity if estuarine fish species are the target species. Therefore fish health assessments based on changes in the activity of this enzyme would have to employ a strict criteria for sampling to ensure this variability was accounted for [94, 97]. Ethoxyresorufin O-deethylase (EROD) and aryl hydrocarbon hydroxylase (AHH). Ethoxyre- sorufin O-deethylase (EROD) activity is a measure of catalytic response in fish, and is a sensitive indicator of the inductive response of the cyt P450 system. EROD activity is indicated by changes in the fluorescence of the metabolic product resorufin. Aryl hydrocarbon hydrolase (AHH) catalytic activity can be used as a measure of the response of the CYP1A isoenzyme, by determining the hydroxylation of benzo[a]pyrene. Both EROD and AHH are sensitive biomarkers when measured in the livers of fish, especially when used in conjunction with other

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biomarkers such as CYP1A and CYP1A mRNA. The other sub units of the cytochrome 450 system are measured using the following assays pentoxyresorufin-O-depentylase (PROD) measures the catalysis of cytochrome P450 CYP2B, 7-benzyloxyresorufin-O-depentylase (BROD) measures the catalytic activity of the cytochrome P450 subfamily CYP3A and 7-methoxyresorufin-O-methoxyresorufin (MROD) cytochrome P450 CYP1A2. A single study assessed EROD activity in the gills of fish (Solea senegalensis) exposed to sediment contaminated with metals and PAHs showed a significant decrease, correlated to heavy metals in preference to PAHs [95]. Although this is a single study, these results suggest that this combination of tissue and biomarker may be suitable for assessing fish health in relation to metals in systems also containing PAHs. EROD activity in the liver of fish significantly increased as a result of metals exposure in 13 of the 21 of the studies reviewed, while six reported no response and six a mixed response (S14 Table). EROD activity in the liver is heavily influenced by PAHs, due to it being a detoxification pathway for these chemicals [96, 97, 99, 100]. The majority of studies reporting a positive response in EROD activity, however, were also contaminated with organics. Therefore, these findings do not necessarily confirm EROD activity is indicative of metal contamination. For example, Oliva et al. [95] reported a significant correlation between Cu labile organic fraction in water and EROD, but also reported a significant correlation between EROD activity and phenanthrene-type metabolites in liver in the same fish. Other studies identified a mixed response, where EROD activity was not necessarily highest at the site with the highest metal concentrations [97, 101–103]. This highlights the complexity of elucidating which particular contaminants are driving EROD activity in systems affected by multiple contaminants, particularly combinations of metals and PAHs. To address this complexity, Fonseca et al. [101] applied generalised linear models (GLMs) to describe the biomarker responses, and was able to separate Hg from the other metals as influencing EROD activity [101]. Finally, similar to CYP1A, EROD activity is influenced by life history characteristics such as sex [97]. Hence, EROD is a potentially a suitable biomarker for assessing fish health in Gladstone Harbour, including L. calcarifer [93], but will require careful examination in a combination of field and laboratory studies, and analysis to interpret the responses. Aryl hydrocarbon hydrolase (AHH) catalytic activity was not used in any of the studies reviewed, and is not further considered. The activity of CYP2B and CYP1A2, but not CYP3A, in liver of Scophthalmus maximus increased following exposure to sediment contaminated with Cu, Pb and Zn [99]. In contrast, CYP3A levels increased in liver tissue of fish exposed to sediment contaminated with a suite of metals and PAHs [104]. This discrepancy may be due in part to the different methods of analyses used (BROD vs real time PCR analysis of liver mRNA). The limited number of studies applying MROD, BROD, and PROD to measure biomarker response following metal exposure precludes a recommendation for their use in assessing fish health in Gladstone Harbour. Cytochrome b5 (cyt b5). Cytochrome b5 (Cyt b5) is ‘involved in the cyt P450-mediated bio- transformations through electron donation by NADH via cytochrome b5 reductase’ [5]. This biomarker is not further considered as none of the studies reviewed used cyt b5. NADPH cytochrome P450 reductase (P450 RED).According to Van der Oost et al. [5], ‘Cyt P450 activity depends on reduction of the heme iron by electron transfer from the flavoprotein P450 RED, and in some cases from cyt b5’. P450 RED supplies electrons from NADPH for use in monooxygenase reaction whereas cyt b5 utilises NADP as the electron source [105]. P450 RED activity was not used in any of the studies reviewed, and is not further considered. Biotransformation enzymes: Phase II enzymes and co-factors. Phase II biotransformation involves the attachment (conjugation) of a polar molecule to the xenobiotic parent molecule. The resultant molecule is more water soluble, of higher molecular weight and is more

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effectively excreted. Depending on the type of xenobiotic compound, certain molecules may be directly metabolised by the phase II system; others may require biotransformation via phase I enzymes first. Reduced and oxidised glutathione (GSH and GSSG).Reduced glutathione (GSH) and oxidised glutathione (GSSG) provide the major pathways for conjugation of electrophilic compounds and metabolites. GSH plays a key role in the detoxification of xenobiotic compounds by reacting with compounds, replacing hydrogen, chlorine and nitro groups among others. Thiol status is the ratio between reduced and oxidised glutathione (GSH/GSSG) and has been used as an indicator of oxidative stress. Five studies investigated GSH and GSSH as biomarkers for assessing impact of metals on fish health (S15 Table). Results from field studies vary with no change in GSH/GSSH, a significant decrease, and no response/a negative response in M. cephalus depending on the age of the fish [106–108]. In laboratory toxicity tests, GSH/GSSH in gills and in whole fish (including M. cephalus) increased following Pb exposures [69], while GSH/GSSG levels varied with tissue and life stage following Cd exposure [109, 110]. Specifically, results from the latter two studies indicate GSH/GSSG is biphasic, with increasing activity occurring up until a certain concentration after which levels decline [109, 110]. The return to normal GSH/GSSG levels may be due to compensation by other enzymes such as superoxide dismutase (SOD). This means the activity of this enzyme does not necessarily reflect contamination levels after a prolonged exposure time. Based on the variability in GSH/GSSH response in relation to metal exposure concentration and time, fish age and tissue type, as well as the conflicting field and laboratory results for M. cephalus, we consider this biomarker currently unsuitable for assessing fish health in Gladstone Harbour. Glutathione S-transferase (GST). Glutathione S-transferase (GST) is a catalyst required for the conjugation of electrophilic compounds by GSH [111]. GST provides essential functions in intracellular transport as well as defence against oxidative damage and peroxidative DNA products. Twenty studies have examined the response of GST activity in fish to elevated metals levels in water or sediment (S15 Table). The response of GST activity was variable, with six reporting no response and the remaining 14 reporting positive (9 studies) or negative (5 studies) responses. Additionally, in one study different responses were reported when the same fish population were exposed to the same sediment but under either laboratory or field conditions [112]. This variability is likely due to species-specific responses [83, 101], tissue-specific responses [113], seasonal and annual variations [114] and responses to contaminants other than metals (e.g. organics [97, 98]). For example, GST activity was generally higher in liver tissue in Cynoglossus arel from polluted locations, but not so in Acanthopagrus latus even though hepatic metal bioaccumulation was confirmed [83]. The confirmed metal contamination of marine sediment suggest that these differences are due to different feeding habits, with C. arel a bottom dwelling fish and A. latus a more pelagic species [83]. In addition, tissue-specific responses are potentially related to differential metal accumulation with the liver accumulating 30 times more Cu than the kidney, and twice the concentration of Zn [113]. The site of accumulation is related to exposure route for the metals, with the liver mainly representing metals accumulated through diet while the kidney would generally reflect metals taken up through the gills [113]. Even within the same tissue (e.g. gills), however, GST levels have shown an increase [115], a decrease [84], and no significant response [83] to environmental metal concentrations. Five studies examined GST response under toxicity testing conditions with Cu only, as well as two with Cd and one with Pb (S15 Table). Similar to field studies the responses to Cu and to Cd, respectively, varied with tissue type and showed a biphasic response meaning it could not

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be used to confirm exposure to higher metal concentrations [109, 110, 116]. In contrast, Pb exposure resulted in a negative response in GST levels in gills and whole juveniles of two different fish species under all concentrations [69]. The absence of a biphasic response to Pb may be due to the metal concentrations not reaching threshold levels for this protein. Overall, GST activity does respond to metal contamination but this response can vary significantly with species, tissue, season, and the presence of other contaminants, under both field and toxicity testing conditions. Importantly, the GST response to contaminants can be low (~2 fold), making it difficult to differentiate increased activity due to contaminant exposure from natural variability in the fish species of interest [97]. Hence, with many factors influencing the induction of GST activity, but in particular the relatively low response of this enzyme when exposed to contaminants, this biomarker was considered unsuitable for assessing fish health in Gladstone Harbour. UDP-glucuronyl transferases (UDPGTs). Uridine 5’-diphosphoglucuronic acid—glucuronyl transferases (UDPGT) are the catalysts responsible for the addition of glucuronic acid to a sub- strate, which is a key pathway for the detoxification and excretion of xenobiotic and endogenous substances. This biomarker is not further considered as none of the studies reviewed used UDPGT. Total glutathione. Five studies used total glutathione to assess fish health when exposed to metals [100, 106, 112, 113, 115] (S15 Table). The response of total glutathione varied with tissue, with no response in the kidney despite confirmed metal accumulation, a positive response in the gills, and a negative one in the liver [113, 115]. The response in either gill or liver was not related with metal accumulation in these tissues [113, 115]. Like GST, total glutathione levels exhibited different responses when the same species was exposed to the same sediment under caged field conditions and controlled conditions [112]. Two other studies found that total glutathione decreased with increasing metal concentrations in the environment, with the same response observed in fish at different life stages [100, 106]. The variability in total glutathione response with exposure conditions and tissues, combined with the low number of studies to assess, would suggest that at this stage total glutathione is not a suitable biomarker for assessing fish health in Gladstone Harbour. Oxidative stress parameters. Oxidative stress parameters are a suite of measures of the oxygen toxicity being suffered by an organism. Oxygen toxicity is caused by reactive oxygen species (ROS) and oxygen free radicals, and is a result of either increased production of these oxidising species, or a significant decrease in production of antioxidants (such as glutathione). Severe oxygen toxicity can lead to enzyme deactivation, lipid peroxidation, DNA damage and eventually apoptosis or cell necrosis. ROS may be produced endogenously, or as a result of the reduction of xenobiotic compounds such as aromatic diols, quinones and hydroxylamines, nitroaromatics, bipyridyls and some transition metal chelates [5]. Superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPOX), are groups of antioxidant enzymes that form part of a cellular defence system that inhibit and detoxify oxyradical formation. SODs are metalloenzymes that catalyse the reaction of superoxide anions into hydrogen peroxide (which is also an ROS). Resultant hydrogen peroxide is then detoxified by CAT and GPOX. CATs enable the conversion of hydrogen peroxide to release molecular oxygen and water and are involved in fatty acid metabolism as well as detoxi- fying xenobiotic ROS. GPOXs facilitate the reduction of a range of peroxides (unlike CAT which only acts on hydrogen peroxide) to alcohols. Where CAT uses a donor hydrogen peroxide molecule in the reduction of another, GPOX requires an additional reductant to achieve this (such a selenium-dependant tetrameric cytosolic enzyme), and commonly utilises reduced glutathione (GSH) as a cofactor. GPOX is crucial in protecting cell membranes from damage via lipid peroxidase (LPOX).

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Superoxide dismutase (SOD). Nineteen studies have examined the influence of metals on SOD activity in fish (S16 Table). One study used a real-time PCR assay and found no significant response of SOD in fish gonads in response to metal contamination [94]. All other studies used bioassays to measure SOD activity in liver, gills, muscle, intestine, kidney, red blood cells, blood, spleen and whole fish. SOD activity in gill tissue decreased in all studies, irrespective of exposure to mixed metals in field studies or single metal in laboratory toxicity tests [49, 84, 110, 117]. Following Cd exposure in toxicity tests, responses in different tissues have been consistent across various tissues (e.g. gill, liver, spleen [117]), as well as inconsistent (e.g. negative in gill, positive in liver [110]). The response of SOD activity to Cd exposure has been described as biphasic [109] as well as potentially polyphasic [110]. Controlled Cu exposures have resulted in decreased SOD activity in gills and liver [116], although responses can differ between tissues in one and the same species (e.g. no response in liver, negative response in gills and intestines) [49]. Activity of SOD in muscle tissue did not change significantly following Cu exposure [116]. In field studies, SOD activity in fish tissue is responsive, albeit variably, to metal concentrations in sediment and water (S16 Table). Four studies reported no significant change in SOD activity, six showed a mixed response and four reported significant positive responses. For example, SOD activity in the muscle of fish increased when exposed to sediment contaminated with As, Cd, Cu and Pb [118]. In M. cephalus, SOD activity was lower in individuals exposed to contaminated water [72]. A range of contaminants, however, can affect SOD activity of different species exposed in the same environment, such as Cd, Cu and Zn in Dicentrarchus labrax and PAHs in S. senegalensis and Pomatoschistus microps [102]. Despite the general variable results in both field and laboratory studies, the consistent results for SOD activity in gill tissue suggest that this biomarker may be suitable to assess fish health in Gladstone Harbour. Catalase (CAT). Twenty eight studies have assessed the response of CAT activity to metal contamination, with seven being toxicity tests and 22 field assessments (S16 Table). Following controlled Cu exposure CAT activity did not change in gill tissues [49, 116]. In contrast, controlled Cd exposure resulted in a decrease in CAT activity in gill, liver and spleen tissue in Synechogobius hasta [117], but no response in gill, kidney or liver tissue in Paralicthys olivaceus [110]. These differences may be due in part to different Cd concentrations being examined rather than species-specific responses. Notwithstanding, differences in CAT activity were identified between M. cephalus and Terapon jarbua following controlled Pb exposure [69]. Results from these toxicity tests show that CAT activity can be species, tissue, metal and concentration dependent [49, 69, 110, 116, 117]. Field studies demonstrate a variety of responses in CAT activity (S16 Table). Sixteen of these studies used bioassays to assess CAT activity in liver tissue, of which only ten reported either a significant increase or decrease in CAT activity at contaminated sites [100, 106, 107, 112, 113, 115, 118–120]. For example, CAT activity in the liver of D. labrax was correlated with Cd, Cu and Zn in sediment [102]. In M. cephalus, hepatic CAT activity showed a significant negative response to contaminated water [72], but was also shown to be influenced by season [114]. In addition to season, the variability in CAT responses documented in field studies is likely due to salinity, location, and species [98, 102, 103, 121, 122]. Duration of exposure also affects CAT activity, with response in the liver of S. maximus correlated with Cd, Hg, Cr, Mn, Ni, Pb and V on day 7 of exposure to sediment, but with Cd, Mn, Ni, Pb and V by day 21 [103]. The variable response of CAT to specific metals or combinations of metals under both field and laboratory conditions suggests that this biomarker is not suitable to assess fish health in Gladstone Harbour. Glutathione peroxidase (GPOX). Twelve studies assessed glutathione peroxidase (GPOX) activity in fish in response to increased metals concentrations (S16 Table). Controlled

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laboratory exposures showed that GPOX activity decreased significantly in the gills, liver and spleen but not the kidneys following exposure to Cd [110, 117]. Of the 13 field studies, five reported no change in hepatic GPOX in different fish exposed to metals, four showed a mixed response, two a positive response, and two showed negative response [72, 83, 96, 102, 106, 112, 113, 115, 123]. In M. cephalus, hepatic GPOX activity decreased significantly following exposure to contaminated water [72]. Like many biomarkers GPOX exhibits seasonal variation and species-specific responses [83, 113, 115]. In addition, GPOX activity in kidneys appears to respond preferentially to Cu concentrations in the sediment [113], which may reduce its effectiveness as a biomarker in cases where Cu is not a contaminant of concern. Based on its inconsistent responses to metals, in combination with seasonal and species variability, this biomarker is not recommended for assessing fish health in Gladstone Harbour. Glutathione reductase (GRED). Glutathione reductase (GRED) maintains homeostasis between the reduced and oxidised forms of glutathione, namely GSH and GSSG, particularly under oxidative stress conditions. GRED uses the oxidation of NADPH to NADP+ to catalyse the reduction of GSSH to GSH. NADPH levels can be measured spectrometrically and can therefore be used as a proxy for GRED activity. Twelve studies in our review examined the response of GRED activity to different metal concentrations (S16 Table). In the one toxicity study, GRED activity showed no response in muscle and gill tissue, and a mixed, biphasic response in liver tissue following Cu exposure [116]. In most of the fish species studied in the field, GRED activity did not significantly change when exposed to contaminated sediment [83, 106, 108, 113]. Only a few field studies reported a significant correlation between hepatic GRED activity and metal concentrations in the environment, including for M. cephalus [72, 122]. In species showing a mixed response there was no correlation between the GRED activity and metal concentrations [83]. A few studies found significant responses to accumulation, such as higher GRED activity in gill tissues with activity levels correlated with Zn, Cd and Cu accumulation on gill tissue [115]. In cases where changes in GRED activity were reported, the response varied with tissue type, exposure time, and seasonality [104, 113, 115]. Based on these mixed findings, GRED was considered unsuitable as a biomarker for assessing fish health in Gladstone Harbour. Non-enzymatic antioxidants. Non-enzymatic antioxidants such as vitamins have been used as biomarkers although relevant studies are very scarce [5]. Indeed, none of the studies reviewed used non-enzymatic antioxidants and these biomarkers are not further considered. Biochemical indices of oxidative damage. Lipid peroxidation (LPOX) is the oxidation of polyunsaturated fatty acids as a consequence of oxidative stress, and can be quantified by the measurement of degradation products such as aldehydes, acetone and malondialdehyde. Twenty three studies using different methods assessed changes in LPOX activity in fish in relation to metal concentrations (S16 Table). Five field studies identified no response in LPOX activity of fish when exposed to contaminated sediment [102, 118, 124–126]. Several field studies have shown a significant increase in LPOX activity in the blood of S. senegalensis, Lutjanus russellii and Pomadasys hasta when exposed to contaminated sediment [120, 127]. This relationship was not observed, however, when the fish were exposed to the same sediment under laboratory conditions [127]. Other field studies showed species- and tissue-dependent responses of LPOX activity, with differences in gill and liver tissues between the pelagic A. latus and the demersal C. arel collected from the same locations [83, 128]. For example, hepatic LPOX activity showed a mixed response to contaminant levels in A. latus but no response in C. arel [83]. Sea- sonal variation in LPOX activity has also been detected in various field studies [115, 122, 124]. The influence of life history stage was observed in Anguilla anguilla with glass eels showing no response and yellow eels showing a significant positive response when exposed to the same contaminated sediment [106].

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Laboratory toxicity tests showed that LPOX activity in gills, liver or spleen significantly increased when fish were exposed to Pb, Cu or Cd [69, 116, 117]. LPOX activity, however, can vary with tissue, with an increase in gill and liver tissue following exposure to Cd or mixed metals in sediment, but not in kidneys [110, 113]. Similar to field studies, LPOX activity varies with life stage following Cd exposure, with juveniles showing a more sensitive response than various larva stages [109]. The relatively consistent changes in LPOX activity in response to metal exposures in a number of studies, including for M. cephalus [69, 72, 114], suggest this enzyme is a potential biomarker for assessing fish health in Gladstone. However, its suitability will depend on whether species-specific and seasonal responses can be differentiated from those resulting from metal contamination. Selenium-dependent glutathione peroxidase (Se-GPx) was assessed in four studies (S16 Table). Two field studies showed either a lower Se-GPx activity in M. cephalus at sites with contaminated sediment [114], or no response in Se-GPx activity in three estuarine fish species [102]. Exposure of A. anguilla to contaminated sediment in the laboratory resulted in a significant increase in Se-GPx activity in two different studies [100, 112]. However, Se-GPx activity decreased following exposure of the same fish to the same sediment under field conditions [112]. Due to the limited number of studies assessing the effect of metals on Se-GPx activity, combined with conflicting results between laboratory and field tests, this biomarker is not recommended for assessing fish health in Gladstone Harbour. Reactive oxygen species (ROS) were only assessed in a single study (S16 Table). Following controlled Cu exposure in the laboratory, ROS activity in the estuarine guppy (Poecilia vivipara) increased in gill and liver tissue, but showed no response in muscle tissue [116]. The low number of studies precludes recommendation of this biomarker for Gladstone Harbour. Total oxyradical scavenging capacity (TOSC) is a standardised assay that provides a method of assessing the capability of samples to neutralise ROS. By introducing different ROS to a sample at a constant rate and assessing the efficiency of antioxidants, its scavenging capacity can be determined. Two studies, both on A. anguilla, used TOSC to assess fish health when exposed to metals (S16 Table). Exposure to contaminated sediment results in no response under field conditions, but a mixed response under laboratory conditions [112]. In contrast, TOSC activity showed a negative response in the same fish species under field conditions [100]. Hence, this biomarker was not recommended for Gladstone Harbour due to inconsistent and insufficient data. Antioxidant capacity against peroxyl radicals (ACAP) were only assessed in a single study, on the estuarine guppy (P. vivipara) (S16 Table). Following controlled Cu exposure, ACAP response varied with tissue with no response in gills, a positive response in liver and a negative response in muscle [116]. Due to lack of data, this biomarker was not recommended for Glad- stone Harbour. Glucose-6-phosphate dehydrogenase (G6PDH) contributes to maintaining the level of the co-enzyme NADPH, thereby maintaining the level of glutathione to protect cells such as erythrocytes from oxidative damage. Only a single study, on Symphodus melops, used G6PDH to assess the effect of metals and found no response in females but a negative response in males [108]. Due to a lack of data, and the likely interaction of sex and G6PDH response, this biomarker was not recommended for assessing fish health in Gladstone Harbour. Additional oxidative stress biomarkers were examined in a few studies only, namely Acyl- CoA oxidase; methemoglobin; nitric oxide synthase; hypoxia inducible factor, lipid hydroperoxide, conjugated diene; carbonyl proteins; 2-Keto-4-methiolbutyric acid, oxidised proteins and DT-diaphorase (S8 Table). Due to the limited data for these biomarkers they were not recommended for Gladstone Harbour.

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Biotransformation products. This biomarker is not further considered as none of the studies reviewed used biotransformation products. Stress proteins, metallothioneins and multixenobiotic resistance. Stress proteins or heat shock proteins (HSP). Stress proteins protect and regenerate cells in response to stress and harmful conditions. The group consists of heat shock proteins (HSPs), which respond (by increasing synthesis) to heat and other physical and chemical stresses, the glucose-regulated proteins (GRPs) which respond to oxygen or glucose deficiency, and the stressor-specific stress proteins which include metallothioneins (MTs) and heme oxygenase proteins. The eight studies that assessed the response of HSP70 to metal contamination used a range of techniques (S17 Table). Two studies using real-time PCR assays of samples from gonads, skin or liver did not observe a response when fish were exposed to contaminated sediment [86, 94]. In contrast, two other studies using real-time PCR saw upregulation of HSP70 in liver or gills of fish exposed to contaminated sediments, which was confirmed using immunohis- tochemistry and in situ hybridisation [104, 129]. This indicates the suitability of all these methods to asses HSP70 activity. In five of the eight studies HSP70 activity was upregulated, with four assessing the response of fish to contaminated sediment and one examining the response after fish had been exposed to Cd [104, 129–132]. This suggest that this biomarker is suitable for Gladstone Harbour, but further field studies are required to validate its use given the reported seasonal variation in HSP70 activity [133], in combination with the strong influence of climate on HSP70 [5]. The two studies that assessed the response of HSP90 to metal contamination used real-time PCR (S17 Table) [86, 96]. One of these studies reported a response of HSP90 in the skin of fish exposed to contaminated sediment but not the liver [86], while the other found no difference between clean and contaminated fish [96]. Based on the relatively low number of studies on HSP90 and metal contamination, this particular biomarker is not considered a priority for assessing fish health in Gladstone Harbour. However, the reported findings for HSPs in general are promising and these biomarkers could be examined in further detail to assess their suitability in the Gladstone context. Metallothioneins (MT). Metallothioneins (MTs) are a family of proteins essential for the regulation, sequestration and detoxification of metals including Cu, Zn, Cd, Hg and have been shown to be induced by Co, Ni, Bi and Ag. MTs act by intercepting and binding metal ions as they are being taken up by the cell and also by the removal of metals by non-thionein ligands. MTs can be measured by quantitative assays of the protein via IEC-AAS, metal substitution assays and polarographic and immunochemical techniques. In fish, MT activity is greatest in tissues that are most closely involved in uptake, storage and excretion (such as the gills, liver and small intestine). MTs are one of the most studied biomarkers for assessing the effect of metal contamination in fish, with a total of 32 studies reviewed (S17 Table). Thirty-one different MT assays were performed on livers from fish exposed to metals in field samples; most of these studies (83%) reported no change or a mixed response. Only a few studies reported a significant change in hepatic MT levels after exposure to contaminated sediment or water [87, 102, 124, 134], and in some of these the response was not observed in all fish exposed to the same contaminants [87, 102]. In contrast, MT levels exhibited a significant positive response in the gills of three different fish species [128, 129, 135], and in the muscle in two other studies [128, 132]. In laboratory toxicity tests, MT levels increased in Aphanius fasciatus, Atherinops affnis, Gobius niger, Sparus aurata and T. jarbua with increasing Cd concentrations [94, 130, 136–139]. However, upregulation of MTs was not sustained over the entire exposure period, with some MTs levels return- ing to normal levels by the last day of the toxicity test [130, 136–139]. Reported changes in MT levels following Cu toxicity testing were not directly related to Cu concentrations [116, 140].

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Controlled exposures to Zn via food resulted in a significant increase in whole juveniles, carcass and viscera of T. jarbua and A. latus but not in their gills [141]. Combined, these results suggest that MT activity in tissues other than liver may be a suitable biomarker to assess fish health in Gladstone Harbour. The copper transporter gene (Ctr1) mediates the transport of copper into eukaryotes for metabolic processes such as a cofactor for enzymes. The single study identified in our review showed that this biomarker in S. aurata did not change following aqueous exposure to Cu, and the response varied with tissue following dietary uptake of Cu [140]. Hence, Ctr1 was not considered a suitable biomarker for Gladstone Harbour. Multixenobiotic resistance (MXR). Multixenobiotic resistance (MXR) prevents the accumulation of both xenobiotic and endogenous compounds inside the cell, by removing them via an energy-dependent transport protein, Transmembrane P-glycoprotein (PGP). This biomarker is not further considered as none of the studies reviewed used MXR. Haematological parameters. Haematological parameters offer the ability to conduct non- lethal or non-destructive tests. Haematological parameters may be less chemical specific than some other biomarkers, but provide other insights into the general health and physiology of the organism. A number of haematological parameters have been used to monitor pollution induced stress in fish, including plasma enzyme activity, erythrocyte counts, haemoglobin content and haematocrit (S18 Table). Serum transaminases. Measurement of enzymes in the serum (extracellular fluid or plasma) is considered a sensitive indicator of cellular damage. Alanine transferases (ALT or GPT) and aspartate transaminase (AST or GOT) are a group of enzymes responsible for the conversion of amino acids and α-ketoacids. One of the two studies identified in our review (S18 Table) showed no change in ALT/AST levels of Gadus morhua and a mixed response in Platichthys flesus when exposed to the same sediment in the field [97]. The other laboratory study reported a significant increase in ALT/AST levels following dietary exposure to Cd in Rachycentro canadum [142]. The lower number of studies, combined with their inconsistent results, means that these biomarker are not recommended for assessing fish health in Gladstone Harbour. Other haematological parameters. The delta-aminolevulinic acid dehydratase (ALAD) gene encodes for the δ-aminolevulinic acid dehydratase enzyme also known as porphobilinogen synthase [143]. The ALAD enzyme catalyses the second step in heme synthesis. ALAD is expressed in all tissues, but the highest levels of expression are found in erythrocytes and the liver. This biomarker is not further considered as none of the studies reviewed used ALAD. Sodium (Na) and chloride (Cl) are the main ions in the extracellular fluid. Maintaining these ion concentrations is necessary so that membrane potential is maintained across the cell. Membrane potential is critical for generating energy, transmission of nerve impulses and muscle movement. Only one study measured Na and Cl in fish after metal exposure and found no significant change [144] (S18 Table). Hence this biomarker is not further considered for Glad- stone Harbour. Haematocrit is the percentage by volume of red blood cells in the blood, whereas erythrocyte counts refer to the absolute number of red blood cells in given volume of blood. Haemo- globin is an iron based oxygen transport molecule that makes up a large proportion of the red blood cell, it can be easily measured and is expressed as g/dL of blood. All of these parameters have been shown to change in the presence of certain contaminants and can be used as indicators of stress or toxicity (S18 Table). Haematocrit results in two field studies were inconsistent though, with one study reporting no response to metal exposure and the other a significant increase [97, 108]. Following dietary exposure in the laboratory, a significant change was observed when fish were fed Cd but not for Se [142, 145]. Erythrocyte counts did not change following exposure to sediment or water contaminated with metals [97, 127, 142]. In contrast,

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haemoglobin levels in fish increased significantly following exposure to Cu in water [126]. Due to the limited number of studies using haematocrit, erythrocyte counts and haemoglobin as biomarkers, and the inconsistent results for haematocrit and erythrocyte counts, these biomarkers are not recommended for assessing fish health in Gladstone Harbour. Alkaline phosphatase (ALP) is an enzyme that catalyses the dephosphorylation (hydrolysis of phosphate group) of a range of molecules such as nucleotides and proteins. Two studies used ALP activity in fish (S18 Table), with a mixed response in ALP levels in a field study [97], and a significant increase in ALP levels of fish fed Cd [142]. Due to the limited data this biomarker was not further considered for assessing fish health in Gladstone Harbour. Intermediary metabolism parameters in the serum, such as glucose, albumin and total protein, are frequently analysed to examine the nutritional status of fish. Two field studies reported different responses for all three biomarkers to metal exposure [97, 120] (S18 Table). Omar et al. [120] identified a significant decrease in total protein and albumin in fish exposed to contaminants. Beyer et al. [97] identified species specific responses in albumin levels and albumin/protein ratio, and reported no significant change in glucose levels in either G. morhua or P. flesus when exposed to water containing metals. Due to the limited data on these biomarkers, combined with apparent species-specific responses, they are not recommended for assessing fish health in Gladstone Harbour. Immunological parameters. Immunological parameters have not been used often as biomarkers in fish exposed to metal contamination, with the most common ones being proliferating cell nuclear antigen (PCNA) [120, 129, 146], calcium binding proteins (CBP), cell respiratory burst (RB) and total complementary activity of serum (TCAS) [126, 129] (S18 Table). Levels of PCNA in fish from contaminated sites varied across studies, with higher levels recorded in Coris julis [129] and D. labrax [146], but lower in P. hasta [120]. Levels of CBP were lower in fish exposed to contaminated sediment while TCAS and RB levels did not show a response [126, 129]. Fasulo et al. [129] concluded that PCNA and CBP were most likely due to physiological adaptation and not a specific response to contaminants. Levels of RB and TCAS D. labrax did not respond to Cu exposure (in antifouling nets) [126]. Other biomarkers did not respond either [126] suggesting a low toxicity of the antifouling nets rather than insensitivity of the RB and TCAS. The inconsistent results of these few studies suggest that these biomarkers are not suitable to assess fish health in Gladstone Harbour. Additional immunological parameters that have been used include % leukocytes and % thrombocytes [97, 127], stability of lysosomes 1 and 2 [100, 147], lysozyme activity [103, 112, 148], macrophage aggregate activity (MAM), macrophage aggregates (MA), proteomics serum (Pro-S), cytokine transforming growth factor (β1 9TCF-b1), melanomacrophage centres (MMC), thymus volume (TV) and cell viability (CV) (S18 Table). The response of lysozyme activity following metal exposure varied across studies, with a decline in juvenile A. anguilla, an increase in P. flesus, and a mixed response in S. maximus [103, 112, 148]. Similarly, the response of % leukocytes and % thrombocytes differed in G. morhua and S. senegalensis following exposure to sediment contaminated with metals, including when exposed to the same sediment under different conditions [97, 127]. In contrast, both the stability of lysosomes 1 and 2 and MMM showed consistent responses, with the former declining following metal exposure [100, 147], and the latter increasing following exposure to contaminated sediment in the field and Cd exposure in laboratory toxicity testing [117, 148]. Only one study each measured changes in TV, TCF-b1, Pro-S, MAM, MA and CV [72, 103, 145, 147, 149]. Due to the limited number of studies using these immunological parameters these biomarkers were not recommended for Gladstone Harbour. Neurotoxic parameters. Cholinesterase (ChE) are enzymes relevant to neural functions; those with a high affinity for acetylcholin (AChE), and those with affinity for butyrylcholin

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(BChE) [5]. Fish muscle contains both, while fish brains only contain the former. Nine studies used ChE as a biomarker in fish exposed to metals under field conditions [93, 98, 100, 104, 106, 123, 128, 146, 147], with variable responses reported (S19 Table). Most studies measuring ChE activity in the liver did not identify a response in fish exposed to contaminated sediment [100, 112, 147], while other studies showed a mixed or negative response [98, 106]. Activity of ChE in L. calcarifer muscle differed across rivers with different levels of contamination, but was not clearly linked with aqueous or sediment metal concentrations [93]. The response of ChE can vary with tissue following exposure to contaminated sediment, with an increase in S. senegalensis in gill and muscle tissue, but no response in brain tissue [128]. The response of ChE levels in muscle and gill tissue was also variable between studies [93, 123, 128, 146]. This variability in ChE response to metal contamination suggests that it is not suitable as a biomarker to assess fish health in Gladstone Harbour. The neurotransmitters choline acetyltransferase (ChAT) and serotonin, as well as the enzymes tyrosine hydroxylase (TH), involved dopamine synthesis and the serotonin receptor (5-HT3) have been used as fish biomarkers [129, 146] (S19 Table). Levels of both ChAT and serotonin declined, and those of TH increased, in D. labrax exposed to sediment contaminated with metals and hydrocarbons [146]. A decline in serotonin levels was also observed in C. julis exposed to contaminated sediment [129]. The response of 5-HT3 in these two studies, however, varied with an increase in C. julis but no response in D. labrax [129, 146]. Given the limited number of studies these biomarkers were not recommended for Gladstone Harbour. Reproductive and endocrine parameters. Pollution stress has well-known effects on reproductive and endocrine parameters in fish [150], however, only a few studies were identified in our review that specifically examined these biomarkers (S19 Table). Vitellogenin (VTG) is a precursor egg yolk protein found in the blood or haemolymph in females of nearly all oviparous species [151]. VTG levels are highly sensitive to endocrine disrupting chemicals (EDCs), and are affected by exposure to some xenobiotics including PCBs and Cd [5]. Only two studies used VTG as a biomarker in fish exposed to metals. Hepatic vtg transcripts assessed on real- time PCR were higher in male A. fasciatus collected from a site contaminated with heavy metals compared to a more pristine site [94]. In contrast, plasma VTG levels in male P. flesus varied considerable between sites including reference sites [98]. The species-specific gene sequence for vtg has been isolated for one of the priority species identified for Gladstone Harbour, namely L. calcarifer [152–154]. Hence, examining the suitability of this biomarker for Gladstone Harbour may be appropriate if a relationship with fish health can be demonstrated. The endocrine receptor, thyroid receptor alpha (Trα) has also been used as a biomarker to assess fish health but the levels varied with exposure time [104]. Given that this biomarker was only used in one study it was not recommended for Gladstone Harbour. Genotoxic parameters. Genotoxic parameters refer to any changes to the genetic material of an organism caused by exposure to a contaminant. A number of genotoxic parameters have been used to monitor pollution induced stress in fish, including DNA adducts, secondary DNA modifications, and irreversible genotoxic events (S20 Table). DNA adducts. A DNA adduct is a form of DNA damage caused by the binding of a chemical to a segment of DNA. The two studies identified in our review showed different responses in DNA adducts in P. flesus exposed to metals, namely an increase [148] and no change [98] (S20 Table). These inconsistent results in a low number of studies preclude recommendation of this biomarker for fish health assessments in Gladstone Harbour. Secondary DNA modifications. Secondary DNA modifications including changes in DNA strand breaks, DNA unwinding, as well as programmed cell death (i.e. apoptosis). DNA strand breaks can be assessed using the comet assay and has been examined in 14 studies [96, 99, 100, 116, 127, 132, 133, 137, 155, 158–162] (S20 Table). Eight out of the eleven field studies reported

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a significant increase in DNA damage following metal exposure [99, 127, 132, 155, 159–162], including two of the potential suitable fish species (Table 2) namely E. coioides [159] and M. cephalus [160]. DNA damage was similar following exposure to metals or to organic pollutants [96]. Seasonal variation in DNA damage has been reported, with the highest damage observed in the post-reproductive season [133]. No change in DNA damage was observed in S. senegalensis following Cd exposure in toxicity testing conditions [137]. Given the field results, assessing DNA damage using the comet assay may be a suitable biomarker for assessing fish health in Gladstone Harbour. However, its specificity for metal contamination as well as potential seasonal variation with reproductive status [133] will need to be further examined. DNA unwinding was assessed in two field studies only, showing no effect of metal contamination in P. flesus [147], but higher DNA unwinding in L. calcarifer exposed to contaminated sediment [93] (S20 Table). The low number of studies and inconsistent results precluded recommendation of this biomarker for assessing fish health in Gladstone Harbour. Apoptosis, also known as programmed cell death, is ‘a physiological and irreversible process in tissue homeostasis that leads to DNA fragmentation of multiples of 180/200 base pairs’ [5]. All four studies reviewed reported increased apoptosis in four different fish species exposed to metals in the field and laboratory [96, 129, 130, 136] (S20 Table). As such, apoptosis may be appropriate as a biomarker, but further species-specific work is required to determine its suitability to assess fish health in Gladstone Harbour. The Fas ligand is a type II transmembrane protein involved in apoptosis, and has been used as a biomarker in three of the studies reviewed [129, 132, 146] (S20 Table). The Fas ligand protein increased in both C. julis and D. labrax exposed to contaminated sediments [129, 146]. However, no changes were detected when using bioassay in the same study [146]. Due to only three studies using Fas ligand, it was not further considered as a potential biomarker for fish health assessments in Gladstone Harbour. Irreversible genotoxic events. Micronucleus and nuclear abnormalities in fish erythrocytes were examined in twelve studies [84, 90, 96, 100, 112, 114, 116, 127, 132, 158, 160, 162] (S20 Table). Eleven of these identified abnormalities when exposed to contaminated sediment in the field or to Cu in toxicity testing [90, 96, 100, 112, 114, 116, 127, 132, 158, 160, 162], including in M. cephalus [114, 160]. The effect of metals in M. cephalus could be identified through- out the year despite seasonal variation in micronuclei frequency [114]. Hence, micronuclei in fish erythrocytes could be a potential biomarker for Gladstone Harbour, but the specificity of this biomarker to certain contaminants needs to be examined. Other biomarkers of exposure. A range of other biomarkers of exposure have been used to assess the response in fish following metal exposure, including osmoregulatory and respiratory markers, and carbohydrate and aerobic metabolism (S21 Table). Osmoregulatory and respiratory markers. Na+ / K+ ATPase (NKA) is an enzyme in the plasma membrane involved in ion transport and osmoregulation, and has been used in five studies to assess the response to metal exposure [84, 106, 135, 144, 163](S21 Table). Exposure to contaminated sediment resulted in an increase in NKA in A. anguilla [106] but not in D. labrax [135]. Activity of NKA showed seasonal variation in fish from Brazilian estuaries likely related to ionic changes in the water rather than changes in metal contamination [84]. In laboratory toxicity testing, NKA activity increased in Fundulus heteroclitus exposed to Zn [163] but not in Squalus acanthias exposed to Pb [144]. Given the variable response of NKA to metal contamination in these studies, this biomarker is not recommended for use in Gladstone Harbour. The enzyme lactate dehydrogenase (LDH) catalyses the conversion of pyruvate to lactate in anaerobic glycolysis, and has been used in five studies [49, 106, 117, 123, 128] (S21 Table). Four of these reported no change in LDH activity following metal exposure in either the field

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or under toxicity testing conditions [49, 117, 123, 128]. The other study found that life history stage affected the response of LDH to metal contamination [106]. Lactate, the end product of anaerobic glycolysis, has also been used to as a biomarker, but no effects on lactate levels were reported [49, 144]. Due to the absence in all but one study of a response in LDH activity and lactate levels these biomarkers were not recommended for Gladstone Harbour. Single studies were identified using other osmoregulatory and respiratory markers, such as 2+ aquaporins (AQPs), Ca -ATPase, trimethylamine oxide (TMAO), urea, PaO2, PaCO2, arterial pH, K, Ca, NH4, Na and Cl [135, 144, 163] (S21 Table). These were not further considered for Gladstone Harbour. Carbohydrate and aerobic metabolism. Pyruvate kinase (PK) is involved in the generation of energy for the cell due to its role in transferring a phosphate to ADP in the final step of glycolysis. Two laboratory toxicity studies examined the effects of Cd exposure (S22 Table), with PK activity increasing in the intestine, but not in hepatic tissue of F. heteroclitus [49], whereas hepatic PK activity increased in S. hasta [117]. These inconsistent results of only two studies suggest that PK activity may not be a suitable biomarker for Gladstone Harbour. Single studies were identified using other carbohydrate and aerobic metabolism biomarkers, namely succinate dehydrogenase (SDH), malic dehydrogenase (MD), hepatic lipase (HL), lipoprotein lipase (LPL), isocitrate dehydrogenase (IDH), hexokinase (HK), citrate synthase (CS), cytochrome c oxidase (COX), 9-cis retinic acid receptor (RXRα) and nuclear receptor binds xenochemical compounds termed estrogen like molecules (Erα) [49, 104, 117, 123] (S22 Table). These were not further considered for Gladstone Harbour. Biomarkers of effect. Histopathology. Histopathology of a range of fish tissues following metal exposure has been assessed in 19 studies [69, 84, 96, 98, 117, 129, 131, 135, 139, 145, 148, 160, 164–170] (S23 Table). Fourteen of these reported an increase in histological alterations when fish were exposed to metals in the field (10 studies) and under toxicity testing conditions (4 studies) [69, 96, 117, 129, 131, 135, 139, 148, 160, 164, 166, 168–170]. This includes three of the 20 species identified as potentially suitable for biomarker studies in Gladstone Harbour, namely M. cephalus, L. calcarifer and E. coioides [69, 160, 164, 166]. While histological changes have been observed following metal exposure, this relationship is not necessarily linear or specific to a particular set of contaminants [169, 170]. Therefore, the suitability of histopathology to assess fish health in Gladstone Harbour will need to be examined for specificity and sensitivity. Gross indices. Gross indices reflect fish condition as determined by morphology, appearance and other gross characteristics [5]. These biomarkers have been used extensively to examine the response in fish exposed to metals both in field and laboratory studies (S23 Table). Liver somatic index (LSI). The hepatic somatic index (HIS) or liver somatic index (LSI) is used to identify possible liver diseases and is estimated using the equation;

LSI ¼ liver weight=body weight Â 100

Eleven studies examined changes in HSI/LSI in fish exposed to metals [83, 93, 97, 98, 108, 117, 120, 145, 148, 161, 171], with most of these reporting no change [83, 93, 97, 98, 108, 148, 161]. This includes a study on one of the potential suitable fish species L. calcarifer [93]. Sea- sonal and intra-specific variation likely confounds any potential effect of metal contamination on LSI [83, 171]. The lack of change in LSI in most studies suggests that LSI is not a suitable biomarker for Gladstone Harbour.

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Gonadosomatic index (GSI). The gonadosomatic index (GSI) is used as an indicator of reproductive condition and is estimated using the equation;

GSI ¼ gonad weight=body weight Â 100

Six of the studies reviewed used GSI to assess fish health following metal exposure [97, 98, 108, 114, 156, 161]. The reported results ranged from a significant decrease in GSI, including in M. cephalus [108, 114, 161], to no change in GSI [97, 98, 156]. These inconsistent results precluded the inclusion of GSI in the suite of potential biomarkers for Gladstone Harbour. Spleen somatic index (SSI). The spleen somatic index (SSI) is an indicator of overall spleen health and is calculated using the equation;

SSI ¼ spleen weight=body weight Â 100

Two studies used SSI to assess fish health following metal exposure, with a decline reported for Symphodus melops and no response for G. morhua [97, 108]. The low number of studies, and inconsistent results meant that this biomarker was not further considered for Gladstone Harbour. Visceral somatic index VSI. The visceral somatic index is an indicator of the overall health of a fish’s viscera. VSI is calculated using the equation;

VSI ¼ spleen weight=body weight Â 100

One toxicity test used VSI and reported no change in fish exposed to Cd [117]. Hence, this biomarker was not further considered for Gladstone harbour. Condition factor (CF). The condition factor (CF) is used to assess the general condition of fish, and is estimated using the following equation;

CF ¼ body weight=length Â 100

Eleven out of the fifteen studies that used CF to assess fish health following metal exposure reported no change or a mixed response [83, 94, 97, 98, 114, 117, 138, 148, 160, 161, 167–169]. Given this inconsistency in results, this biomarker was not further considered for Gladstone Harbour. Fulton’s condition index (K index). The Fulton’s condition index (K index) is a measure of fish health based on standard weight, and is estimated using the equation;

K ¼ 100ðW=L3Þ

Nine studies used the K index to assess fish health following exposure to sediment contaminated with metals [93, 101, 106, 124, 125, 139, 172–175]. Five of the 12 fish species sampled in these studies, including L. calcarifer, showed no change in their K index [93, 101, 106, 124]. One study reported an increase in the K index following exposure to contaminants [125]. Six fish species showed a decrease in the K index in response to metal contamination, of which three were exposed to sediment from the same location [172, 175]. Changes in the K factor are related to exposure time [175], and also vary with life history stage [106]. The variable response of the K index in these metal exposure studies suggests that its suitability and specificity as a biomarker for fish health in Gladstone would have to be assessed in more detail.

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Growth index. Specific growth rates (in % day) in length and weight have been used to esti- mate growth indices for both length and weight, using the following equations;

GL ¼ 100ðlnL2 À L1Þ=t2 À t1

GW ¼ 100ðlnW 2 À W1Þ=t2 À t1

Only one field study used the growth index showing a significant decrease in juvenile D. labrax, but not in S. maximus, caged in a polluted harbour [172]. A controlled laboratory exposure to the same sediment resulted in a mixed response in S. maximus [175]. These inconsistent results suggest that the growth index is not a suitable biomarker for Gladstone Harbour. RNA: DNA ratio. The RNA:DNA ratio is used as an indicator of nutritional condition, but its response to metal exposure is highly variable [93, 98, 101, 102, 125, 172, 174, 175]. Some studies have correlated the response in RNA:DNA ratios to metals under certain scenarios [102, 172]. Two other studies reported a decrease in RN:DNA ratio following exposure to contaminated sediment for two estuarine goby species [101] and in L. calcarifer [93]. In addition, species-specific responses have been observed for the RNA: DNA ratio [102, 172]. Given this variation in response, this biomarker was not recommended for Gladstone Harbour. Other biomarkers of effect. A range of other biomarkers of effect have been used to assess the response in fish following metal exposure, including metabolism and nutrition related biomarkers, parasites, neuromasts in lateral line tissue, prey capture and food intake, phototactic responses and epidermal diseases (S23 Table). Metabolism and nutrition. The lipid storage index has been used to assess fish health based on ratio of the quantity of triacylglycerols (TAG; reserve lipids) to the quantity of sterols (ST; structural lipids) (TAG:ST) [172, 175]. Following exposure to contaminated sediments, TAG: ST declined in juvenile S. maximus under controlled laboratory conditions [103], in juvenile D. labrax and S. maximus caged in a polluted harbour [175], and in P. flesus in field conditions [173]. The total lipid concentration were analysed in three studies [102, 117, 173], with the results in field studies ranging from species-specific responses [102] to a decline [173]. In controlled toxicity tests, the total lipid concentration increased in S. hasta following Cd exposure [117], but declined in E. coioides following Cu toxicity [164]. Total protein concentration in muscle or liver was used in three studies that reported a mixed response [98, 102, 144], while total muscle glycogen levels only started to decline at high Pb concentrations [144]. Total polyunsaturated fatty acids decreased, and monounsaturated fatty acids and total saturated fatty acid increased in juvenile E. coioides following Cu exposure [164]. In the same study, the digestive enzymes protease, amylase and lipase in the liver, stomach and intestine decreased [164]. Collagen levels were higher in A. fasciatus deformed following Cd exposure, compared to normal fish under field conditions, and corresponded with increased collagen fibres [139]. In contrast, collagen levels did not differ in non-deformed fish from reference and contaminated sites [139]. Overall, these results suggest that out of all the metabolism and nutrition biomarkers TAG:ST may be the most suitable to assess fish health in Gladstone Harbour. Parasites. The abundance of parasites has been assessed as a fish biomarker of sediment metal concentrations in the North Sea [147, 167]. The parasite community on P. flesus was influenced by sediment metal concentrations, with the abundance of several parasites nega- tively correlated with the concentration of individual metals [147]. Parasite abundance and diversity correlated significantly with several other biomarkers, including EROD, Choline (brain) activity, macrophage aggregate activity (MAM), hepatic lysosomal stability (LY2) and plasma lysosomal activity (Lys) [147]. No changes in the abundance of the one parasite examined was reported in the second study [167]. The apparent metal specificity of parasite

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abundance in fish [147], and the role of parasites in assessing fish health more broadly, suggests that this may be a suitable biomarker for Gladstone Harbour. However, given that only one study examined responses in parasite community more work needs to be conducted to assess its suitability. Lateral line tissue. The lateral line of fish is composed of neuromasts and is involved in their behaviour such as detecting predators and avoiding obstacles [176]. The one study that examined the response of neuromasts to metal exposure reported localised damage in D. labrax, resulting in a 10% decrease in fish escape rate [176]. Damage to lateral line tissue is thus a potential indicator of detrimental impacts at higher biological levels. However, only one study examined this biomarker and it is not recommended for use in Gladstone Harbour. Food intake and prey capture. Food intake was examined in one laboratory toxicity study, showing a decrease with increasing Cd concentrations in A. affinis [136]. Prey capture is a behavioural biomarker that is associated with reduced fish growth [177]. Fundulus heteroclitus captured in contaminated sites showed a lower prey capture ability in laboratory experiments than fish from reference sites [177, 178].The reduced prey capture ability was strongly correlated with metal accumulation in liver tissue [177, 178]. Given the low number of studies on food intake and prey capture, and the requirement of experimental studies to assess their response, these biomarkers were not recommended for Gladstone Harbour. Phototactic response. The phototactic response of fish larvae can be used as indicator of their physical condition [89]. This biomarker was examined in one laboratory study and showed a lower response in larvae hatched from eggs exposed to contaminated sediments compared to control larvae [89]. Given that this biomarker requires experimental studies to assess its response it was not recommended for use in Gladstone Harbour. Epidermal diseases. The incidence of epidermal diseases in fish exposed to contaminants in mesocosm experimental systems increased in both contaminated and clean mesocosms tanks [148]. However, lymphocystis was higher in the contaminated mesocosm tank [148]. Given that only one study examined this biomarker, with unclear results, it was not recommended for use in Gladstone Harbour. Biomarkers integrating exposure and effect. Traditionally, a suite of individual biomarkers has been used to assess biological impacts of contaminants on fish. New techniques such as microarray, proteomics and RNA sequencing (RNASeq) take fish health assessments beyond individual biomarkers to a global analysis of how all genes or proteins within an individual fish respond to contaminants [3, 149, 156]. Approaches such as microarray and proteomics not only extend the number of biomarkers analysed but also provide knowledge of exposure and pathways of injury, and potentially facilitate the identification of new more suitable individual biomarkers for routine monitoring programs [3, 156]. On the other hand, these approaches have the disadvantage of generating a wealth of information which can be difficult to interpret when there is no obvious toxic effect to the fish [3]. Microarray and proteomics. Our systematic review identified four papers which assessed fish health using microarray or proteomics [104, 149, 155, 156]. Microarray analysis of P. flesus exposed to reference and contaminated sediments showed significant changes in the hepatic abundance of 241 transcripts and 18 metabolites [155], and differential expression of 57 hepatic genes [156]. Endocrine microarray identified upregulation of five genes and downregulation of six genes, including CYP4501A and CYP450, in S. aurata exposed to contaminated sediment [104]. Based on proteomic analysis of plasma proteins from S. aurata exposed to Cu under laboratory conditions, 10 proteins were differentially expressed between controls and Cu exposed fish [149]. The ten differentially expressed proteins included the traditional biomarker proteins COX, ALT and GST, but also three new potential biomarkers of Cu exposure: growth hormone receptor, DNA recombinase complex and warm acclimation physiological

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response proteins [149]. Hence microarray or proteomics may be suitable for assessing fish health in Gladstone Harbour and reveal additional pathway impacted by contamination. How- ever, specific relationships between gene transcripts or metabolic profiles and sediment contamination could not always be established [155], potentially due to the contaminants being present in a non-bioavailable form. Combined with the low number of studies to assess their effectiveness, this precludes the inclusion of these biomarkers for Gladstone Harbour until clear relationships have been established between contaminant exposure, changes in transcripts, metabolites and protein abundance, and early adverse effects in fish. RNASeq. RNASeq is based on measuring the abundance of transcripts, but unlike microarray, is not limited to commercially available array chips or customising expensive individual chips, and does not require pre-existing knowledge of the fish genome [179]. This means that RNASeq can be used to assess responses to contaminants in non-model fish species [179], such as potential suitable fish species in Gladstone Harbour like L. calcarifer [157]. Our review, however, did not identify any papers that applied RNASeq to assess effects of metals on fish health. RNASeq may be a suitable biomarker for assessing fish health in Gladstone Harbour particularly given the existing knowledge of the L calcarifer transcriptome [157]. However, the application of this biomarker needs to be further development to provide clear connections between external levels of contaminant exposure, changes in transcripts abundances, and early adverse effects in organisms.

Assessment of fish health biomarkers for Gladstone Harbour Based on the metals being identified as contaminant of concern (Table 1), our review identified several biomarkers that would be suitable to assess fish health in Gladstone Harbour. These include bioaccumulation markers, biomarkers of exposure (e.g. CYP1A, EROD, SOD, LPOX, HSP70, MT, DNA strand breaks, micronucleus and nuclear abnormalities, apoptosis), and biomarkers of effect (e.g. histopathology, TAG:ST) (Fig 3). In addition, several other potential biomarkers were highlighted as having potential, but generally the number of studies was too low to provide a strong recommendation. These include biomarkers of exposure (e.g. VTG), biomarkers of effect (e.g. K index), and, in particular biomarkers that integrate exposure and effects, (e.g. RNASeq). Many biomarkers, however, exhibit variable responses depending on fish species, type of tissue analysed, location and time of sampling, and in some cases life history characteristics such as age and sex. Hence, targeted field assessments and controlled laboratory experiments need to be conducted with the species and biomarkers identified to verify their applicability and specificity for fish health assessments in Gladstone Harbour. Our review highlighted that the pathway of metal exposure is critical in causing an effect, particularly whether metals are taken up through the water or the sediment. Hence, fish species recommended for Gladstone Harbour need to include both pelagic and demersal species. In addition, several biomarkers have already been developed for certain species identified as potentially suitable (Table 2), albeit not necessarily in the context of metal contamination. These include (i) CYP1A for L. calcarifer [93, 181] and L. carponotatus [182, 183]; (ii) EROD for A. berda [184], L. calcarifer [93, 181], L. carponotatus [182, 183], and M. cephalus [185, 186]; (iii) micronucleus test for M. cephalus [185]; (iv) VTG for L. calcarifer [152–154]; (v) transcriptome for L. calcarifer [157], and (vi) various gross indices for L. calcarifer [93] and M. cephalus [185]. Combined information on life history characteristics relevant to metal contamination (Table 2), and fish genera used in fish biomarker studies globally we recommend that five fish species be further considered for the assessment of fish health in Gladstone Harbour. These are the demersal Acanthopagrus australis (Yellowfin Bream), Lates calcarifer (Barra- mundi), and Mugil cephalus (Sea Mullet), and the pelagic Eleutheronema tetradactylum (Blue Threadfin), and Scomberomorus queenslandicus (School Mackerel).

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Fig 3. Flow diagram showing chemical, biochemical, physiological and other alterations in response to metal exposure. Biomarkers that have been identified in our systematic review as potentially suitable for fish health assessment in Gladstone Harbour are included in italics. For each biomarker, the arrow presents likely up- or downregulation following metal exposure; suitable tissues are given in between brackets: b = blood, g = gill, l = liver, m = muscle, r = gonads. Modified from [180] in [3]. https://doi.org/10.1371/journal.pone.0174762.g003

Conclusion In this study, we present a protocol for identifying suitable biomarkers to assess fish health in coastal and marine ecosystems, using Gladstone Harbour (Australia) as a case study (Fig 1). To ensure that our use of the term ‘biomarker’ is consistent with that used in fish health assessments worldwide [5, 6], we formulated and used clear working definitions of biomarkers based on a review of the global literature [5–7, 9, 52–59] (S1 Table). We identified metals, specifically aluminium (Al), cadmium (Cd), copper (Cu), gallium (Ga), lead (Pb), selenium (Se), and zinc (Zn), as contaminants of concern in water and sediment of Gladstone Harbour (Table 1), based on point and diffuse sources of pollution [15] (S2 and S3 Tables) and available monitoring data[10, 16–31] (S4–S11 Tables). These definitions and contaminants of concern were subsequently used to inform our systematic literature review [8] (Fig 2; S1 Text), structure our findings, and assess the suitability of different biomarkers for monitoring fish health in Gladstone Harbour. Our systematic review has identified various biomarkers that could be suitable for assessing fish health in Gladstone Harbour (Fig 3). In addition, our review has identified five fish species suitable for such biomarker studies, based on (i) abundance information from fisheries dependent [35–39] and independent [40–44] (S12 Table), (ii) exposure and life history information relevant to metal contamination [46–48] (http://www.fishbase.org/; http://australianmuseum.

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net.au/) (Table 2), and (iii) existence of suitable biomarkers [93, 152–154, 157, 181–186]. These five fish species are Acanthopagrus australis (Yellowfin Bream), Lates calcarifer (Barra- mundi), Mugil cephalus (Sea Mullet), Eleutheronema tetradactylum (Blue Threadfin), and Scomberomorus queenslandicus (School Mackerel). For two of these species (L. calcarifer and M. cephalus), many of the potentially suitable biomarkers have already been developed but will need to be verified for Gladstone Harbour conditions. For the other fish species, the potentially suitable biomarkers will need to be developed and subsequently verified for Gladstone Harbour. Our protocol outlines a clear pathway to identify suitable biomarkers to assess fish health in coastal and marine ecosystems, which could be applied to biomarker studies in aquatic ecosystems around the world. Our results demonstrate that, while biomarkers have been used extensively in coastal and marine fish (S13–S23 Tables), none of the biomarkers reviewed have been specifically linked to adverse effects in organisms (i.e. growth, reproduction, and mortality) and populations (i.e. Adverse Outcome Pathways; [187, 188]. Thus, following the identification of suitable biomarkers and prioritised fish species, further targeted field studies and controlled laboratory experiments will need to be conducted to verify their applicability and specificity for fish health assessments in the ecosystem of interest.

Supporting information S1 Table. Definitions of biomarkers. (DOCX) S2 Table. Facilities in the Gladstone region. (DOCX) S3 Table. Total emissions of 46 substances from 26 facilities. (DOCX) S4 Table. Inorganic elements, organometallics, metals and metalloids concentrations in Gladstone Harbour water. (DOCX) S5 Table. Inorganic elements, organometallics, metals and metalloids concentrations in Gladstone Harbour sediment. (DOCX) S6 Table. Polycyclic aromatic hydrocarbon (PAHs) concentrations in Gladstone Harbour sediment. (DOCX) S7 Table. Polychlorinated biphenyls (PCBs), chlorinated hydrocarbons and semi-volatile organic compounds (SVOCs) concentrations in Gladstone Harbour sediment. (DOCX) S8 Table. Total petroleum hydrocarbons (TPHs) and benzene, toluene, ethylbenzene and xylene (BTEX) concentrations in Gladstone sediment. (DOCX) S9 Table. Organochlorine pesticide concentrations in Gladstone Harbour sediment. (DOCX) S10 Table. Organophosphorus pesticide concentrations in Gladstone Harbour sediment. (DOCX)

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S11 Table. Herbicides, carbamate pesticides and insecticides concentrations in Gladstone Harbour sediment. (DOCX) S12 Table. Fish species in Gladstone Harbour. (DOCX) S13 Table. Summary of bioaccumulation studies. (DOCX) S14 Table. Summary of biomarker of exposure studies: biotransformation enzymes, Phase I. (DOCX) S15 Table. Summary of biomarker of exposure studies: biotransformation enzymes, Phase II. (DOCX) S16 Table. Summary of biomarker of exposure studies: oxidative stress parameters. (DOCX) S17 Table. Summary of biomarker of exposure studies: bio transformational products, stress proteins, metallothioneins and metal proteins. (DOCX) S18 Table. Summary of biomarker of exposure studies: haematological and immunological parameters. (DOCX) S19 Table. Summary of biomarker of exposure studies: reproductive, endocrine and neurotoxic parameters. (DOCX) S20 Table. Summary of biomarker of exposure studies: genotoxic parameters. (DOCX) S21 Table. Summary of biomarker of exposure studies: osmoregulatory and respiratory parameters. (DOCX) S22 Table. Summary of biomarker of effect studies: carbohydrate and aerobic metabolism. (DOCX) S23 Table. Summary of biomarker of effect studies: histopathology and gross indices. (DOCX) S1 Text. PRISMA Checklist for Systematic Review. (DOCX)

Acknowledgments Members of the Gladstone Harbour Healthy Partnership Independent Science Panel, Andrew Negri, and two anonymous reviewers have provided valuable comments on previous versions of this manuscript.

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Author Contributions Conceptualization: FK CS SH. Data curation: FK CS SH. Formal analysis: FK CS SH. Funding acquisition: FK CS. Investigation: FK CS SH. Methodology: FK CS SH. Project administration: FK CS. Resources: FK CS SH. Software: FK CS SH. Supervision: FK CS. Validation: FK CS. Visualization: SH. Writing – original draft: CS SH. Writing – review & editing: FK CS SH.

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PLOS ONE | https://doi.org/10.1371/journal.pone.0174762 April 12, 2017 43 / 43 S1 Table. Definitions of biomarkers used in the global scientific literature for assessments of fish health specifically, and aquatic ecosystem health more broadly. General definitions of biomarkers are given, as well as those for biomarkers of exposure, of effect and of susceptibility, respectively. Biomarker definitions References Biomarker (general) Detectable biochemical and tissue‐level changes that indicate altered physiology. [1] (from [2]) Detectable biochemical and tissue responses that represent changes in organisms after exposure to [2] (from [3]) pollutants. A biological response to a chemical or chemicals that gives a measure of exposure, and sometimes, [4] (from [5]) also of toxic effect. Biochemical, anatomical, physiological and behavioural responses that signal exposure to and/or [4] (see [6]) adverse effects of anthropogenic chemicals and radiations. Any measurement reflecting an interaction between a biological system and a potential hazard, [7](from [8]) which may be chemical, physical or biological. A change in a biological response (ranging from molecular through cellular and physiological [7]( (from[6]) responses to behavioral changes) which can be related to exposure to or toxic effects of environmental chemicals. Any biological response to an environmental chemical at the sub-individual level, measured inside [7] (from [9]) an organism or in its products (urine, faeces, hair, feathers, etc.), indicating a deviation from the normal status that cannot be detected in the intact organism. All biological (biochemical, physiological, histological and morphological) indicators measured [7] inside an organism or its products. A xenobiotically-induced variation in cellular or biochemical components or processes, structures, [10] (modified based or functions that is measurable in a biological system or sample. on [11]) A biological response (ranging from the molecular to community structure and even to the function [6] and structure of ecosystems) to a chemical or chemicals that gives a measure of exposure and sometimes, also of toxic effect. A biological response to a chemical or chemicals that gives a measure of exposure and sometimes, [5] also of toxic effect. The types of 'biological responses' that can be considered range from the molecular to species composition. Any biological response to an environmental chemical at the below-individual level, measured [9] inside an organism or in its products (urine, faeces, hairs, feathers, etc.), indicating a departure from the normal status, that cannot be detected from the intact organism. Restrict the term 'biomarker' to biochemical, physiological, histological and morphological (including appearance, pigmentation, surface deformation, etc.) measurements of 'health' and exclude behavioural effects. Almost any measurement reflecting an interaction between a biological system and a potential [8] hazard, which may be chemical, physical or biological. The measured response may be functional and physiological, biochemical at the cellular level, or a molecular interaction. Biochemical, physiological, or histological indicators of either exposure to, or effects of, xenobiotic [12] chemicals at the suborganismal or organismal level. Quantifiable biochemical, physiological, or histological measures that relate in a dose-dependent [3] manner the degree of dysfunction that the contaminant has produced. Indicators signalling events in biological systems or samples. [11]

Biomarker of exposure Show an early response to contaminants and are typically specific to a particular class of [1] contaminants. The internal dose or bioavailability of a particular xenobiotic or its metabolite in an organism. [13] Should be well-characterized responses that take into account pharmacodynamic and physicochemical properties of the biological organism and agent, respectively (e.g. CYP1A, EROD). Markers which indicate that exposure of an individual or organism to a xenobiotic has occurred and [14] to what extent (e.g. particular metabolites or adducts indicating interaction with the biological system). The detection and measurement of an exogenous substance or its metabolite or the product of an [7](from [11], [8]) interaction between a xenobiotic agent and some target molecule or cell that is measured in a compartment within an organism. An exogenous substance or its metabolite or the product of an interaction between a xenobiotic [8] agent and some target molecule or cell that is measured in a compartment within an organism. The identification of an exogenous substance within the system, the interactive product between a [11] xenobiotic compound and endogenous components, or other event in the biological system related to the exposure. Biomarker of effect (also called biomarker of response) Indicators of physiological or biochemical changes as a consequence of exposure. [1] Measurable biochemical, physiological or other alterations within tissues or body fluids of an [7](from [11], [8]) organism that can be recognized as associated with an established or possible health impairment or disease. Markers that can be measured at any point along the continuum from the molecular to the [13] ecosystem level and may vary tremendously in their specificity (e.g. HSPs). Indicators of biochemical change of actual or potential toxicological importance to an organism [14] resulting from exposure to a xenobiotic (e.g. HSPs, cytochrome P450) A measurable biochemical, physiological, behavioural or other alteration within an organism that, [8] depending upon the magnitude, can be recognized as associated with an established or possible health impairment or disease. An indicator of an endogenous component of the biological system, a measure of the functional [11] capacity of the system, or an altered state of the system that is recognized as impairment or disease. Biomarker of susceptibility The inherent or acquired ability of an organism to respond to the challenge of exposure to a specific [7](from [11], [8]) xenobiotic substance, including genetic factors and changes in receptors which alter the susceptibility of an organism to that exposure. In contrast to biomarkers of effect or exposure, biomarkers of susceptibility do not represent stages [13] along the dose-effect continuum, but are conditions that increase the rate of transition between the steps. Markers which indicate that an organism may be more or less susceptible to adverse effects [14] following exposure to a particular xenobiotic. An indicator of an inherent or acquired ability of an organism to respond to the challenge of [8] exposure to a specific xenobiotic substance. An indicator that the health of the system is especially sensitive to the challenge of exposure to a [11] xenobiotic compound (a compound originating outside the organism).

References 1. Hook SE, Gallagher EP, Batley GE. The Role of Biomarkers in the Assessment of Aquatic Ecosystem Health. Integr Environm Ass Manag. 2014; 10: 327-41. doi: 10.1002/ieam.1530 PMID: 000338133500004 2. Smit MGD, Bechmann RK, Hendriks AJ, Skadsheim A, Larsen BK, Baussant T, et al. Relating biomarkers to whole-organism effects using species sensitivity distributions: a pilot study for marine species exposed to oil. Environ Toxicol Chem. 2009; 28: 1104-9. 3. Mayer FL, Versteeg DJ, McKee MJ, l.C. F, R.L. G, D.C. M, et al. Physiological and nonspecific biomarkers. In: R.J. H, Kimerle RR, Mehrle PMJ, Bergman HL, editors. Biomarkers: Biochemical, Physiological, and Histological Markers of Anthropogenic Stress. Boca Raton, Fl, USA: Lewis Publishers; 1992. p. 5-85. 4. Handy RD, Galloway TS, Depledge MH. A proposal for the use of biomarkers for the assessment of chronic pollution and in regulatory toxicology. Ecotoxicol. 2003; 12: 331-43. doi: 10.1023/a:1022527432252 PMID: 000181129000028 5. Peakall DB, Walker CH. The role of biomarkers in environmental assessment (3). Vertebrates. Ecotoxicol. 1994; 3: 173-9. 6. Peakall DB. The role of biomarkers in environmental assessment (1). Introduction. Ecotoxicol. 1994; 3: 157-60. 7. van der Oost R, Beyer J, Vermeulen NPE. Fish bioaccumulation and biomarkers in environmental risk assessment: a review. Environ Toxicol Pharmacol. 2003; 13: 57-149. doi: 10.1016/s1382-6689(02)00126-6 PMID: 000180555100001 8. World Health Organization International Programme on Chemical Safety. Biomarkers and risk assessment: concepts and principles Geneva, Switzerland: World Health Organization; 1993 [cited 2016 25 July]. Available from: http://www.inchem.org/documents/ehc/ehc/ehc155.htm#SectionNumber:1.2 9. Van Gestel CA, Van Brummelen TC. Incorporation of the biomarker concept in ecotoxicology calls for a redefinition of terms. Ecotoxicol. 1996; 5: 217-25. doi: 10.1007/BF00118992 10. McCarty LS, Power M, Munkittrick KR. Bioindicators versus biomarkers in ecological risk assessment. Hum Ecol Risk Ass. 2002; 8: 159-64. PMID: 000173786800014 11. Committee on Biological Markers of the National Research Council. Biological markers in environmental health research. Environ Health Perspect. 1987; 74: 3-9. 12. Huggett RJ, Stegeman JJ, Page DS, Parker KR, Woodin B, Brown JS. Biomarkers in fish from Prince William Sound and the Gulf of Alaska: 1999-2000. Environ Sci Technol. 2003; 37: 4043-51. doi: 10.1021/es0342401 PMID: 000185326700005 13. Schlenk D. Necessity of Defining Biomarkers for Use in Ecological Risk Assessments. Mar Pollut Bull. 1999; 39: 48-53. doi: 10.1016/S0025-326X(99)00015-6. 14. Editor. Editorial. Biomarkers. 1996; 1: 1-2. doi: 10.3109/13547509609079340.

Text S1. Prisma 2009 Checklist for Systematic Review. Section/topic # Checklist Item Reported on Page # TITLE Title 1 Identify the report as a systematic review, meta-analysis, or both. 1 ABSTRACT Structured summary 2 Provide a structured summary including, as applicable: background; objectives; data sources; study eligibility criteria, 2-3 participants, and interventions; study appraisal and synthesis methods; results; limitations; conclusions and implications of key findings; systematic review registration number. INTRODUCTION Rationale 3 Describe the rationale for the review in the context of what is already known. 3-5 Objectives 4 Provide an explicit statement of questions being addressed with reference to participants, interventions, comparisons, N/A outcomes, and study design (PICOS). METHODS Protocol and 5 Indicate if a review protocol exists, if and where it can be accessed (e.g., Web address), and, if available, provide N/A registration registration information including registration number. Eligibility criteria 6 Specify study characteristics (e.g., PICOS, length of follow-up) and report characteristics (e.g., years considered, language, 8-9 publication status) used as criteria for eligibility, giving rationale. Information sources 7 Describe all information sources (e.g., databases with dates of coverage, contact with study authors to identify additional 8-9 studies) in the search and date last searched. Search 8 Present full electronic search strategy for at least one database, including any limits used, such that it could be repeated. 8-9 Study selection 9 State the process for selecting studies (i.e., screening, eligibility, included in systematic review, and, if applicable, included 8-9 in the meta-analysis). Data collection process 10 Describe method of data extraction from reports (e.g., piloted forms, independently, in duplicate) and any processes for 8-9 obtaining and confirming data from investigators. Data items 11 List and define all variables for which data were sought (e.g., PICOS, funding sources) and any assumptions and 8-9 simplifications made. Risk of bias in 12 Describe methods used for assessing risk of bias of individual studies (including specification of whether this was done at N/A individual studies the study or outcome level), and how this information is to be used in any data synthesis. Summary measures 13 State the principal summary measures (e.g., risk ratio, difference in means). N/A Synthesis of results 14 Describe the methods of handling data and combining results of studies, if done, including measures of consistency (e.g., I2) for each meta-analysis. Risk of bias across 15 Specify any assessment of risk of bias that may affect the cumulative evidence (e.g., publication bias, selective reporting N/A studies within studies). N/A Additional analyses 16 Describe methods of additional analyses (e.g., sensitivity or subgroup analyses, meta-regression), if done, indicating which were pre-specified. RESULTS Study selection 17 Give numbers of studies screened, assessed for eligibility, and included in the review, with reasons for exclusions at each 22 stage, ideally with a flow diagram. Study characteristics 18 For each study, present characteristics for which data were extracted (e.g., study size, PICOS, follow-up period) and provide Supp Info the citations. Risk of bias within 19 Present data on risk of bias of each study and, if available, any outcome level assessment (see item 12) N/A studies Results of individual 20 For all outcomes considered (benefits or harms), present, for each study: (a) simple summary data for each intervention N/A studies group (b) effect estimates and confidence intervals, ideally with a forest plot. Synthesis of results 21 Present results of each meta-analysis done, including confidence intervals and measures of consistency. 22-62 Risk of bias across 22 Present results of any assessment of risk of bias across studies (see Item 15). N/A studies Additional analysis 23 Give results of additional analyses, if done (e.g., sensitivity or subgroup analyses, meta-regression [see Item 16]). N/A DISCUSSION Summary of evidence 24 Summarize the main findings including the strength of evidence for each main outcome; consider their relevance to key 62-65 groups (e.g., healthcare providers, users, and policy makers). Limitations 25 Discuss limitations at study and outcome level (e.g., risk of bias), and at review-level (e.g., incomplete retrieval of identified 62-65 research, reporting bias). Conclusions 26 Provide a general interpretation of the results in the context of other evidence, and implications for future research. 64-65 FUNDING Funding 27 Describe sources of funding for the systematic review and other support (e.g., supply of data); role of funders for the In online systematic review submission

From: Moher D, Liberati A, Tetzlaff J, Altman DG, The PRISMA Group (2009). Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. PLoS Med 6(7): e1000097. doi:10.1371/journal.pmed1000097

S2 Table. Facilities in the Gladstone region with emissions reported in the National Pollutant Inventory. Data from the National Pollutant Inventory (www.npi.gov.au).

Registered Business Name Main activities Facility Name Suburb Aurizon Operations Ltd Locomotive fuelling Barney Point Rail Fuelling Facility Gladstone Aurizon Operations Ltd Locomotive servicing and fuelling Callemondah Rail Yard (Fuelling Callemondah Facility) Boyne Smelters Ltd Aluminium smelting (from alumina) BSL Gladstone BP Australia Bulk petroleum storage facility Gladstone Terminal Gladstone Caltex Australia petroleum Pty Ltd Petroleum product wholesaling Caltex Terminal Gladstone Gladstone Cement Australia (Queensland) Pty Ltd Limestone mining, crushing and preparation of raw meal for cement East End Mine Mount Larcom manufacture Cement Australia (Queensland) Pty Ltd Cement Manufacture Fishermans Landing Gladstone Central combined group Pty Ltd Petroleum product wholesaling Gladstone Galdstone Coogee chemicals Pty Ltd Sulphuric acid terminal Coogee Chemicals Pty Ltd, Yarwun Yarwun Earth Commodities Gladstone Pty Ltd Drill & Blast, Load & Haul, Crushing & Screening, Stockpiling and Loading Gladstone Quarry Yarwun Trucks Elgas Ltd n/a Gladstone AU123 Gladstone Gladstone Port Corporation Ltd Wharf operation Port Central Gladstone International Bunker Supplies Pty Ltd Marine fuel oil storage facility only (do not burn any fuel, only store) INTERNATIONAL BUNKER Gladstone SUPPLIES PTY LTD Jemena Queensland gas pipeline (1) Pty Ltd, Gas Pipeline Gladstone Meter Station (Queensland Gladstone and Jemena Queensland gas pipeline (2) Pty Gas Pipeline) Ltd Jemena Queensland gas pipeline (1) Pty Ltd, Natural Gas Metering Larcom Creek (Tee)Check Meter Mount Larcom and Jemena Queensland gas pipeline (2) Pty Station (Queensland Gas Pipeline) Ltd Jemena Queensland gas pipeline (1) Pty Ltd, Natural Gas Metering Orica Meter Station (Queensland Gas Gladstone and Jemena Queensland gas pipeline (2) Pty Pipeline) Ltd Jemena Queensland gas pipeline (1) Pty Ltd, Natural Gas Metering QAL + Boyne Meter Station Gladstone and Jemena Queensland gas pipeline (2) Pty (Queensland Gas Pipeline) Ltd Northern oil refineries Pty Ltd Used oil recycling Northern Oil Refinery Yarwun Registered Business Name Main activities Facility Name Suburb NRG Gladstone operating services Pty Ltd Electricity generation using coal or coal derived products Gladstone Power Station Gladstone Orica Australia Pty Ltd Production & storage of:- Ammonium Nitrate, Nitric Acid, Sodium Cyanide, Yarwun Site Yarwun Via Chlorine, Hydrochloric Acid, Caustic Soda, Sodium Hypchlorite and Expanded Gladstone Polystyrene Beads QC LNG Operating company Pty Ltd LNG Manufacturing Curtis Island LNG Plant Curtis Island QER Pty Ltd Care and maintenance predominantly. Minimal shale mining, retorting using the Stuart Project Gladstone Technology Demonstration Plant Queensland Alumina Ltd Bauxite refining QAL Gladstone RTA Yarwun PTY LTD Alumina refining RTA Yarwun Pty Ltd Gladstone Sibelco Australia Ltd (Extractive Industry) Mining, Crushing and Screening of Limestone to produce Calliope Quarry Calliope Agricultural, Concrete Aggregate and Roadbase Products Trility Pty Ltd Water Treatment Agnes Water / 1770 Water Treatment Agnes Water Plant Wiggins Island Coal Export Terminal Pty Ltd Coal export terminal WIGGINS ISLAND COAL EXPORT Callemondah TERMINAL PTY LTD

S3 Table. Total emissions of 46 substances into air, land and water from 26 facilities located around Gladstone Harbour in 2014/2015. Data from the National Pollutant Inventory (www.npi.gov.au). Discharges from coastal river basins into Gladstone Harbour are not included in ammonia, total nutrient and total phosphorus emissions.

Total emissions (kg) for 2014/15 Contaminant Air Land Water Acetaldehyde 80,000 0 0 Acetic acid (ethanoic acid) 20 0 0 Ammonia (total) 46,000 0 5,300 Antimony and compounds 12 0 0 Arsenic and compounds 300 14 470 Benzene 35,000 0 0.0010 Beryllium and compounds 14 0.048 0 Boron and compounds 14,000 1,300 0 1,3-Butadiene (vinyl ethylene) 0.31 0 0 Cadmium and compounds 100 0.079 26 Carbon monoxide 43,000,000 0 0 Chlorine and compounds 8 0 900 Chromium (III) compounds 1,400 0.11 21 Chromium (IV) compounds 15 0.34 0.67 Cobalt and compounds 120 6.8 0.097 Copper and compounds 2,800 1.0 430 Cumene (1-methylethylbenzene) 870 0 0 Cyanide (inorganic) compounds 110 0 17 Cyclohexane 770 0 0 Ethylbenzene 470 0 0.0020 Fluoride compounds 690,000 430 160,000 Formaldehyde (methyl aldehyde) 200,000 0 0 n-hexane 5,700 0 0 Hydrochloric acid 820,000 0 0 Lead and compounds 700 0.54 28 Magnesium oxide fume 10 0 0 Manganese and compounds 5,100 840 690 Mercury and compounds 160 0.052 0.42 Methyl ethyl ketone 300 0 0 Nickel and compounds 2,100 4.9 39 Oxides of nitrogen 46,000,000 0 0 Particulate matter 10 µm 3,600,000 0 0 Particulate matter 2.5 µm 750,000 0 0 Polychlorinated dioxins and furans 0.0010 0 0 Polycyclic aromatic hydrocarbons (B[a]Peq) 39 0 0.67 Selenium and compounds 60 0 0 Styrene (ethenylbenzene) 0.70 0 0 Sulfur dioxide 40,000,000 0 0 Sulfuric acid 320,000 0 0

Total emissions (kg) for 2014/15 Contaminant Air Land Water Toluene (methylbenzene) 31,000 0 0.0050 Total nitrogen 0 0 155,100 Total phosphorus 0 0 14,950 Total volatile organic compounds 1,400,000 0 0 Trichloroethylene 23,000 0 0 Xylenes (individual or mixed isomers) 4,700 0 0.0080 Zinc and compounds 4,900 9.2 1,000

S4 Table. Inorganic elements, organometallics, metals and metalloids concentrations (µg L-1) in Gladstone Harbour water based on publicly available water quality data.

Apte et al. 2005 [1] Angel et al. 2010± [2] Angel et al. 2012± [3] DEHP 2012 [4] Kroon et al. 2015 [5] Guide- Contaminant line # of samples Concentration # of samples Concentration # of samples Concentration # of samples Concentration # of samples Concentration value* Tested > LOR Min Max Tested > LOR Min Max Tested > LOR Min Max Tested > LOR Min Max Tested > LOR Min Max Chlorine -

Cyanide 4 25 0

Fluoride - 100 100 1,110 1,320

Tributyltin* 0.006 7 5 0.009 0.021

Aluminium 24¥ 100 11 1 34 32 32 1 330 244 39 10 61 ns ns 2.5 560

Antimony - 244 0 nd nd

Arsenic - 100 100 0.51 1.27 32 32 0.68 1.12 244 244 0.9 2.7 ns ns 0.1 4.7

- Barium - Beryllium Cadmium 5.5 100 56 0.004 0.110 30 30 0.002 0.038 32 32 0.003 0.009 244 3 0.2 0.2 ns ns 0.1 0.6 Chromium 24.7 100 0 nd nd 32 0 nd nd 244 4 0.5 0.9 ns ns 0.5 3.1

Cobalt 1 32 32 0.022 0.171 244 23 0.2 0.4 ns ns 0.5 1.3

Copper 1.3 100 100 0.05 1.18 30 30 0.019 0.650 32 32 0.588 1.060 244 29 1 2 ns ns 0.11 6.9 Gallium - 36 14 1 5.8 ns ns 0.5 4.3

Iron - 100 2 15 16 32 5 1.5 12 244 39 5 110 ns ns 0.5 380

Lead 4.4 100 9 0.08 0.77 32 5 0.016 0.085 244 5 0.2 2.1 ns ns 0.5 2.3

Manganese - 100 35 0.6 7 30 21 0.400 7.000 32 29 0.2 6.5 244 226 0.5 82.3 ns ns 0.3 400 Mercury 0.4 244 0 nd nd ns ns 0.03 0.11

Molybdenum - 244 244 0.4 32.4 ns ns 0.5 41

Nickel 70 100 82 0.19 0.66 30 30 0.150 0.910 32 32 0.345 0.823 244 71 0.5 1.7 ns ns 0.5 3.6 Selenium - 100 100 0.04 0.38 244 3 2 2 ns ns 0.5 6

Silver 1.4 244 6 0.1 0.2 ns ns 0.3 5

Strontium -

Tin - 244 3 5 5 ns ns 0.5 3.5

Uranium - ns ns 0.5 5.6

Vanadium 100 244 244 0.9 6.8 ns ns 0.5 11

Zinc 15 100 46 0.22 1.35 30 30 0.055 0.260 32 32 0.109 1.620 244 12 5 25 ns ns 0.5 24 *ANZECC/ARMCANZ 2000 [6];¥ Guideline value from Golding et al. 2015 [7]; Bold font indicates value exceeds guideline value; * units are µg Sn L-1; # = number; ± = samples from The Narrows and Port Curtis only. Abbreviations: LOR = limit of reporting; Min = minimum; Max = maximum; nd = not detected; ns = not specified. 1

References 1. Apte S, Duivenvoorden L, Johnson R, Jones MA, Revill A, Simpson S, et al. Contaminants in Port Curtis: screening level risk assessment. Indooroopilly, Australia: Cooperative Research Centre for Coastal Zone, Estuary and Waterway Management, 2005. 2. Angel BM, Hales LT, Simpson SL, Apte SC, Chariton AA, Shearer DA, et al. Spatial variability of cadmium, copper, manganese, nickel and zinc in the Port Curtis Estuary, Queensland, Australia. Mar Freshw Res. 2010; 61: 170-83. doi: 10.1071/MF09046 3. Angel BM, Jarolimek CV, King JJ, Hales LT, Simpson SL, Jung RF, et al. Metal Concentrations in the Waters and Sediments of Port Curtis, Queensland. Sydney, Australia: CSIRO Wealth from Oceans Flagship, 2012. 4. Department of Environment and Heritage Protection. Update on the quality of sediment from Port Curtis and Tributaries. Brisbane, Australia: Department of Environment and Heritage Protection, 2012 Number 6, ISSN 1834-3910. 5. Kroon FJ, Berry KLE, Brinkman DL, Davis A, King O, Kookana R, et al. Identification, impacts, and prioritisation of emerging contaminants present in the GBR and Torres Strait marine environments. Final Report Project 1.10. Cairns, Australia: Report to the National Environmental Science Programme. Reef and Rainforest Research Centre Limited, 2015. 6. ANZECC/ARMCANZ (Australian and New Zealand Environment and Conservation Council and Agriculture and Resource Management Council of Australia and New Zealand). National Water Quality Management Strategy, Paper No. 4 - Australian and New Zealand Guidelines for Fresh and Marine Water Quality. Canberra, Australia: ANZECC/ARMCANZ, 2000. 7. Golding LA, Angel BM, Batley GE, Apte SC, Krassoi R, Doyle CJ. Derivation of a water quality guideline for aluminium in marine waters. Environ Toxicol Chem. 2015; 34: 141-51. doi: 10.1002/etc.2771

S5 Table. Inorganic elements, organometallics, metals and metalloids concentrations (mg kg-1) in Gladstone Harbour sediment based on publicly available data.

Guideline value€ Apte et al. 2005 [1] Vicente-Beckett et al. 2006 [2] GHD et al. 2009 [3] DERM 2011 [4]

Contaminant low high # of samples Concentration # of samples Concentration # of samples Concentration # of samples Concentration Tested > LOR Min Max Tested > LOR Min Max Tested > LOR Min Max Tested > LOR Min Max Chlorine - - Cyanide - - Fluoride - - Tributyltin* 9.0 70 56 8 36 655 839 3 0.0031 0.9 Aluminium - - 152 152 14200 83551 1024 1044 530 20600 17 17 5900 43500 Antimony 2.0 25 100 96 0.24 1.09 82 82 0.312 0.75 1044 0 nd nd 17 0 nd nd Arsenic 20 70 100 100 5 124 152 152 6.35 34.8 1044 976 1.08 49.5 17 17 7 42 Barium - - 17 17 11 380 Beryllium - - 17 17 0.11 0.83 Cadmium 1.5 10 100 2 0.11 0.24 152 152 0.022 0.226 1044 12 0.1 2.4 17 1 0.9 0.9 Chromium 80 370 100 100 8 251 152 152 14.4 90.5 1044 1044 1.1 33.1 17 17 8 42 Cobalt - - 1024 1024 0.6 77.4 17 17 4.5 16.0 Copper 65 270 100 100 3 45 152 152 8.1 54.2 1044 1041 1.2 171 17 14 3 49 Gallium - - Iron - - 152 152 14600 51300 1024 1024 560 76000 17 13000 73000 Lead 50 220 100 100 3 18 152 152 5.75 22.60 1044 1006 1 43 17 17 4 9 Manganese - - 1044 1033 10 7680 17 17 82 1000 Mercury 0.15 1.0 100 100 0.9 ¶ 55.3 ¶ 82 82 0.020 0.065 1044 526 0.01 0.75 17 0 nd nd Molybdenum - - 17 14 0.5 2.8 Nickel 21 52 100 100 2 35 152 150 8.21 50.9 1044 1033 1.1 49 17 17 4 21 Selenium - - 1024 945 0.1 5.4 17 0 nd nd Silver 1.0 4.0 100 21 0.1 0.5 152 152 0.050 0.744 1024 11 0.1 0.6 Strontium - - Tin - - 17 10 2 4 Uranium - - Vanadium - - 1024 1022 5.6 287 17 17 25 160 Zinc 200 410 100 100 6 113 152 149 24.2 108 1044 1041 2 136 17 17 11 87 * Simpson et al. 2013 [5]; Bold font indicates value exceeds guideline value; # unit is µg Sn kg-1 and values are normalised to total organic carbon; ¶ unit is µg kg-1; Abbreviations: LOR = limit of reporting; Min = minimum; Max = maximum; nd = not detected; ns = not specified.

S5 Table. Continued

Guideline value* Angel et al. 2012± [6] DEHP 2012 [4] Kroon et al. 2015 [7]

Contaminant low high # of samples Concentration # of samples Concentration # of samples Concentration Tested > LOR Min Max Tested > LOR Min Max Tested > LOR Min Max Chlorine - -

Cyanide - -

Fluoride - - 31 31 50 280

Tributyltin# 9.0 70 31 5 1.1 2.0

Aluminium - - 19 19 1720 26900 31 31 1920 20200 ns ns 1100 38600 Antimony 2.0 25 19 19 0.08 0.35 31 0 nd nd

Arsenic 20 70 19 19 7 54 31 31 3.45 25.7 ns ns 2.5 34 Barium - - 19 19 9 82

Beryllium - - 19 19 0.22 0.79

Cadmium 1.5 10 19 19 0.11 0.44 31 0 nd nd ns ns 0.3 3.7 Chromium 80 370 19 19 7 32 31 31 4.3 29.8 ns ns 4 45 Cobalt - - 19 19 6 31 31 31 2.6 15.2 ns ns 2 25 Copper 65 270 19 19 2 22 31 31 1.5 44.4 ns ns 1 52 Gallium - - 19 19 2 10 ns ns 0.3 17

Iron - - 19 19 12300 62600 31 31 8490 34300 ns ns 3800 42700 Lead 50 220 19 19 3 13 31 31 1.6 11.4 ns ns 1.1 26 Manganese - - 19 19 74 1330 31 31 82 766 ns ns 21 1330 Mercury 0.15 1.0 19 3 0.02 0.05 31 14 0.01 1.37 ns ns 0.1 0.1 Molybdenum - - 19 19 0.4 2 ns ns 0.25 5.4

Nickel 21 52 19 19 4 16 31 31 2.1 16.9 ns ns 1 31 Selenium - - 19 19 0.05 0.51 31 31 0.1 0.9 ns ns 0.25 1.6 Silver 1.0 4.0 19 19 0.01 0.07 31 1 0.3 0.3 ns ns 0.25 0.5 Strontium - - 19 19 28 576

Tin - - ns ns 0.25 2

Uranium - - 0.3 3.3

Vanadium - - 19 19 27 114 31 31 14.7 71 ns ns 11 94 Zinc 200 410 19 19 18 57 31 31 7.5 78.2 ns ns 3 100 * Simpson et al. 2013 [5]; Bold font indicates value exceeds guideline value; # unit is µg Sn kg-1 and values are normalised to total organic carbon; ¶ unit is µg kg-1; Abbreviations: LOR = limit of reporting; Min = minimum; Max = maximum; nd = not detected; ns = not specified.

References 1. Apte S, Duivenvoorden L, Johnson R, Jones MA, Revill A, Simpson S, et al. Contaminants in Port Curtis: screening level risk assessment. Indooroopilly, Australia: Cooperative Research Centre for Coastal Zone, Estuary and Waterway Management, 2005. 2. Vicente-Beckett V, Shearer D, Munksgaard N, Hancock G, Morrison H. Metal and polycyclic aromatic hydrocarbon contaminants in benthic sediments of Port Curtis. Indooroopilly, QLD: Cooperative Research Centre for coastal zone, estuary and waterway management., 2006 Technical report 73. 3. GHD Pty Ltd. Gladstone Ports Corporation. Report for western basin dredging and disposal project. Sediment quality assessment. Brisbane, Australia: GHD Pty Ltd, 2009. 4. Queensland Department of Environment and Heritage Protection. Update on the quality of sediment from Port Curtis and Tributaries. 2012. ISSN 1834-3910. 5. Simpson SL, Batley GE, Chariton AA. Revision of the ANZECC/ARMCANZ Sediment Quality Guidelines. Sydney, Australia: CSIRO Land and Water, 2013. CSIRO Land and Water Science Report 08/07. 6. Angel BM, Jarolimek CV, King JJ, Hales LT, Simpson SL, Jung RF, et al. Metal Concentrations in the Waters and Sediments of Port Curtis, Queensland. Sydney, Australia: CSIRO Wealth from Oceans Flagship, 2012. 7. Kroon FJ, Berry KLE, Brinkman DL, Davis A, King O, Kookana R, et al. Identification, impacts, and prioritisation of emerging contaminants present in the GBR and Torres Strait marine environments. Final Report Project 1.10. Cairns, Australia: Report to the National Environmental Science Programme. Reef and Rainforest Research Centre Limited, 2015.

S6 Table. Polycyclic aromatic hydrocarbon (PAHs) concentrations (µg kg-1) in Gladstone Harbour sediment based on publicly available data.

Apte et al. 2005 [1] Vicente-Beckett et al. 2006 [2] GHD Pty Ltd 2009 [3] DEHP 2012 [4] Guideline value* Contaminant # of samples Concentration # of samples Concentration # of samples Concentration # of samples Concentration low high Tested >LOR Min Max Tested >LOR Min Max Tested >LOR Min Max Tested >LOR Min Max 2-Methylnaphthalene 1012 0 nd nd 31 5 6 14

Acenaphthene 16 500 23 0 nd nd 30 1 5 5 997 0 nd nd 31 0 nd nd Acenaphthylene 44 640 23 0 nd nd 30 0 nd nd 997 1 4 4 31 0 nd nd Anthracene 85 1100 23 0 nd nd 30 9 0.48 2 997 3 4 5 31 0 nd nd Benz(a)anthracene 261 1600 23 0 nd nd 30 19 0.61 15 997 9 5 47 31 10 4 10 Benzo(a)pyrene 430 1600 23 0 nd nd 30 17 2 15 997 13 4 42 31 8 5 10 Benzo(e)pyrene 30 19 0.48 19 1009 23 4 35 31 11 5 12

Benzo(b)fluoranthene 997 32 4 64 31 12 4 15

Benzo(k)fluoranthene 997 4 4 20 31 7 4 9

Benzo(b,k)fluoranthene 23 0 nd nd 30 26 1 51

Benzo(g,h,i)perylene 23 0 nd nd 30 21 0.71 13 997 19 4 21 31 6 5 10 Chrysene 384 2800 23 0 nd nd 30 25 0.8 26 997 10 4 51 31 5 6 18 Dibenzo(a,h)anthracene 63 260 23 0 nd nd 30 0 nd nd 997 1 6 6 31 1 4 4 Coronene 1009 0 nd nd 31 0 nd nd

Fluoranthene 600 5100 23 0 nd nd 30 26 1 42 997 19 4 55 31 14 4 17 Fluorene 19 540 23 0 nd nd 30 13 0.61 8 997 0 nd nd 31 0 nd nd Indeno(1,2,3-c,d)pyrene 23 0 nd nd 30 7 0.61 2 997 10 4 18 31 1 4 4 Naphthalene 160 2100 23 0 nd nd 30 19 2 7.8 997 3 6 7 31 23 5 15 Perylene 30 27 2 66.1 1009 246 4 1640 31 11 4 10

Phenanthrene 240 1500 23 0 nd nd 30 23 2 30 997 5 8 31 31 13 6 34 Pyrene 665 2600 23 0 nd nd 30 26 1 32 997 12 4 41 31 16 5 19 Total PAHs 10000# 50000# nd nd 22 22 5 266 997 263 4 1660 31 23 5 169 *ANZECC/ARMCANZ 2000 [5], except for # from Simpson et al. 2013 [6]; Abbreviations: LOR = limit of reporting; Min = minimum; Max = maximum; nd = not detected.

References 1. Apte S, Duivenvoorden L, Johnson R, Jones MA, Revill A, Simpson S, et al. Contaminants in Port Curtis: screening level risk assessment. Indooroopilly, Australia: Cooperative Research Centre for Coastal Zone, Estuary and Waterway Management, 2005. 2. Vicente-Beckett V, Shearer D, Munksgaard N, Hancock G, Morrison H. Metal and polycyclic aromatic hydrocarbon contaminants in benthic sediments of Port Curtis. Indooroopilly, QLD: Cooperative Research Centre for coastal zone, estuary and waterway management., 2006 Technical report 73. 3. GHD Pty Ltd. Gladstone Ports Corporation. Report for western basin dredging and disposal project. Sediment quality assessment. Brisbane, Australia: GHD Pty Ltd, 2009. 4. Queensland Department of Environment and Heritage Protection. Update on the quality of sediment from Port Curtis and Tributaries. 2012. ISSN 1834-3910. 5. ANZECC/ARMCANZ (Australian and New Zealand Environment and Conservation Council and Agriculture and Resource Management Council of Australia and New Zealand). National Water Quality Management Strategy, Paper No. 4 - Australian and New Zealand Guidelines for Fresh and Marine Water Quality. Canberra, Australia: ANZECC/ARMCANZ, 2000. 6. Simpson SL, Batley GE, Chariton AA. Revision of the ANZECC/ARMCANZ Sediment Quality Guidelines. Sydney, Australia: CSIRO Land and Water, 2013. CSIRO Land and Water Science Report 08/07.

S7 Table. Polychlorinated biphenyls (PCBs), chlorinated hydrocarbons and semi-volatile organic compounds concentrations (µg kg-1) in Gladstone Harbour sediment based on publicly available data.

GHD Pty Ltd 2009 [1] Guideline value* Contaminant group Contaminant # of samples Concentration low high Tested >LOR Min Max Arochlor 1221 997 0 nd nd Arochlor 1232 997 0 nd nd

Arochlor 1242 997 0 nd nd

Polychlorinated biphenyls (PCBs) Arochlor 1248 997 0 nd nd

Arochlor 1254 997 0 nd nd

Arochlor 1260 997 0 nd nd

Total PCBs 34 280 997 0 nd nd Chlorinated hydrocarbons Hexachlorobenzene 1029 0 nd nd 2,4,6-trichlorophenol 608 0 nd nd 2,6-dichlorophenol 608 0 nd nd

Semivolatile organic compounds (SVOC) 2-nitrophenol 608 0 nd nd

4-chloro-3-methylphenol 608 0 nd nd

Pentachlorophenol 608 0 nd nd *Simpson et al. 2013 [2]; Abbreviations: LOR = limit of reporting; Min = minimum; Max = maximum; nd = not detected. References 1. GHD Pty Ltd. Gladstone Ports Corporation. Report for western basin dredging and disposal project. Sediment quality assessment. Brisbane, Australia: GHD Pty Ltd, 2009. 2. Simpson SL, Batley GE, Chariton AA. Revision of the ANZECC/ARMCANZ Sediment Quality Guidelines. Sydney, Australia: CSIRO Land and Water, 2013. CSIRO Land and Water Science Report 08/07.

S8 Table. Total petroleum hydrocarbons and BTEX concentrations (mg kg-1) in Gladstone sediment based on publicly available data.

GHD Pty Ltd 2009 [1] DEHP 2012 [2] Guideline value* Contaminant group Contaminant # of samples Concentration # of samples Concentration low high Tested >LOR Min Max Tested >LOR Min Max Benzene 465 8 0.3 0.5 31 0 nd nd Ethylbenzene 465 8 0.3 0.5 31 0 nd nd

Toluene 465 2 0.2 0.3 31 0 nd nd Benzene, toluene, ethylbenzene and xylene (BTEX) Xylene (m & p) 462 0 nd nd 31 0 nd nd

Xylene (o) 462 0 nd nd 31 0 nd nd

Xylene total 465 0 nd nd 31 0 nd nd TPH C6 - C9 465 1 3 3 31 0 nd nd

TPH C10 - C14 465 1 6 6 31 1 4 4

Total petroleum hydrocarbons (TPH) TPH C15 - C28 465 334 3 48 31 27 4 48

TPH C29 - C36 465 307 5 42 31 20 6 37

TPH + C10 - C36 280 550 465 349 7 73.5 31 27 4 89 *Simpson et al. 2013 [3]; Abbreviations: LOR = limit of reporting; Min = minimum; Max = maximum; nd = not detected.

References 1. GHD Pty Ltd. Gladstone Ports Corporation. Report for western basin dredging and disposal project. Sediment quality assessment. Brisbane, Australia: GHD Pty Ltd, 2009. 2. Queensland Department of Environment and Heritage Protection. Update on the quality of sediment from Port Curtis and Tributaries. 2012. ISSN 1834-3910. 3. Simpson SL, Batley GE, Chariton AA. Revision of the ANZECC/ARMCANZ Sediment Quality Guidelines. Sydney, Australia: CSIRO Land and Water, 2013. CSIRO Land and Water Science Report 08/07.

S9 Table. Organochlorine pesticide concentrations (µg kg-1) in Gladstone Harbour sediment based on publicly available data.

GHD Pty Ltd 2009 [1] DEHP 2012 [2] Guideline value* Contaminant # of samples Concentration # of samples Concentration low high Tested >LOR Min Max Tested >LOR Min Max 4,4-Dichlorodiphenyldichloroethylene (DDE) 1.4 7 1029 0 nd nd 31 0 nd nd a-Hexachlorobenzene 1029 0 nd nd 31 0 nd nd Aldrin 1029 0 nd nd 31 0 nd nd b-Hexachlorobenzene 1029 0 nd nd 31 0 nd nd Chlordane (cis) 1029 0 nd nd 31 0 nd nd Chlordane (trans) 1029 0 nd nd 31 0 nd nd d-Hexachlorobenzene 1029 0 nd nd 31 0 nd nd Dichlorodiphenyldichloroethane (DDD) 3.5 9 1029 0 nd nd 31 0 nd nd Dichlorodiphenyltrichloroethane (DDT) 1.2 5 1029 0 nd nd 31 0 nd nd DDT + DDE + DDD 1029 0 nd nd 31 0 nd nd Dieldrin 2.8 7 1029 0 nd nd 31 0 nd nd Endosulfan I 1029 0 nd nd 317 0 nd nd Endosulfan II 1029 0 nd nd 31 0 nd nd Endosulfan sulphate 1029 0 nd nd 31 0 nd nd Endrin 2.7 60 1029 0 nd nd 31 0 nd nd Endrin aldehyde 1029 0 nd nd 31 0 nd nd Endrin ketone 1029 0 nd nd 31 0 nd nd Lindane 0.9 1.4 1029 0 nd nd 31 0 nd nd Heptachlor 1029 0 nd nd 31 0 nd nd Heptachlor epoxide 1029 0 nd nd 31 0 nd nd Methoxychlor 1029 0 nd nd 31 0 nd nd * Simpson et al. [3]; Abbreviations: LOR = limit of reporting; Min = minimum; Max = maximum; nd = not detected. References 1. GHD Pty Ltd. Gladstone Ports Corporation. Report for western basin dredging and disposal project. Sediment quality assessment. Brisbane, Australia: GHD Pty Ltd, 2009. 2. Queensland Department of Environment and Heritage Protection. Update on the quality of sediment from Port Curtis and Tributaries. 2012. ISSN 1834-3910. 3. Simpson SL, Batley GE, Chariton AA. Revision of the ANZECC/ARMCANZ Sediment Quality Guidelines. Sydney, Australia: CSIRO Land and Water, 2013. CSIRO Land and Water Science Report 08/07.

S10 Table. Organophosphorus pesticide concentrations (µg kg-1) in Gladstone Harbour sediment based on publicly available data.

GHD Pty Ltd 2009 [1] DEHP 2012 [2] Guideline value* Contaminant # of samples Concentration # of samples Concentration low high Tested >LOR Min Max Tested >LOR Min Max Azinophos methyl 1009 0 nd nd 31 0 nd nd Bromophos 1009 0 nd nd 31 0 nd nd Carbophenothion 1009 0 nd nd 31 0 nd nd Chlorfenvinphos 1009 0 nd nd 31 0 nd nd Chlorfenvinphos Z 1009 0 nd nd 31 0 nd nd Chlorphyrifos 1009 0 nd nd 31 0 nd nd Chlorphyrifos-methyl 1009 0 nd nd 31 0 nd nd Demeton-S-methyl 1009 0 nd nd 31 0 nd nd Diazinon 1009 0 nd nd 31 0 nd nd Dichlorvos 1009 0 nd nd 31 0 nd nd Dimethoate 1009 0 nd nd 31 0 nd nd Ethion 1009 0 nd nd 31 0 nd nd Fenamiphos 1009 0 nd nd 31 0 nd nd Fenthion 1009 0 nd nd 31 0 nd nd Malathion 1009 0 nd nd 31 0 nd nd Methyl parathion 1009 0 nd nd 31 0 nd nd Monocrotophos 1009 0 nd nd 31 0 nd nd Parathion 1009 0 nd nd 31 0 nd nd Pirimphos-ethyl 1009 0 nd nd 31 0 nd nd Prothiofos 1009 0 nd nd 31 0 nd nd Abbreviations: LOR = limit of reporting; Min = minimum; Max = maximum; nd = not detected.

S11 Table. Herbicides, carbamate pesticides and insecticides concentrations (µg kg-1) in Gladstone Harbour sediment based on publicly available data.

GHD Pty Ltd 2009 [1] Contaminant group Contaminant # of samples Concentration Tested >LOR Min Max 3-Hydrox Carbofuran 30 0 nd nd Aldicarb 30 0 nd nd Bendiocarb 30 0 nd nd Carbaryl 30 0 nd nd Carbamate Carbofuran 30 0 nd nd Methiocarb 30 0 nd nd Methomyl 30 0 nd nd Oxamyl 30 0 nd nd Thiodicarb 30 0 nd nd Atrazine 30 0 nd nd Pronamide 4 0 nd nd Simazine 30 0 nd nd 2,4,5-Trichlorophenoxyacetic acid 30 0 nd nd 2,4,5-Trichlorophenoxypropionic acid (fenoprop, silvex) 30 0 nd nd 2,4-Dichlorophenoxyacetic acid 30 0 nd nd 2,4-Dichlorophenoxybutyric acid 30 0 nd nd 4-Chlorophenoxy acetic acid 30 0 nd nd Herbicides Clopyralid 30 0 nd nd Dicamba 30 0 nd nd Fluroxypyr 30 0 nd nd 2-methyl-4-chlorophenoxyacetic acid 30 0 nd nd 4-chloro-2-methylphenoxybutanoic acid 30 0 nd nd Mecoprop 30 0 nd nd Picioram 30 0 nd nd Triclopyr 30 0 nd nd Abbreviations: LOR = limit of reporting; Min = minimum; Max = maximum; nd = not detected.

References 1. GHD Pty Ltd. Gladstone Ports Corporation. Report for western basin dredging and disposal project. Sediment quality assessment. Brisbane, Australia: GHD Pty Ltd, 2009.

S12 Table. Fish species reported in and around Gladstone harbour from 2005 to 2015. Data were sourced from both fisheries dependent [1- 5] and independent [6-10] studies, as well as from the Queensland Government shark control program [10], published from 2005 onwards.

Type of Study Fisheries Dependent Fisheries Independent References Commercial [3, 5] Recreational [1, 2] [7] [8] [9] [6] [10] Scientific Name Common Name 2005 / 2015 2006 / 2014 2003 ?? 2012 2014 2006 / 2015 N/A Australian Blacktip 9

N/A Blacktip Reef Whaler 360

N/A Bull Whaler 42

N/A Common Blacktip Whaler 62

N/A Creek Whaler 1

N/A Dusky Whaler 0

N/A Hammerhead Shark 1

N/A Long Nose Whaler 1

N/A Sandbar Whaler 10

N/A Scalloped Hammerhead 5

N/A Sharptooth Shark 1

N/A Tawny Shark 1

N/A Tiger Shark 113

N/A Whaler 1

Absalom radiatus Fringe-Finned Trevally 2

Acanthopagrus australis Yellowfin Bream x 47 x 3 58

Acanthopagrus berda Pikey Bream x x 1 32

N/A Bream 18.1 x

Acanthurus fuliginosus Surgeonfish x

Acentronura tentaculata Shortpouch Pygmy Pipehorse x

Achlyopa nigra Black Sole 1

Alepes kleini Razor Belly Scad x

Ambassis marianus Macleay's Glassfish 244 x 1 266

Type of Study Fisheries Dependent Fisheries Independent References Commercial [3, 5] Recreational [1, 2] [7] [8] [9] [6] [10]

Scientific Name Common Name 2005 / 2015 2006 / 2014 2003 ?? 2012 2014 2006 / 2015 Ambiserrula jugosa Flat Head x

Amniataba percoides Barred Grunter x 3 12

Anacanthus barbatus Bearded Leather Jack x

Anyperodon leucogrammicus Grouper x

Apogon fasciatus Striped Cardinalfish 30

Apogon limenus Cardinal Fish x

Argyrosomus japonicus Jewfish 2

Arius graeffei Blue Catfish x 5

Arothron hispidus Puffer Fish x

Arothron manilensis Narrow-Lined Toadfish 6

Arrhamphus sclerolepis Snub-Nosed Garfish 24 1 3

Aseraggodes normani Flat Fish x

Atherinomorus ogilbyi Common Hardyhead 97

Auxis thazard Frigate Mackerel 1

Bathyaploactis ornatissimus Velvet Fish x

Bathygobius fuscus Dusky Frillgoby x

Campichthys tryoni Tryon’s Pipefish x

Carangoides caeruleopinnatus Onion-Ring Trevally 2

Carangoides fulvoguttatus Yellow Spotted Trevally x

Caranx ignobilis Giant Trevally x

Caranx melampygus Blue Fin Trevally x

Caranx sexfasciatus Big Eye Trevally x

Centropogon marmoratus Cobbler/Marbled Fortescue x

Chaetodon tricinctus Three-Band Coralfish 2

Chanos chanos Milkfish 2

Type of Study Fisheries Dependent Fisheries Independent References Commercial [3, 5] Recreational [1, 2] [7] [8] [9] [6] [10]

Scientific Name Common Name 2005 / 2015 2006 / 2014 2003 ?? 2012 2014 2006 / 2015 Chelonodon patoca Milk-Spotted Puffer 1

Choerodon cephalotes Purple Tuskfish 1

Choeroichthys brachysoma Pacific Short-Bodied Pipefish x

Corythoichthys amplexus Fijian Banded Pipefish x

Corythoichthys flavofasciatu Reticulate Pipefish x

Corythoichthys haematopterus Reef-Top Pipefish x

Corythoichthys intestinalis Australian Messmate Pipefish x

Corythoichthys ocellatus Orange-Spotted Pipefish x

Corythoichthys paxtoni Paxton’s Pipefish x

Corythoichthys schultzi Schultz’s Pipefish x

Craterocephalus stercusmuscarum Flyspecked Hardyhead x

Cynoglossus maccullochi Mcculloch's Tongue Sole x

Dasyatis fluviorum Brown Stingray 2

Diodor nichthemerus Porcupine Fish 1

Doryrhamphus excisus Bluestripe Pipefish x

Drepane punctata Spotted Sickelfish 12 x

Eleutheronema tetradactylum Blue Threadfin x 1 x 1

N/A Threadfin 212.0

Elops hawaiensis Ladyfish Or Herring x

Engraulis australis Anchovy- Australian 3

Epinephelus coioides Goldspotted Rockcod x 1 x

Epinephelus maculatus High Fin Grouper Or Rock Cod x

Epinephelus malabaricus Malabar Or Greasy Grouper x

Epinephelus quoyanus Longfin Rockcod x

Erosa erosa Pacific Monkey Fish x

Type of Study Fisheries Dependent Fisheries Independent References Commercial [3, 5] Recreational [1, 2] [7] [8] [9] [6] [10]

Scientific Name Common Name 2005 / 2015 2006 / 2014 2003 ?? 2012 2014 2006 / 2015 Favonigobius exquisitus Sand Goby x

Festucalex cinctus Girdled Pipefish x

Filicampus tigris Tiger Pipefish x

Fraudella carassiops Carp Prettyfin x

Gerres filamentosus Threadfin Silverbelly 23 3

Gerres oyena Oceanic Silverbelly 20 x

Gerres subfasciatus Common Silverbelly 65 x 32

Glossamia aprion Mouth Almighty x 1

Gnathanodon speciosus Golden Trevally x

Gymnothorax favagineus Laced Moray x

Halicampus dunckeri Red-Hair Pipefish x

Halicampus grayi Mud Pipefish x

Halicampus nitidus Glittering Pipefish x

Halicampus spinirostris Spiny-Snout Pipefish x

Harpadon translucens Lizard Fish x

Herklotsichthys castelnaui Southern Herring 489 x 96

Hippichthys cyanospilos Blue-Speckled Pipefish x

Hippichthys heptagonus Madura Pipefish x

Hippichthys penicillus Beady Pipefish x

Hippocampus bargibanti Pygmy Seahorse x

Hippocampus hendriki Eastern Spiny Seahorse x

Hippocampus kuda Spotted Seahorse x

Hippocampus multispinus Northern Spiny Seahorse x

Hippocampus planifrons Flat-Faced Seahorse x

Hippocampus zebra Zebra Seahorse x

Type of Study Fisheries Dependent Fisheries Independent References Commercial [3, 5] Recreational [1, 2] [7] [8] [9] [6] [10]

Scientific Name Common Name 2005 / 2015 2006 / 2014 2003 ?? 2012 2014 2006 / 2015 Hyperolophus translucidus Glassy Sprat 2

Hyporhamphus australis Sea Garfish 1

Hyporhamphus quoyi Short-Nosed Garfish 6 x

Hypseleotris compressa Empire Gudgeon x

Jaydia argyrogaster Cardinal Or Siphon Fish x

Johnius borneensis Croaker/River Dew Fish x

Kuhlia rupestris Jungle Perch x

Kyphosus bigibbus Brown Chub x

Lates calcarifer Barramundi 415.6 x 6

Leiognathus equulus Common Ponyfish 1030 x 10 195

Leiopotherapon unicolor Spangled Perch x 4

Leptobrama muelleri Salmon 30 x 4

Lethrinus genivittatus Threadfin Emperor x

Lethrinus laticaudis Grass Emperor x x

Lethrinus nebulosus Spangled Emperor x

Liocranium praepositum Waspfish x

Lissocampus runa Javelin Pipefish x

Liza argentea Goldspot Mullet 454

Liza dussumieri Flat-Tail Mullet 27

Liza subviridis Greenback Mullet x 51

Liza tade Tade Mullet x

Liza vaigiensis Diamond Scale Mullet x 2

Lubricogobius ornatus Ornate Slippery Goby x

Lutjanus argentimaculatus Mangrove Jack x 1

Lutjanus carponotatus Stripey Snapper x

Type of Study Fisheries Dependent Fisheries Independent References Commercial [3, 5] Recreational [1, 2] [7] [8] [9] [6] [10]

Scientific Name Common Name 2005 / 2015 2006 / 2014 2003 ?? 2012 2014 2006 / 2015 Lutjanus gibbus Humpback Red Snapper x

Lutjanus russellii Russell's Snapper x 5

Marilyna pleurosticta Banded Toadfish 8

Megalaspis cordyla Finny Scad 121

Megalops cyprinoides Indo-Pacific Tarpon Or Herring x

Meiacanthus luteus Yellow Fangbelly x

Melanotoenia splendida Eastern Rainbow Fish 2

Meuschenia sp2 Leatherjacket 42

Microcanthus strigatus Stripey x

Micrognathus andersonii Anderson’s Pipefish x

Micrognathus brevirostris Thorntail Pipefish x

Monodactylus argenteus Diamond Fish 1

Mugil cephalus Sea Mullet x 243

N/A Mullet 147.6

Mugilogobius stigmaticus Blackspot Mangrove Goby x

Muraenesox cinevus Pike Eel 1

Nannocampus pictus Painted Pipefish x

Naso annulatus Whitemargin Unicornfish x

Naso tonganus Bulbnose Unicornfish x

Naso unicornis Bluespine Unicornfish x

Nematalosa come Bony Bream 18

Nematalosa erebi Bony Bream 83

Nemipterus theodorei Yellow-Lip Butterfly-Bream 2

Notesthes robusta Bull Rout x 1

Omobranchus rotundiceps Rotund Blenny x

Type of Study Fisheries Dependent Fisheries Independent References Commercial [3, 5] Recreational [1, 2] [7] [8] [9] [6] [10]

Scientific Name Common Name 2005 / 2015 2006 / 2014 2003 ?? 2012 2014 2006 / 2015 Ophiocara porocephala Spangled Gudgeon x

Opistognathus eximius Harlequin Smiler x

Orbonymus rameus White Spotted Dragonet x

Otolithes ruber Tiger Tooth Croaker x

Paracentropogon vespa Wasp Roguefish x

Parachaetodon ocellatus Ocellate Coralfish 2

Paradicula setifer Sole x

Paramugil georgii Goldspot Mullet x

Paraploactis trachyderma Mosback Velvetfish x

Parapriacanthus ransonneti Pigmy Sweeper x

Parascorpaena mossambica Mozambique Scorpionfish x

Pelates quadrilineatus Trumpeter 53

Pelates sexlineatus Eastern Striped Trumpeter 17

Pentapodus paradiseus Paradise Threadfin Bream 1 x

Periopthalmus koelreuteri Mud-Skipper 1

Petroscirtes variabilis Sabre Tooth Blenny x

Platycephalus arenarius Sand Flathead 1

Platycephalus fuscus Dusky Flathead x 4

Platycephalus indicus Bartail Flathead

N/A Flathead 11.3 x

Plectorhinchus gibbosus Brown Sweetlips 1

Polydactylus multiradiatus Australian Threadfin x

Pomacanthus semicirculatus Blue Angel Fish x

Pomadasys argenteus Speckled Javelin 1

Pomadasys kaakan Barred Javelin x 77 x 1

Type of Study Fisheries Dependent Fisheries Independent References Commercial [3, 5] Recreational [1, 2] [7] [8] [9] [6] [10]

Scientific Name Common Name 2005 / 2015 2006 / 2014 2003 ?? 2012 2014 2006 / 2015 Pomadasys maculatus Saddle Grunt x

N/A Grunter 24.1

Priacanthus macracanthus Red Big Eye x

Pseudomonacanthus elongatus Fourband Leatherjack / File Fish x

Pseudomugil signifer Pacific Blue Eye x

Pseudorhombus argent Flounder 1

Pseudorhombus arsius Large-Toothed Flounder 23 x

Pteragogus flagellifera Cocktail Fish x

Sardinella gibbosa Goldstripe Sardinella x

Saurida undosquamis Large-Scaled Grinner 65

Scatophagus argus Spotted Scat 54

Scolopsis monogramma Rainbow Monocle Bream x

Scomberoides commersonnianus Talang Queenfish 10 x 32 2

Scomberoides lysan Doublespotted Queenfish x

Scomberomorus queenslandicus School Mackerel x 1

N/A Mackerel 319.8

Selaroides leptolepis Smooth-Tailed Trevally 15

Selenotoca multifasciata Striped Butterfish 3 72

Seriola lalandi Yellowtail Kingfish x

Siganus lineatus Gold Lined Rabbitfish 5

Siganus rivulatus Happy Moments 83

Sillago analis Whiting 3 x

Sillago ciliata Sand Whiting x 2 30

Sillago maculata maculata Winter Whiting 15

N/A Whiting 20.2

Type of Study Fisheries Dependent Fisheries Independent References Commercial [3, 5] Recreational [1, 2] [7] [8] [9] [6] [10]

Scientific Name Common Name 2005 / 2015 2006 / 2014 2003 ?? 2012 2014 2006 / 2015 Solegnathus hardwickii Pallid Pipehorse x

Soleichthys heterorhinos Tiger Sole x

Solenostomus cyanopterus Robust Ghostpipefish x

Solenostomus paradoxus Ornate Ghostpipefish x

Sphyraena jello Pick Handle Barracuda x

Sphyraena obtusata Yellowtail Barracuda x

Strongylura strongylura Spottail Needlefish x

Synanceia horrida Estuarine Stonefish x

Syngnathoides biaculeatus Double-End Pipehorse x

Synodus lobeli Lobel's Lizardfish x

Tathicarpus butleri Blackspot Angerlfish x

Terapon jarbua Crescent Grunter 11

Terapon puta Spinycheek Grunter x

Tetractenos hamiltoni Common Toadfish 9 4 7

Tetradontidae Puffer Fish

Thryssa aestuaria Southern Anchovy 10 1

Thunnus albacares Yellowfin Tuna x

Thunnus tonggol Longtail Tuna x

Trachinocephalus myops Painted Grinner x

Trachyrhamphus bicoarctatus Bentstick Pipefish x

Trepaon jarbua Crescent Perch 14

Triacanthus brevirostris Short-Nosed Tripod-Fish 7

Tripodichthys angustifrons Yellow-Fin Tripod-Fish 26

Tylosurus crocodilus Crocodile Long-Tom 2

Tylosurus gavialoides Stout Longtom 2

Type of Study Fisheries Dependent Fisheries Independent References Commercial [3, 5] Recreational [1, 2] [7] [8] [9] [6] [10]

Scientific Name Common Name 2005 / 2015 2006 / 2014 2003 ?? 2012 2014 2006 / 2015 Ulua mentalis Cale Trevally 1

Upeneus australiae Australian Goatfish x

Upeneus tragula Bar-Tailed Goatfish 7

Valamugil cunnesius Longarm Mullet x

Valamugil georgii Fantail Mullet 80

Zenarchopterus buffonis Buffon's River Garfish x

References 1. Sawynok B, Platten JR, Parsons W, Sawynok S. Gladfish 2012. Assessing trends in recreational fishing in Gladstone Harbour and adjacent waterways. Frenchville, Australia: Infofish Australia, 2012. 2. Sawynok B, Platten JR, Parsons W, Sawynok S. Gladfish 2014. Assessing trends in recreational fishing in Gladstone Harbour and adjacent waterways. Frenchville, Australia: Infofish Australia, 2014. 3. Department of Agriculture Fisheries and Forestry. Queensland Fishing (QFish) Brisbane, Australia: Queensland Government; 2016 [cited 2016 April]. Available from: http://qfish.fisheries.qld.gov.au/ 4. Department of Agriculture Fisheries and Forestry. Commercial Catch of Key Species. Gladstone. 2006-2011. Brisbane, Australia: Fisheries Queensland, 2012. 5. Queensland Government. Commercial fishing logbook maps of Queensland Brisbane, Australia: Queensland Government; 2016 [cited 2016 April]. Available from: https://www.business.qld.gov.au/industry/fisheries/commercial- fishing/monitoring-and-reporting/reporting-commercial-fishers/queensland-logbook- maps 6. Sawynok B, Parsons W, Sawynok S. Calliope River Fish Recruitment. Frenchville, Australia: Infofish Australia, 2015. 7. Connolly RM, Currie DR, Danaher KF, Dunning M, Melzer A, Platten JR, et al. Intertidal wetlands of Port Curtis: Ecological patterns and processes and their implications. CRC for Coastal Zone, Estuary and Waterway Management, 2006. 8. Gladstone Port Corporation. Curtis Coast Coastal and Marine Resource Inventory Report. Gladstone, Australia: Gladstone Ports Corporation, 2012. 9. Wilson S, Anastasi A, Hansler M, Stoneham B, Payne M, Charlesworth D. Arrow LNG Plant Supplementary Report to the EIS. Part B: Marine & Estuarine Ecology Report. Gladstone, Australia: Central Queensland University, 2012. 10. Department of Agriculture and Fisheries. Shark catch numbers Brisbane, Australia: Queensland Government; 2016 [cited 2016 April]. Available from: https://www.daf.qld.gov.au/fisheries/services/shark-control-program/catch- numbers

S13 Table. Bioaccumulation studies conducted in the (a) field and (b) laboratory for contaminants of concern on coastal and marine fish. For contaminants measured in fish, only those contaminants that were identified as of concern to Gladstone Harbour (Al, Cd, Cu, Ga, Pb, Se, Zn) are presented; most studies measured additional contaminants in the environment which are also presented. (a) Species Contaminants measured Tissue Method Exposure pathway Life history stage Reference Fish Environment G L M Wf O W S F J A U Acanthogobius hasta, Cd, Cu, Pb, Zn As, Cr, Hg, Mn, ICP, AFS x x x [1] Chaeturichthys sitgmatias, Ni Cyprinus carpio Linnaeus, Hypophthalmichthys molitrix, Lateolabras japonicus, Liza haematocheila Acanthopagrus australis, Cu, Pb, Zn As, Cr, Co, Fe, x x x ICP-MS x x x [2] Arrhamphus sclerolepis, Mugil Mn, Ni cephalus Acanthopagrus australis, Mugil Se - x AAS x [3] cephalus, Platycephalus fuscus Acanthopagrus berda, Elops Cd, Cu, Pb, Se, Zn Ag, As, Fe, Hg, x x AAS, ICP- x x x [4] machnata, Gerres abbreviatus, G. Mn, Ni, MS filamentosus, Leiognathus equulus, Liza affinis, L. macrolepis, L. subviridis, Mugil cephalus, Nematalosa come, Pelates quadrilineatus, Platycephalus indicus, Pseudorhombus dupliciocellatus, Scatophagus argus, Sillago sihama, Sphyraena putnamae, Terapon jarbua, Valamugil cunnesius Acanthopagrus latus, Cynoglossus Cd, Cu, Pb, Zn As, Cr, Hg, Ni, V x x ICP-MS, x x [5] arel ICP-OES Aldrichetta forsteri, Sillago Cd, Cu, Pb - x PDV x x [6] schomburgkii Alosa bulgarica, Dicentrarchus Cd, Cu, Pb, Zn Co, Cr, Fe, Mn, x AAS x x x [7] labrax, Engraulis encrasiocolus, Ni Merlangius euxinus

Species Contaminants measured Tissue Method Exposure pathway Life history stage Reference Fish Environment G L M Wf O W S F J A U Amphilophus robertsoni, Astyanax Cd, Pb, Zn Cr, Ni, V x ICP-AES x x x [8] aeneus, Batrachoides goldmani, Centropomus undecimalis, Cichlasoma salvini, Cichlasoma urophthalmus, Gobiomorus dormitor, Gobiomorus dormitor larvae, Oreochromis niloticusa, Parachromis managuensisa, Paraneetroplus bifasciatusa, Paraneetroplus synspilusa, Petenia splendidaa, Pterygoplichthys pardalis, Rhamdia quelen, Rocio octofasciata, Theraps heterospilusa, Thorichthys helleri, Thorichthys meeki, Thorichthys pasionis Anguilla anguilla Cd, Cu, Pb As, Cr, Fe, Hg, x x AAS x x [9] Mn, Ni, V, aliphatic hydrocarbons, PAHs Apogon kiensis, Argyrosomus Cd, Cu, Pb, Zn As, Cr x AAS x x x [10] argentatus, Brachirus, Cynoglossus sinicus, Eleotriodes Bleeker, Harpodon nehereus, Johnius belengeri, Lagocephalus lunaris, Leiognathus rivulatus, Nibea soldado, Setipinna taty, Siganus oramin, Trypauchen vagina Argyrosomus japonicus, Al, Cd, Cu, Pb, Se, Zn As, Ag, Au, B, x x x ICP-MS x x [11] Gilchristella aestuaria, Lichia Ba, Be, Bi, Co, amia, Mugil cephalus, Pomadasys Cr, Fe, Hg, Mn, commersonnii, Psammogobius Mo, Ni, Pd, Pt, knysnaensis Rb, Sb, Sr, Th, Ti, Tl, U, V, PCBs, PAHs, OCPs Arius parkii, Etroplus suratensis, Cd, Cu, Pb, Zn Fe, Mn, Cr, Ni, x x x x AAS x x x [12] Gerres oyena, Liza parsia, Co Oreochromis mossambicus, Sillago sihama

Species Contaminants measured Tissue Method Exposure pathway Life history stage Reference Fish Environment G L M Wf O W S F J A U Atherina boyeri, Alosa pontica, Cd, Cu, Pb Cr, Ni x AAS x x x [13] Mullus barbatus, Sprattus sprattus Boops boops, Eutrigla gurnardus, Cd, Cu, Pb, Se, Zn As, Co, Cr, Cs, x x x x ICP-MS x x x [14] Micromesistius poutassou, Mugil Fe, Ga, Ge, Hg, cephalus,Mullus surmuletus, Li, Mo, Ni, Rb, Serranus Sr, Tl, Th, U, V, cabrilla Y Cathorops spixii Al, Cd, Cu, Pb, Zn As, Cr, Fe, Hg, Ni x ICP-MS, x x [15] AAS Centropomus parallelus Al, Cd, Cu, Pb, Se, Zn As, Ag, Cr, Fe, x ICP-MS x x x [16] Hg, Mn, Ni Champsocephalus gunnari, Al, Cd, Cu, Pb, Se, Zn Ag, As, Cr, Fe, x x x x ICP-AES x x x [17] Trematomus scotti Mn, Ni, Sr, Ti, V

Channa asiatica, Oreochromis Cd, Cu, Pb, Zn Cr, Mn, Ni x x ICP-OES x x [18] mossambicus Chanos chanos Cd, Cu, Pb, Zn Fe, Mn x x x x AAS x x x [19]

Cichla ocellaris, Geophagus Al, Cd, Cu, Pb, Zn As, Cr, Fe, Hg, x x x ICP-MS x x x [20] brasiliensis, Hoplias malabaricus, Mn, Ni Hoplosternum littorale, Mugil liza, Rhamdia quelen, Tilapia rendalli, Trachelyopterus striatulus Coris julis Cd, Cu, Pb, Zn Co, Cr, Ni, Sn x AAS x x [21] Coris julis Cd, Pb As, Cr, Hg, PAHs, x ICP-MS x x x [22] PCBs, pesticides Dentex spp., Chloroscombrus Al, Cd, Cu, Pb, Se, Zn Ba, Cr, Fe, Mn, x ICP-AES x x [23] chrysurus, Galeodes decadactylus, Ni, Sr, Ti, U, V, Trichurus lepturus Zr, PAHs Dicentrarchus labrax Cd, Cu, Pb, Se, Zn - x x dPSA, dCSP x x x [24]

Dicentrarchus labrax Cd, Cu, Pb, Se, Zn As, Cr, Hg x ICP-OES x x [25] Dicentrarchus labrax, Scophtalmus Cd, Cu, Pb, Zn Al, Cr, Hg, Mn, x ICP-MS x x [26] maximus Ni, V, PAHs, PCBs

Species Contaminants measured Tissue Method Exposure pathway Life history stage Reference Fish Environment G L M Wf O W S F J A U Dicentrarchus labrax, Sparus Al, Cd, Cu, Pb, Se, Zn As, Ba, Co, Cr, x x x x ICP-MS x x [27] auratus Cs, Fe, Ge, Hg, Li, Mo, Ni, Pd, Rb, Sr, V, U, Y Diplodus sargus Cd, Cu, Pb As x x ICP-MS x [28]

Diplodus vulgaris, Engraulis Cd, Cu, Pb, Zn Ni, Hg x AAS x x [29] encrasicholus, Engraulis encrasicholus, Merluccius merluccius, Mullus surmuletus, Pagellus erythrinus, Sardina pilchardus, Scomber scombrus, Sparus aurata, Solea vulgaris, Trachurus trachurus, Thunnus thynnus, Xiphias gladius Engraulis encrasicolus, Sardina Pb - x x x Alpha x x x [30] pilchardus spectrometer Epinephelus coioides, Lethrinus Cd, Cu, Pb, Zn Ag, Al, As, Co, x x ICP-MS x x [31] nebulosus Cr, Fe, Hg, Mn, Ni, Sb, V Fundulus heteroclitus Cd, Cu, Pb, Zn - x ICP-MS x x x [32] Fundulus heteroclitus Cd, Cu, Pb, Zn - x AAS x x x [33] Fundulus heteroclitus Cd, Cu, Pb, Zn Hg, PCBs, PAHs x x AAS x x [34] Gadus morhua L., Platichthys Cd, Cu, Zn Hg, Ti, PAHs, x AAS x x [35] jlesus L. PCBs Genyonemus lineatus, Cd, Pb Hg x x x x AAS x x [36] Hippoglossoides elassodon, Pleuronectes vetulus

Gerres cinereus, Haemulopsis Cd, Cu, Pb, Zn x x AAS x x x x x [37] leuciscus, Lutjanus argentiventris, Mugil cephalus Halobatrachus didactylus Cd, Cu, Pb, Zn Co, Cr, Ni x AAS x x [38]

Species Contaminants measured Tissue Method Exposure pathway Life history stage Reference Fish Environment G L M Wf O W S F J A U Helicolenus dactylopterus, Cd, Pb, Zn - x IPC-MS x x [39] Lepidotrigla cavillone, Merluccius merluccius, Micromesistius poutassou, Scyliorhinus canicula, Trachurus trachurus, Trisopterus luscus Liza aurata Cd, Cu, Zn Ni x PIXE, RBS, x x [40] STIM Liza aurata Cd, Cu, Pb, Zn Cr, Ni, Mn x x ICP-MS x x x [41]

Liza aurata Cd, Cu, Pb, Zn Cr, Ni, Mn x AAS x x [42] Liza aurata Cd, Cu, Pb, Zn As, Hg x ICP-MS x x x [43] Liza dumerelii Al, Cu, Pb, Zn Cr, Fe, Mn x x x AAS x x x [44]

Liza klunzingeri Cd, Cu, Pb, Zn - AAS x x [45] Liza ramada Cd, Cu, Pb, Zn Fe, Mn, Ni x x AAS x x [46]

Liza saliens Cu, Zn - x x x AAS x x [47]

Lutjanus malabaricus, Saurida Cd, Cu, Pb, Zn - x x AAS x x x x [48] undosquamis, Stromateoides argenteus, Trachinotus blochii Lutjanus malabaricus, Trachinotus Cd, Cu, Pb, Zn As, Cr, Hg x x AAS x x x x [49] blochii Mugil cephalus Cd, Cu, Pb, Se, Zn Cr, Fe, Hg, Mn, x AAS x x [50] Ni Mugil cephalus Pb - x ICP-MS x x x x [51] Mullus barbatus Al, Cd, Cu, Pb, Zn Cr, Hg, Mn, Ni x AAS x x [52] Mullus barbatus Cd, Cu, Pb, Zn As, Hg, PAHs, x AAS x x [53] PCBs, DDTs, pesticides Mullus barbatus, Solea vulgaris Cd, Pb Cr, Hg, PAHs AAS x x [52] Notothenia spp., Trematomus Zn Fe, Hg x AAS x x [54] newnesi

Species Contaminants measured Tissue Method Exposure pathway Life history stage Reference Fish Environment G L M Wf O W S F J A U Parablennius sanguinolentus Cd, Pb Cr, Hg, PAHs, x AAS x x x [55] PCBs, pesticides Platichthys flesus Cd, Cu, Pb, Se, Zn As, Cr, Mn, Ni, V x ICP-MS x x [56] Platichthys flesus Cd, Cu, Pb, Zn Al, Cr, Hg, Mn, x x ICP-MS x x [57] Ni, V, PAHs Platichthys flesus Cd, Zn As, Cr, Cu, Hg, x Unknown x x x [58] Ni, Pb, OCPs, PAHs, PCBs, organotonins Platichthys flesus Cd, Cu, Pb Al, Cr, Fe, Hg, x Unknown x x [59] Mn, Ni, Zn, PCBs Platichthys flesus Cd, Cu, Pb, Zn - x x AAS x x [60]

Platycephalus bassensis Se Fe, Hg x HG-AFS x x [61] Pseudorhombus jenynsii Cu, Se, Zn As, Fe, Hg, Mn x ICP-MS, x x [62] ICP-AES Sardinella brasiliensis Se Hg x AAS x x [63] Sciaenops ocellatus Al, Cd, Cu, Pb, Se, Zn Ag, As, Fe, Hg, AAS x x [64] Mn, Ni, Sn Solea senegalensis Cd, Cu, Pb, Zn As, Fe, PAHs x ICP-MS, x x x [65] ICP-AES Solea senegalensis Cd, Cu, Pb, Zn As, Fe, PAHs x x ICP-MS, x x x [66] ICP-AES Solea senegalensis Cd, Cu, Pb, Zn As, Fe x ICP-MS, x x x [67] ICP-AES Solea senegalensis, Sparus aurata Cd, Cu, Pb, Zn As x x x ICP-MS, x x x [68] ICP-AES Solea solea, Solea senegalensis Cd, Cu, Pb, Se, Zn, Se Cr, Fe, Hg x ICP-MS x x [69] Tetractenos glaber Al, Cd, Cu, Pb, Se, Zn As, Co, Fe, Cr, Ni x x x x ICP-MS, x x [70] ICP-AES

Trematomus bernacchii Cd Hg x x x x AAS x x x [71]

Abbreviations: G=gill, L=liver, M=muscle, Wf=whole fish, O=other; W=water, S=sediment, F=food; J=juvenile, A=adult, U=unknown.

(b) Species Contaminants measured Tissue Method Exposure pathway Life history stage Reference Fish Environment G L M Wf O W S F J A U Acanthopagrus schlegeli Cd - x x x x ICP-MS x x x [72]

Acanthopagrus schlegeli, Cd, Zn - x x x ICP-MS x x x [73] Terapon jarbua

Acanthopagrus schlegelii Cu - x x x ICP-OES x x x [74] schlegelii Ambassis jacksoniensis Cd, Se, Zn - x Gamma x x x [75] spectrometer Anguilla anguilla Cd, Cu, Pb, Zn As, Cr, CrIV, Hg, x x x AAS x x [76] Ni, V, PAHs Atherinops affinis Cu, Zn - x x x ICP-MS x x [77] Centropomus parallelus Cu - x AAS x x [78] Dicentrarchus labrax Cu - x x AAS x x [79]

Dicentrarchus labrax Cd - x x AAS x x [80] Dicentrarchus labrax, Cd, Se, Zn Ag, Am, Co, Cr, Gamma x x [81] Psetta maxima, Raja Cs, Mn spectrometry undulata, Scyliorhinus canicula, Sparus aurata, Torpedo marmorata Dicentrarchus labrax, Cd, Se, Zn Am, Co, Cs, Mn x Gamma x x [82], Sparus auratus spectrometer Fundulus heteroclitus Cd As, Cr, Hg x Gamma x x [83] dectector Fundulus heteroclitus Zn - x AAS x x [84]

Fundulus heteroclitus Cu - x x x AAS x x [85]

Galaxias maculatus Cu, Pb, Zn - x ICP–MS x x x [86]

Gobius niger Cd - x dPASV x x [87] Liza microlepis Cd, Cu, Zn Fe, Mn, Ni x x x x x AAS x x x [88]

Species Contaminants measured Tissue Method Exposure pathway Life history stage Reference Fish Environment G L M Wf O W S F J A U Menidia beryllina Cd - x AAS x x [89] Menidia menidia Cd, Cu, Zn Am, Hg x Gamma x x x [90] dectector

Mugil cephalus, Terapon Pb - AAS x x x [91] jarbua Oryzias melastigma Cu - x ICP-MS x x x [92] Paralichthys olivaceus Cd - x AAS x x [93] Paralichthys olivaceus Cd - x x x x AAS x x [94] Platichthys flesus Cd, Cu, Pb, Se, Zn As, Cr, Hg, Ni, x x ICP-MS x x [95] CBs, PAHs, PBDEs, pesticides, organotonins Platichthys flesus Cd, Pb Ni x x x x ICP-OES, x x [96] AAS, ICP-MS

Platichthys flesus Cd, Pb, Zn Hg, PAHs, PCBs x AAS x x [97] Platichthys flesus Cd, Cu, Pb, Se, Zn As, Cr, Hg, Ni, x x ICP-MS x x [98] CBs, PAHs, PBDEs, pesticides, organotonins Poecilia vivipara Cu - x x x x n/a x x [99]

Pogonichthys Se Hg x ICP-MS x x [100] macrolepidotus Pogonichthys Se - x x ICP-MS x x [100] macrolepidotus Rachycentron canadum Cd - x x x x AAS x x [101]

Scophthalmus maximus Cd, Cu, Pb, Se Al, Cr, Mn, Ni, V, x ICP-MS x x [102] Zn, PAHs, PCBs Scyliorhinus canicula Cd, Zn Am, Co, Cs, Mn x Gamma x x [103] spectrometry

Scyliorhinus canicula, Cd, Zn Am, Co, Cr, Cs, x x x Gamma x x [104] Psetta maxima Mn spectrometry

Species Contaminants measured Tissue Method Exposure pathway Life history stage Reference Fish Environment G L M Wf O W S F J A U Seriola lalandi Se - x x AAS x x [105]

Siganus oramin Cu - x x x ICP-MS x x x [72]

Solea senegalensis Cd, Cu, Pb, Zn As, Cr, Ni, PAHs, x ICP-MS x x [106] PCBs, DDTs Sparus aurata Cd, Cu, Pb, Zn As, Fe x x x AAS x x x [107]

Sparus aurata Cu, Cd, Pb As x ICP-AES x x [108] Sparus aurata Cu - x x AAS x x [109]

Sparus aurata Cd, Cu, Zn - x x x x AAS x x [110] Sparus aurata Cu, Zn - x x x AAS x x x [111]

Squalus acanthias Pb - x x x ICP-MS x x [112]

Synechogobius hasta Cd - x x x x ICP-MS x x [113] Terapon jarbua Cd - x x x AAS x x x [114]

Tetractenos glaber Cd, Se - x x x x Gamma x x x [115] spectrometer Abbreviations: G=gill, L=liver, M=muscle, Wf=whole fish, O=other; W=water, S=sediment, F=food; J=juvenile, A=adult, U=unknown.

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S14 Table. Field and laboratory studies on responses of biomarkers of exposure in fish to metals and other contaminants: biotransformation enzymes, Phase I. Most studies measured contaminants in the environment in addition to those identified as of concern for Gladstone Harbour (Al, Cd, Cu, Ga, Pb, Se, Zn); these are also presented for completeness.

Species LHS Tissue Method Laboratory or Metals other Reference Field contaminants

cyt P450 CYP1 A EROD Others Anguilla J liver Bioassay Caged field sed As, Cd, Cr , Cu, Fe, Hg, PAHs + [1] anguilla Mn, Ni, Pb, V, Zn

Lab field sed As, Cd, Cr, Cu, Fe, Hg, Mn, PAHs + [1] Ni, Pb, V, Zn

Lab field sed As, Cd, Cr, Cu, Hg, Ni, Pb, PAH + [2] V, Zn

Aphanius A gonads Real time PCR Field water and sed Cd, Cu, Zn PAHs = [3] fasciatus

Atherina A liver Bioassay Field sed Cd, Hg, Ni, Pb, Zn PAHs +/- [4] presbyter

Dicentrarchus A liver Bioassay Caged field sed Cu, Pb, Zn PAHs = [5] labrax Bioassay Field sed Cr, Cu, Ni, Pb, Zn PAHs +/- [6]

J liver Bioassay Caged field sed Cd, Cr, Cu, Ni, Pb, Zn + [7]

Gadus morhua A liver Bioassay Cage field water Cd, Cu, Hg, Pb, Zn PAHs, PCBs +/- +/- [8] L.

Lates calcarifer A liver Bioassay Field sed Cd, Cr, Cu, Ni, Zn Diuron, PAHs = + [9]

Species LHS Tissue Method Laboratory or Metals other Reference Field contaminants

Others cyt P450 CYP1 A EROD Mullus A liver Bioassay Field sed As, Cr, Cu, Ni, Pb, Zn PAHs, CBs, + [10] barbatus DDT, HCB, trans- nonachlor, Lindane, Dieldrin

Plastichthys A liver Bioassay Cage field water Cd, Cu, Hg, Pb, Zn PAHs, PCBs + +/- [8] flesus Field sed Cd, Hg, Pb, Zn PAHs, PCBs = = [11]

Field sed Cd, Cu, Hg, Pb PCBs + [12]

Field water and sed As, Cd, Cr, Cu, Hg, Ni, Pb, PAHs, PCBs, +/- [13] Zn OCPs

mRNA absorbance Field sed Cd, Hg, Pb, Zn PAHs, PCBs + F [11]

Pomatoschistus A liver Bioassay Field sed Cd, Cr, Cu, Hg, Ni, Pb, Zn PAHs + [14] microps Bioassay Field sed Cd, Hg, Ni, Pb, Zn +/- [4]

Bioassay Field sed Cr, Cu, Ni, Pb, Zn PAHs = [6]

Scophthalmus J liver Bioassay Caged field sed Cd, Cr, Cu, Ni, Pb, Zn + [7] maximus Bioassay Lab field sed Cd, Cd, Cr, Cu, Hg, Ni, Pb, +/- [15] Zn

Species LHS Tissue Method Laboratory or Metals other Reference Field contaminants

Others cyt P450 CYP1 A EROD western blot Lab field sed Cu, Pb, Zn + + BROD [16] =; MROD +; PROD +

Solea A gills Immunohistochemical Field water and sed As, Cd, Cu, Fe, Pb, Zn PAHs = - [17] senegalensis /bioassay

A liver Bioassay Field sed Cr, Cu, Ni, Pb, Zn PAHs = [6]

Immunohistochemical Field water and sed As, Cd, Cu, Fe, Pb, Zn PAHs = + [17] / bioassay

J liver Bioassay Field sed Cd, Cr, Cu, Ni, Pb, Zn PAHs = [18]

Lab and field sed As, Cu, Zn PAHs, PCBs, + [19] DDT

Lab field sed As, Cd, Cr, Cu, Hg, Ni, Pb, PAHs + [20] Zn

Lab field sed As, Cd, Cr, Cu, Ni, Pb, Zn PAHs, PCBs, - [21] DDT

Sparus aurata A liver Real time PCR Lab and field sed As, Cd, Cu, Pb, Zn, + [22]

A skin Real time PCR Lab and field sed As, Cd, Cu, Pb, Zn, = [22]

J liver Bioassay Lab field sed As, Cd, Cr, Cu, Hg, Ni, Pb, PAHs + [20] Zn

Species LHS Tissue Method Laboratory or Metals other Reference Field contaminants

Others cyt P450 CYP1 A EROD J liver Real time PCR Lab field sed As, Cd, Cr, Cu, Hg, Ni, Pb, PAHs CYP3A [23] Se, V, Zn +

Symphodus A liver Bioassay Field water and sed Fe, Pb, Zn = [24] melops

Abbreviations: LHS: life history stage; J: juveniles, A: adults; Lab: laboratory; Sed : sediment; HCB: hexachlorobenzene; OCP: total organochlorine pesticides; CB: chlorinated biphenyls; naph: naphthalenes; PAHs: total polycyclic aromatic hydrocarbons; PCBS: polychlorinated biphenyl; HCH:hexachlorcyclohexane; DDT: dichlorodiphenyltrichloroethane; HCB: hexachlorobenzene; cyt P450: total cytochrome P450; CYP1A: Cytochrome P450 family 1 subfamily A; EROD: 7-ethoxyresorufin O-deethoxylase; + induction; - inhibition; +/- mixed response; = no significant induction; F: Female only; BROD: 7-benzyloxyresorufin O-debenzylase; MROD: 7-methooxyresorufin O-methoxyresorufin; PROD: 7-pentooxyresorufin O-depentylase; AHH: aryl hydrocarbon hydroxylase; CYP3A: Cytochrome P450 family 3 subfamily A.

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their potential use in biological effects monitoring - C. Pollution effects on the parasite community and a comparison to biomarker responses. Helgoland Mar Res. 2003; 57: 262-71. doi: 10.1007/s10152-003-0159-x PMID: 000186604600015 13. Schipper CA, Lahr J, van den Brink PJ, George SG, Hansen P-D, de Assis HCdS, et al. A retrospective analysis to explore the applicability of fish biomarkers and sediment bioassays along contaminated salinity transects. Ices J Mar Sci. 2009; 66: 2089-105. doi: 10.1093/icesjms/fsp194 PMID: 000272080600003 14. Serafim A, Company R, Lopes B, Fonseca VF, Franca S, Vasconcelos RP, et al. Application of an integrated biomarker response index (IBR) to assess temporal variation of environmental quality in two Portuguese aquatic systems. Ecol Indic. 2012; 19: 215-25. doi: 10.1016/j.ecolind.2011.08.009 PMID: 000302891100022 15. Kerambrun E, Henry F, Marechal A, Sanchez W, Minier C, Filipuci I, et al. A multibiomarker approach in juvenile turbot, Scophthalmus maximus, exposed to contaminated sediments. Ecotoxicol Environ Saf. 2012; 80: 45-53. doi: 10.1016/j.ecoenv.2012.02.010 PMID: 000304337300007 16. Hartl MGJ, Kilemade M, Sheehan D, Mothersill C, O'Halloran J, O'Brien NM, et al. Hepatic biomarkers of sediment-associated pollution in juvenile turbot, Scophthalmus maximus L. Mar Environ Res. 2007; 64: 191-208. doi: 10.1016/j.marenvres.2007.01.002 PMID: 000248488500007 17. Oliva M, Gravato C, Guilhermino L, Dolores Galindo-Riano M, Antonio Perales J. EROD activity and cytochrome P4501A induction in liver and gills of Senegal sole Solea senegalensis from a polluted Huelva Estuary (SW Spain). Comp Biochem Phys C. 2014; 166: 134-44. doi: 10.1016/j.cbpc.2014.07.010 PMID: 000342532000015 18. Fonseca VF, Vasconcelos RP, Tanner SE, Franca S, Serafim A, Lopes B, et al. Habitat quality of estuarine nursery grounds: Integrating non-biological indicators and multilevel biological responses in Solea senegalensis. Ecol Indic. 2015; 58: 335-45. doi: 10.1016/j.ecolind.2015.05.064 PMID: 000360776100035 19. Costa PM, Caeiro S, Vale C, Angel DelValls T, Costa MH. Can the integration of multiple biomarkers and sediment geochemistry aid solving the complexity of sediment risk assessment? A case study with a benthic fish. Environ Pollut. 2012; 161: 107-20. doi: 10.1016/j.envpol.2011.10.010 PMID: 000300539300016 20. Jimenez-Tenorio N, Morales-Caselles C, Kalman J, Salamanca MJ, Luisa Gonzalez de Canales M, Sarasquete C, et al. Determining sediment quality for regulatory proposes using fish chronic bioassays. Environ Internat. 2007; 33: 474-80. doi: 10.1016/j.envint.2006.11.009 PMID: 000246315800008 21. Costa PM, Caeiro S, Diniz MS, Lobo J, Martins M, Ferreira AM, et al. Biochemical endpoints on juvenile Solea senegalensis exposed to estuarine sediments: the effect of contaminant mixtures on metallothionein and CYP1A induction. Ecotoxicol. 2009; 18: 988-1000. doi: 10.1007/s10646-009-0373-7 PMID: 000269917200004 22. Benhamed S, Guardiola FA, Martínez S, Martínez-Sánchez MJ, Pérez-Sirvent C, Mars M, et al. Exposure of the gilthead seabream (Sparus aurata) to sediments contaminated with heavy metals down-regulates the gene expression of stress biomarkers. Toxicol Rep. 2016; 3: 364-72. doi:10.1016/j.toxrep.2016.02.006 23. Ribecco C, Baker ME, Sasik R, Zuo Y, Hardiman G, Carnevali O. Biological effects of marine contaminated sediments on Sparus aurata juveniles. Aquat Toxicol. 2011; 104: 308-16. doi: 10.1016/j.aquatox.2011.05.005 PMID: 000293042100017 24. Almroth BC, Sturve J, Stephensen E, Holth TF, Forlin L. Protein carbonyls and antioxidant defenses in corkwing wrasse (Symphodus melops) from a heavy metal polluted and a PAH polluted site. Mar Environ Res. 2008; 66: 271-7. doi: 10.1016/j.marenvres.2008.04.002 PMID: 000257817100006

S15 Table. Field and laboratory studies on responses of biomarkers of exposure in fish to metals and other contaminants: biotransformation enzymes, Phase II. Most studies measured contaminants in the environment in addition to those identified as of concern for Gladstone Harbour (Al, Cd, Cu, Ga, Pb, Se, Zn); these are also presented for completeness.

Species LHS Tissue Meth Laboratory or Metals other contaminants GSH / GS Total Refere od Field GSSH T Glutathione nce

Acanthopagrus latus J gills Bio- Field sed As, Cr, Cu, Ni, Pb, V, Zn PAHs = [1] assay

liver Bio- Field sed As, Cr, Cu, Ni, Pb, V, Zn PAHs = [1] assay

Anguilla anguilla Glass liver Bio- Field sed Cd, Cr, Cu, Hg, Ni, Pb, V, Zn PAH = - - [2] eels assay

J liver Bio- Caged field sed As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, PAHs + + [3] assay V, Zn

Bio- Lab field sed As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, PAHs = - [3] assay V, Zn

Bio- Lab field sed As, Cd, Cr, Cu, Hg, Ni, Pb, V, Zn PAH + - [4] assay toxicity

Yellow liver Bio- Field sed Cd, Cr, Cu, Hg, Ni, Pb, V, Zn PAH - + - [2] eels assay

Atherina presbyter J liver Bio- Field sed Cd, Hg, Ni, Pb, Zn PAHs +/- [5] assay

Centropomus parallelus J gills Bio- Field sed and Ag, Al, As, Cd, Cr, Cu, Fe, Hg, Mn, - [6] assay water Ni, Pb, Se, Zn

liver Bio- Field sed and Ag, Al, As, Cd, Cr, Cu, Fe, Hg, Mn, - [6] assay water Ni, Pb, Se, Zn

Species LHS Tissue Meth Laboratory or Metals other contaminants GSH / GS Total Refere od Field GSSH T Glutathione nce

Cynoglossus arel J gills Bio- Field sed As, Cr, Cu, Ni, Pb, V, Zn PAHs = [1] assay

liver Bio- Field sed As, Cr, Cu, Ni, Pb, V, Zn PAHs + [1] assay

Dicentrarchus labrax J liver Bio- Caged field sed Cu, Pb, Zn PAHs - [7] assay

Bio- Field sed Cr, Cu, Ni, Pb, Zn PAHs = [8] assay

Bio- Lab water Cu antifouling = [9] assay toxicity test

J liver Bio- Caged field sed Cd, Cr, Cu, Ni, Pb, Zn + [10] assay

Gadus morhua L. J liver Bio- Cage field Cd, Cu, Hg, Pb, Zn PAHs, PCBs +/- [11] assay water

Liza aurata J gills Bio- Field water Cd, Cr, Cu, Mn, Ni, Pb + + [12] assay

kidney Bio- Field water Cd, Cr, Cu, Mn, Ni, Pb + = [13] assay

liver Bio- Field water Cd, Cr, Cu, Mn, Ni, Pb = - [13] assay

Lutjanus russellii J blood Bio- Field sed and Cd, Cu, Fe, Pb, Zn + [14] assay water

Mugil cephalus J liver Bio- Field water Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, - - [15] assay Zn

Species LHS Tissue Meth Laboratory or Metals other contaminants GSH / GS Total Refere od Field GSSH T Glutathione nce

J gill Bio- Lab water Pb + - [16] assay toxicity test

whole Bio- Lab water Pb + - [16] assay toxicity test

liver Bio- Field sed and Cd, Cu, Mn, Ni, Pb AHCs, PAHs, PCBs, +/- [17] assay water DDTs, TBT

Paralichthys olivaceus J gill Bio- Lab water Cd - - [18] assay toxicity test

kidney Bio- Lab water Cd = + [18] assay toxicity test

liver Bio- Lab water Cd - - [18] assay toxicity test

whole Bio- Lab water Cd = - [19] assay toxicity test

L meta whole Bio- Lab water Cd (≥12µg L-) + = [19] assay toxicity test

L set whole Bio- Lab water Cd (48 µg L-) + - [19] assay toxicity test

Plastichthys flesus J liver Bio- Cage field Cd, Cu, Hg, Pb, Zn PAHs, PCBs +/- [11] assay water

Bio- Field water and As, Cd, Cr, Cu, Hg, Ni, Pb, Zn PAHs, PCBs, OCPs +/- [20] assay sed

Poecilia vivipara acclimated J gills Bio- Lab water Cu = [21] to saltwater assay toxicity test

Species LHS Tissue Meth Laboratory or Metals other contaminants GSH / GS Total Refere od Field GSSH T Glutathione nce

liver Bio- Lab water Cu +/- [21] assay toxicity test

muscle Bio- Lab water Cu +/- [21] assay toxicity test

Pomadasys hasta J blood Bio- Field sed and Cd, Cu, Fe, Pb, Zn + [14] assay water

Pomatoschistus microps J liver Bio- Field sed Cd, Cr, Cu, Hg, Ni, Pb, Zn PAHs = [22] assay

Bio- Field sed Cd, Hg, Ni, Pb, Zn PAHS +/- [5] assay

Bio- Field sed Cr, Cu, Ni, Pb, Zn PAHs = [8] assay

Scophthalmus maximus J liver Bio- Caged field sed Cd, Cr, Cu, Ni, Pb, Zn + [10] assay

Bio- Lab field sed Cd, Cr, Cu, Mn, Ni, Pb, V, Zn +/- [23] assay toxicity

Solea senegalensis J liver Bio- Field sed Cr, Cu, Ni, Pb, Zn PAHs - [8] assay

Bio- Field water and As, Cd, Cu, Fe, Pb, Zn PAHs = [24] assay sed

J liver Bio- Field sed Cd, Cr, Cu, Ni, Pb, Zn PAHs = [25] assay

Symphodus melops J liver/bl Bio- Field water and Fe, Pb, Zn = = [26] ood assay sed

Species LHS Tissue Meth Laboratory or Metals other contaminants GSH / GS Total Refere od Field GSSH T Glutathione nce

Terapon jarbua J gill Bio- Lab water Pb + - [16] assay toxicity test

whole Bio- Lab water Pb + - [16] assay toxicity test

Abbreviations: LHS: life history stage; A: adult, J = juvenile, L = larvae; Lab: laboratory; meta: metamorphosing; set: settling; Sed: sediment; AHCs: aliphatic hydrocarbons; OCP: total organochlorine pesticides; PAHs: total polycyclic aromatic hydrocarbons; PCBS: polychlorinated biphenyl; TBT: tributyltin; DDT: dichlorodiphenyltrichloroethane; GSH/GSSH: reduced and oxidised glutathione GST: Glutathione S-transferases; + induction; - inhibition; = no significant induction; +/- mixed response.

References 1. Beg MU, Al-Jandal N, Al-Subiai S, Karam Q, Husain S, Butt SA, et al. Metallothionein, oxidative stress and trace metals in gills and liver of demersal and pelagic fish species from Kuwaits’ marine area. Mar Pollut Bull. 2015; 100: 662-72. doi: 10.1016/j.marpolbul.2015.07.058 2. Gravato C, Guimaraes L, Santos J, Faria M, Alves A, Guilhermino L. Comparative study about the effects of pollution on glass and yellow eels (Anguilla anguilla) from the estuaries of Minho, Lima and Douro Rivers (NW Portugal). Ecotoxicol Environ Saf. 2010; 73: 524-33. doi: 10.1016/j.ecoenv.2009.11.009 PMID: 000277103600009 3. Piva F, Ciaprini F, Onorati F, Benedetti M, Fattorini D, Ausili A, et al. Assessing sediment hazard through a weight of evidence approach with bioindicator organisms: a practical model to elaborate data from sediment chemistry, bioavailability, biomarkers and ecotoxicological bioassays. Chemosphere. 2011; 83: 475-85. doi: 10.1016/j.chemosphere.2010.12.064 PMID: 21239037 4. Benedetti M, Ciaprini F, Piva F, Onorati F, Fattorini D, Notti A, et al. A multidisciplinary weight of evidence approach for classifying polluted sediments: Integrating sediment chemistry, bioavailability, biomarkers responses and bioassays. Environ Int. 2012; 38: 17-28. doi: 10.1016/j.envint.2011.08.003 PMID: 21982029 5. Fonseca VF, Vasconcelos RP, Franca S, Serafim A, Lopes B, Company R, et al. Modeling fish biological responses to contaminants and natural variability in estuaries. Mar Environ Res. 2014; 96: 45-55. doi: 10.1016/j.marenvres.2013.10.011 PMID: 000334981600007 6. Souza IC, Duarte ID, Pimentel NQ, Rocha LD, Morozesk M, Bonomo MM, et al. Matching metal pollution with bioavailability, bioaccumulation and biomarkers response in fish (Centropomus parallelus) resident in neotropical estuaries. Environ Pollut. 2013; 180: 136-44. doi: 10.1016/j.envpol.2013.05.017 PMID: 000322425300019 7. Traven L, Micovic V, Lusic DV, Smital T. The responses of the hepatosomatic index (HSI), 7-ethoxyresorufin-O-deethylase (EROD) activity and glutathione-S-transferase (GST) activity in sea bass (Dicentrarchus labrax, Linnaeus 1758) caged at a polluted site: implications for their use in environmental risk assessment. Environ Monitor Ass. 2013; 185: 9009-18. doi: 10.1007/s10661-013-3230-3 PMID: 000325116500018 8. Fonseca VF, Franca S, Serafim A, Company R, Lopes B, Bebianno MJ, et al. Multi- biomarker responses to estuarine habitat contamination in three fish species: Dicentrarchus labrax, Solea senegalensis and Pomatoschistus microps. Aquat Toxicol. 2011; 102: 216-27. doi: 10.1016/j.aquatox.2011.01.018 PMID: 21356184 9. Cotou E, Henry M, Zeri C, Rigos G, Torreblanca A, Catsiki V-A. Short-term exposure of the European sea bass Dicentrarchus labrax to copper-based antifouling treated nets: Copper bioavailability and biomarkers responses. Chemosphere. 2012; 89: 1091-7. doi:10.1016/j.chemosphere.2012.05.075 10. Kerambrun E, Sanchez W, Henry F, Amara R. Are biochemical biomarker responses related to physiological performance of juvenile sea bass (Dicentrarchus labrax) and turbot (Scophthalmus maximus) caged in a polluted harbour? Comp Biochem Phys C. 2011; 154: 187-95. doi: 10.1016/j.cbpc.2011.05.006 PMID: 000293994200007 11. Beyer J, Sandvik M, Hylland K, Fjeld E, Egaas E, Aas E, et al. Contaminant accumulation and biomarker responses in flounder (Platichthys flesus L) and Atlantic cod (Gadus morhua L) exposed by caging to polluted sediments in Sorfjorden, Norway. Aquat Toxicol. 1996; 36: 75-98. doi: 10.1016/s0166-445x(96)00798-9 PMID: A1996VY98200005

12. Pereira P, de Pablo H, Vale C, Pacheco M. Combined use of environmental data and biomarkers in fish (Liza aurata) inhabiting a eutrophic and metal-contaminated coastal system - Gills reflect environmental contamination. Mar Environ Res. 2010; 69: 53-62. doi: 10.1016/j.marenvres.2009.08.003 PMID: 000274773800001 13. Pereira P, de Pablo H, Pacheco M, Vale C. The relevance of temporal and organ specific factors on metals accumulation and biochemical effects in feral fish (Liza aurata) under a moderate contamination scenario. Ecotoxicol Environ Saf. 2010; 73: 805-16. doi: 10.1016/j.ecoenv.2010.02.020 PMID: 000279623800015 14. Omar WA, Saleh YS, Marie M-AS. The use of biotic and abiotic components of Red Sea coastal areas as indicators of ecosystem health. Ecotoxicol. 2016; 25: 253-66. doi: 10.1007/s10646-015-1584-8 PMID: 000370716000001 15. Padmini E, Rani MU. Evaluation of oxidative stress biomarkers in hepatocytes of grey mullet inhabiting natural and polluted estuaries. Sci Total Environ. 2009; 407: 4533-41. doi: 10.1016/j.scitotenv.2009.04.005 PMID: 000267631700019 16. Hariharan G, Purvaja R, Ramesh R. Environmental safety level of lead (Pb) pertaining to toxic effects on grey mullet (Mugil cephalus) and Tiger perch (Terapon jarbua). Environ Toxicol. 2016; 31: 24-43. doi: 10.1002/tox.22019 PMID: 000366585300003 17. Tsangaris C, Vergolyas M, Fountoulaki E, Nizheradze K. Oxidative Stress and Genotoxicity Biomarker Responses in Grey Mullet (Mugil cephalus) From a Polluted Environment in Saronikos Gulf, Greece. Arch Environ Con Tox. 2011; 61: 482-90. doi: 10.1007/s00244-010-9629-8 PMID: 000298500400013 18. Cao L, Huang W, Shan X, Ye Z, Dou S. Tissue-specific accumulation of cadmium and its effects on antioxidative responses in Japanese flounder juveniles. Environ Toxicol Pharmacol. 2012; 33: 16-25. doi: 10.1016/j.etap.2011.10.003 PMID: 000301876600003 19. Cao L, Huang W, Liu J, Yin X, Dou S. Accumulation and oxidative stress biomarkers in Japanese flounder larvae and juveniles under chronic cadmium exposure. Comp Biochem Phys C. 2010; 151: 386-92. doi: 10.1016/j.cbpc.2010.01.004 PMID: 000275627400016 20. Schipper CA, Lahr J, van den Brink PJ, George SG, Hansen P-D, de Assis HCdS, et al. A retrospective analysis to explore the applicability of fish biomarkers and sediment bioassays along contaminated salinity transects. Ices J Mar Sci. 2009; 66: 2089-105. doi: 10.1093/icesjms/fsp194 PMID: 000272080600003 21. de Souza Machado AA, Mueller Hoff ML, Klein RD, Cardozo JG, Giacomin MM, Ledes Pinho GL, et al. Biomarkers of waterborne copper exposure in the guppy Poecilia vivipara acclimated to salt water. Aquat Toxicol. 2013; 138: 60-9. doi: 10.1016/j.aquatox.2013.04.009. PMID: 000322293600007 22. Serafim A, Company R, Lopes B, Fonseca VF, Franca S, Vasconcelos RP, et al. Application of an integrated biomarker response index (IBR) to assess temporal variation of environmental quality in two Portuguese aquatic systems. Ecol Indic. 2012; 19: 215-25. doi: 10.1016/j.ecolind.2011.08.009 PMID: 000302891100022 23. Kerambrun E, Henry F, Marechal A, Sanchez W, Minier C, Filipuci I, et al. A multibiomarker approach in juvenile turbot, Scophthalmus maximus, exposed to contaminated sediments. Ecotoxicol Environ Saf. 2012; 80: 45-53. doi: 10.1016/j.ecoenv.2012.02.010 PMID: 000304337300007 24. Oliva M, Jose Vicente J, Gravato C, Guilhermino L, Dolores Galindo-Riano M. Oxidative stress biomarkers in Senegal sole, Solea senegalensis, to assess the impact of heavy metal pollution in a Huelva estuary (SW Spain): Seasonal and spatial

variation. Ecotoxicol Environ Saf. 2012; 75: 151-62. doi: 10.1016/j.ecoenv.2011.08.017 PMID: 000297088500020 25. Fonseca VF, Vasconcelos RP, Tanner SE, Franca S, Serafim A, Lopes B, et al. Habitat quality of estuarine nursery grounds: Integrating non-biological indicators and multilevel biological responses in Solea senegalensis. Ecol Indic. 2015; 58: 335-45. doi: 10.1016/j.ecolind.2015.05.064 PMID: 000360776100035 26. Almroth BC, Sturve J, Stephensen E, Holth TF, Forlin L. Protein carbonyls and antioxidant defenses in corkwing wrasse (Symphodus melops) from a heavy metal polluted and a PAH polluted site. Mar Environ Res. 2008; 66: 271-7. doi: 10.1016/j.marenvres.2008.04.002 PMID: 000257817100006

S16 Table. Field and laboratory studies on responses of biomarkers of exposure in fish to metals and other contaminants: oxidative stress parameters. Most studies measured contaminants in the environment in addition to those identified as of concern for Gladstone Harbour (Al, Cd, Cu, Ga, Pb, Se, Zn); these are also presented for completeness.

Species LHS Tissue Method Laborator Metals Other Others Referenc

y or Field contami- e

nants - GPx SOD CAT GPOX GRED LPOX Se ROS TOSC Acanthopagru A gills Bioassay Field sed As , Cr , PAHs = +/- +/- + [1] s latus Cu, Ni, Pb, V, Zn

liver Bioassay Field sed As, Cr, PAHs +/- +/- +/- +/- [1] Cu, Ni, Pb, V, Zn

Anguilla Glass liver Bioassay Field sed Cd, Cr, PAH - - = = = [2] anguilla eels Cu, Hg, Ni, Pb, V, Zn

J liver Bioassay Caged field As, Cd, PAHs + - - = [3] sed Cr, Cu, Fe, Hg, Mn, Ni, Pb, V, Zn

Lab field As, Cd, PAHs - + + +/- AOX + [3] sed Cr, Cu, Fe, Hg, Mn, Ni, Pb, V, Zn

Species LHS Tissue Method Laborator Metals Other Others Referenc

y or Field contami- e

nants - GPx SOD CAT GPOX GRED LPOX Se ROS TOSC Lab field As, Cd, PAH + - + + - AOX = [4] sed toxicity Cr, Cu, Hg, Ni, Pb, V, Zn

Yello liver Bioassay Field sed Cd, Cr, PAH = - = = + [2] w eels Cu, Hg, Ni, Pb, V, Zn

Aphanius A gonads Real time Field water Cd, Cu, PAHs = [5] fasciatus PCR and sed Zn

Atherina A liver Bioassay Field sed Cd, Hg, PAHs +/- +/- +/- [6] presbyter Ni, Pb, Zn

Centropomus J gills Bioassay Field sed Ag, Al, - [7] parallelus and water As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, Zn

liver Bioassay Field sed Ag, Al, + +/- [7] and water As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, Zn

Coris julis A gills Immuno- Field sed Cd, Co, NOS - [8] histochemica Cr, Cu, Ni, Pb,

Species LHS Tissue Method Laborator Metals Other Others Referenc

y or Field contami- e

nants - GPx SOD CAT GPOX GRED LPOX Se ROS TOSC l Sb, Zn

Cynoglossus A gills Bioassay Field sed As, Cr, PAHs = = = +/- [1] arel Cu, Ni, Pb, V, Zn

liver Bioassay Field sed As, Cr, PAHs = +/- +/- = [1] Cu, Ni, Pb, V, Zn

Dicentrarchus A liver Bioassay Field sed Cr, Cu, PAHs +/- = +/- = = [9] labrax Ni, Pb, Zn

Lab water Cu = [10] toxicity test antifoulin g

J gills Immuno- Lab field As, Cd, NOS + [11] histochemica sed toxicity Co, Cr, l Cu, Fe, Hg, Mn, Ni, Pb, Sb, V, Zn

Real time Lab field As, Cd, PAHs and HIF-1 + [12] PCR sed toxicity Cr, Cu, PCBs Hg, Ni, Pb, Zn

liver Bioassay Caged field Cd, Cr, - [13] sed Cu, Ni,

Species LHS Tissue Method Laborator Metals Other Others Referenc

y or Field contami- e

nants - GPx SOD CAT GPOX GRED LPOX Se ROS TOSC Pb, Zn

Diplodus A liver Bioassay Field and As, Cd, + + = [14] sargus aquaculture Cu, Pb

muscle Bioassay Field and As, Cd, + + = [14] aquaculture Cu, Pb

Fundulus A gill Bioassay Lab water Cu - = CP = [15] heteroclitus toxicity test

intestin Bioassay Lab water Cu - + CP = [15] e toxicity test

liver Bioassay Lab water Cu = + CP = [15] toxicity test

Limanda A liver Bioassay Field sed Cu, Hg hydrocarbon +/- +/- - DT- [16] limanda s diphoras e =

Liza aurata J gills Bioassay Field water Cd, Cr, + + + + [17] Cu, Mn, Ni, Pb

kidney Bioassay Field water Cd, Cr, + = + = [18] Cu, Mn, Ni, Pb

liver Bioassay Field water Cd, Cr, + + = + [18] Cu, Mn, Ni, Pb

Species LHS Tissue Method Laborator Metals Other Others Referenc

y or Field contami- e

nants - GPx SOD CAT GPOX GRED LPOX Se ROS TOSC Lutjanus A blood Bioassay Field sed Cd, Cu, - + [19] russellii and water Fe, Pb, Zn

Mugil A liver Bioassay Field water Cd, Cr, - - - - + LHP +; [20] cephalus Cu, Fe, Hg, Mn, CD +; Ni, Pb, Se, Zn CP +

Mugil J gill Bioassay Lab water Pb - + [21] cephalus toxicity test

whole Bioassay Lab water Pb - + [21] toxicity test

liver Bioassay Field sed Cd, Cu, AHCs, +/- + +/- [22] and water Mn, Ni, PAHs, Pb, PCBs, DDTs, TBT

Paralichthys J gill Bioassay Lab water Cd - = - + [23] olivaceus toxicity test

kidney Bioassay Lab water Cd - = + = [23] toxicity test

liver Bioassay Lab water Cd + = - + [23] toxicity test

whole Bioassay Lab water Cd (≤12 + = + [24] toxicity test µg L-)

L meta whole Bioassay Lab water Cd (48 µg - +/- +/- [24]

Species LHS Tissue Method Laborator Metals Other Others Referenc

y or Field contami- e

nants - GPx SOD CAT GPOX GRED LPOX Se ROS TOSC toxicity test L-)

L set whole Bioassay Lab water Cd = +/- = [24] toxicity test

Platichthys A liver Bioassay Field water As, Cd, PAHs, +/- +/- +/- KMBA [25] flesus L. and sed Cr, Cu, PCBs, OCPs +/-; OP Hg, Ni, =; Pb, Zn

Poecilia A gills Bioassay Lab water Cu - = = + + ACAP = [26] vivipara toxicity test acclimated to saltwater liver Bioassay Lab water Cu - + +/- + + ACAP + [26] toxicity test

muscle Bioassay Lab water Cu = = = +/- = ACAP - [26] toxicity test

Pomadasys A blood Bioassay Field sed Cd, Cu, - + [19] hasta and water Fe, Pb, Zn

Pomatoschistu A liver Bioassay Field sed Cd, Cr, PAHs = = [27] s microps Cu, Hg, Ni, Pb, Zn

Field sed Cd, Hg, PAHS +/- +/- +/- [6] Ni, Pb, Zn

Field sed Cr, Cu, PAHs +/- +/- = = +/- [9] Ni, Pb, Zn

Rachycentron J liver Bioassay Lab food Cd - [28]

Species LHS Tissue Method Laborator Metals Other Others Referenc

y or Field contami- e

nants - GPx SOD CAT GPOX GRED LPOX Se ROS TOSC canadum

Scophthalmus J liver Bioassay Caged field Cd, Cr, - [13] maximus sed Cu, Ni, Pb, Zn

Lab field Cd, Cr, +/- [29] sed toxicity Cu, Mn, Ni, Pb, V, Zn

Seriola lalandi J RBC Bioassay Lab food Se + [30]

Solea A gill Bioassay Field sed Cd, Cr, - [31] senegalensis Cu, Fe, Hg, Pb, Zn

liver Bioassay Field sed Cr, Cu, PAHs +/- +/- = = = [9] Ni, Pb, Zn

Field water As, Cd, PAHs +/- = +/- +/- [32] and sed Cu, Fe, Pb, Zn

muscle Bioassay Field sed Cd, Cr, + [31] Cu, Fe, Hg, Pb, Zn

J blood Bioassay Caged field Cd, Cr, PAHs, + [33] sed Cu, Ni, PCBs, DDT Pb, Zn

Species LHS Tissue Method Laborator Metals Other Others Referenc

y or Field contami- e

nants - GPx SOD CAT GPOX GRED LPOX Se ROS TOSC Lab field Cd, Cr, PAHs, - [33] sed toxicity Cu, Ni, PCBs, DDT Pb, Zn

liver Bioassay Field sed Cd, Cr, PAHs = = = [34] Cu, Ni, Pb, Zn

Real time Lab and As, Cu, PAHs, +/- +/- [35] PCR field sed Zn PCBs, DDT

Solea solea A gill Bioassay Field sed Cd, Cr, = [31] Cu, Fe, Hg, Pb, Zn

muscle Bioassay Field sed Cd, Cr, = [31] Cu, Fe, Hg, Pb, Zn

Sparus aurata J liver Real time Lab field As, Cd, PAHs +/- [36] PCR sed toxicity Cr, Cu, Hg, Ni, Pb, Se, V, Zn

liver, Real time Food and Cu +/- [37] gill and PCR water lab kidney

Symphodus A blood Bioassay Field water Fe, Pb, Zn = G6PHD [38] melops and sed -M; metHB

Species LHS Tissue Method Laborator Metals Other Others Referenc

y or Field contami- e

nants - GPx SOD CAT GPOX GRED LPOX Se ROS TOSC =; CP +;

Symphodus A liver Bioassay Field water Fe, Pb, Zn = = = [38] melops and sed

Synechogobiu J gill Bioassay Lab water Cd - - - + [39] s hasta toxicity test

liver Bioassay Lab water Cd - - - + [39] toxicity test

spleen Bioassay Lab water Cd - - - + [39] toxicity test

Terapon J gill Bioassay Lab water Pb - + [21] jarbua toxicity test

liver Bioassay Lab food Cd = [40]

whole Bioassay Lab water Pb + + [21] toxicity test

Abbreviations: LHS: life history stage; A: adult, J: juvenile, L: larvae; RBC: red blood cells; meta : metamorphosing; set: settling; Lab: laboratory; Sed : Sediment; SOD: Superoxidase mutase;

CAT: Catalase; GP X : glutathione peroxidases; GR: glutathione reductase; LPO: lipid peroxidation; Se-GPx: selenium-dependent glutathione peroxidase; ROS: reactive oxygen species; TOSC: Total oxyradical scavenging capacity; + induction; - inhibition; = no significant induction; +/- mixed response; AHCs: aliphatic hydrocarbons; OCP: total organochlorine pesticides ; PAHs : total polycyclic aromatic hydrocarbons; PCBS: polychlorinated biphenyl; TBT: tributyltin; DDT: dichlorodiphenyltrichloroethane; AOX: Acyl-CoA oxidase (peroxisomal proliferation); G6PD: Glucose -6-phosphate dehydrogenase; metHB: methemoglobin; NOS: nitric oxide synthase; HIF-1: Hypoxia inducible factor ; ACAP: antioxidant capacity against peroxyls radicals; LHP: lipid hydroperoxide; CD: conjugated diene; CP: carbonyl proteins; KMBA: 2-Keto-4-methiolbutyric acid; OP: oxidised proteins; M: males only.

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S17 Table. Field and laboratory studies on responses of biomarkers of exposure in fish to metals and other contaminants: bio transformational products, stress proteins, metallothioneins and metal proteins. Most studies measured contaminants in the environment in addition to those identified as of concern for Gladstone Harbour (Al, Cd, Cu, Ga, Pb, Se, Zn); these are also presented for completeness.

Species LHS Tissue Method Laboratory Metals Other contaminants HSP70 HSP90 MT Others Reference or Field

Acanthopagrus latus A gills Bioassay Field sed As, Cr, Cu, Ni, Pb, PAHs = [1] V, Zn

liver Bioassay Field sed As, Cr, Cu, Ni, Pb, PAHs +/- [1] V, Zn

J whole Ag-saturation Food lab Zn + [2]

Ag-saturation Lab water Zn + [2] toxicity test

Anguilla anguilla J liver Bioassay Caged field As, Cd, Cr, Cu, Fe, PAHs = [3] sed Hg, Mn, Ni, Pb, V, Zn

Lab field sed As, Cd, Cr, Cu, Fe, PAHs = [3] Hg, Mn, Ni, Pb, V, Zn

Lab field sed As, Cd, Cr, Cu, PAH = [4] toxicity Hg, Ni, Pb, V, Zn

Aphanius fasciatus A gonads/liver Real time PCR Field water Cd, Cu, Zn PAHs = = [5] and sed

J bone tissue Real time PCR Lab water Cd +/- [6] toxicity test

Atherina presbyter A liver Bioassay Field sed Cd, Hg, Ni, Pb, Zn PAHs +/- [7]

Species LHS Tissue Method Laboratory Metals Other contaminants HSP70 HSP90 MT Others Reference or Field

Atherinops affnis L whole Bioassay Lab water Cd + [8] toxicity test

Chanos chanos A muscle, liver Immuno- Field water Cd, Cu, Fe, Mn, + [9] and gills histochemical and sed Pb, Zn

Coris julis A gills Immuno- Field sed Cd, Co, Cr, Cu, Ni, + + [10] histochemical Pb, Sb, Zn

In situ Field sed Cd, Co, Cr, Cu, Ni, + + [10] hybridisaton Pb, Sb, Zn

Real time PCR Field sed Cd, Co, Cr, Cu, Ni, + + [10] Pb, Sb, Zn

liver Western blot Field water As, Cd, Cr, Cu, PAHs, PCBs + +/- [11] and sed Hg, Pb, Zn

muscle Western blot Field water As, Cd, Cr, Cu, PAHs, PCBs + + [11] and sed Hg, Pb, Zn

Cynoglossus arel A gills Bioassay Field sed As, Cr, Cu, Ni, Pb, PAHs = [1] V, Zn

liver Bioassay Field sed As, Cr, Cu, Ni, Pb, PAHs +/- [1] V, Zn

Dicentrarchus labrax A liver Bioassay Field sed Cr, Cu, Ni, Pb, Zn PAHs = [12]

J gills Immuno- Lab field sed As, Cd, Co, Cr, + [13] histochemical toxicity Cu, Fe, Hg, Mn, Ni, Pb, Sb, V, Zn

Real time PCR Lab field sed As, Cd, Co, Cr, + [13] toxicity Cu, Fe, Hg, Mn,

Species LHS Tissue Method Laboratory Metals Other contaminants HSP70 HSP90 MT Others Reference or Field

Ni, Pb, Sb, V, Zn

Gadus morhua L. A liver Bioassay Cage field Cd, Cu, Hg, Pb, Zn PAHs, PCBs = [14] water

Gobius niger A liver Bioassay Lab water Cd + [15] toxicity test

Southern blot Lab water Cd + + [15] toxicity test

testis Bioassay Lab water Cd + [15] toxicity test

Southern blot Lab water Cd + + [15] toxicity test

Liza aurata J liver Bioassay Field water Cd, Cu, Hg, Zn + [16] and sed

Mullus barbatus A liver Bioassay Field sed As, Cd, Cu, Hg, PAHs, CBs, DDT, HCB, = [17] Pb, Zn trans-nonachlor, Lindane, Dieldrin

Parablennius A liver Western blot Field water Cr, Pb +/- [18] sanguinolentus and sed

Plastichthys flesus A liver Bioassay Cage field Cd, Cu, Hg, Pb, Zn PAHs, PCBs = [14] water

Field water As, Cd, Cr, Cu, PAHs, PCBs, OCPs +/- [19] and sed Hg, Ni, PbZn,

Species LHS Tissue Method Laboratory Metals Other contaminants HSP70 HSP90 MT Others Reference or Field

Poecilia vivipara A gills Bioassay Lab water Cu +/- [20] acclimated to toxicity test saltwater liver Bioassay Lab water Cu - [20] toxicity test

Pomatoschistus A liver Bioassay Field sed Cd, Cr, Cu, Hg, PAHs - [21] microps Ni, Pb, Zn,

Cd, Hg, Ni, Pb, Zn PAHS +/- [7]

Cr, Cu, Ni, Pb, Zn PAHs = [12]

Scomber scombrus A liver Bioassay Field water Cd, Cu, Hg, Ni, = [22] Pb, Zn

Solea senegalensis A liver Bioassay Field sed Cr, Cu, Ni, Pb, Zn PAHs + [12]

J liver Bioassay Field sed Cd, Cr, Cu, Ni, Pb, PAHs = [23] Zn

Lab field sed As, Cd, Cr, Cu, Ni, PAHs, PCBs, DDT = [24] Pb, Zn

Lab field sed As, Cd, Cr, Cu, PAHs = [25] toxicity Hg, Ni, Pb, Zn

Real time PCR Lab and field As, Cu, Zn PAHs, PCBs, DDT +/- +/- [26] sed

Electrophoretic Caged field Cd, Cr, Cu, Ni, Pb, PAHs, PCBs, DDT = [27] sed Zn

Solea solea A gill Bioassay Field sed Cd, Cr, Cu, Fe, + [28] Hg, Pb, Zn

Species LHS Tissue Method Laboratory Metals Other contaminants HSP70 HSP90 MT Others Reference or Field

muscle Bioassay Field sed Cd, Cr, Cu, Fe, + [28] Hg, Pb, Zn

Sparus aurata A liver Bioassay Field water Cd, Cu, Hg, Ni, + [22] Pb, Zn

Real time PCR Lab and field As, Cd, Cu, Pb, Zn = = = [29] sed

liver/kidney Chromatography Lab water Cd + [30] toxicity test

skin Real time PCR Lab and field As, Cd, Cu, Pb, Zn = - +/- [29] sed

J liver Bioassay Lab field sed As, Cd, Cr, Cu, PAHs = [25] toxicity Hg, Ni, Pb, Zn

Real time PCR Lab field sed As, Cd, Cr, Cu, PAHs + +/- [31] toxicity Hg, Ni, Pb, Se, V, Zn,

liver, gill and Real time PCR Food and Cu +/- Ctr1 [32] kidney water lab +/-

Symphodus melops A liver/blood Bioassay Field water Fe, Pb, Zn = [33] and sed

Terapon jarbua J carcass Ag-saturation Food lab Zn + [2]

Lab water Zn + [2] toxicity test

digestive tract Bioassay Lab water Cd = [34] toxicity test

Species LHS Tissue Method Laboratory Metals Other contaminants HSP70 HSP90 MT Others Reference or Field

gills Ag-saturation Food lab Zn = [2]

Lab water Zn = [2] toxicity test

Bioassay Lab water Cd = [34] toxicity test

liver Bioassay Lab water Cd + [34] toxicity test

viscera Ag-saturation Food lab Zn + [2]

Lab water Zn + [2] toxicity test

whole Ag-saturation Food lab Zn + [2]

Lab water Zn + [2] toxicity test

Thunnus thynnus A liver Bioassay Field water Cd, Cu, Hg, Ni, + [22] Pb, Zn

Abbreviations: LHS: life history stage; A: adult, J: juveniles; Lab: laboratory; Sed : Sediment; AHCs: aliphatic hydrocarbons; HCB - hexachlorobenzene ; OCP: total organochlorine pesticides; CB: chlorinated biphenyls; naph: naphthalenes; PAHs: total polycyclic aromatic hydrocarbons ; PCBS: polychlorinated biphenyl; TBT: tributyltin; DBT: dibutytin; DDD: 1,1-dichloro-2.2- bis(p-chlorophenyl) ethane; DDE : 1,1-dichloro-2.2-bis(p-chlorophenyl) ethylene; HCH: hexachlorcyclohexane; DDT: dichlorodiphenyltrichloroethane;HCB: hexachlorobenzene; HSP70: Heat shock protein 70; HSP90: Heat shock protein 90; MT: Metallothionein; + induction; - inhibition; = no significant induction; +/- mixed response; Ctr1: copper transporter.

References 1. Beg MU, Al-Jandal N, Al-Subiai S, Karam Q, Husain S, Butt SA, et al. Metallothionein, oxidative stress and trace metals in gills and liver of demersal and pelagic fish species from Kuwaits’ marine area. Mar Pollut Bull. 2015; 100: 662-72. doi: 10.1016/j.marpolbul.2015.07.058 2. Zhang L, Wang WX. Effects of Zn pre-exposure on Cd and Zn bioaccumulation and metallothionein levels in two species of marine fish. Aquat Toxicol. 2005; 73: 353-69. doi: 10.1016/j.aquatox.2005.04.001 PMID: 000230798000003 3. Piva F, Ciaprini F, Onorati F, Benedetti M, Fattorini D, Ausili A, et al. Assessing sediment hazard through a weight of evidence approach with bioindicator organisms: a practical model to elaborate data from sediment chemistry, bioavailability, biomarkers and ecotoxicological bioassays. Chemosphere. 2011; 83: 475-85. doi: 10.1016/j.chemosphere.2010.12.064 PMID: 21239037 4. Benedetti M, Ciaprini F, Piva F, Onorati F, Fattorini D, Notti A, et al. A multidisciplinary weight of evidence approach for classifying polluted sediments: Integrating sediment chemistry, bioavailability, biomarkers responses and bioassays. Environ Int. 2012; 38: 17-28. doi: 10.1016/j.envint.2011.08.003 PMID: 21982029 5. Annabi A, Kessabi K, Navarro A, Said K, Messaoudi I, Pina B. Assessment of reproductive stress in natural populations of the fish Aphanius fasciatus using quantitative mRNA markers. Aquat Biol. 2012; 17: 285-+. doi: 10.3354/ab00482 PMID: 000312247800008 6. Kessabi K, Annabi A, Navarro A, Casado M, Hwas Z, Said K, et al. Structural and molecular analysis of pollution-linked deformities in a natural Aphanius fasciatus (Valenciennes, 1821) population from the Tunisian coast. J Environ Monitor. 2012; 14: 2254-60. doi: 10.1039/c2em30329a PMID: 000306852100028 7. Fonseca VF, Vasconcelos RP, Franca S, Serafim A, Lopes B, Company R, et al. Modeling fish biological responses to contaminants and natural variability in estuaries. Mar Environ Res. 2014; 96: 45-55. doi: 10.1016/j.marenvres.2013.10.011 PMID: 000334981600007 8. Rose WL, Nisbet RM, Green PG, Norris S, Fan T, Smith EH, et al. Using an integrated approach to link biomarker responses and physiological stress to growth impairment of cadmium-exposed larval topsmelt. Aquat Toxicol. 2006; 80: 298-308. doi: 10.1016/j.aquatox.2006.09.007 PMID: 000242776900010 9. Rajeshkumar S, Munuswamy N. Impact of metals on histopathology and expression of HSP 70 in different tissues of Milk fish (Chanos chanos) of Kaattuppalli Island, South East Coast, India. Chemosphere. 2011; 83: 415-21. doi: 10.1016/j.chemosphere.2010.12.086 PMID: 21257190 10. Fasulo S, Mauceri A, Maisano M, Giannetto A, Parrino V, Gennuso F, et al. Immunohistochemical and molecular biomarkers in Coris julis exposed to environmental contaminants. Ecotoxicol Environ Saf. 2010; 73: 873-82. doi: 10.1016/j.ecoenv.2009.12.025 PMID: 000279623800023 11. Tomasello B, Copat C, Pulvirenti V, Ferrito V, Ferrante M, Renis M, et al. Biochemical and bioaccumulation approaches for investigating marine pollution using Mediterranean rainbow wrasse, Coris julis (Linneaus 1798). Ecotoxicol Environ Saf. 2012; 86: 168-75. doi: 10.1016/j.ecoenv.2012.09.012 PMID: 000311064800024 12. Fonseca VF, Franca S, Serafim A, Company R, Lopes B, Bebianno MJ, et al. Multi- biomarker responses to estuarine habitat contamination in three fish species: Dicentrarchus labrax, Solea senegalensis and Pomatoschistus microps. Aquat Toxicol. 2011; 102: 216-27. doi: 10.1016/j.aquatox.2011.01.018 PMID: 21356184

13. De Domenico E, Mauceri A, Giordano D, Maisano M, Gioffre G, Natalotto A, et al. Effects of "in vivo" exposure to toxic sediments on juveniles of sea bass (Dicentrarchus labrax). Aquat Toxicol. 2011; 105: 688-97. doi: 10.1016/j.aquatox.2011.08.026 PMID: 000298120600055 14. Beyer J, Sandvik M, Hylland K, Fjeld E, Egaas E, Aas E, et al. Contaminant accumulation and biomarker responses in flounder (Platichthys flesus L) and Atlantic cod (Gadus morhua L) exposed by caging to polluted sediments in Sorfjorden, Norway. Aquat Toxicol. 1996; 36: 75-98. doi: 10.1016/s0166-445x(96)00798-9 PMID: A1996VY98200005 15. Migliarini B, Campisi AM, Maradonna F, Truzzi C, Annibaldi A, Scarponi G, et al. Effects of cadmium exposure on testis apoptosis in the marine teleost Gobius niger. Gen Comp Endocrinol. 2005; 142: 241-7. doi: 10.1016/j.ygcen.2004.12.012 PMID: 15862569 16. Oliveira M, Ahmad I, Maria VL, Serafim A, Bebianno MJ, Pacheco M, et al. Hepatic metallothionein concentrations in the golden grey mullet (Liza aurata) - Relationship with environmental metal concentrations in a metal-contaminated coastal system in Portugal. Mar Environ Res. 2010; 69: 227-33. doi: 10.1016/j.marenvres.2009.10.012 PMID: 000275342300003 17. Martinez-Gomez C, Fernandez B, Benedicto J, Valdes J, Campillo JA, Leon VM, et al. Health status of red mullets from polluted areas of the Spanish Mediterranean coast, with special reference to Portman (SE Spain). Mar Environ Res. 2012; 77: 50- 9. doi: 10.1016/j.marenvres.2012.02.002 PMID: 000304296700008 18. Tigano C, Tomasello B, Pulvirenti V, Ferrito V, Copat C, Carpinteri G, et al. Assessment of environmental stress in Parablennius sanguinolentus (Pallas, 1814) of the Sicilian Ionian coast. Ecotoxicol Environ Saf. 2009; 72: 1278-86. doi: 10.1016/j.ecoenv.2008.09.028 PMID: 000265767900037 19. Schipper CA, Lahr J, van den Brink PJ, George SG, Hansen P-D, de Assis HCdS, et al. A retrospective analysis to explore the applicability of fish biomarkers and sediment bioassays along contaminated salinity transects. Ices J Mar Sci. 2009; 66: 2089-105. doi: 10.1093/icesjms/fsp194 PMID: 000272080600003 20. de Souza Machado AA, Mueller Hoff ML, Klein RD, Cardozo JG, Giacomin MM, Ledes Pinho GL, et al. Biomarkers of waterborne copper exposure in the guppy Poecilia vivipara acclimated to salt water. Aquat Toxicol. 2013; 138: 60-9. doi: 10.1016/j.aquatox.2013.04.009. PMID: 000322293600007 21. Serafim A, Company R, Lopes B, Fonseca VF, Franca S, Vasconcelos RP, et al. Application of an integrated biomarker response index (IBR) to assess temporal variation of environmental quality in two Portuguese aquatic systems. Ecol Indic. 2012; 19: 215-25. doi: 10.1016/j.ecolind.2011.08.009 PMID: 000302891100022 22. Papetti P, Rossi G. Heavy metals in the fishery products of low Lazio and the use of metallothionein as a biomarker of contamination. Environ Monit Ass. 2009; 159: 589- 98. doi: 10.1007/s10661-008-0725-4 PMID: 000271530400046 23. Fonseca VF, Vasconcelos RP, Tanner SE, Franca S, Serafim A, Lopes B, et al. Habitat quality of estuarine nursery grounds: Integrating non-biological indicators and multilevel biological responses in Solea senegalensis. Ecol Indic. 2015; 58: 335-45. doi: 10.1016/j.ecolind.2015.05.064 PMID: 000360776100035 24. Costa PM, Caeiro S, Diniz MS, Lobo J, Martins M, Ferreira AM, et al. Biochemical endpoints on juvenile Solea senegalensis exposed to estuarine sediments: the effect of contaminant mixtures on metallothionein and CYP1A induction. Ecotoxicol. 2009; 18: 988-1000. doi: 10.1007/s10646-009-0373-7 PMID: 000269917200004

25. Jimenez-Tenorio N, Morales-Caselles C, Kalman J, Salamanca MJ, Luisa Gonzalez de Canales M, Sarasquete C, et al. Determining sediment quality for regulatory proposes using fish chronic bioassays. Environ Internat. 2007; 33: 474-80. doi: 10.1016/j.envint.2006.11.009 PMID: 000246315800008 26. Costa PM, Caeiro S, Vale C, Angel DelValls T, Costa MH. Can the integration of multiple biomarkers and sediment geochemistry aid solving the complexity of sediment risk assessment? A case study with a benthic fish. Environ Pollut. 2012; 161: 107-20. doi: 10.1016/j.envpol.2011.10.010 PMID: 000300539300016 27. Costa PM, Repolho T, Caeiro S, Diniz ME, Moura I, Costa MH. Modelling metallothionein induction in the liver of Sparus aurata exposed to metal- contaminated sediments. Ecotoxicol Environ Saf. 2008; 71: 117-24. doi: 10.1016/j.ecoenv.2007.05.012 PMID: 000258550400014 28. Siscar R, Torreblanca A, Palanques A, Sole M. Metal concentrations and detoxification mechanisms in Solea solea and Solea senegalensis from NW Mediterranean fishing grounds. Mar Pollut Bull. 2013; 77: 90-9. doi: 10.1016/j.marpolbul.2013.10.026 PMID: 000329888600025 29. Benhamed S, Guardiola FA, Martínez S, Martínez-Sánchez MJ, Pérez-Sirvent C, Mars M, et al. Exposure of the gilthead seabream (Sparus aurata) to sediments contaminated with heavy metals down-regulates the gene expression of stress biomarkers. Toxicol Rep. 2016; 3: 364-72. doi:10.1016/j.toxrep.2016.02.006 30. Isani G, Andreani G, Cocchioni F, Fedeli D, Carpene E, Falcioni G. Cadmium accumulation and biochemical responses in Sparus aurata following sub-lethal Cd exposure. Ecotoxicol Environ Saf. 2009; 72: 224-30. doi: 10.1016/j.ecoenv.2008.04.015 PMID: 000260660100028 31. Ribecco C, Baker ME, Sasik R, Zuo Y, Hardiman G, Carnevali O. Biological effects of marine contaminated sediments on Sparus aurata juveniles. Aquat Toxicol. 2011; 104: 308-16. doi: 10.1016/j.aquatox.2011.05.005 PMID: 000293042100017 32. Minghetti M, Leaver MJ, Carpene E, George SG. Copper transporter 1, metallothionein and glutathione reductase genes are differentially expressed in tissues of sea bream (Sparus aurata) after exposure to dietary or waterborne copper. Comp Biochem Phys C. 2008; 147: 450-9. doi: 10.1016/j.cbpc.2008.01.014 PMID: 18304880 33. Almroth BC, Sturve J, Stephensen E, Holth TF, Forlin L. Protein carbonyls and antioxidant defenses in corkwing wrasse (Symphodus melops) from a heavy metal polluted and a PAH polluted site. Mar Environ Res. 2008; 66: 271-7. doi: 10.1016/j.marenvres.2008.04.002 PMID: 000257817100006 34. Dang F, Wang W-X. Assessment of tissue-specific accumulation and effects of cadmium in a marine fish fed contaminated commercially produced diet. Aquat Toxicol. 2009; 95: 248-55. doi: 10.1016/j.aquatox.2009.09.013 PMID: 000272784900009

S18 Table. Field and laboratory studies on responses of biomarkers of exposure in fish to metals and other contaminants: haematological and immunological parameters. Most studies measured contaminants in the environment in addition to those identified as of concern for Gladstone Harbour (Al, Cd, Cu, Ga, Pb, Se, Zn); these are also presented for completeness.

Species LHS Laboratory Metals Other Others Reference

or Field contaminants

T / AST AL Haematocrit EC ALP Total protein Albumin Albumin /Ʃprotein PCNA Anguilla J Caged field As, Cd, Cr, Cu, PAHs Lys - [1] anguilla sed Fe, Hg, Mn, Ni, Pb, V, Zn

Lab field sed As, Cd, Cr, Cu, PAHs Lys - [1] Fe, Hg, Mn, Ni, Pb, V, Zn

Lab field sed As, Cd, Cr, Cu, PAH LY 1and2 - [2] toxicity Hg, Ni, Pb, V, Zn

Coris julis A Field sed Cd, Co, Cr, Cu, + CBP - [3] Ni, Pb, Sb,

Dicentrarchus A Lab water Cu antifouling TCAS =; [4] labrax toxicity test Haemoglobin +; RB =

J Lab field sed As, Cd, Cr, Cu, PAHs and PCBs + PCNA RT + [5] toxicity Hg, Ni, Pb, Zn

Lab field sed As, Cd, Cr, Cu, PAHs and PCBs [6] toxicity Hg, Ni, Pb, Zn

Gadus morhua A Cage field Cd, Cu, Hg, Pb, PAHs, PCBs = = = +/- +/- = +/- % leukocytes = ; [7] L. water Zn % thrombocytes

Species LHS Laboratory Metals Other Others Reference

or Field contaminants

T / AST AL Haematocrit EC ALP Total protein Albumin Albumin /Ʃprotein PCNA +/-; Glucose =

Lutjanus A Field sed and Cd, Cu, Fe, Pb, - - = - [8] russellii water Zn

Mugil cephalus A Field water Cd, Cr, Cu, Fe, CV - [9] Hg, Mn, Ni, Pb, Se, Zn

Plastichthys A Cage field Cd, Cu, Hg, Pb, PAHs, PCBs +/- = +/- +/- +/- = Glucose = [7] flesus water Zn

Field sed Al, Cd, Cr, Cu, PCB,DDD,DDE, MAM -; [10] Fe, Hg, Mn, Pb, HCH Zn, LY1and2 -

Cd, Hg, Pb, Zn PAHs, PCBs Lys +; [11]

MMC +

Pomadasys A Field sed and Cd, Cu, Fe, Pb, - - = - [8] hasta water Zn

Rachycentron J Lab food Cd + + + + Glucose + [12] canadum

Scophthalmus J Lab field sed Cd, Cr, Cu, Mn, TV =; [13] maximus toxicity Ni, Pb, V, Zn TCF-b1 +/-;

Lys +/-;

Species LHS Laboratory Metals Other Others Reference

or Field contaminants

T / AST AL Haematocrit EC ALP Total protein Albumin Albumin /Ʃprotein PCNA MMC =/-

Seriola lalandi J Lab food Se - MA + [14]

Solea J Caged field Cd, Cr, Cu, Ni, PAHs, PCBs, = % leukocytes -; [15] senegalensis sed Pb, Zn DDT % thrombocytes =

Solea J Lab field sed Cd, Cr, Cu, Ni, PAHs, PCBs, = % leukocytes - ; [15] senegalensis toxicity Pb, Zn DDT % thrombocytes =

Sparus aurata J Lab water Cu Pro-S + [16] toxicity test

Squalus A Lab water Pb Na -; [17] acanthias toxicity test Cl -

Symphodus A Field water Fe, Pb, Zn, + [18] melops and sed

Synechogobius J Lab water Cd MMC + [19] hasta toxicity test

Abbreviations: LHS: life history stage; J: juvenile, A: adult: Lab: laboratory; Sed : Sediment; PAHs - total polycyclic aromatic hydrocarbons; PCBS: polychlorinated biphenyl; DDD - 1,1- dichloro-2.2-bis(p-chlorophenyl) ethane; DDE - 1,1-dichloro-2.2-bis(p-chlorophenyl) ethylene; HCH - hexachlorcyclohexane; DDT: dichlorodiphenyltrichloroethane; ALP: Alkaline phosphatase; AST aspartate aminotransferase; EC: Erthrocyte count; PCNA - proliferating cell nuclear antigen; + induction; - inhibition; = no significant induction; +/- mixed response; CBP - calcium binding proteins; RB - respiratory burst ; TCAS - total complementary activity of serum; LY1 and 2 - Stability of lysosomes 1 and 2; Lys - Lysozyme activity; MAM - macrophage aggregate activity ; Pro-S: Proteomics serum; TCF-b1: cytokine transforming growth; factor - β1; MMC: melanomacrophage centres; TV: Thymus volume; CV: cell viability.

References 1. Piva F, Ciaprini F, Onorati F, Benedetti M, Fattorini D, Ausili A, et al. Assessing sediment hazard through a weight of evidence approach with bioindicator organisms: a practical model to elaborate data from sediment chemistry, bioavailability, biomarkers and ecotoxicological bioassays. Chemosphere. 2011; 83: 475-85. doi: 10.1016/j.chemosphere.2010.12.064 PMID: 21239037 2. Benedetti M, Ciaprini F, Piva F, Onorati F, Fattorini D, Notti A, et al. A multidisciplinary weight of evidence approach for classifying polluted sediments: Integrating sediment chemistry, bioavailability, biomarkers responses and bioassays. Environ Int. 2012; 38: 17-28. doi: 10.1016/j.envint.2011.08.003 PMID: 21982029 3. Fasulo S, Mauceri A, Maisano M, Giannetto A, Parrino V, Gennuso F, et al. Immunohistochemical and molecular biomarkers in Coris julis exposed to environmental contaminants. Ecotoxicol Environ Saf. 2010; 73: 873-82. doi: 10.1016/j.ecoenv.2009.12.025 PMID: 000279623800023 4. Cotou E, Henry M, Zeri C, Rigos G, Torreblanca A, Catsiki V-A. Short-term exposure of the European sea bass Dicentrarchus labrax to copper-based antifouling treated nets: Copper bioavailability and biomarkers responses. Chemosphere. 2012; 89: 1091-7. doi:10.1016/j.chemosphere.2012.05.075 5. De Domenico E, Mauceri A, Giordano D, Maisano M, Giannetto A, Parrino V, et al. Biological responses of juvenile European sea bass (Dicentrarchus labrax) exposed to contaminated sediments. Ecotoxicol Environ Saf. 2013; 97: 114-23. doi: 10.1016/j.ecoenv.2013.07.015 PMID: 000325039400015 6. De Domenico E, Mauceri A, Giordano D, Maisano M, Gioffre G, Natalotto A, et al. Effects of "in vivo" exposure to toxic sediments on juveniles of sea bass (Dicentrarchus labrax). Aquat Toxicol. 2011; 105: 688-97. doi: 10.1016/j.aquatox.2011.08.026 PMID: 000298120600055 7. Beyer J, Sandvik M, Hylland K, Fjeld E, Egaas E, Aas E, et al. Contaminant accumulation and biomarker responses in flounder (Platichthys flesus L) and Atlantic cod (Gadus morhua L) exposed by caging to polluted sediments in Sorfjorden, Norway. Aquat Toxicol. 1996; 36: 75-98. doi: 10.1016/s0166-445x(96)00798-9 PMID: A1996VY98200005 8. Omar WA, Saleh YS, Marie M-AS. The use of biotic and abiotic components of Red Sea coastal areas as indicators of ecosystem health. Ecotoxicol. 2016; 25: 253-66. doi: 10.1007/s10646-015-1584-8 PMID: 000370716000001 9. Padmini E, Rani MU. Evaluation of oxidative stress biomarkers in hepatocytes of grey mullet inhabiting natural and polluted estuaries. Sci Total Environ. 2009; 407: 4533-41. doi: 10.1016/j.scitotenv.2009.04.005 PMID: 000267631700019 10. Schmidt V, Zander S, Korting W, Broeg K, von Westernhagen H, Dizer H, et al. Parasites of flounder (Platichthys flesus L.) from the German Bight, North Sea, and their potential use in biological effects monitoring - C. Pollution effects on the parasite community and a comparison to biomarker responses. Helgoland Mar Res. 2003; 57: 262-71. doi: 10.1007/s10152-003-0159-x PMID: 000186604600015 11. Vethaak AD, Jol JG, Meijboom A, Eggens ML, apRheinallt T, Wester PW, et al. Skin and liver diseases induced in flounder (Platichthys flesus) after long-term exposure to contaminated sediments in large-scale mesocosms. Environ Health Persp. 1996; 104: 1218-29. doi: 10.2307/3432916 PMID: A1996VX74000021 12. Liu K, Chi S, Liu H, Dong X, Yang Q, Zhang S, et al. Toxic effects of two sources of dietborne cadmium on the juvenile cobia, Rachycentron canadum L. and tissue- specific accumulation of related minerals. Aquat Toxicol. 2015; 165: 120-8. doi: 10.1016/j.aquatox.2015.05.013 PMID: 000359030300013

13. Kerambrun E, Henry F, Marechal A, Sanchez W, Minier C, Filipuci I, et al. A multibiomarker approach in juvenile turbot, Scophthalmus maximus, exposed to contaminated sediments. Ecotoxicol Environ Saf. 2012; 80: 45-53. doi: 10.1016/j.ecoenv.2012.02.010 PMID: 000304337300007 14. Ky Trung L, Fotedar R. Toxic effects of excessive levels of dietary selenium in juvenile yellowtail kingfish (Seriola lalandi). Aquacult. 2014; 433: 229-34. doi: 10.1016/j.aquaculture.2014.06.021 PMID: 000342529400033 15. Costa PM, Neuparth TS, Caeiro S, Lobo J, Martins M, Ferreira AM, et al. Assessment of the genotoxic potential of contaminated estuarine sediments in fish peripheral blood: Laboratory versus in situ studies. Environ Res. 2011; 111: 25-36. doi: 10.1016/j.envres.2010.09.011 PMID: 000286715300005 16. Isani G, Andreani G, Carpene E, Di Molfetta S, Eletto D, Spisni E. Effects of waterborne Cu exposure in gilthead sea bream (Sparus aurata): A proteomic approach. Fish Shellfish Immun. 2011; 31: 1051-8. doi: 10.1016/j.fsi.2011.09.005 PMID: 000298569700041 17. Eyckmans M, Lardon I, Wood CM, De Boeck G. Physiological effects of waterborne lead exposure in spiny dogfish (Squalus acanthias). Aquat Toxicol. 2013; 126: 373- 81. doi: 10.1016/j.aquatox.2012.09.004 PMID: 000315125600040 18. Almroth BC, Sturve J, Stephensen E, Holth TF, Forlin L. Protein carbonyls and antioxidant defenses in corkwing wrasse (Symphodus melops) from a heavy metal polluted and a PAH polluted site. Mar Environ Res. 2008; 66: 271-7. doi: 10.1016/j.marenvres.2008.04.002 PMID: 000257817100006 19. Liu XJ, Luo Z, Li CH, Xiong BX, Zhao YH, Li XD. Antioxidant responses, hepatic intermediary metabolism, histology and ultrastructure in Synechogobius hasta exposed to waterborne cadmium. Ecotoxicol Environ Saf. 2011; 74: 1156-63. doi: 10.1016/j.ecoenv.2011.02.015 PMID: 000291960600007

S19 Table. Field and laboratory studies on responses of biomarkers of exposure in fish to metals and other contaminants: reproductive, endocrine and neurotoxic parameters. Most studies measured contaminants in the environment in addition to those identified as of concern for Gladstone Harbour (Al, Cd, Cu, Ga, Pb, Se, Zn); these are also presented for completeness.

Species LHS Tissue Method Laboratory or Metals Other ChE Other Reference Field contaminants

Anguilla anguilla Glass eels liver Bioassay Field sed Cd, Cr, Cu, Hg, Ni, Pb, V, Zn, PAH - [1]

J liver Bioassay Caged field sed As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, V, PAHs = [2] Zn

Lab field sed As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, V, PAHs = [2] Zn

Lab field sed As, Cd, Cr, Cu, Hg, Ni, Pb, V, Zn PAH = [3] toxicity

Yellow liver Bioassay Field sed Cd, Cr, Cu, Hg, Ni, Pb, V, Zn, PAH - [1] eels

Aphanius fasciatus A liver Real time PCR Field water and sed Cd, Cu, Zn PAHs VTG + [4]

Coris julis A gills Immuno- Field sed Cd, Co, Cr, Cu, Ni, Pb, Sb, Zn 5-HT3 [5] histochemical +;

5HT -

Dicentrarchus J gills Immuno- Lab field sed As, Cd, Cr, Cu, Hg, Ni, Pb, Zn PAHs and PCBs - ChAT -; [6] labrax histochemical toxicity TH +;

5-HT -

Real time PCR Lab field sed As, Cd, Cr, Cu, Hg, Ni, Pb, Zn PAHs and PCBs - 5-HT3 = [6] toxicity

Lates calcarifer A muscle Bioassay Field sed Cd, Cr, Cu, Ni, Zn Diuron, PAHs - [7]

Species LHS Tissue Method Laboratory or Metals Other ChE Other Reference Field contaminants

Platichthys flesus L. A liver Bioassay Field sed Cd, Cu, Hg, Pb PCBs = [8])

Field water and sed As, Cd, Cr, Cu, Hg, Ni, Pb, Zn PAHs, PCBs, OCPs +/- VTG +/- [9]

Solea senegalensis A brain Bioassay Field sed Cd, Cr, Cu, Fe, Hg, Pb, Zn = [10]

gill Bioassay Field sed Cd, Cr, Cu, Fe, Hg, Pb, Zn + [10]

muscle Bioassay Field sed Cd, Cr, Cu, Fe, Hg, Pb, Zn + [10]

Field water and sed As, Cd, Cu, Fe, Pb, Zn PAHs +/- [11]

Solea solea A brain Bioassay Field sed Cd, Cr, Cu, Fe, Hg, Pb, Zn + [10]

gill Bioassay Field sed Cd, Cr, Cu, Fe, Hg, Pb, Zn = [10]

muscle Bioassay Field sed Cd, Cr, Cu, Fe, Hg, Pb, Zn - [10]

Sparus aurata J liver Real time PCR Lab field sed As, Cd, Cr, Cu, Hg, Ni, Pb, Se, V, Zn PAHs TRα + [12] toxicity

Abbreviations: LHS: life history stage; J: juvenile, A: adult: Lab: laboratory; Sed : Sediment; PAHs - total polycyclic aromatic hydrocarbons; PCBS: polychlorinated biphenyl; OCP: total organochlorine pesticides; ChE: choline esterase; + induction; - inhibition; = no significant induction; +/- mixed response; VTG: Vitellogenin; TH: tyrosine hydroxylase; ChAT: Choline AcetylTransferase; ChE: AcetylCholineEsterase; 5-HT: Serotonin; 5-HT3: Serotonin receptor; TRα: thyroid receptor alpha.

References 1. Gravato C, Guimaraes L, Santos J, Faria M, Alves A, Guilhermino L. Comparative study about the effects of pollution on glass and yellow eels (Anguilla anguilla) from the estuaries of Minho, Lima and Douro Rivers (NW Portugal). Ecotoxicol Environ Saf. 2010; 73: 524-33. doi: 10.1016/j.ecoenv.2009.11.009 PMID: 000277103600009 2. Piva F, Ciaprini F, Onorati F, Benedetti M, Fattorini D, Ausili A, et al. Assessing sediment hazard through a weight of evidence approach with bioindicator organisms: a practical model to elaborate data from sediment chemistry, bioavailability, biomarkers and ecotoxicological bioassays. Chemosphere. 2011; 83: 475-85. doi: 10.1016/j.chemosphere.2010.12.064 PMID: 21239037 3. Benedetti M, Ciaprini F, Piva F, Onorati F, Fattorini D, Notti A, et al. A multidisciplinary weight of evidence approach for classifying polluted sediments: Integrating sediment chemistry, bioavailability, biomarkers responses and bioassays. Environ Int. 2012; 38: 17-28. doi: 10.1016/j.envint.2011.08.003 PMID: 21982029 4. Annabi A, Kessabi K, Navarro A, Said K, Messaoudi I, Pina B. Assessment of reproductive stress in natural populations of the fish Aphanius fasciatus using quantitative mRNA markers. Aquat Biol. 2012; 17: 285-+. doi: 10.3354/ab00482 PMID: 000312247800008 5. Fasulo S, Mauceri A, Maisano M, Giannetto A, Parrino V, Gennuso F, et al. Immunohistochemical and molecular biomarkers in Coris julis exposed to environmental contaminants. Ecotoxicol Environ Saf. 2010; 73: 873-82. doi: 10.1016/j.ecoenv.2009.12.025 PMID: 000279623800023 6. De Domenico E, Mauceri A, Giordano D, Maisano M, Giannetto A, Parrino V, et al. Biological responses of juvenile European sea bass (Dicentrarchus labrax) exposed to contaminated sediments. Ecotoxicol Environ Saf. 2013; 97: 114-23. doi: 10.1016/j.ecoenv.2013.07.015 PMID: 000325039400015 7. Humphrey CA, King SC, Klumpp DW. A multibiomarker approach in barramundi (Lates calcarifer) to measure exposure to contaminants in estuaries of tropical North Queensland. Mar Pollut Bull. 2007; 54: 1569-81. doi: 10.1016/j.marpolbul.2007.06.004 PMID: 000250599700014 8. Schmidt V, Zander S, Korting W, Broeg K, von Westernhagen H, Dizer H, et al. Parasites of flounder (Platichthys flesus L.) from the German Bight, North Sea, and their potential use in biological effects monitoring - C. Pollution effects on the parasite community and a comparison to biomarker responses. Helgoland Mar Res. 2003; 57: 262-71. doi: 10.1007/s10152-003-0159-x PMID: 000186604600015 9. Schipper CA, Lahr J, van den Brink PJ, George SG, Hansen P-D, de Assis HCdS, et al. A retrospective analysis to explore the applicability of fish biomarkers and sediment bioassays along contaminated salinity transects. Ices J Mar Sci. 2009; 66: 2089-105. doi: 10.1093/icesjms/fsp194 PMID: 000272080600003 10. Siscar R, Torreblanca A, Palanques A, Sole M. Metal concentrations and detoxification mechanisms in Solea solea and Solea senegalensis from NW Mediterranean fishing grounds. Mar Pollut Bull. 2013; 77: 90-9. doi: 10.1016/j.marpolbul.2013.10.026 PMID: 000329888600025 11. Oliva M, Antonio Perales J, Gravato C, Guilhermino L, Dolores Galindo-Riano M. Biomarkers responses in muscle of Senegal sole (Solea senegalensis) from a heavy metals and PAHs polluted estuary. Mar Pollut Bull. 2012; 64: 2097-108. doi: 10.1016/j.marpolbul.2012.07.017 PMID: 000310929500028 12. Ribecco C, Baker ME, Sasik R, Zuo Y, Hardiman G, Carnevali O. Biological effects of marine contaminated sediments on Sparus aurata juveniles. Aquat Toxicol. 2011; 104: 308-16. doi: 10.1016/j.aquatox.2011.05.005 PMID: 000293042100017

S20 Table. Field and laboratory studies on responses of biomarkers of exposure in fish to metals and other contaminants: genotoxic parameters. Most studies measured contaminants in the environment in addition to those identified as of concern for Gladstone Harbour (Al, Cd, Cu, Ga, Pb, Se, Zn); these are also presented for completeness.

Species LHS Laboratory Metals Other contaminants Apoptosis / Comet Micronucleus and Others Reference or Field Caspase nuclear abnormalities

Aldrichetta forsteri A Field sed Cd, Cu, Pb + [1]

Anguilla anguilla J Lab field sed As, Cd, Cr, Cu, Fe, PAHs + [2] Hg, Mn, Ni, Pb, V, Zn

As, Cd, Cr, Cu, PAH - + [3] Hg, Ni, Pb, V, Zn

Atherinops affnis L Lab water Cd + [4] toxicity test

Centropomus J Field sed and Ag, Al, As, Cd, Cr, = [5] parallelus water Cu, Fe, Hg, Mn, Ni, Pb, Se, Zn

Lab water Cu - + [6] toxicity test

Coris julis A Field sed Cd, Co, Cr, Cu, Ni, + Fas ligand + [7] Pb, Sb, Zn

Field water As, Cd, Cr, Cu, PAHs, PCBs + + Fpg-comet + [8] and sed Hg, Pb, Zn

Dicentrarchus labrax J Lab field sed As, Cd, Cr, Cu, PAHs and PCBs Fas ligand RT +; [9] toxicity Hg, Ni, Pb, Zn Fas ligand -

Epinephelus coioides J Lab field sed Cu PAHs + [10]

Gobius niger A Lab water Cd + [11] toxicity test

Lates calcarifer A Field sed Cd, Cr, Cu, Ni, Zn Diuron, PAHs DNA unwinding + [12]

Mugil cephalus A Field sed and Cu, Fe, Mn, Pb, Zn + + [13] water

J Field sed and Cd, Cu, Mn, Ni, AHCs, PAHs, PCBs, DDTs, + [14] water Pb TBT

Mullus barbatus A Field sed As, Cd, Cu, Hg, PAHs, CBs, DDT, HCB, + [15] Pb, Zn trans-nonachlor, Lindane, Dieldrin

Parablennius A Field water Cr, Pb PAHs +/- [16] sanguinolentus and sed

Plastichthys flesus A Field sed Al, Cd, Cr, Cu, Fe, PCB,DDD,DDE, HCH DNA unwinding = [17] Hg, Mn, Pb, Zn

Field sed Cd, Hg, Pb, Zn PAHs, PCBs DNA adducts + [18] PAHs

Lab field sed As, Cr, Cu, Hg, Ni, HCB, OCP, CB, naph, + Transcriptomics +; [19] Pb, Zn PAHs, TBT, DBT Metabolomics +

Field water As, Cd, Cr, Cu, PAHs, PCBs, OCPs DNA adducts = [20] and sed Hg, Ni, Pb, Zn

J Lab field sed As, Cr, Cu, Hg, Ni, PAHs, DDT, HCB, CB, Microarray +/- [21] toxicity Pb, Zn TBT, DBT

Poecilia vivipara A Lab water Cu +/- + [22] acclimated to toxicity test saltwater

Scophthalmus J Lab field sed Cu, Pb, Zn + [23] maximus

Sillago schomburgkii, A Field sed Cd, Cu, Pb + [1]

Solea senegalensis J Caged field Cd, Cr, Cu, Ni, Pb, PAHs, PCBs, DDT + + [24] sed Zn

Lab and field As, Cu, Zn PAHs, PCBs, DDT +/- +/- + [25] sed

Lab field sed Cd, Cr, Cu, Ni, Pb, PAHs, PCBs, DDT + + [26] Zn

Cd, Cr, Cu, Ni, Pb, PAHs, PCBs, DDT + + [24] Zn

Sparus aurata A Lab water Cd = [27] toxicity test

J Lab field sed As, Cd, Cr, Cu, PAHs microarray + [28] Hg, Ni, Pb, Se, V, Zn

Abbreviations: LHS: life history stage; J: juvenile, A: adult: Lab: laboratory; Sed: Sediment; HCB: hexachlorobenzene; OCP: total organochlorine pesticides; CB: chlorinated biphenyls; naph: naphthalenes; PAHs : total polycyclic aromatic hydrocarbons; PCBS: polychlorinated biphenyl; TBT: tributyltin; DBT: dibutytin; DDD: 1,1-dichloro-2.2-bis(p-chlorophenyl) ethane; DDE: 1,1- dichloro-2.2-bis(p-chlorophenyl) ethylene; HCH: hexachlorcyclohexane; DDT: dichlorodiphenyltrichloroethane; HCB: hexachlorobenzene; 8-OHdG: 8-hydroxy-2'-deoxyguanosine; + induction; - inhibition; = no significant induction; +/- mixed response; Fpg-comet: formamido pyrimidine glycosylase.

References 1. Edwards JW, Edyvane KS, Boxall VA, Hamann M, Soole KL. Metal levels in seston and marine fish flesh near industrial and metropolitan centres in South Australia. Mar Pollut Bull. 2001; 42: 389-96. doi: 10.1016/s0025-326x(00)00168-5 PMID: 000169401400018 2. Piva F, Ciaprini F, Onorati F, Benedetti M, Fattorini D, Ausili A, et al. Assessing sediment hazard through a weight of evidence approach with bioindicator organisms: a practical model to elaborate data from sediment chemistry, bioavailability, biomarkers and ecotoxicological bioassays. Chemosphere. 2011; 83: 475-85. doi: 10.1016/j.chemosphere.2010.12.064 PMID: 21239037 3. Benedetti M, Ciaprini F, Piva F, Onorati F, Fattorini D, Notti A, et al. A multidisciplinary weight of evidence approach for classifying polluted sediments: Integrating sediment chemistry, bioavailability, biomarkers responses and bioassays. Environ Int. 2012; 38: 17-28. doi: 10.1016/j.envint.2011.08.003 PMID: 21982029 4. Rose WL, Nisbet RM, Green PG, Norris S, Fan T, Smith EH, et al. Using an integrated approach to link biomarker responses and physiological stress to growth impairment of cadmium-exposed larval topsmelt. Aquat Toxicol. 2006; 80: 298-308. doi: 10.1016/j.aquatox.2006.09.007 PMID: 000242776900010 5. Souza IC, Duarte ID, Pimentel NQ, Rocha LD, Morozesk M, Bonomo MM, et al. Matching metal pollution with bioavailability, bioaccumulation and biomarkers response in fish (Centropomus parallelus) resident in neotropical estuaries. Environ Pollut. 2013; 180: 136-44. doi: 10.1016/j.envpol.2013.05.017 PMID: 000322425300019 6. Oliveira BL, Loureiro Fernandes LF, Bianchini A, Chippari-Gomes AR, Silva BF, Brandao GP, et al. Acute copper toxicity in juvenile fat snook Centropomus parallelus (Teleostei: Centropomidae) in sea water. Neotrop Ichthyol. 2014; 12: 845- 52. doi: 10.1590/1982-0224-20140040 PMID: 000347909800020 7. Fasulo S, Mauceri A, Maisano M, Giannetto A, Parrino V, Gennuso F, et al. Immunohistochemical and molecular biomarkers in Coris julis exposed to environmental contaminants. Ecotoxicol Environ Saf. 2010; 73: 873-82. doi: 10.1016/j.ecoenv.2009.12.025 PMID: 000279623800023 8. Tomasello B, Copat C, Pulvirenti V, Ferrito V, Ferrante M, Renis M, et al. Biochemical and bioaccumulation approaches for investigating marine pollution using Mediterranean rainbow wrasse, Coris julis (Linneaus 1798). Ecotoxicol Environ Saf. 2012; 86: 168-75. doi: 10.1016/j.ecoenv.2012.09.012 PMID: 000311064800024 9. De Domenico E, Mauceri A, Giordano D, Maisano M, Giannetto A, Parrino V, et al. Biological responses of juvenile European sea bass (Dicentrarchus labrax) exposed to contaminated sediments. Ecotoxicol Environ Saf. 2013; 97: 114-23. doi: 10.1016/j.ecoenv.2013.07.015 PMID: 000325039400015 10. Tse CY, Chan KM, Wong CK. DNA damage as a biomarker for assessing the effects of suspended solids on the orange-spotted grouper, Epinephelus coioides. Fish Physiol Biochem. 2010; 36: 141-6. doi: 10.1007/s10695-008-9243-0 PMID: 000277710800004 11. Migliarini B, Campisi AM, Maradonna F, Truzzi C, Annibaldi A, Scarponi G, et al. Effects of cadmium exposure on testis apoptosis in the marine teleost Gobius niger. Gen Comp Endocrinol. 2005; 142: 241-7. doi: 10.1016/j.ygcen.2004.12.012 PMID: 15862569 12. Humphrey CA, King SC, Klumpp DW. A multibiomarker approach in barramundi (Lates calcarifer) to measure exposure to contaminants in estuaries of tropical North

Queensland. Mar Pollut Bull. 2007; 54: 1569-81. doi: 10.1016/j.marpolbul.2007.06.004 PMID: 000250599700014 13. Omar WA, Zaghloul KH, Abdel-Khalek AA, Abo-Hegab S. Genotoxic effects of metal pollution in two fish species, Oreochromis niloticus and Mugil cephalus, from highly degraded aquatic habitats. Mutat Res-Genet Tox En. 2012; 746: 7-14. doi:10.1016/j.mrgentox.2012.01.013 14. Tsangaris C, Vergolyas M, Fountoulaki E, Nizheradze K. Oxidative Stress and Genotoxicity Biomarker Responses in Grey Mullet (Mugil cephalus) From a Polluted Environment in Saronikos Gulf, Greece. Arch Environ Con Tox. 2011; 61: 482-90. doi: 10.1007/s00244-010-9629-8 PMID: 000298500400013 15. Martinez-Gomez C, Fernandez B, Benedicto J, Valdes J, Campillo JA, Leon VM, et al. Health status of red mullets from polluted areas of the Spanish Mediterranean coast, with special reference to Portman (SE Spain). Mar Environ Res. 2012; 77: 50- 9. doi: 10.1016/j.marenvres.2012.02.002 PMID: 000304296700008 16. Tigano C, Tomasello B, Pulvirenti V, Ferrito V, Copat C, Carpinteri G, et al. Assessment of environmental stress in Parablennius sanguinolentus (Pallas, 1814) of the Sicilian Ionian coast. Ecotoxicol Environ Saf. 2009; 72: 1278-86. doi: 10.1016/j.ecoenv.2008.09.028 PMID: 000265767900037 17. Schmidt V, Zander S, Korting W, Broeg K, von Westernhagen H, Dizer H, et al. Parasites of flounder (Platichthys flesus L.) from the German Bight, North Sea, and their potential use in biological effects monitoring - C. Pollution effects on the parasite community and a comparison to biomarker responses. Helgoland Mar Res. 2003; 57: 262-71. doi: 10.1007/s10152-003-0159-x PMID: 000186604600015 18. Vethaak AD, Jol JG, Meijboom A, Eggens ML, apRheinallt T, Wester PW, et al. Skin and liver diseases induced in flounder (Platichthys flesus) after long-term exposure to contaminated sediments in large-scale mesocosms. Environ Health Persp. 1996; 104: 1218-29. doi: 10.2307/3432916 PMID: A1996VX74000021 19. Williams TD, Davies IM, Wu H, Diab AM, Webster L, Viant MR, et al. Molecular responses of European flounder (Platichthys flesus) chronically exposed to contaminated estuarine sediments. Chemosphere. 2014; 108: 152-8. doi: 10.1016/j.chemosphere.2014.01.028 PMID: 000337881600020 20. Schipper CA, Lahr J, van den Brink PJ, George SG, Hansen P-D, de Assis HCdS, et al. A retrospective analysis to explore the applicability of fish biomarkers and sediment bioassays along contaminated salinity transects. Ices J Mar Sci. 2009; 66: 2089-105. doi: 10.1093/icesjms/fsp194 PMID: 000272080600003 21. Leaver MJ, Diab A, Boukouvala E, Williams TD, Chipman JK, Moffat CF, et al. Hepatic gene expression in flounder chronically exposed to multiply polluted estuarine sediment: Absence of classical exposure 'biomarker' signals and induction of inflammatory, innate immune and apoptotic pathways. Aquat Toxicol. 2010; 96: 234- 45. doi: 10.1016/j.aquatox.2009.10.025 PMID: 000274950000007 22. de Souza Machado AA, Mueller Hoff ML, Klein RD, Cardozo JG, Giacomin MM, Ledes Pinho GL, et al. Biomarkers of waterborne copper exposure in the guppy Poecilia vivipara acclimated to salt water. Aquat Toxicol. 2013; 138: 60-9. doi: 10.1016/j.aquatox.2013.04.009. PMID: 000322293600007 23. Hartl MGJ, Kilemade M, Sheehan D, Mothersill C, O'Halloran J, O'Brien NM, et al. Hepatic biomarkers of sediment-associated pollution in juvenile turbot, Scophthalmus maximus L. Mar Environ Res. 2007; 64: 191-208. doi: 10.1016/j.marenvres.2007.01.002 PMID: 000248488500007 24. Costa PM, Neuparth TS, Caeiro S, Lobo J, Martins M, Ferreira AM, et al. Assessment of the genotoxic potential of contaminated estuarine sediments in fish peripheral

blood: Laboratory versus in situ studies. Environ Res. 2011; 111: 25-36. doi: 10.1016/j.envres.2010.09.011 PMID: 000286715300005 25. Costa PM, Caeiro S, Vale C, Angel DelValls T, Costa MH. Can the integration of multiple biomarkers and sediment geochemistry aid solving the complexity of sediment risk assessment? A case study with a benthic fish. Environ Pollut. 2012; 161: 107-20. doi: 10.1016/j.envpol.2011.10.010 PMID: 000300539300016 26. Costa PM, Lobo J, Caeiro S, Martins M, Ferreira AM, Caetano M, et al. Genotoxic damage in Solea senegalensis exposed to sediments from the Sado Estuary (Portugal): Effects of metallic and organic contaminants. Mutat Res-Genet Tox En. 2008; 654: 29-37. doi: 10.1016/j.mrgentox.2008.04.007 PMID: 000258049500005 27. Isani G, Andreani G, Cocchioni F, Fedeli D, Carpene E, Falcioni G. Cadmium accumulation and biochemical responses in Sparus aurata following sub-lethal Cd exposure. Ecotoxicol Environ Saf. 2009; 72: 224-30. doi: 10.1016/j.ecoenv.2008.04.015 PMID: 000260660100028 28. Ribecco C, Baker ME, Sasik R, Zuo Y, Hardiman G, Carnevali O. Biological effects of marine contaminated sediments on Sparus aurata juveniles. Aquat Toxicol. 2011; 104: 308-16. doi: 10.1016/j.aquatox.2011.05.005 PMID: 000293042100017

S21 Table. Field and laboratory studies on responses of biomarkers of exposure in fish to metals and other contaminants: osmoregulatory and respiratory parameters. Most studies measured contaminants in the environment in addition to those identified as of concern for Gladstone Harbour (Al, Cd, Cu, Ga, Pb, Se, Zn); these are also presented for completeness.

Species LHS Laboratory or Metals other lactate LDH Na+/K+ - others Reference Field contaminants ATPase

Anguilla anguilla Glass eels Field sed Cd, Cr, Cu, Hg, Ni, Pb, V, Zn PAH - + [1]

Yellow Field sed Cd, Cr, Cu, Hg, Ni, Pb, V, Zn PAH + + [1] eels

Centropomus J Field sed and water Ag, Al, As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, +/- [2] parallelus Pb, Se, Zn

Dicentrarchus labrax J Lab field sed As, Cd, Cr, Cu, Hg, Ni, Pb, Zn PAHs and PCBs = AQPs - [3] toxicity

Fundulus heteroclitus A Lab water toxicity Zn + Ca2+-ATPase [4] test +

Fundulus heteroclitus A Lab water toxicity Cu = = [5] test

Solea senegalensis A Field sed Cd, Cr, Cu, Fe, Hg, Pb, Zn = [6]

A Field water and sed As, Cd, Cu, Fe, Pb, Zn PAHs = [7]

Solea solea A Field sed Cd, Cr, Cu, Fe, Hg, Pb, Zn = [6]

Squalus acanthias A Lab water toxicity Pb = - Gills TMAO =; [8] test = rectal gland Urea +/-;

PaO2 =;

PaCO2 -;

arterial pH +/-;

K -;

Ca =;

NH4 +/-

Na -;

Cl -

Synechogobius hasta J Lab water toxicity Cd = [9] test

Abbreviations: LHS: life history stage; A: adult, J: juvenile, L: larvae: Lab: laboratory; Sed: Sediment; PAHs: total polycyclic aromatic hydrocarbons ; PCBS: polychlorinated biphenyl; + induction; - inhibition; = no significant induction; +/- mixed response; RB: Respiratory burst; AQPs: aquaporins; TMAO: trimethylamine oxide; LDH: lactate dehydrogenase.

References 1. Gravato C, Guimaraes L, Santos J, Faria M, Alves A, Guilhermino L. Comparative study about the effects of pollution on glass and yellow eels (Anguilla anguilla) from the estuaries of Minho, Lima and Douro Rivers (NW Portugal). Ecotoxicol Environ Saf. 2010; 73: 524-33. doi: 10.1016/j.ecoenv.2009.11.009 PMID: 000277103600009 2. Souza IC, Duarte ID, Pimentel NQ, Rocha LD, Morozesk M, Bonomo MM, et al. Matching metal pollution with bioavailability, bioaccumulation and biomarkers response in fish (Centropomus parallelus) resident in neotropical estuaries. Environ Pollut. 2013; 180: 136-44. doi: 10.1016/j.envpol.2013.05.017 PMID: 000322425300019 3. De Domenico E, Mauceri A, Giordano D, Maisano M, Gioffre G, Natalotto A, et al. Effects of "in vivo" exposure to toxic sediments on juveniles of sea bass (Dicentrarchus labrax). Aquat Toxicol. 2011; 105: 688-97. doi: 10.1016/j.aquatox.2011.08.026 PMID: 000298120600055 4. Loro VL, Nogueira L, Nadella SR, Wood CM. Zinc bioaccumulation and ionoregulatory impacts in Fundulus heteroclitus exposed to sublethal waterborne zinc at different salinities. Comp Biochem Phys C. 2014; 166: 96-104. doi: 10.1016/j.cbpc.2014.07.004 PMID: 000342532000011 5. Ransberry VE, Morash AJ, Blewett TA, Wood CM, McClelland GB. Oxidative stress and metabolic responses to copper in freshwater- and seawater-acclimated killifish, Fundulus heteroclitus. Aquat Toxicol. 2015; 161: 242-52. doi: 10.1016/j.aquatox.2015.02.013 PMID: 000352177500026 6. Siscar R, Torreblanca A, Palanques A, Sole M. Metal concentrations and detoxification mechanisms in Solea solea and Solea senegalensis from NW Mediterranean fishing grounds. Mar Pollut Bull. 2013; 77: 90-9. doi: 10.1016/j.marpolbul.2013.10.026 PMID: 000329888600025 7. Oliva M, Antonio Perales J, Gravato C, Guilhermino L, Dolores Galindo-Riano M. Biomarkers responses in muscle of Senegal sole (Solea senegalensis) from a heavy metals and PAHs polluted estuary. Mar Pollut Bull. 2012; 64: 2097-108. doi: 10.1016/j.marpolbul.2012.07.017 PMID: 000310929500028 8. Eyckmans M, Lardon I, Wood CM, De Boeck G. Physiological effects of waterborne lead exposure in spiny dogfish (Squalus acanthias). Aquat Toxicol. 2013; 126: 373- 81. doi: 10.1016/j.aquatox.2012.09.004 PMID: 000315125600040 9. Liu XJ, Luo Z, Li CH, Xiong BX, Zhao YH, Li XD. Antioxidant responses, hepatic intermediary metabolism, histology and ultrastructure in Synechogobius hasta exposed to waterborne cadmium. Ecotoxicol Environ Saf. 2011; 74: 1156-63. doi: 10.1016/j.ecoenv.2011.02.015 PMID: 000291960600007

S22 Table. Field and laboratory studies on responses of biomarkers of effect in fish to metals and other contaminants: carbohydrate and aerobic metabolism. Most studies measured contaminants in the environment in addition to those identified as of concern for Gladstone Harbour (Al, Cd, Cu, Ga, Pb, Se, Zn); these are also presented for completeness.

Species LHS Tissue Method Laboratory or Metals Other PK HK CS COX CS:COS Others Reference Field contaminants ratio

Fundulus A gill Bioassay Lab water Cu - = = - = [1] heteroclitus toxicity test

intestine Bioassay Lab water Cu + = + + = [1] toxicity test

liver Bioassay Lab water Cu = = = = = [1] toxicity test

Solea A muscle Bioassay Field water and As, Cd, Cu, Fe, Pb, PAHs, IDH +\- [2] senegalensis sed Zn

Sparus aurata J liver Real time Lab field sed As, Cd, Cr, Cu, Hg, PAHs Erα +; RXRα [3] PCR toxicity Ni, Pb, Se, V, Zn +

Synechogobius J liver Bioassay Lab water Cd + SDH -; MDH [4] hasta toxicity test +\-; HL +;

LPL +;

Abbreviations: Lab: laboratory; Sed: Sediment; PAHs : total polycyclic aromatic hydrocarbons; PK: pyruvate kinase; HK: hexokinase; CS: citrate synthase; COX: cytochrome c oxidase; + induction; - inhibition; = no significant induction; +/- mixed response; IDH:Isocitrate dehydrogenase; Erα: nuclear receptor binds xenochemical compounds termed 'estrogen like molecules; RXRα: 9-cis retinic acid receptor; SDH: succinate dehydrogenase; MDH: malic dehydrogenase; HL: hepatic lipase; LPL: lipoprotein lipase.

References 1. Ransberry VE, Morash AJ, Blewett TA, Wood CM, McClelland GB. Oxidative stress and metabolic responses to copper in freshwater- and seawater-acclimated killifish, Fundulus heteroclitus. Aquat Toxicol. 2015; 161: 242-52. doi: 10.1016/j.aquatox.2015.02.013 PMID: 000352177500026 2. Oliva M, Antonio Perales J, Gravato C, Guilhermino L, Dolores Galindo-Riano M. Biomarkers responses in muscle of Senegal sole (Solea senegalensis) from a heavy metals and PAHs polluted estuary. Mar Pollut Bull. 2012; 64: 2097-108. doi: 10.1016/j.marpolbul.2012.07.017 PMID: 000310929500028 3. Ribecco C, Baker ME, Sasik R, Zuo Y, Hardiman G, Carnevali O. Biological effects of marine contaminated sediments on Sparus aurata juveniles. Aquat Toxicol. 2011; 104: 308-16. doi: 10.1016/j.aquatox.2011.05.005 PMID: 000293042100017 4. Liu XJ, Luo Z, Li CH, Xiong BX, Zhao YH, Li XD. Antioxidant responses, hepatic intermediary metabolism, histology and ultrastructure in Synechogobius hasta exposed to waterborne cadmium. Ecotoxicol Environ Saf. 2011; 74: 1156-63. doi: 10.1016/j.ecoenv.2011.02.015 PMID: 000291960600007

S23 Table. Field and laboratory studies on responses of biomarkers of effect in fish to metals and other contaminants: histopathology and gross indices. Most studies measured contaminants in the environment in addition to those identified as of concern for Gladstone Harbour (Al, Cd, Cu, Ga, Pb, Se, Zn); these are also presented for completeness.

Species LHS Laboratory Metals Other contaminants HA HIS/ GSI CF K RNA:DNA TLC Other Reference or Field LSI index ratio

Acanthopagrus A Field sed As, Cr, Cu, PAHs - = (1) latus Ni, Pb, V, Zn

Anguilla anguilla Glass Field sed Cd, Cr, Cu, PAH = (2) eels Hg, Ni, Pb, V, Zn

Anguilla anguilla Yellow Field sed Cd, Cr, Cu, PAH - (2) eels Hg, Ni, Pb, V, Zn

Aphanius A Field water Cd, Cu, Zn PAHs (3) fasciatus and sed +/-

Aphanius J Field sed Cd, Cd, Cu, PAHs + = COL1A2 +/- (4) fasciatus Cu, ZnZn (Collagen)

Aphanius J Lab water Cd COl1A2 - (4) fasciatus toxicity test

Atherina A Field sed Cd, Hg, Ni, PAHs = +/- (5) presbyter Pb, Zn

Atherinops affnis L Lab water Cd Food intake - (6) toxicity test

Species LHS Laboratory Metals Other contaminants HA HIS/ GSI CF K RNA:DNA TLC Other Reference or Field LSI index ratio

Centropomus J Field sed and Ag, Al, As, = (7) parallelus water Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, Zn

Chanos chanos A Field sed and Cd, Cu, Fe, + (8) water Mn, Pb, Zn

Coris julis A Field sed Cd, Co, Cr, + (9) Cu, Ni, Pb, Sb, Zn

Cynoglossus arel A Field sed As, Cr, Cu, PAHs = = (1) Ni, Pb, V, Zn

Dicentrarchus A Caged field Cu, Pb, Zn PAHs - (10) labrax sed

Dicentrarchus A Field sed Cr, Cu, Ni, Pb, PAHs - - TPC -; (11) labrax Zn

Dicentrarchus J Caged field Al, Cd, Cr, - - Growth index -; (12) labrax sed Cu, Mn, Ni, TAG:ST - Pb, V, Zn

Dicentrarchus J Lab field sed As, Cd, Co, + (13) labrax toxicity Cr, Cu, Fe, Hg, Mn, Ni, Pb, Sb, V, Zn

Dicentrarchus J Lab water Cd Lateral line (14) labrax toxicity test tissue +

Species LHS Laboratory Metals Other contaminants HA HIS/ GSI CF K RNA:DNA TLC Other Reference or Field LSI index ratio

Epinephelus J Lab water Cu + - Digestive (15) coioides toxicity test enzymes -; TSFA +; MFA +; TPFA -;

Fundulus A Field and lab Cd, Cu, Hg, PAHs, PCBs Prey capture -; (16) heteroclitus sed Pb, Zn

Fundulus L Field sed / lab Cd, Cu, Hg, PAHs, PCBs Prey capture -; (17) heteroclitus Pb, Zn

Gadus morhua L. A Cage field Cd, Cu, Hg, PAHs, PCBs = = = SSI = (18) water Pb, Zn

Gadus morhua L. J Lab water As, Cd, Co, PAHs = (19) toxicity test Cr, CuHg, Mn, Ni, Pb, Zn

Galaxias E Lab water and Cu, Pb, Zn Phototactic (20) maculatus sed toxicity response - test

Lates calcarifer A Field sed Cd, Cr, Cu, Diuron, PAHs = = - (21) Ni, Zn

Lates calcarifer Lab water Cu + (22) toxicity test

Limanda limanda A Field sed Cd, Cr, Cu, PAHs, PCBs = + Parasites =; (23) Hg, Ni, Pb, Zn

Lutjanus russellii A Field sed and Cd, Cu, Fe, - (24) water Pb, Zn

Species LHS Laboratory Metals Other contaminants HA HIS/ GSI CF K RNA:DNA TLC Other Reference or Field LSI index ratio

Mugil cephalus A Field sed and Cu, Fe, Mn, + - (25) water Pb, Zn

Mugil cephalus F Lab water Pb + (26) toxicity test

Mugil cephaus J Field sed and Cd, Cu, Mn, AHCs, PAHs, PCBs, = (27) water Ni, Pb DDTs, TBT

Mullus barbatus A Field sed As, Cd, Cu, PAHs, CBs, DDT, = - - - (28) Hg, Pb, Zn HCB, trans-nonachlor, Lindane, Dieldrin

Plastichthys A Cage field Cd, Cu, Hg, PAHs, PCBs = = (18) flesus water Pb, Zn

Plastichthys A Field sed Al, Cd, Cr, PCB,DDD,DDE, HCH Parasites - (29) flesus Cu, Fe, Hg, Mn, Pb, Zn

Plastichthys J Field sed Al, Cd, Cr, PAHs - - TAG:ST - (30) flesus Cu, Hg, Mn, Ni, Pb, V, Zn

Plastichthys J Field sed Al, Cd, Cr, - - (31) flesus Cu, Hg, Mn, Ni, Pb, V, Zn

Plastichthys J Lab field sed As, Cr, Cu, PAHs, DDT, HCB, = (32) flesus toxicity Hg, Ni, Pb, Zn CB, TBT, DBT

Platichthys flesus A Field sed Cd, Hg, Pb, PAHs, PCBs + lesions = Epidermal (33) Zn +/- disease =

Species LHS Laboratory Metals Other contaminants HA HIS/ GSI CF K RNA:DNA TLC Other Reference or Field LSI index ratio

Platichthys flesus A Field water As, Cd, Cr, PAHs, PCBs, OCPs = = = - +/- TPC +\-; (34) L. and sed Cu, Hg, Ni, Pb, Zn

Pogpmichthys L Lab food MeHgSe + = (35) macrolepidotus

Pomadasys hasta A Field sed and Cd, Cu, Fe, - (24) water Pb, Zn

Pomatoschistus A Field sed Cd, Cr, Cu, PAHs = = (36) microps Hg, Ni, Pb, Zn

Pomatoschistus A Field sed Cd, Hg, Ni, PAHs = +/- (5) microps Pb, Zn

Pomatoschistus A Field sed Cr, Cu, Ni, Pb, PAHs - = TPC +/- (11) microps Zn

Scophtalmus J Caged field Al, Cd, Cr, - = Growth index =; (12) maximus sed Cu, Mn, Ni, TAG:ST - Pb, V, Zn

Scophtalmus J Lab field sed Al, Cd, Cr, - +/- Growth index (37) maximus Cu, Mn, Ni, +\-; TAG:ST - Pb, V, Zn

Seriola lalandi J Lab food Se = - (38)

Solea A Field sed Cr, Cu, Ni, Pb, PAHs +/- +/- TPC +\- (11) senegalensis Zn

Solea J Field sed Cd, Cr, Cu, PAHs + +/- (39) senegalensis Ni, Pb, Zn

Species LHS Laboratory Metals Other contaminants HA HIS/ GSI CF K RNA:DNA TLC Other Reference or Field LSI index ratio

Solea J Lab and field As, Cu, Zn PAHs, PCBs, DDT + (40) senegalensis sed

Solea J Lab field sed As, Cd, Cr, PAHs + + (41) senegalensis toxicity Cu, Hg, Ni, Pb, Zn

Solea J Lab field sed Cd, Cr, Cu, PAHs, PCBs, DDT + (42) senegalensis toxicity Ni, Pb, Zn

Sparus aurata J Lab field sed As, Cd, Cr, PAHs + + (41) toxicity Cu, Hg, Ni, Pb, Zn

Squalus A Lab water Pb TPC liver +/-; (43) acanthias toxicity test glycogen muscle -

Symphodus A Field water Fe, Pb, Zn = - = SSI - (44) melops and sed

Synechogobius J Lab water Cd + + = + VSI = (45) hasta toxicity test

Terapon jarbua F Lab water Pb + (26) toxicity test

Terapon jarbua J Lab food Cd = (46)

Abbreviations: LHS: life history stage; A: adult, E: eggs, F: fingerlings, J: juvenile, L: larvae: Lab: laboratory; Sed: Sediment; HCB: hexachlorobenzene; OCP: total organochlorine pesticides; CB: chlorinated biphenyls; naph: naphthalenes; PAHs : total polycyclic aromatic hydrocarbons; PCBS: polychlorinated biphenyl; TBT: tributyltin; DBT: dibutytin; DDD: 1,1-dichloro-2.2- bis(p-chlorophenyl) ethane; DDE: 1,1-dichloro-2.2-bis(p-chlorophenyl) ethylene; HCH: hexachlorcyclohexane; DDT: dichlorodiphenyltrichloroethane; HCB: hexachlorobenzene; HA: histological alteration; HSI: hepatosomatic index; LSI: liver somatic index; GSI: gonadosomatic index; CF: condition factor; + induction; - inhibition; = no significant induction; +/- mixed response; TAG:ST - lipid storage index; TLC - total lipid content; TPC - total protein content; TPFA - total polyunsaturated fatty acids; MFA - monounsaturated fatty acids; TSFA - total saturated fatty acids; COL1A2: Collagen 1A2; VSI: viscerosomatic index; Vac: Vacuolation; Fibr: Fibrillar structures.

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