Multibiomarker responses to polycyclic aromatic hydrocarbons and microplastics in thumbprint emperor Lethrinus harak from a South Pacific locally managed marine area

To determine the baseline threat of microplastics and polycyclic aromatic hydrocarbons (PAHs) in an important seafood fish from Vueti Navakavu locally managed marine area, a multibiomarker risk assessment was conducted on the thumbprint emperor fish Lethrinus harak. Condition factor, a measure of relative general health condition of fish, was significantly lower in samples from the wet season compared to the dry season but no significant differences were observed for hepatosomatic index, a measure of relative stored energy/nutrition, between seasonal groups. PAHs levels of four metabolites in emperor fish from Fiji waters are reported here for the first time; seasonal groups showed no significant differences, but all samples presented levels of biliary PAHs. Each specimen also contained at least one microplastic in its gastrointestinal system; fibres were the predominant form-type and ingestion levels showed that more than 80% of fragment sizes were below 1.0 mm. Biochemical responses were observed for ethoxyresorufin-O-deethylase and glutathione S-transferase biotransformation activity, oxidative stress (glutathione peroxidase and glutathione reductase activity; lipid peroxidation) and genotoxicity (micronuclei assay). Though there were no statistically significant differences found, there were biological significances that were important to note; relatively low levels of pollutant exposure and low levels of biochemical responses showed enzymes response in thumbprint emperor were as expected to their roles in the body. In this multibiomarker approach, the observation of pollutants presence and histopathological injuries are considered biologically relevant from a toxicological perspective and serve as a baseline for future pollution studies in seafood fishes in Fiji, with site differences and the inclusion of fish species comparison. We recommend adopting a suite of biomarkers in future regional biomonitoring studies to develop holistic baseline information for other marine settings in Fiji and other Pacific Island countries.

www.nature.com/scientificreports/ weighed. Five random sections of the liver were separated for the biochemical parameters and stored in liquid nitrogen until storage in a − 80 °C freezer.
Biomarkers. Fulton's condition factor was calculated as K = total weight/length 3 × 100. The hepatosomatic index was calculated as HSI = liver weight/total weight × 100. The PAH metabolites were determined through fixed wavelength fluorescence (FF) screening method 60 and achieved by diluting the bile (10:1000 µL) in 48% ethanol before being measured spectrofluorometrically (absorbance and fluorescence intensity; double monochromotors) in a multimode reader (Thermo Scientific™ Varioskan™ MIB#5250030) to determine the signals intensity ratios of four biliary PAH metabolite types; phenanthrene (FF 260/380 ), naphthalene (FF 290/335 ), 1-hydroxypyrene (FF 341/383 ), and benzo[a]pyrene (FF 380/430 ) 61,62 . The multimode instrument reader measured at a dynamic wavelength range (emission: 200-1000 nm; excitation 5 nm and 12 nm/12 nm) with an accuracy of 0.003 Abs or ± 2%, at 200-399 nm (0-2 Abs) and 0.003 Abs or ± 1%, at 400-1000 nm (0-3 Abs), which was within the required spectrofluorometric parameters for the fluorescent aromatic compounds (FACs) analysis 63 . The quality assurance and quality control for the four biliary PAH metabolites included analytical standards for each of the PAH metabolites measured, calibration curves, continuing calibration standards, and method blanks in accordance with the technical guidelines described by the International Council for the Exploration of the Sea 60, 64 . To assess the activity of biochemical analysis of EROD, the liver was homogenized in ice-cold buffer (50 mM Tris-HCL, pH 7.4, 0.15 M KCl) 65 . The S9 fraction of the hepatic tissue was homogenized 66 . The EROD activity was evaluated fluorometrically 67 . GST activity was determined by a substrate artificial 1-chloro-2, 4 dinitrobenzene, which was conjugated by GST 68 . GPX activity was determined through the metabolism of H 2 O 2 to water, involving concomitant oxidation of reduced glutathione to its oxidized form glutathione disulfide 63 . GR activity was measured by catalysing the transformation of GSSG to reduced GSH with the concomitant oxidation of NADPH to NADP + 69 . The glutathione group of enzymes were measured at an absorbance of 340 nm. The peroxidative damage to lipids that occurs with reactive oxygen species generation and results in the production of malondialdehyde was assessed by the determination of TBARS. Malondialdehyde was determined by the thiobarbituric acid and was measured at an absorbance of 532 nm 70 . Micronuclei assay was assessed for genotoxicity. The blood smears were treated with methanol for 10 min to fix the cells and left to dry before staining  Microplastics. Extraction techniques for the removal of MPs from the gastrointestinal system (foregut to the hindgut) were done following Avio et al. 72 . In summary, a NaCl hypersaline (1.2 g/cm 3 ) solution was added at three times the volume of the gastrointestinal system and was stirred and decanted twice for ten minutes, followed by filtration through a 63 µm sieve. The filtrate was transferred from the sieve into a 500 mL beaker and digestion of the suspended organic matter was performed by adding 30 mL of hydrogen peroxide (15%) to the sample before incubation at 60 °C for 24 h. The dried samples were resuspended with distilled water and filtered again through 63 µm sieve. Blank samples were analysed concomitantly with field samples, to gauge any potential cross-contamination. Identification of all plastic particles (different sizes and form type) was performed under a dissecting microscope (Olympus SZ-ST) at 10× magnification and an Infinity 1 camera whose images were processed through the Infinity Analyse software. All MPs larger than 0.5 mm were classified into fragment, film, fibre, and microbead based on their visual aspect 73 .
Statistical analyses. Seasonal differences in K, HSI, PAH metabolites and MPs were tested using nonparametric Wilcoxon-Mann-Whitney (WMW) test at a 5% significant level. All statistical tests were performed using the R software 74 .
Ethical statement. Fish specimens and data were collected according to the procedures and protocols approved by the University Research Ethics Committee (UREC) of the University of the South Pacific, in accordance with the policies and guideline stipulated in the Animal Research Ethics Handbook 75 . The processes and procedures of live specimen handling and field transportations were in compliance with Fiji's regulation of animal protection as outlined in the Protection of Animals Act 1954 76 . Euthanasia method on specimens was consistent with the commonly accepted norms of veterinary best practice 77 .

Results
A total of 53 specimens of thumbprint emperor were caught and sampled; 31 from the dry season and 22 from the wet season. Mean total weight was 185.   Figure S1). On average, the ingestion levels were not significantly higher in fish from the wet season compared to those from the dry season (mean ± SE = 4.7 ± 0.9 MP/fish, 3.3 ± 1.3 MP/fish, respectively; WMW test: Z = − 0.62, p = 0.537). The majority of the MP found was less than 1.0 mm in size (Table 3). In particular, MPs ranging 0.1 to 0.4 mm made up 20% of the samples in the dry season and 50% of the samples in the wet season, while MPs ranging 0.5 to 0.9 mm made up 60% of the samples in the dry season and 38% in the wet season. MP form type was dominated by fibres in the wet season (87% of 103 samples), while in the dry season a similar abundance of fibres and fragments (42% and 38%, respectively) was recorded (Fig. 2). Films had an overall lower abundance, although it was significantly higher in the dry season than in the wet season (0.6 ± 0.

Discussion
This study demonstrates a low level of PAHs and MPs in the gastrointestinal tract of mature-sized thumbprint emperor of Vueti Navakavu LMMA in Fiji, South Pacific, both in the wet and dry seasons of the year. All the sampled specimens were of mature size 78 ; the observed differences in K were thus most likely resulting from the reproductive activity. Indeed, the thumbprint emperor reportedly reproduces in the wet season 79 . This is further supported by a lower HSI in the wet season, suggesting a lower general stored energy. Variations in energy reserves based on the two physiological biomarkers can also occur as a result of recent feeding habits that can affect the liver size and bodyweight of the fish 80 . To identify whether reproduction or feeding behaviour resulted in the observed differences, we suggest identification of the maturation stage of the gonads via histology 81 in future ecotoxicological studies.
The potential effects of PAHs on thumbprint emperor are of interest because the species, during its life cycle, occupies key positions in the food web 82 , and also because it is commonly consumed by the villagers of the local community 83 . PAHs enter fishes via food intake and during respiration, are then transported through the bloodstream, and adsorbed to lipid-rich tissues such as liver, muscle tissues, and gonads 84 . The concentration of Table 3. Abundance (mean ± SE) and percent contribution (%) of MPs size classes found in thumbprint emperor sampled at Vueti Navakavu LMMA in the dry and wet seasons in Fiji. www.nature.com/scientificreports/ PAHs found in fishes is normally higher than in the environment from which they were taken 8 . PAH metabolites were present in the bile of all thumbprint emperor sampled, ranging from 0.21 mg/L (benzo[a]pyrene type) to 75.74 mg/L (phenanthrene type) and, altogether, PAHs summed up to 127.10 mg/L. Compared with values found in the Atlantic cod (Gadus morhua) sampled from a PAH-polluted site in western Norway, where the lowest concentration was 66 mg/L (benzo[a]pyrene) and the highest concentration was 2704 mg/L (naphthalene) 85 , the ones found in the locally managed areas likely represent a low level of PAH pollution. However, coastal communities that tend to consume large quantities of fishes 86 could be at greater risk to health issues like growth reduction, endocrine alterations 87 and gastrointestinal infections 88 due to PAH bioaccumulation and biomagnification.
Monitoring of biliary biotransformation products permits detection of polar metabolites, which are proven to be sensitive PAH exposure markers, possibly two orders of magnitude more sensitive than tissue parent compound levels and is therefore ideal in monitoring and environmental risk assessment studies 89 . The majority of the fishes in Fijian rural areas are sourced locally from nearshore and coastal areas 90 and in Vueti Navakavu, 88% of all households are involved in fishing activities for income and subsistence purposes 91 . Vethaak et al. 92 found that marine areas close to industrial harbours have a gradual increase in PAH pollution over time. Site-specific pre-impact assessments are necessary to accurately evaluate before-after, control, impact (BACI) effects 93 . The values identified in this study represent a baseline against which comparison of future levels of biliary PAHs in the thumbprint emperor can be performed to assess environmental improvements in pollution reduction resulting from new policies and procedures. The hepatic expressions for phase I and II biotransformation showed a total EROD and GST activity of 1.05 nmol/min/mg protein and 51.23 nmol/min/mg protein, respectively. A problem that frequently occurs in field studies with biomarkers is the difficulty of finding suitable reference values, which can be overcome with range-value comparisons of wild species to laboratory-conditioned species 94 . In this case, the Mozambique tilapia (Oreochromis mossambicus) exposed to phenanthrene concentration below 4.0 μg/g was found to have EROD activity of 5.4 nmol/min/mg protein, and to exhibit sublethal hepatotoxicity 95 . These biochemical values of EROD response are 5.1 times higher than those found in thumbprint emperor in this study, although without experimental studies in laboratorial conditions the sublethal consequences of this activity cannot be excluded. Compared with literature, GST activity in the present study was much lower than in two flatfishes (English sole Parophrys vetulus: 16-fold; starry flounder Platichthys stellatus: 44-fold) captured from a contaminated site in Puget Sound, Washington, and was related to higher activation and lower detoxification ability of PAHs resulting in hepatic neoplasms and putatively preneoplastic lesions 96 . Although the types of biotransformation reactions are similar between fishes, differences exist in the metabolic handling of chemicals; particularly reactions rates, the relative contribution of a given pathway, and the products formed 97 . From a functional viewpoint, biotransformation reactions can significantly influence the biological properties of chemicals, depending on the nature of the reaction and the rate at which it occurs 98 . In the case of the thumbprint emperor from Vueti Navakavu, phase I and II biotransformation activity were observed although there was no statistically clear relationship between the two pollutants.
Total activity for GPX and GR responses were 10.90 and 25.97 nmol/min/mg protein, respectively. The reaction of biomarkers GPX and GR is naturally inverse within a biological system 69 . The GPX responses in thumbprint emperor (10.90 nmol/min/mg protein) in the present study was lower than the one (11.89 nmol/min/mg protein) found on sterlet sturgeon (Acipenser ruthenus) in a petroleum-polluted site in Novi Sad (Serbia) but higher than the levels reported in a control site (10.30 nmol/min/mg protein) in the same place 99 . Consistently with the expected opposite enzymatic role of GR, the recorded GR activity in thumbprint emperor was 2.4 times higher than the GPX reaction. This is consistent with effective hepatic biochemical responses of the thumbprint emperor under oxidative stress due to oxidative-inducing pollutants, likely as a result of a significant accumulation of hydrogen peroxide 100 . We suggest including variations of antioxidant system response and its regulatory mechanisms under different circumstances (e.g., biological effects from exposure to heavy metals) in further studies, which would provide a valuable assessment of seafood fish quality and health risks from consumption.
A low level of biological damages was identified by LPO concentrations, whereby the levels found in the thumbprint emperor in the present study were 17 times lower than those found in spotted snakehead (Channa punctatus) in Aligarh, India, which were exposed to wastewater and, as a result, reported clear membrane damage. LPO concentrations mainly depend on the availability of polyunsaturated fatty acids and the antioxidants defences 101 . Consistently, the micronuclei assay in thumbprint emperor showed the very low occurrence of abnormal nuclei formation in erythrocytes (0.03%) compared to genotoxic findings on Nile tilapia (Oreochromis niloticus) (0.95%) captured from a heavy metals-polluted site in India 102 .
MPs were found in thumbprint emperors in both seasons, and the most abundant were smaller than 1.0 mm. The size of MPs may be a key factor in determining the range of animals that ingest them 103 , as well as the retention rate; for example, retention in the gastrointestinal system of spiny Chromis (Acanthochromis polyacanthus) increased (with a maximum of 2102 small particles) when the MPs size was reduced from 2 mm to 0.3-0.125 mm 104 . MPs cause oxidative damages in gills, muscle, and an increase in neurotoxic responses of fishes 105 . Low exposure to MPs is supported by both the low numbers of MPs found in thumbprint emperor as well as the low level of oxidative stress identified in the present study. Studies of MPs in seafood fish [106][107][108] have also found that some pollutants like PAHs have a greater affinity to MPs than to water, and as a result MPs ingestion by seafood fishes likely have equal exposure to PAH intake (including other chemical pollutants), leading to bioaccumulation and biomagnification. It is for this reason that MPs and PAHs is recommended herein to be included in monitoring programmes in Fiji and other Pacific Island countries. While this baseline study observes the presence of MPs and PAHs in the thumbprint emperor, it only serves as a reference point for future pollution studies in seafood fishes in Fiji, and does not report risks or make recommendations from a human health perspective.
The PAHs and MPs pollutants quantified in this study represent a baseline for Vueti Navakavu LMMA, and indeed despite not having statistical significance, the interpretation of biologically relevant effects and efforts www.nature.com/scientificreports/ should be carefully considered. It is generally more valuable in ecotoxicology studies to know about the magnitude and certainty of an effect as well as its probability to occur, to ultimately evaluate its biological relevance 109 . Gagnon and Hodson 110 demonstrated that biomarkers may vary in proportion to the extent of exposure to pollutants, and that individual studies might discover different magnitudes and directions of biomarker responses according to the specific situation investigated. In this study, the use of ecotoxicological tools (i.e. biomarkers) allows for direct assessment of the health of the thumbprint emperor within the LMMA since this approach shifts the focus of the assessment from the agents (pollutants) in the environmental compartments to the target species (biological responses) 111 . The thumbprint emperor, as aforementioned, showed relatively low levels of PAHs exposure and MPs ingestion, including relatively low levels of biological damages shown by ENA and LPO concentrations. The biochemical responses of Phase I and II biotransformation, GPX and GR activity were also comparatively low to studies that were identified. As a baseline for pollutants quantification and biochemical responses, it is important to not exclude the prospect of Vueti Navakavu's twenty-long years as an LMMA being of some consequence to the health conditions and low pollutants exposure in the thumbprint emperor. Furthermore, this baseline study does not assume a "normal" biological state being defined for the thumbprint emperor, and the observation of pollutants presence and histopathological injuries that were observed in the fish is considered biologically relevant from a toxicological perspective 112 . It is for this reason that future biomonitoring studies is encouraged to build on the baseline information identified herein and employ a suite of biomarkers to bridge knowledge gaps and enable environmental risk assessment programmes to strengthen conservation efforts in marine spaces. Marine protected areas are legally designated areas where human activities are restricted or managed to ensure sustainability and avoid over-fishing and habitat destruction 113 . Enforcement and management are essential to ensure that marine protected areas fulfil their purpose and do not remain mere polygons on a chart. Moreover, the effectiveness of these areas in protecting the marine and coastal habitat, their resistance and resilience to pollution, and their capacity of providing benefits like ecosystem services and ecological spillover, is related to the size of the MPAs and the place where the MPAs are established 114 .
The Vueti Navakavu LMMA is representative of Fiji's key coral reef and mangrove coastal habitats, but it is relatively small-sized and its location is a few kilometres on the west of the harbour. Reportedly, the LMMA is threatened by chemical and solid waste pollution from the former Lami Dump and Suva city, both of which are located upwind and up-current 115 . It is important noting that the boundaries of the LMMA have cultural significance and are not ecological boundaries, therefore they are neutral and completely permeable to pollution.
Our study shows the presence of PAHs and MPs in all samples of emperor fish, an important seafood fish. Seasonal differences to the multibiomarker responses and pollutants levels were not statistically significant, which suggests that the threat posed by PAHs and MPs in Vueti Navakavu LMMA is all year round. Emerging pollutants (like MPs) and legacy pollutants (like PAHs) move unrestrictedly beyond conservation systems and boundaries such as LMMAs and MPAs. The baseline biological and pollutants parameters, herein, may provide meaningful insights for future biomonitoring studies in Fiji, with site differences and the inclusion of fish species comparison. We also recommend expanding the range and suite of biomarkers to contribute to enhancing a more holistic baseline information for other marine settings in Fiji and Pacific Island countries.

Data availability
All data generated or analysed during this study are included in this published article (and its Supplementary Information files). www.nature.com/scientificreports/