Mullet in the two study areas show measurable biological and biochemical responses for a wide range of endpoints (genetic, biochemical, physiological) confirming that mullet can serve as a suitable sentinel/reference species also in a tropical marine environment. The application of these biomarkers on mullet from the two sites also support the use of mullet as a suitable species for biomonitoring in Fiji, as they reflect variations in environmental conditions and potential pollutant impacts across different sites.
The K factor and HSI of the sampled mullet were relatively lower in Laucala Bay than in the Ba River Delta. The K factor is usually related to food quality and reproductive status, and significant differences in the K factor between sites may be related to the nutritional conditions of the species in those sites, as noted in the morphometric variation between samples from those sites. Samples collected from Laucala Bay weighed much less than samples from the Ba River Delta, despite being similar in average total length. Like the K factor, HSI provides a relative indication of stored energy and may change due to environmental factors shifting more rapidly than the organism-level responses, with compounding factors like nutritional status, pathogenic effects, and toxic chemical exposures causing increased or decreased weight/size. The HSI in the Laucala Bay mullet was relatively lower than in the Ba River Delta. An increase in HSI may indicate an increase in liver size and/or lipid content and is associated with liver function deterioration, as demonstrated in similar studies 27,34,35. The HSI values reflected in samples from the Ba River Delta generally indicate poor environmental quality. Studies on pollutants like PAHs and metal contamination strongly show its correlation to lower HSI and K factor in fishes 36–39. While HSI and K factor is general, relative and non-specific as physiological and morphological biomarkers, several studies from different countries in the Global North and South agree that their low cost, ease and repeatability still make their application a valuable environmental risk assessment tool 26,40−42.
As an essential metal, Cr is widespread but not always as a pure metal, instead in the trivalent state; Cr (III). In this study, the concentration of Cr in the mullet was highest in Ba River Delta (0.33 ± 0.11 mg/kg) than in Laucala Bay (0.20 ± 0.03 mg/kg). These levels were also comparative to a Fijian study on heavy metals in fishes by Ghani and Deo 43 that found the concentration of Cr in Clupea pallasii, Macolor niger and Pristipomoides filamentosus from coast of Lautoka City to be 0.291 ± 0.001 mg/kg, 0.344 ± 0.003 mg/kg and 0.312 ± 0.002 mg/kg, respectively. While the sources of Cr in the study sites are unclear, it is common to find Cr levels higher in water bodies that receives effluents from electroplating, dyeing, or printing industries 43, most of which are found in the Western Division which includes Ba Province. A similar study of Cr levels in mullet from the Maule Region, Chile, found mean concentrations at 2.4 mg/kg (dry weight) [min-max: 0.3 ± 0.1–6.3 ± 0.7 mg/kg dry weight] 44. Comparatively, the findings in this study are in the lower ranges of Cr concentration than what was detected by Tapia, et al. 44. The Food and Agriculture Organisation has not set permissible limits of Cr related to human intake for food or drinking water; however, the Environmental Protection Agency 45 indicates a maximum of 8.0 mg/kg. Cr can enter fish tissues and accumulate to an asymptotic level related to the concentrations in the water. Fish that are first exposed to Cr above 0.5 mg/L have a variety of behavioural changes, including suspended feeding behaviour, uneven swimming, and accelerated opercular beat frequency 46–48. It may cause structural abnormalities in the gill epithelium, such as hypertrophy and paraplegia, weakening the body’s immune system 49. Cr is involved in natural human lipid and protein metabolism at low concentrations, so tiny amounts are needed for normal human life functions 50. Much of the daily intake of Cr, typically about 100 µg, is from grains, fruits and vegetables, potatoes, seafood, mushrooms, and egg yolk. According to the reports by the World Health Organisation (WHO) and the Federal Environmental Protection Agency (FEPA) 51–53, the maximum allowable limit of Cr in fish food is 0.05–0.15 mg/kg. This study found that levels of Cr detected in muscle tissues of the mullet from Laucala Bay were within the permissible limits for human consumption (0.20 ± 0.03 mg/L). However, the Ba River Delta was above the permissible limits (0.33 ± 0.11 mg/kg) reported by WHO and FEPA by a factor of 2.2, which is concerning for coastal communities that regularly consume mullet in the area.
Non-essential heavy metals exert their toxicity through biotransformation responses and oxidative stress/damage. The concentration of Hg and Cd in edible muscle tissues of the mullet in both study sites was below the limits of detection (0.1 mg/kg); however, the concentration of Pb was higher in samples from Laucala Bay (0.14 ± 0.03 mg/kg) compared to Ba River Delta (0.09 ± 0.01). Pratap, et al. 54 found that the heavy metals accumulation and concentration in sediments, especially for metals like Pb, in Laucala Bay are extraordinarily high compared to baseline studies 55–57. Additionally, the multivariate analyses in the study by Pratap, et al. 54 showed that Cu, Cd and Pb were clustered into one component, confirming the discharge of wastewater effluent into Laucala Bay as a significant point source. In a study of mullet in the Southeast Coast of India 58, levels of Pb in muscle tissues of M. cephalus were between 10.25 ± 0.15 mg/kg to 10.75 ± 0.39 mg/kg. While observations in this study did not explore the biological and biochemical effects of Pb concentrations in the muscle tissues of mullet, it is noted that the mullet species serve as a critical bioindicator for the presence of heavy metals in the environment, and the presence of Pb may pose a health risk to humans. The World Health Organization 52 has recommended that dietary Pb should not exceed 0.3 mg/kg (wet weight basis). The United Nations Food and Agriculture Organization 59 set the heavy metals regulation on the mean total lead content, as determined by analysing the edible parts of the fishery products, shall not exceed 0.2 mg/kg of fresh weight. Pb is a ubiquitous heavy metal in the environment, and lead poisoning affects almost every organ in the human body, particularly the nervous system 60. The detected Pb levels in the mullet from Laucala Bay and Ba River Delta are within the safe limits defined by the WHO and UNFAO. This suggests that moderate consumption of these fish would not pose a direct threat to human health. However, continuous monitoring is necessary to ensure long-term safety.
The presence and quantification of PAH metabolites in the mullet found total PAH levels to be greater in Ba River Delta (218.23 ± 19.33 mg/L compared to Laucala Bay (117.82 ± 11.83 mg/L). All the samples from the two sites showed the presence and traces of all four PAH metabolites in the bile, the highest concentrations for each metabolite from samples in the Ba River Delta. The International Agency for the Research on Cancer 61 have classified PAH metabolites into five groups; (1) Carcinogenic to humans; (2A) Probably carcinogenic to humans, (2B) Possibly carcinogenic to humans, (3) Not classifiable as to its carcinogenicity to humans, (4) Probably not carcinogenic to humans. The four metabolites measured in this study are in categories 1 (benzo[a]pyrene), 2B (naphthalene), and 3 (1-hydroxypyrene and phenanthrene). Benzo[a]pyrene was most concerning as it is a class 1 carcinogen which was present at both sites but also statistically significantly higher in the Ba River Delta mullet than in Laucala Bay. While some of the primary sources of PAHs in the Pacific may be from the combustion of fossil fuels and burning of organic matter, it would be more prevalent in places like Ba Province due to the extensive burning of sugarcane in the year 62, and the release of machinery oil into the delta area because of the coastal mining of magnetite 63. PAHs on the thumbprint emperor (Lethrinus harak) had levels of benzo[a]pyrene similar to those of the mullet in the same Fijian study site, Vueti Navakavu (L. harak: 0.21 ± 0.02 mg/L, 24; M. cephalus: 0.26 ± 0.05 mg/L, Varea unpubl. data). However, the values in Laucala Bay and in the Ba River Delta were almost 5-folds higher (M. cephalus: 1.04 ± 0.26 mg/L, this study) and 6-folds higher (M. cephalus: 1.16 ± 0.23 mg/L, this study), respectively. The Ba River Delta also showed much higher levels of naphthalene, phenanthrene and 1-hydroxypyrene in this study than when compared to the study by Varea, et al. 24 on the thumbprint emperor. Except for naphthalene, samples from Laucala Bay in this study also had higher levels of PAHs by 2 folds when compared to samples of the thumbprint emperor from Vueti Navakavu in the study by Varea, et al. 24. Compared to similar studies in other countries like Morrocco, Iceland and Egypt 13,64,65, total PAH concentrations in this baseline study were generally low, although to the authors knowledge, no such biological threshold for “unacceptable effects” exist for mullet. There is a possibility that when PAHs combine with other pollutants or environmental dangers, it can have an impact and potentially harm fishes like mullet 66,67. For this reason, biomonitoring should be continuously utilised because of high fish consumption by Fiji coastal communities.
Many xenobiotic compounds like PAHs and heavy metals are catalysed by the Phase I microsomal monooxygenase enzymes, also known as mixed function oxidase system (i.e., cytochrome p450, cytochrome b5 and NADPH cytochrome reductase). This study evaluated the Phase I inductive response of the cytochrome P450 system through the activity of ethoxyresorufin O-deethylase (EROD), which is the most sensitive catalytic probe 68. Samples collected in Laucala Bay were statistically significantly higher in EROD activity than in the Ba River Delta. Hepatic expression of the phase II enzyme, GST, was measured in mullet, and samples from the Ba River delta were significantly higher than in Laucala Bay (~ 4-fold). The levels of GST activity in mullet from Laucala Bay were also consistent with levels found in L. harak by Varea, et al. 24, while Ba River Delta samples were higher by a magnitude of 3 orders. Studies have demonstrated the reversal of inhibition caused by pollutants like heavy metals on EROD and GST activities 69,70. For an active mining area such as the Ba River Delta, exposure to higher levels of heavy metals in the environment would likely increase the biochemical responses of the biotransformation enzymes in mullet. Monitoring biotransformations in future studies is encouraged since the increased levels of pollutants observed here in, if left unchecked, could lead to higher activation and lower detoxification ability in seafood fish, leading to organelle and systemic biological damages at early stages.
The following antioxidant enzymes (GPX and GR) work as an enzyme cascade, where the product of one enzyme acts as the substrate for the next. GPX catalyses the reduction of hydrogen peroxide and organic hydroperoxides by glutathione, producing glutathione disulfide. Concomitantly, GR catalyses the reduction of glutathione disulfide back to glutathione, using NADPH as an electron donor. The GPX activity in mullet was not significantly different in sample groups from the two sites, while GR activities were significantly higher in samples from Laucala Bay than in the Ba River Delta. Nevertheless, the enzyme cascade between GPX and GR was notably reflected in mullet from both sites based on the levels of pollutants present in the respective environments. Studies show that relatively high levels of organic pollutants like PAHs and essential metals are likely to induce higher levels of oxidative stress responses due to hepatic metabolism and biotransformation efficiency 71,72. In this study, it was observed that mullet from Ba River Delta, which had more polycyclic aromatic hydrocarbons (PAHs) and chromium (Cr), also had higher levels of biotransformation activities.
Conversely, pollutants like metals (e.g. Hg, Pb, and Cd) impair antioxidant defences in fish, especially those involving thiol-containing antioxidants and enzymes. However, noting the low levels of Hg, Pb and Cd found in sample groups across the two sites, it is possible that the oxidative stress responses were due to organic xenobiotics that undergo biotransformation and detoxification. Cell-reducing agents such as GSH and NADPH can reduce Cr (VI) to the pentavalent state (V), which can participate in the Fenton reaction to produce hydroxyl radicals. This transformation is an effective means of detoxifying Cr (VI) in biological systems. The findings demonstrate that the biomarker responses and application of this species for biomonitoring studies align with other ecotoxicological studies in other countries, strengthening the support to incorporate more (suits of) biomarkers in Fiji and the Pacific Island Region, using mullet for biomonitoring studies.
LPO was used to measure biological damages in mullet from Laucala Bay and the Ba River Delta. A radical attack on lipids leads to the formation of lipid peroxides, which can decompose to yield alkanes, ketones, and aldehydes. The aldehydes studied herein are malondialdehyde (MDA). The variety of lipid peroxidation (LPO) by-products can also exert adverse biological effects in exposed animals 73. LPO was found to have statistically significant differences between samples from the Ba River Delta and Laucala Bay. It is important to note that exposure of marine fish to heavy metals or PAHs by environmentally realistic routes can increase LPO, and fish rely on glutathione as a first line of defence against such exposures 74. For example, GPX enzymes are an essential protective mechanism against oxidative damage 75. In this study, sample groups with low activities of GST, GPX and GR (i.e., Ba River Delta) show correspondingly higher levels of lipid peroxidative damages compared to Laucala Bay. This is consistent with studies demonstrating these biochemical linkages 74,76,77. When compared to a similar Fiji study of L. harak by Varea, et al. 24, mullet from Laucala Bay were lower in LPO concentrations (different by 73% of their average value), but Ba River Delta samples were higher in concentration (different by 25% of their average value).
Erythrocyte nuclei abnormalities (ENA), including the formation and presence of micronuclei (MN) in the red blood cells of the mullet, indicate genotoxicity as a reflection of genetic material damage. Samples from the Ba River Delta had significantly higher occurrences of ENA and MN in 2000 erythrocytes compared to samples from Laucala Bay. The presence of abnormal erythrocytes in the mullet induced by exposure to a range of genotoxic materials can include, but not limited to, pollutants like heavy metals 78,79 and PAHs 80,81. In this baseline study, the mullet had various degrees of sensitivity in monitoring genetic and clastogenic damage, as indicated by the variations in the average numbers of micronucleated cells. While the frequency of other nuclear abnormalities (i.e. ENA assay) is considered a relatively less efficacious indicator of genotoxicity than MN assay 82, as a baseline study, it was necessary to include it for comparison with future ecotoxicological studies in Fiji. The findings show that early signs of biological damage at the genetic level were evident in mullet, which are about a quarter of an order of magnitude (~ 0.27) in Laucala Bay samples and about half an order of magnitude (~ 0.60) in Ba River Delta samples when compared to levels in L. harak from a similar Fiji study 24.
When considering the biochemical and environmental parameters in this study, mullet has demonstrated to be a helpful sentinel species for ecotoxicology studies, as the biomarker responses observed correspond to the general condition of the environment and the level of pollution presence quantified in mullet in this study. The findings in this study align with a study by Telli Karakoc, et al. 83 that determines the ability of the mullet to live in contaminated environments and shows that enzyme activities and DNA adduct levels can serve as indicators of marine pollution. Similarly, Pacheco, et al. 84 find that the application of biomarkers like EROD activity, ENA frequencies, as well as biotransformation and genotoxicity biomarkers (as applied herein) could be successfully employed for biomonitoring anthropogenic contamination using mullet, both in the Atlantic and Mediterranean European coastal waters. The findings show that the Ba River Delta is polluted, and mullet are undergoing biological and biochemical stress, which may result from the heavy anthropogenic activity in the area. Laucala Bay also shows early warning signs of pollution effects in mullet at the biological and biochemical levels, likely due to multiple sources of entry of pollutants, like the Rewa River, Kinoya sewerage treatment outfall and the surrounding urban Suva district. With interactions between local and climate change stressors being expected to create particularly damaging synergies 85, this baseline study is a necessary and urgent first step onto which future ecotoxicology studies can be developed in Fiji and the other tropical Pacific Island Countries. We recommend the use of mullet as a sentinel species for future marine ecotoxicological studies and suggest the application of wider suits of biomarkers to elucidate the suitability of a broad inclusion within biomonitoring to strengthen environmental risk assessments on a national and regional level.