Literature Review

Seagrasses are highly specialized marine angiosperms that inhabit shallow coastal waters, remaining fully submerged yet prominently visible in nearshore environments. Although they occupy only about 0.1–0.2% of the ocean's surface, they contribute disproportionately to global carbon dynamics-sequestering approximately 20% of the ocean’s total carbon [1]. Beyond carbon storage, seagrasses provide critical ecosystem services, such as acting as natural filters that trap carbon and stabilize sediments. Importantly, they have emerged as effective bioindicators of heavy metal pollution due to their sessile nature, which allows for accurate, site-specific monitoring of environmental contamination over time.

Numerous studies support the utility of seagrasses in tracing heavy metal contamination. Metal accumulation patterns have been shown to vary based on factors such as metal type, environmental conditions, and sediment characteristics, highlighting the strong interplay between habitat quality and bioaccumulation potential in seagrasses. [2] Demonstrated that species like Zostera capricorni effectively accumulate trace metals such as copper (Cu), lead (Pb), and zinc (Zn), reflecting the surrounding water and sediment quality. Despite the relatively low diversity of seagrass species globally, their ecological roles are significant. They provide habitat for commercially valuable fish and invertebrates, shelter juvenile and endangered marine fauna, stabilize shorelines, contribute to primary productivity and complex food webs, produce oxygen, and act as long-term carbon sinks [3,4]. In addition to their ecological importance, seagrasses are also harvested for commercial and cultural purposes [5, 6] further embedding them in the socio- economic fabric of coastal communities [7]

Their extensive root and rhizome systems play a crucial role in sediment stabilization, reducing erosion and resuspension, and facilitating the deposition of suspended particles [8,9]. These functions underscore their importance to the health of coastal ecosystems and economies.

However, the proximity of seagrass meadows to human activity makes them highly vulnerable to a wide array of anthropogenic pressures including pollution, habitat destruction, overharvesting, and climate change [10,11]. Physical disturbances from vessel groundings, propeller scarring, and coastal infrastructure development are particularly detrimental in shallow-water meadows.

These activities can uproot plants, damage rhizome networks, and create sediment blowholes, leading to long-term degradation of seagrass beds [12,13]. Such disturbances reduce biomass and impair the recovery potential of these ecosystems.

The global decline of seagrass habitats is increasingly well-documented and is largely driven by anthropogenic stressors, including eutrophication, pollution, and sedimentation [14,10].

Physiological responses of seagrasses to heavy metal exposure such as reduced photosynthetic efficiency, inhibited growth, and altered leaf morphology serve as indicators of environmental degradation [15]. These responses enhance their value as early-warning systems in coastal ecosystem monitoring.

As a result, seagrasses are widely acknowledged as sensitive and reliable bioindicators of environmental health. Variations in physiological traits such as pigment content, biomass, and growth rates provide insight into changes in water quality, nutrient load, and pollutant levels [2]. Monitoring these parameters offers a practical tool for assessing the ecological integrity of marine environments and supports informed management and conservation strategies. Preserving seagrass diversity is therefore essential not only for biodiversity but also for maintaining their vital ecological services, including carbon sequestration, habitat provision, and sediment stabilization [16].

Heavy metals defined as elements with a density greater than 5 g/cm³ are persistent environmental pollutants that pose serious ecological and health risks when present in excess of regulatory thresholds. Their toxicity has garnered growing concern due to implications for ecosystem health, human nutrition, and evolutionary processes [17]. In marine ecosystems, prolonged exposure to heavy metals can impair reproductive function, cause neurological and respiratory disorders in aquatic species, and lead to bioaccumulation in the food chain posing severe health threats to human consumers. While certain metals such as iron are essential for biological functions, and others like copper, zinc, and lead play roles in enzymatic activity at trace levels, their accumulation beyond permissible concentrations is hazardous [18]. Chronic exposure to elevated levels of heavy metals such as lead and mercury has been associated with progressive physical, muscular, and neurological deterioration, contributing to conditions like multiple sclerosis, Alzheimer’s disease, and Parkinson’s disease [19].

In the Chwaka Bay ecosystem, evidence has already pointed to reduced reproductive success in marine organisms, impaired mobility, seagrass bed degradation, and increasing health risks linked to bioaccumulation. These findings highlight the urgency of assessing heavy metal concentrations in both seagrasses and commonly consumed fish species from the region, and of identifying the likely sources of contamination to inform mitigation strategies.

Preparation of Seagrass Species for Analysis

The four seagrasses species were identified by our marine biologist Dr. Rashid Juma Rashid named as Enhalus Acoroides, Halophila Ovalis, Cymodocea Rotudanta and Thalassia Hemprichii. These sea grass were sampled in the form of leaves and stems then labelled as CHW-E.A, CHW-H.O, CHW-C.R and CHW-T.H respectively. They were then transported in well dried sealed sample bags to African Minerals and Geosciences Centre (AMGC) laboratory. Seagrass samples were individually placed on clean trays and oven-dried at 100 °C for 45 minutes using a laboratory drying oven, following modified procedures adapted from standard protocols [20,21]. Once dried, the samples were pulverized using a clean mortar and pestle to obtain a homogeneous powder. Approximately 0.5 g of each powdered sample was accurately weighed using an Ohaus PR Series Analytical Balance. For digestion, the samples were placed in Teflon vessels and subjected to microwave-assisted acid digestion using an ETHOS Easy Microwave Digestion System for 15 minutes, following the manufacturer's guidelines and similar methodologies reported [22].After digestion, the contents were quantitatively transferred to 50 mL volumetric flasks and diluted to volume with distilled water. The digested solutions were then filtered using Whatman® No. 1 filter paper (125 mm) by gravity filtration. The final filtrates were analyzed for heavy metal content using an Agilent 7900 Inductively Coupled Plasma– Mass Spectrometer (ICP-MS).

Quality Assurance Procedures

Appropriate quality assurance procedures and precautions were carried out to ensure the reliability of the results. Samples were homogenized and carefully handled to avoid contamination. The procedural blanks, calibration standards and duplicate samples were used for calibration of the instrument, process control and quality assurance, accuracy, precision, limit of quantification, limit of detection and validation of the 7900 ICP-MS method.

Questionnaire Survey

A questionnaire-based survey was conducted to establish the commonly consumed fish species in Chwaka region of Unguja Island, Zanzibar. Questionnaires were administered to 35 randomly selected participants from the Chwaka region. Apart from the locally consumed fishes, it also inquired about basic information on gender, age, education, income level, frequency and ingestion rates of the commonly consumed fish species.

Sampling of Fish

Eight generally purchased and consumed fishes in Ckwaka region of Unguja Island immediately caught were sorted and stored in an icebox, transported to the laboratory where the samples were maintained at −25 °C till further investigation. They were purchased from the market while dead and fish caught for commercial sale do not require obtaining permission from the Local Ethics Commission.

Procedure of Trace Elemental Analysis from the Eight Fish

Fish samples were first allowed to equilibrate to room temperature prior to dissection. Using a clean stainless-steel knife and a sterile ceramic cutting board, approximately 20 g of dorsal muscle tissue was extracted from each specimen. The tissue was rinsed thoroughly with distilled water to remove surface contaminants and then freeze-dried at −80 °C until a constant weight was achieved. Dried muscle samples were subsequently pulverized and homogenized using a mixer mill to ensure uniformity.

An accurately weighed 1.0 g aliquot of each powdered sample was transferred into acid-cleaned Teflon vessels and digested with 10 mL of concentrated nitric acid (HNO?, Supra pure grade); Adolf Plinke GmbH) at room temperature for approximately 3 hours. The vessels were then sealed and heated at 80 °C for 7 hours to enhance digestion.

Afterward, the vessels were opened and the contents were heated further at 100 °C for around 3 hours to evaporate any remaining acid. This digestion process was repeated until only a negligible white residue remained. The dried residue was reconstituted in 80 mL of 2% nitric acid, followed by filtration through Advantec 5C filter paper (110 mm diameter; Advantec MFS, Dublin, CA, USA). The filtrate was then diluted to a final volume of 100 mL with 2% nitric acid. Concentrations of metals including As, Al, B, Mn, Si, Mo, Ni, La, Cu, Cr, and Pb were quantified using Inductively Coupled Plasma–Mass Spectrometry (ICP-MS; PerkinElmer ELAN 6000)according to [23]. Mercury (Hg) concentrations were determined separately using Cold Vapor Atomic Absorption Spectrometry (CV-AAS) on a Bacharach Coleman MAS-50 mercury analyzer.

Quality Assurance Procedures

The accuracy and precision of the analytical procedures were validated through the analysis of certified reference materials (CRMs). For mercury (Hg), ERM-CE278, produced by the Institute for Reference Materials and Measurements (IRMM), was employed. For all other heavy metals, DORM-3, a fish protein reference material supplied by the National Research Council Canada (NRCC), was utilized. These CRMs were processed and analyzed using the same sample preparation and analytical protocols as those applied to the actual fish and seagrass samples. The mean recoveries for the heavy metals in the CRMs ranged from 92% to 102%, indicating satisfactory accuracy of the analytical methods. Method blanks were also subjected to the complete analytical workflow and showed similar recovery rates (92% to 102%). All heavy metal concentrations were reported on a dry weight basis and expressed in milligrams per kilogram (mg/kg) according to [24].

Estimated Daily Intake (EDI) used to assess metal intake in humans, and it is a common method in environmental health and toxicology for evaluating potential exposure to heavy metals or trace elements through food, water, or other sources. It is defined as amount of a substance (like a metal) ingested daily per unit of body weight, typically expressed as:

EDI (mg/kg body weight/day) = (Concentration of metal in food × Daily intake of food) / Body weight. Daily intake is approximately 348 gm/day; average body weight = 63 kg.

The EDI obtained shall be compared with world statutory health organization values. If it exceeds benchmarks set by World Health Statutory Authorities like the WHO, FAO, USEPA, etc., values, it indicate a potential health risk [25,26].

THQ: Target Hazard Quotient

THQ is used to evaluate non-carcinogenic risk from consuming contaminated food (like fish with heavy metals). If it is greater than one, then there is potential health risk and vice versa. Its formula is given as; THQ=EDI/RfD

• EDI = Estimated Daily Intake (mg/kg/day)
• RfD = Reference Dose (safe daily exposure limit set by USEPA/WHO)

HI: Hazard Index

When there are multiple contaminants, we calculate a cumulative risk as:

HI=∑THQi; Where THQi is the THQ for each individual metal. If it is less than one then it is safe while if it is greater than one then combined metal exposure could pose non-carcinogenic health risk

To determine the PI of the elements, statistical analysis was performed on the elemental concentrations in fish samples. The PI is the ratio of element x concentration in the sample to the element’s maximum allowable level [27].

PI(x) = Metal concentration in the sample/Permissible limit or background value

It is generally accepted that if an element’s PI value is more than 1.0, the element is highly likely to have contaminated the sample and may even be dangerous at the amount it is present.

Cumulative Pollution Index CPI = C1×C2×?×Cn) 1/n , Where:

• C1, C2,...,Cn are the concentrations (mg/kg) of individual heavy metals in the fish sample
• n is the number of metals considered

This is the geometric mean of metal concentrations and gives a single number that reflects cumulative contamination.

The table 4.0 shows the series of calibration curves used for the estimate of metals from the sea grasses.

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