Introduction
Fossil fuel hydrocarbons have a negative impact on the environment because of their high aromatic hydrocarbon content and their ability to resist microbial degradation [1,2]. Moreover, following the initial dispersal of the spill, hydrocarbons have the potential to continue seeping or desorbing from sediments, which can result in benthic organisms being exposed to high concentrations of polycyclic aromatic hydrocarbons (PAHs) and alkanes (major components of crude oil) [3,4]. Furthermore, there is a concern that organic substances, particularly petroleum hydrocarbons, could have similar adverse effects on human health [5,6].
Anthropogenic activities heavily impact the North Java Coast, the lack of environmental consciousness among coastal communities, and inadequate enforcement of environmental regulations, leading to a decline in the quality of coastal waters [7–9]. The waste generated by human activities directly and indirectly enters the waters, accumulating in water bodies, sediments, and the organisms inhabiting them [10–12]. This accumulation of organic and inorganic waste can have detrimental effects on aquatic organisms and even pose toxic risks to humans [13].
Studies on organic pollutant pollution in green mussels are still very limited in Indonesia, especially studies regarding the relationship between potential genomic instability and organic pollutants contained in biota. Cilincing Waters in DKI Jakarta are heavily polluted due to the significant industrial, fishing, port, and household activities that contribute to high levels of pollution in the area [14–16]. On the other hand, pollution in Sayung waters has also been reported. The Sayung estuarine is located in Sayung District, Demak Regency, and is home to a mangrove ecosystem area, as well as is surrounded by the Kaligawe industrial area and pond areas. The water quality in this area is considered to be moderately polluted [17]. Meanwhile, the water of Rembang is generally utilized for marine tourism, fishing, residential areas, and aquaculture. This area is classified as lightly polluted [18–20].
Green mussels (Perna viridis), are a valuable species that primarily inhabit river estuaries and are widely distributed in the coastal waters of Indonesia, particularly in the North Java Coast. These benthic organisms possess a remarkable ability to absorb pollutants, making them an important bioindicator species for coastal pollution. The presence of chemical compounds, commonly found in waste, can easily permeate and accumulate within green mussels [21,6]. These compounds are toxic and can cause various forms of genomic instability and alterations in genetic patterns within the population. The extent of genomic instability serves as an indicator of the toxicity level of these compounds in marine animals [22]. Disruptions in DNA polymorphisms in marine animals can have adverse effects on their physiological functions. Marine organisms are commonly used as bioindicators of environmental pollution due to their ability to absorb contaminants and exhibit molecular responses such as genomic instability in the presence of toxic substances [23].
Ecogenotoxicology is an approach that uses genetic techniques to assess the effects of genotoxic substances from environmental pollution on ecosystem health [24]. Research on ecogenotoxicity in marine animals in Indonesia is scarce as studies on green mussels are limited to cell histology, physiology, and morphology [25–27]. The molecular response to chemical compounds is critical, and one method for assessing molecular toxicity is Random Amplified Polymorphic DNA (RAPD) analysis. RAPD analysis is based on polymerase chain reaction (PCR) and involves the random amplification of DNA segments without prior knowledge of the genome under study. This technique uses short, randomly selected primers, requires minimal DNA, and can detect genetic changes in aquatic organisms following contamination with pollutant compounds, as well as intrapopulation polymorphisms [28,29]. By comparing DNA banding patterns, these techniques enable the identification of differences between polluted and non-polluted organisms.
Previous studies used RAPD to assess genomic instability caused by various toxic compounds, including organic compounds, in different shellfish species. Qu et al. [30], Rocco et al. [31], Mat et al. [32], and Yap et al. [33] successfully used RAPD to assess genomic instability in shellfish exposed to toxic compounds. In addition, RAPD has been used to study the influence of chemical compounds on genomic instability in different biota. Pandey et al. [34] examined the genomic instability caused by pesticide compounds in the freshwater fish Channa punctatus. Zhou et al. [35] investigated the genotoxicity of nitrofurazone in the marine ciliate Euplotes vannus, while Atienzar et al. [36] investigated the effects of ultraviolet radiation on the marine macroalga Palmaria palmata. Furthermore, Atienzar et al. [37] studied the toxicity of Cu metal on Daphnia magna, and Atienzar et al. [38] studied the genotoxicity of phenolic compounds in the crustacean Elminus modestus.
Despite the extensive use of RAPD in genotoxicity studies, there is a lack of research on marine biota, particularly green mussels (Perna viridis), in The North Java Coast in particular. It is critical to fill this gap in knowledge, as Indonesia is a hotspot for marine biodiversity, but also at risk for high levels of pollution [39]. Therefore, this study aims to address the research gap by evaluating the differences in environmental quality and how it affects the genotoxicity of green mussels in The North Java Coast.
Materials and Methods
Study Location and Sampling
Green mussel samples were collected from three locations, Cilincing (heavily polluted), Sayung (moderately polluted), and Rembang, representing various pollution levels (Figure 1). Rembang water (lightly polluted) was chosen to serve as a reference site. The green mussels from Jakarta Bay were obtained from TPI Cilincing, while the green mussels from Sayung and Rembang waters were sourced from local fishermen close to Sayung Harbor and Rembang Harbor.
To ensure consistency, samples of green mussels were collected from 20 individuals, all of which had a uniform size with a shell length greater than 6 cm. The green mussels were cleaned on-site using seawater to remove any contaminants both biotic and abiotic. Subsequently, they were stored in a ziplock plastic pouch and labeled for further analysis in the laboratory. For DNA analysis, the shells of the green mussel samples were carefully opened, and aseptic surgery was performed to extract the gill tissue (figure 2). The extracted tissue was then placed in a sample bottle containing a solution of absolute Ethanol (99%) to preserve the DNA. To maintain the integrity of the samples, they were stored in a cool box filled with ice, ensuring a temperature of approximately 10°C, during transportation to the laboratory for further analysis.
Genomic Instability analysis of green mussel
The green mussel DNA extraction process followed the standard protocol of the Qiagen kit (catalog number ID 69506). First, green mussel gill tissue was ground in a 1.5 mL microcentrifuge tube with 180 μL of ATL buffer solution, which served as a tissue lysis buffer. Then, 20 μL of proteinase K was added to the tube and the mixture was incubated overnight at a temperature of 56 °C. After the incubation period, the samples were supplemented with 200 μL buffer AL and 200 μL ET-OH (96%-100%). The resulting mixture was then transferred to a DNA Easy Mini spin column and centrifuged for one minute at a speed of 8,000 rpm. The residue from the centrifugation step was discarded. Next, 500 μL of AW1 buffer was added to the spin column and centrifuged again for 1 minute at 8,000 rpm. The spin column was then placed in a new 2 mL collection tube and 500 μL of AW2 buffer was added. This was followed by another centrifugation step at a higher speed of 14,000 rpm for three minutes. To elute the DNA, 200 μL of AE buffer (elution buffer) was added to the spin column and centrifuged at 8,000 rpm for one minute. The resulting eluate contained the extracted DNA. An IMPLEN nanophotometer was used to assess the concentration and quality of the DNA extract. This instrument provided accurate measurements of both the concentration and quality of the extracted DNA.
DNA amplification was conducted using the Qiagen HotStarTaq Plus Master Mix kit DNA amplification reagent (catalog number ID 2303646) containing dNTPs and Taq DNA polymerase. A total of 15 μL of master mix solution was combined with 1 μL of DNA extract, 1 μL of RAPD 6 primer (5-CCCGTCAGCA-3), RAPD primers OPB07, OPB10, OPC09, OPC13, OPAH-01, 04, 15, 19, OPA-06, OPA-13, and ddH20 solution to reach a final volume of 20 μL. The selection of primers was based on amplification results with a sufficient number of bands, high band definition, and reproducibility. The amplification process involved adjusting the PCR machine temperature settings as follows: pre-denaturation at 95°C for five minutes, 45 cycles of denaturation at 95°C for 1 minute, annealing at 36°C for 1 minute, extension at 72°C for 2 minutes, and post-extension at 72°C for five minutes. The PCR product was visualized using 100v electrophoresis on a 2% agar gel stained with EtBr solution and documented on a UV transluminator after 20 minutes.
Changes in the number of DNA bands are related to changes in the genetic material (Liu et al., 2007). Polymorphic patterns resulting from RAPD-PCR profiles are used to calculate genomic template stability (GTS, %) as follows:
GTS (%) = (1-a/n) x 100
where a is the number of in the DNA from polluted waters detected polymorphic bands is; and n is the total number of bands in the reference site waters. Polymorphisms in the RAPD profile included the disappearance and appearance of new bands in the reference site water DNA.
Identification of organic pollution in green mussels
Approximately 5 grams of green mussels with 2 grams sodium sulphate anhydrate were used for extraction by the Sokhlet technique for 8 hours with the solvents DCM, n-hexane, and tetrachloroethylene with 120 ml volume respectively. The extracted substances underwent a purification procedure involving silica gel 60. Subsequently, the purified substances were condensed to a volume of 3 mL. The Total Petroleum Hydrocarbon (TPH) value was determined using an iD5 Thermo FTIR instrument Nicolet iS5 Thermo with operational system: Accessory: iD1 Transmission; wavenumber range: 4000-400 with max range limit: 4000 cm-1, min range limit: 400 cm-1. The Thermo Scientific™ OMNIC™ TQ Analyst™ software is employed to interpret the data collected from these measurements [41]
Quality control
The calibration standards were initially set up by mixing iso-octane and octanoic acid in equal amounts in tetrachloroethylene (ASTM, 2011). Peak height and resolution were checked by analyzing relevant standards before conducting sample analysis. Prior to analyzing each set of samples, a solvent blank, standard mixture, and procedural blank were processed one after the other to verify contamination levels and ensure precise peak identification and quantification.
Data analysis
Band presence/absence data were summarized at the site level for calculation of GTS. Inferential statistical testing was not applied due to absence of verified population genetic homogeneity. Results are interpreted descriptively and supported by multivariate association analysis. The results were analyzed using principal component analysis (PCA) to investigate further interrelationship between petroleum hydrocarbon pollutants, RAPD PCR, and GST value with PAST: Paleontological Statistics software package for education and data analysis 4.13 version.
Results
RAPD-PCR Banding Pattern in Green Mussels
A total of 20 individuals were obtained from each sampling location. Individuals with relatively uniform shell sizes were selected to minimise the variability associated with age and growth stage. The sampling strategy may restrict the representation of the entire population structure, and consequently, the results viewed as indicative of genotoxic responses within the sampled cohort rather than the entire species population.
Among the ten RAPD primers screened, only primer RAPD-6 consistently produced scorable DNA bands across all green mussel samples (Perna viridis). In RAPD-based ecogenotoxicity studies, multiple primers are used to enhance reproducibility and reduce locus-specific bias. Drawing ecological or toxicological conclusions from a single primer is methodologically flawed. This limitation's existence is acknowledged for this study. RAPD-PCR analysis showed distinct differences in banding patterns based on sampling location. Mussels from Rembang Waters (considered low pollution) produced 7 DNA bands, while samples from Sayung (moderate pollution) and Cilincing (heavy pollution) yielded 4 and 3 bands respectively (Figure 3, Table 1). Gel profiles obtained at each sampling site were used to perform RAPD band scoring. The number of bands and GTS values reported represents the overall RAPD profile rather than variation at the individual level. These band differences reflect genomic variation likely influenced by the environmental quality of each location.
Genomic Template Stability (%GTS)
%GTS values were derived from the RAPD banding profiles and used as a quantitative biomarker of DNA integrity. The highest %GTS was found in Rembang Waters (100%), followed by Sayung Waters (60%) and Cilincing Waters (40%). These values suggest increasing genomic instability corresponding to higher pollution gradients (Table 1). RAPD banding patterns and GTS values varied with the pollution gradient. Findings are presented as descriptive indicators of genomic response patterns.
Total Petroleum Hydrocarbons (TPHs) in Green Mussel Tissue
TPH concentrations in green mussels differed by sampling location. The highest levels were detected in Cilincing Waters (5.16 ± 1.14 mg/kg wet weight), indicating significant hydrocarbon exposure. Intermediate levels were found in Sayung Waters (2.91 ± 0.41 mg/kg), while the lowest TPH concentrations were recorded in mussels from Rembang Waters (2.01 ± 1.07 mg/kg) (Figure 4). These results reflect the varying degrees of hydrocarbon contamination in the different coastal zones.
Correlation Between TPH Concentration and Genomic Instability
To evaluate the relationship between pollutant levels and DNA integrity, principal component analysis (PCA) was performed. Two principal components (PC1 and PC2) with eigenvalues >1 explained 99.91% of total variance (Figure 5). PC1 accounted for 77.76% and was strongly associated with %GTS. PCA results revealed that mussels from Cilincing and Sayung were predominantly impacted by petroleum hydrocarbon pollutants, with a clear inverse relationship observed between TPH levels and %GTS values.
Disccusion
Green mussel genotoxicity test
Primer RAPD 6 proved to be the sole primer among the ten RAPD primers tested that consistently produced bands without any fragmentation. The efficacy of this primer was previously confirmed in the assessment of genomic instability in marine mussels, Mytilus galloprovincialis [43]. The RAPD PCR analysis on green mussels from different sampling locations, such as Jakarta Bay (representing heavily polluted waters), Sayung Waters (moderately polluted), and Rembang Waters (lightly polluted), is shown in Figure 3. In Rembang Waters, 7 bands were observed on green mussels, while 3 bands were detected in Jakarta Bay Waters and 4 bands in Sayung Waters. The %GTS value was calculated to be 40% in Jakarta Bay Waters and 60% in Sayung Waters (refer to Table 1). This study highlights the variations in environmental conditions that can lead to alterations in the DNA bands of green mussels. The differences observed in the RAPD PCR analysis are associated with the quality of the polluted aquatic environment. RAPD band polymorphisms may arise from environmental stress, natural population-level genetic variation, primer–template mismatches, polymerase chain reaction (PCR) artifacts, and differences in DNA quality [44]. Since population genetic identity was not validated, band shifts cannot be exclusively ascribed to genotoxic influences. The findings are interpreted as potential genomic instability associated with polluted environments. Farhadi et al. [45] reporter thar the DNA band patterns of rock oysters (Saccostrea cucullata) from polluted waters differed from those in unpolluted waters. Changes in DNA bands during RAPD PCR analysis are attributed to genomic instability, mutations, and/or complex chromosomal rearrangements induced by genotoxins [46,,29].
Ecogenotoxicity can be determined by determining the %GTS value of various water bodies. A recent study has shown that a significant increase in water pollution leads to a decrease in the %GTS of green mussels (Perna viridis). Similar results were observed in Mytilus galloprovincialis shellfish exposed to a combination of titanium dioxide nanoparticles and cadmium chloride, with increased concentrations of these chemical compounds leading to a reduction in the shellfish's %GTS [43]. The percentage GTS value of green mussels in Jakarta Bay was 40%, while it was 60% in Sayung Waters, indicating that the pollutant concentration in Jakarta Bay is higher compared to Sayung Waters. The assessment of environmental genotoxicity using the Bivalvia group was also carried out by Butrimavičienė et al. [47], who observed an increase in genomic instability in mussels in the Gulf of Riga (Baltic Sea) due to the presence of pollutant compounds in the water that cause genomic changes (genotoxin pollution). The changes in GTS reflect RAPD banding pattern alterations and may indicate genomic instability in organisms exposed to environmental stressors. Decreases and increases in GTS values may represent changes in genomic integrity associated with genotoxic stress, with reductions in GTS more frequently reported as indicators of DNA damage in contaminated environments [29]. Qu et al. [30] also supported the use of RAPD-PCR and %GTS analysis in evaluating the genotoxicity of pollutant compounds on Mytilus coruscus.
Pollutant compounds present in water can infiltrate the body of organisms through the gills during water filtration. Internally, these compounds tend to accumulate in the mantle and digestive glands. According to a study by Banni et al. [48], these chemical compounds have the ability to activate pathways that lead to genomic instability. This activation occurs through their interaction with DNA strands and the formation of DNA adducts. Interestingly, these compounds show a strong preference for binding to the exocyclic amino groups of deoxyguanosine and deoxyadenosine. This interaction can cause various anomalies within organisms. Pratt et al. [49] further support this assumption by explaining that genetic damage can also occur indirectly through the action of chemical compounds. One such mechanism involves the production of reactive oxygen species that can break DNA strands. Kumar et al. [50] have shown that chemical compounds can induce the production of these reactive oxygen species. In addition, these compounds can interfere with the processes of base excision repair and nucleotide excision repair, which are responsible for repairing damaged DNA. This disruption ultimately inhibits the DNA repair machinery. García-Leston et al. [51] have provided evidence to support this claim.
Total petroleum hydrocarbons (TPHs) in green mussels
The areas under study are complex systems where chemical stressors rarely appear in isolation. While the current research highlights Total Petroleum Hydrocarbons (TPH), these environments are defined by a variety of overlapping factors, including heavy metals and runoff from farming. Consequently, any observed genomic changes must be evaluated carefully, as failing to quantify all co-occurring pollutants risks incorrectly crediting TPH for biological responses that emerge from a more complicated toxicological setting. TPH levels in green mussels showed that the highest was detected in green mussels from Cilincing Waters, 5.16 ± 1.14 mg/kg, while the lowest TPH level was detected in green mussels from Rembang Waters, 2.01 ± 1.07 mg/kg, and the TPH level in green mussels from Sayung Waters was 2.91 ± 0.41 mg/kg (Figure 4). TPH levels in marine mussels indicate TPH contamination in marine environments [52]. High anthropogenic activities originate from municipal and shipping activities around Cilincing Waters due to affected TPH pollutants in green mussels [6]. On the other hand, Sayung Water is predominantly inhabited by mangrove ecosystems. Geographically, the location of Sayung Water is neighboring the international port and industrial area of Kaligawe, East Semarang, which is also a source of TPH pollutants that expose green mussels in Sayung Waters [53]. Consequently, green mussels in Sayung Water were exposed to petroleum hydrocarbon substances. In contrast to other locations (Cilincing and Sayung), TPH exposure in green mussels in Rembang Waters was detected. However, petroleum hydrocarbon pollutants were still detected in the green mussels even at low levels. The Rembang Waters are commonly used for marine tourism, fishing, and residential settlements along the coast [18]. The petroleum hydrocarbon pollutants predominantly come from shipping activities, ballast waters, urban/industrial runoff, and atmospheric deposition [54].
Rybakovas [55] showed that organic pollutants correlate with the genotoxicity of bivalve mussels in Lithuanian rivers. Furthermore, Pazi et al. [56] reported a positive correlation between the organic pollutant levels and the genotoxicity of marine mussels in Turkish waters. Either directly or after metabolic activation, organic compounds (including TPH) can interact with DNA and enzymatic complexes, causing the formation of genetic disorders. A malfunctioning DNA repair system can heighten the chances of developmental and degenerative diseases, along with genome instability caused by genotoxic compounds [57]. Genetic abnormalities can lead to a decline in reproductive capacity, decreased survival rates in natural populations, and ultimately, the loss of genetic diversity [58].
Correlation between Genomic Instability with TPH levels in green mussels
Marine mussels are frequently utilized for genotoxic monitoring, specifically evaluating the level of genomic instability in somatic cells such as gills, digestive glands, and hemolymph. The gradual genome destruction in somatic cells can increase dysfunction within individual cells, tissues, and organs, progressing from reversible to irreversible stages, ultimately disrupting life processes and the organism's death.
A study by Slobodskova et al. [59] results play a role in the connection between seawater pollution and DNA integrity. Previous studies have shown significant harmful DNA changes in marine mussels, where anthropogenic activities are situated along the coast [60,61]. Nevertheless, mussels inhabiting Cape Krasny’s waters may undergo DNA degradation not just from contaminants, but also from limited oxygen levels as the environmental oxygen levels reach the minimum threshold. Previous studies have demonstrated that oxygen deficiency can trigger damaging modifications to the DNA of Mizuhopecten yessoensis and Corbicula japonica mollusks, causing genome instability [59].
A study from Rybakovas [55] showed that organic compound pollution is positively correlated with the genotoxicity of bivalve mussels in Lithuanian rivers. The same thing was also reported by Pazi et al. [56], who concluded that there was a positive correlation between the concentration of organic compounds and the genotoxicity of marine mussels in Turkish waters. Either directly or after metabolic activation, organic compounds (including TPH) can interact with DNA and enzymatic complexes, causing the formation of genetic disorders. If the DNA repair system is damaged, genotoxic compounds will increase the risk of developmental and generative diseases, as well as genome instability [56]. Genetic disturbances may further result in the decrease of reproductive potential, and the reduced viability of natural populations and, in the long term, can cause the loss of genetic diversity [58].
Principal component analysis (PCA) was applied to evaluate the interrelationship of petroleum hydrocarbon pollutants and genomic instability in green mussels. In addition, this PCA was also employed as a means of identifying covariance patterns to emphasize its exploratory role, rather than as proof of direct causality. In the PCA biplot, Total Petroleum Hydrocarbon (TPH) levels and genomic changes (measured by the 'Number of bands') are positioned together on the negative side of the first principal component (PC1). This positioning indicates a significant statistical link, implying that genomic shifts and hydrocarbon levels are closely grouped. Consequently, the study utilizes terms like 'correlated' or 'clustered' to describe this relationship, ensuring accuracy while respecting the intricacies of field data.
Based on eigenvalue >1.0, factors analysis retained two factors that explained 99.91% of the total variance (Figure 5). The contribution of PC1 was 77.76% of the total variance and was largely a function of the GTS value in green mussels. It has lower accumulation in the contribution of TPHs in green mussels, particularly in gills. Including the lesser interrelationship between the adsorption of petroleum pollutants in green mussels. Petroleum hydrocarbons pollution is recognized as organic pollutants from diesel or gasoline fuel of ships and boats [62]. The PC2 analysis reveals that green mussels in Cilincing and Sayung waters are predominantly impacted by petroleum hydrocarbons. The relationship between TPH levels and green mussel genomic instability is directly proportional; the higher the TPH level, the more massive the potential for genomic instability. Organic pollutants in the environment have the ability to act as prooxidants in different parts of the body, leading to genomic instability and other harmful effects on biological systems [61].
Conclusions
The application of genotoxicity as a strategy in ecotoxicological studies is a novel approach that can effectively address a range of environmental challenges. The findings of this study indicate that the levels of total petroleum hydrocarbons (TPHs) in green mussels from Cilincing Waters are considerably above those from Sayung and Rembang Waters. This study has demonstrated an association between pollution gradients and potential genomic instability in Perna viridis along the North Java Coast. RAPD-PCR is recommended to be used as an early-warning biomonitoring tool with cautious interpretation. Further research integrating population genetic verification and multi-pollutant analyses is required. Furthermore, the marine mussel model serves as a reliable bioindicator for assessing ecosystem health or identifying adverse impacts in monitoring programs.
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