Evaluation of Sewage Molecular Markers in A Highly Industrialized Zone in Peninsular Malaysia

doi:10.21203/rs.3.rs-2253485/v1

The anthropogenic inputs that have serious effects on riverine and coastline ecosystems could be identified using organic chemical tracers like linear alkylbenzenes (LABs). In order to identify the LABs, sediment samples taken from the Kim Kim river and the Port Dickson coast were collected. The origins of the LABs were discovered and identified with the aid of gas chromatography-mass spectrometry (GC-MS). The Pearson correlation coefficient, Post Hoc Tests, LSD Techniques, and Analysis of Variance (ANOVA) were used to establish the significance of differences in the LABs distribution and concentrations among sampling sites at p <0.05. Internal over exterior (I/E) congeners, homologs of C13 and C12, and long over short chain (L/S) were employed to determine the LABs degradation levels and sewage treatment. According to the statistical analysis of the study findings, LABs concentration in the tested areas ranged between 88.3; 112.0 and 119.0; 256.0 ng∙g−1 dw, respectively. With a high number of C13-LAB homologs in the majority of sampling locations, LAB homologs were substantially different at p < 0.05. The fact that the computed LAB ratios (I/E) fell between 1.7; 2.0 and 2.0; 4.1, showed that the investigated locations received effluents from sources related to primary and secondary treatment. In the locations that were investigated, LAB degradation ranged between 34, 38%, 40, and 64%. In conclusion, the need for ongoing wastewater treatment system upgrades and the strong markers of LABs in tracking sewage pollution.

Sediment

I/E

Sewage pollution

Distribution

Molecular marker

High loads of anthropogenic pollution regularly enter riverine and coastal ecosystems close to metropolitan areas (Halpern et al., 2008). Moreover, the dumping of enormous quantities of industrial as well as domestic wastewater into these places changes the biodiversity in the riverine and coastal habitats. (Zhang et al., 2012). In order to detect the presence of potentially dangerous contaminants and related detrimental effects on the aquatic ecosystem, it is crucial to monitor these ecosystems. This will provide the data needed for effective environmental protection and management (Chaler et al., 2004).

Organic chemical molecules like linear alkylbenzenes (LABs) can be used to measure industrial and domestic pollution. The primary ingredients in detergents (linear alkylbenzene sulfonates; LASs) are LAB with C₁₀-C₁₄ alkyl chains (Ricking et al., 2003).

Due to their great biodegradability and cost-effectiveness, branched alkylbenzene components were completely transformed to LABs for detergent production in the 1960s.

LABs are widespread and plentiful discharge from less and untreated domestic waste and industrialised effluents into the aquatic ecosystem as a result of their inadequate sulfonation (Oller et al., 2011; Alkhadher et al., 2020a). Due to LABs' persistent and great attraction to industrial effluents, they have been employed as anthropogenic markers of pollution of industrial and residential sources in the aquatic ecosystem (Cabral et al., 2020; Alkhadher et al., 2020b).

Due to the nature of the exterior and internal isomers and the varied phenyl substitutions to the straight alkyl chain, LABs are utilised to identify the extent of degradation in sediment and suspended particulate matter (Alkhadher et al., 2015). Additionally, LAB isomers are used to determine the residence time and the types of industrial and domestic wastewater released into marine ecosystem, for example primary and secondary treatments (Takada et al. 1990; Tsutsumi et al., 2002).

The coastal areas in Malaysia have shown high levels of industrial and domestic wastewater contamination due to high population there and there is a positive relation between sewage contamination and illnessesi n Asian countries (Isobe et al., 2002; Sakai et al., 2017). Thus, the marine sediments and their fate impacts should be investigated for possible pollution of industrial and domestic wastewater to enhance water quality and lower illness risk (Wang et al., 2010).

Water contamination Simpang Renggam in Sungai Benut, which mostly affects 75,000 residents in Johor state, was recently published in three local media (Kasimon, 2019). Thus, West and South Malaysia's sources of industrial and home wastewater contamination should be frequently monitored for environmental and human health protection. However, the Kim Kim river and Port Dickson coast are the first places where the recent anthropogenic impacts are investigated. The current research intends to employ LABs as chemical tracers to evaluate impacts of industrial and domestic wastewater in the chosen areas. In the investigated areas, measurements of LAB concentration, distribution, and degradation level were made. Furthermore, the distribution of LAB isomers carried undertaken to improve the performance of current sewage treatment plants (STPs).

2.1Sampling

The investigated study areas are situated in Johor and Sembilan states, alongside of west south of Malaysia (Fig. 1). Table1 provides a description of the research sampling locations with an illustration. In order to assess LABs inputs in these locations, the surface sediments were sampled. Briefly, Each sediment sample's top 4 cm from the Kim Kim river and the coast of Port Dickson were taken with the aid of an Ekman dredge sampler, indicating the current imports of anthropogenic contamination. The samples were then placed inside the stainless steel containers that had been pre-solvent rinsed before being placed within a cooling box, brought to the lab, then kept at 20 °C in a freezer. Prior further analytical procedure, a freeze-dryer was used to remove moisture from the sediments.

2.2 Chemical analysis

In order to analyse the LABs, the sediment extractions carried out using two columns. According to descriptions by researchers in the literature, the first column was used for purification to get rid of the polar compounds, and the second column was used for fractionation to produce the desired organic extracts (Zakaria et al. 2002).

In details, to extract the dried sediments, 250 mL of dichloromethane (DCM) was added to a cellulose thimble with a 10 g of dried sediments inside for 8–10 hours (Vaezzadeh et al. 2014; Masood et al. 2015). Each sample had a predetermined volume of "1-Cn" LABs (50 μL) added before extraction as LABs' surrogate standards (SS) for target LABs' recovery correction. To remove the sulphur that was linked to the sediment samples and prevent it from interfering with the final GC-chromatograms, a half spoon of activated copper was added to each extract. A rotary evaporator was used to reduce an extracted volume before it was placed on top of a chromatography column that included 5% H2O-deactivated silica gel (60-200 mesh size, Sigma Chemical, USA) (0.9 cm i.d, 9 cm height). The extracted hydrocarbons were first eluted with 20 mL of a pure hexane/DCM mixture (3:1, v/v), and then reduced to 1-2 mL. In order to collect the LAB fractions, a fully activated silica gel column (0.47 cm i.d., 18 cm height) was utilised in the second stage with 4 mL of hexane. The reduced LAB fractions were transferred to a 2-mL amber vial, where they underwent further reduction with a moderate stream of nitrogen until dry. Prior to gas chromatography-mass spectrometry (GC-MS) analysis, an internal standard (IS = biphenyl-d10, m/z = 164) was injected into the LAB fraction.

A gas chromatograph from Agilent Technologies in the 7890A series was connected to a C5975 MSD split/splitless injector to detect the LAB compounds. In short, a 30-m fused silica capillary column with a 0.25-mm (i.d.) was utilised, as was a DB-5MS capillary column with a 0.25-m film thickness.

The carrier gas was helium, which had a constant pressure of 60 kg cm−2. A selective ion monitoring (SIM) mode was used to collect the mass spectrum data, and the LAB chromatographs were observed at m/z = 91, 92, and 105. For the ionisation process, which uses an external source (200 °C) as well as the working parameters of the GC-MS were set at 70 eV with an electron multiplier voltage of ~1250 eV. Splitless mode was used to inject the sample, and an injection port was kept at 300 °C at that time. After that, there was a 1-min purge. The column's temperature was held constant at 70 °C for two minutes, 30 °C min-1 to 150 °C for ten minutes, and then 4 °C min-1 to 310 °C for fifteen minutes.

2.3 Quality control and assurance

Surrogate standards (1-Cn LABs) recovered within the permitted range (between 60 and 120%). at a respectable efficiency percentage, display only a minimal of target chemical loss throughout LABs' analytical processes. For all sediment in this analysis, 87-98% was the recovery range of LAB surrogates. Additionally, in order to guard against any potential cross-contamination from different sources during the analytical procedure, each batch (four samples) was accompanied by a blank sample that had all the substances and the standards existing in the typical samples. Freshly made SS, IS, and native standards were spiked into sediment samples at predetermined amounts on a daily basis. The target LAB congeners were located using a GC-MS in the selected ion monitoring mode at m/z = 91, 92, and 105. Using the five concentrations of a LAB standard mixture between 0.25 and 5.0 ppm spiked with the target samples, a 5-point calibration curve was used to quantify the LAB chemicals. The limits of quantitation (LOQ) and detection (LOD) were calculated by dividing the lowest concentration levels of each calibration curve by the mean sample weight (Takada and Eganhouse 1998). It was accordingly between 0.02-0.1 ng g-1 and 0.1-2 ng g-1.

2.4 Statistical analysis

IBM®-SPSS 25 software applied to analyse the statistical process. To show the importance of the fluctuations in LAB concentration and distribution between sample sites at p < 0.05, analysis of variance and Post Hoc Tests, LSD techniques (ANOVA) were utilised.. For sampling stations, a correlation analysis using the Pearson correlation coefficient was utilised. To reduce the number of original variables, the concentration of LAB, as well as to identify a few latent factors, principal component analysis (PCA) was applied. All elements with eigenvalues greater than one were extracted and rotated using the varimax rotation method. Standardized regression coefficients are used to show the probable involvement of numerous LAB sources.

2.5 Total organic carbon analysis (TOC)

Using an LECO CR-412 analyzer, TOC was found in the samples. The sediment samples were dried in an oven for the entire night, set at 60 °C before being pulverised in a mortar and pestle. To eliminate inorganic carbon, 1 M HCl (2 mL) were applied to each weighted dried sediment (1-2 g), until it was totally moist (carbonates). The HCl was subsequently removed by drying the sediment sample at 100 °C for 10 hours. A LECO CR-412 Carbon Analyzer (with a furnace temperature of 1350 °C as well as an O2 boost period of 1 min) was used to calculate TOC%. (Nelson and Sommers 1996; Table 2).

3.1 Distribution and concentration LABs

Congeners structure of the LABs is shown in Fig. 2 as the "n-Cm" LAB, where "n" indicates the benzene ring position on the straight alkyl chain and "m" indicates how many alkyl carbons there are. LAB C₁₀–₁₄ found in all sediment samples under analysis. In the Kim Kim river and Port Dickson Coast, LABs C₁₀–C₁₄ ranged from 88.3–112.0 and 119.0–256.0 ng g-1dw, respectively.

The results of this investigation demonstrate that there was a highly significant Pearson correlation between LABs concentration of the investigated locations (p < 0.05, r = 0.88)), suggesting that LABs may be used as a marker for tracing domestic and industrial contamination (Table 3). Between 88.3 and 256ng∙g−1dw of LAB were present, with a significant concentration at the Port Dickson coast (Fig. 3). This is in line with a significant variation in LAB concentration between study locations at p < 0.05 (Table 4a, b).

Based on the LAB content, the treated effluent runoff and their dilution with organic matter are the most likely causes of LABs' spatial distribution in the riverine environment, which may have an impact on the distribution of LABs on riverine ecosystems (Zeng et al., 1997; Cabral and Martins, 2018a). On the other hand, the spatial distribution of LABs in coastal areas may be explained by high levels of industrialisation, urbanisation, and a lack of piped sewers (Shahbazi et al., 2010; Sakai et al., 2016).

The investigational sites have lower LAB concentrations than Port Klang, the Penang Estuary, Malacca, and the Anzali Wetland (Isobe et al., 2004; Bakhtiari et al., 2018). Additionally, in contrast to those found in Southern Brazil and the Pearl River, the LABs found in this study had high and comparable quantities (Luo et al., 2008; Zacchi et al., 2018).

It was discovered that LAB isomers and homologs were regularly distributed along this trend: the the Kim Kim river < Port Dickson. Thus, it demonstrates that the LAB distribution may be influenced by the geographic placement of sampling locations. Additionally, it showed that in comparison to Malaysia and the rest of the world, the LABs in this research were low to moderate as an indicator of industrial and domestic pollution.

The significant difference between LAB homologs in sample locations is seen in Table 4a, b at p < 0.05. The proportion of homologs and isomers containing, C₁₃ was discovered to be highly significant in the Kim Kim River's LABs, followed by C₁₂ and C₁₄ (Fig.4; Table 4a, b). Contrarily, C₁₀ and C₁₁ homologs showed low quantities, showing that LABs containing C₁₀ and C₁₁ compounds were selectively degraded during the discharge of industrial effluents. It is demonstrated that long-chain LABs made up the majority of the LAB concentration in the River (LC-LABs). According to the chemical compound test, the concentrations of the 6, 5-C₁₃, and 6-C₁₂ isomers are higher than those of others, demonstrating that LABs have long lateral transport. It was discovered that the concentration of C₁₃ homologs in the SKK1 station is high, indicating that these compounds are degrading anaerobically (Dauner et al., 2015; Alkhadher et al., 2020b). Surprisingly, LABs were found to be on the rise in this study compared to what Isobe et al. had noted (2004). Additionally, compared to those with the previous studies, there are many of LABs in the mixture of isomeric and homologous molecules, especially LC-LABs like C₁₃-LAB (Isobe et al., 2004).

The sediments around the Port Dickson coast contain the largest concentration of C₁₃-LAB homologs, whereas C₁₀ homologs have the lowest quantities across all sites in this region. Contrarily, LC-LABs, such as C₁₃ and C₁₄, had higher concentrations than SC-LABs, which included homologs of C₁₀ and C₁₁. Therefore, C₁₃ homologs were the most prevalent and dominant chain length of the LAB distribution, followed by C₁₂, C₁₄, C₁₀, and C₁₁ homologs. The most probable explanation for this LAB chain length compositional range has to do with the increased hydrophobicity of LABs as chain length increases (Sherblom et al., 1992).

When compared to detergents studied by Luo et al., (2008), the homologs distribution in sediments of this region tends to be lower with C₁₀homologs.

The comparison of LABs' LC and SC in the areas under investigation is shown in Fig. 5. The first and second sites had the highest amounts of LC homologs relative to SC ones. The most likely explanation for this is that the SC-homologs (i.e., C₁₀, C₁₁) showed higher rates of selective deterioration in the sediments than the LC ones (Magam et al., 2015; Alkhadher et al., 2016).

3.2 Efficiency Evaluation of LAB Degradation and Effluent Treatment

Primary treatment effluent was found to be the predominant source of LABs of the Kim Kim river, whereas secondary treatment effluents were the principal source in Port Dickson (Figure 6). This is connected to the high STP levels present in the study regions (Alkhadher et al., 2020a).

I/E ratios in the Kim Kim river varied from 1.7 at SKK 2 to 2.0 at SKK 1, with an average of 1.8, indicating that primary treated effluents were discharged to the river water, which is consistent with research by Gustafson et al (2001). L/S ratio ranged from 2.4 to 2.6 on average, as well as it was greater than 1.8 in detergents (Ni et al., 2008), indicating improved LAB biodegradation. In addition, the C₁₃/C₁₂ ratio was in the range of 5.1 to 5.2, on average 5.1, greater than the 1.7 ratio seen in coastal sediments, ranging from (Liu et al., 2013).

In contrast, the I/E ratio in Port Dickson ranged from 2.0 to 4.1, indicating that effluents from secondary treatment were released here.

The I/E ratios were higher than those found in the river of Selangor (0.2–1.0; Masood et al. 2015), according to the results, substantial discharges of pre-treated effluents is anticipated. The LABs degradation also goes through an aerobic condition.

Further evaluation of LAB biodegradation in the examined locations was done using L/S and C₁₃/C₁₂ ratios. The idea that the Port Dickson received untreated effluents, as previously mentioned, is supported by the partial presence of LAB. More than 80 sewage pipelines were observed to discharge untreated effluents and wastewaters from hotels and housing outlets onto the coastlines, resulting in a lack of quality and protection of the coastlines (Hamzah et al., 2011; Thomes et al., 2019).

However, the rates of LAB degradation ranged from low in the Kim Kim river (SKK3; 34%) to high in the Port Dickson (SPD1; 64%), indicating that LABs degraded more quickly along the Port Dickson shore than in the Kim Kim. Because residents use it for recreational and fishing purposes, there has been a high direct discharge from ferries and boats into the river in recent years (Abu Samah et al., 2011). Detergent waste discharge and washing from boats result in an increase in LABs in the river sediments, which to some extent significantly alters molecular indices.

It is clear that raw, primary, and additional secondary treated effluents released into the ecosystem constitute LAB inputs in developing nations like Malaysia.

The study's findings revealed that the Kim Kim river contains more primary industrial effluents and that LABs are discharged from a range of sources, such as municipal or industrial wastewaters. These findings suggest a potential improvement in the movement of LASs from their primary sources through their later transport (Zhang et al., 2012; Harwood, 2014; Cabral et al., 2018b).

3.3 TOC Evaluation

Owing to high hydrophobicity nature, LABs are likely to have a high attach to the organic materials when they enter the aquatic environment. Hence, TOC in the sediments correlated to the LAB concentration (Wang et al., 2001).

A minor correlation between LABs and TOC (R² = 0.42) was found in the Kim Kim when TOC was observed in the sediment samples, in addition, the linear regression was used to evaluate its relationship with LABs. Therefore, TOC did not take into account a determining element in LABs distribution. The LABs input intensity, which are released from many residential and industrial sources, may therefore be the important component. Previous research found that Peninsular Malaysia's Selangor and Perak Rivers have modest LABs-TOC correlations, with R2 values of 0.008 and 0.17. (Masood et al., 2015; Magam et al., 2015).

However, there was a relationship between TOC and LAB concentration in the Port Dickson coast (R² = 0.64), suggesting that TOC may be a determining factor in the LABs' geographical distribution from city and industrial zone. This outcome agreed with that of the Dongjiang River (R² = 0.82), indicating that wastewater treatment plants and industrial waste in the research area are the primary sources of organic matter.

3.4 Source Apportionment

In this investigation, there was a lot of I/E ratio fluctuation. In many sites, readings below 1 indicate that untreated wastewater has been dumped into the aquatic environment. A PCA in Kim Kim river and the Port Dickson shoreline led to the extraction of the two components that together accounted for 89.2% of the variance in the composition of the LABs.

According to it, the first factor—with the exception of 2-Cn and 4-C₁₃—is responsible for 55.4% of all variations (Table 5).

PCA was used to determine the most likely LABs sources in the Kim Kim river and the Port Dickson seashore. By removing the less significant factors with the least amount of data loss, this approach identifies the details of the variables that explain the variation in the data. The two PCs with multiple readings were obtained. PCs through eigenvalue more than one is measured based on the eigenvalues.

The two PCs reveal 78.28% in the sediments' LABs structure. Furthermore, it shown that PCA on the compositions of LABs offered superb correlations between the sampling sites. The importance of diffuse LAB sources in sedimentary LAB loading is demonstrated by strong spatial correlation of LAB compositions across different locales.

The results of PCA demonstrate that the first PCs, which account for 45.8% of the total variance, identify the main contamination sources in Kim Kim and Port Dickson coast. Except for 2-Cn and 4-C₁₃, that laden in second factor which clarifies 38.39% of overall variation, this factor has loading of entire LABs (Table 5). While 2-Cn LABs were less common in wastewaters than other isomers, they were quicker to biodegrade than other isomers. This means that the 2-Cn loadings on second PC could be a sign for LAB coming from raw industrial effluent. While the first PC can indicate the release of LABs from industrial wastewaters that have been treated. Therefore, it is likely that the LABs sources are comparable across entirely locations. Moreover, some areas have an increase in LAB concentration as well as a decline I/E ratio may be caused by those areas' closeness to areas with dense populations, significant industrial activity, and tourism-related activities. Instead, the Kim Kim river has a lower concentration of LABs than the Port Dickson Coast due to areas that are less industrialised, urbanised, and populated close to the river.

LABs concentrations were significantly varied in the sediments of the investigated locations with 88.2 to 255.8ng∙g−1 dw. The Kim Kim river has less LABs than Port Dickson Coast. The SC- homologs have less abundant than LC- ones where less records found in the Kim Kim river. I/E ratios showed that primary treatment effluents are the primary source in Kim Kim river, while the secondary treatment effluents were the main source at the Port Dickson coast. A high concentration of LABs may be caused by inadequate STPs that possibly incapable to handle a large metropolis in the area under investigation. The findings of this study therefore revealed that untreated industrial and municipal wastewater issues might persist for a long period as long as the population grew. In the near future, it is projected that the amount of industrial and urban effluent being discharged into Peninsular Malaysia's rivers and beaches would increase. Therefore, it is important to take the necessary steps to improve the wastewater treatment system. In view of that, the continuous evaluation of the current industrial and municipal contamination should be routinely perform beside improve the present wastewater system to reduce the environmental risks and the people health in the riverine and coastline zones.

Acknowledgments

The postdoctoral scheme grant from University Malaysia Terengganu (UMT) and the Inisiatif Putra Berkumpulan Grant, (UPM) are funding this study (9412401)

Data Availability

The Metadata used to support the findings of this study have been deposited in the University 301 Putra Malaysia repository at http://upm.edu.my/

Abu Samah, B., Yassin, S.M., Shaffril, H.A.M., Hassan, M.S., Othman, M.S, Abu Samah, A., Ramli, S.A., 2011. Relationship to the river: The case of the Muar River community. Am J Environ. Sci. 7: 362-369
Alkhadher, S.A.A., Zakaria, M.P., Yusoff, F.M., Kannan, N., Suratman, S., Keshavarzifard, M., Magam, S.M., Masood, N., Vaezzadeh, V., Sani, M.S.A., 2015. Baseline distribution and sources of linear alkylbenzenes (LABs) in surface sediments from Brunei Bay, Brunei. Mar Pollut Bull. 101(1): 397–403
Alkhadher, S.A.A., Zakaria, M.P., Yusoff, F.M., Kannan, N., Suratman, S., Magam, S.M., Masood, N., Keshavarzifard, M., Vaezzadeh, V., Sani, M. S.A., 2016. Distribution and sources of linear alkylbenzenes (LABs) in surface sediments from Johor Bahru Coast and the Kim Kim River, Malaysia. Environ Forensics. 17 (1): 36-47
Alkhadher, S.A.A., Kadir, A.A., Zakaria, M.P., Al-Gheethi, A., Magam, S.M., Masood, N., 2020a. Monitoring of Sewage Pollution in the Surface Sediments of the Coastal Ecosystems using linear alkylbenzenes (LABs) as Molecular Markers. Journal of Soils and Sediments. https://doi.org/10.1007/s11368-020-02671-7
Alkhadher, S.A.A., Kadir, A.A., Zakaria, M.P., Al-Gheethi, A., Keshavarzifard, M., Masood, N., Alenezi, K.M, Magam, S.M., 2020b. Linear alkylbenzenes in surface sediments of an estuarine and marine environment in peninsular Malaysia. Mar Pollut Bull. 153
Bakhtiari, A.R., Javedankherad, I., Mohammadi, J., Taghizadeh, R., 2018. Distribution of linear alkylbenzenes as a domestic sewage molecular marker in surface sediments of International Anzali Wetland in the southwest of the Caspian Sea, Iran. Environ Sci Pollut Res. https://doi.org/10.1007/s11356-018-1942-1
Cabral, A.C., Martins, C.C., 2018a. Insights about sources, distribution, and degradation of sewage and biogenic molecular markers in surficial sediments and suspended particulate matter from a human-impacted subtropical estuary. Environ Pollut. 241 (2018): 1071-1081
Cabral, A.C., Stark, J.S., Kolm, H.E., Martins, C.C., 2018b. An integrated evaluation of some faecal indicator bacteria (FIB) and chemical markers as potential tools for monitoring sewage contamination in subtropical estuaries. Environ Pollut. 235, 739–749
Cabral, A.C., Dauner, A.L.L., Xavier, F.C.B., Garcia, M.R.D., Wilhelm, M.M., Santos, V.C.G., Netto, S.A., Martins, C.C., 2020c. Tracking the sources of allochthonous organic matter along a subtropical fluvial-estuarine gradient using molecular proxies in view of land uses. Chemosphere 251 (2020): 126435
Chaler, R., Cantón, L., Vaquero, M., Grimalt, J.O., 2004. Identification and quantification
of n-octyl esters of alkanoic and hexanedioic acids and phthalates as urban wastewater markers in biota and sediments from estuarine areas. J. Chromatogr. A 1046,
203–210
Crossland, C.J., Kremer, H.H., Lindeboom, H.J., Marshall., Crossland, J.I.L.e., Tissier, M.D.A., 2005. Coastal Fluxes in the Antropocene: The Land–Ocean Interactions in the Coastal Zone, Project on the International Geosphere-Biosphere Programme Series. Global Change— The IGBP Series. Springer-Verlag, Berlin, 232 pp
Dauner, A.L.I., Hernández, E.A., MacCormack, W.P., Martins, C.C., 2015. Molecular characterisation of anthropogenic sources of sedimentary organic matter from Potter Cove, King George Island, Antarctica. Sci Total Environ 502: 408–416
Gustafsson, Ö., Long, C.M., MacFarlane, J., Gschwend, P.M., 2001. Fate of linear alkylbenzenes released to the coastal environment near Boston Harbor. Environ Sci Technol 35(10): 2040–2048
Halpern, B.S., et al., 2008. A global map of human impact on marine ecosystems. Science
319, 948–952.
Hamzah, A., Kipli, S., Ismail, S.R., Una, R., Sarmani, S., 2011. Microbiological study in coastal water of Port Dickson, Malaysia. Sains Malays. 40: 93–99
Harwood, J.J., 2014. Molecular markers for identifying municipal, domestic and agricultural sources of organic matter in natural waters. Chemosphere. 95 (2014): 3-8
Isobe, K.O., Tarao, M., Zakaria, M.P., Chiem, N.H., Minh, L.Y., Takada, H., 2002. Quantitative application of fecal sterols using gas chromatography-mass spectrometry to investigate fecal pollution in tropical waters: Western Malaysia and Mekong Delta, Vietnam. Environ Sci Technol 36 (21): 4497–4507
Isobe, K.O., Zakaria, M.P., Chiem, N.H., Minh, L.Y., Prudente, M., Boonyatumanond, R., Saha, M., Sarkar, M., Takada, H., 2004. Distribution of linear alkylbenzenes (LABs) in riverine and coastal environments in South and Southeast Asia. Water Res 38(9): 2449–2459
Kasimon, M.N., 2019. Pencemaran Ammonia: 75000 Penduduk Terjejas Bekalan Air. Sinar Harian.
Luo, X.J., Chen, S.J., Ni, H.G., Yu, M., Mai, B.X., 2008. tracing sewage pollution in the Pearl River Delta and its adjacent coastal area of South China Sea using linear alkylbenzenes (LABs). Mar Pollut Bull 56(1): 158–162
Magam, S.M., Zakaria, M.P., Halimoon, N., Masood, N., Alsalahi, M.A., 2012. Aliphatic Distribution of Linear Alkylbenzenes (LABs) in Sediments of Sarawak and Sembulan Rivers, Malaysia. Environ Asia 5 (1): 48-55
Magam, S.M., Halimoon, N., Zakaria, M.P., Aris, A.Z., Kannan, N., Masood, N., 2015. Evaluation of distribution and sources of sewage molecular marker (LABs) in selected rivers and estuaries of Peninsular Malaysia. Environ Sci Pollut Res Int 23(6): 5693–5704
Martins, C.C., Ferreira, J.A., Taniguchi, S., Mahiques, M.M., Bícego, M.C., Montone, R.C., 2008. Spatial distribution of sedimentary linear alkylbenzenes and faecal steroids of Santos Bay and adjoining continental shelf, SW Atlantic, Brazil: origin and fate of sewage contamination in the shallow coastal environment. Mar Pollut Bull 56(7): 1359–1363
Masood, N., Zakaria, M.P., Halimoon, N., Aris, A.Z., Magam, S.M., Kannan, N., Mustafa, S., Ali, M.M., Keshavarzifard, M., Vaezzadeh, V., Alkhadher, S.A.A., Al-Odaini, N.A., 2015. Anthropogenic waste indicators (AWI) particularly PAHs and LABs in Malaysian sediments: application of aquatic environment for identifying anthropogenic pollution. Mar Pollut Bull 102 (1): 160-175
Nelson, D., Sommers, L., 1996. Total carbon, organic carbon and organic matter. In ‘Methods of soil analysis. Part 3: Chemical methods’. (Ed. DL Sparks) Soil Science Society of America: Madison, WI. pp: 961–1010
Ni, H.G., Lu, F.H., Wang, J.Z., Guan, Y.F., Luo, X.L., Zeng, E.Y., 2008. Linear alkylbenzenes in riverine runoff of the Pearl River Delta (China) and their application as anthropogenic molecular markers in coastal environments. Environ Pollut. 154 (2): 348-355
Oller, I., Malato, S., Sánchez-Pérez, J.A., 2011. Combination of advanced oxidation processes and biological treatments for wastewater decontamination, A review. Sci Total Environ. 409:4141–4166
Raymundo, C., Preston, M., 1992. The distribution of linear alkylbenzenes in coastal and estuarine sediments of the Western North Sea. Mar Pollut Bull. 24(3): 138–146
Ricking, M., Schwarzbauer, J., Franke, S., 2003. Molecular markers of anthropogenic activity in sediments of the Havel and Spree Rivers (Germany). Water Res. 37(2003):2607
Rinawati., Koike, T., Koike, H., Kurumisawa, R., Ito, M., Sakurai, S., Togo, A., Saha, M., Arifinc, Z., Takada, H., 2012. Distribution, source identification, and historical trends of organic micro pollutants in coastal sediment in Jakarta Bay, Indonesia. J Hazard Mater. (217–218): 208–216
Sakai, N., Yusof, R., Sapar, M., Yoneda, M., Ali, M.M., 2016. Spatial analysis and source profiling of beta-agonists and sulfonamides in Langat River basin, Malaysia. Sci. Total Environ. 548-549, 43-50
Sakai, N., Shirasaka, J., Matsui, Y., Ramli, M.R., Yoshida, K., Mohd, M.A., Yoneda, M., 2017. Occurrence, fate and environmental risk of linear alkylbenzene sulfonate in the Langat and Selangor River basins, Malaysia. Chemosphere. 172 (2017) 234-241
Shahbazi, A., Zakaria, M.P., Yap, C.K., Tan, S.G., Surif, S., 2010. Use of different tissues of Perna viridis as biomonitors of polycyclic aromatic hydrocarbons (PAHs) in the coastal waters of Peninsular Malaysia. Environ Forensics 11(3): 248–263
Sherblom, P.M., Gschwend, P.M., Eganhouse, R.P., 1992. Aqueous solubilities, vapor pressures, and 1-octanol-water partition coefficients for C9-C14 linear alkylbenzenes. J Chem Eng Data 37(4): 394–399
Takada, H., Ishiwatari, R., 1990. Biodegradation experiments of linear alkylbenzenes (LABs): isomeric composition of C12 LABs as an indicator of the degree of LAB degradation in the aquatic environment. Environ Sci Technol 24(1): 86–91
Takada, H., Eganhouse, R.P., 1998. Molecular markers of anthropogenic waste: Their use in determining sources, transport pathways and fate of wastes in the environment, In. Encyclopedia of Environmental Analysis and Remediation. 5:2883–2940
Thomes, M.T., Vaezzadeh, V., Zakaria, M.P., Bong, C.W., 2019. Use of sterols and linear alkylbenzenes as molecular markers of sewage pollution in Southeast Asia. Environ Sci Pollut Res. https://doi.org/10.1007/s11356-019-05936-y
Tsutsumi, S., Yamaguchi, Y., Nishida, I., Akiyama, K., Zakaria, M.P., Takada, H., 2002. Alkylbenzenes in mussels from South and South East Asian coasts as a molecular tool to assess sewage impact. Mar Pollut Bull 45(1): 325–331
Wang, X.C., Zhang, Y.X., Chen, R.F., 2001. Distribution and partitioning of polycyclic aromatic hydrocarbons (PAHs) in different size fractions in sediments from Boston Harbor, United States. Mar Pollut Bull 42 (11): 1139-1149
Zacchi, F.L., Flores-Nunes, F., Mattos, J.J., Lima, D., Lüchmann, K.H., Sasaki, S.T., Bícego, M.C., Satie Taniguchi, S., Montone, R.C., Almeida, E.A., Bainy, A.C.D., 2018. Biochemical and molecular responses in oysters Crassostrea brasiliana collected from estuarine aquaculture areas in Southern Brazil. Mar. Pollut. Bull 135: 110–118
Zakaria, M.P., Takada, H., Tsutsumi, S., Ohno, K., Yamada, J., Kouno, E., Kumata, H., 2002. Distribution of polycyclic aromatic hydrocarbons (PAHs) in rivers and estuaries in Malaysia: a widespread input of petrogenic PAHs. Environ Sci Technol 36 (9): 1907–1918
Zeng, E.Y., Khan, A.R., Tran, K., 1997. Organic pollutants in the coastal environment Off San Diego, California. 3. Using linear alkylbenzenes to trace sewage-derived organic materials. Environ Toxicol Chem 16(2): 196–201
Zhang, K., Wang, J.Z., Liang, B., Shen, R.L., Zeng, E.Y., 2012. Assessment of aquatic wastewater pollution in a highly industrialized zone with sediment linear alkylbenzenes. Environ Toxicol Chem 31(4): 724–730

Table 1. Sampling sites along the selected areas in Malaysia

Sample Name	Geographical Coordination	Location Type	Weather Condition	Site Description
SKK1	N 01°27'28.2'' E103°57'76.2''	River	Rainy	Urban and industry area
SKK2	N 01°26'77.6'' E103°58'04.6''	River	Rainy	Urban and industry area
SKK3	N 01°25'09.6'' E103°58'3.31''	River	Rainy	Urban and industry area
SPD1	N 02°27'882'' E101°50.687''	Coast	Cloudy	Urban and tourism area
SPD2	N 02° 27'638'' E101°50.7'35''	Coast	Cloudy	Urban and tourism area
SPD3	N 02° 28'047'' E101°50.7'96''	Coast	Cloudy	Urban and tourism area
SPD4	N02°27'943'' E101°50.8'10''	Coast	Cloudy	Urban and tourism area

Table 2. LABs concentration (ng.g^-1.dw) and relative compound ratios in the Kim Kim river and the Port Dickson coast

Compound	^bSKK1	SKK2	SKK3	SPD1	SPD2	SPD3	SPD4
^a C₁₀-LABs(ng.g^{- 1}dw)	9.4	7.7	6.8	17.2	15.0	4.2	6.2
C₁₁-LABs(ng.g^-1dw)	16.3	13.0	12.0	66.7	57.0	19.0	30.7
C₁₂-LABs(ng.g^-1dw)	25.6	20.7	18.6	68.6	61.0	24.0	38.0
C₁₃-LABs(ng.g^-1dw)	41.0	32.6	29.5	86.0	78.4	51.2	63.6
C₁₄-LABs(ng.g^-1dw)	26.8	21.1	21.3	17.4	15.6	13.2	14.8
LABs (ng.g^-1dw)	119	95.2	88.3	256	227	112	153
^cLC-LABs (ng.g^-1dw)	67.8	53.8	51.0	103	94.0	64.5	78.4
^dSC-LABs (ng.g^-1dw)	25.8	20.7	19.0	84.0	71.8	23.2	37.0
^eI/E	2.0	1.8	1.7	4.1	3.7	2.0	2.6
^gL/S	2.4	2.5	2.6	1.6	1.6	1.7	1.6
hC13/C12	5.1	5.2	5.2	9.7	9.5	13.7	14.3
^fLABDegradationa(%)	38	35	34	64	61	40	48
ⁱTOC(%)	1.2	1.0	8.1	2.0	2.3	1.5	1.9
TOC(mg/g)	11.5	9.0	81	19.7	22.7	14.7	18.9

^aC₁₀- LAB. Sum of the 26LAB congeners ranging from 5-C₁₀ to 2- C₁₀

^bSPD1, the first letter indicate the station, the second and third letters represent the first letters of location name, the numbers 1,2,3,4, indicate the first, second, third and fourth station for each location respectively.

^cLC-LABs, sum of LABs ranging from 6-C₁₃ to 2-C₁₄^dSC-LABs, sum of LABs ranging from 5-C₁₀ to 2-C₁₁ ^eI/E (C₁₂-LABs), ratio of (6-C₁₂LAB+5-C₁₂LAB) relative to (4-C₁₂LAB+3-C₁₂LAB+2-C₁₂LAB). ^gL/S,ratio of (5-C₁₃LAB+5-C₁₂LAB) relative to (5-C₁₁LAB+5-C₁₀LAB). ^hC₁₃/C_12, ratio of (6-,5-,4-,3- and 2-C₁₃/(6-, 5-, 4-, 3-, and 2-C₁₂LAB) ^fLAB Degradation (%), LAB deg =81×log (I/E ratio) +15 (r² = 0.96).

ⁱTOC (%), Total organic carbon

Table 3. Pearson correlation coefficients between total LABs concentration in the Kim Kim river and the Port Dickson and, Southern Malaysia. Significant Pearson correlation (p < 0.01)

**.Correlation is significant at the 0.01 level (2-tailed).

Table 4 a, b. Total LABs Concentration in sediments among different locations of the Kim Kim river and the Port Dickson, Southern Malaysia. a) ANOVA, b) Post Hoc Tests

a) ANOVA

	Sum of Squares	df	Mean Square	F	*Sig.**
Between Groups	8410	4	210	7.3	.000
Within Groups	8680	30	289
Total	17100	34

b) Post Hoc Tests

Dependent Variable: LABs Concentration
LSD
(I) Location	(J) Location	Mean Difference (I-J)	Std. Error	Sig.	95% Confidence Interval
(I) Location	(J) Location	Mean Difference (I-J)	Std. Error	Sig.	Lower Bound	Upper Bound
1.00	2.00	-21.2^*	9.1	.03	-39.7	-2.6
	3.00	-27.2^*	9.1	.01	-45.7	-8.6
	4.00	-45.1^*	9.1	.00	-63.7	-26.6
	5.00	-9.1	9.1	.32	-27.7	9.5
2.00	1.00	21.2^*	9.1	.03	2.6	39.7
	3.00	-6.0	9.1	.52	-24.6	12.6
	4.00	-24.0^*	9.1	.01	-42.5	-5.3
	5.00	12.1	9.1	.20	-6.5	30.6
3.00	1.00	27.2^*	9.1	.01	8.6	45.7
	2.00	6.0	9.1	.52	-12.6	24.6
	4.00	-18.0	9.1	.06	-36.5	0.6
	5.00	18.0	9.1	.06	-.53	36.6
4.00	1.00	45.1^*	9.1	.00	26.6	63.7
	2.00	24.0^*	9.1	.01	5.4	42.5
	3.00	18.0	9.1	.06	-.6	36.5
	5.00	36.0^*	9.1	.00	17.4	54.6
5.00	1.00	9.1	9.1	.32	-9.5	27.7
	2.00	-12.1	9.1	.20	-30.6	6.5
	3.00	-18.0	9.1	.06	-36.6	.53
	4.00	-36.0^*	9.1	.00	-54.6	-17.4

* The mean difference is significant at the 0.05 level.

Table 5. Loadings of LABs in the sediment samples of the Kim Kim river and the Port Dickson coast after varimax rotation

** Significant at the 0.01 level (2-tailed).

Evaluation of Sewage Molecular Markers in A Highly Industrialized Zone in Peninsular Malaysia

Status:

Version 1

Abstract

Figures

1 Introduction

2 Methods And Materials

3 Result And Discussion

4 Conclusion

Declarations

References

Tables

Status:

Version 1