Near Real Time Biomonitoring of Mercury Copper and Silver in Port Dickson Coastal Water Using a Ficin Inhibition Assay
DOI:
https://doi.org/10.54987/jebat.v8i2.1186Keywords:
Heavy metals, Biomonitoring, Enzyme inhibition, Ficin, Port DicksonAbstract
Frequent coastal-water surveillance is often constrained by the cost, transport requirements, and turnaround time of element-specific laboratory analysis. This study evaluated a portable ficin-inhibition assay as an effect-based screening tool for near-real-time monitoring of coastal water at Port Dickson, Negeri Sembilan, Malaysia. Ficin-catalysed casein hydrolysis was quantified indirectly using the Bradford dye-binding response. Surface water was collected hourly from 10:00 to 16:00 during a single field campaign and analysed at 30 ± 1 °C with a portable spectrophotometer. Mercury (Hg) was subsequently determined by flow-injection mercury analysis, while copper (Cu) and silver (Ag) were measured by inductively coupled plasma optical emission spectrometry. Mean ficin inhibition remained below 10% at every sampling time and did not reach the prespecified 20% positive-screening threshold. Instrumental metal concentrations varied during the six-hour period, but these changes were not proportional to the enzyme response, consistent with differences between total measured concentrations and the bioavailable fraction interacting with ficin. The assay produced results within approximately 1 h using small sample volumes and simple field equipment. It should, however, be used as a first-tier screening method rather than a substitute for element-specific analysis because salinity, pH, colour, turbidity, metal speciation, and mixture effects may alter enzyme activity. Broader validation across seasons, tidal conditions, sites, and matrix-matched fortified samples is required before routine or regulatory application.
References
1 Briffa J, Sinagra E, Blundell R. Heavy metal pollution in the environment and their toxicological effects on humans. Heliyon. 2020;6:e04691. https://doi.org/10.1016/j.heliyon.2020.e04691.
2 Rainbow PS. Trace metal bioaccumulation: models, metabolic availability and toxicity. Environ Int. 2007;33:576–582. https://doi.org/10.1016/j.envint.2006.05.007.
3 Chapman PM. Determining when contamination is pollution—weight of evidence determinations for sediments and effluents. Environ Int. 2007;33:492–501. https://doi.org/10.1016/j.envint.2006.09.001.
4 Peijnenburg WJGM, Jager T. Monitoring approaches to assess bioaccessibility and bioavailability of metals: matrix issues. Ecotoxicol Environ Saf. 2003;56:63–77. https://doi.org/10.1016/S0147-6513(03)00051-4.
5 Paquin PR, Gorsuch JW, Apte S, Batley GE, Bowles KC, Campbell PGC, et al. The biotic ligand model: a historical overview. Comp Biochem Physiol C Toxicol Pharmacol. 2002;133:3–35. https://doi.org/10.1016/S1532-0456(02)00112-6.
6 Birch GF, Taylor SE, Matthai C. Small-scale spatial and temporal variance in the concentration of heavy metals in aquatic sediments: a review and some new concepts. Environ Pollut. 2001;113:357–372. https://doi.org/10.1016/S0269-7491(00)00182-2.
7 Barkay T, Miller SM, Summers AO. Bacterial mercury resistance from atoms to ecosystems. FEMS Microbiol Rev. 2003;27:355–384. https://doi.org/10.1016/S0168-6445(03)00046-9.
8 Tchounwou PB, Ayensu WK, Ninashvili N, Sutton D. Environmental exposure to mercury and its toxicopathologic implications for public health. Environ Toxicol. 2003;18:149–175. https://doi.org/10.1002/tox.10116.
9 Lemire JA, Harrison JJ, Turner RJ. Antimicrobial activity of metals: mechanisms, molecular targets and applications. Nat Rev Microbiol. 2013;11:371–384. https://doi.org/10.1038/nrmicro3028.
10 Silver S. Bacterial resistances to toxic metal ions—a review. Gene. 1996;179:9–19. https://doi.org/10.1016/S0378-1119(96)00323-X.
11 Belkin S. Microbial whole-cell sensing systems of environmental pollutants. Curr Opin Microbiol. 2003;6:206–212. https://doi.org/10.1016/S1369-5274(03)00059-6.
12 van der Meer JR, Belkin S. Where microbiology meets microengineering: design and applications of reporter bacteria. Nat Rev Microbiol. 2010;8:511–522. https://doi.org/10.1038/nrmicro2392.
13 Harms H, Wells MC, van der Meer JR. Whole-cell living biosensors—are they ready for environmental application? Appl Microbiol Biotechnol. 2006;70:273–280. https://doi.org/10.1007/s00253-006-0319-4.
14 Tecon R, van der Meer JR. Bacterial biosensors for measuring availability of environmental pollutants. Sensors. 2008;8:4062–4080. https://doi.org/10.3390/s8074062.
15 D'Souza SF. Microbial biosensors. Biosens Bioelectron. 2001;16:337–353. https://doi.org/10.1016/S0956-5663(01)00125-7.
16 Daunert S, Barrett G, Feliciano JS, Shetty RS, Shrestha S, Smith-Spencer W. Genetically engineered whole-cell sensing systems: coupling biological recognition with reporter genes. Chem Rev. 2000;100:2705–2738. https://doi.org/10.1021/cr990115p.
17 Su L, Jia W, Hou C, Lei Y. Microbial biosensors: a review. Biosens Bioelectron. 2011;26:1788–1799. https://doi.org/10.1016/j.bios.2010.09.005.
18 Ejeian F, Etedali P, Mansouri-Tehrani HA, Soozanipour A, Low ZX, Asadnia M, et al. Biosensors for wastewater monitoring: a review. Biosens Bioelectron. 2018;118:66–79. https://doi.org/10.1016/j.bios.2018.07.019.
19 Kahru A, Dubourguier HC, Blinova I, Ivask A, Kasemets K. Biotests and biosensors for ecotoxicology of metal oxide nanoparticles: a minireview. Sensors. 2008;8:5153–5170. https://doi.org/10.3390/s8085153.
20 Charrier T, Durand MJ, Affi M, Jouanneau S, Gezekel H, Thouand G. Bacterial bioluminescent biosensors: current applications and prospects. Biotechnol Adv. 2011;29:270–278. https://doi.org/10.1016/j.biotechadv.2011.01.002.
21 Woutersen M, Belkin S, Brouwer B, van Wezel AP, Heringa MB. Are luminescent bacteria suitable for online detection and monitoring of toxic compounds in drinking water and its sources? Anal Bioanal Chem. 2011;400:915–929. https://doi.org/10.1007/s00216-010-4372-6.
22 Amine A, Mohammadi H, Bourais I, Palleschi G. Enzyme inhibition-based biosensors for food safety and environmental monitoring. Biosens Bioelectron. 2006;21:1405–1423. https://doi.org/10.1016/j.bios.2005.07.012.
23 Rodriguez-Mozaz S, Marco MP, López de Alda MJ, Barceló D. Biosensors for environmental monitoring: a global perspective. Talanta. 2005;65:291–297. https://doi.org/10.1016/j.talanta.2004.06.031.
24 Farré M, Kantiani L, Pérez S, Barceló D. Sensors and biosensors in support of EU Directives. TrAC Trends Anal Chem. 2009;28:170–185. https://doi.org/10.1016/j.trac.2008.09.018.
25 Viarengo A, Lowe D, Bolognesi C, Fabbri E, Koehler A. The use of biomarkers in biomonitoring: a 2-tier approach assessing the level of pollutant-induced stress syndrome in sentinel organisms. Comp Biochem Physiol C Toxicol Pharmacol. 2007;146:281–300. https://doi.org/10.1016/j.cbpc.2007.04.011.
26 Uba G, Manogaran M, Gunasekaran B, Halmi MIE, Shukor MYA. Improvement of ficin-based inhibitive enzyme assay for toxic metals using response surface methodology and its application for near real-time monitoring of mercury in marine waters. Int J Environ Res Public Health. 2020;17:8585. https://doi.org/10.3390/ijerph17228585.
27 Botsford JL. A simple assay for toxic chemicals using a bacterial indicator. World J Microbiol Biotechnol. 1998;14:369–376. https://doi.org/10.1023/A:1008813211422.
28 Jung K, Bitton G, Koopman B. Assessment of urease inhibition assays for measuring toxicity of environmental samples. Water Res. 1995;29:1929–1933. https://doi.org/10.1016/0043-1354(94)00353-9.
29 Shukor MY, Baharom NA, Rahman FA, Abdullah MPA, Shamaan NA, Syed MA. Development of a heavy metals enzymatic-based assay using papain. Anal Chim Acta. 2006;566:283–289. https://doi.org/10.1016/j.aca.2006.03.001.
30 Shukor MY, Masdor N, Baharom NA, Jamal JA, Abdullah MPA, Shamaan NA, et al. An inhibitive determination method for heavy metals using bromelain, a cysteine protease. Appl Biochem Biotechnol. 2008;144:283–291. https://doi.org/10.1007/s12010-007-8063-5.
31 Currie LA. Limits for qualitative detection and quantitative determination: application to radiochemistry. Anal Chem. 1968;40:586–593. https://doi.org/10.1021/ac60259a007.
32 Uhrovčík J. Strategy for determination of LOD and LOQ values—some basic aspects. Talanta. 2014;119:178–180. https://doi.org/10.1016/j.talanta.2013.10.061.
33 Thompson M, Ellison SLR, Wood R. Harmonized guidelines for single-laboratory validation of methods of analysis. Pure Appl Chem. 2002;74:835–855. https://doi.org/10.1351/pac200274050835.
34 Taverniers I, De Loose M, Van Bockstaele E. Trends in quality in the analytical laboratory. II. Analytical method validation and quality assurance. TrAC Trends Anal Chem. 2004;23:535–552. https://doi.org/10.1016/j.trac.2004.04.001.
35 Lionetto MG, Caricato R, Calisi A, Giordano ME, Schettino T. Acetylcholinesterase as a biomarker in environmental and occupational medicine: new insights and future perspectives. Biomed Res Int. 2013;2013:321213. https://doi.org/10.1155/2013/321213.
36 de Souza PR, de Souza KS, de Assis CRD, de Araújo MC, Silva KCC, da Silva JFX, et al. Acetylcholinesterase of mangrove oyster Crassostrea rhizophorae: a highly thermostable enzyme with promising features for estuarine biomonitoring. Aquat Toxicol. 2018;197:109–121. https://doi.org/10.1016/j.aquatox.2018.02.002.
37 Sabullah MK, Sulaiman MR, Shukor MS, Yusof MT, Johari WLW, Shukor MY, et al. Heavy metals biomonitoring via inhibitive assay of acetylcholinesterase from Periophthalmodon schlosseri. Rend Lincei Sci Fis Nat. 2015;26:151–158. https://doi.org/10.1007/s12210-014-0359-0.
38 Lopez-Roldan R, Kazlauskaite L, Ribo J, Riva MC, González S, Cortina JL. Evaluation of an automated luminescent bacteria assay for in situ aquatic toxicity determination. Sci Total Environ. 2012;440:307–313. https://doi.org/10.1016/j.scitotenv.2012.05.043.
39 Amaro F, Turkewitz AP, Martín-González A, Gutiérrez JC. Whole-cell biosensors for detection of heavy metal ions in environmental samples based on metallothionein promoters from Tetrahymena thermophila. Microb Biotechnol. 2011;4:513–522. https://doi.org/10.1111/j.1751-7915.2011.00252.x.
40 Eom H, Ashun E, Toor UA, Oh SE. A solid-phase direct contact bioassay using sulfur-oxidizing bacteria to evaluate toxicity of soil contaminated with heavy metals. Sens Actuators B Chem. 2020;305:127510. https://doi.org/10.1016/j.snb.2019.127510.
41 Gadd GM. Metals, minerals and microbes: geomicrobiology and bioremediation. Microbiology. 2010;156:609–643. https://doi.org/10.1099/mic.0.037143-0.
42 Nies DH. Microbial heavy-metal resistance. Appl Microbiol Biotechnol. 1999;51:730–750. https://doi.org/10.1007/s002530051457.
43 Hobman JL, Crossman LC. Bacterial antimicrobial metal ion resistance. J Med Microbiol. 2015;64:471–497. https://doi.org/10.1099/jmm.0.023036-0.
44 Ahmad NI, Noh MFM, Mahiyuddin WRW, Jaafar H, Ishak I, Azmi WNFW, et al. Mercury levels in crustaceans and cephalopods from Peninsular Malaysia. Environ Sci Pollut Res Int. 2015;22:12960–12974. https://doi.org/10.1007/s11356-015-4415-9.
45 Raissy M, Ansari M. Health risk assessment of mercury and arsenic associated with consumption of fish from the Persian Gulf. Environ Monit Assess. 2014;186:1235–1240. https://doi.org/10.1007/s10661-013-3452-4.
46 Tajam J, Kamal ML. Marine environmental risk assessment of Sungai Kilim, Langkawi, Malaysia: heavy metal enrichment factors in sediments as assessment indexes. Int J Oceanogr. 2013;2013:482451. https://doi.org/10.1155/2013/482451.
47 Prabhakaran K, Nagarajan R, Merlin Franco F, Anand Kumar A. Biomonitoring of Malaysian aquatic environments: a review of status and prospects. Ecohydrol Hydrobiol. 2017;17:134–147. https://doi.org/10.1016/j.ecohyd.2017.01.001.
48 Bhat SA, Singh J, Singh K, Vig AP. Genotoxicity monitoring of industrial wastes using plant bioassays and management through vermitechnology: a review. Agric Nat Resour. 2017;51:325–337. https://doi.org/10.1016/j.anres.2017.11.002.
49 Grela E, Kozłowska J, Grabowiecka A. Current methodology of MTT assay in bacteria—a review. Acta Histochem. 2018;120:303–311. https://doi.org/10.1016/j.acthis.2018.03.007.
Downloads
Published
Issue
Section
License
Copyright (c) 2025 Garba Uba, Mohd Izuan Effendi Halmi, Motharasan Manogaran
This work is licensed under a Creative Commons Attribution 4.0 International License.
Authors who publish with this journal agree to the following terms:
- Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0) that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal.
- Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal.
- Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).
