# Off-flavors in Aguacultured Fish: Origins and Implications for Consumers
## 3 Off-Flavor Detection Methods  
Sensory analyzes have been widely used in the assessment of fish and drinking water quality. According to [PERSON] [50], standard methods include the use of tools, such as the odor threshold number (TON) and flavor profile analysis (FPA) for sensory analysis. These methods reflect the maximum level of dilution at which an odor is still perceptible. The revisions cited in [50] are based on gas chromatography coupled to mass spectrometry (GC/MS) with different strategies of extraction techniques that have been used to identify and quantify the types of several off-flavors [51; 52]. Headspace solid-phase microextraction (HSPME) has been recommended as a suitable method for volatile compounds' quantitation in source water, due to its better selectivity towards target compounds [17; 24]. Stir bar sorbent extraction (SBSE) is a technique that is highly sensitive to TOCs and has good reproducibility [53; 54]. In addition, several methods used to analyze TOCs more quickly and accurately are being developed [55; 56].  
Furthermore, molecular biology techniques have been studied to monitor and identify early microorganisms and genes involved in the production of TOCs in aquatic environments. In this sense, the _geo_A gene, which encodes the bi-functional enzyme GEO synthase, has been used as a molecular marker [20; 23; 56; 57]. Additionally, the 16S rRNA gene is often used to identify prokaryotic microorganisms and cyanobacteria and has been combined with other markers, such as _geo_A for molecular analysis (Table 2). In addition, researchers have created a reference database in order to monitor the toxinogenic taxa that form blooms and act as an effective early warning system for the growth of potentially harmful blooms.  
Genes involved in the synthesis of MIB were first identified in actinomycetes [58] and subsequently, these genes were also reported in cyanobacteria [20]. Phylogenetic analysis suggests that the MIB synthase gene was spread by horizontal transfer and that this gene has a common origin in cyanobacteria and actinomycetes. However, the organization of the genes was different in cyanobacteria, suggesting that recombination events may have occurred during evolution [59]. In any case, the biosynthetic mechanism of MIB occurs in two stages, first, a reaction occurs where geranyl diphosphate (GPP) is converted by methyltransferase into methyl-GPP, and then MIB synthase cyclizes methyl-GPP into MIB [58], while the GEO mechanism is related to the cyclization of the precursor sesquiterpene C15.  
Accurate diagnostic tools for detecting GEO and MIB producers are important in monitoring water reservoirs for quality purposes. The monitoring of microbial communities in aquaculture production can provide a tool for future microbial management in order to guarantee stability in fish production performance. In addition, biomolecular methods, such as conventional and quantitative PCR (qPCR), are widely used techniques. Furthermore, qPCR has been a great tool for the early detection and monitoring of cyanobacteria and toxins, especially when they are present in small quantities.