1. INTRODUCTION
Aquatic toxicology is a field of environmental science that studies the effects of chemical pollutants on aquatic organisms, contributing to the biocomplexity with large number of species in various levels of biological organisations including fish, invertebrates, and algae. Traditional aquatic toxicological studies often focus on finding the acute lethal concentrations and chronic, sublethal and subacute impacts of the contaminants on the behaviour, physiology, histology, and ecology of aquatic organisms with a multidisciplinary approach. However, with the rapid advancements in genomic technologies, there has been a shift toward understanding how pollutants affect organisms at the genetic and molecular levels. The field of aquatic toxicology has experienced a significant transformation in the last decades with the emergence of omics technologies, such as genomics, transcriptomics, proteomics, and metabolomics, those technologies offer a comprehensive and high-throughput approach to studying the complex interactions between aquatic organisms and their environment. Within the developments of molecular biology methods, “omics” approaches have been booming worldwide since the early 1990s. Omics refers to a field of study in biology that aims to comprehensively analyze biological systems by examining various molecules or components within an organism or ecosystem. Molecular techniques have been intensively used for mammalians, especially in humans, but its usage in aquatic toxicology is very recent and will provide us with a more clearly understanding of the molecular mechanisms underlying the effects of environmental contaminants on aquatic organisms.
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“Ecotoxicogenomics term describes the studies analyzing the adaptive response to toxic exposure at the transcriptomic, proteomic, and metabolomic levels”. In aquatic toxicology, omics approaches have become increasingly important for understanding the complex interactions between aquatic organisms and their environment with the impacts of contaminants on aquatic organisms and ecosystems. The use of biomarkers (“A biochemical, cellular, physiological, or behavioral variation that can be measured in tissue or body fluid samples or at the level of whole organisms that provides evidence of exposure to and/or effects of, one or more chemical pollutants (and/or radiations)”), early warning indicators of the effects of the pollutants, are always anticipated by early responses at the genome level in the high hierarchical levels (Depledge and Fossi 1994, Van der Oost et al. 2003). The hierarchy of toxicant actions in aquatic systems is summarized in Figure 1. As seen in Figure 1; (1) Toxic chemicals first affect the individual/or their genomes. The effects of the toxicants on population can also be observed if exposure to the toxicant directly or indirectly affects the reproduction or the mortality of individuals. (2) The populations affect the ecosystem, because of species interactions. (3) Individuals’ genomes are affected toxicologically relevant by functional responses of organisms. Hence, toxicants affect individuals of some species biologically, making functional responses of individuals primary in the hierarchy of toxicant effects (Nikinmaa, 2014). 
Figure 9.1. The Toxicant Actions Hierarchy (Source: Nikinmaa, 2014)
The aim of this LO is to present an overview of the latest case studies of aquatic toxicology using omics approach in vitro methods emphasizing the ecotoxicological effects on genomics, transcriptomics, proteomics, and metabolomics changes (Figure 9.2). 
Figure 9.2. The Omics Technologies in Aquatic Toxicology (Source: Kayode-Edwards et al. 2024)
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2. Context (Findings)
2.1. Genomics in Aquatic Toxicology
Genomics in aquatic toxicology allows researchers to investigate the molecular mechanisms underlying toxic responses, offering a deeper and more comprehensive understanding of how pollutants affect aquatic ecosystems. Genomics provides valuable insights into how pollutants influence gene expression, mutation, and overall genomic integrity in aquatic organisms, can be used to determine which genes in an organism’s entire genome are involved in its lifespan. With the development of technology, the genetic codes of many aquatic organisms have been studied (Nam et al. 2023). Initially, fugu (Takifugu rubripes) and pufferfish (Tetraodon nigroviridis) considered as the best genomic models because of their compact genome size compared to the human genome followed by the completion of the genome sequencing of three-spined stickleback (Gasterosteus aculeatus) and zebrafish (Danio rerio) (Aparicio et al. 1995; Baxendale et al. 1995, McKinnon and Rundle 2002, Tickle and Cole 2004, Peichel 2005, Howe et al. 2013, Ahmad et al. 2022). The recent genomics case studies on aquatic toxicology are revised in Table 9.1.
Table 9.1. The recent genomics case studies on aquatic toxicology
| Aquatic Organisms | Toxicant | Results | More learn from References |
| Danio rerio | Cadmium | DNA methylation occurs in cadmium exposure and epigenetic changes occur in estrogen-sensitive genes. | Pierron et al. 2023 |
| Danio rerio | Antimony | Increased non-synonymous single nucleotide polymorphisms (SNPs) in coding gene regions. | Yao et al. 2023 |
| Danio rerio | Dibutyl phthalate | Increased oxidative stress due to substance exposure disrupted mitochondrial functions, resulting in oxidative stress in mitochondria. | Fan et al. 2024 |
| Mytilus galloprovincialis | Polyethylene terephthalate microparticles | Increased DNA methylation and increased toll-like receptor gene expression were observed in mussels with microplastic exposure. | Park et al. 2024 |
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2.2. Transcriptomics in Aquatic Toxicology
Transcriptomics focuses on the studies of an organism’s entire transcriptome, including all the RNA molecules transcribed from its genome studies of the whole transcriptome of organisms. The methods used in transcriptomic analysis include quantitative real-time polymerase chain reaction (PCR) and microarray analysis and the studies initiated back in the early 1990s’ (Solanke and Kanika, 2015). It may be possible to reduce animal testing and speed up chemical screening by using transcriptomics to characterize, categorize, and predict the toxicity of chemicals in vitro. Transcriptomic responses obtained from organisms taken from natural habitats or from organisms in controlled experiments in the laboratory environment are the responses of the cells and therefore the organism to these stress factors (Jeffrey et al. 2023). When combined with other bioindicators, transcriptomic studies also enable the identification of molecular biomarkers that may be employed for effective chemical exposure monitoring in aquatic organisms. The transcriptomic analyses can reflect the physiological changes in organisms at the molecular levels, with the development of large-scale and high-throughput methods (Kayode-Edwards et al. 2024). The recent transcriptomics case studies on aquatic toxicology are revised in Table 9.2.
Table 9.2. The recent transcriptomics case studies on aquatic toxicology
| Aquatic Organisms | Toxicant | Results | More learn from References |
| Cyprinus carpio | Silver nanoparticles (AgNPs) | Transcriptome and metabolome levels of fish gill were triggered by AgNPs after a 24 h exposure. | Xiang et al. 2021 |
| Mytilus edulis | Hypoxic stress | Changes in transcriptomes responsible for organelle activities were observed during the early stages of hypoxic conditions. | Hall et al. 2023 |
| Mytilus galloprovincialis | Decabromodiphenyl ethane | Genes related to cholesterol homeostasis were changed in female and male individuals under the effect of the substance. In addition, effects occurred on reproductive genes. | Wang et al. 2023 |
| Sander vitreus | Hypoxic stress | Hypoxic conditions have been shown to be associated with transcriptomes related to protein catabolism, DNA repair, molecular chaperones, and ion regulation. | Jeffrey et al. 2023 |
| Platax teira | Heat stress | Differentially expressed genes in cell division and metabolism events in fish under heat stress were identified. | Liu et al. 2023 |
| Mytilus trossulus | Norfluoxetine | In females, serotonin synthesis and transport were stimulated, accelerating gamete formation. In males, serotonin levels decreased, delaying sperm maturation. Thus, transcriptomic analyses revealed that the substance had effects on gametogenesis. | Goździk et al. 2024 |
Learn: Fish Transcriptomic Responses in Environmental Toxicology
2.3. Proteomics in Aquatic Toxicology
Proteomic approaches, which allow the examination of post-transcriptional can provide valuable insights into harmful mechanisms of xenobiotics and aid in biomarker discovery. (Wang et al. 2023b). Proteomic studies with three main technologies of electrophoresis, chromatography, and mass spectrometry, obtained the results through interpretation of bioinformatics. Term of “ecotoxicoproteomics” is a trending study area developed recent decades, as a powerful tool for analysing predetermined proteins across different samples (Gajahin Gamage et al. 2022). Proteomics in aquatic toxicology focuses on understanding how exposure to toxicants, such as heavy metals, pesticides, endocrine-disrupting chemicals (EDCs), and pharmaceuticals, affects the proteome the entire set of proteins expressed by an organism at a given time. The recent proteomics case studies on aquatic toxicology are revised in Table 9.3.
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Table 9.3. The recent proteomics case studies on aquatic toxicology
| Aquatic Organisms | Toxicant | Results | More learn from References |
| Chlorella sp. | Alpha-cypermethrin | 53 proteins were identified that showed differential accumulation with substance exposure in important cellular metabolic events such as photosynthesis, carbohydrate metabolism, cell division, and lipid metabolism. | Chanu et al. 2023 |
| Danio rerio (embryo) | Benzyl benzoate | 83 differentially expressed proteins were found to be involved in different biological activities including translation, amide biosynthetic process, lipid transport, stress response, and cytoskeletal activity. | Kwon et al. 2023 |
| Alosa pseudoharengus, Myoxocephalus thompsonii, Salvelinus namaycush | Per- and polyfluoroalkyl substances (PFAS) | PFOS-exposure was found to contain similar serum proteins in all three fish species. Albumin was found to be observed only in Salvelinus namaycush. Apolipoproteins were found to be the primary serum protein for the other two species. | Point et al. 2023 |
| Danio rerio (embryo) | Copper | Caused higher proteome differentiation in fish embryos. Apart from oxidative stress, cell respiratory events and neurotransmission, proline, glycine and alanine amino acids were shown to cause differentially expressed proteins. | Green et al. 2024 |
| Danio rerio | Glyphosate and its metabolits aminomethylphosphonic acid | Proteome changes were observed in cellular respiration events, carbohydrate and lipid metabolism reactions to substance exposure. | Morozov & Yurchenko 2024 |
2.4. Metabolomics in Aquatic Toxicology
Metabolomic analysis can provide insights into the metabolic pathways perturbed by exposure to contaminants, identify metabolic biomarkers of exposure or effect and gain a better understanding of the physiological responses of organisms to pollutants (Figure 9.3). 
Figure 9.3. The schema of the various data associations that link exposures highlights the use of metabolomics to study the molecular reactions to exposure to chemical pollutants. (Source: Bedia 2022)
Metabolomics, the comprehensive analysis of metabolites in biological systems, has become a critical tool in toxicology, especially in aquatic toxicology, for understanding the molecular effects of environmental contaminants on aquatic organisms. Unlike other omics technologies, it is a method close to the cellular phenotype (Olesti et al. 2021). Because it enables the simultaneous profiling of the metabolome (“endogenous metabolites whose levels are altered due to an external stressor”) and the xenometabolome (“chemical xenobiotics and their metabolites accumulated in an organism exposed to environmental contaminants”), environmental (xeno)metabolomics offers a significant advantage over other approaches for the evaluation of aquatic organisms’ exposure to contaminated water. Environmental (xeno)metabolomics has only recently begun to be used in field studies, although this method has been extensively investigated in laboratory exposure experiments (Gil-Solsona et al. 2021). The recent metabolomics case studies on aquatic toxicology are revised in Table 9.4.
Table 9.4. The recent metabolomics case studies on aquatic toxicology
| Aquatic Organisms | Toxicant | Results | More learn from References |
| Daphnia magna | Fenoxycarb | Metabolites occurring during the reproductive cycle change. | Jeong and Simpson, 2020 |
| Danio rerio | Bisphenol A | Lipid derivatives (triglyceride, diglyceride, phosphatidylcholine and phosphatidylinositol) concentrations increased. | Martinez et al. 2020 |
| Danio rerio | Polystyrene microplastics | Substance exposure has been found to have effects on lipid metabolism. | Dimitriadi et al. 2021 |
| Danio rerio | Heat stress Polystyrene microplastics | Substance exposure has been found to have effects on lipid metabolism including arachidonic, oleic and stearidonic acid. | Sulukan et al. 2022 |
| Danio rerio (embryos) | 6-propyl-2-thiouracil | The metabolome provides evidence for the induction of (neuro-)developmental toxicity. 6-Propyl-2-thiouracil (PTU) decreased methionine levels, increased tyrosine, pipecolic acid and lysophosphatidylcholine levels. | Wilhelmi et al. 2023 |
| Daphnia magna | Ti3C2Tx | Caused significant changes in up to 265 and 191 differential metabolites and these metabolites are substances involved in lipid and amino acid metabolism | Xiang et al. 2024 |
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3. ALTERNATİVES
In aquatic toxicity studies, traditional toxicity tests were carried out in vivo models of aquatic organisms. In recent years, alternative models have been developed due to the development of in vitro techniques and computer-based models of systems.  Cell culture-based methods can be useful for assessing the toxic effects of the toxicants, providing high-throughput screening of various chemicals together. These ways can prevent ethical problems, reduce costs and get faster results in a short time. For example, contaminant analyzes can be performed with cell cultures developed from fish organisms. These models, as well as genomics, proteomics, and metabolomics, can redefine aquatic toxicology by providing in-depth insights into the biological reactions of species to pollutants. Thus, these methods enable the identification of new biomarkers and pathways affected by toxic substances. In silico models and machine learning have emerged as crucial instruments for improving predictive toxicological results in last decades without relying on live organisms. In silico approach can estimate toxicity based on chemical structure and known biological interactions, reducing the need for extensive animal testing. In addition, Quantitative structure–activity relationship (QSAR) models and multi-task deep learning algorithms can correlate chemical structure with biological activity, enabling risk assessments based on existing data (Son et al. 2024).
4. SOLUTIONS
4.1. Integration of Omics Technologies
Integration of omics technologies enhances the understanding of complex and adverse responses of toxic effects, allowing for a more holistic view of organism responses to pollutants (Calabrese et al., 2023). Data from different omics technologies are used to comprehensively analyse complex biological interactions (John Martin et al. 2024). The goals of toxicity studies are to prevent or manage adverse health effects on the organism by comprehensively studying the basic mechanisms, especially in xenobiotic-induced toxicity (Joseph 2017). The integration of omics technologies in the environment summarized in Figure 9.4. 
Figure 9.4. Integration of omics technologies in the environment (Source: Colli-Dula et al. 2022)
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4.2. Limitations and challenges of omics in the aquatic toxicology
Although omics techniques have efficiently been used to assess the effects of toxicants on aquatic organisms, improvements are still required for practical application in regulatory and monitoring programs. The main limitations and challenges of omics in aquatic toxicology are as follows:
- Natural variability (biological and environmental variations)
- Interpretation of data
- Limited availability of the reference databases for non-model aquatic species
- Data complexity and the difficulty understanding the interaction between various biological pathways and toxic effects
- Lack of standardization and validation of the omics technologies
- Assessing the complex mixtures of pollutants in the aquatic environments
5. RECOMMENDATIONS (CONCLUSIONS)
Omics technologies have many uses in aquatic toxicology, such as identifying biomarkers for environmental monitoring, understanding species-specific responses to contaminants, and analyzing the effects of complicated pollutant mixtures. These technologies are useful for generating the mechanistic view to the biological responses associated with aquatic animal health and exposure to environmental stressors and conservation measures for endangered aquatic species. The integration of omics data with other environmental data, such as water quality parameters and ecological assessments, will enhance our ability to predict and mitigate the impacts of pollutants on aquatic ecosystems. Advances in omics have important implications for risk assessment practice and regulatory decision-making. Advances in sequencing technologies and bioinformatics tools are making omics analyses more accessible and cost-effective. Well-defined standard protocols that incorporate quality controls and validity testing will be necessary for the future integration of omics data in risk assessment and management.
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