Background

Effect-based monitoring and assessment.  Traditional water quality monitoring and environmental status assessment focus on targeted analysis for priority substances, which only capture a fraction of potentially present chemicals. Moreover, many of these chemicals are often below the detection limit yet contribute to biological effects from exposure to complex low-level mixtures through different routes \cite{Vermeulen_2020}. Hence, the traditional approach alone is not sufficient to assess subtle or complex biological impacts in situ, under a myriad of confounding factors and at varying bio-availability of the environmental contaminants.
Effect-based methods are essential to directly assess the biological impact of contaminants in field and laboratory studies, and, particularly, in environmental surveys and monitoring \cite{Neale2023}. Measuring responses at the molecular or organismal level, offers early detection, identification of mixture effects, and a holistic understanding of ecosystem health, ensuring a more comprehensive assessment of environmental risks. However, there is a lack of biological data suitable for assessing risks associated with adverse effects or ecosystem perturbations caused by most environmental contaminants \cite{Parmar_2016}. Adding to this challenge is the inherent uncertainty in ecological risk assessments, especially concerning chemical mixtures. Hence, there is an urgent call for the development of advanced predictive tools to assess biological effects. Additionally, there is a critical demand for the integration of OMICS technologies (genomics, transcriptomics, proteomics, lipidomics, metabolomics, and adductomics), capable of generating extensive datasets, to facilitate the identification of effective biomarkers.
DNA adductome monitoring: insights from the Swedish experience. Since 2017, DNA adductome analysis was conducted jointly with the Swedish National Marine Monitoring Program (SNMMP) in the Baltic Sea, using amphipods  Monoporeia affinis and Pontoporeia femorata as sentinel species. In the context of SNMMP, the prevalence of embryo aberrations in sediment-dwelling amphipods serves as a key indicator of pollution effects. This indicator is based on the high sensitivity of embryo development to chemical exposure, leading to the manifestation of diverse developmental aberrations observable in gravid females \cite{Reutgard_2014,sea} used to assess the biological effects of contaminants in the Baltic Sea \cite{L_f_2016}. The measured embryo aberration rate serves as a specific health condition when evaluating the female DNA adductome, establishing baselines, and identifying biomarkers linked to an elevated risk of developmental disorders. Furthermore, the association between exposure and specific adductome profile can be categorized based on pollution loads in sediment at monitoring sites. Here, drawing from this experience, we offer insights into how DNA adductome can enhance the current effect-based assessment of biological effects in environmental monitoring.  

DNA adductomics: advancing environmental monitoring

OMICS-based ecosurveillance.  Current discussions center on integrating OMICS-based technologies into ecosurveillance monitoring frameworks. This integration aims to capture the comprehensive biological responses of ecosystems under perturbation \cite{Beale2022,Bahamonde2016}. These technologies and the data they provide have already enhanced our grasp of how environmental chemicals impact ecosystems and human health. However, despite progress, most environmental OMICS are currently in the data collection phase, with crucial gaps in linking toxicity data with OMICS endpoints \cite{Machuca-Sepúlveda2023a}. Future efforts are expected to address real environmental challenges, focusing on issues like chemical mixture toxicity, biomarker identification, baseline variability, and the development of approaches for deriving environmental quality standards from the OMICS data \cite{Henke2023,Machuca-Sepúlveda2023,Ebner2021} towards more effective risk monitoring, and sustainable natural resource utilization \cite{Beale2022}.
DNA adductome - an exposome component.  Adductomics, an emerging research field, provides structural insights into chemical exposures and serves as a platform for discovering biomarkers to identify both the occurrence of exposure and associated effects. DNA adductomics, one of the newest OMICS techniques, is particularly well suited for assessing exposure and effects of environmental contaminants \cite{Lockridge2023} and elucidating genotoxic and epigenetic changes due to chemical stressors. However, whereas the DNA adductome approach is well-established in human toxicology due to its direct relevance to human health \cite{Balbo2014,Villalta2017}, its use in environmental studies has been limited despite the wide acceptance of DNA adducts as exposure biomarkers in wildlife.  
DNA adducts are well-established biomarkers in (eco)toxicology. They are chemical modifications occurring when certain chemicals bind covalently to DNA molecules. Unrepaired DNA adducts can disrupt DNA structure and function, potentially leading to mutations and adverse biological effects \cite{Phillips_2009}. These adducts are associated with health issues, reproductive toxicity, genotoxicity, and epigenetic alterations in humans and wildlife. For the last 50 years, DNA adducts have been used as biomarkers of exposure in environmental toxicology, where the focus has mainly been on the adducts derived from polycyclic aromatic hydrocarbons (PAHs) in fish and mussels as exposure biomarkers \cite{Pampanin2017,Dolcetti2002}. In environmental toxicology, PAHs have been most commonly linked to DNA adduct formation \cite{Skarphédinsdóttir2007,Amat_2004,Meier2020}, both in the laboratory and in field observations after oil spills. However, other contaminants have also been found to induce DNA modifications \cite{Guilherme_2012,letters}. Also, in amphipods, abundant epigenetic DNA modifications have been associated with contaminated environments \cite{Martella2023} and females that carry embryos with various developmental disorders \cite{Gorokhova2020}. Thus, ample evidence supports the informative value of detecting and quantifying DNA adducts in biological samples for assessing contaminant exposure and genomic effects.
Traditional analytical methods for DNA adducts. The detection of DNA adducts has commonly been conducted using immunochemical \cite{Müller1982} and 32P-postlabelling \cite{Poirier2000} assays. Although useful, these assays do not provide chemical or structural information about the adducts to trace back to specific exposures. Moreover, these techniques have significant limitations: the immunochemical method is not sensitive enough to detect DNA adducts at trace levels and requires an antibody to each chemical. Albeit highly sensitive, 32P-postlabelling involves radioactive phosphorus usage, implying safety concerns and regulatory challenges  \cite{Hwa2020}. The method is also labour-intensive and further complicated by highly variable labelling efficiency \cite{Totsuka_1996}. Nowadays, researchers need to adhere to strict safety protocols for handling radioactive materials and waste disposal, which has led to a gradual disappearance of laboratories willing to conduct these measurements. Moreover, key limitations include the scarcity of information on the detected DNA adduct structure and occasional co-migration of adducts on the thin layer chromatography plate \cite{Phillips_2007}. These challenges collectively impede the chemical structural characterization and identification of the adducts.  
A novel approach to measure old biomarker. Nowadays, one of the most powerful techniques for detecting and quantifying DNA adducts is liquid chromatography-mass spectrometry (LC-MS). Using high-resolution mass spectrometry (HRMS), sensitive and selective analytical methods for detecting and identifying DNA adducts in the genome have been developed \cite{Hemeryck_2016} and successfully applied in human health research \cite{Hwa2020,Totsuka2021} and ecotoxicological \cite{Gorokhova2020,Martella2023} diagnostics of adverse effects due to chemical exposure. Instead of analyzing a few adducts from a specific chemical exposure ( (i.e., bottom-up approach), HRMS gives the possibility to screen for DNA adducts from multiple classes of exposure, an “adductomics” approach. Thus, this is a new OMICS approach, with both target and non-targeted analytical methods available to comprehensively investigate the adductome via screening for all known and unknown adducts in the genome (i.e., top-down approach). Various modification types, e.g., bulky PAH-adducts, methylation and oxidation, can be analyzed in a single sample by HRMS, including the determination of their chemical structures, which is useful for exposure diagnostics \cite{Balbo2014,Villalta2017}. Thus, the current capacity for DNA adduct characterization is superior to the classical assays, advocating this OMICS approach to detect and monitor the biological effects of contaminants \cite{Martella2023}.

Methodology

Using various cell types and matrices, several DNA adductomics methods based on LS-HRMS have been developed for applications in human toxicology \cite{Balbo2014a,Guo2018,Hemeryck2017}. These developments are poised to be applicable to any biological sample and allow proposing a workflow for low- and high-mass DNA adduct analysis in wildlife as a part of ecotoxicological surveillance \cite{Martella2023}.  
Test organisms. Any plant or animal tissue is suitable for the analysis, including the whole body (for small-sized planktonic and benthic animals as well as embryos), blood, liver and muscle tissues. If fertilized eggs or embryos are present in small specimens, they should be dissected out and either analyzed separately or not included in the female DNA samples to avoid the natural ontogenetic variability in the epigenetic DNA modifications related to embryogenesis \cite{Gorokhova2020}.  In fish, liver and muscle tissues are commonly used as test tissues, and more work is needed to identify target tissues and organs for specific adducts if we are to improve the diagnostic properties of the method.