Ecotoxicological studies of direct and indirect genotoxicity with Artemia: a integrative review.

Artemia is a brine shrimp genus adapted to extreme habitats like ranges salinity from 5-25 g/L and in temperatures from 9 to 35 °C. It is widely distributed and used as an environmental quality biomarker. Artemia franciscana and Artemia salina species are commonly used in ecotoxicological studies and genotoxicity assays due to their short life cycle, high fecundity rate, easy culture, and availability. Thus, considering the importance of these tests in ecotoxicological studies, the present study aimed to present Artemia genus as a biological model in genotoxicity research. To this end, we reviewed the literature, analyzing data published until July 2023 in the Web of Science, SCOPUS, Embase, and PubMed databases. After screening, we selected 34 studies in which the genotoxicity of Artemia for various substances. This review presents the variability of the experimental planning of assays and biomarkers in genotoxicity using Artemia genus as a biological model for ecotoxicological studies and show the possibility of monitoring biochemical alterations and genetic damage effects. Also highlight innovative technologies such as transcriptomic and metabolomic analysis, as well as studies over successive generations to identify changes in DNA and consequently in gene expression.

Ecotoxicological evaluation of an aqueous phytoextract of Melia azedarach L.

Melia azedarach L. is a Meliaceae that has shown important insecticidal activities. However, few researchers have extensively studied the toxicology of aqueous extracts of M. azedarach (MAE). Therefore, the main objective of this study was to characterize the phyto-eco-toxicological profile of MAE. First, a botanical and phytochemical characterization of MAE was performed using a histological, and metabolomic multi-analytical approach. Second, the toxicological effects on pollinating insects (Apis mellifera ligustica) and soil collembola (Folsomia candida) were evaluated. In addition, acute toxicity was evaluated in zebrafish (Danio rerio) to assess effects on aquatic fauna, and toxicity was determined in human neuroblastoma (SH-SY5Y) and fibroblast (FB-21) cell models. Finally, phytotoxic effects on germination of Cucumis sativus L., Brassica rapa L. and Sorghum vulgare L. were considered. Metabolomic analyses revealed the presence of not only limonoids but also numerous alkaloids, flavonoids and terpenoids in MAE. Histological analyses allowed us to better localize the areas of leaf deposition of the identified secondary metabolites. Regarding the ecotoxicological data, no significant toxicity was observed in bees and collembola at all doses tested. In contrast, severe cardiac abnormalities were observed in zebrafish embryos at concentrations as low as 25 µg/mL. In addition, MAE showed toxicity at 1.6 µg/mL and 6.25 µg/mL in FB-21 and SH-SY5Y cells, respectively. Finally, MAE inhibited seed germination with inhibitory concentrations starting from 5.50 µg/mL in B. rapa, 20 µg/mL in S. vulgare, and 31 µg/mL in C. sativus. Although M. azedarach extracts are considered valuable natural insecticides, their ecological impact cannot be underestimated. Even the use of an environmentally friendly solvent (an aqueous solution), for the first time, is not without side effects. Therefore, the data collected in this study show the importance of evaluating the dosages, modes of administration and production methods of M. azedarach phytoextracts in agricultural settings.

Silence is not always golden: A closer look at potential environmental and ecotoxicological impacts of large-scale dsRNA application.

RNA interference (RNAi) technology has emerged as a pivotal strategy in sustainable pest management, offering a targeted approach that significantly mitigates the environmental and health risks associated with traditional insecticides. Originally implemented through genetically modified organisms (GMOs) to produce specific RNAi constructs, the technology has evolved in response to public and regulatory concerns over GMOs. This evolution has spurred the development of non-transgenic RNAi applications such as spray-induced gene silencing (SIGS), which employs double-stranded RNA (dsRNA) to silence pest genes directly without altering the plant's genetic makeup. Despite its advantages in specificity and reduced ecological footprint, SIGS faces significant obstacles, particularly the instability of dsRNA in field conditions, which limits its practical efficacy. To overcome these limitations, innovative delivery mechanisms have been developed. These include nanotechnology-based systems, minicells, and nanovesicles, which are designed to protect dsRNA from degradation and enhance its delivery to target organisms. While these advancements have improved the stability and application efficiency of dsRNA, comprehensive assessments of their environmental safety and the potential for increased exposure risks to non-target organisms remain incomplete. This comprehensive review aims to elucidate the environmental fate of dsRNA and evaluate the potential risks associated with its widespread application on non-target organisms, encompassing soil microorganisms, beneficial insects, host plants, and mammals. The objective is to establish a more refined framework for RNAi risk assessment within environmental and ecotoxicological contexts, thereby fostering the development of safer, non-transgenic RNAi-based pest control strategies.

Transferring knowledge across aquatic species via clustering techniques to unravel patterns of pesticide toxicity.

In silico modelling takes the advantage of accelerating ecotoxicological assessments on hazardous chemicals without conducting risky in vivo experiments under ethic regulation. To date, the prevailing strategy of one model for one species cannot be well generalized to multi-species modelling. In this work, we propose a new strategy of one model for multiple species to facilitate knowledge transfer across aquatic species. The available lethal concentration values of 4952 pesticides on 651 fish species are aggregated into one toxicity response matrix, purely through which we attempt to unravel fish toxicosis-phylogenesis relationships and pesticide toxicity-structure relationships via clustering techniques including non-negative matrix factorization (NMF) and hierarchical clustering. The clustering results suggest that (1) close NMF weights indicate close species-toxicosis and pesticide-toxicity profiles; (2) and that species toxicosis patterns are related with species phylogenetic relationships; (3) and that close pesticide-toxicity profiles indicate similar atom-pair structural fingerprints. These environmental, chemical and biological insights can be used as expert knowledge for environmentalists to manually gain knowledge about untested species/pesticides from tested species/pesticides, and meanwhile provide support for us to build in silico models from species phylogenetic and pesticide structural points of view. Besides unravelling the mechanisms behind toxicity response, we also adopt stratified cross validation and external test to validate the reliability of using NMF to predict missing toxicity values. Independent test on external data shows that NMF achieves 0.8404-0.9397 R2 on four fish species. In the context of toxicity prediction, non-negative matrix factorization can be viewed as a model based on quantitative activity-activity relationships (QAAR), and provides an alternative approach of inferring toxicity values on untested species from tested species.

Ecological risk assessment methods for oxidative by-products in the oxidation degradation process of emerging pollutants: A review.

The inherent toxicity and persistence of emerging contaminants such as antibiotics and endocrine disruptors pose substantial threats to the environment. Advanced oxidation processes (AOPs) employed for oxidative degradation could yield toxic oxidation by-products (OBPs), including organic acids and aromatic hydrocarbons. Despite their typically low concentrations, OBPs require scrutiny owing to their potential health risks. Although effective assessment methodologies are available, a comprehensive review focusing on the ecological and environmental effects of these pollutants is lacking. This study offers a succinct overview of existing ecotoxicological exposure assessments for emerging organic pollutants. Further, it encapsulates principal dose-response assessment techniques and provides a comparative analysis of several methods. The straightforward assessment factor method evaluates risk based on exposure and species sensitivity and is suitable for preliminary assessments of single pollutants; Species Sensitivity Distribution (SSD) compares species sensitivities to OBPs, emphasizing the importance of species-specific toxicological responses; microcosm and mesocosm methods simulate and predict the effects of OBPs on aquatic life by considering environmental diversity and biological community structures and are ideal for assessing the toxicity of multiple OBPs; the ecological risk analysis model employs mathematical and probabilistic approaches to comprehensively and accurately assess exposures and effects, accounting for the complexities and uncertainties inherent in ecotoxicological evaluations. Different risk characterization techniques are outlined in this study, including the risk quotient (RQ), which is ideal for quantifying and comparing risks; probabilistic ecological risk assessment (PERA), suitable for managing significant uncertainty; and the Environmental Pollution Index (EPI), the preferred method for quantitative assessment of OBP pollution levels. The merits and limitations of each of these quantitative assessment tools are evaluated, providing a comprehensive view of their applications in risk analysis. In addition, pressing contemporary challenges are identified and trajectories and pivotal issues suggested for future research.

Ecotoxicological assessment, in freshwater environment, of wastewater sludge coupled and uncoupled with micro-polyvinyl chloride on algae and water fleas.

In the frame of bioeconomy and circular economy, wastewater sludge (WS) could be a good candidate for its use in agriculture as fertilizer, due to its high content of organic matter, N and P, but on the other hand, it is full of toxicants such as heavy metal, microplastics, detergent, antibiotics, and so on that can reach groundwater and water bodies in leachate form. In this study, we have investigated different sludge concentrations in the eluate form, combined and not with PVC on two different freshwater organisms Selenastrum capricornutum and Daphnia magna, using ecotoxicity tests. At the endpoint, we have evaluated inhibition growth rate, oxidative stress, and pigments production for S. capricornutum, while in case of D. magna, we have assessed organism immobilization and development. From our results, it emerged that at the higher WS concentration, there was not inhibition growth rate, while at oxidative stress, it was higher in algae treated with WS and PVC. Higher Chl-a production was shown for algae treated with 0.3 g/L of sludge coupled with PVC, where higher phaeopigments production were recorded for algae treated with 0.3 g/L of WS. D. magna has shown an opposite trend when compared with algae, where at the highest WS concentrations supplied was corresponding to an increased mortality explaned as the highest immobility percentage. PRACTITIONER POINTS: Wastewater sludge is used in agriculture as fertilizer. PVC microplastic presence and associate ecotoxicity was tested. PVC presence increased oxidative stress in S. capricornutum. D. magna was significantly affected by sludge concentrations supplied.

Oxidation of metazachlor herbicide by ozone in the gas and aqueous phases: mechanistic, kinetics, and ecotoxicity studies.

The atmospheric and aqueous ozonolysis of metazachlor (MTZ) is investigated using high-level quantum chemical and kinetic calculations (M06-2X/6-311 + + G(3df,3pd)//M06-2X/6-31 + G(d,p) level of theory). The ozone (O3)-initiated degradation pathways of MTZ under three different mechanisms, namely cycloaddition, oxygen-addition, and single electron transfer (SET), are explored in the temperature range of 283-333 K and 1 atm pressure. As a result, the cycloaddition reaction at the C16C18 double bond of the benzene ring of MTZ is found to be the most dominant channel in the atmosphere with the standard Gibbs free energy of reaction (ΔrG0g) of - 129.13 kJ mol-1 and the highest branching ratio of 95.18%. In the aqueous phase, the main reaction channel turns into the SET mechanism, which owns the lowest Gibbs free energy of activation (ΔG#aq) of 73.8 kJ mol-1 and contributes 87.8% to the ktotal. Over the temperature range of 283-333 K, the total rate constant (ktotal) significantly increases from 8.42 to 5.82 × 101 M-1 s-1 in the atmosphere and from 4.10 × 102 to 2.40 × 104 M-1 s-1 in the aqueous environment. Remarkably, the ecotoxicity assessment shows that MTZ may be harmful to fish and chronically harmful to daphnia. In contrast, its main ozonolysis products exhibit no acute or chronic toxicity or mutagenic effects.

Multi-task pretrained language model with novel application domains enables more comprehensive health and ecological toxicity prediction.

In silico models for screening substances of healthy and ecological concern are essential for effective chemical management. However, current data-driven toxicity prediction models confront formidable challenges related to expressive capacity, data scarcity, and reliability issues. Thus, this study introduces TOX-BERT, a SMILES-based pretrained model for screening health and ecological toxicity. Results show that masked atom recovery pretraining and multi-task learning offer promising solutions to enhance model capacity and address data scarcity issues. Two novel application domain (AD) parameters, termed PCA-AD and LDS, were proposed to improve prediction reliability of TOX-BERT with accuracy surpassing 90 % and mean absolute error (MAE) below 0.52. TOX-BERT was applied to 18,905 IECSC chemicals, revealing distinct toxicity relationships that align with experimental studies such as those between cardiotoxicity and acute ecotoxicity. In addition to previous PBT screening, 156 potential high-risk chemicals for specific endpoint were identified covering 7 categories. Furthermore, a SMILES-based toxicity site detection approach was developed for structural toxicity analysis. These advancements carry profound implications to address challenges faced by current data-driven toxicity prediction models. TOX-BERT emerges as a valuable tool for more comprehensive, reliable, and applicable predictions of health and ecological toxicity in chemical risk assessment and management.

Ecotoxicity assessment of ashes from calcium-rich fuel combustion: contrasting results and regulatory implications.

The European Union's (EU) regulation for the waste classification based on their ecotoxicological hazard (hazardous property HP14) came into force on 5 July 2018. The regulation advocates the utilisation of computational formulae for the hazard classification of waste associated with hazardous property HP14. Concurrently, ecotoxicological testing remains an alternative. To date, the absence of a consensus regarding test organisms and methodologies has vested EU member states with autonomy in determining the approach for conducting ecotoxicity assessments. The discussions on waste classification are also ongoing globally, namely the discussions under the Basel Convention. This paper endeavours to elucidate whether the widely employed test organisms, Daphnia magna and Aliivibrio fischeri, may serve as suitable indicators for the evaluation of the ecotoxicity of waste. The research is grounded in the examination of ashes derived from a combustion process of calcium-rich fuel. Ecotoxicity testing was conducted on 14 ash samples with a liquid-to-solid ratio of 10:1. The results of the Aliivibrio fischeri testing indicated that all 14 ash samples were non-hazardous in terms of their ecotoxicity. However, the results of the Daphnia magna testing showed the opposite, suggesting that the ash samples may have the potential to be ecotoxic. This study offers valuable insights into ecotoxicity assessment and waste classification, emphasising the need for scientific rigour and comprehensive understanding before making regulatory decisions. It also situates its findings within the broader global context of waste management discussions, particularly those related to international agreements like the Basel Convention.

Ecotoxicological Effects of Microplastics Combined With Antibiotics in the Aquatic Environment: Recent Developments and Prospects.

Both microplastics and antibiotics are commonly found contaminants in aquatic ecosystems. Microplastics have the ability to absorb antibiotic pollutants in water, but the specific adsorption behavior and mechanism are not fully understood, particularly in relation to the impact of microplastics on toxicity in aquatic environments. We review the interaction, mechanism, and transport of microplastics and antibiotics in water environments, with a focus on the main physical characteristics and environmental factors affecting adsorption behavior in water. We also analyze the effects of microplastic carriers on antibiotic transport and long-distance transport in the water environment. The toxic effects of microplastics combined with antibiotics on aquatic organisms are systematically explained, as well as the effect of the adsorption behavior of microplastics on the spread of antibiotic resistance genes. Finally, the scientific knowledge gap and future research directions related to the interactions between microplastics and antibiotics in the water environment are summarized to provide basic information for preventing and treating environmental risks. Environ Toxicol Chem 2024;43:1950-1961. © 2024 SETAC.

Evaluating non-monotonic dose-response relationships in ecotoxicological risk assessment: A case study based on a systematic review of data on fluoxetine.

The environmental presence of pharmaceuticals, including the antidepressant fluoxetine, has become a subject of concern. Numerous studies have revealed effects of fluoxetine at environmental concentrations. Some of these studies have reported non-monotonic dose-response curves (NMDRs), leading to discussion because of the inconsistent detection of subtle effects and lack of mechanistic understanding. Nevertheless, investigating NMDRs in risk assessment is important, because neglecting them could underestimate potential risks of chemicals at low levels of exposure. Identification and quantification of NMDRs in risk assessment remains challenging, particularly given the prevalence of single outliers and the lack of sound statistical analyses. In response, the European Food Safety Authority (Beausoleil et al., 2016) presented a framework delineating six checkpoints for the evaluation of NMDR datasets, offering a systematic method for their assessment. The present study applies this framework to the case study of fluoxetine, aiming to assess the weight-of-evidence for the reported NMDR relationships. Through a systematic literature search, 53 datasets were selected for analysis against the six checkpoints. The results reveal that while a minority of these datasets meet all checkpoints, a significant proportion (27%) fulfilled at least five. Notably, many studies did not meet checkpoint 3, which requires NMDRs to be based on more than a single outlier. Overall, the current study points out a number of studies with considerable evidence supporting the presence of NMDRs for fluoxetine, while the majority of studies lacks strong evidence. The suggested framework proved useful for analysing NMDRs in ecotoxicological studies, but it is still imperative to develop further understanding of their biological plausibility.

Cement pastes containing air entraining agent release the biocide octylisothiazolinone posing ecotoxicological effects.

We investigated whether cement pastes are a possible source of ecotoxicologically potent substances. For this purpose, leaching according to DIN EN 16637-2 was performed on portland cement pastes as well as blast furnace slag cement with and without an air entraining agent (AEA). The AEA, consisting of wood rosin and resin, contained the stabiliser drometrizole and the biocide octylisothiazolinone (OIT), which was confirmed by our non-target screening (NTS). Our ecotoxicological studies (Daphnia magna, Aliivibrio fischeri and Desmodesmus subspicatus) of the pure cement eluates showed no effects at all. In these samples, it was possible to attribute up to 85 % of the dissolved organic carbon (DOC) to acetate, formate and diethylene glycol (DiEG). Eluates from cement pastes with AEA contained up to 70 µg/L octylisothiazolinone (OIT), and no drometrizole was found. Around 90 % of the total OIT release happened within the first 6 h. It was possible to attribute the observed ecotoxicological effects mainly to the OIT concentrations. Additional leaching with elevated sulphate concentrations (800 mg/L) did not influence the release of DOC and OIT or increase the ecotoxicological effects. As a consequence, we advise curing the cement paste for 24 h prior to use, as this largely avoids the release of OIT and the observed ecotoxicological effects.

Assessing the presence of microplastic in agriculture soils irrigated with treated waste waters using Lumbricus sp.: Ecotoxicological effects.

Global water scarcity entailed the use of treated wastewater (TWW) in agriculture, however, this water can vehiculate numerous pollutants into soil and further crops such as microplastics (MPs). To date, few studies had quantified the accumulation of MPs in soils and earthworms after irrigation with TWW as well as their toxicological effects. Hence, the main objective of the present work is to evaluate the toxicity of MPs using Lumbricus sp. earthworms collected from TWW irrigated soils with an increasing gradient of time (5 years, 16 years and 24 years). MPs determination in soil, as well as in earthworms were performed. The intestinal mucus was quantified, and cytotoxicity (Lysosomal membrane stability (LMS), Catalase (CAT) and glutathione-S-Transferase (GST) activities), neurotoxicity (Acetylcholinesterase activity (AChE)) and genotoxicity (Micronuclei frequency (MNi)) biomarker were assessed. Our results revealed that the use of TWW rendered MPs accumulation in earthworms' tissues and induce alteration on the intestinal mucus. An important cytotoxicity time-depending was observed being associated with an increase on genotoxicity. Overall, the present investigation highlights the ecotoxicological risk associated with the use of TWWs as an important driver of MPs and consequently measures are necessary to reduce MPs in wastewater treatment plans to improve this non-conventional water quality.

Unveiling the ecotoxicological effects of azoxystrobin-based fungicides at realistic concentrations on the land snail, Theba pisana.

The ecotoxicological consequences of azoxystrobin on land snails have not yet been addressed. Therefore, the present study aims to provide novel data on the threat of a commercial grade azoxystrobin (AMISTAR) at two environmentally relevant concentrations (0.3 µg/ml) and tenfold (3 µg/ml) on the model species, Theba pisana by physiological, biochemical, and histopathological markers for 28 days. Our results showed a reduction in animal food consumption and growth due to exposure to both azoxystrobin concentrations. It also induced oxidative stress and led to a significant decrease in lipid peroxidation (LPO) levels after 7 days of exposure, while the opposite effect occurred after 28 days. Except for the 7-day exposure, all treated snails had significantly reduced glutathione (GSH) content and increased catalase (CAT) activity at all-time intervals. Glutathione peroxidase (GPx), glutathione-S-transferase (GST) activities, and protein content (PC) were elevated in treated snails at all-time intervals. Moreover, alterations in acetylcholinesterase (AChE) activity between a decrease and an increase were noticed. Additionally, azoxystrobin exerted changes in T. pisana hepatopancreas architecture. Our study suggests that azoxystrobin may have negative ecological consequences for T. pisana and highlights its potential risks to the natural environment.

Ecotoxicological risk assessment of triclosan, an emerging pollutant in a riverine and estuarine ecosystems: A comparative study.

Triclosan (TCS), an antibacterial biocide, pervades water and sediment matrices globally, posing a threat to aquatic life. In densely populated cities like Mumbai, rivers and coastal bodies demand baseline TCS data for ecotoxicological assessment due to the excessive use of personal care products comprising TCS. This pioneering study compares spatiotemporal TCS variations and risks in freshwater and marine ecosystems employing multivariate analysis of physicochemical parameters. Over five months (January to May 2022), Mithi River exhibited higher TCS concentrations (water: 1.68 µg/L, sediment: 3.19 µg/kg) than Versova Creek (water: 0.49 µg/L, sediment: 0.69 µg/kg). Principal component analysis revealed positive correlations between TCS and physicochemical parameters. High-risk quotients (>1) underscore TCS threats in both water bodies. This study furnishes crucial baseline data, emphasizing the need for effective treatment plans for TCS in effluent waters released into the adjacent aquatic systems.

Bioactivity of Eugenol: A Potential Antibiotic Adjuvant with Minimal Ecotoxicological Impact.

Combining commercial antibiotics with adjuvants to lower their minimum inhibitory concentration (MIC) is vital in combating antimicrobial resistance. Evaluating the ecotoxicity of such compounds is crucial due to environmental and health risks. Here, eugenol was assessed as an adjuvant for 7 commercial antibiotics against 14 pathogenic bacteria in vitro, also examining its acute ecotoxicity on various soil and water organisms (microbiota, Vibrio fischeri, Daphnia magna, Eisenia foetida, and Allium cepa). Using microdilution methods, checkerboard assays, and kinetic studies, the MICs for eugenol were determined together with the nature of its combinations with antibiotics against bacteria, some unexposed to eugenol previously. The lethal dose for the non-target organisms was also determined, as well as the Average Well Color Development and the Community-Level Physiological Profiling for soil and water microbiota. Our findings indicate that eugenol significantly reduces MICs by 75 to 98%, which means that it could be a potent adjuvant. Ecotoxicological assessments showed eugenol to be less harmful to water and soil microbiota compared to studied antibiotics. While Vibrio fischeri and Daphnia magna were susceptible, Allium cepa and Eisenia foetida were minimally affected. Given that only 0.1% of eugenol is excreted by humans without metabolism, its environmental risk when used with antibiotics appears minimal.

Impacts of cattle management and agricultural practices on water quality through different approaches: physicochemical and ecotoxicological parameters.

The intensification of livestock farming can pose risks to the environment due to the increased use of veterinary products and the generation of waste in confined areas. The quality of water bodies near livestock establishments (Areco River (A) and Doblado stream (D), San Antonio de Areco, Buenos Aires, Argentina) was studied by physicochemical parameters, metals, pesticides, emerging contaminants, and lethal and sublethal toxicity (neurotoxicity and oxidative stress) in larvae of the native amphibian Rhinella arenarum. Six sites were selected: upstream (S1A and S1D), at the level (S2A and S2D), and downstream (S3A and S3D) from the establishments. A low concentration of dissolved oxygen was observed in Doblado stream (< 2.34 mg/L). Cu, Mn, V, and Zn exceeded the limits for the protection of aquatic life at various sites. Between 24 and 34 pesticides were detected in all sites, with 2,4-D, atrazine, and metolachlor being the most recurrent. In water and sediment, the concentrations of ivermectin (S2A, 1.32 µg/L and 58.18 µg/kg; S2D, 0.8 µg/L and 85.22 µg/kg) and oxytetracycline (S2A, < 1 mg/L and < 1 mg/kg; S2D, 11.8 mg/L and 39 mg/kg) were higher at sites near the establishments. All sites caused between 30 and 38.3% of lethality and produced neurotoxicity and alterations in the reduced glutathione content. Moreover, larvae exposed to samples from all sites incorporated ivermectin. These results demonstrate the degradation of the studied sites in relation to the agricultural activities of the area, highlighting the need to take measures to protect and preserve aquatic ecosystems.

Current research on ecotoxicity of metal-based nanoparticles: from exposure pathways, ecotoxicological effects to toxicity mechanisms.

Metal-based nanoparticles have garnered significant usage across industries, spanning catalysis, optoelectronics, and drug delivery, owing to their diverse applications. However, their potential ecological toxicity remains a crucial area of research interest. This paper offers a comprehensive review of recent advancements in studying the ecotoxicity of these nanoparticles, encompassing exposure pathways, toxic effects, and toxicity mechanisms. Furthermore, it delves into the challenges and future prospects in this research domain. While some progress has been made in addressing this issue, there is still a need for more comprehensive assessments to fully understand the implications of metal-based nanoparticles on the environment and human well-being.

Micronucleus test in reptiles: Current and future perspectives.

Micronucleus (MN) cell counting emerged in 1973-1975 as a valid alternative for characterizing chromosomal damage caused by different agents. It was first described in mammals, but its application was rapidly extended to other vertebrates, mainly fish. However, it was not until 28 years later that this test was implemented in studies on reptiles. Nowadays, reptiles are found to be excellent non-target species from environmental contamination exposure and MN test has become a fundamental tool for analyzing genotoxic effects induced by various xenobiotics. In this article we provide an updated review of the application of the MN test in reptile species, from an ecotoxicological perspective. Therefore, we present (I) a bibliometric analysis of the available research on genotoxic-induced MN formation in reptile species; (II) the use of reptiles as sentinel organisms in ecotoxicological studies; and (III) the strength and weakness of the application of the MN test in this group. With this review, we aim to provide a comprehensive view on the use of the MN test in ecotoxicology and to encourage further studies involving reptile species.